Magnesium incorporation into hydroxyapatite.
ABSTRACT The incorporation of Mg in hydroxyapatite (HA) was investigated using multinuclear solid state NMR, X-ray absorption spectroscopy (XAS) and computational modeling. High magnetic field (43)Ca solid state NMR and Ca K-edge XAS studies of a ∼10% Mg-substituted HA were performed, bringing direct evidence of the preferential substitution of Mg in the Ca(II) position. (1)H and (31)P solid state NMR show that the environment of the anions is disordered in this substituted apatite phase. Both Density Functional Theory (DFT) and interatomic potential computations of Mg-substituted HA structures are in agreement with these observations. Indeed, the incorporation of low levels of Mg in the Ca(II) site is found to be more favourable energetically, and the NMR parameters calculated from these optimized structures are consistent with the experimental data. Calculations provide direct insight in the structural modifications of the HA lattice, due to the strong contraction of the M⋯O distances around Mg. Finally, extensive interatomic potential calculations also suggest that a local clustering of Mg within the HA lattice is likely to occur. Such structural characterizations of Mg environments in apatites will favour a better understanding of the biological role of this cation.
- SourceAvailable from: Luigi Giusto Spagnoli[Show abstract] [Hide abstract]
ABSTRACT: Mammary microcalcifications have a crucial role in breast cancer detection, but the processes that induce their formation are unknown. Moreover, recent studies have described the occurrence of the epithelial-mesenchymal transition (EMT) in breast cancer, but its role is not defined. In this study, we hypothesized that epithelial cells acquire mesenchymal characteristics and become capable of producing breast microcalcifications. Breast sample biopsies with microcalcifications underwent energy dispersive X-ray microanalysis to better define the elemental composition of the microcalcifications. Breast sample biopsies without microcalcifications were used as controls. The ultrastructural phenotype of breast cells near to calcium deposits was also investigated to verify EMT in relation to breast microcalcifications. The mesenchymal phenotype and tissue mineralization were studied by immunostaining for vimentin, BMP-2, beta2-microglobulin, beta-catenin and osteopontin (OPN). The complex formation of calcium hydroxyapatite was strictly associated with malignant lesions whereas calcium-oxalate is mainly reported in benign lesions. Notably, for the first time, we observed the presence of magnesium-substituted hydroxyapatite, which was frequently noted in breast cancer but never found in benign lesions. Morphological studies demonstrated that epithelial cells with mesenchymal characteristics were significantly increased in infiltrating carcinomas with microcalcifications and in cells with ultrastructural features typical of osteoblasts close to microcalcifications. These data were strengthened by the rate of cells expressing molecules typically involved during physiological mineralization (i.e. BMP-2, OPN) that discriminated infiltrating carcinomas with microcalcifications from those without microcalcifications. We found significant differences in the elemental composition of calcifications between benign and malignant lesions. Observations of cell phenotype led us to hypothesize that under specific stimuli, mammary cells, which despite retaining a minimal epithelial phenotype (confirmed by cytokeratin expression), may acquire some mesenchymal characteristics transforming themselves into cells with an osteoblast-like phenotype, and are able to contribute to the production of breast microcalcifications.BMC Cancer 04/2014; 14(1):286. · 3.32 Impact Factor
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ABSTRACT: The aim of this study was to compare the sol-gel and hydrothermally synthesized pure and magnesium doped hydroxyapatite. Calcium nitrate tetrahydrate, magnesium nitrate hexahydrate and ammonium dihydrogen phosphate were used as a precursuor for Ca, Mg and P in both methods. The synthesized powder was sintered at 800ºC. The samples were characterized by Fourier Transform Infrared Spectroscopy for functional group analysis, X-Ray Diffraction for crystalinity and phase purity analysis, Scanning Electron Microscopy coupled with Energy dispersive X-Ray for morphological analysis and (Ca+Mg)/P ratio. Simulated Body Fluid is prepared by using chlorides, carbonates, oxides and sulphates of alkali metals at 37ºC. The bioresorbability of sol-gel and hydrothermally synthesized materials has been examined in vitro by immersing in simulated body fluid and measuring the variation of pH. The results obtained shows that Hydroxyapatite synthesized by both methods are bioresorbable. However, the hydrothermally synthesized pure and Magnesium doped hydroxyapatite revealed a higher resorbablity. The FTIR shows the influence of Mg and XRD results confirmed presence of Mg in the lattice structure of HAP. The crystal size is found to be in the range of 10nm-40nm. Scanning Electron Micrographs confirms the influence of Mg on the morphology and particle size. I. INTRODUCTION Bone tissue has a high regenerative capacity for self repair on damage. This self repairing process often fails when the bone defects are too large or the natural healing capacity is insufficient. The recent strategies for repairing and reconstructing these large bone defects use bone grafting materials such as autografts, allografts and xenografts. However, limitations in those approaches viz., the limited availability, possibility of disease transmission and poor biocompatibility have all increased the necessity of artificial synthetic bone implants incorporating ceramics like calcium phosphate materials on its surface  . The biomineral phase, which is one or more type of calcium phosphates, comprises 65-70% of bone, water accounts for 5-8% and the organic phase, like collagen, accounts for the remainder. The collagen, which gives the bone its elastic resistance, acts as a matrix for the deposition and growth of minerals. Among the calcium phosphate salts, hydroxyapatite is the thermodynamically most stable crystalline phase of calcium phosphate in body fluid  . Hydroxyapatite [HAP] constitutes the main mineral components of bone and teeth. Stoichiometric HAP has the composition Ca 10 (PO 4) 3 (OH) 2 with Ca/P ratio of 1.67  . Thus it is commonly used as a filler to replace amputated bone or coating to promote bone ingrowth over the prosthetic implants. Recently hip replacement and dental implants are coated with HAP. It has been reported that this may promote osseointegration. Porous HAP implants are used for localized local drug delivery in the affected areas of bones  . Due to its growing importance and applications numerous techniques have been reported for the synthesis of HAP  . Important techniques being used are solid state reaction  , co-precipitation  , emulsion(IJIRSE) International Journal of Innovative Research in Science & Engineering. 08/2014; 2(1):2347-3207.
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ABSTRACT: Nano rods of magnesium-doped hydroxyapatite, Ca10−x Mg x (PO4)6(OH)2(Mg x –HA, x = 0–1.0), were successfully synthesized through cetyltrimethyl ammonium bromide assisted hydrothermal synthesis method. X-ray diffraction, infrared spectroscopy, thermogravimetric analysis and transmission electron microscopy, provided experimental evidences about the effects of Mg-doping on the phase assemblage, crystallite size, morphology, specific surface area of Mg-doped hydroxyapatite nanopowders. The replacement Ca2+ ions by smaller Mg2+ ones caused lattice shrinkage and lattice strains that enhanced the solubility and the in vitro bio-mineralisation activity upon immersing sintered samples in simulated body fluid. The severity of these structural changes rose with increasing Mg-doping and enable tailoring the in vitro biological activity enabling selecting the most suitable material for bone grafts and tissue engineering applications.Journal of Biomimetics Biomaterials and Tissue Engineering 10/2013; 3(5):570-580(11).
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Author(s): Laurencin, Danielle, Almora-Barrios, Neyvis, de Leeuw, Nora
H., Gervais, Christel, Bonhomme, Christian, Mauri, Francesco,
Chrzanowski, Wojciech, Knowles, Jonathan C., Newport, Robert J.,
Wong, Alan, Gan, Zhehong and Smith, Mark E.
Article Title: Magnesium incorporation into hydroxyapatite
Year of publication: 2011
Link to published article :
Publisher statement: NOTICE: this is the author’s version of a work
that was accepted for publication in Biomaterials. Changes resulting
from the publishing process, such as peer review, editing, corrections,
structural formatting, and other quality control mechanisms may not
be reflected in this document. Changes may have been made to this
work since it was submitted for publication. A definitive version was
subsequently published in Biomaterials, 32(7), pp. 1826-1837. Doi:
Magnesium incorporation into hydroxyapatite
Danielle Laurencin,a,1,* Neyvis Almora-Barrios,b Nora H. de Leeuw,b,* Christel Gervais,c Christian
Bonhomme,c Francesco Mauri,d Wojciech Chrzanowski,e,2 Jonathan C. Knowles,e,f Robert J.
Newport,g Alan Wong,a,3 Zhehong Gan,h and Mark E. Smitha
a Department of Physics, University of Warwick, Coventry, CV4 7AL, UK
b University College London, Department of Chemistry, 20 Gordon Street, London, WC1H 0AY, UK.
c UPMC Univ Paris 06, UMR CNRS 7574, Laboratoire de Chimie de la Matière Condensée de Paris,
F-75005 Paris, France
d UPMC Univ Paris 06, UMR CNRS 7590 Institut de Minéralogie et Physique des Milieux
Condensés, F-75015 Paris, France
e Eastman Dental Institute, University College London, 256 Gray’s Inn Road, London, WC1X 8LD,
f WCU Research Centre of Nanobiomedical Science, Dankook University, San#29, Anseo-dong,
Dongnam-gu, Cheonan-si, Chungnam, 330-714, South Korea
g School of Physical Sciences, Ingram Building, University of Kent, Canterbury, CT2 7NH, UK
h Center of Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory,
Tallahassee, Florida, FL32310, USA.
1 Present address : Institut Charles Gerhardt de Montpellier, UMR 5253, CNRS-UM2-ENSCM-UM1,
CC 1701, Place Eugène Bataillon, 34095 Montpellier cedex 5, France
2 Present address: The University of Sydney, The Faculty of Pharmacy, Pharmacy and Bank Building
Sydney, NSW, 2006.
3 Present address : CEA Saclay, IRAMIS/SCM/LSDRM, Gif-sur-Yvette 91191, France.
* Corresponding authors. Tel.: +33 4 67 14 38 02; Fax: +33 4 67 14 38 88. Email address:
The incorporation of Mg in hydroxyapatite (HA) was investigated using multinuclear solid state NMR,
X-ray absorption spectroscopy (XAS) and computational modeling. High magnetic field 43Ca solid
state NMR and Ca K-edge XAS of a ~10% Mg-substituted HA were performed, bringing direct
evidence of the preferential substitution of Mg in the Ca(II) position. 1H and 31P solid state NMR show
that the environment of the anions is disordered in this substituted apatite phase. Both Density
Functional Theory (DFT) and interatomic potential computations of Mg-substituted HA structures are
in agreement with these observations. Indeed, the incorporation of low levels of Mg in the Ca(II) site
is found to be more favourable energetically, and the NMR parameters calculated from these
optimized structures are consistent with the experimental data. Calculations provide direct insight in
the structural modifications of the HA lattice, due to the strong contraction of the M∙∙∙O distances
around Mg. Finally, extensive interatomic potential calculations also suggest that a local clustering of
Mg within the HA lattice is likely to occur.
Solid State NMR
X-ray absorption Spectroscopy
Density Functional Theory
Calcium hydroxyapatite is the main mineral component of bone tissue and teeth. Its
composition differs from that of synthetic hydroxyapatite Ca10(PO4)6(OH)2 (HA), due to the presence
of several ionic substitutions in the lattice, such as CO3
2, F , Mg2+ and Na+.[1-3] These minor species
not only alter the space group, morphology, stability, and mechanical properties of the HA structure,
but also play an important role in the biological responses of bone cells. For instance, carbonates have
a strong influence on the growth of apatite crystals, sodium plays a role in bone remodeling,
whereas fluoride prevents the development of dental caries. These ions are distributed
inhomogeneously throughout the tissue, and their concentration changes according to the age and
maturity of the mineral.[1, 6, 7]
Magnesium is known to be an important trace element in bone and teeth. Indeed, despite its
low concentration (generally between ~0.5 and 1.5 wt%), it plays a key role in bone metabolism, in
particular during the early stages of osteogenesis where it stimulates osteoblast proliferation, and its
depletion causes bone fragility and bone loss. Furthermore, relationships have been suggested
between the magnesium content in enamel and the development of dental caries.
Given the biological relevance of magnesium, many research teams have worked on the
preparation of apatite and calcium-phosphate implant materials containing low levels of Mg, which
has been shown to improve their bioactivity.[11, 12] Moreover, much work has been done on synthetic
magnesium-substituted apatites (Mg-HA), in order to try to elucidate the exact structural role of Mg in
bone. According to the literature, the replacement of calcium by magnesium in HA is limited. This is
related to the large size difference between Mg2+ and Ca2+ (~0.28 Å difference in radius according to
the Pauling scale), which leads to strong distortions of the HA lattice and reduces its crystallinity.
These changes have a direct impact on the properties of Mg-HA, compared to their non-substituted
analogues:[12-16] it notably increases their solubility and biodegradability in physiological fluids and
favours their thermal conversion into substituted β-tricalcium phosphate (β-Ca3 xMgx(PO4)2).
Additional studies have also shown that the co-substitution of other ions such as CO3
2− in the structure
may help counteract the destabilization of the apatite phase  and that, according to the synthetic
procedure or the age of the Mg-HA material, higher Mg concentrations at the surface of the HA
crystallites can be observed.[18, 19]
Numerous spectroscopic techniques have been used to learn more about the substitution of
magnesium in HA. Nevertheless, although X-ray diffraction, X-Ray Photoelectron
Spectroscopy, and cathodoluminescence spectroscopy clearly indicate that Mg enters the HA
lattice, several key structural characteristics remain unsolved, and little is known about how exactly
Mg-incorporation affects the bulk structure of apatites. In particular, the position of Mg in the HA
lattice is still an open question: does it occupy one or both of the two crystallographic calcium sites,
referred to as Ca(I) and Ca(II), which present different local environments, as depicted in Figures 1
and 2? Some authors state that Mg enters the Ca(II) site, whereas others the Ca(I) site.[15, 21, 22]
The main difficulty in answering this question in the case of Mg-HA arises from the absence of high
resolution spectroscopic data over an important range of composition, and the significant loss of
crystallinity of Mg-HA compounds above ~ 20% Mg. Thus, in contrast with Sr- or Ba-substituted
HA,[23, 24] Rietveld analyses based on X-ray powder patterns are not conclusive for Mg-HA, and
both the Ca(I) and the Ca(II) sites have been suggested to be the preferential site of
incorporation of Mg. Such discrepancies might be due to differences in synthetic procedures (which
could perhaps alter the final site for incorporated Mg), and/or to the low quality of the XRD patterns
used in the Rietveld refinements. Indeed, it is noteworthy that some of the refined distances are
surprising: for a sample with ~ 30% Mg, an average M∙∙∙O bond distance was found that was almost
identical to the non-substituted compound, which seems contradictory with the fact that average
Mg∙∙∙O distances are generally ~0.4 Å shorter than average Ca∙∙∙O distances and that there is a
contraction of the HA lattice. It thus appears necessary to find other analytical tools not only to
help elucidate the structural and biological role of Mg, but also help shed light on the changes in local
structure around the different atoms in the lattice.
Recently, alternative approaches have started to emerge to investigate in more depth the mode
of incorporation of substituting divalent cations in HA phases. In particular, it has been shown that
much information could be accessed experimentally using spectroscopic techniques which are
sensitive to the local structure around particular cations, such as X-ray absorption spectroscopy and
solid state NMR. For instance, the preferential incorporation of Zn2+ and Pb2+ into the Ca(II) site of
apatites has been demonstrated using Zn K-edge EXAFS and XANES, and 207Pb solid state
NMR, respectively. Furthermore, an increasing number of computational studies are reported
aimed at determining any energetic advantages of incorporating impurity ions into different sites of the
HA lattice, [27-30] and it has been shown that these can bring further insight into the structural
changes in substituted species, and help understand spectroscopic data.
In the case of Mg-substituted apatites, no experimental NMR or XAS data have been reported
so far, and the first computational studies were published very recently. Using Density Functional
Theory (DFT) calculations, Matsunaga et al. showed that the incorporation of Mg2+ is more favourable
in the Ca(II) than in the Ca(I) site, and that it induces strong changes in the position of the coordinated
oxygen atoms. However, no experimental evidence could be referred to to confirm the changes in the
local structure around the different ions in the structure: the experimental data available so far for Mg-
HA are insufficient (i) to validate the computational models developed for these systems, and (ii) to
show how Mg modifies the bulk and surface structure of apatite crystallites and thereby modifies the
biological responses. The purpose of this work was thus to investigate the structure of Mg-HA systems
at the molecular level, using techniques which are sensitive to the local environment of the atoms,
such as multinuclear solid state and X-ray absorption spectroscopy, and to discuss the experimental
and spectroscopic data in view of computational models of Mg-HA. The local structure around all the
ions in the structure, and notably magnesium and calcium, was analysed in order to try to derive sound
conclusions on the structure of these phases.
Materials and methods
Hydroxyapatite Ca10(PO4)6(OH)2 (HA) and magnesium-substituted hydroxyapatite
Ca10-xMgx(PO4)6(OH)2 (Mg10-HA) were prepared following a precipitation method adapted from the
literature. High purity calcium nitrate tetrahydrate (Ca(NO3)2.4H2O, Alfa Aesar), magnesium
nitrate hexahydrate (Mg(NO3)2.6H2O, Aldrich) and ammonium dihydrogen phosphate (NH4H2PO4,
Aldrich) were used as starting materials. All reactions were performed in freshly distilled water and
under an N2 atmosphere, in order to avoid the incorporation of carbonates in the HA lattice.
HA was prepared by heating an aqueous solution of calcium nitrate to 90°C ([Ca2+] = 0.216
mol.L–1, V = 100 mL, pH adjusted to 10.0 with a 1 mol.L–1 solution of NH4OH), and adding dropwise
under stirring an aqueous solution of ammonium dihydrogen phosphate (C = 0.130 mol.L–1, V = 100
mL, pH adjusted to 10.0 by addition of concentrated NH4OH). This leads to the progressive
precipitation of a white solid. The suspension was stirred at 90°C (temperature of the oil bath) for a
total time of 5 hours, during which small amounts of concentrated NH4OH were added, in order to
keep the pH above 9.0. The reaction medium was then left to cool at room temperature, and
centrifuged in order to separate the white precipitate (10000 rpm for 10 min). The solid was washed
four times with freshly distilled water, and then dried at 100°C under vacuum overnight (yield > 85%).
In the case of Mg10-HA, the same synthetic procedure was followed, but using a mixture of calcium
and magnesium nitrate, with 10.0% magnesium with respect to the total cationic concentration.
Elemental analyses of Mg10-HA were performed (i) by ICP-AES by the Service Central
d’Analyse of the CNRS (Vernaison, France), to determine the Ca, P and Mg contents, (ii) by ionic
chromatography on a Dionex 1000 ICS system to measure the Ca and Mg contents, after dissolution of
the samples in sulphuric acid.
SEM and EDXS (for Ca, P and Mg) studies were carried out on a Jeol 6100 Scanning Electron
Microscope coupled to an EDAX Genesis analytical system. TEM and EDXS analyses (for Ca, P and
Mg) were carried out on a Jeol 6100 Transmission Electron Microscope coupled to an EDAX Genesis
analytical system. X-ray diffraction patterns were collected with a Bruker D8 Advance diffractometer
using the CuKα radiation. Room temperature measurements were performed in the 2θ range between
10 and 70°. IR spectra were also recorded from KBr pellets on a Perkin Elmer GX FTIR spectrometer
between 4000 cm−1 and 400 cm−1. XRD, SEM and TEM data can be found in Figure S1 in the
supplementary materials (from hereon, “S” will refer to supplementary information).
Solid state NMR
1H and 31P NMR experiments were carried out at 7.1 T on a Varian Infinity Plus 300
spectrometer, using a Bruker 4 mm MAS probe. Single-pulse magic angle spinning 1H and 31P NMR
spectra were collected at a spinning rate of 10 kHz, using recycle delays of 10 s and 30 s respectively.
In the case of 31P, it was verified that the lineshape remains unaltered when increasing the recycle
delay to 500 s. 1H and 31P chemical shifts were referenced using TMS (Si(CH3)4, resonance at 0 ppm)
and NH4H2PO4 (resonance at 0.9 ppm), respectively.
Natural abundance 43Ca solid state NMR spectra were recorded at 18.8 T on a Varian Infinity
Plus 800 spectrometer using a Varian 4 mm rotor probe spinning at 8 kHz, and at 19.6 T on a Bruker
DRX 830 spectrometer using a 7 mm homebuilt probe spinning at 5 kHz. Recycle delays of 0.1 to
0.25 s were used, and 400000 to 1500000 transients were acquired, with experimental times ranging
from 28 to 48 h (it is noteworthy that changing the recycle delays did not affect the final lineshapes).
For the spectra recorded at 18.8 T, the rotor-assisted population transfer (RAPT) sequence (sets of
+X/−X 2.5 μs pulses with a ~ 15 kHz radiofrequency field strength),[32, 33] which had previously
been optimized for non-substituted hydroxyapatite, was applied for sensitivity enhancement prior
to a 90° pulse selective for the central transition. For the spectra recorded at 19.6 T, a 5 kHz chirp
sweep was applied for sensitivity enhancement at ~250 kHz below the central transition
frequency. 43Ca NMR chemical shifts were referenced using a 1 mol.L-1 aqueous solution of CaCl2 (at
0 ppm).[36, 37] The conclusions from the data recorded at 19.6 T were identical to those at 18.8 T.
Natural abundance 25Mg NMR experiments were carried out at 19.6 T on a Bruker DRX 830
spectrometer using a 7 mm homebuilt probe spinning at 5 kHz. 240000 transients were acquired, with
a recycle delay of 0.3 s. 25Mg NMR chemical shifts were referenced using an aqueous solution of
magnesium sulfate.[38, 39]
Ca K-edge EXAFS and XANES
EXAFS (Extended X-ray Absorption Fine Structure) and XANES (X-ray Absorption Near
Edge Structure) measurements were performed on the XAFS BL11.1 beamline at the Elettra
Sincrotrone, Trieste, Italy. Samples were ground to a fine powder, diluted in polyvinyl pyrrolidone
(average molecular wt 40,000, Sigma-Aldrich, “PVP40”), pressed into pellets, and run at room
temperature in transmission mode using ion chambers before and after the sample in order to measure
incident and transmitted X-ray intensity. Spectra were collected at the Ca K-edge at 4038 eV. The
Elettra ring energy was 2 GeV and the maximum ring current was 200 mA. The incident X-ray energy
on the sample was defined using a two-crystal Si(111) monochromator detuned (by ~50%) to reduce
higher energy harmonics. The instrument was evacuated to ~10
5 Pa in order to reduce X-ray losses
due to attenuation in the air. An energy resolution of ~0.3 eV at the Ca K-edge was achieved, and the
energy was calibrated using CaF2 as a calibrant placed between the transmission and a third ion
chamber. For the XANES spectra, the pre-edge (3900–4017 eV), edge (4017–4100 eV) and post-edge
(4100–4200 eV) regions were scanned in 5.0, 0.1 and 1.0 eV steps respectively, and dwell times per
point of 1.0, 2.0 and 1.0 s respectively. For the EXAFS spectra, the pre-edge (3900–4017 eV), edge
(4017–4100 eV) and post-edge (4100–4800 eV) regions were scanned in 5.0, 2.0 and 1.0 eV steps
respectively, and dwell times per point of 1.0, 1.0 and 2.0 s respectively.
Data reduction was performed using the standard software packages Athena,
Viper2006[41, 42] and EXCURV98. Typically, two XANES and three EXAFS data sets were
collected for each sample, which were respectively averaged and normalized using Athena. All
EXAFS spectra were background-subtracted using Viper2006, with a second order polynomial fitted
to the pre-edge region and a polynomial spline (going through 7 knots) fitted to the post-edge region to
describe the underlying atomic absorption. Conversion of energy to k space followed before k3
weighting of the data. The EXCURV98 code was then used to model the structure from the parameters
of the radial shells of atoms surrounding the central atom. Phase shifts were calculated by ab initio
curved wave theory methods in EXCURV98 for the central atom and for all backscattering elements
in the samples. Multiple scattering effects were not considered, since only the nearest coordination
shells are probed at these low energies. Such a simplification has already been employed for the study
of non-stoichiometric apatites and bioactive glasses and bone by K-edge EXAFS
Computational investigation of the site of incorporation of Mg
The distribution of Mg in the hydroxyapatite lattice was investigated by a combination of
electronic structure techniques and interatomic potential methods.[28-30, 47-50] It is noteworthy that
both approaches have already been used to investigate ionic substitutions in apatite phases. Although
interatomic potential methods are more efficient for dealing with structures containing a very large
number of atoms, it is important first to validate a given method by comparing the results with
electronic structure techniques.
The electronic structure calculations were performed using the SIESTA code , which
employs Density Functional Theory , norm-conserving pseudopotentials and linear combinations
of numerical atomic orbitals (LCAO) to calculate the total energy of the system. We have used the
Perdew-Burke-Ernzerhof (PBE)  generalized gradients approximation (GGA) for the exchange-
correlation functional, whereas pseudopotentials for all atoms were generated in the Troullier-Martins
manner . The basis sets used for all atoms were of the DZP type (double with polarization),
except for the oxygen and hydrogen in the hydroxy group, for which the basis sets were obtained from
the optimization of water at 0.2 GPa [55, 56]. The unit-cell was optimized using a cutoff energy of 250
Ry, sampling was taken at the point and a force tolerance of 0.01 eV/Å was used. The suitability of
cutoff energy, k points and the force tolerance were evaluated by monitoring the convergence of the
total energy with respect to the various parameters and validated against the structural properties of
The larger-scale systems were modeled using the General Utility Lattice Program (GULP)
[57-59], which uses interatomic potential simulation techniques based on the Born model of solids
, which assumes that the ions in the crystal interact via long-range electrostatic forces and short-
range forces, including both the repulsions and Van der Waals attractions between neighbouring
electron charge clouds. The interatomic potential model for hydroxyapatite was taken from the work
of de Leeuw , which includes electronic polarisability via the shell model of Dick and Overhauser
: each polarisable ion (in this case oxygen), is represented by a core and a massless shell,
connected by a spring. The polarisability of the oxygen ions is then determined by the spring constant
and the charges of the core and shell. When necessary, angle-dependent forces are included to allow
directionality of bonding, for example in the covalent phosphate anion. In order to make geometry
predictions, the lattice energies are minimized with respect to the structural parameters, until the forces
acting on the ions are all less than 0.001 eV Å
1. All structures reported are the result of constant
pressure energy minimizations, with an external pressure set to zero, where not only the ionic
positions but also the cell parameters are allowed to vary to find the energy minimum.
The hexagonal crystal structure of hydroxyapatite has the P63/m space group [1, 63-65]. The
4e Wyckoff positions are occupied by two hydroxy oxygen atoms, each with 1/2 occupancy. In
order to translate this structure into a model with full occupancies for the hydroxy groups, as is
required to carry out the calculations, we have assigned alternate 0 and 1 occupancies to these sites
along the hydroxy channels in the c direction, thus changing the space group of the hydroxyapatite
unit cell from P63/m to P63. It is noteworthy that within the channels, the hydroxy groups were all
oriented in the same direction to form OH...OH...OH chains, as this corresponds to the most stable
configuration.[66, 67] However, since there is only one hydroxy channel per hexagonal unit cell, all
the hydroxy groups in the periodic structure are oriented in the same direction, thereby creating a net
electric polarisation per unit cell. In contrast, in the real structure, disorder in the relative orientation of
the parallel OH channels means that electric polarisation is not present in the material. Therefore, in
order to create a more realistic structure, the smallest cell in our simulations is doubled in the b
direction, and the two OH channels of the resulting supercell were assigned opposite directions. The
larger supercells used in our simulations are all multiples of this (1 x 2 x 1) simulation cell. It is
noteworthy that the antiparallel orientation of the hydroxy groups in neighbouring columns, as shown
in Figure 1, has been calculated to be slightly more stable than the parallel configuration  and it
coincides with the arrangement of the hydroxy groups in pure, synthetic HA, which crystallises as an
ordered monoclinic structure (P21/b) with a double unit cell compared to the hexagonal structure [68,
69]. However, given that natural HA and the species synthesized here have the hexagonal structure,
the (1 x 2 x 1) “hexagonal”-derived structure was used as a starting point for all our calculations on
HA and Mg-HA, thus allowing direct comparison of the simulations with experimental data.
Calculations were carried out on the (1 x 2 x 1) simulation cell (which has a total of 20 cation sites),
and the (2 x 2 x 1) and (1 x 2 x 2) supercells (40 cation sites), for the investigation of the incorporation
of Mg into the inequivalent Ca(I) and Ca(II) positions.
DFT calculations of the NMR parameters
NMR calculations were then performed on the various simulation cells described above, in
order to determine the NMR parameters of all the nuclei present in Mg-HA. These first principles
calculations based on the GIPAW method were performed within Kohn-Sham DFT using the
PARATEC code. As previously, the crystalline structures were described as infinite periodic
systems using three-dimensional periodic boundary conditions. The PBE generalised gradient
approximation was used and the valence electrons were described by norm conserving
pseudopotentials in the Kleinman-Bylander form. The core definition for O and Mg is 1s2 and
it is 1s22s22p6 for P and Ca. The core radii are 1.2 a.u. for H, 1.45 a.u. for Ca, 1.5 a.u. for O and 1.59
a.u. for Mg. The wave functions are expanded on a plane wave basis set with a kinetic energy cut-off
of 80 Ry. The isotropic chemical shift
iso is defined as
iso = [ –
ref] where is the isotropic
ref is the isotropic shielding of the same nucleus in a reference system as previously
described for 1H, 31P and 43Ca.[37, 74] For
ref(25Mg), external referencing with respect to crystalline
MgO was chosen, with
iso(25Mg) = 26.3 ppm. For 1H, 31P, 43Ca and 25Mg, the values of
isotropic chemical shifts were calculated. In addition, in the case of 43Ca and 25Mg, the quadrupolar
parameters CQ and η were also analysed. It should be noted that based on previous studies and our
own experience, the error on calculated 1H and 31P isotropic chemical shifts is estimated at ~0.4
and 0.7 ppm, respectively, whereas it is ~3 to 5 ppm on 43Ca and 25Mg isotropic chemical shifts,
and ~0.5 to 2 MHz on 43Ca and 25Mg CQ values,[37, 39] respectively.
Results and discussion
1. Experimental study of Mg-HA
1a. Description of the local environment of the ions in HA
There are two crystallographically different calcium sites in the HA unit cell: Ca(I) and Ca(II).
The four Ca(I) ions in the unit cell are usually referred to as “columnar calcium sites”, because they
form single atomic columns perpendicular to the basal plane. On the other hand, the six Ca(II) ions in
the unit cell are arranged in triangles around the hydroxy groups (OH), thereby forming hexagonal
channels along the c-direction of the structure.
As illustrated in Figure 2, the local environment of calcium in the two sites is very different.
Each Ca(I) is coordinated to nine O atoms belonging to six different PO4
3‒ anions; six of these
oxygens are located at less than 2.55 Å from the cation, whereas the three others are more distant.
Ca(II) cations have a less regular seven-fold coordination: they are bound to six O atoms belonging to
five different phosphates, and to one hydroxy group. When the local environment of the anions is
considered, it appears that PO4
3‒ tetrahedra have five Ca(II) and four Ca(I) sites in their vicinity, in
contrast with the hydroxy groups which are only surrounded by Ca(II) sites.
In order to learn how the incorporation of a small cation like Mg2+ in the HA lattice can affect
the cation and anion environments, solid state NMR and X-ray absorption spectroscopy studies were
carried out. Given that previous reports have shown that it is preferable to keep the Mg concentration
low in order to avoid losing the crystallinity of the HA lattice and forming more amorphous
phases, we prepared a Mg-HA containing ~10% Mg (Mg10-HA). SEM, TEM, and XRD confirm
the crystallinity of this sample and the presence of Mg in the HA lattice (Figure S1). Elemental
analyses show that the actual amount of Mg in the lattice is ~9%, which is slightly less than the ionic
concentrations initially introduced in solution, meaning that the actual formula for Mg10-HA is
Ca9.1Mg0.9(PO4)6(OH)2. The observation of a lower Mg content in the solid is not entirely surprising,
given previous reports on Mg-HA phases prepared according to similar synthetic procedures.