An NMR Study of Biomimetic Fluorapatite – Gelatine Mesocrystals

The mesocrystal system fluoroapatite—gelatine grown by double-diffusion is characterized by hierarchical composite structure on a mesoscale. In the present work we apply solid state NMR to characterize its structure on the molecular level and provide a link between the structural organisation on the mesoscale and atomistic computer simulations. Thus, we find that the individual nanocrystals are composed of crystalline fluorapatite domains covered by a thin boundary apatite-like layer. The latter is in contact with an amorphous layer, which fills the interparticle space. The amorphous layer is comprised of the organic matrix impregnated by isolated phosphate groups, Ca3F motifs and water molecules. Our NMR data provide clear evidence for the existence of precursor complexes in the gelatine phase, which were not involved in the formation of apatite crystals, proving hence theoretical predictions on the structural pre-treatment of gelatine by ion impregnation. The interfacial interactions, which may be described as the glue holding the composite materials together, comprise hydrogen bond interactions with the apatite PO43− groups. The reported results are in a good agreement with molecular dynamics simulations, which address the mechanisms of a growth control by collagen fibers, and with experimental observations of an amorphous cover layer in biominerals.

Scientific RepoRts | 5:15797 | DOi: 10.1038/srep15797 mesocrystallization 11,12 . Since biominerals combine complex morphology and unique functional properties as the result of evolution-optimized processes, comprehensive insights into the biomineralization mechanisms open up promising approaches for bioinspired and biomimetic materials design 11,13 . In addition, adopting the bioinspired approach enables not only production of novel functional materials, but also better understanding of biomeralization processes through controlled synthesis and the in situ characterization methods. Recently, using a combination of in-situ methods the biomimetic nucleation of calcium phosphate, which is a major inorganic constituent of the hard tissues of animals, has been shown to occur through a unifying crystallization process 14 . Practical application of biomimetic strategies has been recently demonstrated for remineralization of human dentine 15 and in vitro repair of a seashell 16 . It was suggested that organic macromolecules play important roles in the repair process and can control the mineralization reactions on several levels, including the formation of prenucleation clusters. In a recent review, Cölfen described the polymers that are useful for this purpose and the experimental conditions suitable for directing a crystallization reaction in the desired direction 17 .
However, a classical epitaxy paradigm, e.g., an epitaxial match between the structural organic matrix and the mineral, has been disproven by a HRTEM study of synthetic aragonite and of Haliotis laevigata gastropod nacre 18,19 . The existence of the amorphous layer around aragonite platelets in nacre is explained by the exclusion of impurities and expulsion of macromolecules throughout the crystallization, which prevents further crystallization. It has been suggested that this layer could also provide better adhesion and mechanical performance of the hybrid material. In our previous studies of synthetic hydroxy-and fluorapatite mesocrystals a disordered (amorphous) layer, which covers the apatite crystalline domains and is coordinated to water and the organic matrix, has been identified by solid-state NMR 20,21 . Thus, a disordered layer between aligned single crystalline nanoparticles embedded in an amorphous organic phase therefore seems to be an intriguing hypothesis for biominerals, which demonstrate mesocrystalline properties. Despite its relevance, little is known about the constituents and the molecular-level structure of this composite material. Therefore in the present work we continue our efforts on the structural elucidation of biomimetic fluoroapatite-gelatine nanocomposites.
The mesocrystal system fluoroapatite-gelatine grown by double-diffusion is a fair illustration of self-organized morphogenesis and a hierarchical structure on the mesoscale [22][23][24][25][26][27][28] . Growth of the nanocomposites starts with an elongated hexagonal prismatic seed (5-20 μ m in length, Fig. 1a), followed by a self-similar branching of dumbbell states (Fig. 1b,c), which finally leads to the development of closed spheres. As proven by electron holography the fractal growth mechanism, which results in the dumbbell-like nanocomposite aggregates, is controlled by the intrinsic electrical dipole fields induced by an aligned polar biomolecule 26,29 . The inner architecture of the young seed, as indicated by high-resolution TEM, is built by a parallel stacking of elongated subunits oriented with their long [001] axes parallel to the seed. X-ray diffraction has revealed the presence of crystalline fluorapatite within the hexagonal prismatic seed, while a tilted mounting of the later crystal generations during the fractal morphogenesis has been observed 30 . On the mesoscopic scale, the so-called mosaic arrangement is suggested, where the periodic mineral domains of a nanocomposite subunit grow around a central protein triple-helix 27 . It has been shown that the domains do not perfectly match, giving rise to a healing layer at the outer borders to match the hexagonal pattern. As evidenced by electron microscopy, the gelatine molecules are surrounded by assemblies of fluorapatite nanoparticles arranged in a honeycomb-like network providing homogeneous intergrowth of the inorganic and organic components. Using solid state NMR we studied the interfacial mineral-organic structure in large spherical aggregates up to 100 μ m in size, which represent the final growth state of the fractal-grown fluorapatite-gelatin composites 20,21 . As it is obvious that the fundamental principles of mineral growth, passivation and stabilization are already included in the early stages of composite growth, in the present work we focus on the study of the hexagonal prismatic seeds and dumbbells, thus the initial states of growth of fluorapatite/gelatin composites.

Experimental section
Samples. Details of synthesis of fluorine-gelatine nanocomposites using the double diffusion technique have been published previously 24,30,31 . A composite aggregate extracted at the early growth stage was ground, washed 3 times for 20 min in distilled water at 40 °C, then centrifuged and finally dried at 40 °C in order to remove a fraction of gelatine, which is only physisorbed on the aggregates' surface and not integrated in the composite. X-Ray diffraction. X-ray powder data were collected in transmission mode using a Huber G670 Image Plate Camera, Cu K α1 radiation (λ = 1.540598 Å) and germanium (111) monochromator. Lattice constants a and c of apatite were calculated by least-squares refinements using LaB 6 (cubic, a = 4.15692 Å) as internal standard. The measurements were performed before and after heating to 250 °C to study the effect of release of hydroxyl groups and water from the crystal structure.
SEM. The morphology was studied by scanning electron microscopy (SEM). SEM investigations were performed by means of a Philips ESEM Quanta 200FEGI system operated in high-vacuum mode at an acceleration voltage of 25 kV (FEI, Eindhoven, NL). For investigation under high vacuum, the samples were coated by a thin gold layer (for 30 seconds), and secondary electron images were recorded.

NMR.
For NMR experiments the samples were filled into zirconia rotors and closed tightly. 1 H and 19 F NMR experiments were carried out on a (11.7 T) Bruker Avance III 500 spectrometer operating at resonance frequencies of 500.1 MHz for 1 H and 470 MHz for 19 F using a 2.5 mm HFX-MAS probehead with a spinning frequency of 30 kHz. For 1 H MAS NMR the 90°-pulse duration of 3 μ s, a recycle delay of 5 s and high-power 19 F TPPM (50 kHz) decoupling were applied. The 19 F MAS NMR experiments were acquired with a 90°-pulse duration of 4.5 μ s, a recycle delay of 40 s, a number of repetitions of 64 and TPPM (50 kHz) decoupling on protons. For the 19 F{ 1 H} cross polarization (CP) measurements contact times in the range of 100 μ s to 1500 μ s and a recycle delay of 5 s were used. The two-dimensional 19 F-1 H HETCOR experiment was performed at 30 kHz MAS and a contact time of 500 μ s. A recycle delay of 5 s and 512 scans per t 1 time increment were applied. A total of 32 t 1 slices with a 50 μ s time increment in the indirect dimension were acquired. 31 P NMR spectra were acquired on a (7 T) Bruker Avance 300 spectrometer operating at resonance frequencies of 300.1 MHz for 1 H and 121.5 MHz for 31 P employing a BL4 HX 4 mm MAS probehead. The 31 P MAS NMR experiments were performed at spinning frequencies of 10 and 14 kHz with either 1 H or 19 F high-power TPPM decoupling, a 90°-pulse duration of 4.5 μ s, a recycle delay of 30 s and 16 repetitions. For 31 P{ 1 H} CP measurements a recycle delay of 5 s and a contact time varied from 100 μ s to 4 ms were used. Two-dimensional 31 P-1 H HETCOR experiments were performed with a spinning speed of 10 kHz and contact times of 1.5 ms and 3 ms. A recycle delay of 5 s, 32 scans per t 1 time increment and a 75.53 μ s time increment in the indirect dimension were used. 31 P{ 19 F} CP MAS (10 kHz) spectra were measured at a contact time in the range of 0.1 ms to 4 ms, 16 repetitions and a recycle delay of 50 s. The 33 P-19 F HETCOR spectrum (10 kHz MAS) was acquired at a contact time of 1.5 ms, 64 scans per t 1 time increment and a 200 μ s time increment in the indirect dimension. All 1 H chemical shifts were referenced to tetramethylsilane (TMS) at 0 ppm using poly(vinylidene fluoride) as an external reference (δ 1 H = 2.9 ppm); 19 F chemical shifts were referenced relative to CFCl 3 at 0 ppm using PTFE as an external reference (δ 19 F = − 122 ppm); powdered ammonium dihydrogen phosphate was used to reference the 31 P spectra at 0.72 ppm relative to 85% phosphoric acid. All spectra were fitted using Dmfit 32 .

Results
SEM. SEM images of the fluorapatite-gelatine composite demonstrate that the sample under study represents a combination of prismatic seeds (Fig. 1a) and dumbbell-like aggregates (Fig. 1b,c). According to the previously published results, this corresponds to earlier stages of morphogenesis, when an initially formed elongated hexagonal prismatic seed starts to grow and split, demonstrating fractal morphogenesis.

19
F NMR. In the 19 F MAS NMR spectrum of nanocomposite recorded by direct polarization (DP), five spectral components are identified (Fig. 2a), whose fit parameters are summarized in Table 1. The chemical shift values δ iso 19 F for four lines are close to those found for the spherical aggregates reported in ref. 21. In the present study a minor high-field shoulder centred at ca. δ iso 19 F = − 86 ppm has been found in addition. Distribution of the component intensities essentially changes in the spectrum obtained by the cross-polarization (CP) showing different 1 H-19 F proximities of corresponding species (Fig. 2b). The peak at δ 19 F = − 103 ppm is no longer visible in Fig. 2b, which indicates lack of spatial correlation to the protons and proves its assignment to pure crystalline fluorapatite 21,33 . Figure 2c shows the different CP build-up behaviour for the spectral components determined in the 19 F{ 1 H} CP MAS spectrum. In contrast to other signals, the 19 F peak at − 104 ppm continues growing up to t CP = 1 ms. Such behavior is characteristic of the crystalline apatite structure. Moreover, its chemical shift, which is very close to the pure crystalline fluorapatite, can indicate that the local apatite structure around this fluorine site is preserved 34 . Faster CP build-up of the remaining 19 F signals, which reach a maximum at t CP = 0.1 ms (Fig. 2c), points to the close proximity to protons, perhaps, indicating covalent binding. In our previous work we attributed the 19 F signal at ca. − 109 ppm to an amorphous layer in close contact to the organic phase, while the signal around − 96 ppm has been tentatively assigned to partial fluorination of gelatine during the formation of the nanocomposite 21 . Here we apply two-dimensional 19 F-1 H heteronuclear correlation (HETCOR) spectroscopy in order to analyse cross-correlation between the 1 H and 19 F signals and to get detailed information on the spatial association between 1 H and 19 F sites.
The 19 F-1 H HETCOR spectrum (Fig. 3) demonstrates strong correlation to the peaks at δ 1 H = 8.2 ppm and δ 1 H = 5.8 ppm, which are attributed to certain molecular fragments of gelatin and water, respectively. The low-field shift (to higher ppm-values) relative to bulk water (δ 1 H = 4.8 ppm) is explained by strong hydrogen bonding of the water molecules to the organic molecule or orthophosphate groups. The  crystalline fluorapatite peak at δ 19 F = − 103 ppm also appears in the HETCOR spectrum, indicating the minor cross-signals at δ 1 H = 2.0 ppm and δ 1 H = 11.4 ppm. The former arises from isolated water molecules in the apatite channels and has been observed in the final growth stage 21 . The latter is assigned to HPO 4 2− groups, which are frequently observed in the spectra of hydroxyapatite 35 and other relevant systems, such as biological hard calcified tissues 36,37 and biomimetic calcium phosphates 21 . The signals in the range of δ 19 F = − 86 to − 100 ppm cross-correlate to the 1 H peaks related to water and the organic matrix. 31 P NMR. The 31 P MAS spectra are presented in Fig. 4. In contrast to the 31 P MAS NMR spectrum of the spherical aggregates 21 , which represents a single fluorapatite 31 P signal at δ 31 P = 2.8 ppm, in the early stage sample, in addition, a second component at δ 31 P = 2.1 ppm appears (Fig. 4a,b). Applying  heteronuclear high-power decoupling enables linewidth narrowing due to suppression of the corresponding heteronuclear dipolar interactions. The effect of two different decoupling channels on the 31 P spectrum ( 1 H-and 19 F-decoupled, Fig. 4a,b, respectively) is evidently indicating that the 31 P site which gives a signal at 2.1 ppm is surrounded by a large proton bath. Indeed, this component is essentially amplified in the 31 P spectrum recorded by cross-polarisation from protons (Fig. 4c) and nearly disappears when measured by cross-polarisation from 19 F (Fig. 4d). The 31 P{ 19 F} CP spectrum (Fig. 4d) proves that the peak at 2.8 ppm arises from a pure fluorapatite structure 21,23,24 . In addition, a very low fraction of a non-apatite origin is visible in the 31 P{ 1 H} CP spectrum at 5.1 ppm. We can thus conclude that besides the dominating contribution from fluorapatite at δ 31 P = 2.8 ppm in the 31 P spectra, there are small fractions of phosphorus-containing species spatially associated to protons. Two-dimensional HETCOR experiments, where 31 P chemical shifts are correlated to either 19 F or 1 H in the indirect dimension enable us to get deeper insight into the structural environment around phosphorous atoms. The 31 P-19 F HETCOR spectrum (Fig. 5a) demonstrates a single asymmetric cross-peak with a maximum intensity at δ 31 P = 2.8 ppm and δ 19 F = − 103 ppm. This is a strong indication of the pure fluorapatite structure and support for the assignment given above. In the 19 F sum projection a low-field shoulder at ca. − 104 ppm is observed, which hints to the presence of another component not well separated in the 2D spectrum. Actually, its 19 F line position and correlation to the peak at δ 31 P = 2.8 ppm points to its apatite origin. It is noteworthy that no other 31 P-19 F cross-peak is observed, demonstrating that the other peaks listed in Table 1 are of non-mineral origin at this growth stage.
In contrast, 31 P-1 H HETCOR shows a more complex spectrum (Fig. 5b). First, the cross-peaks associated with the apatite structure (δ 31 P = 2.8 ppm) are visible at δ 1 H = 5.8 ppm and δ 1 H = 11.4 ppm (dashed vertical line in Fig. 5b). They prove the presence of surface water and hydrogenated phosphate groups, respectively. Similar correlation signals have been observed in synthetic biomimetic nanocomposites 20 , as well as in biominerals such as joint mineralized cartilage 38 , animal bone 39,40 , and rat dentine 41 . Further, two strong cross-signals appear at δ 31 P/δ 1 H of 2.1 ppm/8.4 ppm and 5.0 ppm/8.4 ppm (Fig. 5b). According to the literature these 31 P signals can be assigned to orthophosphate groups located at the mineral surface 21,42,43 or to the side products of a double-diffusion reaction such as NaH 2 PO 4 H 2 O, K 2 HPO 4 · 3H 2 O and NaNH 4 HPO 4 · 4H 2 O, whose 31 P signals are known to appear at 2.3, 2.1 and 5.1 ppm, respectively 44 . The signal at δ 31 P = 5.1 ppm has been observed in the one-dimensional 31 P{ 1 H} CP NMR spectrum (Fig. 4c) and attributed to non-apatite species. In either case, it is evident that these signals can arise from phosphate groups of non-apatite origin interacting with organic matrix. Note, that athough a low fraction of apatite-channel water has been found in 19 F-1 H HETCOR NMR (Fig. 3), the correlation signals at δ 1 H = 0 ppm and δ 1 H = 1.5 ppm, characteristic of apatite OHgroups and apatite channel water, respectively, are absent here.

Discussion
Fitting and quantification of the 19 F MAS NMR spectrum obtained by direct polarization provides information about the relative fraction of each species resolved in the spectra. This allows us to get insight into the structure of the internal and interfacial regions of the early stage nanocomposite aggregates.
As suggested in our previous work for the final growth stage the mineral component is composed of crystalline apatite-like core surrounded by a disordered layer with first motifs of the apatite structure, which interacts with organic matrix 21 . In the earlier stage sample, the dominating contribution (72%) from all fluorine-containing species comes from crystalline fluorapatite (FAp) (Table 1, Fig. 2a). The 2D data (Fig. 3) show that FAp is associated weakly to HPO 4 2groups and isolated water molecules. The latter can be entrapped as structural defects and substitute fluorine ions in the apatite channels. The cross-peaks between FAp and gelatine/water molecules, which would prove the scenario of the periodic FAp domains' growth around a protein triple-helix 27,31 , could be hidden beneath the large peaks due to their low fraction.
In the following, the origin of the 19 F peak at − 104 ppm is discussed. It represents 10% of all 1D 19 F spectral intensity. Correlating this signal to the 31 P dimension in the 2D spectrum (− 104 ppm/2.8 ppm) proves its apatite character with the preserved local arrangement. However, long-range ordering is lacking as demonstrated by the line broadening as compared to crystalline fluorapatite (Fig. 2, Table 1). We anticipate that this species is located in a boundary layer, which "heals" the mismatch of the periodic domains to get a perfect hexagonal shape of the nanoparticles. Indeed, calculation of the intensity ratio for hexagonal geometry yields the thickness of such a layer of fractions of a nm, corresponding to the size of an orthophosphate molecule. Finding an identifiable species, which we will call a boundary layer in the following, is a clue to the hexagonal arrangement of primary apatite nanocrystals observed previously using HRTEM 31 . In general, the interfacial interactions, which may be described as the glue holding the composite materials together, may comprise nonspecific (e.g. van der Waals force driven alkane-surface interactions) and specific adsorption (hydrogen bonding, coulombic ion pairing) or covalent interfacial bridging 45 . Our 2D data (Fig. 3) show that the boundary layer interacts with gelatine and water. As the CP-build up data (green line in Fig. 2c) show that the corresponding fluorine-to-proton distances are too long for the formation of covalent bonds, we suggest that specific adsorption including hydrogen bonding to the apatite PO 4 3− groups is the major interfacial interaction in the present organic-inorganic nanocomposite. The 31 P-1 H cross-peak at 2.8 ppm/5.8 ppm (Fig. 5b) can prove the presence of such hydrogen bonded complexes as PO 4 3− ··· H(w) or PO 4 3− ··· H(org). The appearance of the highly ordered hydrogen bond interactions between citrate and phosphate ions in spherulites of fluorapatite has been recently demonstrated using solid-state NMR 46 . Furthermore, two phosphorous environments different from apatite are found in the 31 P NMR spectra (Fig. 4) at δ 31 P = 2.1 and 5.1 ppm, whose fractions are relatively small compared to the whole phosphorous content. From the 2D HETCOR data it is evident that they are not related compositionally and spatially to FAp domains and the boundary layer (Fig. 5a), but related to the proton-containing organic phase (Fig. 5b). Moreover, the 1 H cross-correlation signal at δ 1 H = 8 ppm proves association of the negatively charged phosphate groups to the positively charged amino groups. Thus, we suggest the existence of another spatially distinguishable phase, which contains organic matrix with incorporated fluorine ions, water molecules and isolated non-apatite phosphate groups. Indeed, our 2D HETCOR NMR measurements (Figs 3 and 5b) provide clear evidence for this claim. The absence of the corresponding peaks in the 31 P-19 F HETCOR experiment (Fig. 5a) is explained by a low concentration of the constituents, longer distances between phosphorous and fluorine atoms in this phase or their high mobility.
In the previous simulation study on this system 47 , which addressed the mechanisms of a growth control by collagen fibers, formation of Ca 3 F motifs has been postulated. According to ref. 47 such a motif is represented by a triangle formed by calcium ions, with a fluoride ion located in the center of the triangle. The Ca···F and Ca···Ca distances range from 2.0 to 2.5 Å and 3.6 to 4.7 Å, respectively, and the angles of the triangles vary by up to 20° from the ideal value of 120°. These are incorporated into the triple helix during the embryonic stage of ion association. Such motifs represent the nucleation seeds for the formation of the oriented apatite crystal structure along the triple helix molecule. We assume here that the 19 F signal observed at − 108 ppm arise from Ca 3 F motifs, expected to occur during aggregation in/at the collagen triple helix. Indeed, its chemical shift is very close to that typical for Ca···F interactions, such as in crystalline CaF 2 32 , although no indication for the presence of CaF 2 has been found in the corresponding X-ray powder diffraction data (Fig. S1, Supplementary information). It is worth noting that the presence of Ca 3 F and the absence of the corresponding CaF 2 X-ray reflections have been also found for the spherical aggregates 21 . In contrast, the formation of calcium fluoride has been found in our previous Scientific RepoRts | 5:15797 | DOi: 10.1038/srep15797 studies of fluorapatite-gelatine composites, when gelatine concentration exceeded 15 wt.%. 30 , as well as more recently, when citric acid was used as a crystal modifier for the preparation of spherical hierarchical structures of FAp 48 . We emphasize that the present work is the experimental proof of the existence of Ca 3 F and PO 4 3− precursor complexes in the gelatine phase, which were not involved in the formation of apatite crystals. Alternatively, the scenario that they were expelled from the single crystalline fluorapatite domain throughout crystallization as suggested in Refs. 18,19 could also be feasible.
Our experimental observations are in a good agreement with previously published molecular dynamics simulations, which provide characteristic binding positions and preferable associations of the involved ions with a gelatine molecule 47,49 . It has been predicted that the phosphate ions are preferentially bound outside the polypeptide strands by forming hydrogen bonds with hydroxyproline side-groups and amino groups. This gives rise to bending of the polypeptide backbone and thus leads to the observed fractal morphogenesis. The same tendency has been predicted for HPO 4 2− ion aggregation. Our NMR data provide clear evidence for the existence of such phosphate groups bound to organic molecules demonstrating the corresponding NMR signals. The 19 F signals in the range of δ 19 F = − 86 to − 100 ppm are attributed to the amorphous organic-related phase, which contains fluorine ions hydrogen bonded to water or/and gelatine molecules.
Finally, when all species have been identified, it is worth considering the locations and interactions of water in the nanocomposite. In this work it has been found that (i) the isolated water molecules are incorporated as structural defects in the crystalline FAp domains; (ii) bound water is present on the mineral surface, and, finally, (iii) mobile water is included in the amorphous organic layer, where it strongly interacts with the gelatine molecules, non-apatite phosphate groups and Ca 3 F motives. Heating of the nanocomposite to 250 °C resulted in weight loss of 0.7 wt.% (Fig. S2, Supplementary information), demonstrating that most water molecules are integrated in the nanocomposite structure. The XRD pattern after heating (Fig. S1) proves no effect on the FAp crystalline structure.
Based on the experimental observations in the present work, a scheme of co-existing nano-structured mineral and a superficial organic layer in the fluorapatite-gelatine nanocomposite is presented in Fig. 6. The mineral part is represented by a mosaic arrangement of the periodic fluorapatite domains (grey circles) surrounded by the boundary layer (black circles), which matches the domains to the hexagonal shape and interacts with water and the organic layer impregnated with Ca 3 F and HPO 4 2− /PO 4 3− species.

Conclusions
A fluorapatite-gelatine nanocomposite at the early growth stage represented by hexagonal prismatic seeds and dumbbell-like aggregates with first sights of fractal splitting has been studied. We have applied solid-state NMR spectroscopy to investigate the structure in this nanocomposite on a molecular level and provide a link between electron microscopy data, electron holography and atomistic computer simulations. Based on our results we propose a model, which demonstrates the presence of a thin boundary layer around the crystallites and of pre-nucleation clusters in the amorphous organic-containing phase. The phosphate groups in the boundary layer are involved in the hydrogen bond interactions with the organic and water molecules in the amorphous layer. Our results are in a good agreement with the theoretical predictions on the structural pre-treatment of gelatine by ion impregnation and experimental observations of an amorphous cover layer in biominerals.