Crystallization of Calcium Phosphate in Agar Hydrogels in the

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Crystallization of calcium phosphate in agar hydrogels in presence of polyacrylic acid under double diffusion conditions Norio Wada, Naohiro Horiuchi, Makoto Nishio, Miho Nakamura, Kosuke Nozaki, Akiko Nagai, Kazuaki Hashimoto, and Kimihiro Yamashita Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01453 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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Cover Page Title: Crystallization of calcium phosphate in agar hydrogels in presence of polyacrylic acid under double diffusion conditions Authors: Norio Wada,*,† Naohiro Horiuchi,† Makoto Nishio,†,‡ Miho Nakamura,† Kosuke Nozaki,† Akiko Nagai,† Kazuaki Hashimoto,‡ and Kimihiro Yamashita† Affiliations: † Department of Inorganic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan ‡

Department of life and Environmental Science, Chiba Institute of Technology, 2-17-1 Tudanuma, Narashino, Chiba 275-0016, Japan

*Corresponding author’s E-mail: [email protected] Tel: +81-03-5280-8015; Fax: +81-03-5280-8015

We studied the precipitation of calcium phosphate (CaP) in presence or absence of polyacrylic acid (PAA) under double diffusion conditions in agar hydrogels. Waiting times and first precipitation positions were measured to analyze the CaP crystallization behavior, with the equivalency rule governing the precipitation in gel for a double diffusion process. We speculated that amorphous dicalcium phosphate (ADCP, CaHPO4·nH2O) nucleates at the first precipitation position via the reaction that Ca2+ ion interact with HPO42− ion, subsequently transforming into octacalcium phosphate (OCP) according to the Ostwald rule of stages and finally being converted to the thermodynamically more stable hydroxyapatite (HAp) via dissolution. The irregularly shaped aggregates formed at an early stage of crystallization were a mixture of OCP and HAp. In the later stage, spherulites with a hierarchical structure precipitated, with their inner part composed of OCP crystals and the outer part composed of HAp crystals aligned with the c-axis. The dissolution of the inner part advanced the growth of the needle-like crystals comprising the outer layer. PAA addition favored the formation of HAp as the final precipitate. We proposed a mechanism for the crystallization of ADCP, OCP, and HAp in presence or absence of PAA under double diffusion conditions. 1

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Crystallization of calcium phosphate in agar hydrogels in presence of polyacrylic acid under double diffusion conditions

Norio Wada,*,† Naohiro Horiuchi,† Makoto Nishio,†,‡ Miho Nakamura,† Kosuke Nozaki,† Akiko Nagai,† Kazuaki Hashimoto,‡ and Kimihiro Yamashita†

†

Department of Inorganic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan

‡

Department of life and Environmental Science, Chiba Institute of Technology, 2-17-1 Tudanuma, Narashino, Chiba 275-0016, Japan

*Corresponding author’s E-mail: [email protected] Tel: +81-03-5280-8015; Fax: +81-03-5280-8015

ABSTRACT: We studied the precipitation of calcium phosphate (CaP) in presence or absence of polyacrylic acid (PAA) under double diffusion conditions in agar hydrogels. Waiting times and first precipitation positions were measured to analyze the CaP crystallization behavior, with the equivalency rule governing the precipitation in gel for a double diffusion process. We speculated that amorphous dicalcium phosphate (ADCP, CaHPO4·nH2O) nucleates at the first precipitation position via the reaction that Ca2+ ion interact with HPO42− ion, subsequently transforming into octacalcium phosphate (OCP) according to the Ostwald rule of stages and finally being converted to the thermodynamically more stable hydroxyapatite (HAp) via dissolution. The irregularly shaped aggregates formed at an early stage of crystallization were a mixture of OCP and HAp. In the later stage, spherulites with a hierarchical structure precipitated, with their inner part composed of OCP crystals and the outer part composed of HAp crystals aligned with the c-axis. The dissolution of the inner part advanced the growth of the needle-like crystals comprising the outer layer. PAA addition favored the formation of HAp as the final precipitate. We proposed a mechanism for the 2


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crystallization of ADCP, OCP, and HAp in presence or absence of PAA under double diffusion conditions.

■ INTRODUCTION Biological organisms produce organic-inorganic composites, i.e., biomaterials such as shells, bones, and teeth. These biomaterials contain organic molecules that significantly affect the nucleation and growth of inorganic materials. Biomolecules with reactive functionalities, such as carboxylate and amino groups, regulate the crystallization of calcium phosphate (CaP).1‒6 In fact, the hard tissues of vertebrates such as bones and teeth consist of hydroxyapatite (HAp) and various proteins. Moreover, the results of earlier studies have shown that organic molecules control the formation and transformation of CaP through specific molecular interactions at inorganic/organic interfaces,7‒9 with an emphasis on molecular complementarity (electrostatic, stereochemical, geometric, etc.) between the surface of an inorganic crystal and the functional groups of the adsorbed organic molecules.8 In general, it is postulated that the soluble organic matrix in solution reduces the rate of crystal growth or delays the onset of inorganic material precipitation, while the same matrix adsorbed on the surface of these materials promotes heterogeneous nucleation.10 Therefore, the soluble organic matrix has opposite functions, depending on whether it is present in solution or adsorbed on a surface. Hence, soluble organic molecules may play a key role in the crystallization of biomaterials.10 The formation and transformation of CaP have attracted the attention of many authors due to their importance in various fields, e.g. environmental sciences, technology, biomedicine, etc. It is well known that CaP phases precipitated in aqueous solutions mainly include amorphous calcium phosphate (CaxHy(PO4)z·nH2O, ACP), dicalcium phosphate dehydrate (CaHPO4·2H2O, DCPD),

octacalcium

phosphate

(Ca8(HPO4)2(PO4)4·5H2O,

OCP),

and

hydroxyapatite

(Ca10(PO4)6(OH)2, HAp).11 HAp, precipitating from neutral to basic solution containing calcium and phosphate ions, is considered the most thermodynamically stable form in a physiological environment. ACP, OCP, and DCPD have been regarded as precursors for HAp formation during the biomineralization of bones and teeth, since their formation is kinetically favorable.12,13 3



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Understanding the effects of biomolecules on the crystallization of biomaterials is of particular importance for the strategies of their controlled synthesis. However, the effects of soluble biomolecules and the nature of precursor phases during biomineralization are still controversial. The aim of this study was to provide useful insights for these issues. We examined the precipitation of CaP in presence or absence of polyacrylic acid (PAA) under double diffusion conditions in agar hydrogels. In general, hydrogels contain a large amount of free water, providing an environment capable of performing biological functions. Agar gel is a porous medium exhibiting a three-dimensional structure with channels, which allows the gel to be used as a transport medium where convection is suppressed, and possesses pores that allow the reactant ions to pass. Hence, in a gel crystallization system, the reactant ions are supplied exclusively by the diffusion process without the convection phenomenon, i.e., volume mass-transfer by diffusion. In addition, the gel is physically, chemically, and thermally stable, with its lower degree of chemical complexity also making it less likely to interact with biomolecules, i.e., agar itself does not influence crystallization. PAA is a weak polyacid capable of binding calcium and exhibiting a single-chain structure with carboxylic side chains and no hydrophobic side chains.14 In addition, PAA plays a significant role in the crystallization of CaP as an epitaxial nucleation template, growth inhibitor, and promoter of crystal aggregation due to its adsorption.14,15 Hence, PAA is of particular interest owing to its use as a model ligand for organic polyacids, which play a major role in many physicochemical processes in aquatic systems.16,17

■ EXPERIMENTAL SECTION Precipitation in gel under double diffusion conditions. Pučar et al. showed that precipitation in gel under double diffusion conditions is described by the equivalency rule rather than by the ionic product law.18,19 The equivalency rule states that precipitation in a gel takes place at the position when CA = CB = CC, where CA and CB are the concentrations of both precipitating components, and CC is the minimum concentration required for nucleation (critical concentration). The most important parameters for understanding the crystallization behavior of a double diffusion system are 4


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the waiting time (defined as the time elapsed from the experiment onset to the first observation of visible precipitation) and the first precipitation position (referring to the position at which the first visible precipitate appears in the gel). The waiting time consists of the following three time periods: (1) the time needed for a reactant solution to reach from an undersaturation to a critical supersaturation, (2) the time needed to the production of the critical nuclei, (3) the time needed to grow for the nuclei to grow to an observable size. In our previous reports,20,21 we demonstrated the relationship between the reactant diffusion time and the position of equivalent concentration to discuss crystallization behavior based on the equivalency rule, and revealed that an increase of waiting time is reflected in a change of the first precipitation position, with a concomitant increase of the equivalent concentration. In other words, a shift of the first precipitation position reflects the change in waiting time. Therefore, we can discuss nucleation behavior by comparing the waiting time and first precipitation position obtained in the presence of PAA with those obtained in its absence. Precipitation by the gel growth method. The crystallization of CaP was studied employing a double diffusion technique and utilizing three acrylic tube chambers containing a Ca2+ source, gel column, and a PO43− source (Figure 1). These tubes were separated by a cellophane dialysis membrane. Solutions of 0.06 M (NH4)2HPO4 (8 mL) and 0.1 M Ca(NO3)2·4H2O (8 mL) were poured into the respective chambers, located on both sides of the gel column (50 mm length; 20 mm diameter) that was prepared using a 0.3% agar solution. The pH values of Ca(NO3)2·4H2O and (NH4)2HPO4 solutions were 5.0 and 8.0, respectively. PAA (sodium salt, MW = 2000) was added in various amounts (2, 4, and 8 mg) to the 0.3% agar solution before gelation. The pH of the gel solutions under all experimental conditions was adjusted to 7.6 using a Tris-HCl buffer. Reagent-grade chemicals were used without further purification, and ultrapure water (18 MΩ cm) was produced using a Milli-Q high-purity water system. The experiments were run in a thermostatic chamber kept at 28 °C for 10 days.

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Figure 1. Photograph (a) and illustration (b) of the apparatus used for crystallization under double diffusion conditions. The scale bar is 1 mm.

To study the nucleation and growth behaviors of CaP, the waiting time was measured in multiples of 0.5 h, and the first precipitation position, practically determined as the distance from the dialysis membrane of the Ca2+ chamber to the center of the first visible precipitation disk, was measured in multiples of 0.2 mm by an optical microscope of 20 magnifications. In addition, pH evolution of the agar hydrogel interior during crystallization was checked using pH indicators (pH test papers) such as Bromocresol green (BCG, pH 3.8‒5.4), Methyl red (MR, pH 4.4‒6.2), Bromothymol blue (BTB, pH 6.0‒7.6), and Cresol red (CR, pH 7.2‒8.8) to determine the dominant phosphate species in the precipitation positions. Characterization of precipitates. At the end of the experiments, the gel column containing CaP precipitates was sliced into sections. The precipitates in these slices were washed with hot-distilled water to remove the excess gel matrices and dried at room temperature. Identification of the precipitated phase was performed by X-ray powder diffraction (XRD) with Ni-filtered Cu Kα radiation (λ = 1.5406 Å) at 40 kV, 40 mA with a step size of 0.02º and a present time of 1 second at each step and Fourier transform infrared (FT-IR) spectroscopy with 400 scans at 2 cm−1 resolution using KBr pellets. The cross-sections of spherulites were provided, and the crystal phase of their interior was characterized by microarea X-ray diffraction (MA-XRD) using a 0.1 mm φ collimator at 6


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40 kV, 40 mA with a step size of 0.03º and a present time of 1.4 second at each step. The morphology of the precipitated CaP was observed by optical microscopy and scanning electron microscopy (SEM).

■ RESULTS AND DISCUSSION Waiting time, first precipitation position, and pH evolution in the gel. The photographs in Figure 2 show the CaP precipitation disk in presence or absence of PAA. The first visible precipitation appeared in a disk perpendicular to the diffusion direction. The CaP crystallization proceeded toward the chamber containing PO43− during the experiment. Moreover, the time-dependent turbidity change of the gel column occurred in the first visible precipitation disk without any change of the turbid zone thickness, regardless of experimental conditions, i.e., the turbidity increased, decreased ca. 3 h later, and increased again. This phenomenon suggests that the precipitates were partially dissolved. When no PAA was added, the first precipitation disk was observed as a broad band (band length of ca. 0.6 mm) in the gel. As the amount of added PAA increased, the first precipitation disk became narrow. The first precipitation positions were not substantially changed under all experimental conditions examined (being equal to 21.0‒21.4 mm), and the addition of PAA increased the waiting time as follows: 21 h without PAA; 31 h with 2.0 mg of PAA; 40 h with 4.0 mg of PAA; 52 h with 8 mg of PAA.

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Figure 2. Photographs of precipitation disks in the gel column: (a) first precipitation disk formed in absence of PAA at a precipitation position (of 21 mm) and a waiting time (of 21 h); (b) first precipitation disk formed with 4 mg of added PAA at a precipitation position (of 21 mm) and a waiting time (of 40 h); (c, d) crystallizations in gel in absence of PAA and in presence of 4 mg of PAA at the end of the experiment (10 days). The right- and left-hand sides of the gel tube were in contact with (NH4)2HPO4 and Ca(NO3)2·4H2O, respectively. Red arrows mark the first precipitation position in (a) and (b). White arrows indicate the direction of precipitation in (c) and (d). The crystallization space of CaP formed in presence or absence of PAA at the end of the experiments was divided into regions A and B in (c). The scale bar is 1 mm.

According to our discussion based on the equivalency rule, an increase in the waiting time must be reflected in a shift of the first precipitation position toward the PO43− chamber. The first precipitation position was observed at approximately the same position for all cases, despite the dependence of waiting time on the amount of added PAA. To explain this apparent discrepancy, it is reasonable to assume that the nucleation of CaP in all experiments occurred at same time, 21 h after the diffusion onset, but the growth of the nuclei was inhibited by PAA, as indicated by the marked increase of waiting time. Figure 3 shows the pH values in CaP containing agar hydrogels at the time of the first visible precipitation and at the end of the experiments, in presence or absence of PAA. Before the experiments were started, the gel column exhibited a homogeneous pH of 7.6. During the experiment, the pH increased in the direction from the Ca2+ side to the PO43− side, as shown in Figure 3, indicating that the reactant diffusion induces a pH gradient in the gel. Moreover, the pH values at different positions in the gel columns were similar at the end of the experiments in presence or absence of PAA, suggesting that pH evolution in the gel column was independent of the amount of PAA added (Figure 3b,d). In the case of 4 mg of added PAA, the pH value of the first precipitation position decreased to 6.6, while the waiting time increased to 19 h compared to the corresponding values obtained in absence of PAA. This pH change is attributed to the continued reactant diffusion during the increased waiting time. As described above, the nucleation onset 8


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occurred at the same diffusion time (21 h) under all experimental conditions (in presence or absence of PAA). The pH value of the first precipitation position in absence of PAA was 7.8, showing that the corresponding dominant phosphate species is HPO42− (pKa = 6.7).

Figure 3. The pH values in CaP containing agar hydrogels at the time of the first visible precipitation and at the end of the experiments in absence of PAA (a, b) and in presence of 4 mg of PAA (c, d). The numerical values in the upper part indicate the distance from the dialysis membrane of the Ca2+ chamber in the gel column.

Initially formed precipitates at the first precipitation position were a small quantity and very fine, therefore, they could not be easily isolated from the gel matrices. Hence, the phase of the precipitates was performed in the state of containing the gel matrices by XRD and FTIR analysises. The samples of the precipitates formed under identical experimental conditions in 3 gel columns were collected for one analysis. Figure 4 shows the XRD patterns of the initially formed precipitates at the first precipitation position in presence or absence of PAA. The obtained patterns are characterized by two broad diffractions centered at 2θ ≈ 32º and 17º. The broad diffraction centered at 2θ ≈ 32º was assigned to the presence of ACP phase.22 The XRD pattern obtained from gel matrices showed the 9



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broad peak centered at 2θ ≈ 17º. The FTIR spectra of the precipitates in presence or absence of PAA show that broad spectra similar to that of ACP, with the peaks attributed to the vibrations of PO43− ions at ca. 550‒590 and ca.1040‒1080 cm–1, and absorbed water peak at ca. 2800‒3600 cm–1.11 It is confirmed that the first formed phase during the precipitation process is an unstable amorphous phase of CaP.23 Since the equivalency rule requires the critical concentrations of calcium and phosphate ions at the nucleation onset to be equal and the dominant phosphate species at the first precipitation position is HPO42−, we speculate that amorphous dicalcium phosphate (ADCP, CaHPO4·nH2O) with an atomic Ca/P ratio of 1.0 nucleates at the first precipitation position via the reaction that Ca2+ ion interact with HPO42− ion during phase of nucleation.19,24,25 PAA (Mw = 2000) exhibiting the calcium-binding ability, possesses 23 equivalent acid groups per molecule, with the dissociation constant of about 5.1.26 The dissociation degree of the COOH groups in solution is pH-dependent. In gel columns, where the pH ranges from 5.8 to 7.8 at the position of precipitation, the COOH groups are primarily in the form of COO−. Therefore, some PAA molecules can form the complexes with Ca2+ ions via electrostatic attraction forces.16 PAA is known to be easily and irreversibly adsorbed on CaP, which has been ascribed to electrostatic interactions and/or hydrogen bonding.27 The decrease of the free calcium concentration can be neglected, since the amount of PAA-Ca2+ complexes formed is small due to the low molecular weight of PAA and its small quantity, resulting in no influence on crystallization. The increase of waiting time in presence of PAA is supposedly due to some of the mobile PAA molecules and PAA-Ca2+ complexes inhibiting the growth of ADCP via their adsorption. Various interpretation models for the inhibition of crystallization by additives propose that below a critical supersaturation level (depending on the concentration of adsorbed additives), the nuclei exist in a so-called dead zone, where their growth does not occur.28,29 Namely, when the supersaturation with CaP in the gel exceeds a critical supersaturation value, the nuclei grow. These speculations support the narrowing of the first precipitation and the increase of the waiting time in presence of PAA.

10


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Figure 4. XRD patterns of the initially formed precipitates at the first precipitation position at different amounts of PAA: (a) no PAA; (b) 4 mg of PAA.

Morphologies and crystal phases of precipitated CaP. The crystallization space of CaP formed in presence or absence of PAA at the end of experiments can be divided into two regions, A and B, under all conditions (Figure 2c), i.e., the turbid zone (region A) and the spherulite precipitation zone (region B). Typical SEM images of CaP precipitated in region A in presence or absence of PAA are shown in Figure 5. The white color of the turbid zone containing precipitates indicates a strong aggregation taking place upon precipitate formation. In absence of PAA, irregularly shaped aggregates consisting of needle-like crystals with lengths up to ca. 50 µm were formed close to the first precipitation position in region A, with the size of their aggregates decreasing in the direction of region B (Figure 5a,b). When PAA was present in the gel, significantly shorter needle-like crystals were formed. This result suggests the inhibition of precipitate growth due to the preferential adsorption of PAA and PAA-Ca2+ complexes. In addition, as the amount of added PAA was increased, the aggregates adopted a spherulite-like shape, which became more pronounced close to the boundary of region B. These phenomena indicate that PAA acted as a binder for CaP crystals.

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Figure 5. SEM images of the aggregates precipitated in region A: (a, b) in absence of PAA; (c, d) in presence of 2 mg of PAA; (e, f) in presence of 4 mg of PAA; (g, h) in presence of 8 mg of PAA.

The XRD patterns of the crystals precipitated in region A are shown in Figure 6. It is difficult to distinguish HAp and OCP phases, since their diffraction peaks are located at similar positions. Therefore, the presence of OCP in precipitates was confirmed by its (010) peak (2θ = 4.7°), which is the highest-intensity one for OCP. Although the obtained XRD patterns were relatively poor, some diffraction peaks were assigned to the reflections of HAp and OCP by comparison with the standard ICDD cards (HAp: No. 9-432; OCP: No. 26-1056), indicating that the precipitates are a mixture of OCP and HAp crystals. As the amount of added PAA increased, the amount of the precipitates formed in the gel decreased and the obtained XRD patterns became poor. In addition, the intensity of the (010) OCP peak decreased with the amount of added PAA, and the (010) OCP peak was not 12


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detected definitely when 8 mg of PAA were added. Unfortunately, it was not possible to distinguish between individual OCP and HAp crystals of the aggregates shown in Figure 5.

Figure 6. XRD patterns of the precipitates formed in region A at different amounts of PAA: (a) no PAA; (b) 2 mg of PAA; (c) 4 mg of PAA; (d) 8 mg of PAA.

CaP precipitates isolated from region A were characterized by FTIR measurements, and their phases were analyzed using the CaP FTIR data in ref. (30). FTIR spectra of each specimen for different amounts of added PAA are presented Figure 7, showing the characteristic bands of CaP and additional characteristic bands due to ager matrices. Designations in Figure 7 are parallel to those reported in Figure 6. The bands indicated by black dots at around 3570 and 630 cm−1 are ascribed to the stretching and liberation of OH− ions, respectively, indicating their presence in the crystal structure. These bands were used to identify the presence of HAp crystals in the specimens, being observed for all of them except specimen (a). Bands between ca.1100 and 960 cm−1 and between ca. 620 and 550 cm−1 are attributed to the vibration of PO43− ions. The bands around 910 cm−1, indicated by red dots, are attributed to the vibrations of HPO42− ions and were used to identify the presence of OCP crystals in the specimens. The bands at around 1660 cm−1 and the small bands between ca.1300 and 1400 cm−1 are due to agar matrices. Therefore, the obtained FTIR data support the result indicated from the XRD data. However, FTIR measurements did not provide information on how the 13



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polymer chains are associated with the precipitated crystals. The FTIR spectra (Figure 7) showed a significant sharpening of the adsorption bands between ca. 620 and 550 cm−1, indicating the good crystallinity of the precipitates.11 On the other hand, the XRD patterns (Figure 6) were poor. It is known that a poor XRD pattern is due to a small quantity of the precipitates and their low crystallinity.11,31 The results of FTIR measurements indicated that the precipitates have a good crystallinity. Therefore, we conclude that the obtained XRD patterns became poor because of a small quantity of the obtained precipitates. In addition, the degree of crystallinity is examined by FTIR. As crystallinity improves, the characteristic splitting of bands between ca. 620 and 550 cm−1 of the FTIR spectra becomes evident.11 It is suggested from the FTIR spectra that the splitting of the bands became clear with increasing PAA, that is, an increase of crystallinity. In general, the presence of organic matrices is expected to reduce crystallinity. However, we cannot explain the reason why the crystallinity of the precipitates increases with PAA and would like to discuss this matter as for the subject of a future study.

Figure 7. FTIR spectra of the precipitates formed in region A at different amounts of PAA: (a) no PAA; (b) 2 mg of PAA; (c) 4 mg of PAA; (d) 8 mg of PAA.

Several studies demonstrated the formation of characteristic CaP spherulites with hierarchical structures and reported that these spherulites consisted of a large number of needle- or plate-like CaP 14


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crystals radiating from a common center, with some of them having a hollow structure.32-36 Herein, we provide a detailed characterization of the CaP spherulites. Figure 8 shows SEM images of the spherulites precipitated in region B in presence or absence of PAA and their surface morphologies, revealing that their surface has a porous structure and consists of packed needle-like

Figure 8. SEM images of the spherulites precipitated in region B at different amounts of PAA and their surfaces: (a, b) no PAA; (c, d) 2 mg of PAA; (e, f) 4 mg of PAA; (g, h) 8 mg of PAA. The right-hand column shows the surface morphologies.

crystals. Furthermore, as the amount of added PAA was increased, the length of the needle-like crystals increased, adopting a fibrous morphology owing to bundles of crystals. This result can be attributed to the preferential adsorption of PAA and PAA-Ca2+ complexes on the surfaces of the developing CaP crystals. We consider that the bundles of CaP crystals are firmly bound to each by 15



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PAA that acts as a binder. Cross-section and fracture SEM images in Figure 9 point toward the existence of a common center core. Moreover, these images clearly reveal that the spherulites have a hierarchical structure, with the inner part (the core) consisting of aggregates formed from closely arranged fine crystals, and the outer part being composed of needle-like crystals radiating from a common center. Some of the SEM images suggest the dissolution of the core (Figure 9d,e). The core shown in Figure 9f is not dissolved. Samples A, B, and C in Figure 9c indicated that the thickness of

Figure 9. SEM images of the fractured spherulites precipitated in absence of PAA (a, b) and the cross-sections of spherulites precipitated in presence of PAA (c) in region B. (d), (e), and (f) show higher-magnification images of the cores of samples A, B, and C in (c), respectively.

the outer part increased with the degree of dissolution. Hence, it is considered that complete dissolution of the core occurs with time, resulting in the formation of the hollow spherulite shown in Figure 10a. The appearance of the hollow spherulite external surface of in Figure 10b,c reveals that the outer part is porous and consists of an interconnected crystal net-work. Figure 10e,d shows a 16


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spherulite in the middle of its growth, suggesting that spherulites with a hierarchical structure are produced by overgrowth to form the outer part on the core surface. Hence, we conclude that the core acts as a substrate for the overgrowth of the outer layer, and its dissolution supplies

Figure 10. SEM images of the cross-sections of a hollow spherulite (a, b, c) and of a spherulite in the middle of growth (d) in region B in presence of PAA. (b) External surface view of the hollow spherulite in the backscattered electron mode. (c) Higher-magnification image of the external surface, indicating porosity.

calcium and phosphate ions for the growth of the outer part.33 The MA-XRD patterns for cross-sections of spherulites precipitated in presence of PAA in region B are shown in Figure 11. The image (a) reveals that the large part of the spherulite has the core structure. The diffraction pattern (a) obtained from the sectioned spherulite shown in the image (a) indicates that the major part of the precipitates consists of OCP crystals. The image (b) shows that the spherulite has the outer part being composed of needle-like crystals radiating from the inner part and the partially dissolved core. The diffraction pattern (b) obtained from the sectioned spherulite shown in the image (b) exhibits the predominant peak corresponding to the (100) HAp plane in addition to some peaks assigned to HAp and some peaks attributed to OCP. Based on these XRD measurements of the sectioned spherulites and the radial growth texture of the outer part observed in the image (a), we conclude that the outer part is composed of aligned bundles of elongated HAp crystals oriented 17



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along the c-axis and the inner part consists of OCP crystals. We suggest that the preferential adsorption of PAA and PAA-Ca2+ complexes parallel to the c-axis produces HAp crystals elongated in the above direction.37

Figure 11. MA-XRD patterns of the cross-sections of spherulites precipitated in region B. Photographs (a) and (b) indicate the presence of an intact core and the partial dissolution of the core, respectively.

Possible crystallization mechanism of OCP and HAp. Based on experimental evidence and the equivalency rule speculation, we propose the following mechanism for the crystallization of OCP and HAp in presence or absence of PAA under double diffusion conditions in agar hydrogels. At the initial crystallization stage in presence or absence of PAA (region A), ADCP precipitates at the same diffusion time and position in the gel column. When PAA is present in the gel, some of the mobile PAA molecules and PAA-Ca2+ complexes inhibit the growth of ADCP, leading to an increase of waiting time. In the next stage, ADCP transforms into OCP via the Ostwald rule of stages,23,24 and the growth of OCP crystals continues as long as their surroundings are kept in a saturated state. The Ostwald rule of stages states that the initially formed more soluble phase subsequently transforms into a less soluble phase. This closed system, under conditions of double diffusion, exhibits a continuous decrease of reactant concentrations as a result of crystallization. Hence, under conditions of this continuous decrease, OCP transforms into the thermodynamically 18


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more stable HAp via dissolution. Many studies reported the transformation mechanisms of CaP.38 Two mechanisms were proposed for the transformation of OCP into HAp: (a) a dissolution-precipitation process, that is, the Ostwald rule of stages;25 (b) a solid-state transformation occurring in the OCP crystals.39,40 The obtained results suggest that the addition of PAA promotes the formation of HAp as the final precipitate, which is explained by the occurrence of two phenomena: (1) PAA inhibits the growth of OCP by adsorption and suppressing the decrease of supersaturation in the vicinity of OCP precipitates; (2) during the suppression, OCP precipitates are transformed into the more stable HAp via dissolution. The precipitation of CaP in region B following the precipitation in region A is accompanied by an increase of diffusion time. The crystallization process involved is the same as that in region A, since the nucleation under double diffusion conditions is governed by the equivalency rule. The fluxes of both reactants continuously decrease, to a great extent than those in region A, owing to crystallization in a closed system (finite diffusion). Hence, ADCP nucleates at a low density and grows. Subsequently, ADCP transforms into OCP, growing into particles with spherical morphology. In the next stage, the needle-like crystals of HAp overgrow the OCP precipitate as a crystallization substrate, leading to a hierarchical spherulite structure. Finally, the dissolution of the core promotes the growth of the needle-like crystals comprising the outer layer and leads to hollow spherulites.

■ CONCLUSION We studied the precipitation of calcium phosphate in presence of polyacrylic acid under double diffusion conditions in agar hydrogels. Based on the equivalency rule, we discussed the crystallization of ADCP, OCP, and HAp precipitated under the experimental conditions. we speculate that amorphous dicalcium phosphate (ADCP, CaHPO4·nH2O) with an atomic Ca/P ratio of 1.0 nucleates at the first precipitation position via the reaction that Ca2+ ion interact with HPO42− ion during phase of nucleation, and subsequently transforming into OCP in accord with the Ostwald rule of stages. Finally, OCP transforms into the thermodynamically more stable HAp via dissolution. Irrespective of the presence of PAA, irregularly shaped aggregates consisting of needle-like crystals 19



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precipitated in region A being a mixture of OCP and HAp, while spherulites with a hierarchical structure precipitated in region B. The inner part of these spherulites (core) is composed of OCP aggregates, while the outer part is composed of aligned bundles of HAp crystals attending the radial overgrowth of needle-like crystals with c-axis orientation. The dissolution of the core promoted the growth of needle-like HAp crystals in the outer layer and finally produced the hollow structures. PAA acted as a binder for OCP and HAp crystals and inhibited the growth of ADCP, OCP, and HAp via adsorption on their surface. The adsorption of PAA onto OCP and HAp crystals also led to their different morphologies. The addition of PAA favored the formation of HAp as the final precipitate. This study may provide new insights and important information for understanding the effects of organic molecules on biomineralization.

■ AUTHOR INFORMATION Corresponding Author

∗ E-mail: [email protected]

Notes The authors declare no competing financial interest.

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For Table of Contents Use Only Crystallization of calcium phosphate in agar hydrogels in presence of polyacrylic acid under double diffusion conditions

Norio Wada,*,† Naohiro Horiuchi,† Makoto Nishio,†,‡ Miho Nakamura,† Kosuke Nozaki,† Akiko Nagai,† Kazuaki Hashimoto,‡ and Kimihiro Yamashita†

SEM image (a) shows the irregularly shaped aggregates formed at an early stage of crystallization. The aggregates were a mixture of OCP and HAp. SEM image (b) and photograph (c) reveal that the spherulites formed at the later stage have a hierarchical structure. Their inner and the outer parts were composed of OCP crystals and HAp crystals, respectively.

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Crystallization of Calcium Phosphate in Agar Hydrogels in the

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