Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture.
Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture.
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DOI：
10.1083/jcb.96.3.639
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发表时间：
1983-03
期刊：
影响因子：
--
通讯作者：
Pereira G
中科院分区：
文献类型：
--
作者：
Ecarot-Charrier B;Glorieux FH;van der Rest M;Pereira G
本文报道了一种不使用消化酶从新生小鼠颅骨分离成骨细胞的方法。该过程基于成骨细胞从骨迁移到小玻璃碎片上的能力（Jones，S. J.，和A. Boyde，1977，细胞组织研究，184：179- 193）。将分离的细胞在补充有10%胎牛血清和50微克/ml抗坏血酸的Dulbecco改良Eagle培养基中培养长达14天。7-d培养物与[3 H]脯氨酸孵育24 h。通过胶原酶消化培养基和细胞层蛋白质，发现相对于总蛋白质的高水平胶原蛋白合成。通过SDS PAGE分析来自相同培养物的胃蛋白酶消化的蛋白质表明，I型胶原主要与少量III型和V型（α 1链）胶原一起产生。在β-甘油磷酸盐存在下生长的成骨细胞能够在培养物中启动矿物质沉积。培养物的电子显微镜分析揭示了与细胞外间隙中的胶原纤维和囊泡相关的针状磷灰石样晶体的存在。在相同条件下培养的小鼠皮肤成纤维细胞未能启动矿化。电子组织化学研究显示存在碱性磷酸酶活性，与成骨细胞膜，基质囊泡和胶原纤维上或附近。因此，这些分离的成骨细胞在培养中保留了其独特的启动矿化的特性，因此代表了体外研究矿化过程的有价值的模型。
A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179- 193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle- shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.