gms | German Medical Science

Objectives: Administration of bone marrow aspirates is already a clinical option to treat cartilage defects. Their genetic modification prior to implantation may improve the healing capacities in sites of lesions. Here, we examined the effects of FGF-2 gene transfer via rAAV on the chondrogenic differentiation of human bone marrow aspirates.

Methods: rAAV were packaged, purified, and titrated as previously described. rAAV-lacZ carries the E. coli β-galactosidase gene and rAAV-hFGF-2 human basic fibroblast growth factor cDNA (480 bp), 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 FGF-2 ELISA. Proliferative activities were determined by measuring the DNA contents (Hoechst 22358 assay) and by assessing the cell densities on histological sections (H&E staining). Biological and chondrogenic activities were monitored by measuring the proteoglycan contents (DMMB blue assay, toluidine blue staining) and those for type-II collagen (immunohistochemistry). Real-time RT-PCR analyses were performed to detect the expression of chondrogenic (COL2A1, ACAN) and hypertrophic (COL1A1, RUNX2) markers. Each 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: FGF-2 expression was significantly achieved in the aspirates treated with rAAV-hFGF-2 for up to 21 days compared with the lacZ control. Significantly increased DNA contents (2.3 ± 1.0 versus 0.9 ± 0.6 µg DNA/mg total proteins, 2.6-fold difference, P = 0.046) and cell densities (4.3 ± 2.3 versus 9.7 ± 3.5 103 cells/mm², 2.3-fold difference, P < 0.01) relative to lacZ were detected upon FGF-2 transduction. FGF-2 gene transfer also stimulated the chondrogenic differentiation processes, as evidenced by the increased proteoglycan contents (49.4 ± 0.9 versus 66.2 ± 9.9 ng proteoglycans/mg total proteins on day 21, 1.3-fold difference, P = 0.018) and higher staining intensities per total cell area for toluidine blue (matrix proteoglycans) and type-II collagen compared with lacZ. An analysis by real-time RT-PCR showed enhanced expression profiles for the chondrogenic markers (ACAN, COL2A1) in the aspirates treated with FGF-2 versus lacZ. Strikingly, administration of rAAV-hFGF-2 also increased expression of the hypertrophic markers (COL1A1, RUNX2).

FGF-2 gene transfer of human bone marrow aspirates by rAAV activated chondrogenic processes for at least 21 days. Development of a hypertrophic phenotype suggests that a tight regulation will be required prior to clinical translation of the approach. Current treatment options for articular cartilage defects may be improved using the promising approach of the transplantation of genetically modified bone marrow aspirates.