Wu Wei - Binnenwerk - complete proef DIGI PDF tissue and the... · Wu Wei. The printing of this...

A D I P O S E

T I S S U E

A N D T H E

K N E EA

DI

PO

SE

TI

SS

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AN

D T

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KN

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Role of inflammation on joint

degeneration and cartilage repair

Wu Wei

Wu

We

i

A D I P O S E

T I S S U E

A N D T H E

K N E E

AD

IP

OS

E T

IS

SU

E A

ND

TH

E K

NE

E



Wu Wei

Wu

We

i

A D I P O S E

T I S S U E

A N D T H E

K N E E

AD

IP

OS

E T

IS

SU

E A

ND

TH

E K

NE

ERole of inflammation on joint


Wu Wei

Wu

We

i

The printing of this thesis was financially supported by:

• DepartmentofOrthopedics,ErasmusMCUniversityMedicalCenter,

Rotterdam

• NederlandseOrthopaedischeVereniging(NOV),‘sHertogenbosch

• AnnaFonds,Leiden

• CentrumOrthopedieRotterdamb.v.,Rotterdam

• LivitOrthopedie,Amsterdam

• Chipsoftb.v.Amsterdam

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ADIPOSE TISSUE AND THE KNEERoleofinflammationonjointdegeneration

and cartilage repair

Vetweefsel en de knieRolvanontstekingopgewrichtsdegeneratie

enkraakbeenherstel

Proefschrift

terverkrijgingvandegraadvandoctoraande

ErasmusUniversiteitRotterdam

opgezagvande

rectormagnificus

Prof.dr.H.A.P.Pols

envolgensbesluitvanhetCollegevoorPromoties.

Deopenbareverdedigingzalplaatsvindenop

vrijdag20oktober2017om13:30uur

door

Wu Wei

geborenteHubei,China

PROMOTIECOMMISSIE

Promotoren: Prof.dr.G.J.V.M.vanOsch

Prof.dr.J.A.N.Verhaar

Overige leden: Prof.dr.J.M.W.Hazes

Prof.dr.S.K.Bulstra

Dr.P.J.Emans

Copromotor: Dr.Y.M.Bastiaansen-Jenniskens

Voormijnouders

致我的父母

CONTENTS

Chapter1 Generalintroduction,aimsandoutlineofthethesis 11

Part I Local effects of adipose tissue

Chapter2 The size of infrapatellar fat pad adipocytes is not influenced by obesity

27

Chapter3 Stimulationoffibroticprocessesbytheinfrapatellarfatpadinculturedsynoviocytesfrompatientswithosteoarthritis: apossibleroleforprostaglandinF2α

37

Chapter4 Theinfrapatellarfatpadfromdiseasedjointsinhibitschondrogenesisofmesenchymalstemcells

59

Chapter5 Anti-chondrogenicandpro-cataboliceffectofinfrapatellarfatpadanditsresidingmacrophagescanbemodulatedbytriamcinoloneacetonide

79

Part II Systemic effects of adipose tissue

Chapter6 StatinsandfibratesdonotaffectdevelopmentofspontaneouscartilagedamageinSTR/Ortmice

99

Chapter7 HighfatdietacceleratescartilagerepairinDBA/1mice 119

Epilogue

Chapter8 Summaryandgeneraldiscussion 137

Addendumfigures 153

Nederlandsesamenvatting 155

中文简介 159

PhDportfolio 161

Listofpublications 165

Abouttheauthor 169

Dankwoord 171

CHAPTER 1

General introduction, aims and outline of the thesis

General introduction, aims and outline of the thesis 13

1GENERAL INTRODUCTION

The normal knee

Thekneejointisoneofthecomplexandheavilyloadedjointsofthehumanbody.

Itisabicondylarsynovialjointwhichisformedbyarticulationsbetweenthetibia,

femurandpatella.Thekneejointallowsflexion,extensionandsomedegreesof

internalandexternalrotations.Tendonsconnectthebonestothemusclesand

togetherwiththeligamentscreatestabilitytothejoint.Thearticulatingendsof

thebonesarecoveredbyarticularcartilageandthefibrocartilaginousmeniscilie

betweenthetibiaandthefemur.

Femur

Tibia

Cartilage

Meniscus Infrapatellar fat pad

Patella

Synovial fluid

Synovial fibroblast

Blood vesselAdipocyte

Macrophage

Collagen fiber

Figure 1 ThenormalkneeThekneejointisformedbyarticulationsbetweenthetibia,femurandpatella.Theinfrapatellarfatpadislocat-edinsidethejointcapsulebutoutsidethesynovialmembrane.Thesynovialmembraneisrichlyvascularized.Itconsistsofalayeroffibroblastsandmacrophagesfollowedbyalayerofconnectivetissue.Theinfrapatellarfatpadconsistsmainlyofadipocytes.

Articular cartilage allows smooth gliding movements and acts as a shock ab-

sorber.Itisaspecializedtissuethatisavascularandaneuralandonlyharborsa

smallnumberofcells.Thesecellsarecalledchondrocytesandtheyproduceand

maintainthearticularcartilageextracellularmatrix(ECM).Thearticularcartilage

ECMconsistsmainlyofcollagentype2fibersandproteoglycans.Thenegatively

chargedglycosaminoglycans(GAG)sidechainsontheproteoglycansattractwa-

ter.TheseGAGsattractwaterinsuchextentthat65%-80%ofthecartilageconsists

ofwater.Duetoitsavascularnature,thearticularcartilagereceivesitsnutrients

14 CHAPTER 1

fromthesynovialfluidor fromthesubchondralboneunderneath thecartilage

through diffusion1.

Thesynoviumfluidisencapsulatedbythesynovialmembrane,whichisalsoan

integralpartofthejoint.Thesynovialmembraneisarichlyvascularizedconnec-

tivetissuethatisresponsiblefortheproductionofsynovialfluidandforfiltering

ofdebrisoutofthesynovialfluid.Themembraneismainlycomposedoftwocell

types:macrophagesandsynovialfibroblasts2.

Macrophagesareatypeofcellsthatcanphagocytizeforexamplecellulardebris,

foreignmaterialandmicrobes.Macrophagesplayanimportantroleininflamma-

tionandwoundhealing.Inreactiontostimulifromtheirmicroenvironmentthese

macrophagescanbecomeactivatedintoaspectrumofdifferenttypes.Roughly,

macrophagescanbecategorizedintopro-inflammatory(oftenreferredtoasM1)

macrophages and anti-inflammatory/wound healing (often referred to as M2)

macrophages3.

SynovialfibroblastsarecellsthatcansynthesizeECMsuchascollagentype1,but

alsohyaluronicacidandlubricinforthesynovialfluid.Duringinflammation,these

synovialfibroblastscanalsobetriggeredtosecreteinflammatoryandcatabolic

factors.Outsidethesynovialmembranebutinsidethejointcapsuleseveralfatde-

potsarelocalizedofwhichtheinfrapatellarfatpad(IPFP)isthelargest.TheIPFP

isalsocalledHoffa’sfatpad,namedafterdr.AlbertHoffawhofirstdescribedit

in19044.TheIPFPfillstheanteriorkneecompartmentanditsposteriorsurfaceis

coveredwithsynovialmembrane.TheIPFPprovidesmechanicalcushioningand

facilitatesthedistributionofsynovialfluids.TheIPFPisrichlyvascularizedand

innervated. Furthermore, it containsmany immune cells such asmacrophages

andstemcells5;6.These immunecellscouldplayanrole in thepathogenesisof

kneeosteoarthritis(OA)andthestemcellscouldbeusedfortissueengineering

purposes.

The diseased knee

ThekneejointisvulnerabletotraumaandthedevelopmentofOA.Accordingto

theUnitedStatesBoneand Joint Initiative, thesocioeconomicburdenof joint

diseasesin2014wasveryhighwithmorethan50%ofpeopleaged18yearsand

olderintheUSreportingamusculoskeletalconditionandallcostsassociatedwith

musculoskeletalconditionwasabout5.7%ofthegrossdomesticproduct7.OAis

themostcommondegenerativejointdisorderandaffects1.1millionintheNeth-

erlands8.KneeOAisadiseaseofthewholejointandismorethan‘wearandtear’


1ofthecartilagealone.ThesymptomsofkneeOAarepain,reducedmovementand

disability.ThereareseveralriskfactorsforthedevelopmentofkneeOA,which

includeage,sex,obesityandpreviousjointinjury.Structurally,thereiscartilage

damage,subchondralbonesclerosis,osteophytesformationandsynovialinflam-

mationandfibrosis9.SomeofthestructuralchangesinOAareassociatedwiththe

degreeofsymptoms.IthasbeenshownthatinflammationoftheIPFPisassociated

tokneepainandthatsynovialfibrosiswithreducedjointmovement.Currently,

thereisnocureforOA.Thereareonlytreatmentsagainstthesymptomsofknee

OA,includingreplacementbyarthroplastyinend-stagekneeOA9.

Itisgenerallyassumedthatarticularcartilagedamageifleftuntreated,eventually

couldleadtoOA.Articularcartilagenormallydoesnothealspontaneouslyoronly

partiallyduetoitsavascularandaneuralnature.Furthermore,itharborsonlya

relativelysmallnumberofchondrocytesthatcouldpotentiallyproducematrix.In

thecaseofcartilagedamage,thesechondrocytesreactbyproducingproteolytic

enzymesat the siteof injury thatdestroy theECMand therebyworsening the

damage10.

Cartilage repair

Treatment strategies

Successful treatment of these cartilage defects remains a challenging clinical

problembecausenoneofthecurrentregenerationoptionsareabletocompletely

restore theoriginalstructuralandbiomechanicalpropertiesof thecartilage. In

theknee,surgicaltreatmentoptionsaremarrowstimulationtechniques,suchas

microfracture,forsmallsymptomaticcartilagelesionsandautologouschondro-

cyteimplantation(ACI)andmosaicplastyforlargerlesions10.

The microfracture procedure, first introduced by Steadman11, is a simple and

low cost method to stimulate the body’s own repair response. In the original

technique, during knee arthroscopy, the cartilage defect is debrided down till

thesubchondralbonelayerandsmallholesare‘fractured’intothesubchondral

bone11;12.Thisinturncreatesa‘bleeding’fromtheunderlyingbonemarrowand

thusstimulationofarepairresponsebybioactivefactorsandmesenchymalstem

cells(MSC)fromtheunderlyingbonemarrow13.Thisprocedurecanpotentiallybe

enhancedbyaddinggrowthfactorsorscaffolds,orbychangingthetechniqueof

creatingtheholesorthesize,numberanddepthoftheholes14-16.Themicrofrac-

turetreatmentisgenerallyusedforsingledefectssmallerthan2.5cm2 and leads

toclinicalimprovementinfunctionandpain16-18,butnottohyalinecartilagerepair.

16 CHAPTER 1

Instead,afibrocartilaginoustissuefillsthedefect,whichisofinferiorbiomechani-

cal properties13;19;20.

TheACIprocedureuseslaboratoryexpandedautologousarticularchondrocytes

harvestedfromhealthypartof the jointtofillupthedefect21.These implanted

articular chondrocytes can potentially repair the defect by laying down a new

hyalinecartilageECM.Itisalsopossibletoenhancetherepairprocessbyaddinga

(bio)scaffold22;23.TheACIprocedureisgenerallyusedforcartilagedefectsgreater

than4cm2ormultipledefectsandthelongtermresultsaregood24.Comparedto

microfracture, theACIprocedure is expensive and requires two surgeries. Fur-

thermore,thereismuchdiscussiononthesuperiorityofthistechniquecompared

tothecheapermarrowstimulationtechniquessuchasmicrofracture17;25;26

BesidesmicrofractureandACIprocedure,anosteochondraltransplantationcan

beperformed.Inthisprocedure,alsocalledmosaicplasty,autologousosteochon-

dralgrafts fromhealthy, lessdemandingpartsof the jointare transplanted.Al-

logenousgraftcanalsobeused.Noevidenceisyetavailableaboutthesuperiority

ofthisprocedureversusmicrofractureandpatientsareleftwithanewdefectat

thedonorsitethatcausesdonorsitemorbidity25;27.

Nexttotheseinterventions,currentresearchisalsofocusedontransplantation

ofspecificengineeredMSC-basedconstructs.Inthesestrategies,anovelgraftis

engineeredin-vitrousingMSCs,scaffoldsandgrowthfactors10.Thesestrategies

couldpotentiallyreducedonorsitemorbidityandleadtoimplantationofahya-

linecartilageimplant28.However,theseareonlyexperimentaltreatmentsandcur-

rentlynorandomizedcontrolledtrialhasbeenpublishedyetonthesestrategies.

Effect of inflammation

Cartilagerepairresultscanbeinfluencedbyseveralfactors.Twentyfourhours

afterjointtrauma,thereisapeakinpro-inflammatorycytokinesintheknee,in-

cludingtumornecrosisfactoralpha(TNF-α),interleuking-1beta(IL-1β),aswellas

matrixmetalloproteinase(MMP)-1andMMP-1329-32. Ithasbeenshownthatthese

pro-inflammatorycytokinescaninhibitchondrogenesisandthereforepotentially

inhibit successful hyaline cartilage repair33-35.

Althoughthelevelofthesepro-inflammatorycytokinesdropsovertime,thelevel

ofinflammatorycytokinesininjuredkneesisstillhigherthaninnon-injuredknees

oneyearafterinjury36.Anycartilagerepairprocedureinthekneethereforetakes

placeinanenvironmentwithsomedegreeof inflammation. Inflammationisnot


1alwaysdetrimentaltosuccessfulwoundhealing,becausesomedegreeofinflam-

mationisnecessaryto initiateandmaintainanyhealingresponse37.Controlling

theamountof inflammationandreducingtheamountofpro-inflammatoryanti-

chondrogeniccytokinesinthekneeisthereforeimportant.

Obesity and adipose tissue inflammation

Obesityisanotherfactorthatcouldinfluencecartilagerepairresultsintheknee.

Obesityisdefinedbyabodymassindex(BMI)above30.Studieshavesuggested

thatahigherBMIcouldadverselyaffecttheclinicaloutcomeofmicrofracture18;38

andACI39inthekneeandpatientswithaBMIabove30arenowgenerallyexcluded

fromtreatment40.

Obesityisawell-knownriskfactorforthedevelopmentofOA41.Atypicalstruc-

turalchange inobesity is the increase inadiposetissuesize.Excessivedietary

intakeoflipidsandcarbohydratesleadstoadipocytehypertrophy,deathandsub-

sequentlyinfluxofinflammatorycellsintotheadiposetissue.Thisinturnleads

toincreasedinflammatorycytokinesecretionbytheadiposetissueandsystemic

inflammation.Theseinflammatorychangesleadstosystemicmetabolicchanges

such as hyperglycemia, hyperinsulinemia and dyslipidemia42-44. Combined with

obesity,thesechangesarecalledmetabolicsyndromeandareallriskfactorsfor

thedevelopmentofOA41.BesidesincreasingtheriskofdevelopingOA,obesityalso

couldexaggeratespost-traumaticarthritis45;46anddecreaseskinwoundrepair46.

Mostoftheadiposetissueinthebodyislocatedsubcutaneously.Therearealso

adiposetissuelocatedintra-articularly.Inthekneejoint,theIPFPisthebiggest.

TheIPFPishowevernotthesameassubcutaneousadiposetissue.OnMRI,total

IPFPvolumeisnotassociatedtoBMI47;48and IPFPadipocytesize inmice isnot

influenced by high fat feeding49.TheIPFPdoesplayanroleinjointhomeostasis.

MRIstudieshaveshownthatalargerIPFPisevenassociatedwithlessOA47;50-52.

Previouslywehaveshownthatthesecretionofpro-inflammatorycytokinesbythe

IPFPcanbeincreasedbyaninflammatorystimulus53andthattheIPFPsecretes

factors that could influence the cartilage in degenerative joint disease54.Other

authors report that the IPFP containsmany inflammatory cells and that it is a

sourceofinflammatoryfactors55;56.

18 CHAPTER 1

AIMS AND OUTLINE OF THE THESIS

Adiposetissuebecomesinflamedinobesityandsecretesfactorsthataffectthe

kneejoint.Thesefactorsoriginatefromadiposetissuelocatedoutsidetheknee

joint and thus influence the knee through systemically secreted factors. There

ishoweveralsotheintra-articularly locatedIPFPthatcandirectly locallyaffect

thekneejoint.Therefore,themainaimofthisthesisistoinvestigatetheeffectof

inflammationinadiposetissueondegenerativejointdiseaseandcartilagerepair.

Morespecifically,weinvestigate:

1. whetheradiposetissueinandoutsidethekneeinfluencesdegenerativejoint

diseaseandcartilagerepairintheknee

2. whattheroleisofadiposetissueresidentmacrophagesindegenerativejoint

diseaseandcartilageintheknee

3. whetherwecanmodulateadiposetissueandmacrophageswithmedicationto

reducedegenerativejointdiseaseandtoimprovecartilagerepair.

Adipocytesizeinsubcutaneousandvisceraladiposetissueisincreasedinobesity.

This increase inadipocyte size is associatedwithadipose tissue inflammation.

However,thereisnoinformationyetavailableaboutwhetherobesityinfluences

theadipocytes inthe IPFP inhumans. InChapter 2we investigatewhetherthe

adipocytes inthe IPFPare increased inobesepatients.Theposterioraspectof

theIPFPiscoveredbysynovialmembraneandthesynovialmembraneisheavily

influencedbyfactorssecretedbythe IPFP.Synovialfibrosis iscommoninknee

OAandcausesjointstiffness.InChapter 3wedeterminewhetherIPFPsecretes

factors that influence fibrotic processes in synovial fibroblasts. Previously, we

haveshownthattheIPFPsecretesfactorsthatinfluencecartilage.InChapter 4 we

examinewhethertheIPFPsecretesfactorsthatalsoinfluenceMSC-basedcartilage

repair.Furthermore,inChapter 4wedescribetheroleofmacrophagesresiding

intheIPFPonMSC-basedcartilagerepair.Toimprovethejointenvironmentfor

cartilagerepair,inChapter 5wefocusontheeffectofdifferentanti-inflammatory

medicationontheIPFPandmacrophagesintheIPFP.

TheIPFPisanintra-articularadiposedepotandmostadiposetissuearelocated

extra-articular.Subcutaneousandvisceraladiposetissuedepotsarelocatedout-

sidethejointandobesityleadstochangesinthesedepots.Thisresultsinsystemic

metabolicandinflammatorychangesthatcouldinfluencedegenerativejointdis-

easeandcartilagerepair.Thesesystemiceffectsarecomplexandthereforein-vivo

studies aremore suitable. InChapter 6 we testwhether commonly used lipid

modifyingmedicationscanpreventdegenerativejointdiseaseandcartilagedam-


1ageinamousemodelofspontaneouscartilagedamagewithmetabolicsyndrome.

However,whencartilagedamagedoesoccurinobesepatientsacartilagerepair

treatmentmightbeneeded.Currently,therearenostudiesavailableexaminingthe

structuralcartilagerepairoutcomeinobesepatientsorexplaininghowobesity

influencescartilagerepair.InChapter 7weinvestigatewhetherahighfatdietcan

influencecartilagerepairinamousemodelofcartilagerepair.Inthisway,Ihope

tofindnewtreatmentstoimprovethejointenvironmenttoreducecartilagedam-

ageandimprovecartilagerepair.Finally,inChapter 8Iwillsummarizeanddiscuss

ourmostimportantfindings.Iwillendwithmyperspectiveonfutureresearch.

20 CHAPTER 1

REFERENCES

1. SophiaFoxAJ,BediA,RodeoSA.2009.Thebasicscienceofarticularcartilage:structure,composi-

tion,andfunction.SportsHealth1:461-468.

2. SmithMD.2011.Thenormalsynovium.OpenRheumatolJ5:100-106.

3. MurrayPJ,AllenJE,BiswasSK,etal.2014.Macrophageactivationandpolarization:nomenclature

andexperimentalguidelines.Immunity41:14-20.

4. HoffaA.1904.Influenceofadiposetissuewithregardtothepathologyofthekneejoint.JAMA:795–

796.

5. ClockaertsS,Bastiaansen-JenniskensYM,RunhaarJ,etal.2010.Theinfrapatellarfatpadshould

beconsideredasanactiveosteoarthriticjointtissue:anarrativereview.OsteoarthritisCartilage

18:876-882.

6. DragooJL,JohnsonC,McConnellJ.2012.Evaluationandtreatmentofdisordersoftheinfrapatellar

fatpad.SportsMed42:51-67.

7. USBJ.2014.UnitedStatesBoneandJointInitiative:TheBurdenofMusculoskeletalDiseasesinthe

UnitedStates

8. ChorusAMDFS.2011.NationalePeilingvanhetBewegingsapparaat2010.

9. LaneNE,BrandtK,HawkerG,etal.2011.OARSI-FDAinitiative:definingthediseasestateofosteo-

arthritis.OsteoarthritisCartilage19:478-482.

10. HunzikerEB,LippunerK,KeelMJ,etal.2015.Aneducationalreviewofcartilagerepair:precepts&

practice--myths&misconceptions--progress&prospects.OsteoarthritisCartilage23:334-350.

11. SteadmanJR,MillerBS,KarasSG,etal.2003.Themicrofracturetechniqueinthetreatmentoffull-

thicknesschondrallesionsofthekneeinNationalFootballLeagueplayers.JKneeSurg16:83-86.

12. MithoeferK,McAdamsT,WilliamsRJ,etal.2009.Clinicalefficacyofthemicrofracturetechnique

forarticularcartilagerepairintheknee:anevidence-basedsystematicanalysis.AmJSportsMed

37:2053-2063.

13. ShapiroF,KoideS,GlimcherMJ.1993.Celloriginanddifferentiationintherepairoffull-thickness

defectsofarticularcartilage.JBoneJointSurgAm75:532-553.

14. GomollAH.2012.Microfractureandaugments.JKneeSurg25:9-15.

15. OrthP,DuffnerJ,ZurakowskiD,etal.2016.Small-DiameterAwlsImproveArticularCartilageRepair

AfterMicrofractureTreatmentinaTranslationalAnimalModel.AmJSportsMed44:209-219.

16. Fischer S, Kisser A. 2016. Single-step scaffold-based cartilage repair in the knee: A systematic

review.JOrthop13:246-253.

17. ErggeletC,VavkenP.2016.Microfractureforthetreatmentofcartilagedefectsinthekneejoint-A

goldenstandard?JClinOrthopTrauma7:145-152.

18. MithoeferK,WilliamsRJ,3rd,WarrenRF,etal.2005.Themicrofracturetechniqueforthetreatment

ofarticularcartilagelesionsintheknee.Aprospectivecohortstudy.JBoneJointSurgAm87:1911-

1920.

19. FrisbieDD,OxfordJT,SouthwoodL,etal.2003.Earlyeventsincartilagerepairaftersubchondral

bonemicrofracture.ClinOrthopRelatRes:215-227.

20. Kaul G, CucchiariniM, Remberger K, et al. 2012. Failed cartilage repair for early osteoarthritis

defects:abiochemical,histologicalandimmunohistochemicalanalysisoftherepairtissueafter

treatmentwithmarrow-stimulationtechniques.KneeSurgSportsTraumatolArthrosc20:2315-2324.

21. BrittbergM.2008.Autologouschondrocyteimplantation--techniqueandlong-termfollow-up.Injury

39Suppl1:S40-49.


122. BendersKE,BootW,CokelaereSM,etal.2014.MultipotentStromalCellsOutperformChondrocytes

onCartilage-DerivedMatrixScaffolds.Cartilage5:221-230.

23. BrittbergM.2010.Cellcarriersasthenextgenerationofcelltherapyforcartilagerepair:areview

ofthematrix-inducedautologouschondrocyteimplantationprocedure.AmJSportsMed38:1259-

1271.

24. PareekA,CareyJL,ReardonPJ,etal.2016.Long-TermOutcomesafterAutologousChondrocyte

Implantation:ASystematicReviewatMeanFollow-Upof11.4Years.Cartilage7:298-308.

25. GracitelliGC,MoraesVY,FrancioziCE,etal.2016.Surgicalinterventions(microfracture,drilling,

mosaicplasty,andallograft transplantation) for treating isolatedcartilagedefectsof theknee in

adults.CochraneDatabaseSystRev9:CD010675.

26. KnutsenG, Drogset JO, Engebretsen L, et al. 2016. A RandomizedMulticenter Trial Comparing

AutologousChondrocyteImplantationwithMicrofracture:Long-TermFollow-upat14to15Years.J

BoneJointSurgAm98:1332-1339.

27. AndradeR,VastaS,PereiraR,etal.2016.Kneedonor-sitemorbidityaftermosaicplasty-asystem-

aticreview.JExpOrthop3:31.

28. AnzAW,BapatA,MurrellWD.2016.Conceptsinregenerativemedicine:Past,present,andfuturein

articularcartilagetreatment.JClinOrthopTrauma7:137-144.

29. BigoniM,SacerdoteP,TuratiM,etal.2013.Acuteandlatechangesinintraarticularcytokinelevels

followinganteriorcruciateligamentinjury.JOrthopRes31:315-321.

30. CatterallJB,StablerTV,FlanneryCR,etal.2010.Changesinserumandsynovialfluidbiomarkers

afteracuteinjury(NCT00332254).ArthritisResTher12:R229.

31. IrieK,UchiyamaE,IwasoH.2003.Intraarticularinflammatorycytokinesinacuteanteriorcruciate

ligamentinjuredknee.Knee10:93-96.

32. SwardP,FrobellR,EnglundM,etal.2012.Cartilageandbonemarkersandinflammatorycytokines

areincreasedinsynovialfluidintheacutephaseofkneeinjury(hemarthrosis)--across-sectional

analysis.OsteoarthritisCartilage20:1302-1308.

33. Heldens GT, Blaney Davidson EN, Vitters EL, et al. 2012. Catabolic factors and osteoarthritis-

conditionedmediuminhibitchondrogenesisofhumanmesenchymalstemcells.TissueEngPartA

18:45-54.

34. WehlingN,PalmerGD,PilapilC,etal.2009.Interleukin-1betaandtumornecrosisfactoralphain-

hibitchondrogenesisbyhumanmesenchymalstemcellsthroughNF-kappaB-dependentpathways.

ArthritisRheum60:801-812.

35. YangKG,SarisDB,VerboutAJ,etal.2006.Theeffectofsynovialfluidfrominjuredkneejointsonin

vitrochondrogenesis.TissueEng12:2957-2964.

36. LieberthalJ,SambamurthyN,ScanzelloCR.2015.Inflammationinjointinjuryandpost-traumatic

osteoarthritis.OsteoarthritisCartilage23:1825-1834.

37. KohTJ,DiPietroLA.2011.Inflammationandwoundhealing:theroleofthemacrophage.ExpertRev

MolMed13:e23.

38. NegrinL,Kutscha-LissbergF,GartlehnerG,etal.2012.Clinicaloutcomeaftermicrofractureofthe

knee:ameta-analysisofbefore/after-dataofcontrolledstudies.IntOrthop36:43-50.

39. JaiswalPK,BentleyG,CarringtonRW,etal.2012.Theadverseeffectofelevatedbodymassindex

onoutcomeafterautologouschondrocyteimplantation.JBoneJointSurgBr94:1377-1381.

40. GomollAH,FarrJ,GilloglySD,etal.2010.Surgicalmanagementofarticularcartilagedefectsofthe

knee.JBoneJointSurgAm92:2470-2490.

41. BerenbaumF,GriffinTM,Liu-BryanR.2017.Review:MetabolicRegulationofInflammationinOsteo-

arthritis.ArthritisRheumatol69:9-21.

22 CHAPTER 1

42. ApovianCM,BigorniaS,MottM,etal.2008.Adiposemacrophage infiltration isassociatedwith

insulin resistance andvascular endothelial dysfunction in obese subjects.ArteriosclerThromb

VascBiol28:1654-1659.

43. GrantRW,DixitVD.2015.Adiposetissueasanimmunologicalorgan.Obesity(SilverSpring)23:512-

518.

44. OuchiN,ParkerJL,LugusJJ,etal.2011.Adipokinesininflammationandmetabolicdisease.NatRev

Immunol11:85-97.

45. Louer CR, Furman BD, Huebner JL, et al. 2012. Diet-induced obesity significantly increases the

severityofposttraumaticarthritisinmice.ArthritisRheum64:3220-3230.

46. WuCL,JainD,McNeillJN,etal.2015.Dietaryfattyacidcontentregulateswoundrepairandthe

pathogenesisofosteoarthritisfollowingjointinjury.AnnRheumDis74:2076-2083.

47. CaiJ,XuJ,WangK,etal.2015.AssociationBetweenInfrapatellarFatPadVolumeandKneeStruc-

turalChangesinPatientswithKneeOsteoarthritis.JRheumatol42:1878-1884.

48. ChuckpaiwongB,CharlesHC,KrausVB, et al. 2010.Age-associated increases in the sizeof the

infrapatellarfatpadinkneeosteoarthritisasmeasuredby3TMRI.JOrthopRes28:1149-1154.

49. BarbozaE,HudsonJ,ChangWP,etal.2017.Pro-fibroticinfrapatellarfatpadremodelingwithout

M1-macrophage polarization precedes knee osteoarthritis in diet-induced obesemice. Arthritis

Rheumatol.

50. DuranS,AksahinE,KocadalO,etal.2015.Effectsofbodymassindex,infrapatellarfatpadvolume

andageonpatellarcartilagedefect.ActaOrthopBelg81:41-46.

51. HanW,CaiS,LiuZ,etal.2014.Infrapatellarfatpadintheknee:islocalfatgoodorbadforknee

osteoarthritis?ArthritisResTher16:R145.

52. PanF,HanW,WangX,etal.2015.Alongitudinalstudyoftheassociationbetweeninfrapatellarfat

padmaximalareaandchangesinkneesymptomsandstructureinolderadults.AnnRheumDis

74:1818-1824.

53. ClockaertsS,Bastiaansen-JenniskensYM,FeijtC,etal.2012.Cytokineproductionbyinfrapatellar

fatpadcanbestimulatedbyinterleukin1betaandinhibitedbyperoxisomeproliferatoractivated

receptoralphaagonist.AnnRheumDis71:1012-1018.

54. Bastiaansen-JenniskensYM,ClockaertsS,FeijtC,etal.2012.Infrapatellarfatpadofpatientswith

end-stageosteoarthritisinhibitscatabolicmediatorsincartilage.AnnRheumDis71:288-294.

55. Klein-Wieringa IR,deLange-BrokaarBJ,YusufE,etal.2016. InflammatoryCells inPatientswith

EndstageKneeOsteoarthritis:AComparisonbetweentheSynoviumandtheInfrapatellarFatPad.

JRheumatol43:771-778.

56. Klein-WieringaIR,KloppenburgM,Bastiaansen-JenniskensYM,etal.2011.Theinfrapatellarfatpad

ofpatientswithosteoarthritishasaninflammatoryphenotype.AnnRheumDis70:851-857.

PART I

Local effects of adipose tissue

CHAPTER 2

The size of infrapatellar fat pad adipocytes is not

infl uenced by obesity

John GarciaWu Wei

Jos RunhaarKarina Wright

Gerjo J.V.M. van OschYvonne M. Bastiaansen-Jenniskens

The data presented in this chapter is accepted as part of the manuscript “Lack of high BMI-related features in adipocytes and infl ammatory cells in the infrapatellar fat pad”

by A.J. De Jong et al in Arthritis Research & Therapy

28 CHAPTER 2

ABSTRACT

Adipocytehypertrophyandtheresultingadiposetissueinflammationarekeyfea-

turesofobesity.However,itisstillunclearwhetherobesitycausestheadipocytes

intheinfrapatellarfatpad(IPFP)tocontributetojointinflammation.Theaimof

thisstudywastodeterminetheeffectofobesityonthesizeofIPFPadipocytes.

IPFPwasobtainedfromend-stageosteoarthritispatientswithameanbodymass

index(BMI)of29.7kg/m2(range21.5-48.47kg/m2).Subcutaneousfatwasobtained

from12 end-stageosteoarthritisdonorswith ameanBMIof 33.0 kg/m2 (range

24.2-48.5kg/m2).Fat tissueswerecryosectionedandstainedwithhaematoxylin

and eosin.The cross-sectional areaof adipocyteswasdeterminedusing image

analysissoftware.NorelationshipbetweenadipocytesizeandBMIwasobservedin

IPFPadipocytes(r=-0.06,p=0.82),whereassubcutaneousadipocytesizepositively

correlateswithdonorBMI(r=0.63,p=0.028).Fornon-obesedonors,sizeof IPFP

adipocyteswasnot significantlydifferent tosubcutaneousadipocytes (p>0.99).

Subcutaneous adipocytes fromobesedonorswere significantly larger than the

IPFPadipocytesfromotherobesedonors(p=0.03).Ourresultsdemonstratethat

obesity does not affect the size of adipocytes in the IPFP,which suggests that

inflammationoftheIPFPisnotinfluencedbyobesityassociatedadipocytehyper-

trophy.

The size of infrapatellar fat pad adipocytes is not influenced by obesity 29

2

INTRODUCTION

Ahighbodymassindexasaresultofexpansionofadiposetissueisawell-known

risk factor in the development of knee osteoarthritis (OA)1. During expansion,

adipocytesinadiposetissuebecomehypertrophic.Largeadipocyteshavebeen

showntoproducesubstantiallymorepro-inflammatorycytokinesandadipokines

thansmalleradipocytes2Thisincreasedsecretionofpro-inflammatorymolecules

such IL-6andtumornecrosis factoralpha(TNF-α)promotestherecruitmentof

macrophagesand theirdifferentiation intoapro-inflammatoryphenotype.This

leadstolow-gradesystemicinflammationandmetabolicchanges,bothofwhich

havebeenassociatedwithOA1.Besidesmechanicaloverloading,obesityrelated

inflammationandmetabolicchangeshavealsobeenassociatedwithOA1.

The infrapatellar fat pad (IPFP) is an adipose tissue located intracapsular yet

extra-synovial.IPFPisapotentialsourceofinflammatoryfactorsinthekneejoint3.

Duetoitsintra-articularlocationandbecauseadiposetissuesgenerallybecomes

inflamedinobesity,ithasbeenlongthoughtthattheIPFPcouldplayamajorrole

in obesity related joint inflammation.Unlike subcutaneous adipose tissue,MRI

studies have suggested that total IPFP volume is not associated to bodymass

index(BMI)4.However,theseMRIstudiesdidnotinvestigatethesusceptibilityof

theIPFPtoobesityrelatedeventsthatleadtoinflammation,suchasadipocytehy-

pertrophy.Theaimofthisstudywastoinvestigatewhetherincreasedadipocyte

size,whichcontributestoadiposetissueinflammation,isassociatedwithBMIin

theIPFP.

METHODS

Tissue sample preparation

Eighteen IPFPs were obtained from end stage OA patients with a mean body

mass index (BMI) of 29.7 kg/m2 (range 21.5-48.47 kg/m2) undergoing total knee

arthroplasty.Subcutaneousadipose tissuewasobtained fromtwelveendstage

OAdonorswithameanBMIof33.0kg/m2(range24.2-48.5kg/m2)undergoingtotal

hiporknee replacement.Consentwasgiven inaccordancewith theguidelines

oftheFederationofBiomedicalScientificSocieties(http://www.federa.org)after

approvalbythe localethicalcommittee(MEC2008-181andMEC2012-267).Do-

norsweresubdividedintoobese(BMI≥30)andnon-obese(BMI<30)foreachof

theadiposetissues(atotalof4groups).Tissuesampleswerecryosectionedand

30 CHAPTER 2

stainedwithhaematoxylinandeosin(H&E)andimagedusinganOlympusSC30

camera(Olympus,Zoeterwoude,Netherlands).

Measurement of adipocyte size

Thecross-sectionalareaoftheimagedadipocyteswascalculatedusingFijiIsJust

ImageJ softwarewith the additionalAdiposoft plugin. Three separate sections,

withaminimumof25adipocytesineachsectionweremeasuredperdonor.The

Adiposoft applicationwas calibrated to identify cellswith a diameter between

30-130µm.Ameasuringscaleof0.33µm/pixelwasalsousedbytheapplication

todetermine the cross-sectional area (size)of eachadipocyte identified in the

images.Amanualinspectionofoutputdatawasperformedtoconfirmtheconsis-

tencyofthemeasurements(Figure1).

Figure 1 IdentificationofadipocytesusingtheAdiposoftpluginforFIJI.A)Haematoxylin and eosin stainingof adipose tissue imaged at 100xmagnification.B) Imageprocessed inAdiposoft,C)Correctedimageaftermanualinspectionofprocessedimaged.Theyellowcontours(BandC)representthecross-sectionalareaofanadipocyte.Scalebar=100µm.

Statistical analysis

TheShapiro-Wilktestwasconductedtoassessthedistributionoftheadipocyte

sizesforeachdonor.Thesizeofadipocytesfromindividualdonorswasnotnor-

The size of infrapatellar fat pad adipocytes is not infl uenced by obesity 31

2

mallydistributed.Spearman’srhowasdeterminedtorelateBMItoadipocytesize

in IPFPandsubcutaneousadipose tissue.TheKruskal-Wallis testwithmultiple

comparisonswasusedtocomparethemediansizeofadipocytesfromIPFPand

subcutaneousfatfrombothnon-obeseandobesedonors.Statisticalsignificance

wasconsideredforp-values<0.05.

RESULTS

Amicroscopicobservationoftheadiposetissuesshowednodistinctdifference

betweenadipocytesintermsofgeneralmorphologyanddistributionacrossdonor

groups,howeverrelativelylargeradipocyteswerenoticeableinsubcutaneousfat

ofobesedonorscomparedtootherdonorgroups(Figure2).Thesizeofadipocytes

in subcutaneous adipose tissuewas correlated todonorBMI (r=0.63,p=0.028),

whichwasnotthecaseintheIPFP(r=-0.06,p=0.82)(Figure3A,B).Fornon-obese

donors(BMI<30), IPFPadipocytesize(1765μm2±163.5)wasnotsignificantly

differenttosubcutaneousadipocytesize(2157μm2±835.6,p>0.99).Howeverthe

sizeofadipocytesfromtheIPFPofobese(1732μm2±292.4)andnon-obesedonors

(1765μm2±163.5)weresignificantlysmallerthansubcutaneousadipocytesfrom

obesedonors(3195μm2±833.9,p=0.03andp=0.04respectively)(Figure3C).

Figure 2 RepresentativeimagesofH&EstainedcryosectionsofadiposetissuefromtheIPFPandsubcutaneousfat(SCF)ofnon-obeseandobesedonors.IPFP:infrapatellarfatpad,SCF:subcutaneousfat.Scalebar=100µm.

32 CHAPTER 2

IPFP

BMI

Adip

ocyt

eSi

ze(µ

m2 )

0 20 40 600

1000

2000

3000

4000

5000A

r=-0.06p=0.82

SCF

BMI

Adip

ocyt

eSi

ze(µ

m2 )

0 20 40 600

1000

2000

3000

4000

5000

B

r=0.63p=0.028

Adip

ocyt

eSi

ze(µ

m2 )

IPFP SCF IPFP SCF0

1000

2000

3000

4000

5000p=0.03

p=0.04C

Non-obese Obese

Figure 3 ObesitydoesnotaffectIPFPadipocytesize.Spearman’stest forcorrelationbetweendonorBMIandadipocytesize intheA) infrapatellar fatpad(IPFP)andB)subcutaneousfat(SCF).Eachdotrepresentsanindividualdonor.C)ComparisonofsizeofadipocytesbetweenIPFPandSCFinobeseandnon-obesedonor.Valuesaremean±SD

DISCUSSION

Theobesity-relatedalterationsthatoccur inadiposetissues,suchas increased

adipocyte size5,arenotwellcharacterisedintheIPFP. Inthecurrentstudy,we

haveshownthatunlikesubcutaneousadiposetissueadipocytes,thesizeofIPFP

adipocyteswasnotassociatedwithBMI.

OurobservationsinhumantissuesprovideevidencethattheIPFPadipocytesdo

notundergothemetabolicalterationsthatoftenengenderscellularhypertrophy6,

andconfirmsapreviousinvestigationthatshowedtheabsenceofhypertrophic

adipocytesintheIPFPofobesemice.7 Subcutaneous adipose tissue is considered

asbothanenergystorageandanendocrineorganthatissusceptibletoinflam-

matorymacrophageinfiltrationduringobesity.TheIPFPisapotentialcontributor

tolocaljointinflammationviatheproductionofadipokinesandcytokines3 that

exacerbatejointdegeneration.IthasbeenshownthatinflammationoftheIPFPis


2

associatedwithkneepainanddisabilityinobeseOApatients.8 Since the adipo-

cytesoftheIPFPitselfarenotaltered,atleastnotinsizeasaresultofobesity,it

couldbeinferredthattheIPFPderivedadipocytesdonotpossessthesamepro-

inflammatoryphenotypeassubcutaneousadipocytes.Hence,ourfindingscould

highlightnovelfunctionaldifferencesbetweentheIPFPandsubcutaneousadipose

tissueinobesity.PerhapstheinflammatoryroleoftheIPFPinthepathogenesis

ofobesityrelatedOAisseparatetothatofsubcutaneousfat.Thecellular,genetic

ormolecularmechanismsthatcausesthisdifferenceinsensitivitytoobesityin

theIPFPandotheradiposetissuesisnotfullyunderstoodandwarrantsfurther

investigation.

It is noteworthyhowever, that recentMRIbased studieshave associated large

IPFPwithareducednumberofabonemarrowlesions,lowwalkingpainandhigher

totalcartilagevolumeinOApatients. 9Furthermore,conditionedmediaof IPFP

adipocytesfromobeseOApatientswasfoundtohavenoeffectonthesecretion

ofTNF-αinlipopolysaccharidestimulatedmacrophagesin vitro,butinterestingly

inhibitedtheproductionofIL-12p40inthesestimulatedmacrophages.10Wehave

alsoshownthattherewerenodifferencesbetweentheamountofsecretedfactors

in conditionedmediaof IPFP fromobeseandnon-obeseOAdonors11, and that

conditionedmedia from OA IPFP explants have a chondroprotective effect on

cartilage.12Inaratmodelforageing,onlysynovialthicknessasoneofthemany

OA features was correlated with IPFP adipocyte size and this correlation was

independentofage.Thesecretionofthepro-inflammatorycytokineTNF-α from

IPFPwasassociatedwithage,butnoassociationbetweenIPFPadipocytesizeand

cytokinesreleasewasreported13.Theprogressiveage-dependantdownregulation

ofgenesrelated toanti-inflammatorymacrophages in IPFPexplants fromthese

ratssuggests thatashift towardsapro-inflammatoryphenotype is likely tobe

mediatedbyimmunecellsratherthanadipocytes.Takingthistogether,itisclear

thatfurtherinvestigationsarerequiredtoclarifywhetherandhowobesityinflu-

encestheIPFP,andifso,whattheimplicationsareforOA.

The use of unmatched IPFP and subcutaneous fat is a limitation to this study.

Assessingabiggersamplesizewithmatched IPFPandsubcutaneous fatwould

further confirm the differences in adipocyte size in the two tissues within a

particular donor. A recent study reported considerably larger adipocyte sizes

forbothIPFP(3708±976µm2)andsubcutaneousfat(6082±628µm2)compared

to our results14. This couldbe attributed to thedifference inmethodsused to

measurecellsize.Analysisofthesecretoryprofilesofindividualadipocyteswould

alsoprovidevaluable insightconcerningthe inflammatorystatusof the IPFP in

34 CHAPTER 2

obeseandnon-obesepatients. Interestingly,ourpreviousexperimentsrevealed

thatthesecretoryprofileofIPFPexplantsandthepresenceofmacrophagesinthe

IPFParenotinfluencedbydonorBMI.3,11Thisprovidessupportingevidencefor

ourhypothesisthatperhapsanincreasedBMIdoesnothavesignificantinfluence

ontheadipocytesintheIPFP.Furthermore,wedidnothavesamplesfromnon-OA

IPFPdonors.We thereforecannotexclude thepossibility that theOAcouldal-

readyhaveinfluencedtheadipocytesizeinourdonors.TheIPFPcanalsobecome

fibroticwhichcouldpossiblylimittheenlargementofadipocytes.

Inconclusion,toourknowledge,thisisthefirstevidencethatIPFPadipocytesdo

notbecomehypertrophicwithobesity.Adifferenceinthephysiologicalrolesof

theIPFPandsubcutaneousfatcouldpossiblyaccountforthedifferenceinsuscep-

tibilityoftheiradipocytestohypertrophy.Thedatapresentedhereaddstothe

growingbodyofliteratureinvestigatingthephysiologicalfunctionoftheIPFPand

itsroleinthedevelopmentOA.


2

REFERENCES

1. ThijssenE,vanCaamA,vanderKraanPM.Obesityandosteoarthritis,morethanjustwearand

tear:pivotalrolesforinflamedadiposetissueanddyslipidaemiainobesity-inducedosteoarthritis.

Rheumatology(Oxford).2015;54(4):588-600.doi:10.1093/rheumatology/keu464.

2. SkurkT,Alberti-huberC,HerderC,HaunerH.RelationshipbetweenAdipocyteSizeandAdipokine

ExHpression and Secretion. J Clin Endocrinol Metab. 2007;92(3):1023-1033. doi:10.1210/jc.2006-

1055.

3. ClockaertsS,Bastiaansen-JenniskensYM,RunhaarJ,VanOschGJVM,VanOffelJF,VerhaarJAN,et

al.Theinfrapatellarfatpadshouldbeconsideredasanactiveosteoarthriticjointtissue:anarra-

tivereview.OsteoarthritisCartilage.2010;18(7):876-882.doi:10.1016/j.joca.2010.03.014.

4. TeichtahlA,WulidasariE,BradyS,WangY,WlukaA,DingC, et al.A large infrapatellar fatpad

protectsagainstkneepainandlateraltibialcartilagevolumeloss.ArthritisResTher.2015;17:318.

doi:10.1016/j.joca.2015.02.517.

5. Hirsch J, Batchelor B. Adipose tissue cellularity in human obesity. Clin Endocrinol Metab.

1976;5(2):299-311.doi:10.1016/S0300-595X(76)80023-0.

6. DeFerranti S,MozaffarianD.Theperfect storm:Obesity, adipocytedysfunction,andmetabolic

consequences.ClinChem.2008;54(6):945-955.doi:10.1373/clinchem.2007.100156.

7. ChangW,DeMoeJ,KentC,KovatsS,GarteiserP,DoblasS,etal.Infrapatellarfatpadhypertrophy

without inflammation in a diet-inducedmousemodel of obesity and osteoarthritis. Osteoarthr

Cartil.2011;19:66.doi:10.1016/S1063-4584(11)60157-X.

8. BallegaardC,RiisRGC,BliddalH,ChristensenR,HenriksenM,BartelsEM,etal.Kneepainandin-

flammationintheinfrapatellarfatpadestimatedbyconventionalanddynamiccontrast-enhanced

magneticresonanceimaginginobesepatientswithosteoarthritis:Across-sectionalstudy.Osteo-

arthrCartil.2014;22(7):933-940.doi:10.1016/j.joca.2014.04.018.

9. HanW,CaiS,LiuZ,JinX,WangX,AntonyB,etal.Infrapatellarfatpadintheknee:islocalfatgood

orbadforkneeosteoarthritis?ArthritisResTher.2014;16(4):R145.doi:10.1186/ar4607.

10. Klein-WieringaIR,AndersenSN,KwekkeboomJC,GieraM,deLange-BrokaarBJE,vanOschGJVM,

etal.Adipocytesmodulatethephenotypeofhumanmacrophagesthroughsecretedlipids.J Im-

munol.2013;191(3):1356-1363.doi:10.4049/jimmunol.1203074.

11. ClockaertsS,Bastiaansen-JenniskensYM,FeijtC,DeClerckL,VerhaarJAN,ZuurmondA-M,etal.

Cytokine production by infrapatellar fat pad can be stimulated by interleukin 1β and inhibited

by peroxisome proliferator activated receptorα agonist. Ann RheumDis. 2012;71(6):1012-1018.

doi:10.1136/annrheumdis-2011-200688.

12. Bastiaansen-JenniskensYM,ClockaertsS,FeijtC,ZuurmondA-M,Stojanovic-SusulicV,BridtsC,

etal.Infrapatellarfatpadofpatientswithend-stageosteoarthritisinhibitscatabolicmediatorsin

cartilage.AnnRheumDis.2012;71:288-294.doi:10.1136/ard.2011.153858.

13. FuY,Huebner JL, KrausVB,GriffinTM. Effect of Aging onAdiposeTissue Inflammation in the

KneeJointsofF344BNRats.JournalsGerontolSerABiolSciMedSci.2015;00(00):1-11.doi:10.1093/

gerona/glv151.

14. MacchiV,PorzionatoA,SarasinG,PetrelliL,GuidolinD,RossatoM,etal.TheInfrapatellarAdipose

Body:AHistotopographicStudy.CellsTissuesOrgans.2016.doi:10.1159/000442876.

CHAPTER 3

Stimulation of fi brotic processes by the infrapatellar

fat pad in cultured synoviocytes from patients

with osteoarthritis: a possible role for prostaglandin F2α

Yvonne M Bastiaansen-Jenniskens Wu Wei

Carola Feijt Jan H Waarsing Jan AN Verhaar

Anne-Marie Zuurmond Roeland Hanemaaijer

Reinout Stoop Gerjo JVM van Osch

Arthritis Rheum. 2013 Aug;65(8):2070-80

38 CHAPTER 3

ABSTRACT

Objective

Stiffeningofthejointisafeatureofkneeosteoarthritis(OA)thatcanbecaused

byfibrosisofthesynovium.Theinfrapatellarfatpad(IPFP)presentintheknee

jointproducesimmune-modulatoryandangiogenicfactors.Thegoalofthepres-

entstudywastoinvestigatewhethertheIPFPcaninfluencefibroticprocessesin

synovialfibroblasts, and todetermine the roleof transforminggrowth factorβ

(TGFβ)andprostaglandinF2α(PGF2α)intheseprocesses.

Methods

Batchesoffat-conditionedmedium(FCM)weremadebyculturingpiecesofIPFP

obtainedfromthekneesof13patientswithOA.HumanOAfibroblast-likesynovio-

cytes(FLS)(frompassage3)wereculturedinFCMwithorwithoutinhibitorsof

TGFβ/activinreceptor–likekinase5orPGF2αfor4days.TheFLSwereanalyzed

for production of collagen and expression of the gene for procollagen-lysine,2-

oxoglutarate5-dioxygenase2(PLOD2;encodinglysylhydroxylase2b,anenzyme

involvedincollagencrosslinking)aswellasthegenesencodingα-smoothmuscle-

actin and type I collagen α1chain.Inparallel,proliferationandmigrationofthe

synoviocyteswereanalyzed.

Results

CollagenproductionandPLOD2geneexpressionbytheFLSwereincreased1.8-

fold(p<0.05)and6.0-fold(p<0.01),respectively,inthepresenceofFCM,relative

tocontrolcultureswithoutFCM.Moreover,themigrationandproliferationofsyn-

oviocyteswerestimulatedbyFCM.Collagenproductionwaspositivelyassociated

withPGF2αlevelsintheFCM(r=0.89,p<0.05),andinhibitionofPGF2αlevelsreduced

theextentofFCM-inducedcollagenproductionandPLOD2expression.Inhibition

ofTGFβsignalinghadnoeffectonthepro-fibroticchanges.

Conclusion

TheseresultsindicatethattheIPFPcancontributetothedevelopmentofsynovial

fibrosis in theknee jointby increasingcollagenproduction,PLOD2expression,

cell proliferation, and cellmigration. In addition,whereas the findings showed

thatTGFβisnotinvolved,themorerecentlydiscoveredpro-fibroticfactorPGF2α

appearstobepartiallyinvolvedintheregulationofpro-fibroticchanges.

Infrapatellar fat pad and synovial fibrosis 39

3

INTRODUCTION

Osteoarthritis(OA)isamultifactorialdiseaseofthearticularjoints,withaninci-

dencethatishigherinwomen,obesesubjects,andolderindividuals1;2.Therole

ofthesynoviuminOApathologyisbecomingmoreevident.Inconjunctionwith

cartilagedamageandbonealterations,thepathologic featuresof inflammation,

hyperplasia,andextensivefibrosisarealsooftenobservedinthesynoviumofOA

joints3-5.

Fibrosis can be seen as an abnormal healing process that is characterized

byexcessivedepositionof extracellularmatrixproteins, inparticular collagen,

which,inturn,resultsinalterationofthestructureofthetissueand,finally,even

lossof functionof this tissue.Fibroticprocessesarearesponse toavarietyof

insults,suchasinfection,trauma,autoimmunity,orinflammation,resultinginan

inflammatoryreactionwithrapidrecruitmentofmonocytesfromthecirculation

ormacrophages resident in the tissue.Thesecells are important sourcesof fi-

broticcytokines,suchastransforminggrowthfactorβ(TGFβ)andplatelet-derivedgrowthfactor,that,inturn,recruitfibroblaststothesiteofinjuryandstimulate

themtoproliferate6.

Fibroblastscanalsodifferentiate towardmyofibroblasts.Myofibroblasts,which

are characterized by the presence of α-smoothmuscleactin,arepresentduring

normaltissuerepair,butwhenthewoundisclosed,theydisappearfromthesite.In

thecaseoffibrosis,myofibroblastspersistinthedamagedtissue7-9.Anadditional

hallmarkoffibrosisistheincreasedlevelofhydroxyallysinecollagencrosslinks,

which is regulatedby lysylhydroxylase2b (LH2b), anenzymeencodedby the

gene for procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2)10. These

hydroxyallysinecollagencrosslinksare found infibrotic tissuesandareassoci-

atedwithirreversibleaccumulationofcollagen11.Recently,itwasdemonstrated

thatPLOD2expressionandthepresenceofLH2bareup-regulatedinthefibrotic

synoviumofmicewithOA12.

TGFβisapotentinducerofPLOD2expressionandLH2bactivity12;13,makingTGFβ

averypotentfibroticgrowthfactorinvolvedinalloftheprocessesseeninfibro-

sis. Inaddition toTGFβ,prostaglandinF2α (PGF2α)wasrecentlydiscoveredasa

cytokinethatfacilitatespulmonaryfibrosisindependentofTGFβ.Micelackingthe

PGF2αreceptordidnotdeveloppulmonaryfibrosisinresponsetobleomycin,and

cellsstimulatedwithPGF2αproducedmorecollagen14;15.

40 CHAPTER 3

Inadditiontocartilage,menisci,ligaments,andsynovium,thekneejointcontains

fatpads.Oneofthelargestofthefatpadsistheinfrapatellarfatpad(IPFP).The

mainroleofthe IPFPisto facilitatedistributionofsynovialfluidanddistribute

mechanicalforcesthroughthekneejoint.Severaladipokinesandcytokines,such

astumornecrosisfactorα,interleukin-6(IL-6),IL-10,leptin,andvascularendothe-

lialgrowthfactor,areknowntobeproducedbytheIPFP16-20.TheIPFPislocated

incloseproximitytothesynoviallayersandcartilagesurfaces,makingtheIPFP

abletoinfluenceinflammatoryprocessesintheknee21.Manysecretedproteinsare

derivedfromthenon-adipocytefractionofadiposetissue(macrophages,Tcells,

andBcells)18;22,andit issuggestedthatmostofthecytokinesproducedbythe

adiposetissuearemacrophage-derived23;24.

Sincesynovialfibrosisisoftenseeninend-stageOA,thegoalofthepresentstudy

was to investigate whether the IPFP and its secreted factors influence fibrotic

processesinsynovialfibroblasts,andtodeterminetheroleof2potentfibroticin-

ducers,TGFβandPGF2α,intherelationshipbetweentheIPFPandsynovialfibrosis.

MATERIALS AND METHODS

Preparation and analysis of fat-conditioned medium

Samples of IPFP were derived from anonymized leftover knee tissue material

obtainedfrompatientswithOAwhohadundergonetotalkneearthroplasty.The

IPFP sampleswereused toproduce FCM.Thepatients implicitly consented to

theuseofthesetissuesforscientificresearch,inaccordancewiththeguidelines

oftheFederationofBiomedicalScientificSocieties(http://www.federa.org),with

approval from the local ethics committee in Rotterdam, The Netherlands (ap-

provalno.MEC2008-181).Themeanageofthedonorswas67.9years(range54–81

years)andthemeanbodymassindex(BMI)ofthedonorswas30.52kg/m2(range

19.6–44.5kg/m2).Theinnerpartsofthefatpads,wherenosynoviumispresent,

werecutintosmallpiecesof±10mgandculturedinsuspensionfor24hoursina

concentrationof50mgtissue/mlinDulbecco’smodifiedEagle’smedium(DMEM)

withGlutamax(GibcoBRL),containinginsulin,transferrin,selenicacid,andalbu-

min(ITS+)(dilution1:100;BDBiosciences)aswellas50µg/mlgentamicinand1.5

µg/mlFungizone(bothfromGibcoBRL).Asacontrolmedium,weusedidentically

composedculturemediumthatdidnotcontainpiecesofIPFP,culturedinparallel.

After24hours,themediumwasharvested,centrifugedat300gfor8minutesto

remove(immune)cells,andfrozenat -80°Cinaliquotsof1.5ml,resultingin29


3

differentbatchesofFCM.Thisincubationtimewaschosenarbitrarily,sinceeach

mediatorhasitsownoptimumregardingreleasekinetics22;25-27.

Isolation and culture of fibroblast like synoviocytes (FLS)

Samples of human synoviumwere also obtained as anonymized leftovermate-

rialfrompatientswithOAwhohadundergonetotalkneearthroplasty(approval

no.MEC2004-322).Onthebasisofitsspecificstructure,thesynoviumcouldbe

distinguishedandremovedfromtheadjacenttissue.Thesynoviumsampleswere

thendigestedinPronase(2mg/ml;Sigma)for2hoursandincollagenaseB(1.5

mg/ml;RocheDiagnostics)overnight.Digestedcellswereplatedoutas3,500cells

per cm2 and expanded in Iscove’smodifiedDulbecco’smediumwith 10% fetal

calfserum(FCS),50µg/mlgentamicin,and1.5µg/mlFungizone(all fromGibco

BRL).Cells frompassage 3were allowed to adhere at adensityof 50,000 cells

percm2inDMEMwithGlutamax,10%FCS,andantibiotics(allfromGibcoBRL).

Afterovernightattachment,theculturemediumwasremovedandthecellswere

washedcarefully3timeswithsaline.FCMwasmixed1:1withfreshDMEMwith

Glutamax,supplementedwith50µg/mlgentamicin,1.5µg/mlFungizone,andITS+

(dilution1:100),andappliedtotheattachedFLS.Thereafter,theFLSwerecultured

for4days.ThemeanageoftheFLSdonorswas67years(range60–81years)and

themeanBMIwas32.4kg/m2(range24.2–42.5kg/m2).

ToinvestigatetheinvolvementofTGFβ1orPGF2α,1µMSB505124(Sigma),an

inhibitorofTGFβ1signaling,or10µMAL8810(CaymanChemical),aninhibitorof

PGF2αsignaling,wasadded1hourpriortoaddingtheFCM;aspositivecontrolfor

theinhibition,1ng/mlTGFβ1or1µMPGF2αwasadded.Concentrationsandtiming

werebasedonthosepreviouslydescribedinastudybyOgaetal14.

Analysis of collagen deposition

CollagenwasdeterminedusingtheQuickzymesolublecollagenassayaccordingto

themanufacturer’sguidelines(QuickZymeBiosciences,Leiden,theNetherlands).

Briefly,culturemediumwasremovedandthecell/matrixfractionwassolubilized

byovernightincubationin0.5Maceticacidat4°C.Theassayisbasedonbinding

ofcollagenwithSiriusRed.

Analysis of gene expression

After culture,monolayers of synoviocyteswere suspended in 350µl RLT buffer

(Qiagen,Venlo,theNetherlands)supplementedwith1%β-mercaptoethanol.RNA

wasextractedandcomplementaryDNAwasanalyzedforgeneexpressionusing

previously described methods28. The primer sequences for the genes were as

follows: for the GAPDH reference gene, forward GTCAACGGATTTGGTCGTATT-

42 CHAPTER 3

GGG, reverse TGCCATGGGTGGAATCATATTGG, and probe FAM-CGCCCAATAC-

GACCAAATCCGTTGAC-TAMRA;forthetypeIcollagenα1chaingene(COL1A1),

forward CAGCCGCTTCACCTACAGC, reverse TTTTGTATTCAATCACTGTCTTGCC,

and probe FAM-CCGGTGTGACTCGTGCAGCCATC-TAMRA; for PLOD2, forward

CCCTCCGATCAGAGATGATTandreverseAATGTTTCCGGAGTAGGGGAGTCTTTTT;

and for the gene encoding α-smoothmuscleactin(ASMA),forwardCGTTGCCCCT-

GAAGAGCATandreverseCCGCCTGGATAGCCACATACA.PrimersforthetypeIIIcol-

lagengene(COL3)werepurchasedfromQiagenAssays-on-Demand(QT00058233).

For analysis of GAPDH and COL1A1, TaqMan 2x Universal Polymerase Chain

Reaction(PCR)MasterMix(AppliedBiosystems)wasused in thereaction.For

analysis of PLOD2 and ASMA, quantitative PCRMasterMix Plus SYBRGreen I

(Eurogentec)wasusedinthereaction.Indeterminingtheoptimalhousekeeping

gene,wecomparedGAPDH,18SRNA,2-microglobulin,andhypoxanthineguanine

phosphoribosyltransferase,andobservedthatGAPDHwasthemoststablehouse-

keepinggeneinourexperiments.

Migration assay

Toinvestigatethemigrationofthesynoviocytesinresponsetosolublefactors,a

scratchwoundassaywasperformed.Synoviocytesfrompassage3wereseededat

100,000cellspercm2in12-wellplatesandallowedtoadhereovernightinDMEM

containing10%FCS.A10-µlpipettetipwasusedtomakeascratchintheconfluent

monolayerofthesynoviocytes,aftermarkingthescratchlocationonthebottom

of the well. When applicable, SB505124 or AL8810 was added 1 hour prior to

makingthescratch.Celldebriswasremovedbywashingwithsaline,andthecell

culturewascontinued inDMEM–1%ITS+mixedwithFCM(1:1)withorwithout

SB505124orAL8810.

Photographic imageswereobtaineddirectlyafterscratchingandat14,16,and

19hoursafter scratching.These timepointswerechosenon thebasisofpilot

experimentsshowingthat,from14hoursonward,migrationofthesynoviocytes

isbestvisualized(resultsnotshown)without interferencefromproliferationof

thecells.ClosurewasmeasuredusingTScratchsoftware(ComputationalScience

&EngineeringLaboratory).Theextentofmigrationispresentedasthepercentage

ofclosureafterwounding.

Proliferation assay

Toanalyzeproliferation,synoviocytesfrompassage3wereseededatadensityof

10,000cellspercm2in12-wellplatesandallowedtoadhereovernightinDMEM

containing10%FCS.ToarrestcellsintheS1phase(whichwasnecessaryinorder


3

toreducevariation),cellswereculturedinDMEMcontaining0.1%FCSfor24hours

afteradherence.Following24hoursofstarvation, theculturewascontinued in

DMEM–1%ITS+mixedwithFCM(1:1)withorwithouttheadditionofSB505124or

AL8810.Whenapplicable,SB505124orAL8810wasadded1hourpriortoadding

theFCM.SamplesforDNAmeasurementwereobtainedafter1,2,3,and4days

bysuspendingthemonolayerinphosphatebufferedsalinewith0.1%Triton.The

amountofDNAineachsamplewasdeterminedusingethidiumbromide,withcalf

thymusDNA(Sigma)asstandard.

Enzyme-linked immunosorbent assay (ELISA) for TGFβ1

TodeterminetheactivityofTGFβ1intheFCM,ahumanTGFβ1QuantikineELISA

kit(R&DSystems)wasusedaccordingtothemanufacturer’sguidelines.Toacti-

vatelatentTGFβ1totheimmunoreactiveform,acidactivationwith1NHCland

neutralizationwith1.2NNaOH/0.5MHEPESwasperformed.

PGF2α measurements using mass spectrometry

TodeterminethelevelsofPGF2αintheFCMsamplesthatremained(n=7),samples

wereanalyzedwith liquidchromatographytandemmassspectrometry(LC-MS/

MS)asdescribedpreviously29.Briefly,topreparethesamplesformeasurement,

FCM samples,with deuteratedPGF2α -d4 (CaymanChemical) added as internal

standard, were extracted with LC-MS–grade methanol (Riedel-de-Ha¨en). The

methanol extract was loaded on an HLB SPE column (Oasis), after which the

sampleswerereconstitutedin100µlethanolcontainingCUDA(CaymanChemi-

cal)asasecondinternalstandard,andimmediatelyusedforLC-MS/MSanalysis

onanAcquityC18BEHUltraPerformanceliquidchromatographycolumncoupled

toaXevoTQ-Smassspectrometer (Waters).Conevoltageandcollisionenergy

wereoptimizedforeachcompoundindividually.Parentandproductmass/charge

(m/z) values of PGF2α were 353.1 and 193.0. Parent and productm/z values of

PGF2α-d4were357.1and313.4.Parentandproductm/zvaluesofCUDAwere339.1

and214.1.Identificationandquantificationofpeakvalueswereperformedusing

MassLynxsoftwareversion4.1.


Experimentsexamining theeffectof FCMwereperformedwith samples from3

differentFLSdonorsand13differentFCMbatchesrepresenting13differentIPFP

donors.ExperimentsexaminingtheeffectofinhibitionofTGFβ1orPGF2α together

withFCMstimulationwereperformedwithsamplesfrom2differentFLSdonors

and8differentFCMbatchesrepresenting8differentIPFPdonors.Allexperiments

wereperformedwithtriplicatesamplespercondition,whichwastakenintoac-

44 CHAPTER 3

countinthestatisticalanalysis.Amixedlinearmodel,followedbyaBonferroni

posthoctest,wasusedtoanalyzegeneexpression,collagendeposition,andcell

migration.Aunivariategenerallinearmodelwasusedtoanalyzetheresultsfrom

theproliferationassays.SincenoteveryFCMbatchwastestedonFLSfromevery

donor,weallowedforthisinthestatisticalanalysisbyaddingasubjectvariable

indicatingtheFLSdonor.Spearman’srhocorrelationsweredeterminedtoexam-

ineassociationsbetweenTGFβ1orPGF2αlevelsandotherparameters.Datawere

analyzedwithIBMSPSSstatisticalsoftware(version20.0).

RESULTS

Induction of fibrotic processes with medium conditioned

by IPFP

Incultureconditionswith8ofthe13FCMbatches(derivedfrom13differentOA

IPFPdonors),collagenproductionbyFLSwas increased1.8-foldafter4daysof

culture, fromamean1.9µg/monolayer in control conditionswithout FCM toa

mean3.4µg/monolayer incultureswithFCM(Figure1A [all 13 included in the

figure]).Inaddition,geneexpressionoftheenzymeinvolvedintheformationof

pyridinolinebasedcollagencrosslinks,PLOD2,wasincreased6.0-foldinthepres-

enceofFCM.Surprisingly,COL1A1expressiononday4was2.5-fold lowerthan

Figure 1 The effect of fat conditionedmedium (FCM)onA) total collagenprodutionby FLSsinmonolayerafter4daysofcultureandB)geneexpressionafter4daysofculturerelativetothecontrolwithoutFCM(setat1)ofgenesdescribingafibroticprocess:Col1(coll1,α1chain),procollagen-lysine,2-oxoglutarate5-dioxygenase2(Plod2)andαsmoothmuscleactin(Asma).Atotalof13differentFCMbatcheswastestedonFLSofthreedifferentdonors.Barsrepresentthemean±standarddeviation.Box-whiskerplotsindicatethe75thand25thpercentilewiththemedian(boxes)andthemaximumandtheminimum(whiskers).*indicatesp<0.05,**indicatesp<0.01.


3

thatincontrolconditionswithoutFCM,andASMAexpressionwas1.7-foldlower

(Figure1B).COL3expressionwasunalteredbytheadditionofFCM(resultsnot

shown).Theobservedincrease incollagenproductionbutdecrease inCOL1A1

expressionmightbeexplainedbythehypothesisthatalteredprocessingofthe

collagenwouldleadtomoreefficienttranslation,butwouldnotalterexpression

ofCOL3.Also,thecollagendepositionrepresentsaccumulationinthetotalculture

for4days,whereasthecollagengeneexpressionisthespecificexpressionatthe

momentofharvest.

Since TGFβ1 is known to be a potent inducer of collagen deposition and of

COL1A1,PLOD2,andASMAexpression,wealsoincluded,asapositivecontrola

cultureconditioninwhichTGFβ1wasaddedinalloftheexperiments.Indeed,irre-

spectiveofwhichFLSdonorwasused,TGFβ1inducedthesamefibroticprocesses

asseeninFLScultureswithFCM(resultsnotshown).

To investigatewhether theBMIof the IPFPdonor influenced theeffectof FCM

onFLSs,weperformedapost hocsubgroupanalysiscomparingtheFCMbatches

madefromIPFPsofdonorswhohadaBMIlowerthan30kg/m2(n=5)withFCM

batchesmadefromIPFPsofdonorswhohadaBMIequaltoorhigherthan30kg/

m2(n=8).NodifferencesincollagendepositionorexpressionofCOL1A1,PLOD2,

orASMAwereobservedbetweenthe2BMIsubgroups.Bothgroupsstillshowed

significantdifferencesingeneexpressionwhencomparedwiththecontrolcondi-

tionwithoutFCM(Figure2).

TodeterminetheeffectsonFLSmigration,weperformedascratchwoundassay

(typicalexamplesrightafterscratchingand19hoursafterscratchingareshown

in Figures3AandB).MigrationofFLS(p=0.005)andproliferationofFLS(p=0.002)

werestimulatedinthepresenceofFCM(Figures3CandD).

Association of FCM effects with the presence of TGFβ1 or PGF2α.

TGFβ1andPGF2αarebothknownaspotentpro-fibroticmediatorsandcandidates

tobe involved in theprocessesseen in theFLS inresponse toFCM.Themean

TGFβ1contentoftheFCMwas37.3pg/ml,rangingfrom0.1pg/mlto74.9pg/ml.

ThemeanPGF2αcontentintheFCMwas6,204pg/ml,rangingfrom560pg/mlto

29,718pg/ml.ThelevelofPGF2αwaspositivelycorrelatedwiththeextentofcol-

lagendepositionbytheFLS(Figure4A)andnegativelyassociatedwithCOL1A1

expression(Figure4B).

46 CHAPTER 3

Figure 2 TheeffectofFCMsubdived innonobese(BMI<30,n=5,1maleand4 female)andobese(n≥30,n8,1maleand7female)donorsonA)collagendepositionB)collagentype1geneexpression,C)Plod2geneexpression,andD)Asmageneexpressionbysynoviocytes.ThedottedlineindicatesthelevelsinthecontrolconditionwithoutFCM.**indicatessignificantlydifferentfrom control conditionwithp<0.01, ***indicates significantly different from control conditionwith p<0.005.Barsrepresentthemean±standarddeviation.

ThelevelofTGFβ1waspositivelyassociatedwithCOL1A1expression(Figure

4C).Nootherassociationswereseen.

To evaluate the effect of TGFβ1 and PGF2α on FLS in our culture system,we

addedthesecompoundstoFLScultureswithoutthepresenceofFCM.Theaddi-

tionof1ng/mlTGFβ1increasedtotalcollagendeposition,COL1A1expression,and

ASMAexpression.Theadditionof1µMPGF2α increasedPLOD2expressionand

totalcollagendeposition(Figures5AandD-F).TGFβ1andPGF2α both increased

themigrationof theFLS,buthadnoeffectonproliferationof theFLS (Figures

5BandC).TheeffectsofPGF2αbestsimulatedtheeffectsofFCMasseeninour

experiments(asdescribedinFigures1and2).


3

Counteraction of the pro-fibrotic effect of FCM via inhibition of PGF2α signaling.

ToexaminetheinvolvementofTGFβ1andPGF2αinthepro-fibroticeffectofFCM,

theFLSwereculturedwithFCMwithorwithoutaTGFβ1receptortypeIkinaseinhibitor, SB505124, or a selective PGF receptor antagonist, AL8810. First, we

verifiedwhetherSB505124couldindeedinhibittheeffectof1ng/mlTGFβ1,andwhetherAL8810couldindeedinhibittheeffectof1µMPGF2α.SB505124blocked

Figure 3 TheeffectofFCMonmigrationandproliferationofFLSdeterminedbyascratch-woundassay.(A)Picturesofthescratchweretakendirectlyaftermakingthescratchand(B)aftermigrationfor19hours.ThelinesatthetopofAandBmarkthebottomofthewellasreferencepointfortakingpicturesduringmigra-tion.TheeffectofFCMon(C)migrationaspercentageopenand(D)proliferationasµgDNApermonolayerwastested.Atotalof9differentFCMbatcheswastestedonFLSofthreedifferentdonors.*indicatesp<0.05regard-ingthetotalmigrationandproliferationcurves.Dataareshownasmeanmigrationorproliferation±standarddeviationof9FCMbatches.

Figure 4 AssociationsbetweenconcentrationofTGFβ1andPGF2αintheFCMandtheeffectofFCMontheFLSinculture.A)apositiveassociationbetweenthelevelofPGF2αintheFCMandcollagendepositionbytheFLS(R=0.89,p=0.028)B)anegativeassociationbetweenPGF2αintheFCMandCol1geneexpression(R=-0.82,p=0.023),andC)apositiveassociationbetweenTGFβ1intheFCMandCol1geneexpression(R=0.77,p=0.012).Thep-valueswerecorrectedformultipletesting.

48 CHAPTER 3

theeffectofTGFβ1oncollagenproduction,COL1A1expression,andASMAexpres-

sion,confirmingtheefficacyoftheinhibitor.AL8810inhibitedtheeffectofPGF2α

oncollagenproductionandtherewasatrendtowardnormalizationofPLOD2

expression(p=0.08)(Figures5AandD–F).Thepresenceof1µMSB505124or10

µMAL8810aloneseemedtohavenoinfluenceoncollagendepositionorCOL1A1,

PLOD2,andASMAexpression(Supplementaryfigure1).

Inhibition of TGFβ/activin receptor–like kinase 5 signalingwith SB505124 did

notalter theFCM-inducedeffectsonFLS, indicating that theeffectofFCMwas

notcausedbyTGFβ.BlockingthePGFreceptorwithAL8810,ontheotherhand,

inhibitedtheincreaseincollagendepositionthathadbeeninducedbyFCM,bring-

ingthecollagendepositionbacktothelevelsseenincontrolconditionswithout

FCM(Figure6A).TheincreaseinPLOD2expressioninducedbyFCMwassimilarly

abrogated,with a return to the levels seen in control conditionswithout FCM,

whentheFLSwereco-incubatedwithFCMandAL8810(Figure6E).

Figure 5 Theeffectof1ng/mlTGFβ1and1µMPGF2αandtheirinhibitorsSB505124andAL8810intheabsenceofFCMonA)collagenproduction,B)migration,andC)proliferationandgeneexpressionofC)Col1α1,D)Plod2andE)AsmabyFLSsrelativetothecontrolwithoutthesead-ditions(setat1).AllanalysesweredoneintriplicatewiththreeFLSdonors.*indicatesp<0.05,**indicatesp<0.01.Barsordotsrepresentthemean±standarddeviation.


3

TheeffectsofFCMonthemigrationandproliferationofsynoviocytesandon

thelevelofASMAexpressionwerenotcounteractedbytheadditionofthePGF

receptorinhibitorAL8810(Figures6B,C,andF).COL1A1expression,whichhad

beendecreasedinFLScultureswithFCM,wasdecreasedevenmoreincultures

withAL8810(Figure6D).ThisisconsistentwiththeminimaldecreaseinCOL1A1

expressionthatwasobservedwhenonlyAL8810wasaddedtotheFLScultures

(Figure5D).

DISCUSSION

OAisadiseaseofthearticularjointsinwhichsynovialfibrosisisoftenseen3,4,12.

Accumulatingdatahavebeenpresented tosuggest thatOA isan inflammatory

Figure 6 TheeffectoftheTGFβ/ALK5(SB505124)orPGF2αsignalling(AL8810)inhibitionontheeffectofFCMinFLSculturesonA)collagenproduction,B)migrationandC)proliferationandD)Col1α1geneexpression,E)Plod2geneexpressionandF)Asmageneexpressionbysynoviocytes.Atotalof8differentFCMbatcheswastestedusingFLSoftwodifferentdonors.Thecontrolwith-outFCMwassetat1andindicatedwithadottedlineforthegeneexpressionanalyses.*indicatesp<0.05,***indicatesp<0.005.Barsordotsrepresentthemean±standarddeviationof8differentFCMbatches.

50 CHAPTER 3

disease inwhichcytokinesand immunecellsplaya role30.Adipose tissuecan,

ingeneral,beconsideredtobeanendocrineorganthatsecretescytokinesand

growthfactorsandthatexhibitssignificantinfiltrationofimmunecells,including

macrophages31-33.Inearlierstudiesconductedbyourgroupandotherinvestiga-

tors, itwas shown that the IPFP is able toproduce cytokines, adipokines, and

growthfactors,andtherebycontributestotheirlevelsinthesynovialfluid18-20;34.

In the current study, we demonstrate thatmedium conditioned by samples of

IPFPobtained from the joints of patientswith end-stageOA stimulates fibrotic

processesinFLS.

Culturing theFLSwithFCM increasedcollagenproduction, theexpressionof

PLOD2encoding for the enzymeLH2b (involved inpyridinoline-basedcollagen

crosslinks),andthemigrationandproliferationofFLS,whicharehallmarksofa

fibrotic process11;13;35.TheseeffectswereindependentoftheBMIoftheIPFPdonor

(BMI<30kg/m2versusBMI>30kg/m2).

TGFβ1,andmorerecently,PGF2α14,havebeensuggested toactaspro-fibrotic

factorsinthejoints.WefoundthatbothTGFβ1andPGF2αwerepresentintheFCM

batchesusedforculturewiththeFLS;thisfindingisinadditiontothepreviously

describedpresenceofmanyother cytokines, adipokines, and growth factors17.

WhenwecomparedthelevelsofTGFβ1andPGF2αintheFCMwithourfunctional

parameters,we foundapositiveassociationbetweenPGF2α levelsandcollagen

deposition,anegativeassociationbetweenPGF2αlevelsandCOL1A1expression,

andapositiveassociationbetweenTGFβ1levelsandCOL1A1expression.These

associations indicate that PGF2αwas responsible for someof the effects of the

FCM.The absenceof associationsbetweenPGF2α levels andPLOD2expression

andbetweenTGFβ1levelsandASMAexpressionmightbeexplainedbythefact

thattheFCMcontains,inadditiontoPGF2αandTGFβ1,manyotherunknownfac-

torsthatcouldalsohaveinfluencedthefibroticprocessesinFLS.

Furthermore,ourexperimentsindicatethattheeffectsofFCMonFLSarecom-

parabletotheeffectsofaddingPGF2αtoFLScultureswithoutFCM,againindicat-

ingthatthepresenceofPGF2αcontributestotheFCMeffect.Theprofibroticeffect

ofFCMmaybeattributablenotonlytothePGF2αpresentintheFCM,butalsoto

thePGF2αthatisproducedbyFLSinresponsetoFCM.Fibroblasts,ingeneral,are

knowntoproducePGF2α36;37.Thismayexplainthediscrepancybetweenourfind-

ingsofPGF2αincreasingPLOD2expressionandAL8810bringingPLOD2expression

backtocontrol levelsandourfindingsoftheabsenceofacorrelationbetween

PGF2αlevelsintheFCMandPLOD2expression.

MessengerRNA(mRNA)andproteinlevelsare,ingeneral,associatedwitheach

other.ThiswastruefortheCOL1A1mRNAandproteinlevelsinthisstudy,when

we cultured the FLSwith TGFβ1.However, collagen deposition is regulated on


3

manylevels,anditsregulationthroughvariationintheamountofmRNAisonly

thebeginning.Forexample,aftersynthesisofthedifferentcollagenchains,post-

translationalmodificationthroughenzymessuchasthelysylandprolylhydroxy-

lasesandlysyloxidasescanoccur,whilecorrectfoldingofthecollagenmolecules

requirestheinvolvementofchaperonessuchasHsp47.Thesechangesnotonly

aredirectlyinvolvedincollagensynthesisbutalsocanindirectlyregulatecollagen

content.Thelevelofcollagencrosslinking,forexample,canhaveaneffectonthe

sensitivityofcollagentodegradationbymatrixmetalloproteinases38.Ofcourse,

degradationofcollagencanhaveamajorroleindeterminingtowhatextentcol-

lagencontentincreasesovertime.Inthisrespect, it isveryexcitingtoseethat

despiteareductionintypeIcollagenmRNA,thepresenceofFCMorPGF2α does

result in increasedcollagendeposition, and that therearedifferencesbetween

stimulationwithPGF2α andstimulationwithTGFβ1.Unfortunately,wewerenot

abletoquantifydepositionofspecifictypesofcollagen.

TofurtherexaminetheinvolvementofTGFβ1andPGF2αpresentintheFCMin

thedifferentfibroticprocesses,weusedaTGFβ1receptortypeIkinaseinhibitor,SB505124, anda selectivePGF2α receptorantagonist,AL8810, togetherwith the

FCMincubation.BlockadeofPGF2αwithAL8810broughtcollagendepositionand

PLOD2expressionbacktothelevelsincontrolconditionswithoutFCM,whereas

thepresenceoftheTGFβ1inhibitorSB505124didnotaltertheFCMeffectonFLS.

Since theadditionofAL8810decreasedPLOD2expression,our results indicate

indirectly that PGF2α levels are associated with PLOD2 expression, in addition

to the already-shown association between PGF2α and collagen production. The

latterisconfirmedbythefactthatinhibitionofthePGF2αreceptorwithAL8810

normalizedcollagendepositioninFCM-treatedFLS.InhibitionofTGFβ1signalingwithSB505124incombinationwithFCMdidnotnormalizecollagendepositionor

PLOD2expression.Fromtheseresults,weconcludethatPGF2αmightbeamore

importantfactorthanTGFβ1intheFCM-inducedfibroticprocessesinFLS.

NoeffectoftheinhibitorswasseenontheFCM-inducedmigrationandprolifera-

tionofsynoviocytes,andco-incubationofFCMwithAL8810decreasedCOL1A1

expressionevenmorethanthatwithFCMalone.Thus,nexttoPGF2α,otherfactors

alsoinfluencedtheparametersoffibrosis,sincenotallprocessesinducedbyFCM

werecounteractedbyAL8810.Inaddition,theextrainhibitionofCOL1A1expres-

sionthatoccurredwhentheFLSwereculturedwithFCMandAL8810couldbe

explainedbythefactthat,inourculturesystem,thereisnodirecteffectofPGF2α

onCOL1A1expressionandthatotherfactorsareinvolvedinthisrelationship.

Likeotherorgans,adiposetissuecontainsaresidentpopulationofcellsofthe

innate immune system, in particular macrophages and T lymphocytes. In our

earlierstudy,wedemonstratedthatmacrophageswerepresentintheIPFP,many

52 CHAPTER 3

ofwhichhaveanM2phenotype39.AlternativelyactivatedM2macrophageshave

ananti-inflammatoryorrepairphenotypeandproduce,predominantly,IL-10but

also growth

factorssuchasTGFβ1andinsulin-likegrowthfactor1,andalmostnoIL-12or

IL-2340.ThepresenceofM2macrophages in the IPFPofpatientswithend-stage

OAmightcontributetotheprofibroticeffectoftheFCMonFLSdescribedinthe

presentstudy.Earlierstudiesalsofoundthatmacrophagesareabletoproduce

PGF2α41;42.Inadditiontomacrophages,adipocytes,Tlymphocytes,orothercells

fromthestromalvascularfractionmightalsocontributetoPGF2α production in

theIPFP43.

Prostaglandins,includingPGD2,PGE2,PGF2α,andPGI2,areproducedwhenphos-

pholipidsarecleavedinresponsetostimuli,resultinginthereleaseofarachidonic

acid,which is thenmetabolizedbycyclooxygenase toproduceprostaglandins.

PGF2α isconsideredtobeamajorandstablemetaboliteofPGE244.Reductionin

PGE2productionistheclassicmodeofactionofanti-inflammatoryagentssuch

asnonsteroidalanti-inflammatorydrugs(NSAIDs),whicharecommonlyusedin

medicalmanagementofOA,andnumerousstudieshavedemonstrateda lower

PGE2concentrationinsynovialfluidfollowingNSAIDtreatment45;46.Morerecently,

PGF2αwasalsofoundinthesynovialfluidofhorses,thelevelsofwhichincreased

afterstimulationofacute inflammationandwhichwereshowntobedecreased

after thetreatmentof inflamedkneeswithanNSAID47.NSAIDsmight, therefore,

alsobeusefulinthepreventionofthesynovialfibrosisseeninOA.

Toourknowledge,thisisthefirststudytoexaminetheeffectofadiposetissue

onthesynoviumandtoassessthepotentialinvolvementoftheIPFPonthedevel-

opmentofthesynovialfibrosisoftenseeninOA3.Theresultsofthisstudyindicate

thattheIPFPinthekneesofpatientswithend-stageOAnotonlyinhibitscatabolic

mediatorsincartilage39butalsoexertspro-fibroticeffectsonthesynovium,and

thesepro-fibroticeffectscanbepartiallyexplainedbythepresenceofPGF2α.How-

ever,sincenotallofthefibroticeffectscanbeexplainedbythepresenceofPGF2α,

otherfactorsmayalsoplayarole.Additionalexperimentsarerequiredtoexamine

theeffectofFCMontheentireOAfibroticprocess,andtoinvestigatewhetherthe

effectthatwefoundisspecifictotheIPFPinend-stageOAorwhethertheIPFP

fromanearlierstageofOAwouldhavethesamepro-fibroticeffect.Futurestud-

iesshouldalsoinvestigatewhetherFLSfrompatientswithOAinanearlierstage

wouldrespondinamannercomparabletothatofFLSfrompatientswithend-stage

OA.Inaddition,itshouldbeexaminedwhetherOApatientshaveincreasedlevels

ofPGF2αintheirsynovialfluidandwhetherthisisassociatedwithseverechanges

intheirsynovium.Thecontinuingexpansionofthisknowledgemighteventually

contributetomoreoptimaltreatmentoreventhepreventionofOA.


3

REFERENCES

1. Yusuf E, Bijsterbosch J, SlagboomPE, Rosendaal FR,Huizinga TW,KloppenburgM. Bodymass

indexandalignmentandtheirinteractionasriskfactorsforprogressionofkneeswithradiographic

signsofosteoarthritis.OsteoarthritisCartilage2011;19:1117–22.

2. ReijmanM,PolsHA,BerginkAP,HazesJM,BeloJN,LievenseAM,etal.Bodymassindexassociated

withonsetandprogressionofosteoarthritisofthekneebutnotofthehip:theRotterdamStudy.

AnnRheumDis2007;66:158–62.

3. Revell PA,MaystonV, Lalor P,MappP.The synovialmembrane in osteoarthritis: a histological

studyincludingthecharacterisationofthecellularinfiltratepresentininflammatoryosteoarthritis

usingmonoclonalantibodies.AnnRheumDis1988;47:300–7.

4. HuttonCW,HintonC,DieppePA.Intra-articularvariationofsynovialchangesinkneearthritis:bi-

opsystudycomparingchangesinpatellofemoralsynoviumandthemedialtibiofemoralsynovium.

BrJRheumatol1987;26:5–8.

5. Loeuille D, Chary-Valckenaere I, Champigneulle J, Rat AC, Toussaint F, Pinzano-Watrin A, et al.

Macroscopicandmicroscopicfeaturesofsynovialmembraneinflammationintheosteoarthritic

knee: correlating magnetic resonance imaging findings with disease severity. Arthritis Rheum

2005;52:3492–501.

6. Gharaee-KermaniM,PhanSH.Roleofcytokinesandcytokinetherapyinwoundhealingandfibrotic

diseases.CurrPharmDes2001;7:1083–103.

7. Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol

2003;200:500–3.

8. DesmouliereA,GeinozA,GabbianiF,GabbianiG.Transforminggrowthfactor-1induces-smooth

muscleactinexpression ingranulation tissuemyofibroblastsand inquiescentandgrowingcul-

turedfibroblasts.JCellBiol1993;122:103–11.

9. Ronnov-JessenL,PetersenOW.Inductionof-smoothmuscleactinbytransforminggrowthfactor-1

inquiescenthumanbreastglandfibroblasts:implicationsformyofibroblastgenerationinbreast

neoplasia.LabInvest1993;68:696–707.

10. VanderSlotAJ,ZuurmondAM,BardoelAF,WijmengaC,PruijsHE,SillenceDO,etal. Identifica-

tionofPlod2astelopeptidelysylhydroxylase,animportantenzymeinfibrosis.JBiolChem2003;

278:40967–72.

11. VanderSlotAJ,ZuurmondAM,vandenBogaerdtAJ,UlrichMM,MiddelkoopE,BoersW,etal.

Increasedformationofpyridinolinecross-linksduetohighertelopeptidelysylhydroxylaselevels

isageneralfibroticphenomenon.MatrixBiol2004;23:251–7.

12. RemstDF,BlaneyDavidsonEN,VittersEL,BlomAB,StoopR,SnabelJM,etal.Osteoarthritis-related

fibrosisisassociatedwithbothelevatedpyridinolinecross-linkformationandlysylhydroxylase2b

expression.OsteoarthritisCartilage2013;21:157–64.

13. VanderSlotAJ,vanDuraEA,deWitEC,DeGrootJ,HuizingaTW,BankRA,etal.Elevatedformation

ofpyridinolinecross-linksbyprofibroticcytokinesisassociatedwithenhancedlysylhydroxylase

2blevels.BiochimBiophysActa2005;1741:95–102.

14. Oga T,Matsuoka T, Yao C, NonomuraK, Kitaoka S, SakataD, et al. Prostaglandin F2α receptor

signalingfacilitatesbleomycininducedpulmonaryfibrosisindependentlyoftransforminggrowth

factor-β.NatMed2009;15:1426–30.

15. OlmanMA.BeyondTGF-β:aprostaglandinpromotesfibrosis.NatMed2009;15:1360–1.

54 CHAPTER 3

16. ChenWP,BaoJP,FengJ,HuPF,ShiZL,WuLD. Increasedserumconcentrationsofvisfatinand

its production by different joint tissues in patients with osteoarthritis. Clin Chem Lab Med

2010;48:1141–5.

17. ClockaertsS,Bastiaansen-JenniskensYM,FeijtC,DeClerckL,Verhaar JA,ZuurmondAM,etal.

Cytokineproductionby infrapatellar fatpadcanbestimulatedby interleukin1and inhibitedby

peroxisomeproliferatoractivatedreceptoragonist.AnnRheumDis2012;71:1012–8.

18. Klein-Wieringa IR, Kloppenburg M, Bastiaansen-Jenniskens YM, Yusuf E, Kwekkeboom JC, El-

BannoudiH, et al.The infrapatellar fatpadofpatientswithosteoarthritishasan inflammatory

phenotype.AnnRheumDis2011;70:851–7.

19. PresleN,PottieP,DumondH,GuillaumeC,LapicqueF,PalluS,etal.Differentialdistributionof

adipokinesbetweenserumandsynovialfluidinpatientswithosteoarthritis:contributionofjoint

tissuestotheirarticularproduction.OsteoarthritisCartilage2006;14:690–5.

20. UshiyamaT,ChanoT,InoueK,MatsusueY.Cytokineproductionintheinfrapatellarfatpad:another

sourceofcytokinesinkneesynovialfluids.AnnRheumDis2003;62:108–12.

21. ClockaertsS,Bastiaansen-JenniskensYM,RunhaarJ,vanOschGJ,VanOffelJF,VerhaarJA,etal.

Theinfrapatellarfatpadshouldbeconsideredasanactiveosteoarthriticjointtissue:anarrative

review.OsteoarthritisCartilage2010;18:876–82.

22. FainJN,MadanAK,HilerML,CheemaP,BahouthSW.Comparisonofthereleaseofadipokinesby

adiposetissue,adiposetissuematrix,andadipocytesfromvisceralandsubcutaneousabdominal

adiposetissuesofobesehumans.Endocrinology2004;145:2273–82.

23. Fain JN. Release of interleukins and other inflammatory cytokines by human adipose tissue is

enhancedinobesityandprimarilyduetothenonfatcells.VitamHorm2006;74:443–77.

24. FainJN,TichanskyDS,MadanAK.Mostoftheinterleukin1receptorantagonist,cathepsinS,mac-

rophagemigrationinhibitoryfactor,nervegrowthfactor,andinterleukin18releasebyexplantsof

humanadiposetissueisbythenon-fatcells,notbytheadipocytes.Metabolism2006;55:1113–21.

25. FainJN,CheemaPS,BahouthSW,LloydHilerM.Resistinreleasebyhumanadiposetissueexplants

inprimaryculture.BiochemBiophysResCommun2003;300:674–8.

26. FainJN,MadanAK.Regulationofmonocytechemoattractantprotein1(MCP-1)releasebyexplants

ofhumanvisceraladiposetissue.IntJObes(Lond)2005;29:1299–307.

27. FainJN,TageleBM,CheemaP,MadanAK,TichanskyDS.Releaseof12adipokinesbyadiposetissue,

nonfatcells,andfatcellsfromobesewomen.Obesity(SilverSpring)2010;18:890–6.

28. Bastiaansen-JenniskensYM,deBartAC,KoevoetW,JansenKM,VerhaarJA,vanOschGJ,etal.

Elevatedlevelsofcartilageoligomericmatrixproteinduring invitrocartilagematrixgeneration

decreasecollagenfibrildiameter.Cartilage2010;1:200–10.

29. BalversMG,VerhoeckxKC,Meijerink J, Bijlsma S, RubinghCM,WortelboerHM, et al. Time-de-

pendenteffectofinvivoinflammationoneicosanoidandendocannabinoidlevelsinplasma,liver,

ileumandadiposetissueinC57BL/6micefedafish-oildiet.IntImmunopharmacol2012;13:204–14.

30. Pelletier JP,Martel-Pelletier J, Abramson SB.Osteoarthritis, an inflammatory disease: potential

implicationfortheselectionofnewtherapeutictargets[review].ArthritisRheum2001;44:1237–47.

31. KershawEE,FlierJS.Adiposetissueasanendocrineorgan.JClinEndocrinolMetab2004;89:2548–

56.

32. FraynKN,KarpeF,FieldingBA,MacdonaldIA,CoppackSW.Integrativephysiologyofhumanadi-

posetissue[review].IntJObesRelatMetabDisord2003;27:875–88.INFRAPATELLARFATPADAND

SYNOVIALFIBROSIS2079

33. GalicS,Oakhill JS,SteinbergGR.Adiposetissueasanendocrineorgan.MolCellEndocrinology

2010;316:129–39.


3

34. DistelE,CadoudalT,DurantS,PoignardA,ChevalierX,BenelliC.Theinfrapatellarfatpadinknee

osteoarthritis: an important source of interleukin-6 and its soluble receptor. Arthritis Rheum

2009;60:3374–7.

35. ShiQ,LiuX,BaiY,CuiC,LiJ,LiY,etal.Invitroeffectsofpirfenidoneoncardiacfibroblasts:prolif-

eration,myofibroblastdifferentiation,migrationandcytokinesecretion.PLoSOne2011;6:e28134.

36. HarksEG,PetersPH,vanDongenJL,vanZoelenEJ,TheuvenetAP.Autocrineproductionofpros-

taglandinF2αenhancesphenotypictransformationofnormalratkidneyfibroblasts.AmJPhysiol

CellPhysiol2005;289:C130–7.

37. OlsonDM,TanswellAK.ProductionofprostaglandinsbyfetalratlungtypeIIpneumonocytesand

fibroblasts.BiochimBiophysActa1989;1003:327–30.

38. VanderSlot-VerhoevenAJ,vanDuraEA,AttemaJ,BlauwB,DegrootJ,HuizingaTW,etal.Thetype

ofcollagencross-linkdeterminesthereversibilityofexperimentalskinfibrosis.BiochimBiophys

Acta2005;1740:60–7.

39. Bastiaansen-JenniskensYM,Clockaerts S, FeijtC,ZuurmondAM,Stojanovic-SusulicV,BridtsC,

etal.Infrapatellarfatpadofpatientswithend-stageosteoarthritisinhibitscatabolicmediatorsin

cartilage.AnnRheumDis2012;71:288–94.

40. VerreckFA,deBoerT,LangenbergDM,vanderZandenL,OttenhoffTH.Phenotypicandfunctional

profilingofhumanproinflammatorytype-1andanti-inflammatorytype-2macrophagesinresponse

tomicrobialantigensandIFN-γ-andCD40Lmediatedcostimulation.JLeukBiol2006;79:285–93.

41. NorwitzER,LopezBernalA,StarkeyPM.Tumornecrosisfactor-αselectivelystimulatesprostaglan-

dinF2αproductionbymacrophagesinhumantermdecidua.AmJObstetGynecol1992;167:815–20.

42. O’Sullivan MG, Chilton FH, Huggins EM Jr, McCall CE. Lipopolysaccharide priming of alveolar

macrophagesforenhancedsynthesisofprostanoidsinvolvesinductionofanovelprostaglandinH

synthase.JBiolChem1992;267:14547–50.

43. IyerA,FairlieDP,PrinsJB,HammockBD,BrownL.Inflammatorylipidmediatorsinadipocytefunc-

tionandobesity.NatRevEndocrinol2010;6:71–82.

44. SamuelssonB.Prostaglandinsandthromboxanes.RecentProgHormRes1978;34:239–58.

45. DeGrauwJC,vandeLestCH,vanWeerenPR.Inflammatorymediatorsandcartilagebiomarkers

insynovialfluidafterasingleinflammatoryinsult:alongitudinalexperimentalstudy.ArthritisRes

Ther2009;11:R35.

46. MastbergenSC,MarijnissenAC,VianenME,ZoerB,vanRoermundPM,BijlsmaJW,etal. Inhibi-

tionofCOX-2bycelecoxibinthecaninegroovemodelofosteoarthritis.Rheumatology(Oxford)

2006;45:405–13.

47. DeGrauw JC, vande LestCH, vanWeerenPR.A targeted lipidomics approach to the studyof

eicosanoidreleaseinsynovialjoints.ArthritisResTher2011;13:R123.

56 CHAPTER 3

Supplementary figure 1TheeffectofTGFβ/ALK5(SB505124)orPGF2αsignalling(AL8810)inhibi-tiononFLSculturesonA)collagenproduction,B)Col1α1geneexpression,C)Plod2geneexpres-sion,andD)Asmageneexpression.AllanalysesweredoneintriplicateononeFLSdonor.Barsrepresentthemean+standarddeviation.

CHAPTER 4

The infrapatellar fat pad from diseased joints

inhibits chondrogenesis of mesenchymal stem cells

Wu Wei Resti Rudjito Niamh Fahy

Jan A.N. Verhaar Stefan Clockaerts

Yvonne M. Bastiaansen-Jenniskens Gerjo J.V.M. van Osch

Eur Cell Mater. 2015 Dec 02;30:303-14

60 CHAPTER 4

ABSTRACT

Cartilagerepairbybonemarrowderivedmesenchymalstemcells(MSCs)canbe

influencedbyinflammationintheknee.Nexttosynovium,theinfrapatellarfatpad

(IPFP)hasbeendescribedasasourceforinflammatoryfactors.Here,weinves-

tigatedwhetherfactorssecretedbytheIPFPaffectchondrogenesisofMSCsand

whetherthisisinfluencedbydifferentjointpathologiesorobesity.Furthermore,

weexaminedtheroleofIPFPresidentmacrophages.First,wemadeconditioned

medium from IPFP obtained fromosteoarthritic joints, IPFP from traumatically

injuredjointsduringanteriorcruciateligamentreconstructionandsubcutaneous

adiposetissue.Additionally,wemadeconditionedmediumofmacrophagesiso-

latedfromosteoarthriticIPFPandofpolarisedmonocytesfromperipheralblood.

WeevaluatedtheeffectofdifferenttypesofconditionedmediumonMSCchon-

drogenesis.Conditionedmedium from IPFPdecreasedcollagen2 andaggrecan

geneexpressionandthioninandcollagentype2staining.Thisanti-chondrogenic

effectwasthesameforconditionedmediumfromIPFPofosteoarthriticandtrau-

matically injured joints. Furthermore, IPFP from obese (BodyMass Index >30)

donorsdidnotinhibitchondrogenesismorethanthatoflean(BodyMassIndex

<25)donors.Finally,conditionedmediumfrommacrophagesisolatedfromIPFP

decreased the expression of hyaline cartilage genes, as did peripheral blood

monocytesstimulatedwithpro-inflammatorycytokines. The IPFPandtheresi-

dentpro-inflammatorymacrophagescouldthereforebetargets for therapiesto

improveMSC-basedcartilagerepair.

Anti-chondrogenic effect of IPFP on MSCs 61

4

INTRODUCTION

Articularcartilagehas limitedself-healingcapabilitiesandsurgical intervention

usingbonemarrowstimulationtechniques,suchasthemicrofractureprocedure,

canbeusedtoactivatemesenchymalstemcells(MSCs)fromtheunderlyingbone

marrow to repair the defect1;2. However, instead of a normal hyaline cartilage

matrix,afibrocartilaginouscartilagematrixfillsupthedefect1;3;4.Anexplanation

why hyaline cartilage production is inhibited could be that the cartilage defect

islocatedinapost-traumaticinflammatoryenvironment5.Inthispost-traumatic

inflammatory environment, circulating inflammatory factors stimulate matrix

degradation and inhibit chondrogenic differentiation6.

Besidessurgicalintervention,researchiscurrentlyfocusedonMSC-basedtissue-

engineered cartilage repair strategies7. In these strategies, repair tissues are

engineered in-vitro using a combination ofMSCs, scaffolds and growth factors

andsubsequentlyimplantedintothecartilagedefect.Whenimplanted,theMSCs

in thesepre-engineeredconstructswillbeexposed to the samepost-traumatic

inflammatory environment as the MSCs after the microfracture procedure. An

inflammatoryenvironmentcaninhibithyalinecartilagematrixproduction8.

AnotherfactorthatcouldinfluenceMSCchondrogenicdifferentiationisobesity.

Theclinicalresultsafterthemicrofractureprocedurearereportedtobeworsein

patients with obesity9.Obesityisknowntocausesystemicmetabolicandinflam-

matorychanges,withincreasedcirculatinginflammatoryfactors10.

Inflammatoryfactorscanbeproducedbydifferenttissuesofthekneejoint,the

mostwelldescribedbeingthesynoviumandcartilage.Theinfrapatellarfatpad

(IPFP) is an adipose tissue located extra-synovially and intra-capsularly. It is

richlyvascularised,innervatedandknowntoalsosecretemanyadipokinesand

cytokines11;12.Adiposetissueinobesepatientshasbeenshowntohaveaninflam-

matoryphenotypewithabundantimmunecellinfiltrationandsecretionofinflam-

matory cytokines13. Furthermore, joint trauma and joint degradation can both

increaseinflammationintheIPFPandthesecretionofinflammatoryfactors12;14-16.

TheIPFPcontainsimmunecellssuchasmacrophages15;17.Macrophagesplayan

importantroleininflammationandwoundhealing.Macrophagescanbecatego-

rizedintopro-inflammatorymacrophagesandanti-inflammatoryorwoundhealing

macrophages18;19.Inflammationorobesityoftenresultinincreasednumbersofin-

62 CHAPTER 4

flammatorymacrophages.Previouslywehaveshownthatonlypro-inflammatory

andnotanti-inflammatorymacrophagesinhibitMSCchondrogenesisinvitro20.

SincetheIPFPfromend-stageOApatientsisasourceofinflammatoryandcata-

boliccytokines12, it istobeexpectedthat IPFPsecretedfactors influenceother

jointtissues.AgainstourexpectationthatthefactorsreleasedfromIPFPwould

stimulate tissue degradation, our previous studies demonstrated that factors

releasedfromIPFPfromend-stageOApatientsinhibitcatabolicmediatorsincarti-

lage15andstimulatefibroticprocessesinsynovialfibroblasts21.Thisindicatesthat

thefactorsreleasedbytheIPFPnotonlyhaveapro-inflammatoryeffectonjoint

environmentandthatthiseffectcouldbedifferentineachtypeofjointtissue.

ConsideringtheIPFP’spotentialtoproducefactorsthatinfluencethejointenviron-

ment,andtheknowledgethatthejointenvironmentinfluencescartilagerepair,we

hypothesizedthatfactorssecretedbytheIPFPaffectMSC-basedcartilagerepair.

Inthisstudy,weinvestigatedwhetherandhowfactorssecretedbytheIPFPaffect

thechondrogenesisofMSCsinvitroandwhetherthiscouldbeinfluencedbyjoint

pathologyorobesity.Furthermore,weevaluatedwhethermacrophagespresentin

theIPFPcouldberesponsiblefortheeffectseenonMSCchondrogenesis.

METHODS

Preparation of adipose tissue conditioned medium (CM)

IPFP tissue was obtained as leftover material from patients with OA who had

undergonea total knee replacement (Age66.1years (53.9-80.2);BMI29.3 (21.5-

43.7)),orfrompatientsduringanteriorcruciateligament(LR)reconstruction(Age

29.3 (17.6-48.0); BMI 22.8 (19.3-29.0)),with time from trauma to reconstruction

5.4 months (2-12 months). Subcutaneous adipose tissue (SAT) from patients

undergoingtotalhipreplacementwasused(Age63.0(50.2-79.1);BMI31.3(24.0-

35.8)). Consentwas given in accordancewith the guidelines of the Federation

of Biomedical Scientific Societies (http://www.federa.org) after approval by the

localethicalcommittee(MEC2012-267).Allculturemediafromnowonwardswere

supplementedwith1.5μg/mLfungizoneand50μg/mLgentamicin(bothGibco).

Togenerateadipose tissueCM,adipose tissuewascut intosmallpiecesofap-

proximately9mm2,washedfortwotimeswithsalineandculturedataconcentra-

tionof100mg/mlinDulbecco’sModifiedEagleMediumwithGlutamax(DMEM-HG;

Gibco)supplementedwith1%insulin-transferrin-selenium(ITS+;BDBiosciences)

for24hoursat37ºC.Afterwards,themediumwasharvested,centrifugedat250G


4

for8minutesandthesupernatantwasstoredat-80ºCforcultureexperiments.

UnconditionedmediumwasgeneratedbyincubationofDMEM-HGsupplemented

withITS+for24hoursat37ºC,centrifugedat250Gfor8minutesandstoredat-80

ºC.

Isolation of macrophages from adipose tissue and preparation of IPFP macrophage CM

Toisolatemacrophagesfromthestromalvascularfractionofadiposetissue,six

OAIPFPsamples(Age66.6(54.1-80.2),BMI28.9(21.9-33.9))wereused.Adipose

tissuesampleswerecutintopiecesofaround9mm2andincubatedovernightat

37ºCwith1mg/mlcollagenaseB(Roche,Germany) inDMEM-HGwith10%FCS.

After centrifugation, the supernatant containing thefloatingadipocytesand fat

was removed, cellpelletwas re-suspendedandfiltered througha100µmfilter

followedbya40µmfilter.Theresultingcellsuspensionwaslayeredontopof15

mlFicoll(Ficoll-Paque™PLUS,GEHealthcare)andseparatedbydensitygradient

following centrifugation at 1,000g for 15minutes. The interphasewas removed

andwashedwithphosphatebufferedsaline(PBS;Gibco)containing2%FCSfol-

lowedbyincubationwithCD45-PE(BDBiosciences)for1hourandincubationin

20µlofanti-mouse-IgGmagneticbeads(MiltenyiBiotec)per10.000.000cellsfor

30minutes inthedarkat4 ºC.CD45positivecellswereseparatedbymagnetic

activatedcellsorting(MACS,MACSSeparationcolumnsLSandMidiMACS™Sepa-

rator,MiltenyiBiotec)accordingtomanufacturer’sinstructions.Flowcytometric

analysiswasperformedtoconfirma>90%CD45+populationpurity.CD45reduced

fractioncontained<10%CD45+cells. IsolatedCD45+cellswereseededatacell

densityof100.000cells/cm2in24wellsplatesandculturedovernightat37ºCin

DMEM-HGsupplementedwith10%FCS.Cellswere thencarefullywashed twice

withPBStoremovenon-attachedcellsandDMEM-HGwith1%ITS+wasadded.

After24hours,IPFPCD45+CMwasharvested,centrifugedat250gfor8minutes

andstoredat-80ºCforfurtherexperiments.

Human monocyte isolation, differentiation and CM preparation

Peripheral blood monocytes were isolated from buffy coats with CD14+ mag-

neticbeads(MiltenyiBiotec)andMACSaccordingtomanufacturer’sinstructions.

Monocyteswerethenseededat500.000cells/cm2,stimulatedtoM(LPS+IFN-ɣ)with

100ng/ml lipopolysaccharide(LPS;SigmaAldrich)and10ng/ml IFN-ɣ (PeproT-

ech)orM(IL-4)with10ng/mlIL-4(Preprotech)19.Subsequently,M(LPS+IFN-ɣ)andM(IL-4)CMweremadeaspreviouslydescribed20;22.Samplesoftheseexperiments

werepreviouslyusedinFahyetal20,butadditionalgeneexpressionanalysiswere

performedforthecurrentstudy.

64 CHAPTER 4

Human dermal fibroblast expansion and CM preparation

Human dermal fibroblast from adult donors (HDFa)were acquired fromGibco

(Cat.no.C-013-5C)andwereseededat5000cells/cm2inDMEM-HGcontaining10%

FCS.Afterreachingsub-confluency,HDFasweretrypsinizedandseededagain.For

HDFaCMpreparation,P4cellswerecultureduntil subconfluency,washedwith

PBSforthreetimesandDMEM-HGwithITS+wasadded.After24hours,CMwas

harvested,centrifugedat250gfor8minutesandstoredat-80ºCforfurtherexperi-

ments.

Isolation and culture of human MSCs

MSCswereisolatedfromheparinizedbonemarrowaspiratesofpatientswithOA

undergoingtotalhipreplacement(9donors;Age61years(32-78)).Patientsgave

informed consent and the studywas approved by the local ethical committee

of theErasmusMC,Rotterdam(MEC2004-142)andAlbertSchweitzerHospital,

Dordrecht(MEC2011-07).Bonemarrowaspirateswereplatedandwashedafter

24hourswithPBS containing 2% fetal calf serum (FCS; Lonza).MSCswere ex-

pandedinMinimumEssentialMedium-Alpha(α-MEM;Gibco)supplementedwith

10%FCS,1ng/mLfibroblastgrowthfactor2(FGF2;R&DSystems)and25μg/mL

ascorbicacid-2-phosphate(Sigma-Aldrich).Afterreachingsub-confluency,MSCs

weretrypsinisedandseededagainatadensityof2500cells/cm2.MSCsofpassage

3or4wereused.

Chondrogenic differentiation of MSCs

MSCs were encapsulated in 1.2% alginate (CP Kelco) in saline at a density of

4.000.000 cells/ml. Beads were formed by purging the MSC-alginate solution

througha23Gneedleandallowingdropletstofallinto102mMCaCl2solution.

Pelletsweremadebycentrifugationof200.000cellsinpolypropylenetubes.Beads

andpelletswereculturedwithDMEM-HGwith ITS+containing25% IPFPCMor

unconditioned medium. As chondrogenic supplements, we used 0.1 µM Dexa-

methasone(Sigma–Aldrich),10ng/mlTGFβ1(R&Dsystems),25μg/mLascorbic

acid-2-phosphate (Sigma-Aldrich),40µg/mlL-Proline (Sigma-Aldrich)and1mM

sodiumpyruvate(Gibco).MSCbeadswereculturedin100µLofmediumperbead

andpelletswereculturedin500µLofmediumperpellet.Mediumwasrefreshed3

timesaweek.Beadsandpelletswereharvestedafter28days.

Fortheexperimentswithmacrophages,obeseversusleanIPFP,osteoarthriticver-

suspost-jointtraumaIPFPandSATversusIPFP,beadswerefirstculturedinDMEM-

HGcontainingITS+andchondrogenicsupplementsfor14days.Fromday14-17,

partsoftheDMEM-HGwithITS+wasreplacedbyconditionedmediumoranequal


4

percentageofunconditionedmedium.FortheexperimentswithIPFPandSATCM,

wereplaced25%oftheDMEM-HGcontainingITS+.Forthemacrophagesexperi-

ments,wereplaced50%oftheDMEM-HGcontainingITS+withCD45CMor20%

withM(LPS+IFNɣ)/M(IL-4)CM.FortheCD45experiments,thelowestcellnumber

usedtogenerateCM(0.23µgDNA)wassetat50%andfortheM(LPS+IFNɣ)/M(IL-4)

experiments,theaveragecellnumberusedtogenerateCM(1.64µgDNA)wasset

at20%.Allsubsequentdonorswerecorrectedaccordinglytoassurethatmedium

conditionedbyanequalnumberofcellswasadded.Chondrogenicsupplements

werealwaysaddedaftermixingthemediumwithCMorunconditionedmedium.In

thisway,allconditionshadthesamefinalconcentrationofallsupplements.Beads

wereharvestedatday17.Allmediawererefreshed24hourspriortoharvest.

Gene expression analysis

MSC alginate beadswere harvested in ice-cold 55mM sodium citrate and dis-

solvedaftergentleagitationat4 ºC.Thesolutionwas thencentrifugedat400g

for8minutesat4ºCandcellpelletswerere-suspendedin650µlRNA-Bee(TelT-

est, USA) per bead. Chloroform (Sigma-Aldrich) was added to all samples at

200µL/mLRNA-Bee.RNAwasfurtherpurifiedusingRNeasyMicroKit(Qiagen).

RNA concentration and quality wasmeasured using NanoDrop ND1000 UV-Vis

Spectrophotometer(IsogenLifeScience).RNAwasreverse-transcribedtocDNA

using theRevertAid First StrandcDNASynthesisKit (Fermentas,Germany) ac-

cordingtomanufacturer’sinstructions.qRT-PCRwasperformedin10µLreactions

on cDNAusing theCFX96Touch™Real-TimePCRDetection System (BIO-RAD,

USA). Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hy-

poxanthinephosphoribosyltransferase1(HPRT1),collagentype IIa1(COL2A1),

aggrecan(ACAN),collagentypeI(COL1A1)andversican(VCAN)weredetermined

with qRT-PCR. The genesGAPDH and HPRT were used as housekeepers. Gene

expressionwasanalyzedaccordingtotheΔΔCtmethod,withtheconditiontreated

withunconditionedmediumusedascontrolandsetat1.0.Expressionlevelsof

otherconditionsareexpressedrelativetothethiscontrolcondition.Theprimer

efficiency for GAPDHwas1.06,forHPRT0.96,forCOL2A11.02,forACAN0.95,for

COL1A10.97andVCAN0.95.ThemeanCtvalueforGAPDHwas22.9andforHPRT

28.8.DifferenceinCtvaluesbetweenthesampleswithineachexperimentwasless

than1Ctandtherewerenosignificantdifferencesbetweentheconditions(data

notshown).Geneexpressionvaluesafternormalizationtohousekeepersareused

to calculate COL2A1/COL1A and ACAN/VCANratios.

Primer sequences were as followed: GAPDH, forward: GTCAACGGATTTGGTC-

GTATTGGG, reverse: TGCCATGGGTGGAATCATATTGG and probe Fam: TGGC-

66 CHAPTER 4

GCCCCAACCAGCC.HPRTforward:TATGGACAGGACTGAACGTCTTG,reverse:CA-

CACAGAGGGCTACAATGTGandprobeFam:CGCCCAATACGACCAAATCCGTTGAC.

COL2A1 forward: GGCAATAGCAGGTTCACGTACA, reverse: CGATAACAGTCTT-

GCCCCACTTandprobeFam:CCGGTATGTTTCGTGCAGCCATCCT.ACAN forward:

TCGAGGACAGCGAGGCC,reverse:TCGAGGGTGTAGCGTGTAGAGAandprobeFam:

ATGGAACACGATGCCTTTCACCACGA. COL1A1 forward: CAGCCGCTTCACCTA-

CAGC, reverse: TTTTGTATTCAATCACTGTCTTGCC and probe Fam: CCGGTGT-

GACTCGTGCAGCCATC. VCAN forward: TGGAATGATGTTCCCTGCAA, reverse:

AAGGTCTTGGCATTTTCTACAACAG,probe:CTGGCCGCAAGCAACTGTTCCTTT.

Quantification of DNA and GAG content

Alginatebeadsweredissolvedinsodiumcitrateanddigestedwithpapain.(Sigma

Aldrich).PelletsweredigestedwithproteinaseK(SigmaAldrich).DNAwassubse-

quentlyquantifiedbyspectrophotometricdetectionofethidiumbromide(Gibco)

bindingat340and590nm.GAGcontentwasdeterminedwithdimethylmethylene

blue(Polysciences)spectrophotometricallyat530nmand590nm23andatapHof

1.75.

Histological evaluation of MSC chondrogenesis in pellet culture

6µm thickparaffinsectionsweremadeof thepelletsand thioninstainingwas

performed. For immunohistochemical staining, sections were incubated with

monoclonalantibodyagainstcollagentype2(DSHB,#II-II6B3)andcollagentype1

(Abcam,#6308)orIgG1isotypecontrol(DakoCytomation,#X0931).Sectionswere

thenincubatedwithLinkandLabel(Biogenex,HK-321-UK)andfreshlyprepared

new-fuchsinwasusedassubstrate.

Evaluation of macrophage subtypes in adipose tissue

10µmcryosectionsweremadeof13samplesofOAIPFP(Age65.3(54.3-74.1),BMI

28(21.5-35.8)),7samplesofLRIPFP(Age29.9(21.3-48),BMI25.6(19.3-38.5))and

9samplesofSAT(Age63.1(±50.2-79.1),BMI32.3(25.9-47.2)).Afterfixationinac-

etone,sectionswereincubatedwithmonoclonalantibodiesagainstCD68(DAKO,

#EMB11),CD11c(Chemicon,#EP1347Y)andCD206(Abcam,#64693).MouseIgG

(Dako)wereusedasisotypecontrols.Subsequently,sectionswereincubatedwith

linkandlabel(BioGenex)andfreshlypreparednew-fuchsinwasusedassubstrate.

Acellwasconsideredpositivewhenstainedred.Sampleswererankedbasedon

theirrelativenumberofpositivecellsinthreesectionspersamplebytwoblinded

observers as previously described15. Ranking was performed for each marker

separatelyandforeachsample,theaveragerankofthetwoobservers(WWand

RR)wasusedintheanalysis.


4


IBMSPSS statistics 21 (IBMCorporation)wasused for statistical analysis.The

Shapiro-Wilk testwasused to testdata fornormalityand theLevene’s test for

homogeneityofvariance.Nodatawerenormallydistributed.Forgeneanalysis

andGAGmeasurement,ifnototherwisestated,theKruskal-Wallistestwasused

followedbyapost-hocBonferronicorrection.Forimmunohistochemicalranking,

theMann-WhitneyUtestwasused.p<0.05wasconsideredstatisticallysignificant.

RESULTS

IPFP negatively impacts chondrogenic differentiation of MSCs

COL2A1 and ACANwereonlydetectableafterTGFβ1wasadded(Figure1).IPFP

fromOAdonorsstatisticallysignificantlyloweredthegeneexpressionofMSCsfor

COL2A1 (p<0.001),ACAN (p<0.001)andVCAN, without affecting COL1A1 (Figure

1A). This resulted in a lowerCOL2A1/COL1A1 (p<0.001) andACAN/VCAN ratio

(p<0.05)(Figure1A).ThiseffectwasabsentwhenCMwasheatedto95°Cfor10

minutesbeforeuse,indicatingthattheeffectsarecausedbyproteinsproduced

by IPFP(Figure1B).GAGcontentof thealginatebeadswere increasedby IPFP

(Figure2A).TofurtherinvestigatetheeffectsofIPFPonmatrixproduction,pellet

culturewasused. IPFPdecreasedthioninand immunohistochemicalstainingof

collagentype2,whereascollagentype1stainingwasnotinfluenced(Figure2C).

Furthermore,IPFPdecreasedGAGcontentofpellets(Figure2D-E).

Effect of obese versus lean IPFP and osteoarthritic versus traumatic joint IPFP on MSC chondrogenesis

Obesity increasesthereleaseof inflammatory factorsbyadiposetissue.There-

fore,wehypothesizedthatCMfromobeseIPFPdonors(BMI33.1(30-35.8))would

reduceMSCchondrogenesismore thanCM from lean IPFP (BMI 23.1 (21.5-24))

donors. However, chondrogenicmarkers were notmore inhibited by CM from

obesethanbyCMfromleandonors(Figure3A).Next,wecomparedtheeffectof

CMfromLRIPFP(BMI22.9(22.0-25.0))withBMImatchedOAIPFP(BMI23.1(21.5-

24.0)).Chondrogenicgeneexpressionwasnotstatisticallysignificantlydifferent

betweenMSCsexposedtoCMfromLRIPFPandCMfromOAIPFP.InterestinglyLR

IPFPCMstatisticallysignificantlyincreasedCOL1A1comparedtocontrolandOA

IPFPCM(p=0.023)(Figure3B).

68 CHAPTER 4

VCAN

0.0

0.5

1.0

1.5

2.0 p<0.05

TGFb1 --

+-

++IPFP CM

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1

0.0

0.5

1.0

1.5 p<0.001

TGFb1 --

+-

++IPFP CM

Gen

eex

pres

sion

com

pare

dto

cont

rol

ACAN

0.5

1.0

1.5 p<0.05

TGFb1 --

+-

++IPFP CM

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL1A1

0.5

1.0

1.5

TGFb1 --

+-

++IPFP CM

p<0.001

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1 / COL1A1

0.01

0.1

1

10 p<0.001

Control IPFP

Com

pare

dto

cont

rol

ACAN / VCAN

0.0

0.5

1.0

1.5 p<0.05

Control IPFP

Com

pare

dto

cont

rol

COL2A1

0.0

0.5

1.0

1.5

TGFb1 +-

++

+- IPFP CM

++

- - +Heating +

p<0.05

ns

Gen

eex

pres

sion

com

pare

dto

cont

rol

ACAN

0.0

0.5

1.0

1.5

2.0

TGFb1 +-

++

+-

++

- - +Heating +

p<0.05 ns

IPFP CM

Gen

eex

pres

sion

com

pare

dto

cont

rol

A

B

Figure 1 IPFPinhibitschondrogenicdifferentiationofMSCs.A)MSCalginatebeadsweretreatedwith25%IPFPCMfor28daysofculture inchondrogenicmediumwithTGFβ1.Thecontrolconditionwastreatedwith25%unconditionedmediumwithchondrogenicmediumwithTGFβ1.RelativegeneexpressionCOL2A1,ACAN,COL1A1andVCANnormalizedtothecontrolconditionandCOL2A1/COL1A1andACAN/VCANratio.CMwerepooledfrom3-5differentIPFPdonorsandusedtotreat3MSCdonors,witheachconditionperformedintriplicate.B)MSCalginatebeadsweretreatedwith25%CMfromIPFPfromday14-17ofchondrogenicstimulation.CMorunconditionedmediumwereheatedfor10minutesat95°CandallowedtocooldowntoroomtemperaturebeforeadditiontoMSCalginatebeads.RelativegeneexpressionofCOL2A1andACANinMSCsaftertreatment,normalizedtothecontrolcondition.OnepoolofCMfrom3IPFPdonorswasusedtotreat1MSCdonorwitheachconditionperformedintriplicate.ThemeanCtvalueinthecontrolconditionforCOL2A1was23.2,forACAN27.3,forCOL1A119.7andforVCAN24.3.Valuesrepresentthemean±SD.


4

Chondrogenic differentiation of MSCs is more affected by IPFP than by SAT

Toinvestigatewhethertheeffectofadiposetissueofthejoint(IPFP)isdifferent

fromsubcutaneousadiposetissue(SAT),weaddedCMmadefromSATtoMSCs

duringchondrogenicdifferentiation. SATstatistically significantly loweredgene

expression of COL2A1 and ACAN (p<0.01), but did not influence the COL2A1/

COL1A1 and ACAN/VCANratio(Figure4A).Toinvestigatewhetherthisisduetoa

differenceintheconcentrationoffactorsreleasedbyIPFPandSAT,weincreased

thepercentageofSATCMaddedtocultureupto50%.Incontrasttotheeffectof

IPFP,theeffectofSATonchondrogenesiswasnotdosagedependent(Figure4B).

Figure 2 IPFPinhibitschondrogenicdifferentiationofMSCs.AllconditionswerechondrogenicallystimulatedwithTGFβ1duringtheentirecultureperiod. MSCalginatebeadsandpelletsweretreatedwith25%IPFPCMwhereasthecontrolconditionwastreatedwith25%uncondi-tionedmediumfor28days.A)TotalGAGcontentandB)totalDNAcontentofalginatebeads.C)Representativeimagesofpelletsfrom1MSCdonorstainedforglycosaminoglycanswiththioninorcollagentype2andcollagentype1usingimmunohistochemistry.D)TotalGAGcontentandE)totalDNAcontentofpellets.DifferentpoolsofCMfrom3differentIPFPdonorswereusedforeachexperiment.FortotalGAGandDNAcontentinbeads(PanelA),3MSCdonorswereusedwitheachconditionperformedintriplicate.Forthepelletcultureexperi-ment(PanelC-E),2MSCdonorwereusedwith5pelletspercondition.Valuesrepresentmean±SD.TheMann-WhitneyUtestwasusedtodeterminestatisticalsignificance.

70 CHAPTER 4

Difference between IPFP and SAT in number and phenotype of macrophages

WehypothesizedthatthedifferenceineffectsofIPFPandSATonexpressionof

chondrogenicandfibroustissuegenescouldbeduetothetypeofmacrophages

in the adipose tissue.We therefore first characterized themacrophages in the

differentadiposetissues. IPFPsamples fromOAdonorsrankedhigheronCD68

(p=0.007andp=0.03)andCD206 (p=0.02andp=0.049)positive staining thanLR

IPFPandSATsamples(Figure5BandD).Wecouldnotdetectdifferencesbetween

obeseandleandonors.

Macrophages in IPFP contribute to the effects seen on MSC chondrogenesis

ToinvestigatewhethermacrophagespresentintheIPFPwereresponsibleforthe

effectseenonMSCchondrogenesis,weaddedCMfrommacrophagesisolatedfrom

IPFPtoMSCsduringchondrogenesis.IPFPmacrophagesstatisticallysignificantly

lowered the COL2A1 and ACANgeneexpression(Figure6A).Sinceweapplieda

verystrictselectionforCD45+cells,theremainderfractionstillcontainedasmall

numberofCD45+cells.ThisCD45reducedfractionappearedtobeabletoinhibit

COL2A1 and ACAN geneexpressionaswell (Figure6B).To investigatewhether

thiseffectwasspecificforcellsisolatedfromtheIPFP,weaddedCMfromhuman

dermalfibroblasts(HDFA)toMSCsduringchondrogenesis.CMfromHDFAsdid

notinfluenceMSCchondrogenesis(Figure6B).

COL2A1

Control IPFP obese IPFP lean0.0

0.5

1.0

1.5 p<0.01

ns

Gen

eex

pres

sion

com

pare

dto

cont

rol

ACAN

Control IPFP obese IPFP lean0.0

0.5

1.0

1.5 p<0.01

ns

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1

Control OA IPFP LR IPFP0.0

0.5

1.0

1.5 p<0.01

nsGen

eex

pres

sion

com

pare

dto

cont

rol

ACAN


0.5

1.0

1.5 p<0.01

nsG

ene

expr

essi

onco

mpa

red

toco

ntro

l

COL1A1

Control IPFP obese IPFP lean0

1

2

3

4

Gen

eex

pres

sion

com

pare

dto

cont

rol

VCAN

Control IPFP obese IPFP lean0

1

2

3

4

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL1A1

Control OA IPFP LR IPFP0

5

10

15p<0.05

Gen

eex

pres

sion

com

pare

dto

cont

rol

VCAN


0.5

1.0

1.5p<0.05

ns

Gen

eex

pres

sion

com

pare

dto

cont

rol

A

B

Figure 3 EffectofobeseversusleanIPFPandosteoarthriticversustraumaticjointIPFPonMSCchondrogenesis.AllconditionswerechondrogenicallystimulatedwithTGFβ1duringtheentirecultureperiod.MSCalginatebeadsweretreatedwith25%IPFPCMfromday14-17.Thecontrolconditionwastreatedwith25%unconditionedme-dium.RelativegeneexpressionofCOL2A1,ACAN,COL1A1andVCANinMSCsaftertreatmentwithCMfromA)obese(BMI>30)orlean(BMI<25)OAIPFPdonorsandB)OAorBMImatchedLRIPFPdonors.GeneexpressionwasnormalizedtothecontrolconditionperMSCdonor.CMfrom6obeseOAIPFP,6leanOAIPFPand6LRIPFPdonorswereused.ThreedifferentpoolsofCMwasmadewitheachpoolaselectionofthreedifferentIPFPdonors.ThreeMSCdonorswereused,witheachconditionperformedintriplicate.Valuesrepresentthemean±SD.


4

FinallyweinvestigatedwhetherthenegativeeffectsofIPFPderivedmacrophages

onMSCchondrogenesiscouldbeexplainedbythephenotypeofthemacrophage.

DuetothelownumberofmacrophageswecouldobtainfromIPFP,weusedPBMCs

that were stimulate to pro- or anti-inflammatory phenotype. MSCs exposed to

CMofM(LPS+IFNɣ)macrophageshad lowerhyalinecartilagegenesexpression

comparedtocontrolandCMofM(IL-4)macrophages(Figure6C)

COL2A1

Control IPFP SAT

0.5

1.0

1.5 p<0.01

p<0.01

Gen

eex

pres

sion

com

pare

dto

cont

rol

ACAN

Control IPFP SAT

0.5

1.0

1.5p<0.01

p<0.05

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL1A1

Control IPFP SAT0

5

10

15

Gen

eex

pres

sion

com

pare

dto

cont

rol

VCAN

Control IPFP SAT0.0

0.5

1.0

1.5p<0.01

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1 / COL1A1

Control IPFP SAT0.0

0.5

1.0

1.5

2.0

2.5

p<0.05

Com

pare

dto

cont

rol

ACAN / VCAN

Control IPFP SAT0.0

0.5

1.0

1.5

2.0

2.5 p<0.05

Com

pare

dto

cont

rol

COL2A1

Control

12.5%

IPFP

25%

IPFP

50%

IPFP

12.5%

SAT

25%

SAT

50%

SAT1

10

100

a

a

a

Gen

eex

pres

sion

com

pare

dto

hous

ekee

pers

ACAN

Control

12.5%

IPFP

25%

IPFP

50%

IPFP

12.5%

SAT

25%

SAT

50%

SAT0.01

0.1

1

10

a a

a

Gen

eex

pres

sion

com

pare

dto

hous

ekee

pers

A

B

Figure 4 MSCchondrogenesisismoreinfluencedbyIPFPthansubcutaneousadiposetissue(SAT).MSCalginatebeadsweretreatedwithCMfromIPFPorSATfromday14-17ofculture.Thecontrolconditionwastreatedwith25%unconditionedmedium.AllconditionswerechondrogenicallystimulatedwithTGFβ1duringtheentirecultureperiod.A)25%ofCMwasusedandCMwerepooledfrom3poolsofdifferent3IPFPor3poolsofdifferent3SATdonorsandusedtotreat3MSCdonors.Eachexperimentwasperformedintriplicate.RelativegeneexpressionofCOL2A1,ACAN,COL1A1,VCAN,COL2A1/COL1A1ratioandACAN/VCANratioinMSCsaftertreatment.GeneexpressionwasnormalizedtothecontrolconditionperMSCdonor.B)DifferentpercentagesofCMwereusedandCMwerepooledfrom3IPFPor3SATdonorsandusedtotreat1MSCdonor,witheachconditionperformedintriplicate.RelativegeneexpressionofCOL2A1andACANcomparedtohousekeepersinMSCsaftertreatmentAllconditionsreceivedthesamefinalconcentrationofallchondrogenicsupplements.Valuesrepresentthemean±SD.ap<0.05versusControlusingtheMann-Whitney-Utest.

72 CHAPTER 4

Figure 5 Macrophageswithdifferentphenotypesarepresentinadiposetissuesamples.13samplesofOA IPFP,7samplesofLR IPFPand9samplesofSATwereused.A)Representative imagesofimmunohistochemcalstainingofCD68,CD11candCD206positivecellsinOAIPFPsamples.Positivecellsarestainedredandindicatedwitharrows.RankingofpositivestaininginadiposetissuesamplesforB)CD68,C)CD11candD)CD206.

COL2A1

Control CD45 positive0.0

0.5

1.0

1.5 p<0.05

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1

Control CD45 HDFa 20% HDFa 10%0.0

0.5

1.0

1.5

2.0

reduced

p<0.05

Com

pare

dto

cont

rol

COL2A1 / COL1A1


0.5

1.0

1.5

2.0p<0.05

reduced

Com

pare

dto

cont

rol

ACAN/VCAN


0.5

1.0

1.5

2.0 p<0.05

reduced

Com

pare

dto

cont

rol

COL2A1

0.0

0.5

1.0

1.5

2.0p<0.05

Control M(LPS+IFNg) M(IL-4)

Gen

eex

pres

sion

com

pare

dto

cont

rol

ACAN

0.0

0.5

1.0

1.5

p<0.05

p<0.05


Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1 / COL1A1

0

1

2

3 p<0.05


Com

pare

dto

cont

rol

ACAN / VCAN

0.0

0.5

1.0

1.5 p<0.05


Com

pare

dto

cont

rol

A

B

C

ACAN


0.5

1.0

1.5

2.0p<0.05

reduced

Com

pare

dto

cont

rol

ACAN


0.5

1.0

1.5 p<0.05

Gen

eex

pres

sion

com

pare

dto

cont

rol

COL2A1 / COL1A1


0.5

1.0

1.5

Com

pare

dto

cont

rol

ACAN / VCAN


0.5

1.0

1.5p<0.05

Com

pare

dto

cont

rol

Figure 6 MacrophagesinIPFPcontributetotheeffectsseenonMSCchondrogenesis.MSCalginatebeadsweretreatedwithCMfromday14-17.Thecontrolconditionwastreatedwith25%uncondi-tionedmedium.AllconditionswerechondrogenicallystimulatedwithTGFβ1duringtheentirecultureperiod.A)Effectof50%CMfromCD45+cellsfromIPFPonMSCchondrogenesis;B)Effect50%CMfromCD45reducedfractionfromIPFPorCMfromadulthumandermalfibroblasts(HDFa)onMSCchondrogenesis;C)Effectof20%CMfromM(LPS+IFNɣ)orM(IL-4)macrophagesonMSCchondrogenesis.RelativegeneexpressionofCOL2A1,ACAN,COL2A1/COL1A1ratioandACAN/VCANratioinMSCsaftertreatment.GeneexpressionwasnormalisedtothecontrolconditionperMSCdonor.CMwerefrom6IPFPdonors,1HDFadonoror3blooddonorsandusedtotreat1MSCdonor.ConditionswithCMderivedfromIPFPwereperformedinsinglewellandallothercondi-tionswereperformedintriplicate.Valuesrepresentthemean±SD.


4

DISCUSSION

IPFPsecretescytokines,growthfactorsandadipokineswhichcouldinfluencethe

jointenvironment11. Inthisstudywehaveshownthatthe IPFPreleasedfactors

that inhibitchondrogenicdifferentiationofMSCsandtheproductionofhyaline

cartilagematrix.Thisanti-chondrogeniceffectwasthesameforpatientswithend

stageOAandpatientswithjointtrauma.Furthermore,theIPFPofobesepatients

didnotinhibitchondrogenesismorethanIPFPofleanpatients.Finally,wehave

indicationsthatmacrophagesresidingintheIPFPmightplayanimportantrolein

thisanti-chondrogeniceffect.

Repairtissueformedaftermarrowstimulationtechniques,suchasthemicrofrac-

tureprocedure,ismainlyfibrocartilagewhichisoflessbiomechanicalqualitythan

nativehyalinecartilage24. Inthepresentstudy,thedecreaseinCOL2A1/COL1A1

and ACAN/VCANratiossuggeststhatIPFPnotonlyinhibitschondrogenesis,but

alsoshiftstheproductionfromhyalinetofibro-cartilage.Fibrocartilagealsocon-

tainsproteoglycans,butinsteadofaggrecanthisisversican.Thiscouldbeanex-

planationwhywefoundnoreductioninglycosaminoglycanproductioninalginate

beadsafterIPFPCMtreatment,despiteadecreaseinaggrecancore-proteingene

expression.However,thiscouldalsobecausedbyinterferenceoftheDMMBassay

byalginateespeciallywhenlowamountofGAGisinvolved23;25.Analysesonmatrix

withDMMBand immunohistochemistrycanbeusedmore reliablyonpellets26.

Thereducedthioninstainingandimmunohistochemicalstainingforcollagentype

2inpelletstreatedwithIPFPCMsupportsthegeneexpressionresultsinalginate

beads.OurfindingsindicatethatfibrocartilageproductionbyMSCs,takingplace

aftermicrofractureprocedure,couldbepartlyduetofactorssecretedbytheIPFP.

ThesecretionoffactorsbyIPFPcouldbeinfluencedbythediseasestateofthe

joint.ThemicrofractureprocedureandMSCbasedtreatmentsarerecommended

forthetreatmentofpost-traumatic,ratherthanend-stageOAcartilagedefects27.

Higherlevelsof inflammatorycytokineswerefoundinpost-traumaticthanend-

stageOAknees28;29.Gandhiet al 16 reported thatpost-traumatic IPFPexpresses

morepro-inflammatorygenescompared to end-stageOA IPFP.We founddiffer-

encesinmacrophagenumberandinparticularCD206positivecellsbetweenIPFP

fromOAandLRknees,albeitfactorssecretedbybothOAandLRIPFPinhibited

chondrogenesis.However,wecannotexcludethattheseeffectsareduetodiffer-

entcytokinesoradipokines.

74 CHAPTER 4

AnotherfactorthatcouldinfluencebothIPFPandtheresultsofmicrofractureis

obesity9.Inobesepatients,macrophagesinadiposetissuearechangedtowardsa

chronicinflammatoryphenotypewithincreasedproductionofpro-inflammatory

cytokines10. An inflamed obese IPFP would possibly be an explanation for the

worseresultsofmicrofractureinobesepatients.Incontrasttothishypothesis,we

didnotdetectanincreasedanti-chondrogeniceffectofIPFPfromobesecompared

toleandonors.ToinvestigatewhethertheOAprocesscouldbeofmoreinfluence

tothefactorssecretedbyIPFPthanbeingobese,experimentswithIPFPdonors

withaloworhighBMIbutwithoutOAwouldberequired.Itishoweverdifficultto

obtainsufficientIPFPfromnon-OAdonors.

Independent from joint pathology or obesity, differences have been reported

betweenIPFPandotheradiposetissue.IPFPexpressesmoreinflammatorygenes,

releasemore inflammatory factorsandcontainsmore immunecells thanSAT17.

Inourstudy,theanti-chondrogeniceffectof IPFPwasstrongerthanthatofSAT

and even after increasing the dosage of SAT CM, the anti-chondrogenic effect

wasnotincreasedtothelevelofIPFP.ThissuggeststhattheIPFPreleasesanti-

chondrogenicfactorsnotproducedbysubcutaneousadiposetissues.

IPFPsecretespro-inflammatoryfactorsandadipokines,suchasTNF-α,IL1β,IL6andadipokines,suchasleptin,resistinandadiponectin12.Thesefactorscouldbe

producedby thefibroblasts, adipocytesand immunecells residing in the IPFP.

A largeproportionof the immunecells in the IPFParemacrophages15;17. Immu-

nohistochemical analysis on IPFP and SAT indicated that IPFP containedmore

macrophages than SAT. Previously we have shown that only pro-inflammatory

macrophages inhibit chondrogenesis20 and in the current study, only the pro-

inflammatory macrophages shifts MSCs to produce fibrocartilage matrix. As a

proofofprinciple,weshowedthatisolatedmacrophagesfromtheIPFPwereanti-

chondrogenic.AlthoughwecouldnotexcludethatothercellsintheIPFPcould

alsosecreteanti-chondrogenicfactors,ourstudydoessuggestthatmacrophages

intheIPFPplayavitalroleininhibitingproductionofhyalinecartilageandthe

shifttowardsfibrocartilagematrixproduction.

NexttodirectlynegativelyaffectingchondrogenesisofMSCs,IPFPcaninfluence

other joint structures and thereby indirectly influence chondrogenesis. IPFP

factors have been shown to stimulate fibrotic processes21 and production of

pro-inflammatoryfactorsinsynovialfibroblasts30.Inthisway,synoviumcanthen

inhibit chondrogenesis20.Incontrasttotheeffectonsynovium,theIPFPinhibits

catabolicmediatorsincartilageexplantsstimulatedwithorwithoutIL1β15.This

showsthatdifferenttissuesinthejointcanreactdifferentlytofactorssecreted


4

bytheIPFPandtheinterplaybetweenalljointtissueswilldeterminetheeventual

effect on cartilage repair. The use of co-cultures of several joint tissueswould

berequiredtoelucidatethisinfuturestudiesandtoshowthepotentialeffectof

modulation.

Insummary,wehaveshownthattheIPFPisasourceoffactorsthatinhibitchon-

drogenicdifferentiationofMSCsandshiftedtheproductionfromhyalinetofibro-

cartilagematrix.Thisanti-chondrogeniceffectofadiposetissuewasspecificfor

IPFPandnotsubcutaneousadiposetissue.Furthermore,thisanti-chondrogenic

effectwasnotmoreinobesedonorsthanleandonorsandwasnotdifferentbe-

tweendonorswithpost-jointtraumaorend-stageOA.Finally,theIPFPandspecifi-

callythemacrophagesinthetissuecouldthereforebetargetsforfuturetherapies

toimprovethejointenvironmentforMSCchondrogenesisandtherebyimprove

theoutcomeofmicrofractureprocedureandfutureMSC-basedtreatmentsforthe

repairofcartilagedefects.

76 CHAPTER 4

REFERENCES



2. SteadmanJR,MillerBS,KarasSG,etal.2003.Themicrofracturetechniqueinthetreatmentoffull-

thicknesschondrallesionsofthekneeinNationalFootballLeagueplayers.JKneeSurg16:83-86.

3. FrisbieDD,OxfordJT,SouthwoodL,etal.2003.Earlyeventsincartilagerepairaftersubchondral

bonemicrofracture.ClinOrthopRelatRes:215-227.

4. Kaul G, CucchiariniM, Remberger K, et al. 2012. Failed cartilage repair for early osteoarthritis

defects:abiochemical,histologicalandimmunohistochemicalanalysisoftherepairtissueafter

treatmentwithmarrow-stimulationtechniques.KneeSurgSportsTraumatolArthrosc20:2315-2324.



6. Heldens GT, Blaney Davidson EN, Vitters EL, et al. 2012. Catabolic factors and osteoarthritis-

conditionedmediuminhibitchondrogenesisofhumanmesenchymalstemcells.TissueEngPartA

18:45-54.

7. HunzikerEB,LippunerK,KeelMJ,etal.2015.Aneducationalreviewofcartilagerepair:precepts&

practice--myths&misconceptions--progress&prospects.OsteoarthritisCartilage23:334-350.

8. WehlingN,PalmerGD,PilapilC,etal.2009.Interleukin-1betaandtumornecrosisfactoralphain-

hibitchondrogenesisbyhumanmesenchymalstemcellsthroughNF-kappaB-dependentpathways.

ArthritisRheum60:801-812.



1920.

10. LumengCN, SaltielAR. 2011. Inflammatory linksbetweenobesity andmetabolicdisease. JClin

Invest121:2111-2117.



18:876-882.




13. GrantRW,DixitVD.2015.Adiposetissueasanimmunologicalorgan.Obesity(SilverSpring).

14. GiermanLM,WopereisS,vanElB,etal.2013.Metabolicprofilingrevealsdifferencesinconcentra-

tionsofoxylipinsand fattyacidssecretedby the infrapatellar fatpadofdonorswithend-stage

osteoarthritisandnormaldonors.ArthritisRheum65:2606-2614.



16. GandhiR,TakahashiM,VirtanenC,etal.2011.Microarrayanalysisoftheinfrapatellarfatpadin

kneeosteoarthritis:relationshipwithjointinflammation.JRheumatol38:1966-1972.

17. Klein-WieringaIR,KloppenburgM,Bastiaansen-JenniskensYM,etal.2011.Theinfrapatellarfatpad

ofpatientswithosteoarthritishasaninflammatoryphenotype.AnnRheumDis70:851-857.

18. MosserDM,EdwardsJP.2008.Exploringthefullspectrumofmacrophageactivation.NatRevIm-

munol8:958-969.




4

20. FahyN,deVries-vanMelleML,LehmannJ,etal.2014.Humanosteoarthriticsynoviumimpacts

chondrogenicdifferentiationofmesenchymalstemcellsviamacrophagepolarisationstate.Osteo-

arthritisCartilage22:1167-1175.

21. Bastiaansen-JenniskensYM,WeiW, Feijt C, et al. 2013. Stimulation of fibrotic processes by the

infrapatellarfatpadinculturedsynoviocytesfrompatientswithosteoarthritis:apossiblerolefor

prostaglandinf2alpha.ArthritisRheum65:2070-2080.

22. GrotenhuisN,VdToomHF,KopsN,etal.2014.Invitromodeltostudythebiomaterial-dependent

reactionofmacrophagesinaninflammatoryenvironment.BrJSurg101:983-992.

23. EnobakhareBO,BaderDL,LeeDA.1996.Quantificationofsulfatedglycosaminoglycansinchondro-

cyte/alginatecultures,byuseof1,9-dimethylmethyleneblue.AnalBiochem243:189-191.

24. Furukawa T, Eyre DR, Koide S, et al. 1980. Biochemical studies on repair cartilage resurfacing

experimentaldefectsintherabbitknee.JBoneJointSurgAm62:79-89.

25. Zheng CH, Levenston ME. 2015. Fact versus artifact: avoiding erroneous estimates of sulfated

glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-

engineeredconstructs.EurCellMater29:224-236;discussion236.

26. YangIH,KimSH,KimYH,etal.2004.Comparisonofphenotypiccharacterizationbetween“alginate

bead”and“pellet”culturesystemsaschondrogenicdifferentiationmodelsforhumanmesenchy-

malstemcells.YonseiMedJ45:891-900.


knee.InstrCourseLect60:461-483.

28. BeekhuizenM,GiermanLM, van SpilWE, et al. 2013.An explorative study comparing levels of

solublemediatorsincontrolandosteoarthriticsynovialfluid.OsteoarthritisCartilage21:918-922.

29. TsuchidaAI, BeekhuizenM,RutgersM, et al. 2012. Interleukin-6 is elevated in synovial fluid of

patientswithfocalcartilagedefectsandstimulatescartilagematrixproductioninaninvitroregen-

erationmodel.ArthritisResTher14:R262.

30. Eymard F, Pigenet A, CitadelleD, et al. 2014. Induction of an inflammatory and prodegradative

phenotypeinautologousfibroblast-likesynoviocytesbytheinfrapatellarfatpadfrompatientswith

kneeosteoarthritis.ArthritisRheumatol66:2165-2174.

CHAPTER 5

Anti-chondrogenic and pro-catabolic effect of

infrapatellar fat pad and its residing macrophages can be modulated by triamcinolone

acetonide

Wu Wei Serdar Capar

Nicole Kops Jan A.N. Verhaar Stefan Clockaerts

Gerjo J.V.M. van Osch Yvonne M. Bastiaansen-Jenniskens

Submitted

80 CHAPTER 5

ABSTRACT

Background

Prolongedinflammationinhibitssuccessfulcartilagerepair.Aninflamedinfrapa-

tellarfatpad(IPFP),andinparticulartheresidingpro-inflammatorymacrophages,

secrete factors that inhibit chondrogenic differentiation and stimulate tissue

degradation.Weaimed to reduce inflammation in the IPFP therebycreating an

environmentmoresuitableforcartilagerepair

Hypothesis

Theanti-chondrogenicandcataboliceffectofIPFPcanbereducedbyinfluencing

macrophagephenotypewithanti-inflammatorymedication

Study design

Controlledlaboratorystudy

Methods

IPFPexplantsobtainedfrom12donorsduringtotalkneearthroplastywerecul-

turedwithcelecoxib,triamcinoloneacetonide(TAA),pravastatinandfenofibrate

for24hours.TheeffectofthemedicationsonIPFPgeneexpressionandmacro-

phagesurfacemarkerswasevaluated.Theeffectsofconditionedmedium(CM)

fromIPFPexplantstreatedwithmedicationsonchondrogenesisofhumanbone

marrowmesenchymalstemcells(MSC)andcatabolicprocessesinosteoarthritic

synovialfibroblastswereevaluated.

Results

TAA significantly decreased gene expression of tumor necrosis factor-alpha

(TNFA),interleukin(IL)-1β(IL1B) and IL6andincreasedexpressionofIL10, cluster

of differentiation (CD)206(CD206) and CD163inIPFPexplants,whereascelecoxib,

pravastatin,andfenofibratehadnoeffectonIPFPinflammation.TAAalsosignifi-

cantlyincreasedanti-inflammatoryCD14+/CD163+andCD14+/CD206+cellsinthe

IPFP.Thedecreaseofcollagentype2(COL2A1)expressioninchondrogenicMSCs

asaresultofCMfromIPFPexplantscouldbereducedbytreatingIPFPwithTAA

duringtheproductionoftheCM.Theincreasedmatrixmetalloproteinase(MMP)1

and MMP13expressioninMSCsandosteoarthriticsynovialfibroblastsasresultof

CMfromIPFPexplantscouldbesignificantlyreducedbytreatingIPFPwithTAA.

Triamcinolone affects infrapatellar fat pad and macrophages 81

5

Conclusions

TAA decreases IPFP inflammation by increasing the anti-inflammatory macro-

phagesinIPFP.TAAcanbeusedtodecreasetheanti-chondrogeniceffectofIPFP

onMSCchondrogenesis.Furthermore,TAAdecreasesthecataboliceffectofIPFP

onMSCsandsynovialfibroblasts.

Clinical relevance

TAAmightbeusedtomodulatetheIPFPandthereforeimprovethejointenviron-

mentforcartilagerepairprocedures.

82 CHAPTER 5

INTRODUCTION

Jointinjuriesarecommonandoftenresultincartilagedefectsandinflammation

of the joint. Joint inflammation in turnmayprevent successful cartilagedefect

repairandwithoutsuccessfulrepair,cartilagedefectscaneventuallyleadtothe

developmentofosteoarthritis(OA)1.Toimprovecartilagerepairandpreventthe

developmentofOA,restorationofthejointhomeostasisisnecessary.

Currently,non-steroidanti-inflammatorydrugssuchascelecoxibandglucocorti-

coidssuchastriamcinoloneacetonide(TAA)areusedtoreduceinflammationand

reduce thesymptomsofOA2.Furthermore, statinsandfibratesarealsoshown

tohaveanti-inflammatorypropertiesbesidestheirmetabolicmodulatoryproper-

ties3-5. The effect of anti-inflammatorymedication on the synoviummembrane,

cartilage and bone are often studied6;7.However,theinfrapatellarfatpad(IPFP)

alsoseemstoplayanimportantroleinjointhomeostasis8.

TheIPFPisanadiposetissuelocatedextra-synovially,butintracapsularlywithin

thekneejoint.TheIPFPsecretesfactorsthatinhibitchondrogenicdifferentiation

ofmesenchymalstemcells(MSCs)9andstimulatefibroticandcatabolicprocesses

insynovialfibroblasts10;11.Furthermore,cytokinesecretionbytheIPFPincreases

in response to inflammation4 and IPFPaggravates furtherdegenerationof trau-

matized cartilage12.The IPFP contains many pro- and anti-inflammatory macro-

phages9;13. Pro-inflammatoryorM1macrophages are indicated to contribute to

theanti-chondrogeniceffectoftheIPFP9.M2macrophagesontheotherhandcan

haveananti-inflammatoryortissuerepairphenotypeandtheydidnotinhibit9;14

andcouldevenenhanceMSCchondrogenesis15.

Toimprovethejointenvironmentforcartilagerepairprocedures,wehypothesized

thatmedicationcanbeusedtomodulatetheIPFPanditsresidentmacrophages.

Inthisway,secretionofanti-chondrogenicfactorsbytheIPFPmightreduce.Fur-

thermore,catabolicandanti-chondrogenicprocessesinsynoviummightalsobe

reducedsincetheIPFPinfluencesthesynovium5;10;11.

Inthecurrentstudy,weinvestigatedtheeffectofcelecoxib,TAA,pravastatinand

fenofibrateonIPFPinflammation.Wechoosethesefourmedicationsduetotheir

wide-spreadclinicaluse.DuetothepresenceofmanymacrophagesintheIPFP

and the importance ofmacrophages in inflammation, we investigatedwhether

macrophagephenotypewithin the IPFPwasmodulatedby themedications. Fi-

nally,weinvestigatedwhethertheanti-chondrogeniceffectofIPFPonMSCsand


5

thepro-fibroticandpro-cataboliceffectonsynovialfibroblastscouldbereduced

bytreatingtheIPFPwiththesemedications.

MATERIALS AND METHODS

Treatment of infrapatellar fat pad and preparation of conditioned medium (CM)

IPFPtissuewasobtainedasleftovermaterialfrom12patientswithOAwhohad

undergoneatotalkneearthroplasty.Consentwasgiveninaccordancewiththe

guidelinesoftheFederationofBiomedicalScientificSocieties(http://www.federa.

org),withapprovalbythelocalethicalcommittee(ErasmusMCUniversityMedi-

calCenter,TheNetherlands,MEC2012-267).TheinnerpartoftheIPFPwascut

into small pieces of approximately 9 mm2, washed two times with saline and

culturedataconcentrationof100mg/ml.Asaresult,eachsamplecontained0.5g

IPFPtissuedividedover10explants.ExplantswereculturedinDulbecco’sModi-

fiedEagleMediumwithGlutamax(DMEM-HG;Gibco™,ThermoFisherScientific,

Waltham,Massachusetts,USA)supplementedwith1% insulin-transferrin-seleni-

um(ITS+;BDBiosciences,FranklinLakes,NewJersey,USA)at37ºC.Allculture

mediawere supplementedwith 1.5 μg/mL fungizone and 50 μg/mL gentamicin

(both Gibco™). Triamcinolone acetonide (TAA; #T6501), celecoxib (#PZ0008),

pravastatin(#P4498)andfenofibrate(#F6020)wereacquiredfromSigma-Aldrich

(St.Louis,Missouri,USA). Dimethylsulfoxide(DMSO;Sigma-Aldrich)wasused

to dissolve all four medications. Used end-concentration are indicated in the

respectivegraphs.Medicationswere furtherdiluted inDMEM-HGandaddedto

IPFPexplantsdirect fromthestartofculture.Theusedendconcentrations for

themedicationswerebasedonliterature16andourpreviouswork17;18.Totakeinto

account anypossible effect ofDMSO,we added extraDMSO to the conditions

where themedicationsweremorediluted.As a result, all conditionshad 0,1%

DMSOintheculturemedium.After24hoursofculture,mediumwasharvested,

centrifugedat250gfor8minutesandsupernatantwasstoredinaliquotsof1ml

at-80ºCforanalysisandcultureexperiments.IPFPexplantswerefurtherusedfor

fluorescence-activatedcellsorting(FACS)analysisorweresnap-frozeninliquid

nitrogenforgeneanalysis.Unconditionedmediumwasgeneratedbyincubation

ofDMEM-HGsupplementedwithITS+for24hoursat37ºC,centrifugedat250g for

8minutesandstoredat-80ºC.

84 CHAPTER 5

Analysis of macrophage phenotype in IPFP

CulturedIPFPexplantsweredigestedfor3hoursat37°CinHanks’BalancedSalt

solution(Gibco™)withCa2+andMg2+,containing2mg/mlCollagenaseIV(Gibco™)

and0.2mg/mlDispaseII(Roche,Penzberg,UpperBavaria,Germany).Weused0.5

gofculturedIPFPpercondition.Afterincubation,thedigestwasneutralizedby

adding fetal calf serum(FCS;Lonza,Basel, Switzerland) toafinal solutioncon-

centrationof5%.Subsequently,thesolutionwasfilteredthrougha100µmfilter

followedbytwicea40µmfilter.Cellswerethencentrifugedfor8minutesat250g,

re-suspendedinFACSflow(#342003BDBiosciences)andcounted.Approximately

200.000cellswerestainedforeachcondition.Cellswerere-suspendedin40μL

ofFACSflowandincubatedfor15minutesatroomtemperatureinthedarkwitha

mixtureofthefollowingantibodiesagainstCD14(APC-H7,#561384),CD206(FITC,

#551135),CD163(PerCP-Cy™5.5,#563887),CD80(PECy™7,#561135),andCD86

(PE, #560957, all BD Biosciences). Cells were fixed in 1.8% paraformaldehyde

for20minutesinthedark.Subsequently,paraformaldehydewasremoved,cells

weresuspendedinFACSflow,andanalyzedwithFACSJazz™(BDBiosciences)and

FlowJoV.10(FlowJoLLC,Ashland,Oregon)

Isolation and culture of human MSCs

HumanMSCswereisolatedfrombonemarrowaspiratesfrom3end-stagehipOA

patientsundergoingtotalhiparthroplasty.Writteninformedconsentwasattained

andthestudywasapprovedbythelocalethicalcommittee(ErasmusMCUniver-

sityMedicalCenter,TheNetherlands,MEC2004-142)andAlbertSchweitzerHos-

pital,TheNetherlands(MEC2011-07).Aspirateswereplatedinexpansionmedium

consistingofMinimumEssentialMedium-Alpha(α-MEM;Gibco™)supplemented

with10%FCS,1ng/mlfibroblastgrowthfactor2(FGF2;R&DSystems)and25μg/

mLascorbicacid-2-phosphate(Sigma-Aldrich).After24hours,plateswerewashed

with PBS containing 2% FCS.MSCswere then expanded in expansionmedium,

trypsinisedafterreachingsub-confluencyandseededagainatadensityof2500

cells/cm2.MSCsofpassage4wereencapsulated in1.2%alginate (CPKelco,At-

lanta,Georgia,USA)insalineatadensityof4.000.000cells/ml.Beadswereformed

bypurgingtheMSC-alginatesolutionthrougha23Gneedleallowingdropletsto

fallinto102mMCaCl2solution.Beadswerefirstculturedfor14dayswith100µL

ofDMEM-HGwithITS+peralginatebeadsupplementedwith0.1µMDexametha-

sone (Sigma-Aldrich), 25 μg/mL ascorbic acid-2-phosphate (Sigma-Aldrich), 40

µg/ml L-Proline (Sigma-Aldrich), and 1mM sodium pyruvate (Gibco™). 10 ng/

mlofTGFβ1(R&Dsystems;Minneapolis,Minnessota,USA)wasusedto induce

chondrogenesis.Mediumwasrefreshed3timesaweek.After14days,beadswere

culturedwith25% IPFPCMorunconditionedmedium.SupplementsandTGFβ1


5

werealwaysaddedaftermixingthemediumwithCMorunconditionedmedium.

Thisway,alltheconditionshadthesamefinalamountofsupplementsandTGFβ1.Beadswereharvestedafter3additionaldaysofcultureandmediumwasrefreshed

24hourspriortoharvest.

Isolation and culture of human fibroblast like synoviocytes

Human synoviumwas obtained as leftovermaterial from 3 end-stage kneeOA

patientswhohadundergonea totalkneearthroplasty (ErasmusMCUniversity

MedicalCenter,TheNetherlands,MEC2008-181).Thesynoviumwasseparated

fromtheunderlyingfat,cutintosmallpiecesanddigestedovernightwith1mg/ml

collagenaseB(Roche,Germany)inIscove’smodifiedDulbecco’smedium(IMDM;

Gibco™)containing10%FCS.Thecellsuspensionwasthenfilteredthrougha100

µmfilter,followedbycentrifugationat250gfor8minutes.Theresultingcellpellet

wasre-suspendedandcellswereplatedat3500cells/cm2inIMDMwith10%FCS.

After reaching sub-confluency, cellswere trypsinised and re-seeded. Fibroblast

like synoviocytes (FLS) of passage 3wereplated asmonolayer at 50.000 cells/

cm2 in IMDM with 10% FCS and allowed to attach. After 72 hours, cells were

washed3timeswithsalineandDMEM-HGcontainingITS+with25%IPFPCMor

unconditioned medium were added. 25 μg/mL ascorbic acid-2-phosphate was

alsoaddedtotheculturemedium.After4daysofculture,mediumwasremoved

andcellswereharvest inRLTbuffer (Qiagen,Venlo,TheNetherlands)with1%

β-mercaptoethanol.Mediumwasrefreshed24hourspriortoharvest.

RNA extraction and gene expression analysis

FrozenIPFPexplantswerehomogenizedwithaMikro-Dismembrator(BraunBio-

techInternationalGmbH,Melsungen,Germany)re-suspendedin1.8mlRNA-Bee

(TelTest,Friendswood,Texas,USA).MSCalginatebeadsweredissolvedinice-cold

55mMsodiumcitrateat4 ºC.Thesolutionwasthencentrifugedat400g for8

minutesat4ºCandcellpelletswerere-suspendedin600µlRNA-Beeperbead.

Chloroform(Sigma-Aldrich)wasaddedtoallsamplesat200µL/mLRNA-Bee.FLS

wereharvestedinRLTbuffer.

RNAfromIPFPexplants,MSCsandFLSwasfurtherpurifiedusingRNeasyMicro

Kit (Qiagen) according to manufacturer’s instructions. RNA concentration and

qualitywasmeasuredusingNanoDropND1000UV-VisSpectrophotometer(Isogen

LifeScience,Veldzicht,TheNetherlands).RNAwasreverse-transcribedtocDNA

usingtheRevertAidFirstStrandcDNASynthesisKit(ThermoFisher)accordingto

manufacturer’sinstructions.qRT-PCRwasperformedin10µLreactionsoncDNA

usingtheCFX96Touch™Real-TimePCRDetectionSystem(Biorad,Hercules,Cali-

fornia,USA).Expressionofglyceraldehyde-3-phosphatedehydrogenase(GAPDH),

86 CHAPTER 5

tumor necrosis factor alpha (TNFA), interleukin-1β (IL1B), IL6, cluster of differ-

entiation (CD)206, CD163 matrix metalloproteinase (MMP)-1, MMP-13, collagen

typeII(COL2A1),aggrecan(ACAN),collagentypeI(COL1A1),procollagen-lysine

2-oxoglutarate5-dioxygenase2(PLOD2),andα-smoothmuscleactin(ASMA)were

determinedwithqRT-PCR.GeneexpressionwascalculatedaccordingtotheΔΔCt

method andGAPDHwas used as housekeeper. Ct values between the samples

withineachexperimentdifferlessthan1Ctandtherewerenosignificantdiffer-

encesbetweentheconditions.0.1%DMSOandtreatmentwithmedicationdidnot

interferewithGAPDHlevel.Unlessotherwisestated,theconditiontreatedwith

unconditionedmediumandDMSOwasusedascontrolandsetat1.0.Expression

levelsofotherconditionsareexpressedrelativetothecontrolcondition.Primer

sequencescanbefoundinthesupplementarymethods,


IBMSPSSstatistics21(IBMCorporation,Armonk,NewYork,USA)wasusedfor

statisticalanalysis.AlldatawasnotnormallydistributedaccordingtotheShapiro-

WilktestandLevene’stestforhomogeneityofvariance.

Ifnototherwisestated,statisticalsignificancebetweenconditionswasdeter-

minedusingtheMann-WhitneyUtest.p<0.05wasconsideredstatisticallysignifi-

cant.

RESULTS

Triamcinolone acetonide reduces IPFP inflammation

Of the four medications tested, only 100 µM TAA significantly reduced TNFA

(p<0.001), IL1B (p<0.001) and IL6 (p<0.001) and increased the expression of

genesencodingforproteinsrelatedtoanti-inflammatoryprocesses,namelyIL10

(p<0.05),CD206(p<0.001)andCD163(p<0.001)inIPFPexplants(Figure1).DMSO

asdiluentof themedicationsdidnot significantly influencegeneexpression in

IPFPexplants(Supplementaryfigure1).

Triamcinolone acetonide reduces IPFP inflammation, even at lower concentrations

BecauseTAAwastheonlymedicationthatsignificantlydecreasedtheexpression

of genes encoding for pro-inflammatory proteins and increased the expression

of genes associatedwith anti-inflammatory processes,we decided to focus on

theeffectsofthismedication.Toreducepossiblenegativeeffectsof100µMTAA,

weinvestigatedwhetheralowerconcentrationofTAAwouldgivethesameanti-


5

inflammatoryresults.Ataconcentrationof0.1µM,TAAstillsignificantlyreduced

TNFA(p<0.05)andIL1B(p<0.05)andincreasedCD206(p<0.05)andCD163(p<0.05)

(Figure2).10and1µMofTAAalsosignificantlyincreasedIL10(p<0.05).IL6expres-

sionwasonlysignificantlyreducedwith10µMofTAAorhigher.TNFA and IL1B

expressionwerenotsignificantlydifferentbetweenthefourconcentrationtested.

TNFA

DMSO0.1

%

Celeco

xib10

mM

Triam. a

cetonide 10

0 mM

Pravas

tatin

50mM

Fenofib

rate 10

0 mM0.0

0.5

1.0

1.5

2.0

2.5

Gen

eex

pres

sion

com

pare

dto

DM

SO

*

IL1B

DMSO0.1

%

Celeco

xib10

mM

Triam. a

cetonide 10

0 mM

Pravas

tatin

50mM

Fenofib

rate 10

0 mM0

1

2

3

4

Gen

eex

pres

sion

com

pare

dto

DM

SO

*

IL6

DMSO0.1

%

Celeco

xib10

mM

Triam. a

cetonide 10

0 mM

Pravas

tatin

50mM

Fenofib

rate 10

0 mM0.0

0.5

1.0

1.5

2.0

Gen

eex

pres

sion

com

pare

dto

DM

SO

*

IL10

DMSO0.1

%

Celeco

xib10

mM

Triam. a

cetonide 10

0 mM

Pravas

tatin

50mM

Fenofib

rate 10

0 mM0

1

2

3

4

Gen

eex

pres

sion

com

pare

dto

DM

SO

*CD206

DMSO0.1

%

Celeco

xib10

mM

Triam. a

cetonide 10

0 mM

Pravas

tatin

50mM

Fenofib

rate 10

0 mM0

2

4

6

8

10

Gen

eex

pres

sion

com

pare

dto

DM

SO *

CD163

DMSO0.1

%

Celeco

xib10

mM

Triam. a

cetonide 10

0 mM

Pravas

tatin

50mM

Fenofib

rate 10

0 mM0

2

4

6

8

10

Gen

eex

pres

sion

com

pare

dto

DM

SO *

Figure 1 TriamcinoloneacetonidereducesIPFPinflammationIPFPexplantsweretreatedfor24hourswithDMSO,celecoxib,triamcinoloneacetonide,pravastatinorfenofi-brate.GeneexpressionofTNFA, IL1B, IL6, IL10, CD206, and CD163inIPFPexplantsaftertreatment,comparedtotheDMSO0.1%treatedcondition.ThegeneexpressionofDMSO0.1%treatedsamplewassetat1.IPFPfrom3donorswereculturedwith2samplesperdonor(n=6).Valuesaremean±SD.*p<0.05versusDMSOtreatedcondition.

Triamcinolone acetonide modulates macrophage phenotype in IPFP

NextweinvestigatedwhetherTAAmodulatesthephenotypeofthemacrophages

in the IPFP,sincemacrophagesareknowntobe important in inflammation.We

investigatedtheeffectof10and1µMonmacrophagesbecausethesetwocon-

centrationsalreadydecreasedpro-inflammatoryandincreasedanti-inflammatory

genesexpression.10µMofTAAsignificantlyincreasedthepercentageofCD14+/

CD206+andCD14+/CD163+cellsintheIPFP,indicatingmacrophageswithanM2-

likephenotype(Figure3).However,TAAdidnotalterthepercentageofCD14+/

CD80+andCD14+/CD86+cellsintheIPFP(Figure3),indicatingthatthepercentage

ofmacrophageswithanM1-likephenotypedidnotchange.

88 CHAPTER 5

TNFA

0.0

0.5

1.0

1.5

Gen

eex

pres

sion

com

pare

dto

DM

SO

10 mM 0.1 mM100 mM 1 mM

+ triamcinolone acetonide

* ** *

0.1%DMSO

IL1B

0.0

0.5

1.0

1.5

Gen

eex

pres

sion

com

pare

dto

DM

SO

10 mM 0.1 mM100 mM 1 mM


* ** *

0.1%DMSO

IL6

0.0

0.5

1.0

1.5

Gen

eex

pres

sion

com

pare

dto

DM

SO

10 mM 0.1 mM100 mM 1 mM


**

0.1%DMSO

IL10

0

1

2

3

Gen

eex

pres

sion

com

pare

dto

DM

SO

10 mM 0.1 mM100 mM 1 mM


**

0.1%DMSO

*

*

CD206

0

2

4

6

8

10

Gen

eex

pres

sion

com

pare

dto

DM

SO

10 mM 0.1 mM100 mM 1 mM


**

*

*

0.1%DMSO

CD163

0

2

4

6

8

10

Gen

eex

pres

sion

com

pare

dto

DM

SO

10 mM 0.1 mM100 mM 1 mM


* * * *

0.1%DMSO

Figure 2 TriamcinoloneacetonidereducedIPFPinflammationindoserangeof0.1-100µMIPFPexplantsweretreatedfor24hourswithtriamcinoloneacetonideintheconcentrations0.1µM-100µM.GeneexpressionofTNFA, IL1B, IL6, IL10, CD206, and CD163inIPFPexplantsaftertreatment,comparedtothecondi-tiontreatedwith0.1%DMSOonly.Geneexpressionin0.1%DMSOonlywassetat1.IPFPfrom2donorswereculturedwith3samplesperdonor(n=6).Valuesaremean±SD.*p<0.05versus0.1%DMSOtreatedsamples

CD80

Perc

enta

geof

CD

14+

cells

com

pare

dto

DM

SO

0

1

2

3

0.1% DMSO 10 mM


1 mM

CD86

Perc

enta

geof

CD

14+

cells

com

pare

dto

DM

SO

0.0

0.5

1.0

1.5

10 mM


1 mM0.1% DMSO

CD163

Perc

enta

geof

CD

14+

cells

com

pare

dto

DM

SO

0

2

4

6

8

10

*

10 mM


1 mM0.1% DMSO

*CD206

Perc

enta

geof

CD

14+

cells

com

pare

dto

DM

SO

0.0

0.5

1.0

1.5

2.0

2.5

*

10 mM


1 mM0.1% DMSO

Figure 3 TriamcinoloneacetonidemodulatesmacrophagesinIPFP.IPFPexplantsweretreatedfor24hourswithtriamcinoloneacetonideintheconcentrations10µMand1µM.Intheseexplants,percentageofCD80,CD86,CD206andCD163positivecellswithintheCD14positivepopulation(indicatingmacrophages)wasdeterminedusingflowcytometry.Relativepercentageofpositivecellsaftertreatment,comparedtotheconditiontreatedwith0.1%DMSOonly.Conditiontreatedwith0.1%DMSOonlywassetat1.Valuesarefrom5IPFPdonorswith1-3samplesperdonor(n=12).Valuesaremean±SD.*p<0.05versus0.1%DMSO.


5

Triamcinolone acetonide reduces anti-chondrogenic and pro-catabolic effects of IPFP

Finally,we investigatedwhetherTAAcan reduce thenegativeeffectof IPFPon

chondrogenesisofMSCsandthefibroticandcatabolicprocessesinfibroblastlike

synoviocytes (FLS). Factors secretedby untreated IPFP significantly decreased

COL2A1(p<0.05)inchondrogenicallystimulatedMSCs(Figure4A).Treatmentof

theIPFPwith1µMand10µMTAAwhilemakingtheCMsignificantlyreducedthisef-

fect,althoughnotentirely.FactorsproducedbyIPFPsignificantlyincreasedMMP1

and MMP13inMSCs(p<0.05)(Figure4A)andFLS(p<0.05)(Figure4B).Treatment

oftheIPFPwithTAAduringmakingoftheCMsignificantlyreducedtheexpres-

sionofthesegenesinMSCsandFLS.SinceTAAwasalsopresentinthemedium

conditionedbyIPFP,wealsoaddedTAAdirectlytothechondrogenicstimulated

MSCalginatebeadsandtheFLS.COL2A1 and ACAN were significantly reduced in

MSCsbyTAA.COL1A1,ASMA,MMP1, and MMP13 were significantly reduced and

PLOD2significantlyincreasedinFLSbyTAAtreatment(Supplementaryfigure2).

Thissuggests thatTAAmainlyactsbyreducing theanti-chondrogeniceffectof

othercellsonMSCsandnotthroughstimulationofchondrogenesisinMSCs.

COL2A1

TGFb1only

+0.1%

DMSO+C

M

+CM

+10mM

TAA

+CM

+1mM

TAA0.0

0.5

1.0

1.5

**

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+10mM

TAA

+CM

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TAA0.0

0.5

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+10mM

TAA

+CM

+1mM

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4

6

8

10

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+10mM

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+1mM

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2

3

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+10mM

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FLS only

+0.1%

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+CM

+10mM

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+CM

+1mM

TAA0.0

0.5

1.0

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+0.1%

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M

+CM

+10mM

TAA

+CM

+1mM

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1

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3

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5

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+0.1%

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* *##

A

B

Figure 4 Triamcinolone acetonide reduces the anti-chondrogenic and pro-catabolic effects ofIPFP.FLSandchondrogenicstimulatedMSCalginatebeadsweretreatedwithCMfromIPFPtreatedwithoutorwithTAAintheconcentrations10µMand1µM.A)COL2A1, ACAN, MMP1, and MMP13expressioninchondrogenicstimulatedMSCalginatebeadsrelativetotheconditiontreatedwithTGFβ1only.B)COL1A1, PLOD2, ASMA, MMP1, and MMP13expressioninFLSrelativetotheconditionwithonlyFLSinculture.GeneexpressioninTGFβ1onlyandFLSonlyconditionsweresetat1.3differentpoolsofCMofeachfrom3IPFPdonorswereusedtotreat3MSCdonorsand3FLSdonors,witheachconditionperformedintriplicate(n=9).Valuesrepresentmean±SD.*p<0.05versusnon-treatedcontrols(TGFb1onlyorFLSonly).#p<0.05versustreatedwithCMonly.

90 CHAPTER 5

DISCUSSION

Inthisstudy,weinvestigatedwhetheranti-inflammatorymedicationscanreduce

IPFP inflammationand therebyprevent thenegativeeffectof IPFPonchondro-

genic MSCs9 and synovial fibroblasts10;11. We have tested four commonly used

medications and found that TAA, a glucocorticoid, reduced IPFP inflammation

andmodulatedIPFPmacrophagephenotype.Furthermore,TAAreducedtheanti-

chondrogeniceffectofIPFPonMSCsandthepro-cataboliceffectonFLS.

Inflammationplaysamajorinhibitoryroleinjointrepair1andmacrophagesplay

animportantroleininflammation19.Nexttothesynovium,whichisoftenstudied

inrelationtojointinflammation,theIPFPcanbecomeinflamed.TheIPFPcontains

a largenumberofmacrophages,andwasproposed tostimulatesynovialfibro-

sis10 and inhibit of chondrogenesis during cartilage repair9.Inthecurrentstudy

study,TAAnotonlydecreasedexpressionofgenesencodingforpro-inflammatory

proteins in IPFPbut also increased the percentage ofmacrophages expressing

CD163andCD206proteinontheirsurfaceintheIPFP.Anti-inflammatoryorM2-like

macrophagesexpressCD163andCD206.Furthermore,theyexpressfactorsthat

reduce inflammationor stimulate tissue repair, such as IL10orCCL1818; 20. The

increaseofM2-likemacrophagesinIPFPcouldeitherdirectlyorindirectlyaffect

theM1-likemacrophages.Asaresult,thechangeinfavourofM2-likemacrophages

mighthavecontributedtothealteredgeneexpressionofinflammatorycytokines.

Inthisway,theanti-chondrogeniceffectofIPFPonMSCandpro-cataboliceffect

onFLScanbereduced.

Theusedmedicationwaschoosenfortheiranti-inflammatoryeffects.However,

celecoxib,pravastatinandfenofibratedidnotinfluenceIPFPinflammationfocus-

ing on genes known to be expressedbydifferentmacrophagephenotypes.We

thereforedecidednottocontinuewithcelecoxib,pravastatin,andfenofibratein

the current study, as inflammation driven by themacrophages seem to play a

large role in the negative effect of IPFP on joint tissues9; 10.We cannot exclude

however that celecoxib, pravastatin and fenofibrate might have an effect on

anti-chondrogenic and catabolic properties of IPFP through inhibition of other

pathwayslessrelatedtoinflammationandviaothercellsthanthemacrophages.

TAAiscurrentlyinjectedintra-articularlyastreatmentofpainandinflammation

inearly-OA.Althoughsymptomaticreliefdoesoccur,nostructuralmodifyingef-

fect on cartilage is seen6.TAAdoesreducecatabolicprocessesinOAsynovium

explantsco-culturedwithcartilageexplants17andin-vivoratstudieshaveshown


5

thatintra-articularinjectionofTAAcanreducepost-traumaticarthrofibrosis21;22.

TAAinjectedintra-articularlydirectlyafterjointtraumacouldalsoreducesyno-

vitis23. Furthermore, unlike other glucocorticoids, it is not toxic to cartilageor

synovium24-26. There are cases of adipose tissue atrophy described after TAA

injection27.AsystematicreviewbyBrinksetalshowedthattheseadverseevents

arerareandifpresent,arerelativelymild28.RepeatedinjectionsofTAAhowever,

increasedcollagentype2andaggrecanreleaseandturnoverinkneecartilageof

horses29.ToreducethenegativeeffectsofTAAoncartilage,adosageaslowaspos-

sibleispreferred.Inourcurrentstudy,expressionofGAPDH was not influenced

bytreatmentwithanyoftheconcentrations,suggestingthattheconcentrations

ofTAAweusedhadnogrossoveralleffectoncellmetabolism.Furthermore,TAA

influencedIPFPinflammationataconcentrationof0.1µM,andreducedtheanti-

chondrogenicandpro-cataboliceffectsofIPFPat1µM.Thissuggeststhatalower

doseofTAissufficienttomodulatetheIPFP.

Inrabbits,asingleintra-articularinjectionoftheglucocorticoiddexamethasone

reducesIPFPcellularityandfibrosis48hoursafterjointtrauma.Howeveritdidnot

reduce IL1BexpressionnorwereeffectsonIPFPcellularityandfibrosisstillpresent

after9weeks30.Therefore,ahigherdosageand/ormultipleintra-articularinjec-

tionsofmedicationmightbeneededbeforeaprolongedeffectivelevelisreached

intheIPFP.MultipleinjectionsthatresultinahigherconcentrationofTAA,might

however,benegativeforcartilageandMSCs31.Toreducethenumberofinjections

andtheside-effectsofahighdosage,aslow-releasedrugdeliverysystemdirectly

in the IPFPcouldbeasolution. Besidesasustaineddeliverysystem, the time

ofdeliveryisalsocrucial.Directlyafterjointtrauma,levelsofpro-inflammatory

factorsinthesynovialfluidpeak32;33.Thelevelofthesepro-inflammatoryfactors

dropsovertime,butarestillhigherininjuredkneesthaninuninjuredkneesupto

1yearafterinjury34.ThetimingofadditionofTAAisthereforeimportantaswell

astherequireddosage,whichcouldchangeovertimedependingontheexpected

levelofinflammation.

Taken together, TAA reduces IPFP inflammation and the anti-chondrogenic and

pro-cataboliceffectoftheIPFPonthejoint.Thisispossiblycausedbymodulation

ofmacrophagesintheIPFP.ThereforeTAAisapromisingmedicationtomodulate

thejointenvironment,therebycreatingabetterenvironmentforsuccessfulcar-

tilagerepair.

92 CHAPTER 5

REFERENCES

1. ZhangY,PizzuteT,PeiM.2014.Anti-inflammatorystrategiesincartilagerepair.TissueEngPartB

Rev20:655-668.

2. BannuruRR,SchmidCH,KentDM,etal.2015.Comparativeeffectivenessofpharmacologicinter-

ventionsforkneeosteoarthritis:asystematicreviewandnetworkmeta-analysis.AnnInternMed

162:46-54.

3. AbeM,MatsudaM,KobayashiH, et al. 2008. Effectsof statinson adipose tissue inflammation:

theirinhibitoryeffectonMyD88-independentIRF3/IFN-betapathwayinmacrophages.Arterioscler

ThrombVascBiol28:871-877.




5. vanEekerenIC,ClockaertsS,Bastiaansen-JenniskensYM,etal.2013.Fibratesastherapyforosteo-

arthritisandrheumatoidarthritis?Asystematicreview.TherAdvMusculoskeletDis5:33-44.

6. EvansCH,KrausVB,SettonLA.2014.Progressinintra-articulartherapy.NatRevRheumatol10:11-

22.

7. ScottiC,GobbiA,KarnatzikosG,etal.2016.CartilageRepairintheInflamedJoint:Considerations

forBiologicalAugmentationTowardTissueRegeneration.TissueEngPartBRev22:149-159.



18:876-882.

9. WeiW,RudjitoE,FahyN,etal.2015.Theinfrapatellarfatpadfromdiseasedjointsinhibitschondro-

genesisofmesenchymalstemcells.EurCellMater30:303-314.

10. Bastiaansen-JenniskensYM,WeiW, Feijt C, et al. 2013. Stimulation of fibrotic processes by the

infrapatellarfatpadinculturedsynoviocytesfrompatientswithosteoarthritis:apossiblerolefor

prostaglandinf2alpha.ArthritisRheum65:2070-2080.




12. He J, Jiang Y, Alexander PG, et al. 2017. Infrapatellar fat pad aggravates degeneration of acute

traumatizedcartilage:apossibleroleforinterleukin-6.OsteoarthritisCartilage25:138-145.






15. SesiaSB,DuhrR,MedeirosdaCunhaC,etal.2015.Anti-inflammatory/tissuerepairmacrophages

enhance the cartilage-forming capacity of human bone marrow-derived mesenchymal stromal

cells.JCellPhysiol230:1258-1269.

16. ToyodaT,KameiY,KatoH,etal.2008.Effectofperoxisomeproliferator-activatedreceptor-alpha

ligandsintheinteractionbetweenadipocytesandmacrophagesinobeseadiposetissue.Obesity

(SilverSpring)16:1199-1207.

17. BeekhuizenM,Bastiaansen-JenniskensYM,KoevoetW,etal.2011.Osteoarthriticsynovialtissue

inhibitionofproteoglycanproductioninhumanosteoarthritickneecartilage:establishmentand

characterizationofalong-termcartilage-synoviumcoculture.ArthritisRheum63:1918-1927.


5

18. UtomoL,vanOschGJ,BayonY,etal.2016.Guidingsynovialinflammationbymacrophagepheno-

typemodulation:an invitrostudytowardsatherapyforosteoarthritis.OsteoarthritisCartilage

24:1629-1638.

19. HamidzadehK,ChristensenSM,DalbyE,etal.2017.MacrophagesandtheRecoveryfromAcute

andChronicInflammation.AnnuRevPhysiol79:567-592.



21. ClarkDD,WeckesserEC.1971.Theinfluenceoftriamcinoloneacetonideonjointstiffnessintherat.

JBoneJointSurgAm53:1409-1414.

22. EfirdW,KellamP,YeazellS,etal.2014.Anevaluationofprophylactictreatmentstopreventpost

traumaticjointstiffness.JOrthopRes32:1520-1524.

23. SiekerJT,AyturkUM,ProffenBL,etal.2016.ImmediateAdministrationofIntraarticularTriamcino-

loneAcetonideAfterJointInjuryModulatesMolecularOutcomesAssociatedWithEarlySynovitis.

ArthritisRheumatol68:1637-1647.

24. ShermanSL,JamesC,StokerAM,etal.2015.InVivoToxicityofLocalAnestheticsandCorticoste-

roidsonChondrocyteandSynoviocyteViabilityandMetabolism.Cartilage6:106-112.

25. ShermanSL,KhazaiRS,JamesCH,etal.2015.InVitroToxicityofLocalAnestheticsandCorticoste-

roidsonChondrocyteandSynoviocyteViabilityandMetabolism.Cartilage6:233-240.

26. SolaM,DahnersL,WeinholdP,etal.2015.Theviabilityofchondrocytesafteraninvivoinjectionof

localanaestheticand/orcorticosteroid:alaboratorystudyusingaratmodel.BoneJointJ97-B:933-

938.

27. ImagawaK,OhkumaS.2010.Acaseoffatinjectionfortreatingsubcutaneousatrophycausedby

localadministrationofcorticosteroid.TokaiJExpClinMed35:66-69.

28. BrinksA,KoesBW,VolkersAC,etal.2010.Adverseeffectsofextra-articularcorticosteroidinjec-

tions:asystematicreview.BMCMusculoskeletDisord11:206.

29. CelesteC,IonescuM,RobinPooleA,etal.2005.Repeatedintraarticularinjectionsoftriamcinolone

acetonidealtercartilagematrixmetabolismmeasuredbybiomarkersinsynovialfluid.JOrthop

Res23:602-610.

30. HeardBJ,SolbakNM,ChungM,etal.2016.Theinfrapatellarfatpadisaffectedbyinjuryinduced

inflammationintherabbitknee:useofdexamethasonetomitigatedamage.InflammRes65:459-470.

31. WylesCC,HoudekMT,WylesSP,etal.2015.Differentialcytotoxicityofcorticosteroidsonhuman

mesenchymalstemcells.ClinOrthopRelatRes473:1155-1164.



33. CatterallJB,StablerTV,FlanneryCR,etal.2010.Changesinserumandsynovialfluidbiomarkers

afteracuteinjury(NCT00332254).ArthritisResTher12:R229.



94 CHAPTER 5

TNFAIL1B IL6

IL10

CD206

CD163

0

1

2

3

4

Gen

eex

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rela

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toco

ntro

l

Control+ DMSO 0.1%+ DMSO 0.01%

Supplementary figure 1DMSOdoesnotsignificantlyaffectgeneexpressioninIPFPexplants.IPFPexplantsweretreatedfor24hourswithDMSO.GeneexpressionofTNFA, IL1B, IL6, IL10, CD206 and CD163 comparedtocontrol.Geneexpressionofcontrolwassetat1.Resultsarefrom3donorswith2samplesperdonor(n=6).Valuesaremean±SD.*p<0.05versuscontrol.

COL2A1

TGFb1only

+0.1%

DMSO

+10mM

TAA

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+0.1%

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+10mM

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+0.1%

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+10mM

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FLS only

+0.1%

DMSO

+10mM

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+1mM

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+10mM

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+0.1%

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+10mM

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+0.1%

DMSO

+10mM

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TAA0.0

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+0.1%

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A

B

Supplementary figure 2TriamcinoloneacetonidehasaneffectonMSCandFLS.FLSandchondrogenicstimulatedMSCalginatebeadsweretreatedwithTAAintheconcentrations10µMand1µM.A)COL2A1, ACAN, MMP1, and MMP13expressioninchondrogenicstimulatedMSCalginatebeadsrelativetotheconditiontreatedwithTGFβ1only.B)COL1A1, PLOD2, ASMA, MMP1, and MMP13inFLSrelativetotheconditionwithonlyFLSinculture.GeneexpressioninTGFβ1onlyandFLSonlyconditionsweresetat1.TAAwasusedtotreat3MSCdonorsand3FLSdonors,witheachconditionperformedintriplicate(n=9).Valuesrepresentmean±SD.*p<0.05versusnon-treatedcontrols(TGFb1onlyorFLSonly).

PART II

Systemic effects of adipose tissue

CHAPTER 6

Statins and fi brates do not affect development

of spontaneous cartilage damage in STR/Ort mice

Wu Wei Stefan Clockaerts

Yvonne M. Bastiaansen-Jenniskens Lobke M. Gierman

Sander M. Botter Sita M.A. Bierma-Zeinstra

Harrie Weinans Jan A.N. Verhaar

Margreet KloppenburgAnne-Marie Zuurmond

Gerjo J.V.M. van Osch

Osteoarthritis Cartilage. 2014 Feb;22(2):293-301

100 CHAPTER 6

ABSTRACT

Objective

Sincestatinsandfibratesarecapableof improving themetabolicprofileofpa-

tientsaswellasdecreasinginflammation,theyareconsideredaspotentialdrugs

forpreventingOA.Thegoalofthepresentstudywastoinvestigatetheeffectof

thesedrugsintheSTR/OrtspontaneousOAmousemodel.

Design

MaleSTR/Ortmicereceivedcontroldietorcontroldietcontainingtwodifferent

dosages of simvastatin or fenofibrate or a combination of both.Micewere eu-

thanizedafter16weeksoftreatmentattheageof24weeks.Serumanalysisfor

metabolicandinflammatorymarkers,histologicOAgradingandμCTanalysisof

subchondralboneplatewereperformed.

Results

Simvastatintreatmentdidnothaveastatisticallysignificanteffectonanyofthe

measuredparameters.Fenofibratetreatedmicegainedlessbodyweightandhad

lowerserumamyloidA(SAA)levels,buthigherIL1αandMIP1αthanothermice.

Micetreatedwith200mg/kgBW/dayfenofibratehadlesssubchondralboneplate

volume thancontrol,butno statistically significant reduction incartilagedam-

age.Inthecombinationtreatmentgroup,bodyweightandSAAwerelowerthan

control.

Overall,bodyweight,synoviummembranecelllayersandSAAlevelscorrelated

to subchondral bone plate changes and subchondral bone plate changes corre-

latedtocartilagedamage.

Conclusions

StatinsandfibratesdidnotaffectdevelopmentofcartilagedamageintheSTR/Ort

spontaneousosteoarthritismousemodel.Fenofibrateshowever,hadaneffecton

bodyweight,seruminflammationmarkersandsubchondralboneplatemorphol-

ogy.

Statins and fibrates in the STROrt mouse model 101

6

INTRODUCTION

Thecurrentnonsurgicaltreatmentoptionsforosteoarthritis(OA)arelimitedto

nonpharmacologicalinterventionssuchaslifestylechanges,exerciseorweight

reduction,withpharmacological treatmentofsymptomsifneeded.Noeffective

diseasemodifyingdrugiscurrentlyonthemarketforOA1.OAisacommonjoint

diseasethataffectstheentirejoint,includingthearticularcartilage,subchondral

boneandthesynovialmembrane.Highageandobesityarebothmajorriskfactors

ofOA.Prevalenceandincidencefiguresareexpectedtorisewithincreasinglife

expectancyandgrowingobesity2;3.

Themetabolic syndrome is a concurrenceofobesity,hyperinsulinemia,dyslip-

idemia and hypertension4.Metabolic OA is considered one of the subtypes of

OAandisassociatedwiththemetabolicsyndrome.Besideshyperglycaemiaand

increasedserumlipids,alsothevascularpathologyassociatedwiththemetabolic

syndromemighthavenegativeeffectsonthecartilage5.

Statins are hydroxylmethylglutaryl coenzyme-A reductase inhibitors that de-

creaseserumcholesterollevels,reducesystemicinflammation,anddecreasethe

amountofcardiovascularevents6;7.FibratesareligandsofPeroxisomeProliferator

ActivatedReceptor(PPAR)α.Thesedrugsdecreaseserumtriglyceridesandalso

exert anti-inflammatory effects on different tissues. Like statins, they decrease

inflammatoryprocessesincartilage,synoviumandintra-articularadiposetissue

inin-vitrostudies8-14.Chroniclowgradeinflammationplaysanimportantrolein

thepathogenesisofOAandmetabolicsyndrome4.Sincestatinsandfibratescan

potentially inhibit inflammatory processes in the joint as well as improve the

metabolicprofileofpatients,theymightofferapotentialtherapeuticorpreven-

tive strategy forOA.Recently,weassociated theuseof statinswithdecreased

incidenceandslowerprogressionofkneeOAintheRotterdamStudyonx-rays15.

KadametalassociatedtheuseofstatinwithdecreasedincidenceofclinicalOA

intheGeneralPracticeResearchDatabasefromtheUnitedKingdoms16.Nodirect

associationbetweentheuseoffibratesandincidenceofOAhasbeendescribed

yet17.Beforestartingaclinicaltrialthatdirectlyaddressesthequestionwhether

statinsandfibratesareabletopreventOAorOAprogression,anin-vivoanimal

studyshouldbeperformed.

IntheSTR/Ortmousestrain,80%ofthemalemicespontaneouslydevelopOAin

thetibiofemoraljointattheageof6month,particularlyatthemedialcondyleswith

similarhistopathologicallesionsasinhumans18.STR/Ortmicealsospontaneously

102 CHAPTER 6

develop obesity19. Furthermore, serum levels of total cholesterol, high density

lipoprotein(HDL)cholesterol,LDLcholesterol, triglycerides,nonesterified fatty

acids,glucoseandinsulinarereportedtobeincreasedinSTR/Ortmicecompared

toC57BL/6Jmice19.InadditionithasbeensuggestedthatSTR/Ortmicedevelop

OAspontaneouslyduetothereductionofendogenousPPARαorPPARγsignalling,andthuspossiblyincreasinginflammationandalteringosteoblastphenotype20.

Forthesereasons,theSTR/Ortmousemodelseemseminentlysuitedtoinvesti-

gatetheeffectsofstatinsandfibratesonOAdevelopment. Inthisstudy,wein-

vestigatedwhetheradministrationofsimvastatinorfenofibrateoracombination

ofbothwouldpreventordelaythedevelopmentofOAinthetibiofemoraljoint.

Weevaluatedtheeffectofthistreatmentonserumcholesterol,triglyceride,the

inflammationmarkerserumAmyloidA(SAA)andtheinflammatorycytokinesin

theblood.Cartilagedamagewasevaluatedwithhistologyandjointbonemorphol-

ogywithmicrocomputedtomography(μCT).

METHODS

Animals

Four-week-oldmaleSTR/Ortmice(n=72)acquiredfromHarlanItaly(Udine,Italy)

were maintained at the animal testing facilities of the ErasmusMC University

MedicalCenter,TheNetherlands.Micewerehousedingroupsofthreemiceper

cageunderstandardconditionsandhadaccesstowaterandfoodadlibitum.The

studyprotocolwasapprovedbytheinstitutionalAnimalCareandUseCommittee

(ErasmusMCUniversityMedicalCenter,TheNetherlands,AEC116-10-01)

Diets and drug intake

Mice were fed semi-synthetic non-irradiatedmice reference diet (AbDiets,Wo-

erden,TheNetherlands)for4weeksbeforefeedingtheexperimentaldiets.Body

weight(BW)wasmeasuredeverytwoweeksandfoodwasreplaced2-3timesper

week.AverageBWpermouseandaverage food intakeper cageperday could

thusbedeterminedandwecouldcalculatetheamountofdrugs(mg/kg)needed

tobemixedwithreferencedietforthemicetoreceivetheintendedtargetdosage

(mg/kgBW/day).Attheageof8weeks,themiceweredividedinto6experimental

groupsofn=12.Group1(control)continuedtoreceivethenon-supplemented

referencediet.Group2and3receivedreferencedietmixedwitheither300or750

mg/kgsimvastatin(Eurogenerics,Brussel,Belgium),whichcorrespondstodos-

agesof40and100mg/kgBW/day.Groups4and5receivedreferencedietmixed


6

witheither600or1500mg/kgfenofibrate(Eurogenerics,Brussel,Belgium),which

correspondstodosagesof80and200mg/kgBW/day.Finally,group6receiveda

combinationofsimvastatin(300mg/kg)andfenofibrate(600mg/kg)withdosages

40and80mg/kgBW/dayrespectively.Targetdosagesofeachdrugwerebased

uponwhatisoftenusedintheliteraturetostudytheanti-inflammatoryorlipid

lowering effects of these drugs 21.Allexperimentaldietswereprovidedandmixed

byABDiets(Woerden,TheNetherlands)andwerestoredat4ºCforamaximumof

26weeks.Attheageof6monthsthemiceweresacrificedviaisofluraneanesthesia

andheartpuncturing.

Onemouse in the simvastatin 40mg/kg BW/day treatment group rapidly lost

weight andwaseuthanized2weeksbefore theendof the study.Nevertheless,

havingcompleted>90%ofthestudyperiod,wedecidedtoincludethedatafrom

thismouseinallanalyses.

Serum analysis

Bloodwascollectedthroughacheekpuncturepriortothestartofexperimental

diet.Atendpoint,bloodwascollectedthroughaheartpuncture.Bloodwasstored

for1houratroomtemperature,followedby30minutesat4ºC,thencentrifuged

for5minutesat12.000rpmandserumwasremovedandimmediatelystoredat

-80ºC. Total plasma cholesterol (RocheDiagnostics kit no.1489437) andplasma

triglyceride(Rochediagnosticskitno.1488872)weredeterminedafterdefrosting.

SerumAmyloidA(SAA)wasalsodeterminedineachsamplebyELISA(Tridelta

development, Maynooth, Ireland) according to the manufacturer’s instruction.

Mouseinflammatorycytokines(FGFbasic,GM-CSF,IFN-γ,Interleukin(IL)-1α,IL-1β,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12p40/p70,IL-13,IL-17,IP-10,KC,MCP-1MIG,MIP-1α,TNF-αandVEGF)weremeasuredusingamagnetic20-plexbeadassay(Invitrogen,

Frederick,MD,USA)accordingtomanufacturer’sinstructions.

Duetotechnicalproblemsduringsampleprocessing,theserumsamplesofone

mouseinthecontrolgroupandtwoofthesimvastatin40mg/kgBW/daytreatment

groupwerelost.

µCT analysis

Thelefthindlegsofallanimalswereexcisedandfixedin4%bufferedformalinfor

µCTandhistology.WeusedtheSkyscan1176X-raymicrotomographandcomple-

mentarydedicatedsoftware(Brukermicro-CT,Kontich,Belgium)toanalyzethe

subchondralbonemorphologyintheproximaltibiaoftheleftknee.Thefollowing

scansettingswereused:voltage100kV;current100µA;filter0.5mmaluminium;

imagepixel size 17.92 μm; exposure time 2360ms; frame averaging 3;with 0.4

degree rotation through 180degrees. Following scanning, datasetswere recon-

104 CHAPTER 6

structedusingNRecon(Bruker-Skyscan).Segmentationwasperformedwithalo-

calthresholdalgorithm22andusingCTAnalyser(Bruker-Skyscan),themedialand

lateralsubchondralboneplate(regionofinterestdepth:1.61mmventro-dorsal,

starting fromtheanterior intercondylararea; regionof interestwidth:adjusted

accordingtototalmedio-lateralwidthineachtibia,thensplitinhalfinthemiddle

oftheintercondylareminence,yieldingalateralandamedialpart)wereselected

asregionsofinterest.Finally,CTAnalyserwasusedtocalculatesubchondralbone

plate bone volume (BV) and subchondral bone plate thickness, as described

previously23.

Histological analysis

AfterμCTscanning,thelegsweredecalcifiedin10%formicacidindistilledwater

for10daysandembeddedinparaffinand6µmthickcoronal(frontal)histological

sectionswere cut at 100µm intervals through the joint. Sectionswere stained

with thionine. Two independent blinded observers scored cartilage damage

atthemedialandlateralsidesofboththefemurandthetibiaaccordingtothe

semi-quantitativescoringsystemdevisedbytheOsteoarthritisResearchSociety

International(OARSI)histopathologyinitiative24.Briefly,thisscoringsystemhas

arangefrom0to6,with0meansnormalcartilage,1smallfibrillationswithout

lossofcartilageand3-6erosionstothecalcifiedcartilageextendingfrom<25%

to>75%.Onlyslideswhereallfour(i.e.themedialandlateralsidesofboththe

femurandtibia) locationswerevisiblewereused.Themeanscoreof threedif-

ferentslideswasdeterminedandincaseofa>0.5scorediscrepancybetweenthe

firstobservers,athirdindependentobserverrescoredtheslides.Todetermine

whether treatment has an effect on synovial inflammation, the number of cell

layersofthesynoviummembraneonthemedialsideofthepatellawascounted.

Themeannumberofcelllayersofthreedifferentslideswasusedinthestatistical

analysis.

Duetotechnicalerrorduringanalysis,thekneesofonecontrolmouseandone

combinationtreatedmousewerelost.

Data analysis

Statistical analysis was performed in IBM SPSS statistics 20 (IBM Corporation,

Armonk,NewYork,USA)andR:Alanguageandenvironmentforstatisticalcom-

puting 3.0.2 (R Development Core Team, Vienna, Austria). Data was tested for

normalityusingtheShapiro-Wilktestandforhomogeneityofvarianceusingthe

Levene’stest.TheonewayANOVAwithpost-hocBonferronicorrectionwasthen

usedforBWatendpoint,serumtotalcholesterolandboneparametersbecause

theseparameterswerenormallydistributedandhadhomogeneityof variance.


6

Forotherserumparameters,foodintakeandhistologicaldata,theKruskal-Wallis

withpost-hocBonferronicorrectionwasused.The95%confidenceinterval(CI)

ofthedifferencebetweenconditionswhentheKruskal-Wallistestwasused,was

calculatedusingthebootstrapmethod.ToassesstherelationshipbetweenBWat

endpoint,serumtotalcholesterolandμCTdata,thePearson’scorrelationcoeffi-

cientwasusedbecausetheseparameterswerenormallydistributed.Forallother

associationanalyses,theSpearman’scorrelationcoefficientwasused.p<0.05was

consideredstatisticallysignificant.

RESULTS

Simvastatin treatment had no statistically significant effect on serum parameters or OA characteristics

Body weightAsexpected,themeanBWofthemiceinthecontrolgroupincreased

during the study, reaching ameanof 36.8 (± 3.2) gramsafter 16weeks (Figure

1). Inthesimvastatinonlytreatedgroups,BWatendpointdidnotstatistically

significantlydifferfromcontrol(95%CI:-3.4to4.0and-2.6to4.7forthedifference

betweencontroland40or100mg/kgBW/dayofsimvastatinrespectively).Food

intakewasnotstatisticallysignificantlyinfluencedbytheadditionofsimvastatin

tothefoodandthemicereceivedamedianof77%oftheintendeddosageofdrug

(Table1).

SerumNostatisticalsignificantdifferencewasobservedinserumtotalcholes-

terol(95%CI:-0.7to0.4and-0.6to0.6),serumtotaltriglyceride(95%CI:-2.4to

1.1and-1.0to1.6)andSAAlevels(95%CI:-286.4to680.1and-570.0to728.4)after

bothdosagesofsimvastatinonlytreatments(Figure2).Usingthemultiplexassay,

IL1α,MIP1αandIL10weredetectableintheserumandcomparedtocontrolmice,

thesewerenotstatisticallysignificantlydifferentaftersimvastatintreatment.

OA cartilage damage Microscopic evaluation demonstrated variability in

cartilage lossandtheamountofsubchondralboneinall thegroups(Figure3).

Cartilagedamagewasmostprofoundinthemedialcompartmentofthejoint.The

medianOARSIscoresinthesimvastatin100mg/kgBW/daytreatedgroupatthe

medialfemurandtibiawerelowerthantheuntreatedcontrolgroup,althoughthis

differencewasnotstatisticallysignificant(95%CI:-0.6to3.8formedialfemurand

-1.8 to4.6 formedial tibiaOARSIscore).Wecouldnotdetectastatisticallysig-

nificantreductionofsynovialhyperplasiainthemiceaftersimvastatintreatment

comparedtocontrols(95%CI:-0.7to1.0and-0.7to1.0forthedifferencebetween

controland40or100mg/kgBW/dayofsimvastatinrespectively;Figure3).

106 CHAPTER 6

Figure 1 Effectoftreatmentonbodyweightandserummetabolicmarkers.T=0weeksrepresentstartofexperimentaltreatmentattheageof8weeks.Controlgroupreceivedreferencedietfor16weeks.Treatmentgroupsreceivedsimvastatinand/orfenofibratemixedintotheirdietfor16weeks.Indicateddosagesareintendeddrugintake/kgbodyweight/day.Combinationgroupreceivedsimvastatin40mgandfenofibrate80mg.A)Bodyweight,B)Serumtriglyceride,C)Serumtotalcholesterol.Valuesindicatemean+SD(n=11-12micepergroup).Horizontaldashedline:meanvalueofT=0.*p<0.01versusControlgroup.**p<0.001versusControlgroup

Table 1 Totalfoodintakeanddrugintakepermouse*

Treatment Group(Intended drug intake)

Average total food intake per mouse in grams**

P Value Actual drug intake per kg body weight per day in mg (% of intended drug intake)

Control 492.1±15.7

Simvastatin40mg 467.1±13.8 1.000 30.7±1.8(77)

Simvastatin100mg 460.2±4.2 0.265 76.2±2.6(76)

Fenofibrate80mg 557.5±18.8 0.009 74.5±2.0(93)

Fenofibrate200mg 524.9±8.6 0.265 189.3±5.5(95)

Combination 484.3±11.8 1.000 33.5±1.4(84)$67.0±2.8(84)#

*Treatmentgroupsreceivedsimvastatinand/orfenofibratemixedwiththeirdietfor16weeks.Indicateddosag-esareintendeddrugintake/kgbodyweight/day.Combinationgroupreceivedsimvastatin40mgandfenofibrate80mg.Dataaremean±SD;Kruskal-Wallistestwithpost-hocBonferonnicorrectionwasperformed;Pvaluesindicatethestatisticaldifferencebetweentreatmentandcontrolgroup;**Averagetotalfoodintakepermousefor16weeks$Simvastatin40mg#Fenofibrate80mg


6

Figure 2 Effectofsimvastatinand/orfenofibrateonseruminflammatorymarkers.Controlgroupreceivedreferencediet.Treatmentgroupsreceivedsimvastatinand/orfenofibratemixedwiththeirdiet for16weeks. Indicateddosagesare intendeddrug intake/kgbodyweight/day.Combinationgroupreceivedsimvastatin40mgandfenofibrate80mg.A)SerumIL1α,B)SerumMIP1α,C)SerumIL10andD)SerumAmyloidAlevels.T=0:valuesbeforetreatmentattheageof8weeks.Horizontaldashedlineindicatesthemeanvalueatthestartoftreatment(T=0).Valuesindicatemean+SD(n=11-12micepergroup)

Twentyonepercentofthemicedevelopedpatellasubluxationandtheprevalence

ofsubluxationwasnotdifferentbetweengroups.Excludingthemicewithpatella

dislocationfromtheanalysesdidnotaltertheresults(Supplementarytable1).

μCT analysisBoththesubchondralbonevolume(95%CI:-0.1to0.1and-0.1to

0.2)andplatethickness(95%CI:-40.6to42.1and-24.3to60.2)didnotstatistically

significantlydifferbetweenmicetreatedwithsimvastatinandcontrolmice(Figure

4).

108 CHAPTER 6

Figure 3 Histologicalanalysisofeffectofsimvastatinand/orfenofibratetreatmentoncartilagedamageinSTR/Ortmice.Thecoronalsectionsofcartilageintheweight-bearingareaofthetibiofemoralcompartmentoftheleftkneewerestainedwiththionineandseverityofcartilagedamagewasscoredonascaleof0-6usingtheOARSIhis-tologicalscoringscale.Controlgroupreceivedreferencedietfor16weeks.Treatmentgroupsreceivedsimvas-tatinand/orfenofibratemixedwiththeirdietfor16weeks.Indicateddosagesareintendeddrugintake/kgbodyweight/day.Combinationgroupreceivedsimvastatin40mgandfenofibrate80mg.A)Representativepictureofscore0:normalcartilageB)Representativepictureofscore6:Verticalclefs/erosiontothecalcifiedcartilageextendingto>75%ofthesurface.Picturesaretakenat40xmagnification.C)QuantificationofOARSIscoresofthemedialfemur.D)QuantificationofOARSIscoresofthemedialtibia.E)Numberofcelllayersinthesynovialmembrane.Eachdotrepresentsanindividualmouse(n=11-12micepergroup)HorizontallineindicatesmedianvaluepergroupinCandD.Mean±SDisshowninE.


6

Figure 4 Effectofsimvastatinand/orfenofibratetreatmentonsubchondralboneplatebonevol-umeandthicknessofthetibiaplateauinSTR/Ortmice.Control group receivedreferencedietfor16weeks.Treatmentgroupsreceivedsimvastatinand/orfenofibratemixedwiththeirdietfor16weeks.Indicateddosagesareintendeddrugintake/kgbodyweight/day.Combinationgroupreceivedsimvastatin40mgandfenofibrate80mg.A)Sub-chondralboneplatebonevolumeB)Subchondralboneplatethickness,Valuesindicatemean+SD(n=11-12micepergroup)

Fenofibrate treatment reduced body weight, SAA and subchondral bone

Body weightMicetreatedwith200mg/kgBW/dayfenofibratehadthelowestmean

BWatendpointcomparedtothecontrolgroup(p<0.001;Figure1).Allfenofibrate

treatedmiceatemorecomparedtothecontrolgroup,althoughonlythefenofi-

brate80mg/kgBW/daytreatedgroupatestatisticallysignificantlymore(Table1).

Nosignsofdiarrhoeawereobserved.Themicereceivedamedianof94%ofthe

intendeddrugdosage.

Serum markersWedidnotdetectastatisticallysignificanteffectoffenofibrate

treatmentonserumtotaltriglyceride(95%CI:-0.5to2.2and-0.8to2.0forthedif-

ferencebetweencontroland80or200mg/kgBW/dayoffenofibraterespectively)

andonserumtotalcholesterol(95%CI:-0.9to0.3and-0.8to0.4;Figure2).SAA

levels in fenofibrate treatedgroupsdidnotchangeafterstartof treatmentand

at end pointwere significantly lower than in the control group (95%CI: 9.5 to

828.0 and10.8 to 829).On themultiplex assayonly IL1α,MIP1α and IL10were

detectableintheserum(Figure2).IL1αandMIP1αlevelsweresignificantlyhigher

infenofibratetreatedmicethanincontrolmice.

OA cartilage damageWecouldnotdetectastatisticallysignificantreductionof

medialfemur(95%CI:-2.8to3.4and-3.5to2.6)andtibia(95%CI:-4.3to4.6and-3.4

to4.3)OARSIscoresandsynovialhyperplasia(95%CI:-1.7to1.8and-0.6to1.3)in

thefenofibratetreatedmicecomparedtocontrols(Figure3).

μCT analysisMicetreatedwith200mg/kgBW/dayfenofibratehadalowersub-

chondralbonevolumeandplatethicknessthanthecontrolgroupalthoughonly

thelowersubchondralbonevolumewasstatisticallysignificant(Figure4).

110 CHAPTER 6

Effects of combination treatment of simvastatin and fenofibrate

ThemeanBWofthemicetreatedwithacombinationofsimvastatinandfenofi-

brateattheendofthestudywaslowerthanthemeanBWinthecontrolgroup

(p=0.003,Figure1).Themicereceived84%oftheintendeddosagesimvastatinand

fenofibrate(Table1).

Only serum amyloid A was statistically significantly different between mice

treatedwithacombinationofsimvastatinandfenofibrateandcontrols(Figures

2,3and4)

Correlation between body weight, serum markers, cartilage damage, synovial hyperplasia and bone changes.

Bodyweight at end point correlatedwith serum total triglyceride (p<0.001), IL1α

(p<0.001)andMIP1α(p=0.001)andsubchondralbonevolume(p=0.01).Innoneofthe

treatmentgroups,seruminflammationmarkerscorrelatedstatisticallysignificantly

withOAcartilagedamage(Table2).SAAlevelsdidnotdifferbetweenmicewithand

without cartilage damage. SAA levels correlated to subchondral bone volume

(p=0.008) Bothsubchondralbonevolume(p=0.002),platethickness(p<0.001)and

synovialhyperplasia(p<0.001)correlatedwithcartilagedamage.(Table2)

Table 2 Correlationbetweenbodyweightserummarkers,OARSIcartilagescores,synovialhyper-plasiaandµCTdata.

Body Weight(end point)

OARSI Score Synovium cell layers

Subchondralbone plate volume

Subchondralbone plate volume

Total Femur Tibia

Body weight (endpoint)

0.14 0.04 0.19 0.32 0.31 0.22

Serum

Total cholesterol 0.09 0.07 0.02 0.05 0.04 -0.26 -0.23

Total triglyceride 0.45 0.12 0.10 0.10 0.28 0.03 0.04

AmyloidA 0.21 -0.01 -0.08 0.08 0.12 0.34 0.19

IL1α -0.52 -0.07 -0.09 -0.04 -0.09 -0.09 -0.13

MIP1α -0.44 -0.09 -0.13 -0.02 -0.05 -0.01 -0.06

IL10 0.11 -0.08 -0.14 0.03 -0.01 -0.01 -0.11

Synoviumcelllayers 0.33 0.62 0.32 0.42

Subchondral bone platevolume

0.31 0.37

Subchondral bone platethickness

0.22 0.49

Serummarkersweremeasuredatendpointafter16weeksoftreatment;OARSIscoresforthetotaljoint,bothfemurandtibialcompartments;Correlationsbetweenbodyweightatendpoint,serumtotalcholesterolandµCTdatawerecalculatedusingthePearsoncorrelationcoefficient;CorrelationsbetweenallotherparameterswerecalculatedusingtheSpearmancorrelationcoefficient;Significantcorrelations(p<0.05)areshowninbold.


6

DISCUSSION

Inthisstudyweinvestigatedwhetherdrugsoftenusedtoreducethemorbidity

andmortality ofmetabolic syndrome and obesity could serve as a therapy to

preventortreatOA.Wedidnotfindastatisticallysignificantlyreductionofthe

severityofcartilagedamageinSTR/Ortmicewithneithersimvastatin,fenofibrate

northecombinationtreatment,althoughfenofibratetreatmentpreventedahigh

subchondralbonevolume.

TheSTR/Ortstrainwascarefullychosenforthisstudybecausethesemicehave

been described to develop OA and metabolic syndrome spontaneously at a

relativelyyoungage19,thusresemblingpatientswithmetabolicOA4.Sincestatins

andfibratesareusedtoimprovethemetabolicprofileofpatients,theuseofthis

strainseemedtomatchthesubgroupofmetabolicOApatientsthatcouldbenefit

fromtreatmentwiththesedrugs.Inadditiontohyperlipidemiaandobesity,other

possiblecausesforOApredispositionintheSTR/Ortstrainhavebeendiscussed,

suchastheroleofpatellasubluxation18;25.Whenpatellasubluxationoccurs,itis

amajorbiomechanicalriskfactoranddiseasemodifyingeffectsofsimvastatinor

fenofibratewouldbehighlyunlikely. Inourstudy,patellasubluxationoccurred

in 21% of the mice throughout all the groups. Although all mice with patella

subluxationhadextensivecartilagedamage,notallmicewithextensivecartilage

damagehadpatellasubluxation.Thissuggeststhatpatellasubluxationisnotthe

maincauseofOAinthesemiceandisnotlikelytomasktheeffectsofsimvastatin

orfenofibrate.

Inaprevioushumancohortstudywefoundthatstatinusewasassociatedwitha

lowerincidenceandreducedprogressionofkneeOAonx-rays15.Inanotherstudy

statinusewasassociatedwithlowerincidenceofclinicalOA16.Inthepresentstudy

withSTR/Ortmice,simvastatindidnotlowertheserumtotalcholesterollevel.This

couldbeexplainedbyadifferentcholesterolmetabolismpathwayinmicethanin

humans26-28.TheabsenceofastatisticallysignificanteffectofsimvastatinonOA

inourstudycouldindicatethatcholesterolandOAmightbelinkedtoeachother

andthatloweringcholesterolbystatinsisimportanttoreduceOAdevelopment.

Furthermore,differenttypesofstatinswereincludedinthehumanclinicalstudies.

Thesedifferenttypesofstatinshavebeendescribedtohavedifferenttherapeutic

effects29.Theserum levelsof totalcholesterol in thecontrolmice inourstudy

werecomparabletoapreviousstudy19.Serumcholesterolcanbeincreasedwitha

highfatdietinmiceanddecreasesaftertreatmentwithstatin30;31.Inaddition,we

havepreviouslyreportedthatstatintreatmentdecreasedthedevelopmentofOA

112 CHAPTER 6

inmiceonahighfatdiet32.Thissuggeststhattheeffectofstatintreatmentcould

alsodependonthetypeofdiet.However,thisconclusionshouldbedrawnwith

caution,becausethestudiesofFraulob31andGierman32usedrosuvastatinandthe

studyofParaskevast30atorvastatin,whereasweadministeredsimvastatin.Aswith

humans, thesedifferent typesof statinshavebeendescribed tohavedifferent

therapeuticeffectsinmice21.

Theresultofourstudythatsimvastatincouldnotstatisticallysignificantlyreduce

OAcartilagedamage is alsonot consistentwith aprevious studybyYudohet

al14,whoreportedastatisticallysignificantdecreaseinOAcartilagescoreinSTR/

Ort mice after 12 and 24 weeks of simvastatin (40 mg/kg BW/day) treatment.

Thisinconsistencycouldbeduetodifferencesinstrains:Yudohandcolleagues

usedSTR/OrtCrljmiceobtainedfromCharlesRiverJapanwhereasweusedSTR/

OrtOlaHsdmiceobtainedfromHarlan,Italy.Responsetosimvastatincouldhave

beendifferentbetweenthesetwopopulationsofmice.Furthermore,inthestudy

byYudohetal,thecartilagedamageintheuntreatedgroupattheageof24weeks

waslessseverethaninthecontrolgroupinourstudy.Thisindicatesthatsimvas-

tatinmightbemoreeffectiveinslowlydevelopingorlesssevereOA.Lastly,Yudoh

etaladministratedthedrugsbydailygavageinsteadofmixingitwiththefoodand

thereforeadifferenttherapeuticlevelmightbereachedinthemice.

Fenofibrateisanotherlipidloweringdrug,widelyusedtolowerserumlipidsin

patientswithhypertriglyceridemia.Serumtriglyceridewasnotloweredaftertreat-

mentwithfenofibrateinourstudy,butthetreatedmicegainedlessweight.BW

washowevermoderatelycorrelatedtoserumtriglyceridesuggestingthatweight

reductioncould reduceserum triglyceride.Controlmicecontinued togainBW

overtime,reachingameanweightof36.8±3.2gramsattheageof24weeks.This

is comparable toprevious studies19;33.The reduction inBWgainby fenofibrate

treatmenthowever,didnotprotectthemicefromdevelopingcartilagedamage.

ThisabsenceofcorrelationbetweenBWandincidenceofcartilagedamagecon-

firmsourearlierfindings32thatobesitydoesnotincreaseOAcartilagedamageby

biomechanicaloverloading.

Inourstudy,serumcholesterolandserumtriglyceridewerebothnotcorrelated

withcartilagedamage.ThissuggeststhatintheSTR/Ortmicelipidmetabolism

isnottheprimarycauseofspontaneousOAcartilagedamagedevelopment.Next

totheireffectonlipidmetabolism,statinsandfibratesareknowntohaveanef-

fectoninflammation8;9;11-13.SAA,amarkerforsystemicinflammation,increasedin

controlmicewithaging.SAAhasbeenassociatedwithinflammatoryprocessesin


6

artherosclerosisandrheumatoidarthritis34-36,isincreasedinobesity37 and clinical

studieshaveshownthatSAAcanbeloweredbyfenofibrateandsimvastatintreat-

ment38-40. InthepresentstudywithSTR/Ortmice,SAAlevelswereonlylowered

after fenofibrate treatment. SAA is described to be induced by the cytokines

IL1β, IL6 andTNF-α and it canactivate theNF-ĸB pathway34;35;41.Kyostio-Moore

et al42 reportedelevated inflammatory cytokines in the serumof STR/Ortmice

includingIL1β,IL12p70andIL5,andthesechangeswerecorrelatedwithcartilage

damage.WewereabletodetectIL1α,MIP1αandIL10intheserumofthemice,

butothercytokineswerenotdetected.Surprisingly,IL1α,andMIP1αlevelswere

significantlyhigherinmicethatreceivedfenofibratetreatment.Thissuggeststhat

differentaspectsofinflammationmightoccurthatpossiblycounteracteachother.

Furthermore, we found no correlation between individual serum inflammatory

markersandsynovialhyperplasia,suggestingthatsystemicinflammationisnot

directlyrelatedtolocalinflammatorychangesinthekneeinourSTR/Ortmice

Fenofibrate lowers lipid levelsby activatingPPARα. It hasbeen suggested thatSTR/Ort mice have reduced endogenous PPARα signalling. This reduction ofPPARα signalling could be associated with increased osteogenic differentiation

andreducedadipogenicandchondrogenicdifferentiationofbonemarrowderived

mesenchymalstemcellsandthusbeassociatedwiththedevelopmentofOAin

thesemice20. Inour study,PPARα activationwith200mg/kgBW/day fenofibrate

did indeed reduce the total bone volume in the subchondral bone. We could

however, not detect a significant reduction of cartilage damage in our study.

ChangesincartilageandsubchondralboneduringOAdevelopmentarethought

to be correlated to each other43andwedidfindamoderatecorrelationbetween

cartilagedamageandsubchondralboneparameters.Theabsenceofastatistically

significantreductionofcartilagedamagemightbeduetothelimitedsensitivity

ofthehistologicalcartilagedamagescoringortothehighvariationinincidence

ofspontaneousOAcartilagedamageinthesemice.Inthepresentstudy,60%of

theanimalshaddevelopedOAcartilagedamageat24weeksofage,whichisless

thanthe80%reportedinotherstudiesthatweusedforourpowercalculation18.

Moreover, since we used a spontaneousmodel of OA development, individual

micedevelopedOAatadifferentspeed25whichmightexplainthelargevariation

incartilagedamageinthecontrolgroup.

Alternatively,itispossiblethatthemicetreatedwithfenofibrateareatadifferent

phaseinOAdevelopment.DuringOAdevelopmentinmice,abiphasicsubchon-

dralboneturnoverprocessistakingplace,withinitiallossofsubchondralbone,

followedbyincreasedvolumeandthickeningofthesubchondralboneplate23;44.

114 CHAPTER 6

Inthefenofibratetreatedmice,initialOAdevelopmentcouldhavebeendelayed

asdemonstratedbythelowersubchondralbonevolumewhereastheothermice

alreadyprogressedtoathickeningofthesubchondralplate.Furtherinvestigation

withtimeseriesisneededtoconfirmthishypothesis.

Possiblesideeffectsofstatinsandfenofibratesaremusclepainandelevatedliver

enzymes. Furthermore, toxicology studies performed onmice showed that ad-

ministrationofveryhighdosagesofsimvastatinorfenofibratefor72weekscould

possiblyinducelivercarcinomas45;46.Inourstudy,wedidnotanalysethelivers

ofourmiceatage20weeksforthepresenceoflivercarcinomas,sowecannot

excludethepresenceofthesetumours.Weincludedonegroupofmicethatwas

treatedwithacombinationofsimvastatinandfenofibrate.Thiscombinationhas

beenshowninclinicalstudiestobemoreeffectiveindecreasingcardiovasculair

mortality47, treating dyslipidemia48 and decreasing inflammation49 compared to

monotherapy.Becausethecombinationisdemonstratedtobemoreeffective47-49,

a lower dose of both drugs can be used and therefore less side effects will occur50.

Unfortunately,we did not see synergistic effects of the combination treatment

inourstudy.Simvastatinand fenofibratearebothmetabolised in the liverand

therebyloweringthelevelsthatarereachedlocallyinthekneeorsubchondral

bone.Methodstoincreasethelevelsofbothdrugslocally,suchasintra-articularly

orsubcutaneouslyinjectionsmightprovideoptionstoincreasethelocaleffects

andreducethesystemicsideeffects.

Insummary,wecouldnotdetectastatisticallysignificantlydecreaseofsimvas-

tatinorfenofibratetreatmentonthedevelopmentofcartilagedamagein6months

old STR/Ortmice.On theotherhand, the effect of fenofibrateon subchondral

bonemorphologyandthecorrelationbetweensubchondralbonemorphologyand

cartilagedamagesuggeststheremightbeamildeffectonOAdevelopmentthatis

maskedbytheinsensitivityofthecartilagescoreorthehighvariationincartilage

damageinmiceof6monthsold.Furthermore,ourdatasuggestthatbiomechani-

calchangesduetooverweightorpatellasubluxationandspontaneousmetabolic

changesarenottheprimarycauseofOAintheSTR/Ortmouse.Complexinflam-

matoryprocessesoccurinthesemicethatdidnotaffectthecartilagedirectly.

Nonetheless,wedonotexcludethatlipidloweringdrugscanstillbebeneficialin

othersubtypesofOAsuchashighfatdietinducedmetabolicOAasindicatedby

ourpreviousstudies.ThisemphasizestheimportanceoffurthersubtypingOAto

findpossibilitiesforpharmacologicaltreatmentofOA.


6

REFERENCES

1. BijlsmaJW,BerenbaumF,LafeberFP.2011.Osteoarthritis:anupdatewithrelevance forclinical

practice.Lancet377:2115-2126.

2. Aspden RM, Scheven BA, Hutchison JD. 2001. Osteoarthritis as a systemic disorder including

stromalcelldifferentiationandlipidmetabolism.Lancet357:1118-1120.

3. OliveriaSA,FelsonDT,ReedJI,etal.1995.Incidenceofsymptomatichand,hip,andkneeosteoar-

thritisamongpatientsinahealthmaintenanceorganization.ArthritisRheum38:1134-1141.

4. ZhuoQ,YangW,ChenJ,etal.2012.Metabolicsyndromemeetsosteoarthritis.NatRevRheumatol

8:729-737.

5. HoevenTA,KavousiM,ClockaertsS,etal.2012.Associationofatherosclerosiswithpresenceand

progressionofosteoarthritis:theRotterdamStudy.AnnalsoftheRheumaticDiseases.

6. DevarajS,ChanE,JialalI.2006.Directdemonstrationofanantiinflammatoryeffectofsimvastatin

insubjectswiththemetabolicsyndrome.JClinEndocrinolMetab91:4489-4496.

7. AbeM,MatsudaM,KobayashiH, et al. 2008. Effectsof statinson adipose tissue inflammation:

theirinhibitoryeffectonMyD88-independentIRF3/IFN-betapathwayinmacrophages.Arterioscler

ThrombVascBiol28:871-877.

8. BakerJ,WalshP,ByrneD,etal.Pravastatinsuppressesmatrixmetalloproteinaseexpressionand

activityinhumanarticularchondrocytesstimulatedbyinterleukin-1β.JournalofOrthopaedicsand

Traumatology:1-5.

9. Clockaerts S, Bastiaansen-JenniskensYM, Feijt C, et al. 2011. Peroxisomeproliferator activated

receptor alpha activation decreases inflammatory and destructive responses in osteoarthritic

cartilage.OsteoarthritisCartilage19:895-902.

10. ClockaertsS,Bierma-ZeinstraSM.2011.Commenton:Statinsandthe joint:multipletarget fora

globalprotection?SeminArthritisRheum40:588.

11. DombrechtEJ,VanOffelJF,BridtsCH,etal.2007.Influenceofsimvastatinontheproductionofpro-

inflammatorycytokinesandnitricoxidebyactivatedhumanchondrocytes.ClinExpRheumatol

25:534-539.

12. LazzeriniPE,CapecchiPL,NerucciF,etal.2004.SimvastatinreducesMMP-3level in interleukin

1betastimulatedhumanchondrocyteculture.AnnRheumDis63:867-869.

13. LazzeriniPE,LorenziniS,SelviE,etal.2007.Simvastatininhibitscytokineproductionandnuclear

factor-kB activation in interleukin 1beta-stimulated synoviocytes from rheumatoid arthritis pa-

tients.ClinExpRheumatol25:696-700.

14. YudohK,KarasawaR.2010.Statinpreventschondrocyteaginganddegenerationofarticularcarti-

lageinosteoarthritis(OA).Aging(AlbanyNY)2:990-998.

15. ClockaertsS,VanOschGJ,Bastiaansen-JenniskensYM,etal.2012.Statinuse isassociatedwith

reducedincidenceandprogressionofkneeosteoarthritisintheRotterdamstudy.AnnRheumDis

71:642-647.

16. KadamUT, BlagojevicM, Belcher J. 2013. Statin Use and Clinical Osteoarthritis in the General

Population:ALongitudinalStudy.JGenInternMed.

17. vanEekerenIC,ClockaertsS,Bastiaansen-JenniskensYM,etal.2013.Fibratesastherapyforosteo-

arthritisandrheumatoidarthritis?Asystematicreview.TherAdvMusculoskeletDis5:33-44.

18. MasonRM,ChambersMG,FlannellyJ,etal.2001.TheSTR/ortmouseanditsuseasamodelof

osteoarthritis.OsteoarthritisandCartilage9:85-91.

19. UchidaK,UrabeK,NaruseK,etal.2009.Hyperlipidemiaandhyperinsulinemiainthespontaneous

osteoarthritismousemodel,STR/Ort.ExpAnim58:181-187.

116 CHAPTER 6

20. WattersJW,ChengC,PickarskiM,etal.2007.Inverserelationshipbetweenmatrixremodelingand

lipidmetabolismduringosteoarthritisprogressionintheSTR/Ortmouse.ArthritisRheum56:2999-

3009.

21. ZadelaarS,KleemannR,VerschurenL,etal.2007.Mousemodelsforatherosclerosisandpharma-

ceuticalmodifiers.ArteriosclerThrombVascBiol27:1706-1721.

22. Waarsing JH,Day JS,WeinansH. 2004. An improved segmentationmethod for in vivomicroCT

imaging.JBoneMinerRes19:1640-1650.

23. Botter SM, van Osch GJ, Clockaerts S, et al. 2011. Osteoarthritis induction leads to early and

temporalsubchondralplateporosityinthetibialplateauofmice:aninvivomicrofocalcomputed

tomographystudy.ArthritisRheum63:2690-2699.

24. GlassonSS,ChambersMG,VanDenBergWB,etal.2010.TheOARSIhistopathologyinitiative-rec-

ommendationsforhistologicalassessmentsofosteoarthritisinthemouse.OsteoarthritisCartilage

18Suppl3:S17-23.

25. NaruseK,UrabeK,JiangSX,etal.2009.OsteoarthriticchangesofthepatellofemoraljointinSTR/

OrtCrljmicearetheearliestdetectablechangesandmaybecausedbyinternaltibialtorsion.Con-

nectTissueRes50:243-255.

26. KleemannR,PrincenHM,EmeisJJ,etal.2003.Rosuvastatinreducesatherosclerosisdevelopment

beyond and independent of its plasma cholesterol-lowering effect inAPOE*3-Leiden transgenic

mice:evidenceforantiinflammatoryeffectsofrosuvastatin.Circulation108:1368-1374.

27. SparrowCP,BurtonCA,HernandezM,etal.2001.Simvastatinhasanti-inflammatoryandantiath-

eroscleroticactivitiesindependentofplasmacholesterollowering.ArteriosclerThrombVascBiol

21:115-121.

28. EndoA,TsujitaY,KurodaM,etal.1979.EffectsofML-236Boncholesterolmetabolisminmiceand

rats:lackofhypocholesterolemicactivityinnormalanimals.BiochimBiophysActa575:266-276.

29. WengTC,YangYH,LinSJ,etal.2010.Asystematicreviewandmeta-analysisonthetherapeutic

equivalenceofstatins.JClinPharmTher35:139-151.

30. ParaskevasKI,PantopoulouA,VlachosIS,etal.2011.Comparisonoffibrate,ezetimibe,low-and

high-dosestatintherapyforthedyslipidemiaofthemetabolicsyndromeinamousemodel.Angiol-

ogy62:144-154.

31. Fraulob JC, Souza-Mello V, Aguila MB, et al. 2012. Beneficial effects of rosuvastatin on insulin

resistance,adiposity,inflammatorymarkersandnon-alcoholicfattyliverdiseaseinmicefedona

high-fatdiet.ClinSci(Lond)123:259-270.

32. GiermanLM,vanderHamF,KoudijsA,etal.2012.Metabolicstress-inducedinflammationplaysa

majorroleinthedevelopmentofosteoarthritisinmice.ArthritisRheum64:1172-1181.

33. SokoloffL,MickelsenO,SilversteinE,etal.1960.Experimentalobesityandosteoarthritis.AmJ

Physiol198:765-770.

34. OkamotoH,KatagiriY,KiireA, et al. 2008. SerumamyloidAactivatesnuclear factor-kappaB in

rheumatoidsynovialfibroblaststhroughbindingtoreceptorofadvancedglycationend-products.

JRheumatol35:752-756.

35. McNiffPA,StewartC,SullivanJ,etal.1995.Synovialfluidfromrheumatoidarthritispatientscon-

tainssufficientlevelsof IL-1betaandIL-6topromoteproductionofserumamyloidAbyHep3B

cells.Cytokine7:209-219.

36. KingVL,ThompsonJ,TannockLR.2011.SerumamyloidAinatherosclerosis.CurrOpinLipidol

22:302-307.

37. TrayhurnP,Wood IS. 2004. Adipokines: inflammation and the pleiotropic role ofwhite adipose

tissue.BrJNutr92:347-355.


6

38. GervoisP,KleemannR,PilonA,etal.2004.GlobalSuppressionof IL-6-inducedAcutePhaseRe-

sponseGeneExpressionafterChronicinVivoTreatmentwiththePeroxisomeProliferator-activated

Receptor-αActivatorFenofibrate.JournalofBiologicalChemistry279:16154-16160.

39. HoriuchiY,HirayamaS,SodaS,etal.2010.Statintherapyreducesinflammatorymarkersinhyper-

cholesterolemicpatientswithhighbaselinelevels.JAtherosclerThromb17:722-729.

40. HuY,TongG,XuW,etal.2009.Anti-inflammatoryeffectsofsimvastatinonadipokinesintype2

diabeticpatientswithcarotidatherosclerosis.DiabVascDisRes6:262-268.

41. RamadoriG,VanDammeJ,RiederH,etal.1988.Interleukin6,thethirdmediatorofacute-phase

reaction,modulateshepaticproteinsynthesisinhumanandmouse.Comparisonwithinterleukin1

betaandtumornecrosisfactor-alpha.EurJImmunol18:1259-1264.

42. Kyostio-MooreS,NambiarB,HuttoE,etal.2011.STR/ortmice,amodel forspontaneousosteo-

arthritis, exhibit elevated levels of both local and systemic inflammatorymarkers. CompMed

61:346-355.

43. MahjoubM,BerenbaumF,HouardX.2012.Whysubchondralboneinosteoarthritis?Theimpor-

tanceofthecartilageboneinterfaceinosteoarthritis.OsteoporosInt23Suppl8:841-846.

44. BotterSM,GlassonSS,HopkinsB,etal.2009.ADAMTS5-/-micehavelesssubchondralbonechanges

afterinductionofosteoarthritisthroughsurgicalinstability:implicationsforalinkbetweencarti-

lageandsubchondralbonechanges.OsteoarthritisCartilage17:636-645.

45. Drugs.com.2013.FenofibrateOfficialFDAinformation,sideeffectsanduses,fromDrugs.com.

46. Drugs.com.2013.SimvastatinorallydisintegratingtabletOfficialFDAinformation,sideeffectsand

uses,fromDrugs.com.

47. TenenbaumA,MedvedofskyD,FismanEZ,etal.2012.Cardiovasculareventsinpatientsreceived

combinedfibrate/statin treatmentversusstatinmonotherapy:AcuteCoronarySyndrome Israeli

Surveysdata.PLoSOne7:e35298.

48. DerosaG,MaffioliP,SalvadeoSA,etal.2009.Fenofibrate,simvastatinandtheircombinationinthe

managementofdyslipidaemiaintype2diabeticpatients.CurrMedResOpin25:1973-1983.

49. WagnerAM,Sanchez-QuesadaJL,BenitezS,etal.2011.Effectofstatinandfibratetreatmenton

inflammationintype2diabetes.Arandomized,cross-overstudy.DiabetesResClinPract93:e25-28.

50. FranssenR,VergeerM,StroesES,etal.2009.Combinationstatin-fibratetherapy:safetyaspects.

DiabetesObesMetab11:89-94.

CHAPTER 7

High fat diet accelerates cartilage repair in

DBA/1 mice

Wu Wei Yvonne M. Bastiaansen-Jenniskens

Mathijs Suijkerbuijk Nicole KopsPieter K. Bos

Jan A.N. Verhaar Anne-Marie Zuurmond

Francesco Dell’AccioGerjo J.V.M. van Osch

J Orthop Res. 2017 Jun;35(6):1258-64.

120 CHAPTER 7

ABSTRACT

Obesity isawell-knownrisk factor forosteoarthritis,but it isunknownwhat it

doesoncartilagerepair.Hereweinvestigatedwhetherahighfatdiet(HFD)influ-

encescartilagerepairinamousemodelofcartilagerepair. WefedDBA/1mice

controlorHFD(60%energyfromfat).Aftertwoweeks,afullthicknesscartilage

defectwasmadeinthetrochleargroove.Miceweresacrificed,1,8and24weeks

after operation. Cartilage repair was evaluated on histology. Serum glucose,

insulinandamyloidAweremeasured24hbeforeoperationandatendpoints.Im-

munohistochemicalstainingwasperformedonsynoviumandadiposetissueto

evaluatemacrophageinfiltrationandphenotype.Oneweekafteroperation,mice

onHFDhaddefect fillingwith fibroblast-like cells andmore cartilage repair as

indicatedbyalowerPinedascore.After8weeks,miceonaHFDstillhadalower

Pinedascore.After24weeks,nomicehadcompletecartilagerepairandwedid

notdetectasignificantdifferenceincartilagerepairbetweendiets.Bodyweight

wasincreasedbyHFD,whereasserumglucose,amyloidAandinsulinwerenotin-

fluenced.Macrophageinfiltrationandphenotypeinadiposetissueandsynovium

werenot influencedbyHFD. Incontrast tocommonwisdom,HFDaccelerated

intrinsiccartilagerepairinDBA/1miceontheshortterm.ResistancetoHFDin-

ducedinflammatoryandmetabolicchangescouldbeassociatedwithaccelerated

cartilagerepair.

High fat diet accelerates cartilage repair 121

7

INTRODUCTION

Traumatic articular cartilage injury, a common pathology of the knee1, results

inconsiderablemorbidityanddisabilitiesandcouldeventuallyleadtoosteoar-

thrithis (OA).Articularcartilagehas lowself-repaircapabilities2andthemicro-

fractureprocedure isused to induce intrinsiccartilage repairbymesenchymal

stemcells(MSCs)3Themicrofractureprocedureleadstosignificantsymptomatic

improvements inmostpatients4;5Theclinical improvementsarereportedtobe

less in patients with obesity6andthesepatientsarenowoftenexcludedfromtreat-

ments7.However,thereisnoinformationaboutthestructureofthenewlyformed

cartilageormechanismsexplainingtherelationshipbetweenobesityandworse

clinicaloutcome.

ObesityisamajorhealthproblemintheWesternsocietyandthenumberofpersons

withobesityisincreasing.Obesitycouldleadtosystemicmetabolicandinflamma-

torychangessuchashyperglycemia,hyperinsulinemia,increasedserumamyloid

Aandinfiltrationofadiposetissuebypro-inflammatorymacrophages8.Obesityis

awell-knownmajorriskfactorforthedevelopmentofOA9.Furthermore,obesity

exaggeratedpost-traumaticarthritis10andrheumatoidarthritis11 and could lead

toimpairedwoundhealingafteranoperation12.However,itisunknownwhatthe

preciseeffectisofobesityonintrinsiccartilagedefectrepair.

Tooptimizecartilagerepairtreatmentresultsinobesepatients,itisthereforees-

sentialtoinvestigatewhetherandhowobesitynegativelyinfluencescartilagere-

pair.Toanswerthisquestion,weperformedanin-vivomousestudytoinvestigate

thedirecteffectofobesityonintrinsiccartilagerepair.Inourstudy,ahighfatdiet

(HFD)wasusedtoincreaseweightandmimichumanobesityinmice13.HFDhas

beenshowntoincreaseweightandtobedetrimentaltocartilageandcauseOA

intheC57/BL6strainofmice12;14.Furthermore,HFDdecreasesthechondrogenic

differentiationpotentialofMSCsfromC57/BL6mice15

OurhypothesiswasthatHFDwouldnegativelyinfluenceintrinsiccartilagerepair.

SinceC57/BL6micehadbeenshownnottopossessintrinsicrepaircapabilitiesof

cartilage defects16,theywereunsuitableforourstudy.Therefore,weusedmale

DBA/1micewhichpossesscapabilitiestorepairafullthicknesscartilagedefect16

andgainweightonaHFD17.Thisstrainofmiceisthereforesuitabletostudythe

possiblenegativeeffectsofaHFDoncartilagerepair.

122 CHAPTER 7

METHOD

Animals and diet

Eight-weeks-old male DBA/1OlaHsd mice were acquired from Harlan UK and

maintainedattheanimaltestingfacilitiesoftheErasmusMCUniversityMedical

Center,TheNetherlands.Micewerehousedingroupsofthreeorfourmiceper

cageunder12hourslight-darkcycleandhadaccesstowaterandfoodadlibitum.

ThestudyprotocolwasapprovedbytheinstitutionalAnimalCareandUseCom-

mittee(ErasmusMCUniversityMedicalCenter,TheNetherlands,AEC116-12-05).

Micewereallowedtoacclimatize,werefedregularchowfortwoweeksandwere

allocatedrandomlytothedietgroups.Attheageof10weeks,micewerefedwith

aHFD(D12492;ResearchDiets,60%kcalfromfat)orcontroldiet(D12450B;Re-

searchDiets,10%kcalfromfat).Bothdietscontained4057kcalperkilogramdry

weight.Micewereweightedeverytwoweeksandthemicecontinuedtoreceive

theirrespectivedietsuntiltheywerekilled.

Operative procedure

Attheageof12weeksmicewereanesthetizedwithanisoflurane/O2mixtureand

afullthicknesscartilagedefectwascreatedaspreviousdescribed16.Thelefthind

legwasshavedanddisinfectedusingalcohol.Anincisionusinganr11microsur-

gicalscalpelwasmademediallyandproximally fromthe insertionof thepatel-

lar tendonon the tibia towards the attachmentof thequadricepsmuscle.The

jointcapsulewasopenedwithasmallincisionandthejointwasfullyextended

todislocatethepatella laterally.Theknee jointwasthen fullyflexedtoexpose

thetrochleargroovearticularsurface.Afullthicknesscartilagedefectwasthen

madeusinga25Gneedlebyscratchingthearticularsurface.Thejointwasflushed

withsalineandclosedusing6-0Vicryl(Ethicon).Theskinwasclosedusing6-0

Ethilon (Ethicon). Themice were given one dose of analgesic subcutaneously

preoperatively(Temgesic,0.05mg/kg).Micewereallowedtomovefreelypostop-

erationandtobefullweightbearing.Miceweresacrificed1,8and24weeksafter

operationwithaheartpunctureunderisoflurane/O2anesthesia.Allmicereached

end-pointwithoutdisease.34%oftheoperatedmicehadapatelladislocationand

wereexcludedfromtheanalysis.Thesemicewereequallydistributedoverboth

groups:patelladislocation ratewas35% for thecontrolgroupand32% for the

HFD fed group.We continued to operatemice until therewere at least 5mice

pergroup.Thetimepointsandminimumnumberofmicepergroupneededwere

basedonpreviousstudybyEltawiletal16.Thefinalnumberofmiceonacontrol

dietincludedforanalysiswas8miceat1week,9miceat8weeksand6miceat24


7

weeks.ForthemiceonaHFD,thenumberofmiceincludedwas6miceat1week,

9miceat8weeksand6miceat24weeks.

Serum collection and analysis

Bloodwascollectedthroughacheekpunctureonedaybeforetheoperationand

throughaheartpunctureatendpoints.Wecollectedthreedropsofbloodduring

thecheekpunctureand1mlofbloodthroughtheheartpuncture.Therewasno

noticeabledifferenceinbleedingtendencybetweenanimals.Micewerefastedfor

4hoursbeforebloodcollection.Bloodwasstoredfor1houratroomtemperature,

followedby30minutesat4°C,thencentrifugedfor5minutesat250g.Serumwas

removedandimmediatelystoredat-80°C.Serumglucosewasdeterminedusinga

bloodglucosemeter(FreeStyleFreedomLite,AbbottLaboratories).Seruminsulin

(Alpco,#80-INSMS-E01)andserumamyloidA(SAA,Alpco,#41-SAAMS-E01)were

determinedbyEnzyme-Linked ImmunoSorbentAssay(ELISA)according to the

manufacturer’sinstructions.

Tissue preparation for histological analysis

Theskinofbothhindlegsofallanimalswasopened.Thepatellawithsurrounding

synoviumwasremoved,placedinTissueTek®(SakuraFinetek,theNetherlands)

andsnapfrozeninliquidnitrogen.6µmthickcryosectionsweremadeofthepa-

tellawithsurroundingsynovium.Subcutaneousadiposetissueof5miceperdiet

attimepoint8weeksaftertheoperationwerefixedin4%bufferedformalinfor1

hour,embeddedinparaffinand6µmthickparaffinsectionsweremade.Theknees

ofallanimalswereexcisedandfixedin4%bufferedformalinfor7days,decalcified

in10%formicacidindistilledwaterfor7daysandembeddedinparaffin.6µm

thickparaffinsectionsweremadeofthekneesasdescribedbyEltawiletal16.

Evaluation of cartilage repair

Paraffin sections were stained with thionin or immunohistochemically stained

withamonoclonal antibodyagainst collagen type2. For immunohistochemical

staining, collagen type 2 antibody (DSHB, # II-II 6B3) or IgG1 isotype control

(DakoCytomation#X0931)werefirstpre-coupledwithbiotin-SPF(ab)2(Jackson,

#115-066-062).Sectionswerethenincubatedwiththecoupledantibodycomplex,

followedbyLabel(Biogenex,HK-321-UK)andfreshlypreparednew-fuchsinwas

usedassubstrate.PicturesweretakenusinganOlympusSC30camera.Thesize

ofthedefectandpercentageofdefectfilledwasquantifiedusingImageJsoftware

(National Institutes ofHealth, Bethesda,MD,USA). The number of cells in the

defectwasdeterminedinImageJandexpressedasper100µm2.Cartilagerepair

wasassessedusingthescoringsystemdevisedbyPinedaetal18,inwhichfilling,

124 CHAPTER 7

reconstruction of osteochondral junction,matrix staining and cellmorphology

were taken into account.ThePineda scoring systemhasa rangeof 0 to 14, in

which0meanscompleterepairand14norepairatall.Osteoarthriticdamagein

thecartilagesurroundingthedefectwasassessedusingamodifiedMankinscor-

ingsystem19.Bothscoringsystemswereperformedbytwoindependentobserv-

ers(WWandMS)whilebeingblindedtothegroupassignment.Themeanscore

of threedifferent slideswasdeterminedand in caseof a >1 scorediscrepancy

betweenthetwoobservers,rescoringwasperformedtogetherbythetwoobserv-

ers.

Evaluation of local and systemic inflammation

Cryosectionofthepatellawithsurroundingsynoviumwerefixedinacetoneand

stainedwith hematoxylin and eosin (H&E). For immunohistochemical staining,

sectionswere incubatedwithmonoclonal antibody against F4/80 (eBioscience,

#14-4801),polyclonalantibodiesagainst iNOS (Abcam,#15323)andCD206 (Ab-

cam,#64693).RatIgG2aantibody(eBioscience,#14-4321-82)wereusedasisotype

controlforF4/80andrabbitIgGantibody(DakoCytomation#X0903)wereusedas

isotypecontrolforiNOSandCD206.Subsequently,F4/80sectionswereincubated

withbiotinylatedrabbit-anti-ratIgG(Vector,#BA-4000)andiNOsandCD206sec-

tionswere incubatedwithRabbit-Link(Biogenex,HK-326-UR).Allsectionswere

afterwards incubated with Label (BioGenex, HK-321-UK) and freshly prepared

new-fuchsinwasusedassubstrate.Acellwasconsideredpositivewhenstained

red.

Synovialinflammationwasevaluatedusingthehistologicalscoringsystemdevised

byKrennetal20.TodeterminewhetherHFDhadaneffectonsynovialmacrophage

phenotype,immunohistochemicallystainedsampleswererankedbasedontheir

relativenumberofpositivecellsinthreesectionspersamplebytwoindependent

observers (WWandMS).Scoringandrankingwereperformed foreachmarker

separatelyandtheaveragerankforeachsamplebetweenthetwoobserverswas

usedintheanalysis.

Paraffinsectionsofsubcutaneousadiposetissueweredeparafinized,treatedwith

proteinase k solution (Sigma-Aldrich) for antigen retrieval and followedby the

same immunohistochemicalstainingprocedure forF4/80asthesynovium.Two

independentobservers (WWandMS)counted thenumberofcrown likestruc-

turesperareaofrandom100adipocytesfor3sectionspersampleandtheaverage

numberbetweenthetwoobserverswasusedintheanalysis.


7


Statistical analysis was performed in IBM SPSS statistics 20 (IBM Corporation,

Armonk,NewYork,USA).TheShapiro-Wilktestwasusedtotestdatafornormality

andtheLevene’stestforhomogeneityofvariance.Histologicaldatawasanalyzed

usingtheMann-Whitney-Utest.Weight,serumdataanddefectfillingwerenormally

distributedandunlessotherwisestated,statisticaldifferencesbetweendietswere

determined using an unpaired T-test. Differences were considered statistically

significant when p<0.05.

RESULTS

High fat diet accelerates cartilage repair early after cartilage damage

Oneweekafterthecreationofthedefect,inmiceonaHFD,spindleshapedcells

hadpartiallyfilledthedefect(Figure1A).Ahigherpercentageofthedefectwas

filledinthemiceonaHFDthanoncontroldiet(Figure1AandD),althoughthis

Cartilage Repair

Control High fat Control High fat Control High fat0

2

4

6

8

10

12

14P = 0.01

Worst

Best

1 week 8 weeks

P < 0.05

24 weeks

Pine

da

Filling of defect

Control High fat Control High fat Control High fat0

20

40

60

80

100

120

1 week 8 weeks 24 weeks

%of

defe

ct

A B

D F

Control

High fat

Non-operatedknee

1 week afteroperation

8 weeks afteroperation

Collagentype 2

Control

High fat

Control

High fat

Non-operatedknee

24 weeks afteroperation

C

Cells in defect

Control High fat0

1

2

3

4

5

Cells

/100

µm2

P < 0.05

1 week

E

Figure 1 Highfatdietfeedingimprovescartilagerepair.10weeksoldmaleDBA/1micewerefedwithcontrolorhighfatdiet.After2weeksoffeeding,fullthicknesscartilagedefectswerecreated.A)Representativepicturesofthioninstainedknees;non-operated,1weeksand8weeksafterdefectcreation,B)immunohistologicalstainingwithantibodiesagainstcollagentype2,8weeksafterdefectcreation.Thisisthefromthesamekneeasthethioninstaining.Thereisanartifactshowninthestaining.C)Representativepicturesofthioninstainedknees;24weeksafterdefectcreationandcorrespondingnon-operatedknees,D)percentageofdefectfilled,E)numberofcells inthedefectper100µm2,F)cartilagerepairevaluatedusingthePinedascoringmethod.Valuesaremean±SD(n=6-9micepergroup).

126 CHAPTER 7

wasnotstatisticallysignificant(Figure1D;95%CI:63-91%versus38-77%;p=0.15).

After8weeks,almostallthemicehadfillingofthedefect(Figure1D),however

onlytheHFDfedmicehaddefectsfilledwithchondrocyte-likecellssurrounded

withpositivethioninandcollagentype2staining(Figure1AandB).Inaddition,

thenumberofcells in thedefectwashigher inHFD fedmicecompared to the

controlmice(Figure1E).Finally,thePinedascorewaslower1week(p=0.04)and8

weeks(p=0.01)afteroperationinmiceonaHFDcomparedtocontroldiet(Figure

1FandTable1).

Table 1 Highfatdietimprovescartilagerepair

Controldiet Highfatdiet

F R M C Total F R M C Total

1week 2.0(±1.1)

2.0(±0.0)

3.4(±0.7)

3.1(±0.9)

10.5(±1.4)

1.3(±0.5)

2.0(±0.0)

2.7(±0.8)

2.3(±0.5)

8.5 (±0.7)

8weeks 0.8(±0.8)

1.9(±0.3)

3.1(±0.3)

2.8(±0.4)

8.7(±1.3)

0.8(±1.3)

1.3(±0.7)

2.0 (±1.1)

2.1(±1.3)

5.3 (±3.0)

24weeks

0.8(±0.8)

1.2(±0.4)

1.9(±0.9)

2.1(±1.0)

6.0(±2.4)

0.8(±0.8)

1.8(0.4)

2.2(0.8)

2.2(0.8)

7.0(±2.0)

CartilagerepairevaluatedusingthePinedascoringmethod.ThedifferentcomponentsofthePinedascoringmethodareshowed.Lowerscorerepresentsbettercartilagerepairresults.F:Fillingofthedefect(-1-4),R:Re-constructionofosteochondraljunction(0-2),M:Matrixstaining(0-4)andC:Cellmorphology(0-4).Valuesaremean(±SD).Boldindicatesp<0.05versusControldiet.

High fat diet does not cause major systemic or local metabolic and inflammatory changes in DBA/1 mice

24hoursbefore theoperation,high fatdiet fedmicewereheavier (26.2grams

versus23.3grams;p=0.004)andhadhigherserumglucose(p=0.01)thanmicere-

ceivingacontroldiet(Figure2A-B).Seruminsulinandamyloidwerenotdifferent

betweenthetwogroups.EightweeksaftertheoperationthemiceonHFDwere

stillheavierandhadhigherfastingserumglucose,althoughtheglucoselevelwas

notdifferentfrom1weekafteroperation.(Figure2B).Seruminsulinandamyloid

AwerenotaffectedbyHFD(Figure2CandD).Asseenonhistology,HFDinduced

adipocytehypertrophy(Figure2E).Therewaslessthanonecrownlikestructure

(CLS)per100adipocytesandtherewasnosignificantdifferencebetweencontrol

andHFDfedmice.Finally,weinvestigatedwhetherHFDinfluencedlocalinflamma-

tioninthejointandspecificallythesynovium.Thesynoviumwasmoreinflamed1

weekaftertheoperationcomparedto8weeks(Figure3A-B).Therewasnodetect-

ablesynovialinflammationinthenon-operatedknees.Nostatisticallysignificant

effectoftheHFDonsynovitisandrankofF4/80,iNOSorCD206positivecellswas

detected(Figure3B-E).


7

Long term effects of high fat diet and cartilage damage in DBA/1 mice

LongtermfeedingwithHFDsignificantlyincreasedtheweightofthemice(Figure

2A,41.1gramsversus30.3grams;p=0.008),butdidnotinfluenceserummetabolic

and inflammatorymarkers(Figure2B-D).Asmall lossof thioninstainingof the

cartilage surrounding thedefect in theoperatedkneeswasvisible (Figure1C).

Therewashowevernomajorcartilagelossorstructuralchangesindicatingos-

teoarthritisafter24weeksintheoperatedknees,notevenincombinationwitha

HFD(Table2,Figure1C).Therewasnocartilagelossinthenon-operatedknees

(Figure1C,Table2)

Body weight

0 10 2020

30

40

50ControlHigh fat diet

24-2Weeks

Gra

ms

** *

* * * ** * * *

E

F4/80

Control High fat

A B

C D

Fasting serum glucose

0

5

10

15

20 ControlHigh fat diet

-2Weeks

mm

ol/L

*

0 1 8

*

24

**

Fasting serum insulin

0

2

4


-2Weeks

mm

ol/L

0 1 8 24

Fasting serum amyloid A

40

50

60

70

80


-2Weeks

mg/m

l

0 1 8 24

Figure 2 Highfatdietincreasesbodyweight,butdoesnotcausemajorsystemicmetabolicandinflammatorychangesinDBA/1mice.Micewerefastedfor4hourspriortoserumcollection.A)Bodyweightofthemice.FastingserumB)glucose,C)insulinandD)amyloidAat1,8and24weeksafteroperation.Timepoint0represents24hoursbeforeoperation.E)RepresentativepicturesofsubcutaneousadiposetissuestainedwithantibodiesagainstF4/80at8weeksaf-teroperation.Positivecellsareindicatedwitharrows.Barvaluesaremean±SD(n=6-9micepergroup).*p<0.01versuscontroldiet.**p<0.05versuscontroldiet.

128 CHAPTER 7

DISCUSSION

Obesity,amajorrisk factor for jointdiseases9,hasbeenreportedtonegatively

influence the clinical results of cartilage repair procedures6;21.Thepresentstudyis

toourknowledgethefirstonethatinvestigatedthedirecteffectofaHFDinduced

weight gain on intrinsic repair of cartilage defects in an experimental mouse

model for cartilage repair. In contrast toourhypothesis,miceonaHFDhada

betterPinedascorethanthemiceonacontroldiet.Thisdifferencewasalready

detectable1weekaftermakingthedefect,butbecamemoreobviousafter8weeks.

Wecouldnotdetectasignificantdifferenceincartilagerepairanymorebetween

thetwodietgroupsafter24weeks,suggestingthatHFDonlyacceleratedandnot

improvedcartilagerepair.

Synovitis

Control High fat Control High fat0

2

4

6

8

1 week 8 weeks

P<0.01

##

# #Scor

e

Nonoperated

F4/80


20

30

40

P<0.01

1 week 8 weeks

# #

# #

Rank

ing

Nonoperated

iNOS


20

30

40

1 week 8 weeks

P<0.05

# #

##

Rank

ing

Nonoperated

CD206


20

30

40

1 week 8 weeks

P<0.01

# #

# #

Rank

ing

Nonoperated

F4/80 iNOS CD206A H&E

1 weekafter

operation

8 weeksafter

operation

B C D E

Figure 3 Highfatdietdoesnotinfluenceoperationinducedsynovialinflammation.A)RepresentativepicturesofsynoviumwithimmunohistochemicalstainingwithantibodiesagainstF4/80,iNOSandCD206,1and8weeksafteroperation.B)SynovitisscoreandrankingscoreofpositivecellsforC)F4/80,D)iNOSandE)CD206.Barvaluesaremean±SD(n=6-9pergroup).#p<0.01versusnon-operatedknees

Table 2: Longtermeffectsofhighfatdietonosteoarthritisdevelopment.

Controldiet Highfatdiet P-Value

ModifiedMankinscore(non-operatedknee)

0(NoOA) 0(NoOA) 1.0

ModifiedMankinscore(operatedknee)

1.5(±0.5) 1.7(±0.5) 1.0

P-Value p<0.01 p<0.01

Osteoarthritisscores24weeksafteroperation.Valuesaremean(±SD)(n=6micepergroup)


7

PreviousreportsonHFDhaveallshowndetrimentaleffectsofanHFDoncartilage.

Wuetal12showedthatHFDreducedearwoundhealingandacceleratedthepro-

gressiontoOAafterdestabilizationofthemedialmeniscus.Loueretal10 showed

thatHFDacceleratedpost-traumaticarthritis.However,thesestudiesusedyoung

adultC57BL/6miceandthisstrainofmice,incontrasttoDBA/1mice,doesnot

possess cartilage repair capabilities16;22;23NexttoDBA/1mice,MRL/MpJmiceare

knowntopossesscartilagerepaircapabilities24;25.Interestingly,thesemicegained

weightand fatmass,butdidnotdevelop themajormetabolicchangesseen in

C57BL/6miceafterbeingfedaHFD17;26.Ourmicegainedweight,evenonlyafter2

weeksofHFDandcontinuedtoincreaseweightduringthe24weeksofthestudy,

butdidnotdevelopmajormetabolicchanges.Thissuggests that thedegreeof

metabolicresponsetoHFDandabilitytorepaircartilagedamagemightbeassoci-

ated.

Individualdifferences in response tobeingobeseare alsoknown inhumans.

Accumulationofmacrophagesincrownlikestructures(CLS)inobesesubjectsis

associatedwithhyperinsulinemiaand increasedsystemic inflammation27; 28 and

metabolicallyhealthyobesesubjectsdonothavetheseCLSintheiradiposetis-

sue.TheabsenceofCLSintheadiposetissueoftheDBA/1miceonaHFDagain

indicatesthattheDBA/1miceareresistanttoHFD-inducedmetabolicandinflam-

matorychanges.

PreviouslywehaveshownthatthelevelofinflammatoryresponsetoaHFDwas

associatedwiththedevelopmentofOA29.Aninitialhigherinflammatoryresponse

inCRP-C57BL/6micetoaHFDwithincreasedserumlevelsofhumanC-reactive

protein(hCRP)wascorrelatedtoOAdevelopment.Furthermore,rosiglitazone,a

peroxisomeproliferator-activatedreceptors(PPAR)γagonistwithanti-inflamma-

tory properties, decreasedHFD associated inflammation andOAdevelopment,

eventhoughitincreasedweightgaininthesemice.Inthepresentstudy,HFDdid

notinducesystemicandlocalinflammatorychangesinmaleDBA/1micebasedon

theserumamyloidAmeasurementsandimmunohistologicalstainingformacro-

phagesinthesynoviumandadiposetissue.Thesemicealsodidnotdevelopmore

OA,eveninthepresenceofacartilagedefect.Thisagainsuggeststhatsusceptibil-

itytodevelopinginflammatorychangesmightbeassociatedtothedevelopment

ofOA.However,thismayonlyaccountforHFDinducedinflammatorychanges,be-

causeinhibitionofspontaneousoccurringobesityrelatedinflammatorychanges

inSTR/OrtmicewithfenofibratesdidnotinhibitthedevelopmentofOA30.

ThemaleDBA/1miceonacontroldietinourstudyshowedcartilagerepairafter

8weeks,albeitnottothesamedegreeasdescribedbyEltawiletal16.Although

130 CHAPTER 7

themiceusedwereofthesameDBA/1OlaHsdstrainfromthesamecompany,we

havemadethedefectsattheageof12weeksinsteadof8weeks,asinthestudy

byEtawiletal.Intrinsiccartilagerepaircapabilitiesmighthavebeenreducedwith

increasingage,howeveryoungerDBA/1micewouldverylikelyhaveadaptedto

aHFDandnotgainedweight11.Thechoicetouse12weeksoldDBA/1micemight

maketranslationoftheresultsofourstudytoolderindividualsdifficult.However,

thecurrentmodelhaslimitedusnottouseoldermice.Finally,weusedD12450B

as the control diet as advisedby thedietmanufacturer (ResearchDiets).This

controldietcontains10%kcal fromfatand35%kcal fromsucrose.Eltawiletal

usedchowwhichhasloweramountofkcalfromsucrose.Wecannotexcludethat

thehighsucrosecouldhavereducedcartilagerepair.

Furthermore,cartilagerepaircouldbeinfluencedbyweightbearingactivities

after surgery31.AlthoughithasbeenshownthatHFDdoesnotinfluenceactivity

levelinC57BL/6mice,wecannotexcludethatHFDcouldhaveinfluencedactivity

levelofthemiceinourstudy12.

Although HFD had no effect on inflammation, it slightly increased the serum

glucosebeforecreationofthedefectandserumglucoselevelsremainedhigher

8weeksafterthedefectwasmade.Thishigherglucoselevelmayhaveimproved

theinitialcartilagerepairprocessaftercartilagedamage,becausedifferentiation

to chondrocytes and synthesis of extracellularmatrix requires a large amount

of nutrients.An abundanceof nutrientsmight alsohave increased the activity

ofmammaliantargetofrapamycin(mTOR),whichisknowntosupportskeletal

growthandextracellularmatrixproduction32.

However,ahighermTORexpressionincartilageisalsocorrelatedtocartilage

damage and mTOR inhibition by rapamycin could prevent post-traumatic OA

development33. IncreasedmTOR expression could have played a role in initial

cartilagerepairinDBA/1mice,butinthelongtermmTORmighthavebeensup-

pressedtoincreaseautophagyandprotectthecartilage.

Nexttohyperglycemia,aHFDcanleadtohypercholesterolemia.Recentlyithas

beenshown thatcholesterolmetabolismandcartilagedevelopmentandrepair

couldberelated.Statinsareusedtoreduceserumcholesterolthroughinhibition

oftheenzymeHMG-CoAandstatinscouldbeapotentialtherapyfordevelopmen-

taldiseasessuchasachondroplasiaandmightstimulatechondrogenicpotential

ofintervertebraldisccells.34;35However,itisknownthataHFDdoesnotincrease

serumtotalcholesterolinmaleDBA/1mice17.ItcouldbethattheenzymeHMG-

CoAoranotherenzymeinthemevalonatepathwayislessactiveinDBA/1mice

andthatthiscouldberelatedtoincreasedcartilagerepairinthismousestrain.


7

Besides serum metabolic changes, cartilage repair could also be directly

influencedbythefatcontentofHFD.HFDcontainsmainlysaturatedandmono-

unsaturatedfattyacidswhichreduces,ratherthanstimulates,thechondrogenic

potentialofMSCsfromC57BL/6mice15.However,MSCsisolatedfromDBA/1mice

havebeenreportedtohaveabetterchondrogenicpotentialthanthoseisolated

fromC57BL/6mice36.TheeffectofHFDonMSCchondrogenesiscouldthereforebe

differentindifferentstrainofmice.

Inconclusion,ourstudyshowsthatHFDandincreasedbodyweightintheDBA/1

mice do not negatively impair cartilage repair, but can even accelerate it. The

precisemechanisminhowHFDinfluencescartilagerepairinDBA/1isunknown.

However, our data suggests that resistance to HFD induced inflammatory and

metabolic changes could be associated with accelerated cartilage repair and

reducedOAsusceptibility.Furtherresearchisnecessarytoelucidatethecomplex

relationshipsbetweenahighfatdiet,metabolicandinflammatorychanges,carti-

lagedegenerationandregeneration.Fortheclinicalpractice,ourstudysuggests

thatnotallobesepatientsshouldbeexcluded fromtreatment. Investigationof

theirmetabolicandinflammatorystatecouldbehelpfulinmakingthedecisionto

startcartilagerepairtreatments.

132 CHAPTER 7

REFERENCES

1. WiduchowskiW,WiduchowskiJ,TrzaskaT.2007.Articularcartilagedefects:studyof25,124knee

arthroscopies.Knee14:177-182.

2. NewmanAP.1998.Articularcartilagerepair.AmJSportsMed26:309-324.



4. MithoeferK,McAdamsT,WilliamsRJ,etal.2009.Clinicalefficacyofthemicrofracturetechnique

forarticularcartilagerepairintheknee:anevidence-basedsystematicanalysis.AmJSportsMed

37:2053-2063.

5. NegrinL,Kutscha-LissbergF,GartlehnerG,etal.2012.Clinicaloutcomeaftermicrofractureofthe

knee:ameta-analysisofbefore/after-dataofcontrolledstudies.IntOrthop36:43-50.



1920.


knee.JBoneJointSurgAm92:2470-2490.

8. LumengCN, SaltielAR. 2011. Inflammatory linksbetweenobesity andmetabolicdisease. JClin

Invest121:2111-2117.


8:729-737.

10. Louer CR, Furman BD, Huebner JL, et al. 2012. Diet-induced obesity significantly increases the

severityofposttraumaticarthritisinmice.ArthritisRheum64:3220-3230.

11. HamaguchiK,ItabashiA,KuroeY,etal.2012.Analysisofadiposetissuesandstromalvascularcells

inamurinearthritismodel.Metabolism61:1687-1695.


pathogenesisofosteoarthritisfollowingjointinjury.AnnRheumDis.

13. BuettnerR,ScholmerichJ,BollheimerLC.2007.High-fatdiets:modelingthemetabolicdisordersof

humanobesityinrodents.Obesity(SilverSpring)15:798-808.

14. GriffinTM,HuebnerJL,KrausVB,etal.2012.Inductionofosteoarthritisandmetabolicinflamma-

tionbyaveryhigh-fatdietinmice:effectsofshort-termexercise.ArthritisRheum64:443-453.

15. WuCL,DiekmanBO,JainD,etal.2013.Diet-inducedobesityaltersthedifferentiationpotentialof

stemcellsisolatedfrombonemarrow,adiposetissueandinfrapatellarfatpad:theeffectsoffree

fattyacids.IntJObes(Lond)37:1079-1087.

16. EltawilNM,DeBariC,AchanP,etal.2009.Anovelinvivomurinemodelofcartilageregeneration.

Ageandstrain-dependentoutcomeafterjointsurfaceinjury.OsteoarthritisCartilage17:695-704.

17. SvensonKL,VonSmithR,MagnaniPA,etal.2007.Multipletraitmeasurementsin43inbredmouse

strains capture the phenotypic diversity characteristic of human populations. J Appl Physiol

(1985)102:2369-2378.

18. Pineda S, PollackA, Stevenson S, et al. 1992.A semiquantitative scale for histologic gradingof

articularcartilagerepair.ActaAnat(Basel)143:335-340.

19. vanderSluijsJA,GeesinkRG,vanderLindenAJ,etal.1992.ThereliabilityoftheMankinscorefor

osteoarthritis.JOrthopRes10:58-61.

20. KrennV,MorawietzL,HauplT,etal.2002.Gradingofchronicsynovitis--ahistopathologicalgrading

systemformolecularanddiagnosticpathology.PatholResPract198:317-325.


7

21. JaiswalPK,BentleyG,CarringtonRW,etal.2012.Theadverseeffectofelevatedbodymassindex

onoutcomeafterautologouschondrocyteimplantation.JBoneJointSurgBr94:1377-1381.

22. MatsuokaM,OnoderaT,SasazawaF,etal.2015.AnArticularCartilageRepairModelinCommon

C57Bl/6Mice.TissueEngPartCMethods.

23. RaiMF,SandellLJ.2014.Regenerationofarticularcartilageinhealerandnon-healermice.Matrix

Biol39:50-55.

24. FitzgeraldJ,RichC,BurkhardtD,etal.2008.EvidenceforarticularcartilageregenerationinMRL/

MpJmice.OsteoarthritisCartilage16:1319-1326.

25. MakJ,LeonardC,FoniokT,etal.2015.Evaluatingendogenousrepairoffocalcartilagedefectsin

C57BL/6andMRL/MpJmiceusing9.4Tmagneticresonance imaging:Apilotstudy.MagnReson

Imaging33:690-694.

26. MullAJ,BerhanuTK,RobertsNW, et al. 2014.TheMurphyRothsLarge (MRL)mouse strain is

naturallyresistanttohighfatdiet-inducedhyperglycemia.Metabolism63:1577-1586.



VascBiol28:1654-1659.

28. vanBeekL,LipsMA,VisserA, et al. 2014. Increasedsystemicandadipose tissue inflammation

differentiatesobesewomenwithT2DMfromobesewomenwithnormalglucosetolerance.Metabo-

lism63:492-501.

29. GiermanLM,vanderHamF,KoudijsA,etal.2012.Metabolicstress-inducedinflammationplaysa

majorroleinthedevelopmentofosteoarthritisinmice.ArthritisRheum64:1172-1181.

30. WeiW,ClockaertsS,Bastiaansen-JenniskensYM,etal.2014.Statinsandfibratesdonotaffectdevel-

opmentofspontaneouscartilagedamageinSTR/Ortmice.OsteoarthritisCartilage22:293-301.

31. HowardJS,MattacolaCG,RomineSE,etal.2010.ContinuousPassiveMotion,EarlyWeightBear-

ing,andActiveMotion followingKneeArticularCartilageRepair:Evidence forClinicalPractice.

Cartilage1:276-286.

32. LaplanteM,SabatiniDM.2009.mTORsignalingataglance.JCellSci122:3589-3594.

33. ZhangY,VasheghaniF,LiYH,etal.2015.Cartilage-specificdeletionofmTORupregulatesautophagy

andprotectsmicefromosteoarthritis.AnnRheumDis74:1432-1440.

34. BushJR,BerubeNG,BeierF.2015.Anewprescriptionforgrowth?Statins,cholesterolandcartilage

homeostasis.OsteoarthritisCartilage23:503-506.

35. ZhangH,LinCY.2008.Simvastatinstimulateschondrogenicphenotypeofintervertebraldisccells

partiallythroughBMP-2pathway.Spine(PhilaPa1976)33:E525-531.

36. PeisterA,MelladJA,LarsonBL,etal.2004.Adultstemcellsfrombonemarrow(MSCs)isolated

fromdifferentstrainsofinbredmicevaryinsurfaceepitopes,ratesofproliferation,anddifferentia-

tionpotential.Blood103:1662-1668.

EPILOGUE

CHAPTER 8

Summary and general discussion

Summary and general discussion 139

8

SUMMARY AND GENERAL DISCUSSION

Themostcharacteristicchangesinobesityareanincreaseinadiposetissuemass

andadipose tissue inflammation.This in turn leads to increased loadingof the

jointsandsystemicmetabolicandinflammatorychanges.Becauseadiposetissue

is amajor contributor to the inflammation in obesity,wehave focusedon the

effectofadiposetissueondifferenttissueinthekneejointandcartilagerepair.

LOCAL EFFECTS OF ADIPOSE TISSUE

The infrapatellar fat pad, an adipose tissue not changed by obesity

Theinfrapatellarfatpad(IPFP)isanintra-articularadiposetissueindirectcontact

withthesynoviummembraneandcanthereforeinfluencethekneejointenviron-

ment. Itcanplayanrole inbiomechanicsof theknee,actasashockabsorber

andisareservoirofinflammatoryandregenerativecells1-3.Adiposetissueingen-

eralfunctionsasareservoirforexcessnutrientsandincreasesinsizeinobesity.

However,MRIstudiessuggestthat,unlikemostotheradiposetissuesinthebody,

IPFPsizeisnotcorrelatedtobodymassindex(BMI)4-6.Furthermore,unlikeinsub-

cutaneousandvisceraladiposetissue,theadipocytesizeinosteoarthritic(OA)

IPFPwasnotcorrelatedtoBMI(Chapter 2).Besidesadipocytehypertrophy,an

increased infiltrationofpro-inflammatorymacrophagesand formationofcrown

likestructuresinadiposetissuearegenerallyconsideredtobecharacteristicsfor

obesity7.However,wecouldnotdetectadifferenceinmacrophageinfiltrationin

IPFPbetweenobeseandnon-obesedonorsinourpreviouswork8 nor in Chapter

4.Anin-vivostudyinhighfatdietfedC57BL/6micehasconfirmedourfindingthat

adipocytesizeandinfiltrationofmacrophagesarenotchangedbyweightgain9.

Obesity increases secretion of pro-inflammatory factors such as TNFα, IL1β,MCP-1,IL6andleptinbyadiposetissue10.However,wehavepreviouslyshownby

multiplexELISAanalysisthatsecretionofcytokinesbyOAIPFPisnotassociated

toBMI11.Finally,alltheeffectstheIPFPhadoncartilage,synovialfibroblastsand

MSCswerenotcorrelatedtoBMI(Chapter 3 and 4)8,.ThisallsuggeststhatIPFP,

unlikesubcutaneousandvisceraladiposetissue,isnotinfluencedbyobesity.

The IPFP and inflammation

Although the IPFPseemsnot tobe influencedbyobesity, it couldstillbecome

inflamedafterjointtraumaandsurgery12-14.InflammatorycytokinessuchasIL1β

canincreasethesecretionofinflammatoryfactorsbyIPFP11.Withmoreinflamma-

torystimuli,thereisanincreasedsecretionofinflammatoryfactorsbytheIPFP.

140 CHAPTER 8

Becausethesynoviumdoesnothaveabasalmembrane,theonlybarrierformol-

eculesbetweenthesynovialfluidandtheIPFPistheextracellularmatrix(ECM)

of thesynovial interstitium. Inthiscase, thesmaller themolecule, theeasier it

canfreelydiffusethroughthesynovialinterstitium15.WeinvestigatedinChapter

4theeffectofIPFPsecretedfactorsoncartilagerepair,andfoundthattheycould

inhibitMSC-basedcartilagerepair.Nexttodirectlyinfluencingtherepairprocess,

factorssecretedby IPFPalso influencethesynoviummembranebystimulating

pro-fibrotic processes (Chapter 3)andpro-catabolicprocessesinsynovialfibro-

blasts16( Chapter 5).Inturn,thiscanleadtoanincreaseininflammatoryfactors

inthesynovialfluidandfurtherdeteriorationofthejointenvironmentforrepair.

Studies have shown an increased amount of pro-inflammatory cytokines such

as IL1α/β, IL6, IL8, andTNFα, in synovial fluids of patientswith focal cartilage

defects17; 18, ACL rupture19-21 or OA22 knees compared to healthy knees. Pro-

inflammatorycytokinessuchasIL1α/β,IL6,IL17,IFNƴ,TNFα,MCP1canallinhibit

MSCchondrogenesis23-26 andincreasecatabolicprocesses inthesynoviumand

cartilage27.ReductionofthesecretionofthesecytokinesbytheIPFPorinhibiting

theeffectsofthesecytokinescouldbepromisingstrategiestoimprovethejoint

environmentandcartilagerepair.Oneshouldbecarefulhoweverwithinhibition

ofinflammation.StudiesinMRL/MpJmice,whopossesssuperhealingcapacities,

haveshownthatinflammationisnecessaryforsuccessfulwoundhealing28;29.Find-

ing the rightbalancebetweensufficient inflammation to initiatewoundhealing

andreducechronicinflammationtoinhibitcatabolicprocessesisnecessaryfor

successfulcartilagerepair.

TheIPFPconsistsmainlyofadipocytes,stemcellsandinflammatorycellssuchas

macrophages.InChapter 4wehaveshownthatpro-inflammatorymacrophages

in IPFPcontributetotheanti-chondrogeniceffectof IPFP.Macrophagesplayan

importantroleininflammationandtissuerepair.Pro-inflammatorymacrophages

secreteTNFα,IL1βandIL630andcouldthereforeinhibitcartilagerepairinvitro,in-

duce catabolic processes in cartilage31andpro-fibroticprocessesinsynovialfibro-

blasts (Addendum figure 1).Ananimalstudyreportsthatablationofallsynovial

macrophagescouldinhibitdevelopmentofOA32.Anti-inflammatorymacrophages

however,donotinhibitcartilagerepair(Chapter 4)33andmightbeevenbeneficial

tocartilagerepairinvitro34.Furthermore,Axolotls,whichareknowntobeable

toregenerate limbs,cannotregeneratewithout thepresenceofmacrophages35.

Therightbalancebetweenpro-andanti-inflammatorymacrophagesseemstobe

crucialforsuccessfulcartilagerepair.InChapter 5weuseddifferentmedications

tomodulatethemacrophagesinsidetheIPFPtowardsamoreanti-inflammatory

phenotype.Ofthemedicationswetested,onlytriamcinoloneacetonide(TAA)was


8

able to reduce inflammation in IPFPandmodulate themacrophages towardsa

anti-inflammatoryphenotype.Theanti-chondrogenicandpro-cataboliceffectsof

theIPFPwerealsoreducedbyTAA.Thissuggeststhatmodulationofmacrophage

phenotypebymedicationmight improve the joint environment to reduce joint

diseaseandbebettersuitableforcartilagerepair.

Although the adipocyte size, macrophage phenotype in and the secretion of

inflammatorycytokinesbyIPFParenotinfluencedbyobesity,theIPFPremains

alargesourceofinflammatoryfactorsthataffectsthejointenvironment.Nextto

inflammatorycytokines,theadipocytesinIPFPsecretemanyadipokinessuchas

leptin36,adiponectin36,resistinandvisfatin11.Leptin,resistinandvisfatinstimulates

catabolic processes in cartilage1,synovium1andmeniscus37.Theroleofadiponec-

tinisnotyetfullyunderstood.AdiponectinincreasesATDC5-cellproliferationand

chondrogenesis38,howeveritisalsoassociatedwithcartilagedamageinOAand

couldincreaseMMP13activityandPGE2releaseinOAchondrocytes39.However

blockingadiponectinand leptindidnot inhibit thepro-cataboliceffectsof IPFP

on OA chondrocytes and synovial fibroblasts36. The effect of these adipokines

onMSCchondrogenesisisstillunknown,butreductionofcatabolicprocessesin

generalwouldeventuallybenefitthejointenvironment.Glucocorticoidsmodulate

adipokinessecretionbyadiposetissue40-42.Therefore,thereductionofcatabolic

processes in Chapter 5by treatmentwithTAAcouldalsohave takenplacevia

modulationofadipokinesecretionbytheIPFP,nexttomodulationofmacrophages

andtheirsecretionprofile.

TheIPFPandadipocytesalsoreleaseprostaglandins.Prostaglandinsareagroup

oflipidsthatplayanimportantroleininflammation43,cartilagemetabolism44;45 and

pathogenesisofOA46;47.InChapter 3wedescribethatprostaglandinF2α(PGF2α)

secreted by IPFP is involved in pro-fibrotic processes in synovial fibroblasts.

However,PGF2αdoesnotinhibitMSCchondrogenesis(Addendum figure 2),but

evenenhanceschondrogenesisinATDC5cells45andmatrixdepositionbyarticular

chondrocytes44.NexttoPGF2α,theIPFPalsosecretesprostaglandinE2(PGE2)48;49,

which stimulatespro-catabolic processes in synovial fibroblasts48. Inhibitionof

prostaglandinsecretion ispossiblethroughinhibitionofcyclooxygenase(COX)

activity.Non-specificCOXinhibitorssuchasnon-steroidanti-inflammatorydrugs

(NSAIDs) and specificCOX-2 inhibitors such as celecoxib are already clinically

usedtoreduceinflammationandthesymptomsofOA.AlthoughCOXinhibition

could be beneficial to reduce pro-fibrotic and catabolic processes47,non-specific

COX-inhibitionalsoinhibitsMSCchondrogenesis50.Ontheotherhand,aspecific

COX-2inhibitorislessdetrimentaltochondrogenesis50-52.Therefore,specificCOX-2

142 CHAPTER 8

inhibitorssuchascelecoxibarepossiblecandidatestoimprovethejointenviron-

mentforcartilagerepair.Surprisingly,celecoxibdidnotreduceIPFPinflammatory

genesexpressioninChapter 5andbecausereductionofgeneralIPFPinflammation

isimportanttoimprovethejointenvironment,wedidnotfurtherinvestigatethe

effectsofcelecoxiboncartilagerepair. InChapter 5wedemonstratedareduc-

tion inanti-chondrogenicandpro-cataboliceffectsof IPFPafter treatmentwith

TAA.TAAisanoftenusedglucocorticoidandglucocorticoidsareknowntoinhibit

prostaglandin synthesis53.However,italsoinhibitsotherpro-inflammatorypath-

waysandthereforetargetingprostaglandinsynthesisalonemightbeinsufficient

toimprovethejointenvironmentforcartilagerepair.

Nexttoprostaglandins,therearealsootherlipidsinthesynovialfluidoftheknee

joint49;54.Onegroupof lipidsare the fattyacidsand these fattyacidsarebeing

extensivelystudied.Fattyacidscanstimulateorinhibitinflammatoryprocesses55

and influence cartilage loss in OA cartilage56. An increased intake of omega-3

fattyacidscoulddecreasechronicinflammation57 and dietary fatty content could

regulatewoundhealinginmice58.Itisthereforeinterestingtoinvestigatewhether

changingdietisapossiblestrategytomodulateIPFPinflammationandthereby

improvecartilage repairprocedures. Furthermore, future researchcouldbe fo-

cusedonusingmedicationstomodifythelevelsofspecifictypesoffattyacidsand

inthatwayinfluencecartilagerepair.InChapter 5weusedstatinsandfibrates,

twooftenusedserumlipidloweringdrugs,toreduceIPFPinflammation.However,

wedidnotseeaneffectonIPFPinflammatorygenesexpression.Thesemedica-

tionsareknowntoreducesystemicinflammationsotheycouldstillbeusefulin

improving thegeneral jointhealthduringcartilagerepairprocedures,although

moreinvestigationisnecessary.

Removal of the IPFP

InsteadofmodulationoftheIPFP,itisalsopossibletosurgicallyremovetheIPFP2.

Duringtotalkneearthroplasty,theIPFPisoftenremovedtoimprovevisibilitydur-

ing the surgical procedure59;60.HowevertheremovaloftheIPFPduringtotalknee

arthroplasty is under debate61 because it is associated with shortened patellar

tendonandanteriorkneepain.Evensubtotalremovalof the IPFPwitharthros-

copy could lead to patellar tendon shortening60;62-64.Thissupportsthetheorythat

theIPFPhasamechanicalfunctionthatisnecessaryforproperjointmovement.

Besidesadipocytesandmacrophages,therearealsoMSCsintheIPFP65-67.These

MSCshavebeenshowntohavechondrogenic66;67aswellasimmunomodulatory68

capacities and some authors have proposed to use theseMSCs inMSC-based


8

cartilage repair procedures66;69.ItmightalsobepossiblethatMSCsfromtheIPFP

migratetoacartilagedefectandcontributetorepair,althoughcurrentlythereis

nostudythathaveshownthis.MSCsfromthebonemarrow70andsynovialmem-

brane71couldmigratetowardsacartilagedefect.Peripheralmononucleatedcells

migratefromthebloodcirculationtoacartilagedefectinrats72,sofutureresearch

mightshowthatMSCsfromIPFPcouldalsodothis.

Furthermore,wehavepreviouslyshownthatOAIPFPconditionedmediuminhib-

itscatabolicmediatorsinbovinecartilage8.ThismeansthattheeffectofIPFPon

theseparatejointstructurescouldbedifferent.Hence,completeremovalofthe

IPFPcouldchangethejointenvironmenttoomuchandremovethebeneficialcells

intheIPFPthatmightcontributetosuccessfulcartilagerepairintheknee.

Other intra-articular adipose tissues

Mostsynovialjointshaveadiposetissuefillingthesynovialfoldsaroundthear-

ticularmargin.Thesefatdepotspossiblyactascushionsduringjointmovement73.

AlthoughtheIPFPisthelargestintra-articularadiposetissue,itisnottheonlyone

intheknee.Inthekneetherearealsothesupra-patellarfatpad,locatedbeneath

thequadricepstendon74;75,andthepre-femoralfatpad,locatedattheanteriorside

ofthefemur.SignalalterationsonMRIofthesefatpadsareassociatedtosynovi-

tis,anteriorkneepainandstructuralabnormalities74;76;77.Duetotheirproximity

totheIPFPandbecausetheyareincontactwiththesamesynovialsynovialfluid,

itmightbeexpectedthatthesameinflammatorysignalsthatwouldinfluencethe

IPFPwouldalsoinfluencethesefatpaddepots.

Besides the knee, the ankle and the hip also have an intra-articular adipose

tissue.IntheankleitiscalledtheKager’sfatpadandislocatedposteriortothe

anklejoint,anteriortotheAchillestendon78.TheKager’sfatpadisalsoknownas

thepre-Achilles fatpad.Traumato thecalcaneusorAchilles tendinopathycan

influencetheKager’sfatpad78;79.Theintra-articularadiposetissueinthehiphas

only been recently described80andissimilartotheIPFPintissularphenotype81.

Notmuch isknownaboutthisadiposetissue,but itcanbecome impingedand

causepainduringhipmovement80.

Withmoreunderstandingoftheroleoftheseotherintra-articularadiposetis-

sues,wemightimproveourunderstandingoftheroleoftheIPFPinthekneejoint.

Furthermore,theseotherintra-articularadiposetissuemightalsobetargetedto

modifythejointenvironment.

144 CHAPTER 8

SYSTEMIC EFFECTS OF ADIPOSE TISSUE

Besidesthelocaleffectsofadiposetissueonjointdiseaseandrepair,thesystemic

effects has alsobeen a focus of investigation. Especially the systemic changes

thatoccurinobesityisbeingextensivelystudied.Obesityincreasesthechance

of developing the metabolic syndrome, which is characterized by low grade

systemicinflammation,dyslipidemia,hyperinsulinemiaandhyperglycemia.Meta-

bolicsyndromehasbeenshowntobeariskfactorforthedevelopmentofOA82;83.

Obesityandtheresultingmetabolicsyndromearecausedbyaninterplaybetween

behavioral, environmental and genetic factors84; 85. Althoughourwork suggests

that the IPFP is possibly not influencedby obesity, subcutaneous and visceral

adiposetissuesbecomeinflamedduetoobesity86.Inadiposetissueinflammation,

there ismore infiltrationofpro-inflammatorymacrophages, formationofcrown

likestructuresandthereisanincreaseinsecretionofinflammatoryfactorssuch

asTNFα,IL1β,MCP-1,IL6andleptin86.Toinvestigatetheeffectofmetabolicandin-

flammatorysystemicchangesonjointdiseaseandcartilagerepair,weperformed

twoin-vivostudieswithmice.

Obesity related metabolic and inflammatory changes on cartilage damage

In Chapter 6weusedtheSTR/Ortstrainofmice.Thesemicedevelopmetabolic

and inflammatorychangesspontaneouslyonanormaldiet (Chapter 6,87).This

isnotcausedbyovereatingascomparedtotheOB/OBstrainofmice,whichdue

totheirleptindeficiency,doesnotstopeating88.Nexttodevelopingspontaneous

metabolicsyndrome89,maleSTR/Ortmicearealsoknowntodevelopspontaneous

cartilagedamage90.Thismousemodel therefore seemed tousverywell suited

to investigate whether development of spontaneous metabolic syndrome and

cartilagedamagearelinkedandwhethercartilagedamagecouldbeprevented.In

Chapter 6wefedmaleSTR/Ortmicechowmixedwithsimvastatinandfenofibrate.

Thesetwolipid loweringmedicationsarecommonlyusedtotreatdyslipidemia

and to reduce cardiovascularmorbidity andmortality91;92. Statin use has been

associatedwithdecreasedincidenceandprogressionofkneeOA93.Furthermore,

statinsandfibratesalsoreducesystemicinflammation94;95.InChapter 5wehave

shownthatstatinsandfibratesdidnotreduceIPFPinflammation,suggestingthat

thesemedicationsmightonlyhavesystemiceffects.Inourin-vivostudy,TheSTR/

Ortmicedevelopeddyslipidaemiaandsimvastatinandfenofibratedidnothave

aneffectondyslipidaemia in thesemice.Fenofibrate reducedweight, systemic

inflammationandvolumeofsubchondralbone,whereassimvastatindidnothave

aneffect.However, incontrast towhatweexpected,cartilagedamagewasnot

reducedbythesetwomedications.Ourresultssuggestthatdevelopmentofcar-


8

tilagedamageinSTR/Ortmiceisnotprimarilylinkedtometabolicinflammation,

butismulti-factorial.Thisshowsthatacombinationofmetabolicinflammatory

changes andgeneticpredisposition are the causeof cartilagedamage in these

mice.Thisalsosuggestthatbeingobesedoesnotalwaysleadtocartilagedamage.

Obesity and cartilage repair

Whencartilagedamagehaveoccurredinobesepatients,weneedtorepairitto

preventthedevelopmentofOA.Theclinicalresultsofcartilagerepairinobesepa-

tientsisworse,butthereisnoinformationonthestructuraloutcomes.InChapter

7weinvestigatedwhetherahighfatdiet(HFD)inducedobesityreducescartilage

repairinDBA/1mice.Surprisingly,theHFDdidnotnegativelyinfluencecartilage

repair,butevenacceleratedit.Thiswasincontrasttocommonknowledgeand

whatwehypothesized.Furthermore,althoughthesemiceareknowntoberesistant

toOAdevelopmentaftercartilagedamage,theydidnotevendevelopOAaftercar-

tilagedamageincombinationwithaHFD.TheDBA/1miceonaHFDgainedweight,

butdidnothaveseruminflammatoryandmetabolicchanges.Furthermore,the

subcutaneousadipocytesofthesemiceonaHFDbecamehypertrophic,butthere

wasnoincreasedinfiltrationofmacrophagesnorformationofcrownlikestruc-

tures.ThisisinstarkcontrasttotheSTR/OrtmiceweusedinChapter 6,which

developed metabolic and inflammatory changes spontaneously. This suggests

thatresistancetoHFDinducedinflammatoryandmetabolicchangesisassociated

withacceleratedcartilagerepairandreducedOAsusceptibility.Inhumans,some

individuals are prone to development ofmetabolic syndrome84, but some also

gainweightandbecomeobese,butdonotdeveloptheaccompanyingmetabolic

and inflammatorychanges.Thisgroupofobese individuals, alsoknownas the

metabolic healthy obese, are often characterized by less serum inflammation,

lessvisceralfat,lessmacrophageinfiltrationinadiposetissueandlessadipocyte

hypertrophy96;97.Itremainsinconclusivewhethertheriskofcardiovascularheart

diseaseisdifferent inthisgroupof individuals97comparedtoobeseindividuals

withmetabolicchanges.Furthermore,itisunknownwhethertheseindividualsare

protectedfromjointdiseaseorhaveimprovedrepaircapabilities.Futureresearch

investigatingwhethertheseindividualsareprotectedagainstjointdiseaseorhave

improvedrepairmightprovideuswithnewtreatmenttargets.

The results of our two in-vivo studies indicate that the link between obesity

relatedmetabolicandinflammatorychanges,degenerativejointdiseaseandcar-

tilagerepair iscomplex.Furthermore,thereisheterogeneityin individualswho

areobese.AstudybyGreenetalhasshownthattherearemultipletypesofobese

individuals,whoallhavetheirspecificcausesforobesityandrelateddiseases98.

146 CHAPTER 8

Theauthorshaveconcludedthattreatmentstrategiesforobesityshouldnottar-

getobeseindividualsasawhole,butsubgroupspecificstrategiesshouldbeused.

Tobetterunderstandhowobesityinfluencesthejoint,itisthereforeimportantto

takeintoaccountthisheterogeneityofobesity.Repetitionofourin-vivostudies

usingdifferentanimalmodelsshouldtakethisheterogeneityintoaccount.

FUTURE PERSPECTIVES

Obesity, adipose tissue and the joint environment

Adiposetissueisnotonlyastoragesiteforexcessenergy,butisalsoanendocrine

organ,whichsecreteslargeamountsofimmunological,endocrineandhormonal

factors.Adiposetissueinflammationplaysanimportantroleinthepathophysi-

ologyofmetabolicdysregulation inobesity.Much isknownabout thechanges

thatoccurinsubcutaneousandvisceraladiposetissueinobesity.Lessisknown,

however,aboutobesityrelatedchangesinintra-articularadiposetissue.Obesity

isawell-knownriskfactorforjointdiseasesandhasbeenextensivelystudiedin

animalandclinicalstudies.Duetotheintra-articularlocationofIPFP,ithaslong

been thought that the IPFPwouldplayan important role inobesityassociated

jointinflammation.However,wehaveshownthattheIPFPmightnotbeinfluenced

byobesityandthatobesitycausesdegenerativejointdiseasesviaotherpathways

andnotviaIPFPinflammation.Thiscouldbethroughincreasedsystemicinflam-

mationormetabolicdysregulationduetoinflammationofthebiggersubcutane-

ousandvisceraladiposetissuedepots.

However,theeffectsofobesitycouldvarybetweenindividuals.Wehavenowshown

inanimalstudiesthatobesityisnotalwaysnegativeortherelationshipbetweenthe

changescausedbyobesityondegenerativejointdiseaseisnotalwayscompletely

clear.Theobesityepidemicwillonlyincreaseinthefutureandresearchwillcon-

tinue inthisfield.Newtargets forstrategiesto improvethe jointenvironment in

obesitymightbefoundinresearchfocusingonsubgroupsofindividualswhoare

lesssusceptibletodevelopingobesityrelatedchanges.Itwouldbeinterestingto

investigatewhethertheseindividualsarealsoprotectedagainstjointdiseases.

Modulation of the infrapatellar fat pad to improve the joint environment for cartilage repair

Irrespectivelytotheeffectsofadiposetissuerelatedsystemicchangeshaveonthe

local jointenvironment, the IPFPsecretesa largeamountofdifferentadipokines

andfattyacids.Theseindividualfactorsmighthaveanabolicorcataboliceffectson


8

differentjointstructures.Within-vitroexperimentsusingIPFPconditionedmedium,

wehaveshownwhattheeffectsareofIPFPsecretedfactorsonthejoint.Further-

more,wehaveshownthatwecanmodulatein-vitrothesecretionoffactorsbythe

IPFPandtheresidentmacrophageswithacommonlyusedglucocorticoid.Thenext

stepcouldbeanin-vivoanimalstudytotestthehypothesisthatusingTAAtheIPFP

canbemodulated tobebeneficial for the joint environment.Most experimental

studiesontheeffectofinflammatorymodulationonjointdiseasearefirstperformed

in rodents.However, due to the small sizeof the rodent knee, specific targeting

theIPFPisdifficult,althoughnotimpossible.Futureexperimentsusingcanineor

ovinemodelscouldbeconsideredtoinvestigatewhetherIPFPmodulationwouldbe

feasibletoimprovethejointenvironmentforcartilagerepair.Itwouldalsobepos-

sibletodirectlyperformahumanstudy,becauseTAAiscurrentlyalreadyinjected

intra-articularly as an anti-inflammatory treatment.Thenext stepwouldonlybe

injectionofadosageofTAAintheIPFPfollowingmicrofracturesurgery.

Currently, theeffectofdifferent intra-articularstrategiesusingpharmacological

drugsandimmunomodulatorcellsisbeingstudiedforusetoimprovethejoint

environmentforcartilagerepairordirectlyinfluencecartilagerepairitself99.Next

to theeffectoncartilage repair, theeffectof these intra-articular strategieson

synoviumisalsoimportant.Thesynoviumcanbecomeinflamedandisasourceof

catabolicandanti-chondrogenicfactors.Ontheotherhand,withtheexperiments

performedinthisthesis,wehaveshownthattheIPFPisanimportanttissuein

thejointenvironmentaswell.TheIPFPcanstimulatecatabolicprocessesinthe

synovium,althoughthiscouldalsobevice-versa,wheresynoviuminflammation

couldstimulatecatabolicprocessesintheIPFP.Nevertheless,itispossiblethat

anyfutureintra-articulartherapyadministeredcouldinfluencetheIPFPandvia

the IPFP influence the synovium. Therefore, IPFP changes should be carefully

monitoredduringexperimentaltreatmentsandreductionofinflammationinthe

IPFPcouldbeconsideredasoneofthesecondaryoutcomes.

Concluding remarks

Inconclusion,thisthesisshowsthattheIPFPisnotanordinaryadiposetissueand

thatitsecretesspecificfactorsthatlocallyinfluencethekneejointandcartilage

repair.Inthefuture,itispossibletousemedicationtospecificallymodulatethe

IPFPtoimprovethejointenvironmentforcartilagerepair.Furthermore,thisthesis

providesevidencethatthereisvariabilityinhowobesityrelatedsystemiceffects

canaffectdegenerative jointdiseaseandcartilagerepair.Futurestudiesonthe

relationshipbetweenobesity,adiposetissueandcartilagerepaircouldfocuson

thisvariabilitytouncovernoveltherapeutictargets.

148 CHAPTER 8

REFERENCES



18:876-882.

2. DragooJL,JohnsonC,McConnellJ.2012.Evaluationandtreatmentofdisordersoftheinfrapatellar

fatpad.SportsMed42:51-67.

3. FontanellaCG,CarnielEL,FrigoA,etal.2016.InvestigationofbiomechanicalresponseofHoffa’sfat

padandcomparativecharacterization.JMechBehavBiomedMater67:1-9.

4. CaiJ,XuJ,WangK,etal.2015.AssociationBetweenInfrapatellarFatPadVolumeandKneeStruc-

turalChangesinPatientswithKneeOsteoarthritis.JRheumatol42:1878-1884.

5. ChuckpaiwongB,CharlesHC,KrausVB, et al. 2010.Age-associated increases in the sizeof the

infrapatellarfatpadinkneeosteoarthritisasmeasuredby3TMRI.JOrthopRes28:1149-1154.

6. DuranS,AksahinE,KocadalO,etal.2015.Effectsofbodymassindex,infrapatellarfatpadvolume

andageonpatellarcartilagedefect.ActaOrthopBelg81:41-46.



VascBiol28:1654-1659.



9. ChangWDC,KentC,KovatsS,GarteiserP,DoblasS,TownerR,GriffinTM.2011.Infrapatellarfat

padhypertrophywithoutinflammationinadiet-inducedmousemodelofobesityandosteoarthri-

tis.OsteoarthritisCartilage19:S53–S236.

10. GrantRW,DixitVD.2015.Adiposetissueasanimmunologicalorgan.Obesity(SilverSpring)23:512-

518.




12. AbreuMR,ChungCB,TrudellD, et al. 2008.Hoffa’s fatpad injuries and their relationshipwith

anteriorcruciateligamenttears:newobservationsbasedonMRimaginginpatientsandMRimag-

ingandanatomiccorrelationincadavers.SkeletalRadiol37:301-306.

13. GandhiR,TakahashiM,VirtanenC,etal.2011.Microarrayanalysisoftheinfrapatellarfatpadin

kneeosteoarthritis:relationshipwithjointinflammation.JRheumatol38:1966-1972.

14. SolbakNM,HeardBJ,AchariY,etal.2015.AlterationsinHoffa’sfatpadinducedbyaninflammatory

responsefollowingidealizedanteriorcruciateligamentsurgery.InflammRes64:615-626.

15. EvansCH,KrausVB,SettonLA.2014.Progressinintra-articulartherapy.NatRevRheumatol10:11-

22.

16. EymardF,PigenetA,CitadelleD,etal.2017.Kneeandhipintra-articularadiposetissues(IAATs)

comparedwithautologoussubcutaneousadiposetissue:aspecificphenotypeforacentralplayer

inosteoarthritis.AnnRheumDis.

17. TsuchidaAI, BeekhuizenM,RutgersM, et al. 2012. Interleukin-6 is elevated in synovial fluid of

patientswithfocalcartilagedefectsandstimulatescartilagematrixproductioninaninvitroregen-

erationmodel.ArthritisResTher14:R262.

18. TsuchidaAI,BeekhuizenM,tHartMC,etal.2014.Cytokineprofilesinthejointdependonpathol-

ogy,butaredifferentbetweensynovialfluid,cartilagetissueandculturedchondrocytes.Arthritis

ResTher16:441.


8





21. MarksPH,DonaldsonML.2005.Inflammatorycytokineprofilesassociatedwithchondraldamage

intheanteriorcruciateligament-deficientknee.Arthroscopy21:1342-1347.

22. BeekhuizenM,GiermanLM, van SpilWE, et al. 2013.An explorative study comparing levels of

solublemediatorsincontrolandosteoarthriticsynovialfluid.OsteoarthritisCartilage21:918-922.

23. HarrisQ,SetoJ,O’BrienK,etal.2013.Monocytechemotacticprotein-1inhibitschondrogenesisof

synovialmesenchymalprogenitorcells:aninvitrostudy.StemCells31:2253-2265.

24. JagielskiM,WolfJ,MarzahnU,etal.2014.TheinfluenceofIL-10andTNFalphaonchondrogenesis

ofhumanmesenchymalstromalcellsinthree-dimensionalcultures.IntJMolSci15:15821-15844.

25. KondoM,YamaokaK,TanakaY.2014.Acquiringchondrocytephenotypefromhumanmesenchymal

stemcellsunderinflammatoryconditions.IntJMolSci15:21270-21285.

26. WeiH,ShenG,DengX,etal.2013.TheroleofIL-6inbonemarrow(BM)-derivedmesenchymalstem

cells(MSCs)proliferationandchondrogenesis.CellTissueBank14:699-706.



28. CanhameroT,GarciaLV,DeFrancoM.2014.AcuteInflammationLociAreInvolvedinWoundHealing

intheMouseEarPunchModel.AdvWoundCare(NewRochelle)3:582-591.

29. GourevitchD,KossenkovAV,ZhangY,etal.2014.InflammationandItsCorrelatesinRegenerative

WoundHealing:AnAlternatePerspective.AdvWoundCare(NewRochelle)3:592-603.

30. MosserDM,EdwardsJP.2008.Exploringthefullspectrumofmacrophageactivation.NatRevIm-

munol8:958-969.

31. UtomoL,Bastiaansen-JenniskensYM,VerhaarJA,etal.2016.Cartilage inflammationanddegen-

erationisenhancedbypro-inflammatory(M1)macrophagesinvitro,butnotinhibiteddirectlyby

anti-inflammatory(M2)macrophages.OsteoarthritisCartilage24:2162-2170.

32. BlomAB,vanLentPL,LibregtsS,etal.2007.Crucialroleofmacrophages inmatrixmetallopro-

teinase-mediatedcartilagedestructionduringexperimentalosteoarthritis:involvementofmatrix

metalloproteinase3.ArthritisRheum56:147-157.




34. SesiaSB,DuhrR,MedeirosdaCunhaC,etal.2015.Anti-inflammatory/tissuerepairmacrophages

enhance the cartilage-forming capacity of human bone marrow-derived mesenchymal stromal

cells.JCellPhysiol230:1258-1269.

35. GodwinJW,PintoAR,RosenthalNA.2013.Macrophagesarerequired foradultsalamander limb

regeneration.ProcNatlAcadSciUSA110:9415-9420.

36. GrossJB,GuillaumeC,Gegout-PottieP,etal.2016.Theinfrapatellarfatpadinducesinflammatory

anddegradativeeffectsinarticularcellsbutnotthroughleptinoradiponectin.ClinExpRheumatol.

37. NishimutaJF,LevenstonME.2015.Meniscusismoresusceptiblethancartilagetocatabolicand

anti-anaboliceffectsofadipokines.OsteoarthritisCartilage23:1551-1562.

38. ChallaTD,RaisY,OrnanEM.2010.EffectofadiponectinonATDC5proliferation,differentiationand

signalingpathways.MolCellEndocrinol323:282-291.

39. FrancinPJ,AbotA,GuillaumeC,etal.2014.Associationbetweenadiponectinandcartilagedegra-

dationinhumanosteoarthritis.OsteoarthritisCartilage22:519-526.

150 CHAPTER 8

40. FainJN,MadanAK,HilerML,etal.2004.Comparisonofthereleaseofadipokinesbyadiposetissue,

adiposetissuematrix,andadipocytesfromvisceralandsubcutaneousabdominaladiposetissues

ofobesehumans.Endocrinology145:2273-2282.

41. FardetL,Antuna-PuenteB,VatierC,etal.2013.Adipokineprofileinglucocorticoid-treatedpatients:

baselineplasmaleptinlevelpredictsoccurrenceoflipodystrophy.ClinEndocrinol(Oxf)78:43-51.

42. HeardBJ,SolbakNM,ChungM,etal.2016.Theinfrapatellarfatpadisaffectedbyinjuryinduced

inflammationintherabbitknee:useofdexamethasonetomitigatedamage.InflammRes65:459-470.

43. RicciottiE,FitzGeraldGA.2011.Prostaglandinsandinflammation.ArteriosclerThrombVascBiol

31:986-1000.

44. JakobM,DemarteauO,SuetterlinR,etal.2004.Chondrogenesisofexpandedadulthumanarticular

chondrocytesisenhancedbyspecificprostaglandins.Rheumatology(Oxford)43:852-857.

45. KimJ,ShimM.2015.ProstaglandinF2alphareceptor(FP)signalingregulatesBmpsignalingand

promoteschondrocytedifferentiation.BiochimBiophysActa1853:500-512.

46. AtturM,Al-MussawirHE,PatelJ,etal.2008.ProstaglandinE2exertscataboliceffectsinosteoar-

thritiscartilage:evidenceforsignalingviatheEP4receptor.JImmunol181:5082-5088.

47. ZweersMC,deBoerTN,vanRoonJ,etal.2011.Celecoxib:considerationsregardingitspotential

disease-modifyingpropertiesinosteoarthritis.ArthritisResTher13:239.




49. GiermanLM,WopereisS,vanElB,etal.2013.Metabolicprofilingrevealsdifferencesinconcentra-

tionsofoxylipinsand fattyacidssecretedby the infrapatellar fatpadofdonorswithend-stage

osteoarthritisandnormaldonors.ArthritisRheum65:2606-2614.

50. PountosI,GiannoudisPV,JonesE,etal.2011.NSAIDSinhibitinvitroMSCchondrogenesisbutnot

osteogenesis: implications formechanismof bone formation inhibition inman. J CellMolMed

15:525-534.

51. WeltingTJ,CaronMM,EmansPJ,etal.2011.Inhibitionofcyclooxygenase-2impactschondrocyte

hypertrophicdifferentiationduringendochondralossification.EurCellMater22:420-436;discus-

sion436-427.

52. CaronMM,EmansPJ,SanenK,etal.2016.TheRoleofProstaglandinsandCOX-EnzymesinChon-

drogenicDifferentiationofATDC5ProgenitorCells.PLoSOne11:e0153162.

53. Goppelt-StruebeM,WolterD,ReschK.1989.Glucocorticoidsinhibitprostaglandinsynthesisnot

onlyatthelevelofphospholipaseA2butalsoatthelevelofcyclo-oxygenase/PGEisomerase.BrJ

Pharmacol98:1287-1295.

54. KimIC,CohenAS.1966.Synovialfluidfattyacidcompositioninpatientswithrheumatoidarthritis,

goutanddegenerativejointdisease.ProcSocExpBiolMed123:77-80.

55. Soto-VacaA,LossoJN,McDonoughK,etal. 2013.Differentialeffectof14 free fattyacids in the

expressionofinflammationmarkersonhumanarterialcoronarycells.JAgricFoodChem61:10074-

10079.

56. Bastiaansen-JenniskensYM,SiawashM,vandeLestCH,etal.2013.MonounsaturatedandSatu-

rated,butNotn-6PolyunsaturatedFattyAcidsDecreaseCartilageDestructionunderInflammatory

Conditions:APreliminaryStudy.Cartilage4:321-328.

57. SimopoulosAP.2002.Omega-3fattyacidsininflammationandautoimmunediseases.JAmCollNutr

21:495-505.


pathogenesisofosteoarthritisfollowingjointinjury.AnnRheumDis74:2076-2083.


8

59. VanBeeckA,ClockaertsS,SomvilleJ,etal.2013.Doesinfrapatellarfatpadresectionintotalknee

arthroplastyimpairclinicaloutcome?Asystematicreview.Knee20:226-231.

60. WhiteL,HolyoakR,SantJ,etal.2016.Theeffectof infrapatellarfatpadresectiononoutcomes

post-totalkneearthroplasty:asystematicreview.ArchOrthopTraumaSurg136:701-708.

61. BosPK.2014.CORRInsights(R):Theeffectofinfrapatellarfatpadexcisiononcomplicationsafter

minimallyinvasiveTKA:arandomizedcontrolledtrial.ClinOrthopRelatRes472:702-703.

62. Pinsornsak P, NaratrikunK, Chumchuen S. 2014. The effect of infrapatellar fat pad excision on

complicationsafterminimallyinvasiveTKA:arandomizedcontrolledtrial.ClinOrthopRelatRes

472:695-701.

63. TakatokuK,SekiyaH,HayashiM,etal.2005.Influenceoffatpadremovalonpatellartendonlength

duringgrowth.KneeSurgSportsTraumatolArthrosc13:706-713.

64. YeC,ZhangW,WuW,etal.2016.InfluenceoftheInfrapatellarFatPadResectionduringTotalKnee

Arthroplasty:ASystematicReviewandMeta-Analysis.PLoSOne11:e0163515.

65. GarciaJ,WrightK,RobertsS,etal.2016.Characterisationofsynovialfluidandinfrapatellarfatpad

derivedmesenchymalstromalcells:Theinfluenceoftissuesourceandinflammatorystimulus.Sci

Rep6:24295.

66. NeriS,GuidottiS,LilliNL,etal.2016.Infrapatellarfatpad-derivedmesenchymalstromalcellsfrom

osteoarthritispatients:Invitrogeneticstabilityandreplicativesenescence.JOrthopRes.

67. WickhamMQ, Erickson GR, Gimble JM, et al. 2003.Multipotent stromal cells derived from the

infrapatellarfatpadoftheknee.ClinOrthopRelatRes:196-212.

68. MaumusM,ManferdiniC,ToupetK,etal.2013.Adiposemesenchymalstemcellsprotectchondro-

cytesfromdegenerationassociatedwithosteoarthritis.StemCellRes11:834-844.

69. TangchitphisutP,SrikaewN,NumhomS,etal.2016.InfrapatellarFatPad:AnAlternativeSourceof

Adipose-DerivedMesenchymalStemCells.Arthritis2016:4019873.



71. Hunziker EB, Rosenberg LC. 1996. Repair of partial-thickness defects in articular cartilage: cell

recruitmentfromthesynovialmembrane.JBoneJointSurgAm78:721-733.

72. OkanoT,WakitaniS,OkabeT,etal.2014.Nucleatedcellscirculatingintheperipheralbloodcon-

tributetotherepairofosteochondraldefectsonlyintheearlyphaseofhealing.JTissueEngRegen

Med8:414-420.

73. FontanellaCG,CarnielEL,FrigoA,etal.2017.InvestigationofbiomechanicalresponseofHoffa’sfat

padandcomparativecharacterization.JMechBehavBiomedMater67:1-9.

74. SchweitzerME,FalkA,PathriaM,etal.1993.MRimagingoftheknee:canchangesintheintracap-

sularfatpadsbeusedasasignofsynovialproliferationinthepresenceofaneffusion?AJRAmJ

Roentgenol160:823-826.

75. StaeubliHU,BollmannC,KreutzR,etal.1999.Quantificationofintactquadricepstendon,quadri-

cepstendoninsertion,andsuprapatellarfatpad:MRarthrography,anatomy,andcryosectionsin

thesagittalplane.AJRAmJRoentgenol173:691-698.

76. TsavalasN,KarantanasAH.2013.Suprapatellarfat-padmasseffect:MRIfindingsandcorrelation

withanteriorkneepain.AJRAmJRoentgenol200:W291-296.

77. WangJ,HanW,WangX,etal.2014.Masseffectandsignalintensityalterationinthesuprapatellar

fatpad:associationswithkneesymptomsandstructure.OsteoarthritisCartilage22:1619-1626.

78. LyJQ,Bui-MansfieldLT.2004.AnatomyofandabnormalitiesassociatedwithKager’sfatPad.AJR

AmJRoentgenol182:147-154.

79. PingelJ,PetersenMC,FredbergU,etal.2015.InflammatoryandMetabolicAlterationsofKager’s

FatPadinChronicAchillesTendinopathy.PLoSOne10:e0127811.

152 CHAPTER 8

80. JayasekeraN,ApratoA,VillarRN.2014.Fatpadentrapmentatthehip:anewdiagnosis.PLoSOne

9:e83503.

81. EymardF,PigenetA,CitadelleD,etal.2016.Kneeandhipintra-articularadiposetissuessharea

commonphenotypeinosteoarthritis.OsteoarthritisCartilage24(2016)S63-S534.

82. BerenbaumF,GriffinTM,Liu-BryanR.2017.Review:MetabolicRegulationofInflammationinOsteo-

arthritis.ArthritisRheumatol69:9-21.


8:729-737.

84. AlbuquerqueD,SticeE,Rodriguez-LopezR,etal.2015.Currentreviewofgeneticsofhumanobesity:

frommolecularmechanismstoanevolutionaryperspective.MolGenetGenomics290:1191-1221.

85. BouchardC.2007.Thebiologicalpredispositiontoobesity:beyondthethriftygenotypescenario.

IntJObes(Lond)31:1337-1339.

86. AbranchesMV,OliveiraFC,ConceicaoLL,etal.2015.Obesityanddiabetes:thelinkbetweenadi-

posetissuedysfunctionandglucosehomeostasis.NutrResRev28:121-132.

87. UchidaK,SatohM,InoueG,etal.2015.CD11c(+)macrophagesandlevelsofTNF-alphaandMMP-3

areincreasedinsynovialandadiposetissuesofosteoarthriticmicewithhyperlipidaemia.ClinExp

Immunol180:551-559.

88. PelleymounterMA,CullenMJ,BakerMB,etal.1995.Effectsoftheobesegeneproductonbody

weightregulationinob/obmice.Science269:540-543.

89. UchidaK,UrabeK,NaruseK,etal.2009.Hyperlipidemiaandhyperinsulinemiainthespontaneous

osteoarthritismousemodel,STR/Ort.ExpAnim58:181-187.

90. MasonRM,ChambersMG,FlannellyJ,etal.2001.TheSTR/ortmouseanditsuseasamodelof


91. FulcherJ,O’ConnellR,VoyseyM,etal.2015.EfficacyandsafetyofLDL-loweringtherapyamong

menandwomen:meta-analysisofindividualdatafrom174,000participantsin27randomisedtrials.

Lancet385:1397-1405.

92. KeeneD,PriceC,Shun-ShinMJ,etal.2014.Effectoncardiovascularriskofhighdensitylipoprotein

targeteddrugtreatmentsniacin,fibrates,andCETPinhibitors:meta-analysisofrandomisedcon-

trolledtrialsincluding117,411patients.BMJ349:g4379.

93. ClockaertsS,VanOschGJ,Bastiaansen-JenniskensYM,etal.2012.Statinuse isassociatedwith

reducedincidenceandprogressionofkneeosteoarthritisintheRotterdamstudy.AnnRheumDis

71:642-647.

94. IzadpanahR,SchachteleDJ,PfnurAB,etal.2015.Theimpactofstatinsonbiologicalcharacteris-

ticsofstemcellsprovidesanovelexplanationfortheirpleiotropicbeneficialandadverseclinical

effects.AmJPhysiolCellPhysiol309:C522-531.

95. ZhangO,ZhangJ.2015.Atorvastatinpromoteshumanmonocytedifferentiationtowardalterna-

tive M2 macrophages through p38 mitogen-activated protein kinase-dependent peroxisome

proliferator-activatedreceptorgammaactivation.IntImmunopharmacol26:58-64.

96. DenisGV,ObinMS.2013. ‘Metabolicallyhealthyobesity’:originsand implications.MolAspects

Med34:59-70.

97. NavarroE,FuntikovaAN,FitoM,etal.2015.Canmetabolicallyhealthyobesitybeexplainedbydiet,

genetics,andinflammation?MolNutrFoodRes59:75-93.

98. GreenMA,StrongM,RazakF,etal.2016.Whoaretheobese?Aclusteranalysisexploringsubgroups

oftheobese.JPublicHealth(Oxf)38:258-264.

99. ZhangY,PizzuteT,PeiM.2014.Anti-inflammatorystrategiesincartilagerepair.TissueEngPartB

Rev20:655-668.

Addendum figures 153

8

ADDENDUM FIGURES

COL1A1

FLS only +M1 CM +M2 CM0.0

0.5

1.0

1.5

2.0

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FLS

only

ASMA

FLS only +M1 CM +M2 CM0.0

0.5

1.0

1.5

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ativ

eto

FLS

only

PLOD2

FLS only +M1 CM +M2 CM0

20

40

60

80

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ativ

eto

FLS

only

*

Addendum figure 1Pro-inflammatoryM1macrophagessecretepro-fibroticfactors.Humanperipheralbloodmonocytes(pBMC)werestimulatedtoM1orM2phenotypeandconditionedmedium(CM)wasmade.Synovialfibroblasts(FLS)weretreatedwithM1orM2macrophageCMfor4days.RelativeexpressionofCOL1A1,ASMAandPLOD2inFLStreatedwith25%CM.GeneexpressionwasnormalizedtotheFLSonlyconditionperdonor.CMfrom3pBMCdonorswereusedtotreat3FLSdonors,witheachconditionperformedintriplicate(n=9).Valuesrepresentmean±SD.*p<0.05versusFLSonly.

COL2A1

TGFb1only

+PGF2a+AL8810

+CM +CM +AL8810

0.0

0.5

1.0

1.5

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TGF b

1on

ly

*

**

ACAN

TGFb1only

+PGF2a+AL8810

+CM +CM +AL8810

0.0

0.5

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1.5

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TGF b

1on

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* **

MMP1

TGFb1only

+PGF2a+AL8810

+CM +CM +AL8810

0

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8

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1on

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*

*

MMP13

TGFb1only

+PGF2a+AL8810

+CM +CM +AL8810

0.0

0.5

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2.5

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TGF b

1on

ly **

*

Addendum figure 2EffectofPGF2αonMSCchondrogenesis.ChondrogenicstimulatedMSCalginatebeadsweretreatedwithPGF2α,AL8810inhibitororCMfromIPFP.Rela-tive expressionofCOL2A1,ACAN,MMP1andMMP13 in chondrogenic stimulatedMSCalginatebeadsaftertreatment.TwopoolsofCMfrom3differentIPFPdonorseachpoolwereusedtotreat2MSCdonors,witheachconditionperformedintriplicate(n=6).Valuesrepresentmean±SD.*p<0.05versusnon-treatedcontrols.

Nederlandse samenvatting 155

NEDERLANDSE SAMENVATTING

Deknieheeftgewrichtsvlakkendiebedektwordendoorkraakbeen.Kraakbeenis

eenbindweefselmeteenelastischekarakter,hetzorgtervoordatdegewrichtsvlak-

kensoepeloverelkaarheenkunnenglijdenenhetgeeftschokdemping.Kraakbeen

kanbeschadigdrakenenindiendekraakbeenschadeniethersteldwordt,kandit

uiteindelijkleidentotartrose.Desymptomenvanartrosezijnonderanderepijn

enverminderdebewegelijkheid.Bijartroseisnietalleenhetkraakbeenaangetast

maarde gehele knie, inclusief het bot, ligamenten enhet synovium.Artrose is

niettegenezen;alleendesymptomenzijntebehandelen.Inheteindstadiumvan

artrosekanhetgehelekniegewrichtvervangenwordendooreenprothese.

Kraakbeenhersteltuitzichzelfnauwelijks.Ditkomtdoordathetgeenzenuwenen

geenbloedvatenbevat.Daarnaastbestaathetkraakbeenvooraluittussencelstof

enmaarvooreenkleingedeelteuitkraakbeencellen.Indienersprakeisvaneen

kraakbeendefect,isermomenteeleenaantalmethodesomhetdefectteherstel-

len.Demeest gangbare isdemicrofractureprocedure.Tijdensdezeprocedure

worden er gaten gemaakt in het bot onder het kraakbeen.Hiermeewordende

mesenchymalestamcelleninhetbeenmerggestimuleerdomhetkraakbeendefect

optevullenmetlittekenweefsel.

De resultaten van deze operaties kunnen door ontsteking negatief beïnvloed

worden.Ineenontstokenkniezijnerveelontstekingsfactorendieervoorzorgen

datstamcellennietgoedveranderennaarkraakbeencellenendaaromnietgoed

nieuwkraakbeenkunnenproduceren.De resultatenvandekraakbeenoperaties

kunnenooknegatiefbeïnvloedwordendoorobesitas.Bijobesitaszijndevetcel-

len vergroot. Daarnaast is ermeer infiltratie van ontstekingscellen, waaronder

macrofagen. Macrofagen zijn witte bloedcellen die een belangrijk rol spelen

inonsafweersysteem.Er zijn inhet lichaampro-inflammatoiremacrofagen en

weefselherstellende/anti-inflammatoiremacrofagen.Hetvetweefselisbijobesitas

ontstoken door onder andere een verhoogde infiltratie van pro-inflammatoire

macrofagenenscheidtdanookmeerontstekingsfactorenuit.Hetmeestevet in

hetlichaambevindtzichonderdehuid.Deontstekingsfactorenbereikendeknie

systemischviadebloedbaan.Echterindekniezelfbevindtzichookvetweefsel.

Hetgrootstevetweefsel indeknieishetinfrapatellairvetweefsel,ofwelHoffa’s

vetweefsel.HetHoffa’svetweefsel ligtdirect tegenhetsynoviumaan.Hetheeft

alsfunctieschokdempingenhethelpenverspreidenvansynovialevloeistof.Het

Hoffa’s vetweefsel kan ontstoken raken en daarbij stoffen uitscheiden die het

kraakbeenbeïnvloeden.

156 Nederlandse samenvatting

In Hoofdstuk 2beschrevenwijofhetHoffa’svetweefselookdaadwerkelijkveran-

derdisdoorobesitas.WijhebbendaarbijstukjesHoffa’svetweefselgebruiktvan

patiënten die een totale knieprothese kregen en stukjes onderhuids vetweefsel

vanpatiëntendieeen totaleheupprothesekregen.Wijanalyseerdendegrootte

vandevetcellenmethistologie.Obesitasleidttotvergrotingvandeonderhuidse

vetcellenenditiseenaanwijzingvoorvetweefselontsteking.Wijkondengeenver-

schilingroottevindentussenvetcellenvanhetHoffa’svetweefselvanpatiënten

met een body mass index (BMI) van <25kg/m2 en >30 kg/m2. Wij concluderen

hieruitdatdevetcelleninhetHoffa’svetweefselwaarschijnlijknietdoorobesitas

beïnvloedworden.DitsuggereertverderdathetHoffa’svetweefselniethetzelfde

isalsonderhuidsvetweefsel.

Obesitas iseenrisicofactorvoorartrose.Eenvandekenmerkenvanartrose is

stijfheid van het gewricht. Dit wordt deels veroorzaakt door fibrosering, ofwel

verlittekening van het synovium. Dit proces ontstaat doordat fibroblasten in

het synoviummeer fibrotischweefsel gaat produceren. De fibroblasten produ-

cerenmeerfibrotischweefseldooraanwezigheidvanontstekingsfactorenofeen

overdaadaangroeifactoren.HetHoffa’svetweefselligtindekniedirecttegenhet

synovium aan. Het synoviumwordt dusmogelijk continue beïnvloed door het

Hoffa’svetweefsel. InHoofdstuk 3hebbenwijbeschrevendat factorendiehet

Hoffa’svetweefseluitscheidtsynovialefibrosekanveroorzaken. Dithebbenwij

onderzochtdooreerstdefactorendiehetHoffa’svetweefseluitscheidtoptevan-

geninkweekmedium.Daarnahebbenwijfibroblastenuithetsynoviumgeïsoleerd

endeze gekweektmethet kweekmedium.Wij zagenhierbij datdefibroblasten

meer fibrotische eigenschappenontwikkelden.Wemerktenookopdat er geen

verschilwas ineffect tussenkweekmediumgemaaktvanHoffa’svetweefselvan

obese en van niet-obese donoren. Verder ontdekten we dat prostaglandine F2a

(PGF2a)uitgeschedendoorhetHoffa’svetweefselmedeverantwoordelijkisvoor

deze reactie.Ditbetekentdusdat eenprostaglandine remmer, zoals celecoxib,

mogelijkgebruiktzoukunnenwordenomontstaanvansynovialefibroseterem-

men.

WewetennudathetHoffa’svetweefselstoffenuitscheidtdathetkraakbeenen

synoviumbeïnvloeden.Indekliniekzijnderesultatenvankraakbeenherstelopera-

tiesnietoptimaal,wanterontstaateensoortlittekenkraakbeen.In Hoofdstuk 4

hebbenwijexperimentenuitgevoerdomtekijkenofhetHoffa’svetweefselook

stoffenuitscheidtdiedekraakbeenvormendecapaciteitvanstamcellenremmen.

Wijgebruiktenhetzelfdesoortkweekmediumwelkeweeerdergebruikthebben

voordesynovialefibroseexperimenteninHoofdstuk 3.Wijzagendatditkweek-

Nederlandse samenvatting 157

mediumkraakbeenvormendecapaciteitvanstamcellenremde.Verdervondenwij

geenverschilinhetremmendeeffectvanHoffa’svetweefseltussenobeseenniet-

obesedonoren,nochtussenartrotischeenpost-traumatischedonoren.Diteffect

waswelHoffa’svetweefselspecifiek,wantsubcutanevetweefselhadgeeneffect

opdestamcellen.Tenslottetoondenweaandatpro-inflammatoiremacrofagenin

hetHoffa’svetweefselbijdragenaanhetkraakbeenvormingremmendeeffectvan

hetHoffa’svetweefsel.Ditbetekentdusdatalswedenegatieveeffectenvanhet

Hoffa’svetweefselwillenverminderen,datweeenmedicijnmoetengebruikendat

depro-inflammatoiremacrofageninhetHoffa’svetweefselbeïnvloedt.

In Hoofdstuk 5probeerdenwevervolgensmetmedicijnenontstekinginhetHoffa’s

vetweefselteremmen.Wegebruiktencelecoxibentriamcinoloneacetonide,twee

vaak gebruikte ontstekingsremmers, en pravastatine en fenofibraat, twee vaak

gebruiktecholesterol-entriglyceridegehalteverlagersmetontstekingsremmende

effecten.WekweektenstukjesHoffa’svetweefselmetdeverschillendemedicijnen.

WevondendatalleentriamcinoloneacetonideontstekinginhetHoffa’svetweefsel

konverlagen.Daarnaastremdehetookspecifiekdepro-inflammatoiremacrofagen.

HierdoorwerdhetHoffa’svetweefselmindernegatiefvoorkraakbeenvormende

capaciteitvandestamcellen.VerderzagenwijookheteffectvanHoffa’svetweefsel

opsynovialefibroblastenveranderde.Ertradennamelijkkraakbeenafbraakpro-

cessenopsynovialefibroblasten.Hetlijkterdusopdattriamcinoloneacetonide

gebruiktkanwordenomspecifiekhetHoffa’svetweefseltemodulerennaareen

meervoorkraakbeenherstelgeschiktestaat.

Hetmeeste vetweefsel in het lichaambevindt zich onder de huid. Bij obesitas

isditonderhuidsevetweefselontstokenendeontstekingsfactorenwordensys-

temischuitgescheidenenbereikendanviadebloedbaandeknie.Hierdoorkan

kraakbeenschadeenuiteindelijkartroseontstaan.Daarnaastisditontstokenmi-

lieumogelijkmindergeschiktvoorkraakbeenhersteloperaties.Naastontsteking

speeltookmetaboleontregelingeenrolbijobesitasenhetontstaanvanartrose.

Omdecomplexeverschillendeveranderingeninobesitasteonderzoeken,ishet

noodzakelijkdierproevenuittevoeren.InHoofdstuk 6gebruiktenwijdeSTR/Ort

muizenstam,diespontaanobesitasenmetabole-eninflammatoireveranderingen

ontwikkelt.Dezemuizenkregengedurendeeenhalfjaardagelijkssimvastatineof

fenofibraatofeencombinatiehiervan.Ditzijntweevaakgebruiktecholesterol-en

triglyceridegehalteverlagersmetontstekingsremmendeeffecten.Naeenhalfjaar

kondenwe geen effect op kraakbeenschade vinden. Fenofibraat voorkwamwel

obesitas,verlaagdesystemischeontstekingenbotsclerose,maarkonkraakbeen-

schadenietvoorkomen.Tenslottewarenmetaboleinflammatoireveranderingen

158 Nederlandse samenvatting

indemuizennietgeassocieerdmethetontstaanvankraakbeenschade.Wijcon-

cludeerdenuitdezemuizenstudiedatobesitasenobesitasgerelateerdemetabole

eninflammatoireveranderingennietdirectdeoorzaakzijnvankraakbeenschade

indezemuizen.Voordepatiënt suggereertdit dat obesitasniet altijd leidt tot

kraakbeenschade.

Alseenobesepatiënteenmaalwelkraakbeenschadeheeft,ishetnoodzakelijkeen

kraakbeenhersteloperatieuittevoerenomdeprogressietotartrosetevoorkomen.

Op ditmomentworden obese patiënten in principe niet geopereerd omdat de

klinischeresultatenminderzijnbijdezepatiënten.Echterishetnognietduidelijk

ofdaadwerkelijkdekwaliteitvanhersteldekraakbeenbeïnvloedwordtdoorobe-

sitasenhoeditgebeurt.Wijhebbengeprobeerddezevraagtebeantwoordenin

Hoofdstuk 7.IndithoofdstukgebruiktenwijDBA/1muizen,waarvanbekendis

datzekraakbeenschadekunnenherstellen.Wijhebbendezemuizeneenhoogvet

dieetgevoerdwaar60%vandeenergieuitvetkomt.Daarnaastmaaktenweeen

kraakbeendefectinhunknie.Dezemuizenwerdendikker,maartegendeverwacht-

ingin,hersteldedegroepdieeenhoogvetdieetkregenbeterdandemuizendie

eencontroledieetkregen.Daarnaastontwikkeldedegroepdieeenhoogvetdieet

kregenookgeenartroseenhaddendemuizengeenmetaboleof inflammatoire

veranderingenopdelangetermijn.Wijconcludeerdennaaraanleidingvandeze

resultatendatresistentietegenhoogvetdieetgeïnduceerdemetaboleeninflam-

matoireveranderingenmogelijkgelinktisaanbeterkraakbeenherstel.Wijweten

nietprecieswelkemechanismeshierineenrolspelen,daarvoorismeeronderzoek

nodig.

Concluderend heb ik met het onderzoek beschreven in dit proefschrift aan-

getoonddathetHoffa’svetweefselniethetzelfde isalsonderhuidsevetweefsel.

HetHoffa’svetweefselvanpatiëntenmetartroseofknieschadescheidtfactoren

uitdiehetkniegewrichtenkraakbeenherstelkunnenbeïnvloeden.Indetoekomst

is het mogelijk met medicijnen het Hoffa’s vetweefsel te moduleren om zo de

gewrichtsomgeving te optimaliseren voor kraakbeenherstel. Verder hebbenwij

aanwijzingengevondendatobesitasnietaltijdleidttotartroseennietperdefinitie

negatiefisvoorkraakbeenherstel.Ombetertebegrijpenwaaromditverschillend

istussenobesepatiëntenzijnermeeronderzoekennodig.Wellichtkunnenwijin

detoekomsthierdoorooknieuwetherapieënvindenomartroseteverminderen

enkraakbeenherstelteverbeteren.

159

中文简介

盖软骨作为减震器，可以使膝盖平滑无痛的运动。软骨可能会受损，一旦受损就不

能恢复，最终可能导致膝关节骨性关节炎。膝关节骨关节炎不能治愈，但通过治疗

可以缓解其症状。在骨性关节炎末期，需要施行关节置换手术更换整个关节。

软骨损伤可以通过软骨修复手术来治疗。但是，炎症会影响手术的效果。在肥胖人

群的脂肪组织多于常人，大多数脂肪组织位于皮下，而且会发炎。在膝盖内部有不

小的一片脂肪组织，称为髌下脂肪垫或霍法氏脂肪垫。本论文的研究表明，这种脂

肪垫与皮下脂肪不同。霍法氏脂肪垫分泌因子可能对膝关节和软骨的修复具有负面

影响。这可能是PGF2α 因子的分泌和促炎性巨噬细胞的存在引起的。添加曲安奈德可

以部分抵消这种负面作用。这意味着将来可以使用药物来调节霍法氏脂肪垫，从而

改善膝关节软骨修复的环境。

除了研究霍法氏脂肪垫引起的炎症之外，本论文还研究了额外关节脂肪组织炎症对

关节疾病和软骨修复的影响。通过小鼠活体实验发现，肥胖和肥胖相关的代谢和炎

症变化并不总是导致软骨损伤，也不总是对软骨修复具有负面作用。研究实验提供

的证据表明，肥胖相关的全身效应对退行性关节疾病和软骨修复影响具有变化性。

关于肥胖，脂肪组织和软骨修复之间的关系的未来研究可以侧重于这种变化性以揭

示新的治疗靶点

中文简介

PhD portfolio 161

PHD PORTFOLIO

NamePhDStudent: WuWei

ErasmusMCDepartment: Orthopedics

ResearchSchool: Postgraduate SchoolMolecularMedicine (Mol-

Med)

PhDTime-span: February2012–December2015

Promotors: Prof.dr.G.J.V.M.vanOsch

Prof.dr.J.A.N.Verhaar

Co-promotor: Dr.Y.M.Bastiaansen-Jenniskens

PhD Training

Year Workload

(ECTS)

In-depth courses

Laboratoryanimalscience(ErasmusMCGraduateSchool)

BiostatisticalmethodsI:Basicprinciples(NIHES)

BiomedicalEnglishwritingcourse(MolMed)

Researchintegritycourse(ErasmusMCGraduateSchool)

BiomedicalEnglishWritingandCommunication(ErasmusMC

GraduateSchool)

ICRSFocusMeeting–TheKnee(ICRS)

Podium presentations

Statinsandfibratestopreventspontaneousosteoarthritis:an

invivostudyinmice

NVMB Meeting, Lunteren

Pro-inflammatorymacrophagesinHoffa’sfatpadreduces

chondrogenicpotentialofmesenchymalstemcells

NOV Jaarcongres, Rotterdam

The infrapatellar fat pad inhibits chondrogenesis of

mesenchymalstemcells

Molecular Medicine Day, Rotterdam

Obesitydoesnotnegativelyinfluencecartilagerepairinmice

NOV Jaarcongres, Rotterdam

2012

2013

2013

2014

2014

2014

2013

2014

2014

2015

4.02.02.00.34.0

1.0

1.0

1.0

1.0

1.0

162 PhD portfolio

PhD Training

Year Workload

(ECTS)

Theinfrapatellarfatpadfromdiseasedjointsinhibits

chondrogenesisofmesenchymalstemcells

ICRS World conference, Chicago, USA

Award for excellence in cartilage research

Theinfrapatellarfatpadofdiseasedjointsinhibits


SEOHS 2015, Leiden

Best abstract nominee

Anti-chondrogenicandpro-cataboliceffectoftheinfrapatellar

fatpadcanbemodulatedbytriamcinoloneacetonide

ICRS World conference, Sorrento, Italy

Poster presentations

Statinsandfibratestopreventspontaneousosteoarthritis:

aninvivostudyinmice


Statinsandfibratesdonotaffectdevelopmentofspontaneous

osteoarthritisinSTR/Ortmice

OARSI World conference, Philadelphia, USA

PPARαsignallingreducessubchondralbonethickeningandinflammation,butdoesnotpreventcartilagedamageinSTR/

Ortmice

OARSI World conference, Philadelphia, USA

The infrapatellar fat pad inhibits chondrogenesis of

mesenchymalstemcells

Matrix Biology Europe Meeting, Rotterdam

HighfatdietacceleratescartilagerepairinDBA/1mice


Theinfrapatellarfatpadfromdiseasedjointsinhibits


OARSI World conference, Seattle, USA

2015

2015

2016

2013

2013

2013

2014

2015

2015

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

PhD portfolio 163

PhD Training

Year Workload

(ECTS)


OARSI World conference, Seattle, USA


ICRSWorldconference,Chicago,USA

Teaching

Tutoraatfirstyearmedicalstudents

Supervisingthirdyearmedicalstudentsattendingtheminor

“OrthopedicSportsTraumatology”

SupervisingMasterstudentMolecularMedicine

Supervisingmedicalstudent

SupervisingJuniorMedSchoolStudents

Winner best presentation

Other

AnnaFondsgrant

Reviewerforinternationaljournals

Cartilage (2x)

Osteoarthritis and cartilage (2x)

Rheumatology

Journal of Biomedical Materials Research Part A

VisitingresearcherAOResearchInstitute

Davos, Switzerland

2015

2015

2012

2012-

2014

2014

2014

2014

2012

2013-

2015

2015

1.0

1.0

1.5

1.0

5.0

0.5

1.0

0.2

4.0

Total 43.3

List of publications 165

LIST OF PUBLICATIONS

1. Wei W,CaparS,KopsN,VerhaarJAN,ClockaertsS,VanOschGJVM,Bastiaan-

sen-JenniskensYM.Anti-chondrogenicandpro-cataboliceffectofinfrapatellar

fat pad and its residing macrophages can be modulated by triamcinolone

acetonide.Submitted.2017.

2. Wei W,Bastiaansen-JenniskensYM,SuijkerbuijkM,KopsN,BosPK,Verhaar

JAN,ZuurmondAM,Dell’AccioF,vanOschG.Highfatdietacceleratescartilage

repairinDBA/1mice.JOrthopRes.2017Jun;35(6):1258-64.

3. DeJongA,Klein-WieringaI,AndersenS,KwekkeboomJ,Herb-vanToornL,De

Lange-BrokaarB,VanDelftD,GarciaJ,Wei W,VanderHeideH,Bastiaansen-

JenniskensY,VanOschG,ZuurmondA,Stojanovic-SusulicV,NelissenR,Toes

R,KloppenburgM, Ioan-FacsinayA.LackofhighBMI-related features inadi-

pocytesandinflammatorycellsintheinfrapatellarfatpad.AcceptedArthritis

ResTher2017.2017.

4. RezaieW,Wei W,CleffkenBI,vanderVliesCH,RoukemaGR.InternalFixation

VersusHemiarthroplastyforDisplacedIntra-CapsularFemoralNeckFractures

inASA3-5GeriatricPatients.OpenOrthopJ.2016;10:765-71.

5. Wei W, RudjitoE, FahyN,Verhaar JA,Clockaerts S,Bastiaansen-Jenniskens

YM,vanOschGJ.Theinfrapatellarfatpadfromdiseasedjointsinhibitschon-

drogenesisofmesenchymalstemcells.EurCellMater.2015Dec02;30:303-14.

6. Siebelt M, Korthagen N,Wei W, Groen H, Bastiaansen-Jenniskens Y, Muller

C, Waarsing JH, de Jong M, Weinans H. Triamcinolone acetonide activates

ananti-inflammatoryand folate receptor-positivemacrophage thatprevents

osteophytosisinvivo.ArthritisResTher.2015Dec05;17:352.

7. Wei W, Clockaerts S, Bastiaansen-Jenniskens YM, Gierman LM, Botter SM,

Bierma-ZeinstraSM,WeinansH,Verhaar JA,KloppenburgM,ZuurmondAM,

vanOschGJ.Statinsandfibratesdonotaffectdevelopmentofspontaneous

cartilagedamageinSTR/Ortmice.OsteoarthritisCartilage.2014Feb;22(2):293-

301.

166 List of publications

8. FahyN,deVries-vanMelleML,LehmannJ,Wei W,GrotenhuisN,FarrellE,van

derKraanPM,MurphyJM,Bastiaansen-JenniskensYM,vanOschGJ.Human

osteoarthriticsynoviumimpactschondrogenicdifferentiationofmesenchymal

stem cells viamacrophage polarisation state. Osteoarthritis Cartilage. 2014

Aug;22(8):1167-75.

9. Bastiaansen-JenniskensYM,Wei W,FeijtC,WaarsingJH,VerhaarJA,Zuurmond

AM,HanemaaijerR, StoopR,vanOschGJ. Stimulationoffibroticprocesses

by the infrapatellar fat pad in cultured synoviocytes frompatientswith os-

teoarthritis:apossiblerole forprostaglandin f2alpha.ArthritisRheum.2013

Aug;65(8):2070-80.

10.Wei W, Akkersdijk GP. Spontaneous aneurysm of the superficial temporal

artery.Lancet.2011Jul09;378(9786):168.

11.Damen TH,Wei W, MureauMA, Tjong-Joe-Wai R, Hofer SO, Essink-BotML,

HoviusSE,PolinderS.Medium-termcostanalysisofbreastreconstructionsin

asingleDutchcentre:acomparisonofimplants,implantsprecededbytissue

expansion, LD transpositions andDIEPflaps. J PlastReconstrAesthet Surg.

2011Aug;64(8):1043-53.

About the author 169

ABOUT THE AUTHOR

WuWei(韦芜)wasbornonMay28th1986inSongzi,

Hubei,China. In1993hemoved to theNetherlands.

AfterobtaininghisVWOdiploma(ChristelijkLyceum

Delft,Delft),hestartedmedicalschoolattheErasmus

UniversityRotterdamin2004.Duringmedicalschool,

he spent a year as full time board member of the

Medical Student Association of Rotterdam (MFVR)

and was active in various other committees of the

study.HealsoworkedasanErasmusambassadorand

attheEmergencyDepartmentasastudent-assistant.

Furthermoreheparticipatedforseveralyearsinthe

ErasmusAnatomyResearchProject,whichincreased

hisenthusiasmforOrthopedics.

In2012heobtained thedegreeofMedicalDoctor. Subsequentlyhe startedhis

PhDprojectentitled‘Adiposetissueandtheknee:roleof inflammationonjoint

degeneration and cartilage repair’ at thedepartmentof orthopedicsunder the

supervisionofprof.dr.GerjoJ.V.M.vanOsch,prof.dr.JanA.N.Verhaaranddr.

YvonneM.Bastiaansen-Jenniskens.

FromJanuary2016tillJune2017,Wuworkedasaresident(VAIOS)attheDepart-

mentofSurgeryattheReinierdeGraafGasthuisinDelft(underthesupervision

ofdr.MaartenvanderElstanddr.MarkR.deVries).FromJuly2017onwardshe

isaresident(AIOS)attheDepartmentofOrthopedicsinErasmusMCUniversity

MedicalCenterinRotterdam(supervisionbydr.PieterK.Bos)

Besideshiswork,Wuenjoysrunning,cycling,foodandcooking.

Dankwoord 171

DANKWOORD

Erwordtaltijdgevraagdofjeietsopnieuwhadwillendoenalsjewederomvoor

dezelfdekeuzestaat.Mijnantwoordopdevraagofikopnieuwvooreenpromo-

tietrajectzouhebbengekozen,isvolmondig“JA!”.Devierjareninhetlabopde

16ezijnvoorbijgevlogen.Ditproefschriftwasnooittotstandgekomenzonderde

steunvananderen.Hierbijwilikinhetbijzondereenaantalpersonenbedanken.

Allereerstmijneerstepromotorprof.dr.VanOsch.BesteGerjo,alinhetvoorjaar

van2010liepikbijjenaarbinnenomtekomenpratenovermijnkeuzeonderzoek.

Ikhadnauwelijkseenideewatfundamenteelwetenschappelijkonderzoekinhield,

nochwatertedoenwasophet lab.Naeenhalfjaar intensievebegeleidingvan

jouenYvonne,wasikheelblijdatjemijdekansboodomeenpromotietrajectte

starten.Jewasaltijdheellaagdrempeligtebereikenvooroverleg,hoedrukjehet

ookhad.Daarnaastwasernogietswaarjeookaltijdtijdvoorvrijmaakte:jouw

goudvissen.Tijdens jeafwezigheidwerdencompleteschema’sopgesteldomte

zorgendatzenietovervoerdwerden.IkvondhetleukdatjeonzegoudvisSep(je

noemdehemmini-Wu)wildeadopteren.Helaasheefthijdefinishvanditboekniet

gehaald.Gerjo,ikbenblijendankbaarjoualspromotortehebben.

Daarnaastmijntweedepromotor,prof.dr.Verhaar.BesteprofessorVerhaar,dank

udatumijdekansgegevenhebtompromotieonderzoektedoenopdeafdeling

orthopedie.Metuwuitgebreidekennisuitdekliniek,hadualtijdkritischevragen

overmijnonderzoek.Uheeftmijgeleerdénuitgedaagdbasaalwetenschappelijk

onderzoekzoklinischrelevantmogelijktemaken.Ikkijkernaaruitommeervan

utelerenindekliniek.

Dr. Bastiaansen-Jenniskens,mijn co-promotor Yvonne. Door de zeer intensieve

begeleidinghebikveelvandefundamenteletechnischezakenvanhetlabvanjou

geleerd.Veelvanjouwwerkvormdedebasisvanditboek.ZelfsviadeWhatsApp

kondenwijhetvaakhebbenoverversedata.Ookbuitenhetwerkhebikveellol

metjegehadmetdedoorjougeliefdeslechteHousemuziek.Naalhetintensief

samenwerkenbenikblijdatiknietjouwvoorkeurvoorrozehebovergenomenen

datjijnietAziaatbentgeworden.

Besteprof.dr.Hazes,prof.dr.Bulstraendr.Emans,ledenvandekleinecommissie.

Hartelijk dank voor het kritisch lezen en beoordelen van dit proefschrift. Dear

prof.dr.Dell’Accio,thankyoufortakingtimetovisitusfromtheUK.Ithankyoufor

teachingmethecartilagedefectmodelandforyourinputandsharingyourview

172 Dankwoord

oncartilagerepair.Prof.dr.VanSaase,prof.dr.Kleinrensinkendr.VanLent,dank

vooruwbereidheidomplaatstenemenindegrotecommissie.

Dr.Clockaerts, beste Stefan, jouwwerk vormde samenmetdat vanYvonnede

basisvoormijnproefschrift.Dankvoorjevoorwerkzodatikdirectkoninstromen.

Wiekonbedenkendaternogzoveelonderzoekuitgevoerdzouwordenoverdat

enestukjevetindeknie?DefilosofeersessiestijdensdeICRScongressenwaren

zeerinspirerendenhopelijkkunnenweindetoekomstnogietssamenopstarten.

Daarnaastwilikdeanderesenioronderzoekersenstafledenvandeafdelingortho-

pediegraagbedanken.Julliewarenaltijdbereidkritischnaarmijnonderzoekte

kijkenenactiefweefselteverzamelen.Devolgendepersonenwilikhierbijspeciaal

benoemen.Bestedr.Bos,vanafhetbeginhebbenwevaakmetGerjogebrainstormd

overmijnpromotieonderzoek.Dankvoorjeideeënhierbij.Max,trouweAjacied,

dankvoorjekritischeblikopmijnmaniervanpresenteren.Erwin,statistiekblijft

bijzonder.Ikwaardeerheelergjevisiehierin.

Graagwilikalleco-auteursbedankenvoorhunexpertiseeninzet,waarbijikde

volgendespeciaalwilbenoemen.LobkeGiermanenAnne-MarieZuurmond,jullie

warenmijngroteTNO-hulplijnen.Jullieexpertise inhigh-fat-dietheeftonverwa-

chteinzichtenopgeleverd.Ikkijknooitmeerhetzelfdenaardikkemuizen.Sander

Botter,bedanktvoorhetdelenvanjeexpertiseinmicro-CTensubchondraalbot

bijvroegartrose.DearNiamh,talkingaboutresearch,macrophagesandgingers

came‘handinhand’withyou.GoodtoseeyouinHollandagain!Resti,youwereso

organizedandpreciseinyourwork.YourMasterprojectmadechapter4possible.

GoodluckinSwedenwithfinishingyourownPhD.Mathijs,ikhebnooitbegrepen

waarjealletijdvandaanhaaldeomallestegelijktedoen,maarhetisjeallemaal

gelukt.Wesprekenelkaarnog indekliniek.Dear John, fellowHoffaenthusiast!

Maybeinthefuturewecanreallystartafanclub?Thankyouforyourcontribu-

tion!Serdar,thanksforculturingtheHoffaandperformingtheFACSanalysisand

goodluckwithfinishingyourownPhD!

Het‘analistentrio’Nicole,WendyenJanneke.Julliezijndevastekrachtenvanhet

lab.Altijdontspannendevenbij tepraten in julliekantoor/kamer/hok.Janneke,

bedanktvoordegezelligemomentenindeML2tijdenshetveleverversen.Wendy,

jijhebtditgroentjeveelbegeleidtijdenshetpipetteren.Aanheteindkonikhet

bestgoed,alzegikhetzelf.Nicole!Hetisjammerdatjenietinhethistologiehok

kuntsnijden,terwijljeaanhetinternetshoppenbentbijdeBijenkorf,zonderde

Dankwoord 173

cakevandeweektemissen,voordeparaffinemachine.Dankvoorhetsnijdenvan

aldieknietjesenuitvoerenvanvelekleuringen.

Sandra,altijdvrolijk.Zonderjouwashetquaformulierennietgoedgekomenbij

mij.Ikbendankbaardatjijmijviademailstructuurhebtgegevenaandeafgelopen

periode.

Theforeignseniorsofthelab.Roberto,Iwastherewhenyoudidyourfirstmara-

thonandonedayyou,MaartenandIwilldoanotheronetogether!Keepupthe

goodworkanddon’tforgettoeatlesswhennotrunning.Eric,thankyouforyour

wisdomaboutFACS,bone,wineandcuredham.Kavitha,gratefulforyouradvice

inthelastmonthsofmytimeinthelab.

Aanallecollegapromovendiopde16e:Callie,Johannes,Marloes,Nienke,Mieke,

Johan,Jasper,Michiel,Anna,Panithi,Rintje,Marjan,Mairéad,Marianne,Lizette,

Caoimhe, Shorouk, Simone, Sohrab en Laurie. Bedankt voor de samenwerking,

koffiedrinken,labdays,cakevandeweek,cursussen,borrels,inpakkenvanmijn

bureau inaluminium folie, selfies,collages,congressen inAmerikaen leerzame

tijd.Deklinischecollega’sookbedanktvoordegezelligheid.

Collega’svandechirurgieinDelft,gedurendedevooropleidingbenikeenéchte

doktergeworden.Hetwaseenwarmbadomintestarten.Ikhebmetpleziermet

julliegewerkt,inclusiefdevoetenpolienproctopok.Dankvoordezeerleerzame

tijdendeskireis.

TeamWhiskey,mijnstudievrienden,waarvaneenkleinedelegatieuiteindelijkook

indetorengepromoveerdisofgaatpromoveren.Ikhoopdatweonzevriendschap

zullenblijvenvoortzettenmetregelmatigetentjes,reizen,festivalsenfietsen.De

SV-mannenenoud-CLD’ersvandeKEB-groep:jullievormdeneenbelangrijkebron

vanafleidingtussenhetschrijvenenpipetterendoor.Niek,denkendeaanhetoude

Sintineltijdperk,wilikjehierbijspeciaaldankenvoordemooieomslag.

ChrisenMaarten:deeenkonzijnbenen inzijnnek leggen,deanderhadhaar

totzijnschoudersenikhadietsmeerwekedelen.Vroegerstondenwemetveel

bierbijGoldfishvooraan,tegenwoordiggewoonrustigmeteenwijntjeaantafel.

Maarten,metjoubenikin2011tegelijkmetdatpipetterenbegonnen,destartvan

eengeweldigeperiode.Eenmuurwasnietdikgenoegomonstescheiden.ikben

blijdatweookdevolgendestapvanonzecarrièretotorthopedischchirurgsamen

kunnenvolgen.Bedanktdatjemijnparanimfwilzijn.

174 Dankwoord

MijnschoonzussenLisaenMalou.Lisa,altijdaanhetlachenengeïnteresseerdin

watikaanhetdoenben.Onzediscussieszijnsomsfel,maardatmaaktmijook

weerscherp.JijenJoosthebbeneenprachtigdochter,Evi.Ikbenblijdatikvoor

heteerstoomgewordenben!Malou,dekleinesportieveling.Jewordtonderschat

doorjelengte,maarzwemmendinhetwater,rennendophetlandenrijdendopde

fiets,benjesupersnel.MisschienzelfsietssnellerdanMichiel?Datpromoveren

gaatjounatuurlijkmakkelijklukken!

Lieve schoonvader Jos, op geneeskundig vlak ben jemisschien een leek,maar

desondanksvindikdatjetochveelvanhetmedischeafweet.Misschienookwel

doordatweaantafelalleenmaarhieroverkunnenpraten.Omditgoedtemaken

zijnerweltweeinjegeliefdestadgepromoveerd,echterdederdemoetjehelaas

aandeconcurrentoverlaten.Jebenteenfijnenzachtpersoonenaltijdwelkom

bijons.LieveschoonmoederJosé,jewasaltijdgeïnteresseerdinwatikdeeden

vroegmijdeorenvanhetlijf.Doorjebetrokkenheidvoeldeikmealsnelthuisbij

jullie.Ikzaljeblijvenherinnerenalseenliefdevolpersoon.

Mijn lieveouders,omdatChineesbinnenonsgezindevoertaal is,zal ikverder

gaan in het Chinees.我亲爱的爸妈，谢谢你们让我成长在这么温暖又充满爱的家庭

里。你们俩一直希望我能够生活幸福事业顺利。妈，你经常说作为一个中国父母，

孩子是最重要的。一直到现在，无论我做什么你们都无条件地支持和帮助我。爸，

我现在跟你一样也是博士了，能够引用你的论文并且由你来当我的paranimf，我十

分高兴也非常很骄傲。除了你们，我也想用这个机会感谢我的全部家人和其他好

友。谢谢你们一直以来对我的照顾与关心，以及对我完成博士论文的支持与鼓励。

MijnallerliefsteMyrthe.Jijbentdemeestveerkrachtigeensterkstepersoondie

ikken.Naalleswatdeafgelopenjarengebeurdisenhoegestrestenchagrijnigik

ookwas,jijbleefaltijdsuperliefengeduldig.Alsstelzijnwijsomseencomplete

chaos,maarmeestalvoelenwijelkaarheelgoedaan.Watweooksamendoen,de

tijdvliegtveeltesnelvoorbij.Ikbenheelgelukkigdatjijinmijnlevenbentenik

weetdatwijeraltijdvoorelkaarzullenzijn.Ikhouvanje.

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