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Antibacterial Titanium Plate Anodized by being Discharged in NaCl Solution Exhibits Cell Compatibility

Department of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan; and
¹ Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan;

View larger version (50K): [in a new window]   Figure 1. SEM micrographs showing small amounts of granular deposits sparsely distributed on the Ti-Cl. The Ti-Cl specimens were prepared by undergoing gold vaporizing with a vacuum evaporation device. Low-magnification SEM micrograph of Ti-Cl (x200; bar = 200 µm) on the left, and high-magnification SEM micrograph of Ti-Cl (x800; bar = 50 µm) on the right.

View larger version (16K): [in a new window]   Figure 2. Surface characterizations of the Ti-Cl. (A) An XRD peak of TiCl3 is detected on Ti-Cl, while the peak of the TiO suboxide layer is increased on Ti-Cl. XRD was operated at 40 kV, 40 mA with a scanning speed of 0.02°/4 sec and a scanning range of 20-50° with CuK radiation. XPS high-resolution spectra of O1s on Ti-Cl before immersion (B) and after 1 hr of immersion in pure distilled water (C). A 20-mA emission current and 8-kV accelerated voltage were applied in the XPS analysis with MgK radiation. The three peaks of the curve fit of the O1s spectra on Ti-Cl are shown. Peak 1 was set at 530.1 eV at TiO2, peak 2 at 531.1 eV for H2O, and peak 3 at 532.3 eV for Ti-OH. The peak 3 indicating Ti-OH increased (p < 0.01) on the surface of Ti-Cl after immersion (C). The results of the XPS data are expressed as the mean ± SD of 6 specimens (n = 6). The findings were analyzed statistically by Student’s t tests. Significant differences are considered to exist when p < 0.01.

View larger version (24K): [in a new window]   Figure 3. Numbers of adherent MC3T3-E1 and L 929 cells on the specimens after 1 wk of cultivation. The number of adherent cells for each cell line on Ti-Cl was significantly higher than those on control specimens. Adherent cells were incubated with 100 µL/mL of assay reagent solution for 1 hr, and then the absorbance at 450 nm was measured. The number of adherent cells was calculated from the activity of the original cell suspension. The results are expressed as the mean ± SD of 6 specimens (n = 6). The findings were analyzed statistically by ANOVA. *Significant differences from respective control values were considered to exist when p < 0.01.

View larger version (118K): [in a new window]   Figure 4. Fluorescence microscope images of adherent MC3T3-E1 (A) and L929 (B) cells on the specimens. The cells were fixed with 3.7% formaldehyde in PBS and permeabilized by treatment with 0.1% Triton X-100 in PBS for 1 min. The cells were then incubated for 3 hrs with a rhodamine-conjugated phalloidin solution. After the cells were washed with water, stress fiber formation and cell morphology were visualized with the use of a fluorescence microscope. The left panel displays adherent cells on a control specimen. No stress fiber formation is observed in cells on the control specimens, and the cells adhere loosely to the surface compared with those on Ti-Cl. The right panel displays adherent cells on Ti-Cl. These adherent cells have already begun to form stress fibers and have widely extended cytoplasm. (C) The adsorbed fibronectin on each specimen was visualized by means of FITC labeling. The specimens were soaked in minimal essential medium containing 10% fetal bovine serum (37°C, 30 min). The specimens were washed with PBS 3 times, blocked with 5% BSA for 1 hr, and immersed in a 2000-fold dilution of monoclonal anti-human fibronectin for 2 hrs. After another 3 washes with PBS, they were immersed in a 32-fold dilution of FITC-conjugated anti-mouse IgG for 1 hr. Following another 3 washes with PBS, the adsorbed fibronectin on each specimen was visualized with the use of a fluorescence microscope. The left panel shows an FITC image of adsorbed fibronectin on a control specimen. The right panel shows an FITC image of adsorbed fibronectin on Ti-Cl. Quantitative analysis of the images (C) was performed on a Windows <model> computer equipped with the public domain program NIH Image (developed at the US National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov from the National Technical Information Service, Springfield, VA; part number PB95-500195GEI). The results are expressed as the mean ± SD of 6 specimens (n = 6). The findings were analyzed statistically by Student’s t tests. Significant differences were considered to exist when p < 0.01. Bars = 10 µm (A,B) and 40 µm (C).