gms | German Medical Science

Objectives: Adult articular cartilage has a limited ability for self-healing in response to injury. Gene transfer using the clinically adapted recombinant adeno-associated virus (rAAV) vector is a powerful tool for cartilage repair but the pre-existence of neutralizing antibodies against viral capsid epitopes in patients may impede gene transfer in vivo. To circumvent such a barrier, we tested the feasibility of delivering therapeutic (chondrogenic sox9 and TGF-β ?) rAAV vectors in reparative human bone marrow aspirates by vector delivery using poly (sodium styrene sulfonate) (pNaSS)-grafted poly (? ε-caprolactone) (PCL) films for implantation in a human osteochondral defect model as a potential scaffold-guided gene therapy option.

Methods: The PCL films were fabricated using a spin-coating method. The PCL films were tested in the following conditions: absence of pNaSS grafting (PCL conditions) versus pNaSS grafting (1.3 x 10-5 mol/g pNaSS; pNaSS-PCL conditions). rAAV-lacZ carries the E. coliβ-galactosidase (lacZ) reporter gene, rAAV-FLAG-hsox9 a human sox9 sequence, and rAAV-hTGF-β a human transforming growth factor beta 1 sequence, all controlled by the CMV-IE promoter/enhancer. Immobilization of rAAV on the films was performed by adding the vectors (40 µl) with 0.002% poly-L-lysine. 150 µl of human bone marrow aspirates (hBMAs) were placed on the films with fibrinogen/thrombin and implanted in 4-mm diameter of human osteochondral defects (hOCDs) and kept in chondrogenic medium for 21 days. The biochemical assays and real-time RT-PCR analysis were assessed after 21 days. T-test was employed with P≤0.05 considered statistically significant.

Results and Conclusion: Treatment with rAAV sox9 and TGF-β enhanced type-II collagen deposition in hBMAs via PCL film-guided gene transfer via implantation in hOCDs. Similar results were noted when measuring the normalized type-II collagen contents and increased the normalized proteoglycan contents but only via pNaSS-grafted films. The DNA contents were also increased by the two treatments. These results were corroborated by real-time RT-PCR of the profiles for COL2A1, ACAN, andSOX9 expression upon sox9 treatment. Interestingly, the sox9 and TGF-? treatments further reduced premature expression of COL1A1 and COL10A1. Overall, the effects reported with the pNaSS-grafted PCL films were more potent than when using ungrafted films.

Discussion: Delivery of rAAV sox9 and TGF-β via pNaSS-grafted PCL films enhances the chondroreparative activities of hBMAs following implantation in experimental human cartilage lesions while reducing premature hypertrophy and terminal differentiation relative to control treatments.

Significance: These results show the potential of pNaSS-grafted PCL film-guided therapeutic rAAV gene transfer in hBMAs as a novel healing platform for implantation in articular cartilage defects.