同种异体胎兔BMSCs为种子细胞构建组织工程骨的实验研究

发布时间：2018-04-18 02:16

本文选题：组织工程 + 同种异体　；参考：《南华大学》2015年硕士论文

【摘要】：骨缺损在临床上的整复治疗备受关注。传统自体骨移植具有不可避免的二次损伤并且伴随着感染、疼痛等风险的可能。人工骨代用品成骨性低、个体差异性大,并且还存在因其抗原性引起的体内长期排斥与降解速度慢造成的异物反应等缺点。组织工程骨有望成为临床修复下颌骨缺损的理想替代品。目的:探索建立以同种异体家兔胎龄期骨髓间充质干细胞为种子细胞构建组织工程骨的可行性。方法:通过人工受精方法,获得孕期3周的孕兔1只,行剖腹产术取出胎兔7只,冲取胎兔骨髓以贴壁培养法进行体外扩增培养。另16只成年新西兰大白兔无差别分为实验组与空白材料对照组,各8只。取P3代胎龄期BMSCs以20×106 cells/ml的浓度接种DBM继续培养诱导。用经诱导的BMSCs-DBM修复实验组家兔桡骨1.5cm节段缺损,用空白DBM材料按同样手术方法修复空白对照组家兔。术后不同时间进行随访与X线观察。术后12周两组家兔均实行空气注射栓塞致死,行取材后大体观察、Micro-CT影像学检测、组织学染色等检测指标评价修复效果。同时对胎龄期家兔BMSCs行相应数据检测,测量P3代胎兔骨髓间充质干细胞的生长曲线,克隆形成率,并进行成骨、成脂及成软骨诱导分化。另外将原代细胞冻存后30天复苏,测量其P3代生长曲线,对冻存前后增殖能力的变化进行观察。扫描电镜观察胎兔骨髓间充质干细胞与DBM形成细胞材料复合物的体外形态。将BMSCs-DBM复合物植入裸鼠皮下,于术后1、3、6月分别取材行HE、VG、Masson染色观察。结果:胎兔来源的骨髓间充质干细胞倒置相差显微镜下观察细胞饱满均匀,呈梭形或倒三角形状;传代后各代细胞形态未发生明显变化,生长曲线相差不大;成骨、成脂以及成软骨诱导观察到钙结节、脂肪空泡、黏多糖。冻存30天后复苏,其P3代生长曲线与冻存前相比未见明显变化。电镜下观察,与DBM复合7天后细胞能均匀紧密贴合于材料上,伸展良好分布均匀。植入裸鼠皮不同时间点取材进行HE、VG、Masson染色,均显示有新生骨组织生成。诱导BMSCs-DBM植入缺损后通过X线发现阻摄影不断增高,12周大体和Micro-CT见骨愈合。结论:1.家兔来源的骨髓间充质干细胞(BMSCs)经过一系列观察与试验,证明是一种易获取、诱导分化能力较强、与生物材料结合较好的骨组织工程种子细胞。2.单纯脱钙骨基质(DBM)作为单纯生物材料不能修复家兔桡骨标准节段骨缺损。3.同种异体胎龄期骨髓间充质干细胞(BMSCs)结合脱钙骨基质(DBM)构建的组织工程骨能修复兔桡骨原位缺损模型。
[Abstract]:The clinical treatment of bone defect has attracted much attention.Traditional autografts have the potential for inevitable secondary injury and associated risks of infection, pain, and so on.Artificial bone substitute has low osteogenesis, great individual difference, and it also has some shortcomings such as long-term rejection and foreign body reaction caused by its antigenicity.Tissue engineered bone is expected to be an ideal substitute for clinical repair of mandibular defects.Aim: to explore the feasibility of constructing tissue engineered bone with allogeneic bone marrow mesenchymal stem cells (MSCs) as seed cells.Methods: 1 pregnant rabbit was obtained by artificial insemination, 7 fetal rabbits were taken out by caesarean section, bone marrow was extracted from fetal rabbit and cultured by adherent culture in vitro.The other 16 adult New Zealand white rabbits were divided into experimental group and blank control group, 8 rabbits in each group.P3 generation of gestational age BMSCs was inoculated with DBM at 20 脳 10 ~ 6 cells/ml for further induction.The radial 1.5cm segment defect was repaired by induced BMSCs-DBM in the experimental group and the blank DBM material was used to repair the defect in the blank control group.Follow-up and X-ray observation were carried out at different time after operation.12 weeks after operation, the rabbits in both groups were killed by air injection embolization. Micro-CT imaging examination and histological staining were used to evaluate the repair effect.The growth curve of bone marrow mesenchymal stem cells (BMSCs) of P3 generation fetal rabbits were measured. The colony formation rate and osteogenesis, adipogenic and chondrogenic differentiation of P3 generation fetal rabbit bone marrow mesenchymal stem cells (BMSCs) were also measured.In addition, the primary cells were resuscitated 30 days after cryopreservation, the growth curves of P3 generation were measured, and the changes of proliferation ability before and after freezing were observed.The morphology of fetal rabbit bone marrow mesenchymal stem cells (BMSCs) and DBM in vitro was observed by scanning electron microscope (SEM).The BMSCs-DBM complex was implanted subcutaneously into nude mice.Results: the bone marrow mesenchymal stem cells from fetal rabbits were observed to be full and uniform, fusiform or inverted triangle under inverted phase contrast microscope.Calcium nodules, fat vacuoles and mucopolysaccharide were observed in adipogenic and chondrogenic induction.After 30 days of cryopreservation, there was no obvious change in P3 generation growth curve.Electron microscope observation showed that the cells combined with DBM for 7 days could fit to the material evenly and spread well.In nude mice skin samples were collected at different time points for HEV GG Masson staining, all of which showed the formation of new bone tissue.After induced BMSCs-DBM implantation, X-ray findings showed that the bone healing was observed at 12 weeks after continuous increase of resistive radiography and Micro-CT.Conclusion 1.Bone marrow mesenchymal stem cells (BMSCs) from rabbits were proved to be a kind of bone tissue engineering seed cells.Simple decalcified bone matrix (DBM) as a simple biomaterial could not repair the standard segmental bone defect of rabbit radius.The tissue engineering bone constructed by bone marrow mesenchymal stem cells (BMSCs) combined with decalcified bone matrix (DBM) at gestational age could repair the rabbit radial in situ defect model.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R687.3;R318.08

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