DISSOCIATIVE EXTRACTION AND RECONSTITUTION OF EXTRACELLULAR-MATRIX COMPONENTS INVOLVED IN LOCAL BONE DIFFERENTIATION
DISSOCIATIVE EXTRACTION AND RECONSTITUTION OF EXTRACELLULAR-MATRIX COMPONENTS INVOLVED IN LOCAL BONE DIFFERENTIATION
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DOI：
10.1073/pnas.78.12.7599
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
REDDI, AH
中科院分区：
文献类型：
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作者：
SAMPATH, TK;REDDI, AH
同种异体大鼠皮下植入脱钙骨干骨基质可诱导软骨内骨分化。3种解离性提取剂，4 M盐酸胍（Gdn·HCl）、8 M尿素/1 M NaCl和1%NaDodSO 4 [十二烷基硫酸钠]（pH 7.4），含有蛋白酶抑制剂溶解骨基质中推定的诱导分子的潜力。用这些浸提剂中的任何一种浸提骨基质均导致骨诱导性能丧失。然后通过对水透析将溶解的提取物与残留物复溶。在植入后第12天，通过定量碱性磷酸酶活性和45 Ca掺入来生物测定各种重构基质的骨诱导潜力。用3种浸提液中的每一种浸提液复溶残留物后，生物活性完全恢复。提取物的聚丙烯酰胺凝胶电泳显示相似的蛋白质谱。在SepharoseCL-4 B上凝胶过滤4 M盐酸钆提取物显示出不均匀的宽峰.当含有<50，000道尔顿的蛋白质的该峰的级分用无活性的4 M Gdn·HCl处理的骨基质重建，然后植入时，诱导了新骨。骨诱导大分子在脱钙骨基质中的解离提取和成功的生物重建被证明。
Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. The potential of 3 dissociative extractants, 4 M guanidine hydrochloride (Gdn.cntdot.HCl), 8 M urea/1 M NaCl and 1% NaDodSO4 [sodium dodecyl sulfate] at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any 1 of these extractants resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 45Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the 3 extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M Gdn.cntdot.HCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins < 50,000 daltons were reconstituted with inactive 4 M Gdn.cntdot.HCl-treated bone matrix and then implanted, new bone was induced. The dissociative extraction and successful biological reconstitution of bone inductive macromolecules were demonstrated in demineralized bone matrix.