Bellows C G, Aubin J E, Heersche J N, Antosz M E
Calcif Tissue Int. 1986 Mar;38(3):143-54. doi: 10.1007/BF02556874.
Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na beta-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and beta-glycerophosphate or ascorbic acid alone, three-dimensional nodules (approximately 75 micron thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3-4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II-V bore a linear relationship to the number of cells plated (r = .99). The results indicate that enzymatically released calvaria cells can form mineralized bone nodules in vitro in the presence of ascorbic acid and organic phosphate.
从胎鼠颅骨连续酶消化获得的单细胞悬液，在抗坏血酸、β-甘油磷酸钠和地塞米松存在的情况下进行长期培养，以确定这些细胞群体形成矿化骨的能力。在抗坏血酸和β-甘油磷酸钠存在下或仅在抗坏血酸存在下培养的成骨细胞样细胞中，汇合后3天可检测到由类似成骨细胞的多边形细胞覆盖的三维结节（约75微米厚）。再过3-4天后，结节变得肉眼可见（直径达3毫米）。只有在有机磷酸盐存在的情况下它们才会矿化。培养基中没有抗坏血酸时结节不会形成。地塞米松导致结节数量显著增加。组织学上，结节类似于编织骨，覆盖结节的细胞碱性磷酸酶染色强烈。用特异性抗体进行免疫标记显示，与矿化相关的I型胶原染色强烈，III型胶原和骨连接蛋白染色较弱，II型胶原染色未检测到。I群细胞不会形成结节，II-V群细胞形成的结节数量与接种的细胞数量呈线性关系（r = 0.99）。结果表明，酶解释放的颅骨细胞在抗坏血酸和有机磷酸盐存在的情况下可在体外形成矿化骨结节。
