Bone Repair Material Made From Deantigenicity Bovine By Enzymolgy

This study aims at repairing the defect bone to the clinic cure. Removing non-collagen albumen and fat-like substances from xenogeneic bone .the treated bone can be used as deantigenicity materials(DM) for reparing the defect in bone clinic cure. The structure properties and cytocompatible of DM were valuated in the present paper. It is implied that DM have good mechanical properties and cytocompatible. The main work and conclusions as follows:Removing non-collagen albumen and fat-like substances from xenogeneic bone: The fat-like substances in the bone were extracted with chlorlform/methanol(1:1, 50℃) for 72h,and the extracted bone were immerged in the solutions with carbamidine hydrochloride(pH in solution 7.4) and CaCl2 for 24h,0.1%Typsin(1:250,Sigma) for 8h(25℃,bone/liquid=1:1). Test of DM by DSC shows that 15%of the organic matter in bone were removed. By testing the content of hydroxyproline in solutions of treated bone,we can find 0.13% of the hydroxyproline in bone were removed.The like-fat substances and non-collagen albumen can be removed in this way, and collagenⅠwould not be significantly destroy in this way.(2) Test the structure properties of DM:a)By testing the tension and compress properties of fresh bovine cortical bone and DM,we can find that ultimate compress properties of two groups is 128.8MPa and 114.0 MPa respectively(n=5,p<0.05),the young's modulus is 7.732GPa and 6.803 GPa respectively(n=5,p<0.05); The compress modulus and compress ultimate is 12.47±7.64 GPa and 76.51±31.77 MPa respectively. So we can conclude that process of removing fat-like substance and albumen would not significantly destroy mechanical properties of DM.b) Characterizing the structure and constitute content of DM with FTIR spectroscopy, X-ray diffraction spectroscopy, scanning electron microscopy ,we can get results which indicate that some holes(diameter 10μm) distributing in treated cortical bone and cancellous bone. Especially for cancellous bone there is not obvious attachment inside the holes and has better pore size distribution which make it more suitable to be used as material for bone tissue engineering; the main inorganic component of DM is HA,there is very small quantity of CaCO3 in DM, the structure of HA was not be destroy.(3)Osteoblasts culture and identification: We separate osteoblasts from cranial bones of Wistar rat. The acheomycin staining osteoblasts were observed with fluorescence ,we can detect some golden mineralized knur.(4) The 3th generational osteoblasts were implanted into DM with blank as control group and compositively cultivated in 24-hole cultivating board. Then we take cell sample after two days,four days,six days and eight days and evaluate their cytotoxicity by MTT respectively. The result shows that there is not obvious cytotoxicity in the DM and is helpful for the attachment and the proliferation of the osteoblasts. Implant the 3th generational osteoblasts into fresh bone with blank as control group and compositively cultivate it in 50ml cultivating bottle. Then take out some cell sample after four day and seven day cultivation respectively. Quantitative analysis of ALP activity and content of extracellular calcium on the cell samples and staining observation with SEM shows that the attachment , proliferation and expression of the osteoblasts in cancellous bone is normal . so we can say that cancellous bone has better cytocompatible than cortical bone.The innovation of our study is that we take a moderate removing albumen method. The method take advantage of typsin enzyme degradation and carbamidine hydrochloride ability to denaturize albumen to remove the non-collagen albumen in the bovine. It has advantage in lightening the immunity response of heterogeneity replantation and is helpful for the attachment , the proliferation and the function expression of the osteoblasts. Also the collagen fibers and crystal structure of HA will not be seriously destroyed and this make the preparing material have high mechanical property and become a bone implantation ma...