Study On Biological Evaluations Of TiO₂ And SiO₂ Surfaces

The surface properties of biomaterials are the key to the interaction with cells.Since the outermost layer of oxide materials is always the oxygen atoms,the surface properties are essentially determined by the state of the surface oxygen atoms.In this thesis,the TiO₂ surfaces of different oxygen atoms were construct by micro-nanostructures,and the SiO₂ macroscopic surfaces with different oxygen atoms were produced by the single crystal quartz and vitreous silica.The main consideration of the oxide surfaces is the oxygen atoms states.The effects of different oxygen atom states on osteogenesis and immune response were systematically studied by surface calcium phosphate（CaP）mineralization,protein adsorption and cells[mesenchymal stem cells（MSCs）and macrophages（RAW 264.7）]interaction.The law of action and mechanism of such effects was also investigated.The main achieved research results are as following:1.Study on the effects of micro-nanostructure TiO₂ surfaces on osteogenesis.Anatase TiO₂ polyhedral microcrystals（AN-TPMCs）with{001}and{101}crystal facets exposed on the surface were prepared by hydrothermal method.There was no different mineralization behavior on AN-TPMCs occurring when fetal bovine serum（FBS）was inexistence,and CaP deposits are uniformly covered on the surface of AN-TPMCs.Whereas the distinct mineralization behavior of facet specific on AN-TPMCs occurred with the existence of FBS,CaP was completely covered on{101}facets,but only a few of CaP were deposited and distributed sparsely on{001}facets.The result was mainly due to the different adsorption behavior of FBS on different crystal faces,which leads to the occurrence of subsequent facet-specific mineralization behavior.The results of MSCs culture showed that AN-TPMCs on the macroscopic had good osteogenesis.Rutile TiO₂ nanorods with{110}crystal facts on the side and{001}crystal facets on the top were prepared by hydrothermal method,and a trace anatase phase is doped therein.After heat treatment at 900℃,the anatase phase disappeared,and the electron binding energy of O1s was increased,the polarization of surface oxygen atoms was increased.After soaking in simulated body fluids（SBF）,it revealed that CaP mineralization was more likely to occur on the surface with weakly polarized oxygen atoms.And the result of cell culture showed it beneficial to the proliferation of MSCs and the early osteogenic differentiation.Titanium sheets were heat-treated at a temperature of 500℃or 900℃to produce a macroscopic surface of TiO₂ with different surface oxidation states.The oxide layers produced by oxidation of titanium sheets were rutile phase.As the treatment temperature increased,the electron binding energy of O1s was increased,and the polarization of surface oxygen atoms was increased.After soaking in SBF,it revealed that CaP mineralization was more likely to occur on the surface with weakly polarized oxygen atoms.The result of MSCs culture showed it beneficial to the proliferation of MSCs and the early osteogenic differentiation.According to the results,CaP mineralization was more likely to occur on the surface with weakly polarized oxygen atoms.Due to the absence of topological factors on the macroscopic TiO₂ surface of the heat-treated titanium sheets,the surface with weakly polarized oxygen atoms was more favorable for promoting MSCs osteogenic differentiation.2.Study on the effects of SiO₂ surfaces on osteogenesis.Compared with the single crystal SiO₂{110}facets,the electron binding energy of O1s on the vitreous silica was smaller.On the vitreous silica,the degree of oxygen atoms polarization was weaker,the surface water contact angle was larger,and theζ-potential was lager under the solution environment.At the same time,compared with TiO₂,oxygen atoms polarization on SiO₂ was stronger,and the surfaces were not prone to mineralization and deposition of CaP soaking in SBF.The experiments of fibronectin（FN）adsorbed on the surface showed that the vitreous silica surface with weak surface oxygen atoms polarization upregulated the exposure of functional sites（HFN7.1&mAB1937）compared to that of the SiO₂{110}surface with stronger surface oxygen atoms polarization.The results of cell culture showed that MSCs cultured on the vitreous silica surface developed a larger spreading area,more actin filaments and more adhesions compared to the SiO₂{110}surface,which may be due to the closely binding of cellular integrins to FN functional sites,and making integrinβ1 andβ3 more active.Meanwhile,it increased the activation of ILK signaling pathway,and up-regulated the expression levels of p-Akt andβ-catenin in MSCs,which significantly enhanced the osteogenic differentiation ability of MSCs.The results also showed that the ability of promoting the MSCs osteogenic differentiation on the vitreous silica was as well as the TiO₂ obtained by heat treatment of titanium sheets.The results of this thesis suggest that the mineralization of CaP on materials surfaces may not be an intrinsic indicator to osteogenic response of oxide biomaterials,the surface oxygen atoms states（polarization）may be a key factor.The weaker degree of oxygen atom polarization on the surface can promote the cellular adhesion,proliferation and osteogenic differentiation of MSCs,by upregulated the exposure of adsorbed protein functional sites.3.Study on the effects of TiO₂ and SiO₂ surfaces on immune response.In view of the important role of immune response in the process of osseointegration,it was investigated that the effects of TiO₂ and SiO₂ surfaces on immune response.The results of TiO₂ surfaces showed that strong or weak oxygen atoms polarization induced RAW 264.7 macrophage to M2 polarization,enhanced the expression of M2-polarized gene and the secretion of anti-inflammatory factors,and significantly promoted MSCs osteogenic differentiation in subsequent culture.Whereas,the intermediate oxygen atoms polarization induced RAW 264.7 macrophage to M1polarization,which gave rise to M1-polarized gene expression and inflammatory factor secretion,and a certain extent inhibited MSCs osteogenic differentiation.The results of crystal and vitreous SiO₂ surfaces showed that strong（SiO₂{110}surface）or weak（vitreous silica）oxygen atoms polarization induced RAW 264.7macrophage to M2 polarization,enhanced the expression of M2-polarized gene and the secretion of anti-inflammatory factors,and significantly promoted MSCs osteogenic differentiation in subsequent culture.Whereas,the intermediate oxygen atoms polarization（SiO₂{001}surface）induced RAW 264.7 macrophage to M1polarization,which gave rise to M1-polarized gene expression and inflammatory factor secretion,and a certain extent inhibited MSCs osteogenic differentiation.Meanwhile,it showed the ability of induced RAW 264.7 to M2 on the vitreous silica was as well as on the TiO₂ nanorods film.This thesis can provide a theoretical guidance for the surface design of medical oxide biomaterials in the future,and is of great significance for improving the osseointegration of bone implants.