本文選題：下頜骨重建 + 同種異體骨髓間充質(zhì)干細(xì)胞��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2017年博士論文

【摘要】：研究背景由于外傷、腫瘤、炎癥等因素所導(dǎo)致的大段下頜骨缺損,臨床上常采用自體骨移植修復(fù)。但是,自體骨移植存在供骨量有限,且可能伴有供區(qū)畸形、疼痛等并發(fā)癥。同種異體下頜骨具有與宿主骨相似的外形輪廓和內(nèi)部結(jié)構(gòu),避免了因獲取自體骨所造成的二次創(chuàng)傷,成為自體骨移植的替代方法。許多研究證實(shí)經(jīng)過物理化學(xué)處理的同種異體骨免疫原性較低,生物力學(xué)強(qiáng)度與宿主骨相接近,是較合適的骨缺損修復(fù)材料[1-2]。許亦權(quán)等應(yīng)用冷凍/凍干同種異體下頜骨修復(fù)犬顳下頜關(guān)節(jié)及下頜骨缺損,所有動(dòng)物均獲得了臨床愈合[3]。李祖兵等應(yīng)用凍干同種異體下頜骨修復(fù)7例大段下頜骨缺損,所有病例均獲得較好的修復(fù)效果[4]。但是,同種異體骨移植后骨融合緩慢,單獨(dú)應(yīng)用同種異體下頜骨常常因感染、骨吸收、免疫排斥等因素影響修復(fù)效果[5-7]。為了提高同種異體骨移植后的成功率,學(xué)者們采用復(fù)合自體骨顆粒、自體骨髓、生長(zhǎng)因子等方法來促進(jìn)同種異體下頜骨移植后的成活[8-10]。有研究采用冷凍同種異體下頜骨復(fù)合自體髂骨松質(zhì)骨顆粒成功修復(fù)了 4例大段下頜骨缺損[10]。但是,自體骨髓來源有限,促進(jìn)骨生長(zhǎng)能力受患者自身狀況影響,其臨床應(yīng)用受到限制。骨組織工程為大段下頜骨缺損的修復(fù)帶來了新的希望。但是,大段下頜骨缺損的修復(fù)重建要求骨組織工程支架具有與缺損區(qū)相同的外形結(jié)構(gòu)和相似的力學(xué)強(qiáng)度,目前所應(yīng)用的支架材料的強(qiáng)度尚無法滿足要求。經(jīng)過處理的同種異體下頜骨可以滿足修復(fù)需求,是較合適的支架材料。鈦合金是臨床上常用的人工骨修復(fù)材料,隨著計(jì)算機(jī)輔助設(shè)計(jì)軟件和3D打印設(shè)備的快速發(fā)展,3D打印技術(shù)制作的網(wǎng)狀鈦合金支架具有個(gè)性化外形結(jié)構(gòu)和可調(diào)控的力學(xué)強(qiáng)度,可能是較合適的骨組織工程支架。那么,這兩種支架材料能否應(yīng)用于下頜骨缺損的臨床治療呢,效果有無差別?這需要我們研究。目的探索和比較3D打印個(gè)性化鈦合金和冷凍干燥同種異體下頜骨修復(fù)下頜骨缺損的可行性,為其應(yīng)用于下頜骨缺損的臨床修復(fù)提供參考依據(jù)。方法1分離培養(yǎng)和鑒定比格犬骨髓來源間充質(zhì)干細(xì)胞。2制備同種異體凍干下頜骨。建立犬下頜骨節(jié)段性缺損模型,隨機(jī)分為3組,分別接受凍干同種異體下頜骨修復(fù);凍干同種異體下頜骨復(fù)合自體骨髓間充質(zhì)干細(xì)胞修復(fù);凍干同種異體下頜骨復(fù)合同種異體骨髓間充質(zhì)干細(xì)胞修復(fù)。采用大體觀察,CT掃描,顯微CT及組織學(xué)評(píng)估修復(fù)效果。3通過細(xì)胞毒性試驗(yàn),溶血試驗(yàn),皮膚刺激試驗(yàn)及皮膚致敏試驗(yàn)評(píng)價(jià)和比較不同3D打印技術(shù)制備的鈦合金試樣的生物相容性。4電子束選區(qū)熔化技術(shù)制作三維網(wǎng)狀鈦合金支架。采用殼聚糖、Bio-Oss骨粉和β-甘油磷酸鈉制備溫敏型水凝膠。骨髓間充質(zhì)干細(xì)胞與水凝膠共培養(yǎng),CCK-8和死活細(xì)胞染色觀察細(xì)胞在水凝膠中的生長(zhǎng)情況。建立比格犬下頜骨體部缺損,隨機(jī)分為3組,分別接受單純網(wǎng)狀鈦合金支架;網(wǎng)狀鈦合金支架復(fù)合殼聚糖/Bio-OSS水凝膠;網(wǎng)狀鈦合金支架復(fù)合殼聚糖/Bio-Oss和同種異體骨髓間充質(zhì)干細(xì)胞修復(fù)。采用大體觀察、CT、顯微CT和組織學(xué)檢測(cè)評(píng)估修復(fù)效果。5建立比格犬包括顳下頜關(guān)節(jié)的半側(cè)下頜骨缺損,采用凍干同種異體下頜骨、3D打印個(gè)性化實(shí)性和網(wǎng)狀鈦合金修復(fù)體修復(fù)。通過大體觀察、CT及組織學(xué)檢測(cè)來比較三種修復(fù)方法的術(shù)中操作、外觀恢復(fù)情況,對(duì)非手術(shù)側(cè)顳下頜關(guān)節(jié)的影響,以及對(duì)肝臟和腎臟組織的影響。結(jié)果1采用密度梯度離心法成功分離培養(yǎng)比格犬骨髓間充質(zhì)干細(xì)胞,所分離細(xì)胞具有良好的增殖能力和多向分化能力。2應(yīng)用同種異體下頜骨移植修復(fù)節(jié)段性缺損后,術(shù)區(qū)完全愈合,外形恢復(fù)良好。術(shù)后CT顯示單純同種異體下頜骨修復(fù)組的移植體吸收不明顯,復(fù)合干細(xì)胞的兩組移植體體積明顯減小。顯微CT顯示復(fù)合同種異體干細(xì)胞組和自體干細(xì)胞組移植體之間的骨相關(guān)參數(shù)無明顯差異(P0. 05),與單純修復(fù)組相比較,兩組移植體新骨形成量明顯多于單純修復(fù)組,差異有統(tǒng)計(jì)學(xué)意義(P0. 05) 。組織學(xué)檢測(cè)顯示,復(fù)合同種異體干細(xì)胞組和自體干細(xì)胞組的移植體有明顯的新骨形成,而單純修復(fù)組新骨形成量較少。3體外細(xì)胞試驗(yàn)結(jié)果顯示電子束選區(qū)熔化和選擇性激光熔化制備的鈦合金試樣對(duì)犬骨髓間充質(zhì)干細(xì)胞的生長(zhǎng)和成骨分化能力沒有明顯影響,兩種試樣之間沒有明顯差異(p0. 05)。溶血試驗(yàn)結(jié)果顯示,兩種鈦合金試樣料的溶血率分別為2. 24%和2.46%,均小于5%,提示兩種鈦合金材料均具有良好的血液相容性。皮膚刺激試驗(yàn)和遲發(fā)型超敏反應(yīng)最大劑量試驗(yàn)結(jié)果顯示,兩種鈦合金材料沒有潛在的皮膚刺激性和致敏性。4制備的殼聚糖/Bio-Oss水凝膠具有溫敏性,在室溫下為溶液,在37℃條件下轉(zhuǎn)化為凝膠,所需時(shí)間約為10-12min。CCK-8和死活細(xì)胞染色結(jié)果顯示,骨髓間充質(zhì)干細(xì)胞在殼聚糖/Bio-Oss水凝膠中生長(zhǎng)良好,活性未受影響。應(yīng)用3D打印網(wǎng)狀鈦合金修復(fù)下頜骨缺損后,移植體未出現(xiàn)松動(dòng)、暴露或折斷,所有動(dòng)物均達(dá)到臨床愈合。術(shù)后CT顯示網(wǎng)狀支架與斷端固位良好,未引起周圍骨吸收。顯微CT顯示,復(fù)合殼聚糖/Bio-Oss/BMSCs水凝膠的網(wǎng)狀鈦合金支架組新骨形成量多于復(fù)合殼聚糖/Bio-Oss組(p0. 05);兩組網(wǎng)狀支架內(nèi)的新骨形成量明顯多于單純網(wǎng)狀支架修復(fù)組(p0.05)。組織學(xué)檢測(cè)顯示,術(shù)后6個(gè)月,3組網(wǎng)狀支架近宿主骨端均有明顯新骨形成,復(fù)合殼聚糖/Bio-Oss/BMSCs和復(fù)合殼聚糖/Bio-Oss組的網(wǎng)眼內(nèi)的新骨量明顯多于單純網(wǎng)狀支架修復(fù)組,復(fù)合殼聚糖/Bio-Oss/BMSCs水凝膠的網(wǎng)狀支架內(nèi)新骨量最多。5凍干同種異體下頜骨、3D打印個(gè)性化實(shí)性修復(fù)體和3D打印個(gè)性化網(wǎng)狀修復(fù)體術(shù)中操作均較方便。同種異體下頜骨術(shù)中需要對(duì)其進(jìn)行塑形,而3D打印實(shí)性和網(wǎng)狀鈦合金修復(fù)體均不需要塑形。3組修復(fù)體修復(fù)后,下頜骨外形輪廓恢復(fù)較好,面部對(duì)稱,咬合關(guān)系良好。術(shù)后CT顯示雙側(cè)顳下頜關(guān)節(jié)位置關(guān)系未見明顯異常。掃描電鏡和組織學(xué)檢測(cè)結(jié)果顯示,三組動(dòng)物非手術(shù)側(cè)關(guān)節(jié)盤均有退行性變。結(jié)論1電子束選區(qū)熔化和選擇性激光熔化制備的鈦合金材料均具有良好的生物相容性,兩者之間沒有統(tǒng)計(jì)學(xué)差異。2凍干同種異體下頜骨和電子束選區(qū)熔化網(wǎng)狀鈦合金均可以作為骨組織工程支架修復(fù)下頜骨缺損。同種異體下頜骨復(fù)合骨髓間充質(zhì)干細(xì)胞后具有較高的成骨能力。復(fù)合干細(xì)胞和成骨材料能夠促進(jìn)網(wǎng)狀鈦合金支架內(nèi)的新骨形成新骨,但是骨再生速度較慢,新生骨量尚不足。今后,還需采用增加促骨生長(zhǎng)因子及促血管生長(zhǎng)因子等方法來改善成骨速度和成骨質(zhì)量。3同種異體骨髓間充質(zhì)干細(xì)胞和自體骨髓間充質(zhì)干細(xì)胞均能提高移植體的成骨速度和質(zhì)量,兩者之間沒有統(tǒng)計(jì)學(xué)差異。同種異體骨髓間充質(zhì)干細(xì)胞有望成為骨組織工程種子細(xì)胞來源。4冷凍干燥同種異體下頜骨和3D打印個(gè)性鈦合金修復(fù)體均具有操作方便,可個(gè)性化修復(fù)的優(yōu)勢(shì)。三組修復(fù)體均能很好地恢復(fù)下頜骨缺損區(qū)的外形和重建顳下頜關(guān)節(jié),是較好的大段下頜骨缺損修復(fù)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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R782

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