【摘要】：目的 研究制備具有成骨活性的仿生性骨膜,植入骨缺損區(qū)修復(fù)骨缺損,為解決骨缺損修復(fù)提供新的思路,進(jìn)而為臨床的實(shí)際應(yīng)用,最終代替自體骨及異體骨移植修復(fù)骨缺損提供實(shí)驗依據(jù)。 方法 應(yīng)用組織工程的技術(shù)方法,以組織學(xué)、免疫組織化學(xué)與動物實(shí)驗學(xué)相結(jié)合的方法進(jìn)行實(shí)驗研究。選定脂肪干細(xì)胞(ADSCs)作為種子細(xì)胞,體外成骨誘導(dǎo)為成骨細(xì)胞,對其生物學(xué)特性進(jìn)行檢測,通過細(xì)胞的形態(tài)學(xué)、堿性磷酸酶活性、茜素紅染色、von Kossa染色等技術(shù)指標(biāo)鑒定其向成骨方向分化。將成骨誘導(dǎo)的脂肪干細(xì)胞與作為支架材料的小腸粘膜下層(SIS)在體外復(fù)合培養(yǎng)構(gòu)建出具有功能的細(xì)胞-材料復(fù)合體,制成仿生性骨膜,植入裸鼠皮下,通過組織學(xué)、透射電鏡、免疫組織化學(xué)觀察成骨情況及兩者的細(xì)胞相容性。最后建立節(jié)段性骨缺損動物模型,將仿生性骨膜植入骨缺損區(qū)修復(fù)骨缺損,通過X線攝片、組織形態(tài)學(xué)檢測等方法鑒定其成骨機(jī)制與自體骨膜移植成骨的相似性,確定仿生骨膜修復(fù)骨缺損的效果。 結(jié)果 體外分離培養(yǎng)擴(kuò)增兔脂肪干細(xì)胞,經(jīng)成骨誘導(dǎo)后可表達(dá)成骨細(xì)胞表型。成骨誘導(dǎo)的脂肪干細(xì)胞與SIS復(fù)合培養(yǎng)可構(gòu)建出具有生理骨膜結(jié)構(gòu)和功能的仿生骨膜,植入裸鼠體內(nèi)可形成具有良好血運(yùn)的新生骨組織。仿生骨膜修復(fù)兔節(jié)段性骨缺損效果良好,其成骨機(jī)制與自體骨膜移植成骨類似。 結(jié)論 兔脂肪干細(xì)胞體外成骨誘導(dǎo)培養(yǎng)可轉(zhuǎn)化為成骨細(xì)胞,可表達(dá)成骨細(xì)胞表型,為脂肪干細(xì)胞作為骨組織工程的種子細(xì)胞提供實(shí)驗依據(jù)。SIS與脂肪干細(xì)胞構(gòu)建的仿生骨膜在動物實(shí)驗的效果,確定了SIS作為支架材料為組織工程骨的新陳代謝提供了良好的環(huán)境,說明兩者具有良好的細(xì)胞生物相容性。仿生骨膜可修復(fù)兔長段骨缺損的實(shí)驗研究,使組織工程骨膜移植有望成為修復(fù)大段骨缺損的有效手段。意義近年來,組織工程技術(shù)的迅速發(fā)展使現(xiàn)代醫(yī)學(xué)走出了組織、器官移植的范疇,進(jìn)入了組織、器官再造的新時代。骨缺損是骨科臨床上常見的疾病,骨缺損的修復(fù)也是臨床治療的難題之一。利用骨組織工程學(xué)技術(shù)制造仿生性骨膜,為解決骨缺損修復(fù)這個難題提供新的思路,寄希望利用患者自身的脂肪干細(xì)胞與SIS構(gòu)建仿生骨膜,應(yīng)用于臨床骨缺損、骨不連的治療,為骨缺損的修復(fù)治療帶來創(chuàng)新性的變革。
[Abstract]:Objective to study the preparation of biomimetic periosteum with osteogenic activity and the implantation of bone defect area to repair the bone defect. The experimental evidence was provided to replace autogenous bone and allogeneic bone graft to repair bone defect. Methods the technique of tissue engineering was used to conduct the experimental study with the combination of histology, immunohistochemistry and animal experiment. Adipose stem cells (ADSCs) were selected as seed cells and osteoblasts were induced into osteoblasts in vitro. The biological characteristics of adipose stem cells were determined by morphology, alkaline phosphatase activity and alizarin red staining. Von Kossa staining and other technical indicators identified its osteogenic differentiation. The osteoblast-induced adipose stem cells were co-cultured with small intestinal submucosal (SIS) as scaffold material to construct a cell-material complex in vitro. The biomimetic periosteum was prepared and implanted into nude mice subcutaneously. The osteogenesis and their cytocompatibility were observed by transmission electron microscope and immunohistochemistry. Finally, the animal model of segmental bone defect was established, and the bone defect was repaired by implanting the biomimetic periosteum into the bone defect area. The mechanism of osteogenesis was identified by X-ray film and histomorphology examination, which was similar to that of autogenous periosteum graft osteogenesis. To determine the effect of biomimetic periosteum in repairing bone defect. Results Rabbit adipose stem cells were isolated and cultured in vitro, and osteoblast phenotype could be expressed after osteogenic induction. The bionic periosteum with physiological periosteum structure and function could be constructed by co-culture of osteogenic adipose stem cells and SIS, and new bone tissue with good blood circulation could be formed in nude mice. Biomimetic periosteum was used to repair segmental bone defect in rabbits, and its osteogenic mechanism was similar to that of autogenous periosteum graft. Conclusion in vitro osteogenic culture of rabbit adipose stem cells can be transformed into osteoblasts and can express osteoblast phenotype. To provide experimental basis for adipose stem cells as seed cells of bone tissue engineering. Effect of bionic periosteum constructed by SIS and adipose stem cells in animal experiments. It was confirmed that SIS as scaffold material provided a good environment for the metabolism of tissue engineered bone, which indicated that the two materials had good biocompatibility. Biomimetic periosteum can be used to repair long bone defect in rabbits, which makes tissue engineered periosteum transplantation an effective method to repair large bone defect. In recent years, with the rapid development of tissue engineering technology, modern medicine has stepped out of the category of tissue and organ transplantation and entered a new era of tissue and organ reconstruction. Bone defect is a common disease in orthopedic department. The repair of bone defect is also one of the difficult problems in clinical treatment. Making biomimetic periosteum by using bone tissue engineering technology provides a new idea for solving the problem of repairing bone defect. It is hoped that the biomimetic periosteum can be constructed by using the adipose stem cells and SIS of the patients themselves, which can be used in the treatment of clinical bone defect and nonunion. For the repair of bone defects to bring innovative changes.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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