[Establishment of osteoblastic cell line (OR-D 3)]

To establish an osteoblastic cell line, new-born mouse calvaria was cultured in alpha MEM with 10% fetal calf serum. Four colonies with marked cell division were formed at the 3rd generation. By the limiting dilution method, eight clones (A1, B1, C1, C2, C3, D1, D2, D3) were obtained. Cells from C3 and D3 had marked alkaline phosphatase activities. After the addition of 10 mM beta-glycerophosphate to the medium, white nodules stained by Kossa's method were formed in D3 dish. Transmission electron microscopy revealed matrix vesicles, collagen fibers and needle-like crystals of hydroxyapatite in these nodules. These findings indicated that D3 clone had the ability of mineralization.