| Surface nanostructure of biomaterials influence cellular behavior greatly, as well as surface biochemical properties. It is one of the key means to enhance cells affinity, specificity recognition and biological function of biomaterials by means of constructing ordered microenvironment which is analogous to cellular growth on biomaterial surfaces. With the rapid development of nanobiology and medical technology, it has been found that nanoscale structures are more similar to the natural environment of the cellular growth than micronscale, so researches in the area of fabrication of nanoscale structures and the influence of nanoscale structures on cellular response are increasing rapidly. Up to now, few studies have been reported about the influence of ordered nano-pore arrays on cellular behavior, and the optimal size for cellular growth remains further study. The porous anodic alumina (PAA) membranes are typical self-assembly nano-porous arrays materials with uniform cylindrical pore size and ordered nano-porous structure, which have certain characteristics such as well controlled pore size, chemical and mechanical properties stability, good biocompatibility and nonbiodegradability, so they can be served as nanoscale structure templates to research the effects of ordered nano-pore arrays on cellular behavior and protein adsorption.PAA membranes were fabricated by two-step anodization procedure in oxalic acid. SEM (JSM-5600LV, JEOL, JPN) was used to observe the pore morphology and pore size distribution, followed by figuring out the pore size, hole centers distance, hole wall thickness, porosity density and porosity with Photoshop. Surface Profiler (Wyko NT9100, USA) and contact angle tester (OCA40Micro, GER) were used to characterize the membranes in terms of surface roughness and hydrophilicity. The surface elemental compositions of PAA membranes were analyzed by EDS (IE300X, Oxford, UK) and XPS (ESCALAB250, Then-no Electron, USA). HUVEC, L929and MC3T3-E1were seeded on PAA to investigate the ability of PAA membranes to enhance cell adhesion, proliferation and differentiation. Finally, the adsorption of serum-protein on ordered PAA membranes was preliminary investigated using BCA assay.The results indicated that ordered PAA membranes with controlled pore size of25,50,65and75nm were successfully fabricated in oxalic acid. With the increase of pore sizes, porosity increased (8.2%,19.1%,28.6%and45.2%) but hole wall thickness decreased (70,50,40and25nm), hole centers distance were kept constant (100nm) and porosity density were0.98×1010,1.04×1010,1.07×1010,1.04×1010pores/cm2, respectively. Another65nm PAA membrane was fabricated by altering technological conditions, which hole centers distance was125nm, hole wall thickness was65nm, porosity density was0.67×1010pores/cm2and porosity was29.8%. Feature analysis of PAA surface showed there was no difference among the roughness of these membranes, which had good water affinity since the contact angles were less than90°(30.53°-80.83°). Elemental composition analysis indicated that PAA membranes mainly consisted of oxygen and aluminum. The cytological experiments exhibited that HUVEC, L929and MC3T3-E1adhere to PAA membranes closely and showed the normal morphologies. MTT assay indicated that after7days of culture, the number of HUVEC on50nm PAA membrane was higher than that of other membranes and had a significant difference with25nm PAA (p<0.05). CCK assay indicated that after4hours of culture, L929on75nm PAA membrane had the strongest adhesion ability, after7days of culture, the number of L929on experiment group was higher than that of blank control,75nm PAA membrane demonstrated higher cell proliferation capacity than that of25,50,65nm PAA and blank control (p<0.05).75nm PAA membrane showed higher cell adhesion potential of MC3T3-E1than25and50nm PAA (p<0.05), which indicated that75nm PAA membrane had higher bioactivity. After7days of culture, the number of MC3T3-E1on experiment group was higher than that of blank control,50nm PAA membrane demonstrated higher cell proliferation capacity than25nm PAA (p<0.05). The ALP results intimated that 75nm PAA and blank control had the most increased biochemical activity of MC3T3-E1than that of25,50and65nm PAA (p<0.05).L929and MC3T3-E1cultured on PAA with smaller porosity density showed better adhesion, proliferation capacity and osteogenic differentiation activity.The adsorption of serum-protein on experiment group was higher than that of blank control, which indicated that ordered nano-pore arrays can enhance protein adsorption.75nm PAA had the greatest ability of protein adsorption. For the same pore size, PAA with smaller porosity density can enhance serum-protein absorption. |
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