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Stepwise formation of crystalline apatite in the biomimetic coating of surgical grade SS 316L substrate: A TEM analysis
Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Stepwise formation of crystalline apatite in the biomimetic coating of surgical grade SS 316L substrate: A TEM analysis Jui Chakraborty a,*, Nina Daneu b, Aleksander Recˇnik b, Manjusha Chakraborty a, Sudip Dasgupta a, Jiten Ghosh a, Somoshree Sengupta a, Sujata Mazumdar a, Mithlesh K. Sinha a, Debabrata Basu a a b
Central Glass and Ceramic Research Institute, Kolkata 700 032, India Jozef Stefan Institute, Jamova-39, S1-1000 Ljubljana, Slovenia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 August 2010 Received in revised form 3 November 2010 Accepted 26 November 2010 Available online 5 February 2011
The present communication reports an interesting observation on stepwise development of crystallinity along with change in elemental composition of the HAp crystals, precipitated on SS 316L substrates (4 mm 5 mm 1 mm) by biomimetic route. The SS 316L substrates were incubated up to 6 days in simulated body fluid (SBF) at 37 8C with a periodic replacement of freshly prepared SBF at 48 h intervals. X ray diffraction (XRD) analysis of coated substrates revealed phase pure hydroxyapatite (HAp) as the only phase present in the coating. Transmission electron microscopy (TEM) images of the coating showed a gradual transformation of core shell type spherical HAp crystal into elongated forms over a period of 6 days of incubation of SS 316L substrate in SBF. The elemental composition of the deposited HAp phase was also changed with increase in exposure time of SS 316L to SBF as indicated by the energy dispersive spectra (EDS) of coated substrates. ß 2011 Published by Elsevier B.V. on behalf of Taiwan Institute of Chemical Engineers.
Keywords: Amorphous Coatings Crystals/crystallization Hydroxyapatite Transmission electron microscopy
1. Introduction Covering biometal surface with hydroxyapatite (HAp) represented a radical innovation, which opened a novel direction in the bone prosthesis development (Hench and Wilson, 1993; Suchanek and Yoshimura, 1998). The integration of such implants with bone takes place largely at the tissue-implant interface (Puleo and Nanci, 1999) that initiates bone mineralization and growth under physiological conditions. The metallic implant suffers from poor osteointegration with bone tissues because of its weak osteoconductivity and bioresorsbability. Coating of metallic implant with apatite phase processed in a biomimetic route has great promise in enhancing the rate of osteointegration of metallic implant with bone cells and tissues. Several processes have been reported to coat apatite phase on metallic surface (Ben-Nissan et al., 1997; Ducheyne et al., 1986; Ong et al., 1992). Apatite deposition on metallic implants through biomimetic route seems to be very promising because the processed apatite phase can nearly mimic the apatite particles present in natural bone. Since the process is carried out at physiological temperature any protein, growth factors or drug can be incorporated in the coating. Moreover, the biomimetically processed apatite phase on metallic substrate exhibits very good osteoconductivity and bioresorbability under physiological condition. Investigation on * Corresponding author. Tel.: +91 33 24733476x3233; fax: +91 33 24730957. E-mail address: [email protected] (J. Chakraborty).
crystallographic structure of bio-apatites has been addressed extensively (Calderin et al., 2003; Chakraborty et al., 2007a,b; Cho et al., 2003; Haverty et al., 2005; Kim et al., 2005; Kokubo et al., 2004; Leventouri, 2006; Mueller et al., 2007; Pasteris et al., 2004; Porter et al., 2004) including transmission electron microscopy (TEM) analyses (Kokubo et al., 2004) although no attempts have been made so far to investigate on the steps of bioapatite formation on a metallic substrate using cross sectional TEM analysis. The objective of this research work is to shed light on the basic phenomenon of nucleation, growth and crystallization of calcium phosphate nanoparticles on a metal substrate that governs the biochemical processes of bone mineralization on implant surface. The present study is primarily focused on characterization of biomimetically processed HAp coating on a SS 316L substrate using TEM. The process reveals an interesting observation that depicts nucleation of HAp mostly in amorphous phase, having few nanocrystals in the size range of 4–5 nm that slowly crystallizes into plate-like structures with crystallite size varying from 30 to 50 nm, after a period of 6 days of immersion of SS 316L in SBF. According to the TEM analysis and EDS spectroscopy a stepwise development of crystallinity accompanied with changes in elemental composition of the apatite crystals was observed and described. 2. Experimental Preparation of the SS 316L substrate: SS 316L sheets (thickness 2 cm) were mechanically roughened (sand blasting, pressure
1876-1070/$ – see front matter ß 2011 Published by Elsevier B.V. on behalf of Taiwan Institute of Chemical Engineers. doi:10.1016/j.jtice.2010.11.008
J. Chakraborty et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687 Table 1 Description of the samples. S. no.
Name of the substrate
Description
1 2 3
A1 A2 A3
SS 316L substrate coated with HAp for 2 days SS 316L substrate coated with HAp for 4 days SS 316L substrate coated with HAp for 6 days
8 kg/cm2, for a period of 40 s), ground (Praga Tools, Secunderabad, India, 451) and cut into the final dimension (4 mm 5 mm 1 mm) in three steps (first step, each block: 10 cm 10 cm 2 cm, second step, each block: 1 cm 1 cm 0.5 cm, third step, each block: 4 mm 5 mm 1 mm). The blocks (12 Nos.) were then cleaned ultrasonically (Microclean-109, Oscar Ultrasonics, Mumbai, India) and biomimetically coated with calcium phosphate as per standard procedure (Chakraborty et al., 2007a). Characterization of biomimetically coated SS 316L substrate: Samples were collected after 2, 4 and 6 days of exposure of SS 316L substrates in SBF (Table 1), washed and dried (Chakraborty et al., 2007a). Phase evaluation of the coated sample was carried out with the [(Fig._1)TD$IG] help of X-Ray diffractometer (X’Pert Pro MPD, Panalytical,
683
Almelo, the Netherlands) operating at 40 kV and 30 mA using CuKa radiation and attached with a Ni filter. The specimens were first scanned in continuous mode over the range of 2u angles of 208 to 708 to obtain a XRD pattern and then XRD data were recorded in step scan mode with a step size of 0.028 and step time of 5 s to obtain more accurate (0 0 4) diffraction line shape. TEM analysis (Jeol, JEM 2100, USA) was carried out using a JEOL 2100 microscope, operated at 200 kV. Two biomimetically coated (HAp) SS 316L substrates were pasted at the coated surface using standard pasting resin and hardener [Gatan, model 601.07, CrossSectional TEM Specimen preparation kit]. Subsequently, slices were cut and the samples were characterized as per standard procedure (Leventouri, 2006). 3. Results and discussion Fig. 1(a) exhibits a cluster of globules with an amorphous outer boundary, forming a core (X)-shell (Y) like structure in sample A1 (Table 1). Analysis of HREM image [Fig. 1(b)] of the amorphous shell (Y) shows the presence of crystallites in the size range of 4.85–10.50 nm with interplanar spacing corresponding to (3 0 0) and (1 0 0) planes of HAp [arrow marked]. The SAED pattern
Fig. 1. 2 days HAp coating (a) Cluster of HAp globules showing core-shell structure (X: nanocrystalline core, Y: amorphous shell) (b) HREM image of amorphous shell (Y) (inset: SAD pattern) (c) HREM image of nanocrystalline core (X).
[(Fig._2)TD$IG]
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J. Chakraborty et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687
Fig. 2. 4 days HAp coating (a) smaller crystals at the center (X0 ) larger crystals outside (Y0 ) (b) SAD of X0 showing characteristic HAp planes (c) HREM image of X0 showing crystal size of 20–39 nm.
exhibits diffused rings due to the presence of calcium phosphate nanocrystals [Fig. 1(b) inset]. At the center of core of the globule, X, the crystallite size was found to be 23.23 nm corresponding to (1 0 0) plane of HAp [Fig. 1(c)]. The SAED pattern in [Fig. 1(a) (inset)] shows a dot pattern suggesting a heterogeneous nucleation of HA single crystal on the substrate after 2 days of exposure to SBF. After 4 days, in sample A2, a change in morphology could be noticed for the amorphous calcium phosphate phase [Fig. 2 (a)]. A HREM image [Fig. 2 (b)] of X0 shows the presence of crystals in the size range of 20–30 nm corresponding to (2 1 1) and (0 0 2) planes of HAp. This in turn was confirmed by the SAED pattern of the same area corresponding to (2 1 1), (0 0 2) and (1 1 2) planes of HAp [Fig. 2(c)]. The rings in the SAED pattern indicates that HA nuclei gradually grew to give polycrystalline HA phase after 4 days of exposure of SS 316L in SBF. Also, the rings in SAED pattern were found to be sharper in A2 [Fig. 1(c)] as compared to the diffused pattern obtained in A1 [Fig. 1(b)] which clearly signifies that crystallinity of HAp increased with increase in exposure time in SBF. These findings are consistent with previous observations (Kim et al., 2005; Kokubo et al., 2004; Mueller et al., 2007; Porter et al., 2004). In the present study, we consider (0 0 4) reflection for single profile analysis to calculate crystallite size and lattice strain of the HAp because (0 0 2) and (0 0 4) reflections are the only two
diffraction lines corresponding to different orders of reflections from the same plane appeared in the XRD pattern and another reason to consider (0 0 4) reflection was to avoid the partial overlapping of peaks. The crystallite size and lattice strain are calculated by well known two Scherrer formula (Scherrer, 1918). Crystallite size ¼ K l=ðB cos uÞ Lattice strain ¼ B=ð4 tan uÞ where B (size) = Bobs Bstd and B ðstrainÞ ¼ Square root ðB2obs B2std Þ: Here B describes the structural broadening, which is the difference in integral profile width between a standard (Bstd) and the experimental sample (Bobs). For this single profile analysis, NIST 660a was taken as the standard sample to encompass the effect of instrumental broadening of the X-ray diffractometer. It was also assumed that this standard did not have any size and/or strain broadening. The values of crystallite size and lattice strain along (0 0 4) crystallographic direction are shown in Table 2. From Table 2, it was observed that size of HAp crystallite gradually increased while lattice strain decreased with increase in exposure time in SBF. This is in conformity with our TEM data as above and confirms the fact that HAp crystallites gradually grew into bigger size with increase in exposure time in SBF.
J. Chakraborty et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687 Table 2 Values of crystallite size and lattice strain of HAp along (0 0 4) crystallographic direction from single profile analysis using Scherrer formula. Sample
Size-strain along (0 0 4) crystallographic direction Crystallite size (nm)
Lattice strain (%)
A1 A3
14.4 17.5
0.647 0.550
Finally, the 6 days sample, A3, comprises HAp crystal plates only [Fig. 3(a)]. No globules could be observed here, HREM image of the area exhibits the presence of (0 0 2) planes of HAp and the size of crystal in the range of 30–50 nm [Fig. 3(b)]. Some researchers reported octacalcium phosphate (OCP) as the bioactive calcium phosphate phase formed on biocompatible [(Fig._3)TD$IG]
685
substrate in simulated body fluid (Lu and Leng, 2004; Szekeres et al., 2005). However, in this case we clearly exclude the presence of OCP due to the absence of diffraction rings corresponding to d1 1 0 (0.938 nm), d2 0 0 (0.932) and d0 0 1 (0.683 nm), which are characteristic for OCP. The XRD data (Fig. 4) reveals HAp as the only phase in the coating and its crystallinity was gradually increased, as exhibited by growth in (2 1 1), (1 1 2), (2 2 2) and (0 0 4) planes with increase in days of exposure of SS 316L substrate to SBF. Sand blasting of SS 316L substrate removed all the organics adsorbed on the surface of metallic substrate and activated M2O3/ MO molecules on the surface, where M stands for Fe, Cr, Mn, Ni, etc. When immersed in SBF solution the oxygen present in the M2O3/ MO molecules attracted Ca2+ ions and formed –O–Ca bond that is more of ionic in nature. The Ca2+ ions in turn bound to HPO42 ions present in the solution which interacted with two molecules of
Fig. 3. 6 days HAp coating (a) Almost monodisperse crystals of HAp in a period of 6 days (b) HREM of X00 showing characteristic HAp planes and crystals in the size range of 30– 50 nm.
[(Fig._4)TD$IG]
(004)
20
30
Intensity (a.u)
substrate (SS316L)
50
50
52
54
56
2 days
50
(004)
(202) (300)
40
(222) (402)
Intensity (a.u)
(002) (102) (210)
(112)
substrate (SS316L)
(211)
6 days
52
2θ (degree)
54
56
6 Days
60
70
2 Days 20
30
40
50
60
70
2θ (degree) Fig. 4. X ray diffraction pattern of HAp on SS 316L substrate, showing the increase in peak intensity after a period of 6 days corresponding to (2 1 1) and (1 2 2) planes.
[(Fig._5)TD$IG]
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Fig. 5. (a) EDX of the A2 shows the presence of both sodium and chlorine. Ca:P molar ratio is 1.2 here. (b) EDX of A3 shows the presence of sodium in the crystals. No chlorine could be observed here. Ca:P molar ratio is 1.6 here.
H3O+ ions in pH 6 solution of SBF. This formed dicalcium phosphate dihydrate (CaHPO42H2O) moiety on the metallic substrate. The H3O+ molecules behaved as a center for accumulation of more HPO42 molecules through H-bonding interaction. The adsorption of Ca2+ and HPO42 ions on the surface created a local scarcity of the same ions in the stagnant liquid layers adjacent to the metal surface. Also the positive and negative charge centers on the metallic surface acted as sites for attraction of more Ca2+ and HPO42 ions to the metal surface. This caused a local transport of Ca2+ and HPO42 ions towards the metallic surface and the stagnant layer on the metallic surface became supersaturated with Ca2+ and HPO42 ions. As a result more number of DCPD nuclei was formed on the metallic substrate. Due non uniform heterogeneous nucleation on the metallic surface a large number of kinks were formed and these acted as an active center for the growth of DCPD nuclei. Again DCPD is thermodynamically unstable and readily converts into more stable hydroxyapatite phase with the consumption of Ca2+ ions according to following equation.
4Ca2þ þ 6CaHPO4 þ 2H2 O ¼ Ca10 ðPO4 Þ6 ðOHÞ2 þ 8Hþ This created a core shell type structure on the metallic substrate with core enriched in HAp phase and shell being predominantly DCPD and the growth of HAp phase occurred through the active transport of Ca2+ ions through porous shell of DCPD. Due to Ca2+ drag from shell to core the HAp core gradually grew bigger with shell becoming increasingly thinner with increase in exposure time of SBF to SS 316L. Crystallization in HAp occurred through the ordered assembly of hexagonal unit cells preferentially along the basal planes of the crystal as it is thermodynamically the most stable direction of growth of hexagonal HAp crystals. That is why
spherical HAp core gradually turned into elongated HAp crystals after 4 days of exposure of metallic substrate to SBF. An interesting change in elemental composition of the deposited HAp phase was also observed within a 2 days of time period, in the coating. In Fig. 5 (a), the Ca:P molar ratio changed from 1.2 in A1 to 1.6 in A3. In SBF, SS 316L behaved as a positively charged surface due to the presence of surface H3O+ groups in DCPD nuclei and preferentially interacted with PO43 groups to nucleate a core enriched in P as reflected in lower Ca:P molar ratio in A2 after 2 days. The predominantly negatively charged P rich core then absorbed more positive ions, i.e., Ca2+ to compensate for Ca deficiency and thereby increased Ca:P molar ratio in A3 after 6 days. This was also reflected in an increase in sodium content (element %: 1.2–8.0) in the HAp crystals from A1 [Fig. 5 (a)] to A3 [Fig. 5 (b)], i.e., from 2 days to 6 days [Fig. 5 (b)]. Another important observation was the presence of chlorine in the core (X) [Fig. 5(a)] which was completely absent in shell (Y). This also reveals the fact that the core was enriched in HAp phase where Cl substituted for OH ions. A number of related works have already been done (Engqvist et al., 2006; Fujita et al., 2003; Kitsugi et al., 1995; Iliescu et al., 2004) although, no reference could be obtained in line with the present study to reveal the transformation of nucleated amorphous phase of the globular HAp to crystalline plates altogether in a period of 6 days in biomimetic coating. In a heterogeneous system of nucleation and growth, diffusion of atoms into cluster occurred very fast at a given temperature. This phenomenon was maximum before the equilibrium was reached. Hence, initially, we saw formation of bigger crystals in the size range of 10–25 nm at the core. Following this, the growth of these crystals was governed by very slow and controlled diffusion and the crystals diffused into each other to form the thermodynamically stable plate like crystals of HAp, for energy minimization at the coating interface. 4. Conclusions The cross sectional TEM study of the biomimetically processed HAp coating on SS 316L substrate was undertaken. A gradual development of crystallinity along with a morphological variation in deposited HAp crystals were investigated over a period of 6 days of immersion of SS 316L substrate in SBF. A predominantly amorphous core shell like globules of HAp phase was observed after a period of 2 days which gradually turned into elongated HAp crystals after 6 days of immersion in SBF with an increase in Ca:P malor ratio from 1.2 to 1.6. A gradual growth in the HAp nanocrystals from crystallite size of 20 nm to a crystallite size of 50 nm was also evident over a period of 6 days in SBF. The observation is in conformity with the principles of heterogeneous nucleation and growth in the system, during development of the HAp coating on SS 316L substrate, immersed in SBF at 37 8C. Acknowledgements The authors are grateful to the Director, Central Glass and Ceramic Research Institute, Kolkata, India for providing his permission to carry on the above work at Jozef Stefan Institute, Slovenia, in a bilateral Indo-Slovenia project program. Thanks are due to Dr. Spomenka Kobe, Head, Nanostructured Materials, JZI, Slovenia for providing all the necessary facilities for carrying out the study. We are indeed indebted to the DST Indo-Slovenia S&T bilateral project No. CLP 0205 for all the kind support and the financial assistance for undertaking this work. References Ben-Nissan, B., C. S. Chai, and K. A. Gross, ‘‘Effect of Solution Aging on Sol–Gel Hydroxyapatite Coatings,’’ Bioceramics, 10, 175 (1997).
J. Chakraborty et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687 Calderin, L., M. J. Stott, and A. Rubio, ‘‘Electronic and Crystallographic Structure of Apatites,’’ Phys. Rev. B, 67, 13406 (2003). Chakraborty, J., M. K. Sinha, and D. Basu, ‘‘Biomolecular Template Induced Biomimetic Coating of Hydroxyapatite on an SS 316 L Substrate,’’ J. Am. Ceram. Soc., 90, 1258 (2007a). Chakraborty, J., M. K. Sinha, and D. Basu, ‘‘Interconnected Hydroxyapatite Nanofibres in the Biomimetic Coating of a Bioinert Ceramic Substrate,’’ J. Am. Ceram. Soc., 90, 3667 (2007b). Cho, G., Y. Wu, and J. L. Ackerman, ‘‘Detection of Hydroxyl Ions in Bone-Mineral by Solid-State NMR Spectroscopy,’’ Science, 300, 1123 (2003). Ducheyne, P., W. Van Raemdonck, J. C. Heughebaert, and M. Heughebaert, ‘‘Structural Analysis of Hydroxyapatite Coatings on Titanium,’’ Biomaterials, 7, 97 (1986). Engqvist, H., G. A. Botton, M. Couillard, S. Mohammadi, J. Malmstrom, L. Emanuelsson, L. Hermansson, M. W. Phaneuf, and P. Thomsen, ‘‘A Novel Tool for High-Resolution Transmission Electron Microscopy of Intact Interfaces between Bone and Metallic implants,’’ J. Biomed. Mater. Res. A, 78, 20 (2006). Fujita, R., A. Yokoyama, Y. Nodasaka, T. Kohgo, and T. Kawasaki, ‘‘Ultrastructure of Ceramic-Bone Interface Using Hydroxyapatite and b-Tricalcium Phosphate Ceramics and Replacement Mechanism of b-Tricalcium Phosphate in Bone,’’ Tissue Cell, 35, 427 (2003). Haverty, D., S. A. M. Tofail, T. K. Stanton, and J. B. McMonagle, ‘‘Structure and Stability of Hydroxyapatite: Density Functional Calculation and Rietveld Analysis,’’ Phys. Rev. B, 71, 094103 (2005). Hench, L. L. and J. Wilson, An Introduction to Bioceramics, p. 223, World Scientific, London, UK, (1993). Iliescu, M., V. Nelea, J. Werckmann, and I. N. Mihailescu, ‘‘Transmission Electron Microscopy Investigation of Pulsed-Laser Deposited Hydroxylapatite Thin Films Prepared by Tripod and Focused Ion Beam Techniques,’’ Surf. Coat. Technol., 187, 131 (2004). Kim, H. M., H. Teruyuki, T. Kokubo, and T. Nakamura, ‘‘Process and Kinetics of Bonelike Apatite Formation on Sintered Hydroxyapatite in a Simulated Body Fluid,’’ Biomaterials, 26, 4366 (2005).
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Kitsugi, T., T. Yamamuro, T. Nakamura, and M. Oka, ‘‘Transmission Electron Microscopy Observations at the Interface of Bone and Four Types of Calcium Phosphate Ceramics with Different Calcium/Phosphorus Molar Ratios,’’ Biomaterials, 16, 1101 (1995). Kokubo, T., T. Himeno, H. M. Kim, M. Kawashita, and T. Nakamura, ‘‘Process of Bonelike Apatite Formation on Sintered Hydroxyapatite in Serum-Containing SBF,’’ Key Eng. Mater., 139, 254 (2004). Leventouri, T., ‘‘Synthetic and Biological Hydroxyapatites: Crystal Structure Questions,’’ Biomaterials, 27, 3339 (2006). Lu, X. and Y. Leng, ‘‘TEM Study of Calcium Phosphate Precipitation on Bioactive Titanium Surfaces,’’ Biomaterials, 25, 1779 (2004). Mueller, F. A., L. Mueller, D. Caillard, and E. Conforto, ‘‘Preferred Growth Orientation of Biomimetic Apatite Crystals,’’ J. Cryst. Growth, 304, 464 (2007). Ong, J. L., L. C. Lucas, W. R. Lacefield, and E. D. Rigney, ‘‘Structure, Solubility and Bond Strength of Thin Calcium Phosphate Coatings Produced by Ion Beam Sputter Deposition,’’ Biomaterials, 13, 249 (1992). Pasteris, J. D., B. Wopenka, J. J. Freeman, K. Rogers, E. Valsami-Jones, J. A. M. van der Houwen, and M. J. Silva, ‘‘Lack of OH in Nanocrystalline Apatite as a Function of Degree of Atomic Order: Implications for Bone and Biomaterials,’’ Biomaterials, 25, 229 (2004). Porter, A. E., C. M. Botelho, M. A. Lopes, J. D. Santos, S. M. Best, and W. Bonfield, ‘‘Ultrastructural Comparison of Dissolution and Apatite Precipitation on Hydroxyapatite and Silicon-Substituted Hydroxyapatite In Vitro and In Vivo,’’ J. Biomed. Mater. Res. A, 69, 670 (2004). Puleo, D. A. and A. Nanci, ‘‘Understanding and Controlling Bone-Implant Interface,’’ Biomaterials, 20, 2311 (1999). Scherrer, P., ‘‘Bestimmung der Gro¨sse und der inneren Struktur von Kolloidteilchen mittels Ro¨ntgenstrahlen,’’ Nachr. Ges. Wiss. Go¨ttingen, 26, 98 (1918). Suchanek, W. and M. Yoshimura, ‘‘Processing and Properties of Hydroxyapatite-Based Biomaterials for Use as Hard Tissue Replacement Implants,’’ J. Mater. Res., 13, 94 (1998). Szekeres, M., G. Fodor, A. Fazekas, M. Radnai, K. Turzo, and I. Dekany, ‘‘Formation of Octacalcium Phosphate by Heterogeneous Nucleation on a Titania Surface,’’ Coll. Polym. Sci., 283, 587 (2005).
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Stepwise formation of crystalline apatite in the biomimetic coating of surgical grade SS 316L substrate: A TEM analysis Jui Chakraborty a,*, Nina Daneu b, Aleksander Recˇnik b, Manjusha Chakraborty a, Sudip Dasgupta a, Jiten Ghosh a, Somoshree Sengupta a, Sujata Mazumdar a, Mithlesh K. Sinha a, Debabrata Basu a a b
Central Glass and Ceramic Research Institute, Kolkata 700 032, India Jozef Stefan Institute, Jamova-39, S1-1000 Ljubljana, Slovenia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 August 2010 Received in revised form 3 November 2010 Accepted 26 November 2010 Available online 5 February 2011
The present communication reports an interesting observation on stepwise development of crystallinity along with change in elemental composition of the HAp crystals, precipitated on SS 316L substrates (4 mm 5 mm 1 mm) by biomimetic route. The SS 316L substrates were incubated up to 6 days in simulated body fluid (SBF) at 37 8C with a periodic replacement of freshly prepared SBF at 48 h intervals. X ray diffraction (XRD) analysis of coated substrates revealed phase pure hydroxyapatite (HAp) as the only phase present in the coating. Transmission electron microscopy (TEM) images of the coating showed a gradual transformation of core shell type spherical HAp crystal into elongated forms over a period of 6 days of incubation of SS 316L substrate in SBF. The elemental composition of the deposited HAp phase was also changed with increase in exposure time of SS 316L to SBF as indicated by the energy dispersive spectra (EDS) of coated substrates. ß 2011 Published by Elsevier B.V. on behalf of Taiwan Institute of Chemical Engineers.
Keywords: Amorphous Coatings Crystals/crystallization Hydroxyapatite Transmission electron microscopy
1. Introduction Covering biometal surface with hydroxyapatite (HAp) represented a radical innovation, which opened a novel direction in the bone prosthesis development (Hench and Wilson, 1993; Suchanek and Yoshimura, 1998). The integration of such implants with bone takes place largely at the tissue-implant interface (Puleo and Nanci, 1999) that initiates bone mineralization and growth under physiological conditions. The metallic implant suffers from poor osteointegration with bone tissues because of its weak osteoconductivity and bioresorsbability. Coating of metallic implant with apatite phase processed in a biomimetic route has great promise in enhancing the rate of osteointegration of metallic implant with bone cells and tissues. Several processes have been reported to coat apatite phase on metallic surface (Ben-Nissan et al., 1997; Ducheyne et al., 1986; Ong et al., 1992). Apatite deposition on metallic implants through biomimetic route seems to be very promising because the processed apatite phase can nearly mimic the apatite particles present in natural bone. Since the process is carried out at physiological temperature any protein, growth factors or drug can be incorporated in the coating. Moreover, the biomimetically processed apatite phase on metallic substrate exhibits very good osteoconductivity and bioresorbability under physiological condition. Investigation on * Corresponding author. Tel.: +91 33 24733476x3233; fax: +91 33 24730957. E-mail address: [email protected] (J. Chakraborty).
crystallographic structure of bio-apatites has been addressed extensively (Calderin et al., 2003; Chakraborty et al., 2007a,b; Cho et al., 2003; Haverty et al., 2005; Kim et al., 2005; Kokubo et al., 2004; Leventouri, 2006; Mueller et al., 2007; Pasteris et al., 2004; Porter et al., 2004) including transmission electron microscopy (TEM) analyses (Kokubo et al., 2004) although no attempts have been made so far to investigate on the steps of bioapatite formation on a metallic substrate using cross sectional TEM analysis. The objective of this research work is to shed light on the basic phenomenon of nucleation, growth and crystallization of calcium phosphate nanoparticles on a metal substrate that governs the biochemical processes of bone mineralization on implant surface. The present study is primarily focused on characterization of biomimetically processed HAp coating on a SS 316L substrate using TEM. The process reveals an interesting observation that depicts nucleation of HAp mostly in amorphous phase, having few nanocrystals in the size range of 4–5 nm that slowly crystallizes into plate-like structures with crystallite size varying from 30 to 50 nm, after a period of 6 days of immersion of SS 316L in SBF. According to the TEM analysis and EDS spectroscopy a stepwise development of crystallinity accompanied with changes in elemental composition of the apatite crystals was observed and described. 2. Experimental Preparation of the SS 316L substrate: SS 316L sheets (thickness 2 cm) were mechanically roughened (sand blasting, pressure
1876-1070/$ – see front matter ß 2011 Published by Elsevier B.V. on behalf of Taiwan Institute of Chemical Engineers. doi:10.1016/j.jtice.2010.11.008
J. Chakraborty et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687 Table 1 Description of the samples. S. no.
Name of the substrate
Description
1 2 3
A1 A2 A3
SS 316L substrate coated with HAp for 2 days SS 316L substrate coated with HAp for 4 days SS 316L substrate coated with HAp for 6 days
8 kg/cm2, for a period of 40 s), ground (Praga Tools, Secunderabad, India, 451) and cut into the final dimension (4 mm 5 mm 1 mm) in three steps (first step, each block: 10 cm 10 cm 2 cm, second step, each block: 1 cm 1 cm 0.5 cm, third step, each block: 4 mm 5 mm 1 mm). The blocks (12 Nos.) were then cleaned ultrasonically (Microclean-109, Oscar Ultrasonics, Mumbai, India) and biomimetically coated with calcium phosphate as per standard procedure (Chakraborty et al., 2007a). Characterization of biomimetically coated SS 316L substrate: Samples were collected after 2, 4 and 6 days of exposure of SS 316L substrates in SBF (Table 1), washed and dried (Chakraborty et al., 2007a). Phase evaluation of the coated sample was carried out with the [(Fig._1)TD$IG] help of X-Ray diffractometer (X’Pert Pro MPD, Panalytical,
683
Almelo, the Netherlands) operating at 40 kV and 30 mA using CuKa radiation and attached with a Ni filter. The specimens were first scanned in continuous mode over the range of 2u angles of 208 to 708 to obtain a XRD pattern and then XRD data were recorded in step scan mode with a step size of 0.028 and step time of 5 s to obtain more accurate (0 0 4) diffraction line shape. TEM analysis (Jeol, JEM 2100, USA) was carried out using a JEOL 2100 microscope, operated at 200 kV. Two biomimetically coated (HAp) SS 316L substrates were pasted at the coated surface using standard pasting resin and hardener [Gatan, model 601.07, CrossSectional TEM Specimen preparation kit]. Subsequently, slices were cut and the samples were characterized as per standard procedure (Leventouri, 2006). 3. Results and discussion Fig. 1(a) exhibits a cluster of globules with an amorphous outer boundary, forming a core (X)-shell (Y) like structure in sample A1 (Table 1). Analysis of HREM image [Fig. 1(b)] of the amorphous shell (Y) shows the presence of crystallites in the size range of 4.85–10.50 nm with interplanar spacing corresponding to (3 0 0) and (1 0 0) planes of HAp [arrow marked]. The SAED pattern
Fig. 1. 2 days HAp coating (a) Cluster of HAp globules showing core-shell structure (X: nanocrystalline core, Y: amorphous shell) (b) HREM image of amorphous shell (Y) (inset: SAD pattern) (c) HREM image of nanocrystalline core (X).
[(Fig._2)TD$IG]
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Fig. 2. 4 days HAp coating (a) smaller crystals at the center (X0 ) larger crystals outside (Y0 ) (b) SAD of X0 showing characteristic HAp planes (c) HREM image of X0 showing crystal size of 20–39 nm.
exhibits diffused rings due to the presence of calcium phosphate nanocrystals [Fig. 1(b) inset]. At the center of core of the globule, X, the crystallite size was found to be 23.23 nm corresponding to (1 0 0) plane of HAp [Fig. 1(c)]. The SAED pattern in [Fig. 1(a) (inset)] shows a dot pattern suggesting a heterogeneous nucleation of HA single crystal on the substrate after 2 days of exposure to SBF. After 4 days, in sample A2, a change in morphology could be noticed for the amorphous calcium phosphate phase [Fig. 2 (a)]. A HREM image [Fig. 2 (b)] of X0 shows the presence of crystals in the size range of 20–30 nm corresponding to (2 1 1) and (0 0 2) planes of HAp. This in turn was confirmed by the SAED pattern of the same area corresponding to (2 1 1), (0 0 2) and (1 1 2) planes of HAp [Fig. 2(c)]. The rings in the SAED pattern indicates that HA nuclei gradually grew to give polycrystalline HA phase after 4 days of exposure of SS 316L in SBF. Also, the rings in SAED pattern were found to be sharper in A2 [Fig. 1(c)] as compared to the diffused pattern obtained in A1 [Fig. 1(b)] which clearly signifies that crystallinity of HAp increased with increase in exposure time in SBF. These findings are consistent with previous observations (Kim et al., 2005; Kokubo et al., 2004; Mueller et al., 2007; Porter et al., 2004). In the present study, we consider (0 0 4) reflection for single profile analysis to calculate crystallite size and lattice strain of the HAp because (0 0 2) and (0 0 4) reflections are the only two
diffraction lines corresponding to different orders of reflections from the same plane appeared in the XRD pattern and another reason to consider (0 0 4) reflection was to avoid the partial overlapping of peaks. The crystallite size and lattice strain are calculated by well known two Scherrer formula (Scherrer, 1918). Crystallite size ¼ K l=ðB cos uÞ Lattice strain ¼ B=ð4 tan uÞ where B (size) = Bobs Bstd and B ðstrainÞ ¼ Square root ðB2obs B2std Þ: Here B describes the structural broadening, which is the difference in integral profile width between a standard (Bstd) and the experimental sample (Bobs). For this single profile analysis, NIST 660a was taken as the standard sample to encompass the effect of instrumental broadening of the X-ray diffractometer. It was also assumed that this standard did not have any size and/or strain broadening. The values of crystallite size and lattice strain along (0 0 4) crystallographic direction are shown in Table 2. From Table 2, it was observed that size of HAp crystallite gradually increased while lattice strain decreased with increase in exposure time in SBF. This is in conformity with our TEM data as above and confirms the fact that HAp crystallites gradually grew into bigger size with increase in exposure time in SBF.
J. Chakraborty et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 682–687 Table 2 Values of crystallite size and lattice strain of HAp along (0 0 4) crystallographic direction from single profile analysis using Scherrer formula. Sample
Size-strain along (0 0 4) crystallographic direction Crystallite size (nm)
Lattice strain (%)
A1 A3
14.4 17.5
0.647 0.550
Finally, the 6 days sample, A3, comprises HAp crystal plates only [Fig. 3(a)]. No globules could be observed here, HREM image of the area exhibits the presence of (0 0 2) planes of HAp and the size of crystal in the range of 30–50 nm [Fig. 3(b)]. Some researchers reported octacalcium phosphate (OCP) as the bioactive calcium phosphate phase formed on biocompatible [(Fig._3)TD$IG]
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substrate in simulated body fluid (Lu and Leng, 2004; Szekeres et al., 2005). However, in this case we clearly exclude the presence of OCP due to the absence of diffraction rings corresponding to d1 1 0 (0.938 nm), d2 0 0 (0.932) and d0 0 1 (0.683 nm), which are characteristic for OCP. The XRD data (Fig. 4) reveals HAp as the only phase in the coating and its crystallinity was gradually increased, as exhibited by growth in (2 1 1), (1 1 2), (2 2 2) and (0 0 4) planes with increase in days of exposure of SS 316L substrate to SBF. Sand blasting of SS 316L substrate removed all the organics adsorbed on the surface of metallic substrate and activated M2O3/ MO molecules on the surface, where M stands for Fe, Cr, Mn, Ni, etc. When immersed in SBF solution the oxygen present in the M2O3/ MO molecules attracted Ca2+ ions and formed –O–Ca bond that is more of ionic in nature. The Ca2+ ions in turn bound to HPO42 ions present in the solution which interacted with two molecules of
Fig. 3. 6 days HAp coating (a) Almost monodisperse crystals of HAp in a period of 6 days (b) HREM of X00 showing characteristic HAp planes and crystals in the size range of 30– 50 nm.
[(Fig._4)TD$IG]
(004)
20
30
Intensity (a.u)
substrate (SS316L)
50
50
52
54
56
2 days
50
(004)
(202) (300)
40
(222) (402)
Intensity (a.u)
(002) (102) (210)
(112)
substrate (SS316L)
(211)
6 days
52
2θ (degree)
54
56
6 Days
60
70
2 Days 20
30
40
50
60
70
2θ (degree) Fig. 4. X ray diffraction pattern of HAp on SS 316L substrate, showing the increase in peak intensity after a period of 6 days corresponding to (2 1 1) and (1 2 2) planes.
[(Fig._5)TD$IG]
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Fig. 5. (a) EDX of the A2 shows the presence of both sodium and chlorine. Ca:P molar ratio is 1.2 here. (b) EDX of A3 shows the presence of sodium in the crystals. No chlorine could be observed here. Ca:P molar ratio is 1.6 here.
H3O+ ions in pH 6 solution of SBF. This formed dicalcium phosphate dihydrate (CaHPO42H2O) moiety on the metallic substrate. The H3O+ molecules behaved as a center for accumulation of more HPO42 molecules through H-bonding interaction. The adsorption of Ca2+ and HPO42 ions on the surface created a local scarcity of the same ions in the stagnant liquid layers adjacent to the metal surface. Also the positive and negative charge centers on the metallic surface acted as sites for attraction of more Ca2+ and HPO42 ions to the metal surface. This caused a local transport of Ca2+ and HPO42 ions towards the metallic surface and the stagnant layer on the metallic surface became supersaturated with Ca2+ and HPO42 ions. As a result more number of DCPD nuclei was formed on the metallic substrate. Due non uniform heterogeneous nucleation on the metallic surface a large number of kinks were formed and these acted as an active center for the growth of DCPD nuclei. Again DCPD is thermodynamically unstable and readily converts into more stable hydroxyapatite phase with the consumption of Ca2+ ions according to following equation.
4Ca2þ þ 6CaHPO4 þ 2H2 O ¼ Ca10 ðPO4 Þ6 ðOHÞ2 þ 8Hþ This created a core shell type structure on the metallic substrate with core enriched in HAp phase and shell being predominantly DCPD and the growth of HAp phase occurred through the active transport of Ca2+ ions through porous shell of DCPD. Due to Ca2+ drag from shell to core the HAp core gradually grew bigger with shell becoming increasingly thinner with increase in exposure time of SBF to SS 316L. Crystallization in HAp occurred through the ordered assembly of hexagonal unit cells preferentially along the basal planes of the crystal as it is thermodynamically the most stable direction of growth of hexagonal HAp crystals. That is why
spherical HAp core gradually turned into elongated HAp crystals after 4 days of exposure of metallic substrate to SBF. An interesting change in elemental composition of the deposited HAp phase was also observed within a 2 days of time period, in the coating. In Fig. 5 (a), the Ca:P molar ratio changed from 1.2 in A1 to 1.6 in A3. In SBF, SS 316L behaved as a positively charged surface due to the presence of surface H3O+ groups in DCPD nuclei and preferentially interacted with PO43 groups to nucleate a core enriched in P as reflected in lower Ca:P molar ratio in A2 after 2 days. The predominantly negatively charged P rich core then absorbed more positive ions, i.e., Ca2+ to compensate for Ca deficiency and thereby increased Ca:P molar ratio in A3 after 6 days. This was also reflected in an increase in sodium content (element %: 1.2–8.0) in the HAp crystals from A1 [Fig. 5 (a)] to A3 [Fig. 5 (b)], i.e., from 2 days to 6 days [Fig. 5 (b)]. Another important observation was the presence of chlorine in the core (X) [Fig. 5(a)] which was completely absent in shell (Y). This also reveals the fact that the core was enriched in HAp phase where Cl substituted for OH ions. A number of related works have already been done (Engqvist et al., 2006; Fujita et al., 2003; Kitsugi et al., 1995; Iliescu et al., 2004) although, no reference could be obtained in line with the present study to reveal the transformation of nucleated amorphous phase of the globular HAp to crystalline plates altogether in a period of 6 days in biomimetic coating. In a heterogeneous system of nucleation and growth, diffusion of atoms into cluster occurred very fast at a given temperature. This phenomenon was maximum before the equilibrium was reached. Hence, initially, we saw formation of bigger crystals in the size range of 10–25 nm at the core. Following this, the growth of these crystals was governed by very slow and controlled diffusion and the crystals diffused into each other to form the thermodynamically stable plate like crystals of HAp, for energy minimization at the coating interface. 4. Conclusions The cross sectional TEM study of the biomimetically processed HAp coating on SS 316L substrate was undertaken. A gradual development of crystallinity along with a morphological variation in deposited HAp crystals were investigated over a period of 6 days of immersion of SS 316L substrate in SBF. A predominantly amorphous core shell like globules of HAp phase was observed after a period of 2 days which gradually turned into elongated HAp crystals after 6 days of immersion in SBF with an increase in Ca:P malor ratio from 1.2 to 1.6. A gradual growth in the HAp nanocrystals from crystallite size of 20 nm to a crystallite size of 50 nm was also evident over a period of 6 days in SBF. The observation is in conformity with the principles of heterogeneous nucleation and growth in the system, during development of the HAp coating on SS 316L substrate, immersed in SBF at 37 8C. Acknowledgements The authors are grateful to the Director, Central Glass and Ceramic Research Institute, Kolkata, India for providing his permission to carry on the above work at Jozef Stefan Institute, Slovenia, in a bilateral Indo-Slovenia project program. Thanks are due to Dr. Spomenka Kobe, Head, Nanostructured Materials, JZI, Slovenia for providing all the necessary facilities for carrying out the study. We are indeed indebted to the DST Indo-Slovenia S&T bilateral project No. CLP 0205 for all the kind support and the financial assistance for undertaking this work. References Ben-Nissan, B., C. S. Chai, and K. A. Gross, ‘‘Effect of Solution Aging on Sol–Gel Hydroxyapatite Coatings,’’ Bioceramics, 10, 175 (1997).
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