gms | German Medical Science

Objectives: Transplantation of genetically modified bone marrow aspirates is an attractive approach to improve the healing processes in articular cartilage defects. In the present study, we examined the potential benefits of sustained overexpression of the critical chondrogenic transcription factor sox9 via rAAV gene transfer on the biological activities of bone marrow aspirates freshly isolated from minipigs.

Methods: All transgenes were under the control of the CMV-IE promoter/enhancer: rAAV-lacZ carries the E. coli β-galactosidase gene (lacZ) and rAAV-FLAG-hsox9 a FLAG-tagged human sox9 sequence. Bone marrow aspirates were obtained from the distal femurs of minipigs, aliquoted, and immediately transduced with rAAV vectors (15 µl) and incubated in chondrogenic medium for up to 21 days. Detection of transgene expression was performed by X-Gal staining (lacZ) and by immunohistochemistry (sox9). The DNA and proteoglycan contents were determined with a fluorimetric assay using Hoechst 22358 and by binding to the dimethylmethylene blue dye, respectively. Chondrogenic differentiation processes in the aspirates were monitored by histological (toluidine blue staining) and immunohistochemical analyses (type-II collagen immunodetection). Real-time RT-PCR was performed to analyze the expression of chondrogenic (SOX9, COL2A1, ACAN) and hypertrophic (COL1A1, COL10A1) markers. Each condition was performed in duplicate in three independent experiments. The t-test was used where appropriate with P ≤ 0.05 considered statistically significant.

Results and Conclusion: Results: Successful lacZ expression was evidenced in the aspirates for up to 21 days upon rAAV-lacZ transduction compared with the untreated control. Conversely, sox9 immunoreactivity was stronger in samples where rAAV-FLAG-hsox9 was provided. Application of the sox9 vector led to enhanced DNA contents in the samples compared with the control lacZ condition. Transduction with rAAV-FLAG-hsox9 also increased the proteoglycan contents in the aspirates at the end of the evaluation period compared with lacZ. Enhanced chondrogenic differentiation was noted in the sox9-treated aspirates versus lacZ, as noted by the more robust toluidine blue staining and type-II collagen deposition over time. These findings were confirmed by the results of a real-time RT-PCR analysis, showing an increase in the expression of chondrogenic markers upon sox9 treatment (SOX9, COL2A1, ACAN), while sox9 gene transfer advantageously decreased that of hypertrophic markers (COL1A1, COL10A1).

Conclusion: Successful rAAV-based sox9 gene transfer and overexpression enhances the chondrogenic differentiation processes in bone marrow aspirates from minipigs compared with control treatment over extended periods of time. Direct, convenient genetic manipulation of bone marrow aspirates via rAAV has strong value in developing novel treatments for articular cartilage lesions.