Biomimetic apatite coatings on micro-arc oxidized titania

Won-Hoon Song ^a, Youn-Ki Jun ^a, Yong Han ^{a, b} and Seong-Hyeon Hong <sup>, , a

a</sup> School of Materials Science and Engineering, Seoul National University, Seoul 151-742, South Korea
^b State-key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 71004, China

Received 16 March 2003;  accepted 21 September 2003.  Available online 2 December 2003.
Biomaterials
Volume 25, Issue 17 , August 2004, Pages 3341-3349

1. Abstract

Biomimetic apatite coatings on micro-arc oxidized titania films were investigated and their apatite-inducing ability was evaluated in a simulated body fluid (1.0SBF) as well as in a 1.5 times concentrated SBF (1.5SBF). Titania-based films on titanium were prepared by micro-arc oxidation at various applied voltages (250-500 V) in an electrolytic solution containing
-glycerophosphate disodium salt pentahydrate (
-GP) and calcium acetate monohydrate (CA). Macro-porous, Ca- and P-containing titania-based films were formed on the titanium substrates. The phase, Ca and P content, morphology, and thickness of the films were strongly dependent on the applied voltage. In particular, Ca- and P-containing compounds such as CaTiO₃,
-Ca₂P₂O₇ and
-Ca₃(PO₄)₂ were produced at higher voltages (>450 V). When immersed in 1.0SBF, a carbonated hydroxyapatite was induced on the surfaces of the films oxidized at higher voltages (>450 V) after 28 days, which is closely related to the Ca- and P-containing phases. The use of 1.5SBF shortened the apatite induction time and apatite formation was confirmed even on the surface of the films oxidized at 350 V, which suggests that the incorporated Ca and P in the titania films play a similar role to the Ca- and P-containing compounds in the SBF.

Author Keywords: Author Keywords: Biomimetic; Micro-arc oxidation; Apatite; Bioactivity; Simulated body fluid

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Micro-arc oxidation

2.2. Immersion of MAO-treated samples in the simulated body fluid

2.3. Characterization

3. Results and discussion

4. Conclusion

Acknowledgements

References

3. 1. Introduction

Titanium and its alloys have been widely used as dental and orthopedic implants because of their excellent mechanical properties and biocompatibility [1 and 2]. The biocompatibility of these materials is a direct consequence of the chemical stability and structural integrity of a titanium oxide film [2]. To improve the bone bonding ability of titanium implants, many attempts have been made to modify the structure, composition, and chemistry of the titanium surfaces including deposition of bioactive coatings [3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16]. Among the various techniques, hydroxyapatite (HA) coatings by a plasma spraying technique onto Ti substrate are widely used [9, 10 and 11]. Despite the impressive clinical success [17, 18, 19 and 20], a high processing temperature and a line-of-sight nature of the plasma sprayed HA coatings have several drawbacks such as poor adhesion between the HA layer and the metal substrate, difficulties in controlling the HA composition and structure, and prevention coatings from being deposited on non-metallic, complex shaped, and porous implants [21].

Several glasses and glass ceramics implanted into bone defects bonds directly to living bone without being encapsulated by fibrous tissue [22 and 23]. The bioactivity of these artificial materials can be attributed to the formation of a biologically active bone-like, carbonate-containing HA layer. Based on an investigation of apatite formation on the surface of silica gel and glasses in a simulated body fluid (SBF) [24 and 25], it has been proposed that HA formation is closely related to the hydroxyl groups (SiOH). After determining that hydrated titania can induce bone-like apatite in the SBF [26 and 27], a biomimetic calcium phosphate (Ca-P) coating method was developed to overcome the restrictions of the plasma spray process [21]. Similar to glasses and glass ceramics, Ti-OH groups are believed to play a key role in inducing HA growth on the titanium surface in the SBF. Several strategies have been used to produce abundant hydroxyl groups on the surface and enhance the bone-bonding ability of titanium implants including NaOH, H₂O₂ treatment, and UV exposure [28, 29 and 30].

Micro-arc oxidation (MAO) (or anodic oxidation) can produce a porous, relatively rough, and firmly adherent titanium oxide film on titanium implants [31, 32, 33, 34, 35, 36 and 37]. The porous nature of the anodized films enhances the anchorage of the implants to the bone and opened up the possibility of the incorporation and release of antibiotics around the titanium implants [38]. The precipitation of HA on these anodized titanium oxide films containing Ca and P ions after a hydrothermal treatment provides an alternative approach for preparing bioactive surfaces [32, 33, 34 and 35], and anodized hydrothermally treated Ti showed good bone apposition and push-out force [37 and 39]. Although the anodized, Ca- and P-containing titanium oxide films appear to induce bone-like apatite in the SBF, no successful results of apatite induction have been reported except the induction by a hydrothermal treatment.

In this study, Ca- and P-containing titanium oxide layers were formed on commercially available titanium substrates by MAO at various applied voltages. In vitro bioactivity of the oxidized specimens was investigated by immersing them into either 1.0SBF or 1.5 times concentrated SBF (1.5SBF) and examining the extent of apatite formation on their surfaces. The more concentrated SBF was used to shorten the apatite induction time [40] and to determine the dependence of the apatite-forming ability on the characteristics of the oxidized layers.

4. 2. Materials and methods

2.1. Micro-arc oxidation

Commercially pure titanium plates (10×10×3 mm³) were used as the substrates for MAO. The plates were ground using #1200 abrasive paper and cleaned with acetone, ethyl alcohol, and distilled water, successively. The Ti plates were anodized in an electrolytic solution containing 0.04 mol/l
-glycerophosphate disodium salt pentahydrate (C₃H₇Na₂O₆P·5H₂O,
-GP) and 0.4 mol/l calcium acetate monohydrate ((CH₃COO)₂Ca·H₂O, CA). MAO was conducted at a fixed applied voltage in the range of 250-500 V using a pulse power supply, and a pulse frequency, a duty circle, and a duration time set at 1000 Hz, 60% and 3 min, respectively. After the MAO treatment, the samples were washed with distilled water and dried.

2.2. Immersion of MAO-treated samples in the simulated body fluid

The MAO-treated samples were immersed in both 1.0SBF and 1.5SBF with ion concentrations almost equal to and 1.5 times those in human blood plasma, respectively. The 1.0SBF was prepared by dissolving reagent-grade chemicals of NaCl, NaHCO₃, KCl, K₂HPO₄·3H₂O, MgCl₂·6H₂O, CaCl₂, and Na₂SO₄ in distilled water and buffered at pH 7.4 with tris-hydroxymethyl-aminomethane ((CH₂OH)₃CNH₂) and hydrochloric acid at 36.5°C [41]. The 1.5SBF was also prepared using the same chemicals with ion concentrations 1.5 times those of the 1.0SBF. Both solutions were stable without the precipitation during immersion for up to 56 days. Each sample was immersed in 50 ml of SBF in a plastic vial and stored in an oven at 36.5°C. After immersing for a pre-determined period of time, the samples were removed from the SBF, washed with distilled water and then dried.

2.3. Characterization

X-ray diffraction (XRD, Model M18XHF-SRA, MAC Science Co., Yokohama, Japan) was carried out using Cu K
radiation (
=0.154 nm) at 50 kV and 100 mA between 2
values of 20° and 50° with a step size of 0.02°. Fourier transform infrared spectroscopy (Model FT/IR-300E, JASCO, Japan) was used to analyze the phase and structure of the MAO-treated and SBF-immersed samples, and the spectra were collected over the range 4000-400 cm⁻¹. The surface morphology of the specimens were observed using scanning electron microscopy (SEM, JSM-5600, JEOL, Tokyo, Japan). The elemental composition and surface chemistry was examined with energy dispersive X-ray spectrometer (EDX) and X-ray photoelectron spectroscopy (XPS, ARIES ARSC 10MCD 150). EDX was performed at an acceleration voltage of 20 kV in an ISIS 300 system (Link Analytical, Oxford Instruments). The photoelectrons generated by Al K
primary radiation (10 kV, 15 mA) were analyzed with a hemispherical analyzer and the core level XPS spectra for Ca2p, P2p, and C1s were measured. Energy calibration was achieved by setting the hydrocarbon C1s line at 284.6 eV. The concentrations of calcium and phosphorous in the SBF were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Model ICPS-7500, Shimadzu, Japan), and the pH change in the SBF solution was monitored by a pH meter (AR15, Fisher Scientific).

5. 3. Results and discussion

The XRD patterns of the micro-arc oxidized samples obtained at various applied voltages are shown in Fig. 1. At a low applied voltage (250 V), the oxidized layer was mainly composed of anatase (TiO₂) (Fig. 1(A)). With increasing applied voltage, a rutile phase (TiO₂) began to appear gradually and the oxidized layer became a mixture of anatase and rutile (Fig. 1(B)). No Ca- and P-containing phases were detected by XRD up to 350 V. Similar phase changes as a function of the applied voltage have been observed in Na₂CO₃ electrolyte-containing solutions [42]. However, with further increases in the applied voltage, the Ti peak was significantly reduced and new Ca, P, Ti, and O containing compounds were formed in addition to rutile and anatase ( Fig. 1(C)). These include
-Ca₂P₂O₇, CaTiO₃,
-Ca₃(PO₄)₂, and Ca₂Ti₅O₁₂, which became the dominant phases at 500 V. The interesting feature of the XRD results is the formation of tricalcium phosphate (TCP) at higher voltages, which is a well-known bioresorbable calcium phosphate phase [23].

(8K)

Fig. 1. XRD patterns of the micro-arc oxidized samples obtained at (A) 250, (B) 350, and (C) 450 V.

The extent of Ca and P incorporation in the oxidized layer was determined by EDS and is shown in Fig. 2. All the oxidized layers contained Ca and P as well as Ti and O irrespective of the presence of the Ca- and P-containing compounds. No Na was detected in the oxidized films. The Ca and P concentrations in the oxidized layers were relatively unchanged up to 350 V and increased rapidly at higher voltage, which occurred concurrently with the compound formation. The Ca to P ratio increased with the applied voltage and then decreased after 450 V. It ranged from 1.3 to 1.8. It was difficult to determine the Ca and P concentrations in the films oxidized at 500 V because it varied from place to place, which is probably due to compound formation and an uneven distribution of Ca and P across the oxide films, as has been suggested previously [31].

(6K)

Fig. 2. Ca, P content and Ca/P ratio of the micro-arc oxidized samples as a function of the applied voltage determined by EDS.

The chemical states of Ca and P were further investigated using XPS and the core level XPS spectra of the MAO treated-specimens are shown in Fig. 3. The Ca2p spectra was not well resolved but exhibited an expected doublet feature at 346.4-347.2 eV (2p_3/2) and 350.0-351.0 eV (2p_1/2) (Fig. 3(A)). These peak positions correspond to a divalent oxidation state (Ca²⁺) in inorganic calcium oxygen compounds. The 2p_3/2 peak positions are observed to shift weakly to a higher binding energy with increasing applied voltage. The observed trend can be interpreted as Ca is increasingly involved in the calcium phosphates [43]. Indeed, the XRD results indicated that Ca₂P₂O₇ and Ca₃(PO₄)₂ phases formed at the higher applied voltages. The P2p spectra showed single peaks at 132.5-133.2 eV corresponding to either a mono-valent (P⁺) or more likely a penta-valent (P⁵⁺) oxidation state (Fig. 3(B)). The P2p peak positions also shifted slightly toward the higher binding energy with increasing applied voltage and the binding energies were similar to those reported for calcium phosphates [43]. Therefore, it can be concluded that Ca and P in the MAO-treated samples exist as a divalent ion and a phosphate, respectively, which eventually have the chemical environments of calcium phosphate compounds at higher applied voltage.

(8K)

Fig. 3. (A) Ca2p and (B) P2p core level XPS spectra of the micro-arc oxidized samples.

The surface morphology and cross-sectional view of the oxidized layers are shown in Fig. 4. The oxide films at low voltages exhibited a porous microstructure with spherical pores. These pores were well separated and distributed homogeneously over the samples. The pore size increased with increasing applied voltage. At higher applied voltage, the oxide layers were slightly cracked and the surfaces became irregular and rough (Fig. 4(C)). From the cross-sectional view, there was no obvious discontinuity between the oxide film and the underlying substrate and some of pores extended to the bottom of the substrate. The thickness of the oxide films increased with increasing applied voltage and was in the range 5-30 
m similar to the previous reports [31].

(86K)

Fig. 4. Surface morphologies of the micro-arc oxidized samples formed at (A) 250, (B) 350, (C) 450 V, and (D) cross-section view of specimen (B).

The changes in the Ca and P concentrations and pH values of the 500 V oxidized samples immersed in the 1.0SBF are shown in Fig. 5. The Ca and P concentrations in the SBF increased slightly for up to 14 days and decreased rapidly thereafter. The pH values continuously increased from 7.4 to 7.6. A similar behavior was observed in the 450 V oxidized samples but only a minor variation was detected in the specimens oxidized at the lower voltages. It can be inferred that the oxide films initially dissolved and released Ca and P ions into the SBF. After 14 days, Ca- and P-containing precipitates were formed on the surface of the oxide films resulting in a reduction of the Ca and P concentrations in the SBF.

(5K)

Fig. 5. Changes in the Ca, P concentration and pH during immersion in the 1.0SBF for the samples oxidized at 500 V.

The phase changes of the oxidized films during immersion in the 1.0SBF were investigated by XRD and FT-IR and are shown for the 500 V oxidized samples in Fig. 6 and Fig. 7, respectively. The films oxidized at 500 V contained rutile,
-Ca₂P₂O₇, CaTiO₃,
-Ca₃(PO₄)₂, and Ca₂Ti₅O₁₂ phases (Fig. 6(A)), and no noticeable changes were observed after up to 14 days of immersion ( Fig. 6(B)). However, an apparently distinct apatite peaks appeared at 28 days and continued to exist at a longer period of immersion ( Fig. 6(C)). Among the other phases, the peak intensity of
-Ca₃(PO₄)₂ and Ca₂P₂O₇ increased and the amount of CaTiO₃ was significantly reduced after 28 days of immersion. Based on the XRD results, CaTiO₃ appears to mainly dissolve and the calcium phosphates were deposited on the surface during the exposure to the SBF. Apatite was also induced on the surface of the 450 V oxidized layer but was not in the other surfaces oxidized at voltages lower than 450 V, which appears to be related to the previous Ca and P concentration variation in the SBF. The FT-IR spectrum of the specimen oxidized at 500 V and immersed in the 1.0SBF for 56 days clearly shows the presence of PO₄³⁻ and OH⁻ along with a CO₃²⁻ absorption band (Fig. 7). It is believed that the induced apatite is a carbonated HA.

(10K)

Fig. 6. XRD patterns of the samples oxidized at 500 V and immersed in the 1.0SBF for: (A) 0, (B) 14, and (C) 28 days.

(5K)

Fig. 7. FT-IR spectra of the sample oxidized at 500 V and immersed in the 1.0SBF for 56 days.

In situ formation of apatite on the surface of the oxidized Ti in the 1.0SBF appears to be closely related to the Ca- and P-containing compounds. SBF is a metastable calcium phosphate solution supersaturated with respect to apatite [25 and 27]. However, it was reported that a barrier for the homogeneous nucleation of apatite is too high and a chemical stimulus is required to induce the heterogeneous nucleation of apatite from the SBF [27]. The surface hydroxyl groups such as SiOH, TiOH, and COOH are known to be efficient inducers of apatite nucleation [24, 25, 26, 27, 28, 29, 30 and 44]. In the films oxidized at higher voltages, CaTiO₃ was expected to undergo hydrolysis to form Ca²⁺, OH⁻, and TiO(OH)₂ in the SBF. The hydroxylated titanium oxide is considered to be insoluble and yields a TiOH surface, which might act as a nucleation site [45]. The hydrolysis of the Ca- and P-containing phases provides Ca²⁺, OH⁻, and HPO₄²⁻ ionic species, which increase the local degree of supersaturation with respect to apatite near the surface. The provision of abundant TiOH groups and the enrichment of calcium and phosphate trigger the nucleation of apatite on the oxidized Ti surface. When the apatite nuclei are formed, they spontaneously grow at the expense of calcium and phosphate ions from the metastable supersaturated SBF solution. In addition to the apatite induction during immersion, calcium and phosphate in the SBF appear to be incorporated into the
-Ca₂P₂O₇ and
-Ca₃(PO₄)₂ phases resulting in the growth of these phases compared to the as-oxidized specimen (Fig. 6). This suggests that more complex dynamic processes such as hydrolysis, dissolution, and precipitation, occur between the oxidized surface and the SBF solution, which require a more thorough investigation.

The surface morphologies and cross-sectional view of the immersed specimens oxidized at 500 V are shown in Fig. 8. The surface of the sample immersed for 14 days exhibited a rough and porous characteristic of the micro-arc oxidized layer (Fig. 8(A)). The exposed surface was slightly modified and there appeared to be some apatite deposition. Unfortunately, it was not clearly identified by XRD, possibly due to the poor crystallinity of the induced apatite. After 28 days of immersion, the porous surface nature completely disappeared and the entire surface was covered with apatite. It is believed that the apatite nuclei were formed after approximately 14 day immersion and they continue to grow, initially fill the pores, and then, spread over the entire surface. In the 56 day immersed sample, many micron-sized globules were observed on the smooth apatite layer, which is believed to be secondary apatite nuclei ( Fig. 8(B)). A high magnification image indicated that the smooth apatite layer was indeed a reticular structure composed of numerous flakes of ~100 nm size (Fig. 8(C)). A similar morphology has been observed previously for the apatite induced in the SBF [21, 30, 46 and 47]. The cross-sectional image of the samples immersed for 56 days showed that a dense apatite layer of ~20 
m thick was formed on the porous oxidized layer (Fig. 8(D)). There was no distinct boundary in the cross-sectional image and the Ca and P line profiles by EDS, which suggests fairly strong bonding between the layers. Apatite was also found in the specimens oxidized at 450 V and immersed for more than 28 days. However, the exposed surface was not fully covered with apatite.

(68K)

Fig. 8. Surface morphologies of the samples oxidized at 500 V and immersed in the 1.0SBF for: (A) 14, (B) 56 days, (C) high magnification of (B), and (D) cross-section view of specimen (B).

The XRD patterns for the specimens immersed in 1.5SBF are shown in Fig. 9. The broad apatite peaks were detected in the oxide films formed at 350 V and immersed for 28 days (Fig. 9(A)). In contrast to the 1.0SBF, apatite was induced on the surface oxidized at lower voltages (350 and 400 V). The broad peak is due to a defective, poorly crystalline apatite formation, and the poor crystallinity probably originated from the higher growth rate in the 1.5SBF. Apatite was induced after 14 days of immersion in the samples oxidized at 450 and 500 V, which was earlier than the 1.0SBF, and a further increase in the immersion time enhanced the crystallinity of the apatite resulting in rather sharp apatite peaks (Fig. 9(B)). The Ca and P concentrations in the 1.5SBF exhibited the same trends as those of the 1.0SBF and decreased rapidly at the point of apatite formation. The surface morphologies of the specimens immersed for 56 days are shown in Fig. 10. All the pores were completely filled with apatite producing the smooth surfaces. There were surface cracks present and a large number of secondary apatite nuclei were observed on the smooth surface (Fig. 10(A)), and the smooth surface was actually composed of ~10 
m sized-grains (Fig. 10(B)). The XPS Ca2p and P2p spectra of the apatite induced specimen, which was oxidized at 500 V and immersed in the 1.5 SBF for 28 days, are presented in Fig. 11. The Ca2p spectrum had a doublet at 347. 3 and 350.7 eV and the P2p spectrum revealed a single peak at 133.0 eV. These values are very close to the previous micro-arc oxidized samples and agree well with the published values for HA [42, 48, 49 and 50].

(8K)

Fig. 9. XRD patterns of the samples: (A) oxidized at 350 V and immersed in the 1.5SBF for 28 days and (B) oxidized at 500 V and immersed in the 1.5SBF for 56 days.

(33K)

Fig. 10. SEM micrographs of the samples oxidized at: (A) 350 V and (B) 500 V immersed in the 1.5SBF for 56 days.

(6K)

Fig. 11. (A) Ca2p and (B) P2p core level XPS spectra of the samples oxidized at 500 V and immersed in the 1.5SBF for 28 days.

The immersion experiments in the 1.5SBF demonstrated that apatite could be induced on the surface of titanium oxidized at lower voltages (350 and 400 V) as well as at higher voltages (450 and 500 V), and the time for apatite induction in the 1.5SBF was significantly shortened compared to that in the 1.0SBF. Based on the immersion results in the 1.0SBF, it was speculated that the induction of apatite is closely related to the Ca- and P-containing phases in the oxidized layers, particularly CaTiO₃. However, the samples oxidized at a lower voltage had no such Ca- and P-containing phases but still induced apatite in the 1.5SBF. The reasons for apatite induction in these samples is possibly due to the fact that a considerable amount of Ca and P ions were included in the oxidized layers even though no Ca- and P-containing compounds were detected by XRD. In addition, XPS analysis showed that the chemical environments of the Ca and P in the oxide films were quite similar irrespective of whether or not the Ca- and P-containing compounds were formed. Therefore, after exposure to the SBF, the incorporated Ca and P ions in the oxidized layers were hydrolyzed to yield Ca²⁺, OH⁻, and HPO₄²⁻ ionic species possibly generating a Ti-OH surface, which is similar to that of apatite nucleation in the 1.0SBF. However, the hydrolysis rate is believed to be slow and the number of ionic species and surface sites are insufficient for an apatite formation. A more supersaturated Ca- and P-containing solution is necessary and the 1.5SBF solution appears to have met the requirements for apatite nucleation. Previously, the deposition of a biomimetic calcium phosphate coating on Ti6Al4V was achieved using a SBF solution concentrated by a factor of 5 (5.0SBF) [40]. Indeed, a more concentrated SBF (1.5SBF) solution allowed the induction of apatite even on oxidized films without any Ca- and P-containing compound, and shortened the induction time from 28 days to 14 days when compared to incubation in 1.0 SBF.

6. 4. Conclusion

A biomimetic apatite coating was successfully achieved on micro-arc oxidized, Ca- and P-containing titania films in the 1.0 and 1.5SBF. The applied voltage during the MAO affects the phase of titania, Ca and P concentration in titania, and Ca- and P-containing compound formation, which result in the differences in the apatite induction. Carbonated HA was formed on the surface of the films oxidized above 450 V after immersing for 28 days in the 1.0SBF. The use of the 1.5SBF shortened the apatite induction time and extended the apatite formation to the films oxidized at as low as 350 V. For the apatite induction in the SBF, Ca- and P-containing compounds such as CaTiO₃,
-Ca₂P₂O₇ and
-Ca₃(PO₄)₂ play a key role. It appeared that the incorporated Ca and P in titania have a similar contribution to the apatite formation. Consequently, it was demonstrated that Ca- and P-containing, micro-arc oxidized titanium implants have the capability to induce bone-like apatite (bioactivity) in the SBF.

7. Acknowledgements

This work was supported by the Ministry of Science and Technology of Korea through 21C Frontier R & D Program.

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