小肠粘膜下层复合成骨诱导的脂肪干细胞体内成骨修复骨缺损的实验研究

发布时间：2018-11-01 11:57

【摘要】：目的研究制备具有成骨活性的仿生性骨膜,植入骨缺损区修复骨缺损,为解决骨缺损修复提供新的思路,进而为临床的实际应用,最终代替自体骨及异体骨移植修复骨缺损提供实验依据。方法应用组织工程的技术方法,以组织学、免疫组织化学与动物实验学相结合的方法进行实验研究。选定脂肪干细胞(ADSCs)作为种子细胞,体外成骨诱导为成骨细胞,对其生物学特性进行检测,通过细胞的形态学、碱性磷酸酶活性、茜素红染色、von Kossa染色等技术指标鉴定其向成骨方向分化。将成骨诱导的脂肪干细胞与作为支架材料的小肠粘膜下层(SIS)在体外复合培养构建出具有功能的细胞-材料复合体,制成仿生性骨膜,植入裸鼠皮下,通过组织学、透射电镜、免疫组织化学观察成骨情况及两者的细胞相容性。最后建立节段性骨缺损动物模型,将仿生性骨膜植入骨缺损区修复骨缺损,通过X线摄片、组织形态学检测等方法鉴定其成骨机制与自体骨膜移植成骨的相似性,确定仿生骨膜修复骨缺损的效果。结果体外分离培养扩增兔脂肪干细胞,经成骨诱导后可表达成骨细胞表型。成骨诱导的脂肪干细胞与SIS复合培养可构建出具有生理骨膜结构和功能的仿生骨膜,植入裸鼠体内可形成具有良好血运的新生骨组织。仿生骨膜修复兔节段性骨缺损效果良好,其成骨机制与自体骨膜移植成骨类似。结论兔脂肪干细胞体外成骨诱导培养可转化为成骨细胞,可表达成骨细胞表型,为脂肪干细胞作为骨组织工程的种子细胞提供实验依据。SIS与脂肪干细胞构建的仿生骨膜在动物实验的效果,确定了SIS作为支架材料为组织工程骨的新陈代谢提供了良好的环境,说明两者具有良好的细胞生物相容性。仿生骨膜可修复兔长段骨缺损的实验研究,使组织工程骨膜移植有望成为修复大段骨缺损的有效手段。意义近年来,组织工程技术的迅速发展使现代医学走出了组织、器官移植的范畴,进入了组织、器官再造的新时代。骨缺损是骨科临床上常见的疾病,骨缺损的修复也是临床治疗的难题之一。利用骨组织工程学技术制造仿生性骨膜,为解决骨缺损修复这个难题提供新的思路,寄希望利用患者自身的脂肪干细胞与SIS构建仿生骨膜,应用于临床骨缺损、骨不连的治疗,为骨缺损的修复治疗带来创新性的变革。
[Abstract]:Objective to study the preparation of biomimetic periosteum with osteogenic activity and the implantation of bone defect area to repair the bone defect. The experimental evidence was provided to replace autogenous bone and allogeneic bone graft to repair bone defect. Methods the technique of tissue engineering was used to conduct the experimental study with the combination of histology, immunohistochemistry and animal experiment. Adipose stem cells (ADSCs) were selected as seed cells and osteoblasts were induced into osteoblasts in vitro. The biological characteristics of adipose stem cells were determined by morphology, alkaline phosphatase activity and alizarin red staining. Von Kossa staining and other technical indicators identified its osteogenic differentiation. The osteoblast-induced adipose stem cells were co-cultured with small intestinal submucosal (SIS) as scaffold material to construct a cell-material complex in vitro. The biomimetic periosteum was prepared and implanted into nude mice subcutaneously. The osteogenesis and their cytocompatibility were observed by transmission electron microscope and immunohistochemistry. Finally, the animal model of segmental bone defect was established, and the bone defect was repaired by implanting the biomimetic periosteum into the bone defect area. The mechanism of osteogenesis was identified by X-ray film and histomorphology examination, which was similar to that of autogenous periosteum graft osteogenesis. To determine the effect of biomimetic periosteum in repairing bone defect. Results Rabbit adipose stem cells were isolated and cultured in vitro, and osteoblast phenotype could be expressed after osteogenic induction. The bionic periosteum with physiological periosteum structure and function could be constructed by co-culture of osteogenic adipose stem cells and SIS, and new bone tissue with good blood circulation could be formed in nude mice. Biomimetic periosteum was used to repair segmental bone defect in rabbits, and its osteogenic mechanism was similar to that of autogenous periosteum graft. Conclusion in vitro osteogenic culture of rabbit adipose stem cells can be transformed into osteoblasts and can express osteoblast phenotype. To provide experimental basis for adipose stem cells as seed cells of bone tissue engineering. Effect of bionic periosteum constructed by SIS and adipose stem cells in animal experiments. It was confirmed that SIS as scaffold material provided a good environment for the metabolism of tissue engineered bone, which indicated that the two materials had good biocompatibility. Biomimetic periosteum can be used to repair long bone defect in rabbits, which makes tissue engineered periosteum transplantation an effective method to repair large bone defect. In recent years, with the rapid development of tissue engineering technology, modern medicine has stepped out of the category of tissue and organ transplantation and entered a new era of tissue and organ reconstruction. Bone defect is a common disease in orthopedic department. The repair of bone defect is also one of the difficult problems in clinical treatment. Making biomimetic periosteum by using bone tissue engineering technology provides a new idea for solving the problem of repairing bone defect. It is hoped that the biomimetic periosteum can be constructed by using the adipose stem cells and SIS of the patients themselves, which can be used in the treatment of clinical bone defect and nonunion. For the repair of bone defects to bring innovative changes.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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