CELL DIFFERENTIATION ON COLLAGEN-APATITE MATRIX

细胞培养技术对现代生物科学的发展产生了巨大的影响，但传统的塑料平皿细胞支架仍停留在二维现象的观察上。由于在任何高等生物中，大多数细胞都被三维细胞外基质所包围，因此我们决定利用胶原蛋白、羟基磷灰石等材料开发三维细胞支持体。我们设计了至少七种不同的细胞支持体，包括多孔羟基磷灰石颗粒（PPHAP）、胶原珠、纤维胶原膜（FCM）和纤维玻璃膜（FGM），并将这些新的细胞支持体与经三步层析纯化的骨形态发生蛋白（BMP）结合，进行了体内和体外实验（S-300部分），并植入大鼠皮下组织。通过软X线摄片、组织学观察、碱性磷酸酶活性、Ⅱ型胶原、骨钙素、DNA和钙含量测定，证明在多孔羟基磷灰石颗粒中植入纯化的BMP（S-300组份），可直接诱导成骨，而无明显软骨形成。另一方面，玻璃纤维膜与BMP组合仅诱导膜内的软骨，同时膜表面上少量的骨形成。这些发现表明，典型的生长因子BMP能够诱导未成熟细胞不仅成为成软骨细胞，而且成为形成骨样结构的成骨细胞，这取决于用于植入该蛋白质的细胞支持物的性质。这些发现也表明基质中的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.