BMP-2微球与骨髓间充质干细胞联合脱细胞羊膜修复兔下颌缺损的实验研究

发布时间：2018-02-15 22:50

本文关键词： 骨髓间充质干细胞脱细胞羊膜基质 BMP-2微球　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景骨缺损是常见的临床问题,通常由创伤、疾病或手术等原因造成。骨缺损的修复方法主要包括自体骨移植、异体骨移植和异种骨移植,但存在不同程度的骨吸收和取骨区并发症、排异及感染等问题。而组织工程骨的尚未在临床广泛应用,还处于实验阶段。上个世纪口腔科提出诱导再生技术中应用诱导成骨膜能够促进骨缺损的修复,其中的膜性材料众多,研究中也发现一些不足之处,比如材料的外露、不可降解、制造工艺复杂等等。本研究从诱导再生技术出发,提出以羊膜基质作为支架,复合BMP-2微球构建了具有缓释BMP-2功能的成骨诱导膜性材料,并探索其体外对骨髓间充质干细胞成骨作用和体内修复下颌骨缺损的可行性。羊膜基质获得和制作方便简单,而且具有许多生物特性,是良好的生物敷料和支架,在组织修复和重建方面应用广泛。将羊膜基质携载BMP-2微球提高了该成骨诱导膜性材料的诱导成骨能力,在修复下颌骨缺损乃至其他部位骨缺损都有良好的应用前景。研究目的构建以脱细胞羊膜基质为支架,复合BMP-2微球形成新型的诱导成骨材料。通过研究其对骨髓间充质干细胞成骨能力的影响和其对颌骨缺损动物模型的修复能力,为修复下颌骨缺损乃至其他部位骨缺损探索一种新的治疗方法。研究方法本研究首先制备人脱细胞羊膜基质,固定后行HE染色,证实细胞成分彻底去除;应用扫描电镜观察人脱细胞羊膜的结构特征;制备BMP-2缓释微球并测定其结构、载药量及缓释曲线;抽取兔股骨骨髓以全骨髓培养法分离培养兔骨髓间充质干细胞,进行成骨及成脂诱导分化及测定,并以脱细胞羊膜基质为支架进行复合培养,对其进行HE染色剂及扫描电镜检测;动物实验分为脱细胞羊膜复合BMP-2微球联合兔骨髓间充质干细胞组(实验组)与空白对照组。结果采用酶消化法制备的脱细胞羊膜基质无细胞残留,且保有致密多孔三维网状结构。明胶琼脂载药微球能够有效地起到缓释药物的作用,并且微球形状均匀饱满。全骨髓培养法分离培养的兔骨髓间充质干细胞具有成骨、成脂的分化能力,并且能够在HAMM表面生长增殖。实验动物分为实验组与对照组,对实验兔下颌骨制作1cm×1cm大小骨缺损模型。实验组的兔下颌骨缺损采用BMP-2微球与BMSCs联合脱细胞羊膜的方法填充修复,通过术后影像学及组织学的检测分析比较发现,该方法能够有效地促进骨缺损的修复。结论脱细胞羊膜基质具有良好的细胞相容性,对细胞的生长增殖及分化无明显抑制作用。兔BMSCs经过成骨诱导培养后表达成骨细胞表型,可作为骨组织工程的理想种子细胞之一。BMP-2微球与BMSC联合脱细胞羊膜可以有效地促进兔下颌骨缺损的修复。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68

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