An injectable meta-biomaterialmassaging8,28, the EPI biomaterial behaves as a weak viscoelastic solid amenable to smooth deformation (Fig. 1b). At rest, the self-healing process is

1

Aninjectablemeta-biomaterial

A.Béduer1,2,§,F.Bonini1§,C.Verheyen2§,P.Burch2,3,T.Braschler1*

1UniversityofGeneva,FacultyofMedicine,DepartmentofPathologyandImmunology.

RueMichel-Servet1,CH-1022Geneva,Switzerland.

2EcolepolytechniquefédéraledeLausanne,EPFL,SchoolofEngineering,LMIS4.BM,

Station17,CH-1015Lausanne.

3Volumina-MedicalSA,RoutedelaCorniche5,CH-1066Epalinges,Switzerland.

§Theseauthorscontributedequally

*Correspondingauthor:[email protected]

Abstract

Wepresentanoveltypeofinjectablebiomaterialwithanelasticsofteningtransition.

Thematerialenablesin-vivoshaping,followedbyinductionof3Dstablevascularized

tissueadoptingthedesiredshape.Weestablishthenecessarygeometricalandphysical

parametersbyextensivenumericalsimulation.Irregularparticleshapedramatically

enhancesyieldstrainforin-vivostabilityagainstdeformation,whilefrictionand

porosityprovidetheelasticsofteningtransitionasanemergentmeta-materialproperty.

Accordingly,wesynthesizeourinjectablemeta-biomaterialasasuspensionof

irregularlyfragmented,highlyporoussponge-likemicrogels.Themeta-biomaterial

exhibitsbothhighyieldstrain,andthedesirednovelelasticsofteningtransitionforin-

author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.01.30.926931doi: bioRxiv preprint

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situshapingandunprecedenteddynamicmatchingofadiposetissuemechanics.Invivo,

predeterminedshapescanbesculptedmanuallyaftersubcutaneousinjectioninmice.

The3Dshapeismaintainedduringexcellenthosttissueintegrationintotheparticle

porespace.Themeta-biomaterialsustainsvascularizedconnectivetissuetotheendof

one-yearfollow-up.

Aging,disease,trauma,congenitaldeformitiesandsurgicalsequelaecanallleadtoaloss

orlackofsofttissuevolume,producingamajorandincreasingmedicaldemandfor

tissuereconstructionstrategies1-4.Ideally,suchproceduresshouldbeminimally

invasivetoreducethepatientburdenanddecreasepotentialsurgicalcomplications5,6.

Forsofttissuereconstruction,thisrequiresatissuebulkingagentthatcanbeinjected

throughathinneedleintoatargetsiteinthebody4.However,afterdeploymentthis

bulkingmaterialmustmaintainastablethree-dimensional(3D)configurationto

provideadequatevolumeforthemissingsofttissue7.Beyondinjectabilityandvolume

maintenance,surgeonsimposetheadditionalrequirementofinsitushapeabilityin

ordertosmoothlymatchthearbitrarygeometryofapatient’stissuedefect8.Evenifthe

threeconditionsofinjectability,shapeabilityandstabilityaresatisfied,thistheoretical

tri-statematerialshouldalsoexhibitahighdegreeofbiocompatibilitywhileclosely

matchingthelocaltissuemechanics9,10.

Inpractice,suchanidealmaterialisexceedinglydifficulttoengineer.Forexample,fluid-

likebehaviorneededforminimally-invasivedeliverythroughaneedlelimitsmechanical

stiffness11.Asaresult,manyofthecurrentlyavailableinjectableagentsaretoosoft11to

matchthelocaltissueproperties12,13.Thoughusefulforsmoothingandenhancing


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existingshapes,theyrequireimprovementstoadequatelyliftandshapethree-

dimensionaltissuevolumes11.Ontheotherhand,solid-likebehaviorinpreformed

implantsinterfereswithin-situshaping,evenifminimallyinvasivedeliveryispossible

bycompressionorfolding14,15.Thisunmetneedhaslaunchednumerouseffortsto

engineerinjectable,shape-fixabletissueimplants,basedonvariousphysicalorchemical

in-situcrosslinkingmechanisms16,17.Emergingstrategiesincludetheuseofpeptide,

DNA,orpolyelectrolyteandothercomplexestoimpartintrinsicself-healingability18-20.

Thoughsuchapproachesprovideimprovedmechanicalproperties,theyalsoimposea

numberofadditionalchemicalconstraintsandraiseconcernsregarding

biocompatibilityandtissueintegration16,21,22.

Here,wereconcileinjectability,shapeability,andlong-termvolumestabilitybya

radicallynovelapproach:aninjectablemeta-material23.Mechanicalmeta-materials

obtainunusualpropertiesbytheirmeregeometry23,24.Wehypothesizeherethat

physicalinterlockingofgeometricallydesignedparticlescanrestoremeta-material

propertiesevenaftertransientliquefactionforinjection25-27.Inthisnovelparadigm,

self-healingdeliversmeta-materialphysicsin-vivo,in-situ,inaminimallyinvasive

fashion.Weengineeratri-statemicrogelmeta-biomaterialthatcombinesinjectability,

solid-statesofteningforshapeability,andtissue-mimickingmechanicalstability12,13.The

materialdramaticallyincreasestheaccessiblestiffnessrangeandforthefirsttime

demonstratesareversibleelasticsofteningtransitioninaninjectable.Itexhibits

excellentbiocompatibilityandiscolonizedtoinduceformationofvascularizedtissue.

Takentogether,wehavesuccessfullyintegratedthedesigncriteriainformedbyclinical

needtoengineeranovelinjectableandshapeablebiomaterialforstable3Dsofttissue

reconstruction.


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Results

Elastic porous injectable (EPI) biomaterial

Fig.1.Elasticporousinjectable(EPI)biomaterial.a)Injectability:Particleliquefaction

andmobilityunderhighstrainenablesminimally-invasivedelivery.b)Shapeability:

Partialmechanicalstabilityduetogeometricparticleconstraintsenablesin-situshaping

underintermediatestrains.c)VolumeStability:Fullyinterlockedparticlestateenables

long-termthree-dimensionalstabilityunderlowstrains.d)Macroscopicdemonstrationof

thematerialproperties,depictingfluid-likeejectionofparticlesthroughacannulaand

solid-like3Dshapestability.Scalebar:4mme)Scanningelectronmicroscopepictureofa

singleporousparticle(brightnessproportionallyenhanced).f)Confocalimageshowing

fourinterlockingporousparticles(maximalintensityzprojection).g)Particleidentityin

Fig.1f,determinedbythresholdingtheindividualcolorchannels,andforthedoubly


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labeledparticle,co-localizationofblueandgreenaftercorrectionforchromatic

aberration.Scalebar:200µm.

Theconceptofourelastic,porous,andinjectable(EPI)biomaterialisillustratedin

Figure1.Underthehighshearimposedbyinjection,thematerialfluidizesandbehaves

asaliquid(Fig.1a).Underintermediateshear,typicalofinsitushapingand

massaging8,28,theEPIbiomaterialbehavesasaweakviscoelasticsolidamenableto

smoothdeformation(Fig.1b).Atrest,theself-healingprocessiscompleteandtheEPI

biomaterialbehavesasasoftsolidwithshearmoduliinthelowerkParangetomatch

thenativetissueproperties12,13(Fig.1c).

TheEPIbiomaterialconsistsofaninterlockingsuspensionofhighlyirregular,sponge-

likemicroparticles.Itsuniquematerialdesignenablesbothfluidicinjectionthrougha

cannulaandshapeablethree-dimensionalstability.Amacroscopicdemonstrationofthe

behaviorisprovidedinFig.1d,whileFig.1eshowstheirregular,porousparticle

morphology.Fig.1fand1gdemonstratemicrosopicparticleinterlocking.

In-silico design

TodesigntheEPIbiomaterial,outlinedinFig.1,wefirstperformedadiscreteelement

simulation.Thisessentialsteptranslatestheclinicalusecriteriaintoasetofdesignrules

thatguidedourmaterialfabricationstrategy.Toreiterate,anidealtissue-filling

biomaterialshoulddisplayinjectability,shapeability,3Dvolumestability,

biocompatibility,andtissue-matchingmechanicalproperties.


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Fig.2.InSilicosimulationanddesignoftheEPIbiomaterial.a)Graphicaloverviewof

thesimulation.b)Elasticstorageandviscouslossmodulus(G’andG’’)foradense

suspensionoffrictionalspheresc)forabulkmaterialformedbyfullcross-linkingofevery

neighboringsphered)foradensesuspensionofdiscrete,compact,irregularparticles

formedfromneighboringspherese)foradensesuspensionofdiscrete,irregularparticles

withalowcrosslinkdensityandfreecontactinterfaces.f)Characteristicrheological

responsewithaccompanyingstoragemodulusandstrainvalues. =low-deformation

limit, =softeningtransition, =softplateaustress, =yieldstrain.g)Overviewofthe

influenceofthemodelparametersonthecharacteristicvaluesdefinedinFig.2F.‡The

frictioncoefficienthasamagnitude-dependenteffect,seeSupplementary5,Fig.S5-3.h)

InfluenceoftheYoungmodulusoftheconstituentmaterialonthelow-strainlimitstorage

modulusforthedifferentparticlegeometries.Errorbars=onestandarddeviation.n.s.=

notsignificant.SamplesizeandstatisticaltestinginformationinSupplementary14,items

1to26.


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AfulldescriptionofoursimulationisprovidedinSupplementary1(mathematical

model),2(softwareusageinstructions),3(implementationdetails),4(testcases),15

(sourcecode),16(APIdocumentation)and17(installguide).Briefly,thesimulation

modelsthephysicalinteractionbetweensphericalparticlesbycentralandfrictional

forces25.Tomeetthelong-standingchallengeofsymmetricalstresstensorevaluationin

granularmedia29,30,wesystematicallyreviewedandcorrectedthepublished

mathematicalframework25andstresstensorevaluation29(Supplementary1).Wethen

addedpermanentcrosslinkstostudybothirregularandporousmicroparticles(Fig.2a).

Analogoustotheempiricalcharacterizationofviscoelasticagentsforsofttissue

reconstruction8,weperformedinsilicooscillatoryshearrheometrybyapplicationof

time-varyingstrain(Fig.2a).Thisprovidesanestimateoftheelasticstiffness(elastic

storagemodulusG’)andtheabilitytodeform(viscouslossmoduliG’’)25,31.

Wefirstsimulatedfourprototypicalscenariosofmicrogelsuspensions:asimple

suspensionoffrictionalsphericalmicrogels(Fig.2b,Supplementaryvideo18),

completelycrosslinkedelasticbulkmaterial(Fig.2c,Supplementaryvideo19),

compactlycrosslinkedirregularparticles(Fig.2d,Supplementaryvideo20)andloosely

crosslinkedparticleswithadecreasedinternalcrosslinkingdensity(Fig.2e;

Supplementaryvideo21).Anelasticsofteningtransitionwasfoundforthefrictional

sphericalmicrogelsuspensionasexpected25(Fig.2b,absentinnon-frictionalcontrol

providedinSupplementary5).Buttheyieldstrainwassubstantiallybelowthe50%

typicallyrequiredforasofttissuefiller8towithstandphysiologicalmovement32.Onthe

otherhand,bulkcrosslinking(Fig.2c)abolishedtheyieldingtransitionaltogether.

Irregularparticlesfinally(Fig.2d)exhibitedyieldstrainwellabove50%,enablingboth


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injectabilityandinvivoshapestability.Thisestablishesourfirstdesignrule:The

materialshouldbeasuspensionofirregularratherthansphericalparticles.

Quantitatively,theelasticsofteningtransitionwasbetterpreservedinloosely(vs.

densely)crosslinkedparticles(Fig.2e).Thesofteningbehavioriscriticalbothfor

shapeabilityandformatchingthemechanicalpropertiesoflocaladiposetissue,which

alsodemonstratestrainsofteningundershear12,13.Wethusobtainourseconddesign

rule:theparticlesshouldhavealowdensityofinternalcrosslinkswithfrictionalintra-

particleporosity.

Withourfirsttwodesignrulesestablished,wegeneralizedthedesiredrheological

behavior(Fig.2f)andperformedsystematicanalysisofthemodel’sparameters(Fig.2g,

Supplementary5).Theanalysisconfirmedthatthegeometricparameters(particle

shapeandporosity)dominatethehigh-strainbehaviors33ofyielding( inFig.2fand

2g)andthesoftplateau( ),importantforshapingandinjection.Conversely,the

mechanicalparameters(friction,packing,andYoung’smodulus)dominatethelow-

strainbehaviorsofsoftening( )andthelow-strainplateaushearmodulus( ),

importantfortissueinteraction.

Specifically,Fig.2hindicatesthatthelow-strainstoragemoduluswasproportionalto

theYoung’smodulusoftheconstituentmicrogelmaterial(linearregression,P=8*10-88),

butindependentoftheparticlegeometry(P=0.50)orcrosslinkingdensity(P=0.29)

(statisticalanalysisinSupplementary14,item27).Ideally,ourmaterial’slow-strain

mechanicalbehaviorwouldmatchthatofnativesofttissue(lowerkParange12,13).Thus

weobtainourthirddesignrule:thebulkprecursorfromwhichwesynthesizeour


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microparticlesuspensionshouldhaveastoragemodulusclosetothelowstrainlimitof

adiposetissue.

Thefirstthreedesigncriteriaproducetheoptimalrheologyforinjectability,

shapeability,andtissue-matching3Dstability.Ourlastguidelinecomesfromthe

establishedrulethatscaffoldsshouldhaveporesofatleast50µmdiametertoensure

vascularizationandcolonization34,35.Thusweobtainourfourthandfinaldesignrule:

themicroparticlesshouldhaveameanporesizeofatleast50microns.

Synthesis and mechanical characterization

Ourinsilicoanalysisrevealedthataninjectable,shapeable,andvolume-stablematerial

couldbeachievedbyadenselypackedsuspensionofelastic,frictionalmicroparticles

withirregularporousgeometry(Fig.2).Tofurtherensurebiocompatibilitywebased

oursynthesisonthecryogelation36ofcarboxymethylcellulose34,37.Thepolymercontent

wasadjustedtoobtainaporousbulkmaterialwithanelasticstoragemodulus(G’)of

2.4kPa+/-0.9kPa(Supplementary6).Wethereforesatisfieddesignrule#3,which

statedthatthebulkprecursorelasticstoragemodulusshouldbeinthelow-strainlimit

ofadiposetissue12,13.


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Fig.3.InvitrocharacterizationoftheEPIBiomaterial.Confocalimagesofa)theEPI

biomaterial(stainedwithrhodamine6G)andb)SephacrylS200(autofluorescence).The

starsdenoteporespace.Scalebars=200microns.c)G’oftheEPIbiomaterialasafunction

ofappliedoscillatorystressforvariousEPIbiomaterialpolymerconcentrations(indicated,

mg/ml).Largesymbols( ):solid-likebehavior(G’>G’’);smallsymbols( ):liquidlike

behavior(G’’<G’).Eachcurverepresentsasinglemeasurement.d)Normalizedmaster

curvesforEPIandSephacryl200,errorlines+/-onestandarddeviation.e)Poresizevs.

low-stressG0’values.Greybox( ):50μmminimalporesizeandaG0’valuebetween2and

3kParequiredformatchingadiposetissue12,13,34,35.f)ComparisonoftheEPIbiomaterial

(26mg/mL)tocommercialdermalfillerJuvedermVoluma(singlesamples)andre-plotted

literatureG’dataonbovineretro-orbitaladiposetissue13g)EPIbiomaterialinjectability.


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h)Uniaxialcompressionanalysisofasamplebeforepassagethroughthecannula,afterthe

firstpassage,andaftertwoconsecutivepassages.i)ComparisonofEandG0’beforeand

after1or2passagesthroughthecannula.j)RecoveryoftheG’valuesafteraperiodof

liquefyingshear(“Stress”).k)LevelofG’recoveredafterdifferenttime-pointsasafunction

ofcontinuousoscillatoryshearstress(samedatasetasforFig.3j).l)Rapidandreversible

self-healingbytransitionbetweenliquidstateandsolidstate.Errorbars=onestandard

deviation.SamplesizesandstatisticaltestingdetailsinSupplementary14,items28-41.

Throughforcefulextrusionwethenfragmentedthehighlyporousbulkmaterial37into

irregularmicroparticleswithadiameterof805+/-363µm(Supplementary7).Thanks

totheintra-particleporosity,theporespaceintheresultingEPIbiomaterialdisplaysan

intricateandwidelyconnectedmorphology(Fig.3a).Thisuniqueporositycontrasts

starklywiththatofatraditionalmicrogelsuspensionlikethechromatographymedium

SephacrylS20038,whereporesarelimitedtotheintersticesbetweenthedensespherical

particles(Fig.3b).Thereforetheabundanceoflargefrictionalporespacesfulfilled

designrules#2and#4,whiletheexceptionalirregularityfulfilleddesignrule#1.Thus

weobtainedanelastic,porous,andinjectable(EPI)biomaterialthatsatisfiedourpre-

defineddesigncriteria.

Withthefabricationphasecompletewebeganextensivephysicalcharacterization.We

firstinvestigatedthepresenceofanelasticsofteningtransition,alongwithhighyield

strain,aspredictedbyoursimulation(Fig.2).Forthis,wesubjectedtheEPIbiomaterial

(atvariouslevelsofpolymerconcentration)toincreasingoscillatoryshearstress(Fig.

3c,Supplementary10).Weindeedobservedastableelasticplateau,auniquesoftening

transition,andyieldingathighstrain(62+/-5%)forawiderangeofpolymer


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concentrations,Supplementary10).Normalizationtothelow-strainplateauvalue(G0’)

39confirmsthatthesefeaturesareconservedfortheEPIbiomaterialacrossalltested

concentrations(Fig.3d).Asexpectedforanessentiallynon-frictional33micro-hydrogel

suspension(Supplementary5,ref.25),nodistinctsofteningbehaviorwasseenwiththe

SephacrylS200material(P=1.7*10-8fordifferenceat1%strain,item29Supplementary

14).Further,inagreementwithournumericalresultsforsphericalparticles,theyield

strainwassignificantlylower(24+/-6%,P=4.8*10-6vs.EPIbiomaterial,Supplementary

10anditem27inSupplementary14).Wethereforesucceededinengineeringanovel

biomaterialthatdisplaysnotonlyelasticbehaviorwithhighyielding,butalsoanew

softeningtransitionthatshouldenablebothshapingandtissuematching.

Ournextaimwasprecisemechanicalmatchingtoadiposetissue12,13,whileconservinga

poresizegreaterthan50μmrequiredforvascularization34,35.Fig.3edemonstrates

indeedaninverserelationbetweenG0’andtheaverageporediameter:Fluidwithdrawal

toincreasepolymerconcentrationsloweredbothtotalporefractionandaveragepore

size(Supplementary8).WiththeEPIbiomaterial,adiposetissuestiffnessG0’=2-

3kPa12,13wasmatchedwhilesuccessfullymaintainingaporesizeof100-120µm(Fig.

3e,Supplementary8and9).Onthecontrary,sufficientstiffnessintheSephacrylS200

materialcouldonlybeachievedatinsufficientporesize,andviceversa.TheEPI

biomaterialdesignthusspecificallyenablesthejointfulfillmentofbothmechanicaland

geometricrequirements.

Wefurtherassesseddynamicadiposetissuematchingbyrheologicalcomparisonofthe

EPIbiomaterialtothepublishedrheologicalresponseofadiposetissue(bovine,

retroorbital13).WefindthattheEPIbiomaterialdynamicallymimickstheoverall


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adiposetissueresponseoverawiderangeofdeformations(Fig.3f).Inaddition,theEPI

biomaterialshowsyieldingbehaviorsimilartotheestablisheddermalfillerJuvéderm

Voluma(Fig.3f).TheJuvédermVolumafillerisknowntoconsistofirregularlyshaped

microparticles40,confirmingthepredictedimportanceofthisfeatureforhighyield

strain(57+/-7%forJuvédermVoluma®,P=1.0vs.EPIbiomaterial,Supplementary10).

ThesofteningtransitionengineeredintotheEPIbiomaterialadditionallyoffersthe

unprecedenteddynamictissuematching.

Forminimallyinvasivedelivery,theEPIbiomaterialmustbeinjectable.Fig.3g(controls

inSupplementary11)showsthattheforcesrequiredforextrusionoftheEPIarewell

belowtheupperboundofapproximately20Nreportedforfacilemanualinjectabilityin

aclinicalsetting20.Fig.3hfurtherindicatesconservationofmaterialproperties

throughouttheinjectionprocess,astheuniaxialcompressionresponsewasidentical

after1or2injectioncyclescomparedtotheoriginalcompressionresponse.TheYoung

modulus(E’)wasconservedoversubsequentinjectioncycles(Fig.3i,P=1.0,item35in

Supplementary14),whereastheelasticstoragemodulusG’actuallyslightlyincreased

(Fig.3i,P=3.4*10-8,item36inSupplementary14).Thisincreasewaslikelydueto

dehydrationduringtransfersteps.Overall,weconcludethattheEPIbiomaterialcan

easilybeinjectedandretainsitsmechanicalpropertiesafterminimallyinvasivedelivery

throughacannula.

Finally,weinvestigatedthephysicalself-healingprocessenablingrestorationofsolid-

likemechanicalintegrityaftertheinjectionprocess.ForFig.3j,self-healingoftheEPI

biomaterialfollowingfluidizationwasassessedunderweak(0.1Pa)tostrong(50Pa)

continuousoscillatorystress.Self-healingwasefficientforallstresslevels,butdifferent


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time-scaleswereinvolved.Forsmallershearstresses,substantiallyhigherG’values

wereeventuallyachieved,butthefinalvaluewasreachedonatimescaleof10minutes

orabove(Fig.3j,Fig.3k,Supplementary12).Forstrongcontinuousshearstress,

relativelylowbutstableG’valueswererapidlyestablished.Usinganabruptswitchof

stressslightlyaboveandbelowyieldstress,wecouldindeeddemonstratenear-

instantaneousrecovery(Fig.3i,additionalcreep-recoverydatainSupplementary13).

Webelievethattheimmediateself-healingafterinjectioncouldstabilizetheinjected

implanttopreventunwantedspreading,followedbyattainmentofamuchhigherelastic

storagemodulustobettermatchthelocaladiposetissueatlongertimescales.

Insummary,wehaveengineeredanelastic,porous,andinjectable(EPI)meta-material

(Fig.1)accordingtothedesignspecificationsderivedfromclinicalcriteriaandinsilico

simulation(Fig.2).TheEPIbiomaterialdisplaysauniquetri-phasicrheology:Yield

stressenablesfacilemanualinjection,followedbyrapidinitialself-healingtoprevent

spreading.Thereversibleelasticsofteningtransitionthenprogressivelyrecoversan

elasticstoragemodulusof2-3kPa,inlinewiththatofthelocalsofttissue12,13.Thatsame

softeningtransitionenablestissue-matchingacrossawiderangeofappliedforces.

In-vivo

Afterthedesign,synthesis,andextensiveinvitrotestingofourengineeredmeta-

material,wetheninvestigatedinvivoperformanceoftheEPIbiomaterial.Withthe

ultimategoalofclinicalimplementation,wespecificallyexaminedminimally-invasive

delivery,insitushapeability,long-term3Dshapemaintenanceandtissueintegration.


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Fig.4.InVivoApplicationoftheEPIBiomaterial.a)Experimentalworkflowb)MRI

imagingoftheEPIimplantation,EPIlabeledinfalsecolor(centerofinjectedmaterialat

hairlinecrossing).Scalebars=1mm.c)A400μLbolusoftheEPIbiomaterialafter

minimally-invasiveinjection.d)EPIarbitrary3Dinsitushapingbyapplicationof

moderateexternalforces.e)Quantificationofthemaintenanceoftheshapebytheaspect

ratiooflengthalongtheinjectiondirectiontowidthoftheimplantforEPIandJuvéderm

Voluma®.f)HistologyoftheEPIafter21daysofimplantation,EPIscaffoldmaterialin

darkpurple(S).Towardstheleftlowercorner:endogeneousadiposetissue.Scalebar=500

µm.g)HistologyoftheEPIat6months.Scalebar=500µm.Notshaped,200μLbolus.h)

HistologyoftheEPIatdecreasedsynthesisconcentrationtoacceleratedegradation,at1

year.Exampleofasmallvesselshowninmagnifiedinset.Scalebar=100µm.Notshaped,

200μLbolus.i)Sameconditionas4h,highermagnificationimage.Degradingscaffold

denotedbystars,E=exampleofeosinophilicregion,H=exampleofregionstainedby


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hematoxylin.Images4hand4iwereadjustedregardingwhitebalanceandluminosityon

cell-freeareasfordirectcomparisonto4g.Errorbars=onestandarddeviation.

Vasculatureoutlinedbyarrows.StatisticaltestingdetailsandsampleinSupplementary14,

items43-46.

Forthis,wemanuallyinjectedEPIbiomaterialintothesubcutaneousspaceofCD1mice

andthenshapeditbyapplicationofgentleexternalforce(Fig.4a).Wefoundthat

fluidizationenabledfaciledeliverythrougha20Gcatheter.MagneticResonanceImaging

(MRI)confirmedthatthematerialbehavedasawell-defined,cohesiveimplantinthe

dermalspace(Fig.4b).

Duringatimewindowofabout20minutesfollowingbolusinjection,thematerialcould

bere-shapedinsitutoproduceanewdesiredshape,retainedspontaneously(Fig.4c,4d,

4e,VideoSupplementary22).Thisindicatesoursuccessinengineeringashapeable

materialthatprovidessubstantialliftingcapacityfor3Dsofttissuereconstruction.For

comparisonwealsoinjectedabolusofthehyaluronicaciddermalfiller(Juvéderm

Voluma®)butwereunabletoperforminsitushaping,sincethematerialwouldspread

ratherthanadoptanewshape.

Aftertheshapingtimewindow,theshapestabilizedandexcessiveforcewasrequiredto

dislodgethemechanicallyinterlockedparticles.Weassessedthelong-termstabilityby

attemptingtoreshapethe3Dvolume(bymassaging)everydayduringthefirstweek,

andthenonceaweekfortheremainderofthe3weekfollow-up.Despitethisgentle

mechanicalchallenge,theshaperemainedremarkablystable(Fig.4e).


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Onthetimescaleofhourstodays,initialstabilizationbymechanicalself-healingismost

likelyrelayedbyirreversiblebiologicalprocessessuchasfibrincoagulation41,and

progressivetissueingrowth(Fig.4f,21days).At6months(Fig.4g,notshaped),we

indeedfindmostoftheimplantporespacecolonized,withanonsetofscaffold

degradation.Wethenassessedbio-integrationatthescaleof1yearinFig.4hand4i

(reactionmixdiluted2:1withdeionizedwatertoenhanceourchancestoobservelate

biodegradationstages,notshaped).WefindareasofadvancedEPIbiomaterial

degradationatoneyear(starsinFig.4i).Afibrovasculartissue(pinkinFig.4hand4i,

examplelabeled“E”inFig.4i)neverthelesspersists,alongwithpresumablyphagocytic

cellsprocessingthedegradingmaterial(lightpurpleinFig.4handFig.4i,example

labeled“H”).Detailedquantificationoflong-termvolumestability,colonizationand

biocompatibilityoftheEPIbiomaterialincomparisonwithcommercialinjectablesisto

bepublishedelsewhere(A.B.,M.Genta,N.Kunz,P.B.,T.B.,inpreparation).

Takentogether,ourinvivoexperimentationdemonstratedthattheuniquerheological

andmorphologicalpropertiesoftheEPIbiomaterialtranslatedintoanovelcapacityto

inject,shape,andstabilizecustomized3Dtissuevolumesfortissueinduction.Giventhe

rangeofconditionsdrivingapathologicallossofsofttissuevolume,weproposethatour

newmeta-materialapproachcouldhaveamajorimpactonclinical3Dtissue

reconstruction.

Discussion

Wepresentthedesign,synthesis,andtestingofanin-vivo3Dshapetissueengineering

materialbaedonmeta-materialphysics.23,24Numericalsimulationallowedusto


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translatetheunmetclinicalneedofashapeablesofttissueimplant8,11,16intoasetof

engineer-ablemechanicalandgeometricparameters.Withtheelastic,porous,and

injectable(EPI)biomaterial,weachieveddesiredshape-stable,softening,andyielding

behaviors,alongwithsubstantialporosityandatissue-matchingmechanicalresponse.

Theresultwasexcellentin-vivoinjectability,shapeability,shapestabilizationaswellas

long-term3Dtissueintegration.

Inthisworkweengineeredandexploitedtheelasticsofteningtransitiontocombine

injectabilitywiththefullmatchingofadiposetissuerheologyoverawiderangeof

strains.Suchanapproachallowsforaminimally-invasivefillerwithunprecedentedin

vivo3Dlifting11andshapingcapacity.Strainsofteningiscommoninductilematerials

(likeplasticsormetalalloys)butitisgenerallyaccompaniedbylarge-scaleplastic

deformation42.Onthecontrary,reversiblestrainsofteningwithnegligibledeformation

israre,buthasbeendescribedpreviouslyinactinorcellulosehydrogels26,27.Thoughtto

becausedbyabucklingofconstituentfibers,thisresponseallowsthenetworkto

maintainrigidityatnearstaticconditionsbutsmoothlyandreversiblyrespondto

moderateforcesbycontrolleddeformationaftersoftening26.Reversiblestrainsoftening

inmicrogelsuspensionshasbeenconjectured25,43,hereweprovideexperimentalproof.

Taughtbythenumericalsimulation,thekeytothisachievementisthecombinationof

porosityandirregularparticleshape.Indeed,sphericalcryogelparticlesarereportedto

displayalowyieldstrainandcompleteabsenceofasofteningregime44,whiledermal

fillerswithirregularhyaluronicacidparticles40havehighyieldstrain8,butdonot

displayreversiblesoftening.


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19

OurfocusongeometricaldesignofthemicrostructurecharacterizestheEPIbiomaterial

asamechanicalmeta-material24.Hereweemployedcarboxymethylcellulose-based

scaffoldchemistryforitsknownbiocompatibility34andobtainedexcellentbio-

integrationupto1year.Nevertheless,ourmeta-materialdesignsuggeststhatother

scaffoldchemistriescouldbeusedtorealizesimilarmaterials.Thusinjectable,

shapeable,porousmaterialscouldbeappliedtofieldsasdiverseasboneengineering,

woundrepair,andregenerativeorganengineering.Bymodulatingthestiffness,

degradation,andbiologicalactivityorcelldelivery14,37,webelieveourapproachiswell

suitedforawiderangeofcustomizedtissueengineeringapplicationsbeyond3Dsoft

tissuereconstruction.

Acknowledgments

ParticularthanksgotoAleksandraFilippovaforhelpwithteamcoordinationandtask

schedulingandDanielLyubenovforinitialhelpwiththesimulation.Wewouldfurther

liketothankBenoitDesboillesforSEMimaging,NicolasKunzforMRIimaging,andSidi

Bencherifforteachingandinitialhelpwiththecryogeltechnology,MartinaGentaand

MarianaMartinsforhelpwithanimalexperiments,andProf.PaulBowenforlettingus

usehisrheometer.Wealsoacknowledgethesupportbythefollowingfacilitiesandtheir

staff:microscopicimagingfacilitiesoftheUniversityofGeneva(BioimagingCoreFacility

oftheFacultyofMedicine)andEPFLLausanne(BioimagingandOpticsPlatform),the

histologycorefacilityatEPFLandparticularlyJessicaSordet-Dessimoz,theCenterof

MicronanotechnologyCMIandtheCIBMfacilityatEPFL,includingitsanimalfacility.

ThesimulationswereperformedontheBaobabClusteroftheUniversityofGeneva.


https://doi.org/10.1101/2020.01.30.926931

20

FundingforthisworkwasprovidedbytheSwissNationalScienceFoundation

(PP00P2_163684andPZ00P2_161347),theGebert-Rüffoundation(GRS-0043/15),and

aWhitakerInternationalFellowship.

Author contributions

A.Béduer,F.Bonini,C.VerheyenandT.Braschlercontributedtothedesignofthisstudy.

Allauthorscontributedtothewritingandproofingofthemanuscript.A.Béduercarried

outandanalyzedthein-vivostudy.F.Boniniprovidedthemorphologicalbiomaterial

characterizationandthedesignofthefiguresinthemanuscript.C.Verheyenperformed

themechanicalcharacterization,andT.Braschlerwrotethesoftware,ranandanalyzed

thesimulations.P.Burchdevelopedandoptimizedbiomaterialfabrication.

Competing interests

A.BéduerandT.BraschlerdeclarefinancialinterestinVolumina-MedicalSA,

Switzerland,P.BurchandA.BéduerarenowemployeesofVolumina-MedicalSA.The

otherauthorsdeclarenoconflictofinterest.

Data availability

Figures2,3and4haveassociatedquantitativerawdata.Therawdataandthescriptsto

producethequantitativefiguresareprovidedattheZenodorepository


https://doi.org/10.1101/2020.01.30.926931

21

(https://zenodo.org/record/2653804#.XNBXFIS7Ldk).Qualitativerawdata(histology,

MRI)isavailablefromtheauthorsuponrequest.

Code availability

Thefullsimulationsourcecodeisavailablefordownload(Supplementary15).Detailsof

themathematicalmodel(Supplementary1),fullusageinstructions(Supplementary2),

implementationdetailsincludingpseudocode(Supplementary3),anautomatically

generatedfullAPIdocumentation(Supplementary16)andunittestcases

(Supplementary4)arealsoprovided.Aquickinstallationguideisavailablein

Supplementary17.ThesourcecodeisalsoincludedintotheZenodorepository

(https://zenodo.org/record/2653804#.XNBXFIS7Ldk)forappropriateversioning.

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40 Ohrlund,J.A.&Edsman,K.L.TheMythofthe"Biphasic"HyaluronicAcidFiller.DermatolSurg41Suppl1,S358-364,doi:10.1097/DSS.0000000000000545(2015).

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Online Methods

Simulation

ThesimulationwasimplementedasacustomPythonlibrary.Itimplementsthephysical

interactionframeworkdefinedbyOtsukiatal.25,correspondingtocentralviscoelastic

interactionandtangentialfrictionbetweensphericalparticles.Toensureexactrather

thanapproximateconservationofangularmomentum,weadjustedtheexpressionof

thetorqueresultingfromfrictionalinteraction(Supplementary1,eq.S1-3).

Tocorrectlyevaluatestresstensorsatlargeamplitudes,weimplementedthestress

tensorevaluationframeworkprovidedbyNicotetal.29.Asunittesting(Supplementary

4)revealednon-symmetricstresstensorsinsomecases,were-evaluatedthe

calculationsbyNicotetal.29.Wefoundaprobableintegrationmistakeregardingthe

inertiamatrixforthecontributionofunbalancedtorquestothestresstensor

(Supplementary1).Thisdirectlyconcernseq.29inref.29,andasresultalsoeq.30-35in

ref.29;weuseacorrectedversionofeq.34inNicotetal.29,suppliedaseq.S1-24in

Supplementary1.Toavoidcompleteinterpenetrationofneighboringparticlesatlarge

shear,weaddedanon-lineartermtotherepulsiveelasticinteraction.Weensuredthat

inthesmallcompressionlimit,werecoverthelinearrepulsionlawusedbyOtsukiet

al.25(Supplementary1,eq.S5b).

Finally,wealsoimplementthepossibilityofpermanentcrosslinksbetweenneighboring

spheres.Thepermanentcrosslinksremainintactregardlessofthegeometrical


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separationofthespheres,generatingattractiveforcesifthespheresareseparated

beyondtouchingdistance.Theyalsodonotallowforfrictionalslippage.Additional

mathematicaldetailsoftheimplementationaregiveninSupplementary1.Thesource

codecanbedownloadedasSupplementary15;softwareusage,implementationdetails

andtestcasesareprovidedrespectivelyinSupplementary2,3and4.ThefullAPI

documentationisavailableasSupplementary16,andquickinstallinstructionsas

Supplementary17,simulationvideosareprovidedasSupplementary18–

Supplementary21.

ThesimulationswererunontheBaobabclusteroftheUniversityofGeneva.

Instructionsonhowtoreplicateoursimulationsanddataanalysisareprovidedin

Supplementary2.

Statistics

StatisticalevaluationandgraphingweredoneusingtheR-Cranfreesoftware,version

3.2.345.Centraltendencywasreportedbyarithmeticmeanvalues,andvariabilityby

indicationofsinglestandarddeviations.Forcomparisonswith5orlessmeasurements

pergroup,individualvalueswerealsographed(Fig.3i,Fig.4e).Linearregressionwas

usedtoassesscontinuouseffects,andpairedorunpaired,Student(t)testswereusedto

compareindividualconditions,withP-valuesreportedafterBonferronimultipletesting

correction46foralltestsperformedpersubfigure.Foralltests,theShapiro-Wilkstest

wasusedtoassessnormalityoftheresiduals.Ifsignificantdeviationwasfound,an

alternativetestwasevaluated:generalizedlinearmodelswithidentitylinkbut

expection-valuedependentvariance(quasi-likelihoodestimators47)toaccommodate

heteroscedasticityinlinearregression,andWilcoxonsignedrank(paired)orMann-

WhitneyU(unpaired)forbinarycomparison.Alltestsperformedarereportedin

Supplementary14,includingtheireffectsizesandnormalitytestingresults.


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Forthestatisticalevaluationofthecharacteristicstrainsandstressesofthesimulated

elasticstoragemodulusandviscouslossmoduluscurves(G’andG’’)asafunctionofthe

modelparameters(Fig.2G)abootstrappingapproachwastaken48.Foragivensetof

modelparameters,5subsetsamongthesimulationscarriedoutforthevariousstrain

amplitudesaredrawnrandomlywitha20%probabilityforagivensimulationtobe

included.Foreachsubset,slightlydifferentestimationsforcharacteristicstressesand

strainsasdefinedinFig.2farethusobtained.Bylinearregressionofthesevalues

againstthephysicalparameterbeingvaried,theP-valuesdepictedinFig.2gare

obtainedafterBonferronicorrection46.ToreducedependenceoftheP-valueonthe

randomdraw,weaveragedtheunderlyingF-statisticsover500bootstrappingruns48.

Reagents

1,4-Piperazinediethanesulfonicacid(PIPES),adipicacidanhydride(ADH),1-ethyl-3-(-

(3-dimethylaminopropyl)carbodiimide(EDC),rhodamine6Ghydrochloride,4′,6-

diamidino-2-phenylindole(DAPI),6-aminofluorescein,37%HClsolution,NaOHpellets

andSephacrylS200HRwereallpurchasedfromSigma-Aldrich.Carboxymethyl

cellulose(AQUALONCMC7LFPH,90.5KDa,DS:0.84)waspurchasedfromAshland.

EDTASolution(0.5M,pH8.0)wasobtainedfromThermoFisherScientific.Saline

solution(0.9%NaCl)waspurchasedfromBichsel.Disposablesterilevacuumfiltration

systems(poresize0.2um,filtercapacity1000mL)werepurchasedfromSigma-Aldrich,

whereascellstrainers(40µm)wereobtainedfromVWR.

Biomaterialsynthesis


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Cryogelscaffoldsweresynthesizedasreported37,withminormodifications.Briefly,

carboxymethylcellulose(13.56g),PIPESBuffer(6.30g),adipicacidanhydride(486mg)

andNaOHpellets(1.20g)wasdissolvedtoafinalvolumeof1000mLindeionizedwater

andthesolutionfilteredat0.2µm.Thissolutioncouldbestoredina4°Cforuptoa

monthpriortouseinafridge.ThenEDC(2.70g)wasadded.Aftermixing,thesolution

wasfilledinto35mLplasticsyringes.Thesyringeswereclosedwithasyringecapand

thenplacedintoafreezersetto-20oC.

After2daysthesyringeswereremovedfromthefreezerandallowedtoreachroom

temperature.TheEPIbiomaterialwasobtainedbyforcefulextrusionthrougha22G

catheter.Onafiltersystem,thebiomaterialwaswashedwith10mMEDTA,followedby

incubationwith2MNaOHsolution(2h),washingwithphysiologicalsaline(3x).Forin-

vivoexperimentsthebiomaterialwassterilizedfor20minat121°C.

Microscopicimaging

ConfocalimageswerecapturedonaconfocalZeissLSM700drivenbyZen2010b

versionservicepack1(Zeiss),usinga10xPlanNeofluarlenswithanumericalaperture

(NA)of0.3ora20xPlanApochromatobjectivewithNA=0.8.Alternatively,wealsoused

aZeissLSM800drivenbyZEN2.3withZENmoduleTiles(Zeiss),alsousingeither10x

PlanApochromatlenswithNA=0.45ora20xPlanApochromatwithNA=0.8.Imagesof

histologicalslideswereacquiredwithaLeicaDM750,withachromatlensesof10x(HI

PLAN,NA=0.25),20x(HIPLAN,NA=0.4)or40xmagnification(HIPLAN,NA=0.65),with

imagestoragedirectlytoanSDcardbyaLeicaICC50HDCamerawithoutadditional

software.Table1belowdetailsthemicroscopesetupusedforeachexperimentorfigure.


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Figure Microscope/Objective Adjustments

1f ZeissLSM700,10x Noadjustment

3a ZeissLSM700,10x Noadjustment

3b ZeissLSM800,20x Noadjustment

4f LeicaDM750,10x Noadjustment

4g LeicaDM750,10x Noadjustment

4h LeicaDM750,20x Adjustmentofwhitebalance

andluminosityoncell-freeareas

tomatchFig.4g

4i LeicaDM750,40x Adjustmentofwhitebalance

andluminosityoncell-freeareas

tomatchFig.4g

Table1.Microscopicimaging.Summarylistofthemicroscopesandobjectivesused.

Furtherdetailsonthemicroscopesandlensesinthetext.

Forvisualizationandmorphologicalquantification,theEPIbiomaterialwasstainedwith

5microgram/mLRhodamine6Ghydrochloride(Fig.1and3a)or4′,6-diamidino-2-

phenylindole(DAPI,byaffinitytothefinalmaterial)or6-aminofluorescein(byinclusion

of10micromolaraminofluoresceinintothesynthesismixture37),orDAPIand

aminofluorescein(Fig.1).Forthereferencematerial,SephacrylS200,auto-fluorescence

images(excitation353nm,emission405nmandabove)wasusedforvisualizationand

quantification.

ScanningelectronmicroscopeSEMimageswerefinallyacquiredwithaZeissMerlin

SEM,equippedwithaGeminiIIcolumnandtheZeissSmartSEMacquisitionsoftware.


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Poresizeandporefraction

Afterstaining(fortheEPIbiomaterial)andconfocalimageacquisition,Fijisoftware49

(ImageJversion2.0.0-rc-44/1.50g)wasusedtovisualizez-stackimages,andtoquantify

poresizeandfractionaswellasparticlesize.Forporefractionquantification,images

wereacquiredataresolutionsothatseveralporesfitacrosstheimage,implying

generallytheuseofa10xobjectivefortheEPIbiomaterialvs.a20xobjectiveforthe

SephacrylS200controlmaterial.TheEPIbiomaterialwasstainedwithrhodamine6G

(excitedat550nm)whereasautofluorescenceimages(excitedat355nm,)were

acquiredfortheSephacrylS200reference.Theimageswerethresholdedautomatically

usingtheLialgorithm50,built-ininFiji.Incaseofevidentgrossmisinterpretationofthe

imagesbythealgorithm,thresholdingwascarriedoutmanuallyinstead.Thewalland

porefractioncouldthenbeestimatedfromthefractionofwhiteandblackpixels;for

poresizeestimation,themaximalspherefittingalgorithmbyBeatMünchetal.51,52was

used(Supplementary8).Foreachpolymerconcentrationandmaterial,between20and

34imageswerequantified.

Particlesizedistribution(Supplementary7)finallywasevaluatedfromlargetile-

stitchedimagesacquiredondiluteparticlesuspensionsusingaZeissLSM800witha5x

PlanApochromatobjective,NA=0.16,afterstainingwithrhodamine6Gasforporesize.

StandardFiji49routineswerethenusedontheresultinglargeimages:binarizationby

manualthresholding,digitalfillingofholes,andparticlesizeanalysis(minimalsize10

micrometers2,circularityatleast0.1).

Forallconfocalimaging,observationchambersavoidingbothevaporationand

mechanicalcompressionwereused.Thesechambersconsistedofa1.5mmhighPerspex


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sheet(Evonik)cuttotheshapeofamicroscopeslide(75mmx25mm)usingalaser

cutter(HobbyLaser,FullSpectrumEngineering,commandeeredbyFullSpectrumLaser

RetinaEngrave3D,Version4.423).Arectangularsamplereservoir(30mmx12.5mm)

wasfurthercutintothesePerspexmicroscopeslides.Amicroscopecoverslidewasglued

permanentlytothelowersideofthePerspexmicroscopeslideusingbathroomsilicone

glue(CoopBricoLoisir,Switzerland).Afterfillingwiththesampletobeobservedby

confocalmicroscopy,theobservationchamberwasclosedbycapillarityusingasecond

microscopecoverslide.

Rheology

RheologicalmeasurementswerecarriedoutonaHaakeRheostressRS1005Ncm

apparatus,usingtheRheoWinsoftware(RheoWinJobManager:version3.61.0005)to

controltheapparatusandacquiredata.Toavoidwallslipping,roughenedsurfaceswere

obtainedbygluingaroughcleaningcloth(Miobrill,MigrosSwitzerland,ref.7065.206/

15.02.2330)withhotglue(UHU,LT110,localhardwarestore)tothesurfacesincontact

withthematerial.Experimentsweregenerallyconductedinacustomcupgeometry

(Supplementary10),exceptforwherethesamplevolumewastoolow(Juvéderm

VolumasampleforFig.3fandtheinjectedsamplesforFig.3i,measuredwithplate-plate

geometry:HaakePP20,ref.222-0586).Inaddition,wemeasuredrepeatedsolid-liquid

transition(Fig.3l)withavanegeometry(HaakeFL-16,ref.222-1326)tolimitsample

movement.Furtherdetailsandcomparisonofthedifferentgeometriesaregivenin

Supplementary10.Asolventtrap(Haakeref.222-0607)wasusedwheneverpossibleto

limitsampleevaporation.


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Withtheexceptionoftheself-healingexperiments,wegenerallypreconditionedthe

materialwithanoscillatorystresssweepfrom1-10Paandbackat0.2Hzbefore

applyingthedesiredshearprotocolstominimizetheimpactofshearhistory.Inrare

casesofverydilutematerial,wefoundthistoinducesignificantshearsofteningoreven

yielding,inwhichcasewechangedtoapreconditioningsweepfrom0.1Pato1Paand

back,stillat0.2Hz.Afterdataacquisition,weexportedthedatatotextfiles(RheoWin

DataManger:version3.61.0005)andcompleteddatatreatmentandgraphinginR-Cran

(version3.2.3).

RheologicalmastercurvesrepresenttheelasticmodulusG’normalizedtotheplateau

valueatlowstress39.TheyareobtainedbynormalizingG’curveswithrespecttothe

low-strainG0’plateauvalue39.Weevaluatethelow-strainlimitG0’astheaverageofthe

G’valuesforthemeasurementpointswithlowappliedshearstress(τ<2Pa).Wefurther

takecaretoexcludepointsshowingalreadyanonsetofsofteningorliquefactionbyalso

imposingG’’(τ)<0.1*G’(τ).WealsonormalizetheappliedstressτtoG0’.Aftervisual

verificationthatindeedallthedifferentG’curvesobtainedatdifferentpolymer

concentrationscollapseontoasinglemastercurveafternormalizationofboththeG’and

τ,weobtainthemainmastercurvebyaveragingofthenormalizedcurves,alongwith

evaluationofthestandarddeviation.Forthemorehorizontalpartofthemastercurve

(G’/G0’>0.5fortheSephacrylS200mastercurve,G’/G0’>0.1fortheEPIbiomaterial)we

performverticalaveragingoftheG’/G0’associatedwithagivenintervalofappliedstress

τ,whereasforthesteepestpartofthemastercurves,weratheraveragetheτassociated

withagivenintervalofG’/G0’values.

Uniaxialcompressionandinjectabilitytesting


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WeperformeduniaxialcompressiontestingonaTextureAnalyzerTA.XTplusmachine

byStableMicrosystems,usingtheTestMakerprogram(Version4.0.6.0)suppliedbythe

manufacturertoprogramthetests,andtheTextureExponent32program(Version

4.0.13.0),alsosuppliedbythemanufacturer,torunthem.Afteracquisition,thedatawas

exportedastextandanalyzedusingR-Cran.

Forcompressionanalysisoftheelasticporousinjectable(EPI)biomaterial,adiskof

20mmdiameterandapproximately6mmheightwasshapedundera20mmdiameter

chuck.Thechuckwasthenmovedbyfeedbacktothezeroforcecondition.Thisdefined

theoriginalsampleheight,usuallyintherangebetween5and6mmdependingonthe

actualamountofsample.Fromthisposition,compressionby50%oftheheightata

speedof0.01mm/swasthencarriedout,acquiringtheforceandpositiondata.The

forcewaslow-passfilteredtoreducenoice,andwasthenconvertedtostressbydividing

throughthecontactarea(circleof10mmradius),whereasdeformationwasexpressed

asdeformationstrainrelativetooriginalsampleheight.

Forinjectabilityanalysis,a1mLsyringe(BD)wasloadedwithtestmaterial(EPI

biomaterial,deionizedwater,orair),andequippedwithabluntdeliverycannula

(ThiebaudBiomedicaldevices,ref.F9020100,outerdiameter2mm,length10cm).The

syringewasthenplacedoncustomholder.Thepistonwasthenmovedbyusingthe

TextureAnalyzerXTPlusmachineatthedesiredrate,whilerecordingforceand

distance.

Animalexperiments


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Allin-vivoexperimentswereapprovedbytheAnimalCareandUseCommitteeofthe

CantonofVaud,Switzerland(AuthorizationVD3063).Female,adultCD1micebetween

12and20weeksofagewereobtainedfromCharlesRiver(BarHarbor,Maine,USA)and

allowedtoacclimatizeintheanimalfacilityforatleast1weekpriortoimplantation.

Roomtemperaturewaskeptat22+/-2°Cwith12hourslight/darkcycleandnormaldiet

adlibitum.

Priortoinjection,animalswereanesthetizedwith4%(2%formaintenance)isoflurane

(AnimalcareLtd),usinganophthalmicgel(Viscotears,Alcon)foreyeprotection.The

areaforinjectionwasshavedanddisinfectedwithbetadine(MundipharmaMedical

Company).Forinjection,asmallaccesswascreatedintheskinwitha18Gneedle,

followedbyinjectionofbiomaterialsamples(2injectionsitesperanimal,ormax.400

μLintosinglesite)througha20Gcatheter(BDBiosciences).Nosutureswererequired.

Animalsweremonitoredweeklythroughoutthestudy.

WhenshapingwasfirstattemptedwiththeJuvédermVoluma®,thematerialwasfound

toflowandredistributeduringtheprocedure,andduringfollow-up,weobservedlarge

swellingresponsesoverthefollowingweeks.Toavoidunnecessarystrainonthe

animals,wethereforelimitedthemaximuminjectionvolumeforJuvédermVoluma®to

200μLandrefrainedfromshaping.Theswellingresponseandsomespontaneous

redistributionstilltookplace,butthisallowedustofollowtheaspectratioovertime

despitetheseunexpectedexperimentaldifficulties.

Atpre-definedtimepointsthemacroscopicdimensionsoftheimplantswereassessed

withacaliper(day0,after3days,after3weeks).Forhistologicalevaluation(at3weeks

forshapedsamples,at6monthsand1yearforunshaped200μLsamples),micewere


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34

sacrificedbyintraperitonealinjectionofsodiumpentobarbital(150mg/kg),followedby

transcardialperfusionwith4%paraformaldehydeinPBS.Harvestedsampleswere

furtherimmersedfor24hoursin4%paraformaldehydeat4°C,washed3xwithPBSand

embeddedinparaffinfollowingroutineprocedures.4µmsliceswerestainedwith

hematoxylin/eosininanautomatedslideprocessor(Prismaspecialstainer,Tissue-Tek;

GlasG2coverslipperfromSakura).

Magneticresonanceimaging

Magneticresonanceimaging(MRI)imageswereacquiredat3dayspost-implantationon

aVarianINOVAconsolewith14.1Tmagnetof26cmhorizontalbore(MagnexScientific,

Abingdon,UK)andagradientcoil(maximum400mT/m)withafixrisetimeof120ms.

Ahome-builtsingleloopsurfacecoilof24mmofdiameterwasusedasatransceiver

positionedontopofthetissuegraft.

Duringmeasurements,eachadultmousewasanesthetizedwith1.5–2%isofluranein

mixtureofairandO2(50/50%)andsubsequentlyplacedsupinewithinanadapted

holder.Bodytemperaturewasmaintainedat37°Cusingathermoregulatedwater

circuit.Monitoringrespirationwitharespirationcushionwasusedfortriggeringduring

allacquisitions.

Spinechointensityimageswereacquiredwitharepetitiontimeof1.5sandechotimes

at14msandagainat25ms(theoverallintensityofthissecondechoisusedforthe

imagesshown).Theacquisitionmatrixwas96×192pixelsforafieldofviewof15×25

mm,with46slicesspacedby0.05mm.Theacquisitiontimewasonaverage12min,with

minordifferencesduetorespiratorygating.

Replication


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35

Unlessexplicitlystatedotherwise,replicatedexperimentswereperformedondistinctly

preparedbiomaterialsamples.Wefurtherusedtwo2separate1Lsynthesisbatches.

Thusthesamplescontainvariabilityassociatedwithsamplepreparation,andtosome

extentchemicalsynthesis.Weusedhoweveridenticalreagentstocks,andsocannot

guardagainstlot-to-lotvariationofthechemicalsobtainedcommercially.

Supplementary10furthercomparesrheologicalcharacterizationintwodistinct

geometries.

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An injectable meta-biomaterialmassaging8,28, the EPI biomaterial behaves as a weak viscoelastic solid amenable to smooth deformation (Fig. 1b). At rest, the self-healing process is