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Scaffolds are thought to be a key element needed for successful cartilage repair treatments, and this prospective extension study aimed to evaluate long-term structural and clinical outcomes following osteochondral defect treatment with a cell-free biphasic scaffold. Structural outcomes were assessed using quantitative 3-D magnetic resonance imaging (MRI) and morphological segmentation to determine the percentage of defect filling and repair cartilage T2 relaxation times, and clinical outcomes were determined with the modified Cincinnati Rating System, and the Knee Injury and Osteoarthritis Outcome Score (KOOS). Seventeen subjects with osteochondral defects in the knee were treated with ChondroMimetic scaffolds, from which 15 returned for long-term evaluation at a mean follow-up of 7.9 &plusmn, 0.3 years. The defects treated were trochlear donor sites for mosaicplasty in 13 subjects, and medial femoral condyle defects in 2 subjects. MRI analysis of scaffold-treated defects found a mean total defect filling of 95.2 &plusmn, 3.6%, and a tissue mean T2 relaxation time of 52.5 &plusmn, 4.8 ms, which was identical to the T2 of ipsilateral control cartilage (52.3 &plusmn, 9.2 ms). The overall modified Cincinnati Rating System score was statistically significant from baseline (p = 0.0065), and KOOS subscales were equivalent to other cartilage repair techniques. ChondroMimetic treatment resulted in a consistently high degree of osteochondral defect filling with durable, cartilage-like repair tissue at 7.9 years, potentially associated with clinical improvement.

Objective The efficacy and safety of BST-CarGel®, a chitosan scaffold for cartilage repair was compared with microfracture alone at 1 year during a multicenter randomized controlled trial in the knee. This report was undertaken to investigate 5-year structural and clinical outcomes. Design The international randomized controlled trial enrolled 80 patients, aged 18 to 55 years, with grade III or IV focal lesions on the femoral condyles. Patients were randomized to receive BST-CarGel® treatment or microfracture alone, and followed standardized 12-week rehabilitation. Co-primary endpoints of repair tissue quantity and quality were evaluated by 3-dimensional MRI quantification of the degree of lesion filling (%) and T2 relaxation times. Secondary endpoints were clinical benefit measured with WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index) questionnaires and safety. General estimating equations were used for longitudinal statistical analysis of repeated measures. Results Blinded MRI analysis demonstrated that BST-CarGel®-treated patients showed a significantly greater treatment effect for lesion filling ( P = 0.017) over 5 years compared with microfracture alone. A significantly greater treatment effect for BST-CarGel® was also found for repair tissue T2 relaxation times ( P = 0.026), which were closer to native cartilage compared to the microfracture group. BST-CarGel® and microfracture groups showed highly significant improvement at 5 years from pretreatment baseline for each WOMAC subscale ( P < 0.0001), and there were no differences between the treatment groups. Safety was comparable for both groups. Conclusions BST-CarGel® was shown to be an effective mid-term cartilage repair treatment. At 5 years, BST-CarGel® treatment resulted in sustained and significantly superior repair tissue quantity and quality over microfracture alone. Clinical benefit following BST-CarGel® and microfracture treatment were highly significant over baseline levels.

Objective The efficacy and safety of BST-CarGel, a chitosan-based medical device for cartilage repair, was compared with microfracture alone at 1 year during a multicenter randomized controlled trial (RCT) in the knee. The quality of repair tissue of osteochondral biopsies collected from a subset of patients was compared using blinded histological assessments. Methods The international RCT evaluated repair tissue quantity and quality by 3-dimensional quantitative magnetic resonance imaging as co-primary endpoints at 12 months. At an average of 13 months posttreatment, 21/41 BST-CarGel and 17/39 microfracture patients underwent elective second look arthroscopies as a tertiary endpoint, during which ICRS (International Cartilage Repair Society) macroscopic scoring was carried out, and osteochondral biopsies were collected. Stained histological sections were evaluated by blinded readers using ICRS I and II histological scoring systems. Collagen organization was evaluated using a polarized light microscopy score. Results BST-CarGel treatment resulted in significantly better ICRS macroscopic scores ( P = 0.0002) compared with microfracture alone, indicating better filling, integration, and tissue appearance. Histologically, BST-CarGel resulted in a significant improvement of structural parameters— Surface Architecture ( P = 0.007) and Surface/Superficial Assessment ( P = 0.042)—as well as cellular parameters— Cell Viability ( P = 0.006) and Cell Distribution ( P = 0.032). No histological parameters were significantly better for the microfracture group. BST-CarGel treatment also resulted in a more organized repair tissue with collagen stratification more similar to native hyaline cartilage, as measured by polarized light microscopy scoring ( P = 0.0003). Conclusion Multiple and independent analyses in this biopsy substudy demonstrated that BST-CarGel treatment results in improved structural and cellular characteristics of repair tissue at 1 year posttreatment compared with microfracture alone, supporting previously reported results by quantitative magnetic resonance imaging.

A fundamental understanding of the in vivo biodegradation phenomenon as well as an appreciation of cellular and tissue responses which determine the biocompatibility of biodegradable PLA and PLGA microspheres are important components in the design and development of biodegradable microspheres containing bioactive agents for therapeutic application. This chapter is a critical review of biodegradation, biocompatibility and tissue/material interactions, and selected examples of PLA and PLGA microsphere controlled release systems. Emphasis is placed on polymer and microsphere characteristics which modulate the degradation behaviour and the foreign body reaction to the microspheres. Selected examples presented in the chapter include microspheres incorporating bone morphogenetic protein (BMP) and leuprorelin acetate as well as applications or interactions with the eye, central nervous system, and lymphoid tissue and their relevance to vaccine development. A subsection on nanoparticles and nanospheres is also included. The chapter emphasizes biodegradation and biocompatibility; bioactive agent release characteristics of various systems have not been included except where significant biodegradation and biocompatibility information have been provided.

Subchondral drilling and microfracture are bone marrow stimulation techniques commonly used for the treatment of cartilage defects. Few studies to date have examined the technical variants which may influence the success of the cartilage repair procedures. This study compared the effect of hole depth (6 mm vs. 2 mm) and hole type (drill vs. microfracture) on chondral defect repair using a mature rabbit model. Results from quantitative histomorphometry and histological scoring showed that deeper versus shallower drilling elicited a greater fill of the cartilage defect with a more hyaline character in the repair matrix indicated by significant improvement (p = 0.021) in the aggregate measure of increased cartilage defect fill, increased glycosaminoglycan and type II collagen content and reduced type I collagen content of total soft repair tissue. Compared to microfracture at the same 2 mm depth, drilling to 2 mm produced a similar quantity and quality of cartilage repair (p = 0.120) according to the aggregate indicator described above. We conclude that the depth of bone marrow stimulation can exert important influences on cartilage repair outcomes. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1178–1184, 2011

Summary Objectives Collagen organization, a feature that is critical for cartilage load bearing and durability, is not adequately assessed in cartilage repair tissue by present histological scoring systems. Our objectives were to develop a new polarized light microscopy (PLM) score for collagen organization and to test its reliability. Design This PLM score uses an ordinal scale of 0–5 to rate the extent that collagen network organization resembles that of young adult hyaline articular cartilage (score of 5) vs a totally disorganized tissue (score of 0). Inter-reader reliability was assessed using Intraclass Correlation Coefficients (ICC) for Agreement, calculated from scores of three trained readers who independently evaluated blinded sections obtained from normal (n=4), degraded (n=2) and repair (n=22) human cartilage biopsies. Results The PLM score succeeded in distinguishing normal, degraded and repair cartilages, where the latter displayed greater complexity in collagen structure. Excellent inter-reader reproducibility was found with ICCs for Agreement of 0.90 [ICC(2,1)] (lower boundary of the 95% confidence interval is 0.83) and 0.96 [ICC(2,3)] (lower boundary of the 95% confidence interval is 0.94), indicating the reliability of a single reader's scores and the mean of all three readers' scores, respectively. Conclusion This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

The repair of articular cartilage has posed a longstanding orthopedic challenge. The concept of marrow stimulation is basically the intentional injury of a subchondral bone below a cartilage lesion to elicit a wound repair response. However, the desire to fill the lesion with a blood clot is at odds with platelet-driven clot retraction, which results in clot shrinkage and detachment. Therefore, BST-CarGel was developed to produce a physically stabilized blood clot that is more voluminous and adherent within a debrided cartilage lesion having access to bone marrow, thus improving existing bone marrow-stimulation procedures. BST-CarGel is a soluble polymer scaffold containing the polysaccharide chitosan, which is dispersed throughout uncoagulated whole blood, and then delivered to a surgically prepared lesion. BST-CarGel allows normal clot formation, reinforces the clot, impedes retraction, increases adhesivity, and ensures prolonged residency of both the clot and critical tissue repair factors from blood. In animals, BST-CarGel was shown to increase the volume and hyaline character of repair tissue, with increased GAG and collagen content, compared with microfracture controls. A patient cohort received BST-CarGel treatment, which encompassed the spectrum of traumatic to degenerative lesions, along with other joint pathologies. Anecdotal evidence demonstrated the potential of BST-CarGel for treating focal cartilage lesions of variable etiology through both mini-open and arthroscopic approaches. Because it requires only a single minimally invasive intervention, BST-CarGel and its unique characteristics are novel in orthopedics. In addition, its use with marrow stimulation provides familiarity with well-recognized surgical techniques, bringing a simple-yet-versatile treatment modality that applies scaffold-guided regenerative medicine to a potentially wide-ranging group of indications.

A novel process for the formation of physical hydrogels of chitosan from injectable aqueous solutions is investigated. Uniform neutralization of chitosan solutions with ammonia generated in situ from enzymatic hydrolysis of urea is shown to produce pH-induced hydrogels with monolithic and homogeneous coherent 3D structure. Hydrogel formation was characterized by rheology. We found that the gelation time decreases as temperature increases from 15 to 45 °C as a consequence of synergistic effect of increased reactant diffusion and increased urease activity (maximum activity at 37 °C). The effects of urea and urease concentration are also presented.

Chitosan is a naturally abundant cationic polysacchaside with unique physicochemical properties resulting in its use in a vast array of biomedical applications, including wound repair, drug delivery, and tissue engineering. However, despite numerous and diverse studies investigating its use, there have been no detailed reports describing specific staining methods for cell culture and histopathological studies using chitosan based materials. Here, we describe methods for staining chitosan in both cell culture and within paraffin sections. Procedural models used included identifying chitosan within in vitro whole blood clots, and culture wells, and in vivo chitosan hydrogel implants in multiple anatomical sites. First, a staining process for identifying chitosan was optimized using 0.03% Cibacron Brilliant Red-3BA. A second sensitive and versatile method using 0.01% Fast Green in conjunction with the known Safranin-O Method was demonstrated and may prove to be a key advancement in the histological ...

Anin vivorat cage implant system was used to identify potential surface chemistries that prevent failure of implanted biomedical devices and prostheses by limiting monocyte adhesion and macrophage fusion into foreign-body giant cells while inducing adherent-macrophage apoptosis. Hydrophobic, hydrophilic, anionic, and cationic surfaces were used for implantation. Analysis of the exudate surrounding the materials revealed no differences between surfaces in the types or levels of cells present. Conversely, the proportion of adherent cells undergoing apoptosis was increased significantly on anionic and hydrophilic surfaces (46 ± 3.7 and 57 ± 5.0%, respectively) when compared with the polyethylene terephthalate base surface. Additionally, hydrophilic and anionic substrates provided decreased rates of monocyte/macrophage adhesion and fusion. These studies demonstrate that biomaterial-adherent cells undergo material-dependent apoptosisin vivo, rendering potentially harmful macrophages nonfunctional while the surrounding environment of the implant remains unaffected.

Biocompatibility of implanted materials is determined by the host foreign-body response, which is comprised of cellular (adherent monocytes and macrophages) and soluble (secreted cytokines) components. Modulating the presence, activity or both of adherent macrophages may increase or decrease the biocompatibility of implants because these cells remain adherent to the implant surface and fuse to form foreign-body giant cells (FBGCs), leading to failure of the implant. An attractive mechanism of eliminating these cells is through the induction of apoptosis; therefore ways of inducing or inhibiting apoptosis of biomaterial-adherent inflammatory cells are being investigated. We hypothesized that interleukin-4 (IL-4) promotes macrophage survival by inhibiting tumor necrosis factor-α (TNF-α)—induced apoptosis. We found that TNF-α induces apoptosis in a time- and dose-dependent manner, whereas IL-4 inhibits TNF-α—induced and spontaneous apoptosis of biomaterial-adherent macrophages. Blocking experiments and evaluation of shedding of soluble TNF receptor type I (TNF-RI) demonstrated that endogenous TNF-α production is responsible for spontaneous apoptosis of biomaterial-adherent cells and that IL-4 inhibits this apoptosis by increasing levels of shedding of soluble TNF-RI. These findings suggest that TNF-α and IL-4 play key roles in determining the fate of biomaterial-adherent cells and that fusion of macrophages into FBGCs is a mechanism for promoting inflammatory-cell survival on implanted materials. (J Lab Clin Med 2002;139:90-100)

Monocytes play a critical role as both phagocytes and mediators of inflammatory responses in the prevention of cardiovascular device-related infections. However, persistent infection of these devices still occurs and may be attributed to deleterious cellular alterations resulting from monocyte interactions with a foreign material in an environment of dynamic flow. Thus, the effects of both shear stress and adhesion to material surfaces on human monocyte apoptosis were investigated. A rotating disk system generated physiologically relevant shear stress levels (0-14 dyn/cm(2)), and shear-related apoptosis occurring in adherent monocytes was characterized. Using annexin V analysis, apoptosis of polyurethane-adherent monocytes under shear for 4 h increased to levels >70% with increasing shear in a near-linear fashion (r2 = 0.713). It was qualitatively confirmed using confocal microscopy that filamentous (F)-actin distribution was altered, that DNA fragmentation occurred, and that activated caspases were involved in shear-induced apoptosis. Static studies determined that spontaneous apoptosis was material-dependent over 72 h by demonstrating marked differences between apoptosis of monocytes adherent to a polyurethane compared to an alkyl-modified glass. Treatment with TNF-alpha augmented this material dependency in a dose-dependent fashion over time. F-actin content of TNF-alpha-treated cells decreased to

A common component of the foreign-body response to implanted materials is the presence of adherent macrophages that fuse to form foreign-body giant cells (FBGCs). These multinucleated cells have been shown to concentrate the phagocytic and degradative properties of macrophages at the implant surface and are responsible for the damage and failure of the implant. Therefore, the modulation of the presence or actions of macrophages and FBGCs at the material-tissue interface is an extensive area of recent investigations. A possible mechanism to achieve this is through the induction of the apoptosis of adherent macrophages, which results in no inflammatory consequence. We hypothesize that the induction of the apoptosis of biomaterial adherent cells can be influenced by the chemistry of the surface of adhesion. Herein, we demonstrate that surfaces displaying hydrophilic and anionic chemistries induce apoptosis of adherent macrophages at a higher magnitude than hydrophobic or cationic surfaces. Additionally, the level of apoptosis for a given surface is inversely related to that surface's ability to promote the fusion of macrophages into FBGCs. This suggests that macrophages fuse into FBGCs to escape apoptosis.

The mechanisms underlying problematic cardiovascular device-associated infections are not understood. Because the outcome of the acute response to infection is largely dependent on the function of neutrophils, the persistence of these infections suggests that neutrophil function may be compromised because of cellular responses to shear stress. A rotating disk system was used to generate physiologically relevant shear stress levels (0–18 dynes/cm2; 1 dyne = 10 μN) at the surface of a polyetherurethane urea film. We demonstrate that shear stress diminishes phagocytic ability in neutrophils adherent to a cardiovascular device material, and causes morphological and biochemical alterations that are consistent with those described for apoptosis. Complete neutrophil apoptosis occurred at shear stress levels above 6 dynes/cm2after only 1 h. Morphologically, these cells displayed irreversible cytoplasmic and nuclear condensation while maintaining intact membranes. Analysis of neutrophil area and filamentous actin content demonstrated concomitant decreases in both cell area and actin content with increasing levels of shear stress. Neutrophil phagocytosis of adherent bacteria decreased with increasing shear stress. Biochemical alterations included membrane phosphatidylserine exposure and DNA fragmentation, as evaluated byin situannexin V and terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) assays, respectively. The potency of the shear-stress effect was emphasized by comparative inductive studies with adherent neutrophils under static conditions. The combination of tumor necrosis factor-α and cycloheximide was ineffective in inducing >21% apoptosis after 3 h. These findings suggest a mechanism through which shear stress plays an important role in the development of bacterial infections at the sites of cardiovascular device implantation.

Infection of implanted cardiovascular biomaterials still occurs despite inherent host defense mechanisms. Using a rotating disk system, we investigated Staphylococcus epidermidis and polymorphonuclear leukocyte (PMN) adhesion to a polyetherurethane urea (PEUU-A′) under shear stress (0–17.5 dynes/cm2) for time periods up to 6 h. In addition, the superoxide (SO) release capacity of PMNs after transient exposure to PEUU-A′ under shear stress was determined. Bacterial adhesion in phosphate-buffered saline (PBS) showed a linear shear dependence, decreasing with increasing shear stress. Overall adhesion in PBS decreased with time. However, bacterial adhesion in 25% human serum was similar for all time points up to 360 min. Adhesion was observed at all shear levels, displaying no shear dependence. In contrast, PMN adhesion demonstrated a strong shear dependence similarly for times up to 240 min, decreasing sharply with increasing shear stress. Although PMNs preexposed to shear stress showed a slightly diminished SO release response compared to fresh cells for all stimuli, it was not statistically significant regardless of the stimulus. We conclude that circulating leukocytes are unable to adhere in regions of high shear which may contain adherent bacteria. In addition, exposure to PEUU-A′ and shear stress (in the range 0–18 dynes/cm2) is insufficient to cause a depression in the oxidative response of PMNs. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 46, 511–519, 1999.

The in vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] hydrogels were assessed in order to investigate the influence of poly(ethylene glycol) molecular weight and copolymer composition. These materials have application as injectable cardiovascular implants; cytotoxicity due to leachable products, as well as inflammation caused by the biomaterial itself, may ultimately affect the biocompatibility of the implant. We utilized a 7-day in vitro cytotoxicity assay to quantify cell density and cellular proliferation in the presence of copolymer films. The copolymer films exhibited slight to moderate cytotoxicity toward cultured endothelial cells, showing 20–86% viability relative to controls. Cell viability increased with an increasing weight percent of PEG or, to a lesser extent, the molecular weight of PEG. In vivo biocompatibility was assessed using a cage implantation model over a 21-day time period. This system was used to characterize the local cellular and humoral inflammatory response in the surrounding exudate, as well as the size and density of macrophages adherent to the material itself. All copolymer formulations exhibited excellent biocompatibility relative to controls with no significant differences in total leukocyte count among the different formulations. The in vivo inflammatory reaction displayed normal wound healing over 21 days as shown by a progressive decrease in both leukocyte concentration and enzymatic activity. The surface coverage of the copolymer films remained relatively constant from 7 to 21 days. There were no cells larger than 0.003 mm2, which was previously shown to be the threshold value for foreign-body giant cells. These data suggest that P(PF-co-EG) hydrogels have potential for use as injectable biomaterials.© 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 46, 22–32, 1999.

Photochemical immobilization technology was utilized to covalently couple polymers to silicone rubber either at multiple points along a polymer backbone or at the endpoint of an amphiphilic chain. The coating variants then were tested in vitro and in vivo for improvement of desired responses compared to uncoated silicone rubber. All coating variants suppressed the adsorption of fibrinogen and immunoglobulin G, and most also inhibited fibroblast growth by 90-99%. None of the coating variants inhibited monocyte or neutrophil adhesion in vitro. However, the surfaces that supported the highest levels of monocyte adhesion also elicited the lowest secretion of pro-inflammatory cytokines. None of the materials elicited a strong inflammatory response or significantly (p< 0.05) reduced the thickness of the fibrous capsule when implanted subcutaneously in rats. Overall, the most passivating coating variant was an endpoint immobilized polypeptide that reduced protein adsorption, inhibited fibroblast growth by 90%, elicited low cytokine secretion from monocytes, and reduced fibrous encapsulation by 33%. In general, although some coating variants modified the adsorption of proteins and the behavior of leukocytes or fibroblasts in vitro, none abolished the development of a fibrous capsule in vivo.

Poly(etherurethane urea) (PEUU) elastomers, with their broad range of mechanical properties and high biocompatibility, are used clinically for medical applications. However, the possibility exists for the ether soft segment of PEUU to degrade in long-term uses. To retard degradation, antioxidants that scavenge reactive oxygen intermediates are added. In this study, we incorporated dehydroepiandrosterone (DHEA), which functions by the alternate mechanism of modulating or down-regulating adherent macrophage activity, to retard the biodegradation of PEUUs. Biocompatibility of PEUU samples containing 1% DHEA, 5% DHEA, and 5% vitamin E (α-tocopherol) by weight were studied in vivo and in vitro. The biocompatibility was initially evaluated by examination of the inflammatory cellular exudate. Compared to PEUU without additives and PEUU with 5% vitamin E, the addition of 5% DHEA to PEUU caused a decrease in the total leukocyte exudate concentration at 4 days. The addition of 5% DHEA also caused lower macrophage adhesion and FBGC formation compared to the other materials at 7 days. Despite these short-term effects, the biocompatibility at later time points (14, 21, and 70 days) was similar for all materials. Transmission infrared analysis of the materials revealed that more than 70% of the DHEA had leached out of the samples by 3 days implantation. Furthermore, through attenuated total reflectance Fourier transform analysis and scanning electron microscopy, it was determined that unlike vitamin E, DHEA did not enhance long-term PEUU biostability. The effect of DHEA on inflammatory cell activity appeared to be dose dependent, with improved biocompatibility in vivo for higher loading levels of DHEA, but the overall effect was limited owing to the rapid diffusion of the water-soluble DHEA from the PEUU. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 192–201, 1998.

Objective: Intra-lesional bony overgrowth (BO) identified during or following cartilage repair treatment is being frequently described through subjective reports focusing primarily on incidence. Our objective was to quantify the exact volume of intra-lesional BO at 12 months post-cartilage repair treatment, to determine if a correlation exists between the extent of BO and clinical outcomes, and to visualize and characterize the BO. Design: MRI scans were systematically obtained during a randomized clinical trial for cartilage repair (Stanish etal., 2013) that compared two microfracture-based treatments in 78 patients. Semi-automated morphological segmentation of pre-treatment, 1 and 12 months post-treatment scans utilizing a programmed anatomical atlas for all knee bone and cartilage structures permitted three-dimensional reconstruction, quantitative analysis, as well as qualitative characterization and artistic visualization ofBO. Results: Limited intra-lesional BO representing only 5.8±5.7% of the original debrided cartilage lesion volume was found in 78 patients with available MRIs at 12 months. The majority (80%) of patients had very little BO (

Bone marrow stimulation techniques such as abrasion arthroplasty [1], Pridie drilling [2], and microfracture [3] attempt to use the natural wound repair response elicited by a blood clot originating from the bone marrow. Channels surgically made in the subchondral bone below the cartilage lesion permit access to marrow blood and blood components including stem cells intended to provide an environment for wound healing that ultimately leads to cartilage regeneration. Microfracture, which has been frequently used as a first-line treatment for small cartilage lesions, has the advantage of being simple and safe, cost-effective, and minimally invasive with a low morbidity rate [4, 5]. On the other hand, the procedure results in a mixed repair tissue with mainly fibrous or fibrocartilaginous properties [6–10], limited collagen type II and glycosaminoglycan (GAG) levels, and poor mechanical properties compared to native hyaline cartilage. Indeed, the long-term durability of this repair tissue has been questioned with many reports showing a failure of repair tissue and a return of associated clinical symptoms starting as early as 24 months posttreatment [8, 11, 12].

Subchondral drilling and microfracture are bone marrow stimulation techniques commonly used for the treatment of cartilage defects. Few studies to date have examined the technical variants which may influence the success of the cartilage repair procedures. This study compared the effect of hole depth (6 mm vs. 2 mm) and hole type (drill vs. microfracture) on chondral defect repair using a mature rabbit model. Results from quantitative histomorphometry and histological scoring showed that deeper versus shallower drilling elicited a greater fill of the cartilage defect with a more hyaline character in the repair matrix indicated by significant improvement (p = 0.021) in the aggregate measure of increased cartilage defect fill, increased glycosaminoglycan and type II collagen content and reduced type I collagen content of total soft repair tissue. Compared to microfracture at the same 2 mm depth, drilling to 2 mm produced a similar quantity and quality of cartilage repair (p = 0.120) according to the aggregate indicator described above. We conclude that the depth of bone marrow stimulation can exert important influences on cartilage repair outcomes.

Background: Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair. Hypothesis: Chitosan–glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone. Study Design: Controlled laboratory study. Methods: Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (±blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37°C were analyzed by proteomics and neutrophil chemotaxis assays. Results: Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1β or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (±blood) implants attracted more neutrophils ( P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls ( P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue. Conclusion and Clinical Relevance: AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.

Bone marrow stimulation is performed using several surgical techniques that have not been systematically compared or optimized for a desired cartilage repair outcome. In this study, we investigated acute osteochondral characteristics following microfracture and comparing to drilling in a mature rabbit model of cartilage repair. Microfracture holes were made to a depth of 2 mm and drill holes to either 2 mm or 6 mm under cooled irrigation. Animals were sacrificed 1 day postoperatively and subchondral bone assessed by histology and micro-CT. We confirmed one hypothesis that microfracture produces fractured and compacted bone around holes, essentially sealing them off from viable bone marrow and potentially impeding repair. In contrast, drilling cleanly removed bone from the holes to provide access channels to marrow stroma. Our second hypothesis that drilling would cause greater osteocyte death than microfracture due to heat necrosis was not substantiated, because more empty osteocyte lacunae were associated with microfracture than drilling, probably due to shearing and crushing of adjacent bone. Drilling deeper to 6 mm versus 2 mm penetrated the epiphyseal scar in this model and led to greater subchondral hematoma. Our study revealed distinct differences between microfracture and drilling for acute subchondral bone structure and osteocyte necrosis. Additional ongoing studies suggest these differences significantly affect long-term cartilage repair outcome.

A new biomaterial for cartilage repair has been developed and investigated in animal studies and in a clinical cohort. The biomaterial is a physiological solution of chitosan (a natural polysaccharide containing glucosamine residues) in a buffer containing glycerol phosphate (GP). The soluble and physiological characteristics of this polymer solution permit its combination with freshly drawn autologous whole blood to form a hybrid polymer-blood mixture that can be applied to cartilage and bone surfaces, to which it adheres and solidifies as a polymer-stabilized clot. Histology and electron microscopy analysis of in vitro-generated chitosan-GP/blood clots revealed the chitosan component to be dispersed among the blood components, to interact closely with platelets, and to impede platelet-mediated clot contraction, thereby maintaining a voluminous bioactive and adhesive clot at the site of application. Experiments in microdrilled cartilage lesions in adult rabbits comparing chitosan-GP/blood clots to controls (microdrilled only) highlighted the ability of chitosan-GP/blood clots to recruit more host cells and to increase subchondral vascularization and bone-remodeling activity during acute and intermediate stages of repair. This led to the establishment of more hyaline repair cartilage that was integrated with a porous subchondral bone plate. Microfractured cartilage defects in adult sheep treated with chitosan-GP/blood clots resulted in a statistically significant increase in tissue fill with a greater proportion of hyaline cartilage compared to controls (microfracture only). Patients with femoral condyle cartilage lesions have received chitosan-GP/blood implants to resurface articular cartilage as part of a compassionate use program for medical devices. Results to date suggest safety and clinical benefit of this approach that is free from both donor site morbidity and suture damage to healthy adjacent cartilage. This single-intervention approach is now the subject of a multicenter, randomized comparative clinical trial designed and initiated to investigate cartilage repair resulting from treatment with chitosan-GP and microfracture vs microfracture alone.

The biocompatibility and biofouling of the microfabrication materials for a MEMS drug delivery device have been evaluated. The in vivo inflammatory and wound healing response of MEMS drug delivery component materials, metallic gold, silicon nitride, silicon dioxide, silicon, and SU-8(TM) photoresist, were evaluated using the cage implant system. Materials, placed into stainless-steel cages, were implanted subcutaneously in a rodent model. Exudates within the cage were sampled at 4, 7, 14, and 21 days, representative of the stages of the inflammatory response, and leukocyte concentrations (leukocytes/microl) were measured. Overall, the inflammatory responses elicited by these materials were not significantly different than those for the empty cage controls over the duration of the study. The material surface cell density (macrophages or foreign body giant cells, FBGCs), an indicator of in vivo biofouling, was determined by scanning electron microscopy of materials explanted at 4, 7, 14, and 21 days. The adherent cellular density of gold, silicon nitride, silicon dioxide, and SU-8(TM) were comparable and statistically less (p0.05) than silicon. These analyses identified the MEMS component materials, gold, silicon nitride, silicon dioxide, SU-8(TM), and silicon as biocompatible, with gold, silicon nitride, silicon dioxide, and SU-8(TM) showing reduced biofouling.

Within the complex environment of an implanted cardiovascular device comprised of dynamic flow and foreign materials, phagocytic neutrophils may be ineffective in combating infection due to cellular responses to shear stress. This may be explained, in part, by our recent reports of apoptosis of biomaterial-adherent leukocytes induced through exposure to shear stress. Here we utilize a rotating disk system to generate physiologically relevant shear stress levels (0–18 dynes/cm2) at the surface of a polyetherurethane urea (PEUU) and investigate neutrophil intracellular pathways involved in shear-induced apoptosis. In situ detection of activated caspases, the enzymatic mediators of the apoptosis cascade, showed qualitatively that these proteases participate in shear-induced apoptosis and are activated in a shear-dependent manner. The involvement of caspase 3 was confirmed through immunoprecipitation and immunoblotting of extracted neutrophil proteins. Comparative studies with neutrophils adherent under static conditions demonstrated time-dependent activation of caspases in TNF-α/cycloheximide-induced apoptosis, for which caspase-3 also was implicated. These findings are the first steps toward elucidation of the mechanisms behind the inappropriate induction of apoptosis by adhesion to biomaterials, which may contribute to the development and persistence of device-related infections. ©2002 Wiley Periodicals, Inc. J Biomed Mater Res 62: 163–168, 2002

Cardiovascular disease processes such as atherosclerosis, restenosis, and inflammation are typically localized to discrete regions of the vasculature, affording great opportunity for targeted pharmacological treatment. Liposomes are potentially advantageous targeted drug carriers for such intravascular applications. To facilitate their use as drug delivery vehicles, we have considered three components of liposome design: (i) identification of candidate cell surface receptors for targeting; (ii) identification of ligands that maintain binding specificity and affinity; and (iii) prevention of rapid nonspecific clearance of liposomes into the reticuloendothelial organs. In this report, we describe our work in developing liposomal surface modifications that address both targeting and clearance. An arginine-glycine-aspartic acid (RGD) containing peptide was used as a model ligand to target liposomes to the integrin GPIIb-IIIa on activated platelets. Additionally, oligodextran surfactants incorporated into liposomes provided insight into the effect of vesicle perturbations on liposome clearance, and the importance of molecular geometry in designing oligosaccharide surface modifications. Together these studies demonstrate the feasibility of using peptides to guide liposomes to desired receptors, and illustrate the influence of vesicle stability on liposome interactions in vivo. Furthermore, they underscore the importance of simultaneously considering both targeting specificity and vesicle longevity in the design of effective targeted drug delivery systems.

Summary Objective This study characterizes collagen organization (CO) in human normal ( n = 6), degraded ( n = 6) and repair ( n = 22) cartilages, using polarized light (PLM) and scanning electron (SEM) microscopies. Design CO was assessed using a recently developed PLM-CO score (Changoor et al. Osteoarthritis Cartilage 2011;19:126–35), and zonal proportions measured. SEM images were captured from locations matched to PLM. Fibre orientations were assessed in SEM and compared to those observed in PLM. CO was also assessed in individual SEM images and combined to generate a SEM-CO score for overall CO analogous to PLM-CO. Fibre diameters were measured in SEM. Results PLM-CO and SEM-CO scores were correlated, r = 0.786 ( P n = 32), after excluding two outliers. Orientation observed in PLM was validated by SEM since PLM/SEM correspondence occurred in 91.6% of samples. Proportions of the deep (DZ), transitional (TZ) and superficial (SZ) zones averaged 74.0 ± 9.1%, 18.6 ± 7.0%, and 7.3 ± 1.2% in normal, and 45.6 ± 10.7%, 47.2 ± 10.1% and 9.5 ± 3.4% in degraded cartilage, respectively. Fibre diameters in normal cartilage increased with depth from the articular surface [55.8 ± 9.4 nm (SZ), 87.5 ± 1.8 nm (TZ) and 108.2 ± 1.8 nm (DZ)]. Fibre diameters were smaller in repair biopsies [60.4 ± 0.7 nm (SZ), 63.2 ± 0.6 nm (TZ) and 67.2 ± 0.8 nm (DZ)]. Degraded cartilage had wider fibre diameter ranges and bimodal distributions, possibly reflecting new collagen synthesis and remodelling or collagen fibre unravelling. Repair tissues revealed the potential of microfracture-based repair procedures to produce zonal CO resembling native articular cartilage structure. Values are reported as mean ± 95% confidence interval. Conclusion This detailed assessment of collagen architecture could benefit the development of cartilage repair strategies intended to recreate functional collagen architecture.