本文關(guān)鍵詞： 近β鈦合金 陽極氧化 表面能 成骨細(xì)胞 細(xì)胞附著 細(xì)胞增殖 細(xì)胞分化 ALP 成骨相關(guān)基因　出處：《第四軍醫(yī)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 為了提高骨內(nèi)種植體的骨結(jié)合性能,研究者們嘗試用不同的方法對(duì)種植體進(jìn)行表面改性。陽極氧化是對(duì)鈦及其合金表面改性的一種簡單而有效的方法,可以獲得具有良好生物相容性及生物活性的納米級(jí)表面形貌。本研究采用陽極氧化方法對(duì)彈性模量較低、不含有毒合金元素的近β鈦合金Ti-5Zr-3Sn-5Mo-15Nb (TLM)表面進(jìn)行處理,并觀察其對(duì)成骨細(xì)胞早期生物學(xué)行為的影響。用穩(wěn)壓直流電源在5V和20V兩個(gè)電壓下分別對(duì)TLM表面進(jìn)行陽極氧化處理,采用場發(fā)射電鏡觀察試樣表面形貌、X射線衍射分析儀(XRD)分析表面晶相、接觸角測量系統(tǒng)測量表面接觸角并計(jì)算表面能、商業(yè)試劑盒評(píng)價(jià)表面蛋白吸附能力、熒光計(jì)數(shù)法檢測成骨細(xì)胞在材料表面附著、噻唑鹽(MTT)方法檢測細(xì)胞在材料表面增殖、掃描電鏡觀察細(xì)胞在試樣表面的形態(tài)、商業(yè)試劑盒檢測總蛋白合成及堿性磷酸酶(ALP)活性、實(shí)時(shí)定量PCR(Realtime-PCR)方法檢測不同處理的表面成骨細(xì)胞成骨相關(guān)基因的表達(dá)。 結(jié)果如下: 1. TLM試樣經(jīng)陽極氧化后,在5V電壓下表面形成鄰接緊密的孔徑約為10 nm的網(wǎng)狀結(jié)構(gòu),在20V電壓下表面形成管間隙較大的管徑約為80 nm的微管結(jié)構(gòu)。 2. XRD結(jié)果顯示TLM試樣表面的晶相為銳鈦礦。 3.通過觀察兩種液體在TLM試樣表面的接觸角發(fā)現(xiàn),20V電壓陽極氧化表面、5V電壓陽極氧化表面和拋光表面的液體接觸角依次增大而表面能依次減小,它們之間的差異具有顯著性(p 0.05)。 4.表面蛋白吸附檢測結(jié)果顯示20V電壓陽極氧化TLM表面的蛋白吸附能力顯著強(qiáng)于5V電壓陽極氧化表面和拋光表面。 5.細(xì)胞培養(yǎng)實(shí)驗(yàn)發(fā)現(xiàn),成骨細(xì)胞在20V電壓下陽極氧化處理形成的TLM納米管表面的早期附著數(shù)量明顯高于拋光表面(p 0.05),細(xì)胞增殖水平與拋光表面接近。5V電壓下形成的納米網(wǎng)表面的成骨細(xì)胞附著顯著低于20V電壓陽極氧化表面(p 0.05),但與拋光表面接近,而細(xì)胞增殖顯著低于另兩種表面(p 0.05)。 6. 20V電壓陽極氧化表面的成骨細(xì)胞總蛋白合成及ALP活性均顯著高于5V電壓陽極氧化表面和拋光表面(p 0.05)。 7.成骨細(xì)胞在試樣表面培養(yǎng)四天后,電鏡觀察顯示成骨細(xì)胞在拋光表面沿拋光紋路伸展且細(xì)胞突起較少。在陽極氧化表面,成骨細(xì)胞伸展較好呈扁平形狀,其中在20V電壓處理表面的細(xì)胞伸出大量的板狀和絲狀偽足。 8. Realtime-PCR檢測結(jié)果發(fā)現(xiàn)陽極氧化處理對(duì)TLM表面成骨細(xì)胞整合素β1(Integrinβ1)和轉(zhuǎn)錄相關(guān)因子-2(RUNX2)的表達(dá)無明顯影響。但在20V電壓陽極氧化表面的成骨細(xì)胞的ALP、骨鈣素(OCN)和骨形態(tài)發(fā)生蛋白-2(BMP-2)的表達(dá)在第三天顯著增高,而在5V電壓陽極氧化表面的成骨細(xì)胞骨橋蛋白(OPN)表達(dá)在第十天顯著增高。 結(jié)論:TLM表面經(jīng)陽極氧化處理后形成納米網(wǎng)或納米管狀的形貌,表面濕潤性和表面能提高,蛋白吸附能力提高,這些效應(yīng)均與陽極氧化電壓有關(guān)。20V電壓下陽極氧化處理可以促進(jìn)成骨細(xì)胞在TLM表面的附著、伸展、總蛋白合成和ALP活性,還可促進(jìn)細(xì)胞的早期分化及成骨相關(guān)基因OCN、ALP和BMP-2的表達(dá),但對(duì)細(xì)胞增殖無明顯影響。而5V電壓陽極氧化處理除了對(duì)成骨細(xì)胞在TLM表面增殖有抑制作用和對(duì)細(xì)胞的晚期分化有一定促進(jìn)作用外,對(duì)細(xì)胞其他的生物學(xué)行為無明顯影響。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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本文編號(hào)：1504297