gms | German Medical Science

Objectives: Gene-based modification of bone marrow aspirates is an attractive approach to treat articular cartilage defects. Here, we examined the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under dynamic versus static culture conditions.

Methods: rAAV were packaged, purified, and titrated as previously described. rAAV-luc carries the Firefly luciferase (luc) gene and rAAV-FLAG-hsox9 a FLAG-tagged human sox9 sequence. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty, immediately transduced with rAAV (40 μl), and maintained either in static culture conditions or in dynamic flow rotating bioreactors for 21 days using chondrogenic medium. Transgene expression was monitored by immunohistochemistry on paraffin-embedded sections of bone marrow aspirates. Sections were stained with safranin O and for type-II, type-I, and type-X collagen. The DNA, proteoglycan, and type-II collagen contents were monitored by Hoechst 22358 assay, binding to DMMB, and ELISA, 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 SOX9, 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 triplicate in three independent experiments. The t-test was employed with P < 0.05 considered statistically significant.

Results and Conclusion: Effective sox9 expression was seen in sox9- versus luc-treated aspirates both in static and dynamic culture conditions that had a more uniform structure. Successful chondrogenic differentiation was observed on day 21 of static or dynamic culture as noted by safranin O staining and type-II collagen immunostaining. Reactivity to type-I and -X collagen was less intense in the presence of sox9. In static culture, sox9 treatment significantly increased the DNA contents relative to luc transduction but no such effects were noted for the proteoglycan and type-II collagen contents. In contrast, both the DNA, proteoglycan, and type-II collagen contents were significantly increased with sox9 in dynamic culture and these effects were more marked in dynamic than in static culture. These findings were corroborated by real-time RT-PCR analysis showing enhanced chondrogenic processes (SOX9, COL2A1) in dynamic versus static culture conditions, with reduced hypertrophic differentiation (COL1A1, COL10A1).rAAV-mediated sox9 gene transfer is a potent approach to genetically modify human bone marrow aspirates with enhanced chondrogenesis in dynamic culture condition. Genetic modification of bone marrow aspirates via rAAV may find value in developing novel treatments for articular cartilage lesions.