gms | German Medical Science

Objectives: Genetic modification of bone marrow aspirates has recently raised increased attention as a means to enhance cartilage repair. Here, we evaluated the effects of TGF-β overexpression upon the chondrogenic differentiation events in freshly isolated human bone marrow aspirates based on the direct administration of the potential rAAV vectors.

Methods: rAAV were packaged, purified, and titrated as previously described. rAAV-lacZ carries the E. coli β-galactosidase gene and rAAV-hTGF-β a 1.2 kb human transforming growth factor β 1 sequence, both controlled by the CMV-IE promoter/enhancer. Bone marrow was obtained from the distal femurs of patients undergoing total knee arthroplasty (n = 3), aliquoted (100 µl), immediately transduced with rAAV vectors (40 µl), and incubated in chondrogenic medium for 21 days. Detection of transgene expression was performed by TGF- β ELISA. 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 (COL2A1, ACAN) and hypertrophic (COL1A1, COL10A1) markers. Each test condition was performed in duplicate in two independent experiments. The t-test was used where appropriate with P ≤ 0.05 considered statistically significant.

Results and Conclusion: Successful transgene expression was detected in the culture medium of the aspirates for up to 21 days upon transduction with rAAV-hTGF-β compared with lacZ. Overexpression of TGF-β increased the DNA contents in the aspirates relative to lacZ (3.6 ± 2.0 versus 0.7 ± 0.6 µg DNA/mg total proteins on day 21, 5.1-fold difference, P = 0.018). These results were confirmed by H&E staining of histological sections, with higher cell densities via TGF-β (not shown). The proteoglycan contents also increased with TGF-β compared with lacZ (64.3 ± 13.8 versus 50.8 ± 2.9 ng proteoglycans/mg total proteins on day 21, 1.3-fold difference, P = 0.047). Histological and immunohistochemical analyses revealed enhanced chondrogenic differentiation in the aspirates after 21 days of TGF-β treatment as evidenced by higher toluidine blue (matrix proteoglycans) and type-II collagen staining intensities per areas covered with cells versus lacZ. Finally, real-time RT-PCR analyses demonstrated enhanced chondrogenic differentiation with TGF-β (ACAN, COL2A1) and decreased hypertrophy (COL1A1, COL10A1).

rAAV-mediated overexpression of TGF-β is capable of activating the chondrogenic differentiation processes in human bone marrow aspirates for at least 21 days, while significantly delaying premature, undesirable terminal differentiation. Genetic manipulation of human bone marrow aspirates is thus a promising approach to further develop novel, convenient treatments for cartilage repair.