University of Nebraska Medical Center University of Nebraska Medical Center
DigitalCommons@UNMC DigitalCommons@UNMC
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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Recommended Citation Recommended Citation Alkahtany, Mazen, "Color Stability, Physical Properties and Antifungal Effects of ZrO2 and TiO2 Nanoparticle Additions to Pigmented Polydimethylsiloxanes" (2016). Theses & Dissertations. 89. https://digitalcommons.unmc.edu/etd/89
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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 AntifungalActivity..........................................................................................................59
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
5.3 AntifungalActivity..........................................................................................................68
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
600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................34
Figure4.4.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after
600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................35
Figure4.5.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after
600hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0
representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,
respectively......................................................................................................................37
Figure4.6.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆L*after
1800hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................39
vii
Figure4.7.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆a*after
1800hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................40
Figure4.8.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after
1800hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................41
Figure4.9.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after
1800hoursofweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0
representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,
respectively......................................................................................................................42
Figure4.10.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆L*after
3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................45
Figure4.11.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆a*after
3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................46
Figure4.12.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after
3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).................................................................47
Figure4.13.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆E*after
3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercase
lettersarenotsignificantlydifferent(p>0.05).Linesdrawnat∆E*=1.1and3.0
viii
representtheminimumthresholdsfor50:50visualperceptibilityandacceptability,
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
lettersarenotsignificantlydifferent(p>0.05).................................................................53
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.
4.1.2 After1800HoursofWeathering
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
Table4.3.∆L*,∆a*,∆b*and∆E*valuesafter1800hoursofweathering(Mean(S.D.),n=5)
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
Figure4.6.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆L*after1800hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).
40
Figure4.7.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆a*after1800hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).
41
Figure4.8.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after1800hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).
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.
4.1.3 After3000HoursofWeathering
∆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
Table4.4.∆L*,∆a*,∆b*and∆E*valuesafter3000hoursofweathering(Mean(S.D.),n=5)
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
Figure4.10.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆L*after3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).
46
Figure4.11.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆a*after3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).
47
Figure4.12.Bargraphdisplayingmeansandstandarddeviations(errorbars)of∆b*after3000hoursweatheringfordifferentgroups(n=5).Meanswiththesamelowercaselettersarenotsignificantlydifferent(p>0.05).
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
4.2.1 ShoreAHardness
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 AntifungalActivity
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
5.2.1 ShoreAHardness
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.
5.3 AntifungalActivity
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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