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
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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。
Part I The Preparation of Antigen-free Bovine Cancellous Bone Carrier and The Structural Analysis
Objective:To prepare the antigen-free bovine cancellous bone (BCB) carrier and evaluate the structural analysis of BCB.
Methods:The fresh bovine proximal humerus cancellous bone was cuts into8×5×5mm3and rinsed with water. Particles were immersed in0.5%Triton X-100and10%sodium chloride to remove cells. After immersion in the mixture of equal volume of methanol and chloroform to degrease, they were deproteinized with30%hydrogen peroxide. Then they were dunked in0.5M hydrochloric acid for partial decalcification. BCB particles were sterilized by Co-60gamma irradiation and stored at-80℃. The macroscopical structure, microstructure by scanning electron microscopy, the pore diameter and the porosity of BCB particles were observed and analyzed.
Results:The macroscopical structure of BCB scaffolds presented uniformly distributed, interconnected reticular network. SEM analysis showed clear porous structure and trabecular structure of BCB scaffolds. The average value of the BCB porosity was (58.1±11.4)%. The average value of The BCB pore diameter was (374.2±69.2) μm.
Conclusion:The antigen-free bovine cancellous bone has the interconnected reticular structure and meets the demands of the porosity and the pore diameter as scaffold material of bone tissue engineering.
Part Ⅱ The Biocompatibility of Antigen-free Bovine Cancellous Bone
Objective:To study the biocompatibility of the antigen-free bovine cancellous bone.
Methods:The five experiments were performed with antigen-free bovine cancellous bone in vivo. Acute toxicity test:antigen-free bovine cancellous bone extracts were injected into the abdominal cavity of mice. Pyrogen test:antigen-free bovine cancellous bone extracts were injected into the ear vein of the rabbits. Hemolysis test: the mixed blood of rabbits were added into the antigen-free bovine cancellous bone extracts, sodium carbonate (positive control), and normal saline (negative control). Intracutaneous stimulation test:antigen-free bovine cancellous bone extracts were injected into subcutaneous tissue of rabbits. The situation was recorded at the instant,6h,24h,48h,72h after injection and primarilystimulation index and average primarily stimulation index were calculated. Muscle embedding test:antigen-free bovine cancellous bone particles were implanted into the thigh muscle of rats.
Results:antigen-free bovine cancellous bone did not induce acute toxic reaction, pyrogen reaction, hemolysis reactions, hemolytic reaction, skin irritation, and not lead to rejection reaction after it was implanted into the thigh muscle pouches of rats.
Conclusion:Antigen-free bovine cancellous bone has the property of good biocompatibility.
Part Ⅲ The Cytocompatibility of Antigen-free Bovine Cancellous Bone Combined with Bone Marrow Mesenchymal stem cells of Green Fluorescent Protein Transgenic Rats in Vitro
Objective:To test the cytocompatibility of antigen-free bovine cancellous bone (BCB) with induced bone marrow msenchymal stem cells of green fluorescent protein transgenic rats (GFP-BMSCs) in vitro and estimate the feasibility of the establishment of GFP-BMSCs/BCB composites.
Methods:BMSCs of GFP-rats were isolated, purified, cultured, identified and multidirectional induced differentiation was performed. Third-passage BMSCs were digested, Cells suspension was dropped into BCB scaffold and cocultured for12days. The morphology, attachment, growth and proliferation of BMSCs were observed. MTT assay were performed to decide the toxicity of BCB extracts to BMSCs.
Results:BMSCs adhered onto the surface of BCB showed the normal morphology and adhered, grew and proliferate vigorously. There was not a statistically significant difference in the OD value between BMSCs cocultured with BCB extracts and control groups (P>0.05). The cytotoxicity of BCB to BMSCs was grade0-1.
Conclusion:BCB scaffolds having no cytotoxicity to BMSCs have the good cytocompatibility to BMSCs. BCB can be applied as practicable scaffolds for BMSCs in tissue engineering for the reconstruction of orbit defect.
Part IV Reconstruction of Segmental Orbital Defects by Implantation of Antigen-Free Bovine Cancellous Bone Scaffolds Combined with Bone Marrow Mesenchymal Stem Cells in Rats
Objective:To reconstruct the segmental orbital rim defects with antigen-free bovine cancellous bone (BCB) scaffolds combined with bone marrow mesenchymal stem cells (BMSCs) in rats.
Methods:BCB was prepared by removement of cells, degreasing, deproteinization and partly decalcification. BMSCs isolated fromGFP-rats were osteogenically induced and seeded onto BCB scaffolds to construct induced BMSCs/BCB composites. An8mm full-thickness defect on the inferior-orbit rim of40rats was established. Induced BMSCs/BCB composites cultured for5days were implanted into the orbital defects as experimental group. Noninduced BMSCs/BCB group, BCB group and exclusive group were set. General condition of the animals after surgery was recorded daily. Spiral CT and3D orbital reconstruction were performed at2,4,8,12weeks after implantation. Histological analysis and histomorphometric analysis were performed at12weeks after implantation.
Results:The diet, activity and eyeball motility of all animals were normal and no infection, implants dislocation or adverse tissue reactions appeared through all the process after operation. In induced BMSCs/BCB group, CT and3D reconstruction showed perfect orbital repair situation. Histological analysis indicated BCB was biodegraded mostly; newly-formed bone and complete synostosis were observed. The percentage of newly-formed bone was (57.12±6.28)%.
Conclusions:Antigen-free bovine cancellous bone has the moderate mechanical strength and good osteoconduction. BCB combined with BMSCs is a promising composite for tissue engineering and can effectively reconstruct the orbit rim defects in rats.
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。
Part I The Preparation of Antigen-free Bovine Cancellous Bone Carrier and The Structural Analysis
Objective:To prepare the antigen-free bovine cancellous bone (BCB) carrier and evaluate the structural analysis of BCB.
Methods:The fresh bovine proximal humerus cancellous bone was cuts into8×5×5mm3and rinsed with water. Particles were immersed in0.5%Triton X-100and10%sodium chloride to remove cells. After immersion in the mixture of equal volume of methanol and chloroform to degrease, they were deproteinized with30%hydrogen peroxide. Then they were dunked in0.5M hydrochloric acid for partial decalcification. BCB particles were sterilized by Co-60gamma irradiation and stored at-80℃. The macroscopical structure, microstructure by scanning electron microscopy, the pore diameter and the porosity of BCB particles were observed and analyzed.
Results:The macroscopical structure of BCB scaffolds presented uniformly distributed, interconnected reticular network. SEM analysis showed clear porous structure and trabecular structure of BCB scaffolds. The average value of the BCB porosity was (58.1±11.4)%. The average value of The BCB pore diameter was (374.2±69.2) μm.
Conclusion:The antigen-free bovine cancellous bone has the interconnected reticular structure and meets the demands of the porosity and the pore diameter as scaffold material of bone tissue engineering.
Part Ⅱ The Biocompatibility of Antigen-free Bovine Cancellous Bone
Objective:To study the biocompatibility of the antigen-free bovine cancellous bone.
Methods:The five experiments were performed with antigen-free bovine cancellous bone in vivo. Acute toxicity test:antigen-free bovine cancellous bone extracts were injected into the abdominal cavity of mice. Pyrogen test:antigen-free bovine cancellous bone extracts were injected into the ear vein of the rabbits. Hemolysis test: the mixed blood of rabbits were added into the antigen-free bovine cancellous bone extracts, sodium carbonate (positive control), and normal saline (negative control). Intracutaneous stimulation test:antigen-free bovine cancellous bone extracts were injected into subcutaneous tissue of rabbits. The situation was recorded at the instant,6h,24h,48h,72h after injection and primarilystimulation index and average primarily stimulation index were calculated. Muscle embedding test:antigen-free bovine cancellous bone particles were implanted into the thigh muscle of rats.
Results:antigen-free bovine cancellous bone did not induce acute toxic reaction, pyrogen reaction, hemolysis reactions, hemolytic reaction, skin irritation, and not lead to rejection reaction after it was implanted into the thigh muscle pouches of rats.
Conclusion:Antigen-free bovine cancellous bone has the property of good biocompatibility.
Part Ⅲ The Cytocompatibility of Antigen-free Bovine Cancellous Bone Combined with Bone Marrow Mesenchymal stem cells of Green Fluorescent Protein Transgenic Rats in Vitro
Objective:To test the cytocompatibility of antigen-free bovine cancellous bone (BCB) with induced bone marrow msenchymal stem cells of green fluorescent protein transgenic rats (GFP-BMSCs) in vitro and estimate the feasibility of the establishment of GFP-BMSCs/BCB composites.
Methods:BMSCs of GFP-rats were isolated, purified, cultured, identified and multidirectional induced differentiation was performed. Third-passage BMSCs were digested, Cells suspension was dropped into BCB scaffold and cocultured for12days. The morphology, attachment, growth and proliferation of BMSCs were observed. MTT assay were performed to decide the toxicity of BCB extracts to BMSCs.
Results:BMSCs adhered onto the surface of BCB showed the normal morphology and adhered, grew and proliferate vigorously. There was not a statistically significant difference in the OD value between BMSCs cocultured with BCB extracts and control groups (P>0.05). The cytotoxicity of BCB to BMSCs was grade0-1.
Conclusion:BCB scaffolds having no cytotoxicity to BMSCs have the good cytocompatibility to BMSCs. BCB can be applied as practicable scaffolds for BMSCs in tissue engineering for the reconstruction of orbit defect.
Part IV Reconstruction of Segmental Orbital Defects by Implantation of Antigen-Free Bovine Cancellous Bone Scaffolds Combined with Bone Marrow Mesenchymal Stem Cells in Rats
Objective:To reconstruct the segmental orbital rim defects with antigen-free bovine cancellous bone (BCB) scaffolds combined with bone marrow mesenchymal stem cells (BMSCs) in rats.
Methods:BCB was prepared by removement of cells, degreasing, deproteinization and partly decalcification. BMSCs isolated fromGFP-rats were osteogenically induced and seeded onto BCB scaffolds to construct induced BMSCs/BCB composites. An8mm full-thickness defect on the inferior-orbit rim of40rats was established. Induced BMSCs/BCB composites cultured for5days were implanted into the orbital defects as experimental group. Noninduced BMSCs/BCB group, BCB group and exclusive group were set. General condition of the animals after surgery was recorded daily. Spiral CT and3D orbital reconstruction were performed at2,4,8,12weeks after implantation. Histological analysis and histomorphometric analysis were performed at12weeks after implantation.
Results:The diet, activity and eyeball motility of all animals were normal and no infection, implants dislocation or adverse tissue reactions appeared through all the process after operation. In induced BMSCs/BCB group, CT and3D reconstruction showed perfect orbital repair situation. Histological analysis indicated BCB was biodegraded mostly; newly-formed bone and complete synostosis were observed. The percentage of newly-formed bone was (57.12±6.28)%.
Conclusions:Antigen-free bovine cancellous bone has the moderate mechanical strength and good osteoconduction. BCB combined with BMSCs is a promising composite for tissue engineering and can effectively reconstruct the orbit rim defects in rats.
引文
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