Color Stability, Physical Properties and Antifungal

University of Nebraska Medical Center University of Nebraska Medical Center

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Theses & Dissertations Graduate Studies

Spring 5-7-2016

Color Stability, Physical Properties and Antifungal Effects of ZrO2 Color Stability, Physical Properties and Antifungal Effects of ZrO2

and TiO2 Nanoparticle Additions to Pigmented and TiO2 Nanoparticle Additions to Pigmented

Polydimethylsiloxanes Polydimethylsiloxanes

Mazen Alkahtany University of Nebraska Medical Center

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ColorStability,PhysicalPropertiesandAntifungalEffectsofZrO2andTiO2

NanoparticleAdditionstoPigmentedPolydimethylsiloxanes

By

MazenAlkahtany

ATHESIS

PresentedtotheFacultyof

TheUniversityofNebraskaGraduateCollege

InPartialFulfillmentoftheRequirements

FortheDegreeofMasterofScience

MedicalSciencesInterdepartmentalArea

(OralBiology)

UndertheSupervisionofProfessorMarkBeatty

UniversityofNebraskaMedicalCenter

Omaha,Nebraska

May,2016

AdvisoryCommittee:

FahdAlsalleehPh.D. DennisFeelyPh.D.

ThomasM.PetroPh.D. YouZhouPh.D.

i

ACKNOWLEDGMENTS

Firstofall,IwouldliketoexpressmysinceregratitudetomyadvisorDr.MarkBeattyfor

thecontinuoussupportofmyMasterstudyandrelatedresearch,forhispatience,motivation,

andimmenseknowledge.Hisguidancehelpedmeinallthetimeofresearchandwritingofthis

thesis.

Mycompletionofthisprojectcouldnothavebeenaccomplishedwithoutthesupportof

therestofmycommitteemembers:Dr.FahdAlsalleehduringhistimeasthepostgraduate

endodonticprogramdirector,Dr.ThomasPetro,Dr.DennisFeelyandDr.YouZhou.Iwouldlike

tothankthemfortheirinsightfulcommentsandinspiration.

IcannotexpressenoughthankstoBobbySimetich.Hisinvaluableexperienceinthematerials

labandhissupportallthetimeofmystudywereagreathelptocompletethisthesis.

Mysincerethanksalsogotoallfaculty,staff,labmembersandinstructorsforyoursupport,

friendshipandencouragement.AlsoIthankmyfriendsintheUniversityofNebraskaMedical

Centerforallthesupportandtimewehavebeensharing.

Iwouldliketothankmyfamily:Mydadwhoplantedtheseedofambitioninmetofulfill

mydreamandcometothegreatcountryoftheUnitedStatesofAmerica.Mom,who

continuouslysupported,encouragedandhelpedmekeepalevelheadduringmystudieshome

andintheUnitedStates.

Lastbutnotleast,thankyoutomywifeforsupportingmeandlivingmyresidencyand

studieswithmethroughoutourstayinthegreatstateofNebraskawherewewereblessedwith

ourtwowonderfulbabies,WasnandFaisal.

ii

ColorStability,PhysicalPropertiesandAntifungalEffectsofZrO2andTiO2Nanoparticle

AdditionstoPigmentedPolydimethylsiloxanes

MazenAlkahtanyB.D.S.,M.S.

UniversityofNebraskaMedicalCenter,2016

Advisor:MarkW.Beatty,D.D.S.,M.S.E.,M.S.D.,M.S.

Introduction:Colorchanges,physicaldegradationandfungalinfectionsarepotentialchallenges

tothelongevityofpolydimethylsiloxane(PDMS)elastomers.Thepurposeofthisstudywasto

evaluatepotentialimprovementsincolorchange,physicalpropertiesandantifungalproperties

ofPDMSloadedwithTiO2andZrO2nanoparticles.

Methods:1%weightof40nmor200nmdiameterTiO2orZrO2nanoparticlesweremixedinto

PDMSwith2%functionalintrinsicyellowpigmentandpolymerized.Controlmaterialscontained

13%weight200nmsilica.Sampleswereexposedtoeither3000hoursofUVBradiation

(200µW/cm2)ordarkness.ColorparametersL*a*b*and∆E*,ultimatetensilestrength,strain,

elasticmodulusandshoreAhardnessweremeasured.Datawereanalyzedusingafactorial

ANOVAwithTukey-Kramerposthoctest(p<0.05).Candidaalbicansgrowthwasmeasuredusing

XTTandconfocalmicroscopyanddataanalyzedwiththeDunnetttest(p<0.01).

Results:TiO2200nmshowedtheleastcolorchangeafter3000hoursofUVBweathering,

followedbyTiO230-40nm(p<0.05).Thesilicacontrolgroupwassuperiorinallphysicalproperty

measurements(p<0.05).TiO2-containingmaterialsexhibitedstatisticallysignificantlylowerC.

albicansgrowth(p<0.01).

Conclusion:40nmand200nmTiO2nanoparticles,whenaddedtopigmentedPDMSat1%

weight,provideimprovedcolorstabilityandlowerC.albicansgrowthcomparedtosilica-and

zirconia-filledelastomers.

iii

TABLEOFCONTENTS

ACKNOWLEDGMENTS......................................................................................................................i

ABSTRACT…..…………….………………………………………….…………………………………………………………..….…….ii

TABLEOFCONTENTS….......……….…………………………………………………………………………………………..….iii

LISTOFFIGURES……………………………………………………………………………………………………………………......vi

LISTOFTABLES……………………………………………………………..……………………………………………………………x

LISTOFABBREVIATIONS.................................................................................................................xi

CHAPTER1: INTRODUCTION..................................................................................................1

CHAPTER2: LITERATUREREVIEW...........................................................................................3

2.1 MaxillofacialProsthesesHistoryandCharacteristics.......................................................3

2.2 FactorsAffectingProsthesisDegradation........................................................................7

2.3 Nanoparticles..................................................................................................................10

2.3.1 Characteristics,MechanicalandOpticalProperties................................................10

2.3.2 AntifungalEffectsofNanoparticles.........................................................................15

2.4 GapinKnowledge...........................................................................................................16

CHAPTER3: MATERIALSANDMETHODS..............................................................................17

3.1 PreparationofSamples..................................................................................................17

3.2 ExposuretoUltravioletWeathering...............................................................................20

3.3 ColorMeasurements......................................................................................................21

3.4 PhysicalPropertiesMeasurements................................................................................22

3.4.1 TensilePropertyMeasurements.............................................................................22

3.4.2 ShoreAHardness....................................................................................................23

iv

3.5 AntifungalActivity..........................................................................................................24

3.5.1 CandidaalbicansandGrowthConditions...............................................................24

3.5.2 BiofilmFormation...................................................................................................24

3.5.3 XTTColorimetricAssay............................................................................................25

3.5.4 ConfocalLaserScanningMicroscopy(CLSM)..........................................................26

3.6 DataAnalysis..................................................................................................................27

CHAPTER4: RESULTS............................................................................................................29

4.1 ColorMeasurements......................................................................................................29

4.1.1 After600HoursofWeathering...............................................................................31

4.1.2 After1800HoursofWeathering.............................................................................36

4.1.3 After3000HoursofWeathering.............................................................................43

4.2 PhysicalProperties.........................................................................................................51

4.2.1 ShoreAHardness....................................................................................................51

4.2.2 TensileProperties....................................................................................................54


4.3.1 XTTColorimetricAssay............................................................................................59

4.3.2 ConfocalLaserScanningMicroscopy(CLSM)..........................................................59

CHAPTER5: DISCUSSION......................................................................................................63

5.1 ColorChange..................................................................................................................63

5.1.1 ColorChangesatBaseline.......................................................................................63

5.1.2 ColorChangesinControlEnvironment...................................................................64

5.1.3 ColorChangeCausedbyUltravioletRadiation.......................................................65

5.2 PhysicalProperties.........................................................................................................66

5.2.1 ShoreAHardness....................................................................................................66

v

5.2.2 TensileProperties....................................................................................................67


CHAPTER6: CONCLUSIONS..................................................................................................70

CHAPTER7: RESEARCHLIMITATIONS...................................................................................71

CHAPTER8: CONSIDERATIONSFORFUTURERESEARCH......................................................72

REFERENCES……………………………………………………………………………………………………………………………..73

vi

LISTOFFIGURES

Figure4.1.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofbaseline

colorparametersL*,a*andb*fordifferentgroups(n=10).Meanswiththesame

lowercaselettersarenotsignificantlydifferent(p>0.05).Nostatisticaldifference

detectedina*ofanymaterials........................................................................................30

Figure4.2.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆L*after

600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase

lettersarenotsignificantlydifferent(p>0.05).................................................................33

Figure4.3.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆a*after



Figure4.4.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after



Figure4.5.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after


lettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0

representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,

respectively......................................................................................................................37




vii








1800hoursofweatheringfordifferentgroups(n=5).Meanswiththesamelowercase



respectively......................................................................................................................42













viii


respectively......................................................................................................................49

Figure4.14.Lineplotsdisplayingmeansandstandarddeviations(errorbars)of∆E*over

time(600,1800and3000hours).....................................................................................50

Figure4.15.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofbaseline

ShoreAhardness(n=5).Meanswiththesamelowercaselettersarenotsignificantly

different(p>0.05).............................................................................................................52

Figure4.16.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofdeltaShore

Ahardnessafter3000hoursofweathering(n=5).Meanswiththesamelowercase


Figure4.17.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofultimate

tensilestrengthatbaselineandafter3000hoursofweathering(n=12).Meanswith

thesamelowercaselettersarenotsignificantlydifferent(p>0.05)................................55

Figure4.18.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofmodulusof

elasticityatbaselineandafter3000hoursofweathering(n=12).Meanswiththesame

lowercaselettersarenotsignificantlydifferent(p>0.05)................................................56

Figure4.19.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofstrainat

breakatbaselineandafter3000hoursofweathering(n=12).Meanswiththesame

lowercaselettersarenotsignificantlydifferent(p>0.05)................................................57

Figure4.20.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofC.albicans

opticaldensitymeasuredspectrophotometricallyat492nmafter48hours.Asterisk

denotessignificantdifferencefrompositivecontrol(p<0.01).........................................61

ix

Figure4.21.ConfocallaserscanningmicrographsofCandidaalbicansstainedwithFUN-1.

a)Ag200-400nm.b)Ag30-40nm.c)TiO2200nm.d)TiO230-40nm.e)ZrO2200nm.

f)ZrO240nm.g)Silica200-300nm.Magnification40x,oil.Scalebar=30µm...............62

x

LISTOFTABLES

Table3.1.NanoparticlesTested………………………………………………………………………………………………..18

Table4.1.BaselineColorParametersValues(Mean(S.D.),n=10)……………………………………………..29

Table4.2.∆L*,∆a*,∆b*and∆E*valuesafter600hoursofweathering(Mean(S.D.),n=5)….….32

Table4.3.∆L*,∆a*,∆b*and∆E*valuesafter1800hoursofweathering(Mean(S.D.),n=5)…...38

Table4.4.∆L*,∆a*,∆b*and∆E*valuesafter3000hoursofweathering(Mean(S.D.),n=5)…...44

Table4.5.TensilePropertiesafter3000hoursofweathering(Mean(S.D.),n=12)……………………58

Table4.6.C.albicansMetabolicActivityafter48hoursMeasuredSpectrophotometrically

at492nm(Mean(S.D.),n=9forsilicaandnanoparticlegroups,n=12forpositive

control)...……………………………………………………………………………………………………………………......60

xi

LISTOFABBREVIATIONS

a* Red-greenaxisofCIEL*A*B*system

AAA AmericanAssociationofAnaplastology

AAMP AmericanAcademyofMaxillofacialProsthetics

ANOVA Analysisofvariance

ASTM AmericanSocietyforTestingofMaterials

ATP Adenosinetriphosphate

b* Yellow-blueaxisofCIEL*A*B*system

C.albicans Candidaalbicans

CA42 C.albicanswild-typeSC5314strain

CIEL*A*B* ComissionInternationaledel’Eclairgecolorsystem

FBS Fetalbovineserum

FUN-1 Fluorescentvitaldyeforyeastandfungi

L* White-blackaxisofCIEL*A*B*system

PBS Phosphate-bufferedsaline

PDMS Polydimethylsiloxane

PVC Polyvinylchloride

PVS Polyvinylsiloxanes

RTV Roomtemperature-vulcanizedsiliconeproducts

UTS Ultimatetensilestrength

UV Ultraviolet

UVB UltravioletB

Vol.% Percentageofvolumeloaded

Weight.% Percentageofweightloaded

xii

XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-([phenylamino]carbonyl)-2H-

tetrazoliumhydroxidecolorimetricassay

YNB Yeastnitrogenbasemedium

ΔE* Overallcolorchange

1

CHAPTER1: INTRODUCTION

Extra-oralmaxillofacialprosthesesareessentialtreatmentoptionsforpatientssuffering

significantdefectsoffacialstructures.Thesedefectscanresultfromtrauma,diseaseorasa

congenitalanomaly,andcanleadtopsychologicalandsocialtrauma.Patientswithsuchdefects

requiremultidisplinarytherapeuticcare,involvingateameffortbetweenthemaxillofacial

surgeon,maxillofacialprosthodontistandreconstructivesurgeon(Lemonetal.,2005),aswellas

follow-uppsychologicaltherapy.Prostheticrehabilitationprovidespsychologicalandfunctional

benefits,asitenhancesaesthetics,speech,swallowing,self-esteemandoverallqualityoflife.

Studieshaveshownthatthesurvivalratesofheadandneckcancerareimproving(Carvahloet

al.,2005),thusmaxillofacialprosthesesareincreasinglyindemand.

Since1960,silicon-basedelastomershavebeenusedtofabricatemaxillofacial

prosthesesduetotheirflexiblemechanicalpropertiesandtranslucentopticalproperties

(Barnhart1960).However,commonclinicalproblemsofthesematerialsincludegradualcolor

lossanddegradationofphysicalpropertiesovertimeinaserviceenvironment(Beattyetal.,

1995,Polyzoisetal.,1999).

Colorchangesandphysicalpropertiesoftheseelastomersaremultifactorial.

Mechanicalloadingandenvironmentalfactorssuchasultravioletexposure,temperatureand

relativehumidityhavebeenidentifiedaspotentialchallengesforanelastomer’sopticaland

physicalstability.Ofthesefactors,ultravioletradiationisconsideredakeyfactorin

decomposingtheelastomer’sopticalandphysicalproperties.Anotherpotentialchallengeisthe

susceptibilitytofungalinfectiononthetissuesideoftheprosthesis(Udagama1987).

Previousresearchindicatedpromisingresultswithadditionofnano-sizedparticlesto

siliconelastomersintermsofphysicalandopticalproperties.Nano-sizedoxideparticlessuchas

2

TiO2andZrO2arecharacterizedbytheirsmallsize,largesurfaceareaandstronginteractions

withtheorganicpolymer.Therefore,theycanimprovetheopticalandphysicalpropertiesofthe

polymer,aswellasitsresistancetoenvironmentalstress-inducedcrackingandaging(Liuetal.,

2005).Also,certainnanoparticles,suchassilver,havebeenshowntoprovideantifungalactivity.

Themechanismofactionisnotfullyunderstood,butbiocidalandanti-adhesiveactivity,and

deliveryofantimicrobialagentssuchasmixedligand-metalcomplexeshavebeensuggested

(Allaker2010).

Candidaalbicansaccountsfor70-80%ofallfungalinfectionsandisincreasingly

associatedwithbiomaterial-relatedinfections.Itispartofthenormalfloraonskinandmucosal

surfaces.Thus,itisofparticularinterestofthisinvestigationtoassesstheantifungaleffectof

incorporatingTiO2andZrO2nanoparticlesintothesiliconelastomer.

ThepurposeofthisthesisistoincorporateTiO2andZrO2nanoparticlesintopigmented

PDMSelastomersandassesschangesincolor,physicalandantifungalproperties.Thecentral

hypothesisofthisstudyisthataddingTiO2andZrO2nanoparticlestopigmentedPDMS

elastomerswillimprovecolorstabilityandphysicalpropertieswhensubjectedtocontrolled

dosagesofultravioletradiation.ThesecondhypothesisisthatC.albicansgrowthonthesurface

ofpigmentedPDMSelastomerswillbereducedbyadditionofTiO2andZrO2nanoparticles.

Thesehypothesesaretestedviaasetofnullhypothesesstatedinthefollowingspecificaims.

Specificaim#1teststhefirstnullhypothesisthatcolorstabilityofsiliconelastomerswillnotbe

affectedbytheadditionofTiO2andZrO2nanoparticles.Specificaim#2teststhesecondnull

hypothesisthatphysicalpropertiesofhardnessandtensilepropertiesofsiliconelastomerswill

notbeaffectedbyaddingTiO2andZrO2nanoparticles.Finally,specificaim#3teststhethirdnull

hypothesisthattheantifungalactivityisthesameforsiliconelastomerswithorwithoutTiO2

andZrO2nanoparticles.

3

CHAPTER2: LITERATUREREVIEW

2.1 MaxillofacialProsthesesHistoryandCharacteristics.

Maxillofacialmaterialsareusedtoreplacemissingfacialpartsthathavebeenlost

throughtrauma,diseaseorcongenitalanomaly.Throughouthistoryseveralmaterialshavebeen

usedtoreplacemissingpartsofthemaxillofacialregionincludingwood,ivory,waxes,and

metals(Andresetal.,1992).Kazanjianetal.,(1932)describedpaintingavulcaniterubberto

matchthecoloroftheskin.Othermaterialshaveincludedlatex,poly(methyl-methacrylate),

vinylchloridepolymers,polyurethane,acrylicresinandsilicone(Bulbulian1942,Chalianetal.,

1974).Inthe1960’spoly(siloxane)rubbermaterialswereintroducedinthemaxillofacial

prosthetictechnologybyBarnhart.Sincethentheseelastomershavebeenthematerialof

choiceforthefabricationofmaxillofacialprosthesis.Nowadays,silicon-basedelastomersare

thematerialsofchoiceinthefieldofmaxillofacialprosthetics,specificallypolydimethylsiloxane

(PDMS)(Azizetal.,2003).A2010surveyshowedthatthemajorityoftheAmericanAssociation

ofAnaplastology(AAA)andAmericanAcademyofMaxillofacialProsthetics(AAMP)members

wereusingroomtemperature-vulcanized(RTV)siliconeproducts(Kiat-Amnuayetal2010).

Cantoretal.in1969suggestedamethodtoevaluateprostheticmaterialsinresponseto

therisingneedatthattimeforanobjective,scientificandreproduciblemethodforevaluating

coloroffacialprostheses.Thismethodutilizedspectrophotometriccurvesanditwas

determinedthatfacialmaterialscouldbeisometricallymatchedtohumanskincolor.Withthe

emergenceofnewmaterialsandtechniquesinthefieldofmaxillofacialprosthesis,the

developmentofaspecificationwasessential.In1972Sweenyetal.developedtheSweeney

specificationbyanalyzingmechanicalpropertiesofpolymerizedpoly(vinylchloride).A

specificationtablewaspresentedandincludedhardness,strength,elasticmodulus,tear

4

strengthandchemicalstabilityunderweatheringconditionsusingaweatherometer.In1974

Lontzetal.,foundthatpolysiloxanespropertiescanbeimprovedthroughmodificationwithoils

andcrosslinkingagentstoapproximatethestress-strainprofilesofhumanaortaandtendons.

Therationalebehindthisapproachwasthatinorderformaxillofacialmaterialstosimulateoral

tissues,thedynamicnatureofthesetissuesmustbeconsidered,astheyareconstantlymoving

andareexposedtotensileandcompressiveforces.Itwasarguedthatlaboratorytestsfor

elastomersshouldincludebothstaticanddynamicproperties,sincetheirstress-strainbehavior

isnon-linearandtheyundergotime-dependentstrain(Lontzetal.,1968,Craig&Koran1975).

Inanefforttoimprovephysicalpropertiesofmaxillofacialprostheticmaterials,mixing

ofdifferentcompoundshasbeenattemptedinordertocombinedesiredpropertiesofeach

componentintotheendresult.InastudyconductedbyFirtelletal.,roomtemperature

vulcanized(RTV)siliconewasmixedwithfoamRTVsiliconetoloweritsdensityandweight.The

resultingmaterialdemonstratedareducedweightbutexhibitedashorterclinicallifeduetoloss

oftearstrength.Anotherapproachwastomodifythepolymer-to-crosslinkingagentratio.This

wasaccomplishedwithpolyurethaneandprovedthatphysicalandmechanicalpropertiessuch

assurfacehardness,modulusofelasticity,strengthandstraincanbeimproved(Gonzalesetal.,

1978andGoldbergetal.,1978).Otherstudiesusedtheconceptofaddingreinforcingfillerssuch

asnylon,glassfiber,silicafibersandtulle.Theseadditionsproducedarigidandheavy

compositematerialthathadimprovedmarginaltearresistance(Karayazganetal.,2003&

Gunayetal.,2008).

Oneofthemostcriticalpropertiesofamaxillofacialprosthesisiscolorstability.Craig

andhisgroupwereoneofthefirsttotestthecolorstabilityoffacialelastomersusingan

artificialweatheringapproach(Craigetal.,1978).Inthisstudy,colorstabilityofpolyvinyl

5

chloride,polyurethaneandsiliconeelastomersformaxillofacialapplicationswasdetermined

afterartificialweathering,usingreflectancespectrophotometry.Measuredparameterswere

luminousreflectance,contrastratio,dominantwavelengthandexcitationpurity,whichwere

calculatedusingacomputerprogrambasedontheC.I.EL*a*b*(CommissionInternationalede

l’Eclairge)colorsystem.Luminousreflectanceisameasureofthetotalamountoflightreflected

bythespecimenandisameasureoflightnessordarkness.Contrastratioistheratioof

luminousreflectancewithablacktoawhitebackground.Dominantwavelengthistheactual

colorofthespecimenwhencomparedtoastandardobserverandissimilartohue.Excitation

purityisameasureoftheamountofcolorpresentinthesampleandissimilartochroma.Their

resultsshowedthatsiliconeelastomerswerethemostpromisingintermsofcolorstability

comparedtotheothertwomaterialsafter900hoursofartificialweathering.

Althoughelastomersshowexcellentaestheticsinitially,colorstabilityisasignificant

challenge.Asurveystudyfoundthat70%ofmaxillofacialprostheticpatientshadtheir

appliancesremadewithinoneyear,ofwhich29%wereduetocolorchange(JaniandSchaaf

1978).Otherstudieshavereportedthattheclinicalservicelifetimeofaprosthesisisusually6

monthsto2years,withanaverageof10months(JaniandSchaaf1978,Chenetal.,1981and

Polyzois1999).

Ideallyamaxillofacialprosthesisshouldpossesshightearstrength,tensilestrengthand

toughness,highflexibility,similarhardnesstosurroundingtissues,lowwatersorptionandgood

surfacewettability(Azizetal.,2003b,Hanetal.,2008).Tearstrengthisespeciallyimportant,

particularlyatthethinmarginssurroundingnasalandeyeprostheses.Inordertomaskthe

presenceofafacialprosthesisithastobefabricatedwiththinmargins.Whenaprosthesisis

peeledawayfromfacialtissues,itissubjectedtotearingatitsthinmargins;consequentlyitis

6

importanttouseamaterialwithhightearresistance.Anotherpropertytoconsiderisflexibility.

Asthematerialispeeledawayfromnasaltissuesithastoelongateenoughbeforeitbreaks.

Otherimportantcharacteristicsincludeeaseofapplication,retention,colorstability,durability,

lackoftoxicity,easeofcleansing,easeoffabricationandphysicalandchemicalinertness(Yuet

al.,1978).

Oneofthemostseriousproblemsfacedbymaxillofacialprosthesisisinfectionofthe

materialsurfacebyCandidaalbicansandothercandidaspeciesrelatedtodenturestomatitis

andgastrointestinalandpneumopulmonarycandidiasis(Udagama,1987;Pingoetal.,1994).C.

albicansareopportunisticfungithatarepartofthenormalfloraoftheskin,oralcavity,

gastrointestinaltractandurogenitaltract.However,candidalinfectionmayoccurwhenhost

defensesareloweredbylocalfactors(suchasprosthesisirritation,xerostomia),medication

(antibiotics,immunosuppressantdrugs),chemotherapy,radiationtherapyandsystemic

disorders(suchasendocrineandimmunedisturbances).C.albicansisresponsibleforabout70-

80%ofallfungalinfections(Hay,1999).C.albicansarealsoassociatedwithinfectionsof

biomaterialssuchasmaxillofacialprostheses,catheters,dentures,heartvalves,implanted

devicesandcontactlenses(Ramageetal.,2001).Biofilmformationiscriticalinthedevelopment

ofcandidiasisandC.albicansbiofilmsarepotentiallyhighlyresistanttothecurrentlyused

antifungalagents(Chandraetal.,2003).C.albicansinbiofilmcanbe100-foldmoreresistantto

fluconazoleand30-foldtoamphotericinBagentsthanplanktoniccells(Kumamoto2002).

Polydimethylsiloxane(PDMS)currentlyisthemostcommonlyusedelastomerfor

maxillofacialprosthesis.Twomaintypesofsystemsareused.Oneisroomtemperature

vulcanizing(RTV),inwhichPDMSterminatedwithhydroxylgroupsundergoespolycondensation

inthepresenceoftinasacatalyst.Thesecondisaheat-curedsysteminwhichunsaturated

7

vinyl-terminatedpolysiloxanesundergofreeradicaladditionwiththeaidofaplatinumcatalyst

(Azizetal.,2003a).

Keyfactorsaffectingmechanicalpropertiesofanelastomerincludemolecularweight

distribution,theincorporationofhydrophobicfillerparticleswithlowsizesandhighsurface

areas,andthedegreeofcross-linkingbetweenpolymerchains.Strategiessuchastheformation

ofbimodalnetworksfromblendinglongandshortchainsofthesamepolymercanresultina

combinationofdesiredmechanicalproperties,suchashightearstrengthandflexibility(Shah&

Winter1996,Bellamyetal.,2003).

Recentlyinteresthasbeengainedinincorporatingnanoparticlesintomaxillofacial

prostheticmaterials.Duetotheirhighsurfaceenergyandchemicalreactivity,nano-oxidestend

toagglomerateinsolutionandcanbedifficulttodisperse(Goiatoetal.,2010).Whenthe

elastomerissubjectedtoexternalforces,theagglomeratednanoparticlesactasstress-

concentratingcenterswithinthesiliconmatrix,therebyloweringmechanicalpropertiesand

affectingdimensionalstabilityovertime(Hanetal.,2008).However,whennanoparticlesare

properlydispersed,physicalandmechanicalpropertiesoftheelastomershouldbeimproved

andresistanceagainstenvironmentalstresscrackingandagingshouldbegained(Lueetal.,

2005).

2.2 FactorsAffectingProsthesisDegradation.

Consideringthatamaxillofacialprosthesisundergoesseveralstepsoffabricationand

colormatchingintheclinicandlaboratory,asingleprosthesiscanbecomeverycostlytothe

patient.Therefore,thelong-termdurabilityofamaxillofacialprosthesisbecomesextremely

important.Theoveralldeteriorationofamaxillofacialprosthesishasbeenattributedtocertain

environmentalfactorssuchasexposuretothenaturalsunlight(specificallytheultraviolet

8

component),wetnessanddrynessoftheelastomer,andsurfaceabrasionduringapplicationand

removaloftheappliance(Keyf2002).

Aginganddeteriorationofanextra-oralprosthesisisaccompaniedbyincreasein

stiffnessandlossofflexibility,leadingtotearingalongestheticalthinmargins.Ultraviolet

radiation,whichenhancescross-linkingandincreasesbreakingdownofthepolymer,ultimately

decomposestheelastomer(Hatamleh&Watts2010).Airpollutionalsohasbeenshownto

affectsiliconecolor(Mohiteetal.,1994).

Asstated,ultraviolet(UV)radiationisakeyenvironmentalfactorthathasalargeimpact

ontheopticalandmechanicalpropertiesofamaxillofacialprosthesis(Beattyetal.,1999,

Mohiteetal.,1994,Hanetal.,2010).UVphotonsinthepresenceofoxygenleadtophoto-

oxidativedegradationoftheelastomer.Energeticphotonsorparticles(gammarays,protons,

electrons),withenergygreaterthanthemolecularbondstrength,degradethepolymerthrough

aprocesscalledradiolysis(Cottinetal.,2000).TheabsorptionofUVphotonsleadsto

degradationofthemoleculeandtheproductionofsmallerpolymerchainsandvolatile

degradationproducts.Duetothepresenceoffreeradicals,acompetitionoccursamong

initiation,propagationandtermination(Rabek1995).Thispolymerizationdisturbanceproduces

changeswithinthemolecularweightdistribution,whichinturnnegativelyaffectsthephysical

andchemicalpropertiesofthematerial(Elenietal.,2011).Therefore,considerableresearchhas

beendirectedtowardsdevelopingaprostheticmaterialthatischemicallystableand

environmentallyresistanttoultravioletradiation.Theidealmaterialisyettoberealized(Eleni

etal.,2011,Kiat-Amnuayetal.,2008,Fangetal.,2006,Polyziosetal.,2000).

Asstatedpreviously,colorstabilityisthemostsignificantchallengetomaintainingthe

longevityofamaxillofacialprosthesis.Studiesconductedduringthepastthreedecadeshave

9

evaluatedthecolorstabilityofpigmentedandnon-pigmentedmaxillofacialsiliconelastomers

byaddinginorganicopacifiersororganicultravioletradiationabsorbers,andexposedmaterials

toartificialand/oroutdoorweathering(Kiat-Amnauyetal.,2006,Tranetal.,2004,Kiat-Amnauy

etal.,2002).However,thisapproachisrestrictedbyseveralfactors,namely,theamountof

additivethatcanbetoleratedbythematerialwithoutchangingitsbaselinecolorandthefact

thatthesolubilitylimitmightbeexceededpriortoreachingadequateultravioletradiation

protection(Hanetal.,2010).Anothercomponentofthecolorstabilityofamaxillofacialsilicone

elastomeristhestabilityofthepigmentitself.Koranetal.,studiedthecolorstabilityofeleven

maxillofacialpigmentsandfoundthatallpigmentsdemonstratedchangesinatleastonecolor

parameterthatwasstatisticallysignificant(1979).Beattyetal.,evaluatedthecolorstabilityof

fivemaxillofacialprostheticpigmentsafter400hourexposuretoultravioletradiationandfound

thatcadmiumyellowmediumandcosmeticredunderwentsubstantialcolorchange(1995).In

theirdiscussionstheyhypothesizedthatshort-termcolorchangemaybeattributedtocolorloss

ofnon-UV-resistantpigments,whereaslong-termcolorchangemaybeattributedtothe

additionalcross-linkingandcontinuedpolymerizationoftheelastomerorbysidereactionsof

impuritiespresentwithinthesilicone.Furthercompositionalanalysiswouldberequiredto

confirmtheirhypothesis.Amorerecentstudyevaluatedthecolorstabilityofoilpigmentsmixed

withdryearthopacifiersafterartificialweathering.TheyfoundthatdrypowderTiwhite

remainedthemostcolorstableovertime.However,theauthorsreportedthatitpossessesan

intensecolor,makingitdifficulttouseintheclinicasittendstoproducewhitersamples(Kiat-

Amnuayetal.,2006).

Twoapproacheshavebeentakentostudytheoveralldeteriorationofmaxillofacial

materials.Oneapproachistoexposematerialstoanacceleratedweatheringprocess.A

commondeviceistheweatherometer,anartificialweatheringchamberfirstdescribedby

10

Sweenyetal.,in1972.Thesecondapproachistoexposematerialstooutdoorweathering

conditionsandmeasurematerialpropertychanges.Studieshaveshownthatartificial

weatheringcanbeusedtoapproximatetheoutdoorperformanceofmaxillofacialelastomersas

meanstopredicttheoveralllifetimeofthesepolymersunderserviceconditions.However,

acceleratedartificialweatheringapparentlyinfluencesthedegradationmechanismsdifferently

withintheelastomer,andspecificcolorchangesoftenarenotreproducedaccurately(Gijsman

etal.,1994,Sampersetal.,2002,Pospissiletal.,2006,Elenietal.,2011).

Garyetal.(2001)measuredcolorstabilityofpigmentedandunpigmentedsilicon

elastomersexposedtooutdoorweatheringintwodifferentlocations;Miami,FLandPhoenix,AZ

basedontheirdiverseclimates.ThemeancolorchangewashigherinPhoenix,AZgroups,

indicatingthatanenvironmentwithlowrelativehumidityandrainfallinduceschangesmore

rapidlycomparedtoamoisterenvironment,suchasinFloridaorNebraska.

2.3 Nanoparticles.

2.3.1 Characteristics,MechanicalandOpticalProperties

Nanoparticle-containingmaterialsincludethosecontainingparticleswithapproximate

sizesof5to100nm,withshapesthatcanbespherical,cubicorneedle-like(Cushingetal.,

2004).Asaparticleisreducedinsizefrommicrometertonanometer,theresultantproperties

canchangedramatically.Forexample,hardness,activesurfacearea,chemicalreactivityand

biologicalreactivityarealtered(Allaker&Ren2008).Thepropertiesofnanoparticlescandiffer

fromthosedemonstratedbytheirbulkformsandinsomecasesgivecompletelyunexpected

physicalandchemicalproperties.Forthisreasonindustriesinvariousfields(e.g.structural,

biomedical,opticalandelectric)haveextensivelyresearchednewprocessesthatincorporate

nanoparticlesintopolymericmatrices(Hanetal.,2008).Carbonnanotubeshavebeenstudied

11

extensively.Studieshaveshownthatcarbonnanotubes,whenincorporatedintocomposite

materialscanproducehighelasticmodulusandincreasedstrength,ascomparedtomicro-fiber-

reinforcedcomposites(Thostensonetal.,2001).Onestudyincorporatedcarbonnanotubesinto

elastomersandobservedincreasedrigidityandshearmodulusascomparedtounfilled

elastomers(Frogleyetal.,2003).Nanoparticlesarewidelyincorporatedinindustrialmaterials

suchastextiles,rubbers,sealants,plastics,cosmetics,fibers,coatings,sunscreens,dental

compositesandtoothpastes.Nanoparticlesareaddedtodentalcompositestoimprovethe

tooth-compositeinterfacecontinuityandadhesivestrength.Zinccitrateisincorporatedinto

toothpastestocontrolplaqueformationandtitaniumdioxideactsasawhitener(Tangetal.,

2006).Inelastomers,enhancedmechanicalpropertiesthroughnanoparticleadditionsmaybe

attributedtoahighersurfaceenergyandchemicalreactivity,whichallowthenanoparticlesto

interactwiththesiliconbackboneandformathreedimensionalnetwork(Watsonetal.,2004).

Silicondioxide(SiO2),orsilica,isusedwidelyasafillermaterialinthemicrometersize

rangetoimprovethemechanicalpropertiesofsiloxaneelastomers.Silicaismostcommonly

foundinnatureasquartzanditisthemajorconstituentofsand.Itismainlyusedinthe

constructionindustrytoproducePortlandcement,butothermajorapplicationsincludeglass

production,sandcasting,anddesiccantsthatareusedinthefoodandpharmaceuticalindustries

(Florkeetal.,2008).Silicaexistsinmanycrystallineforms(polymorphic),butinmostcases

tetrahedralSiO44-unitsarelinkedtogetherwithsharedverticesinvariousarrangements.Silica

nanoparticleshavebeenusedinpaintstocontrolrheologicalpropertiesandalsoserveas

reinforcingfillersinnanocompositesinordertoimprovetensilestrengthandwearandscratch

resistance(Zhengatal.,2003).

Metaloxidenanoparticlesimpartthestrengthandmechanicalpropertiesdesiredfroma

12

metal,andthedecreasedsizeimpartsreducedopacityandimprovedoverallopticalproperties.

Metal-oxidenanoparticles,whenincorporatedintoapolymersystem,canshowcombined

desiredpropertiessuchasstrength,hardness,elasticity,electricalconductivityandimproved

opticalperformance(Abdelsayedetal.,2006;Goldenetal.,1995;Maitietal.,2008.Khanetal.,

2007).Khannaetal.(2007)havereportedthatrutilenano-TiO2isusedcommonlyasawhite

pigmentduetoitshighlight-scatteringeffect,whichoffersprotectionfromultravioletradiation.

Raoetal.,(2005)illustratedtheuseofnano-sizedparticlesasagreatadvantagedueto

theirlargesurfacearea.Thesurfaceareaofparticlesforagivenquantityofamaterialscalesas

1/dwheredistheaveragediameteroftheparticle.Inthenanometricrange,materialsare

expectedtobehavequitedifferentlyfrombothmolecularandbulkstatessincetheratioofthe

numberofsurfaceatomstothenumberofbulkatomsbecomessignificant(Raoetal.,2005,

Mohsenietal.,2012).Asaconsequenceofthisincreasedratio,thenumberofunsaturated

valenceatomsinthenanoparticlesbecomessignificant,leadingtohigherchemicalreactivity.

Severalarticleshavebeenreportedwhichstudiedcompositesfilledwithmicro-sized

particles.Thesestudiesindicatedthatmicroparticlesimprovephysicalpropertiesandincrease

wearresistanceincompositepolymersduetotheirabilitytoformtransferfilmsofthe

compositesonthecounterface,whicharethinanduniformandarestronglybondedtothe

substrate.Compositesfilledwithmicro-sizedcoppercompoundfillers(CuO,CuSandCuF2)

loadedat35vol.%showedincreasedwearresistancecomparedtounfilledcomposites(Bahadur

&Gong1992).SinhaandBrisco(2009)examinedstrengthandmodulusofmicro-filled

compositesandsuggestedthatsmallerfillerparticlescontributemoretostrengtheningthendo

largerparticlesofthesamecompound.

13

Whenincorporatingnanoparticlefillersintoapolymersystem,anessentialcriterionis

thestateofdispersion.Thehighchemicalreactivityandspecificsurfaceareaofnanoparticles

resultsinveryhighattractiveforcesbetweentheparticles,therebyinducingastrongtendency

toagglomerate(Wichmannetal.,2006).Whennanoparticlesagglomerate,theyactasstress-

concentratingcenters,leadingtoadecreaseinmechanicalpropertiesandapotentialchangein

dimensionalstabilityovertimewhenamaterialissubjectedtoexternalforces(Hanetal.,2008).

However,ifoptimumdispersionisobtained,ahomogeneouslynanofilledpolymerisproduced,

withimprovedresistancetoenvironmentalstress-inducedcrackingandaging(Liuetal.,2005).

Anumberofnano-sizedmetaloxideshavebeenstudiedaspolymeradditivesforgaining

improvementsinphysical,mechanicalandopticalproperties.Titaniumdioxideisone,andithas

foundapplicationasapigment,adsorbentandcatalyst(particularlyphotocatalyst)(Khannaet

al.,2007).Titaniumdioxideispolymorphicandexistsinthreeforms;anatase,rutileandbrookite

(ElGorseyetal.,2001).Theanatasecrystallinephaseexhibitsthehighestphotocatalyticactivity

duetoitslargebandgapenergyof3.2eVanditisusedinsolarenergyconversionbecauseofits

highphotoactivity(Xuetal.,2002).Rutile-TiO2iscalledthe“whitepigment”anditprovides

protectionfromultravioletradiationduetoitsscatteringeffect(Khannaetal.,2007).Two

theorieshavebeensuggestedtoexplainthescatteringeffectofTiO2.First,theMielight

scatteringtheorythatpredictstheoptimumparticlesizeofconventionalTiO2toobtain

maximumlightscatteringandopacityinthevisiblespectrum.Thistheorygovernsthescattering

effectoflargerTiO2particlesandexplainstheiropticalproperties.Second,Rayleigh’slight

scatteringtheoryimpliesthatshorterwavelengthsoflightaremoreeffectivelyscatteredby

smallerparticlessuchasultrafineTiO2nanoparticles.Accordingtolattertheory,thepeakof

scatteringintensityisreachedwhentheparticle’sdiameterisequaltoonehalfthewavelength

ofUVlightwhichmeansmoreforwardscatteringandlessdiffusescattering.Thisexplainsthe

14

UVblockingcapabilitiesofTiO2nanoparticles(Allenetal.,2002,Yangetal.,2004).Inadditionto

opticalandUVblockingcapabilities,TiO2nanoparticleadditionshavebeenshowntoimprove

mechanicalproperties.Oneweightpercentadditionstoresinbasedcompositerestorative

materialsincreasedmicrohardnessbyapproximately60%andflexuralstrengthby16%

comparedtounfilledcomposite(Xiaetal.,2008).Forcolorimprovement,0.1to0.25weight

percentadditionsbettersimulatedtheopalescenceofhumanenamel(Yuetal.2009).

Zirconiaisametaloxideceramicknownforsuperiormechanicalproperties,excellent

opticalpropertiesandbiocompatibility.Recentlyzirconiausehasremarkablyincreasedindental

restorativeandprosthetictreatments(Denry&Kelly2008;Thompson2011).Itisnotsolublein

water,isnon-cytotoxic(Dionetal.,1994),doesnotenhancebacterialadhesion(Rimondinietal.,

2002),isopaqueandpossesseslowcorrosionpotential(Denry&Kelly2008).Zirconiais

polymorphicandallotropic,existinginthreecrystallineconfigurationsatdifferent

temperatures:cubic,tetragonalandmonoclinic(Lughi&Sergo2010).Duringcoolingthe

tetragonalphasetransformsintoastablemonoclinicphase.Thisisaccompaniedbya4-5%

crystalvolumeincrease,whichleadstointernalcompressivestressandpotentialcracking.In

ordertopreventthetransformationandstabilizezirconia,itisoftencombinedwithothercubic

oxides,notablyMgO,CaO,Y2O3andCeO2(Zaroneetal.,2011).Zirconia,whenusedasacore

materialforsinglecrownsandfixedpartialdentures,enjoysasuccessrateof93%overthree

years(Ortorpetal.,2009).

Silverionsandcompoundshavebeenextensivelyinvestigatedfortheirantibacterial,

antiviralandantifungalproperties(Moronesetal.,2005;Monteiroetal.,2009).Indentistry,

silverhasbeenusedasanantimicrobialagentindentalcomposites(Herreraetal.,2001).

SeveralstudieshaveshownthatAgnanoparticlespossessencouraginglevelsofantimicrobial

15

activity,asbiocidalactivityhasbeenreportedagainstgrampositive,gramnegativeand

pathogenicbacteria,inadditiontonormalflora(Lietal.,2005;Raietal.,2009;Allaker2010).

Importantly,silverhasrarelycausedresistantmicroorganismstodevelop(Dorjnamjinetal.,

2008).SilvernanoparticlesinhibitC.albicansgrowthat0.2 𝜇g/mlconcentrations,whichisless

thanthatrequiredtoproducetoxiceffectsagainsthumanfibroblasts(30 𝜇g/ml)(Panaceketal.,

2009).Whensilvernanoparticleswereincorporatedintoexperimentalcompositeadhesives,

roughersurfacesresulted,buttheyshowedlessbacterialadhesionandsimilarmechanical

propertiestoconventionaladhesives(Ahnetal.,2009).

2.3.2 AntifungalEffectsofNanoparticles

Nanoparticleshavebeenusedincontrollingoralbiofilmformationthroughdifferent

approaches,includingbiocidal,anti-adhesiveandthroughdeliveryofantimicrobialagentsand

ligands(Allaker2010).Nano-oxides,andparticularlyzirconia,haveshowntobesuitablecarriers

forantifungalagents,duetoahighcoordinationnumberandtheabilitytoformstable

complexes(Malgheetal.,2009).Themechanismofactionbywhichnanoparticlesexertan

antimicrobialeffectisnotfullyunderstood.Certainstudiessuggestthatnanoparticlesprevent

DNAreplicationbyinactivatingenzymesnecessaryforATPproduction(Fengetal.,2000;

Yamanaka,2005).Othershavesuggestedthatelectrostaticattractionbetweenpositively

chargedmetalionsandnegativelychargedmicrobialcellmembraneiscriticalforantimicrobial

activity(Kimetal.,2007).Ithasbeenhypothesizedthatsilverionsinactivatemembrane-bound

respiratoryenzymesofmicroorganisms(Allaker2010).Maxillofacialmaterialsexhibitingthe

highestsurfacehydrophobicityalsodemonstratethegreatestantifungaleffect(Nikawaetal.,

1994).Nanoparticleshavegreatpotentialuseforthisapplication,astheycanincrease

hydrophobicityandsurfacechargeinadditiontochemicalreactivity(Allaker2010).

16

2.4 GapinKnowledge

Todate,mostattemptstoimprovefacialprosthesislongevityhavefocusedonmicro-

sizedadditives,whichhaveproducedminimalimprovements.Morerecentresearchhasfocused

onnano-sizedadditives,whichhaveshownsuccessinimprovingmechanicalandoptical

propertiesintheplastic,glassandpaintindustries,aswellasemergingbiomedicalapplications

(Hanetal.,2010).Agapinknowledgeexistswithregardtotheextentnanoparticlesmayoffer

improvementsinoptical,physicalandantifungalproperties,inadditiontoprolonging

prostheseslifetimes.BecausetitaniumoxideprovidesexcellentUVprotection,zirconiaoffers

enhancedmechanicalpropertiesandbiocompatibility,andsilverimpartsantifungalactivity,

thesethreenanoparticlesarethefocusofthisresearch.Thisstudy’spurposeistoadddifferent

sizesoftitaniumoxideandzirconiananoparticlestopolydimethylsiloxane,andstudycolor

stabilityandselectedphysicalpropertiesfollowingexposuretoUVBradiation.Asecondpurpose

istoassesstheantifungalactivityofpolydimethylsiloxanescontainingTiO2,ZrO2nanoparticles.

17

CHAPTER3: MATERIALSANDMETHODS

3.1 PreparationofSamples

Prototypepigmentedelastomerswereconstructedbycombiningunpolymerized

polydimethylsiloxane,nanoparticles,pigment,crosslinkerandcatalyst,andpolymerizingthe

mixtureunderheat.Twosizes,withaten-foldparticlesizedifference,ofTiO2andZrO2

nanoparticleswerechosenforthisproject.Fourexperimentalgroupsplusonematerialcontrol

groupinadditiontotwoAggroupsusedasnegativematerialcontrolgroupsinantifungal

experimentsTable3.1.

Forelastomerpreparation,1%byweightofeachexperimentalnanoparticlewasmixedwith99

weightpercentvinyl-terminatedpolydimethylsiloxane(PDMS)(V-2K,MW23,000,

polydispersity2.5,Matno.057077,MomentiveMaterials,Tarrytown,NY).Thenanoparticles

wereincorporatedusingarotarymixerat3000rpmforfiveminutes.Anultrasonicmixer

(HielscherultrasoundprocessormodelUP200S,Teltow,Germany)withaS3Sonotrodeat100%

amplitude(460W/Cm2)wasusedtoburstnanoparticleagglomeratesanddispersetheminto

thevinyl-terminatedPDMS.Theultrasonicmixerwashousedinasoundboxtominimizenoise

duringmixingandthemixturewascontainedinastainlesssteelmaltcupthatwascooledinan

icebath.Ultrasonicmixingproceededfortenminutes,theneachmixturewasrotarymixed

againwithaCowlesdisperserfortenminutesat5000rpmtoachieveauniformdistributionof

nanoparticles.Twoweightpercentfunctionalintrinsicyellowpigment(FI-202,lotno.DL101606,

FactorII,Inc.,Lakeside,AZ)wasaddedandrotarymixedat5000rpmforanadditionalfive

minutes.Thisyellowpigmentwaschosenbecauseitwasknowntoundergosubstantialcolor

changewhensubjectedtoenvironmentalweathering(GaryandSmith,1998,Kiat-amnuayetal.,

2009).

18

Table3.1.NanoparticlesTested

Nanoparticletype Size Lotno. Company

TiO230-40nm

54885-040108NanostructuredandAmorphousMaterials

Inc.Houston,TX

TiO2200nm

TI07193RUT11

InframatAdvancedMaterials,

Manchester,CT

ZrO240nm

USHT03

USResearchNanomaterials,

Inc.,Houston,TX

ZrO2200-300

nm5970-061503

NanostructuredandAmorphousMaterials

Inc.,Houston,TX

Ag 30-50nmUSHW09

USResearchNanomaterials,

Inc.,Houston,TX

Ag200-400

nm0124-030413

SkyspringNanomaterials,

Inc.,Houston,TX

SiO4

200-300nm

Unknown CabotCorporation,Boston,MA

19

Priortoinitiatingpolymerization,eachmixturewasrotarymixedat5000rpmfor10

minutestore-dispersethenanoparticlesintothePDMS.Forpolymerization,equimolarratiosof

thenanoparticle-containingvinyl-terminatedPDMSwerecombinedwithpolymethylhydrogen

siloxane(V-XLcrosslinker,batchno.HVDD112906,MomentivePerformanceMaterials,Friendly,

WV)and10ppmplatinumcatalyst(VCAT-RT,lotno.502L031798,OSiSpecialtiesInc.,Sistersville,

WV).Themixturesweremechanicallyspatulatedinapapercupfortwominuteswithawooden

tonguedepressor,andairbubbleswereremovedunder5 ×10-3torrconstantvacuumbyahigh

vacuumpump(WelchVacuumTechnology,Skokie,IL)attachedtoabellchamber.Bubble

removalwasascertainedvisually,andittypicallyrequiredfifteenminutes.Themixtureswere

pouredslowlyintomoldassembliestoallowtheairfrompouringtoescape.Alidunderload

wasplacedontopofthemoldstoextrudeexcessmaterial.Themoldassemblieswereplaced

intoan84 °Cforced-airconventionovenforsixtyminutesforpolymerization.Preliminarytrials

determinedthatmoldassembliescontaininglargenanoparticles(200-400nm)requiredthe

moldstobeinvertedeverytenminutestopreventnanoparticlesfromsettlingononesideof

theelastomer.Forthecontrolgroup,13%loadingweightoffumedsilica(tofollowwhatisused

currentlyinmaxillofacialprosthetics)wasaddedtovinyl-terminatedpolydimethyl-siloxane

(PDMS)under2000rpmrotarymixeruntilfullydissolved,followedby15-20minutesofrotary

mixingat7000rpmstoensureparticledispersion.

Testsampleswithdifferentgeometrieswereusedforcolor,mechanicalandantifungal

activitymeasurements.Disk-shapedmoldswereusedtofabricatesamplesforcolorchange

measurementsandDurometer(ShoreA)hardness.Moldassembliesconsistedofpolyvinyl

chloride(PVC)pipescutinto6mmthicksectionsandsecuredwithmedium-bodypolyvinyl

siloxane(PVS)impressionmaterialtoagypsumslabtomimicwhatisusedindentallabs

fabricatingmaxillofacialprosthesis.Themixturewaspouredslowlyfromonesideintothemold

20

andaglassslabwasplacedtoextrudeanyexcessmaterial.Four-inchviceclampswereusedto

securetheglassslabwithapproximately2-5kgofload.Theresultantpolymerizeddiscshada

diameterof38mmandathicknessof6mm.Fivediscspergroupweremade,asprevious

studiesshowedsignificantdifferencecolorchangescouldbedetectedatanalphalevelof0.01

withpowerof0.8(Beattyetal.,1995).

Fortensileproperties,elastomermixtureswerepouredontogypsummoldsand

coveredwithaclearpolycarbonatesheet(13’’×10’’×0.5’’,USPPlasticCorporation,Lima,OH).

Thepolycarbonatewassecuredwithfour-inchviceclamps,tightenedtodeliverapproximately

2-5kgofloadinordertoextrudeexcessmaterials.Theresultantelastomersheetswere254mm

lengthx165mmwidthx2mmthickness.Dumbbell-shapedsampleswerecutfromthese

elastomersheetsusingadiecutterthatconformedtodieCforASTMStandardD412-08(ASTM

InternationalStandardsWorldwide,http://www.astm.org/Standards/D412.htm).Foreach

experimentalandcontrolgroup,twelvedumbbellswereconstructed.ANikonmeasurescope

(MM-11)withcomputersoftware(Quadra-check200)wereusedtomeasurewidthanddepthof

eachsampleatthedumbbellgagelength.Thisinformationwasusedtocalculatecross-sectional

area,whichwasnecessaryforcomputingstressduringgenerationofstress-straincurves.

3.2 ExposuretoUltravioletWeathering

Eachdumbbellordiscwasplacedonareflectivesurfaceinsideaplywoodenclosure

beneathfour36-inchbulbs(UVBBroadbandLamp,FS40T12,NationalBiologicalCorp.,

Twinsburg,OH)deliveringUVBradiationwithwavelengthrangefrom290-315nm.The

enclosurewashousedinanenvironmentalchamberwithcontrolledtemperatureandhumidity

throughouttheexperiment.Sampleswereplaced12inchesdirectlybelowthebulbs,andthe

surroundingenvironmentwasmaintainedat25 °Cand30%relativehumidity.Underthese

21

conditions,radiationwasdeliveredat0.2mW/cm2whichwasequivalentto720mJ/cm2/hour,

andthesamplesurfacetemperaturedidnotexceed0.5 °Cabovethesurroundingenvironment,

asmeasuredbyathermocouple.AccordingtoBeattyetal.,thisUVBoutputreflectedthe

averagedailyexposurefromnaturalsunlightinLincoln,NEduringsummermeasuredwithUVB

detectoratnoon.(1995)Lightoutputwasmonitoredcontinuouslythroughouttheexperiment

usingalightsensorandadatalogger(UVBsensor,PMA2100logger,SolarLightCo.,

Philadelphia,PA).

Inadditiontoultravioletradiationexposure,materialscontainingeachnanoparticle

typewerestoredinaweatheringcontrolenvironment(darkness,25°C,30%relativehumidity).

Thisprovidedanassessmentofpotentialmaterialchangesoccurringovertimewithouta

weatheringstimulus.MeasurementtimesforcolorchangeandShore-Ahardnesswerechosen

topermitcomparisonwithpreviousresearch,whichwassetat600,1800and3000hours.For

mechanicaltesting,anextrasetoftestsampleswasconstructedtoestablishbaselinevalues,

sincethetensiletestsweredestructiveinnature.Forthissamereason,mechanicaltestscould

notbeconductedatintermediatetimeintervals,makingitnecessarytoobtainmechanical

propertyvaluesonlyatbaselineand3000hours.

3.3 ColorMeasurements

Colormeasurementsweremadeusingacolorreflectancespectrophotometer(CM-2002,

KonicaMinoltaCorp.,Ramsey,NJ)withcomputersoftware(SpectraMagicNX,KonicaMinolta

Corp.,Ramsey,NJ).Colormeasurementsweremadeoneachdiscat0,600,1800and3000

hoursaccordingtotheCIEL*A*B*system(CommissionInternationaledel’Eclairge,2004).The

spectrophotometerdeterminedcoloraccordingtoASTMD2244-07(2007,“StandardPractice

forCalculationofColorTolerancesandColorDifferencesfromInstrumentallyMeasuredColor

22

Coordinates”,ASTMinternational,WestConshohocken,PA,DOI:10.1520/D2244-07).Three

axesdefinedthecolorspace;L*wasthewhite-blackaxis,a*wasthered-greenaxisandb*was

theyellow-blueaxis.Atthebeginningofeachsessionthespectrophotometerwascalibrated

withblackandwhitebackgrounds.Blackcalibrationwasconductedwithbackgroundlights

turnedoffandwhitecalibrationwasachievedwithawhitecalibrationplate.Allmeasurements

weremadewithsamplesrestingonastandardwhitebackgroundplate(no.21633347,Konica

MinoltaCorp.,Ramsey,NJ)using50gweightwithbackgroundlightsturnedon.Eachdiscwas

labeledwithacodeusingpermanentinkscribedontheside(alongthethicknessdimension).

Colormeasurementsweremadeonthediscfaceabovethetopofthescribedcode.The

spectrophotometerwasplacedwiththemeasuringportfacingupwardandthreetongueplates

wereplacedunderneaththebodyinamannerthatthedevicewasparallelwiththefloor.Each

discwasorientedwiththecodeplacedinthesamepositionateachtimeintervalsothatcolor

measurementswouldbetakenatthesamelocation.Oncecolormeasurementswerecompleted,

UVBsampleswereimmediatelyreturnedtotheweatheringchamberandcontrolsamples

placedinthecontrolenvironment.

AfterrecordingL*,a*andb*valuesinanExcelspreadsheet,colordifferences(ΔL*,Δa*

andΔb*)werecalculatedforeachsampleateachtimeinterval.Totalcolorchange(ΔE*)was

calculatedfromtheequationΔE*=[(ΔL*)2+(Δa*)2+(Δb*)2]1/2.

3.4 PhysicalPropertiesMeasurements

3.4.1 TensilePropertyMeasurements

Fortensiletestingauniversaltestingmachine(Instron1123-5500R,InstronCorp.,

Boston,MA)andcomputersoftware(M-Bluehill-K2-ENRevisionA)wereusedtoperformthe

testsandrecorddata.Dumbbell-shapedsamples(n=12pergroup)weremeasuredforthickness

23

andwidth,loadedintogrips,andalong-travelextensometerwitha25mmgaugelengthwas

attached.Eachdumbbellwaselongatedatarateof500mm/minandstressversusstraindata

weregraphicallychartedanddigitallyrecordeduntilfailure.Threepropertiesweredetermined,

ultimatetensilestrength,totalstrainatfailureandmodulusofelasticity.Ultimatetensile

strengthwasconsideredtobethemaximumstressthetestsamplecouldwithstand,which

usuallyoccurredatfailure.Maximumstrainatbreakwasameasureofthetotalamountof

extensionamaterialcouldwithstandpriortofailure.Themaximumtensiledisplacementofa

samplewasdividedbythestartinglengthtoobtainmaximumstrainatbreakandwasexpressed

asmm/mm.Modulusofelasticitywascalculatedastheslopeofalinearportionofthestress-

straincurvebetween50%and100%strain.Tensiletestingwasperformedatbaselineandafter

3000hoursofexposuretobothcontrolandUVBenvironments.

3.4.2 ShoreAHardness

ForDurometerhardnesstests,thesamediscsasforcolormeasurementswereused

(n=5pergroup).AshoreAhardnesstester(InstronDurometerTypeA,ModelDRCL,ASTM

D2240,Instrument&ManufacturingCompanyInc.,Freepost,NJ)wasusedtomeasurehardness

atbaseline(zero),600,1800and3000hoursofstorageincontrolorUVBenvironments.

Hardnessmeasurementsweretakenontheoppositefacefromthatusedtoobtaincolor

measurements.ThisprocedurewasfollowedbecausetheDurometerindenterhadthepotential

todeformtheelastomerandaffectcolormeasurements,whichweretakenatthesamesession.

HardnessmeasurementsweremadefollowingASTMD2240protocol;fivemeasurementswere

madeatrandomlocationsandtheaverageofthesereadingswasconsideredtobethe

representativehardnessvalue.

24

3.5 AntifungalActivity

3.5.1 CandidaalbicansandGrowthConditions

C.albicanswild-typestrain(CA42),formerlyknownasSC5314),wasgrownaerobicallyin

yeastnitrogenbase(YNB)medium(DifcoLaboratories,Detroit,MI)onfreshSabouraud

DextroseAgarplate(DifcoLaboratories,Detroit,MI).Theplatewasincubatedfor24hoursat

37℃onashakerat60rpm(modelclassicC25,NewBrunswickScientific,Edison,NJ).Cellswere

harvestedandwashedthreetimeswith0.15Mphosphate-bufferedsaline(GibcoPBS;pH7.4,

Ca+2andMg+2free,Lifetechnologies,GrandIsland,NY).Cellswereresuspendedin10mlPBS,

countedwithahematocytometerandusedwithin24hours.

3.5.2 BiofilmFormation

Testsamplesconsistedofcirculardisksstampedfromtheremnantsofsiliconelastomer

sheetsthatwereusedtomakedumbbell-shapedsamplesfortensiletests.Acircularpunch(9-

PieceHollowPunchSet,SKUNo.P3838,CentralForge,Pittsburg,PA)wasusedtocreatethese

diskswith2mmthicknessand12.7mmdiameter.Twelvediskspergroupwerecreatedforthe

fourexperimentalgroups(TiO2,ZrO2),positivematerialcontrolgroup(silica),andtwonegative

materialcontrolgroups(Ag).SilvernanoparticleswereaddedtoPDMSataconcentrationof1

𝜇g/ml.Thisconcentrationwaschosenduetoitspotentialantifungalactivitywhileremainingat

aconcentrationbelowpotentialtoxicity(30𝜇g/ml)(Panaceketal.,2009).Disksweresterilized

inanautoclaveat120℃and16psifor30minutes.

ThebiofilmformationprotocolusedinthisstudyfollowedthatdescribedbyKuhnetal.,

2002.Alldiscswerepreconditionedwithfetalbovineserum(GibcoFBS;Lifetechnologies,Grand

Island,NY)ina96-welltissuecultureplate(FalconMicrotestTissueculturePlate,96well,Flat

BottomwithLowEvaporationLid,BectonDickinsonLabware,FranklinLakes,NJ)andincubated

25

at37℃for24hours.FBSwasremovedandgentlywashedwith0.15MPBStoremoveresidual

FBS.200𝜇LoffreshYNBmediumwasaddedtoeachwell,followedby200𝜇LofC.albicanscell

suspensioninaconcentrationof1×108cells/ml,whichyielded20,000cellsperwell.Theplate

wasincubatedfor90minutesin5%CO2at37℃onarockertableat60rpmtodevelopthe

adhesionphaseoftheC.albicansbiofilm.SampleswerewashedwithPBStoremoveunattached

cells,coveredwith200 𝜇LofYNBandincubatedfor48hoursin5%CO2at37℃onarockertable

at60rpmtoprovideasuitableenvironmentforthebiofilmmaturationphase.

Inadditiontopositiveandnegativematerialcontrolgroups,whichconsistedofsilica

andsilvernanoparticles,apositivebiologicalcontrolgroupwasincludedwhichentailed

constructingwellscontainingYNBandC.albicans.Twomethodswereperformedtomeasure

antifungalactivity:XTTcolorimetricassayandconfocallaserscanningmicroscopy(CLSM).

3.5.3 XTTColorimetricAssay

C.albicansbiofilmformationontestsampleswasquantifiedusingatetrazoliumsalt-

based2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-([phenylamino]carbonyl)-2H-tetrazolium

hydroxide(XTT)colorimetricassay,asdescribedbyChandraetal.(2008).Thismethodmeasures

enzymeactivitythatreducesXTTdyetowater-solubleformazandye.TheXTTassayisrapid,

reproducible,non-invasive,non-destructiveandrequiresminimalpost-processingofsamples

(Ramageetal.,2009).ThereductionofXTTtoformazancrystalscanonlytakeplaceinthe

presenceofviablecellsandthenecessaryreductaseenzymes.Therefore,theXTTassay

measurestheopticaldensityofformazancrystalsinsolutionasanindicationofthemetabolic

activityofviableC.albicanscells.Immediatelyafterbiofilmmaturation,YNBwasremoved,test

sampleswerewashedwithPBSandsamplesweretransferredtoanew96-welltissueculture

plate.200𝜇lPBS,50𝜇lXTT(1mg/mlinPBS)and4𝜇lmenodinesolution(1mmol/Linacetone)

26

wereaddedtoeachwellinthenew96-wellplatefortestgroups,andtotheold96-wellplate

forcontrolgroups.Plateswereincubatedindarknessat37℃for5hours.Thesuspensionwas

thenmeasuredspectrophotometrically(Elx808AbsorbanceMicroplateReader,BioTek,

Winooski,VT)at492nm.Atthiswavelengththeabsorbanceofthewater-solubleorange

formazandyeend-productcanbemeasured.

3.5.4 ConfocalLaserScanningMicroscopy(CLSM)

Biofilmformationwasalsoevaluatedqualitativelyusingconfocallaserscanning

microscopy.TheprotocolusedwasdescribedpreviouslybyChandraetal.(2008).Twosamples

ofeachgroupweretransferredcarefullytopreservebiofilmstoa12-wellcultureplateand

incubatedfor45minutesat37℃in2mlPBScontainingthefluorescentstainFUN-1(lot#

745237,excitationwavelength=488nmandemissionwavelength=505nm,Invitrogen,

MolecularProbes,Eugene,OR)thatemitsgreenfluorescencewhendiffusedintocytoplasm,and

isconvertedovertimebymetabolicallyactiveenzymesintoorange-redcylindricalintravascular

structures.Afterincubation,siliconelastomerdiscswereturnedoverandplacedona35mm

diameterglass-bottompetridish(MatTechcorp.,Ashland,MA).AnOlympusFV500systemon

anIX81scope(invertedmicroscope)wasusedwith40×lens,locatedinthemicroscopycore

facilityatUNLcenterforbiotechnology(Dr.YouZhou,UniversityofNebraska-Lincoln).Multiple

photomicrographs(using488nmexcitationlaserlineand505nmemissionfilter)weretaken

fromdifferentareasofeachsampletoevaluatethearchitectureofC.albicansbiofilmgrowthon

elastomerdiscs.

27

3.6 DataAnalysis

Forcolordataanalyses,groupmeansandstandarderrorswerecalculatedfor

dependentvariables∆L*,∆a*,∆b*and∆E*.Independentvariablesinfluencingcolorwere

weatheringenvironment(controlandUVB),fillercontent(13%silica,1%TiO2200nm,1%TiO2

30-40nm,1%ZrO2200nmand1%ZrO240nm)andtime(600,1800and3000hours).Thenull

hypothesisthatcolorchangewasnotaffectedbynanoparticleaddition,weatheringexposure

andtimewastestedbyathree-wayanalysisofvariance(ANOVA),followedbyaTukey-Kramer

posthoctestforpairwisecomparisonsata(p<0.05)levelofconfidence.

ForchangesinShoreAhardness,meansandstandarderrorswerecalculatedas

dependentvariables.Independentvariablesinfluencinghardnesswerethesameasthose

describedforcolor.ThenullhypothesisthatShoreAhardnesschangewasnotaffectedby

nanoparticleaddition,weatheringexposureandtimewastestedbyathree-wayanalysisof

variance(ANOVA)followedbyaTukey-Kramerposthoctestforpairwisecomparisonsata

(p<0.05)levelofconfidence.Fortensileproperties,groupmeansandstandarderrorsfor

ultimatetensilestrength,strainatbreakandmodulusofelasticitywerecalculatedasdependent

variables.Independentvariablesweretestcondition(immediate(baseline),3000hourscontrol

and3000hoursUVB)andfillercontent(13%silica,1%TiO2200nm,1%TiO230-40nm,1%ZrO2

200nmand1%ZrO240nm).Atwo-wayanalysisofvariance(ANOVA)followedbyaTukey-

Kramerposthoctestforpairwisecomparisonsata(p<0.05)confidencelevelwasusedtotest

thenullhypothesisthattensilepropertiesofPDMSelastomerswerenotaffectedby

nanoparticleaddition.

Forantifungalactivity,alinearleastsquaremodelwasconstructed,followedbya

DunnettadjustmenttocomparegroupmeansandstandarderrorsofXTTabsorbancetothe

28

positivecontrolgroupata(p<0.01)confidencelevel.Thenullhypothesistestedwasthat

nanoparticleadditiondidnotaffectantifungalactivityofPDMSelastomer.

29

CHAPTER4: RESULTS

4.1 ColorMeasurements

Baselinecolormeasurements(L*,a*andb*)foreachgrouparepresentedinFig4.1and

Table4.1.Fromthesedata∆L*,∆a*,∆b*and∆E*werecalculatedforeachtimeinterval(600h,

1800hand3000h)ofexposuretocontrolorUVBweatheringconditions.

Athree-wayANOVAdeterminedthatsignificantdifferenceswerepresentamong

nanoparticletype,weatheringconditionandtimeofexposureforeachcolorparameter.

Therefore,ANOVAwasfollowedbyaTukey-Kramerposthoctesttocomparedifferencesand

determinewhichinteractionwassignificant.

Table4.1.BaselineColorParametersValues(Mean(S.D.),n=10)

Group L* a* b*

TiO2200nm 90.31(0.39) -0.62(0.25) 55.60(0.32)

TiO230-40nm 88.73(0.18) 0.85(0.07) 60.06(0.66)

ZrO2200nm 79.17(0.41) 4.41(0.14) 88.76(1.87)

ZrO240nm 79.75(0.31) 4.13(0.12) 91.48(0.87)

Silica200-300nm 79.52(0.27) 4.27(0.07) 93.18(1.41)

30

Figure4.1.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofbaselinecolorparametersL*,a*andb*fordifferentgroups(n=10).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).Nostatisticaldifferencedetectedina*ofanymaterials.

31

4.1.1 After600HoursofWeathering

Inthecontrolgroups,∆L*value(whichrepresent,thewhite-blackaxisintheCIEL*a*b*

colorsystem)for40nmZrO2groupwasstatisticallysignificantlylower(darker)than30-40nm

TiO2andsilicagroups(p<0.05,Fig4.2).UnderUVBweathering,200nmTiO2wassignificantly

lessnegative(whiter)comparedtoallothergroups(p<0.05,Fig4.2).Also,the30-40nmTiO2

groupwaswhiterthanbothZrO2groupsandthesilicagroup(p<0.05,Fig4.2).

Forred-greencolorchange,negative∆a*valuesdenotedincreasedgreeningafter600

hoursofcontrolweathering.The30-40nmTiO2groupwaslessgreenwhencomparedtoother

nanoparticlegroups(p<0.05,Fig4.3)butnotsignificantlydifferentwhencomparedtothesilica

group(p>0.05,Fig4.3).ForUVBweathering,positive∆a*valuesindicatedlessgreenwas

present,with200nmTiO2materialsbeingsignificantlygreenercomparedtotheothergroups

(p<0.05,Fig4.3).30-40nmTiO2∆a*valueswerelowerthanbothZrO2groups,andsilicawas

higher(lessgreen)thanallothergroups(p<0.05,Fig4.3).

For∆b*(yellow-blue),valuesmeasuredinthecontrolweatheringenvironmentwere

lowest(leastyellow)forthe200nmZrO2group(p<0.05,Fig4.4)withnostatisticallysignificant

differencesnotedbetweentheothergroups(p>0.05,Fig4.4).After600hoursofUVBexposure,

bothTiO2groupswerelessnegative(moreyellow)comparedtotheotherthreegroups(p<0.05,

Fig4.4).

32

Table4.2.∆L*,∆a*,∆b*and∆E*valuesafter600hoursofweathering(Mean(S.D.),n=5)

Color

Parameter/

Weathering

TiO2

200nm

TiO2

30-40nm

ZrO2

200nm

ZrO2

40nm

Silica

200-300nm

∆L*

Control -0.71(0.07) -0.31(0.03) -0.72(0.16) -0.88(0.07) -0.32(0.06)

UVB -1.48(0.08) -2.05(0.09) -4.48(0.19) -4.31(0.13) -4.37(0.12)

∆a*

Control -0.53(0.02) -0.25(0.02) -0.68(0.02) -0.67(0.03) -0.32(0.02)

UVB 0.28(0.02) 0.51(0.03) 0.95(0.06) 0.90(0.10) 1.78(0.06)

∆b*

Control -1.24(0.12) -0.46(0.20) -2.62(0.45) -1.56(0.12) -0.39(0.43)

UVB -2.15(0.12) -3.32(0.11) -7.04(0.34) -7.00(0.09) -6.91(0.23)

∆E*

Control 1.53(0.12) 0.71(0.08) 2.85(0.40) 1.91(0.12) 1.02(0.15)

UVB 2.63(0.13) 3.94(0.15) 8.40(0.37) 8.27(0.14) 8.37(0.24)

33

Figure4.2.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆L*after600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

34

Figure4.3.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆a*after600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

35

Figure4.4.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

36

Overallcolorchange∆E*waslowestforthe30-40nmTiO2andsilicagroupsinthe

controlweatheringenvironment(p<0.05,Fig4.5).Bothwerebelowtheperceptiblecolor

changethresholdof1.1,whileallgroupswerebelowtheacceptablecolorchangethresholdof

3.0.IntermsofUVBweathering,200nmTiO2demonstratedthelowestcolorchange,followed

by30-40TiO2group(p<0.05,Fig4.5).Allgroupswerewellabovetheacceptablecolorchange

thresholdexceptfor200nmTiO2,witha∆E*of2.63.


Forthe30-40nmTiO2andsilicagroups,∆L*and∆a*valuesweresignificantlyless

negative(whiterandlessgreen)thantheotherthreegroupswhenstoredincontrolconditions

(p<0.05,Fig4.6and4.7).InUVBweathering,∆L*and∆a*valuesfor200nmTiO2washighest,

thenfollowedbythe30-40nmTiO2group(p<0.05,Fig4.6and4.7).Thesilicagroupshowedthe

highestpositivechangeinthe∆a*parameter(moregreen,p<0.05,Fig4.7).

∆b*valuesafter1800hoursofcontrolweatheringshowedthatthe200nmZrO2group

wasmorenegative(lessyellow)whencomparedtothetwoTiO2groupsandthesilicagroup

(p<0.05,Fig4.8).The30-40nmTiO2groupwaslessnegative(moreyellow)comparedtothe

ZrO2groups(p<0.05,Fig4.8).InUVBweathering,∆b*valuesfor200nmTiO2weretheleast

negative,followedbythe30-40nmTiO2group(p<0.05,Fig4.8).

Withrespecttooverallcolorchangeundercontrolconditions,∆E*valuesfor30-40nm

TiO2incontrolweatheringweresignificantlylowerthanallothergroups,butoverlappedwith

thesilicagroup(p<0.05,Fig4.9).200nmZrO2underwentthehighestcolorchange,whichwas

significantlygreaterthantheothergroupsexcept40nmZrO2(p<0.05,Fig4.9).The30-40nm

TiO2wastheonlygroupbelowtheperceptiblecolorchangethreshold,while200nmZrO2was

theonlygroupabovetheacceptablecolorchangethreshold.WithregardtoUVBweathering

37

Figure4.5.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,respectively.

38


Color

Parameter

TiO2

200nm

TiO2

30-40nm

ZrO2

200nm

ZrO2

40nm

Silica

200-300nm

∆L*

Control -1.00(0.09) -0.42(0.05) -1.41(0.04) -1.19(0.08) -0.60(0.09)

UVB -2.71(0.02) -3.64(0.07) -6.99(0.10) -6.14(0.11) -7.36(0.08)

∆a*

Control -0.55(0.04) -0.22(0.02) -0.83(0.02) -0.74(0.03) -0.31(0.02)

UVB 1.22(0.04) 1.49(0.02) 1.79(0.05) 1.73(0.07) 3.05(0.04)

∆b*

Control -1.65(0.13) -0.60(0.29) -3.09(0.29) -2.43(0.21) -1.30(0.42)

UVB -4.19(0.09) -6.00(0.09) -11.57(0.26) -10.57(0.34) -12.31(0.12)

∆E*

Control 2.01(0.16) 0.89(0.20) 3.50(0.27) 2.82(0.21) 1.58(0.31)

UVB 5.14(0.07) 7.18(0.11) 13.64(0.26) 12.35(0.33) 14.67(0.08)

39


40


41


42

Figure4.9.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after1800hoursofweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,respectively.

43

results,200nmTiO2grouphadthelowestcolorchange,whichwasfollowedby30-40nmTiO2,

40nmZrO2,and200nmZrO2groupsrespectively(p<0.05,Fig4.9).Allgroupswerewellabove

theacceptablecolorchangethreshold.


∆L*resultsafter3000hours’storageincontrolconditionsshowedthat30-40nmTiO2

andsilicawerethemostpositive(whiter),whichandthatwassignificantwhencomparedtothe

ZrO2groups(p<0.05,Fig4.10).∆L*valuesforthe200nmZrO2groupwerethelowest(more

negative,darker),whichwassignificantonlywhencomparedtoTiO2groupsandthesilicagroup

(p<0.05,Fig4.10).UnderUVBweathering,the200nmTiO2groupwastheleastnegative(least

darkest)followedby30-40nmTiO2,40nmZrO2,200nmZrO2,andsilica,respectively.All

differenceswerestatisticallysignificant(p<0.05,Fig4.10).

Thered-greencolorparameter,∆a*,showednosignificantdifferencesamonggroups

undercontrolweatheringconditions(p>0.05,Fig4.11).ForUVBweathering,thesilicagroup

showedthehighest∆a*value(mostred,meaningthehighestamountofgreenfading)butno

statisticalsignificancewasdemonstratedamongthegroups(p>0.05,Fig4.11).

For∆b*valuesafter3000hoursofcontrolweathering,TiO2groupswerethehighest

(mostyellow),butnostatisticalsignificancewasobservedamongthegroups(p>0.05,Fig4.12).

∆b*valuesafter3000hoursofUVBweatheringwasthehighestforTiO2(leastamountofyellow

fading),whichwasstatisticallysignificantonlywhencomparedtothesilicagroup(p<0.05,Fig

4.12).

44


Color

Parameter

TiO2

200nm

TiO230-40

nm

ZrO2

200nm

ZrO2

40nm

Silica

200-300nm

∆L*

Control -1.13(0.14) -0.61(0.07) -1.68(0.06) -1.43(0.08) -0.99(0.08)

UVB -3.13(0.01) -4.27(0.07) -8.50(0.12) -7.97(0.10) -10.82(0.13)

∆a*

Control -0.27(0.38) 0.06(0.42) -0.39(0.66) -0.25(0.66) 0.44(0.90)

UVB 1.20(0.25) 1.27(0.50) 1.70(0.52) 1.62(0.65) 3.23(1.01)

∆b*

Control -2.48(0.63) -2.21(1.26) -5.85(2.10) -5.18(2.10) -5.00(3.20)

UVB -4.30(0.84) -6.28(1.02) -11.49(2.57) -11.89(2.04) -15.12(3.03)

∆E*

Control 2.88(0.59) 2.49(1.24) 6.32(2.05) 5.61(2.06) 5.39(3.21)

UVB 5.61(0.59) 7.87(0.81) 14.98(1.61) 14.74(1.47) 19.51(2.01)

45


46


47


48

Inobservingtheoverallcolorchange(∆E*)after3000hoursofcontrolweathering,no

statisticalsignificantdifferencewasdemonstratedamongallgroups(p>0.05,Fig4.13).Onlythe

TiO2groupsdemonstrated∆E*valuesbelowtheacceptablethresholdofcolorchange.After

3000hoursofUVBweathering,the200nmTiO2groupshowedthelowestcolorchange,which

wassignificantwhencomparedtoZrO2groupsandthesilicagroup(p<0.05,Fig4.13).Thesilica

groupshowedthehighestcolorchangewhichwassignificantcomparedtoTiO2groups(p<0.05,

Fig4.13).Allgroupshad∆E*valueswellabovetheacceptablelevelofcolorchange.

Asummaryof∆E*colorchangeforeachgroupincontrolandUVBweathering

environmentsateachtimeinterval(600h,1800h,3000h)isshowninFig4.14.Generallycolor

changeincreasedovertime,asUVBinducedmorecolorchangeforallgroupsat600,1800and

3000hcomparedtocontrolweathering.TheTiO2200nmgroupdisplayedthelowestcolor

changeandsilicagroupdisplayedthehighestcolorchangeincontrolandUVBenvironments.

Colorchangeincontrolweatheringwasapproximatelyatthesamelevelat600hand1800h,

thenstartedtoincreaserapidlytowardsthe3000htimepoint.ThissuggeststhatthePDMS

sampleswerestableuntilafter1800hofcontrolweathering,thenpossiblepolymerand/or

pigmentdegradationbegantotakeplace.UVBweatheringinducedhigherincreaseincolor

changefrom600hto1800hfollowedbyaslightincreasereachingthe3000hpoint.Thiscan

possiblybeexplainedbyinitialpigmentdegradationfromUVBexposure,followedbypolymer

degradation.Interestingly,errorbarsbecamelargerafter3000hoursofweathering,suggesting

thatthematerialswerelessstableatthattimepoint.

49

Figure4.13.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,respectively.

50

Figure4.14.Lineplotsdisplayingmeansandstandarddeviations(errorbars)of∆E*overtime(600,1800and3000hours).

51

4.2 PhysicalProperties


BaselineshoreAhardnessvaluesarepresentedinFig4.15.Thesilicagrouphadhigher

hardnessvaluesthantheothergroups,whichwasstatisticallysignificantwhencomparedto

bothTiO2groupsandthe40nmZrO2group(p<0.05).Itwasnotsignificantwhencomparedto

the200nmZrO2group(p>0.05).The200nmZrO2groupshowedanintermediatehardness

valuesthatwasstatisticallyhigherthanTiO2groupsandthe40nmZrO2group(p<0.05).It

shouldbepointedoutthatthesilicagroupcontained13%weightfillerloadingcomparedtoonly

1%weightforthetitaniaandzirconiagroups.

After3000hoursofweathering,thechangeinshoreAhardnesswasminimalforthe

TiO2andZrO2nanoparticlegroups,rangingfrom-0.28to+0.22unitswithnosignificant

differencesdetectedamongthenanoparticlegroupsineithercontrolorUVBweathering

environments(Fig4.16).Comparedtotheothernanoparticlegroups,thesilicagroupshoweda

higherincreaseinhardnessthatwasstatisticallysignificant,forbothcontrolandUVB

weatheringenvironments(p<0.05,Fig4.16).

52

Figure4.15.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofbaselineShoreAhardness(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

53

Figure4.16.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofdeltaShoreAhardnessafter3000hoursofweathering(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

54

4.2.2 TensileProperties

Meanultimatetensilestrength(UTS)valuesweresignificantlygreaterforthesilica

group,ascomparedtotheTiO2andZrO2groupsfortestsconductedimmediately(baseline)and

after3000hours’storageincontrolandUVBweatheringconditions(p<0.05,Fig4.17,Table4.5).

ImmediateUTSvaluesforthesilicagroupweresignificantlygreaterwhencomparedtothose

measuredaftercontrolandUVBweathering(p<0.05,Fig4.17,Table4.5).Theweatheredgroups

werenotsignificantlydifferentfromoneanother.

Modulusofelasticitywassignificantlygreaterforthesilicagroupcomparedtothe

experimentalgroupsatalltimeperiodsandweatheringconditions(p<0.05,Fig4.18,Table4.5).

Nosignificantdifferenceswereobservedamonganyoftheexperimentalgroupsatanytimeor

foranyweatheringcondition(p>0.05,Fig4.18,Table4.5).

Forthestrainatbreak,silicagroupsweresignificantlygreaterthantheexperimental

groupsforallthreetestingconditions(p<0.05,Fig4.19,Table4.5).Theimmediatemeanvalue

ofstrainatbreakforZrO2200nmgroupwassignificantlylowerthanbothTiO2groups.After

3000hoursofstorageincontrolandUVBenvironments,thesilicagroupshowedmeanstrength

valuesthatweresignificantlylowerthanimmediatemeanvalues.FortheexperimentalTiO2and

ZrO2groups,strainatbreakvaluesshowedminimalchangesbetweenthethreetesting

conditionsthatwerenotstatisticallysignificant(p>0.05,Fig4.19,Table4.5).

55

Figure4.17.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofultimatetensilestrengthatbaselineandafter3000hoursofweathering(n=12).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

56

Figure4.18.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofmodulusofelasticityatbaselineandafter3000hoursofweathering(n=12).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

57

Figure4.19.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofstrainatbreakatbaselineandafter3000hoursofweathering(n=12).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).

58

Table4.5.TensilePropertiesafter3000hoursofweathering(Mean(S.D.),n=12)

Tensile

properties

Testing

Condition

TiO2

200nm

TiO2

30-40nm

ZrO2

200nm

ZrO2

40nm

Silica

200-300nm

Ultimate

Tensile

Strength

(UTS)

Immediate 0.35(0.01) 0.34(0.02) 0.26(0.01) 0.38(0.01) 1.13(0.04)

Control 0.34(0.01) 0.31(0.02) 0.28(0.01) 0.36(0.01) 0.95(0.10)

UVB 0.30(0.01) 0.32(0.02) 0.31(0.01) 0.33(0.01) 0.87(0.06)

Strainat

Break

Immediate 1.43(.05) 1.41(0.11) 0.97(0.07) 1.20(0.05) 2.61(0.07)

Control 1.36(0.09) 1.39(0.10) 1.24(0.07) 1.23(0.05) 2.10(0.09)

UVB 1.35(0.11) 1.16(0.09) 1.02(0.02) 1.15(0.08) 1.92(0.09)

Modulus

of

Elasticity

Immediate 0.17(0.00) 0.17(0.01) 0.18(0.01) 0.21(0.00) 0.47(0.01)

Control 0.18(0.01) 0.16(0.00) 0.15(0.01) 0.19(0.00) 0.48(0.04)

UVB 0.16(0.01) 0.19(0.01) 0.20(0.01) 0.19(0.01) 0.46(0.02)

59


4.3.1 XTTColorimetricAssay

Theopticaldensityofformazancrystalformationinsolutionwasmeasured

spectrophotometricallyat492nmafter48hoursofexposuretoC.albicans.Thiswasameasure

ofmetabolicactivity(Fig4.20,Table4.6).AgandTiO2nanoparticlegroupsshowedsignificantly

lowerC.albicansmetabolicactivitywhencomparedtothepositivecontrolgroup(p<0.01).The

ZrO2andsilicagroupswerenotsignificantlydifferentthanthepositivecontrolgroup(p>0.01).

4.3.2 ConfocalLaserScanningMicroscopy(CLSM)

Fun-1stainingshowedthatC.albicansbiofilmformationisacomplexphenomenon.The

biofilmwasmultiplecellslayersinthicknessconsistingmainlyofyeastandhyphae.Adjacentto

thesiliconelastomerdiscsurface,yeastcellsweredenselyembeddedinanextracellularmatrix.

InbothAggroupsandTiO2200nmgroups,confocallasermicroscopyimagesshowedyeastcells

scatteredandattachedtotheelastomerdiscswithnohyphaeformationdetected.

PseudohyphaeandmoreyeastcellswithextracellularnetworksweredetectedinTiO230-40nm

groupimagesandmoredenseandthickmaturehyphaeprocessformationswerenoticedin

ZrO2andsilicagroups(Fig4.21).

60

Table4.6.C.albicansMetabolicActivityafter48hoursMeasuredSpectrophotometricallyat492nm(Mean(S.D.),n=9forsilicaandnanoparticlegroups,n=12forpositivecontrol)

Group XTTafter48h

Ag200-400nm 0.31(0.07)

Ag30-40nm 0.31(.04)

TiO2200nm 0.33(0.25)

TiO230-40 0.70(0.23)

ZrO2200 1.00(0.16)

ZrO240 1.01(0.12)

Silica200-300nm 1.13(0.11)

Positivecontrol 1.17(0.00)

61

Figure4.20.Bargraphdisplayingmeansandstandarddeviations(errorbars)ofC.albicansopticaldensitymeasuredspectrophotometricallyat492nmafter48hours.Asteriskdenotessignificantdifferencefrompositivecontrol(p<0.01).

62

Figure4.21.ConfocallaserscanningmicrographsofC.albicansstainedwithFUN-1.a)Ag200-400nm.b)Ag30-40nm.c)TiO2200nm.d)TiO230-40nm.e)ZrO2200nm.f)ZrO240nm.g)Silica200-300nm.Magnification40x,oil.Scalebar=30𝝁m.

63

CHAPTER5: DISCUSSION

ThecentralhypothesisofthisstudywasthattheadditionofTiO2andZrO2nanoparticles

topigmentedPDMSelastomerswouldimprovecolorstabilityandphysicalpropertieswhen

subjectedtocontrolleddosagesofultravioletradiation.Theresultsofthisinvestigation

demonstratedthatTiO2nanoparticlesimprovedcolorstability,butZrO2didnot.Neither

nanoparticleimprovedphysicalpropertieswhenaddedat1%byweight.Thesecondhypothesis

wasthatC.albicansgrowthonthesurfaceofpigmentedPDMSelastomerswouldbereducedby

additionofTiO2andZrO2nanoparticles.TheresultsdemonstratedthatTiO2nanoparticles

permittedlessbiofilmgrowththandidsilicaparticles,butZrO2didnot.Interpretationsofresults

arepresentedinthefollowingsections.

5.1 ColorChange

5.1.1 ColorChangesatBaseline

TiO2nanoparticleadditionscausednoticeablevisualcolorchangeatbaselineas

comparedtosilicacontrols.TiO2producedsampleswhichwerelighterincolorcomparedto

silicacontrols(L*valuesranging88-90forTiO2and79forsilica).Ontheotherhand,ZrO2groups

producedsampleswithsimilarL*valuesascomparedtosilica-filledsamples(bothnearL*=79).

Coloristheresultoftheinteractionofthepigmentcolor,nanoparticlesizeandtherelative

differencebetweentherefractiveindicesofthenanoparticleandthepolymer.Therefractive

indexofTiO2ismorethan2.6whereasZrO2isabout2.1,Silicaisabout1.5andPDMSisaround

1.4.Generally,lightisbentmore,travelsshorterpathsanddoesnotpenetrateasdeeplyin

materialswithhigherrefractiveindices.Therefore,samplescontainingTiO2aremoreefficientat

scatteringlightthanaretheothergroups,leavingsmalleramountsoflighttobeabsorbedby

thepolymerandthepigment.ThescatteringeffectofTiO2nanoparticlesgivesthesamplesa

64

whiterappearance,therebyexplaininghigherL*values.Small,non-significantchangeswere

measuredina*(red-green),whichwassomewhatexpectedsincethepigmentwasyellowin

color.Forb*values,silicafilledsamplesproducedthehighestyellowcolor(b*=93.18)which

wasstatisticallygroupedwithZrO2samples(b*valueswere88.76forZrO2200nmand91.48for

ZrO240nmsamples).ThewhitenesscontributedbyTiO2presumablydetractedfromtheyellow

colorandyieldedlowerb*values(55.60forTiO2200nmand60.06forTiO230-40nm).

5.1.2 ColorChangesinControlEnvironment

Inthecontrolenvironment(darkness,25℃,and30%relativehumidity),moreoverall

colorchangewasnoticedforZrO2groupsandthesilica-filledsamplesafter3000hoursof

storage,ascomparedtoTiO2-containingmaterials(p<0.05).Thismaybeattributedtopigment

breakdown,detachmentoffillerparticles,degradationofthelowermolecularweightsilicon

fluidusedtodispersetheplatinumcatalyst,presenceofimpuritiesorcontinuedcrosslinkingof

thematrix.Additionalcrosslinkingoccurswhenunreactedchainscontinuetopolymerizewith

time,therebychangingtherefractiveindexofthepolymer.ElastomerscontainingbothTiO2

sizesunderwentameancolorchangethatwasbelowthevisualthresholdofacceptablecolor

change(∆E*=3).TheloweroverallcolorchangenoticedwithTiO2maybeduetoitshigher

specificheat,whichmayallowmoreheattransmissiontothepolymer,possiblyinducingmore

polymerizationduringthecuringprocessandtherebyreducingpost-curingpolymerization.Post-

curingpolymerizationmayhaveproducedthecolorchangesnotedforsilicaandzirconia.

However,thetrueunderlyingmechanismisunknown.

65

5.1.3 ColorChangeCausedbyUltravioletRadiation

Earlystudiesdemonstratedthatultravioletradiationnegativelyimpactedopticaland

mechanicalpropertiesinmaxillofacialprostheticmaterials.Acommonexplanationgivenfor

thesechangesisthatoxygen,whenpresent,inducesphoto-oxidativedegradationofthe

polymernetworkinadditiontothedegradationofnonUV-resistantpigments.ResultsfromUVB

weatheringshowedthatTiO2200nmsamplesdemonstratedtheleastcolorchangeovertime,

followedbyTiO230-40nmsamples.Thissuggeststhat200nmTiO2nanoparticlesfunctionedas

UVBblockers,therebyreducingultraviolettransmissiontosurroundingpigmentandpolymer

molecules.ThedifferenceinUVblockingcapabilitiesbetweenthetwoTiO2groupsmaybe

attributedtothedifferenceinthesizeofthescatteringparticles.Forthe200nmTiO2

nanoparticleswhosediameterisclosertoone-halfthewavelengthproducedbytheultraviolet

bulb(290-315nm),theMiescatteringtheoryisapplicableandmoreforwardscatteringwillbe

producedclosertothepeakofthetotalscatteringintensity.Ontheotherhand,thesmaller30-

40nmparticlesmaymorecloselyfollowtheRayleighscatteringtheory,whichshowsthatsmall

particlesproduceequalamountsofbackwardandforwardscattering.Inprinciple,the200nm

particlewouldproducemoreforwardscattering,whereasthe30-40nmparticlewouldpermit

morebackward(diffuse)scatteringtothesurroundingpolymerandpigment.This,inturn,

wouldleadtomaterialdegradationwithinashorterperiodoftime.Itshouldbepointedout

thatalthoughthe∆E*valueforTiO2200nmwas5.6unitsafter3000hoursofUVBexposureand

abovetheacceptablethresholdofcolorchange(∆E=3.0),thisdegreeofcolorchangemaynot

beunacceptabletosomeobservers.Itshouldbenotedthatthemeasurementusedto

determinetheperceptibilityandacceptabilitythresholdsofcolordifferencewasdetermined

fromParavinaetal.,wherecolormeasurementsofdifferentskin-coloredelastomerswere

evaluatedinaviewingboothwithneutralgraywallsandafloorwhichloweredthevisual

66

lightnessthreshold(2009).Thesethresholdsmightnotfullyrepresentamaxillofacialprosthesis

thatisviewedunderdifferentlightsourcesandwithdifferentbackgrounds.Also,apatient

mightnotrejectafacialappliancewithminimalshadechangeaslongasthechangeoccurs

gradually.

Basedonourresults,improvementsincolorstabilitywereobtainedbyadding1%

weightTiO2nanoparticlestosiliconelastomersexposedtoartificialweathering,whichis

consistentwithfindingsreportedbyHanetal.,2010.For1%TiO2incorporatedintoSiliconeA-

2186withyellowpigmentsandexposuretoartificialsolarradiation(450kJ/m2),ΔE*was

measuredat5.2units.Colorstabilityimprovedwithsamplescontaininghigherconcentrations

ofTiO2(2%to2.5%)andmixedpigments,ratherthanonepigment.

5.2 PhysicalProperties


BaselineshoreAhardnessshowedthatsilicawassuperiortoallothergroupsexceptthe

200nmZrO2group.Thefactthatthe1%nano-oxideadditionswereonlyslightlylowerin

hardnesstothe10%silicaadditionsmaybeexplainedbyconsideringthenatureofthepolymer

reinforcingbehaviorofnanoparticleswhendispersedintosilicone.Hardnessisasurface

phenomenonandisaffectedbythesurfaceareaofincorporatedfiller.Thelargetotalsurface

areaofsmallerparticlesmayunderlietheobservationthat1%TiO2andZrO230nmparticles

yieldedsimilarhardnessvaluestothosemeasuredforcontrolmaterialscontaining13%200nm

silicaparticles.Thesurfacearea/weightratioincreasesbyonetotwoordersofmagnitudeas

thenanoparticledecreasesinsizeto10nm(Rothon2003).

OveralltherewerefewdifferencesinShoreAhardnessobservedamongthedifferent

67

materials,andonlysilica-filledelastomersdemonstratedappreciablehardeningfrom

weathering.The1.5unitincreaseinhardnesswasconsideredtobeclinicallyinsignificant.

5.2.2 TensileProperties

ComparedtotheTiO2-andZrO2-filledsamples,ultimatetensilestrength,maximum

strainatbreakandmodulusofelasticityweresignificantlygreaterforthesilica-filledelastomers

atalltimepointsforbothcontrolandUVBweathering(roughly2-3timesgreaterUTS,2times

maximumstrainand2-3timesgreatermodulus).Thiswasprobablyduetotheincreasedfiller

loadingforsilica-containingmaterials.Increasingfillerlevelshavebeenshowntoincrease

mechanicalpropertiesformicrometer-rangefillersloadedintopolymersystems.Inthisstudy,it

wasconsideredpossiblethatten-foldlowerfillersizesloadedinten-foldlowerquantitiesmight

producesimilarmechanicalpropertiestomaterialsloadedwithlargerfillersinlargerquantities.

ThisconsiderationwasbasedonweardatapublishedbyBahadurandGongandmechanical

propertydatapublishedbySinhaandBrisco.Similarresultswerenotobservedinthisstudy

becausewearisamorecomplexphenomenathatisaccompaniedbycompressiveandshear

stress,whichisdifferentthanthetensilebehaviorofthematerial.Wearcanoccurinadhesive,

abrasiveandfatiguemodesandrequiresurfacecontact.Thus,itispossiblethatnano-filled

materialsmayexhibitdifferentbehaviorundertensilestress.

TiO2-andZrO2-nanoparticle-filledsamples’tensilepropertieswerenotsignificantly

affectedbyweatheringcondition.Thisresultshouldberegardedwithcaution,asproperty

valueswereinherentlylowinitially.Thismightbeduetothelowfillercontentusedinthisstudy,

whichdidnotraisethemechanicalpropertiessufficientlytopermitweatheringchangestobe

observed.SimilarfindingswerereportedbyHanetal.,2008.Theyconcludedthatthe

incorporationof0.5%,1%and1.5%loadingfractiondidnotimprovemechanicalproperties,

68

whereas2-2.5%ofTiO2,ZnOandCeO2didimprovetensileandshearstrength,maximum

elongationatbreakandShoreAhardness.Howevertheirresultsarenotdirectlycomparableto

ourfindings,sincetheyincorporatednanoparticlesintoSiliconA-2186,acommercialelastomer

thatcontainssilicainadditiontotheloadednanoparticles.Inthisstudy,prototypeelastomers

containingonlynano-oxidesweretested.

Ultimatetensilestrength(UTS)andmaximumstrainatbreakofsilica-filledsampleswas

significantlydecreasedbytimepassage(control)andUVBradiation,buttherewereno

significantdifferencesbetweencontrolandUVBweathering.Changesfrombaselinemaybe

attributedtocontinuedcrosslinkingandchainscissionwithinthepolymernetworkovertime;

howeverthiseffectwasnotincreasedbyUVBexposure.Themodulusofelasticityforsilica

sampleswasnotaffectedbytimepassageorUVBexposure.Thereasonforthisoccurrenceis

unclear.


Candidaalbicansareopportunisticfungithathavetoovercomethehost’simmunesystemin

ordertoproduceaninfection.Itsabilitytochangefromyeastcellstohyphalcellsisknowntobe

oneofitsvirulentfactors.C.albicansbiofilmdevelopmentischaracterizedbythreedistinct

phases.ThefirstistheadherenceofC.albicanstoitssubstrate(≈0-11hours).Inthe

intermediatephase,cellproliferationandmicrocolonyformationtakesplaceanddepositsan

extracellularmatrix(≈12-14hours).Finally,theformationofadensenetworkoffilamentous

forms(pseudohyphaeandhyphae)encasedinexopolymericmatrixisconsideredthematuration

phase(≈24-72hours)(Chandraetal.,2001,Alsalleehetal.,2016).Forthisreason,C.albicans

wereincubatedfor48hoursbeforeconductingtheXTTcolorimetricassayandconfocallaser

69

scanningmicroscopyexperiments,toallowforthebiofilmformationtoreachitsmaturation

phase.

ResultsfromtheXTTcolorimetricassayshowedthatTiO2groupsdemonstratedsimilar

antifungalactivitytoAg(negativematerialcontrol)withsignificantlylessC.albicansgrowth

comparedtothepositivecontrol.TheprecisemechanismbywhichAgandTiO2maycontrolthe

growthofC.albicansisnotfullyunderstood.EarlystudieshaveshownthatAgionsblock

microbialDNAreplication,inactivatevitalenzymesnecessaryforATPproductionandoxidation

ofglucose,anddamagemicrobialcellwalls,resultingincelldeath(Allaker2010).Similareffects

havebeensuggestedforTiO2,asitoxidatesthecellmembraneofthemicroorganismandalters

CoenzymeA-dependentenzymeactivities,therebyproducingabiocidaleffect.Theproposed

mechanismisthroughtheformationofreactiveOH-andHO2-species,theresultofTi-Osurface

bondsproducedbymismatchesbetweenbulkandsurfaceelectronicproperties(Longoetal.,

2013).NosignificantdifferenceswerenoticedbetweenZrO2andsilicagroupswhencompared

tothepositivecontrol.Consequently,C.albicansgrewandformedmorehyphaeonZrO2and

silicadiscs,asseeninconfocalimages.ThepoorantifungalactivityofZrO2andsilicacouldbe

duetothelackofabilitytoproducefreereactiveradicalsthatcanattackcandidalcellwallsand

essentialenzymes.Therefore,TiO2nanoparticleswereabletolimitC.albicanscellsgrowthon

thesurfaceofPDMSdiscs,butZrO2andSiO2didnot.

70

CHAPTER6: CONCLUSIONS

Thepurposeofthisstudywastocompareelastomersfilledwith1%weightTiO2and

ZrO2nanoparticlesintwoparticlesizes(40nmand200nm)toelastomersfilledwith13%weight

silicananoparticles,toevaluateofcolorstabilityandphysicalpropertiesafterexposureto

controlandUVBweatheringchallenges.Asecondpurposewastoanalyzetheantifungal

propertiesofthesesamePDMSelastomers.Conclusionsofthisstudyareasfollow:

1. PDMSelastomersloadedwith1%weight200nmand40nmTiO2nanoparticles

demonstratedlessoverallcolorchangeafterstorageincontrol(p<0.05)and

UVB(p<0.05)environmentscomparedtosamplesfilledwithZrO2andsilica

nanoparticles.

2. Elastomersfilledwith13%silicashowedhighershoreAhardnessatbaseline

(p<0.05)comparedtoallothergroups,exceptsamplesfilledwith200nmZrO2

nanoparticles.Weatheringonlyaffectedsamplesfilledwithsilica,producinga

1.5hardnessunit’sincrease,whichwasconsideredtobeclinicallyinsignificant.

3. TensilepropertiesofultimatetensilestrengthUTS,maximumstrainatbreak

andmodulusofelasticityweresignificantlyhigherforsilicasamplesin

immediatetesting(p<0.05),control(p<0.05)andUVB(p<0.05)weathering

environments,comparedtoallothergroups.

4. Incorporationof1%loadingweightTiO2nanoparticlesintoPDMSimparted

greaterantifungalactivity(p<0.01)comparedtoZrO2andsilica-filledsamples.

71

CHAPTER7: RESEARCHLIMITATIONS

Thelimitationsassociatedwiththisprojectincludethefollowing:

1. Analysisofotherweatheringenvironmentssuchasheatandhumidityandusing

outdoorweatheringwouldprovidefurtherinsightintothisproject.

2. Onlyonepigment(functionalintrinsicyellow)waschosenforthisstudy.

Differentpigmentswouldproducedifferentbehaviorsofstudiedmaterials.

3. Prototypeelastomerswereusedinthisprojectinordertocontroltheir

compositionsprecisely.Commerciallyavailableelastomersmaybehaveina

differentmanner.

72

CHAPTER8: CONSIDERATIONSFORFUTURERESEARCH

Resultsofthisstudysuggestinvestigatingtheeffectsofaddingsimilarlevelsofloading

ofTiO2,ZrO2,andsilicananoparticlesintoPDMSelastomerstoallowfordirectcomparison

betweenthem.

Anotherconsiderationistostudytheeffectsofaddingacombinationofdifferent

nanoparticlesintoPDMSelastomersatcontrolledcompositions,whichmayproducemore

desirableproperties.

Outdoorweatheringmayproducedifferentmaterialperformance,thereforeoutdoor

weatheringshouldbeconsideredasafutureenvironmentalchallenge.

ThedispersionofthenanoparticleswhenloadedintoPDMSelastomersisanessential

factorinobtainingtheoptimalpropertiesofmaterials.Futureresearchshouldfocusonparticle

surfacecoatingsthatproduceuniformparticlespacing.

Finally,theeffectofweatheringonlong-termantifungalactivityofPDMSelastomers

shouldbestudied.

73

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Documents

Color Stability, Physical Properties and Antifungal