atomic force microscopy and langmuir blodgett monolayer technique to assess contact lens deposits and human meibum extracts

Atomic force microscopy and Langmuir---Blodgettwww.journalofoptometry.org ORIGINAL ARTICLE Sarah Hagedorna , 1, Elizabeth Drolleb , c , 1, Holly Lorentza , e, Sruthi Srinivasana , ∗, Zoy

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Please cite this article in press as: Hagedorn S, et al Atomic force microscopy and Langmuir -Blodgett

www.journalofoptometry.org

ORIGINAL ARTICLE

Sarah Hagedorna , 1, Elizabeth Drolleb , c , 1, Holly Lorentza , e, Sruthi Srinivasana , ∗,

Zoya Leonenkob , c , d, Lyndon Jonesa , b , d

aSchool of Optometry and Vision Science, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

bDepartment of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

cWaterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

dDepartment of Physics and Astronomy, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

eDepartment of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4L8

Received26August2014;accepted9December2014

KEYWORDS

Meibum;

deposits;

Langmuir -Blodgett

Abstract

Purpose: Thepurposeofthisexploratorystudy wastoinvestigatethedifferencesin meibo-mianglandsecretions, contactlens(CL) lipidextracts,andCLsurfacetopographybetween participantswithandwithoutmeibomianglanddysfunction(MGD)

Langmuir -Blodgett(LB)depositionwithsubsequentAtomicForceMicroscopy(AFM) visualiza-tionandsurfaceroughnessanalysis.CL Study:ParticipantswithandwithoutMGDworeboth etaﬁlconAandbalaﬁlconACLsintwodifferentphases.CLlipiddepositswereextractedand analyzed usingpressure-areaisothermswiththeLBtroughandCL surfacetopographies and roughnessvalueswerevisualizedusingAFM

Results: Meibum study:Non-MGDparticipantmeibumsamplesshowedlarger,circular aggre-gates with lower surface roughness,whereas meibum samples from participants with MGD showedmorelipidaggregates,greatersizevariabilityandhighersurfaceroughness.CL Study:

WornCLs fromparticipantswithMGD hadafewlargetearﬁlmdepositswithlowersurface roughness,whereasnon-MGDparticipant-wornlenseshadmanysmalllensdepositswithhigher surfaceroughness.Balaﬁlcon A poredepths wereshallower inMGD participantworn lenses whencomparedtonon-MGDparticipantlenses.IsothermsofCLlipidextractsfromMGDand non-MGDparticipantsshowedaseamlessriseinsurfacepressureasareadecreased;however, extractsfromthetwodifferentlensmaterialsproduceddifferentisotherms

夽 Oneoftheauthors(L.J.)hasreceivedfundingoverthepast3yearsfromthefollowingcompanieswhoeitheraredirectlyinvolvedin

products used in this manuscript or are involved in the manufacture of competing products - Alcon, AMO, B&L, CIBA Vision, CooperVision, and Johnson & Johnson.

∗Correspondingauthor.

1 These two authors contributed equally to this publication.

http://dx.doi.org/10.1016/j.optom.2014.12.003

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Conclusions:MGDandnon-MGDparticipant-wornCLdepositionwerefoundtodifferintype, amount, and pattern of lens deposits Lipids from MGD participants deposited irregularly whereaslipidsfromnon-MGDparticipantsshowedmoreuniformity

PALABRAS CLAVE

Meibomio;

Langmuir -Blodgett

contacto

Resumen

Objetivo:Elobjetivodeesteestudioexploratoriofueeldeinvestigarlasdiferenciasentrelas secrecionesdelasglándulasdeMeibomio,losextractoslipídicosdelaslentesdecontacto(LC),

ylatopografíadelasuperﬁciedelaslentesentrelosparticipantes,conysindisfuncióndelas glándulasdeMeibomio(DGM)

Métodos: Estudio de las Glándulas de Meibomio:Serecogieronlassecrecionesglandularesde todoslosparticipantes,estudiándosemediantepelículadeLangmuir -Blodgett(LB)yposterior visualización, utilizando unmicroscopio defuerza atómica (AFM) y analizandola rugosidad superficial.Estudio de las LC:LosparticipantesconysinDGMusaronlentesdeetafilconAy balafilconAendosfasesdiferentes.Seextrajeronyanalizaronlosdepósitoslipídicosutilizando isotermosdeáreadepresiónconlausaron,yvisualizándoselastopografíasdelasuperficiede

laLCylosvaloresdelarugosidadmedianteAFM

participantessinMGDreflejaronunconglomeradomayorycircularconunasuperficiemenos rugosa,mientrasquelasmuestrasdelassecrecionesdelosparticipantesconDGMreflejaron unosconglomeradosmáslipídicos,conmayorvariabilidaddetamaño,yunamayorrugosidad

enlasuperﬁcie.Estudio de las LC:LasLCdelosparticipantesconDGMmostraronunamayor cantidaddedepósitosdepelículalagrimal,conunasuperﬁciemenosrugosa,mientrasquelas

LCdelosparticipantessinDGMreflejaronunamenorcantidaddedepósitosyunamayor rugosi-dadenlasuperficie.LasprofundidadesdelosporosdebalafilconAeranmenoresenlaslentes

delosparticipantesconDGM,queenlosparticipantessinDGM.Losisotermosdelosextractos lipídicosdelasLCdelosparticipantesconosinDGMreﬂejaronunincrementonosigniﬁcativode

lapresióndesuperﬁcieamedidaquedisminuíaelárea;sinembargo,losextractosprocedentes

delosdosdiferentesmaterialesreﬂejaronisotermosdistintos

Conclusiones:LassecrecionesdelasLCdelosparticipantes,conosinDGM,mostraron diferen-ciasencuantoatipo,cantidadypatróndelosdepósitosdelaslentes.Loslípidosprocedentesde losparticipantesconDGMsedepositarondemodoirregular,mientrasquelosdelosparticipantes sinDGMreﬂejaronmásuniformidad

Introduction

Thetearﬁlmishighlystructuredandisarrangedinto

sev-eralindistinctlayers.1,7 -9Theoutermostlayerismadeupof

lipidsproducedbythemeibomianglandsthatresidewithin

theupperandlowertarsalplates.1,9 -11Thislipidlayeris

fur-therdividedintonon-polarandpolarlipidlayers.4,5,7,9,12,13

The outermost non-polar lipid layer functions to control

the rate of evaporation of the aqueous layer, whereas

theunderlying polarlipid layer functions to stabilizeand

supportthenon-polarlayer.1,4,5,7,12 -14Astablelipidlayeris essential for maintaining ocular surface health and visual acuityanddisruptioninanyofthemanytearfilm compo-nents can cause ocular discomfortand dry eye.1,15 -17 Dry eyesyndromeisoneofthemostcommonoculardisorders andhastwomainmanifestations:aqueous teardeficiency andevaporativedeficiency.18Aqueousteardeficiencyisthe mostcommonandisaresultofadecreaseinlacrimalgland secretion.18Sincethenon-polarlipidlayerfunctionsto pre-venttheevaporationofaqueoustears,evaporativedryeye

isoftencausedbyadeﬁciencywithinthelipidlayer,often

aresultofaconditionknownasmeibomiangland dysfunc-tionorMGD.9,18,19 Somecommonocular symptomsofMGD

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anddryeyeincludeburning,irritation,itching,and

ﬂuctu-atingvision.9,10,19Themeibomianglandsecretion(meibum),

isnormallyaclearoilysubstance,however,themeibumof

MGDpatientsisoftenaviscouswhiteoryellowedsubstance,

withatoothpaste-likeconsistency.9,10

When a contact lens (CL) is placed on the cornea, it

hasadisruptiveeffectonthetearﬁlmandmay alterthe

structure, physiochemical properties, and composition of

thenormal tearﬁlm.6,20 In fact,the presenceof the lens

disrupts the normal tear ﬁlm structure so that the lipid

layer covering the lens is thinner and less stable than in

theabsenceofalens.6,20 Asaresult,thetearﬁlmiseasily

destabilized,allowingthelipidstocomeintocontactwith

thelenses.6,7Thisinteractioncanleadtoabsorptionofthe

tear components, especially proteins and lipids, onto the

CL.6,7,21,22MGDanddryeyepatientstend tohavea

desta-bilized tearﬁlmtobeginwith, duetothepoorqualityof

meibum secretions This may resultin them experiencing

signiﬁcantCLdepositionandCLintolerance.10

Twotechniquesthatcanbeusedtostudyocularlipidsand

CLdeposits,eithertogetherorinisolation,areatomicforce

microscopy(AFM)and theLangmuir -Blodgett(LB)trough

Atomicforce microscopy(AFM) isapowerfultechniqueto

studysoftbiologicalsamplessuchaslipidmembranesand

monolayers.23Itallowsforthree-dimensionalimagingatthe

nanoscale and molecular level as well as permitting the

study of physical properties of lipid ﬁlms.24 -26 In AFM, a

sharp scanning probe (AFM tip)scans the sample surface

and theforces of interactionat each point aremeasured

toproduce an image of the surface morphology.27 -29 AFM

has much higher resolution than optical microscopy and

works well in both air and liquid environments Several

researchgroupshave usedAFMtostudywornandunworn

CLs.30 -35 However,to-datethismethodhas notbeen used

tospeciﬁcally study lenses frompatients with dry eye or

MGD

The Langmuir -Blodgett (LB) monolayer technique is

widelyusedtoproduceandstudythinﬁlmsofamphiphilic

moleculesattheair -waterinterface.36 ALBtroughcanbe

usedtodepositlayersofamphiphilicmoleculesontoa

sub-strate or performsurface pressure -area (/A)isotherms

Thisisothermdataprovidesinformationaboutthestructure,

phasetransitions,compressibility,intermolecularforcesand

interactions between lipid molecules.36 -38 If the surface

pressurebecomeshigherthantheintermolecularforces,the

monolayerwillcollapse(breakapart)andsomemolecules

willbesqueezedouttoformasecondlayer.Thepressureat

whichamonolayercollapsesgivestheobservermore

infor-mationaboutthecompressibilityandintermolecularforces

ofthesubstance.Depositedlipidlayerscanbestudiedwith

the AFM to assess their visual features and integrity.38 -40

Whilesomeresearchgroups haveundertakenexperiments

onhumanmeibumusingtheLBtrough41andrecentlysome

experimentshave been undertakenwith theLB trough to

study tear ﬁlmcomponents,42 usingthis technique is still

relativelynovelfortearﬁlmanalysis

The objectiveofthisexploratorystudy wastouseAFM

andaLBmonolayertechniquetoexploreocularlipidﬁlms

inordertodeterminethedifferences,ifany,betweenthe

contactlensdepositsonhuman wornlenses, contactlens

lipidextractsandmeibomianglandsecretionsbetweenMGD

patientsandnon-MGDparticipants

Methods

Thestudywasstructuredintwoparts:

Meibum study: Meibomian gland secretions were col-lectedfromMGDandnon-MGDparticipantsandstudiedvia

LBandAFM

Contact lens study:MGDandnon-MGDparticipantswore both Acuvue 2 (etafilcon A) and PureVision (balafilcon A) lenses in two consecutive wear phases Worn contact lens (CL) lipids were extracted and studied by measur-ing pressure -area isotherms with the LB Worn CLs were imagedwithAFM tostudy accumulation of lens deposits Etafilcon A and balafilcon A were chosen as the study lenses due to their distinct material properties Previous studies43,44 -47haveshownthatetafilconA(anFDAgroupIV, stronglyionic,relatively hydrophilicmaterial)accumulate lowerlevelsoflipiddepositsthanbalafilconA(anFDAgroup

V, mildly ionic, silicone hydrogel, relatively hydrophobic material)

preparation

ApprovalofthisprojectwasgrantedthroughtheOfﬁceof ResearchEthicsattheUniversityofWaterlooandall proce-duresadheredtothetenetsoftheDeclarationofHelsinki ParticipantswererecruitedattheCentreforContactLens Research(CCLR),SchoolofOptometryandVisionScienceat theUniversityofWaterloo.Informedconsentwasobtained from all participants, following explanation of the pur-pose of the study and procedures tobe undertaken The study was conducted as a two part study (meibum study and CL study) Meibomian gland secretions or patient-worncontactlenseswerecollectedfrompatientsforeach study

Clinical assessment

Thestudy consisted ofa singlevisitat which study varia-bleswerecollected.Screeningandthestudyvisitoccurred sequentiallyonthesameday.Therewasnorandomization

ormaskinginthisstudy.Tennon-CLwearerswererecruited and categorized into 2 groups: a non MGD (non-dry eye) group (n=5) and an MGD (dry eye) group (n=5), based on fourfactors:

1 Symptomassessment:ParticipantscompletedtheOcular SurfaceDiseaseIndex©(OSDI)symptomassessmenttool Broadly,theOSDIscoringisbasedona0 -100scale,with higherscoresrepresentinggreaterdisabilityorlevelsof symptoms

2 Tearﬁlmbreakuptime(TFBUT):Theslitlampwassetat

amagnificationof10 -16×usingcobaltblueillumination andayellowbarrierfilterwasusedwhilerecordingthe TFBUT.Whileholdingtheright eyeopen,theexaminer instilledadrop of2%preservative-freesodium fluores-cein onto the superior bulbarconjunctiva of the right eye The participant was instructed to blink several times tomixthefluoresceinwiththetear film Imme-diately followingthis procedure, TFBUT wasmeasured

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3 consecutive times and the average of these was

recorded asTFBUT The time that elapsed between a

blink andthe ﬁrstsign ofa darkarea wasrecorded as

theTFBUT.Thisprocedurewasrepeatedinthelefteye

3 Fluoresceincornealstaining:Cornealstainingwas

eval-uated approximately 2min following ﬂuorescein dye

instillation Corneal staining was assessed at a

magni-ﬁcationof 10 -16×usingcobaltblueilluminationanda

yellow barrier ﬁlter.Theright eye wasevaluatedﬁrst,

followed by the left eye Staining was graded on a 0

(nonetominimal)to4(severestaining)in5regionsof

thecornea(central,nasal,temporal,inferiorand

supe-rior).Theﬁnalscorewasobtainedbysummingthescores

(0 -20)

4 Meibumsecretionqualityassessment:Meibumsecretion

quality score was assessed in both eyes

Secre-tion quality score was assessed using a 0 -3 grading

scale (grade 0=normal, clear oil expressed; grade

1=opaque, diffusely turbid, normal viscosity; grade

2=opaque, increased viscosity; grade 3=inspissated

(thick, toothpaste-like appearance)) meibum or not

expressible glands.Meibum wasexpressed by applying

digital pressure on the lower lid and viewed at the

slit-lamp All evaluations were conducted by a single

examiner

ParticipantswerecategorizedasMGD(dryeye,symptom

score≥13,tearbreakuptime≤4s,cornealstainingscoreof

≥4,meibumsecretionqualityscoreof≥1(ona0 -3scale)

inatleastonesector(nasal,centralortemporal)oflower

lid)or asnon-MGD(non-dryeye,symptomscore≤12,tear

breakuptime≥5s,cornealstainingscoreof ≤1,meibum

secretionqualityscoreofzero(ona0 -3scale)).Participants

whohadanyotheroculardiseaseandwhousedanytopical

medicationsthataffectedocularhealthwereexcludedfrom

thestudy

Collection of meibum

A Mastrota paddle was placed nasally behind the lower

lid to retract the lower lid away fromthe eye while the

patientwaslooking upward.Using asterile cotton-tipped

applicator, gentle pressure on the lid against the face of

thepaddlewasappliedtoforcetheexpressionofmeibum

fromwithinthemeibomianglands.Whilekeepingthelower

lid retractedwith thepaddle toavoid contact withtears

in the lower cul-de-sac, the expressed meibum was

col-lectedbycarefullyglidinga smalldegreased metalocular

spud(EllisEyeSpud,KatenaPartNo.K2-4100)alongthelid

margintocollect anoilypearlofmaterial.Thismotionof

compression,thencollection,wasconductedbyslidingthe

paddlealongtheinsideofthelowerlid.Approximately3 -4

‘‘collections’’wereperformedforeacheye.Thisprocedure

wasrepeatedfor thelefteye andthe meibum fromboth

eyeswaspooled.Meibumsampleswere placedona glass

coverslipandlaidinabrownglassspecimenjar Nitrogen

gaswasthenblownintothespecimenjar,thejarwascapped

andwasstoredimmediatelyat−80◦Cuntilprocessing.The

spud,paddleandspatulausedformeibumcollectionwere

pre-sterilized by autoclaving and wiping with an alcohol

swab

Screening and fitting n=10 (5 symptomatic of DE and 5 asymptomatic)

Phase 1 : Acuvue 2 or pure vision

(Randomized)

Phase 2 : Acuvue 2 or pure vision

(Randomized)

2 wk visit - collect lenses for AFM and LB trough

Exit study

Clinical assessment

Thiswasaprospective,dispensing,singlemasked,crossover, dailywearstudy(Fig.1).TenCLwearers(5MGDand5 non-MGD)participatedinthisstudy,whowerenotthesameas those in the meibum study described above Participants whoworehydrogelorsiliconehydrogelCLonadailywear basis and a monthly/bi-weekly replacement schedule and whoworeCLforatleastﬁvedaysperweekforaminimum

of10heachdaywereincludedinthestudy.Participantswho worelensesonanextendedwearbasiswereexcludedfrom the study.Participants were categorizedinto MGD (symp-tomaticofdry-eye)andnon-MGD(asymptomaticofdryeye) basedontheirsubjectiveevaluationofsymptomsofdryness (SESOD).48,49 The SESOD isaself-assessment questionnaire thatassessedthesubjects’oculardiscomfortdueto symp-toms of dryness ona 0 -4 scale, ranging from‘‘none’’ to

‘‘severe’’.Participantswhoscored≥2onSESODandhada meibum qualityscoreof>1 ona 0 -3scale, weregrouped

asMGD.Thosewhoscored≤1onSESODandhadameibum qualityscoreof0werenon-MGD

Participants were randomized to wear one of the two lenses(etafilcon Aor balafilconA)bilaterally for thefirst phase(2 weeks)of the study.Duringthe second phase(2 weeks)thelensnotwornonthefirstphasewerewornby

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theparticipants.Participantsweremaskedtothelenstype

during the study A hydrogen peroxide-based disinfection

system(AlconClearCare,FortWorth,TX)wasdispensedto

studyparticipantsduringbothphasesofthestudy

Theuseofartiﬁcialtearsand/orrewettingdropswasnot

permittedduringthestudyforthenon-MGDgroup.However,

theMGDgroupwasallowedtousetheirhabitual artiﬁcial

tearsand/or rewettingdrops.Therewereatotalofthree

studyvisits (Fig.1).Each visitwasseparatedby2 weeks

Participantswererandomlyassignedtooneofthetwolens

groups.Theparticipantswereaskedtoweartheirlenseson

a daily wearbasis for a minimum of eighthoursper day

Overallparticipantpreferenceforthelenstypeusedinthe

studywasalsoassessedona0 -100scale

Lens collection and storage

The studylenseswerecollectedat theendofeach phase

(Fig.1).Study lenses werecarefully removed bythe

par-ticipant,withcleanpowder-freenitrilegloves(SemperCare

NitrilePF)attheendofeachtwo-weekphase.Lens

collec-tionwasrandomized,witheithertheleftorrightlensbeing

collectedforanalysiswithAFMorLBtrough.Thesame

ran-domizationwasusedforbothphasestoallowforpair-wise

phasecomparisonsforeachparticipant.Lensesdeemedfor

AFM analysis were placed into a 20mL glass scintillation

vialcontaining2mLofautoclavedPBS.Bluntmetalforceps

wereusedtomanipulatethelensintoan‘‘open’’position

inthe solution.LenseswerescannedwiththeAFMwithin

48h Lenses deemed for LB trough analysis were placed

into empty 20mL scintillation vials with blunt metal

for-ceps,purgedwithnitrogengas, cappedandstoredfrozen

at−80◦C

The Langmuir -Blodgett (LB) microtrough from KSV NIMA

(BiolinScientiﬁc,Finland)wasusedfor creatingisotherms

for lipid analysis and for preparation of solid-supported

monolayersamplesofmeibum ﬁlms.The lipidsolutionsin

organicsolvent werespread atthe water -airinterfaceof

the trough and let equilibrate for a minimum of 10min

to allow for organic solvent evaporation as lipid

equili-bration.Tocollectpressure -areaisotherms,thelipidﬁlms

werecompressedataspeedof20cm2/minandaminimum

of 3 isotherms were collected for each sample For lipid

ﬁlmssupportedonmica,depositionwasdoneataconstant

barrier-controlledcompressionpressureof10mN/manda

consistentdippingspeedof2mm/min

Meibum study

Monolayer samples were created using vertical

deposi-tion with the LB trough A freshly cleaved mica slide

(ruby,ASTMV-2quality;Asheville-SchoonmakerMica,

New-portNews,Virginia)wasplacedinadipperarmofthetrough

andloweredintothesubphase.Meibumsecretionswere

dis-solvedin 1.0mL ofchloroform; concentrations varieddue

todifferencesinamountofmeibumcollectedfrom

partici-pants.Dissolvedmeibumwasdepositedonthesurfaceofthe

LBtroughandlipidswereallowedtospreadandequilibrate

ontheinterfaceforaminimumof10min,beforebeing

com-pressedviathetroughbarrierstoacompressionof10mN/m

Thispressurewasthenheldwhilethemicawasraisedata constantratethroughtheinterface.Aftera10-mindrying periodinair,themonolayersupportedonmicawasafﬁxed

onaglassmicroscopeslideforAFMimaging

Contact lens study

Lenses worn by study participants were extracted twice using 2.0mL of 2:1 chloroform:methanol These extracts weresubsequently driedwithasoftstreamofinert nitro-gen gas and re-suspended in 2.0mL of chloroform These lipid solutions were then studied using the LB trough

at ambient room temperature (25◦C) to look at their pressure -area isotherms Lenses incubated in an artiﬁ-cial tear solution (ATS) for 2 weeks at 37◦C, containing

a‘‘cocktail’’ of the6 most abundant lipids inthe human tearﬁlm(cholesterolat0.0018mg/ml,cholesteryloleateat 0.024mg/ml,oleicacidat0.0018mg/ml,oleicacidmethyl esterat0.012mg/ml,phosphatidylcholineat0.0005mg/ml, and triolein at 0.016mg/ml), were also extracted and analyzed using the LB trough pressure -area isotherms A pressure -areaisothermofthe‘‘6-lipidcocktail’’itselfwas alsomeasured for thepurposes ofanothercomparison.In this process, the trough is ﬁrst thoroughly cleaned with chloform; then, aliquots of the lipid solution in chloro-formwereaddedtotheair -waterinterfaceofthetrough, withMilli-Qultrapurewater(18.2Mcmresistivityat25◦C)

as a subphase, allowed to equilibrate for 10min, and thencompressedviamoveable barrierarmstocollectthe pressure -areaisotherms

TheAtomicForceMicroscopeutilizesasharpprobeto phys-icallyscanacrossthesurfaceofasampleandgivesanimage

ofthetopographicalfeatureswithnanoscaleresolution.For boththemeibumstudysamplesonmicasubstrateandthe

CLstudysamples,NanoworldNCHPointProbeuncoated can-tilevers(Neuchâtel,Switzerland)withaspringconstantof

42N/mandaresonantfrequency of320kHzwereusedto conductscansusingintermittentcontactmode.Imagingwas conductedusingaJPKNanowizardII (JPKInstrumentsAG, Berlin,Germany)atomicforcemicroscope

Meibum study

MeibumﬁlmsforAFMimagingweredepositedonmicaand glassslidesusingLBdepositionandimagedin airin inter-mittentcontactmode.Thetopographyimagesofsupported lipidﬁlms were analyzed and processed using JPK image processingsoftware(JPKInstrumentsAG).Allimageswere subjectedtothesameprocessing,namely polynomialline

ﬁtandhistogramlineﬁt,inordertoimproveimagequality Quantitativeanalysisofsurfacecoverageandroughness val-ueswasperformedusingtheJPKimageprocessingsoftware andGwyddionanalysissoftwareprograms

Contact lens study

Allofthecontrollensesusedinthisstudyhadapower -3.0 dioptresandadiameterof14.0mm.ThebalaﬁlconAlenses hadaradiusof8.6mmandtheetaﬁlconAlenseshadaradius

of 8.7mm Due tothe curvature of the lens, a spherical glasslensholderwascreatedinordertomaintaincurvature

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ofthesample while doingtheAFMscanning.Lenseswere

scannedinbothliquidandairtoanalyzethedifferencein

imagingtechniquesandtodeterminehowthelenssurface

morphology responded toboth conditions.Imaging

condi-tionsareindicated inﬁgure descriptions.Allimageswere

subjectedtothesameprocessingasthemeibumstudy.20

participant-wornlensesweregatheredintotalforAFM

anal-ysis:10etaﬁlconAand10 balaﬁlconA.Howeveronelens

ofeach material typewasnot abletobe scanneddue to

technicaldifﬁculties,giving atotalof 18 CLsanalyzed by

AFM

Clinical data analysis was conducted using Statistica 9.1

andp-valueswereobtainedviaanindependentt-test.

Oth-erwise,datais reportedasmean±standard deviation.LB

trough pressure -area isotherms were analyzed and

plot-tedusingMicrosoftExcel2007 StatisticalanalysisonAFM

images was performed on the images in order to obtain

informationonroughnessofthesamplesandtheheightsof

surfacefeatures.Foreachsampleseries,20cross-sections

weretakenandthedatacollectedfromthem:10acrossthe

ﬂat,featurelessareasofthesampleand10acrossthelarge

aggregates(forthe meibummonolayers)or pores(forthe

humanwornlenses)

Results

Ten female participants were enrolled in the study, with

a mean age of 55 years (median 56 years, ranging from

40 to 65 years) For the non-MGD group, 5 participants

were enrolled in the study witha mean age of 53 years

(median 53, ranging from40 to62 years).For the group

withMGD,5participantswereenrolledwithameanageof

58years(median57,rangingfrom61to65years).Themean

OSDI (non-MGD=1±1 vs MGD=17±7; p<0.01), TFBUT

(non-MGD=5.8±1svsMGD=2±1s;p<0.01),corneal

stain-ing (non-MGD=0.14±0.09 vs MGD=5.8±3.63; p<0.01),

qualityofmeibum (non-MGD=0.0±0.0vsMGD=1.2±0.2;

p<0.01)andmeibumexpressibility(non-MGD=0.7±0.4vs

MGD=1.8±0.8;p=0.03)scoreswereallsigniﬁcantly

differ-entbetweenthegroups

AFMresultsforthemeibumsamplesinthisstudydidvary

widely,aswould beexpected due togenerallipoidal

dif-ferencesbetween individuals.However,sometrendswere

visibleinthedatacollected

For each participant, several images of the meibum

depositsonmicaweretakenofthesampleatvariousareas

aswellasinvarioussizes,toensureconsistencyinthe

sur-face features observed The common surface features of

the samples collected can be seen in the representative

images illustrated in Fig 2 In general, visual inspection

suggestedthatﬂattersampleswithrelativelyfew(but

rel-ativelylarge)sphericallipidaggregatesweremorecommon

forthenon-MGDparticipants(Fig.2A).Wherepresent,these

lipid‘‘clusters’’tendedtobelargerinheight,withheights

upto525nm Upon takinga closerlook at thesesamples

(Fig.2B), it can beseen that some multilayers were also

present,from3.0to8.0nminthickness.FortheMGD par-ticipants,alargernumberoflipidaggregatesofvaryingsizes weremoreplentifulthanthatseeninthenon-MGDresults, althoughtheseclusterswerenotaslargeintermsoftheir height (Fig.2C) Images of highermagniﬁcation show the presenceofmultilayersaswell,rangingfrom3.0to14nm

inthickness

Inordertonumericallycomparethedifferencesbetween thelipidaggregatesandmultilayersonthesupportedﬁlms betweentheMGDandnon-MGDresults,averageroughness

(R a)valueswereobtainedtogiveaquantitativemethodof comparison.R aisameasurementofthechangesinheightof thesampleinquestion;inthiscase,wecanusethese rough-nessmeasurementsasawayofdeterminingdifferencesin themeibumsamplesofMGDvsnon-MGDparticipants caus-ingchangesinthesurfacefeaturesofthesamples,whether

itbeduetoamountofmeibum,ratiosoflipids,presenceof differinglipids,interactionsamongthelipidspresent,and

soon.AsseeninTable1,R avaluesacrossthelipid multilay-erswereonaveragefrom0.07to1.11nmforthenon-MGD samples,ascomparedwiththeMGDsamples,wheretheR a

ranged from0.10 to 1.37nm, indicating that the samples fromMGDparticipantshad slightlyhigherR a values Simi-larly,R avaluesweretakenacrossthelipidaggregatesand thesametrendwasobserved:non-MGDparticipantsamples hadR avaluesfrom2.14to13.77nmwhileMGDsampleshad

R avaluesfrom2.91to39.18nm.InadditiontoR a, peak-to-valleyroughness (R t) valueswereobtained, whichgives a measure of thechanges in height of thesample between the minimum valley and maximum peak of the sample,

an indication in this case of the largest accumulations of meibumconstituentsonthesolid-support.These measure-ments,asshowninTable1,followedasimilartrendtothat

oftheR a,whereawiderdistributionwithhigherroughness values wereobserved for MGDsamples than for non-MGD samples

Ten participants were enrolled in this study (8 female,

2 male) The mean age of the participants was 32 years (median 30 years, ranging from 21 to 54 years) For the

CLwearinggroupwithno-MGDgroup,5 participantswere enrolled,withameanageof32years(median30,ranging from21to40years).FortheCLwearinggroupwithMGD,

5 participantswereenrolled withameanage of 33years (median27,rangingfrom23to54years)

Asexpected,theMGDgroupscored signiﬁcantlyhigher

on the SESOD questionnaire (MGD group=2.60±0.89 vs non-MGDcontrolgroup=0.60±0.55;p<0.01)andmeibum secretion quality score (MGD=1.2±0.2 vs non-MGD con-trolgroup=0.0±0.0;p<0.01).Themajorityofparticipants (90%)preferredetaﬁlconAlensesover balaﬁlconAlenses during the study For each lens, in at least two dif-ferent areas, AFM images were taken at four different sizes (30␮m×30␮m, 20␮m×20␮m, 10␮m×10␮m, and

5␮m×5␮m), resulting in at least eight images for each lens Fig.3 displays afew representativesamplesof such images

Balaﬁlcon A lenses worn by MGD participants showed fewer deposits, although they were larger and

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(AandB)andMGDparticipants(CandD).Imagesshownarerepresentativeimagesillustratingthecommondifferencesobserved betweenMGDandnon-MGDsampleresults.AandCshowthesampleatawiderscale(30␮m×30␮m).FiguresBandDarezoomed

inimages(10␮m×10␮m)ofareasfromtheircorrespondinglargerscaleimages(AandBarethesameparticipantandsample;

CandDarethesameparticipantandsample)

distributed unevenly across the surface These deposits

also had an irregular stringy or elongated appearance

In comparison, the non-MGD lenses showed a dusting of

small,circulardepositsthatwereevenlydistributedonthe

lens These observations suggest an irregular deposition

and accumulation of tear ﬁlm components on the lenses

wornbyMGD participants.Table2displays measurements

of roughness parameters fromthe lens images The

indi-vidual roughness values from the images in Fig 3 are as

follows:Fig.3Ais abalaﬁlconAnon-dryeye lenswithan

average roughness of 3.200nm Fig 3B is a balaﬁlcon A

dryeyelenswithanaveragerounessof1.422nm.Fig.3Cis

anetaﬁlconAnon-dryeye lenswithanaverage roughness

of0.395nm.Fig.3Dis anetaﬁlconAdryeyelenswithan averageroughnessof0.758nm

The surface roughness of balafilcon A lenses worn by MGDparticipants waslowerthanthesurfaceroughness of non-MGD participant worn lenses on average when mea-suredacrossapore,a‘‘flatarea’’onthelensoraflatand porousregion combined Pore depths in the worn balafil-conAlensesweremeasuredandfoundtobeveryvariable

Onaverage,poresin thelenseswornbyMGDparticipants werenotasdeepastheporesin thelenseswornby non-MGD participants Pore widths were also very variable,

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Table 1 Estimationanalysisforhumanmeibumsamples.Cross-sectionsweretakentogivethreeroughnessvaluesformboth acrossthelargelipidaggregatesaccumulatedatopthemonolayerandacrossthemonolayeritselftodetectsmalldomainsand featuresofthemonolayer

Averageroughness(R a)(nm) Peak-to-valleyroughness(R t)(nm)

Range Average Std.Dev Range Average Std.Dev

Cross sections across ‘‘ﬂat area’’ of the sample

Non-MGD 0.07 -1.11 0.51 0.46 0.42 -4.54 0.62 0.52

Cross sections across large lipid aggregates atop monolayer

MGD 2.91 -39.18 19.21 16.09 11.21 -125.63 21.35 17.30 Non-MGD 2.14 -13.77 10.64 8.16 7.53 -67.17 12.85 9.65

mea-surethesedimensionsontheetaﬁlconAlensesbecausethis

materialisnotporous

Fig.3C and D display AFM images of worn etaﬁlcon A lenses.Thistypeoflensproved tobemuchmoredifﬁcult

toscanthanthebalaﬁlconAlensesastheywerevery frag-ileandtoreeasily.Asaresult,twolenseswereunabletobe

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partic-ipants,respectively.CandDareetaﬁlconAlenseswornbynon-MGDandMGDparticipants,respectively.Thesearerepresentative imagesshowingthecommonfeaturesoftheselenses.Eachimageshowsa5␮m×5␮mareaoftherespectivecontactlens

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Table 2 Summarystatisticsofroughnessvaluesfromboththe‘‘ﬂat’’areasofthelensimagesaswellasacrossthelenspores

ofbalaﬁlconAandetaﬁlconAlenses.Averageroughnessandpeak-to-valleyroughnessareshown.Allroughnessmeasurements areinnanometers

Averageroughness(R a) Peak-to-valleyroughness(R t)

Range Average Std.Dev Range Average Std.Dev

Balaﬁlcon A cross sections across a ‘‘ﬂat’’ area of the lens

Dryeyelensesimagedinliquid 1.51 -10.20 4.83 2.26 7.84 -33.39 20.62 7.35 Dryeyelensesimagedinair 0.42 -2.67 1.19 0.58 3.15 -14.44 6.55 2.73 Non-dryeyelensesimagedinliquid 0.69 -18.74 6.03 5.10 3.13 -57.95 24.19 15.61 Non-dryeyelensesimagedinair 0.64 -9.00 2.02 1.80 3.75 -39.75 11.01 8.14 Controlinair 0.74 -1.20 0.96 0.16 4.35 -8.00 6.06 1.14 Controlinliquid 1.31 -6.69 3.53 1.56 7.44 -19.78 14.07 4.08

Balaﬁlcon A cross sections across a pore in the lens

Balaﬁlcon A cross sections across a pore and ﬂat area combined

Etaﬁlcon A lenses

Dryeyelensesimagedinliquid 0.15 -2.01 0.72 0.65 0.84 -9.20 3.86 3.04 Dryeyelensesimagedinair 0.24 -2.13 0.63 0.33 1.29 -9.43 3.56 1.65 Non-dryeyelensesimagedinair 0.15 -7.69 1.16 1.70 0.78 -24.48 4.86 5.67 Controlinair 0.13 -0.35 0.20 0.06 0.74 -1.55 1.00 0.19 Controlinliquid 0.37 -1.46 0.82 0.27 1.69 -5.73 3.96 1.18

lenses

Table 3 PoreanalysisforbalaﬁlconAhumanwornlenses

Analysis ofthepores for the balaﬁlcon A lenseswas

per-formed to investigate the presence of lipid accumulation

inoraroundtheedges ofthelenspores.Bothporedepth

(viapeak-to-valleyroughnessmeasurements)andporewidth

weretaken

Poredepth range(nm)

Porewidth range(nm) MGDparticipants 8.00 -64.91 239 -1880

Non-MGDparticipants 9.19 -109.10 220 -1310

Controlinair 7.40 -18.47 200 -1270

Controlinliquid 22.20 -48.97 310 -700

increasedasthesurfaceareadecreased.However,thelipid extractsfroman etafilconAlensincubatedinanartificial tearsolutioncontainingthislipidcocktaildid showa col-lapse,whereas thelipid extractsfrom balafilconAlenses didnot(Fig.4)

Fig 5 contains representative surface pressure -area isothermsofbalaﬁlconAandetaﬁlconAlensextractsworn

bynon-MGDandMGDparticipants.Bothgroupsofisotherms appeartobefairlyfeaturelessastheydonotcollapsebut thesurface pressure continues toincrease asthe surface areaofthetroughdecreases.Thereareafewcharacteristic

‘‘bends’’intheisothermcurvebutitcannotbesaidwhether

ornottherearemoreoftheseintheMGDisothermsorvice versa

Less lipid extract was needed to measure the surface -pressure area isotherms for the etaﬁlcon A materialcomparedtobalaﬁlconA.Also,ahighervolumeof MGDlensextractwasrequiredtoproduceanisotherm on averagecomparedtothenon-MGDextract

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Balafilcon A Etafilcon A

6 lipid cocktail

ofthesixlipidcocktail-basedsamples.Isothermsincludedare

thatofthepuresix lipidcocktail,whichisrepresentative of

theocularlipidssecretedbythemeibomianglands,aswellas

thoseoflensextractsfrombalaﬁlconAandetaﬁlconAlenses

thatwereincubatedinanartiﬁcialtearsolutioncontainingthe

sixlipidcocktailforatwoweekperiod

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10-os etafilcon a dry eye 230 ul 7-os etafilcon a non-dry eye 160 ul 3-od balafilcon a dry eye 20 ul 9-od balafilcon a non-dry eye 20 ul

etaﬁlconAlensextractswornbyMGDandnon-MGDparticipants

fortwoweeks.Thevolumeoflensextractusedtoproducethe

isothermisshownnexttotheparticipantnumber

Discussion

ThispreliminarystudyexploredtheuseofAFMandLB

meth-odstoexaminethedifferencesbetweenCLdeposits,CLlipid

extractsandmeibomianglandsecretionsinbothMGDand

non-MGDparticipants

Forthe meibum study,samples of meibum monolayers

supportedonmicafromboththeMGDandnon-MGD

partic-ipantsdepositedataspeciﬁccompressionpressurechosen

tooptimizeconditions.Recent studieshaveshown thatat

ambientroomtemperature(24◦C),meibumisabletoreach

pressuresofover35mN/m.50 Ingeneral,this

compressibil-ityofmeibummayaidinitsfunctioninpreventingthetear

ﬁlmfromevaporating,especiallyinrelationtotheblinking

oftheeye.Uponcompression,lipidsassociatetogetherto

formaggregatesatopthemonolayersample,which allows

themtoenduredecreasesinsurfaceareawithoutdramatic

increasesinpressure.Thisisseeninprotein-lipidmixtures

thataresubjectedtoextremechangesinsurfacearea,like

lungsurfactant.51 Itis possiblethatasimilarcasemaybe

occurringinthemeibumﬁlms,asmeibumhasbeenshown

to contain a number of proteins.52 -54 However, because meibumisverylipidrich,itisalsopossiblethatthelipids present inmeibum areabletoassociatetogethertoform theselargeaggregatesthatarevisualized.52Someofthese aggregates are likely to be multilayers formed from the amphiphilicphospholipidsandsphingolipids presentin the meibum,suchassphingomyelinandphosphatidylcholine.55

It is alsolikely thatsome of the aggregates wereformed duetothepresenceoftriglyceridesinthemeibum.These lipidshavebeenstudiedinmonomolecularﬁlmsandfound

to form multilayers atop the monolayer film, with their threehydrophobicfattyacidtailsallowingthemtoforma close-packingaccumulationstructureatopthemonolayerat higherpressures.56 However,inordertospecifically deter-mine the distributionof the various lipids in themeibum film, a method that could differentiate the components wouldneedtobeused

Wechoseacompressionpressureof10mN/mdueinsmall part tolimited amount of meibum sample available from each participant during the extraction process to spread acrossthesubphaseofthetrough,butprimarilytoensure sufﬁcient meibum wasavailable tomaintainthis pressure duringthedepositionpressure

Differencesintrends wereobservedfor the monolayer samplesofmeibumcollectedfromMGDandnon-MGD par-ticipants.Themaintrendsobservedwerethatthemeibum fromMGDparticipantshadslightlyhigherroughnessvalues andawiderdistributionofroughnessvaluesthancompared

to the non-MGD participants These valuescorrespond to what can be visualized in Fig 2, where there are more lipid aggregatespresent inthe meibum from MGD partic-ipants thanthe non-MGD participants, which ledto more features on the surface of the monolayer sample, and thushigherroughnessvalues.However,thelipidaggregates

on thesurface of the monolayers fromMGD participants, though more plentiful, were smaller in height and width thanthosevisualizedonthesurfaceofthemonolayersfrom non-MGDparticipants.This couldpotentiallybeattributed

tochangesinthelipidcompositionorchangesinrespective lipidamountsthatleadstolipidsintheMGDparticipants’ meibum to not combine well together, resulting in more plentifulbuthomogeneoussmalleraggregates.The reason-ing behind this may also be a contributing factor to the instability of the MGD tear ﬁlm In contrast, samples of the meibum from non-MGD participants’ show fewer but larger aggregates, which is potentially due tothe ability

of thedifferentcomponentsof themeibum beingableto associatetogether,formingfewerbutlargerheterogeneous aggregates

Forthe CL study, the AFMscans and roughness of the participant worn balaﬁlcon A and etaﬁlcon A lenses sug-gest that the non-MGD participant wornlenses contained

amoreuniformspreadofsmallerlipiddepositswithhigher roughnessvaluesthantheMGDlenses.Thismaybebecause thetearﬁlmstructureofapersonwithoutMGDisrelatively stableandordered,enablingfewerlipidandprotein interac-tionswiththeCLonceinserted.7Incomparison,thelenses fromMGDparticipants showedan irregulardistribution of variablesizeddepositswithlowerroughnessvalues.Those withMGDhavean unstabletearﬁlmthatishighly suscep-tibletoevaporationduetothecompromised lipidlayer.57

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