gms | German Medical Science

Objectives: Articular cartilage has a limited ability for self-healing. Gene transfer using the clinically relevant recombinant adeno-associated viral (rAAV) vectors is a powerful tool to enhance cartilage repair. Here, we tested the ability of various poly(sodium styrene sulfonate) grafted poly(ε -caprolactone) scaffolds to deliver rAAV to human bone marrow aspirates, a potential source of reparative cells for cartilage repair.

Methods: The scaffolds were fabricated using a spin-coating method. A poly(ε -caprolactone) (PCL) solution in dichloromethane was dropped on a glass slide and spun for 30 sec at 1,500 rpm using a SPIN150-v3 SPS. For grafting with poly(sodium styrene sulfonate) (pNaSS), the films were suspended under stirring into distilled water at room temperature. The following conditions were tested: no coating, low coating (1.11 x 10-6 mol/g pNaSS), and high coating (1.30 x 10-5 mol/g pNaSS). After ozonation, the films were transferred in a degassed NaSS solution in distilled water. The system was maintained at 45°C for 3 h to allow for graft polymerization. The samples were washed with distilled water, NaCl 0,15 M, and PBS and rinsed for vacuum-drying. rAAV vectors were packaged, purified, and titrated as previously described. rAAV-RFP carries the red fluorescent protein gene (RFP) and rAAV-lacZ the E. coli β -galactosidase gene (lacZ), both controlled by the CMV-IE promoter/enhancer. Vectors were labeled with Cy3 as described. Immobilization of rAAV on the scaffolds was performed by adding the vectors (40 μl) with 0.002% poly-L-lysine. rAAV release from the scaffolds was measured in aliquots of culture medium with the AAV Titration ELISA. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 10). Aspirates (150 µl) were placed on rAAV-coated scaffolds with a mixture of fibrinogen/thrombin(Baxter, Volketswil, Switzerland).The constructs were maintained in DMEM medium at 37ºC for up to 14 days. Transgene expression was monitored by detection of live fluorescence and by X-Gal staining.

Results and Conclusion: Successful immobilization of rAAV on the scaffolds was confirmed by detection of a fluorescent signal from Cy3-labeled vectors coated on the various pNaSS-grafted PCL scaffolds compared with scaffolds coated with unlabeled vectors. Remarkably, the various rAAV-coated scaffolds were capable of releasing the vectors over time, especially the low and highly coated pNaSS constructs. Effective scaffold-mediated rAAV gene transfer was achieved in human bone marrow aspirates over time as evidenced by detection of a strong fluorescent signal upon rAAV-RFP delivery versus control conditions.Similar results were observed when the scaffolds were coated with rAAV-lacZ, revealing intense lacZ expression via X-Gal staining compared with controls.Application of therapeutic rAAV-coated pNaSS-grafted PCL scaffolds may provide effective systems to enhance cartilage repair in patients affected with focal tissue lesions.