gms | German Medical Science

Objectives: Implantation of genetically modified MSCs with polyurethane (PU) scaffolds is an attractive tool to treat articular cartilage defects. Here we evaluated whether rAAV sox9 gene transfer in hMSCs seeded onto PU scaffolds enhances the chondrogenic differentiation processes in dynamic culture conditions.

Methods: rAAV vectors were packaged, purified, and titrated as previously described. rAAV-lacZ carries the E. coli β -galactosidase ( β-gal) marker gene (lacZ) and rAAV-FLAG-hsox9 a human sox9 FLAG-tagged sequence. 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 transduced with rAAV (40 μl) or let untreated. A mixture of fibrinogen/thrombin was added to the cells that were then seeded in PU scaffolds for 21-days culture in dynamic flow rotating bioreactors in chondrogenic medium. Histological and immunohistochemical analyses were performed on paraffin-embedded sections of the constructs (toluidine blue staining; anti-SOX9, type-II/-I/-X collagen immunostaining). The DNA, proteoglycan, and type-II collagen contents were monitored by Hoechst 22358 assay, binding to DMMB, and ELISA, respectively. Total RNA was extracted using the RNeasy Protect Mini kit and reverse transcription was carried out for cDNA amplification via SYBR Green 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 lacZ-treated samples) were measured using the 2^-ΔΔCt method. Each condition was performed in duplicate in two independent experiments. The t-test was employed with P below 0.05 considered statistically significant.

Results and Conclusion: Sustained sox9 expression was noted in sox9-treated hMSCs on day 21 compared with lacZ transduction. Successful chondrogenic differentiation was achieved in the sox9 samples on day 21 as seen by toluidine blue staining and type-II collagen immunostaining, with less intense reactivity to type-I collagen expression compared with lacZ. Biochemical analyses revealed that sox9 treatment significantly increased the proteoglycan and type-II collagen contents versus lacZ while the DNA contents were lower with sox9. The findings were corroborated by a real-time RT-PCR analysis, revealing enhanced chondrogenic processes (SOX9, COL2A1) with sox9 versus lacZ and reduced hypertrophy (COL1A1, COL10A1).hMSCs genetically modified to overexpress sox9 via rAAV seeded in PU scaffolds undergo enhanced chondrogenic differentiation in dynamic culture conditions. This combined cell, gene, and scaffold approach may find value in developing novel treatments for articular cartilage defects.