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. Cytocompatibility 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.

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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 authors.17,24,29−33 For example, Kim et al.33 manufactured meltspun 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 © 2014 American Chemical Society

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. Mechanical 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 treatment,37 plasma treatment,38 and adsorption39 of suitable biocompatible materials, including chitosan,40 biotin,41 collagen,37,42 gelatin,37,43 and alginate.44 Received: January 28, 2014 Revised: March 17, 2014 Published: March 28, 2014 1498

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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 nanocomposite 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.

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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 PLA PLA PLA PLA PLA PLA

PVAc CNWs-65 CNWs-75 CNWs-85 CNWs-95

CNWs (wt%)

PVAc (wt%)

65 75 85 95

100 35 25 15 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 hydrophilized 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

MATERIALS AND METHODS

PLA Fibers Drawing. PLA fibers were produced via a meltdrawing 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 1499

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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.

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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 Nacacodylate, 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.

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 1500

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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.

fibers). The deposition of the coating materials on the fiber surfaces resulted in no significant change in average diameter (P > 0.05) compared with noncoated PLA fibers (as shown in Figure 4). PLA CNWs-95 revealed an average diameter around

increased roughness was observed for the nanowhisker-coated PLA fibers with a relatively uniform distribution for PLA CNW65, PLA CNW-75, and PLA CNW-85. The PLA CNW-95 fibers (Figure 2f) revealed uneven 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 surface morphology with reduced homogeneity of the coating 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 with increasing CNW content. Increasing the CNW content within the PVAc-CNW blend compared with pure PVAc and the formation of inter-CNW hydrogen bonds may lead to the formation of a percolated network and increased viscosity, leading to reduced spreading and thus the formation of clusters and increased surface roughness. The diameters of the coated and uncoated PLA fibers were measured via optical microscopy, and an average diameter of 11.2 μm (±0.9) was obtained (measured from at least 30

Figure 4. Average diameter for coated and uncoated PLA fibers measured using optical microscope.

11.4 μm, which suggested that very little coating had been retained on the fiber surface. The low amount of PVAc thus appeared to restrict the amount of CNWs that could be anchored to the PLA surface. Surface Roughness. The average surface roughness (Ra) of the PLA, PLA PVAc, and various ratios of CNWs-PVAc coated films are presented in Figure 5. The surface roughness imparted onto the films produced were found to be consistent with the results obtained via SEM and fluorescence images of the single fibers, where the rod-like nanowhiskers created increased 1501

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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 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 CNWPVAc-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 roughness 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 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

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 1502

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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

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 PVAcsisal 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 hydrophobic 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

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, 1503

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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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coated PLA fibers within biomedical and tissue engineering applications.

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 macrophages.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 hydroxyapatite-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 CNWPVAc 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).

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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.

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AUTHOR INFORMATION

Corresponding Authors

*I.A.: E-mail: [email protected]. *W.T.: E-mail: [email protected]. Present Address ⊥

W.T.: KU Leuven, Kortrijk Campus, Etienne Sabbelaan 53, 8500 Kortrijk, Belgium. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support provided by the University of Nottingham, Faculty of Engineering (the Dean of Engineering Research Scholarship for International Excellence) and would also like to thank Engineering and Physical Sciences Research Council for financial support (grant EP/ J015687/1).

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REFERENCES

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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 CNWcoated 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 CNW1505

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