Electrochemical Impedance Spectroscopy Study of the

Bonelike apatite formed on the surface of titanium pretreated with NaOH solution after .... The effect of electrolytic oxidation on the electrochemica...
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Langmuir 2002, 18, 7641-7647

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Electrochemical Impedance Spectroscopy Study of the Nucleation and Growth of Apatite on Chemically Treated Titanium C. X. Wang,*,† M. Wang,† and X. Zhou‡ School of Mechanical and Production Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798, Singapore, and Center for Development of Science & Technology, Sichuan University, Chengdu 610065, Sichuan, China Received December 31, 2001. In Final Form: April 24, 2002 Bonelike apatite formed on the surface of titanium pretreated with NaOH solution after having been immersed in simulated body fluid (SBF), while no apatite formed on the surface of untreated titanium. In the present study, electrochemical impedance spectroscopy (EIS) measurement was used to investigate the nucleation and growth of apatite on chemically treated titanium immersed in the SBF solution and the difference between the behaviors of treated and untreated titanium. Appropriate equivalent circuit models were constructed to describe the nucleation and growth of apatite and the thin oxide film formed on the surface of untreated titanium. It was found that EIS is a useful method for investigating the nucleation and growth of bonelike apatite on titanium pretreated with NaOH solution.

1. Introduction Due to their good biocompatibility, excellent mechanical properties, and high corrosion resistance,1-3 titanium and its alloys have been widely used for dental and orthopedic implants under load-bearing conditions. However, because they do not form a chemical bond with bony tissue, the possibility of loosening over a long period may become a critical problem. Calcium phosphate ceramics such as hydroxyapatite (HA)-coated titanium and its alloys are among the most promising implant materials for orthopedic and dental applications; however, problems such as low bond strength between the coating and the substrate and nonuniformity across the thickness of the coating are often encountered with these coatings.4 Recently, it has been reported that chemically treated titanium can induce bonelike apatite formation in vitro and in vivo,5-25 which means that titanium and its alloys have potential bioactivity. The reagents most frequently * Corresponding author: Dr. Changxiang Wang. Current address: School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K. Fax: 44-121-414 5324 Tel: 44-121-414 7874. E-mail: [email protected]. † Nanyang Technological University. ‡ Sichuan University. (1) Calson, S. L.; Rostlunt, T. R.; Abrektsson, B.; Abrektsson, T.; Branemark, P. L. Osseointegration of titanium implant. Acta Orthop. Scand. 1986, 57, 285-289. (2) Bardos, D. I. Titanium and titanium alloys. In Encyclopedia of medical and dental materials; William, D., Ed.; Pergamon Press: Oxford, 1990; pp 360-365. (3) Kasemo, B. Biocompatibility of titanium implant: Surface science aspect. J. Prosthet. Dent. 1983, 49, 832-837. (4) Lacefield, W. R. Hydroxyapatite coatings. In Bioceramics: Material Characteristics Versus In Vivo Behavior; Ducheyne, P., Lemons, J. E., Eds.; Annals of The New York Academy of Sciences, Vol. 523; New York Academy of Sciences: New York, 1988; pp 72-80. (5) Takadama, H.; Kim, H. M.; Kokubo, T.; Nakamura, T. An X-ray photoelectron spectroscopy study of the process of apatite formation on bioactive titanium metal. J. Biomed. Mater. Res. 2001, 55, 185-193. (6) Nishiguchi, S.; Kato, H.; Neo, M.; Oka, M.; Kim, H. M.; Kokubo, T.; Nakamura, T. Alkali- and heat-treated porous titanium for orthopedic implants. J. Biomed. Mater. Res. 2001, 54, 198-208. (7) Han, Y.; Fu, T.; Lu, J.; Xu, K. W. Characterization and stability of hydroxyapatite coatings prepared by an electrodeposition and alkaline-treatment process. J. Biomed. Mater. Res. 2001, 54, 96-101.

employed in the treatments are NaOH5-20 and hydrogen peroxide (H2O2) solution,21-23 and HCl and H2SO4 are also used to etch titanium before alkaline treatment.24,25 Treatment with a NaOH solution produces a sodium titanate gel layer on the titanium surface, while H2O2 treatment produces a titania gel layer. Both gel layers have the ability to induce formation of bonelike apatite during immersion in simulated body fluid (SBF) and thus are considered bioactive. The gel layers can initiate apatite (8) Nishio, K.; Neo, M.; Akiyama, H.; Nishiguchi, S.; Kim, H. M.; Kokubo, T.; Nakamura, T. The effect of alkali- and heat-treated titanium and apatite-formed titanium on osteoblastic differentiation of bone marrow cells. J. Biomed. Mater. Res. 2000, 52, 652-661. (9) Kim, H. M.; Kokubo, T.; Fujibayashi, S.; Nishiguchi, S.; Nakamura, T. Bioactive macroporous titanium surface layer on titanium substrate. J. Biomed. Mater. Res. 2000, 52, 553-557. (10) Nishiguchi, S.; Nakamura, T.; Kobayashi, M.; Kim, H. M.; Miyaji, F.; Kokubo, T. The effect of heat treatment on bone-bonding ability of alkali-treated titanium. Biomaterials 1999, 48, 689-696. (11) Yang, B. C.; Weng, J.; Li, X. D.; Zhang, X. D. The order of calcium and phosphate ion deposition on chemically treated titanium surfaces soaked in aqueous solution J. Biomed. Mater. Res. 1999, 47, 213-219. (12) de Andrade, M. C.; Filgueiras, M. R.T.; Ogasawara, T. Nucleation and growth of hydroxyapatite on titanium pretreated in NaOH solution: Experiments and thermodynamic explanation. J. Biomed. Mater. Res. 1999, 46, 441-446. (13) Nishiguchi, S.; Kato, H.; Fujita, H.; Kim, H. M.; Miyaji, F.; Kokubo, T.; Nakamura, T. Enhancement of bone-bonding strengths of titanium alloy implants by alkali and heat treatments. J. Biomed. Mater. Res. (Appl. Biomater.) 1999, 48, 689-696. (14) Kim, H. M.; Miyaji, F.; Kokubo, T.; Nishiguchi, S.; Nakamura, T. Graded surface structure of bioactive titanium prepared by chemical treatment. J. Biomed. Mater. Res. 1999, 45, 100-107. (15) Kim, H. M.; Miyaji, F.; Kokubo, T.; Nakamura, T. Bonding strength of bonelike apatite layer to Ti metal substrate. J. Biomed. Mater. Res. (Appl. Biomater.) 1997, 38, 121-127. (16) Yan, W. Q.; Nakamura, T.; Kobayashi, M.; Kim, H. M.; Miyaji, F. Bonding of chemically treated titanium implants to bone. J. Biomed. Mater. Res. 1997, 37, 267-275. (17) Kokubo, T.; Miyaji, F.; Kim, H. M. Spontaneous formation of bone-like apatite layer on chemically treated titanium metals. J. Am. Ceram. Soc. 1996, 79, 1127-1129. (18) Kim, H. M.; Miyaji, F.; Kokubo, T.; Nakamura, T. Preparation of bioactive Ti and its alloys via simple chemical surface treatment. J. Biomed. Mater. Res. 1996, 32, 409-417. (19) Yan, W. Q.; Nakamura, T.; Kawanabe, K.; Nishiguchi, S.; Oka, M.; Kokubo, T. Apatite layer-coated titanium for use as bone bonding implants. Biomaterials 1997, 18, 1185-1190. (20) Kim, H. M.; Miyaji, F.; Kokubo, T.; Nakamura, T. Effect of heat treatment on apatite-forming ability of Ti metal induced by alkali treatment. J. Mater. Sci.: Mater. Med. 1997, 8, 341-347.

10.1021/la011877u CCC: $22.00 © 2002 American Chemical Society Published on Web 09/05/2002

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nucleation on itself. Once apatite nucleation occurs, it spontaneously grows by taking calcium and phosphate ions from the surrounding environment. The qualitative observation of nucleation and growth of apatite on pretreated titanium could be done using conventional methods such as X-ray diffraction (XRD), scanning electron microscopy (SEM), and so forth. The main objective of this study was focused on an investigation of the nucleation and growth of apatite by using electrochemical impedance spectroscopy (EIS) measurements. In the meantime, the characteristics of the thin oxide film on the surface of titanium immersed in SBF were also studied. 2. Materials and Methods 2.1. Surface Treatment of Titanium Substrates. A commercially available pure titanium substrate (disks of dimensions ) 15 mm × 3 mm) was mechanically polished and ultrasonically cleaned with acetone and alcohol. These disks were soaked in 5.0 M NaOH solution at 60 °C for 24 h, then gently washed with distilled water, and finally dried at 37 °C for 24 h. 2.2. Immersion of Pretreated Substrates in SBF. An acellular SBF with pH 7.4 and ion concentrations (in millimoles: Na+, 142.0; K+, 5.0; Mg2+, 1.5; Ca2+, 2.5; Cl-, 147.8; HCO3-, 4.2; HPO42-, 1.0; SO42-, 0.5) nearly equal to those of human blood plasma was previously proposed by Kokubo et al.26 and has been extensively confirmed to reproduce in vivo apatite formations on bioactive materials.26-28 The SBF was prepared by dissolving reagent grade chemicals of NaCl, NaHCO3, KCl, K2HPO4‚3H2O, MgCl2‚6H2O, CaCl2, and Na2SO4 into distilled water and buffering at 7.40 with tris(hydrochloromethyl)amminomethane ((CH2OH)3-CNH3) and hydrochloric acid at 36.5 °C. After the alkaline treatment, the treated titanium substrates were immersed in SBF. At regular intervals, the specimens were removed from SBF, washed with distilled water and acetone, and dried at room temperature. Some of the specimens were used for microstructural characterization, and the others were used for electrochemical impedance spectroscopy measurements. 2.3. Microstructural Characterizations. XRD was employed to analyze the structure of the titanium substrate, gel layer, and bonelike apatite. A thin-film (TF) X-ray diffractometer (Rigaku X-ray diffractometer) was used. The morphologies of the specimens were examined under scanning electron microscopy (JEOL JSM 5600LV). 2.4. EIS Measurements. EIS, being a sensitive and nondestructive method, has been widely used in recent decades for the characterization of various kinds of solid electrolyte interfaces. The principle of the method, measuring techniques, and data analysis are described in detail elsewhere.29 Usually, some equivalent circuit based on a certain physical model is used to (21) Ohtsuki, C.; Iida, H.; Hayakawa, S.; Osaka, A. Bioactivity of titanium treated with hydrogen peroxide solution containing metal chloride. J. Biomed. Mater. Res. 1997, 35, 39-47. (22) Wang, X. X.; Hayakawa, S.; Tsuru, K.; Osaka, A. Improvement of the bioactivity of H2O2/TaCl3-treated titanium after a subsequent heat treatment. J. Biomed. Mater. Res. 2000, 52, 171-176. (23) Wang, X. X.; Hayakawa, S.; Tsuru, K.; Osaka, A. A comparative study of in vitro apatite deposition on heat-, H2O2-, and NaOH-treated titanium surfaces. J. Biomed. Mater. Res. 2001, 54, 172-178. (24) Wen, H. B.; de Wijin, J. R.; Cui, F. Z.; de Groot, K. Preparation of bioactive Ti6Al4V surface by a simple method. Biomaterials 1998, 19, 215-221. (25) Wen, H. B.; de Wijin, J. R.; Cui, F. Z.; de Groot, K. Preparation of calcium phosphate coatings on titanium implant materials by simple chemistry. J. Biomed. Mater. Res. 1998, 41, 227-236. (26) Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. Solution able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W. J. Biomed. Mater. Res. 1990, 24, 721734. (27) Filguerias, M. R.; LaTorre, G.; Hench, L. L. Solution effects on the surface reaction of a bioactive glass. J. Biomed. Mater. Res. 1993, 27, 445-453. (28) Ohtsuki, C.; Kushitani, H.; Kokubo, T.; Kotani, S.; Yamamuro, T. Apatite formation on the surface of Ceraivatal-type glass ceramic in the body. J. Biomed. Mater. Res. 1991, 25, 1363-1370. (29) Macdonald, J. R. Impedance spectroscopy: emphasizing solid materials and systems; Wiley: New York, 1987; p 262.

Figure 1. Schematic diagram showing the electrochemical impedance spectroscopy measurement technique. fit the spectra, so that electrical or electrochemical parameters such as resistance and capacitance are obtained from the fitting results. Besides the successful application in the corrosion valuation of coatings for industry, EIS has been proved to be favorable for studying the metallic biomaterials, especially various oxide films on their surfaces,30,31 and in biomedical applications.32 In the present investigation, EIS was used to determine the surface change of the alkaline-treated titanium after having been immersed in SBF according to the resistance of the outmost surface, which directly relates with the nucleation and growth of apatite formation (the amount of apatite or the thickness of the apatite layer). The EIS measurements were made using a lock-in amplifier (model 5210, EG & G Instrument) coupled to a potentiostatgalvanostat system (model 273A, EG & G Parc.), which was connected to a three-electrode electrochemical cell (Figure 1). A platinum foil was used as a counter electrode, and a saturated calomel electrode (SCE) was used as a reference electrode. The treated and untreated titanium specimens (five duplicates) were used as the working electrodes. EIS spectra were obtained at the open-circuit potential of the specimens in SBF, with an amplitude of 10 mV. The frequency span was from 100 kHz down to 1 mHz. Data registration and analysis were performed on an interfaced computer. The spectra were then interpreted using the nonlinear least-squares fitting procedure developed by Boukamp.33 The quality of fitting to the equivalent circuit was judged first by the χ2 value and second by the error distribution versus frequency comparing experimental with simulated data.33

3. Results 3.1. Scanning Electron Microscopy. Figure 2 shows SEM micrographs of the surfaces of titanium substrates that were soaked in 5.0 M NaOH solution at 60 °C for 24 h in comparison to the untreated titanium substrates. It can be seen that a porous network structure was formed on the surface of titanium with the NaOH treatment. Figure 3 shows SEM micrographs of the surfaces of NaOH-pretreated titanium substrates that were immersed in SBF at 36.5 °C for regular intervals. It can be seen that after 1 week of immersion in SBF solution, (30) Pan, J.; Liao, H.; Leygraft, C.; Thierry, D.; Li, J. Variation of oxide films on titanium induced by osteoblast-like cell culture and the influence of an H2O2 pretreatment. J. Biomed. Mater. Res. 1998, 40, 244-256. (31) Mustafa, K.; Pan, J.; Wroblewski, J.; Leygraft, C.; Arvidson, K. Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy analysis of titanium surfaces cultured with osteoblastlike cells derived from human mandibular bone. J. Biomed. Mater. Res. 2002, 59, 655-664. (32) Weikart, C. M.; Matsuzawa, Y.; Winterton, L.; Yasuda, H. K. Evaluation of plasma polymer-coated contact lenses by electrochemical impedance spectroscopy. J. Biomed. Mater. Res. 2001, 54, 597-607. (33) Boukamp, B. A. A nonlinear least-squares fit procedure for analysis of immittance data of electrochemical systems. Solid State Ionics 1986, 20, 31-44.

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Figure 2. SEM micrographs of untreated titanium and titanium treated with 5.0 M NaOH solution at 60 °C for 24 h: (a) untreated titanium substrate and (b) NaOH-treated titanium substrates.

Figure 3. SEM micrographs of the surfaces of pretreated titanium substrates immersed in the SBF solution at 36.5 °C at regular intervals: (a) 1 week (mixture of Ca-titanate and apatite); (b) 2 weeks (mixture of Ca-titanate and apatite); (c) 3 weeks (apatite); (d) 4 weeks (apatite).

apatite nuclei formed on the surface of the pretreated titanium substrates. Then, the apatite nuclei grew and the amount of apatite increased with the extension of immersion time. In the stage of nuclei formation (Figure 3a), there were some spherical apatite islands on the network structure and most of the surface was the porous structure. With the increase in the immersion time, islands of apatite grew and the network structure was gradually

covered by apatite (Figure 3b), and with the further increase in the immersion time, the growing apatite islands coalesced and the network structure was completely covered with apatite (Figure 3c,d). This indicated that this network structure formed on the titanium surface by alkaline treatment could induce the nucleation and enhance the formation of apatite, which made titanium to be bioactive. However, as for the untreated titanium

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Figure 6. TF-XRD patterns of alkaline-treated titanium immersed in the SBF solution at 36.5 °C at regular intervals: (a) 1 week, (b) 2 weeks, (c) 3 weeks, (d) 4 weeks, (e) 6 weeks, and (f) 8 weeks.

Figure 4. SEM micrograph of the surface of untreated titanium substrates immersed in the SBF solution at 36.5 °C for 8 weeks.

Figure 7. TF-XRD patterns of untreated titanium immersed in the SBF solution at 36.5 °C at regular intervals: (a) 0 week, (b) 2 weeks, (c) 4 weeks, (d) 6 weeks, and (e) 8 weeks.

Figure 5. TF-XRD patterns of (a) the untreated titanium substrate and (b) the titanium substrate treated with 5.0 M NaOH at 60 °C for 24 h.

substrates, no apatite was observed on their surfaces even with 8 weeks of immersion in SBF solution (Figure 4). 3.2. XRD Results. Figure 5a shows the TF-XRD pattern of the surface of untreated titanium disks, and Figure 5b shows the TF-XRD pattern of the surface of titanium treated with 5.0 M NaOH solution at 60 °C for 24 h. In comparison to the pattern of untreated titanium substrate, it can be seen from Figure 5b that a broad bump and small peaks at around 24, 28, and 48° were observed, indicating that the surface porous network layer, which was formed by the NaOH treatment, is an amorphous sodium titanate phase.14,18 Figure 6 shows the TF-XRD patterns of alkaline-treated titanium immersed in the SBF solution at 36.5 °C at regular intervals. In comparison to the pattern in Figure 5b, all the new peaks appearing in the patterns in Figure 6 are ascribed to crystalline bonelike apatite, indicating that the network structure formed on the surface of titanium could induce the nucleation and growth of bonelike apatite on titanium. In the stage of nucleation (Figure 6a), the counts of the peaks for apatite were very

low and peaks for titanium substrate were also observed. With an increase in immersion time in simulated body fluid, the counts of the peaks for apatite got higher, and no peaks for the titanium substrate were observed (Figure 6b-f), indicating the growth of apatite and a surface fully covered with apatite. Figure 7 shows the TF-XRD patterns of untreated titanium immersed in the SBF solution at 36.5 °C at regular intervals. There was no difference between the XRD patterns of untreated titanium and untreated titanium immersed in simulated body fluid for regular intervals, which indicated that there was no apatite formed on the untreated titanium immersed in simulated body fluid even for 8 weeks. 3.3. Electrochemical Impedance Spectroscopy. When untreated titanium is exposed to simulated body fluid solution, its EIS spectra exhibit behavior typical of a thin passive oxide film on titanium, that is, a nearcapacitive response illustrated by a phase angle close to -90° over a wide frequency range. Furthermore, this does not change with exposure time (Figure 8). However, when alkaline-treated titanium is exposed to simulated body fluid, the spectra appear very different and vary significantly with exposure time, especially for the spectra at lower frequencies. A set of spectra at different exposure times is shown in Figure 9. The evolution of the spectra may be divided into an earlier stage (exposure to 1 h), nucleation stage (exposure to 1 week, islands of apatite can be seen under SEM observation), and a later stage (exposure to more than 2 weeks). During the earlier stage, the phase angle at higher frequencies was far from -30°, as shown in Figure 9A. With an increase in the exposure time, the phase angle at higher frequencies was getting close to -30°, as shown in Figure 9B (nucleation stage), and over -30°, Figure 9C (later stage). The remarkable change in the spectrum coincided with the nucleation and growth of apatite on the pretreated titanium.

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Z(CPE) ) 1/[Y0(jω)n]

Figure 8. Bode plots for untreated titanium immersed in the SBF solution: (A) 1 h and (B) 8 weeks.

where ω is the angular frequency and Y0 is a constant. In the ideal case, the exponential factor n ) 1, the CPE acts like a capacitor, and Y0 is equal to the capacitance C. In general, the CPE is given as both capacitance C and factor n. The physical meaning of n is not yet clear. Figure 10 shows the equivalent circuit based on a twolayer model of an oxide film, which can be satisfactorily used for fitting the spectra obtained from untreated titanium immersed in simulated body fluid at different periods of time. The fitting results are listed in Table 1. As can be seen, Rb is very high, and Cb is relatively low and decreases slightly with the immersion time, reaching a steady-state value. The slight decrease of Cb may correspond to a slow growth of the titanium oxide film, indicating a long-term stability of the thin passive film in simulated body fluid. On the other hand, Rp is low and increases slightly with exposure time. This indicates that the pores are probably filled only with the solution. Figure 11 shows the equivalent circuit based on a threelayer model (inner layer, hydrogel layer, and apatite layer), which can be satisfactorily used for fitting the spectra obtained from alkaline-treated titanium immersed in simulated body fluid at different periods of time. The fitting results are listed in Table 2. As can be seen, Ra (apatite layer resistance) continuously increases with the exposure time. Because Ra has a direct relation with the amount of the apatite formation or the thickness of the apatite layer formed on the pretreated titanium surfaces, the continuous increase of Ra reflects the whole process of apatite nucleation and growth on the treated titanium surfaces. At the apatite nucleation stage (from 1 h to 1 week), even though apatite could not be clearly seen under SEM observation, calcium and phosphate have been detected on the surface by the energy-dispersive X-ray analysis (EDX) technique; the increase in Ra indicated apatite nucleation. And the growth of apatite corresponded to the continuous increase of Ra. 4. Discussion

Figure 9. Bode plots for alkaline-treated titanium immersed in the SBF solution: (A) 1 h, (B) 1 week, and (C) 8 weeks.

3.4. Analysis of EIS Spectra. For analysis of the impedance data, a software program, Equivalent Circuit, was used. The program used a variety of electrical circuits to numerically fit the measured impedance data. A constant phase element (CPE), Q, is used for the equivalent circuit in this study. The CPE is a general diffusion-related element and has been ascribed to a fractal nature (special geometry of the roughness) of the interface. The impedance representation of CPE is given as

A broad bump in the TF-XRD pattern of titanium treated with NaOH solution at 60 °C for 24 h suggests that the network structure on the surface is an amorphous sodium titanate hydrogel. When the treated titanium disks were immersed in simulated body fluid solution at 36.5 °C, this hydrogel layer could induce the nucleation and growth of apatite on the surface. After about 1 week of nucleation time, islands of apatite were seen on the surface of pretreated titanium under SEM observation, and weak peaks for apatite with peaks for titanium substrate appeared in the TF-XRD pattern. With an increase in immersion time in simulated body fluid, islands of apatite were seen to grow and coalesce on pretreated titanium under SEM observation, and strong peaks of apatite with no peaks for titanium substrates appeared in the TFXRD patterns. The possible mechanism of nucleation and growth of apatite on alkaline-treated titanium immersed in SBF solution has been proposed as the following.5 The sodium titanate layer releases its Na+ ions into the surrounding fluid via an ion exchange with H3O+ in the fluid to form Ti-OH groups as early as 0.5 h after immersion. The Ti-OH groups then immediately interact with the calcium ions in the fluid to form a calcium titanate. The calcium titanate incorporates the phosphate ions, as well as the calcium ions, in the fluid to form apatite nuclei in the SBF solution. At this stage, the crystalline bonelike apatite is

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Figure 10. Equivalent circuits used for the two-layer oxide film on untreated titanium immersed in the SBF solution at 36.5 °C at regular intervals and schematic representation of the oxide film on untreated titanium. Notations: Re is the solution resistance; Cb and Rb are the inner layer capacitance and resistance; Cp and Rp are the outer layer capacitance and resistance.

Figure 11. Equivalent circuits used for alkaline-treated titanium immersed in the SBF solution at 36.5 °C at regular intervals and schematic representation of the apatite layer, hydrogel layer, and inner layer of oxide film on treated titanium. Notations: Re is the solution resistance; Cb and Rb are the inner layer capacitance and resistance; Cp and Rp are the hydrogel layer capacitance and resistance; Ca and Ra are the apatite layer capacitance and resistance. Table 1. EIS Spectral Fitting Results for Untreated Titanium Immersed in the SBF Solution for Various Periods of Time time Rp, Ω Cp, F Rb , Ω Cb, F

1h

2 weeks

4 weeks

6 weeks

8 weeks

63.2 5.6 × 10-9 4.3 × 106 3.3 × 10-5

66.4 6.1 × 10-9 4.0 × 107 1.9 × 10-5

60.8 6.9 × 10-9 8.4 × 107 1.9 × 10-5

67.1 6.3 × 10-9 7.8 × 107 1.8 × 10-5

67.3 6.0 × 10-9 8.3 × 107 1.9 × 10-5

first detected TF-XRD analysis. Once formed, the apatite nuclei grow by consuming the calcium and phosphate ions in the SBF solution. So, for the SEM results, in the nucleation stage (Figure 3a) and in Figure 3b, apatite and porous structure can be seen, indicating that the surface is a mixture of Ca titanate and apatite. In Figure 3c,d, the surface was fully covered by apatite. Hence, at these stages, the surface consists primarily of apatite. Electrochemical impedance microscopy analysis has been shown to be a useful method for investigating the nucleation and growth of bonelike apatite on pretreated titanium. On the basis of the EIS spectra, a three-layer

(inner layer, hydrogel layer, and apatite layer) model was used to interpret the obtained spectra. The results coincided with those obtained from SEM and TF-XRD very well. From Table 2, it can be seen that Ra (apatite layer resistance) continuously increases with the exposure time. In the case of the present study, the increase of Ra happened along with the change of the outmost surface due to the nucleation and growth of apatite. Therefore, the continuous increase of Ra reflects the whole process of apatite nucleation and growth on the treated titanium surfaces. At the apatite nucleation stage, even though no apatite nuclei formed on the surface and apatite could not be

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Table 2. EIS Spectral Fitting Results for Alkaline-Treated Titanium Immersed in the SBF Solution for Various Periods of Time time Ra, Ω Ca, F Rp, Ω Cp, F Rb, Ω Cb, F

1h

1 week

2 weeks

3 weeks

4 weeks

6 weeks

8 weeks

78.4 6.3 × 10-6 2.5 × 103 2.9 × 10-4 1.3 × 107 3.1 × 10-5

167.7 3.6 × 10-4 2.0 × 103 1.7 × 10-4 9.3 × 107 2.2 × 10-5

199.6 2.3 × 10-4 1.2 × 103 1.9 × 10-4 2.7 × 108 2.8 × 10-5

211.3 1.1 × 10-4 5.6 × 104 1.6 × 10-4 1.9 × 107 2.7 × 10-5

346.5 2.9 × 10-4 3.8 × 103 5.7 × 10-4 1.3 × 107 3.8 × 10-5

356.2 2.9 × 10-4 1.1 × 103 5.3 × 10-5 5.4 × 107 3.4 × 10-5

418.1 1.7 × 10-4 6.8 × 102 4.7 × 10-5 2.8 × 108 2.7 × 10-5

clearly seen under SEM observation, calcium and phosphate have been detected on the surface by the EDX technique. The increase of resistance in the outmost surface of pretreated titanium disks, which results from the changes on the surface due to release of Na+ ions and formation of Ti-OH groups, calcium titanate, and apatite nuclei, indicated apatite nucleation. With an increase in immersion time in simulated body fluid, islands of apatite (apatite nuclei) were seen to grow and coalesce on pretreated titanium under SEM. The continuous increase of the resistance of the apatite layer indicated the growth of apatite on the pretreated titanium. As for untreated titanium disks immersed in SBF solution for different periods of time, no apatite was found under SEM and in the patterns of TF-XRD. EIS results confirmed that only a thin passive oxide film formed on the surface. 5. Conclusions Bonelike apatite formed on the surface of titanium pretreated with NaOH solution after the pretreated titanium disks had been immersed in the simulated body fluid solution, while no apatite was found on the surface

of untreated titanium disks that had been immersed in the simulated body fluid solution. EIS has been shown to be a useful method for investigating the nucleation and growth of bonelike apatite on pretreated titanium. At the apatite nucleation stage, even though apatite could not be clearly seen under SEM, the increase in electrical resistance in the outmost surface of pretreated titanium disks indicated apatite nucleation. With an increase in immersion time in simulated body fluid, islands of apatite were seen to grow and coalesce on pretreated titanium under SEM. The growth of apatite corresponded to the increase in electrical resistance of the surface layer. EIS can be used to investigate apatite growth on the surface of pretreated titanium and oxide films on the surface of untreated titanium. Acknowledgment. The authors thank Nanyang Technological University (NTU) for funding the research. Wang Changxiang thanks Nanyang Technological University for providing a research fellowship. Assistance provided by technical staff in the School of MPE, NTU, is gratefully acknowledged. LA011877U