| Objective:Current dental implant research aims at seeking an innovative surface able to promote bioactivity and biocompatibility of titanium implant response to the cells and tissues at the bone-implant interface and to accelerate osseointegration. Anodic oxidation is a promising method on the implant innovative surface.In this paper,we modified the titanium surface using anodic oxidation to form surface morphology and biological characteristics to investigation murine preosteoblast MC3T3-E1 cell secretion and expression of osteoprotegerin m RNA influence.We further sought to characterize the cellular and molecular responses of murine preosteoblast MC3T3-E1 cells to modified titanium surface and correlation between biological characteristics and cell biological behavior.Materials and Methods:1 Medical pure Ti plates in dimensions of 10mm×10mm×1mm were polished with #1000 Si C abrasive paper, and then ultrasonically cleaned in acetone, ethonal and deionized water for 5min respectively.Anodic oxidation experiments were carried out using a direct current voltage source. The Ti plates were used as anodic electrode while graphite(40 mm×40 mm×5 mm) was used as cathodic electrode. The distance between anodic and cathodic electrodes was 40 mm. 1 M Na F solution was used as electrolyte. The anodization process was carried out separately 20 V for 20 minute and 10 V for 1 hour at room temperature.Anodic oxidation samples at 10 V for 1 hour as text group NT and 20 V for 20 minute as text group NP,the as-polished Ti plates without anodic oxidation were used as the control group and each group with 8samples.2 Surface morphology of the obtained samples was observed using scanning electron microscopy(SEM). The crystal structure was examined by X-ray diffraction(XRD) analysis on a RIGAKUD/MAX2500 diffractometer with Cu Ka radiation. The surface roughness was examined by atomic force microscopy(AFM), and the hydrophilicity was assessed from the measurements of the contact angle between the deionized water and sample surface at room temperature.The samples were soaked in simulated body fluid(SBF) two weeks without stirring to evaluate their bioactivity.3 MC3T3-E1 murine preosteoblasts were seeded on the sample surfaces. The cells were cultured in the Dulbecco’s modified Eagle’s medium with 10 % fetal bovineserum and 3 % penicillin/streptomycin. Cultures were maintained at 37 ℃ in an incubator with a fully humidified atmosphere of 5 % CO2. After culturing for 1, 4 and7 days, the cellular proliferation on the sample surfaces was evaluated by the MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma, St.Louis, MO] colorimetric assay according to the manufacturers’ instructions.After culturing for 7 days, the cell morphologies were observed by SEM.4 After cells cultured on the sample for 7 days, total RNA was isolated.To determine the OPG m RNA gene expression levels between different groups by Reverse transcription-polymerase chain reaction(RT-PCR) and Quantitative real-time PCR analysis.Results:1It can be seen that,the as-polished Ti showed an even and smooth surface, while a homogeneous and uniform array of nanotubes and nanopres was separately formed on the surface of anodic oxidation Ti sample, the diameter of the nanotube was about 70 nm and nanopore was about 25 nm. The formation of nanostructures has influence on the surface roughness and hydrophilicity, which are two important factors that affect osteoblast behaviors. The surface roughness and hydrophilicity of the samples were quantified using AFM and contact angle measurements. The root-mean-square surface roughness of Sample NT and NP was separately 200 ± 11 nm and 136 ± 7nm,which is significantly higher than that of the as-polished Ti(around 40 ± 5 nm).The water contact angle was decreased from about 91°±1°to about 32°±2°and72 ° ± 2 °, indicating that the initial hydrophobic surface was changed to hydrophilic surface by anodic oxidation induced nanostructures. The as-polished and anodized Ti samples after immersion in SBF for 2 weeks. It can be seen that there was no obvious difference in surface morphology of the as-polished Ti before and after immersion in SBF, indicating that no apatite was formed on the surface. By contrast, for the two groups anodized samples, bone-like apatite on the surfaces,which covered the initial nanopore and nanotube. There is no obvious on the two test group.2 After culturing for 1, 4 and 7 days, the cellular proliferation on the sample surfaces was evaluated by the MTT,It can be seen that, at the 1-day time point, there was no statistically significant difference(P>0.05) in cell numbers among the three samples.Over the 7-day period, there was a progressive and significant increase in cell numbers. By day 4, a statistically significant difference(P < 0.05) in proliferationwas seen between the as-polished and the anodized Ti surfaces, and the cells proliferated much more quickly on nanotubed surface than those cultured on the nanopored surface. By day 7, the cell number on anodized Ti was about twofold as compared to that on the polished samples, however, the difference between the two anodized Ti samples was not so obvious. The cell number on nanotubes is a little higher than that on nanopores. These results revealed that the anodized Ti promoted osteoblasts proliferation, indicating its better biocompatibility.3 The gene expression level of osteoprotegerin in the MC3T3-E1 cells cultured on anodized titanium surface with nanopores was significantly higher than that on the as-polished titanium(P < 0.05).The test group 1 was about 3.7 times of the control group(P < 0.01) and the test group 2 was about 2.6 times of the control group(P< 0.01).Conclusions:1 A homogeneous and uniform array of nanotubes and nanopores was separately formed on the surface of anodic oxidation Ti sample,The beneficial effect of nanostructures on cells behavior might be attributed to the surface roughness and hydrophilicity,and the nanotubes has better results.The anodized samples mersion in SBF for 2 weeks, indicating that bone-like apatite was formed on the surface,indicating that the anodic oxidation treatment enhanced the bioactivity of Ti.2 Anodic oxidation is a promising method to increase cell attachment and spreading. The enhanced biocompatibility was attributed to the anodization induced nanostructured surface, high surface roughness and hydrophilicity, which have been proved to have positive effect on cell behavior, such as early differentiation and significant bone cell proliferation. Based on these results, it can be concluded that anodic oxidation modified Ti surface showed excellent bioactivity and biocompatibility. The reason that nanotubes accelerate cell proliferation more effectively than nanopores might be attributed to the higher surface roughness and hydrophilicity.3 The gene expression of osteogenic markers OPG was upregulated by anodic oxidation modification,and also favored the adhesion and proliferation of cells. The gene expression of osteogenic markers OPG was upregulated clearly by nanotubes.Increased gene expression of OPG indicates that OPG activation may contribute to the increased osteogenic differentiation, improving the Bioactivity and Biocompatibility of the Ti.4 Anodic oxidation as a promising method on the implant innovative surface canaccelerated osseointegration. |
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