去抗原牛松质骨复合骨髓间充质干细胞重建大鼠眶骨缺损的实验研究

发布时间：2018-05-02 02:09

本文选题：骨髓间充质干细胞 + 牛松质骨　；参考：《天津医科大学》2013年博士论文

【摘要】：第一部分去抗原牛松质骨的制备方法与结构分析目的：探讨去抗原牛松质骨材料的制备方法并分析所制备材料的结构特点。方法：选取新鲜小牛肱骨近端松质骨锯为8mm×5mm×5mm规格颗粒后温水冲洗。分别以0.5%Triton X-100与10%氯化钠脱细胞、甲醇与氯仿脱脂、30%过氧化氢脱蛋白以及0.4M氯化氢部分脱钙处理。双蒸水冲洗后烘干颗粒。钻60辐照后-80℃低温保存。对制备的去抗原松质骨支架进行大体结构和扫描电镜微观结构观察分析以及孔径大小与孔隙率测定。结果：去抗原牛松质骨材料为乳白色疏松多孔结构,孔洞间相互连通成立体网状结构。扫描电镜观察可见彼此相连相互沟通且孔隙大小较为规则的多孔状固态结构。松质骨材料的孔径平均值为(374.2±69.2)μm,孔隙率平均值为(58.1±11.4)%。结论：去抗原牛松质骨材料呈现彼此连通的多孔网状三维立体结构,具有与骨组织工程要求相适应的孔径与孔隙率,具备作为骨组织工程适宜的支架材料应有的基本结构特征。第二部分去抗原牛松质骨载体的生物相容性研究目的：观察与评价经过脱细胞、脱脂、脱蛋白与部分脱钙处理的去抗原牛松质骨支架材料的生物相容性。方法：使用去抗原牛松质骨材料进行以下五个体内实验。一、急性毒性实验：将去抗原牛松质骨材料浸提液与生理盐水分别注入小鼠腹腔,观察记录是否有不良反应或死亡发生。二、热源实验：将去抗原牛松质骨浸提液与生理盐水分别注入家兔耳缘静脉后连续测体温3次。三、溶血实验：将兔血混悬液分别加入去抗原牛松质骨、碳酸钠(阳性对照)、生理盐水(阴性对照)中。观察有无溶血现象并计算溶血率。四、皮内刺激实验：将去抗原牛松质骨、20%乙醇生理盐水分别注射于新西兰白兔皮下,注射后即刻、6h、24h、48h、72h观察记录注射后状况并评分计算局部原发刺激指数和平均原发刺激指数。五、肌袋实验：在大鼠大腿肌袋处植入去抗原牛松质骨颗粒。术后每日观察动物状态并于第7d取材进行组织学分析。结果：去抗原牛松质骨材料植入动物体内后未引起毒性反应、热源反应、溶血反应,未见皮内明显刺激反应产生,且植入大鼠肌肉内后未发生排斥反应。结论：去抗原牛松质骨具有良好的生物相容性。第三部分去抗原牛松质骨载体与绿色荧光蛋白转基因大鼠的骨髓间充质干细胞的细胞相容性目的：探讨去抗原牛松质骨材料与绿色荧光蛋白转基因大鼠的骨髓间充质干细胞的细胞相容性,以及构建去抗原牛松质骨材料与骨髓间充质干细胞复合物的可行性。方法：获取绿色荧光蛋白转基因SD大鼠骨髓,对其中的骨髓间充质干细胞进行分离、纯化、培养、鉴定与多向诱导分化。取第三代骨髓间充质干细胞消化后制备单细胞悬液缓慢滴加于去抗原牛松质骨支架表面后连续培养12d。倒置相差显微镜和荧光显微镜下观察材料表面的细胞形态及其黏附、生长与增殖状态。四甲基偶氮唑盐比色法检测去抗原牛松质骨材料浸提液对大鼠骨髓间充质干细胞的毒性作用。结果：绿色荧光蛋白转基因大鼠骨髓间充质干细胞能粘附于去抗原牛松质骨材料表面上,细胞具有良好形态并发出明亮绿色荧光,生长与增殖状态良好。去抗原牛松质骨与大鼠骨髓间充质干细胞复合培养的12d内,OD值与对照组比较,无显著性差异。去抗原牛松质骨材料浸提液对绿色荧光蛋白转基因大鼠骨髓间充质干细胞的细胞毒性为0～1级。结论：去抗原牛松质骨材料对绿色荧光蛋白转基因大鼠骨髓间充质干细胞无细胞毒性作用,具有良好的细胞相容性；去抗原牛松质骨材料可作为理想支架用于构建细胞-材料复合物。第四部分去抗原牛松质骨复合骨髓间充质干细胞重建大鼠节段性眶骨缺损的实验研究目的：评价去抗原牛松质骨支架复合骨髓间充质干细胞重建大鼠的节段性眶骨缺损的效果。方法：选取经过脱细胞、脱脂、脱蛋白与半脱钙处理后制备的去抗原牛松质骨材料作为支架材料。将绿色荧光蛋白转基因SD大鼠骨髓中分离、提纯、扩增后获取的骨髓间充质干细胞接种于去抗原牛松质骨载体上成功构建细胞-材料复合体,体外成骨诱导培养5天后移植入SD大鼠右侧8mm长的眶骨缺损区域内建立经成骨诱导BMSCs/BCB组；分别设置未经成骨诱导BMSCs/BCB组、单纯BCB组和空白对照组。观察四组动物的伤口愈合与术后一般情况,分别于移植后第2、4、8、12周进行眼眶螺旋CT扫描并进行三维立体眶重建。移植后12周处死动物并收集眶骨移植区域标本制备病理切片,进行眶骨缺损修复的病理学评价与病理形态学分析。结果：细胞-支架复合体移植手术后所有大鼠一般情况良好,未见伤口感染或移植物脱出或排斥反应,局部外观与功能未见异常。经诱导骨髓间充质干细胞复合去抗原牛松质骨移植后第12周,螺旋CT与3D眶重建结果显示植入物能够有效地修复眶缘缺损区域并恢复眶缘的正常完整形态。病理学分析显示去抗原牛松质骨颗粒几乎完全降解,缺损区内大量新骨形成并发生新生骨与原有骨断端之间的骨性连接。病理形态学分析显示移植物植入后缺损区的新骨形成率为(57.12±6.28)%。结论：去抗原牛松质材料具有适宜眶缺损修复的机械强度和骨传导性；去抗原牛松质骨载体复合经诱导的骨髓间充质干细胞能够完整地修复大鼠的节段性眶骨缺损,是一种利用组织工程学方法修复重建眶缺损的有效治疗方案。
[Abstract]:Preparation method and structure analysis of first partial antigen bovine cancellous bone

Objective : To study the preparation method of acellular bovine cancellous bone material and to analyze the structural characteristics of the prepared material .

Methods : After washing with 0.5 % Triton X - 100 and 10 % sodium chloride , methanol and chloroform were degreased , 30 % hydrogen peroxide and 0.4 M hydrogen chloride were removed .

Results : The cancellous bone material of the acellular bovine cancellous bone was a porous structure with a porous structure , and the pores were communicated with each other . The average pore size of cancellous bone material was ( 374.2 卤 69.2 ) 渭m and the average porosity was ( 58.1 卤 11.4 ) % .

Conclusion : The anti - antigen bovine cancellous bone material presents a three - dimensional porous network structure which is in communication with each other , and has the pore size and the porosity which are compatible with the requirements of the bone tissue engineering , and has the basic structural characteristics as a suitable scaffold material for bone tissue engineering .

Biocompatibility of the second partial antigen bovine cancellous bone carrier

Objective : To observe and evaluate the biocompatibility of acellular bovine cancellous bone scaffold material subjected to dedifferentiation , degreasing , deprotein and partial decalcium treatment .

Methods : In order to observe whether there was an adverse effect or a death occurred in the rabbit ' s ear vein after injection of the antigen bovine cancellous bone extract and physiological saline into the abdominal cavity of the rabbits . The results of the experiment were as follows : the antigen bovine cancellous bone , sodium carbonate ( positive control ) and normal saline ( negative control ) were injected into the rabbits .

Results : The antigen bovine cancellous bone material did not induce toxic reaction , heat source reaction , hemolytic reaction , no obvious irritation reaction in skin , and no rejection after implantation in the muscle of rats .

Conclusion : Anti - antigen bovine cancellous bone has good biocompatibility .

Cell Compatibility of Bone Marrow Mesenchymal Stem Cells Derived from the Third Part of Anti - Antigen Bovine cancellous Bone Vector and Green Fluorescent Protein Transgenic Rats

Objective : To investigate the cell compatibility of bone marrow mesenchymal stem cells ( MSCs ) with acellular bovine cancellous bone ( GFP ) and green fluorescent protein ( GFP ) transgenic rats , and to investigate the feasibility of constructing acellular bovine cancellous bone material and bone marrow mesenchymal stem cell complexes .

Methods : The bone marrow of bone marrow mesenchymal stem cells were isolated , purified , cultured , identified and multi - directionally induced by green fluorescent protein transgenic SD rat bone marrow . After the third generation of bone marrow mesenchymal stem cells were digested , the cell morphology and its adhesion , growth and proliferation were observed under the reversed phase contrast microscope and fluorescence microscope .

Results : The green fluorescent protein transgenic rat bone marrow mesenchymal stem cells were able to adhere to the surface of the acellular bovine cancellous bone material . The cells had good morphology and bright green fluorescence , and the growth and proliferation were good . The OD value was not significantly different from that of the control group . The cytotoxicity of the acellular bovine cancellous bone material extract on the bone marrow mesenchymal stem cells of the green fluorescent protein transgenic rats was 0 - 1 .

Conclusion : The anti - antigen bovine cancellous bone material has no cytotoxicity on the bone marrow mesenchymal stem cells of the green fluorescent protein transgenic rat , and has good cell compatibility ;
Anti - antigen bovine cancellous bone material can be used as an ideal scaffold for the construction of cell - material complexes .

Experimental study on the reconstruction of segmental orbital bone defects in rat segmental bone marrow mesenchymal stem cells with the fourth part of acellular bovine cancellous bone

Objective : To evaluate the effect of acellular bovine cancellous bone scaffold composite bone marrow mesenchymal stem cells on segmental orbital bone defects in rats .

Methods : The bone marrow mesenchymal stem cells were isolated , purified and amplified from bone marrow of transgenic SD rats .
After transplantation , orbital spiral CT scanning was performed and three - dimensional orbital reconstruction was performed on the 2nd , 4th , 8th , 12th week after transplantation . The animals were sacrificed at 12 weeks after transplantation and orbital bone graft area specimens were collected to prepare pathological sections .

Results : All the rats were generally in good condition after transplantation of the cell - scaffold complex . There was no abnormal wound infection or graft rejection or rejection , and the local appearance and function were not abnormal . After the induction of bone marrow mesenchymal stem cells , the results of spiral CT and 3D orbital reconstruction showed that the implants were almost completely degraded , and the bony connection between the new bone and the original bone fracture occurred in the defect area .

Conclusion : Anti - antigen bovine cancellous bone material has the mechanical strength and osteoconductivity suitable for orbital defect repair .
Bone marrow mesenchymal stem cells ( MSCs ) induced by acellular bovine cancellous bone can completely repair segmental orbital bone defects in rats , and is an effective treatment scheme for repairing orbital defects by tissue engineering method .

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R777.5

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