Maniatopoulos C, Sodek J, Melcher A H
M.R.C. Programme in Dental Implantology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Cell Tissue Res. 1988 Nov;254(2):317-30. doi: 10.1007/BF00225804.
Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40-43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20-30 days. Cells were cultured in either alpha-minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 micrograms/ml ascorbic acid, or the above medium supplemented with either 10 mM Na-beta-glycerophosphate, 10(-8) M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Gla-protein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both beta-glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Gla-protein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both beta-glycerophosphate and, particularly, dexamethasone.
来自胎儿或新生儿骨骼的细胞能够在体外合成类骨组织。相比之下，成年动物来源的细胞在体外形成类骨组织的情况鲜有报道，且利用骨髓细胞尚未实现。我们探索了骨髓基质细胞在体外形成类骨组织的情况。从40 - 43日龄的Wistar大鼠获取的骨髓基质细胞进行原代培养7天，然后传代培养20 - 30天。细胞在含有15%胎牛血清、抗生素和50微克/毫升抗坏血酸的α - 最低必需培养基中培养，或者在上述培养基中添加10毫摩尔/升β - 甘油磷酸钠、10⁻⁸摩尔/升地塞米松或两者的组合。使用相差显微镜、未脱钙和脱钙组织组织学、组织化学（用于碱性磷酸酶活性）、免疫组织化学（用于胶原类型、骨连接蛋白和骨钙蛋白）、扫描和透射电子显微镜、能量色散X射线微分析以及X射线衍射对培养物进行检查。培养物中形成了具有与骨相似的形态和超微结构特征的胶原矿化结节，但仅在同时存在β - 甘油磷酸钠和地塞米松的情况下。与结节相关的细胞表现出碱性磷酸酶活性。结节的基质主要由I型胶原组成，同时存在骨连接蛋白和骨钙蛋白。X射线微分析显示存在钙和磷，X射线衍射表明矿物质为羟基磷灰石。还对结节进行了骨形态发生蛋白样活性检测。将含有部分脱钙结节和胎儿肌肉的成对扩散小室腹腔内植入大鼠体内。40天后在小室中组织学观察到与肌肉相关的软骨诱导。这一发现为结节的类骨性质提供了进一步的支持。这些观察结果表明，只要培养基中同时含有β - 甘油磷酸钠，特别是地塞米松，年轻成年骨髓培养的细胞就能在体外合成类骨组织。
