Investigation Of The Cellular Effects Nanostructures On Substrate Surface And Compatibility Ionic Liquids

Design of new types of biomaterials is one of the key topics in tissue engineering. New types of scaffolds with special topography and chemical composition can be obtained by materials modification. In this thesis, a series of nanobiological phenomena being elicited by periodic nanostructures on biomaterial surfaces at cellular level were found, and analysis of these results was based on the views of cell biology, which would put up with valuable instructions in theory for smart scaffold design. Under the same cell experimental conditions, the cellular compatibility of ionic liquids (ILs) with their"tuneable"nature, which are named green chemicals, was systematically evaluated. The relationship between the cellular compatibility of ILs and their chemical structures was studied, which would offer some indications in theory for developing new biomaterials based on ILs.Polystyrene is a type of nondegradable macromolecular biomaterials, which is widely used in cell culture and tissue culture in vitro and also an ideal biomaterial for investigation of the influence of the physiochemical properties of biomaterial surfaces on cell behavior. Due to its hydrophobility, polystyrene is often treated using oxygen rich plasma to introduce polar groups to accelerate adherent cells to grow. In the present work, a smooth polystyrene thin film and a smooth polystyrene-like polymeric ionic liquid (PIL) - poly (1-octyl-4-vinylpridinium bromide) (PC8PyBr) were irradiated by a beam of polarized laser, with the wavelength of 266 nm and the energy of 3.0 mJ/cm2. The results of atomic force microscopy (AFM), Fourier transform infrared spectrophotometry (FT-IR) and goniometry indicated that not only hydrophilic carbonyl-containing species formed, but also sub-micrometer laser-induced periodic surface structures (LIPSS) with a periodicity of 250 nm and a depth of 60 nm were produced on polystyrene surface, which resulted in the increase of the surface energy from 43.4 mN/m to 63.1 mN/m (in the direction parallel to LIPSS), and LIPSS with a periodicity of 170 nm and a depth of 50 nm also formed on PC8PyBr surface. The quantity of adsorbed bovine serum albumins on laser-modified polystyrene was more than that on smooth polystyrene. Rat glioma C6 cells and human cervical carcinoma HeLa cells were chosen as the cell models. Compared to the behavior of the cells cultured on smooth polystyrene, their behavior on laser-modified polystyrene was obviously different, such as, firmer cell-substrate adhesion, faster proliferation, slower migration, migration along LIPSS, spreading along LIPSS using filopodia step by step, both the longest axis of the whole cell and its nucleus parallel to LIPSS, and the orientation of division parallel to LIPSS. The results of immunostaining focal adhesion kinases andγ-tubulins indicated that, for cells cultured on laser-modified polystyrene, the areas of focal adhesions were larger, and when cells were in metaphase or anaphase, the line connecting two microtubulin organization centers was parallel to the direction of LIPSS. Therefore, the hydrophilic carbonyl-containing species forming on laser-modified polystyrene might play an important role in accelerating cell adhesion and proliferation, while LIPSS acts as a persistent, directional and mechanical stimulus to induce cells to spread using filopodia, and to migrate and divide along LIPSS. The property of LIPSS inducing cell directional migration and orientated division not only helps to reveal the mechanism of the anisotropic patterned nanostructures on biomaterial surface eliciting specific response in cells, but also to control cell directional distribution, to realize some special physiological function in man-made organs in tissue engineering.ILs are a new class of synthesized salts, which are defined as salts that melt at or near room temperature, and are made up of organic cations and inorganic or organic ions. In this study, 38 ILs and two polystyrene-like PILs were synthesized, which were building blocks in IL chemistry, including four classes of cations, four classes of ions, and five classes of alkyl side chains. Human cervical carcinoma cell line HeLa (HeLa cells) was chosen as the cell model and the cellular compatibility of ILs and two polystyrene-like PILs was systematically evaluated. The cytotoxicity of ILs was the marker of their cellular compatibility. A MTT array was used to obtain median effective concentration (EC50) of ILs towards HeLa cells. The relationship between the cytotoxicity of ILs and their chemical structures was studied. Concentration addition model was used to explore the relationship between the modes of action of ILs and their chemical structures. For the first time, 1-ethyl-3-methylimidium tetrafluoroborate ([emim][BF4]), which was the fist synthesized stable IL in air and water in 1992, was added into the culture medium to induce cells to death, and cell morphological and biochemical changes were observed. The results of the above experiments showed that the cytotoxicity of ILs increased with the longer length of the side chains (ethyl-, butyl- and octyl-), alkyl-triehtylammonium salts were less toxic than their pyridinium and imidazoliu analogues, the modes of action of bromide salts of ILs with the side chains of ethyl- and butyl- were similar while those of bromide salts of ILs with the side chains of benzyl- and octyl- similar, and [emim][BF4] could induce HeLa cells to death by apoptosis with some morphological and biochemical changes, such as, vesicles in the cytoplasm, surface blebs, chromatin condensation, the loss of mitochondrial membrane potential, and the increase of reactive oxygen species and of the concentration intracellular free calcium in the cytoplasm. In conclusion, ammoniums salts and ILs with lower cytotoxicity, whose cations are with shorter side length (such as, ethyl-, propyl- and butyl-), could be interesting candidates for developing new biomaterials. At the same time, water-soluble poly (1-ethyl-4-vinylpyridinium bromide, PC2PyBr) is more toxic towards HeLa cells, while laser modification could improve the cytocompatibility of PC8PyBr.