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Possible failures of autologous chondrocyte implantation (ACI), a cell-based technique for articular cartilage repair, are not always clinically apparent and the underlying mechanisms largely remain unknown. This case report presents the first scenario in the literature highlighting an association of a medium-term partial failure of an advanced ACI procedure (matrix-assisted ACI) in the knee with focal asymptomatic calcium pyrophosphate deposition disease, a common inflammatory pyrophosphate arthropathy. The specific presence of CPPDs, resulting from increased biomechanical stresses in the repair tissue-cartilage and repair tissue-subchondral bone integration sites, together with the absence of cartilage regeneration was identified and possibly contributed to the partial failure.Level of evidence V.

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the potent transforming growth factor beta (TGF-β) and insulin-like growth factor I (IGF-I) to enhance the processes of chondrogenic differentiation in human MSCs (hMSCs) relative to individual candidate treatments and to reporter ( lacZ ) gene condition. The rAAV-hTGF-β and rAAV-hIGF-I vectors were simultaneously provided to hMSC aggregate cultures (TGF-β/IGF-I condition) in chondrogenic medium over time (21 days) versus TGF-β/ lacZ , IGF-I/ lacZ , and lacZ treatments at equivalent vector doses. The cultures were then processed to monitor transgene (co)-overexpression, the levels of biological activities in the cells (cell proliferation, matrix synthesis), and the development of a chondrogenic versus osteogenic/hypertrophic phenotype. Effective, durable co-overexpression of TGF-β with IGF-I via rAAV enhanced the proliferative, anabolic, and chondrogenic activities in hMSCs versus lacZ treatment and reached levels that were higher than those achieved upon single candidate gene transfer, while osteogenic/hypertrophic differentiation was delayed over the period of time evaluated. These findings demonstrate the potential of manipulating multiple therapeutic rAAV vectors as a tool to directly target bone-marrow-derived MSCs in sites of focal cartilage defects and to locally enhance the endogenous processes of cartilage repair.

Background: The addition of a type I/III collagen membrane in cartilage defects treated with microfracture has been advocated for cartilage repair, termed "autologous matrix-induced chondrogenesis" (AMIC)., Purpose: To examine the current clinical evidence regarding AMIC for focal chondral defects., Study Design: Systematic review., Methods: A systematic review was performed by searching PubMed, ScienceDirect, and Cochrane Library databases. Inclusion criteria were clinical studies of AMIC for articular cartilage repair, written in English. Relative data were extracted and critically analyzed. PRISMA guidelines were applied, the methodological quality of the included studies was assessed by the modified Coleman Methodology Score (CMS), and aggregate data were generated., Results: Twenty-eight clinical articles were included: 12 studies (245 patients) of knee cartilage defects, 12 studies (214 patients) of ankle cartilage defects, and 4 studies (308 patients) of hip cartilage defects. The CMS demonstrated a suboptimal study design in the majority of published studies (knee, 57.8; ankle, 55.3; hip, 57.7). For the knee, 1 study reported significant clinical improvements for AMIC compared with microfracture for medium-sized cartilage defects (mean defect size 3.6 cm 2 ) after 5 years (level of evidence, 1). No study compared AMIC with matrix-assisted autologous chondrocyte implantation (ACI) in the knee. For the ankle, no clinical trial was available comparing AMIC versus microfracture or ACI. In the hip, only one analysis (level of evidence, 3) compared AMIC with microfracture for acetabular lesions. For medium-sized acetabular defects, one study (level of evidence, 3) found no significant differences between AMIC and ACI at 5 years. Specific aspects not appropriately discussed in the currently available literature include patient-related factors, membrane fixation, and defect properties. No treatment-related adverse events were reported., Conclusion: This systematic review reveals a paucity of high-quality, randomized controlled studies testing the AMIC technique versus established procedures such as microfracture or ACI. Evidence is insufficient to recommend joint-specific indications for AMIC. Additional nonbiased, high-powered, randomized controlled clinical trials will provide better clinical and structural long-term evidence, thus helping to define possible indications for this technique.

Recombinant adeno-associated virus (rAAV) vectors are well suited carriers to provide durable treatments for human osteoarthritis (OA). Controlled release of rAAV from polymeric micelles was already shown to increase both the stability and bioactivity of the vectors while overcoming barriers, precluding effective gene transfer. In the present study, we examined the convenience of delivering rAAV vectors via poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) polymeric (PEO-PPO-PEO) micelles to transfer and overexpress the transcription factor SOX9 in monolayers of human OA chondrocytes and in experimentally created human osteochondral defects. Human osteoarthritic (OA) chondrocytes and human osteochondral defect models were produced using human OA cartilage obtained from patients subjected to total knee arthroplasty. Samples were genetically modified by adding a rAAV-FLAG-h sox9 vector in its free form or via polymeric micelles for 10 days relative to control conditions (unmodified cells). The effects of sox9 overexpression in human OA cartilage samples were monitored by biochemical, histological, and immunohistochemical analyses. Delivery of rAAV-FLAG-h sox9 via polymeric micelles enhanced the levels of sox9 expression compared with free vector administration, resulting in increased proteoglycan deposition and in a stimulated cell proliferation index in OA chondrocytes. Moreover, higher production of type II collagen and decreased hypertrophic events were noted in osteochondral defect cultures when compared with control conditions. Controlled therapeutic rAAV sox9 gene delivery using PEO-PPO-PEO micelles is a promising, efficient tool to promote the remodelling of human OA cartilage.

Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single-step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) in human peripheral blood aspirates via rAAV-mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type-II collagen, SOX9) activities in the samples relative to control (reporter rAAV-lacZ) treatment over extended periods of time (at least 21 days, the longest time-point evaluated). Interestingly, IGF-I gene transfer also triggered hypertrophic, osteo- and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF-I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Recombinant adeno-associated virus (rAAV) vectors are clinically adapted vectors to durably treat human osteoarthritis (OA). Controlled delivery of rAAV vectors via polymeric micelles was reported to enhance the temporal and spatial presentation of the vectors into their targets. Here, we tested the feasibility of delivering rAAV vectors via poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) (poloxamer and poloxamine) polymeric micelles as a means to overexpress the therapeutic factor transforming growth factor-beta (TGF-β) in human OA chondrocytes and in experimental human osteochondral defects. Application of rAAV-human transforming growth factor-beta using such micelles increased the levels of TGF-β transgene expression compared with free vector treatment. Overexpression of TGF-β with these systems resulted in higher proteoglycan deposition and increased cell numbers in OA chondrocytes. In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted. Delivery of therapeutic rAAV vectors via PEO-PPO-PEO micelles may provide potential tools to remodel human OA cartilage., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed an increase in both the stability and bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with that of free vector treatment without detrimental effects on the biological activity of the cells or their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated, especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current gene-based treatments for human OA.

Alginates are important hydrogels for meniscus tissue engineering as they support the meniscal fibrochondrocyte phenotype and proteoglycan production, the extracellular matrix (ECM) component chiefly responsible for its viscoelastic properties. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to provide the best three-dimensional (3-D) microenvironment and to support proteoglycan synthesis of encapsulated human meniscal fibrochondrocytes in vitro. Biomedical-grade, high mannuronic acid alginate spheres (BioLVM, BioMVM) were the most uniform in size, indicating an effect of the purity of alginate on the shape of the spheres. Interestingly, the purity of alginates did not affect cell viability. Of note, only fibrochondrocytes encapsulated in BioMVM alginate produced and retained significant amounts of proteoglycans. Following transplantation in an explant culture model, the alginate spheres containing fibrochondrocytes remained in close proximity with the meniscal tissue adjacent to the defect. The results reveal a promising role of BioMVM alginate to enhance the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D hydrogel microenvironment. These findings have significant implications for cell-based translational studies aiming at restoring lost meniscal tissue in regions containing high amounts of proteoglycans.

Introduction: Osteoarthritis (OA) is the most prevalent chronic joint disease. Its key feature is a progressive articular cartilage loss. Gene therapy for OA aims at delivering gene-based therapeutic agents to the osteoarthritic cartilage, resulting in a controlled, site-specific, long-term presence to rebuild the damaged cartilage., Areas Covered: An overview is provided of the principles of gene therapy for OA based on a PubMed literature search. Gene transfer to normal and osteoarthritic cartilage in vitro and in animal models in vivo is reviewed. Results from recent clinical gene therapy trials for OA are discussed and placed into perspective., Expert Opinion: Recombinant adeno-associated viral (rAAV) vectors enable to directly transfer candidate sequences in human articular chondrocytes in situ, providing a potent tool to modulate the structure of osteoarthritic cartilage. However, few preclinical animal studies in OA models have been performed thus far. Noteworthy, several gene therapy clinical trials have been carried out in patients with end-stage knee OA based on the intraarticular injection of human juvenile allogeneic chondrocytes overexpressing a cDNA encoding transforming growth factor-beta-1 via retroviral vectors. In a recent placebo-controlled randomized trial, clinical scores were improved compared with placebo. These translational results provide sufficient reason to proceed with further clinical testing of gene transfer protocols for the treatment of OA.

Purpose: Our aim was to evaluate whether nonviral vectors can genetically modify primary human juvenile and adult meniscal fibrochondrocytes at low toxicity in vitro and to test the hypothesis that transfected human meniscal fibrochondrocytes transplanted into longitudinal defects and onto human medial meniscus explant cultures are capable of expressing transgene products in vitro., Methods: Eighteen nonviral gene transfer systems were examined to identify the best suited method for an efficient transfection of primary cultures of juvenile and adult human meniscal fibrochondrocytes using luciferase and lacZ reporter gene constructs and then transplanted to meniscus explant cultures., Results: Gene transfer systems FuGENE 6, GeneJammer, TurboFectin 8, calcium phosphate co-precipitates and GeneJuice led to minimal toxicity in both cell types. Nanofectin 2 and JetPEI resulted in maximal luciferase activity in both cell types. Maximal transfection efficiency based on X-gal staining following lacZ gene transfer was achieved using Lipofectamine 2000, revealing a mean transfection efficiency of 8.6 % in human juvenile and of 8.4 % in adult meniscal fibrochondrocytes. Transfected, transplanted meniscal fibrochondrocytes adhered to the meniscal tissue and continued to express the transgene for at least five days following transfection., Conclusions: Nonviral gene transfer systems are safe and capable of transfecting both juvenile and adult human meniscal fibrochondrocytes, which, when transplanted to meniscal tissue in vitro, permit the expression of selected transgenes to be maintained. These results are of value for combining gene therapy and cell transplantation approaches as a means to enhance meniscal repair.

Purpose: Our aim was to study the effect of three-dimensional (3D) environment and overexpression of human fibroblast growth factor 2 (FGF-2) on meniscal fibrochondrocytes in vitro., Methods: Human meniscal fibrochondrocytes were transfected with expression plasmid vectors carrying the Photinus pyralis luciferase gene, the Escherichia coli β-galactosidase gene or a human FGF-2 cDNA. Modified fibrochondrocytes were cultivated in 3D alginate hydrogel or cell pellets or in 2D monolayer culture., Results: The levels of luciferase activity showed a peak at day two and returned to baseline levels by day 11, regardless of the type of cultivation. Both 3D environments supported the secretion of human FGF-2 protein upon FGF-2 transfection. Overexpression of human FGF-2 by genetically modified human meniscal fibrochondrocytes stimulated proliferation but not glycosaminoglycan synthesis only in 3D culture. Culture in alginate spheres resulted in a larger difference in cell numbers compared with pellet cultures., Conclusions: Three-dimensional alginate spheres are well suited for the culture of genetically modified human meniscal fibrochondrocytes. These data are of value for cell-based approaches to meniscal repair using genetically modified human meniscal fibrochondrocytes overexpressing human FGF-2.

Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes overexpressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-overexpressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects.

Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while up-regulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis.

Adult articular cartilage has a limited capacity for self repair. Reproduction of a native structure and functional integrity in damaged cartilage remains a major problem in orthopaedic surgery. Strategies based on the implantation of genetically modified cells to sites of injury may provide workable options to treat articular cartilage lesions like those resulting from acute trauma or associated with the progression of osteoarthritis. Mesenchymal stem cells have remarkable properties that make them an attractive source of cells to treat cartilage disorders due to their self-renewal capability, stemness maintenance, and chondrogenic differentiation potential. For these reasons, such progenitor cells might be further modified by gene transfer protocols to reinforce their potency and consequently, to enhance the healing processes in damaged tissue following transplantation in sites of cartilage injury. Here, we propose an overview of the current approaches employed for cell- and gene-based treatment of articular cartilage disorders using mesenchymal stem cells.

Purpose: The combination of chondrogenic factors might be necessary to adequately stimulate articular cartilage repair. In previous studies, enhanced repair was observed following transplantation of chondrocytes overexpressing human insulin-like growth factor I (IGF-I) or fibroblast growth factor 2 (FGF-2). Here, the hypothesis that co-overexpression of IGF-I and FGF-2 by transplanted articular chondrocytes enhances the early repair of cartilage defects in vivo and protects the neighbouring cartilage from degeneration was tested., Methods: Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or co-transfected with the IGF-I and FGF-2 gene, encapsulated in alginate and transplanted into osteochondral defects in the knee joints of rabbits in vivo., Results: After 3 weeks, co-overexpression of IGF-I/FGF-2 improved the macroscopic aspect of defects without affecting the synovial membrane. Immunoreactivity to type-I collagen, an indicator of fibrocartilage, was significantly lower in defects receiving IGF-I/FGF-2 implants. Importantly, combined IGF-I/FGF-2 overexpression significantly improved the histological repair score. Most remarkably, such enhanced cartilage repair was correlated with a 2.1-fold higher proteoglycan content of the repair tissue. Finally, there were less degenerative changes in the cartilage adjacent to the defects treated with IGF-I/FGF-2 implants., Conclusion: The data demonstrate that combined gene delivery of therapeutic growth factors to cartilage defects may have value to promote cartilage repair. The results also suggest a protective effect of IGF-I/FGF-2 co-overexpression on the neighbouring articular cartilage. These findings support the concept of implementing gene transfer strategies for articular cartilage repair in a clinical setting.

Alginate is a key hydrogel for cartilage tissue engineering. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to support the in vitro culture and in vivo transplantation of articular chondrocytes. Chondrocytes in all ultrapure alginates maintained high cell viability. Spheres composed of biomedical-grade, low-viscosity, high-mannuronic acid content alginate showed the lowest decrease in size over time. Biomedical-grade, low-viscosity, high-guluronic acid content alginate allowed for optimal cell proliferation. Biomedical-grade, medium-viscosity, high-mannuronic acid content alginate promoted the highest production of proteoglycans. When transplanted into osteochondral defects in the knee joint of sheep in vivo, empty spheres were progressively surrounded by a granulation tissue. In marked contrast with these observations, all alginate spheres carrying allogeneic chondrocytes were gradually invaded by a granulation tissue containing multinucleated giant cells, lymphocytes, and fibroblasts, regardless whether they were based on biomedical- or nonbiomedical-grade alginates. After 21 days in vivo, transplanted chondrocytes were either viable or underwent necrosis, and apoptosis played a minor role in their early fate. The individual characteristics of these alginates may be valuable to tailor specific experimental and clinical strategies for cartilage tissue engineering.

Articular cartilage defects do not regenerate. Transplantation of autologous articular chondrocytes, which is clinically being performed since several decades, laid the foundation for the transplantation of genetically modified cells, which may serve the dual role of providing a cell population capable of chondrogenesis and an additional stimulus for targeted articular cartilage repair. Experimental data generated so far have shown that genetically modified articular chondrocytes and mesenchymal stem cells (MSC) allow for sustained transgene expression when transplanted into articular cartilage defects in vivo. Overexpression of therapeutic factors enhances the structural features of the cartilaginous repair tissue. Combined overexpression of genes with complementary mechanisms of action is also feasible, holding promises for further enhancement of articular cartilage repair. Significant benefits have been also observed in preclinical animal models that are, in principle, more appropriate to the clinical situation. Finally, there is convincing proof of concept based on a phase I clinical gene therapy study in which transduced fibroblasts were injected into the metacarpophalangeal joints of patients without adverse events. To realize the full clinical potential of this approach, issues that need to be addressed include its safety, the choice of the ideal gene vector system allowing for a long-term transgene expression, the identification of the optimal therapeutic gene(s), the transplantation without or with supportive biomaterials, and the establishment of the optimal dose of modified cells. As safe techniques for generating genetically engineered articular chondrocytes and MSCs are available, they may eventually represent new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide an important step toward the unanswered question of articular cartilage regeneration.

Background: Genetically modified chondrocytes embedded in alginate improve cartilage repair in experimental models, and alginates are clinically used for articular chondrocyte transplantation. In the present study, we tested the hypothesis that the alginate system allows for sustained transgene expression in cartilage defects in a preclinical large animal model in vivo., Methods: Primary cultures of ovine articular chondrocytes were transfected with the Photinus pyralis luc or the Escherichia coli lacZ genes in monolayer culture in vitro using eight different nonviral compounds. Optimally transfected chondrocytes were encapsulated in spheres composed of nonbiomedical or biomedical grade alginates for evaluation of luciferase expression, cell numbers and viabilities in vitro. Transfected chondrocytes encapsulated in spheres comprised of the different alginates were then implanted into osteochondral defects in the knee joints of sheep to examine the profiles of transgene expression in vivo., Results: Ovine articular chondrocytes were efficiently transfected with FuGENE 6. Transgene expression was detectable after encapsulation in the alginates over 21 days in vitro. Transplantation of genetically modified chondrocytes to cartilage defects in vivo resulted in maximal transgene expression on day 1 after transfection, with a decrease by day 21, the longest time point evaluated. Remarkably, the reduction in luciferase activity was less pronounced when biomedical grade alginates were employed, compared to nonbiomedical grade alginates, suggesting that such alginates might be better suited to support elevated transgene expression after transplantation of genetically modified chondrocytes., Conclusions: This approach may be of value to study the effects of potential therapeutic genes upon cartilage repair in a clinically relevant setting., (Copyright © 2011 John Wiley & Sons, Ltd.)

Cartilage tissue engineering is the creation of functional substitutes of native articular cartilage in bioreactors by attaching chondrogenic cells to polymer scaffolds. One limitation of tissue engineering is the delivery of regulatory signals to cells according to specific temporal and spatial patterns. Using gene transfer techniques, polypeptide growth factor genes such as the human insulin-like growth factor I (IGF-I) gene can be transferred into chondrocytes. When these modified cells are used for cartilage tissue engineering, the resulting cartilaginous constructs have improved structural and functional characteristics compared to constructs based on nonmodified cells. The combination of cartilage tissue engineering with overexpression of potential therapeutic genes using gene transfer technologies provides a basis for the development of novel molecular therapies for the repair of cartilage defects.

Background: Defects of articular cartilage are an unsolved problem in orthopaedics. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo., Methods: Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cartilage defects in the knee joints of rabbits. In vivo, cartilage repair was assessed qualitatively and quantitatively at 3 and 14 weeks after implantation., Results: In vitro, bioactive FGF-2 was secreted, leading to a significant increase in the cell numbers in FGF-2 spheres. In vivo, FGF-2 continued to be expressed for at least 3 weeks without leading to differences in FGF-2 concentrations in the synovial fluid between treatment groups. Histological analysis revealed no adverse pathologic effects on the synovial membrane at any time point. FGF-2 gene transfer enhanced type II collagen expression and individual parameters of chondrogenesis, such as the cell morphology and architecture of the new tissue. Overall articular cartilage repair was significantly improved at both time points in vivo., Conclusions: The data suggest that localized overexpression of FGF-2 enhances the repair of cartilage defects via stimulation of chondrogenesis, without adverse effects on the synovial membrane. These results may lead to the development of safe gene-based therapies for human articular cartilage defects.

Traumatic articular cartilage lesions have a limited capacity to heal. We tested the hypothesis that overexpression of a human insulin-like growth factor I (IGF-I) cDNA by transplanted articular chondrocytes enhances the repair of full-thickness (osteochondral) cartilage defects in vivo. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or the human IGF-I gene and were encapsulated in alginate. The expression patterns of the transgenes in these implants were monitored in vitro for 36 days. Transfected allogeneic chondrocytes in alginate were transplanted into osteochondral defects in the trochlear groove of rabbits. At three and 14 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. In vitro, IGF-I secretion by implants constructed from IGF-I-transfected chondrocytes and alginate was 123.2+/-22.3 ng/10(7) cells/24 h at day 4 post transfection and remained elevated at day 36, the longest time point evaluated. In vivo, transplantation of IGF-I implants improved articular cartilage repair and accelerated the formation of the subchondral bone at both time points compared to lacZ implants. The data indicate that allogeneic chondrocytes, transfected by a nonviral method and cultured in alginate, are able to secrete biologically relevant amounts of IGF-I over a prolonged period of time in vitro. The data further demonstrate that implantation of these composites into deep articular cartilage defects is sufficient to augment cartilage defect repair in vivo. These results suggest that therapeutic growth factor gene delivery using encapsulated and transplanted genetically modified chondrocytes may be applicable to sites of focal articular cartilage damage.

Background: Genetically modified chondrocytes may be able to modulate articular cartilage repair. To date, transplantation of modified chondrocytes into cartilage defects has been restricted to viral vectors. We tested the hypothesis that a recombinant gene can be delivered to sites of cartilage damage in vivo using chondrocytes transfected by a lipid-mediated gene transfer method., Methods: Isolated lapine articular chondrocytes were transfected with an expression plasmid vector carrying the P. pyralis luciferase gene using the reagent FuGENE 6. Transfected chondrocytes were encapsulated in alginate spheres and implanted into osteochondral defects in the knee joints of rabbits., Results: In vitro, luciferase activity in pCMVLuc-transfected spheres showed an early peak at day 2 post-transfection and remained elevated at day 32, the longest time point evaluated. The number of viable chondrocytes in non-transfected and transfected spheres increased over the period of cultivation. In vivo, luciferase activity was maximal at day 5 post-transfection, declined by day 16, but was still present at day 32. On histological analysis, the alginate-chondrocyte spheres filled the cartilage defects and were surrounded by a fibrous repair tissue composed of spindle-shaped cells., Conclusions: These data demonstrate the successful introduction of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system into osteochondral defects and the sustained expression of the transgene in vivo. This method may be used to define the effects of genes involved in cartilage repair and may provide alternative treatments for articular cartilage defects., (Copyright 2003 John Wiley & Sons, Ltd.)

Successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. In the present study we tested the hypothesis that recombinant adeno-associated virus (rAAV) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. Using an rAAV vector carrying the Escherichia coli beta-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter/enhancer (rAAV-lacZ), transduction efficiency exceeded 70% for isolated normal human adult articular chondrocytes, and osteoarthritic human articular chondrocytes. These were comparable to the transduction efficiency obtained with neonatal bovine articular chondrocytes. Transduction of explant cultures of articular cartilage resulted in reporter gene expression within the tissue of all three cartilage types to a depth exceeding 450 microm, which remained present until 150 days. When rAAV-lacZ vectors were applied to femoral chondral defects and osteochondral defects in vivo in a rat knee model, reporter gene expression was achieved for at least 10 days after transduction. These data suggest that AAV-based vectors can efficiently transduce and stably express foreign genes in articular chondrocytes, including chondrocytes of normal and osteoarthritic human articular cartilage. The data further suggest that the same rAAV vectors are capable of transducing chondrocytes in situ within their native matrix to a depth sufficient to be of potential clinical significance. Finally, the data demonstrate that these rAAV vectors are capable of effectively delivering recombinant genes to chondral and osteochondral defects in vivo.