Osteogenic Property of 20a-Hydroxycholesterol on the Surface of Apatite Layer

Objectives: Osteogenic activity of hydroxycholesterols is expected to be useful in the field of biomaterials as well as treatment of osteoporosis. In this study, we employed biomimetic apatite coating as a method to load an osteogenic hydroxycholesterol onto titanium discs. Methods: 20a-hydroxycholesterol (20a-HC), an osteogenic hydroxycholesterol, was loaded in apatite coat in two different ways. First, Ti discs were immersed in DPBS solution containing 20Ą-HC. Second, Ti discs were immersed in DPBS solution without 20a-HC, washed with distilled water, and dried. The dried discs were dipped in 100 % EtOH containing 20a-HC for 10 sec, and dried. Finally, the discs were immersed again in DPBS for another 1 day. In order to investigate cell differentiation on apatite layer, a mouse embryo fibroblast cell line, C3H10T1/2, was cultured on Ti discs for several days, and ALP activity was measured. Results: When 20a-HC and ascorbic acid were directly supplied into media, C3H10T1/2 cells on apatite-coated Ti discs showed a significant increase of ALP activity. However, addition of 20a-HC into apatite coating solution did not affect ALP activity of cells even in the presence of ascorbic acid. When we used the second loading method described above, a dramatic increase in ALP activity was observed even without ascorbic acid. Concentration of calcium ions greatly affected ALP activity in 20a-HC-treated cells in the absence of ascorbic acid. The effect of 20a-HC on ALP activity in the presence of ascorbic acid was significantly attenuated by L-type calcium channel blocker, verapamil and nifedipine. Conclusion: The increase in ALP activity of C3H10T1/2 cells on the apatite layer indicates that 20a-HC tightly remained on apatite. Apatite plays a role in the osteogenic effect of oxysterol as a resource of calcium ions which can attenuate dependence of the osteogenic signaling pathway of oxysterols on ascorbic acid.