Carbon-Based Hierarchical Scaffolds for Myoblast Differentiation

14 Pages·2017·5.21 MB·English

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CCOORREE SScchhoollaarr
Mechanical and Materials Engineering Faculty
Mechanical and Materials Engineering
Publications
3-1-2016
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Akhil Patel
Shilpaa Mukundan
Wenhu Wang
Wright State University - Main Campus
Anil K. Karumuri
Vinayak Sant
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Patel, A., Mukundan, S., Wang, W., Karumuri, A. K., Sant, V., Mukhopadhyay, S. M., & Sant, S. (2016).
Carbon-Based Hierarchical Scaffolds for Myoblast Differentiation: Synergy between Nano-
Functionalization and Alignment. Acta Biomaterialia, 32, 77-88.
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Akhil Patel, Shilpaa Mukundan, Wenhu Wang, Anil K. Karumuri, Vinayak Sant, Sharmila M. Mukhopadhyay,
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ActaBiomaterialiaxxx(2016)xxx–xxx
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Acta Biomaterialia
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Full length article
Carbon-based hierarchical scaffolds for myoblast differentiation:
Synergy between nano-functionalization and alignment
Akhil Patela, Shilpaa Mukundana, Wenhu Wangb, Anil Karumurib, Vinayak Santa,
Sharmila M. Mukhopadhyayb, Shilpa Santa,c,d,⇑
aDepartmentofPharmaceuticalSciences,SchoolofPharmacy,UniversityofPittsburgh,Pittsburgh,PA15261,UnitedStates
bCenterforNanoscaleMultifunctionalMaterials,Mechanical&MaterialsEngineering,WrightStateUniversity,Dayton,OH45324,UnitedStates
cDepartmentofBioengineering,SwansonSchoolofEngineering,UniversityofPittsburgh,Pittsburgh,PA15261,UnitedStates
dMcGowanInstituteforRegenerativeMedicine,UniversityofPittsburgh,Pittsburgh,PA15219,UnitedStates
a r t i c l e i n f o a b s t r a c t
Articlehistory: Whileseveralscaffoldshavebeenproposedforskeletalmuscleregeneration,multiscalehierarchicalscaf-
Received14September2015 foldswiththecomplexityofextracellularmatrix(ECM)haven’tbeenengineeredsuccessfully.Byprecise
Receivedinrevisedform4December2015 controlovernano-andmicroscalefeatures,comprehensiveunderstandingoftheeffectofmultiplefac-
Accepted5January2016
torsonskeletalmuscleregenerationcanbederived.Inthisstudy,weengineeredcarbon-basedscaffolds
Availableonlinexxxx
withhierarchicalnano-andmicroscalearchitecturewithcontrolledphysico-chemicalproperties.More
specifically,webuiltmultiscalehierarchybygrowingcarbonnanotube(CNT)carpetsontwotypesof
Keywords:
scaffolds, namely, interconnected microporous carbon foams and aligned carbon fiber mats.
Carbonnanotubes
NanostructuredCNTcarpetsofferedfinecontrolovernano-roughnessandwettabilityfacilitatingmyo-
Multiscalehierarchy
blastadhesion,growthanddifferentiationintomyocytes.However,microporousfoamarchitecturefailed
Myogenesis
Carbon-basedscaffolds topromotetheirfusionintomultinucleatedmyotubes.Ontheotherhand,alignedfibrousarchitecture
Alignment stimulated formation of multinucleated myotubes. Most importantly, nanostructured CNT carpets
Interconnectedmicroporousstructure interfaced with microscale aligned fibrous architecture significantly enhanced myocyte fusion into
C2C12 multinucleated mature myotubes highlighting synergy between nanoscale surface features and
Nano-roughness micro-/macroscalealignedfibrousarchitectureintheprocessofmyogenesis.
StatementofSignificance
Duetolimitedregenerativepotentialofskeletalmuscle,strategiesstimulatingregenerationoffunctional
musclesareimportant.Thesestrategiesareaimedatpromotingdifferentiationofprogenitorcells(myo-
blasts)intomultinucleatedmyotubes,akeyinitialstepinfunctionalmuscleregeneration.Recenttissue
engineeringapproachesutilizevariousscaffoldsrangingfromdecellularizedmatricestoalignedbioma-
terialscaffolds.Although,majorityofthemhavefocusedonnano-ormicroscaleorganization,asystem-
atic approach to build the multiscale hierarchy into these scaffolds is lacking. Here, we engineered
multiscalehierarchyintocarbon-basedmaterialsanddemonstratedthatthenanoscalefeaturesgovern
thedifferentiationofindividualmyoblastsintomyocyteswhereasmicroscalealignmentcuesorchestrate
fusion of multiple myocytes into multinucleated myotubes underlining the importance of multiscale
hierarchyinenhancingcoordinatedtissueregeneration.
(cid:1)2016PublishedbyElsevierLtd.onbehalfofActaMaterialiaInc.
1.Introduction due to trauma and tumor ablation procedures result in skeletal
muscledamage.Duetolimitedregenerativepotentialofthemus-
AccordingtoUSBureauofLaborstatistics,about100,000cases cle,therapeuticstrategiesaimedatfunctionalmuscleregeneration
ofmuscleinjuriesarereportedinyear2011[1].Similarly,injuries are gaining importance. Some of the strategies used in the past
suchastransplantofautologousmuscleandinjectionofsatellite
cellshaveshownlimitedsuccessduetodonorsitemorbidity,poor
⇑
Corresponding authorat:3501,TerraceStreet,527 Salk Hall,Pittsburgh, PA
integration and survival of implanted tissue or cells [2]. These
15261,UnitedStates.
problems can be circumvented by designing the regenerative
E-mailaddress:[email protected](S.Sant).
http://dx.doi.org/10.1016/j.actbio.2016.01.004
1742-7061/(cid:1)2016PublishedbyElsevierLtd.onbehalfofActaMaterialiaInc.
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
2 A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx
strategiesthataimtorecreatethemicroenvironmentconducivefor provided electrically conductive and macro-porous surface and
myogenesis. promoted proliferation and differentiation of neural stem cells
Multiscalehierarchicalextracellularmatrix(ECM)architecture [21].
at the macro-, micro- and nanoscale along with biomimetic cell- Inthisstudy,weengineeredmultiscalehierarchyintocarbon-
ECM interactions are some of the most important features to be basedmaterialswithcontrollednanoscale(nano-roughness,wetta-
consideredforthedevelopmentoffunctionalmuscletissuewhich bility) and microscale features (highly interconnected pores vs
itself possesses multiscale hierarchy [3,4]. Cell-interactive highlyalignedfibers).Morespecifically,weprocessedcarbonmate-
ECM-mimicking materials can facilitate essential cellular pro- rials into two types of multiscale hierarchical scaffolds; namely,
cesses for normal muscle growth, development and regeneration interconnectedporouscarbonfoams[24]andalignedcarbonfiber
l
[3,5,6].Specifically,acombinationofbiochemicalandbiophysical mats. The 200–500 m size micro-pores with wall width of
l
stimuliarecriticalforguidingappropriateorientationofprogeni- 50–80 m were structural repeating units in reticulated carbon
l
torcellstopromoteformationofalignedandwell-organizedmus- foamswhilecarbonmicrofibers(diameter5–6 m)wererepeating
clebundles[7].Thesebiophysicalstimuliincludenano-roughness, unitsinalignedcarbonfibermats.Wefurtherintroducednanoscale
topographical guidance and electrically conductive surface. featuresbygrowingtailoredcarpet-likearraysofcarbonnanotubes
Surface topography, especially nanoscale surface roughness can (CNTs)onthemicro-porewallsoffoamsandsurfacesoffibers.Such
be an important feature in improving cell-scaffold interaction surface functionalization offers nanoscale roughness, increased
and cell adhesion [8]. Since skeletal muscle tissue is electrically specific surface area and tailored physicochemical properties like
excitable,electroactivematerialsallowlocaldeliveryofelectrical wettability. In this study, we specifically investigated the
stimulus to enhance cell proliferation, differentiation and tissue integratedeffectsofmultiscalehierarchy,thatis,microscalefeatures
regeneration. Indeed, conductive materials have shown to (interconnected pores vs. aligned fibers) and surface nano-
improve myoblast differentiation into myocytes [9,10]. Surface functionalization (CNT/Si-CNT) on differentiation of mouse myo-
wettabilityisanotherfactorthataffectscelladhesiononthescaf- blastsintomultinucleatedmyotubes.Itwashypothesizedthatthe
fold surface. For example, moderately water-wettable surfaces CNTnano-functionalizationofcarbon-basedmaterialswillfacilitate
with contact angles around 70(cid:3) facilitate better attachment of biomimeticcell-materialinteractionandpromotemyoblastdiffer-
cells to the scaffolds through strong binding of cellular adhesion entiation, while the highly ordered fiber alignment will enhance
proteins[11]. fusionofdifferentiatedmyocytesintomultinucleatedmyotubes.
Apart from nanoscale features, many native tissues including
muscles display multiscale hierarchical structure responsible for
2.Materialsandmethods
their unique architecture and function [3]. Submicron scale
myofibrilsconstitutethebasicstructuralunits,whichfurtherorga-
2.1.Materials
nize into muscle fibers (10–80 microns in diameter), which then
bundletogethertoformmusclefasciculusandeventually,afunc-
Cellculturemedia,Dulbecco’sphosphatebufferedsaline(DPBS)
tional muscle [12]. Thus, to enhance regeneration of a functional
and serum were purchased from MediatechInc. (Manassas, USA)
skeletal muscle, it is desirable to recreate multiscale hierarchical
or Corning Inc. (NY, USA) unless otherwise specified. The C2C12
scaffoldsthatarecomposedofrepeatingsmallstructuralunitsto
cells were a kind gift from Dr. Feinberg at Carnegie Mellon
create a three-dimensional architecture. In addition, macro- and
University, Pittsburgh, PA, USA. CNTs used in this study possess
microscale scaffold architecture provides contact guidance to the l
average length of 17 m and a diameter of 18nm. It consists of
myoblastcells.Thisincludesdifferentmicroscalefeaturessuchas
1%(Atomic%)Fe.
interconnected pores [13] or aligned fibrous structure [14].
Similarly, macro-porous architecture of the scaffold is essential
foreasyexchangeofnutrientsandwasteproductsaswellasfacil- 2.2.Nano-functionalizationofcarbonfoamsandfibers
itating cell infiltration throughout the scaffold volume [13,15].
Honeycomb-likepolymerfoamshavebeeninvestigatedforcardiac Reticulatedvitreouscarbon(RVC)foamsaretypicallyfabricated
muscle regeneration and peculiar interconnected honey-comb fromthepyrolysisofthermosettingpolymer,resultinginvitreous
architecturewasthoughttoreinforcethescaffoldduringthecon- carbonstruts.RVCfoamsselectedinthisstudywerewith80pores
tinuous contraction and relaxation process of myocardial tissue perinch(PPI),providedbyUltrametInc.Carbonnanotubecarpets
[16,17]. Thus, for functional skeletal muscle regeneration, it is were synthesized using a two-step process previously developed
essential to build multiscale hierarchical scaffolds that provide byMukhopadhyaygroup[25,26].Thefirststepinvolvesdeposition
contact guidance to myoblasts through nano-roughness, provide of silicon oxide activation/buffer layer via room temperature
alignmentcuesatmicroscaleandallowcellinfiltrationaswellas Microwave Plasma Enhanced Chemical Vapor Deposition reactor
nutrientexchange. (MPE-CVD). The hexamethyldisiloxane [HMDSO, Sigma Aldrich,
Scaffoldspossessingeachoftheseindividualfeatureshavebeen 99.5%]wasusedasaprecursorinconjunctionwith99.9%pureoxy-
built but combining multiple such features into a single scaffold gen. This is followed by chemical vapor deposition of CNT using
andcomprehensiveunderstandingoftheirinterplayingeneration xylene and ferrocene [Alfa Aesar, 99%] as a carbon and catalyst
ofafunctionalmuscletissueisyettobeexplored.Thisisespecially source,respectively.Silica(SiOx)coatingwasappliedusingmeth-
a daunting task given that all the materials cannot be processed ods published previously [24]. Briefly, acid catalyzed sol-gel pro-
intocontrolledhierarchicalarchitecturewithsimultaneouscontrol cess was utilized in order to produce the silica coating on the
over their physico-chemical properties. Electrically conductive CNT-grafted hierarchical substrate using Tetraethyl orthosilicate
carbon-based scaffolds can be processed to possess multiscale (TEOS, Sigma-Aldrich; reagent grade, 98%) as a precursor. Plain
hierarchyalongwithmacro-porousstructureanddifferentgeome- weave carbon fiber mats were obtained from Hexcel (ACGP206-
tries. They have been widely used as conductive materials in P)andfunctionalizedwithCNTsasdescribedaboveforthefoams.
energy storage-related applications [18]. Recently, carbon-based
materials are gaining popularity in various tissue engineering 2.3.Measurementofcontactangle
applicationssuchasosteogenesis[19],neuralcellgrowth[20,21],
bone implants [22] and drug delivery [23]. For example, Li et al. The contact angles of pristine and functionalized foams and
reported that three-dimensional macro-porous graphene foams fibers were measured using a dynamic contact angle goniometer
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx 3
set-up in the Mukhopadhyay laboratory. Details of this well- 2.8.Immunofluorescencestaining
establishedexperimentalset-uparedescribedinanearlierpaper
[24].Adropofde-ionizedwaterwasaddedonthesamplesusing Cell-seededFoamsorFiberswerefixedwith4%paraformalde-
a 21G needle and the image was captured after 10s. The images hyde for 30min and washed 3 times with DPBS followed by the
wereprocessedandcontactanglewasmeasuredusingSolidWorks. blocking/ permeabilization with DPBS containing 0.1% Triton
X-100and5%BSAfor1hatroomtemperature.Thescaffoldswere
2.4.Scanningelectronmicroscopy(SEM) then incubated with the primary antibody against myosin heavy
chain (MHC), MF-20 (1:50, DSHB, Iowa) overnight at 4(cid:3)C and
2.4.1.Imaging washed with DPBS three times. The scaffolds were then stained
Surface characterization of as-prepared and cell-seeded Foam with secondary antibody (Alexa Fluor 488-conjugated goat anti-
and Fiber scaffolds was carried out using SEM (JEOL 9335 Field mouseIgG,1:1000,SantaCruzBiotechnologyInc.,USA)for1hour
Emission SEM) [27]. Cell-seeded scaffolds were fixed following atroomtemperaturefollowedbythreetimeswashingwithDPBS.
21daysand14daysofcultureforFoamsandFibers,respectively. ThecellnucleiwerestainedwithHoechst33342(ThermoFisher,
SEM was performed following air drying for 24h. Scaffolds were USA).
sputter-coated with 5nm of gold-palladium using Cressington
108 auto sputter coater. Images were obtained using accelerated 2.9.Confocalmicroscopy
voltageof3kVandaworkingdistanceof8mm.
Confocal images were obtained using inverted confocal laser
2.4.2.ElementalAnalysisusingenergydispersivespectroscopy(EDS) scanning microscope (Olympus Fluoview 1000). Lasers of 488-,
Elementalcompositionwasdeterminedusingenergydispersive 559-,and633-nmwavelengthwereused.Objectivelensesof20X
spectroscopy(EDS)at7Kvoperatingvoltage.Whenthesolidtobe and40Xwereusedtoacquirethez-stackimageswith5lmthick-
analyzedisexposedtoabeamofelectronsintheScanningElectron nessofeachzslice.Dataispresentedasmaximumintensitypro-
Microscope, the atoms in the sample emit secondary, backscat- jectionofthez-stack.
teredX-rays,whoseenergiesarecharacteristicoftheatomicele-
ment. By analyzing the peaks of X-ray energies using EDS
2.10.Imageanalysis
detector,elementspresentinthesampleareidentified.
NIH ImageJ software was used for quantification of confocal
2.5.C2C12mousemyoblastculture
images. Percentage MHC-positive cells were calculated by taking
aratioofMHC-positivecellstothetotalnumberofcellsinatleast
The mouse myoblast cells (C2C12) cells were cultured in
3imagesperscaffoldtype.Atleast120totalnucleiwereconsid-
Dulbecco’smodifiedEagle’smedium(DMEM)supplementedwith
ered per scaffold type. Myotube fusion index was calculated by
10% of heat inactivated Fetal Bovine Serum (HyCloneTM) and 1%
takingaratioofnumberoffusedcellscontaining2ormorenuclei
antibiotic (Penicillin and streptomycin). The cells were cultured
tototalnumberofnucleiperimagefortheFiberscaffolds.Number
inT75flasksinahumidifiedincubator(37(cid:3)Cand5%CO2),media
ofnucleipermyotubewasmeasuredmanually.Myotubematura-
was changed every 2days and cells were split 1:3 at 70%
tionindexwasmeasuredfromtheimmunofluorescenceimagesby
confluence.
calculating ratio of myotubes with 5 or more nuclei to the total
numberofmyotubes.ThegraphsareplottedusingGraphPadPrism
2.6.Cellseedingonfoamsandfibers
6andOriginPro2015.
The cylindrical foams of height 0.5cm and diameter 0.5cm
2.11.Statisticalanalysis
weresterilizedfor30minin70%isopropanolunderUV.Thefoam
scaffoldswereseededwithaseedingdensityof1(cid:1)106cells/scaf-
The data are represented as mean±standard deviation
fold. For metabolic activity measurement, the cells were seeded
(n=4–5).Thestatisticalsignificancebetweenthegroupswasana-
and cultured for 14days in growth medium. For differentiation
lyzedusingone-wayortwo-wayANOVAformultiplecomparisons
studies, the scaffolds were cultured in growth media until day 3
followedbyTukeypost-hocanalysis(GraphPadPrism6).pvalues
followed by additional 18days in differentiation media (DMEM
lessthan0.05wereconsideredstatisticallysignificant.
supplementedwith10%heatinactivatedhorseserum,HyCloneTM).
Fibermatsofsize1cm(cid:1)1cmwereseededwithC2C12cellsata
seedingdensityof90,000cells/mat(normalizedbasedonthevol- 3.Resultsanddiscussion
umeofmats).Thestudywascarriedoutoveraperiodof14days.
3.1.Nano-functionalizationoffoamscaffoldsaffectsnano-roughness
2.7.Metabolicactivityofcells andwettability
ThemetabolicactivityofcellsseededonFoamsorFibers(n=3) The carbon-based scaffolds with interconnected porous struc-
were measured using the alamarBlue(cid:4) assay (Thermo Scientific, ture were obtained from Ultramet Inc. They are fabricated from
USA). The assay was performed over a period of 14days. ala- pyrolysis of thermosetting polymer, resulting in vitreous carbon
marBlue(cid:4) solution (10% v/v) was prepared in complete growth struts as described previously [24,25]. Surface functionalization
mediaand1mLofthissolutionwasaddedtoeachwellcontaining with nanoscale features is known to alter wettability and nano-
cell-seededscaffoldandincubatedfor4hat37(cid:3)C.Afterthisstep, roughness[24],which,inturn,mayaffectcelladhesion,spreading
l
100 Lofthesolutionfromeachwellwastransferredto96-well anddifferentiation.Inthisstudy,CNTsarechosenforsurfacefunc-
plate and the fluorescence intensity was measured at excitation/ tionalizationastheyhavebeenshowntopromotemyoblastdiffer-
emission wavelength of 530/590nm using the microplate reader entiation [9,28,29]. Along with electrical conductivity, CNTs also
(Synergy HT, BioTek instruments).The wells containing only ala- impartnano-roughness[24]whichmaybebeneficialforcelladhe-
marBlue(cid:4)solutioninmediawasusedforbackgroundfluorescence sion and spreading. Indeed, when encapsulated in gelatin
correction.Pristine-FoamsandPristine-Fiberswereusedascontrol methacrylate hydrogels, CNTs have promoted spreading and
tocomparetheproliferationonfunctionalizedFoamsandFibers. elongationofcardiomyocytes [30].Hence,multiscalehierarchical
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
4 A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx
carbon foams with interconnected microporous structure were FoamandCNT-Foam.Si-CNTcoatingwasfoundtobeperpendicu-
nano-functionalized with carpets of CNT to modulate nano- lartothesurfaceofthescaffold(Fig.1B7,whitearrow)similarto
roughness and wettability. CNT carpets were synthesized using thatofCNT-Foam(Fig.1B4).AsshowninFig.1B8,uniformfibrous
previously reported two-step process [25,26]; first step involves carpetofSi-CNTconferrednano-roughnesstothesurface,similar
thecoatingofsilicananolayerandthesecondstepinvolvesgrowth to CNT-Foam (Fig. 1B5). In conclusion, Si-CNT-Foam possesses
of CNT carpets by chemical vapor deposition (CVD). This process comparablemacro-porosity,contactangleandwettabilitytothat
ensuresgrowthofimmobilizedCNTcarpetsonthesurfaceofthe ofPristine-Foamwithaddednano-roughnessofCNT-Foam.
scaffolds as shown in earlier papers [24,25]. Non-functionalized Following the characterization of the Foam scaffolds, we
foamscaffoldsandCNTfunctionalizedfoamscaffoldsarehereafter assessedtheircytocompatibilityaswellasabilitytopromotecell
referred as Pristine-Foamand CNT-Foam,respectively(schematic adhesion,spreading,anddifferentiation.
in Fig.1A). Detailed multiscale architecture of nano-
functionalizedfoamsisdepictedinSupplementaryFig.1. 3.2.Effectofnano-roughnessandwettabilityonmyoblastadhesion,
ThewettabilityofPristine-FoamandCNT-Foamwasmeasured proliferationanddifferentiationintomultinucleatedmyotubes
bycontactangleofwaterdropletonthescaffoldsurface.Surface
nano-functionalizationwithCNTisknowntoincreasethecontact AlthoughCNTshavebeenutilizedinmanybiomedicalapplica-
angle of carbon foam surfaces [24]. Indeed, CNT grafting on the tions[28–30],theircytocompatibilitystillremainsaconcern.Cyto-
foam surface increased the contact angle from 65-75(cid:3) to 160(cid:3) toxiceffectsofCNTshavebeenstudiedextensively[36].CNTswere
(Table1), making the surfaces less wettable. Similar increase in foundtobecytotoxicwhentheywerefreelysuspendedinculture
contact angle and decrease in surface wetting was also reported andwereavailableforcellularuptake[37].Wehypothesizedthat
forpoly-L-lacticacidsurfacescoatedwithmultiwallCNTs[8].Such immobilization of CNT carpets on Pristine-Foams through strong
decrease in wettability of CNT-coated poly-L-lactic acid surfaces interaction with underlying silica substrate nanolayer (Supple-
wasattributedtonano-roughness. mentary Fig. 1) will prevent shedding of individual CNTs and
Nano-roughness of CNT-Foam was characterized using scan- minimize their cellular uptake. In our previous study on the
ning electron microscopy (SEM). Fig.1B shows SEM images of physico-chemical characterization of Pristine and CNT-Foams
Pristine-Foam and CNT-Foam. As shown in Figs. 1B1 and B3, [25,26],wehaveshownthatinteractionbetweenCNTandunder-
Pristine-Foam and CNT-Foam exhibited interconnected porous lyingsilicananolayerismuchstrongerthanthatbetweengraphitic
structure with overall similar pore size/porosity [25]. CNT carpet layersofthePristine-Foam.
l
of about 20 m thickness was found to grow perpendicular to We, therefore, tested the hypothesis that immobilized
thesurfaceofPristine-Foamscaffolds(Fig.1B4,whitearrows).Suc- CNTs/Si-CNTswillexhibitexcellentcytocompatibilityandpromote
cessfulCNTfunctionalizationwasfurtherconfirmedbyhighmag- myoblast growth and differentiation into myocytes by virtue of
nification (20,000X) images of the walls of functionalized foam structural cues such as, nano-roughness, wettability, conductive
showing uniform fibrous carpet of CNTs rendering nanoscale surfaces and macro-porous scaffold architecture. We cultured
roughnesstothesesurfaces(Fig.1B5)comparedtosmoothsurface mousemyoblast(C2C12)cellsonpristineandfunctionalizedfoam
observedforPristine-Foamscaffolds(Fig.1B2).Thus,CNTfunction- scaffolds(Fig.2A).InfiltrationofC2C12cellsintothescaffoldswas
alization increased the contact angle rendering the surface less studiedafter21daysofcultureusingSEM.Fig.2B1,B2andB3indi-
wettable and also increased the nano-roughness of CNT-Foams. catedthatcells wereabletoinfiltrateandadhere(whitearrows)
TheseresultsindicatethatCNTfunctionalizationaffectedmultiple uniformly to all the foam scaffolds. In case of Pristine-Foams
factors,suchassurfacewettabilityandnano-roughnessthatwould (Fig. 2B1 and Supplementary Fig. 2a), cells adhered to the foam
furtherinfluencethecell-scaffoldinteraction.Indeed,lesswettable wall, forming a uniform cell sheet over the surface, whereas the
surfacestendtopromotetheadsorptionofproteinssuchaslami- presence of nano-hair like CNTs in the other two scaffolds
ninandfibronectin,whichfurtherdisruptorderedwatermolecule, increased surface roughness and cell-scaffold interaction in the
leading to increased entropy. Such entropy-driven adsorption is functionalizedscaffoldsasevidentfromthecells(pseudocolored
usually strong and irreversible compared to enthalpy-driven and in red, black asterisks) attached on to these hair-like projections
reversible adsorption on easily wettable surfaces [31,32]. Protein (SupplementaryFig.2b,blackarrows).Thus,SEMimagessuggest
adsorption further plays important role in cell adhesion. On the that nano-functionalization with CNT carpets enhanced cell-
other hand, nano-roughness has shown to influence cell attach- Foam interactions in the functionalized foam scaffolds. De novo
ment and proliferation by facilitating better cell-material and matrix synthesis on biomaterial scaffolds is one of the essential
cell-cellinteraction[8,33,34]. steps in tissue regeneration. Production of functional tissue-
CNT-Foamswerefurtherfunctionalizedwithamorphoussilica mimetic structures can be facilitated through the synthesis of
(SiOx)(referredasSi-CNT-Foamhereafter)layer.Silicaisahydro- newECMbytheseededcellsalongwithbiodegradationofthescaf-
philic, inorganic, bio-resorbable and biocompatible material that folds.InastudyreportedbySantetal.,Collagentype1,fibronectin
hasbeenwidelyinvestigatedintissueengineering,specificallyin and laminin were secreted by valvular interstitial cells on poly
e
boneregeneration[35].SilicafunctionalizationonCNTwillchange (glycerol sebacate)-poly ( -caprolactone) nanofibrous scaffolds
the wettability, as demonstrated in an earlier study [24], while [38]. In the present study, Pristine-Foam, CNT-Foam and Si-CNT-
maintaining the nano-roughness same as that of CNT-Foam. This FoamfacilitateddenovoECMsecretiononthesurfaceofscaffolds
willdecoupletheeffectofwettabilityandnano-roughnessoncell asshownbywhitearrowsinSupplementaryFig.3.Thisdatasug-
adhesion/spreading. Successful nano-functionalization of CNTs gest that all three foam scaffolds are bioactive and facilitate cell
with Si was confirmed by the elemental analysis of Si-CNT- adhesion,spreadinganddenovomatrixsecretion.
Foams,whichshowedincreaseinthepercentageofSiatomsfrom The effect of surface nano-roughness and wettability on cell
1.38%(CNT-Foam)to4.32%(Si-CNT-Foam)(Table2).Itcouldalso adhesion and proliferation of C2C12 was further investigated by
beseenthatthewatercontactangleoftheCNTcarpetsdecreased determining their viability with alamarBlue(cid:4) assay (Fig.2C).
from160.3(cid:3)(CNT-Foam)to66.5(cid:3)(Si-CNT-Foam)(Table1)implying AlamarBlue(cid:4) assay measures ability of metabolically active cells
thatSi-CNT-Foamisnolongerhydrophobicdespitethepresenceof toreduceresazurintofluorescentresorufin[27].Allscaffoldssup-
CNT carpet morphology. We characterized nano-roughness of Si- portedgrowthofC2C12asevidentfromthesignificantincreasein
CNT-FoamusingSEM.AsshowninFig.1B6,Si-CNT-Foamexhibited their metabolic activity over a period of 7days. It is noteworthy
interconnectedporousstructurewithsimilarporesizeasPristine that differences between Pristine-Foams and CNT/Si-CNT-Foam
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx 5
Fig.1. Characterizationofpristineandnano-functionalizedfoamscaffolds.(A)Schematicofhierarchicalmultiscalearchitectureofcarbonfoamsfunctionalizedwithcarbon
nanotubes(CNT)andsilica-CNT(Si-CNT);(B)Effectofsurfacenano-functionalizationofcarbonfoamswithCNTandSi-CNTonthemicro-andnanoscalemorphologybySEM;
LowmagnificationimagesshowinghighlyinterconnectedporousstructureofpristineFoam(B1),CNT-Foam(B3)andSi-CNT-Foam(B6);Highmagnificationimagesshowing
smoothsurfaceofPristine-Foams(B2)whileCNT(B4)andSi-CNT(B7)functionalizationconferrednano-roughness(depictedbywhitearrows);CNT(B5)andSi-CNTcoating
(B8)at20,000Xmagnificationshowingroughnanofibrousstructure.ScalebarinB1,B3andB6represents500lm,scalebarinB2,B4,andB7represents25lmandscalebar
inB5andB8represents1lm.Table1demonstratestheeffectofsurfacenano-functionalizationoncontactangleofdifferentfoams.Thecontactangleoffoamsincreasedwith
CNTgrafting,whilesubsequentcoatingofSionCNT-coatedfoamsrestoredthecontactanglesimilartoPristine-Foams.Table2showsenergy-dispersiveX-rayspectroscopy
(EDS)analysisofPristine,CNTandSi-CNT-FoamsshowingatomicpercentageofC,O,SiandFebasedonK(innermost)shell.‘‘ND”(notdetectable)indicatesthatthevaluesfor
theseelementswerebelowdetectionlimitsoftheinstrument.CNTstructuresandfoamarchitectureshownintheschematicsareonlyforthedemonstrationoftheconcept.
Schematicsnottoscale.
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
6 A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx
Fig.2. Effectofsurfacenano-functionalizationonmyoblastmorphology,metabolicactivityanddifferentiationintomyocytes.(A)AschematicshowingC2C12myoblastcells
seededontopristineandfunctionalizedfoams;(B)ScanningelectronmicroscopyimagesshowingcellinfiltrationthroughoutthedepthofPristine-Foam(B1),CNT-Foam(B2),
andSi-CNT-Foam(B3).ScalebarinBrepresents500lm.Whitearrowsindicatecellsadheringtothewallsoffoams;(C)Viability/metabolicactivityofC2C12cellsby
alamarBlue(cid:4)assay(n=3),Statisticalsignificancebytwo-wayANOVAandTukeyposthoctest.⁄⁄denotesp<0.005,⁄⁄⁄⁄denotesp<0.00005;(D)Confocalmicroscopyimagesof
Pristine-Foam(D1,D2,D3),CNT-Foam(D4,D5,D6)andSi-CNT-Foam(D7,D8,D9)showingdifferentiationofC2C12cellsintomyocytesasindicatedbymyosinheavychain
(MHC)staining(green,D2,D5,D8).Thenuclei(blue,D1,D4,D7)andactin(red,D3,D6,D9)werestainedwithHoechstandactin-phalloidin,respectively.ScalebarinD
represents100lm.WhitearrowsshowareasshowingMHCexpressingcellswhilewhiteboxshowscellsonPristine-Foamspositiveforactin,butwithnoMHCexpression.
(E)QuantificationofpercentageMHC-positivecellsafterdifferentiationstudy(n>120cellsfromatleast3imagesand3differentscaffolds).Statisticalsignificancebyone-
wayANOVAandTukeyposthoctest.⁄⁄denotesp<0.005,⁄⁄⁄denotesp<0.0005.
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx 7
werenotstatisticallysignificantonanygivendayofculture,sug- TheseSEMimagessuggestedthatcellsheetspeeledoffalongwith
gesting that CNT/Si-CNT carpets immobilized onto carbon foams the underlying CNT/Si-CNT carpets. We have previously reported
did not significantly affect cell viability and growth. Thus, thepeelingoffofCNTcarpetsasanentirelayerfromthefoamsur-
CNT/Si-CNT-Foams are found to be cytocompatible. Interestingly, face [25]. This was attributed to the much stronger interaction
weobserveddecreaseinthemetabolicactivityofcellsonday14 betweenCNTsandunderlyingsilicananolayerthanthatbetween
in all threescaffolds. This can be attributed to two possiblephe- the graphitic layers in the underlying foam. SEM images in our
nomena. First, we observed that confluent cell sheets peeled off study supports this conclusion and indicate that the cell sheets
from the CNT/Si-CNT-Foam surfaces as shown in representative and CNT/Si-CNT carpets remained together during their detach-
SEM images for CNT/Si-CNT-Foam scaffolds (Supplementary ment,furthersuggestingstronginteractionofcellswithunderly-
Fig. 4, white arrows). SEM images also revealed comparatively ing CNT/Si-CNT carpets. Such strong interaction between the
smooth surfaces of CNT/Si-CNT Foam scaffolds after detachment myoblasts and CNT carpets may be beneficial in two ways. First,
of cell-sheets, similar to smooth surfaces observed in Pristine- the CNT carpets can provide a mechanical support to the native
Foams(Fig. 1B2and SupplementaryFig. 4, denoted by asterisks). myoblastsuntiltheygetanchored,orientedanddifferentiatedinto
Fig.3. CharacterizationofalignedPristineandnano-functionalizedcarbonfibermats.(A)Schematicofhierarchicalmultiscalearchitectureofalignednon-functionalized
carbonfibers(Pristine-Fibers)andfunctionalizedCNT-Fibers;(B)Scanningelectronmicroscopyimagesshowingmacro-tomicroscalemorphologyofPristine-Fiber(B1–B3)
andCNT-Fibers(B4–B6);CNTfunctionalizationincreasedthesurfaceroughness(B5andB6)incontrasttothesmoothsurfaceofPristine-Fibers(B2andB3).ScalebarinB1
andB4represents1mm,scalebarinB2andB5represents50lmandscalebarinB3andB6represents10lm;(C)CNT-functionalizationincreasedcontactangleandthus,
decreasedwettabilityoftheCNT-FiberscaffoldsasindicatedinTable3.
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004
8 A.Pateletal./ActaBiomaterialiaxxx(2016)xxx–xxx
functional multinucleated myotubes. Second, this may also pre- enhancetransmissionofsuchsignalsassuggestedbyothers[40],
vent disaggregation/detachment of individual CNTs and their although we do not have direct evidence in our current study. It
uptakebythecells,thusobviatingthecytotoxicityofCNTs. shouldbe noted that although Si coating on CNT-Foams restored
Thedecreaseinthemetabolicactivityatday14mayalsobedue their contact angle from 160(cid:3) to 66(cid:3) (similar to Pristine-Foams),
to the differentiation of myoblast cells into multinucleated altering the surface wettability did not inhibit the ability of
myotubes[39].Thus,weinvestigatedtheroleofCNTandSi-CNT Si-CNT-Foamstoinducemyoblastdifferentiation.Onthecontrary,
functionalization on myoblast differentiation. C2C12 cells were both CNT/Si-CNT-Foam exhibited comparable percentage of
culturedingrowthmediafor3days,followedby18daysindiffer- MHC-positive cells despite significant differencesin theircontact
entiation media. The cells were then fixed and stained with angles (Fig. 2D5 vs. 2D8 and 2E). This suggests that nano-
Hoechst (nucleus, blue), myosin heavy chain (MHC, green) and roughness, which is a common feature of both CNT-Foam and
phalloidin (actin, red). Pristine-Foam scaffolds displayed attach- Si-CNT-Foamratherthanwettabilityplaysthemoreimportantrole
ment and spreading of C2C12 cells on the foam walls as shown in controlling myoblast differentiation into MHC-positive cells.
by nuclear and cytoskeletal actin staining (Fig. 2D1 and D3, Similarly, Papenburg et al. reported more important role played
arrows).However,veryfewMHC-positivecellswereobservedon by surface roughness on cell attachment and proliferation than
Pristine-Foam(Fig.2D2,whitebox),indicatingtheirlimitedability surfacehydrophobicity[34],whichisinlinewithourobservations
todifferentiateintoMHC-positivemyocytes.Onthecontrary,CNT- in this study. Our results on the role of CNTs in promotingmyo-
Foam (Fig. 2D4-D6) and Si-CNT-Foam (Fig. 2D7-D9) displayed blast differentiation are in accordance with the previous reports
presenceofmuchhighernumberofMHC-positivecells,indicating [28,29].
that CNT/Si-CNT functionalization promoted differentiation of Althoughnano-functionalizedfoamspromotedmyoblastdiffer-
myoblasts into myocytes. Image analysis confirmed these results entiation into MHC-positive myocytes, formation of multinucle-
revealing significantly higher percentage of MHC-positive cells atedmyotubeswasnotevidentinanyofthefoamscaffolds.This
(about 90%) in CNT-Foam and Si-CNT-Foam compared to maybeduetotheporousarchitectureofthefoamscaffolds.Foams
Pristine-Foam (only 30%, Fig.2E). Such enhanced differentiation providedexcellentporosityandsurfaceareathatenhancedmyo-
maybeduetothenano-roughnessprovidedbyCNT/Si-CNT.This blastcellproliferationandadhesiononallthreescaffoldswhereas
mayalsobeduetoenhancedcell-cellcommunicationasmyoblasts CNT/Si-CNT functionalization provided additional structural cues
needelectricallyconductivesurfaceforthedevelopmentoffunc- suchasnano-roughnessand/orconductivity,whichfurtheraided
tional muscle tissue and conductive CNT nanostructures may myoblastdifferentiationintoMHC-positivecells.However,foams
Fig.4. EffectofCNT-functionalizationonmyoblastmorphologyandmetabolicactivity.(A)Viability/metabolicactivityofC2C12cellsonPristine-FiberandCNT-Fibermatsby
alamarBlue(cid:4)assay(n=3).Statisticalsignificancebytwo-wayANOVAandTukeyposthoctest.⁄⁄denotesp<0.005,⁄⁄⁄⁄denotesp<0.00005.(B)Scanningelectronmicroscopy
imagesshowingC2C12cellmorphologyandtheirinteractionwiththeunderlyingPristine-FiberandCNT-Fiberscaffolds.Significantlyincreasednumberofcellscanbe
observedontheCNT-Fibercoveringtheentirearea(B4).B5andB6showcellbodies(blackarrows)interactingwiththenanofibrousCNTs(redarrows)coatedoncarbon
fibers.ScalebarinB1andB4represents500lm,scalebarinB2andB5represents10lmandscalebarinB3andB6represents5lm.(Forinterpretationofthereferencesto
colorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
Pleasecitethisarticleinpressas:A.Pateletal.,Carbon-basedhierarchicalscaffoldsformyoblastdifferentiation:Synergybetweennano-functionalization
andalignment,ActaBiomater.(2016),http://dx.doi.org/10.1016/j.actbio.2016.01.004