Article

pubs.acs.org/Biomac

Effect of Cellulose Nanowhiskers on Surface Morphology, Mechanical

Properties, and Cell Adhesion of Melt-Drawn Polylactic Acid Fibers
Kazi M. Zakir Hossain,† Muhammad S. Hasan,∥ Daniel Boyd,∥ Chris D. Rudd,† Ifty Ahmed,*,†
and Wim Thielemans*,‡,§,⊥
†
Division of Materials, Mechanics and Structures, Faculty of Engineering, ‡School of Chemistry, and §Process and Environmental
Research Division, Faculty of Engineering, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom
∥
Department of Applied Oral Sciences, Faculty of Dentistry, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada
*
S Supporting Information

ABSTRACT: Polylactic acid (PLA) fibers were produced

with an average diameter of 11.2 (±0.9) μm via a melt-drawing
process. The surface of the PLA fibers was coated with blends
of cellulose nanowhiskers (CNWs) (65 to 95 wt %) and
polyvinyl acetate (PVAc). The CNWs bound to the smooth
PLA fiber surface imparted roughness, with the degree of
roughness depending on the coating blend used. The fiber
tensile modulus increased 45% to 7 GPa after coating with 75
wt % CNWs compared with the uncoated PLA fibers, and a
significant increase in the fiber moisture absorption properties
at different humidity levels was also determined. Cytocompat-
ibility studies using NIH-3T3 mouse fibroblast cells cultured onto CNWs-coated PLA surface revealed improved cell adhesion
compared with the PLA control, making this CNW surface treatment applicable for biomedical and tissue engineering
applications. Initial studies also showed complete cell coverage within 2 days.

■ INTRODUCTION
Biopolymers are extensively used in the fields of biomedical and
tissue engineering. Polylactic acid (PLA), a linear aliphatic
thermoplastic polymer that can be derived from renewable
sources (e.g., corn and potato starch), is one of the most widely
investigated biopolymers owing to its biocompatibility,
biodegradability, and suitable mechanical properties.1−3 PLA
can be produced into various architectural forms4−10 such as
fibers, films, plates, rods, and screws, which makes it highly
attractive for use in biomedical applications, for example, as
implants, bone fixation devices, or suture materials.11−16
The transformation of PLA into mono- and multi-filaments
has been achieved by melt, dry, wet, dry-jet-wet, and
electrospinning processes, each resulting in distinct fiber
properties.17−28 Melt spinning provides some advantages over
wet-spinning techniques, as it is a solvent-free process and thus
eco-friendly. The properties of melt-spun PLA fibers with
varying aspect ratios (L-/D-lactide) have been investigated
under different processing conditions by several au-
thors.17,24,29−33 For example, Kim et al.33 manufactured melt-
spun PLA monofilaments at 190 °C and investigated their
tensile properties at a range of temperatures between 25 and 65
°C. They reported that the tensile strength and modulus for
PLA monofilaments containing 4.2 mol % D-isomer decreased
significantly from 71 to 18 MPa and from 2.4 GPa to almost
zero (respectively) with increasing temperatures, which was
suggested to be associated with chain mobility of the polymer
in the glass-transition region. Mezghani and Spruiell31
investigated high-speed melt-spun poly(L-lactic acid) (PLLA)
filaments obtained at 233 °C utilizing a compressed air-drag
device to draw fiber filaments. Values for tensile strength of 320
MPa and modulus of 5.5 GPa were reported with diameters
ranging from 11 to 14 μm obtained at 5000 m minute−1 take-up
velocities. Yuan et al.32 characterized PLLA fibers with
diameters ranging between 269 and 340 μm obtained via a
melt-spinning process between 220 and 240 °C, which were
further reduced to diameters ranging between 110 and 160 μm
by hot drawing at 120 °C. The tensile strength and modulus for
the hot drawn fibers were reported to be 300−600 MPa and 3.6
to 5.4 GPa, respectively. They suggested that these relatively
high mechanical properties were associated with strain-induced
crystallization in the polymer chain, which was also confirmed
via differential scanning calorimetric (DSC) analysis. Mechan-
ical properties aside, the surface of PLA is very hydrophobic
and does not support appropriate cell adhesion, resulting in
inadequate biological activity.34,35 For improvement of the
surface morphology of PLA for tissue engineering and
biomedical applications, it has been functionalized via graft or
copolymerization with other biopolymers,36 chemical treat-
ment,37 plasma treatment,38 and adsorption39 of suitable
biocompatible materials, including chitosan,40 biotin,41 colla-
gen,37,42 gelatin,37,43 and alginate.44
Received: January 28, 2014
Revised: March 17, 2014
Published: March 28, 2014

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Biomacromolecules
Cellulose in the form of nanowhiskers has been extensively
used with different types of biopolymers, such as, PLA,15,45−51
polycaprolactone (PCL),52−54 polyvinyl alcohol (PVA),55−57
and polyvinyl acetate (PVAc)58 due to its biodegradability,59−61
biocompatibility,62 and superior mechanical properties (tensile
modulus ∼105 GPa for cotton-based nanocellulose). 63
Incorporation of cellulose nanowhiskers (CNWs) in PLA
matrices to form composites has already shown significant
improvement of their thermal and mechanical properties. John
et al.64 investigated PLA-based nanocomposite fibers (with
diameters ranging 90−95 μm) incorporating CNWs produced
via a melt-spinning process using a twin screw extruder. They
reported an increase in the surface roughness properties for
fibers containing CNWs. The influence of CNWs on the
thermal behavior for electrospun PLA fibers (average diameter
∼300 nm) was investigated by Liu et al.,65 who stated that the
CNWs acted as a heterogeneous nucleating site, which reduced
the cold crystallization onset temperature from 69 to 65 °C in
nanofiber containing 2.5 wt % CNWs when compared with the
unfilled PLA nanofiber. They also suggested that the nano-
composite fiber investigated should improve the mechanical
properties compared with the PLA fiber; however, no
mechanical test data were reported.
The PLA fibers investigated in this study were manufactured
using a melt-drawn process, and CNWs were obtained from
cotton via an acid hydrolysis process. The aim of this study was
to improve the surface of hydrophobic PLA fibers via coating
with varying blends of CNWs and PVAc to create a roughened
and more hydrophilic surface. To the best of our knowledge,
this is the first time CNWs and PVAc were used to coat the
surface of PLA fibers. The morphological, mechanical, and
moisture absorption properties (at various humidity levels) of
CNW/PVAc-coated PLA fibers are reported in this study. To
demonstrate the cytocompatibility of the coating blends
investigated, NIH-3T3 mouse fibroblast cells were cultured
onto CNW/PVAc-coated PLA film surface and investigated for
their initial cell attachment and spreading.
■
MATERIALS AND METHODS
PLA Fibers Drawing. PLA fibers were produced via a melt-
drawing process. In brief, vacuum-dried (at 50 °C for 48 h) PLA beads
(NatureWorks, Ingeo grade 3251D, average Mw ≈ 90 000−120 000 g
mol−1, density 1.24 g cm−3) were melted at 180 °C in air using a
cylindrical steel mold with attached band heater and with a 2 mm hole
at its base. As the molten polymer exited the base via the hole due to
gravity it was collected on a drum (1 m drum diameter with a collector
distance of ∼50 cm) rotating at 400 m min−1.
Preparation of CNW. CNWs were produced via an acid hydrolysis
process using cotton (purchased from Fisher Scientific, U.K.) and
aqueous H2SO4 (64 wt %) (Fisher Scientific, U.K.) for 45 min.15,66
The hydrolyzed cotton was washed with deionized water with
intermittent centrifugation (three cycles, each at 10 000 rpm for 15
min) and then dialyzed under running tap water for 48 h to ensure
removal of free acid. Sonication was conducted to homogenize the
dispersion, followed by filtering through a fritted glass filter (no. 2
porosity grade) to remove residual aggregates prior to treatment with
Amberlite resin (purchased from Fisher Scientific, U.K.) to remove
non H3O+ cations. Filtration removed the Amberlite resin and a final
sonication was performed before storing the dispersion in the fridge
with a drop of chloroform to inhibit bacterial growth.
PLA Fiber Coating. The coating materials were produced by
blending different ratio of CNWs and PVAc (formulation composition
in Table 1) maintaining 20% w/v suspension in DI water. PVAc was
obtained as Kollicoat SR, a 30% dispersion of PVAc (weight average
Mw ≈ 450 000) stabilized with polyvinyl pyrrolidone and sodium lauryl
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Table 1. Formulations of Coating Materials Prepared Using
CNWs and PVAc for PLA Fibers
coating materials
sample codes used in this study CNWs (wt%) PVAc (wt%)
PLA
PLA PVAc 100
PLA CNWs-65 65 35
PLA CNWs-75 75 25
PLA CNWs-85 85 15
PLA CNWs-95 95 5
sulfate, density 1.045 g cm−3, and viscosity ∼100 mPa·s at 20 °C. The
PLA fibers were coated using a syringe needle containing the coating
suspension and running the syringe tip over the fiber five times
followed by drying of the coated fibers in an oven at 37 °C for 24 h to
ensure deposition of materials on the surface of the fiber.
Characterization. Electron Microscopy. The surface morphology
of the coated and uncoated PLA fibers was characterized using
scanning electron microscopy (SEM - Philips XL30, FEI, USA) at an
accelerating voltage of 10 kV and a working distance of 10 mm.
The shape of the CNWs produced in this study was examined using
transmission electron microscopy (TEM) on a JEOL (JEM-2000FXII,
U.K.) at an accelerating voltage of 80 kV. CNWs were deposited from
dilute aqueous dispersions onto carbon-coated copper grids hydro-
philized by plasma treatment in an oxygen/argon 25/75% atmosphere
for 5 s. The deposited CNWs were imaged after staining with uranyl
acetate (Sigma-Aldrich, U.K.) (2 wt %) for 3 min.
Fluorescence Microscopy. Coated and uncoated PLA fibers were
fluorescently labeled with Rhodamine B (Sigma Aldrich, U.K.) using a
syringe needle. Drops of this aqueous dye solution (125 mg in 50 mL
of distilled water) were applied to the fibers three times and allowed to
dry at room temperature. A fluorescence microscope (Leica DMLB)
was employed to image the surface of the Rhodamine-B-labeled fibers.
Optical Microscopy. Fiber diameters were measured using a
calibrated optical microscope (20× magnifications), and an average
fiber diameter was calculated from the measurement of at least 30
random fibers.
Surface Roughness Determination. PVAc and CNWs-PVAc
blends were coated on the solvent-cast (chloroform) PLA films
(thickness around 0.3 mm), and their surface roughness was analyzed
using a Surftest (SV-600) Mitutoyo system. A diamond stylus tip (5
μm tip radius) with a 0.2 mm s−1 travel speed was used to measure the
surface roughness over a 4.8 mm measurement length. The average
roughness (Ra) was determined by measuring at 35 different positions
of the film surface.
Tensile Tests. Tensile property determination of individual fibers
was conducted using a Q800 from TA Instruments employing a
controlled force deformation of single PLA fibers at room temperature
(25 °C). Single PLA fibers were glued onto a 10 mm gauge length
paper tab and equilibrated at the specified temperature for 10 min
before ramping the force at a rate of 0.01 N min−1 until break. A
minimum of six repeat tests were conducted for each sample.
Fiber Conditioning and Moisture Absorption. Moisture uptake
properties of coated and uncoated PLA fibers were obtained after
conditioning the fibers at different relative humidities (RHs) of 0, 35,
75, and 98% at room temperature by recording the weight gain of the
fibers. Previously weighed PLA fibers (∼30 mm length) were placed in
open glass vials, which were kept in a closed chamber containing the
saturated salt solutions (e.g., P2O5 (0% RH), CaCl2·6H2O (35% RH),
NaCl (75% RH), and CuSO4·5H2O (98% RH)) for 2 weeks to ensure
equilibrium was reached.58
Cell Attachment and Morphology Assessment. NIH-3T3 mouse
fibroblast cells (ATCC) were used to investigate cell attachment on
four types of surfaces (in disc form) with similar surface topography of
the uncoated and coated fibers, namely, PLA (control PLA film), PLA
CNWs-75 (PLA film coated with the blend of 75 wt % CNWs and 25
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Figure 1. (a) Scanning electron microscopy image of PLA fibers obtained at 400 m min−1. (b) TEM image of cellulose nanowhiskers (CNWs) and
(c) schematic of the coating procedure employed on the PLA fiber surface.
Figure 2. SEM images of (a) noncoated, (b) PLA PVAc, (c) PLA CNW-65, (d) PLA CNW-75, (e) PLA CNW-85, and (f) PLA CNW-95 fibers.
wt % PVAc), PLA PVAc (PLA film coated with PVAc), and TCP
(tissue culture plastic).
Samples (9 mm diameter, 0.3 mm thick) were sterilized by
immersing into 70% ethanol for 30 min, washed with sterilized
phosphate buffer saline (PBS) media, and inserted into 48-well plates
(Falcon, USA). Cells at passage 17 were cultured in Dulbecco’s
modified Eagle’s medium DMEM (Sigma Aldrich) supplemented with
10% fetal calf serum at 37 °C in a 10% CO2 environment. Cells were
seeded at a density of 40 000 cell/sample and were incubated for up to
48 h at predetermined time points (4, 24, and 48 h). Samples were
washed with warm PBS at 37 °C and fixed in 3% glutaraldehyde
(Sigma Aldrich) in 0.1 M Na-cacodylate buffer (Sigma Aldrich) for 30
min, after which the fixative was replaced by a 7% sucrose solution
(Sigma Aldrich) in 0.1 M Na-cacodylate/distilled water for overnight
storage at 4 °C. The samples were then washed twice in 0.1 M Na-
cacodylate, and fixed cells were dehydrated through a graded ethanol
series (20, 30, 40, 50, 60, 70, 80, 90, 96, and 100% v/v in water) for ∼5
min each. Samples were then dried via hexamethyldisilazine (HMDS)
(Sigma Aldrich) before being sputter-coated in gold and viewed using
a Hitachi scanning electron microscope operated at 3 kV.
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■ RESULTS AND DISCUSSION
In this study, PVAC/CNW blends with varying amounts of
CNWs were used to coat the hydrophobic surface of PLA
fibers. The effect of CNW dimensions, their hydrophilic nature,
and high mechanical properties on modifying the PLA surface
characteristics, as well as their moisture absorption properties,
were investigated.
Morphological Properties. The surface topography of
PLA fibers produced via a melt-drawn process (at 180 °C, 400
rpm onto a 1 m circumference take up drum) exhibited smooth
and even surfaces with fairly uniform diameters, as seen in
Figure 1a. The TEM image of the CNWs produced is
presented in Figure 1b, revealing rod-like particles.67
Figure 1c represents a schematic of the coating method
employed to apply the coating materials (with varying ratio of
CNWs and PVAc) onto the surface of the PLA fibers using a
syringe needle containing the coating suspensions.
The morphology of PVAc- and CNWs-PVAc-coated PLA
fibers (seen in Figure 2) showed that the deposition process
resulted in a textured surface on the fibers compared with a
smooth surface for the uncoated PLA fibers. Furthermore, an
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Figure 3. Fluorescence images of Rhodamine-B-labeled (a) noncoated, (b) PLA PVAc, (c) PLA CNW-65, (d) PLA CNW-75, (e) PLA CNW-85,
and (f) PLA CNW-95 fibers.

increased roughness was observed for the nanowhisker-coated fibers). The deposition of the coating materials on the fiber
PLA fibers with a relatively uniform distribution for PLA CNW- surfaces resulted in no significant change in average diameter (P
65, PLA CNW-75, and PLA CNW-85. > 0.05) compared with noncoated PLA fibers (as shown in
The PLA CNW-95 fibers (Figure 2f) revealed uneven Figure 4). PLA CNWs-95 revealed an average diameter around
attachment of CNWs on the fiber surface, which was believed
to be due to an insufficient amount of PVAc binder present in
the coating materials to provide homogeneous coverage,
leading to CNW clustering. For the control coating, good
coverage and a uniform coating of the PLA fiber surface was
achieved using only PVAc during application (Figure 2b).
The surface morphology of coated and uncoated PLA fibers
was further characterized via fluorescence microscopy using
Rhodamine B to label the fibers. Similar to the SEM images, a
relatively smooth surface could be seen for the fluorescently
labeled PLA fibers (Figure 3a), although the fluorescence
intensity of the image was quite low compared with that for the
coated fibers. This was suggested to be due to better adsorption
of Rhodamine to PVAc and the PVAc/CNW blend in
comparison with PLA alone.
A homogeneous coverage of the PVAc coating on the PLA
fiber surface was also observed, as can be seen in Figure 3b,
which also indicated good binding between the fluorescent
Rhodamine B probe and the PVAc coating. Figure 3c−f
revealed that the CNW-PVAc-coated fibers had a much rougher
Figure 4. Average diameter for coated and uncoated PLA fibers
surface morphology with reduced homogeneity of the coating measured using optical microscope.
observed with increasing CNW content (observed via the
darker areas) on the fibers, which could be due to reduced
wetting of the PLA fiber surface by the PVAc-CNW blends 11.4 μm, which suggested that very little coating had been
with increasing CNW content. Increasing the CNW content retained on the fiber surface. The low amount of PVAc thus
within the PVAc-CNW blend compared with pure PVAc and appeared to restrict the amount of CNWs that could be
the formation of inter-CNW hydrogen bonds may lead to the anchored to the PLA surface.
formation of a percolated network and increased viscosity, Surface Roughness. The average surface roughness (Ra) of
leading to reduced spreading and thus the formation of clusters the PLA, PLA PVAc, and various ratios of CNWs-PVAc coated
and increased surface roughness. films are presented in Figure 5. The surface roughness imparted
The diameters of the coated and uncoated PLA fibers were onto the films produced were found to be consistent with the
measured via optical microscopy, and an average diameter of results obtained via SEM and fluorescence images of the single
11.2 μm (±0.9) was obtained (measured from at least 30 fibers, where the rod-like nanowhiskers created increased
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Figure 5. Surface roughness of PLA film coated with PVAc and
CNWs-PVAc.
roughness (Ra = 3.0 and 2.8 μm for PLA CNWs-75 and PLA
CNWs-85, respectively) compared with the noncoated PLA
control (Ra = 0.22 μm) and PVAc-coated PLA films (Ra = 0.19
μm).
The greatest increase in surface roughness was observed for
coating blends containing 75 and 85% CNWs. A similar surface
roughness of ∼3.0 μm was recently reported for high CNW
content composites (75 wt % in a hydroxyethyl cellulose
matrix).67 The roughness imparted on the surface of CNW-
PVAc-coated PLA could help to improve cell-adhesion
properties for tissue-engineering applications; Khan et al.68
investigated the influence of surface roughness via chemical
etching on silicon-based biomaterials and showed that rough-
ness promoted cell adhesion and longevity. Similar improved
cell adhesion, growth, and proliferation properties were also
reported by Keshel et al.,69 where roughness had been created
via plasma treatment on polyurethane films.
Mechanical Properties. The tensile properties of PLA
fibers (presented in Figure 6) coated with PVAc and CNWs-
Figure 6. Tensile strength and modulus properties for the single PLA
fibers coated with PVAc and CNWs-PVAc.
PVAc were characterized in tensile mode at 25 °C. The tensile
strength and modulus of the noncoated PLA fibers were 207
MPa and 4.9 GPa, respectively. While comparatively higher
tensile properties have also been reported in the literature for
PLLA fibers, for instance, the tensile strength and modulus of
PLLA fiber with diameter ranges from 11 to 14 μm were
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reported to be around 320 MPa and 5.5 GPa, respectively,31
this was due to the higher L-lactide content in PLLA as
compared with the PLA (NatureWorks−3251D) polymer
investigated in this study.
Attachment of all coating substances onto PLA fibers
revealed no significant change in tensile strength (P > 0.05)
compared with the noncoated PLA fibers tested at room
temperature. For instance, PLA PVAc, PLA CNW-65, and PLA
CNW-75 fibers revealed a 9, 7, and 0.5% decrease, and the PLA
CNWs-85 and PLA CNW-95 fibers exhibited 4 and 0.5%
increase in tensile strength compared with uncoated PLA fibers
(tensile strength 207 MPa).
However, a significant improvement in the tensile modulus
(P < 0.0001) for PLA CNWs-65, PLA CNWs-75, and PLA
CNWs-85 fibers (33, 43, and 45% increase, respectively) was
observed when compared with the noncoated PLA fibers
(tensile modulus 4.9 GPa), suggesting a tangible influence from
the nanowhiskers (tensile modulus = 105 GPa)63 deposited on
the fiber surface. This influence of the deposition layer formed
on the fiber surface has been attributed to the CNW-PVAc as
well as the CNW-CNW percolating interactions, where PVAc
strongly attached the CNWs to the surface of the fibers during
the drying process, as demonstrated in Figure 7. A similar
Figure 7. (a) Deposition pattern of CNWs-PVAc on the surface of
PLA fibers and (b) cross-sectional view of the deposits.
percolated network for high CNW (75 wt %) content thin-film
composites was also suggested when the composite tensile
modulus increased from 0.39 to 8 GPa.67 In addition, PLA
CNW-95 also exhibited a 12% increase in tensile modulus
(which was not seen to be statistically significant P > 0.05)
compared with the uncoated PLA fibers.
The experimental modulus of the coated fibers could be
compared using a simple two parallel system core−shell model,
which predicts the tensile modulus of the coated PLA fibers.
The predicted tensile modulus (E′) can be expressed by
E′ = (x × M1) + (y × M 2) (1)
where x and y are the volume fraction of PLA (core) and
coated materials (shell) and M1 and M2 are their corresponding
tensile moduli, respectively. However, tensile modulus of the
CNWs-PVAc blends with various CNWs contents used was
calculated using the Halpin−Kardos model to predict the
modulus of nanocomposites containing randomly oriented
nanofillers in a plane.67 The densities of the PLA and PVAc
were taken to be 1.24 and 1.045 g cm−3 from the supplier data
sheet, and CNWs were considered to be 1.59 g cm−3 from the
literature.70 The average tensile modulus of the PVAc was
considered to be 2.4 GPa (±0.2), which was measured
experimentally using PVAc films (∼0.2 mm thickness) via a
solvent-casting method after dispersing in deionized water. The
aspect ratio of 20 (average length 200 nm and diameter 10
nm)67 and tensile modulus of 57 GPa63 of cotton-based CNWs
were also used to calculate the predicted modulus of the
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CNWs-PVAc-coated PLA fibers. The experimental modulus of
the PLA fibers coated with various CNWs-PVAc blends were
seen to follow the same trend as the predicted modulus. (See
Figure 8.) However, the experimental modulus of the PLA
Figure 8. Comparison of experimental modulus and predicted
modulus using the two parallel system core−shell model applied to
the coated PLA fibers.
PVAc and PLA CNW-65 fibers was found to be higher as
compared with the predicted modulus. This could be suggested
to be due to the volume fraction of the low modulus PVAc
binder, which alone was not sufficient to reduce the modulus of
the PLA PVAc fibers by as much as expected by theory.
The properties for elongation at break for the coated and
uncoated fibers are shown in Figure 9. The attachment of PVAc
Figure 9. Properties for elongation at break for single PLA fibers only
and coated with PVAc and CNWs-PVAc.
on the PLA fiber surface exhibited a decrease (P < 0.05) in
elongation properties from 78 to 62% compared with the
noncoated fibers, which was suggested to be due to the
formation of a thin rigid layer of PVAc onto the PLA fiber
surface. The CNWs-PVAc-coated PLA fibers revealed that their
elongation at break properties increased from 71 to 78% with
increasing CNW content in the coating materials, indicating
that CNW inclusion in the coating appeared to restore the
elongation at break of the coated fibers to the values for
uncoated fibers with the exception for PLA CNWs-65 fibers,
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which showed the value around 63%, closer to that of PLA
PVAc fibers. This could have been due to the fact that 65 wt %
of CNWs in the coating blend was not sufficient to alter the
elongation properties of the fibers as compared with that of the
PLA PVAc fibers.
Moisture Uptake. The coated and uncoated PLA fibers
were conditioned at various humidity levels (0, 35, 75, and 98%
RH) using saturated salt solutions (see Figure 10a) for 2 weeks
to investigate their moisture absorption.
The moisture absorption of the conditioned coated and
uncoated PLA fibers revealed an increasing trend with an
increase in relative humidity, as seen in Figure in 10b. PLA
CNWs-75 fibers exhibited higher water uptake at 98% RH (6.0
wt %) compared with the PVAc-coated (4.2 wt %) and pure
PLA fibers (2 wt %). However, a lower amount of moisture
uptake (only 2.7 wt % at 98% RH) for PLA CNWs-95 fibers
was also suggested to be due to insufficient amount of PVAc
binder as well as CNWs on the fiber surface. Comparatively
higher quantity of water accumulation was expected in the case
of PLA CNWs-65, PLA CNWs-75, and PLA CNWs-85 fibers
due to the hydrophilic nature of the CNWs. This has previously
been shown by Garcia de Rodriguez et al.,58 who compared the
water take-up of CNW-PVAc composite at different RH. The
accumulation of water at the CNW surface was later shown by
Capadona et al.71 to reduce the reinforcing effect of the CNW
percolated network by disrupting hydrogen bonding. In the
water take-up studies of Garcia de Rodriguez et al.,58 they
reported ∼12 wt % water accumulation at 98% RH for PVAc-
sisal whisker nanocomposites containing 2.5 wt % whisker
content.
An increase in the hydrophilic nature of CNW-PVAc coated
PLA fiber could help to provide better cell attachment in cell
culture media, as suggested by Oh et al.,72 who investigated the
in vitro cell compatibility of hydrophobic poly(lactic-co-glycolic
acid) (PLGA) against a hydrophilic PLGA/poly(vinyl alcohol)
(PVA) blend using human chondrocytes. It was reported that
the PLGA/PVA blend was easily wetted in culture medium
without the requirement of prewetting treatments and had
better cell attachment and growth than the control hydro-
phobic PLGA.
Cell Study. The SEM micrographs of NIH-3T3 mouse
fibroblast cells cultured on the surface of CNW/PVAc-coated
PLA film are presented in Figure 11. The PLA sample surface
was completely covered with cells with continuous proliferation
and spreading, which formed a confluent structure of
multilayered cells after 24 h of incubation. (See Figure 11a.)
Initial cell attachment of fibroblasts on the PLA CNW-75
surface within the first 4 h revealed flat (well-attached and
spread) and rounded cells, extending their filopodia to anchor
onto the surface (Figure 11b). After 24 h of incubation,
multilayered fibroblast cells covered the PLA CNW-75 surface,
and only a few rounded cells were observed. The PLA CNW-75
surface was completely covered by multilayer fibroblast cells
after 48 h of culture, although the morphology of these sheets
was not as flat as on PLA. No viable cells could be seen on the
PLA PVAc surface after seeding (as shown in Figure 11c),
which was suggested to be due to the use of high
concentrations of PVAc, which softened, resembling a sticky
glue like substance when exposed to DMEM. A flat
morphology of the cells attached to TCP (the internal control)
surface was observed, as can be seen in Figure 11d.
Delamination of multilayered fibroblasts on the TCP surface
after 48 h of incubation was seen, which could be due to
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Figure 10. (a) Typical arrangement for fibers conditioned at different humidity levels and (b) moisture uptake of PVAc- and CNWs-PVAc-coated
and uncoated PLA fibers as a function of relative humidity.

Figure 11. Influence of materials and surface roughness on NIH-3T3 mouse fibroblast cell morphology and spreading at varying time points (4, 24,
and 48 h): (a) control PLA, (b) PLA CNW-75, (c) PLA PVAc, and (d) control TCP (tissue culture plastic).

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dehydration caused by the chemical drying process employed in
this study. Previous studies have reported on the cytotoxicity of
CNWs, which was suggested to be dose- or cell-type
dependent.73−75 For example, Dugan et al.73 reported that
CNW suspensions were not cytotoxic to C2C12 myoblasts
evaluated using the MTT viability assay. Conversely, CNW
suspensions were reported to elicit a dose-dependent
cytotoxicity effect that was assessed using J774A.1 macro-
phages.76 In another study, Fang et al.77 compared the behavior
of human bone marrow stromal cells cultured onto neat
bacterial-derived CNWs and hydroxyapatite-coated CNWs for
2 days. They reported adhesion and spreading of cells on both
surfaces, along with better cell attachment on the hydrox-
yapatite-coated CNW, due to their rougher surface. Another
study reported that CNW surfaces supported the adhesion,
spreading, and proliferation of C2C12 myoblasts and adhesion
and spreading of primary human stem cells, confirming their
cytocompatibility.74 In addition, the cells were reported to be
orientated along the direction of the CNWs. Figure 11
suggested that CNWs influenced cell attachment and delayed
cell spreading, which was in agreement with Fang et al.77 and
Dugan et al.73,74 Influence of CNWs on cell alignment could
not be confirmed in this study due to the asymmetrical and
disordered CNW layers. Nevertheless, initial cell functions
(attachment, spreading) suggested that the samples tested were
compatible with mouse fibroblast cells and that the attachment
of cells on CNW-coated PLA was qualitatively better than on
neat PLA.
Apart from the potential advantages associated with
improvement in cell attachment properties for the CNW-
PVAc coated PLA fiber, the accumulated water content could
also influence the degradation rate of the fiber. Kim et al.78
suggested that the improved hydrophilicity of the PLA/PLGA
electrospun nanofibers enhanced their degradation rate to 65%
weight loss in 7 weeks compared with hydrophobic PLA (which
only revealed a ∼10% weight loss).
■
CONCLUSIONS
CNWs were attached to the hydrophobic surface of melt-drawn
PLA fibers using PVAc as a binding agent. The morphology of
the CNWs were seen to be aggregated and played a role in
increasing the surface roughness of PLA fibers. No statistically
significant changes in their tensile strength properties were
observed due to the coating substances applied to the fibers
surfaces. However, up to 45% increase in tensile modulus
properties was obtained for the PLA fibers coated with 75 wt %
CNWs-PVAc blend, respectively, which demonstrated the
influence of CNW-CNW and CNW-PVAc interactions.
Additionally, the presence of CNWs-PVAc blend had a
significant effect in increasing the moisture absorption
properties by up to 6.0 wt % at 98% relative humidity, showing
increased hydrophilicity of the coated PLA fibers when
compared with the hydrophobic noncoated PLA fiber. It was
also suggested that the roughness and hydrophilic surface
created on the PLA fibers via CNW-PVAc coating materials
could impart favorable cell attachment and proliferation and
increase degradation properties. Cytocompatibility studies
using NIH-3T3 mouse fibroblast cells cultured onto CNW-
coated PLA surface revealed better cell adhesion compared with
the PLA control. Initial studies also showed that these cells
spread well on the PLA surface to completely cover it within 2
days. This could potentially enable the use of these CNW-
1505
Article
coated PLA fibers within biomedical and tissue engineering
applications.
■
*
ASSOCIATED CONTENT
S Supporting Information
Additional experimental results including SEM images of
coated PLA films, FTIR-ATR spectra, thermal analysis
(DSC), and thermomechanical (storage modulus) properties
the CNW/PVAc-coated PLA fibers. This material is available
free of charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Authors
*I.A.: E-mail: ifty.ahmed@nottingham.ac.uk.
*W.T.: E-mail: wim.thielemans@kuleuven.be.
Present Address
⊥
W.T.: KU Leuven, Kortrijk Campus, Etienne Sabbelaan 53,
8500 Kortrijk, Belgium.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We acknowledge the financial support provided by the
University of Nottingham, Faculty of Engineering (the Dean
of Engineering Research Scholarship for International Ex-
cellence) and would also like to thank Engineering and Physical
Sciences Research Council for financial support (grant EP/
J015687/1).
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