【作者】 靳小兵；

【导师】 罗卓荆；

【作者基本信息】 第四军医大学 ， 外科学， 2004， 硕士

【摘要】 由创伤、感染等引起的长骨生长板损伤，会引起骨骺与干骺端之间形成骨桥，由此导致的肢体短缩与成角畸形一直是小儿矫形外科医师面临的重大挑战，至今尚无可靠的治疗方案。1967年Lanlgenskiold首次报道用骨桥切除、脂肪填塞的方法治疗生长板部分早闭取得一定效果。自此，人们不断尝试将硅胶、骨蜡、脂肪、游离生长板及血管化生长板等不同移植物用于治疗生长板损伤，取得了一定效果。但这些材料及方法均存在不同的缺点及问题，游离软骨和血管化生长板移植修复存在移植物的来源受限，供、受体匹配难及移植物免疫排斥等问题，非软骨类组织如脂肪、肌肉、骨蜡和硅胶等不能再生修复生长板，只能防止小范围生长板损伤并肢体畸形，不能改善肢体生长发育。改进生长板损伤修复方法及寻找更理想的植入材料仍是小儿四肢矫形外科面临的一大难题。二十世纪九十年代以来，组织工程学科的兴起为治疗生长板损伤提供了契机。本实验采用组织工程技术，尝试用自体组织工程生长板治疗长骨生长板损伤，期望为临床解决生长板损伤修复难题奠定基础。 一 兔髂骨生长板细胞的培养及体内成软骨作用的研究 目的：建立大量生产髂骨生长板软骨细胞的培养及保存方法，观察髂骨生长板细胞在裸鼠体内的成软骨作用，为生长板组织工程提供种子细胞。方法：取二周龄兔髂骨生长板软骨，经机械剪切和化学消化作用后，接种、培养及液氮保存，通过显微镜观察其生物 第四军医大学硕士学位论文学表现，并通过甲苯胺兰、碱性磷酸酶〔alkaline phosphatase，ALP)染色及n型胶原染色对其生物学特性进行鉴定。将生长良好的第二代细胞注入裸鼠背部，于第四周取材，进行组织学观察。结果:细胞可在体外大量增殖并在六代内无明显反分化，但缺乏向肥大细胞进一步分化的能力。冷冻复苏细胞存活率为92%。注射的细胞悬液可在裸鼠体内形成软骨组织，并进一步向肥大细胞转化.结论:成功建立了骼骨生长板细胞的培养及冻存方法，证明骼骨生长板细胞可在体内进一步形成软骨组织并向肥大期细胞转化。二兔骼骨生长板细胞和脱钙骨墓质共培养构建组织工程生长板 目的:探索兔骼骨生长板细胞和同种异体脱钙骨基质(demineralized bone matrix，DBM)共培养构建组织工程生长板的可行性。方法:取三周龄兔骼骨生长板软骨细胞经传代培养后作为种子细胞与制备的脱钙去抗原同种异体DBM于体外联合培养，通过对复合物行扫描电镜(seanning electronie mierosco讲，sEM)观察及固定后切片“HE”染色，了解细胞在材料中的生长情况.结果:体外复合培养24小时，软骨细胞即开始贴附于DBM网架上;复合培养7天，分布于支架材料上的生长板细胞迅速分化增殖，分泌细胞外基质:第21天，细胞长满支架，重叠生长。结论:同种异体DBM和骼骨生长板细胞共同培养体外构建组织工程生长板是可行的。三自体组织工程生长板治疗长骨生长板损伤的实脸研究 目的:探讨自体组织工程生长板在治疗生长板损伤中的作用。方法:自三周龄兔骼蜻处切取部分铭骨生长板软骨经机械剪切和n型胶原酶消化后培养生长板软骨细胞，体外单层培培养扩增后接种到同种异体脱钙骨基质(demine司ized bone matrix，DBM)上，混合培养一周后，植入兔自体右侧胫骨上端内侧生长板模型缺损处 (A组)，分别设单纯DBM植入(B组)及单纯破坏即生长板破坏后无任何植入物(C组)作为对照组，左侧胫骨不作处理。术后动态“X”线摄片测量下肢短缩及成角畸形改变，通过组织切片 第四军医大学硕士学位论文“HE”染色及11型胶原免疫组化(imm~histochemistry)染色观察自体组织工程生长板在体内的转归。结果:术后第二周三组右侧胫骨均出现轻度畸形，A、B组较C组为轻，但三者之间无显著差异(p>0.05)，此后B、C组兔右侧胫骨出现渐进性严重短缩及成角畸形，而A组右侧胫骨畸形则无明显加重，各时间点A组胫骨成角及缩短畸形与B、C组有显著差异(P<0.05)，术后第十六周时组织切片显示，损伤区基本恢复正常生长板结构，H型胶原免疫组化染色阳性，而B、C组损伤区则由新生骨组织修复。结论:自体组织工程生长板植入可有效防止急性生长板损伤后肢体畸形的发生，植入的组织工程生长板可产生柱状排列的结构，细胞可表达n型胶原。四人胚肋软骨静止区细胞的体外培养及生物学特性研究 目的:研究体外培养的人胚肋软骨静止区细胞的生物学特性及分化规律，为组织工程生长板的研究提供种子细胞。方法:采用11型胶原酶消化的方法获取人胚肋软骨静止区细胞，观察细胞形态学变化;测定细胞生长曲线;借助特殊染色、免疫荧光染色的方法了解糖胺聚糖、碱性磷酸酶(alkaline phosPhatase，ALP)、11型胶原酶的合成情况。结果:贴壁后细胞呈多角形，从第六代开始绝大多数细胞转变为成纤维细胞状，细胞培养至第二代开始分泌大量n型胶原。结论:体外单层培养的人胚肋软骨静止区细胞具有正常的生长增殖与分化能力，可分化至成熟期软骨细胞。更多还原

【Abstract】 Bone bridge will form between epiphysis and metaphysis if the growth plate of long bone is injured by wound ,infection and tumor et al. The shortening and angulation of limbs caused by bone bridge is one of the big problems which many orthopedists encounters. In 1967,Langenskiold first reported that the partly early closure of the growth plate can be treated by the method of bone bridge cutting and fat packing .Since that ,people attempted to solve the problem by transplanting different grafts such as silica gel ,bone wax , fat and free growth plate et al. The above methods have some effects ,but they also have many defects. Immune expelling between the supply and the recipient is one of the dilemmas. Fat, muscle and bone wax only can prevent bone bridge forming caused by gentle injury instead of the growth plate regenerating, they also can not improve the growth and development of the limbs. In recent years, people try to solve the problem by the method of tissue engineering. We attempt to treat the injury of long bone growth plate by using the tissue-engineered autologous growth plate established in vitro. The experiment aims to establish the foundation for the clinical solving of the tough problem. 1.The culture of the iliac growth-plate chondrocytes andthe study on its cartilage-formation role in vivo Objective:To establish a kind of iliac growth-plate chondrocyte culture and storage method so that we can obtain the seed cells in large quantity for the usage of engineered growth plate, tissue.To observe the cartilage formation of the cells in vivo. MethodsrThe cells were harvested from the iliac growth-plate cartilage of the two-week old rabbit and cultured. Their biological phenomenons were observed and their biological characteristics were identified by using toluidine blue, alkaline phosphatase staining and immunohistochemistry staining for type II collagen. The second generation cells were harvested and injected into the dorsum of nude mice. Specimens were harvested 4 weeks later for histological examination. Results:The cells could proliferate in vitro and have no apparent de-differentiation within the sixth generation, but they could not differentiate into the hypertrophic cells. The cells revived from being frozen(-196癈) was 92%. The cells injected into the body of the nude mice could form the cartilage-like tissue. The iliac growth-plate chondrocytes can differentiate into hypertrophic cells in vivo. Conclusion:We have established a method of culture and storage of iliac growth-plate chondrocytes and cartilage-formation of the cells in vivo was verified. The cells can differentiate into hypertrophic chondrocytes in vivo.2.Experimental study on the establishment of tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cellsObjective:To investigate the feasibility of establishing tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells. Methods: We obtain the growth plate cells from iliac crest epiphyseal cartilage of the three-week old New Zealand rabbits by dissection and sequential digestion with 0.2% collagenase(type II).After the cells proliferated with monolayer culture in vitro for three weeks.The cells were harvested and cocultured with the scaffold DBM.The cell-material complex was observed by "HE" staining and electronic scanning microscope in order to evaluate the interaction between cells and DBM. Results: Twenty-four hours after coculture, the cells adhered to DBM scaffolds; Seven days later, the growth-plate chondrocytes proliferated in the DBM network and secret the extracellular matrix; The cells grow all formed on the surface of the scaffold after three weeks. Conclusion: We can successfully establish the tissue-engineering growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells .3.The experimental study on the treatment of growth plate injuries with a tissue-engineered autologous growth plate Objective.更多还原