| It is well known that cells live in the three-dimensional extracellular matrix which has a significant influence on cell behavior and function.Many studies have already reported that,as cells adhering and growing on the substrate,they sense and interpret the surface structure which is an important indirect signal that strongly influences the cell behaviors,such as adhesion,polarity,proliferation and differentiation.Although there are many studies on the relationship between cell behavior and nanostructure,it is still difficult to independently control single parameter of the nanostructure due to the limitations of current nanostructure processing technology.The ultimate goal of the studies on the relationship between cell behavior and structure is to accurately manipulate the growth of cell by precisely regulating the structural parameters of the substrate.Thus,it is necessary to find a good model material that can independently control the nanostructure parameters to systematically explore the effect of single nanostructure parameter on cell behavior.In this study,the self-organizing process of anodic aluminium oxide(AAO)during hard anodic(HA)was firstly explored to obtain controllable preparation of alumina with different periods.AAO with different structural parameters(period,pore diameter,porosity,height)were obtained by adjusting the anodization conditions to study the regular effect on the breast cancer cell behaviors.And the Angelica polysaccharides(ASP)was modified on the AAO with different structure parmeters through the hydroxyl group on the surfacet of nanostructure to study the regular effect of ASP cooperating with nanostructure on breast cancer cell behaviors.On this basis,electrochemical anodization were combined with cell culture and cell dehydration to prepare the gradient hierarchical micro-nano structure(GHMNS),which due to the different anodization area caused by cell contraction.The specific research contents are as follows:1.Self-organization process of aluminum oxide during hard anodizationThe self-organization process of the porous anodic alumina(AAO)during hard anodization(HA)was proposed by synthetically investigating the nanopore morphology,the current density and barrier layer evolution of the AAO films,which were anodized in 0.3 M oxalic acid at 150 V(the voltage rising from 40 V to 150 V at the beginning).It was found that,the high enough current density in the stage of voltage rising can induce the fast film growth,which caused the pores to rapidly enlarge themselves and to reach a relatively ordered rearrangement.The barrier layer thickness showed linearly increase in voltage rising stage and then increased with a decelerated speed in the followed constant anodization stage,where the pores with growth advantage moved down straightly and gradually expanded their cell size to replace the inferior pores around.Accordingly,HA is a unequilibrium process and can be divided into two stages: I,the rapid pore enlargement and rearrangement during voltage rising stage,where the oxidation rate is larger than that of dissolution;II,competing growth of the nanopores during the followed constant voltage anodization,where the oxidation and dissolution rates approach to each other due to the thickened barrier layer.2.Regulate the cell behaviors using anodic aluminium oxide(AAO)with custom-tailored structural parametersThe effects of controlled single factors of period,porosity,pore diameter and height of the nanostructure on the behaviors of breast cancer cells have been studied,especially focusing on the cell polarity and cell viability.By adjusting the electrolyte,voltage and oxidation time of anodization,AAO with different period,diameter and height were obtained,and co-cultured with breast cancer cell MDA-MB-231 cells.The cell morphology was observed by scanning electron microscopy and cytoskeleton fluorescence staining.Cell viability was detected by MTT assay.It is found that the period and the cell contact area,also known as the porosity,can obviously affect the cell polarity,pseudopodia and cell viability.With the increasing periods of the nanostructure,the percentage of multipolar cells with thicker filapodia and cell viability decreases.And with the increasing porosities of the nanostructure,the cell contact area decreases,both the percentage of bipolar cells,which has the coexistence of lamellipodia and filapodia,and the cell viability increase.However,with the porosity further increasing until the wall of nanopore break and form the nanonipple,the most of cells are nonpolar sphere and cell viability decreases.While the height of the nanostructure has a little effect on the cell behaviors.3.Effects of angelica polysaccharide(ASP)modification on AAO interface on cell growth regularityThe effects of different surface chemical parameters of AAO membrane on the breast cancer cell behavior were investigated by modifying the angelica polysaccharide(ASP).ASP was directly modified on the surface of nanodent and nanopore structured AAO with different periods.And further modified by KH550 covalently with nanodent,nanopore and nanonipple which has different heights.The morphology and cell viability of MDA-MB-231 cells were observed.The results showed that the viability of breast cancer cells was inhibited by the angelica polysaccharide modified on the surface of nanodent,and the viability of cells decreased with the increase of nanodent period and culture time.The covalent connection of ASP to the membrane by KH550 was stable and could inhibit cell activity more effectively,but did not change cell polarity significantly.The inhibition of ASP combined with nanopore structure on cell viabiltiy was similar to that of nanodent structure.There’s little difference in cell viability on the nanostructure covalently ligated ASP with different periods.4.Fabrication of gradient hierarchical micro-nano structured(GHMNS)by cell culture assisted electrochemical anodizationCell culture and dehydration process were combined with electrochemical oxidation to prepare GHMNS AAO membrane.Based on this template,polymer GHMNS was prepared by thermal polymerization.The effects of cell culture density,cell culture time and cell dehydration temperature on the morphology of GHMNS AAO were systematically studied.The density of the microsteps can be adjusted by cell culture density or culture time.The figure of the microsteps is influenced by cell dehydration temperature which control the cell shrinkage degree.Exemplified by using the AAO as a template to fabricate polymer with GHMNS,the possibility that the GHMNS AAO could become a broad platform for the structural preparation of various materials with GHMNS to develop advanced functions is proved.GHMNS is fabricated by inversely using the cell cultivation to control the contact area between the alumina membrane and the electrolyte in the electrochemical anodization,which only needs simple and inexpensive instruments. |
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