gms | German Medical Science

Objectives: Genetic modification of chondrocytes is a powerful approach to enhance the remodeling of osteoarthritic cartilage. Recombinant adeno-associated viral (rAAV) vectors are strong candidates to achieve this goal as these gene vehicles are highly safe and effective tools for clinical application. Here, we tested the feasibility of delivering rAAV transgenes to human normal and osteoarthritic chondrocytes in a three-dimensional environment that may improve favorably the regeneration of osteoarthritic lesions.

Methods: Human articular chondrocytes, normal and OA, were isolated the femoral head of the knee joint, washed in basal medium (DMEM), and kept in DMEM, 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin (growth medium). Chondrocytes were isolated by digested with collagenase, washing, and cell centrifugation at 1,500 rpm (3x5 min). rAAV vectors were packaged, purified, and titrated as described previous publications. rAAV-RFP carries the Discosoma sp. red fluorescent protein gene (RFP) and rAAV-lacZ the E. coli beta-galactosidase gene (lacZ), both controlled by the CMV-IE promoter/enhancer. Cell pellets (2 x 10⁵ cells per condition) were transduced with rAAV-lacZ or rAAV-RFP (20 or 40 µl each vector) or let untransduced. Pellets were directly plated in U-bottom 96-well plates and kept in culture up to 40 days with growth medium changes every other day. Transgene expression was monitored by live fluorescence microscopy (RFP), Beta-Glo® assay (Promega) as Relative Luminescence Units (RLUs) (lacZ), and X-Gal staining. Cell proliferation was monitored using the Cell Proliferation reagent WST-1 (Roche Applied Science).

Results and Conclusion: Successful, prolonged transgene expression was achieved upon application of rAAV-RFP relative to control (untransduced) conditions for at least 40 days (not shown) with a more intense signal in osteoarthritic chondrocytes (Figure 1 [Fig. 1]).

Effective, durable transduction was also observed when providing rAAV-lacZ compared with control (untransduced) conditions as measured by Beta-Glo® assay or via X-Gal staining. The Beta-Glo® assay revealed a dose-dependent effect of rAAV gene transfer in both types of cells, with higher levels of expression in osteoarthritic chondrocytes.Remarkably, there was no detrimental effect of rAAV transduction in these systems as noted by WST-1 assay, regardless of the vector dose applied. Durable rAAV-mediated gene transfer can be achieved in human normal and especially osteoarthritic chondrocytes in a three-dimensional environment adapted for the regeneration of osteoarthritic cartilage. Administration of those three-dimensional cultures may offer potent translational tools to regenerate osteoarthritic lesions.