The E ﬀ ects of Various Metallic Surfaces on Cellular and Bacterial Adhesion

: The e ﬀ ects of Ti, Nb, Ta, Zr, and Ag on cellular and bacterial adhesion were investigated in this study. Moreover, the relationships between surface compositions, metal ion release behaviors, and biological responses were examined. As a result, MC3T3-E1 cells and S. aureus were able to better attach to Ti and Zr rather than the Nb and Ta specimens. For the Ag specimen, the amount of Ag ions released into Hanks’ solution was the largest among all the specimens. Cellular and bacterial adhesion onto the Ag specimen was inhibited compared with the other specimens, because of Ag ion release. Alternatively, Nb and Ta specimens exhibited speciﬁc biological responses. Cellular adhesion on Nb and Ta specimens was similar to that on Ti, while bacterial adhesion on Nb and Ta specimens was inhibited compared with that on Ti. This study proved that Nb and Ta inhibited bacterial adhesion and exhibited no harmful e ﬀ ects on cellular adhesion. In addition, these results indicate that the passive layer on Nb and Ta plays a key role in the inhibition of bacterial adhesion.


Introduction
Titanium (Ti) and its alloys are widely used for medical and dental implant devices because of their high corrosion resistance, specific strength, and tissue compatibility. In addition, the interface reaction between Ti and bone tissue promotes bone formation and bone binding ability, a well-known advantage of the use of Ti for implant materials compared with other metals [1]. For artificial joints, bone fixators, spinal fixators, etc., the Young's modulus of the metal is greater than that of the cortical bone, so the load is not transmitted to the bone and stress shielding and finally, bone resorption may occur [2]. Therefore, implant devices must be constructed of materials with low Young's moduli that are close to that of cortical bone. Since the 1990s, β-type Ti alloys with low Young's moduli for the prevention of stress shielding in bone plates and stems of artificial hip joints in orthopedics have been developed [3][4][5][6][7][8][9]. β-type Ti alloys are comprised of Ti with Na 2 HPO 4 , 4.41 × 10 −4 mol L −1 KH 2 PO 4 , 7.39 × 10 −4 mol L −1 Mg 2 SO 4 , and 4.17 × 10 −3 mol L −1 NaHCO 3 . The specimens were immersed in the test solution (5 mL) at 310 K for 24 h. The pH value of the test solution was confirmed to be within the range of 7.40 ± 0.05 and was prepared immediately before use. Hanks' solution without organic species with ion concentrations similar to those of extracellular fluid was employed as the test solution [34]. After 72 h immersion, the concentrations of the metal ions were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES, ICPS-7000 ver. 2, Shimadzu Corp., Kyoto, Japan).

Cellular Adhesion Test
As described in our previous work [35], MC3T3-E1 cells (RIKEN BioResource Center, Tsukuba, Japan) were maintained in cell culture medium: alpha modification of Eagle's minimum essential medium (α-MEM; GIBCO, Grand island, CA, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO), 10 U mL −1 penicillin, 100 mg mL −1 streptomycin, and 0.25 mg mL −1 amphotericin B (GIBCO). All specimens were sterilized in 70% ethanol for 20 min and thoroughly rinsed with deionized water before in vitro testing. The cells were seeded on to the sterilized specimens with an approximate initial density of 10,000 cells cm −2 . The cells were incubated at 37 • C in a fully humidified atmosphere under 5% CO 2 .
After the seeding for 24 h, the cells attached to each specimen were harvested with trypsin/EDTA, re-suspended in a cell culture medium, and transferred to 96-well microplates. Then, the Cell Counting Kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was added to each 96-well microplate, which contained cell suspension, and the reaction was continued for 4 h at 37 • C. The absorbance of the samples was measured at 450 nm using a microplate reader (ChroMate Microplate Reader, Awareness Technology, Palm City, FL, USA). The reference wavelength was set at 630 nm. In this evaluation, the tissue culture polystyrene (TCPS) was used as a control specimen for the quantification of the number of attached cells on each specimen. The cells attached on the TCPS were harvested by treatment with Trypsin/EDTA, followed by the re-suspension of cells in the cell culture medium. Cell suspensions were diluted serially. The number of cells in cell suspension harvested from TCPS were counted by trypan blue (Trypan Blue Stain 0.4%; Gibco) using a hemocytometer. Then, standard curves for cell number calibration and for light absorbance were constructed and used for the quantification of the number of attached cells on each specimen.

Bacteria Adhesion Test
The antibacterial activity was evaluated according to the ISO 22196: 2007 method. To examine the antibacterial activity of the specimens, we employed S. aureus (NBRC122135). The experiment was approved by the Pathogenic Organisms Safety Management Committee in Tokyo Medical and Dental University (22012-025c). S. aureus is Gram-positive cocci frequently found in the nose, respiratory tract, and on the skin. S. aureus was cultured in tryptic soy broth (TryptoSoya Broth, Nissui, Tokyo, Japan) at 37 • C for 24 h. The optical density of the bacterial suspensions was measured at 600 nm using an ultraviolet-visible (UV-vis) spectrometer (V-550, JASCO, Tokyo, Japan) and diluted to obtain concentrations of 5.5-10 × 10 5 colony-forming units (CFUs) mL −1 . Prior to the antibacterial activity testing, all specimens were sterilized with 70% ethanol, washed with distilled water, and dried. Drops of bacterial suspension were added onto all specimens, which were subsequently covered with a sterilized plastic film and incubated at 37 • C for 24 h (n = 3). Bacterial cells were then collected from all the incubated specimens; the obtained suspensions were diluted, pipetted onto nutrient agar plates, and incubated overnight at 37 • C. The number of viable bacteria was determined by counting the number of colonies formed. In addition, (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich, Tokyo, Japan) was used as a scavenger to investigate the effects of reactive oxygen species for bacteria. Drops of bacterial suspension with 0.05 mM Trolox were added onto the Nb and Ta specimens, which were subsequently covered with a sterilized plastic film and incubated at 37 • C for 24 h (n = 3). Bacterial cells were then collected from all the incubated specimens; the obtained suspensions were diluted, pipetted onto nutrient agar plates, and incubated overnight at 37 • C. The number of viable bacteria was determined by counting the number of colonies formed.

Statistical Analysis
Data were calculated from five independent specimens. All values are presented as means ± SD, and commercial statistical software KaleidaGraph (Synergy Software, Reading, PA, USA) was used for statistical analysis. One-way analysis of variance was used following multiple comparisons with the Student-Newman-Keuls method to assess the data, and p < 0.05 was considered to indicate statistical significance. Figure 1 shows the XPS survey scan spectra obtained from each specimen. The peaks originating from C 1s and O 1s orbital binding energies were detected from the spectra obtained from all specimens. Moreover, each peak originated from the Ti 2p, Nb 3d, Ta 4f, Zr 3d, and Ag 3d binding energies were detected from each metallic specimen, respectively. The XPS spectra of Ti 2p, Nb 3d, Ta 4f, Zr 3d, Ag 3d, and the O 1s electron binding energy regions obtained from the specimens are shown in Figure 2. The deconvolution of peaks was conducted according to the published data with compatible full-width of half-maximum values [36][37][38][39][40]. As a result, the spectra of Ti, Nb, Ta, and Zr were identified as metallic (Ti 0 , Nb 0 , Ta 0 , and Zr 0 ) and oxidized states (Ti 2+ , Ti 3+ , Ti 4+ , Nb 2+ , Nb 4+ , Nb 5+ , Ta 5+ , and Zr 4+ ). Moreover, the spectra of O 1s obtained from Ti, Nb, Ta, and Zr specimens were composed of three peaks, originating from O 2− , OH − , and H 2 O. Alternately, the spectrum of Ag was identified as only the metallic state (Ag 0 ). The spectrum of O 1s obtained from the Ag specimen showed a low-intensity peak and was composed of OH − and H 2 O. Data were calculated from five independent specimens. All values are presented as means  SD, and commercial statistical software KaleidaGraph (Synergy Software, Reading, PA, USA) was used for statistical analysis. One-way analysis of variance was used following multiple comparisons with the Student-Newman-Keuls method to assess the data, and P < 0.05 was considered to indicate statistical significance. Figure 1 shows the XPS survey scan spectra obtained from each specimen. The peaks originating from C 1s and O 1s orbital binding energies were detected from the spectra obtained from all specimens. Moreover, each peak originated from the Ti 2p, Nb 3d, Ta 4f, Zr 3d, and Ag 3d binding energies were detected from each metallic specimen, respectively. The XPS spectra of Ti 2p, Nb 3d, Ta 4f, Zr 3d, Ag 3d, and the O 1s electron binding energy regions obtained from the specimens are shown in Figure 2. The deconvolution of peaks was conducted according to the published data with compatible full-width of half-maximum values [36 -40]. As a result, the spectra of Ti, Nb, Ta, and Zr were identified as metallic (Ti 0 , Nb 0 , Ta 0 , and Zr 0 ) and oxidized states (Ti 2+ , Ti 3+ , Ti 4+ , Nb 2+ , Nb 4+ , Nb 5+ , Ta 5+ , and Zr 4+ ). Moreover, the spectra of O 1s obtained from Ti, Nb, Ta, and Zr specimens were composed of three peaks, originating from O 2− , OH − , and H2O. Alternately, the spectrum of Ag was identified as only the metallic state (Ag 0 ). The spectrum of O 1s obtained from the Ag specimen showed a low-intensity peak and was composed of OHand H2O.

XPS Measurement
The composition and thickness of the passive layer on each metal specimen calculated from the XPS measurement with a detection angle of 90 are summarized in Table 1. The compositions of the passive layer on Ti, Nb, Ta, and Zr specimens consisted almost entirely of 70 at.% oxygen and 30 at.% metal. Moreover, the thickness of the passive layer was 5.5 to 6.2 nm. However, the surface composition of Ag was different from those of the other specimens. The thickness of the surface oxide layer could not be calculated because of the slight peak of O 1s and the Ag 3d peak composed only of Ag 0 .     The composition and thickness of the passive layer on each metal specimen calculated from the XPS measurement with a detection angle of 90 • are summarized in Table 1. The compositions of the passive layer on Ti, Nb, Ta, and Zr specimens consisted almost entirely of 70 at.% oxygen and 30 at.% metal. Moreover, the thickness of the passive layer was 5.5 to 6.2 nm. However, the surface composition of Ag was different from those of the other specimens. The thickness of the surface oxide layer could not be calculated because of the slight peak of O 1s and the Ag 3d peak composed only of Ag 0 .

Cellular Adhesion Test
The results of cellular adhesion testing using MC3T3-E1 cells after seeding for 24 h are shown in Figure 4. There were no significant differences between the numbers of viable cells attached to Ti, Nb, Ta, and Zr specimens. However, the number of viable cells attached to Ag specimens was significantly lower than those on the other specimens.

Cellular Adhesion Test
The results of cellular adhesion testing using MC3T3-E1 cells after seeding for 24 h are shown in Figure 4. There were no significant differences between the numbers of viable cells attached to Ti, Nb, Ta, and Zr specimens. However, the number of viable cells attached to Ag specimens was significantly lower than those on the other specimens.

Cellular Adhesion Test
The results of cellular adhesion testing using MC3T3-E1 cells after seeding for 24 h are shown in Figure 4. There were no significant differences between the numbers of viable cells attached to Ti, Nb, Ta, and Zr specimens. However, the number of viable cells attached to Ag specimens was significantly lower than those on the other specimens.   Figure 5 shows the normalized CFU count for S. aureus, demonstrating the antibacterial effects of the specimens. The vertical axis represents the normalized bacterial number, defined as the bacterial number on each of the specimens divided by the initial number of S. aureus. A normalized bacterial number that was below 1 (shown as a dashed line in the figure) implied that the tested specimen had antibacterial activity. There were no significant differences between the S. aureus counts on the Ti and Zr specimens. However, antibacterial effects of the Nb, Ta, and Ag specimens were observed; the S. aureus counts on these specimens were significantly lower than those on the Ti specimen. In particular, the Ag specimen was most effective against S. aureus. The Nb and Ta specimens exhibited a sufficient effect compared to the Ti specimen and could inhibit the adhesion of S. aureus.  Figure 5 shows the normalized CFU count for S. aureus, demonstrating the antibacterial effects of the specimens. The vertical axis represents the normalized bacterial number, defined as the bacterial number on each of the specimens divided by the initial number of S. aureus. A normalized bacterial number that was below 1 (shown as a dashed line in the figure) implied that the tested specimen had antibacterial activity. There were no significant differences between the S. aureus counts on the Ti and Zr specimens. However, antibacterial effects of the Nb, Ta, and Ag specimens were observed; the S. aureus counts on these specimens were significantly lower than those on the Ti specimen. In particular, the Ag specimen was most effective against S. aureus. The Nb and Ta specimens exhibited a sufficient effect compared to the Ti specimen and could inhibit the adhesion of S. aureus.    Figure 6 shows the normalized CFU count for S. aureus, demonstrating the antibacterial effects of the specimens in the suspension with and without Trolox. The vertical axis represents the normalized bacterial number, defined as the bacterial number on each of the specimens divided by the number of S. aureus in the suspension without Trolox on each specimen. A normalized bacterial number that was above 1 (shown as dashed line in the figure) implied that the antibacterial activity of the tested specimen was reduced by the Trolox. The S. aureus counts in the suspension with Trolox on Nb and Ta were significantly higher than those without Trolox. Therefore, the antibacterial effects of Nb and Ta were reduced by Trolox.

Discussion
The passive layers composed of approximately 70 at.% oxygen and 30 at.% metallic elements were confirmed for the Ti, Nb, Ta, and Zr specimens. The passive layers on these specimens were of almost the same thickness (Figures 1 and 2, Table 1). Generally, valve metals such as Ti, Nb, Ta, and Zr have good corrosion resistance. However, a small amount of Ti, and Nb ions were released from the specimen in this test condition. Although a relatively large amount of Zr ions was eluted, Ta was not eluted (Figure 3). These metal specimens may release a small amount of their ions in some situations in vivo. Nevertheless, MC3T3-E1 cells were able to attach to Ti, Nb, Ta, and Zr specimens (Figure 4). This indicates that their surface compositions and metal ion release behaviors did not affect the adhesion of MC3T3-E1 cells. This demonstrates that these metals are suitable Ti alloying elements for in vivo use. Yamamoto et al. reported that the IC50s of Ti, Nb, Ta, and Zr ions for MC3T3-E1 cells was 8.7 × 10 −4 mol L −1 (Ti ions), 1.5 × 10 −3 mol L −1 (Nb ions), 2.1 × 10 −3 mol L −1 (Ta ions), and 2.8 × 10 −3 mol L −1 (Zr ions) [41]. Therefore, it is possible that MC3T3-E1 cells were able to attach to each metal because the amount of each metal ion released into Hanks' solution during the 72 h period did not reach the IC50s for MC3T3-E1 cells. In other words, Ti, Nb, Ta, and Zr had no harmful effects on the cellular viability of the osteogenic cells and may exhibit good osteogenic cell compatibility, at least during the initial phase of implantation. Moreover, focusing on the biological properties of Nb and Ta, Matsuno et al. implanted Nb and Ta wires into the subcutaneous tissue of the abdominal region and into the femoral bone marrow of rats for either 2 or 4 weeks. They concluded that Nb and Ta had good biocompatibility and osteoconductivity, based on confirmation of new bone formation [42]. These results provide adequate evidence that Nb and Ta had good osteogenic cell compatibility.
Metals that are not valve metals usually do not passivate, and similar results were obtained from the Ag specimen in this study (Figures 1 and 2, Table 1). The amount of Ag ions released from the specimen into the Hanks' solution was 1.9 × 10 −6 mol L −1 (Figure 3

Discussion
The passive layers composed of approximately 70 at.% oxygen and 30 at.% metallic elements were confirmed for the Ti, Nb, Ta, and Zr specimens. The passive layers on these specimens were of almost the same thickness (Figures 1 and 2, Table 1). Generally, valve metals such as Ti, Nb, Ta, and Zr have good corrosion resistance. However, a small amount of Ti, and Nb ions were released from the specimen in this test condition. Although a relatively large amount of Zr ions was eluted, Ta was not eluted (Figure 3). These metal specimens may release a small amount of their ions in some situations in vivo. Nevertheless, MC3T3-E1 cells were able to attach to Ti, Nb, Ta, and Zr specimens ( Figure 4). This indicates that their surface compositions and metal ion release behaviors did not affect the adhesion of MC3T3-E1 cells. This demonstrates that these metals are suitable Ti alloying elements for in vivo use. Yamamoto et al. reported that the IC 50 s of Ti, Nb, Ta, and Zr ions for MC3T3-E1 cells was 8.7 × 10 −4 mol L −1 (Ti ions), 1.5 × 10 −3 mol L −1 (Nb ions), 2.1 × 10 −3 mol L −1 (Ta ions), and 2.8 × 10 −3 mol L −1 (Zr ions) [41]. Therefore, it is possible that MC3T3-E1 cells were able to attach to each metal because the amount of each metal ion released into Hanks' solution during the 72 h period did not reach the IC 50 s for MC3T3-E1 cells. In other words, Ti, Nb, Ta, and Zr had no harmful effects on the cellular viability of the osteogenic cells and may exhibit good osteogenic cell compatibility, at least during the initial phase of implantation. Moreover, focusing on the biological properties of Nb and Ta, Matsuno et al. implanted Nb and Ta wires into the subcutaneous tissue of the abdominal region and into the femoral bone marrow of rats for either 2 or 4 weeks. They concluded that Nb and Ta had good biocompatibility and osteoconductivity, based on confirmation of new bone formation [42]. These results provide adequate evidence that Nb and Ta had good osteogenic cell compatibility.
Metals that are not valve metals usually do not passivate, and similar results were obtained from the Ag specimen in this study (Figures 1 and 2, Table 1). The amount of Ag ions released from the specimen into the Hanks' solution was 1.9 × 10 −6 mol L −1 (Figure 3). Yamamoto et al. reported that the IC 50 of Ag ions for MC3T3-E1 cells was 2.8 × 10 −6 mol L −1 [41]. Therefore, the amount of Ag ions released into the test solution during 72 h almost reached the IC 50 for MC3T3-E1 cells. Moreover, the adhesion of MC3T3-E1 cells was inhibited on the Ag specimen compared with the other specimens ( Figure 4). These phenomena indicate that the surface composition of the Ag specimen and the Ag ion release behavior have a high potential to be cytotoxic and may affect bone tissue.
Bacterial adhesion of S. aureus was inhibited on the Nb, Ta, and Ag specimens compared with that on the Ti specimen ( Figure 5). In the case of the Ag specimen, the amount of Ag ions released from the specimen was the largest among all specimens. The IC 50 of Ag ions for MC3T3-E1 cells was the lowest compared with those of Ti, Nb, Ta, and Zr ions [41]. Therefore, it is possible that the Ag antibacterial effect against S. aureus was due to Ag ion release. However, the amount of Nb and Ta ions released from each specimen was lower than that of the Ag ion. In general, it is known that the effectiveness of Ag ions against cells is higher than that of Nb and Ta ions [41]. Therefore, Nb and Ta ions released from the specimens did not affect the bacterial adhesion of S. aureus. The results of surface characterization by XPS showed that passive layers were confirmed on the Nb and Ta specimens. Each film was composed of an oxide state (Nb 2+ , Nb 4+ , Nb 5+ , and Ta 5+ ) and a metallic state (Nb 0 and Ta 0 ) (Figures 1 and 2, Table 1). Based on these results, it is possible that each metal oxide, as a passive layer, plays a key role in the development of antibacterial effects against bacterial adhesion. Ziolek et al. reported that reactive oxygen specimens (ROS) intermediates were generated on the surface of amorphous Nb 2 O 5 and Ta 2 O 5 upon interaction with aqueous H 2 O 2 [43]. This study revealed that the antibacterial effects of Nb and Ta were reduced by Trolox ( Figure 6). Since Trolox usually acts as the scavenger of ROS, it is considered that the antibacterial effects of Nb and Ta were reduced by the antioxidant action of Trolox. Therefore, we believe that the antibacterial effects of Nb and Ta specimens were due to the generation of ROS on their surface. Regarding the valences of Nb and Ta in the passive layer, Nb and Ta exist in a maximum pentavalent oxidation state. For this reason, Nb and Ta oxides tend to exhibit the characteristics of an n-type semiconductor. Hence, the antibacterial effects of Nb and Ta are the result of their passive layers. However, a more detailed examination related to the generation of ROS on the Nb and Ta surface is necessary in the future. The balances between cellular viability and the bacterial adhesion on Nb and Ta were superior compared with those on the other specimens. Therefore, Nb and Ta are useful not only as β-stabilizers but also as antibacterial elements without any cytotoxicity. On the other hand, Ag exhibited antibacterial activity with cytotoxicity. Therefore, controlling the concentration of Ag is necessary for its use on the implant surface. Some researchers have reported that Ti-Ag alloys show good corrosion resistance and improved mechanical properties compared to Ti [44,45]. In addition, Kang et al. reported that surface-treated Ti-Ag alloys exhibited antibacterial activity without cytotoxicity [46]. Therefore, suitable amounts of Ag are useful not only as antibacterial elements but also as additive elements for implant materials.
We hope that in the future, the outcome of the present study will be useful in the design of biocompatible implant materials.

Conclusions
The biological responses, surface compositions, and metal ion release behaviors of Ti, Nb, Ta, Zr, and Ag specimens were investigated in this study. MC3T3-E1 cells and S. aureus were able to better adhere to the Ti and Zr specimens than the Nb and Ta specimens. The Ag specimen inhibited both cellular viability and bacterial adhesion, owing to Ag ion release. Alternatively, Nb and Ta specimens inhibited only bacterial adhesion and exhibited no harmful effects on cellular viability. Therefore, Nb and Ta are useful as non-cytotoxic antibacterial elements.