Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep.

OBJECTIVE: To investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. METHOD: Normal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. RESULTS: Multivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface-to-volume ratio (all P ≤ 0.0359 vs. normal). CONCLUSION: The data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

Effects of Recombinant Adeno-Associated Virus-Mediated Overexpression of Bone Morphogenetic Protein 3 on the Chondrogenic Fate of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Implantation of genetically modified chondrogenically competent human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to improve cartilage repair. The goal of this study was to examine the potential benefits of transferring a sequence coding for the bone morphogenetic protein 3 (BMP-3) that modulates bone and cartilage formation, using recombinant adeno-associated virus (rAAV) vectors on the chondroreparative activities of hMSCs. Undifferentiated and chondrogenically induced primary human MSCs were treated with an rAAV-hBMP-3 construct to evaluate its effects on the proliferative, metabolic, and chondrogenic activities of the cells compared with control (reporter rAAV-lacZ vector) condition. Effective BMP-3 expression was noted both in undifferentiated and chondrogenically differentiated cells in the presence of rAAV-hBMP-3 relative to rAAV-lacZ, stimulating cell proliferation and extracellular matrix (proteoglycans, type-II collagen) deposition together with higher levels of chondrogenic sex-determining region Y-type high-mobility group box 9 (SOX9) expression. rAAV-hBMP-3 also advantageously decreased terminal differentiation, hypertrophy, and osteogenesis (type-I/-X collagen and alkaline phosphatase expression), with reduced levels of osteoblast-related runt-related transcription factor 2 (RUNX-2) transcription factor and ß-catenin (osteodifferentiation mediator) and enhanced parathyroid hormone-related protein expression (inhibitor of hypertrophic maturation, calcification, and bone formation). This study shows the advantage of modifying hMSCs with rAAV-hBMP-3 to trigger adapted chondroreparative activities as a source of improved cells for transplantation protocols in cartilage defects.

Axial alignment is a critical regulator of knee osteoarthritis.

Although osteoarthritis (OA), a leading cause of disability, has been associated with joint malalignment, scientific translational evidence for this link is lacking. In a clinical case study, we provide evidence of osteochondral recovery upon unloading symptomatic isolated medial tibiofemoral knee OA associated with varus malalignment. By mapping response correlations at high resolution, we identify spatially complex degenerative changes in cartilage after overloading in a clinically relevant ovine model. We further report that unloading diminishes OA cartilage degeneration and alterations of critical parameters of the subchondral bone plate in a similar topographic fashion. Last, therapeutic unloading shifted the articular cartilage and subchondral bone phenotype to normal and restored several physiological correlations disturbed in neutral and varus OA, suggesting a protective effect on the integrity of the entire osteochondral unit. Collectively, these findings identify modifiable trajectories with considerable translational potential to reduce the burden of human OA.

rAAV-Mediated sox9 Overexpression Improves the Repair of Osteochondral Defects in a Clinically Relevant Large Animal Model Over Time In Vivo and Reduces Perifocal Osteoarthritic Changes.

BACKGROUND: Gene transfer of the transcription factor SOX9 with clinically adapted recombinant adeno-associated virus (rAAV) vectors offers a powerful tool to durably enhance the repair process at sites of osteochondral injuries and counteract the development of perifocal osteoarthritis (OA) in the adjacent articular cartilage. PURPOSE: To examine the ability of an rAAV sox9 construct to improve the repair of focal osteochondral defects and oppose perifocal OA development over time in a large translational model relative to control gene transfer. STUDY DESIGN: Controlled laboratory study. METHODS: Standardized osteochondral defects created in the knee joints of adult sheep were treated with rAAV-FLAG-hsox9 relative to control (reporter) rAAV-lacZ gene transfer. Osteochondral repair and degenerative changes in the adjacent cartilage were monitored using macroscopic, histological, immunohistological, and biochemical evaluations after 6 months. The microarchitecture of the subchondral bone was assessed by micro-computed tomography. RESULTS: Effective, prolonged sox9 overexpression via rAAV was significantly achieved in the defects after 6 months versus rAAV-lacZ treatment. The application of rAAV-FLAG-hsox9 improved the individual parameters of defect filling, matrix staining, cellular morphology, defect architecture, surface architecture, subchondral bone, and tidemark as well as the overall score of cartilage repair in the defects compared with rAAV-lacZ. The overexpression of sox9 led to higher levels of proteoglycan production, stronger type II collagen deposition, and reduced type I collagen immunoreactivity in the sox9- versus lacZ-treated defects, together with decreased cell densities and DNA content. rAAV-FLAG-hsox9 enhanced semiquantitative histological subchondral bone repair, while the microstructure of the incompletely restored subchondral bone in the sox9 defects was not different from that in the lacZ defects. The articular cartilage adjacent to the sox9-treated defects showed reduced histological signs of perifocal OA changes versus rAAV-lacZ. CONCLUSION: rAAV-mediated sox9 gene transfer enhanced osteochondral repair in sheep after 6 months and reduced perifocal OA changes. These results underline the potential of rAAV-FLAG-hsox9 as a therapeutic tool to treat cartilage defects and afford protection against OA. CLINICAL RELEVANCE: The delivery of therapeutic rAAV sox9 to sites of focal injuries may offer a novel, convenient tool to enhance the repair of osteochondral defects involving both the articular cartilage and the underlying subchondral bone and provide a protective role by reducing the extent of perifocal OA.

Hydrogel-Guided, rAAV-Mediated IGF-I Overexpression Enables Long-Term Cartilage Repair and Protection against Perifocal Osteoarthritis in a Large-Animal Full-Thickness Chondral Defect Model at One Year In Vivo.

The regeneration of focal articular cartilage defects is complicated by the reduced quality of the repair tissue and the potential development of perifocal osteoarthritis (OA). Biomaterial-guided gene therapy may enhance cartilage repair by controlling the release of therapeutic sequences in a spatiotemporal manner. Here, the benefits of delivering a recombinant adeno-associated virus (rAAV) vector coding for the human insulin-like growth factor I (IGF-I) via an alginate hydrogel (IGF-I/AlgPH155) to enhance repair of full-thickness chondral defects following microfracture surgery after one year in minipigs versus control (lacZ/AlgPH155) treatment are reported. Sustained IGF-I overexpression is significantly achieved in the repair tissue of defects treated with IGF-I/AlgPH155 versus those receiving lacZ/AlgPH155 for one year and in the cartilage surrounding the defects. Administration of IGF-I/AlgPH155 significantly improves parameters of cartilage repair at one year relative to lacZ/AlgPH155 (semiquantitative total histological score, cell densities, matrix deposition) without deleterious or immune reactions. Remarkably, delivery of IGF-I/AlgPH155 also significantly reduces perifocal OA and inflammation after one year versus lacZ/AlgPH155 treatment. Biomaterial-guided rAAV gene transfer represents a valuable clinical approach to promote cartilage repair and to protect against OA.

pNaSS-Grafted PCL Film-Guided rAAV TGF-ß Gene Therapy Activates the Chondrogenic Activities in Human Bone Marrow Aspirates.

Scaffold-guided viral gene therapy is a novel, powerful tool to enhance the processes of tissue repair in articular cartilage lesions by the delivery and overexpression of therapeutic genes in a noninvasive, controlled release manner based on a procedure that may protect the gene vehicles from undesirable host immune responses. In this study, we examined the potential of transferring a recombinant adeno-associated virus (rAAV) vector carrying a sequence for the highly chondroregenerative transforming growth factor beta (TGF-ß), using poly(ɛ-caprolactone) (PCL) films functionalized by the grafting of poly(sodium styrene sulfonate) (pNaSS) in chondrogenically competent bone marrow aspirates as future targets for therapy in cartilage lesions. Effective overexpression of TGF-ß in the aspirates by rAAV was achieved upon delivery using pNaSS-grafted and ungrafted PCL films for up to 21 days (the longest time point evaluated), with superior levels using the grafted films, compared with respective conditions without vector coating. The production of rAAV-mediated TGF-ß by pNaSS-grafted and ungrafted PCL films significantly triggered the biological activities and chondrogenic processes in the samples (proteoglycan and type-II collagen deposition and cell proliferation), while containing premature mineralization and hypertrophy relative to the other conditions, with overall superior effects supported by the pNaSS-grafted films. These observations demonstrate the potential of PCL film-assisted rAAV TGF-ß gene transfer as a convenient, off-the-shelf technique to enhance the reparative potential of the bone marrow in patients in future approaches for improved cartilage repair.

rAAV-Mediated Human FGF-2 Gene Therapy Enhances Osteochondral Repair in a Clinically Relevant Large Animal Model Over Time In Vivo.

BACKGROUND: Osteochondral defects, if left untreated, do not heal and can potentially progress toward osteoarthritis. Direct gene transfer of basic fibroblast growth factor 2 (FGF-2) with the clinically adapted recombinant adeno-associated viral (rAAV) vectors is a powerful tool to durably activate osteochondral repair processes. PURPOSE: To examine the ability of an rAAV-FGF-2 construct to target the healing processes of focal osteochondral injury over time in a large translational model in vivo versus a control gene transfer condition. STUDY DESIGN: Controlled laboratory study. METHODS: Standardized osteochondral defects created in the knee joints of adult sheep were treated with an rAAV human FGF-2 (hFGF-2) vector by direct administration into the defect relative to control (reporter) rAAV-lacZ gene transfer. Osteochondral repair was monitored using macroscopic, histological, immunohistological, and biochemical methods and by micro-computed tomography after 6 months. RESULTS: Effective, localized prolonged FGF-2 overexpression was achieved for 6 months in vivo relative to the control condition without undesirable leakage of the vectors outside the defects. Such rAAV-mediated hFGF-2 overexpression significantly increased the individual histological parameter "percentage of new subchondral bone" versus lacZ treatment, reflected in a volume of mineralized bone per unit volume of the subchondral bone plate that was equal to a normal osteochondral unit. Also, rAAV-FGF-2 significantly improved the individual histological parameters "defect filling,""matrix staining," and "cellular morphology" and the overall cartilage repair score versus the lacZ treatment and led to significantly higher cell densities and significantly higher type II collagen deposition versus lacZ treatment. Likewise, rAAV-FGF-2 significantly decreased type I collagen expression within the cartilaginous repair tissue. CONCLUSION: The current work shows the potential of direct rAAV-mediated FGF-2 gene therapy to enhance osteochondral repair in a large, clinically relevant animal model over time in vivo. CLINICAL RELEVANCE: Delivery of therapeutic (hFGF-2) rAAV vectors in sites of focal injury may offer novel, convenient tools to enhance osteochondral repair in the near future.

rAAV-Mediated Overexpression of SOX9 and TGF-ß via Carbon Dot-Guided Vector Delivery Enhances the Biological Activities in Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Scaffold-assisted gene therapy is a highly promising tool to treat articular cartilage lesions upon direct delivery of chondrogenic candidate sequences. The goal of this study was to examine the feasibility and benefits of providing highly chondroreparative agents, the cartilage-specific sex-determining region Y-type high-mobility group 9 (SOX9) transcription factor or the transforming growth factor beta (TGF-ß), to human bone marrow-derived mesenchymal stromal cells (hMSCs) via clinically adapted, independent recombinant adeno-associated virus (rAAV) vectors formulated with carbon dots (CDs), a novel class of carbon-dominated nanomaterials. Effective complexation and release of a reporter rAAV-lacZ vector was achieved using four different CDs elaborated from 1-citric acid and pentaethylenehexamine (CD-1); 2-citric acid, poly(ethylene glycol) monomethyl ether (MW 550 Da), and N,N-dimethylethylenediamine (CD-2); 3-citric acid, branched poly(ethylenimine) (MW 600 Da), and poly(ethylene glycol) monomethyl ether (MW 2 kDa) (CD-3); and 4-citric acid and branched poly(ethylenimine) (MW 600 Da) (CD-4), allowing for the genetic modification of hMSCs. Among the nanoparticles, CD-2 showed an optimal ability for rAAV delivery (up to 2.2-fold increase in lacZ expression relative to free vector treatment with 100% cell viability for at least 10 days, the longest time point examined). Administration of therapeutic (SOX9, TGF-ß) rAAV vectors in hMSCs via CD-2 led to the effective overexpression of each independent transgene, promoting enhanced cell proliferation (TGF-ß) and cartilage matrix deposition (glycosaminoglycans, type-II collagen) for at least 21 days relative to control treatments (CD-2 lacking rAAV or associated to rAAV-lacZ), while advantageously restricting undesirable type-I and -X collagen deposition. These results reveal the potential of CD-guided rAAV gene administration in hMSCs as safe, non-invasive systems for translational strategies to enhance cartilage repair.

Enhanced Chondrogenic Differentiation Activities in Human Bone Marrow Aspirates via sox9 Overexpression Mediated by pNaSS-Grafted PCL Film-Guided rAAV Gene Transfer.

BACKGROUND: The delivery of therapeutic genes in sites of articular cartilage lesions using non-invasive, scaffold-guided gene therapy procedures is a promising approach to stimulate cartilage repair while protecting the cargos from detrimental immune responses, particularly when targeting chondroreparative bone marrow-derived mesenchymal stromal cells in a natural microenvironment like marrow aspirates. METHODS: Here, we evaluated the benefits of providing a sequence for the cartilage-specific sex-determining region Y-type high-mobility group box 9 (SOX9) transcription factor to human marrow aspirates via recombinant adeno-associated virus (rAAV) vectors delivered by poly(ε-caprolactone) (PCL) films functionalized via grafting with poly(sodium styrene sulfonate) (pNaSS) to enhance the marrow chondrogenic potential over time. RESULTS: Effective sox9 overexpression was observed in aspirates treated with pNaSS-grafted or ungrafted PCL films coated with the candidate rAAV-FLAG-hsox9 (FLAG-tagged rAAV vector carrying a human sox9 gene sequence) vector for at least 21 days relative to other conditions (pNaSS-grafted and ungrafted PCL films without vector coating). Overexpression of sox9 via rAAV sox9/pNaSS-grafted or ungrafted PCL films led to increased biological and chondrogenic differentiation activities (matrix deposition) in the aspirates while containing premature osteogenesis and hypertrophy without impacting cell proliferation, with more potent effects noted when using pNaSS-grafted films. CONCLUSIONS: These findings show the benefits of targeting patients' bone marrow via PCL film-guided therapeutic rAAV (sox9) delivery as an off-the-shelf system for future strategies to enhance cartilage repair in translational applications.

Thermosensitive Hydrogel Based on PEO-PPO-PEO Poloxamers for a Controlled In Situ Release of Recombinant Adeno-Associated Viral Vectors for Effective Gene Therapy of Cartilage Defects.

Advanced biomaterial-guided delivery of gene vectors is an emerging and highly attractive therapeutic solution for targeted articular cartilage repair, allowing for a controlled and minimally invasive delivery of gene vectors in a spatiotemporally precise manner, reducing intra-articular vector spread and possible loss of the therapeutic gene product. As far as it is known, the very first successful in vivo application of such a biomaterial-guided delivery of a potent gene vector in an orthotopic large animal model of cartilage damage is reported here. In detail, an injectable and thermosensitive hydrogel based on poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO poloxamers, capable of controlled release of a therapeutic recombinant adeno-associated virus (rAAV) vector overexpressing the chondrogenic sox9 transcription factor in full-thickness chondral defects, is applied in a clinically relevant minipig model in vivo. These comprehensive analyses of the entire osteochondral unit with multiple standardized evaluation methods indicate that rAAV-FLAG-hsox9/PEO-PPO-PEO hydrogel-augmented microfracture significantly improves cartilage repair with a collagen fiber orientation more similar to the normal cartilage and protects the subchondral bone plate from early bone loss.

Effective Remodelling of Human Osteoarthritic Cartilage by sox9 Gene Transfer and Overexpression upon Delivery of rAAV Vectors in Polymeric Micelles.

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.