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

骨诱导生物材料的开发（骨诱导因子的纯化及其临床应用基础研究）

基本信息

批准号：
01480451
负责人：
MATSUMOTO Naoyuki
金额：
$ 4.16万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

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.

1、纯化用4 M盐酸胍分离提取脱钙牛骨基质粉，提取水溶蛋白（组分一）。然后，通过肝素-sepharose层析和凝胶排除层析得到部分纯化的部分（部分II）。采用大鼠肌内植入法，对ⅰ和ⅱ组分吸附于大鼠动脉胶原进行了生物测定。通过矿化种植体的碱性磷酸酶活性测量，分数II的活性大约是分数I的680倍。最终馏分采用反相高效液相色谱法纯化。电泳结果显示，该组分的表观分子质量为18 kDa.II。生物测定以下几种方法可以在短短几天内测定骨诱导能力。1)体外共培养系统中含有骨诱导因子和来源于大鼠骨骼肌组织或大鼠颅骨骨膜下的间充质细胞形成硬组织，这些细胞对骨诱导因子有很高的责任。2)在体内，骨诱导活性是通过骨诱导因子与软骨胶原复合物的植入诱导的骨组织的一些组织学或生化参数来证明的。部分纯化的骨诱导因子的植入研究揭示了骨诱导机制的一些新发现。1)骨诱导前骨基质明胶中出现的脱细胞矿物沉积可能对成骨细胞的分化具有重要意义。2)肌卫星细胞会向成骨细胞分化。3)多孔羟基磷灰石块和血管间胶原可作为骨诱导因子的载体。