多孔阳极氧化铝薄膜的制备及其对细胞行为和牛血清蛋白吸附的影响

发布时间：2018-06-06 13:24

本文选题：多孔阳极氧化铝 + L929　；参考：《东华大学》2012年硕士论文

【摘要】：生物材料表面纳米结构和表面生物化学性质一样,也强烈影响和控制着细胞的行为。在生物材料表面构筑类似于细胞生长的有序化微环境,是提高细胞亲和性和特异性识别,增强材料生物功能性的重要手段。随着纳米生物和医学技术的快速发展,人们发现,相对于微米尺度,纳米尺度的结构与机体内细胞生长的自然环境更为相似,因此有关纳米结构的构建以及纳米结构对细胞行为影响的研究迅速增加。其中关于有序纳米孔阵列对细胞行为影响的报道并不多见,且适合细胞生长的最佳尺寸目前尚无确定参数,有待于进一步研究。多孔阳极氧化铝薄膜(Porous Anodic Alumina, PAA)是一种典型的具有统一圆柱形孔径和有序纳米孔结构的自组装纳米孔阵列材料,具有孔径可控、化学和机械性能稳定、生物相容性良好和不易降解等特点,可以作为纳米结构模板来研究其有序纳米孔阵列结构对细胞及蛋白吸附行为的影响。本文首先采用两步阳极氧化法在草酸电解液中制备出有序的PAA薄膜。采用FESEM (JSM-5600LV, JEOL, JPN)观察PAA薄膜表面形貌,在Photoshop软件下分析不同条件下制备的PAA膜的孔径、孔心距、孔壁厚、孔密度及孔隙率等孔结构参数；采用光学轮廓仪(Wyko NT9100, USA)和视频接触角测量仪(OCA40Micro, GER)分析材料表面的粗糙度和亲水性；EDS(IE300X, Oxford, UK)和XPS(ESCALAB250, Thermo Electron, USA)对PAA膜表面元素组成进行了检测。将人脐静脉血管内皮细胞(HUVEC).小鼠成纤维细胞(L929)和前成骨细胞(MC3T3-E1)接种于不同孔结构的PAA表面,观察HUVEC、L929和MC3T3-E1在PAA表面的粘附、增殖和分化等生长情况。最后采用BCA蛋白浓度检测试剂盒初步探索了牛血清白蛋白在不同孔结构的PAA表面的吸附情况。实验结果表明在草酸电解液中能够制备出纳米孔径可控的(25、50、65、75nm)高度有序的PAA薄膜。随着孔径从25、50、65、75nm逐渐增大,孔隙率也随之增大(8.2%、19.1%、28.6%、45.2%),孔壁厚度逐渐减小(70、50、40、25nm),但孔心距保持一致,均为100nm,孔密度分别为0.98×1010,1.04×1010,1.07×1010,1.04×1010个/cm2。同时改变工艺条件制备出另一种孔径为65nm的PAA膜,其孔心距为125nm,孔壁厚为65nm,孔密度为0.67×1010个/cm2,孔隙率为29.8%。材料表面特征分析结果表明这些PAA薄膜的粗糙度无明显差异,表面接触角均小于90°(30.53°-80.83°),即PAA薄膜都具有良好的亲水性。元素测定结果表明这些PAA薄膜主要由A1和O两种元素组成。细胞实验结果表明HUVEC、L929和MC3T3-E1细胞在不同孔径PAA薄膜表面上均发生良好的粘附,细胞形态正常。MTT法测试结果表明培养7d后,HUVEC细胞在50nm孔径PAA膜上的增殖能力最大,且与小孔径(25nm)有显著性差异(p0.05);CCK法测试结果表明培养4h后,L929细胞在较大孔径(75nm)PAA膜上的粘附功能最高,培养7d后,L929细胞在各孔径PAA膜的增殖能力均高于空白对照,较大孔径(75nm)PAA膜上的增殖能力最大,且与其他孔径(25、50和65nm)的PAA膜和空白对照均有显著性差异(p0.05);MC3T3-E1细胞在较大孔径(75nm)PAA膜上的粘附功能明显高于小孔径(25和50nm)的PAA膜(p0.05),表明相对于小孔径(25和50nm)PAA膜,较大孔径(75nm)的PAA膜能够促进MC3T3-E1细胞的粘附,初步显示出较好的生物学活性。MC3T3-E1细胞在各孔径PAA膜的增殖能力均高于空白对照,50nm孔径PAA膜上的增殖能力最大,且与小孔径(25nm)有显著性差异(p0.05)。碱性磷酸酶活性(alkaline phosphatase activity,ALP)测定结果表明MC3T3-E1细胞培养7d后,空白对照和较大孔径(75nm)PAA膜上的ALP活性最大,且与其他孔径(25、50和65nm)的PAA膜均有显著性差异(P0.05),说明空白对照和75nm孔径PAA膜具有较强的早期成骨分化活性。 CCK法测试结果表明L929和MC3T3-E1在孔密度较小的PAA膜表面的粘附、增殖能力以及ALP活性均高于孔密度较大的PAA膜上的细胞。各孔径PAA膜表面吸附的牛血清蛋白量均比空白对照多,说明有序纳米孔阵列结构有利于蛋白的吸附,其中较大孔径(75nm)PAA膜表面吸附的蛋白量最多。孔径相同时,孔密度较小的PAA膜的蛋白吸附能力较高。
[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.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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