Hydroxyapatite Crystal Formation in the Presence ... - ACS Publications

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Hydroxyapatite Crystal Formation in the Presence of Polysaccharide Wancai Fang,†,‡ Hong Zhang,†,∥,‡ Jianwei Yin,† Boguang Yang,† Yabin Zhang,† Junjie Li,*,†,§ and Fanglian Yao*,†,⊥ †

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Science, Beijing 100850, China ⊥ Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China §

S Supporting Information *

ABSTRACT: Natural polysaccharides play an important role in the formation of nanohydroxyapatite (nHA) crystals in biological systems. In this study, we synthesized nHA crystals in the presence of four polysaccharides, i.e., pectin, carrageenan, chitosan, and amylose, referred as PeHA, CaHA, CsHA, and AmHA, respectively. X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscope, and thermogravimetric analysis were used to investigate the formation of nHA crystals. The shape of prepared nHA crystals is needle/rod-like in all cases, whereas the size increases in the order of PeHA, CaHA, CsHA, and AmHA. The presence of polysaccharides induces the heterogeneous nucleation of nHA and further modulates the crystal growth. Our data suggest that the interaction intensity between nHA and polysaccharides is in the decreasing order of PeHA, CaHA, CsHA, and AmHA, resulting in the smallest nHA crystals with pectin. It is also demonstrated that a high polysaccharide concentration and short reaction time are adverse to nHA crystals, especially for the polysaccharides with carboxyl groups. This study can provide insight into the effects of polysaccharides with different chemical functional groups (−COOH, −OSO3H, −NH2, −OH) on the formation of nHA crystals.

1. INTRODUCTION The nanohydroxyapatite (nHA, Ca10(PO4)6(OH)2) crystal, as a main structural material in hard tissues, is the most stable phase among various calcium phosphates in neutral or basic conditions. It has been widely used as biomaterial in the field of repair of bone tissue defects1 and nonviral carriers for drug/ gene delivery.2 To produce stoichiometric nHA, various methods have been explored,3 such as chemical precipitation, solid-state reaction, hydrothermal synthesis, sol−gel synthesis, emulsion technique, and so forth. The control of nHA with desired crystal size and morphology will be of practical value in biomedical application.4,5 For example, Wu et al.6 showed that the nHA crystal with smaller size is beneficial for the adsorption of biomolecules, and can further modulate the cell adhesion and growth factors secretion. Our previous study7 also demonstrated that nHA crystals with appropriate size can improve the osteogenic differentiation of mesenchymal stem cells. So far, attempts have been made to synthesize nHA © XXXX American Chemical Society

crystals by adjusting the crystallization environment. In previous studies, citric acid, amino acids, stearic acid, and ethylenediamine-tetraacetic acid have been used to modulate the formation of nHA crystals.8,9 In addition, some polymers can also act as a template to control the nucleation and growth of nHA crystals. For example, Poursamara et al.10 found that the nHA crystals synthesized in poly(vinyl alcohol) solution are almost rod-like with a mean crystallite size of ∼60 nm in diameter and about ∼150 nm in length. The formation of nHA crystals in biological systems is mainly modulated by collagen fiber.11 Sommerdijk et al.12 showed that the supramolecular assembly and charge distribution of collagen can control the mineralization of nHA nucleation in the presence of inhibitors. The addition of gelatin (the Received: August 26, 2015 Revised: January 27, 2016

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DOI: 10.1021/acs.cgd.5b01235 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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denatured type of collagen) is revealed to retard nHA formation as well as to change the aspect ratio of nHA particles due to strong adsorption of gelatin on the surface of calcium phosphate.13 In a seminal report, Reid et al.14 pointed out that polysaccharide, as a predominant component in the organic−mineral interface of bone, can effectively control the nucleation and growth of nHA crystals. The chemical functional groups (e.g., −COOH, −OSO3H, −NH2, −OH) of polysaccharides can chelate Ca2+ ions and form hydrogen bonding with PO43− and H2O on the surface of the minerals. In the past decade, many polysaccharides, such as chitosan, alginate, heparin, carrageenan, and chondroitin sulfate, which are analogous to one of glycosaminoglycans (GAGs) in structure, have attracted extensive interest in regulating the nHA nucleation and growth. Results showed that the chemical structure of polysaccharides is the main factor to modulate the growth and morphology of nHA crystals. For anionic polysaccharides, Li et al.15 prepared fine rod-like nHA crystals with ∼13 nm in diameter and ∼34 nm in length in the presence of alginate. Coleman et al.16 found that phosphorylated alginate exhibits strong nonspecific binding, whereas heparin displays preferential binding to the (002) face of nHA crystal. For cationic polysaccharides such as chitosan, chitosan can control the nucleation and growth of HA crystals via the interactions between hydroxyl group on C3 and C6 of chitosan chain and HA crystals.17 Our previous study18 found that the amino groups of chitosan play crucial roles for nHA crystal formation on the surface of chitosan−gelatin network films. In addition, we also found that anionic/cationic polysaccharide polyelectrolyte complexes can provide multiple nucleation sites and growth space for nHA crystals under different pH conditions.19 A better understanding of polysaccharide architecture effects on mineralization is of immense value in the fabrication of nHA crystals based biomaterials. However, the use of polysaccharides for controlling the nHA crystals has been sparsely reported. Further, the mechanism of formation of nHA crystals in the presence of polysaccharides with different functional groups has yet to be elucidated. In this study, four kinds of polysaccharides, i.e., pectin, ι-carrageenan, chitosan, and amylose, are applied to investigate the effects of polysaccharides on the formation of nHA crystals, with their chemical structures shown in Figure 1.

All these polysaccharides contain similar saccharide ring structure but with different chemical functional groups. Anionic pectin and carrageenan contain carboxyl groups (−COOH) and sulfonic groups (−OSO3H), respectively. Cationic chitosan has amino groups (−NH2), while only hydroxyl groups (−OH) are present in neutral amylose. The effects of type, concentration, and reaction time of different polysaccharides on the morphology and size of nHA crystals are systematically investigated.

2. EXPERIMENTAL SECTION 2.1. Materials. Pectin obtained from citrus peel (galacturonic acid ≥74%, average molecular weight: 2.0 × 105 g/mol) was purchased from Sigma-Aldrich; ι-carrageenan (average molecular weight: 2.5−3.0 × 105 g/mol) was obtained from TCI development Co., Ltd. Chitosan was purchased from Haihui biological product corporation (Qingdao, China). Its degree of deacetylation is 85%, and the viscous average molecular weight is 2.0 × 105 g/mol. Water-soluble amylose (average molecular weight: 1.5 × 105 g/mol), calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ·4H 2 O), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), acetic acid (CH3COOH), and sodium hydroxide (NaOH) were obtained from Jiangtian Chemical Technology Co., Ltd. (Tianjin, China). All reagents are analytic grade and used without further purification. 2.2. Synthesis of Nanohydroxyapatite in the Presence of Polysaccharides. The synthesis procedure of nHA crystals in the presence of polysaccharides is similar to that of the pure nHA crystals in the previous report.20 Briefly, 0.3 g of pectin, carrageenan, or amylose was dissolved in 250 mL deionized water (1.2 g/L), respectively. Then, 1.2 g of Ca(NO3)2·4H2O were added into this solution. After 12 h, 0.5 g of NaH2PO4·2H2O was added and the pH was adjusted to 11 using NaOH solution (0.1 M). The resulting mixture was stored at room temperature for 168 h (7 days). Subsequently, the precipitate was collected via centrifuging and washed using deionized water several times. In order to prepare the nHA/chitosan, 0.3 g of chitosan was dissolved in 250 mL acetic acid solution (1%, v/v), 0.5 g of NaH2PO4·2H2O and 1.2 g of Ca(NO3)2· 4H2O (molar ratio Ca/P = 1:67) were added in this solution under stirring for 6 h. Then, the pH was adjusted to 11 using NaOH solution and stored at room temperature. After 168 h, the precipitate was collected via centrifuging and washed using 1% acetic acid and deionized water for several times. The resulting nHA/polysaccharide samples were obtained by lyophilization, referred to as PeHA, CaHA, CsHA, and AmHA for synthesis environment of pectin, carrageenan, chitosan, and amylose, respectively. Finally, these nHA/polysaccharide composites were dried under vacuum and at 60 °C for 4 h to remove water. The pure nHA crystals without polysaccharides were obtained from water as the control group. The effect of polysaccharide concentration on nHA crystal formation was investigated in 0.2, 0.4, 0.8, 1.2, and 2.0 g/L pectin solution and 0.4, 1.2, 2.0, 3.0, and 4.0 g/L carrageenan solution for 48 h. The formation process of nHA crystal was characterized in 1.2 g/L pectin solution for various reaction times of 5, 12, 24, 48, 72, 120, and 168 h. 2.3. Characterization. Fourier transform infrared spectroscopy (FTIR) spectra of nHA, pectin, PeHA, carrageenan, CaHA, chitosan, CsHA, amylose, and AmHA were recorded on Magna-560 infrared spectrometer (Nicolet, USA). Briefly, 2 mg sample and 100 mg KBr were mixed and were grounded for 2 min using a ball grinding mill, and then they were transferred to a suitable mold and pressured into a transparent tablet under vacuum. Finally, these tablets were placed on Magna-560 infrared spectrometer to record the FTIR spectra from 4000 to 500 cm−1. X-ray diffraction (XRD) patterns of nHA, PeHA, CaHA, CsHA, and AmHA were measured to confirm the calcium phosphate phase (Rigaku D/max 2500v/pc). Traces were recorded from 10° to 70° in 2θ with a scan rate of 8.0°/min at 36 kV and 200 mA. The size of nHA crystal (L) along the (002) plane was calculated according to the Scherrer formula (1). Where, λ represents the wavelength of X-ray

Figure 1. Chemical structures of (A) pectin, (B) ι-carrageenan, (C) chitosan, and (D) amylose. B



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radiation (0.154056 nm), θ is the diffraction angle, K = 0.89, and β is the full width at half-maximum of the diffraction peak in radians.

L=

Kλ β cos θ

(1)

Transmission electron microscopy (TEM) was applied to investigate the morphology of nHA crystals. Briefly, the samples were added into water under ultrasonic and the suspension was collected on a copper mesh TEM grid. The TEM images were observed on a JEOL 100CX-II instrument with an accelerating voltage of 100 kV. The selected area electron diffraction (SAED) was recorded as well. The morphology of dry nHA and nHA/polysaccharide samples was observed using scanning electron microscopy (SEM, PHILIPS, Netherlands) at an accelerating voltage of 20.0 kV after coating with gold. SEM images were processed into 256 × 256 × 8 bit digital images by Photoshop software; a 256 × 256 × 256 cubic box was then established using the scale of the digital images as the third dimension information. Covered boxes and gray scale imaging of every box were counted, and fractal dimensions (Df) were calculated using the eq 2 according to previous studies.21,22 Whereas Bij is the gray levels of digital images, i and j is the row and columns of digital images, respectively. 255

Df =

Figure 2. XRD patterns of the nHA crystals and nHA/polysaccharide samples.

polysaccharide samples, suggesting that the HA crystal is nanosized and of low crystallinity. The broadness of nHA peaks is attributed to the in situ crystal formation on polysaccharide, which is similar to the natural bone.24 3.2. Interactions between Polysaccharides and nHA Crystals. The interactions between polysaccharides and nHA crystal take very important roles in the formation process of nHA crystals. FTIR was used to determine the formation of nHA crystals and interactions in nHA/polysaccharides composites. As shown in Figure 3, the absorption bands at 563, 602, and 1012 cm−1 are assigned to PO43− stretching vibrations of HA.15,25 The absorption bands for CO32− vibration appear at 875 and 1420 cm−1, indicating that PO43− sites of nHA crystal are partially substituted by CO32− groups.20 These carbonate anions are deemed to be introduced from CO2 in atmosphere or water under alkaline conditions during the synthesis procedure. For biomedical applications, the partially carbonated HA provides a more biomimetic material as the natural apatite contains 3−5% carbonate groups.26 The characteristic absorptions of each polysaccharide, i.e., − COO− (1610 cm−1), SO (1240 cm−1), −NH2 (1498 cm−1), and −CH2− in hydroxymethyl group (2925 cm−1), also appear in nHA/polysaccharide samples, indicating that the nHA crystals can be formed in the presences of polysaccharide. The absorption bands of chemical functional groups of polysaccharide matrix shift to new wavenumbers in nHA/ polysaccharides composites (Figure 3). The −CH3 vibration adsorption at 2929 cm−1 in pectin appears in PeHA as well, as shown in Figure 3A, which demonstrates that the pectin is incorporated in PeHA. However, we find that the −COOH (1753 cm−1) of pectin disappears in PeHA due to the dissociation under alkaline conditions.21 Notably, the absorption bands of −COO− at 1620 cm−1 of pectin shift to a lower wavenumber at 1610 cm−1 in PeHA. We reasoned that the electrostatic interaction between −COO− in pectin and Ca2+ in the HA phase causes the shift in band positions. Similarly, the vibration absorption bands of sulfate groups (SO) move to 1240 cm−1 in CaHA from 1270 cm−1 in carrageenan matrix,27,28 as shown in Figure 3B. The N−H bending at 1540 cm−1 and N−H stretch vibration at 3433 cm−1 from the amine of chitosan move to a lower wavenumber of 1498 cm−1 and a higher wavenumber of at 3442 cm−1 in CsHA,25,29 which is due to coordination interaction between Ca2+ and −NH2, as shown in Figure 3C. In addition, owing to the similar

255

ln ∑i = 0 ∑ j = 0 Bij ln 255

(2)

To analyze the interactions between nHA crystal and polysaccharide, the thermostability of nHA, PeHA, CaHA, CsHA, and AmHA composites was investigated by thermogravimetric analysis (TGA) on a Netzsch thermal analyzer TG (STA 449C) under nitrogen from 40 to 800 °C at a heating rate of 10 °C/min. To evaluate the bonding capacity of pectin and Ca2+, excess Ca(NO3)2·4H2O was added into 50 mL pectin solution (1 wt %) and the pH was adjusted to 11 using NaOH solution (0.1 M). After 12 h, the mixture were dialyzed against water (molecular weight cutoff: 8 kD) for 7 days to remove the unbound Ca2+, and the percentage of calcium on pectin was evaluated using atomic absorption spectrometry after lyophilization. In addition, the viscosity of pectin and pectin/Ca2+ solution with different concentrations was investigated by Rotational Viscometer VISCO STAR Plus.

3. RESULTS AND DISCUSSION 3.1. Formation of nHA Crystal in the Presence of Polysaccharides. Polysaccharides with carboxyl groups (−COOH), sulfonic groups (−OSO3H), amino groups (−NH2), or hydroxyl groups (−OH) are often used as templates to control the formation of nHA crystals, where these GAG-like polysaccharides can further regulate the size and morphology of nHA crystals via the interactions between the chemical functional groups and crystals. Herein, nHA crystals were prepared in the presence of pectin, carrageenan, chitosan, and amylose. XRD patterns of the synthesized samples for 7 days are given in Figure 2; XRD diffraction peaks can be observed with no significant differences in all samples. The peaks at 2θ values of 26.0°, 31.8°, 32.9°, 39.8°, 46.7°, and 53.3° are indexed to (002), (211), (300), (310), (222), and (004) planes, respectively, which verifies the formation of the hexagonal structure of HA crystals. In addition, these diffraction peaks coincide with the database entry of a stoichiometric HA with the formula Ca10(PO4)6(OH)2 and a hexagonal (P63/m) unit cell (JCPDS, Card No. 9−0432), indicating that all samples are predominantly HA rather than other calcium phosphates. The previous study23 shows that characteristic peaks of the (211) and (300) planes of pure HA are separated and sharp. However, these two peaks are overlapped into a broad peak in all nHA/ C



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Figure 3. FTIR spectra of polysaccharide, nHA, and nHA/polysaccharide composites (PeHA, CaHA, CsHA, and AmHA).

Figure 4. Schematic illustration of the interactions between nHA crystal and different polysaccharides.

Figure 5. (A) Fractal dimension values and (B) TGA thermograms of synthesized samples in the presence of different polysaccharides.

incorporated in the nHA crystal. Figure 4 summarized the interaction types between nHA crystals and different polysaccharides. In the cases of PeHA and CaHA, both electrostatic interactions and coordination interactions exist between Ca2+ and −COO− or −OSO3−. For CsHA, the

interactions, the amide II adsorption bands of chitosan at 1385 cm−1 move to a higher wavenumber of 1413 cm−1 in CsHA. For AmHA, the characteristic peaks (2925 cm−1) of −CH2− in the hydroxymethyl group in amylose chain are observed in AmHA as well (Figure 3D), indicating that the amylose is D



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Figure 6. TEM images of synthesized samples at different conditions. (A) nHA crystal, (B) PeHA (1.2 g/L, 168 h), (C) CaHA (1.2 g/L, 168 h), (D) CsHA (1.2 g/L, 168 h), (E) AmHA (1.2 g/L, 168 h), (F) PeHA (0.2 g/L, 48 h), (G) PeHA (0.8 g/L, 48 h), (H) CaHA (2.0 g/L, 48 h), and (I) CaHA (4.0 g/L, 48 h). The inset images in A−E are the SAED diffraction patterns of nHA/polysaccharides composites. Note: the first number in brackets represents the concentration of polysaccharides (g/L) and the second number in brackets is the reaction time (hours).

chemical functional groups (−NH2 and −NHCO−) of chitosan can bind the PO43− via electrostatic interaction or bind Ca2+ via coordination interactions, respectively. For AmHA, the coordination interaction between Ca2+ and −OH is the main driving force to induce the nucleation and growth of nHA in the presence of amylose. The above results suggest that multi-interactions exist between nHA crystal and polysaccharides, which is determined by the type of polysaccharide. To further investigate the differences of the interactions, fractal dimension value, obtained by analyzing SEM images (Supporting Information Figure S1) with a box-counting method, was used to compare the intensity of these interaction forces. Previous study has shown that the lower the fractal dimension value, the higher the stability the composites may have.30 As shown in Figure 5A, the fractal dimension value of pure nHA is significantly higher than that of all nHA/polyscaccharides composites, and the fractal dimension values of nHA/polyscaccharides composites are in the order of PeHA < CaHA < CsHA < AmHA, suggesting that the PeHA is the highest stability composite. Therefore, the interaction intensity between nHA crystals and polysaccharides is in the order of PeHA > CaHA > CsHA > AmHA. This result is consistent with previous study of Li et al.,31 who found that the rate of nucleation and crystal growth depends on the type of surface functional groups, in the order of −COOH > −CONH2 > −OH. The thermal stabilities of nHA/polysaccharide composites are demonstrated using TGA, which is similar to the results of fractal dimension, as shown in Figure 5B. The weight loss in the range of 40 °C to 210−280 °C is mainly due to the initial

decomposition of polysaccharides. The weight loss of all samples accelerates when the temperature rises to 600 °C, because of the further decomposition and evaporation of polysaccharides.32,33 Subsequently, the decomposition of carbonates incorporated into nHA occurs above 600 °C.34 At 600 °C, the residual weight percentage is ∼74% for PeHA, 78% for CaHA, 80% for CsHA, and 82% for AmHA. On the other hand, previous study35 showed that the weight of nHA changed slightly during the heating process from 40 to 800 °C because of its excellent stability. That is to say, approximately 26% pectin, 22% carrageenan, 20% chitosan, and 18% amylose are incorporated into PeHA, CaHA, CsHA, and AmHA, respectively. This phenomenon may be caused by the different interactions between nHA crystals and polysaccharides; the higher interactions between carboxyl/sulfate groups of anionic polysaccharides and nHA crystal make more pectin/carrageenan to introduce into PeHA/CaHA composites. Zhu et al.36 also suggested that the carboxyl or sulfate groups in GAGs contribute to the interface interaction and stabilization between mineral and organic phases. 3.3. Effects of Polysaccharides on the Morphology and Size of nHA Crystals. HA crystal is in the space group P63/m; its unit cell parameters are a = b = 9.43 Å and c = 6.88 Å. The lattice parameters of prepared nHA crystal are calculated from Rietveld refinement using Jade 5.0 software. As shown in the Supporting Information Table S1, the lattice parameters are shown to vary between 9.426 and 9.433 Å for the a, b-axis, and between 6.882 and 6.890 Å for the c-axis. The c/a value of PeHA and CaHA is higher than that of pure nHA crystal. E



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effectively bind Ca2+ to induce a very large number of nuclei for nHA crystal growth, resulting in the formation of nHA crystals with a small size. Sulfate groups also have been shown to be appropriate for binding nHA crystals on polysaccharides, such as chondroitin-4-sulfate and heparin, which are employed as initiators for the heterogeneous nucleation of HA crystals.14,44 Herein, lots of nucleation sites (−OSO3Ca+ and (−OSO3)2Ca) can form on a carrageenan chain, which further facilitates the crystal growth. In addition, the helical structures of carrageenan may result in the specific binding to the (002) face of nHA.16,27 The heterogeneous nuclear capability of carrageenan is lower than that of pectin due to the relatively weak interactions, and thus, fewer nuclei form on carrageenan compared to pectin and the size of CaHA is larger than that of PeHA.45 For cationic chitosan, the electrostatic interaction between PO43− and −NH2 groups, together with coordination of −NH2 to Ca2+, are two main driving forces for the nHA crystal nucleation and growth to aligned chitosan chains. 41 However, these interactions are weaker than that between Ca2+ and anionic polysaccharides, leading to fewer nucleation sites and bigger nHA crystals formed. Among all the polysaccharides discussed here, the coordination interactions between Ca2+ and −OH on neutral amylose is the weakest; therefore, nHA crystals exhibit the largest size in the presence of amylose. Meanwhile, the content of free inorganic ions in solution also depends on the interactions between nHA crystals and polysaccharides as well. In anionic polysaccharide solution, the concentration of Ca2+ drastically decreases, because a large amount of Ca2+ ions are bound on polysaccharide chains via the strong interactions, and thus, the growth rate of nHA crystals is slow. On the contrary, a high content of free Ca2+ in amylose can accelerate the growth of nHA crystals and lead to a relatively larger size of AmHA. 3.4. Effect of Polysaccharide Concentration on the Formation of nHA Crystals. The formation process of the nHA crystals varies depending on the polysaccharide concentration. In this study, the effects of concentration of pectin and carrageenan were investigated. Figure 8 displays the XRD patterns of nHA crystals synthesized in the pectin and carrageenan solutions with different concentration. The characteristic peaks of nHA crystals appear when the pectin concentration is less than 0.8 g/L (Figure 8A), and the intensity of the (002) face rises monotonically with the decrease of pectin concentration. However, the nHA crystals cannot be formed when the concentration of pectin reaches 2.0 g/L. During this reaction process, owing to the existance of the excess −COO−, most of the Ca2+ ions are bound to the pectin chain and the content of free Ca2+ in solution is quite low when the pectin concentration is more than 2.0 g/L. Meanwhile, the viscosity of pectin/Ca2+ mixed solution increases from 3.15 to 4.45 mPa·S when the concentration of pectin ranges from 0.08 g/L to 2.0 g/L (Supporting Information Table S2); the high viscosity (4.45 mPa·S) leads to poor flowability and limited diffusion of PO43−. nHA crystal nucleation and growth do not occur under either condition. In addition, the high viscosity also makes it difficult to remove nitrate (NO3−) and sodium ions (Na+) in this reaction system. As shown in Figure 8A, a diffraction peak of sodium nitrate (NaNO3) appears at 30.2°. Our results suggest that the pectin/Ca2+ ratio is the pivotal factor to modulate the formation of nHA crystals. We find that the highest calcium content in pectin/Ca2+ complex is ∼3.8%, obtained from the atomic absorption spectrometry. That is to say, when the ratio of pectin/Ca2+ is more than 96.2/3.8 (w/ w), i.e., 25.3 (w/w), all Ca2+ ions are adsorbed on pectin chains

However, lattice parameters of CsHA and AmHA changes little compared to nHA crystals. The morphology of nHA prepared in the presence of polysaccharides was observed using TEM. As shown in Figure 6A, the pure nHA are irregular to some extent, whereas after the addition of polysaccharides, uniform needle-like or rod-like nHA crystals with a diameter of 8−10 nm and a length of 100− 120 nm can be obtained, as shown in Figure 6B−E, which is similar to the shape of nHA crystals in a natural bone.37 Moreover, the diameter of these nHA is obviously smaller than that of pure nHA, indicating that the polysaccharides can control the morphology of nHA. Moreover, the SAED patterns exhibit strong concentric rings appointed to the (002), (211), and (300) planes of apatite, confirming that nHA crystals are inclined to grow along the c-axis, corresponding to the (002) reflection peak.38 Previous studies also suggested that the addition of polymer with different chemical functional groups can modulate the morphology of nHA crystals. The addition of alginate containing −COOH is beneficial for the formation needle-like nHA crystals.39 In poly(sodium 4-styrenesulfonate) solution, the prepared nHA crystals exhibit regular rod-like structure and less aggregation.40 Similarly, our previous study19 and that of Rusu et al.41 found that the shape of nHA is rod-like in the chitosan matrix. However, nHA crystals prefer to form a spherical morphology in the presence of PEG containing −OH,42 which is obviously different from our AmHA and Shakir’s report,43 in which they showed that the nHA is rod-like with an average size in the range of 12−17 nm after addition of amylose. It may be caused by the different number of −OH groups on the polymer chain. The average size of nHA crystals was calculated by (002) reflection peak using the Scherrer formula. As shown in Figure 7, prepared nHA crystals range from 0 to 20 nm, which is

Figure 7. Average size calculated from (002) reflection peak of nHA crystal synthesized in the presence of polysaccharide.

similar to the natural nHA in bone. This calculated size is obviously smaller than that observed by TEM. We reasoned that nHA crystals may assemble into larger nanoparticles via surface chemical interactions.44 Our data also show that the size of nHA crystals synthesized in the presence of polysaccharides is smaller than that of pure nHA (19.0 nm). Moreover, the size of nHA crystal depends on the type of polysaccharides, i.e., PeHA (11.0 nm) < CaHA (13.1 nm) < CsHA (15.1 nm) < AmHA (16.3 nm). This order is correlated with the interaction intensity between nHA crystals and polysaccharides. For anionic polysaccharides, carboxyl-rich polymer is affirmed to be of special importance in the nucleation of nHA crystal. Pectin contains lots of −COOH groups that can F



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Figure 8. XRD patterns of the nHA crystals prepared in the pectin (A) and carrageenan (B) solution with different concentration.

Figure 9. Formation of nHA crystal in pectin solution with different concentrations.

and nHA crystals cannot form. At low concentration of pectin, most nHA crystals are formed via homogeneous nucleation in solution, rather than growing along the chitosan chains, even though the heterogeneous nucleation on pectin molecules occurs preferentially over homogeneous nucleation due to the smaller activation energy,46 and the tendency of heterogeneous nucleation behavior gradually increases with pectin concentration. The above mechanism has been illustrated in Figure 9. In the case of carrageenan, the diffraction peaks at 30.2° and 41.1° of NaNO3 can be observed in the CaHA with carrageenan concentration higher than 2.0 g/L. Typical diffraction peaks of nHA crystals at 26.0° and 31.8° appear in all CaHA samples, as shown in Figure 8B, which may be due to the interaction between nHA and carrageenan being relatively weak compared to that of nHA and pectin. Regardless, the intensity of CaHA diffraction peaks decreases with the carrageenan concentration, implying a reduction of crystallinity. The diffraction intensity of sodium NaNO3 crystals increases with the carrageenan concentration. The addition of polysaccharide can modulate the morphology of nHA crystals. TEM images of PeHA (Figure 6F,G) and CaHA (Figure 6H,I) show that the shape of the nHA crystal is not changed with different concentrations. However, the crystal size gradually decreases with the polysaccharide concentration for both pectin (within 0.8 g/L) and carrageenan (within 3.0 g/ L), as shown in Figure 10. With the increase of polysaccharide concentration, an abundant supply of −COOH or −OSO3−

Figure 10. Average size of PeHA and CaHA prepared in different polysaccharide concentrations.

coordination sites are available for complexation with Ca2+, which leads to the formation of a very large number of nucleation sites and the reduction of free Ca2+, and as a result, nHA crystals are formed with smaller size.20 Surprisingly, the size of CaHA rapidly increases to 27.5 nm when the carrageenan concentration reaches 4.0 g/L, which is deemed G



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and short reaction time is adverse for formation of nHA crystals, especially for the polysaccharides with carboxyl groups.

a result from NaNO3 crystals, and should be further investigated. 3.5. Effect of Reaction Time on the Formation of nHA Crystals. In order to investigate the formation process of nHA crystals in the presence of pectin, the phases of pure nHA and PeHA at different reaction times are analyzed by XRD. We can find the characteristic diffraction peaks (26.0°, 31.8°, 39.8°, 46.7°, and 53.3°) of pure nHA crystal at 5 h (Supporting Information Figure S2), and there are few changes within 120 h. The addition of pectin delays the formation of nHA crystal, as shown in Figure 11; the XRD pattern of PeHA is a wide

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01235. SEM photos and density histogram of SEM micrographs of nHA/polysaccharides composites; XRD patterns of pure nHA at different reactive time; lattice parameter of HA crystals prepared in the presences of polysaccharides; viscosity of pectin and pectin/Ca2+ solution with different concentration. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] or [email protected]. Tel: +8610-68166874. *E-mail: [email protected]. Tel: +86-22-27402893. Present Address ∥

Micro/Nano Technology Center, Tokai University, 4−1−1 Kitakaname, Hiratsuka, Kanagawa, 259−1292, Japan. Author Contributions ‡

This manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Wancai Fang and Hong Zhang contributed equally to this work.

Figure 11. XRD patterns of the PeHA at different reaction times.

Notes

The authors declare no competing financial interest.

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amorphous hump between 25° and 35° in 2θ within 24 h, indicating the formation of amorphous calcium phosphate. With increasing reaction time, free access of PO43−, Ca2+, and OH− to the nucleation sites further induces the growth and deposition of nHA crystal on pectin chains. After 72 h, the characteristic diffraction peaks of nHA crystals appear in the XRD spectra of PeHA. Moreover, the diffraction intensity of these peaks is gradually enhanced with the reaction time, indicating that the nHA crystals continue to grow during this period. In addition, the size of nHA crystals reaches ∼13.6 nm from 17 to 120 h, and remained unchanged afterward. It may be that the free PO43− and Ca2+ have been completely reacted and the nHA crystals arrive at a stable state. These results suggest that the introduction of pectin changes the formation process of nHA crystals due to the interaction between −COO− and Ca2+.

ACKNOWLEDGMENTS This work is supported by National Nature Science Foundation of China (51573127, 31271016, 31370975, 31100674 and 81272912) and National High Technology Research and Development Program of China (No. SS2015AA020304).

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REFERENCES

(1) Kutikov, A. B.; Skelly, J. D.; Ayers, D. C.; Song, J. ACS Appl. Mater. Interfaces 2015, 7, 4890−4901. (2) Epple, M.; Ganesan, K.; Heumann, R.; Klesing, J.; Kovtun, A.; Neumann, S.; Sokolova, V. J. Mater. Chem. 2010, 20, 18−23. (3) Sadat-Shojai, M.; Khorasani, M. T.; Dinpanah-Khoshdargi, E.; Jamshidi, A. Acta Biomater. 2013, 9, 7591−7621. (4) Dubey, D. K.; Tomar, V. Mater. Sci. Eng., C 2009, 29, 2133−2140. (5) Yang, Y. S.; Wu, Q. Z.; Wang, M.; Long, J.; Mao, Z.; Chen, X. H. Cryst. Growth Des. 2014, 14, 4864−4871. (6) Wu, F.; Lin, D. D. W.; Chang, J. H.; Fischbach, C.; Estroff, L. A.; Gourdon, D. Cryst. Growth Des. 2015, 15, 2452−2460. (7) Li, J. J.; Dou, Y.; Yang, J.; Yin, Y. J.; Zhang, H.; Yao, F. L.; Wang, H. B.; Yao, K. D. Mater. Sci. Eng., C 2009, 29, 1207−1215. (8) de Leeuw, N. H.; Rabone, J. A. L. CrystEngComm 2007, 9, 1178− 1186. (9) Jee, S. S.; Kasinath, R. K.; DiMasi, E.; Kim, Y. Y.; Gower, L. CrystEngComm 2011, 13, 2077−2083. (10) Poursamar, S. A.; Azami, M.; Mozafari, M. Colloids Surf., B 2011, 84, 310−316. (11) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689−702. (12) Nudelman, F.; Pieterse, K.; George, A.; Bomans, P. H. H.; Friedrich, H.; Brylka, L. J.; Hilbers, P. A. J.; de With, G.; Sommerdijk, N. A. J. M. Nat. Mater. 2010, 9, 1004−1009. (13) Han, G. S.; Lee, S.; Kim, D. W.; Kim, D. K.; Noh, J. N.; Park, J. H.; Roy, S.; Ahn, T. K.; Jung, H. S. Cryst. Growth Des. 2013, 13, 3414− 3418.

4. CONCLUSIONS In this study, the polysaccharides with different chemical functional groups display different effects on the nucleation and growth of nHA crystals. Compared with irregular nHA synthesized in water, the uniform needle/rod-like nHA crystals can be formed in the presence of polysaccharides. The size of the nHA crystals depends on the polysaccharide type, and increases in the order PeHA, CaHA, CsHA, and AmHA. The cause of the morphology and size differences is the different interactions between nHA crystals and polysaccharides. Our results suggest that the interaction intensity is in a decreasing sequence of PeHA, CaHA, CsHA, and AmHA. The presence of polysaccharides induces the heterogeneous nucleation of nHA crystals on polysaccharide chain and further modulates the crystal growth. In addition, a high polysaccharide concentration H



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(14) Wise, E. R.; Maltsev, S.; Davies, M. E.; Duer, M. J.; Jaeger, C.; Loveridge, N.; Murray, R. C.; Reid, D. G. Chem. Mater. 2007, 19, 5055−5057. (15) Wang, Y. M.; Ren, X. X.; Ma, X. M.; Su, W.; Zhang, Y. P.; Sun, X. S.; Li, X. D. Cryst. Growth Des. 2015, 15, 1949−1956. (16) Coleman, R. J.; Jack, K. S.; Perrier, S.; Grøndahl, L. Cryst. Growth Des. 2013, 13, 4252−4259. (17) Chen, J. D.; Nan, K. H.; Yin, S. H.; Wang, Y. J.; Wu, T.; Zhang, Q. Q. Colloids Surf., B 2010, 81, 640−647. (18) Li, J. J.; Chen, Y. P.; Yin, Y. J.; Yao, F. L.; Yao, K. D. Biomaterials 2007, 28, 781−790. (19) Li, J. J.; Zhu, D. W.; Yao, F. L.; Yao, K. D. Mater. Sci. Eng., C 2010, 30, 795−803. (20) Liu, Y.; Huang, J.; Li, H. J. Mater. Chem. B 2013, 1, 1826−1834. (21) Yang, Q. F.; Ding, J.; Shen, Z. Q. Chem. Eng. Sci. 2000, 55, 797− 805. (22) Hu, W. W.; Wang, Z.; Zhang, S. S.; Jiang, L.; Zhang, J.; Zhang, X. N.; Lei, Q. F.; Park, H. J.; Fang, W. J.; Chen, Z. Biomaterials 2014, 35, 7386−7397. (23) Kramer, E. R.; Morey, A. M.; Staruch, M.; Suib, S. L.; Jain, M.; Budnick, J. I.; Wei, M. J. Mater. Sci. 2013, 48, 665−673. (24) Bendtsen, S. T.; Wei, M. J. Mater. Chem. B 2015, 3, 3081−3090. (25) Kumar, R.; Prakash, K. H.; Cheang, P.; Gower, L.; Khor, K. A. J. R. Soc., Interface 2008, 5, 427−439. (26) Neira, I. S.; Kolen’ko, Y. V.; Lebedev, O. I.; van Tendeloo, G.; Gupta, H. S.; Matsushita, N.; Yoshimura, M.; Guitián, F. Mater. Sci. Eng., C 2009, 29, 2124−2132. (27) Li, J. J.; Yang, B. G.; Qian, Y. F.; Wang, Q. Y.; Han, R. J.; Hao, T.; Shu, Y.; Zhang, Y. B.; Yao, F. L.; Wang, C. Y. J. Biomed. Mater. Res., Part B 2015, 103, 1498−1510. (28) Araujo, J. V.; Davidenko, N.; Danner, M.; Cameron, R. E.; Best, S. M. J. Biomed. Mater. Res., Part A 2014, 102, 4415−4426. (29) Lawrie, G.; Keen, I.; Drew, B.; Chandler-Temple, A.; Rintoul, L.; Fredericks, P.; Grøndahl, L. Biomacromolecules 2007, 8, 2533−2541. (30) Batte, H. D.; Marangoni, A. G. Cryst. Growth Des. 2005, 5, 1703−1705. (31) Li, X. L.; Lan, J. L.; Ai, M.; Guo, Y. G.; Cai, Q.; Yang, X. P. Colloids Surf., B 2014, 123, 753−761. (32) Ghaffari, A.; Navaee, K.; Oskoui, M.; Bayati, K.; Rafiee-Tehrani, M. Eur. J. Pharm. Biopharm. 2007, 67, 175−186. (33) Ding, F. Y.; Nie, Z.; Deng, H. B.; Xiao, L.; Du, Y. M.; Shi, X. W. Carbohydr. Polym. 2013, 98, 1547−1552. (34) Chang, C. Y.; Peng, N.; He, M.; Teramoto, Y.; Nishio, Y.; Zhang, L. N. Carbohydr. Polym. 2013, 91, 7−13. (35) Ren, F.; Leng, Y.; Xin, R.; Ge, X. Acta Biomater. 2010, 6, 2787− 2796. (36) Zhu, P. Z.; Xu, J. D.; Sahar, N.; Morris, M. D.; Kohn, D. H.; Ramamoorthy, A. J. Am. Chem. Soc. 2009, 131, 17064−17065. (37) Sadjadi, M. S.; Meskinfam, M.; Sadeghi, B.; Jazdarreh, H.; Zare, K. Mater. Chem. Phys. 2010, 124, 217−222. (38) Chen, H. F.; Clarkson, B. H.; Sun, K.; Mansfield, J. F. J. Colloid Interface Sci. 2005, 288, 97−103. (39) Teng, S. H.; Shi, J. J.; Peng, B. X.; Chen, L. Compos. Sci. Technol. 2006, 66, 1532−1538. (40) He, D.; Xiao, X. F.; Liu, F.; Liu, R. F. Appl. Surf. Sci. 2008, 255, 361−364. (41) Rusu, V. M.; Ng, C. H.; Wilke, M.; Tiersch, B.; Fratzl, P.; Peter, M. G. Biomaterials 2005, 26, 5414−5426. (42) Qiu, C. F.; Xiao, X. F.; Liu, R. F. Ceram. Int. 2008, 34, 1747− 1751. (43) Shakir, M.; Jolly, R.; Khan, M. S.; Irama, N.; Khan, H. M. Int. J. Biol. Macromol. 2015, 80, 282−292. (44) Rees, S. G.; Hughes Wassell, D. T.; Embery, G. Biomaterials 2002, 23, 481−489. (45) Koutsopoulos, S. J. Biomed. Mater. Res. 2002, 62, 600−612. (46) Yokoi, T.; Kawashita, M.; Ohtsuki, C. J. Asian Ceram. Soc. 2013, 1, 155−162.

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