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Cellular Adhesion of Normal and Cancerous Colon Cells: Effects of Surface Topographies

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Title:
Cellular Adhesion of Normal and Cancerous Colon Cells: Effects of Surface Topographies
Creator:
PERRAULT, CECILE MONG TU ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Biomaterials ( jstor )
Cancer ( jstor )
Cell adhesion ( jstor )
Cells ( jstor )
Colorectal cancer ( jstor )
Focal adhesions ( jstor )
Microchannels ( jstor )
Shear stress ( jstor )
Surface roughness ( jstor )
Topography ( jstor )

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University of Florida
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University of Florida
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Copyright Cecile Mong Tu Perrault. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2012
Resource Identifier:
660161435 ( OCLC )

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CELLULARADHESIONOFNORMALANDCANCEROUSCOLONCELLSEFFECTSOFSURFACETOPOGRAPHYByCECILEM^O.NGTUPERRAULTAPHDPRESENTEDTOTHEGRADUATESCHOOLOFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHEREQUIREMENTSFORTHEDEGREEOFPHDUNIVERSITYOFFLORIDA2007 1

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c2007CecileM^o.ngTuPerrault 2

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Tomyfamilyandmyhusband,Nicolas,fortheircontinuoussupportandunderstanding. 3

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ACKNOWLEDGMENTS Thisdoctoraldissertationisthesummaryofanintricateandcomplexjourney.IdoubtthatIwouldhavebeenabletocompleteitwithoutthesupportofmyfamily,friendsandcolleagues.Iamgratefulforthehelpofmanypeople.Unfortunately,IknowthatIwillforgettothankalotofthem.Ihopethattheywillunderstandthatitisnotoutofungratefulness.Tenpagesofacknowledgmentswouldjustbeabittoolong.First,IwouldliketothankmydoctoralcommitteeDr.TonySchmitz,Dr.FranckBovaandmorespecicallyDr.AnthonyBrennan,fortheircontinualsupportandencouragement.Iamalsogratefulformyadvisor,Dr.RogerTran-Son-Tay.SincemyrstyearattheUniversityofFlorida,hehasgivenmetheopportunitiesneededtobringmewhereIstandtoday.Iacknowledgeallofthetime,workandsupportheprovided.TheUniversityofFloridawasalsohometomostofmyfriendships.IwanttoacknowledgeEthanSherman,acolleagueoftenyears.Throughclasses,researchorpersonalstorms,hehasprovidedmewithastrongsupport,andhashelpedmeinmanydesignprojects.Furthermore,IwouldnothavemadeitwithoutthesupportofAnne,mydearfriend.Shewasalwaysthere,alwaysknewhowtocheermeup.IalsothankallofthepeoplefromthelaboratoryofcellularmechanicsandbiorheologyandthedepartmentofBiomedicalEngineeringforprovidingagoodworkingenvironment.Amongmyfellowstudents,IwouldliketogivespecialthankstoJamesSchumacher,Dr.OlojompoMoloyeandTailiThula.Finally,IwouldliketothanklesfrancaisdeGainesville:Benoit,Fabien,Thierry,JulienetNissia,Lan,Victor.Mostimportantly,Iwouldliketothankmyparents,forprovidingmewiththebesteducationonecangiveachildandforbelievingmeinmethroughoutthislongandtumultuousprocess.Ialsowanttothankmybrothersandsister.EventhoughIcan'tspendmuchtimewiththem,Iloveeverymomentwearetogether.Mythoughtsarealsowithmygrandparents,bothFrenchandVietnamese. 4

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Finally,IameternallygratefultomyhusbandNicolas,forsupportingme,enduringmymomentsofdespairandrejoicinginmymomentsofhappiness.Hislovehasprovenunconditional.Iowehimmylastyearsofgraduateschool. 5

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TABLEOFCONTENTS page ACKNOWLEDGMENTS ................................. 4 LISTOFTABLES ..................................... 9 LISTOFFIGURES .................................... 10 ABSTRACT ........................................ 13 CHAPTER 1INTRODUCTION .................................. 15 1.1Rationale .................................... 15 1.2Objective .................................... 17 1.3SpecicAims .................................. 17 2PHYSIOLOGICALBACKGROUND ........................ 19 2.1Cancer ...................................... 19 2.1.1WhatIsCancer? ............................ 19 2.1.2ColorectalCancer ............................ 21 2.2Cell ....................................... 21 2.2.1ColonCells ................................ 24 2.2.2ColonCancerCells ........................... 24 2.3TheCellandItsEnvironment ......................... 24 2.3.1StudyingtheEectsofSurfaceRoughness:MeasuringRoughness . 25 2.3.1.1Height/Depth ......................... 25 2.3.1.2RoughnessWidth ....................... 26 2.3.1.3RoughnessSpacing ...................... 27 2.3.1.4Randomness .......................... 27 2.3.1.5OtherFactors ......................... 28 2.3.2StudyingtheEectsofSurfaceRoughness:ManufacturingTechniques 28 2.3.2.1ChemicalModication .................... 28 2.3.2.2Photolithography,ElectronBeamLithography,LaserHolography 29 2.3.2.3MechanicalTechniques .................... 29 2.3.2.4FilmDeposition ........................ 30 2.3.3SurfaceRoughnessandMuscleTissue ................. 30 2.3.4SurfaceRoughnessandNervousTissue ................ 31 2.3.5SurfaceRoughnessandEpithelialTissue ............... 32 2.3.6SurfaceRoughnessandConnectiveTissue ............... 32 2.3.7ConclusiononSurfaceRoughness ................... 35 2.3.8VariationintheExtracellularEnvironmentanditsRoleinCancer . 36 2.4Intra-Extracellularinterface .......................... 37 2.4.1TheOutside-InMechanism ....................... 37 6

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2.4.2TheInside-OutMechanism ....................... 37 2.5Cellularfunction:adhesion ........................... 38 2.5.1Cell-cellAdhesion ............................ 38 2.5.2Cell-SurfaceAdhesion .......................... 39 3BIOMECHANICALBACKGROUND ....................... 45 3.1Biomechanicalmanipulationsofcells ..................... 45 3.1.1Micromanipulation ........................... 45 3.1.2Centrifugation .............................. 46 3.1.3FluidShearSystems .......................... 46 4MATERIALSANDMETHODS ........................... 48 4.1CellCulture ................................... 48 4.1.1Description ................................ 48 4.2Surfacepreparations .............................. 48 4.2.1Patterns ................................. 48 4.2.2Fabrication ................................ 49 4.3GradientShearFlowChamber ......................... 50 4.3.1Purpose ................................. 50 4.3.2Design .................................. 50 4.3.3FabricationandCellAdhesion ..................... 50 4.3.4ShearStressCalculations ........................ 51 4.3.5Operation ................................ 52 4.3.6DataAcquisition ............................ 52 4.3.7DataAnalysis .............................. 53 4.3.8StatisticalAnalysis ........................... 53 4.4NormalForceAdhesionAssay ......................... 53 4.4.1Purpose ................................. 53 4.4.2CentrifugeSetup ............................ 53 4.4.3NormalForceCalculation ........................ 53 4.4.4SurfacePreparation ........................... 54 4.4.5ExperimentalRun ............................ 55 4.4.6DataAcquisition ............................ 55 4.4.7DataAnalysis .............................. 55 4.4.8StatisticalAnalysis ........................... 56 5RESULTS ....................................... 61 5.1GradientShearFlowChamber ......................... 61 5.1.1ADINAFluidCalculations ....................... 61 5.1.2CellularDetachment .......................... 62 5.2Normalforceadhesionassay .......................... 62 5.2.1CellsOrientationChange ........................ 62 5.2.2RegularColonvs.CancerousColonCellsonFlatSurfaces ..... 62 5.2.3EectsofCellSizeonDetachment ................... 63 7

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5.3Topographyandcelladhesion ......................... 63 5.3.1EectofWidth ............................. 63 5.3.2EectofSpacing ............................ 64 5.3.3EectofDepth ............................. 65 5.4Eectofcell-celladhesiononcell-surfaceadhesion .............. 66 5.4.1EectsofDoubleCellSpreadingonCellDetachment ........ 67 5.4.2EectsofCellJunctiononCellDetachment ............. 67 6DISCUSSION ..................................... 77 6.1GradientShearFlowChamber ......................... 77 6.1.1ModelingAdhesionUnderShearFlow ................. 77 6.1.2ShearFlowChamberChallenge .................... 78 6.1.2.1Gassolubilityinliquids ................... 78 6.1.2.2Pdmsgaspermeability .................... 79 6.1.2.3Fluidicresistance ....................... 79 6.1.2.4Pdmsasavalidsubstratemodel .............. 79 6.2NormalForceAdhesionAssay ......................... 80 6.2.1NormalForceCalculation ........................ 80 6.2.2DetachmentBehavior .......................... 81 6.3Topographicaleectsoncellularadhesion ................... 84 6.4Cell-Celladhesion ................................ 85 7CONCLUSIONSANDFUTURERESEARCH ................... 90 7.0.1ForceAssessment ............................ 91 7.0.2FocalAdhesions ............................. 91 7.0.3CellPositionintheMicrotopography ................. 92 7.0.4FluorescenceCellularImagingofAdhesionProteins ......... 92 APPENDIX ANORMALFORCECALCULATION ........................ 94 BSHEARSTRESSCALCULATIONINTHEGRADIENTSHEARFLOWCHAMBER 95 B.1Principle ..................................... 95 B.1.1DesignCalculations ........................... 98 CFROMSHEARSTRESSTOSHEARFORCE ................... 101 REFERENCES ....................................... 103 BIOGRAPHICALSKETCH ................................ 111 8

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LISTOFTABLES Table page 4-1dimensionsoftherectangularpattern ........................ 49 4-2Relationshipbetweencentrifugalspeedandrelativecentrifugalforce. ...... 56 5-1ComparisonofthecalculatedandADINAwallshearstressvaluesattheentrancewidth1(afterentrancelength)andattheexitwidth2 ............. 61 6-1Tangentialforcesinthegradientshearowchamberatdierentowrate .... 78 6-2Centrifugalspeedsandtheircorrespondingforces ................. 81 B-1Table4.1:dimensionsoftherectangularpattern ................. 99 C-1Shearforcesattheentrancewidthofthegradientshearowchamber ...... 101 C-2Shearforcesattheexitwidthofthegradientshearowchamber ........ 102 9

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LISTOFFIGURES Figure page 2-1Stepsinvolvedinthemetastasisprocess. ...................... 39 2-2Overviewofcellularstructures ............................ 40 2-3Morphologychangecanbeobservedwiththeprogressionofcoloncancer(ImageAnalysisofExtracellularmatrixTopographyofColonCancerCells,R.Anderson,E.Anderson,L.Shakir,S.Glover.2006.CopyrightJohnWiley&SonsLimited.Reproducedwithpermission). ............................ 40 2-4Exampleofparametersofsurfacetopography. ................... 41 2-5Theaverageroughness,Ra,isthearithmeticaverageoftheabsolutedeviationsfromthemeansurfacelevel.Itisrepresentedherebytheshadedarea. ..... 41 2-6Eectofsurfaceroughnessonmusclecells.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. ........... 42 2-7Eectsofsurfaceroughnessonnervouscells.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. ............ 42 2-8Eectsofsurfaceroughnessonepithelialcells.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. ............ 43 2-9Eectsofsurfaceroughnessonconnectivetissue.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. ........ 43 2-10Alterationoftheextracellularmatrixatdierentstageofcoloncancer(ImageAnalysisofExtracellularmatrixTopographyofColonCancerCells,R.Anderson,E.Anderson,L.Shakir,S.Glover.2006.CopyrightJohnWiley&SonsLimited.Reproducedwithpermission.) ............................ 44 4-1Thedenedtopographicalparametersofinterestinthemicrochanneldesignaredepth,widthandspacing. .............................. 56 4-2Thegradientshearowchamberdesignconsistsofaconvergentchannelofentrancewidthw1andexitwidthw2,entrancelengthLeandchannellengthL. ..... 57 4-3Gradientshearowchamberrstmanufacturingprotocol.1)Atransparencycut-outofthedesiredchannelisplacedinanholderandtwopiecesoftubingareplacedtocreatetheentranceandexitports.PDMSisthenpouredoverthemold.2)ThecuredPDMSisremovedfromthetransparencycut-outandputover3)apartiallycuredpieceofatPDMS.4)AstheuncuredPDMSpiececured,itcreatesanirreversiblesealbetweenthetwocomponents. ........ 57 10

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4-4Gradientshearowchambersecondmanufacturingprotocol.1)AatpieceofcompletelycuredPDMSand2)thechannelPDMSpiece,manufacturedasexplainedinsteps1and2ofgure 4-3 ,are3)sandwichedbetweentwopiecesofplexiglasandheldinplacebyscrews. ...................... 58 4-5Forcesactingontheattachedcellsduringcentrifugation. ............. 58 4-6Thepetri-dishholderismadeofaluminumtotthelaboratory'scentrifuge.Cellsareadherenttotheinteriorsideofthepetridish.Acustom-madePDMStopsealsthepetridishtoavoidleakagesanduidmovements. .......... 59 4-7Extractfromatypicalseriesofpicturestakenduringanormaladhesionassayexperiment.Superpositionofthepicturesallowsustofolloweachcellastheydetachfromtheirsubstrate. ............................. 60 5-1TheADINAchartsandgraphofwallshearstressesforowratesof0.002and0.426L/hr. ...................................... 68 5-2UsingtheGSFC,asingle5minuteowexperimentonshort-termadheredcellsshowspartialremovalofthecellsat13dynes/cm2andcompleteremovalofthecellsat40dynes/cm2. ................................ 69 5-3Cellularadhesionofhealthycoloncellsontheatsurface.%cellsdetachedreferstotheproportionofcellsoriginallyattachedthathavedetached ......... 69 5-4Cellularadhesionofcancerouscoloncellsonatsurface.%cellsdetachedreferstotheproportionofcellsoriginallyattachedthathavedetached. ......... 70 5-5Sizeofcellsvs.detachmentspeed.Thespreadingarea(size)of96cellswasmeasuredandrecordedandrelatedtotheirdetachmentspeed.StatisticalanalysiswithANOVAstatesthatthereisnorelationshipbetweenthesizeanddetachmentspeed. ......................................... 71 5-6Eectofwidthonhealthycoloncellsadhesion.Byvaryingthewidthofthemicrochanneltopography,wewereabletoevaluateanyvariationincellulardetachmentofthehealthycells.t-testanalysisfoundnodierencebetweenadhesiononthedierentmicrochannelsandontheatsurface. .................. 72 5-7Eectofwidthoncancerouscoloncellsadhesion.Byvaryingthewidthofthemicrochanneltopography,wewereabletoevaluateanyvariationincellulardetachmentofthecancerouscells.t-testanalysisfoundastatisticaldierencebetweenadhesiononthedierentmicrochannelsandontheatsurface.However,therewasnodierenceontheadhesionbetweenthedierentmicrochannels. ......... 72 5-8Eectofspacingonhealthycoloncellsadhesion.Byvaryingthespacingofthemicrochanneltopography,wewereabletoevaluateitseectoncellulardetachmentofthehealthycells.t-testanalysisfoundnodierencebetweenadhesiononthedierentmicrochannelsandontheatsurface. .................. 73 11

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5-9Eectofspacingoncancerouscoloncellsadhesion.Byvaryingthespacingofthemicrochanneltopography,wewereabletoevaluateitseectoncellulardetachmentofthecancerouscells.t-testanalysisfoundastatisticaldierencebetweenadhesiononthedierentmicrochannelsandontheatsurface.However,thereisnodierenceintheadhesionbetweenthedierentmicrochannels. ............... 73 5-10Eectofdepthoncancerouscoloncellsadhesion.Byvaryingthedepthofthemicrochanneltopography,wewereabletoevaluateitseectoncellulardetachmentofthecancerouscells.t-testanalysisfoundastatisticaldierencebetweenadhesiononthedierentmicrochannelsandontheatsurface.However,thereisnodierenceintheadhesionbetweenthedierentmicrochannels. ............... 74 5-11Comparisonoftheadhesionofsingleanddoubletshealthycoloncells. ...... 75 5-12Comparisonofthesingleanddoubletsofcoloncancercells. ........... 75 5-13Thelengthofthejunctionbetweentwoattachedcells,ordoublecells,aremeasuredandrecordedtoassesstheirimportanceoncell-surfaceadhesion. ......... 76 5-14Eectsofcell-celljunctionlengthoncelldetachment. ............... 76 6-1Freebodydiagramofanadherentcellundershearow.Adhesionbondsresisttotheshearforce(Fs)andtorque(Ts)byatangential(Fa),tensile(F)andcompressive(Fc)forces.ThedominantresistantforceisF. ........... 87 6-2Normalcoloncellsadhesiononmicrochannels. ................... 88 6-3Coloncancercelladhesiononmicrochannels. .................... 89 B-1Sketchofaductwithvaryinggapbetweenparallelplates ............. 97 B-2DesignoftheShearGradientFlowChamber .................... 99 B-3Shearstressvariationalongthegradientshearowchamberunderowrateof0.01L/hr ....................................... 100 12

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AbstractofPhDPresentedtotheGraduateSchooloftheUniversityofFloridainPartialFulllmentoftheRequirementsfortheDegreeofPhDCELLULARADHESIONOFNORMALANDCANCEROUSCOLONCELLSEFFECTSOFSURFACETOPOGRAPHYByCecileM^o.ngTuPerraultMay2007Chair:RogerTran-Son-TayMajor:BiomedicalEngineeringNormalcellularadhesionisakeyfactortomaintainhealthyhumanbiologicalfunctions.Adhesionprocessesareinuencedbymanyfactorssuchasdetachmentforcesandextracellulartopography,butthemechanismsinvolvedarenotwellunderstoodbecauseofalackofwell-dened,simple,experimentalsystemsforlong-termcelladhesion.Theliteraturealsosuggeststhatinthecontextofcancerousorgans,analteredcellularadhesioncanpromotethespreadofmetastases.Therefore,inordertobettercharacterizeandgainnewinsightsintotheeectsofsomekeymechanicalfactors,wehavedevelopedseveralsimple,easytouse,techniquesforevaluatingtherolesofbothnormalandshearforces,andsurfaceroughnessincelladhesion,withafocusoncancercells.Theresultsshowasexpectedthat,astheowrateorthenormalforceisincreased,morecellsaredetachedfromthesurfacesubstrate.However,itisfoundthat,forasmoothsurface,20%ofthehealthycellswilldetachattheonsetofanappliednormalforce,andthattherateofdetachmentisnegligibleafterthat.Incontrast,cancercellscontinuouslydetachasthenormalforceisincreased.Anotherdierenceseenbetweenhealthyandcancercellsisinthebehaviorofdoubletcells.Healthydoubletshaveamuchstrongeradhesionthansinglecells.However,thisdierenceisnotseenwithcancercells.Finally,itisobservedthatsurfaceroughnessproducesdierenteectsontheadhesionofhealthyandcancerouscoloncells.Morespecically,theuseofmicrochannelpatternsonasubstrateshowsthatcancercellsdonotattachwellonaatsurface,andthat 13

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theiradhesionismuchweakerthanforhealthycells.Asmallvariationinthewidthorspacingofthepatternswillmakecancercellsmoreadherentandbehavemorelikehealthycells.Changesinthefeaturedepthyieldamoregradualeectontheadhesionbehaviorofcancercells,withanapparentlymaximumeectforadepthofabout2mm.Ontheotherhand,changesinthetopographicalfeaturesdonotaectverymuchthestrengthofadhesionofhealthycells.Inconclusion,thedevelopedtechniquesforculturedcellsoernewtoolsforstudyinglong-termcelladhesion.Theyprovideameanforinvestigatingtheeectsofbothshearandnormalforces,aswellassurfaceroughness.Ourresultsclearlyindicatethatthetopographyoftheextracellularmatrixwillplayamajorroleontheadhesionofthecells,andhaveamorecriticalimpactontheadhesionofcancercellsthanonhealthycells. 14

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CHAPTER1INTRODUCTION 1.1RationaleIn500BC,Hippocratesrejectedthecommonbeliefthatillnesswascausedbysuperstitions,possessionofevilspiritsordisfavorofthegods,andratherbasedhisreasoningwithphysicalandrationalexplanations.Regardedasthegreatestphysicianofhistime,thismanbecamethefounderofmodernmedicineandhisnameforeverassociateswiththeoathbindingdoctorstothehighestethicalstandards,Hippocraticoath.Settingmedicinefreefromculturalandspiritualbeliefswasamajorsteptowardasystematicunderstandingofthehumanbody,basedonobservations.Takinganewandradicalapproachtomedicineallowedprogresstobreakfreeanddevelopawayfromtherigorous,xedtraditionalapproach.Biomedicalengineersarenowtakinganewapproachtomedicine,improvingitbycementingtheirinformationonquantiedobservationsratherthanqualitativereports.Thispointofviewoersnewandexcitingopportunitiesformedicine.Thecurrentapproachnotonlyapprehendsbodyandcellularfunctionswithobservationalreportsbutalsodevelopsmodelsthatexplaintheintricatebehavioroforgans,cellsorevenproteins.Withthisperspective,thestudyoforganicfunctionsrequiresaconstantdevelopmentofnewtechniquesandequipments.Forexample,thesimplerinsingmethodisthepreferredcellularadhesionassaywithmedicalscientists.However,thismethodlacksprecisionanddidnotprovideenoughinformationonthedetachmentmechanism.Aquantitativeunderstandingofcellularadhesiondemandscontroloverstrengthanddistributionoftheforceappliedonthecells.Hencetheneedtodevelopowchambers.Cell-surfaceadhesionistheresultofbondingbetweenadhesionreceptorsonthecellmembraneandligandsontheextracellularsurface.AcellattachedonasurfaceissimilartoaVELCROballattachedonaVELCROsurface.Ifenoughshearstressisappliedon 15

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thisball,theballcouldstartrollingonthesurface,bondsdetachingasnewbondsarecreated.Visualizationofthecellswouldthusprovideuswithinformationonthedynamicsofbondcreationanddetachment.Now,ifanormalforceisappliedtothissameball,wewouldobtaininformationonthedetachmentofthebonds.Therefore,thetwodierentapproachescomplementeachothertoprovideaglobalviewofadhesion.Althoughshearforceassaysareverypopular,theirnormalforcecounterpartsarerelativelyunder-studied.Thepresentworkthusintendstodeveloptwonoveltechniquestounderstandcellularadhesion.Therstonefocusesonapplyingashearforceoncellsbyuseofmicrouidicowchamber.Thesecondtechniqueaimsatstudyingcelladhesionunderanormalforce.Alongwithforces,surfacefeaturesareamongthenumerousfactorsthataectcellularadhesion.Sincecellsin-vivoexperienceadiversetopographicalsurrounding,itsuggeststhatextracellularroughnesscouldinuenceadhesionandothercellfunctions.However,roughnesslacksauniversalcharacterization.Inaddition,previousliteratureutilizesagreatvarietyoftechniques,surfaces,andcelltypes,whichpreventsthealreadypublisheddatatomergeintoonegreatpicture.Toovercomethelackofstructureintheeld,wewilldesignatestingsurfaceusingasetnumberoftopographicalparameterstoisolatetheireectsoncellularadhesion.Therecentobservationthatextracellularmatrixtopographyisalteredincoloncancerhasraisedaninterestastotheeectofthistopographicalalterationoncoloncelladhesion.Thecoloniscomposedofavarietyofcells,includingepithelialcells.In-vivo,healthyepithelialcellsdonotdetach.However,manydiseasescreateanadhesionalteration,whichisanecessaryfactorfortheirdevelopment.Inthecaseofcancer,thisalterationplaysanimportantpartinmetastasis:cancercanonlyspreadandestablishitselfthroughoutthebodyifcellularadhesionalterationoccurs.Whenatumormetastasizes,itproducescellswiththeabilitytoachieveanumberofsteps.First,theinvadingcellspenetratethevascularorlymphaticchannels.Then,theydetachandtravelwithinthecirculatorysystem.Duringthattransportation,thecellshidefromthe 16

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immunesystemandsurviveturbulencesofthecirculation.Finally,themetastaticcellsreattachtothevesselswall,extrudethemselvesfromthevessels,proliferateandestablishamicrometastasis.Ifoneormoreofthesestepsfails,itresultsinthemetastaticcellelimination.Therefore,themetastaticcellexhibitsacomplexphenotyperegulatedbyalteredproperties.Thedetachmentandre-attachmentstepsofthemetastaticcycleshowtheimportanceofalterationincellularadhesionincancer.Hence,thisresearchseekstobetterestablishthedierencebetweenhealthyandcancerouscoloncells'adhesion. 1.2ObjectiveCellularadhesionhasbeenrecognizedasoneofthekeycellularfunctioninthebodyanditsalterationiscrucialindiseasessuchascancer.Whileitgeneratesalotofinterestinresearch,mostoftheworkconcentratesonthemolecularbasisofadhesion,andneglectsthequantitativeimplicationsofmolecularalterations.Cellularadhesionstrengthcanbeaectedbyanumberoffactors,thatincludetheapplicationofnormalorshearforcesandsurfacetopography.Theresearchproposedhereintendstodesignandtestnovel,easy-to-use,economicalandconvenientcellularadhesionassaysthatmakeuseofnormalorsheardetachmentforces.Similarlytoforces,surfacefeaturesaectcellularadhesion.Thiseecthasrarelybeenstudiedinthecontextofsurfacegeometry.Inourwork,weseektoevaluatetheeectsofsurfacetopographyoncellularadhesionbyestablishingthekeydimensionalparametersofinuence.Finally,sinceitwasobservedthattheextracellularenvironmenttopographyisalteredincoloncancer,wewillascertaintheeectsthatthisalterationcouldhaveoncellularadhesion.Wewillusethetechniquesmentionedearliertoassesscellularadhesioninhealthyandcancerouscoloncells. 1.3SpecicAimsDesignaconvenientandeasy-to-usenormalforceassayDesignaneconomicalandnovelowchamber 17

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DesignatopographicalpatternwithdenedparametersEvaluatethecellularadhesionofhealthyandcancerouscoloncells 18

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CHAPTER2PHYSIOLOGICALBACKGROUNDInordertoprovidethenecessaryinformationtobetterunderstandthiswork,thefollowingsectionwillintroducethebasisofcancerandsurfacetopographyresearch. 2.1CancerCancerhasaectedhumanitythroughoutitshistory,ascantestifytheoldesttumorevidencedatingbacktothetimeoftheAustralopithecus.Thediseasehaschallengedeventhebestphysicians.AttheheightofGreekrepublics,Hippocratesdealtwithcancerandwouldprescribeapples,datesandporridgetocurehispatients.Ourunderstandingofthediseasehasincreasedsincethosetimesbutthecuretocancerremainselusive.AccordingtothelateststatisticsoftheAmericanCancer Society , 2007 ,atotalof1,444,920newcancercasesand559,650deathsfromcancerareexpectedintheUnitedStatesin2007.Whendeathsareaggregatedbyage,cancerhassurpassedheartdiseaseastheleadingcauseofdeathforthoseyoungerthanage85since1999.SuchstatisticsmakecanceramajorpublichealthissueintheUnitedStatesandotherdevelopedcountries,justifyingthelargeeldofcancerresearch.Tofacilitatetheunderstandingofthisthesis,wepresentherethegeneraltheoryofcancer. 2.1.1WhatIsCancer?Cancerdevelopswhencellsinapartofthebodybegintogrowoutofcontrol.Thisabnormalbehaviorresultsfromacombinationofexternal(tobaccoorchemicals,forexample)andinternalfactors(inheritedmutations,hormones).Duringtheearlyyearsofaperson'slife,normalcellsdividerapidlyandthenslowdownasthepersonbecomesanadult.Onceatadulthood,cellsinmostpartsofthebodydivideonlytoreplaceworn-outordyingcellsandtorepairinjuries.Thusanhealthycellgrows,divides,anddiesinanorderlyfashion.Thisnormalcycleofcelldivisionandcellgrowthisregulatedbyanumberofbodycontrolmechanisms.Thedisruptionofthosemechanismsleadtotheimbalanceinthisorganizedsystemandtotheabnormal 19

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propagationofcells.Cancercellsthengrow,invadeanddestroysurroundingtissues.Sincecanceralsoleadstotheimmortalityoftheabnormalcells,theyoutlivenormalcellsandspreadnewabnormalcells.Inadditiontotheirabnormalbehaviors,cancercellshavetheuniquecapabilityoftravelingandsettlingindistantareasofthebody.Thisprocess,calledmetastasis,isthebiggestchallengeinthecureforcancerandthecauseof90%ofsolidtumordeaths( GuptaandMassague , 2006 ).Thepresenceofanumberoftumorsthroughoutthebodyrenderslocaltherapeuticsolutionsuselessand,despiteimprovementinscreeningtechniques,thisistherealityofmanypatients.Approximatively30%ofpatientshaveclinicallydetectable(overt)metastasesatthetimeoftheirinitialdiagnosisandanadditional30%havemicroscopic(occult)metastases( Kufeetal. , 2003 ).Theprocessofmetastasisinvolvesanumberofcriticalsteps(gure 2-1 ).First,thecancercelldetachesfromitsprimarytumor.Onceithasmigratedandreachedthebloodvesselwall,theinvadingcellmustpenetratethevascularorlymphaticchannels.Usingthelymphaticsystemasameanoftransportation,thecelltravelsthroughoutthebody.Duringthattime,itmustbeabletohidefromtheimmunesystemandsurviveturbulencesofthecirculation.Onceithasreachedtheappropriateemplacement,thecancerouscellreattachestothevesselwallandextrudesitselffromthevessel.Nowreadytoestablishamicrometastasis,thecellmustbeabletoproliferateandensureaconstantsupplyofnutrientsthroughangiogenesis.Failuretocompleteoneormoreofthesestepswouldresultintheeliminationofthecells.Asaresult,outofthemillionsoftumorcellsfoundinthecirculationeveryday,lessthan0.01%areabletoestablishsuccessfullyametastasis( Kufeetal. , 2003 ).Whilethisgeneralizedmechanismofcancerappliestoalltypes,eachbodypartdevelopsitsownspecializedformofcancer,characterizedbydierencesinmorphologyandsymptoms.Thosecharacteristicaresospecicthat,evenoncethecancerhasspreadtoanotherregionofthebody,itispossibletorecognizewheretheprimarytumorisbylookingatthemetastasis.Forexample,ifbreastcancerwastometastasizeinthelungs, 20

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theremotetumorwouldhavethesamecharacteristicsastheoriginaltumor,andthuswouldstillbecalledbreastcancer.Inmyresearch,ourinterestliesincoloncancer.Thefollowingsectionisintendedtoprovideaquickoverviewofthisspecictypeofcancer. 2.1.2ColorectalCancerColorectalcancerincludescancerousgrowthsinthecolon,rectumandappendix.ItisthethirdmostcommonformofcancerandthesecondleadingcauseofdeathamongcancersintheWesternworld.In2007alone,itisestimatedthatmorethan153,000peoplewillbediagnosedwiththediseaseandover52,000willdiefromit.Whilethedeathrateduetocoloncancerhasbeendecreasingoverthepast20yearsasaresultofbetterscreeningtechniques,muchcouldbedonetofurtherimproveit.Treatmentofcoloncancerdependsonthestagingofthecancer.Whencolorectalcanceriscaughtatearlystages(withlittlespread),the5-yearsurvivalrateis90%.However,earlystageofcoloncanceronlyconcerns39%ofinitialdiagnosis.Andwhencoloncancerisdetectedatlaterstages(whendistantmetastasesarepresent),the5-yearsurvivalratedropsto10%.Surgeryremainstheprimarytreatment.Butchemotherapyand/orradiotherapymayberecommended,dependingonthepatient'sstageofdiseaseandvariousmedicalfactors.Theimpactofthisdiseaseonthegeneralpopulationhasmadecolorectalcanceroneofthemostintensively-studiedtypesofcancer,evenleadingtomathematicalmodelingoftheinitiationofcancer,orcarcinogenesis( vanLeeuwenetal. , 2006 ). 2.2CellCellsarethebasicunitofalllivingorganisms,andoftenreferredtoasthebuildingblocksoflife.Acellisself-containedandself-maintaining.Itcanabsorbnutrient,convertthemintoenergy,carryoutspecializedfunctionsandreproduceifnecessary.Eachcellstoresitsownsetofinstructionstocarryouteachoftheseactivities. 21

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Thebiologicaluniverseconsistsoftwotypesofcellsprokaryoticcellswhichlackadenednucleusandhaveasimpliedinternalorganizationandeukaryoticcellswhichhaveamorecomplicatedinternalstructureincludingadened,membrane-limitednucleus.Ourinterestlieswitheukaryoticcells,andthusourdescriptionwillconcernonlythose.Eukaryoticcells,unlikeprokaryotes,haveaexiblemembrane,givingthemthechancetoexpandandincreasetheirinternalcomplexity.Thismembraneactsnotonlyasabarriertotheoutsideenvironment,butalsoasalterselectivelypermeabletocertainmolecules.Thisallowsnutrientsandotheressentialelementstoenterthecellandwastetoleavethecell.Oxygen,carbondioxide,waterandothersmallmoleculesareabletopassfreelyacrossthemembrane,butlargermolecules,suchasaminoacidsandsugars,canonlypassundercarefulregulation.Accordingtotheacceptedcurrenttheory,knownastheuidmosaicmodel,theplasmamembraneiscomposedofalipidbilayermixedwithmembraneproteins.Themodelwasrstproposedby SingerandNicolson , 1971 asalipidproteinmodelandextendedtoincludetheuidcharacter( SingerandNicolson , 1972 ).Phospholipidshaveanhydrophilicandhydrophobicside,allowingthemtoorganizeintolayers.Thetransmembraneproteinsactasbarrieragents,andexternalsensors.Insidethemembraneliesakeycellularelement:thenucleus.Thenucleusisonlyfoundineukaryoticcellsandisthemostspecializedorganelleofthecell.Thetwomajorfunctionsofthenucleusistostorethecell'shereditarymaterial,orDNA,andtocoordinatethecell'sactivities,suchasgrowth,intermediarymetabolism,proteinsynthesis,andcelldivision.Adouble-layeredmembrane,thenuclearenvelope,separatesthecontentsofthenucleusfromthecellularcytoplasm.Theenvelopeisriddledwithholescallednuclearpores.Theyallowspecictypesandsizesofmoleculestopassbackandforthbetweenthenucleusandthecytoplasm.Thespacebetweentheplasmamembraneandthenucleusislledbythecytoplasm.Itconsistsofcytosolandthecellularorganelles,exceptthecellnucleus.Thecytosolismadeupofwater,salts,organicmoleculesandmanyenzymesthatcatalyzereactions. 22

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Theinsolublecomponentsofthecytoplasmareorganelles(mitochondria,lysosomes,peroxysomesandribosomes),vacuoles,cytoskeletonlaments,andcomplexcellmembranestructures(e.g.,endoplasmicreticulumandtheGolgiapparatus)(gure 2-2 ).Theendoplasmicreticulummanufactures,processes,andtransportsproteinsthroughoutthecellusingribosomes.Membraneproteinsaredirectlyinsertedinthemembrane,whiletheremainingproteinsaretransportedtotheGolgiapparatus.There,theorganelleactsasthedistributionandshippingdepartment.Itmodiestheproteinsandfatsandpreparesthemforexporttotheoutsideofthecell.Thenucleuscancontroltheshapeandfeaturesofthecellbyinuencingthecytoskeleton.Thisdynamicstructurenotonlymaintainscellshape,butalsoenablescellmotion,andplaysimportantrolesinbothintra-cellulartransportandcellulardivision.Eukaryoticcellscontainthreekindsofcytoskeletallaments:actin,microtubulesandintermediatelaments.Theextracellularmatrix(ECM)surroundsandsupportscells.ECM'smaincomponentsareglycoproteins,proteoglycansandhyaluronicacid.Inanimals,themostabundantglycoproteinintheECMiscollagen.TheECMalsocontainsawiderangeofgrowthfactors,andactsasalocalreservoirforthem.Changesinphysiologicalconditionscantriggerlocalreleaseofsuchdepots.Thisallowsfortherapidandlocalactivationofcellularfunctions,withoutdenovosynthesis.TheECMfulllsanumberofotherfunctions,suchassupportandanchorageforcells,separationofthetissues,andregulationoftheintercellularcommunication.Asasummary,theECMregulatesacell'sdynamicbehavior.Inthehumanbody,eukaryoticcellsaredividedintofourdierenttypes:epithelial,connective,muscleandnervouscells.Epithelialcellscovertheouterandinnersurfacesofthebody.Theycanhaveglandularfunctionstosecretesubstancessuchasmucousandhormones.Epithelialtissuesvarycanbesimple(onelayer)orstratied(comprisedofmultiplelayers).Connectivecellshelptosupportandstructureothertissues.Theycan 23

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bearrangedintoavarietyofelements,rangingfromuid(suchasblood),exible(suchascartilage)orhardmatter(suchasbone).Musclecellsarecomposedofbersandenablemuscletocontract.Thehumanbodycontainsthreetypesofmusclecells:smooth,cardiac,andskeletal.Nervouscellsconsistmainlyofneurons.Theirroleistodetectstimuliandprovidetheappropriateresponsesthroughtheuseofelectrochemicalsignals.Ourworkdealswiththecolon,whichiscomposedofepithelialcells.Thefollowingsectionwillbringmoreinformationonthecellularfunctionofthisorgan. 2.2.1ColonCellsThecolonispartofthedigestivesystem,linkingthececumtotherectum.Itsprimarypurposeistoremovewater,electrolytes,vitaminsandaminoacids,lipids,andcarbohydratesfromfeces.Thecolonislinedwithalayerofsimplecolumnarepithelialcells.Thosecellsareabletowithstandabrasionwhileabsorbingessentialnutrients.Epithelialtissueshaveseveralinterestingcharacteristics.Cellsaretighly-packed,withverylittleintercellularspace.Theyareboundbyoneormoretypesofcelljunctions.Sinceepithelialtissuesareavascular,theygettheirnutrientsbydiusion.Theyalsohaveanhighrenewalcapacity;replacementisdonebycelldivision. 2.2.2ColonCancerCellsColorectalcancerusuallydevelopsoveraperiodofseveralyears.Thecolonoftendevelopsnon-cancerousgrowths,calledpolyps,onthelining.Thosegrowthshavethepotentialtodegenerateintocancer(gure 2-3 ).Oncecancerhasstarted,itspreadstowardintheinsideofthecolonwall.Fromthere,theycaninvadebloodorlymphvessels,givingthecellsthetravelingopportunityformetastasis. 2.3TheCellandItsEnvironmentThecell-surfaceinterfacehaslongraisedquestionsastoitsroleoncellfunctions.Intheearlyyears,thecellmechanosensingmechanismwasoftenignored,butresearchprovedthatthecell'senvironmentisakeyinuenceonitsproperfunctioning.Anumberof 24

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reviewsdealswiththetopic( VogelandSheetz , 2006 ; WangandThampatty , 2006 ; Dalby , 2005 ).Thecellcanrespondtoenvironmentalfeaturesfromthenano-tothemolecularscale.Inordertobetterunderstandtheinteractionbetweensurfaceroughnessandcellfunctions,researchprojectshavebeenconducted.Thefollowingsectionevaluatesanarrayofthoseexperiments'techniques,andtheirresults,accordingtocelltypes. 2.3.1StudyingtheEectsofSurfaceRoughness:MeasuringRoughnessTheeectofsurfaceroughnessoncellbehaviorisincreasinglyappreciated.However,roughnessinitselfhasyettobedenedandwhileanensembleofparametershavebeenusedincorrelationtosurfaceroughness,noguidelineshavebeensetonthesubject.Someofthemostcommonparametersareshowningures 2-4 and 2-5 anddescribedbelow. 2.3.1.1Height/DepthWhenreferringtoroughness,thearithmeticaverageroughness,Ra,isthemostcommonsurfaceparameter.Raroughnessisthearithmeticaveragelengthbetweentheroughnessproleanditsmeanlineevaluatedoverasamplelength,L(seegure 2-5 ).Itisderivedmathematicallyas:Ra=1 LRL0(r(x))dx.Inshort,itreectstheheightchangeonthesurface.Theeectsofheightarethemostdocumentedandrevealthattheeectsofheight/depthvarywithcelltype.Eectsofheightanddepthoncelladhesionhavebeenstudiedinnumerouscelltypes( Lampinetal. , 1997 ; Tezcaneretal. , 2003 ; Milleretal. , 2004 ; Yamakawaetal. , 2003 ; RanucciandMoghe , 2001 , Dalbyetal. , 2004 , RosaandBeloti , 2003 ).Whileitappearedatrstthatsomereportswereconicting,furtherinvestigationrevealedaninterestingbehavior.Thecellsappeartoreactbestatacriticaldepthdimension.Forexample,forosteoblastsonsurfaceswithfeaturesrangingfrom0.1to0.3minheight,theiradhesionwasbestbetween0.2and0.25m( Fengetal. , 2003 ).Similarly,neuralcellsshowedbetter 25

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adhesiononsurfaceswithRavaryingfrom20to50nm( Fanetal. , 2002 ).Fibroblastadhesionisbestonsurfaceswithfeaturesof10to24nminheight( Dalbyetal. , 2003 ).Asaresult,theconictsmentionedearliercouldbeexplainedbythefactthatthedepthrangestudieddidnotoverlapandmighthavebeenatdierentpointsofthecriticaldimension.Sensitivitytosurfacetopographyalsoappearstovarywiththecelltypes.Whileorientationhasbeendemonstratedtobemainlyaectedbyfeaturesdepth,resultsfromtwodierentmacrophagecelllinesshowedthattheP388D1celllinereactedtodepthsasshallowas30nm,whilemacrophagesfromratperitonealstartedreactingat71nm( Wojciak-Stothardetal. , 1996 ).Thisfurthersournotionofcriticaldimension.Depthhasalsoprovedtobeafactorinchangesofproliferation( Chungetal. , 2003 ),migrationarea( Lampinetal. , 1997 ),extracellularmatrixorganization( Anselmeetal. , 2000 ),motility( TanandSaltzman , 2002 ),cellalignmentandgeneexpression( Dalbyetal. , 2003 ). 2.3.1.2RoughnessWidthWhileroughnesshasmainlybeenstudiedasadepth/heightchange,theeectoffeature'swidthcannotbeneglected.Forexample,whengroovesarecreatedonasurface,roughnessneedstobecharacterizedasafunctionofthedepthofthegroovesbutalsoasafunctionofthewidthofthegrooves,and/orthewidthoftheridgesbetweenthegroovesorspacing.Widthhasnotbeencommonlystudiedandasaresult,veryfewstudieshavebeenperformedonthesubjectatthepresenttime.Intermsofroughnesswidth,experimentswithmacrophagesgrownongrooveswithwidthof2or10mshowedabetterorientationofthosecellsalongthenarrowgroovesthenthewiderones( Wojciak-Stothardetal. , 1996 ).However,myocytesgrownongrooveswithvaryingwidthshowednochangeinorientation( Motlaghetal. , 2003 ).Whilemoststudiesontheeectsofsurfaceroughnesswidthoncellsweredonebyobservingcellorientation,onewasdonewithcellmotility.Itfoundthatneutrophilshavebettermotilityongroovedratherthan 26

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atsurfaces.Itisalsointerestingtonotethatforridgeswithheightof5m,motilitywasbestwithroughnesswidthof10m,whileforridgesof3m,motilitywasbestwithroughnesswidthof6m.Thesedemonstratethatmotilityofneutrophilsisaectedbothbydepthandwidthofgrooves.Ingeneral,thecellsweremoreelongatedandconnedinnarrowergrooves(6m)thaninwiderones(14m)( TanandSaltzman , 2002 ).Fromthisinformation,orlackthereof,wecanonlyconcludethateachcelltypereactsdierentlytochangeinwidth. 2.3.1.3RoughnessSpacingAlongwithdepthandwidth,spacingbetweenfeaturesisacommonparameterusedforroughnesscharacterization.However,itwasevenlessstudiedthanroughnesswidthandasaresult,onlytheeectsofsurfacetopographyonmyocyteorientationandmacrophageadhesioncanbereported.Itwasfoundthatmyocytesdidnotshowanychangeinorientationwithvaryingroughnessspacing( Motlaghetal. , 2003 )andthatmacrophagesattachpreferablyonsurfacewithhighfeaturespacing( Riceetal. , 2003 ). 2.3.1.4RandomnessRecently,cellreactiontoorderedanddisorderedtopographieshasbeenquestioned. Bigerelleetal. , 2002 denedanewparametertodescriberoughness,describingitastheorderparameter.Thisparameterisscale-independentandcomparestheorganizationofasurfaceatdierentscalesofrangeandamplitude.Theauthorsfoundthatthelevelofadhesionofosteoblastsvariedlinearlywiththeorderroughnessofthesurface. Curtisetal. , 2001 gaveanotherapproachbycreatingseveraltypesoftopographydieringinregularity.Twotypesofsurfaceswerecreated:onewithpillarsarrangedinanorganizedmannerandonewithrandomdots.Theresultsshowedthatregulartopographyreducedcelladhesioninbroblasts.Aninterestingfactwasthatthefailureofattachmentonorderedsurfacewasreplicatedwithcarboxylatepolystyrenebeads.Theclosesimilaritybetweenthoseobservationsdemonstratesthatphysicalforcesratherthanchemicalonesareatwork. 27

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Thisparametertakesthecomprehensionofroughnesstoanotherlevel,asitdenesfeaturesin3dimensions,whilelookingonlyatdepth,widthandspacingallowsforalesscomplicatedandprobablytoosimplistic,2Dview. 2.3.1.5OtherFactorsInthestudyoftheeectsofsurfaceroughnessoncells,ithasoftenbeenquestionedwhethertheresultsweretheconsequencesofroughnessitselforduetochemicalchangesresultingfromthemanufacturingprocess.Manyexperimentswereperformedtoelucidatethequestion( Liaoetal. , 2003 ; Milleretal. , 2004 ; RanucciandMoghe , 2001 ),buttheresultsareinconsistent.Theimportanceoftopographyrelativetochemistrystillremainsunsolved.Inadditiontothepossiblechemicalinteractionsbetweencellsandsurfaces,onemustbemindfulofthemechanicalpropertiesofthesubstrateused.It'sexibilityhasbeenshowntoinuenceadhesion,migrationandproliferation( JrandWang , 1997 ; Wongetal. , 2003 ).Therefore,itisimportanttokeepinmindthatmaterialcomposition,structure,andprocessingallaectthecellularreactionsandthereforealterationsinanyofthesefactorscouldleadtochangesinmorethanonebiologicalprocess. 2.3.2StudyingtheEectsofSurfaceRoughness:ManufacturingTechniquesAvarietyofmethodshavebeendevelopedtoproducesurfaceroughness.Eachhavetheirstrengthsandweaknesses. 2.3.2.1ChemicalModicationSurfacemodicationthroughchemicalreactionisoneofthersttechniquesusedtocreatesurfaceroughness.Thismethodinvolvesdirectinteractionbetweenthechemicalandthesubstrateandcreatesfeaturesthroughremovalofthesurfaceattheinterface.Forexample,oxygenplasmatreatment,whichcombinestheactionofenergeticparticlesandvacuumultravioletradiation,onpoly-(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV8),resultsinfeatureswithdepthrangingfrom48.67to113.45nm( Tezcaneretal. , 2003 ).Thedepthofthefeaturesrelatesdirectlytothepowerandtimeofexposureduringthetreatment.Anothermethodistoexposepolymerstodierentconcentrations 28

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ofNaOHfordierentamountsoftime.Thapaetal.reportedusedthistechniqueonpoly-(lactic-co-glycolicacid)(PLGA)andpoly-(etherurethane)(PU)andobtainedsurfaceswithfeaturesofdepthrangingfrom50nmto15,000nm( Thapaetal. , 2003 ).Whilethistechniqueallowstotheproductionofawiderangeofdepth,itlackscontroloverwidthorspacingbetweenfeatures.Inaddition,itcreatestopographiesofrandomorientation.Thus,chemicalmodicationprovidescontroloveronlyoneparameter,depth.Furthermore,chemicalmodicationofthesurfaceleadstochemicalinteractionswiththecellsandinterferewiththeeectsoftopography. 2.3.2.2Photolithography,ElectronBeamLithography,LaserHolographyForbestpatternresolution,lithographiesarethemostaccurateandeectivemethod.Forthatreason,thistechniqueiswidelyusedontheeldofsurfacetopography.Theprotocolbeginsbythecreationofamaskwithadenedpattern.Aglassorsiliconsubstrateisthencoatedwithaphotoresist.ExposureofthisphotoresisttoUVlightthroughthemaskresultsinareliefshapedasthedesiredpattern.Thissubstratecanthenbeuseddirectlytoculturecells( Dalbyetal. , 2003 ; Wojciak-Stothardetal. , 1996 ; Fanetal. , 2002 ; TanandSaltzman , 2002 )orcanbeusedasamastermoldontowhichpolymersarecasttoreplicatethepattern( Liaoetal. , 2003 ; Motlaghetal. , 2003 ; Deutschetal. , 2000 ).Thistechniquegivesthebestresolutionsandallowsforcompletecontrolofthetopographiesproduced.However,itsresolutioninthenanoscaleislimited.Thetechniqueisquiteexpensiveandtimeconsuming. 2.3.2.3MechanicalTechniquesMachining,polishingorsandblastingasurfaceisthemostconvenientwaytocreateroughness.Polishedandsandblastedtitaniumresultsinroughnessdepthrangingfrom0.16to3.40m( Anselmeetal. , 2000 ).Sandblastedpoly-(methylmethacrylate)PMMAgivessurfaceswithroughnessdepthrangingfrom0.18to3.34m( Lampinetal. , 1997 ). 29

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Thoughreadilyavailableandeectiveincreatingaroughareawithoutaddingchemicalmodicationofthesurface,thistechniqueisnotidealtostudytopographyparameters.Itlackscontrolovertheroughness'width,spacingandrandomness. 2.3.2.4FilmDepositionThetechniquesdescribedaboveachievesurfaceroughnessthroughetchingoftheupperstratumofasurface.Filmdeposition,asitsnamestates,achievesroughnessbydepositionofpolymericlmorparticles. Riceetal. , 2003 creatednanoscaletopographiesbyadsorbingcolloidalparticlestotitaniumoxidesurfacesusingaluminiumchloridehydroxideasabinder.Anothermethodconsistsofdepositingathinpolymericlmonglasscoverslips.Oncethepolymerhasbeenmixedwithasolvent,itispossibletoeitherletthesolventevaporateunderafumehood,resultinginasmoothsurface,orfreezethepolymerassoonasdeposited,sublimatingthesolvent,resultinginsurfacecoveredbymicropores. RanucciandMoghe , 2001 usedpoly(DL-glycolic-co-lacticacid)onglasscoverslipsandcreatedsurfaceswithdepthof0.005mand0.277m.Anothertechnique,calledpolymerdemixing,usespartiallycompatiblepolymerblendsthatundergophaseseparationduringthespincoating.Concentration,proportionsandmolecularweightofthepolymersallinuencetheresultingtopographies.Forexample,PSandPBrSwereusedatdierentcompositiontocreateislandsofdierentheights(13,27,45and95nm)( Dalbyetal. , 2003 ; Dalbyetal. , 2004 ).Thistechniqueallowsfornanometerfeatureswithoutexpensiveequipmentandminimalcomplexity.However,controlofthedepth,widthandspacingisrudimentaryandrandomnesscannotbecontrolled. 2.3.3SurfaceRoughnessandMuscleTissueSeveralexperimentswereperformedwithmusclecells.Upontestingwithmicrogroovesofdierentfeaturesizes,cellsshowedbetterorientationthanoncontrolsurfaces,withoptimumorientationongroovesofwidth10um,separationof10manddepthof5um( Deutschetal. , 2000 ; Motlaghetal. , 2003 ).Andwhilesynchronousbeatingofrat 30

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myocyteswasnotaectedbychangeoftopographies,expressionofN-cadherinandconnexin43inthosesamecellswasclearlyaectedbythegroovedepth(highestat5mdepthcomparedtocontrolsurfacesand2mgrooves).Whenthosecellswereseededonmicropegs,theirmorphologyborecloserresemblancetoin-vivomorphologythanoncontrolsurfaces,withanincreaseinheightandadhesion( Deutschetal. , 2000 ).Whenthefeatureswerereducedtothenanoscale,cellsshowedbetteradhesionanddensityincreasedonnanostructuredsurfacescomparedtocontrolsurfaces( Thapaetal. , 2003 ; Milleretal. , 2004 ).Wearethenabletosummarizethatmusclecellsorientationaswellasn-cadherinandconnexinexpressionareaectedbymicrogroovepatterning.Micropatterningalsoresultsincloserresemblancetoin-vivomorphologyandnanopatterningincreasesadhesionandproliferation(gure 2-6 ). 2.3.4SurfaceRoughnessandNervousTissueConictingresultshavebeenreportedontheinuenceofsurfaceroughnessonnervecells.Itwasshownthatwhenseededonpillarswithheightof1mwithdiametersrangingfrom0.5to2mandinterpillargapsvaryingfrom1to5m,cellularadhesionincreasedcomparedtosmooth-etchedsurfaces.Similarly,on1-m-deepwellswith.5mdiameterandseparationvaryingfrom0.5to2m,cellularadhesionincreasedcomparedtosmoothetchedsurface( Turneretal. , 2000 ).Butwhenseededonsurfaceswithdierentroughnessvalues(Ravaryingfrom0.23to1.98m),cellattachmentdidnotvarybetweensurfaces.Similarly,cellproliferation,totalproteincontentandALPactivitywereunaected( RosaandBeloti , 2003 ).Whenthesurfacesweremodiedinthenano-scale(Rarangingfrom0.002to0.81m),itappearsthatthecellviabilityandadherencewerenegativelyimpactedbyroughnessbelow0.01mandabove0.07m,whilecellswereabletoadhereandsurviveonsurfacewithroughnessrangingfrom0.02to0.05m( Fanetal. , 2002 ).Thoseobservationsaresummarizedingure 2-7 . 31

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2.3.5SurfaceRoughnessandEpithelialTissueTheepitheliumplaysacriticalroleinourbodydefensesystemasitisindirectcontactwiththeoutsideenvironment.Inaddition,itisalsothemostcommontypeoftissueincontactwithimplants.Asaresults,itisacompellingareaofresearchintermsofreactiontosurfaceroughness.Epithelialcellsseededongrooveswithvaryingpitches(0.4to4m)showedthatalignmentwasgreateronpatternedsurfaces( Teixeiraetal. , 2003 ).Andwhiletheproportionofcellsalignedwasequalonallpatternedsurfaces,testingunderowrevealedthatthecellsonthe0.4msurfacesadheredmoretightlythanonthelargerpatterns( Karurietal. , 2004 ).Adhesionhasrevealedtobeaninterestingparameter,asinthemicroscale,attachmentwasbestonthesmoothersurfaces(forsurfaceswithmeanroughnessvaryingfrom0.04mto0.113m)( Tezcaneretal. , 2003 ).But,inthenanoscale,itwasbestontheroughersurfaces(forsurfaceswithRavaryingfrom0.001to0.040m)( Chungetal. , 2003 ; Milleretal. , 2004 ).Thoseresultswereparalleledwithsurfaceeectonproliferation.Celldensityonnanostructureswasobservedtoincreasefasterthanonmicrostructures( Milleretal. , 2004 ).WhenculturedonexactreplicasofECM,withtypicalfeaturesheightof0.001moccurringoverlateraldistancesofonlyafewm,thecellsspreadmorerapidlyonthereplicasthanoncontroluntexturedsurfaces.Inaddition,theirmorphologyandspreadareaatconuenceweremoresimilartotheonesobservedin-vivo( Goodmanetal. , 1996 ).Asaresult,itcanbeconcludedthatadhesionofepithelialcellsisbestonpatternswiththenanofeatures.Similarly,sodoesproliferation,spreadingandmorphology(gure 2-8 ). 2.3.6SurfaceRoughnessandConnectiveTissueTheconnectivetissue,suchasbone,blood,lymph,tendonsandligaments,playsamajorroleasthesupport,defense,nutritionalandstoragesystemsoftheorganism.Asaresult,anyalterationinthecellfunctionsofthosetissuescanbethesourceofnot 32

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onlymajordiscomfortbutalsodeclininghealthforanindividual.Itisthenimportanttofurtherourknowledgeontheeectofsurfaceroughnessonthosetissuesinordertopreventorcureanyresultingdeciencies.Whenneutrophilswereseededontosmoothandroughenedpolystyrene,theirreactionwasquitecontrasted.Whiletheneutrophilsonthesmoothsurfacesdidnotshowanydamage,theonesontheroughenedpolystyrenediedrapidlyafteradhesion( Changetal. , 2003 ).Whenplantedonglasssurfacewithridges,itappearedthattheirmotilitydirectionwascontrolledbythetopography.Thoseexperimentsalsoshowedthiswastheresultofboththechangeinspacingbetweenthegroovesaswellasthechangeindepth.Velocitywashighestongrooveswith10mspacingandthemotilitydecreasedwithdecreasinggroovedepth.( TanandSaltzman , 2002 ).Whenmacrophageswereseededonarraysofgroovesandridges,severaleectswereobserved.Orientationofcellsincreasedwithincreasingdepthanddecreasingwidth.Whengroovesweredeeperthan0.071m,therewasanincreaseinspreadarea.Bothadhesionandphagocytosisactivitywereincreasedwhencellswereonmicropatternedsurfaceandbothshoweddependenceonthedepthofthefeatures,butnotthewidth.( Wojciak-Stothardetal. , 1996 ).However,whenseededonsurfacescoatedwith0.107mdiameterparticlesatdierentdensities,nostatisticaldierencewasnotedfortheviablecellsadherenceonallsurfaces( Riceetal. , 2003 ).Whenlymphocyteswereseededonplasma-treatedglasswithequallyspacedarraysoflines,itappearedthattheirmotilitywasmorediusiveandlesspersistantindirectiononthesmoothsurfacesthanonmicropatternedglass.( Melloetal. , 2003 ).Osteoblastsseededon0.107mdiameterparticlescoatedsurfacesdidnotshowanydierenceinadherenceonsurfaceswith3,19,30and43%coverage.( Riceetal. , 2003 ).Furthertestingonsurfacesthatwereeithersmooth,sandblastedorplasma-treatedshowedthatcellproliferationwashigheronsmoothsurfaces,whilesynthesisofextracellularmatrixwasmoreabundantinsandblastedandplasma-treatedsurfaces.Inaddition, 33

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analysisofintegrinreceptorsandalkalinephosphataseshowedthatroughsurfacepromotedcelldierentiation.However,cellapoptosisdidnotshowanydierenceonallsurfaces.( Postiglioneetal. , 2003 ).Andwhileitappearsthatroughsurfacesimprovecellproliferationanddierentiation,itwasalsoshownthatitimpairedcellspreadingforatleasttherst24hafterattachment.( Luthenetal. , 2005 ).VascularandcornealexplantcellsweregrownonPMMAsurfacewithRarangingfrom0.07to3.34m.Theresultsshowedthatbothmigrationareaandcelladhesionincreasedwithroughness.( Lampinetal. , 1997 ).WhenspleniccellsweretestedonPMMAsurfacewithRarangingfrom0.001to0.014m,itappearedthattheiradhesionincreasedwithroughness.( Yamakawaetal. , 2003 )FibroblastswereseededonglasssurfaceswithPS/PBrSislandsofdierentsizes.Frommultiplesexperiments,itwasfoundthatislandsof0.013mresultedinanincreaseofinitialadhesion,long-termadhesionandcystoskeletonwhileislandsof0.024malsoshowedanincreaseofinitialadhesion,butadecreaseinlong-termadhesionandreducedcytoskeleton.Islandsof0.035mshowednochangeintheinitialadhesionbutareducedcytoskeleton.Islandsof0.095mresultedindecreasedinitialadhesionandreducedskeleton( Dalbyetal. , 2004 ).WhentestedonislandsofPS/PnBMAwithsizerangingfrom0.01to0.05m,broblastsdidnotshowanydierenceinattachment,spreadingorskeletaldevelopmentonthe0.01islandscomparedtoplanarsurfaces.However,onthe0.05mislands,thecellsdidnotattachandgrewverypoorly( Dalbyetal. , 2003 ).Cornealbroblasts,orkeratocytes,seededonarraysofridgeswithvaryingfeaturedimensionsshowedthatthecellsalignedalongthepatterns.Theorientationofthecellswassignicantlyloweronfeaturesof0.4mthanforpatternswithsizerangingfrom0.8to4m.Intermofadhesion,cellsadheredsignicantlylessonfeaturesof0.4msizethanonsmoothsurfacesandonfeaturesof4m.Cellelongationwassignicantlyhigher 34

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onpatternsthanonsmooth,withnodierencesamongthepatterns.( Teixeiraetal. , 2003 ).Osteoblast-likecellswereseededontoPDMSatsurfacesandPDMSsurfaceswithpyramidswith33mbaseand23mheight.Itwasfoundthatthetopographyclearlypromotedosteoblastdierentiation( Liaoetal. , 2003 ).OntitaniumalloysurfacewithRavaryingfrom0.16to3.40m,itwasobservedthatcelladhesionandcellproliferationwasimpairedonroughersurfaces.Inaddition,theextracellularmatrixwasbetterorganizedandorientedonsmoothcomparedtoroughsurfacesandconuencewasneverreachedonroughsurfaces( Anselmeetal. , 2000 ).However,whenplantedontitaniumsurfaceswithRavaryingfrom0.09to0.3m,roughenedsurfaceswerefavorableforattachmentanddierentiationoftheosteoblasts( Fengetal. , 2003 ).Randomnessofmicropatternedtitaniumandtitaniumalloysurfaceswasassessedinordertoevaluateitsinuenceonadhesionofosteoblasts.Experimentsshowedthatthisadhesionwastwicehigherondisorderedsurfacesthanonorderedones,revealingtheimportantofrandomnessinsurfaceroughness( Bigerelleetal. , 2002 ).Thoseobservationsaresummarizedingure 2-9 . 2.3.7ConclusiononSurfaceRoughnessFromthissummarizedreview,wecanestablishthatsurfaceroughnessdoesaectcellfunctionsandthatitseectsvarygreatlywithcelltype.Itwouldthenbeinterestingtoevaluateifextracellularsurfacetopographyisalteredincancer,whichwouldthenleadtoaectsurroundingcells.Couldwethenestablishapossiblecauseofangiogenesis?Whilethishypothesisisattractivebyitsapparentsimplicity,wemustrememberthatthereviewalsoestablishedawidearrayofcellfunctionaectedbythetopographicalparameters,hintingtothehighcomplexityofthemechanismlinkingtheextracellularsurfacefeaturesandcellreaction. 35

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2.3.8VariationintheExtracellularEnvironmentanditsRoleinCancerThebreakdownoftheextracellularmatrixisantypicalhallmarkofcancer.Thisestablishedobservationallowspathologiststosecureacancerdiagnosis,buttheexacttopographicalinformationhasyettobemeasured.TherstlookintotheexactstructureofatumorousECMisin Andersonetal. , 2006 article,wheredatafromAFMexperimentsshowthestructureoftheECMdepositedbycoloncells,normalandcancerous(gure 2-10 ).WhilefewstudiesdoevaluatethetopographyofECMincancer,alargeramountdealswithsurfaceroughnessandangiogenesis.Fromearlyresearch,ithadbeenestablishedthatimplantedmaterialsofanychemicalcompositioncouldresultintumors,aslongastheypossessedasmoothsurface( BatesandKlein , 1966 ).Furtherexperimentswereperformedthroughtheimplantationoflterswithdierentporessizes( IomhairandLavelle , 1997 , Ferguson , 1977 , RosengrenandBjursten , 2003 ).Itwasestablishedthatthecarcinogenicityoftheimplantsdecreasedasthesizeoftheporesincreased( Ferguson , 1977 ).Whenlterswith0.025,0.05or0.1mporesizewereimplanted,theywereobservedtobetumorigenic.However,whenlterswithporessizeof0.22or0.45mwereimplanted,notumorsdeveloped.Itwasalsoreportedthatthetotalsurfaceareaofthelteraectedthecarcinogenesisofthelters.Whenthesurfaceareawasincreasedfrom0.98mm2to2.95mm2onlterswiththesameporesize,tumorsdevelopmentwentfrom0to16.Thus,itwasconcludedthatsurfaceareawasdominantoverporesizeinlmsarcomagenesis( IomhairandLavelle , 1997 ).Itwasalsoobservedthatwhen0.6mporesizelterswereinserted,fewinammatorycellsenteredthelterandalargeforeignbodycapsuleformationwaspresent.Ontheotherside,when10-and30mporesizelterswereimplanted,largeamountsofmacrophageswerefoundinsidethestructure,andfewinammatorycellswerefoundoutsidethelters.Thecapsulesonbothoftheltersweresignicantlythinnerthanforthe0.6mlters,thusshowingaclearrelationshipbetweenporesizeandtissueorganization( RosengrenandBjursten , 2003 ).Whilethose 36

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experimentsevaluatedtherelationshipofsurfaceroughnesswithcancerdevelopment,itdidnottreattheeectofthesurfaceroughnessonthecellitself.Onestudyevaluatedthemotilityofnormalandmalignantlymphocytesonsurfaceroughness.Theyfoundthatmalignantlymphocytesweresignicantlyslowerandlessdiusiveonplanesubstratesthanongroovedwhilenormalcellsonlyshowedamoderatedropinspeedanddiusionbetweenthetwosurfaces( Melloetal. , 2003 ).Inaddition,itwasobservedthatmalignantcellswouldaligninthedirectionofthegrooveswhilenormalcellswouldnot.Thoseresultshaveclearlydemonstratedthatsurfaceroughnessaectscarcinogenesis.Itwouldnowbeinterestingtofurtherourunderstandingofsurfaceroughnessroleincancerdevelopment.Byvaryingcelltypesandsurfaceroughnessfactors,wecouldbetterassesstheinuenceofroughnessoncancercelldevelopments. 2.4Intra-Extracellularinterface 2.4.1TheOutside-InMechanismComplexnetworksofmoleculesenablecellstoreceivesignalsfromtheirenvironmentandtomodifytheirbehaviorinresponsetothesestimuli.Manysignalsarereceivedatthecellsurfacebyadhesionmoleculesandotherreceptors,suchasreceptortyrosinekinasesandG-protein-linkedreceptors.Cascadesofintracellularmoleculesthentransmitthesignalsbyinuencingeachotherthroughdirectbindingandthroughmodicationssuchasphosphorylationandproteolyticcleavage.Signalscantravelfromthecellsurfacetothenucleus,wheretheyultimatelyaectproteinsynthesis,ortothecytoskeleton,wheretheychangecellshapeandmotility.Itisthroughthesemechanismsthattopographyisabletoaectcellfunctions. 2.4.2TheInside-OutMechanismWhileacellresponsetoextracellularsignalsisachievedthoughtheoutside-inmechanismexplainedearlier,asimilardynamismregulatesthecellreactiontointracellularsignaling.Inside-outsignalingcanbeobservedwithintegrins,transmembraneproteinsthatregulateadhesion.Intheirrestingstate,integrinsnormallybindwithlowanity 37

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tothemoleculesthatactivatethem.Uponintracellularstimulation,asignalinducesaconformationalchangeintheintegrincytoplasmicdomain.Integrinsaretransformedfromalow-toahighanityligandbindingstate( Qinetal. , 2004 ).Thisinside-outmechanism,whichrelatesmainlytoplateletsandleukocytes,explainsthenon-adhesionofthosecellsduringtheirnormal,restingstateandtheirsuddenattachmentabilityonceactivated. 2.5Cellularfunction:adhesionOurbodyiscomposedofbillionsofcells,organizedinorgansandtissues.Inordertoaccomplishthehighlyspecializedfunctionofthoseorgans,cellshaveevolvedintonumerousdistinctcelltypes.Thosecelltypescanbedierentiatedbytheircharacteristicfunctionalproperties,beitcellgrowth,migration,spreadingormorphology.Whileallcellfunctionshavetheirimportance,thisworkdealsprimarilywithcelladhesion.Thefollowingsectionthusdealwiththistopic.Celladhesionismediatedbytransmembraneproteinscalledcelladhesionmolecules(CAMs).ThereexistfourmajorgroupsofCAMs:theselectins,theintegrins,theimmunoglobulin(Ig)superfamily,andthecadherins.IntegrinsplayaroleintheECM-celladhesion,whilecadherinsaremoreinvolvedinthecell-celladhesion.Bothadhesiontypesarepresentedbelow. 2.5.1Cell-cellAdhesionIncolonepithelium,cell-celladhesionisessential,duetoitsimportancewithtissueorganization.Epithelialcellsadheretightlytoeachother,andseveralspecializedadhesiveelementsensuretheintegrityandstrengthofepithelialsheets.Theseadhesivestructuresactasjunctionsbetweentwocellsandcanbeoftwosorts:intermediatelaments(desmosomes)ormicrolaments(adherensjunctionsandtightjunctions).Desmosomesactasananchoringplatform,throughastructurecalledcytoplasmicplaque.Cadherins,atransmembraneprotein,attachtothisplaque,extendthroughthemembraneandbindstronglytocadherinscomingthroughthemembraneoftheadjacentcell.Inthecaseofadherensjunction,theacceptedmodelisthattheylinkactinfromthe 38

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cytoskeletalnetworkofneighboringcellsthroughdirectinteraction.Thisassociationwiththecytoskeletonisessentialforthestabilityofcellcelladhesionandtheintegrationofcellcellcontactswiththemorphologicalchangescharacteristicofepithelialcells(forexample,theircuboidalcellshape). 2.5.2Cell-SurfaceAdhesionCellattachestoanextracellularsurfaceatpreciseadhesionpoints,FocalAdhesions.Thosearegiantproteincomplexes,containingasmuchas50proteins,usuallystructuralandcytoskeletalproteinsbutalsosignalingmolecules.Themajorcellsurfacereceptorsforextracellularmatrixmoleculesareintegrins.Thosetransmembraneproteinsnotonlyattachesthecelltoitsextracellularenvironment,butalsoactasasignaltransducer.Asaresults,integrinsplaycriticalrolesinavarietyofbiologicalprocesses,suchascellgrowth,division,survival,cellulardierentiationandapoptosis. Figure2-1. Stepsinvolvedinthemetastasisprocess. 39

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Figure2-2. Overviewofcellularstructures Figure2-3. Morphologychangecanbeobservedwiththeprogressionofcoloncancer(ImageAnalysisofExtracellularmatrixTopographyofColonCancerCells,R.Anderson,E.Anderson,L.Shakir,S.Glover.2006.CopyrightJohnWiley&SonsLimited.Reproducedwithpermission). 40

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Figure2-4. Exampleofparametersofsurfacetopography. Figure2-5. Theaverageroughness,Ra,isthearithmeticaverageoftheabsolutedeviationsfromthemeansurfacelevel.Itisrepresentedherebytheshadedarea. 41

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Figure2-6. Eectofsurfaceroughnessonmusclecells.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. Figure2-7. Eectsofsurfaceroughnessonnervouscells.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. 42

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Figure2-8. Eectsofsurfaceroughnessonepithelialcells.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. Figure2-9. Eectsofsurfaceroughnessonconnectivetissue.Roughnessisdenedastheoverallscale(nanoscaleormicroscale)oftheroughnessfactor,Ra. 43

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Figure2-10. Alterationoftheextracellularmatrixatdierentstageofcoloncancer(ImageAnalysisofExtracellularmatrixTopographyofColonCancerCells,R.Anderson,E.Anderson,L.Shakir,S.Glover.2006.CopyrightJohnWiley&SonsLimited.Reproducedwithpermission.) 44

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CHAPTER3BIOMECHANICALBACKGROUNDInthepastdecades,interestsinmoremathematicalandphysicalapproachofbiologyhasincreased,givingrisetonewelds,suchascellularmechanics,tissueengineeringorneurocomputing.Thefollowingsectionsseektoexplainthedierentdevicesandtechniquesthathavebeenusedtoestablishthebehaviorofcellsinuidowandcelladhesion. 3.1Biomechanicalmanipulationsofcells 3.1.1MicromanipulationMicromanipulationenableustostudytheeectofforcesonasinglecells.Themostcommonandoldesttechniqueisthemicropipetteaspirationofcells.Thepressureappliedatthetipofthepipettecanbedirectlytranslatedintoaprecisepullingforce.Bypeelingadherentcellsfromsurfaces,itisthenpossibletoobtaininformationontheiradhesionforce.Inaddition,anothersetupallowsthestudyofcell-celladhesionbyplacingtwocells,semi-aspiratedbymicropipettes,intocontactandthenpullingthemapart.However,thistechniquepresentsseveralweaknesses.Theaspirationforceappliedtotheadherentcellisplacedatanangle,whichcomplicatestheanalysis,asanasymmetricforcewillresultinunevenforceonthecell.Also,thesemi-aspirationofcellinthecell-celladhesionstudiesisanon-trivialfactor,asithasbeenshowntoinuencecell'sbehavior.Amorerecentapproachistheuseoflasertweezers.Inthistechnique,cellaretrappedbythepressureforceoflaserradiationandpulledothesurface.Whilethoseexperimentsprovidesensitive,real-timeforcedisplacementmeasurements,theupperrangeoftheachievableforce(fewnN)istoolowtoachievecelldetachment.Morerecently,microplatesweredevelopedtostudysinglecelladhesion( Thoumineetal. , 1999 ).Acellissandwichedbetweentwoverythinplates.Theplatesarethenpulledapart,andtheirdeformationasthecellpullsawayfromoneoftheplatescanbe 45

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relatedtothestrengthofadhesionofthecell.However,theactualset-uponlyallowstostudyshort-termadhesion,asitisimpossibletomovetheset-upfromamicroscopeplatformintoanincubator. 3.1.2CentrifugationCentrifugeassayshavebeenusedtomeasurecell-cellandcell-substrateinteractionssincetheearly1980s( McClayetal. , 1981 ).However,thetechniquealsopresentedmanydisadvantages.Theassaysarelimitedtoshorttermadhesionandonlysupportonesingleforceperrun,whichistypicallysmall( GarciaandGallant , 2003 ).Inearlycentrifugalassayprocedures,thecellswererstlabeledwithuorescentandthencentrifugedontothecellmonolayerorsubstrate,usuallyinamicrotiterplateorcomparableholder.Theplatewastheninvertedandcentrifugedagaintopullthelabeledcellsawayfromthemonolayer.Followingthistheplatewassealedinaninvertedpositioninethanol/dryiceandexamined( McClayetal. , 1981 ).Theexperimentswereruninthecentrifugefor10minutesatasinglespeed,600,1,200,1,8002,400or2,750rpm( Channavajjalaetal. , 1997 ).Inlaterexperiments,holderscontainednumerouswellsthattestedmultiplesubstratesduringoneexperimentalrunandtheprocessofusingethanol/dryicewaseliminatedandinsteadcellcountwasdeterminedwithaninverteduorescentmicroscope( Lietal. , 2002 ).Whilethissimpliedthecentrifugeprotocol,manydisadvantagesremained.Theholdersavailableonlyallowedobservationofsmallcellpopulationsandsingleexperimentalforceapplicationresultedinlargenumberofexperimentalrunsandpossiblecomplicationsinvolvedwithmultiplepopulations. 3.1.3FluidShearSystemsApplyingashearforceappearsascommonsense,sinceitisamostobviousforceexertedoncellsfrombloodow.Awiderangeofcellularphenomenaareaectedbyuidshear,includingintegrin/focaladhesions,proteinsignalingandcytoskeletalremodeling( Davies , 1995 ).Severalapparatihavebeendevelopedanddesignsvarygreatlyfromonelaboratorytotheother. 46

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Thecone-and-platesystem( Jr , 1984 ; Gloveretal. , 2004 )consistsofarotatingconeheldperpendicularlytoaatsurface.Theuidvelocityandseparationbetweentheplateandconevarylinearlywiththeradialposition.Thecongurationallowsaspatiallyhomogeneousuidshearstressontheatsurface.Alargerangeofshearstresscanthereforebeappliedontheadherentcells.However,directvisualizationofthecellsischallenging,andthesetuprequiresalargeamountofuid.Theparallelplateowchambercontainsasubstratesurface,wherecell,particleorbacteriaareobservedastheyattach/detach.Thesurfaceismostcommonlyglassorpolymerssuchaspolystyrene( LawrenceandSpringer , 1993 , 1991 )orpolydimethylsiloxane(reviewedin SiaandWhitesides , 2003 ).Thesubstratesurfaceissubjectedtoaow,createdmechanically( McCannetal. , 2005 )orbyelectropheresis.Themajorityofthedevicesaredesignedaschannelsofconstantcrosssection,producingasetvalueofshearstress.Thesizesandaspectratiosofthechambersvarygreatly( LevesqueandNerem , 1985 ; Hungetal. , 1995 ; Tsengetal. , 1995 , Usamietal. , 1993 ).Theparallelplatetechniqueoersmultiplepracticaladvantages:minimaltraining,easeoffabrication,smallvolumetricuidrequirement.Specialversionofshearowdevicesincludetheradialowchamber,alsocalledspinningdisk.Theradialowchamberusesaxisymmetricradialowoveracross-section,thusresultinginadecreasingshearstresswithincreasingradialposition.However,theset-uphasalargetestingarea,requiringalargeamountofmaterial. 47

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CHAPTER4MATERIALSANDMETHODS 4.1CellCulture 4.1.1DescriptionAllcellsareagiftfromDr.SarahGlover,UniversityofIllinoisatChicago,Chicago,IL.NCM460isanonmalignanthumancolonicepithelialcellline.Thesecellswillbeconsideredasourcontrolcellsasopposedtothosefromthecoloncancercellline,Caco-2.Thecellsareculturedat37Cand5%CO2inOpti-MEMcontainingappropriatemedia.NCM460cellsareculturedinHam'sF-12supplementedwith20%heat-inactivatedfetalbovineserum,insulin(0.5U/ml),hydrocortisone(0.4g/ml)andglutamine(0.58g/ml).Caco2cellsareculturedinDMEMsupplementedwith10%fetalbovineserum(FBS).Cellsareseededinthedetachmentassays.Forshort-termadhesion,theensemblewasincubatedfor30minutes.Forlong-termadhesion,theywereincubatedatleast4hourspriortotheexperiment.Theamountoftimeforcellattachmentwasdecidedaftertheworkpublishedby Gallantetal. , 2005 .Intheirresults,GallantandGarciaestablishedthatadhesionstrengthofcellsincreasesduringtherstfourhoursofattachment,andthenplateaus.Asaresult,weestimatedthatattachmentperiodshouldbeoverfourhours. 4.2Surfacepreparations 4.2.1PatternsAsexplainedearlierinthebackgroundsectionofthisproposal,topographyhasbeendenedbynumerousparameters.Wehavechosentosimplifyourviewoftopographytoa2-dimensionalviewandthustouseamicrochannelpatternwithmainparametersofconcernaswidth,depthandspacing.Inordertoassesstheeectsofeachofthosevariables,thesurfaceswillconsistofmicrochannelswithvaryingwidth,depthandspacing 48

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(asshowningure 4-1 ).Seriesofsurfaceswillbetestedsothatonlyoneparametervaries,thusisolatingit.Sincethewidthofanadheredcellhasbeenestimatedat10m,thewidthandspacingofthefeatureshavebeenchosenaroundthatsize(3,5and10m).Depthwassetat2m.Microchannelswillbedescribedinthisworkas(width)(spacing)(depth),thereforea1052microchannelrefertoamicrochannelwithfeaturesof10mwidth,5mspacingand2mdepth.Thelistofplannedmicrochannelspatternsissummarizedintable 4-1 . Table4-1. dimensionsoftherectangularpattern Width(m)Spacing(m)Depth(m) 5525325102352105210511054 4.2.2FabricationThepatternsofthemicrochannelsarereplicatedonsiliconwaferthroughphotolithography.Thewafersarethenusedasatemplateforreplicationwithpoly(dimethylsiloxane)elastomer(PDMSe)andwithKratonG1657R.PDMSewaschosenduetoanumberofitscharacteristics.Itisacastablesiliconrubberwithexcellentresolution.Itisopticallyclearandbiocompatiblebothinvitroandinvivo( BelangerandMarois , 2001 ).KratonG1657Risaclear,high-performanceelastomer,withhigh-resolutionreplicationcapability.SinceKratonandPDMSedonoattachtoeachother,weareabletouseKratontocreatenegativemoldsfromthesiliconwafer.CastingPDMSeonitprovidesuswithinversepatternsfromtheonesmadefromthesiliconwaferpatterns.ThoseproceduresweredonewiththehelpofJamesSchumacheroftheUniversityofFlorida. 49

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4.3GradientShearFlowChamber 4.3.1PurposeInordertostudytheeectsofowandshearstress,cellshavebeenstudiedwithaparallelplateowchamberorarheoscope.However,inthosesystems,theshearstressinthewholeeldisconstantanddependentontheowrate.Asaresult,tostudyvariousshearstresses,onemustchangetheowrate.Thiscombinationofowandno-owperiodswilloftenaectthecellreaction.Inordertoeliminatethisinconvenience,wehavedesignedaGradientShearFlowChamber(GSFC),whichallowsustocreateeldsofvariousshearstresswiththesameowrate. 4.3.2DesignThechangeinareaoftheGSFCresultsinauidvelocitychange,thusleadingtoshearstressvariation.Thedesignisshowningure 4-2 .Thew1andw2dimensionsweresetbymaterialandvisualizationlimitation.Theminimalwidthsizeachievablewassetastheinnerdiameterofthetubingusedtollthechamber,thus1mm.Thelargestwidthrecordableinthemicroscopeusinga4xobjectiveis1.5mm.Wedecidedtoonlyrecordthemiddlesectionandtosetthemaximalsizeas3mm. 4.3.3FabricationandCellAdhesionTheGSFCchamberwasconstructedusingPDMSe.Thedesignwasprintedandcutoutfromtransparencypaper.Thethicknessofthetransparencywasmeasuredas0.15mm.Oneachendofthechannelpattern,ashortpieceoftubingwaspositioned.PDMSwaspouredonandaroundthetransparencytocreatethechannel.PDMSewasalsopouredonthemoldofthesurfacepatterndesired,tocreatethebottompiece.Allpieceswereplacedonanoven,preheatedto200F.After30minutes,thePDMSeisalmostcompletelycured.Thechannelpieceisseparatedfromthetransparencyandthebottompieceisremovedfromthemold.Botharethencombinedandplacedbackintotheoventonishcuring.Astheycure,bothpiecesadheredtoeachother.ThisprotocolisshowninFigure 4-3 .Oncethechamberwascompleted,itwassterilizedbyllingitwithethanol 50

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andplacingitunderUVlightforatleastanhour.ThechamberwasthenlledwithOpti-MEMovernight.Thenextday,cellsweredetachedfromtheircultureaskswithtrypsinandresuspendedinmedia.Forshort-termadhesionassay,thesolutionistheninsertedinthechannelandplacedina37Cenvironmenttoattachfor30minutes.Long-termadhesionassayspresenteddiculties,discussedinchapter6,whichresultedinmodicationsinthemanufacturingandexperimentalprotocol.ThePDMSchannelpartandthebottompartwerelefttocureentirelyseparately.BothwerethensterilizedbycleaningthemwithethanolandplacingthemunderUVlightforatleastanhour.ThebottompartwasthenplacedinapetridishandsubmergedinOpti-MEMovernight.Thenextday,cellsweredetachedfromtheircultureaskswithtrypsinandresuspendedinmedia.Thesolutionisthendepositedinthebottompartandlefttoattachforover4hours.Thetop(channel)PDMSisthenplacedonthebottom,andsandwichedbetweentwopieceofplexiglas,heldtogetherby6screws.Thisfabricationprotocolissummarizedingure 4-4 . 4.3.4ShearStressCalculationsDuetothefactthatthechannellengthismuchlargerthanthemaximumwidthanddepthofthechannel,andthatthemaximalwidthismuchlargerthanthedepthofthechannel,weareabletoconsidertheowas2-dimensionalandtheshearstressatthewallalongthecenterlineisderivedasshowninappendix B by: w=6Q h2 2(w1w1w2 Lx)(4{1)Thehydraulicdiametersofarectangularductwithadepthof0.15mmandawidthof1and3mmrespectivelyarethen0.26and0.28mm.Thesediametersareequivalenttothoseofarteries.Asaresult,theshearstressesthatwearestudyingarephysiologicallyequaltothoseofhumancoronaryarteries:10to20dynes/cm2( Fung , 1993 )forlong-termadhesion. 51

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InordertoconrmtherangeofshearstressintheGSFCatvaryingowrate,weusedtheAutomaticDynamicIncrementalNonlinearAnalysis(ADINA)system.ToensurelaminarowintheGSFC,theentrancelengthwasestablishedfromtheowrates.Allcalculationsrelatedtothisdesign,aswellasatableofshearstressesachievableinthisGSFCareavailableinAppendix B . 4.3.5OperationThesetupisplacedontheAxiovert100MicroscopeconnectedwithadigitalCCDcamera,amonitor,andaJVCHR-VP780UVHSrecorder.Thetubingportsareconnectedtoasyringepump(KDS100,FDScientic,Holliston,MA),usedtocreatethesetow.Viewingareasaremarkedtorecordcellsinthechannelatwidthof1,2and3mm.Thevaryingwidthofthechambercreatesashearstressgradientinthedirectionofthechannel.Thisgradientisimportantovertheentirelengthoftheowchannel,butneedstobeminimalintheviewingarea.Usinga4xobjective,theviewingareaconsistsofa117.16x79.67mrectangle.Usingthesimpliedequationforshearstress: w=6Q h2w(4{2)themaximalvariationintheviewingareaisfoundtobe2.3%andisconsiderednegligible.Forshort-termadhesion,therecordingsaremadebeforeandatthestartoftheowandafter5minutesofow.Forlong-termadhesion,recordingsaremadebeforeandatthestartoftheowandafter10minutesofow. 4.3.6DataAcquisitionAllimageswererecordedonVHStapes.AMatroxRT2500videocapturecard(PinnacleSystems,MountainView,CA)wasusedtodigitizevideorecordingsfromtherecorderintoaPCcomputerusingtheAdobePremiere4.0(Adobe,MountainView,CA). 52

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4.3.7DataAnalysisFramestakenbeforeandduringtheexperimentsweresuperimposedusingTheGIMPimageprocessingprogram( http://www.gimp.org ).Attachedcellsarecountedineachviewingareaatthestartoftheow.After5minutesforshort-termadhesionand10minutesforlong-termadhesion,attachedcellsarecountedagainandthepercentageofcellsdetachedarethenrecorded. 4.3.8StatisticalAnalysisStudentst-testandANOVAwereusedtoevaluatethestatisticalsignicance.Signicancelevelwassetatp<0:05. 4.4NormalForceAdhesionAssay 4.4.1PurposeThepurposeofthistechniqueistoapplyaforceperpendiculartothecellsubstrate.Acustom-madePDMSecover,ttingtightlythetopofaregularculturepetridishallowsustollitwithOpti-MEMmedia.Thedishisthenplacedverticallyinthecentrifuge,withitscultureareaclosesttotheaxisofcentrifugation.Uponcentrifugation,cellswillexperienceacentrifugalforcedirectlyperpendiculartotheirsubstrate(gure 4-5 ).Therefore,thecentrifugalforceexertsaforcenormaltotheadheredcells.SincethedishislledupwithOpti-MEMandthereisnoleakage,wecanassumethatnoshearstressisactingonthecellsduringtheaccelerationofthecentrifuge. 4.4.2CentrifugeSetupThecentrifugeusedinourexperimentsisaMarathon6K,FisherScientic.Acustom-madedishholderwasfabricatedoutofaluminum,inordertokeepthedishverticalduringcentrifugation(gure 4-6 ). 4.4.3NormalForceCalculationThecentrifugalforceactingoncellsiscalculatedfrom: Fc=mcell!2r(4{3) 53

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withmcellisthemassofthecell,!istheangularvelocityinradians/secandrisdeterminedbythedistancefromtherotortothesurface(inthiscase,33mm).Thelateralsizeofthepetridishpreventsallcelllocationsfrombeingindirectlinewiththecentrifugalrotor.Inordertodeterminewhetherthislocationvariancesignicantlyeectstheforceappliedtothecell,weappliedthefollowingequation: Fc=mcell!2rp r2+x2(4{4)withxbeingthemaximumlateraldistancefromcenterofpetridish(27mm).Themaximumpercentagedierencewasdeterminedusing maxpercentagechangeforFc=(r2+x2)1 xr r(4{5)andwasfoundtobe6.3%whichisconsideredwithintheexperimentalerror. 4.4.4SurfacePreparationPDMSeiscreatedbymixinga10:1ratioofbaseandcuringagent.Themixtureisdegassedandpouredoverthedesiredmold(Kratonorsiliconwafers).ThemoldandPDMSearethenplacedinanoven,preheatedto200Ftocure.Oncethepatternsareset,athinlayerofuncuredPDMSeispouredintoaculturepetridish.Thepatternsarecuttotinthedish,placedonthePDMSeandplacedintheoventocurefor30minutes.ThecuredsurfacesaresterilizedbyllingthedishwithethanolandplacingitunderUVlightforanhour.ThedishisthenrinsedwithHank'sBalancedSaltSolution(HBSS)andlledwithOpti-MEMforaminimumof6hours.Cellsarethendetachedfromtheiraskwithtrypsinandresuspendedinmedia.Thesolutionisdepositedonthesurfacesandlefttoattachovernight.Preciseareasaremarkedatthebottomofthepetridish,allowingustorecordthesameareaandsamecellsthroughouttheexperimentalrun. 54

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4.4.5ExperimentalRunPriortotherstrun,cellsarerinsedand5mLofOpti-MEMisplacedinthedish.Markedviewingareasarethenrecordedas0000RPM.ThedishisthenlledwithOpti-MEMandplacedintotheholder.Afterbeingspunfor10minutes,cellsareagainrinsedandtheviewingareasarerecorded.Thecentrifugeexperimentwererunatspeedsof200,400,600and1200rpm. 4.4.6DataAcquisitionAllimagesarerecordedusinganAxiovert100MicroscopeconnectedwithadigitalCCDcamera,amonitor,andaJVCHR-VP780UVHSrecorder.AMatroxRT2500videocapturecard(PinnacleSystems,MountainView,CA)wasusedtodigitizevideorecordingsfromtherecorderintoaPCcomputerusingtheAdobePremiere4.0(Adobe,MountainView,CA). 4.4.7DataAnalysisFramestakenbeforetheexperimentsandaftereachstepsarethensuperimposedusingTheGIMP 1 imageprocessingprogram( http://www.gimp.org ).Anexampleofasequenceofimagestakenfromthesameareabeforeapplicationoftheforceandaftercentrifugationat200,400,600and1200rpmareshowningure 4-7 .Wesuperposedthoseimagesbycomparingthedierentgroupofcellsandaligningthem.Asaresult,wepinpointedtheexactdetachmentforceofeachcell.Single-anddouble-cellsarecountedineachframeandrecorded.Resultarethenpresentedintermsofrelativecentrifugalforce(RCF),asitiscommonlydoneforcentrifuge-relatedresults.Sincecentrifugalforceisdirectlyrelatedtothelengthofthecentrifugearmholdingthesample,transformingthecentrifugalspeedinRCFallowsustocompareresultscomingfromdierentcentrifuges,sincethelengthofcentrifuge'sholdervarywitheachmodel.Thisvaluecanbecalculatedfromtherotor 1 TheGimp:GNUImageManipulationProgram 55

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speed(N)inRPMandthedistancebetweenthecenterofthecentrifugeandthesample(r),usingtheequation: RCF=1:118x106rN2(4{6)Theexplanationofthevalue1:118x106isgiveninappendixA.TheRCFhasunitsofg,whichstandsfortheforceofgravity. Table4-2. Relationshipbetweencentrifugalspeedandrelativecentrifugalforce. Centrifugalspeed(RPM)2004006001200 RCF(g)2.8811.5225.91103.65 4.4.8StatisticalAnalysisStudentst-testandANOVAwereusedtoevaluatethestatisticalsignicance.Signicancelevelwassetatp<0:05. Figure4-1. Thedenedtopographicalparametersofinterestinthemicrochanneldesignaredepth,widthandspacing. 56

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Figure4-2. Thegradientshearowchamberdesignconsistsofaconvergentchannelofentrancewidthw1andexitwidthw2,entrancelengthLeandchannellengthL. Figure4-3. Gradientshearowchamberrstmanufacturingprotocol.1)Atransparencycut-outofthedesiredchannelisplacedinanholderandtwopiecesoftubingareplacedtocreatetheentranceandexitports.PDMSisthenpouredoverthemold.2)ThecuredPDMSisremovedfromthetransparencycut-outandputover3)apartiallycuredpieceofatPDMS.4)AstheuncuredPDMSpiececured,itcreatesanirreversiblesealbetweenthetwocomponents. 57

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Figure4-4. Gradientshearowchambersecondmanufacturingprotocol.1)AatpieceofcompletelycuredPDMSand2)thechannelPDMSpiece,manufacturedasexplainedinsteps1and2ofgure 4-3 ,are3)sandwichedbetweentwopiecesofplexiglasandheldinplacebyscrews. Figure4-5. Forcesactingontheattachedcellsduringcentrifugation. 58

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Figure4-6. Thepetri-dishholderismadeofaluminumtotthelaboratory'scentrifuge.Cellsareadherenttotheinteriorsideofthepetridish.Acustom-madePDMStopsealsthepetridishtoavoidleakagesanduidmovements. 59

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Figure4-7. Extractfromatypicalseriesofpicturestakenduringanormaladhesionassayexperiment.Superpositionofthepicturesallowsustofolloweachcellastheydetachfromtheirsubstrate. 60

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CHAPTER5RESULTS 5.1GradientShearFlowChamber 5.1.1ADINAFluidCalculationsInordertoconrmcalculationofwallshearstressinthemicrochannels,wecomputedthechanneldesignusingADINA.ADINAperformsitscalculationsthroughniteelementanalysis.Thechambervolumeiscutintosmallpiecesandcalculationareperformedforeachpiece.Foroptimalresults,thegeometryofthepieceshouldbeascloseaspossibletooneofacube.Tominimizethenumberofelements,thevolumeofthechamberisdividedbyfour,withassumptionofasymmetricaluidbehavior.ComparisonofthecalculatedshearstressesandshearstressesfoundusingADINAaresummarizedinthegure 5-1 andintable 5-1 . Table5-1. ComparisonofthecalculatedandADINAwallshearstressvaluesattheentrancewidth1(afterentrancelength)andattheexitwidth2 Qcalculated1calculated2ADINA1ADINA2(L/hr)(dynes/cm2)(dynes/cm2)(dynes/cm2)(dynes/cm2) 0.00212.8938.666190.4262744.778234.3212804077 Thereisalargedierencebetweenthecalculatedvaluesandthevaluesfoundwiththemodelingsoftware.Thiscanbeexplainedbythegeometryofourniteelementmesh.Thedimensionsofthechamberconsistsofalength(4.5cm)muchgreaterthanthebiggestwidth(0.1cm)orthedepth(0.013cm).Asaresult,thiselongatedformwouldrequiretohaveaverylargenumberofcubes.However,thetechnologicallimitationofthecomputationalequipmentcannotaccountforaverylargenumberofelements.Asaresult,thechamberiscutintolargerectangles,andasaresult,thecalculationsarenotaccurate.Themeshdensitywasvariedtoconvergetheshearstressvalues.However,convergencewasnotpossiblewiththecomputersactuallyavailable. 61

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5.1.2CellularDetachmentForshort-termadhesion,weappliedaowrateof0.0024L/hrresultinginashearstressofabout13,20and40dynes/cm2atthe3mm-width,2mm-widthand1mm-widthmark.Flowapplicationfor5minutesresultedinapartialdetachmentoftheattachedcellsatthe3mm-widthand2mm-width(24:4%6:2and69:7%7:8,respectively)andinthecompleteremovaloftheattachedcellsatthe1mm-width(100%0).Theresultsarestatisticallydierent(p<0.05).Typicalviewsoftherecordedareaareshowningure 5-2 .Forlong-termadhesion,appliedowcorrespondingtophysiologicalshearstresslevelsof10to20dynes/cm2( Fung , 1993 )didnotdetachcellsafterforceapplicationof10minutes. 5.2Normalforceadhesionassay 5.2.1CellsOrientationChangeItisknownthatcellsaligninthedirectionofshear( LevesqueandNerem , 1985 ).Thus,inordertoestablishifanyshearstresswasactingonthecells,weobservedtheirorientation.Theorientationofeachgroupofcellswasrecordedastheanglebetweenthelongestlineacrossthegroupofcellsandthex-axis.Ifcellsweresubjectedtoashearow,wewouldexpectthemtorealigninaccordancewiththisshearowandthatthere-orientationanglewouldbedependentontheoriginalorientation.However,wewereunabletondanycorrelationbetweenoriginalorientationandre-orientation.Wecanthenconcludethattheeectofshearowoncellsinthissetup,ifexistent,isminimal. 5.2.2RegularColonvs.CancerousColonCellsonFlatSurfacesExperimentsperformedontheCacoandNCMcellshaveresultedintwoverydierentproles.Figure 5-3 showsthepercentageofcelllostbetweeneachcentrifugerunagainsttherelativecentrifugalforceforthehealthycells.FortheNCM,orhealthycolon,cells,wecanobservethatintherststep,correspondingtoaforceof2:88g,aquitelargeamountofcells(19:03%6:11)werepulledothesurface.However,runsat11:52gand25:91ghavedetachedmuchfeweroftheoriginalcells(19:35%3:48and20:33%3:36, 62

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respectively).Atanhigherforceof103:65g,alargeportionoftheremainingcellswerepulledo(38:31%12:98).TheCaco,orcoloncancer,cellsbehaveddierently(gure 5-4 ).Fromtherstrunat2:88gtothe25:91grun,increasingpercentageofcellswereremovedfromthesurfaces(26:19%7:57at2:88g,55:70%10:24at11:52g,79:09%12:34at25:91g).Forthenalstep,at103:65g,anadditional88:91%5:65oftheoriginalattachedcellswasremovedfromthesurface. 5.2.3EectsofCellSizeonDetachmentInanattempttoexplainthevariationinthegradualdetachmentofthecells,wemeasuredthespreadingareaof96healthycoloncellsandcomparedittotheircentrifugaldetachmentforce.TheresultsareshowninFigure 5-5 .AnANOVAtestwasrunandshowsthattherearenostatisticalrelationshipbetweenthesizeofacellspreadingareaanditsdetachmentforce. 5.3Topographyandcelladhesion 5.3.1EectofWidthBygrowingandtestingcellsonthemicrochannelswithfeaturesizesof352,552and1052,weareabletosegregatetheeectofthewidthontheadhesionofhealthycoloncells.Theresultsaresummarizedingure 5-6 .Thedatashowsthattherearenodierencebetweencellsgrownonatsurfacesandvaryingwidthmicrochannels.At2:88g,thepercentageofcellsthatdetachedfromat,352,552and1052microchannelswere19:03%6:11,11:66%4:80,4:55%3:18and19:12%7:96,respectively.Similarly,thevariationisminimalat11:52g(19:35%3:49,19:90%4:95,25:15%9:83and29:93%2:35,respectively).Nodierenceincelldetachmentwasobservedat25:91g(20:33%3:36,23:88%2:51,34:73%9:86and42:34%6:88,respectively).Finally,at103:65g,therearestillnodierences(38:31%12:98,41:90%5:57,52:77%1:52and56:32%7:51,respectively).Forspeedshigherthan11:52g,thereappearstobeasmallrelationshipbetweentheincreasingwidthandincreasingpercentagedetachment.However,thisrelationshipcouldnotbeprovenstatistically. 63

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Similarlytohealthycoloncells,wegrewandtestedcellsonatsurfaceandmicrochannelswithfeaturesizesof352,552and1052tosegregatetheeectofthewidthontheadhesionofcoloncancercells.Theresultsareshowningure 5-7 .Atthelowestforceof2:88g,thevariationinpercentageofcelldetachmentisnotstatisticallydierentformostsurface,with26:19%7:57ofcellsdetachingontheatsurface,3:36%0:80on352microchannels,4:82%3:93on552microchannelsandnonedetachingonthe1052microchannels.Statisticaldierenceisobservedbetweentheatsurfaceand1052microchannelsat2:88g.At11:52g,thedierencebetweenatsurfaceandthemicrochannelsbecomesclearer.Ontheatsurface,55:70%10:24ofcellsdetached,comparedto15:10%1:56onthe352microchannels,12:41%3:18onthe552microchannelsand5:4%1:95onthe1052microchannels.Thedierencebetweentheatsurfaceandallmicrochannelsisstatisticallydierent(p<0:05).Thisstatisticaldierenceremainsatthehigherforceof25:91g(79:09%12:34onatsurfaces,32:49%8:13on352microchannels,20:13%7:65on552microchannelsand13:64%1:88on1052microchannels)and103:65g(88:91%5:65onatsurface,35:74%10:1on352microchannels,35%10:66on552microchannelsand28:73%8:04on1052microchannels).Therefore,thereisacleardierencebetweenatsurfaceandmicrochannelsfortheadhesionofcoloncancercells.However,thereisnorelationshipbetweentheincreaseinadhesionandthevariationofwidth. 5.3.2EectofSpacingToestablishtheexactroleofvaryingspacingoncelladhesion,wetestedhealthycoloncellsonmicrochannelswithfeaturesizesof532,552and5102.Theresultsaresummarizedingure 5-8 .Statisticalanalysisshowsthattherearenodierenceindetachmentofcellsgrownonatsurfacesorvaryingspacingmicrochannels.At2:88g,19:03%6:11oforiginalcellsdetachedfromaatsurface,comparedto11:66%4:80onthe352microchannel,4:55%3:18onthe552microchanneland19:12%7:96onthe1052microchannel.Similarly,at11:52g,thedetachmentpercentageonat 64

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surface,352,552and1052microchannelsshowedlittlevariation(19:35%3:49,19:90%4:95,25:15%9:83and29:93%2:35,respectively).Thistendencyremainedthesameforaforceof25:91g(20:33%3:37,23:88%2:51,34:73%9:86and42:34%6:88,respectively)and103:65g(38:12%12:98,41:90%5:57,52:77%1:52and56:32%7:51,respectively).Similarly,wetestedcoloncancercellsonmicrochannelswithfeaturesizesof532,552and5102.Ataforceof2:88g,whilethereappearstobeadierencebetweenatsurfaceandthemicrochannels,itisnotstatisticallydierent.Ontheatsurface,26:19%7:57oftheorigincalcellsdetached.Onthe532,552and5102microchannels,2:31%2:31,4:82%3:93and2:87%1:42detachedrespectively.Forspeedsof11:52gandhigher,thedierencebetweenatsurfaceandmicrochannelswasstatisticallydierent(p<0:05).At11:52g,55:7%10:24ofcellsdetached,while7:24%1:09,12:41%3:18and10:11%5:83wereremovedonthe532,552and5102microchannels.Thedetachmentpercentagefor25:91gonat,532,552and5102microchannelsare79:09%12:34,14:79%3:68,20:13%7:65and17:33%8:76,respectively.Ataforceof103:65g,thosesamesurfacesyieldeddetachmentpercentageof88:91%5:65,25:95%7:9,35%10:66and36%4:58,respectively.Thoseresultsareshowningure 5-9 .Whiletherewasacleardierenceincellularadhesionbetweenatsurfaceandmicrochannels,theadhesionvariationwasnotcorrelatedtothespacingvariation. 5.3.3EectofDepthThevariationofmicrochannels'widthorspacingdidnotrelatedtochangesinadhesionofhealthycoloncells.Itappearedthatthemicrochannelpatterndidnotaecthealhycelladhesion.However,therewasacleardierenceintheadhesionofcoloncancercellsonatsurfaceandmicrochannels,butitcouldnotberelatedtothevariationofwidthorspacingofthemicrochannels.Asaresult,wetestedtheadhesionoftheCaco2onmicrochannelswithvaryingdepth,i.e.featuresize5101,5102,5104.The 65

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resultsareshowningure 5-10 .Ataforceof2:88g,26:19%7:57ofthecellsattachedontheatsurfacedetached.Onthe5101,5102and5104microchannels,thepercentageofcellsthatdetachedare4:08%1:44,2:87%1:42and7:35%2:97.Atthisstep,therearenostatisticaldierencebetweenthedierentsurfaces.However,at11:52g,55:7%10:24ofcellsdetachedontheatsurfacewhileonly44%6:78ofcellsdetachedonthe5101microchannels.However,thedierenceisnotstatisticallydierent.Onthe5102and5104microchannels,theproportionofcellsthatdetachedare10:11%5:83and17:53%3:7,respectively.Thecellsonthe5102microchannelsthusshowastatisticaldierenceinadhesionfromthecellsattachedtotheatsurfaceandthe5101microchannels.Thecellsonthe5104microchannelsexhibitedstatisticallystrongeradhesioncomparedtocellsattachedontheatsurfaceand5101microchannels.Thosestatisticaldierencearealsoobservedat25:91g,wheretheproportionofcellsdetachedontheatsurface,5101,5102and5104microchannelsare79:09%12:34,59:37%5:72,17:33%8:76and45:84%7:79,respectively.Finally,at103:65g,thoseproportionsare88:91%5:65,62:87%5:4,36%4:58and61:49%8:3,respectively.Atthatforce,adhesiononthemicrochannelsarestrongerthantheatsurface(theresultsarestatisticallydierent,p<0:05).Then,thecellsattachedtothe5102microchannelsattachedbetterthanon5101and5104microchannels(resultsarestatisticallydierent,p<0:05).Adhesiononthe5101and5104microchannelsarenotstatisticallydierent.Asasummary,itwasshownthatcellularadhesionvariedwithdepthofthemicrochannelsandthatadepthof2micronsprovidesthebestadhesionforallcentrifugalforce. 5.4Eectofcell-celladhesiononcell-surfaceadhesionInordertoevaluatetheexistenceofanycross-talkbetweencadherinsandintegrins,weevaluatedthedetachmentforceofsinglecellsand2cellsattachedtogether,calledheredoublets.InNCMcells,thedoublecellsdetachedlessatspeedsof2:88,11:52,25:91and103:65g(2:3%1:43,2:72%1:58,3:14%1:82and19:22%7:35,respectively),a 66

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dierencestatisticallydierent(p<0:01)atallforceexceptat103:65g.Theresultsareshowningure 5-11 .Ontheotherhand,doubleCacocellsdidnotshowanydierence.Forspeedsof2:88,11:52,25:91and103:65g,theirproportionofdetachedcellsare15:63%5:22,44:95%12:57,70:01%9:92and88:12%4:81,respectively.NodierencebetweensingleanddoubleCacocellswasstatisticallydierent(p>0:05).Theresultsaresummarizedingure 5-12 . 5.4.1EectsofDoubleCellSpreadingonCellDetachmentSpreadingareaofdoublecellswasmeasuredandcomparedtosinglecells.Theaveragespreadingareaofsinglecellswas34:59%7:99m2.Thespreadingareaofdoublecellswasfoundtobe64:1%11:71m2.t-testanalysisshowedthattheresultswerestatisticallydierent.Theaveragespreadingareaofdoublecellsisabouttwicethatofasinglecell.Itisreasonabletoassumethatthemassofadoublecellwillcorrespondtotwicethemassofasinglecell.Therefore,theforceexertedonadoublecellwillbetwicetheforceexertedonasinglecell.Ifweassumethatdoublecellshavetwicethenumberofadhesionbondsofasinglecellandthattheforceonthedoubletsistwicetheforceonasinglecell,thedetachmentprolebetweensingleanddoublecellsshouldnotvary.Thedierenceobservedinthedetachmentprolescannotbeexplainedmechanically. 5.4.2EectsofCellJunctiononCellDetachmentFromtheobservationthatthejunctionoftwocellscouldaectthecell-surfaceadhesion,weinvestigatedtheimportanceofthejunctionlengthwiththecell-surfacedetachmentforce.Junctionslengthfrom91NCMdoublecellsweremeasuredandrecordedwiththeirdetachmentforce,asshowningure 5-13 .Doublecellsdetachingat2:88gwerefoundtohaveajunctionlengthof12:790:49m.Thosethatdetachedat11:52,25:91and103:65ghaveajunctionlengthof11:220:35m,12:740:33mand10:800:30m,respectively.Theresultsaresummarizedingure 5-14 .UsingANOVA, 67

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wewereabletoshownthatthereisnocorrelationbetweenthelengthofthejunctionanddetachmentforcerequiredtodetachthecells. Figure5-1. TheADINAchartsandgraphofwallshearstressesforowratesof0.002and0.426L/hr. . 68

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Figure5-2. UsingtheGSFC,asingle5minuteowexperimentonshort-termadheredcellsshowspartialremovalofthecellsat13dynes/cm2andcompleteremovalofthecellsat40dynes/cm2. Figure5-3. Cellularadhesionofhealthycoloncellsontheatsurface.%cellsdetachedreferstotheproportionofcellsoriginallyattachedthathavedetached 69

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Figure5-4. Cellularadhesionofcancerouscoloncellsonatsurface.%cellsdetachedreferstotheproportionofcellsoriginallyattachedthathavedetached. 70

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Figure5-5. Sizeofcellsvs.detachmentspeed.Thespreadingarea(size)of96cellswasmeasuredandrecordedandrelatedtotheirdetachmentspeed.StatisticalanalysiswithANOVAstatesthatthereisnorelationshipbetweenthesizeanddetachmentspeed. 71

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Figure5-6. Eectofwidthonhealthycoloncellsadhesion.Byvaryingthewidthofthemicrochanneltopography,wewereabletoevaluateanyvariationincellulardetachmentofthehealthycells.t-testanalysisfoundnodierencebetweenadhesiononthedierentmicrochannelsandontheatsurface. Figure5-7. Eectofwidthoncancerouscoloncellsadhesion.Byvaryingthewidthofthemicrochanneltopography,wewereabletoevaluateanyvariationincellulardetachmentofthecancerouscells.t-testanalysisfoundastatisticaldierencebetweenadhesiononthedierentmicrochannelsandontheatsurface.However,therewasnodierenceontheadhesionbetweenthedierentmicrochannels. 72

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Figure5-8. Eectofspacingonhealthycoloncellsadhesion.Byvaryingthespacingofthemicrochanneltopography,wewereabletoevaluateitseectoncellulardetachmentofthehealthycells.t-testanalysisfoundnodierencebetweenadhesiononthedierentmicrochannelsandontheatsurface. Figure5-9. Eectofspacingoncancerouscoloncellsadhesion.Byvaryingthespacingofthemicrochanneltopography,wewereabletoevaluateitseectoncellulardetachmentofthecancerouscells.t-testanalysisfoundastatisticaldierencebetweenadhesiononthedierentmicrochannelsandontheatsurface.However,thereisnodierenceintheadhesionbetweenthedierentmicrochannels. 73

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Figure5-10. Eectofdepthoncancerouscoloncellsadhesion.Byvaryingthedepthofthemicrochanneltopography,wewereabletoevaluateitseectoncellulardetachmentofthecancerouscells.t-testanalysisfoundastatisticaldierencebetweenadhesiononthedierentmicrochannelsandontheatsurface.However,thereisnodierenceintheadhesionbetweenthedierentmicrochannels. 74

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Figure5-11. Comparisonoftheadhesionofsingleanddoubletshealthycoloncells. Figure5-12. Comparisonofthesingleanddoubletsofcoloncancercells. 75

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Figure5-13. Thelengthofthejunctionbetweentwoattachedcells,ordoublecells,aremeasuredandrecordedtoassesstheirimportanceoncell-surfaceadhesion. Figure5-14. Eectsofcell-celljunctionlengthoncelldetachment. 76

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CHAPTER6DISCUSSION 6.1GradientShearFlowChamber 6.1.1ModelingAdhesionUnderShearFlowMicrouidicdevicesareconsideredasthemostreliablemethodtoapplyapreciseandcontrolledforceovercells.Theirusefulnessisbeyonddoubt,asthelargenumberofrelatedliteraturecanprove.Numerousmodelsofcellularadhesionoriginatedfromthestudiesofcellsinowchamber.However,mostarerelatedtothestudyofwhitebloodcellsandtheirattachmentpriortodiapedesis( Bell , 1978 ; Evans , 1985 ; HammerandLauenburger , 1987 ; Demboetal. , 1988 ; WardandHammer , 1993 ; Klobouceketal. , 1999 ; Raetal. , 1999 ).Thesemodelsthusonlydealwithshort-termadhesion,asobservedwithleukocytes.Long-termattachmentrequiresmoreelaboratemodelstoincludeitsinherentcomplexities,suchasreceptorclusteringandfocaladhesionassembly.Inaddition,thelong-termadhesionmodelsarediculttovalidateexperimentally.In1993,WardandHammer( WardandHammer , 1993 )rstinvestigatedtheeectsoffocalcontactformationonadhesionstrength.However,theirmodelreliedonthermalenergy,forwardandreverserateofreceptor-ligandbindingandotherbiophysicalparametersthatarediculttoevaluateexperimentally.Morerecently, GallantandGarcia , 2007 ,approachedtheearliermodelsandre-evaluatedtheminthecontextofexperimentalparameters.Theirresultinganalysisallowsadirectcomparisonoftheoreticalandexperimentalresults.Therststepintheanalysisistobalancetheforcesofacellattachedtoasubstrateundershearow(asshowningure 6-1 ).Theshearforce(Fs)andtorque(Ts)createdbythehydrodynamicowarecounteredbythetangential(Fan)andtensile(F)andcompressive(Fc)forces.ThesolutionofFsandTsonarigidsphereinaowdevelopedby Goldmanetal. , 1967 showsthatthedominantresistantforceisF.Bybalancingtheforces,Fisderivedasafunctionoftheshearstress: 77

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F=32R2s 1+0:8R a2(6{1)whereRistheradiusofthecellandaistheradiusofthespreadingarea.Theaveragevalueofthespreadingareasshowningure 5-5 is34.6m2andtheradiuscanbeestimatedasa=q Aspread .Thetangentialforceexertedonacellbytheshearforcesachievableintheowchamberaresummarizedintable 6-1 ,whereQistheowrate,1representsthewallshearstressattheentrancewidth,2thewallshearstressattheexitwidth.TheF1andF2areforcescalculatedwiththeequationaboveand1and2,respectively. Table6-1. Tangentialforcesinthegradientshearowchamberatdierentowrate Q12F1F2inL/hr(dynes/cm2)(dynes/cm2)(N)(N) 1:001040:641:935:1510101:541091:001036:4419:335:151091:541080:002128938:661:031083:091080:01064:43193:295:151081:541070:02128:86386:591:031073:091070:100644:311932:945:151071:541060:4262744:778234:322:191066:58106 6.1.2ShearFlowChamberChallenge 6.1.2.1GassolubilityinliquidsHenry'slawstatesthatgassolubilityinliquiddecreasesastemperatureincreases.Asaresult,whentherstdesignofchannelwasinsertedintheincubatortoletcellsattachedforover4hours,bubblesappearedovertime.Asasolution,alloftheliquidelementsusedfortheprotocolwereheatedtotemperaturesoforabovetheincubator'stemperature.Thisunsuredthatnobubblewouldbecreatedduringtheattachmentperiodintheincubator.Theadherentcellswerethensuspendedinheatedtrypsin,andheatedOpti-MEMwasadded.Theresultingmixturewasaddedintothechamberandincubatedforatleastfourhourstoensureattachment. 78

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6.1.2.2PdmsgaspermeabilityPDMSisagaspermeablepolymer,andthusaninterestingchoicetocreateacultureowchamber.HoweverthepermeabilityofthePDMSwasnotenoughtoprovidethegasnecessarytoensurethewellnessofthecells.OneofthesolutionwastocreateaconstantminimalowofmediaorOpti-MEMduringtheattachmentperiod,soastoprovidethenecessarysupplyofgas.However,suchasolutionimpliedthatthecellswouldbesubjectedtoasmallshearstressduringtheirattachment.Itwasshownthatcellsreactandadapttoshearstress,andasaresult,theadhesionobservationwouldhavebeenbiasedbythisconditioningtreatment.Therefore,wedecidedonthedesignchangeexplainedinthematerialandmethodsectionofthisdissertation. 6.1.2.3FluidicresistanceFlowrateinmicrochannelsarerelatedtothepressuredropacrossthechannel(P)andthechannelresistance(R)bytheequation:Q=P R.Therefore,increasingtheowrateinthemicrochannelwouldresultinanincreaseinthepressuredropacrossthechannel.Sincetheexitpressureistheatmosphericpressureandremainsconstant,toincreasethepressuredropwouldrequiretoincreaseintheentrancepressure.Therefore,aswetriedtoreachhigherowrate,thepressureincreasewassuchthatthesealbetweenthetubingandjunctionwouldfail.Asaresult,themaximumowrateachievablewas0.426L/hrandfailedtobehighenoughtodetachcells. 6.1.2.4PdmsasavalidsubstratemodelTheintentinthisworkwastoachievecelldetachmentusingshearforcesphysiologicallyrelevant.Therefore,thespecicationsoftheowchamberweresettoreachshearstressesintherangeof10to20dynes/cm2( Fung , 1993 ).However,cellsdidnotdetachatthesevalues.Theowratewasincreasedtoitshighestvalue(0.426L/hr),correspondingtoarangeofshearstressesof2745to8234dynes/cm2.However,cellsdidnotdetachatthosevalueseither.Yet, GallantandGarcia , 2007 ,havereportedcelldetachmentundershearstressesaslowas100dynes/cm2.Thosehighstressescanberelatedbycalculationto 79

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forcesrangingfrom0.875to2.62nNonthecell(calculationsareshowninAppendix C ).Ithasbeenhypothesizedthattheoveralladhesionstrengthofacellis200nN( Gallantetal. , 2005 ; GallantandGarcia , 2007 )andothershaveevaluatewholecellstrengthat180nN( Sagvoldenetal. , 1999 ).Asaresult,itcouldexplainwhycellsdidnotdetachinthegradientshearowchamber.However,achievingsuchdetachmentforceinourgradientshearowchamberwouldmeanachievingashearstressofapproximatively125000dynes/cm2,andtherefore,aowrateof15L/hr.Thoseextremelyhighvaluesthenraisethequestionofphysiologicalrelevancyoftheseadhesionmodels.Indeed,theyarebasedonin-vitroexperimentsandtomyknowledge,wehaveyettoestablishtheadhesionstrengthin-vivo.Thefactthatthecellsdidnotdetachinoursetupthusquestionthevalidityoftheshearforcemodelsestablishedatthistime. 6.2NormalForceAdhesionAssay 6.2.1NormalForceCalculationResultswerepresentedwithrespecttothecentrifugalspeed,withunitsofrpm.Itismorecommonforcentrifuge-relatedresultstobepresentedintermsofrelativecentrifugalforcecalculatedwiththeequation 4{6 56 alreadylaidoutinthematerialsandmethodschapter.IfoneweretodecidetopresenttheresultsintermsofforcewithunitsinNewton,theequationwouldbe Fc=mcell!2r(6{2)wheremcellisthemassofthecellingrams,!istheangularvelocity(inradianpersecond)andristhedistancefromtherotortothesurface(inthiscase,33mm).However,usingthisequationrequirestosetanassumptiononthemassofacell,anassumptionthatIwasnotwillingtoincludeintheresultschapter.However,itisimportanttobeabletorelatethecentrifugalspeedofeachsteptoaforce.Thus,thefollowingtableassociates 80

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eachrpmspeedtoitscorrespondingforce,withassumptionofcellmassasonenanogram( FeinendegenandNeumann , 2005 ). Table6-2. Centrifugalspeedsandtheircorrespondingforces CentrifugalspeedRCFNormalforce(RPM)(g)(nN) 2002.881.7340011.526.9060025.9115.51200103.6562.1 Thosevaluesaremuchgreaterthananyofthenormalforceexpectedinthebody.Intheirstudyofthenormalforceexertedonvascularendothelialcells, WangandDimitrakopoulos , 2006 ,demonstratedthatthenormalforcesinsmallvesselscanbeasmuchasfourtimesmoreimportancethanshearforces.However,theshearforcesinsmallvesselshavebeenestimatedtobeintheorderofnN,whichmeansthatthemaximumnormalforceexistinginthebodyisalsointheorderofnN.Theresultsfromthenormaladhesionforceassayarethereforenotphysiologicallyrelevant.However,thedistinctlycleardierenceexhibitedbythehealthyandcancerouscellsinthisset-upmakesthistechniquehighlyrelevantforthestudyofcelladhesion. 6.2.2DetachmentBehaviorThedetachmentbehaviorofthehealthycoloncellsonatsurfacebroughtoutaninterestingquestion.Whywouldcellsgraduallydetachinsteadofallpullingoatacriticalcentrifugationforce?Onecouldspeculatethat,dependingonthespreadingareaofacell,itcouldexhibitstrongerorweakeradhesionproperties.Ifweweretoassumethat,aclearrelationshipwouldappearbetweenthespreadingareaandthecentrifugalforceneededtoachievedetachment.However,gure 5-5 doesnotexhibitanycorrelationbetweenthosetwovariables.Thiscomesincontradictionwithmanyarticles( Balabanetal. , 2001 ; Galbraithetal. , 2002 ; Gallantetal. , 2005 )thatclearlyrelatecellspreadingwithcelladhesionstrength.However,thersttworeferencescitedhaveestablishedthatalargerspreadingarearesultsintheabilityofacelltopullmorestronglyontheir 81

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substrate,asetupcompletelydierenttoournormalforceadhesionassay.Thethirdreferencetestedcellswithaspinningdiskdevice.Thissystemappliesagradientshearstressusingacentrifuge.Therefore,eventhoughtheyfoundthatthecellspreadingareawasdirectlyrelatedtothedetachmentforce,thiscanbeeasilyexplained.Detachmentthroughshearstresshappensbyexertionofforceonthesideofacell.Ifweweretoassumethatallcellshavesamesize,abiggerspreadingareawouldcorrespondtoasmallerheight.Andtherefore,lesssideareafortheshearstresstoactupon.Thiseasilyexplainstherelationshipbetweencellspreadingareaanddetachmentforce.However,inournormalforceadhesionassay,theforceisacentrifugalforce,actingnormaltothesubstrateandrelatedtothemassofthecell.Ifweweretoassumethatthereisauniformdistributionofthefocaladhesionpointsallaroundthecell'smembrane,abiggeradhesionareawouldresultinmoreadhesioncontact,andthusmoreadhesionstrength.Since,thisisnotobservedinoursetup,wecanconcludethatthefocaladhesionpointsarenotuniformelydistributedthroughoutthemembrane,afactalreadyreportedinmanypublishedarticles( Gallantetal. , 2005 ).Therefore,thecellgeometrywillnotaectthenumberofadhesionbondsandasaresult,cellspreadingwouldnotaectthedetachmentforceofthecell.Anotherexplanationcanbementionedtoexplaintheincreasingproportionofcelldetachment.Ananalogycanbemadebetweencoloncelladhesionandleukocyteadhesion.Asdevelopedin Changetal. , 2000 ,leukocytespresentdierentadhesivebehaviors:fast,transient,andrmadhesion.Similarly,ingure 5-3 ,wecandistinguishtwoslopes,onefrom0to2.88g,andasecondslope,comingdirectlyafteralargeplateau,from25.91to103.65g.Thisleadsustobelievethatcoloncellspresenttwotypesofadhesion:weakandstrong.Theweakadhesioncorrespondstotherstslope,whichclearlyshowsthatitsstrengthcouldnotresistthecentrifugalforceof2.88g,whichrelatestoforcesof1.73nN.Thiswouldcorrespondtoselectins'sadhesion,andthevalueofselectinbondshasbeenestimatedintherangeof25-45pN( ShaoandHochmuth , 1999 ).Thestrongadhesionrelatestothesecondslope,andconcernscentrifugalforcehigherthan25.91g,which 82

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relatestoforceshigherthan15.5nN.Thisforcevalueisobyafactorof10comparedtopreviouswork( GallantandGarcia , 2007 ).Intheirmodel,theadhesivepatchofacellwascalculatedtohaveanadhesionstrengthof200nN.Ourvariationinadhesionstrengthcouldverywellbeexplainedbyanumberoffactor:GallantandGarcia'scalculationsweredesignedtosolvehydrodynamicshearowexperiments,thusaccountingforashearforceandtorque(gure 6-1 ).Inaddition,insuchasetup,theapplicationofforceisnotequalonallbonds,dependingontheirpositionwithrespectwiththeow.Asaresult,thebondsatthefrontalpartofthecellswhichexperiencesthemostshearforcewilldetachrst.Oncetheydetached,thecellgeometrywillbeaected,andthussowilltheshearforceexertedonthecell.Inournormalforceadhesionassay,allbondsexperienceequallythecentrifugalforce.Inaddition,whiletheforcesinGarciaandGallant'smodelinvolvebothaverticalandhorizontaldirection,oursonlyincludeahorizontalcomponentandthevariationcouldbetheresultofapreferenceoftheadhesionbondstocertainspatialdirections.Throughouranalysis,wewereabletoprovethatthecancerouscellsadheredlesstoatsurfacesthanhealthycells.Thisobservationhadbeenmentioned( CavallaroandChristofori , 2001 ),butpreviouspublicationsfailedtoquantifytheexactvariationinadhesionstrengthbetweenhealthyandcancerouscells.Thebehaviorofthecancerouscellsundercentrifugalforceisquitepuzzling,asshowningure 5-4 .Unlikethehealthycells,thereisnoplateau,onlytwoslopes.Therstslopeconcernsthecentrifugalspeedsof2.88,11.52and25.91g.Thesecondgoesforcentrifugalforcehigherthan25.91g.Onecouldsupposethattherstsloperelatestotheincreaseinselectinexpressionthathasbeenobservedincolorectalcancer( Birdetal. , 2006 ).However,atthispoint,ourexperimentaldesigndoesnotprovideuswithenoughinformationtoclearlystatewhetherthevariationinadhesionstrengthcomefromalteredstrengthofselectinsand/orintegrins.Itisimportanttomentionthatthenumberofcelllinepassageswasnottakenintoaccountinourdataacquisition.Cellcultureforextentedperiodsoftimeresultsin 83

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alterationofmultipleproperties,suchasmorphology,growthrate,responsetostimuliandproteinexpressionandsignaling.Theeectsofpassagesoncaco-2cellsarereviewedin Sambuyetal. , 2005 .Atthepresenttime,theinuenceofpassagenumberhasnotbeenreportedforcellularadhesion,butitcouldbeafactor. 6.3TopographicaleectsoncellularadhesionTheadhesionofcoloncellsonthedierentmicrochannelsissummarizedingure 6-2 .Itisinterestingtonotethatnovariationwasnotedbetweencellsattachingtheatsurfaceandtheonesfromthemicrochannels.However,theadhesionofcancercellsonmicrochannelsisquitedierentfromtheirhealthycounterparts.Theresultsaresummarizedingure 6-3 .Theadhesionofthecancerouscellswasgreatlyincreasedbythepresenceofthemicrotopography.Throughourassessmentofeachfeaturecharacteristic,wewereabletondthatvariationindepthwasthemainfactoraectingtheadhesion,andthatamongthevaluesstudied,2micronsisthecriticalsizetoachieveoptimaladhesion.Observingtheslopesonthemicrochannelsgraphsrevealsthatthecellularreactiontothemicrotopographyvarieswitheachmicrochannel.Itisinterestingtonotethatthecellularadhesionofcancercellsonthemicrochannelsissimilartothehealthycells.Whileonatsurfaces,cancercellsdetachmorereadilythanhealthycells(resultisstatisticallydierent,p<0:05),onmicrochannels,thehealthyandcancerouscellsbehaveidentically(exceptformicrochannelsofsize1052atspeedsof11:52and25:91g).Therefore,whilethealteredadhesiononatsurfacecouldleadtomistakethisasintegrinmalfunction,theadhesivepropertyonmicrochannelsimplicatesthatthemechanosensingmechanism,andthustheintegrins,stillworks.CombiningtheadhesionbehavioronmicrochannelswiththeresultsfromAnderson'spaper( Andersonetal. , 2006 ),onecouldspeculateontheeectoftheECMtopographychangethatoccursincancer.Inwelldierentiatedcancer,theECMbecomessmoother.Asaresult,youwouldexpectthecancerouscellstodetachmorereadilyfromits 84

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substrate.Furtheralonginthedisease,poorlydierentiatedcancerdevelopsarougherECMtopography.Asaresult,therewouldbenochangeinadhesioncomparedtohealthycells.Theseobservationsinthecell-surfaceinteractionsarerelevantinthematterofcarcinogenesis.Thetraditionalview,calledthesomaticmutationtheory,statesthatcancertumorsgothroughthreestages:initiation,promotionandprogression.Initiationhappensthroughgenomicmutations.Asaresult,aseriesofevents,promotion,leadstoproliferation.Severalgenescanbeinvolvedintheprocess:oncogenesaregenesthat,oncemutated,leadstoabnormalcellsurvivalanddivision;tumorsuppressorgenesaregenesthatnormallyregulatesabnormalcellgrowth.Oncemutated,theyareunabletocompletetheirtask,allowingtumorstodevelop.Asaresult,whencertaincombinationsofmutationsaremade,itresultsintheabnormalpropagationofcellsanddevelopmentofatumor.However,anewtheoryhasbeenpresentedandnamedthetissueorganizationtheoryofcancer( Bisselletal. , 2002 ; SotoandSonnenschein , 2004 ).Thismodelplacestheoriginofcancerinthecell'senvironment.Asaresult,carcinogenesisistheresultofnotonlygeneticdamagesbutalsodynamicchangesintumorcellinteractionswiththeirmicroenvironment.Disorganizedtissuealtersthecell-surfaceinteractions,resultinginthemodicationofthephenotypeincellssurroundingthedisruptivetissue.Thuswouldbecreatedtumors.Essentialtothisideaisthatcancercanbereversedintheappropriateenvironment.Anumberofdatahasbeenpublishedreportingthisnormalizationofcancercells,asreportedin SotoandSonnenschein , 2004 .Whileboththeorieshaveconvincingarguments,itisimpossibleatthepresenttimetoestablishwhichofthetwo,orpossiblyanother,isthecorrectmodel. 6.4Cell-CelladhesionCelladhesionencompassesbothcell-surfaceadhesionandcell-celladhesion.Recentobservationhaveleadmanytohypothesizetheexistenceofacell-cellandcell-surfaceadhesioninteraction.Inordertotestthishypothesis,wecomparedthecellularadhesionofsinglecellsandoftwojoinedcells,labeledasdoublecells.Theresultsareshownin 85

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gure 5-11 forhealthycells.Itappearsthat,whilesinglecellsexhibitatwo-typeadhesion,double-cellsonlyshowonetypeofadhesion.Itisinterestingtonotethattherstslopeobservedinthesingle-cellsandcorrespondingtoweakadhesionhasdisappearedinthedouble-cellsbehavior.Weakadhesionisoftenassociatedwithselectinbinding,comparedtointegrinbinding.Inaddition,thesecondslopeofthedouble-cellsiscompletelyparalleltotheoneofthesinglecells,meaningthatthecellsdetachedatthesamerate.Thus,wecouldhypothesizethatthecell-celladhesioncrosstalkwithcell-matrixadhesionexiststhrougheitherthereinforcementofselectinadhesion,orthetransformationofselectinadhesionintointegrinadhesion.Themechanismbetweencell-cellandcell-matrixinteractionstilleludesus.Sinceourobservationestablishedtheexistenceofthisrelationship,wesoughttofurtherourunderstanding.Weassumedthatthejunctionlengthbetweencoupledcellsrelatetothenumberofboundcadherin.Aftermeasuringthejunctionandcomparingittothecentrifugalforcerequiredtodetachthecell(gure 5-14 ),wedidnotndanycorrelationbetweenthosetwoparameters.Wecanthenarmthatthecadherineectoncell-matrixadhesiondoesnotmatterontheamountofsignal,butratherontheactualexistenceofthesignal,actingmuchlikeanon/oswitch.Cell-celladhesionproteincadherinhasoftenbeenbelievedtoplayakeyroleinthealteredcell-matrixadhesionofcancercells.Weanalyzedtheadhesivebehaviorofsinglecellsanddoublecellsofthecoloncancercells.Theresultsareshowningure 5-12 .Itisclearthatinthecaseofcancerouscells,thepresenceofacell-celljunctiondidnotstatisticallyaectthecell-matrixadhesion.Sincethecancerouscellsarebehavingdierentlyontheatsurfaceandthemicrochannels,integrinreceptorsareapparentlyfunctioning.Thelackofcell-cellandcell-matrixcrosstalkinthecancerouscellswouldthenbetheresultofacadherinmalfunction.Thisresultconferswith deRooijetal. , 2005 ,thatintegrin-mediated-adhesionwouldaectcadherin-mediated-adhesion,ratherthancadherin-mediated-adhesionaectingintegrin-mediated-adhesion. 86

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Thisworkshowsaclearcell-cellandcell-matrixadhesioncrosstalk.Therationalbehinditremainsevasive.Inordertoassessthekeycommunicationcomponentsofthisphenomenon,additionalworkisrequired.Cadherinandintegrinbeingpresentedhereasthemainproteinsinvolvedinthecell-cellandcell-matrixadhesion,uorescenceimagingofthosewouldprovidewithmoreinsightastotheirrelationship. Figure6-1. Freebodydiagramofanadherentcellundershearow.Adhesionbondsresisttotheshearforce(Fs)andtorque(Ts)byatangential(Fa),tensile(F)andcompressive(Fc)forces.ThedominantresistantforceisF. 87

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Figure6-2. Normalcoloncellsadhesiononmicrochannels. 88

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Figure6-3. Coloncancercelladhesiononmicrochannels. 89

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CHAPTER7CONCLUSIONSANDFUTURERESEARCHFromtheresultspresentedthroughoutthiswork,itcanbeconcludedthatthetechniquesdevelopedtomeasurecellularadhesionresultedinvariousdegreesofsuccess.Itwasshownthatthesheargradientowchamberfailedtoprovidequantitativeinformationoncellularadhesionwhilethenormalforceadhesionassay,whichrequiresminimalequipmentandexpertise,hasbroughttolightsomeveryinterestingphenomenon.Therstandforemostconclusionconcerningcellularadhesioncanbesummarizedbythefollowingsentence;cancerouscoloncellsexhibitanalteredsubstrateadhesionwithrespecttohealthycells.Itwaspossibletofurtherelaborateonthisgeneralconclusionbystudyingcellularadhesiononvarioustopographicalsurfaces.Thesemorespecicconclusionscansummarizedinvemainpoints:1.Intheabsenceofsignicanttopographicalfeatures,cancerouscellsdetachedmorereadily(weakeradhesionstrength)thantheirhealthycounterparts.Theirdetachmentprolesuggeststhatthegroupofadhesionmoleculesinvolvedmightbedierentthanforthehealthycells.2.Observationsrevealedthatcancercellsreactmainlytovariationinthetopographicalfeaturesdepthandwereunaectedbychangesinwidthorspacingofthosefeatures.Itwasdeterminedthattherewasacriticaldepthof2matwhichthecancerouscellsexhibitthestrongestadhesion.Notethatthisbehaviorwasobservedforassayforcesnormaltothesurfacewhichareunaectedbythepartialconcealmentofcellsintothemicrochannels.Consequently,thetopographyimpactoncellularadhesioncannotbeexplainedbytheprotectionofthecellsfromtheappliedforcesandshouldbetheresultofmolecularrearrangement.3.Cancerouscellsadhesionissignicantlyaectedbythepresenceoftopographicalfeaturesintheorderofafewmicronswhilehealthycellsadhesionremainsunchanged.Thisdisparityprovidesadditionalconrmationthatthecellularadhesionofcoloncancercellsisaltered.Moreover,sinceitwasobservedthatcancerouscellsresponddierentlytovarioustopographicalroughness(microchanneldepth),itsuggeststhatthesensingmechanismofthecancerouscellsisstillactive.4.Itwasobservedthatcellularadhesionstrengthissimilarforcancerousandhealthycellsgrownonmicrotexturedsurfaceswithfeaturesofafewmicrons.Thisdierssignicantlyfrombehaviorobservedonatsurfacesasdiscussedearlier.Themechanismbehindthisobservationremainsunknown,butthisapparentreturn 90

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toahealthycellularadhesionisworthyofinvestigation.Itwouldbevaluabletodeterminewhetherothercellularfunctionssuchasspreading,proliferationorgrowthfollowthesametrend.5.Observationsshowedthatcell-celladhesioninhealthycoloncellsresultsinastrongercell-substrateadhesionwhichisaclearproofofacell-cellandcell-substratecrosstalk.Thisconstitutestherstdemonstrationofsuchcrosstalkthroughmechanical,asopposedtochemical,means.Itisinterestingtonotethatthiscross-talkdoesnotappearinthecancerouscells.However,atthepresenttime,itisimpossibletoevaluateifthisisaresultofafailureatthecell-celljunctions,cell-surfacejunctionsand/ortheintracellularpathway.Notethatabetterassessmentoftheforcesexertedinthenormalforceadhesionassaywouldcementsomeoftheassumptionsand,throughtheuseofuorescentdyesseveralmorequestionscouldbeanswered. 7.0.1ForceAssessmentOneofthelimitationsofthenormalforceadhesionassayisthatthestrengthoftheforceappliedonthecellsareestimationsfromestablishedequations.However,wehaveyettoconrmthatthesameforcevaluesarepresentinthetestingapparatus.Thismattercouldbesettledbyusingsphericalbeads,coatedwithadhesionproteinsand/orantibodies.Thesubstratecouldbecoatedwiththecorrespondingligand.Sincethepropertiesofthebead(size,mass,coatingdensity)wouldbeknow,theirdetachmentcouldbepredictedandcomparedtotheactualcentrifugalforcerequiredtodetachthem. 7.0.2FocalAdhesionsInthepreviouschapters,wehavepresentedourresultsontherelationshipofcellspreadingandcellattachmentstrength.Itshowedthattherewasnocorrelationbetweenthosetwofactors.However,wehadtospeculateonthedistributionofthebindingpoints,orfocaladhesions.Fluorescentassayswouldprovideinformationonthevariationofthosebindingpointbetweenhealthyandcancerouscells,aswellastheirmobilityundernormalforce.Furthermore,theirdistributionasthecellsattachtomicrotopographywouldbeinterestingtostudy,especiallywhenweconsiderthelargedierenceincellsreactiontotopographybetweenthehealthyandcancerouscells.Inadditiontotheirdistribution, 91

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theanalysisofthenumberandsizeofthefocaladhesioncouldfurtherourunderstandingofthedierencesbetweennormalandcancerouscells.Focaladhesionareclassiedasmature,immature,andsuper-maturestructures.Comparingtheproportionsofthoseelementsandtheotheradhesionproteinsineachcellcouldberelatedtothedierentslopesobservedinthedetachmentproleofthecells. 7.0.3CellPositionintheMicrotopographyThestep-by-stepimagesprovidedbythenormalforceadhesionassayallowustostudyavarietyofvisualparameters.Amongthemisthepositionofattachedcellsonthemicrochannels.Withouttheneedtoperformmoreexperiments,bysimplyanalyzingthecurrentdataavailable,itwouldbepossibletorecordthedierentpossiblecelllocationswithrespecttothemicrotopography,andcompareittotheiradhesionstrength.Healthyandcancerouscellscouldthenbetestedforanypreferenceanddierenceinthesepositions.Onecouldwonderifcellswilltendtoprefertobeinthemicrochannel,especiallywhenthedepthofthemicrochannelisaroundtheheightofacell(estimatedat5micronsfromthecentralpartofthecelland2micronsfortheperipheryby Satoetal. , 2006 ).Inaddition,uorescentassayscouldrevealthelocationofthebindingpointsonthetopography,andcouldprovidemorecluesastotheroleofsurfacetopographyoncellularadhesion. 7.0.4FluorescenceCellularImagingofAdhesionProteinsTherelationshipbetweenthecell-celladhesionandcell-matrixadhesionthatwaspresentedinthisworkraisesanumberofquestions.Mechanicalapproachesareunabletoprovideinsightintothemolecularcomponentsatplayinthecellularadhesionphenomenon.Ourinterestlieswiththebehaviorofcadherinsandintegrins,thekeyproteinsincell-cellandcell-matrixadhesion.Fluorescenceofcadherinproteinshowsaggregationofcadherinsatthecell-celljunctionsandthusconrmsitsroleofcadherininthecell-celladhesion.Fluorescenceofintegrinscannotprovideaclearviewofitsroleasacell-matrixadhesioncomponents.Integrinsaremembraneproteinsanduorescencewould 92

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onlyrevealtheirlocationofthemembrane,notpreciselyatthecell-matrixinterface.Itwouldthenbemoreappropriatetousethecross-linking/extraction/reversalmethodpresentedby KeselowskyandGarcia , 2005 .Thistechniqueallowsvisualizationofintegrinboundtothesurface,afterremovalofthecell.Itwouldthenbepossibletocomparethenumberanddistributionofintegrinsinsinglecellsanddoublets. 93

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APPENDIXANORMALFORCECALCULATIONAsmentionedinthediscussionchapterofthisdissertation,itispossibletorelatetherotorspeed(N)inRPMandthedistancebetweenthecenterofthecentrifugeandthesample(r)inmmintotherelativecentrifugalforce(RCF)withunitsofg(forceofgravity:9810mm/s),usingtheequation: RCF=0:00001118rN2(A{1)Thisequationcaneasilybederivedasfollowed: RCF=Fcentrifugation Fgravity(A{2) RCF=m!2r mg(A{3) RCF=!2r g(A{4) RCF=2 60N2 g(A{5) RCF=42 3600gN2r(A{6) RCF=1:118x106rN2(A{7) 94

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APPENDIXBSHEARSTRESSCALCULATIONINTHEGRADIENTSHEARFLOWCHAMBER B.1PrincipleConsiderthesteadylaminarowofanirrotationalincompressibleinvisciduidthrougharectangularductwith(x,y,z)asthecartesiancoordinates.ContinuityandNavier-Stokesequationsyields: rV=0(B{1) DV Dt=Fb+1 (rp+V)(B{2)whenneglectinggravity,Navier-Stokesbecome: @V @t+(Vr)V=1 (rp+V)(B{3)Sincewedenedtheuidassteady,wehave: @V @t=0;Vz=0(B{4)Asaresult, B{3 isreducedto: Vx@ @x+Vy@ @yVx=1 @p @x+ Vx(B{5) Vx@ @x+Vy@ @yVy=1 @p @y+ Vy(B{6) 0=1 @p @x(B{7)NoslipboundaryconditionsmeansVx(atz=0;h)=Vy(atz=0;h)=0,becauseweassumethattheuidvelocityvectoratthewalliszero. 95

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Foraslowowandasmallgapbetweentheplates,wecanassumethatthederivativesofV1andV2withrespecttox1andx2arenegligiblecomparedtothederivativeswithrespecttox3.Asaresult,weobtainthefollowingsystem: @p @x=@2Vx @z2(B{8) @p @y=@2Vy @z2(B{9) 0=@p @z(B{10)Fromtheboundaryconditions,weobtain: Vx=1 2@p @xz2 hz (B{11) Vy=1 2@p @yz2 hz (B{12)Theintegral~V1~V2meansandacrossthegapare ~Vx=1 hZh0Vxdz=h2 12@p @x(B{13) ~Vy=1 hZh0Vydz=h2 12@p @x(B{14)Theintegralmeanisthen ~V=h2 12rp(B{15)Thevectorwallshearstressonthelowerplate,atx3=0isdenedby: w=13+23(B{16) 96

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w=@Vx @y++@Vy @x(B{17) w=6 h~V(B{18)Now,assumingthatthewidthofthechannelvariesalongthex-direction: w=w(x)=w1 2"w1 2w2 2 L#x(B{19) FigureB-1. Sketchofaductwithvaryinggapbetweenparallelplates Thevolumeowrateperunitwidththroughtheductis: Q(x)=~VA(x)=~Vhw(x)=~Vhw1 2"w1 2w2 2 L#x!(B{20)Thusfrom B{18 ,theshearstressalongthecenterlineofthex-axisis w=6Q h2w1 2(w1 2w2 2) Lx(B{21) 97

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B.1.1DesignCalculationsTheowchamberdesignwascreatedtoallowmultipleshearstresstobeappliedinasingleexperiment.Thesyringepumpused(KDS100,KDScientic,Holliston,MA)achievesowrate(Q)from0:1L/hr.to426mL/hr.Theowchanneldimensionsare:entrancewidthw1of3mm,exitwidthof1mmandheigthof0.13mm.Inordertoensurelaminarowinourdevice,wecalculatedthereynold'snumber.Avalueunder2000ensuresthattheowislaminar.Sincewearenotusingacircularpipemodel,theReynold'snumberforourowisdependentonthehydraulicdiameterofourchannelandcanbefoundby: Re=VDh (B{22)whereisthedensityofthemediumowingthroughthechannel(1g/ML),istheviscosityofthemediuming/(cms)(0.098g/(cms)at20C).AllmediumpropertieswereobtainedfromtheOptiMEMdatasheet.Vistheuidvelocity(cm/s)andDhisthehydraulicdiameterdenedas: Dh=4wh 2w+2h(B{23)wherewisthewidthofthechannelandhisitsdepth(0.13mm).Thewallshearstressofthechambercanbecalculatedusingequation B{21 developedearlierwhereQistheowrateofthemediumincm3/s.Theentrancelengthofthechannel,necessarytoachievefullydevelopedlaminarow,isfoundthrough: le=0:06ReDh(B{24) 98

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FigureB-2. DesignoftheShearGradientFlowChamber Thedimensionssetare:w1=3mm,w2=1mmandL+Le=46:5mm.Atdierentowrate,weobtainthefollowingnumbers: TableB-1. Table4.1:dimensionsoftherectangularpattern QLeRe1Re2w1w2(L/hr)(cm)(dynes/cm2)(dynes/cm2) 11072:711081:811065:021066:441041:9310311062:711071:811055:021056:441031:9310211052:711061:811045:021046:441021:9310111042:711050:0020:0050:641:9311032:711040:0180:0506:4419:330:0020:0010:0360:112:8938:660:010:0030:1810:50264:43193:290:020:0060:3621:003128:86386:590:10:0281:8115:017644:311932:940:4260:1187:71621:3712744:778234:32 Sinceweareplanningontestingphysiologicalshearstressesrangingfrom10to20dynes/cm2,wehavedecidedtouseaowrateof1103L/hr.Asaresult,wewouldneedanentrancelengthoflessthan0.01mm.However,toavoidrestrictionontheowrate,wecreatedallchannelswithanentrancelengthof5mm.Withaowrateof1103L/hr,wehavethefollowingwallshearstressalongthex-axis(notincludingtheentrancelength): 99

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FigureB-3. Shearstressvariationalongthegradientshearowchamberunderowrateof0.01L/hr 100

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APPENDIXCFROMSHEARSTRESSTOSHEARFORCERelatingashearstressvaluetothecorrespondingshearforcevaluerequiresanumberofassumptions.Sincetheshearstressactsonasurfacearea,therstassumptionrelatetothegeometryofacellandmorepreciselyitssizeasaradius,R,of4m.Now,wealsoassumethatthecellcanbeconsideredasasphericalcap,asWangandDimitrakopoulos(2006)did.Thespreadingofthecellcanbecharacterizedbytheanglebetweenthecell'smembraneandthevessellining,0.Forafully-spreadcell,0wouldbeequalto0;foranon-spreadcell,0wouldbe180.Weassumethatthecellsarepartiallyfully-spread,andthus0isequalto30.ThefrontalsurfaceareaScuponwhichtheshearstressactsrelatestothespreadingarea,Aclxly,wherelxandlyarethelengthandwidthofthecell.Inourcase,lxlysinceweareassumingasphericalshapeforthecell.Thecell'sheightlzlxtan0lx0forsmallangles.ThevolumecanbefoundbyVR3lxlylz.Therefore,R3lxlxlx0l3x0andlxR1 30,lzR2 30andScR22 30=1:661012m2.TheshearforceisthenFshearSc.Thetable C-1 presentstheshearforcesattheentrancewidthforavarietyofowratesinthegradientshearowchamber. TableC-1. Shearforcesattheentrancewidthofthegradientshearowchamber Q1Shearforce1Shearforce1(L/hr)(dynes/cm2)(dynes)(N) 11036:441:0710710710120:00212:892:141072:1410120:0164:431:071061:0710110:02128:862:141062:1410110:1644:311:071051:0710100:4262744:774:551054:551010 Thetable C-2 presentstheshearforcesattheexitwidthforavarietyofowratesinthegradientshearowchamber. 101

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TableC-2. Shearforcesattheexitwidthofthegradientshearowchamber Q2Shearforce2Shearforce2(L/hr)(dynes/cm2)(dynes)(N) 1:01041:933:201083:2010131:010319:333:201073:2010120:00238:666:411076:4110120:01193:293:201063:2010110:02386:596:411066:4110110:11932:943:201053:2010100:4268234:321:361041:36109 102

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BIOGRAPHICALSKETCH CecilePerraultwasborninNice,Franceonthe14thofDecember1979.ShewasraisedinthesmallcityofGrasse,ontheFrenchRiviera.Aftershereceivedherbaccalaureatwithhonors,herfamilymovedtoFlorida.ShestartedhercollegeyearsintheUniversityofCentralFloridaasamicrobiologymajor.Shesoonrealizedthatmathematicsandphysicslackedinhercurriculum,andthatmedicalschool,whichsheplannedtoattendafterherundergraduateyears,wasnotappealingtoheranymore.Whilesearchingforanewdirection,sheheardoftheBiomedicalEngineeringprogramattheUniversityofFlorida.AssheappliedtotheCollegeofEngineeringattheUniversityofFlorida,BachelorEngineeringSciencesmajorwithminorinBiomechanics,shehadthechancetomeetthechairmanoftheAerospaceEngineeringDepartment,Dr.Shyyandoneofthefaculty,Dr.Tran-Son-Tay.Theyexplainedtoherthisnewlycreatedprogramandsherealizedthiswasthecareerthatshelongedfor.Afterheracceptanceintotheuniversity,shequicklystartedresearchinDr.Tran-Son-Tay'slaboratory.ShereceivedherBachelorEngineeringScienceswithminorinBiomechanicsin2001andenteredtheBiomedicalEngineeringprogram.Shortlyafter,thisprogramnallybecameitsowndepartment.ShethenreceivedherMasterofScienceinBiomedicalEngineeringin2003.SheiscurrentlypursuingherresearchinDr.Tran-Son-TayCellularMechanicsandBiorheologyLaboratory. 111