Preparation and Structural Characterization of Free-Standing

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Preparation and Structural Characterization of FreeStanding Octacalcium-Phosphate-Rich Thin Films Brook Nien-Fang Tsai, Chieh Tsao, Shing-Jong Huang, Chung-Kai Chang, and Jerry Chun Chung Chan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11977 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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The Journal of Physical Chemistry

Preparation and Structural Characterization of FreeStanding Octacalcium-Phosphate-Rich Thin Films Brook Nien-Fang Tsai,a Chieh Tsao,a Shing-Jong Huang,b Chung-Kai Chang,c Jerry Chun Chung Chana* a

Department of Chemistry, National Taiwan University, No. 1, Section 4, Roosevelt Road,

Taipei, 10617, Taiwan; b Instrumentation Center, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei, 10617, Taiwan; c National Synchrotron Radiation Research Center Hsinchu 30076, Taiwan

* To whom correspondence should be addressed. Phone: 886-2-33662994. E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT:

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Free standing films of calcium phosphates exhibit many favorable properties

for tissue engineering. In this work, a thin film of calcium phosphate is prepared in a liposome suspension using the method of ammonia gas diffusion. The thickness of the film is about 10 m and the lateral dimensions are on the length scale of millimeter. The results of powder X-ray diffraction and transmission electron microscopy show that the thin films contain the mineral phases of hydroxyapatite and octacalcium phosphate (OCP). Using solid-state NMR spectroscopy, in particular the technique of heteronuclear correlation spectroscopy with variable contact time, the major crystalline phase of the thin film has been confirmed to be OCP.


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INTRODUCTION Biological apatites are the major inorganic components of bone and teeth.1 It has been shown that nanocrystalline apatites resemble closely the main characteristics of biological apatites.2 Thus, apatite-based biomaterials are favorable options for various orthopedic and dental applications.3 In particular, free standing films of calcium phosphate (CaP) exhibit many favorable properties for tissue engineering such as providing support for stem cell attachment, inducing osteogenic differentiation, and facilitating bone regeneration.4 Thus, various apatite-based thin films with thicknesses ranging from submicrometer to tens of microns have been prepared under different conditions.5–7 On the other hand, octacalcium phosphate (OCP) was recently found to have excellent osteoconductive properties so that OCP-based materials have become very promising candidates for biomedical applications.8 Deposition of OCP thin films on metal substrate have been achieved by pulsed laser deposition,9 electrochemical treatment,10 and matrix assisted pulsed laser evaporation.11 Because OCP is meta-stable and it will transform to HAp spontaneously under neutral or alkaline conditions,12 the preparations and studies of free-standing thin films of OCP are relatively scarce in the literature.7,13 Recently, liposomes have been successfully exploited for the development of CaP-based biomaterials.14,15 Thus, it would be interesting to investigate whether liposomes could facilitate the preparation of OCP thin films. Although powder X-ray diffraction (XRD) is a powerful method for studying the mineral phase of CaP, the characteristic diffraction peaks of OCP at low 2 angle could be absent due to preferred orientation.7 This explains why it is difficult to quantify the individual crystalline phase of CaP systems, which usually exhibit structural polymorphism. On the other hand, solid-state NMR spectroscopy is a powerful analytical technique for the study of CaP-based biominerals.16–18 Valuable structural information has been obtained in bone,19–25 teeth,26,20,27–29 and CaP


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materials.30–32 In this work, we showed that a free-standing thin film of CaP could be prepared in liposome suspension on a propylene surface by the gas diffusion method. One the basis of a series of physical methods, with particular emphasis on solid-state NMR, the major mineral phase of the CaP films was found to be OCP. The OCP-rich film had a thickness of ~10 m and the lateral dimension was on the millimeter scale.

MATERIALS AND METHOD Sample Preparation. All chemicals were obtained from Acros unless stated otherwise. A lipid film was prepared by dissolving 0.775 g of L--phosphatidylcholine (PC) from soybean (95%, Avanti) and 0.116 g of cholesterol in 20 mL of dichloromethane (Merck), followed by rotary evaporation at 40 °C for 15 min and then drying in vacuo for 2 h. A volume of 50 mL of the solution mixture of 0.25 M Ca(NO3)2 and 0.15 M of (NH4)2HPO4, of which the pH was adjusted to 2.40 with concentrated HNO3, was prepared to hydrate the dried lipid film. The liposome formation was facilitated by sonication at 55 C for 3 min and the excessive lipids were then removed by centrifugation. After aging for two days, the resulting solution was extruded several times through a polycarbonate membrane of pore size 200 nm (Whatman) using an LiposoFast LF-50 extruder (Avestin), followed by an addition of 400 mL of 0.15 M (NH4)2HPO4 at pH 2.40. Consequently, in the liposome suspension the concentration of Ca2+ inside and outside the liposomes were 250 mM and 27 mM, respectively. CaP precipitates were obtained by a gradual pH increase using the NH3 gas diffusion method33,34 at ambient temperature. Briefly, a 50-mL polypropylene centrifuge tube (Corning) containing ~35 mL of the liposome suspension was placed together with another 110-mL glass vial containing 20 mL of 35% NH3 solution in a 10-


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liter sealed container. A hole of 3 mm in diameter was drilled on the plastic cap of the glass vial to control the gas diffusion rate and thus the rate of pH increased. After a predetermined incubation time, a layer of precipitates deposited on the tube wall was washed thoroughly by deionized (DI) water, followed by lyophilization. Synchrotron X-ray Powder Diffraction. The Synchrotron X-ray diffraction of samples were performed at the BL01C2 beamline of the National Synchrotron Radiation Research Center (NSRRC). The ring of NSRRC was operated at energy 1.5 GeV with a typical current of 360 mA. The wavelength of the incident X-rays was 1.03321 Å (12 keV), delivered from the 5 Tesla Superconducting Wavelength Shifter and a Si(111) triangular crystal monochromator. Two pairs of slits and one collimator were set up inside the experimental hutch to provide a collimated beam with dimensions of typically 0.5 mm  0.5 mm (H  V) at the sample position. The diffraction pattern was recorded with a Mar345 imaging plate detector positioned approximately 430 mm from the sample, which was packed into a 0.5 mm capillary, with a typical exposure duration of 60 s. The one-dimensional powder diffraction profile was converted by cake-type integration with GSAS II.35 The diffraction angles were calibrated by the Bragg positions of the LaB6 standard. Electron Microscopy. Scanning electron microscopy (SEM) images were taken on a JEOL JSM-6700F field emission scanning electron microscope operated at 10 kV. The samples were dispersed on a carbon tape mounted on a metal holder, followed by coating a layer of platinum with low-vacuum sputtering at 10 mA for 90 s. Transmission Electron microscopy (TEM) images and selected area electron diffraction (SAED) were acquired on a JEOL JEM-2100F field emission electron microscope operated at 200 kV. The samples were re-suspended in ethanol by sonication and loaded onto a carbon coated copper grid without staining.


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Solid State NMR. All NMR experiments were carried out at 31P and 1H resonance frequencies of 161.98 and 400.15 MHz, respectively, on a Bruker Avance III spectrometer equipped with a commercial 2.5 mm probe. The measurements were carried out at ambient temperature. Samples were confined in the middle of the rotor volume using Teflon spacers to enhance the RF homogeneity. 31P and 1H chemical shifts were externally referenced to 85% phosphoric acid and tetramethylsilane, respectively, using commercial HAp (Sigma-Aldrich) compound as the secondary reference. The chemical shifts of the HAp standard were set to 0.2 and 2.8 ppm for the 1

H and

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P dimensions, respectively. For the

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P{1H} cross-polarization magic-angle spinning

(CPMAS)36 experiments at a spinning frequency of 13 kHz, the 1H nutation frequency was set to 50 kHz and that of 31P was ramped from 31.7 to 39.6 kHz linearly.37 The CP contact time was set to 1 ms unless stated otherwise. Two pulse phase modulation (TPPM)38 proton decoupling of 70 kHz was applied during the acquisition period. For the

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P{1H} heteronuclear correlation

(HETCOR) measurements based on CP, quadrature detection in the F1 dimension was accomplished by the States-TPPI approach.39 For each t1 increment eight transients were accumulated and a total of 64 increments was acquired at steps of 100 μs. The spectral deconvolution of the 1H projection was carried out using DMFit2011.40 The spectra were measured at a spin rate of 10 kHz and the

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13

C{1H} CPMAS

C chemical shifts were referenced to

tetramethylsilane using adamantane as the secondary standard.

RESULTS AND DISCUSSION Morphology and Crystalline Phases of PC-CaP. Typical images of the sample deposited on the centrifuge tube wall are shown in Figure S1, where CaP sheets with size on the millimeter scale were obtained. Our samples are henceforth referred to as PC-CaPx, where x denotes the incubation


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time. There are two prominent features of our sample preparation protocol. First, a calcium and phosphate concentration gradient across the liposome membrane was prepared in such a way that the supersaturation level with respect to CaP was significantly higher inside the liposomes. Second, the pH of the liposome suspension was slowly increased from 2.5 to 9.0 (Figure S2) as NH3 gradually diffused into the solution. The SEM images of PC-CaP24h are shown in Figure 1. The thickness of the CaP sheet was estimated to be ~10 m. At the first glance, the platelet morphology of the crystallites observed at the highest magnification was rather similar to the crystal morphology of HAp. Figure 2 shows the XRD patterns of the samples of PC-CaP incubated for different periods. For PC-CaP17h, the presence of the OCP phase was evidenced by the diffraction peaks at 4.7, 9.4, and 9.7 (PDF 26-1056). On the other hand, the diffraction peaks at 46.6, 49.5, and 53.4 are commonly observed for HAp (PDF 09-0432). Hence, it appeared that both the crystalline phases of OCP and HAp were present in PC-CaP17h. The minor but very sharp peaks at 11, 21, and 29 were assigned to brushite (CaHPO4·2H2O, PDF 11-293). We believe that the well crystalline phase of brushite was formed via the dissolution-reprecipitation of amorphous calcium phosphate (ACP). The presence of brushite as a very minor phase should have little relevance to the formation mechanism of OCP. Knowing that OCP is meta-stable and it will transform to HAp spontaneously under neutral or alkaline conditions,12,41 it is remarkable that the characteristic diffraction peaks of OCP remained observable for PC-CaP2d, where the pH of the liposome suspension was higher than 8.0 (Figure S2). As expected, the OCP peaks of PC-CaP4d were diminished but the profile of the XRD pattern was still very similar to those of PC-CaP17h, PC-CaP24h, and PC-CaP2d. On the basis of our XRD patterns of PC-CaPx, it is very difficult to quantify the relative abundance of the OCP and HAp phases for the PC-CaPx samples because the


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sheet-like morphology of the samples would cause considerable distortions of the scattered intensity. From the SEM and XRD results, one may infer that the OCP crystallites were covered by a layer of HAp so that the OCP crystallites would not transform to HAp under alkaline conditions. To examine the mineral phase of an individual crystallite, we carried out TEM and SAED measurements for PC-CaP24h (Figures 3). The SAED pattern was indexed with reference to the OCP structure. To our surprise, a single crystal of OCP was also found for PC-CaP24h (Figure S3) but HAp crystallites were not detected in our TEM measurements. Nonetheless, the presence of HAp crystallites could not be ruled out by TEM measurements because of the limited sampling size. Thus, the scenario that an HAp layer was formed on the surface of the OCP crystallites could be ruled out. To identify the major crystalline phase of PC-CaPx, we had carried out a series of solid-state NMR measurements. The

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correlation ridge along the

P{1H} HETCOR spectra of PC-CaPx were shown in Figure 4. The 31

P chemical shift at 0 ppm had been assigned to the hydrogen

phosphate groups of OCP.42,43 The correlation peak at 0.2 and 3.1 ppm of the 1H and

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P

dimensions, respectively, is a well-known spectral marker of HAp.42 Because OCP to HAp transformation is thermodynamically favorable,12 it is not surprising that the HAp spectral feature was observed in the 31P{1H} HETCOR spectrum of OCP.43 For the spectrum of PC-CaP17h, there was a broad correlation peak at 5.8 ppm of the 1H dimension. This correlation peak spanned a range of 31P chemical shift from 0 to 4 ppm. In the study of a series of synthetic apatite minerals, the linewidths of

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P NMR signals were found to be inversely proportional to the sample

crystallinity.44 Thus, we inferred that the OCP structure in PC-CaP17h was relatively disordered because the spectrum of PC-CaP17h in the 31P dimension was much poorer in resolution than that


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of crystalline OCP.42 It is also likely that the spectrum of PC-CaP17h comprised substantial contribution of ACP, which has a correlation peak at 7.1 and 2.6 ppm in the 1H and 31P dimensions, respectively.45 As the incubation time increased to 2 d, the corresponding

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P{1H} HETCOR

spectrum revealed that the structural order of the OCP phase increased. The corresponding 1D slices of the HETCOR spectra of PC-CaP17h and PC-CaP2d are shown in Figure S4. Quantification of Crystalline Phases by Variable Contact Time HETCOR. In principle, it is difficult to quantify the amount of a species based on the signal intensity of a CP-HETCOR spectrum because the efficiency of the polarization transfer for difference species could be very different. To alleviate this difficulty, we employed the strategy of variable contact time. In particular, a series of

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P{1H} HETCOR spectra were acquired for the target sample. The 1H

projection of each two-dimensional spectrum was deconvoluted based on the characteristic 1H signals of apatite, orthophosphate, and hydrogen phosphate.16 The 1H projection spectrum of PCCaP24h and its deconvoluted components are shown in Figure 5a. Similarly, the 1H projections of the spectra acquired at other contact time were analyzed using the same set of NMR parameters, where only the intensities were varied to obtain optimal fitting. The intensities of each spectral component were then fitted by the following equation:16

M  t   M 0 1  exp   t  CP  exp   t T1ρH  where M 0 is the intensity factor of arbitrary unit;  CP and T1ρH characterize the spin dynamics of the CP process. As shown in Figure 5b, the favorable agreement of the experimental data and the fitting curves partly justified the approach of our data analysis. Furthermore, the values of the extracted parameters of  CP and T1ρH were consistent with the literature data.16 Accordingly, the relative abundance of the apatite, orthophosphate, and hydrogen phosphate components were estimated to be 9%, 58%, 33%, respectively. Note that the ratio of the abundance of


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orthophosphate and hydrogen phosphate was close to the value expected for OCP, which has a chemical formula of Ca 8  HPO 4 2  PO 4 4  5H 2O . Therefore, it was unequivocally demonstrated that the major mineral phase in PC-CaP24h was OCP (Table S1). This NMR strategy has been applied in the study of teeth and it may also be useful in bone research.27 In addition, the amount of lipid molecules incorporated into PC-CaP24h was negligible, as demonstrated in the 13C{1H} CPMAS spectrum of PC-CaP24h (Figure S5). Presumably, the lipid molecules were physisorbed on PC-CaP24h surface and could be easily washed away by DI water. As another independent verification that OCP indeed was the major mineral phase in PCCaP24h, the sample was incubated at pH 12 so that the phase transformation of OCP to HAp would be facilitated. A series of variable contact-time 31P{1H} HETCOR spectra were acquired for the samples at different incubation time and the data were analyzed similarly (vide supra). On the basis of this painstaking procedure, the relative M 0 of the spectral components were determined as a function of the incubation time (Figure 6). As expected, the apatite component increased dramatically to over 30% after an incubation of 3 h and eventually the amount plateaued at ~50% (Table S1). The corresponding

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P{1H} HETCOR spectrum of PC-CaP24h incubated for 7 d is

almost identical to that of HAp (Figure S6). This observation further substantiated our conclusion that OCP is the major mineral phase in PC-CaP24h. On the other hand, the corresponding XRD patterns showed little variation among the samples incubated for different periods (Figure S7). Effects of Liposomes on PC-CaP Preparation. To investigate whether the presence of OCP in PC-CaP samples was due to the effects of liposomes, we prepared a series of CaP samples using the same procedure for the preparation of PC-CaP except that the lipids were not added. Counterintuitively, both the XRD pattern and the 31P{1H} HETCOR spectrum of CaP2d revealed that the OCP phase of CaP2d had a higher degree of crystallinity than that of PC-CaP2d (Figures


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S8 and S9). As shown in the SEM image of CaP2d (Figure S10), the OCP clusters of 15 to 20 m in size were loosely aggregated. That is, without the lipid molecules, the thin film formation of OCP was hampered. Furthermore, we also attempted to prepare PC-CaP24h samples on glass surface. In the corresponding SEM image (Figure S11), typical OCP crystallites of blade-like morphology were observed and the crystallites were randomly aggregated. This control experiment indicated that the chemistry on the polypropylene surface was important for the thin film formation. Indeed, it has been shown by simulations that PC-based liposomes are unstable on polypropylene surface,46 which suggested that the release of Ca2+ ions upon the disruption of liposomes would elevate the local supersaturation level of CaP. Hence, we surmise that the fusion of neighboring OCP crystallites as observed for PC-CaPx occurred via the space-filling precipitation of ACP inside the void space between adjacent OCP crystallites. Additional experiments revealed that the NH3 gas diffusion rate was the key factor for the formation of OCP in our experimental setup (data not shown). That is, the pH gradient in our system dictated the phase transformation kinetics of CaP. Formation Mechanism of PC-CaPx. The nucleation and phase transformation of CaP has been described in a cascade of events, viz. [Ca(HPO4)3]4  [Ca2(HPO4)3]2  [Ca6(HPO4)4(PO4)2]2  OCP  apatite, where the species in the second and the third stage were considered as the molecular species of acidic ACP.47,48 Although we were not able to verify whether the OCP phase was formed via the phase transformation of acidic ACP, we surmise that the formation of OCP sheets in PC-CaP24h occurred in the following steps. As the solution pH increased, a thin layer of CaP gradually deposited on the polypropylene surface. Concurrently, the liposomes on the propylene surface of the centrifuge tube became disrupted. The released Ca2+ ions generated a relatively high supersaturation level with respect to ACP and/or OCP. The presence of lipid


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micelles might limit the crystallinity of the OCP phase and/or slowed down the phase transformation kinetics of ACP, which consequently facilitated the coalescence of the disordered OCP crystallites to form a mineral sheet of macroscopic scale. Currently, we do not fully understand why the OCP phase could persist under alkaline conditions because the transformation of OCP to HAp would be facilitated in the presence of hydroxide ions:12

Ca 8  HPO4 2  PO4 4  5H 2 O  2Ca 2  4OH   Ca10  PO 4 6  OH 2  7H 2O OCP

HAp

We speculate that the very slow increase in pH by NH3 gas diffusion allowed the generation of a pH gradient so that the pH on the OCP surface might be lower than that of the bulk solution. In other words, it is likely that the persistence our OCP sheets in alkaline medium is largely a kinetically controlled process. Very recently, the method of gas diffusion has been employed to prepare CaP coatings on mica sheets, where the deposited layers comprised needle-like apatite crystals and small platelets of OCP.13 Using a similar method, we found that the major crystalline phase of CaP formed on the polypropylene surface was OCP.

CONCLUSIONS We found that the use of liposomes, NH3 gas diffusion rate, pH gradient, and the surface of polypropylene are important factors for the preparation of free-standing OCP sheets. The apparent persistence of OCP thin films under alkaline conditions were rationalized as a kinetically controlled event. Although XRD diffraction is not effective in monitoring the phase transformation of our CaP systems, we had demonstrated that solid-state NMR is very effective in providing estimates of the amounts of the crystalline phases of CaPs, provided that the spectral markers of the individual phase are well resolved in the 31P{1H} HETCOR spectra.


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ASSOCIATED CONTENT Supporting Information. Photographs of the CaP sheets, plot of the pH values of the liposome suspension versus incubation time, TEM image of the debris of PC-CaP24h, 31P and 1H spectral slices taken from

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P{1H} HETCOR spectra of PC-CaP17h and PC-CaP2d,

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C{1H} CPMAS

spectra acquired for selected samples, 31P{1H} HETCOR spectrum of of PC-CaP24h incubated at pH 12 for 7 d, XRD patterns of the PC-CaP24h sample incubated in a solution of pH 12 for different periods of time, XRD pattern of the sample CaP2d. The sticks denoted the diffraction peaks of OCP, 31P{1H} HETCOR spectrum of the sample CaP2d, SEM image of the sample CaP2d, SEM image of the sample PC-CaP24h prepared on glass surface, table summary of the parameters characterizing the

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P{1H} CP dynamics. This information is available free of charge via the

Internet at http://pubs.acs.org.

ACKNOWLEDGMENT This work was financially supported by the Ministry of Science and Technology (103-2113-M002-013-MY3). The NMR, TEM, and SEM measurements were carried out at the Instrumentation Center of National Taiwan University, supported by the Ministry of Science and Technology. We thank C.-Y. Chien and S.-J. Ji for their help in TEM and SEM experiments.


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FIGURES

Figure 1. SEM images of PC-CaP 24h. The sample was cracked into pieces because of the mounting procedure. Scale bars: on the left = 20 m, right = 1 m.


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Figure 2. XRD patterns of the PC-CaP samples prepared at different incubation time. The green arrows denote the characteristic peaks of OCP, whereas the red ones indicate the peaks commonly observed for HAp. The asterisks highlight the diffraction peaks of brushite.


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Figure 3. TEM images and the corresponding SAED pattern of the debris of PC-CaP24h. The red cross marks the position at which the SAED pattern was taken. Despite the polycrystalline nature, the assignment of the diffraction spots enclosed by dashed circles could be made with reference to the OCP structure. The dashed and solid circles of the same color represented the Friedel pairs. The zone axis was shown at the lower right corner. Scale bars: on the top left = 500 nm, bottom left = 100 nm, right = 5 nm1.


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Figure 4. 31P{1H} HETCOR spectra of the commercial HAp and PC-CaPx samples at a contact time of 1 ms. The contour levels were increased by a factor of 1.5 successively, where the base level was set to 8  root-mean-square noise. The correlation ridge highlighted by a dashed line is the characteristic spectral feature of OCP.


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Figure 5. (a) 1H projection of the 31P{1H} HETCOR spectrum of PC-CaP24h acquired with a contact time of 1 ms. The projection was deconvoluted into three sets of spectral components, viz.


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apatite, phosphate, and hydrogen phosphate, whose chemical shifts were at 0.2 ppm, 6.7 ppm, and > 10 ppm, respectively. The component of hydrogen phosphate has two contributions. Their sum constituted the total signal of hydrogen phosphate. (b) The relative intensities of the spectral components of apatite, phosphate, and hydrogen phosphate as a function of the contact time. The color code is identical to that of (a).


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Figure 6. Relative intensities of the spectral components of apatite, orthophosphate, and hydrogen phosphate extracted for the as-prepared PC-CaP24h and those incubated at pH 12 for different periods of time.


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TOC GRAPHIC


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Preparation and Structural Characterization of Free-Standing

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