Simvastatin is a 3-Hydroxy-3-methylglutaryl Co-enzyme inhibitor, widely used to reduce lipid levels. Recent studies have demonstrated pleiotropic beneficial effects the skeleton. We aim to demonstrate the effect of Simvastatin on the osteogenic differentiation and proliferation of murine embryonic stem cells.

Tg2a cells were cultured in maintenance medium until confluence and passaged twice into tissue culture flasks. They were then seeded into 6 well and 24 well plates at a density of 10.000 cells/cm2 and cultured for 3 days in maintenance medium mixed at 1:1 with HepG2 conditioned medium. The culture then continued using osteogenic medium with different concentrations of simvastatin for another 16 days. Measurements included Alizarin Red quantification for calcified matrix, ALP assay, RT-PCR for genes expressed during osteogenic differentiation (osteocalcin, Runx2, osterix, Col1a1).

Simvastatin has dose dependent effect on mineralized matrix formation. Alizarin Red quantification assays demonstrated that simvastatin (all dose groups) induced a statistically significant increase in calcified matrix formation on day 11 (P< 0.05) and 16 (P< 0.01) compared to the control group. ALP activity was significantly higher on day 8 in the groups that had a simvastatin concentration of 1nM, 10nM and 100nM (P< 0.05). RT-PCR has demonstrated that simvastatin caused increased expression of all genes measured on differentiation. Statins can induce bone formation when combined with embryonic stem cells.