bioactive titanium metal surfaces with antimicrobial properties prepared by anodic oxidation...

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Science in China Series E: Technological Sciences © 2009 SCIENCE IN CHINA PRESS Springer Sci China Ser E-Tech Sci | Aug. 2009 | vol. 52 | no. 8 | 2269-2274 www.scichina.com tech.scichina.com www.springerlink.com Bioactive titanium metal surfaces with antimicrobial properties prepared by anodic oxidation treatment YUE ChongXia 1,2 , YANG BangCheng 1,2& ZHANG XingDong 1,2 1 Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China; 2 National Engineering Research Center in Biomaterials, Chengdu 610064, China In order to endow titanium metals with bioactivity and antimicrobial properties, titanium plates were subjected to anodic oxidation treatment in NaCl solutions in this study. The treated titanium metals could induce apatite formation in the fast calcification solution, and osteoblasts on the treated titanium surfaces proliferated well as those on the untreated titanium metal surfaces. The treated metals could inhibit S. aureus growth in the microbial culture experiments. It was assumed that Ti-OH groups and Ti-Cl groups formed on the treated titanium surface were responsible for the bioactivity and antimicro- bial properties of the metals. The anodic oxidation treatment was an effective way to prepare bioactive titanium surfaces with antimicrobial properties. titanium, anodic oxidation, bioactivity, antimicrobial property 1 Introduction Titanium metal has been widely used for dental and or- thopaedic implants because of its biocompatibility and anti-corrosion properties. However, this metal could not form bioactive bonding with bony tissue in vivo, so it always fails in situ. Bioactive titanium surface has been widely developed in recent years to overcome this prob- lem. In addition, inflammation caused by the titanium im- plants is also a major problem for clinical application. It is reported that about 5%10% failure of titanium or- thopedic implants, such as hip prosthesis, and about 20% failure of titanium dental implants were caused by inflammation [14] . The microbial effect is one of the major sources for inflammation. Therefore it is of great interest to develop titanium metals with antimicrobial properties. In our previous studies, we have developed bioactive titanium metals via anodic oxidation treatment. When the titanium metals were subjected to anodic oxidation treatment in H 2 SO 4 solution, the obtained surfaces with titania structure could induce bioactive responses in vivo because of Ti-OH groups on the metal surfaces [5] . Ikeda et al. reported that anodic oxidation treatment could also make the titanium metals antimicrobial [6] . When the ti- tanium metals were anodized in NaCl solutions, the ob- tained Ti-Cl groups on the titanium metal surfaces re- sulting from the treatment made the titanium metals an- timicrobial. In order to develop bioactive titanium metals with an- timicrobial properties, anodic oxidation method was employed to treat the metals in NaCl solution in this study. It is expected to get both Ti-OH groups and Ti-Cl groups on titanium metal via this method to make it bioactive and antimicrobial. 2 Materials and methods 2.1 Anodic oxidation treatment and characterization Commercially pure titanium plates in sizes of 10 mm× Received April 30, 2009; accepted May 8, 2009 doi: 10.1007/s11431-009-0223-0 Corresponding author (email: [email protected]) Supported by the National Natural Science Foundation of China (Grant Nos. 50672062 and 30870615), Key Programs for Science and Technology Development of Sichuan Province, China (Grant No. 2008SZ0104) and Sichuan Youth Science & Technology Foundation, China (Grant No. 09ZQ026-033)

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Page 1: Bioactive titanium metal surfaces with antimicrobial properties prepared by anodic oxidation treatment

Science in China Series E: Technological Sciences

© 2009 SCIENCE IN CHINA PRESS

Springer

Sci China Ser E-Tech Sci | Aug. 2009 | vol. 52 | no. 8 | 2269-2274

www.scichina.com tech.scichina.com

www.springerlink.com

Bioactive titanium metal surfaces with antimicrobial properties prepared by anodic oxidation treatment

YUE ChongXia1,2, YANG BangCheng1,2† & ZHANG XingDong1,2 1 Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China; 2 National Engineering Research Center in Biomaterials, Chengdu 610064, China

In order to endow titanium metals with bioactivity and antimicrobial properties, titanium plates were subjected to anodic oxidation treatment in NaCl solutions in this study. The treated titanium metals could induce apatite formation in the fast calcification solution, and osteoblasts on the treated titanium surfaces proliferated well as those on the untreated titanium metal surfaces. The treated metals could inhibit S. aureus growth in the microbial culture experiments. It was assumed that Ti-OH groups and Ti-Cl groups formed on the treated titanium surface were responsible for the bioactivity and antimicro-bial properties of the metals. The anodic oxidation treatment was an effective way to prepare bioactive titanium surfaces with antimicrobial properties.

titanium, anodic oxidation, bioactivity, antimicrobial property

1 Introduction

Titanium metal has been widely used for dental and or-thopaedic implants because of its biocompatibility and anti-corrosion properties. However, this metal could not form bioactive bonding with bony tissue in vivo, so it always fails in situ. Bioactive titanium surface has been widely developed in recent years to overcome this prob-lem.

In addition, inflammation caused by the titanium im-plants is also a major problem for clinical application. It is reported that about 5%―10% failure of titanium or-thopedic implants, such as hip prosthesis, and about 20% failure of titanium dental implants were caused by inflammation[1―4]. The microbial effect is one of the major sources for inflammation. Therefore it is of great interest to develop titanium metals with antimicrobial properties.

In our previous studies, we have developed bioactive titanium metals via anodic oxidation treatment. When the titanium metals were subjected to anodic oxidation treatment in H2SO4 solution, the obtained surfaces with titania structure could induce bioactive responses in vivo

because of Ti-OH groups on the metal surfaces[5]. Ikeda et al. reported that anodic oxidation treatment could also make the titanium metals antimicrobial[6]. When the ti-tanium metals were anodized in NaCl solutions, the ob-tained Ti-Cl groups on the titanium metal surfaces re-sulting from the treatment made the titanium metals an-timicrobial.

In order to develop bioactive titanium metals with an-timicrobial properties, anodic oxidation method was employed to treat the metals in NaCl solution in this study. It is expected to get both Ti-OH groups and Ti-Cl groups on titanium metal via this method to make it bioactive and antimicrobial.

2 Materials and methods 2.1 Anodic oxidation treatment and characterization

Commercially pure titanium plates in sizes of 10 mm× Received April 30, 2009; accepted May 8, 2009 doi: 10.1007/s11431-009-0223-0 †Corresponding author (email: [email protected]) Supported by the National Natural Science Foundation of China (Grant Nos. 50672062 and 30870615), Key Programs for Science and Technology Development of Sichuan Province, China (Grant No. 2008SZ0104) and Sichuan Youth Science & Technology Foundation, China (Grant No. 09ZQ026-033)

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10 mm×1 mm were connected to the anode of an ano-dizing device (assembled by our laboratory) and im-mersed in 1 mol/L reagent grade NaCl (Kelong Chemi-cal, China) solution. Then, they were subjected to anodic oxidation treatment by applying DC voltages of 15, 40 or 65 V, as shown in Table 1, at room temperature with a pure titanium plate in a size of 30 mm×10 mm×1 mm as the counter electrode. The as-received plates were rinsed with ultra-pure water and dried for analysis with X-ray diffraction (XRD, Y-2000, China), scanning electron microscopy (SEM, HITACHI-S4800, Japan), X-ray photoelectron spectroscopy (XPS, XSAM800, UK) and coating thickness measurement (CTM, MINITEST-4100, Elektrophysik, Germany, measuring the thickness of the coatings by an eddy current principle ). The cross sec-tions of the treated metals were subjected to SEM analy-sis to detect the oxidized surface.

Table 1 Parameters for the anodic oxidation treatment and designation of the specimens

Specimen Anodized voltage (V) Electrolyte P-Ti - -

15V-AO 15 1 mol/L NaCl 40V-AO 40 1 mol/L NaCl 65V-AO 65 1 mol/L NaCl

In order to check the response of the surfaces in

aqueous solutions, some treated titanium metals were soaked into ultra-pure water for 12 h at 37℃, and then they were analyzed with XPS.

2.2 FCS soaking

Fast calcification solution (FCS) with ionic concentra-tions of Na+ 137 mmol/L, K+ 3.71 mmol/L, Ca2+ 3.10 mmol/L, Cl− 145 mmol/L, and HPO4

2− 1.86 mmol/L, which had been successfully used for quick tests of the calcification ability of biomaterials[7,8], was prepared by dissolving NaCl, CaCl2·2H2O, KCl and NaH2PO4·2H2O into distilled water, using HCl and Tris to adjust the pH to be 7.4. After being subjected to anodic oxidation treatment, each kind of the treated specimens were soaked into 30 mL of FCS for 6 h at 37℃, with titanium plates without treatment (P-Ti) as control. After being taken out from FCS, the specimens were analyzed with SEM and XRD to study their abilities to induce apatite formation.

2.3 Cell culture

Osteosarcoma cell line, Ros17/28, was cultured in Dul-

becco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% fetal bovine serum and 1% antibiotic under a 5% CO2 atmosphere at 37℃. The titanium met-als treated at different voltages, with P-Ti as control, were put into 24-well plates, and 1 mL of floating cells (about 1×104 cells/mL) was added into each well to be cultured for 2, 4, and 6 days. Medium was changed every 2 days. Then the MTT (3-(4, 5-dimethylthiazol-2- yl)-2, 5-dipheyltetrazolium bromide, Sigma, USA) assay was employed to study the bioactivity of the cells on the treated titanium metals. MTT assay was a rapid colori-metric method to determine viable cell numbers, which was based on the OD values of the purple MTT forma-zan that was produced by mitochondrial[9,10]. The results were statistically analyzed with SPSS11.0 software and the corresponding P-values were considered to be sig-nificant at values less than 0.05. At day 4, SEM was used to analyze the morphologies of the cells on the metal surfaces.

2.4 Bacteria culture

After S. aureus (ATCC 25923) strain was cultured and adjusted to 1×106 CFUs/mL, the titanium metals treated at different voltages, with P-Ti as control, were put into 24-well plates, and 1 mL of floating bacterial suspension was added into each well to be cultured for 6, 12 and 24 h. MTT assay was also employed to study the anti- microbial properties of the treated metals. The results were statistically studied with SPSS11.0 software and the corresponding P-values were considered to be significant at values less than 0.05. At 24 h, SEM was used to analyze the bacteria on the metal surfaces.

3 Results 3.1 Surface characterization

The XRD pattern of 15V-AO, 40V-AO and 65V-AO showed that only peaks of titanium appeared and their height of peaks decreased with the increasing voltages (Figure 1(a)). It indicated that amorphous substances formed on the titanium plates, and the amount of them increased with the increasing voltages. From the SEM photographs, it could be found that some macro pores in the size of about 300 μm in diameter formed on the tita-nium surfaces at the voltage of 40 and 65 V because of the spark discharge, and all the surfaces became rough after the treatment (Figure 1(b)). The cross sections of the metals showed that an oxidized layer formed on the

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YUE ChongXia et al. Sci China Ser E-Tech Sci | Aug. 2009 | vol. 52 | no. 8 | 2269-2274 2271

Figure 1 XRD patterns (a) and SEM photographs of the surfaces (b) and cross sections (c) of the treated titanium metals.

surface, and the thickness of the oxide increased with the increasing voltage (Figure 1(c)).

The result of the amorphous substance thickness measurement demonstrated that the thickness of the amorphous substances increased with the increasing voltages (Table 2). Because the MINITEST-4100 meas-ured the thickness of the amorphous substance by an

eddy current principle, the given data from the equip-ment was double thickness of the substance on both sides of the substrate. It could be calculated that the av-erage thickness of the amorphous substance increased from 6.8 μm to 13.7 μm when the voltages increased from 15 V to 65 V.

The XPS spectra (Figure 2(a)) showed that O and Cl

Table 2 The thicknesses of amorphous substance on the titanium plate treated under different voltages Specimen Measured values(double-layers) (μm) Coating thickness (monolayer) (μm) 15V-AO 12.5 12.9 13.8 13.0 15.3 14.0 13.5 6.8±0.46 40V-AO 24.7 22.4 19.5 16.5 18.6 23.9 20.9 10.5±1.48 65V-AO 28.6 24.0 31.1 26.8 27.1 27.6 26.5 13.7±1.08

Figure 2 XPS spectra of the treated titanium metals (a) and those of the metals after being soaked in ultra-pure water for 12 h (b).

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appeared on the treated surfaces. The binding energy of Cl(2p1/2) at 198.3±0.2 eV indicated that Ti-Cl groups formed on the surfaces and the O1s peak at 531.6±0.2eV was from Ti-OH on the surfaces[11―13]. It indicated that both Ti-Cl and Ti-OH formed on the surfaces after the anodic oxidation treatment. After the treated metals were soaked into ultra-pure water for 12 h, Cl disappeared from the surfaces, and Ti-OH remained (Figure 2(b)).

3.2 FCS soaking

After being soaked into FCS for 6 h, the surfaces of 15V-AO, 40V-AO and 65V-AO were covered with a layer of mineral. The amount of the mineral increased with the increasing voltages. However, only little mineral could be found on the surface of P-Ti. The XRD pattern showed that the mineral was apatite (Figure 3). This meant that the treated titanium could induce apatite for-mation in FCS in 6 h. And the apatite formation ability

increased with the increasing of the treated voltage.

3.3 Cell culture

Figure 4 showed the MTT assay and SEM observation results of Ros17/28 cells cultured on the specimens. Af-ter the osteoblasts were cultured for 2 days, the OD val-ues of the treated specimens were less than that of P-Ti. The OD values of all the specimens increased with the time, and the increasing rate of OD values on all the treated specimens was similar. At day 4 and 6, the OD values of the treated specimens were almost the same as that of P-Ti. And there was no significant difference on all the surfaces (P>0.05). The SEM photographs showed that the osteoblasts on all the specimens exhibited large spreading with characteristic spindle-like morphology. This result indicated that the osteoblasts on the treated specimens proliferated well as those on the untreated titanium metals.

Figure 3 XRD patterns (a) and the SEM photographs (b) of the specimens after being soaked in FCS for 6h.

Figure 4 MTT assay of osteoblasts cultured on the specimens for 2, 4, 6 d (a) and SEM photographs of osteoblasts cultured on the specimens for 4 d (b).

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Figure 5 MTT assay of S. aureus cultured on the specimens for 6, 12, 24 h (a) and SEM photographs of S. aureus cultured on the specimens for 24 h (b).

3.4 Bacteria culture

The MTT assay and SEM results of S. aureus cultured on specimens were shown in Figure 5. Although the OD values of the treated titanium metals and P-Ti had no difference at 6 h, all the OD values of the treated tita-nium metals were significantly lower than that of P-Ti at 12 h (P<0.05). At 24 h, the OD value of 65V-AO was significantly lower than that of P-Ti (P<0.05). It was obvious that the higher the treated voltages, the lower the OD values of S. aureus for the treated titanium met-als at 12 and 24 h (Figure 5(a)). The bacterial growth rate on the treated titanium metals was slower than that on pure titanium metal. After being cultured for 24 h, S. aureus had a scattering distribution on the surfaces of 15V-AO, 40V-AO and 65V-AO specimens, while the bacteria on the P-Ti surface gathered together to become fascicular units (Figure 5(b)). It indicated that the treated titanium plates were unfavorable for the growth of S. aureus.

4 Discussion

Our results showed that the titanium plates, subjected to anodic oxidation in 1 mol/L NaCl solution at 15, 40 and 65 V, could induce apatite formation after being soaked into FCS, and the amount of the apatite was increased with the increasing voltages. In addition, the anodized titanium surfaces were favorable for the adhesion and proliferation of osteoblasts as untreated titanium metals were, but were unfavorable for the adhesion and growth of bacteria.

After anodic oxidation in NaCl solution, Ti-Cl groups formed in the amorphous substances on the titanium surfaces and its amount was enhanced with the increas-

ing voltages because of the increased amount of the amorphous substances. After the treated metals were soaked into ultra-pure water for 12 h, XPS spectra of 15V-AO, 40V-AO and 65V-AO specimens showed no Cl on the surfaces. It implied that after soaking into aqueous solution, the Ti-Cl groups hydrolyzed and gradually released chloride into the solutions. According to the research of Ikeda et al.[6], the chloride released from the anodized titanium surface has antimicrobial ability. So the titanium metals were endowed with an-timicrobial properties by anodic oxidation treatment in the NaCl solutions.

In addition, it was reported that Ti-OH groups on the titanium surfaces could absorb Ca ions and PO4 ions from the solutions to form apatite on the surface[14,15]. Therefore, the Ti-OH groups forming on the titanium surfaces endowed the titanium metals with bioactivity after anodizing in 1 mol/L NaCl solution. The hydroly-sis of the Ti-Cl group might result in more Ti-OH groups on the surfaces. The more the Ti-Cl groups hy-drolyzed, the more chloride with antibacterial properties was released. And the more Ti-OH groups formed, cor-respondingly the higher bioactivity for the titanium metals would be.

The cells on the treated titanium metals proliferated well as those on untreated titanium metals. It indicated that the treatment had no negative effects on the bio-compatibilities of the metals. It was reported that rough surface was beneficial for the adhesion and proliferation of cells, especially for the osteoblasts, because of a faster proliferation rate caused by a smaller foot-print[16,17]. Titanium metals subjected to the anodizing treatment in this study had rough surfaces. Therefore, it might be favorable for the osteoblasts activity on the

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15V-AO, 40V-AO and 65V-AO specimens in clinical applications.

5 Conclusion

In summary, our results suggested that the anodic oxida-tion method in NaCl solution was an effective way to

prepare bioactive titanium metals with antimicrobial properties. A certain amount of Ti-Cl groups gained through the anodic oxidation process was required for the bioactivity and antimicrobial properties.

Many thanks are due to Analytical & Testing Center, Sichuan University, China for providing support of SEM observation.

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