Biomaterials
Volume 24, Issue 18 , August 2003, Pages 3069-3077

Nucleation and growth of apatite on chemically treated titanium alloy: an electrochemical impedance spectroscopy study

C. X. Wang^{, , a}, M. Wang^a and X. Zhou<sup>b

a</sup> School of Mechanical and Production Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798, Singapore
^b Center for Development of Science & Technology, Sichuan University, Chengdu 610065, Sichuan, China

Received 25 September 2002;  accepted 9 March 2003. ; Available online 26 April 2003.

1. Abstract

Bone-like apatite formed on the surface of Ti6Al4V pretreated with NaOH solution after having been immersed in simulated body fluid (SBF), while no apatite formed on the surface of untreated Ti6Al4V. In the present study, electrochemical impedance spectroscopy (EIS) measurement was used to investigate the nucleation and growth of apatite on chemically treated Ti6Al4V immersed in the SBF solution, and the difference between the behaviors of treated and untreated Ti6Al4V. Appropriate equivalent circuit models were constructed to describe the nucleation and growth of apatite, and thin oxide film formed on the surface of untreated Ti6Al4V. It was found that EIS is a useful method for investigating the nucleation and growth of bone-like apatite on Ti6Al4V pretreated with NaOH solution.

Author Keywords: Electrochemical impedance spectroscopy; Alkaline treatment; Titanium and its alloys; Apatite; Nucleation and growth

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Surface treatment of Ti6Al4V substrates

2.2. Immersing of pretreated substrates in simulated body fluid (SBF)

2.3. Microstructural characterizations

2.4. Electrochemical impedance spectroscopy (EIS) measurements

3. Results

3.1. Scanning electron microscopy

3.2. XRD results

3.3. Electrochemical impedance spectroscopy

3.4. Analysis of EIS spectra

4. Discussion

5. Conclusions

Acknowledgements

References

3. 1. Introduction

Calcium phosphate ceramics such as hydroxyapatite (HA) coated titanium and its alloy (Ti6Al4V) have been used intensively in biomedical field due to their excellent biocompatibility, osteoconductivity, and mechanical properties. However, problems such as low bond strength between the coating and the substrate and non-uniformity across the thickness of the coating are often encountered with these coatings [1].

In order to create an implant with both superior mechanical properties and excellent bioactivity, attempts were made to produce bioactive titanium and its alloys. Recently, it has been reported that chemically treated titanium and its alloys can induce bone-like apatite formation in vitro and in vivo [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22], which means that titanium and its alloys have potential bioactivity. This apatite layer does not have the problems associated with conventional coating techniques such as the plasma-spray technique and is an essential requirement for artificial materials bond to living bone. The reagents most frequently employed in the treatments are NaOH [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17] and hydrogen peroxide (H₂O₂) solution [18, 19 and 20], and HCl and H₂SO₄ are also used to etch titanium before alkaline treatment [21 and 22]. Treatment with an NaOH solution produces a sodium titanate gel layer on titanium surface while H₂O₂ treatment produces a titania gel layer. Both gel layers have the ability to induce formation of bone-like apatite during immersing in simulated body fluid (SBF) and thus are considered bioactive. The gel layers can initiate apatite nucleation on itself. Once apatite nucleation occurs, it spontaneously grows by taking calcium and phosphate ions from the surrounding environment.

The sodium titanate hydrogel on the titanium alloy (Ti6Al4V) has become much more popular than pure titanium in orthopedic and dental applications owing to its superior mechanical properties and higher corrosion resistance.

The qualitative observation of nucleation and growth of apatite on pretreated titanium and its alloys could be done using conventional methods such as X-ray diffraction (XRD), scanning electron microscope (SEM), etc. The main objective of this study was focused on an investigation of the nucleation and growth of apatite formed on Ti6Al4V by using electrochemical impedance spectroscopy (EIS) measurements. In the meantime, the characteristics of the thin oxide film on the surface of Ti6Al4V immersed in SBF were also studied.

4. 2. Materials and methods

2.1. Surface treatment of Ti6Al4V substrates

A commercially available titanium alloy (Ti6Al4V) substrate (discs of dimensions
15 mm×3 mm) was mechanically polished and ultrasonically cleaned with acetone and alcohol. These discs were soaked in 5.0 
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. Immersing of pretreated substrates in simulated body fluid (SBF)

An acellular simulated body fluid (SBF) with pH 7.4 and ion concentrations (in m
: 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. [23], and has been extensively confirmed to reproduce the in vivo apatite formations on bioactive materials [23, 24 and 25]. 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-hydrochlomethyl-amminomethane ((CH₂OH)₃-CNH₃) and hydrochloric acid at 36.5°C. After the alkaline treatment, the treated Ti6Al4V 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

X-ray diffraction (XRD) was employed to analyze the structure of Ti6Al4V substrate, gel layer and bone-like apatite. A thin-film X-ray diffractometer (XRD, Rigaku X-ray diffractometer) was used. The morphologies of the specimens were examined under scanning electron microscopy (SEM, JEOL JSM 5600LV).

2.4. Electrochemical impedance spectroscopy (EIS) measurements

EIS, being a sensitive and non-destructive 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 [26]. Usually some equivalent circuit based on a certain physical model is used to 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 [27 and 28] and biomedical applications [29]. In the present investigation, EIS was used to determine the surface change of the alkaline treated Ti6Al4V 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 electrochemical impedance spectroscopy (EIS) measurements were made using a lock-in amplifier (Model 5210, EG & G Instrument) coupled to a Potentiostat-Galvanostat System (Model 273A, EG & G Parc.), which was connected to a three-electrode electrochemical cell (Fig. 1). A platinum foil was used as counter electrode and a saturated calomel electrode (SCE) was used as a reference electrode. The treated and untreated Ti6Al4V specimens (five duplicates) were used as the working electrodes. EIS spectra were obtained at 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 non-linear least-square fitting procedure developed by Boukamp [30]. The quality of fitting to the equivalent circuit was judged firstly by the chi-square value, and secondly by the error distribution vs frequency comparing experimental with simulated data [30].

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Fig. 1. Schematic diagram showing the electrochemical impedance spectroscopy measurement technique.

5. 3. Results

3.1. Scanning electron microscopy

Fig. 2 shows scanning electron microscopy (SEM) micrographs of the surfaces of Ti6Al4V substrates that were soaked in 5.0 
NaOH solution at 60°C for 24 h in comparison to the untreated Ti6Al4V substrates. It can be seen that a porous network structure was formed on the surface of Ti6Al4V with the NaOH treatment.

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Fig. 2. SEM micrographs of untreated Ti6Al4V and Ti6Al4V treated with 5.0 
NaOH solution at 60°C for 24 h (a) untreated Ti6Al4V substrate (b) NaOH treated Ti6Al4V substrate.

Fig. 3 shows SEM micrographs of the surfaces of NaOH pretreated Ti6Al4V substrates were immersed in simulated body fluid (SBF) at 36.5°C for regular intervals. It can be seen that after 1 week's immersion in SBF solution, apatite nuclei formed on the surface of the pretreated Ti6Al4V substrates. Then, the apatite nuclei grew and the amount of apatite increased with the extension of immersion time. In the stage of nuclei formation (Fig. 3(a)), there were some spherical apatite islands on the network structure, and most part 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 ( Fig. 3(b)), and with the further increase in the immersion time, the growing apatite islands coalesced, and the network structure was completely covered with apatite ( Fig. 3(c) and (d)). This indicated that this network structure formed on the Ti6Al4V surface by alkaline treatment could induce the nucleation and enhance the formation of apatite, which made Ti6Al4V to be bioactive. However, as for the untreated Ti6Al4V substrates, no apatite was observed on their surfaces even with 8 weeks' immersion in SBF solution ( Fig. 4).

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Fig. 3. SEM micrographs of the surfaces of pretreated Ti6Al4V 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).

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Fig. 4. SEM micrographs of the surface of untreated Ti6Al4V substrates immersed in the SBF solution at 36.5°C for 8 weeks.

3.2. XRD results

Fig. 5(a) shows the TF-XRD pattern of the surface of untreated Ti6Al4V discs, and Fig. 5(b) shows the TF-XRD pattern of the surface of Ti6Al4V treated with 5.0 
NaOH solution at 60°C for 24 h, respectively. In comparison to the pattern of untreated Ti6Al4V substrate, it can be seen from Fig. 5(b) 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 [11 and 15].

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Fig. 5. TF-XRD patterns of (a) untreated Ti6Al4V substrate, and (b) Ti6Al4V substrate treated with 5.0 
NaOH at 60°C for 24 h.

Fig. 6 shows the TF-XRD patterns of alkaline treated Ti6Al4V immersed in the SBF solution at 36.5°C at regular intervals. In comparison to the pattern in Fig. 5(b), all the new peaks appeared in the patterns in Fig. 6 are ascribed to crystalline bone-like apatite, indicating that the network structure formed on the surface of Ti6Al4V could induce the nucleation and growth of bone-like apatite on Ti6Al4V. In the stage of nucleation ( Fig. 6(a)), the counts of the peaks for apatite were very low, and peaks for Ti6Al4V substrate were also observed. With an increase in immersion time in simulated body fluid, the counts of the peaks for apatite were getting higher, and no peaks for the Ti6Al4V substrate were observed ( Fig. 6(c)-(f)), indicating the growth of apatite and the surface was fully covered with apatite.

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Fig. 6. TF-XRD patterns of alkaline treated Ti6Al4V immersed in the SBF solution at 36.5°C at regular intervals, (a) 1week, (b) 2 weeks, (c) 3 weeks, (d) 4 weeks, (e) 6 weeks, and (f) 8 weeks.

Fig. 7 shows the TF-XRD patterns of untreated Ti6Al4V immersed in the SBF solution at 36.5°C at regular intervals. As can be seen that there was no difference between the XRD patterns of untreated Ti6Al4V and untreated Ti6Al4V immersed in simulated body fluid for regular intervals, which indicating that there was no apatite formed on the untreated Ti6Al4V immersed in simulated body fluid even for 8 weeks.

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Fig. 7. TF-XRD patterns of untreated Ti6Al4V immersed in the SBF solution at 36.5°C at regular intervals, (a) 2 weeks, (b) 4 weeks, (c) 6 weeks, and (d) 8 weeks.

3.3. Electrochemical impedance spectroscopy

When untreated Ti6Al4V is exposed to simulated body fluid solution, its EIS spectra exhibit behavior typical of a thin passive oxide film on Ti6Al4V, i.e., a near-capacitive response illustrated by a phase angle close to −90°over a wide frequency range. Furthermore, this does not change with exposure time (Fig. 8).

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Fig. 8. Bode plots for untreated Ti6Al4V immersed in the SBF solution: (a) 1 h, (b) 8 weeks.

However, when alkaline treated Ti6Al4V is exposed to simulated body fluid, the spectra appears very different and varies significantly with exposure time, especially for the spectra at lower frequencies. A set of spectra at different exposure times is shown in Fig. 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 Fig. 9(a). With an increase in the exposure time, the phase angle at higher frequencies was getting close to −30°, as shown in Fig. 9(b) (nucleation stage) and over −30° in Fig. 9(c) (later stage). It is interesting to note that the remarkable change in the spectrum coincided with the nucleation and growth of apatite on the pretreated Ti6Al4V.

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Fig. 9. Bode plots for alkaline treated Ti6Al4 V 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

Z(CPE)=1/[Y0(j )^n],

where
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.

Fig. 10 shows the equivalent circuit based on a two-layer model of an oxide film, which can be satisfactorily used for fitting the spectra obtained from untreated Ti6Al4V immersed in SBF at different periods of time. The fitting results are listed in Table 1. As can be seen that R_b is very high, and C_b is relatively low and decreases slightly with the immersion time, reaching a steady state value. The slight decrease of C_b 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, R_p is low and increases slightly with exposure time. This indicates that the pores are probably filled only with the solution.

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Fig. 10. Equivalent circuits used for the two-layer oxide film on untreated Ti6Al4V immersed in the SBF solution at 36.5°C at regular intervals, and schematic representation of the oxide film on untreated Ti6Al4V. Notations: R_e is the solution resistance; C_b, R_b are the inner layer capacitance and resistance; C_p, R_p are the outer layer capacitance and resistance.

Table 1. EIS spectra fitting results for untreated Ti6Al4V immersed in the SBF solution for various periods of time

Fig. 11 shows the equivalent circuit based on a three-layer model (inner layer, hydrogel layer and apatite layer), which can be satisfactorily used for fitting the spectra obtained from alkaline treated Ti6Al4V immersed in SBF at different periods of time. The fitting results are listed in Table 2. As can be seen that R_a (apatite layer resistance) continuously increases with the exposure time. Because R_a (apatite layer resistance) has direct relation with the amount of the apatite formation or the thickness of the apatite layer formed on the pretreated Ti6Al4V surfaces, the continuous increase of R_a (apatite layer resistance) reflects the whole process of apatite nucleation and growth on the treated Ti6Al4V surfaces. At the apatite nucleation stage (from 1 h to 1 week), even though apatite could not be clearly seen under scanning electron microscopy (SEM) observation, calcium and phosphate have been detected on the surface by energy dispersive X-ray analysis (EDX) technique, the increase in R_a indicated apatite nucleation. And the growth of apatite corresponded to the continuous increase of R_a.

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Fig. 11. Equivalent circuits used for alkaline treated Ti6Al4V 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 Ti6Al4V. Notations: R_e is the solution resistance; C_b, R_b are the inner layer capacitance and resistance; C_p, R_p are the hydrogel layer capacitance and resistance; C_a, R_a are the apatite layer capacitance and resistance.

Table 2. EIS spectra fitting results for alkaline treated Ti6Al4V immersed in the SBF solution for various periods of time

6. 4. Discussion

A broad bump in the TF-XRD pattern of Ti6Al4V 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 Ti6Al4V discs immersed in simulated boy fluid solution at 36.5°C, this hydrogel layer could induce the nucleation and growth of apatite on the surface. After about 1 week's nucleation time, islands of apatite were seen on the surface of pretreated Ti6Al4V under SEM observation, and weak peaks for apatite with peaks for Ti6Al4V substrate appeared in the TF-XRD pattern. With an increase in immersion time in SBF, islands of apatite were seen to grow and coalesce on pretreated Ti6Al4V under SEM observation, and strong peaks of apatite with no peaks for Ti6Al4V substrates appeared in the TF-XRD patterns.

The possible mechanism of nucleation and growth of apatite on alkaline treated titanium immersed in SBF solution has been proposed as following [2]. 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 bone-like apatite is 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 (Fig. 3(a)), and in Fig. 3(b), apatite and porous structure can be seen indicated that the surface is a mixture of Ca titanate and apatite. In Fig. 3(c) and (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 bone-like apatite on pretreated Ti6Al4V. Based on 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 R_a (apatite layer resistance) continuously increases with the exposure time. In the case of present study, the increase of R_a (apatite layer resistance) happened accompanying with the change of the outmost surface due to the nucleation and growth of apatite. Therefore, the continuous increase of R_a (apatite layer resistance) 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 clearly seen under SEM observation, calcium and phosphate have been detected on the surface by energy dispersive X-ray analysis (EDX) technique. The increase of resistance in the outmost surface of pretreated Ti6Al4V discs, which resulting from the changes on the surface due to releases of Na⁺ ions, 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 Ti6Al4V under SEM. The continuous increase of the resistance of apatite layer indicated the growth of apatite on the pretreated Ti6Al4V.

As for untreated Ti6Al4V discs 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 thin passive oxide film formed on the surface.

7. 5. Conclusions

Bone-like apatite formed on the surface of Ti6Al4V pretreated with NaOH solution after the pretreated Ti6Al4V discs had been immersed in the simulated body fluid solution. While no apatite was found on the surface of untreated Ti6Al4V discs had been immersed in the SBF solution. Electrochemical impedance spectroscopy (EIS) has been shown to be a useful method for investigating the nucleation and growth of bone-like apatite on pretreated Ti6Al4V. At the apatite nucleation stage, even though apatite could not be clearly seen under scanning electron microscopy (SEM), the increase in electrical resistance in the outmost surface of pretreated Ti6Al4V discs indicated apatite nucleation. With an increase in immersion time in SBF, islands of apatite were seen to grow and coalesce on pretreated Ti6Al4V 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 Ti6Al4V, and oxide films on the surface of untreated Ti6Al4V.

8. Acknowledgements

The authors would like to 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.

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Corresponding author. School of Chemical Engineering, University of Birmingham, , Edgbaston, Birmingham B15 2TT, , UK. Tel.: [phone]; fax: [phone]

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