ABSTRACT
Osteoblasts isolated mechanically from newborn mouse calvaria produced a calcified matrix when cultured in the presence of 10 mM beta-glycerophosphate or 3 mM inorganic phosphate. The uncalcified matrix revealed numerous matrix vesicles scattered among collagen fibrils. The calcified matrix showed mineralized collagen fibrils and calcified nodules whose underlying organic matrix was detected after decalcification. These structures resembled those described in fetal and woven bone. In partially decalcified areas, calcification was shown to spread out from these structures along collagen fibrils. Alkaline phosphatase activity was found associated with the plasma membrane and matrix vesicles. X-ray diffraction analysis demonstrated that the mineral phase deposited in culture was hydroxyapatite. These observations which demonstrate that the isolated cells elaborate in culture a mineralized matrix with chemical and ultrastructural properties of woven bone further support the osteoblastic nature of the cells.
Subject(s)
Minerals/analysis , Osteoblasts/analysis , Alkaline Phosphatase/metabolism , Animals , Cells, Cultured , Mice , Microscopy, Electron , Osteoblasts/ultrastructure , X-Ray DiffractionABSTRACT
We have studied the submitochondrial localization of guinea-pig kidney 25-hydroxycholecalciferol 1 alpha-hydroxylase. Treatment of the mitochondrial-enriched fraction with recrystallized digitonin produced mitoplasts bordered by a single membrane and with intact matrix. They contained nearly 90% of the 25-hydroxycholecalciferol 1 alpha-hydroxylase activity and nearly 100% of the cytochrome-c:oxygen oxidoreductase. Amine:oxygen oxidoreductase activity remained mainly in the outer membrane fraction. These data show that 25-hydroxycholecalciferol 1 alpha-hydroxylase has a distribution similar to that of steroid hydroxylases.
