Abstract
Purpose: The use of genetically modified bone marrow aspirates may provide convenient, one-step therapeutic approaches to regenerate injured articular cartilage. Here, we examined the effects of overexpressing the mitogenic and pro-anabolic IGF-I factor via the potent and safe rAAV gene transfer vectors on the reparative activities of human osteoarthritic bone marrow aspirates. 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-hIGF-I versus control (reporter) rAAV-lacZ constructs. Aspirates were transduced with each vector and maintained in chondrogenic culture condition for 21 days. Transgene expression was monitored by immunohistochemistry on paraffin-embedded sections of aspirates that were also stained with toluidine blue and for immunodetection of type-II collagen. The DNA and proteoglycan contents were monitored by Hoechst 22358 assay and binding to DMMB, 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, ACAN, 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 duplicate in three experiments. The t-test was employed with P ≤ 0.05 considered statistically significant. Results: Effective IGF-I expression was noted in rAAV-hIGF-I-treated aspirates vis-à-vis control (rAAV-lacZ) condition. Enhanced chondrogenic differentiation was observed in the aspirates after 21 days upon IGF-I gene transfer versus lacZ as seen by a more robust toluidine blue staining and stronger type-II collagen immunoreactivity. The candidate IGF-I treatment also significantly increased the DNA and proteoglycan contents in the aspirates. These findings were corroborated at the gene expression level by a real-time RT-PCR analysis of SOX9, ACAN, and COL2A1. Of further note, IGF-I gene transfer also increased the expression of hypertrophic markers (COL1A1, COL10A1). Conclusions: rAAV vectors can successfully modify human osteoarthritic bone marrow aspirates to overexpress IGF-I, leading to enhanced metabolic and chondrogenic differentiation activities relative to control treatment. Regulating IGF-I production will be necessary for a tight control of terminal differentiation and hypertrophy before adapting the present strategy to treat damaged articular cartilage.
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