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Hydroxyapatite synthesis from biogenic calcite single crystals into phosphate solutions at ambient conditions
ARTICLE IN PRESS Journal of Crystal Growth 311 (2009) 4219–4225
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Hydroxyapatite synthesis from biogenic calcite single crystals into phosphate solutions at ambient conditions Francesca Marchegiani, Eleonora Cibej, Patrizia Vergni, Giovanna Tosi, Simona Fermani, Giuseppe Falini Dipartimento di Chimica ‘‘G. Ciamician’’, Alma Mater Studiorum Universita di Bologna, via Selmi 2, 40126 Bologna, Italy
a r t i c l e in fo
abstract
Article history: Received 28 December 2008 Received in revised form 26 June 2009 Accepted 6 July 2009 Communicated by S. Veesler
Hydroxyapatite is one of the most important bone substitute biomaterials. Here, it has been successfully overgrown on biogenic seed crystals at ambient conditions. Single crystals of calcite from Atrina rigida, Paracentrotus lividus and Heterocentrotus mammillatus have been soaked in phosphate solution with different concentrations and pHs for 2 months. X-ray powder diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy have been used to characterize soaking precipitates. The results show that the conversion of calcite to hydroxyapatite occurs to an extent which depends on composition and morphology of seed crystals, and starting concentration and pH of phosphate solutions. In the same experimental conditions, synthetic calcite single crystals did not convert to hydroxyapatite. The morphological observations suggest for hydroxyapatite formation, a mechanism that involves a superficial dissolution of calcite and a subsequently overgrowth of hydroxyapatite. Moreover, the final architectural assembly of the hydroxyapatite crystals resembles the shape of the starting biogenic seed crystals. & 2009 Elsevier B.V. All rights reserved.
PACS: 87.85.jf 87.15.Zg 61.05.cp 89.30.Ee Keywords: A1. Biocrystallization A1. Nanostructure A2. Seed crystals B1. Calcium compounds
1. Introduction Hydroxyapatite (HA, Ca5(PO4)3OH) is one of the most important biomaterials utilized as artificial bone substitute because of its biocompatibility, bioactivity, osteoconductivity, lack of toxicity and inflammatory, and immunogenity [1–3]. The synthesis of HA has been carried out by many different chemical routes that involve solid-state, solution and hydrothermal reactions [4–6]. In the latter, biogenic calcium carbonates, aragonite or calcite, have been used as source of calcium ions. Hydrothermal conversion of biogenic aragonite to HA has been successfully obtained using coral [7,8], cuttlefish bone [9,10] and nacre [11,12]. Biogenic calcite from sea urchin spines was hydrothermally converted to HA and betatricalcium phosphate (BTCP) mixtures [13]. Only in few experiments biogenic calcium carbonates have been converted to HA at ambient conditions. Ni et al. have transformed the aragonitic surface of fragments of nacre to HA by soaking in buffer phosphate solutions [14]. Recently, Guo et al.
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E-mail address: [email protected] (G. Falini). 0022-0248/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcrysgro.2009.07.010
have also reported the conversion of powder aragonitic nacre to HA using phosphate solutions at slightly acidic pHs [15]. Biogenic calcium carbonates are composite inorganic–organic materials. In them, an organic matrix controls the deposition of the mineral phase in its composition, structure, polymorphism and crystallography. Biogenic calcium carbonates are generally formed by calcite or aragonite, the two most stable calcium carbonate polymorphs. They can precipitate as polycrystalline materials or single crystals, being the last ones generally made of calcite or magnesium calcite [16]. Echinoderm sea urchin spines consist of single crystals of magnesium calcite. They have smooth, continuously curved surfaces that form a micrometer-size-spaced three-dimensional fenestrated mineral network [17]. This so-called stereom is moulded by organisms in different shapes and sizes and has regions with different content of magnesium ions in the calcitic structure [18]. The external layer of the mollusc shell Atrina rigida is formed by prismatic single crystals of calcite [19]. All these biogenic calcites host an intra-crystalline organic matrix, which is rich in acidic amino acids, aspartic and glutamic, and it is present in low percentages [16–19]. In this research, single crystals of calcite from Atrina rigida (A. rigida) shell and magnesium calcite from Paracentrotus lividus
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(P. lividus) or Heterocentrotus mammillatus (H. mammillatus) sea urchin spines were used as seeds to precipitate HA. The experiment has been carried out by soaking the biogenic seed crystals in phosphate solutions at ambient conditions. The reason of carrying out experiments in this condition was double. (i) This mild method allows a better discrimination in the reaction properties between biogenic calcite and synthetic calcite. Indeed by using harsh conditions, such as hydrothermal ones, both materials convert completely in hydroxyapatite [13]. (ii) This method requires common laboratory tools with respect to the harsh ones, and thus is of wide applicability. X-ray powder diffraction, FTIR spectroscopy and electron microscopy have been employed to characterize the soaking precipitates as a function of seed crystals and phosphate solutions used. Control experiments have been carried out using synthetic single crystals of calcite. The results suggest that the formation of HA could be described by dissolution/precipitation processes occurring on the surface of seed crystals of calcite.
repeatedly powered in a mortar before the measurements. The quantification of the mineral phases was carried out from the diffraction patterns by Rietveld method using the program for quantitative analysis Quanto [20]. Fourier transform infrared (FTIR) spectra were collocated at room temperature by using a Nicolet FTIR 380 spectrometer working in the range of wavenumbers 4000–400 cm1 at a resolution of 4 cm1. Disk was obtained mixing 1 mg of powered sample with 100 mg of KBr IR grade and applying a pressure of 10 t to the mixture. The morphology of samples and the elemental analysis were investigated utilizing a scanning electron microscope (Philips XL 20) equipped with an EDX detector (Philips Xd-4). The observations were carried out using a tension of 20 kV. The samples were glued on an aluminium stub and coated by sputtering with a gold or carbon layer before the analyses. The specific surface area, SBET (m2/g) was obtained by nitrogen adsorption (Brunauer–Emmet–Teller (BET) model, CONTROL 750, Carlo Erba Instruments).
2. Experimental section 3. Results 2.1. Materials 3.1. Biogenic calcite single crystals characterization Biogenic single crystals of calcite were isolated from fragments of the prismatic layer of A. rigida, which was mechanically detached from the nacreous layer of the shell. The fragments were treated with 2.5% (v/v) NaClO solution for 4 days. After this treatment, single crystals of calcite were isolated by several decantation processes from a water suspension. They were washed three times in milliQ water (1.7 mS) and finally dried at 60 1C for 2 h. Spines of biogenic single crystals of magnesium calcite were removed from P. lividus or H. mammillatus sea urchins. They were treated with a 2.5% (v/v) NaClO solution, to remove extraskeletal organic material, for 24 h on a rocking table. The spines were then extensively washed with milliQ water and dried at 60 1C for 2 h, those ones about 10 mm long were selected. The treatment of biogenic crystals with sodium hypochlorite removes only the extraskeletal organic material, leaving unaffected the intra-crystalline organic matrix. Synthetic single crystals of calcite were grown by diffusion of ammonium carbonate vapour in a 10 mM CaCl2 solution for 4 days. The deposited crystals were rinsed in water and dried at 60 1C for 2 h. Sodium phosphate solutions were prepared adding 1.0 M NaOH solution to 1.0 M H3PO4 solution until the pH value reached 7.5 or 9.0. This stock solution was then diluted to the final concentration (60, 150, 300 or 450 mM) and the pH eventually adjusted. 2.2. Biogenic calcite conversion to hydroxyapatite Biogenic crystals (50 mg) were placed into a plastic Petri’s dish (5.8 mm in diameter) containing 10 ml of phosphate solution with pH 7.5 or 9.0 and concentration of 60, 150, 300 or 450 mM. The crystals were not ground. NaN3 was added to each solution to avoid bacteria and fungi contaminations. The dishes were kept at 20 1C for 2 months. Control experiments were carried out in the same conditions using synthetic single crystals of calcite. Each experiment was repeated at least three times. 2.3. Characterization of soaking precipitates X-ray powder diffraction (XRD) patterns were recorded using a Philips X’Celerator diffractometer with Cu Ka radiation ˚ and a Ni filter. The samples were scanned for 2y (l ¼ 1.5418 A) angles between 51 and 601, with a resolution of 0.021. They were
X-ray powder diffraction patterns from biogenic single crystals are reported in Fig. 1A. In them, only the diffraction peaks of calcite are present. For A. rigida crystals the unit cell parameters ˚ c ¼ 17.008 A), ˚ which were calculated from the (a ¼ b ¼ 4.994 A, diffraction pattern, are the same of synthetic calcite. Unit cells ˚ ˚ having a ¼ b ¼ 4.942 A, c ¼ 16.873 A˚ and a ¼ b ¼ 4.963 A, c ¼ 16.973 A˚ were calculated from the diffraction patterns from spines of P. lividus and H. mammillatus, respectively. According to Bischoff et al. [21], these values indicate an average isomorphic substitution of magnesium to calcium ions in the structure of calcite of about 7.3% in P. lividus spines and 3.5% in H. mammillatus spines. The FTIR spectra of the biogenic calcites are reported in Fig. 1B. They show the typical absorption peaks of calcite (n2 ¼ 875 cm1; n3 ¼ 1420 cm1; n4 ¼ 713 cm1). The peak associated to the vibration mode n4 at 713 cm1 is diagnostic for pure calcite. The shift of this peak to 715 or 718 cm1, which has been observed in the spectrum from H. mammillatus or P. lividus spines, respectively, is due to the isomorphic substitution of magnesium to calcium ions [22]. In the FTIR spectra a broad peak at around 3420 cm1 is also present, this is associated to OH stretching modes. The weak band around 1650 cm1 could be associated to organic matrix present in the biogenic crystals. The SEM pictures in Fig. 2 show the morphologies of biogenic and synthetic calcite crystals. A. rigida single crystals of calcite appear as prisms elongated along the crystallographic c-axis. In them, the growing steps form bands normal to the c-axis (Fig. 2a). The presence of rounded particles of nano-meter size on crystal surface is shown by a high magnification view (Fig. 2d). They aggregate to form a nano-porous structure. The spines, from H. mammillatus and P. lividus, are single crystals of magnesium calcite grown along the crystallographic c-axis (Fig. 2b and c) [18]. The images of the cross sections of these spines show as they have smooth, continuously curved, surfaces that form a micrometersize spaced three-dimensional fenestrated mineral network in which many pores and channels are present (Fig. 2e and f). The synthetic calcite crystal shows the typical {1 0 4} faces rhombohedral morphology, while the magnesium calcite crystal shows additional {11¯ 0} faces (insets Fig. 2a and b). The biogenic calcite crystals and the synthetic ones showed a surface area, SBET, of the same order of magnitude. The one from synthetic crystals was of 0.3 m2/g, while the ones from biogenic
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Fig. 1. X-ray powder diffraction patterns (A) and FTIR spectra (B) of (a) spines from H. mammillatus, (b) spines from P. lividus and (c) prisms from A. rigida. The intensity of the diffraction peaks and the absorption of the FTIR bands are reported in arbitrary units (a. u.). The Miller indexes of each diffraction peak are indicated, according to the hexagonal unit cell of calcite (PDF 01-083-0578). The peak at 1420 cm1 corresponds to the n3 absorption band of carbonate ions; the peaks at 876 and 713 cm1 correspond to n2 and n4 absorption bands of calcite, respectively. The dashed line in FTIR spectra highlights the shift of the n4 band.
Fig. 2. Scanning electron microscopy images of single crystal of (a, d) A. rigida, (b, e) P. lividus and (c, f) H. mammillatus. The arrow indicates the calcite crystallographic caxis. The insets in (a) and (b) show single crystals of pure calcite and magnesium calcite, respectively (scale bars 50 mm). In them, the Miller index of the faces are reported.
crystals were 0. 8, 0.6 and 0.7 m2/g for H. mammillatus, P. Lividus and A. rigida, respectively.
3.2. Conversion of biogenic calcite to hydroxyapatite In this work, biogenic calcite single crystals were soaked in different phosphate solutions (60, 150, 300 or 450 mM) at pH 7.5 or 9.0 for 2 months. Synthetic single crystals of calcite were used as control. The crystals were not ground. In general, the X-ray powder diffraction patterns from the soaked precipitates showed the presence of diffraction peaks of HA, together with those one of calcite, only when biogenic calcites were used as seed crystals. They were always absent in XRD patterns when seed crystals of synthetic calcite were used. It is important to note that in the carried out experimental conditions, rich of carbonate ions, the formation of carbonate hydroxyapatite occurs. The substitution of magnesium to calcium ions in the HA structure was not detected (EDS analyses) in HA synthesized using biogenic magnesium
calcites. The XRD patterns of biogenic seed crystals after soaking in 450 mM phosphate solution at pH 7.5 or 9.0 are reported in Fig. 3A and B, respectively. These are examples of diffraction patterns in which a high conversion of biogenic calcite to hydroxyapatite occurred. In them, the main diffraction peaks of hydroxyapatite, at about 321 and 25.81 of 2y, are observable together with those ones typical of calcite. The relative intensity of the diffraction peak at 25.81, the one associated to the (0 0 2) crystalline plane, is higher than that one reported for the reference (PDF 98-009-4278). This observation has been also confirmed during the quantification of the mineral phases by the Rietveld refinement process of the calculated diffraction profile, in which the intensity of the reflection (0 0 2) has been increased imposing a preferential growth of HA crystals along the c-axis. However, these crystals did not show any preferential orientation detectable by single crystal X-ray diffractometric techniques, although the presence of a local preferential orientation cannot be excluded. In Table 1, the relative amount of HA present in the final precipitate and the final pH of each experiment are
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Fig. 3. X-ray powder diffraction patterns of the biogenic calcium carbonate crystals after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5 (A) or pH 9.0 (B). (a) Spines from H. mammillatus, (b) spines from P. lividus and (c) prisms from A. rigida. * And # indicate diffraction peaks of calcite and hydroxyapatite, respectively. The intensity of the diffraction peaks are reported in arbitrary units (a. u.).
Table 1 Relative amount (% w/w) of hydroxyapatite overgrown on biogenic crystals after soaking for 2 months in phosphate solutions having different concentrations and pH 7.5 or 9.0. C (mM)
A. rigida
H. mammillatus
pHi 7.5
60 150 300 450
pHi 9.0
P. lividus
pHi 7.5
pHi 9.0
pHi 7.5
pHi 9.0
%
pHf
%
pHf
%
pHf
%
pHf
%
pHf
%
pHf
24(3) 32(3) 47(3) 43(3)
8.6(2) 8.4(2) 8.4(2) 8.2(2)
– – 12(3) 24(3)
8.3(2) 8.4(2) 8.6(2) 9.0(2)
11(3) 9(3) 30(3) 21(3)
7.9(2) 8.1(2) 7.7(2) 8.2(2)
3(2) 14(3) 14(3) 16(3)
8.5(2) 8.9(2) 9.0(2) 9.0(2)
– – 12(3) 20(3)
8.0(2) 7.6(2) 7.6(2) 7.6(2)
– – 13(3) 13(3)
8.3(2) 8.3(2) 8.8(2) 9.0(2)
The initial and final pH of each experiment are also reported. In parenthesis, the standard deviation is indicated. pHi and pHf indicate starting and final pHs, respectively.
reported. In general, the relative amount of HA overgrown on the biogenic calcitic single crystals at pH 7.5 is higher than that one at pH 9.0 and increases with the phosphate solution concentration. Moreover, HA has been always detected when starting phosphate concentrations above 300 mM were used. At pH 7.5, the highest conversion of A. rigida (47%) and H. mammillatus (30%) spines to HA was observed when a 300 mM phosphate solution was used, while for P. lividus (20%) spines this occurred when a 450 mM phosphate solution was used. At pH 9.0, the highest percentage of HA in the precipitate was always found using a 450 mM phosphate solution. Using 60 mM phosphate solution, HA overgrew on A. rigida (24%) prisms at pH 7.5 and on H. mammillatus spines at pH 7.5 (11%) and at pH 9.0 (trace, 3%). Solution pHs after soaking processes (pHf) varied as function of starting concentration of phosphate solutions and extent of biogenic seed crystals conversion to HA. When starting the pH was 7.5, the final pHs always increased; while when it was 9.0, the final values always decreased. These variations did not show clear trends, but change of pH generally increased with the degree of conversion of calcite to HA and using phosphate solutions having low-starting concentrations. It is worth to note that also when no detectable amounts of HA were found, as in the control experiments, the pH changed. It varied from 7.5 to 7.8 or from 9.0 to 8.6 when 60 or 150 mM phosphate solutions were used. The FTIR spectra of samples treated in 450 mM phosphate solution at pH 7.5 and 9.0 are shown in Fig. 4. In them are present
the characteristic absorption peaks of phosphate (n3 ¼ 1032 cm1, n4 ¼ 604 cm1, 564 cm1) [23] and those ones of calcite. Their relative intensity is function of the ratio amount of the two phases, in agreement with the X-ray data. A weak absorption band at 1650 cm1 is also observed in the spectra. Images of A. rigida crystals covered by HA are reported in Fig. 5a–c (starting pH 7.5) and Fig. 5d–f (starting pH 9.0). Using a starting of pH 7.5 (Fig. 5b), HA formed flower-like aggregates made of plate-like crystals, each having an average length and thickness of about 2 mm and 100 nm, respectively. In Fig. 5c, a region of prism surface with a HA layer partially broken and overgrown at pH 7.5 is shown. In it, HA crystals are packed and are closely associated to the calcitic surface in almost all the interface. The calcitic surface appears etched in the region where the HA layer detached. The HA plate-like crystals overgrown on A. rigida crystals using starting pH 9.0 form also a compact layer of aggregates (Fig. 5e), similar to those ones shown in Fig. 5c. In Fig. 5f, a surface region in which HA plate-like crystals start to grow and calcite surface to be etched is shown. Images of H. mammillatus and P. lividus after soaking in 450 mM phosphate solution at pH 7.5 are reported in Fig. 6. The biogenic crystals appear completely covered by the plate-like crystals of HA and their overall morphology is conserved (Fig. 6a and d). In Fig. 6b and c, a broken layer of HA crystals on the surface of the H. mammillatus and HA crystals at the early stage of overgrowth, respectively, are shown. Also, in these case spine surface is etched
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Fig. 4. FTIR spectra of biogenic calcium carbonate crystals after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5 (A) and pH 9.0 (B). (a) Spines from H. mammillatus, (b) spines from P. lividus and (c) prisms from A. rigida. The intensity of the diffraction peaks are reported in arbitrary units (a. u.). The main absorption bands of HA (564, 604 and 1032 cm1) and calcite (713, 870 and1420 cm1) are reported.
Fig. 5. SEM images from A. rigida prism after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5 (a)–(c) or pH 9.0 (d)–(f). The prism conserves its shape while calcite gradually converts to HA.
in the region where the HA overgrowth occurred. In Fig. 6e and f, a cross-section view at two magnifications of a P. lividus spine after soaking is shown. It is possible to observe etched regions of magnesium calcite which are partially filled with growing HA plate-like crystals. As general observation, the dimension of the HA plate-like crystals increases with their distance from crystal seeds surface.
precipitate of calcium phosphates. Since calcium phosphate phases are less soluble than calcium carbonate this process is thermodynamically favoured. The precipitation of one specific phase of calcium phosphate is controlled by solution pH. HA precipitation involves at least the following two chemical reactions: CaCO3-Ca2++CO2 3
4. Discussion 5Ca2++3PO3 4 +OH -Ca5(PO4)3(OH)
The conversion mechanism of calcium carbonate into HA has been proposed to occur by means of a dissolution/recrystallization reaction [11]. Calcium carbonate after soaking in phosphate solutions starts to dissolve releasing calcium ions in solution. Then, these ions react with phosphate ion species forming a
These reactions take place at the surface of biogenic single crystals, which progressively dissolves and acts as substrate for heterogeneous nucleation of HA. The concentration of the various ionic species present in solution is pH dependent. The solubility of
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Fig. 6. SEM images from single crystal of H. mammillatus (a)–(c) and P. lividus (d)–(f) after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5. Magnesium calcite spine converts in HA by a dissolution/recrystallization mechanism. The shape of spines is conserved in all their regions.
calcium carbonate at pH 7.5 is higher than at pH 9.0, thus more calcium ions are dissolved in solution. The concentration of phosphate (PO3 4 ) and hydroxyl (OH ) ions in solution is higher at pH 9.0 than at pH 7.5. Synthetic calcite crystals are less soluble than biogenic ones. This is because the latter host the organic matrix that destabilizes the crystalline structure of calcite [24]. Moreover, the isomorphic substitution of magnesium to calcium ions also destabilizes the structure of calcite and further increases the solubility of biogenic crystals [25]. However, since magnesium ions inhibit the precipitation of HA, this phenomenon should not favour HA crystallization [26]. The different surface area of calcitic single crystals also influences the deposition of HA. The final pH of the experiment gives information on reactions occurring in solution and on calcitic crystals surface. The dissolution of calcium ions and the consequent precipitation of HA crystals concentration in solution. The decrease of PO3 reduces PO3 4 4 amount alters the equilibrium distribution of phosphate species in 2 solution. At pH 7.5, the main species are H2PO 4 and HPO4 (pKa ¼ 7.2), while at pH 9.0 the species mainly present in solution and PO3 (pKa ¼ 10.2). The pH is also influenced are HPO2 4 4 by the dissolution of calcite, which releases CO2 3 ions in solution. When the starting concentration of phosphate solution is high (300 or 450 mM), the relative decrease of PO3 4 concentration due to the precipitation of HA is low and the pH remains almost constant. The degree of conversion of calcite crystals to HA needs to be discussed in view of the above parameters. Powder X-ray diffraction patterns showed that the conversion reaction calcite—HA did not occur when synthetic crystals of calcite were used. However, when dilute solutions of phosphate were used, the final pH changed (from 7.5 to 7.8 or from 9.0 to 8.6 when 60 or 150 mM phosphate solutions were used). This indicates that chemical reactions occurred, although HA was not detectable by XRD and FTIR. The low solubility of synthetic calcite, compared to that one of biogenic calcites, may be considered the key parameter of its un(low)-reactivity. Moreover, the synthetic crystals do not contain nucleating acidic macromolecules, present in biogenic crystals, that could favour HA heterogeneous nucleation process. At pH 7.5, A. rigida prisms of calcite are highly converted in HA crystals, which represent about 45% of the total mass. This substrate matches favourable conditions. It releases more calcium ions and offers a slightly higher surface area (0.7 m2/g) than the synthetic calcite (0.3 m2/g). Moreover, when compared with the biogenic spines, it does not release magnesium ions in solution. Indeed, the biogenic spines from P. lividus and H. mammillatus, which contain,
respectively, 8.0 and 4.5% of magnesium ions in the structure of calcite and have surface area of 0. 8 and 0.6 m2/g, were able to overgrow an amount of HA crystals up to 20% and 30% of their overall mass, respectively. The observation of SEM images helps to describe processes which take place on calcitic surfaces. The images show etched regions of the surface, on which HA crystal overgrew. In the early stage of HA overgrowth, the crystals plate surface is almost normal to the surface of biogenic calcite. In the further stages of HA growth, the dimension of the crystals increases and the crystal plates do not show any preferential orientation. This observation is more marked at pH 7.5 than at pH 9.0, at which a lower HA overgrowth is present. The starting preferential orientation may suggest a role of the calcitic substrate as nucleating template on the HA (11 0) face [27]. Thus, in this stage, it cannot be excluded that the organic matrix plays a role. Indeed, the regions where the HA nucleation starts are those ones with the highest solubility of calcite and probably the richest in organic matrix. Addadi et al. demonstrated that acid macromolecules extracted from biogenic calcites are able to interact with calcium phosphate salts altering their morphology [28]. Moreover, synthetic calcite crystals, which do not contain acidic macromolecules, do not overgrow HA in the same experimental conditions. The dissolution/precipitation mechanism has also been applied to explain bone-like apatite formation on Bioglass [29,30]. Formation of a HA layer on Bioglass can be explained in terms of release of calcium ions and the action of the hydrated silica surface as nucleating sites. As previously mentioned, the organic matrix may serve as template for crystallization in a similar way. The SEM images show that the surface of the biogenic crystals is completely covered by HA crystals. This is an advantage for biomaterial implant applications. HA coverage is desirable for implants because HA can form strong bonds with natural bone. Moreover, HA cannot be directly used as load-bearing joint implants because it is brittle. The biogenic calcite crystals have outstanding mechanical properties, with fracture toughness 3000 times higher than that one of pure calcite [16,18]. Thus, from the standpoint of mechanic, they are attractive for bone implants, having a potential role similar to that of titanium scaffolds.
5. Conclusions In conclusion, this study shows that HA crystals can be deposited on biogenic single crystals of calcite from phosphate
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solution by surface reactions. In this process, the biogenic crystals have a double role: (i) they are the source of calcium ions and (ii) they act as template to obtain complex architectural assemblies of HA crystals.
Acknowledgments We thank Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, the University of Bologna (Funds for Selected Topics) and Ministero dell’Istruzione dell’Universita e della Ricerca for the financial support. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
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Hydroxyapatite synthesis from biogenic calcite single crystals into phosphate solutions at ambient conditions Francesca Marchegiani, Eleonora Cibej, Patrizia Vergni, Giovanna Tosi, Simona Fermani, Giuseppe Falini Dipartimento di Chimica ‘‘G. Ciamician’’, Alma Mater Studiorum Universita di Bologna, via Selmi 2, 40126 Bologna, Italy
a r t i c l e in fo
abstract
Article history: Received 28 December 2008 Received in revised form 26 June 2009 Accepted 6 July 2009 Communicated by S. Veesler
Hydroxyapatite is one of the most important bone substitute biomaterials. Here, it has been successfully overgrown on biogenic seed crystals at ambient conditions. Single crystals of calcite from Atrina rigida, Paracentrotus lividus and Heterocentrotus mammillatus have been soaked in phosphate solution with different concentrations and pHs for 2 months. X-ray powder diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy have been used to characterize soaking precipitates. The results show that the conversion of calcite to hydroxyapatite occurs to an extent which depends on composition and morphology of seed crystals, and starting concentration and pH of phosphate solutions. In the same experimental conditions, synthetic calcite single crystals did not convert to hydroxyapatite. The morphological observations suggest for hydroxyapatite formation, a mechanism that involves a superficial dissolution of calcite and a subsequently overgrowth of hydroxyapatite. Moreover, the final architectural assembly of the hydroxyapatite crystals resembles the shape of the starting biogenic seed crystals. & 2009 Elsevier B.V. All rights reserved.
PACS: 87.85.jf 87.15.Zg 61.05.cp 89.30.Ee Keywords: A1. Biocrystallization A1. Nanostructure A2. Seed crystals B1. Calcium compounds
1. Introduction Hydroxyapatite (HA, Ca5(PO4)3OH) is one of the most important biomaterials utilized as artificial bone substitute because of its biocompatibility, bioactivity, osteoconductivity, lack of toxicity and inflammatory, and immunogenity [1–3]. The synthesis of HA has been carried out by many different chemical routes that involve solid-state, solution and hydrothermal reactions [4–6]. In the latter, biogenic calcium carbonates, aragonite or calcite, have been used as source of calcium ions. Hydrothermal conversion of biogenic aragonite to HA has been successfully obtained using coral [7,8], cuttlefish bone [9,10] and nacre [11,12]. Biogenic calcite from sea urchin spines was hydrothermally converted to HA and betatricalcium phosphate (BTCP) mixtures [13]. Only in few experiments biogenic calcium carbonates have been converted to HA at ambient conditions. Ni et al. have transformed the aragonitic surface of fragments of nacre to HA by soaking in buffer phosphate solutions [14]. Recently, Guo et al.
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have also reported the conversion of powder aragonitic nacre to HA using phosphate solutions at slightly acidic pHs [15]. Biogenic calcium carbonates are composite inorganic–organic materials. In them, an organic matrix controls the deposition of the mineral phase in its composition, structure, polymorphism and crystallography. Biogenic calcium carbonates are generally formed by calcite or aragonite, the two most stable calcium carbonate polymorphs. They can precipitate as polycrystalline materials or single crystals, being the last ones generally made of calcite or magnesium calcite [16]. Echinoderm sea urchin spines consist of single crystals of magnesium calcite. They have smooth, continuously curved surfaces that form a micrometer-size-spaced three-dimensional fenestrated mineral network [17]. This so-called stereom is moulded by organisms in different shapes and sizes and has regions with different content of magnesium ions in the calcitic structure [18]. The external layer of the mollusc shell Atrina rigida is formed by prismatic single crystals of calcite [19]. All these biogenic calcites host an intra-crystalline organic matrix, which is rich in acidic amino acids, aspartic and glutamic, and it is present in low percentages [16–19]. In this research, single crystals of calcite from Atrina rigida (A. rigida) shell and magnesium calcite from Paracentrotus lividus
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(P. lividus) or Heterocentrotus mammillatus (H. mammillatus) sea urchin spines were used as seeds to precipitate HA. The experiment has been carried out by soaking the biogenic seed crystals in phosphate solutions at ambient conditions. The reason of carrying out experiments in this condition was double. (i) This mild method allows a better discrimination in the reaction properties between biogenic calcite and synthetic calcite. Indeed by using harsh conditions, such as hydrothermal ones, both materials convert completely in hydroxyapatite [13]. (ii) This method requires common laboratory tools with respect to the harsh ones, and thus is of wide applicability. X-ray powder diffraction, FTIR spectroscopy and electron microscopy have been employed to characterize the soaking precipitates as a function of seed crystals and phosphate solutions used. Control experiments have been carried out using synthetic single crystals of calcite. The results suggest that the formation of HA could be described by dissolution/precipitation processes occurring on the surface of seed crystals of calcite.
repeatedly powered in a mortar before the measurements. The quantification of the mineral phases was carried out from the diffraction patterns by Rietveld method using the program for quantitative analysis Quanto [20]. Fourier transform infrared (FTIR) spectra were collocated at room temperature by using a Nicolet FTIR 380 spectrometer working in the range of wavenumbers 4000–400 cm1 at a resolution of 4 cm1. Disk was obtained mixing 1 mg of powered sample with 100 mg of KBr IR grade and applying a pressure of 10 t to the mixture. The morphology of samples and the elemental analysis were investigated utilizing a scanning electron microscope (Philips XL 20) equipped with an EDX detector (Philips Xd-4). The observations were carried out using a tension of 20 kV. The samples were glued on an aluminium stub and coated by sputtering with a gold or carbon layer before the analyses. The specific surface area, SBET (m2/g) was obtained by nitrogen adsorption (Brunauer–Emmet–Teller (BET) model, CONTROL 750, Carlo Erba Instruments).
2. Experimental section 3. Results 2.1. Materials 3.1. Biogenic calcite single crystals characterization Biogenic single crystals of calcite were isolated from fragments of the prismatic layer of A. rigida, which was mechanically detached from the nacreous layer of the shell. The fragments were treated with 2.5% (v/v) NaClO solution for 4 days. After this treatment, single crystals of calcite were isolated by several decantation processes from a water suspension. They were washed three times in milliQ water (1.7 mS) and finally dried at 60 1C for 2 h. Spines of biogenic single crystals of magnesium calcite were removed from P. lividus or H. mammillatus sea urchins. They were treated with a 2.5% (v/v) NaClO solution, to remove extraskeletal organic material, for 24 h on a rocking table. The spines were then extensively washed with milliQ water and dried at 60 1C for 2 h, those ones about 10 mm long were selected. The treatment of biogenic crystals with sodium hypochlorite removes only the extraskeletal organic material, leaving unaffected the intra-crystalline organic matrix. Synthetic single crystals of calcite were grown by diffusion of ammonium carbonate vapour in a 10 mM CaCl2 solution for 4 days. The deposited crystals were rinsed in water and dried at 60 1C for 2 h. Sodium phosphate solutions were prepared adding 1.0 M NaOH solution to 1.0 M H3PO4 solution until the pH value reached 7.5 or 9.0. This stock solution was then diluted to the final concentration (60, 150, 300 or 450 mM) and the pH eventually adjusted. 2.2. Biogenic calcite conversion to hydroxyapatite Biogenic crystals (50 mg) were placed into a plastic Petri’s dish (5.8 mm in diameter) containing 10 ml of phosphate solution with pH 7.5 or 9.0 and concentration of 60, 150, 300 or 450 mM. The crystals were not ground. NaN3 was added to each solution to avoid bacteria and fungi contaminations. The dishes were kept at 20 1C for 2 months. Control experiments were carried out in the same conditions using synthetic single crystals of calcite. Each experiment was repeated at least three times. 2.3. Characterization of soaking precipitates X-ray powder diffraction (XRD) patterns were recorded using a Philips X’Celerator diffractometer with Cu Ka radiation ˚ and a Ni filter. The samples were scanned for 2y (l ¼ 1.5418 A) angles between 51 and 601, with a resolution of 0.021. They were
X-ray powder diffraction patterns from biogenic single crystals are reported in Fig. 1A. In them, only the diffraction peaks of calcite are present. For A. rigida crystals the unit cell parameters ˚ c ¼ 17.008 A), ˚ which were calculated from the (a ¼ b ¼ 4.994 A, diffraction pattern, are the same of synthetic calcite. Unit cells ˚ ˚ having a ¼ b ¼ 4.942 A, c ¼ 16.873 A˚ and a ¼ b ¼ 4.963 A, c ¼ 16.973 A˚ were calculated from the diffraction patterns from spines of P. lividus and H. mammillatus, respectively. According to Bischoff et al. [21], these values indicate an average isomorphic substitution of magnesium to calcium ions in the structure of calcite of about 7.3% in P. lividus spines and 3.5% in H. mammillatus spines. The FTIR spectra of the biogenic calcites are reported in Fig. 1B. They show the typical absorption peaks of calcite (n2 ¼ 875 cm1; n3 ¼ 1420 cm1; n4 ¼ 713 cm1). The peak associated to the vibration mode n4 at 713 cm1 is diagnostic for pure calcite. The shift of this peak to 715 or 718 cm1, which has been observed in the spectrum from H. mammillatus or P. lividus spines, respectively, is due to the isomorphic substitution of magnesium to calcium ions [22]. In the FTIR spectra a broad peak at around 3420 cm1 is also present, this is associated to OH stretching modes. The weak band around 1650 cm1 could be associated to organic matrix present in the biogenic crystals. The SEM pictures in Fig. 2 show the morphologies of biogenic and synthetic calcite crystals. A. rigida single crystals of calcite appear as prisms elongated along the crystallographic c-axis. In them, the growing steps form bands normal to the c-axis (Fig. 2a). The presence of rounded particles of nano-meter size on crystal surface is shown by a high magnification view (Fig. 2d). They aggregate to form a nano-porous structure. The spines, from H. mammillatus and P. lividus, are single crystals of magnesium calcite grown along the crystallographic c-axis (Fig. 2b and c) [18]. The images of the cross sections of these spines show as they have smooth, continuously curved, surfaces that form a micrometersize spaced three-dimensional fenestrated mineral network in which many pores and channels are present (Fig. 2e and f). The synthetic calcite crystal shows the typical {1 0 4} faces rhombohedral morphology, while the magnesium calcite crystal shows additional {11¯ 0} faces (insets Fig. 2a and b). The biogenic calcite crystals and the synthetic ones showed a surface area, SBET, of the same order of magnitude. The one from synthetic crystals was of 0.3 m2/g, while the ones from biogenic
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Fig. 1. X-ray powder diffraction patterns (A) and FTIR spectra (B) of (a) spines from H. mammillatus, (b) spines from P. lividus and (c) prisms from A. rigida. The intensity of the diffraction peaks and the absorption of the FTIR bands are reported in arbitrary units (a. u.). The Miller indexes of each diffraction peak are indicated, according to the hexagonal unit cell of calcite (PDF 01-083-0578). The peak at 1420 cm1 corresponds to the n3 absorption band of carbonate ions; the peaks at 876 and 713 cm1 correspond to n2 and n4 absorption bands of calcite, respectively. The dashed line in FTIR spectra highlights the shift of the n4 band.
Fig. 2. Scanning electron microscopy images of single crystal of (a, d) A. rigida, (b, e) P. lividus and (c, f) H. mammillatus. The arrow indicates the calcite crystallographic caxis. The insets in (a) and (b) show single crystals of pure calcite and magnesium calcite, respectively (scale bars 50 mm). In them, the Miller index of the faces are reported.
crystals were 0. 8, 0.6 and 0.7 m2/g for H. mammillatus, P. Lividus and A. rigida, respectively.
3.2. Conversion of biogenic calcite to hydroxyapatite In this work, biogenic calcite single crystals were soaked in different phosphate solutions (60, 150, 300 or 450 mM) at pH 7.5 or 9.0 for 2 months. Synthetic single crystals of calcite were used as control. The crystals were not ground. In general, the X-ray powder diffraction patterns from the soaked precipitates showed the presence of diffraction peaks of HA, together with those one of calcite, only when biogenic calcites were used as seed crystals. They were always absent in XRD patterns when seed crystals of synthetic calcite were used. It is important to note that in the carried out experimental conditions, rich of carbonate ions, the formation of carbonate hydroxyapatite occurs. The substitution of magnesium to calcium ions in the HA structure was not detected (EDS analyses) in HA synthesized using biogenic magnesium
calcites. The XRD patterns of biogenic seed crystals after soaking in 450 mM phosphate solution at pH 7.5 or 9.0 are reported in Fig. 3A and B, respectively. These are examples of diffraction patterns in which a high conversion of biogenic calcite to hydroxyapatite occurred. In them, the main diffraction peaks of hydroxyapatite, at about 321 and 25.81 of 2y, are observable together with those ones typical of calcite. The relative intensity of the diffraction peak at 25.81, the one associated to the (0 0 2) crystalline plane, is higher than that one reported for the reference (PDF 98-009-4278). This observation has been also confirmed during the quantification of the mineral phases by the Rietveld refinement process of the calculated diffraction profile, in which the intensity of the reflection (0 0 2) has been increased imposing a preferential growth of HA crystals along the c-axis. However, these crystals did not show any preferential orientation detectable by single crystal X-ray diffractometric techniques, although the presence of a local preferential orientation cannot be excluded. In Table 1, the relative amount of HA present in the final precipitate and the final pH of each experiment are
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Fig. 3. X-ray powder diffraction patterns of the biogenic calcium carbonate crystals after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5 (A) or pH 9.0 (B). (a) Spines from H. mammillatus, (b) spines from P. lividus and (c) prisms from A. rigida. * And # indicate diffraction peaks of calcite and hydroxyapatite, respectively. The intensity of the diffraction peaks are reported in arbitrary units (a. u.).
Table 1 Relative amount (% w/w) of hydroxyapatite overgrown on biogenic crystals after soaking for 2 months in phosphate solutions having different concentrations and pH 7.5 or 9.0. C (mM)
A. rigida
H. mammillatus
pHi 7.5
60 150 300 450
pHi 9.0
P. lividus
pHi 7.5
pHi 9.0
pHi 7.5
pHi 9.0
%
pHf
%
pHf
%
pHf
%
pHf
%
pHf
%
pHf
24(3) 32(3) 47(3) 43(3)
8.6(2) 8.4(2) 8.4(2) 8.2(2)
– – 12(3) 24(3)
8.3(2) 8.4(2) 8.6(2) 9.0(2)
11(3) 9(3) 30(3) 21(3)
7.9(2) 8.1(2) 7.7(2) 8.2(2)
3(2) 14(3) 14(3) 16(3)
8.5(2) 8.9(2) 9.0(2) 9.0(2)
– – 12(3) 20(3)
8.0(2) 7.6(2) 7.6(2) 7.6(2)
– – 13(3) 13(3)
8.3(2) 8.3(2) 8.8(2) 9.0(2)
The initial and final pH of each experiment are also reported. In parenthesis, the standard deviation is indicated. pHi and pHf indicate starting and final pHs, respectively.
reported. In general, the relative amount of HA overgrown on the biogenic calcitic single crystals at pH 7.5 is higher than that one at pH 9.0 and increases with the phosphate solution concentration. Moreover, HA has been always detected when starting phosphate concentrations above 300 mM were used. At pH 7.5, the highest conversion of A. rigida (47%) and H. mammillatus (30%) spines to HA was observed when a 300 mM phosphate solution was used, while for P. lividus (20%) spines this occurred when a 450 mM phosphate solution was used. At pH 9.0, the highest percentage of HA in the precipitate was always found using a 450 mM phosphate solution. Using 60 mM phosphate solution, HA overgrew on A. rigida (24%) prisms at pH 7.5 and on H. mammillatus spines at pH 7.5 (11%) and at pH 9.0 (trace, 3%). Solution pHs after soaking processes (pHf) varied as function of starting concentration of phosphate solutions and extent of biogenic seed crystals conversion to HA. When starting the pH was 7.5, the final pHs always increased; while when it was 9.0, the final values always decreased. These variations did not show clear trends, but change of pH generally increased with the degree of conversion of calcite to HA and using phosphate solutions having low-starting concentrations. It is worth to note that also when no detectable amounts of HA were found, as in the control experiments, the pH changed. It varied from 7.5 to 7.8 or from 9.0 to 8.6 when 60 or 150 mM phosphate solutions were used. The FTIR spectra of samples treated in 450 mM phosphate solution at pH 7.5 and 9.0 are shown in Fig. 4. In them are present
the characteristic absorption peaks of phosphate (n3 ¼ 1032 cm1, n4 ¼ 604 cm1, 564 cm1) [23] and those ones of calcite. Their relative intensity is function of the ratio amount of the two phases, in agreement with the X-ray data. A weak absorption band at 1650 cm1 is also observed in the spectra. Images of A. rigida crystals covered by HA are reported in Fig. 5a–c (starting pH 7.5) and Fig. 5d–f (starting pH 9.0). Using a starting of pH 7.5 (Fig. 5b), HA formed flower-like aggregates made of plate-like crystals, each having an average length and thickness of about 2 mm and 100 nm, respectively. In Fig. 5c, a region of prism surface with a HA layer partially broken and overgrown at pH 7.5 is shown. In it, HA crystals are packed and are closely associated to the calcitic surface in almost all the interface. The calcitic surface appears etched in the region where the HA layer detached. The HA plate-like crystals overgrown on A. rigida crystals using starting pH 9.0 form also a compact layer of aggregates (Fig. 5e), similar to those ones shown in Fig. 5c. In Fig. 5f, a surface region in which HA plate-like crystals start to grow and calcite surface to be etched is shown. Images of H. mammillatus and P. lividus after soaking in 450 mM phosphate solution at pH 7.5 are reported in Fig. 6. The biogenic crystals appear completely covered by the plate-like crystals of HA and their overall morphology is conserved (Fig. 6a and d). In Fig. 6b and c, a broken layer of HA crystals on the surface of the H. mammillatus and HA crystals at the early stage of overgrowth, respectively, are shown. Also, in these case spine surface is etched
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Fig. 4. FTIR spectra of biogenic calcium carbonate crystals after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5 (A) and pH 9.0 (B). (a) Spines from H. mammillatus, (b) spines from P. lividus and (c) prisms from A. rigida. The intensity of the diffraction peaks are reported in arbitrary units (a. u.). The main absorption bands of HA (564, 604 and 1032 cm1) and calcite (713, 870 and1420 cm1) are reported.
Fig. 5. SEM images from A. rigida prism after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5 (a)–(c) or pH 9.0 (d)–(f). The prism conserves its shape while calcite gradually converts to HA.
in the region where the HA overgrowth occurred. In Fig. 6e and f, a cross-section view at two magnifications of a P. lividus spine after soaking is shown. It is possible to observe etched regions of magnesium calcite which are partially filled with growing HA plate-like crystals. As general observation, the dimension of the HA plate-like crystals increases with their distance from crystal seeds surface.
precipitate of calcium phosphates. Since calcium phosphate phases are less soluble than calcium carbonate this process is thermodynamically favoured. The precipitation of one specific phase of calcium phosphate is controlled by solution pH. HA precipitation involves at least the following two chemical reactions: CaCO3-Ca2++CO2 3
4. Discussion 5Ca2++3PO3 4 +OH -Ca5(PO4)3(OH)
The conversion mechanism of calcium carbonate into HA has been proposed to occur by means of a dissolution/recrystallization reaction [11]. Calcium carbonate after soaking in phosphate solutions starts to dissolve releasing calcium ions in solution. Then, these ions react with phosphate ion species forming a
These reactions take place at the surface of biogenic single crystals, which progressively dissolves and acts as substrate for heterogeneous nucleation of HA. The concentration of the various ionic species present in solution is pH dependent. The solubility of
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Fig. 6. SEM images from single crystal of H. mammillatus (a)–(c) and P. lividus (d)–(f) after soaking for 2 months in 450 mM sodium phosphate solution at pH 7.5. Magnesium calcite spine converts in HA by a dissolution/recrystallization mechanism. The shape of spines is conserved in all their regions.
calcium carbonate at pH 7.5 is higher than at pH 9.0, thus more calcium ions are dissolved in solution. The concentration of phosphate (PO3 4 ) and hydroxyl (OH ) ions in solution is higher at pH 9.0 than at pH 7.5. Synthetic calcite crystals are less soluble than biogenic ones. This is because the latter host the organic matrix that destabilizes the crystalline structure of calcite [24]. Moreover, the isomorphic substitution of magnesium to calcium ions also destabilizes the structure of calcite and further increases the solubility of biogenic crystals [25]. However, since magnesium ions inhibit the precipitation of HA, this phenomenon should not favour HA crystallization [26]. The different surface area of calcitic single crystals also influences the deposition of HA. The final pH of the experiment gives information on reactions occurring in solution and on calcitic crystals surface. The dissolution of calcium ions and the consequent precipitation of HA crystals concentration in solution. The decrease of PO3 reduces PO3 4 4 amount alters the equilibrium distribution of phosphate species in 2 solution. At pH 7.5, the main species are H2PO 4 and HPO4 (pKa ¼ 7.2), while at pH 9.0 the species mainly present in solution and PO3 (pKa ¼ 10.2). The pH is also influenced are HPO2 4 4 by the dissolution of calcite, which releases CO2 3 ions in solution. When the starting concentration of phosphate solution is high (300 or 450 mM), the relative decrease of PO3 4 concentration due to the precipitation of HA is low and the pH remains almost constant. The degree of conversion of calcite crystals to HA needs to be discussed in view of the above parameters. Powder X-ray diffraction patterns showed that the conversion reaction calcite—HA did not occur when synthetic crystals of calcite were used. However, when dilute solutions of phosphate were used, the final pH changed (from 7.5 to 7.8 or from 9.0 to 8.6 when 60 or 150 mM phosphate solutions were used). This indicates that chemical reactions occurred, although HA was not detectable by XRD and FTIR. The low solubility of synthetic calcite, compared to that one of biogenic calcites, may be considered the key parameter of its un(low)-reactivity. Moreover, the synthetic crystals do not contain nucleating acidic macromolecules, present in biogenic crystals, that could favour HA heterogeneous nucleation process. At pH 7.5, A. rigida prisms of calcite are highly converted in HA crystals, which represent about 45% of the total mass. This substrate matches favourable conditions. It releases more calcium ions and offers a slightly higher surface area (0.7 m2/g) than the synthetic calcite (0.3 m2/g). Moreover, when compared with the biogenic spines, it does not release magnesium ions in solution. Indeed, the biogenic spines from P. lividus and H. mammillatus, which contain,
respectively, 8.0 and 4.5% of magnesium ions in the structure of calcite and have surface area of 0. 8 and 0.6 m2/g, were able to overgrow an amount of HA crystals up to 20% and 30% of their overall mass, respectively. The observation of SEM images helps to describe processes which take place on calcitic surfaces. The images show etched regions of the surface, on which HA crystal overgrew. In the early stage of HA overgrowth, the crystals plate surface is almost normal to the surface of biogenic calcite. In the further stages of HA growth, the dimension of the crystals increases and the crystal plates do not show any preferential orientation. This observation is more marked at pH 7.5 than at pH 9.0, at which a lower HA overgrowth is present. The starting preferential orientation may suggest a role of the calcitic substrate as nucleating template on the HA (11 0) face [27]. Thus, in this stage, it cannot be excluded that the organic matrix plays a role. Indeed, the regions where the HA nucleation starts are those ones with the highest solubility of calcite and probably the richest in organic matrix. Addadi et al. demonstrated that acid macromolecules extracted from biogenic calcites are able to interact with calcium phosphate salts altering their morphology [28]. Moreover, synthetic calcite crystals, which do not contain acidic macromolecules, do not overgrow HA in the same experimental conditions. The dissolution/precipitation mechanism has also been applied to explain bone-like apatite formation on Bioglass [29,30]. Formation of a HA layer on Bioglass can be explained in terms of release of calcium ions and the action of the hydrated silica surface as nucleating sites. As previously mentioned, the organic matrix may serve as template for crystallization in a similar way. The SEM images show that the surface of the biogenic crystals is completely covered by HA crystals. This is an advantage for biomaterial implant applications. HA coverage is desirable for implants because HA can form strong bonds with natural bone. Moreover, HA cannot be directly used as load-bearing joint implants because it is brittle. The biogenic calcite crystals have outstanding mechanical properties, with fracture toughness 3000 times higher than that one of pure calcite [16,18]. Thus, from the standpoint of mechanic, they are attractive for bone implants, having a potential role similar to that of titanium scaffolds.
5. Conclusions In conclusion, this study shows that HA crystals can be deposited on biogenic single crystals of calcite from phosphate
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solution by surface reactions. In this process, the biogenic crystals have a double role: (i) they are the source of calcium ions and (ii) they act as template to obtain complex architectural assemblies of HA crystals.
Acknowledgments We thank Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, the University of Bologna (Funds for Selected Topics) and Ministero dell’Istruzione dell’Universita e della Ricerca for the financial support. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
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