Formation of Hydroxyapatite Skeletal Materials from Hydrogel Matrices

Jun 23, 2015 - alization system, we deduced the following biomineralization mechanism: The matrix hydrogel is a complex gel formed via hydrogen bondin...
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Formation of Hydroxyapatite Skeletal Materials from Hydrogel Matrices via Artificial Biomineralization Takashi Iwatsubo,*,† Ryoichi Kishi,‡ Toshiaki Miura,† Takuya Ohzono,† and Tomohiko Yamaguchi† †

Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan ‡ CNT-Application Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan ABSTRACT: Several kinds of hydrogels were prepared as mimics for the collagen/acidic protein hydrogel employed as the polymer matrix for mineralization in natural bone formation. The hydrogels prepared as mineralization matrices were employed for synthesizing artificial bones. The artificial bone made from a network of poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) prepared by heating (PVA/PAA-h-network) exhibited mechanical properties comparable with those of fish scales. To elucidate the formation mechanism of the artificial bone, we synthesized four further kinds of matrix. Artificial bones were obtained from both a PVA/PAA network prepared by repeated freezing and thawing (PVA/PAA-ftnetwork) and a chitosan/PAA network, in which hydrogen bonding exists between the two constituent polymers, similar to that observed in a natural collagen/acidic protein network. The artificial bone made from the chitosan/PAA network was confirmed to be formed by the phase transformation of a cartilaginous precursor by a process similar to the transformation of cartilaginous tissue to natural bone. In addition, skeletal phase material, i.e., a homogeneous solid phase of hydroxyapatite/polymers, was formed in the cartilaginous phase, i.e., the hypercomplex gel. The skeletal phase grew thicker at the expense of the cartilaginous phase until it formed the entirety of the composite. Artificial bones were also obtained from a gelatin/PAA network and a poly[N-(2-hydroxyethyl)acrylamide]-co-(acrylic acid) network. These experimental results suggested that the coexistence of proton donor and proton acceptor functions in the hydrogel is a key factor for bone formation. The hydroxyapatite content of our artificial bones was almost conterminous with those of natural bones.

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INTRODUCTION Hydroxyapatite (HA), calcium carbonate (CC), and silica are the major mineral components of hard skeletal materials in biological organisms. These skeletal materials are composites of minerals and biopolymers such as proteins, the composition of which vary depending on the species of organism. Biopolymers1−6 are thought to template the formation process of skeletal material as a polymer matrix and therefore endue their characteristic properties. For example, HA nucleation in bone formation is believed to occur in the gap zone between collagen fibrils,7,8 although the idea of HA nucleation presumes the fluid in the mineralization field to be saturated with respect to HA. For the purpose of mimicking formation of skeletal materials, polymer gels have been adopted by many researchers as the polymer matrix for mineralization.9−11 There is much variation in both mineralization methods and polymer gels. Among the polymer matrices constructed to date, cartilage-like double network gels12 and chitin-silk fibroin complex films13 seem to mimic the matrices of biological system, because they comprise two polymer components, similar to biological systems. We have elucidated the common physicochemical features of the template biopolymers1−6 concerned with the three kinds of skeletal materials, such as their hydrogen bonding and © XXXX American Chemical Society

electrostatic properties. This knowledge has been applied to the design of synthetic polymer matrices for the formation of artificial skeletal material. Poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) were found to be suitable for this. A PVA/PAA network was prepared by heat treatment (PVA/ PAA-h-network) and employed as a matrix gel in a mineralization solution. The gel changed to a three-dimensional dense monolithic solid composite in an appropriate salt solution.14,15 From the observation of this artificial biomineralization system, we deduced the following biomineralization mechanism: The matrix hydrogel is a complex gel formed via hydrogen bonding between proton-donating and protonaccepting polymers. In bone formation processes, the hydrogel traps both hydrogen phosphate anions (HPO42−) and calcium cations (Ca2+) through hydrogen bonding and electrostatic interaction, respectively, thus forming a hypercomplex gel. Biomineralization of HA skeletal material takes place in a hydrogel surrounded by a salt solution that is unsaturated with respect to HA. Although the salt concentration appears to be Received: April 2, 2015 Revised: June 17, 2015

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DOI: 10.1021/acs.jpcb.5b03181 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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activated alumina column to remove inhibitors, followed by subsequent evaporation of the acetone. Sample Preparation. PVA/PAA Network Prepared by Heating (PVA/PAA-h-Network). The preparation of this polymer network was performed as previously described.14,15 PVA and PAA were combined by heat treatment under reduced pressure.23,24 PVA/PAA Network Prepared by Repeated Freezing and Thawing (PVA/PAA-ft-Network). PVA (7.5 g) was dissolved in 50 mL of a mixed solvent containing dimethyl sulfoxide and water (3:7 by volume) at 98 °C.25,26 The PVA solution was mixed with 28 g of 25% PAA (Mw around 250 kDa) aqueous solution and stirred at 60 °C. The mixture was poured into a glass cell with an aperture of 200 μm and frozen at −45 °C for 20 h. The frozen mixture was warmed to room temperature for 4 h. After repeating this freezing and thawing cycle 7 times, the polymer solution became a turbid hydrogel with a thickness of 200 μm. Chitosan/PAA Network. An aqueous solution containing 1% chitosan and 3.5% formic acid, and an aqueous solution containing 1% PAA (Mw around 250 kDa) were mixed (2:1 by volume). 27 The homogeneous solution was cast onto polystyrene Petri dishes, and the water and formic acid were allowed to evaporate at room temperature. Finally, the films were dried at reduced pressure. The transparency of this network film indicated the miscibility of chitosan and PAA. The resulting films were easily peeled from the dishes. Gelatin/PAA Network Prepared by Heating (Gelatin/PAAh-Network). PAA-NH3 solution containing 1% PAA (Mw around 250 kDa) was prepared by neutralization of PAA aqueous solution with 1 M NH3 solution, producing a solution in which the NH3 molar quantity was equivalent to that of the carboxylic acid group of PAA. This solution and an aqueous solution containing 1% gelatin were mixed (2:8 by volume) and formed into a dried film by the procedure described above. The film was then heated to 120 °C in a vacuum oven for 1 h. The film was washed twice in a mixed solvent of ethanol and 1 M acetic acid (5:3 by volume) to remove NH3, and then dried at room temperature. The film was then reheated to 120 °C in a vacuum oven for 1 h. Poly[N-(2-hydroxyethyl)acrylamide-co-acrylic acid] Network (P(HEAAm-co-AA) Network). P(HEAAm-co-AA) network was prepared by photoinduced radical polymerization. An aqueous solution of 0.67 M HEAAm and 0.33 M sodium acrylate (AA-Na) containing 4 mol % MBAAm as a crosslinking comonomer and 0.2 mol % OGA as a photoinitiator was deoxygenated with argon gas for 20−30 min and then transferred into a glass cell with an aperture of 80 μm. After irradiation with UV light with a wavelength of 365 nm for 5 h at room temperature, the solution became a hydrogel with a thickness of 80 μm. Mineralization and Measurements. A salt solution (200 mL) containing CaCl2 at 5 mM, KH2PO4, at 1.5 mM, K2HPO4 at 1.5 mM, PAA (Mw around 2 kDa) at 0.14 mM in the repeating unit, NaCl at 140 mM and 9 mL of 1 M Tris buffer (pH = 7.5) was prepared. A piece cut out from the prepared sheet of the polymer network was immersed in the salt solution and kept at 30 °C. After an appropriate time interval, the soaked film was taken out from the solution and then rinsed three times in pure water for 10 min to remove residual soluble ions from the sample. The weight percent of inorganic constituents in the obtained organic/inorganic composite, W, was calculated as W = 100(M − M0)/M, where M0 and M are

higher than the saturation value dissolved in pure water, the surrounding solution is unsaturated because the electrostatic potential of dissociated polyelectrolytes in the solution induces charge separation between positive and negative salt ions, Ca2+ and HPO42−;16,17 therefore, the activity of HA can be suppressed to lower than unity. In the formation of a solid phase of the HA/polymer network, the solid phase begins to form in the precursor hypercomplex gel phase. The solid phase then permeates through the hypercomplex gel phase, replacing it until the former phase occupies the entire interior space. The hypercomplex gel represents the fundamental structure of cartilaginous material, while the solid phase represents that of the skeletal material. Both natural CC and silica skeletal materials are thought to be formed in a manner similar to the above-mentioned mechanism.15 This bone formation mechanism is supported by the fact that natural bone is formed by the transformation of cartilaginous tissue.18 In addition to HA bone, CC skeletal tissue is thought to be formed from its cartilaginous precursor. Addadi et al. reported that a mollusk shell is formed from a noncrystalline precursor in a hydrogel composed of two species of polymer.19 They found that the crystalline fraction grew larger via transformation of the noncrystalline fraction. If the noncrystalline fraction is considered to be a CC cartilage such as squid pen,20−22 their observation also supports our above-mentioned mechanism. However, the PVA/PAA-h-network is not a completely descriptive representation of natural bone matrix. Natural bone contains collagen and acidic proteins.1 The collagen is insoluble in water and abundant in hydroxyproline, which can act as a proton donor through its hydroxyl group, while the acidic proteins can act as proton acceptors via several kinds of acidic group. Hence, acidic proteins can be adsorbed by collagen through hydrogen bonding. This bonding yields a physically cross-linked hydrogel that is assumed to be the matrix for mineralization in bone biosynthesis. This matrix formation is difficult in artificial systems owing to the lack of cosolvent for collagen and acidic protein. In previous studies, PVA was employed as a collagen mimic and PAA was employed as the acidic protein mimic, because of the possible hydrogen bonding interactions through the OH group of PVA and the  COO− group of PAA. In this study, we have developed a range of mimic matrices by varying the component polymer species in order to elucidate the formation mechanism of the artificial bone. In addition, comparison of our artificial biomineralization system and natural ossification systems has been conducted in order to examine the validity of our system as a model for biomineralization systems.

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EXPERIMENTAL SECTION Chitosan (viscosity 5−20 cps by the Brookfield method, 1% solution in 1% acetic acid) with a degree of deacetylation of 75−85%, and PAA with a molecular weight (Mw) of around 2 kDa were purchased from Sigma-Aldrich Corporation. PAA with an Mw of around 250 kDa, PVA (degree of polymerization, around 2000), N,N ′-methylenebis(acrylamide) (MBAAm), 2oxoglutaric acid (OGA), acrylic acid (AA), and gelatin derived from bovine bone were purchased from WAKO Pure Chemical Industries, Ltd. AA was purified before polymerization by distillation. N-(2-hydroxyethyl)acrylamide (HEAAm) was kindly supplied by Kohjin Co., Ltd. It was purified before polymerization by passing its acetone solution through an B

DOI: 10.1021/acs.jpcb.5b03181 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B the weights of the dried sample before and after the mineralization process, respectively. When W was assumed to be saturated for each network after repeated renewing of the solution, it was denoted as Ws. Four sheets of composite made from the PVA/PAA-hnetwork having different values of W were pigmented by a double staining technique for the observation of cartilaginous and skeletal regions at the different stages of mineralization. The sample was first immersed in a mixture of alcian blue 8GX and 0.5 M acetic acid for 2 min and then rinsed in several changes of deionized water. Next, it was immersed in an aqueous solution of alizarin red S for 2 h and then rinsed in several changes of deionized water. For the measurement of the tensile stress−strain curve of artificial bone, a rectangular 0.5 × 2 cm2 sheet of the PVA/ PAA-h-network was immersed in the mineralization solution. Although the sample sheet lost its original flat shape and curved after mineralization, artificial bone was pliable in the wet state. The curved wet bone was placed between Teflon meshes, and then put between porous silica−alumina plates to remove residual water from the sample. The sample became flat and hardly pliable after drying. It could be fixed with clamps to test its mechanical properties using a tensile tester (Autograph AGX, Shimadzu) under a constant crosshead speed of 1 mm min−1 in ambient conditions. The Young’s modulus, fracture stress, and fracture strain were determined from the stress−strain curve. The PVA/PAA-h-network film and the composite with Ws = 78.4% made from the network were frozen in liquid nitrogen and broken into pieces. The cross-sectional images of the samples were observed by SEM (S-4300, Hitachi). The mineralized chitosan/PAA-based composite was frozen by liquid nitrogen and cut into pieces. The pieces were fixed on a stage and carbon-coated to allow the observation of their cross sections by energy dispersive X-ray spectroscopy (EDAX, EDAX Inc.). X-ray powder diffraction was conducted with a MiniFlex II diffractometer (Rigaku Co.).

Figure 1. Composites made from the PVA/PAA-h-network after double staining. (a) W = 38.7%, (b) W = 59.1%, (c) W = 68.2%, and (d) Ws = 81.4%. Circled bar, presented for scale, is 2 mm long.

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Figure 2. A typical tensile stress−strain curve of artificial bone with W = 65.3% made from the PVA/PAA-h-network.

RESULTS AND DISCUSSION The pigmented composites made from the PVA/PAA-hnetwork are shown in Figure 1. Since cartilage looks blue, while bone looks red owing to the double-staining technique,28,29 the change in color in the course of the mineralization process can be interpreted as follows: The sample with W = 38.7% looks blue and therefore consists of cartilaginous material (Figure 1(a)). The sample with W = 59.1% consists of a blue cartilaginous phase and a red skeletal phase, which is partially embedded in the blue phase (Figure 1(b)). In the sample with W = 68.2%, the skeletal phase spreads in an in-plane direction, but has not yet reached saturation (Figure 1(c)). Eventually, the skeletal phase grows thicker and occupies the whole portion of the sample with Ws = 81.4%, which shows no blue portion (Figure 1(d)). In this way, the transformation from cartilaginous material to skeletal material can be visualized. This process mimics the transformation of cartilaginous tissue to natural bone. We examined the mechanical properties of the artificial bone with W = 65.5 ± 1.0% (n = 10), which was synthesized from the PVA/PAA-h-network. Its fracture stress and fracture strain are 80.2 ± 2.3 MPa and 0.039 ± 0.002, respectively. A typical stress−strain curve is shown in Figure 2. The Young’s modulus was calculated as 2.9 ± 0.1 GPa. These values are comparable with those of fish scales.30 The fracture stress and Young’s modulus of fish scales from Pagrus major are

93 ± 1.8 MPa and 2.2 ± 0.3 GPa, respectively (n = 10). The corresponding fracture strain is between 0.04 and 0.05. Hence, the artificial bone displays mechanical properties similar to those of material found in biological systems, and also shows similarities in both the physicochemical properties of the constituent polymers and in the bone formation process.15 We have also recorded SEM images of the PVA/PAA-hnetwork before and after mineralization. The initial network film has a dense homogeneous structure in the dry state (Figure 3 (a)). As shown in Figure 3(b), the artificial bone also has a dense structure and no noticeable heterogeneity, implying an

Figure 3. Cross sectional SEM image of the initial PVA/PAA-hnetwork film (a) and the composite with Ws = 78.4% made from the PVA/PAA-h-network. C

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different mineralization periods using this type of gel were characterized by EDX. For the sample with W = 57.1%, the C and O contents are lower in the center portion than in the outer, while those of Ca and P are higher in the center portion than in the outer (Figure 5(a)), when the total content of Ca, P, O, and C are normalized

almost homogeneous formation of the HA/polymer network composite. As discussed above, natural bone contains collagen and acidic proteins.1 The collagen is insoluble in water and abundant in hydroxyproline31 that can act as a proton donor through its hydroxyl group, while the acidic proteins can act as proton acceptors via several kinds of acid group. Hence, acidic proteins can be adsorbed onto collagen through hydrogen bonding. This bonding yields a collagen/acidic protein hydrogel that is assumed to be the matrix for mineralization in bone biosynthesis. Here, PVA has been employed as a collagen mimic and PAA was employed as the acidic protein mimic. To make an insoluble matrix from water-soluble PVA and PAA, the PVA/PAA network was prepared by heat treatment.23,24 Next, to make an insoluble matrix from PVA and PAA, a PVA/PAA network was prepared by repeated freezing and thawing. Using this PVA/PAA-ft-network, we obtained a bone-like material with Ws = 92.9% (Figure 4(a)).

Figure 5. Composition profiles of C, O, Ca, and P (a), and the [Ca]/ [P] profile (b) in the cross section of artificial bone with W = 57.1% made from the chitosan/PAA network.

to 100%, eliminating other elements such as N. The molar ratio, [Ca]/[P], is close to that of HA (1.67) in the center portion, and higher than that of HA in the outer portion (Figure 5(b)). For the sample with Ws = 72.3% (Figure 6, parts (a) and (b)), the C and O contents are lower while the Ca and P contents are higher than those of the outer portion of the sample with Ws = 57.1%. An almost constant value of [Ca]/[P] of approximately 1.67 has been observed. Thus, the mineralization is saturated in this sample. Since these observations are quite similar to those obtained in the previous artificial mineralization in which the PVA/PAA-h-network was used,14 a qualitatively analogous mineralization process to the previous system is suggested. Therefore, the center portion in Figure 5 is in the skeletal phase of HA with chitosan/PAA, and gives rise to the X-ray diffraction peak, while the outer portion is in the hypercomplex network phase that swells and becomes the cartilaginous phase in the salt solution. However, the whole portion in Figure 6 is in the skeletal phase. A schematic model of the structures of the cartilaginous phase, the skeletal phase, and their transformation process in the present system is shown in Figure 7. Since HPO42− has both proton donating and accepting abilities,33 its OH group donates a proton to the COO− group of PAA, while its other oxygen atoms accept proton from the OH and NH2 of chitosan. Via these hydrogen bonding interactions HPO42− is absorbed into the chitosan/PAA network together with Ca2+

Figure 4. Photograph of artificial bone with Ws = 92.9% made from the PVA/PAA-ft-network (a), artificial bone with Ws = 74.3% made from the chitosan/PAA network (b), artificial bone with Ws = 92.2% made from the gelatin/PAA-h-network (c), and artificial bone with Ws = 96.1% made from the P(HEAAm-co-AA) network (d). The circled bar, presented for scale, is 2 mm long.

The turbidity of the original gel was indicated by the opacity of this composite. The water content of the original hydrogel soaked in water was measured to be about 91%, while that of the PVA/PAA-h-network was measured as about 71%. Mineralization with the latter gel yielded a material with Ws = 80%.15 Therefore, the high Ws obtained for the PVA/PAA-ftgel may originate from the high swelling ratio in water of the original hydrogel. It is interesting to note that an aqueous solution containing only PVA becomes a hydrogel through cycled freezing and thawing.32 Thus, PVA and PAA can also form a hydrogen bonded network between the OH of the insolubilized PVA hydrogel and the COOH of PAA in a fashion similar to a collagen/acidic protein gel. Next we report the results of mineralization based on a chitosan/PAA network. Artificial bone with Ws = 74.3% made from this matrix is shown in Figure 4(b). Since the chitosan is insoluble in water and has proton donating groups (OH and NH2), the combination of the chitosan and PAA results in a hydrogel very similar to a collagen/acidic protein gel. Two samples with W = 57.1% and Ws = 72.3% obtained from D

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PAA composites utilizing a supercritical CO2 fluid.37,38 They induced CC mineralization throughout a polymer matrix fixed on a glass substrate. Here, we fabricated HA skeletal material based on the chitosan/PAA network under mild conditions of ordinary temperature and ordinary pressure, intended to mimic the biological process. We next report the results of mineralization based on a gelatin/PAA-h-network. Mineralization using this matrix yielded a material with Ws = 92.2% (Figure 4(c)). This gelatin is manufactured from bone collagen and is therefore abundant in hydroxyproline.31 Its miscibility with PAA can be ascribed to affinitive interaction via hydrogen bonding between the OH of the hydroxyproline residues and the COO− of PAA. However, since the gelatin is soluble in water, the gelatin/PAAh-network was made using heat treatment to make the polymer matrix insoluble in aqueous solution. In this system, the constituent proton donor polymer (gelatin) can be considered an explicit collagen analogue in the primary polymer structure. PVA, chitosan, and gelatin have been employed individually as matrices of mineralization by several researchers.9,10,39,40 Here, monolithic composites have been developed from matrices combining each of the proton donor polymers with PAA. Elsewhere in this work, matrices in artificial biomineralization systems have been constructed by connecting two kinds of polymers, in analogy to biological systems. However, the combination of two kinds of polymer may not be essential for artificial biomineralization. In the P(HEAAm-co-AA) network, the OH of HEAAm acts as a proton donor while the  COO− of AA acts as a proton acceptor. The water content of this gel was 96.8% and mineralization using this gel as the polymer matrix yielded a material with Ws = 94.7%, although it became brittle when dried under reduced pressure. Figure 4(d) shows the composite with Ws = 96.1%. In this way, coexistence of the proton donor and proton acceptor in the hydrogel is thought to be necessary for mineralization. We prepared a more densely cross-linked P(HEAAm-co-AA) network with 5 mol % MBAAm as a cross-linking comonomer. The water content of this gel was 90.4% and mineralization using this gel as the polymer matrix yielded a material with Ws = 88.6%. Again, we can posit that the high swelling ratio tends to favor high Ws. In this study, the mechanical properties were examined only for the PVA/PAA-h-network. This is because other artificial bones were brittle or hard to flatten, which makes the measurement of mechanical properties extremely difficult. This implies that other artificial bones show poor mechanical properties at present. However, we have not explored the preparation conditions significantly, and the mechanical

Figure 6. Composition profiles of C, O, Ca, and P (a), and the [Ca]/ [P] profile (b) in the cross section of artificial bone with Ws = 72.3% made from the chitosan/PAA network.

that is absorbed via electro-static interaction, forming a hypercomplex gel in the salt solution. In this way, the synergistic effect of hydrogen bonding and electro-static interaction makes HPO42− and Ca2+ bind strongly to the matrix gel. This can be considered as a mimic for the structure of a natural cartilaginous phase. Conversely, the skeletal phase is mimicked by the homogeneous solid of HA/polymer network.15 The skeletal phase grows thicker at the cost of the cartilaginous phase as mineralization proceeds, until it completely occupies the volume of the sample. Since, in this way, there is no intermediate state between skeletal and cartilaginous phases, the discontinuous change from the hypercomplex gel to the solid solution will be a phase transformation process. Thus, the present system is a model of bone formation in which cartilaginous tissue changes into, or is replaced by, bone (cartilaginous or endochondral ossification).18 This change is most obviously observed in the formation of deer antlers that are shed and regrown every year. After the growth period of the antler cartilage, the antlers harden.34−36 Wakayama et al. synthesized CC and chitosan/

Figure 7. Schematics of the structures of the cartilaginous phase, skeletal phase, and the transformation process from the cartilaginous to skeletal phases. E

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than that of the standard sate (μ0), μ < μ0. This leads to μ = μ0 + RT ln a, and a < 1, where R is the gas constant, T is the temperature, and a is the activity of HA. For a solid composite, a < 1 indicates that HA is thermodynamically miscible with the polymer network. Thus, a solid composite can be formed from the hypercomplex gel even in unsaturated solutions. From a physicochemical point of view, the change of cartilaginous tissue to bone is a manifestation of the phase transformation of the hypercomplex gel to a homogeneous solid phase of the HA/polymer network.

properties of these artificial bones could be improved using optimized preparation conditions. We have examined the structure of artificial bones by X-ray diffraction, as shown in Figure 8.

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CONCLUSIONS Biomineralization is controlled in a hierarchy of several stages in an organism, and is fundamentally regulated at the genetic level. However, in the final stage of the formation of skeletal tissue, the physicochemical interaction between biological material (such as proteins) and the ionic species comprising the mineral is the dominant factor. Artificial biomineralization was carried out with several hydrogels designed and synthesized by consideration of the physicochemical properties of biopolymers relevant to biomineralization. Particularly, the PVA/PAA-ft-network and chitosan/PAA network mimicked the formation manner of bone matrices, and the gelatin/PAA-hnetwork mimicked the collagen of matrices. All matrices were employed for synthesizing artificial bones in which the HA content could be varied over the range found in natural bone. X-ray diffraction peaks of artificial bones were broad, similar to those observed for natural bone. Artificial bones synthesized from the PVA/PAA-h-network and chitosan/PAA network were confirmed to be formed by the transformation of cartilaginous to skeletal materials. Artificial bones prepared from the PVA/PAA-h-network displayed mechanical properties similar to fish scales, but other artificial bones were broken by the flattening necessary for mechanical measurement. PAA in the mineralization solution is an analogue of acidic proteins in body fluid. Thus, our method can mimic natural bone mineralization in many respects. The coexistence of proton donors and acceptors appears to allow absorption of both HPO42− via hydrogen bonding and Ca2+ via electrostatic interaction. Through this absorption, the hydrogel changes into a hypercomplex gel that is the precursor for the homogeneous solid of the HA/polymer network. To the best of our knowledge, this is the only method that gives a high HA content polymer matrix under mild conditions, except for biological systems themselves. We propose our artificial biomineralization system to be a candidate system for modeling of biomineralization.

Figure 8. Powder X-ray diffraction patterns of artificial bones synthesized from polymer matrices developed in this work. Intensity was increased by 300k cps. (a) Chitosan/PAA network, k = 0; (b) P(HEAAm-co-AA) network, k = 1; (c) PVA/PAA-ft-network, k = 2; and (d) gelatin/PAA-h-network, k = 3.

Since the X-ray powder diffraction patterns have peaks at Bragg angles corresponding to those of HA, we can confirm that the mineral components of the artificial bones are all HA. These peaks, as well as the peaks for real bone, are broader than the peaks for pure HA,41 indicating low crystallinity in bone HA. For example, the main peak at 2θ = 32° for natural and artificial bones is split into four sharp peaks for synthesized pure HA.41 This broadening can be brought about by the dislocation of HA lattice points, caused by the presence of polymer chains embedded in the crystallites of the solid phase. The observed value of Ws = 67.0% is close to that of deer antler (around 63%), while the value of Ws = 94.7% is close to that of porpoise petrosal (around 99%).42 These values are highly representative of the range of natural bones. Thus, in our artificial biomineralization system, artificial bones having a variety of HA contents similar to natural bones can be synthesized by varying the polymer networks. The salt solution as well as the polymer matrix mimics a biological system in our model. Body fluid contains acidic proteins.43 This fluid is unsaturated with respect to HA, because HA precipitates and causes severe damage to the body if it is supersaturated. However, simulated body fluid, in which the acidic proteins in body fluid are substituted with mainly Cl−, is, in fact, supersaturated.44 This supersaturation is attributed to the removal of acidic proteins, which, as discussed above, cause charge separation and decrease the activity of HA in the original body fluid. Hence, the low molecular weight PAA (degree of polymerization, around 2000) in the salt solution of our model system plays a similar role to the acidic proteins in body fluid. In an unsaturated solution, the chemical potential of HA inside and outside the network (μ) is smaller

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-29-861-4758. E-mail: [email protected] (T.I.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Tsuchihara at the Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), for assistance with the measurements of mechanical properties. We would like to thank Editage (www.editage.jp) for English language editing. F

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(23) Kuhn, P. W.; Hargitay, B.; Katchalsky, A.; Eisenberg, H. Reversible Dilation and Contraction by Changing the State of Ionization of High-Polymer Acid Networks. Nature 1950, 165, 514− 516. (24) Kumeta, K.; Nagashima, I.; Matsui, S.; Mizoguchi, K. Crosslinking Reaction of Poly(vinyl alcohol) with Poly(acrylic acid) (PAA) by Heat Treatment: Effect of Neutralization of PAA. J. Appl. Polym. Sci. 2003, 90, 2420−2427. (25) Shiga, T.; Hirose, Y.; Okada, A.; Kurauchi, T. Bending of Poly(vinyl alcohol)−Poly(sodium acrylate) Composite Hydrogel in Electric Fields. J. Appl. Polym. Sci. 1992, 44, 249−253. (26) Suzuki, M.; Hirasa, O. Advances in Polymer Science. Volume 110, Responsive Gels: Vol. Transitions II; Dušek, K., Ed.; Springer Verlag: Berlin, Germany, 1993. (27) Nam, S. Y.; Lee, Y. M. Pervaporation and Properties of Chitosan−Poly(acrylic acid) Complex Membranes. J. Membr. Sci. 1997, 135, 161−171. (28) Kawamura, K.; Hosoya, K. A Modified Double Staining Technique for Making a Transparent Fish-Skeletal Specimen. Bull. Natl. Res. Inst. Aquaculture 1991, 20, 11−18. (29) Cortés-Delgado, N.; Pérez-Torres, J.; Hoyos, J. M. Staining Procedure of Cartilage and Skeleton in Adult Bats and Rodents. Int. J. Morphol. 2009, 27 (4), 1163−1167. (30) Ikoma, T.; Kobayashi, H.; Tanaka, J.; Walsh, D.; Mann, S. Microstructure, Mechanical, and Biomimetic Properties of Fish Scales from Pagrus Major. J. Struct. Biol. 2003, 142, 327−333. (31) Morris, M. D.; Mandair, G. S. Raman Assessment of Bone Quality. Clin. Orthop. Relat. Res. 2011, 469, 2160−2169. (32) Hassan, C. M.; Peppas, N. A. Structure and Applications of Poly(vinyl alcohol) Hydrogels Produced by Conventional Crosslinking or by Freezing/Thawing Methods. Advances in Polymer Science. Vol. 153, Biopolymers, PVA Hydrogels, Anionic Polymerisation Nanocomposites; Dušek, K., Ed.; Springer Verlag: Berlin, Germany, 2000. (33) Luecke, H.; Quiocho, F. A. High Specificity of a Phosphate Transport Protein Determined by Hydrogen Bonds. Nature 1990, 347, 402−406. (34) Frasier, M. B.; Banks, W. J.; Newbrey, J. W. Characterization of Developing Antler Cartilage Matrix I. Selected Histochemical and Enzymatic Assessment. Calcif. Tissue Res. 1975, 17, 273−288. (35) Newbrey, J. W.; Banks, W. J. Characterization of Developing Antler Cartilage Matrix II. An Ultrastructural Study. Calcif. Tissue Res. 1975, 17, 289−302. (36) Hall, B. K. Chapter 8, Bones and Cartilage: Development and Evolutionary Skeletal Biology; Elsevier: San Diego, U.S.A., 2005. (37) Wakayama, H.; Hall, S. R.; Mann, S. Fabrication of CaCO3− Biopolymer Thin Films Using Supercritical Carbon Dioxide. J. Mater. Chem. 2005, 15, 1134−1136. (38) Wakayama, H.; Hall, S. R.; Fukushima, Y.; Mann, S. CaCO3/ Biopolymer Composite Films Prepared Using Supercritical CO2. Ind. Eng. Chem. Res. 2006, 45, 3332−3334. (39) Zhang, S.; Gonzalves, K. E. Influence of the Chitosan Surface Profile on the Nucleation and Growth of Calcium Carbonate Films. Langmuir 1998, 14, 6761−6766. (40) Kato, T.; Suzuki, T.; Amamiya, T.; Irie, T.; Komiyama, M.; Yui, H. Effects of Macromolecules on the Crystallization of CaCO3 the Formation of Organic/Inorganic Composites. Supramol. Sci. 1998, 5, 411−415. (41) Glimcher, M. J.; Bonar, L. C.; Grynpas, M. D.; Landis, W. J.; Roufosse, A. H. Recent Studies of Bone Mineral: Is the Amorphous Calcium Phosphate Theory Valid? J. Cryst. Growth 1981, 53, 100−119. (42) Lees, S. Considerations Regarding the Structure of the Mammalian Mineralized Osteoid from Viewpoint of the Generalized Packing Model. Connect. Tissue Res. 1987, 16, 281−303. (43) Gamble, J. L.; Ross, G. S.; Tisdall, F. F. The Metabolism of Fixed Base during Fasting. J. Biol. Chem. 1923, 57, 633−696. (44) Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. Solutions able to Reproduce in Vivo Surface-Structure Changes in Bioactive Glass-Ceramic A−W. J. Biomed. Mater. Res. 1990, 24, 721− 734.

REFERENCES

(1) Hunter, G. K.; Hauschka, P. V.; Poole, A. R.; Rosenberg, L. C.; Goldberg, H. A. Nucleation and Inhibition of Hydroxyapatite Formation by Mineralized Tissue Proteins. Biochem. J. 1996, 317, 59−64. (2) Berman, A.; Addadi, L.; Kvick, Å.; Leiserowitz, L.; Nelson, M.; Weiner, S. Intercalation of Sea Urchin Proteins in Calcite: Study of a Crystalline Composite Material. Science 1990, 250, 664−667. (3) Weiner, S.; Traub, W.; Parker, S. B. Macromolecules in Mollusc Shells and Their Functions in Biomineralization [and Discussion]. Philos. Trans. R. Soc. London B 1984, 304, 425−434. (4) Kröger, N.; Bergsdorf, C.; Sumper, M. A New Calcium Binding Glycoprotein Family Constitutes a Major Diatom Cell Wall Component. EMBO J. 1994, 13, 4676−4683. (5) Kröger, N.; Deutzmann, R.; Sumper, M. Polycationic Peptides from Diatom Biosilica that Direct Silica Nanosphere Formation. Science 1999, 286, 1129−1132. (6) Poulsen, N.; Sumper, M.; Kröger, N. Biosilica Formation in Diatoms: Characterization of Native Silaffin-2 and its Role in Silica Morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12075−12080. (7) Glimcher, M. J. Recent Studies of the Mineral Phase in Bone and its Possible Linkage to the Organic Matrix by Protein-Bound Phosphate Bonds. Philos. Trans. R. Soc. London B 1984, 304, 479−508. (8) Landis, W. J.; Hodgens, K. J.; Arena, J.; Song, M. J.; McEwen, B. F. Structural Relations Between Collagen and Mineral in Bone as Determined by High Voltage Electron Microscopic Tomography. Microsc. Res. Technol. 1996, 33, 192−202. (9) Grassmann, O.; Müller, G.; Löbmann, P. Organic−Inorganic Hybrid Structure of Calcite Crystalline Assemblies Grown in a Gelatin Hydrogel Matrix: Relevance to Biomineralization. Chem. Mater. 2002, 14, 4530−4535. (10) Taguchi, T.; Kishida, A.; Akashi, M. Hydroxyapatite Formation on/in Poly(vinyl alcohol) Hydrogel Matrices Using a Novel Alternate Soaking Process. Chem. Lett. 1998, 27, 711−712. (11) Shkilnyy, A.; Gräf, R.; Hiebl, B.; Neffe, A. T.; Friedrich, A.; Hartmann, J.; Taubert, A. Unprecedented, Low Cytotoxicity of Spongelike Calcium Phosphate/Poly(ethylene imine) Hydrogel Composites. Macromol. Biosci. 2009, 9, 179−186. (12) Kiyama, R.; Nonoyama, T.; Nakajima, T.; Kurokawa, T.; Gong, J. P. HAp Biomineralization in Tough DN Hydrogel for Cartilage Regeneration. Proc. MACRO 2014 2014, BTEC-31. (13) Falini, G.; Weiner, S.; Addadi, L. Chitin-Silk Fibroin Interactions: Relevance to Calcium Carbonate Formation in Invertebrates. Calcif. Tissue. Int. 2003, 72, 548−554. (14) Iwatsubo, T.; Sumaru, K.; Kanamori, T.; Shinbo, T.; Yamaguchi, T. Construction of a New Artificial Biomineralization System. Biomacromolecules 2006, 7, 95−100. (15) Iwatsubo, T.; Yamaguchi, T. Hypercomplex Gel Changes to Organic/Inorganic Solid Solution by Phase Transition in an Artificial Biomineralization System. Polym. J. 2008, 40, 958−964. (16) Katchalsky, A.; Lifson, S.; Mazur, J. The Electrostatic Free Energy of Polyelectrolyte Solutions. I. Randomly Kinked Macromolecules. J. Polym. Sci. 1953, XI, 409−423. (17) Marcus, R. A. Calculation of Thermodynamic Properties of Polyelectrolytes. J. Chem. Phys. 1955, 23, 1057−1068. (18) Hall, B. K. Chapter 1, Bones and Cartilage: Development and Evolutionary Skeletal Biology; Elsevier: San Diego, CA, 2005. (19) Addadi, L.; Joester, D.; Nudelman, F.; Weiner, S. Mollusk Shell Formation: A Source of New Concepts for Understanding Biomineralization Processes. Chem.Eur. J. 2006, 12, 980−987. (20) Kawai, Y.; Seno, N.; Anno, K. Condroitin Polysulfate of Squid Cartilage. J. Biochemistry 1996, 60, 317−321. (21) Sivakumar, P.; Chandrakasan, G. Occurrence of a Novel Collagen with Three Distinct Chains in the Cranial Cartilage of the Squid Sepia of f icinalis: Comparison with Shark Cartilage Collagen. Biochim. Biophys. Acta, Gen. Subj. 1998, 1381, 161−169. (22) Kimura, S.; Karasawa, K. Squid Cartilage Collagen: Isolation of Type I Collagen Rich in Carbohydrate. Comp. Biochem. Physiol. 1985, 81B, 361−365. G

DOI: 10.1021/acs.jpcb.5b03181 J. Phys. Chem. B XXXX, XXX, XXX−XXX