Role of Nanoscale Confinement on Calcium Phosphate Formation at

Article pubs.acs.org/crystal

Role of Nanoscale Confinement on Calcium Phosphate Formation at High Supersaturation Anand K. Rajasekharan and Martin Andersson* Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, SE-41296, Sweden S Supporting Information *

ABSTRACT: An important factor controlling bone mineralization is spatial restriction within collagen fibrils and matrix vesicles. Such nanoscale confinements in addition to chemical control restrict the growth and final size of calcium phosphate crystals (CaP). To study the role of spatial control on CaP mineralization exclusively, we investigated formation of CaPs under high supersaturations (0.84 M Ca2+ and 2.1 M Ca2+) within ordered aqueous domains (8−27 nm) of polymerized liquid crystals (PLCs). The mineralization of CaPs was achieved by a pressurized gas induced pH rise. CaPs mineralized within varying degrees of confinements showed contrasting results. Confinements ≤10 nm mediated the formation of CaPs to phase pure calcium apatite (CaAp), while confinements between 11 and 27 nm resulted in a mixture of phases such as CaAp, amorphous calcium phosphates (ACP), and acidic polymorphs such as brushite and monetite. Results indicated that smaller confinements offer a low particle/solution interface effectively forming small nucleation clusters leading to a metastable ACP phase. We suggest that CaP formation from high supersaturations strongly depends on the degree of confinement around the nucleation site to efficiently control the mineral phase purity. Moreover, a possible insight from this study is that synthetic and biological confinements, in addition to spatial restriction, also rely on chemical control to obtain phase pure CaPs such as bone apatite.

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INTRODUCTION

alization to study spatial control independently of other factors is desirable. Recently, different synthetic models have been used to study the effect of confinement on formation of calcium-based minerals. Effectively confined domains of gel-like media and gel−liquid interfaces have been explored where specific assemblies of calcium carbonate and calcium phosphate with controlled hierarchical architectures were formed.13,14 A concern with nonstructured gels is their lack of homogeneous confinements acting as rather unspecific scaffolds.13,15 Another interesting model used to study spatial control on CaAp mineralization was synthetic confinements of track etched alumina membranes (25 nm to 5 μm) free of additives.12,16 These confined volumes stabilized metastable phases like amorphous calcium phosphate (ACP), which gradually converted to CaAp with preferred orientations. Interestingly, the confinements were far larger than actual collagen interstices in bone, which implies that geometric restrictions can independently control the formation and alignment of CaAp. However, the studies were performed in relatively mild supersaturations (9 mM Ca2+) that probably is lower than actual in vivo conditions.17,18 To understand the role of confinements on inorganic mineralization, a good synthetic

Biomineralization of calcium phosphate (CaP) in bone is controlled by chemical, spatial, and morphological factors.1,2 These factors are offered by biological macromolecules that control mineral growth in terms of size, morphology, and polymorph resulting in materials with excellent functional capabilities. The nanostructure of natural bone is a highly organized composite of mostly collagen fibrils and calcium apatite (CaAp).3−5 Biomineralization of CaAp in bone occurs in the presence of a complex chemical environment with collagen, acidic noncollagenous proteins, and citrate molecules chemically modulating CaAp crystal size, chemistry, and morphology.6,7 In combination to chemical control, CaAp crystallization is also spatially coordinated within periodic and confined (nanoscale) interstices of collagen fibrils. These interstices (∼70 nm) provide for mineral deposition and restricted transformation of nanocrystalline CaAp via an amorphous precursor phase.8,9 This mode of crystallization control endows bone-CaAp with unique chemistry and preferential alignment over long length scales. Although chemical control on CaAp formation in bone is explored extensively, the actual spatial effects are relatively sparsely studied.6,7,10,11 This is mainly because of the complex environment in vivo where monitoring the effect of spatial restriction independent of other factors has proved to be difficult.12 As a consequence, a synthetic mimic for biominer© XXXX American Chemical Society

Received: January 31, 2015 Revised: March 29, 2015

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DOI: 10.1021/acs.cgd.5b00139 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Table 1. Recipes Used to Synthesize PLC and PLC-CaP Composites20,23 samplea S1 S2 S3 S4 S5

amphiphile

calcium phosphate precursorb

MF127 MF127 MF127 MF127 MP123

PLC phasec

amphiphile (wt %)

precursor (wt %)

I1 I1 H1 H1 H1

35 35 35 65 45

65 65 50 20 55

2+

0.84 M Ca 2.1 M Ca2+ 2.1 M Ca2+ 2.1 M Ca2+ 2.1 M Ca2+

BuOH (wt %)

15 15

S1 is pure PLC, CS1 is unaged composite, and ACS1 aged composite (see text). bpH (0.84 M Ca2+) = 0.37; pH (2.1 M Ca2+) = −0.26. cI1 (micellar cubic) and H1 (normal hexagonal) denote the geometry of the PLC.

a

Synthesis of Polymerized Lyotropic Liquid Crystals (PLCs). PLCs were formed by mixing the modified amphiphile (MF127 or MP123), calcium phosphate precursor, and butanol (BuOH) as per recipes shown in Table 1. 2-Hydroxy 2-methylpropiophenone was added (1 wt % of the amphiphile) as a photoinitiator. Mixing was performed in 20 mL vials manually using a spatula until a thick, homogeneous and viscous gel formed. The gel was then transferred to a mold and UV-polymerized (90 W, λ = 252 nm) for 10 min to form a flexible polymeric material with a thickness of 4−5 mm. Synthesis of PLC-CaP Composites. The PLC-CaP composite was formed by exposing the as-synthesized PLC containing calcium phosphate precursor to NH3 gas in a sealed gas chamber.20 Pressurized NH3 rapidly increased the pH inside the autoclave consequently raising the pH within aqueous domains of the PLC, which initiates CaP mineralization. The final pH inside the reaction chamber was measured to be 12.9 (pH meter, Metrohm inc.). NH3 pressure within the autoclave was maintained at 2 bar for 3 h. After reaction, the sample had turned white suggesting formation of CaP particles in the PLC. The composites were analyzed with X-ray diffraction immediately after reaction and then aged (dry state) at 22 °C for 24 h before further characterization. Characterization. Small angle X-ray scattering (SAXS) was performed on the PLCs and corresponding PLC-CaP composites. Characterization was accomplished using synchrotron radiation at the I911-SAXS beamline in MAX-Lab (Lund, Sweden). Samples were placed in small slit sealed with KaptonR tapes and supported on a stainless steel sample holder. The data were recorded on a twodimensional MarCCD 165 mm detector, and the wavelength of the Xray was 0.91 Å. X-ray exposure time on samples was 100 s and data was collected over a q-range of 0.1−4.0 nm−1. Powder X-ray diffraction (PXRD) was performed on a Bruker D8 Advance X-ray diffractometer with Cu−Kα radiation (λ = 1.54056 Å). The crystalline phases were qualitatively and semiquantitatively identified (based on reference intensity ratio (RIR) values) in the search/match module of DIFFRAC EVA (Bruker AXS). Scanning electron microscopy (SEM, accelerating voltage 5 kV) coupled with energy dispersive X-ray scattering (EDS, accelerating voltage 10 kV) was performed on the PLC-CaP composites using a LEO Ultra 55 FEG. Prior to imaging, all samples were cut to expose their cross sections and sputter coated with gold for 60 s. TEM was performed on a Tecnai operated at an accelerating voltage of 200 kV. TEM samples were prepared by grinding the composites and dispersing it in ethanol followed by drying the dispersion on a C/Cu grid. Thermogravimetric analysis (TGA, Pyris 1, PerkinElmer) was performed on the PLC-CaP composites at a heating rate of 10 °C/min from 30 to 550 °C.

model must meet specific requirements like chemical inertness towards CaPs, possess uniform nanoscale mineralization domains comparable to collagen interstices and be possible to use at high supersaturation of precursor ions. In the present study, we addressed these issues by investigating the effect of ordered nanoscale confinements (8−27 nm) with different geometries on rapidly induced CaP formation. Specifically, to mimic mineralization of collagen interstices we mineralized CaPs from highly supersaturated acidic precursor solutions within polymerized liquid crystals (PLCs) based on polymerizable derivatives of commercially available triblock copolymers (Pluronic F127 and P123). PLCs are mesoscopically ordered polymers with periodic and nanoscale aqueous domains.19,20 Lyotropic liquid crystals (LCs) and polymerized liquid crystals (PLCs) have previously been used as confined reactors to study CaP formation.20−22 CaPs formed within cubic (I1) and hexagonally ordered (H1) PLCs from supersaturation of 0.84 M Ca2+ were first stabilized as ACPs (30−40 nm) and gradually converted to preferentially oriented and phase pure nanocrystalline CaAp (thickness 1.5−3 nm) resembling bone mineral. Interestingly, in the same systems, when CaPs were mineralized from higher supersaturations (>1.3 M Ca2+), a large number of small crystals including acidic polymorphs like dicalcium phosphate dihydrate (DCPD or brushite) and dicalcium phosphate anhydrate (DCPA or monetite) cooccurred with ACPs.20 The mixture of CaP phases suggested that beyond a certain supersaturation limit, the specific geometric restriction was insufficient to maintain phase purity of minerals. In the present study, we further investigate how kinetically driven CaP nucleation at high supersaturations (0.84 and 2.1 M Ca2+) was affected by varying size and geometry of confinements in PLCs. The rationale behind applying such extremely high supersaturations was to study whether nanoscale confinements have any specific effects on mineral phase purity under these conditions and also obtain insights into forming bone-mimetic nanocomposites with a high and phase pure CaAp content.

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EXPERIMENTAL SECTION

Materials. All chemicals used in this study were provided by Sigma-Aldrich unless specified. The modification of triblock copolymer Pluronic F127 (EO100PO70EO100) to its diacrylate derivative MF127 was previously reported.20 The diacrylate derivative of Pluronic P123 (EO30PO70EO30), MP123 was also synthesized according to previously reported methods for MF127, except for a slightly different molar ratio of P123/triethylamine/acryloyl chloride (0.004:0.008:0.016). Milli-Q water was used in all experiments. Ammonia gas (NH3, 99.999%, Hi-Q 5.0) for the gas reaction was supplied by AGA Sweden. Calcium phosphate precursor was prepared by mixing calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), phosphoric acid (H3PO4, 85%), and Milli-Q water at a Ca/P molar ratio of 1.67. Different precursors with varying calcium concentration and initial pH were prepared as presented in Table 1.

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RESULTS In order to examine the effect of PLC confinement on CaP formation, five different composite samples were prepared (see Table 1). Mesoscopic Structure of PLCs and Composites. SAXS was used to investigate phase structure and quantify the aqueous domain size (daq) of each PLC. SAXS data of each sample (Figure 1Ia−e) showed strong scattering peaks suggesting the presence of mesoscopic order in the PLCs. Specifically, adjacent peak ratios from reflections of sample S1 B



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right shifts observed in primary peaks for the composites (e.g., CS5, Figure 1IIe) in relation to their corresponding PLCs (S5, Figure 1Ie) are probably due to the locally deformed PLC domains following mineralization (e.g., deformation of the “cylinders” of a H1 phase to accommodate the formed CaP particles). However, a direct correlation between particle size and daq values of the composites is difficult to determine directly from the SAXS data. Calcium Phosphate Formation in PLCs. Figure 2 (I and II) shows PXRD diffractograms of the composites and how

Figure 1. SAXS data of (I) PLC samples (a) S1, (b) S2, (c) S3, (d) S4, and (e) S5 and (II) corresponding PLC-CaP composites (a) CS1, (b) CS2, (c) CS3, (d) CS4, and (e) CS5. All SAXS data have been shifted vertically for clarity.

(Figure 1Ia) and S2 (Figure 1Ib) could be identified as 1:21/2:31/2:41/2:51/2 confirming a micellar cubic (I1) structure with an Im3m geometry.24 Both S1 and S2 showed a similar lattice parameter (dhkl = daq 27.81 ± 0.34 nm and 27.33 ± 0.30 nm respectively, see Table S1 and Figure S1 in Supporting Information), which shows that the difference in precursor strength does not affect the mesostructure of PLC. The SAXS data of samples S3 (Figure 1Ic), S4 (Figure 1Id), and S5 (Figure 1Ie) showed adjacent peak ratios of 1:31/2:2:71/2, characteristic of a hexagonally ordered mesostructure (H1).23−25 The lattice parameters of S3, S4, and S5 were 16.08 ± 0.07 nm, 13.60 ± 0.01 nm, and 13.02 ± 0.02 nm, respectively, from which daq values were calculated (S3 daq = 11.72 ± 0.07 nm, S4 daq = 9.21 ± 0.01 nm, S5 daq = 8.63 ± 0.02 nm, see Table S1 and Figure S2 in Supporting Information). It should be noted that the daq values showed a pronounced decrease from S1 to S5, which suggests that aqueous domains become more strictly confined as the geometry or composition of the PLC system is varied. Qualitatively, this effect can be observed by studying the significant right shift of scattering peaks of consecutive samples, i.e., from S1 to S5 where for S1 the primary peak (111) appears at q111 = 0.356 nm−1 and for S5 (100) at q100 = 0.482 nm−1 (see Table S1 in Supporting Information for peak indexing and corresponding qhkl values of all samples). The I1 PLCs (S1 and S2) showed the largest daq due to its three-dimensional mesocopic continuity. In comparison, sample S3 that is a H1 PLC showed a much lower daq of 11.72 ± 0.07 nm due to the one-dimensional freedom of aqueous domains along the length of the “cylinders”. Consequently, by varying MF127/CaP precursor/ BuOH ratio, the H1 PLC of S4 has a stricter geometry (daq = 9.21 ± 0.01 nm), which indicates that the amphiphile packs closer together compared to S3. Furthermore, a H1 PLC formed using a smaller chain amphiphile (sample S5) has a far stricter confinement with daq = 8.63 ± 0.02 nm due to tighter packing of the short chain amphiphile at the mesoscale aqueous interfaces. SAXS data of corresponding unaged composites (samples CS1−CS5 (Figure 1IIa−e) showed only the primary reflection (111) for I1 (CS1 and CS2) and (100) for H1 (CS3− CS5) composites). All other peaks were masked out most probably due to particle induced X-ray scattering. The minor

Figure 2. PXRD diffractograms of (I) PLC-CaP composites immediately after synthesis, i.e., unaged (a−e) corresponding to sample CS1 to CS5, respectively. (II) PLC-CaP composites aged at 22 °C for 24 h (a−e) corresponding to sample ACS1 to ACS5, respectively. (IIIA) TEM image of grounded composite CS3. (IIIB) TEM image of grounded composite ACS3. (IIIC) Selected area electron diffraction (SAED) of sample ACS3. All PXRD diffractograms have been shifted vertically for clarity.

crystallinity and phase purity of CaPs varied within different PLCs. The diffractogram of an unaged composite (CS1, Figure 2Ia) mineralized using low precursor strength (0.84 M Ca2+) showed that the material is completely amorphous, indicating the presence of spherical ACPs.20 Upon aging at 22 °C for 1 day (ACS1), ACP particles converted to nanocrystalline CaAp, as observed by the characteristic diffractogram corresponding to CaAp (Figure 2IIa, matched with reference database of calcium apatite in search/match module of DIFFRAC EVA). However, when composite CS2 was formed using higher precursor strength, i.e., 2.1 M Ca2+, PXRD showed multiple crystalline peaks suggesting the presence of different CaP phases (Figure 2Ib, marked by a star). On qualitatively matching the PXRD using a reference database, CS2 showed the presence of DCPD and DCPA phases. PXRD of aged sample (ACS2) showed the emergence of peaks corresponding to nanocrystalline CaAp in addition to DCPD and DCPA, which indicated the coexistence of multiple CaP polymorphs (Figure 2IIb). The relative ratios of CaAp/DCPD and CaAp/DCPA in ACS2 were roughly estimated to be 1.3 and 3.0, respectively, based on RIR values obtained from PXRD data. To support the results obtained from PXRD, SEM images were taken at the cross section of sample ACS1 and ACS2. ACS1 showed rodlike nanoparticles (Figure 3a, confirmed to be nanocrystalline apatite; see ref 20), C



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Figure 3. SEM images of (a−d) cross-section of aged samples ACS1, ACS2, ACS3, and ACS4 respectively, (e) cross-section of unaged sample CS5, and (f) aged sample ACS5.

Figure 4. SEM images of cross sections of composite (a−b) ACS2, (c−d) CS3, and (e−g) ACS5.

uniformly embedded within the PLC, while ACS2 showed the presence of micron-sized, platelike crystals resembling DCPD and rod-like crystals resembling DCPA, piercing through the PLC (Figures 3b and 4a,b; see also Figure S3a−d in Supporting Information).11,20,26,27 This observation suggests that changes in precursor concentration affect the phase purity of CaPs formed in I1 PLCs. To study the effect of PLC geometry on CaP formation at high precursor strengths (2.1 M Ca2+), H1 PLCs with different daq values were mineralized with CaPs to form corresponding composites (samples CS3 and CS4). Interestingly, PXRD data of an unaged sample, CS3 (Figure 2Ic), showed a marked change in CaP formation with a dominant amorphous phase and less intense peaks corresponding to DCPD (marked by a star). Spherical ACPs with an average diameter of 30−40 nm were observed to form within CS3 (Figure 2IIIA). He et al. have observed formation of spherical ACPs of similar morphology and sizes within H1 PLCs.20 The cross section of CS3 showed the coexistence of spherical particles with micronsized, larger crystallites resembling DCPD (Figure 4c and d). Upon aging the sample (ACS3), broad peaks matching to nanocrystalline CaAp were observed along with minor traces of DCPA (Figure 2IIc, Figure 3c, and Figure S3e in Supporting Information). The apatitic nature of the formed crystals were further clarified from TEM images (Figure 2IIIB) and diffraction patterns of SAED data (Figure 2IIIC), which showed bundles of nanocrystalline CaAp (and diffraction patterns) with crystallite lengths ranging between 30 and 100 nm.20,22 More interestingly, composite CS4 that has a smaller daq (9.21 ± 0.01 nm) showed a purely amorphous PXRD data before aging (Figure 2Id) and on aging (ACS4) gradually convert to phase pure nanocrystalline CaAp (Figure 2IId). This is further supported by the corresponding cross-section of

ACS4 that showed uniformly dispersed, nanosized, and rodshaped particles resembling the morphology of CaAp (Figure 3d).20,22 These observations show that PLC geometry and the domain size (daq) where CaP mineralizes have a high impact on the type of CaP formed. To confirm the effect of daq size on CaP phase purity, composite CS5 (daq = 8.63 ± 0.02 nm) was formed using a shorter amphiphile (MP123) and 2.1 M Ca2+. As predicted, the unaged composite (CS5) showed a completely amorphous diffractogram (Figure 2Ie), and on aging broad nanocrystalline peaks corresponding to CaAp appeared (Figure 2IIe). SEM image presented in Figure 3e shows the cross-section of CS5 where CaP particles with spherical morphologies are uniformly embedded in the polymer matrix. Figures 3f and 4e−g show that the aged sample (ACS5) consisted of nanosized rod-like particles, with a Ca/P ratio between 1.45−1.5, uniformly embedded in the PLC matrix (Figure S4, Supporting Information). The PXRD and SEM data of CS5 and ACS5 correlate well with results from our earlier studies and suggest that spherical particles (in CS5) are ACPs and rod-like particles (in ACS5) are CaAp.20 In addition, the formation of CaP particles in ACS5 was truly homogeneous from the surface throughout the entire depth with no sign of mineralization gradient (Figure S4). Compositionally, the mineral content in sample ACS3 and ACS5 was determined to be roughly 20− 25% by weight of the entire composite. Final polymer to CaP ratio in the composites was approximately 1.75 (Figure S5, Supporting Information). The major component in both composites was water, occupying almost 45% by weight of the samples. D



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DISCUSSION Briefly, results showed that nanoscale confinement has a key effect on CaP formation that is rapidly induced from high supersaturations (2.1 M Ca2+). For confinements ≤10 nm, H1 PLCs govern the formation of CaPs to phase pure ACPs, which gradually converted to nanocrystalline CaAp. Meanwhile mineralization of H1 and I1 PLCs with daq between 11 and 27 nm resulted in a mixture of CaP phases, namely, DCPD, DCPA, ACPs, and CaAp. To explain these results, it is relevant to remember that formation of CaP phases from solution strongly depends on pH, level of supersaturation of ions, and mode of precipitation.28 Although thermodynamically metastable, acidic polymorphs such as DCPD, octacalcium phosphate (OCP) and ACP are the kinetically stable forms of CaPs at near physiological pH and sufficiently high supersaturation.29 Considering these factors, our results can be interpreted with respect to kinetic vs thermodynamic competition of CaP precipitation and phase transformation. As NH3 penetrates the PLC, pH within the aqueous domains begins to increase rapidly which results in instant or burst precipitation of CaP. Since particle formation is fast, the most kinetically stable CaP will form. When this mode of particle growth occurs at precursor strength of 0.84 M Ca2+ in the largest confinement (S1), we observe that only ACPs are formed as the initial phase. In contrast when ionic strength was increased to 2.1 M Ca2+ (sample CS2), a mixture of DCPD and ACP formed in the PLC. This difference can be related to differences in supersaturation of ions in the aqueous domains of S1 and S2. A possible hypothesis is that when supersaturation is very high as in the latter case, the ions are much more sensitive to small pH variations and results in sufficiently large prenucleation clusters which can act as seed for growth of micron-sized metastable crystals like DCPD. Moreover, the aqueous domains in this case offer a sufficiently large particle/ solution interface for an uncontrolled growth of large crystals.13 When NH3 comes in contact with macroscopic surface of the PLC, formation of large crystals might be first observed at the PLC surfaces. This could probably lead to slower pH increase (diffusion limited) deeper in the PLC, consequently producing smaller nucleation clusters, hence stabilizing ACPs. Upon aging, thermodynamics takes control and gradually the ACPs transform to rod-like CaAp particles. However, the aged sample ACS2 still showed the presence of DCPD and DCPA that might be stabilized by the PLC. When confinements were changed to H1 geometry, an interesting change was observed in CaP formation. ACPs and a minor fraction of DCPD was formed in the case of largest daq (11.72 ± 0.07 nm), and phase pure ACPs formed at smaller daq (≤10 nm). The CaP formation in H1 PLCs follow a similar mechanism as for the I1 PLC hypothesized above. However, the more confined, one-dimensional freedom of the H1 structure reduces particle/solution interface and inhibits the formation of large crystals. This was observed in sample CS3 where PXRD peaks corresponding to DCPD was relatively less intense and broader compared to CS2, suggesting that DCPD crystals were possibly much smaller. This was visually evident in Figure 4c where relatively small, platelike crystals resembling DCPD were observed. Furthermore, this theory was experimentally verified where smaller confinements (S4 and S5) showed a stronger effect to restrict CaP crystal growth, fully inhibiting growth of DCPD phase and selectively stabilizing ACPs. Our results can be corroborated with an earlier study by He et al. where it was

shown that a reverse hexagonal LC structure (H2) showed greater favorability for ACP formation due to its highly confined and discrete aqueous domains.22 Although this study was also performed at low supersaturations and under diffusion control, it is possible to conclude that below a certain daq the geometry of confinements might become sufficient to control the CaP formation. A general mechanism can be suggested that might explain the role of nanoscale confinements on CaP formation from high supersaturations. Decreasing the particle/solution interface during kinetic precipitation of CaPs inhibits stabilization of acidic polymorphs such as DCPD while enhancing ACP stabilization. Stricter confinements provide a smaller interface that restrict growth of nucleating clusters beyond a certain size and forces them to precipitate randomly, forming amorphous CaPs (Figure 5). Finally, the slow transformation of ACP to

Figure 5. Schematic of two H1 PLCs after CaP formation depicts differences in mineralization behavior based on the PLC confinement (daq).

CaAp within confinements observed in this study agrees well with results from earlier studies.10,16,22 It is important to note that there is no chemical interaction between CaPs and amphiphilic molecules of the PLC. Therefore, the observed effect over CaP formation is purely related to PLC geometry and confinement size. There are speculations on the presence of acidic or neutral CaP precursors like DCPD and ACP in bone mineralization. Although DCPD is kinetically stable at physiological pH, it has been difficult to observe its presence in in vivo mineralization processes, except for more acidic environments (pathological calcifications).29 One reason for this might be that confined domains of matrix vesicles or collagen fibrils in coordination with chemical control inhibit the formation of DCPD. The in vivo mineral growth, phase transformation, and separation have been linked to the role played by collagen, noncollagenous proteins (NCPs) and citrate bridges.7,30 However, results from our study strongly support that geometric or spatial restrictions can independently modulate the mineral size and respective phase purity even at extremely high ionic precursor supersaturations. A derived insight from our results hints that in the absence of chemical control, confinements might have to be smaller than those present in bone formation to obtain phase pure CaAp crystals.

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CONCLUSIONS In this study it is shown that nanoscale confinements in polymeric matrices control the phase purity of calcium phosphates. When CaP is precipitated in confinements of sizes 11−27 nm, multiple phases including acidic polymorphs coexisted. Contrastingly, confinements smaller than 10 nm produced phase pure ACPs, which on aging converted to E



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(11) Gómez-Morales, J.; Delgado-López, J. M.; Iafisco, M.; Hernández-Hernández, A.; Prat, M. Cryst. Growth Des. 2011, 11, 4802−4809. (12) Wang, Y.-W.; Christenson, H. K.; Meldrum, F. C. Chem. Mater. 2014, 26, 5830−5838. (13) Meldrum, F. C.; Cölfen, H. Chem. Rev. 2008, 108, 4332−4432. (14) Steenbjerg Ibsen, C. J.; Mikladal, B. F.; Bjornholt Jensen, U.; Birkedal, H. Chem.Eur. J. 2014, 20, 16112−161120. (15) Asenath-Smith, E.; Li, H.; Keene, E. C.; Seh, Z. W.; Estroff, L. A. Adv. Funct. Mater. 2012, 22, 2891−2914. (16) Cantaert, B.; Beniash, E.; Meldrum, F. C. Chem.Eur. J. 2013, 19, 14918−14924. (17) Anderson, H. C. Current Rheumatology Reports 2003, 5, 222− 226. (18) Anderson, H. C.; Garimella, R.; Tague, S. E. Front Biosci. 2005, 10, 822−837. (19) Gin, D. L.; Bara, J. E.; Noble, R. D.; Elliott, B. J. Macromol. Rapid Commun. 2008, 29, 367−389. (20) He, W.; Rajasekharan, A. K.; Bagha, A.; Andersson, M. Adv. Mater. 2015, 27, 2260−2264. (21) He, W.; Fu, Y.; Andersson, M. J. Mater. Chem. B 2014, 2, 3214− 3220. (22) He, W.; Kjellin, P.; Currie, F.; Handa, P.; Knee, C. S.; Bielecki, J.; Wallenberg, L. R.; Andersson, M. Chem. Mater. 2012, 24, 892−902. (23) Holmqvist, P.; Alexandridis, P.; Lindman, B. J. Phys. Chem. B 1998, 102, 1149−1158. (24) Holmqvist, P.; Alexandridis, P.; Lindman, B. Langmuir 1997, 13, 2471−2479. (25) Holmqvist, P.; Alexandridis, P.; Lindman, B. Macromolecules 1997, 30, 6788−6797. (26) Mandel, S.; Tas, A. C. Mater. Sci. Eng., C 2010, 30, 245−254. (27) Huelin, S. D.; Baker, H. R.; Merschrod, S. E. F.; Poduska, K. M. Cryst. Growth Des. 2006, 6, 2634−2636. (28) He, W.-X.; Nik, S. M.; Andersson, M. Cryst. Growth Des. 2015, 15, 5−9. (29) Wang, L.; Nancollas, G. H. Chem. Rev. 2008, 108, 4628−4669. (30) Davies, E.; Muller, K. H.; Wong, W. C.; Pickard, C. J.; Reid, D. G.; Skepper, J. N.; Duer, M. J. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E1354−63.

nanocrystalline CaAp. It can be hypothesized that larger confinements offer sufficiently large particle−solution interfaces for uncontrolled growth of metastable DCPD crystals. The effect of confinement was supported further since no chemical complexing was present between the PLC and CaPs. The obtained results support earlier studies on the confinement effect on ACP stabilization and provide new insights on controlling CaP precipitation under high supersaturations. A general hypothesis is proposed that in the absence of chemical control, confinements might need to be smaller than naturally occurring ones to control the mineral phase purity in both synthetic and biological systems. The results also provide insight into designing biomaterials with an ordered structure and high mineral content while maintaining chemical homogeneity.

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ASSOCIATED CONTENT

S Supporting Information *

Additional SAXS results, SEM images, EDS and TGA data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Visiting address: Kemivägen 10. Phone: +46 (0)31-772 2966. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge MAX-Lab, Lund, Sweden, for assistance with SAXS measurements. We thank Dr. Wenxiao He (Dept. of Chemistry and Chemical Engineering, Chalmers University) for assisting us with the TEM and electron diffraction data. We thank the Knut and Alice Wallenberg foundation, Sweden, for funding this research.

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ABBREVIATIONS ACP, amorphous calcium phosphate; CaAp, calciumapatite; DCPD, dicalcium phosphate dihydrate; DCPA, dicalcium phosphate anhydrate; PLC, polymerized liquid crystal

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REFERENCES

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Role of Nanoscale Confinement on Calcium Phosphate Formation at

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