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JournaloftheEuropeanCeramicSocietyxxx(2014)xxx–xxx

Preparation, characterization and in vitro behavior of a new eutectoid bioceramic

V. Rubio

^a

, P. Mazón

^b

, M.A. de la Casa-Lillo

^a

, P.N. De Aza

^a,∗

aInstitutodeBioingeniería.UniversidadMiguelHernández,EdiﬁcioVinalopó.Avda.delaUniversidads/n.03202-Elche,Alicante,Spain

bDepartamentodeCienciadeMateriales,ÓpticayTecnologíaElectrónica.UniversidadMiguelHernández,EdiﬁcioTorrevaillo.Avda.delaUniversidads/n.

03202-Elche,Alicante,Spain Received22July2014;accepted21August2014

Abstract

Anew typeofbioceramichasbeendesignedand obtainedwithin thesub-systemCa2SiO4–7CaOP2O52SiO2.Theselectedcompositionwas thatcorresponding to the eutectoidpoint 69 wt%dicalciumsilicate–31 wt%tricalciumphosphate. Sinteringbehavior, phaseevolutionand microstructuralchangeswereanalyzedbyXRDandSEM.Bio-reactivitywasdeterminedbyimmersionofmaterialsinsimulatedbodyfluidfor severalperiodsoftimeaswellasstudiesinhumanadiposestemcells(hASC).Theinvestigatedmaterialsarebio-acceptablesincenotoxicorother harmfulevidencewasdetected.Acarbonatedhydroxyapatitewasformedonthesurfaceofthe31Rmaterialwithin3days.Cellattachmentassay showedthattheceramicssupportedthehASCcellsadhesionandspreading,andthecellsestablishedclosecontactswiththeceramicsafter24hof culture.Theinfluenceofthemicrostructure(porosity,grainsizeandphasecomposition)ontheinvitrobehavioroftheobtainedbioceramicswas alsoexamined.

Keywords:Bioceramics;Dicalciumsilicate;Bioactivity;Biocompatibility;Electronmicroscopy

1. Introduction

Aseriesof calciumphosphate-basedmaterials(bioglasses, glassceramics,ceramics,cements,coatingsandbioactivecom- posites)haveattractedconsiderableinterestfororthopedic,oral andmaxillofacialapplicationsbecauseoftheirbiocompatibility andtightbonding tobone,resulting inthegrowth ofhealthy tissuedirectlyontotheirsurface.^1–5

Inrecentyears, someSiandMgcontainingceramicshave drawninterestsinthedevelopmentofboneimplantmaterials.^6–8 Previousresearchsuggeststhatsilicon,asanessentialelement inskeletaldevelopment.Calrisle^9–11firstreportedinthe1970s thatsiliconwasuniquelylocalizedintheactiveareasofyoung boneandinvolvedintheearlystageofbonecalcification.Sim- ilarstudiesby Schwarz andMilne¹² haveshownthat silicon

∗Correspondingauthor.Tel.:+34966658485;fax:+34965222033.

E-mailaddresses:[email protected](P.Mazón),[email protected] (M.A.delaCasa-Lillo),[email protected],[email protected] (P.N.DeAza).

deficiencyinratsresultedinskulldeformation,withthecranial bonesappearingflatterthannormal.

Dicalcium silicate (Ca2SiO4) is an important material in the calcium–silica system, which is frequently identified as an important constituent in Portland cement,^13,14 refractory materials¹⁵andcorrosion-resistancecoatingmaterials.^16,17And it has alsobeen wellstudied because of its polymorphism.¹⁸ Recently, Ca2SiO4 ceramics have been investigated as a newtype of bioceramic for hardtissue regeneration.¹⁹ Some studies²⁰ havedemonstratedthat Ca2SiO4 powders, ceramics andcoatingsarebioactiveandcanquicklyinducetheformation of abone-likeapatitelayerontheirsurfaceaftersoaking ina simulatedbodyfluid(SBF).However,poorsinterabilityandthe residualporesinCa2SiO4ceramicsdeterioratethemechanical properties.

Tricalcium phosphate (Ca3(PO4)2=TCP) is one of the most important biomaterials based on phosphates currently recognized as ceramic material that significantly simulate the mineralogical structural of bone.^1–3 When TCP ceramics are implanted in vivo, they are non-toxic, antigenically

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JECS-9801; No.ofPages12

2 V.Rubioetal./JournaloftheEuropeanCeramicSocietyxxx(2014)xxx–xxx

inactive, non-carcinogenic and bond directly to bone with- out any intervening connective tissue later. They show good biological compatibility, safety and osteoconductivity in liv- ing tissues,such that,they areclinicallyused for hard tissue replacement.^21,22 The TCP has three polymorphic forms, ␤,

␣ and␣.The lastonelacks interestsince ittransforms during the cooling into the ␣ form. ␤-TCP is stable to room temperature andreconstructivetransforms to1125^◦C into␣- TCP, which is metastably retained during the cooling until roomtemperature.^23,24Moreover,␣-TCPismoresolubleand biodegradablethanHA,anditisexpectedthatSiionsoccupying PsitesinTCPnetworkaremorelabilethanintheHAnetwork as well.Dissolutionandbiodegradation ratesare intheorder

␣-TCP>␤-TCP>HA²¹andtheycanbestronglyinfluencedby ionsubstitutions.^25–27

As a consequence, Si-substituted silicon-doped calcium phosphates (CaPs) haveattracted the interest of many scien- tistsbecauseSi incorporationisconsideredtobeapromising waytoimprovethebioactivityofCaP-basedbiomaterials.^26–32 According to the above considerations, materials containing phosphorus, calcium and silicon are promising candidates for preparing bioceramics andglass ceramics with improved osteogenicproperties.

Compositions belonging to the system dicalcium silicate (Ca2SiO4=C2S)–tricalciumphosphate(Ca3(PO4)2=TCP)^33,34 are promising candidates for preparing new ceramic bone implants. In this context, the objectives of this work were to fabricate novel ceramics in the sub-system 2CaOSiO2

(C2S)–7CaOP2O52SiO2(A).³⁵Theeffectofthemicrostructure (porosity, grain size and phase composition),on the invitro behavior of the new ceramics in asimulated body fluid and humanadiposestemcellsattachmentandproliferationhasbeen studied.

2. Materialandmethods

2.1. Materialsandprocessingmethods

Chemicals used in the synthesis of tricalcium phosphate and dicalcium silicate were calcium hydrogen phosphate anhydrous (CaHPO4>98.0 wt%, Panreac), calcium carbon- ate (CaCO3>99.0 wt% Fluka) and high-purity silicon oxide (SiO2>99.7 wt%, Strem Chemicals). Stoichiometric quanti- ties of the raw powders to obtain tricalcium phosphate and dicalcium silicate were ground ina laboratory mixing miller (MM301-Retsch)byusingPSZ-zirconiaballs.Inbothsyntheses thepowderwereinitiallycalcinedat950^◦Cfor4htodecom- poseCaCO3followedbymixingmillerwithPSZ-zirconiaballs andisopropyl alcohol as suspensionmedia. The powder was thendriedandsievedthrougha30meshscreen.

ForTCP,thepowderswereheatedinaplatinumcrucibleat 1500^◦Cfor 3h.Thenwas liquid-nitrogenquenched byrapid removal from the furnace. For C2S, the powders were cold isotatically pressed at 200MPa and heat treated at 1525^◦C for 12h, at a heating rate of 8.3^◦C/min followed by cooling rate of 5^◦C/min. The reaction-sintering temperature was selectedbearinginmindtheinformationprovidedbythephase

equilibrium diagram SiO2–CaO evaluated and reported by Erikssonetal.³⁶Thenthepowdersthusobtainedwereground and characterized by X-ray diffraction (XRD-Bruker AXS D8-AdvanceX-rayDiffractometer(Karksruhe,Germany))and particlesizedistributioninpowderaqueoussuspension(Laser diffraction,MastersizerSMalvern).

Amixtureof69wt%C2S/31wt%TCPwasprepared.First, TCPandC2Spowdersweregroundtoanaverageparticlesizeof

∼20␮m,andthedesiredproportionsofeachcomponentwere weighed inan analytical balanceandthoroughly mixedwith PSZ-zirconiaballs.Afterdrying,thesampleswereisostatically pressedinbarsat200MPa.Pelletsobtainedfromthebarswere putintosmallplatinumfoilcruciblesthatweresuspendedfrom a platinumwireinthe hot zoneof an electricalfurnacewith anelectronictemperaturecontroller(±1^◦C).Thepelletswere heatedupto1550^◦Cforatotalperiodof144h(6days),with quenching inliquid-nitrogen, milling, pressing andreheating every24h.

Thefinalstepinthispreparationinvolvedtwodifferentways ofcooling.Thefistone,coolingthesamplefrom1550^◦Cinside the furnaceto512^◦Candheatingat512^◦C/24hfollowedby slow coolingtoroomtemperatureatarate of 3^◦C/min.This combinedheattreatmentprocedurewasrequiredtoensurethat theequilibriumconditionswereachieved.Thesecondmaterial wasquenchedfrom1550^◦Cinliquidnitrogeninordertoobtain atroomtemperatureasamestastablephasethehightemperature phase of the systemCa2SiO4–Ca3(PO4)2.The heat treatment temperatureswerecarefullyselectedbearinginmindinforma- tion providedbythe2CaOSiO2–7CaOP2O52SiO2sub-system existingwithinthebinarysystemofCa3(PO4)2–Ca2SiO4.³⁵The finalsampleswerecutfromthebarsobtained,whichmeasured 7mmindiameterand1.5mminthickness.

2.2. Ceramicscharacterization

Next, the pellets were removed from the platinum foils, embedded inanepoxyresinundervacuumandprogressively polisheddownto0.1␮mdiamondpasteandetchedwithacetic acidwithaconcentrationof0.5%for2s.Then,theyweregently cleanedinanultrasonicbathwithdistilledwater,driedandpal- ladiumcoatedforscanningelectronmicroscope(SEM-Hitachi S-3500N, Ibaraki,Japan)observations. Thechemicalcompo- sition of crystalline grainswas qualitatively determined with anenergydispersivespectroscopy(EDS-INCA-Oxford)system coupledtotheabove-describedelectronmicroscope.

XRDanalyseswerecarriedouttodeterminethecrystalline phasesofthe differentceramicsobtained.XRDpatternswere obtainedusingλCuK␣1radiation(0.15418nm)andasecondary curved graphite monochromator. Data were collected in the Bragg–Brentano(θ/2θ)verticalgeometry(flatreflectionmode) between20^◦and50^◦(2θ)at0.05^◦steps,counting1.5sperstep.

Sampleswererotatedat30rpmduringacquisitionofpatternsin ordertoimproveaveraging.Thediffractometeropticwasasys- temofprimarySollerfoilsbetweentheX-raytubeandthefixed apertureslit.Onescatteredradiationslitof1mmwasplacedafter thesample,followedbyasystemofsecondarySollerslitsand adetectorslitof0.1mm.TheX-raytubewasoperatedat40kV

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at30mA.Diffractogramsofsampleswerecomparedwiththe databaseprovidedbytheJointCommitteeonPowderDiffraction Standards(JCPDS).

2.3. SoakinginSBF

Inordertoestimatethebioactivityoftheceramics,Kokubo etal.³⁷ proposedin2006 the Tris-buffered SBF, withan ion concentrationnearlyequaltothatofhumanbloodplasma.The ceramic disks were placed in polystyrene bottles containing 50mlof SBFat pH=7.40. The solid/liquidweightratio was equalto5×10⁻³.ThebottleswithSBFandsampleswereincu- batedat37±0.5^◦Cinashakingwaterbathforpredetermined intervals.After differentsoaking periods(up to14 days) the diskswereremovedfromSBF,rinsedwithdeionizedwater,and driedatroomtemperature.

Thesamplessurfacesandthecross-sections,aftertheexpo- suretotheSBF,wereexaminedat20keV.Thecross-sections werepreviouslypolishedto1␮mfinish,usingdiamondpaste, gentlycleaned,inanultrasonicbath,andpalladiumcoatedfor SEMexaminationandmicroanalysisbyEDS.Thesamplesevo- lutionwasestablishedbymeasuringthethicknessofthelayer formedintheceramic–SBFinterfacebySEM.

TheSBFwasremovedafterseveralperiodsofimmersionand silicon,calciumandphosphorusweredeterminedintheremoved SBFbyinductivelycoupledplasmaopticalemissionspectrom- etry(ICP-OESPerkin-ElmerOptima2000^TM).ForTEMstudy thesampleswereprepared bycareful removalof thereaction layerfrom sample surfaces using arazor blade, anddispers- ingthe powderon thesurface ofethanol inaPetridish.The powderspecimensafterdryingwerethencollectedoncarbon coatedTEMcoppergridsof300mesh.Electronbeamtranspar- entparticleswerechosenforTEMexaminationbyselectedarea diffraction(SAD)andalsoEDS.

2.4. Celladhesionandproliferationassays

Fortestingbiocompatibilityandadhesionofhumanadipose stemcells(hASC)wereisolatedfromsubcutaneousadiposetis- sueofvolunteerfemaledonorsundergoingelectiveliposuction procedures,afterobtainingtheirinformedconsentaccordingto theproceduresapprovedbytheEthics Committee.³⁸ Samples werecollectedfromthreedifferentpatientsaged25–35years.

The isolation procedure has been previously established by Zuk et al.³⁹ Briefly, the adipose tissue was washed sev- eraltimes withsterilephosphatebuffersaline(PBS).Washed aspirates were treated with 0.075% w/v collagenase in PBS (Sigma-Aldrich,Steinheim,Germany)for30minat37^◦Cunder gentleagitation.Thecollagenasewasinactivatedwithanequal volumeofcontrolculturemediacomposedofDulbecco’sMod- ified Eagle Medium (DMEM) supplemented with 10% fetal bovineserum(FBS)andantibiotics(100U/mLpenicillin and 100␮g/mL streptomycin). This suspensionwas quickly cen- trifuged (172×g for 10min) andthe pellet wasresuspended inacontrolculturemediumandfilteredthroughsterilegauzeto removetissuedebris.Thepercentageoflivingcellswasdeter- minedbytrypanbluestaining.Afterwards,cellswereseededin

adherentdishesatadensityof12000cells/cm²andincubatedin a5%CO2atmosphereat37^◦C.Itwasimportanttowashthecells severaltimeswithPBSafterthe24-hincubation.Thismanipu- lationallowedtheeliminationofnon-adherentcellsthesebeing mainlydeadcells,redbloodcellsandadipocytes.Themedium wasreplacedevery3daysandsplitbytrypsintreatmentwhen culturesreached80–90%ofconfluence.

Inthestudiesofbiocompatibilityandadhesionthecellswere seededdirectlyonthematerialsorwereculturedintheirpres- ence. The increment in the numberof cells on the materials wasevaluatedthroughthereductionoftetrazoliumsalts(MTT) assay.Beforethecellculturestudy,theceramicswerecleaned applyingpressuredairandrinsedseveraltimeswithPBS(pH 7.4).Afterthat,theyweredriedat37^◦Candfinallythepieces were sterilizedbysteamautoclaveat125^◦C.Then,the discs of ceramic were introducedin the wellsof a24-wellculture plate and cells were seeded onto the ceramics at a density of 700cells/mm².hASCwereincubatedwithafinal1mg/ml MTTsolutionwith25%FBSand1×PBSfor3hat37^◦Cand 5%CO2 indark.Cells werewashedwith1×PBSandincu- batedwithDMSO(Sigma,St.Louis,USA)untilthecellswere lisated.Absorbancewasmeasuredat570nmina96-wellplate spectrophotometre(␮Quant,BiotekinstrumentsINC).Control viabilitywashASCgrewoveradherentdishes.Celladherence andgrowthwereanalyzedat24hand7,14and21days.

ThesurfacemorphologyofsampleswasanalyzedbySEM- EDS inordertoevaluate thecellgrowthandadhesion tothe ceramicssurface.Afterincubationfor24hand7,14and21days, thesampleswereremovedfromtheculturewell,rinsedwithPBS andfixedwith3%glutaraldehydeina0.1Mcacodylatebuffer for1.5hat4^◦C.Thentheywererinsedandpost-fixedinosmium tetroxide for 1h, before beingdehydrated through increasing concentrationsofethanol(30,50,70and90vol.%)withfinal dehydratationinabsolutealcohol.Afterthis,theyweredriedby the critical-pointmethodandpalladiumcoatedandexamined bySEM-EDS.

3. Results

3.1. Ceramiccharacterization

Stoichiometricquantitiesofthestartingpowder,correspond- ingtotheeutectoidcomposition69wt%dicalciumsilicate–31 wt%tricalciumphosphate(Fig.1)weremixingmillerwithPSZ- zirconiaballs,for1h,wherebyhomogeneouspowderswithan averageparticlesizeof20␮mwereobtained.

Theceramiccooledinsidethefurnacetoroomtemperature through theeutectoidpoint,called31ACfromnowon,(label asAinFig.2)giveamixtureof␣-C2Sss(JCPDScardno.09- 348)andAss(JCPDScardno.11-0676).Ontheotherhand,the ceramicquenchedfrom1550^◦Cinliquidnitrogen,called31R fromnowon,(labelasBinFig.2)showsmetastablesolidsolu- tionR=(␣-C2S-␣-TCP)ssphase.TheXRDischaracterizedby twostrongdiffractionlinesslightlydisplacedfromthestrongest linesof␣-C2Sand␣-TCP.Asallthephasesaresolidsolutions, thediffractionpeaksareslightlydisplaced withrespecttothe

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Fig. 1.The sub-system 2CaOSiO2 (C2S)–7CaOP2O52SiO2 (A) within the system Ca2SiO4–Ca3(PO4)2.[C2Sss=dicalcium silicate solid solution;

Ass=7CaOP2O52SiO2 solid solution; R=(␣-C2S-␣TCP) solid solution.

C3P=tricalciumphosphate]redrawfromphasediagramproposedbyRubio etal.[35].

correspondingJCPDScards(␣-TCP-JCPDScardno.89-8960 and␣-C2S-JCPDScardno.87-1260).

The31ACceramicspecimensafterchemicaletching(0.5%

aceticacid for 2s)present an irregular eutectoidstructure of lamellaemorphologyas expected (Fig.3A).The samplewas madeupofverythineutectoidplateletsthatconstitutedof␣- C2SssandAssphases.EDSmicroanalysisshowedthatthelight- contrastphasecorrespondedto␣-C2Sss,whilethedarkerphase wasAss.Fig.3Bshowsthemicrostructureofthesample31R.

Thematerialpresentshomogenousmicrostructureofonlyone phasewithirregulargrainsandclosedpores.EDSconfirmedthe presenceofphosphorous,calciumandsilicon.

Foracomprehensivemicrostructuralcharacterizationofthe materials,microstructural parameters, such a crystalline size, porosity,densityandshrinkageofthesamples,havebeenestab- lished(Table1).

Fig.2.X-raydiffractionpatternsoftheeutectoidceramics31AC(A)and31R (B)materialsstudied[=Ass;䊉=␣C2Sss; =Rss].

3.2. InvitrotestinSBF

The surface morphologies of the ceramics after different soaking times inSBFare shown inFig.4.The surface after 3 days of soaking of the 31AC ceramic (Fig.4A) presentsa porouslamellaemorphology,formedbyreactionoftheeutec- toidmaterialwiththeSBFcausingselectivedissolutionofone of thephaseswhichformsthelamellaeoftheeutectoidmate- rialandthesubsequentformationofapatite-likelamellaebya pseudomorphic transformationofoneof thephases(␣-C2Sss

ortheAss)whichformtheceramicmaterial.Thisisconfirmed byEDXmicroanalysesthatshowthattheporouslamellaemor- phology is formed by calcium and phosphorusand traces of silica (datanot shown). The morphology of the singlephase eutectoidmaterial,31R,iscompletelydifferent(Fig.4D).The surfaceispartiallycoveredbysphericalparticlessmallerthan 1.0␮m.Insomeareastheparticlesareformingsmallagglomer- ates.Fig.4BandEshowstheSEMimagesofthesurfaceofboth eutectoidmaterialsaftersoakingfor7days.Afterimmersion, theceramic surfaceswerecoveredbyalayerofagglomerates ofglobularparticles.Themorphologyofprecipitationwasquite similaramongtheceramicsurfacesduringtheinvitroassay;a

Fig.3.SEMmicrographsofchemicaletchedsurfaceofthe31AC(A)and31R(B)ceramics.

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Table1

Physicalcharacteristicoftheeutectoidceramics.

Crystallinephases Crystallitesize(Scherrer)(Å) Porosity(%) Density(g/cm³) Shrinkage(%)

31AC ␣C2Sss+Ass 339.44 9.10 2.53 7.23±0.5

31R Rss 578.95 13.85 2.22 5.63±0.5

Fig.4.SEMimagesofthe31AC(A–C)and31R(D,E)ceramicsurfacesmorphologiesaftersoakingtimesinSBF(A,D)3days,(B,E)7daysand(C)14days.

globularphasegrewgraduallyformingacompactandcontin- uouslayeronthesurface,asaresult,theoriginalmorphology oftheceramicsurfaceswerecompletelychanged(Fig.3).This morphologydoesnotfurtherchangewithsoakingtimealthough withlongerimmersionperiods,theCa–Pspheresbecamebig- ger, with a final average diameter of ∼20␮m after 14 days ofimmersionandjoinedasalargerdenselayer.Somemicro- crackscouldalsobeobservedontheceramicsurfacescaused bytheshrinkage ofthe soakedsamples inair,suggestingthe formationofathickdepositionlayer.Aftersoakingfor14days, moreCa–Pagglomeratesgrewonbothceramicsurfacesandthe Ca–Playerbecamemorecompact(datanotshown).Thehigher magnificationSEMimageasarepresentativeof bothceramic surfaceprecipitatedrevealedthattheagglomeratedwerecom- posedofalargenumberoftinyworm-likecrystalswithasizeof crystallitesabout300–400nminlengthand100nmindiameter (Fig.4C).Thesesmallagglomerateparticlesweredetermined tobeapatite-likealthoughfromSEM-EDSmicroanalysis.The Ca/Pratiooftheapatite-likelayerwasinaverage∼2.4,higher thanthatinHAstoichiometric.ThisfactsuggeststhataCadefi- cienthydroxyapatite(CHA)isformingonthesurfaceof both eutectoidceramics.

Themicrostructureof polishedcross-sectionsof the31AC materialaftersoakinginSBFfor3and7daysareshowninFig.5.

Thelabel^*inFig.5AandCshowsaporouslayerofreaction productsdevelopedonthesurfaceoftheeutectoidceramic,and Fig.5Baclose-upimageofthisreactionzone.Thelayer,about 12(±0.05)␮mthick,wascomposedonaverageof58.45atm%

Ca,32.75atm%Pand1.83atm%Si,andhadCaandSilower

thantheoriginalspecimen(labelas).Forthespecimensoaked inSBFfor7days(Fig.5C)themicrostructuralstudyrevealedthe formationoftwowell-definedsuccessivelayersattheinterface.

AmassiveformationofanewdenseCHAlayerwasobserved attheSBF-sampleinterface(2.08≤Ca/P≤2.14).Adjacentto the dense CHAlayer a porousstructure has been developed paralleltotheinterface(labelas^*inFig.5C)whichisformed bythedissolutionofoneofthephaseswhichformsthematerial (1.66≤Ca/P≤2.06).Ontheotherhand,the31Rceramic(Fig.6) showsat7daysofsoakingadenseprecipitatedofHCAgrowing indirectcontactwiththeceramicsubstrate(1.68≤Ca/P≤2.03).

Table2showsthechangeofthicknessoftheCHAlayeras a functionof the soaking time. It was found that the porous reaction zone in31AC material increased up to about 16.10

Table2

Thicknessofthelayerformedintheceramic-SBFinterfacesasafunctionof soakingtime(error±0.05).

Days/␮m (±0.05)

31AC 31R

Porouslayer Denselayer Denselayer

1 0 0 8.38

3 12.00 0 20.02

5 13.86 25.86 27.78

7 15.00 31.34 30.67

9 15.62 32.35 33.35

11 15.94 36.13 34.25

13 16.04 36.06 34.86

14 16.10 35.10 35.04

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Fig.5.SEMcross-sectionviewtothe31ACbioceramicimmersedinSBFfor(A,B)3daysand(C)7daysandEDSmicroanalysis.

(±0.05)␮mthicknessafter 14 dayssoaking, while the dense CHAlayerreachabout35.10(±0.05)␮mforthesameperiod ofsoaking.Ontheotherhandthe31Rceramicdoesnotpresents an intermediate porouslayer. After 1 and 3 days of soaking thesurfaceof31RsamplewaspartiallydissolvedintheSBF, formingadenselayerparalleltotheSBF–ceramic,andinthe areaswherethelayerispresent,thisisabout8.38(±0.05)␮m thickness,reachingafinalthicknessof35.04(±0.05)␮mafter 14daysofsoaking.

Changesincalcium,phosphorousandsiliconionsconcen- trationsofSBFmeasuredatdifferentdaysofsoakingareshown

inFig.7.BotheutectoidceramicmaterialsreleasedSiionsand removedPionsfromtheSBFinasimilarway,butindifferent amounts. Themaindifference isinrelationtoCaionprofile, indicatingadifferentinvitrobehavior.ThecontinuousSiion dissolutionfromthematerialcontributedtogenerateanincrease ontheSiionconcentrationintheSBF.Thespecificconcentra- tionofSiinSBFincreasedfrom0to0.92and0.52mMolfor 31ACand31R,respectively,whensoakedfor14days.Inaddi- tion,phosphorousionswereremovedfromSBFbybothsintered ceramics,becausetheformationofCHAphaseonthesurface ofthematerialanditsconcentrationdecreasedfrom1.0to0.2

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Fig.6.SEMcross-sectionviewtothe31RbioceramicimmersedinSBFfor7daysandEDSmicroanalysis.

and0.19mMolfor31ACand31R,respectively.After14days ofsoaking,theformationrategraduallyshoweddownduethe depletionofPionconcentrationfromtheSBFsolutionanddiffu- sionprocessesofionsacrossthenewCHAlayer,whichbecomes verydifficult.Incontrast,theCaionconcentrationdecreasefrom 2.5to1.86mMolfor31ACandincreasedfrom2.5to3.90for 31Rceramic.

TEMandSADwereused toexaminethe ultrastructureof the surface product formed after the exposure of the 31AC (Fig. 8A–F) and 31R (Fig. 8G and H) samples to SBF for different soaking times. The results showed the presence of nanocrystalswitha plate-like morphology,mainly composed ofCaandP.

3.3. Celladhesionandproliferationassays.

Fig.9showsthemorphologyofhASCadheringandspread- ing on 31R (A–D) and 31AC (E–H) ceramic samples after culturingfor24h,7,14and21days.Whencomparedwiththe orifinalsurface,beforeexposuretocellculturemedium(Fig.3), itbecameevidentthatthesurfacemorphologywasaltered.

On 24h incubation (Fig. 9A and E) the adhesion was enhanced by means of multiple cytoplasmic digitations that spreadacrossthegranularsurfaceofthematerial,increasingthe contactareawiththesurfaceofthematerial.Atthisperiodthe cellsinalltheceramicsstudiedpresentsasimilarmorphology

showing some cells adhering either individually or in small groupsdispersedacrossthesurfaceofthematerial.After7days exposure, there was a significant increase in the cell growth rate on the ceramic surfaces. The cells formed a monolayer partially covering the 31R surface (Fig. 9B) and produced an extracellular matrix of a fibber network occupying the intercellular gaps. Onthe 31AC surface(Fig. 8F) cell-to-cell contactwasfrequent,withnumerousintercellulargapspresent.

Afteraperiodof14days,thecellsbegantoformamono- layeronalltestedmaterials.Thecellmonolayerappearedalmost continuouswiththecellsexhibitingflattenedandspreadmor- phology.Although inthe 31ACceramic (Fig.9G) itwas not possibletoseethematerialsurfaceunderneath.Attheendof theexperiment(21days)themonolayerfullycoversthesurface of bothmaterials.Inthecaseof31Rceramic(Fig.9D)some rupturedcellswerevisible,whileinthecaseofthe31ACmate- rialscellsshowedatendencytoorientateinthesamedirections (Fig.9H).

Fig.10showsadetailofthemorphologyofthehASCadher- ingandspreadingon31Rceramicsafterincubationfor21days.

Abundantextra-cellularmatrixwasobservedatthistime.The intercellulargapswereoccupiedbyabundantfibrillarmaterial, with deposits of mineral forming and extra-cellular matrix.

Furthermore,thecellsurfaceshowedagranularappearancedue tothepresenceofnumerousnoduleswithawhitishappearance emerging from the surface. To determine the nature of the

Fig.7.Changeincalcium(Ca),phosphorus(P)andsilicon(Si)ionconcentrationwithsoakinginSBFsolution.

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Fig.8.TEMmicrographsandSADofthesurfaceproductfromthe31AC(A–F)and31R(G,H)ceramicsexposuretoSBFfordifferenttimes.(A,D)3days,(B,E) 7daysand(C,F–H)14days.

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Fig.9.SEMimagesofthemorphologyofhASCgrowingonthe31AC(A–D)and31R(E–H)ceramicsurfacesafterdifferentseededtime.

Fig.10.SEM-EDSmicroanalysisofthecytoplasmaticglanulesofthehASCgrowingonthe31Rsurfaceafter21days.

nodules,microanalysisbyEDSwascarriedout.Thisanalysis provedthatitwasacalciumphosphate-basedmaterial.Itisworth notingthatthisEDSspectrumisdifferenttothatofthematerial surface,whichshowedpeakscorrespondingtoSi,CaandP.

MeasuresofMTTcellproliferationassayconfirmedtheSEM observations.Fig.11showsthe proliferationof hASConthe 31Rand31ACceramicsthroughoutthetimeofthestudy.The viabilityvaluesdetectedat24hwereverylow,indicatingthat thenumberofcellsinitiallyadheredtoboththeceramicswere small.However,thegrowthofthosecellsabletoattachtothe biomaterialsprogressedlinearlywithtime.Thenumberofcells growingon31Rceramicwasalwaysslightlylowerthanthose

obtainedon31AC,althoughtherewerenosignificantdifferences betweenthem.

4. Discussion

By controllingthe heat treatment we were able to design twomaterialswiththesamechemicalcompositionbutdiffer- entmicrostructure:Slowcoolingtreatmentproducesaeutectoid ceramicwithlamellaemorphologyof␣-C2SssandAssphases (Fig.3A).Quenchingmethodathightemperatureproduceasin- glepolycrystallinesolidsolutionR=(␣-C2S-␣-TCP)ssphase (Fig.3B)in agreementwiththe temperature of the eutectoid

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Fig.11.GrowthofhASConthe31Rand31ACceramicsatdifferenttimesof studied.

point(Fig.1)establishedbyRubioetal.³⁵ Thethermaltreat- mentsgeneratedshrinkage inboththe materials,as shownin Table1.Thetendencyforthedensitywastoincreasewiththe increasing amounts of shrinkage,in thisway it was possible toobservethat the samplethat showshighest shrinkage,cor- respondingto31AC,showedalsothehighestdensity,although thereisnogreatdifferencesbetweenthem.

Itisobviousfromtheresultsdescribedabovethatboththe ceramicsshowsstronginvitrobioactivebehaviorwhensoaking inSBF.Thepropertyofhavingabioactivesurfaceisrelatedto the formation of aCHA layerwhen the materialcomes into contact with SBF. It is considered that bonelike CHA plays anessentialroleintheformation,growthandmaintenanceof thechemicalbondofthebioactivematerialtothelivingbone.

Inthepresentwork,thecombinedSEM-EDS,TEM-SADand ICP-OES analyses showed that all the studied ceramics pre- sentedinvitrobioactivity.Bothmaterialsarebioactivebutwith differentbehavior.

Inrelationto31ACmaterial,therecordedchangesinthecon- centrationofthedifferentions(Fig.7)showedthatduringthe firststageofsoakingthematerialreleasedCaandSiions.Onthe otherhand,theconcentrationofsiliconwasincreasingcontinu- ouslyduringalltheexperimentreaching0.92±0.04mMolafter 14days.Itmeansthatthephaseorphasesthatweredissolving containedhighcontentof silicon.Thereforethesewillbethe

␣-C2Sssand/ortheAss.Thedegradationofthebothphasesled totheformationofaporousreactionlayerof16.10(±0.05)␮m of thickness after 14 days of soaking (Fig. 5 and Table 2).

Duringthesecondstageofsoakingabout7days,theconcen- trationofCaionsintheSBFdecreasedupto2.0±0.03mMol duetotheprecipitationofaCHAlayerontheceramicsurface (Figs.4A–Cand5).ThePconcentrationalsodecreasedupto 0.4±0.06mMolinthecourseof7days.Thisdecreaseobserved upto7daysofsoakingtoSBFwasmoreprominentduetothe formationoftheHCAlayerofabout31.34(±0.05)␮mthick- ness. Duringthe firststagesofthe reaction,thehighcalcium concentration intheSBFsolution (Fig.7) enhancedthe later nucleationofCHAlayeronthesurfaceofthespecimen,with highCa/P(2.08≤Ca/P≤2.14).The EDS microanalyses pre- sented inFig.5 provideddata for Ca/Pratio calculations for HCAasafunctionofsoakingtime.Thesubsequentchangesin

theionconcentrationintheSBFsolutionsuggestthatthedif- fusion processof theionstookplacethroughthepores ofthe porousreactionlayergeneratedbydegradationofthe␣-C2Sss

and/or Ass atthe 31ACceramic–SBF interface (Fig.5).This explains whyafter 14days period,the Si content within the SBFwasstillincreasing(Fig.7).

TheTEM/SADresultswereessentialinunderstandingthese processes.Fig.8AshowsaTEMimageoftheremovedreaction productfromthe31ACceramicsoakedinSBFfor3days.SAD inFig.8Dclearlyindicates thepreferentialorientationof the CHAcrystalsin[002]direction,inadditiontosomeparticles from the startingmaterial sincesomediffractionspotscorre- spondtotheC2SandAphases.AcomparisonwiththeSADof theremovedsurfaceproductfromtheceramicsoakedinSBFfor 7daysinFig.8EindicatedthattheamountofCHAcrystalswas significantlysmallerthanin14dayssample(Fig.8F),because the (002) arcwaslessprominent.Overall,thesecomplemen- tary TEM/SADdataconfirmedthat aquantityof anewCHA phaseprecipitatedonthesurfacesofthe31ACceramicswasa functionofsoakingtimeintheSBFsolution.

ThemechanismoftheformationoftheCHAlayeron31AC ceramicscanbeasfollows:

Dissolution of ␣-C2Sss in SBFwith release of Ca²⁺ and HSiO4−,as majorityions,andincreaseinCa²⁺,HSiO4−and OH⁻ionicactivitiesattheneighborhoodofthereactingsurface untiltheyexceedthesolubilityproductoftheHA:

Ca2SiO4+H2O → 2Ca²⁺+HSiO4−+OH⁻

Withthereleaseofthecalciumionsfromthedicalciumsil- icate solid solution, many Si–OH groups are formed on the surfacesof31AC.Thesesilanolgroupsinduceheterogeneous nucleationofapatite,andthereleasedcalciumionsincreasethe ionicactivityproductofapatite,enhancingapatitenucleation.

PartialdissolutionofA-phaseinSBFwithreleaseofSiO44−, HPO42−andCa²⁺

Ca7(PO4)2(SiO4)2+2H2O → 2SiO44−+2HPO42−

+7Ca²⁺+2OH⁻

NextthenucleationofaCHAlayertakesplaceonthesurface ofthematerial,byreactionoftheSBFphosphateionswiththe excess of calcium ionsliberated toSBFbythe ceramic. The silicateionsliberatedbytheceramiccanproducesilicon-HA.

Data fromSEM-EDSsuggestaverysmallquantityofsilicon substitutionontheapatitephaseaccordingto61.96/28.88/1.07 (Ca/P/Siatm%):

(6-x)HPO42−+ySiO44−+10Ca²⁺+8OH⁻

→ Ca10−x(HPO4)_x(PO4)6−y(SiO4)_y(OH)2−x+6H2O

Onceapatitenucleiareformedonthesurfaceoftheporous layer,theycangrowspontaneouslybyconsumingcalciumand phosphateionsfromthesurroundingSBFsolution.

TheresultsshowedthatthemorphologygeneratedintheSBF wascontrolledbydifferentphases’solubilitywhichgenerated

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changesof thesurface chemistryandthesurface topography.

Thismechanism leadstothe “in situ” formationof a porous interconnectedstructureofsilicon-HA,whichcouldbeexpected to promote the bone growth when the eutectoid ceramic is implantedintobonedefects.

Inrelationto31Rmaterial,therecordedchangesinthecon- centration of the different ions (Fig. 7) showed that Ca and Siions’concentrationincreasedwiththeincreaseinthesoak- ingtime.Incontrast,thePionconcentrationinSBFdecreased steeplytolevelthatwasonly23%ofthestartingconcentration aftersoakingfor10days,andthenremainedatthislevelafter- wards.TheincreaseinCaandSiconcentrationswasattributedto thedissolutionofcalciumandsilicateionsfromR=(␣-C2S-␣- TCP)ssceramics,andphosphateionsintheimmersionsolution wereconsumedwiththeformationofCHA,whichresultedinthe decreaseofPconcentration.Althoughsomecalciumionswere consumedtoformCHA,morecalciumionsweredissolvedfrom theceramicmatrixthanthosewereconsumed.Itisexpectedthat theprocessof thenewCHAlayerformationatthesurfaceof the31Rceramicwouldcontinueforaslongastheionexchange mechanismbetweenthematerialandtheSBFtakesplace.Itis expectedthattheprocesswillcometotheendwhenthesupply ofthePionsfromtheSBFterminates,orifthediffusionofthe ionsacrosstheinterfaceisstoppedduetothethicknessofthe newCHAlayerreachingacriticalvalue.SADstudyperformed inTEM(Fig.8GandH)showedthattheCHAphaseiscom- posedofnanocrystalswithaplate-likemorphology.Whenthe specimenwasappropriatelyoriented,theSADoftendisplayed (002)and(210)arcsindicatingthepreferentialorientationof theCHAcrystalsinthelayer.

Thedifferencesinmicrostructurebetweenthe31ACand31R eutectoidceramicsareconsideredtoinfluencetheformationof CHAlayerontheirsurfaces.Thedifferencesobservedbetween thesamplesstudiedcanbeexplainedintermsofthedifferences inphase solubility.Theformationof aCHA-layerinthesur- faceis attributed to a higher rate of dissolution of ␣-C2Sss andAssvsR=(␣-C2S-␣-TCP)ssphaseontheinitialsurface ceramics. Although at the end of the SBF testthe thickness ofthedenseCHAlayerisalmostthe samefor bothceramics (35.10±0.05␮mfor31ACand35.04±0.05␮mfor31R),the mechanismis differentsincethe 31ACceramic has aporous CHAinterlayerof16.10±0.05␮mthatthe31Rceramicdoes notpresents.

Regardingthemorphologyofthecellsgrowingonthe31R and31AC ceramics,the cells underwent their morphological changestostabilizethecell-biomaterialinterface,andthecells spreadandestablishedclosecontactswiththeceramics,adapt- ing aflattened morphology andshowing filopodia anchoring thecells tothebioactiveceramics after24h.Thecells grow- inginthepresenceof31Rwereabletoproducesmallmineral depositsofcalciumphosphatebothintheextracellularmatrix coreandintheproximitiesofthecells(Fig.9).Thiseffectwas alsoobservedwithothertypesof bioactiveceramic.^38,39 The resultsobtainedfortheproliferationandgrowthofthehASCon the31Rand31ACceramicsprovidedthatafter21daysculture thedistributionofcellsoverthespecimenswashomogeneous andtheywerewellattached,uniformlycolonizingthesample

surface.Thisindicatesthatthematerialsdevelopedinthisstudy are biocompatible, withahighcolonization andproliferation rate(Fig.11).

It is recognizedthat the cell behavioron artificial materi- alsdependsonthe surfacecharacteristics,such asroughness, improvingcellattachmentandproliferation.hASCdemonstrate significantly higher levels of cell attachmenton rough sand- blasted surfaces withirregular morphologies than on smooth surfaces.^40–42The31ACceramicsamplesstudiedinthepresent work exhibited irregular surface morphology, as shown in Figs. 2Aand4A.Thus,thesurfacepropertiesofthe material canhelptopromotehASCcellattachment.Theinductiveeffect oftheceramicsonhASCadhesionandproliferationshouldbe favoredbythereleaseofSiandCaionsintothemedium(Fig.7) andformationofthepreviouslymentionedCHAlayer(Figs.5 and6).Hence,formationoftheCHAlayerbyreleaseofSiand CaionsintothemediumstimulateshASCproliferation.

Cell culture does not allow ananalysis of possiblediffer- ences in response resulting from the different major phases present in the samples (single polycrystalline Rss phase or lamellar ␣-C2Sss and Ass phases). Under culture conditions thelevels ofviablecelladhesiononcrackfreespecimen sur- faceswerecomparablewithotherSi–CaPsurfacesinthesame medium.^40–44

Thepresentresultsverifythatfeaturessuchasmorphology andmicrostructurecanmodulatecellsresponsesinvitro,includ- ingcellularcolonization,proliferation,attachmentandsurface structureoftheformingcellsmultilayernetwork.Thiseutectoid ceramiccouldserveasapromisingplatformforregenerationof hardtissue.

5. Conclusions

By using the sub-system Ca2SiO4–7CaOP2O52SiO2, we havedesignedandprocessedtwomaterialswithchemicalcom- positionoftheinvarianteutectoidpointanddifferentstructure (singlepolycrystallineorlamellaemorphology).

Acombinationofchemical,structuralandmorphologicalfac- torswereresponsibleforthedifferentinvitrobehaviorofthetwo materials,bothwithacommoncomposition.Theresultsconfirm thattoestablishthepotentialbioactivityinvitroofamaterial, notonlythe physiologicalmediummust beproperlyselected butalsothechemicalcomposition,structureandmicrostructure ofthematerialsmustbestudiedindepth.

The bioactivityand biocompatibilityof allthese ceramics dependedontheirmicrostructure.Wecannottellwhichofthe twomaterialshaveimprovedbioactivityinvitrobecauseboth materialshavealmostthesameCHA-likelayerthicknessatthe end of the study. Cell attachment was slightly higher in the caseofthe31ACceramic,althoughbothceramicspresentedthe cells’spreading,establishedclosecontactswiththesubstrates, andadapted aflattened morphology withnumerous filopodia anchoringthecellstotheceramicsurfaces.

By meansof processingtechnology,it ispossibletotailor ceramicbiomaterialswithcontrolledmicrostructuresinorderto improvetheirosteointegration.

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Acknowledgments

PartofthisworkwassupportedbyMinistryofEconomyand CompetitivenessofSpain(MINECO);contractgrantnumber:

MAT2013-48426-C2-2-R.WeareverygratefultoDr.N.Vicente forhiskindhelpinthecourseofthecellanalyses.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/

j.jeurceramsoc.2014.08.039.

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