异种脱蛋白松质骨作为支架材料的性能分析

Physicochemical properties of xenogeneic deproteinized cancellous bone as stent materials

背景:异种脱蛋白松质骨因其来源广泛和独特的生物学特性有望成为较好的骨组织工程支架,其关键点为免疫原问题,如能解决免疫原问题,则有望开发出一种新型植骨材料。目的:通过理化方法制备脱蛋白松质骨,对其结构、组成及生物安全性能进行分析,探讨异种脱蛋白松质骨作为骨组织工程载体可行性。设计、时间及地点:对比观察实验,于2008-02/2009-01在黑龙江省纤维化生物治疗重点实验室完成。材料:取成年猪和山羊股骨远端松质部分,1∶1氯仿甲醇液脱脂、体积分数30%H2O2脱蛋白,制得脱蛋白松质骨。方法:①通过红外光谱仪、X射线衍射分析仪、氨基酸分析仪、力学测试仪进行载体性能测试。②取第3代人髂骨骨髓间充质干细胞调整浓度为2×108L-1细胞悬液,接种到载体至饱和,倒置显微镜和扫描电镜观察种子细胞黏附及生长增殖情况。③40只BALB/C小鼠随机分为实验组和对照组,每组20只。分离小鼠股后肌肉制成肌袋,实验组植入猪脱蛋白松质骨,对照组直接缝合。术后1,2,4,8周检测分析血清肌苷、谷草转氨酶、谷丙转氨酶水平。④采用免疫酶组织染色方法分析猪脱蛋白松质骨和植入小鼠体内4周脱蛋白松质骨标本的免疫原性。主要观察指标:体外实验分析脱蛋白松质骨理化性能、免疫原性。动物实验分析生物安全性能。结果:脱蛋白松质骨载体无机成分主要是与人骨天然成分相同的羟基磷灰石,经过一系列处理后,胶原蛋白并没有被脱除,这种结构保持了较好的力学性能。脱蛋白松质骨由于脱除了主要的免疫原性物质,其免疫原性较弱,毒性低。人骨髓间充质干细胞接种于载体后生长分化良好。结论:异种脱蛋白松质骨是一种良好骨组织工程载体。

BACKGROUND： Deproteinized cancellous bone can be used as a new kind of bone graft material due to its wide resources and biological characteristics if the problem of immunogen can be solved. OBJECTIVE： To study the properties of deproteinized cancellous bone following preparation with physico-chemical methods, in addition, to explore the feasibility of xenogeneic deproteinized cancellous bone used as scaffolds in bone tissue engineering. DESIGN, TIME AND SETTING： A comparison observation was performed at the Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy between February 2008 and January 2009. MATERIALS： Deproteinized bone was derived from pigs and goals femoral cancellous bone followed by treating with 1：1 methanol extract and 30% H2O2. METHODS： The mechanical properties of vector were examined by scanning electron-microscopy, X-rays diffraction analysis, differential scanning calorimetry, and mechanical experimental instrument. The density of third passage of human bone mesenchymal stem cells （MSCs） of iliac bone was regulated to 2×10^8/L, vaccinated to vector, and the cell adhesion and proliferation was observed by inverted microscope and scanning electron microscope. Forty BALB/C mice were randomly divided into the experimental and control groups, with 20 animals in each group. Muscle behind femur was made muscle pouch, which was implanted pig deproteinized bone in the experimental group, and no other treatment in the control group. The level of serum carnine, glutamic-oxal（o）acetic transaminase （GOT）, as well as glutamic-pyruvic transaminase （GPT） were measured at weeks 1 2, 4, 8 after operation. Meanwhile, the immunogenicity of pig deproteinized bone and specimen of deproteinized bone in mice were detected at the 4 weeks after implantation. MAIN OUTCOME MEASURES： Physicochemical properties and immunogenicity of deproteinized bone were analyzed by in vitro experiment. And the bio-safety was evaluated by animal experiment. RESULTS： The inorganic composition of deproteinized cancellous bone was hydroxyapatite ceramic, which is identical with nature composition of human bone. It showed that deproteinized cancellous bone maintained natural network pore system and kept good mechanical property. The MSCs grew well on deproteinized cancellous bone due to its low immunogenicity and toxicity. CONCLUSION： The xenogeneic deproteinized cancellous bone is an excellent vector in bone tissue engineering.