Research and Development of New Bone Substitute Using BMP with Atelocollagen

1. 骨形态发生蛋白（Bone Morphogenetic Protein， BMP）的纯化：1)从脱钙牛骨中提取粗BMP，经凝胶过滤、阳离子交换层析、肝素亲和层析分离，最后用反相高效液相色谱分离。SDS-PAGE电泳检测最终BMP-fraction显示在非还原条件下16kDa和30-40kDa两条涂片带，在16kDa和18kDa的还原条件下。氨基酸序列分析显示，该蛋白分子量为16kDa，与J. Wazney等人于1988年报道的BMP-3相似。从人牙齿中提取BMP的纯化方法与牛骨相同。通过一系列色谱分离，发现其洗脱模式与牛骨相似。然而，最终的纯化仍在研究中。BMP-复合材料（AC-BMP、HA-AC-BMP）骨缺损植入的研究：1)将胶原-BMP复合材料（AC-BMP）、羟基磷灰石-胶原-BMP复合材料（HA-AC-BMP）（均经冻干压缩）、HA/AC/BMP混合物（凝胶）植入大鼠颅骨缺损。AC-GMP复合材料和HA-AC-BMP复合材料均能在早期诱导最显著的新骨形成。AC-BMP复合材料和HA-AC-BMP复合材料修复缺损最迅速、最彻底。2) HA-AC-BMP复合材料也被植入猴下颌骨。植入3个月后，RA-AC-BMP复合材料植入的缺损完全由新生骨再生，而作为对照的HA-AC复合材料植入的缺损仅在宿主床边缘形成少量新生骨。说明AC-BMP复合材料和HA-AC-BMP复合材料具有良好的骨诱导和骨再生能力。灵长类动物实验结果提示bmp -复合材料（AC-BMP, HA-AC-BMP）临床应用于骨诱导生物材料替代新鲜自体骨的可能性。少

1. Purification of Bone Morphogenetic Protein (BMP): 1) A crude BMP, which was extracted from decalcified bovine bone, was separated with gel filtration, cation exchange chromatography, heparin affinity chromatography, and finally separated with reverse-phase HPLC. SDS-PAGE erectrophoretic examination of the final BMP-fraction showed two smear bands of 16kDa and 30-40kDa under non- reducing conditions, 16kDa and 18kDa tinder reducing conditions. Amino acid sequence analysis revealed the similarity of the protein with a molecular weight of 16kDa to BMP-3 which was reported by J. Wazney et al. in 1988.2) Purification of BMP extracted from human teeth was also performed in the same way as bovine bone. Separation with a series of chromatographies glowed the similarity of elution patterns to that of bovine bone. However, the final purification is under investigation.2. Examination of BMP-composite (AC-BMP, HA-AC-BMP) by implantation into bone- defects: 1)Aterocoliagen-BMP composite (AC-BMP) … More and liydroxyapatite-atelocollagen -BMP composite (HA-AC-BMP) (both were lyophilized and compressed), HA/AC/BMP mixture (gel) were implanted intoskull defects of rats. Both AC-GMP composite and HA-AC-BMP composite consistently induced the most remarkable new-bone formation from early stage. The repair of defects which had been implanted with AC-BMP composite and HA-AC-BMP composite were the most rapid and complete. 2) HA-AC-BMP composite was also implanted into mandible of monkey. Three months after implantation, the defect was completely regenerated by newly formed bone in the implantation of RA-AC-BMP composite, while small amount of new bone was formed only at the rim of the host bed in the implantation of HA-AC composite as control.These results indicate that AC-BMP composite and HA-AC-BMP composite have excellent bone-inducing and bone-regenerating potency. Results from the Primates suggest the possibility of these BMP-composite (AC-BMP, HA-AC-BMP) inclinical use as bone-inductive biomaterials in place of fresh autogenous bone. Less