Objective: We have previously reported that transformation of hydroxyapatite into highly water-solubile calcium phosphates occurred when the hydroxyapatite block was annealed at 1300 degrees C under vacuum, and a polyphasic ceramic which releases alpha-tricalcium phosphate (alpha-TCP) and tetracalcium phosphate (TetCP) could be achieved. The aims of this study were to examine the influence of these soluble calcium phosphates on the proliferation, differentiation, and mineralizing activity of osteoblast, and to determine whether the components released from the polyphasic ceramic could promote osteogenesis, using the culture system of MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in the differentiation medium, to which alpha-TCP or TetCP was added at the concentration of 1 or 10 µM. Cellular proliferation was evaluated by counting the number of cells attached to the culture plate, and the expression of osteopontin, osteocalcin, and type I collagen mRNA was analyzed by RT-PCR, during a 7-day period. Additionally, formation of mineralized bone-like tissue was observed by staining with Alizarin Red S for as long as 14 days. Promotion of mineralization process by the eluates from the polyphasic ceramic was also examined by Alizarin-staining tests. Results: The number of cells was not significantly affected by the addition of either alpha-TCP nor TetCP (Mann-Whitney's U test, p>0.05). However, alpha-TCP enhanced the expression of osteocalcin and type I collagen mRNA. Mineralization process was promoted in all cultures exposed to both soluble calcium phosphates, and the components released from the polyphasic ceramic was confirmed to have the similar effects. Conclusion: These results indicate that alpha-TCP and TetCP could promote mineralization process of MC3T3-E1. Especially, alpha-TCP showed stimulation of the expression of the diffrentiation markes. The polyphasic ceramic has a potential to enhance extracellular matrix formation, subsequently promoting osteogenesis when applied as a bone substitution material.

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