Introduction: Transplantation of genetically modified human bone marrow-derived mesenchymal stem cells (hMSCs) with an accurate potential for chondrogenic differentiation may be a powerful means to enhance the healing of articular cartilage lesions in patients. Here, we evaluated the benefits of delivering SOX9 (a key regulator of chondrocyte differentiation and cartilage formation) via safe, maintained, replication-defective recombinant adeno-associated virus (rAAV) vector on the capability of hMSCs to commit to an adequate chondrocyte phenotype compared with other mesenchymal lineages. Methods: The rAAV-FLAG-hSOX9 vector was provided to both undifferentiated and lineage-induced MSCs freshly isolated from patients to determine the effects of the candidate construct on the viability, biosynthetic activities, and ability of the cells to enter chondrogenic, osteogenic, and adipogenic differentiation programs compared with control treatments (rAAV-lacZ or absence of vector administration). Results: Marked, prolonged expression of the transcription factor was noted in undifferentiated and chondrogenically differentiated cells transduced with rAAV-FLAG-hSOX9, leading to increased synthesis of major extracellular matrix components compared with control treatments, but without effect on proliferative activities. Chondrogenic differentiation (SOX9, type II collagen, proteoglycan expression) was successfully achieved in all types of cells but strongly enhanced when the SOX9 vector was provided. Remarkably, rAAV-FLAG-hSOX9 delivery reduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I and type X collagen, alkaline phosphatise (ALP), matrix metalloproteinase 13 (MMP13), and osteopontin (OP) with diminished expression of the osteoblast-related transcription factor runt-related transcription factor 2 (RUNX2); lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma 2 (PPARG2)), as well as their ability to undergo proper osteo-/adipogenic differentiation. These effects were accompanied with decreased levels of β-catenin (a mediator of the Wnt signaling pathway for osteoblast lineage differentiation) and enhanced parathyroid hormone-related protein (PTHrP) expression (an inhibitor of hypertrophic maturation, calcification, and bone formation) via SOX9 treatment. Conclusions: This study shows the potential benefits of rAAV-mediated SOX9 gene transfer to propagate hMSCs with an advantageous chondrocyte differentiation potential for future, indirect therapeutic approaches that aim at restoring articular cartilage defects in the human population.

Background: Therapeutic gene transfer is of significant value to elaborate efficient, durable treatments against human osteoarthritis (OA), a slow, progressive, and irreversible disorder for which there is no cure to date.Methods: Here, we directly applied a recombinant adeno-associated virus (rAAV) vector carrying a human transforming growth factor beta (TGF-β) gene sequence to primary human normal and OA chondrocytesin vitroand cartilage explantsin situto monitor the stability of transgene expression and the effects of the candidate pleiotropic factor upon the regenerative cellular activities over time.Results: Efficient, prolonged expression of TGF-β achieved via rAAV gene transfer enhanced both the proliferative, survival, and anabolic activities of cells over extended periods of time in all the systems evaluated (at least for 21 daysin vitroand for up to 90 daysin situ) compared with control (reporter) vector delivery, especiallyin situwhere rAAV-hTGF-β allowed for a durable remodeling of OA cartilage. Notably, sustained rAAV production of TGF-β in OA cartilage advantageously reduced the expression of key OA-associated markers of chondrocyte hypertrophic and terminal differentiation (type-X collagen, MMP-13, PTHrP, β-catenin) while increasing that of protective TIMPs and of the TGF-β receptor I in a manner that restored a favorable ALK1/ALK5 balance. Of note, the levels of activities in TGF-β-treated OA cartilage were higher than those of normal cartilage, suggesting that further optimization of the candidate treatment (dose, duration, localization, presence of modulating co-factors) will most likely be necessary to reproduce an original cartilage surface in relevant models of experimental OAin vivowithout triggering potentially adverse effects.Conclusions: The present findings show the ability of rAAV-mediated TGF-β gene transfer to directly remodel human OA cartilage by activating the biological, reparative activities and by regulating hypertrophy and terminal differentiation in damaged chondrocytes as a potential treatment for OA or for other disorders of the cartilage that may require transplantation of engineered cells.

Introduction: The transplantation of genetically modified progenitor cells such as bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the natural healing of articular cartilage defects. In the present study, we examined the potential benefits of sustained overexpression of the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) via gene transfer upon the biological activities of human MSCs (hMSCs). Methods: Recombinant adeno-associated vectors (rAAV) were used to deliver a human IGF-I coding sequence in undifferentiated and chondrogenically-induced primary hMSCs in order to determine the efficacy and duration of transgene expression and the subsequent effects of the genetic modification upon the chondrogenic versus osteogenic differentiation profiles of the cells relative to control (lacZ) treatment after 21 days in vitro. Results: Significant and prolonged expression of IGF-I was evidenced in undifferentiated and most importantly in chondrogenically-induced hMSCs transduced with the candidate rAAV-hIGF-I vector for up to 21 days, leading to enhanced proliferative, biosynthetic, and chondrogenic activities compared with rAAV-lacZ treatment. Overexpression of IGF-I as achieved in the conditions applied here also increased the expression of hypertrophic and osteogenic markers in the treated cells. Conclusions: These results suggest that a tight regulation of rAAV expression may be necessary for further translation of the approach in clinically relevant animal models in vivo. However, the current findings support the concept of using this type of vector as an effective tool to treat articular cartilage defects via gene- and stem cell-based procedures.

Background: Articular cartilage lesions generated by trauma or osteoarthritis are the most common causes of pain and disability in patients. The development of photopolymerizable hydrogels has allowed for significant advances in cartilage repair procedures. Such three‐dimensional (3D) networks of polymers that carry large amounts of water can be created to resemble the physical characteristics of the articular cartilage and be delivered into ill‐defined cartilage defects as a liquid solution prior to polymerization in vivo for perfect fit with the surrounding native tissue. These hydrogels offer an adapted environment to encapsulate and propagate regenerative cells in 3D cultures for cartilage repair. Among them, mesenchymal stem cells and chondrocytes may represent the most adapted sources for implantation. They also represent platforms to deliver therapeutic, biologically active factors that promote 3D cell differentiation and maintenance for in vivo repair.Conclusion: This review presents the benefits of photopolymerization of hydrogels and describes the photoinitiators and materials in current use for enhanced cartilage repair.

Osteochondral defects involve both the articular cartilage and the underlying subchondral bone. If left untreated, they may lead to osteoarthritis. Advanced biomaterial-guided delivery of gene vectors has recently emerged as an attractive therapeutic concept for osteochondral repair. The goal of this review is to provide an overview of the variety of biomaterials employed as nonviral or viral gene carriers for osteochondral repair approaches both in vitro and in vivo, including hydrogels, solid scaffolds, and hybrid materials. The data show that a site-specific delivery of therapeutic gene vectors in the context of acellular or cellular strategies allows for a spatial and temporal control of osteochondral neotissue composition in vitro. In vivo, implantation of acellular hydrogels loaded with nonviral or viral vectors has been reported to significantly improve osteochondral repair in translational defect models. These advances support the concept of scaffold-mediated gene delivery for osteochondral repair.