应用个性化钛合金修复体和同种异体下颌骨修复比格犬下颌骨缺损的比较研究

发布时间：2018-05-28 13:29

本文选题：下颌骨重建 + 同种异体骨髓间充质干细胞　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：研究背景由于外伤、肿瘤、炎症等因素所导致的大段下颌骨缺损,临床上常采用自体骨移植修复。但是,自体骨移植存在供骨量有限,且可能伴有供区畸形、疼痛等并发症。同种异体下颌骨具有与宿主骨相似的外形轮廓和内部结构,避免了因获取自体骨所造成的二次创伤,成为自体骨移植的替代方法。许多研究证实经过物理化学处理的同种异体骨免疫原性较低,生物力学强度与宿主骨相接近,是较合适的骨缺损修复材料[1-2]。许亦权等应用冷冻/冻干同种异体下颌骨修复犬颞下颌关节及下颌骨缺损,所有动物均获得了临床愈合[3]。李祖兵等应用冻干同种异体下颌骨修复7例大段下颌骨缺损,所有病例均获得较好的修复效果[4]。但是,同种异体骨移植后骨融合缓慢,单独应用同种异体下颌骨常常因感染、骨吸收、免疫排斥等因素影响修复效果[5-7]。为了提高同种异体骨移植后的成功率,学者们采用复合自体骨颗粒、自体骨髓、生长因子等方法来促进同种异体下颌骨移植后的成活[8-10]。有研究采用冷冻同种异体下颌骨复合自体髂骨松质骨颗粒成功修复了 4例大段下颌骨缺损[10]。但是,自体骨髓来源有限,促进骨生长能力受患者自身状况影响,其临床应用受到限制。骨组织工程为大段下颌骨缺损的修复带来了新的希望。但是,大段下颌骨缺损的修复重建要求骨组织工程支架具有与缺损区相同的外形结构和相似的力学强度,目前所应用的支架材料的强度尚无法满足要求。经过处理的同种异体下颌骨可以满足修复需求,是较合适的支架材料。钛合金是临床上常用的人工骨修复材料,随着计算机辅助设计软件和3D打印设备的快速发展,3D打印技术制作的网状钛合金支架具有个性化外形结构和可调控的力学强度,可能是较合适的骨组织工程支架。那么,这两种支架材料能否应用于下颌骨缺损的临床治疗呢,效果有无差别?这需要我们研究。目的探索和比较3D打印个性化钛合金和冷冻干燥同种异体下颌骨修复下颌骨缺损的可行性,为其应用于下颌骨缺损的临床修复提供参考依据。方法1分离培养和鉴定比格犬骨髓来源间充质干细胞。2制备同种异体冻干下颌骨。建立犬下颌骨节段性缺损模型,随机分为3组,分别接受冻干同种异体下颌骨修复;冻干同种异体下颌骨复合自体骨髓间充质干细胞修复;冻干同种异体下颌骨复合同种异体骨髓间充质干细胞修复。采用大体观察,CT扫描,显微CT及组织学评估修复效果。3通过细胞毒性试验,溶血试验,皮肤刺激试验及皮肤致敏试验评价和比较不同3D打印技术制备的钛合金试样的生物相容性。4电子束选区熔化技术制作三维网状钛合金支架。采用壳聚糖、Bio-Oss骨粉和β-甘油磷酸钠制备温敏型水凝胶。骨髓间充质干细胞与水凝胶共培养,CCK-8和死活细胞染色观察细胞在水凝胶中的生长情况。建立比格犬下颌骨体部缺损,随机分为3组,分别接受单纯网状钛合金支架;网状钛合金支架复合壳聚糖/Bio-OSS水凝胶;网状钛合金支架复合壳聚糖/Bio-Oss和同种异体骨髓间充质干细胞修复。采用大体观察、CT、显微CT和组织学检测评估修复效果。5建立比格犬包括颞下颌关节的半侧下颌骨缺损,采用冻干同种异体下颌骨、3D打印个性化实性和网状钛合金修复体修复。通过大体观察、CT及组织学检测来比较三种修复方法的术中操作、外观恢复情况,对非手术侧颞下颌关节的影响,以及对肝脏和肾脏组织的影响。结果1采用密度梯度离心法成功分离培养比格犬骨髓间充质干细胞,所分离细胞具有良好的增殖能力和多向分化能力。2应用同种异体下颌骨移植修复节段性缺损后,术区完全愈合,外形恢复良好。术后CT显示单纯同种异体下颌骨修复组的移植体吸收不明显,复合干细胞的两组移植体体积明显减小。显微CT显示复合同种异体干细胞组和自体干细胞组移植体之间的骨相关参数无明显差异(P0. 05),与单纯修复组相比较,两组移植体新骨形成量明显多于单纯修复组,差异有统计学意义(P0. 05) 。组织学检测显示,复合同种异体干细胞组和自体干细胞组的移植体有明显的新骨形成,而单纯修复组新骨形成量较少。3体外细胞试验结果显示电子束选区熔化和选择性激光熔化制备的钛合金试样对犬骨髓间充质干细胞的生长和成骨分化能力没有明显影响,两种试样之间没有明显差异(p0. 05)。溶血试验结果显示,两种钛合金试样料的溶血率分别为2. 24%和2.46%,均小于5%,提示两种钛合金材料均具有良好的血液相容性。皮肤刺激试验和迟发型超敏反应最大剂量试验结果显示,两种钛合金材料没有潜在的皮肤刺激性和致敏性。4制备的壳聚糖/Bio-Oss水凝胶具有温敏性,在室温下为溶液,在37℃条件下转化为凝胶,所需时间约为10-12min。CCK-8和死活细胞染色结果显示,骨髓间充质干细胞在壳聚糖/Bio-Oss水凝胶中生长良好,活性未受影响。应用3D打印网状钛合金修复下颌骨缺损后,移植体未出现松动、暴露或折断,所有动物均达到临床愈合。术后CT显示网状支架与断端固位良好,未引起周围骨吸收。显微CT显示,复合壳聚糖/Bio-Oss/BMSCs水凝胶的网状钛合金支架组新骨形成量多于复合壳聚糖/Bio-Oss组(p0. 05);两组网状支架内的新骨形成量明显多于单纯网状支架修复组(p0.05)。组织学检测显示,术后6个月,3组网状支架近宿主骨端均有明显新骨形成,复合壳聚糖/Bio-Oss/BMSCs和复合壳聚糖/Bio-Oss组的网眼内的新骨量明显多于单纯网状支架修复组,复合壳聚糖/Bio-Oss/BMSCs水凝胶的网状支架内新骨量最多。5冻干同种异体下颌骨、3D打印个性化实性修复体和3D打印个性化网状修复体术中操作均较方便。同种异体下颌骨术中需要对其进行塑形,而3D打印实性和网状钛合金修复体均不需要塑形。3组修复体修复后,下颌骨外形轮廓恢复较好,面部对称,咬合关系良好。术后CT显示双侧颞下颌关节位置关系未见明显异常。扫描电镜和组织学检测结果显示,三组动物非手术侧关节盘均有退行性变。结论1电子束选区熔化和选择性激光熔化制备的钛合金材料均具有良好的生物相容性,两者之间没有统计学差异。2冻干同种异体下颌骨和电子束选区熔化网状钛合金均可以作为骨组织工程支架修复下颌骨缺损。同种异体下颌骨复合骨髓间充质干细胞后具有较高的成骨能力。复合干细胞和成骨材料能够促进网状钛合金支架内的新骨形成新骨,但是骨再生速度较慢,新生骨量尚不足。今后,还需采用增加促骨生长因子及促血管生长因子等方法来改善成骨速度和成骨质量。3同种异体骨髓间充质干细胞和自体骨髓间充质干细胞均能提高移植体的成骨速度和质量,两者之间没有统计学差异。同种异体骨髓间充质干细胞有望成为骨组织工程种子细胞来源。4冷冻干燥同种异体下颌骨和3D打印个性钛合金修复体均具有操作方便,可个性化修复的优势。三组修复体均能很好地恢复下颌骨缺损区的外形和重建颞下颌关节,是较好的大段下颌骨缺损修复方法。
[Abstract]:Autogenous bone graft is often used to repair large segmental mandibular defects caused by trauma, tumor and inflammation. However, autogenous bone graft is limited in bone supply and may be accompanied by complications such as donor site malformation and pain. The allograft mandible has the contour and internal structure similar to the host bone. The two trauma caused by the acquisition of autogenous bone has become an alternative to autogenous bone transplantation. Many studies have confirmed that the physical and chemical treatment of allogeneic bone has low immunogenicity, and the biomechanical strength is close to the host bone. It is a suitable bone defect repair material, [1-2]. Xu Yi right and so on, for the application of frozen / freeze-dried allogenic mandible repair. The canine temporomandibular joint and mandibular defect, all the animals obtained the clinical healing [3]. Li Zubing and other application of the freeze-dried homograft mandible repair 7 cases of large mandible defect, all cases have obtained better repair effect [4]., but the allograft bone graft is slow, the allograft mandible is often infected, bone is often caused by infection, bone The effects of absorption, immune rejection and other factors on the repair effect of [5-7]. in order to improve the success rate of allograft bone transplantation, scholars have adopted the methods of compound autogenous bone, autologous bone marrow, growth factor to promote the survival of [8-10]. after allograft mandible transplantation. 4 cases of large mandible defect [10]. were successfully repaired, but the source of autologous bone marrow was limited and the bone growth ability was affected by the patient's own condition. Its clinical application was limited. Bone tissue engineering brought new hope for the repair of large mandible defect. However, the reconstruction and reconstruction of large mandibular bone defect required bone tissue engineering scaffold. With the same shape structure and similar mechanical strength as the defect area, the strength of the scaffold material is still unable to meet the requirements. The treated allogenic mandible can meet the needs of repair, and it is a more suitable scaffold material. Titanium alloy is a commonly used artificial bone repair material in clinical, with computer aided design software. With the rapid development of 3D printing equipment, the mesh titanium alloy stent made by 3D printing technology has a personalized structure and controllable mechanical strength. It may be a more suitable scaffold for bone tissue engineering. Then, whether these two scaffolds can be applied to the clinical treatment of mandibular defect, the effect is different? This needs our study. To explore and compare the feasibility of 3D printing of individualized titanium alloy and freeze-dried mandibular reconstruction of mandible defect, and to provide reference for the clinical repair of mandibular defect. Method 1 the bone marrow derived mesenchymal stem cells (.2) of beagle dogs were isolated and cultured to prepare the same kind of homologous freeze-dried mandible. The segmental defect model was randomly divided into 3 groups. The freeze-dried allogenic mandible repair was repaired, the freeze-dried allogenic mandible composite autologous bone marrow mesenchymal stem cells were repaired, the freeze-dried allogenic mandible bone marrow mesenchymal stem cells were repaired. General observation, CT scan, microscopical CT and histological evaluation were used to repair the effect of.3 Cytotoxicity test, hemolysis test, skin irritation test and skin sensitization test evaluation and comparison of biocompatible.4 electron beam selective melting technique of titanium alloy samples prepared by different 3D printing techniques to make three-dimensional mesh titanium alloy scaffold. Chitosan, Bio-Oss bone powder and sodium beta glycerphosphate are used to prepare thermosensitive hydrogels. The growth of cells in hydrogels was observed by CCK-8 and dead cells. The defects of the mandible body of the Beagle were divided into 3 groups, which were divided into simple mesh titanium alloy scaffold, reticular titanium alloy scaffold composite chitosan /Bio-OSS hydrogel, and mesh titanium alloy scaffold composite chitosan /Bio-Oss. Repair of bone marrow mesenchymal stem cells and allogeneic bone marrow mesenchymal stem cells. CT, microscopical CT and histological examination were used to evaluate the repair effect of.5. The mandible defect of the TMJ, including the temporomandibular joint, was established by the freeze-dried homoallograft mandible, 3D printing individualized real and reticular titanium prosthesis repair. Through gross observation, CT and histology Test to compare the operation of the three repair methods, the appearance recovery, the effect on the nonoperative temporomandibular joint, and the effect on the liver and kidney tissue. Results 1 the density gradient centrifugation was used to successfully isolate and culture the bone marrow mesenchymal stem cells of the Beagle dog, and the isolated cells had good proliferation ability and multi differentiation ability.2. After the application of allogenic mandible transplantation to repair segmental defect, the operation area was completely healed and the shape recovered well. After the operation, CT showed that the transplantation body of the pure allogenic mandible repair group was not obvious, and the volume of the two groups of transplanted bodies decreased obviously. The microscopic CT showed the transplantation of the same allograft stem cell group and the autologous stem cell group. There was no significant difference in the bone related parameters between the bodies (P0. 05). Compared with the simple repair group, the new bone formation in the two groups was significantly more than that of the simple repair group. The difference was statistically significant (P0. 05). The histological examination showed that the allograft stem cell group and the autologous stem cell group had obvious new bone formation and the simple repair group. The results of.3 in vitro cell test showed that there was no significant effect on the growth and osteogenic differentiation of the canine bone marrow mesenchymal stem cells by electron beam selective melting and selective laser melting, and there was no significant difference between the two specimens (p0. 05). The results of hemolysis test showed that two titanium alloy samples were found. The hemolysis rates were 2.24% and 2.46%, respectively, both less than 5%, suggesting that the two titanium alloy materials had good blood compatibility. The results of the skin irritation test and the delayed hypersensitivity test showed that the chitosan /Bio-Oss hydrogels prepared by the two titanium alloy materials without potential skin irritation and sensitivities.4 were thermosensitive. At room temperature, the solution was converted to gel at 37 centigrade. The time required was about 10-12min.CCK-8 and dead cell staining results showed that the bone marrow mesenchymal stem cells grew well in the chitosan /Bio-Oss hydrogel, and the activity was not affected. After the application of 3D printing mesh titanium alloy to repair the mandibular defect, the transplanted body did not appear loosening, exposure or folding. CT showed that the reticular scaffold and the broken end were in good position and did not cause the absorption of the surrounding bone after the operation. The micro CT showed that the new bone formation of the reticular titanium alloy stent group of the composite chitosan /Bio-Oss/BMSCs hydrogel was more than that of the compound chitosan /Bio-Oss group (p0. 05), and the new bone formation in the two reticular stents was obviously more than that of the composite scaffold. Simple reticular scaffold repair group (P0.05). Histological examination showed that 6 months after operation, 3 groups of reticular scaffolds had obvious new bone formation near the end of the host bone. The new bone mass in the mesh of compound chitosan /Bio-Oss/BMSCs and compound chitosan /Bio-Oss group was more than that of the simple reticular scaffold repair group, and the reticular branch of the composite chitosan /Bio-Oss/BMSCs hydrogel The new bone mass in the frame is.5 freeze-dried homograft, 3D printing individualized solid prosthesis and 3D print individualized reticular prosthesis are easy to operate. Allograft Mandible needs to be shaped in shape, while 3D printing solid and reticular titanium alloy repair body does not need plastic.3 repair body repair, mandible shape wheel CT showed that there were no obvious abnormalities in the position of bilateral temporomandibular joint. The results of scanning electron microscopy and histological examination showed that there were degenerative changes in the non operative side of the three groups of animals. Conclusion the titanium alloy materials prepared by 1 electron beam selective melting and selective laser melting are good. Good biocompatibility, there is no statistical difference between the two.2 freeze-dried mandible and electron beam selected fusion mesh titanium alloy can be used as bone tissue engineering scaffold to repair the mandible defect. The bone allograft bone marrow mesenchymal stem cells have higher osteogenesis ability. Composite stem cells and osteogenic materials can be used. The new bone in the mesh titanium alloy scaffold is promoted to form new bone, but the rate of bone regeneration is slow and the new bone quantity is still insufficient. In the future, it is necessary to improve the bone growth factor and vascular growth factor to improve the osteogenic speed and the quality of osteogenesis,.3 allogenic bone marrow mesenchymal stem cells and autologous bone marrow mesenchymal stem cells can be improved. There is no statistical difference between the bone formation speed and the quality of the transplanted body. The allogenic bone marrow mesenchymal stem cells are expected to be the advantages of the bone tissue engineering seed cell source.4, the freeze-dried allogenic mandible and the 3D print titanium alloy repair body, which can be easily operated and individualized repair. The three groups of restorations are all good. Restoration of the shape of the mandibular defect area and reconstruction of temporomandibular joint is a good method for repairing large mandibular defects.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R782

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