【摘要】：目的：本課題利用新型多孔鈦鈮合金試件為骨組織再生工程構架,觀察復合骨形態(tài)發(fā)生蛋白(Bone Morphogenetic Protein, BMP)的多孔鈦鈮合金試件在體外體內表現出來的特性。進一步為研制新型多孔人工關節(jié)假體或其他骨組織再生骨植入物提供基礎實驗依據。 方法：自行研制出70%孔隙度的鈦鈮合金試件,根據鈦鈮合金試件是否復合骨形態(tài)發(fā)生蛋白(BMP)進行實驗分組：A：單純多孔鈦鈮合金實驗試件；B：復合BMP的多孔鈦鈮合金實驗試件。體外實驗部分：將BMSCS懸液與多孔鈦鈮合金試件按實驗分組復合,觀察BMSCS于合金試件細胞支架貼附后移入相應培養(yǎng)基培養(yǎng)。于不同時間觀察點取材,通過倒置相差顯微鏡、掃描電鏡、成骨細胞表型檢測等方法對合金支架表面及孔洞內BMSC生長以及成骨分化進行觀察鑒定。體內試驗部分：將A、B組實驗試件植入兔大腿肌肉內,分別取10天、20天、30天實驗試件周圍0.5cm范圍內組織,通過組織切片HE染色它們在體內的組織相容性、周圍組織生長分化情況及組織-金屬界面結合情況。 使用SPSS17.0軟件包進行統計分析,計量資料以均數士標準差表示,組間同一時間各指標比較采用組間方差分析。 結果：體外實驗發(fā)現：倒置相差顯微鏡、掃描電鏡等方法顯示兔骨髓間充質干細胞在未復合BMP的合金試件表面及試件孔洞內黏附增殖正常,表現出良好的細胞相容性,其成骨細胞表型檢測呈陰性。而兔骨髓間充質干細胞在復合BMP的合金試件內表現出向成骨細胞轉化,其成骨細胞表型檢測呈陽性。 體內試驗發(fā)現：實驗試件周圍軟組織切片HE染色檢測顯示：10、20、30天肌肉內合金試件周圍0.5cm范圍組織有輕微的炎癥反應和異物反應。其中復合BMP組的周圍組織在10天時鏡下可見有新生血管形成；20天時鏡下可見有多個新生血管形成,局部可見肥大、深染的纖維細胞；30天時鏡下可見組織內有鈣組織生成。 結論： 1、體外兔骨髓間充質干細胞在單純多孔鈦鈮合金材料表面粘附及增殖良好,細胞能沿孔壁向多孔合金試件內部生長,但是未表現出成骨特性。 2、體外兔骨髓間充質干細胞在復合BMP的多孔鈦鈮合金試件中可經誘導向成骨細胞分化,并可在多孔金屬試件內的空間結構中形成鈣組織。 3、多孔鈦鈮合金試件在兔肌肉中中表現出輕微的炎癥反應和異物反應,其材料本身對于周圍組織有誘導血管生成的效果,但是作用效果緩慢。 4、復合BMP的多孔鈦鈮合金植入兔肌肉內可以促進其周圍組織中早期的血管長入、成纖維組織形成及纖維組織的改建,為骨組織的生成創(chuàng)造條件。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R318.08

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