CELL DIFFERENTIATION ON COLLAGEN-APATITE MATRIX

细胞培养技术虽然对现代生物科学的发展产生了巨大的影响，但在传统的塑料培养皿中，细胞培养技术仍停留在对二维现象的观察上。由于在任何高等生物体中，大多数细胞都被三维细胞外基质包围，因此我们决定使用胶原、羟基磷灰石及相关材料来开发三维细胞支架。我们制备了至少7种不同的细胞载体，包括多孔羟基磷灰石颗粒、胶原珠、纤维胶原膜和纤维玻璃膜，并将其与经三步层析纯化的骨形态发生蛋白(S-300组分)结合，并植入大鼠皮下组织中，进行了体内实验。通过软X射线摄影、组织学观察、碱性磷酸酶活性、II型胶原、骨钙素、DNA和钙含量等方法检测骨和软骨的形成情况。结果表明，将纯化的骨形态发生蛋白(S-300组分)植入羟基磷灰石多孔微粒内，可直接诱导成骨，但未见明显的软骨形成。BMP是一种典型的生长因子，它不仅能诱导未成熟细胞成为软骨细胞，还能诱导成骨样细胞，形成骨样结构，这取决于植入BMP所用的细胞载体的性质。这些发现还表明了基质中的基本物理化学因素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.