Enamel Based Composite Layers Deposited on Titanium Substrate with Antifungal Activity

14 Pages·2016·8.13 MB·English

Hindawi Publishing Corporation
Journal of Spectroscopy
Volume 2016, Article ID 4361051, 13 pages
http://dx.doi.org/10.1155/2016/4361051
Research Article
Enamel Based Composite Layers Deposited on Titanium
Substrate with Antifungal Activity
S.L.Iconaru,1A.M.Prodan,2C.S.Turculet,2M.Beuran,2R.V.Ghita,1A.Costescu,3
A.Groza,4M.C.Chifiriuc,5,6P.Chapon,7S.Gaiaschi,7R.Hristu,8
G.A.Stanciu,8R.Trusca,9M.Ganciu,4S.M.Raita,10N.Vineticu,11andC.S.Ciobanu1
1NationalInstituteofMaterialsPhysics,P.O.BoxMG07,077125Ma˘gurele,Romania
2EmergencyHospitalFloreascaBucharest,8CaleaFloresca,014461Bucharest,Romania
3HyperionUniversityofBucharest,169CaleaCalarasilor,030615Bucharest,Romania
4NationalInstituteforLaser,PlasmaandRadiationPhysics,409AtomistilorStreet,P.O.BoxMG36,077125Ma˘gurele,Romania
5MicrobiologyDepartment,FacultyofBiology,UniversityofBucharest,1–3PortocalelorLane,77206Bucharest,Romania
6Earth,EnvironmentalandLifeSciencesSection,ResearchInstituteoftheUniversityofBucharest,
1–3PortocalelorLane,77206Bucharest,Romania
7HoribaJobinYvonSAS,16-18RueduCanal,91165LongjumeauCedex,France
8CenterforMicroscopy-MicroanalysisandInformationProcessing,UniversityPolitehnicaofBucharest,
313SplaiulIndependentei,060042Bucharest,Romania
9SCMETAVResearch&DevelopmentBucharest,P.O.Box22,Bucharest,Romania
10FacultyofVeterinaryMedicine,UniversityofAgriculturalSciencesandVeterinaryMedicine,Bucharest,Romania
11FacultyofPhysics,UniversityofBucharest,405AtomistilorStreet,P.O.BoxMG1,077125Ma˘gurele,Romania
CorrespondenceshouldbeaddressedtoC.S.Ciobanu;[email protected]
Received11July2016;Revised30September2016;Accepted24October2016
AcademicEditor:MarkBaker
Copyright©2016S.L.Iconaruetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
ThegoalofthispaperwastoinvestigatetheantifungalactivityofEnamellayersdepositedontitaniumsubstrate(Ti-Enamel)and
Enamellayersdepositedontitaniumsubstratepreviouslycoatedwithavinylpolydimethylsiloxanelayer(Ti-PDMS-Enamel).The
physicochemicalpropertieswerealsoinvestigated.TheCandidaalbicansbiofilmdevelopmentontheobtainedlayerswasexamined
after24h,48h,and72hbyconfocallaserscanningmicroscopy(CLSM)andscanningelectronmicroscopy(SEM)afterethidium
bromidestaining.AsignificantinhibitionofthefungaladherenceandbiofilmdevelopmentwasobservedonTi-Enamellayers.The
antifungalresultsdemonstratedthattheuseofnewTi-Enamelcompositelayerscouldrepresentapromisingperspectiveforthe
preventionoffungalbiofilmsassociatedimplantinfections.
1.Introduction andsoforth[2].Animportantaspectofthemedicalapplica-
tionascoatingsfororthopedicimplantsconsistsinimproving
Biomaterials[1–6]canbeconsideredthe“buildingblocks”of the biocompatibility and osteointegration of prostheses [7–
modernmedicine,duetotheirmultipleapplications,either 10]. The need for materials that can improve the bonding
forthefabricationofmedicaldevices(heartvalvesprostheses, ability of an implant to bone tissue is at high demand
artificial hip joints, dental implants, intraocular lenses, left because this represents a fundamental requirement for a
ventricular assist devices, wound dressings, artificial skin, successfulimplantinclinicaluse[11,12].Therefore,thesearch
degradable sutures, scaffolds for cell and tissue transplants, for the optimum combination of implant and coating is
arterial stents, etc.) or for diagnostic and theranostic plat- crucial when developing an implantable device. The most
forms,biomoleculesisolation,purification,characterization, used metal in the field of orthopedic implants is titanium,
2 JournalofSpectroscopy
due to the optimal combination of a good biocompatibility depict dental Enamel as being the obvious choice when
and ofsuitable mechanicalproperties.The coatingused for aimingatachievingbothbiocompatibilityandantimicrobial
improving the inert titanium biocompatibility is usually a properties.Enamelisfoundintheouterhardtissuelayerof
bioactive ceramic, such as hydroxyapatite (HAp) or other tooth crowns, is the hardest bioceramic that can be found
similarbioactivematerials[13].Eventhoughtherehavebeen inthevertebratebody,andhasauniquecomplexstructure.
remarkableprogressesmadeintheareaofHApbiomedical This composite contains hydroxyapatite crystals, water, and
applications,therearestillveryimportantissuesthatneedto anorganicmatrix.EventhoughtheavailabledataforEnamel
beaddressed.Oneofthemostimportantonesistheappari- (mainly composed of hydroxyapatite which is well known
tionofpostoperatoryinfectionsandthedifficultyintreating foritsbiocompatibleproperties)isquitelimited,theresults
themwithconventionalantibiotics[14–17].Theemergenceof regardingitsantimicrobialpropertiespresentedbyussuggest
antibioticresistantpathogensrepresentsamajorhealthissue that it can be considered as a coating material for medical
andithasbeenreportedthatmorethan70%ofthebacterial implantabledevices.Nevertheless,thecombinationofmetal
infections cannot be treated with conventional antibiotics mechanical properties and of a bioactive and antimicrobial
[18–20].Inthelast30yearsinfectionscausedbyfungalstrains coating is not sufficient in order to achieve good results in
havebeennotablyincreasinginawiderangeofpatientsfrom themedicalfield.Inclinicaluse,imperfectionssuchasweak
theimmunocompromisedindividualstotheoneswithmild adhesionofthecoatingonthemetalsubstrateorlowscratch
healthproblems.Consideredtobeoneofthemostdangerous resistance could significantly narrow the practical applica-
fungal organisms, the Candida strains are responsible for tions [32, 33]. In their effort to solve these impediments,
theoccurrenceofthemostusuallyacquiredhospitalrelated duringthelastdecadesresearchersworldwideproposedboth
infections [21]. The most representative strain of Candida, the use of various deposition techniques and the use of a
thatis,Candidaalbicans,canbethecauseofvaginal,oral,and polymer with the scope of immobilizing the structure. For
systemic infections in hospitalized patients, causing severe thatpurpose,inourpreviousstudies[34],weproposedthe
and sometimes deadly affections. Recent estimates of the useofapolydimethylsiloxane(PDMS)asaninterlayerfora
National Institutes of Health have reported that more than silver doped hydroxyapatite (Ag:HAp) coating. The studies
80%ofthemicrobialfungalinfectionsarecausedbybiofilm conductedbyPopaetal.[34]revealedthatthePDMSlayer
growingmicroorganisms[21–24].Candidaalbicansbiofilms actedasamatrixfortheincorporationoftheAg:HAp,thus
arehighlycomplexstructuresformedbymultiplecelltypes creatingacrystallineAg:HAp-PDMScompositelayer.
embeddedinanextracellularmatrix[25].Morethanthat,C. The goal of the present research was the fabrication,
albicans is the most frequent microbial strain isolated from characterization,andinvitroevaluationofCandidaalbicans
medical devices such as catheters, pacemakers, mechanical development on Enamel, PDMS, and PDMS-Enamel layers
heart valves, joint prostheses, contact lenses, and dentures depositedontitaniumsubstrate.
associatedwithdevice-relatedinfections.Statisticaldatafrom
United States show that each year more than five million
2.ExperimentalMethod
cathetersareplacedandabiofilminfectionappearsinmore
than50%ofthecases[26].Moreover,theresistanceofthese
2.1.DepositionofEnamelLayeronCommerciallyPureTiDisks
fungalbiofilmstocurrentantibioticshasledtoconsiderable
(Ti-Enamel). The Enamel layer has been deposited on the
effortsintreatingtheinfectionoftenleadingtoacombination
titanium substrate using a thermal evaporation technique
between a supplementary surgery to remove the infected
[34].
device and the administration of high doses of antibiotics.
Most of the times these procedures are dangerous to the
2.2. Deposition of Enamel Based Solid Layers on Titanium
patient, they can be costly, or they could be impossible to
SubstratesPreviouslyCoatedwithaPDMSLayer(Ti-PDMS-
performduetotheprecariousconditionofthepatient[27].
Enamel). Firstly, the PDMS layer was deposited, starting
Inthiscontext,agreatdealofattentionwasaimedtowards
fromvinylterminatedpolydimethylsiloxane(Ti-PDMS)liq-
the improvement of the antimicrobial properties of the
uid precursors, on Ti substrate in atmospheric air pressure
materials used for the fabrication of medical devices. The
corona discharges. The entire deposition procedure was
ability to control bacterial biofilm development would be
depictedindetailin[35,38].Afterthat,theEnamellayerwas
advantageous and helpful in eliminating the risk of biofilm
deposited on the titanium substrate previously coated with
implantassociatedinfectioninpatients.Therefore,therehave
PDMSusingathermalevaporationtechniqueaccordingtoa
been several studies reporting materials with antimicrobial
previouslyreportedpaper[34].
properties that could also be used as coatings for medical
metal devices. The most investigated materials related to
the apparition of infections caused by microbial biofilm 3.CharacterizationMethods
formation are in dentistry. Several studies have reported
bioactive restorative materials which possess antimicrobial 3.1.ScanningElectronMicroscopy(SEM). Themorphological
properties [27–30]. Montanaro et al. [31] have emphasized analysisoftheEnamelbasedcoatingsurfaceinthepresence
in their study that different microbial strains adhesion on and absence of a PDMS layer has been investigated by
dentalrestorativematerialscouldbeinfluencednotonlyby scanning electron microscopy (SEM) using a FEI Inspect S
the particular surface chemistry of the material but also by scanningelectronmicroscopeinbothhigh-andlow-vacuum
thedifferentroughnessofthematerialsurface.Severalstudies modes.
JournalofSpectroscopy 3
3.2.EnergyDispersiveX-RaySpectroscopy(EDS). Elemental dynamics of biofilms developed in the presence of tested
compositionalanalysiswasdoneusingenergydispersiveX- samples.Forthequalitativeevaluationofthefungalbiofilm
ray spectroscopy (EDS). All spectra were acquired with an development, after incubation, the samples were carefully
EDSInc.SiLidetectorattachedinsidethescanningelectron washed with sterile saline buffer to remove any unattached
microscope.Themeasurementswereperformedat10kVin microbialcellsandthenfixedwithcoldmethanolandstained
ordertoavoidthesubstantialpresenceofmetalatomsinthe withethidiumbromide(5𝜇g/mLsolutionindistilledwater).
spectrum. The colonized specimens were submerged in the staining
solution and incubated in dark, for five minutes, at room
3.3.FourierTransformInfraredSpectroscopy(FT-IR). Ti-En- temperature. After incubation, the excess of the staining
amelandTi-PDMS-EnamellayerswereinvestigatedbyFTIR solutionwasremovedusingafilterpaperandthespecimens
spectroscopy using a SP100 IR Perkin Elmer spectrometer wereexaminedimmediately.Thesampleswerevisualizedin
equippedwithavariableanglespecularreflectanceaccessory. reflection and transmission mode by using a Leica micro-
Thespectrawerecarriedoutforanangleofreflectionof30∘. scope(TCS-SPCSLMmodel),equippedwithPLFLUOTAR
(40x NA 0.7, electronic zoom 1), and a He-Ne laser tuned
on633nmwavelength.Alateralresolutionofabout600nm
3.4. Glow Discharge Optical Emission Spectroscopy. The ele-
wasachieved.TheLeicasoftwarewasusedforexaminingthe
mentaldepthprofileanalysisoftheTi-EnamelandTi-PDMS-
surfacetopography[39].
Enamel layers was performed by Glow Discharge Optical
Inordertoexaminetheadherenceofviablecellsonthe
EmissionSpectroscopy.Theexperimentalconditionsusedfor
substratessurface,thesampleswereimmersedinfreeculture
the operation of the GD Profiler (Horiba Company) were
medium inoculated with standard fungal suspension, incu-
650Pa, 35W RF power impulse mode at 1kHz, and a duty
bated in static conditions for 2 hours, and then examined
cycleof0.25.
usingaNikonC2confocallaserscanningmicroscope.Atthe
end of the incubation period, the substrata were removed
3.5. Confocal Laser Scanning Microscopy (CLSM). Confocal
fromthesuspension,gentlywashedwithPBStoremovethe
laserscanningmicroscopy(CLSM)wasperformedtostudy
unattachedfungalcells,andthenstainedwithethidiumbro-
the Candida albicans development on different substrates. mide(5𝜇g/mL)forfiveminutesandexaminedimmediately
The images were obtained using a Leica TCS SP (Leica
inordertohighlighttheadherenceofviablefungalcellson
Microsystems, Germany) confocal laser scanning micro-
thesubstratessurface.
scope. The samples were stained with ethidium bromide
(5𝜇g/mL) for five minutes before microscopic examination
3.8.QuantitativeEvaluationofFungalBiofilmDevelopment
byCLSM.TheimageswerecollectedusingaLeicaHCXPL
FLUORITE40x/0.75NAdryobjective(LeicaMicrosystems,
3.8.1. Total Biofilm Assay. The monospecific biofilm was
Germany)withexcitationwiththeArionlaserat488nmand
grownontheobtainedsampleinsterile6-wellplates(Nunc)
an emission between 580 and 660nm. Under this imaging containing2mLofLBbrothinoculatedwith∼105-106colony
configuration, typical confocal resolution is of the order of
formingunits(CFU)/mLoffungalsuspension.Thesamples
250nm in the lateral direction and 600nm in the axial ∘
wereallowedtoincubateat37 Cforthreetimeperiods(24h,
direction.
48h, and 72h) to assess the temporal dynamics of biofilms
AdditionalimageswereobtainedusingaNikonC2con-
developedinthepresenceoftestedsamples.Afterincubation,
focal laser scanning microscope. The samples were stained
the samples were carefully washed with sterile saline buffer
with ethidium bromide (5𝜇g/mL) for five minutes before
in order to remove the unattached microbial cells and then
microscopic examination by CLSM. The images were col-
immersedin1mLsterilesalinebufferinEppendorftubesto
lected using a Nikon CFI Super Plan Fluor ELWD 40x/0.6
getbiofilmdetachmentbyvigorousvortexing.Thedensityof
NAwithexcitationat488nmandanemissionbetween580
therecoveredsuspensionwasdeterminedspectrophotomet-
and660nm.
rically,bymeasuringtheabsorbanceat600nm.
3.6.AntifungalAssay. Inordertoassesstheantifungalactiv- 3.8.2.ViableCountsAssay. Thesterilesubstratawereadded
ityoftheTi-Enamel,Ti-PDMS-Enamel,andTi-PDMSlayers, inasterilesalineinoculatedwith∼105-106CFU/mLofmicro-
CandidaalbicansATCC26790strainwasused. bialsuspensions.Thesampleswereincubatedunderstirring
∘
at37 Cfor72h.Thenumberoffungalviablecellsdeveloped
3.7.FungalBiofilmDevelopmentontheTi-Enamel,Ti-PDMS, in the presence of the samples was assessed using 10𝜇L of
andTi-PDMS-EnamelLayers. TheC.albicansATCC26790 fungal suspension at 24h, 48h, and 72h. For this purpose
strainwaspurchasedfromAmericanTypeCultureCollection the fungal suspensions were further diluted and 10𝜇L of
(ATCC,US).Themonospecificbiofilmdevelopmentonthe eachserialdilutionwasplatedinduplicateonLBagar.After
∘
obtainedsubstratawasperformedusingsterile6-wellplates 24hofincubationat37 C,viablecellcountswereperformed
(Nunc). Sterile coated and reference specimens were added and the number of CFU/mL for each sample was evaluat-
in a plate well in 2mL of LB broth inoculated with ∼105- ed.
106 colony forming units (CFU)/mL of fungal suspension.
∘
The samples were allowed to incubate at 37 C for three 3.9.ImageAnalysis. TheCLSMimagesacquiredduringthe
time periods (24h, 48h, and 72h) to assess the temporal experimentalsessionwereprocessedusingImageJsoftware.
4 JournalofSpectroscopy
(a) (b) (c)
Figure1:SEMimagesofTi-Enamel(a)andTi-PDMS-Enamel(b)layersandTisubstrate(c).
Ti Ti
O O
Ti
Ba
Ti
Ba
K
Si
Si K
Ti
Ca
P Ti
P Ca
Na
Al Na
K Ti Al K Ti
1 2 3 4 5 1 2 3 4 5
(keV) (keV)
(a) (b)
Figure2:EDSspectrumofTi-Enamel(a)andTi-PDMS-Enamel(b)layers.
The Candida albicans biofilms developed on the surface 4.ResultsandDiscussions
of the layers were investigated using a three-dimensional
(3D) model. The 3D reconstruction of the CLSM images ThesurfacemorphologyoftheTisubstrate,Ti-Enamel,and
was used for the C. albicans biofilm thickness estima- Ti-PDMS-Enamellayerswasexaminedbyscanningelectron
tion. microscopy(SEM).
Thefeaturesoftheinvestigatedsamplesarepresentedin
Figures1(a)–1(c).Itcanbeobservedthatthelayersarehomo-
3.10. Statistical Analysis. All the experiments regarding the geneous with no cracks (Figures 1(a) and 1(b)). Figure1(b)
antifungal properties of the layers were done in triplicate suggests that the presence of the polymer layer on the Ti
and repeated three times. The biofilm development was substrate during the Enamel deposition process determines
estimated by plotting the mean absorbance values of C. amorepronouncedgranulationoftheEnamel.
albicansATCC26790biofilmcellsdevelopedonthesamples. The EDS spectra of Ti-Enamel and Ti-PDMS-Enamel
The statistical analysis was conducted in agreement with coatingsarepresentedinFigures2(a)and2(b).Inbothspec-
other previous studies on surface and biological evaluation tratheelements(Si,Ti,P,Ca,Ba,Na,K,andAl)contained
of hydroxyapatite-based coatings on titanium deposited by bythestudiedlayerscanbeidentified.Moreover,itcouldbe
different techniques presented by Massaro et al. [40]. Fur- observedthatthepeaksassociatedwithSiandOelementsare
thermore,thedataimageanalysiswascarriedoutusingthe moreintenseinthecaseofPDMS-Enamelcoatingsdeposited
Student 𝑡-test and the values of 𝑝 < 0.05 were accepted as onTisubstratecomparedtothoseassociatedwiththeEnamel
significant. layer.
JournalofSpectroscopy 5
Table1:ThemainfunctionalIRgroupspresentinthestructureofTi-EnamelandTi-PDMS-Enamellayers.
−1 IRbandsassignment
Wavenumber(cm )
Ti-Enamel Ti-PDMS-Enamel
3− 3−
491,590,640,685 VibrationsinPO4 ions VibrationsinPO4 ions
750 Si-O/Si-CH3vibrationalgroup[34,35] Si-O/Si-CH3vibrationalgroup[34,35]
796 — Si-OvibrationsinSiO4groups
817 — Si-O-Sibonds[36]
832 Si-Ostretchingvibrations —
860 — Si-CH3rockingvibration[36]
2− 2−
890 VibrationsinCO3 ions VibrationsinCO3 ions
951 SymmetricP-Ostretchingmode —
964 — Si-OHvibrationalgroup
1014,1090 — Si-O-Sivibrations[36]
1042,1052,1063,1094 AsymmetricP-Ostretchingmode AsymmetricP-Ostretchingmode
3− 3−
1130,1179,1216 VibrationsinPO4 ions VibrationsinPO4 ions
1260 — CH3vibrationsinSi-CH3group[36]
2− 2−
1387,1408,1425,1441 VibrationsinCO3 ions[35] VibrationsinCO3 ions[37]
100 100
nce (%) 6800 nce (%) 6800
flecta 40 flecta 40
Re Re 20
20 Ti-Enamel Ti-PDMS-Enamel
0
600 800 1000 1200 1400 600 800 1000 1200 1400
Wavenumber(cm−1) Wavenumber(cm−1)
(a) (b)
Figure3:IRspectraofTi-Enamel(a)andTi-PDMS-Enamel(b)layers.
The molecular structure of the Enamel and PDMS- The elemental depth profiles of the Ti-Enamel and Ti-
Enamel layers uniformly deposited on mirror-like surfaces PDMS-Enamel layers were investigated by Glow Discharge
of Ti substrates have been investigated by reflectance FTIR OpticalEmissionSpectrometry(GDOES)inordertoanalyze
spectroscopy(Figure2)[34,35,38].Asitisalreadyknown, the distribution of Ca, P, O, C, Si, K, Na, Ba, Al, and H
Enamelpowdershaveanapatite-likestructureandthemain atomsintheEnamelbasedlayerinthepresenceorabsence
3− − 2−
vibrationalIRbandsareattributedtoPO4 ,OH ,andCO3 ofthePDMSlayer.Asworkingprincipleofthisinstrument,a
vibrationalgroups[37,41–43]. few millimetres’ squared area from the investigated sample
The specific vibrations of these chemical groups were is sputtered in a pulsed RF Ar plasma. Consecutively, the
identifiedforTi-EnamellayerspresentedinFigure3(a)and intensityofthelightemittedbythesputteredatomsexcited
summarized in Table1. The IR bands from 1042cm−1 and in the plasma is acquired and recorded in real time. This
−1
1090cm aretypicalforapatitephosphorus-oxygenabsorp- is provided as the investigated sample is sputtered from its
tionbandsinEnamelpowders. surface down to the substrate as a function of sputtering
TheobservationofthesebandsinanEnamelbasedcoat- time.Thesputteringratehasspecificvaluesforeachmaterial,
ing certifies that the deposition process was a success. The varying during the elemental depth profiling measurement
640cm−1 vibrational band was previously observed in (acquisition).ThedepthprofilecurvesoftheCa,P,O,C,Si,
synthetic hydroxyapatite and Enamel powders and was K,Na,Ba,Al,andHatomsinthebulkandatthesurfaceof
attributedtohydroxylionsvibrations[37].IntheIRspectrum theTi-EnamellayerarepresentedinFigure4(a).
oftheTi-Enamellayer,theSi-OIRspecificbandswerealso EveniftheelementsspecifictotheEnamelhavesimilar
identified. The Si-O, Si-O-Si, and Si-CH3 specific bonds of profilestotheTi-EnamelandTi-PDMS-Enamellayer,respec-
PDMS layers [36] are also present in the IR spectrum of tively(Figure4(b)),theirintensitiesandwidthsaredifferent.
Ti-PDMS-Enamel presented in Figure3(b). The chemical Atthesametime,inFigure4(b),wecouldnotidentifyasharp
groupsfoundintheTi-PDMS-Enamelspectrumweresum- delimitationbetweentheelementspresentintheTi-Enamel
marizedinTable1. andTi-PDMS-Enamellayer.Differentreasonslikeroughness
6 JournalofSpectroscopy
20 14
18
12
16
10
14
Ti-PDMS-Enamel
u.) 12 u.) 8
a. a.
y ( 10 y (
nsit 8 Ti-Enamel nsit 6
e e
nt nt
I 6 I 4
4
2
2
0
0
0 10 20 30 40 50 0 10 20 30 40 50
Sputtering time (s) Sputtering time (s)
Ca Si Al Ca Si Al
P K Ti P K Ti
O Na H O Na H
C Ba C Ba
(a) (b)
Figure4:GDOESdepthprofilesofTi-Enamel(a)andTi-PDMS-Enamel(b)layers.
ofthelayer/substrateinterfaceasthesignalsrecordedbythe Candida albicans ATCC 26790
GD Profiler are averaged over the investigated zone, crater 1.8
bottom flatness, or a composite material could determine 1.6
such behavior. As the Ti substrate is a mirror-like surface 1.4
and the operating conditions were chosen for providing a 1.2
flat crater bottom, the GD depth profiles from Figure4(b) m 1
n
seemtoindicatetheformationofacompositematerial.Asthe 20 0.8
6
intensityoftheSidepthprofileintheTi-PDMS-Enamellayer 0.6
decreases,theCa,P,O,C,K,Na,Ba,Al,andHdepthprofile 0.4
intensities decrease, indicating the incorporation of the Ti- 0.2
EnamelcomponentsintothePDMSlayer.Theincreaseofthe 0
Tidepthprofilecurvesimultaneouslywiththedecreaseofall 24 48 72
theelementscontainedbytheinvestigatedlayerindicatesthe Time (h)
substrateinterface[34]. Ti-Enamel Ti-PDMS
ThedevelopmentofC.albicansbiofilmonmedicaldevi- Ti-PDMS-Enamel C+
cesisfollowingthreestages:(i)theadherenceofyeastcellsto
Figure5:GraphicalrepresentationoftheabsorbancevaluesofC.
thedevicesurface(0–11h);(ii)thesecretionofextracellular
albicansATCC26790biofilmcellsdevelopedontheTi-Enamel,Ti-
matrixandtheswitchfromyeasttohyphalforms(12–30h);
PDMS-Enamel,andTi-PDMSlayers.
and (iii) the development of a three-dimensional, dense
biofilm containing both yeast and hyphal forms (38–72h)
[25, 44]. Due to the important implications of Candida
albicansstrainsinthepathologyofprostheticdevicesrelated The quantitative results showed that the fungal biofilm
infections, aggravated by the emergence of resistant fungal developmentwasinhibitedbothinearlyphaseofadherence,
strains, using new coatings for titanium implants able to quantifiedafter24h.andinthedevelopmentandmaturation
inhibit the fungal adherence could represent a promising stages, quantified after 48h and 72h of incubation, respec-
perspectiveforthepreventionofbiofilmassociatedimplant tively. The absorbance of microbial suspensions recovered
infections. fromthebiofilmsdevelopedontheTi-Enamellayerswasin
The quantitative results regarding the in vitro biofilm allcasessignificantlyreducedascomparedtothatofbiofilm
formationbyC.albicans26790onthesurfaceofTi-Enamel, cellsdevelopedontheTi-PDMSlayers.
Ti-PDMS-Enamel, and Ti-PDMS layers as determined by Furthermore,theviablecellcount(VCC)assayconfirmed
measuringthedensityofthemicrobialsuspensionrecovered the results of the total biofilm assay, as revealed by the sig-
from the biofilms adhered on the tested samples are repre- nificantdecreaseoftheVCCsrecoveredfromtheTi-PDMS-
sentedinFigure5. Enamel as compared to Ti-PDMS after 24h of incubation
JournalofSpectroscopy 7
C. albicans ATCC 26790 the time exposure. Accordingly, with SEM observation, the
4.5
Enamellayersperformedanimportantroleininhibitingthe
4
developmentofC.albicansbiofilms.Thefungicidaleffectof
3.5
EnamellayerswasnoticedagainstC.albicansbiofilmforma-
3
L tion. The presence of PDMS interlayer (Figures 8(d)–8(f))
m 2.5
U/ 2 causedamajorreductionofthefungicidaleffect.
F
C 1.5 Thestainingoftheviablefungalcellswithethidiumbro-
1 mide has also shown a decreased adherence capacity of the
0.5 fungalcellstothesamplesinthefollowingorder:Ti-Enamel
0 <Ti-PDMS-Enamel<Ti-PDMS(Figure9).
Ti-Enamel Ti-PDMS-Enamel Ti-PDMS
Inrecentyearsithadbecomeevidentthathavinginfor-
24h
mation regarding the biofilm composition and architecture
Figure 6: Graphic representation of the absorbance values of C. hasacrucialroleindevelopingnewmaterialswithantimicro-
albicansATCC26790biofilmcellsdevelopedontheTi-Enamel,Ti- bialactivity[45,46].Therefore,theinvolvementoftechniques
PDMS-Enamel,andTi-PDMSlayers. suchasCLSMcoupledwithimageprocessinghasledtomajor
contributionintheareaofbiofilmdescriptionwhichopened
new opportunities in both the medical and pharmaceutical
field.Figures10(a)–10(i)presentthe3Dreconstructionofthe
(Figure6).However,thenumberofVCCscouldbeassessed CLSMimagesoftheC.albicansbiofilmsstainedwithethid-
onlyafter24hofincubation,asafter48hand72hnoviable iumbromide(5𝜇g/mL)developedondifferentsubstratesat
cellcountscouldberecoveredfromthetestedmaterials. varioustimeintervals(24h,48h,and72h).
TheC.albicansbiofilmmorphologywasobservedusing The3Dreconstructionwasusedtodeterminetheaverage
scanningelectronmicroscopy(SEM)atthreetimeintervals thicknessoftheC.albicansbiofilmsdevelopedonthesurface
(24,48,and72h)(Figures7(a)–7(i)).TheSEMinvestigations of Ti-Enamel, Ti-PDMS-Enamel, and Ti-PDMS layers. The
revealed that the morphology of fungal cells developed on biofilm thickness was determined to be around 60𝜇m in
the Ti-PDMS and Ti-PDMS-Enamel composite layers is thecaseofTi-PDMSlayersandsteadilydecreasedwithtime
characteristictoC.albicansfungalstrain. for the Ti-PDMS-Enamel and Ti-Enamel layers reaching a
The development of C. albicans biofilm on Ti-PDMS minimumofapproximatively30𝜇mfortheTi-Enamellayers
compositelayerswassimilaratalltestedtimeintervals(24, at 72h. The biofilm thickness obtained from three different
48,and72h).Noantifungalactivitywasobservedinthecase experimentsispresentedinTable2.
ofTi-PDMScompositelayers.Onthecontrary,theevaluation Theresultsobtainedforthebiofilmthicknessareinagree-
oftheC.albicansbiofilmdevelopmentonTi-PDMS-Enamel ment with other studies which reported C. albicans biofilm
compositelayersbySEMinvestigationsrevealedanantifun- formationondifferentkindofsubstrates[47].Intheirstudies,
galactivityagainstC.albicansstrain.Theantifungalactivity Chandraetal.reportedabiofilmthicknessof25𝜇mformed
ofTi-PDMS-EnamelcompositelayeragainstC.albicanswas on denture acrylic in vitro [25]. However, Ramage et al. re-
attributed to the Enamel’s antimicrobial properties. On the portedabiofilmthicknessexceedinglygreaterof70𝜇m[48]
otherhand,thereductionofC.albicanscellsprovedtoexhibit andintheirstudies,Andesetal.[47]foundthatmanyareasof
variousgrowthratesatdifferenttimeintervals(Figures7(d)– thebiofilmintheirinvivocathetermodelexceeded100𝜇m.
7(f)). A pronounced antifungal effect against C. albicans Dependingonthetimeofincubationandthesubstrateused,
biofilmdevelopmentonTi-Enamellayerswasalsoobserved studies [25, 47, 48] have reported an average thickness of a
atvarioustimeintervals(Figures7(a)–7(c)). C.albicansbiofilmrangingfrom15to100𝜇m.Theprecedent
The observations regarding the development of C. albi- studies suggested that the difference in thickness may be
cans biofilm suggested that Enamel possesses a significant relatedtothetimeallowedforincubationoradherence.
fungicidal effect (Figures 7(a)–7(c)). SEM examination of More than that, previous studies [25, 49] have shown
theTi-Enamellayersatdifferenttimeintervalsrevealedthat thatfungalbiofilmsgrownondentureandcathetermaterial
Enamel plays a major role in the inhibition of C. albicans become resistant to traditional antifungal agents. Also, it
cellgrowth.AsillustratedinFigures7(a)–7(c),thefungicidal is well known that, similar to their bacterial counterparts,
activityofEnamelincreaseswiththetimeexposureperiod. C. albicans biofilms are highly resistant to antimicrobials.
More than that, the presence of PDMS interlayer (Figures Although drug resistance has been shown in C. albicans
7(d)–7(f))leadstoalowerfungicidaleffectofEnamelagainst [25,49]andbacterialbiofilms[50,51],thisisacomplexstudy
C.albicanscellsgrowth. whichreportsresultsregardingtheantifungalactivityofTi-
Theconfocallaserscanningmicroscopy(CLSM)analysis EnamellayersagainstC.albicansbiofilms.
ofC.albicansbiofilmsdevelopmentonTi-PDMS,Ti-PDMS- In good agreement with the previous studies presented
Enamel,andTi-EnamellayersisshowedinFigures7(a)–7(i). above,ourresultssuggestedthatthethicknessofthebiofilm
The CLSM technique was used for the investigation of isstronglydependentonthesubstrateandalsoontheincu-
C.albicansbiofilmgrowthondifferentlayersatvarioustime bation time. The results regarding the thickness of the C.
intervals (24, 48, and 72h). On the other hand, C. albicans albicans biofilms determined using a 3D reconstruction
cellsmorphologywasobserved.ThedevelopmentofC.albi- modeloftheCLSMimageswerealsoinaccordancewiththe
cans biofilms proved depending on the layer type and on existingstudies.
8 JournalofSpectroscopy
el el el
m m m
a a a
n n n
E E E
Ti- Ti- Ti-
10𝜇m 10𝜇m 10𝜇m
24h 48h 72h
(a) (b) (c)
el el el
m m m
a a a
n n n
E E E
S- S- S-
M M M
D D D
P P P
Ti- Ti- Ti-
10𝜇m 10𝜇m 10𝜇m
24h 48h 72h
(d) (e) (f)
S S S
M M M
D D D
P P P
Ti- Ti- Ti-
10𝜇m 10𝜇m 10𝜇m
24h 48h 72h
(g) (h) (i)
Figure7:SEMimages(10000x)ofC.albicansbiofilmdevelopedondifferentsubstratesatvarioustimeintervals(a)C.albicansdevelopment
onTi-Enamellayerafter24h;(b)C.albicansdevelopmentonTi-Enamellayerafter48h;(c)C.albicansdevelopmentonTi-Enamelcomposite
layerafter72h;(d)C.albicansdevelopmentonTi-PDMS-Enamellayerafter24h;(e)C.albicansdevelopmentonTi-PDMS-Enamellayer
after48h;(f)C.albicansdevelopmentonTi-PDMS-Enamellayerafter72h;(g)C.albicansdevelopmentonTi-PDMSlayerafter24h;(h)C.
albicansdevelopmentonTi-PDMSlayerafter48h;(i)C.albicansdevelopmentonTi-PDMlayerafter72h.
Furthermore,ourresultsregardingtheuseofthesesam- layersresistancegenesandantibioticresistancegeneswillbe
plesindifferentbiomedicalfieldssuchasdentalororthopedic conducted.
implants are in good agreement with previous studies on
nanocompositehydroxyapatiteconductedbyBakeretal.and 5.Conclusions
Grozaetal.[52,53].
The present studies provide the new perspectives of Enamel and PDMS-Enamel layers were deposited on Ti
EnamelandPDMS-EnamellayersonTisubstrateforpoten- substratesusingathermalevaporationtechnique.Themor-
tialuseinmedicalfield.Moreover,thesestudiesarepartof phologyoftheselayerswasinvestigatedbyscanningelectron
the pharmaceutical and medical industry effort to develop microscopy. SEM micrographs revealed that the presence
new materials with antimicrobial properties. Future studies of a polymer interlayer on the Ti substrate during Enamel
regarding genetic linkage of Enamel and PDMS-Enamel depositionledtoapronouncedgranulationofthedeposited
JournalofSpectroscopy 9
el el el
m m m
a a a
n n n
E E E
Ti- Ti- Ti-
24h 48h 72h
(a) (b) (c)
el el el
m m m
a a a
n n n
E E E
S- S- S-
M M M
D D D
P P P
Ti- Ti- Ti-
24h 48h 72h
(d) (e) (f)
S S S
M M M
D D D
P P P
Ti- Ti- Ti-
24h 48h 72h
(g) (h) (i)
Figure8:CLSMimagesofC.albicansbiofilmstainedwithethidiumbromide(5𝜇g/mL)developedondifferentsubstratesatvarioustime
intervals. (a) C. albicans development on Ti-Enamel layer after 24h; (b) C. albicans development on Ti-Enamel layer after 48h; (c) C.
albicansdevelopmentonTi-Enamelcompositelayerafter72h;(d)C.albicansdevelopmentonTi-PDMS-Enamellayerafter24h;(e)C.
albicansdevelopmentonTi-PDMS-Enamellayerafter48h;(f)C.albicansdevelopmentonTi-PDMS-Enamellayerafter72h;(g)C.albicans
developmentonTi-PDMlayerafter24h;(h)C.albicansdevelopmentonTi-PDMSlayerafter48h;(i)C.albicansdevelopmentonTi-PDM
layerafter72h.
powder. The elemental composition of the Ti-Enamel and acompositematerial.FTIRspectroscopyusedtoinvestigate
Ti-PDMS-Enamel layers was investigated using EDS and the chemical structure of the Ti-Enamel and Ti-PDMS-
GDOES analysis. The EDS results confirmed the presence Enamel layers revealed the presence of the Enamel specific
oftheconstituentelementsofbothtypesoflayers(Si,Ti,P, bands in both samples. The presence of these bands in the
Ca, Ba, Na, K, and Al). More than that, the GDOES depth FTIR spectra confirmed that the deposition process was
profilesoftheTi-PDMS-Enamelindicatedtheformationof successful.C.albicansbiofilmdevelopmentontheTi-PDMS,
10 JournalofSpectroscopy
Ti-PDMS Ti-PDMS-Enamel Ti-Enamel
Figure9:CLSMexaminationforviableC.albicansbiofilmcellsstainedwithethidiumbromidedevelopedontheTi-Enamel,Ti-PDMS-
Enamel,andTi-PDMSlayers.
el
m
mel mel na
a a E
En En Ti-
Ti- Ti-
24h 48h 72h
(a) (b) (c)
mel mel mel
na na na
E E E
MS- MS- MS-
D D D
P P P
Ti- Ti- Ti-
24h 48h 72h
(d) (e) (f)
S S S
M M M
D D D
P P P
Ti- Ti- Ti-
24h 48h 72h
(g) (h) (i)
Figure10:Three-dimensionalreconstructionofC.albicansbiofilmsdevelopedondifferentsubstratesatvarioustimeintervalsusingconfocal
microscopy.(a)C.albicansbiofilmdevelopmentonTi-Enamellayerafter24h;(b)C.albicansbiofilmdevelopmentonTi-Enamellayerafter
48h;(c)C.albicansdevelopmentonTi-Enamelcompositelayerafter72h;(d)C.albicansdevelopmentonTi-PDMS-Enamellayerafter24h;
(e)C.albicansdevelopmentonTi-PDMS-Enamellayerafter48h;(f)C.albicansdevelopmentonTi-PDMS-Enamellayerafter72h;(h)C.
albicanslayerafter48h;(i)C.albicansdevelopmentonTi-PDMlayerafter72h.
Ti-PDMS-Enamel, and Ti-Enamel layers was analyzed by regardingtheantifungalactivityexhibitedbytheTi-Enamel
SEM and CLSM at various time intervals (24, 48, and layerswereconfirmedbyquantitativeinvestigations,showing
72h).SEMandCLSMobservationsrevealedthatC.albicans thatthefungalbiofilmdevelopmentwasinhibitedatalltested
biofilmdevelopmentwastimedependent.Thepresentstudies time intervals by Ti-Enamel layers. Moreover, the viable
evidencedthatEnamellayershadafungicidaleffectagainst cellcount(VCC)confirmedthequalitativeandquantitative
C. albicans biofilm formation. The qualitative observations resultsandrevealedasignificantdecreaseofviablecellsinthe