本文選題：多孔陽極氧化鋁 + L929　； 參考：《東華大學(xué)》2012年碩士論文

【摘要】：生物材料表面納米結(jié)構(gòu)和表面生物化學(xué)性質(zhì)一樣,也強(qiáng)烈影響和控制著細(xì)胞的行為。在生物材料表面構(gòu)筑類似于細(xì)胞生長的有序化微環(huán)境,是提高細(xì)胞親和性和特異性識(shí)別,增強(qiáng)材料生物功能性的重要手段。隨著納米生物和醫(yī)學(xué)技術(shù)的快速發(fā)展,人們發(fā)現(xiàn),相對(duì)于微米尺度,納米尺度的結(jié)構(gòu)與機(jī)體內(nèi)細(xì)胞生長的自然環(huán)境更為相似,因此有關(guān)納米結(jié)構(gòu)的構(gòu)建以及納米結(jié)構(gòu)對(duì)細(xì)胞行為影響的研究迅速增加。其中關(guān)于有序納米孔陣列對(duì)細(xì)胞行為影響的報(bào)道并不多見,且適合細(xì)胞生長的最佳尺寸目前尚無確定參數(shù),有待于進(jìn)一步研究。多孔陽極氧化鋁薄膜(Porous Anodic Alumina, PAA)是一種典型的具有統(tǒng)一圓柱形孔徑和有序納米孔結(jié)構(gòu)的自組裝納米孔陣列材料,具有孔徑可控、化學(xué)和機(jī)械性能穩(wěn)定、生物相容性良好和不易降解等特點(diǎn),可以作為納米結(jié)構(gòu)模板來研究其有序納米孔陣列結(jié)構(gòu)對(duì)細(xì)胞及蛋白吸附行為的影響。 本文首先采用兩步陽極氧化法在草酸電解液中制備出有序的PAA薄膜。采用FESEM (JSM-5600LV, JEOL, JPN)觀察PAA薄膜表面形貌,在Photoshop軟件下分析不同條件下制備的PAA膜的孔徑、孔心距、孔壁厚、孔密度及孔隙率等孔結(jié)構(gòu)參數(shù)；采用光學(xué)輪廓儀(Wyko NT9100, USA)和視頻接觸角測(cè)量儀(OCA40Micro, GER)分析材料表面的粗糙度和親水性；EDS(IE300X, Oxford, UK)和XPS(ESCALAB250, Thermo Electron, USA)對(duì)PAA膜表面元素組成進(jìn)行了檢測(cè)。將人臍靜脈血管內(nèi)皮細(xì)胞(HUVEC).小鼠成纖維細(xì)胞(L929)和前成骨細(xì)胞(MC3T3-E1)接種于不同孔結(jié)構(gòu)的PAA表面,觀察HUVEC、L929和MC3T3-E1在PAA表面的粘附、增殖和分化等生長情況。最后采用BCA蛋白濃度檢測(cè)試劑盒初步探索了牛血清白蛋白在不同孔結(jié)構(gòu)的PAA表面的吸附情況。 實(shí)驗(yàn)結(jié)果表明在草酸電解液中能夠制備出納米孔徑可控的(25、50、65、75nm)高度有序的PAA薄膜。隨著孔徑從25、50、65、75nm逐漸增大,孔隙率也隨之增大(8.2%、19.1%、28.6%、45.2%),孔壁厚度逐漸減小(70、50、40、25nm),但孔心距保持一致,均為100nm,孔密度分別為0.98×1010,1.04×1010,1.07×1010,1.04×1010個(gè)/cm2。同時(shí)改變工藝條件制備出另一種孔徑為65nm的PAA膜,其孔心距為125nm,孔壁厚為65nm,孔密度為0.67×1010個(gè)/cm2,孔隙率為29.8%。材料表面特征分析結(jié)果表明這些PAA薄膜的粗糙度無明顯差異,表面接觸角均小于90°(30.53°-80.83°),即PAA薄膜都具有良好的親水性。元素測(cè)定結(jié)果表明這些PAA薄膜主要由A1和O兩種元素組成。細(xì)胞實(shí)驗(yàn)結(jié)果表明HUVEC、L929和MC3T3-E1細(xì)胞在不同孔徑PAA薄膜表面上均發(fā)生良好的粘附,細(xì)胞形態(tài)正常。MTT法測(cè)試結(jié)果表明培養(yǎng)7d后,HUVEC細(xì)胞在50nm孔徑PAA膜上的增殖能力最大,且與小孔徑(25nm)有顯著性差異(p0.05);CCK法測(cè)試結(jié)果表明培養(yǎng)4h后,L929細(xì)胞在較大孔徑(75nm)PAA膜上的粘附功能最高,培養(yǎng)7d后,L929細(xì)胞在各孔徑PAA膜的增殖能力均高于空白對(duì)照,較大孔徑(75nm)PAA膜上的增殖能力最大,且與其他孔徑(25、50和65nm)的PAA膜和空白對(duì)照均有顯著性差異(p0.05);MC3T3-E1細(xì)胞在較大孔徑(75nm)PAA膜上的粘附功能明顯高于小孔徑(25和50nm)的PAA膜(p0.05),表明相對(duì)于小孔徑(25和50nm)PAA膜,較大孔徑(75nm)的PAA膜能夠促進(jìn)MC3T3-E1細(xì)胞的粘附,初步顯示出較好的生物學(xué)活性。MC3T3-E1細(xì)胞在各孔徑PAA膜的增殖能力均高于空白對(duì)照,50nm孔徑PAA膜上的增殖能力最大,且與小孔徑(25nm)有顯著性差異(p0.05)。堿性磷酸酶活性(alkaline phosphatase activity,ALP)測(cè)定結(jié)果表明MC3T3-E1細(xì)胞培養(yǎng)7d后,空白對(duì)照和較大孔徑(75nm)PAA膜上的ALP活性最大,且與其他孔徑(25、50和65nm)的PAA膜均有顯著性差異(P0.05),說明空白對(duì)照和75nm孔徑PAA膜具有較強(qiáng)的早期成骨分化活性。 CCK法測(cè)試結(jié)果表明L929和MC3T3-E1在孔密度較小的PAA膜表面的粘附、增殖能力以及ALP活性均高于孔密度較大的PAA膜上的細(xì)胞。 各孔徑PAA膜表面吸附的牛血清蛋白量均比空白對(duì)照多,說明有序納米孔陣列結(jié)構(gòu)有利于蛋白的吸附,其中較大孔徑(75nm)PAA膜表面吸附的蛋白量最多�？讖较嗤瑫r(shí),孔密度較小的PAA膜的蛋白吸附能力較高。
[Abstract]:The surface nanostructures of biomaterials, as well as the surface biochemical properties, also strongly influence and control the behavior of cells. Building an orderly microenvironment similar to cell growth on the surface of biomaterials is an important means to improve cell affinity and specific recognition and enhance the biological function of the materials. With nanoscale and medical technology Rapid development has been found that the structure of nanoscale is more similar to the natural environment of cell growth in the body relative to the micron scale. Therefore, the construction of nanostructures and the influence of nanostructures on cell behavior have increased rapidly. Porous Anodic Alumina (PAA) is a typical self assembled nanopore array material with uniform cylindrical pore size and ordered nanoscale structure, which has a controllable pore size, stable chemical and mechanical properties and biocompatibility. It can be used as a nanostructure template to study the effect of ordered nanoporous arrays on cellular and protein adsorption behavior.
In this paper, an ordered PAA film was prepared by two step anodization in oxalic acid electrolyte. The surface morphology of PAA film was observed by FESEM (JSM-5600LV, JEOL, JPN). The pore structure parameters of PAA film, pore center distance, pore wall thickness, pore density and porosity were analyzed under different conditions under Photoshop software, and optical contour was used. Wyko NT9100 (USA) and video contact angle measuring instrument (OCA40Micro, GER) were used to analyze the roughness and hydrophilicity of the surface of the material; EDS (IE300X, Oxford, UK) and XPS (ESCALAB250, Thermo) were used to detect the surface elements of the membrane. The cell (MC3T3-E1) was inoculated on the surface of PAA with different pore structure and observed the adhesion, proliferation and differentiation of HUVEC, L929 and MC3T3-E1 on the surface of PAA. Finally, the adsorption of bovine serum albumin on the surface of PAA with different pore structure was preliminarily explored with the BCA protein concentration detection kit.
The experimental results show that the nano aperture controlled (25,50,65,75nm) highly ordered PAA film can be prepared in the oxalic acid electrolyte. As the pore size increases gradually from 25,50,65,75nm, the porosity increases (8.2%, 19.1%, 28.6%, 45.2%), and the pore wall thickness decreases gradually (70,50,40,25nm), but the hole center distance keeps the same, 100nm, pore density, respectively. Another PAA membrane with a pore diameter of 65nm is prepared for 0.98 x 1010,1.04 x 1010,1.07 x 1010,1.04 x 1010 /cm2.. The hole center distance is 125nm, the pore wall thickness is 65nm, the pore density is 0.67 x 1010 /cm2, and the porosity is 29.8%. material surface characteristics analysis results show that the roughness of these PAA films has no obvious difference and surface contact angle. It is less than 90 degrees (30.53 -80.83 degrees), that is, PAA films have good hydrophilicity. The results of elemental determination show that these PAA films are mainly composed of two elements of A1 and O. The results of cell experiments show that HUVEC, L929 and MC3T3-E1 cells have good adhesion on the surface of different pore size PAA thin films, and the cell morphology normal.MTT method test results Biao Mingpei After 7d, the proliferation ability of HUVEC cells on the 50nm pore PAA membrane was the greatest, and there was a significant difference from the small aperture (25nm). The CCK method showed that the adhesion function of L929 cells on the larger aperture (75Nm) PAA membrane was the highest after the culture of 4h, and the proliferation ability of the cells in the pore diameter (75Nm) PAA membrane was higher than that of the blank control and the larger aperture (the larger aperture). 75Nm) PAA membrane has the greatest proliferation ability, and is significantly different from that of other Kong Jing (25,50 and 65nm) PAA membrane and blank control (P0.05), and the adhesion function of MC3T3-E1 cells on the larger Kong Jing (75Nm) PAA membrane is significantly higher than that of small Kong Jing (25 and 50nm) PAA membrane (P0.05). It can promote the adhesion of MC3T3-E1 cells. It is shown that the proliferation ability of.MC3T3-E1 cells with better biological activity in each aperture PAA membrane is higher than that of the blank control, and the proliferation ability on the 50nm pore PAA membrane is the greatest, and there is a significant difference from the small aperture (25nm). The alkaline phosphatase activity (alkaline phosphatase activity, ALP) determination The results showed that after the MC3T3-E1 cells were cultured for 7d, the ALP activity on the blank control and the larger aperture (75Nm) PAA membrane was the most, and the PAA membrane of the other pore size (25,50 and 65nm) had a significant difference (P0.05), indicating that the blank control and 75Nm aperture PAA membrane had a strong early osteogenic differentiation activity.
The results of CCK test showed that the adhesion, proliferation and ALP activity of L929 and MC3T3-E1 on the surface of PAA membrane with smaller pore density were higher than those on the PAA membrane with larger pore density.
The amount of bovine serum protein adsorbed on the surface of the PAA membrane of each aperture is more than that of the blank control. It shows that the ordered nanoscale array structure is beneficial to the adsorption of protein, of which the largest pore size (75Nm) PAA membrane adsorbed the most protein, while the pore density is at the same time, and the protein adsorption capacity of the PAA membrane with smaller pore density is higher.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08

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