Acta BIOMATERIALIA Acta Biomaterialia 1 (2005) 663–669 www.actamat-journals.com
Photoluminescence of annealed biomimetic apatites Cordt Zollfrank *, Lenka Mu¨ller, Peter Greil, Frank A. Mu¨ller Department of Materials Science (Glass and Ceramics), University of Erlangen-Nuernberg, Martensstr. 5, D-91058 Erlangen, Germany Received 21 February 2005; received in revised form 9 June 2005; accepted 13 June 2005
Dedicated to Professor Dr. H. Oel on the occasion of his 80th birthday
Abstract Biomimetic apatite coatings are widely used in orthopaedic applications to provide bioinert material surfaces with bioactive behaviour by means of initiating bone growth at the implant surface. In this study we manufactured biomimetic calcium phosphate coatings consisting of a calcium deﬁcient carbonated apatite by immersing activated titanium platelets into simulated body ﬂuid. The development of the crystal phases was monitored by X-ray diﬀractometry in addition to Fourier-transform infrared spectroscopy. The microstructure of the biomimetic apatites and phase composition was analysed using scanning and transmission electron microscopy as well as attached energy dispersive X-ray spectrometry. The samples were annealed in air yielding in an inherent luminescence of the biomimetic apatite up to temperatures of 600 °C. The photo-induced emission spectra were recorded in the range from 400 to 750 nm at excitation wavelengths ranging 310–450 nm. A blue (437 nm) and a green (561 nm) emission were found between 200 and 600 °C visually appearing white. Photoluminescence of annealed biomimetic apatites might be of interest for histological probing and monitoring of bone re-modelling. The results are discussed in terms of chemical and crystallographic changes in the calcium phosphate layer during heat treatment. Ó 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Photoluminescence; Biomimetic apatite; Calcium phosphate; Simulated body ﬂuid; Electron microscopy
1. Introduction Diseased or damaged tissue of the human body can be replaced by a variety of materials such as metals, polymers or ceramics depending on the mechanical, chemical and biological properties of the tissue to be substituted . The restoration materials are relatively bioinert (i.e. titanium and its alloys), bioactive (i.e. BioglassÒ, hydroxylapatite (HA, Ca10(PO4)6(OH)2) or resorbable (tricalcium phosphate (TCP, Ca3(PO4)2) according to the respective tissue response. Bioactive ﬁxation is deﬁned as interfacial bonding of an implant to * Corresponding author. Tel.: +49 9131 8527560; fax: +49 9131 8528311. E-mail address: [email protected]
the tissue by means of the formation of a biologically active hydroxy carbonated apatite (HCA) layer on the implant surface . It was shown that the formation of a bonelike HCA surface layer and the resultant bonebonding ability of an implant can be evaluated by testing the material in an acellular simulated body ﬂuid (SBF) with ion concentrations nearly equal to those of the inorganic part of human blood plasma [3,4]. Since SBF is supersaturated with respect to HCA at 37 °C, the solution has been widely used for precipitation of nano-scale calcium phosphate powders and for biomimetic coating of implant surfaces under mild conditions [5–8]. Photo-stimulated luminescence of biocompatible materials is an appealing approach in observing the distribution of bioactive compounds in surgery, tissue engineering or bone re-modelling [9–11]. Recently, the
1742-7061/$ - see front matter Ó 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2005.06.004
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near-infrared (NIR) stimulated emission of modiﬁed biocompatible quantum dots has permitted real time imaging of sentinel lymph nodes under the skin and facilitated major cancer surgery . The in vitro behaviour of biomedical calcium phosphates monitored by cathodoluminescence showed that the emission of synthetic apatites is primarily characterised by an intrinsic luminescence, whereas the luminescence of naturally occurring apatites is frequently activated by trace elements . The photo-stimulated luminescence of mineral apatite in a calcite matrix was described for identiﬁcation and quantiﬁcation of major and trace elements . Rare earth elements were identiﬁed as ion luminescence activators for calcium phosphates, i.e. carbonated apatites, mainly in the UV and visible region [15,16]. The luminescence properties point to the interest of doped apatites as biological probes. However, doped apatites are often prepared by sintering at temperatures above 1100 °C. The large crystallites resulting from accelerated growth at these elevated temperatures do not interact well with biological media, and they are not readily internalised by living cells . The inherent luminescence of biomimetic apatites activated at temperatures below sintering might be of speciﬁc interest for histological probing, since rare earth elements and other photo-emission enhancing compounds can be omitted, so that the risk of possible release of toxic dopants can be avoided. In this paper, we report on the manufacturing and properties of biomimetically derived inherent luminescent apatite coatings on titanium metal.
2. Experimental part 2.1. Sample preparation Commercially pure titanium square plates (Timet GmbH, Germany) with an edge length of 10 mm and a thickness of 1 mm were etched in 37 wt.% hydrochloric acid (HCl) under argon atmosphere for 2 h at 50 °C. Subsequently the specimens were soaked in 10 M NaOH aqueous solution at 60 °C for 24 h, washed with distilled water and dried at 100 °C [7,18]. The chemically activated samples were exposed to SBF for 2 weeks in order to obtain homogeneous, dense HCA coatings. The SBF with ionic concentrations equal to human blood plasma was prepared by mixing concentrated solutions of KCl, NaCl NaHCO3, MgSO4 Æ 7 H2O, CaCl2 and KH2PO4 into double distilled water and buﬀered with trishydroxymethyl aminomethan and HCl to pH = 7.4 at 37 °C. Sodium azide (NaN3) was added to the solution to inhibit the growth of bacteria. The composition of the applied SBF solution was the same as described in previous papers [7,18]. The activated Ti-plates and the calcium phosphate coated samples were thermally treated at temperatures between 200 and 800 °C in air.
The applied heating rate was 5 °C/min and the annealing temperature was held for 1 h. Cooling the specimens to room temperature was performed with a rate of 10 °C/min. 2.2. Characterisation Fourier-transform infrared (FT-IR) spectra were measured in transmission using the KBr-technique in the range from 4000 to 400 cm 1 at a resolution of 4 cm 1 (Impact 420, Nicolet Instruments, USA). Approximately 1 mg of the annealed calcium phosphate-coatings was removed from the substrate, mixed with 300 mg of dry KBr powder and ground using an agate mortar and pestle. The resulting mixture was pressed into transparent pellets with a diameter of 13 mm applying a force of 105 N (Perkin–Elmer Hydraulic Press, Germany). The crystalline phases were determined by X-ray diffraction (XRD) using CuKa radiation at a scan rate of 0.75° min 1 over a 2h range of 5–80° (Kristalloﬂex D 500, Siemens, Germany). The microstructures and phase composition of the ceramic coatings were characterised by transmission electron microscopy (TEM) (CM 30, Phillips, The Netherlands) operated at 300 kV. The annealed biomimetic apatite coatings were removed from the substrate, suspended in iso-propanol and ground using an agate mortar and pestle. The resulting suspension was ultrasonicated at a frequency of 50 kHz for 1 min and 7 lL of this suspension were transferred onto a carbon coated copper TEM-grid and allowed to dry. Selected area diﬀraction (SAD) patterns were calibrated against polycrystalline aluminium. The element composition was determined by energy dispersive X-ray spectrometry (TEM-EDS) in the TEM equipped with a SiLi-detector (7370 ISIS 30, Link, United Kingdom). Individual apatite particles were selected in the scanning transmission electron microscopy (STEM) mode for EDS point analysis. The photoluminescence (PL) spectra were recorded with a ﬂuorescence spectrometer (J&M Analytische Mess- und Regeltechnik, Germany) equipped with ﬁbre optics for excitation and detection of the emission. The annealed calcium phosphate coated Ti-plates were illuminated at an angle of 45° and a distance of approximately 5 mm from the platelet surface from 310 nm to 450 nm excitation wavelength. The step width of the monochromator (MLX 300, J&M, Germany) was set to 1 nm and the integration time was 500 ms. The PLsignal was detected (MMS/16, J&M, D) at an angle of 45° relative to the substrate surface between 400 nm and 650 nm. The data sets were evaluated with the Fluoroscan software (FL 3095, J&M, Germany). The amount of trace elements such as manganese in the SBF solutions was measured by inductively cou-
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pled-plasma optical emission spectroscopy (ICP-OES) (Spectroﬂame, Spectro, Germany). The calibration was performed with a standard solution of Mn2+ ions (Merck, Germany), which was diluted to a concentration of 1 and 0.1 ppm Mn2+.
3. Results and discussion The chemically activated titanium samples were exposed to SBF for two weeks to obtain homogeneous, dense HCA coatings with an average thickness of 10 lm. The photoluminescence (PL) measurements were performed on the biomimetic apatite coatings, which were annealed at diﬀerent temperatures between 200 and 800 °C (Fig. 1). In control experiments, chemically
activated titanium plates without apatite coating were annealed under the same conditions. These control samples did not show photo-stimulated luminescence within the whole temperature range (Fig. 1a). The development of a blue (437 nm) and a chartreuse (green-yellow, 561 nm) emission was observed for annealed biomimetic apatites as a function of temperature. The measured PLintensity increased with rising temperature up to a maximum peak intensity at approximately 400 °C. Both emissions decreased at higher annealing temperatures and ﬁnally vanished above 600 °C (Fig. 1b). It should be noted that the PL behaviour appeared to be an irreversible function of the applied temperature. After annealing to temperatures exceeding 600 °C, the PL could not be recovered by heating the same sample to 400 °C.
Fig. 1. Photoluminescence of biomimetic apatites: (a) emission spectra obtained at 371 nm excitation wavelength of activated, annealed titanium plates as function of temperature; (b) annealed, biomimetic apatite coatings as function of temperature; (c) 2D-contour plot of the measured PLemission as a function of the excitation wavelength of biomimetic apatite coating after annealing at 400 °C and (d) development of the blue (437 nm) and the chartreuse (561 nm) emission excited at 371 nm as a function of the annealing temperature. The PL-spectra were background corrected.
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The local maxima of the emission peaks were centred at 437 nm (blue) and 556 nm (chartreuse) as shown by 2D-PL measurements in the excitation wavelength range from 325 to 400 nm (Fig. 1c). The maximum of the background corrected peak for the blue emission (437 nm) after curve ﬁtting was centred at 350 °C (Fig. 1c), whereas the calculated maximum of the emission for the chartreuse band (561 nm) was located at 375 °C. Biogenic carbonate minerals, such as calcite, aragonite and dolomite, often exhibit an intrinsic blue luminescence . It has been discussed how this luminescence might be due to activation with manganese ions at very low concentrations in the range 1–100 ppm. In highly pure calcite or calcites with only minor traces of substituting elements the intrinsic luminescence exhibits peak maxima at approximately 400 nm, 575 nm and 650 nm (manganese activated) [15,20,21]. In our case the measured concentration of manganese in the SBF and biomimetic calcium phosphate precipitations, however, was below 0.8 ppm according to ICPOES. Therefore, manganese ions might be excluded from activating the luminescence observed in the biomimetic apatite coatings. Another explanation refers to the recombination of a self-trapped electron (CO33 ) and a hole, which results in an excited CO23 . The relaxation of the excited CO23 was supposed to give rise to the intrinsic luminescence . The presence of CO23 , which replaces the PO34 (B-type substitution), was identiﬁed for our samples at temperatures below 550 °C. The chartreuse emission (561 nm) might be attributed to excitement and recombination of unpaired electrons on defect centres (e.g. broken bonds between calcium and oxygen ions and also oxygen vacancies ) as might be concluded by the decrease of the (Ca + Mg)/P ratio
with temperature (transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDS) measurements, see below). Halogen containing apatites (so-called halophosphates) are known to be excellent phosphors by the illumination industry [23–25]. A green-blue emission at an excitation wavelength of 254 nm was reported for undoped calcium chlorophosphates [26,27]. A considerable amount of chlorine (2.8 at.%) was detected by TEMEDS in the calcium phosphate coating annealed at 200–600 °C which exhibited pronounced luminescence. The Cl might be incorporated into the crystal lattice of the biomimetic apatites forming chloroapatites by substituting the OH groups. In apatite coatings fabricated at room temperature and not exposed to higher temperatures, however, only 0.1 at.% Cl were found. Since the Ti plates were activated with 37 wt.% HCl, Cl might remain on the surface. During annealing at temperatures above 200 °C, Cl ions diﬀuse into the apatite layer thereby raising the Cl concentration as conﬁrmed by TEM-EDS analysis. At temperatures above 600 °C sublimation of alkali chlorides  is supposed to lead to a depletion of halogen in the biomimetic apatite coating. However, both the blue and the chartreuse emission vanished at temperatures exceeding 600 °C. After annealing to 800 °C the Cl concentration was reduced to 0.1 at.%. FTIR spectra of the biomimetic coatings before and after thermal treatment were recorded in transmission using the KBr-technique (Fig. 2a). A broad absorption band at 3450 cm 1 and the bending mode at 1650 cm 1 proved the presence of H2O in the biomimetic apatite coatings. The asymmetric P–O stretching mode (m3) of the P–O bond of the PO34 group (1200– 960 cm 1) indicated a deformation of the phosphate
Fig. 2. Characterisation of biomimetic coatings precipitated on chemically treated titanium after exposure to SBF for two weeks without and with annealing at 200, 400, 600 and 800 °C for 1 h in air: (a) selected FTIR spectra and (b) X-ray diﬀractograms; RT = room temperature.
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coordination symmetry from their tetrahedral structure . The triple (m4) and double (m2) degenerated bending modes of the O–P–O bonds were found at 604, 567 and 474 cm 1 . A characteristic peak at 875 cm 1 indicated the presence of HPO24 in the crystal lattice. The bands at 1500 cm 1, 1430 cm 1 and 875 cm 1 were assigned to the CO23 group of B-type carbonated apatite. However, the bands at 1490, 1070 and 875 cm 1 can be as well associated with amorphous calcium carbonate . The FTIR spectra of the biomimetic apatite coatings annealed at 200 °C were merely the same as for the untreated specimens. All the bands that are characteristic for carbonated apatites described above were observed in the FTIR-spectra for the specimens prepared at this temperature. The water absorption at 1650 cm 1 disappeared due to evaporation of water at temperatures higher than 200 °C. A new band appeared at 1560 cm 1 after heating to 400 °C indicating an A-type substitution in HCA. At this temperature the presence of pyrophosphate P2 O47 was expected resulting from the decomposition of HPO24 . However, no vibra-
tion of a pyrophosphate group (720 cm 1) was observed even after heating the sample to 600 °C probably due to the reaction of HPO24 with CO23 to form PO34 [17,32]. Further heating to 800 °C led to a partial transformation of HCA to b-TCP. The low transformation temperature indicated that the HCA was Ca-deﬁcient, in contrast to stoichiometric HA, which transforms into tetracalcium phosphate and aTCP at substantially higher temperatures (>1230 °C) . The transformation was conﬁrmed by the presence of bands at 1120, 982, 955 and 557 cm 1 generally assigned to b-TCP. The XRD patterns of chemically activated titanium exposed to SBF with biomimetic apatite coating were identiﬁed as HCA of low crystallinity indicated by peak broadening (Fig. 2b). A preferred orientation in respect to the c-axis of the deposited crystals was implied due to the signiﬁcantly higher relative peak intensity of the (0 0 2) and (0 0 4) planes at 2h = 26° and 53°, respectively, compared to the 100% intensity of the (2 1 1) peak at 2h = 32°. The broad peak at 32° is composed of the reﬂections from (2 1 1) at 31.87°, (1 1 2) at 32.18° and
Fig. 3. TEM bright ﬁeld (BF) micrographs of calcium phosphate precipitates annealed at: (a) 200 °C; (b) 400 °C; (c) 600 °C and (d) 800 °C; the scale bar represents 100 nm; insets are selected area diﬀraction (SAD) diagrams of the depicted region.
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(3 0 0) at 32.87°. The peak intensity ratio I(0 0 2)/I(2 2 1) was calculated applying a peak deconvolution procedure based on Gaussian curve ﬁtting. While for an untextured powder sample this ratio is 0.41, a signiﬁcantly higher value of 2.5 was derived for the biomimetic coating layers. At temperatures below 400 °C the composition of the calcium phosphate layer remained virtually unchanged compared to samples without thermal treatment. At temperatures above 600 °C, a mixture of HCA and magnesium doped tricalcium phosphate (MTCP, Ca2.81Mg0.19(PO4)2) was formed. The diﬀraction peaks of TiO2 were due to the oxidation of the titanium surface at temperatures above 800 °C. Ti peaks were observed in all XRD-patterns. This is due to the fact that the calcium phosphate layers had a thickness of approximately 10 lm, which is lower than the penetration depth of CuKa radiation into the sample. The crystallite size of the HCA-crystals calculated using the Scherrer equation from the XRD-diagrams increased from 7.5 nm after calcium phosphate layer deposition to 8.3, 9.0 and 13.1 nm after annealing the samples to 200, 400 and 600 °C, respectively. From room temperature up to 400 °C, the calcium phosphate precipitate consisted of shapeless nano-aggregates (Fig. 3a and b). The corresponding SAD diagrams proved the polycrystalline microstructure of the biomimetic apatite coatings. Individual particles with an average size of 36 nm were ﬁrst discernible at 600 °C. Increasing the temperature to 800 °C resulted in grain growth and particles with diameters of 100 nm were observed. The particle size observed by TEM is not identical to the crystallite size determined from the XRD data, because an individual particle might be composed of several crystallites. Incipient sintering at temperatures above 600 °C might explain the increase in particle size and that the particle dimensions according to the TEM image analysis are larger than the crystallite sizes obtained from the Scherrer equation. TEM-EDS analyses on several arbitrary selected calcium phosphate particles showed an average (Ca + Mg)/P ratio of 1.52 for the specimens prepared at room temperature. Annealing at 200 °C resulted in a (Ca + Mg)/P ratio of 1.58, which steadily decreased to 1.50 at 400 °C, 1.46 at 600 °C and 1.43 at 800 °C. The gradual decrease of the (Ca + Mg)/P ratio from 1.58 to 1.43 is a result of phase transformations during annealing. This is accompanied by crystallographic rearrangements that might be associated with the formation of calcium vacancies .
4. Conclusion Hydroxy carbonated apatite (HCA) coatings on titanium were obtained by biomimetic mineralisation from SBF. The HCA layers exhibited a pronounced PL with a maximum blue emission (437 nm) and chartreuse
emission (561 nm) after annealing in air between 350 and 375 °C. The PL decreased with increasing temperature and ﬁnally vanished at temperatures exceeding 600 °C. The presence of chlorine was detected by TEM-EDS solely in luminescent specimens giving rise to the formation of halophosphates as potential phosphors. Thus, the observed PL might be a superposition of both the PL of chloroapatites and the recombination of holes and electrons resulting in excited, radiant carbonate centres. To our knowledge, PL of un-doped biomimetically derived apatites is reported for the ﬁrst time. Due to the fact that the biocompatibility of sintered calcium phosphates has already been demonstrated and that biomimetic apatite coatings are applied on commercially available Ti-implants , the observed inherent PL of the annealed apatite coatings might be of speciﬁc interest for histological probing and monitoring of bone re-modelling.
Acknowledgement The TEM work was carried out at the Central Facility for High-Resolution Electron Microscopy of the Friedrich-Alexander of University Erlangen-Nuernberg, Germany. The authors are indebted to Rudolf Weißmann (Glass and Ceramics) for helpful discussions. Financial support of the Deutsche Forschungsgemeinschaft (MU 1803/1) and the University of ErlangenNuernberg is gratefully acknowledged.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version at doi:10.1016/ j.actbio.2005.06.004.
References  Ratner B, Hoﬀman AS, Schoen FJ, Lemons JE, editorsBiomaterials science: An introduction to materials in medicine. Amsterdam: Elsevier; 2004.  Hench LL. Biomaterials: a forecast for the future. Biomaterials. 1998;19:1419–23.  Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solutions able to reproduce in vivo surface-structure changes in bioactive glass–ceramic A/W. J Biomed Mater Res 1990;24: 721–34.  Jona´sˇova´ L, Helebrant L, Sˇanda L. The inﬂuence of simulated body ﬂuid composition on carbonated hydroxyapatite formation. Ceramics–Silika´ty 2002;46:9–14.  Kokubo T. Formation of biologically active bone-like apatite on metals and polymers by a biomimetic process. Thermochim Acta 1996;280/281:479–90.  Habibovic P, Barre`re F, van Blitterswijk CA, de Groot K, Layrolle P. Biomimetic hydroxyapatite coating on metal implants. J Am Ceram Soc 2002;85:517–22.
C. Zollfrank et al. / Acta Biomaterialia 1 (2005) 663–669  Jona´sˇova´ L, Mu¨ller FA, Helebrant A, Strnad J, Greil P. Biomimetic apatite formation on chemically treated titanium. Biomaterials. 2004;25:1187–94.  Mu¨ller FA, Jona´sˇova´ L, Cromme P, Zollfrank C, Greil P. Biomimetic apatite formation on chemically modiﬁed cellulose templates. Key Eng Mater 2004;254-256:1111–4.  Andreoni A. Time-resolved luminescence spectroscopy of photosensitizers of biomedical interest. Photochem Photobiol 1990;52:423–30.  Nicolaides L, Mandelis A, Abrams SH. Novel dental dynamic depth proﬁlometric imaging using simultaneous frequencydomain infrared photothermal radiometry and laser luminescence. J Biomed Opt 2000;5:31–9.  Rice BW, Cable MD, Nelson MB. In vivo imaging of lightemitting probes. J Biomed Opt 2001;6:432–40.  Kim S, Lim YT, Soltesz EG, De Grand AM, Lee J, Nakayama A, et al. Near-infrared ﬂuorescent type II quantum dots for sentinel lymph node mapping. Nature Biotechnol 2004;22:93–7.  Go¨tze J, Heimann RB, Hildebrandt H, Gburek U. Microstructural investigation into calcium phosphate biomaterials by spatially resolved cathodoluminescence. Matwiss Werkstoﬀtech 2001;32:130–6.  Utui RJ, Homman NPO, Yang C, Malmqvist KG, Tembe SS. Nuclear microprobe analysis of Evate (Mozambique) apatites. Nucl Instrum Methods B 1995;104:432–6.  Mason RA, Mariano AN. Cathodoluminescence activation in manganese-bearing and rare earth-bearing synthetic calcites. Chem Geol 1990;88/1-2:191–206.  Mayer I, Layani JD, Givan A, Gaft M, Blanc P. La ions in precipitated hydroxyapatites. J Inorg Biochem 1999;73:221–6.  Doat A, Pelle´ F, Gardant N, Lebugle A. Synthesis of luminescent bioapatite nanoparticles for utilization as biological probes. J Solid State Chem 2004;177:1179–87.  Jona´sˇova´ L, Mu¨ller FA, Helebrant A, Strnad J, Greil P. Hydroxyapatite formation on alkali-treated titanium with diﬀerent content of Na+ in the surface layer. Biomaterials. 2002;23: 3095–101.  Habermann D, Newer RD, Richter DK. Low limit of Mn2+activated cathodoluminescence of calcite: state of the art. Sediment Geol 1998;116:13–24.
 Sippel RF, Glover ED. Structures in carbonate rocks made visible by luminescence petrography. Science. 1965;150:1283–7.  Machel HG, Mason RA , Mariano AN, Mucci A. Causes and emission of luminescence in calcite and dolomite. In: Barker CE, Kopp OC, editors. Luminescence microscopy and spectroscopy: qualitative and quantitative applications. SEPM short course, vol. 25; 1991. p. 9–25.  Calde´rdon T, Aguilar M, Jaque F, Coy-Yll R. Thermoluminescence from natural calcite. J Phys 1984;17:2038–77.  Doherty M, Harrison W. Preparation and characteristics of calcium halophosphates. Br J Appl Phys 1955;6:11–6.  Shionoya S, Yen WM. The phosphor handbook. Boca Raton: CRC Press; 1998.  Blasse G, Grabmaier BC. Luminescent materials. Berlin: Springer-Verlag; 1994.  Campbell NR (patent agent), British Patent 512,154; 1938.  Cornock AF (patent agent), British Patent 578,192; 1942.  Miller RC, Kusch P. Molecular composition of alkali halide vapors. J Chem Phys 1956;25:860–76.  Stoch A, Jastrzebski W, Brozek A, Stoch J, Szatraniec J, Trybalska B, et al. FTIR absorption–reﬂection study of biomimetic growth of phosphates on titanium implants. J Mol Struct 2000;555:375–82.  Koutsopoulos S. Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods. J Biomed Mater Res 2002;62:600–12.  Loste E, Rory M, Wilson M, Seshadri R, Meldrum FC. The role of magnesium in stabilising amorphous calcium carbonate and controlling calcite morphologies. J Cryst Growth 2003;254: 206–18.  Lafon JP, Champion E, Bernache-Assollant D, Gibert R, Danna AM. Thermal decomposition of carbonated calcium phosphate apatites. J Therm Anal Calor 2003;72:1127–34.  Elliott JC. Structure and chemistry of the apatites and other calcium orthophosphates. Amsterdam: Elsevier; 1994.  Ivanova TI, Frank-Kamenetskaya OV, Koltsov AB, Ugolkov VL. Crystal structure of calcium-deﬁcient carbonated hydroxyapatite. Thermal decomposition. J Solid State Chem 2001;160:340–9.  Li P. Biomimetic nano-apatite coating capable of promoting bone in-growth. J Biomed Mater Res 2003;66A:79–85.