构建同种异体组织工程骨及修复犬颅骨临界骨缺损的实验研究

发布时间：2018-06-27 23:59

本文选题：同种异体 + 组织工程骨　；参考：《北京协和医学院》2013年博士论文

【摘要】：目的：组织工程技术为临床骨缺损的治疗带来了新的希望,应用自体骨髓间充质干细胞(autogenic BMSCs, Auto-BMSCs)在体内构建组织工程骨修复骨缺损的方法已基本成熟。然而对于同种异体骨髓间充质干细胞(allogeneic BMSCs, Allo-BMSCs),是否能成功构建组织工程骨,及移植后的免疫原性如何,仍然有很多争论。本课题拟应用同种异体BMSCs与可降解支架复合构建组织工程骨(Tissue engineered bone, TEB),并植入犬背部皮下非受力部位,探索同种异体组织工程骨的异位成骨能力,并检测同种异体BMSCs移植的免疫原性。之后进一步构建犬颅骨临界骨缺损模型,评估同种异体BMSCs复合β-磷酸三钙(β-TCP)修复大型哺乳动物颅骨缺损的可行性。方法： 1.从犬骨髓血中分离纯化BMSCs,体外向成骨、成软骨、成脂三系诱导分化、鉴定。用CM-DiI对成骨诱导至第2代的BMSCs标记后进行体内示踪研究。MTT比色法测定标记前后BMSCs的增殖状况。RT-PCR检测标记细胞中Ⅰ型胶原、骨粘连蛋白、骨形态发生蛋白-2、骨钙素的表达。将标记CM-DiI后的BMSCs复合β-TCP后植入犬背部皮下,8周后取材,荧光显微镜下观察BMSCs体内转归,组织学观察标记BMSCs-TCP复合物异位成骨情况。 2.同种异体BMSCs体内构建异位组织工程骨：成骨诱导至第2代犬BMSCs复合β-TCP分别植入同种异体、自体犬背部皮下,分别作为同种异体组织工程骨组(Allo-TEB组),自体组织工程骨组(Auto-TEB组),单纯β-TCP作为材料对照组(Control组)。手术前及术后3、7、14、28、56天通过流式细胞学分别检测三组的外周血T淋巴细胞亚群的变化,评估全身免疫反应情况。术后24周取材并行HE染色,通过组织学计量分析,量化比较三组的成骨情况。 3.同种异体BMSCs体内构建原位组织工程骨：构建犬双侧颅骨全层临界骨缺损模型,应用同种异体、自体BMSCs-TCP复合物原位移植修复犬颅骨临界骨缺损,分别作分Allo-TEB组,Auto-TEB组,单纯β-TCP作为材料对照组。术后1、3、6、9月通过影像学量化比较三组的颅骨缺损修复情况。术后9月通过大体观察、micro-CT、生物力学和组织学检查,分别评估三组的颅骨缺损修复质量。结果： 1.犬骨髓血中分离得到的BMSCs可以向成骨、成软骨、成脂细胞方向分化,CM-DiI标记BMSCs前后的细胞形态基本一致,两组间细胞的增殖率无显著性差异(P0.05)；标记后RT-PCR可检测到Col-I、BMP-2、BGLAP、SPARC的表达,显示标记对成骨分化无明显影响。标记细胞构建的组织工程骨植入犬皮下8周后取材,标记细胞仍能激发红色荧光,且HE染色证实标记BMSCs构建的组织工程骨可在体内异位成骨。 2.同种异体BMSCs体内构建异位组织工程骨：同种异体、自体BMSCs-TCP复合物均可异位成骨,24周时Allo-TEB组与Auto-TEB组比较,其成骨百分比无显著性差异(P0.05),均显著高于单纯β-TCP对照组(P0.001)。流式细胞学检测显示Allo-TEB组植入第3天、7天时的组内CD4+T淋巴细胞及CD4+/CD8+T高于术前及之后的其他时间点(P0.05),随着时间的延长,Allo-TEB组、Auto-TEB组CD4+/CD8+T细胞的百分比呈先升高后降低的曲线。但Allo-TEB组、Auto-TEB组及Control组组间的CD4+T细胞计数、CD8+T细胞计数、CD4+/CD8+T淋巴细胞的百分比无显著性差异(P0.05)。 3.同种异体BMSCs体内构建原位组织工程骨修复犬颅骨临界骨缺损：术后CT三维重建及计量分析显示Allo-TEB组、Auto-TEB组的组织工程骨骨密度在术后1、3月时有所降低,但在术后6、9月时保持稳定,两组间骨密度无显著性差异(P0.05),材料对照组骨密度随时间延长逐渐降低,在3、6、9月时明显低于上述组织工程骨组(P0.001)。9月时标本大体观察及micro-CT显示同种异体、自体组织工程骨仍能保持颅骨的完整性,抗压能力检测显示Allo-TEB组、Auto-TEB组的组织工程骨之间无显著性差异(P0.05)。组织学检测显示,Allo-TEB组、Auto-TEB组的组织工程骨的类骨质中有大量骨细胞、骨陷窝,在缺损边缘与正常骨之间形成了骨性连接,单纯TCP材料大部分降解,缺损区由纤维组织填充。结论： 1.犬骨髓血中分离得到的BMSCs具备向成骨、成软骨、成脂方向分化的潜能。CM-Dil标记对BMSCs的生长增殖、成骨分化无明显影响。体内示踪实验证实,BMSCs可以在体内至少存活8周,且8周时BMSCs在体内异位构建的组织工程骨的成骨过程中发挥了种子细胞作用。 2.同种异体BMSCs-TCP在体内构建的组织工程骨有异位成骨的作用。同种异体组及自体组的异位成骨能力在24周时无显著性差异。术后两组间的全身免疫反应无显著性差异。同种异体BMSCs没有引起明显的免疫排斥反应。 3.同种异体BMSCs-TCP在体内构建组织工程骨能够原位修复犬颅骨临界骨缺损,成骨速率在早期(3月时)慢于自体组,最终(9月时)两组间无显著性差异。
[Abstract]:Objective:
Tissue engineering technology has brought new hope for the treatment of clinical bone defect. The method of constructing tissue engineered bone defect with autogenous bone marrow mesenchymal stem cells (autogenic BMSCs, Auto-BMSCs) has been basically mature. However, it is possible to construct allogeneic BMSCs (Allo-BMSCs) for allogeneic bone marrow mesenchymal stem cells (Allo-BMSCs). There are still many controversies on how to build tissue engineering bone and the immunogenicity after transplantation. This topic is to construct tissue engineering bone (Tissue engineered bone, TEB) with allogeneic BMSCs and biodegradable scaffold, and to implant the non stressed parts of the dog's back subcutaneous, and explore the heterotopic osteogenesis ability of allograft tissue engineering bone and detect the allograft difference. The immunogenicity of the body BMSCs transplantation was further constructed and the critical bone defect model of the canine skull was further constructed to evaluate the feasibility of the allograft BMSCs compound beta tricalcium phosphate (beta -TCP) for the repair of large mammal skull defects.
Method:
1. isolation and purification of BMSCs from bone marrow blood of dogs, differentiation into osteogenesis, chondrogenesis, and lipid three lines, identification. BMSCs markers induced by osteogenesis to second generations by CM-DiI were traced in vivo. The proliferation of BMSCs before and after the determination of BMSCs by.MTT colorimetric assay was used to detect the type I collagen, osteonectin, and bone morphogenetic eggs in the labeled cells. The expression of white -2, osteocalcin. After labeling the BMSCs compound beta -TCP after CM-DiI, it was implanted subcutaneously in the back of the dog. After 8 weeks, the material was harvested. The changes of BMSCs in vivo were observed under the fluorescence microscope, and the ectopic osteogenesis of the BMSCs-TCP complex was observed by histological observation.
2. the construction of heterotopic tissue engineering bone in BMSCs allograft: osteogenesis induced by osteogenesis to second generation canine compound beta -TCP and subcutaneous allograft in the back of autologous dog, as allograft tissue engineering bone group (group Allo-TEB), autologous tissue engineering bone group (Auto-TEB group), simple beta -TCP as the material control group (Group Control). Before and after operation, the operation and operation were performed. After 3,7,14,28,56 days, the changes of T lymphocyte subsets in the peripheral blood of three groups were detected by flow cytometry, and the whole body immune response was evaluated. 24 weeks after the operation, the three groups were collected in parallel with the HE staining, and the osteogenesis of the three groups was quantified by histologic analysis.
3. in situ tissue engineering bone was constructed in vivo of allogeneic BMSCs: Construction of a canine bilateral cranial critical bone defect model, using allogeneic and autologous BMSCs-TCP complex in situ to repair the critical bone defect of the canine skull, which were divided into Allo-TEB group, Auto-TEB group and simple beta -TCP as the material control group. The image was quantified after 1,3,6,9 months after operation. The three groups of cranium defect repair were compared. In September, three groups of cranial defects were evaluated by gross observation, micro-CT, biomechanics and histological examination.
Result:
1. BMSCs isolated from bone marrow blood could be osteogenic, cartilaginous, and adipocyte differentiation. The cell morphology of CM-DiI before and after BMSCs was basically the same, and there was no significant difference in the proliferation rate between the two groups (P0.05); RT-PCR could detect the expression of Col-I, BMP-2, BGLAP, SPARC, indicating that the markers had no obvious effect on the osteogenesis. The tissue engineered bone constructed by labeled cells was implanted 8 weeks after subcutaneous tissue, and the labeled cells could still stimulate red fluorescence, and HE staining showed that the tissue engineered bone marked by BMSCs could be ectopic osteogenesis in the body.
2. BMSCs allogenic tissue engineering bone was constructed in vivo: Allogenic and autologous BMSCs-TCP complex could be ectopic osteogenesis. At 24 weeks, there was no significant difference in the percentage of bone formation between the Allo-TEB group and the Auto-TEB group (P0.05), which was significantly higher than that in the simple beta -TCP control group (P0.001). The flow cytology test showed that the group of Allo-TEB was implanted for third days and 7 days. The CD4+T lymphocyte and CD4+/CD8+T in the group were higher than the other time points before and after the operation (P0.05). The percentage of CD4+/CD8+T cells in group Allo-TEB and Auto-TEB group increased first and then decreased with the prolongation of time. But the CD4+T cells count, CD8+T cell count, CD4+/CD8+T lymphocytes in group Allo-TEB, Auto-TEB and Control groups were counted. There was no significant difference in percentage (P0.05).
3. BMSCs in situ tissue engineering bone was constructed to repair the critical bone defect of the canine skull. After the operation, CT three-dimensional reconstruction and metrological analysis showed that the bone mineral density of group Auto-TEB decreased at 1,3 months after the operation, but remained stable at 6,9 months after the operation, and there was no significant difference in bone density between the groups (P0.05). The material control was not significant (P0.05). Group bone density gradually decreased with time. At 3,6,9 month, the specimens were significantly lower than that of the tissue engineering bone group (P0.001).9 months, and micro-CT showed the same allograft. Autologous tissue engineering bone still maintained the integrity of the skull. The test of compression ability showed Allo-TEB group, and there was no significant difference between the tissue engineering bone in group Auto-TEB (P0.). 05). The histological examination showed that there were a large number of bone cells and bone lacunae in the tissue engineering bone of group Allo-TEB and Auto-TEB, which formed a bone connection between the defect edge and the normal bone, and the most of the TCP materials were degraded, and the defect area was filled with fibrous tissue.
Conclusion:
1. the BMSCs isolated from the bone marrow blood of the dog has the potential for osteogenesis, cartilage, and adipogenic differentiation..CM-Dil markers have no significant effect on BMSCs growth and proliferation. In vivo tracer experiments confirmed that BMSCs could survive for at least 8 weeks in the body, and at 8 weeks, BMSCs played a role in the osteogenesis of tissue engineered bone constructed in the body. The effect of seed cells.
2. the tissue engineered bone constructed by allogeneic BMSCs-TCP had ectopic osteogenesis. The heterotopic osteogenesis ability of the allograft group and the autologous group had no significant difference at 24 weeks. There was no significant difference in the systemic immune response between the two groups after the operation. The allogenic BMSCs did not cause obvious immunization rejection.
The construction of tissue engineered bone in 3. allogeneic BMSCs-TCP in vivo can repair the critical bone defect of the canine skull in situ, and the rate of osteogenesis is slower in the early (March) than in the autologous group, and there is no significant difference between the two groups at the end of the year.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

【共引文献】