The contribution of matrix and cells to leaflet retraction in heart valve tissue engineering

The contribution of matrix and cells to leaflet retraction in heart

valve tissue engineering

Citation for published version (APA):

Vlimmeren, van, M. A. A. (2011). The contribution of matrix and cells to leaflet retraction in heart valve tissue

engineering. Technische Universiteit Eindhoven. https://doi.org/10.6100/IR716523

DOI:

10.6100/IR716523

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Published: 01/01/2011

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Thecontributionofmatrixandcells

toleafletretractioninheartvalve

tissueengineering

 



 AcataloguerecordisavailablefromtheEindhovenUniversityofTechnologyLibrary  ISBN:978Ͳ90Ͳ386Ͳ2710Ͳ6  Copyright©2011byM.A.A.vanVlimmeren 

All rights reserved. No part of this book may be reproduced, stored in a database or retrievalsystem,orpublished,inanyformorinanyway,electronically,mechanically,by print,photoprint,microfilmoranyothermeanswithoutpriorwrittenpermissionbythe author.  Coverdesign:InekevanVlimmeren  PrintedbyPrintPartnersIpskampB.V.,Enschede,theNetherlands. 

Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefullyacknowledged.



The authors gratefully acknowledge the support of the Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, CultureandScience.

  







Thecontributionofmatrixandcellstoleaflet

retractioninheartvalvetissueengineering

PROEFSCHRIFT

terverkrijgingvandegraadvandoctoraande TechnischeUniversiteitEindhoven,opgezagvande rectormagnificus,prof.dr.ir.C.J.vanDuijn,vooreen commissieaangewezendoorhetCollegevoor Promotiesinhetopenbaarteverdedigen opdonderdag3november2011om16.00uur door  MarijkeAntoniaAdrianavanVlimmeren  geborenteVeldhoven

  prof.dr.ir.F.P.T.Baaijens  Copromotoren: dr.A.DriessenͲMol en dr.ir.C.W.J.Oomens

I

Contents

 Summary III Chapter1:Generalintroduction  ₁ 1.1Thehumanheartvalves 1.2Heartvalvediseaseandreplacements 1.3Heartvalvetissueengineering 1.4Tissuestress,compactionandretraction 1.5Collagenmaturation 1.6Rationaleandoutline 2 5 6 9 12 14  Chapter2:Aninvitromodelsystemtoquantifystressgeneration, compactionandretractioninengineeredheartvalvetissues _17



2.1Introduction 2.2Materials&Methods 2.3Results 2.4Discussion 2.5Acknowledgements 18 20 24 27 31  Chapter3:Passiveandactivecontributionstogeneratedforceand retractioninheartvalvetissueengineering 33 3.1Introduction 3.2Materials&Methods 3.3Results 3.4Discussion 3.5Acknowledgements 34 35 39 45 49 

Chapter 4:Controlling matrix formation and crossǦlinking by

hypoxiaincardiovasculartissueengineering



_51



4.1Introduction 4.2Materials&Methods 4.3Results 4.4Discussion 4.5Acknowledgements 52   54 58 63 66   

II Chapter5:Lowoxygenconcentrationsimpairtissuedevelopment intissueengineeredcardiovascularconstructs



 67 5.1Introduction 5.2Materials&Methods 5.3Results 5.4Discussion 5.5Acknowledgements 68 69 74 81 84 

Chapter 6:The potential of prolonged tissue culture to reduce stressgenerationandretractioninengineeredheartvalvetissues.  85 6.1Introduction 6.2Materials&Methods 6.3Results 6.4Discussion 6.5Acknowledgements 86 87 92 99 102  Chapter7:Generaldiscussion 103 7.1Introductionandmainfindings 7.2Limitationsandconsiderations 7.3Implicationsandfutureperspectives 7.4Conclusion 104 107 108 114  References 115 Samenvatting 131 Dankwoord 133 Curriculumvitae 135 Listofpublications 137  

III

Summary

 Thecontributionofmatrixandcellstoleafletretractioninheartvalve tissueengineering  Heartvalvetissueengineeringisapromisingtechniquetoovercomethedrawbacks of currently used mechanical and prosthetic heart valve replacements. Tissue engineered (TE) heart valves are viable and autologous implants that have the capacity to grow, remodelandrepairthroughoutapatient’slife,withouttheneedofanticoagulationtherapy. Thevalvesaremadebyseedingextracellularmatrix(ECM)producingcells,suchasvascularͲ derived cells, onto a rapidly degrading scaffold material manufactured into the shape of a heartvalve.TEvalvesareculturedconstraintwherebytheleafletsfusetogether.Duringfour weeksofculture,thescaffoldisreplacedbynewlyformedtissue,whilestressisgenerated within the tissue by traction forces exerted by the cells. This stress is beneficial for tissue formationandarchitecture.However, duringcultureitcausestissuecompaction,resulting inleafletflattening,andattimeofimplantation,theleafletconstraintsarereleasedandthe generatedstresscausesretractionoftheleaflets.Duetothisretraction,theleafletsarenot able to fully close during diastole and valvular regurgitation occurs. In this thesis, this phenomenonoftissueretractionwasexaminedandstrategiestodecreaseretractionwere investigated.

The first aim was to unravel and quantify stress, compaction and retraction in developingengineeredheartvalvetissues.Therefore,arepresentativeinvitromodelsystem ofrectangularTEstrips(TEconstructs)wasdevelopedinwhichtheevolutionofstressand compactionduringculture,andtheresultingretractionafterreleaseofconstraints,canbe quantifiedfromasingleTEconstruct.Animportantfindingwasthatduringthefirst2weeks of tissue culture, the scaffold was able to counterbalance the traction forces of the cells, which reveals a key role of the stiffness of the cellular surroundings in compaction. When scaffold degradation started after 2 weeks, stress generation and compaction became evidentandgraduallycontinueduptoweek4.Inaverage,theTEconstructscompacted50Ͳ 65% in width and reached force levels of 15Ͳ55 mN and stress levels of 5Ͳ30 kPa. The resulting retraction 24 hours after release of constraints was 35Ͳ50% in length. These degrees of compaction and retraction thus seriously affect leaflet geometry and need substantialreductiontopreventregurgitation.

Subsequently, the relative contributions of passive and active retraction were examined.Passiveretraction occursthroughpassivestressreleaseinthecellsandECMat releaseofconstraints,whileactiveretractioniscausedbythetractionforcesofthecells.To quantify the active and passive contributions, the active traction forces of the cells were eliminated by Cytochalasin D or an inhibitor of the RhoͲassociated kinase pathway, while bothpassiveandactivecontributionsofthecellswereeliminatedbylysisand/orremovalof

IV

thecells.Amajorfindinginthisstudywasthatthepassivecontributionofcellstoretraction issubstantial.Itwasfoundthatpassivecellretractionaccountsfor45%oftotalretraction, while active cell retraction accounts for 40% of the total retraction. The remaining 15% is attributedtopassiveretractionoftheECM.Thesefindingsillustratetheimportanceofthe cellsintheprocessoftissueretraction,notonlyactivelyretractingthetissue,butalsoina passivemanner.

Finally, we aimed to decrease tissue retraction in order to obtain functional, nonͲ retracting leaflets. As it was hypothesized that a strong and wellͲdeveloped ECM would providemoreresistancetothecelltractionforces,twostrategiestoimprovethemechanical integrityoftheECMwereinvestigated.First,theeffectoftheenvironmentalfactoroxygen concentrationontheECMformationwasinvestigatedatbothcell(2D)andtissue(3D)level. Atcellularlevel,culturingatoxygenconcentrationsof4%andbelow(hypoxia)enhancedthe production of collagen crossͲlink enzymes and to a lesser extent collagen type I and III. Unfortunately, these results did not translate into enhanced collagen deposition and maturation in TE constructs. Tissue properties remained similar at 7% and 4% O2 as compared to 21% O2, while culturing below 4% O2 reduced ECM production and the mechanical integrity of the tissue. From this latter study, it was concluded that hypoxia is notverylikelytocreateamorerobustECM.

Inthesecondstrategy,theECMintegritywashypothesizedtoincreasebyprolonged tissue culture. Collagen content and crossͲlinking remained constant when increasing culturetimefrom4weeksto6and8weeks,butGAGcontentincreased,whichresultedin thicker tissues. Although the generated force remained constant from week 4 on, the increased thickness contributed to a decrease in generated stress. The most important findinginthis study wasthat retraction decreased by~50%at week6 and8compared to week4,likelyduetotheincreasedGAGcontent.Thesefindingsemphasizetheroleofthe ECM in tissue retraction and that changing its composition might represent an important strategytoreducetissueretractionand,thereby,valvularregurgitation.

To summarize, solving the problem of leaflet retraction in heart valve tissue engineering remains a challenge due to the substantial contribution of passive retraction. The cellular surroundings have shown to affect the resulting compaction and retraction. However, improving resistance against cell traction forces by enhancing the compressive stiffnessoftheECMhasproventobedifficult.Therefore,otherpromisingapproacheslikea slower degrading scaffold, adjustment of the valve geometry or decellularization of the tissue are discussed in the present thesis and will be focus of future research. Although these strategies still require extensive research, the new insights into the mechanisms of leafletretractionobtainedwithinthisthesisprovideusefulknowledgeneededtodealwith theprobleminordertodevelopfunctionaltissueengineeredheartvalves.



Chapter1



Generalintroduction

Heart valve tissue engineering is a promising approach to overcome the limitations of current available heart valve replacements. However, there are some challenges that need to be overcome, before safe transition to clinical use is possible. ThepresentthesiswillunraveltheproblemofcellͲtractionmediatedtissueshrinkagein heartvalvetissueengineering.Amodelsystemisdevelopedtoinvestigateandquantify theaspectsoftissueshrinkageandproblemsolvingstrategiesareinvestigated.Before going into details, first some background information about native and tissue engineeredheartvalvesisprovidedinthenextsection.



1.1Thehumanheartvalves

The human heart is an essential muscular organ that regulates the blood flow through the body. Blood is oxygenated in thelungs through the pulmonary circulation andsubsequentlypumpedthroughthesystemiccirculationwhereitdeliversoxygento the organs, tissues and cells and removes carbon dioxide. Unidirectional blood flow within the heart is regulated by four heart valves (Figure 1.1). The atrioͲventricular tricuspidandmitralvalvesprohibitreverseflowfromtheventriclestotheatriaduring systole.Thepulmonaryandaorticsemilunarvalvesprevent retrogradeflowbackfrom the pulmonary and aortic arteries into the ventricles during diastole. Heart valves are complexbiologicaltissuescapableofsustainingthesuccessiveloadingofapproximately 40millionheartcyclesayear,equivalenttoapproximately3billioncyclesoveratypical 75yearlifetime(Schoen,2011).   Figure1.1:Schematicsofthehumanheartanditsfourheartvalves.(A)CrossͲsectionof theheart,anteriorview,withthetricuspidvalvepositionedbetweentherightatriumand rightventricleandthemitralvalvepositionedbetweentheleftatriumandleftventricle. Inaddition,thepulmonaryvalveissituatedbetweentheoutletoftherightventricleand the pulmonary artery, and the aortic valve is situated between the outlet of the left ventricleandtheaorta.(B)CrossͲsectionoftheheart,topͲview,showingthefourheart valvesfromabove.

1.1.1 Basicanatomyandphysiologyofthesemilunarvalves

Thesemilunarvalvesarepositionedbetweentheoutletoftherightventricleand the lungs (pulmonary valve) and the outlet of the left ventricle and the aorta (aortic valve). They are referred to as semilunar valves due to the halfͲmoon shape of their three flexible leaflets (Figure 1.2). The leaflets are attached to a ring of tough fibrous tissue,calledtheannulus.Theattachmentpointsoftwoadjacentleafletstotherootare namedthecommissures.Behindeachleafletaredilatedpocketsinthevesselwall;the sinuses of valsalva. In the middle of the free edge of each leaflet is a fibrous section calledthenoduleofArantius.Coaptationofthethreenodulesensurescompletecentral closureofthevalve.   Figure1.2:Schematicanatomyofoneleafletoftheaorticvalve.(A)CrossͲsectionalview indicatingtheleafletsattachedtotheannulusfibrosuswiththesinusofvalsalvabehind theleaflet.Inaddition,thecoronaryorificeintheaorticwallisshown.(B)FrontͲviewof one leaflet indicating the commissures, nodule of arantius and the annulus fibrosus. (Adaptedfromcardiacsurgeryintheadult,L.H.Cohn)



There are several differences between the aortic and the pulmonary valves, including the size and structure of their leaflets and the fact that the aortic valve has coronaryarteryorificesbehindtwoofitsleafletsthatprovidebloodtotheheartmuscle. Furthermore,themechanicalloadingofthevalvesduringthecardiaccycleisdifferent. The cardiac cycle consists of a period of relaxation called diastole, during which the heartvalvesareclosedandtheheartfillswithblood,followedbyaperiodofcontraction called systole, during which the valves are pushed open. The pressure difference over thevalveleafletsduringthediastolicphaseissignificantlyhigherintheaorticvalvethan inthepulmonaryvalve.Intheadultheart,transvalvularpressureatpulmonaryposition is 10 mmHg just before opening of the valves, while transvalvular pressure over the aorticvalvereaches80mmHg(GuytonandHall,2000).

1.1.2 Theheartvalveleaflets

The mechanical functioning of the semilunar heart valves depends on the elasticity and structural integrity of the thin leaflets. The essential functional componentsoftheleafletscomprisecellsandextracellularmatrix(ECM).Twocelltypes exist, namely, the valvular endothelial cells (VECs) and the valvular interstitial cells (VICs). The VECs form a single layer of cells lining the leaflet surface, providing a protective nonͲthrombogenic surface. VICs are the most abundant cell type in leaflets and are distributed throughout all layers. In healthy adult heart valves, VICs have a quiescent fibroblastͲlike phenotype, but they can differentiate into myofibroblasts to mediateECMremodelingandvalverepair(RabkinͲAikawaetal.,2004;Mendelsonand Schoen,2006;Schoen,2008;Apteetal.,2011).Indevelopingheartvalves,VICshavean activated myofibroblast phenotype as well to mediate growth (RabkinͲAikawa et al., 2004).

The ECM of the leaflets consists of collagen, elastin, and amorphous ECM, composed predominately of glycosaminoglycans (GAGs). The leaflets have a layered architecture,inwhichthreelayerscanbedistinguished(Figure1.3A);theventricularis, the spongiosa and the fibrosa, which have different compositions and mechanical properties (Flanagan and Pandit, 2003; Schoen, 2008; Apte et al., 2011; Bouten et al., 2011). The fibrosa, at the outflow side of the leaflet, provides primary strength to the leafletconsistingmainlyofcollagenfibers.Atthecommissures,thecollagenfibersare predominantlyorientedinthecircumferentialdirection,whilemoredivergingfibersare presentinthecentre(Figure1.3B).



 Figure 1.3: (A) Schematic crossͲsection of the aortic leaflet indicating the fibrosa,

spongiosa and ventricularis (Vesely, 1998). (B) Anisotropic collagen architecture within the aortic valve. Collagen fibers are oriented in circumferential direction at the commissuresandmoredivergentinthecenteroftheleaflet(adaptedfromBalguidetal., 2007).

This natural fiber orientation results in anisotropic behavior of the leaflet with high flexibility in the radial direction and high strength in the circumferential direction (Balguid et al., 2007). The spongiosa forms the central core of the leaflets and predominantlyconsistsofGAGs.GAGshaveasignificantwaterͲbindingcapacity,dueto their highly negative charge (Culav et al., 1999). This GAGͲwater combination of the spongiosa absorbs shear and provides compressive strength to the ventricularis and fibrosis.Bycontrast,theventricularisisanelastincontaininglayerthatensuresflexibility oftheleafletsandrecoilduringsystole(Vesely,1998).



1.2 Heartvalvediseaseandreplacements

Pathological changes associated with heart valves consist largely of four types: (1)divergentvalvearchitectureasincongenitalmalformation,(2)ECMdamageleading to loss of mechanical integrity of the leaflets (e.g. myxomatous disease, infective endocarditis), (3) nodular calcification, (4) fibrotic thickening (e.g. due to rheumatic fever). These disorders can lead to heart valve insufficiency or stenosis. In insufficient heart valves leakage occurs, causing backflow from the artery to the ventricle during diastole (regurgitation). In case of stenosis, the valve opening becomes narrowed and the heart has to increase its contraction force to pump sufficient amounts of blood throughtheaffectedvalve.

Valvular heart disease is a significant cause of morbidity and mortality worldͲ wide.Althoughheartvalverepairisthepreferredtreatment,thisisnotalwayspossible. Worldwide, approximately 290.000 heart valve replacements are performed annually andduetoaneverͲagingworldpopulation,thisnumberhasbeenestimatedtoincrease threefold over the upcoming five decades (Yacoub and Takkenberg, 2005; Pibarot and Dumesnil,2009).

Current available heart valve replacements are either mechanical or bioprostheticinnature.ThefirstavailablemechanicalvalvewastheballͲandͲcagevalve in 1962. Since then, many modifications have been made to this valve to improve its performance. Current mechanical valves are made of carbon and the biͲleaflet mechanical valve is, at present, the most popular design. Mechanical heart valves are longͲlasting and readily available, but vulnerable to thrombus formation, due to high shearstressesandnonphysiologicalflowprofilesthatresultinblooddamage(Dasietal., 2009).Asaconsequence,lifelonganticoagulationtreatmentisrequired,whichinvolves increasedriskofinternalbleeding.Bioprostheticvalvesareeitherofhuman(homograft) or animal origin (xenograft). The homograft valves are intact cryopreserved human valvesobtainedfromdonorsandareclosesttonaturalvalves,buttheiruseisrestrained due to limited availability of donors and high costs. Xenografts are made of gluteraldehydefixatedporcineorbovinematerial.Themajoradvantageofbioprosthetic

valves is that there is no need for anticoagulation therapy and that the mechanical functioningofthesevalvesisverygoodastheyhaveanativeͲlikeshape.However,these valves are prone to structural degeneration and calcification (Pibarot and Dumesnil, 2009;Siddiquietal.,2009),andtheassociatedneedforreoperationsmakesthemless suitable for young patients. Furthermore, the use of xenografts is associated with the riskofzoonoses,whicharehumandiseasescausedbyinfectiousagentsfromanimals.

Theoveralllimitationofthecurrentlyavailableheartvalvereplacementsisthat they are nonͲviable prostheses, unable to adapt to the constantly alternating hemodynamicenvironment.Inessence,thesevalveslackthecapabilityofgrowth,repair and remodeling in the body. These shortcomings restrict the use of these valves in pediatric patients as multiple operations are required to accommodate growth during their childhood. Although both mechanical and bioprosthetic heart valve prostheses significantlyimprovelifeexpectancy,theidealprostheticheartvalve,abletoadapttoits environment,hasyettobedeveloped.



1.3Heartvalvetissueengineering

Tissue engineering aims to develop autologous heart valves able to function a lifetimeinvivo.Severalkeycharacteristicsrequiredforatissueengineeredheartvalve are viability, sufficient strength to withstand repetitive and substantial mechanical stress, and the ability to grow and repair any damage by tissue remodeling. Several tissueengineeringapproachesarebeingexploredtoreachthesegoals.



1.3.1Tissueengineeringapproaches

Tissue engineering approaches can be divided into three distinct strategies; (1) implantation of decellularized valvular material (implanted as such or reͲseeded with cells),(2)formationoftissueinvitrobyseedingcellsontoabiodegradablesyntheticor naturalscaffold,and(3)implantationofa biodegradablepolymerthatisremodeledin

vivobyendogeneouscells.

Implantationofdecellularizedxenograftsorhomograftshastheadvantagethata nativeͲlikegeometryisprovidedwithgoodmechanicalbehaviorandstructure.Varying results have been published on the in vivo functioning of these valves. In sheep, remodeling and growth potential was demonstrated in unseeded decellularized valves without calcification (Erdbrugger et al., 2006; Hopkins et al., 2009). However, in reͲ seededdecellularizedvalvescalcificationhasbeenobserved(Metzneretal.,2010).Two clinicalstudieswithdecellularizedvalveshavebeenperformedinpediatricpatients.In thefirst,decellularizedporcineheartvalvesfailedtofunctionduetodegenerationand rupture (Simon et al., 2003), while in the other study decellularized human valves reͲ

seeded with endothelial progenitor cells have shown good functionality in pediatric patientsupto3.5years(Cebotarietal.,2006).Overtime,thepulmonaryvalvediameter increased, while regurgitation decreased, indicating growth of the valve. In adult, pulmonary valve replacements of decellularized xenografts (Konertz et al., 2005) and homografts (Bechtel et al., 2003) have been successful up to two years. The major concerns of decellularized valves are that total cell removal is needed as remnants of cellscouldinduceanimmuneresponse,andthatpreservationoftheECMarchitecture andcompositionisrequiredasthedisruptionoftheECMmightreducethedurabilityof theleaflets.Furthermore,donorvalvesarerequiredwhichhavealimitedavailability.

The classical tissue engineering approach comprises cellͲseeded biodegradable scaffoldsculturedinvitroinbioreactorsystemsmimickingnativephysiologicalpressures and/or flows. Within this tissue engineering approach many cell sources have been examined(Apteetal.,2011).Neonatalcellsources(Schmidtetal.,2005;Schmidtetal., 2006;Sodianetal.,2006;Schmidtetal.,2007),vascularͲderivedcells(Hoerstrupetal., 2000; Schnell et al., 2001; Mol et al., 2005), endothelial progenitor cells (Sales et al., 2010)andmesenchymalstemcells(Sutherlandetal.,2005;Gottliebetal.,2010)have allshownpotentialinheartvalvetissueengineering.Scaffoldmaterialsofsyntheticand naturaldegradingmaterialshavebeenused.Adisadvantageofnaturalscaffolds,suchas fibrin and collagen, is that the polymer itself is weak and long cultures times are requiredtoobtainmechanicalintegrity(NeidertandTranquillo,2006).Analternativeto natural scaffolds, are synthetic scaffolds that degrade within a few weeks. Tissue engineeredvalveshavebeenmadebasedonvariouscombinationsofpolyglycolic acid (PGA),polylacticacid(PLA),polyͲ4Ͳhydroxybutyrate(P4HB)andpolycaprolactone(PCL).

ProofͲofͲconcept was first demonstrated in 1995 when an autologous tissue engineeredleafletwasimplantedinsheep(Shinokaetal.,1995).Thenextstepwasto developfunctionalthreeͲleaflettissueengineeredvalves.Hoerstrupetal.,Sodianetal. and Stock et al. were the first to report on in vivo studies with pulmonary autologous seeded tissue engineered heart valves based on synthetic scaffolds. These valves showed functionality at the pulmonary position for up to 24 weeks and were fully remodeledintonativeͲliketissues(Hoerstrupetal.,2000; Sodianetal.,2000; Stock et

al., 2000). More recent studies have demonstrated further progression in this field.

Sutherlandetal.haveshowninvivofunctionalityuptoeightmonths(Sutherlandetal., 2005),butusingaverysimilarprotocolGottliebetal.observedincreasingregurgitation starting from week six (Gottlieb et al., 2010). Recently, in vivo functionality was demonstrated after eight weeks of implantation with mobile but thickened leaflets (Schmidt et al., 2010). Autologous fibrinͲbased engineered heart valves have demonstratedhighͲqualitytissueformationinvitro(Jockenhoeveletal.,2001;Flanagan

etal.,2007;Robinsonetal.,2008)andrecently,aninvivostudypresentedfunctionality

in sheep (Flanagan et al., 2009). However, the leaflets demonstrated mild shrinkage afterthreemonthsinvivo,resultinginvalvularinsufficiency.

A relatively new approach of tissue engineering focuses on the direct implantationofscaffoldmaterialsintothebodywithoutinvitroculture.Thisapproachis based on the hypothesis that the scaffold will be populated with endogenous cells, followedbyinvivotissueformation.Theadvantageofthisinsituapproachisthatitis cheap,quickandavailableonͲdemand.However,recruitmentoftheappropriatecellsto remodel the scaffold material is needed. It remains to be determined whether these scaffoldsshouldbepreͲseededwithfreshlyisolatedcellstoattractendogenouscells,or whetherthisisunnecessary.Recently,freshlyisolatedautologousboneͲmarrowderived mononuclear cells were seeded onto a biodegradable synthetic scaffold and immediately implanted in primates (Weber et al., 2011). Although this approach was reportedtobesuccessful,mostoftheseededcellswerenotpresentafterfourweeksin

vivo.Rohetal.observedthathumanbonemarrowmononuclearcells,preͲseededinto

vascularconstructs,disappearedwithinaweek,butinitiatedaninflammationmediated process of vascular remodeling through the attraction of endogenous monocytes. This indicatesthatpreͲseededcellscouldattractendogenouscells(Rohetal.,2010).



1.3.2Currentstatusoftissueengineeredheartvalves

Although different approaches of heart valve tissue engineering have shown promising results in vivo, none have been adopted in the clinic. The current available heart valve prostheses have their disadvantages, but perform relatively well (15 to 20 years),havepredictivebehaviorandprovideagoodqualityoflife.Thetissueengineered heart valves need to function properly and provide added value if they are to be accepted by surgeons. Before safe transition to clinical use, some challenges must be overcome in tissue engineering of heart valves. These challenges include thickening of theleafletsinvivocausinglossofflexibilityandmimickingtheanatomyofthevalveand thethreeͲlayeredstructureoftheleaflet.

This thesis will focus on a specific challenge in heart valve tissue engineering, namelyvalvularregurgitation.Duringtissueculture,stressisgeneratedwithinthenewly formedtissueduetotractionforcesexertedbythecells.Atreleaseofconstraints,this generatedstressandthetractionforcesofthecellscauseleafletshrinkage.Whenthis shorteningoftheleafletsissevere,closureduringdiastoleisimpairedandregurgitation willoccur(Flanaganetal.,2009).Valvularregurgitationisobservedinalmostallinvivo studies, but has always been reported as trivial, minimal, mild and moderate regurgitation (Hoerstrup et al., 2000; Sutherland et al., 2005; Schmidt et al., 2010). However, two recent studies are the first to address leaflet shrinkage as a problem (Flanaganetal.,2009;Gottliebetal.,2010).TissueshrinkageisacellͲmediatedproblem, caused by traction forces exerted by the cells. Within the present thesis, this phenomenonoftissueshrinkageisunraveledandbothpassiveandactivecontributions to it are quantified. Further, the hypothesis that a strong extracellular matrix will

improvetheresistanceagainstthetractionforcesofthecellsisinvestigated.Totestthis hypothesis, strategies to provide maturation of the collagen network, which provides stiffnessandstrengthtothetissueengineeredvalves,wereinvestigated.



1.4Tissuestress,compactionandretraction

This thesis focuses on tissue engineered human heart valves fabricated from vascularͲderived cells, seeded into a PGA scaffold coated with P4HB according to the paradigm illustrated below (Figure 1.4). The cellͲseeded scaffolds are cultured in a bioreactor system where mechanical stimuli are applied to stimulate tissue growth. After 4 weeks of tissue culture, the scaffold is degraded and a completely autologous livingheartvalveisgrown.



 Figure 1.4: Schematic overview of in vitro tissue engineering of human heart valves.

Human vascularͲderived cells are expended in the laboratory and seeded onto a heart valve shaped scaffold. Subsequently, the tissues are cultured in a bioreactor system where the valves are subjected to mechanical loading. Finally, the resulting living autologousheartvalvewouldbeimplantedintothepatient.



Tissue engineered valves are cultured constrained with the leaflets attached to each other. During culture, stress is generated within the tissue by contractile forces exerted by the cells. This stress has shown to be beneficial for tissue formation and architecture (Mol et al., 2005; Neidert and Tranquillo, 2006; Robinson et al., 2008). However,duringculturecellͲtractionmediatedremodelingresultsincompactionofthe tissue(Figure1.5A),representedbyleafletflatteninginheartvalves(Moletal.,2005), decreased width in tissue engineered constructs (Shi and Vesely, 2003) and decreased thicknessingeneral(Neidertetal.,2002;RossandTranquillo,2003;Mol etal.,2005). When the constraints are released, tissue retraction occurs due to release of the preͲ stressinthecellsandECMthatwasgeneratedduringculture(passive)andthetraction forces exerted by the cells (active) (Balestrini and Billiar, 2009). Tissue retraction is

illustrated in figure 1.5B and an example of tissue retraction in an ovine tissue engineeredheartvalveispresentedinfigure1.5C.

TissuecompactionandretractionarecellͲtractionmediatedprocessesregulated throughcellͲmatrixinteractionsthattransmitinformationfromtheextracellularmatrix tothecytoplasmandviceversa.CellͲmatrixinteractionsregulatecellgrowth,migration, differentiation, survival, tissue organization and matrix remodeling and are formed by focal adhesion complexes that bind integrin within the cell membrane to the ECM outside the cell (Geiger et al., 2001; Cukierman et al., 2002). Focal adhesions exist in different sizes, related to the amount of stress they exert on the extracellular matrix surroundingthem(Goffinetal.,2006).TheproteinɲͲsmoothmuscleactin(ɲSMA)isa mechanosensitiveproteinthatgenerateshighcontractileactivityinstressfibers(Hinzet

al.,2001;Hinzetal.,2003).Itispresentinstressfibersunderhightensionandclosely

correlated to focal adhesion size. Therefore, it is often used as a measure for the contractilityofacell(Duginaetal.,2001;Goffinetal.,2006;Hinz,2006;WipffandHinz, 2009).

 Figure 1.5: Schematic representation of compaction (A) and retraction (B) in tissue

engineeredstripsandheartvalves.(A)Compactioncausesadecreaseinwidthintissue engineeredstripsandleafletflatteningintissueengineeredheartvalves.(B)Atreleaseof constraints, tissue retraction reduces the length of both tissue engineered strips and leaflets. (C) An ovine tissue engineered heart valve after 4 weeks of culturing with the leafletsattachedtoeachother(I),immediatelyafterseparationoftheleaflets(releaseof constraints)(II)andthreehourslaterwhenkeptonice(III).

In vivo, cells are usually quiescent and become temporarily activated when

remodeling is required, associated with increased ɲSMA expression (RabkinͲAikawa et

al.,2004).Cellsourcesusedinheartvalvetissueengineeringgenerallyhaveanactivated

remodeling phenotype, which is beneficial for the excessive tissue formation that is needed within a short timeͲframe. However, the accompanying ɲSMA expression (Rensen et al., 2007; Beamish et al., 2010) leads to cellͲtraction mediated problems. Cells need a certain internal stress level to function well and exert traction forces to theirsurroundingstoachievethis.Whenintracellulartensionchanges,cellsadjusttheir tractionforcestoreestablishthepreferredinternalstressbalance(Brownetal.,1998). During culture, cells remodel their environment until they have reached the desired internal stress level, which results in tissue compaction. At release of constraints, the internalstresslevelofthecellsdecreasesandtheywillexerttractionforcestoreachit again,resultinginretraction.CellͲtractionhasbeenwidelyinvestigatedincollagenand fibringels(GuidryandGrinnell,1985;Brownetal.,1998;ShiandVesely,2003;Balestrini and Billiar, 2009; Chieh et al., 2009). Contractile cellͲbehavior differs between cell sources(Eastwoodetal.,1996)andisaffectedbytheboundarystiffnessappliedtothe carriermaterial(Legantetal.,2009;Johnetal.,2010).Furthermore,tissuecompaction increases when cell density increases and decreases when the density of the provided naturalscaffoldmaterialincreases(ShiandVesely,2003;Chiehetal.,2009).

Tostudytheevolutionofcompactionandretractioninhumanheartvalvetissue engineering,theselectedconstructsinthisthesisconsistofthreecomponents;vascularͲ derived cells, a PGA scaffold coated with P4HB and newly synthesized tissue. The balancebetweenthesecomponentschangesduringculture.Thecellswithinthetissue will always develop a certain internal stress and exert traction forces to their extracellular matrix and scaffold surroundings to achieve this. In the early phase of tissue culture, the scaffold stiffness is high enough to withstand the traction forces of the cells. However, after approximately two weeks of culture, the scaffold rapidly degrades(Kloudaetal.,2008),whilenewtissueisbeingformedgraduallyovertime.The changesincelltraction,scaffoldandtissuequalityduringcultureareillustratedinfigure 1.6A.Wehypothesizethatwhenthescaffoldcannolongerwithstandthetractionforces of the cells, while the newly formed tissue is still weak, an imbalance among the components exists, leading to compaction and retraction. The schematic figure 1.6B illustratesthateitherslowingdownscaffolddegradation(Figure1.6BͲI)oranincreasein tissue stiffness (Figure 1.6BͲII) can reduce the development of compaction during culture and the resulting retraction. Moreover, temporarily eliminating cell traction forces (Figure 1.6BͲIII) at the time of implantation could reduce tissue retraction. It is worthy of mention that loads applied during the cardiac cycle might be able to counterbalancethetractionforcesoncethevalveisfunctioninginvivo.This,however, canonlyworkifpassiveretractionissmallcomparedtoactiveretraction.

 Figure 1.6: (A) Illustration of the hypothesized changes in scaffold and tissue stiffness,

and cell traction during four weeks of culture of tissue engineered heart valves. The scaffolddegradesrapidlyaftertwoweeks,tissuestiffnessgraduallyincreasesovertime, andtotaltractionforceofthecellswillquicklyreachitsmaximumandwillstayatthat level.Assoonasthescaffoldlosesitsmechanicalintegrity,whilethetissueisstillweak, cell traction forces cause compaction and retraction. (B) An overview of hypothesized potential strategies to reduce tissue compaction and retraction. Slowing down scaffold degradation (I) or increasing tissue stiffness (II) make the TE constructs less prone to tractionforces,decreasingbothcompactionandretraction.Reducingcelltractionatthe timeofreleaseofconstraints(III)wouldreduceretraction.



1.5Collagenmaturation

CollagenisthemainloadͲbearingcomponentoftheleaflets,withcollagentypeI and type III the most abundant types in native heart valves. To provide mechanical integrity and ensure longͲterm in vivo functioning of tissue engineered heart valves, a mature wellͲorganized collagen network is needed and thus mimicking native collagen architecture might be necessary. Further, it was hypothesized that a wellͲdeveloped extracellularmatrixwouldbelesspronetocelltractionforces,reducingtissueretraction atreleaseoftheconstraints.



1.5.1CollagenstructureandcrossǦlinking

Collagen synthesis starts in the endoplasmatic reticulum where three proͲɲͲ chainsarecombinedintoahelicalmolecule,knownasprocollagen.Thisprocollagenis transported into the extracellular space where collagen fibrils are formed. Collagen fibrilsconsistofunitsoffiveprocollagenmoleculesstaggeredtogetherreferredtoasthe microfibrils. Finally, collagen fibrils are organized by the cells into large collagen fibers (Figure1.7A).

Collagen fibrils are stabilized by intermolecular collagen crossͲlinks. Mature trivalent collagen crossͲlinks are situated between two telopeptides (positioned at the

endofaprocollagenmolecule)andthetriplehelixofanadjacentprocollagenmolecule (Figure 1.7B). Two major forms of mature crossͲlinks have been identified, hydroxylysylpyridinoline(HP)andlysylpyridinoline(LP).ThesecrossͲlinksareformedby hydroxylationofthetelopeptides,wherelysineisconvertedtohydroxylysine,catalyzed by the enzyme lysyl hydroxylase. The hydroxylysine residues serve as a substrate for lysyl oxidase, which transforms some of them into hydroxyallysine. The resulting reactivealdehydecancondensewithlysylorhydroxylysylintobivalentcrossͲlinks.Two bivalent crossͲlinks can form trivalent HP or LP crossͲlinks via a spontaneous chemical reaction. It can take a couple of weeks for this to happen. HP crossͲlinks are derived from three hydroxylysyl residues and are predominantly found in highly hydroxylated collagens. LP crossͲlinks are derived from two hydroxylysyl and one lysyl residues and are found primarily in calcified tissues. The loadͲbearing capacity of collagen is, apart from collagen content and organization, highly dependent on collagen crossͲlinking, becausethecrossͲlinksstabilizethecollagenfibrils(Balguidetal.,2007).Moreover,the presence of mature collagen crossͲlinks makes collagen fibers less susceptible to enzymaticdegradation(PaulandBailey,2003).   Figure 1.7: (A)thehierarchicalstructureofcollagenfromafiberdowntoatriplehelix organization(adaptedfromBiologyofthecell,LewisJ.,1994).(B)Schematicofcollagen crossͲlinkslocatedbetweentwotelopeptidesandatriplehelix.  1.5.2Invitroregulationofthecollagennetwork Tissueengineeringapproachesaimatdevelopingculturemethodstoimprovein vitrocollagensynthesis,architectureandmaturation.TheinvitrodevelopmentofawellͲ

organized collagen network can be affected by mechanical, biochemical and environmental stimuli, from which mechanical loading has been investigated most thoroughly.Forexample,theexposureofdevelopingheartvalvestoincreasingflowand pressure in a bioreactor system improved tissue formation in vitro (Hoerstrup et al., 2000). StrainͲbased mechanical loading of tissue engineered heart valves resulted in

superiortissueformation,nonͲlineartissueͲlikemechanicalproperties(Moletal.,2005) andnativeͲlikecollagenfiberarchitecture(Coxetal., 2010).Furthermore,intermittent strainͲbased loading has been shown to accelerate collagen production and crossͲlink formation (Rubbens et al., 2009). Finally, in collagen and fibrin gelͲbased tissue engineered heart valves, commissural alignment of collagen fibers was induced by mechanicalconstraints(NeidertandTranquillo,2006;Robinsonetal.,2008).

Theenvironmentalfactor,oxygen(O2),iswidelyinvestigatedincancerresearch

andneoͲvascularization,buthasalsoshownpotentialtoenhancecollagensynthesisand crossͲlink formation (Falanga et al., 1993; Agocha et al., 1997; Horino et al., 2002; Brinckmann et al., 2005; Wang et al., 2005). Hypoxia, characterized by low cellular O2

concentrations, has a strong impact on cell biology. The process behind it is not completelyknown,butthetranscriptionfactorhypoxiaͲinduciblefactorͲ1(HIFͲ1)isone ofthekeyregulatorsresponsiblefortheinductionofgenesinhypoxicconditions.HIFͲ1 is a complex consisting of a hypoxically inducible subunit HIFͲ1ɲ and a constitutively expressedsubunitHIFͲ1ɴ.Innormoxia,theHIFͲ1ɲproteinsarerapidlydegradeddueto hydroxylationofprolineintohydroxyproline,resultinginessentiallynodetectableHIFͲ 1ɲprotein.Duringhypoxia,HIFͲ1ɲremainsstableandtranslocatesfromthecytoplasm to the nucleus, where it dimerizes with HIFͲ1ɴ. The HIFͲ1 complex formed then associateswithhypoxiaresponseelementsintheregulatoryregionsoftargetgenesand bindsthetranscriptionalcoͲactivatorstoinducegeneexpression(KeandCosta,2006). HypoxiahasbeenassociatedwithincreasedamountsofthecollagencrossͲlinkenzyme lysylͲhydroxylase2(Hofbaueretal.,2003;Brinckmannetal.,2005)andprocollagentype I and III (Takeda et al., 2000; Chen and Aplin, 2003; Estrada and Chesler, 2009). Furthermore, hypoxia has shown potential in increasing the amount of collagen and collagen crossͲlinks in tissue engineered cardiovascular constructs cultured at 7% O2

(Balguidetal.,2009).



1.6Rationaleandoutline

Constrained tissue culture, in which stress can be generated by the cells within the tissue, is a requirement for good tissue formation and architecture, but it is also associated with undesired processes such as compaction and retraction. To develop a functionaltissueengineeredheartvalve,itisimportanttoobtainabalancebetweenthe beneficial and adverse effects of stress generation within the tissue engineered heart valve. The present thesis focused on the development and adverse effects of stress generation.Theaimwas:

x Toquantifytissuestress,compaction,andretractionduringcultureand afterreleaseofconstraints.

x To decrease tissue retraction by improving tissue strength through maturationofthecollagennetwork.

 First, an in vitro model system was designed to quantify the development of stressanditsresultingcompactionandretractionintissueengineeredstrips(chapter2). In chapter 3, this model system has been used to unravel the passive and active contributions to tissue retraction. Secondly, improving tissue quality by inducing collagenmaturationhasbeeninvestigatedinordertodecreaseretraction.Thepotential of the environmental factor oxygen concentration to enhance and improve matrix formation has been investigated at cellular (chapter 4) and tissue level (chapter 5). Finally,theeffectofprolongedtissuecultureontissuematuration,stressandretraction was investigated in chapter 6. Finally, the most important findings are discussed in chapter7,followedbytheirimplicationsforfutureresearch.







Chapter2



Aninvitromodelsystemto

quantifystressgeneration,

compactionandretractionin

engineeredheartvalvetissue

         

This chapter is based on: Marijke A.A. van Vlimmeren, Anita DriessenͲMol, Cees W.J. Oomens, Frank P.T. Baaijens (2011). “An in vitro model system to quantify stress generation, compaction and retraction in engineered heart valve tissues.” Tissue Eng

2.1Introduction

Endstageheartvalvediseaseisafrequentlyoccurringdisease,mostcommonly treatedwithheartvalvereplacement.Worldwide280000heartvalvereplacementsare performed annually and this number has been estimated to triple over the upcoming five decades (Yacoub and Takkenberg, 2005; Pibarot and Dumesnil, 2009). Current clinical options for heart valve replacement are bioprosthetic and mechanical valves. Bioprostheticvalvesmimicnativevalvebehavior,butarepronetovalvedegeneration, which makes them less suitable for younger patients (Zilla et al., 2008). Mechanical valves can function for a lifetime, but anticoagulation therapy is needed to prevent thromboembolism(Zillaetal.,2008;PibarotandDumesnil,2009).Atissueengineered heart valve is an autologous and viable prosthesis. Therefore, it is able to repair, remodel and grow throughout a patient’s life. Especially for pediatric patients a tissue engineeredheartvalvewouldbebeneficial,asitwouldpreventmultipleoperationsto matchgrowthduringtheirchildhood. Autologoustissueengineeredheartvalvesarefabricatedbyseedingextracellular matrixproducingcellsonadegradablescaffoldmaterialandculturethisinvitro.During thecultureperiodthescaffoldmaterialdegradesandtheextracellularmatrixproduced bythecellstakesover.Invivoresultsofautologousheartvalveshaveshownpromising resultsupto8months(Sodianetal.,2000;Sutherlandetal.,2005;Gottliebetal.,2010). Arterial or venous derived cells are commonly used as cell sources for this approach (Schnell et al., 2001). These cells are associated with high extracellular matrix production,buttheircontractilepropertiesmaybeasourceofproblems.Inourgroup, tissue engineered heart valves are grown with the leaflets attached to each other to assureconstraint tissue culture, which hasshown to be beneficial for tissue formation (Mol et al., 2005). In this constrained situation, the cells are able to generate stress within the tissue, due to their contractile properties. Just before implantation, the leafletsareseparatedtoenableopeningandclosingofthevalveinvivo.Thestressthat hasbeenbuildupwithinthetissueduringculturecausesretractionoftheleafletsonce constraintsarereleased(Figure2.1).Ifthisretractionissevere,theheartvalvewillnot beabletofullycloseduringdiastoleanditsfunctionalityislost.

Tissue retraction is a serious issue within heart valve tissue engineering as dysfunctionalclosingofthevalveleafletsleadstoregurgitation.Valvularregurgitation, related to decreasing leaflet length, has been observed in both natural and synthetic scaffold based autologous heart valves (Flanagan et al., 2009; Gottlieb et al., 2010). Tissue retraction results from an imbalance among the three major components of engineeredheartvalvetissues:thecells,thescaffoldandthenewlyformedtissue.The cellswithinthetissuewillalwaysaimtodevelopacertaininternalstress(Brownetal., 1998) and exert traction forces to the extracellular matrix (ECM) and scaffold surroundingstoachievethis.Intheearlyphase,thescaffoldisstiffenoughtowithstand thecontractileforcesofthecells.However,afterapproximately2weeksofculture,the

scaffoldrapidlydegrades,whilethenewlyformedtissueisnotcapableyettowithstand thecontractileforces.Thechangesincelltraction,scaffoldandtissueduringcultureare illustratedinfigure2.2.Asaresultofthesechanges,twoprocessesoftissueshrinkage canbedistinguished.Duringculture,tissuecompactionoccursbycellͲtractionmediated remodeling of the construct. Upon release of the constraints, retraction occurs immediatelyduetoreleaseofthestressgeneratedwithinthematrixbythecellsduring cultureandslowlycontinuesduetoretractionofthematrixbythecells(Balestriniand Billiar,2009).



 Figure 2.1: An ovine tissue engineered heart valve after 4 weeks of culturing with the

leafletsattachedtoeachother(A),immediatelyafterseparationoftheleaflets(B)and3 hourslaterwhenkeptonice(C).



To solve the problem of tissue shrinkage, it is important to know how the imbalance between scaffold, tissue and cell traction results in stress generation, compactionandtissueretraction.Therefore,theaimofthepresentstudyistoquantify and correlate tissue compaction, retraction and stress generation in engineered heart valvetissueduringacultureperiodof4weeks.Wedevelopedaninvitromodelsystem in which stress generation, compaction and retraction can be measured in a single engineered tissue. Proof of principle of this model system is studied by culturing engineered heart valve tissues based on human vascularͲderived cells seeded onto a rapid degrading scaffold within this model system for 4 weeks. Stress generation, compaction and retraction were quantified during culture and after release of constraints at week 4. Using this model system, we are able to gain insight in the developmentofstress,neededtounravelthebalancebetweentissue,scaffoldandcell traction.Thereforethismodelsystemisofgreatvalueforinvestigatingtheapplicability ofpossiblesolutionstopreventorcompensatefortissueretractiontowardsafunctional tissueengineeredheartvalve.



Figure 2.2: Illustration of the changes in scaffold and tissue stiffness, and cell traction

during 4 weeks culture of tissue engineered constructs. The scaffold degrades rapidly aftertwoweeks,tissuestiffnessgraduallyincreasesovertime,whiletotaltractionforce ofthecellswillquicklyreachitsmaximumandwillstayatthatlevel. 

2.2Material&Methods

 2.2.1ExperimentalsetǦup Aninvitromodelsystemisdevelopedinwhichgeneratedstress,compactionand retractionofonetissueengineeredconstructcanbequantifiedduringcultureandafter releaseofconstraints(Figure2.3A).Themodelsystemconsistsofastainlesssteelframe in which two ultraͲhighͲmolecularͲweightͲpolyethylene (UHMWPE) sliding blocks are positioned opposite from each other. In between the two sliding blocks, the tissue engineeredconstructs(TEconstructs)arecultured.Oneslidingblockcanbeeitherfixed (during culture) or move freely (to measure retraction after culture, Figure 2.3B). The otherisconnectedtothemetalframeviatwoleafspringsandthedisplacementofthis slidingblockisrelatedtothegeneratedforcewithinthetissue(Figure2.3C).Thesliding blocks can be fixed with clamps (Figure 2.3AͲ1,2). To measure force generation during culture, the sliding block of the leaf springs is not fixed. Because the force that is generated by the cells will deform the leaf springs this configuration is called semiͲ constrained.Tomeasuregeneratedforceafterreleaseofconstraint,bothsidesarefixed andthisconfigurationiscalledconstrained.

 Figure 2.3: Photographs (ABCͲ1) and a schematic overview (ABCͲ2) of model system in

whichretraction(R ,B1Ͳ2)andgeneratedforce( F ,C1Ͳ2)canbemeasuredthroughtheL

displacement(v)oftwoslidingblockspositionedoppositefromeachother.(1)Clampto

fixateretraction side.(2)Clampto fixate force side.(3)Dotstomeasureretraction.(4)

Dotstomeasuregeneratedforce.L0andLretrrepresenttheoriginalandretractedlength

oftheTEconstructs.

Bothgeneratedforceandretractionaremeasuredthroughthedisplacementof theslidingblocksbycomparingthedisplacementofablackdotontheslidingblocktoa reference dot on the metal frame. To quantify the displacement during culture (generated force), photographs were made twice a week and analyzed with ImageJ (WCIF ImageJ,NationalInstitutesofHealth,USA).Displacementsatweek4(retraction and generated force after release of constraints) were assessed from pictures taken underneath a stereomicroscope (Zeiss Observer, Zeiss, Göttingen, Germany) and quantifiedwithMatlab(TheMathWorks,Eindhoven,TheNetherlands).



2.2.2Quantificationofthegeneratedstress

Theoretically,theforce generatedbythetissueengineeredconstruct(mN)can becalculatedfromthedisplacementvof theslidingblock(mm)andthepropertiesof theleafspringsfromtheequationforbendingofastraightbeam(Youngetal.,2002): 3 24 L vEI F      (2.1)

where E is the young’s modulus of the beam material (mN/mm2_{), is the moment of}

inertia determined from the width b (mm) and height h (mm) of the leaf spring (I 121bh3,mm

4

),and L isthelengthoftheleafsprings(mm).Forthismodelsystem, E was2x102nN/mm2, I was1.04x10Ͳ5mm4and L was14mm.Sincetheleafspringswere madeandpositionedbyhand,eachmodelsystemwascalibratedwithanindividualfit:

Thiswasdonebymeasuringthedisplacementoftheslidingblockduringthreecyclesof subjection to known applied forces ranging from 0 to 20 mN. Loading was within the linear region of the leaf springs verified by the linearity of the forceͲdisplacement curves.

To translate the generated force to stress

V

(kPa), the crossͲsectional area A (mm2) of the TE constructs was measured. Width was assessed during culture from photographsandunderneathastereomicroscopeatweek4.Theinitialthicknessatday 0 was 1 mm, representing the thickness of the scaffold. Thickness at week 4 was assessed from histological sections (see under tissue formation), assuming a linear shrinkage factor of 1.043 during processing of the tissue for histology (Schned et al., 1996).Thicknessduringculturecouldnotbemeasuredand,therefore,thethicknessat earliertimepointwasinterpolatedassumingachangeovertimewithacoursesimilarto that of the width. The calculated crossͲsectional area was used to determine the generatedstressvia: A F V     (2.3)  2.2.3Tissueculture

VascularͲderived cells were harvested from the human vena saphena magna obtained according to the Dutch guidelines for secondary used materials. These cells have previously been characterized as myofibroblasts (Mol et al., 2006). They show expression of vimentin, but not desmin and a subpopulation of the cells express ɲͲ smooth muscle actin. Cells were expanded using standard cell culture methods in a humidified atmosphere containing 5% CO2at 37°C. Culture medium consisted of advanced Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, USA), supplementedwith10%FetalBovineSerum(FBS;GreinerBioone,Frickenhausen,The Netherlands),1%GlutaMax (Invitrogen),and1%penicillin/streptomycin(Lonza,Basel, Switzerland). Thecellswereseeded ontorectangularshapedscaffolds(18x5x1mm) of rapid degrading nonwoven polyglycolic acid (PGA) (Concordia Manufacturing Inc, Coventry, RI, USA) coated with polyͲ4Ͳhydroxybutyrate (P4HB) (Tepha, Lexington, Massachusetts; received as part of the collaboration with University Hospital Zurich). The scaffolds were attached to the sliding blocks with polyurethaneͲtetrahydrofuran glue(15%wt/vol).Sterilizationwasachievedby70%ethanolincubationfor30minutes. Cellswereseededatpassage7withaseedingdensityof15millioncellspercm3_using

fibrin as acell carrier.The TEconstructs werecultured inrectangularwell plates for 4 weeksinahumidifiedatmospherecontaining5%CO2at37°C.Duringtissueculture,the medium was supplemented with LͲascorbic acid 2Ͳphosphate to promote extracellular matrixproduction(0.25mg/ml;Sigma,St.Louis,MO,USA),andreplacedtwiceaweek.

2.2.4Experimentaldesign

TE constructs were cultured for 4 weeks in constrained (n=5) and semiͲ constrained (n=5) configuration.  During culture, the compacted widthWcomp(μm) of

bothconstrainedandsemiͲconstrainedTEconstructs(n=10)wasassessedtwiceaweek. CompactionCW(%) was defined as the percentage of shrinkage compared to the

originalwidthW0:



1



100

%

0

˜



W_W W comp

C

   (2.4)

Stress generation during culture was measured in the semiͲconstrained TE constructs twice a week. After 4 weeks of culturing, generated stress and retraction were measured in the constrained samples after release of the constraints. First, generatedstresswasmeasuredat3minuteintervalsoveratotalperiodof30minutes, afterwhichretractioninlengthRL(%)wasmeasuredduring24hoursasthepercentage ofshrinkagecomparedtotheoriginallengthL0(μm):



1



L_L0



˜

100

%

L retract

R

   (2.5)

inwhichLretractistheretractedlength(μm)oftheTEconstruct.Retractionwasmeasured

every6minutesinthefirsthalfhour,followedbymeasurementsafter1,2,4,6,16and 24hours.Anoverviewoftheexperimentaldesignisgiveninfigure2.4.



Figure 2.4: Schematic overview of the experimental design. Compaction during culture

was measured in both constrained and semiͲconstrained samples. Retraction and generatedstressweremeasuredatweek4afterreleaseofconstraintsintheconstrained samples.GeneratedstressduringculturewasmeasuredinthesemiͲconstrainedsamples.

2.2.5Tissueformation

Tissue quality was evaluated by histological staining. The TE constructs were fixedin3.7%formaldehyde(Merck)andembeddedinparaffin.Tissuesectionsof10μm werecutandstudiedbyhematoxylinandeosinstainingforgeneraltissuemorphology and MassonͲTrichrome staining for deposition of collagen. The stained sections were evaluatedusinglightmicroscopy(AxioObserver,Zeiss,Göttingen,Germany).Additional sections were stained with Picrosirius red to visualize mature collagen fibers and evaluatedusingpolarizedlightmicroscopy.



2.2.6Statisticalanalyses

All data are presented as mean and their standard error of mean. Linear regression analysis was performed to fit the calibration curves using GraphPad Prism software(GraphPadSoftware,Inc,USA).



2.3Results

2.3.1Calibrationofthemodelsystems

Giventheconstantsofthematerialsandthemodelsystem(seequantificationof generated stress), the theoretical correlation between force ( F in mN) and displacement(vinμm)becomesF 0180. ˜v.Figure2.5showsarepresentativefitand its95% confidenceintervalfor oneofthe20modelsystemsthatwerecalibrated.The averageslopeofallmodelsystemswas0.021±0.003,indicatingthatbendingoftheleaf springs is slightly stiffer than theoretically determined. The averaged 95% confidence intervalofallmodelsystemscomprised8.7%ofthegeneratedforce.



Figure 2.5:  Representative forceͲdisplacement calibration curve. The dotted lines

indicatethe95%confidenceintervals.

2.3.2Tissuecultureinthemodelsystem

TEconstructsweresuccessfullyculturedinthemodelsystemfor4weeksinboth constrained and semiͲconstrained configuration (Figure 2.6AͲC). Newly formed tissue andcompactioninwidthwasclearlyvisibleatweek4(Figure2.6B,C)comparedtoday0 (Figure2.6A).Infigure2.6C itcanbeseenthatthegeneratedstresshasdisplacedthe sliding block attached to the leaf springs. The hematoxylin and eosin staining showed homogeneoustissueformation(Figure2.6D).TheMassonͲTrichromeandPicrosiriusred staining indicate a dense collagen network with mature collagen fibers (Figure 2.6DͲF) after4weeks.

 Figure 2.6: Tissue engineered constructs just after seeding (A) and after 4 weeks of

cultureinconstrained(B)andsemiͲconstrained(C)configuration.MassonͲTrichrome(D), HematoxylinandEosin(E)andPicrosirius Red(F)stainingshowedhomogeneoustissue formationwithadenseandmaturecollagennetwork.Thewhitebarsrepresent200μm.  2.3.3Tissuecompactionandgeneratedstressduringculture TheTEconstructsstartedtocompactafter2weeksandcontinuedtocompactto approximatelyhalfoftheiroriginalwidth(CW=51.2±4.8%)atweek4(Figure2.7A).At day0,tissuethicknesswas1000μmandatweek4athicknessof1067μmwasassessed from histology. It was assumed that tissue thickness increase over time changed to a patternsimilartothatofwidthdecrease,startingafter2weeks(Figure2.7B).Usingthis assumption, the total crossͲsectional area of the TE constructs was determined, which compactedfrom5.44±0.08mm2atstartto2.57±0.17mm2after4weeks(Figure2.7C). ForceandstressgenerationshowedthesametimeͲlapseascompaction,startingafter2

weeksandgraduallyincreasinguptorespectively20.3±2.9mNand8.0±1.3kPaatweek 4(Figure2.8).

 Figure2.7:Tissuecompaction(A)startedaftertwoweeksandcontinueduptoweek4.

Tissue width (B, filled triangles) was measuredduring culture and it was assumed that tissue thickness(B, open triangles)followed the same course to the measured value at week4.Thesevaluesforthicknessandwidthresultedinthe decreasingcrossͲsectional areaovertimeillustratedin(C).



 Figure 2.8:  Force (A) and stress (B) generation started at week 2 and continued to

increaseuptorespectively20.3±2.9mNand8.0±1.3kPaatweek4. 

2.3.4Tissueretractionandgeneratedstressafterreleaseofconstraints

Within5secondsafterreleaseofconstraints,themeasuredgeneratedforceand stresswererespectively8.7±0.5mNand3.3±0.3kPa.After30minutes,generatedforce had gradually increased to 15.1±0.7 mN, which correlated to a generated stress of 5.6±0.6kPa(Figure2.9A,B).Next,retractionwasmeasuredwhichoccurredveryfastin the first 20 minutes after release of constraints. This resulted in a retraction of 16.4±1.3%,whichwasalmosthalfofthetotalretractionafter24hoursbeing35.8±3.3% (Figure2.9C).

 Figure 2.9: Tissue retraction (A) occurred rapidly in the first 20 mintes after which is

graduallycontinuedupto35.8±3.3%after24hours.Generatedforce(B)andstress(C) reachedmaximumvaluesof15.1±0.7mNand5.6±0.6kPaafter30minutes.



2.4Discussion

This paper describes a new in vitro model system for investigating the development of stress, generated by traction forces exerted by the cells, in TE constructs. This cell traction on the one hand is beneficial for tissue maturation and alignment in engineered tissues (Mol et al., 2005; Neidert and Tranquillo, 2006; Robinsonetal.,2008),whileontheotherhandiscausingtissueshrinkageatreleaseof constraints(Figure1).Tissueshrinkageresultsfromanimbalancebetweenscaffoldand tissuestiffness,andthetractionforcesofcells(Figure2).Whentractionforcesexerted by the cells are larger than the forces the scaffold or newly formed tissue can resist,