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’žARTICLE IN PRESS Biomaterials 25 (2004) 2533 2538 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, 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 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. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hydroxyapatite coating; Titania buffer layer; Titanium substrate; Sol gel method; Bonding strength; Cell response; Corrosion resistance 1. Introduction load-bearing implants over a prolonged period of time [7,8]. In practice, the very thin (at most several tens of Titanium (Ti) and its alloys have long been used as nanometers) oxide film on the Ti surface, which is implant materials in dental and orthopedic applications formed in an aqueous environment, plays a decisive role [1]. To improve the implant-tissue osseointegration, much in determining the biocompatibility and corrosion effort has gone into the modification of the Ti surface [2 behavior of the Ti implant [9]. Since the corrosion 4]. Among the various attempts which have been made to resistance is known to increase with the thickness of the improve the osseointegration, hydroxyapatite (HA, oxide layer [10,11], many attempts have been made to Ca10(PO4)6(OH)2) coatings on Ti implants have shown form a thick TiO2 layer on the Ti substrate using various good fixation to the host bone and increased bone methods, such as anodization, thermal oxidation, and ingrowth to the implant [5]. The improved biocompat- the sol gel process [12 16]. ibility provided by the HA coatings is due to the chemical Therefore, this study was performed to fabricate an and biological similarity of HA to hard tissues, and its HA/TiO2 double layer coating on the Ti substrate, in consequent direct bonding to host bones [6]. order to optimize the biocompatibility of the Ti implant. Parallel with this development, titania (TiO2) coatings The HA layer is expected to enhance the bioactivity and on Ti have been used to improve the corrosion osteoconductivity during the initial stage following resistance of Ti, which otherwise restricted its usage in 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 *Corresponding author. Tel.: +82-02-880-7161; fax: +82-02-884- been completely dissolved due to biological processes. 1413. More importantly, the TiO2 layer, placed between the E-mail address: kimhe@snu.ac.kr (H.-E. Kim). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.09.041 ARTICLE IN PRESS 2534 H.-W. Kim et al. / Biomaterials 25 (2004) 2533 2538 HA and the Ti, is expected to improve the bonding pellets were resuspended and disrupted by a process of capability of the HA layer with respect to the Ti repeated freezing and thawing. The cell lysates, which substrate. Since the bonding strength of HA coatings on were obtained, were quantified using a BioRad DC Ti has been reported to be relatively low (20 30 MPa), protein assay kit and assayed colorimetrically by their improving the adhesive properties of the HA/Ti system reaction with p-nitrophenyl phosphate using a spectro- is essential for it to be used in load-bearing implants. As photometer. To observe the corrosion behavior of the regards the coating method, in the current study, the material, a potentiodynamic polarization test was sol gel method was employed for both the HA and TiO2 conducted using a potentiostat (model 273, EG&G layers. The sol gel approach was favored due to the PAR, USA) in physiological saline solution (0.9% chemical homogeneity and fine grain size of the resultant NaCl) at 37 C. Anodic polarization curves were coating, and the low crystallization temperature and obtained by scanning the potential from 0.2 V below mass-producibility of the process itself [17,18]. The the corrosion potential up to 1.8 V at a scan rate of phase and morphology of the HA/TiO2 double layer 5 mV/s. coating on the Ti substrate were characterized. The mechanical and biological performances of the coatings were investigated. In addition, the corrosion resistance 3. Results of the samples was briefly examined. 3.1. Coating phase and morphology 2. Materials and methods Fig. 1 shows the XRD patterns of the HA layer deposited on a Ti substrate after heat treatment at To make the TiO2 sol, 0.5 m titanium propoxide various temperatures for 1 h. Prior to HA coating, the (Ti(OCH2CH2CH3)4, Aldrich) was hydrolyzed in etha- TiO2 was pre-coated onto the Ti substrate at 500 Cf or nol mixed with diethanolamine ((HOCH2CH2)2NH, 1 h. When the HA was heat-treated at 400 C, small Aldrich) (diethanolamine/ethanol=20% v/v), and then apatite peaks began to appear (Fig. 1(A)). When the a small amount of distilled water was added to the heat treatment temperature was increased to 450 C and solution, followed by stirring for 24 h to obtain a clear 500 C(Figs. 1(B) and (C), respectively), the apatite peak TiO2 sol. The HA sol was fabricated from its precursors, intensities increased, indicating that there was an calcium nitrate tetrahydrate (Ca(NO3)2 4H2O, Aldrich) improvement in crystallization. Only the HA, TiO2, and triethyl phosphite (P(C2H5O)3, Aldrich) within an and Ti peaks were detected, regardless of the heat ethanol water mixed solvent, as described previously treatment temperature, suggesting the absence of any [19]. A commercially pure Ti (cp Ti, grade 2) disc was chemical reaction between the different components. used as the substrate after polishing and cleaning. Fig. 2 represents the SEM morphologies of the HA/ Firstly, the TiO2 layer was coated by a spin coating at TiO2 double layer coating on the Ti substrate. When a speed of 3000 rpm for 20 s, and this was followed by TiO2 was coated onto the Ti substrate at a heat heat treatment at 500 C for 1 h. The HA layer was treatment temperature of 500 C, the initial machining subsequently spin coated and heat treated at tempera- tures of 400 500 C. The phase and morphology of the coating layer were analyzed using X-ray diffraction (XRD) and scanning (A) electron microscopy (SEM), respectively. The bonding strength of the coating layer was measured using an adhesion test apparatus [19]. The cellular response to the (B) specimen was assessed in terms of the cell proliferation and the cell differentiation by measuring the alkaline phosphatase (ALP) activity. The details of the proce- (C) dure were described previously [19 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 20 25 30 35 40 of up to 10 days. The cell growth morphology was 2ø [°] observed using SEM, after fixing, dehydrating, and Fig. 1. XRD patterns of the HA coating on the Ti substrate pre- critical point drying of the cells. After detaching the cells coated with TiO2 after heat treatment at various temperatures for 1 h by a trypsinization process, the live cells were individu- in air: (A) 400 C, (B) 450 C, and (C) 500 C. The TiO2 pre-deposition ally counted using a hemocytometer. For the ALP was performed at 500 C for 1 h. The legends are: (J) Ti, ( ) TiO2: assay, the cultured cells were centrifuged and the cell rutile, (&) TiO2: anatase, and ( ) HA. Intensity [Arb. Unit] ARTICLE IN PRESS H.-W. Kim et al. / Biomaterials 25 (2004) 2533 2538 2535 grooves on the Ti substrate could still be observed, 3.2. Bonding properties suggesting the formation of a very thin TiO2 coating layer (Fig. 2(A)). The coating layer appeared to be The variation in the bonding strength of the HA/TiO2 highly dense and uniform. After coating the HA over double layer on the Ti substrate as a function of the heat the TiO2 layer and heat-treating the sample at 500 C, treatment temperature is represented in Fig. 3. The HA the surface changed so as to have a relatively rough and coating directly deposited on the Ti substrate without nano-porous structure (Fig. 2(B)). The cross-section the TiO2 layer was also tested for the purpose of view clearly shows the formation of the HA/TiO2 double comparison. For both coating systems, the bonding layer on the Ti substrate (Fig. 2(C)). The thicknesses of strengths were about the same and were relatively low the HA and TiO2 layers were approximately 800 and (B22 MPa) at the heat treatment temperature of 400 C, 200 nm, respectively. Each layer bonded firmly and had however the values increased steadily with increasing the a uniform thickness throughout on the Ti surface. heat treatment temperature. Notably, for heat treatment Moreover, there were no delaminations or cracks at temperatures above 450 C, the strength of the HA/TiO2 either of the interfaces, suggesting that the bonding double layer coating was much higher than that of the capability of both the HA/TiO2 and TiO2/Ti interfaces HA single coating on the Ti substrate, with the relative was quite good. 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. 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 2 µm µ (A) 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)). The proliferation number and alkaline phosphate (ALP) activity of the HOS cells were quantified after 1 µm (B) 70 60 HA/TiO 2 50 HA HA 40 30 TiO2 20 Ti 200 nm (C) 400 450 500 Heat Treatment Temperature [°C] Fig. 2. SEM images of the various coating systems deposited onto Ti: (A) TiO2 coating surface; (B) HA/TiO2 double layer coating surface; Fig. 3. Bonding strength of HA/TiO2 double layer coating as a and (C) HA/TiO2 double layer coating cross sectional views. The heat function of the heat treatment temperature of the HA coating layer. treatment for each coating was performed at 500 C for 1 h in air. The TiO2 pre-deposition was performed at 500 C for 1 h in air. Bonding Strength [MPa] ARTICLE IN PRESS 2536 H.-W. Kim et al. / Biomaterials 25 (2004) 2533 2538 ALP activity 0.8 Cell numbers 2 0.6 0.4 1 (A) 30 µm 0.2 0 0.0 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. (B) 30 µm µ 1.5 1.0 TiO /Ti 2 0.5 Pure Ti 0.0 -0.5 (C) 30 µm -1.0 1E-12 1E-10 1E-8 1E-6 1E-4 0.01 Fig. 4. SEM images of the HOS cells growing on each sample after Current Density [A/cm2] culturing for 5 days: (A) pure Ti; (B) TiO2 coating on Ti; and (C) HA/ Fig. 6. Potentiodynamic anodic polarization curves of the pure Ti and TiO2 double layer coating on Ti. The heat treatment for each coating TiO2 coated Ti substrates, obtained with the sample placed in a was performed at 500 C for 1 h in air. physiological saline solution at 37 C. culturing them for 5 and 10 days, respectively, as shown namic anodic polarization curves of the pure Ti and the in Fig. 5. The cell proliferation number and ALP level TiO2 coated Ti substrates were obtained under physio- on the bare Ti substrate were larger than those on the logical saline solution at 37 C, as plotted in Fig. 6. Both plastic control. Moreover, the cells on the coated specimens showed an active to passive transition samples (both TiO2 coated- and HA/TiO2 double layer behavior. Apparently, when compared to bare Ti, the coated-Ti) proliferated more and expressed higher ALP TiO2 coated Ti substrate exhibited a reduced corrosion levels compared to those on the bare Ti substrate. There current for all of the potentials measured, confirming the was little difference between the TiO2 coated- and the significantly improved corrosion resistance of the TiO2 HA/TiO2 double layer coated-Ti samples in terms of coated Ti. 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. 4. Discussion 3.4. Corrosion behavior This study was intended to investigate the effects of using a combination of HA and TiO2 coatings in order To observe the effect of the TiO2 coating layer on the to optimize the biocompatibility of the Ti substrate, i.e. corrosion resistance of the Ti substrate, the potentiody- to obtain an HA/TiO2 double layer coating on the Ti [ µ mol p -nitrophenol/hr/mg protein] Alkaline Phosphatase Activity 5 2 Cell Numbers [x 10 /cm ] Potential [V, SCE] i i /T 2 /T 2 Ti TiO HA/TiO Thermanox ARTICLE IN PRESS H.-W. Kim et al. / Biomaterials 25 (2004) 2533 2538 2537 substrate. The purpose of the HA outer layer is to might lessen the strengthening effect resulting from the improve the bioactivity and osteoconductivity during chemical affinity of TiO2 toward HA and Ti. The the initial period following implantation. The TiO2 inner properties of the TiO2 layer, such as its thickness, layer was inserted with the purpose of conferring integrity and crystallinity, are reputed to be particularly corrosion resistance on the Ti substrate, even after the important in determining the bonding strength of the HA layer is completely dissolved due to biological double layer coating, since the degradation of the TiO2 processes. In addition, the TiO2 layer was intended to would result in the bonding failure of the whole double act as a buffer layer, by improving the adhesion layer coating. In practice, an increase in the TiO2 layer properties of the HA layer to the Ti substrate. thickness was observed to result in a decrease in the As expected, the insertion of the TiO2 layer signifi- bonding strength of the double layer coating, due to the cantly improved the bonding strength of the HA layer to thermal mismatch between the Ti and TiO2 layers being the Ti substrate (Fig. 3). The strength of the double layer increased. For this reason, the application of a thinner coating increased to as high as 55 MPa, which con- layer of TiO2 might be favored. However, reducing the stituted an approximately 60% enhancement with thickness of the TiO2 layer decreases its ability to act as a respect to that of the HA single coating (35 MPa). Such barrier against the corrosion of the Ti substrate, since the an improvement was to be expected, given the dense and corrosion resistance is proportional to the thickness of uniform coating structure, as well as the tight bonding the layer [10,11]. Therefore, the TiO2 thickness should be of the TiO2 layer to both the HA layer and the Ti controlled in such a way as to produce a compromise substrate (Fig. 2). The favorable chemical affinity of between the bonding strength and the corrosion proper- TiO2 with respect to HA as well as to Ti, i.e. its tight ties. The TiO2 thickness of B200 nm obtained in this bonding to both HA and Ti, greatly contributed to the study was observed to be highly effective at improving observed improvement in bonding strength. The ex- both the bonding strength and the corrosion resistance. actitude of this postulation was evidenced by the EDS The significantly reduced corrosion current density in the composition analysis, which showed the existence of Ti TiO2 coated Ti substrate (B9.5 10 7 A/cm2) compared element on the failure surface of the double layer to the pure Ti (B4.2 10 5 A/cm2) clearly demonstrates coating, thus showing that the failure occurred at both the improvement obtained in the corrosion resistance. the HA/TiO2 and TiO2/Ti interfaces (data not shown The biological properties of the double layer coating here). Previously, an HA coating on a ZrO2 substrate, system were assessed in terms of their proliferation and rather than on a Ti substrate, was observed to have a differentiation behaviors using osteoblast-like HOS relatively high bonding strength of B70 MPa, when cells. The cells on the TiO2 coated Ti substrate produced by the same sol gel method [21]. The bonding proliferated more actively and expressed ALP activity property of plasma-sprayed HA coatings on Ti-alloy to a higher degree, as compared to those on pure Ti. The was also reported to improve slightly with the insertion presence of a TiO2 coating has been reported to improve of a ZrO2 or TiO2 layer [22,23]. Based on these results, it the biocompatibility of Ti, and this is attributed to the could be confirmed that the bonding strength of the formation of the O H bond in TiO2 in moist conditions coating layer was highly dependent on the substrate [9]. In this study, the HA coating on the TiO2 type. In this study, the HA layer was found to adhere considerably increased the ALP expression of the more strongly to the TiO2 layer than to the Ti substrate. proliferated cells. In practice, on the samples containing It should be noted that the TiO2 layer was able to HA, the HOS cells were frequently observed to express a improve the bonding strength only when the HA layer higher level of ALP activity [19,20]. This higher ALP was highly crystallized, i.e. when the heat treatment expression observed in the double layer coating system process was conducted at temperatures above 450 C. In confirms the enhancement of cell function and activity practice, in the case of the double layer coating heat- at least at an early stage of differentiation [25,26]. For a treated at 400 C, most debonding fractures occurred in deeper understanding of the cell material interactions, conjunction with a failure in the HA layer instead of a in vitro experiments using other differentiation markers, TiO2-related failure. such as osteocalcin (OC), bone-sialo protein (BSP), and At this point, the possible occurrence of thermal type I collagen need to be performed. Moreover, in vivo mismatch relief caused by the insertion of TiO2 needs to tests are needed for the complete evaluation of the be envisaged. However, considering the similar thermal biocompatibility of the double layer coating system. 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 (B200 nm) compared to HA Acknowledgements (B800 nm), there appeared to be little, if any, effects on the bonding strength driven by thermal mismatch [24]. This work was supported by a grant from the Korea Rather, the slight increase in the thickness of the coating Health 21 R&D Project, Ministry of Health & Welfare, layer with the insertion of the TiO2 layer (B200 nm) Republic of Korea (02-PJ3-PG6-EV11-0002). ARTICLE IN PRESS 2538 H.-W. Kim et al. / Biomaterials 25 (2004) 2533 2538 References [14] Haddow DB, Kothari S, James PF, Short RD, Hatton PV, van Noort R. Synthetic implant surfaces: 1. The formation and characterization of sol gel titania films. Biomaterials [1] Adell R, Lekholm U, Rockler B, Branemark PI. A 15-year study 1996;17:501 7. of osseointegrated implants in the treatment of the edentulous [15] Dieudonne SC, van den Dolder J, de Ruijter JE, Paldan H, jaw. Int J Oral Surg 1981;10:387 461. Peltola T, van t Hof MA, Happonen RP, Jansen JA. Osteoblast [2] Ratner BD. New ideas in biomaterials science-a path to differentiation of bone marrow stromal cells cultured on engineered biomaterials. 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