Article
Cooverexpression of transforming growth factor beta and sox9 genes via recombinant adeno-associated virus system enhances the chondrogenic differentiation and metabolic activities in human bone marrow aspirates
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Published: | October 23, 2017 |
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Objectives: Transplantation of genetically modified bone marrow concentrates is an attractive approach to conveniently activate the chondrogenic differentiation processes as a means to improve the intrinsic repair capacities of damaged articular cartilage [1]. Here, we examined the potential benefits of co-overexpressing the pleiotropic transformation growth factor beta (TGF-beta) and the cartilage-specific transcription factor sox9 using recombinant adeno-associated virus (rAAV) vectors upon the chondroreparative processes in human bone marrow aspirates.
Methods: 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. To assess TGF-beta secretion, 30 ul of culture supernatant were collected at the denoted time points 24 h after medium change and TGF-beta production was measured by ELISA as previously described. 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[1,2,3,4].
Results and Conclusion: Successful TGF-beta/sox9 combined gene transfer and overexpression via rAAV was achieved in fresh, chondrogenically induced human bone marrow aspirates for up to 21 days (Fig.1A), 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 (Fig.1B, D). Optimal co-administration of TGF-beta with sox9 also advantageously reduced hypertrophic differentiation in the aspirates (Fig.1C, D). Our results first indicate that combined TGF-beta/sox9 gene transfer (especially at the highest vector doses employed) allowed for the sustained expression of SOX9 as previously noted with single rAAV-FLAG-hsox9 transduction and to a durable production of TGF-beta relative to the control conditions, in the range of those achieved when providing rAAV-hTGF-beta alone[1]. The present data further show that prolonged, effective co-overexpression of TGF-beta and sox9 led to increased levels of cell proliferation, matrix biosynthesis, and chondrogenic differentiation in the aspirates over time (at least 21 days)[3]. Equally important, combined TGF-beta/sox9 transduction advantageously delayed premature hypertrophic differentiation in the aspirates versus control treatments, again in good agreement with the known anti-hypertrophic activities of SOX9 that may counterbalance possible hypertrophic effects of TGF-beta. 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.