Local Environment in Biomimetic Hydroxyapatite−Gelatin

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Local Environment in Biomimetic Hydroxyapatite-Gelatin Nanocomposites As Probed by NMR Spectroscopy Anastasia Vyalikh,† Paul Simon,‡ Theresa Kollmann,‡ R€udiger Kniep,‡ and Ulrich Scheler†,* †

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‡

Leibniz-Institut f€ur Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany Max-Planck-Institut f€ur Chemische Physik fester Stoffe, N€othnitzer Strasse 40, 01187 Dresden, Germany ABSTRACT: The local environment in biomimetic hydroxyapatite (HAp) nanocomposites containing 0 and 33 wt % gelatin was characterized by 31P solid-state NMR spectroscopy. The presence of crystalline HAp and amorphous calcium phosphate phases was found in both materials. The latter can appear at the grain surfaces or at the boundaries of the crystalline phases. In the 31P cross-polarization MAS NMR spectra of HAp containing 33 wt % of built-in gelatin, an additional signal at 0.9 ppm was observed, which demonstrates the spatial correlation to hydrogen atoms of nonmineral origin, as indicated by 31P-1H heteronuclear correlation (HETCOR) NMR spectroscopy. This site is identified as a phosphate species present at the surfaces of the mineral component (HAp) interacting with the surrounding organic matrix.

’ INTRODUCTION Biological mineralization processes, such as the formation of bone and teeth in vertebrates, are highly complex and specific for different kinds of hard tissues. In bone and dentin tissues, the inorganic mineral closely resembles hydroxyapatite and is intimately associated with the organic matrix consisting of a complex assemblage of organic macromolecules, mostly collagenic protein (about 90 wt % of the organic content of bone).1,2 It has been established that apatite, which is the main inorganic constituent of bone and teeth, first nucleates in the gaps between the triple-helical collagen molecules arranged in form of fibrils.3,4 Knowledge of general principles of biomineralization has been implemented in concepts of biomimetics, which find application in the design of biocompatible materials, for example, to replace and regenerate bone.5,6 However, for the fabrication of novel hardtissue scaffolds and implants, deeper understanding of the structure formation of biomimetic organic-mineral nanocomposites is essential. So far, investigations have been extensively focused on microscopic techniques. Scanning probe microscopy has been applied to measure the surface kinetics of mineral surfaces.6 Microscopic variation in the degree of mineralization across a sample has been investigated by transmission electron microscopy.7 Synchrotron X-ray diffraction on individual aggregates has provided information on the orientation of the mineral component of fluorapatitegelatin composites.8 Mostly, the combination of X-ray powder diffraction with different methods of electron microscopy and IR spectroscopy has been used to investigate organic-calcium phosphate composite materials.9-11 However, a detailed study of how the mineral phase of calcium phosphate nanocomposites interacts with the organic phase at a molecular level is lacking. As the nature of the organic-inorganic interfacial interactions is a r 2011 American Chemical Society

key aspect defining how the mineral binds to the organic matrix and, consequently, how to develop biomimetic materials,5 the study of the molecular-level structure at such interfaces is crucial from the point of view of bone and teeth tissue engineering strategies. Unlike other structural techniques, which characterize the “long-range” order, giving an average view of a structure, solid-state nuclear magnetic resonance (NMR) spectroscopy is sensitive to the local environment of a particular nucleus and, therefore, is highly suited to study interfacial phenomena in natural dentine and bone minerals12-19 and their model compounds.20-23 Substitution of the rigid, insoluble collagen by water-soluble gelatin (denatured collagen) enables the reduction of the level of complexity as compared to the natural formation of calcified tissue, because gelatin provides a higher mobility and accessibility and thus eases rearrangements of the protein fibers during the formation of the organic-inorganic nanocomposite. During the past few years, calcium phosphate-gelatin biocomposites have been extensively investigated as potential bone replacement biomaterials.10,24-28 In our previous studies, nanocomposites consisting of gelatin and hydroxyapatite, as well as of gelatin and mixtures of hydroxyapatite and different amounts of octacalcium phosphate, were prepared by using a precipitation process.29 The composites were prepared from aqueous solutions of CaCl2 3 2H2O and (NH4)2HPO4 in the presence of varying amounts of gelatin. It has been shown that prestructuring of gelatin by calcium and phosphate results in different phase compositions and morphologies of the final products. Thus, in samples prepared by means of the Ca prestructuring Received: August 30, 2010 Revised: December 17, 2010 Published: January 18, 2011 1513

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reaction, hydroxyapatite is the only inorganic phase obtained independent of the amount of gelatin, yielding small platelike particles (50 nm  33 nm). Prestructuring of gelatin with (NH4)2HPO4 at low gelatin quantities favors the formation of hydroxyapatite in the form of small, platelike particles (60 nm  35 nm), whereas high gelatin concentrations lead to the formation of octacalcium phosphate causing the development of large foils (∼730 nm  410 nm). This work focuses on molecular-level investigations of composites precipitated from aqueous (NH4)2HPO4 solutions as the prestructuring agent at pH 9 whereby the octacalcium phosphatefree precipitation of hydroxyapatite is assured. 31P solid-state NMR spectroscopy has been applied to determine the nature of the inorganic species at the mineral crystal surfaces. 1H-31P heteronuclear correlation (HETCOR) experiments have been performed to examine the arrangement and order of phosphate, hydroxide groups, and water molecules in the inorganic-organic nanocomposites and to compare the results with its gelatin-free analogue.

’ EXPERIMENTAL SECTION Synthesis of the Samples. Calcium phosphate gelatin nanocomposites were prepared by precipitation from aqueous solutions. CaCl2 3 2H2O (p.a., Merck, Darmstadt, Germany), (NH4)2HPO4 (p.a., Riedel-de Haen, Seelze, Germany), and gelatin (300 bloom, acid bone, technical, DGF Stoess AG, Eberbach, Germany) were used as starting reagents. The experimental setup consisted of a 3-L cylindrical glass reactor, a three-fold impeller, a heating and circulating bath, two slope pumps, a pH controller, and a stirring motor. For the PO4 prestructuring reactions, two solutions were prepared: a gelatin solution at 50 °C (50 g of gelatin in 500 mL of H2O) and a PO4 solution [0.180 mol of (NH4)2HPO4 in 1500 mL of H2O] at ambient temperature. The solutions were poured into the reaction vessel under mild stirring. The pH value of the system was adjusted to 9.0 ( 0.1 using NH3(aq). Then 300 mL of a 1.0 M CaCl2 3 2H2O aqueous solution at pH 9 was dropped into the reaction mixture at a rate of 2.5 mL/min. A final Ca/P ratio of 1.67 was kept for all reaction mixtures in a total reaction volume of 2.3 L. During the entire precipitation process, stirring at 300 rpm was applied. The pH value was controlled at 9.0 ( 0.1, and the temperature was held at 25 °C. After 2 h of precipitation, the milky suspension was left under constant conditions (25 °C, pH 9, 300 rpm) for 21 h. To isolate and clean the resulting precipitate, 1200 g of the aged suspension was centrifuged at 2044 rpm for 15 min, suspended again in 1000 g of deionized water, and washed by stirring at 500 rpm for 10 min to remove salts and excess gelatin. Four cycles of washing and centrifugation were required. The samples for 0 and 50 g of added gelatin were labeled as CaP and gel/ CaP, respectively. Finally, a small portion of the white precipitate was suspended in ethanol for investigation by transmission electron microscopy (TEM; Figure 1). The reference sample of hydroxyapatite was purchased from Merck, Darmstadt, Germany. TEM. High-resolution TEM (HR-TEM) experiments (Figures 1a-d) were carried out at the Special Laboratory Triebenberg for Electron Holography and High-Resolution Microscopy at TU Dresden. A CM 200 FEG/ST-Lorentz field-emission microscope (FEI Company, Eindhoven, The Netherlands) equipped with a Gatan 1  1 k slow-scan CCD camera was used. The analyses of the TEM images were performed using DigitalMicrograph software (Gatan, Pleasanton, CA, USA). For sample preparation, a small

Figure 1. (a, b) TEM overview images of a typical aggregate of the precipitated hydroxyapatite sample without gelatin (CaP), (c) HR-TEM image of the area marked (with a white square) in image b, (d) corresponding Fourier transform of image c. The interplanar distances of 2.77 Å (112) and 3.44 Å (002) indicate the presence of HAp.

amount (spatula tip) of the as-prepared precipitate was diluted in about 10 mL of ethanol. A drop of this suspension was put on a TEM grid (holey carbon film on 300-mesh Cu grid, Plano GmbH, Wetzlar, Germany) that was plasma-etched for 10 s (Plasma Prep II, SPI Supplies, West Chester, PA, USA). Plasma etching was performed to flatten the foil-like particles. NMR Spectroscopy. All NMR spectra were obtained on a (11.7 T) Bruker Avance 500 spectrometer operating at resonance frequencies of 500.1 MHz for 1H and 202.5 MHz for 31P. The 31P NMR spectra were acquired at a spinning frequency of 10 kHz with proton decoupling employing a BL4 HXY 4-mm MAS probehead. For 31P MAS NMR spectra, a single 90° pulse of 4-μs duration, a recycle delay of 80 s, and 16 repetitions was applied. For the 31 1 P{ H} cross-polarization (CP) measurements, a 1H spin-lock 75100% ramp up and a recycle delay of 5 s were used. High-power two-pulse phase modulation (TPPM) proton decoupling of 100 kHz was applied. The contact time was varied in the range of 100-800 μs to concentrate on the strong dipolar couplings from the local environment. The spectra were fitted using Dmfit.30 The 1H MAS NMR spectra were measured with a 90°-pulse duration of 2.5 μs, a spinning frequency of 15 kHz, and a recycle delay of 5 s. The 1H chemical shifts were referenced to tetramethylsilane (TMS) at 0 ppm using poly(vinylidene fluoride) as an external reference; powdered ammonium dihydrogen phosphate was used to reference the 31P spectra at 0.72 ppm relative to 85% phosphoric acid. Two-dimensional 1H-31P HETCOR experiments were performed using frequency-switched Lee-Goldburg (FSLG)31 crosspolarization with a contact time of 200 μs and an LG frequency of 70.7 kHz to avoid spin diffusion during the cross-polarization. A recycle delay of 5 s and 256 scans per t1 time increment were used. A total of 128 t1-slices with a 37.75-μs time delay corresponding to a rotor-synchronized incrementation in the indirect dimension were 1514

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Figure 2. 31P MAS NMR spectra of hydroxyapatite without gelatin CaP (solid line) and with 33 wt % built-in gelatin (gray dashed line) as obtained from the precipitation reaction. The 31P MAS NMR spectrum of pure hydroxyapatite (dotted line) is shown for comparison.

acquired. Prior to Fourier transform, the exponential multiplication with 20- and 120-Hz line broadenings were used in the 31P and 1H dimensions, respectively.

’ RESULTS TEM. The structure of a sample precipitated by PO4 preimpregnation with 0 g of gelatin (CaP) was investigated by highresolution TEM. Parts a and b of Figure 1 show the TEM overview images of typical aggregates of the CaP sample. The marked area (white square) in the overview image (Figure 1b) was examined in detail and is displayed as an HR-TEM image in Figure 1c. Figure 1d represents the corresponding fast Fourier transform (FFT) of Figure 1c, showing reflections corresponding to interplanar distances of 2.77 Å (112) and 3.44 Å (002) of hydroxyapatite. 31 P MAS NMR Spectroscopy and 31P{1H} CP MAS NMR Spectroscopy. Figure 2 shows the 31P MAS NMR spectra of CaP and gel/CaP samples. Both spectra are dominated by a 31P signal at 2.6 ppm similar to the 31P MAS spectrum of pure HAp included in Figure 2 for comparison. Therefore, we attribute the peak at 2.6 ppm to PO43- groups as belonging to hydroxyapatite (HAp). Unlike pure HAp, the lineshape in both gel/CaP and CaP are asymmetric and significantly broader, demonstrating structural inhomogeneities, which can include the presence of an additional phase different from pure crystalline HAp. Moreover, the overall linewidths in gel/CaP and CaP were found to be increased from 1.5 to 2.3 ppm when gelatin was introduced into the mineral matrix. The 31P{1H} CP MAS NMR spectra measured with proton decoupling of 100 kHz are shown in Figure 3. The spectrum of CaP (Figure 3a) is dominated by a 31P peak at 2.6 ppm characteristic of crystalline HAp, whereas the gel/CaP spectrum (Figure 3b,c), where the organic phase is present, additionally shows a sharp signal at 0.9 ppm. Because of the lower content of the species associated with this peak and strong heteronuclear dipolar interactions with the abundant protons, this signal is visible only in the CP experiment at strong proton decoupling. Moreover, variation of its relative contribution to the entire lineshape depending on the contact time (Figure 3b,c) points to a difference in the spatial

Figure 3. 31P{1H} CP NMR spectra of (a) CaP at a contact time of 0.5 ms and (b,c) gel/CaP at CP contact times of 0.3 and 0.5 ms, respectively. Gray lines denote the difference spectrum, fit components, and their sum giving the best-fit spectrum.

proximity of phosphorus species associated with 2.6 and 0.9 ppm signals with regard to neighboring protons. Investigating the lineshape of the signal at 2.6 ppm in both gel/ CaP and CaP, one can note that at least two components contribute to this peak. Deconvolution yields two contributions with close chemical shifts and essentially different line width parameters. The resulting fit data for two components in the gelatin-free sample and three components in the gelatin-containing nanocomposite are presented in Table 1. Based on the chemical shift and line width data, the narrow line at ca. 2.6 ppm is assigned to the crystalline HAp phase. However, to investigate the nature of the broad component, additional information is needed that is available from a heteronuclear correlation experiment, which enables separating 31 P signals associated with different kinds of surrounding protons (see the next subsection). For the quantitative estimation of the corresponding 1H-31P distances, the cross-polarization buildup curves were analyzed with respect to each component (Figure 4). The data for the narrow component at 2.6 ppm show a continuous increase up to 3 ms with the contact time for both samples. This is a typical behavior for the hydroxyl protons of HAp reported in the literature even up to the longer CP times (see, e.g., ref 32). Because a large number of 1515

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Table 1. Fit Data for 31P CP MAS NMR Spectra (Contact Time = 0.5 ms) of CaP and Gel/CaPa sample

δ (ppm)

31

line width (ppm)

relative content (%)

CaP

2.6

1.1

26

gel/CaP

2.9 2.5

4.5 1.3

74 19

3.0

4.9

79

0.9

0.5

2

a

Typical measurement error in the values quoted is (0.2 ppm for δ(31P), (0.2 ppm for line width, and (5% for relative content values (except for the 0.9 ppm peak, whose intensity determination error was (1%.

Figure 4. 31P{1H} CP buildup curves for gel/CaP. The intensities of the narrow HAp peak are shown by filled squares, the broad HAp peak by open squares, and the 0.9 ppm peak by circles. The latter data are scaled for comparison. The lines denote simulation for a single spin pair with a distance of 0.53 nm corresponding to the average close 31P-1H distance in HAp (solid line) and for four 31P-1H spin pairs with the closest distances 2.2 and 2.5 nm (dashed and dotted lines, respectively).

relatively remote protons contribute to this CP efficiency, it seems sufficient to simulate only the initial slope of the cross-polarization dynamics using a single 31P-1H spin pair with an average distance between them of 0.53 nm (considering that two protons nearest to phosphorus are ca. 0.4 nm apart and four other are at ca. 0.6 nm distance). In contrast, the significantly faster growth of the broad components in both gel/CaP and CaP with increasing contact time and the maximal CP efficiency at ca. 1 ms demonstrates the different cross-polarization dynamics. The simulation of the latter yields a 31P-1H distance of ca. 0.2 nm for a spin pair that is essentially closer than the distance from the HAp phosphorus atom to the hydrogen in a hydroxide group. This is a typical distance for hydrogen phosphate units that could also be present in the samples. The assignment of the broad line is discussed in the next section. However, the presence of this component in both samples points to the fact that the structure of the mineral phase is preserved in the organic composite material. Further, cross-polarization behavior similar to that for the broad component was observed for the signal at 0.9 ppm in gel/CaP. This finding seems to be surprising at first glance, particularly taking into consideration the different lineshapes of the latter and the 0.9 ppm signal. The distance between the P site corresponding to 0.9 ppm signal and the neighboring protons was found to be ca. 0.25 nm. This peak was reproduced in

Figure 5. 1H-31P HETCOR spectra of the (a) CaP and (b) gel/CaP nanocomposites. On the right are the 1H MAS NMR spectra and the summed 1H projections of the 2D spectrum. The spectra on the top are (a) 31 1 P{ H} CP NMR spectrum at 0.3-ms contact time, (b) 31P MAS NMR single-pulse spectrum, (c) horizontal summed projection.

the 2D 1H-31P HETCOR experiment (see the next subsection), and its origin will be discussed later. 1 H-31P HETCOR Spectroscopy. The two-dimensional 1H31 P HETCOR spectra of CaP and gel/CaP (parts a and b, respectively, of Figure 5) show spatial correlations between 1H spins (from both organic and inorganic parts) and 31P spins (primarily phosphate species are present in the mineral component). The CaP correlation spectrum (Figure 5a) is dominated by a strong peak at 2.6 ppm in the 31P dimension and at ca. 0.2 ppm in the 1H dimension, which is assigned according to ref 33 to PO43-/OH- groups arising from crystalline HAp. The inhomogeneous broadening of this line clearly observable in the 1H projection originates from structural heterogeneity that results in chemical shift distributions. Actually, all of the 1H signals in the spectrum of CaP correlate to the 2.6 ppm 31P signals that point 1516

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The Journal of Physical Chemistry C out their association with the mineral phase. The signal spread from 4 to 8 ppm in the 1H dimension is assigned to structural/ surface water protons related to the mineral phase. Two distinct dominating contributions at 4.9 and 5.9 ppm are definitely observed within this signal intensity, which merge into a nonresolved peak at 5.4 ppm in a 1H MAS NMR spectrum (on the right of Figure 5a) because of the strong coupling with neighboring protons. Higher resolution in the 2D HETCOR spectrum was provided by applying the FSLG for proton decoupling during the evolution period t1. Actually, the different water structural motifs in CaP manifested in the distinct chemical shifts within this broad signal confirm this material as a nanocomposite with nanoscale structural heterogeneity. In general, similar correlation signals have been observed in the HETCOR spectra in animal bone,12,13 joint mineralized cartilage,16 and rat dentine.22 Two very weak signals at 1.3 and 1.6 ppm are observed in the 1H MAS NMR spectrum, which is quite similar to the 1.1 and 1.5 ppm shifts reported for the protons in an isolated water molecule in the octacalcium phosphate (OCP) structure,33 which the sample of HAp studied in ref 33 was known to contain, and to the 2.0 ppm signal detected in 1H combined rotation and multiple-pulse spectroscopy (CRAMPS) of HAp and attributed to OCP-like water defects.12 However, in the HETCOR spectrum, only the peak at 1.9 ppm is visible, which cannot be unambiguously assigned based on the present data. Finally, the CaP spectrum demonstrates a weak 1H peak at 12.6 ppm, which belongs to acidic protons of the HPO42- group and can result from the protonation of the surface phosphate groups.12 The spectrum of the gel/CaP nanocomposite (Figure 5b) shows two groups of signals, which are separated with respect to their 31P chemical shifts. The correlation signal at 2.6 ppm/0.2 ppm (thereafter corresponds to 31P/1H notification) is attributed to PO43-/OH- groups. The 1H linewidth of the PO43-/OH- signal is significantly narrower than that in CaP, demonstrating the more ordered local proton environment in the organic component because the proton linewidth here is dominated by inhomogeneous broadening. The correlation signal at 2.6 ppm/3.0 ppm is attributed to structural/surface water, respectively. The linewidth in the 31P direction is significantly larger that that in the CaP spectrum, illustrating essential structural inhomogeneities and disorder associated with the presence of water. The considerable 1 H shift from 4.9-5.9 ppm (Figure 5a) to higher field at 3.0 ppm (Figure 5b), when considering gelatin-containing sample, can be explained by a variation of the strength of hydrogen bonds of water molecules. Indeed, a 1H peak at ∼3.0 ppm is quite typical for water structures with lower dimensionality, such as water clusters, surface monolayers, or bilayer water structures in restricted geometries, as confirmed by both experiments34,35 and theoretical calculations.36 Moreover, the 1H signals at a slightly higher field (2.2 and 2.3 ppm) have been even detected in carbonated apatite and deproteinated cortical bone, respectively, and have been assigned to isolated water molecules in the hydroxide ion channels.13 Therefore, one can assume that the peak at ca. 3 ppm replaces the usual water signal at ca. 5 ppm in HAp, which, upon removal of bound water, shows an upfield shift to 3 ppm and a reduced intensity. Additionally, a smaller intensity signal at 3.0 ppm/4.2 ppm is observed, which can also stem from water structures with different strengths of hydrogen bonds. The second group arising at 0.9 ppm in the 31P dimension is correlated to the 1H signals at 3.7 and 7.7 ppm. Because these signals are not observed in the gelatin-free sample, we relate them

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to the appearance of the organic phase and tentatively attribute to the mineral-gelatin interface. It is worth noting that, in the gel/CaP sample, no 1H peak from acidic phosphate protons at ca. 13 ppm is observed.

’ DISCUSSION To understand the structure and composition in the inorganicorganic nanocomposite, the signals have to be carefully assigned. The narrow 31P line at 2.6 ppm found in both the directly-polarized and cross-polarized from protons (CP) spectra of both materials obviously arises from PO43- groups associated with pure crystalline HAp. The slight increase in the linewidth from 1.1 to 1.3 ppm upon introduction of the organic component indicates a less homogeneous phosphate environment in the composite material. The presence of a second (broad) component is clearly visible in 31 P CP MAS spectra. Its chemical shift (around 3 ppm), which is different from the intrinsic hydroxyapatite shift of 2.6 ppm, points to variations in the local structure of the phosphate environment. Furthermore, its cross-polarization dynamics is similar for the two samples, but somewhat different from that in HAp, indicating significantly closer 1H-31P spatial proximity with regard to neighboring protons. The linewidth of the broad component is considerably larger than that of pure reference HAp and can be caused by inhomogeneous broadening due to a distribution in the local phosphorus environments. Its increase from 4.5 ppm in CaP to 4.9 ppm in gel/CaP demonstrates that structural order in the latter is affected even more stronger when the gelatin-containing nanocomposite is considered. This component can be attributed to amorphous HAp-like phases that can appear at the surface of the grains, whose core is formed by crystalline HAp described above, or at the boundaries of the crystalline mineral phases found in related fluoroapatite-gelatin nanocomposite7,37 presenting “highly mosaic-controlled nanocomposite superstructures”. Indeed, some recent NMR studies have suggested the presence of the “nonHAp” calcium phosphate phase as a disordered surface layer around the synthetic or natural hydroxyapatite nanocrystals.19,23,32,38,39 Their chemical composition is dominated by hydrogen phosphate anions and structural water32 or by PO43- ions that are slightly distorted and/or weakly bound to protons.38 Additionally, vacancies due to carbonate substitutions are known to take place in the structure of HAp nanocrystals,23,38 causing line broadening relative to the crystalline HAp signals. Another important piece of evidence for the presence of chemical bonding between the organic and mineral components is given by the signal at 0.9 ppm in gel/CaP. First, its deviation from a pure HAp signal (2.6 ppm) can be linked to a change in the concentration and location of Ca ions, because the 31P chemical shift of the phosphate unit is very sensitive to the Ca content40 or to protonation of the phosphate, which is known to lead to an upfield 31 P shift.41 The similar 31P peak at 0.8 ppm was previously assigned to HPO42- groups in earlier works on bone42 and synthetic Cadeficient apatites,43 as well as to active -PO2H and -PO3Hsurface sites in synthetic carbonate-free fluoroapatite.44 The results of the simulation based on the cross-polarization experiment demonstrate the essentially shorter 1H-31P distance (ca. 0.2 nm) compared to that in crystalline HAp (0.4 nm). The linewidth of the peak was found to be significantly smaller (0.5 ppm), indicating a relatively ordered composition. All of these factors point to the appearance of an additional structural motif located between the mineral phase and the gelatin fibrils. Structurally, it can be represented as a relatively ordered assembly of Ca ions 1517

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The Journal of Physical Chemistry C and PO4 tetrahedra, where the latter is coordinated by organic molecules adjusted to the surface of the mineral crystallites. Further support for the presence of such an interface between the organic and inorganic components is provided by the twodimensional 1H-31P HETCOR spectrum, which provides information about the spatial correlation of 1H containing groups in the mineral and/or in close proximity to the mineral surface and mineral phosphate. Most of the protons of the gelatin matrix, which are located in the bulk phase and not close to 31P, are not observed in this experiment. In contrast, the protons of the organic matrix in close proximity to mineral 31P sites become visible in the HETCOR spectrum, making it an excellent method to visualize the mineral component of a sample and phosphate species in close contact with the organic component. Therefore, the signals observed at 0.9 ppm/3.7 ppm and 0.9 ppm/7.7 ppm in the gel/CaP sample evidence the spatial correlation to the hydrogen atoms of nonmineral origin, as indicated by their 1H chemical shifts, and are attributed to the presence of the mineral-gelatin interface. Notably, the nonappearance of the 1H signal at ca. 13 ppm due to hydrogen phosphate and, in contrast, the emergence of two new signals in the correlation spectrum of the organic composite can be interpreted by a conversion of the surface-related HPO42- units in CaP into a new phosphate group covalently bonded or hydrogenbonded to the protein molecule in gel/CaP.

’ CONCLUSIONS The aim of this work was to use solid-state NMR spectroscopy to study the inorganic-organic interaction at a molecular level in a gelatin-containing hydroxyapatite nanocomposite (gel/CaP) and to compare the results with its gelatin-free analogue as a reference system. The 31P CP MAS NMR spectra of the nanocomposite demonstrate the presence of phosphate species, which (i) have an environment different from that of pure crystalline hydroxyapatite as indicated by a variation of the chemical shift, (ii) are relatively well-ordered according to the narrower linewidth, and (iii) are located in close spatial proximity to hydrogen atoms as demonstrated in the heteronuclear correlation experiment. Moreover, the heteronuclear 1H-31P distances were found to be essentially shorter than those in pure hydroxyapatite. The two-dimensional heteronuclear 1H-31P correlation experiments evidence the spatial correlation of this phosphate group to the hydrogen atoms of the nonmineral origin. Therefore, we identify this new structural motif as phosphate species being present at the boundaries of mineral crystals that interact with the surrounding organic matrix.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) through Grant TRR67. We thank Prof. Scharnweber (TU Dresden) for the reference sample of hydroxyapatite. P.S. likes to thank Prof. Dr. Hannes Lichte for the possibility to perform TEM measurements at the Triebenberg Laboratory (TU Dresden). ’ REFERENCES (1) Currey, J. D. Bones: Structure and Mechanics; Princeton University Press: Princeton, NJ, 2002.

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