X-ray absorption spectroscopy shining (synchrotron) light onto the insertion of Zn 2+ in calcium...

Journal ofMaterials Chemistry B

PAPER

aClermont Universite, ENSCCF, Institut de

F-63000 Clermont-Ferrand, France. E-mail:

4 73 40 70 95; Tel: +33 4 73 40 73 36bCNRS, UMR 6296, ICCF, F-63171 Aubiere,

† Electronic supplementary informa10.1039/c3tb21397h

Cite this: J. Mater. Chem. B, 2014, 2,536

Received 8th October 2013Accepted 14th November 2013

DOI: 10.1039/c3tb21397h

www.rsc.org/MaterialsB

536 | J. Mater. Chem. B, 2014, 2, 536–

X-ray absorption spectroscopy shining(synchrotron) light onto the insertion of Zn2+ incalcium phosphate ceramics and its influence ontheir behaviour under biological conditions†

Sandrine Gomes,ab Amandeep Kaur,ab Jean-Marie Nedelecab

and Guillaume Renaudin*ab

The present study gives a fine description of the Zn2+ location in Zn-doped Biphasic Calcium Phosphate

(BCP) samples heat treated between 500 �C and 1100 �C. Structural considerations were used to explain

the sample interactions with biological fluid (DMEM). X-ray Absorption Spectroscopy (XAS) experiments

were used to characterize the powdered samples. The main results (1) indicate the presence of Zn2+

complexes physisorbed at the HAp surface for a sintering temperature of 500 �C, (2) confirm the

insertion of Zn2+ into the b-TCP phase using a substitution mechanism for a sintering temperature

around 700 �C, and (3) fully describe the insertion of Zn2+ into the HAp phase by an interstitial

mechanism for heat treatment above 900 �C (composition Ca10Znx(PO4)6(OH)2�2xO2x; xmax � 0.25). The

formation of the linear O–Zn–O entity with dZn–O ¼ 1.72(2) A has been clearly evidenced by Fourier

transform amplitude fitting in the R-space (Zn two-fold coordination unambiguously described for the

first time). The mineralisation stimulatory effect of Zn2+ incorporated into BCP has been explained by

two different mechanisms. For samples heat treated between 500 �C and 800 �C, the stimulatory effect

is attributed to the presence of soluble Zn2+ species: Zn2+ physisorbed at the HAp surface for sintering

treatment around 500 �C and Zn2+ incorporated into about 20 wt% (weight percent) of the soluble Zn-

doped b-TCP phase for sintering treatment around 800 �C. A sintering temperature above 900 �C led to

the formation of an extremely weakly soluble and well-crystallized Zn-doped HAp phase which acts by

facilitating the nucleation of a calcium phosphate phase at its surface.

1. Introduction

The mechanism of Zn2+ insertion in Biphasic Calcium Phos-phate (BCP) bioceramics has been recently deeply investigatedby our team thanks to the detailed crystallographic investiga-tions.1 Long range order characterisation has demonstratedthat this mechanism is temperature dependent.2 Up to 600 �C,Zn-doped BCP is mainly composed of undoped hydroxyapatite(HAp, Ca10(PO4)6(OH)2) and a part of the introduced Zn2+

cations is not detectable by the X-ray diffraction technique(either physisorbed at the HAp surface as already mentioned3,4

or incorporated into an amorphous compound). For tempera-tures around 800 �C, Zn-doped BCP is still composed of undo-ped HAp (slightly Zn-doped HAp more precisely) together with

Chimie de Clermont-Ferrand, BP 10448,

[email protected]; Fax: +33

France

tion (ESI) available. See DOI:

545

Zn-doped b-tricalcium phosphate (b-TCP, b-Ca3(PO4)2). The Zndoping mechanism for b-TCP is realized by substitution on theCa4 and Ca5 crystallographic sites of the b-TCP structure, i.e.the low-density column.5 Finally, above 900 �C Zn-doped BCP ismainly composed of the Zn-doped HAp phase with compositionCa10Znx(PO4)6(OH)2�2xO2x (with xmax � 0.25). Starting from1000 �C, the Zn2+ cations are inserted into the HAp structure,thanks to an interstitial mechanism on the 2b Wyckoff site ofthe HAp structure.6 In spite of the extremely similar ionic size,the mechanism of incorporation of Zn2+ cations into the HApstructure is fundamentally different from the one of Mg2+.7 Torealize the 2-fold coordination with the formation of linear O–Zn–O entities2 (a Zn2+ cation located in the 2b Wyckoff site issurrounded by two close hydroxyl crystallographic sites alongthe hexagonal channel), 3d atomic orbitals of the transitionmetal cation are certainly needed. One of the objectives of thepresent study is to conrm the formation of this O–Zn–O entitydescribed for the rst time in Zn-doped HAp.1 X-ray AbsorptionSpectroscopy (XAS) at the Zn K-edge was used to investigate theZn2+ local environment (coordination number and interatomicdistances) in Zn-doped BCP samples containing a low amount

This journal is © The Royal Society of Chemistry 2014

Paper Journal of Materials Chemistry B

of doping element. An amount of about 1 weight percent (wt%)of Zn in the whole sample is inevitably a limiting factor for thepreviously realized Rietveld analyses. Although joint Rietveldrenements have been performed on synchrotron diffractiondata and neutron diffraction data, the exact characterization ofthe local environment of Zn2+ is not allowed by long range orderdescription with statistical disorder around the Zn atomic site.The statistical disorder between one O–Zn–O entity and twohydroxyl groups prevents the exact local structure description bylong range order consideration (similar to the silicate/phosphatedisorder in silicon containing HAp samples also characterized byX-ray and neutron joint Rietveld analyses).8,9 XANES/EXAFSstudies have already been performed on Zn-doped apatiticmaterials in the last ve years with divergent conclusions: (1) Znatoms are localised at the surface and not in the structure ofpathological apatite,3,4 (2) Zn2+ cations substitute Ca2+ into theHAp structure either in the Ca1 site10 or in the Ca2 site,11 and (3)tetrahedral coordinated Zn2+ cations are located in the Ca2 site.12

It is now important to reinvestigate these systems thanks to theXAS technique in the light of the known temperature dependentmechanism of Zn2+ incorporation into BCP. Aerwards theobtained results (i.e. the exact description of the temperaturedependent Zn2+ local environment) will be used to correctlyinterpret the data from the biological uid/Zn-doped BCP inter-action study, which is the main issue of the present study.

The important role of zinc in biological applications hasbeen put forward in the literature.13–15 Uptake and release of Znin the body are strongly mediated by the bone reservoir.15 Whenincorporated into bioceramics, it has been demonstrated thatzinc has a stimulatory effect on bone formation and minerali-zation in vivo and in vitro,16,17 and that Zn incorporation intoimplants promotes bone formation around the material,18–20

improves biological properties,18,21 decreases the inammatoryresponse22,23 and has an antibacterial effect.24 In order to explorein detail the effect of the Zn doping element in promoting boneformation, two series of samples with nominal compositionCa10Znx(PO4)6(OH)2�2xO2x (x ¼ 0.15 and 0.20) have beensynthesized at sintering temperatures between 500 �C and 1100�C (as well as an undoped series, with x¼ 0.00, for comparison).The interactions with biological uid (DMEM) have beeninvestigated. Finally, X-ray absorption spectroscopy (XANES andEXAFS; SAMBA beam line at SOLEIL synchrotron) has been usedto correctly interpret the behaviour of the materials in DMEM.

2. Materials and methods2.1 Sol–gel elaboration of Zn-substituted BCP samples

The sol–gel route previously proposed by the authors was usedto synthesize one undoped and two Zn-doped series of BCPsamples.2 Briey, to produce 2 g of undoped BCP powder, 4.7 gof Ca(NO3)2$4H2O (Aldrich) and 0.84 g of P2O5 (AvocadoResearch chemicals) were dissolved in ethanol under stirringand reuxed at 85 �C for 24 h. Then, this solution was kept at 55�C for 24 h to obtain a white consistent gel. The as-prepared gelwas further oven-dried at 80 �C for 10 h to obtain a whitepowder. The last step consists of the high temperature heattreatment: pellets of crushed white powder were heat treated for


15 h at temperatures between 500 �C and 1100 �C (7 sinteringtemperatures – leading to seven samples per series of compo-sition – were used: 500 �C, 600 �C, 700 �C, 800 �C, 900 �C, 1000�C and 1100 �C). To prepare Zn-substituted samples, therequired amount of Zn(NO3)2$6H2O (Acros Organics) was addedto the solution, simultaneously with Ca(NO3)2$4H2O (Aldrich).Nominal compositions have been calculated assuming theinsertion of Zn2+ cations into an interstitial crystallographicsite, i.e., Ca/P ¼ 1.67 (whatever the Zn added amount). In thefollowing, samples are labelled ‘xZn-T’ with x ¼ 00, 15 and 20for samples with the nominal compositions Ca10(PO4)6(OH)2,Ca10Zn0.15(PO4)6(OH)1.70O0.30 and Ca10Zn0.20(PO4)6(OH)1.60O0.40,respectively. T refers to the sintering temperature: T¼ 500 �C, 600�C, 700 �C, 800 �C, 900 �C, 1000 �C and 1100 �C. Elementalanalyses of the samples by ICP-AES have conrmed the targetednominal compositions.

2.2 X-ray powder diffraction (XRPD) and Rietveld analyses

XRPD patterns were recorded on an X'Pert Pro Philips diffrac-tometer, with q–q geometry, equipped with a solid detector X-Celerator and using Cu Ka radiation (l ¼ 1.54184 A). XRPDpatterns were recorded at room temperature in the interval 3� < 2q< 120�, with a step size of D2q ¼ 0.0167� and a counting time of200 s for each data value. A total counting time of about 200 minwas used for each sample. An XRPD pattern was collected fromthe pure LaB6 NIST standard under the same experimentalconditions in order to extract the instrumental resolution functionto improve the peak prole tting during Rietveld renements.

Rietveld renements of X-ray powder patterns were per-formed for each sample with the program FullProf.2k.25 Theprocedure used (both data-collection and renement strategy)corresponds to the general guidelines for structure renementusing the Rietveld (whole-prole) method formulated by theInternational Union of Crystallography Commission on PowderDiffraction.26–28 The Rietveld strategy has been detailed inprevious related studies.1,2

2.3 X-ray absorption spectroscopy (XAS)

Zn K-edge Extended X-ray Absorption Fine Structure (EXAFS)spectra, simultaneously with the X-ray Absorption Near EdgeStructure (XANES) part of the spectra, were collected on the Zn-doped samples and two reference compounds (ZnO with Zn2+

tetrahedral coordination, and a ZnAl–CO3 layered doublehydroxide phase with Zn2+ octahedral coordination) at theSAMBA beam line at the SOLEIL synchrotron (Saint Aubin,France) in order to determine the electronic state as well as todescribe accurately the coordination spheres of Zn atoms. TheSOLEIL synchrotron was running at 2.75 GeV with an averagecurrent of 400 mA. The X-ray beam was obtained with a twocrystal Si(111) monochromator which offers an energy resolu-tion of DE/E ¼ 10�4 necessary to resolve the XANES structure.Harmonics were rejected from the beam thanks to the use oftwo palladium-coated mirrors tilted at 4.5 mrad. Calibrations ofthe experiments were made with Zn metallic reference foil (K-edge 9659 eV). Experiments were performed at the liquidnitrogen temperature (77 K) and atmospheric pressure. Spectra

J. Mater. Chem. B, 2014, 2, 536–545 | 537

Fig. 1 Mineral composition of the two main phases (HAp and b-TCP)as a function of the sintering temperature for the three series undoped(black squares and dotted lines), 15Zn-T (blue circles and solid lines)and 20Zn-T (red diamonds and solid lines). Lines are guides for eyes.

Journal of Materials Chemistry B Paper

were collected in the energy range between 9400 and 10 650 eV,with the energy step varying from 0.3 eV (XANES part) to 2.0 eV(end of the EXAFS part) and 3 s dwell time per point. Spectrawere obtained three times running in direct absorption modeand six times running in uorescence mode using a SDDdetector.29 The size of the beam was determined by a set of slits(200 mm � 500 mm).

Data treatments have been performed by using the programsAthena and Artemis from IFFEFIT package soware30 bymerging the successively recorded absorption/uorescencespectra. Single scattering theory has been used here. FollowingLengeler–Eisenberg normalization, EXAFS oscillations wereFourier Transformed (FT) using a Hanning window between 3.0and 13.0 A�1. The c(k) function was Fourier transformed usingk3 weighting, and all shell-by-shell tting was done in R-space.Theoretical backscattering paths were calculated successivelyusing ATOMS31 and FEFF6.32

2.4 Interaction of powdered samples with the biologicaluid (DMEM)

The samples synthesized at T ¼ 500 �C, 700 �C, 900 �C and 1100�C were immersed at 37 �C for 1, 2, 6, 10 and 20 days in standardDulbecco's Modied Eagle's Medium (DMEM; VWR Interna-tional SAS, France); pH 7.3 (DMEM without D-glucose andwithout L-glutamine, Biochrome AG Cat. no. F0405). DMEMcontained the following mineral ingredients: NaCl (6400 mgL�1), KCl (400 mg L�1), CaCl2 (200 mg L�1), MgSO4$7H2O (200mg L�1), Fe(NO3)3$9H2O (0.1 mg L�1), MgSO4$7H2O (200 mgL�1) and NaHCO3 (3700 mg L�1). DMEM has been consideredrather than simulated body uid because it matches moreclosely the biological conditions in particular due to the pres-ence of amino acids. Aer determining the Specic Surface Area(SSA) of the samples, thanks to nitrogen sorption isotherms (ona Quantachrom Autosorb-1b instrument), the volume of DMEMhas been calculated for 10 mg of powder and a constant liquid/SSA ratio according to the following equation:

vol. of DMEM (mL) ¼ 5 � SSA (m2 g�1)

At each immersion time, the concentration of the threeelements Ca, P and Zn was determined by ICP-AES in the bio-logical uid aer elimination of the solid using a 0.2 mm lter.The SSA is temperature dependent as densication of thematerials occurs on heating. The values are about 15 m2 g�1, 10m2 g�1, 4 m2 g�1 and 2 m2 g�1 for sintering temperatures of 500�C, 700 �C, 900 �C and 1100 �C, respectively.

3. Results and discussion3.1 Mineralogical analysis of the samples

To correctly interpret the behaviour of our samples, the mineralcompositions have been extracted from the Rietveld treatmentperformed on laboratory X-ray diffraction patterns. Mineralcompositions of the three series of samples are shown in TableS1 (ESI†). In agreement with previous results2 samples heattreated at 500 �C and between 900 �C and 1100 �C are mainly

538 | J. Mater. Chem. B, 2014, 2, 536–545

composed of the HAp phase. The b-TCP phase is stabilized forthe intermediate temperature with the maximum amountobserved at 800 �C. Fig. 1 illustrates these results for the twomain phases HAp and b-TCP. Combined with the two expectedHAp and b-TCP phases, a-CDP (composition Ca2P2O7), CaCO3,CaO and ZnO are temperature dependent impurities. a-CDPand CaCO3 (which transforms into CaO on heating) are presentup to 700 �C and ZnO is observed up to 900 �C.

As deeply described in a previous study,2 Zn2+ cations areinserted in the interstitial 2bWyckoff site into the HAp structureabove 900 �C only. Structural parameters of the HAp phase(Table 1) evidence the late interstitial mechanism of incorpo-ration of Zn, contrary to the substitution mechanism observedfor b-TCP (Table S2, ESI†) at intermediate temperatures. TheHAp lattice parameters show anisotropic variation between 900�C and 1100 �C (contraction of the a lattice parameter andexpansion along the c lattice parameter) combined with anincrease of the Zn occupancy in the interstitial 2b Wyckoff siteto reach the targeted compositions: Ca10Zn0.16(1)-(PO4)6(OH)1.68(2)O0.32(2) and Ca10Zn0.23(1)(PO4)6(OH)1.54(2)O0.46(2)

for the 15Zn-1100 and 20Zn-1100 samples, respectively. Thevariations of lattice parameters for b-TCP agree with a substi-tution mechanism (simultaneous decrease of both a and clattice parameters due to the calcium substitution for a smallerZn2+ cation) which takes place along the low density column ofthe structure.

According to our previous studies1,2 three kinds of samplesare distinguishable in terms of Zn location:

- Samples heat treated at 500 �C and 600 �C with about onethird of Zn2+ contained in ZnO and the other two thirds notdetectable by XRPD (physisorbed at the HAp surface or includedin an amorphous compound),

- Samples heat treated between 700 �C and 900 �C with onepart of Zn2+ contained in ZnO (still about one third) and theother main part incorporated into the b-TCP structure,

- Samples heat treated at 1000 �C and 1100 �C with the wholeZn2+ cations inserted into the HAp structure.


Table 1 Structural parameters of the HAp phase obtained by Rietveld refinements

Samples

HAp structural parameters

a (A) c (A) V (A3) Zn Occa Rened composition

00Zn-500 9.4196(1) 6.88419(10) 528.94(1) — Ca10(PO4)6(OH)200Zn-600 9.4197(1) 6.88410(9) 528.97(1) — Ca10(PO4)6(OH)200Zn-700 9.41874(9) 6.88356(8) 528.846(9) — Ca10(PO4)6(OH)200Zn-800 9.41954(5) 6.88241(4) 528.848(5) — Ca10(PO4)6(OH)200Zn-900 9.42007(3) 6.88100(3) 528.799(3) — Ca10(PO4)6(OH)200Zn-1000 9.42025(2) 6.88127(2) 528.840(2) — Ca10(PO4)6(OH)200Zn-1100 9.42037(2) 6.88176(2) 528.891(2) — Ca10(PO4)6(OH)2

15Zn-500 9.4228(1) 6.88155(9) 529.14(1) — Ca10(PO4)6(OH)215Zn-600 9.42044(9) 6.88233(8) 528.943(9) — Ca10(PO4)6(OH)215Zn-700 9.42057(8) 6.88365(7) 529.058(8) 0.5(3) Ca10Zn0.01(1)(PO4)6(OH)1.98(2)O0.02(2)

15Zn-800 9.41927(5) 6.88273(5) 528.842(6) 0.4(3) Ca10Zn0.01(1)(PO4)6(OH)1.98(2)O0.02(2)

15Zn-900 9.41859(3) 6.88622(2) 529.034(3) 3.2(3) Ca10Zn0.06(1)(PO4)6(OH)1.88(2)O0.12(2)

15Zn-1000 9.41766(2) 6.88989(2) 529.211(2) 5.4(3) Ca10Zn0.11(1)(PO4)6(OH)1.78(2)O0.22(2)

15Zn-1100 9.41740(2) 6.89068(2) 529.242(2) 7.9(3) Ca10Zn0.16(1)(PO4)6(OH)1.68(2)O0.32(2)

20Zn-500 9.4230(1) 6.8814(1) 529.16(1) 2.8(3) Ca10Zn0.06(1)(PO4)6(OH)1.88(2)O0.12(2)

20Zn-600 9.4194(1) 6.88251(9) 528.84(1) 2.2(3) Ca10Zn0.04(1)(PO4)6(OH)1.92(2)O0.08(2)

20Zn-700 9.41829(8) 6.88265(7) 528.726(9) 1.2(3) Ca10Zn0.03(1)(PO4)6(OH)1.94(2)O0.06(2)

20Zn-800 9.41858(5) 6.88280(4) 528.770(5) 2.0(3) Ca10Zn0.04(1)(PO4)6(OH)1.92(2)O0.08(2)

20Zn-900 9.41933(3) 6.88786(2) 529.242(3) 5.0(3) Ca10Zn0.10(1)(PO4)6(OH)1.80(2)O0.20(2)

20Zn-1000 9.41892(2) 6.89540(2) 529.776(2) 10.4(3) Ca10Zn0.21(1)(PO4)6(OH)1.58(2)O0.42(2)

20Zn-1100 9.41704(2) 6.89485(2) 529.522(2) 11.5(3) Ca10Zn0.23(1)(PO4)6(OH)1.54(2)O0.46(2)

a Zn occupancy in the 2b Wyckoff site.


During Rietveld renements the occupancy factors of allcalcium and phosphorus crystallographic sites have beensystematically tested. Whatever the temperature and the Zn-doping level, no vacancies have been observed contrary to theproposed Ca-decient chemical composition for Zn-dopedHAp.11 For both b-TCP and HAp phases the occupancies did notdeviate signicantly from unity (except the half-lled Ca4 site inb-TCP). Aer testing, all occupancies were xed to unity (andCa4 occupancy xed to 1/2 for b-TCP). As already mentioned inour previous studies1,2 no calcium substitution has been char-acterized in the HAp structure, contrary to the b-TCP case andcontrary to the conclusion found in the literature.10–12 On theother hand, an XRPD invisible part of Zn was determined fordoped samples heat treated between 500 �C and 700 �C, inagreement with indication of Zn2+ physisorbed at the HApsurface.3,4 During the last runs of Rietveld renement, Znsubstitution in a-CDP (i.e. x renement in Ca2�xZnxP2O7 solidsolution) was considered. A maximal x value around 0.1 wasobserved (correlated with a weak decrease of the rened latticeparameters; decrease of 0.4% of the unit cell volume comparedwith published undoped a-CDP data from ICSD #22225). Suchan amount of Zn2+ substituted in a-CDP did not allow us toexplain the two thirds of Zn not detectable by long range orderinvestigation. On the other hand, the XRPD invisible part of Znwas not correlated with the a-CDP weight amount and theZn2P2O7 phase (not isostructural to Ca2P2O7) was not observed(the three a-, b-, and g-Zn2P2O7 polymorphs – corresponding tothe ICSD #18315, ICSD #24153 and ICSD #51095 data, respec-tively – have been tested).


3.2 XAS analyses of the 20Zn-T series

The seven samples of the 20Zn-T series have been investigatedthanks to XAS spectra recorded in transmission mode. Thisseries has been chosen because of the single phase character ofthe two last 20Zn-1000 and 20Zn-1100 samples (Table S1† andFig. 1). For the other 15Zn-T series, only the two 15Zn500 and15Zn1100 samples have been measured. A set of two referencematerials have been used: ZnO and ZnAl–LDH (a layered doublehydroxide with composition Zn2Al(OH)6(CO3)1/2$2H2O). Zn2+

presents a tetrahedral coordination in zincite ZnO with fourdistances around 1.97 A,33 whereas it presents an octahedralcoordination in ZnAl–LDH with six distances around 2.00 A.34

3.2.1 Temperature dependence of the spectra. Fig. 2 and 3show the XANES part of the spectra and the EXAFS modulationsfor the 20Zn-T series and for the two reference compounds,respectively. The temperature dependence of the spectra agreeswith previous long range order characterizations.1,2 Modica-tions appear mainly between 600 �C and 700 �C (the tempera-ture at the beginning of the Zn substitution into the b-TCPstructure), and around 900 �C (the temperature at the beginningof the Zn insertion into the HAp structure).

In agreement with PXRD analysis which indicates one thirdof Zn2+ contained in crystallized ZnO (Table S1†) with tetrahe-dral coordination, XANES spectra and EXAFS modulations from20Zn-500 and 20Zn-600 samples are extremely similar to thosefrom ZnO as shown by arrows ‘1’ in Fig. 2 and 3. The Zn2+ partnot detectable by long range order investigation is then char-acterized in XANES spectra by the rst le shoulder at 9664.5 eV

J. Mater. Chem. B, 2014, 2, 536–545 | 539

Fig. 2 Normalized XANES spectra at the Zn K edge (E0 ¼ 9662 eV) forsamples from the 20Zn-T series (solid lines) and the two referencecompounds (ZnO and ZnAl–LDH, dashed lines). Arrows labelled ‘1’, ‘?’and ‘3’ refer to signals from Zn in ZnO, not detectable by XRPD andinserted into HAp, respectively.

Fig. 3 The k-weighted EXAFS signal, c(k), extracted from theabsorption measurements from the 20Zn-T series (solid lines) and thetwo reference compounds (ZnO and ZnAl–LDH, dashed lines). Arrowslabelled ‘1’, ‘?’ and ‘3’ refer to signals from Zn in ZnO, not detectable byXRPD and inserted into HAp, respectively.

Fig. 4 The k-weighted amplitude of the Fourier transform uncor-rected for the phase shift for samples from the 20Zn-T series (solidlines) and the two reference compounds (ZnO and ZnAl–LDH, dashedlines). Arrows labelled ‘1’, ‘2’ and ‘3’ refer to signals from Zn in ZnO,substituted in b-TCP and inserted into HAp, respectively.


(arrow with label ‘?’ in Fig. 2), and in EXAFS modulation byshoulders (also identied by label ‘?’ in Fig. 3) corresponding tothe modulations of octahedral Zn2+ in ZnAl–LDH. This indi-cates that the not detectable part of Zn2+ is certainly in octa-hedral coordination (physisorbed or in an amorphouscompound). Spectra of the 20Zn-800 sample are relativelysimilar to those of the formers, nevertheless we can observe theformation of a new rst le shoulder at 9662.5 eV (arrow ‘3’ inFig. 2). EXAFS modulations are quasi invariant up to a sinteringtemperature of 800 �C. We can only observe that the shoulderidentied by arrow ‘3’ in Fig. 3 is more pronounced. Spectrapresent important modications when Zn2+ is inserted into theHAp structure (i.e. above 900 �C). XANES spectra for samples20Zn-900, 20Zn-1000 and 20Zn-1100 present a rst sharp whiteline at 9662.5 eV (arrow ‘3’ in Fig. 2) followed by supplementarypeaks at 9669 eV and 9677 eV. This remarkable sharp white line

540 | J. Mater. Chem. B, 2014, 2, 536–545

traduces a new symmetry for Zn atoms without modication ofthe effective charge (as E0 is still close to 9662 eV, characteristicof Zn2+). EXAFS modulations are also highly modied fortemperatures above 900 �C, with apparition of the mainmodulation at k ¼ 4.06 A�1 (arrow ‘3’ in Fig. 3). In Fig. 2 and 3arrows with labels ‘1’, ‘?’ and ‘3’ refer to signals from Zn atomsin ZnO, not detectable by XRPD, and inserted into HAp,respectively. In Fig. 4, the arrow with label ‘2’ refers to the Znatom substituted in the b-TCP structure.

Fourier transformed amplitudes (not corrected for phaseshi) are represented in Fig. 4 in the R-space. Radial distribu-tion from the 20Zn-500 sample is similar to that from ZnO withthe rst peak at R¼ 1.52 A (arrow ‘1’) and the second peak at R¼2.90 A. Nevertheless, the intensity ratio between these two rstpeaks for 20Zn-500 did not correspond to the one of ZnO. Thesecond contribution is less intense in 20Zn-500, indicating thata part of Zn2+ in the sample has only one oxygen shell (withoutsecond neighbours). This observation indicates that the Zn2+

part not detectable by XRPD is more probably physisorbed atthe HAp surface, and not incorporated into an amorphouscompound. According to the previous observation on theXANES part and EXAFSmodulations it seems that Zn2+ forms anoctahedral complex physisorbed at the HAp surface. Already forsample 20Zn-600, the radial distribution presents importantmodications, namely the appearance of a distribution at R ¼1.20 A (arrow ‘3’ in Fig. 4). This short contribution becomes theonly rst shell for samples heat treated at 1000 �C and 1100 �C(and the second shell is observed at R ¼ 2.58 A), i.e. the signalcorresponding to Zn2+ inserted into the HAp structure withshort Zn–O distances. This indicates clearly that Zn2+ incorpo-ration into HAp is already weakly realized at 600 �C in agree-ment with a previous study.2 The contribution at 1.52 A (arrow‘1’ in Fig. 4) is shied toward higher interatomic distance forthe intermediate sintering temperature (close to the valueobserved for octahedral Zn2+ in ZnAl–LDH), indicating anincrease of the coordination number in agreement with thesubstitution in Ca4 (unusual 3 + 3 coordination) and Ca5


Table 2 Comparison of the Zn-local environment determined byXRPD Rietveld analysis and fit of the k3-weighted EXAFS raw data for20Zn-1100. Atom labels correspond to structural description reportedby Rodriguez-Lorenzo et al.6

Zn shells XRPD Rietveld EXAFS t Shi

Zn–X CN dZn–X (A) Biso(X) (A2) dZn–X (A) s2 (A2) D(dZn–X) (A)

Zn–O4 2 1.42 ¼ Biso(O1) 1.72(2) 0.001(2) +0.30Zn–O3(*) 6 2.92 ¼ Biso(O1) 2.69(5) ¼ s2 (P1) �0.23(*)

Zn–Ca2 6 2.92 0.83(2) 2.97(2) 0.007(3) +0.05Zn–P1(*) 6 4.01 0.54(4) 3.5(1) 0.015(8) �0.5(*)

Zn–O3(*) 6 4.13 ¼ Biso(O1) 3.3(2) ¼ s2 (P1) �0.8(*)

Zn–O1(*) 6 4.38 0.80(4) 4.0(2) ¼ s2 (P1) �0.4(*)

Zn–O3 6 4.87 ¼ Biso(O1) 4.79(6) 0.006(9) �0.08Zn–O2(*) 6 4.90 ¼ Biso(O1) 4.56(9) ¼ s2 (P1) �0.34(*)

(*): atoms belonging to the 6 neighbouring phosphate groups.


(octahedral coordination) crystallographic sites in b-TCParound 800 �C (arrow ‘2’ in Fig. 4).

3.2.2 Zn2+ insertion into HAp. Samples 20Zn-1000 and20Zn-1100 are single phase, composed of Zn-doped HAp only.Radial distribution functions are ideal data to conrm the localenvironment of Zn2+ inserted into the interstitial 2b site of theHAp structure. The k3-weighted tted Zn K-edge EXAFS data of20Zn-1100 are shown in Fig. 5 and t results are gathered inTable 2. Fit has been performed with the Artemis program inthe range 1 A < R < 4 A, not corrected for phase shis. Eightdirect paths have been considered in this R range. Six multiplepaths (minor contributions to the Fourier transform ampli-tudes, not indicated in Table 2) have been added to slightlyimprove the t. The number of neighbours for each shell hasbeen checked in the rst step, and they were xed to theircrystallographic values (to minimise the number of renedparameters) as the deviations were small. The obtained resultsunambiguously conrm the insertion of Zn2+ cations into the 2bWyckoff site of the HAp structure.1,2 The rst amplitudeobserved for a short R distance agrees with the O–Zn–O linearentity. A comparison with reference materials is available inFig. 4: see arrow ‘3’ at R ¼ 1.21 A for Zn-doped HAp (two-foldcoordinated), arrow ‘1’ at R ¼ 1.52 A for ZnO (four-fold coordi-nated) and arrow ‘2’ at R ¼ 1.66 A for ZnAl–LDH (six-foldcoordinated).

The rened Zn–O distance is 1.72(2) A (Table 2). Thisdistance is comparable to the interatomic distance dNi–O ¼1.68 A for the equivalent O–Ni–O linear entity in K2NiO2.35

Rietveld analyses indicate a Zn–O distance about 1.42 A only(Table 2). The difference of 0.30 A (indicated by ‘shi' in Table 2)between XRPD long range order characterization and localstructure EXAFS analysis is due to statistical disorder aroundthe 2a Wyckoff site along the hexagonal channel in the Zn-doped HAp structure (a unique crystallographic site, O4,

Fig. 5 (Left) k3-weighted amplitude of the Fourier transform uncor-rected for phase shift (black line) and best fit in R-space (dotted redline) for samples 20Zn-1100 (independent points: 19.6, variables: 15and R-factor: 0.022). (Right) Representation of the Zn-local environ-ment; projection along the hexagonal c axis (top) and normal to thehexagonal c axis (bottom). Atom labels correspond to structuraldescription reported by Rodriguez-Lorenzo et al.6 (black centralspheres for Zn atoms, large grey spheres for neighbour O4 oxygenatoms belonging to the linear O–Zn–O entity, blue large spheres forCa2 calcium atoms and pink tetrahedra for phosphate groups).


designs either the hydroxyl groups or oxygen atoms from O–Zn–O entities). EXAFS results allows correct characterization of thisdistance dZn–O ¼ 1.72(2) A. The tted distance of the six calciumcations (from Ca2 site) agrees with Rietveld renement (a shiof only 0.05 A is observed). On the other hand, tted EXAFS dataclearly indicate that the six neighbouring phosphate groupsapproach the inserted Zn2+ cations. The tted distances of theve atoms forming the PO4

3� anions are shied between 0.23and 0.8 A, toward Zn2+, by comparison with long range orderresults from Rietveld analysis (see asterisks in Table 2). FittedDebye–Waller factors (Table 2) are in good agreement with themechanism of Zn insertion into HAp. The small s2 value for O4corresponds to the formation of rigid O–Zn–O entities, whereaslarge s2 values for atoms belonging to phosphate groups areexplained by Zn2+ attraction which led to local disorder. Neigh-bouring Ca2+ cations are not affected by the Zn2+ insertion: theyare not shied from their position in the undoped HAp.

3.2.3 Zn2+ localisation at 500 �C. Samples heat treated at500 �C (and also 600 �C) present two kinds of Zn2+ cations.Rietveld results indicate that about one third of Zn2+ is con-tained in the ZnO phase. The two other thirds are not detectableby XRPD, either physisorbed at the HAp phase or incorporatedinto an amorphous phase. These two possibilities should havedifferent EXAFS signatures. The formation of a Zn2+ complexphysisorbed at the HAp surface will generate one rst shell ofoxygen atoms only (at a distance dZn–O of about 2.0 A). In the caseof Zn2+ contained in an amorphous compound, an equivalentrst oxygen shell should be observed, but almost a second shell(formed by cations Ca2+ or Zn2+) should also be present in theradial distribution (Fourier transform amplitude of EXAFS data).

The superposition of Fourier transform amplitudes of theZnO reference material, 15Zn-500 and 20Zn-500 samples (Fig. 6,top le), evidences that Zn-doped samples heat treated at 500 �Cpresent the two radial contributions from ZnO (R¼ 1.55 A and R¼ 2.90 A) but relative intensities are not respected. The secondcontribution close to R ¼ 2.90 A, relative to the second Zn-shell,is clearly less intense for Zn-doped samples (about half inten-sity). This indicates that about half Zn2+ cations are containedin the ZnO phase. The second half Zn2+ cations present the rstoxygen shell (with R close to 1.55 A) only, corresponding to the

J. Mater. Chem. B, 2014, 2, 536–545 | 541

Fig. 6 (Top left) k3-weighted amplitude of the Fourier transform uncorrected for phase shift for the ZnO reference material (dashed line), 15Zn-500 sample (bold line) and 20Zn-500 sample (black line). (Top right) Plot of the best fit in R-space (dotted red line; independent points: 17.0,variables: 9 and R-factor: 0.014) for the ZnO reference material (black line). (Bottom left) Plot of the best fit in R-space (dotted red line; inde-pendent points: 13.0, variables: 4 and R-factor: 0.021) for 15Zn-500 (black line). (Bottom right) Plot of the best fit in R-space (dotted red line;independent points: 13.0, variables: 3 and R-factor: 0.045) for 20Zn-500 (black line).

Table 3 Results of the k3-weighted EXAFS raw data fit for ZnO, 15Zn-500 and 20Zn-500, x indicates the fitted proportion of Zn2+ from thephysisorbed hydroxyl–aqua complex

Zn shells ZnO 15Zn-500 20Zn-500

Neighbour Zn–X CN dZn–X (A) s2 (A2) dZn–X (A) s2 (A2) x dZn–X (A) s2 (A2) x

Zn–O 4 1.97(1) 0.001(1) 1.97(—) 0.001(—) 1 � x 1.97(—) 0.001(—) 1 � xZn–Zn 12 3.23(1) 0.005(1) 3.23(—) 0.005(—) 1 � x 3.23(—) 0.005(—) 1 � x‘Zn–O complex’ 4 — — 1.94(2) 0.001(—) 0.47(6) 1.84(—) 0.001(—) 0.4(—)

6 — — 1.94(2) 0.001(—) 0.37(6) 1.84(—) 0.001(—) 0.4(—)


physisorbed Zn2+ complex. In the rst step, the Fourier trans-form amplitude of the ZnO reference material was tted in theR-space in order to extract tted parameters for the two rstshells of ZnO (Fig. 6, top right). The obtained tted parametersfor ZnO were used, as xed values, for tting the Fouriertransform amplitude of both Zn-doped samples heat treated at500 �C (Fig. 6, bottom). Physisorbed Zn2+ cations were alsoconsidered during the tting procedure by considering aunique rst shell. Only two parameters were then tted: the Zn–O interatomic distance for the physisorbed Zn2+ complex andthe relative proportion of Zn2+ from ZnO and from the phys-isorbed Zn2+ complex. Fitting results indicate about 60% of Znatoms contained in ZnO and 40% of Zn atoms physisorbed atthe HAp surface in both samples heat treated at 500 �C(Table 3). The large R values (see the Fig. 6 caption) illustratethat these renements brought rough, but highly interesting,

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quantitative estimation of the two Zn2+ populations. Fitting didnot allow us to discriminate the four-fold or six-fold coordina-tion for physisorbed Zn2+ cations. Nevertheless, previousobservation on XANES spectra favours an octahedral coordina-tion. The rst radial contribution for 20Zn-500 is shied towardlow R values indicating certainly that a small amount of Zn2+

cations are already inserted into HAp. In agreement with Riet-veld results,2 the Fourier transform amplitude of samples heattreated at 600 �C already evidences the insertion of Zn2+ intoHAp: see minor R contribution at 1.21 A (Fig. 4) for 20Zn-600.

3.3 DMEM interactions

Undoped HAp series as well as the two Zn-doped HAp serieswere used to follow the interaction with DMEM for 20 days.Samples heat treated at 500, 700, 900 and 1100 �C were


Fig. 7 Zn concentration in DMEM determined by ICP-AES as a function of interaction time for the 15Zn-T series (left) and the 20Zn-T series(right).


considered to evidence Zn2+ leaching in DMEM from Zn-dopedpowder and calcium phosphate precipitation (decrease of theCa and P concentrations in DMEM).

3.3.1 Zn-doped BCP interaction with DMEM. Before inter-preting the role of the Zn2+ cation in the rate of calcium phos-phate precipitation in DMEM, in the presence of Zn-doped BCPpowder, we have to determine the Zn2+ leaching from powder.Fig. 7 shows the evolution of the Zn2+ concentration in DMEMaccording to the interaction time and heat treatment. Fig. 7evidences that the different locations of Zn2+ in the powder ledto radically different rates of Zn2+ leaching. For samples heattreated at 500 �C, 700 �C and 900 �C, Zn2+ cations are present inDMEM since the rst days of the interaction. The Zn2+

concentration observed with 15Zn-500 certainly corresponds tothe part of Zn atoms physisorbed at the HAp surface. Thisconcentration increases for the 15Zn-700 sample in agreementwith the large part of Zn2+ cations which substitutes the highlysoluble b-TCP phase (Table S1†). From 900 �C, the amount ofZn2+ in DMEM decreases considerably to reach a nearly zerovalue for 15Zn-1100. The insertion of Zn2+ into the interstitialsite of the HAp structure makes its dissolution practicallyimpossible. When considering the 20Zn-T series, the samebehaviours are observed for the rst days of interaction with

Fig. 8 Ca (spheres) and P (squares) concentrations in DMEM deter-mined by ICP-AES as a function of interaction time for the undopedseries heat treated at 500 �C (black), 700 �C (green), 900 �C (blue) and1100 �C (red).


higher Zn2+ concentrations. Aer several days of interaction, thetwo series behave differently. Large amounts of Zn2+ in DMEMwith the 20Zn-T series decrease, indicating that a sufficientconcentration of Zn2+ is needed in biological uid to improvethe precipitation of calcium phosphate (i.e. to improve miner-alisation). The two samples heat treated at 900 �C present asimilar behaviour. The Zn2+ concentration in DMEM decreasesfrom day 10 for 15Zn-900 and 20Zn-900. The presence of Zn-doped HAp (already formed in large quantity at 900 �C) seems tostimulate the mineralisation at the HAp surface by improvingnucleation at the HAp/DMEM interface.

3.3.2 Sintering temperature effect on calcium phosphateprecipitation. To correctly interpret the role of Zn2+, it isimportant to characterize before the effect of the sinteringtemperature in the absence of Zn2+. Fig. 8 shows the evolutionof the Ca and P concentrations in DMEM as a function of sin-tering temperature and interaction time. Surprisingly, noprecipitation is observed for the undoped sample heat treated at500 �C. The sample heat treated at 700 �C, which contains thesoluble b-TCP phase, quickly shows a decrease of both Ca and Pelements (i.e. precipitation of calcium phosphate). Thisprecipitation of calcium phosphate is delayed for samplesmainly composed of crystalline HAp (i.e. heat treated at 900 �Cand 1100 �C). The formation of a calcium phosphate layer at thesurface of crystalline HAp occurs in a signicant manner only atday 20, aer several days of incubation necessary for nucleation.

3.3.3 Zn2+ inuence on calcium phosphate precipitation.Fig. 9 shows the role of Zn2+ in the formation of the calciumphosphate layer for the four investigated sintering tempera-tures. For samples heat treated at 500 �C, a large amount of Zn2+

is needed to stimulate the calcium phosphate precipitation.Actually, no effect was observed with 15Zn-500, whereas a lateprecipitation of calcium phosphate is observed at day 20 with20Zn-500. The simultaneous decrease of the Zn2+ concentration(Fig. 7) indicates that the formation of the solid is certainly dueto the incorporation of Zn2+ cations. The kinetics of calciumphosphate precipitation observed for samples heat treated at700 �C (Fig. 8) is affected by the presence of Zn2+. Two expla-nations have to be considered: (1) either the Zn-doped b-TCPphase is less soluble than the undoped b-TCP phase or (2) thelarge amount of soluble Zn-doped b-TCP (about 20 wt%, Table

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Fig. 9 Ca (spheres) and P (squares) concentrations in DMEM determined by ICP-AES as a function of interaction time for the undoped series(black), the 15Zn-T series (blue) and the 20Zn-T series (red) heat treated at 500 �C (top left), 700 �C (bottom left), 900 �C (top right) and 1100 �C(bottom right).


S1†) counterbalances partially the calcium phosphate precipi-tation. The large amount of Zn2+ in DMEM with samples heattreated at 700 �C (Fig. 7) favours the latter explanation.

Finally the presence of Zn2+ in samples heat treated at 900 �Cand 1100 �C has no signicant effect. Nevertheless, Ca and Pconcentrations are slightly smaller in the presence of Zn2+

(namely at day 10 for the samples heat treated at 900 �C, andalready since the rst days for samples heat treated at 1100 �C)indicating a stimulatory effect on mineralisation. Almost noZn2+ cations are detectable in DMEM (Fig. 7) for samples heattreated at 1100 �C, indicating that the stimulatory effect shouldbe attributed to the improvement of the calcium phosphatenucleation sites at the Zn-doped HAp surface.

4. Conclusions

The present XAS analyses of different Zn-doped BCP samplesdenitively conrm the mechanism of Zn2+ incorporation intoBCP samples1,2 and highlight the interaction with biologicaluid (DMEM). XANES (Fig. 2) and EXAFS (Fig. 3 and 4) evidencethe successive location of Zn2+ in the samples according to thetemperature and mineral composition (Table S1† and Fig. 1).Samples heat treated at 500 �C contain a small amount of ZnOphase (about 0.5 wt%) and a part of Zn2+ cations is physisorbedat the HAp surface. The corresponding quantities of phys-isorbed Zn2+ cations are indeed available to interact with bio-logical uid if sufficient amount has been incorporated (Fig. 9,

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top le). From 600 �C a small amount of Zn2+ cations is insertedinto the interstitial sites of the HAp structure (arrow ‘3’ inFig. 4), nevertheless the main part of Zn2+ substitutes calcium inthe b-TCP phase forming a soluble Zn-doped b-TCP phase.Then, the samples heat treated at 700 �C deliver a large amountof Zn2+ cations in biological uid (Fig. 7). The effect on miner-alisation has been determined here due to the compensationeffect between Zn-doped b-TCP dissolution and calcium phos-phate precipitation when considering Ca and P concentrationsin DMEM (Fig. 9, bottom le). Finally the t of the EXAFS datafrom samples heat treated at 1100 �C has allowed us to provide ane description of the local environment of Zn2+ inserted intothe HAp structure. The formation of the linear O–Zn–O entityhas been unambiguously conrmed, and the Zn–O interatomicdistance is now determined (dZn–O ¼ 1.72(2) A, Table 2). The sixphosphate anions, forming the second shell (together with sixCa2+ cations, Fig. 5), are shied toward the central Zn2+ cationof about 0.5 A compared to crystallographic long range orderconsiderations. Zn2+ inserted into Zn-doped HAp is not avail-able during the interaction with biological uid; neverthelessnucleation of a calcium phosphate layer at the HAp surface isfacilitated during the rst days of contact. The positive miner-alisation properties known for Zn-doped BCP powder can beexplained by two different mechanisms according to the sin-tering temperature. For samples heat treated between 500 �Cand 800 �C, the presence of the soluble Zn2+ cation (eitherphysisorbed at the HAp surface for samples heated at 500 �C, or



substituted in the soluble Zn-doped b-TCP phase for samplesheated around 700 �C) allows the precipitation of calciumphosphate containing certainly zinc atoms. For samples heattreated above 900 �C, the formation of a well crystalline Zn-doped HAp phase facilitates the nucleation of calcium phos-phate at the HAp surface.

Acknowledgements

This work was supported by ANR under project NANOSHAP(ANR-09-BLAN-0120-03). We acknowledge SOLEIL for provisionof synchrotron radiation facilities and we would like to thankStephanie Belin for assistance in using beamline SAMBA(proposal 20120148). One of the authors, A. Kaur, wants tothank her originating University – School of Chemistry, VITUniversity, Vellore, India – to have allowed her master thesis inthe French ICCF laboratory.

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