In the process of bone remodeling, mineral phase of bone is dissolved by osteoclasts, resulting in elevation of calcium concentration in micro-environment. This study was performed to explore the effect of high extracellular calcium (Cα²+e) on mineralized nodule formation and on the expression of progressive ankylosis (Ank), plasma cell membrane glycoprotein-1 (PC-1) and osteopontin by primary cultured mouse calvarial cells. Osteoblastic differentiation and mineralized nodule formation was induced by culture of mouse calvarial cells in osteoblast differentiation medium containing ascorbic acid and β-glycerophosphate. Although Ank, PC-1 and osteopontin are well known inhibitors of mineralization, expression of these genes were induced at the later stage of osteoblast differentiation during when expression of osteocalcin, a late marker gene of osteoblast differentiation, was induced and mineralization was actively progressing. High Cα²+e(10 mM) treatment highly enhanced mRNA expression of Ank, PC-1 and osteopontin in the late stage of osteoblast differentiation but not in the early stage. Inhibition of p44/42 MAPK activation but not that of protein kinase C suppressed high Cα²+e- induced expression of Ank, PC-1 and osteopontin. When high Cα²+e (5 mM or 10 mM) was present in culture medium during when mineral deposition was actively progressing, matrix calcifiation was significantly increased by high Cα²+e. This stimulatory effect was abolished by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an alkaline phosphatase inhibitor. In addition, probenecid (2mM), an inhibitor of Ank, suppressed matrix calcification in both control and high Cα²+e- treated group, suggesting the possible role of Ank in matrix calcification by osteoblasts. Taken together, these results showed that high Cα²+e stimulates expression of Ank, PC-1 and osteopontin as well as matrix calcification in late differentiation stage of osteoblasts and that p44/42 MAPK activation is involved in high Cα²+e- induced expression of Ank, PC-1 and osteopontin.

This research was designed to investigate changes of growth factors and bone matrix proteins during the bone healing processes using immunohistochemistry and in situ hybridization. Especially this study was focused on the changes of bone matrix and growth factors around the titanium implant. Threaded implants were introduced into the long bone of tibia. Time dependent changes of several bone associated protein and and its mRNAs were observed. Proteins investigated in this study are collagen, osteonectin(ON), osteopontin(OPN), osteocalcin(OC). Expression of the proteins were measured using immunohistochemistry. VEGF and ON were measured using in situ hybridization, and northen blot technique. Bone regeneration were observed as early as the third day of experiment. Matrix proteins and growth factors observed around implant were identical to the proteins observed in the control group. The expression of the ON, OC and VEGF were observed mainly in the osteoblast-like cell on the surface of new bone around the implant and the cells lining the margin of bone defect apart from the implant. The observation may not result from direct osteoconducting activities of titanium but by passive adsorption of extracellular factors which has bone inducing capacities. These passive adsorption results in the immobilization of the growth factors and consequent prolongation of the activities.