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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;

^* corresponding author, yookun{at}dent.showa-u.ac.jp

	ABSTRACT

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Implant surfaces should be modified to achieve excellent cell compatibility as well as antibacterial activity. Our previous study demonstrated that titanium plates anodized by being discharged in NaCl (Ti-Cl) exhibited high antibacterial activity. Since Ti-Cl was prepared with a NaCl solution, we hypothesized that Ti-Cl would exhibit low toxicity toward cells. The aims of this study were to characterize the surface of Ti-Cl and investigate the cell compatibility (MC3T3-E1 and L929 cells) of Ti-Cl. The results demonstrated that, since the TiCl₃ formed on the Ti-Cl surface was hydrolyzed into HCl, HClO, and TiOH after immersion in pure distilled water, TiCl₃ contributed to the antibacterial activity of Ti-Cl. On the other hand, TiO formed on the Ti-Cl surface enhanced cell extension and cell growth through a larger adsorption of fibronectin compared with the pure titanium control. These findings suggest that antibacterial titanium is a promising material for use in dental implant systems.

KEY WORDS: titanium • anodizing • oral bacteria • antibacterial activity

	INTRODUCTION

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Titanium has been widely utilized in biomaterials fields (Albrektsson et al., 1981; Howlett et al., 1994; Gronowicz and McCarthy, 1996), particularly as applied to dental implants, since it has excellent corrosion-resistance and biocompatibility. However, since titanium implants penetrate not only bone but also gingiva, they are partially exposed in an oral environment that includes oral bacteria. It has been reported that titanium itself has no antibacterial activity compared with other metals (Leonhardt and Dahlén, 1995). Since bacterial accumulation surrounding dental implants has pathogens known to cause peri-implantitis, it is important to prevent colonization of oral bacteria on the surfaces of implants to ensure their long-term clinical success. Indeed, there are a few reports regarding surface modification of titanium implants to prevent colonization of oral bacteria (Yoshinari et al., 2000).

We have already demonstrated that titanium plates anodized by being discharged in various concentrations of NaCl, NaF, and KI solutions acquired antibacterial activity against oral bacteria (Ikeda and Igarashi, 2001). The results showed large decreases in the amounts of Streptococcus mutans, Streptococcus salivarius, Streptococcus sobrinus, Actinomyces viscosus, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea, and Porphyromonas gingivalis on antibacterial titanium plates with increasing contact time with the plates. In particular, titanium plates discharged in 1 M NaCl (Ti-Cl) demonstrated high levels of antibacterial activity. Furthermore, the antibacterial activity of Ti-Cl did not decrease even after 8 weeks’ immersion in pure distilled water. Although Ti-Cl obviously demonstrated excellent antibacterial activity, the details of the mechanism have not yet been elucidated.

On the other hand, implant surfaces should be modified to exhibit excellent cell compatibility as well as antibacterial activity. Commonly, it is believed that antibacterial activity is involved in some form of toxicity toward cells. However, the cell compatibility of Ti-Cl has not yet been evaluated.

In this study, the surface topography of Ti-Cl was observed by scanning electron microscopy (SEM). The surface of Ti-Cl was characterized by thin-film x-ray diffraction (TF-XRD) and x-ray photoelectron spectroscopy (XPS). In addition, the initial adhesion and proliferation of osteoblastic MC3T3-E1 and fibroblastic L929 cells on Ti-Cl were investigated to elucidate the cell compatibility of Ti-Cl. Cell binding protein adsorption onto Ti-Cl was observed by fluorescein isothiocyanate (FITC) labeling.

	MATERIALS & METHODS

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Preparation of Specimens
JIS grade 2 titanium (KS-50, Kobe Steel, Kobe, Japan) was used as the starting material. The surfaces of the titanium plates, 10 x 10 x 1.0 mm, were gradually ground with waterproof polishing papers from #500 to #1200 under running water, and then polished with alumina particles with an average diameter of 0.3 µm. The prepared specimens were cleaned ultrasonically in acetone, detergent solutions (7X, ICN Biomedicals, Aurora, OH, USA) and pure distilled water for 15 min. Specimens were then dried and stored for 24 hrs in a desiccator that maintained a humidity of 50% at 23°C.

Anodizing
Specimens were connected to the anode of a device developed in our laboratory and immersed in 1 M NaCl. A 50 x 100 x 0.1-mm stainless steel plate was used as the counterelectrode. Discharging was generated between the electrolyte and the working electrode through a gas layer on the surface of the electrode. Discharging was maintained for 60 sec. After this process, specimens were washed in pure distilled water and stored in a desiccator for 24 hrs in humidity of 50% at 23°C. Specimens without being anodized were used as controls.

Surface Observation
The Ti-Cl specimens were prepared by undergoing gold vaporizing with a vacuum evaporation device (IB-2, Eiko Engineering, Tokyo, Japan). The surface topographies of the coatings on the specimens were then observed by SEM (S-2360N, Hitachi, Tokyo, Japan).

Surface Characterization
    TF-XRD
The crystal phases of Ti-Cl were detected by TF-XRD (XRD-6100, SHIMADZU, Kyoto, Japan) with CuK radiation. XRD was operated at 40 kV, 40 mA with a scanning speed of 0.02°/4 sec and a scanning range of 20-50°.

    XPS
The surfaces of Ti-Cl specimens were analyzed by XPS (ESCA-3400, SHIMADZU, Kyoto, Japan). High-resolution spectra of Ti2p, O1s, C1s, Na1s, and Cl2p were analyzed with the use of MgK radiation. After analysis, the specimens were immersed in pure distilled water and again subjected to XPS analysis. A 20-mA emission current and 8-kV accelerated voltage were applied in this analysis. The binding energies for each spectrum were calibrated with the use of a C1s spectrum of 285.0 eV.

The results were expressed as the mean ± standard deviation (SD) of 6 specimens (n = 6), and analyzed statistically by Student’s t tests. Significant differences were considered to exist when p < 0.01.

Cell Cultivation
An osteoblastic cell line, MC3T3-E1, and a fibroblastic cell line, L929, were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in minimal essential medium (Gibco, Tokyo, Japan) containing 10% fetal bovine serum (Gibco) and 1% antibiotic (penicillin, Gibco) under a 5% CO₂ atmosphere at 37°C. Cells were suspended in the medium at 1 x 10⁵ cells/mL and used for cell adhesion experiments.

A 1-mL quantity of floating cells was plated onto each of the specimens and incubated at 37°C and 5% CO₂ for 1 wk.

Cell Counting
A cell-counting kit (Dojindo, Kumamoto, Japan) was used for the measurement of cell adhesion. After incubation, each specimen was moved to another well and washed 3 times with phosphate-buffered saline without Ca⁺⁺ and Mg⁺⁺ (PBS; Gibco) to remove non-adherent cells. Adherent cells were mixed with 1 mL of medium and 100 µL of reagent solution. After 1 hr of incubation, the absorbance at 450 nm was measured. The number of adherent cells was calculated from the activity of the original cell suspension. The results were expressed as the mean ± SD of 6 specimens (n = 6), and analyzed statistically by analysis of variance (ANOVA). Significant differences from respective control values were considered to exist when p < 0.01.

Stress Fiber Formation and Cell Morphology
Specimens were placed in 24-well culture plates with 1 mL floating cells each. Subsequently, the specimens were incubated at 37°C in 5% CO₂ for 1 hr. Adherent cells on each specimen after 1 hr of cultivation were dehydrated after being washed with PBS. The cells were fixed with 3.7% formaldehyde in PBS and permeabilized by treatment with 0.1% Triton X-100 (Sigma, Tokyo, Japan) in PBS for 1 min. The cells were then incubated for 3 hrs in a rhodamine-conjugated phalloidin solution. After the cells were washed with water, stress fiber formation and cell morphology were observed with the use of a fluorescence microscope (E-600, Nikon, Tokyo, Japan).

Protein Adsorption
FITC labeling was used for the visualization of adsorbed fibronectin on each specimen. The specimens were soaked in minimal essential medium (Gibco) containing 10% fetal bovine serum (Gibco) at 37°C for 30 min. After incubation, the specimens were washed with PBS 3 times and blocked with 5% BSA (SIGMA) for 1 hr. After being washed again with PBS 3 times, they were immersed in a 2000-fold dilution of monoclonal anti-human fibronectin (TaKaRa, Shiga, Japan) for 2 hrs. After another 3 washes with PBS, they were immersed in a 32-fold dilution of FITC-conjugated anti-mouse IgG (SIGMA) for 1 hr at room temperature. Following another 3 washes with PBS, the adsorbed fibronectin on each specimen was observed with the use of a fluorescence microscope (E-600, Nikon). Quantitative analysis of the images 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 were expressed as the mean ± SD of 6 specimens (n = 6), and analyzed statistically by Student’s t tests. Significant differences were considered to exist when p < 0.01.

	RESULTS

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SEM Observation
Small amounts of granular deposits were sparsely observed on the Ti-Cl surface (Fig. 1).

View larger version (50K): [in this window] [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 this window] [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.

Cell Cultivation
After 1 wk of cell cultivation, the number of adherent cells for each cell line on Ti-Cl was significantly higher than that of control specimens (Fig. 3) (p < 0.01).

View larger version (24K): [in this window] [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 this window] [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).

	DISCUSSION

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The present study characterized the surface and in vitro cell compatibility of Ti-Cl.

In our previous study, we examined the antibacterial activity of titanium plates anodized by being discharged in NaCl, NaF, and KI solutions (Ikeda and Igarashi, 2001). Since titanium plates anodized in NaCl demonstrated much greater antibacterial activity than those of the other specimens, we hypothesized that the chemical state of the chloride formed on the Ti-Cl was HClO. However, in the XPS and XRD studies, TiCl₃ was detected on the Ti-Cl. Since chloride itself has no antibacterial activity, the chemical state of TiCl₃ changed chemically with increasing immersion time. In addition, since TiCl₃ combined with oxygen and Ti-OH increased on Ti-Cl after 1 hr of immersion in the XPS study, the process of the antibacterial effect on Ti-Cl can be explained as follows. The large amounts of chloride ions contained in the electrolyte were adsorbed onto the substrate, while the titanium oxide layer grew with anodizing. TiCl₃ was formed on the substrate. Subsequently, chloride was gradually released into the culture medium and hydrolyzed into HCl, HClO, and TiOH. Since HClO itself was not formed on Ti-Cl, but TiCl₃ was slowly hydrolyzed into HClO with increasing immersion time, the antibacterial effect of Ti-Cl was maintained even after 8 weeks’ immersion (Ikeda and Igarashi, 2001). In addition, HClO was non-toxic, at least at this concentration.

In the cell cultivation test, since cell growth was not inhibited on Ti-Cl compared with the control specimens, Ti-Cl exhibited no toxicity toward the cells. In addition, the numbers of adherent cells on Ti-Cl were significantly greater than those on the control specimens (p < 0.01). Furthermore, in the initial adhesion test, since cells were extended on Ti-Cl compared with those on the control specimens, Ti-Cl demonstrated good compatibility to cells, at least to MC3T3-E1 and L929 cells.

It is well-known that adhesion of osteoblastic cells and fibroblasts to a substrate depends strongly on fibronectin. In addition, many tissue culture cells need the formation of focal adhesions initiated by specific binding of extracellular matrix proteins and receptors to grow and differentiate. In this study, since higher cell extension was observed on Ti-Cl from 1 hr of cultivation, and fibronectin adsorption on Ti-Cl was much greater than that on control specimens, Ti-Cl promoted cell growth and differentiation. The XRD study showed that the peak of TiO increased with processing. Since the TiO suboxide layer has high ionic activity compared with the original TiO₂ layer (Zhu et al., 2001), the forming TiO contributed to the adsorption of cell-binding proteins.

Yoshinari et al.(2001) reported that titanium plates could be modified to be antibacterial by dry process ion plating (Ca⁺, N⁺, F⁺). They also reported that the antibacterial metal has low toxicity to L929 cells. However, this method did not contribute to cell growth. In addition, since the antibacterial metal has been utilized by means of complicated processes, the methods involved are relatively high-cost.

On the contrary, cell growth on Ti-Cl was significantly higher than that on control specimens. In addition, since our anodizing method was processed by means of a simple power supply in a NaCl solution, our anodizing method was relatively low-cost, and appears to be more suitable than other methods reported in earlier studies. From this study, we conclude that Ti-Cl is a promising material for use in dental implant systems.

	ACKNOWLEDGMENTS

 
This study was conducted by the Department of Oral Biomaterials and Technology, Showa University School of Dentistry and Oral Microbiology, Showa University School of Dentistry. The authors gratefully acknowledge the financial support received from a Grant-in-Aid for Scientific Research (B) from The Ministry of Education, Culture, Sports, Science and Technology.

Received December 27, 2002; Last revision August 4, 2003; Accepted November 11, 2003

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