gms | German Medical Science

Objectives: Recombinant adeno-associated viral (rAAV) vectors are clinically adapted gene vehicles to promote articular cartilage repair. Platelet rich plasma (PRP) raised increasing interest for a number of applications in orthopedic surgery as a way to enhance tissue healing while reducing pain as it contains multiple growth factors. However, the possible synergic or deleterious effect of PRP on rAAV-mediated transduction has not been reported so far. Here, we examined the effect of PRP upon rAAV-mediated transduction of human bone marrow-derived mesenchymal stem cells (hMSCs).

Methods: rAAV-lacZ carries the E. coli β-galactosidase gene (lacZ) and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP), both controlled by the CMV-IE promoter/enhancer, both controlled by the CMV-IE promoter/enhancer. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 2). PRP samples (n = 4) were provided by the Transfusion Center from Saarland University (403 x 10³ ± 512 x 10² platelets/ μl). hMSCs were prepared as previously described. Cells (passage 1) were seeded in 96-well plates (5,000 cells/well), transduced with rAAV-lacZ or rAAV-RFP (5, 10, or 20 μl) in the presence of PRP (1:2 and 1:4 ratio), and cultured for up to up to 6 days. Transgene expression was monitored by detection of live fluorescence, X-Gal staining, and using the Beta-Glo® Assay System (Promega) as Relative Luminescence Units (RLUs) normalized to the cell numbers. Cell viability was monitored with the Cell Proliferation reagent WST-1 (Roche Applied Science). Each condition was performed in duplicate in two independent experiments. The t-test was employed with P ≤ 0.050 considered statistically significant.

Results and Conclusion: The presence of PRP in the culture medium resulted in a marked inhibition of rAAV-lacZ-mediated transduction of hMSCs). While at day 1, a decrease of rAAV-mediated transduction of hMSCs was observed in the presence of PRP at all the conditions assessed, statistical significant differences were only reached at the lowest amounts of vector (up to 1.9-fold decrease; P = 0.020). Such inhibitory effects significantly increased over time, resulting in an ~ 11-fold decrease of rAAV-mediated gene transfer efficiency in the presence of the compound (P ≤ 0.040). These results were confirmed when monitoring RFP expression, always showing a weaker signal of fluorescence from rAAV-RFP-transduced hMSCs in the presence of PRP. An analysis of the indices of cell viability over time revealed a statistically significant reduction of cell survival in the presence of PRP (up to 3.4- and 2.3- fold decrease on day 1 and respectively; P ≤ 0.030). Combining PRP with rAAV gene transfer vectors resulted in a marked inhibition of the transduction mechanisms mediated by this class of vectors. The presence of neutralizing antibodies against the rAAV capsid proteins in PRP samples may explain this observation.