K. Tao (Peking, CN)
Peking University People's Hospital,Presenter Of 1 Presentation
23.3.1 - Co-overexpression of FGF-2 and SOX9 via rAAV gene transfer enhances chondrogenic and metabolic processes in human bone marrow aspirates
Abstract
Purpose
Transplantation of genetically modified bone marrow concentrates is an attractive strategy to conveniently activate the chondrogenic differentiation processes as a means to improve the intrinsic repair capacities of damaged articular cartilage. In this study, we examined the potential benefits of co-overexpression of the pleiotropic fibroblast growth factor-2 (FGF-2) and the cartilage-specific transcription factor sox9 using recombinant adeno-associated virus (rAAV) vectors upon the chondroreparative processes in human bone marrow aspirates.
Methods and Materials
Human bone marrow aspirates were obtained from the distal femurs of patients undergoing total knee arthroplasty. Aspirates were aliquoted in 96-well plates and immediately transduced with the rAAV vectors (rAAV-lacZ: 20 or 40 μl) or co-transduced (rAAV-hFGF-2 /rAAV-FLAG-hsox9: 10 or 20 μl each vector) with each aliquot. 60 μl chondrogenic medium was then added per aspirate. To assess FGF-2 secretion, 30 μl of culture supernatant were collected at the denoted time points 24 h after medium change and FGF-2 production was measured by ELISA. Transgene expression, and expression of type-II, -I, and -X collagen were assessed by immunohistochemical analyses. Hematoxylin eosin for cellularity, toluidine blue for matrix proteoglycans, and alizarin red for matrix mineralization were also measured. Each condition was performed in duplicate in three independent experiments.
Results
Successful FGF-2/sox9 combined gene transfer and overexpression via rAAV was achieved in chondrogenically induced human bone marrow aspirates for up to 21 days, the longest time point evaluated, leading to increased proliferation, matrix synthesis, and chondrogenic differentiation relative to control treatments (reporter lacZ treatment, absence of vector application) especially when co-applying the candidate vectors at the highest vector doses tested. Moreover, optimal co-administration of FGF-2with sox9 also advantageously reduced hypertrophic differentiation in the aspirates.
Conclusion
These findings report the possibility of directly modifying bone marrow aspirates by combined therapeutic gene transfer as a potent and convenient future approach to improve the repair of articular cartilage lesions.