Citation
In vitro modeling of interstitial fluid flow relating to pulmonary hypertension

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Title:
In vitro modeling of interstitial fluid flow relating to pulmonary hypertension
Creator:
Delaney, Ryan P. ( author )
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
Physical Description:
1 electroni file (156 pages). : ;

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Subjects / Keywords:
Pulmonary hypertension ( lcsh )
Lungs -- Blood-vessels ( lcsh )
Pulmonary artery ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Review:
Pulmonary Hypertension (PH) is a disease characterized by inflammation and histopathologic changes that are associated with a progressive remodeling of the pulmonary vasculature. The hallmarks of this remodeling are a stiffening and thickening of pulmonary arteries due to an increase in smooth muscle and a change in the protein structure of the vessel. The adventitia or outermost layer of these vessels undergoes profound changes with the infiltration of a large number of leukocytes, and an increase in the expression of collagen and consequent stiffening of the tissue. The pulmonary adventitial fibroblast (PAF) is arguably the most important cell type in this region and is responsible for remodeling the extracellular matrix as well as for the production of proliferative and inflammatory signaling molecules. Fibroblasts in general are very sensitive to mechanical and chemical signals, as well as to the static microenvironment around themselves and will change their behavior based on these factors. The tissue environment that cells are normally exposed to in vivo is very complex. So when this region adapted for in vitro studies a more elaborate culturing setup must be employed to fully recreate the behavior of these cells and understand the complex nature of pulmonary vascular remodeling. As medical studies become more advanced, the physiological recapitulation of tissues becomes a greater necessity in performing cell culture experiments and these studies must employ the growing array of tools available to modern researchers. Fibroblasts grown in standard conditions on flat plates as opposed to those grown on or inside different types of hydrogel mixes will change their behavior drastically. These gels additionally alter the behavior of the cells grown inside them based on their mechanical and chemical properties. Due to this, no single hydrogel culturing setup will be useful for all cell types and gels must be made to recreate the region of residency for each cell type. To facilitate a more complete recreation several imaging and culturing studies were carried out. These studies led to several novel observations, a confocal study of cells seeded into a decellularized lung matrix revealed a preference for cells to seed into diffuse fibrous regions as opposed to dense protein sheets. Based on this and previous studies which determined specific lung mechanical and chemical properties, a hyaluronic acid and collagen based hydrogel was employed. This gel produced a diffuse soft matrix with tunable properties. Culturing of cells inside these gels revealed changes in behavior when the elastic moduli and chemical properties were varied. Slicing the gels onto slides and staining with Hematotoxilin and Eosin revealed that cells grew well within the fibrous matrix and proceeded to remodel the fibers over the course of two weeks. The use of a hydroxyproline assay to study the collagen content of these gels over time showed that this remodeling proceeded with the cells initially increasing the collagen content of the gel, and then returning it to a lower level later on. This suggests that under normal conditions cells will initially increase collagen in a semi-inflammatory, or wound healing type of behavior, due to being in an unfamiliar environment but will then reverse remodel to a lower steady state of collagen content. In PH, adventitial collagen never returns to a lower state so there must be some other forcing factor maintaining the proinflamatory state. Immunofluorescent staining studies showed that there was a dysregulation in the tight junctional barriers of endothelial cells and a migration of fibroblasts and dendritic cells across bronchus associated lymphoid tissues towards vessels and airways. These two factors taken together would suggest that there is a breakdown in the system that regulates fluid flow across the interstitial space to lymphatics. This fluid flow may in fact serve as a guide for fibroblast movement across the lymphatic due to flow. Scanning electron microscopy studies also showed that the adventitial region of large vessel underwent a decrease in overall extracellular matrix void space and preferential realignment of fibers to be perpendicular to interstitial fluid flow. This would indicate that PAFs are attempting to control the increased fluid flow and higher interstitial fluid pressure drop caused by endothelial cell breakdown through the remodeling of the adventitia.
Thesis:
Thesis (M.S.)--University of Colorado Denver. Bioengineering
Bibliography:
Includes bibliographical references.
System Details:
System requirements: Adobe Reader.
General Note:
Department of Bioengineering
Statement of Responsibility:
by Ryan P. Delaney.

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University of Colorado Denver
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|Auraria Library
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904408521 ( OCLC )
ocn904408521

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INVITROMODELINGOFINTERSTITIALFLUIDFLOWRELATINGTOPULMONARYHYPERTENSIONbyRYANPDELANEYB.S.ChemicalandBiochemicalEngineering,ColoradoSchoolofMines,2009 AthesissubmittedtotheFacultyoftheGraduateSchooloftheUniversityofColoradoinpartialfulllmentoftherequirementsforthedegreeofMastersofScienceBioengineering2014

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ThisthesisfortheMastersofSciencedegreebyRyanPDelaneyhasbeenapprovedfortheBioengineeringProgramby MichaelYeager,AdvisorRichardBenninger,ChairKendallHunter November21,2014 ii

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Delaney,RyanP(M.S.,Bioengineering)InVitroModelingofInterstitialFluidFlowRelatingtoPulmonaryHypertensionThesisdirectedbyAssistantProfessorMichaelYeagerABSTRACTPulmonaryHypertension(PH)isadiseasecharacterizedbyinammationandhistopathologicchangesthatareassociatedwithaprogressiveremodelingofthepul-monaryvasculature.Thehallmarksofthisremodelingareastieningandthickeningofpulmonaryarteriesduetoanincreaseinsmoothmuscleandachangeintheproteinstructureofthevessel.Theadventitiaoroutermostlayerofthesevesselsundergoesprofoundchangeswiththeinltrationofalargenumberofleukocytes,andanincreaseintheexpressionofcollagenandconsequentstieningofthetissue.Thepulmonaryadventitialbroblast(PAF)isarguablythemostimportantcelltypeinthisregionandisresponsibleforremodelingtheextracellularmatrixaswellasfortheproductionofproliferativeandinammatorysignalingmolecules.Fibroblastsingeneralareverysensitivetomechanicalandchemicalsignals,aswellastothestaticmicroenviron-mentaroundthemselvesandwillchangetheirbehaviorbasedonthesefactors.Thetissueenvironmentthatcellsarenormallyexposedtoinvivoisverycomplex.Sowhenthisregionadaptedforinvitrostudiesamoreelaborateculturingsetupmustbeemployedtofullyrecreatethebehaviorofthesecellsandunderstandthecomplexnatureofpulmonaryvascularremodeling.Asmedicalstudiesbecomemoreadvanced,thephysiologicalrecapitulationoftissuesbecomesagreaternecessityinperformingcellcultureexperimentsandthesestudiesmustemploythegrowingarrayoftoolsavailabletomodernresearchers.Fi-broblastsgrowninstandardconditionsonatplatesasopposedtothosegrownonorinsidedierenttypesofhydrogelmixeswillchangetheirbehaviordrastically.These iii

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gelsadditionallyalterthebehaviorofthecellsgrowninsidethembasedontheirme-chanicalandchemicalproperties.Duetothis,nosinglehydrogelculturingsetupwillbeusefulforallcelltypesandgelsmustbemadetorecreatetheregionofresidencyforeachcelltype.Tofacilitateamorecompleterecreationseveralimagingandcul-turingstudieswerecarriedout.Thesestudiesledtoseveralnovelobservations,aconfocalstudyofcellsseededintoadecellularizedlungmatrixrevealedapreferenceforcellstoseedintodiusebrousregionsasopposedtodenseproteinsheets.Basedonthisandpreviousstudieswhichdeterminedspeciclungmechanicalandchemicalproperties,ahyaluronicacidandcollagenbasedhydrogelwasemployed.Thisgelproducedadiusesoftmatrixwithtunableproperties.Culturingofcellsinsidethesegelsrevealedchangesinbehaviorwhentheelasticmoduliandchemicalpropertieswerevaried.SlicingthegelsontoslidesandstainingwithHematotoxilinandEosinrevealedthatcellsgrewwellwithinthebrousmatrixandproceededtoremodelthebersoverthecourseoftwoweeks.Theuseofahydroxyprolineassaytostudythecollagencontentofthesegelsovertimeshowedthatthisremodelingproceededwiththecellsinitiallyincreasingthecollagencontentofthegel,andthenreturningittoalowerlevellateron.Thissuggeststhatundernormalconditionscellswillinitiallyincreasecollageninasemi-inammatory,orwoundhealingtypeofbehavior,duetobeinginanunfamiliarenvironmentbutwillthenreverseremodeltoalowersteadystateofcollagencontent.InPH,adventitialcollagenneverreturnstoalowerstatesotheremustbesomeotherforcingfactormaintainingtheproinamatorystate.Immunouorescentstainingstudiesshowedthattherewasadysregulationinthetightjunctionalbarriersofendothelialcellsandamigrationofbroblastsandden-driticcellsacrossbronchusassociatedlymphoidtissuestowardsvesselsandairways.Thesetwofactorstakentogetherwouldsuggestthatthereisabreakdowninthesystemthatregulatesuidowacrosstheinterstitialspacetolymphatics.Thisuidowmayinfactserveasaguideforbroblastmovementacrossthelymphaticdue iv

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toow.Scanningelectronmicroscopystudiesalsoshowedthattheadventitialre-gionoflargevesselunderwentadecreaseinoverallextracellularmatrixvoidspaceandpreferentialrealignmentofberstobeperpendiculartointerstitialuidow.ThiswouldindicatethatPAFsareattemptingtocontroltheincreaseduidowandhigherinterstitialuidpressuredropcausedbyendothelialcellbreakdownthroughtheremodelingoftheadventitia.Toprovethatuidowcanserveasaguideforbroblastmigration,anexperi-mentwassetupwherecellswereseededintoamicrouidicchamberandmediawasoweddirectlyoverthecells.Theirmovementwasobservedinrealtimeduetothenovelnatureoftheculturingapparatus.Thisallowedfornumerouscellstobetrackedatoncethroughtheuseofautomatedtrackingsoftware.Thesestudiesrevealedthatatdierentowconditionsitwaspossibletotrackcellularmovementinrealtimeandthatcellsdidadoptapreferenceformovementundermoderateowconditions.Thebasisforthispreferencewascharacterizedthroughthemeasurementofuidowandtheuseofacomputationaluiddynamicssimulation.Thisrevealedthatthereisaspecicamountofshearforcethatwillallowcellstoguidemovementandoutsideofthisregiondirectionaltravelbecomesrandom.Theuseofthesediverseresearchtechniquesproducedanumberofdierentresultsthatcouldbebroughttogetherintoasingleconclusion.VascularremodelingseeninPHisnotthenormalstateforbroblastsaswasseenfromourculturingexperimentyet,someundenedpathologicmechanismiskeepingtheminaproinamatorystate.Thereisabreakdownintheuidowcontrolmechanismsandariseininterstitialuidpressureinthelungs.Fibroblastscanutilizeuidowasaguideformovementandremodeling.Thissuggeststhatthisincreasedinterstitialuidowisdirectlycontrollingthebehaviorofthesecellsandguidingthevascularremodelingofthevessel.Thisopensupthepossibilityformorecomplexstudiesinthefutureandpotentialtherapiesthattargettheseissues. v

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Theformandcontentofthisabstractareapproved.Irecommenditspublication. Approved:MichaelYeager vi

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DEDICATIONThisthesisisdedicatedtomyfamilyandfriendswhohelpedandencouragedmeallthewayincompletingmywork. vii

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ACKNOWLEDGMENTIwouldliketothank:MyAdvisers:Dr.MichaelYeager,Dr.RichardBenningerandDr.KendallHunter.YeagerLab:KelleyColvin,SarahWilliams,OzusLohaniChildrensHospitalPathologyDepartment:EricWarchowDr.RaduMoldovanMelanieDufvaMyfellowBioengineeringStudents viii

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TABLEOFCONTENTSTables........................................ xii Figures....................................... xiii Chapter 1.Introduction................................... 1 1.1Purpose.................................. 1 1.2PulmonaryHypertension........................ 1 1.3Justication............................... 4 1.4Goals................................... 6 2.Methods..................................... 8 2.1MicrouidicCulture........................... 8 2.2Rat/CellLines.............................. 8 2.2.1RatAdventitialFibroblasts................... 8 2.2.2Staining.............................. 9 2.2.3RatModels............................ 9 2.3HAHydrogel............................... 10 2.4ScanningElectronMicroscopy..................... 12 2.5DecellularizationofLungTissue.................... 14 2.6ConfocalMicroscopyofDecellularizedTissue............. 14 2.7HydrogelCulturingStainingandMorphology............. 16 2.8HydrogelHistology........................... 16 2.9PCR................................... 17 2.10MechanicalStrengthTesting...................... 20 2.11ImmunouorescentStaining....................... 20 2.11.1FibroblastSpecicProtein,SmoothMuscleActinandOX62. 20 2.11.2Claudin-5andAquaporin-1................... 21 2.12CellASICsCultureSystem....................... 23 ix

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2.13CellTracking............................... 27 2.14SpinningDiskFluidDynamicsCharacterization........... 28 2.15SolidWorksSimulation......................... 28 2.16VascularPermeabilityTest....................... 29 2.17HydroxyprolineAssay.......................... 30 2.183DPrinting............................... 30 3.Results...................................... 32 3.1ScanningElectronMicroscopy..................... 32 3.1.1HydrogelImages......................... 32 3.1.2RatTissueImages........................ 32 3.2ConfocalDecellularizedTissue..................... 35 3.3HydrogelCulturingStainingandMorphology............. 36 3.4HydrogelHistology........................... 38 3.5PCR................................... 41 3.6MTSTesting............................... 42 3.7ImmunouorescentStaining....................... 44 3.7.1FibroblastSpecicProtein,SmoothMuscleActinandOx-62 44 3.7.2Claudin-5andAquaporin.................... 46 3.8CellASICCulturingandCellTracking................. 52 3.9SpinningDiskFluidDynamicsCharacterization........... 55 3.10SolidWorksSimulation......................... 60 3.11CellASICDivisionofChamber..................... 64 3.12VascularPermeabilityTest....................... 67 3.13HydroxyprolineAssay.......................... 67 3.143DPrinting............................... 73 4.Discussion.................................... 77 4.1NewCulturingMethods......................... 77 x

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4.2SEMRelationships........................... 79 4.3PHRelationshipsSeeninStaining................... 80 4.4CellASICs................................ 81 4.5ShearForcesonCells.......................... 81 4.6RapidPrototyping............................ 83 4.7StrengthsandLimitations....................... 84 5.Conclusion.................................... 90 References ...................................... 94 Appendix A.MatLabCode.................................. 101 A.1CellTrackingProcedure......................... 101 A.2BeadTrackingProcedure........................ 107 A.3PoreSizeAnalysis............................ 112 A.4MTSTesting............................... 117 A.5OrientationAnalysis........................... 130 B.ExcelCalculators................................ 134 B.1HydroxyprolineAnalyzer........................ 134 B.2HystemCWeightCalculator...................... 135 xi

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TABLESTable2.1WeightpercentagesofdierentcomponentsforthefourhydrogelmixesandtheassociatedelasticmodulusasdeterminedbyJ.L.Vanderhooft.. 13 3.1MeanFreePathdeterminedporesizesintheadventitia.Xdirectionreferstoporesizeparalleltovessel.Ydirectionreferstoporesizeperpendiculartovessel.................................... 34 3.2ModulusofdecellularizedsamplesallunitsinkPA............ 44 3.3PearsoncorrelationcoecientsforOx-62andFSPstaining........ 46 3.4RatioofvesselsseeninIFstainedimagesthatdisplayedabreakdownintheClaudin-5organizationandAquaporinorganizationaroundvessels.. 52 3.5TravelcharacteristicsofcellsinCellASICchamber.DirectionalTravelistheaveragedistancethatthesecellstraveledinmicronswithapositivenumberindicatingmovementwithowandanegativeindicatingmove-mentagainstow.Tracksisthenumberofcellstracked.Directionalpreferenceisindegreeswith0beingdirectlywithow........... 59 3.6AverageowvelocityofbeadstravelingdowntheCellASICchannel... 60 3.7ShearstressesinPascalsofthedivisionsfortheCellASICchamber... 64 3.8DirectionoftravelofcellsintheCellASICchamber.Negativevaluesareagainstowandpositivevaluesarewithow............... 72 xii

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FIGURESFigure1.1Thelayersofavessel.Consistingoftheintima,mediaandadventitia.Theadventitiaandspecicallyresidentbroblastsarethefocusofthisstudy...................................... 2 1.2Anormalpulmonaryvesselandairwayonthelefthandsidewiththinvessellayer.Ahypertensivevesselwiththickenedremodeledlayersthatleadtoincreasedvascularresistance..................... 3 1.3BronchusAssociatedLymphoidTissues(BALTs)innormalandPHmodelrats...................................... 4 2.1Hydrogelusedforcellculturecrosslinkingreaction,CHMA-SisHA,Gtn-DTPHisDCandPEGDA.......................... 12 2.2(a)SEMimagetaken,subsettedtotheadventitialspace,gaussiansmoothedandthenbinarized(b)Poresizeanalysisinthexandydirections.... 15 2.3SlopeInterpolationofStressStrainCurve................. 20 2.4ThecellASICchamberwithGFPcellsgrowninsideofit.Redarrowdesignatedthedirectionofuidow..................... 23 2.5Cellsseededatahighdensityof20x106cellspermL(a)Lightmicroscopyimage200xafteroneweekofculture.Thecellshaveformedlargemasses,shownwithblackarrows,thatareaverydierentbehaviorfromwhatisseenwhencellsarenormallygrownonaatplateandbecomeconuent(b)ImmunouorescentstainingofthechamberrevealsthesecellshavedierentialexpressionofSMA(green)andFSP(red)suggestingthatthecellsmaynotbemaintainingastablephenotype.............. 25 xiii

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2.6Cellsseededatadensityof5x106cellspermL(a)IFmicroscopyimage100xaftertwodaysofculture.Thecellscanbevisuallydierentiatedfromeachotherasindividualswhichallowsforcelltrackingalgorithmstobeappliedtothem.(b)IFstainingofthechamberforsimplyFSPinredshowsthatthecellsmaintainamorebroblasticphenotype........ 26 2.7SnapshotofIcycelltrackingsoftwarescellstracks.Notetheredcircleswhichdesignatethecenterofmassofeachofthesecells......... 27 3.1SEMimagesofdierenthydrogelmixesforqualitativeanalysis(a)Mix1.Stiestmixwithnocollagen(b)Mix2.Stimixwithcollagen.(c)Mix3.Softermixwithlittlecollagen.(d)Mix4.Softestmixwiththemostcollagen.Asthestinessofthegelsdecreasestheporosityofthegelincreasesaswell.Additionallyasthegelsincreasedincollagencontentthesizeofthesheetsofthegelsdecreasedaswell............. 33 3.2Aexampledistributionofmeanfreepathlengthsfoundwhenexaminingtheadventitiaofarat.Withthelengthonthexaxisorcircumferentialdirectionandthenumberofoccurrencesontheyaxisorradialdirection. 34 3.3QQplotsofthemeanfreepathofdierentsamplesfromtheadventitiaofratstakentoshowthatthemeansoftheseporeanalyseswerenormallydistributed.(a)Xdirectionnormalanimals(b)Ydirectionhypertensiveanimals(c)XdirectionPHanimals(d)YdirectionPHanimals...... 35 3.4VoidfractionmeasurementsbetweennormalandPHanimals....... 36 xiv

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3.5Confocalimagesofdecellularizedratlungs.AutouorescenceandresidualGFPingreenandcellsthatwereculturedontothedecellafterwardsmarkedwithPKHinred(a)Cellsindecelltissuearoundseveralvesselswithanevendistributionofcellsgrowing(b)Cellsindecelltissuenearlungpleurawiththepleuramarkedbytheredarrowandnormallungtissuebythebluearrow.Cellshaveapreferencetoattachtothebroadernetworkofbersinthistissueasopposedtothesheetlikemorphologyofthepleura................................... 37 3.6Cellsgrownonastandard16wellplateonthebottomofthechamberstainedwithSMAgreen200x........................ 38 3.7GreenstainSMA,BlueDAPI.All200x.Allcellsculturedontopofgels1weekofgrowth(a)Mix1.Stiestmixwithnocollagen(b)Mix2.Stimixwithcollagen.(c)Mix3.Softermixwithlittlecollagen.(d)Mix4.Softestmixwiththemostcollagen.Asthestinessofthegelsdecreasedtheaspectratioofthecellsbecamelargerandcellselongated....... 39 3.8GreenstainSMA,BlueDAPI,RedFSP.All200x.Allcellsculturedinsideofgels2weekofgrowth(a)Mix1.Cellsremainspherical(b)Mix2.Cellsforadensenetworkofinterconnection(c)Mix3.Cellsformanetworkbetweeneachotherwiththeuseofthindendriticlikeconnections(d)Mix4CellsformabroaderbrousnetworkwithdenseregionsofSMAsuggestingmigratoryactivity.AsstinessdecreasescellsfollowanincreasingtrendtodevelopthindendriticnetworkswithhighSMAcontent......... 40 3.9FibroblastsinltratingthroughpillarsinCellASICplate.ThesepillarsgiveonlyonemicronofspacebetweenPDMSsectionswhichcellsarenotabletodegradedemonstratingtheabilityofcellstomovethroughverysmallregions.................................. 41 xv

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3.10(a)1weekgrowthgelmix1H&E(b)2weekgrowthgelmix1H&E(c)IFstaininggelmix1FSPredSMAred.Cellbodiesinarrows.ThereislittletonochangeinthestructureofthegelandIFstainingrevealsthatthecellsremainstaticasphereinthegel.................... 42 3.11(a)controlgelmix2H&E,(b)1weekgrowthgelmix2H&ECellsshownwithblackarrows.(c)2weekgrowthgelmix2H&ECellsshownwithblackarrows,thickerbersshownwithwhitearrows.(d)IFstaininggelmix2FSPred,SMAgreen,DAPIblueThisshowsthebrousnetworkthatthegelsareformingingreaterdetailandmatcheswiththeH&Estainingshowninb&c............................ 43 3.12GreenFSP,RedOx-62,BlueDAPI(a)1wkMCT(b)1wkSuHx(c)2wkMCT(d)3wkSuHx(e&f)4wkMCT.RegionsofbothFibroblastandimmunecelllocalizationshownwithwhitearrows,airwayshownwithyellowarrowandBloodvesselwithgreenarrow............... 45 3.13GreenSMA,RedFSP,BlueDAPI(a)Normoxic.NotethedelocalizationofPAFsintheBALT.LocalizationofFibroblastsshownwithwhitearrowsandthebreakdownoftheSMAlayeroftheairwaywithyellowarrows.(b)1wkMCTlocalizationofPAFstowardsairwaysshownandSMAdiscontinuity(c)4wkMCTlocalizationofPAFstoaroundtheBALTandgreaterbreakdowninSMAlayerofvesselshownwithyellowarrows 47 3.14RedClaudin-5,BlueDAPI.Twonormoxicratlungsectionsshowingves-sels.Notethedistincttightjunctionalbarriersbetweenendothelialcellswithclearseparationwherecellbodiesarelocatedshownwithwhitearrows 48 3.15RedClaudin-5,BlueDAPI.OneweekafterMCTinjection.NotethedecreaseinthelocalizationofClaudin-5toregionsbetweencells.Claudin-5isinsteadmoreevenlyspreadoutthroughouttheendothelialcellsshownwithwhitearrows............................... 49 xvi

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3.16RedClaudin-5,BlueDAPI.FourweeksafterMCTexposure.NotethelossofdistinctjunctionalbarriersofClaudin-5shownwithwhitearrows. 50 3.17RedClaudin-5,BlueDAPI.ThreeweeksofSugenandHypoxicatmo-sphereexposure.NotethealmostcompletealterationlocalizationofClaudin-5expressiontotheregionsinbetweentheendothelialcellsshownwithwhitearrows............................... 51 3.18GreenAQ1,RedClaudin-5,BlueDAPI.(a)Normoxicsections;notethewellorganizedlayersofAQ1surroundingvesselsandairways(b)4weekMCTrats.ThereisadistinctbreakdownoftheAQ1layeraroundthevesselandairwayanddisorganizationinitslocalization,shownwithwhitearrows.(c)3weekSugenHypoxiarats.ThereisathinningoftheAQ1layeraroundtheairwayandadistinctlossofexpressionaroundthebloodvesselsshownwithwhitearrows....................... 53 3.19Cellsseededatadensityof5x106cellspermLwithnoforcedowappliedtothechannel;twoexperimentsthatwerecarriedoutinthisfashion.(a&b)GFPsnapshotsofthechannelbeforetheexperiments.(c&d)Tracksofcellmovementnormalizedtoastartingcentralposition.(e&f)Polarhistogramofdirectionalpreferenceformovement.............. 54 3.20Cellsseededatadensityof5x106cellspermLwith6895Paofforcedowappliedtothechannel;twoexperimentsthatwerecarriedoutinthisfashion.(a&b)GFPsnapshotsofthechannelbeforetheexperiments.(c&d)Tracksofcellmovementnormalizedtoastartingcentralposition.(e&f)Polarhistogramofdirectionalpreferenceformovement....... 56 xvii

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3.21Cellsseededatadensityof5x106cellspermLwith27580Paofforcedowappliedtothechannel;oneexperimentwascarriedoutathalfthetime(6hours)beforethewastewellslledup.(a)GFPsnapshotsofthechannelbeforetheexperiments.(b)Tracksofcellmovementnormalizedtoastartingcentralposition.(c)Polarhistogramofdirectionalpreferenceformovement................................. 57 3.22SpinningdiskconfocalowcharacterizationoftheCellASICchamber.(a)SnapshotofthecenteroftheCellASICchamberasuorescentbeadsareoweddownthemiddleofthechamber.(b)Snapshotofthemovementtrackingofbeadsintheimagingwindow.Individualpositionsatthetimepointareshownasredcirclesandfullbeadtracksasmulticoloredlines. 58 3.23Histogramsofcellmovement;6895PaappliedowfromCellASICsma-chine(a)Polarhistogramofmovementdirectionofbeadsfromthetrack-ingprogramstoMatLabtrackingprogram(b)Individualpathsofbeadsasthemovefromtheirinitialpositionwhichisnormalizedtoazeropointonthepolaraxis............................... 60 3.24SolidworksrepresentationoftheCellASIC'schamberwithcellbodiesin-sertedasrepresentativeofthoseseenwhencultured.ACFDsimulationwasexecutedofthechamberandthepipesshownarerepresentativeofuidowwiththevelocityrepresentedbythecolorofthepipe...... 62 3.25ThetwodivisionsoftheCellASICchamberthatwereperformedfortheanalysisofshearstressoncellswiththeinletofthechamberatthetopofbottomoftheimage(a)Horizontaldivisions,(b)Verticaldivisions.. 63 3.26Cellsseededatadensityof5x106cellspermLwithnoforcedowappliedtothechanneltwoexperimentsshowningure3.19buttheanalysisofcellmovementisdividedinthechamber(a&b)Theleftsideofthechamber.(c&d)Themiddleofthechamber.(e&f)Therightsideofthechamber. 65 xviii

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3.27Cellsseededatadensityof5x106cellspermLwith6895Paofforcedowappliedtothechanneltwoexperimentsshowningure3.20buttheanalysisofcellmovementisdividedinthechamber(a&b)Theleftsideofthechamber.(c&d)Themiddleofthechamber.(e&f)Therightsideofthechamber................................ 66 3.28Xmovementdirectionalmovementdistributionwherenegativevaluesrep-resentmovementagainstowandpositivevalueswithit.(a)controlofnoappliedpressure(b)6895Paappliedpressure.(c)27580Paappliedpressure.................................... 68 3.29Xmovementdirectionalmovementdistributionwherenegativevaluesrep-resentmovementagainstowandpositivevalueswithitfortheleftthirdofthechannel.(a)controlofnoappliedpressure(b)6895Paappliedpressure.(c)27580Paappliedpressure................... 69 3.30Xmovementdirectionalmovementdistributionwherenegativevaluesrep-resentmovementagainstowandpositivevalueswithitforthemiddlethirdofthechannel.(a)controlofnoappliedpressure(b)6985Paappliedpressure.(c)27580Paappliedpressure................... 70 3.31Xmovementdirectionalmovementdistributionwherenegativevaluesrep-resentmovementagainstowandpositivevalueswithitfortherightthirdofthechannel.(a)controlofnoappliedpressure(b)6895Paappliedpres-sure.(c)27580Paappliedpressure..................... 71 3.32Relativeamountsofcollageningelswhengelsamplesaretakeninmicro-gramsofcollagenpertotalgelsampleweight.1standarddeviationerrorThisshowsnochangeincollagenformixonebutaninitialriseatoneweekandthenasubsequentdropattwoweeksofcellculture...... 74 xix

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3.33Collagenlevelsofallfourmixesofgelsfromthehydroxyprolineassayat1and2weeksofcellsgrowth.Thisshowsthatthegelsexhibitsimilarbehaviorbutatdierentlevelsofcollagen.................. 75 3.343Dprintedpartrepresentingthebersofdecellularizedlungtissue... 76 B.1TheHydroxyprolinecalculatorspreadsheet................. 134 B.2Hystem-Cweightcalculatorfordeterminingmixratios........... 136 xx

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1.Introduction 1.1PurposePHisadiseasethatinvolvestheprogressiveremodelingofthelungvasculatureandtherightheartandthesechangeseventuallyleadtoheartfailure.Theunderlyingcausesofthisprogressionarepoorlyunderstoodandthereforetreatmentstotargetitarediculttodevelop.GainingabetterunderstandingoftheunderlyingfactorsthatdrivevascularremodelingcanprovideuniqueinsightintoPHandanumberofotherdiseasessuchasPulmonaryFibrosisandAsthma.WiththeuseofnewcelltrackingtechniquesitisnowpossibletoobservehowcellsreacttotheenvironmentofPHandthesemethodscanthenbeadaptedforuseinnumerousotherresearchareas. 1.2PulmonaryHypertensionPulmonaryHypertensionisaconditionwherethemainhallmarkisameanpul-monaryarterypressure(MPAP)greaterthan25mmHgatrestand30mmHgde-terminedfromrightheartcatheterization[1].ThereareanumberofdierentformsofthediseaseandtheyfallintovedierentcategoriesbasedonthecurrentWorldHealthOrganizationdenition[2].GrouponeencompassesPHfromdrugandtoxininduction,tissuediseases,heritablePHandmoreidiopathicversionsofthedisease.GrouptwoisPHduetoleftheartdiseases.GroupthreeisPHduetootherlungdiseasesandinductionduetohypoxemia.GroupfourisPHcausedbytheincidenceofchronicthromboembolismsandcanbetreatedthroughtheuseofbloodthinners.GroupveisPHduetoanumberofmultifactorialcauses.Theanimalmodelsthatweutilizedinourstudiesmostcloselyresembledgroup1PH.OneofthemainpathologicalpresentationsofPHistherarefactionofsmallervascularvesselsandtheremodelinginvolvingtheirstieningandthickeninginthecelllayersoftheremainingvessels.Invascularvesselstherearethreedistinctlayersshowningure1.1.Theinnermostistheintimallayermadeupofamonolayerof 1

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Figure1.1:Thelayersofavessel.Consistingoftheintima,mediaandadventitia.Theadventitiaandspecicallyresidentbroblastsarethefocusofthisstudy. endothelialcellsonabasementmembraneoftypeIVcollagen.Belowthisisthemediallayermadeupofathickregionofsmoothmusclescellsthatregulatevesselrigidityanddiameter.Beyondthisistheadventitiawhichislesscelldenseandmainlycomposedofextracellularmatrix(ECM),mainlycomposedofcollagens,elastinsandglycosaminoglycans,withtheprimarycelltypepresentbeingbroblasts[3].InPHthevessellayersundergodistinctchangesknownasremodeling.Thisincludesthethickeningofalloftheselayersofthevesselandanoverallstieningofthevessel[4] 2

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Figure1.2:Anormalpulmonaryvesselandairwayonthelefthandsidewiththinvessellayer.Ahypertensivevesselwiththickenedremodeledlayersthatleadtoincreasedvascularresistance. seeningure1.2.Thisstieningleadstoanincreaseintheresistanceofthevesseltobloodowandafurtherloadontherighthearttocompensate.Theadventitiallayeriskeyintheregulationofremodelingallvessellayersandmayprovetobepotenttargetsitefortherapiesaimedatthereversalofvascularremodeling[3,5].Theprimarycelltypeintheadventitiaisthepulmonaryarterialbroblast(PAF).PAFsarekeysignalingcellstotheotherlayersofthevesselandtheirsecretionofmatrixproteinscontrolsthestructuralandmechanicalpropertiesoftheadventitiaandconsequentlythevessel[3].Hypoxia,acommonfactorcausingPH,hasbeenshowntoturnPAFstoachronicproinammatorystate[6]aswellasconvertingthemintocellsthatkeycontributorstotheprogressivepulmonaryremodelingseeninPHthroughthereleaseofsignalingandstructuralproteins[7].BothhypoxiaandthechronicinammatorystateofhypertensivevesselscausetheactivationofPAFswhichleadsthemtodierentiateintomyobroblasts[8,9].Myobroblastsarekeyintheprocessofinjuryhealingandtheassociatedacuteinammation.Whenbroblasts 3

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arekeptinthisactivatedstateitcanleadtotheprogressionofvascularremodelingandastateofchronicinammationinapositivefeedbackfashion[10]. a Figure1.3:BronchusAssociatedLymphoidTissues(BALTs)innormalandPHmodelrats aColvinK.L.etal Bronchus-associatedlymphoidtissues(BALTs)showningure1.3aredenselym-phoidtissuesfoundintheinterstitialspaceinthelungbetweenvesselsandairwaysandserveasimmunemaintenanceorgansthatmonitorthesterilityofthisarea.BALTshavebeenshowntoincreaseinnumberandsizeinPHandbegintopro-duceanti-broblastantibodiesleadingtoachronicstateofinammationwhichcanfurthercontributetomaladaptivebroblastbehavior[11].ThisfurtherinducestheactivationofbroblastsandtheprogressionofvascularremodelingfoundthroughtheincreasedcontentofproinammatoryproteinssuchInterleukins1and6,platelet-derivedgrowthfactorandmacrophageinamatoryprotein1[12].ThereforeshowingthatBALTsareakeyplayerintheprogressionofPH. 1.3JusticationTheimportanceoftheadventitiallayeranditsresidentPAFshasbeenwelles-tablishedintherealmofPHespeciallyasitpertainstovascularremodeling.ThecommonfactorsbetweenthelargelydierentetiologiesofPHthatleadtothesamepathogenicchangesinbroblastbehaviorhavenotbeenfullyestablished.Abroblast 4

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hastheprimaryroleofmaintainingtheextracellularstructureofthevesselthroughthereleaseofstructuralproteinssuchascollagenandelastinaswellasproteinsthatbreakdowntheseproteinslikematrixmetalloproteinases[3].Thisisnotonlyachem-icalhomeostaticbalancebutamechanicaloneaswellwiththeconcentrationsoftheseproteinspossiblyaectingthestructuralpropertiesofthelocaltissue.Fibroblastsareabletosensetherigidityofthematrixaroundthemselvesthroughtheuseofintegrinswhichattachtotheextracellularmatrixandcomplexwithfocaladhesionkinases(FAKs)[13].WiththeseattachmentstheyareabletogaugethemechanicalpropertiesoftheECMsurroundingthemandcanevenguidemigrationbasedonthis[14].MigrationbasedonthisisguidedinthedirectionofincreasingrigiditythroughmembranelocalizedsignalingofFAKs[15].Inadditiontoguidingcellmovementthemechanicalpropertiesofthematrixsignicantlyaectthephysicalmorphologyofadherentbroblasts[16]withstiermatricesleadingtoattercuboidalshapesandsoftermatriceshavingelongateddendriticlikestructures.Throughthesesamemechanicalsensingmechanismscellsareabletoperceivethetransductionofstressesimpartedbyuidowontothemselvesaswellastheextra-cellularmatrixsurroundingthem[17].Thesemechanicalsensingmechanismsguidecellmovementandleadtothelocalizationofproteinsinvolvedincellularmovementsuchasvinculin,actinandFAKtothepromigratorysideofthecell[18].Whenthereiselevatedarterialpressurevesselsnormallyhavecompensatorypro-teinexpressionmechanismstopreventthisincreasedpressurefromdrivingadditionaluidtodrainintotheintercellularspace[19].Itishoweverindicatedinchronichy-poxiathatthereisanincreaseinthelunguidinterstitialpressureandanincidenceoflungedema[20].Interstitialuidowcanhaveanumberofeectsontissues.Ithasbeenshowntoguidelympangiogenesis[21].InterstitialuidowcanalsobeutilizedbycellstoestablishautologousgradientsofsignalingfactorssuchasthosethatactofCCR7[22],whichhasbeenstudiedextensivelyasamechanismforcancercellhoming 5

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towardslymphaticvessels.ItcanalsoservetoguidecellularreleasedECMdegrad-ingproteasesdownstreamofthecelltofocusactivityinthedirectionofthecellsmigration.BasedonthisitcanbesurmisedthatmechanicalsensingmechanismsofPAFscoupledwiththechemotacticgradienteectsofsignalingfactorswouldcoupletogetherinacompetitivefashionwhereonemechanismwillbecomedominantovertheother.Thisbehaviorhasbeenseeninvitrowithcancercellswheredierentlevelsofuidowandstressforcesledtochangesinthemigratorybehaviorofcells[23].Fi-broblastsdevelopespecicbehaviorpatternsbasedonthemicroenvironmentaroundthemselvesaswellastheinterstitialuidowaectingthem[24]. 1.4GoalsInrecreatingtheinterstitialenvironmentofthelungsinaninvitromodeltwothingsmustbeaccountedforwiththerstbeingthethreedimensionalenvironmentofnativelungtissueandthentheuidpressureforcesthatcellsexperience.Thesespecicchangeswillaectthebehaviorofbroblasts[24].WehypothesizethatthereisincreasedinterstitialuidowinthepulmonaryadventitiainPHduetothehigherpressureheadinthepulmonaryarteriesandthebreakdownoftheendothelialuidbarrierandnormaluidconductionmechanisms.Thisincreaseduidowistrans-ducedasmechanicalstress[17]toadventitialbroblastsandmayguidemigrationofthesecellsaswellasconvertingthemtoanactivatedstate[25]wheretheycontributetothechronicinammationandprogressiveremodelingseeninpulmonaryhyperten-sion[10].Thiswouldoccurinaprogressivefeedforwardfashionwherethegreatertheremodelingthegreaterthestressimpartedonthecellsandthusfurtherincreaseduidow.WeinvestigatedthephysicalandchemicalcharacteristicsofthislungspaceinnormalandPHrats.Withthisinformationathreedimensionalculturingsetupcanbedevelopedthatmorefullyrecreatesthisspaceandthedierencesbetweenthisculturingsetupandmorestandardmethodscanbeevaluated.Finallytheeects 6

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ofinterstitialuidowoncellbehaviorespeciallymovementwillbeevaluatedandrelatedtohowthiswouldpertaintoPH. 7

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2.Methods 2.1MicrouidicCultureTheproblemwithobservingcellbehaviorinrealtimearisesfromtheneedtokeepcellsinaconsistentculturingenvironment,maintainingspeciccontroloverthecellsmicroenvironmentandestablishingaconstantviewwindow.Astandardculturingsetupwouldrequiretheconstructionofanewculturingchamberaroundamicroscopetomaintainaconstantincubationenvironmenthoweverasimplersolutioncanbefoundintheuseofmicrouidicchips.Inthissetupcellscanbeculturedinasmallmicroenvironmentwithveryprecisecontrolovertheculturingcharacteristicsandtheeaseofsimulatinganumberofdierenttissuespreciselyatvariousscales[26].Microuidicplatformshavebeendiculttodevelopconsistentlyandinahighthroughputmannerinthepastbutwiththedevelopmentofsoftlithographyandtheuseofdierentpolymerstherapidprototypingofmicrouidicdeviceshasbecomefarmorefeasible[27,28].Polydimethylsiloxane(PDMS)hasbyfarbecometheeasiesttoworkwithinthistechnique.PDMSisespeciallyattractiveintheapplicationofrealtimeimagingduetoitstransparentopticalpropertiesandrelativelyinertnatureuponpropertreatment[29,30].PDMSchipshaveevenbeendesigneddirectlyforuseinrealtimecellimaging[31].BasedontheseconclusionsaPDMSmicrouidicchipwaschosenastheculturingsetuptoproperlyrecreatethemicroenvironmentoftheadventitialvascularspace. 2.2Rat/CellLines 2.2.1RatAdventitialFibroblastsAgreenuorescentproteinGFPstrainofratswasusedforallPAFcellculturingwheresectionsofadventitiawereremovedfromthepulmonaryarteryandtheresult-ingcellsculturedinamixofDulbeco'sModiedEagleMedia(DMEM),10%FetalBovineSerum(FBS),withL-Glutamine,Penicillin,StreptomycinandNon-essentialaminoacids.Allcellsusedinexperimentswerebetween3and10passages.GFPis 8

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aproteintakenfromthejellyshAequoreavictoriawhichwillproduceandemissionpeakoflightat509nmwhenexcitedat395or475nm.ThisproteinwasstabilytransfectedintotheratswiththeCAGpromoterregion.CAGisasyntheticpromoterthatutilizesthecytomegalovirusenhancerregion,thepromotorofbeta-actinandthespliceacceptregionofglobin.ThisallowsforGFPtobeconstitutivelyexpressedinallcellsintheratCellsnotrequiringGFPexpressionwerefromSpragueDawleyratsculturedinthesamefashion. 2.2.2StainingTissuecryosectionscutat5mwerexedinmethanol:acetone(1:1).Nonspecicbindingwasblockedwithfetalbovineserum:phosphate-bueredsaline(1:1),andsectionswereincubatedforonehourwithprimaryantibodies.Secondaryantibodiesatgreen488andred594wereusedatdilutionsof(1:500).ImmunolabeledsectionsweremountedinVectaShield/DAPI(VectorLaboratories)andexaminedunderaZeissuorescentmicroscopewithanAxioVisiondigitalimagingsystem(CarlZeissMicroImaging,Inc.,Thornwood,NY).PrimaryantibodiesusedwereAquaporin1Anti-mouse(NovusBiologicals)(1:400),SMAAnti-Mouse(Abcam)(1:500),FSPS100A4Anti-Rabbit(Abcam)(1:200),Claudin-5Anti-Rabbit(SantaCruzBiotechnology)(1:300)andOx-62Anti-Rabbit(Abcam). 2.2.3RatModelsIFstainingwasperformedonlungsectionsfromSpragueDawleyrats.InthestudynormalDenverelevationratsaswellastwodierentPHratmodelswereused,monocrataline(MCT)inducedPHandPHinducedwithSugenandexposuretoahypobaricchamber.MCTisanalkaloidderivedfromtheseedsoftheCrotalariaspectabilisplant.Itisadministeredtrhoughasingleinjectiontotherat.Thisalkaloidisactivatedinthe 9

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liveroftheanimalbytheenzymecytochrome-P450intoapyrolicform.Theactivatedreactiveformhasanumberofeectsactingasatoxinthroughoutthebodyinthelungespecially.ItinitiallyactsupontheendotheliallayersofvesselsinthelungbynarrowinganddestroyingthevascularlumenleadingtoanintenseandirreversibleformofPHinrats[32].TheMCTmodelexhibitsadistinctremodelingofthevascularlayersofthevesselthroughintimalhyperplasiaandthickeningofthemediaandadventitia.MCTalsoactstoinducehepaticvenooclusivedisease,MyocarditisandRightVentricularhypertrophythroughthesamemechanisms[33].Sugen5416combinedwithahypoxicenvironmentisanothercommonanimalmodelforstudyingPH.Sugenisavascularendothelialgrowthfactor(VEGF)recep-torinhibitor.AdministeringsugentochronicallyhypoxicratsleadstoprecapillaryendothelialcellproliferationandvascularsmoothmusclecellproliferationleadingtothedevelopmentofpersistentPH.SugenalsohasthesuprisingtraitofonlyaectingVEGFreceptorsinthelungandnototherorgans[34].AlltissuesectionstakenforslicingtoslidesandsubsequentstainingwereobtainedfromSpragueDawleyrats.AllratswhichvascularpermeabilitywastestedwereSpragueDawleyrats. 2.3HAHydrogelThestandardcellculturingsetupwhichallowscellstogrowinaatenvironmenthasprovedessentialinbiologicalresearchfordecades.Itishowevernotafullrecapit-ulationofthethreedimensionalenvironmentthatcellsexperienceinvivoandmaynotentirelyrepresentthetruebehaviorofcellsintheirtrueenvironment.Themostcommonmethodtomorefullyrecreateinvivoconditionsisthroughthesuspensionofcellculturesinpolymerswiththemostcommonsetupbeinghydrogels.Hydrogelsarecross-linkedpolymericnetworkswhoseprimaryweightcomponentiswaterandthereforerepresentaverysparsesoftmatrixwithsimilarmakeuptonaturalsofttis-sues.Theyareanattractivetoolincellcultureowingtotheeasewithwhichcells 10

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canbesuspendedinthemandbiologicalcompoundscanbefunctionalizedintothematrix[35].Themakeupofindividualcomponentsinthegelcanbetailoredtopro-videspecicmechanicalandmorphologicalpropertiesbasedonthedegreeandnatureofpolymercrosslinking[36].Thereareanumberofcommerciallyavailablehydrogelsthathavebeendevelopedandextensivelytested.ForourpurposestostudyPHagelthatrecreatestheadventitialenvironmentofthelungswaschosen.Thelungisahet-erogeneouslooselybroustissuewithsoftmechanicalproperties.IthasbeenshownthatbroblaststhatleadtopathologicalinterstitiallungdiseasesduetobroblastdysfunctionsuchasbrosishavebeendirectlylinkedtohyaluronananditsreceptorCD44[37]aswellasinthetransdierentiationofbroblaststomyobroblasts[38]wetherforechosetouseahydrogelincorporatinghyaluronan.TheHydrogelcho-seninthiscasewasHystem-CtrademarkbyESIBio.Hyaluronanbasedhydrogelshavebeentestedandprovedtobebothbiocompatibleandcapableofsupportingcellgrowthwhenencapsulatedinthegel[39,40,41,42].Thisspecichydrogelhasalsobeentestedforbiocompatibilitywithbroblastsaswellandsupportedcellsgrowthandproliferation[43].TheHystem-ChydrogelhasamakeupofHyaluronicAcid(HA),DenaturedCol-lagen(DC)andCrosslinkingPolyethyleneGlycoldiacrylate(PEGDA).InthissetupbymixingthethreecomponentscrosslinkingwilloccurbetweenthediacrylicgroupsonPEGDAandthiolgroupsonHAandthecysteineresiduesonDC.Thiscomplexmeshingreactionisshownintable2.1andcrosslinksPEGDAtothiolgroupsonHAandDC.Thehydrogelchosenoersthecapabilityofhavingitsthreecomponentsalteredinconcentrationwhichinturnwillalterthemechanicalpropertiesofthegel.Thelocalmechanicalpropertiesvariessignicantlyacrossdierentareasofthedecellularizedlungwhenmeasuredwithatomicforcemicroscopy[44]andtheelasticmodulusofthesimplematrixsecreteddirectlyfrombroblastsinculturecanbeanorderof 11

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a Figure2.1:Hydrogelusedforcellculturecrosslinkingreaction,CHMA-SisHA,Gtn-DTPHisDCandPEGDA aJ.L.Vanderhooftetal magnitudelowerthanthis[45].Basedonthisvariationfourdierentmixeswereusedtotestdieringeectsofbothhydrogelmechanicalpropertiesandchemicalmakeup.Theelasticmodulusofthesedierentmixeswascharacterizedthroughastudyusingrheologicalmeasurementtechniques[46].Thisisshownintable2.1. 2.4ScanningElectronMicroscopyScanningelectronmicroscopy(SEM)isausefultoolforanalyzingtheultrastruc-tureandporesizeoftissuesandhydrogels.Thisismainlyduetothesuperresolutionofelectronmicroscopymakingitpossibletorevealthefeaturesofsampleswithnerdetailthanstandardmicroscopytechniques.Thesemorphologicalcharacteristicsareimportantbecauseananalysisofthespacecanrevealingreaterdetailthechangesthatconsequentlyleadtostructuralchangesandthereforefunctionaldierencesinthevesselbetweenstandardandhypertensivelungtissue.Whenappliedtoinvitro 12

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Table2.1:WeightpercentagesofdierentcomponentsforthefourhydrogelmixesandtheassociatedelasticmodulusasdeterminedbyJ.L.Vanderhooft. Mix HAweight% DCweight% PEGDAweight% ElasticModulus(Pa) 1 0.8 0 0.8 720 2 0.7 0.6 0.6 520 3 0.56 0.24 0.4 270 4 0.8 0.8 0.2 180 a aPropertiesfromJ.L.Vanderhooftetal studiesthisisimportantbecauseHAhydrogelswithlargerporesizeshavebeenshowntosupportincreasedproliferationofbroblastsprovingthattheporosityofthetissuehasasignicanteectofcellularbehavior[47].Poresintheadventitiaaretheopenspacesinthetissuenotmadeupofproteinorcellsandtheresizeandorientationcanindicatethemechanicalpropertiesofthetissueaswellasthebehaviorofthesecells.SEMwascarriedoutwithaJEOLJSM-6010LAmicroscopeforallsamples.Thiswasdoneforthefourmixesofgelstoprovidequalitativecomparisonsforthetissues.Obserevationswerecarriedoutatlowpressureconditionsatamagnicationof100and300x.SEMwasalsocarriedoutonnormoxicandfourweekmonocrotalinelungtissuesections.Allsampleswere5micronthickslicesplacedontoglassslidestreatedwithxyleneforonehourtodeparanize,criticalpointdriedinaLeicaEMCPD300dryer,andsputtercoatedwithgoldbyaLeicaEMACE200coater.TheSEMimagesweretakenoftheadventitialregionsofvesselsat500xandanalyzedforporesizeandmeanvoidfraction.ThiswasdonewithacustomMatLabcode.InthiscodesubsectionsoftheimagesfromtheSEMfocusedinontheadventitialspaceofvesselexcludingallothersectionsoftheimage.A5x5Gaussianlterwasappliedtotheimagetosmoothoutthenoiseandthiswasconvertedtoabinaryimage. 13

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Thisresultingbinaryimageallowedfortheanalysisofvoidfractionoftheregionofinterestasasimpleratioofpixelsthatwererepresentedtissueandthosethatdidn't.Theporesizeoftheimageswasdeterminedusingacodethattracedlinesthroughvoidspacesandmeasuredthesizeofthesegaps.Thiswasconstructedintotheadistributionofmeanfreepathsofthegelsinboththexandydirections.Thisisshowningure2.2.StatisticalanalysiswasperformedintheformofaWelch'st-testforunequalvariancetoanalyzethesignicanceofthesizeofthesedeterminedporesandwhetherthedistributionsofporesizeweredierentinthexandydirectionsaswellasbetweenthenormalanddiseasedanimals.Thecodeforthisanalysisisshownintheappendices. 2.5DecellularizationofLungTissueDecellularizedsectionsofratlungsweregeneneratedinordertostudythebasicscaoldonwhichcellsaregrown.Tobeginwiththewholeheartlungblockwasremovedfromaproperlyeuthanizedrat.Decellularizationswereonlyperformedonleftlungssotherightlungairwaybranchwastiedointheblock.Acannulawasinsertedintothetracheaandwastiedoaboveusingsutures.Thelungwasthenrunin9cyclesofushesconsistingof4ushesofdeionizedwater,4ushesofa4%deoxycholatewatermixand4ushesofDNase-1in1MNaCLwitheachushbeing6mL.Thelungwasthenushed10xwithDMEMandstoredinDMEMat4degreesCelsius. 2.6ConfocalMicroscopyofDecellularizedTissueThedecellularizedlungsectionsthatweremadeearlierwerecutintothinslicesanda10mmbiopsypunchwasusedtoseparatesectionsofthelung.ThesesectionswerewashedrepeatedlyinDMEMandamixtureofcellswereaddedontopandincubatedintothemfor2days.ResidualGFPonthetissueandautouorescenceofelastinandcollagen,theexcitationlaserwas488nmandpeakemisionwavelengthat509nm,allowedfortheimagingofbersleftbehindinthetissue.Cellswerestained 14

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(a) (b) Figure2.2:(a)SEMimagetaken,subsettedtotheadventitialspace,gaussiansmoothedandthenbinarized(b)Poresizeanalysisinthexandydirections 15

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withuorochromPKH26whichallowedforthelivecelltrackingofpositionswasex-citedwithalaserat561nm,withpeakemissionat567nm.700x700x50Micronimagesweretakenwithzplaneimagestakenevery1micronofthetissuewitha3IMAR-IANASinvertedspinningdiskconfocalmicroscopeat100xeectivemagnifacationwitha10xZeissECPlanNeouor0.3NAairobjectivewasused.ThestackimagesofthedecellularizedlungwereloadedintotheprogramScanIPwhichgenerateda3Dimageoftheconfocal.AmaskoftheECMwasgeneratedandsavedasa.STLproject.ThisallowedthemodeltobeconvertedtoaCADlewhichcouldthenbemadeintoascale3Dprintedpart. 2.7HydrogelCulturingStainingandMorphologyFibroblastswereculturedinstandard2Denvironmentonaatplatefortwodaysin10%FBSDMEMat37degreesCelsiusand5percentCarbonDioxide.Cellswereculturedontopofthegeltypesforoneweek.Cellswereadditionallyculturedinsidethesegelsforoneandtwoweeks.Oneandtwoweeksofcellculturetimewerechosenbecausecellsgrowninsidegelstooklongertocoverthefullwindowofviewthanwhenculturedonaatplate.ThesecellswerestainedwithantibodiesforSmoothMuscleActin(SMA)Sigma-AldrichwithsecondaryantibodyGoatAntimouseAlexaFluor488andFibroblastSpecicProtein(FSP)ABCAMandsecondaryantibodyGoatAntirabbit594.Thisallowedforthelocalizationofexpressionandphenotypetobedeterminedthroughuorescencephotography.Thesedyesalsoallowedforthemorphologyofthecellstobecompared.ThesewerealldoneonanAxiovertS100witha10xZeissPlan-NeoFluor0.3NAobjectiveandaFilterCubeSliderModel3FLwithdichroicltercubesfor:excitation405nmwithemissions435nm,excitation480nmwithemissions510nmandexcitation525nmwithemissions595nm 2.8HydrogelHistologyCellsculturedinathreedimensionalhydrogeldonotstaystaticandwillalterthemicroenvironmentaroundthemselvesbothphysicallyandchemically.Thisrela16

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tionshipwasshownwhenvocalcordbroblastsculturedinasimilarHAandcollagengelslowlybrokedownthecollagenandHAinthegel[48].InordertoexaminehowthegrowthofPAFsinhydrogelschangesthestructureofthehydrogelstheywerepreparedinasimilarmannertohowtissuesectionswereexaminedpreviously.Gelswereslicedontoslidestocapturethetwodimensionalstructureofthesegels.Thesecanbestainedandimagedtoexaminethestructuralpatternsofthesegelsascellsembededinsideofthemchangethestructureovertime.Thesegelswereculturedinthesamemannerasothergelsforoneandtwoweeksofcellsgrowthaswasacontrolofthegelwithnocellsinmediafortwoweeks.ThesesampleswerethenxedinTissue-TekOptimalCuttingTemperatureCompoundandfrozenat-80C.ThegelswereslicedatathicknessoftenmicronsandastandardHematatoxilynandEosin(H&E)stainingprocedurewasfollowed.Microscopicexaminationsofthegelswerethencarriedout.ThephysicalcharacteristicsofthegelwereexaminedandcomparedtothetrueinvivoenvironmentofPAFs.ThistwodimensionalrepresentationcanalsobecomparedwiththethreedimensionalstructureofcellgrowthseenthroughIFstaining.ThesewerealldoneonanAxiovertS100witha10xZeissPlan-NeoFluor0.3NAairobjective, 2.9PCRInthesamewaythatcellswillalterthematrixaroundthemselvesinresponsetothissameenvironmenttheymayalsochangetheirtranscriptionprolesofteninamannerthatfacilitatesthischange.TofacilitateananalysisofthesetypesofchangesandhowtheymayrelatetoPHtheexpressionpatternsofseveraldierentspecicproteinswereexamined.Fibroblastspecicprotein(FSP)isamemberofthecalmodulinS100troponinCsuperfamilyandisanimportantmarkerforbrob-lasts.UpregulationofFSPinbroblastshasbeenassociatedwithbleomycininducedpulmonarybrosis,especiallyasitpertainstoincreasedcollagendepositionandlungarchitecturechanges[49].Thisbehavioralpatternwouldleadtoastieningofvessels 17

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throughtheincreasedcontentofcollagenintheadventitialspaceofvesselsandthere-forethedepositionofcollagenshouldalsobeexamined.Collagen1A1wasshowntohavethelargestincreaseinthelungsofmiceexposedtochronichypoxia[50]andaprimarydegradingenzymeofcollagen1A1ismatrixmetalloproteinase-1a(MMP1a).MMPsservethefunctionofregulatingtheturnoverofanumberofdierentcollagensinacycleofcontrolwithtissueinhibitorsofmetalloproteinases(TIMPS).HypoxiaandMonocrotalinehavebothprovedtoaecttheMMP-2/TIMP-1balance.Hypoxiainducestheupregulationofbothandachangeofbroblaststoamoremyobrob-lasticphenotype[51].CountertothisTIMP-1upregulationandconsequentdownregulationofMMPinmonocrotalineinducedhypoxicratsleadstoaremediationofthevascularremodeling[52].Takentogetherthesetwocyclesindicatethatthecontrolofinammationandremediationofvascularremodelingisacomplexprocesswithmanyregulatoryfactors.Sincemyobroblastshavebeenshowntohaveagreaterbroticcapacitywhichwillinturnleadtostiervessels.Theregulationofmarkersforbroblasttomyo-broblastdierentiationneedstobestudied.Twomarkersrelatedtothisaresmoothmuscleactinalpha(SMA)andtransforminggrowthfactorbeta(TGFB).TGFBcanregulatetheexpressionofSMAandthetransitionofbroblaststoamyobroblastphenotype[53,54,55].Smoothmuscleactinhasalsobeenshowntoincreasethecon-tractilecapacityofbroblastsloweringtheircompliancetherebystieningthevesseltheyareassociatedwith[54,56].TheincreasedproductionofSMAhasalsobeenlinkedtogreatercollagenproduction.Thisleadstothedirectstieningoftheasso-ciatedtissuethroughthecollagenitselfaswellasandindirectstieningbyallowingformorecontractileattachmentpointsforthecelltotheextracellularmatrix[56].AdventitialbroblastswerealsoshowntocoexpressTGFBandSMAsuggestingthatTGFBmayplayanautocrineroleinSMAproduction[57].IndirectrelationtoPHhypoxiahasbeenshowntoincreasetheexpressionofSMAinPAFs[58]. 18

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PCRwasperformedonPAFsgrownforoneandtwoweeksinthefourhydrogelmixesalongsidecellsgrowninastandard2Dculturingsetup.ThisanalysiswascarriedouttodeterminewhatchangesPAFsunderwentwhenbeingtransferredfromastandardculturingsetuptoamoreinvivolikeenvironmentandwhethercellsproceededtoremodelthetissuearoundthemselvesinanyfashion.ThiscouldberelatedtoPHbecauseitwouldshowthedirectprocessofhowPAFsalteredtheirmicroenvironment.ToextractRNAfromPAFculturesaQiagenRNeasyMicroKitwasused.AMixof10microlitersofBeta-MercaptoethanolpermilliliterofRLTBuerwasaddedtothecellgelmixesandthenhomogenized.Anequalvolumeof70%ethanolwasaddedtothemixandtheMixturewastransferredtoaRNeasyMinElutespincolumnandcentrifugedfor15secondsat12000g.350microlitersofBuerRWashwasaddedtothecolumnandcentrifugedfor15secondsat12000g.500microlitersofBuerRPEwereaddedandthecolumnwascentrifugedfor15secondsat12000g.500microlitersof80%ethanolwereaddedandcentrifugedfor2minutesat12000g.Thecolumnwasopenedandcentrifugedat15000RPMfor5minutestodry.14microlitersofRNasefreewaterwereaddedandcentrifugedfor1minuteat15000gintoanewtubeforRNAcollection.TheextractedRNAwasturnedintocDNA.ATaqmanqPCRprotocolwasfollowedwithtwosamplesrunforeachmixandPCRprimer.HRPT2/CDC73Paf1/RNApolymerase2complexcomponentwasusedasacontrolforthemixes.ThecDNAmixturewasdilutedtoaconcentrationof50ng/sampleandcombinedwith1microliterofTaqManGeneExpressionAssayagainstthetargetsmentionedpreviouslyand10microlitersofTaqManGeneExpressionMasterMixand9micro-litersofRNase-freewater.Thesampleswererunonastandard96wellplateandrunwiththeprotocolforuorophore6-carboxyuorescein(FAM)andquencherdi-hydrocyclopyrroloindoletripeptideinanAppliedBiosystems7300RealTimePCR 19

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System. 2.10MechanicalStrengthTestingMechanicalstrengthtestingwascarriedoutusingaUniaxialMTSInsightma-chinetodeterminetheModulusofcellularandacellularratlungtissue.ThesesectionsoflungtissuewereharvesteddirectlyfromratlungsamplesharvestedfromnormalSprague-Dawleyrats.Testswereperformedonbothcellularanddecellularizedseg-mentsoflungthathadallpleuraltissuecutoutandsectionscuttotheapproximatedimensionsof5x5x20mm.Themoduluswasdeterminedthroughtheuseofastressstraincurvegeneratedfromthemachine.AMatLabcodedevelopedbyJenniferWagnerwasutilizedtoimportthisdatafromtheMTSmachineintoMatLab.AcustomMatLabcodewaswrittenthatinterpolatedacurvetoaregionofthegraphandfoundthesectionofthecurvewiththeleastchangeinslopeandthusdeterminedtheelasticregionofthecurve.Thisisshowningure2.3. Figure2.3:SlopeInterpolationofStressStrainCurve 2.11ImmunouorescentStaining 2.11.1FibroblastSpecicProtein,SmoothMuscleActinandOX62ThemovementofbroblastsandtheirlocalizationinthelunginPHisofinterestbecauseitindicateshowtheyarelocalizedandwheretheywillbeprimarilyinteract-ingwiththeenvironmentandchangingthematrix.BALTsorcollectinglymphatic 20

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regionshavebeenassociatedwithPHandareofinterestwhenitcomestoPH.Thealsoservethepurposeofcollectinginterstitialuidowofthatexitsvesselssotheirbehaviorintissueswithleakyvesselswouldbeofinterest.AswasstatedpreviouslyFSPisamarkerforbroblastsbutitcanalsoserveasamarkerfordendriticcells.TotrackthemovementofbroblastsFSPcanbeusedasamarkerwhenanothermarkerprovesthethatcellsarebroblastsandnotdendriticcells.ThemarkerchoseninthiscasewasOx-62orintegrinalphaE2whichisanothermarkerfordendriticcellsandisimportantinthemigratoryactivityofthesecells.StainingforFSPandOx-62wasthereforeperformed.ToanalyzethisslideswerecostainedwithFSPandOx-62andtheimage-JpluginforanalyzingMander'sCoecientswasutilizedandthePearsoncorrelationcoecientforthetwowasfound.SMAisamarkerforsmoothmusclecellsandcanallowforthemediallayerofvesselstobeidentied.AlloftheseobservationswerealldoneonanAxiovertS100witha10xZeissPlan-NeoFluor0.3airNAobjectiveandaFilterCubeSliderModel3FLwithdichroicltercubesfor:excitation405nmwithemissions435nm,excitation480nmwithemissions510nmandexcitation525nmwithemissions595nm.OX-62wasstainedforwithABCAMOx-62withsecondaryantibodygoatantimouseAlexaFluor594andFSPABCAMandsecondaryantibodygoatantirabbitAlexaFluor488.SmoothMuscleActin(SMA)Sigma-AldrichwithsecondaryantibodygoatantimouseAlexaFluor488andFSPABCAMandsecondaryantibodygoatantirabbitAlexaFluor594. 2.11.2Claudin-5andAquaporin-1Claudin-5isakeyregulatorofthevascularpermeabilityofbloodvessels.ClaudinstogetherwithVE-cadherinactinconcerttostitchupthejunctionsbetweenendothe-lialandepithelialcellspreventingthemovementofuidpastthesebarriersintointerstitialspaces[59].ForClaudinssimpleexpressionoftheproteinisnotenoughtoregulatevascularpermeability.TheseClaudinsmustalsobelocalizedtotheright 21

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regionsofthecellmembraneietheconnectionpointsbetweentwocells[60].Ifthislocalizationislostthantherewillbeasignicantincreaseinvascularleak.Inthiscasethereeitheradecreaseinproteintranslationoralossoflocalizationofthepro-teintothecorrectregionscanleadtoanincreaseinvascularleak[61,62,63,4,64].AnumberofsignalingfactorssuchasvascularendothelialgrowthfactorcanaectthispermeabilitybutmorespecicallychronicinammationhasbeenshowntodecreasetheexpressionlevelsofClaudin-5[65].A3IMARIANASinvertedspinningdiskcon-focalmicroscopewitha40xZeissECPlanNeoFluor0.75NAairobjectivewasused.Claudin-5wasSantaCruzwithsecondaryantibodydonkeyantirabbitAlexaFluor594.Thesamplewasexcitedwithalaserat561nmwithmaximumemmissionat617nm.Inadditiontotheregulationoftheamountofuidthatpassesfromavesseltotheinterstitialspacethemovementofthisuidthroughtheinterstitialspaceisregulatedbykeyproteinssuchasaquaporin-1(AQP).AQPiswaterchannelthatallowsforfreepassageofuidacrossalipidbilayermembrane.WhilelungfunctionispreservedthroughthedeletionofAQP1and5inmicethereisatenfolddecreaseinthepermeabilityofthelung[66,56].Inhumanswithanaquaporinnullgenotypewhenintroducedtoasalinepulmonarychallengetherewasnochangeinairwaywallthicknesscomparedtoa44%increaseinthicknessforcontrolsubjects[67].Thisindicatesthataquaporinisakeyregulatorofuidtransportinthelung.ThesewerealldoneonanAxiovertS100witha10xZeissPlan-NeoFluor0.3NAobjectiveandaFilterCubeSliderModel3FLwithdichroicltercubesfor:excitation405nmwithemissions435nm,excitation480nmwithemissions510nmandexcitation525nmwithemissions595nm.Aquaporin-1wasNovusbiologicalswithsecondarygoatantimouseAlexaFluor488.AlloftheseIFstainingexperimentswerecarriedouttoevaluatetherelativepermeabilityoflungtissuetointerstitialuidowandobservedierencebetweennormalandPHratlungs 22

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2.12CellASICsCultureSystemToutilizeamicrouidicculturingwithtightlycontrolledconditionsaEMDMilli-poreCellASICsOnixplatformusingtheM04Gplatewaschosen.Withthisplatformuidcanbeoweddirectlydownthechannelbyturningonpressureatthefeedwellsofthechamberandwherecellsarebeingculturedineitheratwodimensionalsetuporathreedimensionalsetup.PAFsareowedintothechamberfromtheseedwellataconcentrationof5x106cells/mLseeningure2.4.Thecellsareincubatedinthesamewaydescribedearlierfortwodays.Aftertwodaysowisturnedondownthechannelshownbythedirectionofthearrowingure2.4.Snapshotsofthecellsaretakeneveryhalfhourforatwelvehourperiodandconstructedintoatimelapsedvideoofcellbehavior.Conditionsofcontrolornoappliedpressure,1PSIofappliedpressureor6895pascalsand4PSIor27580pascalsofappliedpressuresforforcingowwasemployed.Thesepressureswerechosenbecausetheyallowedfortheawiderangeofconditionstobeobserved.ThesewerealldoneonanAxiovertS100witha5xZeissAPlan0.12NAobjectiveandandaFilterCubeSliderModel3FLwithdichroicltercubesfor:excitation405nmwithemissions435nm,excitation480nmwithemissions510nmandexcitation525nmwithemissions595nm Figure2.4:ThecellASICchamberwithGFPcellsgrowninsideofit.Redarrowdesignatedthedirectionofuidow. 23

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Therealtimeculturing,celltrackingandimagingofcellsutilizingtheCellASICapparatuspresentedanumberofdiculties.TherstbeingtheinconsistencyofthePDMSchipsthatweremanufactured.AlargenumberofthechipscontaineddryuidicchannelsorhighsalinityPBShydratingthem.Theseledtoinitiallyinconsistentculturingconditions.TherewereproblemsinembeddingcellsinsidethehydrogelsinsideofthecellASICsystemandgettingthemtogrowastheydidinstandardwellplates.Inordertoaddressthiscellswereowedintotheplatewithoutgelsandallowedtogrowonallfoursidesoftheculturingchamberorstretchedinbetweeninapseudo3Dfashion.Whenseededinextremelyhighdensitiesthesecellswouldformlargecellmassesthatstretchedbetweenthetopandbottomofthechamber.Thistypeofbehaviorisshowningure2.5avialightmicroscopy.In2.5bthissameregionwasstainedforSMAandFSPwhichrevealeddierentialexpressionofbothofthesemarkersthroughoutthechamberindicatingthatthesecellsmayhavedierentiatedintoothercelltypesotherthanPAFS.Thisnewcelltypewasnotknown.Duetothislackofconsistantexpressionandlargecellmassesmakingindividualcelltrackingdicultduetothedicultyindierentiatingthemfromeachotheranoptimalseedingdensityneededtobefound.Thisdensityofseedingwouldhavecellsnotbefullyconuentand/orformmassesbutalsonottodiuseleadingtopoorcelltocellsignaling,interactionandoverallpoorsurvival.Testsat1,5,10and20x106cells/mLwereexecutedtodeterminetheoptimalcellseedingdensity.Thecellseedingdensitythatwasarriveduponasoptimalforculturingthatallowedpropercellstrackingwas5x106cells/mL.Anexampleofthisseedingisshowningure2.6awithgreenbeingGFPandredFSP.Ingure2.6bwithFSPaloneitcanbeseenthatthesecellshaveamoreuniformexpressionpatternofFSPindicatingthattheyarenotmaintainingastablephenotype.Oncethisoptimalseedingdensitywasdeterminedcelltrackingprotocolscouldbeestablished.Pressurewasappliedtothetopofthesystemandthemajorityofthe 24

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(a) (b) Figure2.5:Cellsseededatahighdensityof20x106cellspermL(a)Lightmicroscopyimage200xafteroneweekofculture.Thecellshaveformedlargemasses,shownwithblackarrows,thatareaverydierentbehaviorfromwhatisseenwhencellsarenormallygrownonaatplateandbecomeconuent(b)ImmunouorescentstainingofthechamberrevealsthesecellshavedierentialexpressionofSMA(green)andFSP(red)suggestingthatthecellsmaynotbemaintainingastablephenotype. 25

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(a) (b) Figure2.6:Cellsseededatadensityof5x106cellspermL(a)IFmicroscopyimage100xaftertwodaysofculture.Thecellscanbevisuallydierentiatedfromeachotherasindividualswhichallowsforcelltrackingalgorithmstobeappliedtothem.(b)IFstainingofthechamberforsimplyFSPinredshowsthatthecellsmaintainamorebroblasticphenotype. 26

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pressuredropwasabsorbedbeforethechamber.ThecellASICsoftwareonlyallowedforpressuretobeappliedinunitsofPSIwhere1PSIisequivalentto6895PascalsExperimentscouldonlybeconsistentlyrunatcontrolconditionswithnoappliedowandapressureupto6895Pascals.AnyhigherthanthatthewastewellswouldllupbeforetheexperimentwasoverandcauseabackupofpressurewhichwouldseparatethePDMSchipfromthebaseofthechamber.27580PSItestswereexecutedbutthishighowratelimitedthetimethattheseexperimentscouldberunsothesewereshorterexperiments.Twotestswereexecutedatcontrolowandtwoat1PSIofappliedpressure.Thereasonwhyonlythesetwopressureswereusedwasduetothetimeconsumingnatureofexperimentsandthelackofplates. 2.13CellTracking Figure2.7:SnapshotofIcycelltrackingsoftwarescellstracks.Notetheredcircleswhichdesignatethecenterofmassofeachofthesecells Thetimelapsevideoofcellbehaviorallowedatrackingofcellularmotiontobeperformed.ImageswereconvertedtobinaryimagesandGaussiansmoothedwitha4x4ltertoreducenoiseintheimage.ThiswasdonewiththeopensourcecelltrackingpluginfromICY.ICYutilizesthemethodofmultiplehypothesistrackingandanimageofthecellstrackingisshowningure2.7.ThetrackofcellularpositionsaspreadsheetofthesepositionswasgeneratedandimportedintoMatLab.MatLab 27

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codewasconstructedtolookattheindividualtravelofcellsandconstructananalysisofpreferreddirectionsoftravel.Thisanalysiswasnormalizedtoonlyincludecellswhohadtravelledfartherthanaspeciedminimumdistanceof1microntominimizecellstoremovenonmigratorycellsfromtheanalysis.Statisticalanalyseswerreperformedtodeterminethepreferenceofcellmovementoverallandmorespecicallywithoragainstowasdescribedintheresultssection. 2.14SpinningDiskFluidDynamicsCharacterizationInordertocharacterizetheshearforcesthatcellswereexperiencinginthecham-berthevelocitythatuidwasmovingthroughthesechannelsneedtobedetermined.Todothisuorescentbeadswereplacedinuidinthewellsandowedintothemicrouidicchannels.ThesamecelltrackingsoftwareandMatLabcodeusedforcelltrackingwasadaptedforthisinstancetodeterminethespeedofuidmovementinthechannels.ThebeadsusedinthisinstancewerePolyscienceInc.FluorsbriteMul-tiuorescent0.2micronMicrosphereswithexcitationmaximaat377nmwithemissionmaximaof546nm.Thesmallsizeofthesebeadsminimizedtheirinteractionwiththeirsurroundingenvironmentandallowedthemtotravelthroughsmallspacesinthechamber.A3IMARIANASinvertedspinningdiskconfocalmicroscopewitha40xZeissECPlanNeoFluor0.75NAairobjectivewasused.Theexcitationlaserwasat488nm.Thissetupwaschosenbecauseitallowedforrapidcaptureandthesegmentationofthecapturewindowtoasingleplaneintheaxialdirection. 2.15SolidWorksSimulationFluidshearstressisafactorthatchangescellularbehaviorespeciallyasitpertainstotheinterstitialuidowandcanleadtochangesincellbehavior[68].Itwasthereforeofinteresttodeterminetheshearthatcellsinthemicrouidicchamberwereexperiencing.Withtheuidvelocityestablishedfromthespinningdiskbeadtrackingauidvolumetricowratecouldbedeterminedforthedierentpressuresestablishedby 28

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theCellASICsapparatus.BasedonthegivendimensionsoftheCellASICsplatea3DCADreconstructionofthemicrouidicchannelwasconstructedinSolidWorkswithsimulatedcellsinsertedintothemodel.WiththisreconstructionthecomputationaluiddynamicspackageinSolidworkswasusedtoconstructacomputationaluiddynamicssimulation.ThedensityandviscosityofDMEMbeing0.99g/cm3and0.78centipoise.Withthissimulationandtheowvaluesdeterminedfromthebeadtrackingdonewithspinningdiskconfocaltheowthroughthesystemcouldbesim-ulated.Withtheexamplecellsinsertedinthechambertheinteractionofuidowwiththecellscouldbeobservedandanintegratedshearstressforthecellextracted.ThesimulationwasrunatsteadystateconditionsandutilizedSolidWorks'adaptiveCartesianmeshrenementsystemandasecondordersimulationwithautomaticallyrenedstepsizetobestable.Itconvergedafter112iterations. 2.16VascularPermeabilityTestToestablishthevascularleakinananimalmodelofPHanEvansBlueDyevascularpermeabilitytestwasexecuted.EvansBluedyehasahighanityforserumalbuminwhichisnormallypreventedfromleakingfrombloodvesselstothevascularspace.BymeasuringtheamountofEvansbluedyethatleaksfromvesselsintotissueandcomparingitbetweenanormalandPHratametricofvascularleakcanbeestablished.Inthistest3gramsofEvansBluedyewassuspendedinsterilephysiologcalsaline.Ratswererestrainedandgivenatailveininjectionequivalenttoonemicroliterofbluedyepergramoftherat'sbodyweight.17minutesaftertheinjectiontheratwasgivenalethaldoseofSleepAwayandthechestcavitywasopenedat19minutesandaninjectionof1%PFA,0.05Molar,pH3.5solutionintotherightatriumandlungstoxtheEvansBlueDyeinthelung.Theheartlungblockwasexcisedandthewetweightofthedierentlobesoftherightlungaswellasathreeslicedivisionoftheleftlungwererecorded.Thesetissueswerethenplacedinseparatescintillationvials 29

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lledwith5mLofformamideandincubatedina56degreeCelsiusoven.24hourslaterthetissuewasremoved.Theamountofblueintheformamidewasquantiedasameasureofcolorlevelatawavelengthof620withastandardcurve.TheresultswerecomparedasaratioofthemicrogramsofEvansbluepermLinthetissuetothedryweightofthesample. 2.17HydroxyprolineAssayInordertotesthowcellswouldremodelthedierenthydrogelmixesin3DcultureaHydroxyprolineassaywasutilized.TheHydroxyprolineassaymeasuresthecontentofcollageninasample.Collagenisamajorconstituentoftheextracellulsrmatrixandisproducedbybroblasts.Aswasstatedpreviouslyitsincreasedproductionhasbeenassociatedwithpulmonaryhypertension[50].Cellsweregrowninthefourgelmixesmentionedpreviouslyforoneandtwoweeksaswellascontrolgelswithnocellsculturedinsidethem.Atthetimepointsasampleofthegelwastakenanditsweightmeasured.Thesampleswerethenhomogenizedin100microlitersofwater.100microlitersof12MHClwasthenaddedandthesamplewasincubatedat110degreesCelsiusfor18hours.10microlitersofthissolutionwastakentoa96wellplate.ASigma-AldrichHydroxyprolineAssaykitwasthenutilizedonthesamplesperthegiveninstructions.TheabsorbanceoftheSampleat560nmwasthenmeasuredandtheamountofcollagendeterminedagainstastandardcurveofcollagencontent.Withthemeasurementofcollagencontenttheratioofcollagentototalsampleweightcouldbedetermined. 2.183DPrintingInordertofullltospeedofuptheresearchprocessinthisprojectthemethodof3Dprintingwasutilizedtoconstructpartsthatlledspecicfunctions.3Dprintingwasalsocarriedouttogeneratethreedimensionalrepresentationsoftissuetoallowforaphysicalmodelofimagestobeheld.ThiswasdoneusingtheSolidWorks3D 30

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CADprogram.ThepartsthemselveswereprintedusingtheProJetx60seriesfuseddepositionprinter. 31

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3.Results 3.1ScanningElectronMicroscopy 3.1.1HydrogelImagesAqualitativeanalysisofthedierenthydrogelmixtureswasperformedwithSEMtheonlyqualitativeanalyseswereabletobecarriedout.Thisdidrevealsignicantmorphologicaldierencesinthegelsappearanceasisshowningure3.1.In3.1awithhighstinessandlowcollagencontentthegelismadeofsmoothersheetswithsmallerpores.Asthestinessisdecreasedandthecollagenincreasedasisshownin3.1banddtheporesizesincrease.Whenthecollagenisdecreasedagainbutstillasoftergelitonceagaindevelopssmallerporesasisshowninc. 3.1.2RatTissueImagesInexaminingtheadventitiaofratsthreenormoxicandthreehypertensiveratswithSEMvariousproximallargearterybloodvesseladventitialregionswereseenwiththemethoddescribed.Thisvoidfractiondatafortheregionaswelladistributionxandymeanfreepaths.Withthexmeanfreepathrunningparalleltothevesselandtheymeanfreepathrunningperpendiculartoit.Eachofthesemeanfreepathdistributionscontainedanywherebetween500and3000datapoints.Anexampleofameanfreepathdistributionisshowningure3.2.Fromthisitcanbeseenthatthedistributionofmeanfreepathlengthsfallsintoafairlyuniformnormaldistribution.Whenexaminingthedierencebetweenthexandtheydistributionsitcanbedeterminediftheporeswereofacircularmakeupwereequallyrandominsizeorhadanovalpreferenceofmovement.Thiswasdonewithapairedttest.Ofthe21poreanalysesinallbutonethexandydistributionswereshownbedierentfromeachotherwithaWelchst-test.Whenexaminingthemeanvaluesofthesedistributionsseveralthingscanbelearned.FirstthemeanvalueswereexaminedonQQplotstodetermineiftheywerenormallydistributedasisshowningure3.3.Thisdemonstratesthatthemeans 32

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(a) (b) (c) (d) Figure3.1:SEMimagesofdierenthydrogelmixesforqualitativeanalysis(a)Mix1.Stiestmixwithnocollagen(b)Mix2.Stimixwithcollagen.(c)Mix3.Softermixwithlittlecollagen.(d)Mix4.Softestmixwiththemostcollagen.Asthestinessofthegelsdecreasestheporosityofthegelincreasesaswell.Additionallyasthegelsincreasedincollagencontentthesizeofthesheetsofthegelsdecreasedaswell fromdierentmeasurementswerenormallydistributedandat-testaroundthemeanofthedistributionmeansconrmedthisaswell.WhenaWelch'spairedt-testwasperformedneitherthexandymeansfornormalorPHanimalsbetweenPHand 33

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Figure3.2:Aexampledistributionofmeanfreepathlengthsfoundwhenexaminingtheadventitiaofarat.Withthelengthonthexaxisorcircumferentialdirectionandthenumberofoccurrencesontheyaxisorradialdirection. Table3.1:MeanFreePathdeterminedporesizesintheadventitia.Xdirectionreferstoporesizeparalleltovessel.Ydirectionreferstoporesizeperpendiculartovessel Normal PH NumberofDistributions 11 10 xMean+/-SD 3.08+/-0.27 2.83+/-0.23 yMean+/-SD 2.7+/-0.37 2.02+/-0.2 %dierence 12.4 28.4 normalprovedtobepartofthesamepopulation.Whatcanbetakenfromthisthoughisthattherewasanincreaseinthedierencebetweenthexandymeansofporesizefromnormalanimalstohypertensiveone.Thisrelationshipisdemonstratedintable3.1.Thevoidfractionofthetissuewasalsoshowntodecreaseascanbeseeninthechartingure3.4.Thisdemonstratesanincreaseintheoverallproteinorcellularcontentoftheadventitia.Takentogetherthevoidfractionanalysisandthemeanfreepathanalysisindicatedthatthereisadecreaseinthenumberofporesandvoid 34

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(a) (b) (c) (d) Figure3.3:QQplotsofthemeanfreepathofdierentsamplesfromtheadventitiaofratstakentoshowthatthemeansoftheseporeanalyseswerenormallydistributed.(a)Xdirectionnormalanimals(b)Ydirectionhypertensiveanimals(c)XdirectionPHanimals(d)YdirectionPHanimals spaceintheadventitiaandtheporesthatremainarespreadoutparalleltothevesselsindicatinganincreasingrealignmentofbersandcells. 3.2ConfocalDecellularizedTissueFromtheconfocalimagestakenawell-establishedbrousnetworkcanbeseenwhichdemonstratesthatcellsareabletoreestablishconnectionsandliveinthedecellularizedtissueofthematrixseenifgure3.5.Cellsareshowntoseedwellonto 35

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Figure3.4:VoidfractionmeasurementsbetweennormalandPHanimals. thebroadermeshregionsofgure3.5bshownwiththebluearrowbutnotaswellintoothersectionsofthedecell,specicallythepleurallayershownbytheredarrow.Itcanseenthatinrecreatingtherealenvironmentofthelungswithahydrogelmustimitatetheseregions.TheSEManalysisofthedierenthydrogelmixesdoneearliershowedthatthedierentmixesofgelscreateddieringstructuresthatwouldimitatethistypeoftissue. 3.3HydrogelCulturingStainingandMorphologyTheimagingofcellsgrownindierentconditionsrevealedthattheculturingenvironmentofthecellchangesitsbehavior.StainingforSmoothMuscleActinrevealedadierenceinthelocalizationofexpressionofSMA.In2DandPsuedo3Dcultures,wherecellsaregrownontopofgels,smoothmuscleactinwasseeninbersevenlythroughoutthecellinlamentouspatterns.Thisisshowningure3.6forthestandardculturingsetupofbroblastsculturedonhardatculturingplasticfortwodays.Thecellsspreadoutcoatingtheareaoftheplasticandbecomingconuent.Inthesetupcommonlyknownaspseudo3Dculturingthesamebroblastswereculturedontopofthefourmixesofgelsforoneweek.Thecellsstillspreadoutandgrowtoastageofconuenceacrosstheoutsideofthegel.Thestinessofthegelalsoaectsthemorphologyofthesecells.Inthestiestgelshownin3.7athecellsspreadoutinacuboidalfashionbutarerathernarrow.Whenthestinessofthegelsisdecreasedthecellsspreadoutacrossabroaderareaandbegintoassumea 36

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(a) (b) Figure3.5:Confocalimagesofdecellularizedratlungs.AutouorescenceandresidualGFPingreenandcellsthatwereculturedontothedecellafterwardsmarkedwithPKHinred(a)Cellsindecelltissuearoundseveralvesselswithanevendistributionofcellsgrowing(b)Cellsindecelltissuenearlungpleurawiththepleuramarkedbytheredarrowandnormallungtissuebythebluearrow.Cellshaveapreferencetoattachtothebroadernetworkofbersinthistissueasopposedtothesheetlikemorphologyofthepleura. moreelongatedshape.Thisisshownasthegelsproceedthroughdecreasingstinessfrom3.7btod.Boththe2Dcultureandthepseudo3DcultureshowthatSMAisdistributedthroughoutthecellinverydistinctlinearbersinasingledirection.WhencellsareencapsulatedinthesegelsandthenculturedtheyassumeaverydierentmorphologicalpatternandSMAexpressionbecomesmorehighlylocalizedtobersaroundtheedgesofcellprojectionsindicatingtheprojectionofsmalldendritelikestructuresmaybeguidedbySMAseeningure3.8b-dasopposedtothesimplespreadingofthecellsseeninsimple2DculturewhereSMAisevenlydistributed. 37

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Figure3.6:Cellsgrownonastandard16wellplateonthebottomofthechamberstainedwithSMAgreen200x ThiscapacitytoformnarrowprojectionswasseenintheCellASICplatformwherebroblastsmigratedoutofthecentralchannelpastpillarswith1microngapsthroughtheuseofnarrowprojectionsofthecell3.9indicatingthatthistypeofpatternmayhavebeenindicativeofmigratorybehavior.FSPstainingofthesesamecellsconrmedthattheymaintainedabroblasticphenotype. 3.4HydrogelHistologyExaminationofcellsembeddedinsideoftheHystemCgelmixesthroughtheuseofstandardslideslicingandstainingoffrozensectionsoftheslidesfurtherrevealedaspectsoftheirbehavior.StainingwithH&Eshowedthatthegelshaddistinctmakeupsaswellasdieringeectsoncellsthatwereembeddedinsideofthem.Forinstancetherstgelwhichwasthestiestwithamodulusof720Pascalsandcontainednocollagencellsremainedasspheresinthegelsanddidnotspreadoutorproliferatetoanyrealextentbutstillremainedviable.Thisisshowninthegure3.8awherecellsstainedwithFSPandSMAandembeddedinthisgelmixaftertwoweeksremainspheroidalanddonotspreadout.WhenthissamegelisexaminedonH&Estainedgelslicesitcanbeseeninbothonandtwoweeksofgrowthingure 38

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(a) (b) (c) (d) Figure3.7:GreenstainSMA,BlueDAPI.All200x.Allcellsculturedontopofgels1weekofgrowth(a)Mix1.Stiestmixwithnocollagen(b)Mix2.Stimixwithcollagen.(c)Mix3.Softermixwithlittlecollagen.(d)Mix4.Softestmixwiththemostcollagen.Asthestinessofthegelsdecreasedtheaspectratioofthecellsbecamelargerandcellselongated. 3.10bandcthatcellsaresparseinthegelandtheydonotsignicantlyaectthematrixaroundthemselvesasseenbythelimitedstainingofthegelbyEosinonanyofthetimepointswhencomparedagainstthecontrolslidewithnocellsingure3.10a. 39

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(a) (b) (c) (d) Figure3.8:GreenstainSMA,BlueDAPI,RedFSP.All200x.Allcellsculturedinsideofgels2weekofgrowth(a)Mix1.Cellsremainspherical(b)Mix2.Cellsforadensenetworkofinterconnection(c)Mix3.Cellsformanetworkbetweeneachotherwiththeuseofthindendriticlikeconnections(d)Mix4CellsformabroaderbrousnetworkwithdenseregionsofSMAsuggestingmigratoryactivity.AsstinessdecreasescellsfollowanincreasingtrendtodevelopthindendriticnetworkswithhighSMAcontent. Whenthesamestainingprotocolwasusedongelmixtwo,aslightlysoftergelmix,withcollagenincorporatedintothegelthecellsexhibitamarkedlydierentbehavior. 40

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Figure3.9:FibroblastsinltratingthroughpillarsinCellASICplate.ThesepillarsgiveonlyonemicronofspacebetweenPDMSsectionswhichcellsarenotabletodegradedemonstratingtheabilityofcellstomovethroughverysmallregions. Whencomparedtothecontrolshowningure3.11awheretheEosinsignaloftheslideisrelativelyweakandahoneycombingbrousmeshisseen.Atoneweekofcellulargrowthseeningure3.11bcellscanbeembeddedinthebersofthematrixandthesignalofsampleincreasesindicatingthatthecellsareremodelingthemesh.Attwoweeksseeningure3.11ctheremodelingofthematrixbecomesprofoundwiththeinitiallythinbersbeingrobustlybuiltupandthickenedwithasignicantincreaseinthenumberofcellsinthematrix.Additionallysmallerspindlybersconnectingtheselargercellrichonesbegintodisappearleadingtoalesstightbrousmeshwithcellsinterspersedintodensebersinthematrix.WhenexaminedasawholegelthroughtheuseofIFstainingofSMAandFSPingure3.11dthesebrousmeshescanbeseeninsidethegelsmatrixwhileontheoutsideofthematrixcellsspreadoutinaconuentpatternmorecommonlyseeninstandardtwodimensionalculturing. 3.5PCRPCRwasperformedoncellsgrownforoneandtwoweeksinthefourhydrogelmixes.ThiswascomparedagainstaPCRofcellsgrowninastandardculturing 41

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(a) (b) (c) Figure3.10:(a)1weekgrowthgelmix1H&E(b)2weekgrowthgelmix1H&E(c)IFstaininggelmix1FSPredSMAred.Cellbodiesinarrows.ThereislittletonochangeinthestructureofthegelandIFstainingrevealsthatthecellsremainstaticasphereinthegel. setup.Theapplicationsthatwereperformedwereaftertoofewmanydivisionsfortheresultstobeconsistentlytrusted. 3.6MTSTestingResultswereinconsistentforthetwonormalratlungs.ThismaybeowingtothesizeofthesamplerequiredfortheMTSmachinewherealargesectionoftherat 42

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(a) (b) (c) (d) Figure3.11:(a)controlgelmix2H&E,(b)1weekgrowthgelmix2H&ECellsshownwithblackarrows.(c)2weekgrowthgelmix2H&ECellsshownwithblackarrows,thickerbersshownwithwhitearrows.(d)IFstaininggelmix2FSPred,SMAgreen,DAPIblueThisshowsthebrousnetworkthatthegelsareformingingreaterdetailandmatcheswiththeH&Estainingshowninb&c lungtobeplacedinthemachine.Thisledsectionsoflungtohavedierentmakeupsofcomponents,suchaslargevessel,airways,alveolarspaces,andpleuraltissue.Thedecellularizedlunggavefarmoreconsistentresults.Thisthelikelyoccurredbecauseoftwothings.Theremovalofalldierentcelltypesleftbehindasimplermatrixwhich 43

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Table3.2:ModulusofdecellularizedsamplesallunitsinkPA Sample1 Sample2 Sample3 Sample4 Mean StandardDeviation 13.67 12.66 14.32 14.47 13.78 0.82 hadmoreuniformmechanicalpropertiesandtheswellingofthelungtissueduetotheprocessofdecellularizationallowedforlargersectionsoflungtobeselectedwithoutpleuraltissueorlargevesselsandairways.Theresultsforthisareshownintable3.2.Itwasunfortunatelyimpossibletoremovelargevesselsandairwaysfromthesurroundingtissueduetosizeconstraints.Thisdidnotallowforthemechanicalpropertiesofdierenttissuetypestobemeasuredseparately. 3.7ImmunouorescentStaining 3.7.1FibroblastSpecicProtein,SmoothMuscleActinandOx-62StainingratlungtissuesectionswithFSPandOx-62revealedseveralthings.FirstlookingatthePearsoncorrelationcoecientfortheco-stainingofthetwoproteinsrevealedthatFSPwasnotinfactstainingdendriticcellsbutwasinfactstainingbroblasts.Theresultsfromthisanalysisareshownintable3.3.Thesevalueswhicharecloseto0indicatethereisnocorrelationinthelocalizationofthetwoproteins.ThisindicatesFSPisasucientmarkerforthetrackingofbroblastbehaviorandisnotbeingexpressedbydendriticcells.Thestainingforthesetwomarkersinbothmodelsofthediseaseisshowningure3.12.InbothanimalmodelsafteroneweekitcanbeseenthatbothdendriticcellsandbroblastlocalizeinBALTstowardtheairwaysideoftheBALT.ThistrendofbehaviorcontinuedforthetwoweekMCTanimalbroblastsandalsobegintobelocalizedallaroundtheedgeofthecell.InthreeweekSuHxthesametrendcontinuedwithothercellsequallydistributedthroughout.In4wkMCTintwoserialslicesofaBALTitcanbeseenthatcellslocalizetothesideofavesselnearanairwayaswellasnearabloodvessel.This 44

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indicatesthatbothbroblastsanddendriticcellsarefoundonthesideoftheBALTthatisclosesttoanytypeofairwayorvessel. (a) (b) (c) (d) (e) (f) Figure3.12:GreenFSP,RedOx-62,BlueDAPI(a)1wkMCT(b)1wkSuHx(c)2wkMCT(d)3wkSuHx(e&f)4wkMCT.RegionsofbothFibroblastandimmunecelllocalizationshownwithwhitearrows,airwayshownwithyellowarrowandBloodvesselwithgreenarrow. WithFSPconrmedasareliablemarkerforbroblastsfurtherstainingcanbeperformedandexamined.InthestainingofFSPwithSMAitwasrevealedthatinnormaltissuesBALTsshowningure3.13aareseentobeintheadventitialspaceandnotlocalizedtoanyspecicsideofairwaysorvessels.WhenPHisinducedwithMCThoweveritcanbeseenthattheBALTsmoveclosertothesurroundingairways.ThisinturncausestheBALTtomakethesmoothmusclelayeroftheassociatedairwaydiscontinuousascanbeseenbythelossofSMAexpressionwheretheBALT 45

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Table3.3:PearsoncorrelationcoecientsforOx-62andFSPstaining AnimalModel PearsonCoecient Normal 0.045 1weekMCT -0.184 1weekSu/Hx 0.218 2wkMCT 0.218 3wkSu/Hx -0.09 4wkMCT 0.08 ispushingupontheairwayingures3.13b-c.Fibroblastsinturnmigratetowardtheairwaysideofthevessel.In1weekMCTseeningure3.13bthismigrationishighlylocalizedandtheSMAbreakdownisonlypartial.In4weekMCTthebreakdownoftheSMAlayerisalmostcompletebetweentheBALTandairwaywithalargenumberofFSPpositivecellslocalizedthere.AdditionallymoreFSPpositivecellscanbeseenallaroundtheedgeoftheBALT. 3.7.2Claudin-5andAquaporinAswasstatedearlierClaudin-5andAquaporin-1areimportantregulatorsinuidmovementintheinterstitialspacebetweenthebloodvesselsandlymphatics.WhenstainingforClaudin-5wasperformedonnormalratlungsectionsthetightjunctionalbarriersincellsarereadilyvisibleasisshownin3.14.Thisdistinctdelineationbetweencellsindicatesthatthetightjunctionalcomplexesareintactinthesecellsandproperlypreventingwaterfromleavingthevessel.WhenPHhasbeeninducedinRatsthroughMCTthereisinitiallyabreakdowninthelocalizationofClaudin-5shownatoneweekingure3.15.ThisislikelyduetothetoxiceectsofMCTdirectlyinjuringendothelialcellsandinhibitingtheirabilitytolocalizeexpressionofClaudin-5tothelipidbilayerjunctionbetweencells. 46

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(a) (b) (c) Figure3.13:GreenSMA,RedFSP,BlueDAPI(a)Normoxic.NotethedelocalizationofPAFsintheBALT.LocalizationofFibroblastsshownwithwhitearrowsandthebreakdownoftheSMAlayeroftheairwaywithyellowarrows.(b)1wkMCTlocalizationofPAFstowardsairwaysshownandSMAdiscontinuity(c)4wkMCTlocalizationofPAFstoaroundtheBALTandgreaterbreakdowninSMAlayerofvesselshownwithyellowarrows AfterfourweeksofMCTexposurethelocalizationofclaudin-5becomesevenlessdistinctbetweencellsandtheoverallexpressionlevelsoftheproteinaremuchlessdistinct.Thisisdisplayedingure3.16. 47

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(a) (b) Figure3.14:RedClaudin-5,BlueDAPI.Twonormoxicratlungsectionsshowingvessels.Notethedistincttightjunctionalbarriersbetweenendothelialcellswithclearseparationwherecellbodiesarelocatedshownwithwhitearrows 48

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(a) (b) Figure3.15:RedClaudin-5,BlueDAPI.OneweekafterMCTinjection.NotethedecreaseinthelocalizationofClaudin-5toregionsbetweencells.Claudin-5isinsteadmoreevenlyspreadoutthroughouttheendothelialcellsshownwithwhitearrows. 49

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(a) (b) Figure3.16:RedClaudin-5,BlueDAPI.FourweeksafterMCTexposure.NotethelossofdistinctjunctionalbarriersofClaudin-5shownwithwhitearrows. 50

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InSugenadministeredanimalsinahypoxicenvironmentasimilarbreakdowninlocalizationandexpressionlevelsofclaudin-5wasseeningure3.17 (a) (b) Figure3.17:RedClaudin-5,BlueDAPI.ThreeweeksofSugenandHypoxicat-mosphereexposure.NotethealmostcompletealterationlocalizationofClaudin-5expressiontotheregionsinbetweentheendothelialcellsshownwithwhitearrows. 51

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Table3.4:RatioofvesselsseeninIFstainedimagesthatdisplayedabreakdownintheClaudin-5organizationandAquaporinorganizationaroundvessels. Ratioofphotoswith: Normoxic MCT1wk MCT4wks Su/Hx3wks Claudinbreakdown 0.29 0.70 0.90 0.88 Aquaporindisorganization 0 0.25 0.70 0.42 Aquaporinservesasanotherimportantregulatorofuidmovementinthein-terstitialspaceanditsexpressioncanindicateexactlyhowthemovementofuidisregulatedbetweenvascularvesselsandlymphaticcollectingvessels.Thisisdonebyitallowingthefreeowofuidacrossmembranesthusfacilitatinginterstitialuidow.Organizedexpressionaroundvesselscanbeobservedinnormoxicratsingure3.18(a)indicatingthatthereisawellestablishedmigrationofuidbetweenthevesselandcollectinglymphatics.WhenPHisinducedwithMCTasisshowningure3.18(b)itcanbeseenthatthereisabreakdownintheorganizationofaquaporinandthusapossibledierenceinthewaythatuidinowingintheinterstitialspace.WithSugenhypoxiainducedPHalackofexpressioncanbeseenintheadventitiaaroundvesselsingure3.18(c). 3.8CellASICCulturingandCellTrackingAsnapshotoftheCellASICChamberforthetwocontrolowexperimentsisshowningure3.19a&b.Themovementofcellsinthechamberwhentheirstartingpositionisnormalizedtoazeropositionisshowningure3.19c&d.Theweightedpolarhistogramofthepreferreddirectionofmovementintheexperimentisshowningure3.19e&f.Forthe1PSI/6895Paofappliedforcetothechamberthissameinformationisshowngure3.20withthesnapshotoftheCellASICChambershownina&b.Themovementofcellsinthechamberwhentheirstartingpositionisnormalizedtoazeropositionisshowninc&d.Theweightedpolarhistogramofthecellspreferred 52

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(a) (b) (c) Figure3.18:GreenAQ1,RedClaudin-5,BlueDAPI.(a)Normoxicsections;notethewellorganizedlayersofAQ1surroundingvesselsandairways(b)4weekMCTrats.ThereisadistinctbreakdownoftheAQ1layeraroundthevesselandairwayanddisorganizationinitslocalization,shownwithwhitearrows.(c)3weekSugenHypoxiarats.ThereisathinningoftheAQ1layeraroundtheairwayandadistinctlossofexpressionaroundthebloodvesselsshownwithwhitearrows. 53

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(a) (b) (c) (d) (e) (f) Figure3.19:Cellsseededatadensityof5x106cellspermLwithnoforcedowap-pliedtothechannel;twoexperimentsthatwerecarriedoutinthisfashion.(a&b)GFPsnapshotsofthechannelbeforetheexperiments.(c&d)Tracksofcellmove-mentnormalizedtoastartingcentralposition.(e&f)Polarhistogramofdirectionalpreferenceformovement. 54

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directionsofmovementintheexperimentisshownine&f.Forthe4PSI/27580Patestofappliedpressurethissameinformationisshowngure3.21withthesnapshotoftheCellASICChambershownina&b.Themovementofcellsinthechamberwhentheirstartingpositionisnormalizedtoazeropositionisshowninc&d.Theweightedpolarhistogramofthecellspreferreddirectionsofmovementintheexperimentisshownine&f.Theseresultsindicatethatthemethodofcelltrackingiscapableoffollowingcellmovementinalargethroughputfashion.Thiscantranslatedintoadirectionalmetricwhichcanberepresentedvisually.Thedatarepresentingthisispresentedintable3.5.Thisshowstheaveragetravelofcellsineachofthesegroups,thedirectionaltravelofthecellswhichreferingtotheoverallmovementpreferenceasaweightedaveragethenumberoftracksandthedirectionalpreferenceindegreesrotatingcounterclockwisefromthepositivex-axis.ThisrevealsthatthecellstraveledroughlythesamedistancealthoughmuchfasterforthefourPSItestswhichhadhalfthetime.ThecellsatcontrolandonePSI/6895Paconditionshadapreferencetomovewithow.Themovementofthe4PSI/27580Paappliedpressurecellsastatisticallysignicantdirectionofmovement.Tointerpretwhattheseresultsanothermethodanothermethodofdataanalysiswasrequired. 3.9SpinningDiskFluidDynamicsCharacterizationWiththeuseofspinningdiskconfocalmicroscopyanduorescentmicrobeadswewereabletocharacterizetheuidowbehaviorwithintheCellASICmicrouidicplatform.Thisallowedustoobservedirectlywhatuidowthecellswereactu-allyexperiencinginvitroandcharacterizethespatiallyintegratedshearstressthatthesecellswouldbeexperiencingthroughtheuseofacomputationaluiddynamicssimulation.Thesameowconditionsusedinthetimelapsestudieswererecreatedonthemicroscopewithmediaandaddeduorescentbeads.Theowofthesebeadswas 55

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(a) (b) (c) (d) (e) (f) Figure3.20:Cellsseededatadensityof5x106cellspermLwith6895Paofforcedowappliedtothechannel;twoexperimentsthatwerecarriedoutinthisfashion.(a&b)GFPsnapshotsofthechannelbeforetheexperiments.(c&d)Tracksofcellmove-mentnormalizedtoastartingcentralposition.(e&f)Polarhistogramofdirectionalpreferenceformovement. 56

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(a) (b) (c) (d) (e) (f) Figure3.21:Cellsseededatadensityof5x106cellspermLwith27580Paofforcedowappliedtothechannel;oneexperimentwascarriedoutathalfthetime(6hours)beforethewastewellslledup.(a)GFPsnapshotsofthechannelbeforetheexperiments.(b)Tracksofcellmovementnormalizedtoastartingcentralposition.(c)Polarhistogramofdirectionalpreferenceformovement 57

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(a) (b) Figure3.22:SpinningdiskconfocalowcharacterizationoftheCellASICchamber.(a)SnapshotofthecenteroftheCellASICchamberasuorescentbeadsareoweddownthemiddleofthechamber.(b)Snapshotofthemovementtrackingofbeadsintheimagingwindow.Individualpositionsatthetimepointareshownasredcirclesandfullbeadtracksasmulticoloredlines. 58

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Table3.5:TravelcharacteristicsofcellsinCellASICchamber.DirectionalTravelistheaveragedistancethatthesecellstraveledinmicronswithapositivenumberindicatingmovementwithowandanegativeindicatingmovementagainstow.Tracksisthenumberofcellstracked.Directionalpreferenceisindegreeswith0beingdirectlywithow. Test MeanTravel DirectionalTravel Tracks DirectionPreference Control1 1.74 0.236 307 47.05 Control2 1.96 0.498 91 39.29 6895Pa1 1.76 0.345 143 29.39 6895Pa2 1.56 0.0738 80 259.04 27580Pa1 1.504 0.366 83 67.67 27580Pa2 1.89 0.116 141 116.32 capturedinthecenterofthechamberacrossthebreadthofthechamber.ThevideoofbeadowincoveredasingleplaneofimagecaptureintheverticalmiddleofthecellASICschamberandspanedtheentirewidthofthechamber.Thiswouldallowforthefullowproletobemeasureacrossthewidthofthechamber.ThisAsingleimageofthevideoisofbeadmovementisshowningure3.22a.WiththeICYspottrackingprogramusedforcelltrackingthebeadsweretrackedastheymoveacrossthechamber.Asnapshotofthistrackingwithbeadtracksshownaslinesisshowningure3.22b.Sinceasingleplaneatthecenterofthechamberwasusedforthestudybeadswouldmoveintoanoutofthisplanerandomlyastheyoweddownthechamber.Thismeantthattracksacrosstheentireviewingwindowwererare.Theentiretyoftrackswasconstructedintoasingleaveragevalue.Themovementshowninweightedpolarhistogramsingure3.23showanover-whelmingpreferenceofmovementdownthechannel.Thisservesasavalidationthat 59

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(a) (b) Figure3.23:Histogramsofcellmovement;6895PaappliedowfromCellASICsma-chine(a)PolarhistogramofmovementdirectionofbeadsfromthetrackingprogramstoMatLabtrackingprogram(b)Individualpathsofbeadsasthemovefromtheirinitialpositionwhichisnormalizedtoazeropointonthepolaraxis. Table3.6:AverageowvelocityofbeadstravelingdowntheCellASICchannel AppliedPressure Velocity(m/s) StandardDeviation(m/s) DataPoints Control 0.87 0.16 837 6895Pa 0.91 0.17 820 thetrackingsoftwareandMatLabprogramarefunctional.Thespeedofuidowatcontroland6895Paareshownintable3.5.ThisindicatesthatincreasesinpressureappliedontheCellASICapparatusupto6895PacontributeslittletoanincreaseinuidowintheCellASICchamber. 3.10SolidWorksSimulationWiththevelocitiesdeterminedthroughtheuseofcelltrackingthevolumetricowratecanbecalculated.Thisassumesthattheaveragevelocityfoundinthebeadtrackingexperimentwasthemeanofaparabolicfullydevelopedowproleorhalfofthemax.Thisisnotacompletelycorrectassumption.Ifitwastrulyparabolicthe 60

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standarddeviationofthedatapointsshouldbearound30%ofthemaximumwhileinthiscaseitisaround10%ofthemaximum.Convertingtothreedimensionsanassumptionofaparaboloidvelocityprolecanbemadewhichwhenintegratedwillgivethevolumetricowrate.Thisisabasicconversionoftheequationfordevelopedow.AcVave(3.1)WhereAcisthecrosssectionalareaandVcisthevelocityatthecenter.Usingtheaveragevelocityfoundfromthespinningdiskbeadtrackingexperimentallowsforthisvolumetricowratetobedetermined.Withtheowvelocityof1m/sandthecrosssectionalareaof50x50mthisgivesa2500m3/sToconrmthattheassumptionoflaminarfullydevelopedowinthechamberwasacceptabletheReynold'snumberandentrancelengthofthesystemwerecalculated.Sincethechamberisnoncircularthehydraulicdiameterneedstobeused.Sincethechamberisa50micronsquareboxthehydraulicdiameterissimplethatlength.WiththistheReynoldsnumbercancalculatedusingtheformulainequation3.2whereisdensity,Vavetheaveragevelocityinthechamber,Dhthehydraulicdiameterandmtheviscosity.VaveDh (3.2)Withaowvelocityaround1m/sandthedensityandviscosityofDMEMbeing0.99g/cm3and0.78centipoisetheReynold'snumberwillbeverylowforallowvaluesandontheorderof6x10)]TJ /F4 7.97 Tf 6.58 0 Td[(5.Thisindicatesthattheowislaminar.Todeterminetheentrancelength,equation3.3forlaminarowwasusedwhereReistheReynold'snumberandDhthehydraulicdiameter.Le=0:06ReDh(3.3)Withthisagaintheentrancelengthofthechamberisfarlessthenonemicron.SincetheareaofthechamberthatwasobservedinallCellASICsexperimentswas 61

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notneartheentrancetheowcanbeassumedtobefullydeveloped.WiththisinhandtheSolidworkssimulationcanbedeveloped.TheSolidworksmodelwasanexacttoscalereplicationoftheCellASICchamberwithfalsecellsinsertedasextrudedbodiesinthebaseofthechamber.AComputationalFluidDynamics(CFD)simulationwasexecutedoftheuidowinthechamber.Thischamberwithpipesrepresentinguidowandthescaleofthevelocityofuidowisshowningure3.24. Figure3.24:SolidworksrepresentationoftheCellASIC'schamberwithcellbodiesinsertedasrepresentativeofthoseseenwhencultured.ACFDsimulationwasexe-cutedofthechamberandthepipesshownarerepresentativeofuidowwiththevelocityrepresentedbythecolorofthepipe. Thissimulationallowedfortherepresentativeshearstressesthatcellsexperienceinthechanneltobetested.Theshearontherandomcellsinthechannelsunderaowratebasedonanaveragevelocityof1micronpersecondwasdetermined.Whenaveragedforallofthedrawncellsinthechannelthewas2.41Pascalsor24.1dynes/cmn2.Thisisafairlycommonamountofsheartoplacecellsunder[24].Sincethischannelhasanitesizetherewillberegionsthatexperiencehigherandlowervelocityowandconsequentlydierentshearlevels[69].Thechamberwasdividedupasisshowningure3.25.Forthesedivisionstheaverageshearofthecellswithin 62

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(a) (b) Figure3.25:ThetwodivisionsoftheCellASICchamberthatwereperformedfortheanalysisofshearstressoncellswiththeinletofthechamberatthetopofbottomoftheimage(a)Horizontaldivisions,(b)Verticaldivisions 63

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Table3.7:ShearstressesinPascalsofthedivisionsfortheCellASICchamber Region 1 2 3 4 5 6 ShearStress(Pa) 1.72 2.31 3.25 2.24 2.57 2.45 theregionwasdetermined.Theseresultsareshownintable3.6.Thisdemonstratesthattheshearstresscanbedierentthroughouttheobservedregionsofthechamber.Thenthedivisionisacrossthewidthofthechamberinregions4,5and6theshearisfairlyuniform.Whenthechamberisdividedinthedirectionofowfromtheupstreamtothedownstreamsidesinregions1,2and3thoughthereisabigdierenceintheshearthatthecellsareexperiencing.ThelackofdierenceacrossthechamberthanlikelyisduetotheextremelylowReynoldsnumberoftheuidowinthechamberindicatingthatthevelocityisfairlyuniformacrossthechamber.Thedivisiondownthechamberwithregion1experiencinghalftheshearstressthatthecellsfurtherupthechamberinregion3experienceindicatesthatthereisachangeapossiblechangeinvelocitydownthechamberthiswouldbeoweingtouidowingoutofthecentralchamberascanbeseeninsimulationdiagramofgure3.24. 3.11CellASICDivisionofChamberTheSolidworksCFDsimulationshowedthatcellsinvariousregionsofthecham-berexperienceddierentshearstressesandowcharacteristics.Itwouldthereforebeofinteresttobreakupthechambertoexaminethedierencesinmigratorybehaviorbetweendierentsectionsofthechannel.Thisbehaviordivisionisshownforthecontroltestsingure3.26andingure3.27forthe1PSIforcedowwitha&bbeingtheleftsideofthechamber,c&dbeingthemiddleande&fbeingtheright.Figures3.27and3.26revealthatthedividedregionsofthechamberhavedier-entmigratorycharacteristicsbutmanuallyinterpretingthedierencesbetweentheseregionsisdiculttodo. 64

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(a) (b) (c) (d) (e) (f) Figure3.26:Cellsseededatadensityof5x106cellspermLwithnoforcedowappliedtothechanneltwoexperimentsshowningure3.19buttheanalysisofcellmovementisdividedinthechamber(a&b)Theleftsideofthechamber.(c&d)Themiddleofthechamber.(e&f)Therightsideofthechamber 65

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(a) (b) (c) (d) (e) (f) Figure3.27:Cellsseededatadensityof5x106cellspermLwith6895Paofforcedowappliedtothechanneltwoexperimentsshowningure3.20buttheanalysisofcellmovementisdividedinthechamber(a&b)Theleftsideofthechamber.(c&d)Themiddleofthechamber.(e&f)Therightsideofthechamber 66

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Toenableamorethoroughanalysisofwhetherthecellspreferredtomovewithoragainstowthedisplacementsofthecellsofthetwotestswerecombinedandconstructedintoahistogramofmovementwithpositivemovementbeingwithow,negativemovementagainstitand0nomovement.AGaussiancurvewasttedtothesedistributionsasavisualrepresentationandat-testexecutedtodetermineifthisdistributionwasdierentthanthebasicversioncenteredaround0.Ifthedistributionwasnotcenteredaround0itcouldbedeterminedwhetherthecellsweremovingwithoragainstow.Thehistogramsfortheentirechannelareshowningures3.28,fortheleftsideofthechamber3.29,forthemiddleofthechamber3.30andfortherighthandsideofthechamber3.31.Overallthesedemonstratethatthedistributionsofmovementcanbedescribedasnormalandthatstandardstatisticalanalysismethodscanbeusedtoexaminethem.Thetotalanalysisofwhetherthesecellsweretravelingwithoragainstowisshownintable3.8.Thisindicatestheat27580Patheirisnopreferenceofmovementanywhereinthechannel.Thedivisionofthechamberhadtobedoneforthesecondtestinthiscasebecausetherstonlyhadcellontherightandmiddleofthechamber.AtonePSIthecellsexhibitedastatisticallysignicanttendencytomovewithowespeciallyinthemiddleofthechamber.Thecontrolowconditionhoweveronlyshowedastatisticallysignicantpreferencetomovedirectionallyinthelefthandsideofthechamber. 3.12VascularPermeabilityTestWithtestsdoneonfournormoxicratstheresultsprovedtobeinconsistentandfurtherexperimentswerenotdone. 3.13HydroxyprolineAssayInthehydroxyprolineassaysomeverydistincttrendswereseen.Cellsembeddedinsomeoftheestablishedmixturesofgelsexhibitedsimilarpatternsofbehavior.Intherstgelmixturebetweenthecontrolofnocellcultureandtheoneortwoweeksof 67

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(a) (b) (c) Figure3.28:Xmovementdirectionalmovementdistributionwherenegativevaluesrepresentmovementagainstowandpositivevalueswithit.(a)controlofnoappliedpressure(b)6895Paappliedpressure.(c)27580Paappliedpressure 68

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(a) (b) (c) Figure3.29:Xmovementdirectionalmovementdistributionwherenegativevaluesrepresentmovementagainstowandpositivevalueswithitfortheleftthirdofthechannel.(a)controlofnoappliedpressure(b)6895Paappliedpressure.(c)27580Paappliedpressure 69

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(a) (b) (c) Figure3.30:Xmovementdirectionalmovementdistributionwherenegativevaluesrepresentmovementagainstowandpositivevalueswithitforthemiddlethirdofthechannel.(a)controlofnoappliedpressure(b)6985Paappliedpressure.(c)27580Paappliedpressure 70

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(a) (b) (c) Figure3.31:Xmovementdirectionalmovementdistributionwherenegativevaluesrepresentmovementagainstowandpositivevalueswithitfortherightthirdofthechannel.(a)controlofnoappliedpressure(b)6895Paappliedpressure.(c)27580Paappliedpressure 71

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Table3.8:DirectionoftravelofcellsintheCellASICchamber.Negativevaluesareagainstowandpositivevaluesarewithow Test Mean(microns) p-value FullChamber 27580Pa 0.139 0.252 6895Pa 0.213 0.020 Control 0.212 0.004 LeftThirdofChamber 27580Pa -0.242 0.283 6895Pa 0.207 0.153 Control 0.339 0.002 MiddleThirdofChamber 27580Pa 0.065 0.760 6895Pa 0.368 0.001 Control 0.125 0.38 RightThirdofChamber 27580Pa -0.062 0.779 6895Pa 0.258 0.028 Control 0.112 0.381 72

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cellgrowththerewasanegligibleamountofcollagenmeasuredbetweenallofthese.Thisindicatesthatthecellsembeddedhereexhibitlittleabilitytoremodelingthematrixduetomechanicalorchemicalfactorsofthegel.Thisisshowningure3.32a.TheseresultsreinforcewhatwasseenintheIFstainingofcellsembeddedinthisrsthydrogelwherecellsdidnotproliferateandsimplystayedinastaticsphericalshapeandraisethepossibilitythattheyweredoinglittletoalterthematrix.Mixestwo,threeandfourunderwentasimilarremodelingtoeachother.Inallthreegelmixturesthecollagencontentinitiallyincreasedinthegelsoverthecourseofoneweekofcellulargrowth.Thecollagenthendecreasedtoalevelbelowthecontrolamountofcollagenplacedintothegelasisshowningure3.33.Thisindicatedachangeinthebehaviorofthecellsmidwaythroughtheprocess.Thisispossiblyduetotherapidinitialspreadingthroughthegeltoformattachmentsseenearlier.Thiswouldfunctioninasimilarfashiontotherapiddepositionofcollagenexhibitedbybroblastsinresponsetoastimulusofinjury.Inthiscasetheinjurywouldbetheirsuddenencapsulationinahydrogelthatisnotafullrecreationoftheirnaturalenvironment.Thecellsproceedtochangethematrixrapidlybedepositingcollageninaprocessofremodelinguntiltheyhavereachedacomfortablestatewheretheywillproceedtoreverseremodeltoanormalstateseeninstandardinjuryrecovery.Theywilleventuallyapproachanalsteadystatethatissimilartothenaturalchemistryandmechanicalpropertiesofthetissue. 3.143DPrintingPartsmadeincludeincludedamoldforhydrogelstobecastinandtestedontheMTSuniaxialstrengthtestingmachine,acustommicroscopestageforconfocaltobeperformedonthecellASICmanifoldanda3Dmodeloftheberscapturedintheconfocalofthedecelllungseeningure3.34. 73

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(a) (b) (c) (d) Figure3.32:Relativeamountsofcollageningelswhengelsamplesaretakeninmicrogramsofcollagenpertotalgelsampleweight.1standarddeviationerrorThisshowsnochangeincollagenformixonebutaninitialriseatoneweekandthenasubsequentdropattwoweeksofcellculture 74

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Figure3.33:Collagenlevelsofallfourmixesofgelsfromthehydroxyprolineassayat1and2weeksofcellsgrowth.Thisshowsthatthegelsexhibitsimilarbehaviorbutatdierentlevelsofcollagen. 75

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Figure3.34:3Dprintedpartrepresentingthebersofdecellularizedlungtissue 76

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4.Discussion 4.1NewCulturingMethodsCellculturinghasbeenavitaltoolinthebiomedicalindustryandwithoutstan-dardmethodsofculturingmanymedicalandresearchadvanceswouldnothavebeenpossible.Normalcellculturingtechniquesaregreatforgrowinglargenumbersofcellsandfortestingsimplecellularbiologyquestions.InstudyingadiseasesuchasPHespeciallyasitpertainstothechangesinmicro-architecturethatmaycontributetodiseaseprogressionamorephysiologicalinvitromodelmustbebuilt.ThisassertionwasconrmedthroughtheuseofnovelcombinationsofculturingandIFstainingwhichrevealedthatthebehaviorofPAFsissignicantlyalteredindiseasestateanimalsandwhentheirculturingenvironmentisvaried.Standardculturingtechniquesgrowcellsonhardatplatesofplasticwhichencouragecellstoexpandintoandevenmonolayeracrossthissurface.Whenthecellsareculturedontopofamorephysiologicallysofthydrogeltheybegintochangetheiractivity.Initiallythesecellsstillspreadoutevenlyacrossthesurfaceofhardergelsinamannerverysimilartostandardculturingsetups,butasthestinessofthegelsisdecreasedtheybegintospreadoutthroughnarrowerelongations.Thiscausesanincreaseintheaspectratioofthecells.Theyalsobegintoinltrateinsideofthegelsandbecomemorelikeatrue3Dculture.Inotherexperimentswherecellshavebeengrownonsubstancesofdierentrigiditiesattachmentsitestothematrixservedassignalingfactorsalteringcellularbehavior[14].Thebehaviorseenherewouldindicatethatthecellsaresensingthemechanicalnatureofthegeltheyarebeinggrownon.Whenculturedinsideofadiusegelmatrixofsmallspindlybersthecellsdrasticallyalteredtheirbehavior.Insteadofspreadingoutevenlythecellsfollowthepathofthebersofthegelsandformedlinkingdendriticlikestructurebetweencells.Theymaintainameshlikestructurewhichissimilartowhatwasobservedintheadventitiaofvesselswherecellswerestudiedamongstabroadermeshnetworkofproteins. 77

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Whenthedecellularizedenvironmentofthelungswasexamineditcouldbeseenthattherewasadiusenetworkofbrousmeshthatmadeupthemajorityofthelungaswellasdensesheetsofproteinthatmadeuppleurallayers.Whencellswereseededintothisdecellularizedtissuetheyshowedapreferencetogrowinthebrousregionsasopposedtoattachingtoatsheetsofprotein.Cellsculturedinsideofthehydrogelalsoshowedapreferencetoremodelthebrousmatrixintothickerbers.Whenseededinastimatrixwithnocollagencontentthecellsdidnotexhibitanyabilitytospreadoutoralterthematrix.Thissuggeststhatevenin3Dculturethemakeupofthehydrogelaectsthebehaviorofthecellsandwillguidetheiractivity.Thisprovesthat3Dculturingelicitsdierentresponsesthanstandardcultureandisausefultechniquetomorefullyrecapitulatethenaturalenvironmentwherecellsarenormallycultured.Theuseof3DculturesprocessedwiththehydroxyprolineassayshowedthatPAFswillchangethemakeupofthematrixthattheyareembeddedinit.Thesecellswillinitialincreasethecollagencontentofthegeltheyareembeddedinandthendecreaseitovertime.Thisprocesscanberelatedtothenormalrecoveryofthelungfromaninjurywhereaninitialinammatoryresponseisrequiredforhealingandleadstotheremodelingandproductionofcollagenofwhichbroblastsarekeyplayers[70].Fibroblastsnormallyreturnthetissuetoahealthymakeupaftertheinitialinam-matoryresponseasarecoveryfrominjuryandadeactivationofinammation.Inpulmonaryhypertensionthisrecoveryfromtheinjuryanddeactivationofinamma-tiondoesnotoccur[71].Insteadthestatebecomeschronicasisevidencedbythepersistentpresenceofinammatorycytokines[72].ItisnotknownwhyPHismain-tainedinastateofchronicinammationandpersistentremodeling.ThebreakdownofthebarriersthatcontrolIFowintheformoftightjunctionalbarriers,theevi-dencethatuidshearstresscanleadtobrosis[73]andstudiesthatshowbroblastsplayakeyroleinchronicinammation[74]canprovideaninsight.Allthisevidence 78

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raisesthepossibilitythatincreasedinterstitialuidowismaintainingbroblastsinaprobroticmyobroblaststateandthusfurthercontributingtotheprogressiveremodelingofthevesselandaprogressionofthePHpathophysiology.Possiblether-apeutictargetsincludereestablishingthenormaluidbarrierinendothelialcellsortheblockageofmechanicalsensingmechanismsforPAFstoreturnthemtoanormalstate. 4.2SEMRelationshipsSEManalysisoerstheopportunitytoexaminesampleswithsuperresolutionandcanrevealmorphologicalfeaturesthatarenotinherentlydistinguishablewithstandardimagingtechniques.Thisstudyrevealedafundamentalchangeinthestruc-tureoftheadventitia.InPHthevoidspacesseenintheadventitiadecreased.Itwasnotknownwhetherthisdecreasewasduetohyperplasiaofcells,hypertrophyofcellsorincreasedproteindepositionthough.Thereisevidenceofincreasedcollagendepo-sitioninPHbeingacausativefactorinincreasedvesselstinesshowever[75].Aswasshownearlier,hydrogelswithdierentporesizesalteredthebehaviorofbroblasts..Whenthemeanfreepathoftheseimageswasanalyzeditshowedthattherewasastatisticallysignicantdecreaseinthesizeofporesinthehypertensiveadventitia.Thistswhenconsideredwiththedecreaseinvoidspace.Thereisalsoanincreaseintheaspectratioofthesevoidspacesnearvesselsindicatedthatporeswereelongatingparalleltovesselsbyagreaterdegreethanperpendicular.Thiswouldindicatethatbersandcellsarepreferentiallyaligningthemselvesparalleltoow.Aswasstatedearlierithasbeenfoundthatincreasesininterstitialuidowcausecollagenbersaswellasbroblasttopreferentiallyalignthemselvesperpendiculartoow.Thisallowsforthecellsandproteinstoabsorbagreateramountoftheuidpressuredropbetweenthevesselandcollectinglymphatics.TheseresultswouldsuggestthatthebreakdownintheendothelialcellbarriersseenthroughIFstainingmaybecausing 79

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anincreaseinvesselleak,interstitialuidowandconsequentlyachangeinberalignmentandbroblastbehavior.Thechangesintheadventitiamayworkasthefeedforwardmechanismthatleadstofurtherstieningofthevesselandconsequentlyhigherbloodpressure.Onceagainitappearsthatthematrixaectscellularbehaviorandcellscaninturnaectthematrixaroundthemselves. 4.3PHRelationshipsSeeninStainingInPHtherearemechanismsthatarenotfullunderstoodmaintainingelevatedbloodpressure,inammationandprogressivevascularremodeling.TorecreatetheconditionsofPHinvitrothesemechanismsmustrstbeunderstood.MonocrotalineandothertoxinsassociatedwithanimalPHmodelsarebroadlypnuemotoxictovessels[33].Itcanthenbesurmisedthattheroleofendothelialcellsasbarrierstouidowisnotfunctioningcorrectlyandcontributestodiseaseprogression.Wefoundthatthereisabreakdownoftheorganizedproteincomponentsthatregulatethemovementofwaterfromvesselsthroughtheinterstitialspaceandtolymphatics.Thisisvisibleinthestainingthatwasperformedforclaudin-5.Wefoundthattheexpressionofclaudin-5isdisorganizedandnolongerlocalizedtotightjunctionalregionsbetweencells.Thissuggeststhepossibilitythattheyarenolongercorrectlyperformingthefunctionofblockinguidfromowingoutofthevessel.Abreakdownofthistypewouldleadtoanincreaseinuidow[61,62,63,4,64]andthereisaprovenriseininterstitialuidpressureinhypoxicanimals[20].ThisalterationininterstitialuidmaintenancehasprofoundeectsonPAFs.Themechanicalsensingmechanismsofintegrintofocaladhesionkinasecomplexesallowcellstosensethispressureandthegradientfromvesselstolymphatics[13,14,15].TheevidenceformigrationinPHindirectlyderivesfromthestainingwithFSPandexaminationofBALTs.BALTsarekeylymphaticregionsofthelungwhichnormallycontainbroblastsuniformlydistributedthroughout.InPH,however,atallstagesofpathogenesisthebroblastsadoptapreferentiallocalizationtotheregionofthe 80

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BALTproximaltoavesselorairway.Thissuggeststhatthereisamigrationtowardstheseareas,perhapsduetosignalingfactors,uidow,oracombinationoffactors. 4.4CellASICsTheCellASICsapparatusprovidesanintriguingopportunitytoobservethebe-haviorofcellsinvitroandestablishmetricsforthemovementofcells.Insideofitsmicrouidicchannelitallowedtightcontrolofthemicroenvironmentofthecellsandamorefullrecreationoftherealinvivostateofcells.Theabilitytomaintainnormalculturingconditionsonamicroscopestageprovedtobeveryusefulforobservingthebehaviorofcellsinrealtime.Theresultsthatwerecapturedfromtimelapsedvideosofcellbehaviordemonstratethatthetechniqueofcelltrackingisindeedviableandthemethodissoundfortheanalysisofrealtimecellmovement. 4.5ShearForcesonCellsTheuseofspinningdiskconfocalalsoallowedustoaccuratelytracktheowratesofbeadsinsidethechannel.Wefoundthattherewaslessdierenceintheowratebetweendierentappliedpressuresthanhadbeenpreviouslyexpected.SincethecellswerestillexperiencingowtheseratescouldstillbeusedtoexaminetheamountofshearstressthesecellswereexperiencingwithaCFDsimulation.Thissimulationwasbasedontheaverageowratethatwasfoundaswellasthedimensionsofthechamber.Itwasvalidatedthatowinsidethechannelwaslaminar.TheentrancelengthofthechannelfortheowtobecomefullydevelopedwasshowntobeminisculeincomparisontothesizeofthechamberandthereforestandardCFDsimulationtechniquescouldbeemployed.CellshapeswererandomlyinsertedintothechambersimulationthatwasconstructedinSolidWorks.TheCFDsimulationusedtheowratefromthespinningdiskconfocalbeadtracking.Theaverageshearthatwasacrossthechamberfortheinsertedcellswas2.4Pascals.Sincethischannelhasanitesizetherewillberegionsthatexperiencehigherandlowervelocityowandconsequentlydierentshearlevelsaswasseeninasimilar 81

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study[69].Itwasconsequentlyofinteresttodivideuptheregionsofthechamber.Thisdivisionwasperformedintwodierentways.Firstbydividingthechamberintothirdsdownthelengthofthechannelthenthreedivisionsacrossthelength.Thisindicatedthattherewaslittledierenceintheshearstressthatthecellsexperiencesacrossthewidthofthechannelwith2.2and2.4Pascalsontheedgesofthechanneland2.6Pascalsinthecenter.Thedivisionsdownthelengthofthechannelrevealedadierentpatternofshearstressonthecells.Theshearstressdecreasedtowardstheendofthechamber.Thisindicatesthattheshapeofthechamberslowsdowntheowoftheuidandconsequentlytheshearstressthatthecellsexperiencedownthelengthofthechannelischanging.Thisisexhibitedingure3.24wherethevelocityofuidowisrepresentedbythecolorofthelinesinthesimulationandthereisadeniteslowdownofuidowattheendofthechannelasowmovesoutofthecenterchannelandtothebroaderendofthechannel.Shearstressisanimportantfactorthatdrivescellmigration.Thereisdirectmechanicaltransductionofstressthroughconnectionsthatcellshavefromtheircy-toskeletontocellsurfaceproteinsthatallowsthecelltosensemechanicalforcessuchasuidowactingonthecell'ssurface.Movementisalsoguidedthroughautocrineandparacrinesignaling.Cellsreleasechemokinesignalingfactorswhichfollowtheowofuiddownthechannel[22].Thesesignalingfactorscanguidecellmovementbasedonwheretheyinteractwithcellsandarecommonlyusedasamechanismforcancercellstoguidetheremovementdownstreamtowardslymphatics.Iftheshearstressistoohighonthecellsactivationofmechanicalsensingproteinsworkingcountertochemokinesignalingwillguidecellmigrationagainstow[23].Thismayserveasanexplanationforthedierentialpatternsofmigrationseenwithaltereduidowandregionalshearinthechannelattheseowrates.Thesechangingpatternsofmovementareshownintable3.4,whichshowswhethercellshadstatisticallysigni-cantdierencesinmovementfromrandom.Whenexaminingthewholechamberat 82

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thehigherowvalueof4PSIthemovementofcellswasnotsignicantlydierentfromrandom.Whileatcontrolowaswellas1PSIowthereisastatisticallysignicantmovementofcellswithowindierentregionsofthechamber.Itcanbespeculatedthatatlowerowcellswillmovewithow,likelyduetochemokinesignalingfactor,butathigherowthecompetitionbetweenchemokinesignalingandshearstressonthecellarehighenoughtopreventmovementinanyspecicdirection.Whenthechamberisdividedupalongitslengthasisshownin3.25furtherdierencesinbehaviorbecomeapparent.Forthe1PSIexperimentsthemigrationalbehaviorexhibitedwasthatinthehighshearregionofthechambertherewasnotastatisticallysignicantpreferenceforcellstomigrateinanydirection.Inthetwolowershearregionsofthechambertherewasapreferenceforthecellstomovewithow.Atcontrolowthehighestshearregionshowedastatisticallysignicantpreferenceforcellmigrationwithowdownthechannel.Movingtothetworegionswithlowershearhowevertherewasnostatisticallysignicantpreferencefordirectionalmovement.Thissuggeststhatattheendofthechamberasuidvelocityslowsdownandsheardecreases.Thismaycausecellstoonolongerbeabletoestablishagradientofchemokinesthatisconsistentandwillallowthemtomovedownthechamber.OverallcombiningthedivisionofcelltrackinginthecellASICsapparatusandtheSolidworkssimulationtheconclusionthatthereisaregionofuidshearstressthatleadstostatisticallysignicantmovementofcellswithowcanbemade.Thenaturalcounterargumenttothisassertionwouldbethatowispushingcellsdownthechamber,butinfactmovementdownthechamberdecreasesinthehighestowexperiments. 4.6RapidPrototyping3Dprintinghasprovedtobeausefultoolinrapidlyprototypingtoolsforresearchanditsusewillinthefutureallowfortoolstobemorequicklydevelopedandthepaceofresearchwillbeincreasedduetoitsimplementation.Forourpurposeswitha 83

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fuseddepositionmachinewewereabletoproduceacustommoldforhydrogelstobecuredinbeforemechanicalstrengthtestingwasperformedonthegelmixes.AcustommicroscopestagetoaccommodatethecellASICplatewithmanifoldattachedwasalsoprintedsincethestandardwouldnotttheplateandwasproducedatafractionofthecostofhavingsuchapartmachined.Finally,fromthethreedimensionalimageofthedecellularizedlungmatrixthatwascapturedwithconfocalmicroscopyascaledupthreedimensionalmodeloftheberswasprintedthatallowedforaphysicalmodeloftheparttobebuilt.Thismodelwouldhavebeenalmostimpossibletoconstructwithstandardmachiningtechniquesbutwaseasilydonewith3Dprinting. 4.7StrengthsandLimitationsTheuseofthemicrouidicculturingsystemprovidedanumberofstrengthsandlimitations.Thesystemallowedforthegreatercontrolofthecellculturingenviron-mentandtherealtimeobservationofcellactivitiesintheculturingenvironment.Unfortunatelythesystemalsomadeitdiculttoconsistentlyseedcellsthatwouldsurviveinthechamberandthedensitythatcellsmigratedintothechamberwasnotreliabledespitefollowingthesameseedingprotocoleverytime.Thismayhaveledtoinconsistenciesinthemeasurementcellmigrationbehaviorduetochangesinsignalingbetweencellsfromdensitieschanges.Therewerealsoanumberofissueswiththemanufacturingoftheplates.TheplatesmanufacturedatlowaltitudesdidnotalwaysperformasadvertisedpossiblyowingtothehigherelevationinDenver.Thehydrogelsystemthatwasusedfor3Dculturingprovidedtheabilitytotunebothitsmechanicalandchemicalpropertieswhich,inturn,alteredthebehaviorofthecellsinsideofthem.Itdidnothoweverallowforthemechanicalpropertiestobeincreasedtoveryhighlevels.Thiswassuitableforsoftertissueslikelungbutwouldnotbeapplicableforcellsfromstiertissuessuchascartilage.Thegelalsomadeitdiculttoapplystandardmechanicalstrengthtestingtechniquesduetothesoftnessofthegelandmethodslikeatomicforcemicroscopywouldberequiredforthistype 84

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ofdataacquisition[46].SEMprovidedhighresolutionimagesofthemicroscopicarchitectureoflungtissueandhydrogels.ThemethodsoftreatmenttopreptissuesforSEManalysisprovedtobeveryharshonthetissue.Thecriticalpointdryerusedfortheslidesdegradedthehydrogelsandmadeitsoonlylargeblocksofhydrogelcouldbeusedafterlyophilizationcouldbeusedmakingporesizeanalysisdicultduethelackofatsurfaces.Inaddition,allmethodsofpretreatmenthavetheinherentpossibilityofalteringthetissuewhichcanpossiblyleadtoinaccuraciesinanalysis.Theblackandwhiteimagesthatweretakenwerediculttotunetoallowforimageanalysis.Thecodewrittentoanalyzeporesizesrequiredahighlevelofcontrastbetweenalltissueandthebackgroundtoproperlydistinguishthetwo.Dependingontheproteininaregionthelevelofcontrastwasdierentduetohowtheproteinischargedbytheelectronbeam.SEMadditionallymakesstainingforspecictissueandcelltypesdicult.Itispossibletoattachgoldparticlestoantibodiesbutsuchtechnologiesarerequirespecialequipmentandexpertise.ThechemicalcompositionsofsamplescanbeevaluatedusingEDSbutthisdoesnotprovideproteinorcellularsignaturesunlesstheyareknownapriora.Confocalmicroscopyoftissueswaslimitedbythedepththattheexcitationandemissionlightcouldpenetratethroughthetissues.Decellularizedtissuesarenotverydensesothescatteringoflightislowerthanstandardtissues.Culturingofcellsinsideofhydrogelsallowedforthechangesinbehaviorofthecellstobeobservedandcomparedtostandardculturingtechniques.Stainingofthecellsinthegelhadseverallimitations.Onewasthevariabilityofhowfarsomeoftheantibodieswereabletopenetrateintothehydrogelleadingtodierentialstainingatchangingdepthsinthegel.Truetestingofthisvariabilitywouldrequireconfocalsectioningtoreconstructpenetrationdepthinsteadoftheepiuorescencemicroscopythatwasusedinthiscase. 85

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CuttingfrozensectionsofhydrogelsandstainingthemforH&Efacilitatedthedirectexaminationofatwodimensionalsectionofthegelandtheidenticationofkeystructuralfeaturesofthegel.Therewereanumberofdicultiesinthisprocesshoweverowingtothestructuralpropertiesofthegel.Standardfrozentissueslicingprotocolsproveddiculttocarryout.Thiswasduetothestructurallyweakanddiusenatureofthehydrogel,whichiscomposedmainlyofwater.Samplesforallfourhydrogelsweretakennogrowth,oneweekandtwoweeksofcellgrowthweretaken.Onlythersthydrogel,thestiestandthesecondhydrogelthesecondstiestprovedstrongenoughtoundergotheslicingprotocolwithoutthehydrogelstructurebreakingaparthowever.Thethicknessofthesesliceswasincreasedtopreservetheirintegritybutthisledtoagreaterdegreeoffoldingintissuesectionandinconsistentresults.MechanicalstrengthtestingwithaMTSmachineprovidedtheintriguingoppor-tunityoftestingthemechanicalpropertiesoftissuesaswellasgels.Therehowever,provedtobetomanyissuestomakethismethodoftestingviable.Itrequiredalargepieceofthelungtissueharvestedfromratsontheorderof5x20mm.Thismeantthatanumberofmicrostructuressuchasbloodvesselsandpleuraltissuemayhavebeenincluded.Eachofthesetissueshasdierentmechanicalpropertiesandwouldaltertheoverallresults.Theregionofinterestwastheadventitiaandmaynothavebeentheonlytissueintheregionleadingtoresultsthatwerenotpertinent.SinceMTStestingwasnotmicroscopicallyspecicenoughitbecametoodiculttoimplementonratlungtissue.Decellularizedlungtissuesdidprovetobemorespecicwhenthetissuewastested.Thismayhaveowedtotheremovalofcelltypesthatwouldhavechangedstructuralpropertiesleavingonlyextracellularmatrixbehind.WhenMTStestingwasattemptedonhydrogelsectionsthatwerecuredinacustomsizedmoldallgelmixesprovedtobetogelatinousforpropertestingtobeperformed.WhenthesegelmixeswereplacedintheclampsoftheMTStesterthey 86

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cameapartpreventingtesting.FSPandOx-62wereshowntonotcolocalizewitheachotherwhencostainedshowingthattheywererepresentativeofdierentcelltypesthatexpressedthesetwoproteinsseparately.Thisallowedfortheproperdistinctionbetweenbroblastanddendriticimmunecelltypes,butexpressionlevelscouldnotbetrulyquantied.Inadditiononlyaqualitativeanalysisofthelocalizationoftheseproteinstovesselsandairwayswaspossible.Claudin-5providedausefultooltodeterminethelocalizationoftightjunctionsandwhethertheyarefunctionalornot.Thisanalysiswasqualitativeandtheexpres-sionlevelswerenottrulymeasured.WithClaudin-5howeverexpressionlevelsarelessimportantthanlocalization.UsingthespotdetectionandtrackingsoftwareofIcyimageanalyzerhadseveralbenetsanddrawbacks.Thesoftwarereliedonthemultiplehypothesistrackingalgorithmdescribedearlierwhichtrackedthepositionsofdetectedpointsforseveralframesaheadandbehindthecurrentpoint.Thisdeterminedthemostprobabletrackforthecurrentdetectionpointanddecreasedthemarginoferrorinanoisysystem.Thismethodstillrequiredasucientlyhighcontrastandhighspacebetweencellstoaidtracking.Ifthecellorbeaddensitywastoohightherewouldbeacrossoverindetectionofcellsintheregionsofinterestandleadtoinaccurateresults.Thislimitedthenumberofcellsthatcouldbeseededintoamicrouidicculturingchamberbasedonwhethertheycouldbedistinguishedfromeachother.Inadditionifthedetectedpointsinsuccessiveimagesweretoofarapartthetrackingsoftwarecouldnotdistinguishthem.Thisrequiredanincreaseintheframeacquisitionratetoensurethatthecell/beadimageswerecloseenoughtogetherthatthesoftwarecouldeasilytrack.ThevascularpermeabilitytestrelieduponthepenetrationofEvan'sbluedyeintotissues.Evansbluedyeattachestoalbumenandwillpenetratethroughblood 87

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vesselsintotissues.Afterallowingforthedyetocirculateinthebloodforacertainperiodoftimeitwasthenextractedbysoakingthelunginformamidewhereatestofabsorbancecouldthenbeperformedandaratiometriccomparisonbetweentissuesperformed.Resultsfornormalratsdidnotprovideastatisticallysignicantcorrela-tiontoproceedtocomparisontodiseasedanimals.Thiswasmorethanlikelyduetointeranimalvariabilitymainlytheinabilitytoperformtakedownsandinjectionsofbluedyeinconsistentperiodsoftimeleadingtovariablebluedyepenetrationintotissues.Weusedthehydroxyprolineassaytoanalyzethelevelofcollagencontentinsam-ples.Inthisrespectitprovidedanidealtooltoanalyzehowbroblastswerealteringthehydrogelaroundthemselves.Therewereanumberofcomplicationswiththeassay,however.Thesemainlyowedtotheinconsistentexecution.Hydrogelsarebynaturemainlymadeupofwater.Thismeansthatwhenweighingahydrogel,aconsistentamountofthewatermustberemovedwithoutdehydratingthegel.Thisprovidesvariabilitybetweentestsandsamples.Anotherfailingofthehydroxyprolineassaycamefromtheovernightincubationofsamplesimmersedin5.5molarhydrochloricacidat110degreesCelsius.Thesesampleswerecontainedin1mLpolyethyleneaskswithanadditionalpressuresealerattached.Someasksstillleakedevaporateduid.Samplesthatwerevisibledierentwerediscarded,butothersamplesmayhavelostsmalleramountsofuidwhichwouldagaincontributetovariabilitybetweensamples.Theuseofatightersealingaskwillberequiredforfuturetests.3Dprintingtechnologiesoerrapidprototypingcapabilityandnoveltoolsforresearch,yetcarryaprimarylimitation.Thislimitationisthematerialsthatcanbeusedby3Dprinters.Unlesstheyareexceedinglyexpensivetheywillnotprintcomponentsthatcanbesterilized.Inadditionstandard3Dprintersdonotprintwithresolutionsthateliminatedefectsorallowformicroscaleequipmenttobemade. 88

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ThePCRthatwasperformedoncellsgrowningelsproveddiculttocarryoutforseveralreasons.FirstextractingmRNAfromthegelsprovedtobedicultduetothedicultyinbreakingupthegel.NextcellsgrowninthegelprovedtoproducefarlessusablemRNAandconsequentlycDNAleadingtoamplicationonlyafteralargenumberofcyclesonthemachineandthereforelessconsistentresults. 89

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5.ConclusionToincreasethepaceandecacyofbiomedicalresearchthereisaneedtodirectlylinkinvitroandinvivoexperimentsandtomorefullyrecreatetheactualenvironmentofadiseaseandprovidecloserpathophysiology.Thisrecreationrequirestheproperstudyofthestatusofthediseaseandanunderstandingofthefactorsleadingtoitsprogression.Inthisway,ourstudywasdesignedtomimicaspectsofpulmonaryhypertensioninamorecompletefashion.Whenresearchandrecreatingthelungsofratswithinducedpulmonaryhypertension,thephysicalandchemicalpropertiesoftheregionneedtobeconsidered.Todothisthetissuewasexaminedinitsmostbasicform,asadecellularizedversionofitselfwithonlytheproteinmakeuprepresented.Thisrevealedthatthecellswhenreseededhadapreferencetogrowonthelessdensebroustissueofthelungasopposedtothethickproteinregionofthepleuraltissueofthelung.Withthisinmind,atypeofhydrogelthatrecreatedthelargeopenspacesofthelungsinterstitialspacescouldbeutilizedforabasicinvitromodel.Themechanicalpropertiesofanytissueneedtoberecreatedwhendoingatrue3Dculture.Inthisrespect,thestudyattemptedtotestthemicromechanicalpropertiesofthelungthroughanMTSmechanicalstrengthtestingmachine.Theexperimentsproducedinconsistentresultsbuttheconceptofperforminginvitroculturingexperimentsinbiomaterialswiththetunablemechanicalpropertiestoobservethechangesinbehaviorforbroblastswasproventobeviable.ItwasshownthatPAFswouldaltertheirbehaviorbasedsimplyonculturingenvironment.Cellschangedmorphologydramaticallythroughsimplechangesinchemistryandmechanicalproperties.Themostmarkedexampleofthiswastheformationofdendritelikeconnectionsbetweencellsinadrasticallydierentfashionfromtheconuencyseeninstandard2Dcultures.MostanimalmodelsofPHinducethediseasethroughtheuseofdrugsthatactuponendothelialcellsinthevesselsofthelungs.Inadditionagrowingbodyofmedicalresearchshowsthatthereistoabreakdownoftheintimallayerofvessels 90

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inPH.Oneprimaryfunctionofendothelialcellsistoestablishabarriertopreventuidmovementfromthevesselintotheinterstitialspace.WhenthesebarrierswereexaminedthroughtheuseofimmunouorescentstainingitwasrevealedthattherewasalossoforganizationofClaudin-5,oneoftheprimarytightjunctionalproteinsofthelung.TherewasalsoavisiblelossoforganizationofAquaporin-1,aproteinresponsibleforfacilitatinguidmovementintheinterstitialspacesofthelung.SEMrevealedthatporesandconsequentlyberswerepreferentiallyaligningperpendiculartouidow.Thisatypeofbehaviorthatinothertissueshasbeenassociatedwithincreasedinterstitialuidow.Thisisduetothecellsandbersintheadventitiahavingtoabsorbthelargerpressuredropbetweenthebloodvesselsandlymphat-ics.Paststudieshaveshownthatthereisanincreaseininterstitialuidpressureinhypoxichypertensiveanimals.TakentogetherallofthesestudiespointtotheconclusionthatthereisanincreaseinuidowintheinterstitialspacesofthelungsofthesePHanimals.SEMexaminationalsorevealedthattheadventitiawasundergoingveryspecicchangeswherethetotalamountofvoidspaceandtheoverallporesizeweredecreasingtofacilitatethemabsorbingtheincreasedpressuredrop.InadditiontotheadventiaabsorbingagreaterloadforthepressuredropacrosstheinterstitiumthischangemayalsobeincreasingthestinessofthetissueandconsequentlyitsresistanceleadingtotheprogressionofPH.ThethreedimensionalculturingsetupemployedprovedthatPAFswouldremodelthegeltheywereembeddedinbyinitiallyincreasingthecollageninthegelandthendecreasingit.Thiscouldbeseenasaresponsefromthebroblastsfacinganunfamiliarenvironmentleadingtoaninitialproinamatoryremodelingofthetissuearoundthemselves.Laterthegelswereseentochangeandreverseremodeledbacktoalowercollagenstatewhichmayrepresentamorehealthyphysiologicalsteadystatetissuecomposition.InPHhowever,thisreverseremodelingstepdoesnotoccur, 91

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insteadthevesselsstayintheirinitialproinamatorystateWiththepossibilityoftherebeingincreaseduidowacrosstheinterstitialspacetolymphatics.FibroblastsanddendriticimmunecellsinsideoflymphaticlikeBALTtissueswerestudiedinPH.ThisshowedthatbothcelltypeslocalizetowardsvesselsandairwaysindiseasedPHanimalsindicatingthatthereissomethingguidingthemovementofthesecells.Thereisasizablebodyofevidenceinotherstudiespointingtointerstitialuidowbeingoneofthekeyguidingfactorsleadingtocellmigration.TostudytheeectthatuidowhasonPAFsanexperimentwiththesecellsseededintothemicrouidiccellASICschamberwasutilized.Thisdemonstratedthatatdierentowlevelsandshearlocationsinthechamberthemovementcharacter-isticsofcellschanged.Therearetwofactorsthatguidecellmovement.Therstisshearforcesthatcellssensethroughmechanicaltransductiontoproteinsinsidethecellandthesecondischemokinesignaling.Thesecanworkinacompetitivefashionagainsteachothersuchthatasthelevelofshearisincreasedandthespeedofuidowischangedthecellswillchangebehaviorfrommigratingwithowtorandommovement.Withspinningdiskconfocalthedirectquanticationofthespeedatwhichuidwasowingdownthechannelandthereforethetotalvolumetricowratedownthechannel.Afterthisthecomputationaluiddynamicssimulationcouldbeusedtoquantifyhowmuchshearcellswereexperiencingindierentregionsofthechamber.Takentogetherthisrevealedthattherewasaspecicamountofshearwhichledtostatisticallysignicantmovementofcells.Theseresultstakenalltogetherpointtooneconclusion.Theobserveddisorgani-zationoftheendothelialcellbarrierpointstothepossibilityofincreasedinterstitialuidow.Thereisanobservedchangeinlocalizationofcells.Thereisevidencefromthisstudyandotherspointingtouidowaectingcellbehaviorwithaspe-cialfocusbeingoninterstitialowcausingbroblaststobecomeprobrotic.PAFswereshowntohavethecapabilityofsignicantlychangingtheproteinmakeupofa 92

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hydrogel.TheseresultswouldpointtotheconclusionthattheincreasedinterstitialowwouldbeacausativefactorinthereleaseofcollagenandinmaintainingtheproinamatorystateofPH.ThiswouldleadtoastieningofthevesselandariseinvascularresistanceandtheprogressionofPH.Theexperimentsdoneinthisthesispresentanumberofnewoptionsfortyingtogethertraditionalcellbiologyandengineeringresearchtechniques.TheuseoftraditionalIFstainingandSEMallowedthestudyofhowuidowbarrierswerechanginginPH.Thiscouldthenbeutilizedtosetupanexperimentwherecellswerestressedwithuidshearforcestostudytheirbehavior.Imageprocessingtechniquescouldthenbeutilizedtotrackthisbehaviorquicklyandstudytheuidowinthechamberaswell.Computationaluiddynamicssoftwarewasusedtoquantifytheshearforcesthatthesecellswouldbeexperiencing.Inthefutureutilizingthesemanyaspectsofresearchwillallowformorecompletestudiestobecarriedout. 93

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APPENDIXA.MatLabCode A.1CellTrackingProcedure Cleartemporaryvariables Individualcelltracking clearcloseall%Importthedata[~,~,raw]=xlsread('C:\path\excelfile.xls','Tracks','B1:E10000');%Excelfileoftracks%Replacenon-numericcellswith0.0R=cellfun(@(x)(~isnumeric(x)&&~islogical(x))||isnan(x),raw);%Findnon-numericcellsraw(R)={0.0};%Replacenon-numericcells%CreateoutputvariableTest1=reshape([raw{:}],size(raw));Test1=Test1.*-1; Errorusingxlsread(line129)XLSREADunabletoopenfile'C:\path\excelfile.xls'.File'C:\path\excelfile.xls'notfound.ErrorinCellTrackingOutline(line5)[~,~,raw]=xlsread('C:\path\excelfile.xls','Tracks','B1:E10000');%Excelfileoftracks 101

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Cleartemporaryvariables clearvarsrawR;[nm]=size(Test1);count=1;count2=0;scale=4.76;%micrometersperpixelofchambertscale=.5;%hourspertimepointtravel=4;%amountofpixelstraveledtobeincludedtotTravel=0;fora=2:1:n%extractingthetrackingpointsanddeterminingstart/finishpointsifTest1(a,3)~=0&&Test1(a-1,3)==0%findingstartingpointsofthetrackerxstart=Test1(a,3);ystart=Test1(a,4);tstart=Test1(a,2);endifTest1(a,3)==0&&Test1(a-1,3)~=0%findingtheendpointtfinish=Test1(a-1,2);xfinish=Test1(a-1,3);yfinish=Test1(a-1,4);xdiff=xstart-xfinish;%findingthexdifferenceydiff=ystart-yfinish;%findingtheydifferencetime=tfinish-tstart;%findingthetimedifferencetotal=sqrt(xdiff^2+ydiff^2);iftotal>travel[thetarho]=cart2pol(xdiff,ydiff);%conversionpolarthetaRho(count,:)=[thetarho];%indexingintoamatrixxdisp(count)=xdiff/scale;%findingxdispoftrack 102

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ydisp(count)=ydiff/scale;%findingydispoftrackcount=count+1;%counterforindexingcount2=count2+1;%fortotalnumberoftrackstotTravel=totTravel+sqrt(xdiff^2+ydiff^2);endiftotal
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axisy=0counterclockwiserotation%makingapolarweightedhistogramcount=0;fora=1:length(thetaRho(:,1))%indexthroughallpointsnumber=round(thetaRho(a,2));%theroundedangleoffinalmovementtheta(count+1:count+number)=thetaRho(a,1);%thepixelmovementisaddedtotheangleusedwitheachpixelbeingrecordedasanadditionalpointatthatangleintheindexcount=count+number;endfigure%Productionoftheweightedpolarhistogramaxisoffholdonrose(theta)thetaRho(:,1)=radtodeg(thetaRho(:,1));fora=1:length(thetaRho(:,1))ifthetaRho(a,1)<0thetaRho(a,1)=thetaRho(a,1)+2*pi;endend[hp]=ttest(thetaRho(:,1),0);fprintf('Thepvalueofwhethercellsaretravelinginthesamedirectionis%fwith%idatapoints.\n',p,count2)[hxpx]=ttest(xdisp,0); 104

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fprintf('Thepvalueofwhethercellsaretravelinginthesamexdirectionis%f\n',px)[hypy]=ttest(ydisp,0);fprintf('Thepvalueofwhethercellsaretravelinginthesameydirectionis%f\n',py) 105

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Individualcelltracking figureholdonaxisoffxstart=0;count=1;%forindexingfora=2:1:nifTest1(a,3)~=0&&Test1(a-1,2)==0%testforstartingpointxstart=Test1(a,3);%recordthestartpointofeachtrackystart=Test1(a,4);endifTest1(a,3)==0&&Test1(a-1,3)~=0%testforendingpointxstart=0;%zerostartingpointsforfutureuseystart=0;count=1;polar(poltrack(:,1),poltrack(:,2))%plottheindividualpathofthecellsclearpoltrack%cleartheindividualtrackendifxstart~=0%testfortheexistanceofatrack[RhoTheta]=cart2pol(xstart-Test1(a,3),ystart-Test1(a,4));%convertthenewpositionintoapolarpositionpoltrack(count,1)=Rho;%recordthenewtrackpositionintoitsprogressivepointpoltrack(count,2)=Theta;%andangle 106

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count=count+1;%moveforwardontrackingthecellendend A.2BeadTrackingProcedure clearcloseall[~,~,raw]=xlsread('C:\Filepath\Excelfile.xls','Tracks','B1:E10000');R=cellfun(@(x)(~isnumeric(x)&&~islogical(x))||isnan(x),raw);%Findnon-numericcellsraw(R)={0.0};%Replacenon-numericcellsTest1=reshape([raw{:}],size(raw));Test1=Test1*-1;clearvarsrawR;[nm]=size(Test1);count=1;count2=0;scale=.33333;%micrometersperpixeltscale=.2;%secondspertimepointtravel=4;%amountofpixelstraveledtobeincludedtotTravel=0;fora=2:1:n%extractingthetrackingpointsanddeterminingstart/finishpointsifTest1(a,3)~=0&&Test1(a-1,3)==0%finding 107

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startingpointsofthetrackerxstart=Test1(a,3);ystart=Test1(a,4);tstart=Test1(a,2);endifTest1(a,3)==0&&Test1(a-1,3)~=0%findingtheendpointtfinish=Test1(a-1,2);xfinish=Test1(a-1,3);yfinish=Test1(a-1,4);xdiff=xstart-xfinish;%findingthexdifferenceydiff=ystart-yfinish;%findingtheydifferencetime=abs(tfinish-tstart);%findingthetimedifferencetotal=sqrt(xdiff^2+ydiff^2);iftotal>travel[thetarho]=cart2pol(xdiff,ydiff);%conversionpolarthetaRho(count,:)=[thetarho];%indexingintoamatrixxdisp(count)=xdiff/scale;%findingxdispoftrackydisp(count)=ydiff/scale;%findingydispoftrackvelocity(count)=rho/time;count=count+1;%counterforindexingcount2=count2+1;%fortotalnumberoftrackstotTravel=totTravel+sqrt(xdiff^2+ydiff^2);end 108

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iftotal
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micrometers/second\n',mean(velocity)*tscale/scale)%makingapolarweightedhistogramcount=0;fora=1:length(thetaRho(:,1))%indexthroughallpointsnumber=round(thetaRho(a,2));%theroundedangleoffinalmovementtheta(count+1:count+number)=thetaRho(a,1);%thepixelmovementisaddedtotheangleusedwitheachpixelbeingrecordedasanadditionalpointatthatangleintheindexcount=count+number;endfigure%Productionoftheweightedpolarhistogramaxisoffholdonrose(theta)thetaRho(:,1)=radtodeg(thetaRho(:,1));fora=1:length(thetaRho(:,1))ifthetaRho(a,1)<0thetaRho(a,1)=thetaRho(a,1)+2*pi;endend[hp]=ttest(thetaRho(:,1),0);fprintf('Thepvalueofwhethercellsaretravelingin 110

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thesamedirectionis%fwith%idatapoints.\n',p,count2)[hxpx]=ttest(xdisp,0);fprintf('Thepvalueofwhethercellsaretravelinginthesamexdirectionis%f\n',px)[hypy]=ttest(ydisp,0);fprintf('Thepvalueofwhethercellsaretravelinginthesameydirectionis%f\n',py) Errorusingxlsread(line129)XLSREADunabletoopenfile'C:\Filepath\Excelfile.xls'.File'C:\Filepath\Excelfile.xls'notfound.ErrorinBeadTrackingOutline(line5)[~,~,raw]=xlsread('C:\Filepath\Excelfile.xls','Tracks','B1:E10000'); figureholdonaxisoffxstart=0;count=1;%forindexingfora=2:1:nifTest1(a,3)~=0&&Test1(a-1,2)==0%testforstartingpointxstart=Test1(a,3);%recordthestartpointofeachtrackystart=Test1(a,4);endifTest1(a,3)==0&&Test1(a-1,3)~=0%testforendingpoint 111

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xstart=0;%zerostartingpointsforfutureuseystart=0;count=1;polar(poltrack(:,1),poltrack(:,2))%plottheindividualpathofthecellsclearpoltrack%cleartheindividualtrackendifxstart~=0%testfortheexistanceofatrack[RhoTheta]=cart2pol(xstart-Test1(a,3),ystart-Test1(a,4));%convertthenewpositionintoapolarpositionpoltrack(count,1)=Rho;%recordthenewtrackpositionintoitsprogressivepointpoltrack(count,2)=Theta;%andanglecount=count+1;%moveforwardontrackingthecellendend A.3PoreSizeAnalysis clearcloseallI1=imread('image.tif');%importimagemustbeinsamefolderas.mfilefiguresubplot(2,2,1)imagesc(I1)%RawImagecolormap(gray)scale=10;%inputthescaleoftheimageorthenumberofpixelspermicron 112

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I=I1(1:100,1:100);%Selectasubsectionoftheimagetoanalyzesubplot(2,2,2)imagesc(I)%Subsetofimagecolormap(gray)[mn]=size(I);I=I(:,:,1);%ifitisamultichannelimagethreshold=.3;%chooseathresholdvaluefortheimagebetween0and1I=im2bw(I,threshold);subplot(2,2,3)imagesc(I)%Displaythethresholdedimage%porousareafractionPaf=1-sum(sum(I))/(m*n);fprintf('Theporousareafractionoftheimageis%.2f\n',Paf)%meanfreepathImfa=I;filter=fspecial('gaussian',[33],2);%GaussianSmoothingFilterImfa=round(conv2(double(Imfa),filter));Imfa=Imfa(3:m+2,3:n+2);%filter=fspecial('average',[33]);%Averagingfilter%Imfa=round(conv2(double(Imfa),filter));Imfa2=Imfa;%Imagetodrawlinesonsubplot(2,2,4)imagesc(Imfa)%smoothedsubset 113

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colormap(gray)count=0;%#ofpixelsinvoidcount2=1;rowCount=0;%#oflinesthatwentacrossavoidposition1=[0,0];fora=1:5:m-1%CountinginYdirectionforb=1:n-1ifImfa(a,b)==1&&Imfa(a,b+1)==0%Thestartofavoidspacecount=count+1;position1=[a,b+1];endifb==1&&Imfa(a,b)==0&&position1(1)==0%Thestartofavoidspaceedgeeffectscount=count+1;position1=[a,b];endifImfa(a,b)==0&&Imfa(a,b+1)==1&&count~=0&&b-position1(2)~=0%Findingtheendofavoidspaceposition2=[a,b];ifposition2(1)==position1(1)%ensuringtherowisthesameylength(count2)=position2(2)-position1(2);count2=count2+1;else%iftherowisnotthesamethanithittheedgeoftheimageylength(count2)=m-position1(2); 114

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%recordvoida-1ylength(count2+1)=b;%recordvoudatacount2=count2+2;endposition1=[0,0];%resetthepositiontoaccountforedgesendifImfa(a,b)==0%CountingthetotalnumberofpixelscoveredinvoidsImfa2(a,b)=1;rowCount=rowCount+1;endendendMfpx=rowCount/count;%Findingthemeanareafractioncount=0;count2=1;rowCount=0;position1=[0,0];forb=1:5:n%CountingintheXdirectionfora=1:m-1ifImfa(a,b)==1&&Imfa(a,b+1)==0%Thestartofavoidspacecount=count+1;position1=[a,b+1];endifb==1&&Imfa(a,b)==0&&position1(1)==0%The 115

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startofavoidspaceedgeeffectscount=count+1;position1=[a,b];endifImfa(a,b)==0&&Imfa(a,b+1)==1&&count~=0&&b-position1(2)~=0%Findingtheendofavoidspaceposition2=[a,b];ifposition2(1)==position1(1)%ensuringtherowisthesamexlength(count2)=position2(2)-position1(2);count2=count2+1;else%iftherowisnotthesamethanithittheedgeoftheimagexlength(count2)=m-position1(2);%recordvoida-1xlength(count2+1)=b;%recordvoudatacount2=count2+2;endposition1=[0,0];%resetthepositiontoaccountforedgesendifImfa(a,b)==0%CountingthetotalnumberofpixelscoveredinvoidsImfa2(a,b)=1;rowCount=rowCount+1;endendend 116

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Mfpy=rowCount/count;figureimagesc(Imfa2);%Displaythetracksthatwerecountedcolormap(gray)fprintf('Theaverageporesizeis%.2fmicronsindiameter\n',(Mfpx+Mfpy)/2/scale)%histogramanalysiscutoff=5;figurexlength=xlength(xlength>=cutoff*scale);subplot(1,2,1)hist(xlength)ylength=ylength(ylength>=cutoff*scale);subplot(1,2,2)hist(ylength)fprintf('Theaverageporesizegreaterthan%.1fmicronsis%.2fmicronsindiameter\n',cutoff,(mean(xlength)+mean(ylength))/2/scale) A.4MTSTestingCodeamodifacationofcodethatwasprovidedbyJenniferWagner,UniversityofColoradoDenver.Bioengineering. %ThisscriptloadsuniaxialdatageneratedfromanMTSmachine.%%3rdAugust,2009%CJL 117

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%Preparetheworkspace.clearall;closeall;clc;%Scriptparameters.FileName1='Sample1.txt';FileName2='Sample2.txt';FileName3='Sample3.txt';FileName4='Sample4.txt';FileName5='Sample5.txt';%FileName6='Sample6.txt';%FileName7='Sample7.txt';Spec1='Sample1';Spec2='Sample2';Spec3='Sample3';Spec4='Sample4';Spec5='Sample5';%Spec6='Sample6';%Spec7='Sample7';Name='LungtissueMSTstretchtest1';%Testing='Elongation[%]';Hydrogel_mouse_07062011_Testing='Strain[%]';HeaderLinePosition=8; 118

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Delimiter=',';FLAGS.Print=1;%startinglengthsL01=20.4;L02=18.043;%[millimeters]L03=17.096;L04=12.59;L05=14.48;%L06=12.1;%L07=10;%StartingX-sectionaldimensions/areas(assumesrectangle%specimen1W1=6.69;H1=6.69;%D1=10.00;Sample1CrossSectional=H1*W1;%[millimeterssquared]%Sample1CrossSectional=0.25*(D1^2)*pi;%Specimen2W2=6.38;H2=6.38;%D2=10.09;Sample2CrossSectional=H2*W2;%[millimeterssquared] 119

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%Sample2CrossSectional=0.25*(D2^2)*pi;%specimen3W3=3.9;H3=3.9;%D3=10.04;Sample3CrossSectional=H3*W3;%[millimeterssquared]%Sample3CrossSectional=0.25*(D3^2)*pi;%Specimen4W4=3.59;H4=3.59;%D4=10.01;Sample4CrossSectional=H4*W4;%[millimeterssquared]%Sample4CrossSectional=0.25*(D4^2)*pi;%specimen5W5=3.73;H5=3.73;Sample5CrossSectional=H5*W5;%[millimeterssquared]%%Specimen6%W6=3;%H6=3;%Sample6CrossSectional=H6*W6;%[millimeterssquared] 120

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%%Sample6CrossSectional=0.25*(W6^2)*pi;%[millimeterssquared]%%%specimen7%W7=3.3;%H7=3.3;%Sample7CrossSectional=H7*W7;%[millimeterssquared]%%Sample7CrossSectional=0.25*(W7^2)*pi;%[millimeterssquared]%SlidingAverageFilter%variableforaveragingwindowWindow=5;%Loadthedatafile.Data1.RAW=dlmread(FileName1,Delimiter,HeaderLinePosition,0);fori=Window+1:1:size(Data1.RAW,1)-WindowData1.Extension(i-Window)=mean(Data1.RAW(i-Window:i+Window,3));Data1.Force(i-Window)=mean(Data1.RAW(i-Window:i+Window,1));endData1.Extension=Data1.Extension+abs(Data1.Extension(1));Data1file=[Data1.Extension*100/L01;Data1.Force]; 121

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Data1file=Data1file';Data2.RAW=dlmread(FileName2,Delimiter,HeaderLinePosition,0);fori=Window+1:1:size(Data2.RAW,1)-WindowData2.Extension(i-Window)=mean(Data2.RAW(i-Window:i+Window,3));Data2.Force(i-Window)=mean(Data2.RAW(i-Window:i+Window,1));endData2.Extension=Data2.Extension+abs(Data2.Extension(1));Data2file=[Data2.Extension*100/L02;Data2.Force];Data2file=Data2file';Data3.RAW=dlmread(FileName3,Delimiter,HeaderLinePosition,0);fori=Window+1:1:size(Data3.RAW,1)-WindowData3.Extension(i-Window)=mean(Data3.RAW(i-Window:i+Window,3));Data3.Force(i-Window)=mean(Data3.RAW(i-Window:i+Window,1));endData3.Extension=Data3.Extension+abs(Data3.Extension(1));Data3file=[Data3.Extension*100/L03;Data3.Force];Data3file=Data3file'; 122

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Data4.RAW=dlmread(FileName4,Delimiter,HeaderLinePosition,0);fori=Window+1:1:size(Data4.RAW,1)-WindowData4.Extension(i-Window)=mean(Data4.RAW(i-Window:i+Window,3));Data4.Force(i-Window)=mean(Data4.RAW(i-Window:i+Window,1));endData4.Extension=Data4.Extension+abs(Data4.Extension(1));Data4file=[Data4.Extension*100/L04;Data4.Force];Data4file=Data4file';Data5.RAW=dlmread(FileName5,Delimiter,HeaderLinePosition,0);fori=Window+1:1:size(Data5.RAW,1)-WindowData5.Extension(i-Window)=mean(Data5.RAW(i-Window:i+Window,3));Data5.Force(i-Window)=mean(Data5.RAW(i-Window:i+Window,1));endData5.Extension=Data5.Extension+abs(Data5.Extension(1));Data5file=[Data5.Extension*100/L05;Data5.Force];Data5file=Data5file';%Data6.RAW=dlmread(FileName6,Delimiter 123

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,HeaderLinePosition,0);%fori=Window+1:1:size(Data6.RAW,1)-Window%%Data6.Extension(i-Window)=mean(Data6.RAW(i-Window:i+Window,3));%Data6.Force(i-Window)=mean(Data6.RAW(i-Window:i+Window,1));%end%Data6.Extension=Data6.Extension+abs(Data6.Extension(1));%Data6file=[Data6.Extension*100/L06;Data6.Force];%Data6file=Data6file';%%Data7.RAW=dlmread(FileName7,Delimiter,HeaderLinePosition,0);%fori=Window+1:1:size(Data7.RAW,1)-Window%%Data7.Extension(i-Window)=mean(Data7.RAW(i-Window:i+Window,3));%Data7.Force(i-Window)=mean(Data7.RAW(i-Window:i+Window,1));%end%Data7.Extension=Data7.Extension+abs(Data7.Extension(1));%Data7file=[Data7.Extension*100/L07;Data7.Force];%Data7file=Data7file';%CalculateTrueStress-Strainusingfiltereddatafori=1:1:length(Data1.Force) 124

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Data1.TrueStrain(i)=log(1+(Data1.Extension(i)/L01));Data1.TrueStress(i)=((Data1.Force(i)/Sample1CrossSectional*(1+Data1.TrueStrain(i))));endfori=1:1:length(Data2.Force)Data2.TrueStrain(i)=log(1+(Data2.Extension(i)/L02));Data2.TrueStress(i)=(Data2.Force(i)/Sample2CrossSectional*(1+Data2.TrueStrain(i)));endfori=1:1:length(Data3.Force)Data3.TrueStrain(i)=log(1+(Data3.Extension(i)/L03));Data3.TrueStress(i)=(Data3.Force(i)/Sample3CrossSectional*(1+Data3.TrueStrain(i)));endfori=1:1:length(Data4.Force)Data4.TrueStrain(i)=log(1+(Data4.Extension(i)/L04));Data4.TrueStress(i)=(Data4.Force(i)/Sample4CrossSectional*(1+Data4.TrueStrain(i)));end 125

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fori=1:1:length(Data5.Force)Data5.TrueStrain(i)=log(1+(Data5.Extension(i)/L05));Data5.TrueStress(i)=(Data5.Force(i)/Sample5CrossSectional*(1+Data5.TrueStrain(i)));end%fori=1:1:length(Data6.Force)%%Data6.TrueStrain(i)=log(1+(Data6.Extension(i)/L06));%Data6.TrueStress(i)=(Data6.Force(i)/Sample6CrossSectional*(1+Data6.TrueStrain(i)));%end%%fori=1:1:length(Data7.Force)%%Data7.TrueStrain(i)=log(1+(Data7.Extension(i)/L07));%Data7.TrueStress(i)=(Data7.Force(i)/Sample7CrossSectional*(1+Data7.TrueStrain(i)));%end%CreateDataSetsForFitTool%EngineeringStress-Strain 126

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Data1x=Data1.Extension/L01*25.4;Data1y=(Data1.Force/Sample1CrossSectional);Data2x=Data2.Extension/L02*25.4;Data2y=(Data2.Force/Sample2CrossSectional);Data3x=Data3.Extension/L03*25.4;Data3y=(Data3.Force/Sample3CrossSectional);Data4x=Data4.Extension/L04*25.4;Data4y=(Data4.Force/Sample4CrossSectional);Data5x=Data5.Extension/L05*25.4;Data5y=(Data5.Force/Sample5CrossSectional);%Data6x=Data6.Extension/L06*25.4;%Data6y=(Data6.Force/Sample6CrossSectional);%Data7x=Data7.Extension/L07*25.4;%Data7y=(Data7.Force/Sample7CrossSectional);figureholdoncount=1;fora=71:10:length(Data1x)-71p=polyfit(Data1x(a-70:a+70),Data1y(a-70:a+70),2);d=polyder(p);slope(count)=polyval(d,Data1x(a));point(count)=Data1x(a);count=count+1;plot(Data1x(a-70:a+70),polyval(p,Data1x(a-70:a+70)))endfora=2:1:length(slope)dif(a)=slope(a)-slope(a-1);endfigure 127

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holdonplot(point,slope,'MarkerEdgeColor','k')fprintf('modulus=%fkpaforsample1\n',max(slope))clearpointslopecount=1;fora=71:10:length(Data2x)-71p=polyfit(Data2x(a-70:a+70),Data2y(a-70:a+70),2);p=polyder(p);slope(count)=polyval(p,Data2x(a));point(count)=Data2x(a);count=count+1;endfora=2:1:length(slope)dif(a)=slope(a)-slope(a-1);endfigureholdonplot(point,slope,'MarkerEdgeColor','k')fprintf('modulus=%fkpaforsample2\n',max(slope))clearpointslopecount=1;fora=71:10:length(Data3x)-71p=polyfit(Data3x(a-70:a+70),Data3y(a-70:a+70),2);p=polyder(p);slope(count)=polyval(p,Data3x(a));point(count)=Data3x(a);count=count+1; 128

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endfora=2:1:length(slope)dif(a)=slope(a)-slope(a-1);endfigureholdonplot(point,slope,'MarkerEdgeColor','g')fprintf('modulus=%fkpaforsample3\n',max(slope))clearpointslopecount=1;fora=71:10:length(Data4x)-71p=polyfit(Data4x(a-70:a+70),Data4y(a-70:a+70),2);p=polyder(p);slope(count)=polyval(p,Data4x(a));point(count)=Data4x(a);count=count+1;endfora=2:1:length(slope)dif(a)=slope(a)-slope(a-1);endfigureholdonplot(point,slope,'MarkerEdgeColor','g')fprintf('modulus=%fkpaforsample4\n',max(slope))clearpointslopefigureholdon 129

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count=1;fora=101:10:length(Data5x)-101p=polyfit(Data5x(a-100:a+100),Data5y(a-100:a+100),2);p=polyder(p);slope(count)=polyval(p,Data5x(a));point(count)=Data5x(a);count=count+1;plot(Data5x(a-70:a+70),polyval(p,Data5x(a-70:a+70)))endfora=2:1:length(slope)dif(a)=slope(a)-slope(a-1);endfigureholdonplot(point,slope)fprintf('modulus=%fkpaforsample5\n',max(slope))clearpointslope A.5OrientationAnalysis clearI1=imread('BinaryImage2.tif');I=I1(600:700,900:1000);figureimagesc(I)colormap(gray)[mn]=size(I);fora=1:m*nifI(a)==255I(a)=1; 130

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endendblank=zeros(m,n);figureimshow(blank)holdonpositionUsed=zeros(m,n);count=0;fora=2:1:n-1forb=2:1:m-1boundary=0;surround=I(b-1,a-1)+I(b-1,a)+I(b-1,a+1)+I(b,a-1)+I(b,a+1)+I(b+1,a-1)+I(b+1,a)+I(b+1,a+1);ifI(b,a)==1&&positionUsed(b,a)==0&&surround<8&&surround>5boundary=bwtraceboundary(I,[b,a],'N');endifboundary>0iflength(boundary(:,1))
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(h-1,1),boundary(h,2))=1;endifh~=length(boundary(:,1))positionUsed(boundary(h+1,1),boundary(h,2))=1;positionUsed(boundary(h,1),boundary(h+1,2))=1;positionUsed(boundary(h+1,1),boundary(h+1,2))=1;endifh~=length(boundary(:,1))&&h~=1positionUsed(boundary(h+1,1),boundary(h-1,2))=1;positionUsed(boundary(h-1,1)boundary(h+1,2))=1;endpositionUsed(boundary(h,1),boundary(h,2))=1;blank(boundary(h,1),boundary(h,2))=1;end[centerx,centery]=ait_centroid(blank);blank=imfill(blank,'holes');areaObject(count)=bwarea(blank);plot(boundary(:,2),boundary(:,1))plot(centerx,centery)endendclearcenterxcenterysurroundblank=zeros(m,n); 132

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endendfigureimagesc(positionUsed) 133

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APPENDIXB.ExcelCalculators B.1HydroxyprolineAnalyzerHydroxyprolineAnalyzertakesthevariousmeasurementsfromtheHydroxypro-lineassayandconstructstheweightratioofcollageninthesampletothetotalweightofthesample.TheexcelrepresentationoftheconstructerisshowningureB.1.Itaccountsforatestofallfourgelswithcellsandasacontrolwithoutcells. FigureB.1:TheHydroxyprolinecalculatorspreadsheet. Inthecalculator:Aiswherethemeasuredweightsofthesamplesinmilligramsareentered.Btheabsorbanceofthestandardcurveinduplicateisenteredhere.InCandDthestandardcurveisconstructedintoalineandalinearinterpolationisgenerated.ThisisenteredintothetrendlinedatainE.InFtheabsorbanceofthesamplesisentered.InGtheamountofcollageninthesamplevolumeisdeterminedusingthetrendlinefromthestandardcurve.InHtheweightratioofcollagento 134

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totalsampleweightisdeterminedbytakingthetotalcollagencontentofthe200mLsampleanddividingitbytheweightsinA. B.2HystemCWeightCalculatorHystemCWeightCalculatorisanExcelsheetdesignedtospecifytheappropriatevolumesofwatertobeaddedtodissolveHA,DGandPEGDAaswellastheamountsofthesesolutionstobeusedtomaketheappropriateweightpercentagesofeachcomponenttomakeupthefourhydrogelmixes.TheexcelrepresentationoftheconstructerisshowningureB.2.InthecalculatoratpositionAThevolumesofHydrogelsinmLneedforanexperimentsareentered.Thereareslotsfor5dierentvolumeexperimentstobeentered.AtpositionBthemaxweightpercentageofthegelmixisentered.Thiscanbetunedfordierentgelmixes.AtCthenumberofvialsofHA,DCandPEGDAareenteredthisneedstobeadjustedtoensurethatenoughofthereagentsaremadeforeachexperiment.AtDthevolumeofDegassedwaterthatshouldbeaddedtoeachcomponentvialiscalculated.AtEthevolumeofthehydrogelcomponentthatisneededforeachgelmixiscalculatedanoutput.AtpositionFtheextravolumeofPBSthatneedstobeaddedtobringthesolutiontothedesiredweightpercentagesandvolumesiscalculated. 135

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FigureB.2:Hystem-Cweightcalculatorfordeterminingmixratios. 136