本文選題：同種異體 + 組織工程骨　； 參考：《北京協(xié)和醫(yī)學院》2013年博士論文

【摘要】：目的： 組織工程技術(shù)為臨床骨缺損的治療帶來了新的希望,應(yīng)用自體骨髓間充質(zhì)干細胞(autogenic BMSCs, Auto-BMSCs)在體內(nèi)構(gòu)建組織工程骨修復(fù)骨缺損的方法已基本成熟。然而對于同種異體骨髓間充質(zhì)干細胞(allogeneic BMSCs, Allo-BMSCs),是否能成功構(gòu)建組織工程骨,及移植后的免疫原性如何,仍然有很多爭論。本課題擬應(yīng)用同種異體BMSCs與可降解支架復(fù)合構(gòu)建組織工程骨(Tissue engineered bone, TEB),并植入犬背部皮下非受力部位,探索同種異體組織工程骨的異位成骨能力,并檢測同種異體BMSCs移植的免疫原性。之后進一步構(gòu)建犬顱骨臨界骨缺損模型,評估同種異體BMSCs復(fù)合β-磷酸三鈣(β-TCP)修復(fù)大型哺乳動物顱骨缺損的可行性。 方法： 1.從犬骨髓血中分離純化BMSCs,體外向成骨、成軟骨、成脂三系誘導分化、鑒定。用CM-DiI對成骨誘導至第2代的BMSCs標記后進行體內(nèi)示蹤研究。MTT比色法測定標記前后BMSCs的增殖狀況。RT-PCR檢測標記細胞中Ⅰ型膠原、骨粘連蛋白、骨形態(tài)發(fā)生蛋白-2、骨鈣素的表達。將標記CM-DiI后的BMSCs復(fù)合β-TCP后植入犬背部皮下,8周后取材,熒光顯微鏡下觀察BMSCs體內(nèi)轉(zhuǎn)歸,組織學觀察標記BMSCs-TCP復(fù)合物異位成骨情況。 2.同種異體BMSCs體內(nèi)構(gòu)建異位組織工程骨：成骨誘導至第2代犬BMSCs復(fù)合β-TCP分別植入同種異體、自體犬背部皮下,分別作為同種異體組織工程骨組(Allo-TEB組),自體組織工程骨組(Auto-TEB組),單純β-TCP作為材料對照組(Control組)。手術(shù)前及術(shù)后3、7、14、28、56天通過流式細胞學分別檢測三組的外周血T淋巴細胞亞群的變化,評估全身免疫反應(yīng)情況。術(shù)后24周取材并行HE染色,通過組織學計量分析,量化比較三組的成骨情況。 3.同種異體BMSCs體內(nèi)構(gòu)建原位組織工程骨：構(gòu)建犬雙側(cè)顱骨全層臨界骨缺損模型,應(yīng)用同種異體、自體BMSCs-TCP復(fù)合物原位移植修復(fù)犬顱骨臨界骨缺損,分別作分Allo-TEB組,Auto-TEB組,單純β-TCP作為材料對照組。術(shù)后1、3、6、9月通過影像學量化比較三組的顱骨缺損修復(fù)情況。術(shù)后9月通過大體觀察、micro-CT、生物力學和組織學檢查,分別評估三組的顱骨缺損修復(fù)質(zhì)量。 結(jié)果： 1.犬骨髓血中分離得到的BMSCs可以向成骨、成軟骨、成脂細胞方向分化,CM-DiI標記BMSCs前后的細胞形態(tài)基本一致,兩組間細胞的增殖率無顯著性差異(P0.05)；標記后RT-PCR可檢測到Col-I、BMP-2、BGLAP、SPARC的表達,顯示標記對成骨分化無明顯影響。標記細胞構(gòu)建的組織工程骨植入犬皮下8周后取材,標記細胞仍能激發(fā)紅色熒光,且HE染色證實標記BMSCs構(gòu)建的組織工程骨可在體內(nèi)異位成骨。 2.同種異體BMSCs體內(nèi)構(gòu)建異位組織工程骨：同種異體、自體BMSCs-TCP復(fù)合物均可異位成骨,24周時Allo-TEB組與Auto-TEB組比較,其成骨百分比無顯著性差異(P0.05),均顯著高于單純β-TCP對照組(P0.001)。流式細胞學檢測顯示Allo-TEB組植入第3天、7天時的組內(nèi)CD4+T淋巴細胞及CD4+/CD8+T高于術(shù)前及之后的其他時間點(P0.05),隨著時間的延長,Allo-TEB組、Auto-TEB組CD4+/CD8+T細胞的百分比呈先升高后降低的曲線。但Allo-TEB組、Auto-TEB組及Control組組間的CD4+T細胞計數(shù)、CD8+T細胞計數(shù)、CD4+/CD8+T淋巴細胞的百分比無顯著性差異(P0.05)。 3.同種異體BMSCs體內(nèi)構(gòu)建原位組織工程骨修復(fù)犬顱骨臨界骨缺損：術(shù)后CT三維重建及計量分析顯示Allo-TEB組、Auto-TEB組的組織工程骨骨密度在術(shù)后1、3月時有所降低,但在術(shù)后6、9月時保持穩(wěn)定,兩組間骨密度無顯著性差異(P0.05),材料對照組骨密度隨時間延長逐漸降低,在3、6、9月時明顯低于上述組織工程骨組(P0.001)。9月時標本大體觀察及micro-CT顯示同種異體、自體組織工程骨仍能保持顱骨的完整性,抗壓能力檢測顯示Allo-TEB組、Auto-TEB組的組織工程骨之間無顯著性差異(P0.05)。組織學檢測顯示,Allo-TEB組、Auto-TEB組的組織工程骨的類骨質(zhì)中有大量骨細胞、骨陷窩,在缺損邊緣與正常骨之間形成了骨性連接,單純TCP材料大部分降解,缺損區(qū)由纖維組織填充。 結(jié)論： 1.犬骨髓血中分離得到的BMSCs具備向成骨、成軟骨、成脂方向分化的潛能。CM-Dil標記對BMSCs的生長增殖、成骨分化無明顯影響。體內(nèi)示蹤實驗證實,BMSCs可以在體內(nèi)至少存活8周,且8周時BMSCs在體內(nèi)異位構(gòu)建的組織工程骨的成骨過程中發(fā)揮了種子細胞作用。 2.同種異體BMSCs-TCP在體內(nèi)構(gòu)建的組織工程骨有異位成骨的作用。同種異體組及自體組的異位成骨能力在24周時無顯著性差異。術(shù)后兩組間的全身免疫反應(yīng)無顯著性差異。同種異體BMSCs沒有引起明顯的免疫排斥反應(yīng)。 3.同種異體BMSCs-TCP在體內(nèi)構(gòu)建組織工程骨能夠原位修復(fù)犬顱骨臨界骨缺損,成骨速率在早期(3月時)慢于自體組,最終(9月時)兩組間無顯著性差異。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R318.08

【共引文獻】

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