gms | German Medical Science

Objectives: Adult articular cartilage has a restricted capability for self-repair. Direct gene based modification of bone marrow aspirates is a stunning tool to treat cartilage defects. In these studies, we investigated application of rAAV-FLAG-hsox9 in minipig bone marrow aspirates seeded in woven poly(ε-caprolactone) scaffolds could prolonged gene expression to enhance a chondrogenic cell population for articular cartilage lesions.

Methods: rAAV vectors were packaged, purified, and titrated as previously described. rAAV-RFP carries the Discosoma sp. red fluorescent protein gene (RFP) and rAAV-FLAG-hsox9 a human FLAG-tagged sox9 sequence, both controlled by the CMV-IE promoter/enhancer. Bone marrow aspirates were obtained from the distal femurs of Göttingen minipigs and transduced with rAAV (15μ l) as previously described. A mixture of fibrinogen (17 mg/ml)/thrombin (5 U/ml) (Baxter, Volketswil, Switzerland) was added to the samples in the presence of woven poly(ε -caprolactone) (PCL) scaffolds for further cultivation in chondrogenic medium for up to 21 days. Transgene expression was detected by live fluorescence. Paraffin-embedded histological sections were prepared for toluidine blue staining and type-II and X collagen immunodetection. The DNA and proteoglycan contents were monitored by Hoechst 22358 assay and by binding to DMMB, respectively. Total RNA was extracted and reverse transcription was carried out for cDNA amplification via SYBR Green real-time RT-PCR. Ct values were obtained for each target gene using GAPDH as a control for normalization and fold inductions (relative to RFP-treated samples) were measured using the 2-Δ Ct method. Each condition was performed in duplicate in two independent experiments. Data are expressed as mean ± SD. The t-test was employed with P < 0.05 considered statistically significant.

Results and Conclusion: Successful detection of RFP expression was noted for at least 21 days in rAAV-RFP-transduced minipig aspirates seeded in PCL scaffolds versus sox9 treatment.Treatment with sox9 significantly increased the DNA and proteoglycan contents in the samples relative to RFP (2.5- and 2.2-fold, respectively) Toluidine blue and anti-type-II collagen immunodetection. Real-time RT-PCR analysis revealed enhanced expression levels of chondrogenic markers (SOX9, COL2A1) (between 2- and 4-fold) and decreased hypertrophic expression profiles (COL1A1, COL10A1) (between 1.4- and 2.5-fold) upon sox9 treatment Gene transfer and overexpression of sox9 via rAAV vectors can successfully modify bone marrow aspirates from minipigs seeded in woven PCL scaffolds, allowing to enhance the chondrogenic differentiation processes in the samples. Analyses are ongoing to translate the approach in vivo by transplanting such constructs in the animals.Combining gene therapy with tissue engineering to modify bone marrow aspirates is of potential value for the development of novel treatments for articular cartilage lesions.