gms | German Medical Science

Objectives: Implantation of genetically modified bone marrow aspirates is a promising approach to treat cartilage defects. Here, we tested the benefits of applying an rAAV sox9 vector to human bone marrow aspirates in 3D woven poly(ε-caprolactone) (PCL) scaffolds to stimulate the chondrogenic differentiation processes to enhance cartilage repair.

Methods: rAAV-lacZ carries the E. coli β-galactosidase (lacZ) gene and rAAV-FLAG-hsox9 a human sox9 FLAG-tagged sequence controlled by the CMV-IE promoter/enhancer. Human bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty. Aspirates were transduced with rAAV and fibrinogen/thrombin was added prior to seeding in the woven PCL scaffolds for 21 days in defined chondrogenic medium. Histological/immunohistochemical analyses were performed on paraffin-embedded sections of the constructs (toluidine blue staining; anti-SOX9 and anti-type-II/-I/-X collagen immunostaining). The DNA and proteoglycan contents were monitored by Hoechst 22358 assay and by binding to DMMB, respectively. 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^{- ΔΔCt} 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, over time sox9 expression was noted in the rAAV sox9 aspirates compared with the controls. Chondrogenic differentiation was seen after 21 days especially with rAAV sox9 (toluidine blue staining, type-II collagen immunostaining) that significantly increased the proteoglycan contents (up to 2.2-fold versus controls) while no effects were noted on the DNA contents. Furthermore, type-I and -X collagen immunostaining was less intense with rAAV sox9. These findings were corroborated by real-time RT-PCR (up to 4-fold higher COL2A1 and ACAN expression and up to 15-fold lower COL1A1 and COL10A1 expression with sox9 versus controls, p ≤ 0.001), probably due to increased SOX9 levels (up to 6-fold more with sox9 versus controls, p ≤ 0.001).

Genetic modification of human bone marrow aspirates via rAAV sox9 in woven PCL scaffolds stimulates chondrogenic differentiation processes and delay premature hypertrophy. Such combined cell-, gene-, and tissue engineering-based approach is of value to develop novel, effective treatments for cartilage repair by minimizing the need for complex cell isolation and processing.