Enhanced chondrogenic differentiation of human osteoarthritic bone marrow aspirates modified to overexpress the transcription factor sox9 via rAAV-mediated gene transfer in dynamic versus static culture conditions

Abstract

Purpose: Administration of bone marrow aspirates modified to overexpress reparative genes is a promising approach to remodel damaged articular cartilage. The goal of this study was to test whether gene transfer of the chondrogenic SOX9 transcription factor in human osteoarthritic bone marrow aspirates via the potent, clinically relevant rAAV vector stimulates the chondrogenic differentiation processes under mechanical loading culture conditions. Methods: Bone marrow aspirates were obtained from the distal femurs of osteoarthritic patients undergoing total knee arthroplasty. All patients provided informed consent before inclusion in the study. rAAV vectors were prepared according to a routine protocol using the candidate rAAV-FLAG-hsox9 versus control (reporter) rAAV-luc (Firefly luciferase) constructs. Aspirates were transduced with each vector and placed in mixed fibrinogen/thrombin for over time (21 days) maintenance in dynamic flow rotating bioreactors versus static culture conditions using chondrogenic medium. Transgene expression was monitored by immunohistochemistry on paraffin-embedded sections of aspirates that were also stained with safranin O and for immunodetection of type-II, type-I, and type-X collagen. The DNA, proteoglycan, and type-II collagen contents were monitored by Hoechst 22358 assay, binding to DMMB, and ELISA, respectively. Total RNA was extracted and reverse transcription was carried out for cDNA amplification via real-time RT-PCR. Ct values were obtained for SOX9, COL2A1, COL1A1, and COL10A1, using GAPDH as a control for normalization, and fold inductions relative to luc-treated samples were measured using the 2-ΔΔCt method. Each condition was performed in triplicate in three experiments. The t-test was employed with P ≤ 0.05 considered statistically significant. Results: Effective sox9 expression was observed in rAAV-FLAG-hsox9- versus rAAV-luc-treated aspirates both in dynamic and static conditions. Successful chondrogenic differentiation in the aspirates was observed after 21 days in both types of culture by safranin O staining and immunodetection of type-II collagen while immunoreactivity to type-I and -X collagen was less intense when applying rAAV sox9. The candidate sox9 treatment significantly increased the DNA contents in static culture versus luc but no effects were noted upon the proteoglycan and type-II collagen contents. In contrast, application of sox9 increased both the DNA, proteoglycan, and type-II collagen contents in dynamic culture versus luc, with effects that were more marked than in static condition. All findings were corroborated at the gene expression level via real-time RT-PCR analysis. Conclusions: Human osteoarthritic bone marrow aspirates can be modified via rAAV to overexpress SOX9, leading to enhanced chondrogenesis in dynamic culture conditions, showing the value of such an approach for the future development of novel treatments to repair damaged articular cartilage.