Ti-5Zr-3Sn-5Mo-15Nb合金表面阳极氧化处理对成骨细胞生物学行为的影响

发布时间：2018-02-11 23:50

本文关键词： 近β钛合金阳极氧化表面能成骨细胞细胞附着细胞增殖细胞分化 ALP 成骨相关基因　出处：《第四军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 为了提高骨内种植体的骨结合性能,研究者们尝试用不同的方法对种植体进行表面改性。阳极氧化是对钛及其合金表面改性的一种简单而有效的方法,可以获得具有良好生物相容性及生物活性的纳米级表面形貌。本研究采用阳极氧化方法对弹性模量较低、不含有毒合金元素的近β钛合金Ti-5Zr-3Sn-5Mo-15Nb (TLM)表面进行处理,并观察其对成骨细胞早期生物学行为的影响。用稳压直流电源在5V和20V两个电压下分别对TLM表面进行阳极氧化处理,采用场发射电镜观察试样表面形貌、X射线衍射分析仪(XRD)分析表面晶相、接触角测量系统测量表面接触角并计算表面能、商业试剂盒评价表面蛋白吸附能力、荧光计数法检测成骨细胞在材料表面附着、噻唑盐(MTT)方法检测细胞在材料表面增殖、扫描电镜观察细胞在试样表面的形态、商业试剂盒检测总蛋白合成及碱性磷酸酶(ALP)活性、实时定量PCR(Realtime-PCR)方法检测不同处理的表面成骨细胞成骨相关基因的表达。结果如下: 1. TLM试样经阳极氧化后,在5V电压下表面形成邻接紧密的孔径约为10 nm的网状结构,在20V电压下表面形成管间隙较大的管径约为80 nm的微管结构。 2. XRD结果显示TLM试样表面的晶相为锐钛矿。 3.通过观察两种液体在TLM试样表面的接触角发现,20V电压阳极氧化表面、5V电压阳极氧化表面和抛光表面的液体接触角依次增大而表面能依次减小,它们之间的差异具有显著性(p 0.05)。 4.表面蛋白吸附检测结果显示20V电压阳极氧化TLM表面的蛋白吸附能力显著强于5V电压阳极氧化表面和抛光表面。 5.细胞培养实验发现,成骨细胞在20V电压下阳极氧化处理形成的TLM纳米管表面的早期附着数量明显高于抛光表面(p 0.05),细胞增殖水平与抛光表面接近。5V电压下形成的纳米网表面的成骨细胞附着显著低于20V电压阳极氧化表面(p 0.05),但与抛光表面接近,而细胞增殖显著低于另两种表面(p 0.05)。 6. 20V电压阳极氧化表面的成骨细胞总蛋白合成及ALP活性均显著高于5V电压阳极氧化表面和抛光表面(p 0.05)。 7.成骨细胞在试样表面培养四天后,电镜观察显示成骨细胞在抛光表面沿抛光纹路伸展且细胞突起较少。在阳极氧化表面,成骨细胞伸展较好呈扁平形状,其中在20V电压处理表面的细胞伸出大量的板状和丝状伪足。 8. Realtime-PCR检测结果发现阳极氧化处理对TLM表面成骨细胞整合素β1(Integrinβ1)和转录相关因子-2(RUNX2)的表达无明显影响。但在20V电压阳极氧化表面的成骨细胞的ALP、骨钙素(OCN)和骨形态发生蛋白-2(BMP-2)的表达在第三天显著增高,而在5V电压阳极氧化表面的成骨细胞骨桥蛋白(OPN)表达在第十天显著增高。结论:TLM表面经阳极氧化处理后形成纳米网或纳米管状的形貌,表面湿润性和表面能提高,蛋白吸附能力提高,这些效应均与阳极氧化电压有关。20V电压下阳极氧化处理可以促进成骨细胞在TLM表面的附着、伸展、总蛋白合成和ALP活性,还可促进细胞的早期分化及成骨相关基因OCN、ALP和BMP-2的表达,但对细胞增殖无明显影响。而5V电压阳极氧化处理除了对成骨细胞在TLM表面增殖有抑制作用和对细胞的晚期分化有一定促进作用外,对细胞其他的生物学行为无明显影响。
[Abstract]:In order to improve the bonding properties of the implant bone, the researchers try to surface modification of implant with different methods. The anodic oxidation is a kind of simple and effective method of titanium and its alloy surface modification, can be obtained with nanoscale surface morphology and biological activity of good biological compatibility. This study the anodic oxidation method in low elastic modulus, near beta titanium alloy Ti-5Zr-3Sn-5Mo-15Nb alloy does not contain toxic elements (TLM) surface treatment, and to observe its effect on the behavior of early biological bone cells. With regulated DC power supply in 5V and 20V two respectively under the voltage of the TLM surface anodic oxidation treatment, surface observation the morphology of samples by field emission scanning electron microscopy, X ray diffraction (XRD) analysis of the surface phase, contact angle measurement system for measuring surface contact angle and surface energy calculation, commercial kit evaluation of surface protein absorption The ability of fluorescence, counting method for detecting bone cell attachment on the surface of the material, a tetrazolium (MTT) method for detection of cell proliferation on the material surface, scanning electron microscopic observation of cells on the surface of sample form, commercial kit for detection of total protein synthesis and alkaline phosphatase (ALP) activity, real time quantitative PCR (Realtime-PCR) method for the detection of different surface the osteoblast gene expression in osteoblasts.
The results are as follows:
After anodizing, 1. TLM samples form a closely connected network structure with a pore size of about 10 nm at 5V voltage. At 20V voltage, a microtube structure with a larger tube gap and a diameter of 80 nm is formed on the surface.
The results of 2. XRD show that the crystal phase on the surface of the TLM specimen is anatase.
3., by observing the contact angle of two liquids on the surface of TLM sample, it is found that the contact angle of 5V voltage anodized surface and polished surface increases sequentially, while the surface energy decreases successively, and the difference between them is significant (20V 0.05). The surface contact angle of two kinds of liquids is increased.
The results of 4. surface protein adsorption showed that the protein adsorption capacity of the 20V voltage anodized TLM surface was significantly stronger than that of the 5V voltage anodic oxidation surface and the polished surface.
5. cell culture experiments showed that osteoblasts under 20V voltage anodic oxidation number early in the formation of TLM nanotube surface attachment was significantly higher than that of the polished surface (P 0.05), cell proliferation level formation and polishing surface close to the.5V voltage of the osteoblast surface nano network attached was significantly lower than that of 20V voltage anodic oxidation (P 0.05), but close to the polished surface, and the cell proliferation was significantly lower than that of the other two surface (P 0.05).
The total protein synthesis and ALP activity of osteoblasts on the 6. 20V voltage anodized surface were significantly higher than that of the 5V voltage anodized surface and the polished surface (P 0.05).
7. osteoblasts cultured for four days in the sample surface, electron microscopy showed that the osteoblast cell processes and polishing lines stretching less on the polished surface along. In anodic oxidation, osteoblast spread good flat plate, in which a large number of filopodia and extends to the 20V voltage surface treatment of cells.
8. Realtime-PCR the results showed that the anodic oxidation treatment on the surface of TLM osteoblast integrin beta 1 (Integrin beta 1) and transcription factor -2 (RUNX2) expression was not affected. But in osteoblastic ALP 20V voltage anodic oxidation surface, osteocalcin (OCN) and bone morphogenetic protein -2 (BMP-2) the expression on the third day increased significantly, while in the bone cells of osteopontin into 5V voltage anodic oxidation surface (OPN) was significantly higher in tenth days.
Conclusion: TLM anodized surface after the formation of nanoparticles or nanotubes, surface wettability and surface energy increase, improve the protein adsorption capacity, these effects are anodic oxidation and anodic oxidation voltage on voltage of.20V can promote osteoblast spread on the TLM surface, adhesion, total protein synthesis and ALP activity also, can promote cell differentiation and early osteogenic related gene OCN, expression of ALP and BMP-2, but has no obvious effect on cell proliferation. 5V voltage anodic oxidation treatment in addition to osteoblast inhibition has a role in promoting differentiation and late on cell proliferation on the surface of TLM, had no obvious effect on cell the biological behavior of the other.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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