gms | German Medical Science

Objectives: Gene-based modification of bone marrow-derived mesenchymal stem cells (MSCs)is an attractive approach to treat articular cartilage defects. Here, we tested the benefits of co-delivering rAAV-FGF-2 and rAAV-IGF-I in human MSCs as a tool to enhance the cartilage repair.

Methods: rAAV-lacZ carries the E. coli  β -galactosidase (lacZ) gene, rAAV-hFGF-2 a human FGF-2 sequence, and rAAV-hIGF-I a human IGF-I sequence, all controlled by the CMV-IE promoter/enhancer. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty, washed, and MSCs we prepared as previously described.Cells were (co)transduced with rAAV (FGF-2 + IGF-I: 40  μ l each vector; lacZ: 40  μ l vector) and kept in chondrogenic medium for up to 21 days. Histological and immunohistochemical analyses were performed on paraffin-embedded sections of the constructs (5  μ m) (toluidine blue staining; anti-FGF-2, anti-IGF-I, and anti-type-II/-I/-X collagen immunostaining). The proteoglycan contents were monitored by binding to dimethylmethylene blue dye and the DNA contents by Hoechst 33258 assay. Total RNA was extracted and reverse transcription carried out for cDNA amplification via real-time RT-PCR with GAPDH as control for normalization and the 2- Δ Δ Ct method relative to untreated samples. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p ≤ 0.05 considered statistically significant.

Results and conclusion: Successful overexpression of the two candidate growth factors was observed in hMSC aggregates upon co-delivery of the rAAV-FGF-2 and IGF-I compared with the control (lacZ). Chondrogenic differentiation was evidenced in the cells cotransduced with FGF-2 and IGF-I (intense toluidine blue staining and type-II collagen immunostaining)that significantly increased the proteoglycan contents to the control treatment (2-fold difference; p ≤ 0.001), while no effects were noted on the DNA contents. Remarkably, immunoreactivity to type-I and -X collagen was less intense with the two FGF-2 and IGF-I candidate vectors. These findings were corroborated by the results of a real-time RT-PCR analysis revealing enhanced chondrogenic differentiation with FGF-2 and IGF-I versus control treatment (3-fold difference in COL2A1 expression; p ≤ 0.001) and reduced hypertrophic differentiation (up to 2- and 2.5 -fold difference in COL1A1 and COL10A1 expression; p ≤ 0.001), probably due to increased SOX9 levels (3-fold difference; p ≤ 0.001). Conclusion: Significant, sustained co-overexpression of FGF-2 with IGF-I in hMSC aggregates upon co-transduction via rAAV is capable of stimulating the chondrogenic activities relative to control treatment in vitro.These results demonstrate the possibility of modifying hMSCs by combined therapeutic growth factor gene transfer as a potent, future reparative strategy for implantation in clinically relevant animal models of cartilage defects in vivo.