Formation Process of Hydroxyapatite Granules in Agarose Hydrogel

Feb 23, 2018 - To eliminate this concern, the XRD analysis was also performed, and we confirmed that the transformation was not induced by the electro...
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Formation Process of Hydroxyapatite Granules in Agarose Hydrogel by Electrophoresis Kenshiro Kimura,† Masanobu Kamitakahara,*,† Taishi Yokoi,†,§ and Koji Ioku‡ †

Graduate School of Environmental Studies, Tohoku University, Sendai, Miyagi 980-8579, Japan Faculty of Economics, Keio University, Yokohama, Kanagawa 223-8521, Japan

‡

ABSTRACT: Biomimetic mineralization is useful in obtaining organic−inorganic composite and inorganic materials with controlled sizes and morphologies. The formation of hydroxyapatite (HA) granules with porous structures can be achieved by using electrophoresis processes inside an agarose gel in which the compulsory migration of calcium and phosphate ions are driven by an electric field, causing precipitation of the granules. In the present study, we examined the formation process of HA granules through electrophoresis and the subsequent aging process. Spherical particles of amorphous calcium phosphate (ACP) precipitated, increasing their size with increased running time during electrophoresis; then the ACP granules gradually transformed to the HA granules during the aging process in ultrapure water. When the ACP granules were large, one HA granule was formed from one ACP granule. One HA granule was formed from multiple ACP granules for small-sized ACP granules. The size of ACP granules depends on the gel concentration and pH in the electrophoresis. The dominant transformation process from ACP to HA was the dissolution−precipitation process.

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INTRODUCTION

An electrophoresis process is useful for the rapid synthesis of calcium phosphates in hydrogels because it can induce the directional migration of ions rapidly by imposing an electrical field. Watanabe et al. reported that HA particles can be precipitated by electrophoresis in a hydrogel to obtain an HA/ hydrogel composite.16,17 The electrophoresis was also applied to HA synthesis in poly(hydroxyethyl methacrylate), 18 polyacrylamide,19 and collagen20 hydrogels. Additionally, we also reported the formation of submicron HA granules with unique structures.21,22 The HA granules were composed of a number of fiber-like crystals. However, the formation process of HA granules in hydrogels through the electrophoresis has not been revealed. An understanding of the formation process of HA granules in hydrogels is crucially significant in the design of HA/hydrogel composites and HA granules with controlled sizes and morphologies. Moreover, knowledge surrounding the formation of HA in a hydrogel is important in understanding biomineralization. As mentioned above, the transformation of ACP into the more stable calcium phosphate phase generally occurs, and this transformation reaction should occur in the calcium phosphate synthesis in the hydrogels. However, this transformation behavior of ACP in hydrogel is still not fully understood. Hence, in this study, we clarify the crystalline phase of initially formed calcium phosphate and its transformation behavior. Since the transformation reaction is rapidly

Biomimetic mineralization is useful process in obtaining the organic−inorganic composite and inorganic materials with controlled sizes and morphologies.1,2 The bones of vertebrates are composed of hydroxyapatite [HA, Ca10(PO4)6(OH)2] and collagen, and the formation of HA is controlled by the collagen. The composite, which contains HA and a polymer hydrogel, is the attractive composite for bone repair.3 Moreover, the formation of HA and the other calcium phosphates in hydrogels have been studied for an understanding of biomineralization.4−6 Since the morphology and size of the HA granules formed in a hydrogel are regulated by its 3D network structure, the process might be useful in obtaining the HA granules suitable for drug carriers as HA shows excellent biocompatibility and specific adsorption properties. 7−9 Although there are several methods to prepare the HA nanoparticles with controlled morphologies,10−13 we focused on the process using a hydrogel because this process is easy. The process that uses the natural diffusion of ions and their concentration gradient is widely used for the preparation of composites of HA granules and hydrogels.14,15 However, a long time is required for HA precipitation because of the very slow diffusion speed of ions. Calcium phosphate synthesized in an aqueous solution is often formed first in an unstable phase, such as amorphous calcium phosphate (ACP), and is then transformed into the other phase under Ostwald’s step rule. Therefore, we believe that the transformation process of ACP in hydrogels is a key process in determining the size and morphology of the finally formed calcium phosphate. © XXXX American Chemical Society

Received: August 17, 2017 Revised: February 14, 2018 Published: February 23, 2018 A

DOI: 10.1021/acs.cgd.7b01154 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Figure 1. Schematic drawing of the electrophoresis system. (a) View from above and (b) cross-section.

Figure 2. TEM images of the precipitates inside the gels immediately after electrophoresis for different running periods. The electron diffraction pattern of the precipitates obtained for a 12 min running period is shown in (f). specimen with the maximum volume of the white precipitates obtained after 12 min of electrophoresis was crushed and immersed in an excess volume of ultrapure water for varying periods of up to 3 days. In order to eliminate the effects of residual calcium and phosphate ions or reagents on the transformation reaction, we evaluated the transformation behavior of the precipitates immersed in ultrapure water. These experiments were conducted at room temperature. The precipitates inside each of the specimens were observed by using a transmission electron microscope (TEM, SU-8000 or HF2000, Hitachi, Japan), immediately after both the electrophoresis and immersion in ultrapure water for each case. The gel specimens were crushed, dispersed in ethanol, and loaded on a TEM microgrid (Okenshoji, Japan). The crystalline phase of the precipitates was identified using electron diffraction equipped with TEM. Additionally, the specimens were freeze-dried immediately after the collection, prepared to prevent the transformation, and examined by X-ray diffraction (XRD, RINT2200VL, Rigaku, Japan) to determine the phase of the precipitates. As an additional experiment, we examined the effect of the gel concentration and pH of the buffer solutions. Namely, the gels concentrations were 3 and 6 mass %, and the pH of the buffer solutions were 9 and 10. In these experiments, the electrophoresis running period was 12 min, and the subsequent aging period in ultrapure water was 3 days.

occurring, we used the electrophoresis process for the rapid synthesis of calcium phosphate rather than the natural diffusion process.

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MATERIAL AND METHODS

A buffer solution consisting of 0.04 mol L−1 tris(hydroxymethyl)aminomethane (NacalaiTesque, Japan) and 0.02 mol L−1 of acetic acid (Wako Pure Chemical Industries, Japan) was prepared. The pH of the solution was approximately 8 at 25 °C. A 1 mass % of agarose gel with the wells casted on both the positive and negative sides was prepared from a low-melt agarose powder (NuSieve GTG Agarose, Lonza, USA) and the buffer solution. A volume of 0.70 mL of the 0.20 mol L−1 CaCl2 solution (NacalaiTesque, Japan) was placed in the well on the positive side, and 0.70 mL of the 0.12 mol L−1 of Na2HPO4 solution (Wako Pure Chemical Industries, Japan) was placed in the well on the negative side of the gel. The gel was installed in the middle of the system and immersed in the buffer solution followed by a supply of a potential of 100 V. Figure 1 shows a schematic drawing of the electrophoresis system. After the electrophoresis, white precipitates appeared in the middle part of the gel, and the area of the white precipitates expanded as the electrophoresis running period was increased. In order to understand the formation behavior of the precipitates, the white area with the precipitates and gels were cut out at different electrophoresis running periods. Moreover, to investigate the transformation behavior, a B

DOI: 10.1021/acs.cgd.7b01154 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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RESULTS The TEM images of the precipitates inside the gels immediately after electrophoresis with different running periods are shown in Figure 2. The granules were distributed homogeneously in the agarose gel. The electron diffraction pattern of the precipitates obtained for a 12 min-running period is also shown in Figure 2f. Spherical granules were formed with dispersion inside the gel, and their size gradually increased with running time. After 12 min, the white area became most noticeable, and the diameters of the particles were 200−400 nm. The electron diffraction from the precipitated spherical particles showed a typical hollow pattern, and this indicated that the spherical particles are amorphous. The XRD patterns of the gels after electrophoresis with different running periods are shown in Figure 3. A distinct pattern of HA was not found

Figure 4. TEM images of precipitates inside the gel after immersion in ultrapure water for different periods. The electron diffraction pattern of the precipitates after immersion in ultrapure water for 3 days is shown in (d).

Figure 3. XRD patterns of the gels immediately after electrophoresis for different running periods.

for every electrophoresis running period, and this result also supports the hypothesis that the precipitated granules are amorphous. These results indicated that spherical ACP granules were formed and that their size increased with time during the electrophoresis. The TEM images of the precipitates inside the gels after the immersion in ultrapure water for different periods are shown in Figure 4. The electron diffraction pattern of the precipitates obtained after 3 days of immersion in ultrapure water is also shown in Figure 4d. At first, small needle-shaped particles precipitated from the surface of the spherical granules during the immersion in ultrapure water. As time increased, the spherical particles changed into the aggregates of the fiber-like precipitates. The electron diffraction pattern showed the rings associated with HA. The XRD patterns of the gels after immersion in ultrapure water for different periods are shown in Figure 5. Broad diffraction lines that are associated with HA gradually appeared with increasing immersion time. The broadness of the HA diffraction lines indicates a small crystallite size or low crystallinity. From the TEM observations, the products were composed of very fine fibers, which could explain the broadness of the diffraction lines. These results indicated that the spherical

Figure 5. XRD patterns of the gels after immersion in ultrapure water for different periods.

ACP granules changed to granules composed of the fine HA fibers during the immersion in ultrapure water. In our previous study,22 the formed HA obtained in 1 mass% agarose gel had a Ca-deficient composition with Ca/P molar ratio of 1.58. The TEM images of the precipitates inside the gels with different gel concentrations before and after the immersion in ultrapure water for 3 days are shown in Figure 6. As the specimens before the immersion in ultrapure water did not C

DOI: 10.1021/acs.cgd.7b01154 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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sizes of the HA granules formed after the immersion in ultrapure water were almost the same regardless of the differences in the sizes of the ACP granules before the immersion in ultrapure water.

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DISCUSSION In the TEM observation, there was the concern that the electron beam may have induced the transformation of the ACP to HA granules, due to the high energy of the electron beam. To eliminate this concern, the XRD analysis was also performed, and we confirmed that the transformation was not induced by the electron beam of TEM. Although the most stable calcium phosphate under the pH condition (pH 8) of the buffer solution in the electrophoresis is HA,23 the spherical ACP granules were formed during the electrophoresis. ACP is a metastable phase of calcium phosphate. It is known that ACP is formed when the solutions containing calcium ions and phosphate ions are mixed rapidly under the basic pH solution at low temperature.24 Therefore, the formation of ACP may be due to the rapid reaction of the calcium and phosphate ions because these are moved rapidly by electrophoresis. The formation of ACP in a hydrogel has also been reported in an agar hydrogel by the diffusion process.15 It has also been reported that the ACP particles are formed when the pH of a solution supersaturated with HA is increased.25 Therefore, the formation of ACP is not a special case. The increase in size of the ACP granules was observed over time during the electrophoresis and relatively large ACP spherical granules were formed (Figure 2). The calcium and phosphate ions were consumed immediately by the formation of ACP in the case of the mixed solutions, but the present electrophoresis system provided the gradual supply of calcium and phosphate ions. The nucleation of ACP occurs when the area becomes supersaturated with ACP and then the growth occurs after the supply of calcium and phosphate ions. It is wellknown that the number of the formed nuclei is dependent on the degree of the supersaturation, and the number of the nuclei also affects the size of ACP granules. This speculation is supported by the results in which the size of the ACP granules decreased by increasing the pH as shown in Figure 7. When the pH increases, it is speculated that the degree of supersaturation with respect to calcium phosphate will increase rapidly and large numbers of nuclei of ACP will be formed. As the amounts of calcium and phosphate ions are decided by the solutions applied, the size of the ACP granules is small when large numbers of ACP nuclei are formed. Conversely, the gel structure also affects the size of the formed ACP granules. The size of the ACP granules decreases with increasing gel concentration as shown in Figure 6. It is speculated that an increased agarose concentration will provide a rigid network for the gel structure, and this rigid structure will leave smaller spaces for the growth of ACP granules. Moreover, the results where the ACP granules were not aggregated are due to the gel structure. The transformation from ACP to HA proceeded in ultrapure water after the electrophoresis. The transformation from ACP to HA has been examined by the previous studies.26−29 The transformation process, the dissolution−precipitation process27 and the internal arrangement process28 will now be discussed. From our results, the transformation reaction seemed to begin at the surface (Figure 4). It is speculated that the dissolution of ACP occurred first; then the nucleation of HA after the surrounding conditions became supersaturated with HA, and

Figure 6. TEM images of precipitates inside the gels with different gel concentrations, before and after the immersion in ultrapure water for 3 days.

have peaks in the XRD patterns and the specimens after the immersion in ultrapure water showed HA peaks, the granules before and after the immersion in ultrapure water are speculated to be ACP and HA, respectively, for the case of 1 mass % gel concentration. The size of ACP granules decreased with increasing the gel concentration from 1 to 6 mass %. Conversely, although the size of the HA granules formed after the immersion in ultrapure water showed a tendency to decrease with increasing gel concentration, the size of the HA granules was much larger than that of the ACP granules after the immersion in ultrapure water for the case of 6 mass % gel. Figure 7 shows the TEM images of the precipitates inside the

Figure 7. TEM images of precipitates inside the gels formed under different pH conditions, before and after the immersion in ultrapure water for 3 days.

gels prepared by electrophoresis under different pH conditions before and after the immersion in ultrapure water for 3 days. As the specimens before the immersion in ultrapure water did not have peaks in the XRD patterns and the specimens after the immersion in ultrapure water showed HA peaks, the granules before and after the immersion in ultrapure water are also speculated to be ACP and HA as in the above case. The size of ACP granules decreased with increasing pH. Conversely, the D

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(3) Gkioni, K.; Leeuwenburgh, S. C.; Douglas, T. E.; Mikos, A. G.; Jansen, J. A. Mineralization of hydrogels for bone regeneration. Tissue Eng., Part B 2010, 16, 577−585. (4) Yokoi, T.; Kawashita, M.; Kawachi, G.; Kikuta, K.; Ohtsuki, C. Synthesis of calcium phosphate crystals in a silica hydrogel containing phosphate ions. J. Mater. Res. 2009, 24, 2154−2160. (5) Yokoi, T.; Kawashita, M.; Kikuta, K.; Ohtsuki, C. Crystallization of calcium phosphate in polyacrylamide hydrogels containing phosphate ions. J. Cryst. Growth 2010, 312, 2376−2382. (6) Yokoi, T.; Kawashita, M.; Kikuta, K.; Ohtsuki, C. Biomimetic mineralization of calcium phosphate crystals in polyacrylamide hydrogel: Effect of concentrations of calcium and phosphate ions on crystalline phases and morphology. Mater. Sci. Eng., C 2010, 30, 154− 159. (7) Roy, I.; Mitra, S.; Maitra, A.; Mozumdar, S. Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery. Int. J. Pharm. 2003, 250, 25−33. (8) Hossain, S.; Stanislaus, A.; Chua, M. J.; Tada, S.; Tagawa, Y. I.; Chowdhury, E. H.; Akaike, T. Carbonate apatite-facilitated intracellularly delivered siRNA for efficient knockdown of functional genes. J. Controlled Release 2010, 147, 101−108. (9) Uskoković, V.; Uskoković, D. P. Nanosized hydroxyapatite and other calcium phosphates: Chemistry of formation and application as drug and gene delivery agents. J. Biomed. Mater. Res., Part B 2011, 96, 152−191. (10) Yang, Y.-H.; Liu, C.-H.; Liang, Y.-H.; Lin, F.-H.; Wu, K. C.-W. Hollow mesoporous hydroxyapatite nanoparticles (hmHANPs) with enhanced drug loading and pH-responsive release properties for intracellular drug delivery. J. Mater. Chem. B 2013, 1, 2447−2450. (11) Bastakoti, B. P.; Hsu, Y.-C.; Liao, S.-H.; Wu, K. C.-W.; Inoue, M.; Yusa, S.; Nakashima, K.; Yamauchi, Y. Inorganic−organic hybrid nanoparticles with biocompatible calcium phosphate thin shells for fluorescence enhancement. Chem. - Asian J. 2013, 8, 1301−1305. (12) Bastakoti, B. P.; Inuoe, M.; Yusa, S.; Liao, S.-H.; Wu, K. C.-W.; Nakashima, K.; Yamauchi, Y. A block copolymer micelle template for synthesis of hollow calcium phosphate nanospheres with excellent biocompatibility. Chem. Commun. 2012, 48, 6532−6534. (13) Liang, Y.-H.; Liu, C.-H.; Liao, S.-H.; Lin, Y.-Y.; Tang, H.-W.; Liu, S.-Y.; Lai, I.-R.; Wu, K. C.-W. Cosynthesis of cargo-loaded hydroxyapatite/alginate core−shell nanoparticles (HAP@Alg) as pHresponsive nanovehicles by a pre-gel method. ACS Appl. Mater. Interfaces 2012, 4, 6720−6727. (14) Imai, H.; Tatara, S.; Furuichi, K.; Oaki, Y. Formation of calcium phosphate having a hierarchically laminated architecture through periodic precipitation in organic gel. Chem. Commun. 2003, 1952− 1953. (15) Wada, N.; Horiuchi, N.; Nishio, M.; Nakamura, M.; Nozaki, K.; Nagai, A.; Hashimoto, K.; Yamashita, K. Crystallization of calcium phosphate in agar hydrogels in the presence of polyacrylic acid under double diffusion conditions. Cryst. Growth Des. 2017, 17, 604−611. (16) Watanabe, J.; Akashi, M. Novel biomineralization for hydrogels: Electrophoresis approach accelerates hydroxyapatite formation in hydrogels. Biomacromolecules 2006, 7, 3008−3011. (17) Watanabe, J.; Akashi, M. An electrophoretic approach provides tunable mineralization inside agarose gels. Cryst. Growth Des. 2008, 8, 478−482. (18) Liu, G.; Zhao, D.; Tomsia, A. P.; Minor, A. M.; Song, X.; Saiz, E. Three-dimensional biomimetic mineralization of dense hydrogel templates. J. Am. Chem. Soc. 2009, 131, 9937−9939. (19) Li, Z.; Su, Y.; Xie, B.; Wang, H.; Wen, T.; He, C.; Shen, H.; Wu, D.; Wang, D. A tough hydrogel−hydroxyapatite bone-like composite fabricated in situ by the electrophoresis approach. J. Mater. Chem. B 2013, 1, 1755−1764. (20) Johnson, J. R., III; Meng, L.; Wnek, G. E.; Schiraldi, D. A. Electrophoretic calcium phosphate mineralization of collagen hydrogels. Green Mater. 2015, 3, 71−79. (21) Kamitakahara, M.; Ogata, S.; Tanihara, M.; Ohtsuki, C. Control of calcium phosphate precipitation in hydrogel. Key Eng. Mater. 2007, 330−332, 79−82.

then crystal growth of HA on the HA nuclei occurred with the supply of calcium and phosphate ions from ACP since HA is thermodynamically in a more stable phase than ACP. The dominant transformation process seemed to be the dissolution−precipitation process. Although the sizes of the HA granules corresponded to those of the ACP granules when the gel concentration was 1 and 3 mass %, the sizes of the HA granules were much larger than those of the ACP granules when the gel concentration was 6 mass % and the pH was high. This indicated that the crystal growth of HA was due to the supply of calcium and phosphate from the other ACP granules near the growing HA granules when the size of ACP granules was small. These results also support the dissolution− precipitation process. It has been reported that the rate-limiting step of the transformation is the formation of the initial HA nuclei.26 Due to the existence of the activation energy for HA nucleation, the ACP was relatively stable during the electrophoresis. After the transformation, the formed HA granules were composed of thin fibers. The porous structure constructed of HA fibers can provide high surface areas to load drugs or DNAs. Moreover, the entanglement of the fibers with high aspect ratios produces granules that are predisposed to nonbrittle fracturing.30 As the morphology of the HA crystals strongly depends on the synthesis conditions,31,32 the shape of the particles consisting of HA granules is expected to be controlled by the aging process.

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SUMMARY The formation process of the HA granules in the electrophoresis process was examined. It was revealed that spherical ACP granules were precipitated, and their size increased with increasing running time during electrophoresis. The ACP granules gradually transformed to the HA granules during an immersion in ultrapure water after the electrophoresis. The ACP size was dependent on the gel concentration and pH in the electrophoresis. The dominant transformation process was the dissolution−precipitation process.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-22-795-7375. Fax: +81-22-795-7375. E-mail: [email protected]. ORCID

Masanobu Kamitakahara: 0000-0002-3548-7010 Present Address §

Materials Research and Development Laboratory, Japan Fine Ceramics Center, 2-4-1 Mutsuno, Atsuta-ku, Nagoya 456-8587, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Mr. Miyazaki of Technical Division, School of Engineering, Tohoku University for experimental help in the TEM observations.

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REFERENCES

(1) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, 2001. (2) Xu, A. W.; Ma, Y.; Cölfen, H. Biomimetic mineralization. J. Mater. Chem. 2007, 17, 415−449. E

DOI: 10.1021/acs.cgd.7b01154 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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(22) Kamitakahara, M.; Kimura, K.; Ioku, K. Synthesis of nanosized porous hydroxyapatite granules in hydrogel by electrophoresis. Colloids Surf., B 2012, 97, 236−239. (23) Elliot, J. C. In Structure and Chemistry of Apatites and Other Calcium Orthophosphates; Elsevier Science: Amsterdam, 1994; Chapter 1, pp 1−62. (24) Dorozhkin, S. V. Amorphous calcium (ortho)phosphates. Acta Biomater. 2010, 6, 4457−4475. (25) Hashizume, M.; Nagasawa, Y.; Suzuki, T.; Kawashima, S.; Kamitakahara, M. Effect of preparative conditions on crystallinity of apatite particles obtained from simulated body fluids. Colloids Surf., B 2011, 84, 545−549. (26) Boskey, A. L.; Posner, A. S. Conversion of amorphous calcium phosphate to microcrystalline hydroxyapatite. A pH-dependent, solution-mediated, solid-solid conversion. J. Phys. Chem. 1973, 77, 2313−2317. (27) Eanes, E. D.; Termine, J. D.; Nylen, M. U. An electron microscopic study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite. Calcif. Tissue Res. 1973, 12, 143−158. (28) Kim, S.; Ryu, H.-S.; Shin, H.; Jung, H. S.; Hong, K. S. In situ observation of hydroxyapatite nanocrystal formation from amorphous calcium phosphate in calcium-rich solutions. Mater. Chem. Phys. 2005, 91, 500−506. (29) Bjørnøy, S. H.; Bassett, D. C.; Ucar, S.; Strand, B. L.; Andreassen, J. P.; Sikorski, P. A correlative spatiotemporal microscale study of calcium phosphate formation and transformation within an alginate hydrogel matrix. Acta Biomater. 2016, 44, 254−266. (30) Murakami, S.; Kato, K.; Enari, Y.; Kamitakahara, M.; Watanabe, N.; Ioku, K. Hydrothermal synthesis of porous hydroxyapatite ceramics composed of rod-shaped particles and evaluation of their fracture behavior. Ceram. Int. 2012, 38, 1649−1654. (31) Lin, K.; Wu, C.; Chang, J. Advances in synthesis of calcium phosphate crystals with controlled size and shape. Acta Biomater. 2014, 10, 4071−4102. (32) Kamitakahara, M.; Ohtsuki, C.; Kawachi, G.; Wang, D.; Ioku, K. Preparation of hydroxyapatite porous ceramics with different porous structures using a hydrothermal treatment with different aqueous solutions. J. Ceram. Soc. Jpn. 2008, 116, 6−9.

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DOI: 10.1021/acs.cgd.7b01154 Cryst. Growth Des. XXXX, XXX, XXX−XXX