Preparation and characterization of a bacterial cellulose/silk fibroin sponge scaffold for tissue regeneration

Bacterial cellulose (BC) and silk fibroin (SF) are natural biopolymers successfully applied in tissue engineering and biomedical fields. In this work nanocomposites based on BC and SF were prepared and characterized by scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA).

jo u r n 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 / c a r b p o l

H.G Oliveira Barudb, Hernane da S Baruda,e,∗, Maurício Cavicchiolia,

Thais Silva do Amarala, Osmir Batista de Oliveira Juniorb, Diego M Santosc,

Antonio Luis de Oliveira Almeida Petersenc, Fabiana Celesc, Valéria Matos Borgesc,

Camila I de Oliveirac, Pollyanna Francielli de Oliveirad, Ricardo Andrade Furtadod,

Denise Crispim Tavaresd, Sidney J.L Ribeiroa

a Institute of Chemistry – São Paulo State University – Unesp, P.O Box 355, Araraquara, SP 14801-970, Brazil 1

b School of Dentistry/Unesp, São Paulo State University – Unesp, Rua Humaitá, 1680, Zip code 14801-903, Araraquara, SP, Brazil 2

c Gonc¸alo Moniz Research Center, FIOCRUZ, Av Waldemar Falcão, 121, Zip code 40296-710, Salvador, BA, Brazil 3

d University of Franca, Av Dr Armando Salles de Oliveira, 201, Zip code 14404-600, Franca, SP, Brazil 4

e Laboratório de Química Medicinal e Medicina Regenerativa (QUIMMERA) – Centro Universitário de Araraquara (UNIARA), Araraquara, SP, Brazil

a r t i c l e i n f o

Article history:

Received 1 October 2014

Received in revised form 3 April 2015

Accepted 8 April 2015

Available online 17 April 2015

Chemical compounds studied in this article:

d-Glucose (PubChem CID: 5793)

glycine (PubChem CID: 750)

l-Alanine (PubChem CID: 5950)

MTT (PubChem CID: 64965)

XTT (PubChem CID: 14195569)

Keywords:

Bacterial cellulose

Silk fibroin

Biocompatible materials

Nanocomposites

Scaffold

Tissue engineering.

a b s t r a c t Bacterialcellulose(BC)andsilkfibroin(SF)arenaturalbiopolymerssuccessfullyappliedintissue engi-neeringandbiomedicalfields.InthisworknanocompositesbasedonBCandSFwerepreparedand characterizedbyscanningelectronmicroscopy(SEM),infraredspectroscopy(FT-IR),X-raydiffraction (XRD)andthermogravimetricanalysis(TGA).Inaddition,theinvestigationofcytocompatibilitywas donebyMTT,XTTandTrypanBluedyetechnique.Cellularadhesionandproliferationweredetected additionally.Theevaluationofgenotoxicitywasrealizedbymicronucleusassay.Invitrotestsshowed thatthematerialisnon-cytotoxicorgenotoxic.SEMimagesrevealedagreaternumberofcellsattached

attheBC/SF:50%scaffoldsurfacethanthepureBCone,suggestingthatthepresenceoffibroinimproved cellattachment.ThiscouldberelatedtotheSFaminoacidsequencethatactsascellreceptors facil-itatingcelladhesionandgrowth.Consequently,BC/SF:50%scaffoldsconfiguredanexcellentoptionin bioengineeringdepictingitspotentialfortissueregenerationandcultivationofcellsonnanocomposites

©2015ElsevierLtd.Allrightsreserved

1 Introduction

Tissueengineering hasthepurpose ofdeveloping

therapeu-tic options especially designed to be appliedin special clinical

conditions, aiming to replace or regenerate damaged tissues

∗ Corresponding author at: Institute of Chemistry – São Paulo State University –

Unesp, P.O Box 355, Araraquara, SP 14801-970, Brazil.

E-mail address: hernane.barud@gmail.com (H.d.S Barud).

1 Tel.: +55 16 3301 9500.

2 Tel.: +55 16 3301 9300.

3 Tel.: +55 71 3176 2201.

4 Tel.: +55 16 3711 8871.

using biomaterials The success of the methodology depends

on Biomaterials’ properties that can be manipulated to mimic thethree-dimensionalarchitectureofextracellularmatrix(ECM) native tissues which is regarded as a complex organization of fibrousstructuralproteinssuchascollagensandawidevarietyof proteoglycansandpolysaccharides(Cai&Xu,2011;Shietal.,2012,

2014)

In recent years, dueto climatechanges andthe decreaseof oilsupply,syntheticmaterialsarebecomingincreasingly unfavor-able,enhancingtheneedtofindrenewablegreenalternatives.Itis generallyknownthatnanomaterialsshowunusualproperties,not observedinthebulkmaterials,suchashighsurfacereactivityand abilitytocross-cellmembranes.Thedevelopmentofmaterialswith biomimeticbehaviorisessentialfortissueengineeringpurposes

http://dx.doi.org/10.1016/j.carbpol.2015.04.007

0144-8617/© 2015 Elsevier Ltd All rights reserved.

becausescaffolds based onnanofibres (NFs) mimic the natural

extracellularmatrix and its nanoscalefibrous structure (Barnes

etal.,2008;Hutchens,Benson,Evans,O’Neill,&Rawn,2006)

Celluloseis the mostabundant biopolymer on earth and is

presentinawidevarietyoflivingspeciesbeingharvestedmainly

obtainedfromtreesandcotton.Itcanalsobeobtainedfromthe

bacteria Gluconacetobacter xylinus that produces nanobacterial

cellulose (BC)free of lignin and hemicellulosein a 3-D

hierar-chicalnetwork composedbybundles ofmuch finermicrofibrils

ofnanometricsizerangefrom3.0to3.5␮m(Barudetal.,2011;

Klemm,Heublein,Fink,&Bohn,2005;Svenssonetal.,2005).Since

itsdiscoveryBChasshowntremendouspotentialasaneffective

biopolymerinvariousfields,asthestructuralaspectofBCisfar

superiortothoseofplantcellulose,whichprovideitwithbetter

properties(Ul-Islam,Khan,&Park,2012)

Then,BCisacompletelybiocompatiblepolymeralsousefulas

scaffoldforcellulargrowthandtissueengineering(Bäckdahletal.,

2006;Heleniusetal.,2006;Ramboetal.,2008;Svenssonetal.,

2005).ItisdistinguishedfromtheusualscaffoldsbecauseBC

pos-sessesnaturalrefined3-Dnanofibrilsnetworksbearingashape

similartothatofthecollagennanofibrilsinnaturaltissuesuchas

umbilicalcord(Bäckdahletal.,2006)andbasementmembranein

cornea(Fraseretal.,2008)

DuetothisuniformstructureandmorphologyBCisendowed

withuniquecharacteristicssuchashighpurity,highcrystallinity

andremarkablemechanicalproperties,goodchemicalstability,and

thehighwaterholdingcapacity(Svenssonet al.,2005).Despite

itshighwatercontent,BCshowsagoodmechanicalperformance

andBCcanbeproducedinalmostanyshapebecauseofitshigh

moldabilityduringformation(Ross,Mayer,&Benziman,1991)

BCisusedasawounddressingsinceitprovidesamoist

envi-ronment,resultinginbetterwoundhealingwithnotoxicity(Barud

etal.,2007;Klemm etal.,2005).Besidesthat,BCoffersa wide

rangeofapplications,especiallyinmedicalapplications,artificial

microvessel,andtissueengineeringofcartilageandbone(Fontana

etal.,1990;Klemm,Schumann,Udhardt,&Marsch,2001;Svensson

etal.,2005).Otherstudieswithendothelial,smoothmusclecells

andchondrocyteshaveshownthatthesecellspresentgood

adhe-siontobacterialcellulose(Bäckdahletal.,2006;Heleniusetal.,

2006;Ramboetal.,2008;Svenssonetal.,2005)

However,somecharacteristicsthatwouldlimitBCinmedical

applicationsisthatBCisnoteasilyabsorbedinhumanbody;in

driedstateBCnanofibrilsforma densemeshthatcanlimitcell

adhesionandproliferation(Bäckdahletal.,2006).Besidesthat,BC

hasnoantibacterialpropertiesandactsonlyasaphysicalbarrier

againstinfection(Czaja,Young,Kawecki,&Brown,2007)

Polymer composites have enhanced material and biological

propertiescomparedtopurepolymers.Basedonthenatureand

sizeofthereinforcementmaterial,BCcompositesaresynthesized

throughnumerousroutesaimingtoovercomeitslimitationsand

increaseitsapplications

Literature displaysseveral composites basedon BC, suchas

BC/chitosan (Kim et al., 2011), BC/agarose (Yang et al., 2011),

BC/poly(3-hydroxybutyrate)(PHB)(Barudetal.,2011;Barud,Caiut,

Dexpert-Ghys, Messaddeq, & Ribeiro, 2012), BC/hydroxyapatite

(Hap) (Grande etal., 2009; Saskaet al.,2011)and BC/Collagen

(Luo et al., 2008; Saska et al., 2012) These bacterial cellulose

based materials have been commercialized and recognized as

non-genotoxicandnon-cytotoxic(Jonas &Farah,1998;Schmitt,

Frankos,Westland,&Zoetis,1991).Amongtheinvestigated

mate-rialsthatcouldpossiblybeassociatedwithBC,wehaveoptedto

usesilkfibroin(SF)

Fibroinisanaturalproteinextractedfromsilkcocoonsof

Bom-byxmori silkwormthat canbeprocessed tocreate avariety of

materialssuchashydrogels,ultrathinandthickfilms,3Dporous

matrices, and fibers with controllable diameters (Omenetto &

Kaplan,2010).Thisproteinisalsoapotentialcandidatematerialfor biomedicalapplicationsbecauseithasseveralattractiveproperties, includinggoodbiocompatibility,goodoxygenandwatervapor per-meability,andbiodegradability(Altmanetal.,2003;Wang,Blasioli, Kim,Kim,&Kaplan,2006)thatcanbecontrolledby functionaliza-tionoffibroinorchangingtheprocessingmethods

SFrevealssomeknownapplicationslikethepreparationof scaf-foldsfor bone and meniscusregeneration (Altman et al.,2003; Bhardwajet al.,2011; Kim, Jeong,etal., 2005;Kim, Park, Kim, Wada,&Kaplan,2005;Mandal,Park,Gil,&Kaplan,2011;Mauney

etal.,2007;Zhanget al.,2010), small-diametergraftfor vascu-larsubstitution(Alessandrinoetal.,2008;Cattaneoetal.,2013; Enomotoetal.,2010;Marelli,Alessandrino,Fare,Tanzi,&Freddi, 2009;Marellietal.,2010)andtransparentthinfilmsfor biopho-tonics(Amsdenetal.,2010)

Inaddition,SFasproteinhasaminoacidsthatactascell recep-torsandmediateimportantinteractionsbetweenmammaliancells and extra cellular matrix (ECM) facilitating cell adhesion and growth(Fang,Chen,Yang, &Li,2009; Fang,Wan,Tang,Gao, & Dai,2009)anditpresentsantimicrobialactivity(Lietal.,2011) However,theregeneratedSFhassomedisadvantages,suchas brit-tleness,easyfragmentation,and difficultyincreatinga uniform thickness(Lee,Kim,Lee,&Park,2013)

Some authors have prepared plate BC/SF composites and observedtheimprovementofthemechanicalpropertiesofSF(Choi, Cho,Heo,&Jin,2013)andothersappliedtheminananimalmodel

topromotethecompletehealingofsegmentaldefectsofzygomatic arch(Leeetal.,2013)withoutpreviouslystudyingitinvitro.Despite bothmaterialsbeingseparatelybiocompatible,itisimportantto demonstrateifthisnewcompositecanbesafelyappliedintissue regeneration

Thus, theaimof this studywas toprepareporousscaffolds basedonBCandSFbylyophilizationprocess,inordertomaintain theirpropertiesandcomplementeachotherasacomposite,taking advantageofBC’ssurfacemodificationwithaminoacidsextracted fromSF.Towardmeetingtheseobjectives,theresultant nanocom-positeswerecharacterizedbyphysicochemicaltechniquesandthe cytocompatibilitywasassessedbytheinvestigationofthe cytotox-icityandgenotoxicityofthedevelopedmaterial

2 Experimental

2.1 Materials 2.1.1 Bacterialcellulose Bacterial cellulose membranes were obtained from cultiva-tionoftheGluconacetobacterhanseniistrainATCC23769.Cultures wereincubatedfor96hat28◦Cintrays30cm×50cm, contain-ingmediumcomposedofglucose50gL−1,yeastextracts4gL−1, anhydrous disodium phosphate 2gL−1, heptahydrated magne-sium,sulphate0.8gL−1andethanol20gL−1.Afterthreedaysof incubationhydratedBCpelliclesof3mmofthicknesscontaining

upto99%ofwaterand1%ofcellulosewereobtained.These mem-braneswerewashedina1wt%aqueousNaOHat70◦Cinorder

toremovebacteriaandthenseveraltimesinwater,untilneutral

pH.Pristinebacterialcellulosemembranes(25cm2)wereusedfor nanocompositepreparation

2.1.2 Silkfibroinsolution Silkfibroin(SF)solutionwasobtainedfromsilkcocoons pro-ducedbyBombyxmorisilkwormssuppliedbyBratac,Fiac¸ãode SedaS.A.(Bastos/SP,Brazil).Themethodwasbasedonprevious reportsfromliteratures(Kweon,Ha,Um,&Park,2001;Rockwood

etal.,2011).Rawsilkwasdegummedwith0.02MNa2CO3solution

at100◦Cfor30minandwashedthoroughlywithdistilledwater DegummedsilkwasdissolvedinasolutioncomposedofCaCl2,H2O,

ofthesolvent.Theresultingviscoussolutionwasdialyzedagainst

mili-Qwaterfor48hinordertoremovesalts.A3.7%(w/V)aqueous

fibroinsolutionfreeofimpuritieswasobtainedafterthe

centrifuga-tion(twice)ofthedialyzedsolutionat20,000rpmat4◦Cfor30min

ThefinalconcentrationofaqueousSFsolution(3.7wt.%)was

deter-minedbyweighingthedriedsolids.Thefinalsolutionwasstored

at4◦C beforeuse.Thestockfibroinsolution(3.7%)wasusedto

prepareBC/fibroincomposites

2.1.3 Bacterialcellulose/silkfibroinnanocomposites

Porous composites of BC/SF were prepared by soaking BC

membranes(25cm2)intosilkfibroinsolutionsofdifferent

concen-trations(1%,3%and7%ofSFcontent(w/v)inordertoexchange

waterbythesilkfibroinsolutionintothemicrofibrillarcellulose’s

network.Themembraneswerekeptinfibroinsolutionsfor24h

undershaking,removedandfreezedried

ThefinalSFcontentsinBC/SFnanocompositesweredetermined

bythemasspercentagesofSF.TheremainingSFinsolutionwas

measuredandsubtractedfromtheinitialSFamountinorderto

calculateSFboundtotheBCmembranes.Samplesweretermed

accordingtoSFcontentsBC/SF:25%,BC/SF:50%andBC/SF:75%.They

wereallpackedandsterilizedwitha25kGy gammairradiation

(Embrarad–Brazil)

2.2 Fieldemissionscanningelectronmicroscopy

Scanningelectronmicroscopy(SEM)imageswereobtainedwith

theuseof a Field EmissionScanningElectron MicroscopeJEOL

JSM–7500F.Freeze-driedscaffoldswerecarefullysectionedat

hori-zontalplanewitharazorblade,mountedwithconductiveadhesive

tapeoncopperstubs,andsputter-coatedwithacarbonlayer

2.3 Solubilitytest

BC/SFnanocompositeswerecutintopiecesof1.0cm2,weighed

(mi),immersedin20mLofdistilledwaterand keptat37◦Cfor

24h.Afterthisperiod,thesamplesweredriedandweighedagain

(mf.Thepercentageofsolublemasswasdeterminedbythe

fol-lowingequation.Allexperimentswereperformedintriplicate,and

standarddeviationwascalculated

Solublemass (%)= (mi−mmf)100

i 2.4 Water-uptakecapacity

Theswellingratiowascalculatedbyplacingseparatelypristine

freeze-driedBCandBC/SF:50%samplesof1.0cm2indistilledwater

foraspecifictime.Sampleswereremovedatcertaintimes,initially

at1and5min,followedbymeasurementsevery5minupto30min,

andthen60,360and1440min.Afterremovalfromthedistilled

water,excesssuperficialwaterwasremovedbygentletappingwith

filterpaper;thenthesampleswereweighed.Thecontentofthe

distilledwaterintheswollenscaffoldswascalculatedbythe

fol-lowingequation:wateruptake(%)=[(Ws−Wd)/Ws]×100,where

WdistheweightofthedrymembraneandWsistheweightofthe

swollenmembrane,respectively.Allexperimentswereperformed

intriplicate,andstandarddeviationwascalculated

2.5 MeasurementofSFreleasewithtime

ThefibroinproteinconcentrationwasmeasuredbytheBradford

proteinassayprocedure(Bradford,1976),inordertoinvestigatethe

stabilityofscaffoldsinaqueousenvironment.Samplesof1.0cm2

BC/SF:50%scaffoldswereimmersedin 5mLofdeionizedwater

TheBradfordreagentwasaddedandthesampleswereincubated

at30◦C,inthedark.Thealiquotswerecollectedat5,15,30,60,720 and1440min.TheBradfordassayreliesonthebindingofthedye CoomassieBlueG-250toprotein.Therefore,thequantityofprotein canbeestimatedbydeterminingtheamountofdyeintheblueionic form,usuallyachievedbymeasuringtheabsorbanceofthesolution

at595nm.Bovineserumalbuminwasusedasastandardprotein 2.6 FT-IRspectroscopy

InfraredspectrawererecordedonaSpectrum2000FT-IRPerkin Elmerspectrophotometer,usingsamplespreparedasKBrpellets Thespectrawerecollectedovertherangeof4000–700cm−1with

anaccumulationof32scans,resolutionof2cm−1andintervalof 0.5cm−1

2.7 Thermogravimetricanalysis Thermogravimetry(TG)wasconductedusingdriedsampleson

aThermoanalyzerTG/DTAsimultaneousSDT2960TAInstruments underthefollowingconditions:aluminumcrucible,syntheticair (100mL/min),andaheatingrateat10◦Cperminute,from30to

1000◦C

2.8 PowderX-raydiffractometry X-raydiffractionpatterns(XRD)wereobtainedinaSiemens KristalloflexdiffractometerusingnickelfilteredCuK-␣-radiation from4◦to70◦(2angle),instepsof0.02◦andasteptimeof3s 2.9 Porositystudy

A porosity study wasconducted by examining SEM surface imagesofthecompositethatwillbeusedtoperforminvitrotests applyingImageJsoftware.Thecalculationwasbasedon30 diame-termeasurementsofdifferentporestoestimateaverage,coefficient

ofvariationandconfidencelevelofdataobtained

2.10 Cytotoxicityandgenotoxicityassays These testswere performedonly in case ofBC/SF:50% sam-ples.Cytotoxicitycanbemeasuredbymultipledifferentmethods depending on the cell damage: changes in plasma membrane are detected by using dyes, such as Trypan Blue, and changes related tometabolicfunctionsofmitochondriacan bedetected

by a colorimetric method such as MTT (tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Kim, Yoon,Lee,&Jeong,2009)andXTT (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide),accordingtothe manufacturer’sinstructions

Thus,thecytotoxicityassayrequiresaselectionbasedonthe suitabilityofthecellemployedin thetest method.Thepresent studyusedL929cellsfortheMTTtestwhichisinaccordancewith ISO standards (10993-5:2009) and Chinese hamster fibroblasts (V79cells)fortheXTTmethodaccordingtoISO10993-12:2007 thatgoalinvitrocytotoxicitytestsofbiomedicaldevices

2.10.1 Preparationofthesamplesforinvitrotests

AsmentionedinSection2.1.3,freezedriedBCandBC/SF:50% scaffolds previously sterilized were then soaked in RPMI 1640 medium (Invitrogen)for 1h in 24-well plates to facilitate cell adhesion.L-929cellswereliftedwithtrypsin/EDTA(Invitrogen), washedtwicewithsalinebycentrifugation,seededontothe scaf-folds(10␮L/scaffoldat5×105cells/mL),andallowedtoattachfor

1hat37◦,5%CO2.OnemLofRPMImediumsupplementedwith

2mMl-glutamine,100U/mLpenicillin,100␮g/mLstreptomycin,

10%FCS (all fromInvitrogenTM) and0.05M␤-mercaptoethanol

wasaddedtowellsandcellswerecultivatedfor48h.All

treat-ments,negativecontrolandalsopositivecontrolwereperformed

inquintuplicateandinconditionsofsterility

TheV79celllinewasmaintainedandcultivatedasmonolayers

inplasticcultureflasks(25cm2)containingHAM-F10plusDMEM

(1:1;Sigma–Aldrich),supplementedwith10%fetalbovineserum

(Nutricell)and 2.38mg/mLHepes (Sigma–Aldrich) at37◦C in a

humidified5%CO2atmosphere.Antibiotics(0.01mg/mL

strepto-mycinand 0.005mg/mL penicillin; Sigma–Aldrich) were added

tothemediumtopreventbacterialgrowth.TheBC/SF:50%was

extractedwithHAM-F10plusDMEM(1:1)for72hat37◦C and

sonicatedfor1hbeforetreatment.Immediatelypriortouse,the

culturemediumwastransferredtoanotherflaskandfetalbovine

serumwasaddeduptovolume.Thissolutionwassetasareference

as100%

2.10.2 MTTassay

Afterthepreparation, thesampleswereremovedfromeach

wellandthecultures werewashed with250␮L ofsaline

solu-tion.With thelaminar flow light off, 200␮L of M199 medium

(InvitrogenTM)withoutphenolredsolutionwithMTT(final

concen-tration500␮g/mL)wereplacedperwell.Theplateswereincubated

at37◦Cforatreatmentperiodof4h.Then,thecolorimetric

mea-surementwasverifiedwithspectrophotometerspectraMax190

(MolecularDevices)at570nmand690nm,andtheresultallowsan

OpticalDensitymeasurement(OD)analysis.Thedarkerthecolor,

thehigherthemetabolismofMTTandconsequently,thehigherthe

ODandlesscytotoxicisthematerialtested

2.10.3 TrypanBlueassay

TrypanBlueSolutionisroutinelyusedasastaintoassesscell

viabilityusingthedyeexclusiontest.Thistestisoftenperformed

whilecountingcellswiththehemocytometerduringroutine

sub-culturing,butcanbeperformedanytimecellviabilityneedstobe

determinedquicklyandaccurately.Thedyeexclusiontestisbased

upontheconceptthatviablecellsdonottakeupimpermeabledyes

(likeTrypanBlue),butdeadcellsarepermeableandtakeupthedye

Aftertheinitialpreparation(seeSection2.10.1),thepercentageof

viablecellswasdeterminedbycounting200cellsinatleastfive

randomfieldsusingtheinvertedlightmicroscopeinthepresence

of50␮LofTrypanBlue

2.10.4 XTTassay

For this experiment, 104 cells were plated onto 96-well

microplates.Eachwellreceived100␮LHAM-F10/DMEM

contain-ingdifferentpercentagesofBC/SF:50%rangingfrom0.78%to100%

Thecellswereculturedina5%CO2atmospherefor24hat37◦C

Afterincubation,theculturemediumwasremovedandthecells

werewashed with100␮Lphosphate-buffered saline(PBS) and

exposedto100␮LHAM-F10culturemediumwithoutphenolred

Next,25␮LXTT(RocheDiagnostics)wasaddedtoeachwelland

themicroplateswereincubatedfor17hat37◦C.Absorbanceof

thesampleswasreadin amicroplate reader(ELISA, AsysUVM

340/Microwin 2000) at a wavelength of 450nm and a

refer-encelength of 620nm The amountof soluble product formed

(formazan)wasproportionaltothenumberofviablecells.The

neg-ativecontrolgroupwasdesignatedas100%,andtheresultswere

expressedasapercentageofthenegativecontrol.Cytotoxicitywas

calculatedwiththeGraphPadPrismprogram,plottingcellsurvival

againsttherespectivepercentagesofmaterial.One-wayANOVA

wasusedforthecomparisonofmeans(p<0.05).Theexperiments

wereperformedintriplicate

2.10.5 Celladhesionandproliferationassays Aftertheinitialpreparation,thenumberofcellsseededonthe scaffolds wasdetermined bycounting in a Newbauer hemocy-tometerchamberbytreatmentwithtrypsin/EDTA.Scaffoldswere thencollectedandthecellculturewashedtwicewithRPMI1640 medium(Invitrogen).Cellswerefixedinasolutionof2.5% glu-taraldehydegradeII,2%formaldehydeand2.5mMCaCl2in0.1M sodiumcacodylatebufferpH7.0andprocessedforthepreparation

ofSEM

Regardingcellproliferation,treatmentswereperformedin con-tactwiththecells(atthefollowingexperimentalperiods:16,24,

48 and72h)by placinga diskofabout0.8cm diameterofthe biomaterialsineachwell.Aftertherespectiveperiods,cellswere removedfromthewellswithtrypsin-EDTA0.25%V/V(GIBCO), cen-trifugedat1300×grpmand40◦Cfor10min,resuspendedin20␮L

ofTrypanBlueandthencountedinaNewbauerhemocytometer chamber

2.10.6 Assessmentofgenotoxicity Themicronucleus assayin V79cells was employedto eval-uatethe genotoxicityof BC/SF:50%samples.Therefore, 500,000 cellswereseededintotissue-cultureflasksandincubatedfor24h

in 5mLcompleted HAM-F10/DMEM medium and washed with PBS(pH 7.4).Aftertheseprocedures, thecultures weretreated

inserum-freemediumfor3hwiththreedifferentpercentagesof BC/SF:50%(25%,50%and100%).Weincludedthenegative (with-out treatment) and positive (MMS – methylmethanesulfonate,

44␮g/mL)controlgroups.ThecellculturestreatedonlywithBC (100%)werealsoincluded.Afterthetreatmentperiod,thecells werewashedwithPBSandaculturemediumsupplementedwith fetal bovine serum containing 3␮g/mL of cytochalasin-B (CAS: 14930–96-2; Sigma–Aldrich) and the cells were incubated for

17h Aftertheincubation,thecells wererinsedwith5mLPBS, trypsinizedusing0.025%trypsin-EDTAandcentrifugedfor5minat

900rpm.Thepelletwashypotonizedinsodiumcitrate1%at37◦C andthenhomogenizedwithaPasteurpipette.Thiscellsuspension wascentrifugedagain,thesupernatantwasdiscarded,thepellet wasresuspendedinmethanol:aceticacid(3:1)andhomogenized againwithaPasteurpipette.Afterfixation,thecellswerestained

inaGiemsasolution5%.Thecriteriaemployedfortheanalysisof micronucleuswereestablishedbyFenech(2000).Therefore,1000 binucleatedcellswereanalyzedbycultureinatotalof3000 bin-ucleatedcellspertreatment.Thenucleardivisionindex(NDI)was determinedfor500cellsanalyzedperculture,foratotalof1500 cells per treatmentgroup.Cells withwell-preservedcytoplasm containing1–4nucleiwerescoredandtheNDIwascalculatedusing thefollowingformula(Eastmond&Tucker,1989):

IDN=[M1+2(M2)+N3(M3)+4(M4)]

whereM1–M4isthenumberofcellswith1,2,3,and4nuclei, respectively,andNisthetotalnumberofviablecells

3 Results and discussion

3.1 Infraredspectroscopy FT-IRspectraforBC,SFandallBC/SFnanocompositesareshown

in Fig 1 The spectrum obtained for BC Fig 1(a) shows bands

inthe400–700cm−1rangecharacteristicsoftheOHbending, ␤-glucosidiclinkagesbetweentheglucoseunitsat∼896cm−1 and

C OsymmetricstretchingofprimaryalcoholandC O C antisym-metricbridgestretchingat1040cm−1and1168cm−1,respectively TheC Hdeformation(CH3orO Hinplanebending)isobserved

at1340cm−1andthebandcenteredat1400cm−1isrelatedtoCH2 bendingandOHinplanebending.AccordingtoBarudetal.(2008),

Fig 1.FT-IR spectra: (a) pure freeze dried BC membrane, (b) freeze dried BC/SF

composite (25% SF), (c) freeze dried BC/SF composite (50% SF), (d) freeze dried BC/SF

composite (75% SF) and (e) pure freeze dried SF.

otherbandsarerelatedtoH O Hbendingofadsorbedwater(at

1650cm−1),CHstretchingofCH2andCH3groups(at2900cm−1)

andOHstretching(broadbandat3500cm−1)

The obtained spectra related to pure silk fibroin, Fig 1(e),

presentbandsintheregionfrom1500to1700cm−1 assignedto

absorptionbythepeptidebackbonesofamideI(1700–1600cm−1)

andamideII(1500–1600cm−1),whichhavebeencommonlyused

fortheanalysisofdifferentsecondarystructures offibroin.The

peaksat1610–1630cm−1(amideI)and1510–1520cm−1(amide

II)arecharacteristicofsilkII(␤-pleatedsheet)secondary

struc-ture while the absorptions at 1640–1660cm−1 (amide I) and

1535–1542cm−1areindicativeofsilkI(␣-form)conformation(Lu

etal.,2011;Nazarov, Jin,&Kaplan,2004).Inthepresentstudy

theamideIbandoffreezedriedfibroinshowedstrongpeaksat

1647and1537cm−1correspondingtosilkIstructure.Howevera

shoulderat1620cm−1indicatestheformationofsomesilkII

For all BC/SF composites, cellulose bands in the region

1000–1300cm−1 areexactlyobservedatthesamepositionsand

samerelative intensitiesaspristineBCspectrashows.Thereare

noimportantbandschangingaftersilkfibroinimpregnationinthe

nanocomposites.InfactFTIRspectraforallBC/SFnanocomposites

couldbeconsideredonlyasumoftheBCandsilkfibroinspectra

andnonewcovalentbondsweredetected,accordingtoKweon

etal.(2001)

3.2 X-raydiffractionanalysis

Fig.2 shows XRDpatterns for allsamples.Broad diffraction

peaksareobservedat15◦ and22.5◦ forthepureBCmembrane

Thesepeaksareassignedtothecharacteristicinterplanedistances

ofnativecellulosetype1(Kim,Jeong,etal.,2005;Kim,Park,etal.,

2005)

FreezedriedSFpresentspeaksat11.8◦,19.8◦and22.6◦

corre-spondingtotheSilkIcrystallinephase.Thebroadpeaksshouldbe

duetothefreezedryingprocess(Ming&Zuo,2012)

TypicalcellulosetypeIpatternsarepreservedintheBC/SF25%

nanocomposite,Fig.2(b),andtherearenosignificantchangeswhen

(e )

(d ) (c )

(b )

2 θ(d e gre es)

(a )

Fig 2.XRD diffraction patterns: (a) pristine freeze dried BC, (b) freeze dried BC/SF composite (25% SF), (c) freeze dried BC/SF composite (50% SF), (d) freeze dried BC/SF composite (75% SF) and (e) pure freeze dried SF.

comparedtothepristineBCdiffractogram.Fig.2showsdiffraction patternscharacteristicsofthesuperimpositionofBCandSF pat-ternasobserved.BCdiffractionpatternsoverlaptheSFpatternin thecompositesasobservedbyLeeetal.(2013).So,itisnot pos-sibletoelucidatethetypeofSFcrystalpattern (silkIorsilkII)

inthesecomposites.XRDdiffractogramforBC/SF:75% nanocom-positescomprisesanamorphousprofileprobablyduetothelarge amountofsilkfibroindepositedinsidetheBCporousasobserved belowinSEMimages

3.3 Thermogravimetricanalysis

Fig 3 shows TG/DTG curves The curve obtained for freeze driedBCdisplaystwomasslosses.Thefirstone,occurringfrom room temperature to 200◦C is due to evaporation of the sur-facewater(∼4.6%)(DeSalvi,Barud,Caiut,Messaddeq,&Ribeiro,

2012).Thesecondmoresignificanteventrelatedtoahighmass loss(80%) beginsat about280◦,withmaximumdecomposition (Tonset)at373.7◦C.Thiseventisassociatedtothermaldegradation, relatedtodepolymerizationanddecompositionof dehydrocellu-loseintogases(water,carbonmonoxideandcarbondioxide)(Sofia, McCarthy,Gronowicz,&Kaplan,2001)

TheinitialmasslossforfreezedriedSFisrelatedtowaterloss and startsfromroomtemperatureto120◦C (7.3%).Thesecond eventinvolvingmassloss(52%)occursinthetemperaturerange

of180–500◦Cwithmaximumdecomposition(Tonset)at281.4◦C Thiseventisassociatedwiththebreakdownofsidechaingroups

ofaminoacidresidues,aswellasthecleavageofpeptidebonds (Nogueiraetal.,2009)

TGcurvesobtainedforBC/SFcompositespresentthree most important masslossesand display a compositionof theevents observedfortheindividualBCandSFcomponents

Thefirstoneisacontinuousmasslossofaround7%fromroom temperatureuptoaround200◦Cwhichisassociatedwiththewater lossesandispresentinallBC/SFcurves.Next,twomajoreventsin therangeof200–500◦Carepresentinallsamples.Thefirstoneis attributedtofibroindecompositioncounterpartsandthesecond referstoBCdecomposition

100 200 300 400 500 600

0 20 40 60 80 100

Temperature/ºC

(a)

373.7

0 20 40 60 80

(c)

369.4

0 20 40 60 80 100

Temperature/ºC

(e)

281.4

0

20

40

60

80

100

300,9

(b)

0

20

40

60

80

100

Temperature/ºC

(d)

305.3

Fig 3.TG curves: (a) pristine freeze dried BC, (b) freeze dried BC/SF composite (25% SF), (c) freeze dried BC/SF composite (50% SF), (d) freeze dried BC/SF composite (75% SF) and (e) pristine freeze dried SF.

TheDTGpeakareaisdirectlyproportionaltomassvariations

andcouldbeusedtocomparetheratiosbetweenpeaksheights

forallcomposites.ThefirstDTGpeak,whichisassociatedwiththe

fibroindecomposition,becomesmoreintensewhenSFcontents

increaseinnanocomposite.BC/SF:50%,Fig.3(c)showsthesame

peakheightforthefirstandsecondDTGpeaks;itisquite

rele-vantduetotheBCandSFpercentage ratiois ofBC/SF50:50in

thiscomposite.InthesamewaytheTG/DTGcurveforBC/SF:75%

sample denotes great similarity with the TG/DTG of the

pris-tinefibroin.TheheightofthefirstDTGpeakassociatedwiththe

fibroindecompositionis largerthanthefirstpeakattributedto

BCdecomposition.Theseresultscorroboratewiththegravimetric

measurementswherebythepercentageoftheBC25:75fibroinin

thisnanocompositewasdetermined.Thisbehaviorisduetohigher

SFcontentinthissample

TG/DTG resultsalso indicate that BC and SF decomposition eventsoccurseparately,andweakinteractionsbetweenBCandSF suggestedinFTIRanalysisarethereforeconfirmed

3.4 Fieldemissionscanningelectronmicroscopy(FEG-SEM) SEMimagesarepresentedinFig.4.Surfaceimagesshowthat freeze-dried BCexhibits a 3-D network nanofibrilsin theform

ofaheterogeneousporousstructurethatisobservedinFig.4(a) TheseporousstructuresofBCspongesaresimilartothatof col-lagenspongesreportedbyMizuno,Watanabe,andTakagi(2004)

Fig 4. FEG-SEM images taken at the same magnification (bar – 1 ␮m): (a) pure freeze dried BC, (b) freeze dried BC/SF 25% composite, (c) freeze dried BC/SF 50% composite and (d) freeze dried BC/SF 75%.

ItisimportanttonotethatthemethodemployedforBC

cultiva-tioncouldalterthemorphologyandporosityofthefinalcellulose

membrane

SEM images for BC/SF:25% and 50% nanocomposites reveal

a sponge-like structure where BC nanofibersand fibroin

struc-tures are easily discerned BC/SF samples exhibit a very well

interconnectedporousnetworkstructureformedbyrandom

nano-filaments entangled with each other presenting a large aspect

surface These characteristicsalso suggest that the presence of

fibroinhasmodifiedthesurfaceof BCnanocompositesand this

modificationmaybeinducedbyfibroinconcentration.SEMimage

forBC/SF:75%samplepresentedalessporousstructureduetothe

coatingoftheBCnanofibrilsbyexcessoftheSFsolution

Then,thebestoutcomeobtainedinthisstudywastheonewith

50%ofSFand50%ofBCcontent.Theresultofthisblendof50%of

eachbiomaterialresultsinacompositethatisintendedtopreserve

BCandSFproperties

3.5 Porositystudy

Intotal30diametermeasurementsofdifferentporesby

exam-iningSEMsurfaceimagesofBC/SF:50%compositeswereexamined

Afterstatisticalanalysistheresultsrevealedaporesizerangeof

102±5.43␮matthesurfaceofthescaffolds

Asa scaffoldfortissueengineering,macroporesarerequired

toallowforcellincorporation,migration,proliferationandtissue

growthintothescaffold,accordingtoChenetal.(2002).But,onthe

otherhandtheliteraturealsodemonstratesthatthereisnogeneral

consensusregardingtheoptimalporesizeforcellgrowthand

tis-sueformation.Zeltinger,Sherwood,Graham,Mueller,andGriffith

(2001)foundthatvascularsmoothmusclecellsshowedequalcell

proliferationandECMformationinporesranginginsizefrom38

to150␮m.Zhang et al.(2010) showedthat poresizesranging

from100to300␮mdisplayedhumanbonemarrowmesenchymal

stromalcells(BMSCs)proliferationandECMproductionapplying silkfibroinscaffolds.Theseauthorsalsoobservedthateveninthe presenceofsmallporesof50–100␮mrange therewereBMSCs proliferationandECMproductionoccurred,butinlessquantity

In terms of porosity of BC/SF:50% scaffolds, our findings (102±5.43␮m)areinaccordance withtheseprevious observa-tionsand alsowithBhardwajandKundu(2011).Theyprepared SF/ChitosanandpureSFscaffoldsfortissue regenerationandin termsofporosityresultsinarangeof100–155and90␮mwere identified

Oneobviouschallengeoftissueengineeringisthedesignand fabrication of the 3-D polymer scaffolds composed of refined nanofibrilswithhierarchicalporestructureincludinglargepores and nanoporestomimic theorganizationof ECMs(Kim etal.,

2011).Gaoetal.(2011)producedBCspongesbyfreeze-drying tech-nique,withlargeandnanoporeswithahighsurfaceareaandthey demonstratedthatthematerialexhibitedexcellentcellcompatible

asfibroussynoviumderivedMSCscouldproliferatewellandgrow insidetheBCsponges

Thustheresultsobtainedinthisstudyfurthersupportedbythe literatureleadustoconcludethattheproducedscaffoldscould sup-portBMSCsproliferationandECMproduction,astheporesizesof

ascaffoldmatrixaffectthecelladhesion,proliferationand direc-tionalgrowth,accordingtoBodinetal.(2010)

3.6 Watersolubilitytest PristineSFspongepresentsabout35.4%ofsolubilityinwater,in

aperiodof24h.Ontheotherhand,probablySFbecamemore insol-ubleincontactwithBCnanofibrils.Thus,BC/SF:25%nanocomposite presented0%ofsolubilityindistilledwater,BC/SF:50%,3.0%,and BC/SF:75%,8.6%,aspresentedinFig.5(a).TheincreaseonSF releas-ingmayberelatedtotheexcessofSFattachedtonanocomposites surface,asobservedbySEMimages

Fig 5.(a) Water solubility test and (b) water-uptake capacity, expressed in

per-centage (%).

3.7 Water-uptakecapacity

Thewater-uptakecapacityisimportanttocheckifthematerial

hasthepropertytodiffusewater,sincethewaterdiffusionallows

thetransportofnutrients,andhelpsthegrowthofnewcells.The

resultsshowedthatafter24h,thewateruptakeabilityofthe

pris-tineBCandBC/SF:50%scaffoldswereabout600%(Klemmetal.,

2001)and216%,respectively(Fig.5(b)).Allthescaffoldsabsorbed

waterwithin1min;andtheyweresaturatedwithin1h.This

behav-iorisdue totheuniquechemical andphysical structuresofBC

andSF.Thereisdiminishingontheswellingabilityobservedfor

BC/SF:50%scaffold.Thisdecreaseinthecapacityofabsorptionmay

berelated tothereduction of theamounts of BCporesdue to

SFpresence,thathadcoveredandobliteratedBCsurface,asSEM

imagesshowinFig.4

3.8 MeasurementofSFreleasewithtime TheBradfordassaydemonstratedthatnosignificantamount

ofsilkfibroinproteinwasreleasedtothesolutionintheinitial measurements.After24hthefinalconcentrationofreleased pro-teinwas0.89mg/mL.Regardingtheamountofproteinreleasedwe believethatitmayberelatedtotheexcessofsilkfibroinattachedto thescaffoldsincetheamountofproteininsolutionhasnotchanged after24h,indicating thatBC/SF:50%scaffoldremainedstablein aqueousenvironment.OurresultsareinaccordancewithSahand Pramanik(2010)thatobservedthatafter3htheconcentrationof silkfibroinreleasedinsolutionwasabout1.7mg/mL.Theyshow thattheconcentrationofreleasedproteindidnotchangeafter3h, remainingstable

3.9 Cytotoxicityandgenotoxicityassays The statistical analysis was performed according to Kruskal WallisandDunnsinrelationtoMTTand TrypanBluetestsand MannWhitneyforcelladhesionandproliferation,byapplyingthe statisticalprogramGraphpadPrism5.0.Allinvitrotestsresults werestatisticallysignificantatp<0.05

3.9.1 MTTassay Colorimetric measurements were performed as the method described

In termsof cell viabilityfor48hbyMTT, under the experi-mental conditions appliedin this study,theresultsshowed no statisticallysignificantdifferencebetweenBCandBC/SF:50% Nev-ertheless,bothmaterialsshowednocytotoxicityandtheaverage cellviabilityfoundwas␮BC=91.25%,and␮BC/SF:50%=123.81%, respectively.TheseresultsareinaccordancewithISOstandards (10993-5)

3.9.2 TrypanBlue

Ontheotherhand,thecytotoxicityassays relatedtoTrypan Bluedyetechniqueevidencedastatisticallysignificantdifference (p=0.02).DunnsposttestpointedoutthatBC/SF:50%group pre-sentedasuperiorperformance,indicatinglesscytotoxicityanda greaternumberofviablecellscomparedtothereferences(Fig.6) AlthoughTrypanBlueresultsdifferfromthoseobtainedbythe MTTassay,bothinvitroassaysevidencedahighcellviabilitygreater than90%foralltreatments,indicatingthatthematerialis non-cytotoxicandcouldallowcellattachmentandproliferation.Despite thecytotoxicityresultsbeingmutuallyconsistentthisdifference mayoccurbecauseTrypanBlueassaymeasuresthecytotoxicity

Fig 7. Percentage of cell viability by XTT colorimetric assay in V79 cells BC –

bac-terial cellulose, SF – silk fibroin, DMSO – dimethylsulfoxide *Statistically different

to the negative control.

bycellmembrane integrity,whileMTTmeasurestheactivityof

mitochondrialdehydrogenases

3.9.3 XTTassay

Thepercentageofcellviabilityfoundforeachtreatmentis

pre-sentedinFig.7.TheresultsshowedthatthelyophilizedBC/SF:50%

aswellasBCtreatmentsdo notindicatestatisticallysignificant

differenceswhencomparedtothenegativecontrolinall

concen-trationtested,revealingthelackofcytotoxicity.Cellviabilityofthe

testedmaterialwasgreaterthan95%

3.9.4 Celladhesionandproliferation

Totestthehypothesisthatfibroinincreasescelladhesioninto

cellulosescaffolds,L-929cells wereseededintothepristineBC

scaffoldsandBC/SF:50%nanocomposite.Cellswereliftedupand

countedinhemocytometerchamber.Inrelationtocellproliferation andadhesionassaysthevaluefoundwassignificantatp=0.04 TheimagesofFig.8showthatthecellsseededonpureBCand BC/SF:50%scaffoldssurface(a,b)didnotmigrateintothe mate-rial(c,d).Thisfactwasexpectedbecausethedensestructureof

BCnetworksdisplaysaporesizenotlargeenoughtoallow migra-tionandconsequentlycomplex3Dscaffoldscouldnotbeobtained (Bäckdahletal.,2006).However,thepresenceofSFinduceda sig-nificantincreaseincelladhesiononBC/SF50%nanocompositesin relationtothepurecellulosescaffolds(p<0.05)

Theresultsprovidedbycytotoxicitytestsaddedtothe anal-ysisoftheseimagesindicatethatBC/SF:50%composite displays highercellviabilityincomparisontopureBCintermsoffibroblast adhesion.ThecellsobservedinpureBCscaffoldremained round-shapedwhereasthecellsthat adheredtotheBC/SF50%scaffold arecompletelyspreadoverthesurface,withthepresenceofmany pseudopodiaformingalayer,suggestingthatcellsstretchingtheir morphologywereproliferating

Itisknownthattheporesizesofascaffoldmatrixaffectthe celladhesionandproliferation.Theresultsobtainedinthisstudy relatedtotheporosityofthematerialwereinaccordancewiththe literature;therefore,theBC/SF:50%producedscaffoldscouldact allowingBMSCsproliferationandECMproduction

However,itisimportanttopointthatthisimprovementincells proliferationprobablyoccurredduetothepeculiarcompositionof silkfibroin.Fibroinisaninsolubleproteincontainingupto90%of theaminoacidsglycine,alanine,andserinethatformcrystalline

␤-sheetsinsilkfibers(Fang,Chen,etal.,2009;Fang,Wan,etal., 2009;Fuetal.,2013).Thesetypesofproteinsusuallyexhibitgreat mechanicalpropertiesand,incombinationwiththeir biocompati-bility,provideanimportantsetofoptionsinthefieldofcontrolled release,biomaterialsandscaffoldsfortissueengineeringand med-icalapplications

Fig 8.To test the hypothesis that the addition of silk fibroin to cellulose scaffolds increases cell adhesion (48 h), L-929 cells were seeded in BC and BC/SF scaffolds SEM images of the cells attached to BC (a) and BC/SF (b) scaffolds surface; cross-section SEM images of BC (c) and BC/SF (d) evidenced that the cells did not migrate into the

Table 1

Frequencies of micronuclei (MN) and nuclear division index (NDI) obtained in V79

cell cultures treated with BC/SF 50% and respective controls.

Mean ± SD

NDI b Mean ± SD Negative control 7.33 ± 1.52 1.75 ± 0.02

BC control (100%) 5.66 ± 1.54 1.68 ± 0.01

MMS, methyl methanesulfonate (44 ␮g/mL).

a A total of 3000 binucleated cells were analyzed per treatment group.

b A total of 1500 cells were analyzed per treatment group.

c Significantly different to the negative control group (p < 0.05).

Otherstudies(Chiarinietal.,2003;DalPràetal.,2003;Enomoto

etal.,2010;Petrini,Parolari,&Tanzi,2001)evidencedafewmore

propertiesofsilkfibroinsuchas:it canbechemicallymodified

withadhesionsitesorcytokines,duetotheavailabilityofamine

andacidsidechainsonsomeoftheaminoacids;itpresentsslow

ratesofdegradationinvitroandinvivo,thatisparticularlyusefulin

biodegradablescaffoldsinwhichslowtissueingrowthisdesirable

According to the present findings, BC/SF:50% scaffolds

evidencedpotentialapplicationsintermsofalternativematerials

fortissueregenerationandmedicaldevices.Furtherinvestigation

suchas differentiation,osteogenic and osteoinductivepotential,

improvementsrelated toincrease BC/SFporosityandcontrolled

releaseof active ingredientsshouldbeconducted Additionally,

tests in an animal model to evaluate the performance of the

materialinrelationtospecifictissuesofapplication,areunderway

Future steps point to previously mentioned improvements to

turn the material into a more complex 3D scaffold for tissue

engineering

3.9.5 Assessmentofgenotoxicity

The micronuclei frequency and NDI obtained in V79 cells

treatedwithBC/SF:50%andrespectivecontrolsaredemonstrated

inTable1.Nosignificantdifferenceinthefrequenciesof

micronu-cleiwereobservedbetweentheculturestreated with25%,50%

and100%ofBC/SF:50%whencomparedtothenegativecontrol,

revealingthelackofgenotoxiceffect.In relationtoNDIvalues,

nosignificant differences wereobserved between thedifferent

treatmentsand negativecontrol, demonstrating theabsence of

cytotoxicity

4 Conclusions

Sponge-likenanocompositesbasedonbacterialcelluloseand

silk fibroin (BC/SF:25%, BC/SF:50% and BC/SF:75%) were

devel-oped in this work SEM evaluation results exhibit a very well

interconnectedporousnetworkstructureandlargeaspectofall

nanocompositesproduced.Itcouldbedemonstratedthatthe

pres-enceoffibroininfluencedBC/SFscaffoldssurfacecoveringandthis

aspectwasaffectedby fibroinconcentration.The bestoutcome

obtainedisrelatedto50%fibroincontent,wheretheequalratio

lettoaverygoodsymbioticeffect,preservingthespecific

prop-ertiesofBCandSF.BradfordassaydemonstratedthatBC/SF:50%

nanocompositeisstable.Theresultsforcelladhesionassayshowed

thatthepresenceoffibroininducedasignificantincreaseoncell

adhesion(BC/SF:50%)comparedtopureBCmembranes,duetothe

biologicnatureofSFcoating.Cytotoxicityassaysdemonstratedthat

thematerialis non-cytotoxicandTrypanBlueassociated tothe

SEMimagesrevealedthatBC/SF:50%scaffoldspresenthigherrates

ofcellularviabilitythanpureBC.Further,itwasfoundthatthe

preparedBC/SF:50%scaffoldledtoanimprovedbiocompatibility

comparedtopureBCscaffolds,especiallyconcerning biocompati-bilityandthesuitabilitytoinducecelladhesion.Furthermore,the genotoxicitytestrevealedthatthematerialisnon-genotoxic, indi-catingsafetyformedicalapplications.Generally,thePOCofthe BC/SFcompositescouldbedemonstrated;next,moreadjustments arerequiredtogeneratescaffoldsforcomplextissueengineering

References

Alessandrino, A., Marelli, B., Arosio, C., Fare, S., Tanzi, M., & Freddi, G (2008) Elec-trospun silk fibroin mats for tissue engineering Engineering in Life Sciences, 8(3), 219–225.

Altman, G H., Diaz, F., Jakuba, C., Calabro, T., Horan, R L., Chen, J., et al (2003) Silk-based biomaterials Biomaterials, 24(3), 401–416.

Amsden, J., Domachuk, P., Gopinath, A., White, R., Dal Negro, L., Kaplan, D., et al (2010) Rapid nanoimprinting of silk fibroin films for biophotonic applications Advanced Materials, 22(15), 1746–1749.

Bäckdahl, H., Helenius, G., Bodin, A., Nannmark, U., Johansson, B., Risberg, B.,

et al (2006) Mechanical properties of bacterial cellulose and interactions with smooth muscle cells Biomaterials, 27(9), 2141–2149.

Barnes, C., Elsaesser, A., Arkusz, J., Smok, A., Palus, J., Lesniak, A., et al (2008) Reproducible commet assay of amorphous silica nanoparticles detects no geno-toxicity Nano Letters, 8(9), 3069–3074.

Barud, H., Caiut, J., Dexpert-Ghys, J., Messaddeq, Y., & Ribeiro, S (2012) Transparent bacterial cellulose-boehmite-epoxi-siloxane nanocomposites Composites Part A: Applied Science and Manufacturing, 43(6), 973–977.

Barud, H., de Araujo, A., Santos, D., de Assuncao, R., Meireles, C., Cerqueira, D.,

et al (2008) Thermal behavior of cellulose acetate produced from homogeneous acetylation of bacterial cellulose Thermochimica Acta, 471(1–2), 61–69 Barud, H., Ribeiro, C., Crespi, M., Martines, M., Dexpert-Ghys, J., Marques, R., et al (2007) Thermal characterization of bacterial cellulose-phosphate composite membranes Journal of Thermal Analysis and Calorimetry, 87(3), 815–818 Barud, H., Souza, J., Santos, D., Crespi, M., Ribeiro, C., Messaddeq, Y., et al (2011) Bac-terial cellulose/poly(3-hydroxybutyrate) composite membranes Carbohydrate Polymers, 83(3), 1279–1284.

Bhardwaj, N., & Kundu, S (2011) Silk fibroin protein and chitosan polyelectrolyte complex porous scaffolds for tissue engineering applications Carbohydrate Poly-mers, 85(2), 325–333.

Bhardwaj, N., Nguyen, Q T., Chen, A C., Kaplan, D L., Sah, R L., & Kundu, S C (2011) Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering Biomaterials, 32(25), 5773–5781.

Bodin, A., Bharadwaj, S., Wu, S., Gatenholm, P., Atala, A., & Zhang, Y (2010) Tissue-engineered conduit using urine-derived stem cells seeded bacterial cel-lulose polymer in urinary reconstruction and diversion Biomaterials, 31(34), 8889–8901.

Bradford, M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry, 72, 248 Available at: www.ncbi.nlm.nih.gov/pubmed/942051 Cai, X J., & Xu, Y Y (2011) Nanomaterials in controlled drug release Cytotechnology, 63(4), 319–323.

Cattaneo, I., Figliuzzi, M., Azzollini, N., Catto, V., Fare, S., Tanzi, M., et al (2013).

In vivo regeneration of elastic lamina on fibroin biodegradable vascular scaffold International Journal of Artificial Organs, 36(3), 166–174.

Chen, G., Ushida, T., & Tateishi, T (2002) Scaffold design for tissue engineering macromolecular bioscience Macromolecular Bioscience, 2(2), 67–77.

Chiarini, A., Petrini, P., Bozzini, S., Dal Pra, I., & Armato, U (2003) Silk fibroin/poly(carbonate)-urethane as a substrate for cell growth: in vitro inter-actions with human cells Biomaterials, 24(5), 789–799.

Choi, Y., Cho, S Y., Heo, S., & Jin, H.-J (2013) Enhanced mechanical properties of silk fibroin-based composite plates for fractured bone healing Fibers and Polymers, 14(2).

Czaja, W K., Young, D J., Kawecki, M., & Brown, R M (2007) The future prospects of microbial cellulose in biomedical applications Biomacromolecules, 8(1), 1–12 Dal Prà, I., Petrini, P., Chiarini, A., Charini, A., Bozzini, S., Farè, S., et al (2003) Silk fibroin-coated three-dimensional polyurethane scaffolds for tissue engi-neering: interactions with normal human fibroblasts Tissue Engineering, 9(6), 1113–1121.

De Salvi, D., Barud, H., Caiut, J., Messaddeq, Y., & Ribeiro, S (2012) Self-supported bacterial cellulose/boehmite organic–inorganic hybrid films Journal of Sol-Gel Science and Technology, 63(2), 211–218.

Eastmond, D., & Tucker, J (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human-lymphocytes and an antikinetochore anti-body Environmental and Molecular Mutagenesis, 13(1), 34–43.

Enomoto, S., Sumi, M., Kajimoto, K., Nakazawa, Y., Takahashi, R., Takabayashi, C., et al (2010) Long-term patency of small-diameter vascular graft made from fibroin,

a silk-based biodegradable material Journal of Vascular Surgery, 51(1), 155–164 Fang, Q., Chen, D., Yang, Z., & Li, M (2009) In vitro and in vivo research on using Antheraea pernyi silk fibroin as tissue engineering tendon scaffolds Materials Science and Engineering: C, 29(5), 1527–1534.

Fang, B., Wan, Y., Tang, T., Gao, C., & Dai, K (2009) Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial