【作者】 欧新荣；

【导师】 翦新春；

【作者基本信息】 中南大学 ， 外科学， 2006， 博士

【摘要】 研究背景牙槽嵴裂是一种常见的先天性疾病，伴发于唇腭裂，严重影响患者的面容及功能。一般在唇裂和腭裂治疗后一段时间(9-11岁)进行修复，在这段时间内前颌骨生长发育的变化临床上尚无定论。常规的治疗方法是取自体松质骨(髂骨或下颌骨)植入缺损部位，以恢复上颌牙槽骨连续性，同时使尖牙从植骨部位萌出，并矫正鼻翼基底部畸形。植骨后有相当多的病例存在不同程度的植入骨吸收以及取骨区疼痛，跛行等并发症。近十余年发展起来的组织工程骨技术在修复躯干与四肢的长骨部分缺损上取得了进展，但工程骨的血管化目前仍然是一个悬而未决的问题，在牙槽嵴裂修复中的应用也较少。因此，有必要对唇裂和腭裂修复后前颌骨的生长发育，组织工程骨及血管化的组织工程骨修复牙槽嵴裂后的形态改变等进行研究，为其临床应用提供理论依据。第一部分双侧牙槽嵴裂动物模型的建立及对上颌骨生长发育的影响目的：建立双侧牙槽嵴裂大动物模型，研究其对上颌骨发育的影响。方法：选用8只12W龄的同窝实验犬，随机分成正常对照组和实验动物模型组，对实验组的动物采用外科手术的方法建立裂隙程度一致的双侧上颌牙槽嵴裂，术后12W处死动物，通过实体标本、三维CT对两组动物行头颅测量。结果：实验模型组牙槽嵴裂裂隙为(10.81±0.41)mm，其上颌骨长度、前部宽度和前部高度较正常对照组明显缩小(P＜0.05)。结论：牙槽嵴裂对上颌骨生长发育具有一定的影响。第二部分组织工程骨修复牙槽嵴裂的实验研究目的：探讨骨髓来源的种子细胞复合胶原蛋白海绵构建组织工程骨修复牙槽嵴裂的可行性。方法：16条实验犬被分成4组。于双侧上颌第三切牙处去除15mm牙槽骨形成牙槽嵴裂动物模型；经股骨骨髓穿刺，分离骨髓基质细胞，培养、传代扩增诱导后，与胶原蛋白海绵混合培养72h，植入骨缺损处。饲养12W后处死动物，通过三维CT及组织学检查评价骨缺损修复的效果。结果：实验组牙槽骨断端间形成完整的骨连接，切片可见髓腔通畅，新形成的牙槽嵴宽度与松质骨植入对照组相似(P＞0.05)，但高度不足(P＜0.05)。结论：组织工程骨修复牙槽嵴裂具有一定的可行性，有望成为修复牙槽嵴裂的治疗方法。第三部分血管化组织工程骨修复牙槽嵴裂的实验研究目的：探讨应用经血管内皮细胞生长因子(VEGF)转染的骨髓基质干细胞(BMSC)复合胶原蛋白海绵构建的血管化组织工程骨修复牙槽嵴裂的可行性。方法：经股骨骨髓穿刺获取犬BMSC经分离、培养和鉴定后，转染pAdMV-VEGF165质粒，流式细胞仪、RT-PCR和免疫组化检测；转染细胞与蛋白胶原海绵混合培养72h形成复合体；将复合体植于犬牙槽嵴裂模型的骨缺损处，12W后处死动物，通过大体形态及X线、三维CT重建、组织学检查等方法评价修复牙槽嵴裂的效果。结果：转染组骨缺损完全愈合，骨髓腔通畅，组织工程骨高度及宽度与自体松质骨植入组无明显差异(P＞0.05)。结论：由经VEGF转染的BMSC复合胶原蛋白海绵构建的组织工程骨可以较好地修复牙槽嵴裂，在修复后的形态、结构等方面均与自体松质骨修复相似，是一种牙槽嵴裂修复的良好方法。第四部分内皮前体细胞在组织工程骨修复牙槽嵴裂中的应用目的：探讨血管内皮前体细胞(EPC)联合骨髓基质干细胞(BMSC)复合胶原蛋白海绵构建的血管化组织工程骨修复牙槽嵴裂的可行性。方法：20只实验犬被分成模型组、单纯材料组、BMSC组、实验组、松质骨植入组；经股骨穿刺获得犬骨髓分离BMSC、EPC，差异消化法纯化EPC，经培养和鉴定后，与蛋白胶原海绵混合培养72h形成复合体；将复合体植于犬牙槽嵴裂模型的骨缺损处，12W后处死动物，通过大体形态及X线、三维CT重建、组织学检查等方法评价修复牙槽嵴裂的效果。结果：实验组骨缺损完全愈合，骨髓腔通畅，骨密度、高度及宽度与自体松质骨植入组无明显差异(P＞0.05)；单位面积血管数较对照组明显增加(P＜0.05)。结论：EPC可以促进组织工程骨血管化及骨化，与BMSC联合形成组织工程骨可以较好地修复牙槽嵴裂，有望成为修复牙槽嵴裂的治疗方法。更多还原

【Abstract】 BackgroundAlveolus cleft is a congenital disease accompanied with cleft lip andpalate, which seriously affects the facial complexion and function. Thedefect will be repaired after the restoration of cleft lip and palate (whenpatient are 9-11 years old). There isn’t any clinic verdict of the growth ofthe maxilla during the stage. As a general operative mode, autogolouscancellous (from the ilium or the mandible) is implanted into thedefective part to restore the continuity of the maxilla, to help thegeneration of canine, and to rectify the malformation of the nose. Manycomplications such as pain in the bone taking and bone reception area, etc,are reported during or after the operation. Engineered bone have beenused to restore the bone defect in the limbs during the past 10 years, butthe problem of vascularization remains unsolved, and it is rarely used inthe restoration of alveolus cleft.Therefore, it is necessary to study the growth of pre-maxilla after therestoration of cleft lip and palate, the use of engineered bone, and thechange of form after the use of vascular engineered bone to restorealveolus cleft, and to provide theoretical base for clinical application. Part 1 Influence of surgical induced bilateral cleft alveolus onmaxillary growth in dogsObjective To establish an animal model of bilateral alveolus cleftand study the influence of cleft on maxillary growth.Methods Eight dogs (12 weeks old ) were divided into unoperatedcontrol groups (n=4)and the model group (n=4) .The model dogs wereoperated onto establish a bilateral alveolus cleft. All the animals werekilled and the craniofacial morphology on clean skull was analyzed bythe direct detection and CT.Results The length ,the foreside width and the foreside height ofthe maxilla in the model group were shrunken than those of control group(P＜0.05)Conclusion The alveolus cleft plays an important role in themaxillary aberration.Part 2 An Investigation of Restoration of Alveolus Cleft withEngineered BoneObjective To investigate the feasibility of the restoration ofalveolus cleft with engineered bone constructed by sponge collagenprotein combined bone mesenchymal stem cells (BMSC).Methods Sixteen dogs were divided into 4 groupes, the thirdincisor and alveolus bone with peri0steum in bilateral maxilla wereremoved to form alveolus cleft model. The BMSCs were isolated from dog bone marrow. After being cultured and induced, the BMSCs wereseeded in sponge collagen protein and cultured for 72 hours. Thecomposites of BMSCs and collagen were implanted into the defect ofalveolus cleft. After being fed for 12 weeks, those dogs were killed.Three-dimensional CT and histological examination were used to observethe progress of bone formation.Results The defects healed 12 weeks after BMSCs-collagencomposites were implanted, the width of engineered bone resembledpositive control(implant with autologous cancellous), but the height isless than positive control(P＜0.05).Conclusion The engineered bone can restore the defect ofalveolus cleft, it may be used in the clinical treatment of the restoration ofalveolus cleft.Part 3 An Investigation of Restoration of Cleft Alveolus withVascularised Engineered BoneObjective To investigate the feasibility of the restoration of cleftalveolus with vascularised engineered bone constructed by collagencombined with vascular endothelial growth factor (VEGF) genetransfected bone mesenchymal stem cells.Methods The BMSCs were isolated by gradient densitycentrifugation with Percoll solution from dog bone marrow. After beingcultured and VEGF165 gene transfected, Verified by FCM, RT-PCR and IHC, BMSCs were seeded in collagen for 72 hours. The compositesof BMSCs and collagen were implanted into the defect of cleft alveolusof dog. After being fed for 12 weeks, those dogs were sacrificed. X-ray,three-dimensional CT and histological examination were used to observethe bone formation.Results 52% of BMSCs were transfected with VEGF165plasmid successfully. The defects of cleft alveolus healed at 12 weeksafter being implant collagen and VEGF 165 plasmid transfected BMSCcomposites and the forms of engineered bone resembled those implantedby autologous cancellous bone.Conclusion The vascularised engineered bone can repair thedefect of alveolus cleft effectively. It may be used in the clinic.Part 4 Application of Endothelial Progenitor Cell in Restoration ofCleft Alveolus with Engineered BoneObjective To investigate the feasibility of the restoration of cleftalveolus bridge with vascularised engineered bone which wereconstructed by collagen combined with endothelial progenitor cells(EPC)and bone mesenchymal stem cells (BMSC).Methods Twenty dogs were divided into 5 groups.The BMSC andEPC were isolated from dog bone marrow. EPC were purified bytime-limited digestive method. After being cultured and identified, BMSC and EPC were seeded in collagen for 72 hours. The composites wereimplanted into the defect of cleft alveolus model of dogs. After being fedfor 12 weeks, 20 dogs were sacrificed. X-ray, three-dimensional CT andhistological examination were used to observe the bone formation.Results The alveolus defects healed 12 weeks after beingimplanted composites and the forms of engineered bone resembled thoseimplanted by autologous cancellous bone. The engineered bone had morevessels than control groups in section.Conclusion EPC may promote the vascularization and ossificationof the engineered bone. Combined with BMSC, the vascular engineeredbone can repair the defect of alveolus cleft effectively, and it may be usedin clinical practice.更多还原