Development of Osteoinductive Biomaterials (Purification of Osteoinductive Factor and Its Basic Research for Clinical Application)

1.纯化脱钙牛骨基质粉在4M盐酸胍中解离提取，收集提取液中的水溶性蛋白质(组分I)。通过肝素-琼脂糖层析和凝胶排斥层析，得到部分纯化的组分(组分II)。用大鼠肌肉植入的方法对吸附在侧胶原上的组分I或II进行生物测定。根据矿化植入物的碱性磷酸酶活性测量，组分II的活性大约是组分I的680倍。用反相高效液相色谱法对最终组分进行纯化。这一组分的电泳带显示了一条表观分子质量为18 kda的条带。II.生物测定发展了以下方法来确定在短短几天内的成骨能力：1)在体外，在我们的共培养系统中形成了硬组织，其中含有骨诱导因子和来自大鼠骨骼肌组织或大鼠颅骨骨膜下的间充质细胞，它们对该因素具有很高的敏感性。2)通过骨组织的一些组织学或/和生化参数证明了成骨活性。通过对部分纯化的骨诱导因子的植入研究，揭示了一些关于骨诱导机制的新发现。1)骨诱导前骨基质明胶中出现的脱细胞矿物质沉积可能对成骨细胞的分化有重要意义。2)肌肉卫星细胞向成骨细胞分化。3)多孔羟基磷灰石块和侧胶原作为骨诱导因子的载体是有效的。

I. PurificationDecalcified bovine bone matrix powder was extracted dessociatively in 4 M guanidine hydrochloride and the water soluble proteins of the extract was collected (Fraction I). Then, the partial purified fraction (Fraction II) was obtained from fraction I by using heparin-sepharose chromatography and gel-exclusion chromatography. Fraction I or II adsorbed to aterocollagen were bioassayed by intramuscular implantation in rats. Fraction II had approximately 680 times activity as much as fraction I, as measured by alkaline phosphatase activity of the mineralized implant. Final fraction was purified by using reverse-phase HPLC. Electrophoresis of this fraction revealed a band with an apparent molecular mass of 18 kDa.II. BioassayThe following methods have been developed to decide osteoinduclive ability in just a few days.1) in vitroA hard tissue is formed in our co-culture system which contains the osteoinductive factor and mesenchymal cells derived from rat skeletal muscle tissue or rat calvaria subperiosteum, which have a high responsibility to that factor.2) in vivoOsteoinductive activity is demonstrated by some histological or/and biochemical parameters of bone tissue, which is induced by implantation of the complex of osteoinductive factor and aterocollagen.III. ImplantationSome new findings about the mechanism of osteoinduction has been revealed by the investigations on the implantation of partial purified osteoinductive factor.1) Acellular mineral deposition, which appeared in bone matrix gelatin preceding bone induction may be significant for differentiation of osteoblast.2) Muscle satellite cells will differentiate into osteoblasts.3) Porous hydroxyapatite block and aterocollagen are valid as a carrier for osteoinductive factor.