• Aucun résultat trouvé

Elaboration and evaluation of alginate foam scaffolds for soft tissue engineering

N/A
N/A
Protected

Academic year: 2021

Partager "Elaboration and evaluation of alginate foam scaffolds for soft tissue engineering"

Copied!
11
0
0

Texte intégral

(1)

To cite this version : Ceccaldi, Caroline and Bushkalova, Raya and Cussac,

Daniel and Duployer, Benjamin and Tenailleau, Christophe and Bourin,

Philippe and Parini, Angelo and Sallerin, Brigitte and Girod Fullana, Sophie

Elaboration and evaluation of alginate foam scaffolds for soft tissue

engineering. (2017) International Journal of Pharmaceutics, vol. 524 (n° 1-2).

pp. 433-442. ISSN 0378-5173

O

pen

A

rchive

T

OULOUSE

A

rchive

O

uverte (

OATAO

)

OATAO is an open access repository that collects the work of Toulouse researchers and

makes it freely available over the web where possible.

This is an author-deposited version published in :

http://oatao.univ-toulouse.fr/

Eprints ID : 19792

To link to this article :

DOI : 10.1016/j.ijpharm.2017.02.060

URL :

http://dx.doi.org/10.1016/j.ijpharm.2017.02.060

Any correspondence concerning this service should be sent to the repository

(2)

Elaboration

and

evaluation

of

alginate

foam

scaffolds

for

soft

tissue

engineering

Ceccaldi

Caroline

a,b,1

,

Bushkalova

Raya

a,b,1

,

Cussac

Daniel

b,c

,

Duployer

Benjamin

f

,

Tenailleau

Christophe

f

,

Bourin

Philippe

e

,

Parini

Angelo

b,c,d

,

Sallerin

Brigitte

b,c,d,2

,

Girod

Fullana

Sophie

a,

*

,2

aCIRIMAT,UniversitédeToulouse,CNRS,INPT,UniversitéToulouse3PaulSabatier,FacultédePharmacie,F-31062Toulouse,France bINSERM,UMR1048,F-31432Toulouse,France

cUniversitédeToulouse,UPS,FacultédesSciencesPharmaceutiques,F-31062Toulouse,France dCHUToulouse,PôlePharmacie,F-31432Toulouse,France

eEFS,Laboratoiredethérapiecellulaire,F-31027Toulouse,France fUniversitédeToulouse,CIRIMAT,UPS-INPT-CNRS,F-31062Toulouse,France

Keywords: Alginate

Foam-basedscaffolds Surfactants

Mesenchymalstemcells Softtissueengineering

ABSTRACT

Controlling microarchitecture in polymer scaffolds is a priority in material design for soft tissue applications.Thispaperreportsforthefirsttimetheelaborationofalginatefoam-basedscaffoldsfor mesenchymalstemcell(MSC)deliveryandacomparativestudyofvarioussurfactantsonthefinaldevice performance.Theuseofsurfactantspermittedtoobtainhighlyinterconnectedporousscaffoldswith tunableporesizeonsurfaceandincross-section.Theirmechanicalpropertiesincompressionappeared tobeadaptedtosofttissueengineering.ScaffoldstructurescouldsustainMSCproliferationover14days. Paracrineactivityofscaffold-seededMSCsvariedwiththescaffoldstructureandgrowthfactorsrelease wasgloballyimprovedincomparisonwithcontrolalginatescaffolds.Ourresultsprovideevidencethat exploitingdifferentsurfactanttypesforalginatefoampreparationcouldbeanoriginalmethodtoobtain biocompatiblescaffoldswithtunablearchitectureforsofttissueengineering.

1. Introduction

Forthepastdecades,therehasbeenagrowinginterestinthe useofMesenchymal StemCells(MSCs) toregenerate biological tissuesafterseveral acute andchronic diseases.Afteran initial focusontheircapacitytodifferentiateintomesodermallineage, theyarenowacknowledgedfortheirpositiveeffectsattributedto theirparacrineactivities,whichallowdirectregenerationaswell asindirectmodulatoryeffectsondamagedanddiseasedtissues. MSCs secrete paracrine factors which promote tissue repair, stimulateproliferation anddifferentiationof endogenous tissue progenitors, and decrease inflammatory/immune reactions (Caplan, 2007; Li and Ikehara,2013; Souidi et al., 2013). Such

therapeutic properties are particularly effective in ischemic diseases treatment of the heart (Léobon et al., 2009; Panfilov etal.,2013),kidneys(Alfaranoetal.,2012;Furuichietal.,2012)and lungs(Chenetal.,2012;Yipetal.,2013).Inthesetreatments,MSCs aredeliveredtothetargetedorganbyinjectionintotheperfusing arteryordirectlyintothetissuesurroundingthedamagedarea. Unfortunately,benefitsofsuchtherapeuticapproachesarelimited bypoorcellretentionandearlycelldeathattheinjurysiteafter implantation.Indeed,severalstudieshavereportedthatmorethan 80–90%oftransplantedcellsdiewithinthefirst72hafterinjection (Maureletal.,2005;Tomaetal.,2002).Multiplemechanismsare involved in these early cell losses including hypoxia, local inflammationandmechanicalstressoccurringduringcell admin-istration.Improvementofcellconcentrationandviabilityatthe injury site, in order to promote their therapeutic activity, is becomingapriorityinthefieldofcelltherapy.

OnepromisingstrategyistoassociateMSCswitha biocompat-iblematerialthatprotectsandconcentratesthemonthedamaged area.TheidealscaffoldshouldimproveviabilityofgraftedMSCs, preservetheirparacrineactivityand provideanartificialmatrix

* Correspondingauthorat:CIRIMAT,UniversitédeToulouse,CNRS,INPT,UPS, UniversitéToulouse3PaulSabatier,FacultédePharmaciedeToulouse,35chemin desMaraichers,31062Toulousecedex9,France.

E-mailaddress:sophie.fullana-girod@univ-tlse3.fr(G.F.Sophie). 1 Theseauthorsprovidedequalcontributiontothiswork(firstauthor). 2 Theseauthorsprovidedequalcontributiontothiswork(lastauthor).

(3)

allowingmedium/longtermcellsurvivalaswellastheirsecretion function. In addition,themechanical propertiesof theselected materialmustnotonlybecompatiblewithsofttissuesbutalso appropriateforsurgerymanipulationsduringimplantationonthe damagedtissue.Scaffoldarchitectureisanothercriticalparameter thatcouldaffectthebiologicalactivityofentrappedcellsandthe fateoftheimplanteddevice.Morespecifically,ithasbeenreported that poresizedistributionandporeinterconnectivityaffectcell morphogenesis (Zmora et al., 2002), stem cell behavior and implant'scolonizationbyhostcells(Salemetal.,2002;Souidietal, 2013;Tomaetal.,2002;Zeltingeretal.,2001).

Amongmaterialsusedforcelltherapy,naturalpolymersseem tobeparticularlyadaptedintermsofbiocompatibility(Lee and Mooney, 2001). In that regard, alginates are among the most widelyusedpolymers(Andersenetal.,2015;Bidarraetal.,2014; Giovagnolietal.,2015;RuvinovandCohen,2016;Silvaetal.,2015) due to their low toxicity after purification, gelling properties (underconditionscompatiblewithbiologicalactivities:37!C,pH 7.4 ...), structural resemblance to the extracellular matrix (consideredtobeattheoriginoftheirexcellentbiocompatibility), and relatively low cost. Regarding their origin and chemical structure, alginates are naturally occurring anionic linear (un-branched) polysaccharides, which can be extracted from kelp, brownseaweedandsomebacteria.Theyaresaltsofalginicacid consistingof1,4-linked

b

-D-mannuronic(M)and

a

-L-guluronic(G) residues organized in regions of sequential G units (G-blocks), regionsofsequentialMunits(M-blocks)andregionsofGandM unitsatacticallyorganized.Theirsol–geltransitionpropertiesare based on the formation of a stiff “egg-box” structure due to divalentcationsselectivebindingtotheG-blocksoftwoadjacent polymericchains(Grantetal.,1973).Themajorissuelimitingthe widespread use of alginate hydrogels as tissue engineering scaffolds is the possible exchange of divalent cations with monovalentcationsovertime(BajpaiandSharma,2004),resulting in crosslinks dissociations in the gel's network followed by a mechanicaldegradation.However,alginates’mechanicalbehavior is easilymodifiable bydifferentcrosslinkingor bychangingthe type and/or the molecular weight distribution to match the requiredstiffnessofhosttissues(Augstetal.,2006).Moreover,the degradationratedependsnotonlyonalginates’characteristics,but also on the device's dimensions and implantation site. For example,alginatemicrospheresinjectedundertherenalcapsule were almost intact 4 weeks after implantation (Trouche et al., 2010);itwasalsothecaseforG-typealginatescaffoldsimplanted onratmyocardiumbutnotforM-typealginatescaffolds(Ceccaldi etal.,2012).Thus,anaccuratechoiceofalginatetype/properties couldallowawiderangeofbiomedicalapplications.

Thebiocompatibilityofalginateshasbeenextensivelydescribed in the literature and for the last few decades, the scientific communityhasworkedtoestablishedefficientmethodstoproduce alginates with high purification grades and limited amount of polyphenols,endotoxinsandproteinresidueswhichcanimpactthe inflammatory reaction after implantation (Klock et al., 1997; Leinfelderetal.,2003;Tametal.,2006).Ingeneral,alginatesare notknowntobebiologicallyactive.Infact,proteinadsorptionand cell attachmentare low dueto their high watercontent, dense negativesurfacecharge,andthelackofmolecularrecognitionbycell surfacereceptors(Dvir-Ginzbergetal.,2008;Gandhietal.,2013; Glicklisetal.,2000).Thisparticularityofalginates,combinedwith theirstrictlylocaleffect(ontheapplicationsite),haveallowedthe materialtobequalifiedassafeforhumanapplication.Furthermore, several clinical trials using alginate-based medical devices are currentlyinprogress(AUGMENT-HF:NCT01311791;PRESERVATION 1:NCT01226563;NCT01734733;NCT00521937)orcompleted (GLP-1 CellBeads1: NCT01298830; DIABECELL1: NCT00940173;

NCT01396304),demonstratingthegrowinginterestintheuseof thispolymerforbiomedicalapplications.

Regardingtissueengineeringapplications,macroporous three-dimensional (3D) alginate scaffolds are of particular interest. Indeed,comparedtonon-macroporoushydrogelstheyprovideto cellsabiomimeticenvironment,allowimprovedcellinfiltration, better diffusion of solutes, nutrients and oxygen, as well as enhancedwasteremoval(ShapiroandCohen,1997).Additionally, despitethenon-adhesive natureof alginatepolymers,cells are efficientlyincorporatedandretainedwithin3Dalginatesponges duetotheporousstructureofthematrixwhereastheyarenoton bi-dimensional (2D) alginate films (Dvir-Ginzberg et al., 2008; Glickliset al.,2000).Anumber ofstudies haveshownbenefits when using alginate macroporous scaffolds for 3D cell culture (Sapiretal.,2011;Caplan,2007;LiandIkehara,2013;Shacharand Cohen,2003;ShapiroandCohen,1997;Zieberetal.,2014)andfor softtissuesregeneration (Dvir etal.,2009;Dvir-Ginzbergetal., 2008; Leor et al., 2000). In particular, foaming alginates has allowedobtaininghighlyporousscaffoldswithtunable morphol-ogy and mechanical characteristics according to the type and concentrationofalginateusedaswellasthesourceofgellingions (Andersenetal.,2012,2014a).Inaddition,alginatefoamsappeared tobehighlycompatible for cell entrapment,prolonged 3Dcell cultureandretrievalofNHIK3025andNIH:3T3cells(Andersen et al.,2014b).In ourstudy, we wished toproduce foam-based alginateporousscaffoldsspecificallyadaptedforMSCuseincell therapy,i.e.tailoredforMSCimmobilizationandimprovementof theirsecretionability.Foralginatefoaming,wehavechosentouse surfactantscomingfromthepolysorbates(Montanox1)and the

poloxamers (Pluronic1) families, as they are non-ionic, water

soluble (hydrophilic–lipophilic balance >8), biocompatible, and certifiedforbiomedicalapplications(Andersenetal.,2012;Bueno etal.,2014;Eiseltetal.,2000;Fowleretal.,2002;Inzanaetal., 2014;Tadros,2005;Vashietal.,2008).Moreprecisely,weused fourofthesesurfactantsaswehadobservedthemtobecompatible with MSC culture (based on a preliminaryevaluation of their cytotoxicity and water solubility):Montanox 20, Montanox 80, Pluronic127andPluronic108.Mixingeachoneofthemwithan alginate solution followed by a freeze-drying, permitted the generation of four different foam-based scaffolds. They were characterizedwithregardtotheirarchitecture,porosity, mechani-cal properties and cell-seeding ability with functional MSCs. Finally,cellviabilityaswell ascellsecretionfunctionwerealso investigatedinordertoascertainthemostpromisingformulations forsofttissuecelltherapy.

2. Materialsandmethods

Ultrapure MVG sodium alginate with a M/G ratio of 0.47 (determined by 1H NMR measurement) was purchased from

ProvonaBiopolymerInc.(Novamatrix,Norway).Sodium bicarbon-atewas furnished by Cooper(France). Montanox and Pluronic surfactantswereprovidedbySeppic(France)andBASF Corpora-tion (France),respectively. HEPES (4-(2-hydroxyethyl)-1-pipera-zineethanesulfonicacid)sodiumsaltwaspurchasedformSigma– Aldrich, France. Sodium chloride (NaCl) and calcium chloride dehydrate(CaCl2"2H2O)werepurchasedfromVWR.Reagentsused

for in vitro cell culture were

a

-Minimum Essential Medium (

a

-MEM,Invitrogen,SanDiego,CA,USA)supplementedwith10% fetal calf serum (Hyclone, Logan, UT, USA) and ciprofloxacin (10mgml#1;BayerScheringPharma,Germany).

2.1.Macroporousscaffoldselaboration

Solutions of 3% (w/w) MVG alginate were prepared in iso-osmotic saline solution during 30min at 1800–2000rpm

(4)

(HeidolphRZR-2041, Germany).0.9%(w/w) sodiumbicarbonate and 1%(w/w)surfactant (Montanox80, Montanox 20, Pluronic F127orPluronicF108)wereaddedandstirredduring30minto incorporateairbubblesuntilastablefoamwasobtained.

Three-dimensionalscaffoldsweregeneratedbyafreeze-drying technique.Briefly,aliquots(500

m

l)ofthepolymersolutionswere placed in a 48-well plate, frozen overnight at #20!C, and lyophilized. The constructs were then cross-linked in an iso-osmotic buffer containing calcium ions (150mM NaCl, 0.1M CaCl2"2H2O,10% w/wacetic acid) during 30min. The obtained

scaffoldswerewashed3times(10mineach)inaHEPESbufferand lyophilized again. All studied scaffolds were prepared under aseptic conditions and finally exposed to UV light. The final scaffolds dimensions, used in all experiments, were 10mm diameter$5mmthickness.

2.2.Foamstabilityevaluation

Foamstabilitywasevaluatedbymeasuringthefoamvolumein a graduatedtest tubeat determinedtime intervals. Resultsare expressedasapercentageofthefinalfoamvolumemeasured24h afterpreparation.

2.3.Scanningelectronmicroscopy(SEM)

SEM analyses of surfaces and cross-sections of dried 3D scaffoldswereperformed witha Leo435 VP scanningelectron microscope. Samples were mounted on an aluminum sample mount and sputter-coated with silver. The specimens were observedata10kVacceleratingvoltage.

2.4.ComputedX-raymicro-tomography(micro-CT)

The micro-CTstudyof samples was carried out on Phoenix Nanotom 180 (GE Sensing, Germany) using the following parameters: 30kV voltage, 160

m

A current, no filter material, 0.25!rotationstep,5framesasframeaveraging,1440tomographic projectionsovera 360! scanangle,1sexposuretime.Abinning 2$2wasappliedfortheslicesreconstructionandtheresulting voxel size was 11.5

m

m3. 3D virtual models of scaffolds were

obtainedusingVGStudioMAX2.1.Aregionofinterest(ROI)was drawn within the reconstructed volume and a threshold was definedtoidentify thepolymericphase. Then, a morphometric analysisoftheROIwasperformedtoobtainthetotalporosityand voidsinterconnectivity.Scaffolds’morphologieswereanalyzedon thebasisof2DX-raytomographicslicesusingImageJ(NIH,USA). CalculationsweredoneonaROIdefinedonthesurfaceandinthe cross-sectionofeachscaffold.Feret’sdiameterswereobtainedand poredensitieswerecalculatedasthetotalvoidnumber/ROIarea (n=10slicesperscaffold).Voidsonedgeswereexcluded. 2.5.Evaluationofscaffoldsstabilityuponrehydration

Scaffoldsswellingbehaviorwas evaluatedbyweighingthem every10minafterimmersionincellculturemedium.Theswelling ratiowascalculatedaccordingtothefollowingformula: Swellingratio¼ðWt#W0Þ

W0

whereWtisthescaffoldweightattimetandW0istheweightof

thedriedscaffoldbeforeplacingitinculturemedium.

2.6.Fouriertransforminfraredspectroscopy(FTIR)

DriedsamplesweremixedwithKBr(Fluka,France)andpressed into a pellet. FITR spectra were recorded between 400 and 4000cm#1usingSpectrumOneFT-IRSpectrometer(PerkinElmer,

France).

2.7.Mechanicalpropertiesevaluation

Differential elastic moduli and mechanical behavior of the scaffoldswerefollowedbythreesuccessiveuniaxialcompressive assays(TA-XT2TextureAnalyzer,StableMicrosystems,UK).The apparatus consisted of a mobile probe (314.16mm2) moving

vertically up and down at a constant and predefined velocity (0.5mms#1).Theforceexertedbytheprobeonthescaffoldswas

recordedasa functionofthedisplacement.Then,theforcewas convertedintostressbyreportingtheforcetothesurfaceofforce application and the displacement was converted to a strain percentageincomparisonwiththeinitialdimension.Differential elasticmoduliwerecalculatedfromthestress–straincurvesat50% ofstrainandrepresenttherelativestiffnessofthescaffoldat50% strain.Thedifferentialelasticmoduluswasexpressedasfollows from at least three independent observations: E50%=[(F50%/S)/

Strain]$1000kPa,whereF50%istheforceregisteredat50%strain

(N) andSisthesurfaceofthespecimen(mm2).

2.8.IsolationandcultureofhumanMSCsforinvitroexperiments Human MSCs were isolated from PBS-washed filters used duringbonemarrowgraftprocessingforallogenicbonemarrow transplantation. Cellswerecultured at adensity of 5$ 104cells cm#2in

a

-Minimum EssentialMediumsupplemented with10%

fetalcalfserumandciprofloxacin(10mgml#1).After72hat37!C in5%CO2,non-adherentcellswereremovedandthemediumwas

changed. Cultures were fed every 3–4 days. MSCs were used betweenthe3rdandthe6thpassage.

2.9.MSCseedingandcultivation

Forinvitrostudies,cellswereseededwithinthescaffoldsby centrifugation(400g,1min)afterdropping15

m

lofcell suspen-sioncontaining20,000cellsonthetopofthedriedscaffolds(n=3 foreachexperimentalcondition).Thecell-seededscaffoldswere hydratedbyadding985

m

lofculturemediumtoeachscaffold.The constructswereculturedat37!Cin5%CO

2andthemediumwas

changedevery3–4days.

2.10.LIVE/DEADassayandconfocalmicroscopy

LIVE/DEADassayswereperformedusingthe Viability/Cytotox-icitykit(FluoProbes1,Interchim,France).

Briefly,4hafterseeding, constructs were washed two times with

a

-MEM/physiological serum(1/1)andimmersed(30min,37!C)inthepresenceof2

m

M ethidium homodimer-3 (necrotic marker measuring nucleus membrane integrity) and 1

m

M calcein AM (viability marker measuringtheintracellularesteraseactivity)tostaindeadcellsin red and live cells in green. Aftera washing with physiological serum,scaffoldswereobservedunderaconfocalmicroscope(Zeiss LSM 510)using a $10 objective.Samples were excited with a 488nmArgonlaserand witha 543nm helium–neonlaser.The emitted fluorescence was collected using two separate photo multipliertubeswithaBP500–560nmfilterforcalceindetection andaLP620nmfilterforethidiumhomodimer-3detection.

(5)

2.11.QuantificationofMSCmetabolismactivity

CellmetabolismactivitywasquantifiedbyAlamarBlue1assay

(Molecular Probes, Invitrogen,France).3D scaffoldswere trans-ferred to new wells and incubated with 1ml of

a

-MEM supplemented with 10% of AlamarBlue1 reagent for

1–4h as specifiedbythemanufacturer.Aliquotsof100

m

lweretransferred to a 96-well plate and the fluorescence was measured at an excitationwavelengthof540nmandanemissionwavelengthof 620nmusingaplatereader(Infinite1200Pro,TecanGroup).

2.12.Growthfactorsreleasequantification

After cell seeding, scaffolds werehydrated by adjusting the volumeofculturemediumandculturedat37!Cin5%CO

2for24h

under sterileconditions. The amountof HGF,FGF-2 and VEGF releasedintothemediumwasquantifiedinthesupernatantby xMAPtechnology (Luminex100TM system,LuminexCorp.)with anti-humanHGF,FGF-2andVEGFantibodies(Ozyme,France). 2.13.Statisticalanalysis

Resultsareexpressedasmean(SEM.Statisticalcomparisonof the data was performed using one-way ANOVA and post hoc Bonferroni'stestforcomparisonofmorethantwogroups.Forthe mechanicaltests,atwo-wayANOVAwasusedtoanalyzechanges overtimeamongtheexperimentalgroups.AvalueofP<0.05was consideredsignificant.

3. Results

3.1.Stabilityofalginatefoamspreparedusingvarioussurfactants Alginatefoams wereproducedby mixing3% (w/w)alginate solution with bicarbonate and 4 various surfactants as foam stabilizers:Montanox80,Montanox20,PluronicF127orPluronic F108. Thefoamsstability wasstudiedafter30minof mixingat 1800–2000rpmandFig. 1showsthestabilityofthefoamovertime during6h.Foamswerestableforallformulationstested,showing thatthe4surfactantsselectedforthisstudyareadaptedtoprepare foam-basedscaffoldsaftergelationanddryingsteps.

3.2.Morphologyandporosityofalginatefoam-basedscaffolds Fourdifferentscaffoldswithaconstantalginateconcentration (3% w/w) were prepared in the presence of Montanox 80,

Montanox 20, Pluronic F127 or Pluronic F108 surfactants, as describedinSection2.Controlscaffoldscomposedbypurealginate (withoutsurfactants)werealsoprepared,accordingtothesame procedure. Fig. 2 shows SEM images of the surface and the cross-sectionofcontrolalginatescaffolds(REF-S,Fig.2AandF), andofalginatescaffoldsobtainedinthepresenceofMontanox80 (Mx80-S:Fig.2BandG),Montanox20(Mx20-S:Fig.2CandH), PluronicF127(F127-S:Fig.2DandI)andPluronicF108(F108-S:

Fig.2EandJ).

SEMmicrographspresentedinFig.2revealahighlyporousand interconnectedmorphologybothonsurfaceandincross-sectionof allfreeze-driedscaffolds.Quantitativedataobtainedbymicro-CT showsthat themeanporesize rangedfrom100 to200

m

mon surface,andfrom100to230

m

mincross-section,dependingon the surfactant used in the preparation step. The highest pore density was obtained using Pluronic 108 and the lowest pore densitywasobtainedusingMontanox20andPluronic127,bothon surface and in cross-section (Fig. 2Kand L).The total porosity spannedfrom80%to98%(Table1)andthevoidsinterconnectivity was100%forallscaffolds,meaningthatallporeswereconnected tothe surface (datanot shown). It is interesting tonotice the varietyof3Dporousarchitecturesofthescaffoldsgeneratedby using two different families of surfactants (Montanox and Pluronic)compared toeach otherand tothereferencescaffold withoutsurfactant(Fig.3AandB).

3.3.Presenceofsurfactantresidueswithin3Dscaffolds

Surfactantsareamphiphilicmoleculesandarethereforeableof inserting into the plasma membrane of the entrapped cells. Consequently, it is indispensable to wash the scaffolds after preparationinordertoeliminateallsurfactantresidues.Scaffolds were washed 3 times in HEPES buffer bath and residues of surfactantweretrackedbyFITR.Tothatend,wefirstdetermined wavenumbersof specific infraredpeaks of each surfactant and then checkedtheirabsencein the final scaffoldsspectra. Their characteristicpeaksweresituatedat2859cm#1and1105cm#1for

Montanox 80;2869cm#1,1732cm#1,1460cm#1,1346cm#1and

1100cm#1forMontanox20;2884cm#1,1467cm#1,1341cm#1and

1240cm#1forPluronicF127andF108 (Fig.4A).Concerning the

finalscaffoldsspectra(Fig.4B),theypresentedthecharacteristic peaksofalginateat1033cm#1and1091cm#1,correspondingto

theglucuronic(G)andthemannuronic(M)acidunits,respectively. The ##OH stretching peak was observed at 3407cm#1. The

H##C##H and O##C##H stretching vibration was seen at 1421cm#1. The ##COO# stretch was visible at 1609cm#1. The

peaksat881cm#1and816cm#1indicated

b

-glycosidiclinkages

betweenGandMunitsofalginates.Thus,theFTIRanalysisallowed us to demonstrate that alginate scaffolds spectra were not contaminated by the surfactant specific peaks after the three washingstepsinaHEPESbuffer.

3.4.Scaffolds’behaviorsuponhydration

Swellingbehaviorwasfollowedduringrehydrationin

a

-MEM. Itwasdeterminedevery10minforeachscaffoldduring150min.

Fig.5showsthatswellingrateswerehigher(around20–40times) after2hofimmersionincellculturemediumandremainedstable overtimeforallscaffoldtypes(PanelA).Noscaffolddegradation wasobservedduringtheexperiments.PanelBshowsthatalginate scaffolds preserve their morphology after rehydration. These resultssuggest thatthescaffold integrity waspreservedduring the rehydration in cell culture medium and, therefore, that scaffoldscouldsupport3Dcellculture.

Fig.1.Foamstabilityovertimeofalginatefoamsolutions(Mx80-F,Mx20-F,F127-F andF108-F)preparedusingdifferentstabilizingagents(Montanox80,Montanox 20,PluronicF127andPluronicF108)orwithoutastabilizingagent(REF).

(6)

3.5.Mechanicalpropertiesof3Dscaffoldsundercompression Mechanicalbehaviorsofrehydratedscaffoldswereassessedby 3successive compressionsand theirelasticmoduliwere deter-mined at 50% of strain (Fig. 6). The matrices prepared in the presenceofsurfactantspresentedlowermechanicalpropertiesin the first compression cycle (Mx80-S: 11(2.5kPa; Mx20-S: 22.2(1kPa;F127-S:10.53(2.6kPa;F108-S:15.13(1.3kPa)than controlalginatescaffolds(REF-S:27.13( 1.43kPa).Controlalginate scaffoldsandMx80-Spresentedastableelasticmodulusoverthe successivecompressions,whichsuggestedanelasticbehavior.On thecontrary,elasticmoduliofMx20-S,F127-SandF108-Swere time-dependentandtheirmechanical behaviorsincompression suggestedviscousand/orplasticphenomena.

3.6.MSCseeding,viabilityandmetabolicactivitywithin3Dscaffolds Forinvitroexperiments20,000humanMSCswereseededby centrifugation on each type of scaffold. Fig. 7A shows the distributionofcellsthroughthethicknessoftheF108-Sjustafter seeding(4h).AssuggestedbytheLive/Dead1labeling,cellswere

aliveand wereable tobe seededthrough thethicknessof the scaffolds.Celllabelingandconfocalmicroscopyobservationswere similarforalltheexaminedscaffoldtypes,whichvalidatesthecell seedingprocedure.

Thenweinvestigatedcellmetabolicactivityafter3and14days ofculturewithintheporous scaffoldsusingAlamarBlue Assay.

Fig.7Bshowstherelativefluorescenceintensitymeasuredinthe supernatant of the cell-seeded scaffolds. The results were normalized by the fluorescence intensity measuredfor 20,000 MSCs(theinitialmetabolicactivityatthemomentofseeding).

After3daysofculture,adecreaseincellmetabolicactivitywas observedinalltypesofscaffoldssuggestinganearlycelldeathora decreaseinmetabolicactivity.However,14daysafterseeding,the relative fluorescence intensity in all scaffolds has increased compared to that measured for MSCs on the day of seeding, indicatingthatallformulationssupportedlong-termcell prolifer-ationand/orincreaseinmetabolicactivity.

3.7.Secretionofparacrinefactorsbyscaffold-seededMSCs

Paracrine factors secreted by MSCs play a major role in beneficial effects of cell therapy. Previous studies showedthat HGF,FGF-2andVEGFmayplayanimportantroleinmediatingthe beneficialeffectsoftheMSCsincelltherapyofischemicdiseases (Efthimiadouetal.,2006;Gnecchietal.,2008;Kittaetal.,2003; Miasetal.,2008,2009;Rayssacetal.,2009;Tögeletal.,2007;Xin etal.,2001).Therefore,thefunctionalityofMSCswasinvestigated by the quantification of HGF, FGF-2 and VEGF released in the supernatantofMSCsculturedinalginatemacroporousscaffolds. Analyseswereperformed24haftercellseedingandtheresults werecompared tothesecretionlevel obtainedfromMSCsin a cultureplate.

AsshowninFig.8,wefoundthatthesecretionofHGF,FGF-2 andVEGFbytheMSCsdifferedaccordingtothescaffoldtypeand cultureconditions.Cellshadatendencytodown-regulateHGFand VEGF secretions and to up-regulate FGF-2 secretion when cultivatedin3Dconditionscompared to2Dcultureina culture plate.ExceptedforMx80-S,thesecretionprofilesoftheentrapped MSCs within scaffolds preparedusing surfactant seemed to be improvedincomparisonwithREF-S.Thistrendwasparticularly marked for HGF secretion within Mx20-S and F127-S

Fig.2.Morphologyandporosityofmacroporousscaffoldsobtainedwithvariousstabilizingagents.RepresentativeSEMimagesofscaffoldssurfaces(PanelAtoE)and cross-sections(PanelFtoJ).Scalebarcorrespondsto50mm(magnificationat250$).Determinationofsurface(PanelK)andcross-section(PanelL)porosityandporedensityof macroporousscaffolds(measuresmadeon2Dmicro-CTimages).*:p)0.05and***:p)0.001,basedonAnovaanalysis.

Table1

Porosityofthedifferentalginatescaffolds,asdeterminedbymicro-CTanalysis.The totalporosity(voidvolume/totalvolumeoftheROI)iscalculatedfroma2DROI drawnwithinthereconstructedvolumeoftheentirescaffold.

Samples Porosity(%) REF-S 85.12 Mx80-S 80.06 Mx20-S 97.96 F108-S 91.98 F127-S 88.29

(7)

(1.81(1.28pg/ml within Mx20-S and 1.47(0.69pg/ml within F127-S,p>0.05vs0.22(0.05withinREF-S)andbecame signifi-cant for VEGF secretion (19.31( 3.5pg/ml within Mx20-S and 44.5( 13.04pg/ml within F127-S,p<0.001vs 2.58(0.5 within REF-S).

4. Discussion

Generatinghighporosityinimplantablescaffoldsisbecominga priority in tissue engineering and cell therapy. Indeed, an interconnected porosity has been reportedto beindispensable for promoting good nutriment circulation, entrapped cell

Fig.3. Micro-CTanalysisofscaffolds.(A)3Dmicro-CTreconstructionofaROIofREF-S,Mx20-SandF127-S.(B)2Dmicro-CTimagesofthecross-sectionofthesamescaffolds. Thescalebaris1mmforbothpanels(AandB).

Fig.4. FTIRanalysis.(A)FTIRspectraandcharacteristicinfraredpeaksofsurfactants:Mx80(1),Mx20(2),F127(3)andF108(4).(B)FinalscaffoldsFTIRspectraand characteristicpeaks:Mx80-S(1*),Mx20-S(2*),F127-S(3*),andF108-S(4*).

(8)

migrationandproliferationaswellasforimprovingthelong-term efficacyoftheimplanteddevicebyfavoringtissueintegrationand neovascularization. A consensus has been established on the necessity to generate an interconnected porous structure with poresizerangingfrom50to300

m

m(Mikosetal.,1993;Pittenger andMartin,2004).However,thisrangeneedstobeadaptedtothe typeofthedeliveredcells,tothetargetedorganandtothetreated pathology.

Different strategies havebeen described in theliterature to obtain porosity in alginate scaffolds, particularly by utilizing differentfreezingregimes(Zmoraetal.,2002), addingporogens (Hwang et al., 2010)or usingfoaming techniques (Eiseltet al., 2000;Barbettaetal.,2010;Andersenetal.,2012;Sharmaetal., 2012;Buenoetal.,2014).Tailoringtheporousarchitectureof3D alginate scaffolds by changing the freezing regime holds the advantageofbeingasimplemethodhoweveritdoesnotallowfine controloverporosityparameters(Zmoraetal.,2002).Incontrast, addingporogens(Hwangetal.,2010;Sergeevaetal.,2015)and/or usingfoamingtechniques(Andersenetal.,2012;Barbettaetal., 2010;Buenoetal.,2014;Eiseltetal.,2000;Sharmaetal.,2012)

enabletuningadditionaloperationalparametersoverhydrogels’ porosity. The latter technique appears promising in providing highlymacroporous3Dscaffoldsbutitsrealpotentialisyettobe exploredasthefinalscaffold'sbiocompatibilityandarchitecture maybegreatlyaffectedbyfoamcomposition,foamstabilityand operating conditions. The influence of parameters related to alginate macromolecular properties (Barbetta et al., 2010; Andersenetal.,2012,2014a),gellingtime,concentrationand/or sourceofgellingions(Andersenetal.,2012)aswellastosurfactant concentration (Buenoetal.,2014;Eiseltet al.,2000)havebeen extensivelystudied.However,theinfluenceofsurfactanttypeon scaffold's characteristics has never been explored. Moreover, although globally dedicated to cell culture (Andersen et al., 2014b;Costantinietal.,2016),noneofthepreviouslydescribed foam-basedscaffoldshavebeendesignedforMSCculturetomatch thespecificrequirements ofa giventargetedorganand/orof a givencellsource.Thepresent paperisthefirstonetoreporta comparativestudyofalginatefoam-basedscaffoldsforsofttissue engineering using MSCs. To that aim, we compared alginate matrices with various porosities and mechanical properties (producedusingvarioussurfactants),andshownthatthescaffolds’ architecture and performance can becontrolledby thetype of surfactantused.

Some protocols proposed in the literature avoid using surfactants because of their possible toxicity; however, the obtainedporositywithinthescaffoldmaybedifficulttocontrol in a reproducible manner. Consequently, an improvement of mechanical properties involving the use of a potentially toxic cross-linker(Sharmaetal.,2012)mayberequired.Inourstudy,we selectednon-ionichighlyhydrophilicsurfactants,commonlyused in biomedical applications(Andersen etal., 2012; Bueno etal., 2014;Eiseltetal.,2000;Fowleretal.,2002;Inzanaetal.,2014; Tadros,2005;Vashietal.,2008),andwashedthemfollowingthe preparationsteptopreservethefinalscaffolds’biocompatibility. Surfactantadditiontoalginatesolutionsappearstobeaneffective strategy forstabilizing thefoam and, consequently,acquiringa homogenousporousstructureaftercross-linking.Theuseofthese surfactantsenabledobtainingstablefoams,forthestudiedperiod of 120min, thus permitting the preparation of foam-based scaffoldswithlargelyhomogeneousporosityinallstructures.

Ourscaffolds,preparedusingseveralsurfactants,differedwith regard to their surface and cross-section porosity profiles. Therefore, scaffolds’ microarchitectures can be controlled by varying thesurfactant type. Also, for all scaffold types,surface porosity and pore interconnectivity allowed MSC seeding by centrifugation.

Mechanical resistance is anothercritical parameter affecting the final device's engraftment ability and its invivo fate.In a previousstudyourgrouphasdetermined,invitroandinvivo,the influenceofalginatetypeon3Dalginatescaffolds biocompatibili-ty.WehaveshownthatG-typealginatecanimprovemechanical propertiesofhydrogelswithoutaffectingMSCsecretioncapacity (Ceccaldi etal., 2012). Based onthese results, we havechosen ultrapureG-typealginateandahighpolymerconcentration(3%w/ w) in order to optimize the porous structures’ mechanical properties. Measurements of the differential elastic moduli showedaslightlossofmechanicalproperties,butnotsignificant, when porosity was generated in foam scaffolds (11–22kPa) in comparison tocontrol alginatescaffolds (27kPa).Thisrange of mechanical resistance matches soft tissues presenting elastic modulibetween1and20kPa,dependingontheconsideredorgan (Engleretal.,2006).

Scaffolds’FTIRspectradidnotshowanytraceofsurfactantand theirporositysupportedMSCviability.Thelatterwasmaintained during14days,thusshowinggoodinvitrobiocompatibilityofall alginatefoamscaffolds.Furthermore,weobservedthattheseeded

Fig.5.Swellingbehavior.(A)SwellingratiosofMx80-S,Mx20-S,F127-SandF108-S asafunctionofsampleimmersiontimeincellculturemedium.(B)Scaffolds morphologiesafterrehydrationincellculturemedium.

Fig.6.Mechanical properties ofmacroporous scaffolds. Determination ofthe differentialelasticmoduliofmacroporousscaffoldsobtainedunder3successive compressions(1,2and3)at50%ofstrain.*:p)0.05;**:p)0.01,basedontwo-way Anovaanalysis.

(9)

cellswereretainedwithinallofthetestedscaffolds.Thisindicates thatdespitethenon-celladhesivenatureofthealginatepolymer, alginateporousscaffoldscouldconstituteanexcellentsupportfor MSCdeliveryincelltherapyastheyfavorbothcellsurvivaland retention. In vitroresults of thesecretion levels obtainedfrom MSCs revealedthat growthfactorsreleasedifferedconsiderably dependingontheconditionsofcultureandscaffoldtype.Wefound thatwhenMSCsweregrownwithinthescaffoldsthesecretionof FGF-2increasedandthatthesecretionofHFGandVEGFdecreased, incomparisontocellsgrowninacultureplate.Whencomparing MSCs’secretoryprofileswithinmacroporousscaffolds,wefound thattheygloballyimprovedmorewhenasurfactantwasusedin the preparation step than within control alginate scaffolds. Interestingly,forthethreestudiedgrowthfactors,cellsecretion globallyleveled-upmorewithinMx20-SandF127-Sthanwithin

REF-S.Thesegrowthfactorsarewellknowntobeinvolvedinthe positiveeffectsofMSCsontissueregeneration,particularlyafteran ischemic injury. Indeed, HGF is known to reduce the fibrotic responseand topromote both cytoprotectionand angiogenesis (Espositoetal.,2003;Jayasankaretal.,2005;Tomitaetal.,2003; Wangetal.,2004).FGF-2andVEGFarealsowidelydescribedas beinginvolvedinbloodvesselsformation(Kimetal.,2011;Presta etal.,2005;Simons,2004;Vanderveldeet al.,2005).Since the functionalityof theentrappedMSCs wasbestpreservedwithin Mx20-SandF127-S,theseformulationsappearasmostpromising forregenerativeengineeringapplications.

Giventhatsurfactantsgeneratedvariousporousarchitectures andhaddifferentimpactsonMSCs’paracrineactivity,wefurther examinedtheporous structure ofthedifferentformulationsby reconstructing a representative volume of each of them using micro-CT. The pore sizes of all scaffolds remained within the compatible range for 3D cell culture (50–300

m

m) but notable differences existed in their pore density. Even though MSC metabolicactivitywas similarinallscaffoldtypes,Mx20-Sand F127-Spresentedlowerporedensitiesthancontrolscaffolds,both onsurfaceandincross-section.Furthermore,itwaswithinMx20-S and F127-S that cell secretion of growth factors was better preserved.Althoughthe mechanismsresponsiblefor theMSCs’ paracrine activity changes in scaffolds are unclear, we can speculatethatitcouldberelatedtothematrix3Denvironment. Indeed, numerousresearchers havereportedthat thescaffold's porositycouldaffectthecells’morphology,secretoryfunctionsand fate(Coutuetal.,2009;DadoandLevenberg,2009;El-Ayoubietal., 2008;Zehbeetal., 2010).Eventhoughall authorsagreethat a highlyporousand interconnectedstructure isnecessaryfor the optimaldiffusionofnutrients,gasesandwaste,themechanisms responsibleforthechangesincellcharacteristicsandproperties arenotfullyelucidatedandthereisnocurrentconsensusregarding thescaffold'soptimalporesizeforagivenphysiologicalprocess.A recent study comparing two- and three-dimensional culture conditionshasshownthatthelatterenhancedtheMSCs’paracrine immunomodulatorypotential (Follinet al.,2016).Nevertheless, suchaneffectstronglyvarieswiththescaffold'smorphologyand thisparticularitymakespossiblethedesignoftunable biomate-rialsadaptedtospecificapplication.

5. Conclusion

Our workpresents forthefirst time acomparative studyof varioussurfactantsinassociationwithalginatetogeneratehighly porousscaffoldsmatchingspecificationsrequiredforMSCtherapy. We used various foam stabilizing agents and compared their influenceonmatrixporosity,mechanicalpropertiesandsecretion capacity of human MSCs. Therange of theirdifferential elastic

Fig.7.CellseedingandmetabolicactivityofhumanMSCsculturedwithinalginate macroporousscaffolds.(A)Confocalz-planes(fromthesurface:1tothebottom:10) ofMSC-seededF108-SandaLive/Deadstaining(livecellsingreenanddeadcellsin red)4hafterseeding.Scalebarcorrespondsto200mm(magnificationat10$).(B) AlamarBlueassaysperformed3and14daysaftercellseeding.Resultsarefoldto thefluorescencemeasuredfor20,000humanMSCsinacultureplate(presentedas MSCsinthefigure).*denotesasignificantdifferencecomparedtohumanMSCsona culture plate (**: p)0.01, ***: p)0.001, based on Anova analysis). (For interpretationofthereferencestocolorinthisfigurelegend,thereaderisreferred tothewebversionofthisarticle.)

Fig.8.ParacrineactivityofseededhumanMSCs.QuantificationofHGF,FGF-2andVEGFreleasedinthesupernatantbyhumanMSCsculturedwithinmacroporousscaffolds orinaculturePlate24hpost-seeding.*denotesasignificantdifferencecomparedtohumanMSCsinacultureplate(*:p)0.05;**:p)0.01,***:p)0.001,basedonAnova analysis)andydenotesasignificantdifferencecomparedhumanMSCsculturedwithinREF-S(yyy:p)0.001,basedonAnovaanalysis).

(10)

modulicorrespondstothatofsofttissuesandtheirporositycan supportcellproliferationandsecretionofparacrinefactors.These resultssuggestthattheuseoffoamsforthepreparationofalginate scaffoldsmayimprovetheefficacyofMSCsincelltherapy,asan appropriatesupportforcellpreservationduringimplantation.The in vitro evaluation of their compatibility with MSC viability, metabolicactivityandsecretionfunctionsupportsthepotentialof thisapproachforcelltherapyofsofttissues.

Authordisclosurestatement

Nocompetingfinancialinterestsexist. Acknowledgements

ThisworkwassupportedbyCNRS(CNRSgrantforexploratory projects“InterfaceMatériau/Vivant”),INSERMandRégion Midi-Pyrénéesgrants.WewishtothanktheCellularImagingFacility(T. R.I.GenotoulPlateform,Toulouse,France).

References

Alfarano,C.,Roubeix,C.,Chaaya,R.,Ceccaldi,C.,Calise,D.,Mias,C.,Cussac,D., Bascands,J.L.,Parini,A.,2012.Intraparenchymalinjectionofbonemarrow mesenchymalstemcellsreduceskidneyfibrosisafterischemia-reperfusionin cyclosporine-immunosuppressedrats.CellTransplant.21,2009–2019.

Andersen,T.,Melvik,J.E.,Gaserod,O.,Alsberg,E.,Christensen,B.E.,2012.Ionically gelledalginatefoams:physicalpropertiescontrolledbyoperationaland macromolecularparameters.Biomacromolecules13,3703–3710.

Andersen,T.,Melvik,J.E.,Gaserod,O.,Alsberg,E.,Christensen,B.E.,2014a.Ionically gelledalginatefoams:physicalpropertiescontrolledbytype,amountand sourceofgellingions.Carbohydr.Polym.99,249–256.

Andersen,T.,Markussen,C.,Dornish,M.,Heier-Baardson,H.,Melvik,J.E.,Alsberg,E., Christensen,B.E.,2014b.Insitugelationforcellimmobilizationandculturein alginatefoamscaffolds.TissueEng.PartA20,600–610.

Andersen,T.,Auk-Emblem,P.,Dornish,M.,2015.3Dcellcultureinalginate hydrogels.Microarrays(Basel)4,133–161.

Augst,A.D.,Kong,H.J.,Mooney,D.J.,2006.Alginatehydrogelsasbiomaterials. Macromol.Biosci.6,623–633.

Bajpai,S.K.,Sharma,S.,2004.Investigationofswelling/degradationbehaviourof alginatebeadscrosslinkedwithCa2+andBa2+ions.React.Funct.Polym.59, 129–

140.

Barbetta,A.,Carrino,A.,Costantini,M.,Dentini,M.,2010.Polysaccharidebased scaffoldsobtainedbyfreezingtheexternalphaseofgas-in-liquidfoams.Soft Matter6,5213–5224.

Bidarra,S.J.,Barrias,C.C.,Granja,P.L.,2014.Injectablealginatehydrogelsforcell deliveryintissueengineering.ActaBiomater.10,1646–1662.

Bueno,C.Z.,Dias,A.M.,deSousa,H.J.,Braga,M.E.,Moraes,A.M.,2014.Controlofthe propertiesofporouschitosan-alginatemembranesthroughtheadditionof differentproportionsofPluronicF68.Mater.Sci.Eng.C:Mater.Biol.Appl.44, 117–125.

Caplan,A.I.,2007.Adultmesenchymalstemcellsfortissueengineeringversus regenerativemedicine.J.Cell.Physiol.213,341–347.

Ceccaldi,C.,Fullana,S.G.,Alfarano,C.,Lairez,O.,Calise,D.,Cussac,D.,Parini,A., Sallerin,B.,2012.Alginatescaffoldsformesenchymalstemcellcardiactherapy: influenceofalginatecomposition.CellTransplant.21,1969–1984.

Chen,S.,Chen,L.,Wu,X.,Lin,J.,Fang,J.,Chen,X.,Wei,S.,Xu,J.,Gao,Q.,Kang,M., 2012.Ischemiapostconditioningandmesenchymalstemcellsengraftment synergisticallyattenuateischemiareperfusion-inducedlunginjuryinrats.J. Surg.Res.178,81–91.

Costantini,M.,Colosi,C.,Mozetic,P.,Jaroszewicz,J.,Tosato,A.,Rainer,A.,Trombetta, M., !Swie˛szkowski,W.,Dentini,M.,Barbetta,A.,2016.Correlationbetween poroustextureandcellseedingefficiencyofgasfoamingandmicrofluidic foamingscaffolds.Mater.Sci.Eng.C:Mater.Biol.Appl.62,668–677.

Coutu,D.L.,Yousefi,A.M.,Galipeau,J.,2009.Three-dimensionalporousscaffoldsat thecrossroadsoftissueengineeringandcell-basedgenetherapy.J.Cell. Biochem.108,537–546.

Dado,D.,Levenberg,S.,2009.Cell-scaffoldmechanicalinterplaywithinengineered tissue.Semin.CellDev.Biol.20,656–664.

Dvir,T.,Kedem,A.,Ruvinov,E.,Levy,O.,Freeman,I.,Landa,N.,Holbova,R.,Feinberg, M.S.,Dror,S.,Etzion,Y.,Leor,J.,Cohen,S.,2009.Prevascularizationofcardiac patchontheomentumimprovesitstherapeuticoutcome.Proc.Natl.Acad.Sci. U.S.A.106,14990–14995.

Dvir-Ginzberg,M.,Elkayam,T.,Cohen,S.,2008.Induceddifferentiationand maturationofnewbornlivercellsintofunctionalhepatictissueinmacroporous alginatescaffolds.FASEBJ.22,1440–1449.

Efthimiadou,A.,Lambropoulou,M.,Pagonopoulou,O.,Vakalopoulos,I., Papadopoulos,N.,Nikolettos,N.,2006.Theroleofbasic-fibroblastgrowthfactor (b-FGF)incyclosporine-inducednephrotoxicity.InVivo20,265–269.

Eiselt,P.,Yeh,J.,Latvala,R.K.,Shea,L.D.,Mooney,D.J.,2000.Porouscarriersfor biomedicalapplicationsbasedonalginatehydrogels.Biomaterials21,1921– 1927.

El-Ayoubi,R.,Eliopoulos,N.,Diraddo,R.,Galipeau,J.,Yousefi,A.M.,2008.Designand fabricationof3Dporousscaffoldstofacilitatecell-basedgenetherapy.Tissue Eng.PartA14,1037–1348.

Engler,A.J.,Sen,S.,Sweeney,H.L.,Discher,D.E.,2006.Matrixelasticitydirectsstem celllineagespecification.Cell126,677–689.

Esposito,C.,Parilla,B.,DeMauri,A.,Cornacchia,F.,Fasoli,G.,Foschi,A.,Mazzullo,T., Plati,A.R.,Scudellaro,R.,DalCanton,A.,2003.Hepatocytegrowthfactor(HGF) reducestheexpressionofprofibroticfactorsinhumanisolatedglomeruli.Giorn. Ital.Nefrol.OrganOufficialeSoc.TalianaNefrol.20,376–380.

Follin,B.,Juhl,M.,Cohen,S.,Perdersen,A.E.,Kastrup,J.,Ekblond,A.,2016.Increased paracrineimmunomodulatorypotentialofmesenchymalstromalcellsin three-dimensionalculture.TissueEng.PartBRev.22,322–329.

Fowler,E.B.,Cuenin,M.F.,Hokett,S.D.,Peacock,M.E.,McPhersonIII,J.C.,Dirksen,T. R.,Sharawy,M.,Billman,M.A.,2002.Evaluationofpluronicpolyolsascarriers forgraftingmaterials:studyinratcalvariadefects.J.Periodontol.73,191–197.

Furuichi,K.,Shintani,H.,Sakai,Y.,Ochiya,T.,Matsushima,K.,Kaneko,S.,Wada,T., 2012.Effectsofadipose-derivedmesenchymalcellsonischemia-reperfusion injuryinkidney.Clin.Exp.Nephrol.16,679–689.

Gandhi,J.K.,Opara,E.C.,Brey,E.M.,2013.Alginate-basedstrategiesfortherapeutic vascularization.Ther.Deliv.4,327–341.

Giovagnoli,S.,Luca,G.,Blasi,P.,Mancuso,F.,Schoubben,A.,Arato,I.,Calvitti,M., Falabella,G.,Basta,G.,Bodo,M.,Calafiore,R.,Ricci,M.,2015.Alginatesin pharmaceuticsandbiomedicine:isthefuturesobright?Curr.Pharm.Des.21, 4917–4935.

Glicklis,R.,Shapiro,L.,Agbaria,R.,Merchuk,J.C.,Cohen,S.,2000.Hepatocyte behaviorwithinthree-dimensionalporousalginatescaffolds.Biotechnol. Bioeng.67,344–353.

Gnecchi,M.,Zhang,Z.,Ni,A.,Dzau,V.J.,2008.Paracrinemechanismsinadultstem cellsignalingandtherapy.Circ.Res.103,1204–1219.

Grant,G.T.,Morris,E.R.,Rees,D.A.,Smith,P.J.C.,Thom,D.,1973.Biological interactionsbetweenpolysaccharidesanddivalentcations–egg-boxmodel. FEBSLett.32,195–198.

Hwang,C.M.,Sant,S.,Masaeli,M.,Kachouie,N.N.,Zamanian,B.,Lee,S.H., Khademhosseini,A.,2010.Fabricationofthree-dimensionalporouscell-laden hydrogelfortissueengineering.Biofabrication2,035003.

Inzana,J.A.,Olvera,D.,Fuller,S.M.,Kelly,J.P.,Graeve,O.A.,Schwarz,E.M.,Kates,S.L., Awad,H.A.,2014.3Dprintingofcompositecalciumphosphateandcollagen scaffoldsforboneregeneration.Biomaterials35,4026–4034.

Jayasankar,V.,Woo,Y.J.,Pirolli,T.J.,Bish,L.T.,Berry,M.F.,Burdick,J.,Gardner,T.J., Sweeney,H.L.,2005.Inductionofangiogenesisandinhibitionofapoptosisby hepatocytegrowthfactoreffectivelytreatspostischemicheartfailure.J.Card. Surg.20,93–101.

Kim,S.H.,Moon,H.H.,Kim,H.A.,Hwang,K.C.,Lee,M.,Choi,D.,2011. Hypoxia-induciblevascularendothelialgrowthfactor-engineeredmesenchymalstem cellspreventmyocardialischemicinjury.Mol.Ther.19,741–750.

Kitta,K.,Day,R.M.,Kim,Y.,Torregroza,I.,Evans,T.,Suzuki,Y.J.,2003.Hepatocyte growthfactorinducesGATA-4phosphorylationandcellsurvivalincardiac musclecells.J.Biol.Chem.278,4705–4712.

Klock,G.,Pfeffermann,A.,Ryser,C.,Grohn,P.,Kuttler,B.,Hahn,H.J.,Zimmermann, U.,1997.Biocompatibilityofmannuronicacid-richalginates.Biomaterials18, 707–713.

Lee,K.Y.,Mooney,D.J.,2001.Hydrogelsfortissueengineering.Chem.Rev.101,1869– 1879.

Leinfelder,U.,Brunnenmeier,F.,Cramer,H.,Schiller,J.,Arnold,K.,Vásquez,J.A., Zimmermann,U.,2003.Ahighlysensitivecellassayforvalidationof purificationregimesofalginates.Biomaterials24,4161–4172.

Léobon,B.,Roncalli,J.,Joffre,C.,Mazo,M.,Boisson,M.,Barreau,C.,Calise,D.,Arnaud, E.,André,M.,Pucéat,M.,Pénicaud,L.,Prosper,F.,Planat-Bénard,V.,Casteilla,L., 2009.Adipose-derivedcardiomyogeniccells:invitroexpansionandfunctional improvementinamousemodelofmyocardialinfarction.Cardiovasc.Res.83, 757–767.

Leor,J.,Aboulafia-Etzion,S.,Dar,A.,Shapiro,L.,Barbash,I.M.,Battler,A.,Granot,Y., Cohen,S.,2000.Bioengineeredcardiacgrafts:anewapproachtorepairthe infarctedmyocardium?Circulation102(19Suppl.3),III56–III61.

Li,M.,Ikehara,S.,2013.Bone-marrow-derivedmesenchymalstemcellsfororgan repair.StemCellsInt.UNSP132642.

Maurel,A.,Azarnoush,K.,Sabbah,L.,Vignier,N.,LeLorc’h,M.,Mandet,C.,Bissery,A., Garcin,I.,Carrion,C.,Fiszman,M.,Bruneval,P.,Hagege,A.,Carpentier,A., Vilquin,J.T.,Menasché,P.,2005.Cancoldorheatshockimproveskeletal myoblastengraftmentininfarctedmyocardium?Transplantation80,660–665.

Mias,C.,Trouche,E.,Seguelas,M.H.,Calcagno,F.,Dignat-George,F.,Sabatier,F., Piercecchi-Marti,M.D.,Daniel,L.,Bianchi,P.,Calise,D.,Bourin,P.,Parini,A., Cussac,D.,2008.Exvivopretreatmentwithmelatoninimprovessurvival, proangiogenic/mitogenicactivity,andefficiencyofmesenchymalstemcells injectedintoischemickidney.StemCells26,1749–1757.

Mias,C.,Lairez,O.,Trouche,E.,Roncalli,J.,Calise,D.,Seguelas,M.H.,Ordener,C., Piercecchi-Marti,M.D.,Auge,N.,Salvayre,A.N.,Bourin,P.,Parini,A.,Cussac,D., 2009.Mesenchymalstemcellspromotematrixmetalloproteinasesecretionby cardiacfibroblastsandreducecardiacventricularfibrosisaftermyocardial infarction.StemCells27,2734–2743.

Mikos,A.G.,Sarakinos,G.,Lyman,M.D.,Ingber,D.E.,Vacanti,J.P.,Langer,R.,1993. Prevascularizationofporousbiodegradablepolymers.Biotechnol.Bioeng.42, 716–723.

(11)

Panfilov,I.A.,deJong,R.,Takashima,S.,Duckers,H.J.,2013.Clinicalstudyusing adipose-derivedmesenchymal-likestemcellsinacutemyocardialinfarction andheartfailure.MethodsMol.Biol.1036,207–212.

Pittenger,M.F.,Martin,B.J.,2004.Mesenchymalstemcellsandtheirpotentialas cardiactherapeutics.Circ.Res.95,9–20.

Presta,M.,Dell’Era,P.,Mitola,S.,Moroni,E.,Ronca,R.,Rusnati,M.,2005.Fibroblast growthfactor/fibroblastgrowthfactorreceptorsysteminangiogenesis. CytokineGrowthFactorRev.16,159–178.

Rayssac,A.,Neveu,C.,Pucelle,M.,VandenBerghe,L.,Prado-Lourenco,L.,Arnal,J.F., Chaufour,X.,Prats,A.C.,2009.IRES-basedvectorcoexpressingFGF2andCyr61 providessynergisticandsafetherapeuticsoflowerlimbischemia.Mol.Ther.17, 2010–2019.

Ruvinov,E.,Cohen,S.,2016.Alginatebiomaterialforthetreatmentofmyocardial infarction:progress,translationalstrategies,andclinicaloutlook:fromocean algaetopatientbedside.Adv.DrugDeliv.Rev.96,54–76.

Salem,A.K.,Stevens,R.,Pearson,R.G.,Davies,M.C.,Tendler,S.J.,Roberts,C.J., Williams,P.M.,Shakesheff,K.M.,2002.Interactionsof3T3fibroblastsand endothelialcellswithdefinedporefeatures.J.Biomed.Mater.Res.61,212–217.

Sapir,Y.,Kryukov,O.,Cohen,S.,2011.Integrationofmultiplecell–matrix interactionsintoalginatescaffoldsforpromotingcardiactissueregeneration. Biomaterials32,1838–1847.

Sergeeva,A.,Feoktistova,N.,Prokopovic,V.,Gorin,D.,Volodkin,D.,2015.Designof porousalginatehydrogelsbysacrificialCaCO3templates:poreformation

mechanism.Adv.Mater.Interfaces2,1500386.

Shachar,M.,Cohen,S.,2003.Cardiactissueengineering,ex-vivo:designprinciples inbiomaterialsandbioreactors.HeartFail.Rev.8,271–276.

Shapiro,L.,Cohen,S.,1997.Novelalginatespongesforcellcultureand transplantation.Biomaterials18,583–590.

Sharma,C.,Dinda,A.K.,Mishra,N.C.,2012.Fabricationandcharacterizationof naturaloriginchitosan–gelatin–alginatecompositescaffoldbyfoamingmethod withoutusingsurfactant.J.Appl.Polym.Sci.127,3228–3241.

Silva,K.A.,Juenet,M.,Meddahi-Pellé,A.,Letourneur,D.,2015.Polysaccharide-based strategiesforhearttissueengineering.Carbohydr.Polym.116,267–277.

Simons,M.,2004.Integrativesignalinginangiogenesis.Mol.Cell.Biochem.264,99– 102.

Souidi,N.,Stolk,M.,Seifert,M.,2013.Ischemia-reperfusioninjury:beneficialeffects ofmesenchymalstromalcells.Curr.Opin.OrganTransplant.18,34–43.

Tadros,T.F.,2005.AppliedSurfactants:PrinciplesandApplications,1sted. Wiley-VCH,Berkshire.

Tam,S.K.,Dusseault,J.,Polizu,S.,Menard,M.,Halle,J.P.,Yahia,L.,2006.Impactof residualcontaminationonthebiofunctionalpropertiesofpurifiedalginates usedforcellencapsulation.Biomaterials27,1296–1305.

Tögel,F.,Weiss,K.,Yang,Y.,Hu,Z.,Zhang,P.,Westenfelder,C.,2007.Vasculotropic, paracrineactionsofinfusedmesenchymalstemcellsareimportanttothe

recoveryfromacutekidneyinjury.Am.J.Physiol.RenalPhysiol.292,F1626– F1635.

Toma,C.,Pittenger,M.F.,Cahill,K.S.,Byrne,B.J.,Kessler,P.D.,2002.Human mesenchymalstemcellsdifferentiatetoacardiomyocytephenotypeinthe adultmurineheart.Circulation105,93–98.

Tomita,N.,Morishita,R.,Taniyama,Y.,Koike,H.,Aoki,M.,Shimizu,H.,Matsumoto, K.,Nakamura,T.,Kaneda,Y.,Ogihara,T.,2003.Angiogenicpropertyof hepatocytegrowthfactorisdependentonupregulationofessential transcriptionfactorforangiogenesis,ets-1.Circulation107,1411–1417.

Trouche,E.,GirodFullana,S.,Mias,C.,Ceccaldi,C.,Tortosa,F.,Seguelas,M.H.,Calise, D.,Parini,A.,Cussac,D.,Sallerin,B.,2010.Evaluationofalginatemicrospheres formesenchymalstemcellengraftmentonsolidorgan.CellTransplant.19, 1623–1633.

Vandervelde,S.,vanLuyn,M.J.,Tio,R.A.,Harmsen,M.C.,2005.Signalingfactorsin stemcell-mediatedrepairofinfarctedmyocardium.J.Mol.Cell.Cardiol.39, 363–376.

Vashi,A.V.,Keramidaris,E.,Abberton,K.M.,Morrison,W.A.,Wilson,J.L.,O’Connor,A. J.,Cooper-White,J.J.,Thompson,E.W.,2008.Adiposedifferentiationofbone marrow-derivedmesenchymalstemcellsusingPluronicF-127hydrogelin vitro.Biomaterials29,573–579.

Wang,Y.,Ahmad,N.,Wani,M.A.,Ashraf,M.,2004.Hepatocytegrowthfactor preventsventricularremodelinganddysfunctioninmiceviaAktpathwayand angiogenesis.J.Mol.Cell.Cardiol.37,1041–1052.

Xin,X.,Yang,S.,Ingle,G.,Zlot,C.,Rangell,L.,Kowalski,J.,Schwall,R.,Ferrara,N., Gerritsen,M.E.,2001.Hepatocytegrowthfactorenhancesvascularendothelial growthfactor-inducedangiogenesisinvitroandinvivo.Am.J.Pathol. 158,1111– 1120.

Yip,H.K.,Chang,Y.C.,Wallace,C.G.,Chang,L.T.,Tsai,T.H.,Chen,Y.L.,Chang,H.W.,Leu, S.,Zhen,Y.Y.,Tsai,C.Y.,Yeh,K.H.,Sun,C.K.,Yen,C.H.,2013.Melatonintreatment improvesadipose-derivedmesenchymalstemcelltherapyforacutelung ischemia-reperfusioninjury.J.PinealRes.54,207–221.

Zehbe,R.,Goebbels,J.,Ibold,Y.,Gross,U.,Schubert,H.,2010.Three-dimensional visualizationofinvitrocultivatedchondrocytesinsideporousgelatine scaffolds:atomographicapproach.ActaBiomater.6,2097–2107.

Zeltinger,J.,Sherwood,J.K.,Graham,D.A.,Mueller,R.,Griffith,L.G.,2001.Effectof poresizeandvoidfractiononcellularadhesion,proliferation,andmatrix deposition.TissueEng.7,557–572.

Zieber,L.,Or,S.,Ruvinov,E.,Cohen,S.,2014.Microfabricationofchannelarrays promotesvessel-likenetworkformationincardiaccellconstructand vascularizationinvivo.Biofabrication6,024102.

Zmora,S.,Glicklis,R.,Cohen,S.,2002.Tailoringtheporearchitecturein3-Dalginate scaffoldsbycontrollingthefreezingregimeduringfabrication.Biomaterials23, 4087–4094.

Figure

Fig. 2 E and J).
Fig. 7 B shows the relative fluorescence intensity measured in the supernatant of the cell-seeded scaffolds
Fig. 3. Micro-CT analysis of scaffolds. (A) 3D micro-CT reconstruction of a ROI of REF-S, Mx20-S and F127-S
Fig. 5. Swelling behavior. (A) Swelling ratios of Mx80-S, Mx20-S, F127-S and F108-S as a function of sample immersion time in cell culture medium
+2

Références

Documents relatifs

Langage oral (dire) Lecture (lire) Rédaction (écrire)..

De plus, s'il existe une décomposition étoilée de G avec deux sacs, alors chaque arête de G doit être contenue dans au moins un sac.. Pour décider si cette condition est vériée

generis 639. D’autre part, la notion de domaine public est méconnue de la plupart des communautés indigènes. Dans une communication jointe prononcée par des

With the aim of reducing the health risk for pregnant women and restricting their movements during lockdown, at the same time as the workload of healthcare professionals, while

Cassini would spend several years exploring Saturn and its moons, and Huygens would attempt a soft landing on Titan, Saturn’s largest moon.. The spacecraft were named after

identifying complex thermal behaviour[2], derivative conduction calorimetry analysis (dCCA) helps to identify the presence and extent of different reactions during OPC

The variables include water temperature, iceberg size (waterline length), wind and current velocities, and wave height.. Tests were done using a water temperature of 1.1°C, which

Une approche alternative prometteuse consiste donc à identifier et quantifier les métabolites présents dans le mélange complexe à partir de son spectre et à réaliser