本文關(guān)鍵詞： 骨髓間充質(zhì)干細胞 脫細胞羊膜基質(zhì) BMP-2微球　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景骨缺損是常見的臨床問題,通常由創(chuàng)傷、疾病或手術(shù)等原因造成。骨缺損的修復(fù)方法主要包括自體骨移植、異體骨移植和異種骨移植,但存在不同程度的骨吸收和取骨區(qū)并發(fā)癥、排異及感染等問題。而組織工程骨的尚未在臨床廣泛應(yīng)用,還處于實驗階段。上個世紀口腔科提出誘導(dǎo)再生技術(shù)中應(yīng)用誘導(dǎo)成骨膜能夠促進骨缺損的修復(fù),其中的膜性材料眾多,研究中也發(fā)現(xiàn)一些不足之處,比如材料的外露、不可降解、制造工藝復(fù)雜等等。本研究從誘導(dǎo)再生技術(shù)出發(fā),提出以羊膜基質(zhì)作為支架,復(fù)合BMP-2微球構(gòu)建了具有緩釋BMP-2功能的成骨誘導(dǎo)膜性材料,并探索其體外對骨髓間充質(zhì)干細胞成骨作用和體內(nèi)修復(fù)下頜骨缺損的可行性。羊膜基質(zhì)獲得和制作方便簡單,而且具有許多生物特性,是良好的生物敷料和支架,在組織修復(fù)和重建方面應(yīng)用廣泛。將羊膜基質(zhì)攜載BMP-2微球提高了該成骨誘導(dǎo)膜性材料的誘導(dǎo)成骨能力,在修復(fù)下頜骨缺損乃至其他部位骨缺損都有良好的應(yīng)用前景。研究目的構(gòu)建以脫細胞羊膜基質(zhì)為支架,復(fù)合BMP-2微球形成新型的誘導(dǎo)成骨材料。通過研究其對骨髓間充質(zhì)干細胞成骨能力的影響和其對頜骨缺損動物模型的修復(fù)能力,為修復(fù)下頜骨缺損乃至其他部位骨缺損探索一種新的治療方法。研究方法本研究首先制備人脫細胞羊膜基質(zhì),固定后行HE染色,證實細胞成分徹底去除;應(yīng)用掃描電鏡觀察人脫細胞羊膜的結(jié)構(gòu)特征;制備BMP-2緩釋微球并測定其結(jié)構(gòu)、載藥量及緩釋曲線;抽取兔股骨骨髓以全骨髓培養(yǎng)法分離培養(yǎng)兔骨髓間充質(zhì)干細胞,進行成骨及成脂誘導(dǎo)分化及測定,并以脫細胞羊膜基質(zhì)為支架進行復(fù)合培養(yǎng),對其進行HE染色劑及掃描電鏡檢測;動物實驗分為脫細胞羊膜復(fù)合BMP-2微球聯(lián)合兔骨髓間充質(zhì)干細胞組(實驗組)與空白對照組。結(jié)果采用酶消化法制備的脫細胞羊膜基質(zhì)無細胞殘留,且保有致密多孔三維網(wǎng)狀結(jié)構(gòu)。明膠瓊脂載藥微球能夠有效地起到緩釋藥物的作用,并且微球形狀均勻飽滿。全骨髓培養(yǎng)法分離培養(yǎng)的兔骨髓間充質(zhì)干細胞具有成骨、成脂的分化能力,并且能夠在HAMM表面生長增殖。實驗動物分為實驗組與對照組,對實驗兔下頜骨制作1cm×1cm大小骨缺損模型。實驗組的兔下頜骨缺損采用BMP-2微球與BMSCs聯(lián)合脫細胞羊膜的方法填充修復(fù),通過術(shù)后影像學(xué)及組織學(xué)的檢測分析比較發(fā)現(xiàn),該方法能夠有效地促進骨缺損的修復(fù)。結(jié)論脫細胞羊膜基質(zhì)具有良好的細胞相容性,對細胞的生長增殖及分化無明顯抑制作用。兔BMSCs經(jīng)過成骨誘導(dǎo)培養(yǎng)后表達成骨細胞表型,可作為骨組織工程的理想種子細胞之一。BMP-2微球與BMSC聯(lián)合脫細胞羊膜可以有效地促進兔下頜骨缺損的修復(fù)。
[Abstract]:Background Bone defect is a common clinical problem, usually caused by trauma, disease or surgery. However, the complications of bone resorption and bone extraction, rejection and infection, etc., have not been widely used in clinical practice. In 0th century, the Department of Stomatology proposed that the application of induced periosteum could promote the repair of bone defects, among which there were many membranous materials, and some deficiencies were found in the study, such as the exposure of materials. In this study, based on the technology of induced regeneration, we proposed to construct osteogenic membrane materials with sustained release BMP-2 function by using amniotic membrane matrix as scaffold and BMP-2 microspheres as scaffolds. And to explore the osteogenesis of bone marrow mesenchymal stem cells in vitro and the feasibility of repairing mandibular defects in vivo. Amniotic membrane matrix is easy to be obtained and made, and has many biological properties, so it is a good biological dressing and scaffold. It is widely used in tissue repair and reconstruction. Carrying BMP-2 microspheres on amniotic membrane matrix can improve the osteogenic ability of the osteogenic membrane material. To construct an amniotic matrix scaffold with acellular amniotic membrane as a scaffold for repairing mandibular defects and other bone defects. To study the effect of BMP-2 microspheres on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) and the repair ability of animal models of mandibular defects, a new type of osteogenic material was formed with BMP-2 microspheres. In order to repair mandibular defects and other bone defects, a new treatment method was explored. In this study, human acellular amniotic membrane matrix was first prepared and fixed with HE staining to confirm the complete removal of cell components. The structural characteristics of human acellular amniotic membrane were observed by scanning electron microscope (SEM), the structure, drug loading and sustained release curve of BMP-2 sustained-release microspheres were prepared, and rabbit bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow culture method. Osteogenesis and adipogenic differentiation were measured, and acellular amniotic membrane matrix was used as scaffold for co-culture, and HE staining and scanning electron microscopy were performed. Animal experiments were divided into acellular amniotic membrane combined with BMP-2 microspheres combined with rabbit bone marrow mesenchymal stem cells (experimental group) and blank control group. The gelatin Agar loaded microspheres can effectively play the role of slow release drug, and the shape of microspheres is even and full. The rabbit bone marrow mesenchymal stem cells isolated by whole bone marrow culture method have osteogenesis. The ability of adipogenic differentiation, and the ability to grow and proliferate on the surface of HAMM. The experimental animals were divided into experimental group and control group. The mandibular defects of experimental rabbits were made into 1 cm 脳 1 cm bone defect model. The mandibular defects in the experimental group were repaired with BMP-2 microspheres and BMSCs combined with acellular amniotic membrane. The results of imaging and histological analysis showed that the mandibular defects in the experimental group were repaired with acellular amniotic membrane. Conclusion the acellular amniotic matrix has good cytocompatibility and has no inhibitory effect on cell proliferation and differentiation. Rabbit BMSCs can express the phenotype of osteoblasts after osteogenic induction. It can be used as one of the ideal seed cells of bone tissue engineering. BMP-2 microspheres combined with BMSC can effectively promote the repair of rabbit mandibular defect.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R68

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