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The Journal of Supercritical Fluids
jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / s u p f l u
New insights in the morphological characterization and modelling of poly( -caprolactone) bone scaffolds obtained by supercritical CO 2
foaming
Víctor Santos-Rosales
a,∗, Marta Gallo
b,c, Philip Jaeger
d, Carmen Alvarez-Lorenzo
a, José L. Gómez-Amoza
a, Carlos A. García-González
a,∗aDept.Farmacología,FarmaciayTecnologíaFarmacéutica,I+DFarmaGroup(GI-1645),FacultyofPharmacy,AgrupaciónEstratégicadeMateriales (AeMAT),andHealthResearchInstituteofSantiagodeCompostela(IDIS),UniversidadedeSantiagodeCompostela,E-15782,SantiagodeCompostela,Spain
bDepartmentofAppliedScienceandTechnology,PolitecnicodiTorino,CorsoDucadegliAbruzzi24,10129,Torino,Italy
cUniversityofLyon,INSAdeLyon,MATEISUMRCNRS5510,Bât.SaintExupery,23Av.JeanCapelle,F-69621,Villeurbanne,France
dHamburgUniversityofTechnology(TUHH),EißendorferStr.38,D-21073,Hamburg,Germany
h i g h l i g h t s
•Process-structure-functionalityrela- tionshipsofmanufacturedscaffolds wereelucidated.
•The combined -CT/MIP analysis allowedafullmorphologicalcharac- terizationofscaffolds.
•CO2 soakingtimehadadramatical effectonthescaffoldarchitectures
•3Dmodelsof theporousstructure wereobtainedforthemanufactured scaffolds.
•Scaffoldswerescreenedfrominsilico cellinfiltrationandwaterpermeabil- itytests.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Articlehistory:
Received20April2020
Receivedinrevisedform8July2020 Accepted26July2020
Availableonline7August2020
Keywords:
Supercriticalfoaming 3D-biodegradablescaffolds X-raymicrotomography 3D-modelling Poreinterconnectivity Boneregeneration
a b s t r a c t
Hierarchicallyporoussyntheticbonegrafts(scaffolds)aregainingattentionintheclinicalarena.Scaffolds shouldcombinemorphological(macro-andmicroporosity,poreinterconnectivity),mechanicalandbio- logical(biocompatibility,degradationrate)propertiestofitthisspecificuse.Supercritical(sc-)foaming isaversatilescaffoldprocessingtechnology.However,theselectionoftheoptimumoperatingcondi- tionstoobtainadefinedscaffoldstructureishamperedbythelackofasinglecharacterizationtechnique abletofullyelucidatetheporousfeaturesoftheresultingscaffolds.Inthiswork,theeffectofsoaking time(1,3and5h)onthepreparationofpoly(-caprolactone)(PCL,50kDa)scaffoldsbysc-foaming wasevaluatedbyacombinedX-raymicrotomography(-CT)andmercuryintrusionporosimetry(MIP) 3D-morphologicalanalysis.Mechanicaltestsandinsilicomodellingforcellpenetrationandwaterper- meabilityofthescaffoldswerealsoconducted.Resultsevidencedtherelevanceof-CTandMIPasa synergisticanalyticalduotofullyelucidatethemorphologyofthesc-foamedscaffoldsandthesoaking timeeffect.
©2020TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
Abbreviations: ANOVA,analysisofvariance;BP,batchpressure;BT,batchtemperature;DR,depressurizationrate;FDA,FoodandDrugAdministration;MIP,mer- curyintrusionporosimetry;MSCs,mesenchymalstemcells;-CT,X-ray-basedmicrotomography;PCL,poly(-caprolactone);PLCL,poly(l-lactide-co--caprolactone);PLA, poly(D,L-lacticacid);PLGA,poly(lactic-co-glycolicacid);ROI,regionofinterest;scCO2,supercriticalcarbondioxide;SEM,scanningelectronmicroscopy;SOP,standard operatingprocedures;ST,soakingtime.
∗Correspondingauthors.
E-mailaddresses:victor.santos.rosales@rai.usc.es(V.Santos-Rosales),carlos.garcia@usc.es(C.A.García-González).
https://doi.org/10.1016/j.supflu.2020.105012
0896-8446/©2020TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Boneisthesecondmostcommontransplantationtissue and theharvesting of cancellous bone fromthe patient (allografts) is the current gold-standard surgical procedureto repair bone defectsinthelocomotorsystem.Nevertheless,theriskofinfections, theintervention-associateddamagesandthelimitedavailability of transplantable tissue evidence the need of new therapeu- ticapproaches. Thedevelopment ofinnovativesyntheticgrafts, theso-calledscaffolds,providesapromisingstrategytoregener- atedamaged tissuespromoting theself-healing capacity ofthe patients.Scaffoldsmustdisplaya3D-interconnectedandhierar- chicalporousstructureandamechanicalbehaviorthatareadapted totheanatomicaltargettogetanappropriateperformanceonce implanted[1].
Conventionalmethods for scaffold manufacturingfrequently involve the use of high temperatures and/or organic solvents andmayrequirelongandtedious downstreampathways[2–4].
Theseprocessingconditionsareusuallyincompatiblewithscaf- foldscontaining bioactive compounds (i.e. medicated scaffolds) whichdo notwithstandharsh treatmentsandmaybealsolost duringpurification.The useofmedicated scaffoldsis of partic- ularinteresttoimprovethescaffoldintegrationandtheprecise tissueregenerationortoalleviatepost-surgicalharms.Supercrit- ical(sc-)foamingisaversatilesolvent-freegreentechnologyfor theprocessingofbiodegradable polymericscaffolds.Sc-foaming isbasedonthesorptionanddissolutionofCO2 inthepolymeric matrix of the scaffold for a certain time period (soakingtime;
ST)undercertainpressure(batchpressure;BP)andtemperature (batchtemperature;BT),followedbyapressurerelease(depres- surizationrate;DR).Thelatterstepforcesthepolymerexpansion andCO2removalinordertoinducetheformationofporesinthe polymericmatrix.DuetotheplasticizingeffectofCO2,sc-foaming technologyoperatesundermoderateprocessingtemperatures,i.e.
compatiblewiththeincorporationofthermolabilebioactivecom- pounds such as growth factors [5–7], anti-inflammatory drugs [8–10]orantimicrobialagents[11–13]ofinterestforboneregen- eration.
Thefinecontrolofthemainoperatingvariablesofsc-foaming (BP,BT,ST,DR)allowedthemanufacturingoftunableporousand interconnectedarchitecturesforpolyester-basedscaffolds,suchas poly(D,L-lacticacid)(PLA)[14],poly(lactic-co-glycolicacid)(PLGA) [15,16],poly(-caprolactone)(PCL)[17,18]orpoly(L-lactide-co-- caprolactone)(PLCL)[19].Rationaldesignofpatient-personalized andquality-reproduciblescaffoldsdemands thedevelopmentof standardoperatingprocedures(SOP)forsc-foaming.Compilation ofprotocolsandprocessingparametersisofutmostimportanceto obtainscaffoldsfittedtothetargetgraftingsitedemands,partic- ularlyintermsofporesize,morphology,distribution,throatsize andinterconnectionthatarecriticalforcellcolonizationanddif- ferentiation[20].However,themodellingoftheporeformation mechanismduringsc-foamingprocessisstillchallengingbutabso- lutelyrequiredforthedefinitionofSOPs.
Thereare few studies reportingon theeffects of the foam- ing processing conditions on the resulting polymeric scaffold 3D-architectures[18,21–24].Commonly,morphologicalcharacter- izationiscarriedoutbyscanningelectronmicroscopy(SEM),which allowsfordirectmeasurementsofporesizesandvisualestima- tionsofporeinterconnectivityalthoughrestrictedtotheexposed surfacearea.TheevaluationofscaffoldsusingtheSEMapproach hasseverelimitationsmainlyrelatedtothesample preparation itselfandthemethodofdatatreatment.Aphysicalsectioningofthe scaffoldisrequiredasaprevioussteptoexposetheinnerregions.
Thispreparativestepisusuallyperformedmanuallyusingasur- gicalbladethatmakesthesampletobeundercompressionand shearforces. Theseforces maycausestructuraldamagestothe
structure,suchasporeocclusionanddeformation[25].Moreover, flawedcutsofthescaffoldsarefrequentdependingonthedimen- sionsandthedifficultyofhandlingofthescaffold;theobtained angled areasalsocompromise the qualityand reliability ofthe results. Finally,the datatreatmentof SEMimagestostudythe poresizedistributionconsidersthesizeofa poreequivalentto the cross-section diameter of thepore in theSEM-image. This assumptionusually results in anunderestimation of the actual poresizesinthecaseofsphericalpores.Forthecaseofelongated (cylindrical)pores,severalspecimenscutintheaxialandcoronal planeareneededtogetareliableporesizedistributionfromSEM images,requiringmoreamountofmaterialastheyaredestructive tests.
Mercuryintrusionporosimetry(MIP)isawell-establishedtech- niqueforthecharacterizationofporousmaterials,basedonthe profileofmercuryintrusionintoporeswhensubjectedtoincreas- ingpressures.Oncethemaximumpressureisreached,anextrusion profileisalsoobtainedfromthedepressurizationstep.Fromboth profiles,theporeandthroatsizesofthesamplecanbecalculated, andtheporetortuosity,compressibilityorpermeability maybe inferred[26,27].Despitetheadvantagesofthetechnique,MIPisnot asuitablecharacterizationmethodformaterialswithlargemacro- poresduetoitsupper-limitmeasurableporerange(uptoca.200
m)[28].Inaddition,MIPdoesnottakeintoaccountclosepores andassumesperfectlycylindricalporestocorrelatethevolumeof mercuryintrudedwiththeporesize,whichdonotalwaysrepresent therealityoftheanalyzedsamples[25,29].
X-ray-basedmicrotomography(-CT)isanon-invasivemethod thatprovidesquantitativeandqualitativeinformationregarding the 3D-morphology of samplesand is commonly used for the analysisoftrabecularbone [30,31].Theuseofthis techniqueis encouragingforscaffoldcharacterization[32–37],althoughthere aretwomainaspectstoconsiderwhenperforminga-CTscan:
thedurationoftheanalysisandthestorageoftheobtaineddata.
Indeed,thescanningoflow volumesamplescanlast over20 h andthegeneratedfileshavesizesintheorderofseveralterabytes dependingonthe-CTacquisitionparameters(e.g.voxelsize,rota- tionstep)[38]. Also,theselectedvoxelsizehasadirectimpact ontheimage resolutionfromthe-CTscan and,subsequently, onthelowestvalueofmeasurableporesizeoftheporousmate- rials.Therefore,atrade-offsolutionbetweentheimageresolution andthetime-costand datastoragespaceconsumptionmustbe met.
Overall,thestate-of-the-artofmorphologicalcharacterization ofscaffoldsindicatesthatthereisnotauniversaltechniqueableto fullycharacterizetheporousstructureofpolymericscaffoldsinthe micro-to-macroporousrangeregardingpore-throatdistributions andporeinterconnectivities.
Thecombinationofthesecomplementarytechniques(SEM,MIP and-CT)forthefullcharacterizationofscaffoldporestructure canovercometheindividualartifactsorpitfallsofeachindividual technique.Tothebestofourknowledge,itisthefirsttimethat thecombinationofthesecharacterizationtechniquesisexploited togenerateinformation onprocess-structure-functionalityrela- tionshipsofscaffoldsobtainedbysc-foaming,whichisofutmost interestforthedefinitionofSOPs.Thus,theeffectoftheCO2soaking timeduringthesupercriticalfoamingprocessonthescaffoldmor- phologywasevaluatedtogetthetargetscaffoldfeaturesrequired forsyntheticbonegrafts.Themorphologicalstudyoftheresulting scaffoldsofpoly(-caprolactone)(PCL),abioresorbablesemicrys- tallinepolymer[39–45],wasperformedbasedonthecombination ofSEM, MIP,heliumpycnometry and -CTtechniques.Insilico studiesofcellinfiltrationcapacityandwaterpermeabilityaswell asinvitro mechanicaltests werecarriedout forthesc-foamed scaffoldstopredictthegraftperformanceonceimplanted.
2. Materialsandmethods
2.1. Materials
Poly(-caprolactone)(PCL)inthepowderedform(50kDa,Tm
=61.4◦C,66.7%crystallinity)wassuppliedbyPolysciences(War- rington,PA,USA).CO2(purity>99.9%)wasemployedasfoaming andblowingagentandprovidedbyPraxair,Inc.(Madrid,Spain).
2.2. CO2sorptionkinetics
A thermostized magnetic suspension balance (Rubotherm GmbH,Bochum,Germany)wasusedtoevaluatetheCO2sorption kineticsonthePCLat140barand39◦C,i.e.conditionscloseto bodytemperatureandwherePCLwasmoltenaccordingtoprelim- inaryviewcelltests(notshown).Thepolymericpowderwasdosed (80mg)inaglasscontainerandattachedtothebalancethrougha metalhook.Priortothemeasurements,thepolymerwasmoltenat 80◦CinanoventoensureahomogeneousCO2sorptionalongthe material[46].
2.3. ProcessingofPCLscaffoldsbysc-foaming
PCLpowder was weighed (ca. 1 g) and dosed in cylindrical (length =24.6mm,internal diameter=17 mm)Teflonmoulds (BrandGmbH,Wertheim,Germany),followedbymanualcompres- sion.Mouldswerethenplacedina100mL-stainlesssteelautoclave (TharProcess,Pittsburg,PA,USA)andheatedto39◦C(BT).After- wards,thesystemwaspressurizedwithCO2(5g/min)until140 bar(BP)andmaintainedinthestaticmodeforacertainsoaking period(ST=1,3and5h).Thesystemwasstirredat700rpmdur- ingthewholeprocesstoenhancethemasstransferandtoensure ahomogeneousCO2environment.Theautoclavewasthendepres- surizedataconstantventingrate(DR=1.8g/min)untilatmospheric pressure.Priortotheirstorage,theouterskinofthescaffoldswas carefullyremovedusingasurgicalblade.Scaffoldsweredenoted as39STreferringtotheprocessingtemperature(BT=39oC)andthe subscriptaccordingtothesoakingtimeused(ST=1h,3hor5h).
2.4. Characterizationofscaffolds 2.4.1. Structuralcharacterization
Bulkdensities(bulk)wereobtainedfromtheratiobetweenthe weightandvolumeofeachscaffoldaftersc-foaming.Theskeletal density(skel)of thescaffoldswasdeterminedusing a helium- pycnometer (Quantachrome, Boynton Beach, FL, USA) at room temperature(25◦C)and1.01bar.Valueswereobtainedfromsix replicates.Overallporosity(ε)wascalculatedaccordingtoEq.(1).
ε(%)=
1−bulk skel
x100 (1)The morphological properties of the scaffolds were investi- gatedbyscanningelectronmicroscopy(FESEM,ULTRAPLUS,Zeiss, Oberkochen,Germany)runningatavoltageof10kV.Priortotheir imaging, scaffoldswereslicedwitha scalpel and theniridium- sputtered(10nmthickness).
2.4.2. MIPanalysesandinsilicomodellingofPCLscaffolds
MIPanalysesofthePCLscaffoldswereperformed(AutoporeIV 9500model,Micromeritics,Norcross,GA,USA)atworkingpres- suresrangingfrom0.07−1800bartodeterminetheirporesize distributionsinthe0.01−180mrange.Porosityvalues(εMIP)and poresize(MIP-Meanporesize)weredeterminedfromtheintruded volumeofmercury(VpMIP)inthescaffoldswiththeincreaseofpres- sureusingtheWashburnequation[25].A3D-networkmodelwas
generatedfromtheMIP-cumulativecurveshavingidenticalperco- lationpropertiesasthoseofthemanufacturedPCL-scaffoldsusing PoreXpertv.1.6.567software(PoreXpertLtd,Plymouth,UK).Thisin silicomodelconsistsinacubicstructureformedby1,000pores(of cubicshape)connectedbyupto3,000throatsofarbitrarycylindri- calshape.ABoltzmann-annealedsimplexalgorithmwasusedto estimateandtosimultaneouslyoptimizetheconnectivity(mean numberofthroatsperpore),poreskew,throatskewandcorrela- tionlevelfromtheMIP-cumulativecurves.Waterpermeability(25
◦C,1.03bar)wasestimatedassumingthatPoiseuilleflow(water) occurredinthez-directionaccordingtoEq.(2)
kw=
8ωcell(Farcs)lcell
Acell (2)
wherelcellandAcellrepresentthelengthandthecross-sectional areaoftheunitcell,respectively,andωcell(Farcs)isanaveraging operatoroverthewholeunitcelloperatingontheflowcapacitiesof theporethroat-porearcsparalleltothez-axis.PoreXpertcalculates thetermωcell(Farcs)bymeansoftheDinicnetworkanalysisalgo- rithm.Mesenchymalstemcells(MSCs)infiltrationinthescaffolds wassimulatedusingthefiltrationmodulefromthesoftwareand assumingacellsizeof26.5±5.0m,anaveragevalueforhuman MSCs[47–49].
2.4.3. MicroX-raycomputedtomographyimageacquisition, reconstructionandanalysis
MicroX-raycomputedtomography(-CT)scanswereacquired (in the local mode) using a Phoenix v|tome|x tomograph (GE, Boston,MA,USA)equippedwithaVarianPaxScandetector(1920
×1536pixels).Voxelresolutionwassetequalto12m.
3D images were reconstructed and analyzed using Avizo v.2019.1software(ThermoFisherScientific,Waltham,MA,USA).A generalschemeoftheworkflowfromtheimageacquisitiontocom- plete3DreconstructionandanalysisisdepictedinFig.1.Firstly,a specificregionofinterest(ROI)ofcubicshape(4.8×4.8×4.8mm3) andrepresentativeoftheentirescaffoldwasisolatedtoevaluate theinfluence of theworkingparameters ontheresulting mor- phologies.Afterwards,thecalibrationofthethresholdingofthe grey-scalewasperformedtodifferentiatethevoidfraction(pores andporeinterconnections,i.e.throats)fromthesolidmaterial.This imagethresholdingisconsideredacrucialsteppriorto3Dmod- ellingthatinfluencesthesubsequentanalysisandstructuralplots [50].Oncethevoidvolumewaspreciselyidentified,porosity(-CT) ofthescaffoldswascalculatedintheROIandexpressedasper- centageofvoidvoxelswithrespecttothetotalnumberofvoxels.
Connectedporosityofthescaffoldswascalculated(inpercentage) asthevolumefractionofthelargestgroupofinterconnectedpores withrespecttothetotalporevolumeintheROIofthematerial.
Tortuositywascalculatedfromthemeanpathwaydistanceofapar- ticlemovingfromonefaceoftheROItotheoppositeonedivided bythelengthoftheedgeofthecubicROI(i.e.theshortestpossible pathway).
A3D-networkmodelofballsandstrutswasalsoobtainedfrom the-CTdata,wheretheporesarerepresentedasballsandthe porethroatsascylindricalstruts.Thesizesofballsandstrutswere calibratedaccordingtoacolorscaletovisuallyidentifymorpholog- icaldifferencesbetweenscaffolds.Inaddition,poresizeandpore throatsdistributionsandtheirmeanvalueswereobtainedbasedon theformer3Dmodel.Finally,simulatedMIP-dataandporevolume distributionsofthePCL-scaffoldswereobtainedfromthe3Dmodel generatedfrom-CTusingXlibpluginfromFiji-ImageJsoftware [51,52].
2.4.4. Mechanicalproperties
PCLscaffolds (in triplicate)were subjectedto unidirectional compression tests in a tensile bench with a 30 kg load cell
Fig.1.DataprocessingpathwayusedforthemicroX-raycomputedtomographyimageacquisition,reconstructionandanalysisofthesupercriticalCO2foamedscaffold.
Firstly,aspecificregionisisolatedfromtheentirescaffold.Throughtheimagethresholdingstep,thevoidfractionisconvertedtoa3D-networkmodelofballsandstruts, whosesizesarecalibratedaccordingtoacolorscale,andthesolidmaterialingray.Basedonthe3Dmodel,poresizeandthroatsdistributionplotsareobtained.
Fig.2. CO2sorptionkineticsinPCLatthesc-foamingconditions(140barand39◦C).Paralleldottedlinesindicatethethreesoakingtimes(60,180and300min)chosento evaluatetheinfluenceoftheamountofCO2absorbedontheresultingfoammorphologies.
(TA.XTPlus, Stable Micro Systems, Ltd., Godalming, UK) at a crossheadspeedof1mm/min.Alltheexperimentswereperformed atroomtemperature(25◦C),atmosphericpressureand45%rel- ativehumidity.Elasticdeformationwascalculatedfromtheratio betweentheinitialheightandtheheightofthesamplebearing thehighestappliedphysicalstress.TheYoung´smodulus(E)was calculatedfromthestress-strainplotspreviousconversiontoengi- neeringstressandengineeringstrain.
2.5. Statisticalanalysis
Allresultswereexpressedasmean±standarddeviation.Sta- tisticalanalysesof themechanical results(1-wayANOVA)were performedusingStatisticav8.0software(StatSoftInc.,Tulsa,OK, USA)followedbythepost-hocTukeyHSDmultiplecomparison test.
3. Resultsanddiscussion
3.1. ExperimentalCO2sorptionkineticsinPCL
SolubilityanddiffusivityofCO2withinapolymercanbemod- ulatedbytuningtheworkingtemperatureandpressureleadingto dramaticchangesintheresultingfoammorphologies[53].TheCO2
sorptionprofileinPCLunderanatmosphereofsc-CO2at140bar and39◦CisshowninFig.2.TheamountofCO2absorbedinPCL wasgreatlyinfluencedbythesoakingtimewithafastCO2sorp- tionkineticsfollowedbyaslowerstageafterca.60minofexposure withvaluesinthenear-saturationrange(0.23−0.27gramsofCO2 absorbedpergramofPCL).ThesolubilityvaluesofCO2 at35◦C and130bar,and40◦Cand150barwerepreviouslyreportedto reach0.22and0.40gCO2/gPCLatthesaturationstage,respec- tively[17,54].Thebroadrangebetweenthesetwovaluesindicates theproximityofthephasetransitionandshowsthatthereported
Table1
MorphologicalandtexturalpropertiesofPCLscaffoldsobtainedbysc-foaming.Densityvaluesexpressedasmeanvalues±standarddeviation(n=6).
Scaffold bulk(g/cm3) skel(g/cm3) ε(%) εMIP(%) VpMIP(cm3/g) MIP-Meanpore size(m)
ε−CT(%) -CT-Meanpore sizea(m)
-CT-Meanpore sizeb(m)
391h 0.353±0.007 1.101±0.013 68.0±0.7 30.7 1.70 110.29 67.9 733.2±466.5 1340±341
393h 0.317±0.019 1.110±0.011 71.4±1.7 13.1 1.99 116.23 67.2 733.6±330.1 1096±527
395h 0.298±0.008 1.084±0.009 72.5±0.8 20.2 2.31 117.14 68.3 416.7±123.1 698±430
aValuesobtainedfromthe3D-modelreconstruction.Valuesfollowanormaldistribution(R2>0.99).
bValuesobtainedfromthe2D-imagesofthe-CTanalysisintheaxialplane.Valuesfollowanormaldistribution(R2>0.72).
Fig.3. SEMimagesofcoronalcross-sectionsofthefoamedPCLscaffoldsprocessedatincreasingsoakingtimes:(A,B)1h,(C,D)3hand(E,F)5h.Interconnectedporeswere obtainedinallcases(whitearrowsinB,D,F).Scalebar:200m.
valuesintheliterature[54,55]areinfairagreementwiththiswork, inspiteofthedifferencesinthemolecularweightandcrystallinity oftheusedPCLandinworkingpressures.ItisreportedthatCO2 dissolvestoahigherextentintheamorphousregionsofpolymers andinpolymersoflowermolecularweights[56,57].Finally,CO2
solubilityusuallyincreasesathigherpressures[17,54,58].Accord- ingtotheseresults,thesoakingtimesselectedforfurtherfoaming trialsweresetat1,3and5htoevaluatemorphologicaldifferences dependingontheincreasedamountofCO2 dissolvedinthePCL (0.238,0.261and0.270gCO2/gPCL,respectively)
3.2. Sc-foamingprocessdevelopmentandmorphological characterizationofthescaffolds
CylindricalandhighlyporousPCLscaffolds(=68–72%)were obtainedthroughsc-foaming,matchingthehumantrabecularbone porosityvalues[59,60].Adome-liketopendingwasobservedfor
scaffoldsprocessedwithsoakingtimesabove1hincontactwiththe CO2atthefoamingpressure(FigureS1).Allspecimenspresented anon-porousskinof100−140mthicknessduetotherapidCO2 diffusionupondepressurizationfromthesurfaceofthePCLmatrix [61].Longersoakingtimesfavoredthepolymericexpansionupon depressurization,slightlydecreasingthebulkdensityvalues(bulk) ofthemanufacturedscaffolds(Table1).
The morphological analysis of the scaffolds from SEM microscopyimagesunveiledsmoothsurfaceswithsubtlediffer- encesamongthemregarding theporegeometriesanddensities (Fig.3).391hscaffoldspresentedthelesshomogenousporemor- phologieswithsmallpores(<100m)combinedwithlargerones (Fig.3AandB).Enhancedcelladhesion,migration,proliferationand differentiationarereportedforpolymericscaffoldswithaporous populationinthe20–50mrange[62,63].LongerST(5h)favored thehomogeneity and sphericity ofthe pores(395h scaffoldsin Fig.3EandF).Qualitatively, scaffoldsprocessedduring3hpre-
Table2
PorethroatandinterconnectivitiesofPCLscaffoldsprocessedbysc-foamingobtainedfromMIPand-CTdataanalysis.
MIP -CT
Scaffold MIP-meanporethroatdiameter(m) Interconnectivity(%) Tortuosity -CT-meanporethroatdiameter(m) Connectedporosity(%) Tortuosity
391h 35.26 71.17 1.5 242.3±271.5 88.3 1.4
393h 36.86 79.00 1.2 285.8±198.4 99.2 1.5
395h 35.63 77.83 1.3 148.9±89.87 99.8 1.6
sentedmorevoidspacesalongtheanalyzedarea(393hscaffolds inFig.3C,D)than395hscaffolds.Allscaffoldshadinterconnected poresashighlightedbythepresenceofinnerporeswithinthelarge porecavities(whitearrowsinFig.3B,DandF).
Poreinterconnectivitystronglyinfluencestheperformanceof thescaffoldssincelowlevelscanhamperthecellcolonizationphe- nomenaandthediffusionofnutrientsandremovalofwasteprod- uctsfromthecells[1,4].Theporeinterconnectivitylevelofscaffolds obtainedbysc-foamingcanbemodulatedbymodificationsonthe depressurizationrate[18,64].MIPtechniqueallowedthestudyof openporepopulationsin the0.01−180mrangetocharacter- izethemesoporesandthesmallmacroporepopulationsaswell asdegreeofporeinterconnection.Theopenporosityobtainedby MIP(εMIP)ofthefoamedscaffoldrepresentedvaluesinthe13–30% range,clearlydivergingfromtheoverallporosityvalues(Table1).
Thisdivergenceinvaluescanbeattributedtothepresenceofpores eitherlargerthan180morclosed.Namely,thelowestoverall porositywasobtainedforthe393hscaffold.Themeanporesizecal- culatedfromMIPmeasurementsunveiledthatscaffoldsprocessed atlongerSTpresentedlargerporesincreasingupto6%for395hscaf- fold(Table1).Theporethroatdiameterswerevirtuallyidentical, althoughdifferencesinthedegreeofporeconnection(interconnec- tivity)wereobtained,mainlyrelatedtothedifferenttortuosityof thescaffolds(Table2).Overall,themanufacturedscaffoldspresent goodporeinterconnectivity(above70%)forregenerativemedicine purposes,regardlessoftheworkingparameters.
Fig.4. PoresizedistributionofthefoamedPCLscaffoldsprocessedatdifferentsoak- ingtimes(ST=1,3and5h)obtainedfrom2D-imageprocessingfrommicroX-ray computedtomographydata.LongerSTresultedinlowermeanporediameters.
3.3. -CTimagingofPCLscaffoldsobtainedbysc-foamingusing differentsoakingtimes
Thepresenceofnumerousporeswithdiameterslargerthan200
m(asobservedbySEM)encouragedtheuseof-CTinorder toeffectivelyassesstheeffectofCO2soakingtimeontheresult- ingmorphologies.Porosityvaluesdeterminedfrom-CTimages (ε−CT)wereinthe67–68%range,beingclosetotheoverallporos-
Fig.5.2Dhorizontalslices(top)ofthefoamedPCLscaffoldsprocessedafterincreasingsoakingtimes(fromlefttoright,ST=1,3and5h)withtheircorresponding3D reconstructions(bottom)obtainedfrommicroX-raycomputedtomographydata.Scalebars:2mm(top);arrowlength:2mm(bottom).
Fig.6.CumulativeporevolumedistributionofPCLscaffoldsprocessedat39◦C and140barwithdifferentSTvalues.Distributionswereobtainedbycombination obtainedfromMIPexperimentaldataandfromMIPsimulateddatafrom-CT,with acut-offatadiameterof25m.
ityvaluescalculatedfromEq.(1)(Table1).Althoughallscaffolds hadsimilarporosityvalues,remarkabledifferencesintheirmor- phologywereobserved inthe2D-CTimagesasa functionof theprocessingtime(ST).Anincreaseinthenumberofporesand a reduction of thepore size was recorded asthe ST increased (Figs. 4 and 5). An overestimation of the mean pore size was obtainedfromtheanalysisofthe2Daxial planesectionswhen comparedtothebulkstructure,sincethefoamspresentelongated pores(Table1).Ontheotherhand,thetotalspecificporevolume ofthescaffoldscalculatedfrom-CTdataunderestimatedin5–10
%thevaluesobtainedfromMIPanalysis,sincethevolumecontri- butionofsmallmacroporesandmesoporescannotbeconsidered when-CTisusedduetoresolutionlimitations(Fig.6).
Thearchitectureofeachscaffoldwasfurtheranalyzedfromthe dataofthe3D-modelobtainedfromthelibraryof2Dslices(Fig.7).
Longersoakingtimesledtoa reductionin themeanpore sizes ofthescaffoldsandnarrowerporesizedistribution.Particularly, 395hscaffoldspresentedanarrowporesizedistributionwithvalues fallingintheidealsizerange(1−500m)forbonetissueengineer- ing[65,66].Theseresultsareinlinewithpreviousstudieswith neatPCLscaffoldsshowingaremarkablereductionoftheporesize withtheincreaseofthesoakingtime[18].Conversely,otherstudy [67]reportedtheoppositeeffectduringthesupercriticalfoaming ofpurePCLscaffolds,wherethelowestporediameters(11.75m) wereobtainedafterST=1h.Overall,thereductionoftheporesize ofthefoamedscaffoldsobtainedinthisworkfollowstheclassical nucleation-growththeoryofporeformation[68,69].Atlongerpro- cessingtimes,higherinitialamountsofCO2aredissolvedwithin thepolymer(Fig.2)loweringtheinterfacialtensionofthePCL-CO2 system.Thisreductionininterfacialtensionfacilitatestheforma- tionofmorenucleationsitessincetheinitialcritical nucleation radiusisreduced[70–72].Then,upondepressurization,scaffolds ofhighercelldensitieswithsmallerporesaretypicallyobtained whenprocessedatlongersoakingtimes[18,71].Thereductionin poresizeisaconsequenceofthespatiallimitationtothegrowth ofporesfromthesenucleationsitesduetotheirhighabundance.
Moreover,longersoakingtimesallowforamoreefficientCO2dis- tributionalongthepolymericmatrixresultinginmoreuniformand narrowerporedistributions[32].Botheffectsareeasilyappreciated inFig.7asthesoakingtimeincreased.
Scaffoldsallowingtheinfiltrationofcellswithintheirporous structuresandthetransportofnutrients,metabolitesandwastes through them are needed to match the demands of the bone regenerationprocess.Thesepropertiesarestronglydependenton themorphologicalpropertiesofthescaffoldsregardingporeand throatsizedistributionaswellastheporeinterconnectivity.The
Table3
InsilicopredictedvaluesofwaterpermeabilityandMSCsinfiltrationonsc-foamed PCLscaffolds.
Scaffold Waterpermeability(m2) Cellinfiltration(%)
391h 1.37·10−13 66
393h 1.42·10−13 77
395h 1.84·10−12 93
degreeofconnectedporosityinthescaffoldsandhowtheporesare interlinkedwerecharacterizedbytheporethroatanalysis.These featuresareofutmostimportancesinceinhibitioneffectsonthe tissuedifferentiationprocesswerereportedforporousimplantsof narrowporethroats[73].Theincreaseinthesoakingtimeresulted inscaffoldsshowingareductiononthemeanthroatdiametersand narrowerthroatsizedistribution,followingasimilartrendtothe poresizesdiscussedabove(Table2andFig.7).Furthermore,the decreaseinthemeanthroatsizeresultedinanincreaseonthetor- tuosityvalues,althoughthedegreeofconnectedvoidfractionwas almost100%forthelongersoakingtimetested(395hscaffold).
3.4. InsilicomodellingofwaterpermeabilityandhumanMSCs infiltration
Themicrostructureofscaffoldsplaysacriticalroleincellinfiltra- tionanddistributionofbiologicalfluids[74].Despitetheintrinsic limitationsofMIPtechniquepreviouslypresented,itwidelycovers porepopulationsbelow10m,unreachablebyboth-CT(with theselectedacquisitionparameters)andMIPinsilicosimulation basedon-CTdata(Fig.6).Therefore,thesimulationoftheper- meabilityandcellinfiltrationvaluesoftheobtainedscaffoldswas basedonexperimentalMIPdata.
Waterpermeabilityvaluesof391hand393hscaffolds(Table3) wereofthesameorderofmagnitudetothosereportedinsilicofor PCLscaffoldsobtainedbysc-foamingat37◦Candthesamepressure [75],andinvitroforlowmolecularweight-PLGAscaffolds(ca.10−13 m2)withsimilarporousstructures[76].Theincreaseofsoaking timeaugmentedwaterpermeabilityand395hscaffoldsdisplayed oneorderofmagnitudelargervalues.Overall,theobtainedinsilico permeabilityvaluesforthemanufacturedscaffoldswerecloseto thelowestexperimentaldatareportedforcancellousbone(10-12to 10-8m2)[77,78].Nevertheless,scaffoldpermeabilitywasbasedon awaterflowinasingleaxisdirectionanddifferencesareexpected asthestructureshaveanisotropicproperties.Forinstance,notonly directionalbutalsospatialdifferencesinpermeabilitycalculations werereportedforhuman[79],porcine[80]andbovine[81]cancel- lousbone.Despitetheabovementionedlimitationsoftheinsilico model,permeabilityvalueswereofthesameorderofmagnitudeto thosereportedfornaturalbonebyexperimentalperfusionmethods [78].
ThecellinfiltrationcapacityinthePCLscaffoldwasevaluated throughthespreadcapacityofparticleswithdefinedsizes(cor- respondingtohumanMSCs dimensions).395h scaffold hadcell infiltrationvaluesover 90%,suggestingthat thisstructure may havefullaccessibilityforcellsonceimplanted,allowingahomoge- neoustissueingrowthinsteadofrestrictingittotheoutersurface ofthescaffold.Lowercellinfiltrationvalueswereobtainedfor391h and393hscaffolds.Resultsshouldbesparinglyconsideredsincethe modelinthisworkwasbasedonindividualandnon-interacting particles.Forinstance,theformationofundesiredcellaggregates orclustersduringthecellseedingperiodmayoccur,beingasource ofvariabilityintheinvitrodeterminationofcellinfiltration[82].
Overall, 395h scaffold is the most promising candidate regarding its further biological performance as synthetic bone grafts in terms of water permeability and cell infiltration capacity.
Fig.7.3Dnetworkmodels(left)ofballs(pores)andstruts(porethroats)representingthevoidfractionofthesc-foamedPCLscaffoldsunderincreasingsoakingtimes(from toptobottom,ST=1,3and5h),coupledwiththeircorrespondingsizedistributionsplots(right).ProlongedSTresultedinscaffoldswithlowermeanporeandthroatsizes andwithnarrowersizedistributions.
3.5. Mechanicalproperties
Mechanicalperformanceofscaffoldsdirectlycorrelatestothe structuralmodifications inducedby the variation of processing conditions. During the foaming process, the CO2 was vented in a singledirectionand thepolymer expansionwasforced to occur preferentially in the vertical axis, obtaining PCL foams with elongated pores mimicking the natural bone anisotropy (Fig. 3) [83]. Scaffolds presented an elastic deformation of ca.
25 % (Figure S2) and the obtained Young’s moduli were in the5−8 MPa range (Table4), being in the reported range for
Table4
Mechanicalcharacterizationofthesc-foamedPCLscaffolds.Meanvaluesandstan- darddeviation(n=3).Resultswerestatisticallyidentical(1-wayANOVA;p<0.05).
Scaffold Elasticdeformation(%) Young’sModulus(MPa)
391h 23.8±0.5 6.9±0.1
393h 23.9±1.8 6.3±0.9
395h 25.4±2.1 6.8±0.5
humancancellousbone(1–900MPa)[59]. Despitethemorpho- logicaldifferences andtheincrease incell density,theincrease ofthesoakingtimehadnosignificantimpactonthemechanical
behaviorofthescaffoldswhensubjectedtouniaxialcompression stresses.
4. Conclusions
ThearchitectureofporousPCLscaffoldsproducedbysupercrit- icalCO2foamingcanbetailoredbymodificationsoftheprocessing conditionstoprovidehighlyadaptablescaffolds.Fromthe-CT analysis,realistic scaffold reconstructions were obtained, being particularlyusefultoanalyzetheeffectoftheprocessingcondi- tionsontheresultingmorphologies.Longersoakingtimespermit moreCO2tobedissolvedinthepolymericmatrix,leadingtohigher densityofporeswithlowersizes.Inaddition,morehomogeneous scaffoldsandhigherdegreeofporeinterconnectionwereobtained withlongersoakingtimes.Ontheotherhand,MIPallowedthechar- acterizationofthemeso-andlowmacro-porepopulationandofthe degreeofporeinterconnection.Thisisofparticularinterestsince thegraftperformanceafter implantationcanbehighlyaffected bytheseporepopulations.InthissenseandbasedonMIPmea- surements,theinsilicomodellingofcellinfiltrationcapacityand waterpermeabilityoftheobtainedscaffoldsconstitutedapoten- tialscreeningtoolforfurtherinvitro/invivobiologicaltests.The combinationofcomplementarycharacterizationtechniques(-CT andMIP)coupledtothemodellingofthegenerateddata,offersnot onlybroaderandmorerealisticinformationregardingthemanu- facturedscaffoldsbutalsoregardingtheirpotentialuseassynthetic bonegrafts.Overall,thehereinpresentedsupercriticalCO2foaming processallowsthemanufactureofPCLscaffoldsmeetingthestruc- turalandmechanical requirementsforbonetissue regeneration purposes.Thisworkrepresentsastepforwardtowardstheknowl- edgeonprocess-structure-functionalityrelationshipsinsynthetic bonegraftsforthedefinitionofstandardoperatingproceduresin themanufacturingofpoly(-caprolactone)scaffoldsbysupercriti- calCO2foaming.
DeclarationofCompetingInterest Authorsdeclarenoconflictofinterest.
Acknowledgments
Authorswould liketothankProf.EricMaire andDr. Jérôme Adrien for their fruitful advices on -CT measurements. This research was funded by Xunta de Galicia [ED431F 2016/010], MCIUN[RTI2018-094131-A-I00],AgrupaciónEstratégicadeMate- riales [AeMAT-BIOMEDCO2, ED431E 2018/08], Agencia Estatal de Investigación [AEI] and FEDER funds. C.A. García-González acknowledges to MINECO for a Ramón y Cajal Fellowship [RYC2014-15239].V. Santos-Rosales acknowledgesto Xunta de Galicia(ConselleríadeCultura,Educaci ´oneOrdenaci ´onUniversi- taria)forapredoctoralresearchfellowship[ED481A-2018/014].V.
Santos-RosaleswantstoacknowledgetheCOSTActionCA18125
“AdvancedEngineeringandResearchofaeroGelsforEnvironment and Life Sciences” (AERoGELS), funded by the European Com- mission, for the granted Short Term Scientific Mission (STSM) to perform the magnetic suspension balance measurements in EurotechnicaGmbH.
AppendixA. Supplementarydata
Supplementarymaterial relatedto thisarticle canbe found, intheonlineversion,atdoi:https://doi.org/10.1016/j.supflu.2020.
105012.
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