Stimulation by 1,25-Dihydroxyvitamin D 3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

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Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH) 2D 3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH) 2D 3, and a significant stimulation of 45Ca accumulation was observed with 3 × 10 −10M 1,25(OH) 2D 3. Although 1,25(OH) 2D 3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH) 2D 3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D 3 nor human PTH(1–34) affected mineralization in the presence or absence of 1,25(OH) 2D 3. These results demonstrate that 1,25(OH) 2D 3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH) 2D 3 has a direct stimulatory effect on bone mineralization process. The mechanism of how 1,25(OH) 2D 3 exerts these effects remains to be clarified.