Combinatorial MAPLE deposition of antimicrobial orthopedic maps fabricated from chitosan and biomimetic apatite powders

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O

pen

A

rchive

T

OULOUSE

A

rchive

O

uverte (

OATAO

)

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To link to this article : DOI:10.1016/j.ijpharm.2016.07.015

URL :

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To cite this version : Visan, Anita Ioana and Stan, George E. and

Ristoscu, Carmen and Popescu-Pelin, Gianina and Sopronyi, Mihai

and Besleaga, Cristina and Luculescu, Catalin and Chifiriuc, Mariana

Carmen and Hussien, M.D. and Marsan, Olivier and Kergourlay,

Emmanuelle and Grossin, David and Brouillet, Fabien and Mihailescu,

Ion N. Combinatorial MAPLE deposition of antimicrobial orthopedic

maps fabricated from chitosan and biomimetic apatite powders.

(2016) International Journal of Pharmaceutics, vol. 511 (n° 1). pp.

505-515. ISSN 0378-5173

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Combinatorial

MAPLE

deposition

of

antimicrobial

orthopedic

maps

fabricated

from

chitosan

and

biomimetic

apatite

powders

A. Visan

a

,

G.E.

Stan

b

,

C. Ristoscu

a

,

G. Popescu-Pelin

a

,

M. Sopronyi

a

,

C. Besleaga

b

,

C. Luculescu

a

,

M.C.

Chi

ﬁriuc

c,d

,

M.D.

Hussien

c,d

,

O. Marsan

e

,

E. Kergourlay

e

,

D. Grossin

e

,

F. Brouillet

e

,

I.N.

Mihailescu

a,

*

a

NationalInstituteforLaser,PlasmaandRadiationPhysics,077125Magurele-Ilfov,Romania b

NationalInstituteofMaterialsPhysics,077125Magurele-Ilfov,Romania

c_Department_of_{Microbiology,}_Faculty_of_Biology,_University_of_Bucharest,₀₆₀₁₀₁_Bucharest,_Romania

d_Earth,_{Environmental}_and_Life_Sciences_Division,_Research_Institute_of_the_University_of_Bucharest,₇₇₂₀₆_Bucharest,_Romania e

UniversityofToulouse,CIRIMAT,UPS–INPT–CNRS,ENSIACET,4AlléeEmileMonso,31030ToulouseCedex4,France

Keywords: CombinatorialMAPLE Chitosan Biomimeticapatite Thinﬁlms Antimicrobialaction S.aureus E.coli. ABSTRACT

Chitosan/biomimeticapatitethinﬁlmsweregrowninmildconditionsoftemperatureandpressureby Combinatorial Matrix-AssistedPulsedLaserEvaporationonTi,Siorglasssubstrates.Compositional gradientswereobtainedbysimultaneouslaservaporizationofthetwodistinctmaterialtargets.AKrF* excimer(l=248nm,tFWHM=25ns)lasersourcewasusedinallexperiments.Thenatureandsurface

compositionofdepositedmaterialsandthespatialdistributionofconstituentswerestudiedbySEM,EDS, AFM, GIXRD, FTIR,micro-Raman,and XPS.The antimicrobialefﬁciencyofthechitosan/biomimetic apatitelayersagainstStaphylococcusaureusandEscherichiacolistrainswasinterrogatedbyviablecell countassay.

TheobtainedthinfilmswereXRDamorphousandexhibitedamorphologycharacteristictothelaser deposited structures composed of nanometric round shaped grains. The surface roughness has progressivelyincreasedwithchitosanconcentration.FTIR,EDSandXPSanalysesindicatedthatthe composition of theBmAp-CHT C-MAPLE compositefilmsgradually modifiedfrom pureapatiteto chitosan.

ThebioevaluationtestsindicatedthatS.aureusbiofilmismoresusceptibletotheactionof chitosan-richareasofthefilms,whilsttheE.colibiofilmprovedmoresensibletoareascontaininglesschitosan. Thebestcompromiseshouldthereforego,inouropinion,tozoneswithintermediate-to-highchitosan concentrationwhichcanassurealargespectrumofantimicrobialprotection concomitantlywitha significantenhancementofosseointegration,favoredbythepresenceofbiomimetichydroxyapatite.

1.Introduction

Natural polymersare currently employedto obtain tailored systemsfordrugpassive/activetargetinginordertodecreasethe incidence of the side effects (Kim et al., 2008). The natural polymersexhibitthemajoradvantageofbiodegradabilityinside thehumanbody,notrequiringremovaloradditionalmanipulation (NairandLaurencin,2006).Duetotheirexcellentbiocompatibility and cost-effectiveness they can be used as pharmaceutical excipients(Chiﬁriucetal.,2014).

Amongnaturalpolymersusedfordrugdelivery,chitosan(CHT) is a highlybiodegradable,non-toxic andbiocompatible cationic polysaccharidesynthesizedfromchitinbyalkalinedeacetylation (Vllasaliuetal.,2012;Sogiasetal.,2012;DerakhshandehandFathi, 2012;GanandWang,2007).Thechitosanpotentialtobeusedasan antimicrobial agent (Martins et al., 2014), was stressed upon together withdelivery ofantibiotics, suchas beta-lactams(e.g., penicillins, cephalosporins), aminoglycosides and daptomycin (Noel et al.,2008;Grumezescu et al.,2012; Grumezescu et al., 2011).

Nevertheless, CHT application in anti-infective strategies is limitedbecauseofitslowsolubilityatneutralorbasicpH.Forthis reason, the focus has been recently moved to the design of

* Correspondingauthor.

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antimicrobial ﬁlmsconsisting of CHT and its derivatives.They demonstratedexcellentinhibitoryactivityagainstawidespectrum of Gram-positive and Gram-negative bacteria, including food contaminants(Kongetal.,2010;Duttaetal.,2009;Lecetaetal., 2013;Daietal.,2011).

Recent studies revealedthe potential of CHT/hydroxyapatite (HA)compositestobeusedascoatingsontitaniumsurfaceinorder toincreasethe osseointegrationcapacity of boneimplants(Ma etal.,2014;Lietal.,2015).Thereexisthowever,afewstudiesonly, aimingtoevaluatetheanti-bioﬁlmactivity ofsuchcoatings,an aspect of key importance for the prevention and control of implant-associatedinfections.

Many authors have reportedthe preparation of mixtures of calciumphosphates(CaP)andCHTintheformofpowders(Yoshida etal.,2004),membranes(Itoet al.,1999), scaffolds(Zhang and Zhang,2002),ormicrospheres(Sivakumaretal.,2002). Neverthe-less,onlyfewpublicationswerefocusedondevelopingprocedures allowingthe concomitant preparation of a composite material containingthetwocomponents, whichis expectedtoensurea moreintimatecontactbetweenthem(Huetal.,2004;Davidenko etal.,2010;Thein-HanandMisra,2009).

CHT-CaP composite ﬁlms have been synthesized by several methodslike:pulsedelectrochemicaldeposition(Jiaetal.,2016a), electrophoreticdeposition(Zhongetal.,2015), plasmaspraying (Songetal.,2011),orco-precipitationmethod(Peñaetal.,2006). On the otherhand, over thepast decades,laser techniques provedahighpotentialforthefabricationofantimicrobialcoatings fororthopedicimplantsformedicalapplicationssuchasthebone tissue replacement or treatment of osteoporosis or osteolytic tumors(Jiaetal.,2016b;Simchietal.,2011).

Furthermore,thelaser-basedtechnologiesareexhibitingalotof advantages,astheyallowforthefabricationofawide-rangeof differentbiomaterials,withafairlyuniformspreadingofmaterial overratherlargeareas,controlledfilmthickness(withanaccuracy of1Å),goodadhesiontosubstrate(Blindetal.,2005;Dutaetal., 2013;Mihailescuetal.,2016),andspecificsurfaceproperties(Sima andMihailescu,2013).Moreover,thesedepositionmethodsimply a low material consumption, and ensure the stoichiometry preservationofthegrowingfilms(Eason, 2006;Yuetal.,2014; Cristescuetal.,2012).Inthesametime,remarkableeffortswere recentlypaidtothedevelopmentbycombinatorialprocessingof newbiomaterialswithinnovativeproperties.Usually,the fabrica-tionofacompositelayeriscarriedoutbypremixingofbiopolymer solutionsfollowedbyheatingofcoating(Meredithetal.,2000)or filmcasting/solventevaporation(Lietal.,2012).Thecombinatorial technology for the blending of two different biomaterials (Torricellietal.,2015;Simaetal.,2014;Axenteetal.,2014;Sima etal.,2012)isbasedonMatrix-AssistedPulsedLaserEvaporation (MAPLE)method.Thisnewlydevelopedtechnique– Combinatori-al-MAPLE(C-MAPLE)–standsforasimple,singlestep,fabrication route which can easily limit the time of manipulation and biomaterialsconsumption.

Theaimofthisstudywastosynthesizethincoatingscontaining naturalbiopolymerchitosan combinedwithbiomimeticapatite (Eichertetal.,2007;Grossinetal.,2010;Visanetal.,2014)directly ontitaniumimplantsbyC-MAPLEtechnique.

As known,a biomaterial used for bone substitution should possessasetofineluctableproperties.Theyare:

(i)Anidenticalchemicalcompositiontothenaturalbone(which is a complex structure of organic and inorganic materials (Dorozhkin,2009)).Forthispurpose, thenonstoichiometric biomimeticapatite(BmAp)(Eichertetal.,2007;Grossinetal., 2010;Visanetal.,2014),hasbeenusedasmodelforthebasic constituent of theinorganicpart of thebone, and chitosan (CHT),anaturalbiopolymer,withasimilarchemicalstructure

totheglycosaminoglycan,theprevalentextracellularmatrix of the bone and cartilage, as the organic phase of bone (Vllasaliuetal.,2012).

(ii)A good mechanical strength. The coatings were therefore deposited on titanium (Ti) medical implants, thus harmo-niouslycombingtheexcellentmechanicalfeaturesofTiwith thebiomimeticsoftheorganic-inorganicbiofunctionallayers (Agarwal and García, 2015). This will confer stability and reliabilitytothemedicaldeviceassembly.

(iii) Biocompatibility.Fromthispointofview,bothCHTandBmAp exhibitexcellentcytocompatibilityandremarkable osteocon-ductiveproperties,respectively(Songetal.,2011;VandeVord etal.,2002).

(iv)Resistancetomicrobialcolonization,particularlyduringthe osseointegrationperiod.Inthis regard,CHTshowsahigher antibacterialactivityagainst abroadspectrum ofmicrobial agents (Lee et al., 2009). It is therefore expected that the compositestructuresofpolymer(CHT)/ceramic(BmAp)will exhibit a double function: antimicrobial protection and enhancement of osteoblast cell proliferation, opening new promisingopportunitiesfordevelopinganewgenerationof orthopedicimplants.

To the best of our knowledge, this is the ﬁrst attempt to synthesizeacompositiongradientbetweenCHTandBmApbylaser co-evaporationofthetwodistinctcryogenictargetsfollowedbya co-depositionprocess.

2. Materialsandmethods 2.1.Materials

CHTwithalowmolecularweightwaspurchasedfrom Sigma-Aldrich,whilethebiomimeticapatitepowder,withaparticlesize <25

m

m, was prepared by the co-precipitation method in accordance with a previously described protocol (Visan et al., 2014).Solutionsconsistingof2%CHTand1%BmApindeionized water were prepared. All target solutions were poured into a coppertargetholder,pre-cooledat173K,andsubsequentlyfrozen byimmersioninliquidnitrogenfor15min.

2.2.C-MAPLEdepositionprocess

C-MAPLEtechniquewasusedforthefabricationofa composi-tional mapof thetwo compounds(i.e.,CHTand BmAp).In the experiments,anexcimerlasersource(KrF*,

l

=248nm,

t

FWHM=

25ns)operatedat10Hzfrequencyrepetitionratewasusedforthe cryogenictargetsevaporation.Asdepositionsubstrateswereused: 12mmdiameterTi(grade4)disks,Siwafersorglassslides.Inorder toobtainuniformcoatings,thetargetswerepermanentlyrotated (80rpm).Thisway,thedrillingwasavoidedandtheexpulsionof materials is promoted in a proper way, ensuring a uniform deposition.InC-MAPLEexperiments,thetwotargets(e.g.,CHTand BmAp)weresimultaneouslyevaporatedbythelaserbeam,which wasdividedintotwobeams(Fig.1a)byanopticalsplitter.Thetwo beams werefocused inparallelontothesurface ofeach target, containingthefrozensolutionstobeirradiated.

Theusedmethodallowstocombinetwodifferentbiomaterials andtoselectthebestsolvent/concentrationtobeusedforthetwo compounds. In our experiments, after preliminary studies, deionized water was chosen as solventand the concentrations of 1 and 2wt.% were used for CHT and BmAp substances, respectively.Wealsoperformedaparametricstudyonoptimum laserenergy(70,100,120and150mJ)forwhichthematerialsare depositedunaltered.Theselectedlaserenergywas:100mJinthe caseofCHTtargetandof70mJfortheBmApone.

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Inaconﬁgurationwithadistancebetweentheplasmacenters of 30mm, one obtains a 60mm long deposition with edges consistingofCHTandofBmAPonly,andin-betweendiscreteareas ofCHT-BmApblendedcompositions.Thesoftmixingofthetwo compoundsevaporatedfromthetwodistincttargetscanresultin the deposition of a continuous and uniform ﬁlm with a compositiongradient,asdepictedschematicallyinFig.1b.

Thecoatedsamplesareasdepositedontothesubstratearray composedofﬁveconsecutive12mmTidisksarefurtherdenoted S1toS5;whereS1standsforthecoatingareahavingCHTasmajor component,S5representsthecoatingareawithrichercontentof BmAp.S2!S3!S4seriesindicatesblended coatingareaswith decreasingCHT/BmApratios.Forcomparisonreasons,simpleCHT andBmApﬁlmshave beenalsodepositedonthesametype of substrates.

Depending on the laser parameters and polymeric-ceramic composition,thethicknessofC-MAPLEcoating,asdeterminedby proﬁlometry,wasinthe(1–2)

m

mrange.Theobtainedthickness value was calculated as the average of three independent measurements.

2.3.Physico-chemicalcharacterizationtechniques

(a)The general surface morphologyof the deposited _ﬁlms was investigatedbyscanningelectronmicroscopy(SEM) usinga HitachiS3400ElectronMicroscope.The investigationswere carriedoutinhighvacuumatanaccelerationvoltageof25kV. ThesampleswerecoatedwithathinAuﬁlminordertoreduce electricalchargingduringanalysis.

(b) Further surface morphology insightful analysis has been performedinmultipleareasoftheblendedcoatingbyatomic forcemicroscopy(AFM)innon-contactmodeusinganNT-MDT NTEGRA Probe NanoLaboratory system (NT-MDT NSG01 cantileverwithtipradiusof10nm).

(c) Compositional analyses have been carried out by energy-dispersive X-ray spectroscopy (EDS) with SiLi EDAX Inc. detector(attachmentofSEMapparatus).

(d)ThestructureofthesimpleCHTandBmApMAPLEﬁlmswas investigatedby grazing incidence X-ray diffraction (GIXRD) usinga BrukerD8 Advance diffractometer,inparallel beam setting, with Cu K

a

(

l

=1.5418Å) incident radiation. The incidenceanglewassetto2,andthescatteredintensitywas scannedwithinthe2

u

range(20–45)_,_with_a_step_size_of_0.04_,

and40sperstep.

(e)Fouriertransforminfrared(FTIR)spectroscopywas usedfor analyzingthefunctionalgroupspresentintheC-MAPLEcoating deposited on the 60mm long Ti plate. The analyses were performed in different regions of the ﬁlms, at separation distances of 2mm, with a Perkin Elmer Spectrum BX II

spectrometer,inattenuatedtotalreﬂectionmode(ATR)usinga Pike-MIRaclediamondheadof1.8mmdiameter.Thespectra werecollectedintherange4000–550cm1,byrecording128 individualscansataresolutionof4cm1.

(f)The micro-Raman analysis of the starting powders and C-MAPLEcoatingshasbeenperformedwithaHoribaJobinYvon LabramHR800spectrometer,inthe3800–100cm1range.A laserexcitationwavelengthof632.8nmhasbeenusedinthe caseofpowders.TheC-MAPLEﬁlmdepositedonthe60mm long glass plate has been exposed to a continuous laser radiationprovidedbyanArgondiodelaserat532nmwitha powerof12mW.ThesampleswereplacedinanOlympusBX 41 microscope and focused byan objective 50x with long distancenumericalapertureof0.75,whichgivesthesystema lateralresolutionof1

m

mand anaxialresolutionof4.5

m

m. ThemappingswerecarriedoutusinganXYZmotorizedstage withanaccuracyof0.1

m

m.Thesizeofmappingareaswasof (80100)

m

m2_,_with_a_measurement_pitch_of₁

_m

_m.

Thespectrumofeachpointwasacquiredthrougha600line/ mmgrating,withaspectralresolutionof1cm1andcollectedwith aquantumwelldetectorcooledto60_C_double_Pelletier_’s_effect

(Synapse CCD). A certiﬁed silicon standard allowed calibrating frequencyequipmentusingwiththe_ﬁrstorderofsiliconlineat 520.7cm1. Each point on the map was acquired with an integrationtimeof20sand1accumulation.Abaselinecorrection processingwasperformedusingtheLabspec5software.

X-rayphotoelectronspectroscopy(XPS)analysiswasperformed using a Thermo Scientiﬁc K-Alpha apparatus. Photoemission spectraarerecordedusingAlK

a

=1486.71eVmonochromatized radiation.TheX-rayspotdiameteronthesamplesurfaceisof 400

m

m.Thepassenergywasﬁxedat30eVfornarrowscan,and at170eVforsurveyscans.Thebackgroundsignalwasremoved using theShirleymethod.Atomicconcentrations were deter-mined from photoelectron peak areas using the atomic sensitivityfactors,takingintoaccountthetransmissionfunction of the analyzer. Photoelectron peaks were analyzed and deconvoluted using a Lorentzian/Gaussian (L/G=30) peak ﬁtting.

2.4.Antimicrobialassays

2.4.1.Microbialstrainsandgrowthconditions

StaphylococcusaureusATCC6538andEscherichiacoliATCC8739 werepurchasedfromAmericanTypeCultureCollection(ATCC,US). GlycerolstockswerestreakedonLBagartoobtain24hculturesto beusedforallfurtherstudies.

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2.4.2.Bioﬁlmdevelopment

Monospeciﬁcbioﬁlm developmentwas assessed atdifferent timesofexposure,usingsterile6wellplates(Nunc).Sterilecoated andreferencebareTidiscswereinoculatedontheirsurfacewith 50

m

Lbacterialsuspensionof0.5McFarlanddensity correspond-ingto1–3*107_density._The_samples_were_allowed_to_incubate_at

37Cin humidatmosphere andhavebeenobservedafterthree timeperiods(6h,24hand48h)toassessthetemporaldynamicsof developedbiofilms.Afterincubation,thesampleswerecarefully washed with sterile saline buffer to remove any unattached microbialcellsandthenimmersedin1mLsterilesalinebufferin Eppendorf tubes to reach bio_film detachment by vigorous vortexing.Theresultingbiofilm–detachedcellsuspensions were furtherseriallyten-folddilutedand10

m

Lofeachserialdilution wereplatedintriplicateonLBagar.

After6h,24hand48hofincubationat37C,viablecellcounts wereperformedandthenumberofcolonyformingunits(CFU)/mL foreachsamplewereestimated.

3. Resultsanddiscussion

3.1.SEM,AFM,andEDSobservations

Theoptimalcellularresponse(cellularadhesion,spreading,and proliferation) is of great signiﬁcance for tissue and medical engineering and is dependent on surface morphology and composition(Surmenev,2012;Spencer,2011).

ThegeneralsurfacemorphologyoftheCHT-BmApfilmhasbeen first investigated by SEM. Fig. 2 displays the characteristic topologicalfeaturesoftheC-MAPLEfilminvarioussurfaceregions of interest: far-most CHT rich region (S1), CHT-BmAp blended regions(withCHTdecreasingfromS2toS4),andfar-mostBmAp richregion(S5).

SEMmicrographs(Fig.2)showed thattheC-MAPLEblended coatings have a homogeneous spongy appearance all over the substrate,whichisknowntobebeneﬁcialforcelladhesion(Boyan etal.,1999;ChangandWang,2011;Willershausenetal.,2014;Zhu

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etal.,2015).Although,CHTparticulatespreservetheirspherical morphology,whileelongatedfiliformstructureswerealsonoticed intheblendedregionsofthefilm(i.e.,S2–S4).Itwasstatedthat these threadlike structures could support some toughness improvements similar tothe one provided by fibrereinforcing in the composite materials, ensuring the desired mechanical behaviorfortissuesubstitution(Mikhailovetal.,2014).

Further,theroughness(RRMS)oftheCHT-BmApﬁlmshavebeen

inferredbyAFManalyses.Theevolutionofthegrainsroughness alongthesubstratelengthisdepictedinFig.3.Onenoticesthata progressive increase of roughness (RRMS) occurs with the CHT

concentrationintheC-MAPLEcompositeﬁlms.

Fig.4presentscharacteristicEDSspectracollectedfromvarious regionsofCHT-BmApC-MAPLEblendedﬁlm,startingwiththeCHT richregion.OnecanobservetheprogressivedecreaseofC-K

a

and N-K

a

lines with distance from the CHT rich edge, which is indicativeofitsgradualdecreaseofconcentrationinthesample. TheCa/P molarratiofor each combinatorialsurfacewas in the range 1.3–1.5, thus lower than the stoichiometric theoretical value of hydroxyapatite (i.e., 1.67), pointing to the calcium-deﬁcientstateofbiomimeticapatitesynthesized.

3.2.GIXRDinvestigation

InthecaseofsimpleMAPLEﬁlmstheonlydiffractionmaxima (Fig.5)pertaintotheTisubstrate(ICDD:00-044-1294)andtoa titaniumsub-oxidephase(TiO)(ICDD:01-086-2352),visibledue tothelowthicknessoftheMAPLEﬁlms.ThepresenceofTiOphase ontheTisurfacemightbetheresultofsubstratemanufacturing

process. For BmAp ﬁlm, a fairly pronounced hump has been observedinthe(25–35)₂

_u

_range,_where_crystalline

hydroxyapa-tite[HA,Ca10(PO4)6(OH)2]compoundsexhibittheirmostintense

lines.It is thussuggestedthat theBmApMAPLEthin ﬁlmis in amorphousstatewithinthesensitivitylimitoftheemployedXRD apparatus. No suchhalo hasbeen revealedin the case of CHT MAPLE thin ﬁlm. One may account this to the lower electron density of chitosan with respect to HA, determining a lower scattered intensity, hard to discriminate from the background noise.

3.3.FTIRspectroscopystudies

TheFTIRspectraofBmAp,HA(pureandhighlycrystalline),and CHTpowdersarepresentedcomparativelyinFig.6aandb.Onecan notice thebroaderaspectof theIRbands inthe caseof BmAp powder with respect to the pure and highly crystalline HA material.Besidesthecharacteristic(Markovic etal.,2004;Sima etal.,2010)

n

4bending,

n

1symmetricstretchingand

n

3asymmetric

stretchingvibrationbandsoforthophosphateunitsofHA(Table1), theBmAppowderelicitedsupplementalIRbandspeakingat872, 1418,1480and1635cm1.Thewell-deﬁnedbandat872cm1 couldbeattributedtothesuperimposedcontributionsofthe

n

2

bendingmodesofcarbonategroupsandthevibrationsexhibited by hydrogen phosphate ions (HPO4)2– (characteristic to

non-apatiticdomains).Theweakbandat1635cm1belongstothe bending mode of water molecules. It denotes the existence of hydration,andcorrelateswellwiththeverybroadbandranging from3600to2500cm1,engenderedbythestretchingvibrations ofadsorbedwatermolecules.ThebroadIRabsorbancemaximaof the BmAp powder spectrum, and the absence of the libration (628cm1) and stretching (3571cm1) modes of structural (OH) groups, point towards an apatite-like compound witha reduceddegreeof(short-range)order.

TheIRspectrum(Fig.6aandb)oftheCHTpowderdisplaysan intricate envelope with multiple vibration maxima. The two prominentbands,situatedinthewavenumbersregions(i)3600– 2500cm1 and (ii) 1100–950cm1, are ascribed to the (i) overlapped stretching vibrations of adsorbed water and NH bonds, and to the(ii) symmetric stretching vibrationsof CO bondsingroupslikeCOH,COCandCH2OH,respectively(Kattietal.,

2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012). Thelowintensity,butsharpbands,centredat(iii)1150cm1and (iv) 890cm1, are related to the (iii) asymmetric stretching vibrationsofCO,and(iv)waggingvibrationsofCHbondsof thesaccharidegroupsofchitosan(Paluszkiewiczetal.,2011;Silva et al., 2012). The peaks at 1650, 1591 and 1316cm1 are

Fig.3.Surfaceroughness(RRMS)evolutionwiththedistancefromtheC-MAPLEﬁlm CHT-richedge.

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characteristictothevibrationsofamidesI,IIandIIIgroups,whilst thebandsat1420and1376cm1areassignedtothesymmetric deformationmodesofCH3 (Kattietal.,2008;Yuanetal.,2010;

Paluszkiewiczetal.,2011;Silvaetal.,2012).

AcompleteassignmentoftheFTIRbandsispresentedinTable1.

Fig.6candddisplaythecomparativespectrarecordedforthe pure(CHTandBmAp)layersandblended(CHT-BmAp)thinﬁlm(in

tworegions situatedclosetoCHT-andBmAp-rich edgesof the film)depositedontitaniumsubstrate.Asageneralobservation,the spectra of pure CHT and BmAp layers elicit less defined IR envelopeswithbroaderbandswithrespecttotheparentpowders, thus indicating a decreased structural order. This was to be expected for films synthesized at room temperature in non-equilibrium conditions by a physical vapor deposition method, suchasMAPLE.However,remarkably,allmajorvibrationbandsof CHTand BmAPweredetected inthecase offilms, furthermore havingsimilarintensityratioandfeaturingonlyslightwavelength shifts.Theotherminorvibrationbands(observedmoreclearlyin thecaseofpowders)areassumedtobepresent,butoverlappedto the broader, yet convoluted, major peaks. The conservation of similarlevelsofhydration(forboth typesoffilms) astheones foundintheparentmaterialscanbealsoemphasized.

TheblendedCHT-BmApﬁlmwasanalyzedinvariousregions situated on a median line, starting from the CHT-rich region, takingadvantageoftheATRdiamondheadwithawindowareaof 2.54mm2_._In_this_way_the_{compositional}_evolution_of_the_blended

filmwasunveiled(Fig.7aandb).Onecannoticethateveninthe regionssituated in theBmAp-richeredge of the film,the CHT bandsaredominant (Figs.6and 7).ThissuggeststhattheCHT plume was superior expanded, whilst the BmAp was better confined, due to their different ablation and/or volatilization rates. However,the(large)shiftstointermediatepositionsand theshapeofthepeakssituatedinthe1200–800cm1regionare suggestingthat an interaction ofCHT moleculeswithBmApis

Fig.5.ComparativeGIXRDpatternsofsimpleBmAp/Tiﬁlm,simpleCHT/Tiﬁlm andbareTisubstrate.

Fig.6.ComparativeFTIRspectraofCHT,BmAp,andcrystallineHApowders(a,b),andsimpleCHT,BmAp,andCHT-BmApﬁlms(c,d)intheﬁngerprint(a,c)andfunctional groups(b,d)regions.Forabettervisualevaluation,thespectrawerenormalizedtotheintensityofthemostprominentbandcentredat1040–1010cm1.

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occurring.Asexpected,withtheincreaseofthedistancefromthe BmAp-richeredge of theﬁlm, the BmApvibration bands start fading,whilsttheCHTbandsbecomemoreandmorepronounced, andareprogressivelyshiftedtothepositionsrecordedinthecase of the pure CHT ﬁlm (Fig. 7a and b). Concomitantly, one can observeacontinuouschangeofthevibrationbandsshape,which evolves from a CHT-BmAp intermingled spectrum to an IR envelopeallurecloselyresemblingtheoneofpureCHT(Fig.7a andb).

3.4.Micro-Ramanspectroscopyinvestigations

Similarvibration bands tothe onesrevealed by FTIR inves-tigations(Fig.7aandbandTable1)havebeenalsoidenti_ﬁedby micro-Ramananalyses fortheCHTandBmApsourcematerials. (resultsnotshownhere).However,inthecaseofRamanspectra thesymmetricstretchingbandsarethedominantones.

InperfectagreementwiththeFTIRresults,theRamanspectra evidenced that the BmAp material elicit along combinatorial trajectoryadecreasedordering(indicatedbythebroaderaspectof thebands)andacertainlevelhydrationwithrespecttothepure andcrystallineHA.

3.5.XPSanalysis

XPSanalysisconﬁrmedthechemicalcompositionofthe CHT-BmApC-MAPLEcomposite_ﬁlms.XPSsurveyspectracollectedon thesurfaceofCHT-BmApsamples(notpresented)indicatedthe presence of C 1s, O 1s, N 1s, Ca 2s, Ca 2p, P 2s and P 2p photoelectronpeaks.

Fig.8 shows the atomic ratioN 1s/(N1s+Ca 2p) evolution dependingonthepositionalongtheCHT-BmApblendedsample. Aspredicted,theatomicratio(N1s/N1s+Ca2p)isdecreasingfrom

CHTtoBmApcompounds,evidencingthecompositiongradientof thecombinatorialﬁlms.

3.6.Antimicrobialactivityassay

Composite biomaterials comprising Ap and polymers have shownpromising featuresforbone tissueengineering. Previous studies have proven the high antimicrobial activity of CHT/Ap composite particles (Ignjatovic et al., 2016; Shi et al., 2016). However,themostofthechitosancontainingcoatingsreportedin the literature have been obtained by using electrophoretic deposition or hydrothermal fabrication methods (Seuss et al., 2014;Pishbinetal.,2014;Longetal.,2014).

Duringthisstudies,theantibio_filmpropertiesoftheC-MAPLE depositedcoatingscontainingCHTandBmApinvariousratioshave beentestedusingtwomicrobialstrains,whicharerepresentative for the Gram positive (S. aureus) and Gram negative (E. coli) bacterial infections. Such pathogens could contaminate the orthopedic implants by route of exogenous or endogenous infections, leading to the occurrence of implant-associated infections,whicharehardtotreatandcouldoftenleadtoimplant failure.S.aureus,togetherwithS.epidermidisaccountsfor30%to twothirdsofallimplant-relatedinfections,whileamong Gram-negativebacteria,theetiologyisdominatedbyE.coli(Sendietal., 2011;Campocciaetal.,2006;EspositoandLeone,2008).However, little is known about the virulence features of E. coli strains involvedinprostheticinfections.Arecentstudyhasrevealedthat no specific pathogenic signature or increased ability to form biofilms in vitro were detected in E. coli strains isolated from orthopedicinfections,ascomparedtofecalisolates(Crémetetal., 2012).

Thebioﬁlmformationisamulti-stageprocessstartingwiththe initialattachmentofbacterialcells,followedbycellaggregation

Fig.7.FTIRspectraofsimpleCHTandBmApfilmsincomparisonwiththe“punctual”spectracollectedataseparationdistanceof2mmonthesurfaceofCHT-BmAp C-MAPLEfilm,inthefingerprint(a)andfunctionalgroups(d)regions.Forabettervisualevaluation,thespectrawerenormalizedtotheintensityofthemostprominentband centredat1040–1010cm1.

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andaccumulationinmultiplecelllayers,biofilmmaturationand biofilmcellsdetachment(Arciolaet al.,2015).Inourstudy,the developmentofbiofilmsconsistingoftheGram-positiveS.aureus andtheGram-negativeE.colifollowedadifferentdynamiconthe testedsamples.Thus,bothS.aureusandE.colibiofilmsgrownon thecontrol(bareTisubstrate)revealedasimilardynamic,i.e.,a decreasingtrendofbiofilmembeddedviablecells, quantifiedat thethree consecutive point intervals(Fig.9).These results are suggestingthat thehighsurfaceroughnessof bareTisubstrate requiredforanimproved osseointegrationfavorstherapid and firmadhesionofS.aureusandE.colicells.Thisbehaviorsupports the leading position held by these species in the etiology of implant-associatedinfections.

Fig.9Inthecaseofmicrobialbio_ﬁlmsdevelopmentonthe CHT-BmApsurfaces,amorepronouncedinhibitoryeffectwasgenerally

observedagainsttheGram-positiveS.aureusbiofilm,ascompared toE.coli,forallthethreetestedtimeintervals(Fig.10).TheS2,S3 andS4samplesinhibitedthefirststageofS.aureusinitialmicrobial adherencetothetestedsurfacequantifiedafter6h,whilenoneof thetestedsamplesinhibitedtheadherenceoftheGram-negativeE. coli strain (Fig. 10a). At 24h, all tested CHT-BmAp samples decreased the number of S. aureus biofilm embedded cells (Fig.10b); the most intensive inhibitory effect being observed

Table1

TheassignmentofFTIRbandsofchitosanandhydroxyapatite. Position(cm1) Bandassignment

563–557 601–600

Bendingn4of(PO4)3groups(Markovicetal.,2004;Simaetal.,2010) 628 Librationofstructural(OH)groups(Markovicetal.,2004;Simaetal.,2010) 872–867 Bendingn2of(CO3)2groups(Markovicetal.,2004;Simaetal.,2010)

Stretchingof(HPO4)2(Markovicetal.,2004)

897–895 WaggingofCHbondsofthesaccharidestructureofchitosan(Yuanetal.,2010;Silvaetal.,2012) 963–942 Symmetricstretchingn1of(PO4)3groups(Markovicetal.,2004;Simaetal.,2010)

994–993 1034–1026 1068–1061

SymmetricstretchingofCObondsinCOH,COCandCH2OHgroups(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012)

1018–1002 1096–1087

Asymmetricstretchingn3of(PO4)3groups(Markovicetal.,2004;Simaetal.,2010)

1155–1150 AsymmetricstretchingofCOCbondsofthesaccharidestructureofchitosan(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silva etal.,2012)

1262 Stretchingof(COH)bonds(Yuanetal.,2010) RockingofNH2(Kattietal.,2008)

1317–1315 Stretchingof(CH3)ofamideIIIgroups(Kattietal.,2008;Yuanetal.,2010) 1376–1375

1420–1417

Symmetricdeformationof(CH3)(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012) 1427–1418

1480

Asymmetricstretchingn3of(CO3)2groups(Markovicetal.,2004;Simaetal.,2010)

1591–559 Stretchingof(NH)ofamideIIgroups(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012) 1635–1610 Bendingofwatermolecules(Markovicetal.,2004)

1650–1640 Stretchingof(C¼O)ofamideIgroups(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012)

2871–2870 Symmetricstretchingn2of(CH)bondsin(CH3)groups(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012) 2922 Symmetricstretchingofaliphatic(CH)bonds(Pengetal.,2008)

2977 Asymmetricstretchingof(CH3)(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012) 3288–3286

3357–3355

Stretchingvibrationsof(NH)bonds(Kattietal.,2008;Yuanetal.,2010;Paluszkiewiczetal.,2011;Silvaetal.,2012) 3571 Stretchingofstructural(OH)groups(Markovicetal.,2004)

3600–2500 Stretchingvibrationsof(OH)bondsinadsorbedwatermolecules(Markovicetal.,2004)

Fig.8. TypicalXPSmeasurementalongthecompositeCHT-BmApC-MAPLEﬁlms.

Fig.9.Graphicrepresentationofthenumberofviablebioﬁlmembedded(a)S. aureusand(b)E.colicellsdevelopedonthebareTisubstrateusedascontrol.

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fortheS4sampleinthecaseofS.aureusbiofilm.TheE.colibiofilm developmentwasinhibitedbytheS1–S5samples,butthemost intensiveantibiofilmeffectwasnoticedforS4andS5(Fig.10b).

Anintensiveanti-biofilmeffectwasexhibitedafter48hagainst S.aureusinthecaseofallCHT-BmAptestedsamples(Fig.10c)but themostsignificantactivitywasobservedforS1andS2samples. TheE.colibiofilmwasinhibitedonlybyS3–S5samples,themost intensiveeffectbeingnoticedforS5(Fig.10c).

Takentogether,theresultsregardingtheanti-bio_filmeffectof theobtainedcoatingsexhibitedagainstbothbiofilmtypesandfor the three tested intervals recommend S3 and S4 as the most promisingcompositions,assuringananti-biofilmprotectionona longperiodoftimeagainstbothbacterialspecies(Table2).

AlthoughthemechanismsofantimicrobialactionofCHTarenot elucidated,theliteraturestatesthatitstargetisatthemicrobial cellsurface,assuggestedbytheelectronmicroscopyfindings.They show cellular wall disorganization both in Gram-positive and Gram-negativebacteria,aswellasin fungalstrains, due tothe electrostatic interaction of positively-charged_—CHT with the negatively charged bacterial components (proteins, phospholi-pids)(Cuero,1999;Muzzarellietal.,1990; Noetal.,2002).This action mechanism is responsible for a wide spectrum of the antimicrobial activity of CHT and its derivatives including filamentous fungi,yeasts and bacteria (Raafat and Sahl, 2009). Nevertheless, it seems that CHT is more active against Gram-positive than Gram-negative bacteria, as revealed also by our study. In this regard, S. aureus biofilm proved to be more susceptiblethanE.coli,probablyduetothestrongerinteraction

of CHTwiththeamino acidsrich fractionof theGram-positive bacterialwall(Kumaretal.,2005).Inexchange,theGram-negative strainshaveamorecomplexcellularwallduetothe negatively-chargedlipopolysaccharideswhichcanactasanadditionalbarrier intheinteractionofchitosanwithotherstructuresofthecellular wallandmembrane(Helanderetal.,2001).

4. Conclusions

ThesynthesizedCHT-BmApblendedthinfilmsareamorphous, rough,withamorphologycharacteristictoMAPLEstructures.The compositiongradientofthechitosan-to-biomimetic hydroxyapa-titehasbeenconfirmedlongwisethecombinatorialfilms.

Theantimicrobialactivitywascontrolledbytheconcentration ofchitosan,whiletheblendedstructureswerebetterintegratedfor quasi-equal presence of the two compounds (i.e.,chitosan and biomimetic apatite). The most efﬁcient antimicrobial activity, consideredas long-termprotectionagainst initialadhesion and bio_ﬁlmsdevelopedbyGram-positiveandGram-negativestrains, wasassignedtoS3andS4samples.

C-MAPLE technique used in this study proves to be a prospectiveandviablemethodforthefabricationofbiomimetic andbioactiveantimicrobialorthopedic coatingswhichresemble thenativeboneextracellularmatrix(consistingofwater,minerals, ﬁbrousproteins,proteoglycans)andthus,createafavorableliving environmentforosteogeniccells,whileassuringprotectionfrom microbialcolonization.

Acknowledgements

The authors acknowledge the financial support of UEFISCDI under the France_–Romania bilateral contract 778/2014, the “Ministère des Affaires étrangères” under the PHC BRANCUSI 2015 (N 32648SD) and the National Authority for Scientific Research andInnovationin theframeof NucleusProgramme– contract 4N/2016. The authors thank to Iuliana Urzica for performing the profilometry thickness measurements. G.E.S. acknowledgeswiththanksthesupportofNIMPCoreProgramme PN1648-3/2016.Theauthorsacknowledgewiththanksthehelpof Dr.FelixSimaandDr.EmanuelAxenteforassistanceforthedesign andset-upoftheC-MAPLEexperiment.

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