Hydroxyapatite coating on titanium substrate with titania buffer layer processed by sol-gel method

Hae-Won Kim, Young-Hag Koh, Long-Hao Li, Sook Lee and Hyoun-Ee Kim^,

School of Materials Science and Engineering, Seoul National University, Seoul 151-742, South Korea

Received 31 March 2003;  accepted 4 September 2003. ; Available online 18 November 2003.
Biomaterials
Volume 25, Issue 13 , June 2004, Pages 2533-2538

1. Abstract

Hydroxyapatite (HA) was coated onto a titanium (Ti) substrate with the insertion of a titania (TiO₂) buffer layer by the sol-gel method. The HA layer was employed to enhance the bioactivity and osteoconductivity of the Ti substrate, and the TiO₂ buffer layer was inserted to improve the bonding strength between the HA layer and Ti substrate, as well as to prevent the corrosion of the Ti substrate. The HA layer coated over the TiO₂ showed a typical apatite phase at 400°C and the phase intensity increased above 450°C. The sol-gel derived HA and TiO₂ films, with thicknesses of approximately 800 and 200 nm, respectively, adhered tightly to each other and to the Ti substrate. The bonding strength of the HA/TiO₂ double layer coating on Ti was markedly improved when compared to that of the HA single coating on Ti. The highest strength of the double layer coating was 55 MPa after heat treatment at 500°C. The improvement in bonding strength with the insertion of TiO₂ was attributed to the resulting enhanced chemical affinity of TiO₂ toward the HA layer, as well as toward the Ti substrate. Human osteoblast-like cells, cultured on the HA/TiO₂ coating surface, proliferated in a similar manner to those on the TiO₂ single coating and on the pure Ti surfaces. However, the alkaline phosphatase activity of the cells on the HA/TiO₂ double layer was expressed to a higher degree than that on the TiO₂ single coating and pure Ti surfaces. The corrosion resistance of Ti was improved by the presence of the TiO₂ coating, as confirmed by a potentiodynamic polarization test.

Author Keywords: Hydroxyapatite coating; Titania buffer layer; Titanium substrate; Sol-gel method; Bonding strength; Cell response; Corrosion resistance

2. Article Outline

1. Introduction

2. Materials and methods

3. Results

3.1. Coating phase and morphology

3.2. Bonding properties

3.3. In vitro cellular response

3.4. Corrosion behavior

4. Discussion

Acknowledgements

References

3. 1. Introduction

Titanium (Ti) and its alloys have long been used as implant materials in dental and orthopedic applications [1]. To improve the implant-tissue osseointegration, much effort has gone into the modification of the Ti surface [2, 3 and 4]. Among the various attempts which have been made to improve the osseointegration, hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂) coatings on Ti implants have shown good fixation to the host bone and increased bone ingrowth to the implant [5]. The improved biocompatibility provided by the HA coatings is due to the chemical and biological similarity of HA to hard tissues, and its consequent direct bonding to host bones [6].

Parallel with this development, titania (TiO₂) coatings on Ti have been used to improve the corrosion resistance of Ti, which otherwise restricted its usage in load-bearing implants over a prolonged period of time [7 and 8]. In practice, the very thin (at most several tens of nanometers) oxide film on the Ti surface, which is formed in an aqueous environment, plays a decisive role in determining the biocompatibility and corrosion behavior of the Ti implant [9]. Since the corrosion resistance is known to increase with the thickness of the oxide layer [10 and 11], many attempts have been made to form a thick TiO₂ layer on the Ti substrate using various methods, such as anodization, thermal oxidation, and the sol-gel process [12, 13, 14, 15 and 16].

Therefore, this study was performed to fabricate an HA/TiO₂ double layer coating on the Ti substrate, in order to optimize the biocompatibility of the Ti implant. The HA layer is expected to enhance the bioactivity and osteoconductivity during the initial stage following implantation, by acting as an outer coating layer; the inner TiO₂ layer is designed to prevent the Ti substrate from becoming corroded, even after the HA layer has been completely dissolved due to biological processes. More importantly, the TiO₂ layer, placed between the HA and the Ti, is expected to improve the bonding capability of the HA layer with respect to the Ti substrate. Since the bonding strength of HA coatings on Ti has been reported to be relatively low (20-30 MPa), improving the adhesive properties of the HA/Ti system is essential for it to be used in load-bearing implants. As regards the coating method, in the current study, the sol-gel method was employed for both the HA and TiO₂ layers. The sol-gel approach was favored due to the chemical homogeneity and fine grain size of the resultant coating, and the low crystallization temperature and mass-producibility of the process itself [17 and 18]. The phase and morphology of the HA/TiO₂ double layer coating on the Ti substrate were characterized. The mechanical and biological performances of the coatings were investigated. In addition, the corrosion resistance of the samples was briefly examined.

4. 2. Materials and methods

To make the TiO₂ sol, 0.5 
titanium propoxide (Ti(OCH₂CH₂CH₃)₄, Aldrich) was hydrolyzed in ethanol mixed with diethanolamine ((HOCH₂CH₂)₂NH, Aldrich) (diethanolamine/ethanol=20% v/v), and then a small amount of distilled water was added to the solution, followed by stirring for 24 h to obtain a clear TiO₂ sol. The HA sol was fabricated from its precursors, calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O, Aldrich) and triethyl phosphite (P(C₂H₅O)₃, Aldrich) within an ethanol-water mixed solvent, as described previously [19]. A commercially pure Ti (cp Ti, grade 2) disc was used as the substrate after polishing and cleaning. Firstly, the TiO₂ layer was coated by a spin coating at a speed of 3000 rpm for 20 s, and this was followed by heat treatment at 500°C for 1 h. The HA layer was subsequently spin coated and heat treated at temperatures of 400-500°C.

The phase and morphology of the coating layer were analyzed using X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. The bonding strength of the coating layer was measured using an adhesion test apparatus [19]. The cellular response to the specimen was assessed in terms of the cell proliferation and the cell differentiation by measuring the alkaline phosphatase (ALP) activity. The details of the procedure were described previously [19, 20 and 21]. In brief, the human osteosarcoma (HOS) cell line was plated onto each specimen and onto a Thermanox control at a density of 1×10⁴ cells/ml, and then cultured for periods of up to 10 days. The cell growth morphology was observed using SEM, after fixing, dehydrating, and critical point drying of the cells. After detaching the cells by a trypsinization process, the live cells were individually counted using a hemocytometer. For the ALP assay, the cultured cells were centrifuged and the cell pellets were resuspended and disrupted by a process of repeated freezing and thawing. The cell lysates, which were obtained, were quantified using a BioRad DC protein assay kit and assayed colorimetrically by their reaction with p-nitrophenyl phosphate using a spectrophotometer. To observe the corrosion behavior of the material, a potentiodynamic polarization test was conducted using a potentiostat (model 273, EG&G PAR, USA) in physiological saline solution (0.9% NaCl) at 37°C. Anodic polarization curves were obtained by scanning the potential from 0.2 V below the corrosion potential up to 1.8 V at a scan rate of 5 mV/s.

5. 3. Results

3.1. Coating phase and morphology

Fig. 1 shows the XRD patterns of the HA layer deposited on a Ti substrate after heat treatment at various temperatures for 1 h. Prior to HA coating, the TiO₂ was pre-coated onto the Ti substrate at 500°C for 1 h. When the HA was heat-treated at 400°C, small apatite peaks began to appear (Fig. 1(A)). When the heat treatment temperature was increased to 450°C and 500°C ( Figs. 1(B) and (C), respectively), the apatite peak intensities increased, indicating that there was an improvement in crystallization. Only the HA, TiO₂, and Ti peaks were detected, regardless of the heat treatment temperature, suggesting the absence of any chemical reaction between the different components.

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Fig. 1. XRD patterns of the HA coating on the Ti substrate pre-coated with TiO₂ after heat treatment at various temperatures for 1 h in air: (A) 400°C, (B) 450°C, and (C) 500°C. The TiO₂ pre-deposition was performed at 500°C for 1 h. The legends are: (
) Ti, (
) TiO₂: rutile, (
) TiO₂: anatase, and (
) HA.

Fig. 2 represents the SEM morphologies of the HA/TiO₂ double layer coating on the Ti substrate. When TiO₂ was coated onto the Ti substrate at a heat treatment temperature of 500°C, the initial machining grooves on the Ti substrate could still be observed, suggesting the formation of a very thin TiO₂ coating layer (Fig. 2(A)). The coating layer appeared to be highly dense and uniform. After coating the HA over the TiO₂ layer and heat-treating the sample at 500°C, the surface changed so as to have a relatively rough and nano-porous structure (Fig. 2(B)). The cross-section view clearly shows the formation of the HA/TiO₂ double layer on the Ti substrate (Fig. 2(C)). The thicknesses of the HA and TiO₂ layers were approximately 800 and 200 nm, respectively. Each layer bonded firmly and had a uniform thickness throughout on the Ti surface. Moreover, there were no delaminations or cracks at either of the interfaces, suggesting that the bonding capability of both the HA/TiO₂ and TiO₂/Ti interfaces was quite good.

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Fig. 2. SEM images of the various coating systems deposited onto Ti: (A) TiO₂ coating surface; (B) HA/TiO₂ double layer coating surface; and (C) HA/TiO₂ double layer coating cross sectional views. The heat treatment for each coating was performed at 500°C for 1 h in air.

3.2. Bonding properties

The variation in the bonding strength of the HA/TiO₂ double layer on the Ti substrate as a function of the heat treatment temperature is represented in Fig. 3. The HA coating directly deposited on the Ti substrate without the TiO₂ layer was also tested for the purpose of comparison. For both coating systems, the bonding strengths were about the same and were relatively low (~22 MPa) at the heat treatment temperature of 400°C, however the values increased steadily with increasing the heat treatment temperature. Notably, for heat treatment temperatures above 450°C, the strength of the HA/TiO₂ double layer coating was much higher than that of the HA single coating on the Ti substrate, with the relative increase in strength being as much as 60%. The highest strengths were 55 and 35 MPa for the HA/TiO₂ double layer and HA single coatings, respectively, after heat treatment at 500°C.

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Fig. 3. Bonding strength of HA/TiO₂ double layer coating as a function of the heat treatment temperature of the HA coating layer. The TiO₂ pre-deposition was performed at 500°C for 1 h in air.

3.3. In vitro cellular response

The cellular response to the HA/TiO₂ double layer coating system was assessed by an in vitro culture method using osteoblast-like HOS cells. The bare Ti substrate and Ti coated only with TiO₂ were also tested for the purpose of comparison. Fig. 4 shows the SEM morphologies of HOS cells growing on each sample after culturing them for 5 days. The cells spread and grew in intimate contact with the bare Ti surface (Fig. 4(A)). On the Ti substrate coated with TiO₂, the cells grew in a similar fashion to those on the bare Ti (Fig. 4(B)). Also on the HA/TiO₂ double-layer coated sample, the cells grew in a similar fashion, but a little more actively compared to those on the bare Ti and TiO₂ coated Ti (Fig. 4(C)).

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Fig. 4. SEM images of the HOS cells growing on each sample after culturing for 5 days: (A) pure Ti; (B) TiO₂ coating on Ti; and (C) HA/TiO₂ double layer coating on Ti. The heat treatment for each coating was performed at 500°C for 1 h in air.

The proliferation number and alkaline phosphate (ALP) activity of the HOS cells were quantified after culturing them for 5 and 10 days, respectively, as shown in Fig. 5. The cell proliferation number and ALP level on the bare Ti substrate were larger than those on the plastic control. Moreover, the cells on the coated samples (both TiO₂ coated- and HA/TiO₂ double layer coated-Ti) proliferated more and expressed higher ALP levels compared to those on the bare Ti substrate. There was little difference between the TiO₂ coated- and the HA/TiO₂ double layer coated-Ti samples in terms of proliferation. However, the ALP expression level of the cells on the HA/TiO₂ double-layer coated Ti substrate was higher than that on the TiO₂ coated Ti substrate.

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Fig. 5. Proliferation number and ALP activity of HOS cells cultured on each sample for periods of 5 and 10 days, respectively. The heat treatment for each coating was performed at 500°C for 1 h in air.

3.4. Corrosion behavior

To observe the effect of the TiO₂ coating layer on the corrosion resistance of the Ti substrate, the potentiodynamic anodic polarization curves of the pure Ti and the TiO₂ coated Ti substrates were obtained under physiological saline solution at 37°C, as plotted in Fig. 6. Both specimens showed an active to passive transition behavior. Apparently, when compared to bare Ti, the TiO₂ coated Ti substrate exhibited a reduced corrosion current for all of the potentials measured, confirming the significantly improved corrosion resistance of the TiO₂ coated Ti.

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Fig. 6. Potentiodynamic anodic polarization curves of the pure Ti and TiO₂ coated Ti substrates, obtained with the sample placed in a physiological saline solution at 37°C.

6. 4. Discussion

This study was intended to investigate the effects of using a combination of HA and TiO₂ coatings in order to optimize the biocompatibility of the Ti substrate, i.e. to obtain an HA/TiO₂ double layer coating on the Ti substrate. The purpose of the HA outer layer is to improve the bioactivity and osteoconductivity during the initial period following implantation. The TiO₂ inner layer was inserted with the purpose of conferring corrosion resistance on the Ti substrate, even after the HA layer is completely dissolved due to biological processes. In addition, the TiO₂ layer was intended to act as a buffer layer, by improving the adhesion properties of the HA layer to the Ti substrate.

As expected, the insertion of the TiO₂ layer significantly improved the bonding strength of the HA layer to the Ti substrate (Fig. 3). The strength of the double layer coating increased to as high as 55 MPa, which constituted an approximately 60% enhancement with respect to that of the HA single coating (35 MPa). Such an improvement was to be expected, given the dense and uniform coating structure, as well as the tight bonding of the TiO₂ layer to both the HA layer and the Ti substrate (Fig. 2). The favorable chemical affinity of TiO₂ with respect to HA as well as to Ti, i.e. its tight bonding to both HA and Ti, greatly contributed to the observed improvement in bonding strength. The exactitude of this postulation was evidenced by the EDS composition analysis, which showed the existence of Ti element on the failure surface of the double layer coating, thus showing that the failure occurred at both the HA/TiO₂ and TiO₂/Ti interfaces (data not shown here). Previously, an HA coating on a ZrO₂ substrate, rather than on a Ti substrate, was observed to have a relatively high bonding strength of ~70 MPa, when produced by the same sol-gel method [21]. The bonding property of plasma-sprayed HA coatings on Ti-alloy was also reported to improve slightly with the insertion of a ZrO₂ or TiO₂ layer [22 and 23]. Based on these results, it could be confirmed that the bonding strength of the coating layer was highly dependent on the substrate type. In this study, the HA layer was found to adhere more strongly to the TiO₂ layer than to the Ti substrate. It should be noted that the TiO₂ layer was able to improve the bonding strength only when the HA layer was highly crystallized, i.e. when the heat treatment process was conducted at temperatures above 450°C. In practice, in the case of the double layer coating heat-treated at 400°C, most debonding fractures occurred in conjunction with a failure in the HA layer instead of a TiO₂-related failure.

At this point, the possible occurrence of thermal mismatch relief caused by the insertion of TiO₂ needs to be envisaged. However, considering the similar thermal expansion coefficients of Ti (8.6×10⁻⁶/°C) and TiO₂ (8.3×10⁻⁶/°C), and the quite thin intermediate film obtained in the case of TiO₂ (~200 nm) compared to HA (~800 nm), there appeared to be little, if any, effects on the bonding strength driven by thermal mismatch [24]. Rather, the slight increase in the thickness of the coating layer with the insertion of the TiO₂ layer (~200 nm) might lessen the strengthening effect resulting from the chemical affinity of TiO₂ toward HA and Ti. The properties of the TiO₂ layer, such as its thickness, integrity and crystallinity, are reputed to be particularly important in determining the bonding strength of the double layer coating, since the degradation of the TiO₂ would result in the bonding failure of the whole double layer coating. In practice, an increase in the TiO₂ layer thickness was observed to result in a decrease in the bonding strength of the double layer coating, due to the thermal mismatch between the Ti and TiO₂ layers being increased. For this reason, the application of a thinner layer of TiO₂ might be favored. However, reducing the thickness of the TiO₂ layer decreases its ability to act as a barrier against the corrosion of the Ti substrate, since the corrosion resistance is proportional to the thickness of the layer [10 and 11]. Therefore, the TiO₂ thickness should be controlled in such a way as to produce a compromise between the bonding strength and the corrosion properties. The TiO₂ thickness of ~200 nm obtained in this study was observed to be highly effective at improving both the bonding strength and the corrosion resistance. The significantly reduced corrosion current density in the TiO₂ coated Ti substrate (~9.5×10⁻⁷ A/cm²) compared to the pure Ti (~4.2×10⁻⁵ A/cm²) clearly demonstrates the improvement obtained in the corrosion resistance.

The biological properties of the double layer coating system were assessed in terms of their proliferation and differentiation behaviors using osteoblast-like HOS cells. The cells on the TiO₂ coated Ti substrate proliferated more actively and expressed ALP activity to a higher degree, as compared to those on pure Ti. The presence of a TiO₂ coating has been reported to improve the biocompatibility of Ti, and this is attributed to the formation of the O-H bond in TiO₂ in moist conditions [9]. In this study, the HA coating on the TiO₂ considerably increased the ALP expression of the proliferated cells. In practice, on the samples containing HA, the HOS cells were frequently observed to express a higher level of ALP activity [19 and 20]. This higher ALP expression observed in the double layer coating system confirms the enhancement of cell function and activity at least at an early stage of differentiation [25 and 26]. For a deeper understanding of the cell-material interactions, in vitro experiments using other differentiation markers, such as osteocalcin (OC), bone-sialo protein (BSP), and type I collagen need to be performed. Moreover, in vivo tests are needed for the complete evaluation of the biocompatibility of the double layer coating system.

7. Acknowledgements

This work was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ3-PG6-EV11-0002).

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