Mimicking the Articular Joint with In Vitro Models

Review

Mimicking

the

Articular

Joint

with

In

Vitro

Models

Susanna

Piluso

,

Yang

Li

,

Florencia

Abinzano,

Riccardo

Levato

,

Liliana

Moreira

Teixeira

,

Marcel

Karperien

,

Jeroen

Leijten

,

René

van

Weeren

,

and

Jos

Malda

,

*

Treatingjointdiseasesremainsasignificantclinicalchallenge.Conventionalin

vitro cultures and animal models have been helpful, but suffer from limited

predictive power for the human response. Advanced models are therefore

requiredtomimicthecomplexbiologicalinteractionswithinthehumanjoint.

However,theintricatestructureofthejointmicroenvironmentandthecomplex

natureofjointdiseaseshavechallengedthedevelopmentofinvitromodelsthat

canfaithfullymimictheinvivophysiologicalandpathologicalenvironments.In

thisreview,wediscussthecurrentinvitromodelsofthejointandtheprogress

achievedinthedevelopmentofnovelandpotentiallymorepredictivemodels,

and highlight theapplication ofnew technologiesto accurately emulate the

articularjoint.

TheSynovial Joint:AComplexOrgan

The proper functioning of the joint depends on the maintenance of joint homeostasis, a dynamicequilibrium between anabolic andcatabolic processeswithin all the components ofthejoint[1,2].Thejointisacomplexmultitissueorganencompassingthearticularcartilage, thesubchondralbone,thesynovialmembrane,and,insomejoints,additionalintra-articular structures,suchasligamentsandmenisci(Box1).Thesynoviumisessentialforjoint homeo-stasis;infact,synovialmacrophagesareresponsibleforthemaintenanceofafinebalance betweenproinflammatoryandanti-inflammatorycytokines(seeGlossary)inthesynovialfluid [3].Synovialinflammation is nowrecognized to play a key role inthe progression of joint diseases,withthereleaseofinflammatorycytokinesbeingmediatedbythecrosstalkbetween synoviumandcartilage[4].Further,alterationsinthecompositionandstructureofthe sub-chondralbonecanaffectthebehavioroftheoverlyingcartilage,suggestingtheexistenceofa physical and molecular crosstalk between the two tissues [5,6]. The intricate interaction betweenthesedifferenttissuestructuresandcelltypesmakesitquitechallengingto recapitu-latebothhealthyandpathological[e.g.,inthecase ofosteoarthritis(OA)orrheumatoid arthritis(RA)] jointphysiology inamodel. Clearly,conventional2Dinvitro static cultures cannotaccuratelyrecreatethislevelofcomplexity.

Therefore,tounravel theintricatemechanismsinvolvedinjointhomeostasisanddisease,a widerangeofinvivomodelshavebeenused[7,8].Examplesincludemodelsinsmallanimals(i. e.,inthemouse,rat,rabbit,orguineapig)thatareoftenusedforinitialdrugscreening,asthese aregenerallycheaperandeasiertohandlethan thelargeanimalmodels.Incontrast,large animals(i.e.,inthedog,goat,sheep,pig,orhorse)showmoresimilaritiestohumansintermsof joint anatomy and cartilage morphology, but are more expensive and require specialized facilities[9,10].Nevertheless,animalmodels allowforthe studyofdiseasesinthe naturally

Highlights

Whiledifferentinvitroandinvivo

mod-elsofjointdisordershavebeen

devel-oped,therearenoeffectivetoolsfor

theevaluationofnewtherapiesforjoint

diseases,suchasosteoarthritis(OA).

Recent advances in (bio)fabrication

technologies enable the generation

ofinvitromodelsthancanfurther

reca-pitulatearticular physiology with the

potentialofreplacinganimalmodels.

Thevalidation oftheseadvancedin

vitromodelsiscrucialtoexploittheir

translationalpotential.

1

DepartmentofOrthopaedics,

UniversityMedicalCenterUtrecht,

UtrechtUniversity,Utrecht,The

Netherlands 2

DepartmentofDevelopmental

BioEngineering,TechnicalMedical

Centre,UniversityofTwente,

Enschede,TheNetherlands

3

RegenerativeMedicineUtrecht,

UtrechtUniversity,Utrecht,The

Netherlands 4

DepartmentofEquineSciences,

FacultyofVeterinaryMedicine,

UtrechtUniversity,Utrecht,The

Netherlands

*Correspondence:

j.malda@umcutrecht.nl(J.Malda).

occurringenvironmentofthewholejoint.However,duetospecies-specificdifferences[10,11], manytherapeutictreatmentsfailwhentranslatinganimalstudiestohumanclinicaltrials[12]. Furthermore,ethicalconcernsandthesocietalambitiontoreduceanimalexperimentationhave driventhedevelopmentofadvancedinvitromodelsthatcanmoreaccuratelyrepresentstages ofhumandisease.

Recentadvancesinengineeringandbiologyhaveresultedinthedevelopmentoffunctional microscaleunits ofhuman organsthatare ableto recapitulate humandiseases[13,14].A uniquefeatureofthesesystemsistheirabilitytorecreatethecomplextissue microenviron-mentsandfacilitatecommunicationbetweendifferenttissues,accuratelymimickingtheinvivo situation[15].Invivo,cellsresideinahighlysophisticated3Dmicroenvironmentthatprovides biochemicalandbiomechanicalcuesguidingtheirbehavior,includingmigration,proliferation, anddifferentiation[16].Alterationsofaspecificextracellularmatrix(ECM)componentcan alreadygreatlyimpactthebiochemical–biomechanicalbalance,disruptingtissuehomeostasis andfunction[17,18].

Glossary

3Dbioprinting:theautomated

processofpatterningand

assemblinglivingandnon-living

materialswithaspatiallycontrolled

organization,toproduce

bioengineeredstructuresfor

applicationinregenerativemedicine.

Chondrocytes:cartilagemature

residentcells.Theyproduceand

maintaintheextracellularmatrixthat

keepscartilagehealthyand

functional.

Cytokinesandgrowthfactors:

signalingmoleculesreleasedbycells,

withtheobjectiveofaffectingthe

behaviorofothercells,including

growth,proliferation,migration,

inflammatorystate,and

differentiation.

Explant:extractedpiecesofnative

tissuesororgansthatcanbe

culturedinthelaboratory.

Extracellularmatrix(ECM):dense

networkofmacromolecules,suchas

proteinsandpolysaccharides,that

providesstructuralandbiochemical

supporttothesurroundingcells.

Hydrogel:hydrophilicpolymer

networks,whichabsorbandretaina

largeamountofwater.

Mesenchymalstromalcells

(MSCs):multipotentadultcellsthat

candifferentiateintobone,cartilage,

adipose,andmuscletissue.They

canbeobtainedfrombonemarrow,

adiposetissue,andothersources.

Microfluidics:amultidisciplinary

fieldbetweenchemistry,physics,

engineering,andmicro/

nanotechnologywhichdealswiththe

precisemanipulationoffluidsat

submillimeterscale.

Organ-on-a-chip:amultichannel

microfluidicdevicewithintegrated

microscalecellularco-culturesthat

offersphysiologicalbiochemicaland/

orbiophysicalstimulationtomore

realisticallyrecapitulatethenative

microenvironment.

Osteoarthritis(OA):degenerative

jointdiseaseassociatedwith

cartilagedestruction,inflammationof

thesynovialmembrane,and

subchondralboneremodeling.

Rheumatoidarthritis(RA):a

systemicinflammatorydiseasethat

involvessynovialcellproliferationand

structuraldamagetocartilage,bone,

andligaments.

Box1.TissuesoftheJoint

Articularcartilageisahighlyspecializedconnectivetissuethatcoverstheendsofourlongbones.Itisanavascular,

alymphatic,andaneuraltissuethatconsistsofadenseECMwithalowdensityofcells(about1–2%)[5].Subchondral

boneisthelayerofbonebeneaththecartilage.Togetherwiththecartilageitformsabiocomposite,knownasthe

osteochondralunit,which isspecializedtotransfer loadduringweight-bearingandjoint motion.Thesynovium

constitutestheenvelopeofthearticularjointsand,byactingasaninterfacewiththesystemicbloodcirculation,

ensuresnutrientsupplytothearticularcartilageviathesynovialfluid.Moreover,itsupplementsthesynovialfluidwiththe

keymoleculesnecessaryforjointfunction(i.e.,lubricin,hyaluronan,andimmunomodulatorycytokines)[5].Thesynovial

membraneconsistsoftwocelltypes,synovialfibroblasts,whichconstituteupto75%ofallcellsinahealthysynovium,

andmacrophages.Themacrophagescanbeclassifiedintoclassicallyactivated(M1)andalternativelyactivated(M2).

Thelattersubtypeisinvolvedintheproductionofimmunoregulatoryfactors[e.g.,IL-10andchemokineligand

(CCL)-18],whereasM1macrophagesproduceproinflammatorymediatorssuchasTNF-a,IL-1b,andIL-6,whichplayakey

roleinsynovialinflammation[88].Thenumberofmacrophagesincreasesdrasticallyduringinflammationand,together

withsynovialfibroblasts,secreteproinflammatorycytokinesandECM-degradingenzymes.Theproductionofcytokines

attractinflammatorycells(Tcells,macrophages,monocytes)intothesynovium,resultingintheformationofamassof

inflamedtissueorpannus[3].

Additionalcomponentsofahealthykneejointarethemenisci(i.e.,wedge-shapedtissuesthatperformcomplex

functionsinload-bearing,loadtransmission,stabilizationofthejoint,shockabsorptionduringmovements,nutritionof

articularcartilage,andlubrication[89]).Lubricin,asuperficialzoneprotein,whichhasakeyroleinthemaintenanceof

jointintegrity,ishighlyexpressedinhealthykneemenisci.Theexpressionoflubricinisdownregulatedintheknee

menisciandsynovialfluidofOApatients,leadingtoincreasedfrictioninthejointandcartilagedegeneration[80].Further,

meniscuscellsincreasedtheirproductionofmatrix-degradingenzymes,cytokines,andchemokinesinresponseto

stimulationwithproinflammatoryfactors(IL-1b,IL-6,orfibronectinfragments).Thissuggeststhattheroleofthe

meniscusinOAgoesbeyondthemechanicalaspectandmightbeduetobiologicalinteraction[90].

Tendonsandligamentsaresoftconnectivetissuescomposedofcloselypacked,highlyalignedcollagenfiberbundles

thatjoinbonetomuscleandbonetobone,respectively.Thesestructurestransfertensileloadstoguidemotionand

stabilizethediarthrodialjoint[91].Tendonorligamentfailuremayresultinjointdestabilizationandhenceleadtodamage

byalteringthebiomechanicalbalancebetweenneighboringtissues(e.g.,meniscusandarticularcartilage)inthejoint

[92].Further,afteranteriorcruciateligamentinjuries,thegeneexpressionofkeydegradativeenzymes(MMPs)was

upregulatedintheligamentsandthesynovium,suggestingacloseinteractionbetweenthesetissuesinresponseto

injuriesandakeyroleincartilagedegradation[93].

ThekneejointalsocontainsHoffa’sfatpadorinfrapatellarfatpad(IFP),asofttissueinterposedbetweenthejoint

capsuleandthesynovium.TheIFPisahighlyinnervatedtissueand,therefore,acommonsourceofkneepain.Although

itsphysiologicalfunctioninthekneeremainsstillelusive,earlystudiessuggestthatinflammationofthisadiposetissue

Ideally,invitro models ofthe joint mustrecapitulate the intricate microenvironment ofthe synovialcavityandcapturetheinteractionsbetweenthevariousjointelements(Figure1,Key Figure).Thetissuesthatthejointiscomposedofareextremelysensitivetotheirmechanical environment,andloadingplaysakeyroleinthemaintenanceofjointhomeostasis[19].While moderate mechanicalloading contributes to the maintenance of tissueintegrity, reduced loadingoroverloadingcantriggerpathologicalchangesinjointtissues[20].Therefore,invitro modelsofthejointshouldideallyallowfortheinclusionofmechanicalstimulationand repro-ducetheinteractionsbetweenthedifferenttissues.

Here, we reviewthe existingin vitro models and we propose newavenues for the future developmentofmoresophisticatedmodelsofthearticulatingjoint.

KeyFigure

Schematic

of

the

Synovial

Joint

Femur Femur

Tendon Tendon

Synovial membrane Synovial membrane

Hoffa’s fat pad Hoffa’s fat pad

Tibia Tibia

Ar cular car lage Ar cular car lage

Meniscus Meniscus

Healthy

joint

Damaged

+ diseased

joint

unit-on-chip Hoffa’s fat pad-on-chip

Figure1.Thesynovialjointconsistsofseveraltissuesthatworkcloselytogethertorealizeandmaintainjointfunctionandhomeostasis.Thesetissuescomprise

articularcartilage,subchondralbone,synovium,menisci,tendon,ligamentandfatpad,whichallarecharacterizedbyaspecificstructureandwhichareexposedto

differentbiochemicalandbiomechanicalstimulations.Impairmentoffunctionofonetissueleadstoalteredbehavioroftheothertissues,whichcanleadtojoint

degeneration.Theidealinvitromodelmustrecapitulatethiscomplexenvironment;themostsuitableapproachtorealizesuchamodelisthedevelopmentofseveraljoint

TheState oftheArt:InVitroModelsofTissuesintheJoint

2DMonolayerCultureModels

Todate,limitedinvitromodelsexisttostudytheinteractionsbetweenthedifferenttissuesofthe joint.Withinthemajorityofcurrentinvitromodels,thesimulationofinteractionsisoftenlimited totheintroductionofbiochemicalcuesortoco-cultureofcellsortissues[21].Forexample, interactionswiththesynoviumareoftensimulatedbysupplementingtheculturemediawith cytokines[22],orbyadditionofsynovialfluid[23].Alternatively,fibroblast-likesynoviocytescan beincludedinthemodel[24].Recently,aninvitromodelusingsynovialcellswasdevelopedto studytheresponseofthesynoviumtocartilagewearparticles[25].Themodelconsistedofa densecellsheetoffibroblast-likesynoviocytesinculturewithcartilageparticles.Theco-culture resulted inan increased production ofcytokines and thickeningof the cell sheet through increasedcollagencontent.Interestingly,theseresultscorrelatewithdatafrompreviousanimal studiesshowingthatcartilagedebriscaninducesynovitis[25].

Choosing a cell source that is representative can, however,be a challenge, withprimary chondrocytesand chondrogenicallydifferentiated mesenchymalstromal cells(MSCs) beingthemostcommonlychosencellsforthissimplifiedviewofthejoint[21].Additionally,cells culturedon2Dsurfacesarepronetochangetheirphenotypecomparedwiththeirnativeinvivo milieu.Tounderstandthepathwaysleadingtode-differentiation,changesingeneexpression wereinvestigatedinmonolayerculturesofhumanchondrocytes[26].

Notwithstandingtheirrelative simplicity,thesemodels doprovidevaluableinsightsthatcan enhanceourunderstandingoftheeventsinvolvedinjointdiseases. However,thesesimple models provide information only on isolatedevents within a specific tissue and allow for changingonlyonefactoratatime(cytokines,growthfactors,osmoticpressure,etc.).

Biomaterial-Based3DCultureModels

Themost simplistic 3D culture model is basedon aggregation of cells into spheres [27]. Previousstudieshave,indeed,shownthatchondrospheresoutperformsinglecellsintermsof cartilagematrixproduction[28],allowingthestudyofcell-to-cellandcell-to-matrixinteractions. Thisprovidesapowerfulyetsimpletooltostudycartilageformationandtopreliminarilyscreen drugsfor OA[29].Combiningmicroaggregateswithhydrogels furtherexploits thenatural environmentalcuesto promote growth factor-freechondrogenic differentiation [30].These constructscanalsobesubjectedtomechanicalloadstoinvestigatethecellresponsetothese typesofstimuli[31,32].Mechanicalloadscaninducechangesinthepericellularandterritorial matrixofchondrocytes[33].Further,cellularresponsetomechanicalstimulidependsonthe materialwithinwhichcellsareencapsulated[34].Forexample,hydrogelsprovide3Dmatrices withpropertiessimilartonaturalECMs,suchashighwatercontent,porosity,and biocompati-bility[35].TocapturethebiologicalfeaturesofECM,hydrogelscanbemodifiedto provide bioactivecues,suchasthearginine-glycine-asparticacid(RGD)adhesivemotiforthepeptide bindingmotifofN-cadherinthatmimicscell–cellinteractions,whichmightenhance chondro-genesisofMSCs[36,37].Further,thedynamicremodelingofECMthroughcell-responsive enzymaticdegradation canbereplicatedbycrosslinkinghydrogels withdifunctionalmatrix metalloproteinase (MMP)-degradable peptides [38]. The inclusion of protease-degradable crosslinksinhyaluronicacidhydrogelssignificantlyaffectedthemorphologyofencapsulated MSCs,whichdisplayedanelongatedshape.Meanwhile,cellsremainedroundedinhydrogels thatinhibitedcellularremodeling[38].

Additionally, hydrogel mechanical properties can be reinforced and tailored to match the stiffnessofthetargettissue, forexample,bycombining thehydrogelwithpolymericfibers

[39].Thefiberscouldalsomimictissuetopography,whichisknowntoinfluencecellbehavior. Forexample,compositesofelectrospunfiberswithbioglass-derivedfoamsorbiphasic scaf-foldshavebeenusedtorepairosteochondraldefects[40].Recently,anengineeredscaffold consistingofhydrogelsandelectrospunfiberswasdevelopedtosimulatethemechanicaland topographical characteristics of native tendon tissue [41]. Additionally, hydrogels can be fabricatedto mimic structuralfeatures oftissueinterfaces,suchas thetendon/ligament-to boneinterface,byusingbiphasicscaffoldsthatmimicthealignmentofcollagenmolecules[42]. Further,thecombinationofmicrofluidicmixingtechnologieswithhydrogelfunctionalization approachesenablethepreparationofhydrogelswithacontrolledspatialgradientofcellsand biomoleculesignals[43].

Table1.RepresentativeExamplesofInVitroModelsofJointTissues

Tissue Model Findings Refs

Fibroblast-likesynoviocytes

2Dmonolayer

culture

Treatmentoffibroblast-likesynoviocytesresultedincelldeathandproduction

ofproinflammatorymediators(IL–1b,IL-6,andTNF-a)

[24]

2Dmonolayer

culture

Culturingfibroblast-likesynoviocyteswithcartilagewearparticlesresultedinan

increaseinproliferationandECMcontent,similartothethickeningofsynovial

liningobservedinOApatients

[25]

Periostealstemcells Biomaterial-based3Dculture Encapsulationofmacroaggregatedperiostealstemcellsintobiomaterials

resultedinimprovedinvivocartilagetissueformation,comparedwithsingle

cell-ladenhydrogels

[30]

Cartilage Tissueexplants

CollagenasewasusedoncartilageexplantstoimitateearlyOA;mechanical

loadingaffectedthedegradedcartilagemorethanhealthyexplants

[44]

AgeingaffectedTGFandbonemorphogeneticprotein(BMP)signaling

pathways,whichmightcontributetothedevelopmentofOA

[82]

Combininginsulin-likegrowthfactor-1(IGF-1)anddexamethasone(Dex)

preventedmatrixlossinaninflammatoryenvironment

[45]

Bone Tissueexplants Trabecularboneexplantsweresubmittedtoinducedshearstresstoanalyze

theeffectsonciliaexpressionofbonemarrowcells

[83]

Meniscus Tissueexplants

Cytokinesinhibitedmeniscalrepairofexplantsinvitro [84]

InducingoverexpressionofTGF-bviarecombinantadeno-associatedvirus

(rAAV)-mediatedgenetransferstimulatedinvitrohealingofmeniscusexplants

[85]

Tendon Tissueexplants Cyclicloadinginducedexpressionofinflammatorymarkersontendon

fascicles

[86]

Synovium Tissueexplants Synovialexplantsfromrheumatoidarthritispatientswereusedtoevaluatethe

effectsofbiologicdiseasemodifyingantirheumaticdrugtreatmentinvitro;

theseresultswerecorrelatedtoclinicalperformanceofthedrugs

[87]

Cartilageandsynovium

Co-culture Addingosteoarthritissynoviumtocartilageexplantsinhibited

glycosaminoglycanproduction,andthiseffectwascounteractedbythe

additionoftriamcinolone

[55]

Osteochondralplug Anosteochondralplugmodelwithindependentcompartmentsforcartilage

andbonewasusedtoevaluatetheeffectofcelldistributionwithinahydrogel

forcartilagerepairapplications

[51–53]

Osteochondralmicrotissue Multichamber

bioreactor

TheeffectsofIL-1bwereevaluatedonanosteochondralmicrotissuemodel,

whereMSCsseededwithinahydrogelweremaintainedusingseparate

chondralandosseouscompartmentsinwhichthecellsdifferentiatedinto

cartilageandbone-liketissues

[50]

Osteochondralunitandsynoviallining Multichamberbioreactor ThesynovialliningwasreproducedbyincorporatingMSCsseededwithina

polyethyleneglycolhydrogelontheosteochondralinterface,consistingofa

collagenhydrogel

TissueCultureModels

Tissueexplants,however,keepthecellsintheirnaturalmicroenvironment(Table1).Cartilage explantshaveprovidedvaluableinformationonthewholetissueresponsetoseveralstimuli, recreatingbothphysiologicalandpathologicalenvironmentsindiversejointtissues.Forexample, toemulatethedamageobservedinearlystagesofOA,cartilageexplantswerefirsttreatedwith collagenaseandthen subjectedto repetitive mechanicalstress. This modelprovidednovel informationonthemechanismsassociatedwithcartilagedegeneration(e.g.,mechanicalloading) [44].In anotherstudy to elucidatethe mechanismof actionofdexamethasone, a potential therapeuticdrug,cartilageexplantsweretreatedwith inflammatorycytokinesandsubjected tomechanicalinjurytosimulateearlystagesofOA[45].Amajordrawbackisthatonlyalimited numberofexplantscanbeobtainedfromeachdonorandthereisahighintradonorvariability betweensamples,dependingonthelocationofextractionfromthejoint[46].Thisvariationiseven greaterbetweendonors,presentingachallengeforreproducibility.Explantscommonlyfeature celldeathontheedgeswherethetissuewascut[21].Long-termstudiesarealsocomplicated,as thepropertiesofcartilageandboneexplantschangeovertimeintermsofmechanicalproperties andECMcomposition[47].Moreover,althoughitisrelativelyeasytoobtainexplantsfromanimal sources,availabilityofhealthyhumandonortissueislimited.

Bioreactors

Bioreactorsaredevicesabletoculturecellsunderacontrolledenvironment(e.g.,temperature, pH,nutrientsupply,mechanicalstimuli).Differenttypesofbioreactorshavebeendeveloped basedontissueandapplication,suchasspinnerflasks,rotatingwallvessels,andperfusion bioreactors[48,49].Inarecentwork,abioreactorconsistingoftwoseparatecompartments hasbeenused to recreate anengineered osteochondralunit.Thetwo microenvironments couldbeindividuallycontrolled,thusallowingcontrolofconditionsintheboneandcartilage part,followingexposuretoproinflammatorycytokines[50].Further,thedesignofthis bioreac-torwasfurtheradvancedtoenablecontinuousopticalmonitoringduringculture[48]. Preservingtheintegrity ofanosteochondralunitduring invitro culturehasbeenshown to extendthelifeandqualityofexplants[51].Previousworkonosteochondralexplants demon-stratedthatbioreactorplatforms(six-wellplateformat)providingdistinctmediatothecartilage andboneregionsoftheimplantallowforlong-termcultureoftheosteochondralplugs,while maintainingcartilagetissuecontent,structure,andmechanicalproperties[52].Thisplatform wasalsousedtoinvestigatetheeffectofthespatialchondrocytedistributionincartilagerepair mechanisms[53].Despitethefactthatthisosteochondralplug-basedmodelprovidednew insightintotheinteractionbetweentissues,itisstilllimitedtotheinterplaybetweenboneand cartilageandhasnotbeenyetadaptedtorepresentinflammatoryjointconditions.

Co-CultureofTissueExplants

Theabove-describedinvitromodelsfocusonindividualtissuesofthejointortheosteochondral unit,withoutincludinginteractionswithotherelementsofthejoint.Thisaspectwashighlighted inarecentstudydescribingco-culturesofbovinecartilageexplantswithexplantsoffibrousjoint capsuleandsynovium[54].Specifically,theco-incubationofmechanicallyinjuredcartilagewith fibrousjointcapsuleandsynoviumresultedinincreasedproteolyticdegradationofaggrecanby both MMPs and aggrecanase.This enhanced proteolyticactivity was notobserved when cartilagewasculturedalone[54].Similarly,theco-incubationofexplantsofhumanarticular cartilagewithhumansynoviumresultedintheproductionofMMPsandinflammatorycytokines thatwerenotdetectedinthecartilageexplantmonoculture[55].Thecytokinesanddegradative enzymesdetectedinthe synovium–cartilage co-culturesweresimilarto thosefoundinthe synovialfluidofpatientswithOA,suggestingthatco-cultureshaveahigherpredictivepower

comparedwithmonocultures.Thisfurthersuggeststhatinvitromodelsofthejointmustbe multitissuesystemsinwhichthesynoviumplaysacrucialrole.

TheChallenge:MoreAdvanced InVitroTechnologies forMimickingArticular Function

Although static culture of tissue explants and co-cultures can recapitulate some in vivo functionalitiesofthejointtissues,thesemodelsstilllackmechanicalstimuli,suchastension, compression,andshearstressthatcellsexperienceinvivo.

Advancesinbiofabricationandmicrofluidicstechnologiesprovidetheopportunitytorecreate dynamicflowconditionsandmechanicalstimulation(Figure2)thatmayaidthedevelopmentof more predictive models of the human synovial joint. To this end,a variety of 3D culture techniques,including3Dhumanorganoids,humanorgan-on-a-chip[56],andbiofabricated tissue-likestructures[57],havebeenexploredtomodelphysiologicalandpathologicalhuman conditions.

Anoverviewofthecurrentinvitromodelsusedtoculturearticularjointtissuesisdepictedin Figure 3, highlighting advantages and limitations of each model type, thereby enablinga

Sy nov ia l me mbr ane Os te ochondr al unit

Joint ssue

Main cells

Type of mechanical load

Fibroblast-like synovial cells

Macrophage-like synovial

cells

T and B cells

Chondrocytes

Osteoblasts, osteocytes, and

osteoclasts

Microvascular and nerve cells

Tension

Fluid induced shear strain

Li ga m en t te nd on

Shear stress

Tension

Compression

Ligamentoblast/

ligamentocytes

Tenoblasts/tenocytes

Fibrochondrocytes

Vascular cells

Adipocytes

Vascular and immune cells

Pericytes and mesenchymal

progenitor cells

Fibroblast-like cells

Chondrocyte-like cells

Nerve and vascular cells

Shear, tension, and

compression

Tension

Fluid induced shear

Tension

Fluid induced shear stress

Figure2.MechanicalStimulationoftheSynovialJoint.Illustrativetablewithrepresentativeexamplesofthejoint

Reproducibility and compaƟbility with high-throughput screening Simple and easy to work Models isolated events

ReducƟonisƟc ReducƟonisƟc No Ɵssue–Ɵssue interface No gradients of chemicals and oxygen StaƟc StaƟc

Includes interacƟons with other cells/Ɵssues

Limited 3D environment No direct Ɵssue–Ɵssue interface

Higher predicƟve power compared with monocultures

2D monolayer

Current in vitro models Advantages LimitaƟons

Tissue explants Bioreactors 2D cell culture 3D cell culture Tissue complexity Dynamic culture MulƟƟssue Organs-on-chips Ex vivo mulƟƟssue 3D biomaterial based Limited number of explants from each donor High intra- and

interdonor variaƟon; reproducibility challenge No gradients of chemicals and oxygen No Ɵssue–Ɵssue interface Lacks mechanical actuaƟon

Cells prone to change their phenotype Challenge to select a representaƟve cell source

Semidynamic 3D culturing

Extended culture Ɵme and quality of explants/Ɵssues Enables whole Ɵssue response to several sƟmuli The cells are cultured in their 3D naƟve microenvironment Can provide natural environmental cues Allow the study of cell-to-cell and cell-to-cell-to-matrix interacƟons

(A)

(B)

Figure3.CurrentandEnvisionedModelsoftheArticularJointTissues.(A)Illustrativetablewithmostrelevantin

vitrocell/tissueculturemodelsofthejoint,highlightingtheadvantagesanddrawbacksofeachmodel.(B)Schematic

comparisonbetweenmodels(A).Theevolutionthroughoutthemodelsystemsisillustratedin Figure3B,featuringincreasingcomplexitystepstowardsorgan-on-chipplatforms. Organs-on-chiparemicrofluidicdevicesthatcansimulatephysiologicalfunctionsoftissuesandorgans [14].Numerousorgans-on-chipmodelshavebeendevelopedsofar,however,therearestill issuestobeaddressed.First,weshouldchooseamatrix(e.g.,ahydrogel)withasuitable compositionandtopographytomimictheECMofthedifferenttissues.Forexample,cartilage exhibitsa highly organized structure which is differentfrom the bone or synoviumtissue. Further,the stiffnessof the matrixis also an importantfactor, since themechanicalloads experiencedbythecellsmightbeaffectedbythestiffnessofthematrix(i.e.,shielding)[58].The needtofindsuitableflowratesforeachofthedifferenttissuemodelswillalsobechallenging, especiallywhen connecting the differenttissues.Anotherimportant issue isrelated to the sourceofcells.Themajorityofthemicrofluidicdeviceshavebeenoperatedusingcelllines, eventhoughhuman-derivedprimarycellswouldbeabettermimicofthehumanphysiology. Themainlimitationsforusinghumanprimarycellsarethelimiteddonoravailability,potential variability,andtheircosts[59].

Basedonthefundamentalunderstandingoffunctionalandpathologicalorgans,acombination oftechnologicalapproaches(e.g.,softlithography,microfluidics,3Dbioprinting)isadoptedto simulateinvitrothenative3Dorganizationbyincorporatingbiomaterialscaffolds,continuous vascular-likeperfusion,mechanicalstimulation,andchemicalcueswithinthesameplatform. 3Dbioprintingrepresentsapromisingtechnologyforreplicatingtissue–tissueinterfacesdueto itsinherentcapabilitytodepositheterogeneousbioinks(livingcellsand/orbiomaterials)ina definedspatiotemporalmannerrelevanttobiologicalarchitectures.Biofabricated3Dinvitro modelsofferagreatopportunitytoinvestigatethephysiologicalandpathologicalprocessesof multitissues, as well as to perform drug screening and toxicological studies. Up to now, numerousinvitromodelsoftissuesandorgans,rangingfrombonetomicrovasculature,have beendevelopedwithavarietyofshapes,lengthscales,resolutions,andmechanicalproperties ofbiomaterialsandmostlyusinghuman-derivedcells[56,60].Recently,theperformanceofan osteochondralbioreactorwassignificantlyimprovedbyusing3Dprinting.Specifically,thenew modelenabledthefluidtransportthroughthecentralchambertobemaximizedandallowedfor opticalaccesswithinthe3Dconstruct,whilemaintainingdimensionscompatiblewitha96-well plate[48].Additionally,arecentlydescribed3Dprinterbioreactorallowsfortheprintingof3D constructs directly inside the bioreactor, reducing both contamination risk and the riskof damagetotheconstruct[61].3D-printingtechnologyhasgainedincreasedattentioninthe fabricationofmicrofluidicsystems.Thesedevicesarecommonlyfabricatedusing polydime-thylsiloxane (PDMS), which is easyto mold,biocompatible, transparent,and inexpensive. Additionally,PDMSmoldinghasveryhighresolution.However,thefabricationofthedevices involvesstackingandbondingdifferentlayerstogether,whichincreasesthefinalcostandlimits the 3Dcomplexity that can be achieved[62].Although, 3D-printingis an automated and assembly-freetechnology,withlow-costset-ups,limitedonlybytheresolutionoftheprinting process.Printingtechnologiessuchasstereolithography(SLA)ormeltelectrospinningwriting (MEW) could significantly improve the resolution of perfusable channels. For example, a custom-built projection SLA has been recently used to prepare complex 3D structures withpatterningfeaturesof<5mmresolution[63],whileMEWenablesthefabricationoffibers of4–7mm[64].

Organ-on-ChipTechnology

Microfluidicsystemscanbeconnectedwitheachothertoformmulticompartmentmicrofluidic devices.Animportantchallengewhenconnectingdifferentmicrofluidicdevicesinvolvesthe requirementforacommonmedia,orbloodsubstitute,suitableforeachtissueoftheinteracting

system[65].Thismulticompartmentorganizationisidealforthedesignofinvitromodelsofthe joint,amultitissueorganwherecellsareexposedtodifferenttypesofmechanicalstimuli.For example,withinthesynovialjoint,cellsexperienceahighmechanicalstress,boththrough load-bearingandbyshearforcescreatedbythemotionofthesynovialfluidduringexercise[66].The magnitude,direction,andtypeofstressappliedwasfoundtoaffectthetypeandseverityof inflammationofthejoints[67].However,thebiochemicaleffectsofshearforceonthesynovial membranehavenotbeenstudiedsystematicallyinaninvitroplatform,andamulticompartment microfluidicchamberwouldbeapromisingsolution.

Dependingontheorientationofthecompartmentalizedchannels,mainlytwotypesof con-figurationshave beendeveloped(Figure4C,D)(i.e.,upper–lowerchambersseparatedbya semiporous,stretchablemembraneandlateralchambersobtainedbyanarrayofmicroposts [68–70]oraremovabletemplate[69]).Inaddition,dynamiccompressivebioreactorshavebeen

Figure4.RecreatingMicrophysiologicallyArticularJoint-RelevantEnvironmentswithOrgan-on-Chips.(A)Generationoftheboneperivascularnichefor

studiesofbreastcancercolonization.Humanbonemarrow-derivedmesenchymalstemcells(MSCs)andendothelialcells(ECs)wereculturedinmonolayersandin3D

decellularizedbonematrix.Imagereproducedwithpermission,from[78].(B)Multiple‘organs-on-a-chip’platformtomodelmetastasisfromlungtobrain,bone,and

liverdownstreamorgans.Thismodularstrategycanbeusedtomodelthetissuesfromthearticularjoint.Imagereproducedwithpermission,from[79].(C)Schemeofa

microfluidicvascularizedbonetissuemodelformimickingrealboneangiogenesis.Thebone-mimickingchannelconsistsofamixtureoffibrinwithhydroxyapatite.

Imagereproducedwithpermission,from[69].(D)Schematicdrawingandphotographofthemicrofluidickidneyglomeruluschip,withtheurinaryandcapillary

compartmentsoftheglomerulusseparatedbyapolydimethylsiloxanemembrane.Cyclicstrainisappliedonthesidechanneltomimicthetissuestretch.Itisenvisioned

thatasimilardesigncanbeusedforjointarticulartissues,whichinvolvesimilarmechanicaltension,suchasthesynovialmembraneorthetendons.Imagereproduced

withpermission,from[80].(E)Pictureofacompleteactuatorchipimplementedinmechano-stimuliresponsivestudiesofneuronalcellnetworksonchip.Theimages

representaschematicdrawingofitsmechanismofactuationusinggasflow.Itisenvisionedthatasimilardesigncanbeusedforjointarticulartissuesinvolvingsimilar

developed to screen the effect of biomaterials and of mechanical stimulation on cellular behaviors(Figure4E)[71].

RecapitulatingtheMicrophysiologicalEnvironmentoftheSynovialJoint Thecurrentdevelopmentsdescribedaboveopenupnewwaystorecapitulatewholeorgansor combinationsof multiple organs ina body-on-a-chip fashion. However, the integrationof different organs still faces some challenges, including maintaining the functionality of the differentorgansandthestudyofintertissueresponsestodrugadministration.The develop-mentofthesemulticompartmentsystems,thatcaninteractwitheachotherinaphysiologically relevantmanner,wouldbecrucialforthedevelopmentofmodelsofthejoint,forexamplea joint-on-a-chip.

Organ-on-chiptechnologyprovides a platformto recreate physiologicallyrelevant environ-ments.Althoughthisnoveltechnologyhasbeenusedtodevelopbiomimeticsystemsofseveral organs(lung,kidney,liver),boneistheonlytissueofthejointthathasbeendevelopedonchip thusfar.Tomimictheinvivomicroenvironmentofbone,amicrofluidicapproachwasusedto developa vascularized bone tissuemodel. Fibrin was used as a model of the ECM and combined with various concentrations of hydroxyapatite nanocrystals to mimic the bone structure.Thepresenceofhydroxyapatiteresultedinenhancedangiogenicproperties, induc-ingimprovedsproutlength,sproutspeed,andlumendiameter[72].Otherstudiesfocused mainlyonbonemarrow-on-chip.Forexample,abonemarrow-on-a-chipsystemwas devel-opedbyfirstengineeringnewboneinvivoandthenperfusingitinamicrofluidicdeviceableto reconstitutethehematopoieticnichephysiology[73].Totestthefunctionalityandorgan-level response,theengineeredbonemarrowwasexposedtovaryingdosesofg-radiation,andthe resultscloselymimickedtheeffectsobservedinthebonemarrowoflivemice[73]. Toreconstitutethemicroenvironmentofthearticularjoint,aninvitro3Dmicrosystemmodelof theosteochondralunitwasdevelopedbyfittingamultichamberbioreactorintoamicrofluidic base[50].Tomimicthechondrogenicandosteogenictissues,humanbonemarrowstemcells (hBMSCs)wereseededwithinahyaluronicacid-basedhydrogelandagelatin/hydroxyapatite construct, respectively. The two compartments were supplied by two different medium streamsto reconstitutethechondral andosseousmicroenvironments andpromote tissue-specific differentiationof hBMSCs.In anextstep, theosteochondralunit was exposedto interleukin-1b(IL-1b)toevaluatetheresponsetoproinflammatorycytokinesandthe commu-nicationbetweenthetwocompartments.TheIL-1btreatmentoftheosseouscompartment resultedinastrongcatabolicresponseinthechondrallayer,whichwassignificantlyhigherthan the response observed after local exposure to IL-1b, suggesting an active biochemical communicationbetweenthetwolayers.Tobettermimicthejointenvironment,themicrotissue modelwasimprovedbyincorporatinganosteochondralinterface,consistingofanMSC-laden collagenhydrogel,andasynovialliningproducedwithMSCsseededonapolyethyleneglycol hydrogel[74].Althoughthismicrotissuerepresentsavaluablemodeloftheosteochondralunit, somekeyelementssuchasotherjointcomponents(menisci/tendons)andmechanical stimu-lation(e.g.,compression,shearstress)arestillmissing.

Besidesarticularcartilage,subchondralbone,menisci,tendons,andfatpad,akeycomponent ofthejointorganisthesynovium.Indeed,synovialinflammationisintimatelyassociatedwith jointdegenerationindiseasessuchasOAandRA[3].RecentstudiesindicatethatOAtissue andsynovialfluidcontainhighlevelsofcytokines,andthatchondrocytesandsynovialcellsin OA overproduce several inflammatory mediators, including IL-1b, tumor necrosis factor-a (TNF-a),andnitricoxide,whicharecharacteristicofinflammatoryarthritis.Further,the lipid

metabolismmightalsocontributetothepathogenesisofOAthroughinflammatorymediators, forinstance,adipokinesproducedbyadiposetissuesuchaspresentinHoffa’sfatpad[75].It becomesclearthatthesynoviumtogetherwiththeosteochondralunit,menisci,tendons,and fatpadareessentialcomponentsforthedevelopmentofanadvancedinvitromodelofthejoint. Further,synovialinflammationalsorepresentsaneffectivetargetforthedevelopmentofnovel therapeuticstrategies.However,amajorlimitationsofaristheinabilitytospecificallytarget synovialcells,without affectingthe whole-organismphysiology[3].Thislimitation couldbe overcomebyfabricatingindividualjointcomponentsusingmicrofluidictechnologyonchipto reconstitutethephysiologicalcontextofeachtissue,includingmechanicalandbiochemical stimulation,andcombinetheseindividualtissuesonchipinamultimodulardevice:the joint-on-chip.Thisapproachprovidestheopportunitytounderstandthemolecularpathwaysinvolvedin thephysiology andetiologyofeach tissueindividuallyandmay, asan example,aidinthe discoveryofsynovium-targetingtherapies.Furthermore,bydesigninginterconnectable organ-on-chipmodelsofcartilage,bone,synovium,tendons,andfatpadandarrangingthemasin vivo,itisalsopossibletostudycrosstalkbetweenthevarioustissues.Aninterconnectedmodel of the joint could be valuable for understanding joint homeostasis and to develop novel therapiestopreventandslowtheprogressionofjointdiseases.

ValidationofInVitroModelsoftheJoint

Theseadvancedmodels,duetotheirgreatlevelofphysiologicalmimicry,havethepotentialto replace or be integrated with animal studies for preclinical testing. To achieve this, one importantchallengestillneedstobeaddressedtotranslatethe‘on-chip’resultstotheclinic; thatis,thevalidationofthemodel.

Here,akeystepwillbetodemonstratethepredictivepowerofthemultitissuemodelofthejoint forhumandisease.Therefore,thereisastrongneedtodevelopvalidationmethodsthatcan assessthereproducibility, reliability,and thetranslationalpotential ofthose models[60].A possiblestrategycouldbethetestingofarangeofdrugswithknowneffectagainstRA(inthe caseofthejointmodels)andcomparetheresultswithdataobtainedfrominvitrotissue co-culturemodels, animal studies,and efficacy studiesin patients.Forinstance, celecoxib,a nonsteroidalanti-inflammatorydrugusedforthetreatmentofOAandRA,hasbeenusedto evaluatetheaccuracyofamodelbasedontheco-cultureofatissue-engineeredcartilage constructwithsynovialfibroblastsandmacrophages.Thesystemcouldreplicateonlysomeof theinvivoresponsesofthedrug[76].Animportantlimitation,however,isthatthismodelonly mimicscartilage–synoviuminteractions,whilenumerousstudiesdemonstratedaclose inter-actionbetweencartilage–subchondralboneandvasculature[49].Thisfurtherhighlightsthe necessityto developmultitissue modelsthat can bettermimic the invivo behavior.These multitissuedevicescouldbeadvantageousfordrugscreening,enablingthetestingofvarying concentrationsof adrugor combinationsof drugs,simultaneously andina cost-effective manner.Most importantly,these devicescan beoperated withhuman cells and thusare capableoffurthermimickingthehumanphysiologyandmetabolism[77].

ConcludingRemarksandFuturePerspectives

Understandingthemolecularmechanismsinvolvedinjointhomeostasisanddiseaseis funda-mentalforthedevelopmentofnoveltherapeutictreatments.Amajorchallengesofarhasbeen thelackofmodelsthatcanfaithfullymimicjointphysiologyinhealthanddisease.Thelimited predictivepowerofexistinganimalmodelshasdriventheneedtodevelopadvancedinvitro modelsthatcanmoreaccuratelyrepresenttheinvivo-likeenvironment.Recentadvancesin engineeringandbiologyhave enabledthe developmentofinvitromodels thatrecapitulate

OutstandingQuestions

Canthecomplexmicroenvironmentof

thesynovialjointbefaithfully

recapitu-lated using the organ-on-chip

technology?

Isitpossible tostudyonsetofjoint

diseasesandthesequenceof

patho-logicaleventsrecapitulatingthe

com-plexity of human disease using a

multicompartmentjoint-on-a-chip?

Willsuchahumanizedjointmodelbe

abletopredicttheefficacyandtoxicity

ofnoveltreatmentsforarthritis?

Can a multicompartment

joint-on-a-chipenablethediscoveryofearly

complex 3D organ-level structures with integrated mechanical, biochemical, and physical stimulation(see OutstandingQuestions). This technologyhasthe potential to emulatethe variouscomponentsofthejointorganandtheinterplaybetweenthedifferentelementsofthe joint.Inadditiontoa‘humanized’jointmodel,thedevelopmentofjointmodelsforanimalsis alsoimportantfortheapplicationinveterinarymedicine.Specifically,theuseofhumanand animalmodelsunderasimilarexperimentalsettingcouldbringtolightspecies-specific differ-ences.Ultimately,the developmentofan advancedinvitro modelofthe jointwill assistin reducingorreplacingtheuseofanimalmodelsinbiomedicalresearch,personalizedmedicine, andpharmaceuticalstudies.

Acknowledgments

TheauthorsacknowledgethefinancialsupportprovidedbytheDutchArthritisFoundation(LLP12,LLP22,andLLP25)and

thefinancialsupportofthestrategicallianceprogramentitled:Advancedbiomanufacturing,fundedbytheUniversityof

Twente,UtrechtUniversityandUniversityMedicalCenterUtrecht,projecttitle:Bioprintingfunctionaltissuesfromstem

cellsandenablingbiomaterials.

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