Boccaccini , Csaba R. Balázsi Monika Furko , Viktor Havasi , Zoltán Kónya , Alina Grünewald , Rainer Detsch ,Aldo applications coatings on metallic implants fororthopedic hydroxyapatitebioceramic doped Development and characterization ofmulti-element

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w w w . e l s e v i e r . e s / b s e c v

Development and characterization of multi-element doped hydroxyapatite

bioceramic coatings on metallic implants for orthopedic applications

Monika Furko

^a,∗

, Viktor Havasi

^b

, Zoltán Kónya

^b

, Alina Grünewald

^c

, Rainer Detsch

^c

, Aldo R. Boccaccini

^c

, Csaba Balázsi

^a

aHungarianAcademyofSciences,CentreforEnergyResearch,H-1121Konkoly-Thegestr.29-33,Budapest,Hungary

bUniversityofSzeged,DepartmentofAppliedandEnvironmentalChemistry,RerrichB.sqr1,H-6720Szeged,Hungary

cUniversityofErlangen-Nuremberg,DepartmentofMaterialsScienceandEngineering,InstituteofBiomaterials,Cauerstr.6,91058 Erlangen,Germany

a r t i c l e i n f o

Articlehistory:

Received12June2017 Accepted13September2017 Availableonline9October2017

Keywords:

Coatings Microstructure Corrosion Bioceramics

a bs t r a c t

Multi-elementmodiﬁedbioactivehydroxyapatitebioceramic(mHAp)coatingsweresuccess- fullydevelopedontosurgicalgradetitaniumalloymaterial(Ti6Al4V).Thecoatingswere prepared bypulsecurrentdepositionfromelectrolytecontainingadequate amountsof calciumnitrateandammoniumdihydrogenphosphateat70C.ThepureHAplayerwas doped andco-depositedwithAg,Zn,Mg,Sr ions. Thebiocompatiblepropertiesoflay- erswereinvestigatedbyseedingosteoblast-like MG-63 cellsonto thesamples’surface.

The biocompatiblemeasurementsrevealed enhancedbioactivityofmodiﬁedHApcom- paredtouncoatedimplantmaterialsandpurebioceramiccoating.Themorphologyand structureofcoatingsandcellswerecharacterizedbyscanningelectronmicroscopy(SEM), energy-dispersiveX-rayspectroscopy(EDX)aswellasFT-IRandXRDmeasurements.The biodegradablepropertiesofsampleswereinvestigatedbyelectrochemicalpotentiodynamic measurements.

∗ Correspondingauthor.

E-mailaddress:furkomonika@gmail.com(M.Furko).

https://doi.org/10.1016/j.bsecv.2017.09.003

creativecommons.org/licenses/by-nc-nd/4.0/).

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Desarrolloycaracterizaciónderecubrimientosbiocerámicos dehidroxiapatitadopadosconmulti-elementosenimplantes metálicosparaaplicacionesortopédicas

Palabrasclave:

Recubrimientos Microestructura Corrosión Biocerámicas

r e s u m en

Se handesarrolladocon éxito recubrimientosbiocerámicosde hidroxiapatitabioactiva modiﬁcada conmulti-elementos (mHAp)sobresoportes detitanio de gradoquirúrgico (Ti6Al4V). Los recubrimientos sedepositaron con la técnicade lacorriente pulsada a partirdeelectrólitosconcantidadesadecuadasdenitratodecalcioydihidrogenofosfato deamonioa70^◦C.LacapadeHAppurasedopóyco-depositóconionesAg,Zn,Mg,Sr.

Labiocompatibilidadde lascapas seinvestigómediantesiembra decélulas deMG-63, similaresalososteoblastos,enlasuperﬁciedelasmuestras.Losresultadosdelosensayos debiocompatibilidadrevelaronunabioactividadmejoradadelaHApmodiﬁcadaencom- paraciónconmaterialesdeimplantenorevestidosyderevestimientobiocerámicopuro.La morfologíayestructuradelosrevestimientosylascélulasfueroncaracterizadasmediante microscopía electrónica de barrido(MEB), espectrometría de dispersión de energía de rayosX(EDX),asícomomediantemedicionesdeFT-IRyDRX.Labiodegradabilidaddelas muestrasseinvestigómedianteensayospotenciométricosdinámicos.

Introduction

Great efforts are made to improve the biocompatibility propertiesofcommonlyusedmetallicimplantmaterialsin orthopedicsurgery. Onesolution canbeapplying bioactive coatings such as calcium phosphates. The phase, structure, compositionand morphology ofthe CaPsurfaces are important parameters that must be accurately controlled toinfluencetheirpotentialbiofunctionalitywithrespectto osteoblasts since interaction between calcium phosphate (CaP) thin layers and osteoblastscan be influenced bythe outermostsurfacepropertiesofthosematerials.Hydroxyap- atite(HAp)hasbeenextensivelystudiedduetothestructural andchemicalsimilaritiestothemaininorganicconstituentof bonetissues.However,itiswelldocumentedthatbiological hydroxyapatite, which forms the mineral phases of calci- fiedtissues(enamel,dentinandbone),differfrompureand syntheticallyproducedHAp[1–3].Biologicalapatiteconsists ofa mixture ofcalcium phosphate phases, such astrical- ciumphosphate(TCP),carbonatedhydroxyapatite(CHA)and calcium-deficienthydroxyapatite(CDHA).Inthisregard,syn- theticHApexhibitsaCa/Pratioof1.67,whilebiologicalapatite deviates significantly from this value and its Ca/P ratio is knowntobeaslowas1.5.Onepromisingwaytomodifythe osteoblasticresponseofHApcoatings, bothinvitroand in vivo,couldinvolvetheuseofsubstitutedHAp,incorporating differentions,suchassilicon[3],magnesium[5],zinc[6],silver[7],strontium[8]intotheHAplattice.Numerousresearch worksontheuseofthesesubstitutedmaterialscanbefound intheliterature[3–11].Ontheotherhand,deepinfectionof megaprosthesesisstillaseriouscomplicationinorthopedic surgery.Bacterial adhesionand biofilm formationon these alloyscaneasilycausevarioushumandiseasesaftersurgery [12].Removingbacteriainabiofilmisimpossibleandalocal orsystemic antibiotictreatmentisnoteffective.Therefore,

theinhibitionofbacterialadhesionisthemostcriticalstep inpreventingimplant-associatedinfections[13].

Inviewoftheproblemofbacterialresistancetoantibiotics and antiseptics, nano-structured silver-containing coatings maybeaneffectivewaytopreventdevicerelatedinfections, becauseitshighandpermanentantimicrobialactivitycom- bines witha remarkablylowhumantoxicity[14–16]. Silver and in particular the free silver ion is well known for its broad-spectrumantimicrobialactivityanditslowtoxicityto mammalian cells, but still allowsfor the independent use oftherapeuticantibiotics[13–16].Strontiumhasbeenshown to havethe dualbeneﬁtof promotingboneformation and reducing boneresorption. Furthermore,it has been shown that strontium has the ability to enhance pre-osteoblastic cellreplicationandcanstimulatetheformationofnewbone throughosteogenesisanddifferentiationintoosteoblastsand has theabilitytoinhibit theactivity ofosteoclasts [17–22].

Mg²⁺ dopingcanenhancethe osteoblastadhesionstrength ascomparedtopureHApsinceincorporationofMgintopure calcium HApmakesitclosertothenaturalbone[23]while theZncontentcanpromotethewoundhealingprocessafter implantation.

One of the most promising and cheapest methods to depositcoatingsontometallicsubstratesistheelectrodepo- sition,morespeciﬁcallypulsecurrentdeposition.Themain advantagesofapplyingpulsecurrentinsteadofdirectcurrent arethatmorehomogeneous,uniformcoatingswithsmaller grainsizecanbeachievedthusimprovingthemechanicaland chemicalpropertiesofcoatings.Sofar,manyresearchworks havebeenperformedusingthisnovelmethodforlayerdepo- sition[24–30].Gopietal.[24]havepreparedmineralsdoped hydroxyapatitecoatingbypulsecurrenton andofftimein seconds(from1sto4s)andinvestigatedtheeffectofparam- eterchange.Wangetal.[25],however,appliedpulse-reverse currentforelectrodeposition.Intheirexperimentstheposi- tiveandreversepulsedutycycleswere0.1and0.5,andthe

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positiveand reverse plating times were 10 and 2ms. They foundthatwelladherentcoating couldbeachievedbythis methodwithoutanypost-treatment.Themorphologyofthe suchpreparedcoatingwasmainlyplate-likewiththicknessof around100nm.Inamorerecentstudy,Marashi-Najaﬁetal.

[26]reportedhydroxyapatitecoatingdepositionontoNitinol superelasticalloybypulsecurrentwithdutycycleof0.2at differentcurrentdensities.Theyalsostudiedtheeffectofelec- trolyteconcentrationonthemorphologyofcoatingsandthey revealedthatthestructurechangedfromneedleliketoplate likeastheelectrolyteconcentrationdecreased.Inaddition,it isworthwhiletomentionthatinsomeresearchworksvoltage (pulsedordirect)wasusedfordepositioninsteadofcurrent, accordingtotheauthors’reports[27–30].

Inourpresentresearchworkmulti-element(Ag,Zn,Srand Mg) doped hydroxyapatite coatings havebeen prepared by combinationofpulsecurrentelectrodepositionmethodand surfacepost-treatment.Themorphologyandstructureoflay- ershavebeenstudiedwithSEM-EDXmeasurements.Layers havebeen alsocharacterizedbyFT-IRspectroscopy and X- raydiffractionmeasurements.Thebiocompatibleproperties oflayershavebeenassessedusingMG-63osteoblast-likecells andthebiodegradablecharacteristics ofsampleshavebeen testedinsimulatedbodyﬂuidbyelectrochemicalmethod.

Experimental

Preparationofpureandsubstitutedcalcium phosphate/hydroxyapatitecoatings

Titanium alloy (Ti6Al4V, ISO5832-3, Protetim Ltd.) discs (10mm×1mm)wereusedassubstrates.Onesideofeachdisk wasroughenedusingasandblastingprocedurewitha180-grit aluminumoxidemedia(accordingtothestandardprocedure appliedbythemanufacturersimilarlythan inthe casesof commercialimplantmaterials).Thissurfacepre-treatmentis necessarytoenhancetheadherenceoflayers.

IGTV-4i/6ttypepulsecurrentgeneratorwasusedtoprepare thedifferentbioceramiccoatings.Inthepulsecurrentwave- form ton is the time when current ﬂows and t_off is the relaxationtimewhenthecurrentiszero.Applyingt_offtimein pulsecurrentdepositiongivesthesystemtimetorecoverdur- ingtherelaxationperiods.Theelectrodepositionprocesswas carriedoutinatwo-electrodecellundernormalatmospheric conditions,wherethe anodewasaplatinumsheetandthe metallicimplantdiskwasusedasacathode.Thedeposition parametersaresummarizedinTables1and2.Thethickness oflayerswasaround 1–2␮min allcases(Fig. 1). Themor- phologicalpropertiesofthelayerswerestudiedbySEMand FIBmeasurementswithLEO1540XBCrossbeamworkstation.

ThebeamparametersinSEMimagingmodewere5keVbeam energyand30␮maperturesize,Everhart-ThornleyandInLens secondaryelectrondetectorswereused.Theionbeamparam- etersinFIBmillingmodewere30kVacceleratingvoltageand 5nAbeamcurrent.ForSEM/FIBmeasurementsthesamples weretiltedat36angle.Theelectronbeamparametersforthe EDXwere8and16keVbeamenergy.ARöntecSi(Li)detector andtheBrukerEsprit1.9softwarehadbeenusedfortheEDX measurements.

Table1–Electrodepositionparametersforobtaining purehydroxyapatitelayers.

Electrochemicaldeposition

Electrolyte Concentration/gL

Ca(NO3)2 115.6

NH4H2PO4 33.30

H2O2(30%) 10ml

Depositionparameters

ton/ms 1

toff/ms 10

ip/Acm⁻² 5

Bathtemperature/^◦C 70

pH 4.5

Depositiontime/s 3

Surfacetreatmentafterdeposition 1MNaOHsolution,70^◦C,2h

Table2–Electrodepositionparametersforobtaining modiﬁedHAplayers.

Electrodeposition

Electrolyte Concentration/gL

Ca(NO3)2 115.6

Mg(NO3)2 2.56

Sr(NO3)2 2.10

NH4H2PO4 33.30

H2O2(30%) 10ml

Depositionparameters

ton/ms 1

toff/ms 10

ip/Acm⁻² 5

Bathtemperature/^◦C 70

pH 4.5

Depositiontime/s 3 Surfacetreatmentafter

deposition

Soakinginsolutioncontaining 0.01MZn(NO3)2and0.0025M AgNO3for24handafterwardin 1MNaOHsolutionat70^◦Cfor2h withsubsequentheattreatmentat 150^◦Cfor2h.

FT-IRcharacterization

Torecord FT-IRabsorptionspectraofinvestigatedsamples, specular reﬂection technique was employed. All infrared spectra of the samples were recorded on a Bruker Ver- tex 70 FT-IR spectrometer coupled with Hyperion 2000 IR microscope with 15× (NA=0.4) specular reﬂection objec- tive.Spectrawererecordedover therangeofwave number 4000–400cm⁻¹atroomtemperatureusing128scansat2cm⁻¹ resolution.

X-raydiffractionmeasurements

The crystal structures of the samples were investigated using X-ray diffraction. XRD spectra were recorded at room temperature by Rigaku MiniFlex II diffractometer (Cu K_␣ radiation source, 0.15418nm) equipped with a high count DTEX II detector and operated at 40kV and 40mA. The diffraction patterns were collected over a 2Â range from 10^◦ to 60^◦ with 1^◦/min steps using ﬂat plane geometry.

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a

b

c

d

e

f

500 nm

500 nm 500 nm

500 nm

HAp

o AI

P Ca

Ti V C

0 2 4 6 8 10

500 µm

mHAp KeV

o _AI

P Ca Ti

C V

0 2 4 6 8 10

500 µm KeV

1.896 µm 1.484 µm

2.028 µm

758.4 nm

Ag Zn Zn MgSr

cps / evcps / ev

Figure1–SEMandSEM/FIBmeasurementsonpureHAplayer(a,b)andonmulti-ionmodiﬁedHAp(c,d)aswellasEDX spectraonHAp(e)andmHAp(f).

Electrochemicalcorrosionmeasurements

Thepotentiodynamic polarizationstudies were carried out withZahnerIM6eelectrochemicalworkstation(Zahner,Ger- many). In the electrochemical measurements conventional three-electrode cell was used. The working electrode was ametallicimplant disk(19mm)withand withoutcoatings and platinumnet and Ag/AgCl/KClsat electrodes were used as counter electrode and reference electrode, respectively.

Thepotentiodynamicpolarizationcurveswererecordedwith 1mV/s scanningrate.Simulatedbody ﬂuidwas usedasan electrolyteforalltheelectrochemicalexperiments,whichhas ion concentrations nearly equal to those of human blood plasma and isbuffered at pH7.40 with 50mM trishydrox- ymethylaminomethane and 45mM hydrochloric acid. The compositionofsimulatedbodyﬂuidcanbeseeninTable3.By measuringthecorrosionpropertiesofsamplesitispossibleto tracetheirbiodegradationproperties.Alltheelectrochemical characterizationswerecarriedoutattemperatureof37^◦Cto simulatebodyconditions.

Biocompatiblemeasurementsonpureandmodiﬁed hydroxyapatitelayers

Cellculture

Cells used for the experiments are represented by MG-63 cellline(Sigma–Aldrich,Germany),whichisalineofhuman osteoblast-likecells.Cellsweregrownon75mlflasksandwere detachedbytripsin.MediumwasDMEM(Dulbecco’sModified EaglesMedium)with10%ofFBS(fetalbovineserum,contain- inggrowthfactorsandnutrientstosupportcellgrowth)and 100U/mlpenicillinand100␮g/mlstreptomycintominimize theriskofinfections.Theculturesweremaintainedat37^◦C, 5%CO2inahumidifiedatmosphereinincubator(NewBran- swickGalaxy170S).Theculturemediawerechangedinevery threedays.ThecellswerecountedinaNeubauerchamber.

Table3–Compositionofsimulatedbodyﬂuid[31].

Reagent Amount

(g/L)

Sodiumchloride 7.996

Sodiumbicarbonate 0.350

Potassiumchloride 0.224

Potassiumphosphatetrihydrate 0.228 Magnesiumchloridehexahydrate 0.305

1Mhydrochloricacid 40mL

Calciumchloride 0.278

Sodiumsulfate 0.071

Tris(hydroxymethyl)aminomethane 6.057

CellviabilitymeasurementswithWST-8reagent

For cell viability measurementsthe sampleswere put ina 24-wellmicrotiterplateand1mlofcellsuspensionatconcen- trationof10,000cells/mLwasseededontothesurfaceofeach samples.Thesameamountofculturemediumwithcellswith- outsampleswasusedascontrol.Afteracultivationperiodof 2,7and14days,theculturemediawasremovedfromthe24 wellcultureplateandthecellswerewashedwithsterilePBS.

Afterwashing,1mLofDMEMmediumcontaining1%WST-8 reagentwereaddedtothewellsanditwasincubatedfor3.5h.

Theincubation periodwas followedbyspectrophotometric assayofcoloredproduct.Duringthisincubationperiodviable cellsconvertWST-8toawatersolubleformazandye.Thespe- ciﬁcabsorbanceofformazandye(at450nm)intheMTPcan bedonewithanELISAplatereader(PHomoAutobioAnthos MykrosystemGMbh,Germany).Theabsorbancedirectlycor- relateswiththecellnumber.

ALPactivitymeasurements

ALP enzyme activity wasmeasured after 6and 14 daysof incubation inordertocharacterizethe osteoblasticactivity of the MG-63 cells. The cells were lysed with a cell lysis bufferwhichcontains20mMTRISbufferedsolution(Merck) with0.1wt%TritonX-100(Sigma,Germany),1mMMgCl2and

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Table4–ElementalanalysisofHApandmHApcoatingsderivedfromEDXmeasurements.

Atomicpercent(%)

Spectrum C O Al Ti V Ca P Ag Zn Mg Sr

HAp 11.68 56.18 8.74 9.38 0.47 8.53 5.02 – – – –

mHAp 12.43 56.38 9.77 13.88 0.17 4.08 2.62 0.34 0.06 0.19 0.08

HAp mHAp

Absorbance (a.u)

4000 3500 3000 2000 1500 1000 500

OH-

- -

strech H₂O

H₂O CO₃ abs.

CO3 surf. ads.

HPO

- -

3 4

2 4 960ν1

ν4 sym strech

990 deform 1130 ν3 deform 1400-1500

surf. ads.

875 OH lib 780

PO PO

630-610 1990

3570 and 3700

Wavenumber(cm^-1)

3 4

Figure2–FT-IRspectraofHApcoatingandmodiﬁedHAp coating.

0.1mMZnCl2.Thecelllysatewasincubatedwithareacting solutioncontaining0.1MTrissolution,2mMMgCl2and9mM p-Nitrophenylphosphatefor120min.Afterincubationabsorp- tionwasmeasuredat405nmusingaspectrometer(Specord 40).

Calceinstaining

Forstainingthelivecells,acetoxymethyl(AM)ester(Calcein, MolecularProbes,Germany)wasusedwhichisafluorescent indicator.Thecelldistributiongrowthonthesamplesurface wasanalyzedusingflorescentmicroscope(FM,Scope.A1,Carl Zeiss).Afterthecultivationperiodof48h,theadherentcells werefixedwith3.7vol%paraformaldehydefor10minandper- meabilisedwith0.1vol%TritonX-100(in PBS)for10minat roomtemperature.

DAPI(4,6-diamidino-2-phenylindol)staining

Thenucleioffixedcellswerestainedwiththefluorescence dye4,6-diamidino-2-phenylindol(DAPIRotiVR-MountFluor- Care).Forstainingofthesamples,thematriceswereincubated 15mininthedarkinDAPI-solution(2mLDAPI-stocksolution in1mLDAPIbuffer).Afterstainingward,thematriceswere washedthreetimesinPBStoeliminatethebackground.The nucleiwereimagedbythefluorescencemicroscopewithblue filter.

MorphologicalcharacterizationofMG-63cellsbySEM imaging

Thesamples,seededandculturedwithMG-63cellsfor2days werewashedwithPBS,ﬁxedwithasolutioncontaining3vol%

glutaraldehyde(Sigma, Germany) and3vol%paraformalde- hyde(Sigma,Germany)in0.2Msodiumcacodylatebuffer(pH 7.4),andthoroughlyrinsedwithPBSforSEManalysis(Auriga

CrossBeam,CarlZeissMicroscopyGmbH,Germany).Allsam- plesweredehydratedinethanol,storedin99.8vol%ethanol andcritical-pointdried(EMCPD300,Leica,Germany).

Statistics

Resultsarepresentedusingthemeanvalueandstandarddevi- ationoffourreplicatesofeachsampletype.Allresultswere normalizedtoMG-63cellsgrowthonawellplate(REF=100%).

Thedifferences inanalysis parameters betweenthe differ- entsamplesinvestigatedwereevaluatedbyone-wayanalysis ofvariance(ANOVA).Thelevelofthestatisticalsignificance was definedatp<0.05(Origin 8.6,Origin LabCorporations, USA).Thesignificancelevelwassetas*p<0.05,**p<0.01and

***p<0.001.ForthecomparisonofthemeanvaluestheTukey testwasused.

Results and discussion

Morphologicalinvestigation

Fig.1showstheSEMandFIBmeasurementsonHAplayerand onmodiﬁedHApcoating.ItcanbeseeninFig.1(a)thatthe pulseelectrodepositedHApcoatingaftersurfacetreatmentin 1MNaOHsolutionhasmainlysmallneedle-like andlarger rod-likeparticleswithlengthof100–200nmandwithdiame- terof20–50nm.TheCa/Pelementalratiointhiscaseis1.78 (Table4)whichcanindicatemainlyhydroxyapatitecrystalsin thelayer.TheSEM-FIBcrosssectionalimage(Fig.1b)revealed that thelayerhasavery porous,sponge-likestructureand its thickness is notuniform. The thickness oflayervaried between700nmand2␮m,dependingonthesiteofsamples.

Themetalion-modiﬁedHAplayer(Fig.1b)showssimilar morphology,however,inthiscaseﬂake-likeparticleagglom- erationscan alsobeobserved.TheSEM-FIBcross sectional imageshowssimilarlyporousstructurewithlayerthickness of1–2␮m.OnthecorrespondingEDXspectra,weakpeaksof Ag,ZnSrandMgelementsignalsarealsovisibleprovingthe presenceandincorporationofmetallicionsandparticlesin HAplayer.TheelementalanalysisrevealstheCa/Pelemental ratiotobe1.55whichcanindicatetheHApcrystalstructure disruptionorthepresenceofotherCaPphasesasimpurities.

However,thissmallamountofothercalciumphosphatephase couldnotbedetectedbyXRDmeasurementduetothedetec- tionlimit(Fig.3).ItisvisibleonEDXspectrathatTiandAl andVpeaksalsoappearbecausetheappliedelectronbeam excitedthesubstratematerialalsoduetotheverythinand inhomogeneouscoating.TheappearingveryweaksignalofC ontheEDXspectramightindicatethepresenceofsomecar- bonateimpurities.Thisresultisingoodaccordancewiththe FT-IRmeasurementsinFig.2.

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HAp

mHAp

Intensity (a.u.)

10 20 30 40 50 60

2 Θ

0 0 2 2 2 0 2 1 1 1 1 2 2 2 2 2 1 3 0 0 4

1 1 3

3 0 0 1 3 0

Figure3–XRDmeasurementsonHApcoatingandon modiﬁedHApcoating.

FT-IRanalysisofpureandmodiﬁedcalciumphosphate layers

As Fig. 2 shows, the FT-IR spectra are very identical for both coatings. On the spectraof HAp and mHAp samples peaks at627, 960, 990and 1130cm⁻¹ are related to PO43−

anionicgroupcontent,whilethewideabsorptionpeakinthe 1400–1500cm⁻¹regionisconnectedtoabsorbedCO₃²⁻con- tentofHApphase[32].Weakeroverlappedpeaksat875cm⁻¹ canberelatedtoHPO42−content,suggestingthepresenceof aminorcarbonatedhydroxyapatite(cHAp)phaseincoatings.

However,theslightlyhigherabsorptionofOH⁻groups(OH⁻ stretch vibration)at 3700cm⁻¹ inthe caseof mHAp coat- ingmightbeexplainedbysomeeliminationofcHApphase fromHApowingtotheincorporationofdopingelements.In addition,slightsignsofadsorbedwaterbandsalsoappearon spectrafrom3600cm⁻¹toaround2600cm⁻¹andat3570cm⁻¹. X-raydiffractionanalysis

TheXRDpatternsofpureanddopedHApsamplesareshown inFig.3.BothspectrashowscharacteristicpeaksofHApat 2=31.7^◦(211),32.9^◦(300),25.88^◦(002)inaccordancewiththe JCPDSﬁle09-0432.ThebroadXRDpeaksforHApindicateits nanocrystallinity.Inthecaseofmulti-ionmodiﬁedHAp,very similarpeakscanbeobserved.NootherCaPphasesorphos- phateimpuritiescanbedetectedonthespectraowingtothe detectionlimitandthecomponents’verylowconcentrations.

Inourcase,there isnovisiblelineshifting, peakbroaden- ing andchanging inpeakintensity whenmetallicionsare addedtothehydroxyapatitecoating.However,severalstudies reportedlineshiftingtohigher2valuesduetothereplace- mentoflargersizedCa²⁺(0.099 ˚A)ionswithsmallersizedMg²⁺

(0.69 ˚A)ionsandZn²⁺(0.77 ˚A)ions[33–35].Inotherresearch work,Zianietal.foundbroadeningofthepeaksduetothe reductioninthecrystallitesizeandincreaseinthelatticedis- order,whichtheyattributedtotheMg²⁺ substitutioninthe HAplattice[36].Ontheotherhand,thesubstitutionofstron- tiumandsilvercancausephaseshiftingtolower2Âindicating anincreaseinthelatticeparameters,whichcanbeattributed

Ti6AI4V HAp coating mHAp coating -3

-4 -5 -6 -7 -8

-2 -1 0 1 2

E VS Ag/AgCI / V

log LjL / A

Figure4–Potentiodymanicpolarizationcurvesofuncoated Ti6Al4Valloy(blackline),ofHApcoating(blueline)andof mHAp(greenline)recordedaftertwoweeksimmersionin SBFsolutionat37^◦C.Thepotentialscanningrateis1mV/s.

Table5–Electrochemicalparameters:passivecurrent density(jp),corrosionpotential(Ecorr)andcorrosion current(jcorr)valuesderivedfromthepotentiodynamic curvesinFig.4.

Sample jp/Acm⁻² jcorr/Acm⁻² Ecorrvs Ag/AgCl/mV

Ti6Al4V 0.91 0.26 −190

HApcoating 2.57 1.04 −295

mHApcoating 3.30 1.51 −486

tothehigherionicradiusofSr(1.13 ˚A)andAg(1.15 ˚A),ascom- paredtoCa²⁺[37].

Corrosioncharacterizationbyelectrochemical potentiodynamicmeasurements

Fig. 4demonstratesthepotentiodynamic curvesofimplant material(Ti6Al4V)andHApcoatingandmodiﬁedHApcoating.

ThecurveswererecordedaftertwoweeksimmersioninSBF solution.

As Fig. 4 reveals, large anodic passive regions can be observedontheanodicbranchesofpotentiodynamiccurves inallcaseswithsmallpassivecurrentdensities(jp)andthe shapesofpotentiodynamiccurvesofallsamplesisquitesim- ilar. Inthe caseofuncoatedimplant materialthe onsetof this passive region isaround +100mV vsAg/AgCl and the passive ﬁlm breakdown potential is at +980mV. The pas- siveregiononpotentiodynamiccurves ofpureHApcoating becameslightlywideraftertwoweeksofimmersionthanthat foruncoatedsample,itstartsataround−120mVvsAg/AgCl anditsbreakdownpotentialissimilarlyataround+980mV.

Ontheotherhand,thewidestpassiveregionisobservedin thecaseofmHApcoating,spreadingfrom−280mVtoaround +1VvsAg/AgCl.Theverylargeslopesofanodicandcathodic branchesofcurvesindicatemixedkineticanddiffusioncon- trolledelectrodeprocessesforallsamples.

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***

*** ***

***

**

*

Control Ti6AI4V HAp mHAp 120

100

80

60

40

20

0

% cell viability

Time / days

2. day 6. day 14. day

p=0.94

Figure5–Cellviabilitypercentageontheinvestigated samplescomparedtopositivecontrol.Positivecontrol:

MG-63cellsweregrowninwellplateswithoutsamples.

Thelevelofthestatisticalsigniﬁcanceisgivenbyp-values ascomparedtocontrolandtitaniumsubstrate.Allsamples weremeasuredin6replicateandcalculatedthemean values±standarddeviation.

Control Ti6AIaV HAp mHAp p = 0.094

* *

*

120 **

100

80

60

40

20

0

ALP activity %

6. day 14. day

Time / days

Figure6–ALPexpressionpercentageontheinvestigated samplescomparedtopositivecontrol.Positivecontrol:

MG-63cellsweregrowninwellplateswithoutsamples.

Thelevelofthestatisticalsigniﬁcanceisgivenbyp-values ascomparedtocontrolandtitaniumsubstrate.Allsamples weremeasuredin6replicateandcalculatedthemean values±standarddeviation.

Theelectrochemicalparameters,suchaspassivecurrent densities,corrosioncurrentdensitiesandcorrosionpotentials ofdifferentsamplesaresummarizedinTable5.

Itis visible that the titanium alloy substrate possesses the lowest passive current density (0.91Acm⁻²), whilethe highestvaluebelongs tomulti-elementdopedHApcoating (3.30Acm⁻²). On the other hand, it can also be observed ontheanodicbranchofpotentiodynamiccurvesthatwhile thepassivecurrentsofmHApsamplesslightlydecreasewith potentialscan,thepassivecurrentsofsubstratematerialand HApcoatingarestableandhardlychangetillthebreakdown potential.

Thecorrosioncurrentdensity(jcorr)valuesandcorrosion potentials(Ecorr)canbeobtainedbytheintersectionoflines extrapolatedtothecathodicandanodicbranchofpotentio- dynamiccurves inthe Tafelregion(±50mVfrom corrosion potential).Thetitaniumalloyhasthenoblestcorrosionpoten- tialand lowestcorrosioncurrentdensity whichdenotesits highestcorrosionstability.Ontheotherhand,themostneg- ative Ecorrand the highestjcorrvaluesbelong tothemHAp samples.Thisresultcanprovethatduringimmersioninphys- iological solution, dissolutionprocesses ofdifferentdoping elementsaswellascalciumphosphatecomponentscanoccur.

Thereareseveralresearchworksinvestigatingthedegra- dation processes of hydroxyapatite coatings prepared by differentmethods. Itisreportedthat theporous character- istic (sizeand number of pores present in the coating) of calcium phosphate coatings signiﬁcantlyaffects the corrosion/dissolution rate of hydroxyapatite. The coatings with smallerandfewerporesprovedtobemorecorrosionresis- tantthancoatingswithhigherdegreeofporositybecausethe formercanprovidebetterbarrierproperty[38–40].

Zhangetal.[32]statedthatthecorrosionmechanismof HApcoatingwithporesinvolveshydrogenion(H⁺)generation attheinterfacewherecorrosionoccurs,thusdecreasingthe localpHvalue,andthencausessubsequentdissolutionofHAp inthehighH⁺concentrationarea.ThedissolutionrateofHAp increaseswithdecreasingpH.

Biocompatiblemeasurementsonsamples

CellviabilitymeasurementwithWST-8assay

Fig.5showsthatinallcultureperiodthemHApsamplehad thehighestcellviabilityvalues,after2daysitwas85%while aftertwoweeksitincreasedtoaround90%comparedtopos- itivecontrol.

Thecell viabilitypercentageswere 78% and 85%after2 days,81%and90%after2weeksofcultureonpureHApand multi-ionmodiﬁedHApcoatings,respectively.Foruncoated titanium,theviabilitywas81%at2nddayanditdecreased to71% at14thday. After2daysofculture,the differences betweenthecellviabilityvalueswerenotstatisticallysignif- icant forHApcomparedtotitaniumsubstrate (pvaluewas 0.94), whilethedifference betweenTialloyandmHApwas statisticallydifferent(p<0.01).Itisvisiblethatthereisaslight decrease incell viabilityforeach sampleafteroneweekof incubation.Thisphenomenoncanbeexplainedbycelldiffer- entiation.Severalresearchersprovedthatwhencellsarein thestateofdifferentiation,theyshowlessmetabolicactivity resultinginlowerviabilityvalues[41,42].

After2weeksofcultureinDMEMmediumthedifference between the cell viability on HAp and on mHAp samples become moresignificantly higher than those foruncoated substrate,indicatingthegoodbiocompatible/bioactiveprop- ertiesofbothhydroxyapatitelayers.Itisalsovisiblethatthe multi-elementmodificationadvancedthebiocompatibilityof sample.Thedifferencesbetweenthecellviabilitiesofsam- plesinthistimepointwereallstatisticallyhighlysignificant (p<0.001).Inaddition,it iswell knownthathydroxyapatite coatingfacilitatetheattachmentandgrowthofosteoblastic cellsowingtoitshighhydrophilicproperty[43,44].

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Alkalinephosphataseactivitymeasurements

ALP is one of the ﬁrst osteoblastic markers. Since the osteoblast-likehumanMG-63celllineiscapabletoproduce someosteogenicmarkerssuchasalkalinephosphataseand osteocalcin[45].InourpresentstudyALPexpressionofcells seededonthesurfaceofdifferentsamplesandonculturewell plateasreferencewasevaluated.

It is visible in Fig. 6 that the ALP expressionis higher byaround 25%and30% forpureHApand multi-iondoped HAp,respectively,after6and14daysofculturethanthatfor uncoatedsubstrate.ThelevelofALPactivityincreasedwith culturingtime. After 6 daysof immersion,the ALP values ofbothHApand mHApwere statisticallydifferent(p<0.05) comparedtouncoatedsubstrate,whiletherewasnostatis- tically difference between the calcium phosphate coatings andthe controlgroup.Atthe14thday ofculture,onlythe ALPvaluesofmHApcomparedtoTi alloyandALP expres- sionofcontrolcomparedtoTialloywerestatisticallydifferent (p<0.05).ItisvisiblethatthehighestALPexpressionbelongs tomHApsample.Ontheotherhand,thedifferencesbetween HApand mHApaswell asbetweentitanium substrateand HAp are not statistically different, in the latter case the pvalue is 0.094. Our ﬁndings are ingood agreement with reportsfromliteraturewhereZhaoetal.[46]studiedtheeffect of magnesium-substitutednano-hydroxyapatite coating on implant osseointegration.Intheirresearchtheyfoundthat themagnesiumsubstitutedHAphadhigherALPactivityby twotimesthanthat ofwithoutmagnesium contentafter7 daysofculture.Yangetal.[47]investigatedthebiocompatibil- ityofZnsubstitutedhydroxyapatiteonMurinepreosteoblast cell(MC3T3-E1)cellline.Theyreportedsigniﬁcantincreasein cellproliferationandALPactivityonday7,andosteocalcin

production(p<0.05)were alsoobservedforZn²⁺-containing HAp-coatedsurfacesonday14.Thecoatingswereprepared byelectrochemicalprocessandtheZnwaspresentintheZn- HApcoatingsataZn/(Ca-Zn)molarratioof1.04%.Buenoetal.

[48] studiedthe effectofSr substitution inHApnanocom- positeonthedifferentiationofOFCOLLIIosteoblasts.Other literaturereportshowedthatthepresenceofstrontiuminthe HApstructure(SrHAp) seemstocauseimportanteffectsin osteoblastandosteoclastgrowthandalsofavorstheincrease ofosteoblastALPactivity [49]. Thianet al.[50]investigated theeffectofapatitenanocrystalsontheosteoblastbehavior ofhumanosteoblast(HOB)cellsandtheyfoundthattheALP activityofcellsgrowingonphase-pureapatitenanocrystals wasdetectableonlyafter5daysofculture.

Calcein/DAPIstaining

Directﬂuorescencestainingofcalceinandnucleus(DAPI)of MG-63cells culturedfor2daysontitanium alloy,HApand mHApcoatingsaswellasoncontrolgroup(wellplates)are showninFig.7.

Calceinﬂuorescentstainingisgenerallyusedtoindicate intracellular esteraseactivity present inviable cells.Dense and evenly dispersed multi-layered cells with large nuclei were observedforallsamples,however,inthecaseofHAp andmHApcoatedsamplestherewerelargernumberofliving cells.Theshapeofcellsmainlyelongatedandpolygonalwhich indicateswelladhered,spreadingandproliferatingcells.

MG-63cellmorphologystudy

Theexpressionofthephenotypeofosteoblast-likecells(MG- 63)wasstudiedbySEMafterincubationonuncoatedtitanium

a

c b

50 µm

Figure7–Fluorescencemicroscopyimagesofcalcein-AM(greenﬂuorescent)andnucleus(withDAPI,blueﬂuorescent)and mergedimagesofMg-63cellsculturedfor2daysinDMEMmediumondifferentsamplessuchastitaniumalloy(a)HAp(b) andmHAp(c)coatings.

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a

b

c

10 µm

Figure8–SEMimagesonMG-63cellsgrownontitanium substrate(a)onHApcoating(b)andonmHApcoatingafter 2daysofcultureinDMEMmedium.

alloy,onpureHApcoatingandonion-modifiedHApcoatings for48h.ItisobviousthatthephenotypeofMG-63osteoblast- likecellswerewell-expressedandcellwerespreadedonthe surfacesofallsamplesandwereinflattenedform.Theshape ofcells mainlypolygonalwithfilopodiaorverythin exten- sions.Thecellscoveredthecoatedsamples’surfacesinathick continuousmonolayerandtheMG-63startedtoformalsoa multilayerinsomeareasofthesample.Ontheotherhand,in thecaseofuncoatedsubstrate,thecoveragewasnotperfect.

Insomeplacesthesurfaceofsubstrateisalsovisiblebeside thecells(seeinFig.8a).Thenumberanddensityofcellsaswell astheextentofspreadingseemedtobealittlehigherinthe caseofcalciumphosphatecoatedsamplesthanforuncoated substrate.Nevertheless,thereisnotmuchvisibledifference

incellmorphologyinthecaseofbothHApandmHApcoated samples. Theseresultsmight conﬁrmthat thecoating can advancecelladherencethuspromotingcellproliferationand provetheresultsfromCalcein/DAPIstaining.

Conclusion

TheSEManalysisrevealedthatthemorphologyofHApand mHAp coatings wasmainly needle-like in nanometresize.

The crosssection analysis (FIB)showed the coatings tobe inhighlyporous,sponge-likestructure,whichresemblesthe structureofnaturalbone.TheEDXelementalanalysiscon- ﬁrmedthattheionsdopedHApcoatingcontainedAg,Zn,Sr andMgelementsalsoinunder1At%alongwiththecalcium andphosphorouselements.TheFT-IRspectrashowedsimi- larcharacteristicpeaksofPO₄³⁻ andOH⁻anionicgroupsof calciumphosphatephasesandrevealedcarbonateimpurities inbothsamples.TheXRDmeasurementsalsoconﬁrmedthat thecoatingconsistofmainlynanocrystallinehydroxyapatite phaseandtherewasnovisiblelineshifting,peakbroaden- ingandchanginginpeakintensitywhenmetallicionswere addedtothehydroxyapatitecoating.Accordingtothecorro- sion measurements,the corrosion resistancesofpureHAp andmulti-iondopedHApwerelowerthanthatofuncoated substrateduetothehighlyporouscharacteristicoflayers.

Thebiocompatibletestsshowedthatthecellviabilityval- uesincreasedsigniﬁcantlyinthecasesofbothHApandmHAp samplescomparedtobareimplantmaterialsand thehigh- estvaluesweremeasuredinthecaseofmHAp.TheCalcein andDAPIstainingofsamplesrevealeddense,multi-layered, welladheredlivingcellsonallsampleswithnormalmorphol- ogy.TheinvitroresultspresentedheresupportthatHApand multi-iondopedHApcoatingsadvancethegrowthofMG-63 osteoblast-likecells.

Acknowledgements

Theauthorswouldliketoacknowledgetheﬁnancialsupport ofJECSTrustandtheauthorsaregratefulfortheSEM-FIB/EDX measurementsperformedbyLeventeIllés(MTA-EK,Hungary).

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