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DEVELOPMENTOFSUBSTRATESFORTHEEXVIVOEXPANSION
OFCONJUNCTIVALEPITHELIUM
Thesissubmittedinaccordancewiththerequirementsofthe
UniversityofLiverpoolforthedegreeofDoctorofMedicine
by
ShivaniKasbekar
January2016
i
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.
ii
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.
iii
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
iv
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
v
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
vi
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
vii
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
viii
4.9 TREATMENTOFCICATRISINGEYEDISEASEANDTHEPOTENTIALUSEOFTHESUBSTRATESDEVELOPEDINTHISSTUDY............................................................................................214
5. CONCLUSIONS..............................................................................................217
6. FUTUREDIRECTIONS....................................................................................219
7. APPENDIX....................................................................................................2231.THELIVERPOOLCORNEALANDEXTERNALEYEDISEASECLINICPROFORMA...........................2242.ETHICALAPPROVAL.................................................................................................229
8. REFERENCES.................................................................................................232
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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
x
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
2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDFROMTRIPLICATESAMPLES(N=15WITHINEACHEXPERIMENTALGROUP)...................................................................79
FIGURE19:HISTOGRAMTOSHOWNUMBEROFCELLSCOUNTEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEANDINCREASINGCELLSEEDINGDENSITYONUNTREATEDEPTFE.THISGRAPHSHOWSTHENUMBEROFCELLSCOUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELD(+/-SD)AT20XMAGNIFICATIONOVERSETTIMEPOINTS(DAY1,4AND7).EACHPHOTOGRAPHEDFIELDWAS12,420ΜM
2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDFROMTRIPLICATESAMPLES(N=15WITHINEACHEXPERIMENTALGROUP)...................................................................80
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
ONALLSUBSTRATES.THISGRAPHSHOWSTHENUMBEROFCELLS(+/-SD)COUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELDAT20XMAGNIFICATIONOVERSETTIMEPOINTSDAY1,3,7,10AND14.EACHPHOTOGRAPHEDFIELDWAS12,420ΜM
2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDINTRIPLICATESAMPLES(N=15WITHINEACHGROUP).......................................................................................................84
FIGURE24:REPRESENTATIVEPHOTOMICROGRAPHSOFCULTUREDSUBSTRATESFIXEDANDSTAINEDWITHDAPIAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLB.ALLSUBSTRATESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)UNTREATEDEPTFEC)PET.SCALEBARS100ΜM.....................................84
FIGURE25:HISTOGRAMTOSHOWTHEMEANNUMBEROFCELLPHOTOGRAPHEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETMEMBRANEUSINGMEDIAPROTOCOLC.CELLSWERESEEDEDAT1X105/CM2
ONALLSUBSTRATES.THISGRAPHSHOWSTHENUMBEROFCELLS(+/-SD)COUNTED
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MANUALLYINEACHPHOTOGRAPHEDFIELDAT20XMAGNIFICATIONOVERSETTIMEPOINTSDAY1,3,7,10AND14.EACHPHOTOGRAPHEDFIELDWAS12,420ΜM
2.FIVEAREASPERPHOTOGRAPHEDFIELDWERECOUNTEDINTRIPLICATESAMPLES(N=15WITHINEACHGROUP).......................................................................................................85
FIGURE26:REPRESENTATIVEPHOTOMICROGRAPHSOFCULTUREDSUBSTRATESFIXEDANDSTAINEDWITHDAPIAFTER14DAYSINCULTUREUSINGMEDIAPROTOCOLC.ALLSUBSTRATESWERESEEDEDATADENSITYOF1X105CELLS/CM2:A)AMMONIAPLASMATREATEDEPTFEB)UNTREATEDEPTFEC)PET.SCALEBARS100ΜM.....................................85
FIGURE27:HISTOGRAMTOSHOWTHEMEANNUMBEROFCELLPHOTOGRAPHEDPERPHOTOGRAPHEDFIELDWITHADVANCINGTIMEONAMMONIAPLASMATREATEDEPTFE,UNTREATEDEPTFEANDPETMEMBRANEUSINGMEDIAPROTOCOLD.CELLSWERESEEDEDAT1X105/CM2
ONALLSUBSTRATES.THISGRAPHSHOWSTHENUMBEROFCELLS(+/-SD)COUNTEDMANUALLYINEACHPHOTOGRAPHEDFIELDAT20XMAGNIFICATIONOVERSETTIMEPOINTSDAY1,3,7,10AND14.EACHPHOTOGRAPHEDFIELDWAS12,420ΜM
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
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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
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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
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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
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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
xvi
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
xvii
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
xviii
TABLE15:TABLETOSHOWHYDROXYPROLINE(NG/MG)FOUNDINASSAYSOFPAIREDSAMPLESOFCELLULARANDDECELLULARISEDTISSUE.P=0.74:PAIREDSAMPLEST-TESTBETWEENCELLULARANDDECELLULARISEDDONORTISSUE(N=3INEACHGROUP)...........................................................................................................................143
xix
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
xx
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
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
2
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
3
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.
4
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
5
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)
6
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)
7
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)
8
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)
9
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
10
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.
11
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
12
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.
13
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
14
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
15
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).
16
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
17
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.
18
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)
19
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
20
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,
21
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.
22
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
23
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
24
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
25
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
26
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
27
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)
28
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
29
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.
30
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
31
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
32
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
33
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)
34
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
35
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.
36
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
37
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
38
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
a-d*
StageIII Percentagehorizontalinvolvementbysymblephara
a-d*
*a-d(describessubdivisionswithinIIandIII)
a 0-25%
b 25-50%
c 50-75%
d 75-100%
39
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
40
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%
41
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
42
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)
43
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).
44
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
45
Figure11:Figuretoshowtheoriginalreportedgradingschemes(a)andtheadaptedversions
(b)forthedocumentationofentropionandectropion.Theentropionandectropionscales
weretakenfromandadaptedfromKempetal.(1986)andMoeatal.(2000)respectively.(166,
167)
EctropionGradingScale0 Normaleyelidappearanceand
functionI Normalappearancebutsymptomatic;
eyelidlaxitypresentonexaminationII Scleralshowwithouteversionoflower
eyelidIII Ectropionwithouteversionoflacrimal
punctumIV Advancedectropionwitheversionof
lacrimalpunctumfromlacrimallakeV Ectropionwithcomplication(eg,
conjunctivalmetaplasia,retractionofanteriorlamella,orstenosisoflacrimalsystem)
L PredominantlylateralM PredominantlymedialLM Combinedmedialandlateralr Previousrevision*
a)
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
b)
46
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
47
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.
48
Clinicalapplicationofnovelconjunctivalsubstrates
• Developaproformaforthedocumentationofclinicalsignsanddiseaseactivity
inMMPpatients.
• DescribeMMPdiseasesequelaeinasmallgroupofpatientsattendingcorneal
andexternaleyediseaseclinicsattheRoyalLiverpoolHospital.
49
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).
a) b)
50
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.
51
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
52
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
53
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
54
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
55
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
56
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.
57
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,
a)
b)
58
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
59
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.
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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.
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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
ABCG2
(FITCconjugated)
5D3 Millipore 1:25
p63 ΔN Biolegend 1:50
caspase-3 Asp-175 CellSignalling 1:50
PCNA
(FITCconjugated)
PC10 Santacruz 1:50
Table3:Tableofantibodiesusedforflowcytometrywiththerespectiveclone,supplierand
optimiseddilution
62
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.
63
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
64
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.
65
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.
66
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
67
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
68
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
69
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.
70
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
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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
conjunctivalepithelium
2.7.1 Explantcultureexperimentswithandwithouttheorientationof
basementmembraneofconjunctiva
Initialexperimentswereundertakenusingconjunctivaltissueinwhichtheorientation
andpresenceofbasementmembranehadnotbeenconfirmedormarkedpriortothe
decellularisationprocess.Insubsequentexperiments,theconjunctivawasexamined
72
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
73
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
74
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
75
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.
76
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
ePTFE
6 127.4 135.9 131.2 3.2
Ammonia
plasmatreated
ePTFE
6 67.3 74.8 71.1 2.6
Table6:StaticcontactangleanalysisofuntreatedandammoniagasplasmatreatedePTFE.Six
readingsweretakenfromrandomlyselectedareasonammoniaplasmatreatedanduntreated
ePTFE.10μlwaterwasautomaticallydispensedandcontactanglebetweenthewaterdroplet
andmaterialrecordedbyaninbuiltvideorecorder.Statisticalanalysisshowedadifferenceata
p=0.02levelbetweentreatedanduntreatedePTFE.
77
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.
78
Figure17:Histogramtoshownumberofcellscountedperphotographedfieldwithadvancing
timeandincreasingcellseedingdensityonammoniaplasmatreatedePTFE.Thisgraphshows
thenumberofcellscountedmanuallyineachphotographedfield(+/-SD)at20xmagnification
oversettimepoints(day1,4and7).Eachphotographedfieldwas12,420μm2.Fiveareasper
photographedfieldwerecountedfromtriplicatesamples(n=15withineachexperimental
group).
Cells/pho
tograp
hedfie
ld
Daysinculture
Day1 Day4 Day7
Cellseedingdensity/cm2
1x103
1x104
1x105
100
200
300
400
500
79
Figure18:Histogramtoshownumberofcellscountedperphotographedfieldwithadvancing
timeandincreasingcellseedingdensityonPETmembrane.Thisgraphshowsthenumberof
cellscountedmanuallyineachphotographedfield(+/-SD)at20xmagnificationoversettime
points(day1,4and7).Eachphotographedfieldwas12,420μm2.Fiveareasperphotographed
fieldwerecountedfromtriplicatesamples(n=15withineachexperimentalgroup).
Cells/pho
tograp
hedfie
ld 400
300
200
100
500
Cellseedingdensity/cm2
1x103
1x104
1x105
Daysinculture
Day1 Day4 Day7
80
Figure19:Histogramtoshownumberofcellscountedperphotographedfieldwithadvancing
timeandincreasingcellseedingdensityonuntreatedePTFE.Thisgraphshowsthenumberof
cellscountedmanuallyineachphotographedfield(+/-SD)at20xmagnificationoversettime
points(day1,4and7).Eachphotographedfieldwas12,420μm2.Fiveareasperphotographed
fieldwerecountedfromtriplicatesamples(n=15withineachexperimentalgroup).
Cells/pho
tograp
hedfie
ld
Cellseedingdensity/cm2
1x103
1x104
1x105
Daysinculture
Day1 Day4 Day7
400
300
200
100
500
81
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)
82
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).
83
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)
0
100
200
300
400
500
600
1 3 7 10 14
Cellsperpho
tograp
hedfie
ld
Daysinculture
UntreatedePTFETreatedePTFEPET
84
Figure23:Histogramtoshowthemeannumberofcellphotographedperphotographedfield
withadvancingtimeonammoniaplasmatreatedePTFE,untreatedePTFEandPETmembrane
usingmediaprotocolB.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe
numberofcells(+/-SD)countedmanuallyineachphotographedfieldat20xmagnificationover
settimepointsday1,3,7,10and14.Eachphotographedfieldwas12,420μm2.Fiveareasper
photographedfieldwerecountedintriplicatesamples(n=15withineachgroup).
Figure24:RepresentativephotomicrographsofculturedsubstratesfixedandstainedwithDAPI
after14daysincultureusingmediaprotocolB.Allsubstrateswereseededatadensityof1x105
cells/cm2:a)ammoniaplasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars100μm.
a) b) c)
Daysinculture
0
100
200
300
400
500
600
1 3 7 10 14
Cellsperpho
tograp
hedfie
ld
UntreatedePTFETreatedePTFEPET
85
Figure25:Histogramtoshowthemeannumberofcellphotographedperphotographedfield
withadvancingtimeonammoniaplasmatreatedePTFE,untreatedePTFEandPETmembrane
usingmediaprotocolC.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe
numberofcells(+/-SD)countedmanuallyineachphotographedfieldat20xmagnificationover
settimepointsday1,3,7,10and14.Eachphotographedfieldwas12,420μm2.Fiveareasper
photographedfieldwerecountedintriplicatesamples(n=15withineachgroup).
Figure26:RepresentativephotomicrographsofculturedsubstratesfixedandstainedwithDAPI
after14daysincultureusingmediaprotocolC.Allsubstrateswereseededatadensityof1x105
cells/cm2:a)ammoniaplasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars100μm.
a) b) c)
2
0
100
200
300
400
500
600
1 3 7 10 14
UntreatedePTFETreatedePTFE
PET
Daysinculture
Cellsperpho
tograp
hedfie
ld
86
Figure27:Histogramtoshowthemeannumberofcellphotographedperphotographedfield
withadvancingtimeonammoniaplasmatreatedePTFE,untreatedePTFEandPETmembrane
usingmediaprotocolD.Cellswereseededat1x105/cm2onallsubstrates.Thisgraphshowsthe
numberofcells(+/-SD)countedmanuallyineachphotographedfieldat20xmagnificationover
settimepointsday1,3,7,10and14.Eachphotographedfieldwas12,420μm2.Fiveareasper
photographedfieldwerecountedintriplicate(n=15sampleswithineachgroup).
Figure28:RepresentativephotomicrographsofculturedsubstratesfixedandstainedwithDAPI
after14daysincultureusingmediaprotocolD.Allsubstrateswereseededatadensityof
1x105cells/cm2:a)ammoniaplasmatreatedePTFEb)untreatedePTFEc)PET.Scalebars
100μm.
a) b) c)
0
100
200
300
400
500
600
1 3 7 10 14
UntreatedePTFETreatedePTFE
PET
Cellsperpho
tograp
hedfie
ld
Daysinculture
87
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
a) b)
c)
88
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.
2x105
4x105
6x105
8x105
Num
bero
fcells/cm
2
Day2 Day14 Day21 Day28
Daysinculture
SinglesidetreatedePTFE
Substrate
UntreatedePTFE
DoublesidetreatedePTFE
PETmembrane
89
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).
90
Figure31:Representativephotomicrographsofnuclearandf-actinstainingofHCjE-Gicells
culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter2daysinculture.
CellsizeappearssmalleronPETmembraneshowevercelldensityappearedgreatest.Lowest
celldensitywasapparentonuntreatedePTFEwithcellsmore‘rounded’inappearancethanon
ammoniaplasmatreatedePTFEsubstrates.Scalebars50μm.
DAPI(blue) Phalloidin(red)
SinglesideammoniaplasmatreatedePTFE
DoublesideammoniaplasmatreatedePTFE
PETmembrane
UntreatedePTFE
91
Figure32:Representativephotomicrographsofnuclearandf-actinstainingofHCjE-Gicells
culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter14daysinculture.
CellsizeappearedthesmallestwithgreatestdensityonPETmembrane.Similarmorphology
wasapparentontreatedePTFEandPETmembrane.Lowestcelldensitywith‘rounded’cells
wasapparentonuntreatedePTFE.Culturesweremoreconfluentondouble-sidetreatedePTFE
thansinglesidetreatedePTFE.Scalebars50μm.
DAPI(blue) Phalloidin(red)
SinglesideammoniaplasmatreatedePTFE
DoublesideammoniaplasmatreatedePTFE
PETmembrane
UntreatedePTFE
92
Figure33:Representativephotomicrographsofnuclearandf-actinstainingofHCjE-Gicells
culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter21daysinculture.
LowestcelldensitywasapparentonuntreatedePTFEhoweverthereisgreatervariationinthe
sizeandshapeofthenuclearmaterialthanonanyothersubstrate.Culturesweremore
confluentondoublesidetreatedePTFEthanfollowingsingleside-treatmentandcellsappear
tohavemoreofcobblestonemorphologythanonothersubstrates.Scalebars50μm.
DAPI(blue) Phalloidin(red)
SinglesideammoniaplasmatreatedePTFE
DoublesideammoniaplasmatreatedePTFE
PETmembrane
UntreatedePTFE
93
Figure34:Representativeconfocalz-stackseriesofsinglesideammoniaplasmatreatedePTFE
after28daysofHCjE-Gicellculture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)staining.
Fivesliceshavebeentaken,eachin4.5-5μmintervalswherebythetopandbottomofthez-
stackhadbeenmanuallysetafterthefocuspointsatthetopandbottomofthecellcultures
werelocated.Scalebars50μm.
50μm
50μm
a)
b)
94
Figure35:Representativeconfocalz-stackseriesofdoublesideammoniaplasmatreated
ePTFEafter28daysofHCjE-Gicellculture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)
staining.Fivesliceshavebeentaken,eachin4.5-5μmintervalswherebythetopandbottomof
thez-stackhadbeenmanuallysetafterthefocuspointsatthetopandbottomofthecell
cultureswerelocated.Scalebars50μm.
50μm
50μm
a)
b)
95
Figure36:Representativeconfocalz-stackseriesofPETmembraneafter28daysofHCjE-Gicell
culture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)staining.Fivesliceshavebeentaken,
eachin4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanuallyset
afterthefocuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars50μm.
50μm
50μm
a)
b)
96
Figure37:Representativeconfocalz-stackseriesofuntreatedePTFEafter28daysofHCjE-Gi
cellculture:a)phalloidin(f-actinstaining)(b)DAPI(nuclear)staining.Fivesliceshavebeen
taken,eachin4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanually
setafterthefocuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars
50μm.
50μm
50μm
a)
b)
97
Figure38:RepresentativephotomicrographsofnuclearandUAE-1stainingofHCjE-Gicells
culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter2daysinculture.
LowestcelldensitywasapparentonuntreatedePTFE.OverallgreaterUAE-1(intracellularand
membraneassociated)stainingwasapparentoncellsculturedonePTFEthanPETmembranein
whichstainingappearedmorediscreteandmostlymembraneassociated.Scalebars50μm.
UntreatedePTFE
PETmembrane
DoublesideammoniaplasmatreatedePTFE
SinglesideammoniaplasmatreatedePTFE
DAPI(blue) UAE-1Lectin(green)
98
Figure39:RepresentativephotomicrographsofnuclearandUAE-1stainingofHCjE-Gicells
culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter14daysinculture.
LowestcelldensitywasapparentonuntreatedePTFE.ThegreatestintensityofUAE-1staining
appearedonPETmembraneanddoublesidetreatedePTFE.Stainingofcellmembranes
showedthecellsizewasgreateronePTFEcellculturesthanPETcellcultures.Scalebars50μm.
SinglesideammoniaplasmatreatedePTFE
DoublesideammoniaplasmatreatedePTFE
PETmembrane
UntreatedePTFE
DAPI(blue) UAE-1Lectin(green)
99
Figure40:RepresentativephotomicrographsofnuclearandUAE-1stainingofHCjE-Gicells
culturedonammoniaplasmatreatedePTFE,PETanduntreatedePTFEafter21daysinculture.
LowestcelldensitywasdemonstratedonuntreatedePTFE,whereasthegreatestdensitywas
demonstratedonPETmembraneanddoublesidetreatedePTFE.CellsizewasgreateronePTFE
cellculturesthanPETcellcultures,andwasmorepronouncedondoublesidetreatedePTFE,
especiallyamongstthecellswithgreaterintracellularstaining.Scalebars50μm.
SinglesideammoniaplasmatreatedePTFE
DoublesideammoniaplasmatreatedePTFE
PETmembrane
UntreatedePTFE
DAPI(blue) UAE-1Lectin(green)
100
Figure41:Representativeconfocalz-stackseriesofsinglesidetreatedePTFEafter28daysof
HCjE-Gicellculture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Fivesliceshavebeen
taken,eachin4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanually
setafterthefocuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars
50μm.
50μm
50μm
a)
b)
101
Figure42:Representativeconfocalz-stackseriesofdoublesidetreatedePTFEafter28daysof
HCjE-Gicellculture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Fivesliceshavebeen
taken,eachin4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanually
setafterthefocuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars
50μm.
50μm
50μm
a)
b)
102
Figure43:Representativeconfocalz-stackseriesofPETmembraneafter28daysofHCjE-Gicell
culture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Fivesliceshavebeentaken,eachin
4.5-5μmintervalswherebythetopandbottomofthez-stackhadbeenmanuallysetafterthe
focuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars50μm.
50μm
50μm
a)
b)
103
Figure44:Representativeconfocalz-stackseriesofuntreatedePTFEafter28daysofHCjE-Gi
cellculture:a)UAE-1lectinstaining,b)DAPI(nuclear)staining.Foursliceshavebeentaken,
eachafter3μMwherebythetopandbottomofthez-stackhadbeenmanuallysetafterthe
focuspointsatthetopandbottomofthecellcultureswerelocated.Scalebars50μm.
50μm
50μm
a)
b)
104
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.
105
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
106
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
a)
1:100,30minutes 1:500,30minutes 1:1000,30minutes 1:2000,30minutes
b)
1:100,60minutes 1:500,60minutes 1:1000,60minutes 1:2000,60minutes
c)
107
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
108
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
48.
Figure47:Dotplotofsidescatter-height(SSC-H)againstforwardscatterheight(FSC-H)ofHCjE-
Gi(a)andprimaryconjunctivalcells(b).Thedotsoccurringatthebottomleftoftheplot(low
FSC-HandSSC-H)areparticulates/debris.
a) b)
109
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).
a)
Primaryconjunctivalcells
HCjE-Gicells
CK19
110
b) CK4
CK7c)
HCjE-Gicells
Primaryconjunctivalcells
Primaryconjunctivalcells
HCjE-Gicells
111
e) MUC5AC
HCjE-Gicells
Primaryconjunctivalcells
UAE-1lectind)
Primaryconjunctivalcells
HCjE-Gicells
112
f) PCNA
HCjE-Gicells
Primaryconjunctivalcells
Caspase-3g)
Primaryconjunctivalcells
HCjE-Gicells
113
h) ΔNp63
ABCG2i)
HCjE-Gicells
HCjE-Gicells
Primaryconjunctivalcells
Primaryconjunctivalcells
114
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
Isotypecontrol
Deprivedcellcultures
Healthycellcultures
115
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)
0.54
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).
116
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).
117
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
118
and28daysinculture.Caspase-3expressionwasrelativelylowacrossallsubstratesat
14daysincultureanddidnotincreasewithadvancingtimeoverall:p=0.163(Figure
59).Therewas,however,analmost10-foldgreatercaspase-3expressionat28daysin
cultureonuntreatedePTFEthananyothersubstrate.Caspase-3expressionwas
significantlyhigheronuntreatedePTFEthanallothertestsubstrates;p<0.0001(Table
11).
Figure50:HistogramtoshowpercentageexpressionofCK4onammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
substrates;p<0.0001;timep=0.152.Errorbars+/-SD.
Substrate
DoublesidetreatedePTFE
PETmembraneSinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
CK4
Day14 Day28Daysinculture
4.0
3.0
2.0
1.0
0.0
119
Figure51:HistogramtoshowpercentageexpressionofCK7onammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
substratesandtime;p<0.001.Errorbars+/-SD.
Figure52:HistogramtoshowpercentageexpressionofCK19onammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
substratesp=0.975;timep=0.88.Errorbars+/-SD.
Substrate
DoublesidetreatedPETSinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
CK1
9
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
Substrate
DoublesidetreatedePTFEPET
SinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
CK7
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
120
Figure53:HistogramtoshowpercentageexpressionofMUC5AConammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
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
SinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
UAE
-1lectin
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
Substrate
DoublesidetreatedePTFEPETmembrane
SinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
MUC5
AC
Day14 Day28Daysinculture
0
0.20201
0.40402
0.60603
0.800.88
1.00
121
Figure55:HistogramtoshowpercentageexpressionofCK7andUAE-1lectinco-expressionon
ammoniaplasmatreatedePTFE,untreatedePTFEandPETafter14and28daysinculture.
ANCOVAoverallmodelsubstrateandtimepoint:p<0.0001.Errorbars+/-SD.
Figure56:HistogramtoshowpercentageexpressionofΔNp63onammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
substratesp=0.007;timep=0.096.Errorbars+/-SD.
SubstrateDoublesidetreatedePTFEPETmembraneSinglesidetreatedePTFEUntreatedePTFE
Percen
tageofcellsexpressing
UAE
-1lectinand
CK7
Day14 Day28Daysinculture
80
60
40
20
0.0
Substrate
DoublesidetreatedePTFEPETmembraneSinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
ΔNp6
3
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
122
Figure57:HistogramtoshowpercentageexpressionofABCG2onammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
substratesp=0.003;timep=0.137.Errorbars+/-SD.
Figure58:HistogramtoshowpercentageexpressionofABCG2andΔNp63onammonia
plasmatreatedePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVA
overallmodelsubstratesp=0.001;timep=0.008.Errorbars+/-SD.
Substrate
DoublesidetreatedePTFEPETmembraneSinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcells
expressing
ABC
G2
Day14 Day28Daysinculture
0
100
2010
3015
4020
5025
Substrate
DoublesidetreatedPETmembraneSinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcellsco-expressing
ABCG
2an
dΔN
p63(lo
gscale)
Day14 Day28Daysinculture
0
10
2040
3060
4080
50
123
Figure59:HistogramtoshowpercentageexpressionofCaspase-3onammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodel
substratesp=<0.0001;timep=0.163.Errorbars+/-SD.
Figure60:HistogramtoshowpercentageexpressionofPCNAonammoniaplasmatreated
ePTFE,untreatedePTFEandPETafter14and28daysinculture.ANCOVAoverallmodeltime
p=0.024;substratesp=0.146.Errorbars+/-SD.
Percen
tageofcells
expressing
PCN
A
Substrate
DoublesidetreatedePTFEPETmembraneSinglesidetreatedePTFE
UntreatedePTFE
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
Substrate
DoublesidetreatedPETmembSinglesidetreatedePTFE
UntreatedePTFE
Percen
tageofcellsexpressing
Caspase-3(lo
gscale)
Day14 Day28Daysinculture
0
10
20
3060
124
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.
125
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
126
2x105
4x105
6x105
8x105
10x105
CellCo
untp
ercm
2
Day2 Day14 Day21 Day28
Numberofdaysinculture
DoublesidetreatedePTFE
Substrate
PETmembrane
day14.F-actinstainingofcellsonbothsubstratesdemonstratedthatcellsappeared
moreelongatedwithcellprocessesvisibleonday14whichwerenolongerpresentat
day28whencellsappearedsparsewithamore‘rounded’appearanceonboth
substrates.
Figure61:Histogramtoshownumberofcellspercm2withadvancingtimeincultureondouble
sideammoniaplasmatreatedePTFE(bothsidesexposedtoplasma)andPETmembrane.
Primarycellsusedinthisexperimentwerefromasingledonorandwereseededat1x105/cm2.
Scalebars+/-SD.
127
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
128
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.
129
Figure63:HistogramtoshowpercentageexpressionofCK19inprimarycellsculturedon
doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA
timep=0.079;substratep=0.123.Errorbars+/-SD.
Figure64:HistogramtoshowpercentageexpressionofCK4inprimarycellsculturedondouble
sideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVAtime
0.52;substrate0.753.Errorbars+/-SD.
Day28
Day28
Day14 Day28Daysinculture
Percen
tageofcells
expressing
CK4
DoublesidetreatedePTFE
Substrate
PETmembrane
40
30
20
10
0.0
Percen
tageofcells
expressing
CK1
9
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
130
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
CK7
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
100
2010
3015
4020
5025
Percen
tageofcells
expressing
UAE
-1Lectin
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
2010
4020
6030
8040
100
131
.
Figure67:HistogramtoshowpercentageexpressionofUAE-1lectinandCK7inprimarycells
culturedondoublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28
days.ANCOVAtimep=0.505;substratep=0.263.Errorbars+/-SD.
Figure68:HistogramtoshowpercentageexpressionofMUC5ACinprimarycellsculturedon
doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA
timep=0.124;substratep=0.456.Errorbars+/-SD.
Day28
Percen
tageofcellsexpressing
MUC5
AC
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
5
10
15
20
25
Percen
tageofcellsCK
7an
dUAE
-1lectin
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
100
2010
3015
4020
5025
132
Figure69:HistogramtoshowpercentageexpressionofΔNp63inprimarycellsculturedon
doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA
timep=0.945;substrate0.537.Errorbars+/-SD.
Figure70:HistogramtoshowpercentageexpressionofABCG2inprimarycellsculturedon
doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA
timep=0.753;substratep=0.611.Errorbars+/-SD.
Percen
tageofcellsexpressing
ABCG
2
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
60
40
20
0.0
Percen
tageofcells
expressing
ΔNp6
3
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
20
40
60
80
100
120
133
Figure71:HistogramtoshowpercentageexpressionofΔNp63andABCG2inprimarycells
culturedondoublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28
days.ANCOVAtimep=0.328;substratep=0.787.Errorbars+/-SD.
Figure72:Histogramtoshowpercentageexpressionofcaspase-3inprimarycellsculturedon
doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA
timep=0.138;substratep=0.134.Errorbars+/-SD.
Percen
tageofcellsexpressing
ΔN-p63
and
ABC
G2
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
15
10
5
0.0
Percen
tageofcellsexpressing
caspase-3
DoublesidetreatedePTFE
Substrate
PETmembrane
Day14 Day28Daysinculture
0
5
10
15
20
25
134
Figure73:HistogramtoshowpercentageexpressionofPCNAinprimarycellsculturedon
doublesideammoniaplasmatreatedePTFEandPETmembraneafter14and28days.ANCOVA
timep=0.003;substratep=0.04.Errorbars+/-SD.
3.6 Decellularisationofhumanconjunctivaanditscharacterisation
3.6.1 DNAquantificationofdecellularisedconjunctiva
Acommerciallyavailablekitwasusedtodegradeconjunctivaltissueandisolatedouble
strandedDNAsothatitwaspresentinsolutionforquantificationwithafluorescent
nucleicacidstain.CalfthymusDNAstandardswereusedinknownserialdilutionsto
enabletheacquisitionofastandardcurve(Figure74).Thestandardcurvedisplaysthe
relationshipbetweenthemeasuredabsorbancevaluesandknownconcentrationsof
DNA.Thisenabledcalculationofthegradientandinterceptfromtheequationy=mx+c
sothatDNAcouldbequantifiedfromeachtestsamplefromthemeasuredabsorbance
value.
Percen
tageofcellsexpressing
PCNA
Day14 Day28Daysinculture
DoublesidetreatedePTFE
Substrate
PETmembrane
80
60
40
20
0.0
135
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
136
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.
137
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).
a) b)
200μm 200μm
138
Figure76:RepresentativephotomicrographsoffixedGiemsastainedcellsfromahuman
conjunctivalcellline(HCjE-Gicells)andprimaryhumanskinfibroblastsinculturewith
decellularisedconjunctiva,Steri-stripsTMandcyanoacrylateglueafter48hours.a)
Decellularisedconjunctivawithcellsseenincontactwithtissueb)Steri-stripsTM,experimental
negativecontrolknownnottoexhibitcytotoxicity,seenherewithcellsvisibleincontactc)
cyanoacrylateglue,experimentalpositivecontrolshowingcytotoxicitywithcellulardebrisof
HCjE-Gicellssurrounditandalargezoneoninhibitionapparentwithcellulardebris.Scalebars
200µm.
b)
c)
c)
a)
b)
HCjE-GiCells Primaryhumanskinfibroblasts
c)
139
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).
140
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.
141
Amniotic
membrane
ePTFE
Figure77:Representativehistogramsofload(N)againstadvancingtime(seconds)duringthe
tensilestrengthtest:a)conjunctivab)amnioticmembranec)ePTFE.Eachhistogramalso
demonstratesthesectionofthecurvefromwhichthegradientforthegreatestslopewas
determined(see‘Greatestslope’markedoneachgraphbytheredarrows).
Conjunctiva
a)
c)
b)
142
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
143
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).
144
Figure79:RepresentativephotomicrographsofHaematoxylinandEosinstainedparaffin
embeddedtissuesections:a)decellularisedtissue;b)cellulartissue.Scalebars200µm.
Figure80:RepresentativephotomicrographsofconjunctivatissuestainedwithVanGieson’s
stain:a)decellularisedtissue;b)cellulartissue.Scalebars100µm.
a)
a) b)b)
b)a) b)
145
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.
146
Figure81:Representativephotomicrographsofdecellularisedamnioticmembraneand
conjunctivaltissuesectionsrecellularisedwithconjunctivalepithelialcells.Greatercelldensity
wasevidentqualitativelyfollowingexplantculture(bandd)thanthatfollowingsuspension
culture(aandc).Scalebars100µm.Arrowspointtopurplenucleiincells.
b)
d)
a)
c)
Decellularisedamnioticmembrane
Decellularisedconjunctiva
Suspensionculture Explantculture
b)
d)
147
3.7.2 Explantculturewithattentiontoconjunctivalbasementmembrane
orientation
Confluentcellgrowthwasdemonstratedwithqualitativelygreatercelldensityfrom
explantcellculturefollowingtheconfirmationandorientationofthebasement
membraneincomparisontotissueinwhichthebasementmembranecouldnotbe
identified(Figure82).Theexplantculturesusedinthisexperimentweretakenfrom
donoreye7.
Figure82:Representativephotomicrographsofexplantculturesgrownondecellularisedtissue:
a)basementmembranenotpresentb)basementmembranepresent.Arrowsarepointingto
nuclei(purple).Scalebars100µm.
a)
a) b)
148
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.
149
Tissuedonor9Tissuedonor5Tissuedonor13
Figure84:RepresentativeH&Estainedphotomicrographsofexplantsfromdonor17cultured
ondecellularisedconjunctivafromtissuedonors9/5/13.Nocellsareevidentfromontissue
sectionsderivedfromdecellularisedtissuedonor13.Occasionalareasofepithelialgrowth
weredemonstratedondecellularisedtissuesfromdonor9and5.Inthecentrephotofrom
decellularisedtissuedonor5anexplant(solidarrow)canbeseenwithcellularoutgrowth
(dashedarrow).Scalebars200µm.
Tissuedonor9Tissuedonor5Tissuedonor13
Figure85:RepresentativeH&Estainedphotomicrographsofexplantsfromdonor19cultured
ondecellularisedconjunctivafromtissuedonors9/5/13.Nocellswereevidentfromontissue
sectionsderivedfromdecellularisedtissuedonors9or5(donortissue5showsonlytheexplant
withnooutgrowth;solidarrow).Cellulargrowthwasobservedinmosttissuesectionsderived
fromexplantcultureondecellularisedtissuedonor13(dashedarrow).Scalebars200µm.
150
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).
151
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
a)
b)
152
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)
153
Figure89:Representativephotomicrographsofimmunohistochemicalstainingoftissue
sectionsofdecellularisedconjunctiva21recellularisedusingexplantsfromdonor23.Allbrown
stainingrepresentspositiveimmunolocalisationoftherespectivemarkerstudied.Scalebars
100µm.
CK4
CK7
MUC5AC
CK19CK19
154
Figure90:Representativephotomicrographsofimmunohistochemicalstainingoftissue
sectionsofdecellularisedconjunctiva21recellularisedusingexplantsfromdonor23.Allbrown
stainingrepresentspositiveimmunolocalisationoftherespectivemarkerstudied.Scalebars
100µm.
ABCG2 Caspase-3
PCNA ΔNp63
155
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).
156
Figure91:Representativephotomicrographstissuesectionsofcellular(a)anddecellularised
amnioticmembrane(b)fromthreetissuedonorsstainedwithPAS.Thebasementmembrane
tissuesappearstainedandsimilarbetweencellularanddecellularisedtissuesections
suggestingitwaspreservedfollowingdecellularisation.Scalebars100µm.
a)
b)
a)
Donor1 Donor2 Donor3
157
Figure92:Representativephotomicrographstissuesectionsofcellular(a)anddecellularised
conjunctiva(b)fromthreetissuedonorsstainedwithPAS.ThebasementmembranePAS
staininginallthecellularanddecellularisedtissuesappearedsimilarbetweentissuesections
suggestingitwaspreservedfollowingdecellularisation.Scalebars100µm.
a)
b)
Donor1 Donor2 Donor3
158
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)
159
Figure94:Representativephotomicrographsofcellular(a)anddecellularised(b)amniotic
membranetissuesectionsfromthreeseparatedonorsexaminedforlamininby
immunohistochemistry.Allbrownstainingrepresentspositiveimmunolocalisationoflaminin.
Scalebars100μm.
Donor1 Donor2 Donor3
a)
b)
160
Figure95:Representativephotomicrographsofcellular(a)anddecellularised(b)amniotic
membranetissuesectionsfromthreeseparatedonorsexaminedforfibronectinby
immunohistochemistry.Allbrownstainingrepresentspositiveimmunolocalisationof
fibronectin.Scalebars100μm.
Donor1 Donor2 Donor3
a)
b)
161
Figure96:Representativephotomicrographsofcellular(a)anddecellularised(b)amniotic
membranetissuesectionsfromthreeseparatedonorsexaminedforcollagenIVby
immunohistochemistry.Allbrownstainingrepresentspositiveimmunolocalisationofcollagen
IV.Scalebars100μm.
Donor1 Donor2 Donor3
a)
b)
162
Figure97:Representativephotomicrographsofcellular(a)anddecellularised(b)conjunctival
tissuesectionsfromthreeseparatedonorsexaminedforlamininbyimmunohistochemistry.All
brownstainingrepresentspositiveimmunolocalisationoflaminin.Scalebars100μm.
Donor1 Donor2 Donor3
a)
b)
163
Figure98:Representativephotomicrographsofcellular(a)anddecellularised(b)conjunctival
tissuesectionsfromthreeseparatedonorsexaminedforfibronectinbyimmunohistochemistry.
Allbrownstainingrepresentspositiveimmunolocalisationoffibronectin.Scalebars100μm.
Donor1 Donor2 Donor3
b)
a)
164
Figure99:Representativephotomicrographsofcellular(a)anddecellularised(b)conjunctival
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
Donor1 Donor2 Donor3
b)
a)
165
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.
166
Patientnumber 1;ODand
OS
2;ODand
OS
3;ODand
OS
4;ODand
OS
5;ODand
OS
Conjunctiva
Inflammatoryscore
totals(limbitis)
5;7
(n;n)
5;5
(n;n)
4;2
(n;n)
4;10
(n;n)
7;2
(n;n)
Tauberscore IIcIIIb;IIcIIIb IIdIIId;
IIdIIId
IIcIIIb;
IIbIIIa
IIdIIId;
IIbIIIb
IIcIIIb;
IIaIIIa(nil)
Tauber-Liverpool(%
II/III)
65/30;
70/35
75/100;
75/83
60/25;
50/13
85/83;
50/25
70/46;
0/0
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
(mm)
6/3;
6/4
5/0.5;
6/0.5
6/2;
6/3
0/6;
8/4
6/2;
8/5
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
LIDS
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
n;n
(laxitybut
grade0)
EntropG1
L+M;
EntropG1
L+M
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.
167
Figure100:Photographsoftherighteyeofpatient4.Theextentofhorizontalsymblepharon
involvementwas20mmextendingmediallyfromthelateralcanthus:a)lowerlidheldat
tensionb)upperlidheldattension.Thedistancebetweentheinferiorlimbusatthemidlineto
theedgeofthesubconjunctivalfibrosiswas1.5mm.Thefornixrulercouldnotbeinsertedinto
theinferiorconjunctivalfornix.
a) b)
168
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
169
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
ePTFE
Expandedpolytetrafluoroethylene(ePTFE)isarelativelyhydrophobicmaterial.
Ammoniagasplasmawasusedasatreatmentinthisstudytoincreasethe
hydrophilicityofePTFEtoimproveitsabilitytosupportcelladhesionandproliferation.
170
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-
171
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
172
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
173
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
174
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
175
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
176
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
177
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.
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4.3.2.3 Summaryoftheoptimisationexperimentsforthecultureof
conjunctivalepitheliumonePTFE
Insummary,theseexperimentsdeterminedthatthegreatestcelldensitycouldbe
achievedat14daysinculturewiththeuseofmediaprotocolBandacellseeding
densityof1x105/cm2.AfurtheradvantageofcellcultureprotocolBisthatitissimilar
tothatthatdefinedinotherstudiesusedforprimaryconjunctivalepithelialcells.(97,111,
115)
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
179
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
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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
181
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
182
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.
183
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
184
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
185
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
186
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.
4.5.1.2 Expressionofcytokeratin4
• CK4expressionwasmarkedlyhigherinprimarycellculturesthanHCjE-Gicell
cultures
• HCjE-GiexpressionofCK4waslowerondoublesidetreatedePTFEonPET
membrane
• NosignificanteffectofCK4expressionwasfoundwithrespecttotimeinculture
inprimarycellculturesorHCjE-Gicellcultures
CK4expressionwasalmosta10-foldgreaterinprimarycellsincomparisontoHCjE-Gi
cellsafter28daysinculture.Itshouldbenotedhoweverthatvariancewashighin
primarycellculturesandnosignificantdifferencewasdemonstratedwithrespectto
substrateoradvancingtimeinculture.CK4expressioninHCjE-Gicultureswasgreatest
onPETmembraneanduntreatedePTFEandlowestonsingleanddoublesideammonia
187
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).
188
4.5.1.3 Expressionofcytokeratin7
• 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
189
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
cells
• MUC5ACexpressionwassimilarbetweensubstrates
• MUC5ACexpressionincreasedwithadvancingtimeinHCjE-Gicellcultures
MUC5ACwasexpressedina20-foldorgreaterproportionofprimaryconjunctivalcells
incomparisontoHCjE-Gicellculturesregardlessofsubstrateafter28daysinculture.A
significantincreaseinMUC5ACdetectioninHCjE-Gicellswasfoundwithadvancing
timeinculturebutthesubstratedidnothaveastatisticallysignificanteffect.Inprimary
cellcultures,nostatisticallysignificantdifferencewasdemonstratedinMUC5AC
expressionbetweensubstratesortimepoints.
Angandcolleaguesdevelopedanultrathinpoly(ε-caprolactone)(PCL)membraneand
foundmarginallyhighergobletcelldensityafterseedingprimaryrabbitconjunctival
cellsonPCLmembranetreatedwithsodiumhydroxide.(97)Theysuggestedthatgoblet
190
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,
191
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
192
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
193
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
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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
195
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.
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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.
197
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
198
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
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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
4.7).
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
200
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
201
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
202
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
203
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.
204
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.
205
• 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
206
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)
207
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
208
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.
209
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
210
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.
211
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
212
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
213
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
214
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
215
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
216
constructcouldthenbeattachedtobaresclerawithfibringlueandsecuredwith
suturestotherecessedconjunctivaonthebulbarandtarsalaspectsaspreviously
described.(240)
Thedecellularisedconjunctivalsubstrateislikelytobeavailableinmuchsmaller
sectionsandthereforethiswilllimititsuse.Itcouldbeideal,forexample,infornix
reconstructionfollowinganocularburnonceinflammationhassettled,butthiswould
dependonthesizeofdecellularisedtissueavailableforuse.Adecellularised
conjunctivalsubstratemayalsosuitindicationssuchasthatfollowingtheremovalof
pterygia,conjunctivaltumoursorevenglaucomasurgeryincludingfollowingthe
placementofdrainagevalveimplants.
217
5. Conclusions
ThisstudydeterminedthepotentialofbothammoniagasplasmamodifiedePTFEand
decellularisedconjunctivaassubstratesfortheex-vivoexpansionofconjunctival
epithelium.Thesesubstratesmaybeusedforadifferentrangeofindicationsand
warrantconsiderationforuseintissueengineeringapplicationstoaddresstheclinical
needforconjunctivalreplacement.Thesetherapiescouldleadtosuccessfultreatments
forarangeofsightthreateningandpainfulocularsurfacedisorders.
AmmoniagasplasmatreatedePTFEisaneffectivesubstratefortheexpansionof
conjunctivalepithelium
• AmmoniagasplasmatreatmentofePTFEincreasesitshydrophilicityand
enablesthecultureofconjunctivalepitheliumonthetreatedsurface.Thecell
densitywasfurtherimprovedbytreatmentoftheePTFEmembraneonboth
sides,aneffectmarkedafter14daysinculture.
• Asimilarphenotypeofconjunctivalepithelialcultureswasdemonstratedon
doublesideplasmatreatedePTFEasPETmembrane.
• ThecultureofprimarycellsondoublesideammoniaplasmatreatedePTFE
resultedinadeclineincellnumberafter14daysincultureincontrasttoHCjE-
Gicells,whichincreasedupto28days.Overall,theprimarycellcultureswereof
amoredifferentiatedphenotypewithlessproliferativepotentialdemonstrated
bygreaterCK4,CK7andMUC5ACexpressionandlowerPCNA,ΔNp63and
ABCG2expression.
218
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.
219
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
220
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
221
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
222
surgery,conjunctivalreplacementfollowingresectionofconjunctivaltumoursand
conjunctivalchemicalburns.Theroleofdecellularisedhumanconjunctivamayevenbe
extendedforotherocularcellularreplacementtherapiessuchaslimbalstemcell
transplantation.Indeed,xenogeneicconjunctivalmatrixhasevenbeendemonstrated
asaneffectivescaffoldforcornealepitheliuminarabbitmodeloflimbalstemcell
disease.(92)Thisthereforedemonstratestherangeofcellularreplacementtherapiesin
whichdecellularisedmatricesprovidemajoradvancementsinthefieldoftissue
engineering.Decellularisedhumanconjunctivawarrantsfurtherinvestigationintoits
potentialclinicalapplicationsinocularsurfacedisease.
223
7. Appendix
1.TheLiverpoolcornealandexternaleyediseaseclinicproforma
(pages224-228)
2.Ethicalapproval
(pages229-231)
224
BCVARE………..LE……….Co-existingophthalmicpathology(accountingforreducedVA)………………………………PhotographtakenY NInflammationactivityscore(Grade1-5)RE LE
Totalscore ………… Totalscore …………Limbitis RE Y………quadrants/score N
LE Y………quadrants/score NAnteriorsegmentdrawingsRE
LE
Affixpatientlabel
225
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=………%
Verticalgrading=………%
Horizontalgrading=………%
Horizontalgrading=………%
226
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
227
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
228
RE LE Entropion/
Ectropion Entropion/
Ectropion
Upperlid Lowerlid Upperlid LowerlidGrade0 Grade1 Grade2 Grade3 Medial Lateral Lateral+Medial
229
230
231
232
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