Theextracellular matrix(ECM)playsanimportant regulatory roleinthedevelopment andprogression oftumoral tissue.Itsfunctions andproperties arecrucialindetermining tumor cellbehavior suchasinvasion, migration, andmalignancy development. OurstudyexplorestheroleofcollagentypeIin cancerdevelopment andspreadusingengineered tumormodelslike multicellular spheroids grownincollagen-based hydrogels to simulateearlytumorformation. Weemploymicrofluidic techniques totestthehypothesis that(i)addingLaponitenanoclaytocollagen hydrogels modifiesmechanical andrheological properties and(ii) changing thestiffnessofthecollagenmicroenvironment affects tumorspheroid growth.Ourfindingssupportourtheoriesand suggesttheuseofECMcomponents andengineered tumormodels incancerresearch, offeringabiocompatible andbiomimetic methodtotailorthemechanical properties ofconventional collagen hydrogels. 1.INTRODUCTION Asthemoststudieddisease,cancerstillremainsaleading causeofdeathworldwide. Therefore, furtherresearchisstill crucialtoimprovetheprevention, detection, andtreatment of allcancertypes.Extracellular matrix(ECM)components are knowntoplaysignificant regulatory rolesinthetumoraltissue. Thefunctions oftheECManditsintrinsicfeaturesarekeyin cancertissueby,forexample, triggering tumorcellbehaviors andinvasion,1cellmigration,2andmalignancy development.3 Theimportance ofthetumormicroenvironment hasbeen recognized inrecentyears,4astheinteraction betweenthe biochemical-physical cuesofthetumoranditssurroundings determine cancerpathway signaling. Besides, thismicro- environment iscrucialtounderstand tumorgrowthmecha- nismsand,therefore, isoneofthemainparameters involvedin cancertreatment research.5,6Collagen typeIisthemain component ofthetumoralECM,beinganaturalscaffoldofthe cancercellmicroenvironment.7Thus,changesintheamount ofcollageninthehosttissuecouldplayasignificant rolein promoting thesurvivalandgrowthofdistantmetastases and maysupportsomezone-specific tumorspread.8 Engineered tumormodelshaveprovedtobeusefultoolsin cancerresearch.9Tomimicthemorphological andfunctional featuresofinvivocancertissue,multicellular tumorspheroids aregrownandusedasaninvitromodeltomimicthefirst stagesoftumorformation invivo.10Collagen-based hydrogels arewidelyusedintissueengineering tomimicthe3D physiological microenvironment.11Additionally, collagen allowsthefabrication ofmatriceswithdifferent mechanicalproperties basedontheirinherentcomposition andprepara- tionprocedures.12 Microfluidics enablesminiaturization ofearlystagedtumor growth.13,14Theuseofmicrofluidics intumoralspheroid research constrains thesystemtoasubmillimetric scale, obtaining alargereduction incellnumberrequirements, reagentvolumes, wastes,andworkspace.Besides,itallows morecontroloverthemicroenvironment andpromotion of nutrientandbiochemical diffusionthroughthehydrogel. Thus, cellularculturesembedded incollagen-based hydrogels in microdevices providecellswitha3Denvironment thatcan bettermimicphysiological conditions compared toaconven- tional2Dmonolayer culture,whiletakingadvantage ofthe aforementioned strengths ofmicrofluidics. Laponite nanoclay (LapNC) isalayeredsynthetic silicate composed ofinorganic salts.Thesenanoclays arecharacterized bytheempirical formula: Na (SiMgLi)O(OH)0.7 8 5.5 0.3 20 40.7 [ ]+ (BYKAdditives Ltd.,UK).Gaharwar etal.thoroughly reviewed thebiomedical applications ofLapNC, among others:drugdelivery,tissueengineering, imaging,celladhesion andproliferation, andbiosensors.15Theirhydrophilic charac- Received: March13,2023 Revised: May9,2023 Published: May30,2023 Article pubs.acs.org/Biomac ©2023TheAuthors.Published by American Chemical Society 2879https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−2891 Downloaded via 88.5.1.11 on June 20, 2023 at 19:23:12 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. teristicsandlargesurfacearea(>350m2/g)16facilitatephysical interactions suchasionicinteractions17orhydrogen bonds18 withdifferentmolecules andproteins. Therefore, theyhave beenwidelyinvestigated inthefieldsofregenerative medicine, additivemanufacturing, anddrugdelivery. Individual clays havenanodisk-shaped geometry of20−50nmdiameter and 1−2nmthickness (Figure1).Owingtoitscomposition and size,thenanoclay exhibitsadualchargedistribution: negative onthesurfaceandpositivealongtheedges.Thisproperty makesnanoclay dilutioninliquidmediaauniquegel-like house-of-cards structure whichcanbeimplemented indifferent materials andbiomaterials toenhancestructure, mechanical properties, andgeneralcharacteristics.19 Thisworkwasbuiltupontwohypotheses. First,theaddition ofLapNC tocollagen hydrogels wouldenhance the mechanical properties withoutcollapsing thecollagenmatrix orformingclayaggregations. Thiscouldinvolvethepossibility ofcustomizing themechanical andarchitectural properties of thetumoral invitroECM.Second,LapNCadditioninthe3D collagen microenvironment wouldaffecttumorspheroid growth.Insuchcase,theoutcomes fromourstudywould demonstrate thatcollagen-LapNC hydrogels mayprovidea new3Dcancerculturesystemthatmimicskeyaspectsofthe physiological tumormicroenvironment. Themaingoalofourstudyistoprovide anew biocompatible andbiomimetic methodtoeasilytailorthe mechanical properties ofconvectional collagen-based hydro- gels.Forthispurpose, hydrogels withdifferent collagen concentrations weremodified withLapNCandcharacterized intermsofphysicalandrheological properties. Finally,those matriceswereusedtostudythehomotypic spheroid growth overtime.Neuroblastoma spheroid growthwasstudied according toitssizeandshapeovertimeunderthedifferent microenvironments insidethemicrofluidic device.Toreinforce theconclusions oftheseexperiments, othertumoralcelllines weretested:humanepithelial lungandpancreas carcinoma lines.Eventhoughthestudyofaninvitrocancermodelisout ofthescopeofthiswork,tothebestofourknowledge, thisis thefirstreportontheuseofLapNCtopromote tumoral spheroidgrowthfromasinglecell.2.MATERIALS ANDMETHODS 2.1.Experimental Setup.LapNCwasusedtoenhance mechanical properties ofcollagen-based hydrogels atthreedifferent collagen concentrations (2.5,4,and6mg/mL, respectively). Supplemented andnonsupplemented controlhydrogels were fabricated foralltheprocedures hereinexplained. Asinglechannel microfluidic devicewasusedasaculturesystemtoconductthe15day 3Dcellcultureincubation, whichallowslivetrackingmicroscopy. The experimental setupissummarized inFigure1. 2.2.Laponite Dispersion. LapNC(BYKAdditives Ltd.,UK) powderwas1%w:vdilutedinMilli-Qwater.Thissolutionwas subsequently dispersed inculturemedium1/10tobeapplicable for cellculture,makinga0.1%w:vLapNCconcentration inmedium.The concentrations topreparestocksolutionandtheworkingsolution wereoptimized toavoidclayprecipitates andcollagencollapse.Ithas alreadybeenreportedthatLapNCaretotallycytocompatible atdoses <1mg/mL.20,21Thus,theLapNCconcentration usedinthisworkdid nothindercellularviability. 2.3.TEMInspection. Transmission electronmicroscopy (TEM) wasusedtoimagetheLaponite dilutions andcheckhomogeneous LapNCdispersion (Figure2AandB).A20μLdropofeachsample waspouredonafreshlyglow-discharged (30s,15mA)carbon-coated 200-mesh coppergrid(AgarScientific Supplies,UK).Then,thegrids wereincubated for2min,andtheexcesswasremovedbycontacting thegridedgewithfilterpaperandallowedtoair-dry.Inorderto checkthesizeandmorphology ofnanoclays, theobservation was conducted usingTecnaiT20(FEI,OR,US)microscope at200kV (Laboratory ofAdvanced Microscopy (LMA), University of Zaragoza). 2.4.Hydrogel Fabrication. Collagen hydrogels, withafinal collagenconcentration of2.5,4,and6mg/mL,wereprepared by dilutinginanicebathcollagentypeIsolution(RatTail,stock10.8 mg/mL,Corning, NY,US)inDulbecco’s Modified EagleMedium (DMEM, 4.5g/Lglucose,ThermoFisherScientific, MA,US),10× Dulbecco’s phosphate bufferedsaline(DPBS), and0.5MNaOH (bothfromSigma-Aldrich, Germany) toadjustthepHto7.4−7.6. Supplemented hydrogels werefabricated dilutingthecollagenin0.1% LapNC-DMEM (working solution). Forcharacterization assays,50μLdropsofthehydrogels werelaid inwells(96wellplate).Toconductpermeability andspheroidgrowth tracking,thehydrogels weregentlypipettedinsidethecentralculture chamberofamicrofluidic device(seesubsection 2.5.4,andFigure1). Afterplacement intherespective container, collagensolutions gelled for20mininahumidchamberat37°C.Thetemperature andthepH conditions mentioned aboveinduced aself-assembled gelation Figure1.Experimental setup:Laponite nanoclay wassuppliedinpowderform.Itwasdilutedat1%w:vinMilli-Qwaterand,subsequently, in culturemediaat1/10proportion. Thisworkingsolutionwasusedtofabricate thesupplemented collagenhydrogels atdifferent collagen concentrations. 3Dtumorcellcultureswereincubated inmicrofluidics, andthespheroids wereformedfromsingle-cell proliferation andcell−cell contact.Thehydrogels werecharacterized byscanning electronmicroscopy, andintermsofswellingratioandrheology gelledinwells. Additionally, permeability wascharacterized insidethemicrofluidic platform.Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912880 processofthecollagenhydrogels, wherecollagenfibersarephysically cross-linked.22Duringthegelationprocess,collagenfiberscreatean interpenetrating polymernetworkembedding theresuspended cells.23 2.5.Hydrogel Characterization. 2.5.1.SEMInspection and EDX.First,sampleswerefixedin4%paraformaldehyde for15min. Then,theywererinsedthreetimesinphosphate bufferedsaline (PBS)for5min.Afterthefixationsteps,thesamplesweresubjected tosequential dehydration ingradedseriesofethanol.Subsequently,theycarriedatotalwateradsorption procedure bysubmitting themto acriticalpointdryingstage(LeicaEMCPD300CriticalPointDryer, Germany). Finally,thesampleswerecoatedwitha20nmcarbonfilm beforetheywereexamined byscanning electronmicroscopy (SEM) andenergydispersive X-rayspectroscopy (EDX).Then,sampleswere visualized at10kVand3spotsize,usingafieldemissionSEMInspect F50(FEI,OR,US)inanenergyrangebetween0and30keV.To confirmthechemicalcomposition ofthesamples,EDXwasalsousing Figure2.(A)TEMimagesof1%LapNCstocksolutionand(B)0.1%LapNCworkingsolution.(C)Representative SEMimagesatat25,000× and50,000×magnification ofthecollagenhydrogels (2.5,4,and6mg/mL) with/without LapNC.(D)Representative EDXreportfiguresat 100,000× ofa6mg/mLcollagenhydrogelwith/without LapNC.Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912881 theInspectF50at20kVand4spotsize(LMA,University of Zaragoza). 2.5.2.Rheology. Thegelationprocessofthehydrogels hasbeen analyzedbymeansoflinearoscillatory rheologyusingacontrolstress HAAKERheoStress 1(Thermo FisherScientific, Waltham, MA,US). Therheometer wasconnected toanexternalthermostatic bath.The measurements werecarriedoutusingacone−plate sensorwitha35 mmradiusand1°angle.Oncethehydrogel wasprepared, itwas placedinthelowerplateat20°C.Thentheupperconewaslowered tothemeasurement position,andthetemperature isgradually raised to37°C.Oncethesamplehasbeenequilibrated atthistemperature, thehydrogelgelationwasanalyzedbyapplyingaverylowstressofγ= 0.45Paatafrequency of0.1Hzwithacyclicstrainof0.5%amplitude, whichguarantees atorqueof5μNmandalinearviscoelastic regime. Tomitigatetheevaporation ofwaterfromthehydrogel, aliquidtrap wasused.Storagemoduli(G′)wererecorded asafunctionoftime usingHAAKERheowin software(JobandDataManager). Three sampleswereusedforeachtestcondition. 2.5.3.Swelling Ratio.Theswellingratio(SR)ofthedifferent hydrogels wasmeasured fromthedrymass(Md)andwetmass(Mw) ofthehydrogels following eq1.Mdwasobtained byweighting lyophilized hydrogels (RADWAG-MYA5.4Y microbalance, 1μg readability). Mwwasacquired afterimmersing thesampleinto500 μLofdistilledwaterfor10minatroomtemperature. M M MSR w d w = (1) 2.5.4.Hydrogel Permeability. Theresistance toflowexertedbythe differenthydrogels withinthemicrodevices wasmeasured byexposing themtoapressuregradient.Permeability wassubsequently quantified byusingDarcy’sLaw.Microfluidic deviceswerefabricated asdetailed insubsection 2.6.1,andthehydrogelwasalsoprepared according to theprocedure insubsection 2.4.Thehydrogelwasintroduced inthe centralchannel,andfouremptycolumnswerecoupledinthemedium portsofthedevice.ThewaterinnercolumnswerefilledwithPBSup toaknownheight,creatingapressuregradientthroughthehydrogel. Equation 2provided thetime-dependent relationwithpressureand thematerial(constant c).Afterward, thisconstant wasusedto determine theDarcy’spermeability Kinm2according theeq3. Pt P () ect 0 =· (2) K c LA gAr = ··· ·· (3) with μand ρbeingtheviscosity andthedensityofthefluid, respectively, Lthelengthofthegelthroughwhichthepressuredrop wasestablished, Artheareaofthemediareservoirs, gthegravitational acceleration, and Athecrosssectional areatoflow.Ithasbeen reportedthatthepermeability decreases asthecollagenconcentration increases.24Besides,wehypothesized thatLapNCmayenhancethis effectduetotheirnanoscale biochemical andmorphological properties. 2.6.TumoralSpheroid CultureinMicrofluidics. 2.6.1.Micro- devicesFabrication. Microfluidic deviceswerefabricated inpoly- (dimethysiloxane) (PDMS) bysoftlithography, following the methodology described byShinetal.25Acommercial productwas usedtoproduce thesiliconeelastomer (Sylgard 184Silicone Elastomer Kit,DowChemical, Germany), whichcomprises a polymeric baseandasiliconeresinsolutionascuringagent.The twoliquidpartsweremixedina10:1(base:curing agent)ratioand pouredinamastermadeofanepoxy(SU-8,MicroResistTechnology GmbH, Germany) wherethemicroengineered geometry was patterned withaphotolithography technique (Arago ́nNanoscience andMaterials Institute). Thegeometry consistsofonesingleculture chamber whichisconnected withtworeservoir channels forthe culturehydration. Figure1renderstheinnergeometry ofthe microfluidic device. Thegeometry masterswerethenplacedinavacuumdesiccator for 1h,toremoveairbubblesinthePDMSsolution,andkeptinadryovenovernight tocurethemixture.Later,dermalbiopsypunches wereusedtocreatethereservoirs forcellculturemediaandthe hydrogelinletstotheculturechamber. PDMSdevicesfollowedawet andadryautoclave cyclebeforebeingbondedtoa35mmglass (Ibidi,Germany) byplasmatreatment (PDC-32G BasicPlasma Cleaner,HarrickPlasma,NY,US)undervacuumconditions. They werethencoatedwithPDL(poly-D-lysine;1mg/mLinPBS;Sigma- Aldrich,Germany) andwashedafter4htoenhancematrixadhesion. Beforeuse,thedeviceswereleftinadryovenat80°Cfor48hto restorethehydrophobicity ofthebondedsurfaces.25 2.6.2.3D-CellSeeding. Tumoral cellswerecultured(2D)under standardconditions (5%CO2,37°C)upto70−80%confluence in theirregularexpansion medium: DMEMhighglucose(4.5g/L) supplemented with10%fetalbovineserum(FBS,ThermoFisher Scientific, MA,US),100U/mLpenicillin, 100μg/mLstreptomycin, and2mML-glutamine (allfromLonza,Switzerland). Forcell expansion, cultureswerewashedwithPBS,detached withTrypLE Express(Thermo FisherScientific, MA,US)andplatedinT25cell cultureflasksatadensityof15,000cells/cm2. Cellladencollagen hydrogels (bothsupplemented andnon- supplemented) werefabricated asdetailedabove(subsection 2.4), including cellcontent.Thetumoralcellsweresuspended inexpansion medium ataconcentration of150,000cells/mL inthecollagen solution.Aftergentlypipetting thesolutionintotheculturechamber, itgelledfor20mininahumidchamberat37°C.Oncethehydrogel isgelled,thecultureishydratedthroughthemediaportswithregular DMEM.Thismediawasexchanged every48htoensurenutrients deliverytothecultures. Overtime,sphericalclusterformation fromsinge-cell proliferation intheculturechamber (Figure1)wasexpected inastiffness- dependent manner.Thesespheroid growthweretrackedforthree differentcelllines:Neuroblastoma (PACA), humanlungepithelial carcinoma (A549),andhumanpancreatic ductalcarcinoma (PANC- 1).Moreover, PACAcellswereusedtocomparetheinitialmigration andclusterformation inthesoftesthydrogel. 2.7.Multicellular Structures Morphology Tracking. 2.7.1.Imaging Overtime: Spheroids Growth Quantification. Sampleswereimagedinbrightfield at4×magnification every48h upto15daysofculturewithanopticalmicroscope (LeicaDMIL LED,Germany). Theimageswereanalyzed withacustomsemi- automatic segmentation algorithm basedonactivecontours,26which wasdeveloped inMatlab(Mathworks, Natick,CA,US),toquantify thespheroid areaovertime.FigureS3inSupporting Information showstheshowsascreenshot oftheappwhilesegmenting aparticular image. Bothqualitative (microscopy pictures) andquantitative (area measurement) datawereobtainedforthe3celllines.However, only PACAdatawasshowninthemaintextasrepresentative results. FigureS2inSupporting Information contains theinformation corresponding toPANC-1andA549lines,respectively. 2.7.2.CellMigration. Forstudying 3Dcellmigration, collagen type-Ihydrogels werefabricated following themethodology detailed insubsection 2.4withminormodifications: PACAcellsweremixed with2.5mg/mLcollagentype-I(with/without 0.1%LapNC) ata finalconcentration of2×105cells/mL. Afterfillingthemicrodevice centralchamber, thedeviceswereplacedinhumidified chambers ina CO2incubator toallowthecollagentogelat37°Cfor20min.To promote cellmigration, cellswereserumstarvedwithoutfetalcalf serum.After24h,themedium wasreplaced withDMEM supplemented with10%FBS.Then,time-lapse imagingofthe deviceswascarriedoutwithaD-Eclipse TiMicroscope (Nikon, Japan)witha10×objective, acquiring contrastimagesevery20min for24htostudycellmigration at37°Cinahumidified atmosphere and5%CO2.Thecentralpartofthedevicealongthe Z-axiswas selectedasfocalplanetoensurethatthetrackedcellswereembedded withinthe3Dnetwork.Thus,out-of-focus cellswerenotquantified to minimize artifactsresultingfromtwo-dimensional migration. Atleast50cellsforeachdeviceweremanually trackedalongthe73 time-frames withtheManualTracking pluginfromImageJ(National Institutes ofHealth,MD,US).Subsequently, individual cellmigrationBiomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912882 wasanalyzed withacustomcodedeveloped inMatlab27usedin previousworks.8,28,29Thesetrajectories wereusedtoextractthemean Vmeanandeffective Veffvelocities, definedastheaverageinstantaneous speedincluding alltimesteps,andthespeedcalculated usingonlythe initialandfinalpositions, respectively. Themeansquaredisplacement (MSD)curveofeachtrajectory wasusedtodetermine theglobal diffusioncoefficient (D)asameasureofcellmotilityandmigration persistence.30Additionally, MSDcurveswereusedtofitapowerlaw (MSD(t) =γ×tα)todetermine thekindofmotion(α<1for confined, α=1forBrownian orpurelydiffusive, and α>1for directedmotion,respectively). 2.7.3.NucleiTracking. Forlivenucleivisualization atthe beginning oftheclusterformation, Neuroblastoma spheroids with PACA-Green Fluorescent Protein(GFP)(transfected withaplasmid containing histone2BfusedtoGFP)wereimaged(atdays1and3) withtheLatticeLightsheet 7microscope (ZEISS,Germany) ina mannerthatiscompatible withlong-term fluorescent time-lapse imaging.Imageswereacquiredwitha40×waterimmersion objective lensusingbothheatingandgasincubation system(Ibidi,Germany), toachieveahumidified atmosphere at37°Cwith5%CO2.Fixedand stainedspheroids werealsoimaged,processed (Deconvolution and Deskew), and3Dprojected usingZen3.5Bluesoftware(ZEISS, Germany). 2.8.Statistics. Everycondition wastestedintriplicate. Afterdata normality assessment viaquantile−quantile andnormalprobability plots,analysisofvariance(ANOVA) followedbyposthocTukey− Kramertestswereperformed todetermine statistical significance amongthestudiedcontinuous variables inthedifferentconditions. Non-parametric (Kruskal−Wallis) testsfollowedbyposthocTukey− Kramertestswereusedinsteadwhendatadistribution wasnot normal.Resultspresented inboxplotsshowmedian,quartiles, maximum andminimum values,andoutliers.3.RESULTS ANDDISCUSSION 3.1.LapNCDispersion andHydrogel Microstructure. Figure2showstheLaponite dispersion. Thestockdilutionin waterat1%w:v(Figure2A)showedthepresenceofplatelets ofgroupedclays,asreportedbefore.31Theresultsofdirectly mixingthestocksolutionwithcollageninvolved clayand collagen fiberaggregation. Thus,thestocksolution was subsequently dilutedinculturemedium at0.1%w:vas commented insubsection 2.2.Thisworkingsolution,LapNC inDMEM(Figure2B),wastheoneusedtofabricate the hydrogels (subsection 2.4).TEMimagesevidenced the homogeneous dispersion ofLapNCinDMEMattheselected concentration. Figure2Cshowsrepresentative SEMimagesofevery hydrogelatdifferentcollagenconcentrations with/without the additionofLaponite. Aporousmatrixwasachievedforallthe categories, withawiderangeofporesizes.Thehydrogels were heterogeneous andfibrousnetworks withalargesuperficial areaforcellstoattach.Thepresence ofnano-and micropatterns inthefabricated collagenmatrixisveryrelevant astheyappearinthenaturalECMandcellsarenaturally in vivoincontactwithbothnanoandmicrofeatures.32 Theaddition ofLapNCat0.1%didnotraiseanyclay aggregation orcollagenstructure collapse,whichwereindeed observed inpreliminary testswithmoreconcentrated LapNC stockdispersions. Moreover, thisadditionofLapNCcanbe observed inFigure2Caslittlespeckles stuckalongthe collagenfibers.Thenanodisk attachment tocollagenfibers maybeduetoelectrostatic interaction ofLapNCwiththe Figure3.(A)Singlerepresentative gelationcurvesforeachcollagenconcentration (2.5,4,and6mg/mL). (B)Storagemoduli(G′)afterthe hydrogels gelation. n=3,*p<0.05, **p<0.01, *** p<0.005.Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912883 proteinsidechargedchainsorbecauseoftheformation of hydrogen bonds.17,18Itwasalsoqualitatively evidenced that theincreaseinthecollagenconcentration involvedadenser matrixandareduction inthefibrousporesizes. Thepresence ofLaponite inthehydrogel wascheckedby EDXanalysis.Figure2Dshowsrepresentative spectrafroma gelwithandwithout LapNC. Withtheformula Na (SiMgLi)O(OH)0.7 8 5.5 0.3 20 40.7 [ ]+ ,theappearance ofmagne- sium(Mg),sodium(Na),andsilicon(Si)ionsintheLapNC spectraconfirms thepresence ofthenanoclay. Lithium(Li) couldnotbeobserved possiblyduetoitslowproportion. Inagreement withourresults,Shietal.createdaLaponite supplemented collagenforaleathermatrixfabrication. By usingatomicforcemicroscopy (AFM),theyshowedthatthe primaryandconformational structures ofcollagenmolecules arenotdisturbed bytheintroduction ofLaponite,33similarto ourown.Otherauthorsalsoinvestigated themolecular interactions occurring inLapNCadditiontocollagen-based biomaterials.34Theyshowedthatthenanoclays bindontothe surfaceofcollagenfibrilsandcross-link withthecollagen molecules throughnoncovalent interactions (suchashydrogen andionicbonding). 3.2.Quantification oftheMechanical Properties. 3.2.1.Rheology Confirms theEnhancement oftheMatrix Stiffness. Theenhancement ofthemechanical properties of thehydrogels bytheadditionofnanoclay wasconfirmed by rheology. Storagemodulus (G′)wasmeasured duringthe gelationperiodofthehydrogel. Figure3Bplotsthe G′values afterthegelationperiodofthedifferenthydrogels. Itcanbeobserved thathighercollagen concentration hydrogels involvedastiffermatrix,ascouldbeexpected.35In addition, weobserved significant differences (p<0.01) between supplemented andnonsupplemented hydrogels for thethreecollagenhydrogelconcentrations, whichsupportsour initialhypothesis. Interestingly, bothLapNCsupplemented 2.5mg/mLand control4mg/mL,presentsimilarstoragemodulus(aswellas theLapNCsupplemented 4mg/mLandcontrol6mg/mL pair).Therefore, itseemsfeasibletofabricatehydrogels with thesamestiffness(same G′)buthavingdifferent collagen concentration and,hence,different microstructure (Figure2C).Inagreement withourresults,Lietal.showedthe enhancement ofmechanical properties ofa5mg/mLcattle skincollagen hydrogel atdifferent Laponite concentration addition.Theyshowedthattheelasticmodulus, fracturestress, compression modulus, andrupturepointofhydrogel treated by10%LapNCwere695.2Pa,253.3kPa,13.5kPa,and76.7%, respectively, whichwereabout3.5,5.5,45.0,and1.4times largerthanthoseofregularcollagen-based hydrogel.36 Thisenhancement ofmechanical properties duetothe addition ofnanoclays tothehydrogel couldbeusedfor different purposes. Forinstance, topromote osteogenic differentiation ofmesenchymal stemcells invitro,ithasbeen reported thathumanbonemarrowstemcellsseededonto surfacescoatedwithair-dried Laponite films(2.5mg/mL) possessahigherosteogenic capacity atweek3.21The supplemented collagenhydrogel reportedherecouldbeused topromote osteogenic differentiation ofstemcellsina3D matrix.37Furthermore, thetumoralenvironment hasbeen widelyreportedtobemechanically stifferthanhealthytissue,38 particularly insolidtumors.Therefore, giventhemechanical characteristics ofthetumoralmicroenvironment, thecollagen- basedhydrogeldeveloped inthisstudymaybeasuitabletool forcancerresearch. 3.2.2.Swelling RatioVariation. SRvaluegoesfrom0(no wateradsorption) to1(totalwateradsorption). Ascanbeseen inFigure4A,ahighercollagenconcentration exhibited more waterretention capacity, ascanbeexpected.39Theseresults areinagreement withotherpublished studies.Forinstance, Sawadkar etal.calculated theswellingratioforcollagenand elastinscaffolds, andtheyobtained 92.37%and35.73%, respectively.40Thisindicates thatthesolventabsorbing capacityofelastinscaffoldswasweakerthancollagenscaffolds, asithappened withlowcollagenconcentration hydrogels. According toresultsfromtheliterature, wehypothesized thattheaddition ofLapNCtothecollagenmatrixwould decrease thehydrogel SR.AsitcanbeseeninFigure4A, LapNC supplemented hydrogels (2.5and6mg/mL) presented significant differences. Farahnaky etal.showedthe increaseinagelatinmatrixSRwiththeadditionofdifferent% ofclaynanocomposite (0,2,6,10,14,18%).41Additionally, Nairetal.conducted amechanical behaviorstudyofmodified Figure4.(A)Swellingratios(mean ±SD)ofthedifferenthydrogels: 2.5mg/mL,4and6mg/mLofcollagenconcentration, with(red)and without(blue)theadditionof0.1%Laponitesolution.(B)Darcy’spermeability forcollagenhydrogels, with(red)andwithout(blue)theaddition ofLapNC.Significance for*p<0.05, **p<0.01,and*** p<0.005.Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912884 poly(vinyl alcohol)/laponite nanocomposite membranes. The authorsevidenced thatnanoclay membranes exhibited a decrease inswellingwithanincreaseinlaponitecontent.42 Aggregations ofindividual fibersbythepresence ofLapNC increased stability ofpolymer chainsthrough molecule interactions andthereby reduces theirwateruptake capacity.43,44 3.2.3.Permeability Reduction inSupplemented Hydro- gels.Darcy’spermeability K[m2]wasquantified thankstothe introduction ofawaterpressuregradientthroughthesamples.Figure4BshowsthevaluesofK.Itcanbeobserved that collagenconcentration greatlyaffectedthecollagenous matrix permeability. Additionally, LapNCadditionalsohadasignificant effecton thepermeability regardless ofthecollagenconcentration. As commented previously, LapNCpresentphysicalsurfacearea above350m2/g.45Thus,theLapNCsupplemented hydrogel possessed moretortuous pathsforaparticletomigrate throughthesample,furtherreducingtherateofthemovement ofwatermolecules betweendifferentphases.46 Figure5.(A)Brightfield imagesofarepresentative PACAculturesampleofeachcondition (collagen concentrations with/without LapNC)at4 differenttimepoints.Eachrepresentative imageisaccompanied bythespheroidareamedian(μm2)inthatcondition. ThesymbolXdenotesthat thehydrogelcollapsed atthistimepointorthatnocellsremained in3Dinthiscondition (totalmigration tothebottomsurface).Scalebarinblack 2mm.(B)PACAcellsspheroidarea(μm2)distribution overtime(shownasboxplot):3Dculturesindifferentmatrix:2.5,4,and6mg/mLof collagen;withandwithouttheadditionofLapNC.Conditions notabletoformspheroids atspecifictimepointsaremarkedwithcoloredcrosses.Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912885 Inagreement withourresults,Kanmani etal.indicated that theincorporation ofnanoclaywasabletodecreasewatervapor permeability ofthegelatinfilmmainlybecauseofthepresence ofatortuous pathwayforwatervapordiffusion causedby impermeable silicatelayerednanoclay.47Ourexperiments corroborated this,withtheliquidcolumnexperimental setup used,gettingasignificant reduction inthepermeability value withtheadditionofthenanoclays. Busbyetal.thoroughly characterized differentconcentration ofcollagensolutions assayingrattailtendoncollagentypeI.In agreement withourresults,theyshowedtherelationof collagenconcentration withtheenhancement ofmechanical properties andtheSR,aswellasthereduction ofthe permeability.24Afewprevious studieshavefoundthatthe addition ofLapNCdispersion inthecollagen matrix significantly modified thosephysical properties.24,41,42,48 However, noneofthemappliedthesematrices tocancer research.Regarding therelevance ofthemechanical properties inthetumoralmicroenvironment, theachievedmodification of thematrixproperties couldbeusefulinaculturesystemfor studying invitrocancermodels.Thisoutcome alsohighlights theflairofLapNCincancerresearch, sincetumoraltissue ECMhasbeenshowntoexhibitalowerpermeability thanthe oneofhealthytissue.49 3.3.Spheroid GrowthEvolution Alteredbythe LapNCAddition. Cellcultureswereimagedbybrighfield trackingevery48hupto15daysofculture.Figure5A qualitatively showstheevolution overtimeofPACAcells culturedatdifferentconditions. Itwaspossibletoobservethe creationoftheneuroblastoma spheroids (day15)fromsingle-cellgrowth(day1).Inothermethodstofabricatespheroids, suchashangingdropmethodorultralowattachment culture substrates,50spheroids areformedbythephysicalaggregation ofacellsuspension. Unlikethosemethods, inourexperiments, thecollagen-based matrixproperties (specially itsarchitecture andstiffness)anditsinteraction withthecellswereresponsible forregulating spheroidgrowth.8 Overtime,wegenerally observed thatlargerspheroids were formedinLapNCsupplemented hydrogels. Thedifferences betweensupplemented andnonsupplemented hydrogels are speciallydecisiveinlowcollagenconcentration: wesawthat 2.5mg/mL(withoutLapNC)isanotabletoholdthePACA multicellular structureformation in3D.Thisfactwasprobably duetothelackofstiffness(Figure3)andthelowcollagen densityinthematrix(Figure2).Thus,2.5mg/mLhydrogel withnoclaysresultedinadetachment fromthechannel,cell movement totheglassbottom(2D)ortemporary 3Dculture withcellproliferation butnotspheroid formation. However, LapNCsupplemented 2.5mg/mLhydrogel notonlywas stableupto15daysinthedevice(Figure5A)butwasalsoable toholdsmallspheroidformation. Ontheotherhand,wedid notnoticesignificant differences atfirstsightbetween the control4and6mg/mLcollagencontent. Additionally, wequantified thespheroid growthovertime viaimageanalysis.Figure5Bshowstheevolution ofthe spheroid areainthePACAculturesateverycondition. We observed thatincreasing thecollagenconcentration (4mg/mL over2.5mg/mL)involvedlargeremerging spheroids. Wealso confirmed quantitatively nosignificant differences between control4and6mg/mLmatrix(p>0.16forallthetime Figure6.Evolution overtimeofthedifferentcelllinesateachcondition: 2.5,4,and6mg/mLofcollagenwithandwithouttheadditionofLapNC. Continuous linesshowthemedianvalues,whiledashedlinesrepresent the90%percentile.Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912886 points).Moreover, nostatistical differences inspheroid size werefoundforsupplemented andnonsupplemented 6mg/mL hydrogel(p∼0.9forallthetimepoints)despiteitsvariation ofmechanical properties (Figure3). However, significant changeswereobserved forlowcollagen concentration hydrogels (2.5mg/mL). Thismatrixwithno LapNCexhibited thelowestmechanical properties (Figure3) and,consequently, didnotstand15daysofculture.Infact,no PACAspheroids wereobserved for2.5mg/mLcontrolatany timepoint(Figure5A);thus,noboxesareshowninthefigure. Eitherthecollagenmatrixcollapsed orcellsmovedto2Dby gravity,rapidlyproliferating in2Dovertheglass.Thus,cell incubation longerthan7dayswasnottechnically feasible. Nevertheless, LapNCsupplemented 2.5mg/mLcollagen hydrogel presented amorestablecellcultureplatform. On oneside,supplemented matrixallowedtheincubation for longerperiodsduetoasuperiorstabilityofthehydrogel. On theotherside,ascanbeappreciated inFigure5Asecondrow andB2.5Ldata,cellswereabletoformspheroids embedded inthesupplemented matrix. Differences inspheroid growthat4mg/mLmatrices, compared tosofterones,areeasilyappreciated. Infact,the enhancement ofthemechanical properties ofthematrixwith LapNCleadtothelargestspheroids forallthecelllines (Figure6),highlyevidenced fromday11ofcultureon.This confirms thepossibility toobtainlargemulticellular units embedded inamatrixnotonlybyincreasing collagen concentration butalsobyalternatively addingLapNC. Regarding thehighestcollagenconcentration matrix(6mg/ mL),nolargedifferences inspheroid growthwereobserved comparing LapNCsupplemented andnonsupplemented hydrogels despitetheincreaseinG′(Figure3).Zanotelli etal.suggested thatathighcollagenconcentrations, withsuperior mechanical properties, theproliferation capacityofcellswas highlyreduced,asincubated inthatkindofmicroenvironment, cellswouldonlypossesslowmotilitycapacity.51Thismight explainthatthereisacorrelation betweensubstrate stiffness andspheroidareauptoastiffnessthreshold wherethematrix impedescellproliferation toenlargethespheroid. Furthermore, matriceswithcomparable rigidity,suchas2.5L and4C,aswellas4Land6C,wereinvestigated tocompare cellbehavior, astheyexhibited nostatistically significant differences intermsofstoragemodulus G′(Figure3B). Nevertheless, statistically significant differences wereobserved regarding thegrowthofspheroids amongthesematrices (Figure5B),speciallybetween4L(pink)and6C(darkblue) fromday11on(p=0.0302).Theobserved effectcanbe attributed tothedistinctmicroarchitectures exhibited byeach ofthesehydrogels, asevaluated throughthepermeability measurements (Figure4B). Theresultsobtained forPACAcelllinesarecorroborated withthePANC-1 andA549lines.Thefiguresinthe Supporting Information (S1andS2,respectively) showboth thequalitative andquantitative analysisofbothlinestracking. Nevertheless, toshowafullcomparison, Figure6Ashowsthe spheroidareaevolution forallcollagenconditions andallcell lines.Itisworthnotingthatnewspheroids appeared atany giventimewithinthegelsandthatwedidnottrackthem individually. Thisentailsthatspheroids atdifferentstagesof theirevolution (andtherefore differentsizes)arepresentatthe endoftheexperiments. Toovercome this,thefigureshows boththemedianareavalueandthepercentile 90(Figure6B), whichbetterrepresents thepotential ofeachcondition to producelargespheroids. Supporting Information TableS1 Figure7.PACAcellmigration andbeginning ofclusterformation withinLapNCsupplemented (red)andcontrol(blue)2.5mg/mLcollagen hydrogels: (A)Relativetrajectories ofcellscollected fromcontrolandLapNCsamples(60eventspersample, n=3), Xand Yaxisinμm.(B) ValuesofVmean(left)and Veff(right)ofthecellmigration inbothtypesofsamples.Statistical significance *p<0.05, **p<0.001.(C)Mean squaredisplacement (MSD)ofthetrackedtrajectories. Threedeviceswereanalyzedpercondition usedtostudymorethan150cellsindividually. (D)3Dprojection ofrepresentative PACAcellsatday1and3ofculture.Imageswereprocessed and3Dvolumewasprojected withZen3.5Blue software(ZEISS,Germany).Biomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912887 showsthenumerical valuesatthefinaltimeofthesemedian andp90valuesforthethreecelllines.Ascanbeseen,the LapNCsupplemented 4mg/mLcollagen hydrogel isthe matrixwhichenablesthelargestspheroidformation forallcell lines,butespecially forPACAandPANC-1. This,together withahighermedianvalueovertime,suggeststhatitisnota caseofsinglespheroids growinglarge,butratherthatallof themgrow,overall,faster. 3.4.CellMigration Reduction inSupplemented Matrix.Thisanalysiswasonlyconducted inthesofthydrogel (2.5mg/mL) becauseprevious studieshaveshownthatno relevantmigration isobserved in4and6mg/mLcollagen hydrogels.8InthisworkweobservedthatPACAcellsmigrated significantly slowerwhentheywereembedded withinthe3D networkwithLapNCnanoclays. Boththemean(Vmean)and effective (Veff)velocity (Figure 7B)weresignificantly decreased whenaddingLapNC. TheLapNCadditiontothecollagenhydrogels ledtoa markeddecrease inthemigrated distances ofthecellsas showninFigure7Awhereeachoftherelativecelltrajectories arerepresented. SincetheLapNCsupplemented matrixis significantly stiffer(Figure3),cellsbecomemoreconfined, whichmakemigration morearduous, ultimately leadingto shortercelltrajectories8andlowerspeeds.Thisisfurther confirmed bytheMSDcurves(Figure7C)wherebothLapNC andcontrolconditions werefoundslightlysubdiffusive (confined motion)butwithamuchlowerdiffusivity inthe former(D=0.11 μm2/minandα=0.94) ±0.27,ascompared tocontrol(D=0.77 μm2/min, α=0.93 ±0.29). Theobserveddifference inmotilecapacityforsupplemented andnonsupplemented matrixmaybeexplained bytwo reasons.First,cancercellmigration itshighlydependent on cell−cell adhesion andcell−ECM mechanotransduction.52 Hence,astheLapNCsupplemented matrixisstiffer,cells needtoexerthigherforcestomove.Apartfromthat,LapNC hydrogels possessed moretortuouspathsformigration, which reduced themovement ofindividual cells.Thus,softer networks facilitate cellmigration. Thisoutcome isin agreement withthemechanical characterization ofthe2.5 mg/mLhydrogels: superiormechanical properties (p<0.01, Figure3)wereshownforLapNCsupplemented hydrogel, whichalsopresented ahighlyreducedpermeability (p<0.005, Figure4B). ToexploretheeffectofLapNCintothedevelopment of PACAmulticellular clustersofcellsinmoredetail,wechose theselowstiffnesshydrogels (2.5mg/mL) ofcollagentype-I sincetheywerepreviously characterized with/without LapNC. Then,weimplemented fluorescence time-lapse microscopy withLightsheet microscopy thatdoesnotcompromise cell viabilityandiscompatible withlong-term fluorescent time- lapse.PACAnucleiwererecordedfromday1(singlecells)up today3(Figure7D). Weobserved thatPACAcellsculturesembedded inLapNC supplemented 2.5mg/mLhydrogels inducedahighdegreeof spheroid formation compared withtheiraforementioned controlswithouttheclays(Figure7B).Thisoutcome is, indeed,relatedtothereduction incellmigration. Collecting all theoutcomes fromthelowcollagen concentration, we observed acoherent upshot:control2.5mg/mLhydrogel eitherdidnotallowtheformation ofspheroids orwasnotable toholda15daysincubation (Figures5Band6).However, withtheLapNCaddition, weobserved formation ofseveral smallspheroids (Figure5BorangeandFigure7D).Overall,ourworkindicates thattheuseofLapNCin collagen-based hydrogels canalteritsmechanical properties andmicroarchitecture improving andaccelerating cellular tumorgrowth invitro.Infact,theadditionofLapNCincreases themacroscopic stiffnessofthehydrogel, alsoreducing its permeability. Therefore, itsuseprovides earlierandlarger cellular tumorspheroids athydrogels of4mg/mL supplemented withLapNCinallthetumortypesanalyzed. 3.5.Relevance oftheStudyandOtherConsider- ations.Cellularspheroids canmorecloselymimicthe invivo cellularmicroenvironment compared toconventional 2Dcell culture.Therefore, ithasbecomeapopulartrendtoutilize thosemulticellular structures asinvitromodelsformanytissue engineering research fields.53,54Forexample, Patraetal. createdspheroids fromacellsuspension inone-channel microfludic devicescomposed ofmicrowells (200 ×200μm and300×300μm).55Theysawsignificant differences when applyinganticancer drugstothespheroids and2Dcultures, whichalsohighlighted therelevance of3Dcancercultures. Apartfromthat,othertypesofmicrofluidic devicesare reported intheliterature. Forinstance, otherauthorshave createdspheroids controlling thesize(98−126 μmdiameter) byamicrofluidic droplet-based device.56 Astumormicroenvironment playsakeyroleincellular functions, 3Dhydrogels arewellsuitedtomimic invivoECM forcancercellstudies.38Thesesystemsallowresearchers to gainagreaterunderstanding ofthebiologyanddrug sensitivities ofvarioussolidtumortypes,including pancreatic, prostate,osteosarcoma, glioblastoma multiforme, lung,ovarian, orbreast.57,58 Increased tissuestiffnessisassociated withpathologies, like cancerandfibrosis,amongothers.Thus,ECMstiffnessis emerging asamechanical cuethatprecedesdiseaseanddrives itsprogression byalteringcellularbehaviors.59Targeting ECM mechanics, bypreventing orreversing tissuestiffening or interrupting thecellularresponse, isatherapeutic approach withclinicalpotential. Consequently, beingabletotailorand customize ECMstiffnesscouldbeapowerful methodfor personalized researchandtherapies inmultiplefieldsoftissue engineering. Thisisanapplication thatfitsinthisstudy,aswe wereabletomodifythematrixproperties andarchitecture withouttheneedofincreasing thecollagenconcentration. Additionally, therearealsomanyotheremerging fieldsapart fromcancerresearch, suchastheenhancement ofosteogenic differentiation ofstemcellsforbonetissueengineering. For instance, Xavieretal.embedded LapNC ingelatin methacrylate hydrogels andthenphoto-cross-linked toform acomposite scaffold; whichpromoted ALPactivityand mineralization innormalgrowthmedium without any osteoconductive factors.60Asotherinnovative application, LapNChavebeenalsoinvestigated tosupplement bioinks, eithertoadjusttheirmechanical properties andalsoas nanocarriers asgenedeliverydevices.61,62 Inthiswork,wehavefocusedonthepotential ofthe collagen-based matrices tocreate3Dtumoralmodelsby culturing tumorcellspheroids andthepossibility totailor matrixproperties andarchitecture throughLapNCaddition. Despitethestrongpotential ofthis3Dmodelinstudying cancerdevelopment andprogression, itisessentialtoconsider themodel’slimitations andtheirimpactontheconclusions drawnfromthestudy.First,thethickness ofthemicrofluidic devicesis300 μm,whichcreatesaphysicallimitforthe spheroidsize.Although thickerdevicescanbefabricated withBiomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912888 thistechnology, ouraimistounderstand howtheystarttobe formedandhowtheyself-organize depending onthe microenvironmental conditions. Second, thesespheroids werecreatedfromasinglecelltype,although differentcell typescouldhavebeencocultured. Ourspheroids grewfrom onesinglecell,whichdidnotallowtogrowlargespheroids in shorttimes,andwecouldnotanalyzethenecroticcore characteristic oflargerspheroids. Moreover, thebiological featuresofthespheroids werenot determined here.Inordertoexamine thefunctionality as cancermodel,itwouldbenecessary toconduct further experiments, suchashematoxylin andeosinstaining,TEMor immunohistochemistry63toobserve,forinstance, theinner structureofthecluster.64Additionally, thevariationofcollagen concentration andthehydrogelsupplementation withLapNC affectbothstiffnessandmicroarchitecture. Thus,modifying mechanical properties maintaining hydrogel microstructure wasnotfeasible.Nonetheless, despitetheselimitations, our approach providesamethodology forstudyingtheinitiation of tumorspheroidformation.65 4.CONCLUSIONS Basedonourresults,wecaninferthatincorporating LapNC intoacollagen-based matrixoffersameanstocustomize the mechanical properties ofthehydrogel, regardless oftheinitial amountofcollagenused.Thepresenceofnanoclays resultedin noteworthy alterations inthebiophysical properties, perme- ability,andswellingratioofthehydrogels. Weconducted spheroid growthtrackinginmicrofludic devicestopresenta valuableapplication ofthisinnovative collagen-based culture system.Toevaluate thespheroid growthweusedthree tumoralcelllinesfindingthatthelargestspheroids formedin LapNC-supplemented 4mg/mLcollagenhydrogels. Thedata showedthatapropercombination ofcollagenconcentration andLapNCmayallowcontrolling thespheroid growthover time.Puttogether,ourfindingssuggeststhattheuseofLapNC incollagen-based hydrogels canprovideimproved mechanical properties andmicroarchitecture fortumorgrowthapplica- tions,openingupnewpossibilities forcancerresearchand otherareas. ■ASSOCIATED CONTENT DataAvailability Statement Thedatathatsupportthefindingsofthisstudyareavailable fromthecorresponding authoruponreasonable request. +sıSupporting Information TheSupporting Information isavailable freeofchargeat https://pubs.acs.org/doi/10.1021/acs.biomac.3c00257. Resultsofexperiments conducted withtwoadditional celllines(PANC-1 andA549),atablethatsummarizes thestatisticsofthethreecelllines,andfinally,afigure thatvisuallyandschematically represents howwewere abletoquantifytheparameters usingaMatlabprogram (PDF) ■AUTHOR INFORMATION Corresponding Author PilarAlamán-Díez −Multiscale inMechanical and Biological Engineering, Aragón Institute ofEngineering Research (I3A) &Aragón Institute ofHealthcare Research (IIS Aragón), Department ofMechanical Engineering, University ofZaragoza, Zaragoza 50018, Spain; orcid.org/0000- 0003-1958-4432; Email:alamanp@unizar.es Authors CarlosBorau −Multiscale inMechanical and Biological Engineering, Aragón Institute ofEngineering Research (I3A) &Aragón Institute ofHealthcare Research (IIS Aragón), Department ofMechanical Engineering, University of Zaragoza, Zaragoza 50018, Spain PedroEnriqueGuerrero −Multiscale inMechanical and Biological Engineering, Aragón Institute ofEngineering Research (I3A) &Aragón Institute ofHealthcare Research (IIS Aragón), Department ofMechanical Engineering, University ofZaragoza, Zaragoza 50018, Spain Hippolyte Amaveda −Aragon Institute ofNanoscience and Materials (INMA), University ofZaragoza &CSIC, Zaragoza, Aragon 50018, Spain MarioMora −Aragon Institute ofNanoscience and Materials (INMA), University ofZaragoza &CSIC, Zaragoza, Aragon 50018, Spain JoséMaríaFraile −Institute ofChemical synthesis and Homogeneous Catalysis (ISQCH), University ofZaragoza & CSIC, Zaragoza, Aragon 50009, Spain ElenaGarcía-Gareta −Multiscale inMechanical and Biological Engineering, Aragón Institute ofEngineering Research (I3A) &Aragón Institute ofHealthcare Research (IIS Aragón), Department ofMechanical Engineering, University ofZaragoza, Zaragoza 50018, Spain; Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, University College London, London WC1E 6BT, United Kingdom JoséManuelGarcía-Aznar −Multiscale inMechanical and Biological Engineering, Aragón Institute ofEngineering Research (I3A) &Aragón Institute ofHealthcare Research (IIS Aragón), Department ofMechanical Engineering, University ofZaragoza, Zaragoza 50018, Spain MaríaÁngelesPérez −Multiscale inMechanical and Biological Engineering, Aragón Institute ofEngineering Research (I3A) &Aragón Institute ofHealthcare Research (IIS Aragón), Department ofMechanical Engineering, University ofZaragoza, Zaragoza 50018, Spain Complete contactinformation isavailableat: https://pubs.acs.org/10.1021/acs.biomac.3c00257 Notes Theauthorsdeclarenocompeting financialinterest.■ACKNOWLEDGMENTS Authorswouldliketoacknowledge: JaimeFontDeMoraSainz fromClinicalandTranslational Oncology Research Group (Instituto deInvestigacion LaFe,Valencia, Spain)forproviding thePACAcellline,The Servicio General de Apoyo ala Investigación-SAI (Universidad deZaragoza) fortheuseofthe criticalpointdryerandThe Laboratorio de Microscopías Avanzadas (LMA-Universidad deZaragoza) fortheuseof theelectronic microscopes. Authors alsowouldliketo acknowledge theSpanishMinistry ofEconomy andCom- petitiveness through Projects: PID2020-113819RB-I00, PID2021-122409OB-C21, andPID2021-125762NB-I00. This workhasalsoreceivedfounding fromaHorizon2020�RIA project(TopicSC1-DTH-07-2018, PRIMAGE grantagree- mentno:826494), andtheEuropean Research Council(ERC) undertheEuropean Union’sHorizon 2020research andBiomacromolecules pubs.acs.org/Biomac Article https://doi.org/10.1021/acs.biomac.3c00257 Biomacromolecules 2023,24,2879−28912889 innovation programme (ICoMICS grantagreement No 101018587). PADgratefully acknowledges thesupportofthe Government ofAragon(GrantNo2018-22). EGGisfunded byaRamon&CajalFellowship (RYC2021-033490-I, funded byMCIN/AEI/10.13039/501100011033 andtheEU“Next GenerationEU/PRTR”). ■