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Visan, Anita Ioana and Grossin, David and
Stefan, Nicolaie and Duta, Liviu and Miroiu, Floralice Marimona
and Stan, George E. and Sopronyi, Mihai and Luculescu, Catalin
and Freche, Michèle and Marsan, Olivier and Charvillat, Cédric and
Ciuca, Sorin and Mihailescu, Ion N. Biomimetic nanocrystalline
apatite coatings synthesized by Matrix Assisted Pulsed Laser
Evaporation for medical applications. (2014) Masterials Science and
Engineering : B, vol. 181 . pp. 56-63. ISSN 0921-5107
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Biomimetic
nanocrystalline
apatite
coatings
synthesized
by
Matrix
Assisted
Pulsed
Laser
Evaporation
for
medical
applications
A.
Visan
a,
D.
Grossin
b,
N.
Stefan
a,
L.
Duta
a,
F.M.
Miroiu
a,
G.E.
Stan
c,
M.
Sopronyi
a,
C.
Luculescu
a,
M.
Freche
b,
O.
Marsan
b,
C.
Charvilat
b,
S.
Ciuca
d,
I.N.
Mihailescu
a,∗aNationalInstituteforLasers,Plasma,andRadiationPhysics,409AtomistilorStreet,RO-77125,MG-36,Magurele-Ilfov,Romania
bCIRIMAT–CarnotInstitute,UniversityofToulouse,ENSIACET,4AlléeEmileMonso,31030ToulouseCedex4,France
cNationalInstituteofMaterialsPhysics,RO-077125,Magurele-Ilfov,Romania
dPolitehnicaUniversityofBucharest,FacultyofMaterialsScienceandEngineering,Bucharest,Romania
Keywords:
Biomimetichydratedbioapatites
Thinfilms
MatrixAssistedPulsedLaserEvaporation
(MAPLE)
Coatedimplants
a
b
s
t
r
a
c
t
WereportthedepositionbyMatrixAssistedPulsedLaserEvaporation(MAPLE)techniqueofbiomimetic nanocrystallineapatitecoatingsontitaniumsubstrates,withpotentialapplicationintissueengineering. Thetargetswerepreparedfrommetastable,nanometric,poorlycrystallineapatitepowders,analogous tomineralbone,synthesizedthroughabiomimeticapproachbydoubledecompositionprocess.Forthe depositionofthinfilms,aKrF*excimerlasersourcewasused(=248nm, FWHM≤25ns).The
analy-sesrevealedtheexistence,insynthesizedpowders,oflabilenon-apatiticmineralions,associatedwith theformationofahydratedlayeratthesurfaceofthenanocrystals.Thethinfilmanalysesshowed thatthestructuralandchemicalnatureofthenanocrystallineapatitewasprevalentlypreserved.The perpetuationofthenon-apatiticenvironmentswasalsoobserved.ThestudyindicatedthatMAPLEis asuitabletechniqueforthecongruenttransferofadelicatematerial,suchasthebiomimetichydrated nanohydroxyapatite.
1. Introduction
In recent years, a considerable attention was paid to the developmentofimplantswithbioactivefixation[1,2].Nowadays biomedicalresearchaimsfortheincreaseofsurface biocompati-bilityoftitanium(Ti)orthopedicordentalimplantsbythecoating withbiologicallyactivematerials.Tistandsforthemetallicmaterial ofchoiceinreconstructivemedicineduetoitsexcellent mechan-icalpropertiesinbulk,relativetothelowmassdensity,andhigh corrosionresistance[3].
Calciumphosphate(CaP)ceramicsarecurrentlythemostused biomaterialsformetalimplantcoatingsinordertoincreasethe osseoconductivity and overall bioactivity,thus speeding upthe biointegrationand repairingprocessofbonesor otherhard tis-sues[4].Nevertheless,recentstudieshaveshownthattheactual bioapatitepresentin thehumanbodyis generally nonstoichio-metricandmuchmore bioactivethanpurehydroxyapatite[HA, Ca10(PO4)6(OH)2],whichisconsideredthemodelforthebasic
con-stituentoftheinorganicpartofthebone(65–75wt.%,depending onageandsex)[5,6].Themineralpartofboneisinfactahydrated
∗ Correspondingauthor.Tel.:+40214574491;fax:+40214574243.
E-mailaddress:ion.mihailescu@inflpr.ro(I.N.Mihailescu).
calcium-deficientapatiteoflowcrystallinestatus,containing sev-eralionsubstitutions:Na+,Mg2+cansubstituteCa2+ions;(CO
3)2−
and(HPO4)2−ionscansubstitutephosphateions;(CO3)2−,Cl−and
F−canreplacehydroxylions,aswellasothervarioustraceelements
(Zn,Al,Sr)[7,8].
Researcheffortshavebeenthereforefocusedonthesynthesis ofbioapatitematerialsascloseaspossibletothehumanbone com-positionandstructure,andtheirnextcongruenttransferontothe surfaceofmetallic implants[1,9,10].Variousdopingand differ-entmethodsofdepositionhavebeenemployedinthepastinthe attemptofobtainingasuitableapatiticstructure.Wethusmention: pulsedlaserdepositedmagnesium,manganeseorstrontiumdoped HAcoatings[11–13],magnetronsputteredB-typecarbonatedHA thinfilms[14],andmanganese,strontiumorfluorine(co)-doped electrodepositedHAlayers[15–18].
Oneimportantissueemergingduringmanyphysicaldeposition studiesisthehighlydehydratednatureofthefabricatedHAfilms (thus,contrastingtotheactualmineralphaseofbone),becauseof highvacuumdepositionconditions,hightemperatureprocess,or volatilityofOH−species.Thechemicaldepositiontechniquescan
surpassthisdrawback,butaregenerallyleadingtofilmswithpoor adhesion[19,20].
Inthiscontext,theapatitenanocrystalsobtainedby precipita-tionmethodsinsolutionpresentphysico-chemicalfeaturessimilar
tothose of bone nanocrystals, which make them very promis-ingbiomaterialsforthepreparationofhighlybioactiveceramics [21].Non-stoichiometricnanocrystalline apatite-based biomate-rialsmimicthemineralbonecrystal structureand composition andexhibita controlledreactivity inrespecttotheinteractions withcomponentsofbiologicalfluids(ions,proteins)[22].Recently, apatitenanocrystalshavebeentestedascoatingsandtheyhave shown superior biological behavior [23]. One significant prop-ertyistheirsurfacereactivity thatisrelatedtotheexistenceof a metastable hydratedlayeron thesurface of thenanocrystals inpowders[5].Thesynthesisprocessofnanocrystallineapatites poorlycrystallizedby conventionaltechniques,at high temper-ature,strongly alterstheirphysico-chemicalcharacteristics and biologicalproperties.Thesynthesisprocessesusedinthispaper werethereforeaimedtolimitthesealterations(asgraingrowth, dehydration,evolutiontowardstoichiometry)[24].
Weemphasize upontheadvantagesforprocessingthe pow-dersatlowtemperatureandillustratetheeffectofexperimental parameters’ synthesis on apatite powder characteristics. The non-stoichiometric nanocrystalline apatite bioceramic coatings weredepositedonTisubstratesbyMatrixAssistedPulsedLaser Evaporation (MAPLE) technique, considered convenient for the well-protectivetransferofnovelorganicdelicatemoleculesthin films[25,26].MAPLEensuresgoodthicknesscontrol,patterning facility,andisappropriateforawiderangeofbiomaterialsprone tothedecompositionanddegradationunderdirectintenselaser irradiationandsubsequentexposuretoplasmaaction[25–27].One candepositpatternedfilmsonavarietyofsubstratematerialswith differentgeometricshapes.Themostimportant requirementof MAPLEprocessistominimizeandpossiblyavoidthephotonic dam-age,eitherofthefilmorofmatrixmaterialduringlaserinteraction andtransfer[25–27].MAPLEisanon-contactdepositiontechnique, whicheliminatesmajorsourcesofcontaminationandcanbe inte-gratedwithothersterileprocesses[25–27].
ThismanuscriptitisdedicatedtothedepositionbyMAPLE tech-niqueofbiomimeticnanocrystallineapatitecoatingsontitanium substrates,withpotentialapplicationsinmedicine.Thekeypartof thereportedevidenceistheinvestigationofthelabilenon-apatitic environmentsofmineralionsassociatedwiththehighsurface reac-tivityofbiomimeticapatites whichwasstudiedinourprevious work[28].
2. Materialsandmethods
2.1. Synthesisofbiomimeticapatitepowders
Biomimetic apatites (BmAp), with complex chemical for-mula Ca10−x+u(PO4)6−x−y(HPO42−)x+y(OH)2−x+2u+y, 0≤x≤2 and
0≤2u+y≤x [28], were synthesized at room temperature (RT) andphysiologicalpHbydoubledecompositionmethodbetween adi-ammoniumhydrogenphosphateandacalciumnitrate tetra-hydratesolution[120gof(NH4)2HPO4(CarloErba,analysisquality:
purity:98%)in1500mlofdeionisedwater,52.2gofCa(NO3)2·4H2O
(Merck,analysisquality:purity:99%)in750mlofdeionisedwater]. Thecalciumsolutionwasrapidlypouredintothephosphate solu-tion at 20◦C and stirred for 10min. The excess of phosphate
ionswasdesignedtofixthepHbufferingatahomeostaticvalue of7.4,whichremainconstantthroughoutthesynthesisprocess. Aftermaturationforoneday,theprecipitateswerefilteredunder vacuum and washed with deionised water. Then, the gel was freeze-driedandstoredinafreezertopreventfurthermaturation oftheBmApnanocrystals.Thechosenconditions(physiologicalpH of7.4incloseresemblancetohumanbody)resultedinthe synthe-sisofapoorlycrystallineapatiteanalogoustothemineralpartof neo-formedbone.
Fig.1.MAPLEexperimentalset-up.
2.2. DepositionofBmApthinfilms
TheMAPLEset-upusedinexperimentsisdepicted schemati-callyinFig.1.
ThinBmApfilmshavebeendepositedusingaKrF*excimerlaser source(=248nm, FWHM≤25ns),runningatarepetitionrateof
10Hz.Thecommonlyusedsubstratesinexperimentsweredisks ofpureTiof1.2cmindiameterand0.2cminthickness (Dentau-rumGmbH). For microscopiccharacterizations, somestructures weredepositedonh110isingle-crystallineSiwafers(MEMC Elec-tronicMaterialsInc.).Allsubstratesweredegreasedinacetoneand ethanolinanultrasonicbathfor30minandrinsedwithdeionized water.ForMAPLEexperiments,theslurrywaspreparedby dispers-ing475mgofBmAp,maturatedforoneday,inahydro-alcoholic solution,wherebenzoicacidwaspreviouslydissolved.The result-ingslurrywascarefullystirredatRTandthenpouredinastainless steelcup.Thesolutionwasnextfrozenat77Kbyimmersionin liquidnitrogenindirectcontactwithacoolerinsidethe deposi-tionchamber.Thisway,thetargetevaporationwasinitiallyslowed downtobefinallycompletelystopped.Thetarget-substrate sep-arationdistancewasof5cm.Duringdeposition,thetargetswere rotatedwith0.3Hzandtranslatedalongtwoorthogonalaxesto avoidpiercingandtoensurethedepositionofauniformfilm.The residualworkingpressureinsidethedepositionchamberwasset at∼2.7Pa.Forthedepositionofeachfilm,30,000subsequentlaser pulseshavebeenapplied.
Theselectedvolatilesolvent,thebenzoicacid,ishighly absorb-ingthelaserwavelengthinfrozenstate,butisnotreactingwith thesoluteevenunderlaserexposure.Accordingtoliterature,the dehydrationeffectofBmApafterthesuspensionofpowderina hydro-alcoholicsolutionkeepsnegligible[22].Acomparativestudy ontheincidentlaserfluenceeffectwasconducted.Fourfluence val-uesof0.3J/cm2,0.5J/cm2,0.75J/cm2and1J/cm2havebeenchosen
andtheappliedcriterionwasthepreventionofthepowder decom-position.Finally,thevalueof0.75J/cm2wasselected,asbeingthe
highestlaserfluenceforwhichthefilmsaredeposited stoichiomet-ricandwithoutanydecompositionordenaturationoftheBmAp powder,withthelargestpossiblerate.Thisway,thestructuraland functionalfidelitywaspreservedafterMAPLEtransfer,byavoiding asignificantdirectlaser–biomaterialinteractioninthedeposition chamber.Duetothelowconcentrationofsolute(biomaterial)in thefrozentarget,thelaserphotonspreponderantlyinteractwith thematrix(solvent),whichisvaporized[29].ThemetastableBmAp moleculesarereleasedunalteredand,bymeansofcollisionswith theothermolecules,aredirectedtowardthesubstrate,wherethey formauniformthinfilm.Inthesametime,thevolatilesolventis pumpedawaybythevacuumsystem.
2.3. Characterizationmethods 2.3.1. Nanopowderinvestigation
(i)Calciumconcentrationintheinitialpowderswasmeasured byatomicabsorptionspectroscopy,usingaPerkinElmer AAn-alyst 300 Atomic Absorption spectrometer. The phosphorus concentrationwasdeterminedbyspectrophotometryofthe phospho-vanado molybdenum complex, using a Hitachi V-1100 spectrophotometer at460nm,respectingtheprotocol describedinRef.[30].Thechemicalanalysiswasbasedonthe dissolutionofapatiteinacidicsolutionbeforetheanalysis,for thecalciumandorthophosphateionscontrol,orduringthe analysis,inthecaseofcarbonateions,respectively.
(ii)Thesamplecharacterizationby X-raydiffraction(XRD)was performedusingacurvedcounterdiffractometerINELCPS120 withmonochromaticCoKaradiation(=1.789 ˚A).
(iii)Fourier transform infrared (FTIR) spectroscopy analysis of the powder was carried out in transmission mode, in the (4000–400)cm−1 range,withaPerkinElmer1600◦C
thermo-spectrometerwitharesolutionof4cm−1.TheRamanspectra
ofpowderswererecordedonaJobinYvonHR800spectrometer, inthe(3800–100)cm−1 range,withalaserexcitation
wave-lengthof632.8nm.Theexperimentaldatawerefittedusing theOriginsoftware.
(iv)Transmission electron microscopy(TEM) studieswere con-ductedonaJEOLJEM1011(100–500kV)microscope.
2.3.2. Thinfilminvestigation
(i)ThinfilmthicknesswasmeasuredwithanAmbiosStylus Pro-filerXP-2system,having0.1nmverticalresolution,anoptical deflectionheight-measurementsensorandstyluswith2.5mm radiusand0.1mgforce.
(ii)The identification of crystalline phases was conducted by GrazingIncidenceX-rayDiffraction(GIXRD)usingaBrukerD8 Advancediffractometer,inparallelbeamsetting,equippedwith CutargetX-raytube.Theincidenceanglewassetat2◦,andthe
scatteredintensitywasscannedintherange20–50◦(2),with
astepsizeof0.04◦,and100sperstep.
(iii)TheFTIRspectroscopyanalysisofthethinfilmswasperformed withaPerkinElmerBXSpectrumspectrometer,inattenuated totalreflection(ATR)modeusingaPike-MIRaclediamondhead of0.18cmdiameter.Thespectrawererecordedintherange (4000–550)cm−1,witharesolutionof4cm−1andatotalof
150scansperexperiment.
(iv)TheRamanmeasurementsweremadewiththesamesystem usedforthenanopowdercharacterization.
(v)The morphology of the films was examined by scanning electronmicroscopy(SEM)withaFEIInspectSelectron micro-scope,operatingat20kVaccelerationvoltage,inhighvacuum, undersecondary electronmode. Cross-sectionSEMimages wererecordedonaspecimendepositedonSiwafersin iden-ticalconditionsinordertoinvestigatethefilmhomogeneity indepth.Additionally,higherresolutionmorphological stud-ieswereperformedbyatomicforcemicroscopy(AFM),using anAgilent5500apparatusequippedwithasupersharp TESP-SSNanoworldtip(nominalresonancefrequency320kHzand nominalradiuscurvature2nm).
(vi)TheCa/Pratioofthethinapatiticfilmswasdeterminedby energy dispersive spectroscopy (EDS)analysis, using EDAX Inc.instrumentwithaSiLidetector,operatedat20kV.The analyseswereperformedonfiverelativelylargeregionsof 250mm×250mm,inordertoensurethegoodstatisticofthe measurement.
(vii)TheHAfilmsadherencetothesubstratewastestedby pull-outmethod.Theexperimentalprocedurewasconductedin
Fig.2.XRDpatternsofstoichiometriccrystallineHAandBmAppowder.
accordance withtheASTMD4541andISO4624standards. TheinvestigationswerecarriedoutusingaDFDInstruments AT101PATMICROadhesiontesterequippedwitha0.28cm diameterstainlesssteeltestelements(dollies),gluedtofilms surface witha cyano-acrylateone-component Epoxy adhe-sive, E1100S. Aftergluing, the samples wereplaced in an ovenforthermalcuring(130◦C/1h).Eachtestelementwas
pulled-outverticallywithacalibratedhydraulicpumpuntil detachment.Thebondingstrengthvalueswerecalculatedas theratio betweenthe pullingforce atwhich the adhesive fractureoftheHAfilmoccurredtotheactuallyfilm delam-inatedarea.Wementionthatpriortothefilmtesting,control measurementsregardingthequalityofthebondingadhesive (steel-on-steel)wereperformed.Theaverageadherencevalue estimatedatthestainlesssteeldolly/stainlesssubstrate inter-facewasof∼85MPa.
3. Resultsanddiscussion 3.1. Nanopowders’characterization
Fromtheconcentrationofcalciumandphosphateions,obtained bychemicalanalyses,weinferredtheCa/Patomicratioinorder toassessthechemicalcompositionofsyntheticBmApafterone dayofmaturation.TheobtainedCa/Patomicratiovalueof1.5is noticeablyinferiortothetheoreticalvalueof1.67,characteristic tostoichiometricHA.Thisputsinevidencethenon-stoichiometric, calcium-deficient,biological-likenatureoftheapatitic(HA)phase usedinourexperiments.
The XRD powder patterns of a crystalline pure commercial powderHA(TecknimedSA)andofthesyntheticnanocrystalline apatitematuratedforonedayarepresentedcomparativelyinFig.2. Bothmaterialsexhibited thecharacteristic linesof the hexago-nalHAphase(ICDD:00-009-0432),withsharpandwelldefined peaksinthecaseofcommercialcrystallinematerial,andlargeand overlappingmaximainthecaseofBmApmaturatedpowder.The broadeningoftheBmAppeaksistheindicativeofapoorly crystal-lizednanoapatite,duetothelatticedisorderandveryfinesizeof crystallites,similarlytomineralbone[31,32].
Themeancrystallitesize wasderivedusing thewell-known Scherrerequation[33]:
Lhkl= K∗ b∗cos
HereLhkl isthemeansizeoftheordered(crystalline)domains,
which may besmaller or equal tothe grainsize; shape factor K=0.94;is theX-raywavelength;ˇ isthelinebroadeningat
Fig.3.FTIRspectraofhighlycrystallineHA(aandb)andBmAppowders(candd)inthespectralregions:1800–400cm−1(aandc)and4000–2800cm−1(bandd).
halfthemaximumintensity,aftersubtractingtheinstrumentalline broadening,inradians;andistheBraggangle.
Itwasappliedtodiffractionlines(002)and(310),giving esti-matedvaluesofthecrystallitelengthandanaverageoftheirwidth. Thus,ifoneignoresthestraineffects,basicprofileanalysisleadsto L002valuesof∼20nm,andL310of∼5nm,foramaturationtimeof
oneday.Thesedataconfirmedthenanometersizeofthe crystal-litesconstitutiveofBmAppowder,andindicatedtheirelongated shape.Itshouldbenotedthatpreviousstudiesrevealedby insight-fulmorpho-structuralanalysesthathumanbonealsoexhibitsan elongatedcrystalliteshape, havingananisotropic growthalong the[00l]crystallographicdirection[34,35].FTIRspectrumisgiven inFig.3 oftheBmAppowder (cand d),togetherwiththeone ofthestoichiometriccrystallineHA(aandb),in thefingerprint (1800–400cm−1)andfunctionalgroup(4000–2800cm−1)regions.
AllthecharacteristicabsorptionbandsofHAarepresent.The phos-phatebandpositions correspond totheones oftenreported in specializedliterature(forstoichiometricHA)[36–39].The promi-nentphosphatesbandsarevisible[472(n2bendingmodeofPO43−
groups),561and603(n4 asymmetricbendingofPO43−groups),
875(P–O–HvibrationintheHPO42− group),960(n1 symmetric
stretchingofPO43−groups),1030–1096(n3asymmetricstretching
ofPO43−groups),1151(vibrationsofHPO42−ions),and1250cm−1
(vibrationsofHPO42−ionsandpossible(PO2)−(Q2)species)].The
structuralOH− shallowbandsat638cm−1 (librationmode)and
3570cm−1(stretchingmode)werealsoevidenced(Fig.3candd).
Additionally,oneobservesthepresenceofthebroadwaterbands at(3400–3000)cm−1(stretchingmode)and1630cm−1 (bending
mode),whichindicateahigherdegreeofhydratationofthe mate-rial.Thebroadn3 CO32− asymmetricstretchingbandpeakingat
∼1450cm−1suggeststheslightcarbonationoftheBmAppowder,
duetocontaminationduringpreparationorhandling.
AllIRbandsofBmAppowder(Fig.3c)arebroaderandless con-spicuousthan thecorrespondingonesof thestoichiometricHA
Fig.4.Curvefittingofn4(PO4)3−IRbandofBmAppowder.
(Fig.3a),pointing tothelowerstructuralordering ofthis com-pound,ingoodagreementwiththeXRDobservations(Fig.2).
Moreover,theFTIRbandsoftheBmAppowderhavecomplex shapessuggestingtheoverlapofvariousvibrationcontributions. TheidentificationoftheBmApsub-structurewasattemptedbythe curvefittingofthen4PO43−asymmetricbendingdomain(Fig.4).
Besides thetriply degenerated bands of n4 PO43− groups (561,
572and603cm−1),theHPO
42−band(617cm−1),andthelibration
modeofOH−(638cm−1)[37],disclosedthepresenceofadditional
bands,positionedat535and550cm−1.TheybelongtotheP O
bonds,whicharenotusuallypresentinanapatiticstructureand cannotbeassignedtoanapatiticenvironment[22,28,36].The for-mationofnon-apatiticenvironmentsisassociatedtothesynthesis ofapatitenanocrystalsatphysiologicalpHincloseresemblanceto humanbone[36].
Fig.5.RamanspectraofhighlycrystallineHA(aandb)andBmAppowder(candd)inthespectralregionsofn1(PO4)3−(aandc)andnS(OH)−(candd)bands.Insert:Curve
fittingofn1(PO4)3−RamanbandofBmAppowder.
TheRamaninvestigations(Fig.5)wereinagreementwiththe FTIRresults(Figs.3and4),andwereusedtogetadditional informa-tiononthechemicalcompositionandstructureofBmAppowder. IfinthecaseofthepureHAcrystallinepowderasymmetricalpeak isevidenced(at962cm−1),duetothen
1PO43−vibrations,forthe
BmAppowdertheanalogouspeakhadanasymmetricallure, hint-ingtowardamixbandscontribution.Thecurvefittingevidenced two distinct components (see Fig. 5c-inset): an intense one at 961cm−1 (whichcanbeattributedtoapatiticPO
4), andaweak
oneat 955cm−1 (assignable tonon-apatitic phosphategroups).
Theyarise becauseof thesignificantdifferences in the interte-trahedronP Obondlengthsbetweentherespectivegroups.Such bandsarenotobservableincaseofwellcrystallizedstoichiometric apatites.ThepresenceofnOH−structuralvibrationswasnoticed at∼3571cm−1forbothcrystallineHAandBmAppowders(Fig.5b
andd)
Theagglomerationofaciculargrainshaving(150–250)nmin lengthand(10–15)nminwidthwererevealedbyTEM(Fig.6).The TEManalysesindicatedthehomogeneouscrystallinemorphology oftheBmAppowder,andconfirmeditsnanometricnature.
3.2. Characterizationofdepositedthinfilms
The AFM and step profilometry measurements indicated that the BmAp MAPLE films had an average thickness of ∼1.55±0.15mm.
Thecross-view(Fig.7a)andtop-view(Fig.7b)SEMmicrographs indicatedthatthefilmshadaratheruniform,homogeneousand fairlycompactmorphology,bothindepthandonthesurface.The filmsurfaceconsistsofnanograinshardtodiscriminate, charac-teristictofilmsdepositedbyMAPLEtechnique[26,27].Next,the
Fig.6.TEMmicrographsofBmAppowder collectedattwomagnifications(a:
Fig.7.SEMmicrographsrecordedincross-view(a)andtop-viewmodes(b)forthe
BmApMAPLEfilm.EDSspectrumfortheBmApMAPLEfilm(c).
filmmorphologywasinvestigatedinmoredetailbyhigh resolu-tionAFM.Fig.8 presentsthefilmsmicrostructureas visualized byphase-contrastAFM.Theseresultssupports thehomogenous characterofthenanostructuredMAPLEfilm,consisting predomi-nantlyoffairlypackedgrainswithsizesintherangeof30–50nm. However,rarelocalsurfaceabnormalitieswereobserved(Fig.8c), consistingofclustersofsignificantlylargergrains(150–200nm) withroundedges.Thepresenceofnanoparticulates,is character-istictostructuresdepositedbypulsedlasertechnologies(PLDand MAPLE)[4,27],andcanbeseenasadvantageousintheparticular caseofcoatedimplantsduetothegreaterinteractionoftheactive surfacewiththesurroundingcells.
TheEDSqualitative analysisconfirmedtheuniform distribu-tionofcalciumand phosphorousthroughoutthefilm(datanot shown),whilsttheEDSspectrum(Fig.7c)indicatedtheabsence ofimpurities.ThequantitativeEDSanalysisrevealedaCa/Pratioof 1.48±0.07,closetotheoneofthestartingpowder.
Theshort-rangeorderwasstudiedbyFTIRandRaman spectros-copies,whilstthelong-rangeorderwasinvestigatedbyGIXRD.
Thespectroscopicinvestigationsputinevidencethe preserva-tion,afterdeposition,ofthenon-apatiticenvironmentswhichare believedtoenhancethesurfacereactivity,asmentionedinRef.[36]. TheFTIR(Fig.9)andRaman(Fig.10)spectraoftheBmApMAPLE filmswerequitesimilartotheanalogousspectraofthestarting powder.
UnliketheIRspectrumoftheBmAppowder(Fig.3c),onecan notice intheFTIRfingerprintregionof theMAPLE film(Fig.9a andc)betterdefinedandsharperbands,whichdesignateahigher structuralorder.Thisallowedforabetterdiscriminationofthefilm “sub-structure”.Theshallowphosphateband(at∼552cm−1)
asso-ciatedwiththehydratedlayercoveringthenanocrystals[22,28,36], assignedtothenon-apatiticchemicalenvironments[22,36]and previouslyhinted by theIRpowderspectrum fitting(Fig.4), is observedrather distinctivelyfor thedeposited film, along with allthe othertypical apatiticvibrations, revealedin case ofthe BmAppowder(seeFigs.4and9a,b).Theothernon-apatitic vibra-tionband(positioned in thecase ofthe powderat ∼535cm−1, Fig.4),couldnotbeevidenced,asinfraredspectruminATRmode islimited tothecut-off point at550cm−1,where thediamond
windowabsorbstheinfraredradiation.TheshiftoftheOH−
libra-tionband(Figs.3cand 9a), relativetoitspositionseen forthe
Fig.8. AFMphase-contrastimagesofBmApMAPLEfilmrecordedintheintermittent
contactmode,atdifferentscales:1mm×1mm(aandb);0.5mm×0.5mm(b),on
differentsurfaceregions.
stoichiometricHApowder(Fig.3a),suggeststhatthesestructural unitsaremoredisorderedincaseofBmApmaterials,theirlocations inthe“OHchannel”beingaltered.Aslightcarbonationofthefilm hasbeenalsonoticed(asabroadbandcenteredat∼1450cm−1).
TheRamanspectrum(Fig.10)ofthedepositedfilmdisplayed asimilarasymmetricenvelopeastheoneseenfortheBmAp par-entpowder(Fig.5c),thereforesuggestingitsintimatestructural resemblance.
TheGIXRDpattern(Fig.11)oftheMAPLEfilmindicated the presenceof HA(ICDD: 00-009-0432)asa singlelow crystalline phase, but witha slightly improvedstructural order in respect withtheBmAppowder,which is inagreementwiththe afore-mentionedATR-FTIRobservations.Crystallitesizesestimatedatthe (002)and(310)crystalplanes,byapplyingtheScherrerequation, werealsohigher(L002≈45nm,andL002≈7nm).Theformationof
amorphouscalciumphosphate(ACP),iskineticallypossibleatpH higherthan7,althoughtheHAisthemostthermo-dynamically stable phase. The minor presence of ACP should not be disre-garded,andcouldalsoaccountforthesystematicbandsshiftsof BmApmaterialsrelativetothestoichiometricHAIRpeaks’ pos-itions(Figs.3and9),aswellasfortheirslightlylowerCa/Pratios.
Fig. 9.FTIR spectra of the BmAp MAPLE thin film in the spectral regions:
650–550cm−1(a),1800–830cm−1(b),and3620–3100cm−1(c).
Fig.10.RamanspectrumoftheBmApMAPLEthinfilminthespectralregionofn1
(PO4)3−.
Fig.11. GIXRDpatternoftheBmApMAPLEthinfilm.
TheadherencevalueattheBmApfilm/substrateinterfacewas of44±5.3MPa,asestimatedbythepull-outmeasurements,thus, closetotheone(∼50MPa)imposedbytheinternationalstandards forloadbearingimplantHAcoatings[40].
OurresultsdemonstratedthecongruenttransferoftheBmAp materialbyMAPLEtechnique,theobtainedfilmspreservingthe chemical composition ofthe parentpowder,with onlya slight alterationoftheinitialnanocrystals.
The link between the HA bioreactivity and the presence of hydratedlayerhasbeenproposedinthepast[22,36,41].This super-ficiallayercanintermediatevariousinteractions(ase.g.:proteins bondingoradsorption,ionicexchangesorsharing)withthe bio-logicalenvironment[22,36,41].Ontheotherpart,theOHcontent caninfluencetheinvivodegradabilityoftheHAmaterial,asthe osteoclastsareknowntoinducealocaldropofthepHtobeable toresorbtheapatite[42].ThelowconcentrationofOHcanresult inareducedbufferingcapabilityofthematerial,favoringits disso-lutionandresorbtionbyosteoclasts[41,42].Neverthless,atoolow OHconcentrationcaninduceafastsolubilizationoftheHAcoating, whichcandrasticallyreducetheimplant’sfunctionality.Itshould bementionedthatahigherconcentrationofhydroxylgroupscan leadtoaslowerrateofdissolution,andalsoenablesthebuffering capacityoftheimplantforspecificmedicalapplicationsperforming inhighlyacidicmedia(e.g.dentistry)[41,43].
4. Conclusions
Weperformedthedepositionofadherentbiomimetic nanocrys-tallineapatitethinfilmsbyMAPLEtechniqueontotitaniumand siliconsubstrates.
TheFTIRandRamanspectraofthethin filmswerefoundto behighlysimilarandhadanidenticalsignaturetothespectrum of theinitialpowder.The observed shouldersattributedtothe HPO42− non-apatiticionsconfirmthepreservationofahydrated
phaseinsidethethinfilms.
Averylimitedtransformation oftheinitialnanocrystals was observed,whilsttheoriginalchemicalcompositionofthestarting powderswaspreserved.TheBmApbiomaterialsintheformofthin filmsshowedahighresemblancetothehumanhardtissuemineral structureandcomposition,andarethereforeexpectedtoinsurea betterfunctionalitytometallicimplantcoatings.
WeconcludethattheMAPLEmethodiscapabletomaintain thestructuralfidelityaftertransferofbiomimeticapatitefroma solidfrozentargettoanearbysubstrate,informofthinfilm.We havethusobtainedandputinevidencethecompletetransferofa hydrated,delicatematerialbyMAPLE.
Tothebestofourknowledge,thisisthefirstreportofMAPLE deposition of thin films of poor-crystallized hydrated apatites
synthesizedbythebiomimeticmethod.Weforeseethatthe char-acteristicfeatures–suchascomposition,structureandadherence –ofBmApthinfilmscouldbetailoredinthenextfutureby opti-mizingthedepositionparameters,tothoroughlydemonstratethat theMAPLEtechniqueisapromisingalternativeforfabricationof metallicimplantcoatings.
Acknowledgements
Thisresearch was partiallysupported by ExecutiveUnit for Financing Higher Education, Research, Development and Inno-vation(UEFISCDI)of Romania undertheID304/2011and PCCA 153/2012Contracts.AValsoacknowledgesthesupportofSocrates fellowship.GESacknowledgeswiththanksthefinancialsupportof PNII-RU-TE-2011-3-0164(TE49/2011)researchgrant.
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