骨髓间充质干细胞联合异体骨治疗蒙古羊胫骨缺损的实验研究

发布时间：2019-07-03 13:46

【摘要】：目的：本实验选用体外分离培养的蒙古羊骨髓间充质干细胞接种贴附到同种异体骨上形成载体复合物,植入蒙古羊体内胫骨缺损处,观察细胞-异体骨复合物修复蒙古羊胫骨缺损的疗效,为骨组织工程治疗骨缺损的临床应用提供实验依据。方法：2只1月龄的蒙古羊,在严格无菌条件下穿刺收集股骨骨髓5m1,采用联合培养法分离纯化骨髓间充质细胞,系统地研究其生物学特性以及体外成骨能力。将骨髓间充质干细胞与处理为冻干骨的同种异体骨联合培养24h,制成细胞-异体骨复合物后备用。取9只蒙古羊,体重在50-60kg,用速眠新注射液(0.1m1/kg)麻醉后固定,胫骨处脱毛、碘酒消毒、铺无菌单。手术制造双侧胫骨1cm长的骨一骨膜缺损区,随机选择一侧作为实验组,植入骨髓间充质干细胞与同种异体骨复合物；另一侧为对照组移植单纯同种异体骨。于术后4、8、12周随机选取3只蒙古羊处死做微-CT检查及组织学观察。结果：联合培养法分离得到较高纯度的骨髓间充质干细胞,且细胞形态均一,呈成纤维细胞样,融合后形成明显的细胞生长纹路。骨髓间充质干细胞经地塞米松等可以诱导分化为成骨细胞,碱性磷酸酶、茜素红染色均为阳性。同种异体骨与骨髓间充质干细胞在体外分别联合培养2h、12h、24h、48h,经扫描电镜观察显示异体骨表面及其中心均有细胞贴附生长,且形态多样。证明同种异体骨与骨髓间充质干细胞之间具有良好的相容性。将9只蒙古羊随机分为三组,术后4、8、12周随机抽取一组宰杀取骨,进行微-CT检查及组织学观察。结果显示,各个时期,实验组骨缺损修复效果均优于对照组,骨痂形成量明显多于对照组。这些结果均表明细胞-异体骨复合材料可以治疗蒙古羊胫骨缺损且效果优于单纯异体骨移植。结论： 1.通过密度梯度离心法与贴壁培养法可以分离纯化BMSCs,BMSCs可以作为种子细胞应用到骨组织工程学中。 2. BMSCs与同种异体骨具有良好的生物相容性,二者联合培养可以作为临床治疗胫骨缺损的实验依据。
[Abstract]:Objective: in this experiment, Mongolian sheep bone marrow mesenchymal stem cells isolated and cultured in vitro were inoculated and attached to allogenic bone to form carrier complex and implanted into the tibia defect of Mongolian sheep, and the curative effect of cell-allogenic bone complex in repairing Mongolian sheep tibia defect was observed, so as to provide experimental basis for the clinical application of bone tissue engineering in the treatment of bone defect. Methods: bone marrow 5M1 was collected from 2 1-month-old Mongolian sheep under strict aseptic conditions. Bone marrow mesenchymal cells were isolated and purified by co-culture, and their biological characteristics and osteogenic ability in vitro were systematically studied. Bone marrow mesenchymal stem cells (BMSCs) were co-cultured with allogenic bone treated as freeze-dried bone for 24 hours to make cell-allogenic bone complex and set aside. Nine Mongolian sheep weighing 50 kg and 60 kg were fixed with 0.1m1/kg injection, hair removal in tibia, disinfection with iodine wine and aseptic sheet. The osseous periosteal defect area of bilateral tibia 1cm was made by operation. One side of the experimental group was randomly selected as the experimental group, and the bone marrow mesenchymal stem cells and allogenic bone complex were implanted on the other side, and the control group was treated with simple allogenic bone transplantation. At 4, 8 and 12 weeks after operation, 3 Mongolian sheep were randomly selected for micro-CT examination and histology observation. Results: high purity bone marrow mesenchymal stem cells were isolated by co-culture method, and the cells were uniform in morphology and fibroblasts, and obvious cell growth patterns were formed after fusion. Bone marrow mesenchymal stem cells can be induced to differentiate into osteoblasts by dexamethasone and so on. Alkaline phosphatase and alizalin red staining are positive. Allogenic bone and bone marrow mesenchymal stem cells were co-cultured in vitro for 2 h, 12 h, 24 h and 48 h, respectively. scanning electron microscope (SEM) showed that the allogenic bone surface and its center had cell attachment growth, and the morphology was diverse. It is proved that allogenic bone has good compatibility with bone marrow mesenchymal stem cells. Nine Mongolian sheep were randomly divided into three groups. 4, 8, 12 weeks after operation, a group of slaughtered bones was randomly selected for microCT examination and histology observation. The results showed that the effect of bone defect repair in the experimental group was better than that in the control group, and the amount of eschar formation in the experimental group was significantly higher than that in the control group. These results show that the cell-allogenic bone composite can treat Mongolian sheep tibia defect and the effect is better than that of allogenic bone transplantation alone. Conclusion: 1. BMSCs,BMSCs can be isolated and purified by density gradient centrifugation and adherent culture, which can be used as seed cells in bone tissue engineering. 2. BMSCs has good biocompatibility with allogenic bone, and their co-culture can be used as an experimental basis for clinical treatment of tibia defects.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R681;Q813

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