CELL DIFFERENTIATION ON COLLAGEN-APATITE MATRIX

尽管细胞培养技术对现代生物科学的发展产生了巨大的影响，但它仍然停留在使用传统的塑料培养皿的扁平细胞载体对二维现象的观察上。由于在任何高等生物中，大多数细胞都被三维细胞外基质包围，因此我们决定利用胶原蛋白、羟基磷灰石等相关材料开发三维细胞支撑体。我们在体内和体外设计并检测了至少7种不同的细胞支持物，包括羟基磷灰石多孔颗粒（PPHAP）、胶原微珠、纤维胶原膜（FCM）和纤维玻璃膜（FGM）。将这些细胞支持物与经三步层析纯化的骨形态发生蛋白（BMP） （S-300组分）结合，植入大鼠皮下组织进行体内实验。采用软x线摄影、组织学观察、碱性磷酸酶活性、II型胶原蛋白、骨钙素、DNA和钙含量测定骨和软骨形成情况。结果表明，当羟基磷灰石多孔颗粒植入纯化BMP （S-300部分）时，骨被直接诱导而没有明显的软骨形成。另一方面，结合BMP的玻璃纤维膜在膜内只诱导软骨形成，同时在膜表面形成少量骨。这些发现表明，BMP是一种典型的生长因子，它不仅能够诱导未成熟细胞成为软骨细胞，还能够诱导形成骨样结构的成骨细胞，这取决于用于植入这种蛋白质的细胞载体的性质。这些发现也提示基质中基本理化因子对细胞分化的重要性。

Although cell culture technology has greatly influenced the advance of modern biological sciences, it still remains in observations of two demensional phenomena using traditional flat cell-supporter of plastic dish. Since in any higher organism, most of the cells are surrounded by the three demensional extracellular matrix, we decided to develop three dimentional cell-supporter by using collagen, hydroxyapatite and related materials. We deviced and examined in vivo and in vitro at least seven different cell supporters including porous particles of hydroxyapatite (PPHAP), collagen beads, fibrous collagen membrane (FCM), and fibrous glass membrane (FGM).In vivo experiments were done combining these new cell-supporters with bone morphogenetic protein (BMP) which had been purified by three steps of cromatographic procedure (S-300 fraction), and implanted into subcutaneous tissue of rats. Bone and cartilage formation were assayed by soft X-ray photography, histological observation, alkaline phosphatase activity, type II collagen, osteocalcin, DNA and calcium contents.It was demonstrated that when porous partifcls of hydroxyapatite were implanted with purified BMP (S-300 fraction), bone was directly induccid without noticeable cartilage formation. On the other hand, fibrous glass membrane combined with BMP induced exclusively cartilage within the membrane accompanying a small amount of bone formation on the surface of the membrane.These findings indicate that a typical growth factor, BMP is able to induce immature cells into becoming not only chondrogenic cells but also into becoming osteogenetic cells that form bone-like structures, depending upon the nature of the cell-supporter used for the implantation of this protein. The findings also suggest the importance of basic physics-chemical factors in matrix for cell differentiation.