Adhesion-mediated cell migraton in three-dimensional matrices : effect of spatial gradients of adhesiveness

109 Pages·1999·3.6 MB·English

by  Myles, Jennifer Lee

ADHESION-MEDIATEDCELLMIGRATIONINTHREE-DIMENSIONAL
MATRICES:EFFECTOFSPATIALGRADIENTSOFADHESIVENESS
By
JENNIFERLEEMYLES
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULLFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERISTYOFFLORIDA
1999
Copyright1999
by
JenniferLeeMyles
ACKNOWLEDGMENTS
Firstandforemost,IwishtothankDr.RichardDickinsonforhisguidanceand
support. Secondly,IwishtothankBrianBurgessforthedevelopmentoftracking
algorithmsanddataanalysistechniquesusingthepersistentrandomwalkmodel.
AdditionalhelpwasprovidedbytheInterdisciplinaryCenterforBiotechnologyResearch
attheUniversityofFloridawithpeptidesynthesis(Dr.AlfredChung)andSEM
(RosemaryDavis). IalsowouldliketothankDr.DavidMuirattheUniversityofFlorida
forhelpinestablishingcellcultureandDr.DanielMooradianattheUniversityof
MinnesotaforsupplyingtheK1735M4murinemelanomacells. Additionally,Iwould
liketothankDr.JuliaRossattheUniversityofMarylandatBaltimoreforadviceoncell
adhesionexperiments. Lastly,IwishtoacknowledgetheWhitakerFoundation
BiomedicalEngineeringResearchGrantforfinancialsupport.
in
TABLEOFCONTENTS
page
ACKNOWLEDGEMENTS Hi
LISTOFFIGURES vi
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
2 CELLADHESIONANDMIGRATION 3
2-1 TheRelationBetweenAdhesionandMigration 3
2-2 Receptor-LigandInteractions 6
2-3 DirectedCellMigration 7
2-4 Haptotaxis 8
3 COLLAGEN 11
3-1 StructureandFunction 11
3-2 CellInteractionwithCollagen 12
4 RGDPEPTIDES 14
4-1RGDAdhesiveAminoAcidSequence 14
4-2ModificationofSurfaceswithRGDPeptides 14
5 DIRECTOBSERVATIONOFBIASEDCELLMIGRATION
INTHREE-DIMENSIONALMATRICES 18
5-1 SpecificGoals 18
5-2 ExperimentalTechniques 18
5-2-1 SynthesisofConjugatedCollagen 18
5-2-1-1 Reaction1 19
5-2-1-2 Reaction2 21
5-2-1-3 Quantification 23
5-2-2 AutomatedVideoMicroscopyIncubationSystem 24
5-2-3 CellCulture 25
5-2-4 AdhesionExperiments 26
5-2-4-1 AdhesiononBSACoatedSurfaces 26
IV
1
5-2-4-2AdhesiononCollagenCoatedSurfaces 27
5-2-5 CellMigrationExperiments 28
5-2-6 FormationofSpatiallyAdhesiveGradients 29
5-2-7 PreparationofCollagenGelsforScanningElectronMicroscopy 31
5-3 ExperimentalResults 32
5-3-1 CollagenConjugation 32
5-3-2 CellAdhesionExperiments 35
5-3-3 CellMigrationUnderUniformConditions 39
5-3-3-1 DataAnalysis 39
5-3-3-2 Three-DimensionalCellTracking 41
5-3-4 CellMigrationinSpatialGradientsofAdhesiveness 44
6 MECHANISTICMODELOFAHESION-MEDIATED
CELLMIGRATION 47
6-1 DevelopmentofModel 47
6-2 ModelPredictions 53
6-2-1 ModelPredictionsforUniformConditions 53
6-2-2 ModelPredictionsforSpatialGradients 55
7 DISCUSSION 61
8 SUMMARYANDCONCLUSIONS 68
9 OTHERATTEMPTEDMATRICESANDRECOMMENDATIONSFOR
FUTUREWORK 69
9- ConjugationtoAlternativeMatrices 69
9-2 AdditionalGradientWork 71
9-3 StudyofIntegrinSpecificity 71
9-4 CellularLabeling 72
9-5 CellularForceMeasurements 72
9-6 ExpansionofModeling 72
APPENDICES
A IMAGEANALYSISANDCELLCOUNTINGALGORITHMS 73
B IMAGEANALYSISANDCELLTRACKINGALGORITHMS 76
C ADHESION-MEDIATEDCELLMIGRATIONMODELING 82
PROGRAMS
LISTOFREFERENCES 89
BIOGRAPHICALSKETCH 97
LISTOFFIGURES
Figure page
2-1.Schematicofamotilecell 4
5-1.Thereactionchemistryforthetwostepproteinconjugationreaction 19
5-2.Schematicoftheautomatedvideomicroscopysystemwithincubation 25
5-3 Schematicshowingtheformationofspatialadhesiongradients 31
5-4.Repeatablepeptidesubstitutionlevelsthatwereattainableforthe
reactions 33
5-5.SEMmicrographsofthevariouscollagenmatrices 34
5-6.Celladherencetosurfacescoatedwithvariousamountsofcross-lined
BSA 36
5-7.Celladherencetocollagenandconjugatedcollagencoatedsurfaces 38
5-8.CelladherencetosurfacescoatedwithvariousratiosofRGd-graftedcolagen. 38
5-9.Rateofcelldispersionquantifiedbytherandommotilitycoefficient 42
5-10.Characteristictimeofdirectionalpersistencequantifiedbythedirectional
persistencetime 43
5-11.Tortuosityofthecellpathquantifiedbythepersistencelength 44
5-12.Rateoftranslationalmotionquantifiedbytheroot-mean-square
cellspeed. 45
5-13.Biasedcellmigrationingradientgelsquantifiedbymeasuringthe
driftvelocitytowardthegradientdirection 45
5-14.Thedirectionalorientationbiasisthemeanangleofthedisplacements
relativetothegradientdirection 46
6-1.Forcebalanceonthemodelcell 50
6-2.Schematicshowingtheeffectsofthepolarityparameter,a,thedirection
ofpseudopodextension 51
6-3.Dimensionlessretractionvelocity 54
VI
6-4.Simulationresultsofdimensionlessrandommotilitycoefficient,speed,and
directionalpersistencetimeplottedvs.dimensionlessliganddensity 54
6-5.Simulatedcellpathsforhighgradientsofadhesionatvariousstrengths
ofadhesion 56
6-6.Simulatedcellpathsforlowandmediumlevelsofadhesivenessshowing
theminimumgradientnecessarytoproduceabias 58
6-7Driftvelocitiesforlowandmediumlevelsofadhesivenessshowing
theminimumgradientnecessarytoproduceabias 59
6-8.Simulatedcellpathsformediumlevelsofadhesivenessshowingthe
relativecontributionsoforientationbiasandkinesis 60
vn
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
ADHESION-MEDIATEDCELLMIGRATIONINTHREE-DIMENSIONAL
MATRICES:EFFECTOFSPATIALGRADIENTSOFADHESION
By
JenniferLeeMyles
August1999
Chairman: RichardB.Dickinson
MajorDepartment: ChemicalEngineering
Collagen,eitheraloneorincombinationwithothermaterials,isanimportant
naturalbiomaterialusedinapplicationssuchasvascularprosthesesandbioartificial
organs. Spatiallycontrollingtheadhesivenessofcollagenand/orotherbiomaterialsmay
allowcontrolledlocalizationorredistributionofcells,therebyimprovingtheirchancesof
servingassuccessfulimplants.
Thisdissertationdescribesamethodtocouplepeptideswhichcontainthewell
characterizedarginine-glycine-asparticacid(RGD),adhesionsequence,andarginine-
glycine-glutamicacid(RGE),nonadhesivesequence,directlytocollagenmonomersprior
tofibrillogenesis. Theaveragenumberofpeptidessubstitutedpermonomercouldbe
controlledbychangingthereactionconditions.
Vlll
ScanningelectronmicroscopywasusedtoexaminethemicrostructureofRGD
modifiedcollagengels. Investigationsofporesize,fibrilsizeandfibrilarrangement
showednoqualitativedifferencesincollagengelstructure.
CelladhesionstrengthwasfoundtoincreasewithbothincreasingdegreeofRGD
conjugationandincreasingratioofRGD-modifiedtounmodifiedcollagen. Degreeof
RGEconjugationwasfoundtohavenoeffectoncelladhesion.
Amethodwasdevelopedtotrackindividualcellsinthreedimensionsusingan
automatedvideomicroscopysystemwithimageanalysissoftware. Cellmigrationwas
quantifiedbyanalyzingthereconstructedcellpaths. Parameterssuchascellspeed,
persistencetime,andrandommotilitycoefficientwereobtainedbyfittingthecelltrack
datatoapersistentrandomwalkmodel. Therandommotilityofcellswasfoundtobe
biphasicallydependentonthedegreeofconjugationofRGDpeptides. Nosuch
dependencewasfoundforRGEmodifiedcollagen.
Gelswerealsocastwithspatialgradientsofadhesiveness. Studiesofdrift
velocityandorientationdirectionshowedthatthereisasmallbiasinthedirectionofthe
gradient.
Amechanisticmodelofcellmigrationprovidesqualitativeconfirmationofthe
experimentalobservations.
Thismodifiedadhesivenessdatatakentogetherwiththedatashowingits
influenceoncellmotilitysupportthehypothesisthathaptotaxismaybeapotential
mechanismforthecontrolledlocalizationorredistributionofcells.
IX
CHAPTER
1
INTRODUCTION
In1990aloneover83,000peopleeitherdiedorweremaintainedonlessthan
optimaltherapiesduetothelackofdonatedorgansintheUnitedStates[l]. Thislackof
availabilityandtheriskfordiseasetransmissionassociatedwithtransplantshasledtoa
renewedeffortinfindingviablesyntheticreplacementsforlivingtissue.However,todate
thebiomedicalresearchcommunityisstillverylimitedinthetissuesthatcanbereplaced
withhighsuccessratesduetothecomplicatedhierarchicalstructuresofthesetissuesand
thecomplexbiochemicalinteractionsbetweencellsandtissues[2]. Themajorsourceof
complicationsandfailureswithsyntheticreplacementsispoorbiocompatibilitywithcells
inthetissuesurroundingtheimplant[2]. Properfunctionofsomeimplantsrequires •
infiltrationofsurroundingtissuecellsintotheporoussyntheticscaffoldorontothe
biomaterialsurfaceoftheimplant. Onepossiblesolutiontopromotecellinfiltrationor
ingrowthistomodifythesurfaceofthebiomaterialinordertopromotetheingrowthof
thesurroundingcellsontothebiomaterialsurfaceorintotheporousstructureofthe
biomaterial[3]. Furthermore,bytailoringthespecificadhesivepropertiesofthe
biomaterial,itmaybepossibletodirectthemigrationofonlyaselectedgroupofcellsor
topromotethebiasedmigrationofcellsinaparticulardirectionbycreatingagradientof
adhesiveness. Thephenomenonofbiasedcellmigrationinanadhesiongradientis
knownashaptotaxis[4],andisalsobelievedtobeimportantmechanismofbiasedcell
1