Monetite-Assisted Growth of Micrometric Ca-Hydroxyapatite Crystals

Article pubs.acs.org/crystal

Monetite-Assisted Growth of Micrometric Ca-Hydroxyapatite Crystals from Mild Hydrothermal Conditions Linda Pastero*,†,‡,§ and Dino Aquilano† †

Department of Earth Sciences, University of Torino, Via Valperga Caluso 35, 10125 Torino, Italy NIS Interdepartmental Center for Nanostructured Interfaces and Surfaces, Via Pietro Giuria 7, Torino, Italy § CrisDi Interdepartmental Center for Crystallography, Via Pietro Giuria 7, Torino, Italy ‡

S Supporting Information *

ABSTRACT: Calcium hydroxyapatite, HAp hereinafter, Ca5(OH)(PO4)3, the main component of vertebrate bones and teeth, plays a strategic role mainly in biomedical applications because of its bioactivity, biocompatibility, and slow-degradation rate. It is a critical bioceramic material due to its properties of osteo-conduction, -integration, and -induction. Moreover, HAp has a role in catalysis, agricultural and pharmaceutical products, protein chromatography, and water and soil treatment as well. The bulk of investigations about HAp concerns nanosized crystals, owing to the difficulties encountered when growing large laboratory crystals. Then, deep information about surface and even bulk properties are unavoidably lacking. In this paper, we investigate the relationship between the HAp polymorphism and its growth morphology both from the experimental and theoretical point of view. The micron-sized and well grown crystals we obtained are exploitable for morphological investigations, in order to better understand detailed surface properties determining the crystal reactivity. Further, a clean and effective HAp method of chemical synthesis is proposed, and the involved crystal-growth mechanisms are extensively investigated as well. Finally, the unexpected synergic effect between the low supersaturation of the HAp solution and the templating effect of the monetite (CaHPO4) crystal, used as precursors, is recognized.

1. INTRODUCTION As a strategic material for its multiple applications, the apatite group of minerals has been the subject of a wide variety of studies. The issue of the growth, stability, and crystal quality of apatites has been faced from many points of view: biomedical (bioceramic applications in implantology, dentistry, reconstructive bone applications), catalysis and pharmaceutics, but also environmental and geological (ore deposits, waste remediation, soil science). In order to prepare large calcium hydroxyapatite (HAp) single crystals, many routines have been proposed both by wet and dry methods. Most of these routines involve the mass crystallization of nanoscaled HAp crystals. A wide range of techniques have been applied in order to answer the considerable amount of questions rising from the natural variability of the composition and structure of these crystals, from electron microscopy to many spectroscopic methods. A comprehensive report of the methods for the synthesis of apatites was published in 1951 by the U.S. Geological Survey.1 It represents a complete collection of the experimental work done on the crystal growth of apatites until the 1950s. Only a few attempts have been made toward the growth of clear and large HAp crystals useful for both single crystal diffraction and morphology (bulk-surface) investigations. Among all these studies, for example, in 1932, Schleede, Smith and Kindt2 obtained the HAp from brushite (CaHPO4· © XXXX American Chemical Society

2H2O) hydrolysis working under hydrothermal conditions for 500 h, and, in 1955, Hayek and co-workers3 obtained nonpure HAp (with a few percent of sodium) from nanoscaled HAp, hydrothermally recrystallized under basic conditions. In 1956, Perloff and Posner4 proposed a wet method to obtain pure and large HAp crystals working at low hydrothermal conditions (300 °C and evaluated water vapor pressure of 87 bar for 10 days). They started from a monetite (CaHPO4) suspension in water and obtained HAp crystals about 300 μm long. The monetite/water ratio was constrained to 1/100 in order to obtain a complete conversion of the precursor into HAp. The method resulted in a decrease in pH value during HAp growth. The final pH ranged between 2 and 2.5 due to the phosphoric acid generated during the reaction. In 1968, Kirn and Leidheiser5 suggested a modification to the method proposed by Perloff, leaving an open door to further work. They obtained large HAp crystals from monetite hydrolysis at 350 °C and about 600 bar, starting from a suspension of monetite in a diluted phosphoric acid solution. As Perloff stated, the final pH ranged around 2.5: it was also found that, at this point, the growth reaches its end-point. Received: October 7, 2015 Revised: January 7, 2016

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DOI: 10.1021/acs.cgd.5b01431 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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In 1983, Chiranjeevirao6 and co-workers proposed a very interesting method, ideally able to modulate the carbonate content of the apatites grown from solution at very low temperature. Unfortunately, the crystals they obtained were nanosized. Recently, Ma and Liu7 obtained HAp from the hydrolysis of brushite, as already proposed by Schleede in 1932.2 Their crystals were suitable for TEM analysis, and it was finally shown that monoclinic HAp could grow at low temperature. It must be stressed that one of the main problems to be faced when growing HAp crystals is the lack of reliable solubility data from the literature. Because of the importance of the HAp stability, mainly in oral biology, many studies on its solubility and its dependence from the pH have been carried out, but the values of the solubility product vary over a range of 10 orders of magnitude, as observed by Wazer,8 Larsen,9 and Chen.10 Moreover, the solubility of HAp is incongruent and strongly dependent on the presence of calcium counterions like carbonate, acetate, and so on.11−16 This is a really challenging point to deal with, in order to control the supersaturation of the system, and should be analyzed in depth. In our work, we obtained large and clear crystals useful for bulk morphological studies and surface analysis. Our early experiments were performed modifying the method proposed by Chiranjeevirao, avoiding CO2 in the crystallization environment by bubbling N2 into the system and imposing a positive nitrogen pressure inside the reactor. All hydroxides were substituted with ammonia so as to limit the presence of carbonates from the hydroxide carbonation: pure nanosized HAp crystals were obtained. Then, we followed the suggestion by Kirn and Leidheiser, improving the performance of the growth method first proposed by Perloff and Posner. The effects of the chemistry of the system on the morphology of the HAp crystals were analyzed. From the hydrolysis reaction of the precursor in mild hydrothermal conditions, we obtained large and pure needle-like HAp crystals with well-developed morphology and some interesting surface structures.

chemically inert vessel for microwave applications (PARR Instrument Company) was used. Growth runs were performed in a household microwave oven at 600 W for 5 min. Because of the operating hazards related to the use of such autoclaves, the samples were cooled slowly to room temperature, and then the crystallization product was filtered and dried overnight as well. 2.1. XRPD. X-ray powder diffraction was carried out on all the precursors and all the obtained HAp samples to check the sample purity. A Siemens D5000 diffractometer whit Bragg−Brentano geometry, 2.5° < 2θ < 100°, step size 0.01, 1 s/step scan time was used for the collection of diffraction data useful for phase determination. When committed to peak shape analysis, the XRPD diagrams were collected in the interval 30° < 2θ < 60°, step size 0.005, and 20 s/step. 2.2. SEM Characterization. SEM imaging of the samples was performed using both a Cambridge S-360 scanning electron microscope equipped with secondary electron (SE) and backscattered electron (BSE) detectors. The typical experimental conditions were W filament, EHT 25 kV, probe current 100 pA, working distance 5 mm and a ZEISS SUPRA 40 field emission scanning electron microscope (FESEM) (WD = 3 mm, aperture size = 30.00 μm, EHT = 5 kV) 2.3. TEM Characterization. Transmission electron microscopy was performed on HAp samples showing the smallest size. A JEOL 3010 UHR-HR TEM (300 kV, beam current = 114 μA LaB6 source and 2k × 2k pixels Gatan CCD camera attached). 2.4. Atomic Force Microscopy (AFM) Characterization. AFM measurements were performed using a DME SPM microscope (DME Igloo, Denmark) equipped with a DS95-50E scanner (scan volume 50 × 50 × 5 μm). Data were acquired using MikroMasch Ultrasharp NSC16/Si3N4 Cr−Au back-coated cantilevers with typical resonance frequency 190 kHz, force constant 45 N/m, tip radius lower than 35 nm, and full tip cone angle 40°. All measurements were performed in alternated contact mode. 2.5. Raman Spectroscopy. A high-resolution confocal μ-Raman system by Horiba Jobin Yvon HR800 was used, equipped with two gratings (1800 and 600 grooves/mm), air cooled CCD detector, and a green polarized lasers (solid state Nd, 532 nm, 250 mW). The system is completed by Edge filters (532 nm) and interferential filters. The maximum resolution with the grating 1800 grooves/mm and green laser is 2 cm−1. 2.6. pH, Conducibility and Ion Chromatography. pH and conductivity measurements were obtained using a Thermo Scientific Orion 4-star benchtop pH/ISE meter equipped with an Orion 9107BN Triode 3-in-1 pH electrode for temperature compensated pH measurements and a Orion DuraProbe 4-electrode conductivity ATC cell with nominal cell constant of 0.475 cm−1. Ion chromatography on solutions was performed using a Metrohm 883 Basic IC plus ion chromatograph. 2.7. Equilibria Calculations. Aqueous solution speciation and equilibria were calculated using PhreeqC software using the Minteq v.4 database for thermodynamic data because of the availability of HAp, brushite, and monetite as crystalline phases. 2.8. XRPD Peak Profile Analysis. The profile analysis on powder diffraction peaks was performed by using the Fityk software.18 Because of the instrumental broadening, the PearsonVII profile was chosen. The built-in function was modified in order to calculate both the Kα1 and Kα2 contributions to the peak shape.

2. EXPERIMENTAL SECTION Monetite (CaHPO4), as HAp precursor, was synthesized following the procedure indicated by Perloff4 and already described in a previous paper.17 Analytical grade H3PO4 (85%) from Sigma-Aldrich was diluted 1:5 in ultrapure water 18.2 MΩ. This solution was saturated at room temperature in calcium phosphate using analytical grade Ca3(PO4)2. The solution was heated slightly under the boiling temperature and continuously stirred. Then, the precipitate was filtered, washed with ultrapure water, and dried at 40 °C overnight and then measured by XRPD and Raman spectroscopy to ensure purity. HAp were grown from a monetite/water suspension using 45 mL PFTE lined stainless steel autoclaves (PARR Instrument Company). A small amount (typically 0.1 g) of the precursor was sealed into the PFTE liner with ultrapure 18.2 MΩ water. The precursor/water weight ratio was kept constant at 1/100. In order to avoid CO2 contamination, all the samples were prepared and sealed into the steel autoclaves in N2 atmosphere, the precursor was kept free from gas before use, and the CO2 water content was controlled by bubbling N2. The autoclaves were put in an oven at a constant temperature of 220 °C (the higher limit for the PFTE liners) and autogenic pressure (estimated of 20 bar by water vapor pressure at that temperature). Reaction products were left into the reactors at constant temperature for a time ranging between 5 days and 3 weeks; then, the autoclaves were quenched and the final product was filtered and air-dried overnight. A variation of the routine was performed using microwave heating instead of the traditional convection oven. In this case a 45 mL,

3. RESULTS AND DISCUSSION 3.1. HAp from Monetite Hydrolysis Following the Perloff’s Routine. The routine proposed by Perloff and Posner in 1956 has proven to be a highly effective approach for the growth of large (more than 300 μm) and optically clear HAp crystals with smooth and well developed surfaces. For the sake of clarity, the reference cell used hereinafter for the HAp is the same as that reported in previous papers dealing with the HAp equilibrium shape and twinning17,19 (monoclinic, P21/c, a0 = 9.4214 Å, b0 ≅ 2a0, c0 = 6.881 Å; α = β = 90°, γ ≅120°). B



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Figure 1. (a) Many generations of crystals can be detected in a sample grown from the hydrolysis of monetite at low hydrothermal conditions; (b) ribbon-like crystals belonging to the last generation are commonly found. Usual twinning is parallel to the elongation of the crystal. (c, d) Under these conditions, not completely dissolved crystals of monetite are regularly found.

Figure 2. pH, conductivity, and concentration of free calcium (black dots) and free phosphate species (white dots) of the main species in solution are recorded during a growth experiment. The system reaches the chemical stability between 18 and 56 h. After 56 h, the production of phosphoric acid rises, lowering the pH. The bottom right graph shows the trend of the gap between the concentration of the phosphate and the calcium species in solution.

XRPD and μ-Raman measurements confirmed the purity of the precursor. Synthesis products, obtained by the hydrothermal way and described in Section 2.2, were confirmed by XRPD data.

Monetite (triclinic, P1̅, a0 = 6.91 Å, b0 = 6.627 Å, c0 = 6.998 Å, α = 96.34°, β = 103.82°, γ = 88.33°) as synthesized by the method described in the following shows an usual {010} flattened habit, with an approximately rhombic outline. C



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Figure 3. (a) General behavior of the crystallization of HAp after 3 weeks of growth; (b) pseudohexagonal morphology of the crystals of the first generation and (c) apparent lower symmetry of the second generation of crystals; (d) crystal twinning.

terms of homogeneous nucleation; as a matter of fact, a foreign crystal phase (monetite) can promote the HAp nucleation, remembering that heterogeneous nucleation can be favored as well, in systems unsaturated with respect to the nucleating phase, when good epitaxial conditions between the two phases are fulfilled. The reaction of monetite in water and the crystallization of HAp can be described as a “three-step process”: (i) During the early stages and immediately after the thermal equilibrium is reached, monetite dissolves and HAp heterogeneous nucleation occurs. The pH suddenly decreases from 7.4 to 3.3. At the same time, the conductivity of the solution rises, due to the increasing amount of free ions in solution (mainly hydrogen ions). After 18 h of reaction, large and clear HAp crystals are already present in the sample (first generation of crystals), but monetite is still abundant, as shown in Figure S2a (see Supporting Information). Even if other transient phases, for example, octacalcium phosphate, known as a HAp precursor in enamel and bones formation,22,23 could be taken in consideration during this first step, XRPD measurements confirmed that only monetite and HAp are present. (ii) Both pH and concentration of the free ions in solution reach a plateau after 18 h. HAp nucleates and grows, indicating that the system is substantially far from equilibrium. Three HAp crystal generations can be described, at least, as detailed at the beginning of the section. The presence of multiple generations of crystals evidences an oscillating supersaturation with a maximum (corresponding to the dissolution of the monetite) and a minimum (corresponding to the HAp nucleation and growth. Moreover, between 18 and 72 h of reaction, the concentration of calcium and phosphate ions follows the same trend, with a gap that lingers constant until 56 h of reaction. Inasmuch as the Ca2+/PO43− ratio in monetite is 1 and in HAp is 5/3, the constant gap between calcium and phosphate during the process of dissolution and recrystallization can be obtained only when an oscillatory behavior of the supersaturation is considered.

At the end of each run, it is possible to recognize at least three generations of HAp crystals: (i) The first generation of large crystals optically clear, showing an undeniable pseudohexagonal morphology and frequently twinned, with the original composition plane of the twin (OCP) parallel to the elongation axis of the crystal,20 as can be seen in Figure 1a. (ii) The second generation of smaller crystals with a pseudohexagonal morphology and evident twinning both parallel and perpendicular to the crystal elongation. (iii) The third generation of small crystals, with an average size of a few micrometers and a tape-like (or ribbon-like) morphology similar to that described by Elliott20 for the biogenic HAp, and by Ma7 for the HAp obtained from brushite hydrolysis. Also the third generation is not twin-free, showing the OCP parallel to the crystal elongation (Figure 1b and Figure S1; see Supporting Information). The multiple nucleation of HAp lead to a broad dispersion of crystal sizes, ranging from a few nanometers up to 300 μm. Moreover, partially dissolved crystals of monetite are frequently detected, even for long reaction times, as shown in Figure 1c,d, pointing out the incompleteness of the reaction. In order to obtain a sequence of snapshots of the chemical behavior of the system during HAp precipitation, growth experiments were repeated, and the samples were quenched at 0 °C (at the water triple point) after 4, 15, 18, 21.5, 32, 56, 72, 96, and 168 h. pH, conductivity, calcium and phosphate concentrations were measured. The chemical trends of the crystal/solution system during the crystallization process are reported in Figure 2. The values for the 96 h long run are not reported because of the unsatisfactory quality of the sample. The HAp stability field calculated with the PHREEQ software21 indicates that HAp is stable, starting from pH = 4.5 up to basic values for temperatures higher than 25 °C, while the pH of the experimental system moves toward low pH values. In these conditions, the presence of HAp crystals with well-developed surfaces and sharp edges cannot be explained in D



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As illustrated in Figure 4, the angle of 14° approximates the angle (13.82°) arising between the [010] direction of the monoclinic HAp and the [001] direction of the monetite when the two crystals are epitaxially related. The corresponding geometrical relationship between the two phases is described in Table 1. The crystal forms we considered are confined to the {010} of monetite and to those HAp forms enclosing the pseudohexagonal prism, due to their morphological importance in natural and synthetic crystals 3.1.1. Geometric Epitaxial Relationships between Host (Monetite) and Guest (HAp) Crystal. There are two extreme kinds of coincidence cells. In the first case (see Table 1a along with Figure 5), their multiplicity is decidedly low (especially for the coupling {010}monetite/{001}HAp). Moreover, the angular misfit (obliquity) for the just mentioned coupling does not exceed 1.68°, proving that the density of screw dislocations at this epitaxial interface should be very low. Thus, the epitaxy between the two phases has to be considered fairly good, due to the high short-range interaction across the common interface. In the second case, the multiplicities are fairly high (see Table 1b), proving that long-range interactions are needed at the epitaxial interface, even if the obliquity is null, so the epitaxy could occur. In between the two extreme cases just illustrated, two other families of lattice coincidences can be found at the interface between the {010} form of monetite and the HAp “prismatic” forms, as documented in the Supporting Information (Figure S3). It is worth outlining that in all the cases we explored the angle formed between the elongation axis of HAp with the z axis of monetite is always 13.82°, confirming our preliminary conclusion at the end of Section 3.1. Summing up, the experimental observation shown in Figure 4 along with the just evidenced lattice coincidences, unambiguously proves that HAp and monetite can be intimately related during growth, from low to high supersaturation values (with respect to HAp) in the mother phase. 3.2. HAp from Monetite Hydrolysis Following a Modified Perloff’s Routine. As realized by Perloff and Posner in 1956, the result of the reaction is strictly dependent on the final pH of the solution: “...Apparently the controlling factor for the hydrolysis is the final pH of the liquid. As long as this pH stays above 2.0 to 2.5, the reaction will proceed in the desired direction...” In our batches, the measured chemical quantities follow the trends illustrated in Figure 2, and the final pH reaches a value of 2.2. As stated in the previous paragraph, this value is related to the excess of phosphoric acid during the hydrolysis reaction of monetite in water. Starting from this observation, a modification of the Perloff’s method was introduced by adding small amounts of phosphoric acid to the initial HAp/water suspension, in order to impose a low pH value since the beginning of the experiment. In this way three goals were reached: (i) The dissolution of monetite is almost complete since the early stages of the reaction. (ii) The supersaturation does not fluctuate during the simultaneous processes of monetite dissolution and HAp nucleation. In this way, the multiple nucleation effect is avoided, and only one generation of crystals is obtained. (iii) the supersaturation of the solution, with respect to the HAp, is lowered, so moving the system out of the HAp stability field, and hence to the decreasing of the nucleation rate.

(iii) After 72 h, the concentration of the free phosphate rises, the pH value decreases to 2.2, while the conductivity of the solution suddenly rises, pointing out that the hydrolysis of the monetite becomes the driving process. The lack of information in this interval is due to the low quality of the data obtained from the sample extracted after 96 h of reaction. After a week of growth (Figure S2b,c), a few crystals of monetite are still present in the solution and HAp crystals exhibit their typical pseudohexagonal aspect showing evident composition planes of the twinning both along and perpendicular to the crystal elongation. After a week of reaction, the quality of the HAp surfaces becomes worthy of careful attention. All crystal surfaces show a rough behavior during the first stages of growth, as shown in Figure S2d, mainly if the closure of the crystal tips is considered. After this period, the HAp single crystals in general exhibit a pseudohexagonal morphology, as shown in Figure 3. Two kinds of OCPs twinning can be found: the most frequent parallel to the elongation axis of the twin (as we just described) and the second one coincident with the 010 plane, and hence perpendicular to the previous one. The detailed description of the twinning law can be found in a recent paper.17 A modification of the Perloff’s method was introduced by growing HAp crystals from a monetite/water suspension in a microwave oven. This allowed exclusion of the role of heat transport by convection on the presence of several crystal generations. After 5 min of microwave radiation (600 W), HAp crystals growth is comparable with that obtained after a week in a conventional oven. At the SEM scale, some evidence of growth relationships between the dissolving precursor and the growing crystals is found, as shown in Figure 4.

Figure 4. (a) Evidence of some growth relationships between monetite and HAp. (b) The white dashed line corresponds to the [010] elongation of the HAp crystal. Solid white lines correspond to the [001] direction of the monetite. (c) The geometrical and morphological relationship between the two crystals is shown in the sketch. E



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Table 1. (a) Short-Range and (b) Long-Range Geometric Epitaxial Relationships between Host (Monetite) and Guest (HAp) Crystal crystal form (host) Monetite {010} area of the 2D coincidence cell (Å)2 {010} area of the 2D coincidence cell (Å)2 {010} area of the 2D coincidence cell (Å)2 {010} 2D area {010} 2D area {010} 2D area

2D-lattice (Å) of the host form 4 × [100] = 27.64 [101̅] = 10.946 187.825 5 × d020 = 16.466 8 × [100] = 55.28 [301] = 20.238 375.65 5 × d020 = 16.466 8 × [100] = 55.28 [301] = 20.238 375.65

4[100] = 27.64 [104] = 27.1816 773.25 8[100] = 55.28 [104] = 27.1816 1502.6 4[100] = 55.28 [104] = 27.1816 1502.6

crystal form (guest) HAp (a) Short-Range {001}

{100}

{102}̅

(b) Long-Range {001}

{100}

{10−2}

2D-lattice (Å) of the guest form

misfit %

3 × [100] = 27.9759 [110̅ ] = 11.6305 194.44

+1.21 +6.25 +3.52

2 × d002 = 16.156 3 × [001] = 55.9308 [011]̅ = 19.897 388.736

−1.92 +1.18 −1.71 +3.48

4 × d100 = 16.156 3 × [201] = 55.965 [211] = 19.9076 388.973

−1.92 +1.24 −1.66 +3.54

3[100] 4[010] 777.76 3[001] 4[010] 1555.5 3[201] 4[010] 1555.2

= 27.9759 = 27.8012 = 55.93 = 27.8012 = 55.94 = 27.8012

1.2 2.3 0.6 1.2 2.3 3.5 1.2 2.3 3.5

2D-coincidence cell multiplicity 4 × (010)monetite

8 × (010)monetite

8 × (010)monetite

16 × (010)monetite

32 × (010)monetite

32 × (010)monetite

A simulation of the system was carried out changing the pH by adding a small amount of H3PO4: the PHREEQ software was used along with a Minteq v.4 database with thermodynamic data for HAp, brushite, and monetite. Being acquainted with the low reliability of thermodynamic data and solubilities of phosphates (the problem of the solubility data discussed previously), we are interested in the relative trend of the saturation indices, not in their absolute values. At high temperature, the HAp becomes supersaturated when pH > 4. This trend confirms the experimental results: lowering the pH suspension allows control of both the 3D homogeneous nucleation rate and the final size of HAp crystals. When pH < 2, monetite recrystallizes in well-formed and large crystals. 3.3. Crystal Characterization. A discussion about equilibrium and growth morphology of single and twinned HAp crystals (both experimental and theoretical) has already been presented in our previous works.17,25,26 At first glance, the growth morphology of HAp crystals seems to be hexagonal, with a well-developed {100} six-faces prism and {101} pyramids, but a careful morphological analysis excludes the hexagonal symmetry. Monoclinic single crystals show three pinacoids: {100}, {102̅ }, and {001} belonging to the zone [010] and the related prisms {110}, {112}̅ , and {012}. The pseudohexagonal growth morphology of the single crystals comes from the sharp surface resemblance of the three pinacoids and then to the quasi equivalence of their kinetic behavior. The twinning is generated through a 3-fold rotation around the [010] direction (A3 ≡ 21 axis), as already discussed in the recent literature. The original composition face (OCF) can either belong to one of the three {h0l} pinacoids or to the basal {010} pinacoid. Twinning occurs since the early stages of

Figure 5. Two extreme coincidence cells of monetite (dots) and HAp (crosses) are shown: the short-range coincidence across the common interface (red) and the long-range one (blue).

At low pH values, monetite totally turns to HAp. Moreover, moving out the system to the stability field of the HAp lowers the nucleation frequency, leading to less HAp crystals with a narrower size dispersion and controlled growth morphology. The presence of many generations of HAp as the result of a multiple nucleation process and the presence of monetite as a leftover of the incomplete dissolution process are avoided. The monotonic decrease of the supersaturation allows crystal size selection and very good crystal quality, as can be seen in Figure 6a,b. In order to confirm the effect of the pH on crystal size and morphology, a countercheck was performed growing HAp crystals from a monetite suspension (obtained using a phosphate buffer solution: pH 7.4, Cold Spring Harbor Protocol,24 CSP). The grown crystals are smaller, and their quality is noticeably lower, as shown in Figure 6c,d. F



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Figure 6. (a, b) HAp grown from a low pH (2.5) suspension of monetite in diluted phosphoric acid. (c, d) HAp crystals from a buffered suspension of monetite (CSP buffer, pH 7.4).

Figure 7. (a) The apparent morphological hexagonal symmetry of the crystal decreases to a trigonal pseudosymmetry (white solid arrows). A large stitch runs along the crystal elongation (white dashed arrow). (b) The 3-fold twinning of the crystal is evidenced by the presence of the triangular hole at the crystal tip (white dotted arrow, see also Figure S5) and of the deep stitches running along the crystal (black dotted lines and arrow). (c) A pseudohexagonal double terminated crystal of HAp showing both the original composition faces, parallel and perpendicular to the crystal elongation. (d, e) Twinned crystals with deep stitches. In (e, f) a surface structure with a symmetry not compatible with an hexagonal one is detected. G



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without reason that, up to now, nothing about surface properties and quality has been mentioned. Lowering the pH shifts the crystallizing system at the limit of the HAp stability field, reducing the nucleation frequency and allowing to grow larger crystals. (ii) Moreover, we demonstrated as well that the epitaxial interaction between HAp and monetite used as a precursor phase should favor the 2D heterogeneous HAp nucleation, allowing the HAp to appear beyond its usual growth conditions. The effect of the choice of the precursor on the HAp growth under mild hydrothermal conditions will be described in a forthcoming paper. It is mandatory to bring up a great issue concerning the solubility of the HAp: the values found in the literature show an unbelievable variability, due mostly to the incongruent crystal solubility and to its dependence from the presence of many omnipresent calcium counterions, like carbonate. This is an unavoidable “impasse” when growing HAp crystals because it prevents the control of the operational supersaturation of the system. From the solubility viewpoint, the low pH conditions adopted in this work contribute to simplify the system, avoiding the inadvertent presence of CO2, even working in nitrogen atmosphere. This implies a lower effect of the incongruent solubility of the HAp and a further simplification of the system with respect to the most diffused routine syntheses that require the use of a basis in order to trigger the HAp precipitation. Concerning the HAp growth morphology, new acquisitions have been obtained: −During the early stages of growth, HAp crystals exhibit a ribbon-like monoclinic morphology and are unambiguously twinned. The ubiquitous 3-fold twin axis leads to the typical pseudohexagonal morphology of the crystal, and, during growth, the stitches that characterize the twin shape disappear, leaving a pseudohexagonal crystal morphology, usually misunderstood. The twinning evidence is either aligned with the crystal elongation (with the original composition plane pertaining to the forms {001}, {102̅}, and {100} that limit the pseudohexagonal prism) or across the crystal elongation (with the original composition plane pertaining to the form {010}). Both the twin growth morphology have been described in a previous paper.17 −The pseudohexagonality of the crystals is demonstrated also by some surface patterns detected at the SEM scale. The surface quality of the pseudoprisms has been investigated by AFM. At the AFM scale, the forms {001}, {102̅}, and {100} of the largest crystals are atomically flat and frequently show scars of stitches that have been generated by the twinning and, in turn, are restored through the dihedral angle effect. Finally, we can say that this is the first report of a reproducible, clean, and easy-to-use method to obtain HAp crystals with well-developed morphologies that can be used for both bulk and surface investigations.

growth, as proven by the presence of large stitches resulting from the concave dihedral angle effect.27 In Figure 7a, at the tip of the crystal, the illusory hexagonal symmetry of the crystal decreases to a trigonal pseudosymmetry. Moreover, a deep groove runs along the crystal, pointing out the existence of the twinning. In Figure 7b, the 3-fold twinning is once more demonstrated by the three-tips funnel rising at the crystal termination, whereas a single crystal has no reason to be concave. Figure 7c shows a pseudohexagonal double terminated HAp crystal. Deep grooves can be seen in correspondence of the twinning sutures, both parallel and perpendicular to the crystal elongation. Both twins follow the same law, but in this case the original composition face belongs to the {010} pinacoid. Two twins, viewed along the [010] twin axis, are shown in Figure 7d,e, the mismatch between the two individuals (building each twin) being underlined by an irregular suture. In Figure 7c,d, the pseudobipyramid shows an overlapping structure that will seal the OCF suture. A manifest evidence of the misleading hexagonal pseudosymmetry is shown in Figure 7e,f, where the lack of symmetry is stressed by the surface patterns that are present only on one side of the crystal edge and consequently cannot be ascribable to the hexagonal symmetry. The AFM surface analysis has been carried on the pseudohexagonal prisms of some large and clear crystals. Their surfaces are atomically flat. The stitches between twinned crystals, both along and perpendicular to the crystal elongation, are well detected, and their behavior is shown in Figure S5. A careful analysis of the XRPD pattern of a representative sample can be useful to fully investigate the symmetry of the HAp crystals obtained by the hydrolysis of the precursor. As was already shown17,28 the peak shape analysis is a reliable method to reveal the overlapping of many reflections lying under a seemingly individual peak. The decomposition of the diffraction pattern is reported in detail in Figure S6. Neither smoothing nor filters were applied to the experimental data. The obviousness of the multiplicity of some diffraction peaks is impressive. For example, in the range 31.5° < 2θ < 33.5°, the hexagonal polymorph should show only three diffraction peaks, whereas the monoclinic one should allow 17 diffraction peaks. The decomposition of the XRPD pattern allow us to definitely assign nine diffraction peaks. The loss of multiplicity is related to the twin operation (120° rotation around the [010] axis) of the monoclinic crystals; that illusory increases the symmetry of the phase, from monoclinic to hexagonal.

4. CONCLUSIONS In this work, we modified the Perloff’s routine and showed that large and optically clear HAp crystals can be produced with controlled size. As a matter of fact, the size dispersion, which is usually related to more than one nucleation event, is avoided. Our objectives have been achieved according to the following path: (i) The pH of the reaction has proven to be the most important variable to control, in order to select the crystal size and improve the quality of their surfaces. Most of the methods proposed up to now require high pH values (usually ranging between 10 and 12) to obtain the HAp mass precipitation; under these conditions the supersaturation of the solution is definitely too high to obtain large crystals. Indeed, in most cases, only nanosized HAp crystals are obtained, and it is not

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01431. Figure S1. TEM images of some twinned HAp crystals. Figure S2. CaHAp twinned crystals from monetite hydrolysis after 18 and 24 h of reaction. Figure S3. H



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Article

(23) Suzuki, O. Acta Biomater. 2010, 6, 3379−3387. (24) Cold Spring Harbor Protocols; Cold Spring Harbor Laboratory Press: Woodbury, NY, 2006; pdb.rec8303. (25) Aquilano, D.; Bruno, M.; Rubbo, M.; Massaro, F. R.; Pastero, L. Cryst. Growth Des. 2014, 14, 2846−2852. (26) Pastero, L.; Cámara, F.; Bruno, M.; Rubbo, M.; Aquilano, D. Acta Crystallogr., Sect. A: Found. Adv. 2014, 70, C1115−C1115. (27) Boistelle, R.; Aquilano, D. Acta Crystallogr., Sect. A: Cryst. Phys., Diffr., Theor. Gen. Crystallogr. 1978, 34, 406−413. (28) Pastero, L.; Aquilano, D.; Moret, M. Cryst. Growth Des. 2012, 12, 2306−2314.

The intermediate coincidence cells of monetite and HAp. Figure S4. A schematic representation of a monoclinic HAp crystal twinned around a 3-fold axis parallel to the [010] of the crystal. Figure S5. AFM topography of the pseudoprism surfaces showing some evidence of the closure of the stitches originated by twinning. Figure S6. XRD peak decomposition performed using a Pearson VII peak profile (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by MIUR (GEO-TECH Project, PRIN 2011-2012). The hydrothermal growth facilities and the AFM laboratory at the Department of Earth Sciences, University of Torino were funded by Fondazione CRT (Grant No. 2014.2187 and Grant No. 2014.1042). The authors are deeply grateful to Dr. Enrico Destefanis for chromatographic measurements, and Dr. Ruggero Vigliaturo and Dr. Mauro Giorcelli for FESEM imaging. We would like to thank the anonymous referee for both criticisms and fruitful suggestions.

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REFERENCES

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Monetite-Assisted Growth of Micrometric Ca-Hydroxyapatite Crystals

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