Biological Responses Of Osteoblast Cells On Nitrogen-doped Titanium Dioxide Thin Film By Cathodic Arc Deposition

Part IPreparation and surface characterizations of N-doped titaniumdioxide thin film fabricated by cathodic arc depositionObjective:To prepare the N-doped titanium dioxide thin film by cathodic arc deposition(CAD), and to determine the characterizations of the thin films. Method:N-doped TiO2thin films wereprepared with CAD. The used atmosphere contained pure oxygen and mixture gas of oxygen andnitrogen. There was a direct current superimposed pulsed bias with15%duty ratio applied. The directcurrent voltage was40V and a bias was-500V. The pulse duration of cathodic current was2000μs andthe pulse frequency was7Hz. The deposition time was2h and working pressure was4.0×10-3Pa.Surface morphologies of the films were observed by scanning electron microscopy. X-rayphotoelectron spectroscopy (XPS) was employed to analyze the elements of the films. The contactanglesbetween the water drops and samples were measured by sessile drop method. The Zeta potentialof samples was measured with a Surpass electrokinetic analyzer. Results: N element was successfullydoped into TiO2thin films, which was confirmed by the XPS results. There was no difference ontopography among three samples, which were uniform and smooth. XPS of N-doped TiO2thin filmdisplayed the peak of Ti2p at459eVand N1s at400eV. The contact angle was88.1±1.5°on pure Tisurface,41.5±5.6°on TiO2surface and38.4±5.0°on N-doped TiO2thin film.Conclusion:N-doped TiO2thin film was prepared by CAD. The surface topography was not altered after treatment with CAD. Thecontact angle of N-doped TiO2thin film decreased and showed better hydrophilicity. The Zeta potentialof N-doped TiO2was more negative than that of the other groups under physical pH. Part II The effect of N-doped TiO2thin film prepared by cathodic arcdeposition on adhesion and migration of osteoblast-like MG63cellsObjective:To evaluate the effect of N-doped TiO2thin film fabricated by CAD on adhesionand migration of MG63cells. Methods:MG63cells were cultured on the surfaces of three samples.After culture at the predetermined times, the samples were collected for evaluation. CCK-8Kit andDAPI staining were used to measure the number of cell adhesion. SEM was employed to detect themorphology and spreading of cells on different surfaces. FITC-Phalloidin and DAPI stain wereperformed to observe the dynamic changes of cell spreading and cytoskeleton. Wound healing assaywas performed to evaluate the migration of MG63cells cultured on different surfaces. After culture atthe predetermined times, the samples were collected for evaluation. Gene expression of Integrin-β1were evaluated with reverse transcriptionpolymerase chain reaction (RT-PCR). Results: The CCK-8results showed that there were more MG63cells attaching on the N-doped TiO2thin film after4,6and12hours of culture, which was consistent with the results of DAPI staining. MG63cells on N-dopedTiO2thin filmdisplayed well-defined and complex cytoskeletal actin stress fibers following the maincellular axis as compared with the two other groups. SEM displayed that cells on the N-doped TiO2thinfilm appeared more spreading and flatten than that on the TiO2film. The cell spreading areas were alsolarger, as compared with cells on pure Ti substrate and TiO2film.It was clear that MG63cells on theN-doped TiO2thin film migrated to the center of the wound field. After12h of culture, integrin-β1mRNA expression of cells cultured on the pure Ti substrate was lower than that on the TiO2andN-doped TiO2thin films, however, there was no significant difference between TiO2and N-doped TiO2substrates. Conclusions: It was proved that N-doped TiO2thin film by CAD could enhance the adhesionand migration, which could be caused by the more negative charges and the conformation changes ofadhesive proteins. It is suggested that N doping will be worth further consideration for surfacemodification of joint prosthesis. Part III The effect of N-doped TiO2thin film prepared by cathodic arcdeposition on proliferation of osteoblast-like MG63cellsObjective:To evaluate the effect of N-doped TiO2thin film fabricated by CAD onproliferation of MG63cells. Methods: MG63cells were cultured on the surfaces of three samples.After culture at the predetermined times, the samples were collected for evaluation. CCK-8kit was usedto determine the number of cell proliferation. Acridine orange staining was performed to evaluate theMG63cells proliferation. The cell cycles were analyzed using flow cytometry. Results: The CCK-8andacridine orange staining presented growth in cell number as a function of time for all three groups.Upon4days of culture, cell proliferation on the N-doped TiO2thin film revealed superior to that on thepure Ti-disc, however, no significant differences existed between TiO2and the other two groups. After7days of culture, the difference of cell proliferation was even more obvious. The numbers of cells onN-doped TiO2thin film were significant higher than that on pure Ti disc, and importantly, there was alsosignificant difference between TiO2and N-doped TiO2thin film. The proliferation of MG63cellscultured on three samples were also confirmed using acridine orange staining assay at the predeterminedtime points. Flow cytometric analysis showed that the percentage of MG63cells in S and G2/M phaseson N-doped TiO2thin film was significantly higher than that on the other two groups at4days.Meanwhile, cells in G0/G1phase N-doped TiO2thin film were lower than that on the other two groupsat4days. Conclusion: N-doped TiO2thin film fabricated by CAD could promote the proliferation ofMG63cells, which may be closely related to the better hydrophilicity and more negative surfacecharges. Part IV The effect of N-doped TiO2thin film prepared by cathodic arcdeposition on differentiation of osteoblast-like MG63cellsObjective:To evaluate the effect of N-doped TiO2thin film fabricated by CAD on differentiation of MG63cells. Methods: MG63cells were cultured on the surfaces of three samples.After culture at the predetermined times, the samples were collected for evaluation.Alkalinephosphatase (ALP) activity was measured using ALP kit. Gene expression of ALP and type Icollagen were evaluated with reverse transcriptionpolymerase chain reaction (RT-PCR).Immunofluorescence and Western-blot assay were performed to detect the expression of osteopontin(OPN). Results: After4and7days of culture, cells on TiO2and N-doped TiO2thin film consistentlyexhibited higher ALP activity than those on the pure Ti sample. However, there was no significantdifference on ALP activity between TiO2and N-doped TiO2thin film.After4days of culture, theexpression of ALP gene on N-doped TiO2thin film was high than that on the other groups. On day7, theexpression of ALP gene on the pure Ti substrate was lower than that on TiO2and N-doped TiO2thinfilm, however, there was no difference on expression of ALP gene between TiO2and N-doped TiO2groups. After4days of culture, the mRNA level of type I collagen on TiO2andN-doped TiO2groups washigher than that on the pure Ti group, however, there was no difference between TiO2and N-doped TiO2groups.After7days of culture, there was a little expression of OPN on the three groups. However, onday10, the expression of OPN on N-doped TiO2thin film was higher than that on the pure Ti and TiO2groups. The result of Western-blot about OPN was consistent with that of immunofluorescence.Conclusion: On the N-doped TiO2thin film, the differentiation of MG63cells were enhanced throughthe increase of ALP and OPN expression, up-regulation expression of ALP and type I collagen gene.N-doped TiO2thin film could promote differentiation of MG63cells, which indicated that N-dopedTiO2thin film could be favorable surface modification.