骨形态发生蛋白复合多孔钛铌合金试件的实验研究

发布时间：2018-11-18 09:30

【摘要】：目的：本课题利用新型多孔钛铌合金试件为骨组织再生工程构架,观察复合骨形态发生蛋白(Bone Morphogenetic Protein, BMP)的多孔钛铌合金试件在体外体内表现出来的特性。进一步为研制新型多孔人工关节假体或其他骨组织再生骨植入物提供基础实验依据。方法：自行研制出70%孔隙度的钛铌合金试件,根据钛铌合金试件是否复合骨形态发生蛋白(BMP)进行实验分组：A：单纯多孔钛铌合金实验试件；B：复合BMP的多孔钛铌合金实验试件。体外实验部分：将BMSCS悬液与多孔钛铌合金试件按实验分组复合,观察BMSCS于合金试件细胞支架贴附后移入相应培养基培养。于不同时间观察点取材,通过倒置相差显微镜、扫描电镜、成骨细胞表型检测等方法对合金支架表面及孔洞内BMSC生长以及成骨分化进行观察鉴定。体内试验部分：将A、B组实验试件植入兔大腿肌肉内,分别取10天、20天、30天实验试件周围0.5cm范围内组织,通过组织切片HE染色它们在体内的组织相容性、周围组织生长分化情况及组织-金属界面结合情况。使用SPSS17.0软件包进行统计分析,计量资料以均数士标准差表示,组间同一时间各指标比较采用组间方差分析。结果：体外实验发现：倒置相差显微镜、扫描电镜等方法显示兔骨髓间充质干细胞在未复合BMP的合金试件表面及试件孔洞内黏附增殖正常,表现出良好的细胞相容性,其成骨细胞表型检测呈阴性。而兔骨髓间充质干细胞在复合BMP的合金试件内表现出向成骨细胞转化,其成骨细胞表型检测呈阳性。体内试验发现：实验试件周围软组织切片HE染色检测显示：10、20、30天肌肉内合金试件周围0.5cm范围组织有轻微的炎症反应和异物反应。其中复合BMP组的周围组织在10天时镜下可见有新生血管形成；20天时镜下可见有多个新生血管形成,局部可见肥大、深染的纤维细胞；30天时镜下可见组织内有钙组织生成。结论： 1、体外兔骨髓间充质干细胞在单纯多孔钛铌合金材料表面粘附及增殖良好,细胞能沿孔壁向多孔合金试件内部生长,但是未表现出成骨特性。 2、体外兔骨髓间充质干细胞在复合BMP的多孔钛铌合金试件中可经诱导向成骨细胞分化,并可在多孔金属试件内的空间结构中形成钙组织。 3、多孔钛铌合金试件在兔肌肉中中表现出轻微的炎症反应和异物反应,其材料本身对于周围组织有诱导血管生成的效果,但是作用效果缓慢。 4、复合BMP的多孔钛铌合金植入兔肌肉内可以促进其周围组织中早期的血管长入、成纤维组织形成及纤维组织的改建,为骨组织的生成创造条件。
[Abstract]:Aim: to investigate the characteristics of porous Ti-Niobium alloy specimens combined with bone morphogenetic protein (Bone Morphogenetic Protein, BMP) in vitro using a new porous Ti-NB alloy specimen as a framework for bone tissue regeneration. Further, it provides basic experimental basis for the development of new porous artificial joint prosthesis or other bone tissue regenerated bone implants. Methods: the Ti-Niobium alloy specimen with 70% porosity was prepared and divided into groups according to whether the Ti-Niobium alloy specimen was composed of bone morphogenetic protein (BMP): a: pure porous Ti-Niobium alloy specimen; B: porous Ti-Niobium alloy specimen with BMP composite. In vitro experiment: the BMSCS suspension was combined with porous Ti-NB alloy specimen according to the experimental group, and the BMSCS was attached to the cell scaffold of the alloy specimen and transplanted into the corresponding culture medium. The growth and differentiation of BMSC on the surface and hole of alloy scaffold were observed and identified by inverted phase contrast microscope scanning electron microscope and phenotypic detection of osteoblasts. In vivo experiment part: the specimens of group A B were implanted into the muscle of rabbit thigh, and the tissue around 0.5cm was taken for 10 days, 20 days and 30 days, respectively. The tissue compatibility was stained by tissue section HE staining in vivo. The growth and differentiation of surrounding tissues and the bonding between tissue and metal interface. The statistical analysis was carried out by using SPSS17.0 software package. The measurement data were expressed as the standard deviation of the mean number. The analysis of variance was used to compare the indexes between groups at the same time. Results: in vitro, inverted phase contrast microscopy and scanning electron microscopy showed that the adhesion and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs) were normal and showed good cytocompatibility on the surface of alloy specimens without BMP and in the pores of the specimens. The osteoblast phenotype was negative. However, rabbit bone marrow mesenchymal stem cells (BMSCs) were transformed to osteoblasts in the alloy specimens combined with BMP, and the osteoblast phenotypes were positive in rabbit bone marrow mesenchymal stem cells (BMSCs). In vivo tests showed that there was a slight inflammatory reaction and a foreign body reaction in the tissue around the 0.5cm area of the alloy specimens on the 10th day of 20 ~ 30 days after HE staining in the soft tissue sections around the specimens. In compound BMP group, neovascularization was observed in the surrounding tissues at day 10; at day 20, there were hypertrophic and hypertrophic fibroblasts; at day 30, calcium tissue formation was observed in the tissue. Conclusion: 1. Rabbit bone marrow mesenchymal stem cells adhered and proliferated well on the surface of pure porous titanium-niobium alloy in vitro. The cells could grow into the porous alloy specimen along the pore wall, but showed no osteogenic characteristics. 2. Rabbit bone marrow mesenchymal stem cells can differentiate into osteoblasts and form calcium tissue in the space structure of porous metal specimens in vitro. 3. The porous Ti-NB alloy specimen showed slight inflammatory reaction and foreign body reaction in rabbit muscle. The material itself had the effect of inducing angiogenesis to the surrounding tissues, but the effect was slow. 4. The porous Ti-NB alloy implanted with BMP can promote the early vascular growth, fibroblast formation and fibrous tissue remodeling in the surrounding tissues of rabbits, thus creating conditions for the formation of bone tissue.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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