Nanoclay-Enriched Poly(ɛ-caprolactone) Electrospun Scaffolds for Osteogenic Differentiation of

14 Pages·2014·2.05 MB·English

TISSUEENGINEERING:PartA
Volume20,Numbers15and16,2014
ªMaryAnnLiebert,Inc.
DOI:10.1089/ten.tea.2013.0281
Nanoclay-Enriched Poly(e-caprolactone) Electrospun
Scaffolds for Osteogenic Differentiation
of Human Mesenchymal Stem Cells
Akhilesh K. Gaharwar, PhD,1–3,*,{ Shilpaa Mukundan, BTech,3,4,{ Elif Karaca, MS,3,4
Alireza Dolatshahi-Pirouz, PhD,2–4 Alpesh Patel, PhD,3,4 Kaushik Rangarajan, BTech,3,4 Silvia M. Mihaila, MS,3,4
Giorgio Iviglia, MS,3,4 Hongbin Zhang, PhD,3,4 and Ali Khademhosseini, PhD2–6
Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue
substitutes. Oneapproach to achieve this aim is to develop osteoconductive scaffolds thatfacilitate theformation
offunctionalbonetissue.Wehavefabricatednanoclay-enrichedelectrospunpoly(e-caprolactone)(PCL)scaffolds
for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is
fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases
fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with
nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive
characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In
addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface
interactionsbetweenPCLandnanoclay.Theeffectofnanoclayonthemechanicalpropertiesofelectrospunfibers
is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is
investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion
and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the
electrospunscaffoldsasevidentbyanincreaseinalkalinephosphatesactivityofhMSCsandhigherdepositionof
mineralizedextracellularmatrixcomparedtoPCLscaffolds.Givenitsuniquebioactivecharacteristics,nanoclay-
enriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering.
Introduction play a major role in controlling the human mesenchymal
stem cells (hMSCs) differentiation, and, thus, electrospun
Tissue engineering aims at repairing and regenerating scaffolds represents a promising approach for bone tissues
biologicaltissuestoimprovethefunctionofdiseasedor engineering.10
damaged tissue or organ.1–5 As a direct result, there is an Duringthepastfewdecades,variouspolyestershavebeen
increase in the demand for developing new bioactive scaf- investigated for tissue engineering applications due to their
foldsthatcanfacilitatetheformationoffunctionaltissueby biocompatibility, bioresorbility, and high mechanical
directing stem cell differentiation.6–9 Various processing strength.8,12–14 Among them, poly(e-caprolactone) (PCL), a
techniques such as extrusion, solvent casting, porogen semi-crystalline and hydrophobic polymer with low glass
leaching, and electrospinning are investigated to fabricate transition temperature (-60(cid:2)C) and moderate melting point
different scaffold structures.10,11 Among these techniques, (60(cid:2)C), is an attractive polymer for musculoskeletal tissue
electrospinning is extensively used to fabricate fibrous engineering, as it can be used to fabricate a wide range of
scaffolds, as it can mimic three-dimensional architecture of scaffold materials.10,11 However, some of the problems as-
extracellular matrix (ECM).1,10 The scaffolds architecture sociated with PCL include its slow in vivo degradation rate
1DavidH.KochInstituteforIntegrativeCancerResearch,MassachusettsInstituteofTechnology,Cambridge,Massachusetts.
2WyssInstituteforBiologicallyInspiredEngineering,HarvardUniversity,Boston,Massachusetts.
3CenterforBiomedicalEngineering,BrighamandWomen’sHospital,HarvardMedicalSchool,Cambridge,Massachusetts.
4Harvard-MITDivisionofHealthSciencesandTechnology,MassachusettsInstituteofTechnology,Cambridge,Massachusetts.
5Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University,
Seoul,RepublicofKorea.
6DepartmentofPhysics,KingAbdulazizUniversity,Jeddah,SaudiArabia.
*Currentaffiliation:DepartmentofBiomedicalEngineering,TexasA&MUniversity,CollegeStation,Texas.
{Theseauthorscontributedequally.
2088
NANOCLAY-ENRICHED PCL SCAFFOLDSFOROSTEOGENIC DIFFERENTIATION OF HMSCS 2089
and lack of bioactive characteristics.15 The degradation of electrospun PCL scaffolds was obtained by changing the
PCL occurs due to hydrolytic cleavage of ester groups via concentrations of nanoclay. The effects of nanoclay on
the surface or bulk pathway.11,16 Due to this degradation surface morphology, degradation rate, thermal characteris-
mechanismandhydrophobicnature,PCLtakesmorethan2 tics, mechanical properties, in vitro biomineralization, and
years to completely degrade in vivo.15,16 cellular interactions were evaluated. We hypothesize that
The degradation properties of PCL can be improved by the nanoclay-enriched electrospun structure can support the
incorporating nanoparticles such as hydroxyapatite, silica, osteogenic differentiation of hMSCs, along with the pro-
magnetic nanoparticles, and synthetic clays within the duction of mineralized matrix. The features would enable
polymeric scaffolds11 or by blending PCL with other fast the use of nanoclay-reinforced PCL scaffolds to design
degradingpolymers.17Forexample,Liaoetal.incorporated improved bioactive scaffolds for musculoskeletal tissue re-
hydroxyapatite nanoparticles within PLLA/PCL fibrous generation.
scaffoldstoincreasethebiodegradationrateoftheresulting
constructs.18 Electrospun scaffolds made from hydroxyap- MaterialsandMethods
atite nanoparticles embedded in PCL had higher tensile
Fabrication ofPCL–nanoclay electrospunscaffolds
strength and enhanced bioactivity while supporting osteo-
blast-like cell adhesion and proliferation.19 The addition of PCL-(C H O ) with M *70,000–90,000 Da was
6 10 2 n w
silica (SiO ) nanoparticles within electrospun PCL fibers purchased (Sigma-Aldrich). Synthetic nanoclay (Nano-
2
also enhances the physical and chemical properties of the fil(cid:3)116)wasobtainedfromSouthernClayProducts,Inc.All
fibrous scaffolds.20 Despite interesting physical, chemical, other chemical and reagents are purchased from Sigma-
and biological properties, nanoparticle-reinforced PCL Aldrich. The polymer solution was prepared by dissolving
scaffolds lack osteogenic characteristics. PCL (15% w/v) in 9:1 ratio of anhydrous chloroform and
Nanoclays,alsoknownassyntheticsilicates,haveshown ethanol. We selected a 9:1 ratio of anhydrous chloroform
toimprovephysicalandmechanicalpropertiesofpolymeric and ethanol, as both PCL and nanoclay showed maximum
structures.21,22Thisisduetoanisotropicandplate-like,high solubility, compared to other solvents used for electrospin-
aspect-ratio morphology of nanoclay, which results in high ning.Then,nanoclayindifferentconcentrations(0.1%,1%,
surface interactions between the polymers and nano- and 10% (w/w) with respect to PCL) was added to the so-
particles. Nanoclays are widely used to reinforce thermo- lution. The electrospinning of PCL and nanoclay-enriched
plastic polymers in order to obtain hybrid composites with PCL was carried out using a 21G blunt needle at 12.5kV
hierarchical structure,23,24 elastomeric properties,25 ultra- (Glassman High Voltage) and a flow rate of 2mL/h. The
strong and stiff films,26,27 super gas-barrier membrane,28 collector was a circular plate (diameter 6.5cm) made of
superoleophobicity surfaces,29 flame-retardant structures,30 aluminum and maintained at a constant distance of 18cm
andself-healinghydrogels.31Arecentstudyhasshownthat from the needle. These parameters were chosen based on
nanoclay (synthetic silicates nanoplatelets) can induce os- our previous study on fabrication of PCL scaffolds.41 The
teogenicdifferentiationinhMSCswithoutusinganygrowth electrospun scaffolds were dried overnight in vacuum to
factors.32 These unique bioactive properties of synthetic remove the residual solvent.
silicates may be processed to construct devices such as in-
jectable tissue repair matrices, bioactive fillers, or thera- Microstructureevaluation
peutic agents for triggering specific cellular responses
The microstructure andmorphology of electrospun fibers
toward bone-related tissue engineering approaches.
were evaluated by scanning electron microscopy (SEM)
The addition of these synthetic silicates to polymeric
(JSM 5600LV; JEOL). The electrospun scaffolds were
matrix can be used to control physical and chemical prop-
vacuum dried and then coated with gold/palladium (Au/Pd)
ertiesofnanocompositematrix.33–35Marrasetal.fabricated
for 2min using a Hummer 6.2 sputter coater (Ladd Re-
PCL scaffolds enriched with organically modified nanoclay
search). The images were captured using an accelerating
knownasmontmorillonite(MMT).36Theyobservedthatthe
voltageof5kV,aworkingdistanceof5mm,andaspotsize
incorporation of MMT with PCL matrix enhances me-
of 20. The SEM images were analyzed using the Image J
chanical strength without compromising ductility.36 In a
(NIH) software, and the fiber diameter was calculated from
similar approach, the incorporation of halloysite nanoclay
at least 100 fibers. The distribution of nanoclay within
within electrospun PCL scaffolds increased the mechanical
electrospun scaffold was determined by staining the nano-
strength, protein adsorption, and cell adhesion of the re-
clay particles with food dye (red dye 40). The nanoclay
sulting hybrid materials.37 Despite interesting physical and
stained with dye was separated from the solution by pre-
chemicalpropertiesofclay-enrichednanocomposites,onlya
cipitating in organic solvents. Electrospun scaffolds were
few studies have focused on using clay-based scaffolds for
fabricated using red-stained nanoclay to determine the dis-
biomedical applications.34,38,39 The addition of silicate na-
tribution of clay with the fibers. The optical images were
noclay can be used to tune adhesion and spreading of fi-
obtained using Zeiss Axio Observer Z1 1 (AXIO1; Zeiss)
broblast cells, preosteoblast cells, and hMSCs.34,38 In
that was equipped with Evolve EMCCD 512·512 16mm
another study, Ambre et al. showed that the addition of
pixels.
synthetic silicate to polymeric scaffolds consisting of chit-
osan and polygalacturonic acid promotes osteogenic dif-
ferentiation of hMSCs.40 Acceleratedin vitro degradation
Here, we assessed the use of nanoclay-enriched electro- Electrospun scaffolds were cut into 5mm-diameter cir-
spun PCL scaffolds for adhesion, proliferation, and differ- cular shapes and then subjected to accelerated degradation
entiation of hMSCs. A range of nanoclay-enriched conditions (n=3) by incubation in 5mL of 0.5mM sodium
2090 GAHARWAR ET AL.
hydroxide(NaOH)solutionat37(cid:2)C.Atvarioustimepoints, Protein adsorption
the NaOH solution was carefully pipetted out, and the
Electrospun scaffolds (n=3) that were 3mm in diameter
electrospun scaffold was washed 3·with distilled water.
were washed thrice with phosphate-buffered saline (PBS).
Thesamplesweresubsequentlyfrozenusingliquidnitrogen
The samples were allowed to soak in 500mL of 10% fetal
and then lyophilized. Then, the weight of the degraded
bovine serum (FBS; Gibco) for 24h at 37(cid:2)C. The fibrous
sample(W)wasmeasured,andthepercentagemasslossfor
t samples were washed thrice with PBS to remove any non-
a given sample was calculated as ((W - W)/W ·100),
o t o specific adsorbed proteins. The samples were then treated
based on the initial mass (W ) of the sample before incu-
o with2%SDSsolutionfor6hinashaker(50rpm)toremove
bation.
the adsorbed proteins. The supernatant was collected sepa-
rately by centrifuging the samples, and the eluted proteins
Chemicaland thermalcharacterizations were analyzed using micro Bicinchoninic acid (BCA) pro-
The deposition of minerals (hydroxyapatite and/or cal- tein assay reagent (Pierce BCA; Thermo Scientific) using
cium phosphate) on electrospun fiber after incubating in the manufacturer’s protocol.
10· SBF was evaluated using ALPHA Fourier Transform
Infrared Spectroscopy (FTIR) Spectrometer (Bruker) in the Cell culture studies
wavenumber range of 4000–400cm-1. The thermal prop-
Bone marrow-derived hMSCs (PT-2501; Lonza) were
erties of electrospun scaffolds were investigated using a
cultured in normal growth medium (a-MEM [Gibco], sup-
differentialscanningcalorimeter(DSC)(DSC8500;Perkin-
plemented with 10% of heat-inactivated FBS [Gibco] and
Elmer) and a thermogravimetric analyzer (Pyris 1 TGA;
1% penicillin/streptomycin [100U/100mg/mL; Gibco]), at
Perkin-Elmer).TopreparesamplesforDSC,theelectrospun
37(cid:2)C, in a humidified atmosphere with 5% CO . The cells
2
scaffolds were weighed (between 3–5mg) in standard alu-
were cultured until 70–75% confluence and were used be-
minumpans,sealedwithlids,heatedattherateof10(cid:2)C/min
fore passage 5 for all the experiments. Before the trypsini-
from -70(cid:2)Cto150(cid:2)C,andthencooledfrom150(cid:2)Cto -70(cid:2)C
zation of cells (CC-3232; Lonza), the electrospun scaffolds
using nitrogen as a purge gas. The second cycle (heating
were sterilized using ethanol for 30s and added to well
and cooling) was used to determine melting temperature
plates. Then,thecellswereseededonelectrospunscaffolds
(Tm), melting enthalpy (Hm), crystallization temperature (1·1cm2) at a density of 20,000 cells/scaffold in normal
(T ), and crystallization enthalpy (H ). The crystallinity
c c growth medium, in 24-well plates. After 24h, the medium
(X ) of the PCL in the electrospun fibers was calculated as
c was replaced with the normal or osteoinductive media
DH /DH o (1/m )·100%, where DH o is 136J/g,
m m PCL m (500mL/sample). Osteoinductive media consisted of nor-
whichisthetheoreticalheatoffusionfor100%crystalline
mal growth medium that was supplemented with 10mM b-
PCL,42,43 and m is the mass fraction of PCL in the
PCL glycerophosphate (Sigma Aldrich), 50mg/mL ascorbic acid
nanocomposite fibers (mPCL=1 for PCL fibers and phosphate (Sigma Aldrich), and 10-8 M dexamethasone
m =0.9 for PCL-10% Nanoclay). For TGA, samples
PCL (Sigma Aldrich). The effect of nanoclays on cell prolifera-
wereheated ina ceramic pan at the rateof 10(cid:2)C/min from
tionwasevaluatedusingAlamarBlueAssay(Invitrogen)on
50(cid:2)C to 600(cid:2)C under a constant stream of nitrogen at a
day 3 using standard protocol. The effect of nanoclays on
flow rate of 20mL/min.
the metabolic activity of hMSCs was evaluated using Ala-
mar Blue Assay (Invitrogen) on day 1 and 7 using the
Mechanicaltesting manufacturer’s protocol.
Actin cytoskeleton organization was observed using
The mechanical properties of electrospun scaffolds were
fluorescence microscopy. Cell-seeded scaffolds were fixed
investigated by performing uniaxial tensile tests using an
in 4% paraformaldehyde (PF) solution, and the cell mem-
Instron 5542 mechanical machine (Instron). Electrospun
brane was permeabilized using 0.1% Triton X-100 for
scaffolds (5mm wide and 1cm in length) were tested at a
30min. Subsequently, the samples were blocked in 1%
rate of 10mm/min until fracture. The thickness of fibrous
bovineserumalbumin(BSA)andtheactincytoskeletonwas
scaffolds was obtained between 100 and 200mm. Mechan-
stained using a 1:40 dilution of Alexa Fluor-594 phalloidin
icalpropertiessuchaselasticmodulus(EM),ultimatestrain,
(Abcam) in 0.1% BSA. The cell nuclei were stained with
and fracture stress were calculated from the stress-strain
4¢,6-diamidino-2-phenylindole (DAPI). The fluorescence
curve. The EM was determined as the initial slope of the
images were obtained using Zeiss Axio Observer Z1 1
strain/stress curve, corresponding to 5–15% strain.
(AXIO1) that was equipped with a color camera (Evolve
EMCCD 512·512 16mm pixels).
In vitrobiomineralization
Alkalinephosphatase(ALP)activitywasmeasuredusing
The ability of electrospun scaffold to facilitate biomi- acolorimetricendpointassay(ALPColorimetricAssayKit,
neralization was evaluated using simulated body fluid ab83369), which quantified the conversion of p-nitrophenol
(SBF). The electrospun scaffolds with different concentra- phosphate(pNPP)toyellowp-nitrophenolbyALPenzyme.
tions of nanoclay (0%, 0.1%, 1%, and 10%) were cut into Briefly, at the determined time points, samples were re-
4·4mm squares. The cut scaffolds were incubated with trieved and subjected to osmotic and thermal shocks to
2mL of 10· SBF solution (prepared using a previously collect the cell lysate. The assay buffer solution of 5mM
reported method)44,45 at 37(cid:2)C. After 2h, the solution was pNPPandthesamples(celllysate)wereaddedtoa96-well
removed and scaffolds were washed thrice with distilled plate. After 1h, the absorbance was read at 405nm using a
water. The samples were frozen in liquid nitrogen and microplate reader (Epoch microplate reader; Biotek). A
subsequently lyophilized for SEM imaging and FTIR. standard curve was made from standards (0–20mM)
NANOCLAY-ENRICHED PCL SCAFFOLDSFOROSTEOGENIC DIFFERENTIATION OF HMSCS 2091
prepared with a pNPP solution. Sample and standard trip- PCL chains in solution and resulted in the formation of
licates were analyzed, and sample concentrations were no- beaded microstructures.
ted from the standard curve. The ALP activity was Apart from the decrease in the fiber diameter, surface
normalized with the DNA content. The amount of double- roughnessalsodependsonthenanoclayconcentration.Pure
stranded DNA (dsDNA) was measured using a PicoGreen PCLfibersweresmoothatmicroscale,whiletheadditionof
dsDNA Quantification Kit(P7589;Invitrogen) accordingto nanoclay introduced surface roughness on the electrospun
the manufacturer’s protocol. The expression of ALP was fibers.Theeffectwasmoreprominentinthenanocomposite
also determined using Nitro-blue tetrazolium/indolylpho- scaffolds containing 1% and 10% nanoclay, which showed
sphate (NBT/BCIP) staining. First, the cells were washed higher surface roughness compared to PCL scaffold. A
withPBS,andthen,0.5mLofNBT/BCIPwasaddedtothe similar effect was shown earlier when nanohydroxyapatite
samples. The samples were incubated at 37(cid:2)C in a humid- (nHA) was incorporated in electrospun PCL.46 In a similar
ifiedchambercontaining5%CO for30min.Afterthat,the study, the addition of nHA to collagen type I47 or silk fi-
2
samples were washed with PBS andfixed with 4% PF. The broin48 also resulted in the formation of electrospun fibers
imaging was performed using Zeiss Axio Observer Z1 1 with high surface roughness compared to the fibers con-
(AXIO1)thatwasequippedwithEvolveEMCCD512·512 taining only polymer. These studies also emphasized that
16mm pixels. surface roughness influences physical, chemical, and bio-
The mineralized matrix produced by hMSCs was deter- logical properties of electrospun scaffolds.46–48 In the sub-
mined using Alizarin Red Staining. At 21 days, cells were sequent sections, we investigated the effect of nanoclay on
fixed with 10% formalin (20min) and then washed thrice the biodegradibility, thermal stability, mechanical strength,
with PBS. The fixed cells were further washed with dis- and bioactivity of the scaffolds.
tillatedwaterinordertoremoveanysaltresiduesandthen,a
solutionof2%(wt/v)AlizarinRedS(ARS;SigmaAldrich)
Nanoclaypromotedin vitro degradation
with a pH adjusted to 4.2, was added so that it covered the
offibrous scaffolds
entiresurfaceofthescaffolds.After10minofincubationat
room temperature, the excess ARS was washed with dis- ThedegradationofPCLmainlyoccursthroughhydrolytic
tillated water. The ARS staining was imaged using a Zeiss cleavage of ester groups that can result from either surface
Discovery V8 Stereo Microscope (DISV8). or bulk degradation.11,16 The degradation of PCL is a slow
process and takes more than 2 years under in vivo condi-
tions.16 However, bone regeneration is a relatively fast
Statistical analysis
process and takes 2 to 3 months to fully regenerate small
Experimental data were presented as mean–standard bone defects. So, for bone tissue engineering, an ideal
deviation (n=3 to 5). Statistical differences between the scaffold should have a faster degradation rate and should
groups were analyzed using one-way analysis of variance match the regeneration rate of damaged tissue. Thus, for
using Tukey post-hoc analysis. Statistical significance was PCL-based scaffolds, we would prefer to have a shorter
represented as *p<0.05, **p<0.01, and ***p<0.001. degradation time. The degradation mechanism of electro-
spun PCL is different from its bulk degradation behavior
Results andDiscussion duetothehighsurfacetovolumeratio.Thisismainlydueto
the change in the hydrophobic characteristic and the higher
Effect of nanoclayonfibermorphology
crystallinity of PCL that are induced during the electro-
The fibrous scaffolds of PCL and PCL-nanoclay com- spinning process.11 The degradation of electrospun PCL
posites were obtained by the electrospinning process as scaffold can be investigated by monitoring the weight loss
shown in Figure 1a. The effect of nanoclay on the surface of the scaffolds in accelerated degradation conditions.
morphologyandfiberdiameteroftheelectrospunscaffolds The effect of nanoclay concentration on the degradation
was investigated by using SEM. The results indicated that characteristic of electrospun fibers was investigated in ac-
the scaffolds made of pure PCL showed uniform and celeratedconditionsusing0.5MNaOHsolution(Fig.2a).In
smooth surface morphology of the electrospun fibers (Fig. our experiments, the electrospun PCL scaffolds took 112h
1b). These results were in accordance with the previously to completely degrade. The addition of nanoclay signifi-
reported studies.17,41 However, with the addition of nano- cantly reduces the time required for the complete degrada-
clay, the fiber diameter was decreased and the surface tion of electrospun scaffolds. For example, electrospun
morphologyofthefibersbecamerough.Thefiberdiameter scaffoldscontaining0.1%nanoclayweredegradedin108h,
was quantified using ImageJ, and it was observed that the whereas the scaffolds containing 1% and 10% nanoclay
average diameter of fibers for scaffolds made of pure PCL were degraded in 96h respectively. The complete degrada-
wasaround5.6–0.6mm(n=100)(Fig.1c).Theadditionof tion was considered when the scaffold was dissociated into
1% nanoclay reduced the average diameter of the elec- micro-fragments and the structural integrity was lost com-
trospun fibers to 3.5–0.5mm. A further increase in the pletely. The addition of the nanoclay promotes faster deg-
nanoclay concentration (10%) resulted in the formation of radation of the PCL scaffolds. This may be due to the
beaded structures embedded in the sub-micron fibrous smallerfiberdiameterandthelowerhydrophobicityofPCL-
structure (with an average diameter of 600–50nm). The nanoclayscaffoldscomparedtoPCLscaffolds.Theaddition
decrease in the fiber diameter can be attributed to an in- of nanoclay may facilitate the adsorption of water that
crease in electron charge density of the prepolymer solu- renders PCL chains for hydrolytic degradation. Similar re-
tion due to the addition of nanoclay. In addition, the sults were previously reported, indicating that the addition
nanoclaymayhavedisruptedthemolecularcohesionofthe oftricalciumphosphate toPCLsignificantlyaccelerates the
2092 GAHARWAR ET AL.
FIG. 1. Preparation of electrospun PCL-nanoclay nanocomposite fibers. (a) Schematic representation of the process to
generate PCL/nanoclay composite fibers using electrospinning technique. The amount of nanoclay was varied from 0%,
0.1%,1%,and10%withregardtoPCL.(b)Theeffectofnanoclayonfiberdiameterandsurfacemorphologyoffibers.Pure
PCLfibersshowuniformsize distributionandsmoothsurfacemorphology.The additionofsilicate reducesfiberdiameter
andinducessurfaceroughness.Theeffectappearstobeprominentinelectrospunfiberscontaining1%and10%nanoclay.
(c) The effect of nanoclay on fiber diameter was quantified using image analysis. The average fiber diameter of PCL
scaffolds is 5.6–0.6mm. The addition of 1% and 10% nanoclay significantly reduced fiber diameter to 3.6–0.5 and
0.6–0.5mm. The data represent mean–standard deviation (n=50, ANOVA *p<0.05). The solid bars signify regions
indicating 50% of the distribution of fiber diameter. (d) The distribution of nanoclay within the electrospun scaffold was
determinedbystainingthenanoclaywithareddye.Thescaffoldcontainingnanoclay-formedbeadedstructures.ANOVA,
analysis of variance; PCL, poly(e-caprolactone). Color images available online at www.liebertpub.com/tea
degradation rate of the scaffolds due an increase in the jecting the scaffolds to accelerated degradation conditions
diffusion of media within the scaffolds.49 for 24 and 48h (Fig. 2b). In pure PCL scaffolds, no sig-
To evaluate the effect of nanoclays on the degradation nificant structural damage was observed. However, we ob-
mechanism of the electrospun scaffolds, microstructural servedadecreaseinfiberdiameteraftersubjectingthePCL
changes of electrospun fiber were investigated after sub- scaffold to the degradation medium for 48h. This indicated
NANOCLAY-ENRICHED PCL SCAFFOLDSFOROSTEOGENIC DIFFERENTIATION OF HMSCS 2093
FIG. 2. The effect of nanoclay
addition on the degradation of elec-
trospun PCL scaffolds. (a) Electro-
spun PCL scaffolds degrade via
surfacedegradationasdepictedfrom
thelinearmassloss.Thedecreasein
PCL fiber diameter was observed
after 48h in PCL scaffolds indicat-
ing surface degradation characteris-
tic.Theadditionofnanoclayresults
in the bulk degradation of electro-
spun scaffolds. All the nanocompo-
site scaffolds show breakage of
fibersin24and48h,respectively.In
PCL-10% nanoclay fibers, smaller
fibersquicklydegraded,followedby
bulk degradation of the thicker fi-
bers. (b) The accelerated degrada-
tionoftheelectrospunscaffoldswas
determined by monitoring the
weight loss of the fibrous structure
over a period of 96h. All the scaf-
folds showed a steady weight loss
indicatinggradualdegradationofthe
scaffold. The scaffold containing
10% nanoclay showed enhanced
degradation compared to PCL-only
scaffolds indicating that nanoclay
might promote the adsorption of
waterwithinthestructureaswellas
the bulk degradation of PCL. The
data represent mean–standard de-
viation (n=5).
that the degradation of electrospun PCL was dominated by spectively. This is mainly attributed to an increase in PCL
surface erosion mechanism, similar to previously reported crystallinity induced during the electrospinning process.
studies.50 The addition of nanoclay indicated a distinct The addition of nanoclay to PCL did not result in a sig-
degradation behavior. For example, electrospun fibers con- nificant increase in T , but lowered DH by 4.81J/g due to
m m
taining 0.1% nanoclay showed fiber breakage after 48h, theadditionof10%nanoclay.Thiscanbemainlyattributed
whereas fibers containing 1% nanoclay showed significant toadecreaseintotalPCLcontentintheelectrospunscaffold.
structural degradation after 24h. In electrospun scaffolds We further verified this by determining the effect of nano-
containing 10% nanoclay, thinner fibers were completely clay on the degree of crystallization of polymer fraction in
degraded within 24h. The breakage of thicker fibers in the electrospun fibers. The crystallinity of PCL beads was
nanoclay-enriched electrospun scaffolds indicated bulk determined to be 35.28%. The electrospinning process re-
degradation of the scaffolds. Overall, the addition of nano- sulted in an increase in polymer crystallinity to 49.22%,
clay to PCL significantly accelerates the degradation rate similartothepreviouslyreportedstudies.41Thecrystallinity
and facilitates bulk degradation behavior of the electrospun ofPCLinthenanocompositescaffoldwasdeterminedtobe
scaffolds. around 49–50% (Fig. 3c), which was similar to the electro-
spunPCLscaffold.Aslightincreaseinpolymercrystallinity
wasobservedduetotheadditionofnanoclay,indicatingthat
Nanoclay-improved thermalstability
the nanoclay particle may provide sites for crystallization.
of fibrousscaffolds
TheelectrospunPCLscaffoldsexhibitedacrystallization
The effect of nanoclay on thermal characteristics of the temperature(T )of28.79(cid:2)C,similartopreviousliterature.41
c
electrospun scaffolds was investigated by DSC and TGA The addition of nanoclay significantly increases the crys-
(Fig. 3). The DSC thermograms of heating and cooling tallization temperature of electrospun scaffolds. For exam-
cycles of electrospun PCL and nanoclay-enriched PCL ple, the addition of 0.1%, 1%, and 10% nanoclay results in
scaffolds were shown in Figure 3a (the top panel is cooling crystallization temperatures of 29.75(cid:2)C, 32.33(cid:2)C, and
curve,andthebottompanelisheatingcurve).PCLexhibited 33.18(cid:2)C, respectively. Similarly, the addition of nanoclay
apeakmeltingtemperature(T )of57.26(cid:2)C,andthemelting also results in a significant increase in the crystallization
m
enthalpy (DH ) was calculated to be 47.99J/g, which is enthalpy (DH ). The linear increase in T and DH suggests
m c c c
similar to the trend observed in literature.41 The electro- that the addition of nanoclay restricts chain mobility and
spinning process significantly enhanced T and DH of facilitates heterogeneous nucleation of PCL within the
m m
electrospun PCL scaffold to 59.94(cid:2)C and 66.95J/g, re- scaffolds.
2094 GAHARWAR ET AL.
FIG. 3. Effect of nanoclay on thermal characterization of electrospun PCL fibers. (a) The differential scanning calo-
rimeterthermogramsshowingcoolingcycle(toppanel),theadditionofnanoclay-increasedcrystallizationtemperature(T ),
c
and the enthalpy of crystallization (delta H ) indicate that nanoclay might be restricting polymer chain movements. The
c
electrospinning of PCL results in an increase in polymer crystallinity (w ). The addition of nanoclay results in a slight
c
increase in polymer crystallinity. Similarly, the heating cycles (bottom panel) of electrospun PCL and nanoclay-enriched
PCL scaffolds indicate a significant increase in the melting temperature (T ) and enthalpy of melting (delta H ) when
m m
compared to PCL beads. The addition of nanoclay increased the thermal stability of nanocomposite due to an enhanced
surface interaction between polymer and nanoclay. (b) TGA thermograph and DTGA profile indicate decreased thermal
degradation temperature (T ) due to the addition of nanoclay to PCL. (c) The summary of thermal properties of PCL and
d
nanoclay-enriched PCL scaffolds. Color images available online at www.liebertpub.com/tea
Figures 3b shows TGA thermograms of electrospun PCL degradation of nanoclay. Derivative of TGA thermogram
and nanoclay-enriched PCL scaffolds. Pure PCL scaffolds (DTGA) was determined to investigate the effect of nano-
completely degrade within 440(cid:2)C and negligible residue clay on degradation temperature. A significant shift in
wasobserved,similartothatreported intheliterature.51As DTGA curve was observed due to the addition of nanoclay
expected, with an increase in nanoclay concentration, an asobservedinFigure3bandc.Althoughwedidnotexpect
increaseinresidualmasswasobserved.Similarresultswere anycovalentlinkagebetweennanoclayandPCL,theshiftin
observedbyMarrasetal.,duetotheadditionoforganically DTGA curve might be attributed to an enhanced surface
modified MMT to electrospun polymeric scaffolds.36 The interaction between PCL and nanoclay.
TGA curve for electrospun PCL scaffold showed a single
degradation profile with an inflection point around 420(cid:2)C.
Effect of nanoclayonmechanicalproperties
The addition of 10% nanoclay decreased thermal degrada-
tion temperature of electrospun scaffold to 410(cid:2)C. More- Mechanical properties of scaffolds are important to
over, the addition of nanoclay resulted in two distinct evaluate their suitability for bone tissue engineering. The
degradation slopes. The first slope corresponds to degrada- scaffolds for such application should be able to bear dy-
tion of PCL, and the second slope corresponds to partial namic mechanical loading under in vivo conditions.8
NANOCLAY-ENRICHED PCL SCAFFOLDSFOROSTEOGENIC DIFFERENTIATION OF HMSCS 2095
Moreover, mechanical properties of scaffolds have a sig- these small regions deform locally and lead to fracture. We
nificant influence on hMSCs adhesion, proliferation, and have also evaluated the fiber morphology of the nano-
differentiation.52 The effect of nanoclay on mechanical composite scaffolds (0.1%, 1%, and 10% nanoclay) at their
properties of electrospun scaffolds was evaluated by a uni- extremities. At a lower nanoclay concentration, the fiber
axialtensiletest(Fig.4a,b).TheelectrospunPCLscaffolds alignment was followed by the fiber deformation during the
had an EM of 4.8–0.4MPa, an elongation of mechanical testing. However, at a higher nanoclay concen-
1050%–150%,andafracturestressof2.1–0.3MPathatis tration,nosignificantmechanicaldeformationatthefarendof
consistent with the literature.17,41 The addition of a small the fractured edge was observed. We further evaluated fiber
amount of nanoclay (0.1%) resulted in a decrease in EM to morphology at the fractured site, and found that the fracture
3.65–0.8MPa and an increase in elongation to mainly occurred due to pulling out of fibers and not due to
1160%–200% and fracture stress to 2.8–0.8MPa. This uniform deformation of fibers. This was mainly attributed to
indicates that the addition of a small amount of nanoclay the heterogeneous fiber size and the formation of beaded
results in more stretchable fibrous scaffolds. However, a structures at higher nanoclay concentrations. For designing a
further increase in nanoclay contents significantly reduced scaffold for bone tissue engineering, the scaffolds should be
EM, ultimate strain, and fracture strain. This was mainly able to withstand high mechanical strength. From the me-
attributed to the decrease in fiber diameter due to the ad- chanicaltestingresults,allthescaffoldshavetensilemodulus
dition of nanoclay, as well as the discontinuities along the in the range of 1 to 5MPa and elongation more than 100%.
fibers also weakened the electrospun network. Moreover, These properties are appropriate for deigning scaffolds for
nanoclays within the electrospun fiber might act as stress bone tissue engineering.8 Moreover, these mechanical prop-
concentrators and result in lower mechanical performance. erties are also shown to have a favorable influence on the
The effect of nanoclays on the mechanical deformation osteogenic differentiation of hMSCs.52
wasfurtherinvestigatedbydeterminingfibermorphologyof
the electrospun fibers near the fractured region. In an elec-
Nanoclaypromotedin vitro biomineralization
trospun PCL scaffold, uniform deformation was observed
throughout the scaffold, which had a decrease in the fiber A prerequisite for designing artificial materials for mus-
diameter as seen in Figure 4c. The addition of nanoclay culoskeletal tissue engineering is focused on developing
significantly reduced the elastic deformation of electrospun bone-bonding materials. Previous studies have investigated
scaffolds and resulted in the formation of small stress con- these properties by subjecting artificial biomaterials (or
centration regions. During the mechanical deformation, scaffold) to SBF, an ionic mixture that resembles the body
FIG. 4. Effect of nanoclay on the mechanical properties of electrospun PCL fibers. (a) Stress-strain curve of electrospun
scaffolds subjected to uniaxial tensile stress was shown. (b) The addition of nanoclay significantly reduced elastic modulus,
ultimatestress,andfracturestress.Thisisattributedtothedecreaseinfiberdiameterduetotheadditionofnanoclay.Thedecrease
in elongation was mainly attributed to the deformation of individual fibers. (c) The SEM images indicate that PCL scaffolds
undergouniformdeformationwhensubjectedtotensilestress.Theadditionofnanoclayresultedinaheterogeneousdeformation
and lower mechanical strength. SEM, scanning electron microscopy. Color images available online at www.liebertpub.com/tea
2096 GAHARWAR ET AL.
polyelectrolyte composition.44,53 The in vivo bioactivity reported studies.54 PCL is a bioactive polymer and when
potential of these materials is highly correlated with the exposed to physiological conditions, the surface of PCL fi-
formation of apatite-like depots on the surface of the ma- bers undergoes hydrolysis. This results in the formation of
terialswhenexposedtoSBF.44,53Thismethodisanindirect carboxylic (-COOH) group on the surfaces.55 The nega-
indication of bone-bonding ability of newly designed bio- tively charged functional groups (-COOH) interact with
materials/scaffolds, as it can predict the formation of min- calciumandphosphateionspresentinSBFandfacilitatethe
eral structures that might lead to the integration of the formation and deposition of hydroxyapatite crystals on the
scaffold with the bone.44 The increase in the surface surface of the scaffolds.55 The addition of nanoclays to
roughnessofscaffoldsaidsinthebiomineralizationprocess electrospun scaffolds significantly enhances the amount of
by providing nucleation sites for mineral deposits when mineralized deposits as evident by the increase in the fiber
subjectedtosupersaturatedsolution.44Bearingthisinmind, diameter (Fig. 5a, b). At higher nanoclay concentrations,
the effect of nanoclays on the in vitro biomineralization of mineral depositscovering several fiberswereobserved, and
the electrospun scaffolds was assessed using SBF. at a10% nanoclay concentration, itwasdifficult toidentify
Figure 5a and 5b showed the effect of nanoclays on the individualfibersduetothepresenceofacontinuouslayerof
biomineralization of electrospun PCL scaffolds. As ex- mineral deposits.
pected, on pure PCL scaffolds, the deposition of minerals We analyzed the mineral deposits using FTIR (Fig. 5c).
was observed, which correlates well with the previously Pure hydroxyapatite nanoparticles (positive control) are
FIG. 5. Nanoclay promotes in vitro biomineralization on electrospun scaffold in simulated body fluid (SBF). (a) Since the
prepared PCL scaffolds had uniform and smooth surface morphology, however after subjecting these electrospun scaffolds to
10· SBFfor2h,theformationofmineralizedlayeronthefiberswasobserved.SBFisasupersaturatedsolutionofcalciumand
phosphate, and a bioactive surface when submersed in SBF promotes the formation of mineralized structure. (b) The SEM
imagesindicatedfibermorphologyoftheelectrospunscaffoldsbeforeandaftersubjectingthescaffoldtoSBF.Theadditionof
nanoclay to PCL significantly improved the biomineralization ability due to enhanced surface roughness. (c) The chemical
nature of the deposited mineralized matrix was evaluated using FTIR. Hydroxyapatite has a strong peak at 1045cm-1 that
correspondstotheP-Ostretchingband.BothPCLandPCL-Nanoclaycompositesshowedtheformationofhydroxyapatitewhen
submersedinSBF.FTIR,FourierTransformInfraredSpectroscopy.Colorimagesavailableonlineatwww.liebertpub.com/tea
NANOCLAY-ENRICHED PCL SCAFFOLDSFOROSTEOGENIC DIFFERENTIATION OF HMSCS 2097
characterized by peaks at 1036, 603, and 567cm-1, cor-
respondingtophosphategroups(PO 3-).Weobservedthat
4
afterthetreatmentwithSBF,bothPCLandPCL-nanoclay
fibers show strong phosphate peaks. However, the PCL-
nanoclay scaffolds exhibit uniform coverage of the min-
eralizedlayeroverthescaffoldsurface.Inaddition,dueto
inorganic nature of the nanoclays, a local microenviron-
ment of concentrated inorganic polyelectrolyte is created,
leading to the precipitation/deposition of the mineralized
layer.Furthermore,theirregularfibrillarstructureofPCL-
nanoclay scaffolds provides the base for the formation of
nucleation sites, essential for the growth of inorganic de-
pots, which, in fact, is the prerequisite for fracture-healing
processes.
Adhesion andproliferation ofhMSCs
on nanoclay-enrichedPCLscaffolds
Themultipotentnatureandself-renewalabilityofhMSCs
make them the most clinically relevant cells to address the
healing and reconstruction of tissues or organs.52,56 In a
native microenvironment, the cellular fate of these hMSCs
is strongly dependent on the interaction with the surround-
ing cells, as well as with the ECM in which they are em-
bedded.57 In vitro strategies, aimed at mimicking these
conditions, have employed the seeding or encapsulation of
hMSCs on/in scaffolds aiming at their adhesion and prolif-
eration, along with the triggering of their differentiation
toward the desired lineage.2,4,58 The initial response of
hMSCs when seeded on a fibrous scaffold plays an impor-
tant role in defining the subsequent biological events and FIG.6. AdhesionandproliferationofhMSCsonPCLand
their desired functionality. hMSCs rely on the adhesion on PCL-nanoclay electrospun scaffolds. (a) hMSCs readily
substrates that enable the organization of their cytoskeleton attached and spread on the fibrous structure, and the cell
and sustain their proliferation, further dictating their be- bodieswerestretchedalongthefiberaxis(Day3).However,
havior.35,59 no significant difference in cell attachment was observed
due to the addition of nanoclay. (b) The proliferation of
The interaction of hMSCs with the scaffolds was evalu-
hMSCswasmonitoredusingAlamarBlueonday1and7in
atedbyinvestigatingtheiradherenceandmetabolicactivity.
normal media. hMSCs readily proliferate on PCL and na-
Thecellswereabletoadheretotheelectrospunscaffolds,as
noclay-enriched PCL scaffolds. However, no significant
theyreadilyelongatedtheircytoskeletonalongthefiberaxis effect of nanoclay on cell proliferation was observed. The
(Fig.6a).However,nosignificantinfluenceofnanoclaywas data represent mean–standard deviation (n=3, ANOVA
observed on cellular adhesion. The effect of silicate on the *p<0.05). hMSCs, human mesenchymal stem cells. Color
metabolic activity of adhered cells was evaluated using images available online at www.liebertpub.com/tea
Alamar blue assay. The metabolic activity of cells can be
used as an indicator for cellular proliferation. The result
their fillapodia on the micro-sized rough structure, leading
indicates that cells seeded on the nanoclay-enriched PCL
to an increase in the metabolic activity of adhered cells.
scaffolds exhibited significantly higher metabolic activity
Earlier studies have also indicated a positive effect of na-
when compared to PCL scaffolds (Fig. 6b).
noclay39 and nanoparticles60,61 on enhanced cell adhesion
The adhesion and proliferation of hMSCs are dependent
and proliferation.
on the presence of cell-adhesive proteins as well as on the
surface roughness of electrospun scaffolds. The addition of
NanoclayenhancedALP activityand promoted
nanoclay did not significantly influences protein adsorption
productionof mineralizedmatrix
on the electrospun scaffolds. After 24h, the amounts of
protein adsorbed on the surface of PCL, PCL-0.1% Nano- The effect of nanoclay on the osteogenic differentiation
clay, PCL-1% Nanoclay, and PCL-10% Nanoclay were ofhMSCs wasinvestigated by seedinghMSCs onPCL and
2.7–0.2, 3.1–0.5, 2.5–0.3, 2.4–0.2mg/mL, respectively. PCL-nanoclay scaffolds. The osteogenic differentiation of
This indicated that the adsorption of protein on the fibrous seeded hMSCs wasmonitored by determiningALP activity
scaffold was not the major factor, and, thus, surface overaperiodof21days.TheALPisanearlymarkerforthe
roughnessmayhavebeencontributingtotheincreaseinthe osteogenic differentiation of hMSCs, and its temporal pat-
metabolic activity of hMSCs. The roughness of these scaf- tern consists of an increase in its activity in the first time
folds (Fig. 1b), generated by the heterogeneous distribution points, followed by a dramatic decrease, accompanied by
of fiber diameter and nanoclays within the fiber, the cells themineralizationofthedepositedECM.Wefirstevaluated
caneasilypopulatethesubstratebyanchoringandstretching the distribution of ALP on the substrate, and found that its