Crystallization at Multiple Sites inside Particles of Amorphous Calcium

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CRYSTAL GROWTH & DESIGN

Crystallization at Multiple Sites inside Particles of Amorphous Calcium Phosphate Chen-Guang Wang,†,§ Jia-Wang Liao,‡,§ Bao-Di Gou,† Jian Huang,† Rui-Kang Tang,*,# Jin-Hui Tao,# Tian-Lan Zhang,*,†,‡ and Kui Wang*,†

2009 VOL. 9, NO. 6 2620–2626

Department of Chemical Biology, Peking UniVersity School of Pharmaceutical Sciences, 38 Xueyuan Road, Beijing 100191, P. R. China, Department of Chemical Biology, Capital Medical UniVersity School of Pharmaceutical Sciences, 10 Xitoutiao, You An Men, Beijing 100069, P. R. China, and Department of Chemistry and Center for Biomaterials and Biopathways, Zhejiang UniVersity, Hangzhou, Zhejiang, 310027, P. R. China ReceiVed September 23, 2008; ReVised Manuscript ReceiVed March 9, 2009

ABSTRACT: Calcium phosphates are the main minerals in human bone, enamel, atherosclerosis, and dental calculus. Amorphous precursors may play a key role in biomineralization. We studied the formation and transformation of calcium phosphate particles of amorphous phase by stopped-flow spectrophotometry, simultaneous measurements of particle size and solution pH, and high-resolution transmission electron microscopy. Ion pairs and clusters formed in the first few seconds. They then constituted initial amorphous phase containing protonated phosphates and hydrated calcium ions, which was different from that containing Ca9(PO4)6. Crystalline domains developed at multiple sites inside the primary particles of the amorphous phase. With the consuming of interdomain constituents, these particles partially collapsed, liberating crystallites and inducing rapid precipitation. This study sheds new light on the understanding of crystallization in amorphous phase, as well as the induction period in precipitation kinetics. Introduction Calcium phosphates are the main minerals not only in human bone and enamel, but also in pathological calcifications, such as atherosclerosis and dental calculus.1 The control of cells and proteins on calcium phosphate formation is executed ultimately following chemical and physical principles. Since amorphous nanoparticle precursors are thermodynamically as well as kinetically preferred under the constraint of small particle size, they may play impotent roles in crystallization.2-4 In supersaturated solutions, amorphous calcium phosphate (ACP) usually forms in bulk and then transforms into the thermodynamically stable crystalline phase.5 Besides, in preparation of a supersaturated solution, the mixing of calcium and phosphate solutions often generates very small amount of amorphous precursors.6 Compared with the rich knowledge of the nucleation mechanism in solution, the information is much less about the nucleation associated with ACP.7 Samples from both chemical and biological systems have been shown to contain neutral ion clusters with the formula Ca9(PO4)6.8,9 However, little is known about how such kind of ACP forms and subsequently transforms into crystalline seeds, triggering rapid precipitation in a supersaturated solution. The composition, structure, and phase transition of amorphous nanoparticle precursors are the fundamental aspects in current crystallization studies.10 The aim of the present study was to detect the main events before bulk precipitation in supersaturated solution of calcium phosphate. Effective in rapid mixing and measuring,10 the stopped-flow technique enabled us to mix hundreds of microliters of calcium and phosphate solutions within 2 ms and then follow the spectral change. The simultaneous measurements of * To whom correspondence should be addressed. (T.-L.Z.) Fax: 86-1062015584. Tel: 86-10-82801539. E-mail: [email protected]. (K.W.) Fax: 86-10-62015584. Tel: 86-10-82801539. E-mail: [email protected]. (R.-K.T.) Fax: 86-571-87953736. Tel: 86-571-87953736. E-mail: [email protected]. † Peking University School of Pharmaceutical Sciences. ‡ Capital Medical University School of Pharmaceutical Sciences. # Zhejiang University. § C.G.W. and J.W.L. contributed equally to this work and both should be considered as first authors.

solution pH and particle size provided clues to the composition and structure of ACP particles. And finally, we were able to isolate the solids near the onset of the abrupt pH drop and characterize their structure by high-resolution transmission electron microscopy (HR-TEM). By means of these techniques, we investigated the formation of ACP, the crystal growth in ACP particles, and some factors that affect the as-made seeds in triggering bulk precipitation from supersaturated solution. Materials and Methods CaCl2 · 2H2O and NaH2PO4 were purchased from Sigma. The standard phosphate and calcium species were purchased from Sigma and Fluka, respectively. Other chemical reagents were of analytical grade. Water was deionized and doubly distilled before use. Solutions used in this study were prepared fresh, adjusted to pH 7.40 at 25 °C, filtered twice through 100 nm pore size filters (Millipore), and degassed under lower pressure. In filtration experiments, solutions were passed through a 20 nm pore Anodisc membrane (Whatman). The concentrations of calcium and phosphate stock solutions were determined by inductively coupled plasma mass spectrophotometry (Type 7500, Agilent) analysis. All experiments were conducted at 25 ( 0.1 °C. Stopped-Flow Spectrophotometry. In order to trace the emergence of initial solid, equal volumes of 8.00 mM calcium chloride and 4.80 mM sodium phosphate were mixed on a stopped-flow spectrometer equipped with a 1024 diode array detector (SFM-400, Bio-Logic). The dead-time was 1.6 ms. A special washing procedure was used before each shot to ensure the cleanness of the cuvette, flow path, and mixers. 900 spectra were recorded in each trace. The pH changes in milliseconds-to-minutes time scale were monitored spectrophotometrically on the stopped-flow instrument. Thirty micromolar bromothymol blue (BTB, C27H28Br2O5S, China Chemical) was added into the calcium and phosphate solutions. BTB is a weak acid with two absorption peaks at 418 nm (produced by acid form) and 618 nm (produced by base form). After each shot, the twodimensional (2D) slices (absorbance - time curve) at 418 nm (Curve a) and 618 nm (Curve b) were extracted from a three-dimensional (3D) graph (wavelength - absorbance - time). Define A418 ) (Curve a Curve a′) and A618 ) (Curve b - Curve b′), where Curve a′ and Curve b′ were the corresponding 2D slices from the 3D graph in the absence of BTB. Then the division operation A418/A618 was conducted, giving an absorbency ratio A418/A618 - time curve. An average of three such curves were used to calculate proton concentration

10.1021/cg801069t CCC: $40.75  2009 American Chemical Society Published on Web 04/29/2009

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Figure 1. Initial events after mixing calcium and phosphate solutions as revealed by stopped-flow experiments. The deadtime of the instrument is 1.6 ms. At 25.0 ( 0.1 °C, the reaction was initiated by mixing equal volumes of 8.00 mM CaCl2 and 4.80 mM NaH2PO4, pH ) 7.40. (a) and (e) Changes in light absorption of the solution. (b) A two-dimensional presentation of (a). (c) and (d) Changes in solution pH. according to the equation pH ) 7.00 - 1.05 lg (A418/A618), which was obtained with a series of BTB solutions of known pH (previously calibrated with a pH meter) under exactly the same conditions. The possible effect of BTB on the precipitation kinetics was investigated in a free-drift experiment (Figure S1, Supporting Information). The induction time was slightly prolonged in the presence of BTB, but the profile of the kinetic curve remains unchanged. This fact indicates that the main events that comprise the precipitation mechanism are same as those without BTB. Nevertheless, a pH meter was used, whenever possible, to measure the pH more directly. Simultaneous Measurement of Particle Size and Solution pH. Equal volumes of 8.00 mM calcium chloride and 4.80 mM sodium phosphate were rapidly mixed. One milliliter of the mixture was added to each of two identical polystyrene cells (1 × 1 × 3 cm3). One of the cells was used to monitor the change in solution pH with a pH meter (Orion model 720A). The other cell was put on a Zetasizer Nano analyzer (Malvern model ZS90) to measure particle size based on dynamic light scattering. Each result was the average of six successive measurements in about 1 min. The result was given both numerically as a Z-average size (Zav) and graphically as a histogram showing particle size distribution. The Z-average size is an intensity mean. Although a mass or number mean can also be given, Zav is the most direct property reflected by the dynamic light scattering measurement. The consistency of reaction progress in pH and Zav measurements was checked by exchanging the two cells. In order to monitor the dynamic aggregation process, we sometimes performed continuous measurements and recorded a result for every single run. Transmission Electron Microscopy. Equal volumes of 8.00 mM calcium chloride and 4.80 mM sodium phosphate were rapidly mixed. Thirty milliliters of the mixture was added to a water jacketed Pyrex cell. The solution pH was monitored using a pH meter. At desired times, 2 mL of solution was withdrawn and diluted with 3 mL of water. After

being filtered through a 20 nm pore filter (Anodisc membrane, Whatman; Pop-Top filter, Costar), the solid was washed using 1 mL of water and 3 mL of ethanol successively. A drop of the suspension was placed on a carbon-coated copper grid and then kept in a desiccator at room temperature. The solid on the grid was analyzed by HR-TEM (JEM-2011HR, JEOL, Japan). The fast Fourier transform (FFT) patterns of the crystalline domain in the HR-TEM images were obtained with a program of Digital Micrograph.

Results and Discussion By means of a stopped-flow diode-array spectrophotometer, we detected the formation of an initial solid phase and pH changes in the early stage after mixing calcium and phosphate solutions. In Figure 1a, the colors from deep blue to red represent increasing absorbance. The most striking feature of Figure 1a is the emergence of a solid phase from 3 s, as shown by the prominent absorption of light between 300-400 nm (and possibly in shorter wavelengths). On the left side of the corresponding 2D presentation (Figure 1b), there is a deep blue band across the whole wavelength range. The band indicates that the solution was clearer during 0.65-1.0 s than either before or after this period, which might be a consequence of proton concentration elevation from 3.82 × 10-8 M (pH ) 7.42) at 0.209 s to a maximum of 4.68 × 10-8 M (pH ) 7.33, Figure 1c) at 1.155 s. A possible cause for the pH drop is ion-pair formation. The proton concentration in phosphate solution depends on the molar ratio of H2PO4-/HPO42-. At pH 7.42 and 25 °C, H2PO4-/HPO42- ) [H+]/Ka2 ) 10-7.42/10-7.21 ) 0.62 )

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Figure 2. Temporal changes in Z-average size (0) of the particles and pH (b) of the solution. At 25.0 ( 0.1 °C, the reaction was initiated by mixing equal volumes of 8.00 mM CaCl2 and 4.80 mM NaH2PO4, pH ) 7.40. Each Zav datum was the average of six successive measurements in about 1 min.

62/100, using the secondary ionization constant Ka2 )10-7.21.11 In a solution containing 2.40 mM Pi (total phosphates, mainly H2PO4- and HPO42- under the experimental condition) and 4.00 mM Ca2+, Pi ions form pairs with Ca2+. The dominant pairs under the experimental conditions were CaHPO4 (formation constant, Kpair1 ) 548, at 25 °C12) and CaH2PO4+ (Kpair2 ) 25.6 12), while the existence of CaPO4- or Ca(OH)2(aq) could be negligible. In addition, computer simulation indicates that [Ca2+-(HPO4)2--Ca2+]2+ ion triple could form and subsequently yield [Ca2+-(PO4)3--Ca2+]+ by releasing a proton.13 If the

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increasing proton concentration was solely caused by the ionpair formation, the CaH2PO4+/CaHPO4 molar ratio should be 3.5/100. Because this molar ratio was lower than the original H2PO4-/HPO42- molar ratio of 62/100 at pH 7.42 (here “original” means “before the ion-pair formation”), the ratio of H2PO4-/HPO42-, and thus the proton concentration, increased. This trend could even be strengthened when taking into account the formation of [Ca2+-(HPO4)2--Ca2+]2+. As a consequence of ion pair formation, the solid that had formed previously in the course of solution-mixing dissolved, making a clearer solution as indicated by the deep blue band in Figure 1b. Following the pH drop and accompanying the initial solid formation, a pH elevation was observed. It might be associated with the formation of ion clusters. During the period between 1.155-10 s (Figure 1c), the pH increased from pH ) 7.33 to 7.41. This change could not be attributed to solid formation (Figure 1a,b), since the former was nearly completed at 3 s when the latter just started. Besides, the increase became quite mild beyond 10 s (Figure 1d), which was not compatible with the drastic increase of the solid phase during the same period (Figure 1e). In addition to the dissolution of the mixing-caused solid, ion cluster formation might be another cause for the pH elevation. Because of the insufficient information of the varieties and compositions of ion clusters, we assume that all sorts of

Figure 3. Dynamic changes of particle size distribution in calcium phosphate solution. At 25.0 ( 0.1 °C, the reaction was initiated by mixing equal volumes of 8.00 mM CaCl2 and 4.80 mM NaH2PO4, pH ) 7.40. Measurements, each taking about 11 s, were performed successively and displayed without averaging. The first graphs in (a) and (b) were obtained at 11 and 21 min, respectively.

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Figure 4. Transmission electron micrographs of the solids isolated from calcium phosphate solution. (a) and (b) Solids immediately before the turning point of pH drop. (d) and (e) Solids immediately after the turning point of pH drop. (c) and (f) The FFT analyses of the crystalline domains in (b) and (e), respectively.

clusters formed during this period might be represented by one equivalent cluster, (CaHPO4)m(CaH2PO4+)n, which is defined as in eq 1:

νq{(CaHPO4)m(CaH2PO4+)n} ) ΣνiCi +

(1)

where (CaHPO4)m(CaH2PO4 )n and Ci represent the compositions, and νq and νi show their concentrations, respectively. The cluster compositions may affect proton concentration. A decrease in proton concentration may be expected as long as the value of the ratio n/m in the equivalent cluster is greater than the original H2PO4-/HPO42- molar ratio (76/100 at pH 7.33 and t ) 1.155 s; here “original” means “before the equivalent cluster formation”). A 31PNMR study has revealed the presence of substantial amounts of protonated phosphate in ACP.14 If the decreasing of proton concentration was indeed associated with cluster formation, it might be a hint about the composition and

structure of the clusters. For example, a ratio of n/m approaching unity implies that the H2PO4--favoring hydrogen bond is structurally as important as the HPO42--favoring electrostatic interaction in the cluster. Electrostatic interaction, hydrophobic interaction, and the “cement” effect of water are among the main factors holding a cluster together.15 The cluster formation, along with the resulting pH elevation, may facilitate solid emergence from solution (Figure 1c). The growing amount of solid beyond 10 s caused only a slight alteration in proton concentration (Figure 1d,e), indicating small differences between the solid and the solution in the molar ratios of H2PO4-/HPO42- and CaH2PO4+/ CaHPO4. Therefore, the initial solid must be more acidic and more heavily hydrated than the Ca9(PO4)6-containing ACP,8,9 and might be a precursor of the latter. For changes of the initial solid after the first 3 min, we measured the particle size and solution pH simultaneously. As

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Figure 5. Effect of filtration on the kinetics of calcium phosphate precipitation. Initial concentrations: Ca ) 4.00 mM, Pi ) 2.40 mM; both solutions were passed through a 100 nm filtration membrane respectively. Reactions were conducted at 25.0 °C on a magnetic stirrer. (b) Curve (a): No filtration after mixing Ca and Pi solutions; (4) Curve (b): The mixed solution was passed through a 20 nm filtration membrane at t ) 0 min (0) Curve (c): The solution was filtrated twice at t ) 0 min and t ) 140 min, respectively. Each filtration operation took 2 min.

depicted in Figure 2, a mild decrease preceded an abrupt drop in solution pH. The former, which is often referred to as “induction (or latent) period”, lasted 85 min and exhibited several features. First, the Z-average size (Zav) of calcium phosphate particles kept increasing, though the change in solution pH was quite mild. The absence of apparent change in concentrations of proton16 and calcium6 during the period has been reported previously. These particles were much greater in size than the critical nucleus (less than 5 nm in diameter, see the following discussion on Figure 4b), but they did not induce large-scale crystallization in the solution, indicating the lack of sufficiently ordered structure on particle surface. Second, the Zav-time curve was relatively smooth in the first 30 min, but became rough later. The “roughness” was indicative of particle aggregation in the solution. Even at the early stage of the induction period, aggregation could be observed (Figure 3a). Sometimes larger particles (i.e., the size distribution peak on the right side) formed suddenly from the smaller ones (the peak on the left side) within 11 s (the time needed for one run of size measurement), and grew more rapidly. As a result, the peak of the larger particles moved out of the measurable limit (0-5 µm) of the instrument. Third, there was always a relatively stable size distribution, such as the one with the most populated size around 300 nm in Figure 3a. We may call it “steady size distribution (SSD)”. As the reaction proceeded, SSD shifted toward the right side. After the aggregates grew up beyond the measurable limit, a new peak of size distribution sometimes appeared on the left side of SSD (Figure 3b). These smaller particles might be the fragments derived from collisions of larger particles. A collision could produce two kinds of particles: the larger ones that presented on the right side of SSD, and the smaller ones that could not be detected until growth occurred. The larger and smaller particles generated by collisions were responsible for the observed increases in Zav (Figure 2) and the continuous production of solid material, respectively. It is noteworthy that the smaller particles grew also more rapidly than the SSD particles, and the peaks of the two size distributions finally merged (Figure 3b). Indeed, SSD was the most frequently observed particle size distribution during the induction period, demonstrating its special dynamic stability in the supersaturated solution.

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Along with the continuous increase in size and amount of the solid phase, its structure changed during the induction period. We isolated solid from the solution immediately before the abrupt pH drop. Typically, the solid was “chains-like agglomerates of round particles approximately 100 nm in diameter” (Figure 4a), as described by Brecˇevic´ and Fu¨redi-Milhofer.17 These round particles were the primary ones that composed the SSD particles by agglomeration. Since it took less than 10 min for the particles to grow larger than 100 nm in Zav (Figure 2), agglomeration must be a major factor accounting for the further Zav increase in the rest of the induction time. However, the structural change mainly took place inside those primary particles. The transformation5 of ACP and its increase in internal density18 have been reported previously. Using HR-TEM, we observed crystalline domains in the ACP particles (Figure 4b). The fast Fourier transform (FFT) pattern of the crystalline domain indicates that the d-spacing of the lattice fringes with the highest intensity is ∼0.276 nm (Figure 4c), which is in agreement with that of (112) of hydroxyapatite (HAP). The d-spacings of the other two planes are 0.307 and 0.318 nm, which could be assigned respectively to (210) and (1j02) planes of HAP. The calculated angles between these planes fit well with the measured values, indicating that the crystalline phase of the indexed region in Figure 4b is HAP. It is unlikely that these crystalline domains formed separately in solution and then aggregated, because crystallites aggregated at various stages would not be so uniform in size (about 5 nm in diameter, Figure 4b). Besides, if so many crystallites had been in full contact with the supersaturated solution before aggregation, the solution pH could not be maintained stably (Figure 2). Hence, these crystalline domains might form intrinsically at multiple sites inside the primary ACP particles at roughly the same stage. The presence of crystalline seeds at the onset of pH drop is a logical event as judged by the observations before, after and right at the time point, in addition to the TEM results. Amorphous calcium phosphate formed in a few seconds from the solution mixing (Figure 1a,e). The formation of amorphous calcium phosphate has been reported by other researchers.5,7 The amorphous solid proliferated through growth and aggregation (Figure 3), while the solution pH only slightly decreased (pH curve in Figure 2). These facts imply that the chemical composition (aqueous calcium cations and protonated phosphates) of the initial amorphous solid was similar to that of the solution. Nevertheless, the composition was distinct from that of crystalline calcium phosphate such as OCP and HAP. The sizes of the amorphous particles (Zav in Figure 2) were much greater than the critical nucleus, but they were not capable of inducing rapid precipitation until undergoing compositional and structural changes through the whole induction period (85 min, as shown in Figure 2). In view of the inability of the initial amorphous solid and the crystalline solids detected hereafter (Figure S2, Supporting Information), there must exist, at the onset, some ordered structure that was responsible for triggering the abrupt pH drop. The results from filtration experiments provide further evidence for the crystallization inside amorphous particles (Figure 5). Immediately after mixing calcium and phosphate solutions, removal of the initial solid caused a prolonged induction period (curves (a) and (b) in Figure 5). However, removal of the larger particles at the onset did not prolong the induction period anymore, but reduced the rate of precipitation as indicated by the slope of the kinetic curves (b) and (c). Smaller and much fewer, the remaining crystalline seeds in the solution were still large enough in size and quantity to induce

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Scheme 1. Main Events in the Induction Period of Calcium Phosphate Precipitation

rapid precipitation. These facts imply that the size and quantity of the crystalline seeds presenting previously at the onset were well above the least critical values for inducing rapid precipitation in the supersaturated solution. If so many seeds had not developed inside amorphous particles, they would have induced precipitation immediately after reaching the critical size, and the larger size and excessive number of seeds would not have been observed. Further, the pause in the middle of the abrupt pH drop is an indication of the dissolving of amorphous solid residues. From the onset of pH drop and after, the rapid precipitation on crystalline seeds caused an increase in proton concentration. The latter, along with the reduced supersaturation, tended to slow down the precipitation. When the pH dropped to ∼6.7, the amorphous solid, which had previously formed at pH 7.2 during the induction period, may dissolve. The dissolution tended to elevate the solution pH. In a system without performing filtration (curve (a) in Figure 5), this effect was overwhelmed by the seeded precipitation. However, the removal of larger seeds by filtration greatly reduced the quantity of the seeds, thus weakening the effect of precipitation on pH and making the previously covered dissolution reaction recognizable (curves (b) and (c) in Figure 5). These observations, consistent with TEM results (Figure 4a), indicate the formation of crystalline domains inside amorphous particles. Little is known about how these crystalline domains (Figure 4b) developed in an ACP particle that derived from the initial solid (Figure 1a,e). Dehydration and phosphate deprotonation are essential for the amorphous-to-crystalline transformation. Binding of Ca2+ to the ion pair [Ca2+-HPO42-] can facilitate the proton release from phosphate.13 In the heavily hydrated initial solid, since the released proton should be in hydrated form, the three events (i.e., calcium binding, deprotonation and dehydration) are integrated. A microcalorimetric study has shown the necessity of hydration in ACP transformation.19 Clusters could act as building blocks for crystallites,20,21 and so could pairs for clusters. From the viewpoint of composition and structure, the well-known cluster Ca9(PO4)68,9 is likely an intermediate in the course of crystalline domain formation that started from the initial solid emergence. In addition to the innerparticle formation reported in the present study, HAP nanocrystals might grow from the interparticle phase between the spherical particles within ACP aggregates.16 In colloid science, whether crystals are best grown inside or outside the metastable region is a matter of debate.22 We also isolated solid from solution immediately after the onset of the abrupt pH drop. The characteristic morphology of

the solid appears to be ACP-made “sand beach” with inlaid crystallites of 5 nm in diameter (Figure 4d). No high-electrondensity site was observed in the solids at an earlier stage in the induction time (30 min from the start of the reaction, Figure S2a, Supporting Information). For a crystalline domain in a HRTEM image (Figure 4e), FFT analysis resolves a pattern similar to that of HAP (Figure 4f). The similar sizes and structures of these crystallites immediately before and after the turning point of pH drop are the direct evidence for the same origin. Therefore, the solid in Figure 4d was from the collapse of the primary particles (Figure 4a). The collapsed particles liberated crystallites, triggering the rapid precipitation and causing the abrupt pH drop. Unexpectedly, the change of particle size (Zav) did not decrease at the onset of pH drop (Figure 2). The 15 min lag reveals that a complete disintegration of particles was unnecessary for the wrapped seeds to contact the supersaturated solution. In other words, it was from the partially collapsed particles that the crystalline seeds were liberated and triggered the abrupt pH drop. Because the primary particles were in agglomerated form (Zav . 100 nm), their partial collapse did not lead to an immediate size decrease near the onset of the abrupt pH drop. The complete disintegration of particles in Figure 4d could be caused by sample preparation, but it reflects the extreme fragility of the seed-containing particles at the moment, as a consequence of the compositional and structural changes through the induction period. Our findings and previously published results enabled us to depict a general picture of the main events that take place during the induction period and finally trigger the rapid precipitation of calcium phosphate from the supersaturated solution (Scheme 1). Calcium and phosphate ions form pairs and clusters successively in the first few seconds (Figure 1c). These ions, pairs, and clusters then compose the initial solid phase that is heavily hydrated and contains hydrogen phosphates and absorbs light between 300-400 nm (Figures 1 and 2). Growing and aggregating, the solid increases in size and quantity without affecting the solution pH (Figures 2 and 3). During the induction period, the solid particles exhibit a steady size distribution, which is around 300 nm at the early stage and shifts toward 1000 nm with time. These particles are agglomerates of primary particles of 60-100 nm in diameter (Figure 4a), and are originally amorphous in structure. At multiple sites inside a particle, crystalline domains develop (Figure 4b) from ion pairs and/or clusters by taking up calcium and releasing hydrated proton, possibly through a stage at which the more compact cluster

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Ca9(PO4)6 presents. Since the expansion of crystalline domains consumes surrounding calcium and phosphate ions (or their pairs and clusters) and releases hydrated protons, the mechanic strength decreases in the interdomain regions. Finally, under the action of the shearing strength of the fluid, these primary particles collapse (Figure 4d). The liberated crystallites (Figure 4e) induce the rapid precipitation of calcium phosphates, together with the previously trapped hydrated protons in primary particles, resulting in the abrupt pH drop (Figure 2). Indeed, it is the crystallization at multiple sites inside amorphous particles that finally triggered the rapid precipitation of calcium phosphate from the supersaturated solution. In summary, we have studied the formation and transformation of amorphous calcium phosphate by stopped-flow spectrophotometry, high-resolution transmission electron microscopy, and simultaneous measurements of the particle size and solution pH. Our essential findings are (1) the formation of an initial solid phase that might be a precursor of conventional amorphous calcium phosphate containing the cluster Ca9(PO4)6; (2) the mechanism and consequence of crystalline domain development at multiple sites inside these amorphous particles. This study may shed new light on the understanding of crystallization through phase transition, as well as the induction period in precipitation kinetics. Acknowledgment. This work was supported by National Basic Research Program of China (Grant 2007CB516806), National Natural Science Foundation of China (Grants 20571006 and 20637010), Natural Science Foundation of Beijing (Grant 2062007), and Beijing Talent Project. Supporting Information Available: Figure S1. Influence of BTB on the precipitation kinetics of calcium phosphate. Figure S2. Transmission electron micrograph of the solids isolated from calcium phosphate

Wang et al. solution. This information is available free of charge via the Internet at http://pubs.acs.org.

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