Enhancement of the ALP activity of C3H10T1/2 cells by the combination of an oxysterol and apatite

Biomimetic apatite coating has been used to load osteogenic biomolecules onto the surface of titanium implants. Apatite on the surface of biomaterials is thought to function as a reservoir of biomolecules as well as enhancing osteoconductivity. In this study, 20 alpha-hydroxycholesterol (20 alpha-HC), an osteogenic oxysterol, was used to induce differentiation of a mouse embryo fibroblast cell line (C3H10T1/2) by loading the oxysterol on biomimetically coated apatite of titanium discs. We found that the phosphatase (alkaline phosphatase (ALP)) activity of 20 alpha-HC was significantly higher with ascorbic acid than alone, suggesting a need for ascorbic acid as a co-factor. When 20 alpha-HC was added into the apatite coating solution, the ALP activity of the C3H10T1/2 cells did not increase on the apatite surface, even in the presence of ascorbic acid. However, ALP activity increased dramatically when 20 alpha-HC was loaded by volatilization of EtOH from the apatite coat after dipping discs in 20 alpha-HC-dissolved EtOH. Interestingly, ascorbic acid was not needed for this increase in ALP activity, suggesting a synergistic effect of 20 alpha-HC and apatite. The concentration of calcium ions, a major component of apatite, affected the osteogenic effect of 20 alpha-HC, and the increase in ALP activity was attenuated by L-type calcium channel inhibitors, verapamil and nifedipine. These results demonstrate that calcium ions released from apatite are important in the synergistic effect of 20 alpha-HC and apatite.