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Introduction Articular cartilage of higher animals does not regenerate, but under some circumstances mesenchymal cells can be inducted to behave phenotypically as chondrocytes. Tissue engineering has produced only partial cartilage repair and inconsistent outcomes. This study reports full thickness articular cartilage regeneration induced by freeze-dried particulate cartilage. Methods and Materials Full thickness cartilage defects in the femoral condyles of 22 baboons were filled with freeze-dried cartilage particles. Defects in two animals were filled with frozen cartilage particles. Four animals served as controls. Experimental animals were sacrificed at 2 weeks (2), 6 weeks (6), 9 weeks (1) 12 weeks (3), 14–16 weeks (4) and at 7 and 14 months. The knees were photographed and x-rayed. The condyles were frozen, sectioned, photographed again, fixed, decalcified and processed for histology. To determine if cartilage preparations were osteogenic samples were implanted intramusculary in 16 athymic rats. The specimens were examined at 1,2,4 & 8 weeks. Cartilage particles were also implanted intraosseously into 2 baboons. Regenerating cartilage was graded in accordance with a modified Mankin's method. Results Cartilage regeneration proceeded from the edges of the defect. The 12 week and older defects were covered with smooth and glistening new cartilage. Control defects remained open or contained uneven patches of cartilage. Animals with frozen cartilage implants appeared identical to controls. Cartilage particles implanted into athymic rats remained inert and elicited neither osteogenesis nor chondrogenesis, as did cartilage placed intramedullary in baboons. Conclusions The data demonstrates induction of articular cartilage regeneration by freeze-dried hyaline cartilage particles implanted orthotopically., Introduction Long-term maintenance of cartilage allograft tissue is challenging. This study examines if dexamethasone, a synthetic adrenal corticosteroid, can play a role in maintaining the mechanical properties of cartilage explants in serum-free tissue culture. Methods and Materials In Study 1, middle zone explant disks were harvested from juvenile (4∼6 month) bovine cartilage plugs. In Study 2, full-thickness osteochondral plugs were harvested from mature (1∼2 year-old) bovine femoral condyles. Explants were cultured with continuous dexamethasone (0.1 microM) supplementation; with dexamethasone removed after 2 weeks of culture; or dexamethasone-free throughout 28 days of culture. Results In Study 1, Youngs modulus (Ey) of explants grown with continuous dexamethasone supplementation increased ∼50% from initial values (reaching 2800 kPa) while dexamethasone-free explants decreased ∼80% (down to 400 kPa). Removal of dexamethasone after 14 days resulted in significant Ey reduction of juvenile explants. In Study 2, Ey remained similar (at day 0 levels) for all groups. Modulus changes, when observed, were accompanied by corresponding changes to GAG and collagen levels in some cases. Conclusions Our encouraging findings with the well-established bovine model support the use of serum-free tissue culture as a preservation technique for cartilage grafting. Dexamethasone proved to be necessary in maintaining the mechanical stiffness of juvenile cartilage explants during long-term culture, even improving properties over time, whereas mature bovine explants maintained their initial properties irrespective of steroid supplementation. This outcome suggests that age can be a significant factor in the selection of culture supplements. These findings need to be verified with human cartilage allografts., Introduction The objective of the present study is to investigate the repairing effects of the implanted scaffold-free chondrocyte plate on the osteochondral defects of the articular cartilage. Methods and Materials Thirty eight rabbits were used and 76 osteochondral defects were created. These rabbits were classified into two groups; a chondrocyte plate insertion group (CP group, n=26), and a noninsertion group (defect group, n=12). Histological evaluations were done at 4, 12and 24 weeks after surgery. The histological evaluations contain safranin O staining, immunohistochemical analyses for chondromodulin-I (ChM-I), vascular endothelial growth factor (VEGF) and superficial zone protein (SZP), and the histological scoring using modified O'Driscoll ICRS grading system. Results The repair sites appeared to be filled with cartilaginous tissues and much expressed ChM-I and little VEGF in the CP group 4 weeks after surgery. Conversely in the defect group, the defects were filled with fibrous tissue, and repair sites expressed VEGF and did not expressed ChM-I. The implantation sites kept the phenotype of the articular cartilage in the CP group 24 weeks after surgery. Moreover, SZP was expressed at the surface of repair sites. After 12 and 24 weeks, histological scores in the CP group were significantly higher than those in the defect group. Conclusions The chondrocyte plate contributed to repair the osteochondral defect. The repair site was altered to acquire the cartilage phenotype by implantation of the chondrocyte plate., Introduction Monolayer-grown chondrocytes can keep phenotype only with a limited number of passages. Mechanical stimulus may improve to keep the phenotype of the cultured chondrocytes. The purpose of this study is to investigate the effect of cyclic tesile strain (CTS) on the glycosaminolgycan (GAG) synthesis of primary cultured rabbit articular chondrocytes. Methods and Materials Chondrocytes were isolated from the knee joints of six one-month rabbits. Chondrocytes from each rabbit were divided into three parts and cultured in a Flexercell 4000 strain unit. The cultured cells were loaded with CTS (sinusoidal wave, 0.3Hz) in 0%, 5% and 15% strain for 6 hours per day. The GAG concentrations of supernatants were measured by precipitation with alcian blue at 24, 36, 48, 60h after the beginning of the first CTS loading. (Fig. 1). The data was analyzed with ANOVA of repeated measures. Results The GAG concentrations of the supernatants increased with time (P⇠0.001). It increased with the increase of strain rate (P⇠0.001). And post hoc tests showed the significant difference between different strain rate in any time point (P⇠0.05). (Fig. 2) Conclusions The CTS (sinusoidal wave, 0.3Hz, 6h/d) regimen enhance the GAG synthesis of cultured primary rabbit articular chondrocyte. Higher strain rate produce larger stimulus with more GAG synthesis. The CTS may be an effective way to prevent the dedifferentiation of chondrocytes in monolayer culture., Introduction The aim of this study was to quantify the incidence and microbial spectrum after contamination in the operating room, assess the most effective sterilizing agent, and to identify the effect on bone and cartilage. Fragments were dropped onto the OR floor and cultured. Next, bone fragments were inoculated with three organisms to assess the different sterilizing agents. The contamination rate was 70%, with coagulase negative staphylococcus as the most common organism. Bactericifal agents, and scrubbing were the most effective methods of decontamination. Methods and Materials Phase I, Osteoarticular bone fragments were dropped onto the operating room floor, and permitted to rest on the OR floor for a total of 30 seconds before being cultured. Each positive culture was then speciated by a hospital microbiologist. Phase II, bone fragments were inoculated with different species and exposed to different sterilizing agents based on a protocol. Results Coagulase Negative Staphylococcus: For both betadine and chlorhexidine groups, there was no growth in any group. In the isopropyl alcohol and saline groups, only those rinsed with saline had growth. Bacillus species: There was no growth for betadine, chlorhexidine, or isopropyl alcohol groups. Those rinsed with saline, and all controls had growth. Corynebacterious species: There was no growth in any of the groups, except in the control group. Conclusions We found 70% contamination rate for dropped bone grafts. The most common species in our study was coagulase negative staphylococcus. Bactericidal agents, and scrubbing were most effective. There is no difference in the duration of exposure to sterilizing agent., Introduction The purpose of this study was to evaluate the performance of a novel allograft cartilage scaffold (ACS) in critically sized goat osteochondral defects. ACS is a regionally specific acellular construct comprised of articular cartilage and both demineralized and non-demineralized cancellous bone. Methods and Materials Two (2) 6mm chondral or osteochondral defects, one in the lateral proximal trochlear sulcus (LPTS) and one in the medial femoral condyle (MFC), were created in the knees of skeletally mature goats. The chondral defects were microfractured. The osteochondral defects were filled with ACS, Tru-FitTM, or left empty. Implanted knees were immobilized in a modified Thomas splint for 7 days post-op. After 6 months, gross and histological analyses were done. In addition, stiffness of the chondral-subchondral bone complex was assessed by durometer testing and compare to the contralateral non-operated side. Results Defects treated with either the ACS or Tru-Fit BGS plug had better repair than the empty defect and the microfractured defects. Stiffness results of the joint surface surrounding the MFC lesion sites were generally within the normal range. Histologically, type II collagen repair tissue and positive Safranin-O staining were evident in ACS-filled defects, along with integration to the host. Subchondral bone repair was also observed in ACS-filled defects while cysts were seen with the Tru-FitTM and empty defects. Conclusions This clinically relevant model of osteochondral defect fill demonstrated that the ACS implant is capable of eliciting hyaline or hyaline-like cartilage repair at 6 months, along with endochondral repair of subchondral bone., Introduction Compare the fill of osteochondral defects using a novel allograft sponge with autograft transplants. Methods and Materials Two full-thickness defects (4.5 mm ø, 10 mm depth) were created in both the medial femoral condyle and the lateral patellar groove of adult goats [Lane, 2004]. In each location, one defect was filled with an autograft osteochondral plug while the other was filled with an acellular allograft sponge [Bacterin International]. The sponge is demineralized goat cancellous bone created similarly to commercially-available human allograft products. Animals were splinted for 2 weeks. After 3 and 6 months, the animals were sacrificed and the defects underwent MRI morphological analysis, macroscopic scoring [ICRS] and histological scoring [Sellers, 2000]. Data were analyzed using three-way ANOVA for time, condition and site. Results Sponge-treated defects were filled with white, glistening repair tissue and the quality of repair improved with time. Results achieved using the sponge in the groove were similar to those of the autograft controls in macroscopic appearance and most histological assessments at 6 months. Repairs in the groove were flush with surrounding cartilage (based on histology and MRI). The results in the condyle were less optimal. Remarkably, integration was significantly better in the sponge-treated defects when compared to the autograft controls, and that outcome was true for both sites and both timepoints. Conclusions This study demonstrates the use of a novel allograft sponge to successfully fill osteochondral defects and improve integration with the surrounding cartilage in goats. These results have not been validated in human joints., Introduction Bupivacaine has been shown to be cytotoxic to articular chondrocytes in a dose- and time-dependent fashion in vitro. The purpose of this study was to determine whether a single intra-articular injection of bupivacaine induces chondrolysis in vivo. Methods and Materials Thirty-six Sprague-Dawley rats received a 100μl single-intrarticular-injection of saline and 0.5% bupivacaine or 0.6% MIA (positive control) per IACUC approved protocols. After 1, 4, 12, and 24 wks, 6 rats were euthanized and femurs were harvested. Cartilage health was assessed by chondrocyte viability staining, gross and india ink imaging, gross histology, modified Mankin score, and quantitative histological analysis for cell density. Data were analyzed by one-way ANOVA with Bonferroni post-test. Results Chondrocyte viability was similar to saline following bupivacaine injection (p⇢.05). MIA injections acutely reduced viability 78% compared to saline (p⇠0.01). Gross histological analysis showed no difference with bupivacaine injection vs. saline. However, at 24 weeks MIA treated knees had elevated total Mankin scores and clear cartilage degeneration compared with saline (p⇠.001). Histology post-bupivacaine injection demonstrated a 43–50% reduction in cell density (p⇠0.05) at 24 weeks, but an intact articular surface. In contrast, MIA injected knees exhibited marked cartilage loss with replacement by fibrous scar. Conclusions No chondrolysis was seen with bupivacaine injection in an in vivo rat model. A subtle reduction in overall chondrocyte density was observed six months post-bupivacaine suggesting possible chondrocyte injury. In contrast, MIA injection had acute chondrocyte necrosis followed by degeneration/chondrolysis. These results demonstrate minimal toxic effects of a single intra-articular injection of 0.5% bupivacaine on articular chondrocytes in vivo., Introduction Osteoarthritic change is one of the major complications associated with joint reconstruction using frozen osteoarticular grafts after tumor resection. We employed a frozen autologous whole rabbit knee joint graft model to investigate the influence of freezing on joint components. We then modified a vitrification method utilized for embryo cryopreservation and tested its ability to protect cartilage from cryoinjury. Methods and Materials (Study 1) 30 rabbit knee joints immersed in liquid nitrogen (F) or saline (C) were replanted. Histological and histochemical findings were assessed after 4, 8 or 12 weeks. (Study 2) Full-thickness articular cartilage harvested from rabbit femoral condyles was immersed in liquid nitrogen with and without vitrification. Histologic findings, ultrastructure and chondrocyte viability were examined before and 24hrs after culture. Results (Study 1) Both groups exhibited bone healing. In group F, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed, and cartilage deterioration progressed over time. (Study 2) Vitrified cartilage cell viability was ⇢85% that of fresh cartilage. Transmission electron microscopy demonstrated preservation of original chondrocyte structure. Conclusions Cryoinjury to chondrocytes of articular cartilage causes osteoarthritic changes following joint reconstruction with frozen osteoarticular grafts. To prevent osteoarthritic changes, articular cartilage needs to be protected from cryoinjury during freezing. Our vitrification method is effective for protecting chondrocytes from cryoinjury leading to cartilage deterioration. Reconstructing joints with osteoarticular grafts containing living cartilage may avert osteoarthritic changes. Vitrification is useful for long-term storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumor., Introduction The aim of this study was to establish an in vitro quantitative magnetic resonance (MR) imaging technique of 3D tissue engineered cartilage with a combination of delayed gadolinium-enhanced MR imaging of cartilage (dGEMRIC), transverse relaxation time (T2) mapping, and fat-suppressed three-dimensional spoiled gradient-recalled (3D-SPGR) MR imaging. Methods and Materials Chondrocytes were isolated from knee cartilage of rabbits. Tissue engineered cartilage with different thickness of collagen scaffold and chondrocyte concentration were prepared, and quantitative MR imaging techniques was performed with a custom made micro-imaging coil, which could kept the samples almost similar conditions to that used for cultivation while the MR imaging. To investigate whether the T2 of tissue engineered cartilage had dependence of T2 on the angles relative to the static magnetic field (B0), which normally observed in native articular cartilage, T2 measurements with the samples oriented at different angles relative to B0 were performed. Histological and biochemical assessments of samples were then performed. Results The GAG concentration and histological findings of the tissue engineered cartilage correlated with the findings assessed by dGEMRIC. The water concentration of the tissue engineered cartilage correlated with the findings assessed by T2 mapping. On the other hand, variation of T2 due to the orientation-dependent dipolar interaction was not observed. The thickness of the tissue engineered cartilage evaluated with histology well correlated with the findings assessed with 3D-SPGR MR imaging. Conclusions A combination of dGEMRIC, T2 mapping and 3D-SPGR MR imaging can be a useful in vitro evaluation method for the quality and morphology of the tissue engineered cartilage., Introduction The purpose of this study was to evaluate the effect of splinting time on the retention of allogenic cartilage particle preparations implanted into full thickness cartilage defects. Methods and Materials Three full thickness critical sized cartilage defects were created in the trochlear sulcus and one in the medial femoral condyle. Subchondral bone was subjected to microfracture procedure. Post operative splinting was either 3 days, 7 days, or 14 days. One of four cartilage particle preparations (allogenic cartilage particles in either phosphate buffered saline (PBS), hyaluronan, hyaluronan and proprietary growth factor, or hyaluronan and insulin) was implanted. At 6 weeks, gross and histological analyses were performed to evaluate degenerative changes. Results Slightly increased degenerative articular cartilage changes corresponded to longer casting interval, regardless of treatment. Test article retention was highest in the distal trochlear sulcus site, followed by the MFC site, with the least retention in the middle trochlear site. Microfracture brought bone marrow and blood oozing into the allograft particle preparation filled defects, though no benefit was detectable. Normalized gross scores and histological evaluation demonstrated the best healing occurred with 7 day splint time. Conclusions Four defects per animal were a severe test. At this early 6 week time point, all subjects exhibited some degree of circumferential healing which varied depending on splinting time and treatment. The optimal splinting time was 7 days. The best treatment preparations were the allogenic cartilage particles in PBS and allogenic cartilage particles hyaluronan plus proprietary growth factor., Introduction Medical and industrial attempts for the treatment of articular cartilage pathologies have been increasing. But, they are still a major locomotor problem. Our aim was to determine the effects of synovium on cartilage proliferation as “in-vivo” culture medium and to anticipate a new, biological and cheap treatment method Methods and Materials In this study, 12 New Zealand male rabbits were used. Cartilage samples were taken from both knees: right (osteochondral, 4×4×6mm), left (chondral, 4×4×4mm). Two groups were formed: In group I (synovium group), the cartilage samples were placed into the synovium in the supracondylar groove, and in group II (intraarticular group), behind the patellar tendon. After 4 months, we sized and analyzed samples histologically with the camera lucida method to count the chondrocyte numbers. Mann-Whitney U-Test and regression analysis were used. Results For both groups, the chondrocyte numbers in the osteochondral samples were found to be higher than chondral samples (p⇠0.05) and the chondrocyte number increase was only found in the osteochondral samples, compared with preoperative samples (p⇠0.05). For the osteochondral samples, the chondrocyte numbers were found to be significantly increased more in the group I than in the group II (p⇠0.05). A logarithmic correlation was found between size and chondrocyte numbers (p⇠0.05). Conclusions This is the first study showing histologically the chondrocyte number increase with the camera lucida method. It also showed firstly that the synovium is an excellent chondrogenetic “in-vivo” culture medium. Arthroscopy-assisted implantation of “in-vivo” produced cartilage would probably be a new, cheap and biologically efficient treatment alternative for this clinical problem., Introduction The purpose of the present study was the evaluation of the effect of autologous chondrocyte transplantation placed on non-absorbable, polysulphonic and absorbable collagen mebrane in the treatment of leasion in hyaline cartilage in rabbits. Methods and Materials 80 White New Zealand rabbits, 4 month old, 2- 3.5 kg of weight were used in this research. Articular cartilage defects penetrating into the subchondral bone and superficial were created on the patellar groove of the femur. The defects were filled with chondrocytes placed on polysulfonic or collagen membrane. The expression of gene encoding procollagen type II that is a molecular marker of chondrocytes was evaluated. To estimate a weight of tissue grown on the polysulphonic membrane the burning analysis was performed. The reparative tissue was analyzed macroscopically and histologically after 12, 25 and 52 weeks since the performance of the operation. Results The RT - PCR analysis of the mRNA isolated from the cells detached from the bottom of the culture flask as well as polysulphonic membrane revealed the expression of type II procollagen. The burning analysis of polysulphonic membrane slices after two weeks of culture with chondrocytes revealed the concentration 0.23 mg of tissue on one part of membrane. Conclusions As far as macroscopic and microscopic evaluation is concerned the quality of regenerated tissue was similar after transplantation of cells placed on polysulphonic and collagen membranes. The regenerated tissue in full thickness as well as superficial defects reached maturity after 12 weeks and revealed morphology of hyaline - like cartilage even after 52 weeks., Introduction Meniscus loss leads to degenerative arthritis attributed to load distribution changes. Thus, there is a substantial need to protect the articular cartilage by either repairing or replacing the menisci. A novel Polycarbonate-Urethane (PCU) meniscal-implant was developed to comply with these requirements. Our goal was to elucidate whether a PCU-meniscal-implant is able to provide adequate protection to delay degenerative changes post-meniscectomy. Methods and Materials Seven ewes underwent full medial meniscectomy and were implanted with the PCU-meniscal-implant. Animals were euthanized at 3, 6 and 9 months. Range-of-motion (ROM), macroscopic and histological evaluations were performed post-implantation. The contralateral knee served as control. Results In general, periodic examination showed full ROM, no weight loss or signs of distress. The PCU-implant was durable and remained well-secured throughout 9-month trial. Gross and microscopic examinations of the implant's surfaces did not reveal any structural or material property changes. The PCU did not appear to exert any negative effect on cartilage and the degree of inflammation was minimal and primarily associated with the presence of foreign matter. The main pathological changes seen at 6 and 9 months post-implantation were similar to 3-months post-implantation observations. Cartilage in direct contact with the implant was preserved well and did not show significant degeneration. Conclusions To conclude, PCU did not exert any adverse effects and our results show that PCU-meniscal transplantation may protect, but not completely prevent degenerative changes of the medial articular cartilage. We believe that a PCU-meniscal-implant could counter the occurrence of major degenerative cartilage changes following meniscectomy., Introduction To determine the profile of Coll2–1, Coll2–1NO2 and myeloperoxydase (MPO) serum concentrations in experimental knee OA induced in the dog by transection of the anterior cruciate ligament. Methods and Materials Surgical transection of the ACL of the right knee was performed on 16 adult crossbred dogs. The dogs were sacrificed 8 weeks after the surgical procedure. Coll2–1, Coll2–1NO2 and MPO were measured by specific immunoassays in 16 dogs at baseline and every 2 weeks during the 8 weeks. The results were expressed as median (range). Results Immunostainings with D3 and D37, the antiserum recognizing Coll2–1 and Coll2–1NO2, respectively, labelled extracellular matrix in the superficial layer of fibrillated cartilage. After the transection of the ACL, the concentration of 3 biomarkers increased significantly (Friedman test: p⇠0.001). The concentrations of Coll2–1 and MPO were significantly increased at week 2 compared to baseline [Coll2–1 baseline: 281.57 (131.02–384.67) nM vs Coll2–1 week 2: 345.52 (181.15- 589.25) nM (p⇠0.01) and MPO baseline: 5.16 (⇠0.4–14.7) ng/ml vs MPO week 2: 14.54 (3.28–31.50) ng/ml (p⇠0.001)] and remained stable until week 8 [Coll2–1 week 8:318.89 (117.95–492.28) nM and MPO week 8: 11.55 (2.87–42.94) ng/ml]. The Coll2–1NO2 concentration increased significantly at weeks 6 and 8 compared to baseline [Coll2–1NO2 baseline: 0.54 (0.29–1.48) nM vs Coll2–1NO2 week 6: 0.64 (0.40- 1.9) nM (p⇠0.001) vs week 8: 0.61 (0.37–1.79) nM]. Conclusions These findings suggest that Coll2–1 is a relevant marker for the detection of early structural changes in OA dogs. Interestingly, MPO and Coll2–1NO2 are increased in OA dogs indicating that an oxidative stress occurs in this OA model., Introduction This study aims to set up and validate a protocol for 3D high resolution imaging of rabbit knee joints based on the equilibrium partitioning of a contrast agent with μCT. Accurate segmentation and 3D quantification require consistent scan and threshold parameters. Methods and Materials Five rabbit knee joints were obtained from unrelated studies, dissected and immersed in a 40% /60% solution of Hexabrix/PBS at room temperature, rinced in normal saline solution before scanning. EPIC-μCT scans were performed with a GE Locus μ-CT before immersion and at day 2, 4 and 6. Histology was then performed. Results 20μm isotropic resolution was achieved. Scanning parameters were 50kV, 450mA, 2000ms. The best contrast between subchondral bone and cartilage was found after 4 days of immersion in the contrast agent, showing a clear bimodal distribution of both cartilage and sub-chondral bone Hounsfield Units. An appropriate threshold was selected to perform the cartilage segmentation and 3D morphology modelisation using the GE Micro-View software. Quantification of the 3D local cartilage thickness was obtained and displayed with a color scale related to this parameter, and finally confirmed by histology. Conclusions In this preliminary study, we present the effectiveness of the EPIC-μCT for the quantitative 3D morphometric analysis of the rabbit knee joint. It is a promising complementary non invasive technique to monitor surface morphology, and obtain local and global 3D quantitative information of rabbit articular cartilage since accuracy and resolution are improved by 4 in the plane and by 20 in slice thickness with respect to common 7T μ-MRI acquisitions., Introduction The Göttingen Minipig is a well established large animal model for preclinical evaluation of novel articular cartilage repair procedures. Bone marrow derived mesenchymal stem cells hold great promise for future tissue engineering based articular cartilage repair strategies. The purpose of this study was to identify optimal cell culture conditions during MSC expansion in order to maximize cell proliferation and osteo- and chondrogenic cell differentiation. Methods and Materials Mesenchymal stem cells were isolated, cryopreserved and expanded from 3 adult Göttingen Minipigs. Cells were expanded until passage 2 using three different standard culture media with or without additional FGF-2 supplementation (10ng/ml): DMEM-LG+10%FCS, alpha-MEM+10%FCS and Verfaillie's Media (DMEM-HG, 2 % FCS, MCDB, Transferrin, Selenite, Dexamethason, A2P, Insulin, 10ng/ml EGF and PDGF). MSCs were evaluated for their potential to differentiate into chondro-, osteo- and adipognic cell lineages. Results During cell expansion each culture media displayed a distinct type of cellular morphology. With additional FGF-2 supplementation cell proliferation rate was significantly higher using alpha-MEM and DMEM-LG when compared to Verfaillie's media. Furthermore, FGF-2 did significantly stimulate chondrogenic and osteogenic differentiation in all three media with significantly more GAG and calcium deposition found in alpha-MEM expanded MSCs. Conclusions Our data suggests that using FGF-2 supplemented alpha-MEM media for MSC-expansion in the Göttingen Minipig model does maximize cell yield and boosts chondro- and osteogenic tissue formation., Introduction Frozen osteoarticular grafts treated with liquid nitrogen are often utilized for joint reconstruction after tumor resection, but the joint may develop osteoarthritic changes. Cryoinjury is one of the major complications of frozen osteoarticular grafts. In this study, we modified a vitrification method utilized for embryo cryopreservation in order to preserve articular cartilage from cryoinjury, and investigated the prognosis of the vitrified cartilage in rabbit model. Methods and Materials Osteochondral plugs (2.7mm diameter) were obtained from the medial condyle and patellar groove of the distal femur. These grafts were divided into 3 groups (control group, frozen group, and vitrified group), and were implanted as autografts. Histological findings, ultrastructure, and chondrocyte viability were examined 6 and 12 weeks after implantation. Results Histological findings and chondrocyte viability of the vitrified group showed no significant difference from those of the control group. Viable cells were absent in frozen group. Transmission electron microscopy demonstrated preservation of the original chondrocyte structure in the vitrified group. Conclusions Cryoinjury of chondrocytes from articular cartilage causes osteoarthritic changes following joint reconstruction with frozen osteoarticular grafts. To prevent osteoarthritic changes, the articular cartilage must be protected from cryoinjury during freezing. Our vitrification method was effective in protecting chondrocytes from cryoinjury leading to cartilage deterioration. Reconstructing joints with osteoarticular grafts containing living cartilage may help to avert osteoarthritic changes. Our vitreous preservation method is useful for long-term storage of living cartilage for allografts or, as in Asian countries, for reconstruction with frozen autografts containing tumor. In addition, the preserved grafts can maintain cartilaginous tissue., Introduction PVA-PEG theta hydrogel is a candidate material for synthetic articular cartilage. The strength of PVA-PEG theta hydrogel can be increased by increasing its crystallinity by dehydration followed by annealing to meet the requirements of the targeted application. The crystallization kinetics of PVA-PEG thetagels during high temperature annealing is not well understood; especially the changes that take place during the steps of dehydration, annealing, and rehydration. In this study we used wide angle x-ray diffraction (WAXD) to investigate these changes. Methods and Materials PVA-polyethylene glycol (PEG) hydrogels were prepared by theta gel method. One group was used in their ‘as-gelled’ form (AG). Second group was studied in ‘dePEGed’ form (DP) after removing the PEG in DI water. The crystallinity of the gels were measured during (i) gelation (ii) DePEGing, (iii) dehydration under vacuum at 25°C (iv) high temperature annealing under Ar at 160°C (v) rehydration subsequent to equilibrium dehydration and annealing using a PANalytical X'Celerator powder diffractometer. Results DePEGing process decreased the crystallinity. However, crystallinity of DP gel was increased markedly after dehydration and annealing. After rehydration, crystalline peaks of the dried DP disappeared and DP annealed gel did not show any difference in the WAXD. AG gels exhibited strong diffraction peaks after dehydration and annealing. AG annealed gel showed higher crystallinity than D P. Crystallinity of AG annealed gel decreased after rehydration. Conclusions Annealing and dehydration increased crystallinity of the PVA-PEG theta gels. Annealing resulted in a more stable crystalline network and mechanically stronger gels., Introduction This study was undertaken to evaluate muscle and cartilage damage after different running distances by marathon and ultra-marathon runners. Methods and Materials Ten male marathon and ten male ultra-marathon runners participated in the study. Serum cartilage oligomeric matrix protein (COMP) was measured as a marker of cartilage metabolism and/or damage. Plasma CPK and hs-CRP activities were measured as markers of muscle damage and inflammation. Results Serum COMP was increased 1.6-fold at 10km during a marathon race. In contrast serum COMP was increase 1.9-fold after a 200km race and this level was maintained until day 3 of recovery, only returning to the pre-race level on day 6. Plasma CPK was increased at 10km of the marathon race and up to 3-fold at the end of the race. This was further increased after 1 day of recovery, only returning to pre-race level on day 6. Plasma CPK was increased 35-fold at the end of the 200km race and remained significantly increased until day 5 of recovery. There was no change in plasma hs-CRP during the marathon race, but this was increased 3.4-fold by day 1 of recovery, returning to the pre-race level on day 4. Plasma hs-CRP increased 40-fold by the end of the 200km race and was still increased on day 6 of recovery. Conclusions Long distance running may induce more impact-stress on muscle than on cartilage at a given running distance. Further, the required time for recovery may vary with running distance and the tissue type, e.g. cartilage or skeletal muscle as in this case., Introduction Polyvinyl alcohol) (PVA) has been advanced as a biomaterial to be used as synthetic articular cartilage. Key material requirements for such devices are high creep resistance and high water content to maintain a lubricious surface to minimize damage of the cartilage counterface during articulation. The creep resistance of PVA hydrogels can be increased by high temperature annealing; however this process collapses the pores, reducing the water content and lubricity of the hydrogel surface. In this study we added hydrophilic poly(acrylamide) (PAAm) to prevent the pore collapse by filling the pores during annealing resulting in a highly lubricous hydrogel while still maintaining a high creep resistance. Methods and Materials PVA-AAm interpenetrating networks hydrogels were prepared by the combination of freeze-thaw and thermal polymerization. PVA-polyethylene glycol (PEG) hydrogels were prepared by theta gel method. Coefficient of friction (COF) of the hydrogels was determined using a rheometer. The equilibrium water content (EWC) of the gels determined by using Thermogravimetric Analyzer. The total creep strain was determined on a custom made mechanical tester. Results The equilibrium water content of the PVA-AAm gels showed an increase with increasing AAm content both before and after annealing. The creep resistance of the PVA-AAm gels was similar to PVA-PEG gels. The COF of the PVA-AAm gels was lower than that of the PVA-PEG gels. Conclusions Incorporation of AAm component in to PVA networks substantially improved the lubricity of the resulting gels while maintaining high creep resistance. PVA-AAm hydrogels are promising candidates for cartilage resurfacing., Introduction Fabricating the biphasic scaffold to benefit both bone and cartilage regeneration is potentially an optimal technique for osteochondral repair. This study describes our continuing effort to optimize the osteochondral lesion repair from adipose derived stem cells toward engineering articular cartilage repair in vivo. Methods and Materials We developed the integrated biphasic scaffold, of which the upper part is an extracellular matrix-derived of cartilage by combining a decellularized procedure with ice crystal orientation and freeze-drying technique and the lower part is true bone ceramic. The upper layer the scaffold was loaded with chondrogenically induced adipose-derived stem cells (ADSCs). MTT assay showed no cytotoxicity of the biphasic scaffolds. The structure of biphasic scaffolds were characterized by scanning electron microscopy and micro-computed tomography. Osteochondral defects (4.2 mm diameter ×6 mm deep) were created in the patellar groove of rabbit femurs, and the defects were filled with constructs of a biphasic scaffold with chondrogenically induced ADSCs (Group A, 2×107/ml, n=12) or cell-free scaffolds (Group B, n=12) or nothing (Group C, n=12). Results All of the defects of Group A showed good osteochondral repair, and macroscopic and histologic scores were superior to those of Group B and Group C. The subchondral bone showed mature trabecular bone regularly formed in Group A and Groups B at both 6 and 12 weeks. But the subchondral bone formation was not good in Group C. Conclusions The novel biphasic scaffold loaded with chondrogenically induced ADSCs successfully filled the osteochondral defects of rabbits with hyaline cartilage that is similar to normal cartilage biomechanically and biochemically., Introduction Polyvinyl alcohol) (PVA) is a candidate for synthetic articular cartilage to treat osteochondral defects. However PVA gels are not strong enough to serve this purpose. Annealing has been proven to increase the strength of PVA hydrogels; however it reduces the water content and consequently reduces the lubricity of the hydrogel. Keeping the articulating surface of the hydrogel lubricious is a key requirement to prevent damage against opposing cartilage. In this study we have investigated the effect of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonic acid (AAMPS) on the lubricity of the PVA hydrogels by measuring coefficient of friction (COF) of these gels. We also investigated effect of lubricant on COF while testing these hydrogels in deionized water, saline, and bovine serum based storage media. Methods and Materials PVA-AAM PS, PVA-AAm IPNs and PVA hydrogels were prepared by the combination of freeze-thaw and thermal polymerization. PVA-polyethylene glycol (PEG) hydrogels were prepared by theta gel method. COF of the hydrogels was determined at 40°C using a rheometer. Results AAm and AAMPS addition increased EWC of PVA gels. PVA only gels showed the lowest EWC after annealing. Annealing increased the COF values in all types of gels. PVA-AAm and PVA-AAM PS hydrogels showed lower COF values than PVA only and PVA-PEG hydrogels. COF values measured in saline were lower than other media. Conclusions The addition of AAm and AAMPS in to PVA decreased the friction of the PVA hydrogels significantly. The lubricant in which the gels were tested has a strong influence on the COF., Introduction The purpose of the present study was to explore the surgical possibilities for replacement of the medial tibial plateau by a metal implant and to examine the implications for the opposing cartilage. Methods and Materials In 6 goats, the medial tibial plateau of one knee was replaced by a cobalt-chromium (CoCr) implant, using PMMA bone cement for fixation. The anterior part of the medial meniscus was dissected, without damaging the knee ligaments, and the femoral cartilage. The un-operated knee served as a control. After 26 weeks the animals were killed. Joints were evaluated macroscopically. Cartilage quality was analysed macroscopically, and microscopically. GAG content, synthesis, and release were measured in tissue and medium. Results Knees were loaded without limitations by all animals. Macroscopic articular evaluation scores showed worsening 26 weeks after inserting the implant (p⇠0.05). Macroscopic and microscopic scores showed more cartilage degeneration of the opposing medial femoral condyle in the experimental knee compared to the control knee (p⇠0.05). Higher GAG synthesis was measured in the experimental knees (medial femoral condyle) (p⇠0.05). GAG content and release were not different (p⇢.05). Conclusions Replacing the medial tibia plateau by a custom designed metal implant is surgically feasible, however this induces considerable damage of the opposing medial femoral cartilage. Given the results obtained, the current model is a viable tool in the evaluation of bearing materials for implants. However, the introduction of CoCr tibial implants in a human clinical setting for the treatment of post-meniscectomy cartilage degeneration of the medial tibial plateau does not seem a realistic option currently., Introduction The purpose was to investigate the feasibility of the application of defect-size femoral implants for treating localized cartilage defects and compare this treatment in terms of joint degeneration with microfracturing in a model of established cartilage defects. Introduction The purpose was to investigate the feasibility of the application of defect-size femoral implants for treating localized cartilage defects and compare this treatment in terms of joint degeneration with microfracturing in a model of established cartilage defects. Methods and Materials In 9 goats, a medial femoral condyle defect was created in both knees. After 10 weeks, the knees were treated by microfracturing or by placing an oxidized zirconium implant, and 26 weeks after surgery animals were killed. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry. Cartilage repair (after microfracturing) was scored microscopically. Cartilage quality was analysed macroscopically, and microscopically. GAG content, synthesis and release were measured in tissue and medium. Methods and Materials In 9 goats, a medial femoral condyle defect was created in both knees. After 10 weeks, the knees were treated by microfracturing or by placing an oxidized zirconium implant, and 26 weeks after surgery animals were killed. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry. Cartilage repair (after microfracturing) was scored microscopically. Cartilage quality was analysed macroscopically, and microscopically. GAG content, synthesis and release were measured in tissue and medium. Results Bone-implant contact was 14.6±5.4%. Healing of the microfractured defects was 18.4±0.4 (of 24). Macroscopically no differences were seen. Microscopically, more degeneration (medial tibial plateau) after microfracturing was seen as compared to joints with implants (p⇠0.05). Similarly, a higher GAG content, higher synthetic activity and decreased GAG release of the medial tibial plateau cartilage of implant-treated knees was measured (p⇠0.05). Microscopic degeneration was also found in cartilage of the lateral tibia plateau and condyle, but not different between treatments (p⇢0.05). Results Bone-implant contact was 14.6±5.4%; the amount of bone surrounding the implant was 40.3±4.0%. Healing of the microfractured defects was 18.38±0.43 out of 24. Macroscopically no differences were seen. Microscopically, more degeneration (medial tibial plateau) after microfracturing was seen as compared to joints with implants (p Conclusions Both microfracturing and using implants as a treatment for established localized cartilage defects in the medial femoral condyle caused degeneration in directly articulating cartilage as well as in more remote sites in the knee. Although this study shows that small metal implants may be more suitable than microfracturing, the generalised degeneration found for both treatments should be addressed first. Conclusions Both microfracturing and the use of implants as a treatment for established localized cartilage defects in the medial femoral condyle caused degeneration in directly articulating cartilage as well as in more remote sites in the knee. However, metal implants caused less damage than microfracturing. Although this study shows that small metal implants may be more suitable than microfracturing, the generalised degeneration found for both treatments should be addressed first, Introduction Hemiarthroplasy has been used widely in various joints since the 1950's. Histological studies on dogs confirmed that implants made of rigid materials cause progressive cartilage wear, while the histological study of a Polycarbonate-Urethane (PCU) implant against cartilage in sheep showed very little cartilage degeneration. We believe that PCU performance could be improved by a lubricious-polystyrene-brush-coating. Our goal was to evaluate whether this coating could be beneficial in reducing wear in PCU hemiarthroplasty. Methods and Materials Acetabular components of porcine hip joints (N=3) were placed in a rotating cell. The femur was fixed to the load axis and the artificial PCU buffer was tested by replacing the natural acetabular component (N=6). Simulations of 70,000 load-cycles were conducted on natural joints and hemiarthroplasties of the various PCU buffers. Using India-ink staining, scratches were measured and the total injured area in each specimen was calculated. Last, the difference in injured area between groups was evaluated and the moment developed in the fulcrum axis was measured. Results We found that cartilage-on-polystyrene-coated-PCU and cartilage-on-cartilage groups were significantly indistinguishable (p⩾0.05). In addition, microscopical examination of an intact femoral cartilage after 70,000 cycles showed negligible scratching. Interestingly, the moment measured for the coated-PCU was found to be ∼1.6-times lower, and less affected by lubricant depletion versus the non-coated-PCU. Conclusions To conclude, we evaluated the amount of cartilage damage associated with articulation of cartilage against polystyrene-brush-coated-PCU and showed that PCU performance can be improved further by an additional coating. Coated-PCU demonstrates exceptional wear results, comparable to those attained for articulation of cartilage-on-cartilage., Introduction Joint surface incongruities in the ankle can have a great response on stress gradients and contact area (CA). However, the response of small lesions seems limited. This study aims to determine the changes in contact area characteristics after a limited cartilage biopsy at the postero-medial rim of the talar dome. Methods and Materials Ten specimens were tested before and after harvest of a biopsy of 5 × 11 mm; using pressure sensitive flm; loading in neutral (N), plantar flexion (PF) and dorsiflexion (DF) while applying (sub-) physiological loads. The stains were further analyzed in the ImageJ environment. The surface area of the talar dome, the normalized tibio-talar CA (% of talar dome area), and the centroïd position of the CA were determined. Significant changes were checked using 95% Confidence Intervals. Results The mean changes in normalized CA were non significant. However, when considering the single specimens a significant change was observed 4 times in N and 3 times in PF. For the centroïd position, again, none of the mean changes was significant, although some single specimens showed a significant change especially for the antero-posterior direction in PF. Conclusions In general, harvesting a limited cartilage biopsy at the postero-medial rim of the talar dome does not result in a significant change in contact area characteristics. However, due to a great individual variability some specimens show significant changes. Future studies will have to determine the clinical importance of these changes and the long-term safety of the proposed biopsy site., Introduction Cartilage defect repair (CDR) is an attractive treatment option for osteochondral defects. In the design of such therapies, it is important to understand the very complex tribological regime of the natural joint, as well as the tribology of the CDR. Methods and Materials The friction and wear of CDR were investigated using our previously described tribological simulation of the medial compartmental knee [McCann, L.(2008)Tribology International(41),p:1126]. CDR using a cartilage-bone plug was compared to a metal pin, a blank defect in the femoral condyle, and the natural medial compartment. Friction was determined over 3600 cycles. Peak contact stresses were measured using prescale Fuji flm. A Talysurf Proflometer measured surface roughness. High resolution micro-MRI scans were taken to quantify wear. Results The frictional coefficient (u) was significantly lower (P⇠0.05) for the control bearing (u=0.07), than for the three defect bearings (u≥0.11). Overall, we found elevated frictional coefficient, friction shear stress and surface damage, for the defect bearings in comparison to the intact medial compartment. In the presence of a defect the biphasic nature of the joint was disrupted. The rehydration, which takes place during unloading in a natural joint could not take place, increasing the direct solid-to-solid contact which is known to cause an increase in frictional coefficient. Conclusions This investigation is the first tribological simulation of the knee examining CDR. It supports the use of biphasic materials for CDR, to minimize the solid-solid contact and rise in friction and shear stress. The experimental model has the potential to examine any potential CDR device - synthetic or biological., Introduction The biopsy site best used for an ACI at the talar dome remains an issue of debate. This study aims to determine the changes occurring in the pressure profile across the postero-medial rim of the talar dome after a biopsy has been harvested locally. Methods and Materials Ten specimens were (sub-) physiologically loaded pre- and post-biopsy; in neutral position, 10° of plantar-flexion and 10° of dorsi-flexion. Quantitatively, the pressure profile plots of each pre- and post-biopsy situation were analyzed. Qualitatively, the coverage of the biopsy by the tibial plafond, and changes in the shape of the contact area were analyzed. Finally, all parameters per specimen were compared to cross-check for consistency of the results. Results A lower pressure across the biopsy site after harvest was noted twice in N and 5-times in PF. No peak pressures near to the biopsy site developed. The biopsy site resulted (partially) covered by the tibial plafond 3-times in N and 8-times in PF. A change in the shape of the contact area was seen 7-times in PF. After comparing all results, a limited effect of the biopsy on the pressure profile was seen 6-times in PF and 2-times in N. Conclusions Only a part of the specimens showed a change in the local pressure profile after harvesting the biopsy. Nearly all changes were in PF. In this position the joint is loaded only a short time and in a limited way during the stance phase. Therefore, the investigated biopsy site seems safe to be used in clinical practice., Introduction Treatment of cartilage defects follows recommendations depending on defect-size. Exact sizing of defects is hard to achieve in daily practice. The critical size of a defect leading to pain, functional deficits and osteoarthritis is still unknown and depends on thickness/ stability of the surrounding cartilage, joint containment, loading forces and other factors. Methods and Materials The purpose of this study is to identify additional factors influencing the outcome of cartilage repair after a certain treatment by finite-element simulation with the following standards were used: a circular defect (diameter 5 and 10 mm)cartilage-thickness: 5mmCartilage Elasticity-Modulus: 18.8 MPaAxial-loading F=5000N (gait/running) Deformation and contact pressure were simulated for intact cartilage, cartilage defects with a diameter of 0.5 cm/1.0 cm and a defect filled with repair tissue with a reduced elasticity modulus of 14.8 MPa. Results Max.deformation (mm) and max.contact pressure (kg/mm sec2) was calculated for intact cartilage with 3.074 × 10−1 and 1.592 × 103,cartilage defect ø 5 mm with 4.504 × 10−1 and 1.831 × 103,cartilage defect ø 10 mm with 5.523 × 10−1 and 2.259 × 103,filled cartilage defect with 3.894 × 10−1 and 1.057 × 103. Edge-loading increases with increasing defect-size and decreases even with a defect-fill of inferior quality. Conclusions The model of finite-elements enables the simulation of various factors possibly affecting the outcome of cartilage repair (defect-size, cartilage thickness, elasticity, shape of joint-partners, loading/shear forces and others). Simulation-based individual treatment options after MRI-analyis could enhance the quality of medical recommendations., Introduction The purpose was to examine whether A-mode ultrasound signal intensity (SI, a measure of superficial cartilage integrity) detect fragility of superficial layer of reparative articular cartilage-like tissue in a full-thickness cartilage defect of rabbits (D-model). Methods and Materials A defect of 5-mm in diameter was created in the left trochlea groove in the D-model (n=18). A full-thickness osteochondral plug of 6-mm in diameter was removed from the right trochlea groove and grafted into the defect in an autologous osteochondral grafting model (OCG-model, n=36). Reparative tissue of the D-model and articular cartilage of the plug of the OCG-model was assessed by histology and an ultrasound system (center frequency 10 MHz) at 2-, 4-, 8-, 12-, 24- and 52-week postoperatively and was compared to normal control cartilage. Results In the D-model, the defect was repaired with fibrocartilage. SIs of control, 2-, 4-, 8-, 12-, 24- and 52-week were 3.29 (arbitrary unit), 0.75, 0.35, 0.65, 0.97, 0.96 and 0.23, respectively. SIs of 2-through 52-week were significantly lower than SI of control (P ⇠ .001). Cartilage of the plugs of the OCG-model retained features of hyaline cartilage throughout 52 weeks. SIs of control, 2-, 4-, 8-, 12-, 24- and 52-week were 3.18, 2.17, 2.04, 1.23, 1.01, 1.95 and 3.94, respectively. SIs of 8- and 12-week were significantly lower than SI of control (P ⇠ .001). There was no significant difference in SI between control and 2-, 4-, 24- or 52-week. Conclusions Low SI during 24 weeks might predict fragility of superficial layer of reparative cartilage-like tissue., Introduction There is a resurgence of interest in bone grafting procedures for patients with shoulder instability. We sought to investigate the alterations in glenohumeral contact forces to determine changes in pressure with proud, flush, and recessed Latarjet or ICBG augmentation, and to determine the optimal orientation of the coracoid bone graft. Methods and Materials Twelve fresh-frozen cadaveric shoulders were tested in abduction and ER with a compressive load of 440N. Glenohumeral contact area, mean pressure, and peak pressure were determined with a Tekscan sensor for: 1) intact glenoid, 2)30% defect, 3) 30% glenoid defect treated with Latarjet bone block placed 2mm proud, flush, and 2mm recessed to the glenoid, 4) 30% glenoid defect with ICBG placed 2mm proud, flush, and 2mm recessed to the glenoid, and 5) Latarjet bone block placed flush but oriented with either the lateral (Latarjet-LAT) or inferior (Latarjet-INF) surface of the coracoid as the glenoid face. Results Bone grafts in the flush position restored mean contact pressure to 85% (ICBG, p⇠0.04), 80% (Latarjet-INF, p=0.03), and 65% (Latarjet - LAT, p=0.02) of normal. Latarjet-LAT demonstrated statistically higher peak pressures than the ICBG and Latarjet-INF at all positions (p⇠0.02). With bone grafts in a proud position, contact pressure increased an additional 40% (p⇠0.01) in the anteroinferior quadrant with a concomitant 100% (p⇠0.01) increase in the posteroinferior glenoid pressure indicating a shift posteriorly. Bone grafts placed in a recessed position had high edge-loading. Conclusions The optimal restoration of glenohumeral forces was with the ICBG and the Latarjet-INF when the inferior aspect of the coracoid becomes the glenoid surface. Bone grafts placed in a proud position not only increased the peak pressure anteroinferiorly, but also shifted the articular contact forces to the posteroinferior quadrant. The optimal restoration of glenoid joint surface favors an optimally placed ICBG and inferior surface of the coracoid for glenoid bone augmentation., Introduction The tibio-talar contact area has been widely investigated to monitor biomechanical changes due to articular incongruities or an altered loading. This study aims to investigate for the first time in a systematic way the extent of the inter-specimen variability of the tibio-talar contact area, and its repercussions when analyzing data concerning this parameter. Methods and Materials Ten specimens were loaded to record the tibio-talar contact characteristics by use of pressure sensitive film. The size of the talar dome area, the size of the (normalized) tibio-talar contact area, the position of the tibio-talar contact area, and the shape of the latter were determined and analyzed. Inter-specimen variability was expressed as the Coefficient of Variation and was calculated for the datasets of previous studies as well. Results The size of the tibio-talar contact area showed a very high inter-specimen variability, in agreement with the range found in previous studies. This high variability persisted when a normalized tibio-talar contact area was calculated. The shape of the tibio-talar contact area showed some basic characteristics, but a high variation in details could be observed. Conclusions Every specimen can be considered to have its own “ankle print”. By this variability, articular incongruities are expected to have a different effect on local biomechanical characteristics in every single individual. Therefore, every single case has to be evaluated and reported for significant changes. In case of modeling, this also underscores the need to use subject specific models fed by sets of parameters derived from a series of single specimens., Introduction Microfracture is a marrow stimulation technique thought to produce less bone necrosis than drilling. Our study tested this hypothesis and investigated acute osteochondral characteristics, repair responses and effect of hole depth in adult rabbits. Introduction Microfracture is a marrow stimulation technique that is speculated to produce less bone necrosis than drilling. Our study tested this hypothesis and investigated acute osteochondral characteristics, repair responses and the effect of hole depth in a an adult rabbit model. Methods and Materials Trochlear cartilage defects were prepared and 0.9 mm diameter cylindrical drill holes were made either 2 or 6 mm deep (under cooled irrigation), or conically shaped microfracture holes were made 1mm diameter and 2mm deep. Sacrifices were at 1, 14 and 21 days, with assessments by micro-CT and histology. Methods and Materials Cartilage defects were prepared in rabbit trochleas (N=14) and further treated by marrow stimulation with 0.9 mm diameter cylindrical drill holes (2 or 6 mm deep, under cooled irrigation), or with conically-shaped microfracture holes (1 mm diameter and 2 mm deep). Rabbits were sacrificed at 1, 14 and 21 days, and defects assessed by micro-CT and histology. Results Microfracture induced compaction of surrounding bone, essentially sealing holes off from bone marrow; whereas drilling removed bone and debris from holes to provide access to marrow stroma. Microfracture induced substantial bone necrosis indicated by more empty osteocyte lacunae in adjacent bone around microfracture than drill holes, which were done under cooled irrigation. In our rabbit model, drilling deeper to 6 mm penetrated the epiphyseal scar and led to greater subchondral hematoma with increased access to marrow stroma. At 14 days post-operation, advanced new bone formation and remodelling were seen in drilled defects, in contrast to prolonged resorption of fractured bone around microfracture holes which impeded repair. New bone synthesis and chondrogenesis were observed in both deep and shallow drill holes on Day 21 post-operatively. Results Compaction of bone around microfracture holes essentially blocked connection with bone marrow. Drilling, however, removed bone and debris from holes to provide channels to marrow. Significantly more empty osteocyte lacunae (bone necrosis) were detected in bone lining the microfracture holes, compared to the drill holes generated under cooled irrigation. Deep drilling to 6 mm penetrated the epiphyseal scar in rabbits and led to greater subchondral hematoma and increased access to marrow stroma. At 14 days post-surgery, advanced new bone formation and remodelling were seen in drilled defects, in contrast to prolonged resorption of fractured bone around the microfracture holes, which impeded repair. New bone synthesis and chondrogenesis were observed in both deep and shallow drill holes at 21 days post-surgery. Conclusions Acute subchondral fractures and repair responses were distinct comparing microfractured and drilled defects. Surgical technique and hole depth may affect the patterns and connectivity of subchondral bone marrow channels, and influence long term cartilage repair properties. Conclusions Acute subchondral fractures and repair responses were distinct between microfractured and drilled defects. Surgical technique and hole depth may affect the patterns and connectivity of subchondral bone marrow channels, thus influencing cartilage repair processes and long-term outcomes., Introduction The understanding of cartilage development and its relation to chondrocyte function had been attained principally by biochemical and electronmicroscopic methods. Cartilage has been often viewed as a uniform structure, with ECM divided into pericellular, territorial and interterritorial zones, described by Benninghof in 1925, who accorded a central role to pericellular ECM investing one or a cluster of chondrocytes and forming units termed chondrons. In this scheme chondrocytes were viewed as static entities. In this investigation chondrocytes in cell culture and chondrocytes in hyaline articular and tracheal cartilage were studied to detect cell to cell communications and demonstrating their uniqueness. Methods and Materials Cell cultures were initiated from adult human cartilage using explant techniques. Cell cultures were viewed continuously in an incubated inverted microscope with time lapse photographs obtained from 5 to 15 minutes, utilizing Hoffman modulation contrast. Thin sections of adult human articular cartilage and those of cartilage from rat tracheas were studied utilizing structural coloration as previously described (Histochem Cell Biol 109:1, 1999) and high resolution transmitted light microscopy. Results Time lapse photography demonstrated intermittent contacts between chondrocytes by means of intercellular bridges of various configurations. Transmitted light microscopic studies of cartilage sections showed the presence of intrachondral tubules and intercellular bridges. Conclusions Chondrocytes in vivo and in-vitro demonstrated cell to cell communications by well recognized anatomic structures, suggesting chondrocytes are active dynamic cells, and not static structures., Introduction Honokiol is a small molecular weight ligand originally isolated from the Chinese medicinal herb Magnolia officinalis, a plant used in traditional Chinese and Japanese medicine. A previous study, the effect of honokiol was known as anti-angiogenic, anti-invasive and anti-proliferative activities in a variety of cancer. Methods and Materials MTT assay Western blot analysis Alcian Blue staining Imunohistochemistry Results we investigated whether honokiol inhibited NO-induced apoptosis in articular chondrocyes using by phase-contrast microscope, MTT assay and Western blot analysis. A previous study, SNP induced activation of p53 via activation of p38 kinase in articular chondrocytes. Treatment of honokiol inhibited SNP-induced activation of p38 kinase in articular chondrocytes. Inhibition of p38 kinase with SB203580 rescued SNP-induced apoptosis. Honokiol reduced p53 expression in dose-dependent manner. Also, treatment of honokiol was suppressed NO- induced dedifferentiation in articular chondrocytes as determined by the accumulation glycosaminoglycan by Alcian Blue staining, immunohistochemistry and Western blot analysis. Conclusions Our results collectively suggest that honokiol inhibited NO-induced apoptosis and dedifferentiation of rabbit articular chondrocytes via different mechamixms., Introduction Microtubule-interfering agents (MIAs), paclitaxel have been attributed in part to interference with microtubule assembly, impairment of mitosis, and changes in cytoskeleton. But the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. In this study, we investigated the effects of paclitaxel determined that is the mechanism of paclitaxel-induced differentiation and COX-2 expression in rabbit articular chondrocytes. Methods and Materials Chondrocytes were treated with various concentration of paclitaxel. And cells were determination of chondrocyte pheotype by western blotting, Immunofluorescence Microscopy, and detected cell cycle by flow cytometry of propidium iodide stained cells. Results Microtubule damages inhibit dedifferentiation, and increase COX-2 expression and Prostaglandin E2 production in chondrocytes by paclitaxel. The involvement of three major MAPK family members (ERK, p38 kinase and JNK), PKC and PI3/AKT kinase in the paclitaxel signaling to promote differentiation and COX-2 expression in chondrocytes. Activation of ERK-1/2 is suppressed the differentiation, whereas activation of p38 kinase signaling upregulated the differentiation. Interestingly, activation of ERK-1/2 and p38 kinase increased COX-2 expression by paclitaxel treatment in chondrocyte. In addition, paclitaxel suppressed is dedifferentiaion is necessary for PI3/Akt kinase activation, and COX-2 expression required for blocked the JNK signaling. Conclusions Therefore, these results collectively indicated that ERK-1/2, p38 kinase oppositely regulate paclitaxel-induce differentiaion but not COX-2 expression in chondrocytes., Introduction Leflunomide is an oral immuno-modulatory agent which is considered effective disease modifying anti-rheumatic drug(DMARD) in RA. Leflunomide known as a regulator of iNOS synthesis which largely increases NO production in diverse cell type. However, the effect of leflunomide on chondrocyte is still poorly understood. In our previous studies, we have shown that direct production of NO by treating chondrocytes with NO donor, sodium nitroprusside (SNP), causes apoptosis, dedifferentiation and COX-2 expression via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation and extracellular signal-regulated kinases pathway. In this study, we characterized the molecular mechanism by which A77 1726 supress apoptosis, dedifferentiation and COX-2 expression through activation of Phosphoinositide 3-kinase signaling pathway. Methods and Materials We used that detection of NO-induced apoptosis by MTT assay and DNA fragmentation and FACS. Also, detection of inhibited dedifferentiation and COX-2 expression by A77 1726 were determined by Western blot and Alcian blue stain, Reverse transcription - polymerase chain reaction. Results We found that A77 1726 inhibited NO-induced apoptosis. Also, A77 1726 inhibited dedifferentiation and COX-2 expression. Inhibition of PI3K signaling pathway by LY294002, PI3K specific inhibitor, these effects of A77 1726 were completely inhibited. Thus, these data indicate that A77 1726 supress NO-induced apoptosis, dedifferentiation and COX-2 expression via PI3K signaling pathway. Conclusions Finally, our results suggest that A77 1726, active metabolite Leflunomide is benefit to prevent degradation of cartilage caused by OA or RA., Introduction 2-deoxy-D-glucose is a non-metabolizable glucose analogue in which the hydroxyl group at the second position carbon is replaced by a hydrogen. It is known as inhibitor the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level. Glucose is generally believed to be an various crucial role of the numerous cell including the essential precursor in glycosaminoglycan biosynthesis. The purpose of this study was to characterize the effects of 2DG on dedifferentiation and expression of cyclooxygenase-2 (COX-2) in rabbit articular chondrocytes, focusing on the roles of ER stress. Methods and Materials 2DG reduced type â…¡ collagen and COX-2 in time- and dose- dependent manner as indicated by western blotting, prostaglandin E2 production, alcian blue staining and immunofluorescence. Results 2DG induced growth arrest and DNA damage induced-153 (GADD153) expression. in rabbit articular chondrocytes. Also, 2DG treatment inhibited activation of glucogen synthase kinase 3β (GSK3β) in dose and time dependent manner, accompanied by GADD153 expression. Inhibition of GSK3βwith Na3VO4 recovered 2DG induced N-glycosylation inhibited COX-2 but not effect on dedifferentiation. inactivation of GSK3β induced accumulation of β-catenin. Accumulation of β-catenin by the inhibition of GSK3beta with LICl recovered 2DG induced N-glycosylation inhibited COX-2. whereas addition of glucose recovered type â…¡ collagen and reduced GADD153 expression, but not N-glycosylation inhibited COX-2. Conclusions Our results indicate that 2DG-induced dedifferentiation and inhibition of N-glycosylation COX-2 by modulating ER stress pathway in rabbit articular chondrocytes., Introduction Time-lapse cinematography allows for the exploration of the dynamic features of cell populations in vitro. In the present study this technique was utilized to observe chondrocytes in the primary and low passage cultures. The aim of the study was to document unique features of articular cartilage chondrocytes propagated in vitro. Methods and Materials Cell cultures were initiated by explants of cartilage from cadaver donors. Cells were propagated in tissue cultures medium CMRL 1066 with 10% FCS. Only primary or low passage cultures were used. Chondrocyte cultures were also stained with fluorescent and non fluorescent vital stains. Fluorescent stains were acridine orange, and carboxy fluorescein ester. Non-fluorescent-neutral red, Janus green B and Nile blue sulfate. Cell culture, were viewed in a Nikon inverted microscope with Hoffman modulation contrast optics. Photographs were obtained from every 5 to 15 minutes for 24 to 96 hr period. Images were recorded using Windows version 4.1 and Insight Spot Advances programs. Results Chondrocyte cultures contained large cells with numerous projections. Single nucleated cells with bitemporal filopodia were migrating through the observation fields. When filopodial projections came into contact with chondrocytes the projections and their mother cells become united with the cells with which they established contacts. Cytonemeta were also formed after completion of cytokinesis by dividing cells. Vital stains showed normal patterns of fluorescent and vital stain uptake seen with viable cells. Conclusions Chondrocytes in culture establish cell to cell contacts by filopodia, cytonemeta and other types of intercellular bridges, and have unique morphologic characteristics., Introduction Evaluate the feasibility and efficacy of using autologous synovium tissues in chondral defect repair. Methods and Materials Synovium tissue fragments were harvested from the caprine knee joint using a custom made device. Cell viability in the fragments was assessed using the Live/Dead assay. Chondrogenesis of the synovium was assessed in an explant culture and in a caprine chondral defect repair model. In the explant culture, harvested synovium tissue was embedded in agarose, cultured in TGF-beta 3 supplemented medium for 28 days, and stained with Safranin O. In the repair model, a 4×5×0.45 mm chondral defect was created on the trochlear groove. Microfracture was performed in seven animals. In the other seven animals, synovium fragments were implanted into the defect following microfracture. Defects were covered by a devitalized fascia flap. Repair of the defect was evaluated histologically at 6 weeks post surgery. Results More than 90% of the cells in the harvested synovium appeared viable. Cells in synovium explant culture differentiated into chondrocytes with positive Safranin O staining. In the repair model, flap delamination was observed in 28% of control and 43% of synovium implant group, resulting in minimal tissue filling in the defect. Although defects with intact flaps were filled predominately with fibrous tissue, clusters of chondrocytes were observed in the defect with synovium implant. Conclusions We demonstrated the chondrogenic potential of synovial cells in vitro and the feasibility of harvest and implantation of autologous synovium tissue in a large animal model. Further investigation is required to optimize the repair efficacy mediated by synovium implant., Introduction Stem cell therapies are evaluated as a promising alternative for cartilage regeneration. Since adipose tissue provides a rich source of mesenchymal stem cells (ASCs) which can be harvested in clinically relevant quantities within a short time frame, we investigated whether a one-step surgical procedure for osteoarthritic treatment can be devised with ASCs from the infrapatellar (“Hoffa”) fat pad. Methods and Materials Infrapatellar fat pads were harvested with informed consent from patients undergoing knee arthroplasty. Colony-Forming Unit assays were performed, a.o. to calculate the percentage of stem cells and their multidifferentiation potential. Growth kinetics and the surface marker expression profile of ASCs were determined. Finally, according to the one-step surgical procedure, ACSs were induced into the chondrogenic lineage in a 3D poly-lactic acid-co-caprolactone (PLA-CPL) scaffold, and analyzed using RT-PCR, immunohistochemistry and glycosaminoglycan formation. Results The frequency of Colony-Forming Units in the stromal vascular fraction (SVF) of these fat pads was 2.6 ± 0.6%. These CFUs showed multilineage differentiation towards the osteogenic and adipogenic lineage. The population doubling time of approximately two days and the surface marker expression profile matched that of ASCs. Seeded in a 3D PLA-CPL scaffold material, freshly isolated stromal cells showed chondrogenic differentiation potential. Conclusions Due to the high ASC quantity in the stroma of the infrapatellar fat pad, the favorable proliferation rate and the high chondrogenic differentiation potential, this stroma is a suitable candidate for a one-step surgical procedure to regenerate cartilage tissue., Introduction Mesenchymal stem cells (MSCs) have been preferred to as immunological tolerant cells. Previously we demonstrated that both juvenile and adult articular chondrocytes were non-alloreactive and that they inhibited the mitogenic activity of activated CD4+ T cells by cell-to-cell contact (ORS 2004, 2006). Furthermore adult chondrocytes preserved their immunological characteristics independent of their osteoarthritic condition (ICRS 2007). Cultured articular chondrocytes (ACs) had immune characteristics that could be used for tissue repair; it is likely that allogeneic MSCs will be used as well. We hypothesize that mesenchymal-lineage cells preserve common immunological characteristics. The purpose of this study is to compare immunological properties of ACs with other mesenchymal-lineage cells derived from various origins in the same persons. Methods and Materials ACs and bone marrow stromal cells (BMSCs), meniscus cells (MCs) and synovial cells (SCs) were isolated from 5 knee joints when arthroplasty was performed. Each of the cells were cultured and expanded. To explore surface antigens on each cell, flow cytometric analysis was performed. To determine the allogeneic reaction, MLR assay and a proliferation assay in mitogenic activating CD4+T cells were performed. Results Each of the cells expressed MHC class I(+), MHC class II(−), CD34(−), CD45(−), CD73(+), CD90(+), CD105(+) and CD166(+). These expression patterns were the same as on MSCs. ACs, BMSCs, MCs and SCs failed to stimulate MHC mismatch response in MLR assay and they did not inhibit the proliferation of activated CD4+ T cells without cell-to-cell contact. Conclusions We demonstrated that mesenchymal-lineage cells isolated from various types of tissues preserved common immunological characteristics., Introduction We hypothesized that human cartilaginous constructs can be engineered from chondrocytes derived from osteoarthritic cartilage. Methods and Materials Chondrocytes were isolated from the cartilage removed from patients (n=5, 57±12 years) during total knee arthroplasty for osteoarthritis. Using culture conditions from previous studies, cells were expanded ∼20X over 2 weeks from a plating density of 5,000 cells/cm2 (medium with 2% human serum (HS), TGF-β1, FGF-2, and PDGF-bb), and then redifferentiated over 2 weeks in alginate beads (medium with 20% HS). Cells with associated matrix were released from alginate and seeded into 3.2 mm and 6.5 mm diameter cell culture inserts for 2 weeks to form constructs. Medium was serum-free or with 10% HS, and ±TGF-β1, ±IGF-I, ±BMP-14, individually and in combination. Constructs were evaluated by appearance, wet weight (WW), cell number, sGAG and collagen content. Large constructs were also measured for thickness and compressive modulus. Data were analyzed by 2-way ANOVA. Results During construct formation, inclusion of 10% HS in culture medium led to increased collagen/WW (+120%, p⇠0.05), decreased WW (−56%, p⇠0.001), decreased sGAG/construct (−47%, p⇠0.001), and decreased sGAG/cell (−39%, p⇠0.05). For large constructs, inclusion of 10% HS led to decreased construct thickness (−58%, p⇠0.05), and increased compressive modulus (+176%). Addition of growth factors, in particular the combination of TGF-β1, IGF-I and BMP-14, resulted in an increase in both sGAG/construct (+29%, p⇠0.05) and collagen/construct (+27%, p⇠0.05). Conclusions Using culture medium including 10% HS and additional growth factors including TGF-β1, cartilaginous constructs can be fabricated from culture-expanded chondrocytes that originate from osteoarthritic cartilage., Introduction Purpose: We hypothesized that co-culture of chondroprogenitor cells with chondrocytes could enhance their chondrogenic differentiation. The aim of this study was to investigate the efficacy of simply co-culturing synovial cells with articular chondrocytes and to assess the cells biochemically. Methods and Materials Materials and Methods: Rabbit articular chondrocytes, synovial cells and tenocytes(as control) were used in this study. Co-culture of synovial cells and articular chondrocytes were conducted either with or without cell-to-cell contact. PG content and the expression of mRNA of Sox-9, Aggrecan and type-II collagen were assessed. Results Synovial cells were shown to express chondrogenic phenotypes when grown in chondrogenic medium that contained essential growth factors for cartilage differentiation. When synovial cells were co-cultured with chondrocytes without cell-to-cell contact, their expression of chondrogenic proteins, such as Sox-9, aggrecan and type II collagen, was increased even in the absence of growth factors. furthermore, in the situation of cell-to-cell contact, expression of Sox-9 and type-II collagen in the synovial cells-chondrocytes mixture was significantly higher than that of the control. Conclusions These results suggest that co-culture of synovial cells with chondrocytes has a promotive effect on synovial cell chondrogenesis. It also implies that combined implantation of articular chondrocytes with isolated synovial cells could be an efficient method for repair of large chondral lesions, which need abundant implanted cells., Introduction The aim of this study is to characterize the temporal gene expression levels of different markers in order to determine its potential use as monitors of in vitro differentiation of 3D scaffolds. Methods and Materials Human articular chondrocytes were isolated, culture expanded in monolayer and seeded in Hyaff 11 scaffolds. During expansion DMEM-F12 supplemented with 10% human serum was used. During differentiaton in Hyaff 11 scaffolds DMEM high glucose, ITS, Linoleic acid, human serum albumin, 10 ngTGFβ1, dexamethasone and ascorbic acid was used. The constructs were cultured for 1, 7, 14 and 21 days. Alcian blue-Van Gieson staining was performed to evaluate GAG production. Real time PCR was used to evaluate the gene expression of the following markers: Cathepsin B, EGR1, SerpinA1, SerpinA3, MMP 1 and 13, SOX 9, Collagens I, IIa and IIb, Versican and COMP. Results Histologically it was possible to see increased GAG production within time. Cathepsin B and EGR1 were upregulated during dedifferentiation of chondrocytes. SERPINA1, SERPINA3, SOX 9, Collagen IIa and IIb, and COMP were upregulated during redifferentiation. SOX 9 had the highest expression level before the activation of the Collagen IIa gene. Versican was downregulated during redifferentiation. Conclusions Cathepsin B, EGR1 and SerpinA3 are potential markers to monitor the differentiation status of 3D tissue engineered constructs. Detection of these markers in the media would help to monitor the differentiation stage of the constructs and determine release criteria for clinical tissue engineering., Introduction Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. The current study investigated the ability of implanted chondrocytes to attach at the site of injury and incorporate into the devitalized host matrix adjacent to a defect in an in vitro human explant model. Methods and Materials Human osteochondral dowels received a standardized injury and were seeded with PKH 26 labeled passage 3 chondrocytes and compared to two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Results Seeded chondrocytes adhered to the standardized defect and areas of lamina splendins disruption but did not migrate into the acellular region adjacent to the defects. There was a difference in viable cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present, demonstrating cellular activity. Conclusions This work demonstrated that cultured human chondrocytes do adhere to regions of AC matrix injury but appear incapable of migration into the host tissue despite the presence of viable cells capable of generating a paracrine stimulus. Additionally, the explant model was viable during long-term culture and could be used to study the interaction of implanted cells and host tissue, providing insight to better optimize defect preparation and graft integration., Introduction The results after Autologous Chondrocyte Implantation (ACI) are evaluated using several outcome measures. The histological analysis of the repair tissue after ACI is an important and objective outcome measure to assess the quality of the characterization and the distribution of cells. Methods and Materials The characteristics of the repair tissue after ACI of 97 patients were evaluated histologically. All patients had a second look arthroscopy where a biopsy was taken. Biopsy specimens were obtained from the center of the defect in 97 patients, fixed in 4% formaldehyde for 24 hours, embedded in paraffin, cut into 8-μm sections and placed onto microscope slides. After deparaffinization the sections were stained with Alcian blue van Gieson, hematoxylin and eosin, Alcian blue, or safranine O. To perform a semiquantification of the repair tissue the ICRS visual score was used. Briefy the aspect of the surface, extracellular matrix, subchondral bone, cell distribution and presence of calcified matrix was evaluated by three experienced phisicians. Clinical correlation of the biopsies and the clinical outcome were performed when available. Results After evaluation the surface showed a mean score of 2.60, the matrix 2.02, the subchondral bone 2.57, the cell distribution 0.90, the viability 2.97 and the calcified cartilage 2.95. The maximum score of all the items is 3. Conclusions The quality of the repair tissue after ACI shows a hyaline like or a mixture of fibrohyaline and hyaline like tissue. The normal distribution of the cells was not restored; however in some cases the normal columnar distribution was restored over time., Introduction Autologous chondrocyte implantation has been a widely used clinical strategy in the repair of damaged cartilage. Follow-up showed good clinical results together with the formation of a new tissue with many hyaline features. To explore a new approach to treat also early degenerative lesions to hyaline cartilage in osteoarthritis patients (OA), we proposed to transplant in an experimental animal model of OA, a scaffold seeded with autologous mesenchymal stem cells, obtained from bone marrow and expanded in culture. Methods and Materials Rabbit knee joints were bilaterally subjected to Anterior Cruciate Ligament Transection (ACLT) to surgically induce OA. After 8 weeks, necessary to the development of cartilage surface damage, animals were treated with mesenchymal stem cells seeded onto Hyaff®-11 scaffold in the left condyle and unseeded Hyaff®-11 in the controlateral knee. Untreated rabbits were used as control. All the animals were sacrificed at 3 and 6 months after surgery. Histological, histomorphometric and immunohistological evaluations were performed. Results OA changes developed in all animals subjected to ACLT. The predominant macroscopically observed OA changes were mild (lateral femoral condyle) or moderate (medial femoral condyle) ulcerations. Statistically significant differences in the quality of the regenerated tissue were found between the implants with scaffolds carrying mesenchymal stem cells compared to the scaffold alone or controls in particular at 6 months. Conclusions From the observations, it is possible to demonstrate that Hyaffò-11, a hyaluronan-based scaffold, provides promise for mesenchymal stem cell implantation and that may have application for the treatment of early OA in humans., Introduction There is a need for diagnostic and predictive biomarkers in musculoskeletal disorders generally; in autologous chondrocyte implantation (ACI) it would be useful if a serological marker tested early post-treatment could predict long term success. We have measured levels of 2 molecules commonly investigated in arthritis studies, cartilage oligomeric matrix protein (COMP) and hyaluronan (HA), in synovial fluid and blood of ACI-treated patients. Methods and Materials Plasma (P) and synovial fluid (SF) were obtained from 4 patients (aged 26–41 years) at 2 timepoints: three immediately pre-op and at 8–14 months post-ACI and one at 19 and 31 months post-treatment. COMP, HA and urea were measured via ELISA; COMP and HA in SF were normalised to urea to allow for its dilution factor due to sampling or pathology. Results Levels of HA in plasma were 39±34ng/ml and in SF 10±4mg/ ml, with little difference in SF pre- and post-op (23±15, 55±44ng/ml respectively (plasma); 10±5, 9±4mg/ml (SF)). COMP levels, however, increased post-operatively in both plasma (613±206, 727±290ng/ml respectively) and SF (298±185, 569±438μg/ml). Levels in the fourth patient showed similar trends between sampling times. Conclusions Although patient numbers are small, this study suggests that ACI results in altered composition of the synovial fluid for some time post-treatment, with increased COMP at the second timepoint. Whilst serum levels have been shown to increase similarly for 3–6 months post-arthroplasty, the increase is sustained longer post-ACI. How this relates to COMP's function of organising collagen fibrils and binding other molecules within the repair tissue remains to be determined., Introduction Bone tendon healing in shoulder surgery provides 50% failure rate after repair. The first aim of this study was to validate a small animal model, and the second was to evaluate the effect of chondrocytes injections on healing. Methods and Materials Under general anesthesia, Achilles' tendon of thirty 3 month-old wistar rats were detached from the calcaneum and enthesis was destroyed mechanically. The tendon was then reattached to the bone by transosseous 4/0 non absorbable suture, skin was closed (G1). Cell therapy group consist of the same surgical procedure with local delivery of 4 Millions rat chondrocytes suspended in fibrin glue (G2, n=30). Animals were sacrificed at 15, 30 and 45 days to evaluate global healing rate, biomechanical and histological data. Results The global healing rate increased from 47% (G1) to 70% (G2) (p=0,11). At 45 days, failure to load was 82 N in G2 versus 62 N in G1, with a statistically significant difference (p⇠0,05). Typical aspect of chondrocytes in chain was found, from 15 days in G2 but never in G1. Immunostaining demonstrates local production of type II collagen at the bone tendon junction only in G2. Conclusions The current study validate a small animal model of tendon to bone repair with 47% healing rate, close to the value reported in human shoulder cuff surgery. Injection of chondrocytes induces type II collagen production, and is an efficient therapy to stimulate bone tendon healing by increasing global healing rate and providing a statistically significant difference in failure to load at 45 days compare to surgery without cell transplantation., Introduction To develop an accurate, reproducible lot release assay to evaluate cell viability of advanced cell therapy and tissue engineering products like MACI, which contain cells seeded in 3-D matrices. Methods and Materials A novel enzymatic reaction-based method was invented by correlating viability to relative levels of enzyme activity in cell-containing and non cell-containing fractions (conditioned media) of the culture. Results The new method shows excellent accuracy (15%), precision (15%), linearity (R2⇢.95), and specificity (Signal/Noise⇢3) over a large range of cell viabilities (e.g. 0%-100%) and cell densities (e.g. 8.75 e4 to 2.8 e6 per cm2 membrane for MACI). The working cell density range and applicable medium type of the new method can be easily adjusted using phenol red to meet the needs of different products. The new method does not require cell recovery or control cells, and is not limited by donor variability or matrix type. It can be applied to both 2-D and 3-D cultures of human or animal cells in all media types. Conclusions The new method solved a long-standing technical challenge for tissue engineering products such as MACI. Viability can be accurately determined without interference from the matrix material. The method has been developed and validated for use with MACI, and further enhances our rigorous quality control and assurance program for advance cell therapies., Introduction We developed an injectable implantation system, using a novel in-situ forming material based on alginate, which can be arthroscopically performed. We have demonstrated that this system enhances cartilage repair in a rabbit model. The aims of this study were to evaluate the reparative tissue in a canine model and to determine whether this material can be arthroscopically implanted in cadaveric knees. Methods and Materials ⇠Canine model⇢Two osteochondral defects (95×5mm) were created in the patella groove of beagle dogs. The defects were divided into three groups: no treatment group, material (material without cells) group, and material with BMSCs group. Dogs were euthanized at 16 weeks postoperatively. The sections were evaluated using macroscopic and histological scoring. The mechanical properties were measured by an indentation test. ⇠Cadaver model⇢A cartilage defect (10×20 mm) was made at the medial femoral condyle. A dyed material was arthroscopically implanted and evaluated after 24 hours of manual mobilization. Results ⇠Canine model⇢ The material groups were repaired mainly with fibrocartilage. The material with BMSCs group exhibited normal cartilaginous tissue with rich GAG matrix. The macroscopical and histological scores of the treatment groups were significantly higher than the defect group (p ⇠0.05). The compressive modulus in the material with BMSCs group was significantly higher, compared to that in other groups (p ⇠0.05). ⇠Cadaver Model⇢The implanted material maintained its initial shape. Conclusions The current study showed that the implantation of BMSCs using our novel system induced matured hyaline-like cartilage repair in a canine model. We successfully established an arthroscopic implantation technique using human cadaveric knees., Introduction The aim of this study was to determine the appropriate in vitro culture conditions that increase the in vivo chondrocyte differentiation. Methods and Materials Human articular chondrocytes were isolated, culture expanded in monolayer and seeded in Hyaff 11 scaffolds. Three different media conditions were used. The different media were Media A: DMEM-F12 supplemented with 10% human serum, Media B: DMEM high glucose, ITS, Linoleic acid, human serum albumin, 10 ng TGFβ1, dexamethasone and ascorbic acid, Media C was a composed of Media B and 10% human serum. After 14 days the constructs were transplanted into human osteochondral plugs which were then implanted into the subcutaneous tissue of nude mice for 6 weeks. An empty scaffold was used as negative control. In order to determine if the preculture had an effect on the in vivo differentiation two scaffolds were implanted after 1 or 28 days of in vitro culture. The results were semiquantified using the Bern score. Results The preculture affected the differentiation in vivo. Samples implanted 1 day after seeding showed no differentiation after 8 weeks in vivo whether precultured samples showed significantly higher differentiaiton grades. The media composition affects the in vivo differentiation capacity. In this study the formulations B and C containing TGFβ1 affected the differentiation grade and the integration to the surrounding cartilage. Empty scaffolds did not show differentiation in vivo in this model. Conclusions Tissue engineered constructs should be cultured in vitro with media formulations containing TGFβ1 in order to increase the in vivo differentiation and integration capacity., Introduction Monolayer cultures of chondrocytes as well as in vitro tissues using cells and scaffolds have been widely used to understand the mechanisms involved in cartilage matrix turnover and cell differentiation. However, detailed studies have been hampered by a number of factors such as the scarce availability of human tissue, the limited proliferative capacity of cultured chondrocytes and the enormous donor dependent variances in the differentiation status of cells. It is frequently reported that primary chondrocytes in monolayer culture undergo a phenotypic drift including down regulation of cartilage markers like type II collagen and aggrecan. Methods and Materials In order to establish a nontransformed human articular chondrocyte cell line possessing a remarkable extended life span, we transfected human articular chondrocytes with selected proliferation genes enhancing the replicative capacity. The successful transfection as well as the expression of markers characteristic for hyaline cartilage was verified on the mRNA level by RT-PCR and on the protein level by immunofluorescence. Results This chondrocyte cell-line which has the capacity to escape the Hayfick limit of cell proliferation and also displays a cartilage typical protein expression profile (type II collagen and aggrecan) may provide an ideal cell source for numerous donor independent studies. Conclusions Especially the possibility to bypass the dilemma of donor variances nominate this human chondrocyte cell line as suitable basis for pharmacological test systems relevant to screen the physiological behaviour of cartilage cells in healthy tissue and in arthritic diseases, particularly in response to putative therapeutical agents., Introduction Matrix metalloproteinases (MMPs) are catabolic enzymes involved in the remodeling of connective tissues. In osteoarthritis (OA), dysregulation of MMPs in joints results in the change from physiologic remodeling to pathologic destruction of cartilage. The mechanisms involved in regulating MMP activity are therefore attractive targets for therapeutic intervention. We determined whether MMP expression can be modulated in cytokine activated chondrocytes by the combination of avocado soybean unsaponifiables (ASU), glucosamine hydrochloride (GLU) and chondrotin sulfate (CS). Previous studies have shown the benefit of using ASU alone, or GLU and CS in the management of OA. Methods and Materials Chondrocytes (5×105) in serum free media were activated with IL-1β (10 ng/ml) and TNF-α (1 ng/ml) on days 1 and 4. Cells were next incubated on day 7 with: control media or the combination of ASU (NMX1000® 8.3 μg/ml), GLU (FCHG49®, 11 μg/ ml), and CS (TRH122®, 20 μg/ml). Secreted MMPs were analyzed by zymography and Western blots on day 8. Results Non activated chondrocytes enzymatically express latent and active forms of MMP 9. Cytokine activation induced a shift in the ratio of the latent form of MMP 9 (∼92 kD) to the active form (∼82 kD). The combination of ASU, GLU and CS inhibited the cytokine induced conversion from latent to active forms of MMP 9. Conclusions Our finding suggests that the combination of ASU, GLU and CS inhibits cytokine induced shift from latent to active forms of MMPs. Suppression of MMP activation may minimize cartilage breakdown that is beneficial in the management of OA., Introduction Plasma-mediated bi-polar radiofrequency is a low temperature radio-frequency based technique used to remove soft tissue. In arthroscopic surgery, it is used when treating partial-thickness defects in cartilage, where the method aims to supply a smooth and stable joint surface. Previous studies have shown that plasma-mediated bi-polar radiofrequency effects interleukin production from porcine disc cells in vitro and in vivo in a way that is suggested to promote the healing response of the tissue. The aim of the present study was to evaluate the effect of plasma-mediated bi-polar radiofrequency on human chondrocyte proliferation. Methods and Materials Surplus human chondrocytes from four patients (mean age 23yrs) undergoing autologous chondrocyte transplantation were cultured in 3D-alginate gel in chondrogenic media. IL-1α-supplemented media was also used to stimulate an inflammatory response. After four weeks the cells were exposed to a plasma-mediated bi-polar radiofrequency wand (ArthroCare Corp.) for five seconds, or a temperature sham, with a similar temperature as the wand, approx 50°C. The amount of DNA in the gels was determined three and six days post treatment. Results A significant (p=0.05) increase in cell proliferation was observed in the plasma-mediated bi-polar radiofrequency treated group with unstimulated cells, whereas no such effect was seen in the IL-1α stimulated cultures. Conclusions Our results suggest that the plasma-mediated bipolar radiofrequency treatment induces a proliferative response of chondrocytes in alginate gel culture. The increase in proliferation observed might have positive clinical effects on a tissue healing response. Continued studies are ongoing to further investigate the response on a cellular as well as gene regulatory level., Introduction Oxidative stress plays an important role in the pathogenesis of osteoarthritis. Epigallocatechin gallate has potent antioxidant activity. Oral curcumin efficacy has been shown for many conditions characterized by oxidative damage and inflammation. Although the protective effects of these two agents have been reported in various models, there are few studies about their protection against cartilage damage in vitro. Methods and Materials Articular cartilage was isolated from carpal joints of bovine. Cartilage fragments were cultured in 1ml of DMEM/F12 supplemented with 10% foetal bovine serum, 100units/ ml penicillin/streptomycin. 24h later medium was changed and different cultures were produced: first one with IL-1b, second added with IL-1b and EG, third made of IL-1b and CR, fourth with IL-1b, EG and CR; two concentrations of EG and CR (6+5 mg/ml and 3+2,5 mg/ ml respectively) were used for a new set of cultures. GAG and NO release from cultured tissues were assayed. Results The studied compounds showed a significant inhibition of the release of GAG when the cartilage was challenged with IL-1b; the association of the two components (EG-CR) induced a more powerful and significant effect at both concentrations. Our results also indicate that NO is inhibited with less efficacy, but the effect of the association is more evident when compared with the effect of single product. Only CR and EG+CR are statistically significant. Conclusions Combined use of EG and CR seemed to be effective in cultured bovine cartilage tissues in reducing NO and GAG production when tissues were challenged with IL-1b., Introduction Autologous Chondrocytes Implantation relies on chondrogenic capacities of implanted cells. Markers allowing for selection of human articular chondrocytes (HAC) with better chondrogenic potential would represent a major advantage. In this study we investigated HAC intrinsic chondrogenic capacities with surface marker expression and cell population doubling (PD). Methods and Materials Expression of surface marker CD90, CD166, CD14, CD54, CD105 and CD49c was monitored in HAC during early monolayer expansion via flow cytometry. For cell sorting HAC (n=4) were expanded for 5 days and labelled with anti-CD90 monoclonal antibody. Unsorted, CD90-positive and CD90-negative sorted cells were further expanded for up to 33 days. At weekly passages cumulative PD was determined, flow cytometry analysis performed and micromass pellet cultures prepared and incubated in serum-free medium with and without the addition of chondrogenic factors. Results Changes in surface marker expression correlated with early dedifferentation process, with a marked upregulation of CD105 and CD49c after 3 days, and CD166 and CD90 after 10 days of expansion. CD90 expression distinguished two chondrocyte subpopulations at days 3–5 with a complete shift from CD90-negative to CD90-positive cells at day 14. Sorted CD90-negative cells proliferated slower, reaching a 3.2 PD threshold later, and produced pellets with more mature neocartilagenous tissue compared to CD90-positive or unsorted cells. However, sorted CD90-negative cells became CD90-positive within 26 days of further expansion, and beyond PD 3.2 also produced immature fibrocartilagenous tissue. Conclusions HAC lacking CD90 expression have more intrinsic chondrogenic potential to produce cartilage-like matrix. Furthermore, higher chondrogenic potential correlates with a slower proliferation rate., Introduction Chondrocyte viability is essential for the maintenance of articular cartilage as they constitute the only cell type in articular cartilage. Apoptosis is processed by specific intracellular signaling cascades and thus has been an attractive therapeutic target for diseases in which apoptosis is an integral part of its pathogenesis. Transglutaminase2(TGase2) has been shown to be induced and activated during apoptosis. We have previously shown that TGase2 expression is increased in human chondrocytes undergoing apoptosis. We also have explored the role of TGase2 in chondrocyte apoptosis by using monodansylcadaverine(MDC). RNA interference(RNAi) is a process of sequence-specific gene silencing mediating specific mRNA degradation and thus allows the understanding of the function of specific gene of interest. The purpose of this study was to explore the role of TGase2 in human chondrocyte apoptosis by using RNAi. Methods and Materials Chondrocyte apoptosis was induced by treating with H2O2 for 24 hours. Apoptosis was assessed by DNA fragmentation ELISA and Annexin-V FACS analysis. The expression and enzyme activity of TGase2 was examined with Western blot and immunocytochemistry TGase2 was inhibited by MDC. siRNA targeting TGase2 was constructed with sequences of 5A′-GGGCGAACCACCUGAACAATT-3A′ and 3A′-TCCCGCUUGGUGGACUUGUU-5A′. Results MDC increased the amount of apoptosis, measured by DNA fragmentation ELISA, in H2O2-treated cells in a dose-dependent manner. Transfection of siRNA into human chondrocytes resulted in downregulation of TGase2 protein as shown by the Western blot and immunocytochmistry. The enzyme activity of TGase2 was also down-regulated in siRNA-transfected cells. Apoptosis in siRNA-transfected chondrocytes was increased, measured by Annexin-V FACS analysis, when compared to control-siRNA transfected chondrocytes. Conclusions These results implicate a protective role of TGase2 against apoptosis in human chondrocytes. These results provide new insights into the possibility of TGase2 as a potential modulator of osteoarthritis by protecting against chondrocyte apoptosis., Introduction The purpose of this work is to evaluate the maturation in vitro and in vivo of an engineered cartilaginous tissue obtained by isolated swine articular chondrocytes embedded in fibrin glue at different experimental times. Methods and Materials Isolated swine articular chondrocytes were embedded in fibrin glue. Sample groups were divided as follow: Some samples were left in standard culture conditions for one, five and nine weeks. Some others were implanted in nude mice for the same time schedule. The remaining samples were cultured in vitro for one and five weeks and then implanted in subcutaneous pouches of nude mice for four additional weeks. Gross evaluation, biochemical analysis (DNA and GAGs content) and gene expression (collagen type I and type II, aggrecan and Sox 9) were performed. Results Generally, samples retrieved from nude mice experienced shrinkage and mass reduction; they also resulted in an higher content of DNA and GAG. Collagen type II was higher in the 5- and 9-week samples with respect to those cultured in vitro. Opposite finding was recorded for aggrecan expression, while Sox9 was not significantly different from in vitro and in vivo. Conclusions The results of this study demonstrate that in vivo implantation of engineered cartilage composite results in increasing the cell proliferation and matrix formation. Pre-culturing the samples before implantation does not seem to interfere with the capacity of cell proliferation and synthesis, but, on the other hand, does not appear to ameliorate the quality of the engineered samples in this model., Introduction To repair cartilage, the source of cells used in tissue engineering includes human native chondrocytes, mesenchymal stem cells (hMSCs) and hMSCs-derived chondrogenic cells. The mechanical properties of these cells are important predictors of chondrocyte function. To distinguish the mechanical properties of these different cell sources from the same patient, an atomic force microscope (AFM) was used to probe the surface ultrastructure and to measure the adhesion force and stiffness. Methods and Materials The hMSCs-derived chondrogenic cells were induced by TGF-β1 from hMSCs. The chondrogenic specific marker, type II collagen, was evaluted by RT-PCR and immunohistochemical staining. We directly employed the AFM to image a single cell and to quantitatively measure the dimensions of the cells. The cellular surface proteins were monitored by flow cytometric analysis. Results The profiles of shapes, sizes and structures were different across these 3 groups. The mean adhesion forces of native, hMSC and induced groups were 6.86±2.91, 13.31±5.16 and 4.54±1.14 nN, respectively. The stiffness values were also different (0.134±0.029, 0.164±0.017 and 0.109±0.017 N/m, respectively). The expression of type II collagen and integrin β1 were higher in native chondrocytes than those in hMSCs-derived chondrogenic cells. Conclusions Human native chondrocytes had stronger mechanical properties than those of hMSCs-derived chondrogenic cells. The differential properties might be the results of different expression amounts of cell matrix proteins. This study is the first one to directly characterize the mechanical properties between native chondrocytes and hMSC-derived chondrogenic cells., Introduction In patients with Osteoarthritis, the intra-articular temperature elevates to higher degree due to local inflammation. It was reported that heat stress on chondrocytes were induced apoptosis, but it is still clearly unknown how chondrocytes apoptosis were induced by heat stress. In the present study, we investigated the responses of chondrocytes to heat stress, and how chondrocytes apoptosis were induced by heat stress. Methods and Materials To apply heat stress to NHAC-kn cells(human normal chondrocytes), they were cultured for 1 days in incubator at 37℃. And then, they placed in water bath set at 43℃ or at 37℃ as a control temperature for 30min, 1hour, and 2hours. After the heat stress, they were removed from the water bath and moved to incubator at 37℃. Chondrocytes apoptosis were detected by TUNEL staining and western blotting. To explore the function of p53, NHAC-kn cells were pre-treated with pifithrin-alpha which is inhibitor of p53. Results Apoptotic cells were increased by heat stress in a time depend manner. The expression levels of p53, p53AIP1 and cleaved caspase-9 were increased after induction of heat stress. Apoptotic cells were decreased when chondrocytes were incubated with pifithrin-alfa. The expression levels of p53, p53AIP1 and cleaved caspase-9 were decreased when chondrocytes were incubated with pifithrin-alpha. Conclusions Our results showed that expressions of p53 and p53AI P1 were increased by heat stress, and apoptosis were inhibited when chondrocytes were pre-incubated with pifithrin-alpha which is inhibitor of p53. These indicated that apoptosis by heat stress in chondrocytes were dependent on p53 pathway., Introduction Tissue-engineering may offer promising future alternative to obtain extra autologous cartilage for reconstructions in otorhinolaryngology. Expanded chondrocytes or mesenchymal stem cells (MSCs) could be used for this purpose. The aim of this study was to study the performance of auricular and nasal-septum chondrocytes, and compare tissue-engineered constructs of these cells with those of the more frequently studied MSCs and articular chondrocytes, concentrating on subtype and stability of the tissue-engineered constructs. Methods and Materials Expanded chondrocytes and MSCs were chondrogenically differentiated in vitro. Subtype of the tissue-engineered construct was evaluated using immunohistochemistry, and their stability was tested both in vitro and in vivo. Results Auricular and nasal-septum as well as articular chondrocytes and MSCs produce a collagen II containing matrix. Although auricular and nasal-septum chondrocytes produce collagen X, both form cartilage matrix that is stable and does not mineralize when challenged in-vitro or after subcutaneous implantation in vivo. This in contrast to cartilage produced by MSCs that does mineralize, making it unsuitable for cartilage reconstructions. Although articular chondrocytes produce a true hyaline-like cartilage, without collagen type X, this source is not preferred in otorhinolaryngology. Comparing the performance of auricular and nasal-septum chondrocytes, it was obvious that auricular chondrocytes produce more cartilage-like matrix. Although direct comparison is difficult because nasal septum chondrocytes proliferate faster, auricular chondrocytes have our preference because of the robustness of results and the ease to harvest. Conclusions Our results indicate that auricular cartilage is a suitable candidate to generate stable cartilage for future reconstructions with tissue-engineered cartilage., Introduction Chondrosarcoma classification relies upon a combination of tissue morphology evaluation and clinical features. Analysis of cell membrane proteins may help to better define disease subtypes or uncover clinically and/or therapeutically relevant biomarkers. In this study we have combined flow cytometry with cluster analysis to compare the immunophenotypic profile of chondrosarcoma cells from primary tumors (CS), chondrosarcoma cell lines (CCL), bone-marrow derived mesenchymal stem cells (MSC), normal articular chondrocytes (HAC), and primary fibroblasts (FIB). Methods and Materials Human CS, CCL, MSC, HAC and FIB cultured in monolayer were analyzed by flow cytometry for the expression of 20 cell surface markers. Data expressed as mean fluorescence intensity ratio over that of the control were clustered with wCLUTO (http://cluto.ccgb.umn.edu/cgi-bin/wCluto/wCluto.cgi). Results Selection of a panel of 9 cell surface markers allowed separation of HAC, MSC, and FIB in three well differentiated clusters. CS clustered mainly with either MSC or HAC. CD14 was selectively expressed on HAC while CH2879 was the only other analyzed cell type showing significant expression of CD14. The three CCL analyzed showed decreased or lost expression of the mesechymal marker CD90, and aberrant expression of β-4 integrin, normally associated with cells of epithelial origin. Conclusions Flow cytometry combined with cluster analysis provides a tool to identify optimal combinations of antibodies allowing differentiation between closely related cell lineages. Preliminary data with chondrosarcoma cells suggest the possibility of using the presented approach to discriminate between different chondrosarcomas based on their degree of proximity to MSC or HAC clusters. This knowledge is relevant for further understanding of these neoplasms., Introduction Passaging chondrocytes approaching confluence has been standard practice. The effect of passaging cell cultures at various levels of confluence on chondrocyte gene expression has not been fully investigated. The objective of this study was to determine the effect of cell density during passaging on chondrocyte gene expression. We hypothesized that passaging cells at lower cell densities would alter gene expression of chondrocytes compared to higher densities. Methods and Materials Outerbridge grade 0 and 1 articular cartilage was collected from three patients undergoing total knee arthroplasty. Chondrocytes were isolated and plated in chondrocyte growth medium until confluence was reached (P0). These cells were trypsinized and plated (P1) in five dishes at 20% confluence and allowed to grow until densities of 30%, 50%, 70%, 90% and 100% confluence were obtained. The cells were released from the dish and total mRNA was isolated. RT-PCR for collagen types I and II, aggrecan and GAPDH were performed. Results There was no difference in collagen I and II gene expression at any cell density although more variation occurred at 90% and 100% confluence. The gene expression of aggrecan was also not significantly different between samples. There was a statistically significant difference between the results of each patient resulting in the variability at each cell density. Conclusions The results of this study suggest that the level of confluence up to 70% does not alter chondrocyte gene expression of collagen types I and II. At densities higher than 70% confluence, more variability occurs. There is much variability between patients., Introduction The nature of hyaline cartilage has imposed constraints on its study by transmission light microscopy. Most histochemical techniques are based on the staining characteristics of cartilage matrix. Techniques allowing for direct observation by light microscopy of subcellular structure of chondrocytes would enhance the understanding of chondrocyte biology. Such a technique, based on interference of light transmission by laminated specimens (Progr Histochem Cytochem 34:163; 1999) has now been applied to articular cartilage. Methods and Materials Pieces of normal human articular cartilage was fixed in 4% paraformaldehyde and 2% gluteraldehyde in 0.05M cocodylate buffer. For comparative studies Carnoy's fixative and 10% formalin were used. Dehydrated tissue embedded in LR white resin was cut at 1 to 2 microns and processed as previously described. Parafin embedded tissue was cut at 5 microns. Results The method was applicable to articular cartilage. Many structural characteristics of articular and tracheal chondrocytes were similar, but there were also marked differences. In the former chondronal arrangement of chondrocytes was more pronounced then in latter. There were also differences in the nuclei with typically large birefringent inclusions absent in the articular chondrocytes. The nuclei of the latter contained densely packed birefringent granules. These also stained densely for DNA and were surrounded by tannophilic protein membranes. Cell to cell contracts by intrachondral tubules were not observed. Conclusions Technique of structural coloration is applicable to articular cartilage. Cytological details of articular chondrocytes are different from their tracheal counterparts., Introduction There is extensive data on chondrogenesis utilizing animal tissue. However, there are many unknown aspects using human chondrocytes for neocartilage formation and lesion repair. We hypothesize that human cartilage can be engineered with morphological, biochemical and biomechanical properties compared to native cartilage. Methods and Materials A cartilage-cell/gel-cartilage construct (n=36) and cell/gel nodules (n=30) were done for this study. Articular tissue from healthy (non-arthritic) donors was enzymatically digested for 16–18 hours with dilute collagenase. Cells were collected, washed in phosphate buffered saline, and placed in culture. After expansion, the cells were mixed with a fibrin saline solution. The cell/fibrin mixture was polymerized with an equal volume of bovine thrombin resulting in a final cell concentration of 60×10e6 cells/ml. Two 6mm discs of native devitalized human articular cartilage were placed on the top and bottom of the fibrin gel, creating a cartilage-cell/gel-cartilage construct. The same cell concentration was used to create human cell/gel nodules. Constructs were implanted into a subcutaneous pocket on the dorsum of nude mice for 6, 12 and 18 weeks. Results Constructs harvested at six weeks were examined histologically for the presence of neocartilage formation. New cartilage matrix formation was noted in all of the specimens with a positive stain for glycosaminoglycans and collagen type II. A tight interface between neocartilage and native cartilage was present. Conclusions Chondrogenesis, heavily documented in animal models, has been demonstrated using human cells in a predictable and reliable manner. Engineered cartilage integrates to devitalized native cartilage, creating a tight interface., Introduction SIRT1, a mammalian homologue of longevity factor sir2, is known to inhibit apoptosis and promotes cell survival in cardiac myocytes and neuron. Yet there have never been reported the expression of SIRT1 in human primary chondrocytes (HCs). The purpose of this study is to investigate the localization of SIRT1 in HCs and to elucidate the relations of SI RT1 with apoptosis of HCs. Methods and Materials Expression of SIRT1 in HCs was examined by RT-PCR, immunoblotting, and immunohistology. HCs were transfected with SIRT1-GFP plasmid constructs by electroporation method and cellular localization of SIRT1 were examined in confocal microscopy. The expressions of SIRT1 under stresses, such as oxidative, mechanical and SIRT1 inhibitors, were examined by immunoblotting. siRNA for SIRT1 was also performed in HCs. TUNEL staining and immunoblotting of cleaved PARP were performed to examine whether SIRT1 is related with apoptosis in HCs. Results Expression of SIRT1 was detected by RT-PCR, western blotting, and immunohistology in HCs. Human GFP tagged SIRT1 was detected in the nuclei in HCs. Oxidative and mechanical stresses inhibited SIRT1 expressions in HCs. SIRT1 inhibitors suppressed SIRT1 expression in dose-dependent manner. SIRT1 inhibitors and siRNA for SIRT1 significantly induced apoptosis in HCs in TUNEL staining and immunoblotting of cleaved PARP. Conclusions SIRT1 was expressed in the nucleus of HCs. The results of this study indicated that SIRT1 regulates the apoptosis in HCs. Further research of SIRT1 might contribute to resolve the pathogenesis of osteoarthritis. f, Introduction Current techniques for autologous chondrocyte transplantation (ACT) require large numbers of cells to provide effective defect coverage. Common in vitro cellular dedifferentiation may deteriorate the chondrocyte in vivo performance. The aim of this study was to compare the gene expression profiles of chondrocytes from different topographical regions of the knee joint to characterize specific population behaviour during monolayer expansion. Methods and Materials Bovine knee chondrocytes from eight different topographical locations, weight-bearing medial(MF)/lateral(LF) femoral condyle and tibia(MT, LT), patella(P)/trochlea(T) and non-weight-bearing femoral notch(B) and proximo-medial femoral condyle(X), were cultured until passage (P) 7 under monolayer conditions. The mRNA expression levels for Collagen-1,-2,-10, COMP, Aggrecan, Sox9, PRG-4, PTHrp and MMP-1,-3,-13 were analysed for native cartilage, P0, P1, P2, P3, P5 and P7 and normalized to P0, respectively. Results From the first passage, significant differences in gene expression levels were observed for Col1, Col10, COMP and all MMPs over all passages cultured. While for Col2, Sox9, Aggrecan, PRG4 and PTHrp differences appeared to be exclusively significant for passages 1 and 2. Collagen 1 expression levels, for example, were representative of a striking difference in their upregulation, being consistently strongly increased for the regions MF, LF, P, T compared to an apparent lesser increase for both tibia locations and more pronounced for both non-weight-bearing areas. Conclusions Cartilage specific gene expression patterns among the eight locations revealed differences in regulation levels depending on the gene and/or passage number. These results, demonstrating significantly different behavior during monolayer expansion, could be embraced to identify favorable locations for cartilage biopsies when using ACT., Introduction Stromal cell derived factor (SDF-1), a chemoattractant member of the intercrine-α family, induced by pro-inflammatory stimuli, is synthesized by mesenchymal precursors and synovial fibroblasts. Chondrocytes express its receptor, CXCR4, suggesting oriented signaling upon the onset of inflammatory events in the joint; indeed attempted repair of damaged cartilage by resident chondrocytes, via cell-cycling re-induction, is insufficient to restore the tissue ad integrum. However, a better chondrogenic differentiation was demonstrated in vitro by culturing chondrocytes in serum-free conditions. Our aim was the evaluation of the SDF-1/ CXCR4 ligand/receptor system in this process. Methods and Materials To this purpose human primary chondrocytes were expanded in control (CN; 10% serum), in serum-free (SF), and in TGFp-1-supplemented SF medium (TGF). The SDF-1 mRNA levels were assayed by real-time quantitative PCR. Immunohistochemistry and FACS evidenced cytoplasmic distribution of CXCR4. Immunocytostainings were performed on micromass cultures prepared using CN-, SF- or TGF-expanded cells. Results SF chondrocytes displayed a 6.4±0.08 (mean±SD) fold-increase in SDF-1 with respect to controls, calling for restoration of a “mesenchymal precursor-like” phenotype. 3D cultures were homogenously positive for CXCR4 and matrix components (type-II collagen, aggrecan, proteoglycans). Conversely, TGF-administration brought back the chemokine expression to CN levels (0.96±0.1). However, in micromass cultures, loss of matrix components, positivity for IH H, type X collagen and apoptosis revealed a sustained presence of pre-hypertrophic cells. Conclusions These chondrocytes are CXCR4-negative, indicating that cells undergoing TGF-induced terminal differentiation turn off the expression of both ligand and receptor, acquiring a more “unresponsive” phenotype. Studies are in progress to ascertain the molecular cascades that trigger these events., Introduction Along with the development of tissue engineered products follows a need for storage and preservation. The aim of this work was to develop a cryopreservation technique for tissue engineered cartilage constructs. Methods and Materials A hyaluronan scaffold (Hyaff11, Fidia Advanced Biopolymers, Italy) was seeded with in vitro expanded human articular chondrocytes and cultured for 14 days in a modified differentiation medium. The cell-scaffold construct was frozen with a cooling rate of 1°C/min and then stored at −152°C. The freezing medium used was DMEM/F12 supplemented with 20% human serum and 10% DMSO. After quick thawing, half the construct was analyzed directly to evaluate morphology, viability (Live/Dead staining), histology (Ab/vG) and handling properties. The other half was further cultured for 28 days. Results The cell-scaffold construct showed a high viability directly after thawing. The extracellular matrix was intact after thawing and additional growth was observed during the in vitro culture. Furthermore the cells continued to metabolize the culture medium which was seen as a significant acidification of the medium during further culture. No difference in histological appearance, handling properties and overall appearance was seen between the cryopreserved and the non cryopreserved controls. Conclusions This method used for cryopreservation of cell-scaffold constructs seems promising given that the properties of the construct are kept unchanged during the freezing-thawing process. A great benefit with this technique is that it could be implemented in a near future since the freezing medium with DMSO is already commonly and successfully used for cell preservation., Introduction In pediatric traumatology fracture healing is known to exert a stimulus to the nearby epiphyseal cartilage possibly leading to severe limb-length-discrepancy during childhood. The exact underlying mechanism of increased growth post-trauma has not been clarified, yet. To investigate growth plate changes post-fracture, we determined physeal chondrocytes' proliferation- and apoptosis-rate in a certain time interval in a living rat-model. Methods and Materials Male Sprague-Dawley-rats (1 month, ∼110g) sustained a unilateral closed diaphyseal tibial fracture according to a standardized guillotine protocol. After euthanasia (days 3, 10, 14, and 29 post-fracture), physeal chondrocyte-proliferation was analysed by BrdU-Labelling. Chondrocyte-apaoptosis was determined by TUNEL-staining. Proliferation and apoptosis rates were statistically evaluated. A p-value⇠0.05 was seen as statistically significant. All animal tests were approved by the Austrian Federal Ministry of Science and Research. Results Chondrocyte-proliferation was significantly higher in the growth-plates of the fractured versus contra-lateral bones on days 3, 10, and 14, with maximum rates on day 3 (p-value 0.018). This strong physeal turnover of the fractured bone was accompanied by a significant higher apoptosis rate, valid on all evaluated days, with maximum levels on day 29 (p-value 0.018). Interestingly, no significant differences in proliferation and apoptotis rates were detected between the contra-lateral and control bones. Conclusions This study clearly demonstrates that a diaphyseal fracture locally stimulates the cell turn-over at the nearby growth plate, emphasizing that accelerated growth is at least partly caused by a higher proliferation and apoptosis rate of physeal chondrocytes. Nevertheless, the driving stimulus still needs to be identified., Introduction Linear bone growth is a highly complex process regulated by a multiplicity of endocrine, paracrine and autocrine factors within the growth plate. Knowledge of developmental and local regulation of growth plate function has been derived mostly from rodents that differ from humans in many growth-related aspects. Furthermore in vitro analyses were performed under variable conditions leading to inconsistent results. The aim of this project was to develop a culture model for the analysis of growth plate chondrocytes, which is similar to the human physiology and easy to handle. Methods and Materials Porcine growth plate and articular chondrocytes were isolated from 6–8 weeks old piglets. After one week monolayer culture the cells were cultivated in alginate beads for four weeks. The expression of differentiation factors and the availability of growth factor receptors were assessed in comparison between native samples, monolayer culture and alginate beads by real-time PCR and immunofluorescence. Results Whereas in monolayer culture the differentiation marker Col2, Col10 and aggrecan dramatically decreased, a similar expression to the native tissue was observed in alginate culture. The estrogen receptors Esr1 and Esr2 showed similar characteristics. However, GHR, IGF1R and IGF2R were expressed at higher levels in monolayer than in alginate culture or native tissue. In comparison between growth plate and articular chondrocytes in particular the different expression patterns of estrogen receptor subtypes were striking. Conclusions In conclusion, the porcine alginate model is promising as defined by tissue availability, expression of relevant hormonal receptors and comparability to human conditions, in particular in the area of basic growth research., Introduction Chondrocytes synthesize and degrade components of articular cartilage connective tissue. In osteoarthritis (OA), the balance between synthetic and degradative processes is disrupted by overproduction of pro-inflammatory mediators such as prostaglandin E2 (PGE2) and its cyclooxygenase 2 (COX-2) regulator. PGE2 induces cartilage degrading enzymes and joint pain in OA. We determined whether PGE2 production and COX-2 expression can be down-regulated in cytokine activated chondrocytes by natural products known to have anti-inflammatory activity: Avocado Soybean Unsaponifiables (ASU) and epigallocatechin gallate (EGCG). Methods and Materials Chondrocytes (5×105) were incubated for 24 hrs with: control media; ASU (NMX1000®); EGCG; or combined ASU and EGCG. Chondrocytes were activated with IL-1β (10 ng/ml) and TNF-α (1 ng/ ml) for 24 hrs to assay PGE2 or; 1 hr for COX-2 mRNA real time qPCR. Data analysis by ANOVA used p⇠0.05. Results Activation significantly increased PGE2 production and COX-2 expression. ASU (8.3 μg/ml) significantly reduced PGE2 production but not EGCG (400, 40, 4 ng/ml). The combination of ASU and EGCG synergistically reduced PGE2 production. ASU (4 μg/ml) also showed synergy with EGCG (40 ng/ml). COX-2 expression was similarly inhibited by the combination of ASU (8 μg/ml) and EGCG (40 ng/ml) but not with either agent alone. Conclusions The combination of ASU and EGCG synergistically inhibits PGE2 production and COX-2 expression. ASU is used for the management of OA. EGCG, a major component of green tea polyphenol catechins is being evaluated for the management of neurodegenerative and cardiovascular diseases associated with inflammation. The synergistic interaction between ASU and EGCG could minimize disruption of cartilage homeostasis., Introduction 2D cultivated chondrocytes dedifferentiate towards a fibroblastic phenotype in vitro. With the proceeding dedifferentiation, collagen type I takes over the chondrocyte specific collagen type II expression. To follow the state of dedifferentiation, we now introduce a combination of collagen I and collagen II reporter plasmids. Methods and Materials The collagen I plasmid contains the red fuorescent protein dsRed driven by a collagen I alpha 1 enhancer/ promoter combination. The collagen II reporter contains a CMV-enhancer element for signal amplification linked to a collagen II-promoter fragment containing chondrocyte specific cis-acting activator and repressor recognition sequences. In contrast to the collagen I reporter, the collagen II plasmid includes the fuorescent protein EYFP. Therefore both markers can be detected separately. Furthermore, the differentiation kinetics can be monitored for both directions, de- and redifferentiation. We achieved the design of a reporter-vector system simultaneously indicating collagen expression-levels of transfected cells by cloning of the two reporter-cassettes into one plasmid. Results Initial transfection of porcine BMSC with the separate reporter-vector systems confirmed the functionality of the designed chimeric transcription activating elements by fluorescence microscopy. The collagen-expression specific response of the designed reporter-vector systems is still under investigation in human adipose derived stem cells. Conclusions The described systems will be suitable for investigating collagen I and collagen II expression of human mesenchymal stem cells and articular chondrocytes in vitro. This work was supported by the European projects Hippocrates (NMP3-CT-2003-505758) and Expertissues (NMP-CT-2004-500283), Introduction Bupivacaine has been shown to be cytotoxic to articular chondrocytes in a dose-/time-dependent fashion and to disrupt mitochondrial membrane potential. The goals of this study are to determine whether alterations in mitochondrial membrane potential and function contribute to bupivacaine-induced toxicity and if these alterations are reversible. Methods and Materials Bovine articular chondrocytes were cultured in DMEM/F12. Chondrocytes were labeled with a fuorescent-probe for calcium fux and mitochondrial membrane potential, placed in saline, and exposed to bupivacaine over 14 min (37°C). Peak concentrations ranged from 0.25%-0.0625% occurring for one minute (t=7min), followed by saline inflow. Time-lapse confocal microscopy was performed once a minute for 60 min and images were analyzed. Mitochondrial function was also determined by XTT assay. Results Chondrocytes exposed to 0.25% bupivacaine exhibited reduced mitochondrial membrane potential, membrane blebbing and failure, and calcium influx at peak exposure (7min). In contrast, 0.125% bupivacaine decreased mitochondrial potential during bupivacaine inflow, which then stabilized during outflow. No increase in calcium fux was seen and cell membranes remained intact. XTT assay also demonstrated that bupivacaine reduced mitochondrial function in a dose-dependent fashion (p⇠0.001). Mitochondrial function, potential, and calcium fux following exposure to 0.0625% bupivacaine were similar to saline (p⇢.05). Conclusions These results show that bupivacaine reduces chondrocyte mitochondrial membrane function in a dose-dependent fashion and that progressive loss of mitochondrial membrane potential coincided with calcium influx and chondrocyte death. This provides both a potential mechanism for bupivacaine induced chondrotoxicity and a potential explanation for the importance of dilution in reducing the potential for chondrotoxicity in the clinical setting., Introduction Chondrocytes isolated from its original extracellular matrix and cultured in vitro, may change the phenotype and therefore the gene expression profile. Therefore, after several passages, dedifferenciated cells are similar to fibroblasts. The purpose was to study along the time the dynamic of type I and II collagens (COL1, COL2) and aggrecan expression by qRT-PCR in monolayer chondrocyte cultures. Methods and Materials Articular cartilage from 16 patients. Chondrocytes were isolated after collagenase digestion and cultured in DMEM (Passage 0: P0). Cells were trypsinized at 80% of confluence and subcultured (Passage 1: P1). The procedure was repeated twice, so the cells were subcultured until P3. Total RNA was isolated from P0, P1, P2 and P3 and cDNA synthesis was carried-out. Relative expression of COL1, COL2 and aggrecan expression was performed using the expression of the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene as the reference. Results In the basal samples (P0), the median expression of aggrecan and COL2 were 0.39 (range: 0.04 − 8.00) and 0.08 (range: 1.92 × 10–5 − 4.68) respectively. The median basal expression of COL1 was 1.45 (range: 1.45 − 2.08). The median expression of aggrecan and COL2 is decreasing during the different passages, while that of type I increased during the passages. Aggrecan and COL2 were downregulated from P1 to P3 for aproximately 6-fold and 14-fold, respectively, while COL1 was upregulated 3-fold. Conclusions Although aggrecan and COL2 genes are downregulated in cultured human articular chondrocytes the relative expression of these genes is mantained until the end of culture., Introduction Articular chondrocytes acquire a fibroblastic-like phenotype when cultured in monolayer, characterized by different cell morphology and by expression of non-cartilage specific genes such as type I collagen. We focused on some of the events which could affect the chondrocytes phenotype in vitro: the cell density and the time of culture. Methods and Materials Swine articular chondrocytes were isolated and seeded at different cell densities (from 12,000 cell/ cm2 to 100,000 cells/cm2). Samples were cultured for 6 and 8 days. Phenotype was evaluated in terms of aggrecan and collagen type I and II expression, while proliferation was assessed by cell counting. Results Chondrocytes proliferated more at lower cell densities and did not show any proliferation at highest cell density; moreover, no further proliferation was observed when chondrocytes were kept in culture for 8 days. Chondrocytes seeded at 50,000 and 100,000 cells/cm2 were able to maintain better level of differentiation, characterized by higher expression of type II collagen and aggrecan and lower expression of type I collagen, but in all conditions a significant loss of chondrocyte phenotype from 6 to 8 days of culture was observed. Conclusions Cell density influences chondrocyte capability of maintaining a differentiated phenotype in vitro; moreover, the proliferation is allowed only at low cell densities. Some other important modulators of proliferation and differentiation, such as growth factors and growth substrates, will be valuated in future studies in order to better modulate proliferation and the loss of phenotype in vitro., Introduction Parathryoid hormone-related peptide (PTHrP) induced chondrogenic differentiation of hBMSCs while inhibiting hypertrophic differentiation (Kafenah 2007). PTH and PTHrP bind to the same PTH/PTHrP receptor. As blocking hypertrophic differentiation is desirable for autologous chondrocyte transplantation, and increased chondrogenic differentiation may lead to higher proteoglycan (PG) content, we hypothesized that addition of PTH to in vitro cultured chondrocytes positively influences proteoglycan metabolism. Methods and Materials Healthy femoral cartilage of 9 human individuals was digested in 0.1% collagenase. Following expansion, chondrocytes were cultured on collagen type II- coated Millipore culture inserts. During the 28-day culture, PTH (Brunschwig) was added at concentrations of 0.1 or 1.0 μM from days 0 (d0), 9 (d9) or 21 (d21) onwards, or no PTH was added (control). After 28 days, PG content and release were measured. Results Addition of PTH to in vitro cultured chondrocytes seemed to result in decreased PG content, although this effect was only significant for 0.1μM PTH added at d9 compared to PTH added from d21, (p=0.015). Moreover, PG release was higher when PTH was added later (1.0μM PTH from d21 vs 1.0μM PTH from d9, p=0.037), or when PTH was not added at all (control vs 1.0uM PTH from d0, p=0.01). Conclusions Addition of PTH to in vitro cultured chondrocytes did not result in increased PG content, but did decrease PG degradation. Although PTH and PTHrP are assumed to exert analogous effects, signalling in BMSCs by PTHrP may differ from PTH in chondrocytes. Analysis of the hypertrophic markers collagen type-X and Runx-2 may provide definite answers., Introduction Chondral injuries of the knee are commonly seen at arthroscopy, yet there is no consensus on the most appropriate treatment method. However, untreated cartilage injury predisposes to osteoarthritis contributing to pain and disability. For cell-based cartilage repair strategies, an ex vivo expansion phase is required to obtain sufficient cells for therapeutic intervention. Although recent reports demonstrated the central role of oxygen in the function and differentiation of chondrocytes, little is known of the effect of physiological low oxygen concentrations during the expansion of the cells and whether this alters their chondrogenic capacity. Methods and Materials Initial studies of chondrocyte expansion were performed in mature mice, with cells expanded at either atmospheric oxygen tension (21%) or 5% 02 in monolayer cultures. Chondrogenic differentiation was subsequently assessed via micromass culture. Having determined that oxygen tension influences murine chondrocyte expansion and differentiation, similar studies were conducted using adult human chondrocytes taken from knee arthroplasty off-cuts, with analysis of select genes involved in the chondrogenic program analyzed by q-PCR Results Cellular morphology was improved in hypoxic culture, with a markedly more fibroblastic appearance seen after greater than 2 passages in 21% O2. Micromass cultures maintained in hypoxic conditions demonstrated stronger staining with Alcian blue, indicating stronger expression of cartilaginous glycosaminoglycans. Collagen type II mRNA was twofold higher in monolayer cultures expanded at 5% compared to expansion at 21% O2. Micromass cultures grown at 21% O2 showed up to a twofold increase in the tissue content of glycosaminoglycans when formed with cells expanded at 5% instead of 21% O2. However, no differences in the levels of transcripts and in the staining for collagen type II protein were observed in these micromass cultures. Hypoxia (5% O2) applied during micromass cultures gave rise to tissues with low contents of glycosaminoglycans only. Conclusions In vivo, the chondrocytes are adapted to an avascular hypoxic environment. Accordingly, applying 5% O2 in the expansion phase in the course of cell-based cartilage repair strategies may more closely mimic the normal chondrocyte microenvironment and may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans., Introduction To engineer articular cartilage tissue that more closely resembles the organization of the native tissue, several tissue engineering approaches utilize chondrocytes from the different zones of cartilage. However, controversy remains about the existence of inherent differences between these cell populations. Hence, we investigated zone-related differences between chondrocytes from the superficial, middle and deep zones, during in vitro expansion and subsequent redifferentiation. Methods and Materials Equine articular chondrocytes from the three zones were expanded in monolayer cultures (8 donors). Subsequently, cells were redifferentiated in pellet and alginate bead cultures for up to 4 weeks. GAG and DNA were quantified, and immunohistochemistry was performed to assess expression of various (zonal) markers, including various collagens, COMP and clusterin. Results Cell yield varied between zones, but proliferation rates did not show significant differences. Staining for collagens II, VI and IX, clusterin and COMP was lost after expansion in all cultures, but gradually re-appeared during redifferentiation. Staining for collagen type I was found in all cultures after 4 weeks. Interestingly, COM P and collagen IX were detected mainly in deep and middle zone cultures, whilst clusterin re-appeared in particular in superficial zone cultures. In pellet cultures we observed more intense safranin-O staining in pellets with deep-zone cells, a trend confirmed by a quantitative GAG assay. Conclusions Differences exist indeed between cells from the different zones. Furthermore, differences can be maintained or re-induced after in vitro culture. Appreciation of these zonal differences could lead to important advances in cartilage tissue engineering., Introduction We aimed our study at characterizing the post-expansion cartilage-forming capacity of chondrocytes harvested from the detached fragments of osteochondral lesions of ankle joints (Damaged Ankle Cartilage Fragments, DACF). Chondrocytes derived from normal ankle cartilage (NAC) were used as control cells. Methods and Materials DACF were obtained from 6 patients (mean age: 35years) with osteochondral lesions of the talus, while NAC from 10 autopsies (mean age: 55years). Chondrocytes isolated from the tissues were expanded for two passages and then cultured in Hyaff®-11 meshes (FAB, Italy) for 14 or 28days. Resulting tissues were assessed histologically, biochemically (glycosaminoglycan-GAG-, DNA and collagenI I) and biomechanically. Results DACF contained significant lower amounts of DNA (3.0-fold), GAG (5.3-fold) and collagen II (1.5-fold) and higher amounts of collagen I (6.2-fold) as compared to NAC. Following 14 days of culture in Hyaff®-11, tissues generated by both cell sources were faintly stained for Safranin-O, contained similar amounts of GAG and collagenI I and had similar biomechanical properties. After 28 days of culture, tissues generated by NAC chondrocytes were more intensely stained for Safranin-O and collagenI I, contained higher amounts of GAG (1.9-fold) and collagen II (1.4-fold) and exhibited superior biomechanical properties (1.7-fold and 3.3-fold equilibrium modulus and dynamic pulsatile modulus, respectively). Conclusions We showed for the first time that DACF-chondrocytes have inferior cartilage forming capacity as compared to NAC-chondrocytes, possibly resulting from environmental changes occurring during and after trauma/disease. The study opens some reservations on the use of DACF-derived cells for the repair of ankle cartilage defects, especially in the context of tissue engineering-based approaches., Introduction While intra-articular analgesia is generally effective for pain management during arthroscopic surgery, infusion of amino amides (i.e. bupivacaine) can lead to chondrolysis. The present study evaluates effects of a potential alternative, highly purified capsaicin, on glenohumeral articular cartilage using a rabbit rotator cuff repair model. Methods and Materials Six New Zealand White rabbits underwent a unilateral supraspinatus transection and repair with a single injection of capsaicin into the glenohumeral joint (GHJ). Animals were euthanized at one week and humeral head articular cartilage harvested for biochemical (proteoglycan synthesis and content), cell viability and histological assays. Results Cartilage wet weight, total proteoglycan content (matrix and media combined) and percentage of viable cells was similar (p⇢.05) between treated and untreated shoulders. Chondrocyte proteoglycan synthesis for capsaicin-treated shoulders was 147±45% (p⇠0.05) of that for contralateral, untreated joints. The histopathological score (out of 20) for the shoulders receiving the capsaicin drug was 6.7±1.5 compared to 6.3±1.9 for the unoperated shoulders (p⇢0.3). Conclusions One week following surgery, the cell viability and proteoglycan content of capsaicin-treated shoulders were similar to untreated controls. However, the metabolic activity (proteoglycan synthesis) of the chondrocytes from the treated shoulders was significantly elevated suggesting perhaps a direct effect of the drug or a manifestation of post-surgical inflammatory responses. In contrast to previous reports of bupivacaine infusion in rabbit shoulders, the current results indicate that a single injection of highly purified capsaicin into the GHJ does not induce a deleterious response with regard to matrix metabolism and cell viability of glenohumeral cartilage in the early post-operative period., Introduction Therapies based on in vitro expanded primary cells are more frequently used for tissue regeneration. To proof that the cells stay at the site of implantation, it is necessary to be able to trace the cells in vivo. By iron oxide-labeling the cells can be detected by MRI in live organisms. Methods and Materials Chondrocytes were isolated from biopsies and incubated in growth medium with either 0, 25, 50 or 100 μg/ml very small iron oxide particles (VSOPs) for 90 minutes at 37°C, 5% CO2. Verification of VSOP uptake was performed by Prussian blue staining and the impact of VSOP-labeling on cell proliferation was investigated by cell count after 24, 48 and 72 hours of culture. Furthermore, VSOP-labeled (50 μg/ml) chondrocytes were embedded in alginate beads in three groups with concentrations of 2×106, 5.0×106, or 8.0×106 cells/mL These groups of beads and a negative control of unlabeled cells were placed in growth medium and were scanned with 1.5T MRI using a 5 cm diameter coil. A spin echo sequence with TR=1000 ms, TE=10 ms, FOV=70×70 mm, slice thickness of 3 mm, and a total scan time of 1:56 minutes was used. Results Iron oxide-labeling of human chondrocytes was verified by Prussian blue staining. The proliferation of cells was unaffected by VSOP-labeling. An immediate recognition of the three different cell concentrations was achievable from the images themselves. Quantification of the depression of signal is pending. Conclusions Iron oxide-labeling of chondrocytes was achieved, enabling in vivo detection of cells after implantation., Introduction Platelet-rich plasma (PRP) is an osteoinductive therapeutic approach that is used in treatment of bone and cartilage healing processes. The purpose of this study was to investigate the effect of PRP on human chondrocytes. Methods and Materials Human articular chondrocytes were collected from young patients, single chondrocytes obtained by enzymatic digestion were polled and expanded in vitro for several weeks in Ham's F12 medium plus 10% FBS or in Serum Free Medium or in 10% PL (platelet lysate). In parallel, we performed in vitro pellet culture, to test the capability to maintain chondrogenic potential. Proliferation and differentiation analysis was performed at different time during the in vitro expansion. To evaluate the rate of cell proliferation, at regular intervals the cells were counted. The number of cell duplications was calculated as ratio to starting value. Cell viability was assessed by MTT analysis. To test the ability to maintain cartilage phenotype, chondrocyte expanded in monolayer, was successive cultured in pellet culture. Results Preliminary results, shown that cells proliferation and number of cell doublings were drastically enhanced in cultures supplemented with (PL) compared to cultures performed in FBS or in Serum Free. Histological analysis shown that chondrogenic differentiation was maintained. Biochemical and molecular analyses are in progress to demonstrate that PRP did not markedly affect the chondrogenic potential and the cells remained phenotypically stable. Conclusions PRP may be useful to stimulate the proliferation of human chondrocytes cells. PRP is an effective substitute for FBS to support in vitro expansion of human cells and subsequent tissue-engineering applications., Introduction ACI and MACI® implants have been shown to be effective treatments for the repair of articular cartilage defects. Dell'Accio have reported gene markers that correlated to chondrogenic potential of an ACI product in a nude mouse implantation model1. This study evaluates expression of these molecular markers and a novel marker for their ability to predict chondrogenic potential of MACI® implants. Methods and Materials Gene expression was measured by qPCR in expanded human chondrocytes and chondrocytes that had re-differentiated after long-term culture of the MACI® implant. Human dermal fibroblasts were used as the negative control. The genes monitored were COL2A, FGFR3, BMP2, ALK1 and Hyaline1. Results PCR data indicated that expression of COL2A1, FGFR3, BMP2 and ALK1 did not differ significantly between expanded chondrocytes and fibroblasts. Hyaline1 levels were 121-fold higher in chondrocytes versus fibroblasts. After 28 days of chondrogenic differentiation culture, expression of COL2A1 increased more than 162-fold in chondrocytes, but decreased 30% indermal fibroblasts. These data suggest that the chondrocytes began the process of chondrogenesis while the dermal fibroblasts were non-chondrogenic. Conclusions Previous reports found that expression of COL2A1, FGFR3, BMP2 and ALK1 were predictive of chondrogenesis in a nude mouse intramuscular implantation assay.1 Although we found that COL2A1 levels were much higher in chondrocytes, these results were not statistically significant due to large variation between strains. While the levels of FGFR3, BMP2, and ALK1 were not associated with MACI® implant re-differentiation, a strong association was observed with Hyaline1 expression. The results of this study suggest Hyaline1 as a predictor of MACI product potency., Introduction Children Salter's type III and IV growth plate injuries always induce the skeletal deformity because of bony bridge formation. The purpose of this investigation was to understand the molecular mechanisms of bony bridge formation. Methods and Materials The growth plate injuries of 24 male 6-week-old Swiss Webster mice were generated in the left proximal tibias by drill-hole approach. On days 1, 3, 7, 10, 14, 28 post-surgery, groups of mice (n=4) were killed for specimen collection and further analyses. Results Consisted with the histological changes, both Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and in situ hybridization experiment using Col2a1 probe showed that there was the sub-injury cartilage region adjacent to the original injury site. The chondrocytes within this region showed the dislocation with the cartilage lacunas and these chondrocytes didn't express Col2a1 mRNA, further confirming they were dead cells. Along with the degradation of sub-injury cartilage, some fibroblast-like cells presented to the cartilaginous region between the sub-injury region and uninjured cartilage. In situ hybridization experiment for Patched 1 (Ptch1), indicator of Indian Hedgehog (IHH) signaling, indicated these fibroblast-like cells could respond to Hh signaling. Conclusions These results suggest that the bony bridge formation involves series of changes of chondrocytes and Ihh signaling may be involved in the formation of the transient perichondrium-like structure between sub-injury cartilage and normal cartilage, and partially contribute to the bony bridge formation. Investigating the underlying cellular and molecular changes after the transphyseal injury will contribute us to explore a prevention treatment in the future clinic., Introduction To investigate the apparent fibroblastic shift in tissue specific DNA methylation patterns observed during chondrocyte monolayer expansion. Methods and Materials The upper and lower zones from full thickness articular cartilage slices were sampled. Chondrocytes were cultured for up to 5 passages in unsupplemented and growth factor supplemented media. Analysis of DNA methylation in articular cartilage, clonal cultures, and polyclonal cultures was performed. Polyclonal cultures were also analyzed for gene expression. Results The DNA methylation patterns of both clonal and polyclonal cultures were observed to shift slightly, but significantly, to a more fibroblastic profile during monolayer expansion in all media. While gene expression levels showed significant variability, no significant differences were observed in the DNA methylation levels between media conditions. High interclonal variability in methylation levels of FGFR1, KRT8, and ROPN1L was observed, while HIF1a, FMOD, and C15orf27 methylation levels were characteristically chondrocytic in most clonal cultures. Analysis of articular cartilage revealed that the upper zone displayed a slightly more fibroblastic methylation pattern than the lower zone, with FMOD, FGFR1, KRT8, and ROPN1L genes showing significant differences between zones. Conclusions Articular cartilage appears to harbor a homogeneous population of HIF1a/FMOD/C15orf27 undermethylated cells, and subpopulations with variable FGFR1, KRT8, and ROPN1L methylation. Chondrocytes taken from the lower zone possessed a more chondrocytic DNA methylation profile than those from the upper zone. The shift in DNA methylation levels during monolayer expansion was neither random nor global, but directed to a more fibroblastic profile in a gene specific manner., Introduction Recent clinical and basic science investigations have revealed chondrotoxicity of local anesthetics, especially those containing epinephrine, administered via intra-articular pain pump. However, exact mechanism of toxicity is unknown. This study evaluates the chondrotoxicity of pH, epinephrine and preservatives found in commonly used local anesthetics. Methods and Materials Human chondrocytes were harvested and cultured in a custom bioreactor designed to mimic metabolism of medication. Pain pumps were used to infuse one of the following medications into the culture system: control media, media titrated to pH 4.5, media with 1:100,000 or 1:200,000 epinephrine only, media with the preservative sodium meta-bisulfte only, 0.25% bupivacaine, 0.25% bupivacaine with epinephrine, 1% lidocaine, 1% lidocaine with epinephrine. Cultures were perfused for 24 and 48 hours, stained with Live/Dead Cell Vitality Assay, examined by fluorescence microscopy, counted, and the percentage of cell death was calculated. Results Cultures containing media titrated to pH 4.5, the preservative sodium meta-bisulfte (found only in anesthetic medications containing epinephrine) and all medications containing epinephrine had high cell death rates compared to controls at all time points (p⇠0.001), while cultures containing 1:100,000 and 1:200,000 epinephrine had no increase death rate. The percentage cell death was not significant for 1% lidocaine (12.5%) and 0.25% bupivacaine (16.5%) at 24 hours, but became significant at 48 hours (61.5% and 55.6%, respectively). Conclusions The marked chondrotoxicity of local anesthetics containing epinephrine appears to be due to the preservative sodium meta-bisulfite and low pH, as these medications are titrated to pH 4.5–5.5 for product stability. 1% lidocaine and 0.25% bupivicaine were chondrotoxic if used for greater than 24 hours. Caution should be exercised when using intra-articular pain pumps, especially for greater than 24 hours., Introduction Chondrocyte culture is a standard technique used for investigation and treatment of articular cartilage defects. Surprisingly, numerical description of cell multiplication during chondrocyte culture has not been documented. Documentation of cell multiplication rate is important to help standardize culture techniques so results can be comparable across different laboratories. The objective of this experiment was to numerically describe chondrocyte counts during cell culture from initial plating until maximal confluence. Methods and Materials Articular cartilage of Outerbridge 0 and 1 was collected from three patients undergoing total knee arthroplasty (age 46–69 years). The cells were isolated and plated in chondrocyte growth medium until confluence (P0). The cells were trypsinized and plated at 20% confluence and allowed to grow until maximal confluence. Images were taken from two standardized points in each plate at daily intervals, cells were manually counted and cell densities calculated. Results Initial cell density (approximately 27 cells/mm2) rose gradually for the first five days. Thereafter, there was an exponential increase in cell density that plateaued at 10 days at approximately 1674 cells/mm2. There was no statistically significant difference in the rate of increase in cell density between the three donors. Conclusions This is the first study that documents the numerical description of chondrocyte multiplication during cell culture and can be used to accurately define levels of confluence so future experiments can be compared more precisely. This data was obtained from articular cartilage retrieved from patients undergoing knee arthroplasty and the rate of replication may not be extrapolated to cells taken from normal knees., Introduction The state-of-the-art in cartilage tissue engineering techniques is autologous chondrocyte transplantation. It employs autologous chondrocytes cultivated in monolayer culture, which enhances proliferation compared to the native cells with a limited division capacity. The disadvantage of this cultivation is the alteration to a fibroblast-like appearance with the time accompanied by an alteration in extracellular matrix. This dedifferentiation process is as well characterized by a dramatic decrease of BMP-7 mRNA transcription. Therefore, we investigated the involvement of BMP-7 in the dedifferentiation process. Methods and Materials To maintain, respectively enhance, the BMP-7 production in human monolayer chondrocytes we chose to transfect a constructed plasmid, encoding the human BMP-7 cDNA controlled by a CMV promoter, into the cells by magnetofection and compared it with the application of recombinant protein. For the determination of differentiation state, the ratio of collagen type II and type I expression was determined. Furthermore, the population multiplication was calculated. Results The results revealed a 10-fold increase of the differentiation index by transfecting 0.5μg plasmid per 100.000 cells and 38-fold with 2.0μg after 14 days monolayer cultivation. The retention of the chondrogenic differentiation was accompanied by a dose-dependent decrease of proliferation, resulting in 68%, respectively 48%, population multiplication in ratio to the control. Recombinant BMP-7 showed different impact on the cells than the overexpressed protein. It accelerated the dedifferentiation process in low concentrations and less in higher compared to the untreated cells. Interestingly, the proliferation was reduced as well in dose dependent manner. Conclusions BMP-7 obviously plays a role in the dedifferentiation process of monolayer chondrocytes., Introduction The durability of cartilage repair tissue depends on good bone-cartilage repair tissue integration, but little is known about what controls integrated repair. During remodeling, osteoclasts attract and promote adhesion of pluripotential stem cells (pro-osteoblasts) to bone. We tested the hypothesis that treatment of microdrilled cartilage defects with chitosan-stabilized clot implant elicits more osteoclasts and a more integrated repair, compared to drilling alone. Methods and Materials Microdrilled bilateral full-thickness cartilage trochlear defects were created in skeletally mature rabbits, and treated or not with chitosan-glycerol phosphate/blood implant. After 1 (N=4), 2 (N=4), or 8 weeks (N=7), histological sections from between, the edge, and through the drill holes of the defect were used to quantify osteoclast density (Tartrate Resistant Acid Phosphatase enzymatic activity) and repair tissue histomorphometric parameters (Safranin O stain). Intact femurs (N=8) served as controls. Results Osteoclasts accumulated at the edges of the drill holes after 1 week, at which time osteoclast activity was stimulated 2.5 to 4-fold by chitosan-GP/blood implants, throughout the subchondral trabecular bone. After 8 weeks of repair, osteoclast density subsided below all defects and was limited to the repairing bone plate. At 8 weeks defects treated with implant, compared to drilling alone, demonstrated more complete trabecular bone repair of the drill holes (p⇠0.05), and a repair tissue that was significantly more integrated with the defect base (∼5% vs ∼37% detached repair, p⇠0.001). Conclusions Chitosan-GP/blood implants elicit an acute influx of osteoclasts following marrow stimulation without inducing bone resorption. Rapid remodeling of drilled subchondral bone was associated with signficantly more repair cartilage-bone integration., Introduction Collagen structure in human osteochondral biopsies was assessed by scanning electron microscopy (SEM). Qualitative scales for orientation and density of collagen in different zones and quantitative measurements of fibre diameter were applied and statistically analysed. Methods and Materials Human osteochondral biopsies (N=10) containing normal, degraded or repair cartilage were embedded in paraffin, sectioned, and post-fixed prior to gold coating and SEM imaging. Images were taken in superficial, transitional, and deep zones of each specimen and were scored twice by 3 readers for collagen orientation and density using an ordinal scale of 0, 1, or 2. Inter-reader and intra-reader reliability were analysed using the Intraclass Correlation Coefficient for agreement, ICC(2,1). Fibre diameters were measured. Results Normal human cartilage displayed the expected zonal dependent collagen orientation with fibre diameters in the range 50–140nm. Degraded tissue revealed less evident vertical orientation in the deep zone. Repair tissue arising from bone marrow frequently contained vertically oriented fibers in the deep zone, tangentially oriented fibres in the superficial zone, and an abundance of thinner fibers of ∼50 nm in diameter. The inter-reader and intra-reader reliability (N=10) were 0.84 and 0.92 for the orientation score and 0.86 and 0.95 for the density score, indicating excellent inter-reader and intra-reader agreement. Conclusions Novel methods to characterize collagen structure in biopsies of cartilage repair tissue have been developed using SEM. Qualitative scoring of zonal dependent collagen orientation and density displayed good reproducibility between readers and will be useful in assessing the quality of tissue produced by different cartilage repair procedures., Introduction Under suitable culture conditions, human bone marrow stem cells (hMSCs) can produce a cartilage-like tissue with a matrix based on type II collagen. To what extent the fibrillar matrix is normal, in terms of the assembly and cross-linking of type II collagen into heterofibrils, is not characterized. This study investigated the ability of hMSCs undergoing chondrogenesis to assemble a type II collagen-based cross-linked network characteristic of developing cartilage. Methods and Materials hMSCs were isolated, expanded, centrifuged into transwell filter units and maintained for 7, 14, 28 days as we have published. Neo-cartilage samples were assayed for collagen and hydroxylysyl pyridinoline (HP) cross-link content with human fetal cartilage as a control. Mass spectrometry identified collagen chains. Results A progressive increase in collagen content from week 1 to week 4 was observed. The collagen content (1% of wet wt.) by week 4 compares well with human fetal cartilage (3% of wet wt. at 15 weeks in utero). High levels of HP cross-links are a characteristic of cartilage collagen. At 1 wk HP cross-links were barely detectable. At 2 weeks the HP content of the neo-cartilage (0.23 moles/mole collagen) approached that of human fetal cartilage (0.23) and was maintained over the next 14 days. Conclusions The neo-cartilage collagen framework showed a high and stable cross-link content by 14 days. Since only a modest wet weight gain between 14 and 28 days was observed despite a 95% increase in proteoglycan content, this strongly suggested that the cross-linked collagen framework restricted proteoglycan-driven swelling and limited further gain in wet weight., Introduction Cartilage Oligomeric Matrix Protein (COMP) is a protein present in the cartilage matrix and is expressed more abundantly in OA cartilage than in healthy cartilage. The present study was designed to investigate the effect of growth factors on COMP deposition and the influence of COMP on collagen biochemistry. Methods and Materials Bovine chondrocytes in alginate beads were cultured with or without 25 ng/ml IGF1, TGFβ2 or FGF2. Human COMP (hCOMP) was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP protein production, collagen and proteoglycan deposition, and collagen fibril thickness were determined. Results Addition of TGFβ2 resulted in more COMP mRNA and protein than the control condition without growth factors or with addition of IGF1. FGF2 resulted in less and partially degraded COMP. Lentiviral transduction with hCOMP resulted in elevated gene expression of hCOMP and increased COMP levels in the alginate bead and culture medium compared to untransfected cells. Overexpression of COMP did not affect the deposition of collagen, collagen crosslinking, proteoglycan deposition or the mechanical properties. Stimulating COMP production by either TGFβ2 or lentivirus resulted in collagen fibrils with a smaller diameter. Conclusions Taken together, COMP deposition can be modulated in cartilage matrix production by addition of growth factors or by overexpression of COMP. Inducing COMP protein expression resulted in collagen fibrils with a smaller diameter. Since it has been demonstrated that the collagen fibril diameter is associated with mechanical functioning of the matrix, modulating COMP levels could therefore contribute to successful cartilage regeneration strategies., Introduction The aim of this study is to prospectively examine the effect of intraarticular ascorbate injections on the healing process of acute articular osteochondral defects. As the articular cartilage tissue is hypocellular, avascular, alymphatic and aneural; it does not contain mesenchymal stem cells thus has a minimal repair potential. Besides present treatment protocols have a poor prognostic value in the long term outcome of osteochondral defects and are very expensive. Methods and Materials After creating full thickness, 2 mm wide osteochondral defects bilaterally in the knee joints of 48 Sprague Dawley male rats, a 5% concentration of ascorbate solution was administered intraarticularly every 3 days in 3 weeks' time into one joint and the contralateral knee was used as a control with saline solution injected. Articular cartilage regeneration was immunologically evaluated with TSP-1 and COMP elisa assays; histopathologically with TGF-Beta-1 and Aggrecan immunohistochemistry, toluidin-blue and hematoxylene-eosin staining; and biochemically the ration of hydroxyproline/proline and hydroxylysine/lysine has been determined with the HPLC method. For the pathological grading, the modified OARSI system was used. Results When compared to saline injections, the results show that ascorbate has slightly enhanced cartilage healing with TSP-1 (p=0.007), aggrecan (p=0.249) and TGF-Beta-1 (p=0.036) expressions upregulated; HP/P and HL/L (p=0.031) ratios increased and finally COMP (p=0.006) concentrations decreased distinctively. Conclusions In conclusion, ascorbate positively influenced the articular cartilage molecular biology during the healing process whereas it did not affect the pathological grading in the early onset., Introduction IL-1ra blocks IL-1 function and decreases pain in patients with OA (Yang et al., 2008). Autologous protein serum (APS) rich in IL-1ra can be collected from blood within 30 minutes. However, it is unknown if sustained delivery from a carrier increases the residence time of IL-1ra in the joint space, thereby increasing the potential therapeutic effect. The purpose of this study is to determine if a plasma matrix alters the release of IL-1ra. Methods and Materials From 5 consented human donors, 120cc of anticoagulated blood was collected. Platelet-rich plasma (PRP) was prepared using GPS®III disposables (Biomet Biologics). PRP was loaded into modified plasma concentration devices (Plasmax®device, Biomet Biologics) and processed. The output was divided into 4 groups; IL-1ra in concentrated plasma with and without thrombin activation (1000U/ml in 1MCaCl2), or cell-free IL-1ra with and without thrombin activation. I L-1ra was measured using ELISA (R&d Systems) over time. Results Unclotted APS produced an average of 47.1±2.1ng over 24hrs (p=0.34). The cell-free samples produced 33.7±1.5ng without changing over 24hrs(p=0.38). Once clotted, the elution of IL-1ra was slowed, with only 28% being eluted after 10 hours. Release in the cell-free samples was also delayed, but eluted 100% of available IL-1ra after 10 hours. Conclusions The plasma matrix delays IL-1ra release, is biocompatible, resorbable, and autologous, making it an appealing carrier selection. While cell-free samples do contain and elute IL-1ra, more IL-1ra is obtainable in the APS containing cells. Further work will determine if delayed release of I L-1ra will translate into improved pain relief for patients suffering from OA., Introduction Pro-inflammatory cytokines have been linked to cartilage destruction and chondrocyte death in OA. In many cells pro-inflammatory cytokines may cause mitochondrial dysfunction/ apoptosis by increasing reactive oxygen/ nitrogen species. The purpose of present study was to investigate if mitochondrial dysfunction/damage are involved in human chondrocyte responses to IL-1β and TNF-α. Methods and Materials Primary chondrocyte cultures were generated from OA patients undergoing TKA. Cells were treated with IL-1β and TNF-α for 24, 48, 72 hrs. Total DNA was isolated and subjected to quantitative Southern blot analysis to study mitochondrial DNA (mtDNA) damage. ATP levels were measured by ATP bioluminescence kit. Mitochondrial protein levels were evaluated by Western blot analysis. Apoptosis was measured by flow cytometry, DAPI staining, Western blot analysis was performed to study cytochrome c release and activation of caspases 3 and 9. Results Following 24 hr of exposure, both TNF-α and IL-1β induced dose-dependent mtDNA damage. ATP levels were decreased 34%, 57%, 43%, and 66% for the same doses of cytokines compare to untreated controls. Mitochondrially-encoded subunit I of cytochrome oxidase levels were down-regulated following cytokine exposure. Mitochondrial dysfunction correlated with apoptosis appearance. 10 μg of I L-1β did not cause apoptosis after 72 hrs, 50 μg of I L-1β caused 35% apoptosis, 50 μg of TNF-α caused 44% apoptosis, and 100 μg of TNF-α induced 59% apoptosis. Pro-inflammatory cytokine exposure induced cytochrome c release from mitochondria into cytosol and both caspase 3 and 9 cleavage. Conclusions In conclusion, pro-inflammatory cytokines may cause chondrocyte death by induction of mitochondrial dysfunction/ damage, and activation of mitochondrial led apoptosis., Introduction In vitro investigation of chondrocytes in hypoxic environment has been increasingly studied in cartilage research. Quantitative RT-PCR is a powerful approach used in a wide range of scientific methods, with the use of stable reference genes - house keeping genes (HKG). Stable HKGs are one of the hallmarks of reliable results using RT-PCR. The aim of this study was to determine optimal HKGs in human chondrocyte cultures in both normoxia and hypoxia environment. Methods and Materials Cartilage biopsies were obtained from six healthy patients after written consent. After preparation and isolation, the chondrocytes were divided into two groups and grown in 21% and 1% oxygen respectively. RNA was isolated from the cells at baseline, and then from the two groups after 1, 2, and 6 days. We assed the gene expression at these time-points of 8 HKGs: GAPDH, 18s, B2M, RPL13A, beta-actin, RPII, TBP, UBC, and HPRT1. Results The inter-individual coefficient of variance (CV%) between the patients was lowest for HPII (1.7) and UBC (2.1). The intra-individual CV% between the groups and time-points was lowest for HPII (1.2) followed by RPL13A (1.3), B2M (1.4), and HPRT1 (1.5). The total CV% was lowest for RPII (1.7). Commonly used HKGs such as 18s, GAPDH and beta-actin had a total CV% of 5.6, 4.3, and 5.5 respectively. Conclusions We revealed the importance of selecting H KGs according to the study setup. RPII was the most stable in all conditions while commonly used HKGs such as 18s and beta-actin were the most unstable., Introduction To understand repair effects of anti-VEGF combined with fibrin graft interposition with a proximal tibia transphyseal injury murine model, and to assess the reliability of treatment to physeal injury with the compound. Methods and Materials The growth plate injury model was created in 48 skeletally immature rats. Consisted with the histological changes, and in situ hybridization experiment using VEGF-Flt probe were used to study the bony bridge formation, expressing of VEGF and the changes of chondrocytes. The length and metaphyseal-diaphyseal angle of the grafted tibia were compared. Micro-CT assessment and histological staining were used to compare the bony bridge formation under different interventions. Results At injury site, VEGF signaling appeared on day 10 and increased as time went by. VEGF signaling may be involved in the formation of the bony bridge. The deformity angle and medial length of the tibia were significantly different between grafted tibias and non-grafted tibias at 4, 16 and 24 weeks postoperatively (p⇠0.01). There was no significant difference between compound-graft and fat-graft tibias (p⇢.05). There was no significant difference of the Bone Mineral Density (BMD) between anti-VEGF-Flt grafted group or FS combined with anti-VEGF-Flt grafted group and fibrin-graft or fat-graft tibias (p⇢0.05). Conclusions The long-term prevention effects of compound-graft was significantly better than that of others including fibrin-graft and anti-VEGF-Flt graft group, and little better than that of fat-graft group. Fibrin and anti-VEGF-Flt compound graft interposition would play a role in reducing growth disturbance in growth plate injury., Introduction Recombinant human PDGF-BB is both chemotactic and mitogenic for cells of mesenchymal origin (including chondrocytes) and has the potential to enhance cartilage tissue healing in osteochondral defects. Biocompatible scaffolds, combined with growth factors such as rhPDGF-BB, can help to stimulate and guide the regeneration and repair of cartilaginous tissues. Scaffolds composed of collagen have been developed for use in cartilage repair procedures. The current study was performed to evaluate the stability and release of rhPDGF-BB from a regionally specific, collagen/glycosaminoglycan/calcium phosphate plug. Methods and Materials A volume of 450μl rhPDGF-BB (1.0 mg/ml) was combined with the 8.5mm × 8mm plug. The rhPDGF-BB was eluted from the scaffold using elution buffer of varying salt concentrations. Size exclusion high performance liquid chromatography (SEC-HPLC) was performed to evaluate the rhPDGF-BB contained in the eluate. Total rhPDGF-BB released and the binding efficiency to the PDGF-receptor were quantified over a 24 hour time course using an ELISA (PDGF-BB DuoSet, R&d Systems). Results The observed, salt dependent, release profile included an initial bolus release of 51% after ten minutes, followed by a slower phase of release over the remaining 23 hour study period. The cumulative release of rhPDGF-BB was 70–75% after 24 hours. Finally, the PDGF-receptor binding efficiency, as determined by non-linear regression, of the released rhPDGF-BB was equivalent to that observed for the control rhPDGF-BB. Conclusions After 24 hours, a cumulative total of 70–75% of the rhPDGF-BB was recovered from the scaffold and the eluted rhPDGF-BB was biochemically stable. Studies characterizing cellular response are ongoing. Confidential-Review Purposes Only, Introduction The aim of this study was to ascertain whether the addition of thrombin is required to achieve platelet activation and sustained growth factor release in-vitro, when PRP is applied to a collagen based osteochondral scaffold. Methods and Materials Equal combined volumes of test substances were added to collagen/glycosaminoglycan scaffolds (n=3): 500μl PRP; 375μl PRP + 125μl autologous thrombin; 455μl PRP + 45μl bovine thrombin. One ml of DMEM/F12 medium was added to each scaffold and changed completely at 12/24 hours, and 3/10 days, following which release of TGF-β1, PDGF-AB and bFGF were measured using ELISA. Secondly, 500μl of PRP was added to equal sized collagen/ glycosaminoglycan and polylactide co-glycolide scaffolds (n=3) from which only PDGF-AB was assayed. Results A similar cumulative release profile in all growth factors was found over the 10 day period. An increase in growth factor release was seen in the PRP only group at all time points, particularly with PDGF-AB (p⇠0.006). These findings remained apparent when a correction for volume was made (p⇠0.028) suggesting a particular role of collagen in platelet activation. This was shown in the second experiment, in which a significantly increased cumulative volume of PDGF-AB was released from the collagen/glycosaminoglycan scaffold without thrombin activation (p⇠0.04). Conclusions This study shows that collagen is a potent activator of platelets, requiring no further addition of thrombin to achieve satisfactory growth factor release when applied clinically. These results suggest that if PRP is combined with polymer scaffolds, it should be activated with thrombin to achieve optimum growth factor release., Introduction Platelet-rich plasma (PRP) has been reported to stimulate cell proliferation and extracellular matrix synthesis among ligament, tendon and cartilage. We investigated the effect of PRP gel on the metabolism of chondrocytes in alginate beads. Methods and Materials PRP and platelet-poor plasma (PPP) were isolated from Rabbit's blood using platelet concentration system, SymphonyTM (DePuy). Chondrocytes were isolated by enzymatic digestion from the cartilage tissue of rabbits' knee, and cultured in alginate beads at a density of 4×104cell/bead in three different medium PRP and PPP and FBS5% for one week. Concentration of basic growth factors (TGF-β1 and PDGF) were measured using western blotting. DNA, proteoglycan (PG) and collagen content within the beads were measured at day 3, 7. Results Western blotting showed higher concentration of the basic growth factors in the PRP compard with PPP fraction. DNA content at day 3 did not show significant difference among the groups, however at day 7 the DNA content in the PRP group increased significantly. PG content per bead was increased in the PRP group at both time points, however PG per DNA was less in the PRP group at day 7.PRP had no significant on collagen content, however collagen content per DNA was decresead in the PRP group compared with FBS group at both time point. Conclusions PRP has enhancing effect on three-dimensionally cultured chondrocytes concerning cell cloning and total PG production. These results suggests that filling PRP gel into subchondral bone defect followed by chondrocyte implantation could be a new strategy for refractory large osteochondral lesions., Introduction The current study was designed to compare the Mankin (HHGS) and the OARSI Osteoarthritis Cartilage Histopathology Scoring Systems (OARSI) for the purpose of evaluating a new mid-infrared spectroscopic cartilage analysis tool. A good correlation between these scoring systems has been previously demonstrated both between and within scorers (rs⇢.80, p⇠.01). While the HHGS scoring system was reported to be a valid scoring system to differentiate normal versus severe OA cartilage, it is also reported to have low sensitivity to differences in OA severity. The OARSI, although a newer scoring system, was specifically selected due to reported advantages over the HHGS including greater linearity, a wider range of scores for milder cases of OA, and high reliability, reproducibility, and variability. Methods and Materials Punch biopsies and mid-infrared spectra (n=117) were taken from freshly excised tibial plateaus from knee arthroplasty patients. Following histological processing the slides were scored on the both the HHGS scale and OARSI scale by two raters. The scoring results were compared using Pearson's Correlation Coefficient. Results In our set of specimens the proportion of variation in OARSI score that can be accounted for by variation in Mankin Score was 0.49 (Rs = 0.70). Conclusions This good correlation between the scores is expected to form a suitable base for building a predictive model of histology scores from mid-infrared spectra. Further, we expect that greater sensitivity of the OARSI scoring system in the middle-grade OA cases provides a more suitable base for predictive models such as ours for evaluation of promising instruments., Introduction The intervertebral disc (IVD) is a highly avascular structure occupied by specialized nucleus pulposus cells (NPC) that have adapted to survive within an oxygen concentration of between 2 and 5%. Most studies concerning disc biology have utilized in vitro conditions of 21% O2 and are relatively short-term and often use mono-layer culture. Reports concerning the delivery of anabolic/ matrix protective factors to the disc have claimed effective restoration of disc height in vivo and/or increased expression of desirable genes such as aggrecan and collagen II. Nucleus pulposus cells assume a fibroblastic phenotype in monolayer culture-very different from that found in the in vivo setting. Here for the first time we present the effects of long-term hypoxic and normoxic tissue culture of non-chondrodystrophic canine notochordal cells-cells that appear to protect the disc NP from degenerative change. Methods and Materials Non-chondrodystrophic canine notochordal cells were seeded into alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions using DMEM/F-12 media supplemented with penicillin, streptomycin and fungizone (PSF) and 8% fetal calf seurm (FCS). The cultures were maintained for 5 months following which we used histological, immunohistochemical, scanning electron microscopy and histomorphometric methods to compare differences in extracellular matrix production and viability of the cellular/extracellular matrix. Results Hypoxia induces notochordal cells to produce a highly complex and organized 3D cellular construct rich in aggrecan and collagen II whereas cells cultured under normoxia fail to produce an organized matrix and do not appear viable over long-term tissue culture. Histomorphometric assessment revealed statistically significantly larger cell area under long term hypoxic culture (P=.0001) with the cells retaining an intact cell membrane, nucleus, clear cytoplasm and classic ‘physaliferous’ appearance. Conclusions Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold-other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition-robust aggrecan and type II collagen production. Normoxic tissue culture conditions lead to a failure of these cells to thrive and a lack of extracellular matrix and significantly smaller cells. It is suggested that future studies of nucleus pulposus and in particular, notochordal cells should be cultured under hypoxia in order to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions., Introduction Collagen network structure is the main determinant of cartilage load bearing properties, and can be assessed using Polarized Light Microscopy (PLM). We have developed qualitative and quantitative PLM methods to characterize collagen organization in cartilage biopsies. Methods and Materials Human osteochondral biopsies obtained arthroscopically and equine cartilage were prepared for PLM. Using a newly developed qualitative scoring system, 3 readers rated PLM images on a 0 to 5 ordinal scale describing the range from a totally disorganized tissue (score=0) to ideal hyaline collagen organization (score=5). Inter-reader agreement was analysed using the Intraclass Correlation Coefficient for Agreement (ICC). Quantitative measurements of zonal thicknesses and areas were made by image analysis. Results Normal equine cartilage received perfect hyaline scores of 5, having 2 uniform birefringent zones, representing superficial and deep zones, separated by a non-birefringent transitional zone; proportions of total thickness for deep, transitional and superficial zones were ∼89%, ∼7.5% and ∼3.5%. Normal human cartilage received a score of 4 due to non-uniform zonal interfaces and irregular birefringence patterns; zone proportions were ∼68%, ∼25% and ∼7%. Repair cartilage scores ranged from 0 to 3, reflecting variable levels of collagen orientation achieved with different marrow stimulation procedures. Samples that scored 3 contained a vertically oriented deep zone, evidence of a transitional zone, and a superficial zone that had a predominantly horizontal orientation. The ICC for the qualitative score was 0.89 indicating excellent inter-reader reproducibility. Conclusions A novel method to assess global collagen orientation in osteochondral biopsies has been developed, providing one means to assess cartilage repair tissue quality., Introduction Many cartilage repair procedures rely on subchondral bone marrow to synthesize repair tissue. Since these repair processes also involve bone remodeling and new bone synthesis, it is important to characterize subchondral bone structure in biopsies of cartilage repair. Here we developed a method to measure pore volume density (Vv) and pore surface area density (Sv) in subchondral bone and evaluated reader agreement. Methods and Materials Paraffin sections from ten human osteochondral biopsies (normal, OA, repair) were stained with Safranin-O/Fast-Green. Bioquant Osteo II was used to obtain Sv and Vv in the bone plate and in deeper regions by tracing the contour of reader-defined zones and the bone-pore interfaces. Three readers performed the analysis twice, and Intraclass Correlation Coefficients for Agreement (ICCs) were calculated to assess intra- and inter-reader agreement. Results The thickness of the subchondral bone plate in normal tissue samples ranged from 0.05 to 0.4 mm. Repair tissue could display a thicker remodeled bone plate, in one case up to 2.7mm. Vv (porosity) in these remodeled bone plate was half that of deeper zones (∼0.32 vs ∼0.71), and the range of Sv (pore surface area) was from 4.0 to 8.6 mm-1. Intra-reader ICCs (⇢0.6) were good while inter-reader ICCs (⇠0.6) were fair. Inter-reader differences were mainly related to the definition and delimitation of the remodeled zone and to artifactual structures caused by imperfect section integrity. Conclusions This novel method provides estimates of subchondral bone structure, Sv and Vv, and the thickness of the remodeled zone in human osteochondral biopsies., Introduction Meniscus tear is a common knee injury, which leads to changes in knee load distribution and degenerative arthritis. In such cases, there is a need to protect the cartilage by replacing the menisci. A floating Polycarbonate-Urethane (PCU) meniscal-implant with internal fibers is proposed for pain relief and improvement of pressure distribution. Our goal was to use finite-element (FE) modeling to develop an implant whose ability to distribute pressure is similar to natural meniscus. Methods and Materials The model geometry was based on MR-scans of a cadaveric specimen and analyzed under load-bearing conditions. Several reinforcement configurations were tested. For each configuration, peak/average tibial-plateau (TP) contact pressures, and peak/average von-Mises and tensile stresses were calculated. The model was validated by comparing computational results to experimental TP contact pressures in cadaveric knees. Results Peak and average TP contact pressures across all simulation cases, were 5.5 and 1MPa, respectively. Similarly, PCU peak and average von-Mises stresses were 4.5 and 0.8MPa, respectively. Focusing on the fibers, peak values were predicted in the superior conduit, where fewer fibers could be located due to practical molding reasons. However, in the case of only three conduits containing nine fibers each, peak tensile stress (444MPa) was reduced by ∼20%. Conclusions To conclude, contact pressures developed on the TP cartilage were comparable to those measured under an intact natural meniscus. Safety-wise, strain/stress values in both the PCU and fibers remained within the allowed limits. We believe that the current device will lead to optimal TP pressure distribution, decrease pressure on the cartilage and reduce pain., Introduction When a meniscal lesion occurs, meniscus cells are generally unable to synthesize a valid reparative tissue, which could withstand physiological forces which the meniscus is subjected to. The aim of the study is to evaluate an in vivo model for the meniscal repair. Methods and Materials Swine menisci and articular chondrocytes were harvested from young pigs. Radial slices of menisci were obtained and regularized. Chondrocytes were suspended in fibrinogen and the resulting suspension was placed over a meniscal slice. Immediately after adding thrombin to the cellular suspension, a second slice was placed over the polymerizing cellular hydrogel to form a tri-phasic sandwich. Samples were embedded in acellular fibrin glue in order to provide mechanical protection and to avoid cellular penetration from mouse, and then implanted in subcutaneous tissue of nude mice for 4 weeks. As controls, tri-phasic sandwiches were prepared with acellular fibrin glue between the meniscal samples. All samples were assessed grossly and histologically. Results Experimental samples demonstrated a gross bonding at probing with a pair of forceps while none of control samples showed signs of adhesion between the meniscal slices. Histology analysis demonstrated a continuous hypercellular fibro-cartilaginous tissue at the interface between the two meniscal slices. Moreover, some penetration buds are evident from the cellular fibrin glue to the meniscal tissue. Conclusions The results obtained suggest that cellular fibrin glue has a potential to improve repair of meniscal tears. Further orthotopic in vivo studies are needed to asses the potential for a clinical application of this method., Introduction Arthroscopic partial meniscectomy is a commonly performed procedure to alleviate pain, swelling and mechanical symptoms associated with unstable meniscal tears. Some tears, such as the unstable flap tear and the unstable vertical tear, do not propagate through the vascular zone, and partial meniscectomy is performed. Many chronic meniscal tears, if unstable non-displaced, have pristine cartilage adjacent, and second look arthroscopy after partial meniscectomy reveals varying degrees of chondromalacia in the adjacent cartilage. Methods and Materials In initial testing, eight knees were randomized to two groups. The first group received non-displaced vertical tears of 2 centimeters in length, 3 millimeters from the periphery. The second group received bucket-handle meniscus tears. Knees were loaded in extension with the meniscus intact, with a non-displaced tear, displaced tear (bucket-handle group only) and after partial meniscectomy. The peak contact pressures were compared. Results Initial results showed no significant difference in peak pressures for knees receiving vertical tears between the medial and lateral menisci, or whether the meniscus was intact, torn, or partially meniscectomized. In the bucket-handle tear group, no significant difference was shown between peak pressures in the medial and lateral menisci. However, the peak pressures across the different testing conditions (intact meniscus, non-displaced tear on meniscus, displaced tear on meniscus, partial meniscectomy) were concluded to have a statistically significant difference. Conclusions Partial meniscectomy may negatively alter contact pressures and increase the risk of degenerative arthritis with tears that have not displaced. Clinicians should counsel patients regarding the risk/benefit ratio as it relates to symptom relief versus degenerative arthrosis., Introduction The term insufficiency fracture or spontaneous osteonecrosis of the knee (SONK) is used for patients who have sudden onset of severe knee pain and focal subchondral bone changes. This condition can also occur as an apparent complication of arthroscopy. We hypothesized that this was not a primary disorder of bone or specific to arthroscopy but due to focal force concentration resulting from meniscal deficiencies. Methods and Materials Preoperative MRI studies were reviewed on six subjects with subchondral insufficiency fractures that develop after arthroscopy. Risk factors for secondary osteonecrosis, mensical pathology, body mass index (BMI), bone mineral density (BMD), and demographics were reviewed. These data were also collected on 32 subchondral insufficiency fracture patients with no pre fracture arthroscopy. The two groups were compared. Results None of the five arthroscopy patients had risk factors for osteonecrosis. Three were male and 3 female. Four had posterior horn degeneration and all six had anterior and/or medial meniscal extrusion on MR images. BMI was 25 to 33 except for one 61. Age range was 43 to 74. Two females had BMD one standard deviation above normal for age and one was below. BMD was not available on the males. This population compares with our 32 patient spontaneous onset population. Conclusions Meniscal extrusion appears to be an important risk factor for the development of post arthroscopy subchondral insufficiency fracture formally called spontaneous osteonecrosis of the knee. Hoop stress competency of the meniscus is important in force concentration and is an important consideration for any articular repair procedure., Introduction Geometrical similarity dictates the degree of conformity between the menisci and the condyles, and it has been shown that even very small changes in allograft meniscal implant sizes demonstrate a dramatic change in stress levels. Methods and Materials A set of 12 geometrical parameters, representing typical lengths and widths of the meniscus and joint bone contours were measured in 130 MRI scans of a mixed population of males and females (62±10 yrs., “The Osteoarthritis Initiative” (OAI) database). Mean values of this data were calculated in order to define the most prevalent (‘reference’) implant size in the general population and a novel Poly-Carbonate-Urethane (PCU) meniscal implant was then created by means of an MRI-based reconstruction of femur and tibia geometrical surfaces of a cadaver knee, which corresponded to the reference size definitions. Experimental and computational (finite element modeling) methods were employed to assess the effect of correct matching between a meniscal implant size and a candidate knee on cartilage pressure distributions. Results Both methods confirmed that a meniscus implant performs equally well in distributing joint compressive loads on the tibial plateau surface in a 5% range around the ‘true’ joint space. The meaning of this being that a relatively lenient safety-range exists for the choice of implant by the surgeon. Conclusions The current findings, together with our statistical analysis of the natural distribution of sizes in the general population, imply that an array of nine implant sizes would adequately accommodate the needs of ∼90% of the population, both female and male., Introduction The objective of this study was to assess the in vivo performance of a biodegradable porous polyurethane scaffold. Our hypothesis was that scaffold implantation would promote tissue ingrowth without detrimentally affecting adjacent articular cartilage. Methods and Materials 50 skeletally mature ewes underwent unilateral partial surgical excision of the lateral meniscus. The defect was left unfilled in 20 animals; in the other 30, the defect was filled with an 80% porous aliphatic polyurethane. Animals were evaluated at 3, 6 and 12 months. Outcome measures included histological appearance of the tibial plateau and femoral condyle and histological assessment of the meniscus. Results There was no difference between groups in the percent fill of the meniscal defect. The tissue that filled the empty defect was generally translucent in appearance whereas the tissue fill with the scaffold tended to be dense and fibrous. At 3 months, 38% of scaffold knees showed no damage on the tibia versus 10% of controls. At 6 months, 50% of scaffold knees showed no damage while all the controls had some damage. At 12 months, 10% of scaffold knees showed no damage. On the femur, 75% of scaffold knees showed no damage at 3 months versus 50% of controls; at 12 months 50% of scaffold knees had no damage versus none of the controls. The scaffold was evident histologically at 12 months, infiltrated with cells and surrounded by abundant matrix. Conclusions Implantation of a polyurethane scaffold promoted the ingrowth of dense fibrous tissue into a meniscal defect without adversely affecting adjacent articular cartilage health., Introduction To determine the mRNA expression levels of selected proinflammatory cytokines and matrix metalloproteinases in synovial fluid (SF) cells from osteoarthritic knee joints compared to healthy controls. Methods and Materials Synovial fluid was obtained from 36 patients undergoing total knee arthroplasty due to symptomatic and radiographic evident osteoarthritis (OA) as well as from 10 healthy controls. Expression levels of TNFalpha, IL1beta, MMP1 and MMP3 were assayed among both groups performing realtime qPCR. For subgroup analysis, patients were configured concerning age, gender and BMI. Results All assayed biochemical markers showed significantly higher expression levels among the OA group compared to control, where these markers were not detectable. Furthermore, strong correlation appeared between expression levels of MMP1 and MMP3 among OA patients (r=0,856) while no correlation was found between age, gender or BMI and the expression levels investigated. Conclusions Expression patterns of the examined cytokines and proteinases among SF cells were significantly elevated in OA patients, while no such expression was detected within healthy appearing joints. Consequently, SF cells expressing cytokines and proteinases, known to be crucial in OA pathophysiology, may play a relevant role in the progression of articular cartilage destruction. Considering the fact that SF in an OA-joint comprehends an abnormal amount of bioactive proteins delivering potential detrimental effects to the articular cartilage, temporary clearance, dilution or suppression by means of arthroscopic lavage or disease-modifying medication may represent a therapeutic tool to constitute an interimistic relief or even postpone disease progression due to a decreased inflammative and degrading activity within the human articular environment, Introduction Several studies have shown that chondrocyte apoptosis is positively associated with degree of cartilage matrix damage. On the other hand, SIRT1 (silent information regulator two ortholog 1) has an important effect on regulation of longevity in association with inhibition of apoptosis. Nevertheless, the role of SIRT1 in human cartilage is unknown. To elucidate the role of SIRT1 in the pathogenesis of OA, we examined the effect of SIRT1 inhibition on gene expression changes in chondrocytes. Methods and Materials Human cartilage samples obtained from femoral condyles of patients undergoing TKA and Normal Human Articular Chondrocytes-knee (NHAC-kn) cells (Cambrex) were used as OA chondrocytes and normal human chondrocytes respectively. We investigated whether SIRT1 is up- or down-regulated in OA chondrocytes. We next examined the effect of SIRT1 inhibition by siRNA and chemical inhibitors to investigate whether SIRT1 modulates chondrocyte gene expression associated with OA. Results The expression of SIRT1 was lower in OA chondrocyte than NHAC-kn cells. The inhibition of SIRT1 by siRNA significantly decreased the expression of COLLAGEN2A1 and AGGRECAN and increased the expression of COLLAGEN10A1 compared with the transfection of control siRNA in NHAC-kn cells. Similarly AGGRECAN was significantly decreased and COLLAGEN10A1 and LEPTIN was increased in OA chondrocytes. Consistently, the treatment of chemical inhibitors also increased the expression of LEPTIN and COLLAGEN10A1 and decreased the expression of AGGRECAN. Conclusions Inhibition of SIRT1 induced OA-like chondrocyte gene expression. Our findings suggest that SIRT1 is necessary for preventing chondrocytes from osteoarthritic change and degeneration and that SIRT1 plays an important role in the pathogenesis of OA., Introduction The articular cartilage in Osteoarthritis (OA) is characterized by chondrocyte hypertrophy, apoptosis and calcification. PiT-1, a novel sodium/phosphate cotransporter, has been implicated in this pathologic process. PiT-1 is overexpressed in OA cartilage. Aim: Evaluate if the synovial fluid (SF) from OA patients induce PiT-1 mRNA and protein expression. Methods and Materials ATDC5 cells were cultured with DMEM/F12, FBS 5%, 10 μg/ml human transferrin and 3X10–8 M sodium selenite. These cultures were considered controls and its results were used to normalize the experimental groups results. 10% of SF from patients with OA or healthy donors was added to the cultures. In order to evaluate PiT-1 mRNA and protein expression, Real Time PCR and Western blots were performed at different times: 0, 0.5, 1, 3 and 7 days. Results Real Time PCR: in cultures exposed to 10% of SF from OA patients, PiT-1 mRNA increased 3.4 and 15.9 times compared to the control at day 3 and 7 respectively. Western Blot: in cultures exposed to 10% of SF from OA patients, PiT-1 protein expression increased 1.25 and 2.5 times compared to the control at day 3 and 7 respectively. No changes were detected in cultures exposed to 10% of SF from healthy donors either in mRNA or western blot. Conclusions The SF from patients with OA induces the PiT-1 expression in ATDC5 cells. Because PiT-1 has been implicated in chondrocyte hypertrophy and apoptosis, this cotransporter could be involved in the OA pathogenesis., Introduction Osteoarthritis affects millions of Americans. Optical Coherence Tomography (OCT) is a novel imaging technology that can detect early degenerative cartilage changes prior to surface breakdown, when changes are potentially reversible. Glycosaminoglycan (GAG), a key component of the cartilage matrix, is upregulated during early degeneration. We hypothesized that OCT changes were predictive of GAG content in human cartilage. Methods and Materials Forty-two osteochondral cores were harvested from central and submeniscal regions of human tibial plateaus. Cores were imaged with an OCT scanner and graded as follows: A–obvious birefringence, B–intermediate birefringence, C–no birefringence, D–irregular surface. GAG content was determined using a dimethylmethlene blue assay. Results Cores with OCT grade B-D had a 29% higher GAG content compared to OCT grade A cores (p⇠0.001). The central region had a 30% higher GAG content compared to the submeniscal region (p=0.039). OCT grade had a similar regional variation with a mean grade of 0.74 in the submeniscal region compared to 2.42 in the central region (p⇠0.001). All of the cores with an OCT grade of A were found in the submeniscal region. Conclusions In this study, GAG was elevated in the central weight bearing region, consistent with studies showing regional GAG differences. GAG was also elevated in cores without clear OCT birefringence. As GAG increases during early cartilage repair, this study suggests that loss of OCT-form birefringence is a marker of early cartilage degeneration even when the surface appears normal. Detection at this potentially reversible stage could lead to new treatments to prevent or delay osteoarthritis., Introduction Post-joint injury arthritis affects millions of Americans. Optical Coherence Tomography (OCT) is a nondestructive imaging technology that detects degenerative cartilage changes prior to articular surface breakdown, when potentially reversible changes are occurring. This study tests the hypothesis that OCT can detect acute cartilage changes after impact injury at levels insufficient to produce visible damage. Methods and Materials Forty osteochondral cores were harvested from fresh porcine knees. Cores were scanned with an OCT scanner and then divided into control, low (0.175 J), intermediate (0.35 J) or high-impact groups (0.7 J). Cores underwent impact injury using a computer-controlled impact tower, followed by repeat imaging. OCT image intensities from pre and post-impact images were compared, and the ratio of superficial and deep layer intensity was obtained and analyzed using custom software (Matlab). No change was seen in controls. Results Cartilage prior to impact exhibited a low intensity (dark) superficial layer, followed by a high intensity (bright) deep layer. The intensity of the deep layer decreased after impact. The OCT signal intensity ratio increased by 14% at 0.175J of impact (p⇠0.001), by 17% at 0.35J of impact (p⇠0.001) and by 54% at 0.7J of impact (p=0.02). Conclusions This study shows that OCT can detect acute cartilage changes after impact injury at levels insufficient to cause visible surface damage, supporting the clinical utility of OCT to detect cartilage damage not visualized during standard arthroscopic examination. The ability to identify early changes at potentially reversible stages suggests that OCT could assist in developing treatments to prevent or delay post-traumatic osteoarthritis., Introduction Main consequence of ACL rupture might be development of early osteoarthritis. ACL reconstruction (ACLR) does not prevent OA. To date no one proved that any form of treatment more effectively prevents its development. Some studies demonstrated that ACLR limits the severity of OA, whereas others suggest that it may be even increased by this surgery. The correlation between conservative treatment and development of OA is also unclear. The aim of this study was to compare development of OA during the long-term follow-up in operated and non-operated patients. Methods and Materials 127 patients with an acute isolated total ACL rupture were followed during a 20 year period. In 66 cases ACLR was performed and in 61 cases patients were treated non-operatively. All patients were examined clinically, radiologicaly and evaluated with use of Lysholm and IKDC scores. Radiological assessment was performed according to Kelgren-Lawrence scale. Results According to Lysholm and IKDC objective score operative group (OP) performed better, but difference was not significant (p⇢0.05). Patients of non-operative (NON OP) group demonstrated significantly better results according to IKDC subjective score (p⇠0.05). Osteoarthritic changes were observed in both groups OP (47%), NON OP (51%). In (OP) group 39% patients developed Grade 2 and more of OA, in NON OP (35%) (p⇢0.05). In both groups more severe changes correlated with further injuries. Conclusions Management of ACL rupture may influence development of OA. However, none of methods (operative, non-operative) more effectively prevents OA and limits its severity. ACL reconstruction may in certain circumstances increase both: appearance and severity of OA., Introduction The goal of this project was to determine if Early Intervention Procedures (EIP), such as a Unicompartmental Knee Replacement (UKR), could be expanded, even at the time of TKA. Methods and Materials 87 bone resection samples with predominantly medial wear were obtained after TKA. The proximal tibia, distal femur and posterior femur were photographed and a visual analysis of the lateral cartilage performed using the grading scale described by Weidow et al., 2002. We also obtained Hematoxylin and Eosin stained sections of the lateral tibia and lateral femur and graded them based on the OARSI scale. The operative report was then reviewed for cases determined to have healthy lateral cartilage. Results The visual and histological analyses revealed 49 cases with healthy lateral cartilage. Of these cases, 23 cases presented with severe PF arthritis, 5 cases with a deficient or absent ACL, 1 case with both a deficient ACL and severe PF arthritis, and 20 cases presented with a damaged medial meniscus. Conclusions The remaining cases with healthy lateral cartilage, intact ACL and PCL, and lack of PF arthritis, represented 24% of the 87 cases studied which could have been treated with an EIP. This percent is potentially even higher if patients were treated earlier. These results suggest that EIPs could be used more frequently, and that more in-depth studies are required to investigate further the diagnosis, indications and types of treatment suitable. In addition, the clinical data suggests the meniscus may play a larger role in the development of osteoarthritis than currently thought., Introduction Increasing findings suggest that adipokines which are adipose-derived proteins, can be involved in cartilage destruction during osteoarthritis (OA). However, contradictory data have been found for the effects of adipokines on cultured chondrocytes. The present study investigated in human OA chondrocytes, the effects of phenotypic instability on the expression of leptin, adiponectin and their receptors. We determined also whether their expression is associated with cartilage-specific markers. Methods and Materials The expression of leptin, adiponectin and their receptors (Ob-R, AdipoR1, AdipoR2), as well as collagens type 1, 2A and 2B, aggrecan, Sox9 and MMP-13 was examined by realtime RT-PCR in chondrocytes obtained from patients with OA either directly after cells harvest or after culture in monolayer or in alginate beads. Results Our results showed that leptin and adiponectin are expressed in freshly isolated OA chondrocytes. AdipoR1 and Ob-R are also found while AdipoR2 is barely detected. Interestingly, AdipoR1 is related to aggrecan, collagen type 2A and Sox9. Beside, mRNA levels for adiponectin and leptin are associated with MMP-13. Major changes in the gene expression pattern occurred after culture in monolayer with a shift from the adipokines to their receptors. By contrast, chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in alginate beads, but ceased expressing their receptors. Conclusions In conclusion, this study indicates that experimental conditions are determinant for the expression of adipokines and their receptors. Our data provide also further insights for a potential dualistic role of adipokines in both matrix synthesis and cartilage destruction during OA., Introduction The Acute Anterior Cruciate Ligament Rupture (AACLR) has been described as a high risk factor for osteoarthritis (OA) development. Several studies had linked the inflammation secondary to AACLR as responsible for OA onset. Aim: Evaluate if the synovial fluid (SF) from AACLR induce an OA pattern in ATDC5 cells. Methods and Materials Group 1 (control): ATDC5 cells were cultured with DMEM/F12, FBS 5%, 10 μg/ml human transferrin and 3X10–8 M sodium selenite. These cultures were considered controls and its results were used to normalize the experimental groups results. Group 2: 10% of SF from healthy donors was added to the cultures. Group 3: 10% of SF from patients with AACLR was added to the cultures. In order to assess the OA pattern, we evaluated the phenotypic (nodules apparition) and molecular (collagen II and X) changes by optical microscopy and real time PCR respectively at day 3, 7 and 11 of cultures. Significant differences (p⇠0.05) were determinate by T-student. Results Phenotypic changes: in Group 3, nodules apparition was observed at day 11. This was not observed in Group 1 or 2. Molecular changes: Collagen II: at day 3 the mRNA was in Group 2 1,12 (SD: +0,015) and in Group 3 1,27 (SD: +0,053) times (p⇠0.05). Collagen X: at day 11 the mRNA was in Group 2 1,03 (SD: +0,03) and in Group 3 1,07 (SD: +0,06) (p⇠0.05). No significant differences were observed at others evaluated times Conclusions The SF from patients with AACLR induced an OA pattern in ATDC5 cells. These results suggest that the inflammation secondary to AACLR play a role in the pathogenic mechanism., Introduction Impact injury to articular cartilage can lead to posttraumatic osteoarthritis. This study tested the hypotheses that (1) chondrocyte injury can occur post-impact without visible surface damage, and (2) chondrocyte injury patterns vary with impact energy, time after injury, and cartilage thickness. Methods and Materials 160 fresh bovine osteochondral cores were randomly divided into: (1) Control, (2) 0.35J, (3) 0.71J, (4) 1.07J, (5) 1.43J impact groups and subjected to computer controlled impact loading. Following impact, full-thickness sections were prepared and incubated in DMEM/F12 at 37°C. Contiguous sections were harvested 1 and 4 days post-impact for fluorescent viability staining and microscopy. The area of dead and living chondrocytes was quantified using custom image analysis software (VIS) as a percentage of total cartilage area. Results The highest impact energy of 1.43J fractured the cartilage in all cores (n=17). 73% and 68% of the cores remained intact after being subjected to 0.71J/1.07J impacts, respectively. Cores that fractured were thinner (p⇠0.01) than those remaining intact. Cell death increased significantly (p⇠0.05) with increasing impact energy and with greater time post-impact, while controls showed limited death. A progressive increase in dead cells near the bone/cartilage interface and at the surface was consistently observed. Conclusions These data showing progressive chondrocyte death after impact injury show a potential need for using chondroprotective agents immediately post-joint injury as a strategy to delay or prevent the onset of osteoarthritis. Such treatments may be especially important for individuals with thinner articular cartilage such as women who are also known to have overall higher incidence of osteoarthritis., Introduction Mechanical properties of articular cartilage depend on the content, arrangement and interactions of tissue components, i.e. collagen, proteoglycans (PGs) and interstitial water. Potentially, minor changes in the content and arrangement of tissue components may transmit to significant changes in cartilage biomechanics. Quantitative microscopic imaging techniques enable characterization of tissue structure. In magnetic resonance imaging (MRI) of articular cartilage, T1 and T2 relaxation time measurements may be used to highlight the spatial PG content and arrangement of collagen fibrils, respectively. Methods and Materials We have combined quantitative microscopic and biochemical information with composition-based fibril-reinforced poroviscoelastic finite element model (FEM) of articular cartilage, and have evaluated the feasibility of the model to predict, without mechanical testing, the mechanical response of normal and degenerated human articular cartilage. Results The model analysis was successful in unconfined com pression, as the theoretical stress-relaxation curves agreed closely with the experimental tests. Further, the fibril-reinforced poroviscoelastic cartilage model simulated successfully the changes in tissue mechanical properties during maturation of rabbit knee cartilage. By emerging MRI and FEM we could address closely the PG and collagen specific mechanical properties of bovine and human articular cartilage. Conclusions These studies suggest that specific information on tissue composition and structure might enable assessment of cartilage mechanics without mechanical testing. Especially, the quantitative MRI, when combined with FEM, establishes a functional imaging technique that may be more sensitive and more specific to OA changes than the current clinical methods. Furthermore, the functional MRI could provide means to monitor mechanical maturation of repair tissue non-invasively., Introduction Osteoarthritis of the knee has consistently been linked to obesity, defined as a body mass index (BMI) ⇢30 kg/m2. It has been hypothesized that obesity may lead to osteoarthritis through increased joint pressure, accumulated microtrauma, and disruption of normal chondrocyte metabolism. These changes in chondrocyte metabolism have not been thoroughly investigated, and it is the purpose of this study to identify a relationship between BMI and altered chondrocyte metabolism in osteoarthritic tissue. Methods and Materials Osteochondral explants were harvested from the femoral condyles of patients after total knee arthroplasty, and digested at day 1 and 5 post-surgery. Glycosaminoglycan (GAG) content was measured in both the digestion and media with a dimethyl-methylene blue assay and normalized to DNA content using a PicoGreen® assay. Studies have reported GAG's to be a reliable measurement of chondrocyte metabolism and osteoarthritis progression. Results Our results show a significant linear relationship of increasing BMI and increasing GAG content on both day 1 and 5 (P=0.004 and P=0.0087 respectively). In addition, GAG content from obese individuals (BMI ⇢30 kg/m2) was 3 fold higher than non-obese individuals (BMI ⇠30 kg/m2) on day 1 (P⇠0.01), and 2 fold higher on day 5 (P⇠0.01). Conclusions The study results reveal significant relationships between GAG content and BMI in this population of osteoarthritic patients. The significant difference in GAG content between the obese and non-obese patients supports the connection between osteoarthritis and obesity previously reported. Higher patient BMI (⇢30 kg/m2) may be similar to dynamic compression injuries which cause increased GAG synthesis in response to cartilage damage., Introduction The articular cartilage in Osteoarthritis (OA) is characterized by chondrocyte hypertrophy, apoptosis and calcification. These processes are similar to those observed in endochondral ossification. For that reasons, It has been postulated that one possible pathological mechanism in the OA is the articular cartilage return to its embryologic differentiation pattern. Aim: Evaluate if the synovial fluid (SF) from OA patients induce osteogenesis in ATDC5 cells. Methods and Materials ATDC5 cells were cultured with DMEM/F12, FBS 5%, 10 Î1/4g/ml human transferrin and 3X10–8 M sodium selenite. These cultures were considered controls and its results were used to normalize the experimental groups results. 10% of SF from patients with OA or healthy donors was added to the cultures. In order to assess the osteogenesis induction, we evaluated the phenotypic (nodules apparition) and molecular (collagen II and X) changes by optical microscopy and real time PCR respectively. Results Phenotypic changes: in cultures exposed to 10% of SF from OA patients, nodules apparition was observed at day 3. This was not observed in the cultures exposed to SF from healthy donors. Molecular changes: in cultures exposed to 10% of SF from OA patients, collagen II and X mRNA increased 61 and 13,5 times compared to the control at day 3. No changes were detected in cultures exposed to 10% of SF from healthy donors. Conclusions The SF from patients with OA induced osteogenesis in ATDC5 cells. These results suggest that in OA the SF could play a role in the pathogenic mechanism., Introduction Hyaluronic acid (HA) exerts poorly understood chondroprotective effects in osteoarthritis. Human chondrocytes produce reactive species capable of causing cellular dysfunction and death. Evidence indicates that mitochondrial DNA damage plays a role in conditions linked to generation of oxygen free radicals. The purpose of the current study was to evaluate the chondroprotective effects of HA through the preservation of mitochondrial function and amelioration of mitochondria-driven apoptosis. Methods and Materials Primary chondrocyte cultures, generated from cartilage from patients undergoing total knee replacement, were exposed for 30 min to peroxynitrite and hypoxanthine. After 30 min, cells were lysed for dose-response experiments or rinsed and placed in normal culture medium. Prior to treatment, some cells were incubated for 24 h with sodium hyaluronate (100 to 1000 μg/ml), with anti-CD44 antibody, or in a combination of HA and anti-CD44. To mimic oxidative stress, 1o human chondrocytes were preincubated with HA and treated with IL-1β or TNF-α. Following exposure, cells were collected and evaluated for mtDNA repair/damage, ATP synthesis, and apoptosis. Results When primary human chondrocyte cultures were exposed to reactive oxygen or nitrogen generators, mitochondrial DNA damage/ dysfunction and mitochondria-driven apoptosis accumulated. Cytokine treated 1o chondrocytes showed increased levels of mitochondrial DNA damage. Pretreatment with hyaluronic acid caused a decrease of mitochondrial DNA damage, enhanced mitochondrial DNA repair/cell viability, preservation of ATP levels, and apoptosisamelioration. Conclusions The current results demonstrate that enhanced chondrocyte survival and improved mitochondrial function were noted despite oxidative injury. This observation appears to be one therapeutical mechanism for the beneficial actions of hyaluronic acid in osteoarthritis., Introduction The best known cartilage lesion classification was developed by Outerbridge. This classification consists of four grades and is easy to understand and to use for physicians. However, more objective data regarding cartilage lesion and more accurate methods to evaluate the clinical outcomes are required, because new therapies are developed. The aim of this study is to investigate whether human articular cartilage can be evaluated quantitatively by a spectrocolorimeter. Methods and Materials We studied 79 human articular cartilage specimens retrieved from patients who underwent total knee arthroplasty. 79 human articular cartilage specimens were analyzed using a spectrocolorimeter after macroscopic evaluation and the cartilage characteristics on the L*a*b* colorimetric system, the spectral reflectance distribution, and the yellow/red spectral reflectance percentage(Y/R SRP)were examined. Results There were significant difference among four grade in the L*, a* values and Y/R SRP. The spectral reflectance distribution of grade 1 cartilage showed a gradual increase in spectral reflectance ratio along the increase of wavelength. The spectral reflectance curve of grade 2 to 4 cartilage had dip around 580 nm in wavelength. Across all the measurement wavelengths, there was lower reflectance ratio with the progression of cartilage degeneration. Conclusions The present study is the first to clearly demonstrate the relationship between spectrocolorimetric evaluation and the Outerbridge classification of human articular cartilage. The spectrocolorimeter may be a new quantitative evaluation tool for articular cartilage with clinical potential., Introduction We aimed our study at determining whether a pre-culture time of chondrocytes-seeded matrices before their combination with subchondral layers would modulate extent of cartilage differentiation and integration among the two layers of resulting osteochondral (OC) constructs. Methods and Materials Human articular chondrocytes (HAC) isolated from 5 donors (mean age 57years) were expanded in monolayer and then seeded onto collagenI/III membranes (Chondro-Gide®, Geistlich) (70×106cells/cm3) in a fibrinogen solution (Tisseel®, Baxter). The bony scaffolds (Tutobone®, Tutogen) were pre-wetted in a thrombin solution and then combined to the cell-seeded membranes immediately (group-A) or after 3days (group-B) or 14days (group-C) of preculture of the chondral layers. Constructs were cultured with chondrogenic supplements for a total time of 5weeks and assessed histologically (Safranin-O), biochemically (GAG, DNA). Additionally, the mechanical strength of integration was quantitatively assessed using a “90°-peel-off” test. Results Safranin-O positive matrix was limited to the cartilage phase of group-A constructs and extended to the cartilage/bony interface of group-B and group-C constructs. Peak force and total energy of integration in group-A and group-B constructs were significantly higher than group-C constructs (up to 2.5- and 3.2-fold respectively). Biochemical analysis of the delaminated cartilaginous layers after “90°-peel-off” test demonstrated higher DNA and GAG contents in group-B and group-C constructs as compared to group-A constructs (up to 2.3- and 3.1-fold respectively). Conclusions Our study indicates that functional OC grafts can be generated using HAC and scaffolds currently used in clinical practice. Pre-incubation of HAC for 3days in the chondral scaffold allows increasing cartilaginous matrix formation without reducing integration between the two layers., Introduction Osteochondral grafts are being commonly used to repair articular surface defects. The purpose is to achieve the normal architecture of hyaline cartilage with secure and seamless incorporation into recipient sites. However, the details of the incorporation of these grafts have not yet been completely elucidated. The expectation is that graft union would involve cellular proliferation and/ or migration as well as secretion of matrix and fibers into the graft-host cleft. The aim of this study was to observe any changes in graft architecture with time and to determine the sequence of events during graft incorporation into the host bed. Methods and Materials The medial femoral condyle (MFC) of 12 adult New Zealand White rabbits were studied. A cylindrical 4mm diameter and 4mm long osteochondral graft was obtained from the MFC and reinserted into the same site. At weekly intervals, the specimens were fixed in 10% buffered formalin for a week and decalcified in Kristensen's solution for another week. Next, they were dehydrated and critical point dried. Finally, the specimens were mounted and sputter coated with Gold/Palladium before viewing in the Hitachi S-300H scanning electron microscope. Results Cartilage-to-cartilage healing was not observed at any time interval. Where cartilage union appeared to have occurred, this was due primarily to press fit or ‘surface weld’. In some cases, the adjoining graft and host surfaces revealed superficial fractures. There was bony union at the base in all cases and this union had crept up towards the joint surface in the later time intervals. The materials in the cleft between the graft and recipient bed ranged from fibrous to bony elements. The graft surfaces were smooth like the surrounding normal articular cartilage at 1 and 2 weeks but fibrillated at 3 and 4 weeks. Conclusions These results appear to suggest that direct cartilage-to-cartilage healing may not occur following osteochondral grafting. Bone-to-bone healing appears to be universal and rapid and, materials from this source may be responsible for gap healing. The results also raise the possibility that the articular surfaces may deteriorate with time but the reasons are not apparent from this study., Introduction Study purpose: Measurement of precision comcerning angle of insertion and depth accuracy in navigated autologous osteochondral transplantation in comparison to the conventional free hand technique. Methods and Materials Articular surfaces of 6 cadaveric condyles (medial – lateral) were used. Knee referenced by the navigation system. Pins carrying the navigation detectors were positioned to the femur and to the tibia. The grafts were taken from the donor site (measurement I) with the special instrument which carried the navigation detectors. The recipient site was prepared, the navigation detectors were attached to the insertion instrument and the osteochondral grafts were forwarded into the repair site under the control of the navigation system in an angle of 90° to the articular surface (II). The same procedure took place without navigation. The articular surface congruity was measured with the probe (measurement III) Results Angle of recipient plug removal (measurement I) with navigation: 3,27° (SD 2,05°; 0°-9°) deviation of the 90° axis. Conventional technique: 10,73° (SD 4,96°; 2°-17°). Mean difference between navigation and conventional technique was 7,46° (p⇠0.0001). Navigated recipient plug placement (measurement II): mean angle of 3,6° (SD 1,96°; 1°-9°) Conventional technique: mean angle of 10,6° (SD 4,41°; 3°-17°). Significant difference between navigation and conventional technique (p=0,0001). Navigated depth measurements (III):mean depth of 0,25mm (SD 0,19mm; 0mm-0,6mm). conventional technique:0,55mm (SD 0,28mm; 0,2mm −1,1mm). Significant difference for the navigation and conventional technique (p=0,0034). Conclusions Computer navigated assistance in autologous osteochondal transplantation provides more accurate positioning of the grafts and better results concerning the articular surface congruity., Introduction Determine effects of temperature and serum concentration in medium on efficacy of 28-day allograft storage as indicated by chondrocyte viability, especially in the superficial zone, and cartilage matrix content. Methods and Materials Adult goat osteochondral cores (n=65) were freshly isolated or stored (14, 28 days) in MEM at 4°C including 10% FBS or at 37°C including 0%, 2%, or 10% FBS. Cartilage portions were analyzed using Live/Dead® fluorescence assay to determine viability (percentage live) and cell density at the articular surface en face (E) and in the vertical profile (Overall and by layer (Superficial, Middle, Deep)). The remaining cartilage portions were analyzed for sulfated-glycosaminoglycan and collagen. Storage effects were assessed by ANOVA with Tukey post-hoc tests. Results Viability, live and dead cell density varied with storage group (p⇠0.001), without effects on total cell density. After 28 days, 37°C samples had a higher chondrocyte viability than 4°C samples, especially at the articular surface. At 4°C, viability was markedly affected by storage duration (p⇠0.001). After 28 days, viability at the articular surface was reduced by 4°C storage (E:⇠50%, S:⇠20%), but maintained by 37°C storage (E:∼80%, S:∼60–70%) (each p⇠0.001). M viability was higher following 37°C storage (p⇠0.05), whereas D viability was not. With 37°C storage, viability was similar for 0–10% FBS samples. Cartilage thickness, glycosaminoglycan content and collagen content in stored samples had similar levels to fresh controls. Conclusions 37°C storage of osteochondral grafts maintains long-term chondrocyte viability, especially at the articular surface, and may therefore be used to extend acceptable storage duration and improve treatment outcomes., Introduction One of the most important factors for a successful clinical outcome after transplantation of osteochondral allografts is viability of chondrocytes after preservation. In previous studies, several kinds of culture medium have been tested for suitability for preservation of osteochondral allografts. However, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, the standard for cold preservation of most organs have not been tested. Methods and Materials Osteochondral tissues (OCTs) harvested from distal femora of Sprague-Dawley rats were preserved in Dulbecco's modified Eagle's medium (DMEM), saline, EC solution and UW solution for 7 days. Tetrazolium assay was used to estimate relative viable cell number in OCTs. Lactate dehydrogenase (LDH) activity released from damaged cells into supernatants of preservation solutions was measured to estimate cytotoxicity. Fresh OCTs and OCTs preserved for 7 days were evaluated histologically. Results After 7 days' cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of LDH. In histological evaluation, chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, was observed partially or predominantly in articular cartilage preserved in saline, EC solution and DMEM. In contrast, chondrocyte morphology in articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. Conclusions This study demonstrates that UW solution is better than the other three solutions tested for cold preservation of rat OCTs. If UW solution can protect human osteochondral allograft against damage caused by the cold preservation, the duration of storage of osteochondral allografts may be prolonged., Introduction The purpose of this work is to create an in vitro model of tissue engineered osteochondral composite by combining calcium phosphate scaffold and neocartilaginous tissue produced by isolated swine articular chondrocytes embedded in fibrin glue. Methods and Materials Swine articular chondrocytes were isolated and embedded in fibrin glue. Immediately before gel polymerization, the fibrin glue was placed in contact with the calcium phosphate scaffold. Similar control cylinders were prepared using acellular fibrin glue as chondral element of the composite. The osteochondral composites were left in standard culture conditions and retrieved after 1 and 5 weeks. Samples were macroscopically analyzed and prepared for histological and biomechanical analysis (distraction test). Results Data showed a macroscopic integrity of the osteochondral samples. Histology showed cartilage like tissue maturing within the fibrin glue scaffold, and the presence of GAG between the fibrin glue and the cylinders, infiltrating the scaffold trabeculae in the experimental samples. Control specimens demonstrated inferior biomechanical consistency by gross testing and acellular fibrin glue without infiltration in the calcium phosphate trabeculae. Preliminary distraction test showed first a deformation of the chondral element and then the complete division of the two components. Conclusions The results of this study demostrate that isolated chondrocytes, seeded onto fibrin glue, produce a cartilage-like matrix that integrates with a cylinder of calcium phosphate. This tissue engineered osteochondral composite could represent a valuable model for further in vivo studies on the repair of osteochondral lesions., Introduction Advantages of osteochondral grafting are that the defect is immediately filled with mature hyaline articular cartilage and that stable fixation can be achieved without additional sutures or adhesives. However, a major factor affecting long-term success of osteochondral grafting is poor integration between host and donor cartilage. Methods and Materials Medial and lateral femoral condyles were aseptically harvested from fresh bovine knees. Recipient defects 6mm in diameter were created using an OATS instrument set Osteochondral donor grafts in one of three diameters were harvested: 5.5mm (loose ft), 6.0mm (line-to-line fit) and 6.5mm (tight fit). The 6.5mm donor graft was gently tamped into the recipient site to create a tight press-fit. Ten-mm diameter concentric explants containing the grafted sites were harvested and cultured for 3, 6, or 12 weeks. At each time point, the strength of graft integration with host tissue was assessed by a mechanical tensile test. A full-thickness chondral specimen was cut from the center of each explant, mounted, and tested to failure. Results As expected interface tensile strength increased with time in culture. The tight-fit osteochondral graft design showed the greatest tensile strength after 12 weeks in culture. The greater tensile strength reflects greater chondral integration between the host and graft specimens. Conclusions A tight-fit gap decreases the graft-host interface gap by approximately 21 microns from the current surgical recommendation (line-to-line). We speculate that a tight-fit graft reduces the gap required for matrix production and integration, as well as preventing in-vivo flow of inhibitory molecules such as lubricin, resulting in greater integration., Introduction We evaluated the potential of porous bioactive glass (BG) 13–93 as a subchondral substrate and media supplement for tissue-engineered bi-layered osteochondral (OC) constructs. Methods and Materials In vitro: The effects of BG on the chondrogenic capacity of juvenile bovine chondrocytes (from carpometocarpal (CMC) joints) were studied by assessing biochemical and biomechanical outcomes associated with BG cylinders placed in media of chondrocyte-seeded agarose hydrogel constructs after 28 days of culture and compared to controls 14 days later. In vivo: Rabbit mesenchymal stem cells cultured in monolayer were seeded into poly(ethylene glycol) (PEG) hydrogel and bonded to BG to create tissue-engineered OC constructs. Similar constructs created using allograft bone were used as controls. The OC constructs (3mm × 6mm) were implanted into defects in the medial femoral condyles of rabbit knees and assessed after 12 weeks. Results BG in the culture media resulted in peak values of EY: 743kPa, GAG: 7.6%ww, G*: 2.8MPa and Collagen: 6.2%ww which were each significantly better than controls. These values for BG-treated constructs compare favorably to intact bovine CMC cartilage. The in vivo study showed that bi-layered BG OC constructs histologically appeared to have better integration and significantly greater collagen II expression than allograft bone constructs. Conclusions These data suggest that BG has potential as a media supplement for tissue engineering osteochondral constructs for resurfacing articular cartilage defects and as a subchondral substrate for these grafts. Further studies will be required to optimize the protocol for construct creation and to delineate the long term effects of BG for clinical application., Introduction The development of non-invasive cartilage assessment methodology would permit evaluation of therapeutics without requiring biopsy. Therefore, we evaluated the potential of near infrared (NIR) and mid-infrared (mid-IR) spectroscopy to characterize full-depth matrix changes in the collagen and proteoglycan components of engineered cartilage over time and in response to an anabolic intervention. Methods and Materials Articular bovine chondrocytes were cultured in a collagen type I gel scaffold for 3 or 5 weeks (n = 10). Pulsed low intensity ultrasound was applied to half of the constructs for 20 minutes/day, 5 days per week. A Nicolet Continuum FT-IR Microscope (Thermo Electron Corp) was used to acquire NIR and mid-IR data from the constructs in the spectral region of 800 − 7000 cm-1. Results were compared to biochemical assays for sulfated glycosaminoglycan (sGAG). Results We found that the integrated area of the 850 cm-1 mid-IR peak increased with ultrasound treatment over a time course of 5 weeks, and correlated well with sGAG content (R2 = 0.89). This in agreement with our previous studies that demonstrated correlation of this peak with proteoglycan content. There was no increase in the NIR peak at 4250 cm-1, a spectral feature that arises from type II collagen, with ultrasound treatment, consistent with previous in vitro studies of collagen development under ultrasound intervention. Conclusions These results provide motivation for further development of infrared spectral parameters for full-depth evaluation of cartilage constructs and intact tissues., Introduction We postulate that the biochemical and biomechanical characteristics of articular cartilage are joint and age specific. To verify our hypothesis, we investigated the gene-expression profiles of articular cartilage tissue that was derived from three different joints of differently-aged bovine cows. Methods and Materials Articular cartilage was harvested from the metacarpal (basal region), shoulder (humeral head) and knee (tibial plateau) joints of 4-month-old bovine calves, 15- to 20-month-old (young adult) bovine cows and 3- to 9-year-old (aged) bovine cows within 24 hours of slaughtering. The structural maturity of the tissue was ascertained histologically. The gene-expression profiles of the samples were quantified by a real-time PCR analysis. Results The postnatal development of bovine calves into young adult cows is characterized by a structural evolution of the articular cartilage tissue from an immature (isotropic) to a mature (anisotropic) type. Aging of the animals is not accompanied by any overt change in the structural organization of the cells. In immature articular cartilage, the activity levels of the genes for collagen types IX and X, and Sox9 were similar in each of the three joints. However, in the articular cartilage of young adult and aged animals, the gene-expression profiles of these components were joint specific. Moreover, the gene-activity levels of the collagens were very much lower in the older animals than in the young adults. Conclusions In mature bovine articular cartilage, the gene-activity profiles of key components are joint specific. These differences may be manifested also at the protein-expression level and, consequently, could impact the tissues' biomechanical properties., Introduction The objective of this study was to determine the influence of chondral defect size on defect rim stress concentration, peak rim stress, and load redistribution to adjacent cartilage over the humeral head. Methods and Materials Eight fresh-frozen cadaveric shoulders were mounted at 45° abduction with 5° of external rotation in a materials testing machine. Digital pressure sensors were placed over the humeral head contacting the glenoid. Each intact shoulder was loaded and held while dynamic pressure readings were recorded throughout. Loading was repeated for circular defects (6, 8, 10, 12, 14, 16, 18, 20mm) centered at the contact apex as determined by the initial control load. Results Stress concentration around the rims of defects 10mm and smaller was not demonstrated. For defects 12 mm and greater, distribution of peak pressures followed the rim of the defect with a mean distance of 4.2mm. Load redistribution was observed as the radius to peak value increased above a rim size of 12mm to a mean of 11.8mm compared to 7.9mm for defects 10mm and below. Peak rim pressure did not increase significantly as defects were enlarged from 10mm to 20mm. Conclusions Rim stress concentration was demonstrated for chondral defects 12mm and greater in size. This altered load distribution has important implications relating to long-term integrity of cartilage adjacent to chondral defects in the shoulder. Our understanding of chondral defects in the shoulder, a non-weightbearing joint, is evolving. While the decision to treat is multifactorial, a size threshold of 12mm may serve as a useful guide to clinical decisionmaking., Introduction Epiphyseal cartilage trauma, accidently or caused intra-operatively is known to initiate bone-bridge formation possibly causing full or partial premature physeal closure. The aim of this study was to investigate hypervascularity as one hypothesized underlying mechanism for bone bridge formation using qRT-PCR and 3-Tesla MRI in a living-animal-model. Methods and Materials Male Sprague-Dawley-rats (4 weeks, ∼100g) were subjected to a unilateral transphyseal drilled lesion of 1.2 mm diameter of the proximal tibial physis. Physeal vascularisation and bone bridge formation were investigated by 3-Tesla MRI (n=5; intravenous application of Dimeglumingadopentetat, Gadovist® 0.2ml/100g body weight) while expression profiles of HIF1a, VEGFa, VEGF receptor 1 and 2, and CD 31 were established by qRT-PCR (n=8) on days 1, 3, 7, 14, 28, and 82 post-lesion. Results In the traumatized growth plate, mRNA expression of Hif1a showed a slight upward trend from day 1 on with maximum levels on day 3 while VEGFa, VEGFR1 and 2 showed highest expression levels on day 7. Comparing expression levels of VEGFa, VEGFR1, HIF1a, and CD31 between the lesioned and the contra-lateral physis significant changes were only observed on day 28 (p⇠0.05) giving indirect support for existing bone bridges (observed by 3-Tesla MRI on day 28) as they represent highly vascularised tissue which could further be documented by increased perfusion levels evaluated by 3-Tesla MRI. Conclusions This study gives evidence for HIF1a and VEGF expression to precede angiogenesis and formation of bone bridges at the site of physeal lesion which was clearly documented by qRT-PCR and 3-Tesla MRI., Introduction The objective of this study was to evaluate the mechanical behavior full-thickness osteochondral defects of articular cartilage in the human knee. In addition, change in behavior was measured after a cylindrical lesion was converted to a beveled defect. Also, what part of the lesion size is more critical for diagnosis: the outer or inner diameter? Methods and Materials Ten cadaveric knees were mounted on a load frame. Pressure sensors were placed in the medial and lateral compartments. Each knee was loaded and held. Pressure readings were recorded throughout. Loading was repeated over full-thickness osteochondral defects with vertical walls (6, 8, 10, 12, 14, 16mm). The well-shouldered lesions were then transformed into beveled lesions and loading was repeated. The center of defect to peak pressure distance was calculated. Results An important comparison is the difference in center of defect to peak pressure distance between well-shouldered and beveled defects, using outer diameter as a metric. In the lateral condyle, 4 of 6 test pairs showed increased distance from well-shouldered to beveled lesions, with average distance of 0.2174mm. In the medial condyle, 5 of 6 test pairs showed increased distance from well-shouldered to beveled lesions, with average distance of 0.5231mm. Beveled lesions acted more like well-shouldered lesions of a larger size (10mm beveled acted like 12mm well-shouldered). Conclusions The outer diameter of beveled defects in articular cartilage is a better predictor of pressure distribution on surrounding cartilage than inner diameter. Beveled defects of articular cartilage in the knee should be evaluated and treated based on their outer diameter., Introduction Some preliminar studies concerning cell therapy have been carried-out in animal models to treat the rupture of anterior cruciate ligament (ACL) with two main approaches: a) the use of different fibroblasts or mesenchymal cells for autologous implantation and b) the use of new biomaterials combined with cultured cells. Our purpose was to study the features and behaviour of monolayer cultured fibroblasts isolated from ACL of patients with different ages and type of lesion. Methods and Materials ACL biopsies from 34 patients with ACL rupture. Twenty-eight of 34 patients had acute ACL rupture (elapsed time from rupture to surgery shorter than 1 month), 9 had chronic ACL (elapsed time from rupture to surgery longer than 1 month). Normal ACL samples from 28 patients. The median age of these 62 patients was 32 years (range: 15 – 74 years). Fibroblasts were isolated, the number of cells was estimated and cultured in monolayer. Results The fibroblast culture could be established in 43 cases. First passage was done at the day 15th and the mean growth rate was 21.2 ± 15.5. Negative correlation was observed between the age of the patient and the growth rate. Although without statiscal significance (probably due to the low sample size), the mean growth rate was higher in patients with acute lesion (n=18; 28.3 ± 19.8) followed by normal ACL (n=7; 17.1 ± 8.9) and by those with chronic ACL rupture (n=18; 13.5 ± 9.4). Conclusions The growth rate of ACL fibroblast in culture is higher in young patients with acute ACL rupture., Introduction Mechanical forces play a critical role in nearly all aspects of cell biology. Within synovial joints, they are prerequisite for maintaining cartilage matrix properties whereas excessive mechanical forces leads to a loss of cartilage and the onset of osteoarthritis. How cells sense mechanical stimulation, however, is largely unknown. Our purpose is to develop a new experimental system to study the effects of mechanical loading and the mechanism of mechanotransduction. Methods and Materials We produced a new porous collagen sponge scaffold that has inter-connected pores for 3-D cell culture and a custom-designed and -built apparatus for loading experiments. We isolated human synovial cells and meniscus cells from surgical specimens and incorporated these cells into scaffolds. The 3-D construct was applied to load stimulation and used for the analysis. Firstly, we examined the cell viability, then compared the morphology and the cytoskeleton of cells seeded in monolayer or in 3-D scaffolds. Results Cells in the constructs were viable throughout the culture period of 1 week. The cells were amoeboid in shape in 2-D and spheroid in 3-D culture. Actin was seen to form stress fibers only in 2-D and showed a punctate staining pattern in 3-D culture. The organization of the vimentin meshwork was a more circumferential arrangement in 3-D culture. Conclusions We developed a new 3-D cell culture system in which the morphology and general cytoskeletal disposition of cells are similar to their native condition, i.e. in their natural extracellular matrix. This culture system provides a powerful tool for the study of mechanotransduction and drug screening., Introduction The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI)-derived cells was studied to ascertain the healing potential of PEMFs. Methods and Materials MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibro-blastic phenotype occurred, indicating the success of the surgical im plantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. Results The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. Conclusions The results show that MACI membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions., Introduction The purpose was to examine whether a chondrogenic medium and/or mechanical stress is necessary to differentiate human bone marrow stromal cells (hBMSC) into chondrocyte-like cells in a biological osteochondral matrix stimulated in a bioreactor system. Methods and Materials HBMSC were harvested, density centrifugation was performed prior to resuspension and three cell passages. For 7 days a predifferentiation culture was initated by replacing FGF-2 with 100 ng/ml IGF-1 and 5 ng/ml TGF-β2. Afterwards cells were seeded into the biologic hybrid scaffold out of CaReS® (rat collagen I, Arthrokinetics) and Tutobone® (bovine spongiosa, Tutogen Medical) with a concentration of 1×106 cells/ml. The constructs were exposed to a cyclic compression protocol (10 % compression, 0.5 Hz) under continuous perfusion in a mechano-bioreactor for 14, 21 and 28 days. Effects were evaluated using light microscopy and collagen 2, 3, and 10 staining. GAG and DNA were quantified. Biomechanical characterization was conducted using a confined compression quasi-static loading setup. Results GAG quantification showed differences between mechanical and static stimulation after three and four weeks (p⇠0.05). Mechanical tests showed no difference over four weeks, but the mechanical groups were stiffer compared to the static control (p⇠0.05). The quality of tissue was not improved by IGF/TGF in our system focusing histology, biochemistry and mechanical properties of these tissues. Conclusions Media supplements to foster the quality of the tissue showed no progress in our system although it is well known that those are important to induce a chondrogenic phenotype. It could be postulated that those could be left out in such a system., Introduction No medical intervention has yet been demonstrated to effectively restore damaged cartilage in large clinical trials in OA. It could be speculated that study subjects generally had reached a “point of no return” where medical stimulation of cellular repair mechanisms are insufficient to restore the structure damage. To study this, we performed a series of biochemical assessments of bovine cartilage explants undergoing sequential catabolic and anabolic stimulation. Methods and Materials Bovine cartilage explants were cultured in the presence of OSM+TNFα at different duration followed by IGF1. Release of peptide fragments into the supernatant was monitored by MMP and aggrecanase-dependant neo-epitope specific immunoassays. Results Seven days of OSM+TNFα stimulation of bovine cartilage explants induced a massive loss of proteoglycans as observed by histology. Transferring these explants to anabolic stimulation (IGF1) for another 14 days, reversed the loss of proteoglycans. Assaying the supernatants for presence of neo-epitopes demonstrated that (1) OSM+TNFα induced massive release of aggrecan fragments mediated by aggrecanases in the early phase, and changing to anabolic conditions after 7 days did not change this profile; (2) In contrast, aggrecan and type II collagen fragments carrying MMP-dependant neo-epitopes were released only in the late phase, i.e. after day 11; (3) If catabolic stimulation was discontinued after 7 days, MMP-generated fragments was not released into the supernatant. Conclusions These biochemical data strongly suggest that cellular repair mechanism in bovine explants cultures can be at least partly restored after short-term catabolic stimulation, and that biochemical profiling of the supernatants effectively reflect these metabolic changes., Introduction Subchondral bone is important to maintain quality of articular cartilage. The objective of this study is to investigate the importance of subchondral bone remodeling for articular cartilage regeneration. Methods and Materials Fifty male Japanese white rabbits were used. A 6mm cylindrical osteochondral defect was made in left patellar groove. The rabbits were randomly assigned to control, Alendronate (ALN) (0.14mg/kg/week subcutaneously) and ALN-S group (ALN injection for first 8 weeks). Animals were sacrificed at 4, 8, 24 and 52 weeks. The quality of the cartilage was evaluated histologically (Wakitani Score) and with our ultrasound method. Bone morphology was evaluated with micro-CT. Results At 4 weeks, ALN group showed drastically early remodeling of subchondral bone and regeneration of cartilage-like-tissue filling the defect. However, no cartilage-like-tissue was seen in control group. In Wakitani score, 4 weeks ALN group was superior to control group. In 52 weeks, ALN-S group seem to have thicker and more stained cartilage than ALN or control group. However there was no significant deference among the score of three groups. Ultrasound analysis showed the signal intensity of both ALN and ALN-S group was significantly higher than control group in 4, 24 and 52 weeks, suggesting better quality of the cartilage. Micro CT analysis showed the superior remodeling of continuity of subchondral bone and recovery of BV/TV in 8 weeks ALN group. Conclusions Eight weeks injection of ALN had positive effect on subchondral bone remodeling and following cartilage regeneration. The result showed an importance of the early subchondral bone remodeling for cartilage regeneration., Introduction We investigated the potential of periosteal cells to infiltrate PCL nanofiber scaffolds in vivo and subsequently produce cartilage in vitro. Methods and Materials PCL nanofiber scaffolds, with or without chitosan coating were implanted under the periosteum on the medial proximal tibia of six-month old rabbits. TGF-β1 (200 ng) or vehicle was injected into each implant site. After 1, 3, 5 or 7 days, scaffolds were removed, separated from the periosteum, and both the scaffolds and periosteal explants from the implant site were cultured separately for six weeks. Results Cell infiltration was observed in all the scaffolds. Cartilage formation in the uncoated scaffolds increased with duration of implantation (maximum at 7 days). Cells in the uncoated scaffolds implanted for 7 days produced significantly higher levels of both GAG (560 ±412 vs. 228 ±61 μgGAG/μgDNA) and cartilage yield (9 ±11 vs. 0.02 ±0.04%) compared to chitosan-coated scaffolds (p⇠0.007). There was no significant difference in GAG content or cartilage yield between the TGF-β1-injected and vehicle-injected scaffolds. However, significantly more calcium deposition was detected in TGF-β1-injected scaffolds compared to vehicle-injected scaffolds (p⇠0.0001). Cartilage yield from the implant-site periosteum was significantly increased in all the TGF-β1 injected groups compared to vehicle (p⇠0.0014), with the exception of day 1 and 5 of the chitosan-coated scaffolds. Conclusions This study demonstrates that it is possible to seed PCL nanofiber scaffolds with periosteal cells in vivo and subsequently produce engineered cartilage in vitro. These periosteal cell-seeded nanofiber scaffolds may be useful alone or in combination with the periosteal graft for cartilage regeneration., Introduction We've previously reported the development of scaffold free cell delivery system, and showed good regeneration of rabbit osteochondral defect until more than two years by implantation of molded mesenchymal stem cells (MSCs) construct without use of exogenous factors. To build more complex shaped, multi-lineage cell construct without biomaterials, we invented a new simple method to build larger scaffold free constructs by building up cell aggregates one by one. In this pilot study, we examined our new system, named “Bio rapid prototyping method”, to be abed to build scaffold free cell construct. Methods and Materials For initial experiments, we used primary rabbit MSC, which were divided two groups, labeled with Qtracker 655(Invitrogen, US) as red, and 565ã as green, under monolayer condition. The labeled cell aggregates were placed as pre-designed position one by one with fine micro forceps under stereoscopic microscope. After building process, the constructs were cultured under standard condition for 2–3 days. Results We obtained cell constructs as almost expected shape. Snap frozen section showed no necrotic formation in rough histological observation. Conclusions Until now, we tried hMSC, HepG2, or beating cardiomyocyte as cell source for the construct by our method, and showed good results. In this study, the construct are build by our hands under microscope, it takes much hours to build just only 5mm cubic shaped cell only construct. Near future combination of the robotic technology and the bio technology, we may able to build joint cartilage, meniscus, etc, in accordance with patients 3d body data., Introduction The goal of this study was to evaluate ultrasound as a safe, novel imaging modality for the non-invasive monitoring of the healing response of an osteochondral defect that was treated with implantation of a preconditioned bioactive scaffold, and to correlate the ultrasound imaging with histological observation. Methods and Materials Bilateral osteochondral defects were surgically created in the trochlear groove of the knee in fifteen adult male New Zealand white rabbits, under IACUC approval. Twenty-four defects were filled with PLLA scaffolds preconditioned with either IGF-1 or BMP-7, and eight were left as unfilled controls. The knees were then harvested at 3, 6, and 12 weeks post-surgery, and evaluated with histological evaluation, as compared to ultrasound, using FlexScan image processing software. Results Osteochondral defects were clearly identified in each ultrasound performed. Imaging of knees containing an unfilled osteochondral defect demonstrated an empty gap in the trochlear groove, whereas those with a scaffold implant exhibited filling of the defect. Ultrasound images correlate with histologic evaluation in demonstrating a progressive increase in the healing response around osteochondral defects filled with preconditioned bioactive scaffolds for the three, six and twelve week samples. Conclusions Ultrasound was able to distinguish the site and borders of both the osteochondral defect and the implanted scaffold, and was accurate in measuring the dimensions of the osteochondral defect and the scaffold within it. Progressive changes in these measurements allow monitoring of the repair tissue in a temporal fashion. Lateral edge integration in the osteochondral defect was also observed with ultrasonography, correlating with histological results., Introduction The lack of inflammatory response and the feasibility for controlling mechanical, morphological and degradation properties are attractive features of poly (L-lactic acid) (PLLA) for cartilage repair. Our aim was to determine the biocompatibility between Mesenchymal Stromal Cells (MSC) and PLLA and their effect on the mechanical properties of the scaffolds. Methods and Materials MSC were cultured in PLLA films to study adherence and proliferation. We loaded 10×106 and 2×106 MSCs into PLLA scaffolds (1 and 6 mm in thickness), either by injection or aspiration. Scaffolds were maintained in expansion or differentiation media for 21 days. Seeding efficiency and cell distribution and viability were analyzed after the seeding while chondrocyte differentiation was assessed at 21 days. Results Results showed an adhesion of MSC to PLLA films of 27 ± 0,15 and 34 ± 9,0 % of seeded cells at 1 and 4 hours. Proliferation was detected, but ceases at day 10. An efficiency of 80,8 ± 4,1 and 71,5 ± 9,4 % for 1 and 6 mm scaffolds was detected after the seeding. Retaining of cells were more efficient in the 6 mm scaffolds, where cells were poorly spread. Nevertheless, cells homogeneously saturated 1 mm scaffolds. Final results show the production of matrix composed by collagens I, and X, that produced a 2,6-fold increase in the Young modulus. Conclusions We have developed biocompatible PLLA scaffolds that can be efficiently loaded with MSCs. We also show that differentiation of MSCs can be successful in PLLA and that it modify the mechanical properties of the PLLA itself., Introduction Electro-wetting on Di-Electric (EWOD) printing can create droplets of less than 10μm-diameter and work with a variety of hydrogels, cells and growth factors during scaffold fabrication. EWOD has never been used in tissue engineering and can enable control of droplet size and precise deposition of droplets compared to existing micro-droplet technology. Methods and Materials EWOD uses the electrocapillary principle: surface-tension (ST) is a function of electric potential across an interface. The change in liquid-solid ST changes the contact angle at the liquid-solid-gas interfaces. By applying sequential voltages to electrodes under the dielectric layer, four fundamental droplet manipulations can be established: creation, cutting, joining, and transporting. The system will integrate an EWOD array design with a computer-controlled motion system to position the EWOD chip for hydrogel, crosslinker, growth factor and cell printing based on CAD models. Projected built rates are 1cm/min with droplet diameters on the order of 10μm for picoliter-volume droplets. Results Amounts ranging from 0.2–2% (w/v) chitosan were mixed with acetic acid. The maximum viscosity EWOD can handle is 250cP. High-molecular weight chitosan was shown to behave as a non-Newtonian fluid with a non-linear relationship between shear rate and shear stress; Low-molecular weight chitosan is Newtonian with a linear relationship. After EWOD printing, human fetal osteoblast cells were shown to have a success rate of 94%. Conclusions Rheological testing and droplet operations were completed and studied on chitosan hydrogels and crosslinker solutions. EWOD on-chip crosslinking and cell manipulation were demonstrated, which shows the system is capable to make micro array printing for tissue construct., Introduction We analyzed the early structural characteristics and adhesive response of rabbit chondrocytes seeded into uncoated PCL scaffolds (pPCL) or composite PCL scaffolds containing hyaluronan (PCL/ HA), chitosan (PCL/CS), fibrin (PCL/F), and type I collagen (PCL/COL1) and cultured statically. TEM (x7500, 1h after seeding) and anti-CD44 and anti-integrin-β-1 immunofluorescence (1h and 24h after seeding) analyses were performed. Methods and Materials The surface of random chondrocytes (n=7 each) was analyzed using a grid (parallel lines at 1.1765 μm intervals in TEM). The number of lines intersecting the chondrocyte profiles, and the number of intersections with the cell surface contours were recorded in two orthogonal orientations. Roughness, diameters and horizontal-to-vertical ratios were calculated. Results PCL/HA (2.66±0.43) and PCL/COL1 (4.16±1.75) contained significantly fewer “rough” chondrocytes compared to the other scaffolds (p⇠0.05). No significant differences in diameter and horizontal-to-vertical ratio could be found between the different groups. Integrin-β-1 and CD44 were detected in PCL/COL1 scaffolds. In the other hybrids, integrin-β-1 could only be detected 1h after seeding. CD44 immunoreactivity was found in PCL/HA only 24h post-seeding. PCL/ CS, PCL/F and pPCL lacked CD44 immunoreactivity. Chondrocytes in PCL/HA showed the least prominent surface modifications compared to controls, and an increasing CD44 expression. Cells in PCL/COL1 had more prominent yet not significantly different surface modifications, and a constant expression of CD44 and integrin-β-1. Conclusions Previously, we found that PCL/HA and PCL/COL1 supported cartilage ECM synthesis better than PCL/F and PCL/CS. These functional differences may be related to the early structural observations in this study. Future studies will be needed to decipher this relationship., Introduction Collagen gels are weak cell scaffolds. Can photochemically crosslinking collagen gel permit: 1) cell survival; 2) neo-cartilage formation; 3) neo-cartilage integration with the adjacent native cartilage? Methods and Materials Cartilage was collected from swine and healthy human cadaveric tissue and chondrocytes were obtained by enzymatic digestion. Cells (40–60 million/cc) were mixed in 0.8% solubilized rat tail type I collagen liquid. Different concentrations of riboflavin and irradiation doses of blue light were tested to photochemically crosslink collagen I gel resulting in favorable cell survival as determined by live-dead assay. Hydrogels constructs with cells were implanted into nude mice to assess the capacity of neo-cartilage formation in vivo. Cells in gel were also placed into the center of devitalized swine or human articular cartilage (ring model) or between two disks of devitalized swine or human articular cartilage (sandwich model) and implanted into nude mice to assess the capacity for integration and healing after 6, 12 and 18 weeks. Results 0.25mM riboflavin and 40 seconds irradiation permitted high cell viability and minimal gel contraction. Specimens placed in mice showed neo-cartilage formation as evidenced H&E, Toluidine blue, Safranin-O staining, and immunostaining for type II collagen. Neo-cartilage filled native cartilage ring model showed good integration and neo-cartilage between cartilage disks formed tight bonds with existing cartilage. Conclusions Human, as well as swine, chondrocytes have the capacity to form neo-cartilage in this novel crosslinked hydrogel scaffold, and the cartilage can integrate and heal to adjacent native cartilage. Riboflavin and blue light are benign crosslinkers allowing favorable cell survival and neo-cartilage formation., Introduction Biologic scaffolds can augment the repair of tendon defects by enhancing the endogenous reparative response. PDGF-β and IGF-1 are potent mitogens, shown to improve tendon healing in various models. The current study investigates the effects of a tissue engineering method that involves preconditioning biologic scaffolds with gene-enhanced tenocytes transduced to produce either IGF-1 or PDGF-β. Methods and Materials Adult male Sprague-Dawley rats were used to isolate tendons from the rotator cuff, and tendon fibroblasts (RTF's) were transduced with the gene for either PDGF-β or IGF-1, using retroviral vector plasmids. PLLA scaffolds were preconditioned for 3, 6, or 9 weeks by seeding with wild-type RTF's, or RTF's transduced with either growth factor. After lyophilization, wild-type RTF's were then seeded onto either the preconditioned or control scaffolds. Following 3 or 7 days in culture, samples were pulse-labeled with H Proline and H Thymidine to assess collagen and DNA synthesis. Results Our results demonstrate that the cells grown on scaffolds that were preconditioned with RTF's had greater DNA and collagen synthesis than scaffolds that were not preconditioned. Scaffolds preconditioned for longer durations demonstrated significant increases in DNA synthesis over untreated controls. Similar trends were seen for collagen synthesis, with increases compared to control scaffolds for all preconditioning treatments after both 3 and 7 days of culture. Conclusions This study demonstrated improved collagen and DNA synthesis in scaffolds preconditioned with RTF's. These findings may potentially be attributed to the increased levels of proteins released by both growth factor-transduced RTF's and wild-type RTF's, as directly related to the duration of preconditioning., Introduction The purpose of this study was to compare the histologic appearance of chondral repair tissue at 18 months following treatment with TruFit® plugs or a standard microfracture technique in an ovine model. Methods and Materials Single, 5.1 mm diameter osteochondral or full-thickness chondral defects were created in the medial femoral condyle of 12 adult sheep. The osteochondral defects were repaired with a 5.3 mm diameter X 5 mm long, press-fit, TruFit® plug (n=6) and the full-thickness chondral defects were treated with a standard microfracture technique (n=6). The histologic appearance of the repair tissue was evaluated in each group at 18 months using a modified O'Driscoll scoring system and compared using a t-test. Results At 18 months, both groups showed evidence of organized fibrocartilage repair tissue filling the defects. The repair surfaces were smooth, congruent, and there was excellent lateral integration to the adjacent hyaline cartilage in both groups. Both groups demonstrated thickening of the subchondral bone immediately beneath the repair tissue with the lower phase of the TruFit® plug being replaced with cancellous bone. There was no statistical difference (p⇢0.05) in the quality of the fibrocartilagenous repair tissue between the TruFit®and microfracture groups. Conclusions In this model, both TruFit® and microfracture treatment of focal chondral defects produced a smooth, congruent, fibrocartilagenous repair tissue that was histologically similar at 18 months. The results of this study suggest that, in isolated chondral lesions, the histologic quality of the cartilage repair tissue produced following treatment by either TruFit® plugs or microfracture at 18 months is the same., Introduction New developed interventions for cartilage regeneration, with the application of 3D-scaffolds, require a further evaluation of the surgical technique. This study compared different scaffold fixation techniques in a human cadaver knee model. Methods and Materials A 2cm2 cartilage defect was created at the weight-bearing portion of the medial and lateral condyles of 20 human cadaver knees. The fixation techniques, Tissuecol® (Baxter, Utrecht), transosseous fixation, SmartNail® (ConMed Breda) and continuous cartlilage sutures (Vicryl®6.0, Ethicon, Amersfoort), were randomly assigned to the defects to implant a custom-printed porous PEGT/ PBT 1000/70/30 scaffold. Subsequently, the knees were closed and subjected to a loaded (35N) continuous-passive-motion protocol at vertical orientation. After 60 and 150 cycles the knees were reopened and the implantation sites photographed for fixation evaluation, focusing on area-coverage, outline-detachment and scaffold integrity. After 150 cycles an endpoint fixation test was performed. Differences in quality of fixation technique were analyzed by a non-parametric Kruskal-Wallis test followed by a multiple comparison test and Bonferroni correction. Results Individual evaluation of the fixation techniques revealed marginal differences for area-coverage and outline-detachment after 60 and 150 cycles. The Tissuecol® scored superior for scaffold integrity compared to transosseous (p⇠0.05) and cartilage sutures (p=0.01). Endpoint fixation was highest for the cartilage sutures whereas Tissuecol® showed a weak final fixation strength (p=0.01). Conclusions This study showed that the quality of 3D-matrix fixation is a combination of scaffold properties and mechanical stability of the fixation technique applied. Special attention devoted to scaffold properties, in relation to the fixation technique, will result in an improvement of scaffold fixation., Introduction We have been developing a new tissue-engineering technique involving scaffold-free tissue engineerd construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). In the present study, we investigated the frictional properties of TEC-repaired mature cartilage-like tissue. Methods and Materials Synovium-derived mesenchymal stem cells (MSCs) were cultured through 4 to 7 passages. After an addition of ascorbic acid 2-phosphate, the cells were allowed to undergo active contraction for 8 hours to develop TEC. A round-shaped, cartilaginous defect was created in the medial condyle of the femur of 12 month-old mature pigs. A TEC mass was allografted with the TEC. Six months after surgery, a cylindrically-shaped repaired specimen was extracted and subjected to a reciprocating friction test. The surface of the specimen was rubbed against a glass at the speed of 20 mm/s with the load of 70, 140 and 280 kPa. Results Laser microscopic observation indicated that the surface roughness of the TEC-repaired cartilage was slightly smaller than that of normal cartilage. The repaired tissue was covered by adhesive substance having fewer tubercles of micro size than normal cartilage. Immediately and 60s after the application of compressive force, the coefficient of friction of the TEC-repaired cartilage was slightly decreased with the increase of load. Conclusions The coefficient of friction was decreased closes to normal cartilage in the mature group. This implies that the restoration of lubrication properties is enhanced in the TEC-repaired mature tissue in accordance with the low roughness and adhesive substances at the surface. (Supported, in part, by NEDO(106001904-0) & MEXT(BERC)), Introduction In vitro expansion of hMSCs is important in cartilage tissue engineering. Our hypothesis was nanotopography enhances hMSC chondrogenic differentiation leading to additional cell sources for cartilage tissue engineering. Methods and Materials Novel nanotopographies with randomly distributed 11 nm high islands were fabricated by polymer demixing of polystyrene and polybromostyrene. Control surfaces were flat polystyrene. RT-PCR quantification of aggrecan and Collagen II mRNA was completed after hMSCs were cultured 21 days on nanotopographic surfaces in chondrogenic differentiation media(10ng/mL TGFβ1 and 10 ng/mL BMP6) or on nanotopographic surfaces in proliferation media. ANOVA and post hoc Tukey-Kramer Multiple Comparisons Test were completed. Results Steady-state levels of Collagen II mRNA were significantly increased in hMSCs cultured on 11 nm high surfaces in differentiation media(p⇠0.0001 versus hMSCs cultured on flat surfaces in differentiation or proliferation media and 11 nm high surfaces in proliferation media; n=3). Steady-state levels of aggrecan mRNA were significantly increased in hMSCs cultured on 11 nm high surfaces in differentiation media (p⇠0.001 verses hMSCs cultured on fat surfaces in differentiation or proliferation media but not 11 nm high surfaces in proliferation media; n=3). Conclusions hMSCs cultured on 11 nm high nanotopagraphy and in differentiation media containing TGFβ-1 and BMP6 expressed significantly higher levels of collagen II and aggrecan compared to hMSCs cultured in differentiation media on fat surfaces. Collagen II and aggrecan are phenotypic markers of early chondrocytic differentiation suggesting that nanotopography has a direct influence on stem cell differentiation toward the chondrocyte phenotype. Thus, nanotopographic surfaces represent a novel enabling technology for cartilage tissue engineering., Introduction Articular cartilage shows poor intrinsic repair, leading to progressive joint damage. Therapies like marrow stimulation or tissue engineering of cartilage depend on chondrogenesis of progenitor cells. However, chondrogenesis needs to take place in a diseased joint. We postulate that catabolic factors in diseased joints will inhibit chondrogenesis of progenitor cells. Methods and Materials We examined the effect of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNFα) on human mesenchymal stem cells (hMSC) undergoing chondrogenesis. Osteoarthritic (OA) synovium derived conditioned medium was added to hMSC undergoing chondrogenesis. Chondrogenesis was examined by determining mRNA levels of chondrocyte specific matrix molecules. Proteoglycan deposition was analyzed by safranin O staining of histological sections. Results Chondrogenesis was completely abolished on day 14 by adding IL-1 from day 0, 3, and 7. The expression of type II and X collagen and aggrecan mRNA was blocked completely. Addition on day 10 resulted in only partial inhibition of chondrogenesis. TNFa showed similar effects but appeared to be less potent than IL-1. OA-synovium conditioned medium strongly inhibited chondrogenesis of hMSC. However, blocking IL-1 or TNFα did not inhibit completely prevent the inhibitory activity of synovial fluid on chondrogenesis. These results show that in general chondrogenesis of hMSC is inhibited by factors produced by OA synovium. Conclusions Chondrogenesis of hMSC is blocked by single catabolic factors as well as by factors produced by OA synovium. Our findings have major implications for therapies that depend on repair by mesenchymal stem cells. Successful cartilage regeneration will fail if the catabolic environment of a damaged joint is not altered., Introduction Mesenchymal stem cells are developed for cell-based regenerative therapies. Synovium-derived stromal cells(SSCs) are promising as a source for cartilage regeneration. In vivo and ex vivo procedures required for gene therapy expose cells to considerable oxidative stress. Selenium has been shown to be an essential trace element with antioxidant properties. We hypothesized that selenium can improves stem cell potency via an increase in the proliferation and self-renewal potency of SSCs. We explored the ability of selenium that increase the proliferation of SSCs with the upregulation of stemness gene expression and the effect of selenium on the acquisition of active migration ability in SSCs pretreated with selenium. Methods and Materials Isolated SSCs were analyzed by flow cytometry. To investigate the effect of selenium on the proliferation of SSCs, we performed a MTS assay and BrdU incorporation assay. The expression of stemness genes and cell migration related genes was analyzed by RT-PCR. The wound healing and cell transwell migration assay were performed. Results The SSCs in each population were negative for CD14, CD31, CD34, CD45 and positive for CD44, CD90, CD105. The number of BrdU-labeled cells was increased in response to 5 ng/ml selenium, which is consistent with the observed increase in the MTS assay. Selenium treatment exerted apparent effects on the overexpression of stemness genes (Oct4, Nanog, c-Myc, Rex1, and Klf4) and migration-associated factors. Selenium-treated cells significantly increased the migration efficiencies in a time dependent manner, as compared to control SSCs. Conclusions We demonstrated that selenium increase proliferation, migration and stemness genes expression in synovium-derived stromal cells., Introduction There is great potential for the use of cell-based therapies for repairing cartilage lesions caused by osteoarthritis. Bone marrow stromal cells (BMSCs) are a heterogeneous population of multipotent stem cells which can be differentiated to generate a three-dimensional hyaline cartilage using a defined combination of growth factors. However, there are currently no markers available for identifying those BMSCs which have the greatest chondrogenic capacity. Methods and Materials A sterile FACS sorter was used to deliver single BMSCs into individual wells of 96-well plates and each clone was proliferated in the presence of FGF, seeded onto PGA scaffolds and differentiated to produce cartilage using a tissue engineering approach. The quality of the cartilage produced by each individual clone was measured using biochemical assays for collagen types I and II and proteoglycan. Results Several BMSC clones were expanded sufficiently, without signs of senescence, to use in cartilage tissue engineering. Of those, 2 clones produced a macroscopically good tissue engineered cartilage (mean wet weight 16.8mg) with proteoglycan and type II collagen comprising 29.0% and 6.1% of the dry weight respectively. Cartilage from the other 5 clones had a mean wet weight of only 1.9mg and contained significantly lower amounts of proteoglycan and type II collagen. Conclusions We have demonstrated for the first time that different clonal populations of BMSCs have different chondrogenic capacities. We are currently using gene array technology to identify unique cell surface markers present on highly chondrogenic clones. Such markers would be used to identify those BMSCs which would be most effective for cartilage regeneration., Introduction A current challenge in mesenchymal stem cell (MSC)-based cartilage repair is to solve the donor and tissue-dependent variability of MSC cultures and to prevent chondrogenic cells from terminal differentiation like in the growth plate. The aim of this study was to select the best source for MSC which could promise stable cartilage formation in the absence of hypertrophy and ectopic in vivo mineralization. We hypothesized that MSC from synovium (SMSC) are superior to bone-marrow- (BMSC) and adipose-tissue-derived MSC (ATSC) since they are derived from a joint tissue. Methods and Materials MSC were characterized by flow cytometry. MSC pellets were cultured under chondrogenic conditions and differentiation was evaluated by histology, gene expression analysis, and determination of alkaline phosphatase activity (ALP). After chondrogenic induction, pellets were transplanted subcutaneously into SCID mice. Results BMSC, ATSC, and SMSC revealed similar COL2A1/COL10A1 mRNA levels after chondrogenic induction and were positive for collagen-type-X. BMSC and ATSC showed significantly higher ALP activity than SMSC, in which low ALP-activity before transplantation correlated with marginal calcification of explants. At elevated MMP mRNA levels, some transplants had specifically lost their collagen-type II, but not proteoglycan in vivo, or were fully degraded. Conclusions In conclusion, lower donor-dependent ALP activation and reduced mineralization of SMSC-derived heterotopic transplants surprisingly did not lead to a more stable ectopic cartilage but correlated with specific degradation of collagen-type II, fibrous dedifferentation or complete degeneration of spheroids. This emphasizes that beside appropriate induction of differentiation, locking of MSC in the desired differentiation state is a major challenge for MSC-based repair strategies., Introduction By their limited availability and their rapid dedifferentiation during expansion, chondrocytes seem not the best candidates for cartilage tissue engineering, but other autologous cell sources are actually promising. The aim of this work was to study the influence of growth factors or culture supplements (hyaluronate) (1) on the differentiation of human mesenchymal stem cells (MSC) towards chondrocytes in 3D collagen sponge culture and (2) on the extracellular matrix synthesis profile. Methods and Materials MSCs were isolated from human bone marrow and expanded in monolayer. After passage 3, MSCs were seeded into collagen sponges and cultured for 28 days in vitro with different mediums containing ITS (Insulin Transferin Selenium), FBS (fetal bovine serum), +/- hyaluronate, and TGFβ1 followed or not by BMP-2. Extracellular matrix (ECM) production and chondrogenic differentiation of MSCs during 3D culture were evaluated at D28. Chondrogenic gene expression (Coll2, Coll1, Coll3, Coll10, Aggrecan, Versican, COMP, Sox9, Osteocalcin, Alkaline Phosphatase) were investigated by real-time quantitative RT-PCR. Newly synthesized ECM was assessed histologically and immunohistochemically (Coll1 and Coll2). Results Hyaluronate alone did not promote chondrocyte differentiation of MSCs. TGFβ1 alone or associated to hyaluronate or/and sequential BMP-2 provoked chondrocyte differentiation of MSCs and an ancillary important ECM production rich in proteoglycans and Coll2. On the other hand, the association of hyaluroniate with TGFβ1 and sequential BMP-2 showed similar results associated with small calcium-rich deposits characteristic of an osteoblastic differentiation. Conclusions Chondrogenic differentiation of MSCs seeded in collagen sponges is promoted by the sequential contact with TGFβ1 switched by BMP2 thus leading to local Coll2 synthesis., Introduction Recently, there have been several reports using various SPIO nanoparticles to label stem and other cells for tracking their distribution or accumulation at the target lesion. For magnetic targeting of labeled cells, it is important to determine the effect that the labeling procedure may have on the differentiation capacity of cells. In this study, two FDA-approved agents, Resovist® and protamine sulfate (PS) were used to label human synovium-derived cells and chondrocytes. We examined the effect of labeling on differentiation capacity of cells. Methods and Materials Cells were isolated by sequential digestion and labeled for 24h with 500μg/ml Resovist® and 5μg/ml PS. Chondrogenic differentiation was induced using a pellet culture. The synovium-derived cell pellets were cultured in chemically defined chondrogenic medium with 10ng/ml TGF-β1 and 100ng/ml BMP-2 and the chondrocyte pellets were cultured in standard culture medium. The synthesized GAG was determined by binding to DMB dye. Pellets were stained with prussian blue (PB) for iron detection and with Safranin-O for proteoglycan detection. The expression of collagen I and II was detected by immunofluorescence staining. The cartilage-specific ECM genes expression was analyzed by RT-PCR. Results Synovium-derived cell pellets was larger than chondrocyte pellets without regard to labeling. The GAG production did not show a siginificant difference between the labeled and unlabeled cell pellets. Chondrogenic differentiation in labeled and unlabeled synovium-derived cell pellet cultures over 4 weeks exhibited chondrocyte-like cells embedded in a proteoglycan-rich ECM, as evident in the safranin-O staining. PB staining confirmed the presence of iron-oxide particles within the labeled cells. Chondrocyte pellets also showed positive staining. Positive immunofluorescent staining for collagen II was shown in the ECM of both groups. RT-PCR revealed no difference between labeled and unlabeled pellets of both cells. Conclusions Resovist® and PS were used to effectively label human synovium-derived cells and chondrocytes with no adverse effects on chondrogenic differentiation and phenotypic expression. Magnetic labeling may be applicable for targeting cells for cartilage repair., Introduction Mesenchymal stem cells (MSC) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. We here analysed the early healing response in experimental full-thickness cartilage defects, asking whether MSC can differentiate into chondrocytes in an orthotopic environment, without the induction of hypertrophic markers. Methods and Materials Cartilage defects in knees of minipigs were covered with a collagen-type I/III membrane, and half of them received transplantation of expanded autologous MSC. At 1, 3 and 8 weeks, morphological and molecular aspects of repair were assessed. Results At 1 and 3 weeks after surgery only marginal Col2A1 and no AGC expression were detectable in both groups. At 8 weeks Col2A1 and AGC levels had significantly increased. The orthotopic environment triggered a spatially organized repair tissue with upper fibrous, intermediate chondrogenic, and low layer hypertrophic differentiation of cells and a trend to more safranin-O and collagen type II-positive samples after MSC transplantation at 8 weeks. Compared to in vitro chondrogenesis, Col10A1 and MMP13 mRNA levels were suppressed. Conclusions These data indicate that, as opposed to in vitro chondrogenic induction of MSC, the in vivo signalling molecules and biomechanical stimuli provide an appropriate environment for progenitor cells to differentiate into collagen type X-negative chondrocytes. Thus, until better in vitro induction protocols become available for chondrogenesis of MSC, their pre-differentiation prior to transplantation may be unnecessary., Introduction Cell therapy approaches for cartilage repair rely on isolated chondrocytes which lose their chondrogenic potential during expansion. Mesenchymal stem cells (MSC) represent an alternative if their default pathway towards osteogenesis can be prevented. In this study, mixed pellet cultures containing different ratios of chondrocytes and MSC were prepared to examine possible cellular crosstalk leading towards chondrogenesis. Methods and Materials Chondrocytes from human articular cartilage expanded for 8 days in monolayer culture and human bone marrow MSC were labeled with PKH 67 and 26, respectively. Cells were mixed at different ratios and chondrogenesis was induced in pellet cultures in serum free medium with and without TGF-β1 and dexamethasone. Proteoglycan deposition, collagen II, collagen X synthesis and S100 protein were assessed via histology and immunohistochemistry. DNA/GAG content was quantified using CyQuant kit and DMMB assay. Results Fluorescence of labeled cells indicated an equal distribution throughout cocultured pellets. 100% chondrocyte pellets produced neocartilage rich in proteoglycans, collagen II, and had high expression of S100. 100% MSC pellets did not produce any chondrogenic specific proteins. In 50–75% MSC pellets proteoglycan and collagen II were detected and most of cells expressed S100 protein. Proteoglycan production was confirmed via GAG/DNA measurements. Collagen X, indicative of osteogenic differentiation, was detected only in 100% MSC pellets, indicating that presence of only 25% chondrocytes stabilized chondrogenic phenotype of MSC. Conclusions Detection of cartilage matrix proteins in cocultured chondrocytes and MSC pellets incubated in absence of external chondrogenic factors suggests crosstalk from chondrocytes to MSC inducing chondrogenesis and preventing further differentiation of MSC towards osteogenesis., Introduction Apoptosis is defined as programmed cellular death. This biological phenomenon has been described by some authors, when studying the articular cartilage after a traumatic event. It observes also, that some mediators (Interleukin-1, Nitrous Oxide and TNF-α), when in contact with chondrocytes, they are capable to perpetuate the effect of programmed death, culminating in osteoarthritis. On the other hand, apoptosis can be interrupted by substances such: diacerhein, glucosamine and osteogenic protein-1 (OP-1). Purpose: to evaluate if the injection of intra-articular platelet-rich plasma (PRP) can reduce impact-induced chondrocyte apoptosis. Methods and Materials A double-blind study was developed with four knees of two adult rabbits. Each knee was injured 3 times with 1 kg weight released from the top of a cylinder of one-meter length. Subsequently, 1ml PRP was injected in the right knees and 1ml of normal saline in the left knees. The animals were euthanized ten days after the intervention. All cartilage was removed from the 4 knees and prepared for analysis in electronic microscopy (EM). The individuals involved in the preparation and analysis of the grids were blind to the experiment. Seven to ten meshes of EM were obtained for each knee. The sum of all apoptotic cells in each mesh was registered. Results The PRP-injected knees summed apoptosis 47,62% (50/105) and 48,36% (59/122), respectively. NS-injected showed 56.67% (17/30) and 70.40% (88/125) of apoptosis. PRP injected knees had statistically significant less apoptosis 48.02% than NS injected 67.74% (p Conclusions Immediately post-traumatic intra-articular injection of PRP reduces impact-induced chondrocyte apoptosis in rabbits., Introduction Mesenchymal stem cells (MSC) are a promising source for cell-based tissue engineering of cartilage. The purpose of this study was to evaluate the chondrogenic potential of MSC in two different 3-D cultures, a scaffold-free high-density diffusion-culture (DC) using 106 cells as well as a pellet-culture (PC) using 2,5 × 105 cells, where optimal combinations of growth factors were identified. Methods and Materials MSC were expanded in medium containing FGF-2 and cultured under serum-free conditions in DC or PC. Except for control cultures the medium contained TGF-β3 and IGF-1, TGF-β3 and FGF-2 or TGF-β3. Cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for cartilage markers. Results Except for controls all cultures showed positive staining for proteoglycan and type II collagen. Type II collagen, aggrecan and SOX-9 mRNA were detected in all differentiated cultures with higher amount in PC than DC. The highest expression of type II collagen and aggrecan was found in PC with TGF-β3 and either IGF-1 or FGF-2. An increased type X collagen expression could be shown whenever FGF-2 was added. Conclusions The study shows that an effective chondrogenic differentiation can be obtained in DC. Collagen type II expression and aggrecan content of DC showed results which do not rank far behind PC. Therefore DC, which show an appropriate graft size and cartilage like structure, might be an interesting tool to be used for clinical cartilage repair in regenerative medicine., Introduction Differentiation of BM-MSCs into adult chondrocytes represents a complex physiological process. Full characterization of each individual stage of chondrogenesis through CD cell surface antigens has failed to provide a reliable and reproducible tool. The physiological micro-environment of chondrocytes is hypoxic, which triggers over-expression of SLC2A proteins (GLUTs) in their membranes. This could provide a new generation of functional and morphological markers of chondrogenesis. Methods and Materials BM-MSCs were cultured and differentiated along with adult chondrocytes in hypoxic (3% O2 tension) and normoxic (20% O2) conditions. These cell populations were screened for the presence of the 12 GLUT genes and for the variation of their transcription and translation during chondrogenesis. Functional characterization of the GLUTs expressed in the cellular membrane was carried out using radio-isotope labeled hexose fluxes according to the substrate specificity and kinetic properties particular to each SLC2A isoforms. Results The functional genotype and phenotype of the adult chondrocyte and hypoxic BM-MSCs comprised a mosaic expression of SLC2A transporters. Moreover, similarities in functional “GLUT signature” between BM-MSCs cultured in hypoxic conditions and adult chondrocytes were identified. Investigation of the uptake of a panel of five individual sugars (glucose, fructose, 2-deoxy-glucose, 3-orthomethyl-glucose, and galactose) in these cellular populations, in the presence and absence of a GLUT1-specific inhibitor, showed that SLC2A class II transporters (GLUT 5, 7, 9, 11) play a more important role in chondrogenesis compared to the ubiquitously-expressed GLUT1. Conclusions Identification of the chondrogenic BM-MSC “GLUT signature” will enable selection of those chondrocyte precursors that have the best potential for producing viable articular cartilage., Introduction We Isolated Peripheral blood mononuclear cells and stromal adipose tissue cells from adult horses. Adult stem cell (ASC) are undifferentiated cells commonly present in functional tissues able to renew themselves, when injured, can develop mature functional tissues. We used the surface specific markers CD34, CD45, CD47, CD14, CD117, CD73 and CD90 to identify MSC by flow cytometry analyses. The aim of this study was to investigate the possibility to isolate pluripotent precursor cells from equine peripheral blood and fat tissue, to compare the surface antigen groups Methods and Materials We obtained mononuclear cells from horses by Ficoll gradient centrifugation of peripheral blood. We also isolated fibroblastic cells from fat tissue, incubated the two samples with monoclonal antibodies for flow cytometry analysis to determine if in harvested cells were found specific precursor cells surface antigens. With the positive cells we conducted plasticity trials to evaluate the aptitude of the precursors isolated to generate different tissues. We stimulated our both cells colonies to generate cartilage, glial cells and fat tissue. Results Peripheral blood precursors and stromal cells from adipose tissue were harvested from horses. The presence of specific stem cell precursor surface markers were proved using antibodies synthesized to recognize human surface markers From the horse blood we got a CD34+, CD90+, CD 73+, CD45-, CD14-, CD117- phenotype and the fat cells CD90+, CD117+, CD14-, CD47-, surface markers were lost at the end of the cloning phase Conclusions Indicates cross antigenicity and let us presume proteic sequences homology between horse and human receptors., Introduction For healing articular cartilage damage, synovium-derived cells has been studied as a useful cell source because the cell has several advantages. Our previous study showed that co-treatment of TGFβ-1, BMP-2 and dexamethasone promoted chondrogenesis of synovium-derived cells. To analyze underlying detail mechanisms of the promoted chondrogenesis, we conducted microarray analysis at each time point of chondrogenic process. Methods and Materials 1) Human synovium-derived cells were isolated from knee joint and cultured in DMEM with 10% FBS. 2) Passage 2 synovium-derived cells were centrifuged to make pellets and then cultured with chondrogenic medium containing dexamethasone, BMP-2 and TGF-β1. 3) Total RNA from the pellets were analyzed by RT-PCR and microarray. Illumina's Sentrix HumanRef-8 Expression BeadChips were used in this experiment. 4) GAG amount in pellets were determined by DMB analysis. 5) Pellet specimens were subjected to Safranin-O staining for detection of proteoglycan. Results The protein level of proteoglycan and GAG and the RNA level of type 1,2,10-collagen, SOX-9, aggrecan were increased during chondrogenesis. In microarray analysis, genes showing minimal variation were excluded(p-value⇠0.0001, 5204 genes). We could divide all 5204 genes into 19 clusters showing similar patterns of gene level change. Among the genes showing increased expressions, genes relating organ development (especially relating extracellular matrix) and glycolate metabolism were remarkable. Among the genes showing decreased expressions, genes relating cell proliferation and cell death were notable. Conclusions In this study, we could analyze the expression changes in several genes during chondrogenesis of synovium-derived cells. Further experiment will follow to enlighten detail mechanism of these genes on chondrogenesis., Introduction Human embryonic stem (hES) cells are pluripotent cells and have the capability to differentiate to various cell types. The hES cells are suggested as the ultimate source for cell based therapies and may represent an alternative cell source for the treatment of cartilage defects. Differentiation of hES cells to chondrogenesis by direct co-culture is the purpose of this study. Methods and Materials Here we studied how co-culture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers and differentiation abilities of hES cells. Undifferentiated hES cells were co-cultured with neonatal or adult articular chondrocytes in a pellet mass system. Results Our results demonstrate that unlike undifferentiated hES cells co-cultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Co-culture further resulted in a more homogenous pellet and significantly increased cartilage matrix production. Co-cultured cells also formed colonies in agarose suspension culture demonstrating differentiation towards chondroprogenitor cells, while no colonies were detected in the hES cells. Co-culture also resulted in a significantly decreased osteogenic potential. Conclusions Our results confirm the potential of the culture micro-environment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings. These hES cells share some qualities that are characteristic for chondrocytes. So far all results point out that direct co-culture could have a strong chondrogenic effect on undifferentiated hES cells. This direct co-culture model can be useful for studying early chondrogenesis, Introduction Hypertrophy of transplanted chondrogenic cells is thought to limit cell-based cartilage repair, as it leads to apoptosis and ossification. The present study compares gene transfer of the transcription factor SOX-9 and BMP-2 as agents of chondrogenesis in human MSCs maintained as pellet cultures Methods and Materials Cultures of human marrow-MSCs were infected with 5 × 102 particles/cell of Ad.SOX-9, or Ad.BMP-2, seeded into aggregates and cultured for three weeks in serum-free medium. Untransduced cells or cultures transduced with marker genes (GFP/ Luc) served as controls. Expression of SOX-9 was determined by western blot and BMP-2 by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by real time RT-PCR for chondrogenesis and hypertrophy. Results Levels of SOX-9 or BMP-2 (35–55 ng/mL) in the media were initially high and declined thereafter. SOX-9 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-2 modified aggregates showed a strong tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, immunohistochemical staining for type X collagen protein, and lacunar size. Conclusions SOX-9 and BMP-2 were strongly effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by SOX-9 gene transfer showed less evidence of hypertrophic differentiation than that triggered by the BMP-2 cDNA, suggesting that SOX-9 may be a more suitable for use in cartilage repair., Introduction To determine whether the implantation of alginate beads containing human mature allogenic chondrocytes is feasible and safe for the treatment of symptomatic cartilage defects in the knee. Methods and Materials A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of chondral and osteochondral lesions in the knee. Twenty-one patients were clinically prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and a Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 13 had consented to the taking of a biopsy for investigative purposes from the area of implantation at 12 months of follow-up, allowing histological assessment of the repair tissue. Results A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. Adverse reactions to the alginate/fibrin matrix seeded with the allogenic cartilage cells were not observed. Histological analysis of the biopsy specimens rated the repair tissue as hyaline-like in 15.3 %, as mixed tissue in 46.2 %, as fibrocartilage in 30.8% and as fibrous in 7.7%. Conclusions The results of this short term pilot study show that the alginate-based scaffold containing human mature allogenic chondrocytes is feasible and safe for the treatment of symptomatic cartilage defects of the knee. The described technique provides clinical and histological outcomes equal to those of other cartilage repair techniques., Introduction Hallux rigidus is characterized by limited range of motion at the first metatarsophalangeal joint (I MTPJ) with pain and disability. Treatment options in severe hallux rigidus are arthrodesis or prosthetic replacement, although significative drawbacks of these techniques are reported. Bipolar fresh osteochondral allograft have been successfully used in the treatment of arthritis of the ankle joint and demonstrated to be able to provide new joint surfaces with cartilage able to survive the transplantation and satisfactory clinical results. Aim of this study was to present the applicability of bipolar fresh allograft to the I MTPJ and to describe the results. Methods and Materials 3 patients (4 feet), mean age 49yrs, affected by hallux rigidus grade III, underwent fresh bipolar osteochondral allograft of the MTPJ. Patients evaluation included clinical and X-Rays evaluation at different established follow-up (up to 3 yrs). Results Before surgery patients complained pain and no range of motion at the I MTPJ. Mean AOFAS score was 48±6, while it was 80±4 at follow-up (p⇠0.005). Good consolidation of the graft, with satisfying clinical result in term of stable pain free join with a good range of motion was resumed in 2 patients (one bilateral) at follow up. One graft non-union occurred as post-operative complication and required revision. Conclusions The presented technique demonstrated a new possible application of fresh osteochondral allograft. Although the number of cases is still exiguous this report may offer an interesting opportunity for further research and application in the controversial topic of grade III hallux rigidus treatment., Introduction DeNovo® NT Natural Tissue Graft Purpose: To evaluate the clinical application of particulated allograft cartilage. Preclinical animal studies demonstrated the potential for particulated articular cartilage to fill focal articular cartilage lesions of the knee with hyaline-like cartilage. Methods and Materials A prospective clinical case study of DeNovo NT Graft (particulated human juvenile cartilage allograft) was initiated in 2007 after obtaining the local IRB approval. This is a preliminary report on the first 7 patients who were treated with DeNovo NT Graft. A major inclusion criterion was the presence of one or two persistently symptomatic untreated focal contained femoral articular cartilage lesions of a normally aligned stable knee with intact menisci. Results During 2007- 2008, 7 patients were enrolled (1 female and 6 males) with ages ranging from 20 to 49 with a mean age of 35. The 7 patients have current follow up of between 1 month and 1 year. The small subject number does not allow for statistical analysis, but improvements are noted from the baseline using the KOOS score (and KOOS subscales), IKDC subjective and health assessments, Marx and VAS. Conclusions The early MRI evaluations show filling of the defect and reveal no transplant dislodgment. Patient enrollment will continue and outcomes will be monitored until all patients reach 2-year follow up. This initial report of DeNovo NT Graft demonstrates early clinical improvement in a small cohort., Introduction The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects of the knee. MRI was used for the morphological analysis of cartilage repair. The correlation between MRI findings and clinical outcome was also studied. Methods and Materials A alginate-based scaffold containing human mature allogenic chondrocytes was used for the treatment of symptomatic (osteo)chondral lesions in the knee. Twenty-one patients were prospectively evaluated with use of the WOMAC and VAS for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 12 had consented to follow the postoperative MRI evaluation protocol. MRI data were analyzed based on the original MOCART and modified MOCART scoring system. The correlation between the clinical outcome and MRI findings was evaluated. Results A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. One of the two MRI scoring systems that were used, showed a statistically significant deterioration of the repair tissue at one year of follow-up. We did not find a consistent correlation between the MRI criteria and the clinical results. Conclusions Two MOCART-based scoring systems were used in a longitudinal fashion and allowed a practical and morphological evaluation of the repair tissue. However, the correlation between clinical outcome and MRI findings was poor. The promising short-term clinical outcome of the allogenic chondrocytes/alginate beads implantation was not confirmed by the short-term MRI findings., Introduction Limitations of fresh osteochondral (FOC) allograft transplantation are, scarce supply and short shelf-life. A new cartilage repair technology with off-the-shelf, processed OC grafts (Chondrofix®, a proprietary product of Zimmer Orthobiologics) has been developed and evaluated in an equine model. Methods and Materials OC plugs harvested from equine femoral condyles were machined into either mushroom (MOC) or cylindrical (COC) shape and then processed before implantation. With IACUC approval, four defects were created in the medial trochlea in 6 horses and randomly repaired with MOC, COC, FOC or left untreated. All horses were allowed load-bearing immediately after surgery and underwent controlled strenuous exercise on a high-speed treadmill starting at 4 months postoperatively. Three animals were euthanized at 9 months and the remaining 3 at 18 months post-operatively. Arthroscopic examinations were performed at 3, 6 and 9 months for all animals and at 12 and 18 months for those 3 animals sacrificed at 18 months. Repair tissue was evaluated by MRI, histology, and immunohistochemistry. Results Arthroscopically, all MOC and COC retained smooth surfaces and were stiff when probed. MOC maintained the surface level better with the surrounding native cartilage than COC, which could be improved with a better clearance ft. Both MOC and COC performed better histologically than FOC treatments. Subchondral bone cysts were observed for FOC, but not for MOC and COC. Type II collagen and aggrecan were labeled in all repair tissue. Conclusions Durable cartilage repair was achieved by implantation of both MOC and COC grafts after 18 months, even with immediate load-bearing of the grafts., Introduction Osteochondral lesions of the ankle result from rotational injuries of the ankle in athletes. The purpose of this study is to evaluate the results of arthroscopic treatment of ankle osteochondral lesions and to define the clinical and arthroscopic factors influencing prognosis. Methods and Materials From 1993 to 2002 a total of 108 patients underwent 132 arthroscopic procedures for diagnosis and treatment of osteochondral lesions of the ankle. The evaluations included a pre and postoperative clinical scoring, radiographic, CT and MRI of the ankle. Operative techniques included: microfructures technique, fixation of the lesions using polylactic acid “Biofix” rods, diagnostic arthroscopy followed by cartilage lesions shaving, retrograde drilling of the lesion to the subchondral bone (12 patients) bone graft filling of subchondral cysts and osteochondral autografts. Results Traumatic etiology of the lesions was found to be associated with postero-medial Talar lesions. Significant clinical and radiographic improvements comparing pre and post operative CT and clinical score. Clinical improvements where found to correlate directly with CT grading. Tibial and Talar “kissing lesions” correlated with poor pre operative clinical score. Lesions with sclerosis and cyst on CT, appeared to have less clinical improvement with surgery. Conclusions Ankle x-rays and CT plays a limited role in planing the intra-operative procedure. Findings like sclerosis and subchondral cyst carry less favorable prognosis. Arthroscopy is a valuable tool for evaluation and treatment of ankle osteochondral lesions. The operative technique should be selected according to arthroscopic findings and the surgeon should be prepared to tailor the different types of treatment to each lesion., Introduction We report on the arthroscopic MACI technique for the treatment of chondral defects of the ankle. This bioscaffold can be implanted into the defect using exclusively fibrin glue, allowing the possibility to perform the procedure in articular sites, in which putting stitches for the periosteal patch is impossible. Methods and Materials Five patients suffering from large (⇢1.5cm2) cartilage lesions of the ankle, underwent MACI procedure. The implantation procedure was performed through traditional arthroscopic portals. After the debridment, the membranes were introduced through a cannula and spreaded onto the lesions by a probe. Keeping the correct graft orientation, the seeded membranes were then fixed with fibrin glue at optimized thrombin dilution, temporarily excluding water flow. Clinical outcomes were assessed by ICRS evaluation package. The graft integration was evaluated by MRI at 6 and 12 months after surgery. Results Filling of the defect was shown 12 months after surgery by MRI, which showed in 4 cases out of 5, a hyaline-like cartilage signal. Improvement was found subjectively and in ankle function levels at 12 months after the operation. At 18 months after implantation a biopsy was arthroscopically obtained from one patient. It showed smooth articular surface, hyaline-like matrix cartilage, cluster cell distribution and a nearly normal tide-mark. Conclusions Clinical improvement and hyaline-like appearance of the repair tissue suggest that arthroscopic MACI implantation is an effective technique for the treatment of large lesions of the articular cartilage of the talus. The arthroscopic approach allowed an optimal view of the lesion, avoiding arthrotomy or malleolar osteotomy., Introduction The aim of this study was to explore the feasibility of T2 mapping after matrix associated autologous chondrocyte implantation with Hyalograft C in the ankle joint. Methods and Materials Ten patients (25.5 ± 5.3 years) were included in the study. Mean defect size was 1,31 ± 0,37 (0,8–2) cm2 and the mean follow up period was 4.1 ± 1,8 (1.5–7.25) years. All defects were singular and located on the talar dome. MR examinations were performed on a 3T MR unit with a flexible coil using a multi-echo spin-echo technique. T2 maps were obtained using a pixel wise, mono-exponential non-negative least squares (NNLS) fit analysis. Region of Interest (ROI) analysis was carried out for the repair site and reference region of intact cartilage for comparison. T2 of the repair sites and of the cartilage reference regions was statistically analysed with a double-tailed, unpaired t-test. Results Global T2 values of the repair tissue (mean 30 ms, ± SD 8) were significantly lower (p = 0.03) compared to global T2 values of native cartilage (36 ms, ± 9). Conclusions High resolution capabilities at 3.0T make T2 mapping feasible in the ankle and demonstrated a slight difference in collagen and water content between Hyalograft C repair tissue and healthy hyaline cartilage., Introduction Studies have shown encouraging early results of Autologous Chondrocyte Implantation (ACI) in the ankle. We present a case series with clinical results up to 10 years. Methods and Materials 23 patients who had chondral or osteochondral defects of the talar dome underwent treatment with ACI between June 1998 and September 2005. Chondrocyte harvest was taken from the ipsilateral knee. Patients were evaluated prospectively using the Mazur ankle score and Lysholm knee score pre-operatively and at yearly intervals. Arthroscopic and histological evaluation was performed at 1 year. Results 16 males and 7 females were treated with a mean age of 37 years (17–61 years.) Median follow-up was 6 years (3–10 years.) Mean pre-operative Mazur ankle score was 54.0. At 1 year, 2 years and last follow-up, mean ankle score was 63.7, 68.0 and 66.5. Mean preoperative Lysholm knee score was 92.3. At 1 year, 2 years and last follow-up, mean knee score was 87.1, 85.4 and 89.4. Conclusions The good early clinical results of ACI in the ankle are maintained at longer term follow-up. Harvesting chondrocytes from the ipsilateral knee may lead to a reduction in knee score at early follow-up, but in general this recovers towards baseline in longer follow-up. Currently we are taking chondrocyte harvest from the ipsilateral ankle in order to eliminate any potential morbidity in the knee., Introduction The treatment algorithm for osteochondral lesions of the talus (OLT) remains controversial. We present a prospective case series of 42 patients with OLTs surgically treated between 2000–2006 using a new algorithm based on the ICRS or international cartilage repair society classification of cartilage defects and the size of the defect. Methods and Materials A total of 42 patients with an average age of 34.9 years (SD 12.1) who had failed previous non-operative or operative intervention were prospectively assigned into 3 groups (based on the size and the depth of the lesion) to receive either drilling (N=25; antegrade or retrograde), Single plug osteochondral autograft (N=8) or mosaicplasty / allograft reconstruction (N=9). Approximately 54.8% (23/42) were female, and 78% (32/42) reported antecedent trauma. Mean follow-up was 18 months and median duration of symptoms was 24 months. Results There was a statistically significant improvement (p ⇠ 0.0001, Wilcoxon Signed Rank test) in American Orthopaedic Foot and Ankle Society scores (average 24.8 points), Visual Analog Scale (VAS) Function scores (average 3.51 points), and decrease in VAS Pain scores (average 4.49 points) after surgery regardless of the procedure. 78.9% reported satisfaction with their treatment. Age, BMI (body mass index), duration of symptoms and length of follow-up did not correlate with the outcomes of post VAS Pain, VAS function, or AOFAS. Conclusions Using a combination of drilling, autograft plugs and allograft is successful in treating talus osteochondral lesions. Our treatment algorithm based on the ICRS staging of cartilage defects produced results comparable to published data., Introduction Qualitative evaluation of postoperative outcome in cartilage repair techniques is an issue due to morbidity of bioptic second look. T2 mapping is becoming increasingly popular in the knee, providing information about the histological and biochemical contents of healthy or reparative tissue. Nevertheless, little is known about its applicability to the ankle due to thinner cartilage layer. Aim of this study was to investigate the validity of T2 mapping in ankle cartilage characterization. Methods and Materials 20 healthy volunteers and 30 patients affected by osteochondral lesions of the talus and previously treated by different cartilage repair techniques, were evaluated by T2 mapping. Reparative procedures included microfractures, Autologous Chondrocyte Implantation (open or arthroscopic) and Bone Marrow Derived Cell's Transplantation. DPFSE with or without fat suppression, T2FSE with or without fat suppression, 3D SPGR and T2-Mapping using a dedicated phased array coil and 1.5 T MR scanner were used as MRI acquisition protocol. MRI results were correlated with clinical score (AOFAS) in the cases who received a cartilage reconstruction treatment. Results A statistically significant correlation (p⇠0.05) was shown between MRI and clinical results. A reduced mean T2 value, suggestive for fibrocartilage features, was shown at repair sites in microfractures, whereas no significative differences with healthy hyaline cartilage mean T2 value, were found in other repair techniques with good to excellent clinical score. Conclusions T2 mapping demonstrated to be adequate in characterizing cartilage tissue in the ankle. Further studies are required to implement a tool which may over time be a valuable noninvasive alternative to bioptic evaluation., Introduction Synthetic osteochondral grafts offer advantages over other treatments for osteochondral defects as they are single procedures with no donor site morbidity. Tru-fit™ is a biphasic osteochondral biodegradable graft consisting of a matrix of calcium triphosphate and the cartilaginous substitute of poly DL-lactide-co-glycolide. Methods and Materials Five patients with traumatic osteochondral lesions of the talus and one patient with a lesion of the distal tibia were treated with Tru-fit™ osteochondral plugs. All had failed previous arthroscopic debridement and microfracture and had a cystic bone defect beneath the fibrocartilage layer. Four patients required medial maleolar osteotomy for access. The superficial fibrous cartilage was removed from the defect and cores of subchondral bone were made to accommodate the Tru-fit™ plug. The patients were kept non weight bearing for 6 weeks but mobilised their ankles from day 2. Results At 12 months the postoperative AOFAS, AOS and SF36 scores had significantly improved and all patients were satisfied. There were no surgical complications but one patient injured his peroneal tendons playing football at 9 months post operatively. All patients returned to work and sports. Follow up MRI scans demonstrate good incorporation of the plugs with reduced surrounding bone marrow oedema but continue to show high signal within the plugs themselves. T-2 cartilage mapping demonstrates incorporation of cartilage but at 1 year the signal remains different from the surrounding hyaline cartilage Conclusions The preliminary clinical and radiological results of this procedure are encouraging with excellent improvement in symptoms but more patients and longer follow up are required before firm conclusions can be drawn., Introduction Microfracture is a frequently used technique for the repair of cartilage defects. Here, we introduce a cell-free cartilage implant used for covering of defects pre-treated with microfracture (Mfx) that attracts mesenchymal progenitors (MSC), improves defect filling and leads to a more hyaline-like repair tissue compared to Mfx. Methods and Materials The implant based on a resorbable polyglycolic acid scaffold, hyaluronan and serum attracts MSC as evaluated using a 96-well chemotaxis assay. Cartilage repair tissue formation was shown in high-density MSC cultures after stimulation with hyaluronan and in an ovine joint defect model by gene expression analysis and immunohistochemistry. Clinically, the cell-free implant was used for treating a traumatic and a degenerative articular cartilage defect. Cartilage repair was evaluated by MRI at 12 month after implantation. Results Human serum from whole blood significantly recruits MSC. Gene expression analysis showed that hyaluronan supports the induction of chondrogenic marker genes. In the ovine model, at 6 months, the repair tissue formed by the cell-free implant was hyaline-like and showed a cartilage matrix rich in type II collagen. Clinically, at 12 months, the traumatic and the degenerative defect showed a good to excellent defect filling with a hyperintense repair tissue signal and smooth peripheral integration. Conclusions Covering of cartilage defects with the cell-free implant after Mfx treatment improves repair tissue formation by guiding MSC into the defect and supporting chondrogenic differentiation. First clinical results suggest that defect covering with a resorbable polymer-based implant may be a good treatment option in microfracture., Introduction From 2004 we used MACI for treating focal chondral defects of the knee. We reviewed the two-year outcomes with clinical evaluation, second-look arthroscopy and core biopsies. Methods and Materials From March 2004 to August 2006, 132 consecutive knees with focal chondral defects (grade III or IV by modified Outerbridge) were treated with MACI technique. An initial arthroscopy was carried out. After one month we implanted cells with a parapatellar arthrotomy approach. A standardized postoperative rehabilitation protocol was carried out. We reviewed 43 patients with 3 yrs follow-up with clinical evaluation, ICRS and IKDC evaluation form. A second-look arthroscopy and core biopsies were performed in 8 of them (12 months follow-up). Results Improvement in mean subjective IKDC score from pre-operative (36.5) to 12 months (57.2) post-MACI were observed. Knee function levels also improved (88 % ICRS III/IV to 64.2 % I/II) from pre-operative period to 12 months post-implantation. Objective pre-operatively IKDC score of A were observed in 2%, score B in 87% and score C in 11%. This improve to 93% score B and 7% score A at 36 months post-implantation. Second-look arthroscopy carried out in 8 knees demonstrated all grafts to be normal / nearly normal based on the International Cartilage Repair Society (ICRS), and core biopsies demonstrated 3 (43%) grafts to have hyaline / hyaline-like tissue, 4 (57%) hyaline-like tissue / fibrocartilage; Conclusions Improvement in clinical, second-look arthroscopy and core biopsies indicate that the knees treated with MACI had improved function and focal defects resurfaced., Introduction Both, matrix-assisted chondrocyte transplantation (MACT) and osteochondral autograft transplantation (OCT), are applied for treatment of articular cartilage defects. While previous clinical studies have compared the outcome between these procedures, there is no information investigating the respective ultrastructural composition using T2 mapping comparing cartilage T2 values of the repair tissue (RT). Methods and Materials A total of 18 patients that underwent MACT or OCT for treatment of cartilage defects at the knee joint (9 MACT, 9 OCT) were matched for gender (1 female, 8 male pairs), age (33.8), BMI (28.3), defect localization, and postoperative interval (41.6 months). T2 assessment was accomplished by T2 maps, while the clinical evaluation included the Lysholm and Cincinnati knee scores, a visual analogue scale for pain (VAS), the Tegner activity scale, and the Short Form-36. Results Global T2 values of healthy cartilage (HC) were similar among groups, while T2 values of the RT following MACT (46.8 ms, SD 8.6) were significantly lower when compared to RT T2 values after OCT (55.5 ms, SD 6.7) (P ⇠ 0.05). MACT values were also lower in comparison to HC (52.5 ms, SD 7.9) within MACT patients (P ⇠ 0.05), while OCT values were higher compared to HC (49.9 ms, SD 5.1) within OCT patients (P ⇠ 0.05). Only the Lysholm score revealed a significant difference in the clinical outcome (MACT 77.0, OCT 66.8; P ⇠ 0.05). Conclusions These findings indicate that MACT and OCT result in a different ultrastructural outcome, which is only partially represented by the clinical picture., Introduction Various approaches have been proposed to treat articular cartilage lesions; nevertheless opinions on indications and clinical efficacy of these techniques are still controversial. In this prospective clinical study our goal is to evaluate second generation ACI technique for treatment of femoral condyles cartilage lesions at a medium term follow up. MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. Methods and Materials Prospective evaluation of 40 cases with full-thickness knee chondral lesions (mean value 2,5 cm2) treated with arthroscopic Autologous Chondrocyte Implantation technique. In 23 patients associated procedures were performed, whereas 15 patients had undergone previous surgery. At 5 years follow up ICRS form, Tegner were used for clinical evaluation. All the patients were also evaluated with MRI and analyzed with MOCART score. Results ICRS objective evaluation showed 90% of patients rated good and excellent results at 5 years follow up and the subjective score (mean value 83) demonstrated a marked improvement from preoperative to 5 years of follow up. Also the Sport activity level evaluated with Tegner score showed a statistical significant improvement at 5 years follow up (5). The mean total MOCART score obtained at 5 years was 73,5, with a significant correlation with the clinical outcome. Conclusions This arthroscopic surgery appears to be a valid solution for treatment of grade III-IV cartilage defects. MRI is reliable, reproducibile and accurate tool for assessing cartilage repair tissue., Introduction The objectives of this study was to evaluate the clinical outcome and the safety of BIOSEED-C in consecutive patients treated for symptomatic articular cartilage defects of the knee joint at different times −2weeks, 1, 3, 6, 12, 24 months-post implantation Methods and Materials 50 patients −57% males, 14.3% females with mean age 35.18 years-were followed for 24 months. The defects were localized on femoral condyles (62%), patella (28%) and troclea (10%); 52 % had simple lesions, 31% complex and 17 % salvage procedure. Mean defect size was 4.6 in simple, 4.8 in complex, 9.3cm2 in salvage lesions; 56% of defects were on the femoral condyle, 4.4% in patella, 20 % in tibia plate and 20% in the troclea. The clinical outcome was determined with standard scores (ICRS-IKDC) and 1.5T high resolution magnetic resonance imaging (MRI) by the MOCART score. Results IKDC objective scoring system at 24 months showed a normal or nearly normal knee in 86.2%.; IKDC subjective knee evaluation was 84%; In MRI evaluation 24 months after implantation a complete filling of the defect was found in 78% and a complete integration of the border zone to the adjacent cartilage in 83% with intact subchondral lamina and bone present in 85%. No product-specific adverse events were recorded. We recorded no graft failures, but three cases of delamination have been noticed. Conclusions Based on the results obtained, we conclude that BIOSEED-C® is a successful and safe therapeutic option for the treatment of cartilage lesions of the knee., Introduction Osteonecrosis of the knee is a serious potential complication of corticosteroid therapy with limited treatment options in young, active patients. We report on outcomes of fresh osteochondral allografting for high-grade steroid-associated osteonecrosis of the femoral condyles. Methods and Materials Between 1984–2006, osteochondral allografting was performed in 19 patients (25 knees) with symptomatic modified Ficat stage 3/4 osteonecrosis secondary to corticosteroid therapy; 13 females, 6 males with mean age of 27.8 years (range16–68). 5 had bilateral surgery (total 22 knees). 16 knees had unicondylar (12 lateral, 7 medial), 6 bicondylar lesions. 11 (44%) had previous surgery (average 1.5, range 1–5). Clinical evaluation included modified D'Aubigne-Postel (18-point), IKDC pain, IKDC function, Knee Society (KS) function scores. Subjective outcome measures included questionnaires evaluating pain, function, and satisfaction. Results Mean graft surface area was 11.0 cm2 (range 5.3–19.0). 15/25 (60%) required additional bone grafting. Mean follow-up was 67.1 months (range 25–235). 5 patients had further surgery, 24/25 had avoided arthroplasty, graft survival rate was 88% (22/25), 16 (73%) of which were considered successful (18-point score ≥ 15). Mean score improved from 11.0 to 15.7 (p⇠001), mean IKDC pain score from 7.3 to 3.0 (p⇠0.05), mean IKDC function score from 3.5 to 8.1 (p⇠0.05), mean KS function score from 58.6 to 90.0 (p⇠0.005). 10 patients completed questionnaires; all reported improved pain, function and overall satisfaction. Conclusions Fresh osteochondral allografting led to significantly improved pain and function, and avoided arthroplasty, in a majority of patients. Osteochondral allografting is a reasonable salvage option or management strategy for osteonecrosis of the femoral condyle., Introduction Despite the improvement in the treatment procedures, articular cartilage lesions remain affected by long recovery time that preclude the athlete competition for long time. The rehabilitative challenge is to optimize the achievement of the goals without jeopardizing the graft vitality. In this prospective comparative study we evaluated the results in a group of athletes treated with an intensive rehabilitation protocol after second generation arthroscopic autologous chondrocyte implantation (Hyalograft C). Methods and Materials 31 competitive male athletes with grade III-IV cartilaginous lesions of the medial or lateral femoral condyle or trochlea were evaluated at 1, 2 and 5 years follow up. We also compared the results obtained to a homogeneous group of 34 non-sportive patients. All the 31 sportive patients followed the intensive rehabilitation protocol, but 11 were also treated with isokinetic approach and rehabilitation on field. Results When comparing the 2 groups, a higher improvement in the athletes group was achieved at 5 years follow up (p=0.037) in the EQ-VAS and in the IKDC subjective evaluation either at 12 months and at 5 years of follow up (p=0.001, p=0.002, respectively). Analyzing the resumption of sports activity, 80,6% of the athletes returned to previous activity level in 12.4 months (s.d. 1.6); patients treated with isokinetic approach and rehabilitation on field had faster recover and an even earlier return to competition (10.6 months, s.d. 2.0). Conclusions For optimal results, ACI rehabilitation needs to not only follow but also to facilitate the process of graft maturation. A proper rehabilitation may safely allow a faster return to competition and influence positively also the clinical outcome at medium term follow up., Introduction The chondrocyte's cells culture provides within few weeks autologous cartilage in 3 dimensions, in carrier fleeces of 6cm 2 with 2mm thickness. The carrier fleeces are resistant with a certain degree of elasticity. We can cut them and fix them on the sub-chondral bone. Methods and Materials This technique has been applied for over 5 years in the arthrosis of the knee on 20 patients in 34 sites. In 11 cases kissing lesions were treated. The technique used is the one of BioSeedC: preparation of the graft bed and a press-fit fixation of the graft with trans-bone pulley threads, auto-blocked in the channels by knots. In 5 cases a patella re-alignment was performed and in 8 cases a HTO. 1 case of astragalo-tibial graft has been realised. 2 meniscus grafts have been associated, one at the same time and the other one deferred. Results A MRI control has been systematically performed around the 8th month allowing the demonstration in all cases of good growth and fixation of the graft. arthroscopic controls have shown a good cartilage; one biopsy of the grafted zone has histologically shown a normal hyaline cartilage. The clinical score was clearly improved from the 6th month except in 2 cases. A good result in the ankle is encouraging, avoiding the subastragalar arthrodesis. Conclusions The middle term results of the autologous 3D chondrocyte graft “BioSeed -C” in the arthrosis of the knee are encouraging as they show a good cartilage growth and fixation of the graft., Introduction The Cartilage Autograft Implantation System (CAIS) is being developed as an alternative articular cartilage surgical treatment, which provides a chondrocyte-based repair in a single surgical procedure. The CAIS technique first uses a proprietary device for the preparation of morselized, autologous cartilage uniformly dispersed on a 3-dimensional scaffold. This construct is then fixed in the articular cartilage defect. Preclinical studies show that the embedded chondrocytes migrate onto the scaffold and form hyaline-like matrix. Methods and Materials Two prospective, randomized pilot clinical studies are underway: one in the US and the other in Europe. Both studies were designed to assess safety and initial performance of CAIS. Twenty-nine (29) patients at 5 investigative sites were randomized in a 2:1 schema (CAIS:microfracture) in the US and 24 patients at 5 enrolling sites in Europe. Subjects were clinically evaluated, including MRI, and asked to complete clinical outcome questionnaires. Short-term follow up occurred at 1 & 3 weeks and 2, 3, 6, and 12 months post-surgery with long-term follow-up scheduled every 6 months up to 4 years. Results Preliminary data from both studies indicate that CAIS is safe at 18 months in the US study and 6 months in the European study. The CAIS device has demonstrated safety and improvement over baseline in treated subjects. Safety and clinical outcome results will be reported through 18 months from the US pilot Study and 6 months from the European Pilot Study. Conclusions The instrumentation enabled the successful preparation and fixation of a minced autologous cartilage tissue-loaded implant in a single intraoperative setting., Introduction The purpose of this study was to compare the outcomes of microfracture (MF) and ACT Bioseed-C (C) procedures for the treatment of the articular cartilage defects of the knee joint in active professional athletes. Methods and Materials Between the 2004 and 2006, a total of 49 professional athletes with a mean age of 25.8 years (14 to 36) and with a symptomatic focal lesion of the articular cartilage in the knee underwent microfracture or ACT-BIOSEED-C procedure. There were 20 athletes in MF group and 29 athletes in C group. The mean follow-up was 2 years (range 6ms to 4 years). Patients were evaluated by ICRS, MOCART, KOOS scoring. Follow up examination have been made after six month, and every year after surgery. Results The ICRS functional and objective assessment showed that69% after microfracture had excellent or good results compared with91% after ACT (C). In 1, 2, 3 and 4 years after the operationsthe ICRS did not showed statistically significant differences betweenthe groups (p=0.453). 58% of athletes following MF and 64% athletesfollowing C returned to sports activities at the pre-injury level atan average of 7.5 months after the operations (NS), but only 37%after MF compared with 46% after C procedure maintained sportsactivities at the pre-injury level after 4 years. Conclusions At 4 years follow-up, our prospective clinical study in athletes has shown significant worst outcome of the microfracture vs ACT for the repair of articular cartilage defects in the knee., Introduction Bipolar chondral lesions are generally considered a contraindication to cartilage repair techniques, leaving few treatment options besides arthroplasty. We report on outcomes of fresh osteochondral allografting as a salvage option for bipolar cartilage injuries of the knee. Methods and Materials Between 1984–2006, fresh osteochondral allografting was performed in 52 patients (54 knees) with bipolar chondral lesions of the knee. Clinical evaluation included an 18-point scale, post-operative Knee Society function score, and reoperation. Subjective outcome measures included patient questionnaires evaluating pain, function and satisfaction. Results 25 males, 29 females had a mean age of 39 years (range 15–66). 39 lesions involved the tibiofemoral, 15 the patellofemoral joints. Patients had an average of 3.4 previous surgeries (range 1–8). Mean allograft area was 18.2 cm2. Mean follow-up was 78 months (range 24–236), 7 patients were lost to follow-up. 33 patients had further knee surgery; 4 had repeat allografts, 15 underwent conversion to arthroplasty (2 partial and 13 total) at a mean of 41 months (range 6–119). 89% of the surviving grafts were considered successful (18-point score ≥ 15). Mean 18-point score improved from 11.9 to 16.2 (p⇠0.001). Mean post-operative Knee Society function score was 82 (range 45–100). 24 patients completed questionnaires; 88% were satisfied, 92% reported less pain, and 96% reported improved function. Conclusions Fresh osteochondral allografting successfully avoided arthroplasty in 72% of knees and resulted in significantly improved function, reduced pain, and high patient satisfaction. Fresh osteochondral allografts are a reasonable salvage option in the challenging patient population presenting with symptomatic bipolar cartilage lesions of the knee., Introduction The purpose of this study is clinical evaluation of a novel II generation autologous chondrocyte implant, BioCart™II for cartilage repair. Methods and Materials BioCart™II is a matrix-assisted, fibrin hyaluronic acid-based implant containing autologous chondrocytes propagated with a unique growth factor to maintain their chondrogenic potential. The scaffold enabling a three dimensional distribution of the cells. 20 implantations in 18 patients aged 17–50, with cartilage lesions 15 in the knee and 3 in the ankle, diagnosed by CT MRI and/or arthroscopy underwent biopsy from the intercondylar notch and from the distal margin of the talus respectively. Two to three weeks later, BioCart™II was implanted through a small longitudinal parapatellar incision or a longitudinal antero-medial incision of the ankle. Deep lesions were treated with two layers. Rehabilitation included 3 weeks of non-weight bearing and CPM, followed by 3 weeks of pwb. Full activities were resumed at 4–6 months and follow-up was 10–36 months. Results At diagnosis all patients scored under 4 points in the subjective ICRS questionnaire improving to over 6 post operation. The IKDC score improved from grades C and D before the operation to A or B post operation. Second look arthroscopy and biopsy on two patients, showed excellent coverage, full integration and new hyaline-type cartilage. Post operative MRI on 10 patients showed good integration of the graft with bone edema in some. Conclusions BioCart™II is safe, effective and user-friendly both for the patient and the surgeon. The short time from biopsy to implantation and good to excellent clinical outcome further encourage the continued use of this technique and product., Introduction Hyaluronan was introduced successfully in 1998 to transplant cultured chondrocytes in cartilage defects: this study presents the first ten years clinical result using the Hyalograft technique Methods and Materials 290 patients were treated with Hyalograft C in the knee joint in the last decade (1998–2008): 72.4% were male; 27.6 % had simple lesions, 31% complex and 41.4 % salvage procedure. Mean defect size was 4.9 in simple, 4.8 in complex, 9.3cm2 in salvage lesions. 55.6% cases showed defects on the femoral condyle, 4.4% had patellar defects, 20 % tibial coin lesion and 20% had a troclear defect. Clinical follow up was performed at 1,3,5 and 7/10 years using the ICRS-IKDC scores as well as MOCART score. EuroQol EQ-5D evaluated the patient quality of life. 2nd look biopsy have been made in 26 pts. Results At 7-years follow-up subjective IKDC mean Score passed from preop. value of 34.3 to 79.3; 84% of patients experienced a significative improvement in knee function and symptoms: better results have been evaluated in simple (100%) and in salvage lesions (81.8%); 91 % of treated knee classified as normal or nearly normal. None Hyalograft-C related adverse event have been found; we had found 30 graft failures due to graft delamination, insufficient regenerative cartilage, disturbed fusion; 11 patients resorted to TKA due to progressing osteoarthritis. Conclusions Hyalograft-C is a useful treatment option for cartilage defects with good outcome that is actually comparable to other ACT. In patients with osteoarthritis critical indication should take place., Introduction The aim of this study was to develop a technique to decellularize a porcine cartilage-bone construct with a view to using this as a biological scaffold for transplantation into human osteochondral defect. Methods and Materials Decellularization was based on a modification of the basic technique of Booth et al (2002). Cartilage-bone matrix (n=9) were decellularized by exposing the tissue to freeze-thaw cycles. This was followed by subjecting the tissues to hypotonic (10mM tris-HCl, pH8.0) buffer, ionic detergent (0.1% [w/v] sodium dodecyl sulfate [SDS]), in the presence of protease inhibitors. This was followed by incubation in nuclease solution containing DNase and RNase. Fresh and decellularized cartilage were compared histologically using hematoxylin and eosin, sirius red and alcian blue staining. Immunohistochemistry staining for galactose-α-1,3-galactose (α-gal), collagen I, II and VI was performed for fresh and decellularised samples. The biochemical composition of fresh and decellularised cartilage was examined. This included DNA, hydroxyproline and DMB sulphated sugar assays. The biphasic property of fresh and decellularized cartilage was determined using a pin on plate indentation test. Results H&E staining revealed absence of visible whole cells. Analysis of DNA revealed the absence of genomic DNA in comparison to fresh tissues. The decellularisation process had minimal effect on the collagen content of the cartilage. Nevertheless there was a significant loss of GAG following decellularization. The biomechanical testing showed a significant change in comparison to the fresh cartilage. Conclusions In conclusion, this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair., Introduction To assess P-ACI for articular cartilage lesions of the knee, a study was carried out on patients with minimum 5 year follow up with clinical assessment and second look arthroscopy when indicated. Methods and Materials Between October 2000 and August 2003 the author carried out P-ACI on 116 patients, 120 knees, 168 lesions included in this study. There were 88 single, 34 double and four triple lesions. Lesion aetiology was traumatic in 93, degenerative in 65 and OCD in 17. Previous surgery was frequent, predominantly arthroscopic debridement (78), meniscal surgery (65), arthroscopic microfracture (19) and ACL reconstruction (13). Results Results were assessed using the ICRS Cartilage Repair Evaluation Package 2000. Significant improvement was seen in Activity Level, Objective Knee Examination and SF-36 Physical and Mental Component Scores. IKDC subjective assessment improved by average 29.6 points. “Second look” arthroscopy was carried out on 79 knees with 109 lesions at average 26 months predominantly for graft hypertrophy/extrusion presenting as painless mechanical symptoms. “Third look” arthroscopy occurred in 37 knees with 55 lesions at average 44.4 months from index implantation. “Fourth look” arthroscopy occurred in 10 knees with 14 lesions at average 59 months from index implantation. Four for advancing degenerative change requiring TKR. ICRS VCRA was predominantly recorded Grade II at subsequent arthroscopy. Conclusions This study demonstrates good clinical outcome and satisfactory repair when assessed arthroscopically. Subsequent arthroscopic surgery was frequently required, predominantly related to the periosteal patch in the first year, after which progressive degenerative change became more significant., Introduction The matrix-associated autologous chondrocyte transplantation (MACT) for articular cartilage defect healing has been clinically applied with a variety of 3D-scaffold types. The present study shows the differences between four different MACT-matrices (scaffolds). Methods and Materials Four transplant-types (Biogide(R), CaReS(R), Novocart(R) and Hyalograft(R) C) were analysed at the time of transplantation (t0) and after four further weeks (t = 4w) of in vitro culture. Histology, electron microscopy and molecular biology were used to evaluate the cell distribution, phenotype of the chondrocytes (differentiated or fibroblast-like), cell-scaffold interactions, cell vitality and metabolic activity. Results In dependence of the architecture of the scaffold type, chondrocytes showed very different distribution patterns at t0; differences were also evident between the patients. In the course of four weeks of prolonged cultivation the cells proliferated and changed their distribution. Chondrocytes showed high vitality and metabolic activity in all transplant materials, especially at the 4w stage. Chondrocyte gene expression indicated a tendency of increased collagen type II synthesis after four weeks of in vitro culture. The sites of differentiation varied according to the carrier materials but were generally located in matrix-rich cell accumulations. Conclusions The scaffold architecture influenced the cell distribution while seeding as well as the movement of the cells on the scaffold, and has therefore influence on the cell distribution in the defect. The increase in collagen type II seems to be initiated by the 3D-conditions in matrix-rich cell accumulations. The tendency towards differentiation in vitro presents an indication for successful cartilage regeneration in vivo., Introduction Histological features of successful autologous chondrocyte implantation(ACI) have been widely reported, but the repair characteristics of revised and clinically failed grafts are not well understood. Methods and Materials We examined 39 cases of revised MACI and patients received total knee replacement after MACI for the mechanical properties, histology and the matrix profiles of repair biopsies. Results Histologically, the majority of these biopsies (n=39) were hyaline-like (HLC) and fibrocartilage (FC) in both the revised (30% and 38% respectively) and failed (34% and 22% respectively) cases. Compositionally, more revised cases were positive for aggrecan, collagens VI and IX, and Ki67 compared to failed cases, but not quantitatively different (P⇢0.05). More HLC biopsies were positive for aggrecan and collagen II (compared to the FC group), with diffuse and often colocalized matrix distribution. The majority of H LC biopsies stained positive for Sox-9, whereas FC cases were negative. Most (75%) FC biopsies were positive for Ki-67, compared to the HLC group with 25%. MMP-13 was negative in all biopsies. Qualitatively, reduced collagen II and IX, and increased Ki67 production was noted in FC biopsies (P⇠0.05). An intact repair site showed FC with 30% greater stiffness in the inferior portion compared to the superior, with an associated proteoglycan content increase. Conclusions Revised and failed biopsies display predominantly hyaline-like and fibrocartilage in repair type, are histologically dissimilar to healthy cartilage, but do not differ in composition. Hyaline-like repairs show lower proliferation but improved matrix to fibrocartilage repairs. Our study furthers knowledge into failed and revised cartilage repair by MACI., Introduction The purpose of this study was to assess outcomes of autologous chondrocyte implantation (ACI) in the patellofemoral (PF) joint and determine a treatment algorithm for tibial tuberosity realignment and appropriate articular cartilage treatment. Methods and Materials Prospectively collected data of sixty-three patients who underwent an ACI of the PF joint between 2002 and 2006 were retrospectively reviewed. The primary endpoint was survivorship of ACI. Secondary endpoints included validated outcome scores and subsequent surgical procedures. Results Fifty-two (83%) patients with a mean age of 31.8 years were followed up at an average 4 years (minimum, 2 years). The mean lesion size was 4.19 cm2 and was located on the trochlea in over 50% of cases. Mean improvement in the preoperative to postoperative scores was significant (p0.05). Patients undergoing anterior medialization (AMZ) fared better than those without realignment. Fifty percent of patients with ACI needed a subsequent procedure. There were 5 clinical failures (9%), which were defined as progression to arthroplasty or conversion to osteochondral allograft transplantation. Conclusions ACI of the PF joint provides a significant improvement in symptoms and function in patients with or without a history of a failed prior cartilage procedure. Combined ACI with AMZ improves outcomes more than ACI alone. ACI of the PF joint is associated with a high re-operation rate and an appropriate surgical plan is necessary to successfully treat these patients., Introduction Cartilage lesions represent a significant clinical problem because of their limited ability to regenerate. Recent advances in our understanding of the functions of mesenchymal stem cells (MSC) have shown its chondrogenic potential. The use of autologous MSC represent an improvement on the currently available techniques for cartilage transplantation avoiding the first surgery for cartilage biopsy and chondrocyte cultivation. Methods and Materials We followed prospectively five patients with Gr.III or Gr. IV chondral lesions treated with standard arthroscopic approach and implanted with concentrated MSC from the iliac crest with or without the use of a scaffold. All patients followed the same specific rehabilitation program after MSC implantation. IKDC, KOOS, Lysholm and Tegner were collected at pre-op and every 6 months post-operatively. Results Five patients with a mean age of 46.6 have been treated in our institution in the last 2 years with this technique for grade 3 and 4 cartilage knee lesions. At a mean follow-up of 24 months, patient showed improvements in all scores. Mean pre-op values were IKDC subjective 53.74, KOOS Scores: P=83/S=87.2/ADL=86.6/SP54/ QOL= 44; Lysholm 74 and Tegner 4.5; at final follow-up mean scores were IKDC subjective 73.0, KOOS Scores: P=89.7/S=90.7/ADL=97/SP=71.7/QOL=75; Lysholm 85 and Tegner 6. No adverse reaction or post-op complication were noted in all patients. Conclusions This preliminary report shows that Mesenchymal Stem Cell Implantaion can be a viable technique in the treatment of Gr. III and Gr. IV chondral lesions., Introduction Retropatellar cartilage defects are difficult to treat. A regeneration of pristine hyaline cartilage is desirable but yet impossible. The AMIC-procedure (Autologous Matrixinduced Chondrogenesis) is an option to faciliate regeneration of substitute cartilage. These case reports document the successful cartilage regeneration in patients with chondral and osteochondral retropatellar defects where other treatment options have failed. The aim of this case control study is to evaluate the value of the AMIC-procedure in retropatellar cartilage defects. The operative procedures are introduced. Methods and Materials A case control study introduces 2 cases in which retropatellar AMIC was performed. One was a chondral, one an osteochondral defect. The follow up is 12 months. Pain reduction evaluating the Visual Analogue Scale (VAS) and range of motion of the affected joint were evaluated. Results In both cases a pain reduction in the VAS from 10 to 2 could be achieved. The Range of motion improved in flexion from 90 to 130 degrees. X ray and MRI document almost complete regeneration of bone and cartilage in both cases. Conclusions The AMIC-procedure was successfully carried out in these patients where other options had failed. Matrixinduced chondrogenesis may be an alternative in retropatellar chondral defects which are otherwise difficult to treat. Further studies are encouraged., Introduction Ideal treatment of osteochondral lesions of the talus (OLT) is still controversial. Aim of this study is to review the 10 years follow-up clinical and MRI results of open field Autologous Chondrocyte Implantation in the treatment of OLT. Methods and Materials From December 1997 to November 1999, 10 patients, age 25.8 ± 6.4 years, affected by OLT, received open field Autologous Chondrocyte Implantation. The mean size of the lesions was 3.1 cm2 (2.2 − 4.3). All patients were evaluated clinically (AOFAS score), radiographically and by MRI pre-operatively and at established intervals up to a mean follow-up of 119 ± 6.5 months. At the final follow-up MRI T2 mapping evaluation was performed. A bioptic sample was harvested in 5 cases during hardware removal 12 months after implantation. Results Before surgery the mean score was 37.9 ± 17.8 points, at 24 months it was 93.9 ± 8.5, while at final follow-up it was 92.7 ± 9.9 (p⇠0.0005). Bioptic samples showed cartilaginous features at various degrees of remodelling, positivity for collagen type II and for proteoglycans expression. No degenerative changes of the joint at follow-up were found radiographically. MRI showed well-modelled restoration of the articular surface. The regenerated cartilage showed a mean T2 mapping value of 46 msec with no significant difference compared to that of normal hyaline cartilage. Conclusions The clinical and histological results have confirmed the validity of the technique utilized with a durability of the results over time. T2 mapping was adequate in detecting the quality of the regenerated tissue coherently with the bioptic results., Introduction To compare the chondrogenic potential of mesenchymal stem cells (MSCs) from bone marrow or adipose tissue to cartilage-derived chondrocytes in pursuit of an alternative source for cell therapy for cartilage regeneration. The pluripotential nature of stem cells is widely considered to make them the ideal choice for any cell therapy. Autologous chondrocyte implantation, has shown much promise but remains a two step procedure requiring the harvest of cartilage followed by culture in vitro and subsequent implantation. This procedure has also not yet been indicated for osteoarthritis (a common cartilage pathology). Autologous or allogeneic stem cells could, theoretically, provide a single step “off the shelf” solution for regeneration of cartilage defects. Our study, (30 subjects), was designed to compare the chondrogenic potential of chondrocytes taken from healthy or osteoarthritic cartilage, with that of MSCs prepared from bone marrow of the same subject. MSCs isolated from human adipose tissue were also compared Methods and Materials Chondrocytes isolated from different areas of joints affected with osteoarthritis were compared to bone marrow MSCs from the same subject undergoing total knee replacement. Cells were isolated and expanded under optimized culture conditions including autologous serum, with or without a fibroblast growth factor (FGF) variant. Proliferative capacity, chondrogenic potential and proteoglycan production were analyzed. Results Our results indicate a clear advantage for FGF-directed cartilage derived chondrocytes, even from osteoarthritic joints over the MSCs from either source. Conclusions Chondrocytes under optimized conditions maintain a higher chondrogenic potential than MSCs and could serve as a legitimate source for cartilage repair in osteoarthritic patients., Introduction Arthroscopic debridement represents a relatively simple and effective treatment in the management of glenohumeral degenerative joint disease. The purpose of this study was to review the outcomes of patients who have undergone arthroscopic debridement procedures for isolated degenerative joint disease. Methods and Materials Patients that had an arthroscopic debridement performed by one of four surgeons at a single institution were retrospectively identified. All patients were confirmed to have significant (at least grade 3) articular damage from the operative report. Any patient with adhesive capsulitis or a concomitant rotator cuff or SLAP repair was excluded. Preoperative simple shoulder test (SST) and American Shoulder Elbow Score (ASES) scores were compared against postoperative outcomes with at least a 1 year follow-up. Postoperative Constant scores and range of motion were also recorded. Results Ninety patients were retrospectively identified and 72 (80%) were available for follow up. The average follow up was 27 months (12 − 90 months). Of the 72 patients, 17 (23.6%) went on to total shoulder replacement at an average of 10.2 months. In patients that did not progress to arthroplasty, SST scores improved from a mean of 6.0 preoperatively to 9.0 postoperatively (P ⇠0.001), ASES improved from 52.0 to 76.2 (P ⇠0.001). The average postoperative constant score was 72.4, and 80% of the patients stated that they were satisfied with the surgery and would do it again. Conclusions Arthroscopic debridement of the glenohumeral joint is an option that has the potential to delay more extensive procedures and provide a significant amount of pain relief., Introduction Autologous Chondrocyte Implantation in patellofemoral compartment has unpredictable outcome. Controversy arises when patellofemoral ulcer is a part of the tricompartmental disease. We conducted a study to correlate radiological findings with clinical presentations in patients with patella subluxation and knee pain. Methods and Materials A prospective study was conducted on patients presenting with knee pain or clinical subluxation of the patella. All patients underwent plain knee anteroposterior, lateral and skyline radiographs. Unenhanced axial ct scans of both knees performed at 0, 10o and 20o of flexion. Sulcus and Laurin's patellofemoral angles were measured. Tricompartmental knee arthritis was evaluated on radiographs. Results A total of 62 patients (124 knees) were included in this study. Patients were divided into 3 age groups; Group 1: 18 and below, Group 2: 19 to 40 and Group 3: above 40. Average age was 14.3, 24.9 and 48.9 respectively. Mean values of angles were calculated at 00, 100 and 200 of flexion. In Group 1, 83.3% had patella subluxation on CT. None had any radiological features of degeneration. In Group 2, 52.4% had patella subluxation on CT while 9.52% had degenerative changes. In Group 3, 47.8% had patella subluxation on CT, 56.5% had degenerative changes and 21.7% had cartilage ulcers. Conclusions In younger patients, patella subluxation was associated with anatomical abnormaltities such as trochlear dysplasia, while in older patients, it was part of the tricompartmental disease., Introduction Analysis of hyaline cartilage of the ankle joint is technically demanding, due to the high congruency and thin cartilage layer; therefore, biochemical MR techniques have been developed for quantitative analysis of articular cartilage. The purpose of the present study was to assess T2 and T2* mapping as well as Diffusion-Weighted-Imaging (DWI) for evaluation of cartilage and cartilage repair tissue. Methods and Materials Ten healthy volunteers (mean 32.4 years) and twelve patients after MACI of the ankle joint were included in the study. Morphological imaging was performed in order to identify the cartilage repair tissue. Biochemical imaging was performed at 3T by using a 2D multi-echo spin-echo sequence for T2 mapping, a 3D gradient-echo for T2*-mapping and a 3D partially balanced, steady-state gradient-echo pulse sequence with and without diffusion weighting for DWI. T2, T2*, and DWI values were assessed using a Region-of-interest (ROI) analysis. Results Quantitative assessment of hyaline cartilage of healthy volunteers (T2: 51.1±4,6ms; T2*: 16,6±3,7ms; DWI (diffusion constant): 1.27±0.16) and healthy seen control cartilage sites in patients after MACI (T2: 48.4±7.5ms; T2*: 16.1±4.1ms; DWI: 1.28±0.17) showed no significance difference (p=0.05). Within the cartilage repair tissue T2 and T2* values showed similar results (T2: 49.3±6.6ms (p=0.767); T2*: 16.9±4.2ms (p=0.505)), whereas DWI showed a significant increase (1.49±0.32 (p=0.039)). Conclusions High-resolution T2, T2*, and DWI techniques were performed in-vivo in a clinical feasible scan time, thus demonstrating new possibilities in the evaluation of articular cartilage on the ankle. Valuable parameters for the characterization of cartilage repair tissue could be gained., Introduction Better MRI imaging has improved the noninvasive evaluation of cartilage, however, arthroscopy is still considered the gold standard. Anecdotal evidence suggests that transplanted lesion size is commonly larger than expected by preoperative imaging. While not an issue with 1st generation ACI, where cells can be diluted to accommodate for larger lesions, 2nd generation procedures utilize membranes with size limitations. In light of these developments, we conducted a study comparing size estimates based on MRI and arthroscopy with final defect size at the time of ACI. Methods and Materials Patients were included if they had undergone MRI, arthroscopy and ACI within 18 months to avoid confounding by defect progression. A radiologist measured all defects on MRI; sizes recorded during arthroscopy and implantation were obtained from surgical notes, and compared with paired t-tests. Results 38 patients were enrolled with 53 defects of the condyles and trochlea; defect size averaged 4.5 cm2. Defect locations demonstrated significant trends towards larger sizes between MRI, arthroscopy and ACI. Final sizes were larger than the MRI estimates by 88% (p=0.02) and 95% (p=0.03) for the medial and lateral femoral condyles, and by 50% (p⇠0.001) for the trochlea. Conclusions Our study compared the size of cartilage defects measured by a cartilage radiologist based on high-resolution MRI, and compared this with surgical measurements by an experienced surgeon. Even in this best-case scenario, final defect sizes were significantly larger than those estimated preoperatively. Our findings can be used as a guideline when ordering biologic implants for cartilage repair, especially when using size sensitive membrane technologies., Introduction Aim of this study is the investigation of lower limbs biomechanics before and after meniscectomy. Methods and Materials Materials and methods: motion analysis of the knee before surgery, six months and one year after was performed on ten patients that underwent partial medial menisectomy. Ten healthy volunteers acted as a control group Data were acquired by means of Vicon motion analysis system Results In gait patterns investigation, joint kinematics does not show significant modifications before and 6 months after surgery, 12 months after surgery hip and knee show a greater flexion. The dynamic analysis showed alterations in sagittal moment. Before surgery the knee flexion moment is reduced. After partial meniscectomy the knee flexion moment increases in both the limbs. In squatting investigation, main focus was on repeatability. Before surgery high inter subjects variability affects knee joint angle; while after surgery high variability affects also hip and ankle. Conclusions After meniscectomy, gait and squatting patterns are still altered. Before surgery, the joint mechanical structure is not highly altered and modifications are mainly due to pain avoidance schemas; after partial meniscectomy, pain disappears and the new joint behaviours are probably caused by the new mechanical asset and/or proprioceptive mechanisms., Introduction Femoro-Acetabular Impingement (FAI) is a frequent clinical pathology in the young adult hip. Frequently, athletes are severely handicapped by this disease. FAI is due to mechanical abnormalities of the proximal femur or the acetabulum resulting in rapid focal destruction of acetabular cartilage, most frequently in the antero superior quandrant of the acetabulum. The purpose of this study was to simulate the mechanical effects of a loaded focal acetabular cartilage defect in function of cartilage thickness and ICRS-Classification in a Finite Element-Model. Methods and Materials In the Finite Element-Model. The following assumptions were made: - a rectangular chondral defect: 30mm × 10mm × 2mm in size - femoral head diameter: 50mm - cartilage defect depth: 2mm - ICRS Grades: o, I, II - Cartilage Elasticity Modulus: 18.8 MPa (Gradeo), 11.1 MPa (Grade I), 10.5 MPa (Grade II) -Specialattention was made to see if the femoral head touches the subchodral bone of the acetabulum in this size of chondral defect. Results The simulation shows different mechanical patterns in normal (healthy) cartilage (ICRS o) and degenerative cartilage (ICRS I, II). At 2500 N and 5000 N loading (Gait, Running), it is interesting to note that no femoral head contact with the subchondral bone of the acetabulum has been seen in any of the defects. At 20 000 N the femoral head hits the subchondral bone of the acetabular chondral defect. Conclusions In average weight patients a 3cm2 acetabular chondral defect cannot be considered as a severe lesion. However in heavy weight althletes these lesions have serious clinical implications., Introduction Previous clinical literature has shown that intraarticular (IA) injection of hyaluronic products are safe and effective for the treatment of hip osteoarthritis (OA), but the available casuistry is still numerically limited showing also a short follow up. Aim of the study: to assess the safety and efficacy of administering Jointex® (Chiesi SpA 800–1.200 KD, 8 mg/ml) on painful hip OA in a large cohort of patients for a long follow-up. Methods and Materials Adult, outpatients suffering from symptomatic hip OA (Kellgren & Lawrence Grade 1, 2 or 3) were injected with one syringe of 4 ml (2 vials) of Jointex® under ultrasound guidance, repeated after six months and when clinically necessary an adjunctive injection was performed. Patients were assessed at baseline and at every three months to 12 months for Lequesne index, pain (evaluated by VAS) and NSAID consumption (number of days patients assumed NSAID in the last month). Results 229 patients were enrolled, 99 M and 125 F, mean age 63. 62 had bilateral hip OA. 90 patients were followed up for 12 months, 415 injections were performed. No local or systemic adverse events were reported. A statistically significant reduction (p⇠0.05) was observed at all time points versus baseline for all assessment criteria: Lequesne index, pain VAS, NSAID consumption. Conclusions The data from our cohort study seem to demonstrate the long-term efficacy and safety of intra-articular ultrasound guided treatment with Jointex® in symptomatic hip osteoarthritis. They do, however, need to be confirmed by further data to be collected after longer follow-up times., Introduction Autologous chondrocyte implantation (ACI) has been used most commonly as a treatment for cartilage defects in the knee and there are few studies of its use in other joints. There is only one published report of its use in the hip. Is there a role for ACI of the hip? Methods and Materials We describe 14 consecutive patients studied prospectively with chondral or osteochondral lesions of the femoral head that underwent ACI and were prospectively reviewed with a follow up of 5 years (mean 30 months). 3 patients presented with Perthes and five with AVN. Six had chondral loss following trauma and one presented with an area of bone loss in a hip with congenital dysplasia. Defect size was a mean 6.2 cm2. Pre-operatively hip function was assessed by the patient using the Harris Hip Score and MRI. Post-operatively these were repeated at 1 year and hip scores repeated annually. Hip arthroscopy and cartilage biopsy provided cells for culture in a GMP laboratory where passage numbers were limited to two. Three weeks later by open surgery, all unstable cartilage was excised, the base was debrided or excised and bone graft applied, and suture of a membrane of periosteum or collagen membrane over the defect undertaken. A mean 5.2 million chondrocytes were inserted beneath this patch following a test of the seal. Results Ten of the fourteen patients improved at one year, with a mean rise in Harris Hip score from 57 to 63 points. Five patients underwent arthroscopic examination at one year and in four there was evidence of good integration of the new cartilage. In one patient arthroscopy was difficult due to previous trauma. One patient developed AVN as a post-operative complication following a posterior approach. Four patients have progressed to hip replacement or resurfacing but it is of note that all these patients had cyst formation pre-operatively. Conclusions The short-term results of ACI for osteochondral lesions of the hip suggest that if good early results are obtained they are observed to continue out to 5 years. There is a high failure rate in those with pre-operative cyst formation in the hip., Introduction Hemi-arthroplasty is a more conservative option used for elderly patients who have suffered femur neck fractures and femoral head osteonecrosis. This study investigated the friction and wear of cartilage in the natural acetabulum articulating against a metallic femoral head under constant load with different clearances. We hypothesise that the outcome of hemi-arthroplasty could be improved if the clearance is optimised. Methods and Materials Porcine acetabulums were dissected from 12-month old animal's hip joints and mounted in PMMA at 450 with respect to the loading, and then a silicon rubber replica was made to assess dimensions. The cobalt chrome alloy head diameters were 34, 35, 36, or 37mm with 0.008um roughness (Ra). Clearances were small (≤0.7mm), medium (⇢0.7 & ≤1.4mm), and large (−41.4mm). Two-hour 400 N constant load friction tests were conducted using a pendulum motion friction simulator with a flexion-extension motion of ±15° in 25% bovine serum. The friction coefficient (mefff) was measured throughout testing and wear damaged assessed visually at the end of the test and classed as zero, mild and severe. Results The friction coefficient (the friction factor at o° position) was not significantly different in testing with different clearances, and it was approximately 0.27 following 7200 cycles (range±0.025). However, if wear and friction were correlated: the friction coefficient of zero-wear group (mefff=0.20) was significantly lower (T test p=0.0037) than both mild and severe wear groups (mefff =0.28). Conclusions The friction of hemi-arthroplasty under constant load was dependent on both the clearance and the acetabulum geometry, cartilage is damage led to elevated friction., Introduction There is a controversy in the literature concerning the impact of full thickness cartilage lesions in ACL-injured knees. Methods and Materials Hypothesis: A full-thickness cartilage lesion at the time of the ACL-reconstruction does not influence the knee-function measured by KOOS. Study Design: Cross-sectional study. Methods: Of the 4849 primary ACL-surgery cases in the Norwegian National Knee Ligament Registry per 12.12.2007, thirty patients met the following inclusion criteria: A full thickness cartilage lesion (International Cartilage Repair Society (ICRS) grade 3 and 4), age less than 40 years, no associated pathology or meniscus injury and less than one year between the knee-injury and the ACL-reconstruction. To each of the 30 patients in this study group, two controls with no cartilage lesion were matched from the registry. Preoperatively all patients completed the Knee Injury and Osteoarthritis Outcome Score (KOOS). Peroperatively the surgeon graded the cartilage injury according to ICRS, and also recorded the location and size of the lesion. Results There was no significant difference between the groups for any of the five subscales according to KOOS-score. A cartilage lesion was located in the medial compartment in 67 % of the cases, in lateral compartment 20 % of the cases and in the patello-femoral joint 13 % of the cases. Conclusions The combination of a full thickness cartilage lesion and an ACL-rupture did not result in an inferior knee function at the time of the ACL-reconstruction as measured by KOOS., Introduction Cartilage defects in the knee comprise a socioeconomic burden as it may cause lifelong nonfatal disability at young age. Studies have found defects in up to 60 % of knee arthroscopies. In clinical trials the majority of these patients are excluded. This study investigates whether patients included in RCT represent a selected group and if this might causes bias. Methods and Materials Randomized clinical trials (RCT-level 1) on cartilage repair studies were identified and analyzed in order to define common inclusion criteria. These were applied to all patients (n=70) referred to a cartilage repair center during seven months in 2008. Lysholm knee score, occurrence of previous surgery and localization of cartilage defects were noted. Ratio of previous cartilage repair was 45/70 patients. Medial age was 37 years (range 15–51 years) and we included 22 women and 48 men. Results Common inclusion criteria were; single femoral condyle lesion, age range 18–40 years and size of lesion range 3.2–4.0 cm2. 5/70 patients matched the RCT. Previous cartilage repair stated lower Lysholm score 53 (±13.7) in comparison to no previous cartilage repair 69 (±13.7), p-value ⇠ 0.001. Other parameters such as age, localization and size of defect showed no significant difference. Conclusions Cartilage injuries of the knee represent a heterogenic patient group. Even RCTs show considerable variation regarding inclusion criteria, which might persuade selection bias. New clinical trials should document patients eligible for cartilage repair and flowchart of patients not included, to better evaluate treatment of cartilage defects in the knee., Introduction This study presents an assessment of cartilage repair with the Medicarb implant using post-operative long-term arthroscopic imaging. Methods and Materials A total of 128 knees treated by carbon fiber resurfacing rods for grade 3 and 4 articular cartilage lesions were assessed arthroscopically, with an average follow-up of 22.6 months (9- 48 mos.). Mean age of patients was 37.4 years (18- 60 yrs.) Cartilage repair scores were determined by an independent observer, using the ICRS scale for cartilage repair assessment. Results Mean repair assessment scores using the carbon rods were: 10.5/12 for medial femoral condyle repairs, 9.76/12 for lateral femoral condyle repairs, 9.9/12 for trochlea repairs and 9.4/12 for patella repairs. Grade I and II scores were recorded for 95.1% of medial femoral condyle repairs; 76.0% of lateral femoral condyle repairs; 86.1% of the trochlea repairs, and 89.7% of patella repairs. Patellar resurfacing was combined with a realignment and anteriorization procedure in 77 knees. A grade of I and II repairs were seen with rods on the patella in 96.7%, when combined with a mechanical correction, compared to 66.0%, when used on the patella without a combination procedure. There was no difference in repair scores in patients who were ⇠ 40 yr-old nor ⇢40 yr-old. Conclusions Results from the study show that carbon fiber resurfacing is an effective method of treatment. Results are improved by correction of knee deformity in conjunction with treating the defect. Evidence of recovered synovium was also observed as an additional benefit., Introduction In cases where the subchondral bone is exposed in the mirror area, there is no consensus about the effectiveness of mosaicplasty. The objective of this study is to examine whether the treatment of the femoral osteochondral lesions with autologous osteochondral transplantation even in the setting of exposed subchondral tibial bone in the same joint compartment is successful. Methods and Materials From August 2001 to July 2007, there were 17 cases 18 knees who were treated in the above methods. The mean age at surgery was 60.0 years, and the mean follow-up period was 36.5 months (range 16 to 86 months). There were 6 cases in osteonecrosis and 11 cases 12 knees in osteoarthritis. Eleven patients had second-look arthroscopy at about 11 months after mosaicplasty. We assessed IKDC subjective score, the area of the lesion and exposing subchondral bone of tibia, the ability of straight sitting in Japanese style and IKDC evaluation at second-look. Results The mean area of the lesion and exposing subchondral bone were 562 mm2 and 496 mm2. The mean preoperative IKDC subjective score was 41.0, and follow-up one was 80.5. There was significant difference. However, there were only 5 cases who had no symptoms. In preoperative period, there was one patient who could sit straightly, but 6 patients in the follow-up period. There were no significant differences in the other points. Conclusions There were some problems to leave minor symptoms and not to know long-term results, but this operative technique was successful in short-term., Introduction We present our experience of a microfractured defect covered with a collagen matrix (Chondrogide, Geistlich Biomaterials, Switzerland)c alled Autologous Matrix-Induced Chondrogenesis (AMIC) originally described by Behrens combined with discharching osteotomies. The idea behind is to maintain the stem cells that are mobilised through bleeding in the defect are maintained under the matrix to help to form a regenerate. Methods and Materials From August 2003 to July 2007 a number of 56 patients have been treated by AMIC in Fribourg by the senior author. Patients were treated for chondral and osteochondral lesions (OCD)at the knee joint and for OCD of the talus. 38 patients (40 knee joints) with a minimum follow-up of 1 year (ø follow-up 2.5 years, range 1–4 years) underwent retrospective analysis using clinical scores (ICRS, Lysholm). 17 patients had MRI which was analysed by Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. 11 second look arthoscopies were reviewed using the ICRS Cartilage Repair Assessment and Oswestry Arthroscopy Score. 5 biopsies have been examined histologically. Results 23 men and 17 women with a mean age of 36 years (range 14 − 64) underwent the AMIC procedure. Patients were treated for OCD (11), femoropatellar (20) and posttraumatic (9) lesions. Defects were located on the medial femoral condyle (16), lateral femoral condyle (3) and femoropatellar defects (21) with a mean size of 3.87cm2 (range 0.72 − 12). ICRS and Lysholm scores improved especially for OCD and femoropatellar lesions. MRI showed 18% complete fillings and 24% by hypertrophy; complete integration to the border zone was observed in 47%, but surfaces were only intact in 12%. Arthroscopically we found good fillings with some hypertrophies, but repair tissue was somewhat softer. ICRS Arthroscopy Score showed a mean of 9/12 and Oswestry a mean of 6/10 points. Histologically all biopsies showed fibrocartilage with some hyaline-like elements. Conclusions AMIC improved the clinical outcome and decreased pain in the cartilage defects treated in this study. Arthroscopy showed good fillings with some hypertrophies. In the MRI the AMIC zone was well integrated to the border zone but filling was mostly incomplete and surfaces damaged. Histology showed fibrocartilage with some hyaline-like elements. Especially in OCD and femoropatellar patients, but less in the purely cartilagineous lesions of the femoral condyle the AMIC procedure is an interesting, one step technique., Introduction The aim of our study was to prospectively assess the biochemical properties of cartilage repair tissue after matrix-associated autologous chondrocyte transplantation (MACT) with Hyalograft C® via delayed Gadolinium Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) and T2 mapping over a period of one year. Methods and Materials Ten patients after MACT were examined at two different times of follow-up with a delay of one year. The mean age was 32.2 ± (SD) 11.0 years and the mean defect size was 3.9 ± 1.9 cm2. The mean follow-up period after MACT surgery was 4.4 ± 2.0 range 2 to 7 years. Besides dGEMRIC and T2 mapping, clinical scores were assessed. Results The clinical scores did not change significantly from the first to the second evaluation. δR1 (1/sec) was 1.06 ± 0.58 and 0.84 ± 0.41 in repair tissue and 0.56 ± 0.27 and 0.51 ± 0.23 in healthy reference cartilage. The relative ΔR1 (rΔR1; ratio between RT and healthy cartilage) for repair tissue were 1.89 and 1.65 respectively. The T2 values were 43.8 ± 8.4 ms and 45.0 ± 8.7 ms for RT and 45.5 ± 4.5 ms and 44.9 ± 5.9 ms for healthy cartilage tissue. Conclusions We found both techniques to indicate that the properties of the RT were more similar to native cartilage at the second MRI visit. The ΔR1 decreased, which suggests a slight increase in the glycosaminoglycane concentration of repair cartilage compared to healthy cartilage. Hyalograft repair tissue apparently remained stable at mid-term follow up in our subjects., Introduction Purpose: The aim of this presentation is to demonstrate the presence of mesenquimal stem cells in the knee joint area after the microfractures technique and to demonstrate the regeneration of hyaline cartilage lesion grade IV treated by mechanical shaving and microfracture technique plus intrachondral allograft insertion of growth factor delivered by plasma rich in platelets. Methods and Materials During the knee video arthroscopy, a mechanical shaving was performed until the bone layer; after this, a microfracture technique was performed in multiple sites of the lesion, just after of the microfracture technique the bone marrow was exposed; the isotonic solution was removed and 10 mL delivered bone marrow fluid was collected for steam cells search by immunohistochemistry techniques; the activated plasma rich growth factor (PRGF) was placed into the chondral lesion environment. Another knee video arthroscopy was performed for histological evaluation of chondral tissue four months later. Results Mesenquimal stem cells were detected in the bone marrow fluid collected. Four months after the procedure, a recuperation of hyaline cartilage was observed at macroscopic level. The histological analyses of the hyaline cartilage demonstrated a homogeneous stroma colored by eosin and the presence of chondrocytes in multiple points of biopsy with more than one nucleus suggesting the regeneration of the cartilage. Conclusions This is a technique that presents promising results. In the present case, the patient returned to the practice of physical activity in four months after the first surgical procedure, presenting a good evolution and avoiding others procedures and/or complications as osteoarthrosis., Introduction Anterior cruciate ligament (ACL) rupture has been associated with early occurrence of osteoarthritis, which was originally believed to be due to instability derived from ACL insufficiency. However, some studies suggested that the initial injury to the articular cartilage may also play a role in the development of osteoarthritis. The aim of this study is to evaluate the relationship between acute ACL rupture and cartilage lesion by use of quantitative magnetic resonance (MR) imaging technique, T2 mapping. Methods and Materials 50 knees of 50 patients (23 women and 27 men, mean age 27.3±5.7 years) with acute ACL injury were studied with a 1.5 Tesla MR imaging system. T2 mapping in the coronal plane were performed (1500 msec TR, 8 TEs of 12.4–99.2 msec, 140×140 mm field of view, 3.0-mm slice thickness, and 384×384 matrix), and T2 of the cartilage at medial and lateral condyle was measured. The relationship between T2 of the cartilage and the presence or absence of bone bruise was investigated. In 33 patients, operative findings of cartilage were evaluated and compared with the MRI findings. Results Of the 50 patients, 24 had a bone bruise at lateral femoral condyle. In the patients without bone bruise, there was no significant difference in the mean T2 between lateral femoral condyle and medial femoral condyle, 32.3 ms and 31.5 ms, respectively. The mean T2 of cartilage with bone bruise was significantly higher than that without bone bruise, 38.1 and 32.6 ms, respectively. The operative findings of cartilage were well correlate with the MRI findings. Conclusions It is known that T2 value increases with the loss of collagen anisotropy and increase in water content observed in the damaged or deteriorated cartilage. Thus the presence of bone bruise at the femoral condyle after acute ACL rupture was thought to indicate the presence of cartilage deterioration at that site. Deterioration of articular cartilage associate with bone bruise should be taken into account in clinical treatment for acute knee injury., Introduction Fixation of an osteochondral fragment to an osteochondral defect has often been performed in the treatment of patients with osteochondritis dissecans (OCD). However, the effect of this procedure remains controversial. The purpose of this study was to evaluate the histologic changes of the internally fixed osteochondral fragments in the knee. Methods and Materials Ten knees of 10 patients with OCD of the knee underwent open reduction and internal fixation of the unstable fragments, and histologic changes before and after internal fixation were evaluated. The patient age ranged from 11 to 22 years (mean 15 years). The procedure was performed either with bioabsorbable pins only, or with a combination of an autologous osteochondral plug and bioabsorbable pins. A needle biopsy was done at the time of fixation, and the time of second-look arthroscopy at a mean of 7.8 months (6 to 9 months) after surgery. Results The biopsy specimens at the second-look arthroscopy improved significantly in the histologic grading score compared with the specimens before fixation. In the specimens at the second-look arthroscopy, extracellular matrix was stained more densely than at the time of fixation, especially in the middle to deep layer. Conclusions Comparison of the biopsy samples obtained from lesions before and after fixation enabled us to confirm the improvement of histologic findings of fixed sites., Introduction Autologous chondrocyte implantation (ACI) is widely used to treat symptomatic articular cartilage defects of the knee. On the other hand, the fibrin matrix Autologous chondrocyte implantation (ACI) is a new tissue-engineering technique for the treatment of deep cartilage defects, in which autologous chondrocytes are seeded on a three-dimensional scaffold provided by a gel type fibrin matrix. Methods and Materials The clinical results after ACI with fibrin gel using sequential patient evaluation are reported 30 patients diagnosed with chondral defect of the knee from June 2005 to May 2007, (mean age, 36.6years). Clinical and functional various score systems for the knee, Magnetic resonance imaging (MRI) evaluation, the International Cartilage Repair Society (ICRS) Cartilage Repair Assessment was performed at 12 months postoperatively. Diagnostic histological examination of the graft was performed in only 10 patients. The biopsy was stained with hematoxylin and eosin, Masson's trichrome, and safranin-O, and immunostained with Col type I, II antibodies. Results Clinical and functional statuses and the ICRS showed significant improvement after surgery (p⇠0.01). The mean scores of Henderson classification (MRI evaluation) significantly improved from 7.86 ± 2.12 points to 14.31 ± 1.45 points (p=0.001) and no significant graft-associated complications were encountered. The fibrin matrix ACI showed hyaline-like cartilage or mixed hyaline-like and fibrocartilage in 67.9 % of biopsies. Conclusions Fibrin matrix ACI offers the advantages of technical simplicity, minimal invasiveness, a short operating time, and easier access to difficult sites than classical ACI. Based on the findings of this clinical pilot study, fibrin matrix ACI offers a reliable means for the treatment of articular cartilage defects of the knee., Introduction The incidence of childhood obesity is rapidly increasing worldwide. Especially in industrialized nations the obesity epidemic is predominantly evident. Overweight and obesity are afflictions, which lead significantly to an increased risk of high blood pressure, high cholesterol values, diabetes, hepatic steatosis, sleep apnoea, asthma, and joint problems and in general to a poor health status as well. Methods and Materials 20 morbidly obese patients, BMI higher 99.5 percentile, mean BMI 39.3 kg/m2, mean age 14.3 years (9–19 years), who were suffering from chronic knee pain have been included in the study. Radiographic investigations and a MRI scan (1.0 Tesla) of the painful knees have been performed in a special open MRI tool. Results All patients presented a lesion of the cartilage at least in one region of the knee, although retropatellar cartilage lesions have been found in 19 knees. 10 cartilage lesions grade I, and 4 grade 2 have been watched narrowly after MRI scan in the lateral compartment of the knee, whereas the medial compartment showed in 8 cases a grade I, in 13 cases a grade II and even in 2 cases a grade III defect of the cartilage. Even two adipose young patients showed changes in the sense of an incipient osteoarthritis. Conclusions It is concluded, that a couple of morbidly adipose children and teenagers show pathological changes of their cartilage. Whether obesity is responsible by itself for the development of the pattern of diseases or other factors are causally involved will show further research., Introduction Autologous Matrix Induced Chondrogenesis (AMIC®) combines microfracturing with application of a cell-free collagen scaffold. No cultured chondrocytes are necessary. It was established for focal cartilage repair in the knee as a cost-effective one-step procedure. Methods and Materials 38 focal chondral/osteochondral defects (ICRS III-IV°) of the femoral condyle, trochlea and/or patella in 35 patients (27 male, 8 female, mean age 35.8 (18–52) years) were treated by standardized microfracturing and application of a cell-free collagen type-I/III scaffold (Chondro-Gide®, Geistlich Biomaterials). The mean defect size was 3.8 (1.0–10.8 cm2). The results were evaluated prospectively by functional outcome scores, subjective clinical ratings and MRI with an average follow-up of 30.4 months (range 24–54 months). Results Significant improvements were seen in the Cincinnati-Score (50.4 to 87.3) as well as in the Lysholm-Score (59.6 to 88.6, each p⇠0.001). Pain on a 10-point VAS decreased significantly from 6.2 to 1.8 while subjective knee function improved from a mean of 4.6 to 7.5. In 5 revision cases at 4–26 months the repair tissue revealed reasonable results with regards to surface formation, filling and integration. The MRI follow-ups showed an adequate filling of the defect, no prolonged effusion occured. 85% (30/35) of the patients were satisfied with the functional results (ICRS I°+II°). Conclusions The AMIC® procedure is a minimal invasive effective one-step therapy for focal chondral or osteochondral cartilage defects in the knee. It was shown to provide stable results at mid-term follow-ups., Introduction Many treatment options for full-thickness cartilage defect repair in the knee show good short-term clinical outcomes. The mid-term clinical scores of Autologous matrix-induced chondrogenesis (AMIC®) show a “Plateau Effect” after 3 years. Methods and Materials AMIC® enhances the microfracture technique by stabilizing the blood clot with a collagen type I/III matrix (Chondro-Gide®, Geistlich, Switzerland). Mesenchymal stem cells are activated by microfracturing and the matrix is glued onto the defect with a partially autologous fibrin glue. A retrospective study was carried out to investigate the objective and subjective clinical outcome over a period of up to 4 years post-operatively. 49 patients (mean age 36±10 years) with focal chondral defects of the knee joint (22 retro patellar, 23 femoral condyle, 4 trochlea) were treated with AMIC®. The average defect size was 3.6 cm2 (2–8 cm2). The patients were followed-up clinically with MRI and different knee scores. 30 patients were included into the 2-year, 9 into 3-year and two patients into 4-year evaluation. Results The scores showed significant improvement (p=⇠0.05) in all postoperative evaluations except after 4 years compared to preoperative value. There was no significant improvement or decline within postoperative values at 1, 2 and 3 years. Both 4-year results showed a decline compared to the 3-year postoperative value but higher values than the pre-operative score. Conclusions The results indicate that AMIC is a treatment option for local cartilage defects in the knee which obtains good mid-term results. It shows a “plateau-effect” of the clinical scores after 2 years with no further improvement of subjective outcome., Introduction Subtotal menisectomy is associated with early degeneration of the knee. Meniscus transplant is one approach to this problem. Purpose: This is an evaluation of ten-year clinical outcomes of twenty-two meniscal allografts. Methods and Materials Methods: Records of twenty-one patients with twenty-two meniscal allografts were reviewed. Maximum follow-up time is 13.25 years and the minimum is 9.85 years. Lysholm scores were recorded for pre-op, best ever and present level of function. Patients were followed in clinic or by telephone interview by a person independent of the care team. Joint space on x-rays was evaluated using the non-operative side as a control if asymptomatic. Results Lysholm scores were available for nineteen patients. Comparison of Lysholm scores showed significant improvement. Two patients were not significantly improved. Three patients progressed to unicompartmental arthroplasty at an ten years post transplant. Two have poor Lysholm scores and fourteen have good to excellent scores. X-ray data were available for fourteen of the nineteen patients. Difference in joint space in the fourteen was 1.2 mm. Six patients had second looks. One meniscus failed primarily and was successfully reimplanted. There were 3 retears and 3 had increasing chondromalacia. The majority continue to do well. Workers comp did equally well. Conclusions Meniscal allograft prolongs the functional life of meniscus minus knee by being chondroprotective. Degree of chondromalacia at the time of implant is prognostic of longevity. The three patients who went on to unicompartmental arthroplasty all returned to work and had long periods of improvement before failure, Introduction Osteochondritis dissecans (OD) often negatively affects the articular surface of the knee. There is not yet an optimal treatment intervention defined due to the limited amount of prospective research available. The main limitation of previous studies is heterogeneity of the cartilage defects. We evaluated the medium-term outcome of osteochondral autograft transplantation (OATS) in homogeneous OD-lesions. Methods and Materials We evaluated 7 male patients (mean age 33.4) with 8 classical OD lesions at the lateral boundary of the medial femoral condyle. An average of 3.6 osteochondral plugs was used. Patients were evaluated preoperative, at 6 months and 1 year postoperative. The ICRS-evaluation package, International Knee Documentation Committee (IKDC) and Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaires were used for clinical evaluation. Prospective follow-up MRI was performed using a semi-quantitative scoring system. Results The IKDC subjective score improved significantly from preoperative to 1-year follow-up (p=0.001). The KOOS evaluation showed a significant improvement on all subscales; pain (p=0.002), symptoms (p=0.003), activities-of-daily-living (p=0.019), sport and recreation (p=0.014) and quality-of-life (p=0.001). MRI showes good surface congruency, no edema or protuberance of the cylinders, good similarity of cartilage thickness and a non-complete osseous integration. No correlation could be found between MRI findings, percent of coverage and the patients' satisfaction. Conclusions OATS remains a valid treatment option in selected cartilage defects. A subgroup of osteochondral defects on the lateral border of the medial femoral condyle improve significantly following OATS. Future research should focus on identifying the appropriate choice of treatment in well described cartilage lesions, instead promoting one superior technique., Introduction Purpose: To evaluate the incidence and risk factors for knee cartilage and ligament injury in elite college football players Introduction Each year at the NFL combine, approximately 330 of the nation's best collegiate football players are invited to display their skills and physique. In this environment, MRI is used liberally to evaluate any worrisome history or physical findings. The low threshold for ordering MRI is this select group of athletes offers an outstanding opportunity to examine chondral injury patterns. Methods and Materials Methods: From 2005–2007, all players entering the NFL combine were screened for knee injuries(N=980 players). Height, weight, and BMI measurements were taken at the start of the Combine. MRI and plain film findings were recorded into a database along with position, height, weight, BMI, and surgical history. Results During the 3-yr period reviewed, a total of 980 players were available for analysis. The total number of chondral injuries was 199 (20.3%) of all players. 82 players(8.4%) had medial compartment chondral injuries. 104 players (10.61%) had lateral compartment chondral injuries, 118 players (12.04%) had patellofemoral compartment chondral damage, and 79 players (8.06%) had chondral injuries in more than one compartment. Players with a weight greater than 222.5 lbs, or a BMI greater than 30.5 had a significantly higher risk or cartilage injury. Height of the players was not a significant risk factor. Conclusions Discussion: This study is the first of its kind, highlighting the high prevalence (20.3%)of chondral injuries in the N FL recruitment classes of the past 3 years., Introduction We determined if meniscus tear type correlated with function and activity levels two years after meniscectomy. Methods and Materials 206 patients underwent partial medial meniscectomy and 117 underwent partial lateral meniscectomy (average age=49, range, 18 to 80). At index surgery, type of meniscus tear was recorded. Tears were designated bucket handle/ vertical longitudinal (BV), flap/radial (FR), complex (C), or horizontal (H). Patients undergoing microfracture or ACL reconstruction were excluded. Patients were followed for a minimum of two years (average=4.6 yrs) after meniscectomy. Patients completed questionnaires including Lysholm and Tegner scores to assess function and activity. Results For medial meniscus, there was significant correlation between tear type and patient age. BV (n=35) group was significantly younger than the FR (n=65) and C (n=193) groups (p⇠0.01). BV group had significantly higher Lysholm (88) scores versus FR (79) and C (78) (p⇠0.01). BV group also had higher Tegner activity levels (5.4) than C (4.6) (p=0.04). For lateral meniscus, BV (n=15) and FR (n=37) were significantly younger than complex (n=45). FR had higher Lysholm and Tegner scores, but there were no significant differences. 28% of BV medial meniscus tears required further surgery while less than 15% of other types of medial or lateral tears required further surgery. Average time to second surgery was 2.4 years. Conclusions In this series, BV medial meniscus tears had better function and activity at least two years post-meniscectomy, perhaps due to younger age. This group also required more reoperations the first two years after index meniscectomy. Tear type did not influence outcomes after lateral meniscectomy., Introduction Meniscus suture repairs have a high rate of repeat surgery. We determined what factors lead to failure of meniscus suture repair. Failure was defined as repeat surgery within 2 years of initial repair. Methods and Materials 283 meniscus suture repairs were performed by one surgeon. Average age was 31 years (range, 18–71) with 177 males and 106 females. All repairs used an inside-out suture technique. 137 had ACL reconstruction and meniscus repair (93 concurrent reconstructions and 44 two-staged reconstructions). 181 medial menisci and 102 lateral menisci were repaired. Of medial repairs, 80% were posterior third of the meniscus, 11% middle third, 1% anterior third, and 8% extended to all areas. Of lateral meniscus repairs, 49% were posterior third, 26% middle third, 22% anterior third, and 3% extended to all areas. Results Twenty-eight (28) patients (10%) required repeat surgery on the repaired meniscus within 2 years and were considered failures. Average time to repeat meniscus surgery was 12 months (range, 2.5–24 months). There were no differences based on age, gender or location. Thirteen percent (13%) of medial repairs and 4% of lateral repairs failed (p=0.012). Medial repairs failed significantly earlier (5.6 months) than lateral repairs (12.9 months) (p=0.001). For patients who had ACL reconstruction and meniscus repair, factors associated with failure included age (failed age=22; non-failure age=29; p=0.013), and concurrent ACL reconstruction (two-staged repair failures=2%; concurrent repair failures=11%; p=0.04). Conclusions Failure of index meniscus suture repair within two years is more likely to occur in medial repairs and in young patients with concurrent ACL reconstruction., Introduction We determined anatomic location of partial medial meniscectomy (PMM), then correlated location with function and activity two years post-meniscectomy. We hypothesized that posterior third loss would decrease function and activity more than loss of middle or anterior thirds. Methods and Materials 120 patients (18–60 years) underwent PMM as controls in a device study. There were 74 acute (no prior PMM) and 46 chronic (1–3 prior PMM) patients. At index surgery, location of meniscus removed was documented and categorized as posterior (A), middle (B), or anterior (C) third. Patients were followed clinically for ⇢2 years after meniscectomy. Patients completed validated questionnaires including Lysholm and Tegner scores for function and activity. Results 17 patients had isolated A meniscectomies, 83 had combined AB thirds, 10 had isolated C, and 10 had combined ABC PMM. Patients with isolated A meniscectomies had significantly lower Lysholm scores (78) versus all other patients (88) (p=0.01) and significantly lower Tegner scores (3.5) versus all other patients (4.5) (p=0.03) two years after PMM. Based on actual measurements, isolated posterior PMM group averaged 41% meniscus removed, well below the 50% loss threshold previously reported as predictive of decreased function and activity. Conclusions Function and activity levels were significantly decreased two years after surgery in patients who had isolated posterior third PMM versus all other patients with meniscus loss in other anatomic locations. Decreased function and activity occurred even though ⇠50% meniscus was removed. We confirm the importance of preserving the posterior medial meniscus. Potential positive benefits of replacing or regrowing lost meniscus tissue are supported., Introduction The purpose of this study is to evaluate the outcome of the microfracture procedure in treating full-thickness cartilage lesions of the knee in a large patient sample using a random-effects model for longitudinal data analysis. Methods and Materials 350 patients (avg. age=47.6 year(range, 12–76), 65% male; 55% female) who underwent the microfracture procedure by a single surgeon were identified for analysis. Subjects who underwent concomitant ligament or meniscus surgery were excluded. Data were analyzed with a random-effects model for longitudinal analysis. Outcome variables were Lysholm Score(LYS) and the Tegner Activity Scale(TAS). Independent variables included gender, age, degenerative versus traumatic lesion, and years since surgery(YSS). Average follow-up was 4.3 yrs(range 1 to 12). Results For gender, there was no significant difference in the trajectory of the plots over time. [LYS-squared=4954+435(YSS)–45(yrs-squared)+408(gender)–10.6(yrs*gender)+1.3(yrs-squared*gender); p=0.88] [TAS=3.73–0.001(YSS)-0.002(yrs-squared)+0.7(gender)+0 .035(yrs*gender)+0.0006(yrs-squared*gender)p=0.99]. Although there is a trend favoring the outcome of traumatic over degenerative lesions there was no significant difference in the trajectory of the plots. [LYS-squared=5539–3.4(age)+456(YSS)–47(yrs-squared)–333(djd)-85.6(years*djd)+8.8(yrs-squared*djd) p=0.328] [TAS=6.0–0.03(age)-0.04(YSS)+0.002(yrs-squared)–0.539(djd)+0.123(years*djd)–0.011(yrs-squared*djd) p=0.272] Subjects were divided into two age groups, ⇠= 45 years and ⇢45 years. Subjects with traumatic lesions demonstrated a significant difference in the trajectory of LYS scores over time by age group. [LYS_squared=5194+607(YSS)-58.2(YSS squared)+598(age)–336(YSS*age)+24.5(YSS*age) p=0.0375] Conclusions We found no significant differences in the trajectory of outcome over time between genders, or between degenerative/ traumatic chondral lesions. We did identify age-dependent differences in outcome over time. Subjects with traumatic lesions demonstrated a significant difference in the trajectory of Lysholm scores over time by age group., Introduction Microfracturing is a common primary cartilage repair technique for focal cartilage defects in the knee but sometimes fails. In this prospective study, second-line treatments of focal chondral or osteochondral defects in the knee after failed microfracturing by the use of a scaffold ACI are compared with first-line scaffold ACI treatments. Methods and Materials 28 patients (30.7 (17–51) years, defect size 4.5 (2.1–9.4) cm2) with failed microfracturing of focal cartilage defects in knee (ICRS III-IV°) were treated secondarily by scaffold ACI (MACI®). Their outcome was compared to primary treatments of equal defects of 28 matched patients (31.2 (14–49) years, defect size 4.4 (2.1–8.9) cm2) by scaffold ACI. The follow-ups were 27.1 (12–43) vs. 25.2 (12–39) months. Gender, defect origin, BMI and concomitant therapies were comparable. Results The Lysholm scores improved for second-line ACI (61.1 to 82.1) as well as for first-line ACI (60.9 to 86.4, both p⇠0.001). On a VAS pain decreased in second-line ACI (5.5 to 3.2) and in first-line ACI (4.8 to 2.6). Subjective knee function improved from 4.8 to 6.2 in second-line ACI and from 4.7 to 7.1 in first-line ACI (all p⇠0.001). No statistical differences between both groups were found. In revision cases 4/6 (second-line ACI) and 3/4 (first-line ACI) arthroscopic aspects of the repair tissue revealed normal or nearly normal results according to ICRS classification. MRI follow-ups showed an adequate filling of the defects, no prolonged effusion occurred. Conclusions Scaffold ACI is an efficient therapeutic option even for second-line cartilage repair after failed microfracturing for focal cartilage defects in the knee., Introduction Microfracture is an effective surgical treatment for isolated, full thickness cartilage defects. This study aimed to examine the clinical outcomes of patients who have undergone arthroscopic microfracture for chondral defects of the glenohumeral joint. Methods and Materials From March 2001 to August 2007, 18 patients who underwent arthroscopic microfracture of the humeral head and/or glenoid surface were retrospectively reviewed. All patients were examined by an independent, blinded examiner and completed surveys containing the Simple Shoulder Test (SST), American Shoulder and Elbow Score (ASES), and Visual Analog Scale (VAS). Preoperative scores were compared to postoperative outcomes at a minimum follow-up of one year. Results Three (16.67%) patients went onto shoulder arthroplasty and were considered failures, while two were lost to follow-up, for a total follow-up rate of 88.89%. Of the final thirteen patients, the mean age was 37.03 years (range, 18 to 55) with an average follow-up of 27.8 months (range, 12.1 to 89.2). Microfracture was performed on the humeral head in 8 cases, on the glenoid surface in 4 cases, and on both surfaces in 1 case. The average size of humeral and glenoid defects was 5.07 cm2 and 1.66 cm2, respectively. There was a statistically significant decrease (p⇠0.002) in the VAS following surgery as well as statistically significant improvements (p⇠0.002) in SST and ASES data. Twelve (92.3%) patients were satisfied with the surgery and would have the same procedure again. Conclusions Microfracture of the glenohumeral joint provides a significant improvement in pain relief and shoulder function in patients with isolated, full thickness chondral injuries., Introduction The purpose of this study was to determine the effectiveness of the microfracture technique according to function, pain control and activity level, in the treatment of knee cartilage lesions. Methods and Materials Thirty nine patients were treated with microfracture for ICRS grade III and IV cartilage lesions of the knee. Functional outcome was prospectively evaluated with a minimum 3-year follow-up (range, 36 to 79 months) by subjective rating, visual analogue scale and activity-based outcome scores, tested by paired t test and U Mann Whitney test. p⇠.05 was considered statistically significant. Results The mean age of our patients was 40.5 years (range, 19 to 74), 17 women and 22 men. Articular lesions involving medial or lateral compartment were 89.7% and 10.3% patellofemoral compartment. Partial meniscectomy were done in 24 patients (61.5%) and ACL reconstruction in 15 patients (39.5%). The mean postoperative Lysholm score was 80.9 (sd±17.7), there was a non statistically significant reduction in VAS score from 5.3 to 4.3 (p= 0.173), with a decrease in Tegner activity scale scores from 4.6 before injury to 3.6 postoperatively (p ⇠0.0001). We found no significant differences in Lysholm score between patients older or younger of 40 years old (p = 0.343) neither in patients with or without concomitant meniscectomy (p = 0.3). Conclusions According to our results, moderate functional results, non statistically significant reduction in pain relief and a decrease in activity level score, are obtained after three to six years follow up, with microfracture technique in the treatment of cartilage lesions., Introduction Pro-inflammatory cytokines play a pivotal role in bone tunnel widening after ACL reconstructive surgery. A new treatment option is to administer Autologous Conditioned Serum (ACS/Orthokine) containing endogenous cytokines including IL-1Ra and growth factors like IGF-1, PDGF and TGF-β produced from venous patients blood. The trial purpose was to establish whether the osteoclastic effect could be affected by ACS/ Orthokine, representing a better postoperative outcome. Methods and Materials In a prospective, randomized, double-blind, placebo-controlled trial with two parallel groups 62 patients were treated. Bone tunnel width was measured by CT-scans and efficacy was assessed by WOMAC and IKDC 2000 in patients receiving ACS (Group A) or Placebo (Group B) one year following ACL-reconstruction using autografts of m. semitendinosus and gracilis tendons (HS), as well as the patellar ligament (BTB). Results Tunnel widening during the first year was significantly lower in Group A than in Group B. The increase within the ACS group was significantly lower in the HS (ACS vs. Saline 6 months: p=0.032, 12 months: p=0.048) and in the BTB group (ACS vs. Saline 6 months: p=0.061 [n.s], 12 months: p=0.001). Clinical outcome (WOMAC, IKDC 2000) was better in patients treated with ACS at all data points and outcome parameters and significant in WOMAC stiffness subscale after 1 year. Conclusions The significant reduction of bone tunnel enlargement, the improvement of stiffness together with the consistently higher improvement of most other parameters demonstrates that ACS clearly induces a biological response different from saline treatment and warrant future investigations into the possible protective effects of ACS/ Orthokine., Introduction There is a lack of prospective, multicenter randomized clinical trials comparing cartilage repair products in development to surgical controls, making it difficult for surgeons to confidently develop treatment algorithms. BST-CarGel® is a new medical device being investigated for the repair of focal articular cartilage lesions for which a controlled clinical trial has been designed and implemented. Methods and Materials Eighty subjects (40 subjects/group) are being randomized to BST-CarGel® + microfracture or microfracture alone. The sample size was derived from large animal efficacy studies attempting to reach p⇠0.05 significance and 90% power. Eligible subjects from 18 to 55 years have single Grade 3 or 4 cartilage lesions up to 10 cm2 on the medial or lateral femoral condyles. Both groups follow identical 12 week post-operative physiotherapy programs and are non-weightbearing for 6 weeks. The primary endpoint is cartilage repair at 12 months assessed by blinded MRI for % lesion filling, and T2 MRI and dGEMRIC for repair tissue collagen characteristics and proteoglycan content, respectively. The secondary endpoint is improvement in pain and physical functioning at 12 months as assessed by the WOMAC questionnaire and tertiary endpoints are safety and quality-of-life by SF-36. Optional 13-month biopsies may provide additional quality information via microscopic methods Results A prospectively planned interim analysis was conducted at 6 months follow-up on 23 patients. Results indicated evidence of a positive effect of BST-CarGel® treatment on cartilage structure when compared to the microfracture control, and comparable safety to the control. Conclusions This study represents an important step toward developing evidence-based treatment algorithms for cartilage repair., Introduction Ideal treatment for osteochondral lesions of the knee (OLK) is still controversial. Although good results were obtained by Mosaicplasty and Autologous Chondrocytes Implantation, still well-known drawbacks are inevitable. The One step repair technique with bone marrow derived cells was previously presented in the ankle with satisfactory results. Aim of this study is to present the application of the “one-step” technique in the knee joint and evaluate the results. Methods and Materials From April 2006 to May 2007 13 patients with OLK underwent the One-step procedure. 7 cases affected the medial condyle, 2 cases the lateral condyle and 4 cases the patella. The condylar lesions underwent a completely arthroscopic procedure, while the patellar were treated by open access. Bone-marrow was harvested from the posterior iliac crest, and the cells were concentrated in the operating room and implanted at the lesion site on a hyaluronan-based scaffold. Platelet Rich Fibrin was added providing growth factors. Results The mean preoperative IKDC score was 34±13. The IKDC at 6 months follow-up was 68±15, at 12 months was 81±8 and at 24 months was 88±3 (8 patients). The control MRI at 12 and 24 months follow-up showed a good regeneration of the subchondral bone and the cartilagineous tissue. A biopsy of the regenerated tissue performed at 12 months showed a cartilagineous tissue in organization and a newly formed subchondral bone. Conclusions These results demonstrated that the one-step technique represents a good option for osteochondral lesion repair in the knee joint, overcoming the major drawbacks of previous techniques., Introduction Osteochondral Defect (OD) has a poor spontaneous regenerative capacity. To our knowledge there are no reports about therapy in humans using mesenchymal stem cells (MSCs) induced in vitro to chondrogenic differentiation over a 3D membrane. Aim: To describe the clinic, radiological, histological and molecular results at two-year follow-up. Methods and Materials Bone Marrow (BM) MSCs were obtained from a 32 years old patient carrying an OD ICRS4⇢10cm2 on the external femoral condyle of the left knee. MSCs were cultured and then repleate over a collagen type I membrane. 3 days before the surgical procedure, the cells were supplemented with TGF-b1 (10 ng/ml). After that, the membrane carrying cells was implanted over the top of the OD. Clinical and radiological follow up was performed every 3 months. 2 years after the surgical procedure a second-look arthroscopy was performed and biopsy specimens were obtained from the OD area. Results At two-years follow-up the patients have recovered articular functionality. The Lysholm score improved 80 points (15 to 95). In radiological studies was observed complete scaffold integration. The cartilage was normal at second-look arthroscopy. The biopsy histology (H-E and safranin-O) was normal. Western blot and real time PCR for SOX-9 and collagen II from biopsy were similar to normal cartilage. Conclusions We demonstrated successfully repair of OD ICRS4 ⇢10cm2 with autologous MSCs induced to chondrogenic differentiation over a 3D collagen type I membrane at two-year follow-up. We propose this technique as a new alternative in the treatment of OD ICRS⇢4., Introduction In degenerative arthritis, a full-thickness Outerbridge IV lesion is considered end-stage. However, an in-depth pathological study is not available. The Outerbridge IV lesion may have potential for repair in the proper environment. Cartilage regrowth has been reported in hips and in knees following high-tibial osteotomy. Our study purpose was to examine gross and microscopic characteristics and validate the potential for the cartilage aggregate as a source of repair. Methods and Materials Osteochondral specimens from the lesions were harvested from TKA patients. Multiple disc-shaped samples were prepared for tissue culture. Specimens were stained with Safranin-O (without fixation), which selectively stains the aggregates and adjacent intact cartilage. This technique quantitated the size of cartilage aggregates in live specimens and permitted monitoring of growth in culture and subsequent histology. Results Stained gross specimens showed cartilaginous aggregates on the surface and multiple small depressions. On microscopy, the cartilaginous aggregates were found to stain positive for glycosaminoglycans, type II collagen, and lubricin. Depressions were attributed to aggregate erosion, vascular rupture, and bone fragmentation. Cartilage aggregates grew over the surface of the exposed bone in culture. Histologic examination at 3 and 6 weeks revealed abundant cellular proliferation of spindle- and oval-shaped cells covering the surface. Conclusions The multiple small depressions could house various cell therapies. Cartilage aggregates proliferated in culture, confirming the hypothesis that they contribute to cartilage repair following reduction in joint pressure on the lesion. This experimental model is valuable to evaluate various therapeutics to enhance cartilage repair., Introduction ChonDuxTM is a biomaterial guided cartilage repair system consisting of (1) a photopolymerized hydrogel and (2) a biological adhesive that are used in conjunction with microfracture to enhance the environment for endogenous stem cells to repair cartilage. The purpose of this study was to evaluate the safety and feasibility of ChonDux in treating patients with symptomatic, focal chondral lesions. Methods and Materials Fifteen patients (age 27–57 yrs) without general osteoarthritis who had a single, symptomatic cartilage defect 2–4 cm2 in size on the medial femoral condyle in a stable knee were treated with ChonDux. Pain and knee function were assessed by the IKDC form at baseline, 3, 6, and 12 months. Repair tissue fill was determined by MRI. Data collected to date are presented. Results A significant improvement in knee function was observed. The average IKDC score increased from 38.4 at baseline to 69.0 at 6 months (n=14). The severity of pain dropped from 54% at baseline (n=15) to 25% at three months (n=15) and 24% at six months (n=14). The frequency of pain also progressively decreased from 76% (baseline) to 36% and 27% at 3 and 6 months, respectively. This coincided with significant tissue fill by MRI, averaging 83% at 3 months (n=9) and 85% at 6 months (n=12). Conclusions This study demonstrates Chondux is safe, easy to use, and provides clinical benefits (stable defect fill, reduction in pain, and improvement in knee function). Further studies will evaluate efficacy against microfracture controls. ChonDux may offer an effective, single surgery, off-the-shelf treatment option for cartilage repair., Introduction We need several approaches to improve the clinical results for cartilage defects. Methods and Materials 1. Tissue-engineered cartilage: We have been performing transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints (108 cases) as a second generation of chondrocyte transplantation since 1996. 2. Articulated Distraction Arthroplasty: Bone marrow stimulating procedure has two potential weak points to induce hyaline cartilage. One is compressive overload on the drilled or microfractured area at the early stage. In order to reduce the overload, we have developed external fixators which allow almost full ROM with joint distraction for clinical cases based on an animal study. 3. Future direction for cartilage repair with minimally invasive tissue-engineering technique: The most optimal procedure to repair cartilage defects is just injection of cytokines or growth factors and cells. Our completely novel approach is to use cell delivery system using an external magnetic field. Results 1. Sixty knees who had received transplantation of tissue-engineered cartilage for cartilage defects were followed up for at least 5 years. 2. This apparatus has been effective for clinical cases, although the number of the patients and the follow-up period were limited. 3. This magnetic system was demonstrated to be effective for animal studies. Conclusions Our new approaches are introduced., Introduction This paper assesses clinical and radiological outcome following cartilage repair in the knee using TruFit plugs, with a view to determining safe return to activity. Methods and Materials Prospective analysis of 31 patients undergoing articular cartilage repair using TruFit CB plugs (Smith & Nephew) for symptomatic chondral defects in the knee. Clinical and MRI analysis was performed post op at 6, 12, 18 and 24 months. 14 have minimum 12 months follow up and form the study group. Results 4 female and 10 male patients with mean age of 35.3 were included. 1 − 4 plugs were used in 6 Left and 8 Right knees, 3 for osteochondral lesions and 11 purely chondral. 4 procedures were performed mini-open and 10 arthroscopic. The trochlea was repaired in 3 and femoral condyle in 11 (medial 7, lateral 4). 6 had undergone previous microfracture and 1 previous OATs surgery. Mean IKDC improved from 42.6 pre op to 66.1 at 12 months and 78.9 at 24 months. The Tegner activity score improved from 3.2 preop to 4.6 at 12 months and 5.3 at 24 months. Similarly the Lysholm score also improved, from 53.2 preop to 72.7 and 81.8 (p⇠0.05). MRI evaluation showed that oedema like signal surrounding the plugs resolves around 6 months and the subchondral lamina is seen to reform. At 12 months T2 Mapping indicates that the neo-cartilage shows similar signal to native cartilage. Conclusions TruFit plugs appear to provide good results for repair of small lesions and MRI imaging including T2 mapping relates to clinical improvement., Introduction Recent knowledge regarding tissue biology highlights a complex regulation of growth factors in reaction to tissue damage. Platelet Rich Plasma (PRP), containing a natural pool of growth factors, can be obtained in a simple, low cost and minimally invasive way and be applied to the lesion site. In this prospective comparative study we evaluated if the treatment with PRP injections can reduce pain and increase function in cases of chronic refractory jumper's knee. Methods and Materials We treated 15 patients affected by chronic jumper's knee, who had failed previous nonsurgical or surgical treatments, with multiple PRP injections and physiotherapy, and we compared the clinical outcome with an homogeneous group of 16 patients primarily treated exclusively with the physiotherapic approach. Results No significant differences were obtained with the EQ VAS score and pain level evaluation at 6 months of follow up, as with time to recover and patient satisfaction, whereas a higher improvement of the sport activity level was achieved in the PRP group. Conclusions The clinical results are encouraging, indicating that PRP injections may have the potential to increase the tendon healing capacity in difficult cases with chronic refractory tendinosis., Introduction Pro-inflammatory cytokines play a pivotal role in osteoarthritis as well as after ACL reconstructive surgery. Therefore, controlling the harmful intra-articular influence of elevated interleukin (IL)-1β could be useful. A new treatment option is to administer Autologous Conditioned Serum (ACS/Orthokine) containing elevated amounts of Interleukin-1 receptorantagonist (IL-1Ra) produced from venous patients blood. The study objectives were to measure level and dynamics of IL-1β concentrations in two treatment groups in three post-operative periods. Methods and Materials In a prospective, randomized, double blind, saline-controlled group study we measured the IL-1β concentrations following ACL-reconstruction periodically in 3 synovial fluid and 4 serum samples in 20 patients receiving an injection series with either ACS (Group A) or Saline (Group B). Results The decrease of the IL-1b synovial fluid concentration was pronounced in the ACS Group and values were significant lower in the ACS Group at day 10 (p=0.017). In eight Group A patients serum IL-1β was detected on day 6. In four of them whose synovial fluid levels were over 10 pg/ml on day 6, serum I L-1β was detected on day 10. Correlation between serum and synovial fluid IL-1b appearance persists in patients after ACL surgery and ACS application. At the same time there was no such correlation in Group B. Conclusions The intraarticular application of ACS tends to result in a decrease of IL-1β and indicates a possible influence of ACS on the ACL healing process influencing the IL-1β levels on the basis of the serum IL-1β detection and warrant future investigations into the possible protective effects of ACS/Orthokine., Introduction Joint distraction as treatment of severe osteoarthritis has demonstrated progressive prolonged clinical benefit. The present study describes the first results on changes in tissue structure induced by joint distraction of end stage knee osteoarthritis. Methods and Materials Young patients (⇠60 yrs) with severe osteoarthritis of the tibio-femoral joint, considered for joint replacement, were treated with joint distraction. An external fixation frame bridging the knee joint was placed, distracting the joint 5 mm for a period of two months. In addition to clinical outcome, serum and urine samples were analyzed for biomarkers of cartilage turnover, radiographs were scored digitally for multiple OA related characteristics (KIDA), and MRIs were evaluated for area covered with cartilage, cartilage thickness and volume characteristics. Results The first thirteen patients (48±3yr) have reached a follow-up of 1 year. Clinical condition improved significantly and progressively. Radiographs demonstrated a significant increase in minimum joint space width. These results are corroborated by a gradual increase in cartilage synthesis markers (PIIANP and CS846) and decrease in breakdown markers (CTX-II, COMP) after an initial huge change in cartilage turnover during distraction. MRIs demonstrated a significant increase in cartilaginous tissue volume and covered bone area. Conclusions Absence of mechanical stresses on cartilage, in combination with maintained intermittent intra-articular fluid pressure, and significant increase in peri-articular bone turnover as induced by joint distraction, may be the underlying mechanisms for cartilage repair (demonstrated by different surrogate markers) being involved in the clinical benefits of this very promising treatment., Introduction Until now there are not well established therapies able to prevent the development of osteoarthrits. Methods and Materials We had in our clinical observations a group of young adults under 30 years old, who practiced different sports (athletics, running, ski, basketball, handball, football, gymnastics, tennis, bodybuilding, martial arts) during childhood and/or teenage, now presenting osteoarthritis-like symptomatology of the knee. Therapy with analgesics and/or nonsteroidal anti-inflammatory drugs were not efficient and physiotherapy was slightly helpful in some patients. We have no proof that some sports may trigger a premature osteoarthritis, except the therapeutical evidence that an early treatment with SYSADOA is beneficial by improving rapidly and significantly the osteoarthritis-like symptomatology mainly from the patient's point of view. Results Based on our hypothesis that there is a pre-arthritis state due to micro-lesions of the cartilage sustaining a repeated and intense effort during different sports, we recommended a combination of condroitin 1200 mg and glucosamine 1500 mg divided in 2 doses daily. The osteoarthritis-like symptomatology (crunches, stiffness, pain) was resolved within one month, but the therapy was continued for another month. We monitored the beneficial effect of SYSADOA for a period of 12 months. Conclusions An early therapy with SYSADOA might be a solution for the prophylaxis of knee osteoarthritis in young people after giving up sports. We intend to develop a long term study in order to monitor the effects of exercise on cartilage in the intact human joint in two situations: with physiologic loading conditions and with overloading during different sports., Introduction Although pharmacologic treatment remains the mainstay for treating rheumatoid arthritis, there is an increasing need for a method that biologically regenerates arthritic knee lesions as the patient's life span increases. Methods and Materials Case Presentation A 35-year-old Korean woman with RA was admitted for right knee joint pain. Plain radiographs revealed progression of arthritis with lateral joint space narrowing comparing with radiographs obtained four years previously. The cartilage fragment was sent to a commercial cell culturing facility (SW Cellontech, Seoul) for processing. Autologous chondrocyte implantation was performed six weeks after her initial surgery when 48′106 chondrocytes had been cultured. postoperatively, lateral joint space of the knee became wider and the patient could walk without pain. Results We treated rheumatoid arthritis of the right knee in a thirty-five-year-old Korean female patient by autologous chondrocyte implantation. Twelve months following surgery, the patient could walk without pain. Conclusions Therefore, we believe that autologous chondrocyte implantation appears to be effective for treating rheumatoid arthritis of the knee., Introduction The pathogenesis of osteoarthritis (OA) remains to be elucidated, partly due to limitations in investigative methods. We evaluated the microdialysis technique as a possible in vivo method to obtain dialysates from the intraarticular space and from the synovium of the knee. The technique enables local measurements of cytokines and biomarkers supposedly involved in the pathogenesis and understanding of OA. Methods and Materials Seven patients undergoing planned arthroscopy of the knee due to degenerative changes were included in the study. Following anaesthesia, 2 microdialysis catheters were positioned under ultrasonographic guidance, intraarticularly in the suprapatellar pouch and in the medial parapatellar part of the synovium. The microdialysis catheters were perfused with a solution of Ringer-acetate containing radioactively labelled glucose allowing for determination of relative recovery as an indicator of membrane permeability. The microdialysis catheters were left in situ to determine the actual position during arthroscopy and then removed peroperatively. Results From the perfusion 91% (SD 14%) of the volume, was recovered in the dialysate from the catheters placed in the synovium and 117% (SD 6%) from the intraarticular catheters. Relative recovery for intraarticular catheters was on average 55% (SD 17%) and for the catheters in the synovium 65% (SD 8%). Conclusions Microdialysis of the knee has previously been tried peroperatively2, but has not been methodologically evaluated. We have shown that it is possible to place microdialysis catheters in the synovium of the knee and intraarticularly by ultrasonographic guidance and furthermore to perform microdialysis with a relative recovery comparable to that of other tissues., Introduction There remain some debates on osteochondral autogenous transfer (OAT; mosaicplasty) for an early stage of osteoarthritis in knee joints (OA). The objectives of this study are to investigate effectiveness and validation of OAT for the early OA. Methods and Materials Consecutive series of eighteen cases (eighteen knees) were retrospectively reviewed. Six males, and 12 females, and average age at the surgery was 57.8±12.7 years old. Inclusion criteria was those patients who had isolated chondral lesion (ICRS grade-3 and −4) at medial femoral condyle, whose standing lateral femoro-tibial angle (FTA) was less than 180 degrees, and whose radiographic OA grading was less than 3 (Kellgren-Lawrence (K/L)). Several numbers of osteochondral plugs were transferred from lateral and medial edges of femoral groove. IKDC subjective score, FTA, and K/L grading were assessed pre-operatively and at the final follow-up (18.5±9.9 months in average). Results Post-operative IKDC score improved comparing to preoperative one. FTA significantly increased from 178±2.2 degrees pre-operatively to 179.5±3.0 degrees post-operatively (P=0.0030). In some cases where the pre-operative FTA was close to 180 degrees, the post-operative FTA increased being over 180 degrees. And there was moderate negative correlation between pre-operative IKDC score and the increase in FTA. Regarding K/L grading, nine cases did not change, while 9 cases deteriorated. Conclusions The present study showed favorable short-term clinical outcomes after OAT for the early knee OA where FTA was less than 180 degrees, although OAT cannot improve lower limb alignment. Further long-term clinical study is essential to evaluate the influence of increased FTA on the clinical outcomes., Introduction Mosaic arthroplasty is recognized as viable reconstructive option for cartilage lesions in the 2–4+ cm2 and range. Results have been comparable with more costly and complex cell based techniques, but donor site morbidity and the accuracy of graft harvesting and delivery remain problematic. Restoration of the joint surface relies on complex geometrical fitting of several cylindrical osteochondral plugs that can be re-arranged or rotated around their longitudinal axis to match the geometry of the recipient site. Complex, small radius or re-curved surfaces are difficult to reconstruct accurately resulting in gaps between grafts, protruding or recessed grafts; all of which are associated with a less favorable outcome. The objective of this work was to create a computerized planning and surgical guidance system that could improve outcomes in complex cases. Pilot studies are described here. Methods and Materials A patient-specific 3D virtual knee model is created by registration of CT and MRI imaging studies. Custom surgical planning software is used to define the diameter, location, surface geometry and depth of each plug, then these fitting parameters can be tested in the recipient site. A patient specific template incorporating multiple harvesting and insertion guides is formed in plastic using a rapid prototype printer and then sterilized. This surface template fits in a unique location on the articular surfaces, and provides accurate positioning of mosaic arthroplasty harvesting chisels, and drill guides as well as orientation of the grafts. Results This system has been tested in 8 sheep with impact-induced cartilage defects requiring 3–5 grafts and one patient with a medial condyle cartilage defect requiring 9 grafts. In all cases the immediate postoperative appearance was very good, operative time was similar to traditional surgeries and short term reduction in pain with improvement in mobility appear promising. Long term studies are ongoing. Conclusions Imaging and creation of patient-specific knee models allowed an unprecedented level of preoperative planning as well as production of plastic templates that allowed fast and precise transplantation of osteochondral grafts in areas of the knee with complex curved geometry., Introduction Autologous Osteochondral Transplantation (OCT) is an established method to treat articular cartilage defects in the knee. However, the potential for donor site morbidity remains a concern. Both the restoration of the original cartilage defect and the evolution of the donor site defects can be evaluated by bone scintigraphy. Thus, prospective bone scintigraphic evaluation was performed in patients who were treated with OCT. Methods and Materials In a group of 13 patients with a symptomatic articular cartilage defect bone scintigraphies were obtained pre-operatively, one year after osteochondral transplantation and finally at an average follow-up of 4 years (31–65 mnths). The evolution of bone scintigraphic activity was evaluated for both the recipient and the donor site. Parallel, clinical scoring was performed using the The Lysholm knee scoring, the Cincinnati knee rating system and the Tegner activity score. Results The bone scintigraphic uptake was elevated at the involved femoral condyle preoperatively and gradually decreased to normal levels in most cases after four years. The originally normal uptake at the trochlea, increased significantly one year after transplantation. Then, a gradual decrease in uptake occurred again at this donor site to remain significantly elevated at the final bone scintigraphy. A significant correlation was found between elevated bone scintigraphic activity and the presence of retro-patellar crepitus. Conclusions Elevated bone scintigraphic activity from an osteochondral lesion can be restored with OCT. However, increased scintigraphic activity is introduced at the donorsite, which reduces again with longer follow-up. Number and size of the harvested plugs appear to correlate with retropatellar crepitus and scintigraphic activity., Introduction Fresh osteochondral allograft transplant (OAT) to the distal femora is currently managed post-operatively with a limited weight-bearing (WB) protocol. This study prospectively assesses clinical and radiographic results of an immediate WB as tolerated protocol following fresh OAT with single or multiple cylindrical grafts to the femoral condyle. Methods and Materials Patients with grade IV ICRS defects treated with OAT were allowed immediate full WB. Validated outcome evaluations were collected pre- and post-operatively. CT scans were assessed for degree of osseous incorporation within modified Cahill/Berg zones. Results Six month data from 32 patients (age= 35.2, 15 to 66) with femoral lesions (5.8 cm2, range 0.8 to 19.6 cm2) secondary to focal arthritis (10), OCD (19) and AVN (2) is reported. Statistically significant, p⇠0.05, improvement was observed in all measures versus baseline: KOOS (Pain 57 to 77, Symptoms 58 to 70, ADL 68 to 89, Sports 41 to 65, QOL 23 to 46), IKDC (45 to 56) and SF-36 (56 to 72). CTs indicate grafts implanted to direct WB regions had ⇢75% incorporation (21 of 27 grafts) compared to ⇠50% incorporation in the indirect WB regions (9 of 16 grafts). Greater improvement was seen with single grafts (SG) n=19, compared to multiple grafts (MG) n=13. Indirect WB region inclusive grafts were larger (10.3cm2 vs. 4.2cm2) requiring MG. Incorporation of ⇠50% was associated with fragmentation. Conclusions Fresh OAT with full WB shows osseous incorporation and improved short term clinical outcomes. SG implantation is associated with stable incorporation. MG treatment, which includes indirect WB regions, can lead to fragmentation., Introduction Osteochondral autograft transfer (OAT) procedures involve taking small cylindrical grafts from areas of decreased weight bearing in the knee and transferring them to accurately prepared recipient defect sites. The morbidity of the donor sites, which are selected at the periphery of the articular surfaces, from areas of relatively low load per unit area, has been poorly studied. Methods and Materials We report on a competitive athlete who underwent an autologous osteochondral transplantation for a full-thickness osteochondral defect of the right medial femoral condyle. The patient developed significant morbidity in the donor site. During arthroscopic revision surgery, biopsy of the donor site showed a foreign-body giant cell reaction. Results A revision arthroscopic autologous osteochondral transplantation was performed with good postoperative outcome. The symptoms resolved unremarkably. Physical examination revealed negative patello-femoral signs. The patient has a negative step up-step down test, a negative patellofemoral grinding test, and a negative patellofemoral compression test. The patient returned to sports activity, but not at the previous competitive level. When last reviewed 2 years post-operatively, he was fully active and walked normally with a Lysholm score of 99. Conclusions OATS certainly has a role in managing OCD lesions of the knee, the potential donor-site morbidity and its potential impact on the functional status of the patient have to be taken into consideration when counseling patients about this procedure. Further investigations are necessary to clarify the donor-site morbidity that can occur after OAT., Introduction Large osteochondral defects in the weight-bearing zones of femoral condyles in young and active patients were treated by autologous transfer of the posterior femoral condyle (MegaOATS). This salvage procedure aims at painfree mobility of patients. Methods and Materials 18 subsequent patients were included betweeten July 1999 and December 2000. 16 patients (4 females, 12 males) were evaluated using the Lysholm score and x-rays. A random test of 8 individuals underwent MRI analysis. The average age at the date of surgery was 37,4 (15–59) years, the mean followup 55,2 (46–62) months. The mean defect size was 5,4 cmÂ2 (3,1–7,1). Trauma or osteochondrosis dissecans were pathogenetic in 81%. Results The Lysholm score showed significant increase from preoperatively median 65.0 to postoperative median 86.0 points (p=0.001). 15 patients returned to sport activities. X-rays showed a rounding of the osteotomy edge in 12 out of 14 patients and a partial remodelling of the posterior femoral condyle in 11 patients. Preoperative osteoarthritis in 9 patients was related to a significant lower increase of the Lysholm score (p=0,038). All MRI examinations showed vital and congruent grafts. Conclusions Patients significantly improved in the Lysholm score, in daily life activity levels and often returned to recreational sports. 15 patients were comfortable with the results and would undergo the procedure again. Thus, MegaOATS is recommended as a salvage procedure for young individuals with large osteochondral defects in the weight-bearing zone of the femoral condyle., Introduction Osteochondral autografts (OCA) brings hyaline cartilage when treating full thickness patellar chondral lesions, offering a good alternative for its management. The objective of the present study is to evaluate clinical and functional results of full thickness patellar chondral lesions treated with OCA. Methods and Materials Consecutive series of ten cases were treated in a seven year period evaluated with Lysholm and International Knee Documentation Committee (IKDC) scores. Average follow up 37,3 (12 to 89) months. All patients were diagnosed with patellar instability, six with associated patellofemoral malalignement (PFM). Results Eight cases treated with mosaicplasty (six PFM, two traumatic injuries), two treated with Osteochondral Autograt Transfer System (OATS) (one PFM, one traumatic injury). Average associated procedures for each patient 1,4 (1 to 4). Eight OCA performed in the medial facet, one in the lateral facet, one in the central patellar area. Average chondral lesion area 1,2 (0,9 to 2) cm2. Autograft harvested from superior lateral trochlea in eight cases and from superior medial trochlea in two. Average of 1,9 (1 to 4) grafts used in each patient. Average graft diameter 6,95 (4,5 to 8) mm, average graft length 10,8 (10 to 12) mm. Average post op scores: Lysholm 95 (90 to 100) points, 60% excellent (6 cases) and 40% good (4 cases). IKDC 93,6 (92 to 96) points. No postoperative complications were registered until conclusion of this review. Conclusions Patellar OCA is a good surgical alternative for the treatment of full thickness patellar chondral lesions, offering good and excellent clinical results in the midterm follow up., Introduction The purpose of this study were to evaluate the time course change of glycosaminoglycan concentrations after high tibial osteotomy using delayed gadolinium enhanced MR imaging of cartilage. Methods and Materials A fifty year old male patient who had HTO due to grade IV secondary osteoarthritis was examined using 1.5T MRI. dGEMRIC was performed before and 1,3,6,9 months after the operation. dGEMRIC index which represent GAG concentrations were calculated and color images of articular cartilage layers were described. ICRS grading of the articular cartilage was performed arthroscopically at the time of opening wedge HTO using TomoFix system. Results The articular cartilage of lateral femoral condyle was divided into two layers; low GAG content superficial layer and high GAG content deep layer. Arthroscopic findings revealed medial compartment to be grade IV and lateral compartment to be grade II and III, which were observed in MRI in the same way. Before the operation, dGEMRIC index was 431m, and decreased to 385ms after 3 months, then recovered to 427ms after 6 month, and maintained to 421ms after 9 months. Conclusions dGEMRIC had the potential to evaluate the extracellular matrix of articular cartilage after high tibial osteotomy non-invasively. Temporary decrease and recovery of GAG content was observed after surgery, which indicates extracellular matrix may have self remodeling process after biomechanical changes due to high tibial osteotomy., Introduction The aim of this study was to determine to what extent combination treatment with high tibial osteotomy (HTO) plus chondroabrasion and microfractures could improve clinical symptoms in patients with medial compartmental osteoarthritis in genu varum. Methods and Materials The study population was 40 patients, 20 of which underwent HTO alone (group A) and 20 others received combination treatment with HTO plus chondroabrasion and microfractures (group B). Final assessment was conducted at 5 years after the operation. Assessment included clinical response as measured by the International Knee Documentation Committee (IKDC) and Lysholm scores and patient satisfaction as evaluated on a satisfaction scale. Results A statistically significant improvement in Lysholm scores (P⇠0.05) was noted in both groups but no statistically significant difference in pre- and postoperative IKDC scores was found between the two groups: 80% of group A and 85% of group B patients scored A at postoperative IKDC. Regarding the Satisfaction Score, there was no significative difference between the two groups in preoperative self assessment, while postoperative subjective satisfaction was significantly higher in patients belonging to group A (p=0,004). Conclusions In patients with medial chondropathy, correction of the mechanical axis by osteotomy remains the treatment of choice in those wishing to continue accustomed levels of sports activities. Because it is a biological choice, osteotomy performed together with chrondoabrasion and microfractures allows a fibrocartilagenous layer to regenerate with characteristics very similar to cartilage in areas where the cartilage is still undamaged., Introduction Combined High Tibial Osteotomy (HTO) with Matrix Induced Autologous Chondrocyte Implantation (MACI) has not yet been described in the literature. We have conducted a prospective study of combined procedures in patients with end stage knee osteoarthritis and have investigated the cartilage in-fill on the weight bearing surface of the medial femoral condyle in the hostile environment of the arthritic knee. Methods and Materials There were 18 combined surgical procedures in 15 patients aged between 27 and 58. Functional evaluation using the 5 KOOS-domains independently was compared with preoperative values. MRIs after 3 months 1, 3 and 5 years were scored according to Marlovits. Results There was significant improvement in all KOOS-domains when 1-year results were compared with preoperative values (p⇠0.05). These improvements were maintained at 3 and 5 years. Two patients have been offered salvage Total Knee Arthroplasty after graft failure at 5 years. Histological investigation after the death of one patient (unrelated cause) 18 months after surgery demonstrated full-thickness hyaline-like articular cartilage. MRI improvements were modest from a mean Marlovits-score of 2.2 to 2.4 with subtotal or complete in-fill in only 6 of 18 knees. Conclusions One third of the patients had good cartilage in-fill indicating the possibility of combining HTO with the MACI-technique. However, there was no convincing MRI evidence of substantial in-fill in 12 of the 18 knees. Based on our findings it is difficult to justify the addition of MACI to HTO for young patients with unicompartmental osteoarthritis. The improvements in clinical outcome could be readily attributed to the osteotomy alone., Introduction Our aim was to use a minimally invasive method for management of the young active patient with medial compartment osteoarthritis, without the risk of serious complications. Methods and Materials Prospective data of 28 consecutive knees who underwent Medial opening wedge high tibial osteotomy for medial compartment osteoarthritis using monolateral external fixator, combined with microfracturing was analysed. Mean age was 47 years, follow up was 23 months (range 7 to 61). Results Mean Lysholm scores improved from 63.6 (42 to 85) to 81.6 at latest follow-up (46.5–100) (p⇠0.001). Similarly, the mean Tegner activity scale improved from 1.7 to 3.3 (p⇠0.001). Average IKDC score at last follow-up was 68 (35.6 − 100). Mean pain score improved from 5.5 to 3.5. (p⇠0.001). All but 4 patients reported improved pain scores. The mean mechanical axis angle was improved from 6.07 degrees of varus, to 3.39 degree valgus. The average joint space improved from 2.4 mm to 2.61 mm after surgery. The mean increase was 2.1 mm. Superficial pin site infection occurred in 6 patients (21.4%) and settled with oral antiobiotics in all cases. One patient had persistent patellofemoral (PF) pain. Conclusions The use of hemicallotasis for high tibial osteotomy (HTO) in association with extensive microfracturing of the medial compartment provides a viable, minimally invasive method for management of the young active patient with medial compartment osteoarthritis, without the risk of serious complications. In the short term even with advanced full thickness cartilage damage, this provides effective pain relief, helps put off more major arthroplasty alternatives and improves activity levels., Introduction Aim of this study was to introduce an improved 3D-MOCART score using the possibilities of isotropic 3D-MRI in the post-operative evaluation of patients after matrix-associated autologous chondrocyte transplantation (MACT) as well as to compare the results to the conventional 2D-MOCART score using standard MR sequences. Methods and Materials One hundred consecutive MR scans in sixty patients at standard follow-up intervals of 1,3,6,12,24, and 60 months after MACT of the knee joint were prospectively included. The mean follow-up interval of this cross-sectional evaluation was 21.4±20.6 months; the mean age of the patients was 35.8±9.4 years. MRI was performed at a 3.0 Tesla unit. All variables of the standard 2D-MOCART score where part of the new 3D-MOCART score. Furthermore additional variables and options were included with the aims to utilize the capabilities of isotropic MRI, to include the results of recent studies, and to adapt to the needs of patients and physician in a clinical routine examination. Standard MR sequences were performed to assess the 2D-MOCART score; an isotropic 3D-TrueFISP sequence was prepared to evaluate the new 3D-MOCART score. Results The correlation between the standard 2D MOCART score and the new 3D MOCART was for the eight variables defect fill, cartilage interface, surface, adhesions, structure, signal intensity, subchondral lamina, and effusion; highly significant correlation with a Pearson coefficient between 0.566 and 0.932. The variable bone marrow edema correlated significantly with a Pearson coefficient of 0.257. Conclusions In the clinical routine follow-up after cartilage repair, the 3D MOCART score, assessed by only one high-resolution isotropic MR sequence, provides comparable information than the standard 2D MOCART score, with the possible advantages of isotropic 3D MRI., Introduction There is a clear need for a patient-based questionnaire to measure the outcome of cartilage therapy. The KOOS has been designed for active patients and validated for several stages of osteoarthritis. The aim of this study was to evaluate the clinimetric properties of the KOOS for regenerative cartilage therapy. Methods and Materials A total of 40 patients treated with regenerative cartilage therapy were used in a test-retest setup, with an intermediate of two days. The patients were asked to complete the Dutch KOOS and complementary questionnaires (SF-36, Lysholm, EQ-5D) to evaluate the clinimetric properties of the Dutch KOOS in terms of internal consistency (Crohnbach's alpha), reliability (IntraClassCorrelation (ICC) and Bland Altman plots), construct validity (Spearman's Rank correlation), and floor and ceiling effects. Results For the KOOS subscales and total score the Crohnbach's alpha ranged from 0.74–0.96. The overall ICC of the KOOS was 0.97 while the subscales ranged from 0.87–0.95 and the Bland Altman plots showed a small individual variance between the two assessments in time for each subscale of the KOOS. The Spearman's Rank correlation between the subscales of the KOOS and representative subscales of the SF-36, Lysholm and EQ-5D ranged from 0.59–0.70 with all p-values lower than p⇠0.001. We observed no floor effects while the largest ceiling effect was 10.3%. Conclusions This is the first study evaluating the clinimetric properties of the KOOS for cartilage therapy. We conclude that the validity and reliability of the KOOS is suitable to measure the clinical condition of patients after regenerative cartilage therapy., Introduction There are many different techniques in use to measure the success of cartilage repair. These range from patient self-assessment scores (such as WOMAC) to semi-quantitative imaging scores (such as MOCART). By standardizing image acquisition techniques and using phantoms with known values, it is possible to obtain consistent quantitative measurements using MRI. Without performing a biopsy, it is difficult to assess the composition of the repair tissue. MR imaging biomarkers (T1 and T2 decay times) can serve as proxies for tissue composition. Methods and Materials To obtain structural measurements in cartilage two gradient echo sequences can be used; one which yields good delineation between bone and cartilage (T1-weighted SPGR TR=39ms, TE=7ms, FA=20°, Spacing=1.5mm), and a second that provides good delineation between cartilage, fluid and soft tissue (T2*-weighted GRE TR=29ms, TE=15ms, FA=40°, Spacing=1.5mm). dGEMRIC techniques can be utilized to obtain T1 maps of the repair tissue and surrounding cartilage. Multi-echo sequences are used to obtain T2 maps of the repair tissue and the surrounding cartilage. Results Structural measurements such as defect volume, repair tissue volume and thickness and surrounding cartilage thickness can be obtained from standardized MR imaging sequences. Tissue composition or quality measurements can be obtained utilizing dGEMRIC or T2 imaging sequences. Conclusions Structural measurements determine how much repair tissue has grown, but not the composition of the tissue. The use of tissue-composition biomarkers (T1 and T2) provide further insight into the quality of the repair tissue. Choosing the right biomarkers depends upon several factors including the trial phase, size, cost and anticipated outcome., Introduction Autologous chondrocyte transplantation (ACT) is an increasingly used procedure cartilage defects in the knee. In this cell based technique, it is felt that increasing time results in maturation of the tissue and improved mechanical, histological, and clinical properties. A prospective study of 118 patients was performed to assess the improvement in clinical outcome (IKDC) related to the follow-up interval. Methods and Materials 118 patients (80m, 38f) underwent matrix assisted ACT (NOVOCART™3D, B. Braun - Aesculap, Germany) from 11/03 −12/06 and were prospectively evaluated using IKDC scoring. The total sample was divided into two subgroups: ⇠24 month followup (n= 63, mean 14,7 month, range 3–24); and ⇢24 months(n=55, mean 36 month, range 25–48). Results The subgroup I (⇠24 month, mean 14,7) shows an improvement in IKDC score of 13,7 pts (31,7%). Within the total sample (avg. follow-up: 24,6 months) the IKDC increased 19 pts (47,5%). Subgroup II (⇢24 months, mean 36,1) improved 25,4 pts (68,3%) compared to the preoperative status. A relationship between increasing followup and increasing improvement was identified. Conclusions Our data suggests that the clinical improvement following matrix-ACT increases depending on the follow-up period. Matrix-ACT remains a technique for cell transplantation, and the initial graft does not demonstrate the mechanical and histological properties of articular cartilage. However, as the tissue matures it gains in strengths and durability within the years after transplantation. This process seems to continue over at least 3 years. Our clinical observation correlates with histological data, showing an improvement of tissue quality between 1 and 3 years after ACT [Roberts S Arthritis Res Ther. 2003]., Introduction Osteoarthritis (OA) is a chronic disease, characterized by gradual loss of articular cartilage and functional limitations. A few studies have compared a muscular strength of subjects with OA degrees I and II. The aim of this study was verify the effectiveness of exercise therapy in these patients. Methods and Materials Were selected 10 men (52,9 ± 6,52 years) who performed a test on the isokinetic dynamometer (Biodex System 3) to assess the maximal isometric torque (MIT), the maximal concentric (MCIT) and eccentric isokinetic torque (MEIT) of the quadriceps femoral and the MCIT of the hamstrings muscles. The individuals have undergone a rehabilitation program for 11 weeks and reassessed at the 5th and 11th week. To determine the effect of treatment were analysed used Friedman test (p≤0,05). Results The result showed statistically significant difference in MCIT of the hamstrings, at 90°/s before and after the 11 weeks (p=0.06) and between the 5th and 11weeks (p=0.0005); at 180°/s, for the same torque, was found differences for the same periods (p=0.005 and p=0.02). For the MIT of the quadriceps, MCIT and MEIT wasn't found statistical differences, however, we can observe a qualitative increase of 20% in the MIT of quadriceps (178.8 to 216.7Nm) before and after 11 weeks and 13%, over the same period, for the MEIT at 90°/s (217,55 to 246,51Nm) showing a progress report. Conclusions The rehabilitation program was effective in improving concentric and eccentric strength especially after the sensory-motor training. It is suggested that the rehabilitation training for OA should emphasize strengthening and training sensory-motor., Introduction For a successful outcome after cell based cartilage repair in the knee an optimal patient selection and accompanying physiotherapy planning seems fundamental. In contrast to athletes, patients encompass difficulties to achieve preset goal settings in rehabilitation. Methods and Materials This qualitative study was performed to obtain improved insights on the rehabilitation processes after cartilage repair in the knee. Physiotherapists, specific experienced in guiding patients after local cartilage repair in the knee, attended 4 panel discussions on physiotherapy planning over a 2 year period. Results Experience based suggestions are: 1/Patients have better adherence if there is specific communication on rehab goals and methods every 3 months following surgery. 2/Physiotherapists can set realistic goals if they are well informed on exact location, size and contours of the repair, concomitant procedures and previous pathologic knee conditions. 3/Since patient and therapist often over-estimate the local load ability of the repair zone, rehab protocols with fixed timelines for progression are not recommended. 4/Patient tailored rehab approach is needed to gain knee function progressive and safe. Moreover it allows the physiotherapist to focus systematically on the quality of movements of the operated knee. Conclusions Experienced based recommendations are: The progress in exercise demands should be paced by the biological process that is on-going in the knee joint. Exercises that improve proprioception and neuromuscular control need to be accentuated in low and moderate load conditions. These two movement quality aspects are believed to be most important. Strengthening exercises, which can overload the repair zone, are specifically planned in later phases of rehabilitation., Introduction The purpose of this study was to analyze the morphological characteristics and incorporation of the TruFit CB scaffold plugs used for OATS donor lesion backfill with cartilage-sensitive MRI and T2 mapping. Methods and Materials Twenty-six patients (mean age 28.72 yrs) underwent OATS for chondral defects of the knee or talus with Trufit plug backfill of donor site lesions. 43 cartilage-sensitive MRI's and 25 T2-mapping studies were performed at various postoperative intervals (range: 1–39 months, mean 16.4 months). The donor sites were assessed for plug morphology, displacement, hypertrophy, subchondral edema, bony overgrowth, percentage fill, degree of incorporation, ICRS score of the adjacent and opposing articular cartilage, and T2 mapping of the repair tissue. Results Longitudinal analysis revealed favorable plug appearance at early follow-up (≤6months) with deterioration at intermediate follow-up (ff12months). Plug appearance substantially improved, however, with longer follow-up (≥16 months). Hyperintense signal was associated with a short postoperative interval (p=0.02) or multiple plug configuration (p=0.01). Abnormal morphology was associated with a short postoperative interval (p=0.02) or large plug size (p=0.003). Incomplete defect fill and poor plug incorporation were both associated with intermediate postoperative duration (p=0.02 and p=0.006 respectively). T2 mapping scores significantly improved with increasing postoperative duration (p⇠0.004). Interface T2 scores were better with single compared to multiple plug configurations (p=0.03). Conclusions The MRI appearance of Trufit CB plugs used for filling an osteochondral defect of the knee demonstrates a predictable pattern of postoperative maturation that reflects biological incorporation. Plug appearance significantly improves with increasing postoperative duration with T2-mapping scores that approach native articular cartilage., Introduction The orthopedic community has not found a satisfactory treatment for articular cartilage defects of the knee. The purpose of this retrospective clinical study was to evaluate the short-term results of the treatment of knee osteochondral defects with a synthetic resorbable biphasic implant (TruFit Plug; Smith & Nephew, San Antonio, TX). Methods and Materials Fifteen skeletally mature patients with symptomatic, full-thickness cartilage lesions of the femoral condyles, between 1 and 2.5 cm in diameter, were treated with implantation of bioabsorbable implant. Implants were press-fit into holes drilled into the defect. All patients were evaluated both preoperatively and postoperatively with the Lysholm knee score, International Knee Documentation Committee (IKDC) Standard Evaluation Form, and magnetic resonance imaging of the joint. Results Fifteen patients of a mean age of 39 years were followed-up for a mean time of 9.1 months. The mean Lysholm score improved from 43.6 preoperatively to 87.5 postoperatively. Excellent or good outcome was accomplished in 12 patients. Using the IKDC assessment, 11 patients reported their knee as being normal or nearly normal. Congruency of the articular surface was restored in 13 patients on magnetic resonance imaging. Abnormal marrow signal in the subchondral bone beneath the region of implant was present in all patients. Conclusions The TruFit implant is an effective and safe method of treating symptomatic full-thickness chondral defects of the femoral condyles in appropriately selected cases. However, further studies with long-term follow-up are needed to determine if the implanted area will maintain structural and functional integrity over time., Introduction We have been developing a new tissue engineering technique for cartilage repair which involves a scaffold-free tissue engineered construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). The objective of the present study was to perform an atomic force microscopic analysis and observation on the surface structure and stiffness of mature and immature porcine cartilage-like tissues repaired with the TEC. Methods and Materials Synovium-derived MSCs from the immature porcine knee joints were cultured though 4 to 7 passages. After an addition of ascorbic acid 2-phosphate, the cells were allowed to undergo active contraction for 8 hours to develop the TEC. A cylindrically shaped, cartilaginous defect created on the medial condyle of immature and mature pigs. The TEC was allografted to the defect. Six months after surgery, a cylindrically shaped specimen of repaired tissue was extracted and subjected to a surface observation and micro-indentation test using an atomic force microscope. Results The surface of the TEC-repaired immature and mature cartilage exhibited tubercles of approximately 2–4 mm in height, which was significantly smaller than those observed in the non TEC-repaired cartilage. The stiffness of the immature and mature normal cartilage were 23.8 and 15.0 (x 10–3 N/m), respectively, with a significant difference between two groups. The stiffness of the TEC-repaired cartilage was significantly lower than those of the normal cartilage. Conclusions The present study suggested that the TEC did not enhance the healing of the superficial layer of cartilage-like tissues in both immature and mature porcine femoral cartilage. (Supported, in part, by NEDO (06001904-0) & MEXT (BERC)), Introduction The present study was performed to determine the static and dynamic compressive properties as well as permeability of a cartilage-like tissue repaired with a tissue engineered construct (TEC) (Ando. Biomat. 2007). Methods and Materials Synovium-derived cells from porcine knee joints were cultured though 4 to 7 passages. After an addition of ascorbic acid the matrices were allowed to undergo active contraction to develop TEC. The TEC was allografted to a round-shaped, cartilaginous defect in the porcine femur. Six months after surgery, the static and dynamic compression tests as well as permeability test were performed for a cylindrically shaped repaired tissue specimen extracted from the femur. Results Histological observation indicated that the defect was filled with TEC-repaired cartilage-like tissues. The modulus of the specimens was similar (820 kPa) to that of normal cartilage (620 kPa), and was significantly higher than that of TEC-untreated tissues. However, the accumulated strain was significantly increased (11%) as compared with normal cartilage (8%) at 40s. The permeability of the surface layer of the TEC-repaired tissue was significantly larger (19×10–15 m4/Ns) than that of normal cartilage. Conclusions It is suggested that the surface layer restricts the interstitial fluid outflow in normal cartilage, which contributes adequate compressive properties to the tissue. Although the TEC-repaired tissues exhibited better compressive properties than TEC-untreated tissues, the dynamic compressive property was slightly deteriorated. This may be attributable to a 5-fold higher permeability measured in the surface layer in the TEC-repaired tissues (Supported, in part, by NEDO (06001904-0) & MEXT(BERC))., Introduction The purpose of the present study is to evaluate the efficacy of synthetic resorbable scaffolds transplantation of the talar dome with MRI and clinical short-term follow-up. We therefore present surgical technique steps and early results at one year obtained with preformed in shape and size bone graft substitutes in repair of III and IV degree full thickness osteochondral defects of the talus. Methods and Materials The utilized implant is a cylinder composed of poly(D,L-lactide-co-glycolide) to which calcium sulphate and surfactant are added to enhance bone in-growth and make implant's surface more hydrophilic. The three-dimensional porous cylindrical implant with interconnected pores is press fit into the site for close apposition and encourage migration of repair tissue as blood and marrow into the scaffold. The two layer construct of the implantmimics the mechanics of the surrounding tissues, bone and cartilage, in order to facilitate from the beginning the nature of the repair tissue that will be formed. The plugs are available in different sizes (5,7,9 mm) are preformed in order to match the talus dome surfaces. The first 15 patients were included in the study (7 women, 8 men). Every patient has underwent arthroscopic ankle asse ssment to evaluate size, location and degree of defects and has underwent implantation of TrufitTM cylindrical resorbable scaffold. Majority of synthetic bone substitutes implanted were 7 mm in diameter. Results All surgical procedures have been completed uneventfully. Patient have been controlled clinically and by serial ankle MRI's and showed statistically significant improvement of AOFAS scores associated to healing of defect and integration of bone plugs in absence of adverse reactions. Conclusions Preliminary results enable us to conclude that porous, resorbable scaffolds can be used in treatment of cartilage defects offering a secure support to secondary bone in-growth with the advantage of being applied in one single step procedure, enabling patients to quickly move back to previous daily and sport activities, Introduction A cohort of young patients has recently been described with end-stage glenohumeral arthritis following shoulder stabilization surgery. This study investigates the demographics of patients with post-arthroscopic glenohumeral arthritis and elucidates the factors associated with the development of their condition. Methods and Materials Between 2003 and 2008, 20 patients (mean age 21.8 years) were referred for management of glenohumeral arthritis developing after stabilization surgery. Records were reviewed for initial injury, surgical treatment, and symptom development. Standardized shoulder surveys were provided at referral and surgical follow-up. Results Post-surgical pain pump usage was a contributing factor for degenerative glenohumeral changes in 16 patients, with the remainder related to failed anchor placement (2) and radiofrequency device usage (2). All patients had a recurrence of pain at 5.6 months post-operatively, which was accompanied by a decreased range of motion in 15 cases. Grade 4 chondral damage encompassed over 50% of the humeral head, while glenoid involvement was variable (0% to 100%). Of the 20 patients, 18 have undergone a post-referral surgery, including 13 biologic resurfacing procedures. Nine patients with glenohumeral resurfacing have completed a shoulder function survey at their most recent follow-up (mean 2.5 years). A statistically significant improvement was achieved on both the SST and ASES scales. Conclusions Severe glenohumeral arthritis in young adults is a devastating complication of stabilization surgery. Although not a universal finding, the use of postoperative glenohumeral pain pumps is concerning. The use of biologic resurfacing is promising for this cohort, but further long-term follow-up data is required to determine on the efficacy., Introduction This study tested the hypothesis that MAP kinase inhibitors enhance chondrogenesis and suppress hypertrophic changes during chondrogenesis from MSCs. The effect of PD98059,(an ERK1/2 inhibitor), and SB203580,(a p38 inhibitor), were tested on bone marrow-derived mesenchymal stem cells (BMMSCs) and adipose-tissue-derived mesenchymal stem cells (ATMSCs). Methods and Materials In vitro pellet cultures were carried out using 2.5 × 105 MSCs in chondrogenic medium containing 5 ng/ml of TGF-p2 for BMMSCs, and 5 ng/ml of TGF-p2 and 100 ng/ml of BMP-7 for ATMSCs. From the 14th day of culture, subsets of the pellets were additionally treated with PD98059 [0, 1mM, 10mM] or SB203580 [0, 1mM, 10mM]. After two more weeks of in-vitro culture, pellets were harvested for analysis. Results Treatment of PD98059 increased DNA contents and GAG amounts in both BMMSCs and ATMSCs. Real time PCR analysis showed COL1A1 mRNA decresed to almost a quarter in PD98059 treated BMMSCs, but did not chang in ATMSCs. The mRNA levels of SOX-9 and COL2A1 increased several fold in BMMSCs and ATMSCs after PD98059 treatment. The gene expression of Runx-2, and, to a lesser degree, COL10A1, decreased after PD98059 treatment in both BMMSCs and ATMSCs, whereas, SB203580 elevated their expressions in both cell types. Type I collagen expression was significantly declined by PD98059 in both BMMSCs and ATMSCs, but was not significantly changed by SB203580 in both cells type. Safranin-O and type II collagen expression were increased in both BMMSCs and ATMSCs by PD98059. Whereas SB203580 had moderate increase in Safranin-O and type II collagen expression in BMMSC and no observable effect in ATMSCs. On the other hand type X collagen and Runx-2 protein expressions were reduced by PD98059 in both BMMSCs and ATMSCs, but were slightly increased by SB203580 in both cells type. Conclusions Our study demonstrates the usefulness of the ERK 1/2 inhibitor, PD98059 for the promotion of chondrogenesis and the suppression of hypertrophic changes. This finding could be helpful for cartilage tissue engineering from MSCs.

The soccer athlete has a greater incidence of overuse and acute knee injuries and as a consequence is at most risk for chondropenia and osteoarthritis. Chondropenia conceptually defines the complex nature of the multivariable processes over time including acute and chronic injury, modulators and the aging process. The role of the Sports Medicine team is to prevent injury, restore the joint and ultimately return the athlete to sport while preventing Osteoarthritis. The clinical consequences of full thickness Articular Cartilage defects are pain, swelling, mechanical symptoms athletic and functional disability and osteoarthitis. In the soccer athlete it is articular cartilage that confers the highest levels of performance. It is the fact that any partial or full thickness loss results in loss of day to day resilience and a spectrum of soreness, stiffness, pain and swelling and most importantly. These injuries may be career- ending In this group of high demand participants any increase in activity and loading beyond the articular cartilage threshold for injury, results in a clinical overuse response with the potential negative adaptive consequences of chondropenia and an increased risk of developing osteoarthritis. Lesions to articular cartilage are common acutely but are associated with ACL injuries and long-term chondropenia and Osteoarthitis. The challenge of ACL injury reduction, articular cartilage repair and regeneration and osteoarthitis prevention in the soccer athlete continues to be significant despite significant recent advances. The purpose of this lecture is to present the current concepts with respect to the Athlete's Articular Cartilage the histological, biochemical and clinical implications and contemporary treatments from a historical and an evolutionary prospective as applied during or after their competitive years., Use and abuse of joints, as well as joint trauma can influence the development of osteoarthritis (OA). Soccer is a sport that combines high joint loading and a considerable risk for injuries, especially knee injuries. Since soccer is the most popular sports activity in the world with about 40 million participants it is a useful model for studying OA. An increased risk of hip and knee OA has been shown in former top level soccer players regardless of injuries. There has been much focus on sports related knee injuries and the risk of developing posttraumatic knee OA early in life after these injuries. A knee injury in sports is often associated with an anterior cruciate ligament (ACL) injury, and this injury has been studied extensively both concerning the short and long term effects. The incidence of an ACL injury is almost 1 injury per 1000 of the physical active population1. According to the national ACL registers in the Scandinavian countries some 50% of the injured patients will require an ACL reconstruction. In the US this figure is higher and up to 200 000 reconstructions are performed each year. The indications for surgical intervention are to ensure a return to preinjury activity level or to reduce the risk of osteoarthritis. However, there is not much scientific data supporting either the ability to return to sport or reducing the risk of OA with surgery. Many of the studies have an uncontrolled design and other methodological shortcomings making the interpretation difficult. Top level athletes are usually not taking part in the studies. On group level most studies on ACL reconstruction ends up with an activity level of 6–7 on the Tegner scale compared to a preinjury level of 9. A literature research on OA after ACL injury showed that regardless of treatment about 50% of the patients had radiographic OA 15 years after the injury2. However there is a wide range, from 10–90 %. Many studies point out that an associated meniscus tear increases the risk for OA. The meniscus problem is complex since it is not fully understood if the meniscus tear is entirely due to a trauma or if it is a part of an OA process. If it is a part of the OA process, it is not surprising that it is associated with a higher risk of OA. A reduced risk for secondary meniscus tears has been reported after ACL reconstruction compared to non reconstructed patients. This has also been suggested as an important factor in favour for surgery. However, the lowest prevalence of OA after ACL injury has been noted in a cohort of 100 non-surgically treated patients, and especially in the subgroup of patients without meniscus injury. At follow up more than 75 % had stayed with the non operative treatment3. The 22 who underwent surgery during the follow up had severe problems with instability or a repairable meniscus tear. In the whole group the activity level was 4.5 (median) after 15 years which is comparable to an uninjured population at the same age (45 years). In a recent report from the Danish ACL registry the activity level was 5 on the Tegner scale one year after ACL reconstruction. The mean age was below 30 years of age. Obviously the risk for OA differs among patients after an ACL injury. The risk seems to be influenced by the trauma itself. Compression forces differ depending on the type of trauma, and in a report from 1995 it was shown that ACL injury after soccer had more associated meniscus injuries than knees with an ACL injury after alpine skiing4. Many studies report that bone marrow edemas (BME) are associated with an ACL injury, depending on the MR technique in up to 90% of the cases. The BME may act as a footprint of the trauma. With quantified MR technique one study indicate that a greater trauma is associated with a lager BME. The study showed that a larger BME was related to compression fractures, mainly laterally located in the knee joint. Structural cartilage changes were also seen, and these changes were more pronounced in the surgically treated patients compared to non surgical treated5. With the contrast enhanced MR technique, there are data showing persistent GAG loss in the cartilage up to 2 years after an ACL injury, which verifies the results from studies on joint fluid markers6. A low GAG content could indicate that the matrix is not normalized and probably have a reduced capacity of load absorption, thus making it more vulnerable much longer after a trauma or a surgical procedure than was known previously. An early return to high demanding sports could in this perspective be quite harmful for the cartilage. A normal time frame at present, regarding return to sports after an ACL injury is 6 months, while these findings eventually indicate that the cartilage is not ready for high demanding sports until much later in the course after an injury. In the published studies with a lower prevalence of OA it seems to have been a very controlled rehabilitation or even a slow return to demanding activities. If there will be a fast return to sports activities in combination with persisting biomechanical abnormalities and neuromuscular deficits, this may strongly contribute to the OA risk after an ACL injury. Although it seems clear that some patients do benefit from an ACL reconstruction, there is no generally accepted treatment algorithm based on evidence from the literature. A problem is that the natural course is not known. There is a hope that the new technically demanding double bundle reconstruction shall be the solution. It seems like this technique will decrease the rotational instability compared to the traditional single bundle technique. This fact may not improve the patient satisfaction in the short run, but may, theoretically, have positive impact on the risk of OA. The main problem after an ACL injury certainly is the high risk of OA. It is known that females have an increased risk to sustain an ACL tear. This means that many of the young girls with ACL tears already as teenagers will have OA very early in life with well known consequences. There are possibilities to reduce the risk of OA after a sports related knee injury, the main solution today is probably not surgery. For the future it seems extremely important to continue the work on ACL injury prevention, but also to find methods for the identification of the patients at risk to develop early OA after such an injury. The increased understanding of the post trauma OA process and its determinants is of greatest importance to be able to find methods to reduce the risk., The field of cartilage repair suffers from a lack of randomized studies and thus a consensus on treatment or product efficacy. Unfortunately, while post-operative rehabilitation is recognized as a critical component for achieving outcome success in cartilage repair, it too suffers from little or no comparative data, suggesting that greater efforts are needed in studying rehabilitation concurrent with cartilage repair. This is particularly important for cartilage repair clinical trials being used to meet regulatory requirements, since regulatory agencies are now expecting to see standardized rehabilitation data in their submissions. Such standardization and control of rehabilitation offers unique challenges, particularly in multicenter studies of large or varied geographic scale. Therefore, the design of studies should make careful consideration in the choice, implementation and follow-up of post-operative rehabilitation. Physiotherapy has not been well-studied with regards to cartilage repair, and in some cases orthopaedic surgeons have developed their own preferred programs with little or no scientific basis. But for the most part, patients are relegated to follow programs established by the therapist themselves. Therefore, the standardization of the rehabilitation within a trial must begin with the selection of the appropriate therapeutic modalities and post-operative timeframe, such as the period of restricted weightbearing, or the use of continuous passive motion (CPM). Furthermore, clinical trials which compare cartilage repair therapies that differ in their repair mechanisms- as with an osteochondral graft device versus microfracture-might call for different programs altogether. Close consultation between the investigators and physiotherapists in designing the program is needed. Selection and training of physiotherapist is a critical step in successfully implementing a standardized study-wide rehabilitation program. International studies offer the challenge of multiple languages and may need to employ therapists with differing educational backgrounds including athletic, physical and rehabilitation therapists, for example, who have appropriate experience. Direct training and communication with all physiotherapists is critical, especially with regards to treatment, lesion size and location or other unique study components. Furthermore, the logistics and financial management of such a relationship with the clinic or hospital providing the rehabilitation service needs to be well established in advance of patient treatment. The objective of a solid standardized trial rehabilitation program should try to avoid the situation where compliance becomes a function of willingness or ability to pay. Patients should be well informed regarding the importance of compliance to both the health of their knee as well as the clinical study. Encouragement should come from the investigator, the clinical site as well as the recruited physiotherapists. A randomized clinical trial for cartilage repair is an excellent opportunity to also understand the influence of rehabilitation on repair outcomes. Even simple variables regarding the patient compliance, the progression of range of motion and weightbearing, could be informative as well as more complex data obtained from functional tests. The extent of the data and the method of collection should be established with the physiotherapists prior to treatment. Finally, consideration should be made as to the role that physiotherapy plays within a study protocol. Regulatory requirements have not been clear regarding the level of statistical analysis and correlation to treatment outcomes which must be carried out. This issue should be clarified-particularly if poor rehabilitation compliance is to be viewed as a protocol deviation, and negatively affects the final sample size in study analyses. The need for standardized rehabilitation in cartilage repair clinical trials which will support regulatory submissions now requires a more concerted effort to be put into the management and tracking of post-treatment care. A well-implemented program with experienced physiotherapists will certainly serve to normalize cartilage repair outcome data, while at the same time focusing more attention on the specific rehabilitation modalities currently used but for which there is little scientific support. And although regulatory rules do not explicitly describe the analyses required for rehabilitation data, any correlations to repair outcomes obtained during these regulated trials will bring valuable insight for the clinical practice of cartilage repair., Rehabilitation is generally accepted as being an important component of cartilage repair surgery. However, the degree to which the rehabilitation process has the potential to influence the outcome of cartilage repair surgery, either positively or negatively, has not been fully elucidated to date. Can the rehabilitation process influence the quality and composition of the cartilage repair tissue; optimise repair tissue maturation; maximise repair tissue durability; increase patient satisfaction; and/or facilitate faster return to work and activity for patients? These are just a selection of the many questions to be asked with a view to being answered. The primary goals of a cartilage repair rehabilitation programme are the local adaptation and remodelling of the repair tissue; a return to function; and the reduction in the risk of further degeneration in later life.[1] Cartilage repair tissue maturation is a long process that has been shown to take more than 18 months to be completed.[2] Consequently, the postoperative rehabilitation programme needs to balance getting each patient back to function as quickly as possible whilst respecting the time that it takes for the body to return to homeostasis and the repair tissue to adapt. The evolution of cartilage repair procedures over the last twenty years has resulted in an ever-increasing range of surgical procedures with chondral repair tissues that vary in the timescales needed to accept loading. Despite this variability in timescales each person undergoing cartilage repair progresses through the three rehabilitative phases of: protection, function and activity. These are not discrete phases and at any one point therapy may be split over more than one phase. Differing therapeutic interventions and modalities can be utilised to work with the patient to help them progress through the postoperative period of protection, to assist in the restoration of function and prepare and support the patient for a return to activity. Rehabilitation protocols are the frameworks that guide patients and their therapists in the formulation of the individual rehabilitation programme. Cartilage repair patients are a very heterogeneous group and postoperative rehabilitation programmes need to be individualised based on the surgical procedure, the nature and history of the lesion and the individual patient characteristics. The translation of emerging basic science to the clinical environment necessitates an understanding of the fundamental principles of the biology of healing, clinical biomechanics, exercise programming and cartilage maturation timescales. The challenge for the therapist is to apply these principles to construct an individualised rehabilitation programme that has a load bearing-mechanical match to the status of the repair tissue at any postoperative point in time. Factors that have been shown to influence the rehabilitation process and therefore need to be incorporated into the rehabilitation programme design process include: lesion size; lesion location; concomitant procedures; preoperative duration of symptoms; age; preoperative baseline condition; and individual patient motivation and goals. In considering the importance of the rehabilitation process to the overall outcome there are areas of controversy, a situation that is compounded by the historical poor reporting of rehabilitation in published studies on articular cartilage repair.[3] Graft delamination is frequently cited as one of the main concerns following autologous chondrocyte implantation (ACI) procedures and, where this has materialised, it is often anecdotally attributed in single case reports to over zealous rehabilitation and/or too early return to sports activity. Consequently, initial rehabilitation protocols tended to be focused on minimising the risk of graft delamination by placing restrictions on weight bearing and range of movement with the aim of protecting the graft from deleterious forces. This was reflected in rehabilitation protocols advising restrictions on weight bearing and ranges of movement that sometimes lasted in excess of 12 weeks. [1] However, the most common adverse events following ACI that are currently reported in the literature are not always graft failure and delamination. Recent studies have reported arthrofibrosis/joint adhesions and graft hypertrophy as being the most common serious adverse event.[4, 5] Additionally, the STAR study reported that the most frequent surgical intervention performed in the first 6 months after ACI was lysis of adhesions.[4] This relatively high incidence of arthrofibrosis and joint adhesions could well be at least partially attributed to postoperative rehabilitation guidance that was too restrictive regarding joint mobility. An additional consideration is that therapists with limited experience of working with cartilage repair patients may well take even more of a conservative approach in their implementation of the rehabilitation protocols. In 2008 Ebert et al. published the results of a study on a traditional vs. an accelerated approach to post-operative rehabilitation following matrix-induced autologous chondrocyte implantation (MACI).[6] The traditional approach entailed the first 6 postoperative weeks being non-weight bearing whereas the accelerated approach incorporated earlier partial weight bearing and a more graduated series of progressions to full weight bearing. One of the key findings of their study was that regardless of the rehabilitation protocol employed, no patient suffered any adverse effect to the implant. We have been indoctrinated by the ‘form follows functionÂ’ argument but how relevant is that to cartilage repair? Do chondrocytes need the biomechanical stimuli and if so in what form, over what time period, with what progressions? These are the questions that therapists are asking of basic science and where the therapists are looking for the translation of the science into the clinical arena. Lateral integration of the new cartilage repair tissue has been identified as a chronic problem in cartilage repair.[7] Physical demands need to be placed on the neocartilage repair tissue but there is a delicate balance between exposing the repair tissue to sufficient loading to stimulate chondral matrix production and minimising the exposure to levels and types of loading that could lead to mechanical failure or poor integration of the repair tissue. Recent research in an animal model has shown that rehabilitative joint motion in the form of continuous passive motion stimulates chondrocyte PRG4 metabolism.[8] However, it is not know whether rehabilitative joint motion in the form of active motion in vivo has similar effects on PRG4 metabolism. There is a higher incidence of graft hypertrophy following ACI in periosteal and patella ACI repairs. One explanation given for the patella repairs exhibiting higher levels of hypertrophy is that higher patellar shear forces provide stimuli for hypertrophy.[9] Minimisation of shear forces requires not only a precise knowledge of the repair location but also the ability to select and adapt rehabilitative exercises to suit the location of the repair and the joint arthrokinematics. [10] The counter argument that could be postulated is that early postoperative range of movement restrictions and reduced weight bearing could facilitate the graft hypertrophy. Finally, but by no means least, rehabilitation is important to the patients themselves. The regular input from therapists during the long rehabilitation process provides a valuable personal support mechanism. The therapist has an important role to fulfil in not only guiding (progressing and/or regressing) the rehabilitation but also in keeping the patient motivated and adherent to their rehabilitation throughout its duration. In conclusion, there is still limited direct evidence to support the importance of rehabilitation in cartilage repair but that is increasing. There is a need for further research to evaluate optimal rehabilitative practice for existing cartilage repair procedures and to determine the key factors that can be optimised to improve the overall results for current patient cohorts. Concurrently there is also a need to track new surgical developments to anticipate future rehabilitation requirements as issues such as whether or not mesenchymal stem cell based cartilage repairs will need different rehabilitation protocols to chondrocyte repairs emerge., ICRS Rehabilitation and Sports Committee Injuries of the articular cartilage surfaces of the knee are observed with increasing frequency in athletes. Particularly participation in pivoting sports such as football, basketball, and soccer has been associated with a rising number of sports-related articular cartilage injuries with higher injury rates at the competitive and professional level. Injuries of the articular cartilage surface of the knee in the athlete can often occur in association with other acute injuries such as ligament or meniscal injuries, traumatic patellar dislocations, and osteochondral injuries and have been described in up to 50% of athletes undergoing anterior cruciate ligament reconstruction. Besides acute traumatic injury, articular cartilage injury can develop in the high-impact athletic population from chronic pathologic joint loading patterns such as joint instability or axis deviation. While intact articular cartilage adjusts to the increasing weightbearing activity in athletes by increasing cartilage volume and thickness in a linear dose-response relationship, recent studies indicate that this dose-response curve reaches a threshold and that activity beyond this threshold can result in maladaptation and injury of articular cartilage. High-impact joint loading above this threshold has been shown to decrease cartilage protoglycan content and to increase levels of degradative enzymes and chondrocyte apoptosis. Over time the integrity of the functional weight bearing unit is lost and a chondropenic response is initiated that can include loss of articular cartilage volume and stiffness, elevation of contact pressures, and development or progression of articular cartilage defects. The limited spontaneous repair following acute or chronic articular cartilage injury is well documented. Recent reports demonstrated that hyaline cartilage defects in athletes resulted in significant pain and swelling and were associated with marked life-style changes and limitation of athletic activity. Some long-term data in athletes with isolated severe chondral or osteochondral damage in the weightbearing condyles showed a 75% initial return to sport initially, but a significant decline of athletic activity was observed over time with development of radiographic evidence of osteoarthritis in the 45–60% of athletes 14–34 years after the injury. These results are supported by the up to 12 fold increased risk of knee osteoarthritis in high-impact athletes established by the National Institute of Health (NIH) and other independent studies. Untreated articular cartilage defects have been shown to result in significantly worse long-term joint function. The high demands on the joint surfaces in athletes make treatment of articular cartilage injuries and restoration of the injured joint surfaces critically important to facilitate continued athletic participation and to maintain a physically active lifestyle. The documented detrimental effect of high-impact articular loading in the athletic population requires cartilage surface restoration that can effectively withstand the significant mechanical joint stresses generated during high-impact, pivoting sports. Besides reducing pain, increasing mobility and improving knee function, the ability to return the athlete to sport and to continue to perform at the pre-injury athletic level presents one of the most important parameters for a successful outcome from articular cartilage repair in this challenging population. Treatment of articular cartilage injuries in the athletic population has traditionally presented a significant therapeutic challenge. However, development of new surgical techniques has created considerable clinical and scientific enthusiasm for articular cartilage repair. Based on the source of the cartilage repair tissue, these new surgical techniques can generally be categorized into three groups: marrow stimulation based techniques, osteochondral transplantation techniques, and cell-based repair techniques. Several studies have evaluated the microfracture technique specifically in the athletic population. These studies included recreational and professional athletes with follow-up ranging from 2–6 years. Activity scores and knee function scores increased significantly after microfracture in these athletes. Athletes were able to successfully return to high-impact, pivoting sports including football, soccer, alpine skiing, basketball, rugby, and tennis. Return to sports was reported at an average of 6.5–10 months. However, there was marked variability in the ability to return to sport after microfracture. The ability to return to athletics at the preoperative level also varied significantly. Return to professional and competitive level sports was much better than to recreational athletics. A recent study showed that while return to sports participation after microfracture can be achieved rapidly, performance and playing time will increase gradually to full participation. Return to high-impact sports after microfracture has been found to be higher in younger athletes with small lesion size, shorter preoperative symptoms, and without prior surgical intervention. A decline of initial improvement of postoperative sports participation was observed in some studies after microfracture in athletes and occurred between 24–37 months, however, activity levels and functional scores were still better than at baseline. Osteochondral mosaicplasty has also been specifically evaluated in athletes. Up to 95% good or excellent results with improved functional scores and MRI rating have been reported. Return to full athletic activity was reported in 61–93% of athletes at an average of 6.5 months. Longer preoperative symptoms and increased athlete age resulted in delayed return to sport after mosaicplasty. Preoperative radiographic or clinical evidence of joint degeneration predicted a return to sport at a lower level or even retirement from competitive sports following mosaicplasty. Prospective randomized comparison of mosaicplasty and microfracture in athletes reported significantly better results with mosaicplasty at an average of 36 months. While studies have evaluated autologous osteochondral transfer in athletes, no specific information has been reported on this technique using allograft in this population. Autologous chondrocyte transplantation has recently been evaluated in the demanding athletic population. Good to excellent results were demonstrated in 72–96% with significant improvement of activity score. Best results were obtained with single cartilage lesions of the medial femoral condyle. Return to high impact-athletics was higher in younger, competitive athletes, with short preoperative intervals while return in recreational athletes was less predictable. The time to return to sport was shorter in competitive level athletes. Athletes often returned to the same skill level and a high portion of returning athletes maintained their ability to perform 52 months after chondrocyte implantation. Return to athletics was better with fewer prior surgeries but return to sports was successful also with autologous cartilage transplantation as a salvage procedure. Combined pathology such as malalignment, ligamentous instability, or meniscal injury and deficiency is frequently encountered by the surgeon treating articular cartilage defects in the athletic knee. Surgically addressing these concomitant pathologies is critical for an effective and durable articular cartilage repair. Recent data demonstrated that isolated or combined adjuvant procedures have no significant negative effect on the ability to return to athletics after microfracture, mosaicplasty, or autologous chondrocyte transplantation. In conclusion, articular cartilage repair in athletes is aimed at returning the athlete to the pre-injury level of athletic participation without increased risk for long-term arthritic degeneration. Several surgical techniques have been shown to improve function and athletic activity after articular cartilage repair in this population but the rate of improvement and ability to return to athletic activity is dependent on several factors. The choice of repair technique should be tailored to individual patient and lesion characteristics using an established treatment algorithm. Long-term studies in this population will determine the efficacy of articular cartilage repair to reverse chondropenia and to prevent development of secondary arthritic degeneration., Introduction Chondropathies of the acetabulum and the femoral head are a frequent cause of pain and functional limitation. The incidence of acetabular cartilage damage is estimated to be of 74% in a total of 736 hip arthroscopies. Furthermore there is an association between cartilage damage and lesions of the acetabular labrum in 81% of the cases. Currently, treatment of hip cartilage pathologies is based exclusively on arthroscopic debridement, microfractures, multiple femoral head perforations or fibrine glue injection for chondral delamination. The purpose of this study was to report the results obtained in treating hip chondropathies using the arthroscopic ACT or AMIC technique. A comparison between the two techniques and results was made to evaluate advantages and disadvantages of these two procedures, Materials and Methods A controlled retrospective randomized study was carried out on 182 patients affected by a hip chondropathy of 3rd and 4th degree, according to the Outerbridge classification, extended 2cm2 or more. 120 of these patients underwent arthroscopic autologous chondrocyte transplantation (ACT), while the other 62 underwent arthroscopic autologous matrix induced chondroplasty (AMIC). The surgical treatment, in those cases treated by ACT, was always carried out in two steps. The first was a diagnostic arthroscopy used to evaluate the chondral damage and to take a cartilage biopsy from the area surrounding the pulvinar. In the second step the transplant was implanted by arthroscopy. On the contrary the AMIC procedure was carried out as a one step procedure. Once the chondral defect was located, the area was cleaned and microfractures were performed. Than the collagen menbrane was applied to cover the defect. In that cases treated with the ACT procedure, the chondrocyte culture was carried out on a polymer scaffold, which is a reabsorbable composite material of polyglactin 910 and poly-p-dioxanone, in 65 cases and on a Hyaluronic acid scaffold in 55 cases. In all the cases treated with the AMIC procedure a suine collagen membrane, added with autologous growth factors, was applied to cover the chondral defect. The two groups were similar in age, gender, degree and location of the pathology. The mean follow-up was 23.8 months (36 to 12) in the group of patiets treated with the ACT procedure and 22.6 (36 to 12) in the group of patients treated with the AMIC procedure. The mean size of the defects was 2.6 cm2 (2.0 – 4.8) in the ACT group and 2.8 cm2 (2.0 – 5.0) in the AMIC group. All the patients were assessed before and after the procedure with the Harris Hip Score (HHS). Postoperatively all the patients underwent physiokinesitherapy Exercises began from the 1st postoperative day. Patients were discharged on the 2nd day and were subject to both active and passive physiotherapy to regain complete range of motion without putting any weight on the articulation for 4 weeks. Partial load was allowed after 4 weeks, when exercises on a gym bike and swimming were recommended. After 7 weeks, crutches were no longer required and the patients were allowed to return to normal work activity. Jogging was allowed only after 6 months, while a complete return to sports activities was recommended only one year after the surgical procedure. Results The mean preoperative HHS in the group of patients treated with the ACT procedure was 52 (32 – 60), similar to that of the patients treated with the AMIC procedure that was 48 (28 – 56). Mean post operative HHS results in both groups were also similar: ACT = 86 (58 – 92); AMIC = 88 (56 – 98), showing no significant difference. In both groups, unsatisfactory results were recorded in those patients suffering from a cartilage defect on the femoral head or where standard x-rays showed a reduced or compromised articular space. Discussion Knee arthroscopy has for some time now been able to show the present of chondral lesions and has allowed for the development of the current surgical techniques used for treating these lesions. Even hip arthroscopy, although considerably less common, has allowed for chondropathies in this area to be detected. The therapeutic approach is different, however, since the hip is a deep articulation surrounded by large muscular masses that make surgical access difficult. Hip arthrotomy exposes the articulation to the serious risk of aseptic necrosis of the femoral head, along with being a significantly invasive procedure. The arthroscopic approach to the treating hip chondropathies, therefore, solves the serious problem regarding arthrotomy. The AMIC procedure have several advantages compared to the ACT. Fist of all it is a one step procedure, with no need to expose the patient to a second operation. The other advantage is that there is no need for a logistic support to the procedure, having no external laboratory support. Considering that the post operative results obtained with the two procedures showed no significant differences, the AMIC appears to be much less invasive and more cost effective compared to the ACT. The cartilage defects located on the acetabulum can be treated with athroscopic ACT or AMIC procedure. This study shows the effectiveness of the AMIC procedure respect to the ACT., ChondroCelect, a cell therapy product for the repair of damaged cartilage, has been developed according to the requirements of a medicinal product. This encompasses the definition of quality attributes for the manufacturing and release of the product, preclinical demonstration of safety and proof of concept, as well as demonstration of clinical safety and efficacy through a controlled clinical trial. Development of cell therapy products according to the medicinal product requirements represent a series of challenges, and necessitates a specific translation of these requirements adapted to the nature of these products and their clinical evaluation. Manufacturing of medicinal products requires demonstration of consistency of manufacturing as well as the establishment of stringent product release criteria. Given their complex and biological nature, cell therapy products represent a more difficult type of products to manufacture and characterize when compared to chemical or other biological products. Moreover, autologous products represent an additional challenge since they consist each time of a unique product derived from a defined autologous source, with inherent biological variability. Demonstration of manufacturing consistency of autologous cell products requires therefore a well-defined manufacturing process and knowledge of its associated variability. Definition of the product release criteria necessitates a good understanding of the variability ranges of the product quality attributes, whilst meanwhile clearly establishing the minimal criteria to ensure product safety and quality. Testing of a cell therapy product in non-clinical models has also its particular challenges and limitations. Two specific issues are to be noted in this respect. The first is the relevance or representativity of the animal model. Indeed, whilst large animal models exist for studying cartilage repair by cell implantation, the differences in cartilage biology as well as the difficulty to control the post-operative rehabilitation represent major limitations towards its relevance for the situation in humans. The second potential issue relates to the fact that the medicinal product itself, i.e. human cartilage cells, is of xenogenic nature to the animal and can thus elicit an immune response. Despite these inherent limitations, the non-clinical models can provide important information on the proof of concept of the therapy (by using autologous animal cells) as well to document key safety aspects like cell dispersion and adverse events. A key aspect in the development of medicinal products, being it classical drugs, biologicals like monoclonal antibodies or vaccines, or advanced therapy medicinal products for cell therapy, is the demonstration of safety and efficacy in well-controlled clinical trials. The gold standard of such trials is a prospective, randomized, controlled clinical trial, comparing the new product to an established therapy or placebo. With respect to performing such trials in the field of cartilage repair, a series of particular issues can be recognized. A first one is the blinding of the patient's treatment. Indeed, ACI is often compared to first-line treatment microfracture; given the two vs one step procedure, respectively, it is obvious to both patient and physician to which treatment arm one was allocated. A second relates to the long duration of obtaining stable tissue repair. Cartilage repair clinical studies can generally enroll only a limited number of patients, and maintaining all patients in the study over years periods represent a major challenge. This impacts consequently the calculations of long term efficacy and safety, and their statistical power and significance. Thirdly, cartilage repair recommendations follow a series of possible treatment algorithms, and generating cohorts of similar patient groups for the respective arms of a comparative trial might not be in line with these recommendations. For instance, patients with small lesions would rather be treated with microfracture than ACI, which will not be the case when obligatory enrolling similar patient groups in typical comparative trials. Fourthly, the definition of the most relevant clinical read-outs for a given trial, the validation of the measurement of these outcomes, the statistical criteria to be applied (non-inferiority vs superiority), as well as the clinical relevance of a given statistical results are non-negligible challenges when designing and running cartilage repair clinical trials. Lastly, documentation of the safety profile is a critical hallmark in developing medicinal products. In the case of ACI products, adverse events due to the surgical intervention are common and expected. Such events are not necessarily related to the medicinal product itself, i.e. the cells, and make definition of the product safety profile more difficult than for classical medicinal products. The development of ChondroCelect has anticipated the above regulatory expectations and requirements. The manufacturing process has extensively been elaborated, and product quality attributes have been defined based on the regulatory requirements for medicinal products. Non-clinical evaluation has demonstrated the proof of concept of good cartilage repair as well as the non-clinical safety profile. A prospective, randomized, controlled clinical trial comparing ACI with ChondroCelect to microfracture has demonstrated its clinical efficacy, i.e. clinical superiority of ChondroCelect over microfracture at 36 months, as well as its safety profile. This comprehensive data package for ChondroCelect has been submitted to the European Medicines Agency (EMEA) for evaluation of the product following the centralized procedure. The registration fle is currently being reviewed and the experience gained during the review will exemplify the current regulatory expectations cartilage cell therapy medicinal products are expected to meet., Introduction New legislation has come into operation in the European Union (EU) - Regulation (EC) No 1394/2007[i] on Advanced Therapy Medicinal Products (ATMPs) in Europe. ATMPs are defined as gene and cell therapy medicinal products and tissue engineered products. This long awaited legislation provides clarity on the legal and regulatory framework for this novel class of product, and especially for tissue engineered products, which are considered as medicinal products. Regulation (EC) No 1394/2007 on Advanced Therapies The Regulation defines a Tissue engineered product (TEP) as a product that contains or consist of engineered cells or tissues that is presented as having properties for, or is used in or administered to humans with a view to regenerating, repairing or replacing a human tissue. Engineering is defined as substantial manipulation of the cells or tissues so that biological characteristics, physiological function or structural properties for the intended regeneration, repair or replacement are achieved, or when the cells or tissues are not intended to be used for the same essential function or functions in the recipient as in the donor (art. 2 of the Regulation). Many of the products commonly referred to as ‘Regenerative medicines’ would fall within the definition of a TEP. Other keypoints from the Regulation are that All marketing authorization applications for ATMPs will be reviewed via the centralized procedure and will get a marketing authorization valid for the entire EU (art. 8);The requirements for combined ATMPs (ATMPs containing, as anintegral part, a medical device or implantable medical device) and the involvement of notified bodies of medical devices have been clarified (art. 6 and 9);A new expert Committee, the Committee for Advanced Therapies, with expertise specific to ATMPs will be established (art 20 – 23);Incentives are provided to companies developing ATMPs, including: fee reductions for Scientific advice (art. 16), scientific recommendation on ATMP classification (art. 17) and evaluation and certification of quality and non-clinical data (this certification procedure is available for Small and Medium Size enterprises only) (art. 18);ATMPs legally on the markets of Member States will have to comply with the new legislation by 2011 or 2012 (for tissue engineered products) (art. 29);Follow-up of safety and efficacy, risk management as well as traceability is considered to be a crucial aspect of the regulation, protecting public health and their confidence in advanced therapies (art. 14 and 15);In well defined conditions, Member States can authorize custom-made ATMPs for an individual patients on their national market (art. 28). Regulation (EC) No 1394/2007 entered into operation on 30 December 2008. Implementation of the Regulation on Advanced Therapies Over the last two years, EMEA has been actively implementing the new Regulation on Advanced Therapies. These activities include, but are not limited to: Setting up of the Committee for Advanced therapies (CAT), including the development of the CAT Rules of Procedure;Development of procedures for the evaluation on ATMPs, including the procedure on the involvement of notified bodies in the evaluation of combined ATMPs.Development of procedures for the classification of ATMPs and for the Certification procedure;Scientific contribution to the European Commission on the revision of Annex I to Directive 2001/83/EC (technical requirements for ATMPs), GCP and GMP for ATMPs and Traceability;Development of a Guideline on Post marketing follow-up and risk management plans for ATMPs[ii]. Scientific guidelines in the field of gene and cell therapy and tissue engineering are developed by EMEA Working Parties. Multidisciplinary guidelines covering the pharmaceutical development, non-clinical testing and clinical investigations of ATMPs and guidelines on aspects specific to one of the classes of ATMPs are already in place. Additional, specific scientific guidelines are under development. All scientific and procedural guidelines are published on the EMEA Website for public consultation before finalization. The Committee for Advanced Therapies The Committee for Advanced Therapies (CAT) is an expert Committee composed of: Five members (or co-opted members) from the Committee for Human Medicinal Products (CHMP) and 5 alternates to those membersOne member and one alternate from the Member States not represented by the members/alternates appointed by CHMP;Two members and two alternates representing clinicians;Two members and two alternates representing patient organizations. Following expertise, relevant to ATMPs, is represented in the CAT: medical devices, tissue engineering, gene therapy, cell therapy, biotechnology, surgery, pharmacovigilance, risk management and ethics. CAT will be the main Committee involved in the initial evaluation of applications for marketing authorization for ATMPs: they will be responsible for the scientific review and will prepare a draft opinion on the quality, safety and efficacy of an ATMPs for final approval by CHMP. Other main activities of the CAT will be: Scientific evaluation of quality and non-clinical data for the Certification ProcedureContribution to scientific advices for ATMPsScientific recommendation on ATMP classification The CAT held it inaugural meeting on 15–16 January 2009[iii]. Under the chairmanship of Dr Christian Schneider[iv], it meets on a monthly basis (11 times a year) for a two day meeting, generally set in the week before the CHMP meeting. Authorisation of cartilage repair products in Europe Cell-based products for cartilage repair, such as chondrocytes in suspension or imbedded in three-dimensional structures, are currently under development, in clinical trials or even in clinical use in Europe. These products fulfill the EU definition of a tissue engineered product: the expansion of cell obtained from a biopsy in culture flasks or in a fermentor is considered as a substantial manipulation step. As a consequence, a formal marketing authorization application will have to be submitted and an approval obtained before they can be put on the market. Companies developing such products should consult the EMEA Guideline on Cell-based medicinal products[v], which provides guidance on the quality requirements, the non-clinical testing and the clinical trial requirements for cell therapy and tissue engineered products. It is acknowledged that the non-clinical and clinical testing for a tissue engineered products might pose challenges to developers and authorities (for example shall the primary endpoint for a clinical trial of a cartilage repair product be based on a structural repair, or can this be based on a surrogate endpoint, such a MRI?). The newly established CAT will be a very valuable forum for such discussion and for interactions with applicants (developers) of tissue engineered products. The approval of clinical trials is the responsibility of the member state where the trial will be conducted. This is outside of the scope of the Regulation on Advanced Therapies. Nevertheless, it is highly recommended that developers already in this stage of development consult the above mentioned guideline and/or seek for scientific advice from the EMEA. If the developer is a Small and Medium size Enterprise, he might ask for an evaluation of the quality and non-clinical data by the CAT, for certification. Regarding cell-based cartilage repair product currently on the member state markets, it should be stressed that these products will have to comply with the above mentioned Regulation by end of 2012 at the latest. This implies that a formal marketing authorization application will have to be submitted at least one year before, demonstrating quality, safety and efficacy of the product. It should be noted that clinical data generate from the use of this product (not arising from controlled trials) might not be sufficient to demonstrate the clinical efficacy and safety of the product. Any company having such product on the market in one of the EU member states should contact the EMEA[vi]., ACI-The First generation of Autologous Chondrocyte Implantation Cartilage has a limited capacity for self-repair after trauma which has led to many different surgical attempts to improve the repair of injured articular cartilage surfaces during the 5–6 decades. The first example of clinical cartilage tissue engineering was performed in 1987 when a knee with an articular cartilage defect on the femoral condyle was treated by implanting the patient's own chondrocytes that had been expanded in vitro and then implanted into the defect in combination with a covering mechanical membrane—the periosteum. This, technology is either termed autologous chondrocyte transplantation (ACT) or autologous chondrocyte implantation (ACI). Today, there exist many modifications of the technique, from the first generation to now second and third generations of chondrocyte implantation. This paper discusses the first generation of ACI, the scientific base and results. The articular chondrocytes are responsible for the unique features of articular cartilage, they keep the cartilage alive, and they alone maintain it and regulate it. Therefore it seems rational to use true committed chondrocytes to repair a cartilaginous defect. From a piece of arthroscopically harvested cartilage, chondrocytes can be isolated by enzymatic digestion and in in vitro culture expanded 20–50 times the initial amount of cells. The cells are cultured in so called monolayer and during that time the cells dedifferentiate. The dedifferentiated chondrocytes have a similarity to primitive mesenchymal cells and an implantation of a high density of those in vitro expanded primitive immature chondrocytes could imitate the prechondrogeneic cell condensation and cartilage formation In 1982 Lars Peterson and co-workers developed a rabbit model to treat cartilage defects in the rabbit patella with autologous chondrocytes (Peterson et al, 1984) The same rabbit model has since then been used and further developed by our group at the Göteborg University (Brittberg et al, 1996). The cultured cells are injected into a premade cartilage defect in the patella of the rabbit and covered with a flap of periosteum, functioning as a biological membrane. This method resulted in a high degree of healed rabbit patellar defects and the repair tissue had a similarity to the original cartilaginous tissue. Furthermore, in the rabbit work, the patellar defects were treated with autologous chondrocytes together with a covering periosteal graft on one side and the contra-lateral side was treated with periosteum alone. The defects were deep, reaching down to the calcified zone but with no opening of the subchondral space. In a defect of this type without any treatment, there was an intrinsic repair of 29% of the total defect area, primarily by what we call matrix flow from mitotic activity at the edges of the defect (Brittberg et al, 1996). This level of repair should be compared with the mean repair area of 30% one year after periosteal grafting alone and significantly different from the 87% repair area with chondrocytes and periosteum. More than 20 year's ago, in October 1987 the technique was first used to treat patients with chronic disabling symptoms of the knee joint with cultured cartilage cells from their own cartilage. The first 23 patients (mean age 27) were presented as pilot study in the New England Journal of Medicine in 1994(Brittberg et al, 1994). Those patients had local deep cartilage injuries that had been treated with conventional methods without any healing and 16 defects were located on the so called femoral surface and 7 on the patella. In all, 16 patients had ..good or ..excellent.. knee function at mean three years postoperatively. The best results were found in the femoral group compared with the patella patients that were less successful. The technique appeared to be most successful in patients that had injuries on the femoral surfaces producing a single, localized deep cartilage lesion. This is important to note as opposed to the gradual wear and tear of advancing age. The disappointing outcome of the patella group might have resulted from mechanical misalignments of the patella that were not corrected at the time of transplant surgery. In a long-term durability study (Peterson et al 2002, Brittberg et al, 2003), the degree of patients still belonging to the group of good-excellent was 84 %. Notably, all ACI failures occurred in the first two years and patients showing good to excellent improvement at two years had a high percentage of good results at long-term follow-up. Results are available with up to 16 years' follow-up, and more than 80% of the patients have shown improvement with relatively few complications (Jones and Peterson, 2006). Since 1987 more than 30000 patients worldwide have been treated with ACI techniques. In most published randomised studies (Horas et al 2003, Dozin et al 2005, Knutsen et al 2007, there are no differences found between the different cartilage repairs methods evaluated. In all those studies significant improvement of patient's symptomatology have been found. However, in a recent study by Saris et al. 2008, one year after treatment, ACI with periosteum was associated with a tissue regenerate that was histologically superior to that after microfracture. However, the use of first generation ACI today has become less common because different variants of chondrocyte transplantation have appeared. Due to repeat problems with hypertrophy of the periosteum needing second surgeries for trimming of grafts, risk of uneven distribution of cells and an open surgery change of the initial technique has been required. Second generation ACI has been presented with a collagen membrane used instead of the periosteum (Russlies et al, 2002) and third generation with cells on a carrier(MACI) (Zheng et al, 2007) or with cells precultured in a 3-D scaffold such in hyalyronic acid (Hyalograft –C) (Marcacci et al, 2005). Even the Saris et al- study with ACI + periosteum was different compared to the first generation of ACI as they used a well characterized chondrocyte cell population (called ChondroCelect). This presentation describes the history of chondrocytes used for cartilage repair with a periosteal membrane; the first generation of ACI, more than 20 year's experience and start of cartilage tissue engineering plus an outlook of the evolution of the technique for the future., The management of chondral lesions is still a challenging problem for the orthopaedic surgeon due to the cartilage's poor capacity to heal. The challenge to restore the articular cartilage surface is a multidimensional task faced by both basic scientists in the laboratory and orthopaedic surgeons in the operating room. In the last 30 years, different techniques to address articular cartilage injuries and defects have emerged as valid therapeutic options. While many treatments are mostly directed to the recruitment of bone marrow cells to obtain potential cartilage precursors and allow to form a reparative tissue, the bioengineered approach aims to regenerate the damaged tissue, restoring a biologically and biomechanically valid hyaline-like cartilage surface. The clinical use of first generation autologous chondrocyte transplantation reported encouraging clinical results, especially in the femoral condyle. However, they have to be weighed against the number of problems that can be observed with the standard ACI methods, such us surgical complexity and biological problem related to the cell culture. To address these problems, the so-called second generation ACI techniques have been developed. The second generation ACI used a tissue-engineering technology to create a cartilage-like tissue in a three-dimensional culture system with the attempt to address all the concerns related to the cell culture and the surgical technique. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation onto the defect site. Whereas chondrocytes in two-dimensional cell cultures alter their phenotype and dedifferentiate to fibroblast, cells that no longer posses the capacity to produce collagen type II and proteglycans, the use of three-dimensional scaffolds has been shown to favour the maintenance of a chondrocyte differentiated phenotype. Scaffolds composed of synthetic or natural materials in a variety of physical forms (fibers, meshes, gels) have been applied to cartilage tissue engineering. Commonly used synthetic materials are the polylactides, like polylactic (PLA) and polyglicolic (PGA) acids. Natural materials used to produce scaffolds include agarose, alginate, hyaluronic acid, gelatin, fibrin glue, collagen derivatives and acellular collagen matrix; they have impeccable biocompatibility, can be processed in a reliable and reproducible way and may enhance cell performance. Matrixes mainly used in clinical practice in Europe are collagen or hyaluronic acid based. In the USA there is not FDA approval for matrix-assisted chondrocyte transplantation in human application, yet. The use of a three-dimensional scaffold with open surgery already permits a reduction of joint exposure because it avoids periosteal harvesting and suturing. Furthermore, the easy handling of some of the scaffolds allowed to develop arthroscopic implantation techniques. Autologous chondrocyte transplantation on a three-dimentional matrix was introduced in clinical practice from 1998–1999, so it is very difficult to obtain medium or long-term clinical findings. The clinical outcome is documented for most of the scaffolds at short term follow up, whereas only a few papers report results at follow-up greater than three years. 2ND GENERATION TECHNIQUES In 1998 Behrens et al performed the first transplantation of auotologous chondrocytes using a porcine collagen I/III matrix (Chondro-Gide) as a scaffold. The collagen membrane was utilized as substrate for the so-called matrix-associated autologous chondrocyte transplantation (MACT, MACI®). Since the introduction of the MACI® technique in 1998, more than 3000 patients have been treated across Europe and Australia1. This simple surgical technique obviates periosteal harvest, is generally suture free, is less invasive than traditional treatment methods and allows an early mobilization of the joint. All MACI® studies showed significant improvement in each of the different scoring methods employed at short term follow up, and results have been confirmed at 5 years follow up, with 81.8% of good or excellent results obtained in the knee objective evaluation2. In 1999 a hyaluronic acid based scaffold was introduced into clinical practice in a number of European countries for the treatment of full-thickness cartilage defects. This scaffold, entirely based on the benzylic ester of hyaluronic acid (HYAFF® 11), consists of a network of 20-μ m-thick fibers with interstices of variable sizes and has been demonstrated to be an optimal physical support to allow cell-cell contacts, cluster formation and extracellular matrix deposition. Seeded on the scaffold the cells are able to re-differentiate and retain a chondrocytic phenotype even after a long period of in vitro expansion in monolayer culture3. The cells expanded and then seeded onto the scaffold create the tissue-engineered product Hyalograft C®, that can be implanted by press-fitting directly into the lesion, avoiding suturing to surrounding cartilage and obviating the need for a periosteal flap. No implant related complications were reported, and even in cases where more than 2 patches were used with overlapping of the grafts, no symptoms related to overgrowth or hypertrophy were observed. The features of this device have also permitted the development of an arthroscopic approach, reducing patient morbidity, surgical time and recovery and complications related to open surgery. Some papers report a satisfactory clinical outcome at medium term follow up: Nehrer4 report interesting results at 5 years follow up and Marcacci5 and Kon6 demonstrated a high percentage of subjective and objective improvement at 2 years, maintained at the 4 years follow up. Despite the promising results of this bioengineered approach, there is no agreement about the effective superiority of Second Generation ACI on the other classic techniques. Therefore, our group decided to compare microfracture with arthroscopic second-generation ACI7. In this recently published study better clinical results, assessed with objective and subjective IKDC scores at medium-term follow-up, were found in the group treated with arthroscopic autologous chondrocyte transplantation. Also, no decrease in the resumption of sports activity from 2 to 5 years was observed in patients treated with autologous chondrocyte transplantation, whereas the decrease in sports activity was detected in the group treated with microfracture from 2- to 5-year follow-up. The clinical results obtained using Hyalograft C® are shown to be better and more lasting than the ones obtained with this classic bone marrow stimulation technique. We also applied this scaffold for the treatment of patellofemoral articular cartilage lesions via minimally invasive or arthroscopic approach, and we obtained lower (with respect to femoral condyle cartilage lesions) but satisfactory results even in this complex location at 2 and 5 years of follow up8,9. Another bioengineered product, Bioseed C®, has been introduced into the clinical practice from 2001: it combines autologous chondrocytes with the tissue development-promoting properties of gel-like matrices in an initially mechanically stable bioresorbable polymer scaffold. The cartilage tissue engineering graft Bioseed C® is a polyglactin/poly-p-dioxanon fleece with a standard sizing of 2cm × 3cm or 2cm × 1cm. Autologous chondrocytes are expanded ex vivo and therefore loaded on a 2 mm thick porous scaffold using a fibrin glue to distribute cells, providing a three-dimensional environment and obtaining the bioengineerized tissue for cartilage replacement. To ensure the fixation a transosseus fixation technique is available, moreover in posttraumatic or degenerative defects without intact surrounding cartilage, with high endpoint fixation strength. Thus, the rationale of this fixation approach is to allow a safer and shortened rehabilitation period, without endangering the success of the procedure. Ossendorf applied this bioengineered tissue for the treatment of chondral knee defects and reports a low failure rate and interesting promising results at 2 years follow up10. Numerous other cartilage substitutes have been studied and applied in clinical trials. CaReS® is composed by autologous chondrocytes seeded on 3D collagen type-I gel. Cells are isolated from the patients biopsy, mixed with the collagen gel and after the complete gelling and two-weeks culturing in patient's serum cultivation medium the chondrocyte-loaded gel is available for transplantation. Diameter and thickness of the transplant can be chosen individually depending on the nature of the defect. The transplantation is performed by mini-open technique and a thin layer of fibrin glue is applied to the defect bottom and marginal ridge to secure the graft stability. Clinical results published on a limited number of patients at 2 and 3 years follow up are encouraging, even in patello-femoral defects. Cartipatch® (TBF Banque de tissues, France) is an autologous chondrocyte implant on hydrogel composed of Agarose and Alginate. This vegetal origin hydrogel, mixed with isolated autologous cell suspension, can be modulated at 37° C into complex shape implants which solidify at approximately 25° C. Alginate provides matrix elasticity, making it easy to handle in the O.R. The transplantation is performed by mini-open technique using a dedicated instrumentation. The clinical phase II multi-centre trial was started in France in 2002 and presently Encouraging clinical and histological results are reported in the first 20 patients analyzed at 3–24 months. A different scaffold strategy involves the development of a bifasic scaffold. Novocart 3D® is a autologous chondrocyte implant on collagen-based bifasic scaffold, where a specific protective dense layer was developed to cover the collagen sponge to prevent synovial cells from invasion and improve the mechanical properties of the structure. The transplantation is performed by mini-open technique using a dedicated instrumentation and resorbable mini-pins can be used for the fixation of the graft. The implant is applied in clinical practice from 2003 and promising clinical results have been reported in literature in the first patients treated at short term follow up. Finally, some clinical studies report the utilize of autologous chondrocytes cultured on fibrin glue with significant better performance respect to the chondroabrasion technique, and some case reports report the utilize of autologous chondrocytes cultured on Atelocollagen gel. In this case, this technique cannot be properly defined as second generation autologous chondrocyte implant, considering that the implant is performed with the use of periosteal flap to cover the chondrocyte culture, but the results reported by Japanese colleagues with this bioengeneered approach are interesting. Conclusions In conclusion, the advantages related to the use of a three-dimensional scaffold seeded with chondrocytes can be summarized as follows: a minimally invasive arthroscopic implantation procedure can be used; there is great stability of the implant; and the cells tend to maintain their original phenotype. On the basis of published results, the autologous chondrocyte implant on three-dimensional scaffolds guarantees results comparable, even better of the traditional ACI technique, but reduces the morbidity of the procedure and avoids the use of a periosteal flap with marked advantages from a biological and surgical point of view. It has to be emphasized that presently none of existing second generation autologous chondrocytes products are indicated in generalized degenerative joint disease, yet. Several improvement are soon expected, as the result of the rapidly growing knowledge on cell culture and chondrocyte behaviour, leading to a more reliable surgical technique and better clinical outcome even in larger degenerative lesions., Autologous osteochondral transfer is a popular surgical technique aiming to provide hyaline or hyaline-like cartilage repair for small and medium sized focal chondral and osteochondral defects of weight bearing surfaces. Initial experimental and clinical experiences with autogenous osteochondral grafting have shown consistent survival of the transplanted hyaline cartilage.1, 2 However, two important problems have been encountered in the process: the donor sites must be taken from surfaces that do not bear much weight, which limits the procurement field, and the use of large grafts can cause incongruity at the recipient site, which permanently alters the biomechanics of the joint and represents limited clinical outcome in the long term.3, 4 Mosaicplasty technique is a new way of autologous osteochondral grafting to respond these practical problems.5 Theoretical considerations to eliminate the above mentioned problems suggested to resurface the defected area by several small diameter cylindrical grafts instead of one big osteochondral block. After some technical preparation cadaver studies were done to develop a precise instrumentation for an ideal and standardized way to manage the problems of optimal graft harvest as well as to standardize a safe and efficient way of a mosaic-like graft implantation. Initially, the mosaicplasty concept was tested in German Shepherd dogs and horses and in cadaver studies. Macroscopic and histological evaluations of the resurfaced areas and the donor sites showed: 1)consistent survival of the transplanted hyaline cartilage2)formation of a composite cartilage layer consisting of ï, / 80% transplanted hyaline cartilage and ï, / 20% fibrocartilage ingrown from the prepared bony base of the defect3)deep matrix integration of the transplanted osteochondral cylinders at the recipient site4)donor site filling to the surface with cancellous bone capped by fibrocartilage by 8–12 weeks.6, 7 Fibrocartilage coverage of the donor holes seemed to be acceptable gliding surface for these less weight bearing areas. Preclinical experimental works and evaluations of initial clinical experiences promoted to develop the present recommendations for the most effective rehabilitation algorithms.8 These considerations involve instructions for postoperative course of mosaicplasties combined with procedures to restore the normal biomechanics, such as femorotibial realignment osteotomies, ACL reconstruction, meniscus surgery, patellofemoral corrections etc. The main features of the proper rehabilitation are the following elements: immediate range of motion exercises to provide proper nutrition of the transplanted graftsfew weeks postoperative non weight bearing period to protect the bony part of the grafts from necrosis or collapse due to extreme initial loadingindividually determined partial loading period after initial non weight bearing to promote fibrocartilage ingrowth between the transplanted grafts and in donor areasfull weight bearing period combined with well adapted proprioception training to promote the tolerance of shear forces These main features of rehabilitation protocols should be individually determined to promote the best repair of the defected area, to have less donor site problem and support effectively the concomitant procedure to restore the biomechanics.9 Autologous osteochondral mosaicplasty involves obtaining small-sized cylindrical osteochondral grafts (2.7, 3.5, 4.5, 6.5, and 8.5 mm in diameter) from the minimal weight-bearing periphery of the femoral condyles at the level of the patellofemoral joint and transplanting them to prepared defect sites on the weight-bearing surfaces. Combinations of different graft sizes allow 80% to 100% defect filling rate. Fibrocartilage grouting, stimulated by abrasion arthroplasty or sharp curettage of the underlying bone at the base of the defect, is expected to complete the new surface. Mosaicplasty can be done as an open procedure, through a miniarthrotomy or –in most of femoral condylar defects - arthroscopically. Arthroscopic implantation of the graft requires optimal location and size of the defect as well as special surgical skill of the operating surgeon.10 Clinical application was begun on February 6, 1992. During the following 17 years, clinical results published by various authors matched the animal results, and since 1995, the procedure has been used with equal success at numerous institutes and clinics throughout the world.11, 12, 13, 14 These results were identical with the authors' follow up. According to our experiences in a series of more than one thousand cases involving several diarthrodial joints with varying function and biomechanical loads, the composite results have been in the good to excellent range with a low complication rate. Emphasizing the age limitations of the procedure (patients should be younger than 50 years), it is not surprising that patients who are older (older than 35 years) have faired less well. More than hundred cases of high professional athletes treated by knee or ankle mosaicplasties gave similar clinical outcome as normal population −83% of them were able to return to high professional sports activity. Beside initial clinical application of this technique for femoral condylar and patellofemoral defects, the use of mosaicplasty was extended to tibial damages as well. After first promising results of knee mosaicplasties ankle application was also started to treat talar osteochondritis dissecans defects.15, 16 This is still the most frequent application outside the knee, but femoral head lesions, capitulum humeri lesions of the elbow and humeral head lesions were also treated as rare indications. Some authors also published mosaicplasties on different small joints. Concerns of donor site morbidity remain integral parts of current evaluations. In cases of complaints related to donor sites usually transient symptoms were observed and only in few patients suffered from long lasting patellofemoral pain. The authors think that the full restoration of the donor site centers on the peripheral position of the donor area and the small size and proper spacing of the individual grafts. These elements allow the joint to reconstitute structurally to reaccept the relatively low loads in these parts of the knee. Excessive bleeding from empty donor sites caused painful intraarticular haemorrhage in a limited number of patients. In spite of the fact that this is a relatively rare complication, many experimental studies were done to test different biodegradable materials to provide an optimal filling of donor tunnels preventing such enormous bleedings.17, 18 From these encouraging results from an increasingly large series and similar results from other centers, it seems that autologous osteochondral mosaicplasty – beside cell culture techniques and biodegradable repair options - may be still a useful alternative treatment for localized full-thickness cartilage damage of the weight bearing surfaces of the knee and other weight bearing synovial joints., History Articular surface damage is a common finding in traumatic knee injuries. The limited ability of articular cartilage defects for spontaneous repair was described by William Hunter in 1743 (1). Because of this, chondral injury can lead to progressive, irreversible degenerative changes in the knee. Microfracture of the subchondral bone is a bone marrow– stimulation technique developed by Steadman et al. for the treatment of chondral defects (2). The main indication of this procedure is full-thickness articular cartilage defects caused by acute trauma or chronic repetitive microtrauma (3). It provides a suitable environment for tissue regeneration with pluripotent mesenchymal stem cells from the subchondral bone marrow. Indications and Contraindications The most common indication for microfracture is full-thickness chondral loss in the weight-bearing surface of the femur or tibia or on the articular surfaces of the patella or trochlear groove (2,3). Also it is indicated for unstable, full-thickness cartilage flaps overlying subchondral bone (4). Although microfracture alone has limited indications for degenerative lesions and osteochondritis dissecans, the most significant contraindication of this technique is a malaligned knee (5). Inflammatory arthropathies, systemic cartilage conditions, and septic or neoplastic disorders are other contraindications (6). Patient not willing to participate in the strict postoperative rehabilitation regimen may also be a relative contraindication. Preoperative Planning Preoperative planning for the patient with knee pain begins with a careful history and physical examination. The patient's social status and expectations for the postoperative course as well as short- and long-term functional outcome should be assessed thoroughly. Chondral lesions can present with pain localized to the involved articular surface or with more diffuse pain. A chondral flap can cause a patient to face popping, clicking, or even a feeling of instability. An effusion is often present with an acute chondral injury. With a chondral lesion located on patellofemoral joint, pain can be elicited with compression of the patella. Ligamentous stability should be tested. Physical examination should also determine patient body weight index because an excessive body weight has been associated with limited functional improvement. All patients presenting with a potential chondral lesion should undergo a series of radiographs including bilateral longstanding radiographs, standard AP and lateral radiographs of both knees, and patellar views to evaluate the patellofemoral joint. For further evaluation, we use magnetic resonance imaging. Surgical Technique (7) We perform a thorough diagnostic arthroscopy involving a systematic evaluation of all areas of the knee including the suprapatellar pouch, the patellofemoral joint, the medial and lateral gutters, the intercondylar notch, the anterior interval, the femoral condyles, the tibia plateaus, the entirety of the medial and lateral menisci, and the posterior compartment. All other necessary intraarticular procedures are performed before the microfracture technique to eliminate the cloudy visualization caused by the fat droplets and blood from the microfracture holes. Regions of full-thickness cartilage loss are thoroughly probed and evaluated for a cartilage flap. At the site of the lesion, any unstable cartilage flaps are debrided with a curette out to the edge of viable and stable cartilage. This is followed by the curettage of the necrotic subchondral bone. The presence of two different subchondral tissues is detected macroscopically in the necrotic area: white necrotic tissue at the superficial zone, and pink cancellous tissue that did not seem necrotic but was found not to be healthy in the deeper zones. The curettage is stopped at the second zone and multiple holes were prepared in that “seminecrotic” area using an arthroscopic awl. The holes are approximately 3 to 4 mm apart (3 or 4 holes per cm2) as suggested by Steadman et al. In regards to the depth of the holes, we prefer 4 mm, but in some cases it may be necessary to make them 6 to 7 mm deep. Treatment is considered adequate when fat droplets are observed flowing from all of the holes. The surgically induced marrow clot provides the basis for the optimal environment for the pluripotent cells to mature into fibrocartilagenous repair tissue. We do not use drain because it may inhibit formation of the surgically induced clot within the defect. A compression dressing and ice is applied. The involved cartilage area is not a handicap for this procedure, and some of our cases had 900 mm2 of necrotic lesion. Although there is no limitation as to the area of the necrosis, the depth is an important factor for the indication. We limit the procedure only to lesions that were no more than 10 mm deep from the joint level. For deeper necrosis, debridement is performed as a salvage procedure until subsequent replacement surgery. When the microfracture is completed, all instruments are removed from the joint and the knee is evacuated of fluid. Passive and active range of motion is started the day of surgery. A continuous passive motion machine is used, if available, for 2 weeks. Patients are encouraged for assisted passive motion when the machine could not be used. Weight bearing is not allowed for 6 to 8 weeks regarding to the size of the lesion, and then the patients are progressed to a aggressive rehabilitation program with full weight bearing. For those patients whose lesion area is smaller than 100 mm2, weight bearing is allowed as tolerated in the early postoperative period. Clinical improvement is not observed in the early postoperative period but patients are satisfied after 6 months. The follow-up clinical examination included assessment of pain, range of motion, effusion, instability, and atrophy of the muscles for all patients. Lysholm and Cincinnati Activity scores are used to evaluate the clinical results. Weight-bearing anteroposterior and lateral radiographs are taken of all knees for radiologic follow-up. These imaging studies are assessed for staging, healing, or worsening of the lesion according to Koshino's criteria. In this staging system, stage-1 knees have a normal appearance. Stage-2 knees have a radiolucent oval shadow in the subchondral area with distal sclerosis. In stage-3, there is a calcified plate with radiolucency surrounded by a definite sclerotic halo and collapse of the subchondral bone. Stage-4 knees have osteophytes and osteosclerosis in both ipsilateral femoral and tibial condyles. Our Experience (7) We performed a retrospective clinical study to evaluate the results of arthroscopic subchondral microfracture treatment for patients with primary osteonecrosis (ON) (group 1) or secondary ON (group 2) of the knee joint. ON of the knee may be either primary (spontaneous), or secondary to steroid therapy or chronic illness, such as systemic lupus erythematosus, vasculitis, and hemoglobinopathies. Primary ON of the knee is typically seen with unilateral involvement in elderly patients. Secondary ON generally occurs in younger patients and the necrotic areas are larger than the lesions seen in spontaneous ON. Bilateral and multifocal involvements are possible. Group 1 included 26 patients (mean age, 48 years) who had spontaneous ON. Group 2 included 15 patients (mean age, 32 years) with ON secondary to inflammatory disease or steroid therapy. Seventy-six percent of the chondral defects were located in the medial femoral condyle. The average defect size in group 1 was 162 mm2 and in group 2 was 362 mm2. After debridement of the necrotic tissues, multiple perforations were placed into the subchondral bone to obtain revascularization. There was an increase in the average Lysholm scores from 57 to 90 in group 1 after 27 months of mean follow-up (P⇠0.05); 71% of patients could participate in strenuous sports with no or minimal limitation. The mean activity level in the group1 according to Cincinnati Knee Rating System was 6 preoperatively and 13.54 postoperatively. For group 2, the average scores showed significant improvement and patient satisfaction after surgery (preoperative and postoperative average Lysholm scores were 41 and 75, respectively, with mean follow-up of 37 months). Average activity level in group 2 increased from 2.67 to 11.73. Control magnetic resonance imaging scans of the cases revealed the continuity of normal cartilage with cartilage-like tissue in the treated areas. However, an increase of the size of ON in the subchondral bone was detected in 27% of the knees. In conclusion, the microfracture technique is safe, simple, and cost-effective, and may be an alternative procedure for treatment of ON of the knee, especially in young patients, before possible subsequent replacement surgery. Arthroscopic articular cartilage repair using a resorbable matrix scaffold TruFit® CB Plug (Smith & Nephew, San Antonio, TX) is a synthetic resorbable biphasic implant which provides a scaffold for tissue repair that is both biologically friendly and biomechanically stable. It is a composite of polylactide-co-glycolide, calcium sulfate and polyglycolide fibers and can be fabricated into products such as granules, blocks, wedges and other preformed shapes. It supports the local migration of chondrogenic or osteogenic cells that ultimately synthesize new ground substance. Preclinical studies demonstrated restoration of hyaline-like cartilage in a goat model with subchondral bony incorporation at 12 months (8). Our Experience Fifteen skeletally mature patients with symptomatic, full-thickness cartilage lesions of the femoral condyles, between 1 and 2.5 cm in diameter, were treated with implantation of bioabsorbable implant. Implants were press-fit into holes drilled into the defect. All patients were evaluated both preoperatively and postoperatively with the Lysholm knee score, International Knee Documentation Committee (IKDC) Standard Evaluation Form, and magnetic resonance imaging of the affected joint. Fifteen patients of a mean age of 39 years were followed-up for a mean time of 9.1 months. The mean Lysholm score significantly improved from 43.6 preoperatively to 87.5 postoperatively (P⇠.001). Excellent or good outcome was accomplished in 12 of the patients (80%). Using the IKDC assessment, 11 of the patients (73%) reported their knee as being normal or nearly normal. Congruency of the articular surface was restored in 13 of 15 patients (87%) who underwent magnetic resonance imaging examination. Abnormal marrow signal in the subchondral bone beneath the region of implant was present in all patients. In conclusion, the TruFit CB implant is an effective and safe method of treating symptomatic full-thickness chondral defects of the femoral condyles in appropriately selected cases. However, further studies with long-term follow-up are needed to determine if the implanted area will maintain structural and functional integrity over time., Since patients requiring joint replacement are heavier, more physically active and live more than 25% longer than several decades ago, other modalities have an increasingly important role in the treatment of cartilage pathology of the knee. Cartilage does not have an inherent reparative capability. The natural history of isolated chondral injuries is not fully delineated. It is however accepted that a chondral injury on a weight bearing area of the knee may lead to the development of degenerative joint disease as well as deterioration in knee functional scores. Cartilage and osteochondral defects of the knee are commonly encountered in orthopaedic practice and their incidence is higher in the young active population. With the increasing understanding of the physiology and biomechanics of the musculoskeletal system as well as the progress made in surgical techniques, this area of orthopaedic surgery has evolved into a specialist field. Hyaline cartilage has superior biomechanical properties and is more durable than fibrocartilage. Of the various treatment modalities, only osteoarticular autograft transfer, osteoarticular allograft, and autologous chondrocyte implantation can provide hyaline repair cartilage. In the management of knee cartilage defects, the main factors that affect technique selection are the diameter of the chondral defect, the depth of the bone defect and the knee alignment. When bone is not involved and the chondral defect is less than 3cm in diameter, microfractures, autologous chondrocyte transplantation, osteochondral autografts or periosteal grafts may be offered as treatment options. Osteochondral defects of less than 3cm in diameter and 1cm in bone depth can be addressed with autologous chondrocyte transplantation, osteochondral autografts or periosteal grafts. Articular defects more than 3cm in diameter and 1cm in bone depth require osteochondral allografts. Allografts are indicated also in cases of uncontained defects but should be reserved only if the lesion is beyond the other modalities. Realignment osteotomy is a useful adjunct to all these techniques, in the case of increased loading in the pathological compartment. For osteochondral defects great than 3cms in diameter and 1cm in depth, osteochondral allografts are a reconstructive solution for young, active patients with large osteoarticular defects of the knee. They are useful where implants or an arthrodesis are not desirable. Although the use of allografts has a risk of disease transmission similar to blood transfusion, there are significant advantages since there is no donor site morbidity, the bone stock can be restored, and the allografts can be adjusted in size and shape to fit the defect exactly. There is an 85% long-term survival rate of osteochondral allografts in carefully selected patients, who present with osteochondritis dissecans or traumatic defects of the knee. This talk discusses the indications and contraindications of osteochondral allografts, outlines the peri-operative management of patients, and reviews our long-term results. Our present surgical technique will also be presented. This talk will also cover our long-term retrieval studies. We examined histologic features of 35 fresh osteochondral allograft specimens retrieved at the time of subsequent graft revision, osteotomy, or TKA. The graft survival time in our samples ranged from 1 to 25 years based on their time to reoperation. Histologic features of early graft failures were lack of chondrocyte viability, and loss of matrix cationic staining. Histologic features of late graft failures were fracture through the graft, active and incomplete remodeling of the graft bone by the host bone, and resorption of the graft tissue by synovial inflammatory activity at graft edges. Histologic features associated with long-term allograft survival included viable chondrocytes, functional preservation of matrix, and complete replacement of the graft bone with the host bone. Given chondrocyte viability, long-term allograft survival depends on graft stability by rigid fixation of host bone to graft bone. With the stable osseous graft base, the hyaline cartilage portion of the allograft can survive and function for 25 years or more. Recently we have started a new programme for posttraumatic hip defects, and preliminary results will be presented., The treatment of large or complex lesions involving articular cartilage is extremely challenging and requires a more carefully considered approach. Large or complex lesions are generally defined by any or all of the following criteria: Greater than 2 centimetersSubchondral bone involvementMultifocal or bipolar lesions or presence of arthritisFailed previous repair treatmentPatellofemoral or tibial lesionsMeniscal or ligamentous deficiencyLimb malalignment Currently available techniques for treatment of these lesions include autologous chondrocyte implantation (ACI) and osteochondral allograft transplantation (OCA). Because many patients have abnormalities of the joint or limb in addition to the articular cartilage lesion, the surgical skill set for appropriate management include techniques for ligament reconstruction, meniscus allografting, osteotomy and patellofemoral realignment procedures. The technique of ACI is well described (1,2). The basis of the procedure involve ex vivo cell manipulation and subsequent delivery of chondrocytes to the injury site with coverage by a periosteal patch to contain the cells and potentially provide growth factors. The so called sandwich procedure has been described to address deeper osteochondral lesions with an underlying bone graft covered by a second periosteal patch. ACI requires 2 separate surgeries, the first to arthroscopically harvest articular cartilage for chondrocyte expansion and culture and the second, requiring an arthrotomy, to prepare the lesion, harvest and suture the periosteal patch and inject cells. The technique of OCA has also been well described (3). The basis of the procedure is transplantation of an intact osteochondral tissue with living chondrocytes and mature hyaline cartilage. Most commonly dowels or plugs 15 −30 mm are utilized, however often shell or small fragment grafts are necessary for lesions that have difficult access or exceptionally large. The procedure is performed through an arthrotomy of variable size Clinical Evaluation Defining the lesion size, location and depth is of primary importance. It is also useful to determine the etiology or pathogenesis of the disease process. Is the lesion the result of Osteochondritis dissecans or osteonecrosis or is the process purely chondral? Is the lesion acute or chronic? What previous treatments have been attempted? Does the lesion correlate with the patient's symptoms? What other background factors are present? Is the patient realistic in his or her goals? Large and complex lesions are often associated with significant joint dysfunction and overall disease burden, which may compromise outcome. It can be very helpful to use some type of scoring system (IKDC, Cincinnati, Lysholm, KOOS, Chondropenia severity score, etc.) to quantify the overall status of the joint. Imaging should be comprehensive, including “cartilage series” radiographs: standing AP, PA flexion, lateral, Patellofemoral view and long standing alignment images (hip-knee-ankle). Missed malalignment is perhaps the most common error of omission in complex cartilage restoration. Magnetic resonance (MRI) is important to evaluate both bone and soft tissues and a relatively recent arthroscopic evaluation may be necessary to rule out occult chondral injuries. In cases of enigmatic pain, a bone scan may be useful. For patients that have a contraindication to MRI, computerized tomography arthrography (intra-articular contrast) is a useful study. Diagnostic Categories The clinical evaluation of the patient described above should lead the surgeon to a diagnosis that falls into one of the following six categories. These characteristic categories help to define treatment and predict outcome. Salvage of previous chondral surgery (microfracture, OAT, ACI)Osteochondritis dissecans (OCD)OsteonecrosisOsteochondral fracture or malunionDegenerative chondral lesionsEstablished osteoarthritis Salvage of Previous Cartilage Surgery These patients may be of any age group and a revision cartilage procedure is perhaps the last chance at biological repair. The critical issue is to determine why the previous treatment failed. Is it a biological, mechanical or technical issue that can be overcome? Is there a need for adjunctive procedure? ACI is generally indicated for chondral surface lesions in younger patients (4) and OCA may be a better choice for older patients or those with compromised subchondral bone (5). Osteochondritis Dissecans OCD represents one of the best indications for cartilage repair. Patients with OCD are generally young (15–30 years old) and have a focal lesion of the medial or lateral femoral condyle (often large) with little or no underlying pathology except occasional malalignment. Type III or IV OCD represents cases where the fragment is not salvageable are ideal for osteochondral allografting as this technique uniquely restores both the osseous and chondral deficit. Autologous chondrocyte implantation is also effective for smaller or shallow lesions (⇠8 mm deep) (6). Autologous bone grafting may be necessary for very deep lesions. Treatment outcome in these patients is excellent with 80–90% success (7,8). Osteonecrosis It is important to distinguish two variants of osteonecrosis (9). Spontaneous osteonecrosis of the knee (SONK) is generally characterized by a focal osseous lesion or cyst which may be a prodrome of early osteoarthritis in a middle aged patient. True osteonecrosis is a more diffuse, multifocal pattern of necrosis which may be idiopathic or often associated with steroid treatment in younger individuals. SONK can be effectively treated with bone grafting of the cystic lesion with or without ACI, or with OCA. Particular attention should be made to correcting malalignment as this is often a predisposing factor. Diffuse osteonecrosis is best treated by small or large fragment allografts, particularly if the patient is young and there is a desire to avoid prosthetic arthroplasty (10). Osteochondral Fracture or Malunion This condition is generally the result of high energy trauma such as motor vehicle accident or falls or occasionally the result of patellar dislocation. The typical case involves malunion of a tibial plateau fracture where joint incongruity results or, on the femoral side, where a non salvageable fragment is associated with a sub-acute or chronic osteochondral injury. In this setting ACI is rarely indicated and OCA is usually the best option as anatomic bone restoration is the most critical component of treatment. Realignment osteotomies are commonly employed as well as meniscal transplantation in the case of tibial plateau grafts (11). Degenerative Chondral Lesions These lesions typically occur in older patients (35–55) years old and represent a chronic disease state, resulting from repetitive trauma and surgery. The distinction between degenerative lesions and osteoarthritis is probably only a matter of degree and represent the same disease continuum. These knees are characterized by multiple lesions with meniscus and ligament compromise. Both ACI and OCA can be utilized but multiple grafts that may be necessary are technically challenging and staged or combination procedures (realignment) are common. Results from treatment with either modality are fair to good, but patients must be willing to accept arthroplasty as a salvage (12,13,14) Established Osteoarthritis Established osteoarthritis in the young adult, characterized by significant joint derangement and radiographic changes such as joint space loss should be considered one of the major unsolved problems in orthopaedics. Prosthetic arthroplasty has not yet demonstrated excellent functionality or durability in this population. Cartilage restoration should not be routinely used except in highly selected cases. When appropriate, osteotomy alone as a first line treatment should be considered. Summary Treatment of large and complex cartilage lesions requires a broader and more thoughtful approach than the more common small focal lesion. Patient evaluation should include a comprehensive clinical assessment, with particular attention to biomechanics of the limb and joint. Diagnostic categories appear to be more useful than lesion size in clinical decision making. Current cartilage restoration options are limited to autologous chondrocyte implantation and osteochondral allografting, with few exceptions. Use of adjunctive procedures is critical to success. Future innovation will provide more tools and better outcomes in treating this difficult clinical entity., Introduction “Doc isn't there something you can put in my knee to keep me playing sports?” Seventy million Americans have osteoarthritis leading to 300,000 artificial joint implants annually. The limitations on the patient, the complications, the short lifespan of the implants, and the cost to society all drive the search for biologic joint replacement rather than artificial joint replacement. The two cartilage components of the knee joint, the meniscus and the articular cartilage were traditionally irreparable and irreplaceable. Both can now be repaired and replaced. The methodology and the source of the cells and tissues that make up this new biologic joint replacement era is the subject of the ICRS meeting. The future may lie in new tissue sources from animals as the barriers to transplantation, immunology, source, sterilization and healing are resolved. The potential for superior outcomes, unlimited availability, and sterility may drive this science. Biological vs. Prosthetic: The true unmet clinical need in osteoarthritic knees is to replace the damaged articular cartilage and the missing meniscus cartilage with new tissue. Artificial materials all eventually fail and all limit activities and motion. The holy grail of biologic knee replacement is normal meniscus replacement and complete retreading of the worn surface with hyaline articular cartilage. The meniscus cartilage can today be partially re-grown through the collagen meniscus scaffold and replaced in arthritic knees with allograft menisci [1]. The re-growing technique is limited to pristine knees and the allograft source is limited to young tissue in short and expensive supply. CURRENT TECHNIQUES OF CARTILAGE REGENERATION Biological articular cartilage replacement may most simply be divided into the cellular regeneration of tissue versus implantation of biologic prostheses. The cellular group includes both endogenous and exogenous cellular stimulation. The endogenous group includes drilling, microfracture stimulation of fibrous repair and the paste graft application of cartilage and bone matrix to morselized arthritic lesions [2]. The exogenous cellular group, autologous cartilage implantation and its variants, depend on cellular expansion followed by remodeling of cartilage. The biologic prostheses group follows the original shell allograft transplantation experience and diversified into OATS and Mosaicplasty. Table I provides a brief summary or current techniques for cartilage repair and replacement. Table 1: Current Techniques for Cartilage Repair and Replacement Cartilage Repair Technique Description Debridement / Chondroplasty Debridement and reshaping of the damaged area of the joint by cutting, scraping, heating, or burring the arthritic joint surface. Potential for superficial cartilage reshaping Microfracture Systematically debriding and perforating an traumatic lesion through surface cartilage layer but not through the subchondral plate. Repair tissue consists of a mixed fibrous repair. Autologous Chondrocyte Implantation (ACI) This open surgical (as opposed to arthroscopic) procedure is performed in a two-stage procedure. The first surgery harvests chondrocytes (live cartilage cells) from the patient's knee. The cells are then cultured, expanded and re-implanted during a second surgery. Periosteal tissue, is harvested and sutured into place over the cartilage repair with cultured chondrocytes injected. No bleeding is induced. Matrix Induced Chondrocyte Implantation (MACI) This arthroscopic procedure is similar to ACI, but uses a bio-absorbable cover instead of periosteal tissue. The cover is a biopolymer matrix layer that requires no suturing. Osteochondral Autograft Transfer (OATS) / Mosaicplasty Osteochondral (articular cartilage + attached bone) plugs are harvested from one area of the patient's knee, and transplanted into the damaged portion of the joint. Osteochondral Allograft Transplantation Articular cartilage and the attached bone is transplanted from a cadaver donor to a damaged region of the recipient's joint. Articular Cartilage Paste Grafting Arthritic lesions are extensively microfractured and morselized through to subchondral bone. A core of cartilage and bone is taken from the intercondylar notch and crushed into a paste. The paste is impacted into the lesion and the progenitor cells contained in the paste and recruited from the bleeding fractured lesion bed aid the healing process. These surgical techniques produced variable degrees of success clinically and are limited to focal, or small to moderately sized, lesions in articular cartilage. Treatment becomes more difficult as the arthritic lesions increase in size, as is the case in severe arthritis. The future must be to provide these tissues on a cost effective off-the-shelf basis customizable for each knee. The tissues must be adequately effective immediately and able to undergo remodeling without significant degeneration over time. They must be able to undergo biologic healing in a degradative chemical environment and a mechanically abnormal physical environment without early tearing or loosening. These goals will most probably be met by transplantation of intact cartilage loaded with progenitor cells. CONCEPTS IN ORTHOPAEDIC XENOGRAFTING The Animal to Human Problem: The effectiveness of cartilage regeneration typically depends on endongenous or exogenously embedded and nurtured cells. This problem is compounded in the xenograft case where animal cells may be implanted into human. The use of animal derived cells in humans is strictly prohibited due to the risk of zoonotic disease transmission by agents such and endogenous retroviruses or in the case of bovine species, spongiform encephalopathy. Although several accellular xenograft bioscaffolds are commercially available for musculoskeletal reconstruction, few studies have addressed the primary host antibody reaction to a xenogeneic cell and matrix surface carbohydrate antigen called the ï,μ-galactosyl epitope (alpha-Gal). Humans and Old World primates lack the alpha-Gal epitope, but all other mammals produce and incorporate ïi-Gal epitopes into cellular and extracellular structures using the alpha1,3-galactosyl transferase enzyme [3]. To compound the problem, humans and Old World primates continuously produce anti-Gal antibodies constituting about 1% of circulating immunoglobulins, and are therefore not immunotolerant towards grafts presenting with alpha-Gal epitopes. Previous studies where grafts were tested in lower order species did not show the rejection response. To date, no decellularization, washing or sterilization processing technique has been shown to remove the Gal epitope leaving xenografts with short residence times when transplanted to humans. Our investigations have characterized the immunological response elicited by porcine tissues and examined alpha-Gal epitope enzymatic cleavage methods as applied to porcine fibrocartilage, articular cartilage, bone, tendon, and soft tissue grafts [4]. Due to the above noted concerns about non-human cells, current strategies for the use of xenogeneic materials in human orthopaedic reconstruction are limited to re-assembled scaffolds and devitalized tissues. The collagen meniscal implant and immunochemically modified porcine patellar tendon are relevant examples of applied research to xenograft development. Collagen Meniscal Implant: The collagen meniscal implant scaffold (CMI) was specifically designed to foster regeneration by optimizing the porosity of the scaffold to be cell migration friendly, to chemically cross link the device with glycosaminoglycans and vapor aldehyde to delay the degradation time and to permit tissue remodeling. The device was also designed to be an off the shelf implantable and customizable at the time of surgery. It met the criteria for safety and implantability in 1993 and subsequently approved in the U.S. in 2008 for meniscus repair [5]. The design process serves as an excellent example for tissue engineering for a defined application and unfortunately the approval process highlights the difficulties facing biologic solutions to joint problems. Immunochemically Modified Porcine Patellar Tendon: An immunochemically modified and sterilized porcine ACL reconstruction device was developed with an understanding of the failure modes of prior ACL replacement products. Early on, investigations demonstrated the significance of the major antigen that causes rejection of pig cartilage and ligament xenografts in primates. Later investigations developed techniques to attenuate this rejection without sacrificing graft durability and without using high concentrations of glutaraldehyde, thus optimizing cellular infiltration and remodeling. The clinically used device consists of porcine bone-patellar tendon-bone treated with a-galactosidase to eliminate the Gal epitope, a low level of glutaraldehyde and sterilized with 17.8 kGy irradiation. Pilot clinical studies of ACL reconstruction have shown the utility of the technology with formal clinical studies in development toward commercialization. FUTURE DIRECTIONS FOR CARTILAGE XENOGRAFTS Xenogeneic cartilage as a scaffold represents an idealized biologic prosthesis. If intact cartilage can be transplanted and revitalized with human progenitor cells then true bio-resurfacing of the joint may be possible. The composite may lower the biomechanical barrier and present only the immunologic and cellular challenges to joint reconstitution. Stem cells and their progenitor variants may present the pathway to both immunologic suppression and anabolic stimulation for cartilage remodeling. Our work pushes forward in this direction at this time., Arthroscopic assessment provides important information on the outcome of cartilage repair procedures. There are currently two semiquantitative scoring systems available for systematic evaluation of the repair sites. Both the International Cartilage Repair Society (ICSR) cartilage repair assessment and the Oswestry Arthroscopy Score (OAS) have been validated (Smith et al. 2005, van den Borne et al. 2007). Mechanical properties of the repair tissue reflect the compositional and structural characteristics of the tissue and thus may describe the biological success of the repair process. Visual outcome measures Both the ICRS and OAS scoring systems were designed to evaluate the macroscopic outcome of cartilage repair and to focus the scoring system according to the clinical needs (Brittberg and Winalski 2003, Smith et al. 2005). The ICRS macroscopic evaluation grades the repair site in terms of the degree to which the defect is filled with repair tissue, integration to the border zone and macroscopic appearance of the surface. Each of these three estimates is classified into five levels (0–4 points) and the overall repair assessment has four grades: normal (12 points), nearly normal (8–11 points), abnormal (4–7 points), and severely abnormal (0–3 points). To evaluate the filling of the defect site after ACI or microfracture, the depth of the repair tissue is assessed. When estimating the outcome after osteochondral transfer, which immediately fills the defect, the survival of the initially grafted surface is evaluated. The OAS evaluation grades the repair tissue in terms of graft level with the surrounding cartilage, integration with the surrounding cartilage, appearance of the surface, color of the graft and mechanical stiffness to manual probing. Each estimate consists of three levels (0–2 points) and the points from the five estimates are added together to give an index with a range of 0 to 10 points. The ICRS macroscopic score and the OAS score showed a good inter- and intra observer reliability and the correlation between both scoring systems was excellent (van den Borne et al. 2007, Smith et al. 2005). Thus both scoring systems were considered valid research tools for cartilage repair surgery. The main difference between these scoring systems is that the OAS score takes into account hypertrophy of the repair tissue and tactile probing of the graft, which are not evaluated in the ICRS score. Although the arthroscopic evaluation of articular cartilage and repair tissue is the gold standard investigation to evaluate alterations in articular cartilage, it has also certain disadvantages. Quantitative measurements are seldom possible and evaluation of the changes is mostly qualitative. When trying to estimate, e.g., distances in the knee joint, disagreements of the arthroscopic measurements are usually greater than 34 % (Diaz and Albright 2008). The ICRS and the OAS scoring systems both improve the arthroscopic evaluation of the repair tissue by offering semiquantitative data of filling of the lesion, integration of repair tissue with adjacent cartilage and the macroscopic appearance of the repair site. The advantage of an arthroscopic scoring system is that it provides information of the whole repair area. A biopsy specimen for histology or biomechanical indentation usually provides more local information at a small part of the repair site. Mechanical outcome measures The goal of repair procedures is to produce functionally competent tissue that can withstand the high loads the joint is subject to. Therefore, knowledge on the biomechanical properties of the repair tissue would be important when evaluating new methods of cartilage repair. Manual probing of cartilage surfaces is a routine procedure during arthroscopies. Probing may reveal some information on the resiliency of cartilage and repair tissue, especially when the tissue is much softer or harder than normal cartilage. However, the procedure is a highly subjective and insensitive method, and thus this technique provides information with limited value, especially for research purposes. Laboratory studies have shown that the quantitative stiffness measurement by the indentation technique is a sensitive indicator of cartilage structural integrity (Jurvelin et al. 1989), possibly even more sensitive than the qualitative histological evaluation (Lane et al. 1979, Armstrong and Mow 1982). For example, after long-term immobilization, macroscopically healthy looking cartilage may show significantly impaired functional properties predisposing cartilage to further injury (Jurvelin et al. 1986, Arokoski et al. 2000). Further, studies with human cartilage have shown that indentation stiffness predicts histopathology of cartilage (Franz et al. 2001, Bae et al. 2003, Appleyard et al. 2007). Our group has previously presented and validated an arthroscopic indentation instrument that is capable of measuring compressive stiffness of articular cartilage in vivo (Lyyra et al. 1995). Later, few other indentation instruments have been introduced for in vivo use (Niedeauer et al. 2004, Appleyard et al. 2001). The studies have shown that instruments can detect functional changes in articular cartilage during tissue degeneration (Bae et al. 2003, Lyyra-Laitinen et al. 1999). The mechanical indentation instrument developed by our group consists of a handle and a measurement rod, equipped with a mechanical indenter and an integrated force gage at the tip (Lyyra-Laitinen et al. 1999). The force by which the tissue resists the constant indentation of articular cartilage is a measure for cartilage stiffness. The latest version of this instrument and measurement technique, enabling more controlled, rapid and reproducible measurements, has been recently introduced (Timonen et al. 2009.) Earlier studies have shown that different cartilage surfaces in healthy joints show significant variation in their mechanical properties (Laasanen et al. 2003, Kiviranta et al. 2006, Froimson et al. 1997). It has also been reported that the site-dependent variation exists within one cartilage surface (Samosky et al. 2005, Kiviranta et al. 2008). Due to the site-dependent variation, accurate site-matched reference values would be needed to assess cartilage integrity with this approach. As for now, there are no comprehensive normal values of normal cartilage stiffness. Therefore, values of the healthy adjacent cartilage are used for reference when measurements of cartilage repair tissue are conducted (normalized stiffness) (Vasara et al. 2005). A clinical follow-up study of thirty patients operated with autologous chondrocyte transplantation has shown that the indentation stiffness of the repair tissue improved to 62 % of the adjacent cartilage one year after the cartilage repair procedure (Vasara et al. 2005). The repair tissue stiffness in eight patients reached the same level as the adjacent cartilage stiffness, but there was great variation in the biomechanical properties of the grafts. The osteochondritis dissecans lesions generally had softer grafts suggesting that the deep lesions may need longer maturation time (Vasara et al. 2005). The delayed gadolinium-enhanced MRI of cartilage analysis of four patients showed that the proteoglycan (PG) concentration of the repair tissue had replenished during the one-year follow-up. However, the same grafts had very low stiffness values, indicating that PG concentration alone does not necessarily fully characterize biomechanical integrity of the graft (Vasara et al. 2005). Other studies have shown that after the mean follow-up of 1.8 or 4.5 years after autologous chondrocyte transplantation the normalized stiffness of the cartilage repair tissue was 104 % and 94 %, respectively (Henderson et al. 2007, Peterson et al. 2002). The same studies have revealed that grafts with histologically verified hyaline repairs exhibit indentation stiffness values comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs. These data demonstrate that arthroscopic indentation can be used successfully to distinguish functionally and structurally appropriate tissue from unsatisfactory repairs. Thus, mechanical indentation of the repair tissue may be considered a biological outcome measure, supporting the use of arthroscopic indentation as a method for assessment of cartilage repair tissue., Introduction Many factors influence the evaluation of outcome following cartilage procedures. The outcome is influenced by the patient, the nature of the lesion, the procedure performed and the outcome measure utilized. All of these factors must be independently considered in great detail to appropriately evaluate any treatment or procedure for a cartilage lesion. Each of these factors will be considered separately to present the reader with an organized approach to evaluating the outcome for a patient who has undergone a cartilage regenerative or restorative procedure.1 The Patient Many factors must be considered when evaluating the patient. These factors will have a very large effect on the outcome of treatment and must be documented in detail. The age as well as the height and weight of the patient have an important effect on the outcome. The occupation will also affect outcome do to the relationship with activity level. Whether the patient is male or female should be documented. Perhaps one of the most important prognostic factors following cartilage surgery is the level of activity of the patient. This is a critical variable since with a decreased level of activity many patients can tolerate significant knee pathology. The measurement of patient activity is complex and can be difficult. The Tegner rating scale evaluates patients based on their participation in various sports2. While this scale has been used extensively in the past, it has limitations with respect to patients who do not participate in specific sports measured by the scale. Therefore individuals who are active but do not participate in one of the sports evaluated in this rating scale may be incorrectly rated as having a lower activity level. A rating scale that measures patients' activity independent of specific sports is desirable. One such scale has been published that was developed with patient input with respect to activities that are important and difficult for them to perform3. This rating scale asks patients four questions about the frequency with which they perform four activities. The activities are running, cutting (changing directions while running), decelerating (coming to a quick stop while running) and pivoting (turning the body with the foot planted…). This scale has been evaluated for reliability and validity in separate groups of patients3. The Lesion The characteristics of the cartilage lesion that is repaired have an important impact on the outcome after treatment. These characteristics should be documented in detail prior to surgery to allow an accurate evaluation of the results in light of what was actually treated. The size of the lesion and whether the lesion involves only cartilage or bone should also be determined since lesions involving subchondral bone generally require a more involved reconstruction. The diagnosis will also have an important effect on treatment in many cases. Avascular necrosis leading to a cartilage problem will affect underlying subchondral bone and may be related to systemic health problems. Osteochondritis Dissecans also involves the underlying subchondral bone and will often lead to large defects. The alignment of the lower extremity can also affect outcome depending on the location of the lesion. Alignment is ideally evaluated radiographically using three foot standing x-rays to determine the anatomical alignment and the mechanical axis. Other interarticular disease must also be evaluated, particularly the opposing articular surface. In general, if the opposing surface is degenerative, the patient would be diagnosed with arthritis and cartilage resurfacing may not be appropriate. Therefore, the articular surface opposing the cartilage injury site as well as the articular surfaces elsewhere in the knee must be evaluated. The Procedure When evaluating the results of surgery there are several factors that should be considered in addition to the actual type of operation performed. The indication for surgery should be documented. In general, the indication for surgery is pain. However, if the indication is not pain and the surgeon is performing the operation to avoid future problems in the knee, this should be explicitly indicated. If the patient has had a prior procedure this also should be documented. The type of procedure performed previously and the number of operations done should also be evaluated. The postoperative rehabilitation may have an important influence on the outcome. Factors such as use of continuous passive motion, weight bearing and strengthening exercises, as well as the timing of their incorporation can affect the result of the procedure. While it is obvious that the surgeon has a critically important effect on the outcome, this should be evaluated in studies comparing two treatments. In certain cases two procedures will be evaluated in a head to head comparison. If one surgeon performed all of the operations for the patients in one group while a different surgeon performed a different operation for the patients in a different group, the study is inherently biased. In this case, the study may be comparing the surgeons rather than comparing the procedure itself. In situations such as this, it is important to remember that the investigator must track not only what was done, but by whom. Evaluation of Outcome: Objective There are several “objective” measures of outcome such as physical examination, imaging and tissue biopsy. While these are generally very important to the surgeon, they may not be of any relevance to the patient. Patients are generally more concerned with their subjective complaints and function. Nevertheless, objective measures are important and often give critical information. Physical exam is a routine part of follow-up after surgery. For cartilage procedures about the knee physical exam includes an evaluation of gait, pain on palpation, effusion, range of motion and stability of the knee. Imaging is also an important part of the evaluation. Radiographs can demonstrate the progression of degenerative disease such as osteophytes, subchondral sclerosis, subchondral cysts and joint space narrowing. Change in alignment may also be related to degenerative osteoarthritis. An evaluation of the repair tissue itself is useful to determine the quality. Routine histology as well as immunohistochemical evaluation have been performed6,7. While the information gained by biopsy is valuable, many patients will not consent to this procedure. Despite the potential lack of patient interest in this approach, some authors have been able to evaluate patients with this methodology8. Evaluation of Outcome: Subjective There many factors that are termed “subjective” with respect to patient evaluation. They are termed as such because they are difficult to evaluate or measure quantitatively. Nevertheless, issues such as pain and function are of paramount importance to patients who are recovering from cartilage procedures. Symptoms and disabilities are generally evaluated using validated rating scales. There are many that have been published for use in this patient population9,10. The goal of using rating scales to measure patient outcome is to evaluate concepts that are critical to patients and to do so in a time efficient manner. Therefore, relatively shorter questionnaires are preferred to limit responder burden. Of the available knee rating scales, several will be discussed below with respect to their usefulness for this patient population. The modified Lysolm scale2 is an eight item questionnaire which was initially designed to evaluate patients after knee ligament surgery. It has 25 points attributed to knee stability, 25 to pain, 15 to locking, 10 each to swelling and stair climbing and 5 each to limp, use of support and squatting. It has been used extensively for clinical research studies mainly for the anterior cruciate ligament. However, it has recently been evaluated and found to be acceptable for chondral disorders of the knee11. The activities of daily living scale of the knee outcome survey is a useful instrument for cartilage patients and we have distributed this questionnaire to evaluate patients at our institution12. It was developed based on a review of relevant instruments with clinician input. It is designed for patients with disorders of the knee ranging from ACL injury to osteoarthritis. Therefore, it is generally applicable to most cartilage patients. The questions range from relatively simple basic functions to more advanced activity. It has been found to have excellent psychometric properties10. The international knee documentation committee (IKDC) developed a rating scale for “objective” parameters relating to knee function. These parameters include effusion, motion, ligament laxity, crepitus, harvest site pathology, radiographic findings and one-leg-hop tests. Patients were given a grade as normal, nearly normal, abnormal or severely abnormal for each. The lowest grade for a given group is the patient's final grade. The IKDC has subsequently developed a questionnaire relating to “subjective” factors16. While this questionnaire has not specifically been validated for patients with articular cartilage disorders, it is likely that it is a useful instrument. The knee injury and osteoarthritis outcome score (KOOS), was developed using input from patients who underwent meniscal surgery in the past14. Five separate scores are calculated for pain, symptoms, activities of daily living, sport and recreational function, and knee related quality of life. This scale is useful since the Western Ontario and McMaster University's osteoarthritis index (WOMAC) is incorporated into the KOOS15. The WOMAC involves 24 questions with 5 relating to pain, 2 to stiffness and 17 to difficulty with activities of daily living. It is mainly for patients with lower extremity osteoarthritis and therefore can be useful for patients with cartilage disease. The KOOS is a wide ranging scale since it applies to patients with degenerative disease, but also has questions about sport participation. This makes it an attractive alternative for evaluating outcome follow cartilage procedures., Microfracture is one of many methods available to treat articular cartilage lesions. The technique has been elaborated by Steadman et al. and applied chiefly in young athletes and in young patients generally1. There are some inherent advantages. The microfracture technique is minimally invasive and the costs are minimal since expensive cell cultures are not necessary. In comparison to an abrasion chondroplasty the subchondral bone plate is not completely destructed but partially preserved between the microfracture holes improving load-bearing characteristics following healing2. Unlike osteochondral or periosteal autograft procedures the problem of a harvest site morbidity is excluded. The intervention leads to a spontaneous repair response, which is based upon therapeutically induced bleeding from the opened subchondral bone spaces and subsequent blood-clot-formation3. However the technique has also some inherent disadvantages. Microfracture promotes resurfacing with predominantly fibrocartilaginous repair tissue of inferior quality. In this context a fibrous type of cartilage tissue was found in rabbits or canines treated with Pridie drilling or with microfracture4. Animal studies investigating the durability of fibrous tissue revealed a progressive failure of apparently well healed cartilage. Reason for the worse durability was the lack of physical and chemical bonding between the macromolecular components of the repair tissue and the residual adjacent cartilage following micromotion and macromotion between them. Furthermore the repair tissue over the re-established prominent tidemark was only half as thick as the surrounding original cartilage. These results were confirmed by clinical studies with MRI evaluation, that revealed osseous overgrowth between 25–50% of the patients and persistent gaps between the native and repair tissue in most of the microfracture repairs. Further MRI analysis showed an incomplete defect fill under the level of the intact adjacent hyaline cartilage5–7. While no validated treatment algorithm exists for treating articular cartilage lesions in the knee, the arthroscopic microfracture technique is commonly used as a first-line option and frequently serves as the standard technique against which other cartilage repair procedures are compared. A recent analysis of cartilage repair techniques has pointed out the methodological limitations of the available literature on articular cartilage repair8. This may be a reason for the heterogenity in outcome between worse and excellent results, reported in clinical studies with a follow up to more than 10 years after surgery. To provide a better understanding in the indications and limitations of the microfracture technique we performed a comprehensive analysis of the clinical literature on articular cartilage repair in the knee by this technique. For this purpose literature was searched for human studies reporting clinical, histological and MRI results after microfracturing chondral lesions. The used search engines were MEDLINE, EMBASE, CINAHL and Cochrane Central Register of Controlled Trials. The quality of the existing studies was analysed using modified Coleman Methodology scores. Clinical effectiveness of articular cartilage repair was evaluated by systematic analysis of short- and long-term functional outcome scores, macroscopic and microscopic repair cartilage quality, and findings of postoperative magnetic resonance imaging. Twenty-eight studies describing 3122 patients were included in the review. Average follow-up was 41 months with only five studies reporting follow-up of 5 years or more. Six studies were randomized-controlled trials and mean Coleman Methodology Score was 58 (range 22–97). During the first 24 months microfracture effectively improved knee function in all studies. Afterwards there are conflicting results regarding the long term durability. This heterogenity in long term outcome may be explained by several factors, influencing the final result. In a randomised trial comparing microfracture and ACI more fibrous as hyaline cartilage was found in the histological evaluation of biopsy specimens taken during a second-look arthroscopy after microfracture. The histological results were graded with 1 to 4 points. Most of the failures by 5 years occured with fibrocartilage and none of the patients with a failure had the best-quality cartilage9. The factors leading to more hyaline or to more fibrous cartilage are still unknown. However the quality of the repair tissue seams to play a major role in the long term durability of the repair tissue and subsequent good clinical outcome. The importance of the tissue quality was also addressed by another study comparing microfracture and ACI using characterized chondrocytes. After 12 months ACI revealed significant better histological results and after 36 months significant better clinical results could be detected with the ACI procedure10. We could find some other important factors, that may influence the clinical outcome or the histological result. In this context a good defect fill with improved clinical scores were identified primarily with defects on the femoral condyles. In contrast trochlear or retropatellar defects revealed in most cases a deterioration between 18 and 36 months after surgery7. Furthermore young patients under 40 years had the best clinical results6. The worse results of patients over 40 years compared to younger patients may be explained by the reduced regeneration capacity of the repair tissue. As stem cells age their mitochondrial function and synthetic activity decline. Therefore chondrocyte senescence decreases the efficacy of cartilage repair. Another important factor was the amount of defect fill on MRI, which was highly variable and correlated with functional outcome. Macroscopic repair cartilage quality positively affected long-term failure rate. Furthermore better clinical results could be detected in young sportive patients with a Tegner score over 4, a short duration of symptoms, a small lesion size, a low body mass index and the first surgical intervention. In all prospective clinical randomised controlled trials the lesion size was limited to 4cm2. Larger lesions are predominantly treated with autologous chondrocyte implantation. For these large lesions there are only few and not sufficient data with the microfracture procedure. In conclusion microfracture is a minimal invasive and cheap method, that provides effective short-term functional improvement of knee function especially in young active patients with small chondral defects. Only few data is available on its long-term results. Shortcomings of the technique include limited hyaline repair tissue, variable repair cartilage volume, and possible functional deterioration. Further well-designed prospective randomised controlled trials with an adequate randomisation procedure, power analysis, patient inclusion and exclusion criteria, validated outcome measures, independent investigators, clinical histological and MRI evaluation with a continuous follow up, a detailed rehabilitation protocol and a high Coleman Methodology Score are needed to determine the long-term effectiveness of microfracture and to define its specific clinical indications compared to other cartilage repair techniques., Repair processes often mimic or recapitulate developmental processes quite closely although not exactly. Thus, an understanding of developmental processes is essential if we wish to derive rational strategies in attempting to biologically augment articular cartilage repair. Interestingly, whilst we know a great deal about the signalling molecules that regulate the transition of phenotypic states within the epiphyseal growth plate, we know very little about those that regulate the growth and development of articular cartilage. However, first we must compare the similarities and differences between these two tissues. It is often considered that both the epiphyseal growth plate and articular cartilage are remnants of the embryonic cartilaginous rudiment. Whilst this still holds for the growth plate, it is now known that articular cartilage derives from a sub-set of cells that reside at the periphery of the embryonic epiphysis adjacent to the interzone (Hyde et al., Dev Biol) In terms of the mechanisms of growth, then there are certainly similarities in that they both grow by apposition with stem/ progenitor at the ‘top’ of the tissue, differentiating then proliferating and eventually terminally differentiating to be replaced by bone. Seminal work by Vortkamp elucidated the BMP/IHH/PTHrP pathway that regulates the transition of cell state in the growth plate and she continues to refine these pathways (refs. Science and more recnt) In contrast to the growth plate, little if anything is known of the signalling pathways that regulate the transition of cells in articular cartilage. Current evidence suggests that it is not the same as the growth plate but this is an area that requires urgent attention. In terms of the regulation of the proliferation of stem cells that gives rise to appositional growth, we know that the Delta/Notch pathway plays an important role in articular cartilage as about 70% of the cells at the articular surface are positive for Notch1 and inhibition of Notch signalling abolishes clonality of the progenitor population (Dowthwaite et al., 2004). At the same time, Notch1 is also expressed by the cells immediately above the terminally differentiating cells and is similarly placed above pre-hypertrophic cells in the growth plate where Notch1 has an inhibitory effect of progression to terminal differentiation. Lastly, I will speculate further on other aspects of the development of articular cartilage that may be useful in better understanding repair and pathological processes., Articular cartilages provide within a diarthrodial joint, together with the lubricants lubricin and hyaluronan secreted also by the synovium, an almost frictionless articulating surface capable of handling the stresses, strains and dissipation of loading that is required over many decades of use. It is a truly unique tissue the properties of which are determined by its extensive and very specialized extracellular matrix. What is interesting is how the properties and responses of this tissue to the environment, be it physiological or pathological, can vary according to the site within a joint and between joints. A variety of information collected over many years is now giving us better insights into the variability in cartilage form, function and response to injury. Some of this will be reviewed focussing on the ankle, knee and hip joints. The overall determining factor in cartilage thickness would appear to be the biomechanical loading that the joint experiences. From studies of animals thickness appears to be directly related to the to body size and is in a linear logarithmic relationship to body weight1. In the case of the human lower limb the mass of the donor is significantly correlated with the mean cartilage thickness for ankle2, knee2,3 and hip joints2. Height is also correlated with thickness in the hip and knee joints whereas body mass index and cartilage thickness are correlated in the ankle joint2. The latter studies have also indicated an inverse relationship between cartilage thickness and its compressive modulus. The knee has thicker cartilage than the ankle and hip while hip cartilage is usually thicker than the ankle2. This thickness may be determined by the congruence of the joint as thicker cartilage is found in incongruent joints4. Thus it has been suggested that congruent joints with thin cartilage only deform a small amount yet the area of contact is large enough to distribute load and maintain an acceptable level of stress. In incongruent joints deformation of the thicker cartilage increases the contact area between joint surfaces to decrease the stress. Talar (ankle) cartilages have a higher proteoglycan content and lower water content, consistent with the higher equilibrium modulus and dynamic stiffness found in these cartilages5. Anatomical, structural and biomechanical differences in cartilages are also seen within a joint. The surface layer is very different to the deeper layers with its high tensile stiffness and fracture stress. This decreases progressively with increased depth as proteoglycan aggrecan content increases and cell density decreases6. Even the matrix around cells varies in structure with the distance from the cell6. The fattened cells of the cell surface layer express distinct proteins such as lubricin7. The superficial cells of the ankle and knee cartilages are also quite different in their arrangement. In the knee chondrocytes are present as single cells or doublets. Whereas in the ankle they are arranged in planar clusters containing multiple cells within chondrons8. Adjacent to focal ankle lesions the clusters increase in size. This differential response to degenerative focal lesion development is of special interest. This is because biochemical studies of knees versus ankles have revealed that in knees the reponse of articular cartilage in an early lesion emphasizes degradation of type II collagen. In contrast, in the ankle the emphasis is on increased synthesis of this collagen and increased turnover of the proteoglycan aggrecan with no increase in matrix degradation9. Moreover the changes that occur in the ankle are not restricted to the lesion site as in the knee10. They are seen throughout the joint11. Together these observations demonstrate that there is a strong reparative response to focal damage throughout the ankle joint, something not seen in the knee where matrix destruction is emphasized. Chondrocytes in the knee are more sensitive than those in the ankle to the harmful effects of IL-1, a cytokine considered to be important in cartilage pathology in osteoarthritis12 the superficial cells being most sensitive with a greater number of high affinity receptors for IL-113. These structural, metabolic and mechanical differences could account for the much higher incidence of osteoarthritis in the knee compared to the ankle. Regional differences within joints also exist. In the case of the adult bovine knee joint significant differences exist in femoral condyles for proteoglycan content, this being higher in the lateral femoral condyle than the medial and femoral condylar groove14. Collagen content is higher in the groove than in condylar cartilage as is pyridinoline cross-linking. This is reflected in higher tensile strength in the groove although failure strains are similar in these different joint sites. All these differences no doubt reflect the different mechanical environments acting upon chondrocytes and determine how they may respond in different joint locations to create a matrix that is best suited to that environment. The marked differences between the ankle and knee articular cartilages provide insights that can better help us understand the development of osteoarthritis., Introduction Focal cartilage damage in the knee is most commonly caused by trauma and osteochondritis dissecans. The choice of treatment of such a cartilage lesion depends on a variety of factors. Operative cartilage repair techniques usually consisted of debridement and microfracturing the defect (1,9). Autologous chondrocyte implantation (ACI) and osteochondral autograft transplantation (OAT) are also advocated (1–4). These different techniques all claim substantial clinical improvement and the available literature does not provide evidence to declare one technique superior to another. On the contrary, the few randomized controlled studies available comparing these three techniques conclude with conflicting results (1,4,9). We focus on the options and limitations of osteochondral autograft transplantation in an attempt to facilitate appropriate patient selection for this specific treatment option. Biomechanical Aspects The technique of osteochondral autograft transplantation is based on the concept of transplanting vital hyaline articular cartilage from a minor weight bearing area of the (knee) joint to a chondral defect in the weight bearing area. Thus, the clinical problem of a large articular defect in an important area of the joint is addressed with a solution where a larger number of relatively small defects in a less symptomatic part of the joint is accepted. Since the fixation options of hyaline articular cartilage to the subchondral bone remain insufficient, the cartilage has to be transplanted as an osteochondral plug. There are several biomechanical dilemmas' to address with this technique. For example, the osteochondral plug has to be long enough to allow stable fixation in the recipient hole. Without a stable press fit fixation the plug will not incorporate in the host bone with eventual failure of the transplant. In addition, the articular surface has to be reconstructed as anatomically as possible. Various cylindrical plugs are used to resurface the joint (mosaicplasty). Careful positioning of the transplanted osteochondral plugs is mandatory to achieve a congruently reconstructed joint surface. Protruding plugs may introduce peek stresses on the graft and thus lead to early degeneration. On the other hand, it is equally important not to place the plugs below the recipient joint surface, since this will lead to peak stresses on the border. Surgical technique is thus critical to achieve an optimal reconstruction of the lesion with intact hyaline cartilage and a congruent joint surface to allow an adequate load distribution. We performed cadaver studies on the optimal surgical technique for osteochondral autologous transplantation in human knees. One study focussed on the optimal length of the osteochondral plug to achieve adequate press fit stability (6). In addition, it was hypothesized that transplanted plugs would have more intrinsic stability when the length of the graft was matched exactly with the depth of the recipient hole. This way the plug was ‘bottomed’ in the defect and would therefore be less susceptible for subsidence. Different lengths of (un)bottomed osteochondral plugs were tested using compressive forces from a loading apparatus. It appeared indeed that longer plugs needed higher forces to begin displacement. At flush level, bottomed plugs needed significantly higher forces than unbottomed plugs to become displaced below flush level (mean forces of 404 N and 131 N, respectively), especially when short plugs were used. In clinical practice we therefore recommend to use short bottomed plugs. If, however, unbottomed plugs are still chosen, the longer the plug the higher the resulting stability will be because of higher frictional forces. In a second biomechanical cadaver study surface congruency after osteochondral transplantation was evaluated (7). Restoration of surface congruency and stability of the reconstruction may be jeopardized by early mobilization. To investigate the biomechanical effectiveness of osteochondral transplantation, we performed a standardized osteochondral transplantation in eight intact human cadaver knees, using three (un)bottomed cylindrical plugs. Surface pressure measurements with Tekscan pressure transducers were performed after five conditions. In the presence of a defect the border contact pressure of the articular cartilage defect significantly increased to 192% as compared to the initially intact joint surface. This was partially restored with osteochondral transplantation (mosaicplasty), as the rim stress subsequently decreased to 135% of the preoperative value. Following weight bearing motion two out of eight unbottomed mosaicplasties showed subsidence of the plugs according to Tekscan measurements. This study demonstrates that a three-plug mosaicplasty is effective in restoring the increased border contact pressure of a cartilage defect, which may postpone the development of early osteoarthritis. Unbottomed mosaicplasties may be more susceptible for subsidence below flush level after (unintended) weight bearing motion. Histological aspects Various studies have shown good clinical results from mosaicplasty of the weight-bearing part of the femoral condyle (1–4). After 10 years of follow-up, good-to-excellent results have been described in 92% of 597 treatments with mosaicplasties (2). Despite these enthusiastic reports on mosaicplasty, there is still concern about the fate of transplanted cartilage and the repair potential of the donor site defect (11). Most studies have reported sufficient repair of both the original cartilage defect and the donor site defects on (occasionally performed) repeated arthroscopy (2). Unfortunately, these studies have been based mainly on data from subjective clinical scores. Histology is of course difficult to obtain for ethical reasons. Occasionally, limited histology from a needle biopsy of the graft has been described, with promising results. We report on a good quality full-thickness histological specimen from an entire osteochondral transplantation (5). The sample could be obtained for histological evaluation because of a total knee arthroplasty performed 3 years after the mosaicplasty. Histology of the recipient site showed vital grafts with excellent incorporation in the subchondral bone. The transplanted cartilage had retained its hyaline structure and seemed to provide a good resurfacing of the joint. The donorsite defect, however, showed only limited repair with fibrous tissue filling of a persistent subchondral defect. In clinical practice the donor site defects are commonly left empty, and spontaneous repair with fibrocartilagenous tissue is assumed. From earlier animal experiments on donor site defects, this potential for spontaneous repair appears to be very limited (11). In an attempt to stimulate the repair of the donor site defect an animal experiment was repeated in the goat. A standardized osteochondral donor defect was created in the knee. Defects were addressed by transplanting an extra-articular osteo-periosteal plug from the proximal tibia to the donor site defect. Empty defects and defects with a plug without covering peristeum served as controls. Incorporation of this graft into the subchondral bone was observed; however, no chondrogenesis from the covering periosteum could be detected. In this animal model no substantial benefit could be detected from this approach, except perhaps some structural support to the adjacent subchondral bone, preventing early collapse. We believe that the repair potential of the donor site defect, as well as the value of an additional osteoperiosteal plug from the proximal tibia, must not be overestimated. A second animal model in the goat focussed on the vitality of transplanted osteochondral plugs and the susceptibility of the transplanted plugs to subsidence. A standardised two-plug (ø 6 mm) osteochondral transplantation was performed in the goat knee. Histological evaluation is currently performed on fuorochrome vitality staining of the transplanted articular cartilage cylinder, trochlear donor site defect repair and subsidence of bottomed versus unbottomed plugs. The overlying articular cartilage clearly survives the transplantation procedure of the osteochondral plug, as appears from the diffuse green staining of the chondrocytes. No red staining, representing cell death, was observed at the (cutting) edges or at the surface. Some subsidence of the plug could be observed in a number of unbottomed plugs (personal data). Clinical aspects Osteochondral lesions in the knee remain a clinical challenge and do not not heal spontaneously in adults. There is not yet an optimal treatment intervention defined due to the limited amount of prospective research available. Various studies have reported on good results in a larger number of patients using different techniques (1–4). The main limitation of these studies is the heterogeneity of the treated cartilage defects. We treated a small subgroup of patients with homogenuous OD lesions with OCT and also reported on a significant improval (p⇠0.003) on various clinical scoring systems (ICRS package) (10). Prospective follow-up MRI was also performed using a semi-quantitative scoring system. Magnetic resonance imaging evaluation at 1-year follow-up showed good surface congruency, no edema or protuberance of the cylinders, good similarity of cartilage thickness and a non-complete osseous integration. Despite these promising clinical results, the potential for donor site morbidity remains a concern with OCT. Prospective bone scintigraphic evaluation both for the donor and the recipient site was performed in patients who were treated with OCT (8). In a group of 13 patients with a symptomatic articular cartilage defect bone scintigraphies were obtained pre-operatively, one year after osteochondral transplantation and finally at an average follow-up of 4 years (31–65 mnths). We learned that elevated bone scintigraphic activity from an osteochondral lesion in the knee could be restored with OCT. However, increased scintigraphic activity was introduced at the donorsite, which activity reduced again with longer follow-up. The use of fairly large osteochondral plugs appeared to correlate with retropatellar crepitus and increased scintigraphic activity and therefore is not recommended. Conclusions We conclude that osteochondral transplantation remains a valid treatment option in selected cartilage defects. A subgroup of osteochondral defects on the lateral border of the medial femoral condyle with a limited size improves significantly following osteochondral transplantation. Donor site morbidity remains a concern and clear limitation with this technique. Donor site problems can be controlled using a limited amount of plugs (maximum 3–4) with a diameter of no more then 8 mm. Future research should probably focus on identifying the appropriate choice of operative treatment (OATS, chondrocyte transplantation or microfracture) for well defined subtypes of articular cartilage lesions, instead of searching for one superior technique for all., Cartilage is widely distributed throughout the human body and is comprised of a combination of connective (or skeletal) tissue cells and extracellular matrix [1]. The specific organization of the various cartilaginous tissues is related directly to the temporal and spatial functional demands on the tissue, both static and dynamic. Generally, these functional demands pertain to the following: (1) the protection and support of related non-skeletal tissues and organs, (2) the articulations between skeletal elements, and (3) the dynamic processes related to skeletal growth [1]. Articular cartilage is an extremely important mechanical entity in articulating joint function where it provides a wear-resistant surface for one diarthrodial element to slide over the other [2]. Other types of cartilage tissues fulfill mechanical function as well, although different from that of the articulating surfaces of joints. Cartilage of the intervertebral disc acts as a load transmitter and shock absorber between bony vertebral bodies [3]. The functional roles of cartilage in the trachea, nose, ribs, ears, and pharynx involve maintaining form and resisting deformation, while providing some degree of flexibility [4]. Because of these differences among the cartilage tissues, the extracellular matrix, which possesses a defined biochemical composition and confers specific biomechanical properties, is composed differently. Whereas a large body of work has focused on repair and regeneration of articulating hyaline cartilage, a growing body of research on repair and regeneration of craniofacial and other cartilages offers other paradigms for cartilage repair with some similarities as and differences to be discussed. Cartilage of all types is a relatively simple, but highly specialized, connective tissue consisting of chondrocytes embedded in an extracellular matrix composed primarily of proteoglycans, collagen, and water[1]. Since cartilage has no internal vascular network and, therefore, possesses limited innate ability for repair and regeneration [5–7], injury to cartilage often results in scar formation and permanent loss of structure and function. Nutrition by diffusion rather than through a vascular network, however, allows cartilage to be easily transferred to repair sites and to be used in a multitude of ways. For example, autologous cartilage can be sculpted into delicate structures like an ear, or fill defects and restore contour in areas throughout the face [8–11]. One possible solution for providing quality structural tissue could be to engineer cartilage tissues to meet the anatomic requirements for the repair. As such, the material properties of synthetic or natural compounds could be manipulated to allow the delivery of an aggregate of dissociated cells into a host in a manner that will result in the formation of new functional tissue [12]. Using a sufficient quantity of cells combined or composited with a polymer(s) and transplanted into the defect site could restore normal function. To achieve the desired result, however, one must consider both the properties of the tissue native to the site and the properties of the polymer(s) being used to generate cartilage repair tissue. Although cartilage is a relatively simple tissue containing only one cell type-chondrocytes—the cartilage from different anatomic areas have different structural and functional demands. Entrapped within the extracellular matrix, the chondrocytes continuously produce various macromolecules such as collagen and sulfated glycosaminoglycans to replenish the extracellular matrix according to these anatomical differences. Cartilage can be divided into categories according to the composition of the matrix, and its biological role in the body. Hyaline cartilage, which is rich in type II collagen, can be found in the ribs, trachea, and covering the articulating surfaces of bones where it functions as a gliding surface and shock absorber for skeletal elements[13]. Elastic cartilage, which contains elastin, occurs in tissues such as the external ear, the epiglottis, and portions of the larynx [6]. Fibrocartilage, which is rich in type I collagen fibers, can be found in tissues that are subjected to tensile forces like the outer portion of intervertebral discs, the knee menisci, and in certain ligament and tendon attachments to bone [14]. Specialized cartilage tissues, like those in the epiphyseal growth centers of long bones, contain highly specialized chondrocyte elements that precisely control elongation and mineralization of growing bones [13]. Therefore, the structural and normal biological function of each of the diverse cartilages should be considered when attempting to engineer replacement tissue. The biochemical composition is closely related to the mechanical factors to which the specific cartilage structures are subjected. Hyaline cartilage in the diarthrodial joints are under constant shear and compression forces, while simultaneously providing low friction across the interface [5,7,13]. The extracellular matrix is designed to absorb compression and return cartilage to a normal state once the force is removed. By contrast, the elastic cartilage in the ear has internal structural support and different extracellular matrix molecules to give the ear an external shape and flexibility [14]. In healing and repair of cartilage, the tensile forces can be a measure of integration of the engineered cartilage with the native cartilage at the defect site [15–17]. Anderson and Athanasiou have reported on generating cartilage for replacement of the temporomandibular joint, which in itself has specific mechanical demands [18]. Consideration of the mechanical demands affecting the tissue will help guide approaches to engineer new cartilage specific to the defect. Placing chondrocytes in three-dimensional matrices, similar to their natural environment, can permit the cells to retain their native phenotype and produce their extracellular components. Using polymers that undergo a controllable bulk erosion process in vivo, the polymer can be made to resorb at a rate proportional to the rate at which cartilaginous extracellular matrix is being deposited into the intercellular spaces. A critical element for engineering cartilage is finding or developing suitable scaffold materials that permit or accelerate the formation of new extracellular matrix according to the cartilage tissue desired. Using polymers, both natural and synthetic, that undergo controllable bulk erosion or resorption can be favorable for engineering cartilage tissues in vitro or in vivo. For example, polymers that degrade at a rate proportional to which cartilaginous extracellular matrix is being deposited into the intercellular spaces could be employed to generate cartilage in situ. Several scaffolds, both natural and synthetic, have been tested in animal models for engineering cartilage. Xu et al. have reported on the formation of cartilage from different anatomical sources and differences in mechanical properties of the neocartilage [19]. Investigators have demonstrated numerous techniques for improving the biological and biomechanical properties of tissue-engineered cartilage including techniques to: 1) to improve the bioproperties of extracellular cartilaginous matrix, 2) provide internal support to tissue-engineered cartilage, and 3) add external (pseudoperichondrium) support to tissue-engineered cartilage. One objective is to improve the flexibility of tissue-engineered cartilage framework, particularly in tissues other than joint cartilage. Some possibilities to enhance the neocartilage matrix properties by incorporating non-resorbable materials to meet the needs for reconstruction will be discussed. Cao et al. engineered cartilage in the shape of human auricles in nude mice using articular chondrocytes and a biodegradable internal PGA/PLLA scaffold to attain the desired shape of an ear [20] demonstrating that tissue-engineered cartilage could be generated with a resorbable endoskeletal scaffold. There have been no reports of successfully generating complex three-dimensional cartilage structures using PGA or PLLA in immune-competent animals, however, possibly due to intense inflammatory responses. Recent work by Kusuhara et al. has improved on the generation of three-dimensional human ear shapes using a co-polymer of poly(L-lactic acid) and poly(e-caprolactone) and costal, articular and nasal septal chondrocytes from bovine sources [21]. Arevalo-Silva and colleagues [22] investigated the use of nonbiodegradable endoskeletal scaffolds made from the following materials: 1) high-density polyethylene, 2) soft acrylic, 3) polymethylmethacrylate, 4) extrapurified silastic and 5) conventional silastic. They concluded that using a permanent biocompatible endoskeleton demonstrated success in limiting the inflammatory response to the scaffold, especially the high–density polyethylene, acrylic, and extrapurified silastic. Our laboratory examined the mechanical function of perichondrium in ear cartilage. We found that intact perichondrium prevented fracture of ear cartilage tested [14]. From these studies we concluded that providing a perichondrial layer was important for confer flexibility to engineered cartilage tissue intended for craniofacial reconstruction. To simulate a perichondrial layer, we investigated expanded polytetrafuoroethylene and lyophilized perichondrium as structural components for supporting the engineered cartilage. Recent work by our group has evaluated the use of rigid nondegradable materials, such as porous polyethylene, enhance the structural stability of ear cartilage. Similar hybrid approaches could be employed for generating tissues like the meniscus or vertebral discs. In summary, the primary goal of engineering cartilage as a therapeutic approach is to restore the physiological conditions of an affected or defective tissue in the body and the neotissue should possess the organization related to the specific structural and mechanical demands of a particular anatomical region., Introduction Blood has been considered an armful factor for the articular cartilage. Previous studies have demonstrated that recurrent intra-articular bleedings represent a negative factor for articular cartilage, inducing a deterioration of the tissue. This could ultimately lead to degenerative osteoarthritis, even though the mechanism is not yet entirely understood (1–3). Other authors have investigated the effect of peripheral blood on articular cartilage both in vitro and in vivo: Roosendal and Hooiveld have shown that a short-term exposure of human articular cartilage to whole blood in vitro induced an irreversible dose-dependent inhibition of proteoglycan synthesis and it was accompanied by cell apoptosis (4,5). However, when a short-term exposure was performed in vivo after injection of autologous blood into the canine knee, the initially adverse changes in cartilage proteoglycan synthesis turned into normalization after 10 weeks (6). Recently, the same group has tested the threshold of blood exposure time and concentration that lead to irreversible joint damage (7). However, many current surgical procedures for articular cartilage repair, like subchondral bone drilling (8), abrasion artrhoplasty (9) and microfracture (10), are based on the capacity of bone marrow cells to produce a fibrocartilaginous tissue when migrated in a joint environment (11). In contrast, in the performance of the techniques based on the transplantation of autologous chondrocytes, the presence of blood has been considered a disturbing factor for the development of the new cartilage tissue (12). The more recent techniques for cartilage repair and reconstruction utilize autologous chondrocytes seeded onto a biocompatible scaffold, in which they can duplicate, mature and produce new cartilage matrix in vitro and in vivo after surgical implantation. Also in this approach, it is recommended care in protecting the reparative cells from the contact with blood, which could derive from the subchondral bone or any other part of the joint injured during the surgical implantation (13), both in open and arthroscopic approach. However, the nature of engineered cartilage differs from that of cartilage explants examined in the studies mentioned above. Therefore, we believe that the influence of the contact of peripheral blood to the engineered cartilage represents an important but still unclear issue that needs to be investigated. The engineered cartilage, however, is structurally and biologically different from native articular cartilage, as it is supposed to complete the maturation in vivo. Therefore, it is probably more susceptible to the adverse effects of an articular bleeding, as indicated by studies of other authors, who investigated the effect of blood on immature joint (14). The effect of blood on engineered cartilage was only recently investigated (15). We have developed an in vitro model to investigate the effect of blood contact on the tissue-engineered implant and demonstrated that a 3-day exposure of cartilage to 50% (volume/ volume) blood results in a temporary and reversible effect on engineered cartilage tissue obtained from chondrocytes seeded onto collagen scaffold. However, some important issues remain to be clarified: could the different blood concentration negatively affect the chondrocytes' vitality and synthetic properties? Is the negative effect of blood on the engineered cartilage due to the toxic effect of the peripheral blood or to the lack of nutrients occurring during the exposure to blood? The aim of this study was to investigate the effect of different concentrations of blood and of the lack of nutrients on the morphological, biochemical and biomechanical properties of engineered cartilage, synthesized by articular chondrocytes seeded onto a biological scaffold. Additionally, we have analyzed the effect of the main pro-inflammatory chemokine IL-1p on engineered cartilage based on human articular chondrocytes cultured for two different culture times. Experimental data Tissue engineered cartilage was developed combining expanded chondrocytes with a collagen membrane scaffold. Two sets of experiments were performed: one testing swine chondrocytes and allogeneic blood as inflammatory factor (“Blood study”); the other testing human adult chondrocytes and IL-1β (0.05 ng/ml) (“IL-1 study”). In the “Blood study”, articular chondrocytes were isolated from swine joints, expanded in monolayer culture, seeded onto collagen membranes and cultured for 2 weeks. During this period, an immature extracellular matrix, produced by cells, stabilized the chondrocytes to the biological membrane. This method generally duplicates the protocol for membrane seeding used in current clinical practice. After 2 weeks (t0), some samples were retrieved for analysis; others were exposed for 3 days to the contact with swine peripheral blood diluted with culture medium at 2 different concentrations (B50% group = 50% blood / 50% medium; B80% group = 80% blood / 20% medium); others were exposed for 3 days to the contact with a PBS solution. Following these 3 days (t3), some samples were retrieved for analysis, others were returned to standard culture conditions for 21 additional days (t3+21), in order to investigate the “long term effect” of the blood contact. For all the listed experimental times, some samples belonging to the control group were left in standard culture conditions without having any contact with blood or PBS. All groups of seeded membranes were analyzed grossly, by optic microscopy (OM), biochemically, histologically, and by biomechanical test. In particular, for morphological analysis, samples were analyzed macroscopically and by OM at all experimental times. Samples were weighed and sized with a calliper (products of axis) upon retrieve from culture. The edge of membranes was evaluated by OM analysis. For biochemical analysis, rate of cellular proliferation was evaluated by mitochondrial redox reaction to the tetrazolium salt (MTT) at all experimental times. For histological analysis, samples were processed and stained with safranin-o. Few sections (only at the time t3+21) were processed for immunohistochemical analysis and stained for type II collagen. For biomechanical analysis, samples were tested under unconfined geometry for compression using an electromagnetic testing machine. In the IL-1 study, human articular chondrocytes were harvested post-morted, expanded in monolayer, seeded onto collagen I/III scaffolds and cultured for 2 and 4 weeks. IL-1 was added during the last 3 days of culture. Samples were analyzed with histology, immunohistochemistry for collagen II, and biochemistry. In the “Blood study”, all seeded samples showed an increase in the weight and an evident cartilage-like matrix production. The evaluation of the samples with an optic microscope showed similar results for all study groups. It demonstrated the presence of spherically-shaped cells homogeneously distributed around the edge of the samples and firmly attached to the membranes. A specific concentration-dependent reduction of the mitochondrial activity due to blood contact was evident at the earlier culture time, followed by a partial recover at the longer culture time. An initial reduction of the biomechanical properties of the membranes, followed by a late stabilization, was recorded, regardless the presence of blood. In the “Il-1 study”, all samples exposed to IL-1 demonstrated a reduction in the intensity of Safranin-o and collagen II staining and in the quality of the biochemical composition. This reduction was more marked for the samples cultured for 2 weeks only, while samples cultured for 4 weeks had a better response to the pro-inflammatory stimulus. Conclusion The results from this study demonstrated that isolated chondrocytes could be seeded onto a biological scaffold, producing cartilage-like matrix with tissue specific morphology, composition, and biomechanical integrity. The blood contact seemed to produce a delay in the weight increase of the samples with respect to the control group. The analysis of the mitochondrial activity seemed to indicate a negative effect of the blood contact on the seeded membranes. In fact, samples exposed to a medium diluted with 80% and 50% blood recorded a depression of the MTT values with respect to group C. The same negative effect was recorded for PBS group where the lack of medium nutrients was probably the cause of the reduction of the chondrocytes' vitality. However, the negative effect of the blood contact was specific, because the samples exposed to a medium diluted with different quantity of blood showed different depression of the MTT values. At the longest time period (t3+21), the cellular activity of the blood groups increased almost reaching the values achieved before the blood contact. This indicates that the toxic effect on the chondrocytes was temporary and reversible. The analysis of the biomechanical data did not support these evidences, because the biomechanical results were not affected by blood contact. It could be hypothized that exposure time was not long enough to produce differences in the biomechanical properties of the constructs. We can conclude that the negative effect of the blood on the engineered cartilage is evident at the cellular level. However, it does not seem to be perceptible at the (engineered) tissue level, with the model utilized here. Three days of exposure did not entirely devitalize the tissue cells and did not seem to influence the synthetic properties of the chondrocytes and the biomechanical integrity of the immature cartilage at the longest time point. In the “IL-1 study” the negative effect of IL-1 seemed to be more marked than that of blood in the “Blood study”, although a direct comparison of the 2 model results very speculative. Moreover, this negative effect was clearly dependent on the level of maturation of the construct. A further in vivo study is probably needed to investigate the potential facilitation of the biological environment in reversing the negative effects of bleeding on the cells of the engineered cartilage as shown by other authors for native articular cartilage (14). Future studies are also desirable to test the effect of the different level of maturation of the engineered cartilage on its capacity of surviving and integrating in a joint environment exposed to bleeding., Lesions of the meniscus are frequently observed in orthopedic practice. Injury or loss of meniscal tissue potentially leads to pain, knee dysfunction, and osteoarthritis at long term. [4, 16] In cases of extensive destruction and complete loss of the meniscus, only two methods are available in clinical practice today for meniscal substitution: allograft transplantation and collagen meniscus implantation. However, their long-term success, durability, safety, and chondroprotective effects are still uncertain.[10] Several materials have been tested as partial meniscus substitutes in animal models. Total meniscus substitution remains difficult and its chondroprotective effect has been poorly described in the literature. A polyvinyl alcohol-hydrogel meniscus in rabbits showed promising results in terms of chondroprotection, but problems persisted, such as durability of the polymer, the fixation method, and complete tissue regeneration in a material that does not adhere to tissue.[3, 5] Welsing and van Tienen et al.[11, 15] published a 2-year follow-up study of a degradeable poycaprolacton-polyurethane meniscus implant in dogs. Although they showed promising results in respect of tissue formation a final remodelling into a neomeniscus was not possible because the polymer was still present after 24 months. Cartilage degeneration was not prevented. Tissue engineering with application of cells has recently been proposed as a possible solution for meniscal regeneration aiming at a more stable construct, thus preventing cartilage degeneration more effectively.[8, 9, 12–14] Walsh et al.[13] used a collagenous sponge loaded with mesenchymal stem cells to heal a partial meniscus defect in rabbits and reported that the presence of cells augmented the repair process but did not prevent degenerative osteoarthritis. Martinek et al.[8] reported better macroscopic and histological results in CMI implants seeded with meniscal fibrochondrocytes in comparison to cell-free implants in sheep. However, the tissue-engineered meniscus was biomechanically unstable and the implant size reduced during the 3-months observation period. Different cell sources have also been analyzed in vitro to find the most suitable source for cell augmentation of tissue-engineered meniscus.[2, 7] The surgical technique used in animal models for total meniscal replacement has also not been highly investigated and consistent, as authors use different animal models (rabbit, dog, and sheep) and diverse surgical techniques. We tried to develop a novel approach towards meniscus substitution and developed a new implant made from polycaprolactone and hyaluronan. In a pilot study, the implant was tested for total and partial meniscal substitution in sheep for 6 weeks. Tissue integration between the joint capsule and the implant was observed with tissue formation, cellular infiltration, and vascularization. At this early timepoint no cartilage protection was observed.[1] Our hypothesis was that the application of a tissue engineering approach, using cells seeded onto this scaffold, would offer some benefits in patients submitted to total meniscectomy by increasing the biological response and remodeling processes. Therefore, the aim of the following study was to investigate the feasibility of using this novel material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with autologous articular chondrocytes expanded ex vivo. The secondary aim was to evaluate two different surgical scaffold implantation techniques in an animal model: suture to the capsule and to the meniscal ligament, with or without transtibial fixation of the horns. The animals were sacrificed after 3 and 12 months. All implants showed excellent capsular ingrowth at the periphery. Macroscopically, no difference was observed between cell-seeded and cell-free groups. Better implant appearance and integrity was observed in the group without transosseous horns fixation. Using the latter implantation technique, lower joint degeneration was observed in the cell-seeded group with respect to cell-free implants on a macroscopic level. The histological analysis indicated cellular infiltration and vascularization throughout the implanted constructs. Cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Mankin Scores were not significantly different between cell-seeded and non-cell-seeded implants and they were not better in treated sheep than those of control sheep.[6] To date there is no evidence that meniscus substitution is capable of preventing osteoarthritis. However, the studies show promising results and it became obvious, where developments need go. Enhancement of biomechanical stability, proper selection of cell sources and cell augmentation techniques as well as special surgical techniques will have to be the goal of future research., Articular cartilage has been notorious for its limited healing potential for centuries. In order to enhance the cartilage repair the tissues engineering (TE) concept was introduced into the orthopedic surgery 15 years ago. The classical technique of autologous chondrocyte implantation (ACI) and its 2nd and 3rd generation upgrades proved to be successful in the treatment of small to mid-sized traumatic or osteochondritis dissecans lesions. The outcome of TE methods in degenerative cartilage lesions was inferior, therefore only small and contained degenerative lesions may be treated at present1, 2. As the number of young and active patients with debilitating joint disease is increasing steadily, the currently available cartilage regeneration or substitution techniques need further development3. The possible future applications will be reviewed in the lecture. When addressing a degenerative cartilage lesion the surgeon is dealing with a disease of all joint structures (cartilage, subchondral bone, menisci, ligaments, synovia, and capsule) due to genetic and environmental factors4. A successful cartilage regeneration or substitution must be focused also to the inflammation and mechanical issues together with the joint resurfacing3. Persistent high levels of synovial fluid markers after cartilage repair in non-degenerative lesions lesions, which may indicate graft remodeling or early degeneration, suggest that the resurfacing alone cannot entirely stop the disease progression5. At the final point the cartilage regeneration or substitution needs to promote the anabolic events over the catabolic degenerative mechanisms. Certain conservative and joint reconstruction strategies for the treatment of degenerative joint surfaces have been used for decades. Thank to these strategies the degenerative joint process may be transiently slowed down. They need also to be applied prior or together with the next cartilage resurfacing by regeneration or substitution to provide as stimulating environment as possible3. First, the treated joint has to be systemically prevented from the repetitive injuries or overuse by weight balance, activity modification, and regular exercise programs. Second, the joint reconstruction includes ligament repair/reconstructions, meniscus reconstruction, and unloading procedures6. Since the degeneration is an ongoing process, the treatment requires a proper timing. Any cartilage TE requires a cell source. Today the mature chondrocytes from outside the injured area are used as gold standard. If the same technology is transferred into the degenerative milieu, the source cells will also be diseased. The chondrocytes in OA are lower in number, they have different capacity to proliferate in vitro, their response to growth factors is different, and they express significantly higher levels of matrix degrading enzymes3. In spite of these drawbacks Tallheden et al. demonstrated that OA chondrocytes retain their differentiation potential upon isolation and proliferation in vitro which could make them suitable for cartilage TE7. Mesenchymal stem cells (MSC) were able to differentiate into different tissues in animal studies, but the processes could not be exactly controlled. The clinical evidence of MSC usage in cartilage is limited and no specific applications for degenerative lesions are available. Scaffolds represent another key component in cartilage regeneration or substitution. They are of different origins and can be divided into four chemical classes: protein-based polymers, carbohydrate-based polymers, artificial materials, and combinations. A scaffold needs to be sterile, biocompatible, biodegradable, and mechanically stable to protect seeded cells during the implantation and early postoperative rehabilitation. Numerous scaffolds were successfully used in clinical settings for focal lesions showing comparable results to classical ACI, but enabling quicker and less invasive operative procedures8. Unfortunatelly, no comparative studies between scaffolds are available and none of the scaffold has been specifically designed for the degenerative joint lesions. Growth factors are not routinely used in the cartilage repair surgery today, but the evidence on their positive influence is growing. They will be unavoidable in the reconstruction of a degenerated joint. BMP-7 has been most extensively studied and it has the highest clinical potential at the moment. It demonstrated a strong pro-anabolic and an anti-catabolic activity and a good safety profile in animal studies9. Other mixtures of growth factors will be combined in the future. They could be delivered as recombinant proteins with cells/on the scaffold or as genes in genetically modified cell. To summarize, the degenerative lesions are not an isolated problem, but they constitute a piece in the spectrum of a whole joint disease. There are no successful cartilage regeneration or substitution methods available at present for degenerative lesions. It seems that many old and new strategies will be combined for a successful and stable outcome. However, any planned strategy for the degenerative knee lesions has to meet patient's expectations and agreement on the post-operative protocol., Introduction When confronted with a young patient who has an old knee several parameters have to be assessed: the alignment of the limb, the stability of the knee joint, the status of the menisci and the extent of articular cartilage damage. For each deranged parameter, a validated surgical approach exists. Malalignment can be corrected with osteotomy, stability can be restored by ligament-reconstruction, and meniscal transplantation or cartilage repair surgery can reestablish the load distribution and shock absorption of the knee joint. When all these interventions fail their goals, unicondylar or total knee arthroplasty is the ultimate alternative. Alignment In the young active patient, high tibial osteotomy (HTO) can protract the implantation of a total knee arthroplasty. The frequency of HTO has diminished due to the rise in total knee arthroplasties. Few data are available about the long-term follow-up of HTO. Gstottner et al presented their results of 134 lateral closing wedge osteotomies with survival rates of 94%, 79.9%, 65.5% and 54.1% at respectively 5,10,15 and 18 years. Age was the only parameter with significant influence on survival contrary to gender and mechanical axis. 10 The long-term results in an older population (mean age 69 years) of 76 HTO showed the best survival of 90% at 10 years (with arthroplasty as an end point) when X-ray valgus angle at 1 year postop was between 8–16°. The overall survival at 10 years was 74%. 15 A survival analysis on 67 knees showed a cumulative survival probability of 89.5% at 5y, 74.4% at 10 years and 66.9% at 15 and 20 years. More than 90% of patients had an improvement in pain score and would have the operation again.16 Another well-established indication for HTO is medial, unicompartmental cartilage degeneration in patients with varus malalignment. Studies have led to 8–10° of valgus alignment as widely accepted. A recent laboratory study indeed showed a decrease in contact pressures and contact area when the alignment was shifted from varus to valgus. All contact pressure is shifted to the lateral compartment between 6–10° of valgus. This is regardless of condylar width, baselinge alignment, body weight or chondral defect size. 11 Based on these findings, one could say that HTO could be combined with autologous chondroctye implantation (ACI) for patients with varus malalignment and chondral defects. A small cohort study of 8 patients who underwent ACI and medial opening wedge HTO, showed favorable results at 28 months.7 Stability Anterior cruciate ligament (ACL) reconstruction has been performed for more than 20 years. It is a widely accepted procedure to restore the stability of the knee joint. Some authors hypothesized that a concomitant meniscal allograft transplantation (MAT) could improve the results in patients with an injured medial meniscus and ACL. Short-term results of ACL-reconstruction + MAT were promising and not different from MAT alone in a stable knee. 14, 18 Long-term results of small patient numbers showed a significantly improvement of knee function in symptomatic meniscus-deficient knees. The addition of an ACL-reconstruction probably improved these results.9 ACL-reconstruction combined with ACI results in sustained improvement in pain and function at short-term. ACL-reconstruction + ACI gives better results than ACI after a successful ACL-reconstruction.1 Shock absorption To restore the impaired function of damaged menisci or articular cartilage, both meniscal allograft transplantation (MAT) and autologous chondrocyte implantation (ACI) are procedures with good to excellent short to long-term follow-up. MAT for articular cartilage treatment is not comparable to ACI and gives worse results on long-term. 3 Recently some reports have evaluated the results of a combined MAT+ACI procedure. Short-term results for the combined intervention showed statistically significant improvements in outcome scores, but not always better results compared to the procedures in isolation. 6, 13 This is readily explained by the indication to perform a combined procedure, which allows to operate on patients with worse defects that would otherwise be contra-indications for one of the procedures in isolation. Ultimate alternative In some young patients, even combined procedures are not an option or fail prematurely. Total knee arthroplasty is then the only way out. The unicondylar knee arthroplasty (UKA) can be used in selected patients with a well functioning stability system of the m uscles and ligaments and of course an isolated medial gonarthrosis. It has been shown that a deficient ACL results in higher failure rates after UKA. 5 Combined ACL-reconstruction and unicondylar knee arthroplasty, restoring the knee stability and keeping the advantages of uni versus total knee arthroplasty, yield encouraging short-term results.17 Long-term results of TKA show reliable and durable results at 18 year follow-up with an estimated survival of 100% at 15 years and 93.7% at 20 years in one study on patients less than 55 years with rheumatoid arthritis. All patients received a cemented condylar prosthesis. 2 Another study showed survival rates of 96% at 10 years and 85% at 15 years in 52 patients with OA, all 55 years or younger. A press-fit condylar knee system was used. 4 A series of 80 knees in 63 patients who received a mobile bearing knee prosthesis showed a cumulative survival rate of 96% at a 12-year maximum follow-up. 12 Finally a prospective follow-up of 1047 patients of 55 years or younger in a community registry showed the best survival rate for cemented TKA with 85% survival at 14 years. Cementless designs and unicondylar knee arthroplasty independently increased the revision risk.8 Conclusions HTO has the ability to protract the implantation of a TKA and to improve the patient's quality of life with high satisfaction rates. Combined HTO + ACI seems to be a viable option for medial chondral defects. MAT+ACL-reconstruction may probably improve stability and even provide protection for the cartilage. The anterior cruciate ligament and the medial meniscal allograft seem to protect each other both ways. ACL-reconstruction + ACI gives better results than ACI after a successful ACL-reconstruction Combined MAT+ACI could neutralize some contraindications for MAT or ACI in isolation and offers a safe alternative with acceptable results on short-term. Patients in which none of the interventions above are optional, knee arthroplasty remains a final solution. UKA should only be done in stable knees or in association with an ACL-reconstruction. The long-term results of TKA in young patients are good to excellent., The predominant functions of articular cartilage are mechanical in nature, providing both protection for the subchondral bone and, in association with the synovial fluid, an highly efficient low friction articulation during joint activities. Consequently researchers have attempted to provide a complete mechanical characterization of articular cartilage in heath and disease, with seminal studies having been reported over 60 years ago. This research has been enlivened by the research interest in providing a design template for mechanical parameters critical for the evaluation of tissue engineered repair strategies for articular cartilage, which will provide long-term functionality post implantation. Early laboratory-based tests revealed that the nature of the testing certainly influenced the mechanical responses. As an example, to ensure simulation of physiological situations, cartilage had to be tested in an hydrated state. In addition, the heterogeneous structure of the tissue contributes to the variations in mechanical properties with cartilage depth, as evidenced by tensile testing of thin cartilage specimens, sectioned parallel to the articular surface. These depth-dependent variations also had a major influence on the boundary conditions associated with compression testing. This has led to two separate approaches involving osteochondral constructs, full-depth cartilage constructs with thin section of subchondral bone, being tested in either confined or unconfined compression. The former approach involves fluid movement restricted to one direction through the porous loading platen. Nonetheless it lends itself to modeling using an assumption of isotropic biphasic behaviour, which yields material parameters, namely Young's modulus, aggregate modulii and hydraulic permeability. By contrast, unconfined compression permits lateral fluid movement through the sides of the cylindrical constructs and barreling between the impermeable loading platens. Similar values of Young's modulus have been estimated, approximately 0.80 MPa, from cartilage explants from the bovine humerus (Korhonen et al., 2002). The use of isolated constructs also permits the examination of structure-function relationships, often with the use of proteolytic enzymes to selectively degrade the ECM components of articular cartilage. To minimize the changes associated with sample excision from its natural state, an alternative in vitro approach involves examining the response of articular cartilage to indentation. This testing mode has also proved problematic, particularly in ensuring that the axis of indentation was perpendicular to the articular surface. Two geometries of indenter have generally been employed, using either a fat ended or an hemispherical probe. An analysis of each was provided in a seminal paper, which assumed that the loading was transmitted to an infinite isotropic elastic material on a rigid substrate (Hayes et al. 1972). Nonetheless, the complex nature of the internal strain field within the heterogeneous tissue under an indenter necessitates the determination of structural parameters associated with force-deformation behaviour. To covert to material properties both the local thickness of the cartilage and the exact nature of the cross-sectional area of the tissue under the indenter need to be determined. Another important consideration in mechanical testing involves the inherent viscoelastic behaviour of articular cartilage. Accordingly both stress relaxation and creep tests have been performed. However, the long tem creep experiments to determine the equilibrium or aggregate modulus do not simulate the normal dynamic loading patterns in joints. This could by overcome using an underdamped mechanical loading system, where the short-term loading response of unconfined cartilage constructs could yield both the elastic and viscous parameters separately (Bader et al. 1994, 2000). This approach has demonstrated that the elastic response of the loaded cartilage is controlled by the collagen network. It also revealed values of initial compressive modulus of approximately 10 MPa. With the emergence of arthroscopic examination of joint structures, it soon became clear that any testing device that could be used arthroscopically, would enable the in vivo assessment of early tissue changes in localized areas. This was addressed by the seminal work from Jurvelin's group in Kuopio who designed an arthroscopic-based indenter (Lyyra-Laitinnen et al., 1999), which was developed into a commercial product (Artscan Oy, Helsinki, Finland). It consists of a handheld tool, equipped with a mechanical indenter and an integrated force gage at the tip. The force by which the tissue resists the constant indentation is a measure of the cartilage stiffness. Since then a few groups have presented and validated instruments involving indentation during arthroscopy that are capable of measuring compressive stiffness of articular cartilage in vivo (Appleyard et al. 2001; Bae et al. 2003; Niederauer et al., 2004). These instruments have been shown to be sensitive in detecting superficial changes in degenerated tissue. A later development by the Finnish group involved the integration of a miniature ultrasound (US) transducer into the probe tip of the indenter. This enabled measurements of original cartilage thickness, thus permitting strain measurements and enabling a realistic estimation of compressive modulus (Laasanen et al., 2002). With this instrument, cartilage thickness, dynamic modulus and US reflection coefficient of cartilage surface (RUS) can be determined during short-term, clinically applicable measurements. These parameters are sensitive indices to early cartilage degeneration. US reflection at cartilage surface has been shown to be a sensitive and specific measure of the quality of superficial cartilage tissue (Kiviranta et al. 2007). A recent study demonstrated, that RUS was able to discern degeneration of the samples with high sensitivity (0.77) and specificity (0.98) (Kirivanta et al. 2008). US reflection measurement shows potential for diagnostics of early OA, obviating the need for site-matched reference values. In addition, the high linear correlations between indentation and reference measurements suggest that these arthroscopic indentation instruments can be used for quantitative evaluation of cartilage repair post-surgery. The use of such arthroscopic-based techniques, however, is limited by the invasive nature of the procedure, and restricted to joint areas accessible by the probe. However in the last decade the emergence of quantitative magnetic resonance imaging (MRI) techniques probing macromolecular composition and structure has provided the means to indirectly assess the mechanical properties of normal, early degenerated, and surgically manipulated tissue in vivo. Quantitative MRI techniques have successfully been developed to provide measures of the macromolecular environment within cartilage, made possible via the interaction between interstitial water and the macromolecular constituents of cartilage that affect the nuclear magnetic relaxation properties. Indeed the anisotropic behavior of T2 relaxation time of MRI has been correlated with the 3D arrangement of the collagen network and thus might serve as a parameter for assessing the integrity of the collagen network (Nieminen et al. 2000). Indeed, an increase in T2 relaxation time was observed in early symptomatic degeneration of human articular cartilage in vivo (Mosher et al. 2000). An alternative MRI approach has been recently demonstrated to be sensitive to cartilage proteoglycans. This involves a delayed Gadolinium-enhanced MRI technique (dGEMRIC), which yields a value of the relaxation parameter, T1, which can be used in conjunction with T2 to describe up to 87% of the variations in specific biomechanical properties in cartilage (Nieminen et al., 2004). It is evident that this combined approach can provide important information on the mechanical properties of articular cartilage (Kiviranta et al., 2007). The results are encouraging with respect to functional imaging of cartilage, although in vivo applicability may be limited by the inferior resolution of clinical MRI instruments and, of course cost and limited availability., Introduction Limb skeleton develops through the process called endochondral bone formation. In this process, mesenchymal cells condense and differentiate into proliferative chondrocytes that then differentiate to hypertrophic chondrocytes. Hypertrophic cartilage is gradually replaced by bone. Bone morophogenetic proteins (BMPs) have been thought to play important roles in this process. BMP signals are transduced intracellularly by Smad proteins and non-Smad proteins. Along these pathways, several regulatory mechanisms work. Antagonists such as noggin antagonize BMPs extracellularly, and inhibitory Smads such as Smads 6 and 7 inhibit activation of Smad proteins. It was recently reported that articular cartilage expresses inhibitory Smads in arthritic conditions. Methods and Materials To clarify roles of BMPs and their inhibitory factors in cartilage formation, we generated transgenic mice that overexpress BMPs or related molecules in cartilage under the control of the Col11a2 promoter/enhancer. Results Overexpression of BMP4 or GDF5/CDMP1 expanded cartilage and accelerated chondrocyte hypertrophy. Noggin overexpression severely inhibited cartilage formation, suggesting that BMP signals are essential for cartilage formation. Smad6 overexpression delayed chondrocyte hypertrophy but did not affect cartilage formation, suggesting possibilities that non-Smad pathways transduce BMP-mediated cartilage formation. To examine BMP signals in various steps during endochondral bone formation, we generated conditional transgenic mice for Smad7 using Cre / loxP system. Smad7 overexpression inhibited both initial cartilage formation from mesenchyme and later chondrocyte differentiation to hypertrophy. Our in vivo and in vitro results suggested that Smad7 inhibit cartilage formation, at least in part, by down-regulating BMP-activated MAP kinase pathways. Conclusions BMP signals regulate cartilage formation and differentiation at multiple steps., Scaffolds for cartilage tissue engineering serve as a template to guide cells and extracellular matrices to organize into new tissues, provide the tissues with initial mechanical strength, control tissue shape and size, and promote their integration with adjacent tissues. The scaffolds should be biocompatible and biodegradable, allow cell attachment, proliferation and differentiation, facilitate gas exchange, nutrient diffusion and waste metabolism, and be processible into designed shapes. A number of three-dimensional porous scaffolds fabricated from various kinds of biodegradable materials have been developed and used for cartilage tissue engineering. Especially, polymer materials have received increasing attention and been widely used for cartilage tissue engineering. The polymers used to prepare scaffolds for cartilage tissue engineering include biodegradable naturally derived polymers such as collagen, hyaluronic acid, fibrin and alginate, and biodegradable synthetic polymers such as poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone. Despite various scaffolds have been developed, significant challenges remain in the development of functional scaffolds. Especially, improvements on mechanical properties, cell seeding efficiency and interconnectivity of scaffolds are strongly desired. Two kinds of collagen porous scaffolds with controlled pore structures and interconnectivity were prepared by template method. One is collagen mesh that was prepared by using a PLGA knitted mesh as a template. At first, a hybrid mesh of PLGA and collagen was prepared by forming web-like collagen microsponges in the openings of a PLGA knitted mesh. And then, the hybrid mesh was immersed in an alkaline aqueous solution and incubated at 37 ° C for 24 h to selectively dissolve the PLGA knitted mesh to obtain the collagen mesh. The interconnected porous structure of the collagen mesh was confirmed by SEM observation. Another collagen scaffold is collagen sponge with open surface structure that was prepared by using embossing ice particulates as a template. Ice particulates were formed on a plate surface and collagen aqueous solution was poured onto them. Collagen sponge was prepared after freeze-drying. SEM observation demonstrated that the collagen sponge had a surface structure of open large pores and a bulk porous structure of small pores. The surface large pores and bulk small pores were interconnected. The open surface and interconnected porous structures facilitated cell seeding and homogeneous cell distribution. The synthetic and naturally derived polymers have their respective advantages and drawbacks. The biodegradable synthetic polymers can be easily formed into designed shapes with relatively high mechnical strength. However, the scaffold surface of these polymers is relatively hydrophobic, which is not good for cell seeding. On the other hand, naturally derived polymers have specific cell interaction peptides, and their scaffolds have hydrophilic surfaces, which are beneficial to cell seeding and cell attachment. However, naturally derived polymers are mechanically too weak. Hybridization of naturally derived polymers with synthetic polymers has been developed to to combine their advantages and to avoid their drawbacks by forming sponge or microsponges of a naturally derived polymer in the openings of a synthetic polymer skeleton. The synthetic polymer skeleton enabled easy formation into the desired shapes and provided the appropriate mechanical strength, while the enclosed collagen sponge or microsponges facilitated cell seeding and cell attachment. Several kinds of such hybrid scaffolds were developed. One example is a hybrid sponge prepared by introducing collagen microsponges in the pores of a PLGA sponge. The PLGA-collagen hybrid sponge was prepared by immersing a PLGA sponge in a bovine collagen type I acidic solution under negative pressure, freezing at – 80 Ë š C, and freeze-drying. The hybrid sponge was further crosslinked by treating with glutaraldehyde vapor and washing with glycine aqueous solution and water. Collagen microsponges were formed in the pores of the PLGA sponge. The ultimate tensile strength, the modulus of elasticity, and the static stiffness of the PLGA-collagen hybrid sponge were higher than those of PLGA and collagen sponges, in both dry and wet states. The second example is a PLGA-collagen hybrid mesh. This was prepared by forming collagen microsponges in the openings of a knitted PLGA mesh. SEM observation confirmed that web-like collagen microsponges were formed in the openings of the PLGA mesh. The hybrid mesh exhibited a significantly higher tensile strength than did the collagen sponge alone, and was similar to the PLGA mesh. The hybrid mesh was used to culture chondrocytes for cartilage tissue engineering. Cartilage with high mechanical strength was regenerated and the size of the engineered cartilage could be controlled. Histological examination of these specimens using hematoxylin and eosin stains indicates a uniform spatial cell distribution throughout all the implants both radially and longitudinally. The chondrocytes showed a natural round morphology in all the implants. The bright safranin-O-positive stain indicated that glycosaminoglycans (GAG) were abundant and homogeneously distributed throughout the implants. Toluidine blue staining demonstrated the typical metachromasia of articular cartilage. Immunohistological staining with an antibody to type II collagen showed a homogeneous extracellular staining for type II collagen. The third example is a cylinder-type PLLA-collagen hybrid sponge prepared by enclosing collagen sponge in a PLLA porous cylinder. The PLLA sponge cylinder was prepared using a method of porogen leaching using a Teflon mold. SEM observation showed that the pores were distributed evenly in the wall and bottom sections of the PLLA sponge cylinder. The inner and outer surfaces had smaller pores than did the cross-section. This might be due to a contact effect of the NaCl particulates and wall of the Teflon mold. The porosity and pore size of the PLLA sponge cylinder were 86.8 ± 0.8% and 84.3 ± 5.5 μ m, respectively. The PLLA sponge cylinder was then filled with aqueous collagen solution by introducing collagen solution into the pores of the cylinder and the central space. The collagen solution-filled PLLA sponge cylinder was freeze-dried and cross-linked to form a PLLA-collagen hybrid sponge. The PLLA-collagen hybrid sponge had the same shape as that of PLLA sponge cylinder. SEM observation demonstrated that collagen sponge was formed in the center of the PLLA sponge cylinder and collagen microsponges were formed in the pores of the wall of the PLLA cylinder sponge. The collagen sponge in the center space was connected with the collagen microsponges in the pores of cylinder wall by collagen fibers that passed through the interstices of the PLLA sponge). The interconnection protected the shrinkage of the central collagen sponge. Micrographs of horizontal and vertical cross-sections of the central collagen sponge indicated its tubelike structure in the vertical direction. The porosity of the PLLA-collagen hybrid sponge was 91.7 ± 3.6%, significantly higher than that of the PLLA sponge cylinder (86.8 ± 0.8%). Introduction of collagen sponge in the central space of the PLLA sponge cylinder increased the total porosity of the hybrid sponge. The PLLA-collagen hybrid sponge showed significantly higher Young's modulus than did that of the PLLA sponge cylinder and collagen sponge alone. These results indicate that the PLLA sponge cylinder served as a mechanical skeleton and reinforced the hybrid sponge. The hybrid sponge was used for the cell culture of bovine articular chondrocytes. Most of the seeded cells were trapped within the hybrid sponge. The seeding efficiency was 96.1 ± 2.1%. The special porous structure with central collagen sponge surrounded with a PLLA-collagen sponge cylinder is thought to be advantageous for cell seeding. The outer layer of the PLLA-collagen hybrid sponge cylinder might protect against cell leakage during cell seeding. The chondrocytes adhered on both the central collagen sponge and the collagen microsponges of the PLLA-collagen hybrid sponge cylinder. The collagen sponge facilitated cell adhesion in the hybrid sponge. Biochemical analysis indicated that the DNA and sulfated glycosaminoglycans (GAG) contents increased with culture time. HE staining indicated that the chondrocytes showed round morphology. Safranin O-positive staining indicated the existence of glycosaminoglycans (GAG), which was abundant and homogeneously distributed around the cells. Toluidine blue staining demonstrated the typical metachromasia of cartilage. Immunohistological staining with antibodies to type II collagen and cartilage aggrecan showed that the type II collagen, and cartilage aggrecan were positively stained. These matrices surrounded the cells. Gene expression analysis by real-time PCR indicated that theã € € chondrocytes expressed type II collagens and aggrecan. The hybrid sponge promoted the formation of cartilaginous tissue when bovine chondrocytes were cultured. To summarize, a few kinds of porous scaffolds of collagen with controlled pore structures and PLGA-collagen hybrid porous scaffolds with high mechanical strength and cell seeding efficiency were developed by template and hybridization methods. These porous scaffolds facilitated cell seeding, cell adhesion, distribution and proliferation, and promoted cartilage tissue formation. These scaffolds will be useful for cartilage tissue engineering., Philosophy Scaffold-free TEC is feasible to cartilage and meniscal repair with advantages in various aspects such as safety issues, cost effectiveness, minimal surgical invasion and quick surgical time, with comparable repaired tissue quality with other cell-based therapies in cartilage repair. Background Immune-tolerance of MSCs Safety issues regarding the implantation of animal-derived or chemical materials in clinical settings High medical cost with the use of scaffold Trends in promoting minimally invasive surgery Risk of complications by long surgery In vitro characterization of the TEC -human study- The purpose of this study was to characterize a tissue engineered construct (TEC) generated with human synovial mesenchymal stem cells (MSCs). MSCs were cultured in medium with ascorbic acid 2-phosphate (Asc-2P) and were subsequently detached from the substratum. The detached cell/matrix complex spontaneously contracted to develop a basic TEC. The volume of the TEC assessed by varying initial cell density showed it was proportional to initial cell densities up to 4×105 cells / cm2. Assessment of the mechanical properties of TEC using a custom device showed the load at failure and stiffness of the constructs significantly increased with time of culture in the presence of Asc-2P, while in the absence of Asc-2P the constructs were mechanically weak. Thus, the basic TEC possesses sufficiently self-supporting mechanical properties in spite of not containing artificial scaffolding. TEC further cultured in chondrogenic media exhibited positive alcian blue staining with elevated expression of chondrogenic marker genes. Based on these findings, such human TEC may be a promising method to promote cartilage repair for future clinical application. Large animal studies on cartilage repair The objective was to in vitro generate a mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC) to facilitate in vivo repair in a porcine chondral defect model. Porcine synovial MSCs were cultured in monolayer at high density and were subsequently detached from the substratum. The cell/matrix complex spontaneously contracted to develop a basic TEC. Immunohistochemical analysis showed that the basic TEC contained collagen I and III, fibronectin, and vitronectin. The basic TEC exhibited stable adhesion to the surface of a porcine cartilage matrix in an explant culture system. The TEC cultured in chondrogenic media exhibited elevated expression of glycosaminoglycan and chondrogenic marker genes. The TEC were implanted in vivo into chondral defects in the medial femoral condyle of 4-month-old pigs, followed by sacrifice after 6 months. Implantation of a TEC into chondral defects initiated repair with a chondrogenic-like tissue, as well as secure biological integration to the adjacent cartilage. Histologically, the repair tissue stained positively with Safranin O and for collagen II. Biomechanical evaluation revealed that repair tissue exhibited similar properties similar to those of normal porcine cartilage in static compression test but the TEC-repaired tissue had lower micro-friction properties than normal articular cartilage. We also conducted the same surgical model study using mature (12m-old-) pigs and there was no significant difference in the modified ICRS histological scoring and biomechanical properties except for lubrication paroperties. This technology could potentially be a unique and promising method for stem cell-based cartilage repair. Large animal study on meniscal repair The meniscus is not entirely vascularized and has limited healing capacity. When the lesion involves the avascular region, complete reparative process cannot be expected. Recent studies revealed that the mesenchymal stem cells (MSCs) from synovial membrane have the potential to differentiate into a variety of connective tissue cells. With the synovial MSCs, we have developed a novel scaffold-free tissue engineered construct (TEC). Although scaffold-free, in vitro study demonstrated that the TEC possesses sufficiently self-supporting mechanical properties to withstand surgical handling. Moreover, the TEC is adhesive to the cartilage matrix and have chondrogenic differentiating capacity. Further, we have reported the feasibility of the TEC to repair partial thickness cartilage defect in a porcine model. Likewise, the TEC might also be applicable to incurable meniscal legions. The purpose of this study is to test the feasibility of the TEC to meniscal repair. The synovial MSCs were isolated from the synovial membrane of the skeletally matured Crown miniature pig's knee, and cultured to be confluent with 0.2mM ascorbic acid-2 phosphate (Asc-2P). The monolayer cell-matrix complex was detached from the substratum at confluent condition as reported. The detached complex actively contracted to develop a thick, three-dimensional tissue (the TEC), which was implanted to 4mm diameter cylindrical defect at the anterior horn of medial meniscus. All animals were put a cast on bilateral legs for 1 week and then back to the free-caged activity. The implamted menisci were evaluated grossly and microscopically The meniscal defects treated with the TEC were securely filled with fibrous tissue with good tissue integration. In contrast, there were none, or only partial, repair observed in the untreated defects. Histomorphometrical evaluation revealed that the meniscal defects was significantly better filled when treated with TEC (79% vs 33%) and integration ratio to adjacent meniscal tissue was also significantly better in the treated defects with the TEC (83% vs 51%). Furthermore, SO staining was decreased in 70% of the untreated menisci at the central portion, while SO staining was preserved in the TEC-treated menisci. Notably, focal articular erosions as mirror changes were observed at both the femoral and tibial articular surfaces in the 30% of the untreated knees (n=3), while no mirror lesion was developed in any TEC-treated knee. The present study demonstrated that the TEC implantation resulted in secure filling and repairing the incurable meniscal defect with good integration to the adjacent meniscal tissue. Moreover repair of the defect resulted in the prevention of body degeneration of meniscus as well as of chondral damage facing the meniscal lesion. Taking into account that the TEC does not require any extrinsic (biological or chemical) material for scaffold, this unique implant could be a promising therapeutic tool for the repair of incurable meniscal lesions. Clinical Experiences Clinical Trial has been registered and started in Spring 2009 at Osaka University. https://center.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&recptno=R000000960&type=summary&language=E, Transplantation of Tissue-engineered cartilage We have been performing transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints (108 cases) as a second generation of chondrocyte transplantation since 1996. Sixty knees who had received transplantation of tissue-engineered cartilage for cartilage defects were followed up for at least 5 years. Although the clinical results were satisfactory, we need the surgical approaches to treat large cartilage defects with less invasive technique. Articulated Distraction Arthroplasty Bone marrow stimulating procedure has been a well-accepted procedure for a large osteochondral defect. However, there are two potential weak points to induce hyaline cartilage. One is compressive overload on the drilled or micro fractured area at the early stage. In order to reduce the overload, we have developed external fixators, which allow almost full ROM with joint distraction for animals. Although joint distraction has been used to treat osteoarthritis, there has been little basic research on articulated joint distraction for the repair of osteochondral defects. We examined the effects of joint distraction with range of motion after drilling on a fresh osteochondral defect at the weight bearing area of the rabbit knee joint. A full thickness osteochondral defect was experimentally created at the weight bearing area of both medial femoral condyles of an adult Japanese white rabbit. After drilling to the defect, the experimental knee joint was distracted for 1.5 mm using a pair of external fixators to decrease compression force. The contralateral knee joint was used as a control with no apparatus. Gross findings and histological evaluation were assessed to study the morphology of the repaired cartilage. A partial repair with cartilage-like tissue was observed in the joints of the experimental group at 4 weeks. While cartilage-like tissue stained with Safranin O was found in the experimental group at 8 and 12 weeks, there were destructive changes in the control joints. Morphological changes were evaluated using the histological grading scale. There was no significant difference between experimental and control groups at 4 weeks. However, the mean scores of the experimental groups at 8 and 12 weeks were significantly better than those of the control groups at the same time points. Between the experimental groups, the scores at 8 and 12 weeks were both significantly better than those at 4 weeks. In conclusion, a combination of subchondral drilling, joint motion and distraction by an articulated external fixator promoted repair of a fresh osteochondral defect at the weight bearing area. Although distraction for 4 weeks was not a long enough period to repair the defect, distraction for 8 and 12 weeks resulted in a good outcome (Kajiwara and Ochi et al. JOR in 2005)1. Then we developed a new articulated device which permits smooth exercising of the joint during fixation for human. We evaluated the clinical results of a new distraction arthroplasty device after arthroscopic bone marrow-stimulating technique for the treatment of osteoarthritis of the knee. We developed a new distraction arthroplasty device that allows active ROM and compared preoperative and postoperative findings for 6 knees. The age ranged from 42 to 58 years. The fixation period for the distraction device ranged from 7 to 13 weeks, and the follow-up period was over 1 year. The Japanese Orthopaedic Association knee score, range of motion, and joint space values were significantly improved in all six cases at the latest follow-up (P ⇠.05). Scores on a visual analog pain scale were also significantly improved (P ⇠.05). We conclude that treatment using this new arthroplasty device in combination with a bone marrow-stimulating method was effective for osteoarthritic knees in middle-aged patient (Deie and Ochi et al. Arthroscopy in 2007)2. Another weak point is a small number of mesenchymal stem cells obtained from drilled holes, a new approach is injection of cultured MSCs in the joint after drilling. This injection was demonstrated to be effective for a cartilage defect, although this study was an animal study (Nishimori and Ochi et al. Br JBJS in 2006)3. This approach should be a new one for a large defect. Future direction for cartilage repair with minimally invasive tissue-engineering technique The most optimal procedure to repair cartilage defects is just injection of cytokines or growth factors and cells. Our completely novel approach was to use cell delivery system using an external magnetic field (Kobayashi and Ochi et al. Arthroscopy in 2008)4. We investigated whether it is possible to successfully accumulate magnetically labeled mesenchymal stem cells (MSCs), under the direction of an external magnetic force, to the desired portion of osteochondral defects of the patellae after intra-articular injection of the MSCs. MSCs were cultured from bone marrow and were labeled magnetically. Osteochondral defects were made at the center of rabbit and pig patellae, and magnetically labeled MSCs were injected into the knee joints either under the direction of an external magnetic force or with no magnetic force applied. In the rabbit model we evaluated the patellae macroscopically and histologically, and in the pig model we observed the patellae arthroscopically. Accumulation of magnetically labeled MSCs to the osteochondral defect was demonstrated macroscopically and histologically in the rabbit model and was demonstrated by arthroscopic observation to be attached to the chondral defect in the pig model. Thus, we could show the ability to deliver magnetically labeled MSCs to a desired place in the knee joint. In the clinical setting, our novel approach is applicable for human cartilage defects and may open a new era of repairing cartilage defects caused by osteoarthritis or trauma with a less invasive technique., Cartilage defects are a major clinical challenge because of the very limited regenerative capacity of articular cartilage. The solution to this might be regenerative medicine, a newly emerging area that aims to restore tissue function by applying principles of engineering and life sciences to develop biological substitutes. Cartilage was proposed as an ideal tissue for tissue-engineering treatments, being a “simple” tissue with one cell type (chondrocytes), a homogeneous matrix, and devoid of blood vessels and nerves. However, cartilage has proved difficult to engineer, and despite the significant efforts (∼1600 PubMed articles) over the past 15 years, the translation to the clinic has been limited. In fact, it may be the (oversimplified view of cartilage where the problem lies. Articular cartilage is a highly organized tissue with distinct structural patterns depending on the scale at which the tissue is examined. Over the range of nanometers to micrometers, the cartilage extracellular matrix is arranged as a network of collagen fibers and proteoglycans that allow for cell adhesion and mechanical support, and transduction of chemical and mechanical signals from the surrounding tissue to enter the cell [1]. Over the range of micrometers to millimeters, there are topographical differences in cartilage thickness and matrix content across the surface of the joint, which are associated with the level of loading [2, 3]. Further, the properties of articular cartilage change with depth from the articular surface, resulting in a “zonal” structure that is typically divided into three levels: superficial (surface to 10–20% of thickness), middle (20%-70%), and deep (70%-100%). Cells of each of the different zones of articular cartilage are organized distinctly and express zone-specific markers. In the superficial zone, cell density is high and cells are relatively fattened and clustered in a horizontal fashion.[4, 5] Here, the cells secrete proteoglycan 4 (PRG4) [6, 7] and clusterin [8], a molecule that is overexpressed in osteoarthritis [9]. In mature bovines, Notch1 is expressed solely in the superficial zone of articular cartilage [10], however this distribution appears to be species-, development-, and disease-specific [11, 12]. In the middle zone, cells are more spherical and are randomly oriented. Cartilage intermediate layer protein is a potential marker for the middle-deep zones[13], with apparent autoimmune activity and positive correlation with expression and progression of osteoarthritis[14] and chondrocalcinosis[15]. In the deep zone, chondrocytes are larger and organized in vertical columns. Several markers exist in adult cartilage including members of the Notch-Delta signalling pathway, which are expressed throughout cartilage during development, but are localized to the deeper layers in mature mouse tissue[12], in particular Jagged 1 [16]. Additionally, collagen type X, a marker for chondrocyte hypertrophy and indicator of endochondral ossification during development, is expressed in the deepest layers of articular cartilage [17]. To artificially mimic this zonal organization, an increasing number of investigations is currently directed at the development of a zonal tissue-engineered cartilage implants [18]. To achieve this, we are employing organ printing technology or “bioprinting” which combines the deposition of specific cell populations with the simultaneous deposition of biomaterials [19]. This allows the development of zonal cartilaginous grafts and by using hydrogels, a more physiological environment can be created. Cell suspensions can be mixed into in situ cross-linkable hydrogels (e.g., gelatin, agarose, alginate or PEG) in a cartridge and subsequently printed following a programmed 3D pattern[20–22]. Also, these water-based “bioinks” can contain biologically active components, such as proteins, peptides, DNA, hormones, extracellular matrix molecules and natural or synthetic polymers[23] to further enhance and direct the behavior of the cells. We characterized the use of bioprinting technologies to design and build heterogeneous cell-laden 3D structures. More specifically, we tested the design of various heterogeneous scaffolds, and tailored the porosity and elastic modulus of the grafts by modulating the distance between the strands and their configuration. Further, we are currently evaluating this technology for the printing of layered cartilaginous constructs using cells from the different zones of the articular cartilage. To this end we first investigated if zonal differences between chondrocytes isolated from the deep, middle and superficial zones of the cartilage are maintained during in vitro expansion and redifferentiation. For this study we elected to use equine chondrocytes, since this animal model is becoming increasingly important in the preclinical evaluation of orthopaedic treatments. Chondrocytes were isolated from cartilage of the patella-femoral groove of fresh equine cadavers (7–14 yrs). The separation into the different zones was confirmed using histology. Significantly more cells per gram tissue were isolated from the superficial zone compared to the deeper zones. During the expansion, the proliferation rate did not differ significantly between the cells of the different zones, whilst the expression of collagen types I, II and VI was lost in all zonal populations. After expansion in monolayers, cells were redifferentiated in alginate bead and pellet culture for up to 4 weeks. During redifferentiation staining for these markers re-appeared and was most intense after 4 weeks of culture. Interestingly, collagen type IX staining was also lost during expansion, but only re-appeared gradually in the middle and deep zone cultures. Similarly, COMP could not be immunolocalized after expansion and staining did only re-appear in the middle and deep zone cultures. Finally, we investigated the presence of clusterin [8]. In healthy articular cartilage clusterin is present within the superficial zone of the cartilage. Indeed, after isolation of the cells from the tissue, staining for clusterin was only found in association with cells from the superficial zone and it disappeared after expansion. During (re)differentiation in alginate, clusterin was present in cultures of all zones, but more pronounced within the cultures derived from the superficial zones of the cartilage. This indicates that clusterin would be a useful additional zonal marker, besides e.g. PRG-4, collagen type X and cartilage intermediate layer protein, (CILP), rather than an exclusive one, to further characterize tissue-engineered cartilage constructs with biomimetic zones. Thus, we have observed differences between zonal cell populations during in vitro culture, suggesting that the use of cells from the different zones could yield a tissue-engineered construct that better mimics the zonal native tissue. We have recently combined chondrocytes and osteogenic progenitors in printed constructs, yielding macroscopically heterogeneous grafts with distinct tissue formation. Printed zonal cartilage cells remained viable in alginate, in which they expressed cartilaginous markers including collagen types II and VI, as well as proteoglycans. However current culture experiments will demonstrate if printing cells of the different zones of the cartilage yields a construct with distinct zonal differences. Clearly, appreciation of zonal differences in the cartilage tissue could lead to important advances in cartilage tissue engineering., Despite satisfying clinical results autologous chondrocyte transplantation shows some technical disadvantages: insufficient initial mechanical stabilityuncertain cell distribution within the defectFixation of the periosteal flap with sutures penetrating healthy cartilageNecessity of intact cartilage shoulder surrounding the defectChance of periostal hypertrophy Various research groups are in the process of developing three-dimensional cell-carriers to improve techniques for cartilage repair. Specially designed scaffolds are one of the key components in tissue engineering. Research is focused on developing bioresorbable scaffolds that exhibit optimal physical properties coupled with excellent biocompatibility. Scaffolds act as shape and guidance templates for in vitro and in vivo tissue development. For cartilage and bone tissues, a suitable scaffold provides initial mechanical stability and supports even cell distribution. Various bioresorbable materials are currently used in various forms and shapes: Collagens of animal origin (mostly collagen type I and III)HyaluronanPolymers (PLA, PGLA)and others These matrices may be fixed by auto-adhesion, with fibrin glue, sutured or anchored transosseously. Biomecanical and preclinical studies showed that the stability of fixation varies tremendously with obvious clinical implications. Arthroscopic implantation of autologous chondrocytes on bioresorbable cell-carriers is feasible. Using these scaffolds, more even cell distribution within the defect may be achieved with operative handling being improved at the same time. Various techniques are currently used for the implantation of matrices (availability may vary from country to country): After debriding the defect, a size-matching scaffold is sutured or glued into the defect. Different materials may simply be attached by adhesion forces.Examples Hyaluronan Matrix (Fleece) of animal origin / Hyalograft® (Fidia)Collagen-Gel / CaRES® (ArthroKinetics AG)Collagen matrix / MACT® (CellTec)Collagen matrix / MACI® (Genzyme AG)After exact determination of the defect size a matching implant is prepared. The implant will then be pre-armed with resorbable threads (eg. Vicryl) which are to be knotted using a special technique. Anchoring holes will be placed anterogradely or tibially using a guide instrument. After insertion of pulling threads the pre-armed matrix is anchored within the defect by pulling the knots into the holes. Polyglactin/poly-p-dioxanon Fleece/Bioseed-C® (Biotissue-Technologies)Stable matrices enable a fast and stable but more costly fixation with intraosseous pins (Smart Nail®) Poly-glycolic-acid (PGA) Fleece/Chondrotissue® (Biotissue-Technologies) One of the first studies to examine the stability of implants for cartilage repair was done by Driesang in 2000. He applied autologous chondrocyte transplantation with a periosteal flap in goats and discovered that all sutured flaps (n = 6 animals) became detached from nonimmobilized joints during the recovery period. The purpose of this study was further to ascertain whether postoperative restriction of joint movement could prevent the delamination of flaps. Partial-thickness defects were created in the knee joint cartilage of 27 goats. These defects were then filled with a fibrin matrix and covered with periosteal (n = 6) or fascial (n = 21) flaps, which were sutured with simple, interrupted stitches to the surrounding tissue. The joints were immobilized by means of a modified Robert Jones bandage for periods of 2–6 weeks, after which time they were inspected for the absence or presence of flaps. In four animals, joint immobilization for 3 weeks was followed by free movement for a similar period. Four of the six periosteal flaps and two of the 21 fascial ones became delaminated after the period of immobilization. In the four goats permitted 3 weeks of free joint movement following a similar period of joint immobilization, all flaps (which had been retained up to the end of the immobilization period) became detached. These findings indicated that joint immobilization hinders the delamination of flaps but that this restriction of movement must be sustained for an undefined period of time. The nature of the tissue used for flaps also influenced the rate of retention by immobilized joints. Drobnic published a paper in 2006 about the comparison of four techniques for the fixation of a collagen scaffold in human cadaveric knee. Four fixation techniques for a fibrinogen and thrombin coated collagen fleece, used as a scaffold in the cartilage repair, were compared simulating the initial postoperative period in the cadaveric knee joints. Full-thickness chondral lesions were made on the medial femoral condyles of seven human cadaveric inferior extremities. Four scaffolds without seeded chondrocytes were implanted into each lesion using four fixation techniques consecutively: self-adhesion without additional material (SA), fibrin sealant (FS), bone sutures (BS), and periosteal cover (PC). After each implantation 150 cycles of continuous passive motion (CPM) were performed. Two cases were additionally exposed to 50 cycles of 10 and 20 kg loading each after the completion of CPM. The scaffolds were evaluated after every 30 cycles, and the fixation strength was tested after the motion was completed. All the SA scaffolds were detached prior to reaching 60 cycles. The other scaffolds remained stable throughout the testing with only minor disruptions. The endpoint fixation strength was higher for BS and PC than for the FS scaffolds. The FS scaffolds were detached as a result of additional load cycles, while the BS and PC scaffolds showed substantial deformations. SA of tested scaffold did not provide sufficient fixation. The FS fixation was easy to perform and assured satisfactory scaffold stability. BS and PC provided excellent scaffold stability, but the techniques were difficult and caused additional injuries. Regardless of the fixation technique used, the tested collagen scaffold may not be exposed to loading in the initial postoperative period. Hunziker found that surgical suturing of articular cartilage induces osteoarthritis-like changes. In clinical tissue-engineering-based approaches to articular cartilage repair, various types of scaffolds are used to retain an implanted construct within the defect, and they are usually affixed by suturing. The authors established a large, partial-thickness defect model in the femoral groove of adult goats. The defects were filled with bovine fibrinogen to support a devitalized flap of autologous synovial tissue, which was sutured to the surrounding articular cartilage with single, interrupted stitches. The perisutural and control regions were analyzed histologically, histochemically and histomorphometrically shortly after surgery and 3 weeks later. Compared to control regions, chondrocytes were lost from the perisutural area even during the first few hours of surgery. During the ensuing 3 weeks, the numerical density of cells in the perisutural area decreased significantly. The cell losses were associated with a loss of proteoglycans from the extracellular matrix. Shortly after surgery, fssures were observed within the walls of the suture channels. By the third week, their surface density had increased significantly and they were filled with avascular mesenchymal tissue. The authors concluded that the suturing of articular cartilage induces severe local damage, which is progressive and reminiscent of that associated with the early stages of osteoarthritis. This damage could be most readily circumvented by adopting an alternative mode of scaffold fixation. Knecht et al. performed an in vitro biomechanical testing of fixation techniques for scaffold-based tissue-engineered grafts. In this study, the authors have mechanically tested the fixation stability of four commonly used biomaterials for ACI attached by four different fixation techniques (unfixed, fibrin glue, chondral suture, and transosseous suture) in situ. Scaffolds based on polyglycolic acid (PGA) and polyglycolic acid and poly-L-lactic acid (PGLA), collagen membranes, and a gel-like matrix material were fixed within rectangular full-thickness cartilage defects of 10 × 15 mm(2) and loaded in tension until failure. Fibrin glue fixation of PGLA-scaffolds withstood a load of 2.18 6 +/- 0.47 N, chondral sutured PGA-scaffolds of 26.29 6 +/- 1.55 N, and transosseous fixed PGA-scaffolds of 38.18 6 +/- 9.53 N. The PGA-scaffold could be loaded highest until failure for all fixation techniques compared to the PGLA-scaffold and collagen membrane. The findings might serve as basis for selecting the most suitable fixation technique for scaffold-based tissue-engineered grafts according to the expected in vivo loads. Marlovits et al. examined the early postoperative adherence of matrix-induced autologous chondrocyte implantation for the treatment of full-thickness cartilage defects of the femoral condyle with MRI. In this clinical pilot study, a Matrix-induced Autologous Chondrocyte Implantation (MACI) technique with a three-dimensional collagen type I-I II membrane was used for the treatment of full-thickness, weight-bearing chondral defects of the femoral condyle in 16 patients. The cell-scaffold construct was implanted in the debrided cartilage defect and fixed only with fibrin glue, with no periosteal cover or further surgical fixation. All patients were followed prospectively and the early postoperative attachment rate, 34.7 days (range: 22–47) after the scaffold implantation, was determined. With the use of high-resolution magnetic resonance imaging (MRI), the transplant was graded as completely attached, partially attached, or detached. In 14 of 16 patients (87.5%), a completely-attached graft was found, and the cartilage defect site was totally covered by the implanted scaffold and repair tissue. In one patient (6.25%), a partial attachment occurred with partial filling of the chondral defect. A complete detachment of the graft was found in one patient (6.25%), which resulted in an empty defect site with exposure of the subchondral bone. Interobserver variability for the MRI grading of the transplants showed substantial agreement (kappa=0.775) and perfect agreement (kappa(w)=0.99). The authors concluded that implantation and fixation of a cell-scaffold construct in a deep cartilage defect of the femoral condyle with fibrin glue and with no further surgical fixation leads to a high attachment rate 34.7 days after the implantation, as determined with high resolution MRI. Petersen and his group described arthroscopic techniques for the fixation of a three-dimensional scaffold for autologous chondrocyte transplantation and examined the structural properties in an in-vitro model. The aim of the study was to evaluate the structural properties of matrix-associated autologous chondrocyte implantation (Bioceed-C®/ BioTissue Technologies) with multiple fixation techniques implanted in fresh porcine knees after they had undergone load to failure. The ultimate failure load, yield load, and stiffness of 3 different techniques for the fixation of a 2-mm thick polymer fleece was evaluated: (1) fixation with biodegradable polylevolactide pins (Smart Nail®), (2) a transosseous anchoring technique, and (3) conventional suture fixation. Techniques 1 (pin) and 2 (transosseous anchoring) can be used arthroscopically. Maximum load and yield load were significantly higher in the group 1 (pin fixation) and group 2 (transosseous anchoring) compared to group 3 (conventional suture). Stiffness was significantly higher in group 1 than in groups 2 or 3. This biomechanical dataset showed that two fixation techniques (pin fixation and transosseous anchoring) have a higher ultimate load, yield load, and stiffness than the conventional suture technique at time point zero. The described techniques provide an outstanding fixation strength with arthroscopic techniques for autologous chondrocyte transplantation There is still uncertainty about the importance and the best technique for the fixation of scaffolds in cartilage repair. Extensive research will have to be done to establish new techniques with new scaffolds to ensure biologic performance AND biomechanical stability., The use of biomaterials for cartilage repair has strongly increased in the last decade, due to promising results obtained with the development of new therapeutic options for the treatment of articular cartilage lesions. Actually in clinical practice there are two main concepts for biomaterials application for cartilage repair: cartilage regeneration promoted by cultured autologous chondrocytes, supported by the 3D scaffold (so-called second generation autologous chondrocyte transplantation) or implant of various biomaterials for “ in situ” cartilage repair which exploits bone marrow stem cess differentiation induced by the scaffold properties. The use of classic ACI (first generation) has been associated with several limitations related to the complexity and the morbidity of the surgical procedure. To address these problems-called second generation ACI techniques have been developed. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation onto the defect site. Essential properties of these scaffolds include biocompatibility and biodegradability through safe biochemical pathways at suitable time intervals. It is known that chondrocytes in two-dimensional cell cultures alter their phenotype and dedifferentiate to fibroblast cells that no longer posses the capacity to produce collagen type II and proteglycans. The use of three-dimensional scaffolds has been shown to favor the maintenance of a chondrocyte differentiated phenotype1,2. The clinical application of this second generation tissue engineered approach is well documented for different types of scaffold with an evaluation of the clinical outcome at short and medium-term follow up3,4,5,6. Autologous chondrocyte transplantation on a three-dimensional matrix was introduced in clinical practice in Europe from 1998–1999, so it is very difficult to obtain long-term clinical findings. Matrixes mainly used in clinical practice in Europe are collagen or hyaluronic acid based. In the USA there still no FDA approval for matrix-assisted chondrocyte transplantation in human application. Considering that from a surgical and commercial standpoint, an ideal graft for chondral or osteochondral defect repair would be an off-the-shelf product; thus, some new biomaterials were recently proposed to induce “ in situ” cartilage regeneration after direct transplantation onto the defect site. The possibility to create a cell-free implant to be sufficiently “ intelligent” to bring into the joint the appropriate cues to induce orderly and durable tissue regeneration is still under investigation in numerous animal studies7,8,9,10,11, and/but only few of these have been introduced into the clinical practice12,13. Scaffolds composed of synthetic or natural materials in a variety of physical forms (fibers, meshes, gels) have been used for cartilage regeneration. Solid scaffolds provide a substrate upon which cells may adhere, while gel scaffolds function to physically entrap the cells. Commonly used synthetic materials are the polylactides, like polylactic (PLA) and polyglicolic (PGA) acids. The mechanical properties and the degradation of synthetic biomaterials are more easily modified that for the natural polymers, but their degradation products may cause damage to native tissue or implanted cells. However new chemistry of these materials has improved their biocharacteristic and biocompatibility. Natural materials used to produce scaffolds include agarose, alginate, hyaluronic acid, gelatin, fibrin glue, collagen derivatives and acellular collagen matrix. They have impeccable biocompatibility, can be processed in a reliable and reproducible way and may enhance cell performance. The materials mostly used actually in clinical practice are protein-based (collagen or gelatine), but the use of polysaccharides is in rapid growth. There are several studies pointing to the critical role of saccharide moieties in cell signalling schemes and their importance in cartilage regeneration14,15. One of the more important properties of polysaccharides in general is their ability to form hydrogels. Hydrogel formation can occur by a number of mechanisms and is strongly influenced by the types of monosaccharide involved: for example thermal gellation is tipical for agarose and pH-dependent gellation for chitosan. Chitosan (partially de-acetylated derivative of chitin, found in arthropod exoskeletons) seems to have an important potential in stimulating chondrogenesis15 and there are some chitosan-based gels and scaffolds under clinical investigation for “ in situ” cartilage regeneration after direct transplantation onto the defect site. The treatment of osteochondral lesions is biologically challenging since two different tissues are involved (bone and articular cartilage) with a distinctly different intrinsic healing capacity. There some composite materials in pre-clinical and clinical experimentation for tissue engineered and “ in situ” regeneration approach for cartilage repair (Martin), but only one is widely commercialized for this application. This is bilayer porous PLGA-calcium-solfate biopolymer (TruFit) proposed for direct application into the defect site. Although pre-clinical experimentation is promising12 there still no clinical results available in the literature of the use of this material. RIZZOLI EXPERIENCE We utilized osteochondral nanostructured biomimetic scaffold (Fin-Ceramica S.p.A., Faenza - Italy) with a porous 3-D tri-layer composite structure, mimicking the whole osteochondral anatomy. The cartilaginous layer, consisting of Type I collagen, has a smooth surface to favour the joint flow. The intermediate layer (tide-marklike) consists of a combination of Type I collagen (60%) and HA (40%), whereas the lower layer consists of a mineralized blend of Type I collagen (30%) and HA (70%) reproducing the sub-chondral bone layer. In vitro and animal studies showed good results in terms of both cartilage and bone tissue formation. We observed same macroscopic, histological and radiographic results when implanting scaffold loaded with autologous chondrocytes or scaffold alone. The scaffold was able to induce an in situ regeneration through stem cells coming from the surrounding bone marrow16,17,18. We have performed clinical pilot study on 30 patients where the newly developed scaffold was used for the treatment of chondral and osteochondral lesions of the knee joint in order to evaluate the safety and the reproducibility of the surgical procedure and in order to test the intrinsic potential of the device. The clinical outcome of all patients was analyzed prospectively, at 6 moths and 1 year and an high resolution MRI. 29 of 30 patients (mean age of 29.3 years) were prospectively evaluated at 6 and 12 months follow up(one patient lost at follow up). In 29 patients 35 lesions were treated, average size of the defects was 2.8 cm2 (range: 1.5– 5.9 cm2). Statistical analysis demonstrated a significant improvement (Non Parametric paired Wilcoxon test, p⇠0.0005) from pre-operative to 12 months follow up. IKDC objective score showed preoperatively 46.1% of normal or nearly normal knees and 79.3% of normal or nearly normal knees at 12 months. Statistical analysis showed a significant improvement in the IKDC subjective score from preoperative (37,5± 14,6) to 12 months follow up (82,4± 11,9) (Non Parametric paired Wilcoxon test, p⇠0.0005). MRI evaluation showed complete filling of cartilage defect was noted in 86.2% of the patients and the congruency of the articular surface was seen in same patients. Subchondral bone changes (edema or sclerosis) were noted in 53.3% of patients. This scaffold, composed of Type-I collagen and nanostructured hydroxyapatite was designed for the treatment of cartilaginous and osteocartilaginous defects and have demonstrated to stimulate in situ bone and cartilage regeneration. Obviously, this short follow-up does not allow us to draw conclusions about the clinical effectiveness and histological quality of cartilage repair tissue in the long-term follow-up of this procedure, but clinical and MRI analysis allowed us to study and better understand the potential of this novel developed scaffold. The ability of the scaffold to induce orderly osteochondral tissue repair without necessarily including autologous cells makes it attractive (i) from a practical standpoint, since it could be used as an off-the-shelf graft in a one-step surgical procedure, (ii) from a surgical standpoint, since due to its flexibility it could inserted under minimally invasive conditions. Previous animal study highlighted the good potential of the graded biomimetic osteochondral scaffold in promoting by itself bone and cartilage tissue restoration, probably by inducing selective bone marrow stem cell differentiation in osteogenic and chondrogenic lineages18. Further systematic evaluation is necessary to determine the clinical and morphological outcome, especially compared to other treatment options such as bone-marrow stimulation techniques, mosaicplasty, and autologous chondrocyte transplantation., Purpose Focal articular cartilage lesions can be treated by surgical techniques that puncture holes in the base of the defect to induce bleeding and to generate conduits for stem cell migration into the defect 1–4. An optimal hyaline cartilage repair tissue contains type II collagen and high levels of GAG 5, however “marrow stimulation” techniques frequently elicit fibrocartilage or fibrous repair tissue that contains type I collagen and low levels of glycosaminoglycan (GAG), which is mechanically unstable and fails under load-bearing 3,6,7. Since marrow stimulation is a relatively simple and inexpensive out-patient-based surgery, a genuine interest has developed around finding methods that stimulate a more hyaline, marrow-derived repair. We previously showed in preclinical animal models that more hyaline repair cartilage is formed after microfracture, when the defect is covered with a hybrid polymer-blood clot implant 8,9. Hybrid clots are formed by mixing liquid autologous whole blood with a cytocompatible solution of chitosan, a polysaccharide biomaterial, which then coagulates to form a more adhesive and stable clot 9,10. Hybrid clots were shown to promote hyaline repair by attracting neutrophils and by stimulating transient subchondral angiogenesis and bone repair 9,11, however the step-wise cellular events linking these processes remain unclear. Blood clots 12,13, and chitosan particles 14,15 can attract innate immune cells to skin wounds. When macrophages become activated, they release angiogenic factors 16,17. However “classically” activated macrophages also release cytokines that can trigger cartilage breakdown (TNF-a, IL-1b), whereas “alternatively” activated macrophages express arginase-1 and release wound-healing factors such as IGF-1 18. In this study, we investigated the role of chitosan and blood clot in eliciting neutrophils and activating macrophages both in vitro and in vivo. Materials and Methods Experiments involving healthy, non-fasting human subjects and animals were carried out with institutionally-approved protocols. Hybrid clots were formed by aseptically mixing 3 volumes human or rabbit whole blood with 1 volume sterile liquid chitosan-glycerol phosphate (chitosan-GP: 1.6% w/v chitosan, 100 mM GP pH 6.6, Bio Syntech Inc, QC, Canada), or 1.5% w/v alginate (FMC, QC, Canada), hydroxyethyl cellulose (Spectrum, QC, Canada) or hyaluronic acid (Sigma, ON, Canada) in phosphate-buffered saline. Chitosan was biodegradable (80% degree of deacetylation, DDA, ie 80% glucosamine and 20% N-acetyl glucosamine, medium viscosity: “80M”) or non-biodegradable (95% DDA, ie 95% glucosamine and 5% N-acetyl glucosamine, “95M”). To track scaffold fate, some chitosan-GP samples contained fuorescent rhodamine B isothiocyanate–chitosan (RITC-chitosan) tracer with identical DDA and molecular mass 19. Clots were solidified in sterile glass tubes and cultured for up to 6 hours at 37°C, and exuded serum was analyzed for platelet and inflammatory cytokines, and ability to attract purified human neutrophils using a trans-well migration assay 20. In vivo implants were generated in rabbit dorsal subcutaneous sites (using autologous rabbit blood), and recovered after 1 day (N=7) or 7 days (N=6). In a separate experiment, implants were delivered to knee trochlea full-thickness articular cartilage defects with four, 0.9 mm microdrill holes (N=16 rabbits, analyzed at 1, 2, or 8 weeks of repair), using thrombin to accelerate in situ solidification 10. Control defects were treated with thrombin alone. Implants and decalcified femur ends were sectioned and stained with Safranin O-Fast green-iron hematoxylin, arginase-1, or collagen type I, and analyzed by stereology, or by histomorphometry (Empix, ON, Canada). Neutrophils were identified by nuclear morphology and CD14 expression, and macrophages by RAM-11, CD68, and arginase-1 expression. ANOVA was used to test for significant differences (p⇢0.05) induced by chitosan or time. Results Biodegradable chitosan elicits neutrophils and alternatively activated macrophages in vivo. Neutrophils strongly accumulated around subcutaneous implants containing 80M chitosan while few neutrophils were attracted to 95M chitosan, blood clot, LPS/blood, and hybrid clots incorporated with other polysaccharides (p⇠0.0005, Fig. 1A). After 1 week in vivo, neutrophils further accumulated and degraded 80M chitosan particles as shown by a depletion of RITC-chitosan (Fig. 1B & C). Neutrophils more easily infiltrated chitosan-GP/blood implants (Fig. 1C) compared to chitosan-GP implants (Fig. 1B). Altogether, these data demonstrated that the combination of blood and biodegradable 80M chitosan particles in hybrid clot implants promoted neutrophil chemotaxis, invasion of the implant, and cell-mediated chitosan particle clearance. After 1 day in vivo, macrophages were scarcely detected around subcutaneous implants. However after 1 week in vivo, macrophages expressing phagocyte markers RAM-11 and CD68 collected specifically at the edges of implants with neutrophils. As macrophages migrated into the neutrophil-dense implant, they phagocytosed chitosan, lost the RAM-11 epitope, and expressed arginase-1+ (Fig. 1D) which indicated that macrophages elicited by chitosan were alternatively activated. These data are important because they demonstrated that biodegradable chitosan particles could induce macrophages to adopt an angiogenic, wound-repair phenotype in vivo, through a phase requiring strong neutrophil attraction. Mechanisms of neutrophil attraction. IL-8 is highly chemotactic for neutrophils in vitro 21, and proteomic analysis of serum revealed that out of 25 inflammatory chemokines, IL-8 and MCP-1 were the dominant factors released from leukocytes in whole blood and in chitosan-GP/blood, while IL-6 and IL-1b were the dominant cytokines released from LPS/blood clots. These data showed that chitosan structurally stabilized blood clots without stimulating blood leukocytes to secrete catabolic cytokines. By transwell migration assay, chitosan-GP/blood clots released more potent neutrophil chemotactic factors to serum than blood clot alone. These data suggested that blood leukocytes in contact with biodegradable chitosan generated de novo neutrophil chemotactic factors distinct from IL-8 that recruited neutrophils to 80M chitosan. Chitosan attracts alternative macrophages to granulation tissues in drilled subchondral bone. Chitosan-GP/blood implants solidified over microdrilled cartilage defects doubled the level of arginase-1+ macrophages in granulation tissues formed in the trabecular drill hole at 1 week (p⇠0.05, Fig. 2A). After 2 weeks of repair, arginase-1+ cells persisted in neovascularized granulation tissues below treated defects, while arginase-1+ macrophages diminished in control repair tissues (Fig. 2A). After 8 weeks of repair, RITC-chitosan particles were completely cleared, arginase-1+ macrophages were no longer present, and drill holes below treated defects were repaired with more trabecular bone compared to control defects (p⇠0.05, Fig. 2B). Conclusions Biodegradable chitosan stimulated the release of neutrophil chemotactic factors by blood leukocytes and strongly attracted neutrophils in vivo. Neutrophils attracted to chitosan particles elicited and alternatively activated more macrophages than blood clot alone. When applied to marrow-stimulated articular cartilage defects, chitosan-GP/blood implants recruited significantly more arginase-1+ macrophages than blood clot alone. Increased density of arginase-1+ macrophages was associated with more vascularized granulation tissues. After 8 weeks of repair, chitosan particles, neutrophils, and arginase-1+ macrophages were no longer present, and trabecular bone was more completely restored, indicating that the therapeutic and transient effect was directly related to timely clearance of the biodegradable biomaterial by innate immune cells. Our novel findings generate a new paradigm for using biodegradable biomaterials to attract and activate macrophages in order to stimulate revascularization and repair of damaged subchondral bone., Introduction Osteochondral Defect (OD) has a poor spontaneous regenerative capacity and they can present with a variety of complaints from blister cartilage to full-thickness articular cartilage lesions with subchondral bone exposition. Since articular cartilage defects can progress to osteoarthritis (OA) in some cases1,2, surgical measures should be considered. Large ostheochondral defects (ICRS 4 ⇢ 4–10cm2) are difficult to treat, but several treatment options are available. In our country surgical options include drilling, microfracturing, and transplantation of osteochondral plugs but are often insufficient for the treatment of large defects1. From the last 7 years members of our laboratory has been working to find new alternatives to treat this kind of lesions using Biological Therapies. The works had been principally in the harvest process, expanding and differentiating mesenchymal stem cells (MSCs) into cartilage and bone. As these cells are easy to isolate, culture, and manipulate in vitro and have great plasticity3 they have become an important tool in cell replacement therapy and are considered as candidates for clinical applications4, we has been trying to development techniques to use in clinical setting. THE BEGINNING: ANIMAL MODEL STUDIES. The first work with MSCs were performed in New Zealand rabbits. The aim of this work were to obtain, culture and differentiate rabbit Bone Marrow derived MSCs in vitro to chondral lineage in the latinamerican reality. An iliac puncture was performed percutaneously over liac crests to obtain the bone marrow samples. By differential centrifugation the mononuclear cell level was obtained. After the expansion of MSCs we exposed the cells in micropellet for 21 days to TGF-b1 as described by Johnson et al5. We evaluated the quality of differentiation by histomorphometric analysis performed in the Mayo Clinic. In our hands, the MSCs expanded easily in vitro: at day 3 the cells in all cultures were adherent, at day 6 in all cultures was visible cells with spindle-shaped morphology (fibroblast-like morphology), characteristic of MSCs and in average at day 18 (15–19 day) the cells achieved 70% of confluence. After 21 days of culture with TGF-b1 the MSCs achieved an optimal differentiation quality (Figure 1). With this work we demonstrated that was possible achieve the isolation, culture and chondrogenic differentiation of rabbit bone marrow MSCs in the latinamerican reality. These results were submitted for publications and were presented in congress6. Figure 1: Chondrogenic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells. A: Aliquots of rabbit BMMSCs forming a spherical pellet in differentiation medium containing TGF-β1. B: Safranin-O/Fast Green stain showing chondrogenic differentiation of rabbit BMMSCs in a 21 day pellet culture with TGF-β1 (10 ng/ml). C: Quality of chondrogenic differentiation of rabbit BMMSCs. 10 pellet (83%) showed score 3, 1 pellet (8.5%) score 2 and 1 pellet score 1. In our next work with animals we compared the results of repair full-thickness cartilage defects in a rabbit model with a collagen I/ III matrix seeded with autologous chondrocytes or MSCs. After the harvest process, the cells were cultured in vitro and then the cells were seeded over the matrix at concentration of 20.000 cells/cm2. After 5 days, the collagen I/III matrix seeded with cells were implanted over a full-thickness defects produced in the femoral condyle of New Zealand rabbits. After 6 weeks the rabbits were euthanasied and the quality of repair was evaluated by gross and histologic examination with hematoxylin-eosin and safranin/O staining. The quality of tissue reparation was significantly better in defects treated with collagen I/III matrix seeded with chondrocytes or MSCs. In defects treated with MSCs the tissue appeared to have better integration and the histology showed hyaline-like tissue than did repaired by chondrocytes. These results were presented in congress and are being written to be sent for publication. MOLECULAR APPROACH In the next years our laboratory started studies performed only in vitro to evaluate our capacity to harvest human MSCs from different tissue sources and the quality of MSCs differentiation by techniques of molecular biology. In this context, we harvest human MSCs from bone marrow, umbilical cord blood and adipose tissue. Because the adipose tissue is a promising source of MSCs and they can be obtained by a minimal invasive method and in large quantities we tried to reproduce the two principal published methodology to harvest MSCs from this tissue but we fail, so we propose a new method based in mononuclear cells from adipose tissue. This new methodology to isolate human adipose tissue MSCs is easier than those published and the cells are expanded faster with similar differentiation capacity to that reported. This methodology is currently being patented. With the idea to use clinically the MSCs we evaluated the ability to obtain human bone marrow derived MSCs in a surgical room by density gradient with the same methodology described for the animal studies. In a surgical time an iliac punctures were performed and bone marrow aspirated were collected (15 ml, 3 patients). The density gradients were performed and the mononuclear cell levels were obtained. To determine the presence of MSCs in the mononuclear cell levels, this portion was expanded and differentiated to osteoblastic and chondrogenic lineage in our laboratory. The osteogenic differentiation was evaluated by alkaline phosphatase histochemistry and the chondrogenioc differentiation was evaluated by Western blot for collagen II (Figure 2). We demonstrated that was possible to obtain MSCs by density gradient in a surgical room and these MSCs had osteogenic and chondrogenic capacity7. Figure 2: Osteoblastic and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Isolated in a Surgical Room Osteogenic differentiation evaluated by alkaline phosphatase histochemistry and chondrogenic differentiation evaluated by Western blot for collagen II. CLINICAL PILOT STUDIES In the last 3 years and as a result of presentations of researches performed in our laboratory in different meetings has consulted several patients with large osteochondral defects (around 10cm2) that had no other chance of treatment. The first 3 patients with the informed consent were operated in 2004 and all they were carrying osteochondral defects ICRS 4 (2 in talus and 1 in femoral head). In the 3 patients we concentrate bone marrow cells harvested in the same surgical time by centrifugation. The defects were debrided and the size of defect was measured after debridement. Then a periosteal flap was extracted according to the defect size. From the iliac crest autologous cancellous bone was gained to restore the osseous part of the defect and then the periosteal flap was sutured to the cartilage rim of the chondral defects. The concentrate bone marrow cells were injected into the defects and then the flaps were sealed with fibrin glues. After 2 years of follow-up the 3 patients are satisfied with the results regarding pain during full weight bearing. The 2 patients with talus lesions improved the articular functionality according with AOFAS score in 37 points without evidences of progression of the osteochondral defects. These results were presented in congress and are being written to be sent for publication8,9. 4 years ago a 30 years old patient was diagnosed with an autoimmune disease and underwent cronic corticosteroid therapy. After 2 years of continuos corticosteroid medication the patient started complaining of bilateral knee pain, more severe on the left knee. Weightbearing plain radiographs showed a radiolucent area in the femoral lateral condyle and magnetic resonance imaging revealed an extense area of bone necrosis (⇢10 cm2) and subchondral edema in the weight bearing area of the lateral femoral condyle. At this point the Lyshom score was 15 points. For these reasons a treatment of the osteochondral defect with MSCs was decided with the informed consent of the patient. Briefy, an iliac puncture was performed and bone marrow aspirated was collected (15 ml). Five hundred ml of the patient's blood were collected and 230 ml of autologous serum were obtained. MSCs were obtained from bone marrow aspirated by a density gradient and they were plated at concentration of 106 cells/ml and incubated at 37° C/5% CO2 in expansive medium with 10% of autologous serum. When MSCs achieved 80% of confluence, they were tripsinizated and replated over a collagen type I/III matrix (Chondro-gide®) at 37° C/5% CO2 in expansive medium for 14 days. To induce chondrogenic commitment, 3 days before the surgical procedure the expansive medium was supplemented with TG F-b1 and ITS+Premix. The surgical procedure consisted on open debridement. Subchondral bone defect was filled with autologous cancellous graft and the MSCs in a Chondro-gide® matrix were positioned over the top of the lesion and sutured to the cartilage rim of the chondral defect. Clinical and radiological follow-up was performed every 3 months and 2 years after the surgical procedure a second-look arthroscopy was performed and biopsy specimens for histology and molecular analysis were obtained from the osteochondral defect area. At two-years follow-up the patients have recovered articular functionality. The Lysholm score improved 80 points (15 to 95) and in the radiological studies was observed complete scaffold integration. The cartilage was normal at second-look arthroscopy (Figure 3). The biopsy histology (H-E and safranin-O) was normal. Western blot and real time PCR for SOX-9 and collagen II from biopsy were similar to normal cartilage. The patient is currently asymptomatic of the left knee. These results were presented in congress and are being written to be sent for publication10,11. Figure 3: Treatment of osteochondral defect ICRS 4 ⇢ 10 cm2 with Mesenchymal Stem Cells induced to chondrogenic lineage. Osteochondral defect ICRS 4 ⇢ 10 cm2 and MSCs in a collagen type I/III matrix (Chondro-gide®) sutured to the cartilage rim of the chondral defect. CONCLUSIONES Since Wakitani et al12. demonstrated that bone marrow MSCs had chondrogenic potential and because MSCs-based cell therapy is clearly promising, several clinical trials have been developed. In countries with few economic resources staggered studies appear to be a good option. Although clinical applications of MSC are promising, the clinical use should be carefully evaluated., Chondrocytes and fibrochondrocytes adapt to changes in their biomechanical environment. In this presentation, the mechanoresponsiveness of condrocytes and fibrochondrocytes under normal and inflammatory conditions in vitro is presented. The effects of Continuos passive motion (CPM) on arthritic cartilage and arthritic meniscus fibrocartilage is also demonstrated in vivo (rabbit). Fibrochondrocytes and condrocytes from rat meniscus were exposed to Cyclic Tensile Strain (CTS) in vitro at various magnitudes and frequencies. The mRNA and protein analyses revealed that CTS at magnitudes of 5% to 20% did not induce proinflammatory gene expression. IL-1b induced a rapid increase in the iNOS mRNA. CTS strongly repressed IL-1b-dependent iNOS induction in a magnitude-dependent manner. Exposure to CTS resulted in 90% suppression of IL-1b-induced mRNA within 4 h and this suppression was sustained for the ensuing 20 h. The mechanosensitivity of fibrochondrocytes was also frequency dependent and maximal suppression of iNOS mRNA expression was observed at rapid frequencies of CTS compared with lower frequencies. Like iNOS, CTS also inhibited IL-1b-induced expression of proinflammatory mediators involved in joint inflammation. The examination of temporal effects of CTS revealed that 4- or 8-h exposure of CTS was sufficient for its sustained anti-inflammatory effects during the next 20 or 16 h, respectively. Fibrochondrocytes constitutively also expressed low levels of RANKL and RANK but marked levels of OPG. IL-1b upregulated expression and synthesis of RANKL and RANK significantly, whereas expression of OPG was unaffected following 4 and 24 h. When fibrochondrocytes were simultaneously subjected to CTS and IL-1b, expression of RANKL and RANK was significantly downregulated as compared to that of IL-1b-stimulated unstretched cells. The inhibitory effect of CTS on the IL-1b-induced upregulation of RANKL and RANK was sustained as well as magnitude and frequency dependent. The arthritic menisci and cartilage from rabbits subjected to CPM or immobilization were investigated for glycosaminoglycans (GAG), interleukin-l b (IL-1 b), matrix metalloproteinase-1 (MMP-I), cyclooxygenase-2 (COX-2), and interleukin-10 (IL-10) were determined by histochemical analysis. Within 24 h, immobilized knees exhibited marked GAG degradation. The expression of proinflammatory mediators MMP-I, COX-2, and IL-I p was notably increased within 24 h and continued to increase during the next 24 h in immobilized knees. Knees subjected to CPM revealed a rapid and sustained decrease in GAG degradation and the expression of all proinflammatory mediators during the entire period of CPM treatment. More importantly, CPM induced synthesis of the anti-inflammatory cytokine IL-10. These in vitro and in vivo studies explain the molecular basis of the beneficial effects of mechanical stimulus observed on fibrocartilage and articular cartilage and suggest that mechanical stimulus suppresses the inflammatory process of arthritis., Cartilage tissue has very low capabilities to respond an injury and in the best scenario it recreates a repair tissue far away from the original mechanical resistance, therefore, cartilage has being seen as a great challenge and numerous approaches has being attempted in order to leave as much of this precious tissue in place, or even regenerate it (1)-When performing an arthroscopic surgery, it is commonly foundcartilage injuries that present as fibrillated surface and loose fragments or unstable pieces hanging from the subchondral bone by atiny area of contact, degenerative changes derived by trauma, overuse or systemic diseases.(2,3). Traditionally, when we see such an scenario, cleaning the house cometo mind as the first step of operative procedures. Mechanical shavingand lavage with removal of fragment and fibrous synovial tissue arethe golden standard(4). As technology evolution, other tools likeRadiofrequency appear to solve same problems with apparent advantages over mechanical shaving in chondroplasty procedures (5). Radiofrequency has being launched as a resource to help the surgeoncut, coagulate, marketed as something rather recent from the 90'sdecade, however, RF is as common as the electrical scalpel that we use in every operating room, or at least, the principles are the same. Developed by Dr. Harvey Cushing and a Harvard Physicist William Bovie back in 1920. A glimpse in basic science behind RF When charged particvles such as electrons are accelerated, anelecrtomagnetic field is creatd. Electricity is a flow of electronstraveling through a conductor and a force must push this flow to theentire length and this force is called Voltage which in turn iscomposed of units called volts. The larger number of electrons thelarger the voltage traveling through the conducting material. Resistance to the passage of the current by the medium is calledimpedance which is reflected as Ohm unit. If one volt is sent throughone Ohm of resistance the resultant is an Ampere (Amp.). The electriccurrent also depends on its alternance switching from one pole to theother, therefore when one cycle is ccompleted in one second isreferred as one Hertz (Htz). Radiofrequency starts from 10,000 Htz to 30 Mega Hertz. RF is basically a thermal energy when applied to tissues. When in monopolar mode, the current flows from the dispersive electrode know as the skin patch through the body and leaves the active elecrode known as the one leaves the energy in the tissues to affect. Bipolar meantime does not drive the energy across the tissues since both electrodes are in the immediate neighborhood, ti does close the circuit by mean of the saline conduction properties and those tissues nearby get the thermal influence (6). Tissue response to RF Thermal modulation on collagena, has being studied by Arnockzky(7) The temperatures required to alter the molecular bonding of collagen and thus cause tissue shrinkage (65 degrees C to 70 degrees C) are also known to destroy cellular viability. Therefore, thermally modifiedtissues are devitalized and must undergo a biologic remodelingprocess. Meniscal tissue is capable of full recovery after exposicionto RF thermal energy pulses and has being shown to help heal someavascular inuries in Rabbits and human After the exposicion themeniscal tissue goes through a known curve of cell death, followed bycell repopulation and full histologic recovery after three months dueto vascular neoformation and fibroblast production of collagen (8,9,10). The expression of heat-shock proteins in human chondrocytes and ratfemoral head cartilage following heat shock was analyzed by Westernblotting, and red-blood-cell-induced chondrocyte death was assessed by cell viability and apoptosis by flow cytometry. Heat-shock inducedexpression of heat-shock protein 70 (HSP70) (rat and human) and HSP32 (human). Blood and blood products reduced rat cartilage proteoglycan synthesis and human chondrocyte viability, and induced human chondrocyte apoptosis at concentrations considerably lower than those reported previously. The induction of HSP70 in rat cartilage was ineffective in reducing chondrocyte death in the absence or presence of red blood cells or red cell products. Heat shock to humanchondrocytes reduced low levels of apoptosis (⇠20%) and cell deathinduced by low levels of blood products, but not higher levels(11) Radiofrequency in Chondroplasty Cartilage injuries are quite frequent findings, Curl et als, showed an incidence of 63% in a serie of 31,516 arthroscopies (12). A recent serie on 25,124 concur with the former one showing a prevalence of 60% as for total cartilage injuries (13). When a chondral injury is spoted, several aspects must taken in account such as depth, extension and stability. Chondroplasty goal is to regularize the borders, eliminate unstable fragments and smooth the surface. Traditionally mechanically shaving has being for a long time the method of choice due to it's availability and ease of use (14) One of the main concerns is the prevention or diminishing in reactivesynovitis caused by loose cartilage tissue in the joint, that in turnmay cause the appearance of the feared Matrix Metalloproteinases, MMP, that digest cartilage matrix (15). At the down of the first studies of thermal energy, the initialconcerns were either to use monopolar o bipolar and the depth oftissue penetration. After in vitro study of fresh bovine cartilageexposed to three RF systems two bipolar and one monopolar, the results were t smoothing of the surface and chondrocyte death in all three and that bipolar Rf device got 78% to 92% deeper than monopolar(16).A in vitro study with cartilage confirmed the chondrocyte death and the deeper penetration of the BPRF system(17). Therefore, the general feeling among surgeons was that these systemswhere not suitable for a safe chondroplasty. Furhter studies, on despite were carried on and as better understanding of the RF principles, interactions with the tissue and time of application, rendered more promising results. In a prospective study of mechanical vs BPRF after two year of FU in patella chondral injuries, the group with BPRF has better score Fulkerson-Shea (18) Gambardella in a study in sheeps compared the Cartialge injuries treated with RF, Mechanical shaving and microfracture. He found identical histological findings inthe RF and Microfracture group regarding the filling of the defect, and in the mechanical treated group, there was no filling. Kaplan et alshowed an interesting study on chndorcyte recover after severalthermal expositions of 45,50 and 55. He found that chondrocyte would recover up to 50 degree exposure but will not on 55 degrees Celcius(19) thus a new parameter emerged, the temperature. As time went by and more studies were published a new concern was born, Osteonecrosis apparently related to RF (20). However, there seemed to be a bias and a coincidence because Avascular Osteonecrosismay be also related to repeated trauma to subchondral bone due to alack of meniscus, and a meniscetomy was performed in some cases thatEventually developed thid complication due to tissue overload not tothermal damage(21,22,23). In a clinical study comparing Monopolar RFand shaver in chondroplasty in grade III femoral condyle cartilagelesion. They found no avascular necrosis, and the endpoint for bothgroups was an equal outcome in pain control. Another clinical studycarried out by Voloshin (24) of arthroscopic evaluation ofradiofrequency chondroplasty of the knee in humans Only 3 of 25lesions demonstrated progression. More than 50% showed partial orcomplete filling of the defect. Bipolar radiofrequency chondroplastyis an effective way to treat partial-thickness cartilage lesions;however, long-term effects of this treatment on cartilage remainunknown. A study with basic science evidence compared Monopolar chondroplasty vs mechanical debridement in a human patella. Pre and post treatment samples were incubated with viability stain and examined for smooth surface. The tissue depth affectation for Mechanical debridement was 385 micron meanwhile the RF group was 286 microns. Also the surface was visually smoother (25). In summary RF is a thermal source that produce some tissue response. Before theknowledge of how RF interact with tissue, large areas of chondrocytedeath were the common byproduct of thermal application for Chondroplasty. Modern and actual science stress the new tip probessurface contact, speed of probe passage over the defect or affectedtissue, water flow, temperatures on the range of 50 degreesCelsius. There is a decrease of stiffness of 71% of the tissue comparedwith non treated healthy cartilage. Depth of damage of mechanicalshaver about 386 microns compared with MRF of 286 microns. Someclinical studies shows second look with total and partial filling of the partial thickness defect whereas mechanical debridementarthroplasty has a null response. Some studies show in a short termbetter scores for the RF chondropasty than for the Mechanicalones. Osteonecrosis does not seems to be closely related to RF forthere are several factors that may be of more importance likesubchondral and meniscal trauma, providing the RF was properly applied. As we go further in research, we will have more proof of the total knowledge on safety for long term bases. The actual evidence warrant exciting horizons and promising clinical evidence goes to encourage it's use with careful trust. We must remember that a knowledgeable surgeon must fully understand the RF principles, the biological bases of thermal application like temperature, duration and probe passage over the cartilage tissue as well as power settings which are quite different from Monopolar toBipolar. We are not yet sure wether a RF treated chondromalacicArticular Cartilage will sustain at long term an initial stabilizationjust because we see and want instant gratification, there is the needto think ahead in time, the tissues will not be the same over themonths or years to come and they may have some degree of recovery or destruction. Chondral smoothening by radiofrequency has beingremarked, but it is really important to the clinic, how much does italters the friction coefficient.?, I look forward to see some studies. The field of the Heat Shock proteins may have an important role in Chondral protection, a subject to research. “Not all the new things are good, but the good things were once new”., PRP System: evidence for its use in tissue regeneration Over the last decade, a different approach has emerged to repair demaged cartilage or to fully regenerate it. Currently, the possible tecniques are: cells (bone marrow stem cells or differentiated cells)biomaterial mimicking extracellular matrix (scaffold)growth factors (GF) as regulatory signals. The therapeutic effect of these three individual components is enhanced when they are used together. Scaffolds are cell carriers playing an important role in maintaining cells in defect sites. However, physiological slowdown of cell proliferation may occur according to the scaffold constitution. GF help recreating a microenvironment favourable to cell proliferation and differentiation. In 1998, Marx demonstrated the effectiveness of platelet rich plasma (PRP) as a natural cocktail of growth factors acting in concert to accelerate healing and restoration of damaged tissues. Many in vitro studies show mitogenic effects of PRP on various type of cells (osteoblasts, chondrocytes, endhotelial cells, fibroblasts, marrow derived stem cells). PRP supplies of platelet-derived growth factor (PDGF), transforming growth factor (TGF) - β, insulin-like growth factor (IGF), vascular endhotelial growth factor (VEGF) and basic fibroblast growth factor (b FGF) which influence cell proliferation and differentiation. The tissue healing properties of PRP-gel are linked to some fundamental preparation features: the number of platelets in PRP must not be lower than 1 ×109 mLplatelets in PRP shouldn't be activated, since platelet stress induces increases in the release of GF in the supernatant platelet poor plasma (PPP) that is discarded after platelet concentration. PRP must be activated to form a gel. There are several reports about the use of platelet gel or PRP in dental implant, bone lesions or musculoskeletal injuries. One of the goals of PRP-gel is to improve cohesion of organic fragments (bone, biomaterial and cells) thus creating biologically active growth-factor enriched 3D structure. Platelet gel production follows a two-step procedure. First, hyper-concentrated platelets are prepared through platelet sedimentation (differential centrifugation of whole blood); and second, the gel is formed by adding fibrinogen cleaving agents (Ca ++, thrombin, or batroxobin) to PRP, leading to fibrin formation and polymerization, platelet activation and growth factor release. PRP can be prepared either in a blood bank environment, using good laboratory or manufacturing practice (GLP/GMP), or in the clinical point-of-care, using platelet sedimentation devices (tubes & centrifuge). Various devices are commercially available to prepare PRP in the clinic and the gel can be prepared in proximity to the patient. Some devices are also on the market for PRP gelification. Among these there are a few which devices can perform both phases (platelet sedimentation & PRP gelification). Which kind of PRP should we opt for? The answer is very difficult because each method has its own intrinsic property: DevicesProcessed blood (mL)PRP volume (mL)Platelet recovery(%)ActivatorPRGF Kit209.5 ± 4.1(%) 35 ± 16Autologous thrombinPRP (Landesberg)6010.6 ± 2.430 ± 10Ca++, thrombinAG Curasan157.6 ± 1.633 ± 10Ca++, thrombinPCCS508.5 ± 3.568 ± 9Ca++, thrombinHarvest5010.0 ± 067 ± 10Ca++, Autologus thrombinVivostat1205.0 ± 017 ± 6Neutralization of acidified batroxobinRegen Kit105.0 ± 0.590 ± 5Autologous thrombinFibrinet84.4 ± 0.265 ± 10Ca++, high speed centrifugationPlateltex85.0 ± 0.479 ± 7Ca++, batroxobinMazzucco HM608 ± 495 ± 5Ca++, Autologous thrombin Certainly the home made (HM) preparation of PRP gel using good laboratory or manufacturing practice is the best, because quality concentration (4 – 6 folds) and purity of preparation (no red cells) are manteined. Moreover this allow us to prepare thombin without haemolysis and cryoprecwipitate (if higher gel texture is required). The drawbacks are the time of preparation that is longer and the standardization of the processing because there is a “variety” of protocols of preparation and each laboratory uses its own parameters. Currently the clinical protocols about the use of PRP and PRP gel are based on the concept that platelet growth factor lead the healing processes; autologus use of these hemocomponent has made it safe to apply and combine different cell types, starting from the assumption that there are no adverse reactions and that it is not necessary to make quantitative checks of GF. We have evaluated three commercially available devices and one manual procedure (HM) with respect to resulting platelet concentration, growth factor (PDGF-BB, TGF-β1, b-FGF, VEGF, EGF, IGF-I) content and the kinetics of growth factor release from gel. Our studies showed (currently in print), the amount of GF made bioavailable to tissues depends on the amount of the factors that are contained in the platelets, in the plasma moiety, absorbed in the gel, and released in time both from platelets and gel. The results have shown several phenomena: a lack of correlation between platelet count and growth factors content in the PRP; procedure-dependent and procedure-independent features in growth factors release; process-dependent differences in the kinetics of the bioavailability of growth factors to tissues. Since lesions of almost all tissues are said to benefit from topical application of platelet-derived factors and since different tissues are formed by particular cells and by particular extracellular matrix composition. It is likely that different kinetics of growth-factors bioavailability might be more or less appropriate to treat different kind of lesions or different kind of tissues., Members of the Stem Cell Research Center (SCRC) have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by means of the cells' adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Although most of these cell populations have displayed characteristics similar to those of skeletal muscle satellite cells, we also have identified a unique population of muscle-derived stem cells (MDSCs). The MDSCs exhibit long-term proliferation abilities, elevated self-renewal rates, increased resistance to stress, and they are multipotent and can differentiate toward a variety of tissue types including: muscle (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages, both in vitro and in vivo. In contrast to other myogenic cell types, MDSCs show very efficient engraftment and regeneration of various musculoskeletal tissues due to their ability to highly survive post-implantation through the high anti-oxydant expression by MDSC. Interestingly, it has been observed that female MDSCs (F-MDSCs) can more efficiently regenerate the dystrophic skeletal muscle of mdx mice (a mouse model of Duchenne muscular dystrophy) than their male MDSCs (M-MDSCs) counterparts. MDSCs are influenced by environmental cues released within dystrophic or injured skeletal muscle which has been shown to negatively impact MDSCs and cause the cells to differentiate toward a fibrotic cell lineage and hence produce scar tissue rather than healthy skeletal m uscle fibers. Potential strategies are being explored to prevent the formation of scar tissue within injured skeletal muscle by blocking the action of TGF-b1. Finally, blood vessels contain several cell types, including myo-endothelial cells and pericytes, and are likely the place of origin of the murine MDSCs discussed above. The results outlined above open new avenues by which researchers could use muscle stem cell-based gene therapy and tissue engineering to improve tissue regeneration., Many focal cartilage defects can be effectively treated with autologous chondrocyte implantation, a two-step procedure requiring cartilage harvest at the index surgery, cell expansion, and subsequent reimplantation. In an effort to provide easier surgical delivery of autologous chondrocytes to repair chondral defects, minced cartilage may be an option. In this single-stage option, cartilage tissue, either processed intraoperatively (autologous) and loaded onto a scaffold or processed in advance (allogeneic) and available “on the shelf,” can treat chondral defects. In the lab, cartilage was harvested from both human and bovine trochleas, minced into small fragments (∼1mm3), and loaded onto PGA/PLA nonwoven felt or PGA/PCL foam reinforced with PDS, and cultured. The minced cartilage with scaffold samples were then implanted in mice for 4 weeks to assess chondrocyte migration and growth. The study showed that there is an inverse relationship between cartilage fragment size and amount of outgrowth (smaller size, more chondral growth) and the highest level of cellular activity is localized at the edge of the minced cartilage. Further, we tested the effectiveness of the minced cartilage on goat specimens. A 7mm trochlear defect was created and randomized to one of three treatment options: no treatment (empty), scaffold alone, and scaffold with minced autologous cartilage fragments. All treatments generated tissue within the defect. However, the scaffold with minced fragments produced the best results: whiter tissue with better congruency, more intense staining for proteoglycans, zonal architecture, and had a higher collagen type II to type I ratio. A Phase I FDA clinical study icompleted enrollment in 2007 and are now enrolling patients as part of a prospective comparison of this Cartilage Autograft Implantation System® (Mitek, Inc, Rayham, MA) in a Phase III multi-center study. The purpose of this study is to determine the safety and efficacy of CAIS compared to microfracture at 12 months post-treatment, with the primary efficacy assessment based on an analysis of non-inferiority of CAIS to microfracture for reduction in knee pain., CCI to stage autologous cartilage regeneration ACI has been shown to be an effective method of restoring articular cartilage and joint function in recent years several publications have shown the capability to regenerate a tissue which to some extent histologically resembles functional articular cartilage long-term results of clinical patient outcome are good if the hurdles in initial restoration as for example in the first two postoperative years are passed without complications. However there is a clear need for a better understanding of how cellular technology can be optimized to improve the reliability of patient outcome. Factors which come into play are aspects such as quality of the initial biopsy the number of passages and expansion volume of cells to be used. Also preoperative intra-articular joint homeostasis will be of influence to cartilage culture quality and the outcome of integration and tissue restoration after the transplantation surgery has been completed. Characterized chondrocytes are an autologous cartilage cell population consisting of phenotypically stable cells. As expansion of autologous chondrocytes leads to dedifferentiation and loss of chondrogenic capacity manufacturing methods needed to be developed with the aim of preserving as much as possible the phenotypic characteristics of the expanded cell population. ChondroCelect (TiGenix N.V., Haasrode, Belgium) is an autologous cell therapy product introducing the patented concept of a chondrogenic potential score (patent number WO2008061804) whereby a gene marker profile is used in the identification of in vivo cartilage forming ability. A controlled and consistent manufacturing process optimizing a number of parameters including enzymatic release, seeding density, time and procedure at harvest and shipping conditions is based on ectopic cartilage formation. The correlation between the marker profile and the ectopic cartilage forming activity shows an R2 value of 0.608 The total amount of cells administered is dependent on the size (surface in cm2) of the cartilage defect. The dose used is 0.8 to 1 million cells/cm2, corresponding with 80 to 100 microliter of product/cmÂ2 of defect. Characterized chondrocyte site implantation is the next generation of ACI it builds upon the established principle of culturing autologous chondrocytes but provides the surgeon and patient with a panel of molecular profiling which predicts the stability of cartilage tissue formed. While the method still involves a biopsy surgery and second stage procedure for implantation arthrotomy it has the additional benefit of a reliable cellular products being implanted. We have previously shown that after one year patients that have been treated by CCI have a histological tissue regenerate which is superior to that of a microfracture repair. The clinical outcome has been shown to be beneficial with a statistical superior results for relevant parameters of patient described clinical outcome using KOOS. However this study also indicates further challenges for clinical application of this regenerative technique. These lie in the need to better understand which patients are optimal candidates for such biological reconstruction. How come we predefined the success or failure of the biological regeneration process, which patients are eligible for reconstruction and which joints have passed the point of no return towards osteoarthritic degeneration for which cellular therapy at this moment has not been shown to be a viable option. CCI makes a difference in quality of tissue regeneration it's provides better clinical outcome three years post surgery however improvements are needed for matrix-based implantation patient profiling arthroscopic implantation and eventually maybe even one stage surgical interventions., Articular cartilage lesions, with their inherent limited healing potential, remain a challenging problem for orthopaedic surgeons. Various techniques, both palliative and reparative have been used to treat this pathology with variable success rates. In recent years regenerative techniques, such as ACI, have emerged as a potential therapeutic option. Recent studies [1,2] suggest the durability of this treatment, especially at long-term follow-up, due to its ability to produce hyaline-like cartilage that is mechanically and functionally stable, and also allows integration with the adjacent articular surface. However, despite the favourable clinical results obtained by many authors, the use of classic ACI (first generation) has been associated with several limitations related to the complexity and the morbidity of the surgical procedure, as well as the frequent occurrence of periosteal hypertrophy. Also some recent randomised studies [3,4] report controversial results regarding the better performance of the first generation ACI technique compared to other procedures used for cartilage repair. To address these problems Second generation ACI has been developed and biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation onto the defect have become widely used. Second generation ACI represents a modern and viable technique for cartilage full thickness chondral lesion repair [5,22,23]. However second generation ACI is a two step procedure which includes an arthroscopic biopsy for cell culture and implantation. Aside from the risk of harvest site morbidity and two surgical procedures, the total cost of the operation, scaffold and chondrocytes cultivation is still very high. Future directions in cartilage repair are moving towards the possibility to performing one step surgery. These could include the use of stem cells and growth factors. The use of autologous mesenchymal stem cells (MSC) and growth factors represents an improvement on the currently available techniques as this avoids the primary surgery for cartilage biopsy and subsequent chondrocytes cultivation and seeding on a scaffold. Many authors have recognized that nucleated cells found in bone marrow are a useful source of cells for restoration of damaged tissue [6,7]. Once MSC are cultured in the appropriate microenvironment, they can differentiate to chondrocytes and form cartilage. The onset of chondrogenesis requires a chemically defined serum free medium supplemented with dexamethasone, ascorbic acid and growth factors such as TGF-B [8]. In conjunction with appropriate scaffolds, these has been demonstrated that cells can be used to regenerate cartilage in a variety of applications [6]. However, some animal and laboratory studies have shown the chondrogenic potential of MSC but only few clinical human studies have been published [9,10]. Wakitani et al. [11] used autologous culture of expanded bone marrow for repair of cartilage defects in osteoarthritic knees; they chose 24 knees of 24 patients with knee OA who underwent a high tibial osteotomy; patients were divided into cell transplanted group and cell free group. After 16 months follow-up, they concluded that MSC were capable of regenerating a repair tissue for large chondral defects. Ochi et al. [12] observed that in a rat model the injection of cultured MSC combined with bone marrow stimulation can accelerate the regeneration of articular cartilage; they noted that this cell therapy was a less invasive treatment for cartilage injury. In their animal study [13] they introduced a MSC delivery system with the help of an electromagnetic field, enhancing the proliferation of cartilage inside the chondral defect after intra-articular injection, decreasing ectopic cartilage formation. Fortier et al. [14] concluded in animal studies that development of patient-side configuration techniques for intra-operative stem cell isolation and purification for immediate grafting have significant advantages in time savings and immediate application of an autogenous cell for cartilage repair. In order to find a more effective and simpler technique for cartilage repair we have attempted to use high concentration MSC combined with biologic scaffolds for osteochondral defect repair. Our technique for this operative procedure, consists firstly in preparation of the osteochondral defect with debridement by arthroscopy, and extraction of bone marrow from iliac crest. High concentration MSC are obtained by harvesting 40 −60 mL of bone marrow aspirate from the iliac crest with aspiration kit and a centrifugations system (Harvest Smart PreP2 System - Harvest Technologies, Plymouth, MA, USA). Implantation of the MSC are performed after adding batroxobin obtaining a sticky clot material that is implanted into osteo-chondral defect. Bilayer porcine collagen type I/ III matrix (Chondro-Gide; Geistlich Pharma AG, Wolhusen, Switzerland) is then applied and sutured or fixed with fibrin glue to the surrounding cartilage tissue to cover the entire defect and the BMC gel. In our first group, we prospectively followed grade 3 and 4 cartilage knee lesions implanted with concentrated MSC from the iliac crest with or without the use of a scaffold. All patients followed the same specific rehabilitation program after MSC implantation. With a mean follow-up of 12 months, patients showed improvements in all scores. No adverse reaction or post–op complication were noted in all patients to this date. Simplicity and low cost are the two major advantages of one step M.C.I.. This technique does not require cartilage harvesting, transportation to a G.M.P. laboratory and subsequent cells cultivation, seeding on the scaffold and re-implantation. Though more patients and longer follow up is needed to confirm our results. This one-step procedure permits a significant reduction of operating time and the related costs. And could very well be the future procedure of choice in cartilage repair., Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. In addition to bone marrow stimulating procedures such as microfracturing surgical therapeutic efforts to treat cartilage have focused on delivering new cells capable of chondrogenesis into the lesions. In the classic autologous chondrocyte transplanation (ACT) technique chondrocytes are isolated from small slices harvested from a minor weight-bearing area of the injured knee. The extracted cells are then cultured and once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect using a periosteal patch over the defect as a method of cell containment. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix associated autologous chondrocyte transplantation (MACT). These materials secure the cells in the defect area and enhance their proliferation and differentiation. MR imaging as a non-invasive technique is the method of choice in the preoperative evaluation and follow-up of patients with these different surgical cartilage repair techniques. MR imaging of the morphology of cartilage and cartilage repair tissue has significantly improved in recent years due to the development of clinical high-field MR systems operating at 3 Tesla. The improved performance has also been achieved as a result of the higher gradient strengths and the application of dedicated coils with modern configuration such as phased array coils. MR should be performed with cartilage sensitive sequences such as fat-suppressed PD/T2-FSE or three-dimensional (3D) GRE sequences, which provide a good signal to noise ratio (SNR) and contrast to noise ratio (CNR). High spatial resolution is mandatory and can be best achieved with dedicated coils at 3T. High resolution imaging is necessary for a better visualization of graft morphology, in particular for the evaluation of transplant integration to the adjacent hyaline cartilage and bone. MR imaging also helps to evaluate the filling of the defect by repair tissue, the surface and structure of repair tissue, the signal intensity of repair tissue with respect to the time interval to surgery and the status of the subchondral bone. Complications such as periosteal hypertrophy, incomplete and complete delamination, arthrofibrosis and adhesions, incongruencies of the cartilage surface at the repair site, graft failure and reactive changes of the joint such as effusions and synovitis can be visualized. The evaluation of the success of cartilage repair procedures requires particular grading systems, one of which is “Magnetic resonance Observation of CARtilage repair Tissue” or MOCART. The MOCART scoring introduced by Marlovits and Trattnig is postulated to allow subtle and suitable assessment of the articular cartilage repair tissue. Indeed in many recent original articles, review articles and book chapters, the MOCART score is used and discussed in the follow-up after different cartilage repair procedures. In contrast, new isovoxel sequences (GRE and well as FSE) have the potential for high-resolution isotropic imaging with a voxel size down to 0.4mm3, and can thus be reformatted in arbitrary planes without any loss of spatial resolution. Using this possibility of multi-planar reconstruction (MPR), the cartilage repair tissue could be visualized three-dimensional in every plane and its classification and grading by an MR-based scoring system might benefit. Recently, an improved MOCART scoring system using the possibilities of 3D MPR in the post-operative evaluation of cartilage repair tissue after MACT was developed (3D MOCART). In addition to morphological MR imaging of cartilage repair tissue, an advanced method to non-destructively and quantitatively monitor parameters reflecting the biochemical status of cartilage repair tissue is a necessity for studies which seek to elucidate the natural maturation of ACT and MACT grafts and the efficacy of the technique. For example glycosaminoglycans (GAG) are known to be responsible for stiffness properties of cartilage, which gains even more importance with cartilage implants and the content and organization of the collagen network reflects further mechanical properties of cartilage. Therefore, several MR techniques were developed, which allow detection of biochemical changes that precede the morphological degeneration in cartilage. To date, the most promising technique for visualizing the loss of GAG seems to be the delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC). It is based on the fact that GAG molecules contain negatively charged side chains which lead to an inverse proportionality in the distribution of the negatively charged contrast agent molecules with respect to the concentration of GAG. Consequently, T1 which is determined by the Gd-DTPA2- concentration becomes a specific measure of tissue GAG concentration. Earlier clinical studies of early cartilage degeneration showed that the differences of the pre-contrast T1 values between degenerative cartilage and normal cartilage were so small that they could be neglected; however, this is not true for cartilage repair tissue. For a correct evaluation of glycosaminoglycan concentration in cartilage repair tissue the pre-contrast T1 values have to be calculated, too. If a quantitative T1 analysis is also performed prior to contrast administration, it is possible to calculate the concentration of Gd-DTPA in cartilage repair tissue. The concentration is represented by ï„ R1, that is the difference in relaxation rate (R1=1/T1) between T1precontrast and T1postcontrast. This places time limitation problems on the patient evaluation since both pre-contrast MR imaging and delayed post-contrast MR imaging must be performed in cartilage repair patients. Furthermore, standard Inversion Recovery sequence for T1 mapping is time consuming. To overcome these problems a fast T1 determination by using different excitation flip angle values in gradient echo based sequences was optimized for dGEMRIC technique. For the follow-up of cartilage implants quantitative T1 mapping based on dual flip angle excitation pulse GRE technique allows in plane resolution of 0.3 × 0.3 mm with a slice thickness of 3mm and a scan time of about 4 minutes and was validated in vitro and in vivo. This dGEMRIC technique can be applied to patients following cartilage repair surgery as a way to obtain information related to the long-term development and maturation of grafts. While GAG content reflects stiffness properties of repair tissue, the organisation of the collagen matrix in repair tissue over time is important too, as failure within the collageneous fibre network is considered to entail further cartilage breakdown. The extracellular matrix of native articular cartilage is shaped by a highly organized collagen network, which is the basis of the histologic zones. Under ideal circumstances cartilage repair tissue produced following ACI and MACT, or other repair techniques, should, over time, develop a collagen network with a similar shape and collagen concentration to normal hyaline cartilage. Quantitative T2 mapping has been reported to be sensitive to collagen content and organization Using quantitative T2 mapping of patients at different post operative intervals after MACT surgery significantly higher T2 values in cartilage repair tissue in the early stage (3–6 months) compared to native hyaline cartilage were found with a decrease in repair tissue T2 values over time with the T2 values becoming similar to native healthy cartilage by approximately 10 to 13 months. With high resolution MR T2 mapping it is possible to assess zonal variations within the cartilage layer and use the measurement of the organization of articular cartilage as an additional tool to differentiate between cartilage repair tissues. This is based on the fact that the collagen fibers in the deep portion of cartilage are running perpendicular to cortical bone with consecutive dipolar effect and less mobility of protons resulting in a decrease of T2 values. Since the collagen fibers are randomly oriented in the superficial cartilage zone T2 values are longer. No differences between deep and superficial aspects within cartilage repair tissue and in total shorter T2 values compared to healthy cartilage after microfracture therapy was found indicating disorganized and more fibrous tissue. After MACT, zonal variation with T2 mapping could be measured, however compared to healthy cartilage sites the increase from deep to superficial zones was less pronounced. These findings may indicate that after MACT, cartilage repair tissue is, in terms of organization, more hyaline like. Quantitative T2 mapping may therefore help to better differentiate between normal maturation and development of abnormality in ACI and MACT. This technique may further help in the non-invasive, non destructive follow-up of patients operated on new generations of matrix-associated ACT using new scaffold or carriers. Better knowledge on the macromolecular organization in the implants may help in the planning of rehabilitative procedures after cartilage repair surgery. One encouraging alternative to these above mentioned sequence modalities for the evaluation of cartilage microstructure is the use of diffusion weighted sequences. Diffusion Weighted Imaging (DWI) is based on molecular motion that is influenced by intra- and extra-cellular barriers. Consequently, it is possible, by measuring of the molecular movement, to reflect biochemical structure and architecture of the tissue. To avoid long scan times and susceptibility changesassociated with spin-echo and echoplanar imaging sequences diffusion imaging can be based on steady state free precession sequences (SSFP) which realize a diffusion weighting in relatively short echo times. For the assessment of diffusion weighted images, a three-dimensional steady state diffusion technique, called PSIF (which is a time reversed FISP (Fast Imaging by Steady State Precession) sequence), has been used For evaluation, the quotient image (non-diffusion weighted / diffusion-weighted image) was calculated on a pixel-by-pixel basis. Recently, a quantitative SSFP based diffusion-weighted sequence was developed, but has to be validated in clinical studies. Most recent developments in MR imaging of cartilage repair comprise Magnetization Transfer imaging (MT) and its special variant: Chemical Exchange Saturation Transfer (CEST), the use of ultrahigh MR operating at 7T in vivo and the biomechanical MR imaging of cartilage repair tissue using unloading technique., Introduction Articular cartilage lesions are a common pathology of the knee joint and many patients could benefit from cartilage repair. Untreated, however, cartilage defects may lead to osteoarthritis (OA). Thus, surgical treatment options may offer a possibility for patients with cartilage defects to avoid OA or to delay the progression of OA. Therefore, cartilage repair techniques require sophisticated follow-up, if possible non-invasively. Although clinical findings are the primary criteria, a more objective outcome measure would possibly have the ability to provide predictive values. Advanced magnetic resonance imaging (MRI) is able to depict the morphological and biochemical condition of the cartilage repair tissue and the surrounding structures [1–4]. Histological samples are still the gold standard to gain information on the ultrastructure of cartilage repair tissue, however their impact is limited due to the required reoperation and the fact that a small biopsy may not provide information on the whole cartilage transplants, its borders and the relationship to the adjacent native cartilage. A sophisticated MR protocol in the follow-up after cartilage repair procedures nevertheless is able to visualize the morphological and the biochemical properties of the whole cartilage transplant and its adjacent cartilage non-invasively as a “virtual biopsy”. An ideal MRI protocol for articular cartilage and cartilage repair should provide accurate assessment of cartilage thickness and volume, reveal information about signal changes within cartilage, and demonstrate clear delineation of the cartilage surface, the cartilage and bone interface, as well as the subchondral bone and also provide information about the biochemical composition of articular cartilage and cartilage repair tissue. To evaluate such MRI protocols, cartilage repair procedures, such as arthroscopic or open surgical approaches as well as marrow-stimulation techniques, osteochondral grafting, and chondrocyte implantation/transplantation could be used. The uniqueness of cartilage repair in the use of advanced morphological and especially biochemical MR methodologies is bases in the fact that these procedures include defined areas of repair cartilage in addition to an often-intact surrounding cartilage in mostly young patients. Thus, modified and healthy cartilage can be compared within one subject. Nevertheless, the aim of an ideal MRI protocol must be its ease of implementation in scientific studies as well as day-to-day clinical routine. The combination of high-resolution morphological MR evaluation with biochemical assessment in a clinically applicable scan time needs to exploit high-field MRI together with advanced imaging techniques and sophisticated coil technology. Aim of this manuscript is to demonstrate advanced MRI techniques to depict the morphological and especially the biochemical constitution of cartilage repair tissue as a multiparametric approach in the follow-up of cartilage repair procedures. Methods and Discussion Histology -what do we expect? Native cartilage Native articular cartilage is a complex connective tissue, basically composed of water(∼75%), collagen(∼20%), and proteoglycan aggregates(∼5%). Water either freely moves throughout the matrix or is bound to macromolecules. Collagen in hyaline cartilage is largely type II, creating a stable network throughout the cartilage. The negatively charged proteoglycan is composed of a central core protein to which glycosaminoglycans (GAG) are bound. Hyaline articular cartilage is stratified primarily according to the orientation of collagen within a three-dimensional network [5, 6]. This complex network is stratified and arranged in zones. Cartilage repair In available studies a after MFX, OAT, ACI/ACT, and MACI/MACT histological evaluation show varying results; nevertheless, some trends are visible and may help to characterize the constitution of cartilage tissue. Gudas et al. [7], in a comparison of MFX and OAT, described all biopsies after OAT as hyaline cartilage, whereas after MFX, fibrocartilage (57%) and soft fibroelastic tissue(43%) was found. Bentley et al. [8], who compared OAT with ACI, reported biopsies obtained after ACI as hyaline-like(37%), mixed hyaline-like/fibrocartilage(37%), and fibrocartilage(28%), whereas after OAT hyaline cartilage is usually present within the different plugs, however problems are described in between the plugs. Horas et al. [9], on the other hand, reports histological samples after ACI as hyaline-like near the base of repair tissue, but with no tidemark and rather fibrous tissue in the central and superficial layers. After OAT, biopsies demonstrated the original tidemark and did not differ from surrounding cartilage. Bartlett et al. [10] compared ACI and MACI with nearly identical biopsies of hyaline-like cartilage(∼30%), mixed hyaline-like and fibrocartilage(∼10%), and fibrocartilage(∼60%). Other studies have reported much higher numbers(up to 75%) of biopsies with hyaline-like cartilage after ACI or MACI. When looking at one of the most widely recognized studies in this field, by Knutsen et al. [11], who compared ACT and MFX, two very important points become clear: i) Biopsies obtained from 67 patients showed all histological grades (1= predominantly hyaline; 2= fibrocartilage-hyaline mixture; 3= fibrocartilage; 4= no repair tissue) for both procedures and no significant difference between the cartilage repair techniques. Hence the ultrastructure of the repair tissue is not (only) dependent on the respective cartilage repair procedure and MFX might not only produce fibrocartilage and ACT might not only produce hyaline-like cartilage. ii) However, a comparison of the histological quality of the repair tissue in patients with and without treatment failure revealed that none of the patients with treatment failure in this study had hyaline-like cartilage. This finding suggests that repair cartilage, which is predominantly hyaline, may reduce the risk of subsequent failure [11]. Hence after all cartilage repair procedures the quantity and the quality of the repair tissue have to be assessed and together they might provide an objective outcome measure and a possible predictive value. MRI - what can we visualize? Morphological MRI The morphological shape of the repair tissue, the degree of repair filling (1), the integration of the cartilage repair tissue to the border zone (2), the structure of the surface (3), the structure of the whole repair tissue (4), the signal intensity (5), the constitution of the subchondral lamina (6), the the constitution of the subchondral bone (7), possible adhesions (8), and possible effusion (9) can be combined in the nine variables of the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system [12] which is claimed to allow subtle and suitable assessment of the articular cartilage repair tissue. The MR assessment of the MOCART score is based on standard MR sequences, also recommended by the International Cartilage Repair Society [13]. Depending on the locality of the area of cartilage repair, the MR evaluation of the cartilage repair tissue is performed on sagittal, axial or coronal two-dimensional planes using high spatial resolution together with a slice thickness of 2–4 mm. Following the current standard procedure, the recommended MR sequences by the ICRS and the recommended sequences for the MOCART scoring visualize the area of cartilage repair and the adjacent cartilage as well as the surrounding structures in 2D. In contrast, new isovoxel sequences have the potential for high-resolution isotropic imaging with a voxel size down to 0.4mm3, and can be reformatted in every plane without any loss of spatial resolution. Hence the cartilage repair tissue can be visualized three-dimensionally (3D) in every plane, its subsequent classification and grading by an MR-based scoring system benefits and a new 3D-MOCART score can be presented at the ICRS 2009. Biochemical - dGERMIC and others Of the major macromolecules, glycosamnioglycans (GAG), are important to the cartilage tissueâ ™s biochemical and biomechanical function. GAG are the main source of fixed charge density (FCD) in cartilage, often decreased in reparative cartilage after cartilage repair [14]. Intravenously administered gadolinium diethylenetriamine pentaacetate anion, (Gd-DTPA2-) penetrates the cartilage through both the articular surface and the subchondral bone. The contrast equilibrates in inverse relation to the FCD, which is, in turn, directly related to the GAG concentration; therefore, T1, which is determined by the Gd-DTPA2- concentration, becomes a specific measure of tissue GAG concentration, suggesting that T1 mapping enhanced by delayed administration of Gd-DTPA2- (T1 dGEMRIC) has the potential for monitoring GAG content of cartilage in vivo [15, 16]. Besides standard inversion recovery (IR) evaluation, a new approach for fast T1 mapping has shown promising results and is increasing the clinical applicability of the dGEMRIC technique [17]. Since GAG content is responsible for cartilage function, particularly its tensile strength, the monitoring of the development of GAG content in cartilage repair tissues may provide information about the quality of the repair tissue. A recent study by our group showed dGEMRIC to be able to differentiate between different cartilage repair tissues with higher delta R1 values, and thus, lower GAG content for cartilage repair tissue after MFX compared to MACT [18]. As the mapping of the GAG concentration is desirable in the follow-up of cartilage repair procedures and the presented dGEMRIC technique has the limitation of contrast agent administration and a time delay before post-contrast MRI, a recently described technique for the assessment of GAG concentration in vivo by chemical exchange-dependent saturation transfer (CEST) may have potential in future applications on articular cartilage [19]. At high fields and ultra-high fields, sodium MR imaging might become a gold-standard in future approaches. Sodium MRI has been validated as direct and quantitative method of computing FCD and, hence, proteoglycan content. Achieving high enough signal-to-noise ratio, the sensitivity of sodium MRI is high enough for detecting small changes in proteoglycan content. Biochemical - T2 and others Changes in water and collagen content and tissue anisotropy are most frequently analyzed using the transverse relaxation time (T2) of cartilage [20]. The collagen fiber orientation, its three-dimensional organization and curvature can be reliably visualized by cartilage T2 mapping, however influencing its appearance at 55deg; (with respect to the main magnetic field (B0)) resulting in the magic angle [5, 21]. In healthy articular cartilage, an increase in T2 values from deep to superficial cartilage layers can be observed based on the anisotropy of collagen fibers running perpendicular to cortical bone in the deep layer of cartilage [22]. Histologically validated animal studies have shown this zonal increase in T2 values as a marker of hyaline or hyaline-like cartilage structure after cartilage repair procedures within the knee [23, 24]. To visualize this zonal variation is essential for cartilage T2 mapping and high enough spatial resolution has to be provided. In cartilage repair tissue, full-thickness T2 values, as zonal evaluation have been shown able to visualize cartilage repair maturation [25, 26]. Another study by our group further showed the ability of zonal T2 evaluation to differentiate cartilage repair tissue after MFX and MACT [27]. In addition to standard 2D multi-echo spin-echo T2 relaxation, T2*- weighted 3D gradient-echo articular cartilage imaging has shown reliable results in the evaluation of chondromalacia of the knee [28]. In recent studies, T2* mapping, with its potentially short scan times, was correlated to standard T2, and showed information comparable to that obtained for articular cartilage in the knee, but with overall lower T2* values (ms) [29, 30]. Furthermore, also for T2*, a clear zonal variation between deep and superficial cartilage layers can be shown. Another advanced approach to combine morphological and biochemical T2 cartilage imaging is based on a Double-Echo-Steady-State (DESS) sequence, which generates two signal echoes that are characterized by a different contrast behavior where the underlying T2 can be calculated. Further emerging biochemical MR techniques that are gaining more and more importance are, besides others, diffusion weighted imaging (DWI), magnetization transfer contrast (MTC) or T1rho. The cartilage composition or component visualized by these methodologies is not yet clearly validated. However initial studies are showing very promising results and additional information besides dGERMIC and T2 can be gained also in patients after cartilage repair. Concluding, a combination of morphological and biochemical MRI might, besides a sophisticated non-invasive follow-up examination, provide the chance to gain a predictive value for the future development and performance of the cartilage repair tissue and the joint., Today, there are several existing techniques for the treatment of cartilage injuries but still the evaluation of the quality of such repairs has not been good enough. Also, the classification of cartilage lesions in order to diagnose an early osteoarthritis is yet to date not satisfying. Today, one may classify OA by clinical symptoms and/or the degree of joint space narrowing and number and size of existing osteophytes (Altman 1991). An evaluation system that can in a more objective and qualitative way, describe the nature of the cartilage repair and cartilage morphology would be of great importance. An objective more secure way of OA classification would also be of great importance. Magnetic resonance (MR) imaging is a 3D imaging technique that can permit a direct delineation of cartilage structure. With MRI one may estimate the volume and thickness of cartilage securely and make compositional imaging of cartilage using quantitative MR as well as for semi quantitative scoring of cartilage disease For the surgeon it is possible with MRI to estimate the size, nature and location of lesions Preoperatively and post-surgery at certain intervals also evaluate the quality regarding filling of the defect, tissue integration and subchondral bone reaction. Is it possible with a cartilage repair to stop an osteoarthritic development? MRI may give such an answer and could be used to follow the structure of the surrounding cartilage post-repair comparing the cartilage in such a repaired joint with a joint with similar lesions but without cartilage defect repair. However, with the most MRI techniques used by normal hospitals worldwide, the MRI technique is not yet good enough. High qualities MRI are mostly found at university centres. Figueroa et al, 2007, studied one hundred ffteen chondral lesions in 82 patients which were found during the arthroscopic procedure. Most of them were single lesions (72%) located on the medial femoral condyle (32.2%) or medial patellae (22.6%); 62.6% of the lesions were classified as ICRS type II or II I-A, with an average surface of 1.99 cm2. The authors found a significant direct correlation between the patient's age and the size of the lesion. MRI sensitivity was 45% with a specificity of 100%. The sensitivity increased with deeper lesions (direct relation with the ICRS classification). The authors concluded that even though unenhanced MRI using a 1.5-Tesla magnet with conventional sequences (proton density-weighted, T1-weighted, and T2-weighted) is most accurate at revealing deeper lesions and defects at the patellae, that study shows that a considerable number of lesions will remain undetected until arthroscopy, which remains the gold standard. A step-forward have been the development of the MOCART classification system (Magnetic Resonance Observation of Cartilage Repair Tissue)(Marlovits et al, 2006). The system is an attempt of transferring the ICRS arthroscopic macroscopic post repair evaluation system to the evaluation of defect repair as seen on the MR images. The radiologists and the arthroscopists are subsequently focusing on the same issue; the success of the repair attempts. Two MR imaging techniques that are important in the evaluation of early structural damage in articular cartilage are quantitative T2 mapping and T1 mapping after Gd-DPTA2- administration (dGEMRIC). dGEMRIC technique can demonstrate the glycosamino-glycan(GAG) component of articular cartilage while T2 mapping focuses on changes noticed in the water and collagen content and the different tissue layers. With such techniques it is possible to both better compare the repair with the surrounding native cartilage and also follows the maturation process, especially as the maturation process is important when deciding for the rehabilitation protocols. Still today, when randomised study protocols are developed, biopsies of the repair tissues are included. However, such biopsies show a very little part of the repair tissue and with today's imaging techniques; biopsies should be avoided being invasive procedures with risk of morbidity for the patient. Important is the finding that normal articular cartilage demonstrates an increase in T2 values from the subchondral bone to the articular surface that has been correlated with type II collagen fibre matrix organization (anisotropy) in those zones (White et al, 2006). Perfect agreement was seen between organized T2 and histological findings of hyaline cartilage and between disorganized T2 and histological findings of fibrous reparative tissue. A significant trend of increasing T2 values (from deep to superficial) was found in hyaline cartilage while fibrous tissue sites had no significant change from bone to surface(White et al, 2006). Kurkijarvi et al (2007) found that according to T2 measurements, ACI repair tissue at 10–15 months differs from normal cartilage and probably lacks the preferential collagen arrangement of normal cartilage, while according to dGEMRIC a varying degree of proteoglycan replenishment takes place. Combining these two quantitative magnetic resonance imaging techniques enables a more comprehensive characterization of cartilage repair than before and in the future it will be of great interest to see randomised studies including such non-invasive methodologies in the evaluation of repairs from bone marrow stimulation techniques versus from different cell seeded scaffolds. Furthermore, for the diagnosis of osteoarthritis there is now the possibility to use spin-lock (T1RHO) magnetic resonance imaging (regatta, 2006). T1RHO is highly sensitive to changes in macro-molecular content, which may allow it to be used as a magnetic resonance marker of molecular changes in cartilage degeneration. T1rho has a higher dynamic range to measure even small macromolecular changes for detecting early pathology and subsequently useful to detect early OA compared to T2. The future will involve the above described imaging techniques for both the orthopaedic surgeons following the outcome of their surgeries as well as for the rheumatologists following their results of OA and RA treatments with chondroprotecive drugs and other pre-prosthetic treatments. New systems for cartilage injury classifications, cartilage repair assessments and OA definitions will be developed for the monitoring of success of treatments and for understanding of the epidemiology of disease. Furthermore to look forward for in the future is the use of high field systems (3 T and above) that significantly improve the sensitivity and specificity of imaging studies for the assessment of cartilage and cartilage disorders., In cartilage repair, similarly to other knee injuries and knee osteoarthritis, the relation of structural changes and clinical outcome is generally poor. It seems that pain and other clinical outcomes may be related to other factors than structures such as cartilage, ligaments and the menisci. Studies in knee osteoarthritis have shown quadriceps weakness and psychosocial factors to have a stronger association to pain than radiographic changes [1]. Similarly, impaired function seems to be largely explained by other factors than radiographic changes. McAlindon [2] found impaired function to be explained by quadriceps weakness, knee pain and older age and not by severity of radiographic changes. Traditionally, we consider pain as a result of an underlying patho-anatomic change. In knee injury and knee osteoarthritis causality is weak. Possible reasons for this weak association in knee osteoarthritis include radiographic changes being the result of a process and not mirroring the process in itself, the examination being performed in such a way that changes have not been registered, that radiographic changes which we think constitute the disease actually do not represent the full spectrum of the disease, or that the radiographic changes are not the reason for the symptoms. Much the same is seen in cartilage repair. Structural improvement is not necessarily followed by clinical improvement, and vice versa; clinical improvement may be registered without and structural improvement. In addition to lack of validity in evaluation measure, other underlying pain mechanisms than patho-anatomical lesions, reasons for the discrepancy seen in interventions such as cartilage repair include placebo effects. Placebo is very effective in the treatment of knee osteoarthritis. The size of the placebo effect is determined by the strength of the active treatment, the baseline disease severity, the route of delivery and the sample size of the study [3]. Surgery, as in cartilage repair, is associated with a larger placebo effect than a drug which is turn is associated with larger placebo effects than from non-pharmacological and non-surgical interventions. Although there is a correlation of increasing radiographic changes and pain at a group level in people with osteoarthritis, the association for the individual is weak. In population-based studies about half report knee pain [4–6]. In one of these studies, based on the Framingham cohort, only 15% of the individuals who reported knee pain had radiographic changes of osteoarthritis [6]. Since the prevalence of osteoarthritis increase with age, one could expect a stronger association with older age. However, only 21% of the subjects aged 51–74 who reported knee pain had radiographic osteoarthritis [6]. It has been suggested that the insensitivity of radiographic examination to detect cartilage changes and low sensitivity of the pain questions used could explain the weak correlation of radiographic osteoarthritis and pain. In population-based studies, pain has most often been defined as pain during most days of a month [2, 6]. In studies on osteoarthritis patients, most commonly a self-reported questionnaire evaluating pain during different activities during the last week is used. Although differently formulated questions have different ability to detect radiographic osteoarthritis [7] the differences are relatively small and most likely do not explain the discrepancy between pain and structure in osteoarthritis. In summary, the correlation of structure and pain is weak in cartilage repair as well as in other knee problems such as ligament deficiency, and in knee osteoarthritis. In design of clinical trials there is consensus within the communities of medicine, orthopedics and sports medicine that the primary outcome is the patient's self-report of pain and function. This is important to acknowledge also in the field of cartilage repair when interpreting the sometimes diverging results of evaluation of structure and clinical outcome., Articular cartilage is specialized connective tissue forming smooth surfaces between articulating bones in joints. Cartilage function is extremely demanding since human hip or knee joints should sustain loads up to several times body weight during normal daily activities, such as walking or stair climbing (Mow et al. 1992). The mechanical properties of cartilage are determined by the content, arrangement and interactions of the tissue constituents, i.e. three-dimensional collagen network, proteoglycans (PGs) and interstitial water (Mow et al. 1991). The balance between the mechanical demands and material properties of the tissue is essential for normal joint function. This homeostasis is disturbed in diseases such as osteoarthritis (OA, arthritis), where the balance between anabolic and catabolic activities of cartilage cells – chondrocytes – is distorted, and the collagen network and PGs become degraded with the consequence of impaired functional properties of the tissue. OA causes a high economical burden to the society nationally and internationally. Potentially, minor changes in the content and arrangement of tissue components may transmit to significant changes in cartilage biomechanics. Quantitative microscopic imaging techniques, e.g. polarized light microscopy (PLM) or magnetic resonance imaging (MRI) enable characterization of tissue structure. MRI is a clinical tool for the diagnosis of OA (Nissi et al. 2004). The limitation of MRI is the inability to directly quantitate functional (mechanical) changes of cartilage occurring, e.g., in early OA. Instead, theoretical modelling is needed to describe tissue function (DiSilvestro et al. 2001, Korhonen et al. 2003b). A realistic in vivo estimation of cartilage functional characteristics, stress/strain states and possible failure points in a joint would necessitate that the MRI-derived, depth-dependent tissue structure is incorporated into a theoretical model. Combination of MRI with realistic mechanical model could reveal sensitively early cartilage pathologies. Further, successfully conducted remodeling algorithm, showing for e.g. the development of collagen fibril network during OA progression, could provide a valuable tool for the evaluation of the effect of loading on cartilage development, progression of OA, and ideally prevention of OA. In previous studies we have developed anisotropic cartilage models and applied them for the 1)prediction of the mechanical behaviour of osteoarthritic articular cartilage and differentiation of PG loss from collagen degradation (Korhonen et al. 2003a),2)investigation of the role of superficial layer on the indentation response of cartilage (Julkunen et al. 2008a),3)investigation of the effect of anisotropy (mainly due to collagen) of the extracellular matrix on the mechanical signals experienced by chondrocytes (Korhonen et al. 2006),4)characterization of articular cartilage by combining microscopic analysis and finite element modelling (Julkunen et al. 2007)5)estimation of the mechanical properties of articular cartilage from MRI images (Julkunen et al. 2008b) Specifically, we found that the collagen and PG-specific MRI parameters correlated significantly with the corresponding mechanical parameters of articular cartilage, i.e. the fibril network modulus (collagen) and the non-fibrillar matrix modulus (PGs) (Julkunen et al. 2007). The study was based on the fibril-reinforced poroelastic model of cartilage, which has been shown to be one of the most realistic approaches to describe cartilage mechanics. We further developed the model and used only information on tissue composition to describe the mechanical behavior of cartilage (Julkunen et al. 2008c). Based on tissue fixed charge density, collagen content and fluid fraction the model was able to simulate stress-relaxation responses of cartilage. We believe that a technique which could predict cartilage development under certain loading conditions, or the progression of OA after a local cartilage damage would be important. A potential way to approach this problem is to 1) image joint and cartilage composition and structure using magnetic resonance imaging (MRI), 2) incorporate this information into a finite element model to predict tissue function from its composition, and, further, to estimate changes in tissue architecture, especially in collagen orientation, in loaded articular cartilage, and ultimately 3) simulate joint loads to investigate stresses and strains in cartilage, possible risk locations, and the development of collagen orientation in normal, degenerated or tissue-engineered cartilage. Prediction of cartilage development and progression of OA under spesific joint loads is a challenging task. Combining MRI analyses of tissue solid and fluid contents with the composition based theoretical model of cartilage enables estimation of cartilage mechanics from its composition. This would make a functional imaging technique for sensitive diagnosis of OA. Further, with succesfully conducted remodeling algorithm, it could be even possible to estimate cartilage development and progression of OA after a local damage several years ahead. This approach would help in decision making of clinical treatments and interventions for the prevention of OA. Importantly, the economic burden both for the society and people would be reduced significantly., The menisci of the knee joint are fibrocartilaginous structures that play critically important functions in load distribution, joint stability, shock absorption, and lubrication. The meniscus is classified as a fibrocartilage because of the rounded or oval shape of most of the cells in the inner tissue, and the partly fibrous appearance of the extra-cellular matrix (1). The meniscus has some ability to mount tissue repair processes. In striking contrast to hyaline articular cartilage, cells can and do migrate through the matrix of the meniscus in response to wounding (2,3). This suggests that the wounded meniscus can signal to the specialized cells in the environment to migrate towards the wound, and that the matrix of the tissue contains corridors that permit and may even promote cell migration. The meniscus has a vascular supply. How far the blood vessels penetrate into the meniscus, however, depends on the stage of development of the tissue. Essentially all of the tissue has a vascular supply in the newly born. At maturity, however, only about 15% of the outer radial distance has a vascular supply. Immunostaining and confocal microscopy demonstrates that type I collagen is found throughout the entire tissue of the immature and mature meniscus. Type II collagen, however, is restricted to the non-vascular, inner zone of the canine meniscus (4). No type II collagen was demonstrable in the juvenile meniscus, which had blood vessels throughout its structure. The dominant structural motifs in the meniscus are the circumferential bundles of collagen fibrils with occasional radial structures referred to as “tie fibers” or “tie sheaths”. Electron microscopic studies of normal canine meniscus demonstrates two separate matrix systems in the main body of the meniscus: (a) the “bundles” of circumferentially arranged bundles of collagen fibrils; and (b) a new system that we are calling the “peri-matrix” that is a part of and radiates out of the “tie fibers and that separates and enwraps the circumferential collagen fibrils. Cells reside in and appear to migrate through the peri-matrix. In the outer meniscus, the cells have a fibroblast appearance and show extending processes that enwrap the bundles of collagen fibrils. The wounded canine meniscus does have the capacity to emit signals that activate exogenous human mesenchymal stem cells. Taken together, these data show that the meniscus has a distinctive fibril architecture and perhaps signaling system that should facilitate repair of wounds in the tissue., Introduction The menisci are regarded as important structures for knee stability, shock absorption, and load distribution. Their location in the knee and the extreme forces that they can be subjected to, make them frequently susceptible to injury, especially in contact-sport activities. Due in large part to the limited vascularity of the meniscus, this tissue has little innate ability to heal spontaneously. Partial meniscectomy is still indicated if the lesion cannot be satisfactorily stabilized with sutures or anchors. Particularly troublesome are lesions to the inner portions of the meniscus where the tissue is not vascularized. Engineering tissue to repair an injured meniscus is an emerging strategy for restoring form and function of meniscal fibrocartilage. Specific considerations must be given to the type of cells necessary, the scaffold, and the physical forces within the microenvironment in which the meniscus is located. Although many efforts have been made for engineering articular cartilage, relatively few studies have been reported for engineering meniscal tissue or attempt to repair this construct by a cell-based strategy. Important considerations for engineering meniscal tissue include the material properties of the meniscal substance, the geometry of the construct, and the functional integrity of meniscal attachments. Cell Sources for Tissue Engineering The primary obstacle for engineering cartilage of any type is acquiring the appropriate numbers of chondrogenic cells for generating the tissue. The seeding density of cells capable of chondrogenesis in or onto a polymer carrier is a critical ingredient for successfully engineering cartilaginous tissues, but few would risk morbidity to normal meniscus to obtain cells for engineering new meniscal tissue. It is evident that the type and the number of cells and the physical environment, into which they are placed, are critical elements for successfully engineering meniscal cartilage tissue. Chondrocytes from other areas of the body could be used to generate meniscal tissue or used to induce meniscal repair. We have shown that articular chondrocytes embedded in fibrin glue are capable of inducing repair in meniscal lesions in an explant model in nude mice (1,2); also that articular chondrocytes are able of forming bonds between cartilage explants and meniscal lesions (3–6). Extending these findings to a large animal model, we have shown that articular chondrocytes seeded onto a devitalized cartilage matrix are capable of inducing a healing process in a bucket-handle lesion of the meniscus in swine (7). Other alternative cartilage sources for obtaining cells include the cartilage portion of ribs, which are expandable, or possibly small biopsies of ear cartilage. Chondrocytes from these sources have been shown to generate new cartilage matrix and are capable of forming bonds between cartilage discs (8). These alternative cell sources could be used to engineer reparative meniscal tissue. The use of other noncartilage cells with chondrogenic potential may permit the generation of cartilage given the correct polymer and appropriate conditions as well. Multiple experiments have shown that, under controlled in vitro conditions, mesenchymal stem cells (MSCs) can differentiate into bone, fat, tendon, muscle and cartilage-like tissues. MSCs can be used to regenerate connective tissues through tissue engineering techniques; additionally, they have a natural capacity to home to injured tissues and to participate to tissue healing. This second feature seems particularly interesting as long as MSCs not only provide a cell source for regeneration but they also secrete paracrine factors that enhance the potential for tissue repair, acting as “trophic mediators” (9). One of the first studies evaluating the potential of MSCs for meniscal repair was performed by Port et al. using MSCs cultured in fibrin clot. However, the use of MSCc in that study did not enhance meniscal healing (10). This was probably due to the absence of a scaffold providing a suitable environment to MSCs; and to the lack of a pre-culture time, which is known to improve the capacity of the engineered tissue to withstand the weight bearing forces (11). More recently, MSCs were delivered as a suspension by intra-articular injection after total meniscectomy and ACL resection in a study by Murphy et al., to develop an osteoarthritic knee (12). In this study, authors report a positive effect of MSCs to the injured joint with significant formation of neomeniscal tissue. This study suggests that there may be a therapeutic benefit associated with intra-articular injection of stem cells following traumatic injury to the knee, with a possible longer term effect determining a reduction or delay in the progression to osteoarthritis (12). MSCs seeded onto a hyaluronan-collagen-based scaffold were recently used to repair a critical-size defect in a rabbit model, with a tissue engineering approach (11). In their study, the authors removed the pars intermedia of the medial meniscus and replaced the resected section with an acellular biocompatible scaffold, or with the scaffold loaded with cultured MSCs. Menisci repaired with the acellular scaffold showed a fibrous, scar-like repair tissue, while those repaired with the engineered tissue demonstrated a significantly better filling and meniscal regeneration. Another interesting option to be exploited is the use of chondrocytes from allogeneic or xenogeneic sources for engineering cartilage. Whereas the use of allogeneic and xenogeneic cartilage “en bloc” did not always encounter secure clinical success, the use of isolated chondrocytes from these sources may perform satisfactorily. We have recently tested the potential of allogeneic ear and articular chondrocytes for repairing swine knee meniscus lesions. Ear autologous and allogeneic chondrocytes were seeded onto vicryl mesh and tested for meniscus repair in orthotopic lesion in the swine model. Articular autologous and allogeneic chondrocytes were also evaluated. It was demonstrated that all four cell populations possess repair potential for these lesions (13). Scaffolds for meniscus tissue engineering The other critical element for engineering meniscal cartilage is finding or developing suitable scaffold materials that permit or accelerate the formation of new extracellular matrix. A biomaterial used as scaffold for tissue engineering purposes should present many features: it should be biocompatible and possibly biodegradable allowing to be replaced by biologic tissue; it should have enough resiliency and resistance to withstand weigh-bearing while cells produce matrix; it should maintain structure, volume and shape in an in vivo orthotopic environment; it should promote cell differentiation and proliferation if seeded with cells, or promote cell migration if cell-free; it should allow diffusion of nutrients and catabolic remnants; it should be easily manageable and implantable by the surgeon (14). According to these features, many biomaterials have been evaluated for meniscal repair and tissue engineering both natural or synthetic (15). Natural materials used include: periosteal tissue, perichondral tissue, small intestine submucosa, and meniscal tissue. While these tissues have high biocompatibility, they cannot be adapted for tissue engineering techniques as they do not allow varying structure geometry and initial mechanical properties (15). Isolated tissue components such as collagens and proteoglycans (16) maintain the high biocompatibility of the natural tissues while allowing the creation of custom-made scaffold with definite pore dimension and geometry and, consequently, biomechanical features. However, these scaffolds have usually low biomechanical properties (15). On the other hand, biomimetic synthetic materials can be manufactured in custom-made shapes of any geometrical structure and porosity depending on the characteristic of the host tissue and of the cells seeded. In particular, it has been shown that optimal ingrowth and incorporation of a meniscal scaffold, macropore sizes must be in the range of 150–500 μm (17). To date, the most used synthetic materials include the following: polyglicolic acid (PGA), poly(L) lactic acid (PLLA), polyurethane, polyester, polytetrafuoroethylene, polycaprolactone (PCL), and various combinations of these polymers and other materials even the natural ones. The latter approach, namely coupling synthetic (PCL) and natural (hyaluronic acid) materials, has been recently evaluated in two large animal studies on partial and total meniscus tissue engineering with promising results (18,19). Cell-free techniques The rationale of using a cell-free biomaterial to replace part of the meniscus is based on scaffold repopulation by host cells from the synovium and the meniscal remnants, and subsequent tissue ingrowth. In fact, this approach could be considered to become cell-based after implantation. The collagen meniscus implant (CMI) is the first regenerative technique applied to meniscal repair in clinical practice (20–22). As long as an outer rim of menisci tissue is needed for CMI implantation, it is indicated only as partial meniscus regeneration and not as total meniscus regeneration. Satisfactory clinical results have been reported (23) while results from the imaging and histological standpoints are controversial as the CMI shrinks over time and showed no histological remnants 5 to 6 years after implantation (22) leaving predominantly scar tissue instead of fibrocartilage (24). However, CMI results in satisfactory improvement in pain, can be implantable arthroscopically, and it is the only method used thus far in the clinical setting. Small intestinal submucosa (SIS) has been applied to menisci regeneration in a dog model in a 12 months follow-up study (25). In that study, menisci receiving SIS in a posterior vascular lesion had more meniscus-like tissue filling the defects and significantly less cartilage damage than menisci receiving no implants. Conclusions The geometric complexity of the meniscus, the composition of the tissue, and the forces on the meniscus present many obstacles for engineering a substitute. Finding suitable cell sources, providing a blood supply from the periphery, and developing new polymers that can withstand the rigor of the mechanical forces in the knee are key to successfully engineering meniscal tissue. This requires the interaction and collaboration of several disciplines such as biology, chemistry, molecular biology, bio-engineering, material-and mechanical-engineering, genetics and surgery; that all need to focus on a common target for understanding the mechanisms occurring in the natural environment and for restoring or substituting the damaged tissue. At the present time, successful strategies are being developed using tissue-engineering approaches to heal meniscal tears. Clinical application of these approaches appears to be in the very near future.

We report a biologic approach to improve medial collateral ligament healing using growth factors normally expressed in healing tissue. Our previous in vitro work demonstrated that platelet-derived growth factor-BB and transforming growth factor-beta 1 promoted fibroblast proliferation and matrix synthesis, respectively. There-fore, these growth factors were used in vivo to determine whether they could improve medial collateral ligament healing, whether this effect was dose-dependent, and if combinations of growth factors could improve healing more than individual growth factors. Thirty-seven rabbits had various doses of growth factors applied to the ruptured right medial collateral ligaments using a fibrin sealant delivery vehicle. The five groups consisted of 1) two groups receiving two doses of platelet-derived growth factor-BB, 2) two groups receiving two doses of this growth factor plus transforming growth factor-beta 1, and 3) one group receiving fibrin sealant only. After sacrifice at 6 weeks, biomechanical and histologic evaluations of the healing ligament were performed. Femur-medial collateral ligament-tibia complexes of the knees given the higher dose of platelet-derived growth factor-BB had ultimate load, energy absorbed to failure, and ultimate elongation values that were 1.6, 2.4, and 1.6 times greater than the same complexes of the control group. Adding transforming growth factor-beta 1 did not lead to any further increase in the structural properties of the complex compared with treatment with platelet-derived growth factor-BB. These encouraging results suggest that use of platelet-derived growth factor-BB may improve the quality of the healing medial collateral ligament, and that it may also have a similar potential for promoting healing of other ligaments.