Structural Properties and Antifungal Activity against Candida albicans Biofilm of Different

22 Pages·2016·11.75 MB·English

polymers
Article
Structural Properties and Antifungal Activity against
Candida albicans Biofilm of Different Composite
Layers Based on Ag/Zn Doped
Hydroxyapatite-Polydimethylsiloxanes
AndreeaGroza1,CarmenStelutaCiobanu2,CristinaLianaPopa2,3,SimonaLilianaIconaru2,
PatrickChapon4,CatalinLuculescu1,MihaiGanciu1andDanielaPredoi2,*
1 NationalInstituteforLaser,PlasmaandRadiationPhysics,409AtomistilorSt.P.O.BOXMG36,
Magurele077125,Romania;andreea@infim.ro(A.G.);catalin.luculescu@inflpr.ro(C.L.);
ganciu@infim.ro(M.G.)
2 NationalInstituteforMaterialsPhysics,405AAtomistilorStreet,P.O.BoxMG07,Magurele077125,
Romania;[email protected](C.S.C.);[email protected](C.L.P.);
[email protected](S.L.I.)
3 FacultyofPhysics,UniversityofBucharest,405AtomistilorStreet,P.O.BoxMG1,Magurele077125,Romania
4 HoribaJobinYvonS.A,16-18,rueduCanal,LongjumeauCedex91165,France;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:JianxunDing
Received:29February2016;Accepted:30March2016;Published:9April2016
Abstract: Modernmedicineisstillstrugglingtofindnewandmoreeffectivemethodsforfighting
offviruses,bacteriaandfungi. Amongthemostdangerousandattimeslife-threateningfungiis
Candidaalbicans. Ourworkisfocusedonsurfaceandstructuralcharacterizationofhydroxyapatite,
silver doped hydroxyapatite and zinc doped hydroxyapatite deposited on a titanium substrate
previously coated with polydimethylsiloxane (HAp-PDMS, Ag:HAp-PDMS, Zn:HAp-PDMS) by
different techniques: Scanning Electron Microscopy (SEM), Glow Discharge Optical Emission
Spectroscopy (GDOES) and Fourier Transform Infrared Spectroscopy (FTIR). The morphological
studies revealed that the use of the PDMS polymer as an interlayer improves the quality of the
coatings. Thestructuralcharacterizationsofthethinfilmsrevealedthebasicconstituentsofboth
apatitic and PDMS structure. In addition, the GD depth profiles indicated the formation of a
compositematerialaswellasthesuccessfulembeddingoftheHAp,Zn:HApandAg:HApintothe
polymer. Ontheotherhand,invitroevaluationoftheantifungalpropertiesofAg:HAp-PDMSand
Zn:HAp-PDMSdemonstratedthefungicidaleffectsofAg:HAp-PDMSandthepotentialantifungal
effectofZn:HAp-PDMScompositelayersagainstC.albicansbiofilm. Theresultsacquiredinthis
researchcompletepreviousresearchonthepotentialuseofnewcomplexmaterialsproducedby
nanotechnologyinbiomedicine.
Keywords: compositelayers;Ag/Zndopedhydroxyapatite;PDMS;C.albicansbiofilm
1. Introduction
Inanerawhenscienceandtechnologyhavereachedapointwhentheimpossiblewastransformed
intoreality,whencarslearntodrivethemselvesandintelligentrobotslearnhowtowalkashuman
beingsandtointerprethumanemotions,themedicalfieldisstillstrugglingtofindnewandmore
effectivemethodsforfightingoffviruses,bacteriaandfungi. Outofalmost611,000speciesoffungi,
only600speciesareconsideredtobehumanpathogens[1–3]. Amongthemostdangerousandat
timeslife-threateningfungiisCandidaalbicans,whichintheUnitedStatesisconsideredtobethefourth
Polymers2016,8,131;doi:10.3390/polym8040131 www.mdpi.com/journal/polymers
Polymers2016,8,131 2of22
mostfrequentlyencounteredcauseofsystemicinfectionsacquiredfromhospitals[1,4,5]. Thiswas
possibleduetotheincreasingresistancetoantibioticsofdifferenttypesoffungiandalsoduetothe
limited number of antifungal drugs. Candida species is one of the most dangerous human fungal
pathogensresponsiblefordeeptissueandmucosalinfections,especiallyintheoralcavity. Inaddition,
itwasprovedthatupto50%ofhospital-relatedC.albicansinfectionsaredeadly[1,4,5]. Accordingto
Samaranayakeetal.[6]infectionscausedbyCandidaspeciesareassociatedtobiofilmgrowth. Inthis
context,itisimperativetofindnewsolutionsforfightingofftheinfectionscausedbythisfungus.
Althoughnanotechnologyhasbroughtimprovementstothefieldofnanomedicine,providing
improvedmaterialsthatareabletomimicthehumanbodytissuestoacertainpoint,therearestill
manychallengestoovercome. Oneofthemedicalfieldsthathasbeenimprovedinthelastdecades
duetotheadvancesmadeintheareaofbiomaterialsisthefieldoforthopedics. Inordertoimprovethe
metallicprosthesisthatareusedinorthopedicsurgeries,differentbiocompatiblematerialshavebeen
usedascoatings. Inthiscontext,agreatdealofattentionwasdirectedtobioceramics. Theirability
topromotenewboneformationandtheircapacitytofacilitatetheadherencetotissuesaswellasto
inducegrowthofperipheraltissues[7]havemadethemexcellentcandidatesforvariousapplications
inthebiomedicalfield. Inthiscontext,synthetichydroxyapatite(HAp)hasattractedtheattentionof
researchersandmedicsalikeduetoitssimilaritytotheinorganiccomponentofthehumanbone[8].
Currentlyitisusedasadsorbentfordifferentproteinsandenzymes[8]orashardtissuesubstitute
inorthopedicanddentalsurgeries[9]. Hydroxyapatiteisusedindifferentshapesandsizes, from
granules, porous or solid matter to fine nanoparticles [8]. However, although hydroxyapatite has
remarkablebiocompatibleandbioactiveproperties[10–12],itwasdemonstratedthatHApcoatings
promotebacterialgrowthalongwithitsevidentosteoconductivity[10,13]. Itwasobservedthatcoated
devicesaremorepronetoperi-implantitisthanuncoatedones[10,13].
Asolutiontoincreasetheantimicrobialactivityofhydroxyapatitebasedcoatingswastodope
HApwithdifferentmetalionswhichpossessspecialantimicrobialproperties. Inthiscontext,agreat
dealofattentionwasdirectedtosilverandzincions. Silver(Ag)isawell-knownbiocidalagentused
invariousmedicalfields,whichsucceedsineradicatingalargenumberofbacteriaandfungi[14,15].
Accordingtopreviousresearch[14,16],silverdisruptstherespiratoryfunctionofbacteriabyattaching
tothebacterialcellmembraneandmodifyingitspermeability[14,16]. Anotherexplanationforits
ability to destroy bacteria is that very small Ag nanoparticles have the capacity to penetrate the
bacterialcellandtobindwiththeirDNA[14,15,17,18]. Zinc(Zn)isoneofthetraceelementswhich
is naturally found to some extant in the composition of the human hard tissue (in the enamel of
theteethandinthebonetissue)[19,20]. Accordingtoseveralresearchers,zinccouldbeconsidered
one of the most important element in medicine due to its vital role in the proper functioning of
almost200enzymes[19,21]. DopingHApwithZn2+ionswasfoundnaturalalsoduetoitsabilityto
promoteboneformation[22,23]. Therefore,embeddingeithersilverorzincionsinthestructureof
synthetichydroxyapatiteleadstoasuperiormaterialmoreadequateforbeingusedindifferentmedical
applications. Anotherdrawbackofhydroxyapatiteisitsbrittleness[24,25]. Inordertoimprovethe
brittle nature of HAp, scientists have found a solution by combining it with various biopolymers.
Polydimethylsiloxane(PDMS)hasattractedtheattentionofscientistsduetoitsbiocompatibility. Ithas
beenusedinvariousbiomedicalimplantssincethe1960s[26]. Differentworksdemonstratedthatmost
ofPMDSremarkablepropertiesarisefromthesiloxanebond[27–30]. Amongtheseproperties,itslow
glasstransitiontemperatureat´123˝C,hydrophobicity,anditslowsurfaceenergy,itschemicaland
thermalstabilityaswellaslowelectricalconductivity,physiologicalinertnessanditshighpermeability
togasses[27,28]aresomeofitsadvantages. Recently,YalingLinetal.[31]revealedtheantimicrobial
activitiesofpolysiloxane-containingquaternaryammoniumsaltsagainstbacteriaandphytopathogenic
fungi,consideringthatpolysiloxanesprovedhighstaticanddynamicflexibilityinmanysolvents,high
permeabilityandspecialsurfaceproperties.Inaddition,PDMSlayersbecameapreferredsoftsubstrate
forculturingdifferenttypesofcells[32]duetotheirbiocompatibility[33],non-toxicitytowardmany
speciesoforganismsandtheirbiodegradability[34].
Polymers2016,8,131 3of22
Differenttechniqueslike: sol–gelmethods,radio-frequencymagnetronsputteringplasma-spray
technique,pulsedlaserdepositionorelectrodepositionarenowusedforthedepositionofamorphous
HAp coatings on different types of substrates [35,36]. As these coatings are further crystallized at
elevated temperatures, during these thermal treatments, many cracks are formed, as the bonding
strengthofthecoatinglayerwiththesubstrateisaffectedduetothethermalexpansionmismatch
betweenthecoatingandthemetalsubstrate[36]. Asaresult,differenttypesofinterlayersarenow
usedasreinforcementagentsforimprovingthedelaminationofHApcoatings. Asanexample,due
to their own excellent compatibility with the living tissues, and their high chemical inertness [37]
SiO layershavebeenusedforthispurpose.
2
In this context, in our previous papers [38] we analyzed the physico-chemical influences of a
polydimethylsiloxanefilmusedasinterlayerforanAg:HApcoating. GiventhattheX-raydiffraction
spectraevidencedthecrystallineformofthehydroxyapatitedopedwithsilverintheAg:HAp-PDMS
compositelayerandtheFourierTransformInfraredSpectroscopymeasurementsindicatedtheSiO 4´
4
ionsformation,wesupposedthattheSiO 4´/PO 3´ ionssubstitutionmechanismispossibletotake
4 4
place. In addition, it was shown [39] that the hardness (H) of the Ag:HAp layer increased in the
presenceofthePDMSlayerwhileitsYoung’smodulus(Y)decreased.
Thegoalofthisresearchwasaninvitroevaluationoftheeffectsofvariouscompositelayersbased
onsilverorzincdopedhydroxyapatite/polydimethylsiloxane(Ag:HAp-PDMSandZn:HAp-PDMS)
orhydroxyapatite/polydimethylsiloxane(HAp-PDMS)onCandidaalbicans(C.albicans)adhesionto
layerssurfaces. Thesecompositelayersthatcovercommerciallytitaniumsubstrateswerepreparedby
combiningcoronadischargeatatmosphericpressureandsol-geldipcoatingmethod.
Asaresultofthefactthattitanium-basedimplantscouldpotentiallyberesponsibleC.albicans
for infections [40,41], our studies presented in this work may bring new specific information on
the interactions between fungal cells and complex layers (Ag:HAp-PDMS, Zn:HAp-PDMS and
HAp-PDMS).Despitethefactthattheantimicrobialpropertiesofsilverandzinchavebeenextensively
studied,scientificknowledgeofthecomplexinteractionsbetweencomplexlayersbasedonsilverand
zincareverylimited.
In this context, the research has focused on studies of the Ag:HAp-PDMS, Zn:HAp-PDMS
and HAp-PDMS complex layers surface and structure. These composite layers deposited on Ti
substrates were analyzed by different techniques such as Scanning Electron Microscopy (SEM),
GlowDischargeOpticalEmissionSpectroscopy(GDOES)andFourierTransformInfraredSpectroscopy
(FTIR).Furthermore,theinvitroevaluationoftheantimicrobialeffectofthevariouscomplexlayers,
whichappearedtobeactiveagainstC.albicansbiofilmembeddedcellsatdistinctintervalsoftime,
ispresented.
2. MaterialsandMethods
2.1. Materials
Calcium nitrate tetrahydrate [Ca(NO ) ¨4H O, Aldrich St. Louis, MO, USA], phosphorus
3 2 2
pentoxide(P O ,98%,Merck,Kenilworth,NJ,USA),silvernitrate(AgNO ,99.9%,AlfaAesar,Ward
2 5 3
Hill, MA, USA) and zinc nitrate hexahydrate (Zn(NO ) ¨6H O, 99%, Alfa Aesar, Ward Hill, MA,
3 2 2
USA) were used as precursors for the synthesis of HAp, Ag:HAp and Zn:HAp thin films. All the
precursorswereusedwithoutfurtherpurification. Vinylpolydimethylsiloxane(PDMS,M «25,000,
w
Sigma Aldrich, St. Louis, MO, USA) liquids were used as precursors for PDMS layers generation.
Commerciallypuretitanium(Ti,1.0mmthick,AlfaAesar,WardHill,MA,USA)foilwascutintodisks
witha20mmdiameterandwereusedassubstrates. Allthedisksweredegreased(fivetime)inan
ultrasonicbathwithacetone. Aftereachdegreasingthediskswererinsedwithdeionizedwater.
Polymers2016,8,131 4of22
2.2. DepositionofPDMSLAyersonTiSubstrates
ThePDMSlayersweredepositedonTisubstratesinatmosphericairpressurecoronadischargesin
apointtoplaneelectrodeconfiguration. Undercoronachargeinjection(mainlynegativeoxygenions)
theliquidprecursorofvinylterminatedpolydimethylsiloxanesthatliesontheanode(Tisubstrate),
is transformed into a solid polymer layer after two hours. The entire deposition procedure was
describedindetailin[42,43].
2.3. DepositionofHApLayersonaTitaniumSubstratePreviouslyCoatedwithaPDMSLayer
Hydroxyapatite was synthesized by sol-gel method using calcium nitrate tetrahydrate
(Ca(NO ) ¨4H O) and phosphorus pentoxide (P O ) as Ca and P precursors. On the one hand,
3 2 2 2 5
a designed amount of Ca(NO ) ¨4H O was dissolved in anhydrous ethanol. On the other hand,
3 2 2
aproperamountofP O wasdissolvedsimilarlyinanhydrousethanol. Thephosphorussolutionwas
2 5
addeddropbydropintotheCasolutionundervigorousstirring. Theobtainedsolutionwasstirredfor
30minat40˝C.Theresultedsolswereagedfor72h. Finally,theobtainedsolswereclearandstable.
Furthermore,thetitaniumsubstratepreviouslycoatedwithaPDMSlayerwasimmersedintoHApsol.
Eachcoatingwasdriedat80˝Cfor30minandthecoatingprocedurewasrepeatedfivetimes. Inthe
endthesampleswerethermallytreatedat600˝Cfor2hinair,withaheatingrateof10˝C/min[44].
2.4. DepositionofAg:HApandZn:HApLayersonaTitaniumSubstratePreviouslyCoatedwith
aPDMSLayer
The composition ratios in the Ag:HAp (Ca Ag (PO ) (OH) , x = 0.3) and Zn:HAp
10´x x 4 6 2 Ag
(Ca Zn (PO ) (OH) , x = 0.3) sols were adjusted so that the [Ca + Ag]/P and [Ca + Zn]/P
10´x x 4 6 2 Zn
ratioswouldbeequalto1.67[45–47].
InordertoobtainAg:HAp,silvernitratewasusedforthesubstitutionofCawithAgionsinthe
hydroxyapatitestructure.Firstly,AgNO andCa(NO ) ¨4H Oweremixedanddissolvedinanhydrous
3 3 2 2
ethanol.Ontheotherhand,aproperamountofP O wasdissolvedsimilarly.Thephosphorussolution
2 5
wasaddeddropbydroptothe(Ag+Ca)solutionundervigorousstirring. Theobtainedsolutionwas
stirredfor30minat40˝C.Theobtainedsolswereagedfor72h. Finally,theobtainedsolswereclear
andstable. Furthermore,thetitaniumsubstratepreviouslycoatedwithaPDMSlayerwasimmersed
into the Ag:HAp sol. Each coating was dried at 80 ˝C for 30 min and the coating procedure was
repeated five times. In the end the samples were thermally treated at 400 ˝C for 2 h in air with a
heatingrateof10˝C/min[39,48].
ThesameprocedurewasfollowedforobtainingZn:HApsol. Thistime,forthesubstitutionof
CaionswithZnionsinthestructureofHAp,zincnitratehexahydrate(Zn(NO ) ¨6H O),wasused.
3 2 2
2.5. StructuralCharacterizations
RoughnessmeasurementsoftheTisubstrateswereperformedusingaMahrperthometer(Göttingen,
Deutschland).ForeachTisubstratethearithmeticmeandeviationR ofroughnessprofile,meanpeakto
a
valleyheightR androotmeansquaredeviationR oftheroughnessprofileweredetermined.
z q
ThemorphologicalfeaturesofthehydroxyapatitecoatingsurfaceswereinvestigatedbyScanning
ElectronMicroscopy(SEM)usingaFEIInspectSscanningelectronmicroscope(Hillsboro,OR,USA)
inbothhigh-andlow-vacuummodes.
TheelementaldepthprofileanalysisoftheAg:HAp-PDMS,Zn:HAp-PDMSandHAp-PDMS
coatings were performed by Glow Discharge Optical Emission Spectroscopy (Horiba Company,
Longjumeau,France). TheexperimentalconditionsusedfortheoperationoftheGDProfilerwere:
650Pa,35WRFpowerimpulsemodeat1kHzandadutycycleof0.25.
Polymers2016,8,131 5of22
TheIRspectraoftheAg:HAp-PDMS,Zn:HAp-PDMSandHAp-PDMScoatingsobtainedonTi
substratewereacquiredusingaSP100IRPerkinElmerspectrometer(Waltham,MA,USA)equipped
withavariableanglespecularreflectanceaccessory. Themeasurementswerecarriedoutforanangle
ofreflectionof300. Accordingto[10],thesecondderivativeIRspectrawereacquiredafterperforming
a5-pointsmoothingoftheIRabsorbancespectra. Forthe450–2000cm´1 spectralranges,thepeak
fittinganalyseswerecarriedoutusingproceduresdescribedbyIconaruetal.[10].
2.6. InVitroAntifungalActivity
ThebacterialstrainusedinthebiofilmformationwasC.albicansATCC10231. Inordertoassess
thebiofilmformationonthesurfaceondifferentsurface(Ti,PDMS,HAp-PDMS,Zn:HAp-PDMSand
Ag:HAp-PDMS)ofcompositelayers,0.5McFarlandmicrobialsuspensioninsterilesalineobtained
from24hmicrobialculturesweregrownonthesurfaceofthethinfilmsinaliquidyeastpeptone
glucose(YPG)medium. Every24,48and72hthethinfilmswereremovedfromtheculturemedium.
Theywerewashedusingsterilesalinesolutioninordertoremovethenon-adherentmicrobialcellsand
reintroducedintosterilesaline. Afterthat,thesampleswerevortexedforsuspendingthemicrobial
cellsembeddedinthebiofilmformedonthethinfilmsspecimens. Thisentireprocedurewasrepeated
forthethinfilmscolonizedwithfungalbiofilmsat48and72h.
ThebiofilmformationofC.albicansondifferentsurface(Ti,PDMS,HAp-PDMS,Zn:HAp-PDMS
andAg:HAp-PDMS)ofcompositelayers,wasinvestigatedusingconfocallaserscanningmicrocopy
(CLSM).FortheCLSMobservation,thecellswerestainedfor2minwithpropidiumiodide,washed
2timeswithwater,airdriedandthenvisualizedinreflectionandfluorescencemodesbyusingaTCS
SPconfocalmicroscope,equippedwitha10XHCXPLFLUORITEobjective,withanumericalaperture
NAof0.3. Inordertoacquirebothreflectionandfluorescence,anArionlaser(488nm)wasused.
The biofilm morphology of the samples was analyzed using scanning electron microscopy.
ForSEMobservation,theC.albicansbiofilmswerepreviouslyfixedonthethinfilmsusing200µLcold
absolutemethanolfor5min. Themethanolexcesswasremovedandthefilmswereallowedtoairdry.
Thesampleswereplaceonasampleholderusingcarbontapeandthenanalyzed.
3. ResultsandDiscussions
The results presented in this paper were focused on evaluation of the antifungal activity of
differentsurfaces(Ti,PDMS,HAp-PDMS,Zn:HAp-PDMSandAg:HAp-PDMS)againstC.albicans
onbiofilmdevelopment. Inaddition,westudiedthemorphologicalandstructuralcharacteristicsof
differentsurfacessuchasPDMS,HAp-PDMS,Zn:HAp-PDMSandAg:HAp-PDMS.
3.1. ScanningElectronMicroscopy
Depending on the Ti substrate surface roughness (R), the characteristic features of Ag:HAp,
Zn:HApandHApcoatingswereanalyzedwithorwithoutapolymerinterlayerbyscanningelectron
microscopy. ThemorphologyofaPDMSlayerdepositedonaTisubstratewithanopticallypolished
surface,(R =0.084µm,R =0.489µm,R =0.108µm)inthecoronadischargeinairatatmospheric
a z q
pressureisshowninFigure1.
Asthepolymercoveredonlyacircularareaof10mmindiameteronthecenterofthesubstrate
surfaceandtheHApwasdepositedontheentireTidisksurface(20mmindiameter),theinterfacezone
betweentheAg:HAp-PDMS,Zn:HAp-PDMS,HAp-PDMSandAg:HAp,Zn:HAp,HAprespectively
willbefurtherinvestigated.
Polymers 2016, 8, 131 5 of 22
for an angle of reflection of 300. According to [10], the second derivative IR spectra were acquired
after performing a 5-point smoothing of the IR absorbance spectra. For the 450–2000 cm−1 spectral
ranges, the peak fitting analyses were carried out using procedures described by Iconaru et al. [10].
2.6. In Vitro Antifungal Activity
The bacterial strain used in the biofilm formation was C. albicans ATCC 10231. In order to assess
the biofilm formation on the surface on different surface (Ti, PDMS, HAp-PDMS, Zn:HAp-PDMS
and Ag:HAp-PDMS) of composite layers, 0.5 McFarland microbial suspension in sterile saline
obtained from 24 h microbial cultures were grown on the surface of the thin films in a liquid yeast
peptone glucose (YPG) medium. Every 24, 48 and 72 h the thin films were removed from the culture
medium. They were washed using sterile saline solution in order to remove the non-adherent
microbial cells and reintroduced into sterile saline. After that, the samples were vortexed for
suspending the microbial cells embedded in the biofilm formed on the thin films specimens. This
entire procedure was repeated for the thin films colonized with fungal biofilms at 48 and 72 h.
The biofilm formation of C. albicans on different surface (Ti, PDMS, HAp-PDMS,
Zn:HAp-PDMS and Ag:HAp-PDMS) of composite layers, was investigated using confocal laser
scanning microcopy (CLSM). For the CLSM observation, the cells were stained for 2 min with
propidium iodide, washed 2 times with water, air dried and then visualized in reflection and
fluorescence modes by using a TCS SP confocal microscope, equipped with a 10X HCX PL
FLUORITE objective, with a numerical aperture NA of 0.3. In order to acquire both reflection and
fluorescence, an Ar ion laser (488 nm) was used.
The biofilm morphology of the samples was analyzed using scanning electron microscopy. For
SEM observation, the C. albicans biofilms were previously fixed on the thin films using 200 μL cold
absolute methanol for 5 min. The methanol excess was removed and the films were allowed to air
dry. The samples were place on a sample holder using carbon tape and then analyzed.
3. Results and Discussions
The results presented in this paper were focused on evaluation of the antifungal activity of
different surfaces (Ti, PDMS, HAp-PDMS, Zn:HAp-PDMS and Ag:HAp-PDMS) against C. albicans
on biofilm development. In addition, we studied the morphological and structural characteristics of
different surfaces such as PDMS, HAp-PDMS, Zn:HAp-PDMS and Ag:HAp-PDMS.
3.1. Scanning Electron Microscopy
Depending on the Ti substrate surface roughness (R), the characteristic features of Ag:HAp,
Zn:HAp and HAp coatings were analyzed with or without a polymer interlayer by scanning
electron microscopy. The morphology of a PDMS layer deposited on a Ti substrate with an optically
Ppoolylmisehrse2d0 1s6u,8r,fa13c1e, (Ra = 0.084 μm, Rz = 0.489 μm, Rq = 0.108 μm) in the corona discharge in 6aoirf 2a2t
atmospheric pressure is shown in Figure 1.
Polymers 2016, 8, 131 6 of 22

As the polymer covered only a circular area of 10 mm in diameter on the center of the substrate
Figure 1. SEM image of Polydimethylsiloxane (PDMS) layer deposited on Ti substrate in negative
Figure1. SEMimageofPolydimethylsiloxane(PDMS)layerdepositedonTisubstrateinnegative
surface and the HAp was deposited on the entire Ti disk surface (20 mm in diameter), the interface
corona discharge.
coronadischarge.
zone between the Ag:HAp-PDMS, Zn:HAp-PDMS, HAp-PDMS and Ag:HAp, Zn:HAp, HAp

respectively will be further investigated.
InFigure2areshowntheimagesofAg:HAp-PDMS,Zn:HAp-PDMSandHAp-PDMSlayers,
In Figure 2 are shown the images of Ag:HAp-PDMS, Zn:HAp-PDMS and HAp-PDMS layers,
depositedonTisubstrateswithopticallypolishedsurfaces,attheirinterfacewiththeAg:HAp,Zn:HAp
deposited on Ti substrates with optically polished surfaces, at their interface with the Ag:HAp,
orHAplayers. Thecoatingsseemtobecompactandhomogeneouswithnocracks,thepolymeracting
Zn:HAp or HAp layers. The coatings seem to be compact and homogeneous with no cracks, the
likeamatrixthatallowsthehydroxyapatiteembedding.
polymer acting like a matrix that allows the hydroxyapatite embedding.

Figure 2. The SEM images of the interface zone between: (a) Ag:HAp-PDMS (darker zone) and
Figure2.TheSEMimagesoftheinterfacezonebetween:(a)Ag:HAp-PDMS(darkerzone)andAg:HAp
Ag:HAp (lighter zone); (b) Zn:HAp-PDMS (darker zone) and Zn:HAp (lighter zone); (c) HAp-PDMS
(lighterzone);(b)Zn:HAp-PDMS(darkerzone)andZn:HAp(lighterzone);(c)HAp-PDMS(darker
(darker zone) and HAp (lighter zone).
zone)andHAp(lighterzone).
It is well known that for biological applications, the physical properties of the coatings like:
It is well known that for biological applications, the physical properties of the coatings like:
uniformity, delamination or cracking are very important issues that depend not only on deposition
uniformity,delaminationorcrackingareveryimportantissuesthatdependnotonlyondeposition
procedure but also on the roughness of the substrate surface. A rough substrate surface will
procedurebutalsoontheroughnessofthesubstratesurface. Aroughsubstratesurfacewilldetermine
determine a non-uniform deposited layer, the possibility of coating delamination and cracking being
anon-uniformdepositedlayer,thepossibilityofcoatingdelaminationandcrackingbeingincreased.
increased. Therefore, we supposed that the use of a polymer interlayer for the HAp coating could
Therefore,wesupposedthattheuseofapolymerinterlayerfortheHApcoatingcouldovercomeits
overcome its delamination even when cracks appear. Thus, in the following, we extend our SEM
delaminationevenwhencracksappear. Thus,inthefollowing,weextendourSEMinvestigations
investigations to the case of HAp based coatings deposited on rough surfaces in the presence and the
tothecaseofHApbasedcoatingsdepositedonroughsurfacesinthepresenceandtheabsenceofa
absence of a PDMS layer laying on the substrate surface.
PDMSlayerlayingonthesubstratesurface.
On a Ti rough surface (Ra = 0.520 μm, Rz = 3.064 μm, Rq = 0.643 μm) partially coated with a
OnaTiroughsurface(R =0.520µm,R =3.064µm,R =0.643µm)partiallycoatedwithaPDMS
PDMS layer, we depositeda by sol-gel dizp coating metqhod a Zn:HAp layer. The SEM images
layer, wedepositedbysol-geldipcoatingmethodaZn:HAplayer. TheSEMimagespresentedin
presented in Figure 3 indicate the embedding of the Zn:HAp coating into the PDMS layer.
Figure3indicatetheembeddingoftheZn:HApcoatingintothePDMSlayer.

Figure 3. SEM images of the interface zone between Zn:HAp-PDMS and Zn:HAp coating.
Polymers 2016, 8, 131 6 of 22
As the polymer covered only a circular area of 10 mm in diameter on the center of the substrate
surface and the HAp was deposited on the entire Ti disk surface (20 mm in diameter), the interface
zone between the Ag:HAp-PDMS, Zn:HAp-PDMS, HAp-PDMS and Ag:HAp, Zn:HAp, HAp
respectively will be further investigated.
In Figure 2 are shown the images of Ag:HAp-PDMS, Zn:HAp-PDMS and HAp-PDMS layers,
deposited on Ti substrates with optically polished surfaces, at their interface with the Ag:HAp,
Zn:HAp or HAp layers. The coatings seem to be compact and homogeneous with no cracks, the
polymer acting like a matrix that allows the hydroxyapatite embedding.

Figure 2. The SEM images of the interface zone between: (a) Ag:HAp-PDMS (darker zone) and
Ag:HAp (lighter zone); (b) Zn:HAp-PDMS (darker zone) and Zn:HAp (lighter zone); (c) HAp-PDMS
(darker zone) and HAp (lighter zone).
It is well known that for biological applications, the physical properties of the coatings like:
uniformity, delamination or cracking are very important issues that depend not only on deposition
procedure but also on the roughness of the substrate surface. A rough substrate surface will
determine a non-uniform deposited layer, the possibility of coating delamination and cracking being
increased. Therefore, we supposed that the use of a polymer interlayer for the HAp coating could
overcome its delamination even when cracks appear. Thus, in the following, we extend our SEM
investigations to the case of HAp based coatings deposited on rough surfaces in the presence and the
absence of a PDMS layer laying on the substrate surface.
On a Ti rough surface (Ra = 0.520 μm, Rz = 3.064 μm, Rq = 0.643 μm) partially coated with a
PolymePrsD2M01S6, 8la,1y3e1r, we deposited by sol-gel dip coating method a Zn:HAp layer. The SEM images7 of22
presented in Figure 3 indicate the embedding of the Zn:HAp coating into the PDMS layer.

Figure 3. SEM images of the interface zone between Zn:HAp-PDMS and Zn:HAp coating.
Figure3.SEMimagesoftheinterfacezonebetweenZn:HAp-PDMSandZn:HApcoating.
Polymers 2016, 8, 131 7 of 22

Whenthesamplewastiltedatanangleof45˝,(enlargedimagesfromFigure3),thesmoother
When the sample was tilted at an angle of 45°, (enlarged images from Figure 3), the smoother
surfaceoftheZn:HAp-PDMSwasbetterevidencedincomparisonwiththeZn:HApcoating. Similar
surface of the Zn:HAp-PDMS was better evidenced in comparison with the Zn:HAp coating. Similar
morphologiestotheZn:HApcoatingswereobtainedfortheothertwostudiedsamples(Ag:HApand
morphologies to the Zn:HAp coatings were obtained for the other two studied samples (Ag:HAp
HanAdp HlaAype rlsa)y.ers).
TThhee SSEEMM aannaallyyssiiss ooff tthhee ttrraannssvveerrssaall ccrroossss sseeccttiioonn ooff aa ZZnn::HHAApp--PPDDMMSS llaayyeerr,, FFiigguurree 44,, iinnddiiccaatteedd
iittss tthhiicckknneessss ((aarroouunndd 335500 nnmm)) aass wweelll laas sththe erorolel eofo fthteh epoploylmymere prrperseesnecnec oeno tnhteh seusbusbtrsattrea tseursfuarcfea.c Iet.
Ictocuoldu lbdeb oebosbersverevde hdohwo wtheth peoplyomlyemr ecrovcoevrse rusnuifnoirfmorlmy ltyhet huenuevneenv esnubsustbrsattrea steursfuarcfea.c Teh.uTsh,u its ,ciotuclodu blde
ba eparopmroomteor tfeorrf tohret hinecirnecarseea osef ZofnZ:HnA:HpA cpoactoinatgi nagdhaderheenrceen ctoe ttohet hreouroguhg shubsustbrsattrea. te.

Figure 4. SEM images of the transversal cross section of a Zn:HAp-PDMS layer deposited on a rough
Figure4.SEMimagesofthetransversalcrosssectionofaZn:HAp-PDMSlayerdepositedonarough
Ti surface substrate.
Tisurfacesubstrate.
On a scratched and rough (Ra = 0.462 μm, Rz = 2.349 μm, Rq = 0.550 μm) surface of Ti substrate
Onascratchedandrough(R =0.462µm,R =2.349µm,R =0.550µm)surfaceofTisubstrate
partially coated with the polymer,a we deposited az HAp layer. In qFigure 5a,b can be observed that the
partiallycoatedwiththepolymer,wedepositedaHAplayer. InFigure5a,bcanbeobservedthatthe
scratches from the Ti surface affect the HAp coating morphology and layer uniformity, its
scratchesfromtheTisurfaceaffecttheHApcoatingmorphologyandlayeruniformity,itsdelamination
delamination being promoted. Even if the coating is non-uniform, delaminated and cracked, its
beingpromoted. Evenifthecoatingisnon-uniform,delaminatedandcracked,itsembeddinginto
embedding into the polymer could prevent it from further deterioration. The Ag:HAp and HAp
thepolymercouldpreventitfromfurtherdeterioration. TheAg:HApandHApcoatingshadsimilar
coatings had similar morphologies when deposited on Ti surfaces.
morphologieswhendepositedonTisurfaces.

Figure 5. SEM images of the interface zone between HAp-PDMS (darker zone) and HAp (lighter
zone) for different magnification: (a) ×2000; (b) ×10,000.
Polymers 2016, 8, 131 7 of 22
When the sample was tilted at an angle of 45°, (enlarged images from Figure 3), the smoother
surface of the Zn:HAp-PDMS was better evidenced in comparison with the Zn:HAp coating. Similar
morphologies to the Zn:HAp coatings were obtained for the other two studied samples (Ag:HAp
and HAp layers).
The SEM analysis of the transversal cross section of a Zn:HAp-PDMS layer, Figure 4, indicated
its thickness (around 350 nm) as well as the role of the polymer presence on the substrate surface. It
could be observed how the polymer covers uniformly the uneven substrate surface. Thus, it could be
a promoter for the increase of Zn:HAp coating adherence to the rough substrate.

Figure 4. SEM images of the transversal cross section of a Zn:HAp-PDMS layer deposited on a rough
Ti surface substrate.
On a scratched and rough (Ra = 0.462 μm, Rz = 2.349 μm, Rq = 0.550 μm) surface of Ti substrate
partially coated with the polymer, we deposited a HAp layer. In Figure 5a,b can be observed that the
scratches from the Ti surface affect the HAp coating morphology and layer uniformity, its
delamination being promoted. Even if the coating is non-uniform, delaminated and cracked, its
Polymeersm2b0e16d,d8i,n1g31 into the polymer could prevent it from further deterioration. The Ag:HAp and HAp8 of22
coatings had similar morphologies when deposited on Ti surfaces.

Figure 5. SEM images of the interface zone between HAp-PDMS (darker zone) and HAp (lighter
Figure5.SEMimagesoftheinterfacezonebetweenHAp-PDMS(darkerzone)andHAp(lighterzone)
zone) for different magnification: (a) ×2000; (b) ×10,000.
fordifferentmagnification:(a)ˆ2000;(b)ˆ10,000.
Polymers 2016, 8, 131 8 of 22
The texture of Ag:HAp, Zn:HAp and HAp coatings deposited on mirror like surfaces, in the

The texture of Ag:HAp, Zn:HAp and HAp coatings deposited on mirror like surfaces, in the
presenceandabsenceofthepolymerlayer,wasobtainedperforminga3Dsurfaceplot(Figure6)of
presence and absence of the polymer layer, was obtained performing a 3D surface plot (Figure 6) of
theirSEMimagesusingaprocessingimageusingImageJsoftware(https://imagej.nih.gov/ij/).
their SEM images using a processing image using Image J software (https://imagej.nih.gov/ij/).

Figure6. 3Dsurfaceplotof: (a)PDMS(SEMimagefromFigure1);(b)Ag:HAp(SEMimagefrom
Figure 6. 3D surface plot of: (a) PDMS (SEM image from Figure 1); (b) Ag:HAp (SEM image from
Figure 2a); (c) Ag:HAp-PDMS layer (SEM image from Figure 2a); (d) Zn:HAP (SEM image from
Figure 2a); (c) Ag:HAp-PDMS layer (SEM image from Figure 2a); (d) Zn:HAP (SEM image from
FigurFeig2ubr)e; 2(be)); Z(en) :ZHnA:HPA-PP-DPMDMSS( S(SEEMM iimmaaggee frforomm FiFgiugrue r2eb)2; b(f)); H(fA)pH (ASEpM( SimEMagei mfroamge Fifgruorme 2Fci)g; (ugr)e 2c);
(g)HAHpA-pP-DPDMMSS( S(SEEMMi mimaaggee ffrroomm FFiigguurree 22cc)); ;(h(h) )ZZnn:H:HAAp p(S(ESME Mimiamgea gfreofmro FmiguFrige u3r);e (3i)) ;Z(ni):HZAnp:H-PADpM-PSD MS
(SEM(SimEMag iemfargoem froFmig uFirgeu3r)e; 3()j;) (Hj) AHpAp(S (ESEMMi mimaaggee ffrroomm FFiigguurree 55a)a; )(;k()k H)AHpA-PpD-PMDSM (SSEM(S EimMagime fargome from
FigurFeig5uar)e. 5a).
3.2. Glow Discharge Optical Emission Spectrometry (GDOES)
3.2. GlowDischargeOpticalEmissionSpectrometry(GDOES)
Glow discharge optical emission spectrometry (GDOES) is a chemical analytical method often
Glowdischargeopticalemissionspectrometry(GDOES)isachemicalanalyticalmethodoften
used for the evaluation of constituent elements distributed throughout the coatings. It gives
used for the evaluation of constituent elements distributed throughout the coatings. It gives
information on a macroscopic scale (usually the diameter of the investigated surface is around 4
informmamti)o anboount athme adcerpotshc opproicfislicnagl eo(f utshuina lfliylmths ethdaita cmoveteerr moefttahlleici nsuvrefsatciegsa, thedavsinugr fashcoerits mareoausunrdin4g mm)
abouttimthee adndep htihghp sreonfislitiinvgityo. fInth GinDOfiElmS,s thteh aemt cisosvioenr imnteentaslitliiecs souf rtfhaec eelse,mheanvtsin cgonsthaionretdm ine aa scuoraitningg time
are measured as function of the sputtering time of the sample in a glow discharge plasma. GDOES
depth profiles of the chemical elements contained in the Ag:HAp, Zn:HAp, and HAp films
deposited on substrates previously covered with a PDMS layer are presented in Figure 7.

Figure 7. GDOES depth profiles of: (a) Ag:HAp-PDMS; (b) Zn:HAp-PDMS; (c) HAp-PDMS
composite layers.
Polymers 2016, 8, 131 8 of 22
The texture of Ag:HAp, Zn:HAp and HAp coatings deposited on mirror like surfaces, in the
presence and absence of the polymer layer, was obtained performing a 3D surface plot (Figure 6) of
their SEM images using a processing image using Image J software (https://imagej.nih.gov/ij/).

Figure 6. 3D surface plot of: (a) PDMS (SEM image from Figure 1); (b) Ag:HAp (SEM image from
Figure 2a); (c) Ag:HAp-PDMS layer (SEM image from Figure 2a); (d) Zn:HAP (SEM image from
Figure 2b); (e) Zn:HAP-PDMS (SEM image from Figure 2b); (f) HAp (SEM image from Figure 2c); (g)
HAp-PDMS (SEM image from Figure 2c); (h) Zn:HAp (SEM image from Figure 3); (i) Zn:HAp-PDMS
(SEM image from Figure 3); (j) HAp (SEM image from Figure 5a); (k) HAp-PDMS (SEM image from
Figure 5a).
3.2. Glow Discharge Optical Emission Spectrometry (GDOES)
Glow discharge optical emission spectrometry (GDOES) is a chemical analytical method often
used for the evaluation of constituent elements distributed throughout the coatings. It gives
Polymers2016,8,131 9of22
information on a macroscopic scale (usually the diameter of the investigated surface is around 4
mm) about the depth profiling of thin films that cover metallic surfaces, having short measuring
taimnde haingdh hsiegnhs isteivnistiyt.ivIintyG. DInO GEDS,OtEhSe, etmhei sesmioinssiinotne ninstiteinessitoifesth oef ethleem eelenmtsecnotsn tcaoinnteadinienda inco aa tcionagtianrge
amree amsueraesduraesdf uans cftuionnctoiofnth oef stphuet tsepruinttgertiimnge toimfteh eofs athmep sleaminpaleg ilno wa gdlioscwh adrigsechpalargsme pa.laGsmDOa.E GSDdOepEtSh
dperopfithle sporofftihlees chofe mthicea lcehleemmiecnatl secloenmtaeinntes dcionntthaienAedg :HinA tph,eZ nA:gH:HAAp,pa, nZdnH:HAAppfi, lmansdd eHpAospi tefdilmons
dsuepbsotsriateteds opnr esvuibosutsrlaytecso pvreerevdiowusiltyh caoPvDerMedS wlaiyther aa PreDpMreSs elanyteedr ainreF pigreusreen7te.d in Figure 7.

Figure 7. GDOES depth profiles of: (a) Ag:HAp-PDMS; (b) Zn:HAp-PDMS; (c) HAp-PDMS
Figure 7. GDOES depth profiles of: (a) Ag:HAp-PDMS; (b) Zn:HAp-PDMS; (c) HAp-PDMS
composite layers.
compositelayers.
TheCaandPdepthprofiles,themainelementsinaHApbasedcoating,hadsimilarbehaviorin
allinvestigatedsamples. Accordingtoourpreviousstudies[42],inaGDOESdepthprofilespectrum
ofaPDMSlayer,wemarkedthesurfaceandthepolymer/substrateinterfacebytheSidepthprofile
curvebehavior. TheSi,CaandPdepthprofilecurvesbeginandendedsimultaneously,thusindicating
thattheHApwasembeddedintothepolymer.
Thebroadeningofthedepthprofilecurvesassociatedwiththechemicalelementsidentifiedin
thespectrafromFigure7werealmostthesameinallthesamples,beingnocleardelimitationbetween
themasinthecaseofmultilayercoatings. Differentreasonslikeroughnessofthelayer/substrate
interfaceasthesignalsrecordedbytheGDProfilerareaveragedovertheinvestigatedzone,crater
bottomflatnessoracompositematerial,coulddeterminesuchofbehavior. Sincethesubstratesare
mirrorlikesurfacesandtheoperatingconditionswerechosenforprovidingaflatcraterbottom,the
GDdepthprofilesfromFigure7seemtoindicatetheformationofacompositematerial.
By reaching the coating/substrate interface, the steep rise in the Ti depth profile curve is
accompanied by the decrease of all signals specific to the elements contained in the investigated
sample[42].
3.3. FourierTransformInfraredSpectroscopy(FT-IR)
TheIRspectraoftheHAp-PDMS,Ag:HAp-PDMSandZn:HAp-PDMSlayersthatcoveruniformly
the mirror like surfaces of Ti substrates were obtained by reflectance spectroscopy. The FTIR
absorbancespectratogetherwiththeirsecondderivative(inthespectralregionof450–2000cm´1)
arepresentedinFigure8. IntheIRabsorbancespectraofthestudiedsamples,themainvibrational
bands were attributed to the Si–O–Si (about 1004 and 1071 cm´1) [49], SiO 4´ (around 490 and
4
695cm´1)[44,48,50–54],Si–OandSi–C(approximatively862cm´1)[55]functionalgroupspresentin
thePDMSlayer. Inourpreviousstudies[42,43],weshowedthatduringthepolymerizationtimein
negativecoronadischarge,inaPDMSlayer,SiO likestructuresweregenerated,theSiO 4´groups
2 4
beingpredominantatthepolymersurface. Asaresultofthethermaltreatmentperformedonthe
samplesafterthedepositionofHAp, Ag:HApandZn:HAponthetitaniumsubstratespreviously
coatedwiththePDMSlayer,somesubstitutionsofthePO 3´ fromtheapatiticstructurebySiO 4´
4 4
werepossible.[38,52,53,56–58]. Furthermore,theappearanceofSiO 4´groupsinthespectralregion
4
of450–650cm´1at498(inHAp-PDMSlayer)respectively511cm´1(inZn:HAp-PDMSlayer),was
previouslyattributedtoapartiallossofphosphategroupsand/orofthesymmetryatthesitecaused
bysubstitutionofsilicatespeciesinSi-HAp[51,59,60].
Polymers2016,8,131 10of22
Polymers 2016, 8, 131 10 of 22

FFiigguurree 88..F TFIRTIsRp ecstpreacatnrad saencodn dsedceorinvda tivdeesriovfatthiveeHs Aopf- PDthMe S,HAAgp:H-PADpM-PSD, MASga:nHdAZpn-P:HDAMpS-P DaMndS
Zconm:HpAopsi-tPeDlaMyeSr sc.omposite layers.
TThhee IIRRb bananddssid iednetinfiteifdieadt aabto uabto60u0t a6n0d0 1a0n5d3 c1m0´531 ccomrr−e1 spcoornrdestpoothned PtOo 3t´heg rPoOup43s− tghraotuapscse rtthaaint
4
athsceeHrtaAinp stthreu cHtuArep[ 4s8tr].uTcthuerec h[a4r8a]c. teTrhiset icchpaeraakcstearsisstoicci apteedaktso tahsesoHciaOte,dO Hto, Sthi–eO HH2Ovi,b rOaHtio, nSsi–wOeHre
2
vidibernattiifioends nweaerre1 6id00encmtif´ie1d[ 6n1e]a,rw 1h6il0e0t hcmep−1e a[6k1f]r,o wmh1il2e6 0thcem p´e1aiks forfotemn 1a2tt6r0ib cumte−d1 itso othfteeCn –aHttrbiobnudtefdro tmo
tthhee CSi––HC Hbongdro furpom[6 2t]h.eO Sni–tCheHo3 tghreoruhpa n[6d2,]t.h Oenv itbhrea toiothnearl bhaanndds, tfhoeu nvdibaratt7i0o0narel sbpaencdtisv feolyun7d37 act m70´01
3
raersepsepceticvifielcyt 7o3C7 –cHm−v1i abrrea tsiponeciinficC tHo Cg–rHou vpisbrtahtaiotnbe ilno nCgHt3o gSrio–uCpHs th[4a9t ,b6e3l]o.ng to Si–CH3 [49,63].
3 3
In Table 1 are summarized the main IR vibrational bands observed in the FTIR spectra of
In Table 1 are summarized the main IR vibrational bands observed in the FTIR spectra of
HAp-PDMS, Ag:HAp-PDMS and Zn:HAp-PDMS composite layers.
HAp-PDMS,Ag:HAp-PDMSandZn:HAp-PDMScompositelayers.
A powerful tool usually used for the determination of the weak absorption bands and for
improvement of the resolution of overlapped bands is the second derivative of FTIR spectra. The
second derivative spectrum of each sample is presented in the Figure 8.
The formation of SiO44− groups [44,48,50–54] was also evidenced in the second derivative
spectrum of the investigated samples. Furthermore, in the case of HAp-PDMS layer, the presence of
bands characteristics to the ν4 PO43− vibrations was noticed. These bands are characteristic to the
hydroxyapatite structure and are located at 547 and 599 cm−1 respectively [10,48]. The IR band