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Stem cells are known to have excellent regenerative ability, which is primarily facilitated by indirect paracrine factors, rather than via direct cell replacement. The regenerative process is mediated by the release of extracellular matrix molecules, cytokines, and growth factors, which are also present in the media during cultivation. Herein, we aimed to demonstrate the functionality of key factors and mechanisms in skin regeneration through the analysis of conditioned media derived from fetal stem cells. A series of processes, including 3D pellet cultures, filtration and lyophilization is developed to fabricate human fetal cartilage-derived progenitor cells-conditioned media (hFCPCs-CM) and its useful properties are compared with those of human bone marrow-derived MSCs-conditioned media (hBMSCs-CM) in terms of biochemical characterization, and in vitro studies of fibroblast behavior, macrophage polarization, and burn wound healing. The hFCPCs-CM show to be devoid of cellular components but to contain large amounts of total protein, collagen, glycosaminoglycans, and growth factors, including IGFBP-2, IGFBP-6, HGF, VEGF, TGF β3, and M-CSF, and contain a specific protein, collagen alpha-1(XIV) compare with hBMSCs-CM. The therapeutic potential of hFCPCs-CM observes to be better than that of hBMSCs-CM in the viability, proliferation, and migration of fibroblasts, and M2 macrophage polarization in vitro, and efficient acceleration of wound healing and minimization of scar formation in third-degree burn wounds in a rat model. The current study shows the potential therapeutic effect of hFCPCs and provides a rationale for using the secretome released from fetal progenitor cells to promote the regeneration of skin tissues, both quantitatively and qualitatively. The ready-to-use product of human fetal cartilage-derived progenitor cells-conditioned media (hFCPCs-CM) are fabricated via a series of techniques, including a 3D culture of hFCPCs, filtration using a 3.5 kDa cutoff dialysis membrane, and lyophilization of the CM. hFCPCs-CM contains many ECM molecules and biomolecules that improves wound healing through efficient acceleration of M2 macrophage polarization and reduction of scar formation.

After an injury, soft tissue structures in the body undergo a natural healing process through specific phases of healing. Adhesions occur as abnormal attachments between tissues and organs through the formation of blood vessels and/or fibrinous adhesions during the regenerative repair process. In this study, we developed an adhesion-preventing membrane with an improved physical protection function by modifying the surface of chondrocyte-derived extracellular matrices (CECM) with anti-adhesion function. We attempted to change the negative charge of the CECM surface to neutral using poly-L-lysine (PLL) and investigated whether it blocked fibroblast adhesion to it and showed an improved anti-adhesion effect in animal models of tissue adhesion. The surface of the membrane was modified with PLL coating (PLL 10), which neutralized the surface charge. We confirmed that the surface characteristics except for the potential difference were maintained after the modification and tested cell attachment in vitro. Adhesion inhibition was identified in a peritoneal adhesion animal model at 1 week and in a subcutaneous adhesion model for 4 weeks. N-CECM suppressed fibroblast and endothelial cell adhesion in vitro and inhibited abdominal adhesions in vivo. The CECM appeared to actively inhibit the infiltration of endothelial cells into the injured site, thereby suppressing adhesion formation, which differed from conventional adhesion barriers in the mode of action. Furthermore, the N-CECM remained intact without degradation for more than four weeks in vivo and exerted anti-adhesion effects for a long time. This study demonstrated that PLL10 surface modification rendered a neutral charge to the polymer on the extracellular matrix surface, thereby inhibiting cell and tissue adhesion. Furthermore, this study suggests a means to modify extracellular matrix surfaces to meet the specific requirements of the target tissue in preventing post-surgical adhesions. This article is protected by copyright. All rights reserved.

In this study, we have investigated whether human fetal cartilage progenitor cells (hFCPCs) have anti-inflammatory activity and can alleviate osteoarthritis (OA) phenotypes in vitro.hFCPCs were stimulated with various cytokines and their combinations and expression of paracrine factors was examined to find an optimal priming factor. Human chondrocytes or SW982 synoviocytes were treated with interleukin-1β (IL-1β) to produce OA phenotype, and co-cultured with polyinosinic-polycytidylic acid (poly(I-C))-primed hFCPCs to address their anti-inflammatory effect by measuring the expression of OA-related genes. The effect of poly(I-C) on the surface marker expression and differentiation of hFCPCs into 3 mesodermal lineages was also examined.Among the priming factors tested, poly(I-C) (1 µg/mL) most significantly induced the expression of paracrine factors such as indoleamine 2,3-dioxygenase, histocompatibility antigen, class I, G, tumor necrosis factor- stimulated gene-6, leukemia inhibitory factor, transforming growth factor-β1 and hepatocyte growth factor from hFCPCs. In the OA model in vitro, co-treatment of poly(I-C)-primed hFCPCs significantly alleviated IL-1β-induced expression of inflammatory factors such as IL-6, monocyte chemoattractant protein-1 and IL-1β, and matrix metalloproteinases in SW982, while it increased the expression of cartilage extracellular matrix such as aggrecan and collagen type II in human chondrocytes. We also found that treatment of poly(I-C) did not cause significant changes in the surface marker profile of hFCPCs, while showed some changes in the 3 lineages differentiation.These results suggest that poly(I-C)-primed hFCPCs have an ability to modulate inflammatory response and OA phenotypes in vitro and encourage further studies to apply them in animal models of OA in the future.

Background: We recently analyzed the joint capsule adjacent to the medial meniscus and found that the perimeniscal joint capsule has collagen fiber orientation similar to that of circumferential meniscal fibers, potentially playing a role in preventing extrusion. Purpose: To analyze the meniscal extrusion prevention potential of the circumferential rim augmentation suture around the perimeniscal capsule in a rabbit root tear model and analyze the biomechanical function in a porcine cadaveric knee. Study Design: Controlled laboratory study. Methods: Rabbit medial meniscus root tear models were divided into 3 experimental groups: root tear, root tear and suture repair, and root tear and circumferential rim augmentation suture. As for the circumferential rim augmentation suture procedure, a suture was placed to circumscribe the outer rim of the medial meniscus and passed through bone tunnels located at the tibial insertion of each root. After 4 and 8 weeks, meniscal extrusion was analyzed by micro–computed tomography, gross morphology, and histologic analysis of the medial femoral cartilage. For biomechanical analysis, porcine knees were divided into groups similar to rabbit experiments. Tibiofemoral contact parameters were assessed using a pressure mapping sensor system after applying a load of 200 N on the knee joint. Results: The root tear and circumferential rim augmentation suture group showed less meniscal extrusion, less gap within the tear site, and less cartilage degeneration compared with other groups after 4 and 8 weeks of surgery in the rabbit root tear model. Biomechanical analysis showed the root tear and circumferential rim augmentation suture group had larger contact area and lower peak contact pressure compared with root tear and root tear and suture repair groups. Conclusion: The circumferential rim augmentation suture reduced the degree of meniscal extrusion while restoring meniscal function, potentially preventing progression of arthritis in a rabbit root tear model and porcine knee biomechanical analysis. Clinical Relevance: The circumferential rim augmentation suture may be a novel augmentation option during root tear treatment.

BACKGROUND: The extracellular matrix (ECM) of articular cartilage has an inhibitory effect on vascularization, yet clinical utilization has been technically challenging. In this study, we aimed to fabricate a biologically functional ECM powder suspension from porcine articular cartilage that inhibits neovascularization (NV). METHODS: The digested-cartilage acellular matrix (dg-CAM) was prepared by sequential processes of decellularization, enzymatic digestion and pulverization. Physicochemical properties of dg-CAM were compared with that of native cartilage tissue (NCT). Cellular interactions between human umbilical vein endothelial cells (HUVECs) and dg-CAM was evaluated with proliferation, migration and tube formation assays compared with that of type I collagen (COL) and bevacizumab, an anti-angiogenic drug. We then investigated the therapeutic potential of topical administration of dg-CAM suspension on the experimentally induced rabbit corneal NV model. RESULTS: The dg-CAM released a significantly larger amount of soluble proteins than that of the NCT and showed an improved hydrophilic and dispersion properties. In contrast, the dg-CAM contained a large amount of collagen, glycosaminoglycans and anti-angiogenic molecules as much as the NCT. The inhibitory effect on NV of the dg-CAM was more prominent than that of COL and even comparable to that of bevacizumab in inhibiting the HUVECs. The therapeutic potential of the dg-CAM was comparable to that of bevacizumab in the rabbit corneal NV model by efficiently inhibiting neovessel formation of the injured cornea. CONCLUSION: The current study developed a dg-CAM having anti-angiogenic properties, together with water-dispersible properties suitable for topical or minimally invasive application for prevention of vessel invasion.

The aim of this study was to develop a fetal cartilage-derived progenitor cell (FCPC) based cartilage gel through self-assembly for cartilage repair surgery, with clinically useful properties including adhesiveness, plasticity, and continued chondrogenic remodeling after transplantation. Characterization of the gels according to in vitro self-assembly period resulted in increased chondrogenic features over time. Adhesion strength of the cartilage gels were significantly higher compared to alginate gel, with the 2-wk group showing a near 20-fold higher strength (1.8 ± 0.15 kPa vs. 0.09 ± 0.01 kPa, p in vivo remodeling process analysis of the 2 wk cultured gels showed increased cartilage repair characteristics and stiffness over time, with higher integration-failure stress compared to osteochondral autograft controls at 4 weeks (p p

Osteoarthritis (OA) is an incurable joint disease affecting 240 million elderly population, and major unmet medical needs exist for better therapeutic options for OA. During skeletal development, Nkx3.2 has been shown to promote chondrocyte differentiation and survival, but to suppress cartilage hypertrophy and blood vessel invasion. Here we show that Nkx3.2 plays a key role in osteoarthritis (OA) pathogenesis. Marked reduction of Nkx3.2 expression was observed in three different murine OA models. Consistent with these findings, analyses of surgery-induced and age-driven OA models revealed that cartilage-specific post-natal induction of Nkx3.2 can suppress OA progression in mice. These results suggest that Nkx3.2 may serve as a promising target for OA drug development.

Defects in bone are some of the most difficult injuries to treat. Biomimetic scaffolds represent a promising approach for successful bone tissue regeneration. In this study, a three-dimensional (3D) scaffold with osteo-inductive functionality was designed and assayed both in-vitro and in-vivo. Bone formation peptide-1 (BFP1), an osteo-promoting specific peptide, was covalently bound to a 3D printed polycaprolactone (PCL) scaffold using polydopamine (DOPA). The amount of BFP1 immobilized on the surface was found to increase depending on the BFP1 concentration of the loading solution. To observe the biological effects of the 3D scaffolds, human tonsil-derived mesenchymal stem cells (hTMSCs) were isolated. The cells were cultured on the scaffolds and observed to rapidly differentiate into osteoblast-like cells with osteo-promoting capabilities. The scaffolds were implanted in a rabbit calvarial defect model for 8 weeks and successfully stimulated both vessel and bone regeneration. Osteo-promoting 3D scaffolds may provide a safer and more efficient approach for bone repair and remodelling in regenerative medicine.

BACKGROUND: Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. METHODS: The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2 days and 7 days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. RESULTS: The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. CONCLUSION: These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00317-w) contains supplementary material, which is available to authorized users.

Decellularized extra-cellular matrix (ECM) has been studied as an alternative to anti-adhesive biomaterials and cartilage acellular matrix (CAM) has been shown to inhibit postoperative adhesion in several organs. This study aimed to evaluate the suitability of glutaraldehyde (GA) crosslinked CAM-films as anti-adhesion barriers for peripheral nerve injury. The films were successfully fabricated and showed improved physical properties such as mechanical strength, swelling ratio, and lengthened degradation period while maintaining the microstructure and chemical composition after GA crosslinking. In the in vitro study of CAM-film, the dsDNA content met the recommended limit of decellularization and more than 70% of the major ECM components were preserved after decellularization. The adhesion and proliferation of seeded human umbilical vein endothelial cells and fibroblasts were significantly lower in CAM-film than in control, but similar with Seprafilm. However, the CAM-film extract did not show cytotoxicity. In the in vivo study, the peri-neural fibrosis was thicker, adhesion score higher, and peri-neural collagen fibers more abundant in the control group than in the CAM-film group. The total number of myelinated axons was significantly higher in the CAM-film group than in the control group. The inflammatory marker decreased with time in the CAM-film group compared to that in the control group, whereas the nerve regenerative marker expression was maintained. Moreover, the ankle angles at contracture and toe-off were higher in the CAM film-treated rats than in the control rats. GA-crosslinked CAM films may be used during peripheral nerve surgery to prevent peri-neural adhesion and enhance nerve functional recovery.

To protect unwanted tissue adhesions occurring after surgeries, we aimed to fabricate an anti-adhesive film using cartilage acellular matrix (CAM) with anti-vascular inhibition activity. Additionally, to fabricate anti-adhesive films with tunable swelling, mechanical, and biodegradation properties, a biodegradable polyester (PEP) with N-hydroxysuccinimide (NHS) in the chain end position was synthesized as a cross-linker. CAM/PEP (CP) films were prepared with various CAM: PEP ratios in the wide size with repeatable reproducibility, and then, cross-linked CP (Cx-CP) were obtained by the interpenetrating cross-linking reaction between the amine group on CAM and the NHS group on PEP cross-linkers under thermal treatment. The biodegradation, wettability, swelling, and mechanical properties of the prepared anti-adhesive Cx-CP films were controlled by varying the CAM:PEP ratio. The degradation half-life, contact angle, elastic moduli and toughness of Cx-CP films increased according to the increasing PEP content. Additionally, Cx-CP films significantly inhibits the attachment and proliferation of HUVECs. Cx-CP film prepared by varying the CAM:PEP ratio can be tailored to meet individual requirements for in vivo injured tissues. In animal experiments, anti-adhesive Cx-CP films implanted between the peritoneal wall and the cecum significantly suppressed tissue adhesion between them. Additionally, good adhesion effect observed at anti-adhesive film maintained for proper time period at injured tissues. Taken together, in this work, we successfully achieved strategy for the development of anti-adhesive barrier with tunable swelling, mechanical, and biodegradation properties.

This study introduces an implantable scaffold-free cartilage tissue construct (SF) that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16-week old) and 3 week-cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold-free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold-free engineered cartilage. When implanting tissue-engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.

Cartilage extracellular matrix contains antiadhesive and antiangiogenic molecules such as chondromodulin-1, thrombospondin-1, and endostatin. We have aimed to develop a cross-linked cartilage acellular matrix (CAM) barrier for peritendinous adhesion prevention. CAM film was fabricated using decellularized porcine cartilage tissue powder and chemical cross-linking. Biochemical analysis of the film showed retention of collagen and glycosaminoglycans after the fabrication process. Physical characterization of the film showed denser collagen microstructure, increased water contact angle, and higher tensile strength after cross-linking. The degradation time in vivo was 14 d after cross-linking. The film extract and film surface showed similar cell proliferation, while inhibiting cell migration and cell adhesion compared to standard media and culture plate, respectively. Application of the film after repair resulted in similar tendon healing and significantly less peritendinous adhesions in a rabbit Achilles tendon injury model compared to repair only group, demonstrated by histology, ultrasonography, and biomechanical testing. In conclusion, the current study developed a CAM film having biological properties of antiadhesion, together with biomechanical properties and degradation profile suitable for prevention of peritendinous adhesions.

The use of chemoattractants to promote endogenous stem cell-based in situ tissue regeneration has recently garnered much attention. This study is the first to assess the endogenous stem cell migration using a newly discovered substance P (SP) analog (SP1) by molecular dynamics simulations as an efficient chemoattractant. Further, a novel strategy based on electrostatic interaction using cationic chitosan (Ch) and anionic hyaluronic acid (HA) to prepare an SP1-loaded injectable C/H formulation without SP1 loss is developed. The formulation quickly forms an SP1-loaded C/H hydrogel in situ through in vivo injection. The newly discovered SP1 is found to possess human mesenchymal stromal cells (hMSCs) migration-inducing ability that is approximately two to three times higher than that of the existing SP. The designed VEGF-mimicking peptide (VP) chemically reacts with the hydrogel (C/H-VP) to sustain the release of VP, thus inducing vasculogenic differentiation of the hMSCs that migrate toward the C/H-VP hydrogel. Similarly, in animal experiments, SP1 attracts a large number of hMSCs toward the C/H-VP hydrogel, after which VP induces vasculogenic differentiation. Collectively, these findings indicate that SP1-loaded C/H-VP hydrogels are a promising strategy to facilitate endogenous stem cell-based in situ tissue regeneration.

Meniscus is a fibrocartilage composite tissue with three different microstructual zones, inner fibrocartilage, middle transitional, and outer fibrous zone. We hypothesized that decellularized meniscus extracellular matrix (DMECM) would have different characteristics according to zone of origin. We aimed to compare zone-specific DMECM in terms of biochemical characteristics and cellular interactions associated with tissue engineering. Micronized DMECM was fabricated from porcine meniscus divided into three microstructural zones. Characterization of DMECM was done by biochemical and proteomic analysis. Inner DMECM showed the highest glycosaminoglycan content, while middle DMECM showed the highest collagen content among groups. Proteomic analysis showed significant differences among DMECM groups. Inner DMECM showed better adhesion and migration potential to meniscus cells compared to other groups. DMECM resulted in expression of zone-specific differentiation markers when co-cultured with synovial mesenchymal stem cells (SMSCs). SMSCs combined with inner DMECM showed the highest glycosaminoglycan in vivo. Outer DMECM constructs, on the other hand, showed more fibrous tissue features, while middle DMECM constructs showed both inner and outer zone characteristics. In conclusion, DMECM showed different characteristics according to microstructural zones, and such material may be useful for zone-specific tissue engineering of meniscus.

In this work, we prepared an electrospun small intestinal submucosa/poly(e-caprolactone)-ran-poly(l-lactide) (SIS/PCLA) sheet onto which substance P (SP) was loaded, and this was employed as a cell-free scaffold for wound healing through the mobilization of human mesenchymal stem cells (hMSCs). SP release from the SP-loaded scaffold was 42% at 12 h and 51% at 24 h due to an initial burst of SP, but after 1 day, it exhibited a linear release profile and was released at a sustained rate for 21 days. The SP-loaded SIS/PCLA sheet exhibited higher in vitro and in vivo hMSC migration than did the PCLA and SIS/PCLA sheets. Large hMSCs injected into the tail vein of mice models migrated towards the wound to a greater extent in the presence of the SP-loaded SIS/PCLA sheet than with the PCLA and SIS/PCLA sheets, as confirmed by the CD44 and CD29 markers of recruited hMSCs. In animal wound models, significantly higher wound contraction (∼97%) in the group treated with the SP-loaded SIS/PCLA sheet was observed compared with the PCLA (∼74%) and SIS/PCLA (∼84%) groups at 3 weeks. In addition, SP-loaded SIS/PCLA-treated animals showed significant epidermal regeneration and collagen density (56%) in the mature granulation tissue at 3 weeks compared to the PCLA and SIS/PCLA groups. The wound area after SP-loaded SIS/PCLA sheet treatment also showed high blood vessel formation at the early stage, resulting in enhanced wound healing. Furthermore, the SP-loaded SIS/PCLA group exhibited a lower macrophage count (2.9%) than did the PCLA (7.7%) and SIS/PCLA (3.4%) groups. It was thus confirmed that the use of SP-loaded SIS/PCLA sheet as a cell-free scaffold could effectively enhance wound healing through MSC recruitment.

BACKGROUND: To regenerate tissue-engineered cartilage as a source of material for the restoration of cartilage defects, we used a human fetal cartilage progenitor cell pellet to improve chondrogenesis and modulation of the immune response in an in vivo bioreactor (IVB) system. METHODS: IVB was buried subcutaneously in the host and then implanted into a cartilage defect. The IVB was composed of a silicone tube and a cellulose nano pore-sized membrane. First, fetal cartilage progenitor cell pellets were cultured in vitro for 3 days, then cultured in vitro, subcutaneously, and in an IVB for 3 weeks. First, the components and liquidity of IVB fluid were evaluated, then the chondrogenesis and immunogenicity of the pellets were evaluated using gross observation, cell viability assays, histology, biochemical analysis, RT-PCR, and Western blots. Finally, cartilage repair and synovial inflammation were evaluated histologically. RESULTS: The fluid color and transparency of the IVB were similar to synovial fluid (SF) and the components were closer to SF than serum. The IVB system not only promoted the synthesis of cartilage matrix and maintained the cartilage phenotype, it also delayed calcification compared to the subcutaneously implanted pellets. CONCLUSION: The IVB adopted to study cell differentiation was effective in preventing host immune rejection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-019-00236-5) contains supplementary material, which is available to authorized users.

The aim of this study was to design a click-crosslinked hyaluronic acid (HA) (Cx-HA) depot via a click crosslinking reaction between tetrazine-modified HA and trans-cyclooctene-modified HA for direct intra-articular injection into joints affected by rheumatoid arthritis (RA). The Cx-HA depot had significantly more hydrogel-like features and a longer in vivo residence time than the HA depot. Methotrexate (MTX)-loaded Cx-HA (MTX-Cx-HA)-easily prepared as an injectable formulation-quickly formed an MTX-Cx-HA depot that persisted at the injection site for an extended period. In vivo MTX biodistribution in MTX-Cx-HA depots showed that a high concentration of MTX persisted at the intra-articular injection site for an extended period, with little distribution of MTX to normal tissues. In contrast, direct intra-articular injection of MTX alone or MTX-HA resulted in rapid clearance from the injection site. After intra-articular injection of MTX-Cx-HA into rats with RA, we noted the most significant RA reversal, measured by an articular index score, increased cartilage thickness, extensive generation of chondrocytes and glycosaminoglycan deposits, extensive new bone formation in the RA region, and suppression of tumor necrosis factor-α or interleukin-6 expression. Therefore, MTX-Cx-HA injected intra-articularly persists at the joint site in therapeutic MTX concentrations for an extended period, thus increasing the duration of RA treatment, resulting in an improved relief of RA.

Here, we describe combinational chemotherapy via intratumoral injection of doxorubicin (Dox) and 5-fluorouracil (Fu) to enhance the efficacy and reduce the toxicity of systemically administered Fu and Dox in cancer patients. As the key concept in this work, mixture formulations of Dox-loaded microcapsules (Dox-M) and Fu-loaded Pluronic(®) hydrogels (Fu-HP) or Fu-loaded diblock copolymer hydrogels (Fu-HC) have been employed as drug depots. The in vitro and in vivo drug depot was designed as a formulation of Dox-M dispersed inside an outer shell of Fu-HP or Fu-HC after injection. The Dox-M/Fu-HP and Dox-M/Fu-HC formulations are free flowing at room temperature, indicating injectability, and formed a structural gelatinous depot in vitro and in vivo at body temperature. The Fu-HP, Fu-HC, Dox-M/Fu-HP, Dox-M/Fu-HC, and Dox-M formulations were easily injected into tumor centers in mice using a needle. Dox-M/Fu-HC produced more significant inhibitory effects against tumor growth than that by Dox-M/Fu-HP, while Fu-HP, Fu-HC and Dox-M had the weakest inhibitory effects of the tested treatments. The in vivo study of Dox and Fu biodistribution showed that high Dox and Fu concentrations were maintained in the target tumor only, while distribution to normal tissues was not observed, indicating that Dox and Fu concentrations below their toxic plasma concentrations should not cause significant systemic toxicity. The Dox-M/Fu-HP and Dox-M/Fu-HC drug depots described in this work showed excellent performance as chemotherapeutic delivery systems. The results reported here indicate that intratumoral injection using combination chemotherapy with Dox-M/Fu-HP or Dox-M/Fu-HC could be of translational research by enhancing the synergistic inhibitory effects of Dox and Fu on tumor growth, while reducing their systemic toxicity in cancer patients.

We have designed and characterized an injectable, electrostatically bonded, in situ–forming hydrogel system consisting of a cationic polyelectrolyte [(methoxy)polyethylene glycol-b-(poly(e-caprolactone)-ran-poly(L-lactic acid)] (MP) copolymer derivatized with an amine group (MP-NH2) and anionic BMP2. To the best of our knowledge, there have been hardly any studies that have investigated electrostatically bonded, in situ–forming hydrogel systems consisting of MP-NH2 and BMP2, with respect to how they promote in vivo osteogenic differentiation of human turbinate mesenchymal stem cells (hTMSCs). Injectable formulations almost immediately formed an electrostatically loaded hydrogel depot containing BMP2, upon injection into mice. The hydrogel features and stability of BMP2 inside the hydrogel were significantly affected by the electrostatic attraction between BMP2 and MP-NH2. Additionally, the time BMP2 spent inside the hydrogel depot was prolonged in vivo, as evidenced by in vivo near-infrared fluorescence imaging. Biocompatibility was demonstrated by the fact that hTMSCs survived in vivo, even after 8 weeks and even though relatively few macrophages were in the hydrogel depot. The osteogenic capacity of the electrostatically loaded hydrogel implants containing BMP2 was higher than that of a hydrogel that was simply loaded with BMP2, as evidenced by Alizarin Red S, von Kossa, and hematoxylin and eosin staining as well as osteonectin, osteopontin, osteocalcin, and type 1α collagen mRNA expression. The results confirmed that our injectable, in situ–forming hydrogel system, electrostatically loaded with BMP2, can enhance in vivo osteogenic differentiation of hTMSCs.

Hongsik Cho,1,2,* Byoung Ju Kim,3,4,* Sang-Hyug Park,5 Karen A Hasty,1,2 Byoung-Hyun Min3,4,6 1Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center–Campbell Clinic, 2Veterans Affairs Medical Center, Memphis, TN, USA; 3Department of Molecular Science and Technology, Ajou University, 4Cell Therapy Center, Ajou University Hospital, Suwon, 5Department of Biomedical Engineering, Pukyong National University, Nam-Gu, Busan, 6Department of Orthopedic Surgery, School of Medicine, Ajou University, Suwon, Republic of Korea *These authors contributed equally to this work Background: Early stage osteoarthritis (OA) is clinically asymptomatic due to the avascular and the aneural nature of the cartilage tissue. Nevertheless, early detection of cartilage tissue is critical in order to impede the progression of OA. Hence, in order to develop effective preventive therapy for OA, diagnosis in the early stages is necessary.Methods: To achieve this goal, we have developed targeted, fluorescent nanosomes conjugated with monoclonal anti-type II collagen antibodies (MabCII) for diagnosis of early OA. The MabCII-coated nanosomes (targeted-nanosomes) bind to the damaged cartilage explants in vitro and in vivo in an OA mouse model that mimics early stage OA. The OA mouse model was induced by destabilization of the medial meniscus (DMM) in 9-10 weeks old C57Bl/6 mice.Results: The targeted-nanosomes enhanced the binding specificity to the cartilage tissue according to the severity of damage.Conclusion: We show that MabCII-nanosomes can precisely detect early stage OA in the DMM mouse model. Thus, MabCII-nanosomes have the potential to be used as a non-invasive method for diagnosing the early osteoarthritic lesions. Keywords: osteoarthritis, nanosome, diagnosis, OA score, destabilization of the medial meniscus, matrix metalloproteinases, monoclonal anti-type II collagen antibody&nbsp

In this work, we chose cartilage acellular matrix (CAM) as a promising antiadhesive material because CAM effectively inhibits the formation of blood vessels, and we used electrospinning to prepare antiadhesive barriers. Additionally, we synthesized N-hydroxysuccinimide (NHS)-poly(caprolactone-co-lactide-co-glycolide)-NHS (MP) copolymers (to tune degradation) as a cross-linking agent for CAM. This is the first report on the development of electrospun cross-linked (Cx) CAM/MP (CA/P) nanofiber (NF) (Cx-CA/P-NF) with a tunable degradation period as an antiadhesive barrier. Compared with the CA/P-NF before cross-linking, the electrospun Cx-CA/P-NF after cross-linking showed different biodegradation. Cx-CA/P-NF significantly inhibited the in vitro attachment and proliferation of human umbilical vein endothelial cells (HUVECs), as confirmed by an MTT assay and scanning electron microscopy images. Cx-CA/P-NFs implanted between a surgically damaged peritoneal wall and cecum gradually degraded in 7 days; this process was monitored by NIR imaging. The in vivo evaluation of the anti–tissue adhesive effect of Cx-CA/P-NFs revealed little adhesion, few blood vessels, and negligible inflammation at 7 days determined by hematoxylin and eosin staining. ED1 staining of Cx-CA/P-NFs showed infiltration of few macrophages because of the inflammatory response to the Cx-CA/P-NF as compared with an untreated injury model. Additionally, Cx-CA/P-NFs significantly suppressed the formation of blood vessels between the peritoneal wall and cecum, according to CD31 staining. Overall, Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Thus, it is reasonable to conclude that the Cx-CA/P-NF designed herein successfully works as an antiadhesive barrier with a tunable degradation period. Statement of Significance The cartilage acellular matrix (CAM) can inhibit the formation of fibrous tissue bridges and blood vessels between the tissue at an injured site and the surrounding healthy tissues. However, CAM has not been rigorously investigated as an antiadhesive barrier. In this manuscript, the cross-linked CAM nanofiber (Cx-CA/P-NF) designed herein successfully works as an antiadhesive barrier. Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Moreover, we demonstrated the suitable properties of Cx-CA/P-NF such as easy cross-linking by maintaining the antiadhesive properties, controllable biodegradation, and in vivo antiadhesive effect of Cx-CA/P-NF.

Cartilage tissue engineering typically involves the combination of a biodegradable polymeric support material with chondrocytes. The culture environment in which cell-material constructs are created and stored is an important factor. The aim of the present study was to investigate the effects of combined stimuli on cartilage zonal organization which is important to maintain cartilage functions such as lubrication and cushion. For that purpose, we developed a joint mimicking loading system which was composed of compression and shear stress. To mimic the joint loading condition, we manufactured a stimuli system that has a device similar to the shape of a femoral condyle in human knee. The fibrin/hyaluronic acid mixture with chondrocytes were dropped into support made of silicon, and placed under the device. The cartilage explants were stimulated with the joint mimicking loading system for 1 hour per day over the course of 4 weeks. The amounts of GAG and collagen in the stimulated tissue were more than that of the static cultured tissue. Cells and collagen were arranged horizontally paralleled to the surface by stimuli, while it did not happen in the control group. The results of this study suggests that mechanical load exerting in the joint play a crucial role in stimulation of extracellular matrix (ECM) production as well as its functional rearrangement.

Mastoidectomy, the removal of infected mastoid bones, is a common surgical procedure for the treatment of chronic otitis media. Persistent and recurrent otorrhea and accumulation of keratin debris following open cavity mastoidectomy are still bothersome issues for both patients and otologists. In this study, we used human ear adipose-derived stromal cells (hEASCs) in combination with polycaprolactone (PCL) scaffolds and osteogenic differentiation medium (ODM) to regenerate temporal bone defects. The hEASCs showed stem cell phenotypes, and these characteristics were maintained up to passage 5. Mastoid bulla and cranial bone defects were induced in Sprague-Dawley rats using AgNO

Recently, computer-designed three-dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer-designed 3D scaffold to drive new bone formation in a bone defect. Poly-L-lactide (PLLA) and bioactive β-tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer-by-layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG-63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full-thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG-63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG-63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG-63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.

Objectives Tracheal reconstruction with tissue-engineering technique has come into the limelight in the realm of head and neck surgery. We intended to evaluate the plausibility of allogenic chondrocytes cultured with porcine cartilage-derived substance (PCS) scaffold for partial tracheal defect reconstruction. Methods Powder made from crushed and decellularized porcine articular cartilage was formed as 5 mm × 12 mm (height × diameter) scaffold. Chondrocytes from rabbit articular cartilage were expanded and cultured with PCS scaffold. After 7 weeks culture, the scaffolds were implanted on a 5 mm × 10 mm artificial tracheal defect in six rabbits. Two, four and eight weeks postoperatively, the sites were evaluated endoscopically, radiologically, histologically and functionally. Results None of the six rabbits showed any sign of respiratory distress. Endoscopic examination did not show any collapse or blockage of the reconstructed trachea and the defects were completely covered with regenerated respiratory epithelium. Computed tomography showed good luminal contour of trachea. Postoperative histologic data showed that the implanted chondrocyte successfully formed neo-cartilage with minimal inflammatory response and granulation tissue. Ciliary beat frequency of regenerated epithelium was similar to those of normal adjacent mucosa. Conclusions The shape and function of reconstructed trachea using allogenic chondrocytes cultured with PCS scaffold was restored successfully without any graft rejection.

The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. To increase the efficacy and reduce the toxicity of systemically administered anticancer 5-fluorouracil (5-Fu) treatments in patients, intratumoral administration of an injectable hydrogel has been evaluated in the current work. The MPEG-b-(PCL-ran-PLLA) diblock copolymer (MCL) containing 5-Fu existed in an emulsion-sol state at room temperature and rapidly gelled in vivo at the body temperature. MCL acted as in vivo biodegradable drug depot over a defined experimental period. A single injection of 5-Fu-loaded MCL solution resulted in significant suppression of tumor growth, compared with repeated injection of free 5-Fu as well as saline and MCL alone. For both repeated injections of free 5-Fu and single injection of 5-Fu-loaded MCL, most of the 5-Fu was found in the tumor, indicating the maintenance of therapeutic concentrations of 5-Fu within the target tumor tissue and the prevention of systemic toxicity associated with 5-Fu in healthy normal tissues. In conclusion, this work demonstrated that intratumoral injection of 5-Fu-loaded MCL may induce significant suppression of tumor growth through effective accumulation of 5-Fu in the tumor.

Summary Objective To investigate whether granulocyte macrophage-colony stimulating factor (GM-CSF) can be used to increase the number of mesenchymal stem cells (MSCs) in blood clots formed by microfracture arthroplasty (MFX) and whether it can improve the therapeutic outcome for cartilage repair. Methods Thirty-six New Zealand white rabbits were divided into four groups: (1) control, (2) GM-CSF, (3) MFX, and (4) GM-CSF + MFX. GM-CSF was administrated intravenously (IV) at 10 μg/kg body weight 20 min before the MFX surgery. The repaired tissues were retrieved and examined by histological observation, quantitative assessment, and biochemical assays at 4, 8, and 12 weeks after treatment. The number of MSCs was measured in the blood clots by the colony forming unit-fibroblast (CFU-F) assay. The kinetic profile and distribution of GM-CSF in vivo was also evaluated by near-Infrared (NIR) fluorescence imaging and enzyme-linked immune sorbent assay. Results In the histological observations and chemical assays examined at 4, 8, and 12 weeks, the MFX after GM-CSF administration showed better cartilage repair than the one without GM-CSF. The CFU-F assay showed a significantly larger amount of MSCs present in the blood clots of the GM-CSF + MFX group than in the blood clots of the other groups. The blood concentration of GM-CSF peaked at 10 min and decreased back to almost the initial level after a couple of hours. GM-CSF was distributed in many organs including the bone marrow but was not observed clearly in the joint cavity. Conclusion Intravenous administration of GM-CSF together with MFX could be a promising therapeutic protocol to enhance the repair of cartilage defects.

In this study, human dental pulp stem cells (hDPSCs) are examined as a cellular source for bone tissue engineering using an in vivo-forming hydrogel. The hDPSCs are easily harvested in large quantities from extracted teeth. The stemness of harvested hDPSCs indicates their relative tolerance to ex vivo manipulation in culture. The in vitro osteogenic differentiation of hDPSCs is characterized using Alizarin Red S (ARS), von Kossa (VK), and alkaline phosphatase (ALP) staining. The solution of hDPSCs and a methoxy polyethylene glycol-polycaprolactone block copolymer (PC) is easily prepared by simple mixing at room temperature and in no more than 10 s it forms in vivo hydrogels after subcutaneous injection into rats. In vivo osteogenic differentiation of hDPSCs in the in vivo-forming hydrogel is confirmed by micro-computed tomography (CT), histological staining, and gene expression. Micro-CT analysis shows evidence of significant tissue-engineered bone formation in hDPSCs-loaded hydrogel in the presence of osteogenic factors. Differentiated osteoblasts in in vivo-forming hydrogel are identified by ARS and VK staining and are found to exhibit characteristic expression of genes like osteonectin, osteopontin, and osteocalcin. In conclusion, hDPSCs embedded in an in vivo-forming hydrogel may provide benefits as a noninvasive formulation for bone tissue engineering applications.

In this work, the in vivo biodegradation of, biocompatibility of, and host response to various topographic scaffolds were investigated. Randomly oriented fibrous poly(L-lactide) (PLLA) nanofibers were fabricated using the electrospinning technique. A PLLA scaffold was obtained by salt leaching. Both the electrospun PLLA nanofibers and the salt-leaching PLLA scaffolds formed three-dimensional pore structures. Cytotoxicity studies, in which rat muscle-derived stem cells (rMDSCs) were grown on electrospun PLLA nanofibers or the salt-leaching PLLA scaffolds, revealed that the rMDSCs cell count on the PLLA nanofibers was slightly higher than that on the salt-leaching PLLA scaffolds. An in vivo study was carried out by implanting the scaffolds subcutaneously into rats to test the biodegradation, biocompatibility, and host response at regular intervals over 0–4 weeks. The degradation of the PLLA nanofibers 1, 2, and 4 weeks after initial implantation was more extensive than that observed for the salt-leaching PLLA scaffolds. PLLA nanofibers seeded the growth of larger fibrous tissue masses due to in vivo cellular infiltration into the randomly oriented fibrillar structures of the PLLA nanofibers. In addition, the inflammatory cell accumulation in PLLA nanofibers was lower than that in the salt-leaching PLLA scaffolds. These results indicate that the electrospun PLLA nanofibers may serve as a good scaffold to elicit fibrous cellular infiltration, to minimize host response, and to enhance tissue–scaffold integration. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Scaffolds composed of synthetic, natural, and hybrid materials have been investigated as options to restore intervertebral disk (IVD) tissue function. These systems fall short of the lamellar features of the native annulus fibrosus (AF) tissue or focus only on the nucleus pulposus (NP) tissue. However, successful regeneration of the entire IVD requires a combination approach to restore functions of both the AF and NP. To address this need, a biphasic biomaterial structure was generated by using silk protein for the AF and fibrin/hyaluronic acid (HA) gels for the NP. Two cell types, porcine AF cells and chondrocytes, were utilized. For the AF tissue, two types of scaffold morphologies, lamellar and porous, were studied with the porous system serving as a control. Toroidal scaffolds formed out of the lamellar, and porous silk materials were used to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm, and a height of 3 mm (the interlamellar distance in the lamellar scaffold was 150-250 μm, and the average pore sizes in the porous scaffolds were 100-250 μm). The scaffolds were seeded with porcine AF cells to form AF tissue, whereas porcine chondrocytes were encapsulated in fibrin/HA hydrogels for the NP tissue and embedded in the center of the toroidal disk. Histology, biochemical assays, and gene expression indicated that the lamellar scaffolds supported AF-like tissue over 2 weeks. Porcine chondrocytes formed the NP phenotype within the hydrogel after 4 weeks of culture with the AF tissue that had been previously cultured for 2 weeks, for a total of 6 weeks of cultivation. This biphasic scaffold simulating in combination of both AF and NP tissues was effective in the formation of the total IVD in vitro.

We have developed a method to detect cartilage oligomeric matrix protein (COMP) as a specific biomarker of osteoarthritis (OA). In pathological conditions of the cartilage, COMP is released first into the synovial fluid (SF) and from there into the blood. Thus, measurement of COMP in the blood and SF facilitates OA diagnosis. To determine COMP, we developed a fluoro-microbead guiding chip (FMGC)-based immunoassay. The FMGC has four immunoreactive regions, each with five patterns, to allow multiple assays. A COMP-specific capture antibody was immobilized to the FMGC surface to create a self-assembled interfacial layer. SF or serum samples from patients with OA possessing the target COMP were applied to the COMP-sensing monolayer. To generate binding signal, COMP detection antibody-conjugated fluoro-microbeads were applied and the numbers of fluoro-microbeads bound specifically were counted to determine COMP concentrations. This FMGC-based immunoassay clearly distinguished immunospecific from nonspecific binding by comparing optical signals from inside and outside of the patterns. The optical signals showed linear correlations with serum and SF COMP concentrations. Optical detection and quantification of COMP using fluorescence microscopy correlated well with results from commercial enzyme-linked immunosorbent assay (ELISA). This FMGC-based immunoassay offers a new approach for detecting a clinically relevant biomarker for OA in human blood and SF.

Background: For the treatment of traumatic brain edema, an efficient modality has not yet emerged. There have been many studies to date which have reported the employment of low-frequency ultrasound for blood-brain barrier disruption (BBBD). However, the authors have observed that low-intensity ultrasound increases water permeability without cellular damage in cartilage cells. We have therefore attempted to observe the effects of applying this low-intensity ultrasound to an experimental animal model. Methods: A traumatic brain injury rat model was established according to the weight drop method developing the traumatic brain edema. The degree of BBBD was measured by the changes in the water content and spectrophotometric absorbance of Evans blue dye in the cerebrum after low-frequency ultrasound. Results: The cerebral water content levels showed that the BBBD gradually increased after impact and thereafter decreased after 6 h. After low-frequency ultrasound exposure, the values of water content and spectrophotometric absorbance of Evans blue dye were the lowest at 0 h, and were increased at 2 and 5 h of ultrasonic exposure (after impact). Conclusion: We suggest that traumatic brain edema in the rat model may be alleviated by low-frequency ultrasound, and low-frequency ultrasound might be proposed as a novel treatment modality for brain edema.

We present a non-invasive fluorescence method for imaging of scaffold degradation in vivo by quantifying the degradation of porcine cartilage-derived extracellular matrix powder (PCP).Three-dimensional porous scaffolds should be biocompatible and bioresorbable, with a controllable degradation and resorption rate to match tissue growth. However, in vivo scaffold degradation and tissue ingrowth processes are not yet fully understood. Unfortunately, current analysis methods require animal sacrifice and scaffold destruction for the quantification of scaffold degradation and cannot monitor the situation in real time. In this study, Cy3, a fluorescent dye, was used for visualizing PCP and a real-time degradation profile was obtained quantitatively by a non-invasive method using an imaging system in which the reduction in fluorescence intensity depended on PCP scaffold degradation. Real-time PCP scaffold degradation was confirmed through changes in the volume and morphology of the scaffold using micro-computed tomography and microscopy. Our results suggest that extracellular matrix degradation was induced by collagen degradation because of the binding between Cy3 and collagen. This non-invasive real-time monitoring system for scaffold degradation will increase our understanding of in vivo matrix and/or scaffold degradation.

Scaffold designs are critical for in vitro culture of tissue-engineered cartilage in three-dimensional environments to enhance cellular differentiation for tissue engineering and regenerative medicine. In the present study we demonstrated silk and fibrin/hyaluronic acid (HA) composite gels as scaffolds for nucleus pulposus (NP) cartilage formation, providing both biochemical support for NP outcomes as well as fostering the retention of size of the scaffold during culture due to the combined features of the two proteins. Passage two (P2) human chondrocytes cultured in 10% serum were encapsulated within silk-fibrin/HA gels. Five study groups with fibrin/HA gel culture (F/H) along with varying silk concentrations (2% silk gel only, fibrin/HA gel culture with 1% silk [F/H+1S], 1.5% silk [F/H+1.5S], and 2% silk [F/H+2S]) were cultured in serum-free chondrogenic defined media (CDM) for 4 weeks. Histological examination with alcian blue showed a defined chondrogenic area at 1 week in all groups that widened homogenously until 4 weeks. In particular, chondrogenic differentiation observed in the F/H+1.5S had no reduction in size throughout the culture period. The results of biochemical and molecular biological evaluations supported observations made during histological examination. Mechanical strength measurements showed that the silk mixed gels provided stronger mechanical properties for NP tissue than fibrin/HA composite gels in CDM. This effect could potentially be useful in the study of in vitro NP tissue engineering as well as for clinical implications for NP tissue regeneration.

Cartilage tissue engineering using cells and biocompatible scaffolds has emerged as a promising approach to repair of cartilage damage. To date, however, no engineered cartilage has proven to be equivalent to native cartilage in terms of biochemical and compression properties, as well as histological features. An alternative strategy for cartilage engineering is to focus on the in vivo regeneration potential of immature engineered cartilage. Here, we used a rabbit model to evaluate the extent to which the maturity of engineered cartilage influenced the remodeling and integration of implanted extracellular matrix scaffolds containing allogenous chondrocytes. Full-thickness osteochondral defects were created in the trochlear groove of New Zealand white rabbits. Left knee defects were left untreated as a control (group 1), and right knee defects were implanted with tissue-engineered cartilage cultured in vitro for 2 days (group 2), 2 weeks (group 3), or 4 weeks (group 4). Histological, chemical, and compression assays of engineered cartilage in vitro showed that biochemical composition became more cartilagenous, and biomechanical property for compression gradually increased with culture time. In an in vivo study, gross imaging and histological observation at 1 and 3 months after implanting in vitro-cultured engineered cartilage showed that defects in groups 3 and 4 were repaired with hyaline cartilage-like tissue, whereas defects were only partially filled with fibrocartilage after 1 month in groups 1 and 2. At 3 months, group 4 showed striking features of hyaline cartilage tissue, with a mature matrix and a columnar arrangement of chondrocytes. Zonal distribution of type II collagen was most prominent, and the International Cartilage Repair Society score was also highest at this time. In addition, the subchondral bone was well ossified. In conclusion, in vivo engineered cartilage was remodeled when implanted; however, its extent to maturity varied with cultivation period. Our results showed that the more matured the engineered cartilage was, the better repaired the osteochondral defect was, highlighting the importance of the in vitro cultivation period.

Using a complete spinal cord transection model, the present study employed a combinatorial strategy comprising rat bone marrow stem cells (rBMSCs) and polymer scaffolds to regenerate neurological function after spinal cord injury (SCI) of different lengths. SCI models with completely transected lesions were prepared by surgical removal of 1 mm (SC1) or 3 mm (SC3) lengths of spinal cord in the eighth-to-ninth spinal vertebrae, a procedure that resulted in bilateral hindlimb paralysis. A cylindrical poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold 1 or 3 mm in length with or without rBMSCs was fitted into the completely transected lesion. Rats in SC1 and SC3 groups implanted with rBMSC-containing scaffolds received Basso-Beattie-Bresnahan scores for hindlimb locomotion of 15 and 8, respectively, compared with ∼3 for control rats in SC1-C and SC3-C groups implanted with scaffolds lacking rBMSCs. The amplitude of motor-evoked potentials recorded in the hindlimb area of the sensorimotor cortex after stimulation of the injured spinal cord averaged ∼100 μV in SC1-C and 10-50 μV in SC3-C groups at 4 weeks, and then declined to nearly zero at 8 weeks. In contrast, the amplitude of motor-evoked potentials increased from ∼300 to 350 μV between 4 and 8 weeks in SC1 rats and from ∼200 to ∼250 μV in SC3 rats. These results demonstrate functional recovery in rBMSC-transplanted rats, especially those with smaller defects. Immunohistochemically stained sections of the injury site showed clear evidence for axonal regeneration only in rBMSC-transplanted SC1 and SC3 models. In addition, rBMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating cell survival in SCI. Collectively, our results indicate that therapeutic rBMSCs in a poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold induced nerve regeneration in a complete spinal cord transection model and showed that functional recovery further depended on defect length.

Extracellular matrix (ECM) materials have diverse physiological functions by themselves and can also act as reservoirs of cytokines and growth factors, so that they can affect the cell phenotype, attachment, migration and proliferation of cells. In this study, an ECM scaffold derived from porcine cartilage was evaluated for whether it can support and maintain chondrogenesis of rabbit mesenchymal stem cells (rMSCs) in vitro and in the nude mouse model in vivo. The porcine ECM scaffold was compared to a polyglycolic acid (PGA) scaffold and an MSC pellet as a control group. In an in vitro environment until 4 weeks, the ECM scaffold evoked chondrogenic differentiation of rMSCs earlier and produced more cartilaginous tissues than the PGA scaffold. Next, rMSCs in each scaffold were preconditioned with chondrogenic media in vitro for 1 week and implanted in the backs of nude mice for 6 weeks. The initially formed cartilaginous tissues turned into bone matrix with time centripetally from the outside of the region as observed by Safranin-O and von Kossa stains. This phenomenon progressed much more rapidly in the PGA group than in the ECM group. In the ECM group, the chondrogenic phenotypes of rMSCs were also maintained longer than in the PGA group. The loss of chondrogenic phenotypes was accompanied by the calcification of matrix, and hypertrophic changes by immunohistochemistry for osteocalcin and collagen type I and X. Blood vessel invasion took place more deeply and intensively in the PGA group. These results suggest that the ECM scaffold not only strongly supports chondrogenic differentiation of rMSCs, but also helps maintain its phenotype in vivo. We speculate that the ECM scaffold provides rMSCs with a favorable, native cartilage-like environment and therefore can be a promising tool for cartilage tissue engineering.

In situ-forming gel systems have drawn increasing attention for their potential use in a variety of biomedical applications. Here, we examined an in situ-forming gel system comprised of MPEG- b -PCL and MPEG- b -(PCL- ran -PLLA) diblock copolymers with different PLLA contents (0–10 mol%) in the PCL segment. The crystalline region of the PCL- ran -PLLA segment decreased with increasing PLLA content. The MPEG- b -(PCL- ran -PLLA) diblock copolymer solutions were liquid at room temperature and only MPEG- b -(PCL- ran -PLLA) diblock copolymer solutions with a PLLA content ≤5 mol% in the PCL segment showed a sol-to-gel transition as the temperature was increased. The viscosity change associated with sol-to-gel phase transition depended on the PLLA content in the PCL segment. A MPEG- b -PCL diblock copolymer solution incubated in vitro showed increasing viscosity without degradation, whereas the viscosity of MPEG- b -(PCL- ran -PLLA) diblock copolymer solutions continuously and sharply decreased with increasing PLLA content in the PCL segment. As the amount of PLLA increased, the size of in vivo-formed MPEG- b -(PCL- ran -PLLA) gels after initial injection tended to gradually decrease because of hydrolytic degradation of the PLLA in the PCL- ran -PLLA segment. An immunohistochemical examination showed that in vivo MPEG- b -(PCL- ran -PLLA) diblock copolymer gels provoked only a modest inflammatory response. Collectively, our results show that the MPEG- b -(PCL- ran -PLLA) diblock copolymer gel described here could serve as a minimally invasive, therapeutic, in situ-forming gel system that offers an experimental window adjustable from a few weeks to a few months.

The scaffold-free cartilage fabrication system we have reported previously for human application with nonpassaged cells has a big limitation in securing the enough cell number. Therefore, autologous chondrocytes from small biopsy of cartilage tissue inevitably have to be expanded through multiple passages, which result in poor engineered cartilage in terms of quality and quantity. This study applied basic fibroblast growth factor (bFGF) to overcome the limitation and produce an engineered cartilage tissue with clinically applicable size and quality. Porcine articular chondrocytes were expanded until passage 2 in the absence or presence of 5 ng/mL bFGF, and then subjected to the two-stage scaffold-free cultures for 21 days again in the absence or presence of 5 ng/mL bFGF. The fabrication of cartilage tissues was evaluated along with time in comparison with the constructs from unpassaged chondrocytes. Expansion of chondrocytes in the presence of bFGF increased accumulation of cartilage extracellular matrices and resultantly fabricated the larger size of cartilage tissues in the subsequent stages, being comparable to those of unpassaged cells. In contrast, bFGF showed no positive, but adverse, effects on the cartilage tissue formation, when treated additionally during the scaffold-free fabrication stages. These results suggested that use of bFGF during the expansion stage of chondrocytes could overcome the limitation of previous two-stage scaffold-free cartilage fabrication system, and provided a novel three-stage system to construct a clinically applicable quality of cartilage tissues.

Numerous treatment methods have been applied for use in cartilage repair, including abrasion, drilling, and microfracture. Although chondrocyte transplantation is the preferred treatment, it has some shortcomings, such as difficulty of application (large and posterior condylar regions), poor chondrocyte distribution, and potential cell leakage from the defect region. The cell delivery system of the tissue engineering technique can be used to overcome these shortcomings. We chose fibrin/hyaluronan (HA) composite gel as an effective cell delivery system to resolve these issues. Both components are derived from natural extracellular matrix. In the first trial, fortified fibrin/HA composite gels with rabbit chondrocytes were tested by implantation in nude mice. At 4 weeks, glycosaminoglycan contents in the fibrin/HA composite (0.186 +/- 0.006 mg/mg) were significantly higher than those in the presence of fibrin alone (0.153 +/- 0.017 mg/mg). As a next step, we applied the fibrin/HA composite gel to animal cartilage defects using full thickness cartilage defect rabbit models. The fibrin/HA composite gel with rabbit chondrocytes (allogenic) was implanted into the experimental group, and the control group was implanted with the fibrin/HA composite gel alone. Implanted chondrocytes with the fibrin/HA composite showed improved cartilage formation. In conclusion, the key to successful regeneration of cartilage is to provide the repair site with a sufficient supply of chondrogenic cells with a suitable delivery vehicle to ensure maximal differentiation and deposition of the proper extracellular matrix. This study suggests the feasibility of tissue-engineered cartilage formation using fibrin/HA composite gel.

Object This study investigated the effects of granulocyte macrophage-colony stimulating factor (GM-CSF) on the scar formation and repair of spinal cord tissues in rat spinal cord injury (SCI) model. Methods Sprague–Dawley male rats (8 weeks old) were randomly divided into the sham-operated group, spinal cord injury group, and injury with GM-CSF treated group. A spinal cord injury was induced at T9/10 levels of rat spinal cord using a vascular clip. GM-CSF was administrated via intraperitoneal (IP) injection or on the dural surface using Gelfoam at the time of SCI. The morphological changes, tissue integrity, and scar formation were evaluated until 4 weeks after SCI using histological and immunohistochemical analyses. Results The administration of GM-CSF either via IP injection or local treatment significantly reduced the cavity size and glial scar formation at 3–4 weeks after SCI. GM-CSF also reduced the expression of core proteins of chondroitin sulfate proteoglycans (CSPGs) such as neurocan and NG2 but not phosphacan. In particular, an intensive expression of glial fibriallary acidic protein (GFAP) and neurocan found around the cavity at 4 weeks was obviously suppressed by GM-CSF. Immunostaining for neurofilament (NF) and Luxol fast blue (LFB) showed that GM-CSF preserved well the axonal arrangement and myelin structure after SCI. The expression of GAP-43, a marker of regenerating axons, also apparently increased in the rostral grey matter by GM-CSF. Conclusion These results suggest that GM-CSF could enhance long-term recovery from SCI by suppressing the glial scar formation and enhancing the integrity of axonal structure.

The purpose of this study is to evaluate the feasibility of human amniotic membrane (HAM) as a chondrocyte carrier by assessing cell proliferation and maintenance of phenotype in vitro and cartilage regeneration in vivo. Intact HAM was treated with 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA) for 15 min and the epithelial cells removed to make a denuded HAM. Rabbit articular chondrocytes were then seeded on three different HAM substrates: the epithelial side of intact HAM (IHE), basement side of denuded HAM (DHB), and stromal side of denuded HAM (DHS). These cell-substrate specimens were cultured for up to 4 weeks, and cell proliferation rate and phenotypic stability were examined at weeks 1 and 4. While chondrocytes grew in monolayer fashion on the surface of IHE and DHB substrates, the cells seeded in DHS penetrated and spread into the whole thickness of the stromal layer. The proliferating activity of chondrocytes in DHB was continuously up-regulated. A similar proliferating activity was observed in DHS in the first week, which remained stable for up to 4 weeks. The expression of type II collagen gradually increased with time in the DHS group, while it gradually decreased in the DHB group or was not detected at all in the IHE group. These results suggested that denuded HAM was able to support chondrocyte proliferation and maintenance of phenotype in vitro, seemingly more favorable when DHS was used. Based on this data, the DHS with chondrocytes was used to cover rabbit osteochondral defect with the stromal side facing in. The defect area was successfully regenerated with hyaline cartilage in the Safranin-O stain and International Cartilage Repair Society (ICRS) scoring after 8 weeks of implantation. In conclusion, our findings suggest that denuded HAM could be one of the ideal cell carrier matrices for cartilage regeneration.

Autologous mesenchymal stem cell (MSC) transplantation therapy for repair of myocardial injury has inherent limitations due to the poor viability of the stem cells after cell transplantation. Adhesion is a prerequisite for cell survival and also a key factor for the differentiation of MSCs. As a novel prosurvival modification strategy, we genetically engineered MSCs to overexpress tissue transglutaminase (tTG), with intention to enhance adhesion and ultimately cell survival after implantation. tTG-transfected MSCs (tTG-MSCs) showed a 2.7-fold and greater than a twofold increase of tTG expression and surface tTG activity, respectively, leading to a 20% increased adhesion of MSCs on fibronectin (Fn). Spreading and migration of tTG-MSCs were increased 4.75% and 2.52%, respectively. Adhesion of tTG-MSCs on cardiogel, a cardiac fibroblast-derived three-dimensional matrix, showed a 33.1% increase. Downregulation of tTG by transfection of small interfering RNA specific to the tTG resulted in markedly decreased adhesion and spread of MSCs on Fn or cardiogel. tTG-MSCs on Fn significantly increased phosphorylation of focal adhesion related kinases FAK, Src, and PI3K. tTG-MSCs showed significant retention in infarcted myocardium by forming a focal adhesion complex and developed into cardiac myocyte-like cells by the expression of cardiac-specific proteins. Transplantation of 1 × 106 MSCs transduced with tTG into the ischemic rat myocardium restored normalized systolic and diastolic cardiac function. tTG-MSCs further restored cardiac function of infarcted myocardium as compared with MSC transplantation alone. These findings suggested that tTG may play an important role in integrin-mediated adhesion of MSCs in implanted tissues.Disclosure of potential conflicts of interest is found at the end of this article.

The effectiveness of systemic rheumatoid arthritis (RA) treatments is limited by difficulties in achieving therapeutic doses within articular joints. We evaluated the ability of intra-articular administration of injectable formulations to synergistically enhance repair of RA joints. Methotrexate-loaded hyaluronic acid (Met-HA), dexamethasone-loaded microcapsules (Dex-M) and Dex-M dispersed inside Met-HA were prepared as viscous emulsions and injected into articular joints using a needle to form a drug depot. By near-infrared (NIR) fluorescence imaging, we confirmed the local release of NIR from the depot injected into the articular joint over an extended period. In comparison with the subjects treated with Met-HA or Dex-M alone, subjects treated simultaneously with Met-HA and Dex-M exhibited faster and more significant RA repair. Collectively, these results indicated that the drug depot formed after intra-articular injection of Met-HA/Dex-M induced long-lasting drug release and allowed Met and Dex to effectively act in the articular joint, resulting in enhanced RA repair.

Fetal cartilage-derived progenitor cells (FCPCs) could be a useful cell source in cell-based therapies for cartilage disorders. However, their characteristics can vary depending on the developmental stages. The aim of this study was to compare the characteristics of rat FCPCs from the hind limb on embryonic day 14 (E14), E16 and E20 regarding proliferation, pluripotency, and differentiation. Morphologically, rat fetal cartilage tissue showed an increase in cartilaginous differentiation features (Safranin-O, type II collagen) and decrease in pluripotency marker (Sox2) in the order of E14, E16 and E20. E14 FCPCs showed significantly higher doubling time compared to E16 and E20 FCPCs. While the E14 FCPCs expressed pluripotent genes (Sox2, Oct4, Nanog), the E16 and E20 FCPCs expressed chondrogenic markers (Sox9, Col2a1, Acan). E20 FCPCs showed the highest ability to both chondrogenic and adipogenic differentiation and E14 FCPCs showed relatively better activity in osteogenic differentiation. Further analysis showed that E20 FCPCs expressed both adipogenic (C/ebps) and osteogenic (Runx2, Sp7, Taz) transcription factors as well as chondrogenic transcription factors. Our results show an inverse relationship overall between the expression of pluripotency genes and that of chondrogenic and lineage-specific genes in FCPCs under development. Due to its exceptional proliferation and chondrogenic differentiation ability, fetal cells from epiphyseal cartilage (E20 in rats) may be a suitable cell source for cartilage regeneration.

A computer-designed, solvent-free scaffold offer several potential advantages such as ease of customized manufacture and in vivo safety. In this work, we firstly used a computer-designed, solvent-free scaffold and human dental pulp stem cells (hDPSCs) to regenerate neo-bone within cranial bone defects. The hDPSCs expressed mesenchymal stem cell markers and served as an abundant source of stem cells with a high proliferation rate. In addition, hDPSCs showed a phenotype of differentiated osteoblasts in the presence of osteogenic factors (OF). We used solid freeform fabrication (SFF) with biodegradable polyesters (MPEG-(PLLA-co-PGA-co-PCL) (PLGC)) to fabricate a computer-designed scaffold. The SFF technology gave quick and reproducible results. To assess bone tissue engineering in vivo, the computer-designed, circular PLGC scaffold was implanted into a full-thickness cranial bone defect and monitored by micro-computed tomography (CT) and histology of the in vivo tissue-engineered bone. Neo-bone formation of more than 50% in both micro-CT and histology tests was observed at only PLGC scaffold with hDPSCs/OF. Furthermore, the PLGC scaffold gradually degraded, as evidenced by the fluorescent-labeled PLGC scaffold, which provides information to tract biodegradation of implanted PLGC scaffold. In conclusion, we confirmed neo-bone formation within a cranial bone defect using hDPSCs and a computer-designed PLGC scaffold.

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-beta) treatment under the 3-dimensional (3-D) culture condition. However, more efficient and versatile methods for chondrogenic differentiation of MSCs are still in demand for its clinical application. Recently, low-intensity ultrasound (LIUS) was shown to enhance fracture healing in vitro and induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of LIUS on the chondrogenesis of rabbit MSCs (rMSCs) in a 3-D alginate culture and on the maintenance of chondrogenic phenotypes after replating them on a monolayer culture. The LIUS treatment of rMSCs increased: (i) the matrix formation; (ii) the expression of chondrogenic markers such as collagen type II, aggrecan, and Sox-9; (iii) the expression of tissue inhibitor of metalloprotease-2 implicated in the integrity of cartilage matrix; and (iv) the capacity to maintain the chondrogenic phenotypes in a monolayer culture. Notably, LIUS effects were clearly shown even without TGF-beta treatment. These results suggest that LIUS treatment could be an efficient and cost-effective method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering.

Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-s3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-s3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair.

High molecular weight (MW) hyaluronan (HA) preparation is considered to be more biologically active than HAs of lower MWs. However, many of the HA preparations currently used to treat osteoarthritis (OA) have lower MWs by the enhanced penetration of HA molecules into the synovial lining cells. In this study, we determined the effectiveness of sonophoresis on the delivery of high MW HA into synovial membrane using an animal model of OA. A total of 1000 kDa (HA1000) and 3000 kDa (HA3000) HA were labeled with fluorescein and injected into the knees of rabbits. Low-intensity continuous ultrasound at 1 MHz, 400 mW/cm2 was applied to the knees for 10 min treatment bid. Synovial fluid analysis revealed increased absorption and fluorescence microscopy showed deeper penetration of both HA1000 and HA3000, more so with the latter. Histologic examination indicated that ultrasound treatment resulted in no apparent damage to the synovial membrane. These results suggest that simultaneous sonication with HA injection might compensate for the short half-life of HA. Consequently, this dual treatment would render HA a far more effective tool in the management of OA.