epithelial mechanobiology skin wound healing and the stem cell niche

Author's Accepted ManuscriptEpithelial mechanobiology, skin wound healing, and the stem cell niche Nicholas D.. Oreffo, Eugene Healy, Philipp J.. Oreffo, Eugene Healy, Philipp J.. Thurne

Author's Accepted Manuscript

Epithelial mechanobiology, skin wound

healing, and the stem cell niche

Nicholas D Evans, Richard O.C Oreffo,

Eugene Healy, Philipp J Thurner, Yu Hin

Man

PII: S1751-6161(13)00155-0

DOI: http://dx.doi.org/10.1016/j.jmbbm.2013.04.023

Reference: JMBBM856

To appear in: Journal of the Mechanical Behavior of Biomedical Materials

Received date:5 November 2012

Revised date: 23 April 2013

Accepted date:

29 April 2013

Cite this article as: Nicholas D Evans, Richard O.C Oreffo, Eugene Healy, Philipp J Thurner, Yu Hin Man, Epithelial mechanobiology, skin wound healing, and the stem cell niche, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2013.04.023

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www.elsevier.com/locate/jmbbm

CentreforHumanDevelopment,StemCellsandRegeneration,InstituteforDevelopmentalSciences, UniversityofSouthamptonSchoolofMedicine,TremonaRoad,SouthamptonSO166YD

3

Dermatopharmacology,SirHenryWellcomeLaboratories,Mailpoint825,LevelF,SouthBlock,Universityof SouthamptonSchoolofMedicine,SouthamptonGeneralHospital,SouthamptonSO166YD

*Correspondingauthor;n.d.evans@soton.ac.uk



1.AbstractandIntroduction

Skinwoundhealingisavitalprocessthatisimportantforreestablishingtheepithelialbarrierfollowingdiseaseorinjury.Aberrantordelayedskinwoundhealingincreasestheriskofinfection,causespatientmorbidity,andmayleadtotheformationofscartissue.Oneofthemostimportanteventsinwoundhealingiscoverageofthewoundwithanewepitheliallayer.Thisoccurswhenkeratinocytesatthewoundperipherydivideandmigratetorepopulatethewoundbed.Manyapproachesareunderinvestigationtopromoteandexpeditethisprocess,includingthetopicalapplicationofgrowthfactorsandtheadditionofautologousandallogeneictissueorcellgrafts.

Themechanicalenvironmentofthewoundsiteisalsooffundamentalimportancefortherateandqualityofwoundhealing.Itisknownthatmechanicalstresscaninfluencewoundhealingby

affectingthebehaviourofcellswithinthedermis,butitremainsunclearhowmechanicalforcesaffectthehealingepidermis.Tensileforcesareknowntoaffectthebehaviourofcellswithin

epithelia,however,andthematerialpropertiesofextracellularmatrices,suchassubstratestiffness,havebeenshowntoaffectthemorphology,proliferation,differentiationandmigrationofmanydifferentcelltypes.

Inthisreviewwewillintroducethestructureoftheskinandtheprocessofwoundhealing.Wewillthendiscusstheevidencefortheeffectoftissuemechanicsinreepithelialisationand,inparticular,



2.Skinstructureandfunction

Theskinisthelargestorganinthebody.Itfulfilsavarietyoffunctions,mostimportantlyasabarrierseparatingtheinternalorgansofthebodyandtheexternalenvironment.Criticaltothisfunctionisitsphysicalrobustness–ithasevolvedtowithstandthemanymechanical,chemicalandbiologicalinsultsorganismsfacefromtheoutsideworldeveryday,includingheat,friction,radiation,

pathogenicmicroorganisms,andtoxicchemicalsandmaterials.Toachievethis,ithasevolvedatough,elasticstructurewhichcanremodelandadapttoexternalconditionsandtoquicklyrepairintheeventofinjury.

Inmammals,thetwomostprominentcomponentsoftheskinarethedermisandtheepidermis,whichareattachedto(andseparatedfrom)eachotherbyathinlayerofextracellularmatrix(ECM)proteinscalledthebasementmembrane.Thedermis,whichisusuallymuchthickerthanthe

epidermis,islargelycomposedofECMproteinssuchascollagentypeIandelastin,whichare

responsibleforitsmechanicalstrength.Scatteredthroughoutthedermisarecellscalledfibroblastswhichregulatetheorganisationofthefibrillardermalmatrix.Theepidermis,primarilycomposedofkeratinocytes,consistsofmultilayeredpolarisedepitheliumincloseappositiontotheunderlyingdermis.Cellsinthebasallayer,whichcontactthebasementmembrane,continuallydivideduringthelifetimeoftheorganism,providingasourceofcellswhichprogressivelymigrateupwardsthoughtheepidermis,differentiatingandstratifyingtoformthebarrierlayeroftheskin.

Thehigherorderstructureoftheskinvariesconsiderablybetweenspeciesandanatomicallocation.Forexample,theskinofthepalmsofthehandsandthesolesofthefeetinhumansisclearly

distinguishedfromthatofthetrunkorscalp.Thelatterischaracterisedbythepresenceofhairfollicleswhiletheformerischaracterisedbytheabsenceofhairfolliclesandbypatternsof

bifurcatedreteridgeswhichprojectdeepintothedermis.Thesedifferingstructuresareadaptationsforthefunctionstheareaofskinhasevolvedtoperform–heavilyfollicularisedareasinmammalsareadaptationstominimiseheatlossandlimitUVexposure,whereaspalmskininhumansishighlyinnervated–anadaptationforimprovingtouchsensitivityandtactility.



Regardlessofanatomicallocationorfunction,allbreachesintheskinsurfacemustberepairedquickly,notonlytopreventpathogensorharmfulmaterialsgettingintothebodybutalsotopreventfluidloss.Therefore,inmatureorganismstheprocessofwoundhealingisintimatelyassociatedwithbloodclottingandtheactivityoftheimmunesystem.Thishasbeenreviewedelsewhereindetail(SingerandClark,1999).Althoughwoundhealingisacontinuous,seamlessprocess,many

researchershavefounditconvenienttodividetheprocessofwoundhealingintophases,includinginflammation,tissueformationandtissueremodelling.

Immediatelyafterabreachintheskinsurface,clottingfactorsarereleasedintothewoundbedtopreventlossofbloodandtoprovideahardfibrousmatrixtopreventtheingressofpathogens.Inflammatorycellsarethenrecruitedtothewoundsitebyavarietyofchemotacticsignalsandengulfforeignparticles,includingbacteria.Asthisprocessprogresses,fibroblasticcellsareattractedtothewoundbedandbegintosecretecollagenousECMknownasgranulationtissue,which

graduallyreplacesthefibrineschar(scab).Concurrently,epithelialcellsfromtheepidermis

neighbouringthewoundsitebegintomigrateoverthesurfaceofthewoundbed–insomecasesburrowingapathbeneaththehardscabandtheunderlyinggranulationtissue–untilthetwoepithelialtonguesmeetinthecentreofthewound,providinganewepithelialcoverage(Figure1).Soonafter,whenthenascentepitheliumreachesmaturity,thefibrousclotseparatesfromtheunderlyingepitheliumandisshed.Insomemanagedskinwounds,theescharmayberemovedsothatwoundhealingoccursintheabsenceofafibrousescharandtheepitheliallayermigratesonthesurfaceofthegranulationtissue.Infact,someevidencesuggeststhatskinwoundhealingmaybeexpeditedandimprovedbytheprovisionofsuchamoistenvironment(FieldandKerstein,1994).

Followingandduringreepithelialisation,manyfibroblasticcellsinthegranulationtissueundergoprogrammedcelldeath(apoptosis),whileothersdifferentiate.ThesecellsremodeltheECMoftheskinleadingtotheformationofacollagenousmatrix.Insuperficialwounds,theskinhealstoformatissuethatislargelyindistinguishablefromtheintactskin,butifawoundissufficientlydeepand/orlarge,thewoundwillhealwiththeformationofascar.Scarsarecharacterisedbyacompleteabsenceofskinappendages,suchashairfolliclesandsweatglands,andbyacollagenousmatrixthatdiffersinstructurefromthatofintacttissue.Ingeneral,typeIcollagenfibrilsforma‘basketweave’patterninintactskin,whereasinscarredskin,thesefibrilstendtobeorganisedlongitudinally(van

Zuijlenetal.,2003).Forthesereasons,scarredskintissueisinferiortointactskinandmay

contributetopathology.Forexample,scarsmaybecomehypertrophicleadingtotissuecontracture,

makingitdifficultforaffectedpatientstomovejointsinnearbyaffectedareas(Tredgetetal.,1997).

4.Mitigatingskinwounding

Skinwoundingresultsinhugeeconomicandsocietalburdens,andthereareavarietyofdifferentchallengeswithrespecttodifferentpathologies.Forthisreasonavarietyofapproachesareneededfortheirmitigation.Severeburnwoundsoftenleadstosuchacomprehensivelossoftissuethatintheshorttermcoverageofthebodyiskeytothesurvivalofthepatient.Here,graftingofeitherautogenicskin(fromanothersiteonthesamepatient,ifenoughtissueremains)isthepreferredtreatment,oralternativelyallogenicmaterial(skinorcellsfromadonorindividual)maybeused

(Balasubramanietal.,2001).Othertechnologiesincludedressingsbasedonsyntheticorhumanor

animalderivedmatrixproteins,whichcanbetemporarilyappliedatthewoundsitetopreventfluidlossandinfection,ortissueengineeredsubstitutes,consistingofamaterialanda(usuallyallogeneic)cellsourceculturedinvitrobeforebeingappliedatawoundsite(MetcalfeandFerguson,2007;

Placeetal.,2009).Differentstrategiesmaybeemployedforotherpathologies,suchaschronicskin

wounds.Diseasesorsyndromesthataffectthemicrocirculation,suchasdiabetesorneuropathy,mayleadtoskinulcers–areaswhereskintissuehasundergonenecrosisandbecomelost.Likeacuteskinwounds,infectionmustbecontrolledbycoverageofthewoundwithasuitabledressing.However,thekeyhereistoensurethatwoundhealingactuallyoccurs–oftenthesewoundsarerefractorytohealingduetotheunderlyingcondition(nutrition,metaboliccontrol,persistent

pressureetc),soingeneralthefirstcourseofactionistoalleviatetherootcause–forexamplein

diabetesbytacklingpersistenthyperglycaemia(Cavanaghetal.,2005).

Thereremainveryfewdrugsforexpeditingorimprovingwoundhealing.Epidermalgrowthfactor,whichstimulatesthedivisionandmigrationofkeratinocytesinvitro,wasconsideredatherapywith

highpotentialafterpromisinginitialclinicalresults(Brownetal.,1989),butthispromisefailedto

translateintolongtermclinicalsuccess.Todate,theonlygrowthfactorthathasreachedtheclinicisplateletderivedgrowthfactorBB(PDGFBB),whichhashadapprovalforuseintreatingdiabeticfoot

ulcer(marketedasRegranex(Wieman,1998;Wiemanetal.,1998)).Thiswasshowntostimulatethe

divisionofdermalcellsatwoundsites,speedinguptheformationofgranulationtissue,althoughawarningissuehasrecentlybeenissuedafteritwasshowntoelevatecancerrisk(PapanasandMaltezos,2010).

Similarly,therearenodrugsormoleculesthathavebeenshowntomitigatescarring.Amemberofthetransforminggrowthfactorfamily(TGF3,namedAvotermin)hadhadsomeinitialencouraging

drugfailedatphaseIII(Pharmatimes.com,2011).

Asthewoundhealingmarketisestimatedtobewortharound$6.7billionworldwide(VisionGain,2011)andthescarreductionmarketisestimatedtobeworthgreaterthan$4billionintheUSalone,thereisgreatimpetustodevelopnovelwaysofimprovingtheseconditions.Onepromisinglineofinvestigationisbymodifyingthemechanicalenvironmentofthewoundsite.



5.Mechanicalpropertiesofskinanditsimportanceinwoundhealing

Intactskinissubjecttotensilestress.Thiscanbedemonstratedbytheobservationthatwhenasmallwoundismadewithaspike,theskinrelaxestoformawoundofagreaterdiameterthantheincision.Thistensilestressislargelyanisotropic,andcircularwoundstendtoelongateinthe

directionofthegreateststress.KarlLanger,anineteenthcenturyGermananatomist,cataloguedthepatternofthesestrainsovertheentirehumanbodybypainstakinglyprickingtheskinofacadaverandbymeasuringtheskin’srelaxation.Hethenusedtheelongationoftheincisionstoindicatethedirectionandrelativemagnitudeofthisstress(Langer,1861).DespitetheextensivecitationofLanger’swork,therehasbeensurprisinglyscantresearchtoquantifytheabsolutemagnitudeoftherestingstressinskin,withestimationsintheavailablestudiesofrestingtensionsbetween12–36

Nm1andprestressesof5.4–24kPa(deJong1995;Diridollouetal.2000;Jacquetetal.2008).Ina

morerecentstudy,Flynnetalshowedthattherestingtensionintheposteriorpartofthearmof

humanvolunteersincreasedbyapproximatelyafactorof5duringflexion(inthedirection

longitudinaltoflexion),withforearmvaluesrangingfrom1.8–11.4Nm1(Flynnetal.2011),andmorerecentlythesamegrouphasestimatedprestressesinskintorangefrom28–92kPa.Themagnitudeoftensioninotherregionsoftheskin’ssurfaceremainunexamined,butphysiologicalvaluesmustliebelowtensionslowerthanthatpermittedbythetensilestrengthofhumanskintissue,whichhasbeenmeasuredvariouslybetween5and30MPainvariousstudies(Annaidhetal.2010;reviewedinEdwardsandMarks1995),althoughsomeauthorshavesuggestedthatpre

stressesmaybeasgreatas1MPa(Silveretal.2003).Inparticular,whenskinisstretched,for

exampleduringgrowthofunderlyingtissueduringpregnancyorweightgain,theskinadaptsto

reducethisincreaseinmechanicaltensionbyincreasingitsownmass,volumeandareabyaprocess

ofgrowth.Thisphenomenonof‘biologicalcreep’isexploitedbyplasticsurgeons,whereskincanbeexpandedoveraperiodofseveralweeksusingasubcutaneouslyimplantedinflatabledevice(tissueexpander)atoneanatomicalsitetoprovideskintissueforautograftingataremotesite.(Radovan,

suchdevicesexertintheshorttermhasbeenmodelledbyKuhlandcolleagues(Zöllner2012;Tepole2012),butthereislittledatatosuggestthequantitativeincreaseintensionthatpromotestissuegrowth.Astrainof10%hasbeenadoptedasacriticalthresholdtopromotetissuegrowthinZöllner

etal’ssimulations,butthereislittledataonwhatstressesorstrainsarenecessaryinvivotoelicit tissuegrowth.Clearlymoreworkisnecessarytoquantifysuchreallifeparameterstoensure

skinhavebeenshowntovaryfrom0.02MPato57MPa,afactorofalmost3000(Diridollouetal.,

2000).Confoundingfactorssuchasthethicknessoftheskin,thesurfaceareabeingtested,thetypeof forces applied as well as the hydration level of the sample are thought to give rise to the large

differencesseen(Bhushanetal.,2010;Diridollouetal.,2000;LiangandBoppart,2010).Toaddress

theseproblemsPaillerMatteietal.developedanovelskintribometerdevicetomeasuretheelasticmechanical properties of the skin with a series of indentations to the inner aspect of the forearm

(PaillerMatteietal.,2008).Bysimplifyingthecomponentsoftheskintothedermisandhypodermis

and underlying muscle layer (acting as a rigid substrate) PaillerMattei et al. were able tomathematicallymodelthelayersasthreespringsconnectedinserieseachwithdifferentstiffnesses

andthusdeducingthatfromtheirexperimentsthedermishasanE=35kPa,hypodermisE=2kPa and muscle E = 80 kPa. These values however are not fixed but are dynamic depending on the

positioningoftheforearmasshownbyIivarinenetal.wherebydifferentstiffnessesoftheforearmwas measured at rest, at isometric flexor and extensor loading and with venous occlusion. Theirstudyfoundarestingelasticmodulusof210kPathatchangedaccordingtothedifferentconditions;

atisometricflexorloadingE=446kPa(112%increase),isometricextensorloadingE=651kPa(210% increase)andwithvenousocclusionE=254(21%increase)(Iivarinenetal.,2011).Notonlydoesthe

skin adjust its elastic modulus by the active or passive state of the underlying muscle but is alsosubjectedtoshearforcesduringcontactwitheverydayobjectormaterialsintheformoffriction.Anextensive review on the friction coefficient of human skin has been done elsewhere (Derler andGerhardt, 2012). Derler and Gerhardt concluded that adhesion friction is the main frictionmechanism experienced by the skin and that a minimum shear modulus of 13.3 kPa is observed

Theskin’stensilestressisalsoofgreatimportanceinwoundhealing.Severalstudieshave

establishedthatskinwoundswhichareundermechanicaltensionaremorepronetohealwiththeformationofascar.Thisdatahasledtotheprevailingpractice,wheresurgeonsaimtoreducetensionatincisionsitespostsurgery.Simplemethodsfortensionreduction,suchasadhesivetape,haveprovedtobesuccessfulinimprovingwoundhealing,andmorehitechdevicesarecurrentlyin

preclinicaldevelopment(Atkinsonetal.,2005;Gurtneretal.,2011).

Thattheskin’stensilestresshassuchaprofoundeffectonwoundhealingindicatesthattheremustbeacellularmechanismatthewoundsitebywhichstressissensedandtransducedtoa

physiologicalresponse.Mostevidencepointstocellsintheconnectivetissueofthewoundbed

fibroblastsortheirrelations,themyofibroblastsasmediatorsofthismechanism(see(Sarrazyetal.,

2011)forarecentreview).Thesecellsactduringthecourseofnormalwoundhealingtoactivelycreatetensioninordertodrawtheedgesofthewoundtogether.Thisisespeciallytrueinlooseskinnedmammals(whichcomprisethemajorityofmammalianspecies,suchasrodents),butthisalsooccursinmammalswherethedermaltissueisattachedmorefirmlytotheunderlyingfascia

(suchashumansorpigs).Anumberofinvitroandinvivomodelshavedemonstratedthatincreased tensionpromotestheproliferationofthesecells(Webbetal.,2006),inhibitstheirapoptosis(Aarabi

etal.,2007),andactivatesmanysignallingpathwaysthatmaypromotetheirregulardepositionof

ECM.Forexample,Hinzetal.preventedwoundcontractureinaratmodelofwoundhealingby usingaplasticsplint(Hinzetal.,2001).Theyshowedelevatedsmoothmuscleactinexpressionin

woundfibroblastsandmyofibroblasts–presumablyacellularresponsedirectedatovercomingthesplintandclosingthewound.ThiselevatedcontractileresponseisalsolinkedtotheexcessivedepositionofECM,scarringandaberrantscarring,suchashypertrophicscarringanditsoccasionalcorollary,keloidscarring.Becauseofthis,somedevicesthatfunctionbymechanicallyoffloading

tensionatwoundsitesareinpreclinicaldevelopment(Gurtneretal.,2011).Inaddition,itisthought

thatsomeofthesuccessofvacuumassisteddevicesmayrelatetotheireffectinreducingwoundtension,aswellastheirothereffects(e.g.reducingofswellingandwoundexudate)(OrgillandBayer,2011).Futureworkmayshedmorelightonthis.

Whiletheroleoffibroblastsandcellsofthedermaltissueinintactandwoundedskinisbecoming

wellestablished,thereislessdataonhowthemechanicalenvironmentoftheepidermisaffectsskin

woundhealing.

6.Epidermalmechanics

Theepidermisoftheskinisacertainlyamechanosensitivetissue.Aswehavealreadyseen,theskinhastoexpandtoprovidecoverageforagreatervolumeoftissueduringgrowthanddevelopment,orduringartificialtissueexpansion,anddataconfirmsthatthisincreaseintensionissensedby

humanepidermaltissuesbyanincreaseinmitoticactivity((Oleniusetal.,1993),afterAustad’s experimentsusingGuineapigs(Austadetal.,1982)).Butitwasnotuntilthelate90sthatthe

molecularmechanismsofthiswereinvestigated.Thiswasachievedusingdevicesforcyclicallystretchingcellsinculturetomeasuretheeffectoftensilestrainoncellbehaviour–atechnologythat

hadbeenusedtoinvestigatearangeofothercelltypes,includingendothelialcells(Letsouetal., 1990),lungcells(Liuetal.,1994)andmesangialcellsofthekidney(Harrisetal.,1992).Thisinvolves

culturingcellsonflexible(oftensilicone)surfaceswhichcanbestretchedeitherisotropicallybyamechanicaldevice,orbyapplyingaperiodicpartialvacuumtotheundersideofthesilicone

substrate.Sincethesestudieshadoftenreportedanincreaseinproliferationinresponsetostretch,andastissueexpandersclearlyworkedinpromotingskinexpansion,itwassomewhatunsurprising

whenTakeietal.reportedalargeincreaseinDNAsynthesisandcelldivisioninhumankeratinocytes subjectedtocyclicalstretchwithamaximumof10%strain(Takeietal.,1997).Butthisinvitro

systemalsoprovidedaconvenientmethodforanalysingtheeffectofmechanicalstimulationonintracellularmolecularsignallingpathways.Inparticular,thisstudyandothersfoundthatstretchingactivatedenzymesinvolvedinseveralsignallingpathways,suchasmitogenactivatedproteinkinase

differentiation.Mechanicallystimulatedcellswereshowntoincreaseproductionofcytokeratin6andreducethatofcytokeratin10,indicatingthestimulationofdivisionratherthandifferentiation

(Yanoetal.,2004).Morerecently,thisgrouphasshownthatproteinkinaseB(Akt)isactivatedby stretching,whichpreventsapoptosis(Yanoetal.,2006),whileanothergrouphasdemonstrated

thatkeratinocytesthatarestretchedproducemorematrixmetalloprotease9(MMP9)–anenzyme

necessaryforkeratinocytemigration–thatthosethatarenot(Renoetal.,2009).

7.Epithelialmechanicsandstemcellniches

Inadditiontodynamicstressesexperiencedbycellsintheepidermis–suchascyclingstretching–itisalsoprobablethatcellsexperienceavarietyofstaticmechanical‘microenvironments’dictatedbythestructureoftheskin.Thesemicroenvironmentsarelikelytobeveryimportantindeterminingthepatterningofdifferentcelltypesintheskin–i.e.specifyingwhichcellsappearwhere.Of

particularinterestistheputativestemcell‘niche’.

Theskinepitheliumdoesindeedhaveaphysicalstructurewhichislikelytodeterminethe

mechanicalenvironmentthatacellexperiences.Forexample,inthepalmoplantarregionsofhumanskinthereisapatternofreteridges,whichmarktheboundaryoftheepidermisanddermis.Theseformapatternofpeaksandtroughswheretheepitheliumoverlaysstructuresthatprojectoutformthedermiscalleddermalpapillae(Figure2B&D).(Noteherethat,byconvention,thepeaks–the

‘tips’–ofreteridgesarethoseareaswheretheepitheliumprojectsmostdeeplyintothedermisanddonotrefertotheareaswherethedermiscomesclosesttotheskinsurface.)Theseridgesareresponsiblefordermatoglyphs,whichwerelyonforgeneratingfingerprints.Buttheymayalsocontainmechanicalinformationaboutwherestemcellsshouldreside.LavkerandSunnoted

differentmorphologiesandratesofcelldivisioninthekeratinocytesofthebasallayerofthe

epidermis,dependingonwhethertheywerefoundinthetipsorthetroughsofthereteridges,speculatingthatstemcellsmayinhabitthetipsofdeepreteridges(LavkerandSun,1982).Innonpalmoplantarskin,ontheotherhand,stemlikecellshavebeenfoundtoinhabitthediametricallyoppositeregion–i.e.attroughsofthereteridgeswherethedermiscomesclosesttotheskin

surface(Jensenetal.,1999).Thesestudiesdemonstratethatcellpopulationsmaybepatterned

accordingtothetopographyofatissuesurface.Itisofcourseprobablethatsignallingbysolublemolecules,suchasgrowthfactors,initiallyregulatesthisphysicalpatterning.Threshold

concentrationsofsignallingmoleculeshavebeenknownformanyyearstoregulatethepositioningandpatterningofanorganism’stissues.AlanTuringandotherearlytheoreticiansinitiallyproposedsuchamechanismtoregulatemorphogenesisinplantsandanimals(Turing,1952)anditisknownthatthis‘reactiondiffusion’mechanismisresponsibleforpatterning,forexample,thespacingof

hairfolliclesintheskin(Sicketal.,2006)andbodysegmentationininsects(Kauffmanetal.,1978).

Butitisalsopossiblethatthemechanicalcharacteristicsofthesetopographicallypatterned

environmentsthemselvesthatcaninformresidentcellshowtobehave,andsomeresearchnowsupportsthisidea.Gutepitheliumconsistsofanundulatingtopographicalpatternofvilli,which

gut(Figure2A&C).Hannezoetal.invokedbucklinginstabilitytoillustratehowsuchapatterncould

arisespontaneouslybytheexertionofnegativetension(causedbyadividingepithelium)onan

elasticunderlyingtissue(Hannezoetal.,2011).Thistheoreticaldescriptionhashadbeengivenextra biologicalrelevancebyarecentexperimentalstudybyBuskeetal.(Buskeetal.,2012),whichbuilds

onearlierworkbyHansCleversandhiscolleagues.TheCleversgrouphaddemonstratedthatstemcellsarereproduciblylocatedinveryspecificregionsonlyatthebaseofthecryptsoftheintestine

(Barkeretal.,2008;Barkeretal.,2007).Progenyofthesecellsmigrateupthewallsofthecryptto

giverisetoallofthemanydifferentiatedcelltypesthatlinethegut.Thissuggeststhatthebaseof

thecryptformsatightlyregulatedstemcellniche.Buskeetal.,bymodellinganinvitroorganoid

systemofcryptformation,havesuggestedthatthisnicheismechanicallyselforganised.Theyprovideindirectevidencethatthecurvatureofthesurfacetowhichtheepithelialcellsareattachedregulatescelldifferentiation,withacelltypecalledPanethcellsbeingconfinedtoareasofhighpositivecurvature.SincePanethcellsaresuspectedtonurturestemcells,itispossiblethat

mechanicallydefinedenvironmentsinvivomaycloselyregulatestemcellbehaviour.

Itisunknownwhethersuchphysicallydefinednichesintheskinmayaffectcellsinasimilarway.Butthereisampleevidencetosuggestthatkeratinocytesrespondtothemorphologyoftheirgrowth

environment.Inpioneeringexperiments,Wattetal.showedthatwhenkeratinocyteswereconfined

tosmallsubstrateislandstheydifferentiatedmoreanddividedlessthanthoseonlargerislands

(Wattetal.,1988).Asdetachmentfromthebasementmembraneisanintegralpartofkeratinocyte

differentiation,itwasspeculatedthatreducedadhesionandcellspreadingmightbeeitherthecauseorconsequenceofcelldifferentiationintheskin.Similarworkhasbeencarriedoutmorerecentlyby

McBeathetal.onmesenchymalstemcells(McBeathetal.,2004).Here,confinementonsmall

islandspromotedthedifferentiationofadipocytes,whilegrowthonlargeislands,wherecellswereallowedtospread,promotedthedifferentiationofosteoblasts.TheyfoundthatthisprocesswasinsomepartregulatedbytheRhokinase(ROCK)signallingpathway,indicatingthatcellspreadingcanbetransducedtoanintracellularmolecularsignal,whichcanthendirectcellspecification.More

recently,Nelsonetal.showedsimilareffectsbutinmulticellularsheetsratherthaninsinglecells (Nelsonetal.,2005).BypatterningcellcoloniesonislandsofECM,theywereabletoshowselective

celldifferentiationdependinguponthepredictedmechanicaltensionofareasofthecellsheet.Againusingmesenchymalstemcells,theydemonstratedadipocytespecificationinareasoflowtensilestressandosteoblasticdifferentiationinareasofhightensilestress.Theseexperimentshave

notyetbeenextendedtoepithelialcelldifferentiation,butConnellyetal.recentlyextendedWatt’s

earlierworkbydemonstratingthatcellshape,ratherthanislandsizealone,isanotherimportant

keratinocyteswereculturedonellipsoidcellislandswithidenticalareas,butwithdifferentaspectratios.Thosewiththehighestaspectratio(i.e.stretchedellipsoids)maintainedstemnesstoagreaterdegree.

Theseresultsraisetheinterestingpossibilitythatkeratinocytesinthebasallayeroftheskinmaybeacutelyinfluencedbytissuemorphology,whichultimatelytranslatesintochangesinmechanicalproperties.Topographicalpropertiesofthebasementmembraneandtheunderlyingdermisonlengthscalesgreaterthanthatofanindividualcellmaycreatemechanicalgradientsthatspecifywherecertaincellsshouldreside,ashasbeenimplicatedinthegutepithelium.Cellspreadinginkeratinocytesculturedintwodimensionspromotes‘stemness’,butitisunknownhowbasal

spreadingcombinedwithapicalconstriction(ortheopposite),whichmayoccurinepithelialcellsonacurvedsurface,mayaffectcellbehaviourintheskin.Experimentstodeterminehowepithelialsheets,ratherthanindividualcells,respondtosubstratetopologyandECMmechanicswillhelpustoanswerthesequestions.



8.Epithelialmechanicsinwoundhealing

Sincekeratinocytesaremechanosensitivecellsthatareintrinsicallyinvolvedwiththeprocessofwoundhealing,onemayaskanobviousquestion:whatisthemechanicalenvironmentofthehealingwoundandhowdoesthisaffectand/orsignaltothekeratinocyteandepidermalcellswhichmustacttoclosethewound?

Asdiscussedearlier,woundhealinginvolvesthemigrationofkeratinocytesfromthewound

peripherytowardsthewoundcentre,combinedwithanincreaseinthedivisionofepidermalcellsorkeratinocytesinproximitytothewound.BothoftheseactivitiesaredependentontheabilityofkeratinocytestointeractandadheretotheECMatthesurfaceofthewound.Theimportanceofthisisdemonstratedbymicethatlacksomeoftheproteinsthatareresponsibleforcellmatrix

attachment–deficiencyinmembersoftheintegrinfamilyinexperimentalmice,suchasintegrin1

(Groseetal.,2002),leadstoseveredefectsinwoundhealing.Andaswehavealreadyseen,these

ECMreceptorsaretransducersofmechanicalforce.Themechanismbywhichkeratinocytemigrationoccurs,andthesignalsthatregulateit,arestillacontentiousissue,however.Somemodelssuggestthatkeratinocytesatthewoundedgeprogressivelyfallovereachotherontothewoundsurfacewithminimalcellmigration(Krawczyk,1971)–aprocessthathasbeenreferredtoas‘leapfrogging’

(Lambertetal.,1984).Otherssuggestthatkeratinocytesactivelycrawloutoverthewoundbedby

2003),orbyrowsofcellscollectivelymigratingtowardsthewoundcentreatratesthatdiminishasa

functionofdistancefromthewound(FarooquiandFenteany,2005;Matsubayashietal.,2011).This

latterideahasbeenexaminedinmoredetailbygroupsledbyFredbergandTrepat.Theseauthorshavesuggestedthatepithelialcells–whicharecharacterisedbystrongcellcelladhesions–acttogetheratverylargedistancefromthewoundedgetoproduceanetmovementofcellstowards

thecentreofthewound,eventhoughindividualcellmovementsmaybeheterogeneous(Tambeet

al.,2011;Trepat,2009).Theyusetheevocativeanalogyofa‘mosh’atarockconcerttodescribethis

phenomenon,withsomecellspulling,somecellspushing,butwithultimatelyanetmovementofcellsintoaspace(TrepatandFredberg,2011).Inotherexperiments,forexampleonembryonic

tissueorcertainepithelialsheetsinvitro,amodelofwoundhealingknownas‘pursestring’healing

hasbeenputforward(MartinandLewis,1992).Inthesestudies,acircularactincablebecomesestablishedaroundtheperipheryofthewoundaftermechanicalwounding,whichgradually

shortens,leadingtowoundclosurehencethe‘pursestring’analogy.

Regardlessofthemechanismsofwoundhealing,themolecularsignallingprocessesthatgovernkeratinocytemigrationandwoundclosurehavebeeninvestigatedextensively.Growthfactorssuchasfibroblastgrowthfactors(includingkeratinocytegrowthfactor)andepidermalgrowthfactorsexertstrongchemotacticandmitogeniceffectsatwoundsitestoensurerapidtissuegrowthandwoundclosure(adiscussionofthisisbeyondthescopeofthisreview,butpleaseseea

comprehensivereviewbySantoro&Gaudino(SantoroandGaudino,2005)forfurtherinformation).Butitisalsolikelythatwoundmechanicsmayplayanimportantifoftenoverlookedroleinthisprocess.

Duringwoundhealing,keratinocytesarelikelytoexperienceachangeintheirmechanical

environment,mostlikelyexperiencingtensileforces.Inallofthesemodels,thereisanetmovementofcellstowardsthewound–thismovementislikelytobetranslatedtoa‘tug’oncellsbehindthem.Aswehavealreadyseen,tensileforcesacttopromotekeratinocyteproliferation,soonewouldexpectthisforcetobetranslatedintoanincreaseincelldivision,whichthenwouldacttoproducetissuetoreplacethatlostfollowingthewoundingevent.Inaddition,contractionoftheunderlyinggranulationtissueanddermiswouldalsobetranslatedtoastrainintheoverlyingepidermis,

exertingasimilareffect.Directevidenceforaroleforepithelialmechanotransductioninwoundhealingisskanty,however.Whereas,thestudiesontissueexpandersandsimilarworkby

Pietramaggiorietal.(Pietramaggiorietal.,2007)hasdemonstratedthatappliedforcestimulatescell

divisionandskinremodelling,thereisapaucityofstudiesthatdirectlymeasurethetensilestrainsat

Sofar,wehaveonlydiscussedhowappliedforce–suchastensilestrain–affectscellsandtissues.Butthereisalargebodyofevidencetosuggestthatcellsdonotjustpassivelyrespondtoforce–theyactively‘feel’theirenvironmentbyexertingforceonitthemselvesandbyresponding

accordingly.TheirabilitytodothisisrelatedtotheintrinsicmaterialpropertiesofthecellsandECMwithwhichtheyinteract.Onesuchpropertyofthesubstratethatcellsrespondtoistissuestiffness,

relatedtotheelasticmodulus(E)ofthematerial(s)ofwhichthetissueiscomposed.



9.Substratestiffnessandwoundhealing

ThestiffnessoftheECMcanhaveprofoundeffectsonthebehaviourofthecellsthatinteractwithit.PelhamandWangwerethefirsttodirectlytestthisideabyculturingfibroblastcellsonthesurfaceofpolyacrylamidegelsfunctionalisedbycovalentbindingofcollagentypeI(PelhamandWang,1997).Theyrealisedthattheelasticmodulusofpolyacrylamidecouldbevariedsimplybychangingtheratiosandconcentrationsofthemonomerandcrosslinkerusedtoformthesehydrogels.(Notethatstiffnessandelasticmodulusareoftenusedinterchangeably,butthattheyarenotdirectlyequivalent.Elasticmodulusreferstotheintrinsicpropertyofamaterialandisscaleindependent,whereasthestiffnessofamaterialmaydependonitsdimensions.)Indoingso,theywereabletoshowthatcellsonstiffersubstratesspreadouttoagreaterdegreeandformedmorefocaladhesionsthanthoseonsoftsubstrates.Subsequently,anumberofgroupsshowedrelatedeffectsonother