In vitro antibacterial activity and cytocompatibility of bismuth doped micro-arc oxidized titanium

Abstract

Chemical manipulations of the implant surface produce a bactericidal feature to prevent infections around dental implants. Despite the successful use of bismuth against mucosal and dermis infections, the antibacterial effect of bismuth in the oral cavity remains under investigation. The aim of this study was to evaluate the antibacterial activities of bismuth compounds against Actinobacillus actinomycetemcomitans, Staphylococcus mutans, and methicillin-resistant Staphylococcus aureus (MRSA), and to investigate the antimicrobial effects of bismuth doped micro-arc oxidation (MAO) titanium via an agar diffusion test. Cell viability, alkaline phosphatase activity, and mineralization level of MG63 osteoblast-like cells seeded on the coatings were evaluated at 1, 7, and 14 days. The results demonstrate that bismuth nitrate possess superior antibacterial activity when compared with bismuth acetate, bismuth subgallate, and silver nitrate. The bismuth doped MAO coating (contained 6.2 atomic percentage bismuth) had good biological affinities to the MG63 cells and showed a higher antibacterial efficacy against Actinobacillus actinomycetemcomitans and MRSA, where the reduction rates of colony numbers is higher than that of the control group by 1.5 and 1.9 times, respectively. These in vitro evaluations demonstrate that titanium implants with bismuth on the surface may be useful for better infection control.

Keywords

Bismuth antibacterial activity micro-arc oxidation titanium implant

Introduction

Modifications to biomaterial surfaces allow for the programming of cell-to-substratum events; this diminishes infection by enhancing tissue compatibility or integration or by directly inhibiting bacterial adhesion.¹ Nevertheless, It has been reported that implant patients with a history of periodontitis are more susceptible to peri-implant infections, even if stable osseointegration appears to have taken place.^2,3 Moreover, microbial cells can accumulate/breed in the complex geometrical sites, such as interfaces of implant/abutment⁴ or ball-and-socket joints,⁵ and penetrate into deep pockets when proper oral hygiene is not maintained. Thus chemical manipulation of the implant surface to obtain a self-sterile or bactericidal feature has become an interesting strategy to prevent infections around implants.

Titanium surface chemical modifications including various techniques, it is generally divided into two categories: dry methods, such as sputtering, ion implantation, thermal decomposition, and ion beam mixing^5–10 and wet methods, such as anodize, sol–gel, electrophoresis.^11–18 Yoshinari et al.⁷ used dry processes, to fabricate various surfaces on polished titanium. Their results indicate that pure titanium with implanted F- ions possesses better antibacterial activity against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) when compared to polished pure titanium. By magnetron co-sputtered hydroxyapatite (HA) and Ag coating, Chen et al.⁸ indicated that there is a significantly reduced number of Staphylococcus epidermidis and Staphylococcus aureus (S. aureus) on the Ag–HA surface compared to titanium and HA surfaces. In addition, no significant difference in in vitro cytotoxicity was observed between HA and Ag–HA surfaces. However, some of these processes were cost- and technique-dependent, and their results were obtained from treatment on a relative smooth surface, some of these techniques may not suitable to be applied to a rougher surface for more stable integration consideration.¹⁹ By wet method, it is relatively easier to produce a layer on a rougher surface although a weaker interface between the coatings and substrate may associate with this wet process. Deng et al.¹⁸ using a series of electrolytes containing chlorine ions to anodize titanium. Their results demonstrate that the peroxidation effects of HClO generated from the TiCl₃ formed on the titanium surface anodized in various chloride solutions efficiently killed adherent S. mutans on the surface. Alternatively, micro-arc oxidation (MAO) is an effective method for producing the porous anatase TiO₂ layer, which controls oxide thickness. By the MAO process, elements in the electrolyte can be doped into the oxide layer in different levels depending on the time, voltages, temperature, pH, and electrolyte composition/concentration.^11,13,14 Recent study showed that titanium oxidized in the AgNO₃ containing electrolyte solutions obtained a significant antibacterial efficacy against S. aureus and Escherichia coli.¹⁵

Bismuth salts have been widely used for treating gastrointestinal disorders, duodenal ulcers, general skin wound convergence and even lymph node cancer.^20,21 Despite the successful use of bismuth compounds against mucosal and dermis infections, the antibacterial effect of bismuth in the oral cavity remains under investigation. Bismuth is less toxic than its neighbor elements in the periodic table. In our previous study, we found that the in vitro culture of 3T3 fibroblast cells, which were conditioned by extraction of pure bismuth powder, did not induce cytotoxicity.²² In addition, bismuth oxide has also been used as a radiopacifier additive in some root canal filling materials (endodontic sealers) over a decade.²³ Recently, a bismuth compound has been considered to be an antibacterial additive in calcium phosphate cement. The agar diffusion test results showed antimicrobial activities of bismuth-doped cements against S. aureus.²⁴

The aim of this study was to understand the antibacterial activities of bismuth compounds and to evaluate the antibacterial efficacy of bismuth doped MAO titanium. The effects on the cell proliferation and differentiation abilities are also evaluated by measuring the cell viability, alkaline phosphatase activity, and mineralization level of human osteosarcoma MG-63 cells cultured on a bismuth-doped, anodic titania layer for 1, 7, and 14 days. Hopefully we can produce a bismuth-doped titania layer on pure titanium implants, the implants would be biocompatible and antibacterial and thus able to prevent biofilm production.

Materials and methods

Anodic oxidation sample preparation

Commercial pure titanium disks (ASTM grade 2, 12 mm in diameter and 1 mm in thickness) were mechanically polished to 0.5 µm under standard metallurgical methods. In order to obtain an uniform smooth surface, the titanium disks were chemically polished in a mix of nitric acid and hydrofluoric acid (HNO₃:HF = 3:1) for 15 s.

The electrolytes composed of 0.2 M calcium acetate (Katayama Chemical Co., Osaka, Japan) and 0.04 M β-glycerol phosphate disodium (CALBIOCHEM, Giessen, Germany) and superaddition of 0.001 M of either bismuth acetate, bismuth nitrate or silver nitrate were prepared for experiment groups (BA, BN or AN groups), respectively. The same electrolyte without superaddition was set as control group (AO group). Sandpaper (#600) grinded titanum discs (MT group) were prepared for the comparison of the in vitro cell proliferation, differentiation and mineralization tests.

The anodic oxidation was performed using a double-layer reaction cell; the outer layer was connected to a water cyclic cooling/heating system to control the constant temperature of 25°C. The polished titanum disk was then fixed in a custom Teflon sample holder to control a constant exposed area. A platinum cathode was placed in the reacting cell 1 cm away from the anode disk. The MAO process was performed at a galvanostatic mode under a constant current density of 10 mA/cm² to achieve a final voltage of 280 V by a DC power supply; this continued for 3 min at a pH value around 6 with moderate electrolyte agitation. Samples were ultrasonically cleaned and were dried in a 55°C oven overnight.

Antibacterial activities of bismuth compounds

Zone of inhibition. The antimicrobial activity of bismuth compounds, including bismuth subgallate (98%, Alfa Aesar, Ward Hill, MA, USA), bismuth acetate (98%, Alfa Aesar), bismuth nitrate (98%, Panreac, Barcelona, Spain), and silver nitrate (99%, Merck, Darmstadt, Germany), was assessed by determining the zone of inhibition on an isolated 6 mm filter paper (containing 0.25 mg each). Gram-positive Staphylococcus mutans (S. mutans, ATCC 25175), MRSA (methicillin-resistant Staphylococcus aureus, ATCC 33591) and Gram-negative A. actinomycetemcomitans (ATCC 33384) were selected for the antibacterial activities test. The bacteria were pre-cultured and then dilution to a final concentration of 10⁵ cells/mL monitored by a spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Arlington Heights, IL, USA). One hundred microliter aliquots of bacterial suspension were homogeneously spread onto the surface of the agar plates (1.5% agar, Difco) followed by each conditioned filter paper being put on the center of a 6-cm plate. After incubating the agar plates at 37°C for 24 h, the zones of inhibition were determined by measuring the mean diameter of the clear zone. Each assay was carried out in triplicate.

Bacteriostatic effects. Test solutions containing bismuth acetate, bismuth nitrate, and silver nitrate (0.1% w/v) were stirred at room temperature away from light for 1 h, and 3 aliquots (10 mL) of each were transfered to 15 mL tubes. A. actinomycetemcomitans (10⁷ cells/mL) was added to each test medium and then co-incubated for 0.2, 24, 48, 72 h under aerobic conditions (37°C). Bacterial suspension without selected chemicals was set as the control group. The optical density (OD₆₀₀) value of the supernatant of the suspension was measured and was normalized to the control group. The bacteriostatic effects of each compound was evaluated and compared.

Surface character

The surface chemical composition of the oxide layer was studied by an X-ray photoelectron spectroscopy (PHI 5000 VersaProbe/Scanning ESCA Microprobe, ULVAC-PHI, Chigasaki, Japan) using a microfocused (100 µm, 25 W) Al X-ray beam with a photoelectron takeoff angle of 45°. The spectra were calibrated on C 1 s binding energy of 284.5 eV, the quantified chemical composition of the samples was evaluated by computing the weighted area of each element (peaks of C 1 s, O 1 s, Ti 2 p, P 2 p, Ca 2 p, Ag 3 d and Bi 4f) through the corresponding sensitivity factor.

The surface roughness was measured by a profilometer (Surfcorder SE 1200, Kosaka Laboratory Co, Chiyoba-Ku, Tokyo, Japan). Five disks were measured from each group to obtain an average roughness value Ra. Six individual measurements were made on each specimen. Wettability (hydrophilicity) of the MAO surface was determined using a contact angle analyzer (FTA-125, First Ten Angstroms, Portsmouth, VA, USA). After a 10 µL distilled water drop on the MAO titanium disc at room temperature, a continue record CCD was triggered and pictured. The contact angle of each water drop on the picture was measured automatically by a non-spherical fitting approach. Each reported contact angle is the mean of at least three independent measurements.

In vitro cytocompatibility

MG63 human osteosarcoma cells were purchased from Bioresource Collection and Research Center (BCRC, Taiwan). The cells were grown in a minimum essential medium (α-MEM, Gibco, USA) containing nonessential amino acids and supplemented with 10% fetal bovine serum (FBS), 100 µg/mL of streptomycin, 100 U/mL of penicillin. Cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. The culture medium was renewed twice per week. The cells were seeded at a density of 5 × 10⁴ cells per well onto the autoclaved titanium discs and incubated in a 24-well plate for 1, 7, or 14 days for either MTT proliferation assay, alkaline phosphatase (ALP) quantization or detection of mineralization. Cell differentiation and mineralization was induced by the previous culture medium with osteogenic supplements, 1 mM of β-glycerophosphate (Sigma, St. Louis, MO, USA), and 50 µg/mM of L-ascorbate (Sigma, USA). Prior to each test, the titanium discs with attached cells were changed to a new 24-well plate to eliminate the effects from cells outside the disk.

Cell proliferation. Cell proliferation was evaluated using a tetrazolium dye (MTT) assay. The culture medium with unattached cells was carefully removed from the wells by a pipette. After incubation for 4.5 h at 37°C, the medium was replaced with thiazolyl blue to dissolve formazan. The optical density (OD) value of the dissolved solute was then measured by an ELISA (enzyme-linked immunosorbent assay) reader (Synergy HT, BioTek, Winooski, VT, USA) at a 570 nm wavelength. Measurements were performed in triplicate. Cell viability was calculated by the mean OD of three individual wells in the same group and expressed as percentage of control (MT group).

Cell differentiation. Total protein level and alkaline phosphatase (ALP) activity were detected after 1, 7, and 14 days using the cell contact test. The attached MG63 cells in the 24-well plates reacted with 400 µL of cell lysis solution (500 µL TRIS-HCl, 1 M stock solution, pH = 7.4, 49.45 mL of distilled water, 50 µL TX-100 and MgCl₂ powder 2.38 mg) at −80°C for over 20 h. Twenty-five microliter aliquots were transferred to a 96-well plate used to estimate alkaline phosphatase activity and total protein level. Total protein synthesis in the cell lysates was determined using a commercially available kit (BCA protein assay kit, Pierce Chemical Co., Rockford, Illinois, USA). Two hundred microliter of working reagent was added to each well for 30 min to incubate at 37°C. The absorbance was read using an ELISA reader at 562 nm and was correlated to a standard protein curve. The amount of enzyme ALP of the lysed cells in the 96-well plate was quantified using a colorimetric assay with 200 µL of p-nitrophenylphosphate (Sigma) as substrate. After incubation at room temperature for 30 min, 25 µL of 3 M NaOH was added into the wells to stop color changing; the absorbance was determined at 405 nm. The values of ALP activity were calculated as the ratios of OD₄₀₅ values by ALP assay/OD₅₆₂ values by BCA assay, and the values were expressed as percentages of control (MT group). Triplicate samples from each group were analyzed.

Detection of mineralization. Alizarin Red S cytochemistry was used to detect the mineralization of MG63 cells.¹⁶ Cells in 24-well plates were washed twice with PBS and fixed in 10% (v/v) formaldehyde at room temperature for 15 min. The cells were then washed three times with distilled water. Four hundred microliter of working solution containing 1% Alizarin Red S (Sigma, USA), pH = 4.2, was added to react at room temperature for 5 min. After being washed twice, the cells were incubated in 400 µL of CPC buffer (10% cetylpyridinium chloride in 10 mM Na₂PO₄, pH = 7.0) (Alfa Aesar) for 1 h on the shaker in order to extract Alizarin Red S. The dye was then removed, and 100 µL aliquots were transferred to a 96-well plate prior to the reading at 550 nm. Mineralization levels were expressed as percentages (%), which were normalized to the OD₅₅₀ values of Alizarin Red S assay to the MT group.

Antibacterial efficacy of MAO titanium

MAO specimens were immersed in constant volume of agar suspensions with A. actinomycetemcomitans and MRSA (10⁴ cells/mL) on 6-mm dishes and grown aerobically at 37°C for 48 h. The bacteria colonies on the titanium discs and the colonies in the surrounding areas were counted within the same 10-mm diameter circular area by measuring the colony numbers on 10×-magnified pictures on a computer screen. Antibacterial efficacy of the AN, BA, BN groups were estimated by calculating the reduction rate of A. actinomycetemcomitans bacteria colonies on the MAO samples; they were then compared to the control AO group.

Statistic evaluation

We analyzed data by overall one-way ANOVA followed by Tukey's test for individual between-group comparisons. The significant differences were set to p < 0.05 if not specified.

Results

Antibacterial activities of bismuth compounds

As shown in the Figure 1, bismuth acetate, bismuth nitrate, and silver nitrate possess antibacterial activity against A. actinomycetemcomitans, S. Mutans, and MRSA; however, bismuth subgallate has no inhibition on the selected bacteria. The zones of inhibition are compared in Table 1. The zones of inhibition of bismuth acetate and bismuth nitrate against A. actinomycetemcomitans are larger than the zone of inhibition for silver nitrate. Bismuth nitrate has the highest antibacterial activity against S. mutans and MRSA among other compounds; the bismuth nitrate has a zone of inhibition of 46 cm, which was more than two times that of the other compounds.

Figure 1.

Zones of growth inhibition showing antibacterial activity for 0.25 mg of bismuth subgallate (1^st column), bismuth acetate (2^nd column), bismuth nitrate (3^rd column), and silver nitrate (4^th column) against A. actinomycetemcomitans (1^st row), S. mutans (2^nd row), and MRSA (3^rd row).

Table 1.

Zone of inhibition of four selected chemicals (0.25 mg) against A. actinomycetemcomitans, S. mutans, and MRSA on the 6-cm agar plates after 24h (mean ± SD, n = 3).

Bacterium	Zone of inhibition (mm, including 6 mm diameter filter paper) for
Bacterium	Bismuth subgallate	Bismuth acetate	Bismuth nitrate	Silver nitrate
A. actinomycetemcomitans	NI	26 ± 3	24 ± 5	14 ± 4
S. mutans	NI	10 ± 2	24 ± 3	18 ± 2
MRSA	NI	17 ± 2	46 ± 6	18 ± 5

NI: no inhibition.

Figure 2 shows that bismuth acetate and bismuth nitrate inhibit the growth of A. actinomycetemcomitans at 0.1% w/v. The bacteriostatic effects of 0.1% w/v bismuth acetate and bismuth nitrate were superior to the effect of silver nitrate at 24, 48, and 72 h (marked by an asterisk). The normalized OD₆₀₀ values of bismuth acetate and bismuth nitrate significantly decreased with increasing incubation time (p < 0.05). However, the 0.1% silver nitrate does not show bacteriostatic effects against A. actinomycetemcomitans for up to 72 h of incubation time.

Figure 2.

Bacteriostatic effects of 0.1% w/v bismuth acetate, bismuth nitrate, and silver nitrate tested with A. actinomycetemcomitans. The bacteriostatic effects were determined by normalizing the OD₆₀₀ values of each group to the control group (without chemicals). ★: means p < 0.05 between the two data; a: means significant difference of the data at 0.2 h; b: means significant difference of the data at 24 h; c: means significant difference of the data at 48 h; and d: means significant difference of the data at 72 h.

Surface characterization

The X-ray photoelectron spectrum of BA, BN, AN, and AO discs were compared in Figure 3. All spectrums present highest peak at 530 eV which belong to O 1 s, demonstrate that the MAO produce an oxide layer on the titanium surface. The Ti, Ca and P peaks that originally exist in the control group spectrum (AO) are also observed in BA, BN, and AN spectrums. The Bi 4f (∼160 eV) and Bi 4 d (∼440 eV) peaks demonstrate that the bismuth is successfully doping to the MAO titania layer of BA and BN. The possible composition of the doped bismuth are reducing bismuth (Bi⁰: 157 eV, 162 eV), Bi₂O₃ (Bi³⁺:158.8 eV, 163.7 eV), and Bi₂Ti₂O₇ (Bi³⁺ : 159.5 eV, 164.9 eV)²⁵ by deconvolution of the split Bi 4f peak (data not shown). In the AN spectrum, two small peaks around 370 eV belong to the Ag⁰ 3 d 5/2 (368.2 eV) and Ag⁰ 3 d 3/2 (374.2 eV) bands²⁶ demonstrate the existence of silver on the AN surface. The quantitative determination of the surface compositions of the MAO samples are shown in Table 2. The BA and BN contained 2.3% and 6.2% of bismuth on the surface, respectively. The silver is, however, only 0.8% on the surface obtained by this present MAO condition. The roughness of these MAO titanium oxide layers are statistically the same, and the Ra values are all around 0.5 µm (Table 3), however the MT group presents a higher Ra value of 1.25 µm. The MT group presents highest contact angle of 75° when compared to the BA, BN, AN, and AO groups. The differences of contact angles between MAO titanium discs are not significant.

Figure 3.

The surface chemical composition (by XPS) of MAO titanium produced by electrolyte doped with 0.001 M bismuth acetate (BA), bismuth nitrate (BN), silver nitrate (AN), and of the control group (AO).

Table 2.

Atomic concentrations (in %) of MAO titanium discs doped with 0.001 mol/L bismuth acetate (BA), bismuth nitrate (BN), silver nitrate (AN), and control group (AO) analyzed by X-ray photoelectron spectroscopy.

Sample groups	Atomic concentrations of element by XPS (in %)
Sample groups	C	O	P	Ca	Ti	Bi	Ag	Ca/P
BA	22.0	53.0	6.2	5.7	10.9	2.3	N/A	0.9
BN	6.8	57.6	8.1	5.9	15.5	6.2	N/A	0.7
AN	24.0	53.1	6.2	5.9	10.0	N/A	0.8	1.0
AO	25.5	53.2	5.7	4.6	11.0	N/A	N/A	0.8

Table 3.

Roughness and contact angle of MAO titanium discs doped with 0.001 mol/L bismuth acetate (BA), bismuth nitrate (BN), silver nitrate (AN), and control group (AO) (mean ± SD).

Surface characters	MAO titanium discs				Machined
Surface characters	BA	BN	AN	AO	MT
Roughness, Ra (µm), n = 5	0.56 ± 0.07	0.52 ± 0.02	0.53 ± 0.07	0.53 ± 0.04	1.25 ± 0.08
Contact angle, n = 3	52° ± 1	45° ± 1	50° ± 1	54° ± 2	75° ± 2

In vitro cytocompatibility

The proliferation of MG63 cells that were cultured on BA, BN, AN, and AO groups were expressed as cell viability percentages (mean ± SD%) to the MT group (i.e., the OD₅₇₀ values were normalized to the OD₅₇₀ values of cells cultured on the machined titanium disc) as shown in Figure 4. ANOVA data demonstrates that the cells cultured on BA, BN, AN, and AO discs reveal analogous viabilities when the cells are cultured for the same time periods of up to 14 days. The cell viability percentages of AN and AO groups at the first day were significantly higher than that of the 14^th day.

Figure 4.

Proliferation of MG63 cells (by MTT assay) on the 1^st, 7^th, and 14^th day. 5 × 10⁴ cells were cultured on MAO titanium discs doped with bismuth acetate (BA), bismuth nitrate (BN), silver nitrate (AN), and control group (AO). The OD₅₇₀ values were normalized to the MT group (machined titanium disc). a: means significant difference of the data on the 1^st day; b: means significant difference of the data on the 7^th day; and c: means significant difference of the data on the 14^th day.

The comparison of normalized ALP activity of the BA, BN, AN, and AO groups at the 1^st, 7^th, and 14^th day are shown in Figure 5. The ALP activities of cells cultured on these MAO groups were higher than that of cells on the MT group at day 1 and 7. The ALP activity of the BN group was higher than that of the AN group on the 7^th day (p < 0.05). At day 1 and 14, the ALP activities were statistically the same among BA, BN, AN, and AO groups.

Figure 5.

Alkaline phosphatase activity of MG63 cells on the 1^st, 7^th, and 14^th day. 5 × 10⁴ cells were cultured on MAO titanium discs doped with 0.001 mol/L of bismuth acetate (BA), bismuth nitrate (BN), silver nitrate (AN), and control group (AO). The ratios of OD values by ALP assay/BCA assay were normalized to the MT group (machined titanium disc). ★: means p < 0.05 between the two data sets; a: means significant difference of the data on the 1^st day; b: means significant difference of the data on the 7^th day; c: means significant difference to the data on the 14^th day.

Figure 6 shows the normalized mineralization level of the MG63 cells cultured on the BA, BN, AN, and AO discs after 1, 7, and 14 days (set MT group as 100%). The mineralization level of the MG63 cells cultured on these MAO groups was apparently higher than that of the cells on the MT group at the 1^st, 7^th, and 14^th day. The mineralization levels of the BA and AO groups were higher than that of the BN and AN groups on the 1^st day (marked by an asterisk, p < 0.05; and double asterisks, p < 0.01). The mineralization level of the BN group was higher than that of the AN group on the 14^th day (p < 0.05). The significant differences among incubation times were marked by letters a, b, and c.

Figure 6.

Mineralization level (Alizarin Red S assay) of MG63 cells at the 1^st, 7^th, and 14^th day. 5 × 10⁴ cells were cultured on MAO titanium discs doped with 0.001 mol/L of bismuth acetate (BA), bismuth nitrate (BN), silver nitrate (AN), and control group (AO). The OD values of the Alizarin Red S assay were normalized to the MT group (machined titanium disc). ★: means p < 0.05 between the two data; ★★: means p < 0.01 between the two data; a: means significant difference of the data on the 1^st day; b: means significant difference of the data on the 7^th day; and c: means significant difference of the data on the 14^th day.

Antibacterial efficacy of MAO titanium discs

Figure 7 shows the antibacterial efficacy of the aerobic bismuth- and silver-doped MAO specimens against A. actinomycetemcomitans and MRSA at 37°C for 48 h. Apparently, the A. actinomycetemcomitans and MRSA growth on the BN group are significantly inhibited when compared to the control AO group. The reduction rates (%) of the bacteria colony numbers of Bi- and Ag-doped MAO specimens were compared with the control AO group in Table 4. Apparently, the bismuth-doped BN possess the highest antibacterial activity against A. actinomycetemcomitans; the colony reduction rate of the BN group is higher than that of BA, AN, and AO groups by 1.4, 1.4, and 1.5, respectively. The reduction rate of MRSA colonies on the BN group is also higher than that of BA, AN, and AO groups by 1.7, 1.4, and 1.9, respectively.

Figure 7.

Bacteria colonies on MAO specimens against A. actinomycetemcomitans (a) BA, (b) BN, (c) AN, (d) AO, and against MRSA (e) BA, (f) BN, (g) AN, and (h) AO grown aerobically at 37°C for 48 hours.

Table 4.

Reduction rates (%) of colony numbers of A. actinomycetemcomitans and MRSA growth on Bi- and Ag-doped MAO specimens compared to the AO group (mean ± SD, n = 3).

Bacteria colonies	Reduction rate (%)^*
Bacteria colonies	BA	BN	AN	AO
A. actinomycetemcomitans	63 ± 9	88 ± 16	65 ± 15	59 ± 12
MRSA	52 ± 10	89 ± 18	65 ± 13	47 ± 9

Reduction rate = 100% × (B-A)/B, where A equals the numbers of colonies on the titanium disc and B equals the numbers of colonies on the surrounding agar.

Discussion

The antibacterial activity of bismuth acetate, bismuth nitrate, bismuth subgallate, and silver nitrate against A. actinomycetemcomitans, S. Mutans, and MRSA were compared under aerobic conditions. Bismuth nitrate possesses superior antibacterial activities when compared to other bismuth compounds and silver nitrate. The bismuth-modified titania layers have similar biological affinities to the osteoblast-like MG63 cells. However, the layers show a higher antibacterial efficacy against A. actinomycetemcomitans and MRSA. The reduction rates of colony numbers on the BN group is higher than that of the control group by 1.5 and 1.9, respectively.

It was reported that the antimicrobial effects of bismuth compounds (Bi³⁺) span a wide range of bacteria and affect the inhibition of bacterial growth at 2–10 mM levels, which is deemed therapeutically achievable in the digestive tract.²⁷ The total 0.1% w/v bismuth nitrate that makes up 4.8 mM seems to agree with the previous results mentioned above. The Bi+ groups in complex bismuth salts are known to ionically bind to anionic surfactants such as glycerolipids and glycoproteins, the predominant component of glycocalyx.²⁸ This binding of Bi+ groups may displace divalent cations; the physiochemical properties of polycationic bismuth salts might explain the bismuth compounds’ antimicrobial activity against these selected oral bacteria.^29–31 However, bismuth subgallate, which has been used to treat Helicobacter pylori infection²⁰ and aid in wound therapy³², reveals no antibacterial activity at this concentration (no zone of inhibition). This is probably due to the low solubility of bismuth subgallate in water.³³ Another possibility is that bismuth subgallate inhibits the growth of Helicobacter pylori under anaerobic conditions. The little differences in bacteriostatic effects between bismuth acetate and bismuth nitrate are reflective of the different dissolution concentrations. The 0.5% silver nitrate is effective in prophylactic use for second- and third-degree burns.³⁴ The effects of silver nitrate may result from silver ions readily combining with sulphydryl, carboxyl, phosphate, amino, and other biologically important chemical groups. However, in the present study, 0.1% is below the minimum inhibitory concentration against A. actinomycetemcomitans.

In our study, the XPS data show that the surface of MAO samples contained mainly O, Ti, and C the ratio of which were similar to previous analyses of dental implants by other authors.³⁵ The thickness of bismuth-doped measured by the Scanning electron microscopy (SEM) cross-section of the sample demonstrated that the thickness of the titania film is not affected by Bi-doping treated for 3 min (∼5 µm), however the thickness increases and become weak when prolonging the anodic oxidation time to 9 min (∼8 µm). In the BA and BN group, bismuth was doped into the titania layer by 2.3% and 6.2%, respectively. Other elements such as Ca and P were also found on the surface during the high-voltage plasma process and also confirmed by previous reports.¹¹ Interestingly, the P and Ca elements on the MAO surface were not crowd out by super addition of bismuth compounds in the electrolytes. These P and Ca ions may subsequently release and give a high concentration in the local area making the in vitro environment bioactive. This could promote the osseointegration process and result in possible biochemical bonding between bone and oxidized titanium implant in vivo.³⁶

Biocompatibility and antibacterial effects are largely affected by the surface roughness and wettibility.^17,37,38 The present results show that the surface roughness and hydrophilicities are analogous among AO, BN, AN, and BN and are in line with previous studies. It is notable that oxidized titanium has a lower contact angle (higher surface energy) when compare to the machined titanium, this may be due to the micro-submicron porous titania layer on the oxidized titanium surface or contribute from the titania nano crystalline.¹⁸ The cell proliferation test results demonstrate that neither BA nor BN will cause cytotoxicity of the MG63 cell line; these results confirm our preliminary cell activity results of fibroblastic-like 3T3 cells cultured in pure bismuth powder extractions.²² The MG63 cells cultured on the bismuth-doped MAO surface revealed analogous proliferation and alkaline phosphatase activity when compared to the control AO group. The alkaline phosphatase activity of the BN group on the 7^th day was even higher than that of the AN group (marked by an asterisk in the Figure 5). It was found that the induction of heme oxygenase-1 modulates bismuth oxide (or bismuth salts)-induced cytotoxicity in human dental pulp cells (or the human intestinal epithelial cell line DLD-1).^39,40 In the present study, there might be the same self-protective mechanism, the cytotoxic effect of bismuth may be reduced by the production of heme oxygenase-1. There was a relatively low mineralization level of the osteoblast-like cells on the BN group on the first test day when compared to that of the AN and AO group. However, the mineralization levels of all the groups were similar after day 7, and the mineralization level of the BN group on the 14^th day was even higher than that of the AN group. Although it has been reported that the proliferation of the MG63 cells were inhibited on MAO coatings contains 0.21–0.45% of Ag (by EDS detection)¹⁵, in this study the MG63 cells cultured on the AN group revealed similar abilities to proliferate when compared to the control AO group. The alkaline phosphatase activity of the AN group on the 7^th day was lower than that of the BN group (p < 0.05). It was therefore suggested that MAO doped with bismuth possesses less effect on the cell differentiation and mineralization when compared to MAO doped with silver. It has been reported that there may have occurred the risk of neurotoxicity, and acute renal failure when the bismuth concentration in blood over 0.1 ppm.⁴¹ In the present bismuth/or silver ion doped MAO titanium, the released bismuth and silver is absent in extractions of the samples up to 4 weeks, detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (the detection limit is 0.1 ppm, data not shown). Perhaps the doped bismuth would have a persisting and local effect on the attached microbial as well as to the cells.

The BN group containing 6.2at% Bi doped in the titania surface appears to have more antibacterial efficacy than that of the BA group (Table 4). Although bismuth acetate comparably inhibits A. actinomycetemcomitans and MRSA, as shown in Table 1, less bismuth on the surface of the BA group does not seem to inhibit the growth of the attached bacteria. It is notable that our results are tested in aerobic conditions, and the results may not be applicable in anaerobic conditions. Further research is needed to clarify the antibacterial effects of these bismuth-doped MAO coatings in anaerobic conditions.

For AN group, there is only little antibacterial activity against MRSA and no antibacterial activity against A. actinomycetemcomitans. The results indicate that the doped silver in the MAO coating may not be enough to inhibit the bacterial growth. It has been reported that titanium oxidized in AgNO₃-containing electrolyte solutions under 400 V obtain a significant antibacterial efficacy (>99.8%) against S. aureus and Escherichia coli.¹⁵ It is reasonable that under higher voltage MAO process can fuse more silver into the titania layer and produced a local high silver concentration on the surface to have higher antibacterial efficacy. Although the in vitro data show that the MG63 cell viability and their differential ability (alkaline phosphatase activity) were the same, the mineralization level of the AN group was lower than that of the BN group as well as that of the control AO group. These in vitro evaluations provide evidence that titanium implants with bismuth elements on the surface can improve infection control; however, more studies are needed to determine the effects of clinical use.

Infection around oral implant caused by pathologic bacteria during plaque/biofilm acumination is a major complication leading to potential implant failure. Alternative to the approaches to incorporate antibacterial agents as a bactericide onto the MAO surface, using UV illumination to sterilizing the contaminated surface (to trigger photocatalytic reactions on samples covered with relatively thick titanium dioxide) of titanium has been recently suggested by several researchers.^17,42,43 Developing an implant surface combining these two strategies would be a good direct to provide a more promised successful rate of dental implant in the future.

Conclusions

Chemical modifications of an implant surface can provide antibacterial feature to inhibit bacteria growth on the surface and obtain better infection control. Our study shows that bismuth-doped MAO titania layers possess similar physical properties on the surface; a 6.2at% bismuth on the titania surface would inhibit the growth of A. actinomycetemcomitans and MRSA bacteria.

Footnotes

Acknowledgements

The authors also thank Prof. Chih-Ho Lai (Department of Microbiology, China Medical University) for generously providing bacterial strain (methicillin-resistant Staphylococcus aureus) as a gift.

Funding

This work was supported by grants from the National Science Council, Taiwan, R.O.C. (NSC 98-2221-E-039-004), and from China Medical University (CMU97-300).

References

Gristina

. Biomaterial-centered infection: microbial adhesion versus tissue integration. Science 1987; 237: 1588–1595.

Tonetti

. Risk factors for osseodisintegration. Periodontol 2000 1998; 17: 55–62.

Van der Weijden

Van Bemmel

Renvert

. Implant therapy in partially edentulous, periodontally compromised patients: a review. Munksgaard International Publishers, 2005, pp.506–511.

Quirynen

van Steenberghe

. Bacterial colonization of the internal part of two-stage implants. An in vivo study. Clin Oral Implants Res 1993; 4(3): 158–161.

Gil

Canedo

Padrós

Sada

. Enhanced wear resistance of ball-and-socket joints of dental implants by means of titanium gaseous nitriding. J Biomater Appl 2002; 17(1): 31–43.

Xianghuai

Zhihong

Zhuyao

Nan

Ping

Guanjun

. Surface modification of titanium based biomaterials by ion beam. J Biomater Appl 1996; 10(4): 330–337.

Yoshinari

Oda

Kato

Okuda

. Influence of surface modifications to titanium on antibacterial activity in vitro. Biomaterials 2001; 22: 2043–2048.

Chen

Liu

Courtney

Bettenga

Agrawal

Bumgardner

. In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating. Biomaterials 2006; 27: 5512–5517.

Inoue

Rodriguez

Takagi

Katase

Kubota

Nagai

. Effect of a new titanium coating material (CaTiO3-aC) prepared by thermal decomposition method on osteoblastic cell response. J Biomater Appl 2010; 24: 657–672.

10.

Chang

Lai

Hsu

Tang

Liao

Huang

. Antibacterial properties and human gingival fibroblast cell compatibility of TiO2/Ag compound coatings and ZnO films on titanium-based material. Clin Oral Invest 2010; in-press.

11.

Ishizawa

Ogino

. Formation and characterization of anodic titanium oxide films containing Ca and P. J Biomed Mater Res 1995; 29: 65–72.

12.

Zhitomirsky

Gal-Or

. Electrophoretic deposition of hydroxyapatite. J Mater Sci Mater Med 1997; 8: 213–219.

13.

Zhu

Ong

Kim

. Surface characteristics and structure of anodic oxide films containing Ca and P on a titanium implant material. J Biomed Mater Res 2002; 60: 333–338.

14.

Lin

Chen

Liu

. Structural evolution and adhesion of titanium oxide film containing phosphorus and calcium on titanium by anodic oxidation. J Biomed Mater Res A 2008; 85: 378–387.

15.

Song

Ryu

Hong

. Antibacterial properties of Ag (or Pt)-containing calcium phosphate coatings formed by micro-arc oxidation. J Biomed Mater Res A 2009; 88: 246–254.

16.

Chiang

Chiou

Yang

Hsu

Yung

Tsai

. Formation of TiO2 nano-network on titanium surface increases the human cell growth. Dental Mater 2009; 25: 1022–1029.

17.

Chrzanowski

Valappil

Dunnill

Abou Neel

Lee

Parkin

. Impaired bacterial attachment to light activated Ni-Ti alloy. Mat Sci Eng C 2010; 30(2): 225–234.

18.

Deng

Arimoto

Shibata

Omori

Miyazaki

Igarashi

. Role of chloride formed on anodized titanium surfaces against an oral microorganism. J Biomater Appl 2010; 25: 179–189.

19.

Huang

Hsu

Fuh

Lin

Chen

. Biomechanical simulation of various surface roughnesses and geometric designs on an immediately loaded dental implant. Comput Biol Med 2010; 40: 525–532.

20.

Gorbach

. Bismuth therapy in gastrointestinal diseases. Gastroenterology 1990; 99: 863–875.

21.

Tiekink

. Antimony and bismuth compounds in oncology. Crit Rev Oncol Hematol 2002; 42: 217–224.

22.

Lee

Lin

Yin

Chuang

Lin

. In-vitro and in-vivo evaluation of a new Ti-15Mo-1Bi alloy. J Biomed Mater Res B Appl Biomater 2009; 91: 643–650.

23.

Coomaraswamy

Lumley

Hofmann

. Effect of bismuth oxide radioopacifier content on the material properties of an endodontic Portland cement-based (MTA-like) system. J Endod 2007; 33: 295–298.

24.

Chen

Liu

Mao

. Bismuth-doped injectable calcium phosphate cement with improved radiopacity and potent antimicrobial activity for root canal filling. Acta Biomater 2010; 6: 3199–3207.

25.

Morgan

Stec

Van Wazer

. Inner-orbital binding-energy shifts of antimony and bismuth compounds. Inorg Chem 1973; 12(4): 953–955.

26.

Han

Chen

Ding

Wang

. Optical resonant absorption of Ag-Ti co-sputtered composite films. Mater Lett 2006; 60(4): 467–469.

27.

Manhart

. In vitro antimicrobial activity of bismuth subsalicylate and other bismuth salts. Rev Infect Dis 1990; 12(Suppl 1): S11–S15.

28.

Lambert

. Pharmacology of bismuth-containing compounds. Rev Infect Dis 1991; 13(Suppl 8): S691–S695.

29.

Domenico

Reich

Madonia

Cunha

. Resistance to bismuth among Gram-negative bacteria is dependent upon iron and its uptake. J Antimicrob Chemother 1996; 38: 1031–1040.

30.

Stratton

Warner

Coudron

Lilly

. Bismuth-mediated disruption of the glycocalyx-cell wall of Helicobacter pylori: ultrastructural evidence for a mechanism of action for bismuth salts. J Antimicrob Chemother 1999; 43: 659–666.

31.

Bland

Ismail

Heinemann

Keenan

. The action of bismuth against Helicobacter pylori mimics but is not caused by intracellular iron deprivation. Antimicrob Agents Chemother 2004; 48: 1983–1988.

32.

Serena

Parnall

LKS

Knox

Vargo

Oliver

Merry

. Bismuth subgallate/borneol (suile) is superior to bacitracin in the human forearm biopsy model for acute wound healing. Adv Skin Wound Care 2007; 20: 485–492.

33.

Udalova

Logutenko

Timakova

Afonina

Naydenko

Yukhin

. Bismuth compounds in medicine. In: Strategic Technologies, 2008 IFOST 2008 Third International Forum on Novosibirsk-Tomsk 2008, pp.137–140.

34.

Moyer

Brentano

Gravens

Margraf

Monafo

Jr . Treatment of large human burns with 0.5 per cent silver nitrate solution. Arch Surg 1965; 90: 812–867.

35.

Kang

Sul

Lee

Albrektsson

. XPS, AES and SEM analysis of recent dental implants. Acta Biomater 2009; 5: 2222–2229.

36.

Sul

. The significance of the surface properties of oxidized titanium to the bone response: special emphasis on potential biochemical bonding of oxidized titanium implant. Biomaterials 2003; 24: 3893–3907.

37.

Zitelli

TenHuisen

Libera

. Differential response of Staphylococci and osteoblasts to varying titanium surface roughness. Biomaterials 2011; 32: 951–960.

38.

Duarte

Reis

de Freitas

Ota-Tsuzuki

. Bacterial adhesion on smooth and rough titanium surfaces after treatment with different instruments. J Periodontol 2009; 80: 1824–1832.

39.

Cavicchi

Gibbs

Whittle

. Inhibition of inducible nitric oxide synthase in the human intestinal epithelial cell line, DLD-1, by the inducers of heme oxygenase 1, bismuth salts, heme, and nitric oxide donors. Gut 2000; 47: 771–778.

40.

Min

Chang

Bae

Park

Hong

Kim

. The induction of heme oxygenase-1 modulates bismuth oxide-induced cytotoxicity in human dental pulp cells. J Endod 2007; 33: 1342–1346.

41.

Cengiz

Uslu

Gok

Anarat

. Acute renal failure after overdose of colloidal bismuth subcitrate. Pediatr Nephrol 2005; 20: 1355–1358.

42.

Riley

Bavastrello

Covani

Barone

Nicolini

. An in-vitro study of the sterilization of titanium dental implants using low intensity UV-radiation. Dent Mater 2005; 21(8): 756–760.

43.

Suketa

Sawase

Kitaura

Naito

Baba

Nakayama

. An antibacterial surface on dental implants, based on the photocatalytic bactericidal effect. Clin Implant Dent Relat Res 2005; 7(2): 105–111.