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Biomimetic apatite coatings—Carbonate substitution and preferred growth orientation
Biomolecular Engineering 24 (2007) 462–466 www.elsevier.com/locate/geneanabioeng
Biomimetic apatite coatings—Carbonate substitution and preferred growth orientation Lenka Mu¨ller a, Egle Conforto b,1, Daniel Caillard c, Frank A. Mu¨ller a,* a
University of Erlangen-Nuernberg, Department of Materials Science – Biomaterials, Henkestr. 91, D-91052 Erlangen, Germany b Swiss Federal Institute of Technology, Lausanne (EPFL), CH-1015 Lausanne, Switzerland c CEMES-CNRS, BP 4347, F-31055 Toulouse Cedex, France
Abstract Biomimetic apatite coatings were obtained by soaking chemically treated titanium in SBF with different HCO3 concentration. XRD, FTIR and Raman analyses were used to characterize phase composition and degree of carbonate substitution. The microstructure, elemental composition and preferred alignment of biomimetically precipitated crystallites were characterized by cross-sectional TEM analyses. According to XRD, the phase composition of precipitated coatings on chemically pre-treated titanium after exposure to SBF was identified as hydroxy carbonated apatite (HCA). A preferred c-axis orientation of the deposited crystals can be supposed due to the high relative peak intensities of the (0 0 2) diffraction line at 2u = 268 compared to the 100% intensity peak of the (2 1 1) plane at 2u = 328. The crystallite size in direction of the c-axis of HCA decreased from 26 nm in SBF5 with a HCO3 concentration of 5 mmol/l to 19 nm in SBF27 with a HCO3 concentration of 27 mmol/l. Cross-sectional TEM analyses revealed that all distances correspond exactly to the hexagonal structure of hydroxyapatite. The HCO3 content in SBF also influences the composition of precipitated calcium phosphates. Biomimetic apatites were shown to have a general formula of Ca10xyMgy(HPO4)xz (CO3)z(PO4)6x(OH)2xw(CO3)w/2. According to FTIR and Raman analyses, it can be supposed that as long as the HCO3 concentration in the testing solutions is below 20 mmol/l, only B-type HCA (0 < z < 1; w ¼ 0) precipitates. At higher HCO3 concentration, it can be assumed that AB-type HCA (z = 1;0 < w < 1) is formed. # 2007 Elsevier B.V. All rights reserved. Keywords: Apatite; Biomimetic; SBF
1. Introduction Since biomaterials interact with the body through their surfaces after implantation, the properties of the outer surface of a material are critical features in controlling the biological response between the biological and the artificial system (Williams, 1987). Implants in direct contact to bone should bond to the osseous environment without the formation of an interface layer consisting of fibrous tissue. Several surface modifications of bioinert materials (e.g. titanium and its alloys) including physico-chemical (Ratner and Hoffmann, 2004), morphologic (Jansen and von Reum, 2004) and biochemical ones (Hoffman and Hubbell, 2004) were investigated to
* Corresponding author. Tel.: +49 9131 8525543; fax: +49 9131 8525545. E-mail address: [email protected] (F.A. Mu¨ller). 1 Present address: Universite´ de La Rochelle, Centre Commun d’Analyses, F17071 La Rochelle Cedex 9, France. 1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2007.07.011
overcome these problems of integration by creating osteoconductive as well as osteoinductive surfaces. Calcium phosphate coatings are of particular interest, since the bone is composed of collagen, hydroxy carbonated apatite (HCA) and water. Electrolyte solutions, referred to as simulated body fluids (SBF), reproduce the inorganic part of human blood plasma. Therefore, it can be assumed that the structure of coatings precipitated from SBF will be close to that one of biological apatites present in human bone. The interest in applying SBF for preparation of biomimetic apatite coatings extremely increased within the last 10 years (Barrere et al., 2002; Rambo et al., 2006; Mu¨ller et al., 2006). SBF solutions mentioned in literature differ mostly in the concentrations of HCO3 and Cl compared to human blood plasma. The aim of the present study was to analyse the influence of the HCO3 content on the apatite structure and composition using Fourier transform infrared (FTIR) and Raman spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and crosssectional transmission electron microscopy (TEM) analyses.
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2. Experimental 2.1. Experimental procedure and sample preparation Bioactive titanium samples were prepared according to a procedure reported earlier (Jonasova et al., 2004). Briefly, the titanium surfaces were etched in 37% HCl at 50 8C followed by an activation in 10 mol/l NaOH at 60 8C. Biomimetic apatite coatings were deposited on the surface of chemically pre-treated titanium samples by soaking in SBF solutions under static conditions at 37 0.4 8C for 14 days. Five SBF solutions labelled as SBF5, SBF10, SBF15, SBF20 and SBF27 with a HCO3 content ranging from 5 to 27 mmol/l and a constant (Cl + HCO3) content of 136 mmol/l were prepared by pipetting calculated amounts of concentrated stock solutions of KCl, NaCl, NaHCO3, MgSO47H2O, CaCl2, TRIS (tris-hydroxymethyl aminomethane), NaN3 and KH2PO4 into double-distilled water (Mu¨ller and Mu¨ller, 2006). The theoretical total concentration of ions in each SBF solutions is shown in Table 1. The pH of SBF was adjusted between 7.3 and 7.4 at 37 8C. The samples were freely suspended in a volume of model solution roughly corresponding to a sample surface to volume ratio S/V = 0.05 cm1. After exposure, the samples were washed in double distilled water.
2.2. Sample surface characterization Raman spectra were measured in the range from 4000 to 400 cm1 using a dispersive Raman spectrometer (Nicolet Almega Thermo, USA) equipped with a 780 nm laser. Fourier transform infrared (FTIR) spectra were collected in transmission using the KBr technique in the range from 4000 to 400 cm1 at a resolution of 4 cm1 (Impact 420, Nicolet Instruments, USA). The crystalline phases were determined by X-ray diffraction (XRD) analysis using Cu Ka radiation at a scan rate of 18 (min)1 over a 2u range of 5–708 (D 500, Siemens, Germany). The microstructure and elemental composition of the ceramic coatings were characterized by scanning electron microscopy (SEM; Philips XL-30 FEG) and transmission electron microscopy (TEM; Philips EM-430 and JEOL 2010). For cross-sectional TEM analysis, two parts, that were cut perpendicular to the surface, were joined with epoxy glue, surface against surface. Subsequently, slices were cut, mechanically polished and thinned to perforation by ion milling at a low angle in order to obtain the largest possible thin area, from the substrate up to the surface. Selected-area electron diffraction (SAED) patterns were obtained from cross-sectional specimens.
Fig. 1. Representative SEM micrograph of biomimetic coatings precipitated on chemically pre-treated titanium after exposure to SBF10 for 14 days (inset: corresponding EDX analysis).
and phosphorus. The assumed substitution of Ca2+ ions by Mg2+ and Na+ is consistent with the composition of biological apatites in human bone, which are non-stoichiometric with trace components, such as Mg2+, Na+, CO32, HPO42, F or Cl (Tadic et al., 2002). The phase composition of the coatings on chemically pretreated titanium after exposure to different SBF solutions was identified as hydroxyapatite of low crystallinity by XRD (Fig. 2). A preferred c-axis orientation of the deposited crystals can be supposed due to the high relative peak intensities of the (0 0 2) and (0 0 4) diffraction lines at 2u = 268 and 538, respectively, compared to the 100% intensity peak of the (2 1 1) plane at 2u = 328. The broad peak at 328 consists of the reflections from (2 1 1) at 31.878, (1 1 2) at 32.188 and (3 0 0) at 32.878. The crystallite size in the c-direction of the HCA crystals were calculated from the (0 0 2) peak broadening at 268 in the XRD-diagrams using the Scherrer equation (Mu¨ller and Mu¨ller, 2006) and decreased from 26 nm in SBF5 to 19 nm in
3. Results Fig. 1 shows a representative micrograph (SEM) accompanied by the EDX spectrum of the biomimetic coating precipitated on the samples surface within 2 weeks soaking in SBF10. The layer is composed of spherical aggregates with a diameter up to 20 mm, which are characteristic for coatings precipitated from SBF (Jonasova et al., 2004; Kokubo and Takadama, 2006). A small fraction of magnesium and sodium were found by EDX analysis (inset in Fig. 1) among calcium Table 1 Concentration of ions in SBF (mmol/l)
+
Na K+ Ca2+ Mg2+ Cl HCO3 SO42 HPO42
SBF5
SBF10
SBF15
SBF20
SBF27
142.0 5.0 2.5 1.0 131.0 5.0 1.0 1.0
142.0 5.0 2.5 1.0 126.0 10.0 1.0 1.0
142.0 5.0 2.5 1.0 121.0 15.0 1.0 1.0
142.0 5.0 2.5 1.0 116.0 20.0 1.0 1.0
142.0 5.0 2.5 1.0 109.0 27.0 1.0 1.0
Fig. 2. X-ray diffraction analyses of biomimetic coatings precipitated on chemically treated titanium after exposure to SBF5, 10, 15, 20 and 27 for 14 days.
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Fig. 3. Biomimetic growth of carbonated apatite on chemically pre-treated titanium: (a) cross-sectional TEM view from the titanium hydride (left) to the HCA layer (right) and (b) corresponding SAED pattern taken from the top of the hydroxyapatite layer.
SBF27. Ti and TiH2 (resulting from the acid etching in HCl) peaks were observed in all XRD patterns. The cross-sectional TEM view in Fig. 3a shows the transformations during all treatment steps. Sodium in the hydrated sodium titanate gel formed after treatment in NaOH is replaced by calcium during soaking in SBF (Jonasova et al., 2004; Takadama et al., 2001). A biomimetic apatite coating grew on the gel layer by attachment of Ca2+ and PO43 ions from SBF. The corresponding SAED pattern (Fig. 3b), taken from the area at the top of Fig. 3a, exhibits a large number of rings typical of a nanocrystalline structure. All distances correspond exactly to the hexagonal structure of HAwith the parameters a = 0.944 nm and c = 0.688 nm according to JCPDS 9-432. The Raman spectra of a sample exposed to SBF5 for 14 days is shown in Fig. 4. The band at 3567 cm1 in the spectrum is assigned to the OH– group in hydroxyapatite. The vibrations located around 1040 and 950 cm1 were assigned to the PO43 group. Similar spectra were recorded for Ca–P layers precipitated in SBF10, SBF15 and SBF20. In SBF27 (Fig. 4), the peak at 3567 cm1 disappeared, probably due to (CO3)2
substitution for OH (A-type), and the shoulder at 880 cm1 indicates HPO42 incorporation into the structure. At the same time, the intensity ratio of the bands at 1050 and 950 cm1 decreases with increasing carbonate content in the solution as a consequence of (CO3)2 increasing substitution for (PO4)3 (B-type) (Krajewski et al., 2005; Wilson et al. 2005). The chemical composition of the precipitated layers can be evaluated more specifically using Fourier transform infrared (FTIR) spectroscopy (Fig. 5). Vibrations located in the range from 1200 to 960 cm1 and at 604, 567 and 474 cm1 are associated with the (PO4)3 group. The bands detected at 1460, 1420 and 875 cm1 were assigned to the (CO3)2 group of Btype carbonated apatite. However, characteristic peaks at 875 and 959 cm1 indicate as well the presence of (HPO4)2 in the crystal lattice (Koutsopoulos, 2002). The absence of the OH vibration at 3570 cm1 suggests that carbonate also substitutes for OH (A-type). However, there is no evidence that (CO3)2 substitutes for OH as the characteristic absorption band at 1545 cm1, associated with A-type substitution, was not observed. It is known that Ca-deficient HA shows weaker stretching and librational bands compared to stoichiometric apatite (Wilson et al., 2005).
Fig. 4. Raman spectra of biomimetic coatings precipitated on chemically pretreated titanium after exposure to SBF5, 15 and 27 for 14 days.
Fig. 5. FTIR analyses of biomimetic coatings precipitated on chemically pretreated titanium after exposure to SBF5, 10, 15, 20 and 27 for 14 days.
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Fig. 6. (a) Cross-sectional TEM view of the precipitated apatite layer containing and (b) corresponding auto-correlation calculation (diagram of auto-correlation intensity (middle) and diagram of corresponding longitudinal and transversal intensities (left and right)).
4. Discussion Since the composition of SBF is similar to that of the inorganic part of human blood plasma, it can be assumed that the structure of the crystalline phase precipitated on the surface of bioactive materials would be close to biological apatite present in human bone. The FTIR, Raman and EDS spectra revealed that the layers are composed of Ca-deficient HA with (CO3)2 and Mg2+ substitution. A general formula of Ca10xyMgy(HPO4)xz (CO3)z(PO4)6x(OH)2x, with 0 x, y, z 1, can be assumed. For the maximum carbonate substitution (z = 1), the theoretical carbonate content is equal to 6.4 wt.%. The ratio of the absorption bands at 1420 cm1 (A(CO32)) and 604 cm1 (A(PO43) can be used to calculate the amount of (PO4)3 ions substituted by (CO3)2 ions in the precipitated apatites (LeGeros, 1991). The carbonate content in apatite layers calculated from A(CO32)/A(PO43) was 2.5, 3.2, 5.7 and 6.2 wt.% for SBF5,10,15 and 20. Using FTIR and Raman analysis, it was shown that with increasing amount of (HCO3) in the solution, the substitution of (CO3)2 for OH occurred and thus the formula can be written as: Ca10xyMgy(HPO4)xz(CO3)z (PO4)6x(OH)2xw(CO3)w/2. The theoretical maximum amount of carbonate substitution in this AB-type HCA for w ¼ 1 is equal to 10 wt.%. In our case, the maximum carbonate content calculated from A(CO32)/A(PO43) amounts to 9.1 wt.% in the layer precipitated from SBF27. Hence, it can be assumed that the A-sites are occupied after the B-sites are completely substituted. An interesting comparison can be drawn with bone mineral that contains carbonated apatite with a (CO3)2 substitution of up to 8 wt.% (Krajewski et al., 2005). The peaks in all X-ray diffraction patterns (Fig. 2) are not sharp and not well resolved. However, the higher relative peak intensity of the (0 0 2) and (0 0 4) diffraction lines at 2u = 268 and 538 can be attributed to hexagonal HA crystals with a
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preferred c-axis orientation of the deposited crystals. The preferential crystallite alignment can also be observed on the high-magnification image of Fig. 6a. This image was analysed by an auto-correlation method, with the results shown in Fig. 6b. To realize this calculation, Fig. 6a was superposed with its copy translated by vectors of various orientations and lengths. The ‘‘intensity’’ of each correlation, namely the reciprocal of the difference between the two images, was then plotted as a function of the corresponding translation vector (the origin being at the center of the diagram). This yields a map where the brightest regions correspond to the highest degree of correlation. As expected, the highest auto-correlation intensity is obtained for small translation vectors, namely near the diagram centre. However, the highest auto-correlation intensity is also expected for displacement vectors parallel to the average growth direction. Therefore, the elongation of the bright zone in the vertical direction perpendicular to the surface indicates that the crystallites are elongated preferentially in this direction (Fig. 6b). 5. Conclusions According to X-ray and TEM-SAED, biomimetically derived HCA coatings show a preferred growth orientation in the direction of their c-axis and perpendicular to the surface of chemically treated titanium. The FTIR, Raman and X-ray analyses of the precipitated layers revealed that the (HCO3) content in SBF influences the composition and structure of the resulting calcium phosphates. It can be supposed that as long as the (HCO3) concentration in the testing solution is below 20 mmol/l, only B-type HCA precipitates. At higher (HCO3) concentration, it can be assumed that as well A-type HCA forms. References Barrere, F., van Blitterswijk, C.A., de Groot, K., Layrolle, P., 2002. Influence of ionic strength and carbonate on the Ca–P coating formation from SBF 5 solution. Biomaterials 23, 1921–1930. Hoffman, A.S., Hubbell, J.A., 2004. Surface-immobilized biomolecules. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., Lemons, J.E. (Eds.), Biomaterials Science: An Introduction to Materials in Medicine. second ed. Elsevier, Amsterdam, pp. 225–233. Jansen, J.A., von Reum, A.F., 2004. Textured and porous materials. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., Lemons, J.E. (Eds.), Biomaterials Science: An Introduction to Materials in Medicine. second ed. Elsevier, Amsterdam, pp. 218–225. Jonasova, L., Mu¨ller, F.A., Helebrant, A., Strnad, J., Greil, P., 2004. Biomimetic apatite formation on chemically treated titanium. Biomaterials 25, 1187– 1194. Kokubo, T., Takadama, H., 2006. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27, 2907–2915. Koutsopoulos, S., 2002. Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods. J. Biomed. Mater. Res. 62, 600–612. Krajewski, A., Mazzocchi, M., Buldini, P.L., Ravagliolo, A., Tinti, A., Taddei, P., Fagnano, C., 2005. Synthesis of carbonated hydroxyapatites: efficiency of the substitution and critical evaluation of analytical methods. J. Mol. Struct. 744–747, 221–228. LeGeros, R.Z., 1991. Calcium Phosphates in Oral Biology and Medicine. Karger, Basel.
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Mu¨ller, L., Mu¨ller, F., 2006. Preparation of SBF with different HCO3 content and its influence on the composition of biomimetic apatites. Acta Biomater. 2, 181–189. Mu¨ller, F.A., Mu¨ller, L., Hofmann, I., Greil, P., Wenzel, M.M., Staudenmaier, R., 2006. Cellulose-based scaffold materials for cartilage tissue engineering. Biomaterials 27, 3955–3963. Rambo, C.R., Mu¨ller, F.A., Mu¨ller, L., Sieber, H., Hofmann, I., Greil, P., 2006. Biomimetic apatite coating on biomorphous alumina scaffolds. Mater. Sci. Eng. C26, 92–99. Ratner, B.D., Hoffmann, A.S., 2004. Physicochemical surface modification of materials used in medicine. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., Lemons, J.E. (Eds.), Biomaterials Science: An Introduction
to Materials in Medicine. second ed. Elsevier, Amsterdam, pp. 201– 218. Tadic, D., Peters, F., Epple, M., 2002. Continuous synthesis of amorphous carbonated apatites. Biomaterials 23, 2553–2559. Takadama, H., Kim, H.M., Kokubo, T., Nakamura, T., 2001. An X-ray photoelectron spectroscopy study of the process of apatite formation on bioactive titanium metal. J. Biomed. Mater. Res. 55, 185–193. Williams, D.F., 1987. Tissue-biomaterials interactions. J. Mater. Sci. 22, 3421– 3445. Wilson, R.M., Elliot, J.C., Dowker, S.E.P., Rodriguez-Lorenzo, L.M., 2005. Rietveld refinements and spectroscopic studies of the structure of Cadeficient apatite. Biomaterials 26, 1317–1327.
Biomimetic apatite coatings—Carbonate substitution and preferred growth orientation Lenka Mu¨ller a, Egle Conforto b,1, Daniel Caillard c, Frank A. Mu¨ller a,* a
University of Erlangen-Nuernberg, Department of Materials Science – Biomaterials, Henkestr. 91, D-91052 Erlangen, Germany b Swiss Federal Institute of Technology, Lausanne (EPFL), CH-1015 Lausanne, Switzerland c CEMES-CNRS, BP 4347, F-31055 Toulouse Cedex, France
Abstract Biomimetic apatite coatings were obtained by soaking chemically treated titanium in SBF with different HCO3 concentration. XRD, FTIR and Raman analyses were used to characterize phase composition and degree of carbonate substitution. The microstructure, elemental composition and preferred alignment of biomimetically precipitated crystallites were characterized by cross-sectional TEM analyses. According to XRD, the phase composition of precipitated coatings on chemically pre-treated titanium after exposure to SBF was identified as hydroxy carbonated apatite (HCA). A preferred c-axis orientation of the deposited crystals can be supposed due to the high relative peak intensities of the (0 0 2) diffraction line at 2u = 268 compared to the 100% intensity peak of the (2 1 1) plane at 2u = 328. The crystallite size in direction of the c-axis of HCA decreased from 26 nm in SBF5 with a HCO3 concentration of 5 mmol/l to 19 nm in SBF27 with a HCO3 concentration of 27 mmol/l. Cross-sectional TEM analyses revealed that all distances correspond exactly to the hexagonal structure of hydroxyapatite. The HCO3 content in SBF also influences the composition of precipitated calcium phosphates. Biomimetic apatites were shown to have a general formula of Ca10xyMgy(HPO4)xz (CO3)z(PO4)6x(OH)2xw(CO3)w/2. According to FTIR and Raman analyses, it can be supposed that as long as the HCO3 concentration in the testing solutions is below 20 mmol/l, only B-type HCA (0 < z < 1; w ¼ 0) precipitates. At higher HCO3 concentration, it can be assumed that AB-type HCA (z = 1;0 < w < 1) is formed. # 2007 Elsevier B.V. All rights reserved. Keywords: Apatite; Biomimetic; SBF
1. Introduction Since biomaterials interact with the body through their surfaces after implantation, the properties of the outer surface of a material are critical features in controlling the biological response between the biological and the artificial system (Williams, 1987). Implants in direct contact to bone should bond to the osseous environment without the formation of an interface layer consisting of fibrous tissue. Several surface modifications of bioinert materials (e.g. titanium and its alloys) including physico-chemical (Ratner and Hoffmann, 2004), morphologic (Jansen and von Reum, 2004) and biochemical ones (Hoffman and Hubbell, 2004) were investigated to
* Corresponding author. Tel.: +49 9131 8525543; fax: +49 9131 8525545. E-mail address: [email protected] (F.A. Mu¨ller). 1 Present address: Universite´ de La Rochelle, Centre Commun d’Analyses, F17071 La Rochelle Cedex 9, France. 1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2007.07.011
overcome these problems of integration by creating osteoconductive as well as osteoinductive surfaces. Calcium phosphate coatings are of particular interest, since the bone is composed of collagen, hydroxy carbonated apatite (HCA) and water. Electrolyte solutions, referred to as simulated body fluids (SBF), reproduce the inorganic part of human blood plasma. Therefore, it can be assumed that the structure of coatings precipitated from SBF will be close to that one of biological apatites present in human bone. The interest in applying SBF for preparation of biomimetic apatite coatings extremely increased within the last 10 years (Barrere et al., 2002; Rambo et al., 2006; Mu¨ller et al., 2006). SBF solutions mentioned in literature differ mostly in the concentrations of HCO3 and Cl compared to human blood plasma. The aim of the present study was to analyse the influence of the HCO3 content on the apatite structure and composition using Fourier transform infrared (FTIR) and Raman spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and crosssectional transmission electron microscopy (TEM) analyses.
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2. Experimental 2.1. Experimental procedure and sample preparation Bioactive titanium samples were prepared according to a procedure reported earlier (Jonasova et al., 2004). Briefly, the titanium surfaces were etched in 37% HCl at 50 8C followed by an activation in 10 mol/l NaOH at 60 8C. Biomimetic apatite coatings were deposited on the surface of chemically pre-treated titanium samples by soaking in SBF solutions under static conditions at 37 0.4 8C for 14 days. Five SBF solutions labelled as SBF5, SBF10, SBF15, SBF20 and SBF27 with a HCO3 content ranging from 5 to 27 mmol/l and a constant (Cl + HCO3) content of 136 mmol/l were prepared by pipetting calculated amounts of concentrated stock solutions of KCl, NaCl, NaHCO3, MgSO47H2O, CaCl2, TRIS (tris-hydroxymethyl aminomethane), NaN3 and KH2PO4 into double-distilled water (Mu¨ller and Mu¨ller, 2006). The theoretical total concentration of ions in each SBF solutions is shown in Table 1. The pH of SBF was adjusted between 7.3 and 7.4 at 37 8C. The samples were freely suspended in a volume of model solution roughly corresponding to a sample surface to volume ratio S/V = 0.05 cm1. After exposure, the samples were washed in double distilled water.
2.2. Sample surface characterization Raman spectra were measured in the range from 4000 to 400 cm1 using a dispersive Raman spectrometer (Nicolet Almega Thermo, USA) equipped with a 780 nm laser. Fourier transform infrared (FTIR) spectra were collected in transmission using the KBr technique in the range from 4000 to 400 cm1 at a resolution of 4 cm1 (Impact 420, Nicolet Instruments, USA). The crystalline phases were determined by X-ray diffraction (XRD) analysis using Cu Ka radiation at a scan rate of 18 (min)1 over a 2u range of 5–708 (D 500, Siemens, Germany). The microstructure and elemental composition of the ceramic coatings were characterized by scanning electron microscopy (SEM; Philips XL-30 FEG) and transmission electron microscopy (TEM; Philips EM-430 and JEOL 2010). For cross-sectional TEM analysis, two parts, that were cut perpendicular to the surface, were joined with epoxy glue, surface against surface. Subsequently, slices were cut, mechanically polished and thinned to perforation by ion milling at a low angle in order to obtain the largest possible thin area, from the substrate up to the surface. Selected-area electron diffraction (SAED) patterns were obtained from cross-sectional specimens.
Fig. 1. Representative SEM micrograph of biomimetic coatings precipitated on chemically pre-treated titanium after exposure to SBF10 for 14 days (inset: corresponding EDX analysis).
and phosphorus. The assumed substitution of Ca2+ ions by Mg2+ and Na+ is consistent with the composition of biological apatites in human bone, which are non-stoichiometric with trace components, such as Mg2+, Na+, CO32, HPO42, F or Cl (Tadic et al., 2002). The phase composition of the coatings on chemically pretreated titanium after exposure to different SBF solutions was identified as hydroxyapatite of low crystallinity by XRD (Fig. 2). A preferred c-axis orientation of the deposited crystals can be supposed due to the high relative peak intensities of the (0 0 2) and (0 0 4) diffraction lines at 2u = 268 and 538, respectively, compared to the 100% intensity peak of the (2 1 1) plane at 2u = 328. The broad peak at 328 consists of the reflections from (2 1 1) at 31.878, (1 1 2) at 32.188 and (3 0 0) at 32.878. The crystallite size in the c-direction of the HCA crystals were calculated from the (0 0 2) peak broadening at 268 in the XRD-diagrams using the Scherrer equation (Mu¨ller and Mu¨ller, 2006) and decreased from 26 nm in SBF5 to 19 nm in
3. Results Fig. 1 shows a representative micrograph (SEM) accompanied by the EDX spectrum of the biomimetic coating precipitated on the samples surface within 2 weeks soaking in SBF10. The layer is composed of spherical aggregates with a diameter up to 20 mm, which are characteristic for coatings precipitated from SBF (Jonasova et al., 2004; Kokubo and Takadama, 2006). A small fraction of magnesium and sodium were found by EDX analysis (inset in Fig. 1) among calcium Table 1 Concentration of ions in SBF (mmol/l)
+
Na K+ Ca2+ Mg2+ Cl HCO3 SO42 HPO42
SBF5
SBF10
SBF15
SBF20
SBF27
142.0 5.0 2.5 1.0 131.0 5.0 1.0 1.0
142.0 5.0 2.5 1.0 126.0 10.0 1.0 1.0
142.0 5.0 2.5 1.0 121.0 15.0 1.0 1.0
142.0 5.0 2.5 1.0 116.0 20.0 1.0 1.0
142.0 5.0 2.5 1.0 109.0 27.0 1.0 1.0
Fig. 2. X-ray diffraction analyses of biomimetic coatings precipitated on chemically treated titanium after exposure to SBF5, 10, 15, 20 and 27 for 14 days.
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Fig. 3. Biomimetic growth of carbonated apatite on chemically pre-treated titanium: (a) cross-sectional TEM view from the titanium hydride (left) to the HCA layer (right) and (b) corresponding SAED pattern taken from the top of the hydroxyapatite layer.
SBF27. Ti and TiH2 (resulting from the acid etching in HCl) peaks were observed in all XRD patterns. The cross-sectional TEM view in Fig. 3a shows the transformations during all treatment steps. Sodium in the hydrated sodium titanate gel formed after treatment in NaOH is replaced by calcium during soaking in SBF (Jonasova et al., 2004; Takadama et al., 2001). A biomimetic apatite coating grew on the gel layer by attachment of Ca2+ and PO43 ions from SBF. The corresponding SAED pattern (Fig. 3b), taken from the area at the top of Fig. 3a, exhibits a large number of rings typical of a nanocrystalline structure. All distances correspond exactly to the hexagonal structure of HAwith the parameters a = 0.944 nm and c = 0.688 nm according to JCPDS 9-432. The Raman spectra of a sample exposed to SBF5 for 14 days is shown in Fig. 4. The band at 3567 cm1 in the spectrum is assigned to the OH– group in hydroxyapatite. The vibrations located around 1040 and 950 cm1 were assigned to the PO43 group. Similar spectra were recorded for Ca–P layers precipitated in SBF10, SBF15 and SBF20. In SBF27 (Fig. 4), the peak at 3567 cm1 disappeared, probably due to (CO3)2
substitution for OH (A-type), and the shoulder at 880 cm1 indicates HPO42 incorporation into the structure. At the same time, the intensity ratio of the bands at 1050 and 950 cm1 decreases with increasing carbonate content in the solution as a consequence of (CO3)2 increasing substitution for (PO4)3 (B-type) (Krajewski et al., 2005; Wilson et al. 2005). The chemical composition of the precipitated layers can be evaluated more specifically using Fourier transform infrared (FTIR) spectroscopy (Fig. 5). Vibrations located in the range from 1200 to 960 cm1 and at 604, 567 and 474 cm1 are associated with the (PO4)3 group. The bands detected at 1460, 1420 and 875 cm1 were assigned to the (CO3)2 group of Btype carbonated apatite. However, characteristic peaks at 875 and 959 cm1 indicate as well the presence of (HPO4)2 in the crystal lattice (Koutsopoulos, 2002). The absence of the OH vibration at 3570 cm1 suggests that carbonate also substitutes for OH (A-type). However, there is no evidence that (CO3)2 substitutes for OH as the characteristic absorption band at 1545 cm1, associated with A-type substitution, was not observed. It is known that Ca-deficient HA shows weaker stretching and librational bands compared to stoichiometric apatite (Wilson et al., 2005).
Fig. 4. Raman spectra of biomimetic coatings precipitated on chemically pretreated titanium after exposure to SBF5, 15 and 27 for 14 days.
Fig. 5. FTIR analyses of biomimetic coatings precipitated on chemically pretreated titanium after exposure to SBF5, 10, 15, 20 and 27 for 14 days.
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Fig. 6. (a) Cross-sectional TEM view of the precipitated apatite layer containing and (b) corresponding auto-correlation calculation (diagram of auto-correlation intensity (middle) and diagram of corresponding longitudinal and transversal intensities (left and right)).
4. Discussion Since the composition of SBF is similar to that of the inorganic part of human blood plasma, it can be assumed that the structure of the crystalline phase precipitated on the surface of bioactive materials would be close to biological apatite present in human bone. The FTIR, Raman and EDS spectra revealed that the layers are composed of Ca-deficient HA with (CO3)2 and Mg2+ substitution. A general formula of Ca10xyMgy(HPO4)xz (CO3)z(PO4)6x(OH)2x, with 0 x, y, z 1, can be assumed. For the maximum carbonate substitution (z = 1), the theoretical carbonate content is equal to 6.4 wt.%. The ratio of the absorption bands at 1420 cm1 (A(CO32)) and 604 cm1 (A(PO43) can be used to calculate the amount of (PO4)3 ions substituted by (CO3)2 ions in the precipitated apatites (LeGeros, 1991). The carbonate content in apatite layers calculated from A(CO32)/A(PO43) was 2.5, 3.2, 5.7 and 6.2 wt.% for SBF5,10,15 and 20. Using FTIR and Raman analysis, it was shown that with increasing amount of (HCO3) in the solution, the substitution of (CO3)2 for OH occurred and thus the formula can be written as: Ca10xyMgy(HPO4)xz(CO3)z (PO4)6x(OH)2xw(CO3)w/2. The theoretical maximum amount of carbonate substitution in this AB-type HCA for w ¼ 1 is equal to 10 wt.%. In our case, the maximum carbonate content calculated from A(CO32)/A(PO43) amounts to 9.1 wt.% in the layer precipitated from SBF27. Hence, it can be assumed that the A-sites are occupied after the B-sites are completely substituted. An interesting comparison can be drawn with bone mineral that contains carbonated apatite with a (CO3)2 substitution of up to 8 wt.% (Krajewski et al., 2005). The peaks in all X-ray diffraction patterns (Fig. 2) are not sharp and not well resolved. However, the higher relative peak intensity of the (0 0 2) and (0 0 4) diffraction lines at 2u = 268 and 538 can be attributed to hexagonal HA crystals with a
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preferred c-axis orientation of the deposited crystals. The preferential crystallite alignment can also be observed on the high-magnification image of Fig. 6a. This image was analysed by an auto-correlation method, with the results shown in Fig. 6b. To realize this calculation, Fig. 6a was superposed with its copy translated by vectors of various orientations and lengths. The ‘‘intensity’’ of each correlation, namely the reciprocal of the difference between the two images, was then plotted as a function of the corresponding translation vector (the origin being at the center of the diagram). This yields a map where the brightest regions correspond to the highest degree of correlation. As expected, the highest auto-correlation intensity is obtained for small translation vectors, namely near the diagram centre. However, the highest auto-correlation intensity is also expected for displacement vectors parallel to the average growth direction. Therefore, the elongation of the bright zone in the vertical direction perpendicular to the surface indicates that the crystallites are elongated preferentially in this direction (Fig. 6b). 5. Conclusions According to X-ray and TEM-SAED, biomimetically derived HCA coatings show a preferred growth orientation in the direction of their c-axis and perpendicular to the surface of chemically treated titanium. The FTIR, Raman and X-ray analyses of the precipitated layers revealed that the (HCO3) content in SBF influences the composition and structure of the resulting calcium phosphates. It can be supposed that as long as the (HCO3) concentration in the testing solution is below 20 mmol/l, only B-type HCA precipitates. At higher (HCO3) concentration, it can be assumed that as well A-type HCA forms. References Barrere, F., van Blitterswijk, C.A., de Groot, K., Layrolle, P., 2002. Influence of ionic strength and carbonate on the Ca–P coating formation from SBF 5 solution. Biomaterials 23, 1921–1930. Hoffman, A.S., Hubbell, J.A., 2004. Surface-immobilized biomolecules. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., Lemons, J.E. (Eds.), Biomaterials Science: An Introduction to Materials in Medicine. second ed. Elsevier, Amsterdam, pp. 225–233. Jansen, J.A., von Reum, A.F., 2004. Textured and porous materials. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., Lemons, J.E. (Eds.), Biomaterials Science: An Introduction to Materials in Medicine. second ed. Elsevier, Amsterdam, pp. 218–225. Jonasova, L., Mu¨ller, F.A., Helebrant, A., Strnad, J., Greil, P., 2004. Biomimetic apatite formation on chemically treated titanium. Biomaterials 25, 1187– 1194. Kokubo, T., Takadama, H., 2006. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27, 2907–2915. Koutsopoulos, S., 2002. Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods. J. Biomed. Mater. Res. 62, 600–612. Krajewski, A., Mazzocchi, M., Buldini, P.L., Ravagliolo, A., Tinti, A., Taddei, P., Fagnano, C., 2005. Synthesis of carbonated hydroxyapatites: efficiency of the substitution and critical evaluation of analytical methods. J. Mol. Struct. 744–747, 221–228. LeGeros, R.Z., 1991. Calcium Phosphates in Oral Biology and Medicine. Karger, Basel.
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