Formation of Bone-like Nanocrystalline Apatite Using Self

The initial slope presented before the first-order peak (7, 18, 30, 48, and 72 h) can be attributed to scattering from the presence of solid CaP parti...
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Formation of Bone-like Nanocrystalline Apatite Using Self-Assembled Liquid Crystals Wenxiao He,† Per Kjellin,‡ Fredrik Currie,‡ Paul Handa,‡ Christopher S. Knee,§ Johan Bielecki,|| L. Reine Wallenberg,^ and Martin Andersson*,† †

Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden Promimic, SE-41292 Gothenburg, Sweden § Department of Chemistry, University of Gothenburg, SE-41296 Gothenburg, Sweden Department of Applied Physics, Chalmers University of Technology, SE-41296 Gothenburg, Sweden ^ nCHREM, Department of Chemistry, Lund University, SE-22100 Lund, Sweden

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bS Supporting Information ABSTRACT: A two-step process using liquid crystalline phases combined with controlled postcrystallization for the preparation of bone-like apatite has been developed. First, amorphous calcium phosphate (ACP) spherules with a diameter of 10.8 ( 1.4 nm and specific surface area (SSA) in the range of 150 170 m2/g were synthesized within a reverse hexagonal liquid crystalline (LC) phase. Second, the ACP spherules were dispersed and aged in Milli-Q water, where they crystallized into poorly crystalline apatite (PCA). The addition of heparin during aging was explored, which was shown to retard the ACP PCA conversion. The particle formation within the LC phase was monitored using synchrotron small-angle X-ray scattering, and the formed materials were characterized by X-ray diffraction, conventional and high-resolution transmission electron microscopy, nitrogen adsorption, thermogravimetry with infrared-coupled analysis, and Raman spectroscopy. The PCA formed using the LC aging route presented bone-resembling features, such as Ca2+ and OH deficiency, CO32- substitution, poor crystallinity, and ultrahigh SSA of 356 m2/g. The resulting particles were compared to hydroxyapatite synthesized via a conventional water-based precipitation method. The LC aging route exhibited excellent controllability over the CaP crystallization, which enabled facile tailoring of the resulting material properties for different types of application. KEYWORDS: bone, apatite, liquid crystalline phase, nanoparticles, calcium phosphate

’ INTRODUCTION The mineral phase in bone is made of nanocrystalline apatite (ranging from 30 80 nm in length, 15 30 nm in width, and 1.5 10 nm in thickness), which is Ca2+ and OH deficient, CO32-substituted (4 6 wt %), and poorly crystalline.1 4 As the analogue of bone apatite (Ca10 x/2 z/2[(HPO4)z(PO4)6 x z(CO3)x][(OH)2 2y(CO3)y]),5 synthetic calcium phosphate (CaP) materials with prominent biodegradability, biocompatibility, bioactivity and osteoconductivity,6,7 have been extensively used within the field of biomedical and hard tissue engineering as drug carriers for controlled release, implant coatings, composite components, and injectable cements as well as scaffolds for bone reconstruction.1,6,8,9 Various synthesis routes have been employed to obtain nanosized CaPs, including wet chemical synthesis,10 hydrothermal synthesis,11 mechano-chemical processes,12 sol gel techniques,13 etc. However, plenty of drawbacks exist with these synthetic routes, for instance, the critical synthesis condition (e.g., high-temperature, high-pressure environment for hydrothermal synthesis), long reaction times, and difficulties obtaining reproducible results. More importantly, the resulting materials are usually relatively large in crystal dimensions and r 2011 American Chemical Society

highly crystalline, and, therefore, they might have limited bioefficacy (e.g., resorbability). For obtaining a better compositional and morphological control (or even orientational control in composites) over the final products, CaPs have also been synthesized using various biological templates and synthetic templates. The former involves the utilization of simulated body fluids (SBF) with self-assembled collagen fibrils,3,14 noncollagenous proteins,15,16 enzymes,17 bacteriophages18 or biopolymers,19 etc.,2 in order to mimic the organic matrix in bone. The synthetic templates include self-assemblies of synthetic substances to create spatial confinements for CaP mineralization. In particular, the extensive use of surfactants to form versatile lyotropic phases20 23/emulsions,24 surfactant assisted liquid-solid-solution strategies,25 functionalized dendrimers acting as “artificial proteins”,26 synthetic liposomes as structuredirecting reagents,27 mineralization within hydrogel scaffolds,28,29 Special Issue: Materials for Biological Applications Received: April 14, 2011 Revised: September 2, 2011 Published: September 29, 2011 892

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to mention a few, have been reported to produce CaP materials with delicate architectures. More than a static process, bone mineralization is a dynamic evolution with several unique features. Amorphous calcium phosphate (ACP) is most likely to be the first formed transient mineral phase, according to its great tendency to first form in rapid precipitation in vitro1 3,30 34 together with indirect in vivo evidence (electron-dense granules observed in skeletal tissue specimens).35 37 The ACP then crystallizes, forming “needlelike” to platelet shaped crystals, which increase in carbonate content during its maturation.1 The ACP also functions as a calcium/ phosphate reservoir and can later crystallize into bone apatite.37 The instability induced structural and compositional flexibility of ACP renders an excellent bioactivity and makes it an interesting precursor material for biomimetic mineralization as well as a useful component in nanocomposites for bone substitution.7,37 To utilize these biomineralization properties, the present study adopted a two-step synthetic route for bone-like apatite synthesis: first, the preparation of ACPs with a narrow size distribution in a selfassembled liquid crystal (LC) templating system; second, the subsequent transition of these amorphous particles under a controlled aging process to yield bone-like apatite with well-defined and reproducible particle sizes and morphology. The as-formed apatites have been calcined and applied onto titanium implant surfaces for in vivo investigations, as reported elsewhere.38 44 In this study details of the initial formation processes within the LC template and the subsequent conversion to PCA are presented, along with indepth characterization of the obtained CaP phases. Furthermore, the impact of adding glycosaminoglycans (GAGs) during the aging process has been studied. The electrostatic interactions between the anionic sites (carboxyl or sulfate groups) on GAGs and the cationic sites (calcium) on hydroxyapatite (HAp) have been predicted to have an effect on the CaP crystallization.45

Table 1. Preparation Condition and Specific Surface Area of CaPsa,b

a

Heparin added to the salt solution before ACP formation. b Heparin added in an aging medium after ACP had formed.

Characterization. Small angle X-ray scattering (SAXS) experiments were performed on the prepared LC gel during the reaction (stayed in NH3 atmosphere for 0, 7, 18, 30, 48, and 72 h, respectively), using the synchrotron radiation (λ = 1.1 Å) and a two-dimensional Mar165 CCD detector at the I711 beamline at MAX-lab in Lund, Sweden.48,49 Samples were sealed in a slit (1 mm thick) enclosed by two parallel transparent cellophane films within a stainless steel sample holder mounted in an evacuated sample chamber, 1.5 m from the detector. The data were collected for 180 360 s, and the used q-range (q = 4πsinθ/λ) was 0.1 4.0 nm 1. X-ray powder diffraction (XRD) was done on a Bruker D8 Advance X-ray diffractometer (Cu Kα1 radiation, λ = 1.54056 Å) with a 2θ range of 20 60, step size 0.050, and data collection time 30 min. Transmission electron microscopy (TEM) was done on a JEOL 1200EX II microscope operated at 120 kV. High resolution TEM (HRTEM) was performed using a JEOL 3000F analytical (S)TEM operating at 300 kV with a field emission gun and an Oxford Instruments Inca XEDS system. The TEM specimens were prepared on a holey carbon coated copper grid by evaporating a drop of nanoparticles/ethanol suspension. Scanning electron microscopy (SEM) was carried out by a LEO ULTRA 55 FEG at an accelerating voltage of 5 kV. Specific surface area was measured by BET nitrogen gas adsorption using a Micromeritics Tristar 3000 surface area and porosity analyzer. Approximately 0.3 0.5 g samples were placed into the sample cell and degassed under vacuum at 120 C for 3 h prior to each measurement. Thermogravimetric/differential thermal analysis (TG/DTA) was conducted on a NETZSCH STA 409 PC from room temperature to 850 C at a heating rate of 10 C/min under a flow of N2. Thermogravimetry with Infrared-Coupling (TG-IR) was performed using a TG 209F1 Netzsch TGA coupled to a Bruker Vector 22 FT-IR spectrometer for the evolved gas analysis. A heating rate of 10 C/min was employed, and the flow rate of the He carrier gas through the heated transfer line was 30 mL/min. IR spectra between 600 and 5000 cm 1 with a resolution of 2 cm 1 were collected from selected samples using a Bruker IFS66v FT-IR spectrometer. The signal was obtained through a diamond crystal in single reflection attenuated total reflectance geometry. Raman spectra were collected at room temperature by a liquid-nitrogen cooled CCD detector connected to a Dilor XY spectrometer in microconfiguration using a 40 objective with the 514.5 nm line from an Ar+ laser as excitation source. The laser power was kept at ∼6 mW with a spot size of 4 μm. When compared with a laser power of 1 mW, no changes in spectra due to laser heating was observed. Due to strong luminescence, it was necessary to laser-bleach the samples for 1 h before measuring.

’ EXPERIMENTAL SECTION All chemicals were of analytical grade, purchased from Sigma-Aldrich, and used without further purification. Experiments were conducted at room temperature (22 ( 1 C), unless otherwise stated. Preparation of ACP Using a LC Phase.46 Calcium nitrate tetrahydrate [Ca(NO3)2 3 4H2O] and 85% phosphoric acid (H3PO4) with a Ca/P molar ratio of 1.67 were dissolved in Milli-Q water at an initial concentration of 0.43 M Ca2+. Then by mixing the prepared solution with the surfactant Pluronic L64 (EO13PO30EO13) and p-xylene, a reverse hexagonal (H2) LC phase (15 wt % salt solution, 70 wt % L64, and 15 wt % p-xylene) was formed.47 After being equilibrated for 24 h, the LC gel was applied onto a clean glass plate (gel thickness ∼6 mm) and placed in an ammonia atmosphere (ammonium hydroxide, 35 wt % aq) to increase the pH, thus, to initiate the reaction within the water domains. After 72 h, the reaction was stopped, and the obtained material was washed repeatedly using Milli-Q water and 95% ethanol (the total water-washing time was ca. 0.5 h), in order to remove the surfactants and the p-xylene. The final product was freeze-dried for 3 h and then vacuum-dried at 120 C. ACP Aging Process. As-prepared ACP powders were dispersed in Milli-Q water (0.5 wt %) and aged at room temperature for different time periods. The particles were subsequently filtered, washed with ethanol, and dried as described above. Additional ACP aging was performed together with certain amounts of heparin (sodium salt, cat. no. H3393).

Preparation of CaP via Water-Based Precipitation (WBP). The same salt solution as used for the LC synthesis, as described above, was stirred at 500 rpm and directly exposed to an ammonia atmosphere for 72 h. The purification process for WBP samples was the same as for the LC aging route, as described above.

’ RESULTS In order to compare how the LC aging route differed from conventional WBP of CaPs, as well as the possible influence of 893

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Figure 1. (I): Synchrotron SAXS spectra of LC gels exposed to NH3 atmosphere for 0, 7, 18, 30, 48, and 72 h (0 h* is for LC gel prepared without Ca and P sources. The spectra were shifted vertically for clarity). (II): Illustration of the formed H2 LC structure showing the dimensions of the domains as calculated from the SAXS data.

GAGs on CaP mineralization, seven samples were prepared and defined as A, B, C, D, E, F, and G (Table 1). Investigation of LC Gel Nanostructure. In order to elucidate the nanostructure of the LC phases and how it was affected by the ACP formation, SAXS analysis was performed during the reaction. As shown in Figure 1(I), the obvious reflections corresponding to the relationship 1:31/2:2 can be observed, which clearly shows that LC phase has a hexagonal structure (H2), which is preserved even in the presence of NH3. The initial slope presented before the first-order peak (7, 18, 30, 48, and 72 h) can be attributed to scattering from the presence of solid CaP particles. These results strongly indicate that the H2 structure was maintained during the particle formation. Furthermore, the LC gel with and without CaP solid phase showed a similar lattice parameter, dhex = 8.94 ( 0.12 nm (i.e., the distance between two adjacent cylindrical domains, dhex = ((4π(n)1/2)/((3)1/2qn)), where n = h2 + hk + k2) and a cross section radius of the water channels of Rcyl = 2.96 ( 0.04 nm, as obtained by Rcyl = dhex((((3)1/2)/(2π))(1 f))1/2 (f = Φo + 0.62Φp for the Pluronic L64 system, where Φo and Φp is the volume fraction of oil and polymer phase).47 With the calculated data, a schematic illustration of the as-formed H2 phase can be obtained as depicted in Figure 1(II). These water domains with a diameter of approximately 6 nm were the locations where the CaP formation took place. Characterization of the Nanoparticles. The crystalline structure of the CaPs prepared via the LC aging route was examined by X-ray diffraction (XRD), as shown in Figure 2(I). The particles examined directly after being synthesized via the LC route were amorphous, which could be confirmed by the characteristic halo at 2θ ≈ 30 in the diffractogram (Figure 2(I) a).34 A phase conversion was observed when simply aging the ACP particles in Milli-Q water. For particles that were aged for 3 h, the presence of the small narrow peaks amidst the diffuse background in the XRD pattern (Figure 2(I) b) indicates the emergence of a crystalline phase in the amorphous bulk, which could be attributed to brushite (CaHPO4 3 2H2O). When prolonging the aging time, the former peaks were weakened

Figure 2. (I): XRD patterns of CaPs synthesized by the LC phase with different post procedures: (a) freeze-dried, (b e) subsequently aged in H2O for 3, 18, 23, 27 h, respectively, then freeze-dried, and (f) air-dried. Bottom: XRD profile of HAp (black, JCPDS Card no. 09-0432) and brushite (blue, JCPDS Card no. 72-0713). (II): XRD patterns of A, B, C, and D (listed in Table 1) before and after the calcination at 850 C for 2 h.

after 18 h and disappeared after 23 h (Figure 2(I) c, d). Meanwhile an apatitic structure was emerging, which was further refined after four more hours (Figure 2(I) e). Furthermore, the broad and diffuse peaks with typical apatitic features (d, e) suggest that the resulting materials were poorly crystalline apatite (PCA). However, the replacement of a controlled aging and freeze-drying process of the purified CaPs by air-drying led to sharpened and more narrow peaks in the diffraction curve (Figure 2(I) f), indicating the formation of HAp with an increased crystal size and promoted crystallinity. The XRD patterns of samples A D before and after calcination are shown in Figure 2(II). Prior to the calcination, it can be seen that all the samples are of apatitic structure. The LC aged samples (A, B) 894

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present the broad diffuse peaks implying that they are PCA and show great resemblance to the diffraction pattern of natural bone.3,6,50 On the other hand, the sharper diffraction peaks of WBP samples (C, D) approach those of commercial synthetic HAp.51 Furthermore, calcination at 850 C further enhanced the crystallinity of samples C and D, as shown by the increase in intensity and decrease in peak width of the XRD peaks. Unlike the WBP samples, the apatitic peaks of the LC aged samples (A, B) vanished after calcination, and peaks identified to be tricalcium phosphate (TCP, Ca3(PO4)2) were present. TEM was utilized to investigate the morphological changes regarding the conversion brought by the aging process of the CaPs particles in aqueous media. Specifically, Figure 3a shows an aggregate of spherical particles with diameters of 10.8 ( 1.4 nm, which were ACPs (according to XRD, Figure 2(I) a) directly

synthesized from the LC template. The electron-lucent center of the ACP spherules was probably caused by electron beam damage.52 After being dispersed in aqueous media for 7 h, the ACPs evolved into spherules having a larger size distribution (Figure 3b). After 18 h of aging in H2O, elongated crystallites being ∼1.5 nm thick (XRD, Figure 2(I) c) with a large aspect ratio started to emerge (Figure 3c), among the electron-dense ACP spherules. Further aging led to the coexistence of fused ACPs and crystallites (Figure 3d, 21 h). Figure 3 (e f, 23 25 h) presents an aggregation of elongated platelets with extremely small crystal widths (30 50 nm long, 1.5 4 nm wide), implying that the majority of ACPs were consumed for the crystallization. An apparent growth of the crystallite’s width was observed as shown in Figure 3 (g h, 30 40 h). For the air-dried sample (Figure 3i), plate-like crystals with a size of (60 80 nm)  (8 12 nm) were observed. High-resolution TEM (HRTEM) images of elongated PCA platelets (Figure 4a) synthesized via the LC aging route revealed an apatite-like structure. Specifically, the lattice planes of 4.7 Å and 3.2 Å, corresponding to the (110) and (102) apatite plane, forming an angle of 70, as well as the lattice planes (210) of 3.1 Å, (121) of 2.8 Å spacing, forming a 32 angle (Figure 4b, insert) were identified. Note that the crystallites grew along the Æ001æ direction (indicated by arrows, Figure 4b) while exposing facets like (110) and (210). Two d-spaces at 3.1 and 2.8 Å corresponding to the (210) and (112) reflection forming a 54 angle, as shown in Figure 4c, further confirmed the apatitic structure of the as-grown crystallites. ACPs originating from the same batches as described above were also aged in Milli-Q water in the presence of heparin (E, F, and G in Table 1 and Figure 5). Large amounts of fused ACPs (dashed circle) still remained in F after aging for 23.5 h with a heparin concentration of 0.429 mg/mL, whereas the ACPs in sample E that aged for 23 h without GAGs were mostly converted into crystallites. The prolongation of aging time to 25 h and a decrease in heparin concentration to 0.257 mg/mL could just fulfill the ACP to PCA conversion, yielding elongated crystallites with an even higher aspect ratio (G, 70 90 nm long, 1.5 3 nm wide). Furthermore, energy dispersive X-ray spectroscopy (XEDS) analysis performed using a scanning electron

Figure 3. TEM images of CaPs synthesized by the LC phase with different post procedures: (a) freeze-dried, (b h) subsequently aged in H2O for 7, 18, 21, 23, 25, 30, and 40 h, respectively, then freeze-dried and (i) air-dried. Scale bar: (a h) 15 nm and (i) 30 nm.

Figure 4. HRTEM of PCA crystallites synthesized by the LC aging route. 895

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microscope (SEM) revealed that the crystallites in G were calcium deficient (Ca/P = 1.507, average atomic percentage: O 65.0%, Ca 20.8%, P 13.8%, and S 0.4%). Note that the presence of sulfur was brought by heparin. Figure 6 shows the CaP particles prepared via the WBP route (C, D), which have a plate-like morphology (width ∼ 15 nm) and a considerably larger crystal size compared with samples A and B obtained by the LC aging route (width ∼ 5 nm). Heat-driven crystal growth was observed for WBP sample (C calcined at 850 C); however, the calcined LC aged sample was transformed into electron-dense bulky agglomerates, which were shown to be TCP according to the XRD analysis (Figure 2(II), A calcined at 850 C). Coinciding with the structural and morphological changes presented during the aging of ACPs in H2O (Figure 2(I) and Figure 3), an apparent trend in the specific surface area (SSA) could be observed as presented in Figure 7. Initially (0 14 h), the SSA of the ACP spherules was maintained in the range of 150 170 m2/g. Then, a pronounced increase of SSA appeared after 18 h. The SSA reached a peak of 356.1 m2/g at 25 h but gradually declined afterward. In addition, the SSA of samples A, B, C, D and E, F, G is listed in Table 1. Stepwise mass losses could be observed from thermogravimetric/differential thermal analysis (TG/DTA) curves of samples

A, B, C, and D (Figure S1), as listed in Table 2. Upon heating, the first mass loss is due to release of adsorbed water, which occurs between room temperature and 250 C. Then from 250 to 550 C, the release of strongly linked intracrystalline water and residual surfactant as well as the dehydration of HPO42- groups, indicated by the exothermic peaks at around 420 C in DTA curves of C and D, could be observed. This was followed by a third mass loss stage for samples C and D occurring between 550 and 700 C, corresponding to the crystallization of the ACP moiety in the bulk (exopeaks at 570 C) and HAp crystal growth. While for A and B, the heating process from 650 to 850 C facilitated the reaction of P2O74- and OH ions in apatite, which transformed into TCP giving rise to the exopeaks at ∼730 C in the DTA curves. In order to further investigate the occurring mass losses in the TG of the LC aged samples, thermogravimetry with infraredcoupling (TG-IR) analysis was performed on samples B, E, and F (Figure 8 and Table 1). As shown in Figure 8, the H2O stretch peak at ∼100 C (blue line) consolidates the TG-DTA analysis that the mass loss below 250 C was attributed to the loss of adsorbed water. Additionally, the PCA to TCP transformation for the LC aged samples is linked to the IR signal of H2O and CO2 (red) at ∼730 C. This CO2 signal shown in B, E, and F

Figure 5. TEM images of CaPs synthesized by the LC aging route in the absence and presence of heparin (a) E, (b) F, (c) G, and (d) SEM image of G (Table 1). Scale bar: (a, b) 15 nm, (c) 30 nm, and (d) 200 nm.

Figure 7. Effect of different aging times on the specific surface area of CaPs prepared by the LC aging route.

Figure 6. TEM micrographs of (A, B) LC aged and (C, D) WBP samples before and after calcinations at 850 C. Scale bar: (A (A calcined) 120 nm, (C calcined) 60 nm. 896

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Table 2. Thermal Decomposition of CaPs Prepared via LC Aging and WBP Route

In addition, weaker signals from OH substituted CO32- are only observed in the LC aged samples (deformation: 878 cm 1, stretch: 1455, 1545 cm 1), again showing their similarity to natural bone. Figure 10 shows room temperature Raman spectra in the region of PO43- vibrations.57 As the surface area (Table 1), and hence disorder in the crystal structure increases, the vibrational peaks broaden. This is manifested as a loss of detailed structure in the ν2, ν3, and ν4 modes, while the ν1 mode becomes more asymmetric. This asymmetry grows as the disorder increases. We interpret this as a consequence of two “types” of ν1 peaks: one “crystalline” at ∼960 cm 1 and one “amorphous” at ∼950 cm 1. Samples A, B, C, D, E, and F contain a mixture of the two, resulting in an asymmetric peak, while the ACP peak only contains the amorphous part, as is seen by its symmetric shape (it is perfectly fitted with a Gaussian after removal of background). The disorder probed by the PO43- vibrations in Figure 10 is predominantly of the internal, short-range, type and reflects disorder inside the PO43- structure due to for example OH deficiency. The correlation between short-range disorder and OH deficiency can be seen by comparing the PO43- vibrational modes with the O H stretch mode in Figure 11. The general trend is that sharp PO43- modes imply large OH signal and vice versa. Long-range disorder, on the other hand, can be probed by the external lattice modes, which reflects collective vibrations of the PO43-/Ca2+ ions in the samples.58 External lattice modes are observed between 200 300 cm 1 in single crystalline HA samples.58 As can be seen in Figure 11, samples C and D display weak peaks in this region, while the other samples only show the fluorescent background. We conclude that the large surface area of the synthesized apatite leads to both OH deficiencies as well as a loss of long-range order, possibly due to the small crystal sizes.

Figure 8. TG-IR of LC aged samples B, E, and F.

indicates that they were carbonated apatite. However, for the heparin aged sample F, an extra CO2 peak was presented at ∼610 C, implying the existence of some ACPs in the sample. Finally, we note that the presence of CO2 signals in the T range 250 575 C for all 3 samples probably reflects the loss of residual surfactant. Figure 9 shows IR spectra of CaP samples (ACP and A F in Table 1) in the carbonate deformation and stretch regions. Previous studies have shown that the carbonate ions substitute either on the PO43- (B-type) or on the OH site (A-type) in the apatite structure, with corresponding signatures in the IR spectra.53 The deformational mode shows up as a peak at either 875 cm 1 (PO43- substitution) or 880 cm 1 (OH substitution).54 Similarly, the stretching modes are observed at ∼1460 and 1420 cm 1 for PO43- substitution as well as ∼1550 and 1460 cm 1 for OH substitution.55 In general, naturally occurring bone apatites show both types of substitution, while geological apatite only shows PO43- substitution.55,56 In all samples except ACP, strong signatures of PO43- substituted CO32- can be seen in both the deformation (875 cm 1) and stretch (1416, 1455 cm 1) region (Figure 9).

’ DISCUSSION A schematic illustration of the two-step LC aging route as well as the WBP synthesis of CaPs is presented in Figure 12. Formation of ACP Spherules in the LC Phase. Since the H2 phase was maintained during the ACP formation, as shown by the synchrotron SAXS results (Figure 1(I)), the confined H2 water domain with a diameter of ∼6 nm was the site for ACPs to form and grow. Nevertheless, the diameter of resulting ACP spherules was 10.8 ( 1.4 nm (Figure 3a), which is 3 6 nm larger than that of original water domain, suggesting that the LC structure was swelled by ACPs during particle growth. Since 897

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Figure 9. IR spectra in the carbonate deformation/stretch regions. Clear indications of PO43- substitution are observed in all samples except ACP. OH substitution is only observed in the LC aged samples A, B, E, and F.

Figure 10. Raman spectra of ACP prepared via a typical LC route and samples A, B, C, D, E, and F in the frequency regions of internal PO43- vibration. Spectra are of the same intensity scale.

ACPs are prone to convert into HAp in aqueous media, the “squeezed” surfactant chains seemed to play a stabilizing role on ACPs when equilibrium is reached, preventing them from crystallization and yielding spherules with a narrow size distribution and a high specific surface area (150 170 m2/g).59 In contrast, conventional wet chemical methods yield ACPs in the range of 30 100 nm and with a large size distribution.34 Therefore, the H2 LC phase had an effective regulation on ACPs’ dimension. Moreover, there might be certain local atomic ordering of the attained ACPs as it has been claimed that ACP could be composed of the Posner’s clusters [Ca9(PO4)6] with a dimension of 9.5 Å, which is too small to be detected by the X-ray analysis (Figure 2(I) a).1,34,60,61 Controllable ACP PCA Conversion in H2O. When dispersing the prepared ACP spherules in aqueous media, a phase transition was observed as monitored by electron microscopy and X-ray diffraction as well as the BET surface area. The transition could be divided into three stages as suggested by the trends in the SSA (Figure 7) as a function of aging time

(in Milli-Q water, room temperature). First the induction period (0 14 h) where the CaPs remain in their amorphous state, with a SSA in the range of 150 170 m2/g, accompanying the dissolution of the particles (Figure 3 a, b); then the crystallization period (∼14 25 h) with a strong increase in SSA (peaked at 356.1 m2/g), owing to the elongation of the particles with a narrowing in thickness and width (1.5 4 nm as shown in Figure 2(I), 3 c e); finally the crystal growth (>26 h), with a decline in SSA and a growth in crystal size (especially in width), accompanying the sharpening and refinement of the apatitic peaks in the diffraction pattern (Figure 2(I), 3) with increased aging time. Note that at the early crystallization period, a transient phase of brushite was present (Figure 2(I) b,c). This local occurrence of brushite-like phase has been reported earlier by Kanapathipillai et al.22 in CaPs templated by ionic block copolymers, and it might be due to the imperfect ACP PCA transition. Furthermore, the coexistence of electron-dense ACPs and elongated nanocrystallites at this stage (Figure 3 c, d) has also been observed in bone nodule.62 Interestingly, the decrease 898

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Figure 11. Raman spectra of ACP prepared via a typical LC route and samples A, B, C, D, E, and F in the frequency regions of external lattice modes and O H stretch modes. Spectra are of the same intensity scale.

conversion rate is slow (still, can be regulated by several factors, such as temperature, pH and additives, etc.59), all the particles obtained in the aging media are more or less at the same stage of the ACP PCA conversion. Furthermore, the aging can be terminated at any specific stage by simply extracting the water media and the particles can be used for characterization or further application. While for the WBP route, the particle formation process is less controlled, which generates particles with a large size distribution and a great discrepancy in the ACP PCA conversion/HAp crystal growth stage. The product is usually a mixture of HAp with relatively large crystal size and a small amount of ACP (e.g., sample C: according to TG-DTA results, Figures 2(II), 6, and 8). Similar to the utilized WBP route, Nassif N et al. reported the preparation of HAp nanocrystals with platelets morphology by NH3 vapor diffusion into a CaCl2 NaH2PO4 mixed solution.63 The obtained particles were relatively large and had diverse morphologies. While for the H2 LC system, NH3 could penetrate ∼6 mm gel within 2 days. With the restriction of surfactants, the earlier-formed ACPs were stabilized and ceased growing, until all the ACPs were formed, stabilized, and harvested. Therefore, the LC system could eliminate the effect of the pH gradient. Besides, ACP particles kept their size and morphology for up to 8 days in the H2 LC phase under proper initial salt concentration. Influence of Heparin in CaPs Mineralization. Heparin was chosen for the investigation of GAG CaPs interaction because of its relative high density of anionic sites (sulfate groups) and conformational flexibility due to its iduronic acid-rich molecular structure.64 These two features might render heparin more prone to bind on CaPs surfaces. For the samples prepared by the same route under the same condition (A vs B, C vs D), the heparinsamples (B, D) presented relatively less well-defined and broadened XRD peaks (Figure 2(II)), and a higher SSA (Table 1), suggesting poorer crystallinity and smaller crystal size as compared to their no-GAG counterparts. Besides, the heparin aged sample F was observed to have a large amount of amorphous moiety (Figures 5b and 8), whereas sample E, with no GAG addition, was mainly composed of crystallites, even after a shorter

Figure 12. Scheme of comparison between LC aging and the WBP route for the synthesis of CaPs. LC aging: (I) ACP formation in H2 LC phase; (II) controllable aging process of ACPs with a narrow size distribution in aqueous media; and WBP: uncontrollable precipitation of CaPs in aqueous solution.

in peak width of (310) reflection (the crystallographic a axis of calcium apatite, 2θ ≈ 39.8) was more prominent than that in (002) reflection (c axis, 2θ ≈ 25.9) in diffraction patterns of PCA during aging (Figure 2(I) c f). This anisotropic change in the diffractograms has also been observed in bone maturation, which would be related to the growth in width of PCA during aging.1 Besides, the intense (002) reflection (2θ ≈ 25.9) in the X-ray diffractograms (Figure 2(I) d f) corresponded well with the HRTEM observations, that the crystallites grew along the Æ001æ direction (indicated by arrows in Figure 4b). According to the XRD and TG-DTA results (Figure 2(II), 8), LC aged samples A and B transformed to TCP at ∼730 C (also after the calcination at 850 C). This apparent inferior thermostability of LC aged samples was related to their immature crystalline structure and small crystal dimensions as shown by XRD and TEM analysis (Figures 2(II) and 6). Since the starting ACP spherules have a uniform nanometric morphology and a narrow size distribution together with that the 899

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Figure 13. Comparison of Raman spectra with data of various synthetic and biological CaP samples.65

aging time (Figure 5 a, b). Sample G, with a lower concentration of heparin and longer aging time, reached the state when almost all the ACPs evolved into elongated crystallites. This apparent retardation in ACP PCA conversion suggests the interference of heparin molecules during CaPs crystallization. The effect of the heparin may possibly be linked to its adsorption onto the ACPs disturbing the rearrangement of the Posner’s clusters and thus hindering the crystallization process. The electrostatic repulsion between the heparin chains may also keep the ACP spherules from coalescing, generating a better colloidal stability in the aging suspension. Furthermore, heparin molecules might incorporate into the apatite structure during the aging process, leading to local packing disorders, which was indicated by the broadening of PO43vibrations in Raman spectra (Figure 10 E, F). Bone-Resembling Features of the Prepared Apatite. As stated in the Introduction, bone apatite is Ca2+ and OH deficient, CO32- substituted, and poorly crystalline. While for the PCA prepared by the LC aging route, the calcium deficiency with a Ca/P ratio of ∼1.5 was shown by SEM-EDS (Figure 5G); the carbonate substitution was confirmed by TG-IR (Figure 8 B, E, F), and the broad and diffuse diffraction peaks also confirmed the poor crystallinity (Figure 2(I) d, e; (II) A, B). In addition, IR data on the LC aged samples (A, B, E, and F) revealed that the carbonate substitution occurred on both the PO34- and OH sites (Figure 9), while Raman measurements gave further evidence of the low crystallinity and OH deficiency (Figure 10). In contrast, the WBP samples (C, D) were only carbonate substituted on the PO34- sites and exhibited narrower vibrational peaks, consistent with their lower SSA and higher degree of crystalline order. The PCAs prepared by the LC aging route were elongated platelets (30 50 nm long, 1.5 4 nm wide, Figure 3 e, f) and with a SSA as high as 356.1 m2/g (Figure 7, 25 h). More interestingly, Pasteris J. D. et al. have investigated the differences in Raman spectra of biological, geological, synthetic nanocrystalline and well-crystallized apatites.65 They noticed that for biological apatite, two particular expressions in Raman spectra correspond to the features of less perfect atomic ordering and hydroxyl deficiency: the peak width of the PO43- vibration peak at ∼960 cm 1 and the ratio between the areas of this peak and the O H stretch peak. Figure 13 shows the comparison between LC aged and WBP samples based on the Raman spectra data collected by Pasteris J. D.65 In this case, sample B (LC aged, with heparin) stands out from other synthetic apatites and shows a great resemblance to bone apatite. Notice that, by this measurement, the most bone-like sample does not correspond to the sample with highest SSA. This might indicate an optimal range between 250 and 300 m2/g of the SSA when the aim is to synthesize bone-like apatite.

Additional Comments on Postprocessing of CaPs. The inherent instability of ACP and PCA confers them considerable osteoconductivity and resorbability, meanwhile they are sensitive toward postprocessing procedures and storage. Even though, the formation of synthetic CaPs is a research area of high interest, there are a limited amount of studies where well-defined nanosized CaPs with ultrahigh SSA have been reported. The SSA of HAp prepared by conventional WBP at room temperature is normally in the range of ∼70 100 m2/g.66 While for synthesis via templates, HAp powder with a SSA of 130 m2/g can be synthesized in reverse micelles.67 The relatively small increase in SSA for templating systems here is probably due to the neglect of careful regulation of the purification and drying procedure. Arbitrary water-washing and heating of CaPs results in uncontrolled ACP PCA conversion and PCA crystal growth during the postsynthesis treatment, and destroys the delicate structure (confirmed by the comparingly large crystal size of water-washed and air-dried sample shown in Figures 3i and 2(I) f). Padilla S. et al. precipitated HAp with a SSA of 300 m2/g by dropwise addition of aqueous precursors, and the sample was purified with abundant lukewarm water (the specific time and temperature of purification was not mentioned) followed by freeze-drying.68 Uota M. et al. used three surfactant systems to synthesize calcium stearate-encapsulated HAp and pure HAp crystals, and the samples were washed with distilled water and then ethanol and finally air-dried (with SSA of 364 m2/g, 160 m2/g, 8 m2/g for 500 C-calcined samples).21 They attributed the HAp with ultrahigh SSA to the combined effect of Tween 60 and C12EO9 (mixed surfactant system).21 However, our experience suggests that drawing definitive conclusions with unregulated postprocessing of samples inevitably introduces a level of uncertainty since the CaPs may evolve to different morphologies (therefore different SSA) with different waterwashing time. In addition, the presence of residual water may facilitate uncontrolled aging during the process of air-drying (even at the early stage of calcification). A conclusion confirmed by the morphology and SSA changes during the maturation of CaPs in water and the large crystal size of the air-dried sample as shown in Figures 3 and 7. Lopez-Macipe et al.69 prepared CaPs using microwave and reflux heating method, and they found that the washed products showed a significant increase in SSA (147 186 m2/g) comparing to the SSA of unwashed products (78 95 m2/g). The authors attributed this finding to the elimination of the coprecipitate. However, it might also be linked to the waterwashing induced evolution of CaPs. To stop the ACP PCA conversion and further crystal growth, water needs to be completely extracted from the samples due to the H2O-mediated nature of the process.59 Consequently, strict control of the water-purification 900

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over time, temperature and pH, etc. is necessary. In our system, 0.5 h of water-washing is far smaller than the induction time (∼14 h at room temperature) of the conversion. For sample drying, lyophilization and further vacuum drying are required.

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’ CONCLUSION Inspired by mammalian mineralization, we have successfully synthesized bone-like apatite using liquid crystalline (LC) phases as templates. By mimicking the confined space provided by the collagen matrix, the H2 LC phase has shown impressive regulation on the morphology and dimension of calcium phosphate (CaP), resulting in spherical amorphous calcium phosphate (ACP) with a narrow size distribution of 10.8 ( 1.4 nm in diameter. Interestingly, the ACP particles gradually convert to crystalline apatite in aqueous media (∼25 h at room temperature in Milli-Q water). We have also shown that it is possible to control the aging process by the addition of heparin. The thorough investigation of ACP to apatite conversion by XRD, TEM, BET, TG-IR, and Raman spectroscopy provide solid in vitro information for a better understanding of biomineralization. The prepared apatites were elongated platelets (1.5 4 nm wide), with a specific surface area as high as 356.1 m2/g, in addition to exhibiting bone-resembling features, namely Ca2+ and OH deficiency and CO32- substitution as well as poor crystallinity. More importantly, the excellent controllability over the CaP crystallization facilitates the formation of CaP materials with properties that can be tailor-made depending on the application. ’ ASSOCIATED CONTENT

bS

Supporting Information. Thermogravimetric/differential thermal analysis (TG/DTA) curves of samples A, B, C, and D. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The Swedish Research Council (VR) is acknowledged for financial support. The authors are grateful to Annemarie Wagner, Dept. of Applied Physics, Chalmers, for assistance with the TGIR measurements. L.R.W. also acknowledges funding from the Knut and Alice Wallenberg Foundation. ’ REFERENCES (1) Dorozhkin, S. V.; Epple, M. Angew. Chem.,Int. Ed. 2002, 41 (17), 3130. (2) Palmer, L. C.; Newcomb, C. J.; Kaltz, S. R.; Spoerke, E. D.; Stupp, S. I. Chem. Rev. 2008, 108 (11), 4754. (3) Olszta, M. J.; Cheng, X. G.; Jee, S. S.; Kumar, R.; Kim, Y. Y.; Kaufman, M. J.; Douglas, E. P.; Gower, L. B. Mater. Sci. Eng., R, 2007, 58 (3 5), 77. (4) Kaplan, F. S.; Lee, W. C.; Keaveny, T. M.; Boskey, A.; Einhorn, T. A.; Iannotti, J. P. Form and Function of Bone; American Academy of Orthopaedic Surgeons: Rosemont, 1994. (5) Tampieri, A.; Celotti, G.; Landi, E. Anal. Bioanal. Chem. 2005, 381 (3), 568. 901

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