DEVELOPMENT OF SUBSTRATES FOR THE EX VIVO EXPANSION … · 2017. 12. 13. · The conjunctiva is a...

DEVELOPMENTOFSUBSTRATESFORTHEEXVIVOEXPANSION

OFCONJUNCTIVALEPITHELIUM

Thesissubmittedinaccordancewiththerequirementsofthe

UniversityofLiverpoolforthedegreeofDoctorofMedicine

ShivaniKasbekar

January2016

Abstract

Theconjunctivaisamucousmembraneliningtheocularsurfaceandiscrucialto

ocularsurfacehomeostasis.Ocularsurfacediseasesleadtoapoortearfilm,

irreversibleconjunctivalscarringandcontinualcornealdesiccationthatmayresultin

painfullossofvision.Conjunctivalintegrityandatearfilmwithappropriate

consituentsarecrucialtothesurvivalofcornealandlimbalstemcelltransplants.I

hypothesisedthattwonovelsubstratescouldbedevelopedfortheexvivoexpansion

ofconjunctivalepitheliumtoaddressarangeoftransplantationrequirements:1)a

degradablebiologicalsubstratefromthedecellularisationofhumanconjunctivaand2)

asyntheticnon-degradablesubstratefromexpandedpolytetrafluoroethylene(ePTFE).

Thisstudydemonstratedthatconjunctivalepithelialcells(HCjE-Gicellline)were

supportedatagreatercelldensityonePTFEsubjectedtoammoniagasplasmatreatment.

Flowcytometrydeterminedthephenotypeofconjunctivalepitheliumdevelopedon

treatedePTFEwassimilartothatdevelopedonanestablishedcellcultureproduct;

Thincert,achemicallymodifiedpolyethyleneterephthalate(PET)membrane.Primary

conjunctivalepitheliumwasalsoexpandedexvivoonammoniaplasmatreatedePTFE,

however,thecelldensitydeclinedafter14daysinculture.Nosignificantdifferenceswere

foundintermsofintracellularmarkerexpressionbetweenprimaryconjunctival

epitheliumdevelopedonammoniaplasmatreatedePTFEandthepositivecontrol(PET

membrane).Humanconjunctivawassuccessfullydecellularised(99%DNAremoval).

Therewasnodemonstrablecytotoxicity,evidenceofcollagendenaturation,changein

tensilestrengthorchangeinthequalitativedetectionofextracellularmatrixproteins

collagenIV,lamininandfibronectin.Thedevelopmentofstratifiedconjunctival

epitheliumofanappropriatephenotypewasalsodemonstratedfollowingexplant

cultureonafreshlydecellularisedconjunctivaltissuesubstrate.

ThisisthefirststudytodevelopdecellularisedconjunctivaandplasmamodifiedePTFE

assubstratesfortheexvivoexpansionofconjunctivalepithelium.Novelconjunctival

constructsdevelopedfrombothsubstratesmaybefurtherdevelopedtoaddressa

rangeoftransplantationrequirements.

Acknowledgements

IwouldliketothankmysupervisorsProfessorsKayeandWilliamsandDrStewartfor

theirdirectionalongthisjourneyobtainingexternalfundingandundertakinga

DoctorateinMedicine.Theendlessencouragementandexpertguidancetheyhave

givenmehasbeencrucialtotheundertakingofthiswork.Iwouldliketogratefully

acknowledgetheirhardwork.

IwouldalsoliketothanktheMedicalResearchCouncil,RoyalCollegeof

OphthalmologistsandNovartisforthejointlyfundedClinicalResearchTraining

FellowshipIwasawardedtoenablemetoundertakethisresearch.

IwouldalsoliketothankDrPaulRooneyandstaffattissueservicesatNHSBloodand

Transplant(NHSBT)fortheirguidanceandassistanceundertakingexperimentalwork.I

amalsogratefultoNHSBTnursingstaff,eyeretrievalcoordinators,mortuarystaff,

donorsandtheirrelatives,withoutwhomthesestudieswouldnothavematerialised.I

amalsogratefultoProfessorDarleneDarttattheHarvardMedicalSchool,Bostonfor

hostingmyvisittotheirlaboratories,duringwhichIreceivedtrainingincellcultureof

primaryconjunctivathatwascrucialtothisresearch.IwouldalsoliketothankDr

GabriellaCzannerforherexpertadviceonstatisticalmethods,MrJimBuckhurstfor

producingmaterialsthatwereusedinexperimentalworkandDrSimonBiddolphfor

hisadviceonundertakingimmunohistochemistry.IwouldalsoliketothankDrIlene

GipsonforthegiftoftheHCjE-Gicellline.Iamalsogratefultotheresearchstaffand

studentsattheDepartmentofEyeandVisionSciencefortheirguidanceand

companionship.

Imustalsogratefullyacknowledgetheencouragementandsupportfrommyhusband,

Anand,andfamilythroughthehighsandlowsofthelastfewyears.Thisworkwas

possiblebecauseoftheirunrelentingsupportandwisdom.

TableofcontentsABSTRACT...............................................................................................................I

ACKNOWLEDGEMENTS..........................................................................................II

LISTOFFIGURES....................................................................................................IX

LISTOFTABLES..................................................................................................XVII

ABBREVIATIONS..................................................................................................XIX

1. INTRODUCTION...............................................................................................11.1 THEOCULARSURFACE............................................................................................1

1.1.1 Thecorneaandlimbus..............................................................................................21.1.2 Thehumanconjunctiva.............................................................................................41.1.3 Protectionoftheocularsurfaceandthetearfilm....................................................7

1.2 CONJUNCTIVALDISEASEINHUMANS........................................................................10

1.2.1 Medicalstrategiesincicatrisingconjunctivaldisease.............................................131.2.2 Surgicalocularsurfacereconstructionstrategiesandtheclinicalneedfornovelconjunctivalequivalents......................................................................................................16

1.3 SUBSTRATESFORCONJUNCTIVALREGENERATION........................................................18

1.3.1 Anidealconjunctivalsubstrate...............................................................................181.3.2 Theuseofbiologicalsubstratesfortheexvivoexpansionofconjunctivalepithelium...........................................................................................................................18

1.3.2.1 Decellularisationofhumantissues..............................................................................................191.3.1.2 Decellularisationofhumanamnioticmembrane.........................................................................21

1.3.2 Syntheticsubstratesforex-vivoexpansionofconjunctivalepithelium...................221.3.2.1 Expandedpolytetrafluoroethylene(ePTFE).................................................................................231.3.2.2 AmmoniagasplasmatreatmenttoalterePTFEsurfacechemistry.............................................251.3.2.3 Determinationofthehydrophilicityofmaterialsthroughcontactangleanalysis.......................26

1.4 CULTURECONDITIONSFORTHEEX-VIVOEXPANSIONOFHUMANCONJUNCTIVALEPITHELIALCELLS 271.5 CHARACTERISATIONOFHUMANCONJUNCTIVALEPITHELIUM.........................................28

1.5.1 Gobletcellmarkers.................................................................................................291.5.2 Progenitorcellmarkers...........................................................................................301.5.3 Cytokeratins............................................................................................................311.5.4 Proliferationversusapoptoticmarkers...................................................................33

1.6 FLOWCYTOMETRY...............................................................................................34

1.6.1 Flowcytometryfortheanalysisofconjunctivalepithelialcells..............................35

1.7 MUCOUSMEMBRANEPEMPHIGOID.........................................................................351.7.1 Gradingsystemstodetectprogressionofcicatrisationinocularmucousmembranepemphigoid.......................................................................................................371.7.2 AssessmentoftheinvolvementoftheocularsurfaceandeyelidstogradediseaseprogressioninMMP............................................................................................................41

1.8 AIMSANDOBJECTIVES..........................................................................................47

2. METHODS......................................................................................................492.1 EXPANDEDPOLYTETRAFLUOROETHYLENE(EPTFE)......................................................49

2.1.1 AmmoniaplasmatreatmentofePTFE....................................................................492.1.2 ContactangleanalysisofePTFE..............................................................................50

2.2 CELLSANDTISSUES..............................................................................................51

2.2.1 Cultureofahumanconjunctivalcellline................................................................512.2.1.1 Passageofconjunctivalepithelialcells........................................................................................522.2.1.2 Cryopreservation..........................................................................................................................52

2.2.2 Retrievalofhumanconjunctivaltissueandcultureofprimaryconjunctivalcells..532.2.2.1Retrievalofcadavericconjunctiva.....................................................................................................532.2.2.2 Explantcultureofprimaryhumanconjunctivalcells...................................................................542.2.2.3 Cultureofcellsondecellularisedtissuesusingexplantsandisolatedcellsuspensions..............54

2.3 CELLCULTUREONSUBSTRATES...............................................................................55

2.3.1 Cultureofcellsoncellcultureinserts......................................................................552.3.2 CultureofcellsonePTFEmembrane.......................................................................56

2.4 CELLCULTUREANDCHARACTERISATIONEXPERIMENTSTOASSESSSYNTHETICSUBSTRATES...57

2.4.1 Cellseedingdensity.................................................................................................572.4.2 Optimisationofmediaprotocol..............................................................................582.4.3 ComparingcellcountsonePTFEwithammoniaplasmatreatmentononeandbothsides 582.4.4 Fixationofsubstrateculturesandstainingwithfluorescentmarkers....................592.4.5 Determiningcelldensityusingahaemocytometer.................................................602.4.6 Flowcytometry.......................................................................................................602.4.7 ValidationexperimenttoensureappropriateuseoftheHCjE-Gicellline..............622.4.8 Assessingthephenotypeofcultureswithadvancingtimeandbysubstrate..........62

2.5 DECELLULARISATIONOFHUMANCONJUNCTIVAANDITSCHARACTERISATION....................63

2.5.1 Decellularisationofhumanconjunctiva..................................................................632.5.2 DNAextractionandquantification.........................................................................652.5.3 Collagendenaturation............................................................................................662.5.4 Invitrocontactcytotoxicitytesting.........................................................................662.5.5 Biomechanicaltesting.............................................................................................67

2.6 HISTOLOGYANDIMMUNOHISTOCHEMISTRYDECELLULARISEDTISSUESANDRECELLULARISEDCONSTRUCTS...............................................................................................................68

2.6.1 Preparationoftissuesforhistologyandimmunohistochemistry............................682.6.2 Histology.................................................................................................................692.6.3 Immunohistochemistry...........................................................................................70

2.7 RECELLULARISATIONOFDECELLULARISEDCONJUNCTIVAWITHPRIMARYCONJUNCTIVALEPITHELIUM................................................................................................................71

2.7.1 Explantcultureexperimentswithandwithouttheorientationofbasementmembraneofconjunctiva...................................................................................................712.7.2 Comparisonofconjunctivalepithelialculturesusingtissuefromdifferentdonorsforexplantsanddecellularisedsubstrates..........................................................................72

2.8 STATISTICALANALYSIS..........................................................................................722.9 DETECTIONANDMONITORINGOFOCULARMUCOUSMEMBRANEPEMPHIGOIDPATIENTS....73

2.9.1 Developingaproforma..........................................................................................732.9.2 AssessingMMPusingtheMMPproforma.............................................................74

CHAPTER3:RESULTS.............................................................................................763.1 OPTIMISATIONOFCULTUREMETHODSFORTHEEXVIVOEXPANSIONOFCONJUNCTIVALEPITHELIUMONSYNTHETICSUBSTRATES............................................................................76

3.1.1 AmmoniaplasmatreatmentofePTFE....................................................................763.1.2 Cellseedingdensityanalysis...................................................................................773.1.3 EffectofmediaonHCjE-Gicellproliferation...........................................................81

3.2 EXVIVOEXPANSIONOFCONJUNCTIVALEPITHELIUMONSYNTHETICSUBSTRATES................87

3.2.1 ComparisononcelldensitybetweensubstratesincludingammoniaplasmatreatmentofoneorbothsidesofePTFE.............................................................................873.2.2 Morphologyofconjunctivalculturesdevelopedonsyntheticsubstrates...............89

3.3 RETRIEVEDHUMANCONJUNCTIVA.........................................................................1043.4 PRELIMINARYOPTIMISATIONANDVALIDATIONOFTHEHCJE-GICELLLINEANDFLOWCYTOMETRY..............................................................................................................105

3.4.1 Optimisationofantibodystainingforflowcytometry..........................................1053.4.2 CharacterisationofprimaryHCjE-Giconjunctivalcelllinewithflowcytometry...1083.4.3 Determiningtheutilityofcaspase-3asamarkerfortheidentificationofapoptoticcellsinconjunctivalepithelia.............................................................................................1143.4.4 Validationofthecellline.......................................................................................115

3.5 ANALYSISOFCONJUNCTIVALEPITHELIALPHENOTYPEWITHADVANCINGTIMEONSYNTHETICSUBSTRATESBYFLOWCYTOMETRY.................................................................................116

3.5.1 HCjE-Gicellphenotypewithadvancingtimeincultureonsyntheticsubstrates...116

3.5.2 CelldensityandmorphologyofprimarycellculturesondoublesideammoniaplasmatreatedePTFEandPETmembrane.......................................................................1253.5.3 PhenotypeofprimarycellsexpandedondoublesideplasmatreatedePTFEandPETmembranebyflowcytometry...........................................................................................127

3.6 DECELLULARISATIONOFHUMANCONJUNCTIVAANDITSCHARACTERISATION..................134

3.6.1 DNAquantificationofdecellularisedconjunctiva.................................................1343.6.2 Contactcytotoxicityofdecellularisedconjunctivaltissue.....................................1373.6.3 Tensilestrengthtesting.........................................................................................1393.6.4 CollagenDenaturationAssay................................................................................1423.6.5 Histologyofdecellularisedconjunctiva.................................................................143

3.7 CULTUREOFPRIMARYHUMANCONJUNCTIVALEPITHELIALCELLSONDECELLULARISEDCONJUNCTIVA............................................................................................................145

3.7.1 Cellcultureondecellularisedtissuesubstrateswithprimaryconjunctivalepithelialcellsusingexplantandsuspensioncultures......................................................................1453.7.2 Explantculturewithattentiontoconjunctivalbasementmembraneorientation1473.7.3 Comparisonofconjunctivalepithelialculturesusingtissuefromdifferentdonorsforexplantsanddecellularisedsubstrates........................................................................1483.7.4 Furtherconjunctivalexplantculturesonfreshlydecellularisedtissues................1503.7.5 Characterisationofthecellularphenotypeofconjunctivalepitheliumculturedondecellularisedconjunctiva.................................................................................................151

3.8 IDENTIFICATIONANDCHARACTERISATIONOFBASEMENTMEMBRANESOFHUMANCONJUNCTIVAANDAMNIOTICMEMBRANE..........................................................................................155

3.8.1 CharacterisationofbasementmembranewithPAS.............................................1553.8.2 Characterisationofcellularanddecellularisedtissuewithlaminin,collagenIVandfibronectin.........................................................................................................................158

3.9 CHARACTERISATIONOFPATIENTSWITHOCULARMMP.............................................164

4 DISCUSSION..................................................................................................1684.1 OVERVIEW.......................................................................................................1684.2 AMMONIAGASPLASMATREATMENTINCREASESTHEHYDROPHILICITYOFEPTFE.............1694.3 EXPERIMENTALUSEOFTHEHCJE-GICELLLINE........................................................172

4.3.1 CharacteristicsoftheHCjE-Gicellline..................................................................1724.3.2 SurfacemodifiedePTFEallowshumanconjunctivalcellattachmentandproliferationwithanappropriatecellseedingdensityandcellculturemedia.................173

4.3.2.1 Determinationoftheoptimalcellseedingdensity....................................................................1734.3.2.2 Determinationoftheoptimalculturemedia.............................................................................1744.3.2.3 SummaryoftheoptimisationexperimentsforthecultureofconjunctivalepitheliumonePTFE 178

4.3.3 Consistencyofmarkerexpressionbetweenpassagesanddemonstrationofcaspase-3upregulationinHCjE-Gicells............................................................................178

4.3.3.1 TheHCjE-Gicelllineisconsistentintheexpressionoftherangeoftestedmarkersbetweenpassages2-28..............................................................................................................................................1784.3.3.2 Caspase-3expressionincreasesinresponsetoenvironmentalstress.......................................179

4.4 CULTUREOFHCJE-GIANDPRIMARYCONJUNCTIVALCELLSONAMMONIAPLASMATREATEDEPTFE.....................................................................................................................180

4.4.1 HCjE-GicelldensityisgreateronePTFEsubjectedtoammoniagasplasmaonbothsides 1804.4.2 PrimarycellcultureissimilarondoublesideammoniaplasmatreatedePTFEandPETmembrane..................................................................................................................183

4.5 PHENOTYPEOFCONJUNCTIVALEPITHELIALCULTURESDEVELOPEDONEPTFEANDPETMEMBRANES.............................................................................................................184

4.5.1 Differentialexpressionofcytokeratins,UAE-1lectinandMUC5ACinHCjE-Gicellsandprimaryhumanconjunctivalcells...............................................................................185

4.5.1.1 Expressionofcytokeratin19......................................................................................................1854.5.1.2 Expressionofcytokeratin4........................................................................................................1864.5.1.3 Expressionofcytokeratin7........................................................................................................1884.5.1.4 ExpressionofMUC5AC...............................................................................................................189

4.5.2 Differentialexpressionofmarkersofprogenitorcells,proliferationandapoptosisintheHCjE-Gicelllineandprimaryhumanconjunctivalcells...............................................191

4.5.2.1 ExpressionofΔNp63..................................................................................................................1915.2.2.2 ExpressionofABCG2andco-expressionwithΔNp63................................................................1924.5.2.3 Expressionofcaspase-3.............................................................................................................1944.5.2.4 ExpressionofPCNA....................................................................................................................196

4.6 DECELLULARISEDHUMANCONJUNCTIVA.................................................................197

4.6.1 Decellularisationandcytotoxicityofhumanconjunctiva.....................................1974.6.2 Quantificationofcollagendenaturation...............................................................1994.6.3 Tensilestrengthofconjunctiva,amnioticmembraneandePTFE.........................2004.6.4 Characterisationoftheextracellularmatrixcomponentsandbasementmembraneofcellularanddecellularisedtissues.................................................................................202

4.7 CULTUREOFPRIMARYHUMANCONJUNCTIVALCELLSONDECELLULARISEDCONJUNCTIVAANDAMNIOTICMEMBRANE................................................................................................204

4.7.1 Cellcultureexperimentsandcharacterisationofthedevelopedtissueconstructs 2054.7.2 Limitationsofthestudy........................................................................................208

4.8 CHARACTERISATIONOFPATIENTSWITHMMPANDPOTENTIALOCULARSURFACERECONSTRUCTIONSSTRATEGIES.....................................................................................209

4.8.1 Developmentofaproformatoassessmucousmembranepemphigoidpatients2094.8.2 Characterisationofthepatientexaminedusingthenovelproforma..................2114.8.3 Pilotexercisetodeveloprecommendationsforaproforma................................211

4.9 TREATMENTOFCICATRISINGEYEDISEASEANDTHEPOTENTIALUSEOFTHESUBSTRATESDEVELOPEDINTHISSTUDY............................................................................................214

5. CONCLUSIONS..............................................................................................217

6. FUTUREDIRECTIONS....................................................................................219

7. APPENDIX....................................................................................................2231.THELIVERPOOLCORNEALANDEXTERNALEYEDISEASECLINICPROFORMA...........................2242.ETHICALAPPROVAL.................................................................................................229

8. REFERENCES.................................................................................................232

Listoffigures

FIGURE1:SCHEMATICDRAWINGILLUSTRATINGACROSSSECTIONOFTHEGLOBEANDEYELIDS.TAKENFROMSTEWARTRMK.(2013)IDENTIFICATIONOFPROGENITORRICHSITESINTHECONJUNCTIVA.PHDTHESIS.UNIVERSITYOFLIVERPOOL;ADAPTEDINPARTFROMPAULSENANDBERRY(2006).(2).................................................................................1

FIGURE2:PHOTOMICROGRAPHOFTHENORMALHUMANCORNEA.PARAFFINEMBEDDEDTISSUESECTIONWASSUBJECTEDTOSTAININGWITHHAEMATOXYLINANDEOSIN.SCALEBAR50ΜM.TAKENFROMTAKENFROMSTEWARTRMK.(2013)IDENTIFICATIONOFPROGENITORRICHSITESINTHECONJUNCTIVA.PHDTHESIS....................................................4

FIGURE3:FLOWDIAGRAMTOILLUSTRATETHATTHEMANAGEMENTOFOCULARDISEASEINVOLVESTREATMENTOFTHEDISEASEPROCESSALONGWITHSUPPORTIVETHERAPIESANDRESTORATIVETREATMENT.*DISEASETREATMENTMAYINVOLVEREMOVALOFTHEINCITINGAGENT,ANTIMICROBIALS,IMMUNOSUPPRESSANTSORANTI-INFLAMMATORYAGENTSASDESCRIBEDINEARLIERPARAGRAPHS(SECTION1.2.1)....................................................................................15

FIGURE4:ILLUSTRATIONOFTHESURFACECHEMICALCHANGEINEPTFETHROUGHAMMONIAGASPLASMATREATMENTANDIMMERSIONINDISTILLEDWATER.ENERGYFROMGASPLASMABREAKSSOMEOFTHESURFACECARBONFLUORINEBONDS,WHICHAREREPLACEDBYHYDROXYLFUNCTIONALGROUPSONCONTACTWITHWATER...............................26

FIGURE5:ILLUSTRATIONOFADROPLETONASOLIDSURFACEWITHTHEMEASUREMENTSREQUIREDINYOUNG’SEQUATION.LGDEFINESTHEGAS-LIQUIDINTERFACIALENERGY(SURFACETENSION),SGDEFINESTHESOLID-VAPOURINTERFACIALENERGYANDSLDEFINESTHESOLID-LIQUIDINTERFACIALENERGY.THEEQUILIBRIUMCONTACTANGLECANBEDERIVEDFROMTHERELATIONSHIPBETWEENTHESEVARIABLESTHROUGHYOUNG’SEQUATION:ΓSG-ΓSL-ΓLGCOSΘC=0.(108)..27

FIGURE6:ILLUSTRATIONOFTHEPRINCIPLEOFFLOWCYTOMETRY.AHETEROGENEOUSPOPULATIONOFCELLSLABELLEDWITHFLUORESCENTANTIBODIESANDMARKERSINSOLUTIONISDIRECTEDINTOSINGLEFILEWITHINASTREAMOFFLUID.LASERLIGHTSOURCESEXCITECELLSANDTHELIGHTSCATTERINGCHARACTERISTICSANDEMITTEDFLUORESCENCEISDETECTEDTOENABLEQUANTITATIVEANALYSISOFHETEROGENEOUSCELLPOPULATIONS.(134)...............................................34

FIGURE7:DIAGRAMDEMONSTRATINGTHEMEASUREDAREASFORTHEROWSEYSCORINGSYSTEM.THEDIAGRAMABOVESHOWSTHETHREEMEASUREMENTSTHATARETAKENFROMTHELIMBUSTOTHELIDMARGINAT5,6AND7O’CLOCKFROMTHECORNEALLIMBUS.TAKENFROMROWSEYETAL.ARCHOPHTHALMOL.2004;122:179-184.................39

FIGURE8:APHOTOGRAPHICEXAMPLEOFTHELIVERPOOL-TAUBERGRADINGSYSTEM.TAKENFROMREEVES.G.GRAEFESARCHCLINEXPOPHTHALMOL(2012)250:611–618.ANEXAMPLEOFGRADINGISSHOWNINTHEABOVEPHOTOGRAPHSWITHMEASUREMENTS(MM)ASFOLLOWS:A)VERTICALGRADING(10-5)X10=50%;B)HORIZONTALGRADINGE.G.(27-(6+1+1+4))/27X100=56%.........................................................................................40

FIGURE9:OCULARINFLAMMATIONGRADINGSYSTEMBYSAWETAL.THISGRADINGSYSTEMDEMONSTRATESA5-POINTGRADINGOFOCULARINFLAMMATIONFROM‘MINIMAL’TO‘LIMBITIS’.TAKENFROM:SAW,V.P.DART,J.K.RAUZ,S.ETAL.(2008)IMMUNOSUPPRESSIVETHERAPYFOROCULARMUCOUSMEMBRANEPEMPHIGOIDSTRATEGIESANDOUTCOMES.OPHTHALMOLOGY.115(2):253-261.......................................................................................42

FIGURE10:PHOTOGRAPHSFORTHEGRADINGOFOCULARSURFACEMANIFESTATIONSOFSTEVENS-JOHNSONSYNDROMEASREPORTEDBYSOTOZONOETAL.(2007).THESEIMAGESWERETAKENFROMTHEORIGINALPUBLICATION:SOTOZONOETAL.(2007)NEWGRADINGSYSTEMFORTHEEVALUATIONOFCHRONICOCULARMANIFESTATIONSINPATIENTSWITHSTEVENS-JOHNSONSYNDROME.OPHTHALMOLOGY.114:1294–1302.THECONJUNCTIVALISATION,NEOVASCULARIZATIONANDOPACIFICATIONCOMPONENTSOFTHEGRADINGSYSTEMWEREUSEDINTHEMMPPROFORMA.................................................................................................................................................44

FIGURE12:FIGURETOSHOWTHEOXFORDGRADINGSCHEMEASDESCRIBEDBYBRONETAL.TAKENFROMBRONETAL.(2003)GRADINGOFCORNEALANDCONJUNCTIVALSTAININGINTHECONTEXTOFOTHERDRYEYETESTS.CORNEA.22(7):640-50.....................................................................................................................................46

FIGURE13:PHOTOGRAPHSDEMONSTRATINGTHECELLCROWNCELLCULTUREINSERTSWITHEPTFEMOUNTEDWITHINIT.A)TWOCOMPONENTSOFTHECELLCROWN.ONTHELEFT,THECELLCROWNISSHOWNUPSIDEDOWNWITHTHEEPTFEMEMBRANEPLACEDACROSSIT(SOLIDARROW).THERINGONTHERIGHTHANDSIDE(DASHEDARROW)FITSOVERTHEMEMBRANE,SECURINGITINTOPLACE.B)THISPHOTOGRAPHSHOWSTHEEPTFEMEMBRANEMOUNTEDINTHEBASEOFTHECELLCROWN.EACHOFTHESEWASPLACEDWITHINAWELLOFASTANDARD12-WELLCULTUREPLATEFORCELLCULTUREEXPERIMENTSAFTERSTERILISATION(SECTION2.3.2)........................................................................49

FIGURE14:DIAGRAMTOILLUSTRATETHEPROCESSOFAIRLIFTINGCELLCULTUREINSERTS.THEINSERT(BLUE)CANBEPLACEDINACELLCULTUREWELL(BLACK)ANDISSUPPORTEDSUCHTHATITISSUSPENDEDWITHINTHEWELL.THEMEDIA(YELLOW)ISINSERTEDANDSHOWNHERETOCORRESPONDTOTHEAIR-LIQUIDINTERFACEOFTHECELLULARLAYER(PURPLE).....56

FIGURE15:PHOTOGRAPHSOFTHERINGDEVICEANDTHEPLACEMENTOFTHESERINGDEVICESWITHINCULTUREPLATESTOENABLEAIRLIFTINGOFCULTURES.A)THISRINGSHAPEDDEVICEWASDESIGNEDANDMADEBYUNIVERSITYOFLIVERPOOLWORKSHOPSERVICES(JB).THISWASSIZEDTOENABLETHECELLCROWNTOBEPOSITIONED6MMABOVETHEBASEOFTHEWELLPLATE.MEDIAWASDISPENSEDWITHINTHISGAP,FILLINGTHEWELLTOTHEAIR-LIQUIDINTERFACEFORTHEAIRLIFTINGOFCULTURES.B)THISPHOTOGRAPHSHOWSA12-WELLPLATEWITHTHERINGDEVICEINEACHWELL.THISHOLDSTHECELLCROWNS6MMABOVETHEBASEOFEACHWELL.THESEWEREUSEDFORCELLCULTUREEXPERIMENTSINVOLVINGTHEEPTFESUBSTRATES...........................................................................................................57

FIGURE16:PHOTOGRAPHOFTHEPERSPEXFORNIXRULER.EACHGRADATIONCORRESPONDSTO1MM.THEGRADEDSECTIONISINSERTEDINTOTHEUPPERANDLOWERFORNICES.THETHICKNESSOFTHEPERSPEXMEASURINGARMOFTHEFORNIXRULERIS1MM.......................................................................................................................................75

FIGURE17:HISTOGRAMTOSHOWNUMBEROFCELLSCOUNTEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEANDINCREASINGCELLSEEDINGDENSITYONAMMONIAPLASMATREATEDEPTFE.THISGRAPHSHOWSTHENUMBEROFCELLSCOUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELD(+/-SD)AT20XMAGNIFICATIONOVERSETTIMEPOINTS(DAY1,4AND7).EACHPHOTOGRAPHEDFIELDWAS12,420ΜM

2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDFROMTRIPLICATESAMPLES(N=15WITHINEACHEXPERIMENTALGROUP)...................................................................78

FIGURE18:HISTOGRAMTOSHOWNUMBEROFCELLSCOUNTEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEANDINCREASINGCELLSEEDINGDENSITYONPETMEMBRANE.THISGRAPHSHOWSTHENUMBEROFCELLSCOUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELD(+/-SD)AT20XMAGNIFICATIONOVERSETTIMEPOINTS(DAY1,4AND7).EACHPHOTOGRAPHEDFIELDWAS12,420ΜM

FIGURE19:HISTOGRAMTOSHOWNUMBEROFCELLSCOUNTEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEANDINCREASINGCELLSEEDINGDENSITYONUNTREATEDEPTFE.THISGRAPHSHOWSTHENUMBEROFCELLSCOUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELD(+/-SD)AT20XMAGNIFICATIONOVERSETTIMEPOINTS(DAY1,4AND7).EACHPHOTOGRAPHEDFIELDWAS12,420ΜM

FIGURE20:REPRESENTATIVEPHOTOMICROGRAPHSOFCULTUREDSUBSTRATESFIXEDANDSTAINEDWITHDAPI(BLUEFLUORESCENTNUCLEARSTAIN)AFTER7DAYSINCULTURE.ALLMEMBRANESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)PETMEMBRANEC)UNTREATEDEPTFE.SCALEBARS100ΜM............................................................................................................................................................81

FIGURE21:HISTOGRAMTOSHOWMEANNUMBEROFCELLSCOUNTEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETMEMBRANEUSINGMEDIAPROTOCOLA.CELLSWERESEEDEDAT1X105/CM2

ONALLSUBSTRATES.THISGRAPHSHOWSTHENUMBEROFCELLS(+/-SD)COUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELDAT20XMAGNIFICATIONOVERSETTIMEPOINTSDAY1,3,7,10AND14.EACHPHOTOGRAPHEDFIELDWAS12,420ΜM

2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDINTRIPLICATESAMPLES(N=15WITHINEACHGROUP).......................................................................................................83

FIGURE22:REPRESENTATIVEPHOTOMICROGRAPHSOFDAPISTAINEDCELLSAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLA.ALLSUBSTRATESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)UNTREATEDEPTFEC)PET.SCALEBARS100ΜM............................................................................83

FIGURE23:HISTOGRAMTOSHOWTHEMEANNUMBEROFCELLPHOTOGRAPHEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETMEMBRANEUSINGMEDIAPROTOCOLB.CELLSWERESEEDEDAT1X105/CM2

FIGURE24:REPRESENTATIVEPHOTOMICROGRAPHSOFCULTUREDSUBSTRATESFIXEDANDSTAINEDWITHDAPIAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLB.ALLSUBSTRATESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)UNTREATEDEPTFEC)PET.SCALEBARS100ΜM.....................................84

FIGURE25:HISTOGRAMTOSHOWTHEMEANNUMBEROFCELLPHOTOGRAPHEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETMEMBRANEUSINGMEDIAPROTOCOLC.CELLSWERESEEDEDAT1X105/CM2

ONALLSUBSTRATES.THISGRAPHSHOWSTHENUMBEROFCELLS(+/-SD)COUNTED

MANUALLYINEACHPHOTOGRAPHEDFIELDAT20XMAGNIFICATIONOVERSETTIMEPOINTSDAY1,3,7,10AND14.EACHPHOTOGRAPHEDFIELDWAS12,420ΜM

FIGURE26:REPRESENTATIVEPHOTOMICROGRAPHSOFCULTUREDSUBSTRATESFIXEDANDSTAINEDWITHDAPIAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLC.ALLSUBSTRATESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)UNTREATEDEPTFEC)PET.SCALEBARS100ΜM.....................................85

FIGURE27:HISTOGRAMTOSHOWTHEMEANNUMBEROFCELLPHOTOGRAPHEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETMEMBRANEUSINGMEDIAPROTOCOLD.CELLSWERESEEDEDAT1X105/CM2

2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDINTRIPLICATE(N=15SAMPLESWITHINEACHGROUP).......................................................................................................86

FIGURE28:REPRESENTATIVEPHOTOMICROGRAPHSOFCULTUREDSUBSTRATESFIXEDANDSTAINEDWITHDAPIAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLD.ALLSUBSTRATESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)UNTREATEDEPTFEC)PET.SCALEBARS100ΜM.....................................86

FIGURE29:REPRESENTATIVEPHOTOMICROGRAPHSOFHCJE-GICELLSAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLBONAMMONIAPLASMATREATEDEPTFE(A),UNTREATEDEPTFE(B)ANDPETMEMBRANE(C).GREENSTAININGISPHALLOIDIN(F-ACTIN)ANDBLUENUCLEARSTAININGISTHERESULTOFDAPIUPTAKE.CONFLUENTMORPHOLOGYWASDEMONSTRATEDONBOTHTREATEDEPTFEANDPETANDSPARSEGROWTHFOUNDONUNTREATEDEPTFE.PHALLOIDINSTAININGWASABUNDANTHOWEVERINDIVIDUALFIBRESWEREDIFFICULTTOVISUALISEWITHTHISSTAINONPETMEMBRANE.NUCLEIAPPEARSMALLERINSIZEONTHEPETCOMPAREDWITHTREATEDEPTFESUBSTRATES.SCALEBARS50ΜM.................................................................................................................................................87

FIGURE30:CELLDENSITYOFHCJE-GICELLSGROWNONAMMONIAPLASMATREATEDEPTFE,PETMEMBRANEANDUNTREATEDEPTFEWITHADVANCINGTIME.CELLSWERECULTUREDUSINGMEDIAPROTOCOLBSEEDEDAT1X105/CM2.DATAHASBEENLOGTRANSFORMEDTOALLOWPARAMETRICSTATISTICALANALYSISBYANOVA.THEOVERALLMODELWASSIGNIFICANTP<0.001FORTHEEFFECTOFBOTHTIMEPOINTANDSUBSTRATE.BONFERRONIPOST-HOCTESTSOFTHEDIFFERENCEBETWEENPAIRS(BOTHTIMEPOINTSANSUBSTRATE)INANYCOMBINATIONWEREALSOHIGHLYSIGNIFICANT;P<0.001............................................................................................................................88

FIGURE31:REPRESENTATIVEPHOTOMICROGRAPHSOFNUCLEARANDF-ACTINSTAININGOFHCJE-GICELLSCULTUREDONAMMONIAPLASMATREATEDEPTFE,PETANDUNTREATEDEPTFEAFTER2DAYSINCULTURE.CELLSIZEAPPEARSSMALLERONPETMEMBRANESHOWEVERCELLDENSITYAPPEAREDGREATEST.LOWESTCELLDENSITYWASAPPARENTONUNTREATEDEPTFEWITHCELLSMORE‘ROUNDED’INAPPEARANCETHANONAMMONIAPLASMATREATEDEPTFESUBSTRATES.SCALEBARS50ΜM...............................................................................................................90

FIGURE32:REPRESENTATIVEPHOTOMICROGRAPHSOFNUCLEARANDF-ACTINSTAININGOFHCJE-GICELLSCULTUREDONAMMONIAPLASMATREATEDEPTFE,PETANDUNTREATEDEPTFEAFTER14DAYSINCULTURE.CELLSIZEAPPEAREDTHESMALLESTWITHGREATESTDENSITYONPETMEMBRANE.SIMILARMORPHOLOGYWASAPPARENTONTREATEDEPTFEANDPETMEMBRANE.LOWESTCELLDENSITYWITH‘ROUNDED’CELLSWASAPPARENTONUNTREATEDEPTFE.CULTURESWEREMORECONFLUENTONDOUBLE-SIDETREATEDEPTFETHANSINGLESIDETREATEDEPTFE.SCALEBARS50ΜM.................................................................................................................................................91

FIGURE33:REPRESENTATIVEPHOTOMICROGRAPHSOFNUCLEARANDF-ACTINSTAININGOFHCJE-GICELLSCULTUREDONAMMONIAPLASMATREATEDEPTFE,PETANDUNTREATEDEPTFEAFTER21DAYSINCULTURE.LOWESTCELLDENSITYWASAPPARENTONUNTREATEDEPTFEHOWEVERTHEREISGREATERVARIATIONINTHESIZEANDSHAPEOFTHENUCLEARMATERIALTHANONANYOTHERSUBSTRATE.CULTURESWEREMORECONFLUENTONDOUBLESIDETREATEDEPTFETHANFOLLOWINGSINGLESIDE-TREATMENTANDCELLSAPPEARTOHAVEMOREOFCOBBLESTONEMORPHOLOGYTHANONOTHERSUBSTRATES.SCALEBARS50ΜM........................................................................................92

FIGURE34:REPRESENTATIVECONFOCALZ-STACKSERIESOFSINGLESIDEAMMONIAPLASMATREATEDEPTFEAFTER28DAYSOFHCJE-GICELLCULTURE:A)PHALLOIDIN(F-ACTINSTAINING)(B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM................93

FIGURE35:REPRESENTATIVECONFOCALZ-STACKSERIESOFDOUBLESIDEAMMONIAPLASMATREATEDEPTFEAFTER28DAYSOFHCJE-GICELLCULTURE:A)PHALLOIDIN(F-ACTINSTAINING)(B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM................94

FIGURE36:REPRESENTATIVECONFOCALZ-STACKSERIESOFPETMEMBRANEAFTER28DAYSOFHCJE-GICELLCULTURE:A)PHALLOIDIN(F-ACTINSTAINING)(B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM...................................................95

FIGURE37:REPRESENTATIVECONFOCALZ-STACKSERIESOFUNTREATEDEPTFEAFTER28DAYSOFHCJE-GICELLCULTURE:A)PHALLOIDIN(F-ACTINSTAINING)(B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM...................................................96

FIGURE38:REPRESENTATIVEPHOTOMICROGRAPHSOFNUCLEARANDUAE-1STAININGOFHCJE-GICELLSCULTUREDONAMMONIAPLASMATREATEDEPTFE,PETANDUNTREATEDEPTFEAFTER2DAYSINCULTURE.LOWESTCELLDENSITYWASAPPARENTONUNTREATEDEPTFE.OVERALLGREATERUAE-1(INTRACELLULARANDMEMBRANEASSOCIATED)STAININGWASAPPARENTONCELLSCULTUREDONEPTFETHANPETMEMBRANEINWHICHSTAININGAPPEAREDMOREDISCRETEANDMOSTLYMEMBRANEASSOCIATED.SCALEBARS50ΜM...............................................................97

FIGURE39:REPRESENTATIVEPHOTOMICROGRAPHSOFNUCLEARANDUAE-1STAININGOFHCJE-GICELLSCULTUREDONAMMONIAPLASMATREATEDEPTFE,PETANDUNTREATEDEPTFEAFTER14DAYSINCULTURE.LOWESTCELLDENSITYWASAPPARENTONUNTREATEDEPTFE.THEGREATESTINTENSITYOFUAE-1STAININGAPPEAREDONPETMEMBRANEANDDOUBLESIDETREATEDEPTFE.STAININGOFCELLMEMBRANESSHOWEDTHECELLSIZEWASGREATERONEPTFECELLCULTURESTHANPETCELLCULTURES.SCALEBARS50ΜM.......................................................................98

FIGURE40:REPRESENTATIVEPHOTOMICROGRAPHSOFNUCLEARANDUAE-1STAININGOFHCJE-GICELLSCULTUREDONAMMONIAPLASMATREATEDEPTFE,PETANDUNTREATEDEPTFEAFTER21DAYSINCULTURE.LOWESTCELLDENSITYWASDEMONSTRATEDONUNTREATEDEPTFE,WHEREASTHEGREATESTDENSITYWASDEMONSTRATEDONPETMEMBRANEANDDOUBLESIDETREATEDEPTFE.CELLSIZEWASGREATERONEPTFECELLCULTURESTHANPETCELLCULTURES,ANDWASMOREPRONOUNCEDONDOUBLESIDETREATEDEPTFE,ESPECIALLYAMONGSTTHECELLSWITHGREATERINTRACELLULARSTAINING.SCALEBARS50ΜM................................................................................99

FIGURE41:REPRESENTATIVECONFOCALZ-STACKSERIESOFSINGLESIDETREATEDEPTFEAFTER28DAYSOFHCJE-GICELLCULTURE:A)UAE-1LECTINSTAINING,B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM.......................................100

FIGURE42:REPRESENTATIVECONFOCALZ-STACKSERIESOFDOUBLESIDETREATEDEPTFEAFTER28DAYSOFHCJE-GICELLCULTURE:A)UAE-1LECTINSTAINING,B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM.......................................101

FIGURE43:REPRESENTATIVECONFOCALZ-STACKSERIESOFPETMEMBRANEAFTER28DAYSOFHCJE-GICELLCULTURE:A)UAE-1LECTINSTAINING,B)DAPI(NUCLEAR)STAINING.FIVESLICESHAVEBEENTAKEN,EACHIN4.5-5ΜMINTERVALSWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM..............................................................102

FIGURE44:REPRESENTATIVECONFOCALZ-STACKSERIESOFUNTREATEDEPTFEAFTER28DAYSOFHCJE-GICELLCULTURE:A)UAE-1LECTINSTAINING,B)DAPI(NUCLEAR)STAINING.FOURSLICESHAVEBEENTAKEN,EACHAFTER3ΜMWHEREBYTHETOPANDBOTTOMOFTHEZ-STACKHADBEENMANUALLYSETAFTERTHEFOCUSPOINTSATTHETOPANDBOTTOMOFTHECELLCULTURESWERELOCATED.SCALEBARS50ΜM..............................................................................103

FIGURE45:FLUORESCENCECHANNELHISTOGRAMSOFHCJE-GICELLSDEMONSTRATINGTHERESULTINGFLUORESCENCEAFTERSTAININGWITHVARIOUSCONCENTRATIONSOFUAE-1LECTINOVER30(B)AND60(C)MINUTES.THEDASHEDLINEONALLTHEHISTOGRAMSANDARROWONTHEHISTOGRAMOFTHEUNSTAINEDCONTROLSAMPLE(A)INDICATESTHEFLUORESCENCEBEYONDWHICHSTAININGWASREGARDEDASPOSITIVE.THESEHISTOGRAMSDEMONSTRATETHATTHEREWASLITTLEEFFECTOFINCUBATIONTIMEANDTHEREFORE30MINUTESWASSUFFICIENT.ATALLTHEANTIBODYDILUTIONSSTUDIED,THEREWASMARKEDSEPARATIONOFTHEHISTOGRAMFROMANUNSTAINED(CONTROL)SAMPLEOFCELLS,HOWEVER,THE1:500DILUTIONWASOPTIMAL.................................................................................106

FIGURE46:THEHISTOGRAMSABOVESHOWTHERESULTSATTHEOPTIMALDETERMINEDDILUTIONSOFTHEPRIMARYΔNP63ANTIBODYANDSECONDARYANTIBODY(1:50PRIMARYAND1:1000SECONDARY)COMPAREDWITHANISOTYPECONTROLANDTHESECONDARYANTIBODYINISOLATION.OTHERVARIABLESTESTEDBUTNOTDISPLAYEDABOVEINCLUDED:PRIMARYANTIBODYDILUTIONS1:100,1:250:INALLCOMBINATIONSWITHSECONDARYANTIBODYDILUTIONS1:100,1:500,1:1500.THEPOSITIVELYSTAINEDPEAKWASDETERMINEDFROMPRIMARYANDSECONDARYANTIBODYCOMBINATIONS,WHICHEXCEEDEDTHEFLUORESCENCEOFBOTHTHEISOTYPECONTROLANDSECONDARY

ANTIBODYINCOMBINATIONANDTOTHESECONDARYANTIBODYALONEATTHESAMEDILUTIONS.IMPORTANTLY,THESHIFTINTHEHISTOGRAMCURVEWASSUCHTHATTHEREWASMINIMALOVERLAPWITHTHEHISTOGRAMCURVEOFTHECONTROLSAMPLESDESCRIBED.THEDOTTEDLINESANDARROWSINDICATETHEAREAOFHISTOGRAMFROMWHICHALOGICAL‘GATE’WASPLACEDTODETERMINETHEPERCENTAGEOFCELLSTHATWEREDEEMEDPOSITIVEFORΔNP63EXPRESSION.........................................................................................................................................107

FIGURE47:DOTPLOTOFSIDESCATTER-HEIGHT(SSC-H)AGAINSTFORWARDSCATTERHEIGHT(FSC-H)OFHCJE-GI(A)ANDPRIMARYCONJUNCTIVALCELLS(B).THEDOTSOCCURRINGATTHEBOTTOMLEFTOFTHEPLOT(LOWFSC-HANDSSC-H)AREPARTICULATES/DEBRIS.....................................................................................................................108

FIGURE48(DISPLAYEDBELOWANDOVERPAGES110-114):HISTOGRAMSTOSHOWTHEFLUORESCENCEDETECTEDINPRIMARYCONJUNCTIVALEPITHELIALCELLSANDHCJE-GICELLSAFTERSTAININGASDESCRIBEDINSECTION2.4.6:A)CK19,B)CK4,C)CK7,D)UAE-1LECTIN,E)MUC5AC,F)PCNA,G)CASPASE-3,H)ΔNP63,I)ABCG2.THEDOTTEDLINESANDARROWSINDICATETHEREGIONTHE‘GATES’WERESETSUCHTHATCELLSEMITTINGFLUORESCENCEABOVETHATDETECTEDINTHERELEVANTISOTYPECONTROLSWEREDEEMEDPOSITIVELYSTAINED.THEPERCENTAGEOFTHECELLSPOSITIVELYTHATWERESTAINEDFORTHEANTIGENOFINTERESTWERESUBSEQUENTLYDETERMINEDUSINGANANALYTICALSOFTWARETOOLFORFLOWCYTOMETRYDATA(FLOWING2.5).....................................................109

FIGURE49:HISTOGRAMSOFFLUORESCENCEDETECTEDFOLLOWINGSTAININGWITHCASPASE-3INHEALTHYANDDEPRIVEDCULTURES.H2INDICATESTHE‘GATES’SETUSINGAFLOWCYTOMETRYANALYSISSOFTWAREPROGRAMSUCHTHATCELLSEMITTINGFLUORESCENCELEVELSABOVETHATDETECTEDINTHEISOTYPECONTROLS(INTHERANGEINDICATEDBYH2)WEREREGARDEDASPOSITIVELYSTAINED.THISENABLEDTHEQUANTIFICATIONOFTHECELLPOPULATIONINTERMSOFTHEPERCENTAGEOFTHECELLSPRESENTINTHISGATEDREGION.....................................................................114

FIGURE50:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK4ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATES;P<0.0001;TIMEP=0.152.ERRORBARS+/-SD................................................................................................................118

FIGURE51:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK7ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATESANDTIME;P<0.001.ERRORBARS+/-SD..............................................................................................................................119

FIGURE52:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK19ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATESP=0.975;TIMEP=0.88.ERRORBARS+/-SD..................................................................................................................119

FIGURE53:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFMUC5ACONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATEP=0.033;TIMEP<0.0001.ERRORBARS+/-SD.......................................................................................................120

FIGURE54:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFUAE-1LECTINONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATEP=0.001;TIME:P<0.641.ERRORBARS+/-SD........................................................................................................120

FIGURE55:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK7ANDUAE-1LECTINCO-EXPRESSIONONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATEANDTIMEPOINT:P<0.0001.ERRORBARS+/-SD............................................................121

FIGURE56:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFΔNP63ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATESP=0.007;TIMEP=0.096.ERRORBARS+/-SD...............................................................................................................121

FIGURE57:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFABCG2ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATESP=0.003;TIMEP=0.137.ERRORBARS+/-SD................................................................................................................122

FIGURE58:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFABCG2ANDΔNP63ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATESP=0.001;TIMEP=0.008.ERRORBARS+/-SD..........................................................................................122

FIGURE59:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCASPASE-3ONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELSUBSTRATESP=<0.0001;TIMEP=0.163.ERRORBARS+/-SD......................................................................................123

FIGURE60:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFPCNAONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETAFTER14AND28DAYSINCULTURE.ANCOVAOVERALLMODELTIMEP=0.024;SUBSTRATESP=0.146.ERRORBARS+/-SD................................................................................................................123

FIGURE61:HISTOGRAMTOSHOWNUMBEROFCELLSPERCM2WITHADVANCINGTIMEINCULTUREONDOUBLESIDEAMMONIA

PLASMATREATEDEPTFE(BOTHSIDESEXPOSEDTOPLASMA)ANDPETMEMBRANE.PRIMARYCELLSUSEDINTHISEXPERIMENTWEREFROMASINGLEDONORANDWERESEEDEDAT1X105/CM2.SCALEBARS+/-SD.....................126

FIGURE62:REPRESENTATIVEPHOTOMICROGRAPHSOFPRIMARYCONJUNCTIVALCELLSONDOUBLESIDEAMMONIAPLASMATREATEDEPTFE(D)ANDPET(P)MEMBRANEATAFTER14AND28DAYSINCULTURE.SCALEBARS50ΜM.PHALLOIDIN(F-ACTINSTAINING):RED.DAPI(NUCLEARSTAINING):BLUE........................................................127

FIGURE63:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK19INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.079;SUBSTRATEP=0.123.ERRORBARS+/-SD................................................................................................129

FIGURE64:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK4INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIME0.52;SUBSTRATE0.753.ERRORBARS+/-SD....................................................................................................................129

FIGURE65:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCK7INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIME0.647;SUBSTRATEP=0.25.ERRORBARS+/-SD.ERRORBARS+/-SD......................................................................................130

FIGURE66:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFUAE-1LECTININPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIME0.39;SUBSTRATEP=0.712.ERRORBARS+/-SD...............................................................................................................130

FIGURE67:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFUAE-1LECTINANDCK7INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.505;SUBSTRATEP=0.263.ERRORBARS+/-SD.................................................................................131

FIGURE68:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFMUC5ACINPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.124;SUBSTRATEP=0.456.ERRORBARS+/-SD................................................................................................131

FIGURE69:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFΔNP63INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.945;SUBSTRATE0.537.ERRORBARS+/-SD....................................................................................................132

FIGURE70:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFABCG2INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.753;SUBSTRATEP=0.611.ERRORBARS+/-SD................................................................................................132

FIGURE71:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFΔNP63ANDABCG2INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.328;SUBSTRATEP=0.787.ERRORBARS+/-SD.................................................................................133

FIGURE72:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFCASPASE-3INPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.138;SUBSTRATEP=0.134.ERRORBARS+/-SD................................................................................................133

FIGURE73:HISTOGRAMTOSHOWPERCENTAGEEXPRESSIONOFPCNAINPRIMARYCELLSCULTUREDONDOUBLESIDEAMMONIAPLASMATREATEDEPTFEANDPETMEMBRANEAFTER14AND28DAYS.ANCOVATIMEP=0.003;SUBSTRATEP=0.04.ERRORBARS+/-SD..................................................................................................134

FIGURE74:STANDARDCURVEOFFLUORESCENCEABSORBANCEVALUESWITHINCREASINGDNACONCENTRATIONINCONTROLSAMPLES(KNOWNDILUTIONSOFCALFTHYMUSDNA).THESTANDARDCURVESHOWNENABLEDCALCULATIONOFTHEGRADIENTANDYINTERCEPTINTHEEQUATIONY=MX+CTODETERMINETHEDNACONTENTINEXPERIMENTALSAMPLES..........................................................................................................................................................135

FIGURE75:REPRESENTATIVEPHOTOMICROGRAPHSOFDEPARRAFINISEDTISSUESECTIONSSTAINEDWITHDAPI.BRIGHTFLUORESCENTBLUESTAININGINDICATESTHEPRESENCEOFDNA/NUCLEIA)CELLULARCONJUNCTIVALCELLULARTISSUE:B)CONJUNCTIVALTISSUEDECELLULARISEDWITH0.05%SDS(W/V)..............................................................137

FIGURE76:REPRESENTATIVEPHOTOMICROGRAPHSOFFIXEDGIEMSASTAINEDCELLSFROMAHUMANCONJUNCTIVALCELLLINE(HCJE-GICELLS)ANDPRIMARYHUMANSKINFIBROBLASTSINCULTUREWITHDECELLULARISEDCONJUNCTIVA,STERI-STRIPS

TMANDCYANOACRYLATEGLUEAFTER48HOURS.A)DECELLULARISEDCONJUNCTIVAWITHCELLSSEENIN

CONTACTWITHTISSUEB)STERI-STRIPSTM,EXPERIMENTALNEGATIVECONTROLKNOWNNOTTOEXHIBITCYTOTOXICITY,SEENHEREWITHCELLSVISIBLEINCONTACTC)CYANOACRYLATEGLUE,EXPERIMENTALPOSITIVECONTROLSHOWINGCYTOTOXICITYWITHCELLULARDEBRISOFHCJE-GICELLSSURROUNDITANDALARGEZONEONINHIBITIONAPPARENTWITHCELLULARDEBRIS.SCALEBARS200µM............................................................................................138

FIGURE77:REPRESENTATIVEHISTOGRAMSOFLOAD(N)AGAINSTADVANCINGTIME(SECONDS)DURINGTHETENSILESTRENGTHTEST:A)CONJUNCTIVAB)AMNIOTICMEMBRANEC)EPTFE.EACHHISTOGRAMALSODEMONSTRATESTHESECTIONOFTHECURVEFROMWHICHTHEGRADIENTFORTHEGREATESTSLOPEWASDETERMINED(SEE‘GREATESTSLOPE’MARKEDONEACHGRAPHBYTHEREDARROWS)................................................................................141

FIGURE78:STANDARDCURVEOFCOLORIMETRICABSORBANCEVALUESWITHINCREASINGHYDROXYPROLINECONCENTRATIONFROMKNOWNCONCENTRATIONSOFTHECONTROLSTANDARD.THESTANDARDCURVESHOWNENABLEDCALCULATIONOFTHEGRADIENTANDYINTERCEPTINTHEEQUATIONY=MX+CTODETERMINETHEHYDROXYPROLINECONTENTOFEACHOFTHEEXPERIMENTALSAMPLES..............................................................................................................142

FIGURE79:REPRESENTATIVEPHOTOMICROGRAPHSOFHAEMATOXYLINANDEOSINSTAINEDPARAFFINEMBEDDEDTISSUESECTIONS:A)DECELLULARISEDTISSUE;B)CELLULARTISSUE.SCALEBARS200µM..............................................144

FIGURE80:REPRESENTATIVEPHOTOMICROGRAPHSOFCONJUNCTIVATISSUESTAINEDWITHVANGIESON’SSTAIN:A)DECELLULARISEDTISSUE;B)CELLULARTISSUE.SCALEBARS100µM................................................................144

FIGURE81:REPRESENTATIVEPHOTOMICROGRAPHSOFDECELLULARISEDAMNIOTICMEMBRANEANDCONJUNCTIVALTISSUESECTIONSRECELLULARISEDWITHCONJUNCTIVALEPITHELIALCELLS.GREATERCELLDENSITYWASEVIDENTQUALITATIVELYFOLLOWINGEXPLANTCULTURE(BANDD)THANTHATFOLLOWINGSUSPENSIONCULTURE(AANDC).SCALEBARS100µM.ARROWSPOINTTOPURPLENUCLEIINCELLS..................................................................................146

FIGURE82:REPRESENTATIVEPHOTOMICROGRAPHSOFEXPLANTCULTURESGROWNONDECELLULARISEDTISSUE:A)BASEMENTMEMBRANENOTPRESENTB)BASEMENTMEMBRANEPRESENT.ARROWSAREPOINTINGTONUCLEI(PURPLE).SCALEBARS100µM......................................................................................................................................147

FIGURE83:REPRESENTATIVEH&ESTAINEDPHOTOMICROGRAPHSOFEXPLANTSFROMDONOR15CULTUREDONDECELLULARISEDCONJUNCTIVAFROMTISSUEDONORS9/5/13.THEPHOTOMICROGRAPHOFDECELLULARISEDTISSUEDONOR5HASCAPTUREDTHEPRESENCEOFEXPLANT(SOLIDARROW)WITHCONJUNCTIVALEPITHELIUMTHATHADDEVELOPEDONTHELEFTHANDSIDEOFTHISPHOTOMICROGRAPH(DASHEDARROW).INCONTRASTMINIMALANDNONUCLEIWEREVISIBLEINPHOTOSFROMDECELLULARISEDTISSUEDONORS9AND13.SCALEBARS200µM..............148

FIGURE84:REPRESENTATIVEH&ESTAINEDPHOTOMICROGRAPHSOFEXPLANTSFROMDONOR17CULTUREDONDECELLULARISEDCONJUNCTIVAFROMTISSUEDONORS9/5/13.NOCELLSAREEVIDENTFROMONTISSUESECTIONSDERIVEDFROMDECELLULARISEDTISSUEDONOR13.OCCASIONALAREASOFEPITHELIALGROWTHWEREDEMONSTRATEDONDECELLULARISEDTISSUESFROMDONOR9AND5.INTHECENTREPHOTOFROMDECELLULARISEDTISSUEDONOR5ANEXPLANT(SOLIDARROW)CANBESEENWITHCELLULAROUTGROWTH(DASHEDARROW).SCALEBARS200µM........149

FIGURE85:REPRESENTATIVEH&ESTAINEDPHOTOMICROGRAPHSOFEXPLANTSFROMDONOR19CULTUREDONDECELLULARISEDCONJUNCTIVAFROMTISSUEDONORS9/5/13.NOCELLSWEREEVIDENTFROMONTISSUESECTIONSDERIVEDFROMDECELLULARISEDTISSUEDONORS9OR5(DONORTISSUE5SHOWSONLYTHEEXPLANTWITHNOOUTGROWTH;SOLIDARROW).CELLULARGROWTHWASOBSERVEDINMOSTTISSUESECTIONSDERIVEDFROMEXPLANTCULTUREONDECELLULARISEDTISSUEDONOR13(DASHEDARROW).SCALEBARS200µM...................................149

FIGURE86:REPRESENTATIVEH&ESTAINEDPHOTOMICROGRAPHSOFEXPLANTSFROMDONORS15/17/19CULTUREDONADECELLULARISEDAMNIOTICMEMBRANESUBSTRATE(SAMEDONOR).CELLULARGROWTHISEVIDENTFROMALLTHEEXPLANTS;HOWEVER,QUALITATIVELYTHEGREATESTDENSITYOFCELLSWASEVIDENTFROMEXPLANTSDERIVEDFROMDONOR19.SCALEBARS200µM.............................................................................................................150

FIGURE87:REPRESENTATIVEPHOTOMICROGRAPHSOFSTAINEDTISSUESECTIONSFOLLOWINGCONJUNCTIVALEXPLANTCULTUREUSINGDONOR23(A)AND25(B)ONDECELLULARISEDCONJUNCTIVA(FROMDONOR21)ANDAMNIOTICMEMBRANE.STRATIFIEDCELLSWEREDEMONSTRATEDONDECELLULARISEDCONJUNCTIVAINCONTRASTTOMONOLAYERFORMATIONONAMNIOTICMEMBRANE.QUALITATIVELY,THECELLDENSITYANDMORPHOLOGYOFTHEEPITHELIUMAPPEAREDSIMILARBETWEENTHEDONORS(23AND25)ONBOTHTISSUESUBSTRATES.SCALEBARS100ΜM.........151

FIGURE88:REPRESENTATIVEPHOTOMICROGRAPHSOFTISSUESECTIONSOFCONJUNCTIVALTISSUE(A)MOUSEIGGISOTYPECONTROL,(B)RABBITIGGISOTYPECONTROLSTAINEDWITHMAYERSHAEMATOXYLINONLY.THESEREPRESENTATIVEIMAGESDISPLAYTHELEVELOFBACKGROUNDSTAININGDETECTEDFORIMMUNOHISTOCHEMISTRYSAMPLESINTHISSECTIONANDARETHENEGATIVECONTROLS.SCALEBARS100ΜM.................................................................152

FIGURE89:REPRESENTATIVEPHOTOMICROGRAPHSOFIMMUNOHISTOCHEMICALSTAININGOFTISSUESECTIONSOFDECELLULARISEDCONJUNCTIVA21RECELLULARISEDUSINGEXPLANTSFROMDONOR23.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFTHERESPECTIVEMARKERSTUDIED.SCALEBARS100µM.............153

FIGURE90:REPRESENTATIVEPHOTOMICROGRAPHSOFIMMUNOHISTOCHEMICALSTAININGOFTISSUESECTIONSOFDECELLULARISEDCONJUNCTIVA21RECELLULARISEDUSINGEXPLANTSFROMDONOR23.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFTHERESPECTIVEMARKERSTUDIED.SCALEBARS100µM.............154

FIGURE91:REPRESENTATIVEPHOTOMICROGRAPHSTISSUESECTIONSOFCELLULAR(A)ANDDECELLULARISEDAMNIOTICMEMBRANE(B)FROMTHREETISSUEDONORSSTAINEDWITHPAS.THEBASEMENTMEMBRANETISSUESAPPEARSTAINEDANDSIMILARBETWEENCELLULARANDDECELLULARISEDTISSUESECTIONSSUGGESTINGITWASPRESERVEDFOLLOWINGDECELLULARISATION.SCALEBARS100µM................................................................................................156

FIGURE92:REPRESENTATIVEPHOTOMICROGRAPHSTISSUESECTIONSOFCELLULAR(A)ANDDECELLULARISEDCONJUNCTIVA(B)FROMTHREETISSUEDONORSSTAINEDWITHPAS.THEBASEMENTMEMBRANEPASSTAININGINALLTHECELLULARANDDECELLULARISEDTISSUESAPPEAREDSIMILARBETWEENTISSUESECTIONSSUGGESTINGITWASPRESERVEDFOLLOWINGDECELLULARISATION.SCALEBARS100µM.................................................................................................157

FIGURE93:REPRESENTATIVEPHOTOMICROGRAPHSOFTISSUESECTIONSOFCONJUNCTIVALTISSUE(A)ANDAMNIOTICMEMBRANE(B)INCUBATEDWITHISOTYPECONTROLSANDSTAINEDWITHMAYER’SHAEMATOXYLINONLY.THESEREPRESENTATIVEIMAGESDISPLAYTHELEVELOFBACKGROUNDSTAININGDETECTEDFORIMMUNOHISTOCHEMISTRYSAMPLESINTHISSECTION.ALLPRIMARYANTIBODIESWERERAISEDINRABBIT.SCALEBARS100ΜM.....................158

FIGURE94:REPRESENTATIVEPHOTOMICROGRAPHSOFCELLULAR(A)ANDDECELLULARISED(B)AMNIOTICMEMBRANETISSUESECTIONSFROMTHREESEPARATEDONORSEXAMINEDFORLAMININBYIMMUNOHISTOCHEMISTRY.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFLAMININ.SCALEBARS100ΜM.................................159

FIGURE95:REPRESENTATIVEPHOTOMICROGRAPHSOFCELLULAR(A)ANDDECELLULARISED(B)AMNIOTICMEMBRANETISSUESECTIONSFROMTHREESEPARATEDONORSEXAMINEDFORLAMININBYIMMUNOHISTOCHEMISTRY.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFFIBRONECTIN.SCALEBARS100ΜM...........................160

FIGURE96:REPRESENTATIVEPHOTOMICROGRAPHSOFCELLULAR(A)ANDDECELLULARISED(B)AMNIOTICMEMBRANETISSUESECTIONSFROMTHREESEPARATEDONORSEXAMINEDFORCOLLAGENIVBYIMMUNOHISTOCHEMISTRY.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFCOLLAGENIV.SCALEBARS100ΜM...........................161

FIGURE97:REPRESENTATIVEPHOTOMICROGRAPHSOFCELLULAR(A)ANDDECELLULARISED(B)CONJUNCTIVALTISSUESECTIONSFROMTHREESEPARATEDONORSEXAMINEDFORLAMININBYIMMUNOHISTOCHEMISTRY.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFLAMININ.SCALEBARS100ΜM.................................162

FIGURE98:REPRESENTATIVEPHOTOMICROGRAPHSOFCELLULAR(A)ANDDECELLULARISED(B)CONJUNCTIVALTISSUESECTIONSFROMTHREESEPARATEDONORSEXAMINEDFORFIBRONECTINBYIMMUNOHISTOCHEMISTRY.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFFIBRONECTIN.SCALEBARS100ΜM...........................163

FIGURE99:REPRESENTATIVEPHOTOMICROGRAPHSOFCELLULAR(A)ANDDECELLULARISED(B)CONJUNCTIVALTISSUESECTIONSFROMTHREESEPARATEDONORSEXAMINEDFORCOLLAGENIVBYIMMUNOHISTOCHEMISTRY.ALLBROWNSTAININGREPRESENTSPOSITIVEIMMUNOLOCALISATIONOFCOLLAGENIV.SCALEBARS100ΜM...........................164

FIGURE100:PHOTOGRAPHSOFTHERIGHTEYEOFPATIENT4.THEARROWSHOWSTHEEXTENTOFHORIZONTALSYMBLEPHARONINVOLVEMENT(20MMEXTENDINGMEDIALLYFROMTHELATERALCANTHUS):A)LOWERLIDHELDATTENSIONB)UPPERLIDHELDATTENSION.THEVERTICALARROWSHOWSTHEDISTANCEBETWEENTHEINFERIORLIMBUSATTHEMIDLINETOTHEEDGEOFTHESUBCONJUNCTIVALFIBROSIS.THEFORNIXRULERCOULDNOTBEINSERTEDINTOTHEINFERIORCONJUNCTIVALFORNIX........................................................................................................167

Listoftables

TABLE1:TABLEOFPOTENTIALCAUSESOFCICATRIZINGCONJUNCTIVITIS.THISLISTHASBEENADAPTEDFROMSAWETAL.(2008)ANDILLUSTRATESANON-EXHAUSTIVELISTOFTHEDISEASESPROCESSESTHATMAYLEADTOCONJUNCTIVALCICATRISATION.(38)..................................................................................................................................13

TABLE2:DETAILSOFFLUORESCENTSTAININGAGENTSWITHTHEIROPTIMISEDDILUTIONSUSEDFORTHEANALYSISOFCELLCULTURESDEVELOPEDONSYNTHETICSUBSTRATES........................................................................................59

TABLE3:TABLEOFANTIBODIESUSEDFORFLOWCYTOMETRYWITHTHERESPECTIVECLONE,SUPPLIERANDOPTIMISEDDILUTION...........................................................................................................................................................61

TABLE4:TABLEOFREAGENTSUSEDFORDECELLULARISATIONANDTHEIRSOURCE.........................................................64TABLE5:TABLEOFANTIBODIESUSEDFORIMMUNOHISTOCHEMISTRYWITHTHERESPECTIVECLONE,SUPPLIERANDOPTIMISED

DILUTION..............................................................................................................................................71TABLE6:STATICCONTACTANGLEANALYSISOFUNTREATEDANDAMMONIAGASPLASMATREATEDEPTFE.SIXREADINGSWERE

TAKENFROMRANDOMLYSELECTEDAREASONAMMONIAPLASMATREATEDANDUNTREATEDEPTFE.10ΜLWATERWASAUTOMATICALLYDISPENSEDANDCONTACTANGLEBETWEENTHEWATERDROPLETANDMATERIALRECORDEDBYANINBUILTVIDEORECORDER.STATISTICALANALYSISSHOWEDADIFFERENCEATAP=0.02LEVELBETWEENTREATEDANDUNTREATEDEPTFE.................................................................................................................................76

TABLE7:TABLEOFTHEAGEANDGENDEROFTISSUEDONORSTOGETHERWITHTHEPOST-MORTEMRETRIEVALTIME.THEDONOREYESWEREDESIGNATEDNUMBEREDSEQUENTIALLY,LEFTEYEFOLLOWEDBYRIGHTEYE............................104

TABLE8:TABLETOSHOWPERCENTAGEEXPRESSIONOFCONJUNCTIVALMARKERSINHCJE-GICELLSOFPASSAGE2AND28.TRIPLICATESAMPLESFOREACHMARKERWITHINEACHCOHORT(PASSAGE2ANDPASSAGE28)WEREANALYSEDBYFLOWCYTOMETRY.SIMILARLEVELSOFMARKERSINTERMSOFTHEPERCENTAGEOFCELLSDETECTEDWASAPPARENTBETWEENCELLSOFPASSAGE2AND28WITHNODIFFERENCESCONFIRMEDBYSTATISTICALANALYSIS..................................115

TABLE9:TABLEOFCELLMARKERSINWHICHASTATISTICALLYSIGNIFICANTCHANGEWASDEMONSTRATEDWITHADVANCINGTIMEINCULTURE.RAWPVALUESFROMTHEANCOVAAREDISPLAYED.ONLYPVALUESSTATISTICALLYSIGNIFICANTFOLLOWINGCORRECTIONBYHOLM-BONFERRONIFORMULTIPLETESTINGHYPOTHESESAREDISPLAYED..................124

TABLE10:TABLEOFCELLMARKERSINWHICHASTATISTICALLYSIGNIFICANTDIFFERENCEWASDEMONSTRATEDBETWEENSUBSTRATES.RAWPVALUESFROMTHEANCOVAAREDISPLAYED.ONLYPVALUESSTATISTICALLYSIGNIFICANTFOLLOWINGCORRECTIONBYHOLM-BONFERRONIFORMULTIPLETESTINGHYPOTHESESAREDISPLAYED..................124

TABLE11:TABLEOFCELLMARKERSINWHICHASTATISTICALLYSIGNIFICANTDIFFERENCEWASDEMONSTRATEDBETWEENSUBSTRATES:U=UNTREATEDEPTFE,D=DOUBLESIDETREATEDEPTFE,S=SINGLESIDETREATEDEPTFE,P=PETMEMBRANE.RAWPVALUESFROMTHEANCOVAPOST-HOCCONTRASTTESTSAREDISPLAYED.ONLYPVALUESSTATISTICALLYSIGNIFICANTFOLLOWINGCORRECTIONBYHOLM-BONFERRONIFORMULTIPLETESTINGHYPOTHESESAREDISPLAYED...........................................................................................................................................125

TABLE12:TABLETOSHOWDNACONTENTOFCELLULARTISSUESANDDECELLULARISEDTISSUESUSINGSDSOFVARYINGCONCENTRATION.THEOVERALLANOVAMODELWASSIGNIFICANT;P<0.001.DATAWASLOGTRANSFORMEDTOENABLEPARAMETRICDATAANALYSISANDANOVAMODELWASSATISFIEDFOLLOWINGLEVENE’STESTOFEQUALITYOFVARIANCE(P=0.1).BONFERRONIPOSTHOCTESTSBETWEEN0.05%/0.1/0.5%SDS(W/V)TREATMENTGROUPS;P≥0.1................................................................................................................................................136

TABLE13:TABLETOSHOWDNACONTENTOFCELLULARTISSUESANDDONORTISSUESDECELLULARISEDWITH0.05%SDS(W/V).OVERALLANOVAMODELWASSIGNIFICANTP<0.001.DATALOGWASTRANSFORMEDTOENABLEPARAMETRICDATAANALYSISANDANOVAMODELSATISFIEDFOLLOWINGLEVENE’STESTOFEQUALITYOFVARIANCE(P=0.28).BONFERRONIPOSTHOCTESTSBETWEENDONORA/B/C;P≥0.1.....................................................................136

TABLE14:TABLEOFRESULTSFROMTENSILESTRENGTHTESTINGOFEPTFE,CELLULARANDDECELLULARISEDCONJUNCTIVAANDAMNIOTICMEMBRANE.DATASHOWNINTHISTABLEWASNORMALISEDBYLOGTRANSFORMATIONPRIORTOANOVAANALYSIS.OVERALLANOVAMODELFORBOTHULTIMATETENSILESTRENGTHANDYOUNG’SMODULUSBETWEENTISSUES;P<0.0001.NOSIGNIFICANTDIFFERENCESWEREFOUNDINBOTHPARAMETERSSTUDIEDBETWEENCELLULARANDDECELLULARISEDTISSUES;P=0.354ULTIMATETENSILESTRENGTH;P=0.561YOUNG’SMODULUS.................140

TABLE15:TABLETOSHOWHYDROXYPROLINE(NG/MG)FOUNDINASSAYSOFPAIREDSAMPLESOFCELLULARANDDECELLULARISEDTISSUE.P=0.74:PAIREDSAMPLEST-TESTBETWEENCELLULARANDDECELLULARISEDDONORTISSUE(N=3INEACHGROUP)...........................................................................................................................143

Abbreviations

ABCG2 ATP-bindingcassettesub-familyGmember2BCVA BestcorrectedvisualacuityBPE BovinepituitaryextractBSA BovineserumalbuminCK CytokeratinDAPI 4',6-diamidino-2-phenylindoleDMEM Dulbecco’sModifiedEagleMediumDMSO DimethylsulphoxideDNA DeoxyribonucleicacidECM ExtracellularmatrixEDTA EthylenediaminetetraaceticacidEGF Epidermalgrowthfactor ePTFE Expandedpolytetrafluoroethylene FCS FoetalcalfserumHEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacidHCjE-Gi Humanconjunctivalepithelialcellline(Gipsonlaboratories)HCl HydrochloricacidH&E HaematoxylinandEosinHPAlectin HelixpomatialectinMMP MucousmembranepemphigoidmRNA Messengerribonucleicacid

MUC1/4/7/16 Mucin1/4/7/16MUC5AC Mucin5ACNaOH SodiumhydroxidePAS PeriodicacidSchiffPBS PhosphatebufferedsalinePCNA ProliferatingcellnuclearantigenPET Polyethyleneterephthalate p63 Tumourproteinp63PGLA poly(lactic-co-glycolicacid)RFGD Radiofrequencyglowdischarge RNAse RibonucleaseRPMImedium RoswellParkMemorialInstitutemediumSCCM StandardcubiccentimetresperminuteSDS SodiumdodecylsulfateTRIS 2-Amino-2-hydroxymethyl-propane-1,3-diolTBS TrisbufferedsalineTFF Trefoilfactorfamily3T3 Murinefibroblastcellline;3-daytransfer,inoculum3x105cellsUAE-1lectin UlexEuropaeuslectin1VA VisualacuityZO-1 Zonulaoccludens-1Z-stack Focusstacking/focalplanesections

1. Introduction

1.1 Theocularsurface

Theocularsurfacecomprisesthecornea,conjunctivaandthetearfilm.Thecorneaand

conjunctivalinetheanteriorsurfaceoftheglobe.Thecorneaisatransparentconvex

structureandisthemainrefractivecomponentoftheeye.Itissurroundedbyandis

contiguouswiththeconjunctiva,whichcoverstheanteriorepiscleraltissue.The

conjunctivaextendsfromthecorneallimbusandcoversthetarsal(inner)surfaceofthe

eyelids.Itisamoisttranslucentmembranewithredundantfoldsthataremost

prominentintheforniceswhichareanatomicalspacesbetweentheglobeandeyelids

thatenablefreeocularmovement.(1)Thetearfilmandconjunctivaareessentialforthe

homeostasisofthecornea.Theassociatedtearfilmiscrucialtothemaintenanceof

cornealclarity,andtothepreventionofinfection.(1)Theocularsurfaceisalsoprotected

fromdrying,desiccationandinjurybytheupperandlowereyelidsanditscomponents

includingtheeyelashesandsebaceoussecretionsfrommeibomianglands.(1)

Figure1:Schematicdrawingillustratingacrosssectionoftheglobeandeyelids.Takenfrom

StewartRMK.(2013)Identificationofprogenitorrichsitesintheconjunctiva.PhDThesis.

UniversityofLiverpool;adaptedinpartfromPaulsenandBerry(2006).(2)

Caruncle

PlicaSemilunaris

PalpebralConjunctiva

Accessorylacrimalglands

Fornicealconjunctiva

Orbitalconjunctiva

Tarsalconjunctiva

Cornealepithelium

Bulbarconjunctiva

Tarsalplatewithmeibomianglands

Corneallimbus

1.1.1 Thecorneaandlimbus

Thecorneaisatransparentstructurethatenablestheentryoflightandisthemain

refractivecomponentoftheeye.Theaverageverticalandhorizontaldiametersinan

adultare10.6mmand11.77mm,respectively.(1)Fromsuperficialtodeep,thecorneais

composedofanepitheliallayerandbasementmembrane,ananteriorlimitinglamina

(Bowman’slayer),thesubstantiapropria(stroma),aposteriorlimitinglamina

(Descemet’smembrane)andanendotheliallayer,whichiscontactwiththeaqueous

humouroftheanteriorchamberoftheeye.

Thecornealepitheliumisastratifiedsquamousnon-keratinisingepitheliumthatis5-6

celllayersdeep.Thebasalepithelialcellsareassociatedwiththebasallaminaviatype

VIIandVIcollagen.(3)Continuousturnoverofcornealepithelialcellsoccursinwhich

transientlyamplifyingcellsarisefromthecornealstemcellnicheatthebasallimbus

andmigratecentripetally.(4)Thecornealepitheliumisdenselyinnervatedbysensory

butalsobyasmallerproportionofsympatheticfibres.Thesensorynervescanrespond

totemperature,chemicalandmechanicalstimuli.(5)Epithelialdefectshealrapidly

throughtheamoeboidmovementofcornealepithelialcells.(6)Theapicalcorneal

epithelialcellspossessaglycocalyx,microplicaeandmicrovillithatinteractwithand

stabilisethetearfilm.(7)Thecornealepitheliummeetstheconjunctivalepitheliumat

thelimbus,atransitionzoneattheedgeofthecornea.

CornealandconjunctivalepitheliahavesimilarfeaturesintermsofABGC2andΔNp63

expressioninprogenitorcellpopulationsbutdifferincytokeratinexpression.(8-10)

Differentiatingfeaturesincludetheabsenceofgobletcellsinnormalcorneal

epitheliumanddifferentialcytokeratinexpression.(11)Cornealstemcellsarefoundin

thebasalcryptsoflimbalepithelium.Notablefeaturesofthelimbusareradialpapillae

(termedpalisadesofVogt),densevascularisationandanepitheliallayeraround10cells

deep.(1)Inthistransitionzone,conjunctivalepithelialcellsendalongwithitsblood

vessels.Thevascularsupplyoftheperipheralcorneaoriginatesfromthemarginal

cornealarcadesandthatofthelimbusisfromanepiscleralplexusderivedfromthe

anteriorciliaryarteriesthattravelanteriorlyfromtherectusmusclesdeeptothe

conjunctiva.(1)Thebasementmembranecompositionofthelimbusdiffersfromthe

cornealbasementmembranesuchthatlamininsα1,β1andγ1arefoundingreater

abundancetogetherwithcollagenIVα2.(12)Inlimbalstemcelldiseaseconjunctival

epithelialisationofthecorneaoccurs,resultinginalossofcornealclarity.(1)

Bowman’slayerisamodifiedlayerofthecornealstromacontainingcollagenstypeI,III,

VandVIandterminatesatthelimbus.Thecornealbasementmembraneatthecorneal

limbusiscomposedmainlyofcollagenIVandVII,butalsoextracellularmatrixproteins

laminin(subtypes332andγ2),fibronectinandanumberofglycoproteins:perlecan,

nidogen,fibrillin,clusterin.(3)ThecornealstromaconsistsofcollagenI,III,VandVIin

regularlyarrangedlamellae,thespacingofwhich,isdeterminedbyintervening

glycosaminoglycansandproteoglycans.(1)Themaintenanceofcornealclarityis

dependantupontheregulararrangementanddiameterofcollagenfibrilsandthetight

regulationofhydration.Inthehealthycorneanobloodvesselsarefoundinwithinthe

cornealstromaapartfromthemarginalcornealarcades.Nervefibresarepresentin

theanteriorstroma.Thenervesupplyofthecorneaisviathelongciliarynervesthat

originatefromtheophthalmicdivisionofthetrigeminalnerve.(1)Descemet’s

membraneisbasementmembraneofthecornealendotheliumandiscontinuouswith

thetrabecularmeshworkperipherally.Thecornealendotheliumisamonolayerof

continuouspolygonalsquamousepitheliumrichinmitochondria.(1)Thesecellsare

responsibleforactivetransportregulatingthefluidandelectrolytebalanceofthe

corneaandthereforeitsclarity.

Figure2:Photomicrographofthenormalhumancornea.Paraffinembeddedtissuesectionwas

subjectedtostainingwithHaematoxylinandEosin.Scalebar50μm.TakenfromTakenfrom

StewartRMK.(2013)Identificationofprogenitorrichsitesintheconjunctiva.PhDThesis.

1.1.2 Thehumanconjunctiva

Theconjunctivalisathin,translucentvascularisedmembranethatlinesthescleraand

theinneraspectoftheupperandlowereyelids.Itisastratifiedepitheliumsupported

byabasementmembranethatoverliesathinlayerofvascularisedlooseconnective

stromaltissue.(1)Betweentheconjunctivalconnectivetissueandthescleraliesthe

tenon’scapsule,whichisathinlayeroffibroustissuethatformsadhesionswiththe

underlyingepiscleraandalsoservesaprotectivefunction.

Theconjunctivaisastratifiednon-keratinisedsquamousandcolumnarepitheliumand

containsgobletcells,whichbearsimilaritiestoglandularepithelia.Theconjunctiva

variesbetween3and9celllayersdeepandextendsfromtheedgeofthecorneal

limbus,tothemucocutaneousjunctionoftheeyelidmarginwherethetransitionzone

fromnon-keratinisingtokeratinisingepitheliumoccurs.(1)Theconjunctivaisreflected

Epithelium

Bowman’smembrane

Stroma

Descemet’smembrane

Endothelium

fromtheanteriorscleratothetarsal(inner)surfaceoftheeyelidstoforman

anatomicalspacebetweentheupperandlowereyelidsandtheglobeknownasthe

conjunctivalfornices.Theexcessconjunctivaltissueseenasfoldsintheseareas,allows

afullrangeofocularmovement.Thevolumeofthisspacewiththeeyelidsclosedhas

beenestimatedatapproximately7μl.(1)

Theconjunctivacanbedividedanatomicallyintothreeregions:thebulbar,forniceal

andpalpebralconjunctiva.Thebulbarconjunctivacoversandislooselyadherenttothe

globe.Incontrast,thepalpebralortarsalconjunctivaistightlyadherenttothetarsal

plateontheinneraspectoftheeyelids.Thefornicealconjunctivaformstheblind

pouchbetweentheeyelidsandglobewhereitisthrownintofolds.Thesubepithelial

connectivetissueissparseinthepalpebralconjunctiva.Thelacrimalpunctiopeninto

thelidmarginmedially.Thesearesmallopeningsofthenarrowpassagesthatconnect

theeyelidstothenasalcavityallowingthedrainageoftears.Theconjunctival

epitheliumiscontinuouswiththatliningtheinferiormeatusofthenasalcavity.The

fornicealconjunctivaisalsocontinuouswiththemedialandlateralcanthiandcontains

accessorylacrimalglands(Krause’sglands).

Thesecretionsfrommostoftheaccessorylacrimalglandsinadditiontothosefromthe

mainductofthelacrimalglandopenintothesuperolateralfornix.Thefacialsheathsof

therectusmusclesintheupperandlowereyelidsandlevatorpalpebraesuperiorisin

theupperlidareassociatedwiththeconjunctiva.Thebulbarconjunctivaactsacoating

forthesclera,whichisalsocoveredwithepiscleraandTenon’scapsuleandalsocovers

theextraocularmuscles.Theconjunctivaislooselyadherenttoepiscleraltissueover

thebulbarconjunctivabutistightlyadherentatthelimbus.Atthemedialfornixthe

bulbarconjunctivaformsasemilunarfold(plicasemilunaris)ofthickenedtissuethatis

highlyvascularised,richinimmunocompetentandmucinproducinggobletcells.(1)

Medialtothisisanodulartissueknownasthecarunclethatcontainsdenselypacked

sebaceousglands,accessorylacrimalglandsandlymphoidcells.(1)

Theconjunctivalepithelialcellsaremostlycuboidalorpolyhedralinshape.Gobletcells

canbefoundinterspersedwithinlayersofstratifiedconjunctivalepitheliaeitherin

isolationorinclustersliningcrypts.Theyarefoundwithinapicallayersofepithelium

andcanbeeasilyrecognisableastheyareoftenmoreroundedinappearancethanthe

surroundingepithelia.(13)Gobletcellstendtobelargeandarecharacterisedbyan

apicalportionthatisbroadincomparisontotheirbasalproportions.Thesecellsare

characteristicallyrichinintracellularorganellesowingtotheirsecretoryandstorage

capacityforthegelformingmucinMUC5AC.(11)

Otherthangobletcellsecretions,theconjunctivaalsocontainsaccessorylacrimal

glandsknownasKrause’sglandsintheconjunctivalfornicesandglandsofWolfring

locatedintheupperborderofthetarsus.Theseglandscontributetothebaselinetear

productionandareinnervatedbysympatheticnerves.(1)Theconjunctivaisalsoknown

tohavelymphoidtissueknownasconjunctivaassociatedlymphoidtissue(CALT)where

MHCIIdendriticcellstransportinternalisedantigensandsignalstomountanadaptive

immuneresponse.(1)Inaddition,intraepithelialCD3+velymphocytesarearecognised

featureofconjunctivaandoccasionallyB-lymphocytesaspartoftheimmunedefence

systemoftheconjunctiva.Scatteredmelanocytescanalsobefound.(1)

Thesensorysupplyoftheconjunctivaisderivedfromtheophthalmicbranchofthe

trigeminalnerve.Parasympatheticandsympatheticnervesarisefromthe

pterygopalatineganglionandsympatheticplexusrespectivelyandcoursealong

branchesoftheophthalmicartery,terminatingaroundgobletcells.Thevascularsupply

oftheconjunctivaisinthesubstantiapropria,deliveredbytheanteriorconjunctival

branchesoftheanteriorciliaryarteryandthepalpebralbranchesoftheophthalmic

andlacrimalarteries.Thesevesselsanastomosewiththemarginalcornealarcadesand

thelimbalplexus.Venousdrainageoccursintoavenousplexusthatleadstothe

superiorandinferiorophthalmicveins.Lymphaticvesselsarealsofoundwithina

networkinthebulbarandpalpebralconjunctiva.(14)

Biochemicalcomponentsoftheconjunctivalbasementmembranezonehavebeen

characterisedandincludecollagensI/IV/VII,lamininsandalsofibronectin.(15)Collagen

IVandVII,however,arefoundwithgreatestabundancewithlamininsspecificallyof-

332,α3,γ1,γ2and5:integrinβ4,fibronectin,nidogen,perlecan,andclusterin.(3)

Althoughthesecomponentsareidenticaltothoseofthecornealandlimbalbasement

membranes,theproportionsofsomeofthesecomponentsvarybetweenthe

aforementionedtissues.(3)

Thereisacontinualturnoverofconjunctivalepithelium,whichhasagreatregenerative

capacity.Thisappliestocellsofbothanepithelialandgobletphenotype.(11,16,17)

Evidenceforthiswasdeterminedfromcloningstudiesinwhichitwasalso

demonstratedthatgobletcellswerenotterminallydifferentiated.(18)Markers

associatedwithprogenitorcellsweremostlyfoundinbasalepitheliumandwerefound

ingreatestabundanceattheinferiorfornixandmedialcanthus.(19)

1.1.3 Protectionoftheocularsurfaceandthetearfilm

Therearephysicalbarrierstochemical,environmentalandinfectiousinsultssuchas

theblinkreflex,eyelidsandeyelashes.Theconjunctivacoveringthemajorityofthe

ocularsurfaceprovidesabarrierfunctionandplaysaroleinmucosalimmunity.The

eyelidscontainmeibomianglandsthatarefoundwithinthefibroustarsalplatesand

secretethelipidcomponentofthetearfilm(Figure1).Blinkingisareflexactionthat

redistributesthetearfilmandtogethereyelashespreventtraumaandtheentryof

foreignbodies.Themeibomianglandsecretionsformthesuperficiallayerofthetear

film.Itisacomplexlipidlayercomprisingheterogeneousspeciesofpolarandnon-

polarlipids,13-100nmindepththatstabilisesthetearfilmandpreventsevaporation

fromitsaqueousphase.(20)

Thetearfilmiscrucialtothenormalfunctionoftheocularsurfaceincludingthe

maintenanceofcornealclarity.Severalmodelsofthetearfilmhavebeensuggested.It

isgenerallyacceptedhowever,thattearfilmcomprisesanoutermeibomianlipidlayer,

agelphasecomprisingaqueoussecretionincludingelectrolytes,antimicrobialfactors

andgelformingmucin,andaninnermucinlayerdirectlyincontactwiththeapical

epitheliaofbothcornealandconjunctivalcells.(7)Thestructureoftheprecornealtear

filmhasmorerecentlybeenconsideredasmattressstructurecomprisingthreelayers;

anepithelialcellsurfaceglycocalyxlayer,anaqueous/mucinlayerandasuperficiallipid

layer.(7)

Theaqueouscomponentofthetearfilmisproducedprimarybythelacrimalgland,

whichislocatedinthelacrimalfossainthesuperotemporalorbit.Therearealso

contributionsfromtheaccessorylacrimalglands.Theaqueoussecretionscontain

lactoferrin,immunoglobulinA,defensin,betalysin,lysozyme,lipocalinandthe

antibody-complementsystemofproteinsthatserveanantimicrobialfunction.(7,21)

Mucinsareacriticalcomponentofthetearfilmandareproducedbyconjunctival

epithelialgobletcells.Theseareglycosylatedcomplexhighmolecularweight

glycoproteinsthatarehydrophilicinnatureandcapableofforminggels.Mucinsserve

tocreateamucin-aqueouscomplexthatlubricatesthesurfaceofocularsurface

epitheliaandpreventsdrying.Theroleofmucinalsoextendstoaroleintheprimary

defenceagainstmicroorganismsasgobletcellsalsointeractwithantimicrobial

constituentsofthetearfilm,suchasdefensinsandperoxidase.(17,22)Mucinshavebeen

recognisednotonlytobindtobacterialcellwallsbutalsotoparticipateintheinnate

immuneresponsethroughinteractionswithneutrophilsandleucocytesvia

oligosaccharideepitopesfoundonmucins.(23)Inadditiontomucin,gobletcellsalso

secretethetrefoilfamilypeptides(TFF)TFF1and3.(2)Thesepeptidestogetherwith

mucinpromoteepithelialcellmigrationandhaveanti-apoptoticproperties.(24)

Themucinsontheocularsurfacecomprisemembrane-associatedmucinsonthe

microplicaeofapicalconjunctivalandcornealepitheliumandformtheglycocalyxthat

supportsinteractionsatthecell-tearinterface.(1)ThelargegelformingmucinMUC5AC

isproducedexclusivelybyconjunctivalgobletcellsbyexocytosis.MUC5ACanda

furthersmallsolublemucinMUC7producedbythelacrimalglandarethemajormucin

componentsoftheaqueouslayerofthetearfilmandareresponsibleforitslubricating

andvisco-elasticproperties.(7)Mucinsalsoformaprotectivelayerwithinthetearfilm

reducingevaporationoftheaqueouscomponentsofthetearfilm,therebymaintaining

moistureoftheconjunctivaandcornea.

Ocularsurfacediseasecanbeassociatedwithboththeoverproductionand

underproductionofmucin.(17)Oftheseconditionshowever,mucindepletionleadsto

moreseverediseaseandgobletcelldepletionisassociatedwithmostocularsurface

diseasesandincludeocularmucousmembranepemphigoid,neurotropickeratitisand

chemicalinjury.(25,26)Intheseveredryeyestatethecontinualmicrotraumaofthe

corneaoccursleadingtodesiccation,opacity,ulcerationandeventuallypainful

blindness.Insuchconditions,theconjunctivalepitheliumalsoundergoessquamous

metaplasiawithlossofgobletcellsandpropensitytoinfection.(27)

Theregulationofmucinproductioniscomplexandnotfullyunderstood.Mucinsare

producedbyconjunctivalsquamousepithelialcells(MUC1,4,16)andarefoundas

transmembraneglycoproteinsbuthavealsoassecretedcomponentsinthetearfilm.(28)

Theprecisepathwaysandmechanismsbywhichthesheddingofthesemucinsoccurs

arestillunderinvestigation.Incontrast,MUC5ACsecretionfromgobletcellsisknown

tooccurthroughanapocrinemechanismmediatedbygrowthfactors,inadditionto

parasympatheticandsympatheticnervousstimulation.Purinergicandmuscarinic

agoniststogetherwithacetylcholine,carbacholandvasoactiveintestinalpeptidehave

beenshowntostimulategobletcellmucinsecretion.(17)Inaddition,epidermalgrowth

factor(EGF)stimulatesgobletcellsandmayoccurfollowingreleasefrombacteria,

plateletsandnerves.(17)

1.2 Conjunctivaldiseaseinhumans

Homeostasisoftheocularsurfaceisdependentuponnormalconjunctivalfunction

includingmucinproduction.Theconjunctivalepitheliummaybecomeirreversibly

destroyedbyinfective,inflammatory,neoplasticconditionsandtraumaticinjury.This

mayleadtokeratinisation,lossofgobletcells,cicatrisation,fornicealcontractionand

cornealulcerationleadingtopainfullossofvision.(29)Thesefactorsmayresultinlimbal

stemcelldeficiencyandvisualdeterioration.

Anumberofacquiredandautoimmunediseasesmaycausethesechanges.

Pathologicalchangeintheconjunctivaincludestheformationofneoplasticlesions,

bothmalignantandbenignincludingtheformationofpterygium,proposedtooccur

fromultravioletdamage.Inflammationcanarisethroughautoimmunedisease,

infectiousdiseasesincludingthosefromviralandbacterialpathogens,allergyandalso

inducediatrogenicallyfromtopicalagentssuchmedicationsusedtotreatglaucoma.

TrachomaisaneyediseasecausedbytheintracellularbacteriaChlamydiatrachomitis.

Itisanimportantinfectiouscauseofcicatrisingeyediseaseandisthesecond

commonestcauseofblindnessindevelopingnations.Anestimated1.3millionpeople

areblindworldwidefromthiscondition.(30)Thehighestprevalenceoftrachomaisin

sub-SaharanAfrica,MiddleEastandSoutheastAsia.Althougheasilytreatablewithoral

tetracyclines,theglobalburdenofthisdiseaseissignificantasmanydonothaveaccess

totreatmentandthereforeahighproportiondevelopendstagecicatrisingconjunctival

diseasewhichleadstochronicpainandcornealblindness.

Traumamayoccurfromavarietyofinjuriesincludingthermalinjuryandchemical

burns.Ocularburnsconstitute7-18%ofocularinjuriesseeninemergencydepartments

and17.3%ofbattlefieldinjurieshavebeenrecordedinIraqandAfghanistan.(31,32)

Visualdisabilityoccurredin33%andblindnessin15%ofpatientswithbattlefield

injury.(31)

Autoimmunediseasesoftheconjunctivaincludemucousmembranepemphigoid,

Stevens-Johnsonsyndrome,linearIgAdiseaseandgraft-versus-hostdisease.

Autoimmunediseaseinparticular,mucousmembranepemphigoid,canbeoneofthe

mostchallengingconditionstotreatgiventhechronicityoftheinflammationthatleads

toirreversiblecicatricialeyedisease.Theincidenceofmucousmembranepemphigoid

hasbeenreportedbetween1.13and1.78permillionperyearinEuropeancountries

withocularinvolvementin60-95%ofpatients.(33)TheincidenceofStevensJohnson

syndromeisestimatedat2-3permillionperyearinEuropeandtheUSAofwhich43-

81%haveocularinvolvementand35%havepermanentvisualdisability.(34)Developing

countriessuchasIndiaarerecognisedtohaveasignificantnumberofStevens-Johnson

syndromecases,thoughttobetheresultofwidespreadavailabilityandunprescribed

useofantimicrobialdrugsincludingsulphonamidecontainingdrugsand

fluoroquinolones.(35)TheincidenceofcicatrisingconjunctivitisoverallintheUKhas

beenestimatedat1.3permillionperyear,however,theauthorsofthissurveillance

studystatedthatthisfigurewaslikelytobeanunderestimation.(36)Incontrastto

developingnations,theburdenindevelopedcountriesislargelyduetoautoimmune

conjunctivaldisease,particularlyocularmucousmembranepemphigoid.Diagnosisof

thelattermayattimesbeconsideredatarelativelylatestageinthediseaseprocess,

andeveninthosediagnosed,under-recognisedexacerbationsbetweenfollowup

periodsdespitetreatmenthavebeennoted.(36)

Ocularsurfacedisordersleadtosquamousmetaplasiaandcicatrisationcharacterised

byprogressivefibrosisandscarring.Cicatrisationmaybedefinedbythepresenceof

limbalstemcelldeficiency,fornicealshorteningandsymblepharon.(27)Thehistological

changesinconjunctivalsquamousmetaplasiahavebeendescribedinthefollowing

threestagesbyimpressioncytology:a)lossofgobletcellsb)increaseincellular

stratificationand/orenlargementofthesuperficialcellsandc)keratinisation.(37)Many

ocularsurfacediseasesleadtosquamousmetaplasiaandcicatrisingeyediseasebothin

advancedstagesofchronicinflammationandbyasevereinsultsuchachemicalinjury.

Ocularsurfacedisordersareanimportantclinicalproblemasirreversiblescarringand

keratinisationoftheconjunctivaleadstoanabnormalepitheliumwithlossofsecretory

andmembraneboundmucinandthereforecompromiseddefenceagainstinfection.(38)

Itisalsoaccompaniedbydepletionofgobletcellsandsecondarytearfilmdeficiency.

Whensevere,alongwithfornicealshortening,symblepharonandankyloblepharon,lid

deformitiesalsooccursuchasentropionandectropion.Thisresultsintrichiasis,

exposurekeratopathyandrecurrentcornealdesiccation.Thesefactorsincombination

mayleadtocornealblindnessduetolimbalstemcelldeficiencyresultinginulceration

orprogressiveconjunctivalisationandopacification.

Table1:Tableofpotentialcausesofcicatrizingconjunctivitis.Thislisthasbeenadaptedfrom

Sawetal.(2008)andillustratesanon-exhaustivelistofthediseasesprocessesthatmayleadto

conjunctivalcicatrisation.(39)

1.2.1 Medicalstrategiesincicatrisingconjunctivaldisease

Crucialtothemanagementofcicatrisingconjunctivaldiseaseisearlydiagnosisand

treatmenttoremovetheincitingagentifpresent,reduceorterminatethe

inflammatoryprocessandencourageepithelialregeneration.Thetreatmentstrategy

dependsuponthenatureandcauseoftheinflammation.Forexample,themedical

treatmentofocularburnscomprisesacuteirrigationtoremovetheincitingagent,

tetracyclinesandcitratetoreducemetalloproteinaseactivity,steroidstoreduce

inflammation,tearsupplements,antimicrobialstopreventinfectionandascorbateto

promotehealing.Intrachoma,theinflammationiscausedbyChlamydiatrachomitis

andthereforerespondstooraltetracyclines.

CausesofcicatrisingconjunctivitisTrauma: Chemical,thermal,radiation,physical

Infection: Trachoma,membranousconjunctivitis,chronic

mucocutaneouscandidiasis

Allergiceyedisease Atopickeratoconjunctivits

Druginducedcicatrisation Benzalkoniumchloridecontainingsolutions

Mucocutaneousdisorders: Stevens-Johnsondisease,graftversushostdisease,

lichenplanus

Immunobullousdisease: Mucousmembranepemphigoid,linearIgAdisease,

bullouspemphigoid,paraneoplasticpemphigus

Neoplasia: Sebaceouscellcarcinoma,squamouscellcarcinoma

Stevens-Johnsonsyndromemaybediagnosedthroughskinbiopsyratherthanany

specificserologicaltesttogetherwithclinicalfindings.ThetreatmentofStevens-

Johnsonsyndromeinvolvescessationoftheincitingagent,intravenous

immunoglobulin,systemiccorticosteroidsandthemanagementofthesystemic

sequelaeofthissevereillness.(40)Noneoftheimmunomodulatorytreatmentshowever

havebeenfoundtoinfluencetheocularoutcomes.(41,42)Similarly,otherautoimmune

conjunctivaldiseasesincludingocularmucousmembranepemphigoidmaybe

diagnosedthroughtheirclinicalfeatures,biochemicalfeaturesofautoimmunedisease

andthedirectimmunofluorescenceassayofamucousmembranebiopsywhichmaybe

takenfromtheconjunctiva.(39)Anegativebiopsy,however,isnotconclusive.

Immunosuppressivetherapyisonlyrequiredwhentheautoimmunediseaseprocessis

active.Firstlinetherapyinvolvestheuseofdapsone,sulfasalazineormethotrexate.(39)

Secondlineagentsincludeazathioprine,mycophenolatemofetiland

cyclophosphamide.(39)Adjunctiveuseofcorticosteroidsincludingpulsedintravenous

methylprednisolonemayalsobeconsideredfortheshort-termcontrolofsevere

inflammation.Theuseofbiologicsincludinganti-tumournecrosisfactoragents

etanerceptandinfliximab,andtheanti-CD20antibodyrituximabhavebeen

successfullyusedtotreatsevererefractorydisease.(43-45)Itisrecognised,however,that

symblepharonformationmaystillprogressdespitetheapparentcontrolofocular

inflammationinautoimmuneconjunctivitis,particularlyinocularmucousmembrane

pemphigoid.(36)Inmanycasesofcicatrisingconjunctivitis,irreversibleandsignificant

conjunctivaldamageoccurssuchthatmedicalstrategiesalonemaybeinsufficientto

enabletheregenerationofhealthyocularsurfaceepithelia.

Inallformsofcicatrisingeyedisease,supportivetherapyincludesmanagementofthe

factorsthatexacerbatethecondition.Thisincludesmanagementofinflammation

associatedwithco-existentocularsurfacedisease.Forexample,meibomiangland

dysfunctionandblepharitismayco-existwiththecicatrisingdiseaseprocess.Indeed,

blepharitiscanleadtocolonisationwithbacterialpathogensandthereforerepresents

ariskfactorformicrobialkeratitis.(39)Inastudyoflidpathogens,85%ofmucous

membranepemphigoidpatientsweredemonstratedascarriersforsuchpathogensin

contrastto49%inthecontrolgroup.(46)Dryeyeduetomucinandteardeficiencycan

occurinamanycicatrisingeyediseasesduetosparsegobletcellsandalsoother

dysfunctionofothercomponentsincludingthemeibomianglands.Inturn,this

predisposestopoorepithelialintegrityandcompromisedimmunedefence.

Managementstrategiesincludetearsupplementsincludingautologousserum,punctal

occlusion,topicalcyclosporineorsteroidtoaddresskeratoconjunctivitissicca,andlid

surgerytoaddresslidmalpositionssuchasentropionwithtrichiasisor

lagophthalmos.(39)

Diagnosis

DiseasecontrolDiseasetreatment*

Supportivetherapy Restoration

-Treatmentofassociatedocularsurfacedisease -Correctionoflidmalposition

-Treatmentofdryeye -Fornixreconstruction

-Treatmentoftrichiasis -Conjunctivalreplacement

-Treatmentofpersistentepithelialdefects -Limbalstemcelltransplant

-Avoidanceoftoxicity -Cornealtransplant

Figure3:Flowdiagramtoillustratethatthemanagementofoculardiseaseinvolvestreatment

ofthediseaseprocessalongwithsupportivetherapiesandrestorativetreatment.*Disease

treatmentmayinvolveremovaloftheincitingagent,antimicrobials,immunosuppressantsor

anti-inflammatoryagentsasdescribedinearlierparagraphs(section1.2.1).

1.2.2 Surgicalocularsurfacereconstructionstrategiesandtheclinicalneedfor

novelconjunctivalequivalents

Poorconjunctivalintegrityandadnexalabnormalitiesincludingdryeye,entropionand

trichiasisareprognosticfactorsforcornealandlimbalstemcelltransplantfailure

leadingtopoorvisualoutcomesinthesepatients.(29)Itisparamounttherefore,that

strategiesforocularsurfacereconstructionincludethecorrectionofliddeformities

includingentropionandalsotrichiasis,whichwouldotherwiseresultincontinual

abrasionandtraumatoocularsurfaceepithelia.Liddeformitiestogetherwith

improvementsinconjunctivalfunctionmustthereforebeaddressedwithsurgical

interventionstoenablethesuccessofapotentialcornealorlimbalstemcelltransplant.

Currentstrategiesforthereplacementofconjunctivallossaremostcommonly

conjunctivalautograftsoramnioticmembranegrafts.Conjunctivalautograftshave

beenusedfortherepairofsmalldefectswithrelativesuccess.(47,48)Thesecanbetaken

fromthefelloweyeorfromthesameeye,usuallythesuperiorbulbarconjunctiva.This

approach,however,cannotbeusedfortherepairoflargerdefectsincludingfornix

reconstruction.Furthermore,thetraumatothedonorsiteinretrievingaconjunctival

autograftwouldbedetrimentalinautoimmunediseasesleadingtofurther

inflammationandscarring.

Mucosaefromthenasalturbinatesandoralcavityhavebeeninvestigatedasdonor

tissuesforocularsurfacereconstruction.Itisrecognised,however,thatthesegrafts

areassociatedwithpoorcosmesisandrecurrentscarring.(49)Thehumanoralmucous

membranehasbeenusedforthereconstructionofconjunctivalforniceswithsome

success.(50,51)Similarly,nasalturbinatemucosalgraftshavealsobeendemonstratedin

thesuccessfulreconstructionofconjunctivalfornicesinpatientswithchemicalinjury

andcicatricialmucosaldisease.(52,53)Anincreaseinmucousproductionwas

demonstratedinassociationwithareductioninsymptomsforthemajorityofpatients

thatreceivedthistreatment.Thisapproach,however,islimitedbydifferencesin

appearanceincludingthecolourofthetissue,whichcanappearunsightlyin

comparisontothetranslucentappearanceofamnioticmembraneandnative

conjunctiva.(54)Afurtherconsiderationismorbiditytothegraftdonorsite,whichmay

bealimitationforitsuseinpatientswithmucousmembranepemphigoiddueto

possiblediseaseinvolvementoftissueattheseextra-ocularmucosalsites.

Todate,themostcommonlyusedsubstrateforocularsurfacereconstructionhasbeen

amnioticmembrane,exemplifiedbyitsextensiveclinicalusage.Itismostcommonly

usedinthetreatmentoflargeconjunctivalepithelialdefectswhereitpromotes

epithelialisationandreducesscarring.(55)Theuseofamnioticmembraneforthe

reconstructionofconjunctivalfornices,however,hasbeenlesssuccessful.Inpatients

withongoinginflammationrecurrentsymblepharonwasdemonstratedin10-44%of

patientsandlossinfornicealdepthof50%ormoreoccurredfourmonthsafter

surgery.(56,57)Therefore,outcomesoffornicealreconstructionusingamniotic

membranegraftsarepoorduetoshrinkageandcicatrisationresultinginrecurrent

fornicealloss.(58)

Theseverityofoculardamagetotheconjunctivainmucocutaneousandsystemicand

inflammatorydiseaseswarrantthedevelopmentofgraftstoregeneratetheocular

surfaceandreconstructthefornices.Novelrobustconjunctivalconstructswould

enablevisualandfunctionalrehabilitationinpatientswithsevereocularsurface

diseaseinwhomprognosisispoorduetoalackofavailabletherapies.Novel

conjunctivalequivalentsmayalsoassistinsurgicalproceduresassociatedwith

conjunctivallossincludingconjunctivalneoplasia,pterygiaandglaucomasurgery.

1.3 Substratesforconjunctivalregeneration

1.3.1 Anidealconjunctivalsubstrate

Anidealsubstratefortheexpansionofconjunctivalepitheliumshouldbeelasticto

enablefreeocularmovement,integrateinthehostwithoutcausinginflammationand

supportaself-renewingconjunctivalepitheliumwithasub-populationofprogenitor

andgobletcells.Ifasmalldefectrequirestreatment,aconjunctivalequivalentthat

degradesmaybesuitable,suchthatitiseventuallyreplacedbyhostconjunctiva.Fornix

reconstructionhowevermaybenefitfromanon-degradablesubstratethatiscapable

ofmaintaininglong-termfornicealsupport.

1.3.2 Theuseofbiologicalsubstratesfortheexvivoexpansionofconjunctival

epithelium

Otherthanautologoustissuesincludingautologousconjunctiva,nasalandoral

mucosae,theprincipalallogeneicbiologicalsubstrateinvestigatedforconjunctival

regenerationhasbeenamnioticmembrane.Amnioticmembraneistheinnermostlayer

ofthemembranethatsurroundsthefoetusinutero.Itsuseinophthalmicsurgeryhas

beenestablishedforover70yearsanditplaysamajorroleinocularsurface

reconstructionincurrentclinicalpractice.(59)Desirablefeaturesincludeitslackof

immunogenicity,antibacterialandanti-inflammatoryproperties.(60-62)Theuseof

amnioticmembranefortheprotectionofocularsurfacedefectssuchaslargecorneal

epithelialdefectsandchemicalburnswhereitpromoteshealinghasbeenlong

established.(63,64)Itcanthereforeplayamajorroleinthehealingprocessofocular

surfacedefects.Amnioticmembranebeenusedinbothacellularanddecellularised

form.Decellularisationmayreducetheriskofdiseasetransmissionbutmaydisrupt

extracellularmatrixcomponentsandtheultrastructureofthetissue.(65)

Thepresenceofprogenitorandgobletcellshavebeendemonstratedinrabbit

conjunctivalepithelialcellculturesdevelopedonanamnioticmembranesubstrate.(66)

Stratifiedconjunctivalepithelialconstructshavealsobeensuccessfullydemonstrated

onanamnioticmembranesubstrate.(67)Studiesofex-vivoexpandedhuman

conjunctivalepitheliumonamnioticmembranehavenothowever,shownthepresence

ofgobletcells.(54,68)Humanamnioticmembranehasbeentransplantedintotheeye

bothwithandwithouttheex-vivoexpansionofconjunctivalepithelium.Thesuccessful

reconstructionofconjunctivaldefectswithex-vivoexpandedconjunctivalepithelium

onamnioticmembranehasbeendemonstratedforsmalldefectsincludingthose

followingtheremovalofpapillomataandpterygiainhumans.(69,70)Studieshaveshown

thatamnioticmembranetransplantationresultsinhealingwithoutsignificant

inflammationandcanresultintheregenerationofconjunctiva.(54)

Thesuccessofamnioticmembranetransplantation,however,dependsonthe

underlyingdiseaseprocess.Fornixreconstructioninparticularinthepresenceofactive

inflammatorydiseaseisassociatedwithrecurrenceofsymblepharaandforniceal

loss.(54)Inkeepingwiththis,adescriptivestudyreported12of17eyesrecovered

successfullyfollowingamnioticmembranereconstructionofshortenedfornices.

Clinicaloutcomeswerefavourableineyeswithsymblepharaduetotraumaticinjuryin

contrasttopatientswithsymblepharaduetomucousmembranepemphigoid.(71)Other

thanamnioticmembraneandoral/nasalmucosa,noothercellularordecellularised

humanallogeneictissueshavebeeninvestigatedasconjunctivalequivalents.(52,54)

1.3.2.1 Decellularisationofhumantissues

‘Biological’scaffoldsderivedfromdecellularisedtissueshavebeensuccessfully

transplantedinhumansforamultitudeofregenerativetherapiesincludingskin,heart

valves,vascularrepair,bladderandeventrachea.(72-75)Extracellularmatricesor

decellularisedscaffoldscanbepreparedbytheremovalofcellularcomponentsof

tissuesandorgans.Extracellularmatricesarerelativelyconservedacrossspeciesand

confertheoptimal3Denvironmentprovidingaplatformforrecellularisationwiththe

patient’sowncells,demonstratedthroughavarietyoforganmodels.(75,76)The

decellularisationprocessrenderstissuesimmunologicallyinerttherebyminimisingthe

immunologicalrejectionriskandenablestheuseofalargedonorpool.Thekey

advantagesofacellulartissueoversyntheticsubstitutesarethatthescaffolds

inherentlypossesstheultrastructureandgrossanatomicstructurenativetothetissue

oforigin.Theprecisenatureofthe3-dimensionalarrangementofmatrixproteinsmay

influencecelladhesionwithmoreappropriateligandspecificity.(77)Furthermorethe

biomechanicalpropertiesofthenativetissuesarealsoretainedandthereforehavethe

potentialtoinfluencefunction.

Decellularisedtissuesarecomprisedmainlyofextracellularmatrix,whichisthe

secretedproductofcellsnativetoaparticularorganortissue.Itthereforeprovidesa

uniqueopportunityforregenerationwithautologouscellsinwhichtheuniquecues

fromextracellularmatricesmayprovidetheoptimalconditionsforcellgrowth.The

tissuespecificityoftheextracellularmatrixisproposedtoinfluenceandmaintainthe

cellularfunctionandphenotypedemonstratedthroughavarietyoforganandtissue

modelsandevenincludesnerveallografts,whichhavebeenusedtorepairsensory

defectsinthehand.(78)Interactionswiththeextracellularmatrixhavebeenshownto

influencetheproliferationandchemotaxisofcells,demonstratedinmanytissuesand

includeendothelialandprogenitorcellmigration.(79)Theappropriatedifferentiationof

embryonicstemcellsandregenerationoftissueswithappropriatefunctionhasbeen

demonstratedinstudiesofseveralorgansincludingkidney,lungandtheadrenal

gland.(80-82)Differentiationoftissuesalongtheappropriatelineagetogetherwiththe

appropriatetissueremodellingresponseshavealsobeendemonstratedinavarietyof

tissuesasdescribedinmodelsincludingskeletalmuscleandvocalcord.(77,83,84)

Otherthantheextracellularmatrix,ithasalsobeenproposedthatthesurfacetopology

andthe3-dimensionalultrastructurearrangementalsoinfluencecellinteraction,

functionandphenotype.(77)Itiscrucialtherefore,thatthereisminimaldisruptionto

acellularscaffoldduringthedecellularisationtreatment.Decellularisationcanbe

achievedbyseveralmeansincludingphysical(e.g.temperature,forceandpressure),

enzymaticandchemicalprocesses.Themosteffectiveagentdependsuponfeaturesof

thetissueincludingitscellularity,densityandlipidcontentandmustbebalanced

againsttheadverseeffectsofextracellularmatrixdisruption.(77)Chemicalagentsused

inthedecellularisationoftissuesincludealcoholsandothersolvents,hypertonicand

hypotonicagents,acidsandbasesanddetergents.Enzymaticprocessesofteninclude

nucleases,collagenaseanddispase.(77)Oftheseagents,trypsinandcollagenasein

particulararerecognisedtoresultinsignificantextracellularmatrixdisruption.(77,85,86)

Collagenaseinparticularisgenerallyusedforapplicationsinwhichtheultrastructureof

thetissueisnotcritical.

1.3.1.2 Decellularisationofhumanamnioticmembrane

Thehumanamnioticmembranebearshistologicalsimilaritiesinitsgrossstructureto

humanconjunctiva,asitalsocomprisesanepitheliallayer,abasementmembraneand

substantiapropria.Aprotocolforthedecellularisationofhumanamnioticmembrane

hasbeendevelopedusingacombinationofchemicalandenzymaticprocesses.(87)In

thisprotocol,hypotonicandhypertonicsolutionsareusedwhichcausecelllysisand

thedissociationofDNAfromproteins,respectively,whilstcausingminimaldisruption

totheECM.Thedetergentsodiumdodecylsulphate(SDS)isusedtosolubilisecell

membranes,dissociatesDNAfromproteinsandisknowntobeahighlyeffective

decellularisationagent.(77,88,89)Finally,theprotocolalsoincludesbenzonase,an

endonuclease,whichcleavesnucleicacidsandeffectstheremovalofresidual

nucleotides.(90)Ofthesedescribedprocesses,thedetergent(SDS)stephasthegreatest

potentialfordisruptingtheextracellularmatrixandthereforealoweffective

concentrationshouldbesoughtandtheresidualtissuecharacterisedandtestedfor

cytotoxicity.

Ananimalstudyusingallogeneicandxenogeneicdecellularisedcorneaand

demonstratedsuccessfulintegrationofcornealtissuewithmaintenanceofcorneal

clarityandcellularingrowthincludingnerves(91)Thedecellularisationofporcinebut

nothumanconjunctivahasbeenreportedinliterature.Inthelatterstudy,

decellularisedporcineconjunctivawasusedasasubstrateforthetransplantationof

limbalstemcellsinarabbitmodel.(92)Todate,thedecellularisationofhuman

conjunctivahasnotbeenreported.

ThecollaboratorsofthisstudyatNHSBTalreadyproduceclinicalgradeacellulardermal

matrixdistributedthroughouttheUKandhaveexperienceindecellularisation,

characterisationandex-vivocellularexpansiononhumanamnioticmembrane.An

existingprotocolforthedecellularisationofamnioticmembranewillthereforebe

optimisedtoenablethedecellularisationofhumanconjunctiva.

1.3.2 Syntheticsubstratesforex-vivoexpansionofconjunctivalepithelium

Numeroussyntheticsubstratesforthedevelopmentofcornealgraftshavebeen

studiedincomparisontoconjunctivalsubstratesinwhichrelativelyfewreportsexist.

Theuseofaporousglycosaminoglycanco-polymermatrixhasbeendescribedforthe

repairoffullthicknessconjunctivaldefectsinrabbitsandthefornicealdepthcompared

withanun-graftedwoundinthefelloweye.(93)Asimilarstudydescribestheuseof

porouspoly(lactideco-glycolide)(PGLA)scaffoldmodifiedbyhyaluronicacidand

collagen.(94)Inthesestudies,fornicealshorteningwassignificantlygreaterintheun-

graftedeyes.Inbothstudies,theun-graftedeyeshadirregularlyarrangedcollagenin

keepingwithscartissueincomparisontoamoreregularcollagendeposition

demonstratedinthegraftedeye.(93)Bothsubstratesareexamplesofadegradable

matrix.Itshouldbenotedhoweverthatbothsubstrateswereinelasticandlackedan

epithelium.(73)Incontrastconjunctivalepitheliumculturedoncollagenmatriceswas

demonstratedtodevelopwithanappropriatecellpolarityandevenabasement

membrane,butwasonlyanorganisedmonolayer.(95)Thelatterstudyconfirmedthat

substratemodulationinfluencesthegrowthcharacteristicsofhumanconjunctiva.

Amorepromisingelasticsubstrateforconjunctivalregenerationhasbeentheuseof

plasticcompressedcollagen.(96)Laboratorystudieshavedemonstratedconfluent

growthofconjunctivalepitheliumincludingasubpopulationofprogenitorcells

identifiedbyputativestemcellmarkersandstudiesofcolonyformingefficiency.The

authorsofthisstudyreporttheultimatetensilestrengthofthesubstratesandhanding

wassimilartoamnioticmembrane.(96)Anotherelasticresorbablepolymer

demonstratedtosupportthegrowthofconjunctivalepitheliumincludinggobletcellsis

ultrathinPoly(ε-Caprolactone).(97)Bothsubstratesarebiodegradable.

Todatetherehasnotbeenanon-degradablesubstratedevelopedfortheregeneration

ofconjunctiva.Thismayprovideanidealsolutionfortheepithelialisationfollowing

fornixreconstructionandlong-termmaintenanceoffornixdepth.

1.3.2.1 Expandedpolytetrafluoroethylene(ePTFE)

Polytetrafluoroethylene(PTFE)isasimplepolymercomposedofcarbonandfluorine

andisknownforitsthermalstabilityanditshydrophobicity.Theregulararrangement

offluorineatomsaroundthecarbonbackboneconferslackofpolarityandtherefore

chemicallyinertness.(72)Amicroporousstructuremanufacturedthroughheatingand

expansioniscalledexpandedPolytetrafluoroethylene(ePTFE).(98)Themicroporous

structureallowsthefreemovementofgasandfluiddependingupontheporesize,

whichalsodeterminesitsflexibility.(72)Ithasbeenwidelyusedasabiomaterialasitis

non-reactivewithsurroundingtissuesandisnondegradableinabiological

environment.Degradationstudiesofnon-expandedPTFEtooxidationandacid

hydrolysisfoundPTFEtoberelativelyunaffected.(72)Indeed,degradationstudiesusing

ePTFEarelackinghowevernoevidenceofsystemicorlocaltoxicityhavebeenreported

asaresultofePTFEdegradationinsofttissueapplications.(99)Furthermore,thereare

noknownallergicorteratogenicresponsestothismaterial.(72,73)Thereisconsiderable

scopetomodifythesurfacechemistryofePTFEthoughanumberofmeansincluding

gasplasma,whichcanrenderthematerialmorehydrophilic.Itcanalsobeimpregnated

withanumberofagentsthatcanpreventbacterialcolonisatione.g.withsilverand

chlorhexidine.(73)

Theapplicationofexpandedpolytetrafluoroethylene(ePTFE)hasgrownoverthelast

30yearsandithasbeenwidelyusedinarangeofsurgicalproceduresincludingmesh

inherniarepairs,gynaecologicalsurgery,cardiacandvascularsurgery.Anexampleis

thePRECLUDERmembraneusedintheclosureandreconstructionswithinthe

pericardialspace.Adhesions,collagendepositionorcellularinfiltrationcouldnotbeen

demonstratedfollowingtheuseofthisbiomaterial.(100)Thisisanexampleinwhich

adhesionswiththehosttissuewereundesirablebutanon-degradableandnon-

immunogenicmaterialwasrequiredfortheclosureofadefect.ThePRECLUDER

membraneisanexampleofanePTFEmembraneinwhichtheporesize<1μmhasbeen

citedtolimittissueingrowthandthereforeadhesions.(100,101)

AnumberofreportsalsodescribetheuseofePTFEinophthalmicsurgery.Expanded

PTFEhasbeensuccessfullyusedasa‘stent’fortheshort-termfornicealmaintenancein

anophthalmicpatients.(104)Similarly,ithasalsobeenusedasa‘spacer’deviceforthe

excisionofseveresymblepharoninwhichrecurrencecanbepreventedbyblockingthe

physicalreappositionofthedenudedbulbarandtarsalsurfaceswhenre-

epithelialisationoccursinthepostoperativeperiod.(105)TheePTFEimplant,PRECLUDER

hasalsobeenusedwithlowdosemitomycin-Casanadjuvantinpreventingexcessive

blebleakageinpenetratingglaucomasurgery.(106)Inthelatterstudy,ePTFEwasused

asadevicetopreventexcessivefiltrationofaqueoushumourinaprocedurethat

createsanaqueousdrainingvalvethroughthescleratocontrolintraocularpressure.

Thedevicewaswelltoleratedandreducedtherateofearlyhypotony(excessive

reductioninintraocularpressure),acommoncomplicationfollowingfiltrationsurgery.

Intheseapplicationshowever,ePTFEwasusedasameansofphysicalsupportrather

thanasascaffoldforcellseedingandepithelialisation.Inalaboratorystudy,the

attachmentandproliferationofretinalpigmentepithelialcellshasbeendemonstrated

onammoniagasplasmatreatedePTFEformingamonolayeroffunctionalcells

expressingtightjunctionalproteincomponentsincludingZO-1andoccludin.(107,108)

Theseexamples,togetherwithreportsofendothelialcellepithelialisationdemonstrate

thepotentialforePTFEasasubstrateforcellularexpansionintissueengineering

applications.ThegrowthofconjunctivalepitheliumonePTFEhasnotpreviouslybeen

reported.

1.3.2.2 AmmoniagasplasmatreatmenttoalterePTFEsurfacechemistry

Plasmacomprisesionisedgaswithpositiveandnegativelychargedparticles.Ammonia

gasplasmacanbedevelopedaslowtemperatureplasmaandrequiresavacuum

chamber.Thegasplasmaresonatorisadeviceinwhichgasplasmacanbecreatedand

comprisesanairtightchamberconnectedtoavacuumpump,gasflowandelectric

powersupply.Theairwithinthechamberisevacuatedbythevacuumpumpbefore

ammoniagasisallowedtoenterthechamberatlowpressure.Thegaswithinthe

chamberisthenenergisedbyanelectricalcurrent,whichcreatesaglowdischargeand

avarietyofenergeticspecies.Theseincludeions,electrons,photonsandfreeradicals.

Surfacesincontactwithplasmaaresubjectedtoenergytransferfollowingcollisions

fromtheenergeticparticles.Thesechangesoccuronlyatthesurfaceofthematerial

andthereforedonotaffectitsbulkproperties.ThesurfacechemistryofePTFEcanbe

alteredsuchthataproportionofsurfacecarbon-fluorinebondsarebroken.Following

plasmatreatment,immersionofthesamplesintodistilledwaterleadstotheformation

ofhydroxylfunctionalgroupsinplaceofbrokencarbon-fluorinebonds,whichrenders

thematerialmorehydrophilic(Figure4).

TherearemanyapplicationsinwhichePTFEhasbeenchemicallymodifiedtopromote

adhesionandproliferationofepithelia.Cellularproliferationcanbepromotedby

surfacemodificationsincludingcoatingsthatarespecifictotheantigeniccuesrequired

bythespecificcelltype.ThesuccessfulendothelialisationofvascularePTFEgrafts,for

example,havebeendemonstratedfollowingtheuseofePTFEcoatedwith

heparin/collagenfilmscomprisingfunctionalisedCD133.(102)Similarly,gasplasma

modificationofePTFEincorporateshydroxyl(-OH)groupsonthesurfacerenderingit

hydrophilicandconducivetocellattachment.Furthermore,ithasalsobeenfoundthat

proteinadsorptionincreaseswiththe-OHgroupdensityfoundonthePTFEsurface.(103)

Whenthesurfacechemistryofthesematerialsismodified,thereispotentialfortissue

ingrowthe.g.meshinherniarepairinwhichtissueingrowthwithininternodalspaces

hasbeendemonstratedhistologicallyandlowherniarecurrenceandinfectionrates

reported.(73)

Figure4:IllustrationofthesurfacechemicalchangeinePTFEthroughammoniagasplasma

treatmentandimmersionindistilledwater.Energyfromgasplasmabreakssomeofthesurface

carbonfluorinebonds,whicharereplacedbyhydroxylfunctionalgroupsoncontactwithwater.

1.3.2.3 Determinationofthehydrophilicityofmaterialsthroughcontactangle

analysis

Thecontactangleistheangleatthermodynamicequilibriumofaliquiddropletwhere

itmeetsasolidinterfaceandagas/vapourinterface.Thisallowsestimationofthe

wettabilityofthemeasuredsurfacebytheYoungequationanddefinesthemolecular

interactionsofliquid,gasandsolidmaterial(Figure5).TheYoung-Laplaceequationcan

ascertaintheshapeoftheliquid/gasinterfacethroughYoung’sequation.(109)

OH H20 Ammoniagasplasma

Figure5:IllustrationofadropletonasolidsurfacewiththemeasurementsrequiredinYoung’s

equation.LGdefinesthegas-liquidinterfacialenergy(surfacetension),SGdefinesthesolid-

vapourinterfacialenergyandSLdefinesthesolid-liquidinterfacialenergy.Theequilibrium

contactanglecanbederivedfromtherelationshipbetweenthesevariablesthroughYoung’s

equation:γSG-γSL-γLGcosθc=0.(109)

1.4 Cultureconditionsfortheex-vivoexpansionofhumanconjunctival

epithelialcells

Traditionalcellculturemethodsfortheexpansionofconjunctivalepithelialcells

includeco-culturewithinactivatedmurine3T3feederlayers(immortalisedmouse

fibroblasts)andtheuseoffoetalcalfserum(FCS).(110)Althoughtheseculturemethods

improvecellstratificationandmorphology,theyarenotideallysuitedtoclinical

transplantapplicationsgiventheriskoftransferofxenobioticproteinsandinfective

agents.Someresearchgroupshavedemonstratedthatsuccessfuldevelopmentof

stratifiedconjunctivalepitheliumcanbeachievedthroughtheuseofautologousor

cordbloodserum.(111)

Therehavebeennumerouspublishedprotocolsforthecultureofconjunctivalepithelia

usingbasalmediavaryingfrombronchialepithelialgrowthmedium,RPMI,DMEM/F12

andkeratinocyteserumfreemedia.(11,70,112-114)Thevastmajorityofrecentstudies

avoidingtheuseofserumandmurine3T3’s,however,haveinsteadusedabasal

mediumofkeratinocyteserumfreemedia.Indeed,overrecentyears,thesuccessful

developmentofconjunctivalepitheliumhasbeenpossibleusingserumfree

alternatives.(67,97,115,116)Keratinocyteserumfreemediadoeshoweverrequirebovine

pituitaryextract(BPE)topromoteepithelialproliferation.Interestingly,positive

detectionofMUC5ACmRNAhasbeendemonstratedinstratifiedrabbitconjunctival

epitheliumdevelopedinkeratinocyteserumfreegrowthmedia.(116)Studieshavealso

showncomparableproliferativepotential,stratificationanddifferentiation

characteristicsincludingagobletcellsubpopulationdemonstratedthroughpositive

MUC5ACstainingofprimaryconjunctivalepithelialcellswhenculturedinbasalmedia

DMEM/F12withhumanserum,BPEorFCS.(117)Inmostcultureprotocols,thecell

primarycellcultureswereinitiatedwithaDMEM/F12mediacontainingFBSand

epidermalgrowthfactor(EGF)beforechangingtothekeratinocyteserumfree

media.(67,97,111)

Thereisnoconsensusontheoptimalgrowthmediaforconjunctivalepithelium,

particularlyinthecontextoftherequirementforanoptimalsubpopulationof

progenitorandgobletcells.Markedsimilaritieswerealsofoundbetweenculturemedia

protocolsestablishedbothfortheexpansionoftheHCjE-Gicelllineandprimary

humanconjunctivalepithelium.(67-70,97,113,118)

1.5 Characterisationofhumanconjunctivalepithelium

Thehumanconjunctivalepitheliumisderivedfrombipotentprogenitorcells,which

differentiateintostratifiedsquamousepithelialkeratinocytesinadditiontogobletcells

thatresembleglandularepithelia.Proposedmarkersfortheidentificationof

conjunctivaepitheliumbasedonmarkersofdifferentiationaredescribed.

1.5.1 Gobletcellmarkers

Gobletcellshavebeendemonstratedtooriginatefrombipotentprogenitorcellswhich

alsohavethepotentialtodifferentiateintokeratinocytes.(18)Cytokeratin7(CK7)and

theintracellulargelformingmucinMUC5AChavebeenproposedasmarkersofgoblet

cells.(11)Onlygobletcellsamongstallocularsurfaceepitheliasecretethelargegel

formingmucinMUC5AC.(28)MUC5ACisfoundinthetearfilmandistheresultof

secretionthroughtheprocessofexocytosisbygobletcells.(17)MUC5ACisahigh

molecularweightglycoprotein.

Alternativemethodsofidentifyinggobletcellsbasedontheirmucincontentinclude

UEA-1(UlexEuropaeusAgglutinin-1)lectin,HPAlectin(Helixpomatiaagglutinin),

PeriodicAcidSchiff(PAS)andAlcianBlue.(11)Lectinsareagroupofproteinsthatbind

specificcarbohydrategroups.UEA-1bindstoglycoproteinsandglycolipidscontaining

α-linkedfructoseresiduesandglycoconjugates.Cellshighinmucinsthereforecanbe

identifiedbythebindingofthesemoleculesandhavebeendemonstratedinstudiesto

localisetogobletcellsinculture.(11)PASisastainusedtoidentifyglycolipids,

glycoproteinsandmucins.Similarly,Alcianbluealsoidentifiespolysaccharidesand

mucopolysaccharides.AllofthesestainingmethodsotherthanMUC5ACdetection

thereforeidentifytransmembranemucinspresentonconjunctivalepithelial

keratinocytesinadditiontogobletcellscontainingMUC5AC.PASalsoidentifies

basementmembranes.

1.5.2 Progenitorcellmarkers

Arapidturnoverofconjunctivalepitheliumoccursthroughoutlife.Anearlierstudy

proposedtheauniformdistributionofprogenitorcellsthroughouttheconjunctiva

baseduponclonogenicabilityoffornicealandbulbarconjunctiva.(18)Morerecently,

however,Immunohistochemicalanalysisofprogenitorcellmarkersandcolony-forming

efficiencydeterminedthatprogenitorcellsoccuringreatestfrequencywithinthe

inferiorconjunctivalfornixandmedialcanthus.(19,119)

Conjunctivalstemcellsmaybeidentifiedbyanumberofcellmarkersthrough

immunologicaldetectionandpolymerasechainreaction(PCR).Arelativelywell-studied

markerisp63,interestinwhichinitiallyaroseinthe1990’swhenitwasfoundthatthe

absenceofp63haddeleteriouseffectsontheregenerativepotentialofepidermisand

stratifiedepithelia.(120)Itwassincesuggestedthatadefectinp63resultedinadefectin

stemcellrenewal.Althoughthepreciseroleofp63inthedifferentiationandapoptotic

pathwaysisunclear,cumulativeevidencesuggeststhatp63hasaroleinepidermal

stemcellrenewal.Furthermore,areductioninp63mRNAexpressionhasbeen

correlatedwithareductionintheproliferativecapacityofhumanepidermal

epithelium.(121)Itsroleinprogenitorratherthandifferentiatedcellsisalsosupported

bytheabsenceofp63transcriptionfactorsinparaclonesoflimbalandepidermalcells

thatwerepresentinholoclones.(122)Thereare6isoformsofp63andtheisoformmore

extensivelystudiedinepithelialcellsisΔNp63α.(9)Indeed,p63detectsbasal

conjunctivalepithelialcellsingeneralwhereasΔNp63αisfoundinamuchsmaller

populationofcellswithstemcelllikecharacteristics.(122,123)Ofthep63isoforms,the

ΔNp63αisoformhasbeenidentifiedasthepredominantsubtypeinocularsurface

epithelia.(9)

TheATPbindingcassettesub-familyGmember2(ABCG2)istheATPbindingand

transporterproteinandisalsoacommonlyusedmarkerfortheidentificationof

progenitorcellsinocularsurfaceepithelia(corneaandconjunctiva).Budakand

colleaguesidentifiedABCG2asacandidatestemcellmarkeranddemonstratedthe

activeeffluxofthedyeHoechst33342wasmediatedbytheABGC2transporter.(8)This

populationofcellscouldbeseparatedasasidepopulationrepresenting<1%ofcellsby

fluorescenceactivatedcellsorting.Thecellsdemonstratedbehaviourinkeepingwith

progenitorcellsincludingslowcyclingandclonogeniccapacityinvitro.(8)Budakand

colleaguesalsodemonstratedbyimmunohistochemistrythepresenceofABCG2basal

conjunctivalepithelialcellsfoundinclusters.(8)

PutativestemcellmarkersinoftheconjunctivaincludeABCG2andΔNp63.Thereare

nodefinitivestemcellmarkersoftheconjunctivaandthesemarkersmaydetect

transientlyamplifyingcellsinadditiontotruestemcellsandthereforewillbebroadly

regardedinthisstudyasprogenitorcellmarkers.

1.5.3 Cytokeratins

Cytokeratinsarepolypeptidesthatformtheintermediatefilamentsysteminepithelia

ofwhichthereare30relatedpolypeptides.Theyarebroadlydescribedwithintwo

groups:type1(neutral-basic)andtype2(acidic).Epitheliamaybecharacterisedbythe

specificpair(type1withtype2)ofcytokeratinsthatareexpressedinthecells.

Cytokeratinexpressionmaybealteredindiseasestates,differentiationandare

differentiallyexpressedbetweenepithelia.Furthermore,withinconjunctival

epithelium,cytokeratinexpressioncanbeusedtoidentifyconjunctivalepithelialcells

fromgobletcellsandthereforecanberegardedmarkersofdifferentiation.The

differentialexpressionofcytokeratinsincornealandconjunctivalepitheliahasbeen

recognised.ThecytokeratinpairCK3/12havelongbeenrecognisedasmarkersof

cornealepitheliawhereasCK13,CK19,CK7andMUC5AChavebeenconsidered

markersofconjunctivalepithelia.(10,124-127)ThespecificityofsomemarkerssuchasCK3

andCK19forcornealandconjunctivalspecificity,however,hasbeenquestionedbya

numberofauthors.(124,128,129)Inarecentimmunocytochemicalcharacterisationstudyit

wasdeterminedthatCK13andCK7couldbelocallisedtoallconjunctivalepithelial

layersandsuprabasallimbalepitheliumwhereasMUC5ACwasspecificonlyto

conjunctiva.(124)CK19,however,wasfoundinallconjunctivallayersbutwasalso

detectedinthesuprabasallimbusandperipheralcornealepithelia.(124)Qiand

colleagueslocallisedCK4andCK7tosuperficiallayersofbulbarconjunctiva,whereas

bothmarkerswereabsentincornealepithelium.(130)Baseduponthisevidence,this

studywillthereforeutiliseCK4,CK7,MUC5ACandCK19asmarkersofconjunctival

epithelium.

Cytokeratin7(CK7)ischaracteristicofglandularepitheliaandisfoundinlacrimaltissue

amongstotherapocrineglandsandtrachealepithelium.(125)Studiesinanimaland

humanconjunctivalepitheliumhavefoundthatCK7stainingcellshavemorphological

featuresincludingsecretoryvesiclesinkeepingwithgobletcells.Ithasbeensuggested

thattheCK7filamentmayfacilitatetheexocytosisofmucinbyinteractingmore

specificallywiththecontractileapparatusofthegobletcell,whichisnototherwise

presentinconjunctivalcellsofanepithelialphenotype.(125)Indeed,theupregulationof

CK7hasbeendemonstratedoninductivedifferentiationofgobletcellsinconjunctival

epithelialcultureswithaγ-secretaseinhibitor.CK7expressingcellsinthelatterstudy

alsoexpressedMUC5ACandexhibitedmorphologicalcharacteristicsinkeepingwith

gobletcells.(131)

ConflictingresultshavebeenreportedovertheexpressionofCK7betweencellsofa

gobletcellphenotypeandconjunctivalkeratinocytes.(11,132)Thereasonbehindthis

relatestothecloneofantibodyusedinwhichtheOV/TLclonebindstoallconjunctival

epithelialcellswhereastheRCK105clonehasgreaterspecificity.(133)Cytokeratin4

(CK4)expressionhasbeenfoundinconjunctivalkeratinocytesthatexhibitan

epithelioidratherthanaglandularcellmorphologysuggestingCK4expressing

conjunctivalepitheliaarekeratinocytesthatdifferbothmorphologicallyand

phenotypicallyfromconjunctivalgobletcells.CK4positivecellshavebeenlocallisedto

conjunctivalepitheliumandoftenreportedwithinitssuperficiallayers.(134)CK19

howeverisarecognisedmarkerofconjunctivalepitheliumanditsexpressionhasbeen

reportedthroughouttheconjunctivalepitheliuminbothinvivoandinvitrostudies.(10,

124,127,130,135)CK19isthereforeusedinthisstudyasapan-conjunctivalepithelial

marker.

1.5.4 Proliferationversusapoptoticmarkers

Thereareamultitudeofcandidatemarkersthatmaybeusedtostudyproliferationand

apoptosis.Proliferatingcellnuclearantigen(PCNA)andCaspase-3expressionhowever

havebeenwelldocumentedinconjunctivalepithelia.

Caspaseisaproteolyticenzymeinvolvedintheapoptosisofmammaliancells.

Subgroupsofcaspasesalsohavearecognisedroleininflammation(caspase-1,-4,-5,-

12).Caspasescanalsobegroupedbyfunctioneitherasinitiatorsorexecutorsof

apoptosis.(136)Ofthese,caspase-3isknownforitsroleintheexecutionofapoptosis.

TheactivationofapoptosisoccursviatheB-celllymphoma2(Bcl-2)familyofproteins

andisinitiatedinresponsetocellularstressfromavarietyofstimuliincludingcytotoxic

drugsorirreparableDNAdamage.(136)Caspase-3hasbeenexploitedinnumerous

studiesasamarkerofapoptosisinconjunctivalepithelialcellsincludinghuman

conjunctivalcelllinesandprimaryconjunctivalepithelialcells.(49,137-139)

Proliferatingcellnuclearantigen(PCNA)isfoundinallreplicatingeukaryoticcellsand

playsaroleinthereplicationofDNA.PCNAhadacentralroleinthes-phaseofthecell

cycleduringwhichthisproteinprovidesaplatformfortheprotein-proteinandDNA-

proteininteractionsthatoccurduringDNAreplication,repair,chromatinformationand

remodelling,andthecohesionofsisterchromatids.(140,141)Theimmunological

detectionofPCNAhasbeenextensivelyusedforthecharacterisationofhumancells

includingconjunctivalepithelium.(133,134)

1.6 Flowcytometry

Flowcytometryisascientificapplicationthatenablesthequantitativeanalysisofcells

insuspension.Theprincipleofflowcytometryisthatparticledetectionisbasedonthe

lightscatteringpropertiesofcellswhenexcitedbylaser.(142)Thefluidicswithinthis

systemisdesignedtocarryastreamoffluidwithcellsinsinglefiletothelaserswithin

thesystem.Multiplelasersexistwithintheflowcytometer,illuminatingtheparticlesat

varyingfrequency.(142)Anyscatteredlightandemittedfluorescenceisfocussedand

detected(Figure6).Thetechniquemostlyrequiresthefluorescentlabellingofcellsto

identifythecellassociatedmarkerbeingstudied,wherebythefluorescenceemittedby

cellsisdetectedafterexcitationfromtherespectivelaserattherequiredabsorbance

wavelength.Thetechniquethereforeallowsmultiparametricanalysisofcell

populationsfromseverallaserssimultaneously.

Figure6:Illustrationoftheprincipleofflowcytometry.Aheterogeneouspopulationofcells

labelledwithfluorescentantibodiesandmarkersinsolutionisdirectedintosinglefilewithina

streamoffluid.Laserlightsourcesexcitecellsandthelightscatteringcharacteristicsand

emittedfluorescenceisdetectedtoenablequantitativeanalysisofheterogeneouscell

populations.(142)

Excitationlasers/laserlightsource

Fluorescenceemission

Forwardandsidescatterproperties

Stainedcellsinsuspension

Hydrodynamicfocussingofcells

Bothindirectanddirectantibody-stainingmethodshavebeenemployedintheanalysis

ofconjunctivalepithelia.Monoclonalorpolyclonalantibodiescanbedirectedtothe

antigenofinterestandafluorochromeconjugatedsecondaryantibodytargetedtothe

specificimmunoglobulinandspeciessubtypeoftheprimaryantibody.Flowcytometric

analysiscanbeundertakenassingleormulticolouranalysisinvolvinglaserexcitation

frommultiplechannels.Thefluorochromesarechosenatextremesofwavelengthsto

eachotherinmulticolouranalysistoavoidoverlapindetectedfluorescence.

1.6.1 Flowcytometryfortheanalysisofconjunctivalepithelialcells

Flowcytometryhasbeenusedfortheanalysisofinflammatorymarkersindryeye

diseaseandrosacea.(143,144)Inamurinelaboratorycellculturestudy,conjunctival

gobletcellswereenumeratedbyflowcytometryusingcytokeratinspecificantibodies

CK4(conjunctivalepithelialcells),CK7(gobletcells)andMUC5AC(gobletcells).(145)In

anotherstudyofconjunctivalbrushcytologyspecimens,thesidescatterandforward

scatterpatternofhumanconjunctivalepithelialcellshavebeencharacterisedandCK7

usedasamarkerofconjunctivalgobletcells.(146)Theapplicationofflowcytometryfor

thequantitativeanalysisofintracellularmarkerscharacterisingprogenitorcellsin

conjunctivahasnothoweverbeenpreviouslyreported.

1.7 Mucousmembranepemphigoid

Ofthecicatrisingeyediseases,ocularmucousmembranepemphigoid(MMP)is

encounteredwithgreatestfrequencyinpatientsunderreviewinUKcornealand

externaleyediseaseclinics.(36)Furthermore,theriskofsightthreateningdiseasewith

advancingocularmucousmembranepemphigoidhasbeenregardedas‘highrisk’inan

internationalconsensusreview.(33)Forthisreason,itwillbeconsideredhereasa

prioritytreatmentgroupanddescribedingreaterdetail.

MMPencompassesagroupofacquiredautoimmunediseasescharacterisedbyatypeII

hypersensitivityresponseattheepithelialbasementmembranezone.Thereis

recognisedvariationinbothclinicalpresentationandcirculatingautoantibodiesto

targetauto-antigensinMMP,whichsuggestssignificantdiseaseheterogeneity.(147)

Suspectedantigensincludebullouspemphigoidantigens1and2,β4integrin,typeVII

collagenandlaminins5and6,presentinlaminalucidatransmembrane

hemidesmosomes.(33,148-150)Clinically,thisresultsininflammation,blisteringand

ulceration,whichultimatelyresultinfibrosisandscarring.MMPaffectsmucous

membranesinvaryingfrequencyandpatterninvolvingthemouth,upperairwaytracts,

oesophagus,conjunctiva,anusandgenitalia.(147)

Ocularinvolvementistypicallybilateralandcharacterisedbyconjunctivalinjection,

blisteringandulceration,whichleadtosubepithelialscarringandfibrosis.Thisresults

infornicealshortening,furtheradvancingcicatrisingchangeandmayultimatelyleadto

ankyloblepharon(adhesionsbetweentheeyelids)and‘frozenglobe’(adhesion

betweentheeyelidsandglobetogetherwithankyloblepharon).Thereiswidevariation

inthepresentationofMMPinwhichitmaybeseenintheclinicalsettingwithsignsof

acuteinflammationincombinationwithchroniccicatrisation.Delayedpresentationis

typicalhoweverwithlow-gradeinflammationresultinginslowlyprogressive

cicatrisation.(151,152)Patientstypicallypresentwithestablishedcicatrisingeyedisease

andsignsincludingfornicealshortening,conjunctivalkeratinisationandsymblephara.

Thediagnosisisalsooftenmissedduetothenon-specificnatureofocularirritationdue

todryness,conjunctivalinjectionandsub-epithelialfibrosisthatissubtleinearly

disease.(33)Alongitudinalstudyofocularmucousmembranepemphigoidpatients

foundyoungerpatientswithearlyonsetdiseasehadmoresignsofconjunctival

inflammationandrapidlyadvancingdisease.(36)Itisalsoofinteresttonotethat42%of

patientsdemonstrateddiseaseprogressionwithoutdetectablesignsofconjunctival

inflammationbasedonthedegreeofconjunctivalinjectionmeasuredatclinicvisits.(36)

Thisdemonstratestheimportanceofassessmentofdiseaseactivitybothintermsof

conjunctivalinflammationandcicatrisation,whichrepresentschronicchange.Early

diagnosisandaccurateassessmentofdiseaseactivityandprogressionistherefore

crucialtothemanagementofthischallengingcondition.

Aproformawasthereforedevelopedforuseincornealandexternaleyediseaseclinics

atStPaul’sEyeUnit,RoyalLiverpoolUniversityHospital.Anewproformawas

designedwiththeintentionofincludingrelevantaspectsoftheexaminationthatwould

assisttheclinicianintheassessmentofbotha)diseaseprogressionandb)disease

activity.Componentsofexistinggradingsystemshavebeenusedoradaptedandtheir

pertinentfeaturesdiscussedwithinsections1.7.1and1.7.2.

1.7.1 Gradingsystemstodetectprogressionofcicatrisationinocularmucous

membranepemphigoid

TheaccuratedocumentationandstagingofocularMMPisfraughtwithdifficultydueto

lackofstandardisationofstagingmethodsbetweenclinicians,observererrorsinthe

quantificationofcicatricialprogressionandtheinsidiousnatureofthediseaseprocess

itself.Ithasbeenrecognisedthatcicatrisationmayadvanceintheabsenceof

measureableconjunctivalinflammatorysigns.ItfollowsthereforethatocularMMP

warrantsassessmentbothintermsofconjunctivalinflammationandcicatricialfeatures

todeterminediseaseactivityandprogression.Severalgradingsystemsforcicatrisation

havedevelopedtogradetheseverityofclinicalfeaturesandaredescribedinthis

section.

TheFostergradingsystem(153)

ThestagingsystembyFostercategorisedthediseasesubjectivelyintostagesI-IV.

Fosterstages

I Subconjunctivalscarringandfibrosis

II Fornixforeshortening(ofanydegree)

III Presenceofsymblepharon(ofanydegree)

IV Ankyloblepharon,frozenglobe

TheMondinoandBrowngradingsystem(46)

MondinoandBrowndevelopedagradingsystemthatdescribedfornicealshrinkagein

termsofthepercentagelossoffornicealdepth.

Mondinostages

I 0-25%lossofinferiorfornixdepth

II 25-50%lossofinferiorfornixdepth

III 50-75%lossofinferiorfornixdepth

IV 75-100%lossofinferiorfornixdepth

Taubergradingsystem(154)

TheTaubergradingsystemutilisedFosterstagingI-IVbutincludedsub-divisionsfor

stagesIIandIIItodescribethedegreeoffornixforeshortening(asdescribedby

MondinoandBrown)anddegreeofinvolvementbysymblepharonrespectively.

StageII Percentagelossinferiorfornixdepth

StageIII Percentagehorizontalinvolvementbysymblephara

*a-d(describessubdivisionswithinIIandIII)

a 0-25%

b 25-50%

c 50-75%

d 75-100%

Rowseygradingsystem(155)

Rowseydevelopedasystemtoobjectivelymeasuremoresubtlechangesinocular

mucousmembranepemphigoidthanpreviousmethods.Thiswasbaseduponthree

measurementstakenat5,6and7o’clockfromthelimbustotheposteriorlidmargin

withtheeyeindextroelevation,upgazeandlaevoelevation(Figure7).The

measurementsaretakenwitharuletothenearestmillimetrewiththelowerlidheld

undermaximaltractionbeforetheglobepositionisaffected.Anaggregatescorewas

thencalculatedwithamaximalscoreof45wherebythenormalscorewas15mmfor

eachmeasurement.

Figure7:DiagramdemonstratingthemeasuredareasfortheRowseyscoringsystem.The

diagramaboveshowsthethreemeasurementsthataretakenfromthelimbustothelidmargin

at5,6and7o’clockfromthecorneallimbus.TakenfromRowseyetal.ArchOphthalmol.

2004;122:179-184.

Tauber-Liverpoolmethod(156)

Thegradingsystemsdescribedsofar(Foster,MondinoandTauber)gradelossof

fornicealdepthbyestimationofthepercentagereductioninthefornixdepth,whichis

thedistancefromtheposteriorlowerlidmargintothefornix.Difficultiesinthis

estimationmayariseduetothepoordefinitionofthefornixitselfduetothepresence

ofsubtarsalfibrosisandconjunctivalcorrugations.Furthermore,thesestagingsystems

wouldnotdetectsubtlechangegiventhelevelsofstagingrepresentmarkedchangesin

fornixdepth.TheTauber-Liverpoolmethodisanadaptionofthesystemspreviously

describedbyTauberandMondinoandmeasuresboththeverticalfornicealdepthand

horizontalinvolvementbymeasurementstakenalongthebulbarconjunctivalsurface.

Verticalfornicealdepth

Thisismeasuredtothenearestmillimetrebetweenthelimbusat6o’clockandthe

superioredgeofthefibrosiswiththelidheldgentlyundertractionandtheeyeheldin

upgaze.Thisvalueisthensubtractedfrom10andmultipliedby10togivethe

percentagefornixshorteningfromwhichgradea-d(Tauberstaging)isdetermined.

Horizontalgrading

Thehorizontalshorteningismeasuredobjectivelybyusingaclearruleandmeasuring

thehorizontaldistancefromthemedialandlateralcanthus2mmabovethestartofthe

inferiorfibrosis.Thecombinedwidthofanysymblepharonpresentisthensubtracted

fromthetotalconjunctivalhorizontalwidthtodefinethepercentagehorizontal

shortening.

Figure8:AphotographicexampleoftheLiverpool-Taubergradingsystem.TakenfromReeves.

G.GraefesArchClinExpOphthalmol(2012)250:611–618.Anexampleofgradingisshownin

theabovephotographswithmeasurements(mm)asfollows:a)verticalgrading(10-5)

x10=50%;b)horizontalgradinge.g.(27-(6+1+1+4))/27x100=56%

ThemainadvantageoftheTauber-Liverpoolsystemisthatitwouldallowsmall

changestobedetected.IncontrasttotheRowseysystem,theLiverpool-Taubersystem

attemptstoquantifythedistanceofthebulbarconjunctivabetweenthelimbusand

theedgeofthesubtarsalfibrosisusing10mmasanaveragereferencevalue.This

removespotentialvariationbetweenobserversintermsofthedegreeoftraction

appliedtotheeyelidwhilstenablingthedetectionofsmallerdegreesofchangethan

thetraditionalFoster,MondinoandTaubersystems.

Giventhevariationinthedescribedmethodsthusfarandlackofconsensusthusfaron

theappropriategradingsystemtouseforMMPpatients,allwillbeincludedwithinthe

proformaforcomparison.Afornixrulerwillalsobeusedtomeasurethecentralfornix

depthofboththeupperandlowerfornicesaspreviouslydescribedbyWilliamsand

colleagues.(157)Stagingsystemsdescribedthusfaronlymeasurecicatricialchangein

thelowerfornix.Theupperfornixmeasurementinparticularmaybeofsignificant

clinicalvaluegiventhatitisotherwisenotoriouslydifficulttoassessandquantify

objectively.

1.7.2 Assessmentoftheinvolvementoftheocularsurfaceandeyelidsto

gradediseaseprogressioninMMP

Thedescribedstagingsystemsgradecicatrisationbutnotthelevelofinflammatory

activitypresentatthetimeofexamination.Severalmethodshavebeenreportedfor

thegradingofbulbarconjunctivalhyperaemiaincludingtheOxfordgradingsystem,

Efron,andInternationalEyeResearchgrading.(158-161)Agradingschemeforconjunctival

inflammationspecifictoocularMMPhasbeenadaptedbySawetal.fromanoriginal

reportdescribinginflammatoryactivitybyElder.(162)Theadaptedgradingschemehas

beenusedinresearchincludingclinicaltrialsofocularMMPpatients.(163,164)This

gradingsystemisafive-pointscalebasedonstandardphotographs,whichmaybeused

togradeinflammationineachsectorofbulbarconjunctiva(Figure9).Thishas

thereforebeenusedaspartoftheLiverpoolproformafortheassessmentofMMP

patients(Appendix).

Figure9:OcularinflammationgradingsystembySawetal.Thisgradingsystemdemonstratesa

5-pointgradingofocularinflammationfrom‘minimal’to‘limbitis’.Takenfrom:Saw,V.P.Dart,

J.K.Rauz,S.etal.(2008)Immunosuppressivetherapyforocularmucousmembrane

pemphigoidstrategiesandoutcomes.Ophthalmology.115(2):253-261.(163,164)

Therearenoknownvalidatedscoringsystemsforthegradingofcornealorlid

involvementinMMPpatients.Agradingsystemfortheassessmentofcornealdisease

wassoughtfromtheliteraturetogradecornealinvolvementappropriatetoMMP

patients.Sotozonoandcolleaguespublishedagradingmethodforthedocumentation

ofdiseaseseverityinStevens-Johnsonsyndrome,whichincludedgradingof

conjunctivalisation,neovascularisation,opacification,keratinisationand

symblepharon.(165)Anobservermaygradediseaseseveritybycomparingstandard

photographstotheclinicalassessmentofthepatientundertakenattheslitlamp

(Figure10).AsStevens-Johnsonssyndromeisaninflammatoryocularsurfacedisease

withasimilarocularsignstothatfoundinMMP,itwasfeltthatthisgradingsystem

wouldbeappropriatetouse.Thecornealconjunctivalisation,neovascularisationand

opacificationcomponentsofthisgradingschemewerethereforeincludedthepro

forma.Thegradingofliddeformitieswasalsoincludedintheproformaandwasan

adaptationofexistinggradingschemesforentropion/ectropion.(166,167)Unfortunately,

nogradingschemesspecifictocicatrisingeyediseasecouldbefoundintheliterature.

Theadaptedentropion/ectropiongradingsystemsaresimplifiedversionsofthat

previouslydescribedintheliteratureandincludedocumentationofwhetherthe

medialorlateralaspectoftheupperorlowereyelidsareinvolved(Figure11,

Appendix).

Figure10:PhotographsforthegradingofocularsurfacemanifestationsofStevens-Johnson

syndromeasreportedbySotozonoetal.(2007).Theseimagesweretakenfromtheoriginal

publication:Sotozonoetal.(2007)Newgradingsystemfortheevaluationofchronicocular

manifestationsinpatientswithStevens-Johnsonsyndrome.Ophthalmology.114:1294–1302.(165)Theconjunctivalisation,neovascularizationandopacificationcomponentsofthegrading

systemwereusedintheMMPproforma.

Grade0 Grade1 Grade2 Grade3

Conjunctivalisation

Neovascularisation

Opacification

Keratinisation

Symblepharon

Figure11:Figuretoshowtheoriginalreportedgradingschemes(a)andtheadaptedversions

(b)forthedocumentationofentropionandectropion.Theentropionandectropionscales

weretakenfromandadaptedfromKempetal.(1986)andMoeatal.(2000)respectively.(166,

EctropionGradingScale0 Normaleyelidappearanceand

functionI Normalappearancebutsymptomatic;

eyelidlaxitypresentonexaminationII Scleralshowwithouteversionoflower

eyelidIII Ectropionwithouteversionoflacrimal

punctumIV Advancedectropionwitheversionof

lacrimalpunctumfromlacrimallakeV Ectropionwithcomplication(eg,

conjunctivalmetaplasia,retractionofanteriorlamella,orstenosisoflacrimalsystem)

L PredominantlylateralM PredominantlymedialLM Combinedmedialandlateralr Previousrevision*

Adaptedectropiongradingscale

• 0-normalappearance• 1-scleralshowwithouteversionof

lowerlid• 2-ectropionwitheversionoflidbut

withouteversionoflacrimalpunctum• 3-advancedectropionwitheversionof

lacrimalpunctumfromlacrimallake

M=Medial,L=Lateral

EntropionGradingScaleMinimal -Apparentmigration

meibomianglands-Conjunctivalisationoflidmargin-Lash/globecontactonup-gaze

Moderate -Apparentmigrationofmeibomianglands-Conjunctivalisationoflidmargin-Lash/globecontactinprimaryposition-Thickeningoftarsalplate-Lidretraction

Severe -Grossliddistortion

-Metaplasticlashes-Presenceofkeratinplaques-Lidretractioncausingincompleteclosure

Adaptedentropiongradingscale

• 0-normalappearance• 1-posteriorlidborderinverted

(apparentmigrationofmeibomianglands)

• 2-posteriorlidmargininvertedtointermarginalstriporlidretraction

• 3-wholelidmarginincludinganteriorborderinvertedorlidretractionresultinginincompleteclosure

M=Medial,L=Lateral

ThegradingofoculardrynessisalsogreatimportanceinMMP(pleaseseesection

1.2.1).AgradingschemeforthemeasurementofoculardrynessspecifictoMMP

patientscouldnotbefound,however,anumberofgradingschemesforoculardryness

wereobtainedfromtheliterature.(158-160,168)Oneofthemostwidelyusedschemesis

theOxfordgradingscheme.Thiswasincludedintheproformainitsunadaptedform

(Figure12).(158)Itsadvantagesincludetheapparenteaseinitsadministration;

especiallygiventhepictorialcomparisonsseenbelowandalsothatitgradesboth

cornealandconjunctivalepithelialinvolvement.Itfollowsthereforethatthiscouldbe

appropriateforuseinMMPpatients.

Figure12:FiguretoshowtheOxfordgradingschemeasdescribedbyBronetal.Takenfrom

Bronetal.(2003)Gradingofcornealandconjunctivalstaininginthecontextofotherdryeye

tests.Cornea.22(7):640-50.

0 Absent

I Minimal

II Mild

III Moderate

IV Marked

V Severe

1.8 Aimsandobjectives

Apliable,degradablegraftderivedfromdecellularisedconjunctivaltissuemaysuit

indicationsforaconjunctivaltransplantwherestructuralrigidityisnotarequirement,

forexamplefollowingexcisionofconjunctivallesions.Decellularisedconjunctivawould

offeralltheadvantagesofanextracellularmatrixscaffold,suchthatitclosely

resemblesnativetissuebuthastheadvantageofreducedantigenicity.Incontrast,a

non-degradableconjunctivalconstructderivedfromePTFEcapableofgreaterphysical

supportmaysuitapplicationssuchasfornicealreconstruction.

Thisstudywilldeveloptwosuchsubstrates,eachwithdifferingphysicaland

biochemicalpropertiesthatmayaddressarangeoftransplantationrequirements.The

aimsandobjectivesofthisprojectarebroadlycategorisedbelow.

TodevelopePTFEasasubstrateforconjunctivalepithelialexpansion

• Investigatetheeffectofammoniagasplasmatreatmentontheproliferation

andphenotypeofahumanconjunctivalcellline.

• InvestigatethepotentialofammoniaplasmatreatedePTFEtosupportprimary

conjunctivalepithelium

Todevelopdecellularisedhumanconjunctivaasabiologicalsubstrateforconjunctival

expansion

• Developanovelprotocolforthedecellularisationofhumanconjunctiva.

• Determinecytotoxicity,biochemicalalterationandDNAcontentof

decellularisedtissue.

• Cultivateconjunctivalepitheliumondecellularisedconjunctivaltissueand

characterisethemechanicalpropertiesandhistologyofthetissuein

comparisontoamnioticmembraneandePTFE.

Clinicalapplicationofnovelconjunctivalsubstrates

• Developaproformaforthedocumentationofclinicalsignsanddiseaseactivity

inMMPpatients.

• DescribeMMPdiseasesequelaeinasmallgroupofpatientsattendingcorneal

andexternaleyediseaseclinicsattheRoyalLiverpoolHospital.

2. Methods

2.1 Expandedpolytetrafluoroethylene(ePTFE)

2.1.1 AmmoniaplasmatreatmentofePTFE

CommerciallyavailableePTFE(0.64µmthickness,0.4µmporesize)wasobtainedfrom

GoodfellowsLtd(Cambridge,UK),dividedinto2x2cmsamplesandmountedintissue

culturescaffolds;CellCrowninserts(ScaffdexLtd);Figure13.TheePTFEsheets,

therefore,derivedsupportfromthecellcrownratherlikeascaffold.Itwasalso

straightenedsuchthatitwasheldasaflatsheetonwhichthesurfacechemistrywas

subsequentlyalteredbyexposuretoammoniagasplasma.Larger3x3cmsectionsof

ePTFEwerealsotakenofwhichsomewereleftuntreatedandsomeweresubjectedto

ammoniagasplasmatreatmenttoenabletheacquisitionofcontactangle

measurementstomeasurehydrophilicity.

Figure13:PhotographsdemonstratingthecellcrowncellcultureinsertswithePTFEmounted

withinit.a)Twocomponentsofthecellcrown.Ontheleft,thecellcrownisshownupside

downwiththeePTFEmembraneplacedacrossit(solidarrow).Theringontherighthandside

(dashedarrow)fitsoverthemembrane,securingitintoplace.b)Thisphotographshowsthe

ePTFEmembranemountedinthebaseofthecellcrown.Eachofthesewasplacedwithinawell

ofastandard12-wellcultureplateforcellcultureexperimentsaftersterilisation(section2.3.2).

AmmoniaplasmatreatmentofsampleswasachievedbyplacingtheePTFEmounted

cellcrownsinsidethereactionchamberofhelicalplasmaresonatorsystem,builtin-

houseusingapreviouslydescribedmethod.(108)Theplasmaresonatorsystembriefly

comprisedahalfwavehelicalresonatorplasmasystemassembledbyaglasstube

aroundwhicha100-turncopperwirewasdirectlywound.Thesystemhadaresonant

frequencyof13.6MHzandpower<1W.(169,170)Samplesweretreatedataflowrateof

ammoniaof85sccmfortwominutesat1.2x10-1mbarpressure.(108,171,172)Some

sampleswereplacedonthestagewiththeundersideoftheePTFEmembraneside

flushagainstthestagesuchthatonlytheinsideofthecellcultureinsertwasexposed

toammoniagasplasma(singlesidedtreatment).Samplesdesignatedforammonia

plasmatreatmentonboththeinsideofthecellcultureinsertanditsundersidewere

placedonthestageontheirsidetoallowexposuretogasplasmaonbothsidesofthe

membrane(doublesideammoniaplasmatreatment).

Afterremovingthesamplesfromthereactionchamber,sampleswereimmediately

immersedindistilledwateratroomtemperaturefor24hours.Thecellcrownswiththe

mountedePTFEwereallowedtodryatroomtemperaturebeforetheyweresterilised

usingaultraviolet(UV)cross-linkingmachine,CL-1000U.V.Crosslinker,(UVP,

Cambridge,UK)for5minutes(1500w)eachsidepriortouseincellculture

experiments.

2.1.2 ContactangleanalysisofePTFE

StaticcontactangleanalysiswasundertakenonammoniatreatedanduntreatedePTFE

todetermineachangeinwettabilityusingtheDSA100KrussDropShapeAnalyser

(Kruss,GmbH).Thisisanopticalcontactanglemeasurementdevicethatestimatesthe

wettingpropertiesoflocalisedareasonasolidsurface.

TheDSAsoftwaresuppliedwiththeequipmentwasused.Priortoanalysisthestage

wasappropriatelysetanda‘normalsessiledrop’selectedforanalysisfromthe

softwareoptions.Aliveimagewasvisualisedonthecomputer’smonitorandthe

dispensingneedlevisualisedwithinthecentreoftheimage.A10μLdropwasdispensed

andanimageofthedroplettaken.Thecontactanglewasdeterminedafterselecting

thecontactanglesettingsforasessiledropfittingforcontactanglesgreaterthan90

degreesandheight/width-methodforangleslessthan90degrees.Thiswasrepeated

sixtimesonrandomlyselectedareasoftheammoniaplasmatreatedePTFEand

untreatedePTFE.

2.2 Cellsandtissues

AllcellculturewasundertakenunderasepticconditionsinaclassIIbiologicalsafety

cabinet(Walker)cleanedwithVirkon®(DuPont)and70%ethanol.Cellincubationwas

withina37°Cincubator(NewBrunswick)maintainedwithinairwith5%CO2.

2.2.1 Cultureofahumanconjunctivalcellline

AhumanconjunctivalcelllinedonatedbyGipsonlaboratories(HCjE-Gi),SchepensEye

ResearchInstitute,HarvardMedicalSchool,Boston,USAwasculturedusinganadapted

protocolfromtheirlaboratories.Cellswereseededattherequiredcelldensityby

dispensingacalculatedvolumeofasolutionofevencellsuspension.Cell

number/densitywasdeterminedusingahaemocytometer.

Atanearlystageofculture,cellsweregrowninan‘earlygrowformula’(Media1):

Keratinocyteserumfreemedium(K-SFM,Invitrogen),2ng/mlepidermalgrowthfactor

(Invitrogen),0.25%bovinepituitaryextract(Invitrogen),1%penicillin-streptomycin

(Sigma),0.4mMcalciumchloride(Sigma).Approximately90%oftheexistingmediawas

removedandreplacedevery2-3daysusingmediathatwaspre-warmedat37°C.Cell

culturesweregrownin75cm2tissuecultureflasks(Greiner)inincubatorsat37°Cinair

with5%carbondioxide(CO2).

At70-100%confluence,Media2wasusedandcompriseda50:50mixoftheearlygrow

formulaandDMEM:F12(Invitrogen)with1%penicillin-streptomycinand10%foetal

calfserum(FCS)andcultureswereairliftedsuchthatthemediafluidlevelwasatthe

air-liquidinterfaceasdescribedinsection2.3.

2.2.1.1 Passageofconjunctivalepithelialcells

Passagingofcellcultureswasundertakenat70-100%cellconfluence.Anyremaining

mediainthecultureflaskswasremovedandreplacedwithTrypLE™(1xsolution,

Invitrogen).Thisagentwasusedtodissociatecellsfromthetissuecultureplatesby

incubationintheTrypLE™solutionat37°Cinairwith5%CO2for10minutesoruntil

completedissociationwasapparentonphasecontrastmicroscopy.Aneutralising

mediawasaddedtothedissociatedcellsuspensiontocreatea50:50mixand

centrifugedat1000rpm/180gfor5minutes.Theneutralisingmediacompriseda50:50

mixofDMEMandF12media(Invitrogen),10%FCS,3.5g/lHEPES,1%penicillin-

streptomycin(Sigma).Theresultingcellsuspensionwasresuspendedinmedia1

(section2.2.1)forfurthercultureorstorageasrequired.

2.2.1.2 Cryopreservation

AnysurplusHCjE-Gicellswerestoredlong-terminliquidnitrogen.Cellswere

dissociatedfromtissuecultureflasksusingTrypLE™(Invitrogen)asdescribedinsection

2.2.1.1andresuspendedin900μlmedia1and100μldimethylsulphoxide(DMSO)

(Sigma)bycontinuousmixingofthetubeandplacedincryovials(Starlab).Allcryovials

weresubsequentlyplacedinisopropanolcontainers(Nunc)inafreezerat-80oC.The

isopropanolsolutionsubjectsthecryovialstogradualtemperaturedeclinefromroom

temperatureto-80oCatarateof1oCperminute.After24hours,thecryovialswere

placedinliquidnitrogendewarsforlong-termstorage.

2.2.2 Retrievalofhumanconjunctivaltissueandcultureofprimary

conjunctivalcells

2.2.2.1Retrievalofcadavericconjunctiva

EthicalapprovalwasobtainedfromtheNationalHealthService(NHS)HealthResearch

Authority(NREScommitteeNorthWest)titled‘Isolation,characterisationand

expansionofhumanocularsurface(cornealandconjunctival)stemcells’;REC

reference11/NW/0766protocolnumber4182.Theethicalapprovalenabledthe

retrievalofhumanconjunctivaltissuefromdeceasedpatientsattheRoyalLiverpool

UniversityHospitalinwhomconsenthadbeenobtainedfromthenextofkinforthe

useoftissuesinresearch.ConsentwasobtainedeitherthroughtheUniversityof

Liverpool’sResearchEyeBankorbytheNationalRetrievalCentreofNHSBloodand

Transplant,basedinSpeke,Liverpool.AllpracticeswereinkeepingwithTheHuman

TissueAct(HumanTissueAct2004),theEuropeanUnionTissuesandCellsDirective

(TissuesandCellsDirective2004)andtheDataProtectionAct(DataProtectionAct,

1998).

Methodsforthedissectionofconjunctivaweredevelopedfollowingtrialofseveral

variationstothetechnique.Insummary,asinglelongsectionoftissuewasretrievedby

performinga360°peritomyfollowedbyalongtemporalrelaxingincisionandexcision

oftissueinaclockwiseoranticlockwisedirectionextendingasfarintotheconjunctival

fornicesaspossibletoremoveallbulbarandasmuchfornicealconjunctivaltissueas

possible.ThismethodwasinkeepingwiththeRoyalCollegeofOphthalmologists

standardsfortransplantationandresearch.Conjunctivalepitheliumwasretrievedfrom

botheyesfromeachdonorassoonaspossibleafterdeath.Tissueswerelogged

consecutivelyaccordingtothenumberingsystemofTheResearchEyebank,University

ofLiverpool.Theageandgenderofthedonorwasrecordedtogetherwiththenumber

ofhoursthatlapsedbetweentimeofdeathandretrieval.Retrievedtissuewaseither

storedin2-3mlphosphatebufferedsaline(PBS;Invitrogen)at-40°Cordissectedfor

cultureimmediately.

2.2.2.2 Explantcultureofprimaryhumanconjunctivalcells

Freshhumanbulbar/fornicealconjunctivaltissuewasdissectedfromitsunderlying

Tenon’scapsulelayeranddividedintoapproximately1x1mmtissuesectionsforuseas

explants.Fiveexplantswereseededondecellularisedconjunctivaltissuesections

mounted(section2.5)withintissuecellcrowns(ScaffdexLtd.)orientatedsuchthatthe

basementmembranesidefacedthesubstrate.Thetissueexplantswereculturedfor

thefirst24hoursinDMEM/F12mediawith10%fetalcalfserum,1%

penicillin/streptomycin(Invitrogen).Thesubsequent11daysinculturewereinthe

followingserumfreemedia:K-SFM(Invitrogen),0.2ng/mlEGF(Invitrogen),25μg/ml

BPE(Invitrogen),0.4mMCaCl2,1%penicillin/streptomycin.After12daysinculture,the

tissueswereairlifted(aspersection2.3)andthefollowingmediauseduptoday28in

culture:K-SFM(Invitrogen),10ng/mlEGF,1.2mMCaCl2,1%penicillin/streptomycin,

transferrin5µg/mlInsulin5µg/ml,triiodothyronine1.4ng/ml,adenine12µg/ml,

hydrocortisone0.4µg/mlepidermalgrowthfactor10ng/ml.Medialevelswere

monitoreduptotwicedailyandmediareplenishedasrequiredtoensurethatthe

liquidvolumemettheair-liquidinterface.

2.2.2.3 Cultureofcellsondecellularisedtissuesusingexplantsandisolated

cellsuspensions

Cellsfromthesametissuedonorwereusedtorepopulatedecellularisedamniotic

membraneandconjunctivalsubstrates.Decellularisationofsubstrateswascompleted

usingthedescribedprotocolinsection2.5.1using0.05%SDS(w/v)andmountedinto

CellCrowns(ScaffdexLtd.).Theconjunctivaltissuewasdividedintoexplantswithin

hoursofeyeretrieval.Fiverandomlyselectedexplantswerecultureddirectlyonthe

decellularisedtissues.Theremainingexplantswereseededontissuecultureplatesto

allowexpansionofconjunctivalepitheliumover10dayssothatadequatecellnumbers

developedtoenabletheseedingof1x105cells/cm2isolatedcellsinsuspensiononto

decellularisedsubstratesatalaterdate.After5daysinculture,theexplantswere

removedfromthecultureplatesandthenewlydevelopedepitheliumcontinuedto

divide.After10days,theconjunctivalepitheliumwasdissociatedwithTryPLEfor7

minutes.Agitationofthecellsuspensionwasundertakenbyusingapipettetotakeup

andreleasethesuspensionrepeatedlysuchthatcellsdisaggregatedina50:50mixof

neutralisingmediaaspreviouslydescribed(section2.2.1.1).Boththeexplantsand

isolatedcellswereseededinDMEM/F12mediawith10%fetalcalfserum,1%

penicillin/streptomycin(Invitrogen).After24hours,themediawasreplacedwith

serumfreemediaasdescribedinsection2.2.1.Theexperimentswerecompletedin

triplicateusingbothdecellularisedamnioticmembraneanddecellularisedconjunctiva.

2.3 Cellcultureonsubstrates

2.3.1 Cultureofcellsoncellcultureinserts

Thincerts(Greiner)wereusedasapositivecontrolinexperimentsofsynthetic

substrates.Thesecellcultureinsertsareproducedfromclearpolystyrenehousings,

andpolyethyleneterephthalate(PET)capillaryporemembranes.Thiswasusedasa

positivecontrolincellcultureexperimentswereitisreferredtoasPETmembrane.As

statedbythemanufacturer,themembraneculturesurfacehasundergonea‘physical

surfacetreatment’toimprovecelladherenceandgrowth.Severalporesizediameters

areavailable,however,the0.4�mporesizespecificationwasusedforconsistency

betweenthePETandtheePTFEtestedintheseexperiments.Thecellcultureinserts

weremanufacturedtofitincultureplatessuchthattherewasaspacebetweenthe

cultureinsertandbottomofthecultureplatewherethemediafluidlevelcouldbe

controlled(Figure14).Theprocessof‘airlifting’requiredthemedialeveltobeadjusted

totheleveloftheair-liquidinterfaceatthemembrane.Thismethodrequiredregular

monitoringofthefluidlevelstoensurethatthefluidlevelwasmaintainedattheair-

liquidinterface.

Figure14:Diagramtoillustratetheprocessofairliftingcellcultureinserts.Theinsert(blue)can

beplacedinacellculturewell(black)andissupportedsuchthatitissuspendedwithinthe

well.Themedia(yellow)isinsertedandshownheretocorrespondtotheair-liquidinterfaceof

thecellularlayer(purple).

2.3.2 CultureofcellsonePTFEmembrane

InsubstrateexperimentsinvolvingtheePTFEmembrane,anovelmethodforairlifting

wasdevelopedwherebythemembranemountedcellcrownwasplacedontopofa

ringshapeddevicethatallowedahighervolumeofmediainthecultureplatewhilst

airlifting(Figure15).Inpreliminarywork,itwasfoundthatalthoughregular

replacementofmediawasrequired,thecultureswerelesslikelytodryoutusingthe

ringdevicesthanwithoutgiventhatalargervolumeofmediacouldbedispensedwhen

airlifting.TheringdevicesweredesignedandmadebyJB,attheUniversityofLiverpool

workshopservices.Duringtheairliftingculturephaseofexperiments,mediawas

replacedoncetotwiceperday.

Figure15:Photographsoftheringdeviceandtheplacementoftheseringdeviceswithin

cultureplatestoenableairliftingofcultures.a)Thisringshapeddevicewasdesignedandmade

byUniversityofLiverpoolworkshopservices(JB).Thiswassizedtoenablethecellcrowntobe

positioned6mmabovethebaseofthewellplate.Mediawasdispensedwithinthisgap,filling

thewelltotheair-liquidinterfacefortheairliftingofcultures.b)Thisphotographshowsa12-

wellplatewiththeringdeviceineachwell.Thisholdsthecellcrowns6mmabovethebaseof

eachwell.ThesewereusedforcellcultureexperimentsinvolvingtheePTFEsubstrates.

2.4 Cellcultureandcharacterisationexperimentstoassesssynthetic

substrates

2.4.1 Cellseedingdensity

Cellseedingdensityexperimentswereundertakentodetermineanoptimalseeding

densityforallsubsequentexperimentalwork.CellcultureprotocolsusedforHCjE-Gi

cellsdescribeachangeinmediaafter7daysinculturewhencellsare75-100%

confluent.Thiswasthereforetheoptimalgoaltoreach.Thesubstratesusedforthis

experimentwereePTFEthatwasammoniaplasmatreatedontheinnersurface(cell

culturesurface),untreatedePTFEandPET(polyethyleneterephthalate)membranein

triplicate.Cellswereseededontriplicatesofsubstratesatadensityof1x103,1x104and

1x105cells/cm2.Cultureswerefixed,stainedwithDAPI(4',6-diamidino-2-phenylindole,

Sigma),andphotomicrographstaken(NikonTiE,Japan)after1,4and7daysinculture.

Thenumberofcellsperareafrom5randomareasofeachsampleweremanually

countedforeachofthesubstratesintriplicateandexpressedasthemeanpersample

persubstrate.Eachphotographedfieldwasacquiredusingthex20magnification

microscopelenswhichproducedaphotographwithasurfaceareaof12,420μm2.

2.4.2 Optimisationofmediaprotocol

EstablishedcellcultureprotocolsusingHCjE-Gicellsdescribeachangeinmediafrom

an‘earlygrow’(media1)toa‘lategrow’(media2)formulationonce75-100%

confluent.Giventhatthesubstratesusedwereopaque,thecelldensitycouldnotbe

assessedwithoutriskofdamagetocells.Anumberofcellcultureprotocolswere

thereforetestedusingHCjE-Gicellsseededat1x105/cm2ontheammoniaplasma

treatedePTFE,untreatedePTFE(negativecontrol)andPETmembrane(positive

control)intriplicatetodetermineasuitablemediaprotocolforthecultureofHCjE-Gi

cellsonePTFE.Mediaprotocolswere:A)media2only;B)media1for7daysfollowed

bymedia2;C)media1for3daysfollowedbymedia2;D)media1with1%BSA

(Sigma).CultureswerefixedandstainedwithphalloidinandDAPIafter1,3,7,10and14

daysinculture.Thenumberofcellsperareafrom5randomareasofeachsamplewere

manuallycountedforeachofthesubstratesintriplicateandexpressedasthemean

persamplepersubstrate.

2.4.3 ComparingcellcountsonePTFEwithammoniaplasmatreatmenton

oneandbothsides

Cellcountswererepeatedonafurtherexperimentthatwasrepeatedonthree

separateoccasionswithtriplicatesampleswithineachexperimentalgroup.Thiswas

undertakentodeterminewithgreateraccuracythecelldensitythatwoulddevelopon

eachofthetestsubstratesoveraperiodof28days.Itwasalsounknownwhether

exposingtheePTFEtoammoniagasplasmawithinthegasplasmaresonatoronone

sideonlyorbothsideswouldresultcouldinfluencethecelldensity.Thisvariablewas

alsothereforeintroduced.Cellcountswereundertakenfollowing2,14,21and28days

incultureusingthehaemocytometermethodtodeterminethenumberofcellsfound

onthesubstratepercm2followingdisassociation.Cultureswerealsofixedandstained

withDAPI,phalloidin,andUAE-1(UlexEuropaeuslectin;Genetex)atalltimepoints

studiedandrepresentativephotomicrographstakenusingafluorescencemicroscope

(Nikon)andconfocalimagingsystem(ZeissLSM510).

2.4.4 Fixationofsubstrateculturesandstainingwithfluorescentmarkers

Samplesusedforstainingwereculturedintriplicateandfixedatthetimepoints

measuredintheexperiment.Optimalmethodsforfixationandpermeabilisationwere

determinedtogetherwiththeoptimalconcentrationofstainingagents:UAE-1lectin,

phalloidinandDAPI(Table2).Fixationwasundertakenwith4%paraformaldehyde

(Sigma)for10minutespriortostainingsampleswithphalloidin.Sampleswerealso

stainedwithice-coldmethanol(Sigma)for10minutespriortostainingwithUAE-1

lectin(Genetex).Substrateswerewashedthreetimesinphosphatebufferedsaline

(PBS;Invitrogen)priortoincubationwithphalloidinorUAE-1lectinstainsatvarying

concentrationsasinTable2,bothfor1houratroomtemperature.Substrateswere

subsequentlywashedthreetimesinPBSandstainedwithDAPIfor10minutes.Stained

cellswereimagedusingconfocalmicroscopy(ZeissLSM510).

Stainingagent Supplier Dilution

DAPI Thermofisher 1:1000

Phalloidin Abcam 1:1000

UAE-1Lectin Vectorlabs 1:500

Table2:Detailsoffluorescentstainingagentswiththeiroptimiseddilutionsusedforthe

analysisofcellculturesdevelopedonsyntheticsubstrates.

2.4.5 Determiningcelldensityusingahaemocytometer

Cellsweredissociatedfromthesubstratesusing0.05%trypsin-EDTA(Sigma)under

incubationat37°Cwith5%carbondioxidefor5minutes.Theremovalofallcellsfrom

thesubstrateswasconfirmedbytheabsenceofDAPIstainingofsubstratesfollowing

theremovalofdissociatedcells.Cellssuspendedinaknownvolumewerecounted

usingahaemocytometer.A20μlsolutionwasplacedwithinthehaemocytometer

countingchamber,viewedunderaphasecontrastmicroscopeandcountedwithineach

ofthefour-1mm2cornersquaresofthegridandthemeannumberofcellsin1mm2

calculated.Allcellswithinthegridwerecountedtogetherwiththoseincontactwith

thetopandleftsideofallbordergridlines.Cellsincontactwiththebottomandright

sideofthebordergridlineswerenotincludedasperstandardpractice.

2.4.6 Flowcytometry

Thedatacollectedbyflowcytometrycanbepresentedinhistogramformatofasingle

parameter,emissionwavelengthsorindotplotsordensityplotsthatdisplaythe

correlationbetweentwoparameters.Thesoftware(Flowing2.5,TurkuCentrefor

Biotechnology)allowstheselectionofoneormorecellpopulationsofinterestfor

quantificationintermsofthepercentageofcellsthatitrepresents.Allthecellmarkers

usedinthisstudywereintracellularmarkers.TheHCjE-Gicellsrequireddissociation

fromtheirculturesubstratewithTryPLEfor10minutesat37oC.Followingdissociation,

thecellswerefixedwith2%paraformaldehydefor10minutesandthenwashedthree

timesbymixingthecellsinwashbuffer(gentlevortex)andcentrifugation.Thewash

buffercomprised1%BSA(w/v)and0.1%sodiumazide(w/v)andcentrifugationat

1000rpm/180gfor5minuteswasundertaken.Cellswerethenresuspendedinice-cold

methanolfor30minutesat4oC.Cellsweresubsequentlyblockedusing10%goatserum

for30minutesatroomtemperature.

Primaryantibodyincubationwasin1%goatserumfor1hourat4oC.Allincubation

stepswerefor30minutesat4oC.FollowingprimaryincubationwiththeCK19,CK7,

MUC5ACandCK4antibodies,cellswerestainedwiththesecondaryantibody

(secondaryincubationstep)withgoatanti-mouseIgGH&LAlexafluor405(ab175660,

Abcam)atadilutionof1:500.CellswiththeprimaryantibodiesΔNp63andcaspase-3

werestainedwiththedonkeyanti-rabbitAlexafluor647(Poly4064,Biolegend)

secondaryantibodyatadilutionof1:1000.AnadditionalincubationstepusingFITC-

conjugatedUAE-1lectin(Vectorlabs)atadilutionof1:500wasundertakenforall

samplesstainedwithcytokeratinandMUC5ACprimaryantibodies.Cellswerewashed

threetimesbetweeneachofthestepsdescribed.Theoptimaldilutionforeach

antibodywasinitiallydeterminedbytestingarangeofprimaryandsecondaryantibody

dilutionsalongwiththeisotypecontrolatcorrespondingdilutionstothatofthe

primaryantibody;rabbitIgGisotypecontrol(ThermoFisherScientific),mouseIgG1

isotypecontrol(ab91353,Abcam),MouseIgG2isotypecontrol(ab91361,Abcam).

Antibody Clone Supplier Dilution

CK19 Ab52625 Abcam 1:100

CK7 RCK105 Abcam 1:100

CK4 6B10 Abcam 1:50

MUC5AC 45MI Abcam 1:25

(FITCconjugated)

5D3 Millipore 1:25

p63 ΔN Biolegend 1:50

caspase-3 Asp-175 CellSignalling 1:50

(FITCconjugated)

PC10 Santacruz 1:50

Table3:Tableofantibodiesusedforflowcytometrywiththerespectiveclone,supplierand

optimiseddilution

2.4.7 ValidationexperimenttoensureappropriateuseoftheHCjE-Gicellline

Apreliminaryexperimentwasundertakentoensurethatimmunologicaldetectionof

thecaspase-3antibodybyflowcytometrywouldincreaseinHCjE-Gicellsgrownunder

environmentalstress.CulturesofHCjE-Gicellsgrownontissueculturethathadbeen

deprivedoffreshmediaforoneweekandleftoutsidetheincubatoratroom

temperaturefor24hourswerecomparedwithHCjE-Gicellsculturedunderoptimal

conditions(mediareplacedevery2-3daysandkeptinacellcultureincubatorinair

with5%CO2).Quantitativeassessmentofcaspase-3expressionbyflowcytometrywas

undertakenusingmethodsdescribedinsection2.4.6.

Thecelllinewasalsovalidatedtoensurethattheexpressionofallthecellmarkers

usedinthisstudydidnotvarysignificantlywiththepassageofcellsused.Cellsof

passage2and28wereanalysedbyflowcytometryusingthefullpanelofantibodies

(section2.4.6,Table3)after14dayscultureusingstandardcultureprotocolsthat

describedinsection2.2.1.Thecellswereculturedontissuecultureplatesintriplicate

foreachcellmarkerstudied.

2.4.8 Assessingthephenotypeofcultureswithadvancingtimeandby

substrate

AnexperimentwasundertakentodeterminethephenotypeofHCjE-Gicellsandtheir

subpopulationswithadvancingtimeincultureontissueculturepolystyreneandontest

substrates.Thetestsubstratesstudiedwere:treatedePTFEexposedtoammoniagas

plasma(section2.1.1)ona)oneside(singlesidetreatment)andb)bothsides(double

sidetreatment),PETmembrane(positivecontrol)anduntreatedePTFE(negative

control).HCjE-Gicellswereseededatadensityof1x105/cm2.Sampleswereanalysedin

threeseparateexperimentalsessionsforeachmarkerstudiedafter14and28daysin

culture.

Anequivalentsimilarexperimentwasalsoundertakenusingprimaryconjunctivalcells.

Thecellswereculturedfromconjunctivalexplantsexpandedontissueculture

polystyreneasdescribedforexpansionondecellularisedtissue(section2.2.2.1)overa

10-dayperiod.Cellsweresubsequentlydissociatedandseededatadensityof

1x105/cm2ondoublesidetreatedePTFEandPETsubstrates.TheuntreatedePTFEand

singlesideammoniaplasmatreatedePTFEfrompreviousexperimentscouldnotbe

usedinthisexperimentowingtothelowcellnumberavailableforthisexperimentand

thenecessityforsignificantcellnumberspersamplerequiredtoobtainreliableresults

byflowcytometry.Theexperimentwasundertakenonthreeseparateoccasionsusing

tissuefromadifferentdonorforeachexperimentalrun.

Thecellswerefixedandanalysedbyflowcytometryforarangeofconjunctival

epithelialmarkersafter14and28daysinculture.Co-stainingofCK7withUAE-1lectin

andABCG2withΔNp63(seesection2.4.6)wasquantifiedbysettingtheappropriate

voltagegate.Thegatedeterminedthepercentageofcellswithdualfluorochrome

stainingviaa2-parameterdotplotwherethegatesweredeterminedwithprior

analysisofsingleparameterhistograms.Forallothersamplesinwhichtherewasonlya

singleantigenofinterest,histogramswereusedforquantification.Flowingsoftware

version2.5wasusedforallanalyses.

2.5 Decellularisationofhumanconjunctivaanditscharacterisation

2.5.1 Decellularisationofhumanconjunctiva

Aprotocolforthedecellularisationofhumanconjunctivawasdevelopedbyadaptation

ofanexistingprotocolbyvariationofarangeofsodiumdodecylsulphate(SDS)

dilutions.Theoriginalprotocolbasedonapreviouslyoptimisedprotocolforthe

decellularisationofhumanamnioticmembraneatNHSBloodandTransplant.(87)Fresh

orfrozenconjunctivaltissue(at-40°C)wasallowedtothawatroomtemperatureand

washedindistilledwaterat200rpmat25°Candplacedinahypotonicbuffer(0.1%

EDTA,10mMTRIS,1%penicillin-streptomycin)at200rpmat4°Cfor21hours.The

tissuewasthenplacedinadetergentbuffer(10mMMgCl2,10mMTRIS,0.05-0.5%SDS,

0.1%EDTA,pH8,1%penicillin-streptomycin)at25°Cfor24hours,agitatedinanorbital

incubator(Table4).Tissuewerewashedthreetimesinawashbuffer(PBS,0.1%EDTA)

andplacedinanucleasebuffer(1U/mlbenzonase,10mMMgCl2,50mMTRIS,pH8,

1%penicillin-streptomycin)for37°Cfor3hoursat200rpm(Table4).Tissueswere

washedtwicefor20minutesat200rpmat25°Cinthewashbufferandfinallytwiceat

20minutesinPBSwith1%penicillin-streptomycin.

Reagent Source

Sterilewaterforirrigation Baxter

Trisbufferedisotonicsaline(TBS) Inverclyde

NaCl Sigma

EDTAdisodiumsalt VWR

TRIS VWR

MgCl2 VWR

HCl37% VWR

NaOH Sigma

Penicillin/streptomycin Sigma

Benzonase Novogen

Table4:Tableofreagentsusedfordecellularisationandtheirsource.

2.5.2 DNAextractionandquantification

A5mmtrephine(5mm)wasusedtocuttriplicatesamplesoftissueandeachsample

weighed.DNAextractionwasundertakenusingacommerciallyavailablekit(Easy-

DNATMgDNApurificationkit,Invitrogen,UK)usingapreviouslydescribedprotocol

usingEasy-DNAsolutionsandchloroform.(173)Inbrief,sampleswereincubatedinan

orbitalincubator(225rpm)for20hoursat60°Cwithinaproteindegradationsolution.

ThesupernatantfollowingcentrifugationwasusedforDNAprecipitation.Thisinvolved

using100%and80%ethanolsuspensionsandchloroformwithrepeatedcentrifugation

stepsusingtheupperaqueousphasepriortodigestionwithRNAse(EasyDNATMkit,

Invitrogen).DNAquantificationwasachievedusingacommerciallyavailablekit

(PicoGreen,Invitrogen)andastandardcurvewasdrawnfollowingthepreparationofa

rangeofcalfthymusDNAstandards,alsostainedwithPicoGreen.TheDNAincellular

anddecellularisedsampleswasquantifiedfollowingtheacquisitionoffluorescence

readings(FLX800microplatefluorescencereader,Biotek,UK)wherebyabsorbance

unitswereconvertedtotheequivalentcorrespondingDNAconcentrationusingthe

standardcurvefromthesameexperimentalrun.

DNAquantificationwasundertakentodeterminetheDNAincellularconjunctivaand

theresultingtissuesubjectedtothreevariationsinthedecellularisationprocessbased

upontheSDSdilutionused.Tissuesampleswereobtainedfromeachexperimental

group:cellular(untreated)conjunctivaltissueandtreatmentgroups:0.05%,0.1%and

0.5%SDS(w/v).Threeexperimentalrepeatswereusedforeachofthetissuessections,

whichinturnwerealsotakenintriplicatefromeachexperimentalgroup(n=9foreach

testgroup).Thestandardcurvemeasurementandcalculationswererepeatedfora

subsequentexperimentusingonlythe0.05%(w/v)SDSdecellularisationprotocolwith

conjunctivaltissuefromthreeseparatedonorsintriplicatewithexperimentalrepeats

asdescribedearlier.Thiswasundertakentoensurereproducibilityofthe

decellularisationprocessusing0.05%(w/v)SDS.

2.5.3 Collagendenaturation

Acollagendenaturationassaywasundertakenwherebyhydroxyproline,aproductof

collagendenaturation,wasdetectedthroughcolorimetricabsorbancevaluesusinga

previouslydescribedtechnique.(173)Inbrief,threetissuesamplesfromeachtestgroup

wereusedwithtriplicateexperimentalrepeatsfromeachtissuesample(n=9foreach

testgroupstudied).A5mmtrephinewasusedtocuttissuebiopsies.Asubgroupof

samplesweretreatedwith0.1NNaOHfor16hoursat25°Candusedasthepositive

controlwhereassamplestreatedwith50mMgluteraldehydeat25°Cwereusedas

negativecontrolsforhydroxyprolineestimation.Thetestsamplesweretreatedwith

10mg/mlalpha-chymotrypsinfor24hoursat37°C.Denaturedcollagenresultsinthe

releaseofhydroxyprolineintothesupernatantalongwithacorrespondingcolour

changeusingElrich’sreagent.Controlswithpre-determinedhydroxyproline

concentrationsandsamplesweresimilarlytreatedasfollows:12NHCLwasaddedtoa

sampleofeachsupernatantandautoclavedfor60minutesat121°C18psi.Chloramine

Treagentwasthenaddedtoeachsampleat25°Cfor25minutes.Thesubsequent

additionofElrich’sreagentat65°Cfor20minutesresultsinacolourchangethat

correspondstotheconcentrationofhydroxyprolineinsolution.Quantificationof

hydroxyprolinewasundertakenusingamicroplatereader(ELX808,Biotek,UK)

wherebyastandardcurvewasdrawnfromknownsimilarlytreatedserialdilutionsof

thehydroxyprolinecontrol.

2.5.4 Invitrocontactcytotoxicitytesting

Astandardmethodfordeterminingcontactcytotoxicitywasemployedasoutlinedby

theBiologicalevaluationofmedicaldevices-part5(2009);ISO10993-5.Conjunctival

tissuedecellularisedwith0.05%SDS(w/v)(section2.5.1)wastestedforevidenceof

cytotoxicityinvitrousingacultureofprimaryhumanskinfibroblasts(kindlydonated

byS.Rathbone,NHSBloodandtransplant,Liverpool)andahumanconjunctivalcellline

(HCjE-Gicells)intriplicate.Adisposable5mmtrephinewasusedtodividesectionsof

decellularisedtissue,whichwereattachedto24-wellplateswithSteristripsTM(3M,UK).

Cyanoacrylateglue(RScomponents,UK)andSteri-stripsTMalonewereusedasnegative

andpositivecontrolsrespectively.Allsamplesweretestedintriplicateandagainsttwo

celltypes:primaryhumanskinfibroblastsandHCjE-Gicells.1x105cellswereseededin

eachwellandincubatedfor48hoursinacellcultureincubatorwithairand5%CO2at

37°C.HCjE-Gicellsculturewasundertakenusingearlierdescribedmethods(section

2.2.1).PrimaryhumanskinfibroblastswereculturedinDMEM(Sigma),10%Foetalcalf

serum(Sigma),1%penicillin/streptomycin(Invitrogen).After48hoursinculturethe

sampleswithfixedinserialethanoldilutions:50%,70%,and100%,eachfor5minutes

atroomtemperature.Afterremovalofthe100%ethanol,thesampleswereallowedto

airdrybefore20%Giemsasolution(Sigma)wasaddedfor5minutesatroom

temperature.Thesampleswerethenrinsedindistilledwaterandphotographedonan

invertedmicroscope(Leica090-135-002,UK).

2.5.5 Biomechanicaltesting

Tensilestrengthtestingisanimportanttestinthefieldofmaterialscienceinwhicha

materialissubjectedtotensionatacontrolledrateuntilthematerialisdeformed

beyondrepairandeventuallybreaks.Thetensionisappliedbyplacingthesample

betweentwoclampsholdingthetissuethatmoveapartatacontrolledrate.The

elongationofthematerialismeasuredagainsttheforceapplied.Thechangeingauge

lengthisusedtocalculatetheengineeringstressσthroughthefollowingequation:

σ=Force(newtons)/Crosssectionarea(m2)

Strain(ε)isdefinedasthedeformationofthematerialduetostrainandcanbe

expressedasthefollowingequation:

ε=changeinlength(extension)/originallength

Young’smodulusofelasticityistheresistancethatagivenmaterialhastodeformation

inanelastic(non-permanent)mannerwhenatensileforceisapplied.Itisdetermined

bythestressandstraindefinedbythefollowing:

E=σ/ε

TheLloydInstrumentsUniversalTestingmachine(LRXplus,Lloydsinstruments,UKwas

usedtodeterminethetensilestrengthoftissuesamples.Conjunctivalsectionswere

dividedintoapproximately15mmx3mmsectionsandheldwithinclamps.The

thickness,lengthandwidthofeachsamplewereindividuallymeasuredwithVernier

callipers(DigimaticCD-6”C,MitutoyoUK;resolution0.01mm).Threemeasurementsof

widthandthicknessweretakenfromeachsampleandtheaveragethickness

calculated.Eachsamplewaspulledwitha5Nloadcellanddataincludingstress,strain

andelasticmodulusweregeneratedbyNexgensoftware(Canada).Aminimumof4

samplesweretestedfrom3differentdonors(n=15andn=14respectively)fromeachof

thecellularanddecellularisedtissuetestgroups.

2.6 Histologyandimmunohistochemistrydecellularisedtissuesand

recellularisedconstructs

2.6.1 Preparationoftissuesforhistologyandimmunohistochemistry

Tissuesampleswereunfoldedinto1x1cmtissuebiopsycapsules(CellSafe,Leica)and

fixedbyimmersionin4%paraformaldehydefor24hours.Thetissuebiopsycapsules

werethenplacedintotissuehistologycassettesandwereprocessedthrougha

histologytissueprocessor(Citadel2000,ThermoShandon,UK)overa20hourcycle

comprisingserialimmersionin3.7%neuralbufferedformaldehyde(Sigma),100%

ethanol,xylene(Leica)andFormulaRparaffin(Surgipath,UK).Tissuesampleswere

removedfromthetissuebiopsycapsulesfollowingtissueprocessingandembeddedin

aparaffin-embeddingunit(Shandon,UK).Followingwaxembedding,5-10μmsamples

werecutusingamicrotome(Finesse325,ThermoShandon,UK).Sectionsof5-10μm

wereusedinpreferencetothinnersections.Mountingandstainingwiththinner

sections(2-5μm)wasinitiallyattempted,however,problemswereencountered

includingpooradhesiontoslides,shearingandlossofthetissue,whichprevented

furtheranalysis.Triplicatesamplesweretakenfromatleastthreeseparateareasofthe

tissuesections.Tissuesectionsweremountedontosilanisedglassslides(Dako)after

placingthetissuesectionsinanelectrothermaltissuefloatbath(Cole-Parmer)at40°C.

Wetmountedslideswereplacedontoanelectrothermalslidedryingbenchat45°Cfor

10minutesbeforeleavingovernightinadryingovenat37°C.

2.6.2 Histology

Tissuesectionsweredeparaffinisedusingastandardmethodofserialimmersioninto5

minutesinxylene(Leica)andthen100,90and70%ethanolwashes.Stainingwith

HaematoxylinandEosin(H&E,Leica)wasperformedfor10minutes,rinsedinrunning

tapwater,dippedin1%acid-alcohol(Leica),rinsedinrunningtapwaterandimmersed

inEosin(Leica)for5minutes.Theslideswererinsedagaininrunningtapwater,and

dehydratedbyserialimmersionintoethanol70,90and100%in30secondintervals

priortofurtherplacementintoxylenefor10minutesbeforemountingontoglassslides

withDPX(Sigma)andglasscoverslips(ThermoScientific).

StainingwithPeriodicacidSchiff(Sigma)wasundertakenbyde-parrafinisationas

describedabovebeforeslideswereimmersedinPeriodicAcidSolutionfor5minutes,

rinsedthreetimesindistilledwater,immersedinSchiff’sreagentfor15minutesand

finallywashedinrunningtapwater.Glasscoverslipsweremountedaspreviously

described.

StainingwithvanGieson’sstainwasundertakenondeparaffinisedanddehydrated

tissuesections.SlideswereplacedinVanGieson’sstain(Surgipath,UK)for5minutes

andthenrinsedinrunningtapwater.Similarly,stainingwithDAPI(4',6-diamidino-2-

phenylindole)wasundertakenfollowingdeparaffinisationanddehydration,before

mountingtheslideswithfluorescentmountingmedium(Dako).

TissuesectionswereexaminedandimagedusinganOlympusBX60microscope

(Olympus,UK)andphotomicrographstakenofrepresentativesectionsfroma

minimumof9stainedtissuesectionsfromeachtissueblockstudied.

2.6.3 Immunohistochemistry

Severalantigenretrievalmethodsweretestedontissuesforeachstudiedantibodyto

identifytheoptimalmethod.Themethodsincludedincubationwithtrypsinfor30

minutesat37oC,incubatingwith0.01McitrateacetatebufferpH6(Sigma)ina

microwavefor5-15minutes,andimmersionintargetretrievalsolution(Dako)for5-20

minutesinawaterbathat95oC.Theoptimalmethodforallantibodiesstudiedwasthe

targetretrievalsolutionmethod.Slideswerewashedbetweeneachstepinawash

buffercomprising0.05%tween(Sigma),8.76%NaCl(Sigma),6.05%TRIS(VWR),pH

7.6.ImmunohistochemicalstainingwasundertakenusingtheEnvisionTMkitHRPanti-

mouse/rabbit(Dako).Slideswereincubatedwithaperoxidaseblock(0.03%hydrogen

peroxide,EnvisionTMkit)for10minutes,followedbyblockagewith20%goatserumto

preventnon-specificbindingfor30minutes(Dako)priortoincubationwithprimary

antibodiesatvaryingdilutions(Table5)for3hoursatroomtemperature.The

appropriatesecondaryanti-mouse/rabbithorseradishperoxidase(EnvisionTMkit)was

placedontissuesamplesfor30minutesatroomtemperature.Thechomogen,3-

amino-9-ethylcarbazole(AEC,EnvisionTMkit)producedpigmentationoftheimmune-

positiveareasontissuesamplesover5-10minutes.SlidesweremountedinAquatex

mountingmedia(MerckMillipore)andimagedonanOlympusBX60microscope.A

representativephotographwastakenfromthecentreofeachstainedtissuesection

fromaminimumof9tissuesections.Eachsectionwastakenfromtriplicatetissue

sampleseachfromthreeseparateareasoftheparaffinembeddedtissueblock.

Antibody Clone Supplier Dilution

CK19 Ab52625 Abcam 1:400

CK7 RCK105 Abcam 1:500

CK4 6B10 Abcam 1:200

ABCG2 BXP-21 Millipore 1:50

p63 ΔN Biolegend 1:50

Caspase-3 CPP-32 CellSignalling 1:50

PCNA PC10 Santacruz 1:50

Laminin L9393 Sigma 1:200

Fibronectin F3648 Sigma 1:250

CollagenIV Ab19808 Abcam 1:400

Table5:Tableofantibodiesusedforimmunohistochemistrywiththerespectiveclone,supplier

andoptimiseddilution

2.7 Recellularisationofdecellularisedconjunctivawithprimary

2.7.1 Explantcultureexperimentswithandwithouttheorientationof

basementmembraneofconjunctiva

Initialexperimentswereundertakenusingconjunctivaltissueinwhichtheorientation

andpresenceofbasementmembranehadnotbeenconfirmedormarkedpriortothe

decellularisationprocess.Insubsequentexperiments,theconjunctivawasexamined

underamicroscopetoconfirmthepresenceofconjunctiva(ratherthantenon’sor

adiposetissue)andwasmarkedbyplacing5/0nylonsuturesontheshiniersideofthe

membraneinanattempttoorientatethebasementmembranefollowing

decellularisation.Fiveexplantsweretakenfromasingledonorseededoneach

substrate(i)basementmembranepresentand(ii)basementmembranenot

present/noteasilyidentifiable,intriplicate.

2.7.2 Comparisonofconjunctivalepithelialculturesusingtissuefrom

differentdonorsforexplantsanddecellularisedsubstrates

Thisexperimentwasundertakentoexplorethevariabilityinexplanttissueoutgrowth

andwhetherthedecellularisedtissueitselfhasanyinfluenceonthedegreeof

outgrowth.Decellularisedtissuesthathadbeenstoredat-40oCwerethawedforusein

thisexperiment.Experimentswerecompletedusingtriplicatesamplesinwhich

conjunctivalexplanttissuefromthreeseparatedonors(15/17/19)wasusedoneachof

thedecellularisedtissuesamplesfromthreeotherdonors(9/5/13).Following28days

inculture,thetissueswerefixedandparaffinembeddedasdescribedinsection2.6.1.

Aminimalof3tissuesectionswastakenfrom3areasineachwaxparaffinblock.

Representativephotographsfromthecentreofthetissuesampleweretaken.Amniotic

membranewasusedasacontroltissueonwhichexplantsfromeachofthedonor’s

conjunctivalexplantswerecultured.

2.8 Statisticalanalysis

AllstatisticalanalysiswasundertakenusingSPSS22.0(IBM).Datawastestedfor

variancehomogeneityanddistribution.Alogtransformationofthedatawasusedto

meetthevarianceassumptionsfortheANOVAtest.Thiswasusedtoanalysedatafrom

thefollowingsets;contactangleanalysis,cellcount,DNAcontent,andtensilestrength

testing.Bonferronipost-hoctestswereappliedtoenableamoredetailed

interpretationoftheinteractionsbetweenindividualpairsoftreatmentgroupsinthe

model.Thepairedt-testwasusedtoanalysehydroxyprolinecontentinthecollagen

denaturationtestbetweencellularanddecellularisedsamples.

TheANCOVAmodelforchangewasusedtodeterminetheeffectofadvancingtimeand

substrateonnumerouscellmarkersassessedbyflowcytometryinconjunctivalcells.

Thechangeinpercentageexpressionofagivenmarkerbetweenday14and28wasthe

dependentvariable,andtheinteractionscalculatedfortheeffectofsubstrateandtime

withthevalueatday14astheco-variancefactor.TheHolm-Bonferronicorrectionwas

applied.ThisaccountedforthetotalnumberofANCOVAtestsperformedandwas

baseduponaninitialαvalueof0.05.Theuncorrectedpvalueshavebeenpresented

butonlythepvaluessignificantfollowingHolm-Bonferronicorrectionaredisplayed.In

testswherestatisticalsignificancewasdetermined,post-hoccontrastswere

ascertainedandafurtherHolm-Bonferronicorrectionwasappliedtodetermine

differencesbetweenthesubstratesstudied.Allstatisticalanalysesundertakenwere

verifiedfortheirappropriatenessbyastatistician(GC)inTheDepartmentofEyeand

VisionScience,InstituteofAgeingandChronicDisease,UniversityofLiverpool.

2.9 Detectionandmonitoringofocularmucousmembrane

pemphigoidpatients

2.9.1 Developingaproforma

Aproformawasdevelopedforthedocumentationofclinicalsignsinocularmucous

membranepemphigoid(MMP)patientstofacilitateimproveddocumentation.Itwas

developedinconsultationwiththeCornealandExternalEyeDiseaseteamatStPaul’s

EyeUnit,RoyalLiverpoolUniversityHospitalNHSTrust.Theproformamayenable

clinicianstostandardisethegradingofclinicalsignsandalsoprovidesopportunitiesto

establishadatabaseofpatientswhomaybenefitfromfuturecellularreplacement

therapies.Basedontheliteraturereview,theTauber-Kayemethodwasused.Ithas

proveninter-raterandintra-raterreliabilityandoffersadditionalquantitative

informationtothoseofTauber,MondinoandFoster.(156)Theproformaalsoincluded

thetraditionalTauber,MondinoandFostergradingsystemstogetherwith

photographyandfornixdepthmeasurementusingafornixrulertodeterminewhether

thereisanyadditionalvaluefromthesemethods.

Alltheaforementionedmethodsdonotincludeanydocumentationofthepresenceof

eyelidsigns,conjunctivalinflammationorcornealinvolvement.Methodspreviously

usedinclinicalstudiesofconjunctivalinflammationandcornealinvolvementandhave

beenincludedintheproforma.(33,163)Therehavebeennovalidatedgradingschemes

forcicatricialliddiseaseandthereforeadocumentationschemewasdevelopedfrom

other(non-cicatrising)entropionandectropiongradingsystemssothatitsseverityand

location(medial/lateral)couldbedocumented.(166,174)Thepresenceorabsenceof

lagophthalmos,trichiasis,co-existingocularpathologyandvisualacuitywasalso

included.

Theaimofthispartofthestudywastoallowasmallgroupofpatientswithocular

MMPtobeassessedtoenablediscussionofpotentialtreatmentstrategiesforocular

surfacereconstruction.Italsoprovidedanopportunitytocollectpilotdatathatcould

helpinformfurtherdevelopmentoftheproformaforuseinfutureclinicalstudies

(Appendix1).

2.9.2 AssessingMMPusingtheMMPproforma

ThepurposeofthecaseserieswasnottoobtainarepresentativesampleofMMP

patientsattendingcornealandexternaleyediseaseclinicsingeneral,buttoobtaina

smallsamplewithanyocularinvolvementtoenableasmallpilotstudytohelpdevelop

theMMPproforma.Thecasesacquiredforthisstudywererecruitedopportunistically

overa3-monthperiod.Patientsattendingacornealandexternaleyediseaseclinic

(ProfessorSBKaye,StPaul’sEyeUnit,RoyalLiverpoolUniversityHospital)were

examinedafterthepurposeoftheexaminationwasexplainedandconsentobtained.

PatientswereexaminedataslitlampandmeasurementsweretakenfortheFoster,

Tauber,andTauber-Liverpoolgradingschemesusingadisposableruler.Corneal

drynessgradingscoresweretakenaccordingtotheOxfordgradingscheme.(158)The

cornealandlidgradingwasundertakenafterexaminationaccordingtheproforma.A

perspexfornixrulerwasdesignedandproducedbyUniversityofLiverpoolworkshop

services(JB):Figure16.Fornixdepthmeasurementsweretakenaftertheapplicationof

proxymetacaine0.5%(w/v)drops(Minims).Measurementsweretakenongentle

insertionofthefornixruletothemaximumdepthoftheupperandlowerfornixatthe

midline.

Figure16:PhotographofthePerspexfornixruler.Eachgradationcorrespondsto1mm.The

gradedsectionisinsertedintotheupperandlowerfornices.ThethicknessofthePerspex

measuringarmofthefornixruleris1mm.

Chapter3:Results

3.1 Optimisationofculturemethodsfortheexvivoexpansionof

conjunctivalepitheliumonsyntheticsubstrates

3.1.1 AmmoniaplasmatreatmentofePTFE

UntreatedePTFEasreceivedfromthemanufacturerisknowntobehydrophobic.This

wasapparentfromtheformationofasphericalwaterdropletonitssurfacewhena

knownvolumewasdispensed.Thiswasincontrasttoawaterdropletofthesame

volumedispensedonammoniaplasmatreatedePTFE,whichassumedaflattershape,

exhibitinggreaterspreadonthesurfaceofthematerial.Thisindicatedincreased

wettabilityofthetreatedePTFEandwasconfirmedthroughstaticcontactangle

analysis(Figure6).Thecontactangleofadropletofwaterformedonammoniatreated

ePTFEwassignificantlymoreacutethanthosethatwereuntreated(71.08ovs.131.22o

respectively;p=0.02).

N Minimum Maximum Mean +/-SD

Untreated

6 127.4 135.9 131.2 3.2

Ammonia

plasmatreated

6 67.3 74.8 71.1 2.6

Table6:StaticcontactangleanalysisofuntreatedandammoniagasplasmatreatedePTFE.Six

readingsweretakenfromrandomlyselectedareasonammoniaplasmatreatedanduntreated

ePTFE.10μlwaterwasautomaticallydispensedandcontactanglebetweenthewaterdroplet

andmaterialrecordedbyaninbuiltvideorecorder.Statisticalanalysisshowedadifferenceata

p=0.02levelbetweentreatedanduntreatedePTFE.

3.1.2 Cellseedingdensityanalysis

Thehighestcellseedingdensitywasdemonstratedonallsubstratesfollowing7daysin

culturebyseeding1x105cells/cm2cells(Figures17-19).Thehighestcelldensitywas

notedonthePETmembrane(Figure18)andlowestontheuntreatedePTFE(Figure

19).LowcelldensitywasobservedonuntreatedePTFE,whichwasapproximately25%

oftreatedePTFE,evenatthehighestcellseedingdensityafter7daysinculture.Cell

densityonuntreatedePTFEwasalsocharacterisedbymarkedlyhighvarianceowingto

patchygrowthwherebycellswerevisualisedonsomeareaswithinaphotographed

field,whereasthecompleteabsenceofcellswasobservedinotherareas.Therewasan

approximately4-foldgreatercelldensityafter7daysincultureonammoniaplasma

treatedePTFEwhencellswereseededat1x105/cm2comparedto1x104/cm2.Thisisin

contrasttoPETmembranewherebythecelldensityafter7daysinculturewhencells

wereseededat1x104/cm2wasapproximately75%ofthatseededat1x105/cm2.The

representativephotomicrographsdisplayedevenlyspacedcellnucleiatahighdensity

ontheplasmatreatedePTFEatday7(Figure20).Thechosencellseedingdensityfor

subsequentexperimentswasthereforedeterminedas1x105/cm2forsubsequent

experimentalwork.StatisticalanalysisbyANOVAfoundasignificantassociation

betweencellsperphotographedfieldandtime(p<0.001),andcellsperphotographed

fieldandcellseedingdensity(p=0.006)overallacrossalltestsubstrates.

Figure17:Histogramtoshownumberofcellscountedperphotographedfieldwithadvancing

timeandincreasingcellseedingdensityonammoniaplasmatreatedePTFE.Thisgraphshows

thenumberofcellscountedmanuallyineachphotographedfield(+/-SD)at20xmagnification

oversettimepoints(day1,4and7).Eachphotographedfieldwas12,420μm2.Fiveareasper

photographedfieldwerecountedfromtriplicatesamples(n=15withineachexperimental

group).

Cells/pho

tograp

hedfie

Daysinculture

Day1 Day4 Day7

Cellseedingdensity/cm2

timeandincreasingcellseedingdensityonPETmembrane.Thisgraphshowsthenumberof

cellscountedmanuallyineachphotographedfield(+/-SD)at20xmagnificationoversettime

points(day1,4and7).Eachphotographedfieldwas12,420μm2.Fiveareasperphotographed

fieldwerecountedfromtriplicatesamples(n=15withineachexperimentalgroup).

Cells/pho

tograp

hedfie

ld 400

Daysinculture

Day1 Day4 Day7

timeandincreasingcellseedingdensityonuntreatedePTFE.Thisgraphshowsthenumberof

cellscountedmanuallyineachphotographedfield(+/-SD)at20xmagnificationoversettime

points(day1,4and7).Eachphotographedfieldwas12,420μm2.Fiveareasperphotographed

fieldwerecountedfromtriplicatesamples(n=15withineachexperimentalgroup).

Cells/pho

tograp

hedfie

Daysinculture

Day1 Day4 Day7

Figure20:RepresentativephotomicrographsofculturedsubstratesfixedandstainedwithDAPI

(bluefluorescentnuclearstain)after7daysinculture.Allmembraneswereseededatadensity

of1x105cells/cm2:a)ammoniaplasmatreatedePTFEb)PETmembranec)untreatedePTFE.

Scalebars100μm.

3.1.3 EffectofmediaonHCjE-Gicellproliferation

Fourmediaprotocolsweretestedtodetermineanoptimalprotocolforthecultureof

HCjE-Gicellsandareoutlinedbelow.Media1wasan‘earlygrow’formulawhereas

media2wasusedatalaterstagewhen70-100%confluenttoencouragestratification

anddifferentiation(pleaseseesection2.2.1and2.4.2).

MediaprotocolA:Media2used.

MediaprotocolB:Media1usedandchangedtoMedia2after7days.

MediaprotocolC:Media1usedandchangedtoMedia2after3days.

MediaprotocolD:Media1supplementedwith1%BSAused.

StatisticalanalysisbyANOVAfoundsignificantassociationsbetweencellsper

photographedfieldanddaysinculture(p<0.001),anddaysinculture(p<0.001)and

mediaprotocol(p<0.001)overallacrossalltestsubstrates.Thehighestcelldensitywas

demonstratedwhencellswereculturedonPETmembraneregardlessofthemedia

protocolused(Figures21,23,25,27).Cellsgrewatthelowestdensityusingmedia

protocolD(K-SFMmediasupplementedwith1%BSA)acrossallsubstrates(Figure27).

c)a) b) c)

CelldensitywasalsolimitedwiththeuseofmediaprotocolA(media2only)inwhich

celldensitydidnotriseabove150cells/photographedfieldafter14daysincultureon

anysubstrate(Figure21).Furthermore,theuseofmediaprotocolsAandDresultedin

anoveralldeclineincelldensityincontrasttoprotocolsBandCinwhichanincrease

wasdemonstratedwithadvancingtimeacrossallsubstrates.Celldensitywasrelatively

stablefromthestartingpointatdayoneonPETmembranewithuseofmediaprotocol

A,whereasthecelldensityontreatedanduntreatedePTFEdeclinedfromday1to14.

ThisisincontrasttocelldensityonPETusingmediaprotocolDinwhichahighercell

densitywasdemonstratedatday1,however,amorerapiddeclineincelldensity

occurredincomparisontotreatedanduntreatedePTFEwherecelldensitywaslower

butmorestablewithadvancingtimeinculture(Figure27).

MediaprotocolBandC(media1changedto2atafter7and3daysrespectively)

resultedinagreatercelldensitythanwiththeuseofmediaprotocolsAandDatall

timepointsthatincreasedwithadvancingtimeinculture(Figure23,25).Thegreatest

celldensitydevelopedfromtheuseofmediaprotocolBinwhichcelldensityatday14

was449cells/photographedfieldonammoniaplasmatreatedePTFEand522

cells/photographedfieldonPETmembranerespectively(Figure23).Celldensityon

untreatedePTFEreachedapproximately2/3thatachievedusingammoniaplasma

treatedePTFEwithmediaprotocolB(Figure23).Celldensitywaslowerusingmedia

protocolCacrossallsubstrates,however,increasedwithadvancingtimeincultureon

treatedePTFEandPETmembranes(Figure25).Adeclineinmeancelldensitywas

apparentafter7daysinculture,however,onuntreatedePTFE(Figure25).Qualitative

resultsfromnuclearstainingundertakeninparallelwithcellcountsareinkeepingwith

thedescribedobservations.Qualitatively,cellsappearevenlyspreadacrossthe

materials,demonstratedbyevenspacingbetweennucleidemonstratedbyDAPI

staining(Figures22,24,26,28).CellsgrownonammoniaplasmatreatedePTFEandPET

membraneatday14appearedconfluentasdemonstratedbytheconfluenceoff-actin

stainingincontrasttothesparsecellgrowthobservedonuntreatedePTFE(Figure29).

Figure21:Histogramtoshowmeannumberofcellscountedperphotographedfieldwith

advancingtimeonammoniaplasmatreatedePTFE,untreatedePTFEandPETmembraneusing

mediaprotocolA.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe

numberofcells(+/-SD)countedmanuallyineachphotographedfieldat20xmagnificationover

settimepointsday1,3,7,10and14.Eachphotographedfieldwas12,420μm2.Fiveareasper

photographedfieldwerecountedintriplicatesamples(n=15withineachgroup).

Figure22:RepresentativephotomicrographsofDAPIstainedcellsafter14daysincultureusing

mediaprotocolA.Allsubstrateswereseededatadensityof1x105cells/cm2:a)ammonia

plasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars100μm.

a) b) c)

1 3 7 10 14

Cellsperpho

tograp

hedfie

Daysinculture

UntreatedePTFETreatedePTFEPET

Figure23:Histogramtoshowthemeannumberofcellphotographedperphotographedfield

withadvancingtimeonammoniaplasmatreatedePTFE,untreatedePTFEandPETmembrane

usingmediaprotocolB.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe

after14daysincultureusingmediaprotocolB.Allsubstrateswereseededatadensityof1x105

cells/cm2:a)ammoniaplasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars100μm.

a) b) c)

Daysinculture

1 3 7 10 14

Cellsperpho

tograp

hedfie

UntreatedePTFETreatedePTFEPET

usingmediaprotocolC.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe

after14daysincultureusingmediaprotocolC.Allsubstrateswereseededatadensityof1x105

cells/cm2:a)ammoniaplasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars100μm.

a) b) c)

1 3 7 10 14

UntreatedePTFETreatedePTFE

Daysinculture

Cellsperpho

tograp

hedfie

usingmediaprotocolD.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe

photographedfieldwerecountedintriplicate(n=15sampleswithineachgroup).

after14daysincultureusingmediaprotocolD.Allsubstrateswereseededatadensityof

1x105cells/cm2:a)ammoniaplasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars

100μm.

a) b) c)

1 3 7 10 14

UntreatedePTFETreatedePTFE

Cellsperpho

tograp

hedfie

Daysinculture

Figure29:RepresentativephotomicrographsofHCjE-Gicellsafter14daysincultureusing

mediaprotocolBonammoniaplasmatreatedePTFE(a),untreatedePTFE(b)andPET

membrane(c).Greenstainingisphalloidin(f-actin)andbluenuclearstainingistheresultof

DAPIuptake.ConfluentmorphologywasdemonstratedonbothtreatedePTFEandPETand

sparsegrowthfoundonuntreatedePTFE.Phalloidinstainingwasabundanthoweverindividual

fibresweredifficulttovisualisewiththisstainonPETmembrane.Nucleiappearsmallerinsize

onthePETcomparedwithtreatedePTFEsubstrates.Scalebars50μm.

3.2 Exvivoexpansionofconjunctivalepitheliumonsynthetic

substrates

3.2.1 Comparisononcelldensitybetweensubstratesincludingammonia

plasmatreatmentofoneorbothsidesofePTFE

Thecelldensityatday28washigherwhentheePTFEwasexposedtotheammoniagas

plasmaonbothsidesofthematerialratherthantreatmentonlyonthesideintended

forcellseeding;p<0.001(Figure30).Thisdifferencewasmarkedafter14daysin

culture.CellcountsatalltimepointsstudiedwerelowestontheuntreatedePTFEand

highestonthePETmembrane(Figure30).Statisticallysignificantdifferenceswere

foundbetweenallsubstratesstudied(P<0.0001).

Figure30:CelldensityofHCjE-GicellsgrownonammoniaplasmatreatedePTFE,PET

membraneanduntreatedePTFEwithadvancingtime.Cellswereculturedusingmediaprotocol

Bseededat1x105/cm2.Datahasbeenlogtransformedtoallowparametricstatisticalanalysis

byANOVA.Theoverallmodelwassignificantp<0.001fortheeffectofbothtimepointand

substrate.Bonferronipost-hoctestsofthedifferencebetweenpairs(bothtimepointsan

substrate)inanycombinationwerealsohighlysignificant;p<0.001.

fcells/cm

Day2 Day14 Day21 Day28

Daysinculture

SinglesidetreatedePTFE

Substrate

UntreatedePTFE

DoublesidetreatedePTFE

PETmembrane

3.2.2 Morphologyofconjunctivalculturesdevelopedonsyntheticsubstrates

Alongwithcelldensity,themorphologyofcultureswasalsostudied.F-actinstaining

demonstratedbyredfluorescentstainingwithphalloidintogetherwiththeblue

nuclearstainingfromDAPIshowsconfluentgrowthonPETandtreatedePTFEtowards

themidtolatetimepoints(Figures31-37).Sparsegrowthwasdemonstratedon

untreatedePTFEatalltimepointsstudied.Phalloidinstainingwassomewhatdifficult

toassessinPETcultures,astheactinfibreswerenotvisibleasdiscretelyastheywere

withcellsimagedonePTFEsubstrates.ThiswasovercomebyUAE-1lectin,which

demonstratedthecellmorphologyofHCjE-Gicellsonallthesubstratesclearlyby

stainingofcellmembranestogetherwithintracellularmucin(Figures38-44).The

morphologyofcellsonPETmembranedemonstratedthatculturedcellsweresmaller

insizewithproportionallysmallernucleiincomparisontothoseculturedonammonia

treatedePTFEindicatedbytheoutlineofthecellsthroughUAE-1lectinstaining(Figure

40).Theshapesofthecellswereepithelioidandassumed‘cobblestone’morphology

onallthesubstrates.AlthoughthecellsandtheirnucleiweresmalleronPET

membrane,noothersignificantmorphologicaldifferencesinHCjE-Gicellmorphology

werequalitativelydemonstratedbetweenPETandallotherePTFEsubstratecultures.

ThiswasdemonstratedthroughUAE-1lectinandphalloidinstaining(Figures31-44).

AlthoughthecellsonuntreatedePTFEappeared‘rounded’inappearanceinitially,they

becamemoreepithelioidinappearancewithadvancingtimeincultureasthecell

densityincreased.ThecelldensityappearedthelowestonuntreatedePTFEandlower

onsinglesidedtreatedthandoublesidetreatedePTFE,qualitatively,inkeepingwith

thecelldensityanalysis(Figure30).

Figure31:Representativephotomicrographsofnuclearandf-actinstainingofHCjE-Gicells

culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter2daysinculture.

CellsizeappearssmalleronPETmembraneshowevercelldensityappearedgreatest.Lowest

celldensitywasapparentonuntreatedePTFEwithcellsmore‘rounded’inappearancethanon

ammoniaplasmatreatedePTFEsubstrates.Scalebars50μm.

DAPI(blue) Phalloidin(red)

SinglesideammoniaplasmatreatedePTFE

DoublesideammoniaplasmatreatedePTFE

PETmembrane

UntreatedePTFE

CellsizeappearedthesmallestwithgreatestdensityonPETmembrane.Similarmorphology

wasapparentontreatedePTFEandPETmembrane.Lowestcelldensitywith‘rounded’cells

wasapparentonuntreatedePTFE.Culturesweremoreconfluentondouble-sidetreatedePTFE

thansinglesidetreatedePTFE.Scalebars50μm.

PETmembrane

UntreatedePTFE

LowestcelldensitywasapparentonuntreatedePTFEhoweverthereisgreatervariationinthe

sizeandshapeofthenuclearmaterialthanonanyothersubstrate.Culturesweremore

confluentondoublesidetreatedePTFEthanfollowingsingleside-treatmentandcellsappear

tohavemoreofcobblestonemorphologythanonothersubstrates.Scalebars50μm.

PETmembrane

UntreatedePTFE

Figure34:Representativeconfocalz-stackseriesofsinglesideammoniaplasmatreatedePTFE

after28daysofHCjE-Gicellculture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)staining.

Fivesliceshavebeentaken,eachin4.5-5μmintervalswherebythetopandbottomofthez-

stackhadbeenmanuallysetafterthefocuspointsatthetopandbottomofthecellcultures

werelocated.Scalebars50μm.

Figure35:Representativeconfocalz-stackseriesofdoublesideammoniaplasmatreated

ePTFEafter28daysofHCjE-Gicellculture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)

staining.Fivesliceshavebeentaken,eachin4.5-5μmintervalswherebythetopandbottomof

thez-stackhadbeenmanuallysetafterthefocuspointsatthetopandbottomofthecell

cultureswerelocated.Scalebars50μm.

Figure36:Representativeconfocalz-stackseriesofPETmembraneafter28daysofHCjE-Gicell

culture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)staining.Fivesliceshavebeentaken,

eachin4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanuallyset

afterthefocuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars50μm.

Figure37:Representativeconfocalz-stackseriesofuntreatedePTFEafter28daysofHCjE-Gi

cellculture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)staining.Fivesliceshavebeen

taken,eachin4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanually

setafterthefocuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars

50μm.

Figure38:RepresentativephotomicrographsofnuclearandUAE-1stainingofHCjE-Gicells

LowestcelldensitywasapparentonuntreatedePTFE.OverallgreaterUAE-1(intracellularand

membraneassociated)stainingwasapparentoncellsculturedonePTFEthanPETmembranein

whichstainingappearedmorediscreteandmostlymembraneassociated.Scalebars50μm.

UntreatedePTFE

PETmembrane

DAPI(blue) UAE-1Lectin(green)

LowestcelldensitywasapparentonuntreatedePTFE.ThegreatestintensityofUAE-1staining

appearedonPETmembraneanddoublesidetreatedePTFE.Stainingofcellmembranes

showedthecellsizewasgreateronePTFEcellculturesthanPETcellcultures.Scalebars50μm.

PETmembrane

UntreatedePTFE

LowestcelldensitywasdemonstratedonuntreatedePTFE,whereasthegreatestdensitywas

demonstratedonPETmembraneanddoublesidetreatedePTFE.CellsizewasgreateronePTFE

cellculturesthanPETcellcultures,andwasmorepronouncedondoublesidetreatedePTFE,

especiallyamongstthecellswithgreaterintracellularstaining.Scalebars50μm.

PETmembrane

UntreatedePTFE

Figure41:Representativeconfocalz-stackseriesofsinglesidetreatedePTFEafter28daysof

HCjE-Gicellculture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Fivesliceshavebeen

50μm.

Figure42:Representativeconfocalz-stackseriesofdoublesidetreatedePTFEafter28daysof

HCjE-Gicellculture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Fivesliceshavebeen

50μm.

Figure43:Representativeconfocalz-stackseriesofPETmembraneafter28daysofHCjE-Gicell

culture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Fivesliceshavebeentaken,eachin

4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanuallysetafterthe

focuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars50μm.

Figure44:Representativeconfocalz-stackseriesofuntreatedePTFEafter28daysofHCjE-Gi

cellculture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Foursliceshavebeentaken,

eachafter3μMwherebythetopandbottomofthez-stackhadbeenmanuallysetafterthe

focuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars50μm.

3.3 Retrievedhumanconjunctiva

Humanconjunctivawasretrievedfrom26eyesof13tissuedonors.Sixdonorswere

maleand7donorswerefemalewithameanageof84.2years(range65-92).Themean

timetoretrievalwas20.4hours(range7-32hours).

Donornumber Gender Age Timetoretrieval(hours)

1,2 Male 84 19

3,4 Male 87 7

5,6 Female 96 29

7,8 Male 76 26

9,10 Female 80 16

11,12 Female 90 12

13,14 Male 92 24

15,16 Male 77 9

17,18 Female 88 19

19,20 Female 65 23

21,22 Female 85 28

23,24 Male 84 22

25,26 Female 91 32

Table7:Tableoftheageandgenderoftissuedonorstogetherwiththepost-mortemretrieval

time.Thedonoreyesweredesignatednumberedsequentially,lefteyefollowedbyrighteye.

3.4 PreliminaryoptimisationandvalidationoftheHCjE-Gicelllineand

flowcytometry

3.4.1 Optimisationofantibodystainingforflowcytometry

Manyoftheantibodiesidentifiedforthisstudywerenotconjugatedtoa

fluorochrome.Testingtheoptimaldilutionforboththeprimaryandsecondary

antibodywasundertakenbeforeanyexperimentalworkwasconducted.The

incubationperiodwasalsooptimised.Theoptimalprotocolwasdeterminedwhena

shiftinfluorescencewasclearlydetectablewithminimaloverlapfromanon-stained

population.Forexample,severalconcentrationsofUAE-1lectinweretestedwith

variationsinlengthofincubation,however,thisdidnothaveasignificanteffect(Figure

45).Incontrast,greaterseparationofcellpopulationswasobservedbyvariationinthe

dilutionofUAE-1lectin.Anoptimalconcentrationof1:500atanincubationtimeof30

minuteswasdetermined.

Thedetectedfluorescenceofsamplestreatedwithunconjugatedprimaryand

secondaryantibodieswerecomparedagainsttheirrespectiveisotypecontrols.In

addition,thesecondaryantibodyalonewasalsotestedatvaryingdilutionstofindan

optimalbalancebetweenbrightstainingandnon-specificbinding.Thepercentageof

thecellpopulationthatrepresentedapositivelystainedpopulationwasdeterminedby

quantitativeanalysisofthecellpopulationthatemittedfluorescenceatagreater

intensitytothecontrolsamples:i)therespectiveisotypecontrolincombinationwith

itssecondaryantibodyii)thesecondaryantibodyalone.Experimentsforeachantibody

werethereforeundertakenusingsecondaryantibodyaloneinadditiontowitheach

dilutionoftheprimaryandthesecondaryantibodycombinationandthecorresponding

concentrationofisotypecontrol.Theexampleofantibodyoptimisationshownin

Figure46demonstratesadegreeofnon-specificantibodybinding.Thisoptimisation

stepdeterminedthatthegreatestseparationincellpopulationswasdetectable(and

thereforeleastnonspecificbindingoccurred)whenthedilutionofprimaryand

secondaryantibodywasusedat1:50and1:1000respectively.

Figure45:FluorescencechannelhistogramsofHCjE-Gicellsdemonstratingtheresulting

fluorescenceafterstainingwithvariousconcentrationsofUAE-1lectinover30(b)and60(c)

minutes.Thedashedlineonallthehistogramsandarrowonthehistogramoftheunstained

controlsample(a)indicatesthefluorescencebeyondwhichstainingwasregardedaspositive.

Thesehistogramsdemonstratethattherewaslittleeffectofincubationtimeandtherefore30

minuteswassufficient.Atalltheantibodydilutionsstudied,therewasmarkedseparationof

thehistogramfromanunstained(control)sampleofcells,however,the1:500dilutionwas

optimal.

Unstained

1:100,30minutes 1:500,30minutes 1:1000,30minutes 1:2000,30minutes

1:100,60minutes 1:500,60minutes 1:1000,60minutes 1:2000,60minutes

Figure46:Thehistogramsaboveshowtheresultsattheoptimaldetermineddilutionsofthe

primaryΔNp63antibodyandsecondaryantibody(1:50primaryand1:1000secondary)

comparedwithanisotypecontrolandthesecondaryantibodyinisolation.Othervariables

testedbutnotdisplayedaboveincluded:primaryantibodydilutions1:100,1:250:inall

combinationswithsecondaryantibodydilutions1:100,1:500,1:1500.Thepositivelystained

peakwasdeterminedfromprimaryandsecondaryantibodycombinations,whichexceededthe

fluorescenceofboththeisotypecontrolandsecondaryantibodyincombinationandtothe

secondaryantibodyaloneatthesamedilutions.Importantly,theshiftinthehistogramcurve

wassuchthattherewasminimaloverlapwiththehistogramcurveofthecontrolsamples

described.Thedottedlinesandarrowsindicatetheareaofhistogramfromwhichalogical

‘gate’wasplacedtodeterminethepercentageofcellsthatweredeemedpositiveforΔNp63

expression.

Isotypecontrolwithsecondaryantibody1:1000

Secondaryantibodyonly1:1000

ΔNp631:50,secondaryantibody1:1000

3.4.2 CharacterisationofprimaryHCjE-Giconjunctivalcelllinewithflow

cytometry

Theantibodyoptimisationexperimentsshowninpreviouslydescribedsection(3.4.1)

enabledthedeterminationofoptimalprimaryandsecondaryantibodystaining

protocolstoundertakeflowcytometry.Optimisationwasundertakenforallthe

antibodiesstudiedandrepresentativeexperimentshavebeendisplayedtoillustrate

howthiswasconducted.Characterisationofthepreparedsamplebeganwithadjusting

theforwardandsidescattervoltagesandcompensationtoobtainoptimalscatter

characteristicsfromwhichthecellpopulationwasidentified(Figure47).Thesame

voltagesettingswereusedforallsubsequentexperimentalwork.Figure47displaysthe

cellpopulationthatwasgatedforsubsequentanalysis.Eachstainedcellsamplewas

analysedinconjunctionwithitsrelevantisotypecontrol.Anisotypecontrolofa

correspondingdilutionwasincludedineachexperimentalrunforeachantibodythat

wasused.Theantibodystainingcharacteristicstogetherwiththerelevantisotype

controlsareshowninhistogramsforbothprimaryconjunctivalcellsandtheHCjE-Gi

cellline(Figure48).Thegatingcriteriaregardedaspositiveantibodystainingwas

determinedfromthepointonthexaxisatwhichthehistogramcurvefortheisotype

controltaperswithincreasingfluorescence.IllustrativeexamplesareshowninFigure

Figure47:Dotplotofsidescatter-height(SSC-H)againstforwardscatterheight(FSC-H)ofHCjE-

Gi(a)andprimaryconjunctivalcells(b).Thedotsoccurringatthebottomleftoftheplot(low

FSC-HandSSC-H)areparticulates/debris.

Figure48(displayedbelowandoverpages110-114):Histogramstoshowthefluorescence

detectedinprimaryconjunctivalepithelialcellsandHCjE-Gicellsafterstainingasdescribedin

section2.4.6:a)CK19,b)CK4,c)CK7,d)UAE-1lectin,e)MUC5AC,f)PCNA,g)caspase-3,h)

ΔNp63,i)ABCG2.Thedottedlinesandarrowsindicatetheregionthe‘gates’weresetsuchthat

cellsemittingfluorescenceabovethatdetectedintherelevantisotypecontrols(allhistograms

onthelefthandside)weredeemedpositivelystained.Theredarrowsonthehistogramson

therighthandsidedemonstratethepositiveantibodystainedpopulationdeterminedby

settingalogicalgatebasedontheemittedfluorescenceoftheisotypecontrolsamples

illustratedonthehistogramsonthelefthandsidewithbluearrows.Thepercentageofcells

positivelystainedfortheantigenofinterestwerequantifiedusingananalyticalsoftwaretool

forflowcytometrydata(Flowing2.5).

Primaryconjunctivalcells

HCjE-Gicells

b) CK4

HCjE-Gicells

e) MUC5AC

HCjE-Gicells

UAE-1lectind)

HCjE-Gicells

f) PCNA

HCjE-Gicells

Caspase-3g)

HCjE-Gicells

h) ΔNp63

ABCG2i)

HCjE-Gicells

3.4.3 Determiningtheutilityofcaspase-3asamarkerfortheidentificationof

apoptoticcellsinconjunctivalepithelia

Figure49showstheshiftinfluorescencealongtheAPC(far-red)channelofthecell

populationstainedwithcaspase-3withtheisotypecontrols.Thisrevealedgreaterthan

100-foldexpressionofcaspase-3inthedeprivedcells(43.5%)comparedtothosethat

hadbeenmaintainedinoptimalcultureconditions(0.36%).

Figure49:Histogramsoffluorescencedetectedfollowingstainingwithcaspase-3inhealthy

anddeprivedcultures.H2indicatesthe‘gates’setusingaflowcytometryanalysissoftware

programsuchthatcellsemittingfluorescencelevelsabovethatdetectedintheisotypecontrols

(intherangeindicatedbyH2)wereregardedaspositivelystained.Thisenabledthe

quantificationofthecellpopulationintermsofthepercentageofthecellspresentinthisgated

region.

Isotypecontrol

Deprivedcellcultures

Healthycellcultures

3.4.4 Validationofthecellline

Quantitatively,theexpressionlevelsofallcellmarkerswereverysimilarbetweenHCjE-

Gicellsofpassage2and28atarrivalfromsource(donatedcellline),andno

statisticallysignificantdifferencesweredemonstrated.Basedonthisdata,HCjE-Gicells

betweenpassages2and28weredeemedsuitableforuseinsubsequentexperiments.

Passage2mean(+/-

SD)percentageofcells

Passage28mean(+/-

SD)percentageofcells

pvalue

CK4 0.22(0.07) 0.23(0.09) 0.89

CK7 2.63(0.78) 2.58(1.11) 0.95

CK19 98.8(0.61) 99.1(0.21) 0.47

MUC5AC 0.18(0.04) 0.17(0.08) 0.87

Lectin 44.59(2.61) 46.3(3.35) 0.52

�Np63 54.9(11.2) 53(15.6) 0.78

ABCG2 25.4(3.63) 22.2(4.5) 0.39

ABCG2+

�Np63 24(3.29) 21.7(4.75)

Caspase3 0.15(0.03) 0.12(0.03) 0.41

PCNA 98.9(0.02) 98.3(0.12) 0.41

Table8:TabletoshowpercentageexpressionofconjunctivalmarkersinHCjE-Gicellsof

passage2and28.Triplicatesamplesforeachmarkerwithineachcohort(passage2and

passage28)wereanalysedbyflowcytometry.Similarlevelsofmarkersintermsofthe

percentageofcellsdetectedwasapparentbetweencellsofpassage2and28withno

differencesconfirmedbystatisticalanalysis(ANOVA).

3.5 Analysisofconjunctivalepithelialphenotypewithadvancingtime

onsyntheticsubstratesbyflowcytometry

3.5.1 HCjE-Gicellphenotypewithadvancingtimeincultureonsynthetic

substrates

AlmostallHCjE-GicellswerefoundtoexpressCK19(≥96%)acrossallsubstratesat14

and28daysinculture.NostatisticallysignificantdifferencesinCK19orCK4expression

wereobservedwithrespecttoadvancingtime.ThehighestexpressionofCK4was

demonstratedonPETanduntreatedePTFEsubstrates,wherebysignificantpost-hoc

testdifferenceswerefound:PEThadgreaterCK4expressionthandoublesidetreated

ePTFE,anduntreatedePTFEhadgreaterCK4expressionthandoublesidetreated

ePTFE;p<0.01(Figure50,Table11).CK7expressionincreasedwithadvancingtimein

culturetogetherwiththeproportionofcellsco-expressingCK7andUAE-1lectinacross

allsubstratesstudiedwithsignificantdifferencesdemonstratedbetweentheuntreated

ePTFEandallothersubstratesstudied;p<0.0001(Figures51and55).CK7/CK7+UAE-1

lectinexpressiononuntreatedePTFE(mean78%)wasmorethan3-foldhigherthan

thatondoublesideammoniaplasmatreatedePTFE(mean25%)andmorethandouble

thatonPETandsinglesideammoniatreatedePTFE(mean32%and38%respectively):

Figure55.

UAE-1lectinexpressionvariedsignificantlywithsubstratebutnotwithadvancingtime:

p=0.001,p=0.64respectively(Figure54).After28daysinculture,highestUAE-1

expressionwasfoundondoublesidetreatedePTFEanduntreatedePTFE.Significant

differenceswerefoundinpost-hoctestsbetweenuntreatedePTFEandbothPET

membraneandsinglesideammoniaplasmatreatedePTFE(p<0.007).Significant

differenceswerealsodemonstratedbetweendoublesideammoniaplasmatreated

ePTFEandPETmembrane;p<0.007(Figure54).

MUC5ACdetectionwaslowatbothday14andday28onallsubstrateswherebyit

representedlessthan0.75%ofallcells(Figure53).Nostatisticallysignificant

differencesinMUC5ACdetectionweredemonstratedbetweenthesubstratesstudied

howeverexpressionsignificantlyincreasedwithadvancingtimeincultureoverall;

p=0.33,p<0.001respectively.ThehighestMUC5AClevelsweredetectedondoubleand

singlesideammoniatreatedePTFEatday28,whichwerealmosttwicethatexpressed

inPETmembranecultures.

ΔNp63wasexpressedinlargeproportionofcells(≥90%)andthislevelremainedhigh

withadvancingtimeinculture(Figure56).Nosignificantvariationwithrespectto

substrateoradvancingtimeinculturecouldbedemonstratedafterHolm-Bonferroni

correction:p=0.096,p=0.007respectively.Incontrast,theABCG2expressionwaslower

thanΔNp63atday14andappearedtodeclinewithadvancingtimeinculture,however

thiswasnotstatisticallysignificantandvariancewasrelativelyhigh(Figure57).

SignificantlyhigherABCG2expressionwasfoundondoublesidetreatedePTFEthan

thatonsinglesidetreatedePTFEanduntreatedePTFEafter28daysinculture:p=0.008

andp=0.004betweendoublesideammoniaplasmatreatedePTFEanduntreated

ePTFEanddoublesideammoniatreatedePTFEandsinglesideammoniatreatedePTFE

respectively(Figure57,Table11).NosignificantdifferenceinABCG2expressionwas

demonstratedbetweenculturesfromdoublesideammoniaplasmatreatedePTFEand

PETmembranes.Asignificantdifferencewashoweverdemonstratedintheco-

expressionofABCG2andΔNp63(Figure58).ThehighestABCG2/ΔNp63co-expression

wasdemonstratedondoublesidetreatedePTFE.Thiswassignificantlyhigherthanon

anyothersubstratestudied;p<0.01inpost-hoctestsbetweendoublesidetreated

ePTFEandallothersubstratesstudied(Table11).

PCNAlevelsdidnotchangesignificantlywithadvancingtime:p=0.024(Figure60).No

statisticallysignificantdifferencesinPCNAexpressionweredemonstratedbetween

substrates(p=0.146),howeverthelowestlevelswerefoundonuntreatedePTFEat14

and28daysinculture.Caspase-3expressionwasrelativelylowacrossallsubstratesat

14daysincultureanddidnotincreasewithadvancingtimeoverall:p=0.163(Figure

59).Therewas,however,analmost10-foldgreatercaspase-3expressionat28daysin

cultureonuntreatedePTFEthananyothersubstrate.Caspase-3expressionwas

significantlyhigheronuntreatedePTFEthanallothertestsubstrates;p<0.0001(Table

Figure50:HistogramtoshowpercentageexpressionofCK4onammoniaplasmatreated

ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel

substrates;p<0.0001;timep=0.152.Errorbars+/-SD.

Substrate

PETmembraneSinglesidetreatedePTFE

UntreatedePTFE

Percen

tageofcells

expressing

Day14 Day28Daysinculture

substratesandtime;p<0.001.Errorbars+/-SD.

substratesp=0.975;timep=0.88.Errorbars+/-SD.

Substrate

DoublesidetreatedPETSinglesidetreatedePTFE

UntreatedePTFE

Percen

tageofcells

expressing

Substrate

DoublesidetreatedePTFEPET

UntreatedePTFE

Percen

tageofcells

expressing

Figure53:HistogramtoshowpercentageexpressionofMUC5AConammoniaplasmatreated

substratep=0.033;timep<0.0001.Errorbars+/-SD.

Figure54:HistogramtoshowpercentageexpressionofUAE-1lectinonammoniaplasma

treatedePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel

substratep=0.001;time:p<0.641.Errorbars+/-SD.

Substrate

DoublesidetreatedePTFEPET

UntreatedePTFE

Percen

tageofcells

expressing

-1lectin

Substrate

DoublesidetreatedePTFEPETmembrane

UntreatedePTFE

Percen

tageofcells

expressing

0.20201

0.40402

0.60603

0.800.88

Figure55:HistogramtoshowpercentageexpressionofCK7andUAE-1lectinco-expressionon

ammoniaplasmatreatedePTFE,untreatedePTFEandPETafter14and28daysinculture.

ANCOVAoverallmodelsubstrateandtimepoint:p<0.0001.Errorbars+/-SD.

Figure56:HistogramtoshowpercentageexpressionofΔNp63onammoniaplasmatreated

SubstrateDoublesidetreatedePTFEPETmembraneSinglesidetreatedePTFEUntreatedePTFE

Percen

tageofcellsexpressing

-1lectinand

Substrate

DoublesidetreatedePTFEPETmembraneSinglesidetreatedePTFE

UntreatedePTFE

Percen

tageofcells

expressing

Figure57:HistogramtoshowpercentageexpressionofABCG2onammoniaplasmatreated

Figure58:HistogramtoshowpercentageexpressionofABCG2andΔNp63onammonia

plasmatreatedePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVA

overallmodelsubstratesp=0.001;timep=0.008.Errorbars+/-SD.

Substrate

UntreatedePTFE

Percen

tageofcells

expressing

Substrate

DoublesidetreatedPETmembraneSinglesidetreatedePTFE

UntreatedePTFE

Percen

tageofcellsco-expressing

p63(lo

gscale)

Figure59:HistogramtoshowpercentageexpressionofCaspase-3onammoniaplasmatreated

substratesp=<0.0001;timep=0.163.Errorbars+/-SD.

Figure60:HistogramtoshowpercentageexpressionofPCNAonammoniaplasmatreated

ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodeltime

p=0.024;substratesp=0.146.Errorbars+/-SD.

Percen

tageofcells

expressing

Substrate

UntreatedePTFE

Substrate

DoublesidetreatedPETmembSinglesidetreatedePTFE

UntreatedePTFE

Percen

Caspase-3(lo

gscale)

Marker Pvalue

CK7 0.0001

CK7/UAE-1lectinco-

expression

0.0001

MUC5AC 0.0001

Table9:Tableofcellmarkersinwhichastatisticallysignificantchangewasdemonstratedwith

advancingtimeinculture.RawpvaluesfromtheANCOVAaredisplayed.Onlypvalues

statisticallysignificantfollowingcorrectionbyHolm-Bonferroniformultipletestinghypotheses

aredisplayed.

Marker Pvalue

CK7 0.0001

CK7+UAE-1lectin 0.0001

UAE-1lectin 0.001

CK4 0.0001

Caspase-3 0.0001

ABCG2 0.003

ABCG2+NP63 0.001

Table10:Tableofcellmarkersinwhichastatisticallysignificantdifferencewasdemonstrated

betweensubstrates.RawpvaluesfromtheANCOVAaredisplayed.Onlypvaluesstatistically

significantfollowingcorrectionbyHolm-Bonferroniformultipletestinghypothesesare

displayed.

Marker Post-hoccontrasts;pvalueandpairsofdata

CK7 p<0.0001:Uvs.D;Uvs.S;Uvs.P

CK7+UAE-1lectin p<0.0001;Uvs.D,Uvs.S,Uvs.P

UAE-1lectin p<0.007:Dvs.P,Uvs.S,Uvs.P

CK4 p<0.01:Dvs.P,Dvs.U

Caspase-3 p<0.0001:Dvs.U,Uvs.P,Uvs.S

ABCG2 p=0.008Dvs.Up=0.004Dvs.S

ABCG2+ΔNp63 p≤0.001Dvs.U,Dvs.S,Dvs.P

Table11:Tableofcellmarkersinwhichastatisticallysignificantdifferencewasdemonstrated

betweensubstrates:U=untreatedePTFE,D=doublesidetreatedePTFE,S=singlesidetreated

ePTFE,P=PETmembrane.RawpvaluesfromtheANCOVApost-hoccontrasttestsare

displayed.OnlypvaluesstatisticallysignificantfollowingcorrectionbyHolm-Bonferronifor

multipletestinghypothesesaredisplayed.

3.5.2 Celldensityandmorphologyofprimarycellculturesondoubleside

ammoniaplasmatreatedePTFEandPETmembrane

Primarycellsfromasingledonor(donor10)wereexpandedandseededat1x105/cm2

onammoniaplasmatreatedePTFE(doublesidetreated)andPETmembrane(positive

control)intriplicate.Thecelldensityexpandedupto14daysinculture,afterwhicha

declineoccurred(Figure61).Atalltimepointsstudied,thecelldensityonPET

membranewasmorethandoublethatfoundonePTFE.Thiswasinkeepingwiththe

qualitativeappearanceofnuclearstainingdensityobservedinDAPIstainedsamples

(Figure62).Morphologicallyhowever,thecellsappearedlargerinculturesdeveloped

onePTFEthanPETmembraneatday14givingtheimpressionofasimilardegreeofcell

confluencedespitealowercelldensityontheePTFEsubstrate(Figures61and62).

Othernotablemorphologicalfeaturesincludegreatervariationinnuclearsizeand

shapeincellsdevelopedonePTFEsubstrateincomparisonthoseonPETmembranesat

10x105

CellCo

Day2 Day14 Day21 Day28

Numberofdaysinculture

Substrate

PETmembrane

day14.F-actinstainingofcellsonbothsubstratesdemonstratedthatcellsappeared

moreelongatedwithcellprocessesvisibleonday14whichwerenolongerpresentat

day28whencellsappearedsparsewithamore‘rounded’appearanceonboth

substrates.

Figure61:Histogramtoshownumberofcellspercm2withadvancingtimeincultureondouble

sideammoniaplasmatreatedePTFE(bothsidesexposedtoplasma)andPETmembrane.

Primarycellsusedinthisexperimentwerefromasingledonorandwereseededat1x105/cm2.

Scalebars+/-SD.

Figure62:Representativephotomicrographsofprimaryconjunctivalcellsondoubleside

ammoniaplasmatreatedePTFE(D)andPET(P)membraneatafter14and28daysinculture.

Scalebars50μM.Phalloidin(f-actinstaining):red.DAPI(nuclearstaining):blue.

3.5.3 Phenotypeofprimarycellsexpandedondoublesideplasmatreated

ePTFEandPETmembranebyflowcytometry

Theconjunctivalepithelialcellsusedinthisexperimentwerederivedfromexplants

fromthreeseparatetissuedonors(tissuedonors4,8and12:Table7).Themajorityof

cells(≥97%)expressedcytokeratin19(Figure63).Nosignificanteffectwasfoundeither

withrespecttotimeinculture(day14versusday28)orthesubstrateonwhichcells

weregrown(doublesideammoniatreatedePTFEorPETsubstrate);p=0.079,p=0.123

respectively.Similarly,nostatisticallysignificantdifferencescouldbefoundinCK7,CK4,

UAE-1lectinorco-expressionofCK7/UAE-1lectinwithrespecttoadvancingtimeor

substrate(Figures64-67).ThehistogramsofCK7expressionaloneandCK7andUAE-1

lectinco-expressionwerequantitativelysimilar(Figures65and67).UAE-1lectin

expressionalone,however,wasgreaterthanCK7expressionalone(Figure66).The

expressionofbothMUC5ACandUAE-1lectinwereonaveragehigherat28daysthan

14daysinculture,however,thiswasnotstatisticallysignificant;p=0.12,p=0.51

respectively(Figures66and68).Nosignificantdifferencescouldbecouldbe

demonstratedinMUC5ACorUAE-1lectinexpressionbetweencellsculturedonPET

andePTFEsubstrates;p=0.46,p=0.26respectively.

NostatisticallysignificantdifferencesinΔNp63,ABGC2expressionorco-expressionof

ΔNp63andABGC2weredemonstratedwithadvancingtimeincultureorbetween

substrates(Figures69-71).TheproportionofcellsexpressingeitherABCG2orΔNp63,

however,lessthanhalvedbetweenday14andday28,andtheproportionofcellsco-

expressingABCG2andΔNp63alsodiminishedmarkedly.Itshouldbenotedthat

varianceinthetestsampleswasrelativelyhigh.Similarly,caspase-3expression

appearedtomorethandoublewithadvancingtimeandhigherlevelswerefoundon

ammoniaplasmatreatedePTFE(Figure72).Thevariance,again,washighand

thereforenostatisticallysignificantdifferenceswithrespecttoadvancingtimein

cultureorsubstrateweredemonstrated.PCNAexpressiondeclinedwithadvancing

timeincultureandwasfoundtobestatisticallysignificantfollowingHolm-Bonferroni

correction;p=0.003(Figure73).LevelsofPCNAexpressionweresimilarbetween

doublesideammoniaplasmatreatedePTFEandPETmembrane.

Figure63:HistogramtoshowpercentageexpressionofCK19inprimarycellsculturedon

doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA

timep=0.079;substratep=0.123.Errorbars+/-SD.

Figure64:HistogramtoshowpercentageexpressionofCK4inprimarycellsculturedondouble

sideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVAtime

0.52;substrate0.753.Errorbars+/-SD.

Percen

tageofcells

expressing

Substrate

PETmembrane

Percen

tageofcells

expressing

Substrate

PETmembrane

Figure65:HistogramtoshowpercentageexpressionofCK7inprimarycellsculturedondouble

sideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVAtime

0.647;substratep=0.25.Errorbars+/-SD.Errorbars+/-SD.

Figure66:HistogramtoshowpercentageexpressionofUAE-1lectininprimarycellscultured

ondoublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.

ANCOVAtime0.39;substratep=0.712.Errorbars+/-SD.

Percen

tageofcells

expressing

Substrate

PETmembrane

Percen

tageofcells

expressing

-1Lectin

Substrate

PETmembrane

Figure67:HistogramtoshowpercentageexpressionofUAE-1lectinandCK7inprimarycells

culturedondoublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28

days.ANCOVAtimep=0.505;substratep=0.263.Errorbars+/-SD.

Figure68:HistogramtoshowpercentageexpressionofMUC5ACinprimarycellsculturedon

Percen

Substrate

PETmembrane

Percen

tageofcellsCK

-1lectin

Substrate

PETmembrane

Figure69:HistogramtoshowpercentageexpressionofΔNp63inprimarycellsculturedon

timep=0.945;substrate0.537.Errorbars+/-SD.

Figure70:HistogramtoshowpercentageexpressionofABCG2inprimarycellsculturedon

Percen

Substrate

PETmembrane

Percen

tageofcells

expressing

Substrate

PETmembrane

Figure71:HistogramtoshowpercentageexpressionofΔNp63andABCG2inprimarycells

culturedondoublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28

days.ANCOVAtimep=0.328;substratep=0.787.Errorbars+/-SD.

Figure72:Histogramtoshowpercentageexpressionofcaspase-3inprimarycellsculturedon

Percen

ΔN-p63

Substrate

PETmembrane

Percen

caspase-3

Substrate

PETmembrane

Figure73:HistogramtoshowpercentageexpressionofPCNAinprimarycellsculturedon

3.6 Decellularisationofhumanconjunctivaanditscharacterisation

3.6.1 DNAquantificationofdecellularisedconjunctiva

Acommerciallyavailablekitwasusedtodegradeconjunctivaltissueandisolatedouble

strandedDNAsothatitwaspresentinsolutionforquantificationwithafluorescent

nucleicacidstain.CalfthymusDNAstandardswereusedinknownserialdilutionsto

enabletheacquisitionofastandardcurve(Figure74).Thestandardcurvedisplaysthe

relationshipbetweenthemeasuredabsorbancevaluesandknownconcentrationsof

DNA.Thisenabledcalculationofthegradientandinterceptfromtheequationy=mx+c

sothatDNAcouldbequantifiedfromeachtestsamplefromthemeasuredabsorbance

value.

Percen

Substrate

PETmembrane

Theeffectivedecellularisationoftissueswasdemonstratedinalltreatmentgroups

definedbySDSdilutioninthedecellularisationprocess(p<0.001).Nodifferencecould

bedemonstratedbetweenSDStreatmentgroupsinBonferronipost-hoctests(p>0.1)

indicatingtherewasnodifferenceinefficacywithvariationindilutionofSDS(Table

12).Thereproducibilityofdecellularisationusingthe0.05%SDS(w/v)concentration

(p<0.001)wasconfirmedbynosignificantdifferenceinDNAlevelsdetectedbetween

tissuesobtainedfrom3separatedonors(Table13).Inalltreatmentgroups,98.9%

(mean99.1%)orgreaterDNAremovalwasachieved.Effectivedecellularisationwas

alsoindicatedbytheabsenceoffluorescentnuclearstaining,otherwisedetectableby

DAPI(4',6-diamidino-2-phenylindole,dihydrochloride)indecellularisedtissue(Figure

75).Incontrast,abundantbluefluorescentstainingofnucleiwasdemonstratedinthe

cellular(control)tissuesamples.

Figure74:StandardcurveoffluorescenceabsorbancevalueswithincreasingDNA

concentrationincontrolsamples(knowndilutionsofcalfthymusDNA).Thestandardcurve

shownenabledcalculationofthegradientandyinterceptintheequationy=mx+ctodetermine

theDNAcontentinexperimentalsamples.

Absorbance

DNAconcentrationng/ml

Treatmentgroup DNAng/mg(SD) PercentageDNAremoval

Cellularcontrol 54.5 (10.4) -

0.05% 0.4 (0.2) 99.3

0.1% 0.2 (0.1) 99.7

0.5%SDS 0.3 (0.3) 99.4

Table12:TabletoshowDNAcontentofcellulartissuesanddecellularisedtissuesusingSDSof

varyingconcentration.TheoverallANOVAmodelwassignificant;p<0.001.Datawaslog

transformedtoenableparametricdataanalysisandANOVAmodelwassatisfiedfollowing

Levene’stestofequalityofvariance(p=0.1).Bonferroniposthoctestsbetween

0.05%/0.1/0.5%SDS(w/v)treatmentgroups;p≥0.1.

Tissue DNAng/ml(SD) PercentageDNAremoval

Cellularcontrol 42.3(7.4) -

Donora 0.4 (0.2) 98.9

Donorb 0.3 (0.1) 99.2

Donorc 0.3 (0.1) 99.3

Table13:TabletoshowDNAcontentofcellulartissuesanddonortissuesdecellularisedwith

0.05%SDS(w/v).OverallANOVAmodelwassignificantp<0.001.Datalogwastransformedto

enableparametricdataanalysisandANOVAmodelsatisfiedfollowingLevene’stestofequality

ofvariance(p=0.28).Bonferroniposthoctestsbetweendonora/b/c;p≥0.1.

Figure75:Representativephotomicrographsofdeparrafinisedtissuesectionsstainedwith

DAPI.BrightfluorescentbluestainingindicatesthepresenceofDNA/nucleia)cellular

conjunctivalcellulartissue:b)conjunctivaltissuedecellularisedwith0.05%SDS(w/v).

3.6.2 Contactcytotoxicityofdecellularisedconjunctivaltissue

PhotomicrographstakenoffixedGiemsastainedculturesdemonstratedbothHCjE-Gi

cellsandprimaryhumanskinfibroblastswerepresentincloseproximity,seenwithin

andarounddecellularisedtissueintheabsenceofazoneofinhibitionoranycellular

debristhatwouldhaveindicatedthepresenceofnon-viablecells(Figure76).In

contrast,cellulardebrisindicatingnon-viablecellsweredemonstratedsurroundingthe

cyanoacrylategluesamplethatservedtheroleofapositive(cytotoxic)controlinthis

experiment.Alargezoneofinhibitionwasfoundwhenprimaryhumanfibroblastswere

culturedwithcyanoacrylateglue(Figure76).

200μm 200μm

Figure76:RepresentativephotomicrographsoffixedGiemsastainedcellsfromahuman

conjunctivalcellline(HCjE-Gicells)andprimaryhumanskinfibroblastsinculturewith

decellularisedconjunctiva,Steri-stripsTMandcyanoacrylateglueafter48hours.a)

Decellularisedconjunctivawithcellsseenincontactwithtissueb)Steri-stripsTM,experimental

negativecontrolknownnottoexhibitcytotoxicity,seenherewithcellsvisibleincontactc)

cyanoacrylateglue,experimentalpositivecontrolshowingcytotoxicitywithcellulardebrisof

HCjE-Gicellssurrounditandalargezoneoninhibitionapparentwithcellulardebris.Scalebars

200µm.

HCjE-GiCells Primaryhumanskinfibroblasts

3.6.3 Tensilestrengthtesting

Thetensilestrengthofcellularanddecellularisedconjunctivaweretestedin

comparisontoePTFEandcellularanddecellularisedamnioticmembrane(Table14).

ThemeanultimatetensilestressvaluesweregreatestforePTFE;however,therewas

nosignificantdifferencebetweenePTFEandcellularordecellularisedamniotic

membrane.Theultimatetensilestrengthwasleastforcellularanddecellularised

conjunctivaandsignificantdifferencesweredemonstratedbetween:conjunctivaand

amnioticmembrane(p<0.0001);andconjunctivaandePTFE(p=0.01).

DecellularisedamnioticmembranehadthegreatestYoung’smodulus(12.0MPa),

whichwasmorethan3-foldhigherthanePTFEanddecellularisedconjunctiva,

indicatinggreaterstiffness.Young’smoduluswassimilarbetweenePTFEand

conjunctiva:2.7and3.0MParespectively(p=1).Markedvarianceinconjunctivaland

amnioticmembranetensilestressdatawasdemonstratedbyhighstandarddeviation

valuesinallmeasuredparameters.Nosignificantdifferenceinultimatetensilestressor

Young’smoduluscouldbedemonstratedbetweencellularanddecellularised

conjunctivaorbetweencellularanddecellularisedamnioticmembrane,whichsuggests

nochangeinstiffnessfollowingdecellularisation(Figure77).

TissueUltimatetensilestress

MPa(+/-SD)

Young’smodulusMPa

(+/-SD)

ePTFE 1.2(0.01) 2.7(0.2)

Cellularconjunctiva 0.7(0.5) 3.9(5.6)

Decellularisedconjunctiva 0.5(0.9) 3.0(3.6)

Cellularamnioticmembrane 1.7(1.1) 11.5(6.1)

Decellularisedamnion 1.8(0.7) 12.0(5.1)

Table14:TableofresultsfromtensilestrengthtestingofePTFE,cellularanddecellularised

conjunctivaandamnioticmembrane.Datashowninthistablewasnormalisedbylog

transformationpriortoANOVAanalysis.OverallANOVAmodelforbothultimatetensile

strengthandYoung’smodulusbetweentissues;p<0.0001.Nosignificantdifferenceswere

foundinbothparametersstudiedbetweencellularanddecellularisedtissues;p=0.354ultimate

tensilestrength;p=0.561Young’smodulus.

Amniotic

membrane

Figure77:Representativehistogramsofload(N)againstadvancingtime(seconds)duringthe

tensilestrengthtest:a)conjunctivab)amnioticmembranec)ePTFE.Eachhistogramalso

demonstratesthesectionofthecurvefromwhichthegradientforthegreatestslopewas

determined(see‘Greatestslope’markedoneachgraphbytheredarrows).

Conjunctiva

3.6.4 CollagenDenaturationAssay

Astandardcurvefromthemeasuredabsorbancevaluesofknownconcentrationsofa

hydroxyprolinestandardenabledthedeterminationofthegradientandinterceptfrom

theequationy=mx+c(Figure78).Thesevaluesallowedhydroxyprolinequantification

fromtestsamples.Minimalcollagendenaturationwasobservedinalltissuesamples

studieddemonstratedbylowhydroxyprolinevalues(range3.12-6.24hydroxyproline

ng/mg).Thenegativecontrolvalueswere2.82hydroxyprolineng/mg.Nodifferencein

hydroxyprolinewasfoundbetweenpairedsamplesofcellularanddecellularised

conjunctivaltissuesuggestingcollagenwasnotdenaturedbydecellularisation(p=0.74,

pairedt-test).

Figure78:Standardcurveofcolorimetricabsorbancevalueswithincreasinghydroxyproline

concentrationfromknownconcentrationsofthecontrolstandard.Thestandardcurveshown

enabledcalculationofthegradientandyinterceptintheequationy=mx+ctodeterminethe

hydroxyprolinecontentofeachoftheexperimentalsamples.

Absorbance

Hydroxyprolineng/mgtissue

Hydroxyprolineng/mg SD

Positivecontrol 196.8 31.9

Negativecontrol 2.8 0.9

Cellulartissuedonora 3.1 0.9

Decellularisedtissuedonora 4.0 1.1

Cellulartissuedonorb 4.2 1.2

Decellularisedtissuedonorb 3.6 0.8

Cellulartissuedonorc 6.2 4.6

Decellularisedtissuedonorc 5.2 1.6

Table15:Tabletoshowhydroxyproline(ng/mg)foundinassaysofpairedsamplesofcellular

anddecellularisedtissue.P=0.74:pairedsamplest-testbetweencellularanddecellularised

donortissue(n=3ineachgroup).

3.6.5 Histologyofdecellularisedconjunctiva

HaematoxylinandEosin(H&E)stainingofcellularanddecellularisedtissues

demonstratedmarkedsimilaritiesintheEosinstainingpatternsandthenotable

absenceofbasophilicHaematoxylinstaininginkeepingwiththeabsenceofnucleiin

decellularisedtissue(Figure79).VanGieson’sstaindemonstratedtheabsenceof

brownstainingindecellularisedtissueindicatingtheabsenceofnuclearmaterialand

preservedcollagenextracellularmatrixarchitecture,asshownbythered-purple

staining(Figure80).

Figure79:RepresentativephotomicrographsofHaematoxylinandEosinstainedparaffin

embeddedtissuesections:a)decellularisedtissue;b)cellulartissue.Scalebars200µm.

Figure80:RepresentativephotomicrographsofconjunctivatissuestainedwithVanGieson’s

stain:a)decellularisedtissue;b)cellulartissue.Scalebars100µm.

a) b)b)

b)a) b)

3.7 Cultureofprimaryhumanconjunctivalepithelialcellson

decellularisedconjunctiva

3.7.1 Cellcultureondecellularisedtissuesubstrateswithprimaryconjunctival

epithelialcellsusingexplantandsuspensioncultures

Theconjunctivalexplantsusedforthisexperimentweretakenfromdonoreye5.The

isolatedcellsculturedonamnioticmembraneweresparseandgrewinconcentrated

areasincomparisontoexplantculturesinwhichamoreconfluentcellculture

developed(Figure81).Similarresultswereobtainedusingdecellularisedconjunctivain

whichexplantculturesresultedinaqualitativelyhighercelldensityincomparisonto

thecultureofanisolatedcellsuspensionusingcellsfromthesamedonor.This

differencewasmorenotableonamnioticmembranewherebycellswerealso

qualitativelyfoundingreaterdensitythanonconjunctivaoverall.Thebasement

membrane,however,inthedecellularisedtissueappearedtobeabsentbasedonthe

H&Estaininginthesephotomicrographs.

Figure81:Representativephotomicrographsofdecellularisedamnioticmembraneand

conjunctivaltissuesectionsrecellularisedwithconjunctivalepithelialcells.Greatercelldensity

wasevidentqualitativelyfollowingexplantculture(bandd)thanthatfollowingsuspension

culture(aandc).Scalebars100µm.Arrowspointtopurplenucleiincells.

Decellularisedamnioticmembrane

Decellularisedconjunctiva

Suspensionculture Explantculture

3.7.2 Explantculturewithattentiontoconjunctivalbasementmembrane

orientation

Confluentcellgrowthwasdemonstratedwithqualitativelygreatercelldensityfrom

explantcellculturefollowingtheconfirmationandorientationofthebasement

membraneincomparisontotissueinwhichthebasementmembranecouldnotbe

identified(Figure82).Theexplantculturesusedinthisexperimentweretakenfrom

donoreye7.

Figure82:Representativephotomicrographsofexplantculturesgrownondecellularisedtissue:

a)basementmembranenotpresentb)basementmembranepresent.Arrowsarepointingto

nuclei(purple).Scalebars100µm.

3.7.3 Comparisonofconjunctivalepithelialculturesusingtissuefrom

differentdonorsforexplantsanddecellularisedsubstrates

Conjunctivaltissuesfromdonoreyes9,5and13weredecellularisedforuseinthis

experiment.Theexplantsculturedonthesesubstratesweretakenfromtissuedonor

eyes15,17and19.Resultsfromtheseexperimentsshowedthatgrowthwassparse

fromallthreeexplantdonors.Thebestresultsqualitativelybaseduponconfluenceand

theapparentdensityofcellswasdemonstratedusingdecellularisedtissuefromdonor

13andexplantsfromdonor19(Figure83-86).Consistentcellularexpansionfrom

explantdonor19howeverwasnotevidentacrossalldecellularisedtissuesamples.

Donor19,however,alsoproducedthegreatestdensityofcellsqualitativelyonthe

amnioticmembranecontroltissue(Figure86).Insometissuesections,theexplant

itselfwasvisiblewithminimaloutgrowthfromitsedge.

Tissuedonor9Tissuedonor5Tissuedonor13

Figure83:RepresentativeH&Estainedphotomicrographsofexplantsfromdonor15cultured

ondecellularisedconjunctivafromtissuedonors9/5/13.Thephotomicrographof

decellularisedtissuedonor5hascapturedthepresenceofexplant(solidarrow)with

conjunctivalepitheliumthathaddevelopedonthelefthandsideofthisphotomicrograph

(dashedarrow).Incontrastminimalandnonucleiwerevisibleinphotosfromdecellularised

tissuedonors9and13.Scalebars200µm.

ondecellularisedconjunctivafromtissuedonors9/5/13.Nocellsareevidentfromontissue

sectionsderivedfromdecellularisedtissuedonor13.Occasionalareasofepithelialgrowth

weredemonstratedondecellularisedtissuesfromdonor9and5.Inthecentrephotofrom

decellularisedtissuedonor5anexplant(solidarrow)canbeseenwithcellularoutgrowth

(dashedarrow).Scalebars200µm.

ondecellularisedconjunctivafromtissuedonors9/5/13.Nocellswereevidentfromontissue

sectionsderivedfromdecellularisedtissuedonors9or5(donortissue5showsonlytheexplant

withnooutgrowth;solidarrow).Cellulargrowthwasobservedinmosttissuesectionsderived

fromexplantcultureondecellularisedtissuedonor13(dashedarrow).Scalebars200µm.

Explantdonor15Explantdonor17Explantdonor19

Figure86:RepresentativeH&Estainedphotomicrographsofexplantsfromdonors15/17/19

culturedonadecellularisedamnioticmembranesubstrate(samedonor).Cellulargrowthis

evidentfromalltheexplants;however,qualitativelythegreatestdensityofcellswasevident

fromexplantsderivedfromdonor19.Scalebars200µm.

3.7.4 Furtherconjunctivalexplantculturesonfreshlydecellularisedtissues

Furtherconjunctivalexplantcultureswereundertakenusingdecellularisedtissues

storedinphosphatebufferedsalinesupplementedwith1%penicillin/streptomycin,

usedwithinoneweekofdecellularisation.Inpreviousexperiments,decellularised

conjunctivaltissueswerestoredat-40oCandallowedtothawpriortouse.The

decellularisedtissuesfromthesamedonor(21)wereseededwithexplantsacquired

withinhoursoftissueretrieval.Thiswasundertakentwiceusingtwoseparatedonors

(23and25).Boththeseexperimentalrunsresultedinmoreconfluentgrowthof

conjunctivalepitheliumacrosstheentiresectionedtissuesamplesonboth

decellularisedconjunctivaandtheamnioticmembrane.Theformationofamulti-

layeredepitheliumwasalsoevidentonthedecellularisedconjunctivabutnotonthe

amnioticmembrane(Figure87).

DecellularisedamnioticmembraneDecellularisedconjunctiva

Figure87:Representativephotomicrographsofstainedtissuesectionsfollowingconjunctival

explantcultureusingdonor23(a)and25(b)ondecellularisedconjunctiva(fromdonor21)and

amnioticmembrane.Stratifiedcellsweredemonstratedondecellularisedconjunctivain

contrasttomonolayerformationonamnioticmembrane.Qualitatively,thecelldensityand

morphologyoftheepitheliumappearedsimilarbetweenthedonors(23and25)onbothtissue

substrates.Scalebars100μm.

3.7.5 Characterisationofthecellularphenotypeofconjunctivalepithelium

culturedondecellularisedconjunctiva

Anembeddedtissuewaxblockfromthepreviousexperiment;donor21withexplants

fromdonor23wasfurthersectionedtoallowimmunohistochemicalidentificationof

markersassociatedwithconjunctivalepithelialcells.Figure89demonstratesabundant

expressionofCK19throughoutthetissuesample.Incontrast,CK7andCK4were

expressedinaqualitativelylowerproportionofcells.MUC5ACexpressionwas

qualitativelysparse,howeverdetectable,withinthetissuesectionsexamined.Markers

ofprogenitorcellsΔNp63andABCG2werealsopresentinlesserfrequency,andABGC2

expressionappearedlessabundantthanΔNp63(Figure90).LevelsofPCNAexpression

appearedsimilartothatofΔNp63.Caspase3,amarkerofapoptoticcellswasalso

presentandappearedtobeexpressedinapicalratherthanbasalcells,incontrastto

theothercellmarkersstudiedwhichappearedmoreevenlydistributed.

Figure88:Representativephotomicrographsoftissuesectionsofconjunctivaltissue(a)mouse

IgGisotypecontrol,(b)rabbitIgGisotypecontrolstainedwithMayershaematoxylinonly.

Theserepresentativeimagesdisplaythelevelofbackgroundstainingdetectedfor

immunohistochemistrysamplesinthissectionandarethenegativecontrols.Scalebars100μm.

CK4a) b)

Figure89:Representativephotomicrographsofimmunohistochemicalstainingoftissue

sectionsofdecellularisedconjunctiva21recellularisedusingexplantsfromdonor23.Allbrown

stainingrepresentspositiveimmunolocalisationoftherespectivemarkerstudied.Scalebars

100µm.

MUC5AC

CK19CK19

Figure90:Representativephotomicrographsofimmunohistochemicalstainingoftissue

sectionsofdecellularisedconjunctiva21recellularisedusingexplantsfromdonor23.Allbrown

stainingrepresentspositiveimmunolocalisationoftherespectivemarkerstudied.Scalebars

100µm.

ABCG2 Caspase-3

PCNA ΔNp63

3.8 Identificationandcharacterisationofbasementmembranesof

humanconjunctivaandamnioticmembrane

3.8.1 CharacterisationofbasementmembranewithPAS

Conjunctivafromthreetissuedonorsandamnioticmembranefromthreetissuedonors

werestainedwithperiodicacidSchiff(PAS)staintoidentifyglycoproteinswithin

basementmembranes.PASalsostainstransmembraneandgobletcellassociated

mucinsinconjunctivalepithelium.Thethicknessofthebasementmembraneand

intensityofstainingwerequalitativelysimilarbetweenpairedsamples(cellularand

decellularisedtissue)ofbothamnioticmembraneandconjunctiva(Figure91and92).

Theoutlineofcellswerealsovisualisedanddemonstratedoncellulartissuesamplesof

bothconjunctivaandamnioticmembrane.Thiswasnotablyabsentindecellularised

tissuesamples.Representativephotomicrographstakenfromthecentreoftheslide

mountedstainedtissuesectionareshown(Figures91and92).

Figure91:Representativephotomicrographstissuesectionsofcellular(a)anddecellularised

amnioticmembrane(b)fromthreetissuedonorsstainedwithPAS.Thebasementmembrane

tissuesappearstainedandsimilarbetweencellularanddecellularisedtissuesections

suggestingitwaspreservedfollowingdecellularisation.Scalebars100µm.

Donor1 Donor2 Donor3

Figure92:Representativephotomicrographstissuesectionsofcellular(a)anddecellularised

conjunctiva(b)fromthreetissuedonorsstainedwithPAS.ThebasementmembranePAS

staininginallthecellularanddecellularisedtissuesappearedsimilarbetweentissuesections

suggestingitwaspreservedfollowingdecellularisation.Scalebars100µm.

3.8.2 Characterisationofcellularanddecellularisedtissuewithlaminin,

collagenIVandfibronectin

Deparaffinisedtissuesectionsfromthreeconjunctivalepithelialdonorsandthree

amnioticmembranedonorswerecharacterisedforcollagenIV,fibronectinandlaminin

byimmunohistochemicalstaining.Bothcellularanddecellularisedtissueswerestained

toascertainwhetherdecellularisationqualitativelyaffectedtheimmunohistochemical

detectionofextracellularproteins.Threedifferentdonorshavebeenstudiedforeach

tissuetype.Theimages(Figures93-99)demonstratethatcollagenIV,lamininand

fibronectinweredetectableintheepithelialtissuebasementmembranesandstromal

tissue.Thestrongeststaining,qualitatively,waspresentinthebasementmembranes.

Overall,theredidnotappeartobeanysignificantdifferenceinthedetectionofthese

proteinsbetweendonors.Furthermore,decellularisationdidnotreducetheintensity

ordistributionofstainingbyqualitativeobservation.Thegrossmorphologyofthe

tissue,specificallythethicknessandappearanceofunderlyingstromaltissue,however,

appearedtodiffermoresignificantlybetweentheconjunctivaldonortissues(Figure

97-99)thantheamnioticmembranetissues(Figure94-96).

Figure93:Representativephotomicrographsoftissuesectionsofconjunctivaltissue(a)and

amnioticmembrane(b)incubatedwithisotypecontrolsandstainedwithMayer’shaematoxylin

only.Theserepresentativeimagesdisplaythelevelofbackgroundstainingdetectedfor

immunohistochemistrysamplesinthissection.Allprimaryantibodieswereraisedinrabbit.

Scalebars100μm.

a) b)a) b)

Figure94:Representativephotomicrographsofcellular(a)anddecellularised(b)amniotic

membranetissuesectionsfromthreeseparatedonorsexaminedforlamininby

immunohistochemistry.Allbrownstainingrepresentspositiveimmunolocalisationoflaminin.

Scalebars100μm.

membranetissuesectionsfromthreeseparatedonorsexaminedforfibronectinby

immunohistochemistry.Allbrownstainingrepresentspositiveimmunolocalisationof

fibronectin.Scalebars100μm.

membranetissuesectionsfromthreeseparatedonorsexaminedforcollagenIVby

immunohistochemistry.Allbrownstainingrepresentspositiveimmunolocalisationofcollagen

IV.Scalebars100μm.

Figure97:Representativephotomicrographsofcellular(a)anddecellularised(b)conjunctival

tissuesectionsfromthreeseparatedonorsexaminedforlamininbyimmunohistochemistry.All

brownstainingrepresentspositiveimmunolocalisationoflaminin.Scalebars100μm.

tissuesectionsfromthreeseparatedonorsexaminedforfibronectinbyimmunohistochemistry.

Allbrownstainingrepresentspositiveimmunolocalisationoffibronectin.Scalebars100μm.

tissuesectionsfromthreeseparatedonorsexaminedforcollagenIVbyimmunohistochemistry.

AllbrownstainingrepresentspositiveimmunolocalisationofcollagenIV.Scalebars100μm.

3.9 CharacterisationofpatientswithocularMMP

Fivepatientswereexaminedandaredescribedinthissection.Twooffivepatients

examinedhadvisualacuitiesof6/9Snellenacuityorlowerinoneorbotheyes.Inboth

casestherewasco-existingocularpathologythataccountedforthevisualimpairment.

Patient1hadnormaltensionglaucomaandvisualacuityof6/18and6/9rightandleft

eyesrespectively.Patient2hadagerelatedmaculardegenerationwithgeographic

atrophyandvisualacuityof6/12and6/60rightandlefteyerespectively.Fourpatients

hadaninflammatoryscore≥5inoneorbotheyes.Thescore“0”and“1”weredifficult

todifferentiatewhenassessedatslitlampexamination.Allpatientshadoneorboth

eyesgradeIIIinvolvementbyFoster.(153)AssessmentbyTaubergradingfoundonly1/10

eyeshadnoinvolvementandthiswasinkeepingwiththeTauber-Liverpoolscore.(154,

156)Allpatientshadinoneorbotheyes≥60%verticalinvolvementand≥50%horizontal

involvement.FourpatientshadaRowseyscore≤50%ofthetotalscore.(155)Allpatients

hadgradeIIorlessoxfordgradingscore(mild).Noeyeshadcornealinvolvement

exceedinggrade1onanyofthemeasuredparameters(conjunctivalisation/

neovascularisation/opacification).Noeyeshadlagophthalmosoranyupperlid

deformity.Onlyonepatienthadalowerliddeformity,whichwasagrade1lateraland

medialentropionthatwasinkeepingwiththedegreeoftheirbilateralconjunctival

cicatrisation.Eightofteneyeshadtrichiasisofanydegree.

Patientnumber 1;ODand

2;ODand

3;ODand

4;ODand

5;ODand

Conjunctiva

Inflammatoryscore

totals(limbitis)

Tauberscore IIcIIIb;IIcIIIb IIdIIId;

IIdIIId

IIcIIIb;

IIbIIIa

IIdIIId;

IIbIIIb

IIcIIIb;

IIaIIIa(nil)

Tauber-Liverpool(%

II/III)

65/30;

75/100;

60/25;

85/83;

70/46;

Rowsey(score/45) 22;24 16;19 29;36 14;29 28;45

Foster III;III III/III III/III III/III III/0

Fornixupper/lower

5/0.5;

Cornea

Oxforddrynessscore 2;2 1;1 2;2 2;1 1;2

Conjunctivalisation/

neovascularisation

1/1;1/0 1/0;1/0 0/0;1;0 1/1;0/0 1/0;0/0

Opacification

peripheral/central

0/0;0;0 0/0;0/0 0/0;0/0 1/0;0/0 1/0;0/0

Lagophthalmos n;n n;n n;n n;n n;n

Lashes T;T T;n T;T T;T T;n

Upperliddeformity

andgrade

n;n n;n n;n n;n n;n

Lowerliddeformity

andgrade

(laxitybut

grade0)

EntropG1

n;n n;n n;n

Table16:Tabledisplayingconjunctival,cornealandlidinvolvementinocularMMPpatients

examinedusingthenewproforma:OD=righteye,OS=lefteye,n=none/noinvolvement,T=trichiasis

(anydegree),Entrop=entropion;G=gradeL=lateral,M=medial.Seeappendix1forfulldetailsof

grading.

Figure100:Photographsoftherighteyeofpatient4.Theextentofhorizontalsymblepharon

involvementwas20mmextendingmediallyfromthelateralcanthus:a)lowerlidheldat

tensionb)upperlidheldattension.Thedistancebetweentheinferiorlimbusatthemidlineto

theedgeofthesubconjunctivalfibrosiswas1.5mm.Thefornixrulercouldnotbeinsertedinto

theinferiorconjunctivalfornix.

4 Discussion

4.1 Overview

Theconjunctivaisamucousmembranethatisanimportantcomponentoftheocular

surface.Inarangeofconjunctivaldiseasesleadingtocicatrisation,itmaybecome

irreversiblydamaged.Thisleadstodrynessanddesiccationoftheocularsurfaceand

mayultimatelyleadtocornealblindness.Torestorevision,anypotentialcornealor

limbalstemcelltransplantislikelytofailinthepresenceofsignificantconjunctival

disease.Thisstudysoughttodevelopnovelsubstratesonwhichconjunctival

epitheliumcouldbeexpandedforuseasconjunctivalgrafts.

Twonovelsubstrateshavebeendeveloped,eachwithdifferingpropertiestosuita

differentrangeofclinicalindications.Aconjunctivalconstructdevelopedfromtheex

vivoexpansionofconjunctivalepitheliumonadecellularisedconjunctivalsubstrateis

degradableandthereforemaysuitindicationswheresmallergraftsarerequired.Thisis

incontrasttoaconjunctivalconstructdevelopedfromtheexpansionofconjunctival

epitheliumonePTFE,whichisnon-degradableandavailableinlargequantitiestosuit

indicationswherealargergraftisrequired.Inparticular,thismaysuitforniceal

reconstructionwherebythesubstratewouldremaininsitutopreventrecurrent

fornicealloss.

DescribedinthefollowingsectionsistheprocesswherebyePTFEanddecellularised

conjunctivalsubstrateshavebeendevelopedasculturesubstrates.Ammoniagas

plasmamodificationofePTFEanditseffectonhydrophilicityisdescribedalongwithan

explorationoftheoptimalculturemediaforthegrowthofconjunctivalcells.Thecell

densityofculturedcellshasalsobeencomparedwhenePTFEistreatedononeorboth

surfaces(surfaceandunderside).Finally,thephenotypeofconjunctivalcellsdeveloped

onplasmatreatedePTFEiscomparedwithuntreatedePTFEandanestablishedcell

culturematerial,Thincert,inwhichthecellculturesurfaceisachemicallymodifiedPET

membrane.Inallexperiments,PETmembraneisusedasapositivecontrolgiventhatit

isanestablishedproductdesignedforcellculture.ChemicallymodifiedPETis

sucessfullyutilisedinmedicalapplicationssuchascapillarymembranesfordialysisand

implantablemedicaldevicessuchasvascularstents.(175)Itwouldnotserveasa

potentialsubstrateforconjunctivaltransplantation,however,duetoitsrigidity.In

contrast,ePTFEcanbemanufacturedintothinanddurablesheetswithahighdegree

ofelasticityandmakingitideallysuitedtosoftbiologicaltissueapplicationswith

demonstratrablesuperioritytootherpolymers.(175,176)PETmembraneistherefore

utilisedasapositivecontrolinthisstudyratherthanbeinginvestigatedasan

alternativesubstrateforconjunctivalepithelialexpansion.

Thedecellularisationofhumanconjunctivahasnotbeenpreviouslyreported.

Describedwithinthefollowingsectionsarethedevelopmentofasuitableprotocolfor

thedecellularisationofhumanconjunctivaanditscharacterisationintermsofDNA

content,collagendegradation,tensilestrength,basementmembranecompositionand

contactcytotoxicity.Severalattemptshavealsobeenmadetodevelopconjunctival

culturesonthedecellularisedsubstratewithsomesuccesswhentheexplantcultures

aredevelopedondecellularisedtissuewithanintactbasementmembrane.The

expressionofarangeofconjunctivalmarkersinthedevelopedconjunctivalconstructis

alsodescribed.

4.2 Ammoniagasplasmatreatmentincreasesthehydrophilicityof

Expandedpolytetrafluoroethylene(ePTFE)isarelativelyhydrophobicmaterial.

Ammoniagasplasmawasusedasatreatmentinthisstudytoincreasethe

hydrophilicityofePTFEtoimproveitsabilitytosupportcelladhesionandproliferation.

Indeed,ammoniagasplasmahasbeenpreviouslyshowntoincreasethesurface

wettabilityofarangeofpolymersincludingPTFEandPDMS.(108,177)Theresultsfrom

thisstudyconfirmthatradio-frequencyglowdischarge(RFGD,alsoreferredtoasgas

plasma)followedbythepost-treatmentmodificationindistilledwaterresultedina

significantreductioncontactangleto71o,whichis54%oftheoriginalstaticcontact

angleofuntreatedePTFE.Thesecontactanglemeasurementsareinkeepingwith

previousstudiesontheammoniaplasmatreatmentofePTFE,whichalsodemonstrated

thatthesubstratesupportedthegrowthoffunctionalretinalpigmentepithelium.(107)In

general,contactangles<90oareconsideredhighlywettablesurfacesinwhich

significantspreadofadropletofwatercanbeobserved,whereasthosewithcontact

angles>90oareconsideredtobelesswettable.(109)

Apotentiallimitationinthisstudyistheuseofstaticcontactanglemeasurements

ratherthandynamiccontactanglemeasurements.Potentialadvantagesofthedynamic

contactanglesaretheassessmentofbothadvancingandrecedingmeasurementsof

thecontactangle.Theseactassurrogatemeasuresofhydrophobicityand

hydrophilicityrespectively.(109)Thisallowsadditionalinformationonthehysteresisand

thereforetheroughnessofthematerial.(109)Dynamiccontactanglemeasurements

havehowever,beenobtainedpreviouslyforammoniagasplasmamodifiedePTFEinan

earlierstudy.(178)Themainremitofcontactanglemeasurementinthepresentstudy

wastoensurethatthetreatmenthadthedesiredeffectonhydrophilicity.Featuresto

optimisereliabilityofstaticcontactanglemeasurementinthepresentstudyincludeda

motoriseddropdispensertoensureadropletwasdispensedinacontrolledrateand

volume,anenclosedchambertoreduceairbornecontamination,opticalmeasurement

baseduponanimagetakenbyanin-builtcameraandautomatedprocesstoreduce

problemswithobserverdeterminedtangentlines.(109)

TheRFGDfollowedbythepost-treatmentmodificationindistilledwaterhasbeen

demontratedbyx-rayphotoelectronspectroscopytoresultinbreakageofthecarbon-

fluorinebondswhicharereplacedbyhydroxylgroups(OH).(179)Defluorinationhasalso

beenshowntoresultintheadditionofothernitrogenandoxygencontainingmoieties,

whichalsoresultinanincreaseinsurfacewettabilitythoughammoniagasplasma.(108,

171,172,180)

Thespecificchemicalcompositionofthepolymer,includingthe‘functional’groups

presentonitssurfaceasaresultofplasmatreatmentandtheresultingwettability

greatlyinfluencethenatureofinteractionswithbothcellsandproteins.(108)Studies

haveshownthatmaterialswithterminalfunctionalitiesincludinghydroxyl

(-OH)groupsandsubstrateswithincreasedwettability,ingeneral,adsorbpeptidesto

thematerialsurfacemoreeasilyandpositivelyinfluencecellattachmentand

proliferation.(181,182)Alternativegasplasmamodificationsincludemodificationwith

oxygenandair.(108)Thesewerenotexploredinthisstudyasammoniagasplasma

modificationwasshowntobesuccessfulinsupportingretinalpigmentepithelium.(178)

Giventhatammoniagasplasmatreatmentalsoincreasedthecelldensityofcultured

conjunctivalepithelium,furthermodificationswerenotattempted.Investigationof

alterativegasplasmashowevermaywarrantconsiderationinfuturework.

Anidealsyntheticsubstrateshouldallowtheadherenceofcellsandsupportcell

proliferationanddifferentiationalonganappropriatelineage.Tothisend,amultitude

offactorsareresponsibleforoptimalcellcultureinvitroincludingmediaandits

supplements,thesurfacetopology,roughnessandstiffnessofthesubstrate,porosity

andsurfacechemistry.(77,183-185)ScanningelectronmicroscopyofePTFE,suggestedthat

RFGDmayresultinamilddegreeofsurfaceetchingofthesurfaceridgesthatwasnot

apparentonePTFEwhenexaminedas-received.(108,171,172,180)Theeffectofthis

togetherwithothersurfacetopologyfeatureswarrantconsideration.

Othermodificationstoconsiderinfutureworkincludetheimmobilisationand

adsorptionofextracellularmatrixproteinsonthematerialsurfacetomimicnative

basementmembranes.(183)Tothisend,differentialsignallinginconjunctivalepithelial

cellshavebeendeterminedwhenadherenttodifferentisoformsoflaminin.(186)This

thereforeconfirmsthepotentialforcell-matrixinteractionsofconjunctivalepithelial

cellsandtheirinfluenceoncellularphenotypeandbehaviour.(187)

Theproductionofabasementmembranehasbeenreportedinmatureendothelialcell

culturesinvasculargraftsdevelopedfromsyntheticsubstrates.(185,188)Ithasbeen

proposedthatbasementmembranedepositionwouldoccurfrommostcelltypeswhen

culturedonsyntheticsubstrates.(189)Althoughbeyondtheremitofthepresentstudy,it

wouldbeofinteresttodeterminethetimescalerequiredforthedevelopmentofa

basementmembranefromconjunctivalepitheliumonePTFEandtoexplorethespecific

componentsdepositedincomparisontoconjunctivalbasementmembrane.

4.3 ExperimentaluseoftheHCjE-GiCellline

4.3.1 CharacteristicsoftheHCjE-Gicellline

TheHCjE-Gicelllinewaschosenforthisstudygivenitssimilaritytoprimaryhuman

conjunctivaintermsofitskeratinrepertoireandmucingeneexpressionincluding

gobletcellspecificMUC5AC.(190)AlternativessuchastheIOB-NHCcelllineandChang

celllinewereexcludedastherewereconcernsthattheIOB-NHCwasshowingsignsof

senescence(personalcommunicationswithDr.Diebold)andtheChangcelllinewas

recognisedtohaveHeLacellcontaminantsdisplayingamorefibroblasticphenotype.

CharacterisationstudiesbytheGipsongroupacknowledgedthatHCjE-Giepitheliawere

similartonativeconjunctiva,buttheydidnotachievethenormalmorphologic

differentiationobservedinnativeconjunctiva.AsmallsubpopulationofHCjE-Gicells

expressedMUC5ACmRNAwhendevelopedontype1collagenandfibroblastco-

culture,however,therewasnomorphologicalevidenceofgobletcellpresenceor

secretedmucinwithincultures.DespitethecultureofHCjE-Gicellsunderthekidney

capsuleofSCIDmice,thecelllinestilldidnotdevelopmaturegobletcells.TheGipson

groupsuggestedthatitwaslikelythatthedifferentiationpathwayhadbeen

deleteriouslyalteredsuchthatgobletcelldifferentiationwouldnottakeplace,in

keepingwithotherreportsonthephenotypicoutcomeofcelltransformation.(191)

Similartothepresentstudy,theHCjE-Gicelllinehasalsobeenusedinstudiesof

substratedevelopmentforconjunctivalexpansionandinstudiesofmucingene

expression.(190)Despiteevidencethatthecelllinemaynotfullydifferentiate,theHCjE-

Gicelllinewasusedforthepresentstudyastheupregulationandthereforethe

relativeincrease/decreaseinconjunctivalepithelialmarkersmayserveasthebest

availableindicationoftheexpectedresponseinprimaryconjunctivalepithelium.Inthis

study,consistencyandrepeatabilityofcellularresponsewasofparticularimportance

giventhatnumeroussubstratesandexperimentalrepeatswererequiredfor

experiments.Thealternativeofusingprimaryconjunctivalepithelialcellsfromasingle

donorwouldhavebeeninsufficient.ThedetectionofMUC5ACbyflowcytometryhad

notbeenpreviouslyreportedintheHCjE-Gicelllineandwasthereforealso

investigated.

4.3.2 SurfacemodifiedePTFEallowshumanconjunctivalcellattachmentand

proliferationwithanappropriatecellseedingdensityandcellculturemedia

4.3.2.1 Determinationoftheoptimalcellseedingdensity

Determinationofanoptimalcellseedingdensityiscrucialinthecontextof

repopulatingsyntheticscaffoldswithcells.Studiesonthecellseedingdensityof

primaryendothelialcellsfoundthatbetween20-70%ofcellsthatinitiallyadheredto

syntheticmaterialswerelostafteronehour.(192)Thisislikelytooccurinmostcelltypes

anddependsuponthesubstrateusedforculture.(185)Theresidualcellsmaypartially

replacecelllossbyproliferationandmigrationbutthiswoulddependonthedensityof

residualcells.(185)Itfollowstherefore,thatinitialcellseedingexperimentsenablethe

developmentofcultureswithinasuitabletimeframeandratebutimportantly,ensure

thatthedevelopmentofconfluentcellgrowthispossible.

Inthepresentreport,itwasnotedthatthecellseedingdensitywasmorecrucialfor

culturesseededonammoniaplasmatreatedePTFEthanthoseonPETmembrane

(polyethyleneterephthalate;Thincert).Acellseedingdensityof1x104cells/cm2versus

1x105cells/cm2onplasmatreatedePTFEledtoamorethan3-folddifferenceincell

densityafter7daysinculture.ThiscontrastswithPETmembraneinwhichthe

differenceincelldensityat7daysafterseedingat1x104cells/cm2wasapproximately

30%lessthananinitialseedingdensityof1x105cells/cm2.Theobserveddifferences

suggestasuperiorabilityofPETtosupportgreatercelladherencefollowingseeding.

Inmodelsofboneregeneration,thecellseedingdensitywasregardedasacritical

factortothesynthesisofextracellularmatrixandsubsequentinductionofnew

bone.(193)Theroleofcellseedingdensityhasalsobeenshowntoinfluencethe

differentiationofpluripotentstemcellsandinparticular,theexpressionoftight

junctionalproteins.(194)Itseemslikelytherefore,thatthecelldensityisofcritical

importancetoensurecell-cellcontactandsignalling.(185)ThiswasprovenbyChenand

colleagues,whodemonstratedthatproliferationofcellsinculturewasdependent

uponcell-cellcontact,andnotjustsolublefactorswithinmedia.Thiswas

demonstratedthroughexperimentsdesignedtocontrolthespaceandcontact

betweenpairsofcells.(195)

4.3.2.2 Determinationoftheoptimalculturemedia

TheoriginalprotocolforcultureofthehumanconjunctivalcelllineHCjE-Gihad

describedtheuseof‘early’and‘late’growmediaformulae,whichwerereplicatedin

thepresentstudy.(190)Thepurposeoftestingachangeinmediafromthe‘early’to

‘late’formulaafter3days(protocolC)and7days(protocolB)inculturewereto

ascertainwhichprotocolwouldleadtoagreatercelldensity.Furthermore,itwasalso

ofinteresttoquantifycelldensityusingthe‘lategrow’mediaalonetodetermine

whethertheinitialearlygrowmediawasrequired.Afurthercohortalsoincludedthe

‘earlygrow’mediawith1%BSA(w/v)intheplaceofbovinepituitaryextractand

epidermalgrowthfactor.The1%BSAcohort(protocolD)wasincludedtodetermineits

effectoncelldensitysincetothebestofmyknowledge,ithasnotbeenpreviously

reportedasamediaconstituentusedinthecultureofepithelialcells.

Gipsonandcolleagueshaddescribedaprotocolinwhichthe‘earlygrow’mediawas

changedtothe‘lategrow’formulawhenHCjE-Gicellswere70-100%confluent.(190)As

theePTFEsubstrateisopaque,phasemicroscopycouldnotbeusedwhenan

experimentisinprogresstodeterminecellularconfluence.Theuseofmediaprotocols

AandDledtoanoveralldeclineincelldensityincontrasttoprotocolsBandC,in

whichthecelldensityincreasedwithadvancingtimeinculture.InprotocolsBandC,

themediawaschangedfrom‘early’tothe‘lategrow’mediaafter7and3days

respectively.Theexperimentdeterminedthatthegreatestcelldensitycanbeachieved

ifthemediaischangedfroman‘earlygrow’tothe‘lategrow’formulaonday7

(protocolB)whencellsareseededat1x105cells/cm2.Thiswasinkeepingwithexisting

protocolsforthecultureoftheHCjE-Gicelllineinwhichthelategrowformulawas

usedoncecellswere70-100%confluent.(190)Thiswasevidentandinkeepingwiththe

celldensityandspacingdemonstratedonDAPIstainedphotomicrographs,which

demonstrateconfluentculturesontreatedePTFEandPETmembranebutsparsecell

growthonuntreatedePTFE.Interestingly,evenuntreatedePTFEsupportedaslowly

increasingpopulationofHCjE-GicellsinculturewithmediaprotocolB.Thiswasin

contrasttocultureinmediaprotocolC,inwhichadeclineincelldensitywas

demonstratedafter7daysinculture.ThePETmembranewasdemonstratedtosupport

asteadycelldensitywithmediaprotocolAincomparisontoePTFEsubstratesinwhich

amoreobviousdeclineisnotablewithadvancingtime.Itfollowstherefore,thatthe

PETmembranemaybesupplyingfactorsthatenableahigherrateofinitialcell

adherenceandmaintenanceofahighercelldensityincomparisontocultures

developedonePTFE.ThesedataalsodemonstratethatuntreatedePTFEsupportsa

verylimitedpopulationofcellsandtheresultingcelldensityisinfluencedbyfactorsin

theculturemedia.ItisunlikelythatuntreatedePTFEwouldserveasausefulsubstrate

incellcultureapplicationsgiventhesparsecelldensityofcellsafter14daysinculture.

Foroptimalcellgrowthinvitro,theosmolalityandpHofthemediamustbetightly

controlled.Inaddition,themediamustcontainsugars,salts,aminoacids,growth

factorsandhormonesspecifictothecelltypebeingcultured.(185)Inavarietyofmedia,

growthfactorsandhormonesaresuppliedthroughtheadditionoffoetalcalfserumto

constitute10%ofthemedia.(185)Disadvantagesofthisapproacharethepotentialfor

thetransmissionofinfectionandthepotentialvariabilityinbatchesofserum.(185)

Bovineserumalbumin(BSA)iscommonlyusedinmediastudiesofgametesincluding

theovineoocytesmodel(0.04%w/v)andspermatogonalstemcells.(196,197)Media

usedinthecultureofspermatogonalstemcellscontains0.2%BSAandanumberof

cytokineconstituentshavebeenidentifiedincludingfibroblastgrowthfactor-2,glial

cell-linederivedneurotrophicfactorandGDNFfamilyreceptoralpha1.(198,199)Indeed

othermediadesignedforstemcellculturehavebeendevelopedforhaemopoietic

stemcellculturewhichinclude0.5%BSAinStemPro34-SFMmedia,butthisalso

contains1%foetalcalfserum.(200)

GiventheaboveexamplesoftheuseofBSAinmedia,itseemstohavegreaterutilityin

thecontextofmaintainingundifferentiatedcellsandthereforeitsuseinthepromotion

ofproliferationanddifferentiationofepitheliamaybelimited.Inotherexperimental

modelsBSAunderwentareactionwithglucosetoproduceadvancedglycationend

products,whichwerefoundtodrivelymphocytestowardsapro-inflammatory

response.(201)Indeedinthepresentreport,incontrasttotheothermediaprotocols

tested,thecelldensitywithuseofmediaprotocolDresultedinadeclineincelldensity

acrossallsubstrateswithadvancingtime.Thecelldensityontreatedanduntreated

ePTFEwassimilarbutfour-foldgreateronPETmembranesuggestingfargreaterinitial

adherenceonPETmembrane.Itwouldbeofbenefittoascertainthenatureofthe

surfacechemistryontheThincertPETmembranehoweverthishasbeenprotected

fromthepublicdomaingiventhatitisacommercialcellcultureproduct.

TheinitialcelldensityissimilaronPETmembraneregardlessofthemediaused

suggestingthattheinitialadherenceissuperiorduetoitssurfacemodificationrather

thanmediafactors.Thisstudyalsoconfirmedthatcelldensityisgreateronammonia

plasmatreatedthanuntreatedePTFEregardlessofthemediausedsuggestingthat

superioradherenceoccursontreatedePTFEincomparisontountreatedePTFE.These

differencesincelldensitybecameincreasinglymarkedwithadvancingtimeinculture.

Epitheliaarearrangedasasheetofcellsonabasementmembrane.Ingeneralepithelia

arerecognisedtoberelativelyshortinverticalheightwhereastheyarerelativelywide

andthereforeflatinshape.(185)Thecellmorphologydemonstratedfollowingculturein

mediaprotocolBontreatedePTFEandPETwasthereforeinkeepingwiththeexpected

appearanceofepithelialcellsincontrasttocellsimagedonuntreatedePTFE,which

weresparse.Thephalloidinstaining,however,waspoorwithdiffusestainingofcells

culturedonthePETmembrane.Itmaybethatthereisanunknownchemical

component,whichpreventseffectivephalloidinstainingorthatthechemical

compositionofthemembraneitselfresultsinahighdegreeofbackgroundstaining.

Thismaybeduetothepresenceofproteinsonthepolymersurfaceaspartofthe

surfacemodificationprocess.ThecellmorphologyonbothtreatedePTFEandPET

membranedemonstratedcellularconfluence.Anotabledifferencehoweverwasthe

sizeofboththenucleiandthesizeofthecellsoverallwherebyHCjE-Gicellsculturedon

treatedePTFEwereconsiderablylarger.

4.3.2.3 Summaryoftheoptimisationexperimentsforthecultureof

conjunctivalepitheliumonePTFE

Insummary,theseexperimentsdeterminedthatthegreatestcelldensitycouldbe

achievedat14daysinculturewiththeuseofmediaprotocolBandacellseeding

densityof1x105/cm2.AfurtheradvantageofcellcultureprotocolBisthatitissimilar

tothatthatdefinedinotherstudiesusedforprimaryconjunctivalepithelialcells.(97,111,

4.3.3 Consistencyofmarkerexpressionbetweenpassagesanddemonstration

ofcaspase-3upregulationinHCjE-Gicells

4.3.3.1 TheHCjE-Gicelllineisconsistentintheexpressionoftherangeof

testedmarkersbetweenpassages2-28

TheHCjE-Gicelllinewasusedatseveralpassagesinthisstudy.Passages2and28(from

arrivalofthedonatedcellline)weredevelopedunderthesameexperimental

conditionsandthephenotypeanalysedthroughflowcytometry.Thiswasundertaken

toensurethattherewouldbeconsistencyinexpressionoftherangeofphenotypic

markersusedinthisstudybetweenpassages.

Therearemanyreportsofphenotypicallystablecelllineswithadvancingserial

passage,whichcanbepropagatedindefinitely.Thisenablesustoexploitthe

consistencyandproliferativecapacityofcelllinesinexperimentalwork.Thiswasof

particularimportanceinthepresentstudyinwhichitwasnotpossibletoderivelarge

cellnumbersfromthesameprimarycellsource,especiallyasnumerousreplicatesand

variableswererequired.Therearealsoreportsofcelllinessuchasahumanbronchial

epithelialcellline,whichbecamespontaneouslytumorigenicatpassage184invitro-

culture.(202)Furthermore,otherthanamarkedtransformation,moresubtledifferences

inphenotypeincludingthoseduetoepigeneticchanges(e.g.architectureofchromatin)

arerecognisedcaveatsoftheuseofcelllinesinexperimentalconditions.(203)

Analterationintelomeraseactivityisregardedasakeycomponentinestablishing

immortalityincellslines.(204)Indeed,theHCjE-Gicelllinewastelomerasetransformed

withhTERT,atelomeraseholoenzymereportedtoinducereplicativeimmortality

withoutshorteningtelomeresandwithoutlossofpotentialtodifferentiate.(190,204,205)

GipsonandcolleaguesdevelopedtheHCjE-Gicelllineandconfirmedtheexpressionof

cytokeratins,membraneassociatedmucinsandMUC5ACmRNAdeterminingthatthis

celllinecouldbeusefulinstudiesofconjunctivalmucingenerepertoire.(190)

Thecytokeratinmarkers,togetherwithprogenitorcellmarkers,MUC5AC,PCNAand

caspase-3hadnotbeencharacterisedintheHCjE-Gicellline.Thesepreliminary

experimentsthereforesoughttodeterminethe‘baseline’levelofexpressionofthese

markersafter14daysincultureandtheproportionofcellsexpressingthesemarkers

withadvancingpassage.Theexpressionofarangeofmarkersassociatedwith

proliferation(PCNA),apoptosis(Caspase-3),progenitorcellmarkers(ΔN-p63and

ABCG2),cytokeratins19/7/4andthegobletcellmarkerMUC5ACweretestedincellsof

passage2and28toensureconsistency.Indeed,nosignificantdifferencebetweencells

ofpassage2andpassage28weredemonstrated.Itwasthereforedeterminedthatall

subsequentworkcouldbeundertakenwiththeHCjE-Gicelllinewithinthisrangeof

passagewithoutconcernsrelatingtophenotypicchange.

4.3.3.2 Caspase-3expressionincreasesinresponsetoenvironmentalstress

Caspase-3isinvolvedintheapoptoticpathwayandcanbefoundinthecytoplasmof

cellsdestinedforapoptosis.(136)Thedownregulationofcaspase-3hasbeenreportedin

thecontextofcancerincludingbreastcarcinoma.(206)Caspase-3hasbeenusedas

markerofapoptosisininavarietyofcellsincludingcelllines.(207,208)Nevertheless,since

theuseofcaspase-3asamarkerofapoptosishadnotbeenpreviouslyreportedinthe

HCjE-Gicellline,thisexperimentsoughttocharacteriseitsexpressionpriortousein

subsequentexperimentalwork.

Toensurethatcaspase-3wouldbeausefulmarkertoassesstheviabilityofHCjE-Gi

cultures,stainingwascomparedinhealthycellswiththoseexposedtoenvironmental

stresstoensurethatcaspase-3wasupregulated.(209)Thefindingsofthisexperiment

determineda100-foldincreaseinexpressionofcaspase-3withenvironmentalstress.

Thismarkerwasthereforeappropriatelyupregulatedinthecelllineandwasusedasa

markerofapoptosisinsubsequentexperiments.

4.4 CultureofHCjE-Giandprimaryconjunctivalcellsonammonia

plasmatreatedePTFE

PreliminaryexperimentsdeterminedthatammoniaplasmatreatedePTFEcould

supportagrowingpopulationofconjunctivalepithelialcellsasdescribedinsection

4.3.2.Initialexperimentswerealsoundertakentodeterminetheoptimalseeding

densityandmediathatwouldenablethis.Theworkdescribedinthissectiondescribes

howHCjE-GicelldensitycanbeinfluencedbytheexposureofePTFEtoammoniagas

plasmaonbothsidesofthematerial.

4.4.1 HCjE-GicelldensityisgreateronePTFEsubjectedtoammoniagas

plasmaonbothsides

Anexperimentwasundertakentodeterminehowammoniaplasmatreatmentonone

orbothsides(referredtohereassingleordoublesidetreated)oftheePTFEsubstrate

wouldcomparewithPETmembraneanduntreatedePTFEaspositiveandnegative

controlsrespectively.Thiswasundertakenasitwashypothesisedthatadditional

ammoniagasplasmamodificationontheundersideoftheculturesurfacecould

improvethepassageofnutrientsfrommediathroughtheePTFEmembrane.Thecell

densitywasdeterminedtogetherwithcellmorphologyandanalysisofthecellular

phenotypebyflowcytometry.Thelatterisdescribedinsection4.5.1.

Theresultsofthisexperimentdemonstratedagradualincreaseincelldensitywith

advancingtimeacrossallsubstrates,whichwasgreatestonPETmembrane.

Significantlyhighercelldensityhowevercouldbeculturedondouble-sideammonia

plasmatreatedePTFEthansinglesideammoniaplasmatreatedePTFE.Another

exampleofahydrophobicpolymertreatedbyammoniagasplasmatreatmentonboth

sidesofthematerialtoenablecellculturewaspoly(L-lactide).Astudyinvestigated

thedepthofplasmatreatmentsuchthatitpenetratedbothsidesofthematerialand

foundthatthelatterenabledtheproliferationofcellsandpromotedproliferation

withinthescaffolditself.(210)Thismaybemorerelevanttothisparticulartypeof

scaffold,whichisbiodegradableandthereforecellgrowthandinfiltrationfromboth

sidesandwithinthebodyofthematerialwasdesirable.Itwashypothesisedinthe

presentstudy,however,thattreatmentonbothsidesoftheePTFEmaterialwould

resultinimprovedtransportofwater,solutesandproteinsacrosstheporesfromthe

basalportionoftheePTFEincontactwithculturemediaandtheapicalportion

supportingepithelia.Thiswasmostlikelytohaveaneffectaftertheprocessofairlifting

hadbegun.Indeed,amarkeddifferenceincelldensitybetweensingleanddoubleside

treatedePTFEwasapparentbutonlyafter14daysinculturewhencultureswereinthe

airliftingphaseofthecultureprotocol.

Thecellcytoskeletoncomprisesintermediatefilaments,microtubulesand

microfilaments.(185)Themicrofilamentsarecomposedofactinwhereasintermediate

filamentsrepresentcytokeratinsinepithelialcells.(185)Thecellcytoskeletoniscrucialto

cellfunctionincludingdivisionandmovementandthereforealsoinfluencestheshape

ofthecell.(185)Themicrofilamentsconsideredthemostabundantproteininanimal

cells,comprisedofactinpolymersandarearrangedindifferentwaysbasedupontheir

functione.g.asacontractileringincelldivision.(185)Phalloidinstainsf-actinandwas

thereforeusedtoassessthecytoskeletalstructureincells.Unfortunatelythephalloidin

stainingonthePETmembranedidnotstainthecytoskeletalfibresasclearlyasthose

onePTFEsubstrates.PreliminaryexperimentsinthepresentstudyfoundUAE-1stained

theHCjE-Gicelllinesuchthatthecelloutlinebecameclearlyvisibleandwasalso

accompaniedbyvariableintracellularstainingonbothePTFEandPETsubstrates.Itwas

thereforewasusedasasurrogatemarkertoqualitativelyassesscellmorphologyin

additiontophalloidinstaining.

ThemorphologyofcellsonPETmembraneshowthatculturedcellsweresmallerinsize

andassumeanepithelioid‘cobblestone’morphologyafter14daysinculturethatarein

keepingwithtypicalconjunctivalepithelium.(211)Inearliertimepointswhenthedensity

waslowercellsappearmore‘rounded’acrossallthesubstrates.Thiswouldbe

expectedgiventhiswasatatimewhenthecellpopulationwasexpandinge.g.day2in

culture.Indeed,ithasbeenobservedthatmammaliancellsbecomeroundandmay

evenalmostdetachastheydivide.(185)Thekeymorphologicaldifferences

demonstratedweretheappearanceoflargercellswithproportionatelylargernucleion

ePTFEcomparedwithPETmembranesbyqualitativeassessmentofrepresentative

photomicrographs.Largercells,highnuclearcytoplasmicratio,andheterogeneityin

thenuclearappearancehavebeenreportedasmeasuresofsquamousmetaplasiain

impressioncytologyspecimens.(37,211,212)Tothisend,thecellsdevelopedonePTFE

substrateswerelargerandalthoughthisfeaturemayberegardedtoconstituteasa

featureofsquamousdifferentiation,thenuclearcytoplasmicratiosdidnotappear

qualitativelygreater.Furthermore,heterogeneityinnuclearappearancewasnot

apparentonePTFEorPETmembranesamples.Insquamousmetaplasia,thenucleiare

alsoconsideredtobecomepathologicallyalteredanddescribedaberrationsinclude

doublenuclei,nuclearfragmentationand‘snakelike’chromatin.(211)Again,these

featureshavenotbeennotedinHCjE-Gicellsonanyofthesubstrates.

Allofthegradingschemesdevelopedtodatearebaseduponimpressioncytologyand

thereforeitmustbetakenintoaccountthatthedescriptionsmaybebasedonloosely

adherentcellsonthemostsuperficiallayersoftheconjunctivalepitheliumand

thereforemaynotberepresentativeoftheconjunctivalepitheliumoverall.(37,211,212)

Therearenoknowngradingsystemsfortheassessmentofthecrosssectionof

conjunctivalepitheliumorcytoskeletalstaining.Nevertheless,theappearanceoflarger

cellsmayrepresentagreaterdegreeofsquamousmetaplasiaintheculturesdeveloped

onammoniaplasmatreatedePTFE.Tocorroboratethesefindingsitwouldbe

imperativeinfutureworktoassesstheexpressionofinvolucrin,amarkerofterminal

differentiation,squamousmetaplasiaandkeratinisation.(213)

4.4.2 Primarycellcultureissimilarondoublesideammoniaplasmatreated

ePTFEandPETmembrane

ThroughuseoftheHCjE-Gicellline,itwasdemonstratedthatgreatercelldensitycould

befoundonePTFEexposedtogasplasmaonbothsidesofthematerialratherthan

treatmentoftheculturesideonly.Thiswasrepeatedusingprimaryconjunctival

epithelialcellstodeterminehowculturesdevelopedondoublesideammoniaplasma

treatedePTFEwouldcomparewiththePETmembrane.Thephenotypeintermsofa

rangeofintracellularconjunctivalepithelialmarkerswasalsoundertakenandis

describedinsection4.5.2.Thisexperimentwasundertakentodemonstratethe

potentialofdoublesideammoniaplasmatreatedePTFEasacellculturesubstratein

comparisontoPET,acommerciallyavailablecellcultureproductusedinthis

experimentasapositivecontrol.Alimitationofthisexperimentwastheinabilityto

includesinglesideammoniaplasmatreatedanduntreatedePTFEsubstrate(negative

control)cohortsduetolackofavailablecellnumber.Unfortunately,onlyalimited

numberofcellscouldbederivedfromasingledonor.Indeeditwasimperativetouse

cellsfromasingledonorforconsistencyandcomparability.Theexperimentshowed

thatthetrendincelldensitydevelopedontreatedePTFEmirroredthatofPET

membrane,inwhichbothculturessupportedadegreeofproliferationfromday2to

day14inculture,followingwhichadeclineoccurred.Theprimarycellsmaytherefore

requirefurtherexogenousfactorsandimprovedculturetechniquestoenhancetheir

proliferativepotentialandmaintainapopulationofprogenitorcells.Possiblemethods

mayincludetheuseoffibroblastconditionedmediaand/orenrichmentwithhuman

serum,bothofwhichwereshowntoincreasecellnumberswithadvancingtimein

contrasttoEGFenrichedmediashownbyGarcia-Posadasandcolleagues.(132)

ThemorphologyofculturesondoublesidetreatedePTFEdemonstratedcellsofsimilar

sizeandelongatedcellprocessesvisibleatday14thatarenolongerpresentatday28

whencellsappearmorerounded.Notably,howevertherewasgreatervariationin

nuclearsizeandshapeonePTFEincomparisontocellsonPETmembrane.This

suggeststhatthesecellswereundergoingsquamousdifferentiationbasedupon

conjunctivalcytologygradingclassificationsdescribedintheprevioussection(4.4.1).(37,

211,212)Anotherstudycomparingprimaryhumanconjunctivalcellswithacellline

reportedtheappearanceofelongatedshapestobeinkeepingwitha‘fibroblastic

morphology’.(214)Alimitationofthepresentstudywasthatanalysisofthepresenceof

fibroblast-associatedmarkerswasnotundertaken.Giventhatflowcytometry

demonstratedthatof96%orgreatercellsexpressedCK19(ofthesamecellsinthis

experiment:section4.5.2)itseemsunlikelythatthesecellsrepresentfibroblastsperse

ratherthanconjunctivalepithelia.

4.5 PhenotypeofconjunctivalepithelialculturesdevelopedonePTFE

andPETmembranes

FlowcytometrywasundertakenusingtheHCjE-Giandprimarycellculturesdeveloped

onePTFEandPETmembraneasdescribedinsections4.4.1and4.4.2.Thiswas

undertakentoenablequantitativeassessmentoftheexpressionofarangeof

conjunctivalintracellularmarkerstodeterminethepresenceofgobletandprogenitor

cellsandtodeterminewhethercellswereactivelyproliferatingorundergoing

apoptosis.Thedescribedanalysiswasundertakenwithrespecttothesubstrateused

andadvancingtimeinculture.Comparisonshavealsobeenmadewithregardtooverall

levelsofexpressionofmarkersbetweenHCjE-Gicellsandprimaryconjunctivalcells.

Thiswasundertakenasthesedifferenceswereunknownpriortotheseexperiments

andwouldbeimportantforfuturework.

4.5.1 Differentialexpressionofcytokeratins,UAE-1lectinandMUC5ACin

HCjE-Gicellsandprimaryhumanconjunctivalcells

Analysisofprimarycellcultureswaslimitedtothosedevelopedondoubleside

ammoniaplasmatreatedePTFEandPETmembranewhereastheHCjE-Gicelllinewas

additionallyculturedonuntreatedePTFEandsingle-sideammoniaplasmatreated

ePTFE.Thiswasduetoalimitationinthenumberofcellsavailableforprimarycell

culturesinceallcellswerederivedfromasingletissuedonor.Theexperimentwas

repeatedonthreeoccasionswithcellsfromadifferentdonor.Determinationofthe

celldensityandmorphologywasundertakenusingthesamecohortofHCjE-Gi/primary

conjunctivalcellsasthatdescribedinthissectionandhasbeendiscussedinsections

4.4.1and4.4.2.

4.5.1.1 Expressionofcytokeratin19

• ThemajorityofHCjE-GiandprimaryconjunctivalcellsexpressedCK19

• NodifferencesinCK19expressionweredeterminedbetweensubstratesor

advancingtimeinculture

Inthepresentstudy,detectionoftheCK19antigenwasdemonstratedin96%or

greaterofthegatedcellpopulationsdemonstratedbyflowcytometryforbothHCjE-Gi

andprimaryconjunctivalcellsonallsubstrates.Therewasnosignificantdifferencein

CK19expressionbetweenthetimepointsorsubstratesstudied.Thisindicatesthatthe

cellswereofaconjunctivalepithelialphenotypeasCK19isarecognisedmarkerof

conjunctivalepithelia,andisnotexpressedbyconjunctivalfibroblasts.(125,132)Astudy

ofexplantculturesongelatinspongesfoundthatCK19wasnotexpressedbythebasal

layerofcells,whichexpressedingreaterfrequency,markersassociatedwitha

progenitorphenotype.(215)Incontrast,otherstudieshavefoundCK19expressioninall

epitheliallayerswhendevelopedwithserumona3T3feederlayer.(49)Thedescribed

differencesintheliteraturearelikelytobeduetodifferencesincultureprotocols.

GiventhatasmallproportionofcellsdidnotexpressCK19inthepresentstudy,itmay

provideanexplanationforthelessthan100%CK19expressionifitcouldbefurther

verifiedthatprogenitorcellsdonotexpressCK19.Itwouldalsobeinkeepingwiththe

proportionofcellsexpressingABCG2aloneorABCG2/ΔNp63co-expressiondiscussed

inthenextsectionofthisthesis(section4.5.2).Multichannelflowcytometrymay

providefutureopportunitiestoverifytheseobservationsandachieveabetter

understandingofconjunctivalcelldevelopmentalbiology.

• CK4expressionwasmarkedlyhigherinprimarycellculturesthanHCjE-Gicell

cultures

• HCjE-GiexpressionofCK4waslowerondoublesidetreatedePTFEonPET

membrane

• NosignificanteffectofCK4expressionwasfoundwithrespecttotimeinculture

inprimarycellculturesorHCjE-Gicellcultures

CK4expressionwasalmosta10-foldgreaterinprimarycellsincomparisontoHCjE-Gi

cellsafter28daysinculture.Itshouldbenotedhoweverthatvariancewashighin

primarycellculturesandnosignificantdifferencewasdemonstratedwithrespectto

substrateoradvancingtimeinculture.CK4expressioninHCjE-Gicultureswasgreatest

onPETmembraneanduntreatedePTFEandlowestonsingleanddoublesideammonia

plasmatreatedePTFE.Statisticallysignificantdifferenceswereonlydemonstrated

howeverbetweendoublesideammoniaplasmatreatedePTFE,untreatedePTFEand

PETmembraneinposthocanalyses.CK4isusedasamarkerfornon-goblet

conjunctivalepitheliaandisconsideredtobespecific.(11)Therehavehoweverbeen

variablereportsinrelationtothepatternsofCK4expressioninconjunctivalepithelium.

EidetandcolleaguesfoundnoimmunoreactivitytoCK4inprimaryhumanconjunctival

cellcultureswhentestingaserumfreecryopreservationstorageprotocol.(139)Garcia-

PosadasandcolleaguesalsousedtheCK4markerinthecharacterisationof

conjunctivalculturesbutdidnotcommentonitsqualitativeorquantitative

detection.(132)Incontrast,SchraderandcolleaguesreportedCK4expressionthroughall

butthebasalepitheliallayersincryopreservedepithelialculturesdevelopedinserum

containingmediaonagrowtharrested3T3layer,whereasQiandcolleaguesreported

CK4insuperficiallayersofbulbarconjunctiva.(49,130)Differencesinthefrequencyof

CK4detectionbetweenthepresentstudyandthatbySchraderandcolleaguesmaybe

duetosubstraterelatedfactors,differencesinmedia,thespecificityoftheantibody

cloneusedwithinexperimentsoracombinationofthese.Todate,therehasnotbeena

studythathascharacterisedthefrequencyandspatialdistributionofCK4throughout

nativeconjunctivalepithelium.Therefore,itisnotpossibletocommentonhowthe

culturesdevelopedonsubstratesinthepresentreportcomparewithexpression

patternsinvivo.Furthermore,thefunctionalrelevanceoftheproportionalofcells

expressingCK4isunclearfromtheexistingbodyofliterature.Ifregardedasamarker

ofdifferentiation,itisnotablethatasignificantlygreaterproportionofprimarycells

expressCK4.TheHCjE-Gicelllinedatawouldsuggestthatculturesmaybemore

differentiatedwhenculturedonuntreatedePTFEandPETmembranethanammonia

plasmatreatedePTFE.Thisisinkeepingwithandisinverselyproportionaltothe

proportionofprogenitorcellsandthoseofaproliferatingphenotypedescribedinthe

nextsection(4.5.2).

• CK7expressionwassimilarbetweenHCjE-Giandprimarycellcultures

• AsignificantincreaseinexpressionofCK7andCK7/UAE-1lectinco-expression

wasnotedwithadvancingtimeinHCjE-Gicellcultures

CK7expressionwassimilarinprimaryconjunctivalcellsandHCjE-Gicellsafter28days

inculture.InHCjE-Gicellcultures,however,thepercentageofCK7andCK7/UAE-1co-

expressingcellsdemonstratedonuntreatedePTFEwasmorethandoublethatofother

substrates.InHCjE-Gicellcultures,butnotprimarycellcultures,astatistically

significantincreaseinexpressionofCK7andCK7/UAE-1lectinco-expressionwasfound

withrespecttoadvancingtimeinculture.Theabsenceofastatisticallysignificant

increasewithadvancingtimeinprimarycellculturesmayrelatetothehighlevelsof

varianceasshownbystandarddeviationvalues.IncontrasttoHCjE-Gicellcultures,no

significantdifferencewasfoundbetweensubstratesinvestigatedinprimarycellculture

experiments.Forbothcelltypes,asimilarproportionofcellsco-expressedUAE-1/CK7.

Interestingly,thesevaluesareremarkablysimilartothepercentageexpressionofCK7

aloneinbothHCjE-Giandprimarycells.ThiswouldsuggestthatmostCK7positivecells

expressUAE-1lectinbutnotviceversa.Indeedthisisinkeepingwithexisting

knowledgethatmucinispresentinallconjunctivalepithelia(e.g.transmembrane

mucinsonepithelialcells)andisnotlimitedtogobletcells.(17)

DarttandcolleagueshavelocalisedthepresenceofCK7withUlexEuropaeusLectin-1

(UAE-1).(11)UAE-1isaglycoproteinthatisrecognisedtobindwitholigosaccharideson

themembranesofbloodgroupOerythrocytesbutalsoonhumanepithelialcells.Given

theresultsbyDarttandcolleaguestogetherwiththepresentstudy,itappearsthatCK7

andUAE-1lectinlocalisetothesamecellsmakingithighlylikelythatthesecellsareof

agobletcellphenotype.Indeed,DarttandcolleagueshypothesisethatCK7+/UAE-1

negativecellsmayrepresentapopulationofimmatureandactivelyproliferatinggoblet

cellsintheirstudyofthemigrationpatternsfromconjunctivalexplantoutgrowthand

differentiation.(134)Inthisstudy,thepercentageofconjunctivalcellsco-expressing

CK7/UAE-1lectinincreaseswithadvancingtimeinculture,suggestingthematuration

ofasubsetofgobletcellswasinprogress.Itwasalsodeterminedthatdoubleside

ammoniaplasmatreatedePTFEappearstosupportasubpopulationofimmature

gobletcellssimilartoculturesdevelopedonPETmembrane.Improvementsinculture

techniques,optimisationofbiopsysitelocationandcellpurificationmayresultin

improvedcelldensityandthedevelopmentofmaturegobletcellsonammoniaplasma

treatedePTFEinfuturestudies.Furtherapplicationofmultichannelflowcytometryand

cellsortinginfuturestudieswouldalsoenableverificationandfurtheranalysisof

specificsubpopulationsofcellsandfurtherourunderstandingofconjunctivalcell

biologyandthedevelopmentofgobletcellsinconjunctivalepithelialcultures.

4.5.1.4 ExpressionofMUC5AC

• MUC5ACexpressionwasmarkedlygreaterinprimarycellculturesthanHCjE-Gi

• MUC5ACexpressionwassimilarbetweensubstrates

• MUC5ACexpressionincreasedwithadvancingtimeinHCjE-Gicellcultures

MUC5ACwasexpressedina20-foldorgreaterproportionofprimaryconjunctivalcells

incomparisontoHCjE-Gicellculturesregardlessofsubstrateafter28daysinculture.A

significantincreaseinMUC5ACdetectioninHCjE-Gicellswasfoundwithadvancing

timeinculturebutthesubstratedidnothaveastatisticallysignificanteffect.Inprimary

cellcultures,nostatisticallysignificantdifferencewasdemonstratedinMUC5AC

expressionbetweensubstratesortimepoints.

Angandcolleaguesdevelopedanultrathinpoly(ε-caprolactone)(PCL)membraneand

foundmarginallyhighergobletcelldensityafterseedingprimaryrabbitconjunctival

cellsonPCLmembranetreatedwithsodiumhydroxide.(97)Theysuggestedthatgoblet

cellnumbersweresimilartothatdevelopedonamnioticmembrane(2-3%ofcells),but

lowerthanthatobservedinvivo(21%).(97)Bycomparison,thefindinginthepresent

studythat15%ofcellsdevelopedondoublesideammoniaplasmatreatedePTFE

expressMUC5AC,isthehighestreportedgobletcelldensitydevelopedonasynthetic

orbiologicalsubstratewiththeuseofhumanprimaryconjunctivalcells.Itshouldbe

takenintoaccounthoweverthatthevarianceinthesemeasurementswashigh,which

suggeststhatconsistencymaybeaproblemassociatedwiththeuseofprimarycell

cultures.GiventhatvarianceismarkedlylowerinHCjE-Gicells,itwouldsuggestthat

varianceisduetothecellsthemselvesratherthansubstrateormediafactors.Itwould

beofinteresttoinvestigatetheeffectofmediaconstituents,timeandculture

conditionstoimprovegobletcelldensity.Furthermore,itislikelythatthelocationof

originoftheexplantsseededonthesubstratemayinfluencetheresultinggobletcell

density.(134)Itispossiblethereforethattherandomselectionofexplantscontributedto

thehighvarianceinprimarycelldatainthisstudy.

TsaiandcolleaguesfoundthatmucinantigenandPeriodicAcidSchiffwasdetectedin

culturesofprimaryrabbitconjunctivalcellsdevelopedoncollagenandmatrigelbutnot

onplasticandglass.(95)Thelatterstudyconcludedcollagenandmatrigelaspermissive

environmentsforgobletcelldifferentiationbydemonstratingmucindetectedusingan

anti-mucinantibodybutdidnotquantifyMUC5AC.Adistinctionmustbemade

betweenmucinsoverallandgobletcellspecificmarkerstoenableinvestigationof

gobletcelldifferentiation.Indeedthisisanareathatneedsdevelopinginfuturestudies

andinvestigationintoalternativemarkersofgobletcellsarewarrantedgiventhat

immaturegobletcellsmaynotbeidentifiedusingMUC5AC.(11,134)

Itisknownthatairwaygobletcelldifferentiationisinfluencedbythetranscription

factorsSAM-pointeddomain–containingETS-likefactor(SPDEF)andforkheadortholog

A3(FOXA3)resultinginaninflammatoryresponseandgobletcellhyperplasia.(216)The

roleofSPDEFinconjunctivalgobletcellproliferationhasbeenpartlycharacterised,

howeveritsroleinchronicinflammationmaydiffertothatobservedinairwayepithelia

giventhatSPDEFmRNAwasdeficientinpatientswithSjogrensdiseaseincomparison

tonormalsubjects.(217)Infuturestudies,investigationoffactorsthatdrivegobletcell

differentiationwouldalsobeimportantforconjunctivaltissueengineeringapplications.

4.5.2 Differentialexpressionofmarkersofprogenitorcells,proliferationand

apoptosisintheHCjE-Gicelllineandprimaryhumanconjunctivalcells

4.5.2.1 ExpressionofΔNp63

• ExpressionofΔNp63wasmarkedlygreaterinculturesofHCjE-Gicellsthan

primaryconjunctivalcellsafter28daysinculture

• Nosignificanteffectwasfoundwithrespecttosubstrateoradvancingtimein

culture

ΔNp63expressionwasalmost3-foldgreaterinHCjE-Gicellculturesthanprimarycell

culturesat28daysandnosignificantdifferencewasdemonstratedwithrespectto

substrateoradvancingtimeinculture.Similarly,inprimarycellcultures,nosignificant

differencescouldbedemonstratedinexpressionofΔNp63expressionbetween

substratesoradvancingtimeinculture.ΔNp63hasbeenstudiedasamarkerof

progenitorcellsinprimarycellculturesbutnotintheHCjE-Gicelllinetothebestofmy

knowledge.(18,19)ThegreaterpercentageΔNp63expressioninHCjE-Gicellcultures

overallmaybeaccountabletotheoriginaltelomerasetransformationintheproduction

ofthecelllineandthereforeitsupregulatedreplicativeability.

Indeed,ΔNp63maybeamarkerthatcanbeidentifiedinbothprogenitorcellsin

additiontotransientlyreplicativecells.Thiswouldexplainthemarkedlyhigherlevelsof

expressioninthecelllinethanprimaryconjunctivalcells.Furthermore,itmayalso

explainthehigherthanexpectedlevelsofΔNp63inprimaryconjunctivalcellsafter28

daysinculture.Indeed,themajorityoftissuedonorsfromwhichtheprimarycells

originatedwereelderlyandthereforewouldbeexpectedtohavealowerproportionof

progenitorcells.Inkeepingwiththis,ithasbeenshownbyStewartandcolleaguesthat

thecolonyformingefficiencywasinverselycorrelatedtodonorage.(19)

ItwouldthereforebeofinteresttofurtherinvestigatetheappropriatenessofΔNp63

aloneasamarkerofprogenitorcells.ItmaybethatΔNp63mustbeusedin

combinationwithothermarkerstoidentifywithgreateraccuracyatruepopulationof

progenitorcells.Inviewofthis,theco-expressionofΔNp63togetherwithABCG2has

beendescribed(section5.2.2.2).Futurestudiesusingmultichannelflowcytometrymay

beusedtodeterminethetruenatureofΔNp63byanalysisofotherco-expressed

markersofdifferentiation/proliferation.Thismayalsobeconfirmedinfuturework

throughclonalanalysisofindividualsubgroupsofcellsdefinedbytheirmarker

expressionafterfluorescenceactivatedcellsorting.

5.2.2.2 ExpressionofABCG2andco-expressionwithΔNp63

• ABCG2expressionandco-expressionwithΔNp63wassimilarinHCjE-Giand

primaryconjunctivalcells

• ABCG2expressionandco-expressionwithΔNp63inHCjE-Gicellcultureswas

highestondoublesidetreatedammoniaplasmatreatedePTFEafter28daysin

culture

IncontrasttoΔNp63,ABCG2wasexpressedatsimilarfrequencyinboththeHCjE-Gi

andprimaryconjunctivalcellsat14and28daysinculture.Nosignificanteffectof

substrateortimeinculturecouldbedeterminedwithrespecttoABCG2expressionin

primarycellcultures.AmongstHCjE-Gicellcultures,ABCG2expressionwashigheston

doublesideammoniatreatedePTFEandstatisticallysignificantdifferenceswerefound

betweenthis,untreatedePTFEandsingle-sideammoniatreatedePTFEinposthoc

analyses.Inbothcelllineandprimarycultures,ABCG2/ΔNp63co-expressionwas

remarkablylowat1%orlessafter28daysinculture.Nostatisticallysignificant

differencewasfoundwithregardtoABCG2/ΔNp63co-expressioninprimarycultures

withrespecttoadvancingtimeorsubstrate.InHCjE-Gicellcultureshowever,the

percentageofcellsco-expressingABCG2/ΔNp63washighestondouble-sideammonia

plasmatreatedePTFEandstatisticallysignificantdifferenceswerefoundbetweenthis,

untreatedePTFE,singlesidedammoniaplasmatreatedePTFEandPETmembraneon

posthocanalysis.AlthoughthispatternmirrorsthatofABCG2expressionalonein

HCjE-Gicells,itshouldbenotedthattheproportionofcellsco-expressingABCG2and

ΔNp63ismarkedlylowerthanthatexpressingABGC2aloneinbothprimaryandHCjE-

Gicultures.ItfollowsthereforethatnotallABCG2expressingcellsalsoexpressΔNp63.

Theresultsfromourstudywouldsuggestthatthehighestproportionofprogenitor

cellsdevelopedondoublesideammoniatreatedePTFE,howeverthiswasconfirmed

onlywithcelllinedata.Varianceishighintheprimarycelldataandislikelytobedue

todifferencesintheseededexplantsintermsofthebiopsysitefromwhichtheexplant

originated.Furtherstudiesusinggreaternumbersofdonorsandconsistencyinthe

tissuebiopsysitearewarrantedtocorroboratethefindingsofthisexperiment.The

optimisationofcultureconditionstogetherwithimprovementsintechniquestoisolate

andseedapurifiedsampleofconjunctivalepithelialcellsmayenableprimarycell

culturesinthefuturetobedevelopedonammoniaplasmatreatedePTFEwitha

greaterproportionofprogenitorcellsandpotentialforself-renewal.

ABCG2andΔNp63havebeenstudiedtogetherinastudyofthecolonyefficiencyassays

ofconjunctivaltissuesfromvarioussites.(19)Thelatterreportdemonstratedhighly

significantcorrelationsbetweentheclonogenicabilityofconjunctivalexplantsandthe

levelsofbothABCG2andΔNp63expression.(19)Inkeepingwiththis,ithasbeen

observedinotherstudiesthatthedensityofcellsexpressingthesemarkersreduce

withadvancingpassage.(49)Althoughtherearenodefinitivemarkersofconjunctival

stemcells,themarkersusedherewithinthepresentstudieshavebeenthemost

extensivelystudied.Asfindingsfromthisstudyshow,notallcellsexpressingABCG2

appeartoexpressΔNp63.Theuseofbothmarkers,however,mayidentifywithgreater

sensitivity,asubpopulationofcellsthatrepresentsatrueprogenitorcellpopulation.

Furthermore,italsosuggeststhatABCG2alonemaybesuperiortoΔNp63initsability

todetectprogenitorcells.Inkeepingwithresultsinthepresentstudy,ABCG2

expression,butnotΔNp63hasbeenshowntocorrelatesignificantlywiththecolony

formingefficiencyofprimaryconjunctivalepithelialculturesandisinverselycorrelated

todonorage.(19)ItisofinteresttonotethattheΔNp63antibodywouldidentify

ΔNp63αthatisotherwiseconsideredastemcellmarker.(9)Theresultsofthepresent

studyhowevertogetherwithobservationsbyStewartandcolleagueswouldsuggest

otherwise.(19)Toinvestigatestemcellmarkerexpressionandcorroboratethe

observationsofthepresentstudy,theclonogenicabilityofculturesofcellsinrelation

totheexpressionandco-expressionofcandidatestemcellmarkerswarrantsfurther

study.Furthermore,theuseofamorespecificantibodyclone,forexampleΔNp63α

insteadofΔNp63,whichdetectsallisoforms(α/β/γ),shouldalsobeundertakento

determinethepreciselocationandphenotypeofprogenitorcellsandtherelative

frequencyandrelevanceofcellsofeachoftheΔNp63isoforms.

4.5.2.3 Expressionofcaspase-3

• Caspase-3expressionisgreaterinprimaryconjunctivalcellcultures

• Greatercaspase-3expressionwasdemonstratedonuntreatedePTFEinHCjE-Gi

cultures

Caspase-3expressionwasmorethandoubleinprimaryconjunctivalcellsoverall

comparedtoHCjE-Gicellsatthetimepointsstudied.Inprimaryconjunctivalcell

cultures,nosignificanteffectcouldbedemonstratedwithrespecttoadvancingtimein

cultureorsubstrate.Similarly,therewasnosignificantdifferenceincaspase-3

expressioninHCjE-Gicellcultureswithadvancingtimeinculturebutsubstratehada

significanteffectwherebythehighestlevelsofcaspase-3expressionwerefoundon

untreatedePTFE.Giventhatstatisticallysignificantdifferenceswerefoundonly

betweenuntreatedePTFEandallothersubstratesstudiedinpost-hoctestsforHCjE-Gi

cells,itwouldsuggestthatallsubstrateswiththeexceptionofuntreatedePTFEwere

equallycapableofsupportingconjunctivalcellcultureswithoutinducinghighlevelsof

apoptosis.

AlimitationofthisworkisthattheuntreatedePTFEsubstratewasnottestedusing

primarycellculturesowingtoashortageofavailablecellstouse.Itisthereforenot

possiblethereforetocommentontheexpressionoftheapoptosismarkeronprimary

cellsculturedonuntreatedePTFE.Thesedata,however,suggestthattherewasno

significantdifferencebetweenprimaryconjunctivalepitheliadevelopedontreated

ePTFEandthePETcellculturemembrane.Thisrepresentsproofofconceptthatdouble

sideammoniaplasmatreatedePTFEcouldbeaneffectivesubstrateforprimary

conjunctivalcellexpansionandwarrantsfurtherinvestigation.Tothebestofmy

knowledge,therearenootherreportsintheliteraturedescribinguseofcaspase-3in

thedevelopmentofconjunctivalsubstrates.Thereisthereforenoavailablecomparison

todeterminetherelativecaspase-3expressioninthepresentstudy.Astudyofthe

effectofhypoxiaandstaurosporineonendothelialcellshoweveralsodeterminedthe

levelsofcaspase-3asapercentageofthecellpopulationbyflowcytometry.(218)This

studydemonstrated5.9%caspase-3expressioninahealthypopulation,comparedwith

9%inendothelialcellsexposedtohypoxiaand24%incellsexposedtostaurosporine

(aninhibitorofproteinkinasesusedasaresearchtooltoinduceapoptosis).Similarly,

inthepresentstudyaround20%ofHCjE-Gicellsexpressedcaspase-3at28daysin

culturewhendevelopedonuntreatedePTFEcomparedwith<5%expressingcaspase-3

ontreatedePTFEandPETmembrane.Theseobservationswouldalsosuggestthat

caspase-3wouldbeanappropriatemarkerofapoptosistouseinfuturestudiesof

conjunctivalepithelialcultures.

4.5.2.4 ExpressionofPCNA

• PCNAexpressionismarkedlyhigherinHCjE-Gicellculturesatday28

• PCNAexpressiondeclinesinprimaryconjunctivalcellsbutnotHCjE-Gicellswith

advancingtimeinculture

NosignificantdifferencewasfoundwithrespecttothepercentagePCNAexpressionin

HCjE-Gicellcultureswithadvancingtimeincultureorbetweensubstrates.Similar

levelsofPCNAexpressionwerefoundinprimaryconjunctivalcellculturesdeveloped

ontreatedePTFEandPETsubstratesafter28dayssuggestinganequivalentabilityof

doublesidetreatedePTFEtosupportcellproliferation.IncontrasttoHCjE-Gicells,

however,astatisticallysignificantdifferenceinPCNAexpressionoccurredwhereby

expressionlevelsapproximatelyhalvedfrom14and28daysinculture.Thepercentage

PCNAexpressioninHCjE-Gicellswasapproximatelydoublethatofprimarycellcultures

at28daysinculture.

TheobservedtrendofPCNAexpressioninHCjE-Gicellswithadvancingtimewassimilar

tothatofΔNp63.Thereasonsoftheobserveddifferencesintheprimarycellsandthe

HCjE-Gicelllinemaybeduetoreasonsmentionedinearliersections.Itwouldbeof

interestinfuturestudiestocorrelatePCNAexpressiontomarkersassociatedwithstem

cellstodeterminemethodstodistinguishtransientlyamplifyingcellpopulationsfrom

‘true’stemcells.

EidetandcolleaguesdescribedPCNAexpressionbyimmunohistochemistryin

conjunctivaltissuesamplesinapproximately¾ofcellsthroughasemi-quantitative

analysis.(139)Thisstudywasdesignedtodeterminetheeffectofstoragefreemedia

after4and7days.Itmustbenotedhoweverthatthetimepoints,mediausedand

techniqueused(immunohistochemistry)differstothepresentstudy.Itfollows

thereforethattheoptimalorbaselinefrequencyofPCNAexpressioninconjunctival

epithelialcellcultureshasyettobeestablishedinthecontextofdevelopingsubstrates.

Nevertheless,thefindingofsimilarlevelsofPCNAexpressionbetweendoubleside

ammoniaplasmatreatedePTFEandPETmembraneeveninprimarycellcultures

suggeststhattheformerwarrantsfurtherinvestigation.

4.6 Decellularisedhumanconjunctiva

Decellularisedtissueshavebeenusedasscaffoldsforthere-populationofcellsina

varietyoftissueandorganmodels.Thedecellularisationofhumanconjunctiva,

however,hasnotbeenpreviously.Describedinthissectionisthedevelopedprotocol

forthedecellularisationofhumanconjunctivaandcharacterisationoftheresulting

tissueintermsofitscytotoxicity,DNAcontent,collagendenaturation,basement

membraneandtensilestrength.Comparisonshavebeenmadebetweenthebasement

membraneofamnioticmembraneandhumanconjunctiva.Thetensilestrengthof

conjunctivaisalsocomparedwithamnioticmembraneandePTFE.

4.6.1 Decellularisationandcytotoxicityofhumanconjunctiva

Decellularisationcanbeachievedusingavarietyofchemical,enzymaticandphysical

processes.Sodiumdodecylsulphate(SDS)wastheagentofchoicetooptimiseinthe

presentstudy.Thisstepwaspartofanestablisheddecellularisationprotocolusedfor

thedecellularisationofamnioticmembrane.Decellularisationofamnioticmembrane

hasbeenpreviouslydescribedbyphysicalremovalaloneorincombinationwith

chemicalagentsanddetergentsincludingammoniumhydroxide,SDS,sonicationand

trypsin.(219-222)Ofthesemethods,onlySDSachievedhighlevelsofcellularremovalfrom

thetissuematrix.(77)Thisdetergenthasbeenalsobeenreportedtosuccessfully

decellulariseothertissuesincludingporcineheartvalves.(223)Thelackofobservedcell

mediatedorhumoralimmuneresponseintherecipienttodecellularisedtissuehas

beenproposedtobeduetotheremovalofsolubleproteinsduringthe

decellularisationprocess.(224)

Inthisreport,thedecellularisationprotocolthatutilisedSDSataconcentrationof

0.05%(w/v)removed99%orgreaterDNAwithreproducibilityacrossdonors.Thiswas

alsoconfirmedinhistologyspecimens,wherebytheabsenceofnuclearDAPIstainingin

decellularisedtissuesectionswasdemonstrated.Adequatedecellularisationisof

paramountimportanceasthepresenceofDNAhasbeendirectlycorrelatedtoadverse

responsesintherecipient,aneffectthatismostnotableinxenogeneic

transplantation.(225,226)

DecellularisedtissuepersehasnotbeenstrictlydefinedintermsofresidualDNA

content.Resultsfrominvivostudiesinwhichtissueremodellinghasbeen

demonstratedintheabsenceofanadversehostimmunologicalresponsesuggest90-

95%decellularisationisadequate.(77,173)Itfollowsthereforethatadecellularisation

protocolusing0.05%SDS(w/v)todecellulariseconjunctivaltissuemeetsthese

previouslydefinedstandards.(77)Amnioticmembranecloselyresemblesconjunctiva

giventhatitalsocomprisesanepithelialcelllayer,basementmembraneandathin

layerofunderlyinglooseconnectivetissue.(54)Amnioticmembranecanbe

decellularisedwith0.03%SDSwhichremoves95%oftheDNA.(227)Thisiscomparable

toDNAremovalshowninthepresentstudy.Alimitationofthepresentstudy,

however,wasthatSDSataconcentrationlowerthan0.05%wasnotstudied.Infuture

work,itwouldbeofinteresttotestlowerSDSconcentrationsandcorrelatetheDNA

removalagainstadditionalparameterssuchaselastinandglycosaminoglycan

quantification.

Inthepresentstudy,decellularisationwasachievedthroughacombinationof

chemical,detergentandenzymaticsteps.Inthefirststagecelllysiswasachieved

throughosmosisusingahypotonicbuffer.Ithasbeensuggestedthatthisoccurswith

minimalalterationinthearchitectureofthetissue.(89)Inthesecondstage,dissolution

ofthelipidbilayerofthecellmembraneandremovalofcellularresidueswasachieved

throughtheuseofSDS.Inthefinalstage,Benzonasewasusedtoremoveanyresidual

nuclearmaterialfromthetissuematrix.Hypotonicandhypertonicreagentsareknown

tobeeffectiveinlysingcellsbutmayleavecellularresidues.(77)Theremovalofcellular

residueshoweverisknowntobeeffectedbySDS.(77)Ithasbeendemonstratedto

effectivelyremovenuclearandcytoplasmicremnantsfromevendensetissues,buta

disadvantageisthatitmaydamagecollagenmatrices.(77,228)Ofthesedescribedstages,

theSDSisknowntohavethegreatestpotentialforcellularcytotoxicity.(77)Indeed,

multiplewashesandrinsesofthetissuetakeplaceaspartoftheprotocoltoleachas

muchoftheSDSoutoftheresidualtissueaspossible.Theresultsfromthecontact

cytotoxicityexperimentsinthisstudydemonstratedthatbothprimaryhumanskin

fibroblastsandthehumanconjunctivalcellsproliferateinclosecontactwiththe

decellularisedtissuedemonstratingthatthetissueslackcytotoxicity.Inlatersections,

thisisalsodemonstratedbythecultureofexplantsondecellularisedtissues(section

4.6.2 Quantificationofcollagendenaturation

Totestwhetherthedecellularisationprocesswoulddisruptconjunctivalcollagen

matrices,anassaytotestcollagendenaturationwasundertaken.Thisrationalefor

undertakingthisexperimentwasthatexposuretoSDSisknowntodisruptcollagen

matrices.(77,228)Ourstudyfoundhoweverthatataconcentrationof0.05%(w/v)SDS,

therewasnoincreaseinhydroxyprolinewithinthesupernatantinthecollagen

denaturationassay.Thisindicatedthattherewasnodisruptionofconjunctivalcollagen

matrixasaresultofdecellularisation.Hydroxyprolinewasdetectableinthisassay

followingexposuretoα–chymotrypsin,anenzymethatselectivelydenaturesonly

degradedcollagen.(229)ThesedatathereforesuggestthatanSDSconcentrationof

0.05%(w/v)mayrepresentafavourablebalancebetweendecellularisationandtissue

disruptioninhumanconjunctivaltissue.Thiscollagendegradationassayisusefulasit

providesquantitativedatatosupportthequalitativeexaminationofotherextracellular

matrixproteinsthroughhistology,describedinlatersections(section4.6.4).More

detailedstudiesinvestigatingthedegradationofothercomponentssuchaselastinand

glycosaminoglycancontentarewarrantedinfuturework.Importantly,examinationsof

theultrastructuralfeaturesofthetissuearealsowarrantedtoconfirmtheintegrityand

examinethe3-dimensionalarchitecturefollowingdecellularisation.Theultrastructure

maybestudiedbyscanningelectronmicroscopyinfuturework.

4.6.3 Tensilestrengthofconjunctiva,amnioticmembraneandePTFE

Theeffectofdecellularisationonthetensilestrengthofconjunctivaltissuewas

determinedtogetherwithdataofthetensilestrengthofamnioticmembraneand

ePTFEforcomparison.Tensilestrengthresultsdemonstratednosignificantdifference

intheultimatetensilestress(MPa)orYoung’smodulus(MPa)betweencellularand

decellularisedtissuesuggestingthatbiomechanicalstrengthisnotalteredbythe

decellularisationtreatment.Thisisinkeepingwithothertissuemodelscomparing

cellularanddecellularisedtissue.(173,230,231)Thetensilestrengthmeasurementsare

relativelylowforbothconjunctivaandamnioticmembraneintermsoftheirabsolute

valuesincomparisontoothercharacterisedtissuessuchasdermisandcartilage.(173,230,

231)Nootherpublishedworkcanbefoundinwhichthetensilestrengthofconjunctiva

hasbeencharacterised.

Stressatfailuremeasurementshavebeenpreviouslyreportedforamnioticmembrane

andwereapproximatelyhalfthereportedvaluesinthisstudy.(227)Itisrecognisedthat

differencesbetweenobservedvaluesmaybederivedwhenusingdifferentequipment,

andthatsuchdifferenceshavenotbeenentirelyexplainedinotherreports.(232)There

areahowever,anumberofcrucialdifferencesinthetestingmethod,inthepresent

studycomparedtothatbyWilshawetal.Ingeneral,themethodologyfortensile

strengthtestinginvolvesthecuttingofdumbbellshapedsectionsthataretherefore

constantinlengthandalsowidth,andthewiderportionatbothendsareinsertedinto

theclamps.Thismethodwasnotpossiblefortheassessmentofbothconjunctivaland

amnioticmembraneinthepresentstudyasthestandarddumbbellshapesweresmall,

leavingthinfriablesectionsoftissuethatwouldbreakorbecomedamagedduringthe

processofinsertionintothetestclampsorwouldslidethroughthetestclamps

altogether.Toenabletestingtotakeplace,largerrectangularsectionsoftissuewere

dividedandmeasuredforthetest.Thetestsinthisreportmeasuredthethicknessof

theamnioticmembraneat3differentpointsbyplacingthetissueflatbetweentwo

glasscoverslips.Thismethodresultedinmeanthicknessvaluesforamnioticmembrane

of0.055mminthisstudy,whichcontraststothatbyWilshawandcolleagues,who

measuredmeanthicknessvaluesofamnioticmembraneas0.16mm.Althoughthe

stressmeasurementswouldnormalisetheeffectofthickness,themethodusedto

measurethicknessofthetissuesdiffersbetweenthepresentstudyandthatby

Wilshawandcolleagues.Inaddition,thespeedoftensiletest,whichwasnotreported,

mayhavedifferedinadditiontootherfactorsincludingthetypeofgripsusedtohold

thematerial.Itispossiblethatthesefactors,inparticularthethicknessmeasurements

ofamnioticmembranemayexplaintheobserveddifferences.

Theyoung’smodulushasnotbeenpreviouslycharacterisedforamnioticmembrane.

Young’smodulusisameasureofstiffnessofanelasticmaterial.Thestiffnessofa

materialitselfhasbeenrecognisedtoinfluencethecellularphenotype.(233)Itisof

interesttonotea3-foldgreaterYoung’smodulusofamnioticmembraneincomparison

toconjunctivaindicatinggreaterstiffness.Indeedithasbeenrecognisedthatamniotic

membranehasathickbasementmembrane,whichmayaccountforthese

observations.(49)

Itwouldbeofinteresttoundertakefurthertensilestrengthtestingtoascertainthe

suturepulloutstrength.Thismaybeofparticularrelevancegiventhatthematerials

investigatedareintendedforeventualsurgicaluse.Tensilestrengthcharacterisationis

usefulgiventhecurrentscientificclimateinwhichsyntheticmaterialsarealsounder

investigationasconjunctivalsubstrates.(97)Importantly,thestiffnessofmaterialsis

knowntoinfluencecellattachment,proliferationanddifferentiation.(228,233,234)The

precisesubtypesofextracellularmatrixcomponentsandstiffnessofthedecellularised

conjunctivaarenotgreatlyalteredbydecellularisation.This3-dimensionalscaffold

wouldotherwisebedifficulttoreplicateinsyntheticmaterials.Itmightbeexpected

therefore,thatdecellularisedconjunctivawouldpossessthegreatestpotentialto

supportconjunctivalepitheliumwithasubpopulationofprogenitorandgobletcellsby

providingthemostappropriatenicheforthesecells.

4.6.4 Characterisationoftheextracellularmatrixcomponentsandbasement

membraneofcellularanddecellularisedtissues

Decellularisationmayreducetheriskofdiseasetransmissionbutmaydisrupt

extracellularmatrixcomponentsandtheultrastructureofthetissue.(77)Thehistology

andimmunohistochemistryqualitativelydemonstratethatthereisnodisruptionofthe

extracellularmatrixasaresultofthedecellularisationprocess.

HistologyofthetissuesectionsstainedwithH&EandVanGieson’sstaindemonstrate

thatthereispreservationofthearchitectureofthetissueincludingthemajor

structuralproteinselastinandcollagen.Glycosaminoglycanswerealsostrongly

localisedtothebasementmembranesandconservedfollowingdecellularisation.

Theextracellularmatrixcomponentslaminin,fibronectinandcollagenIVdonotappear

changedfollowingdecellularisation.Thisisapparentqualitativelyintermsofthe

distributionoflaminin,fibronectinandcollagenIVstainingthrough

immunohistochemistry.Theabsenceofcollagendegradationisalsosupported

quantitativelyintermsofthedetectionofdenaturedcollagenasdescribedinthe

previoussection(section4.6.2).Laminin,fibronectinandcollagenIVwasfoundinthe

basementmembranezoneandepitheliumofconjunctivaltissues,astainingpattern

thathasbeenpreviouslydescribedbyimmunohistochemistry.(15)ThePASandH&E

stainingalsoconfirmedthatthebasementmembranelayerappearsunchangedby

decellularisation.

Tissuesamplesfromthreedifferentdonorswereexaminedfordistributionofcollagen

IV,lamininandfibronectininbothconjunctivaltissueandamnioticmembrane.This

qualitativelydemonstratedthattherewerenosignificantdifferencesinthedistribution

oftheseproteinsineithertissuebetweendifferentdonors.Differenceswereobserved,

however,inthedistributionofcollagenIV,lamininandfibronectinbetween

conjunctivaandamnioticmembrane.Thebasementmembraneofamnioticmembrane

hadgreaterqualitativelystainingthanthesubstantiapropriaforallthreeproteins

studied.Alltheinvestigatedproteinswerepresentinthebasementmembraneof

conjunctiva;however,theirpresenceinthesubstantiapropriawaspresent

qualitativelytoagreaterdegreethanamnioticmembrane.Lamininappearsmostly

withinbasementmembranesofconjunctivawhereasfibronectinappearsalmostas

abundantwithinthebasementmembraneasthesubstantiapropriaofconjunctiva.The

distributionoftypeIVcollagenandlamininisoformsinamnioticmembranehavebeen

studiedandreportedtobesimilartothatofconjunctivabutdifferfromcorneal

basementmembrane.(3)Ithasthereforebeensuggestedthatamnioticmembranemay

thereforeactasasuitablesubstrateforconjunctivalexpansion.(12)Thelatterstudyby

Fudakaandcolleagueshighlightspotentiallyimportantdifferencesinextracellular

matrixcompositionbetweenamnioticmembraneandconjunctiva.Inparticular,the

greaterfibronectincompositionofthesubstantiapropriaoftheconjunctivacould

serveasapreferentialenvironmenttotheco-cultureofconjunctivalfibroblasts.(235)

Theresultsinthisstudyalsodemonstratedthatthereisfargreatervariabilityinthe

tissuethicknessandarchitectureoftheconjunctivalsamplesincomparisontoamniotic

membranebetweenthedonorsamplesstudied.Ultimately,thismaylimititsfuture

utilitygivenlimitationintheavailabilityoflargesectionsoftissueofanoptimalquality.

Thequalitativemethodsusedforthecharacterisationofextracellularmatrixhavetheir

meritsastheyprovideinformationaboutthedistributionpatternsandmicroscopic

appearanceoftissues,whichquantitativeassayssuchasthecollagendegradation

assaycannotprovide.Itisimperativethereforethatbothqualitativeandquantitative

analysisoccursinparallel.Alimitationoftheimmunohistochemicalmethodsarethe

variabilityofstainingthatisobservedunderthesameexperimentalconditionsand

evenalongthesametissuesection.Thegradingoftissuesectionsforstrengthof

stainingthereforemaybeunreliable.Itfollowstherefore,thatimmunohistochemical

stainingservesmoreutilityinbeingabletoidentifytheabsenceorpresenceofstaining

incomparisontoanegativecontrol.Itdoeshoweverprovideusefulinformation

regardingthedistributionoftheantigenofinterest.Asstatedpreviously,amore

detailedstudyoftheultrastructure,degradationofothercomponentssuchaselastin,

glycosaminoglycancontentandimportantly,ultrastructuralfeaturesarewarranted.

Furthermore,onlythemajorgroupsofextracellularproteinshavebeencharacterised

inthisstudy.Thereareopportunitiesinfutureworktocharacterisepreciseisoforms

e.g.oflamininincomparisontootherbiologicalsubstratesandamnioticmembrane.

4.7 Cultureofprimaryhumanconjunctivalcellsondecellularised

conjunctivaandamnioticmembrane

Theprotocolforthedecellularisationofhumanconjunctivawasdevelopedandthe

resultingtissuecharacterised(section4.6).Thetissuewasnotcytotoxicandthe

extracellularmatricesandtensilestrengthwerenotsignificantlyalteredby

decellularisation.Techniquesforthecultureofhumanconjunctivalepitheliumon

decellularisedconjunctivaweredevelopedinthisstudy.Experimentalfindingsare

discussedtogetherwiththelimitationsofthestudyandopportunitiesforfurther

research.Thekeyfindingsfromthisstudyaresummariseoverleaf.

• Seedingdecellularisedtissueswithconjunctivaltissueexplantsresultedin

qualitativelygreatercelldensityincomparisontoseedingacellsuspension.

• Confluentepithelialgrowthoccurredwhenexplantswereculturedincontact

withthebasementmembraneofdecellularisedconjunctivaltissue.

• Therewasmarkedvariabilityintheconjunctivalepithelialcellgrowthfrom

explantsfromdifferentdonorsanddecellularisedconjunctivafromdifferent

sources.

• Astratifiedconjunctivalepitheliumwasdevelopedfromexplantcultureon

freshlydecellularisedconjunctivaltissue.

• Thestratifiedconjunctivalepithelialconstructwasdemonstratedtoexpress

conjunctivalepithelialmarkersincludingCK7,sparseMUC5ACstainingand

progenitorcellmarkers

4.7.1 Cellcultureexperimentsandcharacterisationofthedevelopedtissue

constructs

Preliminaryprimarycellcultureexperimentsfoundthatseedingdecellularisedtissue

withconjunctivalexplantsresultedinconfluentepithelialcellgrowthwhereasseeding

thedecellularisedtissuewithacellsuspensionresultedinsparsecellgrowth.Indeed,

theisolatedcellsuspensionhadbeeninitiallyexpandedontissuecultureplatesfrom

explants.Thecellswerethereforeatamoreadvancedpassageandseededafter7days

incultureincomparisontoexplants,whichwereseededwithinhoursofretrieval.One

ofthereasonsthattheexplantculturesdevelopmoresuccessfullythanisolatedcells

maybethatanystemcell‘niche’mayhavebeenpreservedinitsnativestateandwas

thereforemorecapableinsupportingproliferationanddifferentiation.

Theorientationoftissuewascrucialsuchthatcellularexpansionoccurredmore

effectivelywhencellswereculturedindirectcontactwiththebasementmembrane.In

theearliersection,thedistributionoflaminin,collagenIVandfibronectinwas

describedtolocalisestronglytothebasementmembranewithlessexpressioninthe

substantiapropria(section4.6.4).Cell-extracellularmatrixinteractionsarethereforeof

paramountimportanceforproliferationanddifferentiation.(185)Inkeepingwiththis,

differentialsignallingpatternshavebeenidentifiedinconjunctivalepitheliabasedupon

interactionswithspecificisoformsoflaminin.(187)

Resultsfromthisstudycomparingdecellularisedtissuefromthreedonorswithexplant

tissuefromthreefurtherdonorsdemonstratedvariabilityinthepotentialofexplants

todevelopondecellularisedconjunctiva.Thevariabilitymaybeexplainedby

differencesinthedonorage,postmortemretrievaltime,andlocationfromwhichthe

conjunctivalexplantsoriginated.Studiesonbiopsylocationinrelationtothecolony

formingefficiencyhavebeenundertakenanditisofinteresttonotethatincreasing

lengthofthepost-mortemretrievaltimeandadvancingdonoragesignificantlylower

boththeclonogenicabilityandexpressionofprogenitorcellmarkersinculture.(19)

Nevertheless,themajorityofdonorsusedwithinthisstudywereelderlyandgiventhe

smallsamplesize,itisnotpossibletodetermineiftheseweremajorcontributing

factors.Itwashoweverofinteresttonotethatthetwoofthelatercultureattemptsin

whichfreshsamplesofdecellularisedtissuewereusedresultedincultureswithgreater

celldensity,confluenceandevenbecamemulti-layeredconstructs.Astudy

investigatedtheeffectoffreezingpriortodecellularisationofhumanumbilicalcord

anddemonstratedthatcondensationoftheextracellularmatrixleadtogreater

residualDNAfollowingdecellularisation.(236)Reportsonothertissuetypesinwhich

tissuewasfrozenfollowingdecellularisationcouldnotbefound.Itwouldbeofinterest

infutureworktodeterminewhetherfreezingchangestheultrastructureand

potentiallyinfluenceculturepotential.

Immunohistochemistryusingoneofthelatermulti-layereddevelopedconjunctival

constructsconfirmedthattheresultingstratifiedepitheliumwasofaconjunctival

phenotypeexpressingCK19,CK7andCK4typicaltoconjunctivalepithelium.(10,127,134)

CK7,regardedasgobletcellmarkerwasfoundthroughoutthetissuesections

suggestingthepresenceofgobletcellswiththepotentialtoproducemucin.(11,134)Dartt

andcolleagueshypothesisethatCK7+/UAE-1negativecellsmayrepresentapopulation

ofimmaturegobletcellsthatmayhaveproliferativecapacityandpotentialtodevelop

intomaturegobletcellsintheirstudyofthemigrationpatternsfromconjunctival

explantoutgrowthanddifferentiation.(134)Unfortunately,UAE-1lectinwasnotusedas

amarkerintheseimmunohistochemistrystudiesinadditiontoCK7andMUC5AC.

MUC5ACisagelformingmucinfoundexclusivelyinmaturegobletcells.(11)Its

expressionwassparsebutdetectable,whichmaysuggestthepresenceofimmature

gobletcellsgiventhedegreeofCK7staining.(133)Tothebestofourknowledge,thisis

thefirstconjunctivalsubstrate-culturestudyinwhichCK7positiveconjunctivalcells

havebeendemonstrated.Furthermore,exvivocultivatedhumanconjunctival

epithelialgraftsonamnioticmembranehavenotbeenshowntosupportmucin

producingconjunctivaandthereforedecellularisedconjunctivamaybesuperior

substratethanamnioticmembraneforconjunctivalexpansion.(58,68)

MarkersofprogenitorcellsΔNp63andABCG2werealsopresentinlesserfrequency,

withABGC2expressionlessabundantthanΔNp63.Thiswouldbeinkeepingwiththe

quantitativeanalysisofconjunctivalepithelialcellculturesonsyntheticsubstrates

describedinsection5.2.2.LevelsofPCNAexpressionappearedsimilartothatof

ΔNp63.Caspase3,amarkerofapoptoticcellswasalsopresentandappearedtobe

expressedinapicalratherthanbasalcells,incontrasttotheothercellmarkersstudied

whichappeartobemoreevenlydistributed.Thiswouldbeinkeepingwiththe

assumptionthatoldercellsthatwouldbelostaremoreapicalandwouldbereplaced

bybasalcells,whichmaybeatanearlierstageofdifferentiation.Indeedthe

distributionofthesemarkersinhealthyconjunctivaispoorlycharacterisedinthe

literature.Itwouldbeofinterestinfutureworktherefore,tocharacterisethe

distributionofthemarkersinvestigatedinthisstudyinhealthyconjunctivaand

betweendifferentsites.Furthermore,aquantitativeanalysisofmarkerexpressionis

alsowarrantedforbothconjunctivaltissuesinadditiontoconstructsexpandedfrom

explants.Theevidencefromthisexperiment,however,wouldsuggestthatthe

conjunctivalconstructhadpotentialforself-renewalandsupportedapopulationof

activelyproliferatingcells.

4.7.2 Limitationsofthestudy

Oneoftheproblemsassociatedwithseedingprimaryconjunctivalcellson

decellularisedtissueisthelackofcellnumberwhencellsareisolatedfromthetissues

priortocultureanddirectlyseeded.Thiswascircumventedbytheuseofconjunctival

explantsinthepresentstudy.Fromtheoutsetoftheexperiments,however,thecell

numbersseededorthegrowthpotentialoftheexplants(determinedbyfactorssuchas

theanatomicallocationoforiginfromtheconjunctivaanddonorage)werenot

controlled.Thiscouldbeaddressedinfutureworkifagreatersamplenumberis

available.

Alimitationofthisstudywasthatdirectcomparisonswerenotmadebetweenthe

phenotypeofconjunctivalculturesdevelopedondecellularisedconjunctivaand

amnioticmembrane.Thepotentialbenefitsofamnioticmembranecannotbeignored.

Theyoung’smodulusofamnioticmembraneishigherthanthatofconjunctivaand

thereisgreaterconsistencyinthetissuearchitectureofamnioticmembranethanthat

demonstratedbetweenconjunctivalsamples.Giventhatconjunctivawouldbederived

fromacadavericsource,mostdonorswillbeelderlyinwhomtheeffectsoftissue

degenerationcannotbeignored.Humanamnioticmembraneisthoughttopromote

celladhesionanddifferentiation.(237)Itisalsorecognisedthatthestromalmatrix

containsanabundanceoffoetalhyaluronicacid,whichaidsinthereductionofscarring

viaaTGF-ϒpathwayandalsosupressespro-inflammatorycytokines.(237)Itisyettobe

determinedhowtransplanteddecellularisedconjunctivawillcompareinthisregard.

Decellularisationmayreducetheriskofdiseasetransmissionbutmaydisrupt

extracellularmatrixcomponents,theultrastructureofthetissueandremovesoluble

proteinsincludinggrowthfactors.(65)Theresultsfromthisstudyhoweverdonot

suggestanyalterationinthedistributionofthemajorextracellularmatrixproteins,

glycosaminoglycancontentbyPASstainingordenaturationofcollagenthrougha

quantitativeassay.Furtheranalysisofotherextracellularmatrixcomponentsandtissue

ultrastructurehoweveriswarranted.

4.8 CharacterisationofpatientswithMMPandpotentialocular

surfacereconstructionsstrategies

Mucousmembranepemphigoidcanleadtopainfullossofvisionandinitsmostsevere

formcanbelifethreatening.TheonsetofMMPisnotoriouslyinsidious,canbedifficult

todiagnoseandoftenthepresentationisdelayed.(36)Treatmentinvolving

immunosuppressioniscrucialinactivediseaseandtopreventorreducetheriskof

diseaseprogression.Tothisenditisimportantthatobjectiveandrobustgrading

systemsaredevelopedandusedtoi)identifypatientsinneedoftreatmentii)monitor

progressionandtreatmentresponse.Inthisstudy,anovelproformawasdevelopedas

atoolforuseincornealandexternaleyediseaseclinicstoassesspatientswithmucous

membranepemphigoid.

4.8.1 Developmentofaproformatoassessmucousmembranepemphigoid

patients

Anumberofmethodshavebeendescribedforthegradingofocularmucous

membranepemphigoidwithoutanyconsensusontheoptimalmethodforuse.To

comparethescoresofthegradingsystems,allwell-knownmethodsincludingthatby

Rowsey,MondinoandFoster,TauberandtheTauber-Liverpoolwereincludedinthe

proforma.(46,153-156)Itshouldbenotedthatthesemethodsgradecicatrisationand

thereforethesequelaeofdiseasewithoutanyassessmentofthedegreeof

inflammatoryactivity.Toovercomethis,Sawandcolleaguesdevelopedamethodfor

assessingdiseaseactivitybasedonthedegreeofconjunctivalandlimbal

inflammation.(163,164)Thiswasthereforealsoincludedaspartoftheproforma

developedandusedwithinStPaulsEyeUnitinconsultationwiththeCornealand

ExternalEyeDiseaseteam.Alsoincluded,aspartoftheproformawerephotographs,

fornixrulemeasurements,gradingofliddeformities,presence/absenceoftrichiasis,

presence/absenceoflagophthalmos,anddocumentationofdrynessaccordingtoThe

Oxfordgradingscheme.(158)

Theadaptedversionsofexistinggradingschemesusedfortheassessmentoflid

deformitiesincludinggradingofectropion,entropion,thedocumentationoftrichiasis

andlagophthalmosasshowninthedevelopedproformahavenotbeenvalidatedfor

useasameasurementtools(Appendix).Furthermore,othercomponentsofthepro

formasuchasdocumentationwithphotographsinadditiontoanteriorsegment

drawingsalsorequireformalvalidationstudies.Theaimofthisprocesswastodevelop

andpilotaproformathatcouldbedevelopedfurtherforuseintheclinictoidentify

patientsthatmaybenefitfromocularsurfacereconstructionandadjunctivetherapies

toreducetheriskofcicatrisation.Tothisend,itwouldbeofinteresttoundertake

validationstudiesinthefuturetoassesstheinter-raterandintra-raterreliabilityofthe

gradingscalesdevelopedforuseinthisproforma.Inaddition,itwouldbeofinterest

toformallyassessthetimetakenforcompletionoftheproformaandalsotheutilityof

anteriorsegmentdrawingsincombinationwithphotographicevidenceforthe

documentationofdiseaseactivityandprogressioninMMP.

4.8.2 Characterisationofthepatientexaminedusingthenovelproforma

Thepatientsexaminedaspartofthispilotschemewereopportunisticallysoughtfrom

cornealandexternaleyediseaseclinicsatStPaul’seyeunit.Allthepatientswere

thereforeunderactivefollowupforocularmucousmembranepemphigoidorwere

beingmonitoredastheywerereceivingimmunosuppression.Noneofthepatientsseen

weretreatedforanycicatricialdiseaseprocessotherthanocularmucousmembrane

pemphigoid.Thiswouldbeinkeepingwiththeknownincidenceofconditionsclassed

ascicatrisingconjunctivitisinwhichmucousmembranepemphigoidaffectsthe

greatestproportionofpatients.(36)Furthermore,manyotherconditionse.g.ocular

burnsundergoanactiveperiodoffollowupfollowing,whichtheymaybedischargedif

stable,incontrasttoocularmucousmembranepemphigoid.

AlltheexaminedpatientsweregradedasIII(Tauber)inoneorbotheyes.Allpatients

alsohadoneorbotheyes≥60%verticalinvolvementand≥50%horizontalinvolvement,

andfouroutoffivepatientswithaRowseyscore≤50%ofthetotalscore.Thegrading

schemesuggestednoneofthepatientshadsignificantcornealinvolvementand

dryness(oxfordgradingscheme)wasmild.Trichiasiswashoweverasignificant

problemaffectingeightofteneyesexamined.Intermsofliddeformities,onlyoneeye

hadalowerliddeformity.Noupperliddeformitieswererecordedandthiswasin

keepingwiththepatternofcicatrisationinocularmucousmembranepemphigoid.

Thesedatashowthatthegroupofpatientsexaminedhadmoderatetosevereocular

mucousmembraneinvolvement.

4.8.3 Pilotexercisetodeveloprecommendationsforaproforma

Someoftheproblemswiththeproformabecameapparentduringthepilottests.The

fornixrulewasapproximately1mmthick.Itbecameapparentthereforethatit

underestimatedthetruedepthofthefornix.Forexample,inthepatientphotographed

inFigure97,theinferiorfornixdepthwasmeasuredas0mm.Thefornixruler

measurementswouldhoweverproveuseful,particularlyintheassessmentofthe

superiorfornices,whichareotherwisedifficulttoassessandrarelydocumented.(157)

Recommendationsfromthispilotarethereforetodevelopanalternativerulerthatis

sufficientlythin,longandcurvedtobeinsertedcomfortablywithinfornicesorusethat

designedbyWilliamsandcolleaguesthathasalreadyundergonevalidationtests.(157)

Theinflammatoryscorewaseasytoassessandwastimeefficient,takingonaverage

around2minutestocomplete.Somedifficultwasnotedhoweverindifferentiating

between‘minimal’and‘mild’inflammatorygrades,however,advancedgradeswere

easytoscore.Thisgradingsystemhasbeenusedinclinicaltrialshowevervalidation

andfurtherinvestigationintoitsinter-raterandintra-raterreliabilitymayprovide

furtheropportunitiesforitsimprovement.(163,164)Myrecommendationwouldbe

thereforetousethescoringsystemfordiseaseactivityfollowingformalvalidationas

therearenootheravailablevalidatedscoringsystems.Furthermore,itisofparamount

importancetoincludeatoolforgradingconjunctivalinflammation,asitistheonly

availablemeasurementofdiseaseactivity.Incontrast,allothergradedfeatures

measurediseasesequelae.Inendstageornearendstageoculardisease,however,it

maybedifficulttoassessdiseaseactivity.Thiswasdemonstratedinthepresentstudy

(patient4,Figure101).Althoughactivityatalternativesitessuchastheoropharynx

couldbeused,itisunclearwhetherthiswouldmirrorocularactivity.Inthestudyby

Reevesandcolleagues,nocorrelationwasfoundbetweenthegradedscoresforthe

eyesandoropharynx.Thescorefortheoropharynx,however,gradedactivitywhereas

thescorefortheocularinvolvementmeasuredcicatricialandthereforestructural

change.Futureworktocompareactivityscoresatmultiplesiteswouldthereforebe

warrantedinpatientswhohaveextra-ocularmanifestationsofmucousmembrane

pemphigoid.

Thepresenceorabsenceoftrichiasisandlagophthalmoswereincludedinthepro

formaandcouldbeassessedwithinminutes.Similarly,theOxfordgradingschemefor

dryness,gradingofliddeformityandcornealinvolvementwererelativelyquickto

score.Theeaseofadministrationoftheproformawasdeterminedbycomparingthe

scoringtoolwithatooldesignedtodocumentamuchgreaterlevelofdetaildesigned

byRauzandcolleagues,BirminghamandMidlandsEyeCentre,Birmingham.Thelatter

tooltookapproximatelyandhourtocompletewhereasthedevelopedproformatook

approximately20minutesintotal.Componentsoftheinformationgatheringprocess

includingeyelids,adnexainadditiontooculardrynessareimportanttonoteinthe

assessmentofMMPpatientsastheymayalertthecliniciantoinitiatesupportive

interventionsinatimelymannertopreventordelaythedesiccationofocularsurface

epithelia.Thedocumentationofcornealsignsprovidesanindicationofthevisual

impairmentandneedformedicalorsurgicalmanagement.Thetreatmentofacentral

cornealdefect,forexample,mayberequired,whereasperipheralcornealsigns

pathologysuchaslimbitisorcornealvascularisationindicatesapotentialneedfor

futurelimbalstemcelltransplantation.Thisinformationisimportantwhenplanning

thesequenceofocularsurfacereconstruction.

Therewereinsufficientnumbersofpatientsexaminedaspartofthispilottocomment

onthecorrelationandagreementbetweenthevariousmethodsofgrading

cicatrisation.Ithasbeendeterminedhoweverthatgoodlevelsofagreementexist

betweentheRowseyscoreandtheTauber-Liverpool.(156)Rowseyandcolleagueshave

alsodemonstratedthattheirgradingschemeisconcordantwiththatofTauberand

MondinoandBrown.(46,154,155)Duringthispilotexperiment,whenusingallthe

describedgradingschemesitbecameapparentthattherewassomeoverlap.Some

gradingsystemspromptmoredetailedassessmentofcicatricialfeaturese.g.the

TauberschemeprovidesfurthersubdivisionstothatbyMondinoandBrown,andthe

Liverpoolschemeprovidesmoreaccuratequantificationthatbuildsupontheexisting

Tauberscheme.(46,154,156)TheLiverpool-Tauberschememaydetectmoresubtle

structuralprogressionthantheTaubergradingschemealonealthoughtheTauber

gradingschemeissensitivetotarsalconjunctivalchange.Myrecommendationbased

onthepilotworkthereforewouldbetogradecicatrisationasdescribedonpage2of

theproformawhichincludestheTauber-Liverpoolmeasurementstogetherwiththe

Rowseyschemeonthefollowingpage(Appendix1).Bydoingso,therewouldbeno

needtoalsoincludetheFosterandMondinoandBrowngradingschemeasthereis

alreadyoverlapbetweenthesemeasurements.

4.9 Treatmentofcicatrisingeyediseaseandthepotentialuseofthe

substratesdevelopedinthisstudy

Thereisnoconsensusonthedegreeofcicatrisationthatwouldspecificallywarrant

surgicalocularsurfacereconstruction.Somestudieshavesuggestedthat‘pathogenic’

symblepharamaybedefinedbydryeyefrominterruptionofspreadoftears,blink

relatedmicrotraumafromanirregulartarsalsurface,cicatricialentropion,exposure

duetolagophthalmosandrestrictionofocularmotility.(240)Furthermore,itis

imperativethatthediseaseprocessiscontrolledpriortoanysurgicalintervention

otherwiseamarkedinflammatoryresponseinthepost-operativeperiodwould

compromisethesuccessofreconstruction.

Ofthepatientsstudied,therighteyeofpatient4wouldwarrantocularsurface

reconstructiongiventhedegreeofsymblepharonandalmostcompleteobliterationof

theinferiorfornixintherighteye.Thesurgeonwouldneedtobewaryofthefactthat

signsofactivediseasewerepresentinthefelloweyeanddelaytreatmenteventhough

theactivityscoresuggestsdiseasequiescenceintherighteye.Presumingthatdisease

quiescencecouldbeachievedthroughimmunosuppression,surgicalinterventioncould

beundertaken.Inthefirstinstancethepatientshouldundergolashelectrolysisto

ensurepermanentremovalofmisdirectedlashes.Asnootherliddeformitieswere

present,thenextstagewouldinvolvelysisofsymblepharaandforniceal

reconstruction.Asdescribedinearliersections,amnioticmembranewouldbethe

currentavailablestandardforfornicealconstructionbutispronetorecurrentscarring

andfornicealshortening.(54)

Suchpatientsmaybetreatedinthefuturewithnovelconjunctivalgraftsdeveloped

throughtheex-vivoexpansionofconjunctivalepitheliumondecellularisedconjunctiva

orePTFE.Amajorproblemtoapproachinthisscenarioistheautologousbiopsy

requiredtodeveloptheconstruct.Withthisregard,theriskofscarringand

inflammationissignificantandthereforediseasequiescenceattheoutsetoftreatment

inadditiontoimmunosuppressionintheperioperativeperiodwouldbeofparamount

importance.Culturemethodsandthesubstratesthemselvesneedtobedeveloped

suchthatadequateconjunctivalexpansioncouldbeachievedfromsmallbiopsiestaken

fromthepatient.Otherresearchgroupsalsoadvocatetheuseofintraoperative

mitomycinC(MMC)orβ-irradiationtopreventre-adhesion.(240,241)Itisunlikelythata

constructdevelopedfromdecellularisedconjunctivawouldaffordanyadvantageover

amnioticmembraneforfornicealreconstructionasitmaybeequallyproneto

recurrentscarringgivenitwillalsodegrade.IhypothesisethatsurfaceoptimisedePTFE

maybemoresuitableforfornicealreconstructiongiventhatitisnotbiodegradableand

mayi)providelong-termfornicealsupportandii)actasasubstratefortheexvivo

expandedconjunctivalepithelium.Ideallythedevelopedepithelialculturewould

compriseasuitableproportionofmucinproducinggobletcellsthatcouldimprovethe

tearfilmoftheeyeandasubsetofprogenitorcellsthatwouldenabletheepithelium

toselfrenew.

Surgeryhasbeendescribedtoinvolvecicatrixlysisviaacircumlunarincisionofthe

conjunctivaalongthecorneallimbusfollowedbyrelaxingincisionstowardsthefornix

alongthebordersofthesymblepharonwithdissectionofsubconjunctivalfibrovascular

tissue.Followingdissection,MMCexposurecouldbeinitiatedviasoakedspongesand

thefornixextensivelyrinsedfollowingtheirremoval.Thisadditionalstepmayprevent

thesubsequentovergrowthoffibroblasts.Theex-vivodevelopedePTFEconjunctival

constructcouldthenbeattachedtobaresclerawithfibringlueandsecuredwith

suturestotherecessedconjunctivaonthebulbarandtarsalaspectsaspreviously

described.(240)

Thedecellularisedconjunctivalsubstrateislikelytobeavailableinmuchsmaller

sectionsandthereforethiswilllimititsuse.Itcouldbeideal,forexample,infornix

reconstructionfollowinganocularburnonceinflammationhassettled,butthiswould

dependonthesizeofdecellularisedtissueavailableforuse.Adecellularised

conjunctivalsubstratemayalsosuitindicationssuchasthatfollowingtheremovalof

pterygia,conjunctivaltumoursorevenglaucomasurgeryincludingfollowingthe

placementofdrainagevalveimplants.

5. Conclusions

ThisstudydeterminedthepotentialofbothammoniagasplasmamodifiedePTFEand

decellularisedconjunctivaassubstratesfortheex-vivoexpansionofconjunctival

epithelium.Thesesubstratesmaybeusedforadifferentrangeofindicationsand

warrantconsiderationforuseintissueengineeringapplicationstoaddresstheclinical

needforconjunctivalreplacement.Thesetherapiescouldleadtosuccessfultreatments

forarangeofsightthreateningandpainfulocularsurfacedisorders.

AmmoniagasplasmatreatedePTFEisaneffectivesubstratefortheexpansionof

• AmmoniagasplasmatreatmentofePTFEincreasesitshydrophilicityand

enablesthecultureofconjunctivalepitheliumonthetreatedsurface.Thecell

densitywasfurtherimprovedbytreatmentoftheePTFEmembraneonboth

sides,aneffectmarkedafter14daysinculture.

• Asimilarphenotypeofconjunctivalepithelialcultureswasdemonstratedon

doublesideplasmatreatedePTFEasPETmembrane.

• ThecultureofprimarycellsondoublesideammoniaplasmatreatedePTFE

resultedinadeclineincellnumberafter14daysincultureincontrasttoHCjE-

Gicells,whichincreasedupto28days.Overall,theprimarycellcultureswereof

amoredifferentiatedphenotypewithlessproliferativepotentialdemonstrated

bygreaterCK4,CK7andMUC5ACexpressionandlowerPCNA,ΔNp63and

ABCG2expression.

Decellularisedhumanconjunctivaisaneffectivesubstratefortheexvivoexpansion

ofconjunctivalepithelium

• Humanconjunctivalwassuccessfullydecellularisedbyoptimisingapreviously

developedprotocolforthedecellularisationofamnioticmembrane.

• ThedecellularisationprotocolwaseffectiveintheremovalofDNA.Thetissue

didnotexhibitcytotoxicity,therewasnoevidenceofcollagendenaturationand

thehistologydemonstratednosignificantchangeinthegeneraltissue

architecture,elastinorglycosaminoglycandistribution(basementmembrane)

demonstratedbyH&E,VanGieson’sandPASstainsrespectively.

• Decellularisationdidnotaffectthedistributionofextracellularmatrixproteins

laminin,collagenIVorfibronectin.

• Therewasgreatervariabilityinthearchitectureoftheconjunctivaltissuein

comparisontoamnioticmembranebetweendonors.

• Thedevelopmentofstratifiedprimaryconjunctivalepithelialcultureon

decellularisedconjunctivawasdemonstratedforthefirsttime.

Immunohistochemicalstainingalsoconfirmedthatthedevelopedepithelium

wasofaconjunctivalphenotypewithasubpopulationofprogenitorcells,

proliferatingcellsandCK7positivecells.

6. Futuredirections

Thereareopportunitiesinfutureresearchtoinfluencethestemandgobletcell

populationsinculturesofconjunctivalepitheliumthroughthefurtherinvestigationand

optimisationofcultureconditions.Importantimprovementsinculturetechniqueshave

takenplacesuchthatserum-freemediahasbeendevelopedandtheuseofanimal

feederlayershasnotbeenrequiredinrecentwork.Furtheroptimisationand

investigationshouldleadtoanimprovedunderstandingofmediaandculture

requirementsaimingtofurtherreduceoreliminateanimalproductstoreducetherisk

ofzoonoticinfection.Indeed,aserumfreesystemhasbeendevelopedinanattemptto

simulatetheconjunctivalstemcellnichebyco-cultureofconjunctivalepitheliumwith

subconjunctivalfibroblasts.(238)Itwasfoundthatthatthisallowedconjunctivalcells

withprogenitorcharacteristicstodevelopandhasbeenproposedasanidealmethod

toallowexpansionofepithelialprogenitorcellsinvitro.(238)Inaddition,ithasbeen

demonstratedthatacollagengelcontaininghumanfibroblastsresultedingreater

numbersofgobletcells,whichwerenotobservedoncollagengelsseededwithSwiss

3T3cells.(239)

Seedingexplantsfromconjunctivalsitesknowntoberichinstemcellsandgobletcells

andthedevelopmentofmethodstodevelopenrichedculturesofprogenitor/stemcells

couldresultinoptimised,self-renewingepithelialconstructs.(19)Decellularisedtrachea,

oneofthemostsuccessfulmodelsoftissueengineeringinwhichgreatsuccesshas

beenachievedinpatients,cruciallyrequiredanexvivoepithelialculturerichinstem

cellswiththeappropriateangiogenesisanddifferentiationpromotinggrowth

factors.(74)Tothisend,techniquestoexpandandsupportculturesrichinconjunctival

stemcellsareacutelywarrantedandcurrentlylacking.

Examinationoftheproliferativecapacityofconjunctivalexplantstakenfromseveral

locationsrevealedthatfornicealexplantsexhibitthegreatestproliferativepotentialin

bothratandrabbit.(16,133)Inkeepingwiththis,ithasbeensuggestedthatthehuman

forniceshouseconjunctivalstemcells.(18)Inparticular,theinferiorfornicesandthe

medialcanthushavebeenfoundtohavethegreatestdensityofcellsexpressing

markersinkeepingwithconjunctivalprogenitorcells.(19)Ithasbeenalsobeen

proposedbyPellegriniandcolleaguesthatgobletcellsarisefromacommonbipotent

cellfromwhichconjunctivalepithelialcellsariseandthatcommitmenttogobletcell

differentiationcanoccurlateinthedifferentiationpathway.(18)Pellegriniand

colleaguesfoundtransientlyamplifyingcellsatspecifictimesintheirlifecyclepriorto

senescenceandsuggestedthatdifferentiationintoagobletcelllineagewasinfluenced

bya‘celldoublingclock’.(18)Itfollowstherefore,thatmethodsofexplantcultureusing

humanconjunctivashouldbeoptimisedwithrespecttothebiopsylocationsiteand

withanunderstandingofthefactorsthatinfluencegobletcelldifferentiation.

Giventhatexplantsarisingfromthefornicesandmedialcanthushavebeenshownto

havethegreatestgrowthpotential,itmaybepossibleinfutureworktodevelopatwo-

stageculturesystemifthereisinsufficienttissueforthedirectseedingofexplantsfrom

biopsies.Indeed,atwo-stageprocedurehasbeenreportedforothertissueengineering

modelse.g.endothelialcellseedinginvasculargraftstomaximisethecelldensityof

cells.Potentialdisadvantagesarethepotentialforinfectionduringthisextended

periodofcultureandalsoofpotentialalterationsinthecellphenotype.(185)Alternative

methodsforgreaterefficiencyofcellpropagationincludetheuseoffibroblast

conditionedmediaandsupplementationofmediawithhumanserum.(132)

Tsaiandcolleaguesalsodemonstratedthatconjunctivalepithelialcellcultureon

collagengelscontaininghumanfibroblastsresultedinepitheliumstratifiedupto8cells

thickincomparisontoanacellularcollagenmatrixonwhichonlyamonolayerof

epitheliumwasdemonstrated.(239)Indeed,theepithelialculturesinthepresentstudy

couldbeimprovedbyagreaternumberofcelllayersandstratification.Thisinturnmay

promotethedifferentiationofgobletcells.Alternatively,thedifferentiationofgoblet

cellsandstratificationmaybestimulatedbyacommonfactorsuchasthepresenceof

fibroblasts.Theroleofsubconjunctivalfibroblastswarrantsfurtherstudyincludingthe

potentialforinitialculturewithinthesubstantiapropriapriortoseedingconjunctival

epitheliaonthebasementmembraneside.Itwouldalsobeofinteresttodetermine

whetherexplantculturesdevelopsuccessfullyduetothepresenceofastemcell‘niche’

thatpotentiallyincludesasmallpopulationofconjunctivalfibroblasts.

Furtheroptimisationofmediaconstituentsarewarrantedtopromoteconditionsthat

supportthematurationofgobletcellsandmucinproduction.Thiscouldbeachieved

throughabetterunderstandinganduseofthemostappropriategrowthfactorsand

possiblyalengthenedtimeinculturetoupregulateMUC5ACexpression.(17)Alternative

methodstopotentiatethecultureofgobletcellscouldincludetheuseofbronchial

epithelialgrowthmedium,epidermalgrowthfactormediatedsignallingorγ-secretase

asstudiedbyotherresearchgroups.(112,131,242)

TheePTFEisapromisingsubstrateforcellularexpansionthatwarrantsfurther

optimisationofsurfacechemistrytoensurethatafavourableconjunctivalphenotype

canbedevelopedonitssurface.Itmayprovetobeanidealsubstrateforforniceal

reconstructionandactasalong-termscaffoldforconjunctivawhilstmaintaining

fornicealdepth.GiventheexampleofothertissueengineeringapplicationsofePTFE

use,itisunlikelythatavascularsystemwouldberequiredforthemaintenanceof

conjunctivaltissuegiventhatisonlyafewcelllayersdeep.(185)Ultimatelythenumber

ofcelllayersmaybelimitedbyfactorsrelatingtotherateatwhichsupplyofnutrients

andabilitytoremovewasteoccurs.(185)Followingfurtheroptimisationofthesurface

chemistryonePTFE,earlyanimalstudiesarewarrantedtoinvestigateit’spotentialfor

conjunctivalgrowthandfornixreconstructioninvivo.

Asoft,pliablegraftderivedfromdecellularisedconjunctiva,withorwithoutexvivo

expandedconjunctivalepithelium,maysuitarangeofindicationsincludingglaucoma

surgery,conjunctivalreplacementfollowingresectionofconjunctivaltumoursand

conjunctivalchemicalburns.Theroleofdecellularisedhumanconjunctivamayevenbe

extendedforotherocularcellularreplacementtherapiessuchaslimbalstemcell

transplantation.Indeed,xenogeneicconjunctivalmatrixhasevenbeendemonstrated

asaneffectivescaffoldforcornealepitheliuminarabbitmodeloflimbalstemcell

disease.(92)Thisthereforedemonstratestherangeofcellularreplacementtherapiesin

whichdecellularisedmatricesprovidemajoradvancementsinthefieldoftissue

engineering.Decellularisedhumanconjunctivawarrantsfurtherinvestigationintoits

potentialclinicalapplicationsinocularsurfacedisease.

7. Appendix

1.TheLiverpoolcornealandexternaleyediseaseclinicproforma

(pages224-228)

2.Ethicalapproval

(pages229-231)

BCVARE………..LE……….Co-existingophthalmicpathology(accountingforreducedVA)………………………………PhotographtakenY NInflammationactivityscore(Grade1-5)RE LE

Totalscore ………… Totalscore …………Limbitis RE Y………quadrants/score N

LE Y………quadrants/score NAnteriorsegmentdrawingsRE

Affixpatientlabel

Cicatrisationgrading(Tauber-Liverpool)

RE LEHorizontalwidth ……….mmSymblephara ……….mm

Horizontalwidth ……….mmSymblephara ……….mm

ISubconjunctivalscarringandfibrosis

IIFornixforeshortening

a 0-25%

b 25-50%

c 50-75%

d 75-100%

ISubconjunctivalscarringandfibrosis

IIFornixforeshortening

a 0-25%

b 25-50%

c 50-75%

d 75-100%

III Presenceofsymblepharon

a 0-25%

b 25-50%

c 50-75%

d 75-100%

III Presenceofsymblepharon

a 0-25%

b 25-50%

c 50-75%

d 75-100%

IV Ankyloblepharon/frozenglobe IV Ankyloblepharon/frozenglobe

Verticalgrading=………%

Horizontalgrading=………%

CicatrisationbyFostergradingschemeRE LEIsubconjunctivalscarringandfibrosis

IIfornixforeshortening(anydegree)

IIIsymblepharon(anydegree)

IVankyloblepharon/frozenglobe

Isubconjunctivalscarringandfibrosis

IIfornixforeshortening(anydegree)

IIIsymblepharon(anydegree)

IVankyloblepharon/frozenglobe

CicatrisationbyRowseygradingschemeRE LE

Score……./45 Score……/45

FornixmeasureRE Upper mm LE Upper mm

Lower mm Lower mm

Cornealinvolvement Grade0 Grade1 Grade2 Grade3RE Conjunctivalisation Neovascularisation Opacification-

peripheral

Opacification-central

LE Conjunctivalisation Neovascularisation Opacification-

peripheral

Opacification-central

Oculardrynessscore(oxford)RE 0 1 2 3 4 5LE 0 1 2 3 4 5EyelidsLagophthalmos Y……mm N……mmLashes notrichiasis trichiasis acquireddistichiasis

RE LE Entropion/

Ectropion Entropion/

Ectropion

Upperlid Lowerlid Upperlid LowerlidGrade0 Grade1 Grade2 Grade3 Medial Lateral Lateral+Medial

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