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Characterization of coating produced on titanium surface by a designed solution containing calcium and phosphate ions
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Materials Chemistry and Physics 109 (2008) 429–435
Characterization of coating produced on titanium surface by a designed solution containing calcium and phosphate ions C.X. Resende a , J. Dille b , G.M. Platt c , I.N. Bastos c , G.A. Soares a,∗ a
Metallurgical and Materials Engineering Department, Federal University of Rio de Janeiro, P.O. Box 68505, Rio de Janeiro, RJ 21941-972, Brazil b Universit´ e Libre de Bruxelles, Belgium c State University of Rio de Janeiro, IPRJ/UERJ 97282, Nova Friburgo, RJ 28630-050, Brazil
Received 8 May 2007; received in revised form 4 December 2007; accepted 8 December 2007
Abstract The biomimetic process of coating metals has been used to transform bioinert surfaces into bioactive ones. Simulated body fluid (SBF) has been classically used and has a composition similar to that of body fluid. The solution we used also has a composition closely similar to that of body fluid and is less complex than SBF. In this work, titanium surfaces were coated with a designed simplified solution whose chemical composition is based on the ions necessary to form calcium phosphates, e.g., calcium and phosphate ions in aqueous medium. This fluid was called solution for bioactivity evaluation (SBE). Besides the advantage of having a less complex composition, SBE presents fast deposition when compared with conventional SBF. We characterized the calcium phosphate layer by using grazing incidence X-ray diffraction, fourier-transformed infrared spectroscopy (FTIR), scanning electron microscopy and transmission electron microscopy. A uniform coating layer with 15 m thickness was obtained after 7 days of soaking in this simplified solution. This coating exhibited low-dense morphology and a single-phase octacalcium phosphate (OCP) structure. © 2007 Elsevier B.V. All rights reserved. Keywords: Biomaterials; Coatings; Microstructure; Electron microscopy
1. Introduction Calcium phosphate (CaP) coatings are commonly applied to metallic materials in order to combine suitable mechanical properties (from the metallic substrate) with the bioactive behavior of CaP materials. Among coating processes, the biomimetic has received particular attention especially due to its similarity with in vivo biomineralization, to its simplicity, low temperature and low cost when compared with the plasma spray process. A large number of papers discuss the use of solutions containing calcium and phosphate ions to coat metallic substrate under physiological pH or to test the bioactivity of metallic surfaces [1–3]. There is a consensus that pure titanium and titanium alloys should be activated before coating by exposing samples to surface treatments. The most common surface pre-treatment employs the exposure of Ti samples to an NaOH solution [4].
∗
Corresponding author. Tel.: +55 21 2562 8511/8500; fax: +55 21 290 6626. E-mail address: [email protected] (G.A. Soares).
0254-0584/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2007.12.011
After the pre-treatment the metal is immersed in a fluid capable of promoting apatite precipitation. Several acellular simulated body fluids (SBF) have been developed [1,3,5] for calcium phosphate precipitation. These solutions are applied to test biomaterials as some authors consider calcium phosphate precipitation on materials surface an indicative of good in vivo behavior [1,6]. In general, all investigators use the solution with a composition closely similar to that of body fluid. The disadvantage of using the biomimetic process is the relative long time necessary for coating, on the order of some weeks. To overcome this problem some authors [3,7,8] have increased solution concentration and/or included a nucleating agent such as sodium silicate [8] to accelerate the formation of a CaP layer. Indeed, one or 2 weeks are typically considered as the time necessary to obtain a uniform layer of calcium phosphate on titanium surface [8]. Another disadvantage is that non-homogeneity of coatings is likely to occur because CaP nucleation and growth processes are heterogeneous and highly dependent on surface properties. In this paper, we propose an alternative way to accelerate deposition. By using a “solution for bioactivity evaluation”
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(SBE), we can eliminate, as much as possible, the ions not fundamental to the precipitation reaction. It can also be applied to evaluate the bioactivity of biomaterials or coat metal surfaces. SBE has a composition containing mainly the elements necessary to form calcium phosphate and, consequently, offers several advantages over usual SBF; among these are simplicity and low cost. Besides its thermodynamic properties, such as high ion activity and more negative free Gibbs variation energy, it produces a higher nucleation driving force than that of SBF [9] under physiological conditions. These advantages make it possible to reduce the time involved in coating metallic titanium. Our work thus focuses on titanium surfaces coated with this new designed solution. Characterization was performed by using standard and some non-standard techniques, namely grazing incidence small angle X-ray diffraction and focused ion beam (FIB) machining of cross-section. 2. Experimental procedure Titanium samples of 8 mm × 8 mm were cut from a commercially pure titanium (Ti) sheet. Ti samples were cleaned and their surfaces were activated by using a double treatment that consisted of immersion in a NaOH 5 M solution at 60 ◦ C for 24 h, followed by a heat treatment at 600 ◦ C for 3 h. This double treatment produced a sodium titanate layer on titanium surface, and this layer seemed to be fundamental to induce calcium phosphate precipitation during the subsequent step [3]. SBE was designed by using a computational simulator named SimSE [10]. Solution was prepared with the following reagents: NaHCO3 (0.3525 g L−1 ), K2 HPO4 (0.2305 g L−1 ), CaCl2 (0.2890 g L−1 ) and tris-hydroxymethyl aminomethane (TRIS, 6.118 g L−1 ), which resulted in the following ionic concentrations (in mmol L−1 ): 2.5 Ca2+ , 5.0 Cl− , 4.2 Na+ , 4.2 HCO3 − , 2.0 K+ and 1.0 HPO4 − . The sequence of addiction followed the order showed above. The temperature of solution must be approximately 37 ◦ C during all preparation and ultimate pH should be around 7.4, which was adjusted by addition of 1.0 M HCl solution. Ti samples were exposed to 16 mL of SBE solution at 37 ◦ C for 1, 7, 14, 21 and 28 days. The precipitation process that produced the substrate coating was monitored by the chemical analysis of calcium and phosphate content in solution. All experiments were performed in quintuplicate, by running five parallel independent tubes. After each exposition time, calcium content was determined by using atomic absorption spectrometry (AAS), while phosphate concentration
was determined by using phosphomolybdate/UV spectroscopy. For comparison, titanium samples were also exposed to conventional SBF [1]. For these experiments, the sample area/solution volume ratio, temperature and exposition time were kept unchanged for both solutions. Coating morphology was evaluated by scanning electron microscopy (SEM) using a JEOL JSM-6460-LV microscope operating at 20 kV. To evaluate the possibility of deposition on the walls of the tube, a blank test was carried out using both solutions. This test, performed without the metallic sample, revealed a rather small amount of precipitation. The coating of titanium sample exposed to SBE for 7 days was further analyzed by environmental scanning electron microscopy (ESEM), fouriertransformed infrared spectroscopy (FT-IR), grazing incidence X-ray diffraction (XRD) and transmission electron microscopy (TEM). A FEI Quanta 200 3D dual beam was employed for ESEM examination of the cross-section machined in situ by focused ion beam. Energy dispersive spectrometry (EDS) was also performed to examine the distribution of calcium and phosphorus along the deposit thickness. A FT-IR spectrometer (ABB Bomem Inc.) was used to identify the vibration modes of several species. The spectra were collected at room temperature at a nominal resolution of 4.00 cm−1 with number of scans equal to 100. XRD patterns were obtained with a Siemens D5000 diffractometer used in grazing incidence X-ray diffraction conditions. For TEM analysis, the coating layer was partially removed by immersing the sample in an ethanol bath with ultrasonic vibration. Few drops of this solution were deposited on a copper grid coated with an amorphous carbon film. The analysis of precipitates was conducted in a JEOL 2000FX electron microscope, operating at 200 kV.
3. Results and discussion Fig. 1 shows calcium and phosphorus contents in solution as a function of exposure time for experiments using SBE and SBF. The decrease in these ion contents with time is related to precipitation on the materials surface. Both solutions are supersaturated in relation to their equilibrium concentrations for hydroxyapatite and OCP at pH 7.4, as shown in theoretical simulations [10]. Moreover, the experimental results presented in Fig. 1 indicate faster kinetics for SBE than for SBF solution. Indeed, for 7 days, the calcium measured in aliquots of SBE (Fig. 1a) is approximately 20 ppm below the correspondent value of SBF. A similar result is observed for phosphorus (Fig. 1b). The reasons for the fast growth rate observed with SBE could be related to differ-
Fig. 1. (a) Calcium content in solutions for different exposure times and (b) phosphorus content in solutions for different exposure times.
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ences in the kinetic process due to the more negative Gibbs free energy, which acts as a driving force for precipitation, and to the absence of magnesium ions in SBE solution. According to Kapolos and Koutsoukos [11], the growth rate of phosphate crystal (Rg ) is related to relative supersaturation (σ) by Rg = kσ n
(1)
where k is the precipitation rate constant and n indicates the apparent growth reaction order. Relative supersaturation can be evaluated by Gibbs free energy variation (between supersaturated and saturated solutions): σ = Ω1/ i νi − 1 (2) νi ai Ω= (3) KSP RT G = − ln(Ω) i νi
(4)
In the previous equations, ai refers to the activity of chemical species i, νi is the stoichiometric coefficient in the precipitation reaction, Ω is the supersaturation ratio and KSP is the thermodynamic solubility product. SimSE program was used to evaluate all activities and thus G [9]. The G and Ω values act as driving force for CaP precipitation. At pH 7.4, the simulated results for SBE are G and super-saturation index
431
with values of −9343.6 J mol−1 and 42.4 for OCP, respectively; and −18343.5 J mol−1 , and 1641.3 respectively for HA. Some authors [5,7] have reported the effect of magnesium in retarding or inhibiting the crystallization of several calcium phosphate phases, so its absence in SBE can also have a contribution to the fast decrease of Ca and P contents seen in Fig. 1. Some SEM images of Ti exposed to SBE and SBF are shown in Figs. 2 and 3, respectively. In the micrograph with 500× magnification, some small rounded uncoated areas can be observed (Fig. 2a) for 1 day exposure sample to SBE. After 7 days, a homogeneous layer already coats the whole surface (Fig. 2b), in good agreement with data shown in Fig. 1. For SBF, the surface is coated only after a 14 day-exposure (Fig. 3c). Differences in coating morphology are also observed. SBF coating exhibits a dense morphology with small crystals, resulting in some cracks during the drying process. This morphology and the cracked appearance are largely reported in several papers [6,7,12] and are associated with poorly crystallized carbonated apatite or calcium-deficient apatite. The designed solution (SBE) produces a porous plate-like calcium phosphate structure that, after 7 days, completely covers the titanium surface. Similar morphology was called flake-like by Leng et al. [13] and Xin et al. [14], and they have identified such phase as octacalcium phosphate (OCP). Qualitatively, good adhesion was obtained for both solutions.
Fig. 2. SEM of samples immersed in SBE solution for: (a) 1day; (b) 7 days; (c) 14 days; (d) 21 days.
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Fig. 3. SEM of samples immersed in SBF solution for: (a) 1 day; (b) 7 days; (c) 14 days; (d) 21 days.
The cross-section image of the sample coated for 7 days with SBE (Fig. 4a), obtained with ESEM, shows a low-dense structure consisting of a phase with plate-like morphology and a thickness of approximately 15 m. On the metallic surfaces, small crystals appear, but as the precipitated layer grows the
plates become larger and oriented. Line scans obtained by EDS can be observed in Fig. 4b, which shows titanium, calcium and phosphorus profile along vertical line of Fig. 4a. SEM images with EDS spectra do not allow full characterization of apatites rich in co-precipitation phases [2]; consequently
Fig. 4. FIB machined cross-section analysis of the sample exposed to SBE for 7 days: (a) ESEM image; (b) EDS line scan for titanium, calcium and phosphorus elements.
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other techniques should be used. Some cases of misinterpretations concerning hydroxyapatite and octacalcium phosphate have been reported [13,15]. In the present study, the reason for further investigation is mainly due to the crucial effect of octacalcium phosphate on the biological system. In this sense, octacalcium phosphate (OCP) is considered as an intermediate phase or, sometimes, precursor phase of hydroxyapatite due to its high similarity with crystals present in bone and teeth [2,15–17]. According to Liu et al. [16], OCP crystal assemblies seem to enhance osteocalcin expression, which is an important marker of osteoblast phenotype. This fact can be a consequence of the in vivo conversion of OCP to apatite by hydrolysis [17]. Other authors have pointed out that it is possible to have stable OCP crystals in physiological environments [15]. Consequently, a route allowing homogeneous coating of monophasic OCP has great importance for implant performance. FTIR of immersed samples into SBE and SBF for 7 and 21 days, respectively, are shown in Fig. 5. Both spectra exhibited phosphate bands (PO4 2− ) at 1146, 999, 670-610 and 582 cm−1 and peaks corresponding to the HPO4 2− vibration at 523 cm−1 . However, typical bands of OCP structure related to HPO4 2− at 1108 and 1070 cm−1 were observed only for coating obtained with SBE [18]. The 1447–1408 cm−1 bands were assigned to the C–O vibration bands characteristics of group carbonate (CO3 2− ). The presence of this molecular group can arise from chemical reagents or be related to the processing under air atmosphere. Bands at 1652 cm−1 , in both coatings, were identified as physically adsorbed water molecules. The grazing incidence X-ray diffraction patterns of Ti exposed to both solutions (SBE for 7 days and SBF for 21 days) are shown in Fig. 6. The low 2θ angle (4.7◦ ) peak corresponds to the (100) OCP peak and does not exist on XRD patterns of HA or other calcium phosphates. Consequently OCP is the main compound, although the presence of a small quantity of HA cannot
Fig. 5. FT-IR of samples immersed in SBE (for 7 days) and SBF (for 21 days).
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Fig. 6. Grazing incidence X-ray diffraction of the samples immersed in both solutions (SBE for 7 days and SBE for 21 days).
be excluded. The thin layer between Ti sheet and OCP crystals observed by SEM cross-section could be polycrystalline HA phase. Fig. 7a shows the TEM bright field image of an individual plate-like precipitate, and Fig. 7b and c shows, respectively, the energy dispersive spectrum and the electron diffraction pattern of precipitate from Fig. 7a. Besides copper (from grid), the plates were composed by only calcium and phosphorus elements. This diffraction pattern was assigned to B = [1 1 0] of the OCP structure and all particles (around ten) examined exhibited similar shape and electron diffraction pattern with the same zone axis. The similarity of OCP (Ca8 (HPO4 )2 (PO4 )4 .5H2 O, triclinic cell) and HA (Ca10 H2 (PO4 )6 .5H2 O, hexagonal cell) provides geometrically favorable conditions to in vivo conversion of OCP into HA [14,15]. However, it makes difficult the identification of phases [13,15]. The intensity of 26◦ peak in XRD pattern of sample exposed to the SBE designed solution (Fig. 6) is much higher than the sum of contributions from HA and OCP peaks. Another contribution may come from the (0 0 2) texture identified by electron diffraction and mentioned by Barr`ere et al. [5]. The differences of phases observed when using SBE or SBF could be related to differences in solution composition. According to Barr`ere et al. [5,7], one hypothesis is that Mg2+ is adsorbed on the metallic surface. Thus, magnesium ions would favor heterogeneous nucleation, affecting (or delaying) the CaP crystal growth process [7]. In fact, in the absence of magnesium and carbonate ions, OCP with high crystallinity grows epitaxially on titanium alloy surface [5]. In addition, the role of TRIS in these solutions has been poorly discussed. TRIS is known to form complexes with metallic ions, reducing the calcium free to precipitate on the metal surface [9]. As reported by Sigel et al. [19], the stability order for alkaline earth TRIS complexes is Mg2+ > Ca2+ >Sr2+ >Ba2+ . Accordingly, using SBF, TRIS would form a complex preferentially with magnesium, resulting in a Ca(free)/P ratio higher than 1.33. Thus, the solution composition may be adjusted to obtain the desired calcium phosphate, as can be seen in the theoretical and experimental results presented here.
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Fig. 7. Sample immersed in SBE for 7 days: (a) TEM bright field image of OCP precipitate; (b) EDS spectrum of OCP precipitate, showing calcium and phosphorus elements and copper (from grid); (c) electron diffraction pattern with B = [1 1 0].
4. Conclusions
Acknowledgements
The proposed solution (SBE) to coat metallic samples with octacalcium phosphate showed fast response. The FIB machined cross-section shows a low-dense plate-like morphology while EDS elemental mappings reveal a homogeneous composition of calcium and phosphorus along the deposit thickness. These results, together with those of grazing incidence X-ray diffraction and electron diffraction, indicate that the deposit is mainly composed of octacalcium phosphate (OCP). Therefore, contrary to the current trend, searching for fluids almost identical to the biological one should not necessarily be the best way to coat metallic surfaces, not only because of the difficulty to obtain monophasic calcium phosphate but also due to the exposure time necessary for adequate precipitation.
The authors acknowledge the financial support of CNPq (CT-Sa´ude 504.808/2004-4), CAPES and FAPERJ Brazilian agencies and also the CNPq/FNRS international cooperation program. We also acknowledge T.Segato from ULB.
References [1] T. Kokubo, H. Takadama, Biomaterials 27 (2006) 2907. [2] S.V. Dorozhkin, J. Mater. Sci. 42 (2007) 1061. [3] F. Li, Q.L. Feng, F.Z. Cui, H.D. Li, H. Schubert, Surf. Coat. Technol. 154 (2002) 88. [4] Q.L. Feng, H. Wang, F.Z. Cui, T.N. Kim, J. Cryst. Growth 200 (1999) 550.
C.X. Resende et al. / Materials Chemistry and Physics 109 (2008) 429–435 [5] F. Barr`ere, P. Layrolle, C.A. van Blitterswijk, K. de Groot, Bone 25 (1999) 107S. [6] T. Kokubo, H.M. Kim, M. Kawashita, T. Nakamura, Materials exhibit bonebonding? in: J.E. Davies (Ed.), Bone Engineering, EM2, Toronto, Canada, 2000, p. 190. [7] F. Barr`ere, C.A. van Blitterswijk, K. de Groot, P. Layrolle, Biomaterials 23 (2002) 2211. [8] E.C.S. Rigo, A.O. Boschi, M. Yoshimoto, S. Allegrini, B. Konig, M.J. Carbonari, Mater. Sci. Eng. C 24 (2004) 647. [9] I.N. Bastos, G.M. Platt, M.C. Andrade, G.D. Soares, J. Mol. Liq. 139 (2008) 121. [10] C.X. Resende, G.M. Platt, I.N. Bastos, G.D.A. Soares, Mat´eria. 12 (2007) 358.
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[11] J. Kapolos, P.G. Koutsoukos, Langmuir 15 (1999) 6557. [12] L. Jon´aov´a, F.A. M¨uller, A. Helebrant, J. Strnad, P. Greil, Biomaterials 25 (2004) 1187. [13] Y. Leng, J. Chen, S. Qu, Biomaterials 24 (2003) 2125. [14] R. Xin, Y. Leng, N. Wang, J. Cryst. Growth 289 (2006) 339. [15] X. Lu, Y. Leng, Biomaterials 25 (2004) 1779. [16] Y. Liu, P.R. Cooper, J.E. Barralet, R.M. Shelton, Biomaterials 28 (2007) 1393. [17] O. Suzuki, S. Kamakura, T. Katagiri, M. Nakamura, B. Zhao, Y. Honda, R. Kamijo, Biomaterials 27 (2006) 2671. [18] C.X. Resende, G.M. Platt, J. Dille, I.N. Bastos, G.D.A. Soares, Key Eng. Mater. 361–363 (2008) 665. [19] H. Sigel, K.H. Scheller, B. Prijs, Inorg. Chim. Acta 66 (1982) 147.
Materials Chemistry and Physics 109 (2008) 429–435
Characterization of coating produced on titanium surface by a designed solution containing calcium and phosphate ions C.X. Resende a , J. Dille b , G.M. Platt c , I.N. Bastos c , G.A. Soares a,∗ a
Metallurgical and Materials Engineering Department, Federal University of Rio de Janeiro, P.O. Box 68505, Rio de Janeiro, RJ 21941-972, Brazil b Universit´ e Libre de Bruxelles, Belgium c State University of Rio de Janeiro, IPRJ/UERJ 97282, Nova Friburgo, RJ 28630-050, Brazil
Received 8 May 2007; received in revised form 4 December 2007; accepted 8 December 2007
Abstract The biomimetic process of coating metals has been used to transform bioinert surfaces into bioactive ones. Simulated body fluid (SBF) has been classically used and has a composition similar to that of body fluid. The solution we used also has a composition closely similar to that of body fluid and is less complex than SBF. In this work, titanium surfaces were coated with a designed simplified solution whose chemical composition is based on the ions necessary to form calcium phosphates, e.g., calcium and phosphate ions in aqueous medium. This fluid was called solution for bioactivity evaluation (SBE). Besides the advantage of having a less complex composition, SBE presents fast deposition when compared with conventional SBF. We characterized the calcium phosphate layer by using grazing incidence X-ray diffraction, fourier-transformed infrared spectroscopy (FTIR), scanning electron microscopy and transmission electron microscopy. A uniform coating layer with 15 m thickness was obtained after 7 days of soaking in this simplified solution. This coating exhibited low-dense morphology and a single-phase octacalcium phosphate (OCP) structure. © 2007 Elsevier B.V. All rights reserved. Keywords: Biomaterials; Coatings; Microstructure; Electron microscopy
1. Introduction Calcium phosphate (CaP) coatings are commonly applied to metallic materials in order to combine suitable mechanical properties (from the metallic substrate) with the bioactive behavior of CaP materials. Among coating processes, the biomimetic has received particular attention especially due to its similarity with in vivo biomineralization, to its simplicity, low temperature and low cost when compared with the plasma spray process. A large number of papers discuss the use of solutions containing calcium and phosphate ions to coat metallic substrate under physiological pH or to test the bioactivity of metallic surfaces [1–3]. There is a consensus that pure titanium and titanium alloys should be activated before coating by exposing samples to surface treatments. The most common surface pre-treatment employs the exposure of Ti samples to an NaOH solution [4].
∗
Corresponding author. Tel.: +55 21 2562 8511/8500; fax: +55 21 290 6626. E-mail address: [email protected] (G.A. Soares).
0254-0584/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2007.12.011
After the pre-treatment the metal is immersed in a fluid capable of promoting apatite precipitation. Several acellular simulated body fluids (SBF) have been developed [1,3,5] for calcium phosphate precipitation. These solutions are applied to test biomaterials as some authors consider calcium phosphate precipitation on materials surface an indicative of good in vivo behavior [1,6]. In general, all investigators use the solution with a composition closely similar to that of body fluid. The disadvantage of using the biomimetic process is the relative long time necessary for coating, on the order of some weeks. To overcome this problem some authors [3,7,8] have increased solution concentration and/or included a nucleating agent such as sodium silicate [8] to accelerate the formation of a CaP layer. Indeed, one or 2 weeks are typically considered as the time necessary to obtain a uniform layer of calcium phosphate on titanium surface [8]. Another disadvantage is that non-homogeneity of coatings is likely to occur because CaP nucleation and growth processes are heterogeneous and highly dependent on surface properties. In this paper, we propose an alternative way to accelerate deposition. By using a “solution for bioactivity evaluation”
430
C.X. Resende et al. / Materials Chemistry and Physics 109 (2008) 429–435
(SBE), we can eliminate, as much as possible, the ions not fundamental to the precipitation reaction. It can also be applied to evaluate the bioactivity of biomaterials or coat metal surfaces. SBE has a composition containing mainly the elements necessary to form calcium phosphate and, consequently, offers several advantages over usual SBF; among these are simplicity and low cost. Besides its thermodynamic properties, such as high ion activity and more negative free Gibbs variation energy, it produces a higher nucleation driving force than that of SBF [9] under physiological conditions. These advantages make it possible to reduce the time involved in coating metallic titanium. Our work thus focuses on titanium surfaces coated with this new designed solution. Characterization was performed by using standard and some non-standard techniques, namely grazing incidence small angle X-ray diffraction and focused ion beam (FIB) machining of cross-section. 2. Experimental procedure Titanium samples of 8 mm × 8 mm were cut from a commercially pure titanium (Ti) sheet. Ti samples were cleaned and their surfaces were activated by using a double treatment that consisted of immersion in a NaOH 5 M solution at 60 ◦ C for 24 h, followed by a heat treatment at 600 ◦ C for 3 h. This double treatment produced a sodium titanate layer on titanium surface, and this layer seemed to be fundamental to induce calcium phosphate precipitation during the subsequent step [3]. SBE was designed by using a computational simulator named SimSE [10]. Solution was prepared with the following reagents: NaHCO3 (0.3525 g L−1 ), K2 HPO4 (0.2305 g L−1 ), CaCl2 (0.2890 g L−1 ) and tris-hydroxymethyl aminomethane (TRIS, 6.118 g L−1 ), which resulted in the following ionic concentrations (in mmol L−1 ): 2.5 Ca2+ , 5.0 Cl− , 4.2 Na+ , 4.2 HCO3 − , 2.0 K+ and 1.0 HPO4 − . The sequence of addiction followed the order showed above. The temperature of solution must be approximately 37 ◦ C during all preparation and ultimate pH should be around 7.4, which was adjusted by addition of 1.0 M HCl solution. Ti samples were exposed to 16 mL of SBE solution at 37 ◦ C for 1, 7, 14, 21 and 28 days. The precipitation process that produced the substrate coating was monitored by the chemical analysis of calcium and phosphate content in solution. All experiments were performed in quintuplicate, by running five parallel independent tubes. After each exposition time, calcium content was determined by using atomic absorption spectrometry (AAS), while phosphate concentration
was determined by using phosphomolybdate/UV spectroscopy. For comparison, titanium samples were also exposed to conventional SBF [1]. For these experiments, the sample area/solution volume ratio, temperature and exposition time were kept unchanged for both solutions. Coating morphology was evaluated by scanning electron microscopy (SEM) using a JEOL JSM-6460-LV microscope operating at 20 kV. To evaluate the possibility of deposition on the walls of the tube, a blank test was carried out using both solutions. This test, performed without the metallic sample, revealed a rather small amount of precipitation. The coating of titanium sample exposed to SBE for 7 days was further analyzed by environmental scanning electron microscopy (ESEM), fouriertransformed infrared spectroscopy (FT-IR), grazing incidence X-ray diffraction (XRD) and transmission electron microscopy (TEM). A FEI Quanta 200 3D dual beam was employed for ESEM examination of the cross-section machined in situ by focused ion beam. Energy dispersive spectrometry (EDS) was also performed to examine the distribution of calcium and phosphorus along the deposit thickness. A FT-IR spectrometer (ABB Bomem Inc.) was used to identify the vibration modes of several species. The spectra were collected at room temperature at a nominal resolution of 4.00 cm−1 with number of scans equal to 100. XRD patterns were obtained with a Siemens D5000 diffractometer used in grazing incidence X-ray diffraction conditions. For TEM analysis, the coating layer was partially removed by immersing the sample in an ethanol bath with ultrasonic vibration. Few drops of this solution were deposited on a copper grid coated with an amorphous carbon film. The analysis of precipitates was conducted in a JEOL 2000FX electron microscope, operating at 200 kV.
3. Results and discussion Fig. 1 shows calcium and phosphorus contents in solution as a function of exposure time for experiments using SBE and SBF. The decrease in these ion contents with time is related to precipitation on the materials surface. Both solutions are supersaturated in relation to their equilibrium concentrations for hydroxyapatite and OCP at pH 7.4, as shown in theoretical simulations [10]. Moreover, the experimental results presented in Fig. 1 indicate faster kinetics for SBE than for SBF solution. Indeed, for 7 days, the calcium measured in aliquots of SBE (Fig. 1a) is approximately 20 ppm below the correspondent value of SBF. A similar result is observed for phosphorus (Fig. 1b). The reasons for the fast growth rate observed with SBE could be related to differ-
Fig. 1. (a) Calcium content in solutions for different exposure times and (b) phosphorus content in solutions for different exposure times.
C.X. Resende et al. / Materials Chemistry and Physics 109 (2008) 429–435
ences in the kinetic process due to the more negative Gibbs free energy, which acts as a driving force for precipitation, and to the absence of magnesium ions in SBE solution. According to Kapolos and Koutsoukos [11], the growth rate of phosphate crystal (Rg ) is related to relative supersaturation (σ) by Rg = kσ n
(1)
where k is the precipitation rate constant and n indicates the apparent growth reaction order. Relative supersaturation can be evaluated by Gibbs free energy variation (between supersaturated and saturated solutions): σ = Ω1/ i νi − 1 (2) νi ai Ω= (3) KSP RT G = − ln(Ω) i νi
(4)
In the previous equations, ai refers to the activity of chemical species i, νi is the stoichiometric coefficient in the precipitation reaction, Ω is the supersaturation ratio and KSP is the thermodynamic solubility product. SimSE program was used to evaluate all activities and thus G [9]. The G and Ω values act as driving force for CaP precipitation. At pH 7.4, the simulated results for SBE are G and super-saturation index
431
with values of −9343.6 J mol−1 and 42.4 for OCP, respectively; and −18343.5 J mol−1 , and 1641.3 respectively for HA. Some authors [5,7] have reported the effect of magnesium in retarding or inhibiting the crystallization of several calcium phosphate phases, so its absence in SBE can also have a contribution to the fast decrease of Ca and P contents seen in Fig. 1. Some SEM images of Ti exposed to SBE and SBF are shown in Figs. 2 and 3, respectively. In the micrograph with 500× magnification, some small rounded uncoated areas can be observed (Fig. 2a) for 1 day exposure sample to SBE. After 7 days, a homogeneous layer already coats the whole surface (Fig. 2b), in good agreement with data shown in Fig. 1. For SBF, the surface is coated only after a 14 day-exposure (Fig. 3c). Differences in coating morphology are also observed. SBF coating exhibits a dense morphology with small crystals, resulting in some cracks during the drying process. This morphology and the cracked appearance are largely reported in several papers [6,7,12] and are associated with poorly crystallized carbonated apatite or calcium-deficient apatite. The designed solution (SBE) produces a porous plate-like calcium phosphate structure that, after 7 days, completely covers the titanium surface. Similar morphology was called flake-like by Leng et al. [13] and Xin et al. [14], and they have identified such phase as octacalcium phosphate (OCP). Qualitatively, good adhesion was obtained for both solutions.
Fig. 2. SEM of samples immersed in SBE solution for: (a) 1day; (b) 7 days; (c) 14 days; (d) 21 days.
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Fig. 3. SEM of samples immersed in SBF solution for: (a) 1 day; (b) 7 days; (c) 14 days; (d) 21 days.
The cross-section image of the sample coated for 7 days with SBE (Fig. 4a), obtained with ESEM, shows a low-dense structure consisting of a phase with plate-like morphology and a thickness of approximately 15 m. On the metallic surfaces, small crystals appear, but as the precipitated layer grows the
plates become larger and oriented. Line scans obtained by EDS can be observed in Fig. 4b, which shows titanium, calcium and phosphorus profile along vertical line of Fig. 4a. SEM images with EDS spectra do not allow full characterization of apatites rich in co-precipitation phases [2]; consequently
Fig. 4. FIB machined cross-section analysis of the sample exposed to SBE for 7 days: (a) ESEM image; (b) EDS line scan for titanium, calcium and phosphorus elements.
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other techniques should be used. Some cases of misinterpretations concerning hydroxyapatite and octacalcium phosphate have been reported [13,15]. In the present study, the reason for further investigation is mainly due to the crucial effect of octacalcium phosphate on the biological system. In this sense, octacalcium phosphate (OCP) is considered as an intermediate phase or, sometimes, precursor phase of hydroxyapatite due to its high similarity with crystals present in bone and teeth [2,15–17]. According to Liu et al. [16], OCP crystal assemblies seem to enhance osteocalcin expression, which is an important marker of osteoblast phenotype. This fact can be a consequence of the in vivo conversion of OCP to apatite by hydrolysis [17]. Other authors have pointed out that it is possible to have stable OCP crystals in physiological environments [15]. Consequently, a route allowing homogeneous coating of monophasic OCP has great importance for implant performance. FTIR of immersed samples into SBE and SBF for 7 and 21 days, respectively, are shown in Fig. 5. Both spectra exhibited phosphate bands (PO4 2− ) at 1146, 999, 670-610 and 582 cm−1 and peaks corresponding to the HPO4 2− vibration at 523 cm−1 . However, typical bands of OCP structure related to HPO4 2− at 1108 and 1070 cm−1 were observed only for coating obtained with SBE [18]. The 1447–1408 cm−1 bands were assigned to the C–O vibration bands characteristics of group carbonate (CO3 2− ). The presence of this molecular group can arise from chemical reagents or be related to the processing under air atmosphere. Bands at 1652 cm−1 , in both coatings, were identified as physically adsorbed water molecules. The grazing incidence X-ray diffraction patterns of Ti exposed to both solutions (SBE for 7 days and SBF for 21 days) are shown in Fig. 6. The low 2θ angle (4.7◦ ) peak corresponds to the (100) OCP peak and does not exist on XRD patterns of HA or other calcium phosphates. Consequently OCP is the main compound, although the presence of a small quantity of HA cannot
Fig. 5. FT-IR of samples immersed in SBE (for 7 days) and SBF (for 21 days).
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Fig. 6. Grazing incidence X-ray diffraction of the samples immersed in both solutions (SBE for 7 days and SBE for 21 days).
be excluded. The thin layer between Ti sheet and OCP crystals observed by SEM cross-section could be polycrystalline HA phase. Fig. 7a shows the TEM bright field image of an individual plate-like precipitate, and Fig. 7b and c shows, respectively, the energy dispersive spectrum and the electron diffraction pattern of precipitate from Fig. 7a. Besides copper (from grid), the plates were composed by only calcium and phosphorus elements. This diffraction pattern was assigned to B = [1 1 0] of the OCP structure and all particles (around ten) examined exhibited similar shape and electron diffraction pattern with the same zone axis. The similarity of OCP (Ca8 (HPO4 )2 (PO4 )4 .5H2 O, triclinic cell) and HA (Ca10 H2 (PO4 )6 .5H2 O, hexagonal cell) provides geometrically favorable conditions to in vivo conversion of OCP into HA [14,15]. However, it makes difficult the identification of phases [13,15]. The intensity of 26◦ peak in XRD pattern of sample exposed to the SBE designed solution (Fig. 6) is much higher than the sum of contributions from HA and OCP peaks. Another contribution may come from the (0 0 2) texture identified by electron diffraction and mentioned by Barr`ere et al. [5]. The differences of phases observed when using SBE or SBF could be related to differences in solution composition. According to Barr`ere et al. [5,7], one hypothesis is that Mg2+ is adsorbed on the metallic surface. Thus, magnesium ions would favor heterogeneous nucleation, affecting (or delaying) the CaP crystal growth process [7]. In fact, in the absence of magnesium and carbonate ions, OCP with high crystallinity grows epitaxially on titanium alloy surface [5]. In addition, the role of TRIS in these solutions has been poorly discussed. TRIS is known to form complexes with metallic ions, reducing the calcium free to precipitate on the metal surface [9]. As reported by Sigel et al. [19], the stability order for alkaline earth TRIS complexes is Mg2+ > Ca2+ >Sr2+ >Ba2+ . Accordingly, using SBF, TRIS would form a complex preferentially with magnesium, resulting in a Ca(free)/P ratio higher than 1.33. Thus, the solution composition may be adjusted to obtain the desired calcium phosphate, as can be seen in the theoretical and experimental results presented here.
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Fig. 7. Sample immersed in SBE for 7 days: (a) TEM bright field image of OCP precipitate; (b) EDS spectrum of OCP precipitate, showing calcium and phosphorus elements and copper (from grid); (c) electron diffraction pattern with B = [1 1 0].
4. Conclusions
Acknowledgements
The proposed solution (SBE) to coat metallic samples with octacalcium phosphate showed fast response. The FIB machined cross-section shows a low-dense plate-like morphology while EDS elemental mappings reveal a homogeneous composition of calcium and phosphorus along the deposit thickness. These results, together with those of grazing incidence X-ray diffraction and electron diffraction, indicate that the deposit is mainly composed of octacalcium phosphate (OCP). Therefore, contrary to the current trend, searching for fluids almost identical to the biological one should not necessarily be the best way to coat metallic surfaces, not only because of the difficulty to obtain monophasic calcium phosphate but also due to the exposure time necessary for adequate precipitation.
The authors acknowledge the financial support of CNPq (CT-Sa´ude 504.808/2004-4), CAPES and FAPERJ Brazilian agencies and also the CNPq/FNRS international cooperation program. We also acknowledge T.Segato from ULB.
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