[Cell mediated calcification in three-dimensional collagen gel culture of the osteoblast-like cells derived from rat calvaria].

Osteoblast-like cells were obtained by sequential enzymatic digestion of 19-day fetal rat calvaria and cultured within native type 1 collagen gels in alpha-MEM supplemented with 10% fetal bovine serum and 10 mM beta-glycerophosphate for 3 weeks. Cells were also cultured as monolayers in the usual manner and used as controls. The collagen gel discs contracted by one tenth in diameter after 4 days. The cells in the collagen gels seemed to proliferate at a low rate and to differentiate immediately into round cells, spindle-like cells, and adipocytes. In the monolayer cultures, calcifications were recognized after 2 weeks only in the high-density confluent areas where cells were disposed in multiple layers and alkaline phosphatase activity was high. In the collagen gels, some cells showed high alkaline phosphatase activity after 1 day of culture and calcifications were observed after 1 week around the cells with intense alkaline phosphatase activity. In collagen gel cultures, the degree of alkaline phosphatase activity and of calcification correlated with the density of the cell suspension at the time of seeding. The round cells were thought to be osteoblasts since they showed high alkaline phosphatase activity and were closely related to initial calcifications. Some of the spindle-like cells also showed high alkaline phosphatase activity and were apparently involved in the calcification process in the latter phase of culture, so that some of them seemed to be resting osteoblasts or lining cells. In electron-microscopical studies, initial calcification was related to the so-called matrix vesicles, and hydroxyapatite crystals were detected in mitochondria. Gap junctions which are specific to osteocytes were observed mainly between the round cells, but also between the round cells and the other cells. This culture system in which cells form a three-dimensional network is suitable to investigate osteoblastic functions and cell mediated calcification in vitro.