Intermediate water on calcium phosphate minerals: its origin and role

Feb 18, 2019 - Water molecules are known to play crucial roles both in the formation and biological function of materials. Herein, we show the presenc...
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Intermediate Water on Calcium Phosphate Minerals: Its Origin and Role in Crystal Growth Masahiro Okada,† Emilio Satoshi Hara,† Daisuke Kobayashi,† Shoki Kai,† Keiko Ogura,‡ Masaru Tanaka,‡,§ and Takuya Matsumoto*,†

ACS Appl. Bio Mater. Downloaded from pubs.acs.org by MACQUARIE UNIV on 02/25/19. For personal use only.

†

Department of Biomaterials, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan ‡ Soft Biomaterials Research Center, Frontier Center for Organic Materials, Frontier Center for Organic Material Systems, Frontier Center for Organic System Innovations, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan § Soft Materials Chemistry, Institute of Material Chemistry and Engineering, Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan S Supporting Information *

ABSTRACT: Water molecules are known to play crucial roles both in the formation and biological function of materials. Herein, we show the presence of “intermediate water” on an inorganic solid material, hydroxyapatite. In vitro experiments revealed that Mg substitution of apatite significantly enriched the amount of intermediate water, possibly due to the proton transfer to a hydrogen-bonded network of water around HPO42− on divalent-cationdeficient apatite surfaces. The intermediate water formation related to a markedly suppressed protein adsorption on apatite. Analysis of bone apatites suggested that the intermediate water on minerals could play crucial roles in regulating crystal growth. KEYWORDS: bone apatite mineral growth, water hydrogen-bonded network, intermediate water, differential scanning calorimetry, infrared spectroscopy

P

has been demonstrated to have fundamental biological importance in suppressing protein adsorption on the organic material’s surface.5,6 In this study, we investigated the water structure on an inorganic solid material, hydroxyapatite (apatite), which is the main mineral component of bone. Bone constituted of both inorganic and organic materials is a highly sophisticated tissue that has inspired material scientists and engineers to mimic such structures and properties, although precise manipulation of bone tissue formation and maturation has not been achieved. In in vivo microenvironment, bone minerals are formed in the extracellular matrix previously occupied by organics and water molecules. Water molecules have been reported to interact with apatite surfaces4,7 and to play a key role in crystal growth including ion transportation to supply ions and to maintain pH8 and in the structuring of crystals.9 Nevertheless, the water structure on apatite crystals and its roles in the adsorption behavior of proteins, which modulate crystal growth both in vitro and in vivo, remained unknown. Herein, we show the first evidence of the presence of the three types of water structure on apatite. First, we characterized Mg-free apatite (Mg(−)-HAp) and Mg-substituted apatite (Mg(+)-HAp) particles synthesized by

hysico-chemical phenomena occurring at the material/ water interface are controlled by an interfacial layer, whose properties may deviate from those of the respective bulk material and water phases.1 Thus, the water structure (i.e., hydration state and hydrogen-bonded (H-bonded) network of water) on material surfaces is thought to play crucial roles in the formation and function of materials in aqueous media including biological environments. For instance, the protein adsorption, which is a critical determinant of materials’ biological properties, on materials is discussed in terms of the hydration state changes of the protein molecules and materials,2 and water-mediated hydrophobic forces.3 Additionally, minerals of biogenic origin are formed in aqueous environments containing many components that modulate the activity of the water and the hydration state.4 The water structure on organic soft materials has been characterized into three distinct types:5 free water interacting scarcely with organic polymer chains, which has a small nuclear magnetic resonance (NMR) correlation time (τc) value of 10−12−10−11 s similar to bulk water, and crystallizes at 0 °C; freezing-bound water (i.e., intermediate water) interacting weakly with polymer chains, which has lower mobility with a τc value of 10−10−10−9 s, and exhibits melting/crystallization at temperatures below 0 °C; and nonfreezing bound water interacting strongly with polymer chains, which has much lower mobility with a τc value of 10−8−10−6 s, and does not crystallize even at −100 °C. Particularly, intermediate water © XXXX American Chemical Society

Received: January 7, 2019 Accepted: February 18, 2019 Published: February 18, 2019 A

DOI: 10.1021/acsabm.9b00014 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Bio Materials

Figure 1. DSC heating curves for (a) Mg(−)-HAp and (b) Mg(+)-HAp with different water contents after being frozen at −100 °C. Each heat flow was normalized by the dried weight of apatite. The amounts of (c) intermediate and (d) nonfreezing bound waters of apatites with different water contents. Each water content was normalized by the surface area of each apatite type.

in vitro precipitation method. Mg is the most abundant divalent cations substituted in biological apatite.10 Since Mg substitution changes the particle size,11 the precipitation method was controlled to obtain similar sizes ranging from 50 to 100 nm (Figure S1a). Other crystal phases such as octacalcium phosphate (OCP) were not detected in XRD patterns (Figure S1b). Both apatites were deficient in divalent cations (i.e., (Ca +Mg)/P molar ratio was less than 10:6 that is the stoichiometric composition of Ca10(PO4)6(OH)2), and the degrees of the deficiency of bulk and surface compositions were respectively larger in the case of Mg(+)-HAp (Table S1). Of note, the bulk composition of Mg(+)-HAp was similar to that of young mouse bone mineral (Mg/Ca = 0.03 in molar ratio; (Ca+Mg)/P = 1.53 in molar ratio).12 Transmission Fourier transform infrared (FT-IR) spectra suggested the presence of HPO42−, and a more broadened tail due to HPO42− was observed in the case of Mg(+)-HAp (Figure S1c).

To estimate the presence of different water structure on apatite, differential scanning calorimetry (DSC) was carried out at different water content. In the cases of the supernatant of apatite dispersion, endothermic peaks were observed at a temperature above 0 °C (Figure S2), which indicates that the ions slightly dissolved in the supernatant of apatite dispersion were negligible. At low water contents below around 5 wt %, endothermic peaks were not observed, indicating the presence of only nonfreezing bound water on apatite. By increasing water content, peaks were observed below 0 °C, indicating the presence of freezing-bound water (i.e., intermediate water) on apatite (Figure 1a,b), as well as above 0 °C, indicating the presence of free water, above 20 wt % of water content. These results indicate the presence of the three types of water on inorganic apatite surfaces. The amounts of intermediate and nonfreezing bound waters varied significantly with ion substitution of apatite and total water content (Figure 1c,d). The amount of each water was B

DOI: 10.1021/acsabm.9b00014 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Bio Materials

Figure 2. Water structure on apatite estimated from diffuse reflectance FT-IR spectra without KBr dilution. (a) Mg(−)-HAp and Mg(+)-HAp at 10 wt % water contents, where free water was not detected by DSC measurements. Each intensity was normalized by the peak area ranging 2500− 4000 cm−1. (b) Mg(−)-HAp before and after adsorption of HPO42−. The sample was prepared by drying Mg(−)-HAp dispersion on a sample stage for diffuse reflectance method, and the same sample stage was immersed in 10 mM K2HPO4 at different pH value (8.5, 7.5, or 6.5) followed by removing the excess liquid by N2 blowing. Each intensity was normalized by the peak intensity of OH− in HAp (3570 cm−1).

Figure 3. Quantitative analysis of protein adsorption on Mg(−)-HAp and Mg(+)-HAp. Mg(−)-HAp and Mg(+)-HAp were immersed in NaCl 0.9% (w/v) aqueous solutions containing different concentrations of (a) bovine serum albumin or (b) cytochrome c.

nonfreezing bound waters. The O−H stretching vibration in Mg(+)-HAp displayed a broad tail that extends below 3000 cm−1 similar to the water participating in strongly H-bonded network.15 Together with the DSC data showing that Mg(+)HAp had larger intermediate water amount compared with Mg(−)-HAp, the increased absorption region below 3000 cm−1 reflected the strongly H-bonded state of intermediate water. Mg2+ substitution increases the divalent cation deficiency of apatite (i.e., decreases the (Ca+Mg)/P molar ratio), which relates to an increase in the amount of HPO42− (Figure S1 and Table S1) for maintaining its electrical neutrality.11,16 HPO42− could be a major proton donor, and the proton transfer could strengthen the H-bonded network of water around HPO42− on divalent-cation-deficient apatite surfaces. To investigate the effect of HPO42− alone without Mg substitution, Mg(−)-HAp was immersed in K2HPO4 solution at different pH values (Figure 2b). By increasing the amounts of HPO42− (low pH), the peak intensity at around 3200, 3020, and 2770 cm−1 clearly increased, which was more evident at pH 6.5. This result indicated that at least three states of water participating in the strongly H-bonded network increased around HPO42− on apatite surfaces. Taking in account the assignments for O−H stretch absorptions of “ice-like” water17,18 and an aqueous solution containing high concentrated protons,15 the peaks at 3200, 3020, and 2770 cm−1 could attribute to fully H-bonded water molecule, Zundel complex (H(H2O)2+), and Eigen complex (H3O(H2O)3+),

calculated based on equations reported previously for polymer materials6 and normalized by the surface area (Table S1). The intermediate water on Mg(+)-HAp was detected at lower water content (at around 0.7 mg/m2), and its amounts were notably larger than those of Mg(−)-HAp at any water contents (Figure 1c). The amount of nonfreezing bound water on both Mg(−)-HAp and Mg(+)-HAp was directly proportional to the water content until the intermediate water could be detected and subsequently reached plateau values (Figure 1d), which suggested that a saturated concentration of nonfreezing bound water existed on the surface of each apatite (0.6 mg/m2 for Mg(+)-HAp and 1.3 mg/m2 for Mg(−)-HAp). Considering the cross-sectional area of a water molecule as ∼10.6 Å2/ molecule (i.e., ∼0.28 mg/m2) at 24 °C,13 the nonfreezing bound water was estimated to be in multilayers of 2.1 and 4.6 layers in Mg(+)-HAp and Mg(−)-HAp, respectively. Note that Mg2+ with smaller ionic radius14 shows stronger interactions with water compared with Ca2+ dissolved in aqueous solutions. Hence, it was expected that the amount of nonfreezing bound water would increase by Mg substitution in apatite. Interestingly, however, the amount of nonfreezing bound water decreased, whereas that of intermediate water increased by Mg substitution in apatite. To obtain more information about the water structure on apatite, further FT-IR measurements were performed (Figure 2a) without KBr dilution to eliminate the effect of K+ and Br− ions on water structure. The water content was set to 10 wt %, which based on the DSC data present only intermediate and C

DOI: 10.1021/acsabm.9b00014 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 4. Analysis of minerals formed in embryonic mice calvaria isolated from embryos in pregnant mice fed with Mg(−) and Mg(+) diets. (a) Optical microscope images after Alizarin red staining of the isolated calvaria. (b) SEM images of E14.5 minerals after removal of organic matrices by sodium hypochlorite treatment. (c) Calcospherite size (N = 50) determined from low-magnification SEM images. (d) Crystallite size (N = 50) determined from high-magnification SEM images. Italic letters (a, b, and c) on bars in panels c and d indicate significant difference (p < 0.05), and 0 indicates that there are no minerals. (e) Diffuse reflectance FT-IR spectra of minerals in parietal bones of embryonic mice at E14.5 after removal of organic matrices by sodium hypochlorite treatment.

normal diet (Mg(+) diet mice) or low Mg diet (Mg(−) diet mice), and bone minerals were collected from the embryonic mice calvaria, as reported.22 Calvaria mineralization in normal Mg(+) diet mice started at embryonic day 14.5 (E14.5), whereas in Mg(−) diet mice, it initiated earlier at embryonic day 13.5 (E13.5) (Figure 4a,b). Elemental analysis of bone minerals confirmed a significantly higher content of Mg and divalent cation deficiency in minerals from normal Mg(+) diet mice (Mg/Ca = 0.043 ± 0.005; (Ca+Mg)/P = 1.50 ± 0.03) compared to those from Mg(−) diet mice (Mg/Ca = 0.030 ± 0.003; (Ca+Mg)/P = 1.60 ± 0.08). Interestingly, in accordance with the in vitro findings, FT-IR spectra of the minerals formed at E14.5 showed larger peaks near 3020 and 2770 cm−1, indicating the enhanced formation of strongly Hbonded intermediate water on the bone mineral surface in the normal Mg(+) diet mice (Figure 4e). Therefore, since the presence of intermediate water reflects a decreased protein adsorption capacity of apatite, the crystal morphology between Mg(+) and Mg(−) diet mice would be expected to be different. Indeed, the single crystal size of minerals formed in normal Mg(+) diet mice was markedly smaller than those in Mg(−) diet mice (Figure 4d), which is in accordance with previous findings in femora and tibiae of the rats.23 The quantitative analysis of calcospherites (flower-like spherical aggregations of crystals growing radially) also revealed that the calcospherite size (500 nm on average) in bone minerals from normal Mg(+) diet mice was markedly

respectively. Interestingly, the similar experiment using Mg(NO3)2 solution at pH 6.5, however, showed no significant changes in the FT-IR spectra of O−H stretching vibration (Figure S3), reiterating the importance of HPO42− in forming the strongly H-bonded intermediate water on apatite. Next, we demonstrated that alterations in the water structure on apatite reflected its protein adsorption properties (Figure 3). The amount of the proteins adsorbed onto Mg(+)-HAp was remarkably smaller than that onto Mg(−)-HAp, demonstrating the weak interaction of Mg(+)-HAp with proteins, independently of the acidic (bovine serum albumin) or alkaline (cytochrome c) characteristic of the proteins. These results help to explain the reduced protein (including collagen) adsorption capacity of Mg-substituted apatites reported previously.19,20 Intermediate water on organic soft materials can be assigned to hydrating water molecules with moderate mobility due to a weak interaction with swollen polymer side chains6,21 or with self-assembled monolayer (SAM) terminal groups,5 and the adsorption amount of proteins is reversely proportional to the amount of intermediate water on organic material surfaces. 6 Therefore, although the origin of intermediate water formation on inorganic solid materials was different from that on organic soft materials, the mobility (i.e., freezing or melting behavior) of intermediate water and its role in suppressing protein adsorption were identical. To confirm the ion substitution effect on water structure and apatite crystal growth in vivo, pregnant mice were fed with D

DOI: 10.1021/acsabm.9b00014 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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smaller than that (700 nm on average) of Mg(−) diet mice (Figure 4c and Figure S4), indicating that the growth of calcospherites depended on the growth of each crystal. The smaller sizes of synthetic Mg(+)-HAp, and biological mineral crystals and calcospherites formed in Mg(+) diet mice could be explained in part by the presence of strongly H-bonded intermediate water, which would reduce the water dynamics, ionic solute diffusion and chemical reactions around the apatite surfaces, and delay crystal growth. Interestingly, in vivo calcospherites presented narrow size distributions (i.e., the standard deviations for the calcospherite sizes in normal Mg(+) diet mice were smaller than in Mg(−) diet mice at E14.5 and E15.5), which could be related to an inhibition of its growth by the surrounding proteins.24,25 Of note, SEM observation of the initial minerals formed in mouse femur epiphysis also revealed that calcospherites presented similar spherical structure with narrow size distribution (Figure S5). A possible explanation for this phenomenon could be related to the gap between crystals at the initial stages of calcospherite formation and the presence of intermediate water around the crystals. Initially, this gap would be too narrow for proteins to penetrate in so that only ions, such as calcium and phosphate ions, could penetrate into this nanoconfined space and allow crystal and calcospherite growth. At a later stage, as the crystals grow radially, the gap between crystals becomes considerably larger, and proteins can then interact and inhibit crystal growth, eventually limiting the overall size of calcospherites. This consideration is also supported by our previous data showing that the adsorption of fibrous proteins on the surface but not in the inside of calcospherites.26 The changes in the amount of intermediate water during the different stages of bone formation, maturation, and aging can also be estimated. Formation of apatite accompanies the consumption of OH− ions and hence the local microenvironment around the growing apatite would become acidic, which in turn also causes the protonation of phosphate on apatite surface (increase in intermediate water; see Figure 2b), especially in the initial stages of bone formation characterized by the lack of blood circulation. Following blood vessel invasion into the tissue, the blood flow would neutralize the microenvironment of mineralizing tissues, which could cause a reduction in protonation of phosphate on apatite. Additionally, the overall degree of Mg substitution,27 divalent cation deficiency,27 protonation of phosphate,28 and water contents7,29 in bone tissues are known to decrease during mineral maturation and aging. These changes would induce a decrease of intermediate water on apatite crystals and would enhance the interaction of apatite crystals with the surrounding organic matrix proteins, which should be important after the initial steps of bone formation to mechanically strengthen the mineralizing tissues, as the mechanical properties of composite materials are determined by both the filler content and its interaction with matrix. Collectively, these findings not only improve our understanding of the roles of water in the complex process of bone formation, but also are valuable for designing new apatiterelating materials by controlling the physicochemical properties of apatite for diverse applications such as bone tissue engineering, gene transfections, molecule purifications, and catalysis supports.30

Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00014.

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Experimental details and supplementary figures (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Masahiro Okada: 0000-0001-9441-3284 Emilio Satoshi Hara: 0000-0001-7374-3487 Masaru Tanaka: 0000-0002-1115-2080 Takuya Matsumoto: 0000-0002-9804-4786 Funding

This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grant Nos. JP15K15723, JP25293402, and JP18H05254, and by the Matching Planner Program (MP27115663113) from Japan Science and Technology Agency. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Shigeaki Morita, Department of Engineering Science, Osaka Electro-Communication University (Osaka, Japan) for the helpful advice on FT-IR measurements and analysis.

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DOI: 10.1021/acsabm.9b00014 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX