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m apatite
Materials Science & Engineering C 104 (2019) 109966
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HRTEM study of individual bone apatite nanocrystals reveals symmetry reduction with respect to P63/m apatite
T
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Viktória Kovács Kisa, , Zsolt Czigánya, Zsolt Dallosa,b, Dávid Nagyb, István Dódonyb a b
Centre for Energy Research, Hungarian Academy of Sciences, H-1121 Budapest, Hungary Department of Mineralogy, Eötvös Loránd University, H-1119 Budapest, Hungary
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone apatite Nanocrystals Atomic structure HRTEM Biomineralization
In this study we present the first crystal structure model for bone apatite based on the analysis of individual nanocrystals by high resolution transmission electron microscopy (HRTEM). Crystallographic image processing of the obtained HRTEM images from different projections indicates symmetry reduction with respect to P63/m stoichiometric apatites and the presence of threefold symmetry along the c axis. Based on HRTEM observations and the measured Ca/P = 2 ratio we propose a structural model with phosphate-to-carbonate substitution and O vacancies localized along c axis, which explains the observed loss of 63 screw axis parallel, and the shift of mirror plane perpendicular to the c axis. Also, the presence of non-equivalent (010) surfaces has been proven. These results on the atomic structure of bone apatite nanocrystals contribute to the understanding of their biochemically controlled nucleation processes.
1. Introduction While synthesis and characterization of calcium phosphate biomaterials for biomedical applications relies on their similar composition to bone and teeth mineral apatite [1,2], the atomic structure and nucleation processes of the biological calcium phosphates themselves are poorly known. In situ experiments modelling biomimetic calcium phosphate precipitation [3] support the formation of an amorphous precursor phase (aCP), in agreement with analytical results [4–9]. High resolution structural studies of mineralized tissues elucidate macroscopic properties, e.g. the aCP regions in biological apatite (bioapatite), play an important role in mechanical properties and plastic behaviour, like in case of rodent tooth enamel [9] or the continuously remodeling trabecular bone [10]. On the other hand, our knowledge on the atomic structure of bone apatite, the ca. 2 × 25 × 30 nm sized [11] crystalline building blocks of bone, which form an organic-inorganic nanocomposite with collagen to make up the hierarchical architecture [12,13], is very scarce. The apatite crystal structure, determined by Náray-Szabó [14] (Ca5(PO4)F, P63/m), is built up by chains of Ca-oxide polyhedra parallel to c axis. The chains are interconnected by PO4 tetrahedra forming channels of hexagonal cross section. Two subsequent P containing atomic layers are related by inversion which results the 63 screw axis along c. The mirror plane (m) is located at the P containing layer at
⁎
z = ± 0.25. At these heights the channels may host larger anions like OH−, Cl−, F−, CO32−, which can be released or exchanged quite easily. The ordering, iso- and altervalent substitutions in channel and tetrahedral positions together with cationic solid solution series result a number of structural derivates of the prototype P63/m structure [15]. In the apatite group, CO32− can substitute OH− in the channel sites along c axis (A-type) and PO43− in the tetrahedral sites (B-type). According to spectroscopic and X-ray studies, in apatites of biological origin both types of substitutions occur simultaneously, however the degree of CO32− substitution and the ratio of A- to B-type carbonate may vary from enamel to dentin, bone or renal stone apatite [16,17]. For instance, bone apatite practically lacks OH− and CO32− incorporates into the apatite structure [18,19]. Charge neutrality is maintained by coupled substitution of monovalent cations (Na+, K+) and/or vacancies [20,21]. These data on bone apatite rely on investigations of bulk bone material, because the extremely small crystallite size and nanocomposite nature with collagen precludes conventional structure refinement from diffraction data. However, in the case of a special bioapatite, conodont albid tissue, single crystal X-ray diffraction was successfully applied for crystal structure refinement [22]. Transmission electron microscopy is a powerful tool for studying structures on the nanoscale. The atomic structure of individual tooth enamel apatite, which have much less organic content (< 5%), has been successfully investigated by HRTEM [23,24] and nanoscale
Corresponding author. E-mail address: [email protected] (V.K. Kis).
https://doi.org/10.1016/j.msec.2019.109966 Received 27 February 2019; Received in revised form 25 June 2019; Accepted 9 July 2019 Available online 09 July 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 104 (2019) 109966
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electron diffraction techniques [25]. The latter provided evidence for the non-centrosymmetry of the enamel apatite nanocrystals. Furthermore, powder neutron diffraction results allowed specifying the location of H ions in enamel forming hydroxylapatite [26]. However, no HRTEM based atomic level structural information has been published on bone apatite nanocrystals up to now. In this work we have investigated individual nanocrystals of bone apatite of geological origin by HRTEM and applied crystallographic image processing to extract structural information from HRTEM images. This method allows to analyze amplitude and phase relations present in HRTEM images and to improve signal-to-noise ratio of the experimental image by applying the best fitting 2D crystallographic symmetry [27]. Crystallographic image processing is usually applied when the nanostructured nature of the material do not allow the use of other quantitative diffraction techniques. The background of this technique is detailed by Hovmöller [27].
Fig. 1. [10−2] HRTEM image of a bone apatite nanocrystal. The right part is the original image, the middle part was filtered using Fourier maxima and the left part is the simulated HRTEM image (thickness 101 Å, defocus 200 Å, angle of divergence 0.3 mrad, defocus spread 40 Å, crystal tilt 7 mrad at 133° azimuthal angle). The inset is the Fourier transform. The structure model shows the position of the calcium (blue) and phosphorus (orange) atoms, other atoms are omitted for clarity. Pair of arrows on the original image indicate positive and negative (010) terminations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2. Results and discussion 2.1. Chemical composition According to the TEM-EDS data (Table 1), high degree of fluoridization is observed, what is in good agreement with the geological age of the bone sample [20,22]. Fluoridization, i.e. the OH−/CO32− to F− substitution, happens through the hexagonal channels in the apatite structure [2,28]. The Ca/P atomic ratio is close to 2, which is significantly higher than in stoichiometric apatite (1.67). Iron and sulphur were also detected, which can be related to pyritization process of geological environment of the studied bone sample [29]. Based on the measured Ca/P ≈ 2 ratio, ca. 0.5 carbonate ion per formula unit is inferred and the Ca10(PO4)5(CO3)(F,X) composition is proposed, which is in agreement with the measured B-type carbonate substitution in another paleontological bioapatite [22]. This, together with the detected Na and Mg, and trace amounts of Sr is in agreement with the metabolical origin of the apatite nanocrystals. To maintain charge neutrality, we propose a simultaneous O2− substitution in channel sites, leading to the final formula (Ca, Fe, Na, Mg, Sr)10[(P, Si)O4]5[SO4, CO3](F,Cl,O).
P sites, which implies enhanced contrast difference. So, we conclude that the contrast difference marked by arrows on Fig. 1. reveals differences between the surface chemistry of (0k0) and (0k0) faces of the bone apatite nanocrystal. To investigate how the observed polarity along in bone apatite nanocrystal manifests in crystal structure, [010] zone HRTEM image was analyzed (Fig. 2a). The lattice parameters refined using CRISP (a0 = 9.37 Å, c0 = 6.89 Å) are in good agreement with our (Fig. S2) and published [30] X-ray powder diffraction (XRD) data. After correction for contrast transfer function, the measured amplitudes and phases served as basis of the symmetry analysis. The main points of the symmetry analysis are the following. (1) The presence of l = odd reflections contradict 63 screw axis. Pendellösung plots show that in case of P63/m fluorapatite the amplitude of l = odd reflections along [001]* axis, forbidden by the 63 screw axis, remains zero up to at least 80 nm of crystal thickness (Fig. S3). Thus, the measured non-zero amplitude does not arise from multiple scattering but should have implication for the crystal structure. (2) Amplitude and phase relations support plane group pm with mirror plane perpendicular to crystallographic [001] axis (Fig. 2b). The residuals after imposing pm(m⊥y) symmetry were significantly lower (residual amplitude and phase, RA = 4.4%, and phiR = 5.4°) than in case of imposing pmg symmetry (RA = 8%, and phiR = 22°), which should be the plane group for [010] zone projection of P63/m apatite (Fig. 2c). These observations imply that instead of 63 screw axis, a simple rotation axis (6- or 3-fold) is present in bone apatite. To check the symmetry along the crystallographic c axis, [001] zone HRTEM image was analyzed (Fig. 3a and b). While amplitude residuals are practically the same for p3 and p6 symmetry (RA = 33%), for phase residuals significant difference has been detected (18° and 49.4° after imposing p3 and p6 symmetry, respectively). By comparing the symmetry imposed image (Fig. 3c) and the calculated HRTEM image of P63/m apatite (Fig. 3d) contrast difference is evident. This observation supports that the symmetry of the experimental image (Fig. 3a, b and c) is different from that of P63/m apatite (Fig. 3d). Thus, we may conclude that the studied bone apatite nanocrystal has 3-fold rotation axis along crystallographic c axis, which is in agreement with the observed the pm (m⊥y) symmetry of [010] zone image, and also with the results in Fig. 1.
2.2. HRTEM of individual bone apatite nanocrystals HRTEM images of individual bone apatite nanocrystals have been − taken from several zone axes. Viewed along [102] direction the HRTEM image (Fig. 1) reveals contrast difference between the (010) and the
−
(010) facets of the nanocrystal. According to the Pendellösung plots calculated for the [102] projection and its misoriented counterpart (7 mrad in the 133° azimuth values deduced based on the Fourier transform on Fig. 1) (Fig. S1) the amplitude ratios do not change significantly up to ca. 120 Å thickness, which is larger than the estimated thickness of the measured nanocrystals (also see Ref. [11]). The simulated HRTEM image for the experimental conditions shows contrasts with satisfactory match to the experimental HRTEM image (Fig. 1). According to the structure model inserted in Fig. 1, the apatite structure in [102] projection can be interpreted as three consecutive slabs ABB, two of which (denoted as B) have Ca/P ratio 2:1 and the third one (denoted as A) has Ca/P ratio 1:1. A and B slabs correspond to different contrast fringes on the simulated HRTEM image. Carbonate substitution decreases overall atomic mass of Table 1 Chemical composition of the studied bone sample measured by TEM-EDS (10 measurements on different aggregates are averaged).
at.% StdDev
O
F
Na
Mg
Si
P
S
Cl
Ca
Fe
59.1 2.59
3.3 1.09
0.3 0.17
0.1 0.04
0.2 0.15
12.2 1.02
0.2 0.05
0.1 0.01
24.1 2.95
0.4 0.37
2
Materials Science & Engineering C 104 (2019) 109966
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Fig. 2. (a) [100] HRTEM image of bone apatite nanocrystal with its Fourier transform inserted in the lower left corner. Fourier components forbidden by P63/m space group symmetry are encircled. (b) Enlarged symmetry imposed image, mirror planes (m) are marked. The inset is the structure model of P63/m apatite for 2 × 2 unit cells. (c) Calculated [100] HRTEM image of P63/m F-apatite with 2 Å resolution. The 2D symmetry elements, mirror plane (m) and glide plane (g), are marked. The inset is the structure model of P63/m apatite for 2 × 2 unit cells. Calcium (orange) and phosphorus (blue) atoms are shown, other atoms are omitted for clarity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
plane is located in the P sheets (Fig. 2c), the latter implying symmetrical Ca1O layers sandwiching each P sheet. The arrangement of the CO3 triangle in the PO4 tetrahedron site in synthetic carbonated apatites has been discussed in a number of publications. Based on neutron diffraction measurements it was proposed that CO3 substituting a PO4 tetrahedron lies on the mirror plane [32], however another study concluded that the CO3 occupy rather the mirror symmetry related faces of the PO4 site [33]. An X-ray diffraction study specifies a third configuration, namely CO3 occupying adjacent faces of a PO4 tetrahedron that are parallel to the c axis [34]. All these structure refinements were done in P63/m space group, and in his leading opinion, Leventouri [35] underlines the indirect nature of these results and the lack of a convincing model for B-type carbonate substitution. Different approach has been used by Marisa et al. [36] who, based on total scattering and pair distribution function analysis, concluded that CO3 incorporation coupled with random displacement of PO4 tetrahedra results in P1 structure. Our HRTEM results on individual bone apatite nanocrystals definitely indicate the reduction of the P63/m space group, and implies trigonal symmetry, however, the relatively high amplitude and phase residuals after imposing p3 symmetry in [001] projection raise the possibility of an even lower, p1 symmetry of this projection.
2.3. Proposed structure model for bone apatite Crystallographic analysis of HRTEM and electron diffraction data on tooth enamel apatites revealed systematic appearance of l = odd reflections along [00l]* which was related to disordered OH− ions in the channels [31]. In another study, the presence of the 63 screw axis was supported and the P63 space group proposed [25]. On bone apatite, however, there have no crystal structural data been reported. Based on our observations, namely (1) Ca/P = 2, (2) 3-fold symmetry along c axis and (3) l = odd reflections along [00l]*, we propose the following structural model for the studied bone apatite nanocrystals (Fig. 4). We are assuming that (CO32−) substitution is not completely random, but localized to one of the two P-layers, e.g. in Ca2P(C) layer at z = 0.75 as shown in Fig. 4a. This arrangement is described by 2/3 and 1/3 occupancies for P and C respectively, in P positions at z = 0.75 (Table S1). In this case, according to the planar triangle geometry of carbonate, one of the 12 oxygen position necessary for three PO4 tetrahedra at z = 0.75 will be vacant. If the vacancy is localized in one of the oxygen layers out of the z = 0.75 plane, either at z = 0.57 or at z = 0.93 (Ca1O(v) at z = 0.93 in Fig. 4a and e), the 63 screw axis will be reduced to a threefold rotation axis (Fig. 4e), which is in agreement with the experimental [001] HRTEM image (Fig. 3b). As Ca1O and Ca1O(v) are no longer equivalent, this model explains the loss of glide plain in [100] HRTEM image, and also the preservation of the mirror plane, which is the consequence of the alternation of Ca1O and Ca1O(v) layers. Correspondingly, in the HRTEM image of bone apatite nanocrystal, mirror plane is located between the P sheets (at the actual resolution, see Fig. 2b), while in case of P63/m apatite mirror
2.4. Implications for biomineralization Our findings are based on the analysis of bone nanocrystals of paleontological origin, which elemental composition and crystal chemistry may depend on the conditions and geological location in nature.
Fig. 3. (a) [001] HRTEM image of bone apatite nanocrystal with the Fourier transform of the encircled area in the lower right corner. (b) Lattice averaged image using amplitudes and phases extracted from the Fourier transform in (a). (c) Symmetry imposed image using the obtained best fit p3 (RA = 33%, PhiR = 18°). (d) Calculated HRTEM image for the P63/m apatite (using experimental parameters of HRTEM in the (a) image). The differences in the contrast between P63/m apatite (d) and bone apatite (b and c) are evident, so the symmetry of the measured bone apatite nanocrystal is trigonal. 3
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Fig. 4. Proposed structural model for carbonated F-apatite to interpret HRTEM observations on bone apatite nanocrystals, 2 × 2 unit cells. Representation of atoms: blue – Ca (dark and light blue are Ca1 and Ca2 positions, respectively), orange – P, black – C, red – O, pale red – O vacancies, green – F. (a) [100] zone projection. (b)–(e) atomic layers at different z positions, [001] zone projections. (b) Ca2P layer at z = 0.25 height, (c) Ca1O layer is between z = 0.07 and 0.43, (d) Ca2P(C) layer at z = 0.75, one C atom substitutes randomly in the P positions, C and P occupancies are 1/3 and 2/3, respectively. (e) Ca1O(v) layer between z = 0.57 and 0.93, O occupancy at z = 0.93 is 2/3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
propose a structure model for B-type carbonate substitution with O vacancies localized along c axis. Moreover, experimental evidence of (0k0) faces with different surface chemistry is given. The knowledge of the atomic structure of biogenic apatite nanocrystals promotes understanding their morphogenesis and designing new biomimetic material.
Previous crystal structure investigation of paleontological bioapatite proved the nanostructured nature of the apatite-organic composite material [22]. Present results, besides symmetry reduction, indicate polarity along [010] direction of bone apatite which implies chemically distinct (0k0) surfaces. Such surfaces in synthetic hydroxylapatite nanocrystals were reported by Ospina et al. [37] and, based on an earlier first principles study [38], it was related to different water adsorption capacities of the two types of surfaces. Bone apatite nanocrystals, which are platy on (010) [39], have relatively large (0k0) surfaces, which according to our results, are either Ca or P rich. Ca release in exchange for two hydrogens, which proved to be energetically very favourable [38], promotes surface hydratation. Therefore we propose that the relative orientation of (0k0) facets has prominent role in resorption processes and also in the adhesion of apatite nanocrystals to the collagen fibers. Up to now there is no consensus regarding the nature of the binding between the nanocrystals and the collagen [13,40]. Our results highlight the function of surface chemistry and indicate that biochemically controlled crystallization produces highly functionalized nanocrystals. Further comparative HRTEM investigations of both paleontological and recent bone nanocrystals will provide additional structural details of bone apatite crystallization.
4. Materials and methods The material used in this study is a fragment of a 85 Ma old turtle plate sample from Iharkút, Hungary [29,41]. For (HR)TEM study, the sample was gently crushed under ethanol in an agate mortar, and a drop of the resulting suspension was deposited onto a lacey carbon covered Cu grid (Ted Pella). EDS analyses were done with a Philips CM20 TEM operating at 200 keV and equipped with a Bruker XFlash 5030T SDD detector. HRTEM analysis was carried out using a JEOL 3010 TEM with LaB6 cathode, equipped with GIF (0.17 nm point-topoint resolution, UHR pole piece). The experimental setting was the following: 300 keV, angle of divergence 0.3 mrad, defocus spread 40 Å. For data processing Digital Micrograph (Gatan Tridiem) and CRISP (Calidris) softwares, for simulations JEMS (Stadelmann) and Cerius2 softwares were used. Actual defocus values which were used for HRTEM simulations were determined directly from the experimental images using CRISP (Calidris).
3. Conclusion
Acknowledgements
In this study the atomic structure of individual bone apatite nanocrystals was studied by direct imaging down to 2 Å using HRTEM for the first time. By crystallographic image processing we show the loss of sixfold symmetry along the c axis. To explain our observations, we
This work was supported by the National Research, Development and Innovation Fund Office, Hungary under the Project “Investigation 4
Materials Science & Engineering C 104 (2019) 109966
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of the nanostructural background of functionality in case of biogenic and biocompatible mineral apatite”, Grant number K-125100. V.K.K. is indebted to the János Bolyai Fellowship of the Hungarian Academy of Sciences.
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
HRTEM study of individual bone apatite nanocrystals reveals symmetry reduction with respect to P63/m apatite
T
⁎
Viktória Kovács Kisa, , Zsolt Czigánya, Zsolt Dallosa,b, Dávid Nagyb, István Dódonyb a b
Centre for Energy Research, Hungarian Academy of Sciences, H-1121 Budapest, Hungary Department of Mineralogy, Eötvös Loránd University, H-1119 Budapest, Hungary
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone apatite Nanocrystals Atomic structure HRTEM Biomineralization
In this study we present the first crystal structure model for bone apatite based on the analysis of individual nanocrystals by high resolution transmission electron microscopy (HRTEM). Crystallographic image processing of the obtained HRTEM images from different projections indicates symmetry reduction with respect to P63/m stoichiometric apatites and the presence of threefold symmetry along the c axis. Based on HRTEM observations and the measured Ca/P = 2 ratio we propose a structural model with phosphate-to-carbonate substitution and O vacancies localized along c axis, which explains the observed loss of 63 screw axis parallel, and the shift of mirror plane perpendicular to the c axis. Also, the presence of non-equivalent (010) surfaces has been proven. These results on the atomic structure of bone apatite nanocrystals contribute to the understanding of their biochemically controlled nucleation processes.
1. Introduction While synthesis and characterization of calcium phosphate biomaterials for biomedical applications relies on their similar composition to bone and teeth mineral apatite [1,2], the atomic structure and nucleation processes of the biological calcium phosphates themselves are poorly known. In situ experiments modelling biomimetic calcium phosphate precipitation [3] support the formation of an amorphous precursor phase (aCP), in agreement with analytical results [4–9]. High resolution structural studies of mineralized tissues elucidate macroscopic properties, e.g. the aCP regions in biological apatite (bioapatite), play an important role in mechanical properties and plastic behaviour, like in case of rodent tooth enamel [9] or the continuously remodeling trabecular bone [10]. On the other hand, our knowledge on the atomic structure of bone apatite, the ca. 2 × 25 × 30 nm sized [11] crystalline building blocks of bone, which form an organic-inorganic nanocomposite with collagen to make up the hierarchical architecture [12,13], is very scarce. The apatite crystal structure, determined by Náray-Szabó [14] (Ca5(PO4)F, P63/m), is built up by chains of Ca-oxide polyhedra parallel to c axis. The chains are interconnected by PO4 tetrahedra forming channels of hexagonal cross section. Two subsequent P containing atomic layers are related by inversion which results the 63 screw axis along c. The mirror plane (m) is located at the P containing layer at
⁎
z = ± 0.25. At these heights the channels may host larger anions like OH−, Cl−, F−, CO32−, which can be released or exchanged quite easily. The ordering, iso- and altervalent substitutions in channel and tetrahedral positions together with cationic solid solution series result a number of structural derivates of the prototype P63/m structure [15]. In the apatite group, CO32− can substitute OH− in the channel sites along c axis (A-type) and PO43− in the tetrahedral sites (B-type). According to spectroscopic and X-ray studies, in apatites of biological origin both types of substitutions occur simultaneously, however the degree of CO32− substitution and the ratio of A- to B-type carbonate may vary from enamel to dentin, bone or renal stone apatite [16,17]. For instance, bone apatite practically lacks OH− and CO32− incorporates into the apatite structure [18,19]. Charge neutrality is maintained by coupled substitution of monovalent cations (Na+, K+) and/or vacancies [20,21]. These data on bone apatite rely on investigations of bulk bone material, because the extremely small crystallite size and nanocomposite nature with collagen precludes conventional structure refinement from diffraction data. However, in the case of a special bioapatite, conodont albid tissue, single crystal X-ray diffraction was successfully applied for crystal structure refinement [22]. Transmission electron microscopy is a powerful tool for studying structures on the nanoscale. The atomic structure of individual tooth enamel apatite, which have much less organic content (< 5%), has been successfully investigated by HRTEM [23,24] and nanoscale
Corresponding author. E-mail address: [email protected] (V.K. Kis).
https://doi.org/10.1016/j.msec.2019.109966 Received 27 February 2019; Received in revised form 25 June 2019; Accepted 9 July 2019 Available online 09 July 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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electron diffraction techniques [25]. The latter provided evidence for the non-centrosymmetry of the enamel apatite nanocrystals. Furthermore, powder neutron diffraction results allowed specifying the location of H ions in enamel forming hydroxylapatite [26]. However, no HRTEM based atomic level structural information has been published on bone apatite nanocrystals up to now. In this work we have investigated individual nanocrystals of bone apatite of geological origin by HRTEM and applied crystallographic image processing to extract structural information from HRTEM images. This method allows to analyze amplitude and phase relations present in HRTEM images and to improve signal-to-noise ratio of the experimental image by applying the best fitting 2D crystallographic symmetry [27]. Crystallographic image processing is usually applied when the nanostructured nature of the material do not allow the use of other quantitative diffraction techniques. The background of this technique is detailed by Hovmöller [27].
Fig. 1. [10−2] HRTEM image of a bone apatite nanocrystal. The right part is the original image, the middle part was filtered using Fourier maxima and the left part is the simulated HRTEM image (thickness 101 Å, defocus 200 Å, angle of divergence 0.3 mrad, defocus spread 40 Å, crystal tilt 7 mrad at 133° azimuthal angle). The inset is the Fourier transform. The structure model shows the position of the calcium (blue) and phosphorus (orange) atoms, other atoms are omitted for clarity. Pair of arrows on the original image indicate positive and negative (010) terminations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2. Results and discussion 2.1. Chemical composition According to the TEM-EDS data (Table 1), high degree of fluoridization is observed, what is in good agreement with the geological age of the bone sample [20,22]. Fluoridization, i.e. the OH−/CO32− to F− substitution, happens through the hexagonal channels in the apatite structure [2,28]. The Ca/P atomic ratio is close to 2, which is significantly higher than in stoichiometric apatite (1.67). Iron and sulphur were also detected, which can be related to pyritization process of geological environment of the studied bone sample [29]. Based on the measured Ca/P ≈ 2 ratio, ca. 0.5 carbonate ion per formula unit is inferred and the Ca10(PO4)5(CO3)(F,X) composition is proposed, which is in agreement with the measured B-type carbonate substitution in another paleontological bioapatite [22]. This, together with the detected Na and Mg, and trace amounts of Sr is in agreement with the metabolical origin of the apatite nanocrystals. To maintain charge neutrality, we propose a simultaneous O2− substitution in channel sites, leading to the final formula (Ca, Fe, Na, Mg, Sr)10[(P, Si)O4]5[SO4, CO3](F,Cl,O).
P sites, which implies enhanced contrast difference. So, we conclude that the contrast difference marked by arrows on Fig. 1. reveals differences between the surface chemistry of (0k0) and (0k0) faces of the bone apatite nanocrystal. To investigate how the observed polarity along in bone apatite nanocrystal manifests in crystal structure, [010] zone HRTEM image was analyzed (Fig. 2a). The lattice parameters refined using CRISP (a0 = 9.37 Å, c0 = 6.89 Å) are in good agreement with our (Fig. S2) and published [30] X-ray powder diffraction (XRD) data. After correction for contrast transfer function, the measured amplitudes and phases served as basis of the symmetry analysis. The main points of the symmetry analysis are the following. (1) The presence of l = odd reflections contradict 63 screw axis. Pendellösung plots show that in case of P63/m fluorapatite the amplitude of l = odd reflections along [001]* axis, forbidden by the 63 screw axis, remains zero up to at least 80 nm of crystal thickness (Fig. S3). Thus, the measured non-zero amplitude does not arise from multiple scattering but should have implication for the crystal structure. (2) Amplitude and phase relations support plane group pm with mirror plane perpendicular to crystallographic [001] axis (Fig. 2b). The residuals after imposing pm(m⊥y) symmetry were significantly lower (residual amplitude and phase, RA = 4.4%, and phiR = 5.4°) than in case of imposing pmg symmetry (RA = 8%, and phiR = 22°), which should be the plane group for [010] zone projection of P63/m apatite (Fig. 2c). These observations imply that instead of 63 screw axis, a simple rotation axis (6- or 3-fold) is present in bone apatite. To check the symmetry along the crystallographic c axis, [001] zone HRTEM image was analyzed (Fig. 3a and b). While amplitude residuals are practically the same for p3 and p6 symmetry (RA = 33%), for phase residuals significant difference has been detected (18° and 49.4° after imposing p3 and p6 symmetry, respectively). By comparing the symmetry imposed image (Fig. 3c) and the calculated HRTEM image of P63/m apatite (Fig. 3d) contrast difference is evident. This observation supports that the symmetry of the experimental image (Fig. 3a, b and c) is different from that of P63/m apatite (Fig. 3d). Thus, we may conclude that the studied bone apatite nanocrystal has 3-fold rotation axis along crystallographic c axis, which is in agreement with the observed the pm (m⊥y) symmetry of [010] zone image, and also with the results in Fig. 1.
2.2. HRTEM of individual bone apatite nanocrystals HRTEM images of individual bone apatite nanocrystals have been − taken from several zone axes. Viewed along [102] direction the HRTEM image (Fig. 1) reveals contrast difference between the (010) and the
−
(010) facets of the nanocrystal. According to the Pendellösung plots calculated for the [102] projection and its misoriented counterpart (7 mrad in the 133° azimuth values deduced based on the Fourier transform on Fig. 1) (Fig. S1) the amplitude ratios do not change significantly up to ca. 120 Å thickness, which is larger than the estimated thickness of the measured nanocrystals (also see Ref. [11]). The simulated HRTEM image for the experimental conditions shows contrasts with satisfactory match to the experimental HRTEM image (Fig. 1). According to the structure model inserted in Fig. 1, the apatite structure in [102] projection can be interpreted as three consecutive slabs ABB, two of which (denoted as B) have Ca/P ratio 2:1 and the third one (denoted as A) has Ca/P ratio 1:1. A and B slabs correspond to different contrast fringes on the simulated HRTEM image. Carbonate substitution decreases overall atomic mass of Table 1 Chemical composition of the studied bone sample measured by TEM-EDS (10 measurements on different aggregates are averaged).
at.% StdDev
O
F
Na
Mg
Si
P
S
Cl
Ca
Fe
59.1 2.59
3.3 1.09
0.3 0.17
0.1 0.04
0.2 0.15
12.2 1.02
0.2 0.05
0.1 0.01
24.1 2.95
0.4 0.37
2
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Fig. 2. (a) [100] HRTEM image of bone apatite nanocrystal with its Fourier transform inserted in the lower left corner. Fourier components forbidden by P63/m space group symmetry are encircled. (b) Enlarged symmetry imposed image, mirror planes (m) are marked. The inset is the structure model of P63/m apatite for 2 × 2 unit cells. (c) Calculated [100] HRTEM image of P63/m F-apatite with 2 Å resolution. The 2D symmetry elements, mirror plane (m) and glide plane (g), are marked. The inset is the structure model of P63/m apatite for 2 × 2 unit cells. Calcium (orange) and phosphorus (blue) atoms are shown, other atoms are omitted for clarity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
plane is located in the P sheets (Fig. 2c), the latter implying symmetrical Ca1O layers sandwiching each P sheet. The arrangement of the CO3 triangle in the PO4 tetrahedron site in synthetic carbonated apatites has been discussed in a number of publications. Based on neutron diffraction measurements it was proposed that CO3 substituting a PO4 tetrahedron lies on the mirror plane [32], however another study concluded that the CO3 occupy rather the mirror symmetry related faces of the PO4 site [33]. An X-ray diffraction study specifies a third configuration, namely CO3 occupying adjacent faces of a PO4 tetrahedron that are parallel to the c axis [34]. All these structure refinements were done in P63/m space group, and in his leading opinion, Leventouri [35] underlines the indirect nature of these results and the lack of a convincing model for B-type carbonate substitution. Different approach has been used by Marisa et al. [36] who, based on total scattering and pair distribution function analysis, concluded that CO3 incorporation coupled with random displacement of PO4 tetrahedra results in P1 structure. Our HRTEM results on individual bone apatite nanocrystals definitely indicate the reduction of the P63/m space group, and implies trigonal symmetry, however, the relatively high amplitude and phase residuals after imposing p3 symmetry in [001] projection raise the possibility of an even lower, p1 symmetry of this projection.
2.3. Proposed structure model for bone apatite Crystallographic analysis of HRTEM and electron diffraction data on tooth enamel apatites revealed systematic appearance of l = odd reflections along [00l]* which was related to disordered OH− ions in the channels [31]. In another study, the presence of the 63 screw axis was supported and the P63 space group proposed [25]. On bone apatite, however, there have no crystal structural data been reported. Based on our observations, namely (1) Ca/P = 2, (2) 3-fold symmetry along c axis and (3) l = odd reflections along [00l]*, we propose the following structural model for the studied bone apatite nanocrystals (Fig. 4). We are assuming that (CO32−) substitution is not completely random, but localized to one of the two P-layers, e.g. in Ca2P(C) layer at z = 0.75 as shown in Fig. 4a. This arrangement is described by 2/3 and 1/3 occupancies for P and C respectively, in P positions at z = 0.75 (Table S1). In this case, according to the planar triangle geometry of carbonate, one of the 12 oxygen position necessary for three PO4 tetrahedra at z = 0.75 will be vacant. If the vacancy is localized in one of the oxygen layers out of the z = 0.75 plane, either at z = 0.57 or at z = 0.93 (Ca1O(v) at z = 0.93 in Fig. 4a and e), the 63 screw axis will be reduced to a threefold rotation axis (Fig. 4e), which is in agreement with the experimental [001] HRTEM image (Fig. 3b). As Ca1O and Ca1O(v) are no longer equivalent, this model explains the loss of glide plain in [100] HRTEM image, and also the preservation of the mirror plane, which is the consequence of the alternation of Ca1O and Ca1O(v) layers. Correspondingly, in the HRTEM image of bone apatite nanocrystal, mirror plane is located between the P sheets (at the actual resolution, see Fig. 2b), while in case of P63/m apatite mirror
2.4. Implications for biomineralization Our findings are based on the analysis of bone nanocrystals of paleontological origin, which elemental composition and crystal chemistry may depend on the conditions and geological location in nature.
Fig. 3. (a) [001] HRTEM image of bone apatite nanocrystal with the Fourier transform of the encircled area in the lower right corner. (b) Lattice averaged image using amplitudes and phases extracted from the Fourier transform in (a). (c) Symmetry imposed image using the obtained best fit p3 (RA = 33%, PhiR = 18°). (d) Calculated HRTEM image for the P63/m apatite (using experimental parameters of HRTEM in the (a) image). The differences in the contrast between P63/m apatite (d) and bone apatite (b and c) are evident, so the symmetry of the measured bone apatite nanocrystal is trigonal. 3
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Fig. 4. Proposed structural model for carbonated F-apatite to interpret HRTEM observations on bone apatite nanocrystals, 2 × 2 unit cells. Representation of atoms: blue – Ca (dark and light blue are Ca1 and Ca2 positions, respectively), orange – P, black – C, red – O, pale red – O vacancies, green – F. (a) [100] zone projection. (b)–(e) atomic layers at different z positions, [001] zone projections. (b) Ca2P layer at z = 0.25 height, (c) Ca1O layer is between z = 0.07 and 0.43, (d) Ca2P(C) layer at z = 0.75, one C atom substitutes randomly in the P positions, C and P occupancies are 1/3 and 2/3, respectively. (e) Ca1O(v) layer between z = 0.57 and 0.93, O occupancy at z = 0.93 is 2/3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
propose a structure model for B-type carbonate substitution with O vacancies localized along c axis. Moreover, experimental evidence of (0k0) faces with different surface chemistry is given. The knowledge of the atomic structure of biogenic apatite nanocrystals promotes understanding their morphogenesis and designing new biomimetic material.
Previous crystal structure investigation of paleontological bioapatite proved the nanostructured nature of the apatite-organic composite material [22]. Present results, besides symmetry reduction, indicate polarity along [010] direction of bone apatite which implies chemically distinct (0k0) surfaces. Such surfaces in synthetic hydroxylapatite nanocrystals were reported by Ospina et al. [37] and, based on an earlier first principles study [38], it was related to different water adsorption capacities of the two types of surfaces. Bone apatite nanocrystals, which are platy on (010) [39], have relatively large (0k0) surfaces, which according to our results, are either Ca or P rich. Ca release in exchange for two hydrogens, which proved to be energetically very favourable [38], promotes surface hydratation. Therefore we propose that the relative orientation of (0k0) facets has prominent role in resorption processes and also in the adhesion of apatite nanocrystals to the collagen fibers. Up to now there is no consensus regarding the nature of the binding between the nanocrystals and the collagen [13,40]. Our results highlight the function of surface chemistry and indicate that biochemically controlled crystallization produces highly functionalized nanocrystals. Further comparative HRTEM investigations of both paleontological and recent bone nanocrystals will provide additional structural details of bone apatite crystallization.
4. Materials and methods The material used in this study is a fragment of a 85 Ma old turtle plate sample from Iharkút, Hungary [29,41]. For (HR)TEM study, the sample was gently crushed under ethanol in an agate mortar, and a drop of the resulting suspension was deposited onto a lacey carbon covered Cu grid (Ted Pella). EDS analyses were done with a Philips CM20 TEM operating at 200 keV and equipped with a Bruker XFlash 5030T SDD detector. HRTEM analysis was carried out using a JEOL 3010 TEM with LaB6 cathode, equipped with GIF (0.17 nm point-topoint resolution, UHR pole piece). The experimental setting was the following: 300 keV, angle of divergence 0.3 mrad, defocus spread 40 Å. For data processing Digital Micrograph (Gatan Tridiem) and CRISP (Calidris) softwares, for simulations JEMS (Stadelmann) and Cerius2 softwares were used. Actual defocus values which were used for HRTEM simulations were determined directly from the experimental images using CRISP (Calidris).
3. Conclusion
Acknowledgements
In this study the atomic structure of individual bone apatite nanocrystals was studied by direct imaging down to 2 Å using HRTEM for the first time. By crystallographic image processing we show the loss of sixfold symmetry along the c axis. To explain our observations, we
This work was supported by the National Research, Development and Innovation Fund Office, Hungary under the Project “Investigation 4
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of the nanostructural background of functionality in case of biogenic and biocompatible mineral apatite”, Grant number K-125100. V.K.K. is indebted to the János Bolyai Fellowship of the Hungarian Academy of Sciences.
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