Development of matrix-culture system for osteoblastic cells using active-form ascorbic acid

骨形成是一个复杂的过程，包括多种细胞，它们与生长因子或分化因子相互作用。体外细胞培养系统是研究这一过程的有效工具。我们尝试建立一个大规模的成骨样细胞培养体系。使用克隆成骨样细胞系（小鼠）MC 3 T3-E1作为材料。作为初步研究，细胞系在常规塑料皿上培养。细胞增殖、堆积、结节形成、钙化。将细胞培养在直径为0.5- 1.0mm的球形胶原凝胶珠上，细胞增殖并覆盖珠的整个表面，形成最多7层细胞层，将细胞培养在用4种交联剂交联的胶原凝胶上。在这些试剂中，六亚甲基二异氰酸酯（HMDIC）和长链环氧试剂被发现是有效的，以制备用于培养的未收缩凝胶。第三种方法是胶原包埋培养法。在此条件下，细胞增殖并表现出碱性磷酸酶活性增加，这是成骨细胞的特征。当胍/盐酸提取物的骨，粗骨形态发生蛋白，被纳入该系统中，细胞响应碱性磷酸酶活性显着增加。

Osteogenesis is a complex process containing several kinds of cells, which interact each other with growth factors or differentiation factors. In vitro cell culture system is a useful tool for the research on this process. We attempted to establish a large-scale culture system of osteoblast-like cells. A clonal oste*oblast-like cell line (of mouse), MC3T3-E1, was used as a material. As a preliminary study, the cell line was cultured on convensional plastic dishes. The cells proliferated and were stacked in mult-layers, then, formed noduli and finally calcified. The active-form ascorbate was usefui in sustaining cell growth under serum dificient condition.The cells were cultured on spherical beads of collagen gel having a diameter of 0.5-1.0 mm. The cells proliferated and covered the entire surface of the beads forming at most 7 cellular layers.The cells were cultured on collagen gels cross-linked with 4 cross-linking agents. Among these agents, hexamethylenediisocyanate ( HMDIC ) and a long chain epoxy agent were found to be effective to prepare uncontracted gels for culture. The cells pro-liferated and were calcified on the gels.The third method was to culture cells embeded in collagen gels. Under this condition, the cells proliferated and exhibited to increase alkaline phosphatase activity, which is a characteristics of osteoblastic cells. When guanidin/ HCL extracts of bone, crude bone morphogenetic proteins, were incorporated in this system, the cells responded with significant increase in alkaline phosphatase activity.