The matrix reloaded: the evolution of regenerative hydrogels

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The

matrix

reloaded:

the

evolution

of

regenerative

hydrogels

Esmaiel

Jabbari

1,8,

*

,

Jeroen

Leijten

2,3,8

,

Qiaobing

Xu

4,8,

*

and

Ali

Khademhosseini

2,3,5,6,7,

*

1_Biomimetic_Materials_and_Tissue_Engineering_Laboratory,_Department_of_Chemical_Engineering,_University_of_South_Carolina,_Columbia,_SC,_USA 2_Harvard-MIT_Division_of_Health_Sciences_and_Technology,_{Massachusetts}_Institute_of_Technology,_Cambridge,_MA_02139,_USA

3_Department_of_Medicine,_Biomaterials_Innovation_Research_Center,_Brigham_and_Women’s_Hospital,_Harvard_Medical_School,_Cambridge,_MA_02139,_USA 4_Department_of_Biomedical_Engineering,_Tufts_University,_Medford,_MA_02155,_USA

5_Wyss_Institute_for_Biologically_Inspired_Engineering,_Harvard_University,_Boston,_MA_02115,_USA

6_Department_of_{Bioindustrial}_{Technologies,}_College_of_Animal_Bioscience_and_Technology,_Konkuk_University,_{Hwayang-dong,}_Gwangjin-gu,_Seoul_143-701,

RepublicofKorea

7_Department_of_Physics,_King_Abdulaziz_University,_Jeddah_21569,_Saudi_Arabia

Cell-laden

hydrogels

can

regenerate

lost,

damaged

or

malfunctioning

tissues.

Clinical

success

of

such

hydrogels

is

strongly

dependent

on

the

ability

to

tune

their

chemical,

physico-mechanical,

and

biological

properties

to

a

specific

application.

In

particular,

mimicking

the

intricate

arrangement

of

cell-interactive

ligands

of

natural

tissues

is

crucial

to

proper

tissue

function.

Natural

extracellular

matrix

elements

represent

a

unique

source

for

generating

such

interactions.

A

plethora

of

extracellular

matrix-based

approaches

have

been

explored

to

augment

the

regenerative

potential

of

hydrogels.

These

efforts

include

the

development

of

matrix-like

hydrogels,

hydrogels

containing

matrix-like

molecules,

hydrogels

containing

decellularized

matrix,

hydrogels

derived

from

decellularized

matrix,

and

decellularized

tissues

as

reimplantable

matrix

hydrogels.

Here

we

review

the

evolution,

strengths

and

weaknesses

of

these

developments

from

the

perspective

of

creating

tissue

regenerating

hydrogels.

Introduction

Biologicaltissuesoftencontainhighlycomplexhydrogels[1,2]. Theycontaindynamic,heterogeneousandspatiallydefined mix-turesofcelltypes,growthfactors,nutrients,andintricate extra-cellularmatrices(ECMs)[3].Importantly,thematricesofnatural tissueshavecomplex structuresthatstartwiththedefined ar-rangementof amino acids thatcompose ECM proteins at the nanoscale,to theformation of fibrilsandfiber bundlesat the microscale,andtothealignmentoffibersinaspecificdirection andcrosslinkingofthefibersatthemacroscale[4].The hierarchi-calstructureoftheECMnotonlycontrolsthetissue’sbiochemical andphysico-mechanicalproperties,butalsotheconcentration,

locationanddistributionofcellsandgrowthfactors,cytokines, and hormones within the tissue.The ECM thus acts as a key elementininducing,orchestratingandmaintainingthe multi-facetedprocessesthatgoverntissuephenotype,function,andfate [5–8].Naturallyderivedhydrogelsareused inengineered con-structstosupportthegrowthandmaturationofimplantedcells, but lack the minimum stiffness required to resist soft tissue compression [9,10]. Conversely, synthetic hydrogels provide therequiredmechanicalsupportbutlacktheintricate arrange-mentofligands thatregulatecellfate.Notsurprisingly,much efforthasbeendedicatedtorecreateorincorporateECM–ortheir derivativesorbiomimeticcounterparts–inhydrogels.Here,we reviewtheevermoresophisticatedapproachestointegrateECMs in hydrogels by orthogonal conjugation of cell-interactive li-gands, copolymerization with functionalized ECM molecules, dopingwithdecellularizedECM,orhybridizationwithdigested

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Review

*Correspondingauthors:Jabbari,E.(jabbari@mailbox.sc.edu),

Xu,Q.(Qiaobing.Xu@tufts.edu),Khademhosseini,A.(alik@rics.bwh.harvard.edu)

8_Shared_first_authors.

1369-7021/ß2015TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).http://dx.doi.org/10.1016/

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biological sources that include amongst others collagen [13], gelatin [14], silk [15], alginate [16], hyaluronic acid [17] and dextran[18].Hydrogelsofnaturalsourcestypicallysupport cell adhesionandproliferation,butaremechanicallyweakand pro-vide little control overremodeling. Advanced modifications or processing strategies are therefore often required to match the biomaterialwiththeinjuredtissue.Inaddition,minorchangesin sequencedistributionofnaturalgelscandramaticallyaffectthe fateandfunctionoftheencapsulatedcellsinthematrixgivingrise tobatch-to-batchvariation[19,20].

Synthetichydrogelssuchaspolyethyleneglycol(PEG), polyvi-nylalcohol(PVA),andpolyvinylpyrrolidone(PVP)have advan-tages that include well-defined compositionand easily tunable physiochemicalproperties[21–23].Synthetichydrogelsgenerate matriceswithenormousrangeofphysical,mechanical,and chem-ical properties for regeneration of complex multiphase tissues [3,24–27].Reinforcementwithfillers,nanofibers,nanotubesand optimization ofnetworkstructure canimprove the mechanical properties ofhydrogelsby several orders ofmagnitude [28–31]. Synthetichydrogelsareoftencharacterizedbyslowdegradation ratesunlessproteolyticallydegradablepeptidesareincorporated in the gelnetwork [32].However,additionalmodificationscan remedy this challenge.For example, short hydroxy acid (HAc) segmentscanbepolymerizedtoPEGchainstogenerate asymmet-ric HAc-chainextendedPEGgels withtunable resorptiontimes

resemblenaturalECMontheabstractlevel,theydonot incorpo-rate the biological complexity derivedfrom the vast variety of distinctECMmolecules[35].Torecapitulatethis,hydrogelsare commonlydecoratedwithoneorafewmatrixmoleculetypes[36– 38].Theseincludeamongstothershyaluronicacids[38],collagens [36,37],laminins[39],elastins[40],vitronectinsandfibronectins [41]. It is well established that such modifications affect the function,proliferationandmigrationofcells.Inaddition,most of these ECM molecules can affect the biomaterials’ porosity, swellingordegradationcharacteristic.Inconsequence,thisoften increasesthedifficultyofcontrollingthehydrogel’sbehavior[42]. As an alternative, numerous bioactive peptide sequences have been identified and conjugated to the polymer chains in the hydrogelnetwork[28,43–45].Forexample,cell-adhesive, vasculo-genicandosteogenichydrogelsweregeneratedby copolymeriza-tion of PEG macromonomers with acrylamide-terminated GRGDpeptide(IP),propargylacrylateand4-pentenal(aldehyde moiety)monomers [46].Aminooxy-functionalized vasculogenic SVVYGLRKpeptide (VP) derivedfrom osteopontin proteinwas conjugatedtothePEGnetworkbyanaminooxy-aldehydereaction whereastheazide-functionalizedosteogenicKIPKASSVPTELSAI STLYLpeptide(OP)derivedfromrecombinanthumanbone mor-phogeneticprotein-2(rhBMP-2)wasconjugatedbya propargyl-azidereaction(Fig.2)[46].Functionalizationofthehydrogelswith IP, IP+OP, and IP+OP+VP significantly increased osteogenic

FIGURE1

RepresentationoftheSPEXA(X=L,G,CorD)macromonomer.BeadsSPEGc(yellow),EO(green),G(blue),D(pink),L(orange),C(purple)andAc(red) representstarPEGcore,ethyleneoxiderepeatunit,glycolide,p-dioxanone,lactide,e-caprolactonerepeatunit,andacrylatefunctionalgroup,respectively.(b) SimulationoftheeffectofdegradableG(blue),L(orange),D(pink),andC(purple)monomersonthedistributionofwaterbeadsaroundthemicelles’core. Redandlightbluebeadsinbarewaterandreactiveacrylatebeads,respectively.(c)EffectofmonomertypeG(red),L(orange),D(green),andC(blue)on themeasuredmasslossofSPEXAhydrogelswithincubationtime.ThepurplecurveinCisthemasslossofPEGDAhydrogelwithoutchainextensionwitha hydroxyacid.Reproducedwithpermission[27].

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differentiationandmineralizationofmarrowstromalcellsseeded inthehydrogels(Fig.2)[46].Inaddition,IP+OP+VPconjugation increasedthe expressionofvasculogenicmarkersPECAM-1and VE-cadherinbytheseededstromalcellswhereastheIP+BP con-jugationonlyincreasedtheexpressionofa-SMA[46].

Althoughtheabilitytoincorporateoneorafewmatrix mole-culetypesintobiomaterialsrepresentsasignificantimprovement, itstilldoesnotrivalthesophisticatedcomplexitythatispresentin naturalECMs.Specifically,itdoesnotrecapitulatethevarietyof cell-interactiveligands presentin naturaltissues and thus only supportspartofitsexpectedfunctions.Apracticalsolutionwas foundinprocesseddecellularizedmammaliantissues[47–53].This

processyieldsapurifiedmatrixthatsubsequentlycanbedigested and processed intoaconcentrated liquidor ground intoa fine hydrophilicpowder.The extractedECMcanthenbecombined with hydrogels to generate hybrids with tunable physical and biologicalproperties(Fig.3).Forexample,asolublematrixderived from cartilage,meniscus, and tendon tissues by digestionwith pepsin was successfully methacrylated by reaction with methacrylicanhydride[54,55].Theresultingmethacrylated ma-trixcouldbecovalentlyboundintoaphotocrosslinkingnatural hydrogelsuchasgelatinmethacryloyl(GelMA)toproducehybrid hydrogels[54].Moreover,thephotocrosslinkingapproachallows theproductionofmicropatternedscaffoldswithcontrolled

topog-FIGURE2

RGD(blue)isconjugatedtoadegradablehydrogel(green)byco-polymerizationofPEGprecursormacromonomerwithacrylamide-terminatedRGD. Propargylacrylateand4-pentenalareaddedtothecopolymerizationreactiontoformaRGDconjugatedgelwithpropargyl(triplebond)andaldehyde moieties,respectively.Avasculogenicpeptide(orange)wasgraftedtothehydrogelbytheaqueousreactionbetweentheaminooxyonthepeptidewiththe aldehydemoietyinthehydrogel.Anosteogenicpeptide(red)wasgraftedorthogonallytothehydrogelbytheaqueousreactionbetweentheazidemoiety onthepeptidewiththepropargylmoietyinthehydrogel.Theorthogonalreactionsledtotheformationofahydrogelwithacell-adhesiveRGDpeptide,a vasculogenicpeptideandanosteogenicpeptide.TheextentofmineralizationwithincubationtimebyMSCsseededinthehydrogelwashighestwhenall threepeptidesweregraftedtothehydrogel(greenbars,IP+OP+VP)ascomparedtothecelladhesivepeptideonly(bluebars,IP)orcelladhesiveplus osteogenicpeptide(redbars,IP+OP).Reproducedwithpermission46.

FIGURE3

AtypicalprocessfortheproductionofECMderivedhydrogels.Thetissueiscutandmincedintosmallerparticlesanddecellularized.ThedecellularizedECM ismixedwithahydrogelprecursorsolutiontoformadecellularizedECM-dopedhydrogelcomposite(3).ThedecellularizedECMisenzymaticallydigestedto produceadecellularizedECMsolution.ThedecellularizedECMsolutionisphysicallycrosslinkedbytheself-assemblyofproteinsandpeptidestoforma hydrogel(1).ThedecellularizedECMsolutionisfunctionalizedwithmethacryloyloracryloylgroupstoproduceadecellularizedECMhydrogelprecursor solution(2).TheprecursorsolutionismixedwithPEGorcrosslinkeddirectlywithUVtoproduceahydrogelbasedondecellularizedanddigestedECM.

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choiceofincorporatedECMalsodeterminesthepanelofgrowth factors presentin the hydrogel [59]. It is reported that hybrid hydrogelscomposedofnativeheartmatrixinducedcardiac dif-ferentiationofhumanembryonicstemcellswithout supplemen-ted growth factors [60]. This hydrogel was prepared from decellularizedECMofporcineheartsbymixingECMandcollagen typeIatvaryingratios.ThehighECMcontent hybridgels pro-motedcardiacmaturationandimprovedcontractilefunctionof cardiaccells.Inadditiontoservingasareservoirforthereleaseof biologicallyactivemolecules,thedecellularizedECMalso provid-edananchoringpointforotherbiologicalmolecules,forexample heparin-bindinggrowthfactorscouldbindtothesulfatedGAGin thedecellularizedECM[61,62].Inthatregard,hydrogelsproduced frompericardialmatrix wereutilizedforthe bindingofgrowth factorssuchasbasicfibroblastgrowthfactor(bFGF)[63].Binding ofbFGFtopericardialmatrixincreaseditsretentionbothinvitro and invivoinischemicmyocardiumascomparedtodeliveryof bFGFinacollagenmatrix[63].

Despitethenumerousadvantages,thesemethodsstillrequire furtheroptimization.Specifically,groundmatricestypically con-sistofcoarsemicrometersizedparticlesanddigestionphysically cleavesthematrix,whichcanresultinthepartiallossof micro-structureandfunction[64].Anotherchallengeliesincontrolling the batch-to-batch variations, whichotherwise couldconfound clinicaloutcomes.Althoughincorporationofgroundordigested ECMeffectivelyprovidesanaturalvarietyofECMs,itdoessoat matrixdensitieslowerthanthosefoundinnaturaltissues.Thus the intensity of the ECM stimuli from these approaches will remainsignificantlylowerthanthosepresentin naturaltissues. ThisrelativelylowECMconcentrationisaninherentand unavoid-ablecharacteristicofthisapproachasitrequiresahydrogel-bulkto providetheimplantwithitsmechanicalstability.

Hydrogels

derived

from

decellularized

tissues

Arecentapproachthathasbeenreceivedwithgreatinterestisbased onproducinghydrogelspurelyofwholedecellularizedtissues.Such hydrogels areexpected to display superiorbiocompatibility and bioactivityascomparedtoconventionalhydrogels[9].Hydrogels solelyderivedfromdecellularizedECMhaveseveraldesirable char-acteristicsfortherapeuticapplications.Theseincludetargeted de-liverybyminimallyinvasivetechniques,easeofrepeateddelivery, abilitytoquicklyfillanirregularlyshapedspace,polymerizationto form a support structure suitable for host cell infiltration and remodeling,andtheinherentbioactivityofnativematrix[65,66]. Manytissuesincludingskin[67],muscle[68],bladder[67],tendon [69],cartilage [70], heart[71],liver[72],bone [73],fat[74]and

narrow[57].Chemicalcrosslinking,forexamplewith glutaralde-hyde,canbeusedtoincreasethestiffnessoftheECMhydrogels, while slowing the rate of degradation and cellular migration throughthehydrogel[77].ECMhydrogelsremaininjectablevia acatheterfollowingchemicalcrosslinking.Otherchemical cross-linkers such asethyldimethylaminopropyl carbodiimide and N-hydroxysuccinimidehavealsobeenusedtogeneratechemically crosslinkedhydrogelsfromECM[75].Despitethelimitedcontrol overthehydrogel’sphysicalcharacteristics,severalstudiesreport promisingdatathat supportthepotential clinicaltranslationof injectable ECMderived hydrogels [70,71,74,78]. For example, a myocardial-specifichydrogelprecursorsolutionderivedfrom decel-lularizedventricularECMsuccessfullygelledbyself-assemblyafter deliveryviatrans-endocardialinjectioninalargeanimalmodel[78]. Thisincreasedtheendogenouscardiomyocytespresentinthe in-farctareawithoutthe inductionofarrhythmias[78].Regardless, althoughthesemethodsallowtheformationofpureECM hydro-gels,they donotprovideanyspatialorganization.Instead,they generateahomogeneousconstructwithoutanyzonalor organo-typicstructure,whichareimportanttoorganfunction.

Spatially

organized

ECM

hydrogels

Naturaltissues haveamultilayered ororganotypicorganization withgradientsinmatrixstiffness,celldensity,andgrowthfactors [79–81].Thesecomplextissuearchitecturesareimperativetotheir respectivetissuefunctions[82].Forexample,articularcartilageis composedofmultiplezonesincludingsuperficial,middle,deep, andcalcifiedzones,whichbyactingtogetherallowforthe absorp-tionofmechanicalstressgeneratedbymovements[83].Eachzone is characterizedby distinct cellularphenotypes, ECM composi-tions,andgrowthfactors[84].Moreover,eachzoneis mechani-callyuniqueandcontainsadifferentcollagenfibermorphologies thatrangesfromthintothick thatrunparallel,obliqueor per-pendicularfromthesuperficialzonetothecalcifiedzone, respec-tively[85].Thechangeintissuecompositionandcollagenfibers orientationsubstantiallyincreasesthecompressivemodulusfrom 80kPainthesuperficialzoneto320MPainthecalcifiedzone[86]. HydrogelsentirelycomposedofdigestedcartilageECMhavelost this important instructive organization. In consequence, ever-moreattentionisdedicatedtothegenerationofzonalor organo-typic structures [87,88]. For example, to recapitulate the microstructureand compressive propertiesof thecalcifiedzone ofarticularcartilage,asuspensionofgel-coatedandaligned nano-fibermicrosheets, transforming growth factor-b1 (TGF-b1) and humanmesenchymalstemcells(hMSCs)wascrosslinkedintoa disk-shapematrixsuchthatthealignednanofiberswereoriented

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perpendiculartothedisk’ssurface(Fig.4)[30].hMSCs encapsu-latedinthefibroushydrogelwith320MPacompressivemodulus expressed markers of hypertrophic chondrocytes found in the calcified zone (COLX and ALP) whereas compliant gels with a relativelylow modulus of 80kPawithout nanofibers expressed early markers of chondrogenesis found in the superficial zone (SOX9 and SZP) of articular cartilage [30]. Remarkably, hMSCs encapsulated in the fibrous hydrogel were oriented along the directionofnanofibers[30].Similarly,carbonnanotubes embed-dedinGelMAhydrogelsnotonlycontrolledthehydrogel’s visco-elasticity and electrical conductivity, but they also aligned myoblastsinaspecifieddirection[89,90].

Anotherapproachtorecreatethenaturalorganizationoftissues canbefoundinthe three-dimensional(3D)printingof decellu-larized ECM, which is recently pioneered [91]. Various tissues includingheart,cartilageandfatweredecellularized,processed, ladenwithstemcellsand3Dprinted[91].Asexpected,stemcells within 3D printed tissue-specific ECM expressed significantly higherlevelsofbiomarkersspecificto thematrixofthe chosen tissue[91].TheuseofsuchbiologicalECM-basedinksispredicted to enhance our capabilities and accelerate the development of man-madeorgan-likeimplants.

Together,thesestudiesunderlinetheimportanceofrecreating thezonalandorganotypicstructuresasfoundinnaturaltissues. Unfortunately,thesetechniquesareunabletopresentthematrix moleculesintheirnaturalconformation.Naturalmatriceshavea tightlyorchestratedorganization,whichallowsforpresentationof therightmotifattherightplaceatthemicrometerlevel.Tissue digestionirrevocably removesthis organization,even whenthe macrolevelisrecapitulatedintheformofzonalandorganotypic structures.

Intact

decellularized

ECM

as

hydrogels

Decellularized tissues effectively are crosslinked ECM hydrogelswith a highlycontrolledspatial organization [92–94].

Decellularizationofatissuecanbeachievedusingphysical, chemi-calorenzymaticapproaches[95,96].Awell-knownclinicalexample ofthisapproachisthecreationofacellulardermalmatricesforthe treatmentofburnwoundsandcosmeticsurgery[97]. Decellulariza-tionisnotonlycompatiblewithtissues,butalsowithwholeorgans. Uniquely, this yields implantable hydrogels that contain truly organotypicstructures[98].TheresultingdecellularizedECM con-structs allow forreseeding with cells [92–94]. This can produce hybridimplantsthatarecomposedofallogeneicECMand autolo-gouscells,whichcouldreducethechanceoforganrejectionwhile improvingimplantfunction.Inrecentyears,severalstudieshave indeedreportedonthefunctionalityofthistypeofimplantfor amongstotherslung,heartandtrachea[99–101].

Despiteitsmanyadvantages,decellularizationofatissuecan alterpropertiesofthematrixasitalsocandamageorremovepart of the organ’smatrix [102].To counter thisdetrimental event, decellularizedECMhasbeencombinedwithothertypesof hydro-gelstoonceagainformECM/hydrogelcomposites[103,104].In one report, the decellularized bladder matrix was seeded with bladder smooth muscle cells and a crosslinkable form of the non-sulfated glycosaminoglycan hyaluronan (HA) [103]. The HA-modifiedbladdercellularmatrixdisplayedanotableincrease inmatrixcontractionandtriggeredahigherlevelofcell-secreted gelatinaseactivitycomparedtotheunmodifiedbladderacellular matrix [103]. In another report, human myocardium was first decellularized with retainedbiological elements of the ECMas wellastheunderlyingmechanicalproperties[104].The decellu-larized humanmyocardiumwasthensliced intosheets,coated withMSCsinafibringelandlaminatedtoformafullybiological compositescaffoldforcardiovascularrepair[104].Implantationof thecompositeimplantontothemyocardialinfarctbedinanude ratmodelenhancedtherecoveryofbaselinelevelsofleft ventric-ular systolic dimensions and contractility [104]. Assuch, these approachespartlyreversethetraditionalrolesofbiomaterialsand ECMsintissueengineering;thebiomaterialsupportstheimplant’s

FIGURE4

hMSCsareencapsulatedinasoftgelwith80kPacompressivemodulus(a,lightblue)tosimulatethesuperficialzone(SZ)ofarticularcartilageorinastiff gelwithnanofibersalignedinthedirectionperpendiculartotheplaneofthegellayerwith320MPacompressivemodulus(b,green)tosimulatethe calcifiedzone(CZ).hMSCsinthesoftgelareuniformlydistributedinthegel(greendotsinC)whereashMSCsinthestiffgelclusteredaroundthealigned nanofiberswithacolumnarmorphology(greendotsind).hMSCsinthesoftgeldifferentiatedtothesuperficialzonechondrocytesandsecretedamatrix richinsuperficialzoneprotein(SZP,bluebarsinb)whereashMSCsinthestiffgelwithalignednanofibersdifferentiatedtothecalcifiedzonechondrocytes andsecretedamatrixrichincollagenX(orangebarsinf ).Reproducedwithpermission[30].

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functionandtheECMprovidesamechanicallystable microenvi-ronment.

In conclusion, the ultimate decision of which one of these approacheswill bemost suitablefora specifictherapy strongly correlateswiththedesiredbalancebetweencontroloverphysical andmechanicalproperties,requiredECMamount,spatial organi-zationoftheECMandtheenvisionedapplicationmethod(Table 1).

Future

challenges

and

perspectives

Conventionalhydrogelsbasedonmatrix-likepolymernetworks allow for well controlled tuning of physical and mechanical characteristics.However, theincorporation ofa widevariety of bioactivestimuliatappropriateconcentrationstoachieveproper tissue-likefunctionhasremainedamajorchallengeandareaof intensiveresearch.HydrogelscontainingdecellularizedECM pres-ent a naturalselection ofstimulating matrix molecules, but at unnaturallylowdensities.HydrogelsderivedfromECMdisplaya naturalvarietyofmatrixmoleculesatappropriateconcentrations, but provide little control over their physical and mechanical characteristics.Althoughhydrogelsderivedfromdigested decel-lularizedtissues lackthedesiredmechanical properties,the pri-maryandtosomeextentthesecondarystructureoftheoriginal ECMispreserved.Therefore,hybridmatricesbasedonsynthetic gelsfortuningphysico-mechanicalpropertiesanddigestedtissue gels for controlling cell function arevery promising ascellular scaffolds in regenerative medicine. Hydrogels of decellularized intact tissues retain both the composition and complex nano-andmicrostructuresofthenaturaltissue.Thisprovidesan exqui-sitely biomimetic microenvironment for soft tissue repair and regeneration,butofferschallengeswith regardsto effectivecell seedingandiscurrentlynotcompatiblewithminimallyinvasive strategies.Regardless,withtheexceptionofdecellularizedintact tissues,ithasremainedatruechallengetoprovidetheappropriate

stimuli at the appropriate place. Instead, ECM and associated growth factors are typically presented in a homogeneous and unnaturalmanner.Micro-andnanoscaletechniquesareexpected to generate the intricate patterns of cells, growth factors, and complex ECM structures that are essential for the function of naturaltissues.Hybridmatricescombinedwithmicroscale tech-nologies aretherefore expected to lead to the development of biomimeticmatriceswithbalancedandtunable physico-mechan-ical, biochemical,and cellular propertiesfor applicationsin re-generativemedicine.

Acknowledgements

ThisworkwassupportedbyresearchgrantstoE.Jabbarifromthe NationalScienceFoundationunderGrantNos.DMR1049381, IIP-1357109andCBET1403545,andtheNationalInstitutesofHealth (NIH)underGrantNo.AR063745.Q.Xuacknowledgesthe supportofPewScholarforBiomedicalSciencesprogramfromPew CharitableTrustsandNIHunderGrantNo.1R03EB017402-01.Q. XuthanksYujiTakedaforpreparationofFig.3.J.Leijtenwas supportedbyapost-doctoralmandateoftheFlandersResearch FoundationunderGrantNo.1208715N.A.Khademhosseini acknowledgesfundingfromtheNationalScienceFoundation (EFRI-1240443),IMMODGEL(602694),andtheNational InstitutesofHealth(EB012597,AR057837,DE021468,HL099073, AI105024,AR063745).E.JabbarithanksS.Moeinzadehfor preparationoffigures.

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