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BiomimeticfabricationofantibacterialcalciumphosphatesmediatedbyLuciaForte,PaolaTorricelli,FrancescaBonvicini,ElisaBoaniniGiovannaAngelaGentilomi,GigliolaLusvardi Correspondingauthorat:DepartmentofChemistryG.Ciamician,UniversityofBologna,Italy.E-mailaddress:(E.Boanini). formedunderslightlybasicconditionsbytheoxidativepolymerizationofdopamine,whichcreatesastablelayerthatisadherenttothesurfaceofmaterialsmaterials.StablePDAlmswithcontrolledthicknesscanbedepositedonvirtuallyanysubstrate.Moreover,PDAisbiocompatibleandexhibitsseveralfunctionalgroups,suchascatechol,amineandiminegroups,whichareabletoreactwithawiderangeofmoleculesmolecules.Inparticular,thecatecholgroupcanoxidizeintothecorre-spondingquinonegroupandtriggerreductionprocessesofmetallicmetallic.ThepeculiarcharacteristicsofPDApromptedanumberofstudiesthatyieldedanincreasingnumberofnewapplicationsofPDAbasedmaterials,spanningindields,fromenergytoenviron-ment,tobiomedicalsciencescience.Hereinweoptimizedtheexperi-mentalconditionstopreparePDAfunctionalizedOCPandTCPatincreasingAgNPscontents,andweinvestigatedtheinuenceofthepresenceofAgNPsontheantibacterialpropertiesofthecompositematerialsagainstGrampositiveandGramnegativereferencebacterialstrains,aswellasagainstantibiotic-resistantclinicalisolatesrecoveredfrompatientswithboneorprostheticjointinfections.Moreover,wetestedtheabilityofthematerialstosupportinvitrogrowthanddif-ferentiationofosteoblast-likecellsMG63.MG63activityanddientiationwereinvestigatedusingthemostcommonmarkersofosteo-blastmetabolism,whichweretestedbothonthesupernatantsofcellcultureandbymeansofquantitativePolymeraseChainReaction2.Materialsandmethods2.1.MaterialssynthesisandcharacterizationThesynthesisofOCPwascarriedoutaspreviouslyreportedreported.Briey,250mlof0.04MCa(CHOwereaddeddropwiseinto750mlofaphosphatesolutioncontaining5mmolofNaand5mmolofNaHOpreviouslyadjustedtopH5withCOOH.Thereactionwascarriedoutat60°Cundermechanicalstirring.After15mintheprecipitatewasltered,repeatedlywashedwithdistilledwateranddriedat37°C.TCPwasobtainedbysolid-statereactionofamixtureofCaCOandCaHPOOinthemolarratioof1:2at1300°Cfor5hh.Thesolidproductwascarefullygroundandsieved(40m)beforebeingsubmittedtofurthertreatment.Functionalizationwithpolydopaminewasobtainedthroughim-mersionof100mgofOCPorTCPinto50mlofasolutionofdopaminehydrochloride(2mg/ml)atpH8.5(TRISbuer),understirringstirring,fordierentperiodsoftime(1,2and18h)atroomtemperature.Then,thesampleswereltered,repeatedlywashedwithdistilledwateranddriedat37°Covernight.Inthefollowing,thesamplesfunctionalizedwithPDAareindicatedasOCPdandTCPd,respectively.Depositionofsilvernanoparticleswasobtainedbysimultaneousadditionof20mlofAgNOsolution,atdierentconcentrations(1,5and10mM),and20mlofa13mMsodiumcitratesolution,tothePDAfunctionalizedphosphates(100mg)atroomtemperatureunderstir-ring.After1h,thesolidsampleswereltered,repeatedlywashedwithdistilledwateranddriedat37°Covernight.ThetwoseriesofsampleswerelabeledrespectivelyOCPdAgXandTCPdAgX,whereXindicatestheconcentration(mM)ofAgNOX-raydiractionanalysiswascarriedoutbymeansofaPANalyticalX'PertPROpowderdiractometerequippedwithaX'Celeratordetector(40mA,40kV).Forphaseidenticationthe2rangewasinvestigatedfrom3to602°withastepsizeof0.1andtime/stepof100s.ThermogravimetricanalysiswascarriedoutusingaPerkin-ElmerTGA-7.Heatingwasperformedinaplatinumcrucibleinair(20cm/min)atarateof10°C/minupto700°C.Thesamplesweightswereintherange510mg.ResultsfromthisanalysisrepresentthemeanvalueofdeterminationsforthreedierentsamplesofeachMorphologicalinvestigationsofcrystalswereperformedusingaPhenomProXdesktop-scanningelectronmicroscopeatbeamaccel-erationvoltageof10kV.Thesampleswereobservedaspreparedandnotsputtercoatedbeforeexamination.ThequanticationofAgwasperformedusingaquadrupolemassspectrometerwithplasmasourceICPMSXSeriesThermoFisherc.Thesampleswerepreparedbydissolving10mgofpowdersinwater/methanolsolution1/1V/V.5mlweretakenandaddedin50mlof5%nitricacidsolution.Staticcontactanglemeasurementswereperformedondiskshapedsamples(Ø=13.0mm).Eachdiskwaspreparedbypressing100mgofpowderintocylindricalmouldsbyusingastandardevacuablepelletdie(Hellma).AKSVCAM101instrumentwasusedunderambientcondi-tionsbyrecordingthesideprolesofdeionizedwaterdropsforimageanalysis.Theshapeofthedropwasrecordedinatimerangeof060s,bycollectinganimageevery0.033s.Atleastsixdropswereobservedforeachsample.Invitrocellandantibacterialtestswereperformedondisk-shapedsamples(Ø=6.0mm).Eachdiskwaspreparedbypressing40mgofpowderintocylindricalmouldsbyusingastandardevacuablepelletdie(Hellma),andsterilizedusinggammarays(Cobalt-60)atadoseof25kGy.AFManalysisofthedisk-shapedsampleswasperformedusingaVeecoNanoscope3Dinstrument.ThesampleswereanalyzedintappingmodeusinganEscanner(maximumscansize15m)andphosphorus(n)dopedsiliconprobes(springconstant2080N/m;resonancefre-quency250290kHz;nominaltipradius10nm).Roughnesspara-meters,namelyarithmeticmeanroughness(Ra),root-squareroughness(Rq),andtheverticaldistancebetweenthehighestandlowestpointswithintheevaluationlength(Rt),wererecorded.Silverreleasewasmeasuredinthemediumusedforcellcultureerentiation(seeSection2.2.2.).Thesurnatantswereremovedfromthewellsat1,2and7daysandAgcontentwasanalyzedusingaquadrupolemassspectrometerwithplasmasourceICPMSXSeriesThermoFisherScienti2.2.Cellulartests2.2.1.CytotoxicitytestsHumanosteoblast-likecellsMG63(OB,IstitutoZooproSperimentaleIZSBS,Brescia,Italy),wereculturedinDMEMmedium(Dulbecco'sModiedEagle'sMedium,Sigma,UK)supplementedwith10%FCS,andantibiotics(100U/mlpenicillin,100g/mlstrepto-mycin).Cellsweredetachedfromcultureasksbytrypsinization,andcellnumberandviabilitywerecheckedbyErythosineBdyeexclusiontest.OBcellswereplatedatadensityof5×10cells/mlin24-wellplatescontainingsteriledisk-shapedsamples(Ø=6.0mm)(ofthefollowingbiomaterial:OCPdAg1,OCPdAg5,OCPdAg10,TCPdAg10.Wellsfornegative(CTR,DMEMonly)andpositive(CTR+,DMEM+0.05%phenolsolution)controlswerealsoprepared.Plateswereculturedinstandardconditions,at37±0.5°Cwith95%humidityand5%±0.2COupto72h.Thequantitativeevaluationofcytotoxicitywasperformedbymeasuringcellviability,lactatedehydrogenaseenzyme(LDH)release,Interleukin-6(IL-6)andCaspase3activity.Cellproliferationandvia-bilityat24and72hwasassessedbyWST1(WST1,RocheDiagnosticsGmbH,Manheim,Germany)colorimetricreagenttest.Theassayisbasedonthereductionoftetrazoliumsaltintoasolubleformazansaltbyareductaseofthemitochondrialrespiratorychain,activeonlyinviablecells.100lofWST1solutionand900lofmedium(naldi-lution:1:10)wereaddedtothecellmonolayer,andthemulti-wellplateswereincubatedat37°Cforafurther4h.Supernatantswereedspectrophotometricallyat450nmwithareferencewave-lengthof625nm.ResultsofWST1arereportedasopticaldensity(OD)anddirectlycorrelatewiththecellnumber.Proliferationpercentre-lativetoCTRarealsoreported.AttheendofexperimentaltimesthesupernatantwascollectedfromallwellsandcentrifugedtoremoveL.Forteetal. particulates,ifany,forLDHmeasure(LDHenzyme-kinetictest,Roche,D)andIL-6(ELISAkit.BosterBiologicalTechnology,Ca,USA)ac-cordingtomanufactureinstruction.Celllysatefromallgroupswerecollectedat72hforthedetectionofCaspase3activitybyanim-munoenzymatictest(Caspase-eELISAkit,Invitrogen,CA,USA).AqualitativeanalysisforcellmorphologywasperformedbyNeutralRed(NR)vitalstaining.A0.033%solutionofNRstaining(Sigma,UK)inculturemediumwasaddedtoeachgroupsamplesattheendofex-perimentaltime,forfurther90min.Cultureswereexaminedbyopticalmicroscopyfortheevaluationofcellmorphology.2.2.2.BioactivitytestsOBwereexpandedinDMEMsupplementedwith10%FCS,1%an-tibiotics(100U/mlpenicillin,100g/mlstreptomycin),ophosphate(10M)andAscorbicacid(50g/ml),countedandplatedonmaterialsamples(OCP,OCPdAg5,TCPdAg5)ataconcentrationof2×10cells/wellin24wells-plates.OBwerealsoseededinemptywellsforcontrol(CTR).Plateswereincubatedat37°Cinahumidied95%air/5%COatmosphere(standardcondition)upto7days.Mediumwaschangedwithfreshmediumtoallwellsafter3daysofculture.2.2.2.1.Cellviability.At7dayscellviabilitywasobservedbytheLive/assay(MolecularProbes,Eugene,OR,USA),accordingtothemanufacturer'sinstructions.Sampleswerevisualizedusinganinvertedmicroscopeequippedwithanepiuorescencesetup(EclipseTiU,NIKONEuropeBV,NITALSpA,Milan,Italy):excitation/emissionsettingof488/530nmtodetectgreenuorescence(livecells)and530/580nmtodetectreduorescence(deadcells).OBproliferationandviabilitywasquantitativelyevaluatedbyWST1attheendofexperimentaltimeaccordingtothemethoddescribed2.2.2.2.Osteoblastactivity.Attheendofexperimentaltime,after7daysofculture,thesupernatantandcelllysatewerecollectedfromallwells.AliquotsweredispensedinEppendorftubesforstorageat70°Candassayedwiththefollowingimmunoenzymatickits:AlkalinePhosphatase(ALP,Cloud-CloneCorp,Wuhan,China),andOsteocalcin(OSTC,Cloud-CloneCorp),Caspase3(LifeTechnologies,Frederick,MD,USA).AllresultswerenormalizedbyCTRgroup.2.2.2.3.Osteoblastmorphology.Attheendofexperimentaltime,osteoblastswerexed(2.5%glutaraldehydeinPBSpH7.4for1h),dehydratedinagradedethanolseries,andthentreatedwithhexamethyldisilazane.SEMimageswerecollectedusingaHitachiS-2400instrumentoperatingat15kVonPdcoatedsamples.2.2.2.4.QuantitativePolymeraseChainReaction(qPCR).TotalRNAwasextractedfromallsamplesattheendofexperimentaltimeusingPureLinkRNAMiniKit(LifeTechnologies,Carlsbad,CA,USA).PuriRNAwasreversetranscribedwithSuperscriptVILOcDNASynthesiskit(Invitrogen,LifeTechnologies,Carlsbad,CA,USA),followingmanufacturer'sinstructions.Eachsample(10ng)wastestedinduplicate.qPCRanalysiswasperformedinaLightCyclerInstrument(RocheDiagnosticsGmbH,Mannheim,Germany)usingtheQuantiTectSYBRGreenPCRkit(Qiagen,Hilden,Germany).Theprotocolincludedadenaturationat95°Cfor15,40cyclesofamplication(95°C15appropriateannealingtemperatureforeachtargetasdetailedinTableS1for20and72°Cfor20)andameltingcurvetocheckforampliconcity.Thethresholdcycle(Ct)wasdeterminedforeachsample.Relativegeneexpressionwascalculatedwiththe2method,usingthecontrolsamplesascalibrator.2.2.3.StatisticalanalysisStatisticalevaluationofdatawasperformedusingthesoftwarepackageSPSS/PC23.0(SPSSInc.,Chicago,ILUSA).Theresultspresentedarethemeanofsixindependentvalues.Dataarere-portedasmean±standarddeviations(SD)atasignicancelevelofp0.05.Afterhavingveriednormaldistributionandhomogeneityofvariance,aone-wayANOVAwasdoneforcomparisonbetweengroups.Finally,aposthocmultiplecomparisontestswasperformedtodetectsignicantdierencesamonggroups,andPearsontestwasperformedtodetectcorrelationbetweendata.2.3.BacterialstrainsandantibacterialsusceptibilitytestinginvitroantibacterialactivityoftheOCPdandTCPdsamplesloadedwithdierentamountsofAgwasevaluatedagainstapanelofGrampositiveandGramnegativereferencebacterialstrainsincludingStaphylococcusaureus(ATCC25923),StaphylococcusepidermidisEnterococcusfaecalis(ATCC29212),EscherichiacoliKlebsiellapneumoniae(ATCC9591)andPseudomonasaeruginosa(ATCC27853).Subsequently,havingdenedboththeoverallspectrumofantibacterialactivityandthecytotoxicproleofthetestedsamples,theOCPdAg5andTCPdAg5materialswereassayedtowards14clin-icalisolatesrecoveredfrompatientswithboneorprostheticjointin-fections(7and7strains).ClinicalstrainswereisolatedonBDColumbiaAgarwith5%sheepblood(BectonDickinson,GmbH,Germany)andconrmedbyMALDI-TOFMSanalysis(BrukerDaltonics,GmbH,Germany)Germany).Theirantibioticsuscept-ibilitywasdeterminedbyusingtheVitek2semi-automatedsystem(bioMerieux,France);EUCASTcriteriawereusedfortheinterpretationofresultsandcategorizationofstrains(Sensitive,IntermediateorRe-sistantstrains)().Isolatesresistantto3antibioticgroupstestedwereconsideredmulti-drugresistant(MDR).TheeectivenessofthecompositematerialstoinhibitbacterialgrowthwasdeterminedbymeansofstandardizedsensitivitytestsbasedonKirby-Bauer(KB)diusionmethodandfollowingtheproceduresestablishedbyanumberofcommitteescommittees.Briey,thesurfaceofMueller-Hintonagarplate(MHA)(Sigma-Aldrich)wasinoculatedwiththebacterialsuspensionat0.5McFarland,preparedinsterile0.9%salinesolutionandgammarayssterilizeddisks(=6.0mm)wereplacedontheagarplates.IncompliancewiththeInternationalgui-dancedocumentsinsusceptibilitytesting,diskscontaininggentamicin(GMN10g)and/orimipenem(IPM10g)(OxoidSpA,Italy)wereincludedasreferencecontrols.After24hofincubationat37°Ctheagarplatewasobservedandthediameteroftheinhibitionzone(corre-spondingtothebacterial-freezonearoundthedisk-shapedsample)wasandexpressedinmillimeters(mm).Allexperimentswereperformedonduplicateindierentdays.3.Resultsanddiscussion3.1.MaterialscharacterizationTheamountofPDAdepositedonOCPandTCPcanbeevaluatedthroughthermogravimetricanalysis.Infact,PDAundergoesthermaldecompositionbetweenabout250and650°C,anditsrelativecontentcanbedeterminedasdierencebetweenthetotalweightlossoffunctionalizedsamplesandthatofthepristinecalciumphosphates(Fig.S1).ThedatareportedinTable1indicatethattheamountofdepositedPDAincreaseswithimmersiontime,inagreementwithpreviousstudiesstudies.However,theamountofPDAdepositedontoTCPisalwayscantlyhigherthanontoOCP,mostlikelybecauseofthedimorphologyofthetwocalciumphosphates.Infact,SEMimagesofOCPTCPimmersedindopaminesolutionfordierentperiodsoftime(Fig.S2)conrmthegreateramountofthepolymerdepositedontoTCPporousparticlesthanontoOCPatcrystals.However,PDAontoTCPappearsclusteredintoaggregateswhereasitseemsmorespreadinclosecontactwiththelargesurfaceofOCPcrystals.Moreover,thesamplesobtainedafterimmersionfor18hshowamassivedopaminepolymerizationthatoccurswithoutacloseL.Forteetal. relationshipwiththecalciumphosphates.Onthisbasis,depositionofAgNPswascarriedoutonPDAfunctionalizedsamplesobtainedusinganimmersiontimeof2h.ThedepositionofPDAprovokesalsovariationsinthevaluesofcontactangleandtimerequiredforwatercompletespreadingonthesurface,whichincreasewithPDAcontent(Table1).ThisndingissomewhatoppositetowhatusuallyveriedinotherstudieswherePDAdepositioninducesadecreaseofthecontactanglewithrespecttothatofthesupportsupport.However,atvariancewiththesupportsusedinthosestudies,bothOCPandTCParehighlyhydrophilic,inagreementwiththeirlowcontactanglesandwaterspreadingtimes(Table1).ThemodestincreaseofthevaluesoftheseparametersonincreasingPDAcouldbeascribedtothelesshomogeneoussurfacesofthecomposite Fig.1.XRDpatternsofthedierentsamples.Themaindif-fractionpeakduetoAgisindicatedwith().Therelativein-tensityofthisreectionincreasesonincreasingtheconcentra-tionofthesilvernitratesolution. Fig.2.SEMimagesof(a)OCPdAg1,(b)OCPdAg5,(c)OCPdAg10,(d)TCPdAg1,(e)TCPdAg5,(f) Table1Polydopaminecontent(determinedthroughthermogravimetricanalysis)ofthedisamplesasafunctionoftime.Thevaluesofcontactanglesandwaterabsorptiontimearealsoreported.SubstrateTimePDAcontentStaticcontactangleAbsorptiontime28±12OCPd1143±211OCPd22.1±0.545±2&#x-298;&#x.2 0;15OCPd1810.4±1.065±323±21TCPd12.4±0.532±11TCPd26.5±0.753±23TCPd1825.7±2.255±212L.Forteetal. samplesduetothepresenceofthepolymer.TheXRDpatternsofthefunctionalizedcalciumphosphatesafterAgNPsdepositionshowthecharacteristicreectionsofOCP(PDF26-1056)andTCP(PDF9-348)respectively,togetherwithareectionatabout38.1°of2correspondingtothemostintensepeakofsilver(PDF4-783)(Fig.1).TherelativeintensityofthisreectionincreaseswiththeconcentrationoftheAgNOsolutionusedforthedeposition.ThepresenceofAgNPsontothesurfaceofOCPdandTCPdisappreciablealsointheSEMimagesofthecompositematerials.Inparticular,AgNPsappearmorehomogeneouslydistributedontheOCPdcrystalssurfacesthanonTCPdparticles(Fig.2TheresultsofICPinvestigationreportedinTable2indicatethattheamountofAgNPsassociatedtoOCPdandTCPdincreaseswithsilverconcentrationinsolutionuptoabout11.6and6.8wt%,respectively.ThegreaterdepositionofAgNPsontoOCPdcannotbeascribedtoagreaterpresenceofPDA,sinceitscontent(2.1%)itssmallerthanonTCPd(6.5%),anditmightbeduetothemorphologyofthebigplate-likecrystalsofoctacalciumphosphate,whichfavorsamorehomo-geneousdepositionofPDA,and,asaconsequence,ofAgNPs.3.2.InvitrotestsInvitroosteoblastcytotoxicity,viabilityanddierentiation,aswellasantibacterialtestswereperformedondisk-shapedsamples,whichwerepreparedbypressingthesamplepowders,asreportedinandmethods.ThedierentmorphologyofOCPandTCParestillap-preciableonthesurfacesofthedisks,asresultsfromtheAFMimagesreportedinFig.3.ThemeanvaluesofroughnessparametersareRq=43±4nm,Ra=30±4nm,Rt=390±41nmforOCP,andRq=75±8nm;Ra=55±6nm;Rt=600±63nmforTCP.NoappreciabledierenceintheroughnessparametershasbeendeterminedafterAgNPsdepositiononbothsubstrates,althoughthesurfacemorphologyappearslessdened(Fig.3)mostlikelybecauseofthepresenceofPDA,whichgivesadierentinteractionwiththeAFMprobeincomparisonwiththeharderinorganicphases.SilverreleasefromOCPdAg5andTCPdAg5duringinvitrocelltestswasmeasuredincellculturemedium.ThecumulativereleasefromOCPdAg5andTCPdAg5increaseswithtimeandreachesvaluesofg/mland5.2g/mlrespectivelyafter7days,asshowninFig.4TheamountofsilverreleaseisgreaterfromTCPdAg5thanfromOCPdAg5,mostlikelyduetotheirdierentroughness,butitishoweververysmall,1wt%oftheinitialcontent.Thestructureandmorphologyofthediskshapedsamplesafter7daysimmersionincellmediumarecomparedwiththoseofthestartingsamplesinFigs.S3andS4.XRDpatternsofOCPdAg5andTCPdAg5donotshowanyappreciablemodicationsafterincubation.However,atvariancewithOCPdAg5,whichdoesnotshowanyap-preciablemorphologicalchange,theSEMimageofthesurfaceof Fig.3.AFMimagesofthesurfaceofdisk-shapedsamplesusedforin-vitrotests. Table2Agcontent(wt%)determinedthroughICPanalysisofsolidsamplesobtainedafterimmersionofOCPdandTCPdintosolutionsatdierentconcentrationofAgNOSamplesOCPdAg1OCPdAg5OCPdAg10Agcontent1.6±0.18.2±0.211.6±0.20.8±0.14.7±0.16.8±0.2L.Forteetal. TCPdAg5afterimmersiondisplaysthepresenceofsmallcrystals,mostlikelyduetodepositionfromthesupersaturatedmedium.Itiscon-ceivabletosupposethattheamountofdepositionisunderthedetectionlimitofXRDtechnique.3.2.1.CellcytotoxicityMG63areanosteoblastcelllinewellcharacterizedandwidelyusedinvitrostudies.Eveniftheydisplaysomedierencesincomparisontoprimaryosteoblastinmorphologyandproliferationrate,theyex-pressthemainmarkersofosteoblastdierentiationusefulforinvitrostudies,havetheadvantageofastandardbehaviourandarechosentoassessbiomaterialbiocompatibility.Attheendofexperimentaltimes,scheduledtestswereperformedtoanalyzecytotoxicityafterOBculturewithsamplesofOCPandTCPaterentAgNPscontent.ThepercentageofviabilityincomparisontoCTR-(consideredas100%)reportedinFig.5showsthatallsamplesvaluesarelowerthanCTR-,bothat24and72hofculture(p0.0005).Nevertheless,asamaterialisconsideredcytotoxicwhenitsviabilityislessthan70%incomparisontoCTR-,onlysamplesOCPdAg10andTCPdAg10maybeconsideredcytotoxiccytotoxic.Asex-pected,CTR+wassignicantlowerthanallexperimentalsamplesandCTR-.FurtheranalysisofpossiblecytotoxiceectswasperformedthroughevaluationofLDH,Caspase3andIL-6.LDHisanintracellularenzymeanditsdetectioninthesupernatantofcellcultureisconsideredasignofcytotoxicityasaconsequenceofcytoplasmaticmembranemembrane.At24hsignicantLDHlevelwasdetectedonlyinCPdAg10whencomparedtoCTR-.At72hOCPdAg10andallAgNPsTCPgroupsweresignicantlyhigherthanCTR-.CTR+wassignicantlydierentfromallgroupsbothat24and72h.Apoptosismechanism,expressedthroughextrinsicandintrinsicpathways,involvestheactivationofCaspase3throughacascadere-action.Thisproteaseactivityhasanessentialroleinproteindegrada-tionandcelldeathdeath.Moreover,OBinresponsetoacytotoxicagentproducedierentsolubleinammatorymediatorsasIL-6,thatplaysan Fig.5.ViabilityandcytotoxicitytestedonOBgrownonOCPorTCPwithdierentconcentrationofAg,after24or72hofculture.Statisticalsignicance:(*0.05;**p0.005;***p0.0005):WST1:***CTR+andCTR-vsallexperimentalsamplesLDH:***CTR+vsallatboth24and72h;***OCPAg10andTCPdAg10vsCTR-(72h);**TCPdAg5vsCTR-(72h);**TCPdAg10vsCTR-(24h);*TCPdAg1vsCTR-(72h)IL-6:***CTR+vsallatboth24and72h;*TCPdAg10vsCTR-(24h)CASPASE3:**CTR+vsall;*TCPdAg10vsCTR-. Fig.4.Silverionsreleasefromdisk-shapedsamplesofOCPdAg5andTCPdAg5afterincubationinthemediumusedforcellculturedierentiationupto7days.L.Forteetal. importantroleinlocalregulationofboneturnoverturnover.Caspase3(72h)andIL-6(24h)weresignicantlyhigherthanCTR-onlyinItisworthwhiletonoticethatAgNPscontentofTCPdAg10islowerthanthatofOCPdAg5andOCPdAg10,asshowninTable2.ItfollowsthattheabovereportedresultssuggestthatthecombinationofAgNPswithOCPprovidesmorebiocompatiblematerialsthanthoseobtainedusingTCPassubstrate.Representativepicturesofculturedcellsat24hafterNeutralRedstainingareshowedinFig.6.Allsamplesshowasignicantuptakeofdye,eveniftheydisplaydierentcellconcentration,Inparticular,thecellsonOCPdAg10andTCPdAg10displayirregularshape,atvariancewiththeothersamples,whichexhibitnormalcellmorphology.3.2.2.CellbioactivityOnthebasisoftheresultsofthecytotoxicitytests,furtherin-vestigationonbiocompatibilityandbioactivitywasperformedonOCPdAg5andTCPdAg5,aswellasonOCPandTCP.BothOCPandTCPareknowntobeeectiveinimprovingosteoblastactivityactivity–37].TheresultsofcytotoxicitytestsindicatethatthepresenceofAgNPsinOCPdAg5andTCPdAg5shouldnotinterferewithcellularactivity,whileexertingitslocalactiontopreventbacterialinfectionsinfections.3.2.2.1.Osteoblastviability.Theevaluationofcellproliferationandviabilitywasperformedafter7daysofculture,byLive/DeadstainingandWST1test(Figs.7and8respectively).EvenifahighernumberofcellswasobservedontoOCPincomparisontoTCP,picturesofosteoblastsdemonstratedthatcellsculturedontobiomaterials,withorwithoutAg,grewandproliferatedregularly,showinganormalmorphology,asconrmedbySEMimages,whichshowcellswellspreadandrichofphilopodia.Dierencesoncellnumberareappreciable,inagreementwithviabilityandproliferationtest(Fig.S5).CelladheredtothedierentsubstratesandtheiradhesionwasnotectedbythepresenceofAgNPs(Fig.7TheseresultswereconrmedbytheanalysisofWST1test:resultsshowedsignicanthighervaluesforOCPandOCPdAg5whencom-paredtobothTCPandTCPdAg5(Fig.8),conrmingthediuenceofOCPandonviability.Theviabilitypercentageat7daysofallgroups(OCP120%,OCPAg5111%,TCP73%,78%)resultedhigherthan70%incomparisonwithCTRgroup(con-sideredas100%),demonstratingabsenceofcytotoxicitycytotoxicity.More-overnodierencesinviabilitywherefoundbetweenOCPandOCPdAg5,andbetweenTCPand3.2.2.2.Osteoblastactivityandgeneexpression.Tocompletetheassessmentoftheeectsofthestudiedbiomaterialsonosteoblasts,theactivatedCaspase3,asakeyrolemoleculeinthemainpathwayofof,wasmeasured.TheproductionofALPandOSTCwasalsomeasuredat7days,astheyrepresentcommonmarkersoftheactivityofdierentiatedosteoblastsosteoblasts.After7daysofculturenoerencewasfoundforCaspase3neitherbetweenmaterialswithorwithoutAgNPs,norbetweenmaterialsandCTR(consideredequalto1)Fig.8).StatisticalanalysisfoundthatCaspase3valueswereinverselycorrelatedwithWST1data(p0.005),andinagreementwithLive/Deadstaining.Infact,asaverylownumberofreddeadcellswasobservedinallsamples(indicatedbyarrowsinFig.7),thelowernumberofcellsonTCPsamplesmayberelatedtoalowerrateofproliferationratherthantocelldeathforapoptosis.BothALPandOSTCdataafter7daysofcultureshowedsignicantdierencesincomparisonwithCTR(normalizedto1,p0.05),butnoerenceswerefoundamongmaterials(Fig.8).ResultsofALPactivityandOSTCproductiondemonstratedthatbothOCPandstimulatedosteoblastactivityanddierentiation,andthatthepresenceofAgNPsdidnotinterferewithculture.Inparticular,theresultsobtainedonOCPsamplestwellwiththoseofapreviousinvivovivothatdemonstratedthatOCPpromotesboneregenerationattheinitialboneappositionstageandundergoesrapidbiodegradation,suggestingthatOCPandOCPdAg5shouldpromotefastboneformationandbioavailabilityoftheantibacterialagent.Themeasureofgeneexpressionfortheproteinsdetectedinsuper-natantbyimmune-enzymaticassays,namely,and(OSTC),wasalsoperformedattheendofexperimentaltime(Fig.9genewasinvestigatedtoverifyifosteoblastmain-tainedtheirdidi.Theresults,comparedtoCTR,de-monstratedthatgeneexpressiondidnotdierfromCTRandthattherewerenodierencesamongbiomaterialsforAlthoughnodierenceswerefoundforOSTCproductiondetectedinculturesupernatant,expressioninOBculturedontoOCPatthesametimepointshowedasignicanthigherlevelincomparisonwithTCPandTCPdAg5.Toconcludetheanalysisofdata,werefoundtodirectlycorrelate(p0.005),asacon-rmationofdataconsistency. Fig.6.NeutralRedstainingofOBculturedfor72honOCP,TCPandCTRs.Livingcellsactivelyincorporatedreddye,andgrowadheringtothesubstrateshowinganormalmorphology.Deadcells,asCTR+image,didnotuptakedye,appearedroundanddetachedfromthesubstratesurface.L.Forteetal. Fig.8.Osteoblastviabilityandactivityafter7daysofcultureonOCP,OCPdAg5,TCP,andTCPdAg5.(*p0.05;**p0.005;***p0.0005).WST1.***OCPvsTCPdAg5;**OCPdAg5vsTCP;*OCPdAg5vs Fig.7.Live/deadstainingofosteoblastculturedfor7daysonOCP,OCPdAg5,TCP,andTCPdAg5.Cellsproliferatedregularlyontothesurfaceofallsamplesandadheredtothesurfaceofbiomaterials,showingnormalmorphology.Arrowspointoutdeadredcells.L.Forteetal. 3.3.AntibacterialsusceptibilitytestingagainstbacterialstrainsInapreliminarysetofexperiments,theinvitroantibacterialactivityofOCPdandTCPdsamplesloadedwithdierentcontentsofAgNPswasdeterminedagainstthreeGrampositive()andthreeGramnegative()referencestrains.Resultsdemonstratedthatallcompositema-terials,regardlesstheirAgNPscontent,inhibitedbacterialgrowthasaclearbacterial-freezonewasobservedaroundthedisksfollowinga24h-incubation.Inparticular,amongGrampositivebacteria,thehighestinhibitoryactivitywasdisplayedagainstwhiletheectivenessofthecompoundswasremarkabletowardsalltheGramnegativebacteriaanddidnotdieramongtestedstrains.Table3portsthediameterofthebacterial-freezonearoundthedisk-shapedsamplesofOCPdAg5andTCPdAg5(8.2and4.7%contentofAgNPs).Thesematerials,whichwereselectedastheydemonstratedtobeectiveagainstbacterialgrowthwithoutcytotoxicityonosteoblast,wereassayedagainstclinicalisolatesofpresentingdierentantibioticsusceptibilities.Inparticular,thepanelofsamplesincludedmethicillin-resistant(MRSE)and.Theantibiotic-resistanceproleofeachclinicalstrainandtheantibacterialactivitiesofthecompositematerialsarereportedinTable4BothOCPdAg5andTCPdAg5displayedantibacterialpropertiesagainstallthetestedclinicalisolatesandthecomparisonofthedia-metersoftheinhibitionzoneindicatesagreateractivityofOCPdAg5incomparisontoTCPdAg5,possiblyduetothehighercontentofAgNPs(8.2vs.4.7%).Notably,theseselectedbiomaterialsatthisAgNPscontentdidnotinterferewithosteoblastproliferationandactivityin-dicatingaspecicantibacterialproperty.Bothsamplesprovedtobeectivetowardsmethicillin-sensitiveandMRSEstrainsthatwereresistanttoerythromycinorgentamicinorlevo-oxacinortrimethoprim/sulfamethoxazole,inadditiontooxacillinandallother-lactamandcephalosporinantibioticsthatsharestructuralsimilaritywithmethicillin.Ofnote,testedsampleswerealsoactiveagainsttwoisolatesthatwereresistanttotheGMN/IPMreferencediskcontrolsintheKBdiusionmethod,andthatweresusceptibleonlytotetracyclineasdeterminedbyEUCASTclinicalzonediameterbreak-points.Consideringtheselectedstrains,bothOCPdAg5TCPdAg5showedaremarkableinhibitorypropertytowardsbothsensitiveandheterogeneousMDRphenotypes.4.ConclusionsTheresultsofthisstudyindicatethatpolydopaminecanbeusedasatooltotriggerthedepositionofsilvernanoparticlesontocalciumphosphates.ThedierentmorphologyofOCPandTCPplaysanim-portantrolebothonpolydopaminefunctionalizationandonAgNPsdeposition.Infact,PDAformsahomogeneouslayeronthesurfaceofthebigplate-likeOCPcrystalsandaggregatesontheTCPparticles.ThedierentarrangementofPDAcouldjustifythegreateramountofAgNPsloadedonpolydopaminefunctionalizedOCPcomparedtosurfaces.Moreover,thetwocalciumphosphatesupportsplayakeyroleonosteoblastbehaviortowardsnalcompositematerials:theamountofAgNPsthatdoesnotgiveanycytotoxicityissignicantlygreateron Table3Antibacterialactivity:diameteroftheinhibitionzone(inmillimeter)againstATCCre-ferencestrains.ReferencestrainsOCPdAg5TCPdAg5GMN10aureusATCC259237±17±120±1epidermidisATCC1222813±111±125±1faecalisATCC292126±17±112±1coliATCC2592212±112±119±1pneumoniaeATCC959110±19±118±1aeruginosaATCC2785312±112±119±1Allexperimentswereperformedonduplicate,ondierentdays.Diskscontaininggentamicin10g(OxoidSpA,Italy)wereusedaspositivecontrols. Fig.9.Geneexpressionofosteoblastculturedfor7daysonOCP,OCPdAg5,TCP,andTCPdAg5.(*p0.05;**p0.005;***p0.0005).BGLAP.***OCPvsTCPdAg5p0.0005.L.Forteetal. functionalizedOCP(8.2wt%)thanonfunctionalizedTCP(4.7wt%).UptotheseAgNPscontents,thedevelopedcompositematerialsprovidesuitablesupportsforosteoblastadhesion,proliferationanddientiation,whereastheydisplayaremarkableantibacterialactivityto-wardsthemainrelevantsensitiveandmulti-drugsresistantbacteria,demonstratinggoodpotentialityforthetreatmentofboneorprostheticjointinfections.Octacalciumphosphate-tricalciumphosphate-tricalciumphosphateCalciumphosphatesSilvernanoparticlesPDAfunctionalizedOCPPDAfunctionalizedAtomicForceMicroscopyScanningelectronmicroscopyX-raydiInductivelycoupledplasmamassspectrometryDulbecco'smodiedEagle'smediumTetrazoliumsaltLactatedehydrogenaseNeutralRedAlkalinephosphataseIL-6Interleukin6QuantitativePolymeraseChainReactionAlkalinephosphataseliver/bone/kidneyisozymeCaspase3Runtrelatedtranscriptionfactor2MALDI-TOFMSMatrix-assistedlaserdesorption/ionizationtimeof-ightmassspectrometryMulti-drugresistantTheauthorsaregratefultoRizzoliOrthopaedicInstitute(funds5×1000year2014,cod.6562)andtotheUniversityofBologna(RFO2015/16).AuthorsthankDr.LauraSicuroforinvitroexperimentsup-A.SupplementarydataSupplementarydatatothisarticlecanbefoundonlineatatO.Suzuki,Octacalciumphosphate:osteoconductivityandcrystalchemistry,ActaBiomater.6(2010)33793379O.Suzuki,S.Kamakura,T.Katagiri,M.Nakamura,B.Zhao,Y.Honda,R.Kamijo,Boneformationenhancedbyimplantedoctacalciumphosphateinvolvingconver-sionintoCa-decienthydroxyapatite,Biomaterials27(2006)26712671H.Imaizumi,M.Sakurai,O.Kashimoto,T.Kikawa,O.Suzuki,Comparativestudyonosteoconductivitybysyntheticoctacalciumphosphateandsinteredhydro-xyapatiteinrabbitbonemarrow,Calcif.TissueInt.78(2006)4545K.Kobayashi,T.Anada,T.Handa,N.Kanda,M.Yoshinari,T.Takahashi,O.Suzuki,Osteoconductivepropertyofamechanicalmixtureofoctacalciumphosphateandamorphouscalciumphosphate,ACSAppl.Mater.Interfaces6(2014)2260222602E.Boanini,P.Torricelli,M.Fini,F.Sima,N.Serban,I.N.Mihailescu,A.Bigi,Magnesiumandstrontiumdopedoctacalciumphosphatethinlmsbymatrixas-sistedpulsedlaserevaporation,J.Inorg.Biochem.107(2012)6565R.J.Dekker,J.D.deBruijn,M.Stigter,F.Barrere,P.Layrolle,C.A.vanBlitterswijk,Bonetissueengineeringonamorphouscarbonatedapatiteandcrystallineocta-calciumphosphate-coatedtitaniumdiscs,Biomaterials26(2005)52315231A.Bigi,E.Boanini,M.Borghi,G.Cojazzi,S.Panzavolta,N.Roveri,Synthesisandhydrolysisofoctacalciumphosphate:eectofsodiumpolyacrylate,J.Inorg.Biochem.75(1999)145145A.Bigi,E.Boanini,G.Falini,S.Panzavolta,N.Roveri,Eectofsodiumpolyacrylateonthehydrolysisofoctacalciumphosphate,J.Inorg.Biochem.78(2000)227227M.S.Tung,B.Tomazic,W.E.Brown,Theeectsofmagnesiumanduorideonthehydrolysisofoctacalciumphosphate,Arch.OralBiol.37(1992)585585O.Suzuki,H.Yagishita,M.Yamazaki,T.Aoba,Adsorptionofbovineserumalbuminontooctacalciumphosphateanditshydrolyzates,CellMater.5(1995)4545N.Miyatake,K.N.Kishimoto,T.Anada,H.Imaizumi,E.Itoi,O.Suzuki,Eectofpartialhydrolysisofoctacalciumphosphateonitsosteoconductivecharacteristics,Biomaterials30(2009)10051005Y.Shiwaku,T.Anada,H.Yamazaki,Y.Honda,S.Morimoto,K.Sasaki,O.Suzuki, Table4Antibacterialactivity:diameteroftheinhibitionzone(inmillimeter)againstclinicalisolates.TCPdAg5GMN10gIPM10gAntibiotic-resistanceproIsolate110±18±19±152±1CM,LVX,OX,TE,SXTIsolate210±17±127±124±1,LVX,OX,TE,SXTIsolate310±18±137±1,TE,SXTIsolate410±17±128±1,TEIsolate510±125±112±1CM,GMN,LVX,TEIsolate610±18±1,TEIsolate710±18±1,TEIsolate18±17±116±125±1AN,FEP,CAZ,CIP,GMN,IPM,MEM,TZPIsolate211±19±116±126±1,FEP,CAZ,CIP,GMN,IPM,MEM,TZPIsolate310±19±117±125±1,FEP,CAZ,CIP,GMN,IPM,MEM,TZPIsolate410±17±111±1,CSIsolate59±18±1NA11±1,FEP,CIP,CSIsolate67±17±1NA11±1,CIP,CSIsolate711±18±1,CIP,CSAN=Amikacin;CM=Clindamicyn;E=Erythromycin;FEP=Cefepime;CAZ=Ceftazidime;CIP=Ciprooxacin;GMN=Gentamicin;IPM=Imipenem;MEM=Meropenem;TZP=Piparacillin/Tazobactam;TGC=Tigecycline;CS=Colistin;LVX=Levooxacin;OX=Oxacillin;TE=Tetracycline;SXT=Trimethoprim/Sulfamethoxazole.R=Resistant;S=Susceptible;I=Intermediate,asdenedfollowingtheEUCASTguidelines.NA,notappearing.speciesresistanttooxacillinweredeclared,byconvention,methicillin-resistant.speciesdenedMDRasresistanttoatleastthreeagentsfromavarietyofantibioticclasses.L.Forteetal. Structural,morphologicalandsurfacecharacteristicsoftwotypesofoctacalciumuoride-containingapatiticcalciumphosphates,ActaBiomater.8(2012)44174417E.Boanini,S.Panzavolta,K.Rubini,M.Gandol,A.Bigi,Eectofstrontiumandgelatinonthereactivityofa-tricalciumphosphate,ActaBiomater.6(2010)(2010)M.Frasnelli,V.M.Sglavo,EectofMgdopingonbetaalphaphasetransitionintricalciumphosphate(TCP)bioceramics,ActaBiomater.33(2016)283283A.Bigi,S.Panzavolta,K.Rubini,Settingmechanismofabiomimeticbonecement,Chem.Mater.16(2004)37403740M.Bohner,A.K.Malsy,C.L.Camire,U.Gbureck,Combiningparticlesizedistribu-tionandisothermalcalorimetrydatatodeterminethereactionkineticsofa-tri-calciumphosphatewatermixtures,ActaBiomater.2(2006)343343K.Chaloupka,Y.Malam,A.M.Seifalian,Nanosilverasanewgenerationofnano-productinbiomedicalapplications,TrendsBiotechnol.28(2010)580580E.Boanini,P.Torricelli,M.C.Cassani,G.A.Gentilomi,B.Ballarin,K.Rubini,F.Bonvicini,A.Bigi,Cationic-anionicpolyelectrolyteinteractionasatooltograftsilvernanoparticlesonhydroxyapatitecrystalsandpreventcytotoxicity,RSCAdv.4(2014)645645H.Lee,S.M.Dellatore,W.M.Miller,P.B.Messersmith,Mussel-inspiredsurfacechemistryformultifunctionalcoatings,Science318(2007)426426Y.Liu,K.Ai,L.Lu,Polydopamineanditsderivativematerials:synthesisandpro-misingapplicationsinenergy,environmental,andbiomedicalelds,Chem.Rev.114(2014)50575057V.Ball,I.Nguyen,M.Haupt,C.Oehr,C.Arnoult,V.Toniazzo,D.Ruch,There-ductionofAg+inmetallicsilveronpseudomelaninlmsallowsforantibacterialactivitybutdoesnotimplyunpairedelectrons,J.ColloidInterfaceSci.364(2011)(2011)E.Boanini,M.Gazzano,K.Rubini,A.Bigi,Collapsedoctacalciumphosphatesta-bilizedbyionicsubstitutions,Cryst.GrowthDes.10(2010)36123612A.Bigi,E.Boanini,R.Botter,S.Panzavolta,K.Rubini,-Tricalciumphosphatehydrolysistooctacalciumphosphate:eectofsodiumpolyacrylate,Biomaterials23(2002)18491849A.Croxatto,G.Prod'hom,G.Greub,ApplicationsofMALDI-TOFmassspectrometryinclinicaldiagnosticmicrobiology,FEMSMicrobiol.Rev.36(2012)380[25]EUCAST:TheEuropeanCommitteeonAntimicrobialSusceptibilityTesting,BreakpointTablesforInterpretationofMICsandZoneDiameters,Version6.0,6.0,ClinicalandLaboratoryStandardsInstitute,PerformanceStandardsforAntimicrobialSusceptibilityTesting;Twenty-fthInformationalSupplement,CLSIdocumentM100-S25(2015)(2015)E.Ko,K.Yang,J.Shin,S.-W.Cho,Polydopamine-assistedosteoinductivepeptideimmobilizationofpolymerscaoldsforenhancedboneregenerationbyhumanadipose-derivedstemcells,Biomacromolecules14(2013)32023202T.S.Sileika,H.D.Kim,P.Maniak,P.B.Messersmith,Antibacterialperformanceofedpolymersurfacescontainingpassiveandactivecompo-nents,ACSAppl.Mater.Interfaces3(2011)46024602UNIEN,ISO10993-5-BiologicalEvaluationofMEDICALDEVICESPart5:TestsforInVitroCytotoxicity,(2009)(2009)A.Degterev,M.Boyce,J.Yuan,Adecadeofcaspases,Oncogene22(2003)(2003)R.Olivares-Navarrete,S.L.Hyzy,P.J.Slosar,J.M.Schneider,Z.Schwartz,B.D.Boyan,Implantmaterialsgeneratedierentperi-implantinammatoryfactors:poly-ether-ether-ketonepromotesbrosisandmicrotexturedtitaniumpromotesosteogenicfactors,Spine40(2015)399[32]S.Morimoto,T.Anada,Y.Honda,O.Suzuki,Comparativestudyoninvitrobio-compatibilityofsyntheticoctacalciumphosphateandcalciumphosphateceramicsusedclinically,Biomed.Mater.7(2012)045020,,,M.Kamitakahara,C.Ohtsuki,T.Miyazaki,Reviewpaper:behaviorofceramicbiomaterialsderivedfromtricalciumphosphateinphysiologicalcondition,J.Biomater.Appl.23(2008)197197E.Boanini,P.Torricelli,M.Gazzano,M.Fini,A.Bigi,Crystallinecalciumalen-dronateobtainedbyoctacalciumphosphatedigestion:anewchanceforlocaltreatmentofbonelossdiseases?Adv.Mater.25(2013)46054605E.Boanini,P.Torricelli,L.Forte,S.Pagani,N.Mihailescu,C.Ristoscu,I.N.Mihailescu,A.Bigi,Antiresorptionimplantcoatingsbasedoncalciumalen-dronateandoctacalciumphosphatedepositedbymatrixassistedpulsedlasereva-poration,ColloidsSurf.B:Biointerfaces136(2015)449449U.Mayr-Wohlfart,J.Fiedler,K.P.Gnter,W.Puhl,S.Kessler,Proliferationanderentiationratesofahumanosteoblast-likecellline(SaOS-2)incontactwitherentbonesubstitutematerials,J.Biomed.Mater.Res.57(2001)132132A.Ehara,K.Ogata,S.Imazato,S.Ebisu,T.Nakano,Y.Umakoshi,Eectsofalpha-TCPandTetCPonMC3T3MC3T3TetCPonMC3T3proliferation,dierentiationandmineralization,Biomaterials24(2003)831831M.B.Greenblatt,J.N.Tsai,M.N.Wein,Boneturnovermarkersinthediagnosisandmonitoringofmetabolicbonedisease,Clin.Chem.63(2017)464464M.Shahi,A.Peymani,M.Sahmani,Regulationofbonemetabolism,Rep.Biochem.Mol.Biol.5(2017)73L.Forteetal.