Potent chondrogenic differentiation of bone-marrow stem cells by fluocinolone acetonide

Methods: Screening of an FDA-approved drug library was performed by subsequent analysis of a luciferase assay using a reporter plasmid encoding the type-II collagen (col2) promoter, and analysis of expression levels of aggrecan (acan), col2 and Sox-9 by real time RT-PCR and/or western blot, using a pre-chondrogenic cell line (ATDC5 cells). The chondrogenic effect of the selected drug alone or in combination with TGFβ3 (5 ng/mL) was investigated in 21-day micromass cultures of hBMMSCs by means of safranin-O staining for glycosaminoglycans (GAGs) and immunohistochemical (IHC) analysis against col2. Cell cycle analysis was performed with propidium iodide (PI) and antibody against the cell proliferation marker ki-67. The glucocorticoid receptor antagonist (mifepristone) was used for inhibition assays.

Results: Fluocinolone acetonide (FA) was selected as the drug that increased expression of col2 promoter by more than 2 times, and yielded the highest increase in the expression of cartilage marker genes (acan and col2) and Sox-9 in ATDC5 cells. FA strongly enhanced TGFβ3-chondrogenic effect, as demonstrated by a great increase in the expression of Sox-9, col2 and acan, GAG deposition (safranin-O) and IHC-detected col2 in hBMMSC culture treated with both FA and TGFβ3. FA induced cell cycle arrest in G0 phase on the first day, but proliferation rate returned to normal levels on day 3. Treatment with mifepristone partially inhibited GAG formation, and completely inhibited col2 synthesis in hBMMSC cultures.

Conclusions: FA potentiated initial hBMMSC commitment towards chondrogenic lineage by upregulating Sox-9 levels and arresting cell cycle, and strongly enhanced the chondrogenic effect of TGFβ3 in hBMSCs.