Uniaxially Aligned Electrospun All-Cellulose ... - ACS Publications

Jan 9, 2014 - State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute at Sichuan University, Chengdu 610065, China ..... Han...
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Uniaxially Aligned Electrospun All-Cellulose Nanocomposite Nanofibers Reinforced with Cellulose Nanocrystals: Scaffold for Tissue Engineering Xu He,† Qiang Xiao,‡,# Canhui Lu,† Yaru Wang,† Xiaofang Zhang,† Jiangqi Zhao,† Wei Zhang,*,† Ximu Zhang,*,‡,# and Yulin Deng§,∥ †

State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute at Sichuan University, Chengdu 610065, China State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China # Department of Preventive Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China § School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0620, United States ∥ Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, Georgia 30332-0620, United States ‡

S Supporting Information *

ABSTRACT: Uniaxially aligned cellulose nanofibers with well oriented cellulose nanocrystals (CNCs) embedded were fabricated via electrospinning using a rotating drum as the collector. Scanning electron microscope (SEM) images indicated that most cellulose nanofibers were uniaxially aligned. The incorporation of CNCs into the spinning dope resulted in more uniform morphology of the electrospun cellulose/CNCs nanocomposite nanofibers (ECCNN). Polarized light microscope (PLM) and transmission electron microscope (TEM) showed that CNCs dispersed well in ECCNN nonwovens and achieved considerable orientation along the long axis direction. This unique hierarchical microstructure of ECCNN nonwovens gave rise to remarkable enhancement of their physical properties. By incorporating 20% loading (in weight) of CNCs, the tensile strength and elastic modulus of ECCNN along the fiber alignment direction were increased by 101.7 and 171.6%, respectively. Their thermal stability was significantly improved as well. In addition, the ECCNN nonwovens were assessed as potential scaffold materials for tissue engineering. It was elucidated from MTT tests that the ECCNN were essentially nontoxic to human cells. Cell culture experiments demonstrated that cells could proliferate rapidly not only on the surface but also deep inside the ECCNN. More importantly, the aligned nanofibers of ECCNN exhibited a strong effect on directing cellular organization. This feature made the scaffold particularly useful for various artificial tissues or organs, such as blood vessel, tendon, nerve, and so on, in which cell orientation was crucial for their performance.

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INTRODUCTION Nowadays, the growing environmental awareness throughout the world has triggered a paradigm shift toward fabricating environmentally friendly materials.1,2 Cellulose, representing about 1.5 trillion tons of the total annual biomass production, is considered as one of the world’s most abundant natural and renewable resource of raw material for the increasing demand for environmentally friendly and biocompatible products.3,4 Natural cellulose based materials (wood, hemp, cotton, linen, etc.) have been used in our lives as engineering materials for thousands of years and their use continues today as corroborated by the enormity of the worldwide industries in forest products, paper, textiles, and so on.5 Recently, the production of nanocellulose materials has received increasing attention due to their high strength and stiffness combined with small size, low density, renewability, and biodegradability.6−8 There are several polymorphs of crystalline cellulose (I−IV), among which the crystal structure of cellulose I exhibits the highest axial elastic modulus.5,9 Typically, cellulose nanocrystals © 2014 American Chemical Society

(CNCs) with cellulose I crystal structure have attracted considerable interests in the nanocomposite field.10,11 CNCs are mainly prepared from native cellulose fibers through acid hydrolysis to remove the amorphous region and preserve the crystalline region. The theoretical Young’s modulus for ideal CNCs is estimated to be 167.5 GPa, which is even theoretically stronger than steel and similar to that of Kevlar.12 The elastic moduli of CNCs from tunicate and cotton have been experimentally confirmed to reach up to 105 and 143 GPa, respectively.13 These excellent mechanical properties make CNCs very promising as an effective reinforcement for polymer nanocomposites. To date, CNCs have been successfully incorporated into a wide range of polymer matrices to produce high performance nanocomposites, such as poly(diallyldimethylammonium chloride)/CNCs,14 polymethyl Received: November 8, 2013 Revised: December 26, 2013 Published: January 9, 2014 618

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methacrylate (PMMA)/CNCs, 15 polycaprolactone-based waterborne polyurethane (WPU)/CNCs,16 poly(vinyl alcohol) (PVA)−poly(acrylic acid) (PAA)/CNCs,17 polyoxyethylene (PEO)/CNCs,18,19 and all-cellulose nanocomposites.20−24 One-dimensional (1D) nanostructures have been an intensive research subject due to their amazing properties and attractive applications in many fields. A large number of synthetic and fabrication approaches have been suggested for generating 1D nanostructures in the form of fibers,25 such as drawing,26 template synthesis,27 phase separation,28 selfassembly,29 electrospinning,30 and so on. Among these methods, electrospinning is considered as an efficient method that can be further developed for mass production of one-byone continuous nanofibers from various polymers.30 Due to the unique structures, electrospun fibers possess fascinating characteristics, such as very large surface area to volume ratio, flexibility in surface functionalities, and so on.31,32 A wide range of potential applications have been demonstrated for electrospun nanofibers, such as protective clothing,33 battlefield dressings,34 and tissue engineering scaffolds.35,36 For many of these applications, mechanical strength and toughness are extremely important as even partial failure could have dire consequences.37 However, electrospun nonwovens usually exhibit poor mechanical performance, representing one of the limitations for the materials.38 It is conceivable that a high degree of fiber orientation along a preferential direction could increase the stiffness and strength of the nonwoven. For example, Greiner et al. reported that the Young’s modulus and the maximum deformation stress of electrospun copolyamide 6/6T (PA6/6T) nanofibers increased from 100 and 20 MPa (for nonwovens with randomly packed nanofibers) to 900 and 70 MPa (for nonwovens with nanofibers aligned along the elongation direction), respectively.32 Another potential method to strengthen electrospun nanofibers is to incorporate robust nanoparticles into polymer matrices.19,39,40 If the two methods were integrated, the electrospinning technique could become a desirable approach for manufacturing nanofibers with excellent mechanical properties. Cellulose is formed out of glucose-based repeat units, connected by 1,4-β-glucosidic linkages. Unlike starch, which has a very similar glucose-based structure but with alpha linkages, cellulose-based materials exhibit very low water solubility, therefore, allowing for better control over scaffold design.41 In vitro and in vivo applications of cellulose-based materials have demonstrated only negligible foreign body and inflammatory response reactions.42−44 A limited number of studies, however, have used aligned nanofiber scaffolds, which mimic the structure of some aligned tissues, for example, parallel collagen fibers in tendon tissue45 or nerve.46 Creating tissue engineered structures with aligned fibers similar to that of native tendon or nerve could enable the healing tissue to be directed and formed into this optimal orientation. However, to induce cellular organization orderly is not easy for ordinary scaffolds, which only support cells for attachment and proliferation. Recently, Fujie et al. reported a microcontact printing technique to create fibronectin micropatterns on a polystyrene nanomembrane.47 Enhanced cell elongation and overall alignment of the myoblasts along the micropattern were observed. The objective of this work was to fabricate uniaxially aligned electrospun nanofiber nonwovens from cotton cellulose and evaluate their potentials in tissue engineering applications. In order to overcome the drawbacks of poor mechanical

properties, CNCs were incorporated into the spinning dope to strengthen the scaffolds. It is worth to note that so far most studies on cellulose electrospinning used cellulose derivatives as the raw materials. This is because cellulose cannot be dissolved in common solvents due to its strong inter- and intramolecular hydrogen bonds.48 Owing to the intrinsic properties of cellulose solutions and dielectric requirements for this technique, only a few solvents have been successfully explored for electrospinning of nonderivative cellulose,49 known as N-methyl-morpholine Noxide solution/water (NMMO/H2O),50 lithium chloride/ dimethyl acetamide (LiCl/DMAc),51 ionic liquids,52 and ethylene diamine/salt.53 In this study, the solvent system of LiCl/DMAc, which almost does not cause degradation of cellulose,54,55 was adopted for the electrospinning of nonderivative cellulose. Cotton cellulose solutions with well dispersed CNCs, which were also originated from cotton, were prepared as the spinning dope. During the electrospinning process, a rotating steel drum was used as the fiber collector and a high rotating speed was necessary to induce uniaxial alignment of cellulose nanofibers. The dispersion and orientation of CNCs in cellulose nanofibers, mechanical properties, crystalline structures, and thermal stability of the electrospun cellulose/CNCs nanocomposite nanofibers (ECCNN) would be analyzed. In addition, the cytotoxic effect of ECCNN was tested. Since our future research work will focus on the use of ECCNN scaffolds for periodontal tissue engineering, human dental follicle cells (hDFCs) were selected in this study. The proliferation of cultured hDFCs was explored and the cell morphology was revealed by SEM and confocal laser scanning microscopy (CLSM).

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EXPERIMENTAL SECTION

Materials. Purified cotton (medical level, Health Materials Co., Ltd., Xuzhou, China) was used as the raw material. Analytical grade N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF), concentrated sulfuric acid and lithium chloride (LiCl) were purchased from Kelong Chemicals Co., Ltd. (Chengdu, China). Distilled water was used throughout the experiment. For cell culture study, Dulbecco’s modified eagle’s medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2, diphenyl tetrazolium bromide (MTT, 98%) reagent, trypsin, fetal bovine serum (FBS), 40,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Alexa-fluor 568 antibodies were purchased from Invitrogen (Molecular probes, Carlsbad, CA, U.S.A.). Glutaraldehyde solution (25 wt %) was obtained from Zhongshan Fine-Chem Ltd. (Guangdong, China). All the other reagents were of analytical grade and were used without further purification. Preparation of CNCs Suspensions in Water. Aqueous suspensions of CNCs were prepared following a modified method in literature.56 Briefly, cotton cellulose fibers were first ground into small fragments using a crusher. Next, the cellulose fibers were hydrolyzed with 64 wt % sulfuric acid at 45 °C for 70 min. Typically, 40 g of cotton was treated with 700 mL of sulfuric acid. Immediately after hydrolysis, the suspension was diluted with 10-fold of water to terminate the reaction. The suspension was then washed repeatedly with water and centrifugation (10000 rpm, 10 min for each cycle) until the pH of the supernatant was higher than 1. The sample was subsequently dialyzed against water for 3 days. Finally, a colloidal CNCs suspension was obtained after sonication. The solid concentration of CNCs aqueous suspension was 1.0 wt %. Figure S1a showed a representative atomic force microscope (AFM) image of the as-prepared CNCs, which were generally 150−250 nm in length and 15−25 nm in width. Preparation of CNCs Suspensions in DMF. To prepare well dispersed suspension of CNCs in DMF, the aqueous suspension of CNCs was solvent-exchanged into a DMF dispersion by vacuum619

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Figure 1. Scheme of the main experimental procedure in this study. assisted rotary evaporation.57 A total of 150 mL of aqueous dispersion of CNCs was poured into a round-bottom flask, and 150 mL of DMF was added under agitation. Water and a small portion of DMF in the mixture were subsequently evaporated at 60 °C using a rotary evaporator until the amount of distillate was around 170 mL. The final concentration of CNCs in DMF was adjusted to 0.9 wt %. The general experiment procedure in this study was schematically presented in Figure 1. Preparation of Well Dispersed Cellulose/CNCs Suspensions. To dissolve cellulose, an activation pretreatment was required in order to weaken the polymer chain interaction to get a relaxed conformation. Otherwise, cellulose cannot be dissolved in the LiCl/DMAc solvent.54 The pretreatment included the following processes: first, cellulose was immersed in water for 1 h and then water was removed by vacuum filtration; second, solvent exchange with methanol for 1 h to eliminate residual water followed by vacuum filtration; third, solvent exchange with DMAc for 1 h to help expel the residual methanol; finally, the cellulose was filtrated and dried at 70 °C. To prepare 8 wt % LiCl/ DMAc solvent, DMAc was heated to 105 °C to get rid of any residual water. Eight g dry LiCl was added to 92 g warm DMAc (80 °C) under magnetic stirring until absolutely dissolved, and then cooled down to room temperature. A typical preparation procedure for 2 wt % cellulose solutions was as follows: 2 g activated cellulose was added to 98 g as-prepared LiCl/DMAc solvent and stirred at room temperature until complete dissolution. A typical 5% cellulose/CNCs spinning dope was prepared by mixing a CNCs suspension in DMF (containing 0.05 g CNCs) with a cellulose solution (containing 1 g cellulose). After that, the mixture was subjected to magnetic stirring and sonication (100 W, 10 min) to form a well dispersed CNCs suspension in cellulose solution. Cellulose/CNCs spinning dopes with CNCs concentrations of 5, 12.5, 20, and 25% (w/w, in respect to the weight of dissolved cellulose) were prepared in parallel and stored in a refrigerator before use. Electrospinning Process. A computer controlled automatic electrospinning equipment (FM-12, Fuyouma Technology Co., Beijing, China) was used to fabricate ECCNN. Cellulose/CNCs spinning dopes were loaded in a 5 mL plastic syringe with a 0.8 mm stainless steel needle. The needle was connected to a high voltage power supply, which generated positive voltages up to 50 kV (20 kV was used in this study). The flow rate (0.03 mL/min) of the solution was controlled by a micro pump. A steel rotating collector (6 cm in diameter) wrapped with aluminum foil was placed 10 cm away from the tip of the nozzle. The rotating collector was grounded to help collect fibers. The tangential velocity of the collector was set at 300 m/ min so as to induce the alignment of nanofibers. To prepare randomly oriented nanofibers, the tangential velocity was set at 50 m/min. The

rotating collector was partly immersed in a water coagulation bath to thoroughly remove the solvent from electrospun fibers and produce dimensionally stable fibers. The obtained nonwovens were subsequently dried under ventilation and then kept in a desiccator. Cell Cultures. Primary human dental follicle cells (hDFCs) were obtained from patients aged 14 and 20 years after obtaining informed consent as reported previously.58 Briefly, normal human impacted third molars were surgically removed and collected. Dental follicle tissues were digested in a solution of 0.1 U/mL collagenase type I and 1 U/mL Dispase (Roche, Mannheim, Germany) for 1 h at 37 °C. Attached hDFCs were cultured at 37 °C in 100 mm dishes using MSC growth medium (GM; consisting of MSC basal medium supplemented with fetal bovine serum, L-glutamine and penicillin/streptomycin; Lonza, Walkersville, MD) in a humidified incubator (CO2 incubator MCO-175M; Sanyo, Osaka, Japan) in the presence of 5 vol% CO2 in air at 37 °C. In this study, passage 3 to 5 hDFCs were used. Seeding of hDFCs on ECCNN scaffolds. The ECCNN scaffolds used in this study were about 0.04 mm in thickness and were cut into discs of 13 mm in diameter. Before seeding cells, the scaffolds were dehydrated by passing them through ethanol with gradient concentrations (20, 40, 60, 80, and 100%, v/v) for the duration of 15−20 min for each concentration. Subsequently, in order to remove the traces of ethanol, scaffolds were washed in 0.1 M PBS (pH 7.4) three times (5 min for each wash). Scaffolds were then treated with UV to ensure the proper sterilization. For appropriate cell attachment, scaffolds were saturated in complete DMEM by incubating them in 1 mL of media for 1 h.59 Then, a 0.5 mL aliquot of cell suspension was seeded at variable densities depending on the type of experiment, followed by the incubation of the scaffolds at 37 °C in 5 vol% CO2 at 80−90% humidity. Seeding was done on both sides of the samples. Samples were removed at specific intervals for the examination of cell proliferation via MTT, SEM, and CLSM. MTT Assay. Proliferation capacity of the fibroblast cell lines was checked by seeding the scaffolds with the cell suspension with a density 1 × 105 cells/mL. Cell proliferation and scaffold cytotoxicity were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell seeded scaffolds were cultured in 6 well plates. As a negative control, cells were seeded at the same density (1 × 105 cells/mL) without scaffold. As a positive control, Toxol (Paclitaxel: plant-derived chemotherapeutic anticancer drug from Taxus brevifolia L.) was used. MTT assay was performed after every alternate day. On the day of the test (days 1, 3, 5, and 7), media was removed from both test and control followed by washing with cold PBS (pH 7.4). MTT solution at the concentration of 0.5 mg/mL (0.5 mL) was added to both test and control wells followed by incubation for 4 h at 37 °C in humid environment with 5 vol% CO2. According to 620

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Figure 2. SEM images and fiber diameter distributions of ECCNN: (a) 0% ECCNN, (b) 5% ECCNN, (c) 12.5% ECCNN, (d) 20% ECCNN. Scale bar: 1 μm.

Table 1. Tensile and Thermal Properties of ECCNN sample CNCs 0% ECCNN 5% ECCNN 12.5% ECCNN 20% ECCNN

tensile strength (MPa)

elastic modulus (GPa)

elongation at break (%)

average fiber diameter (nm)

Tib (°C)

Tdc (°C)

± ± ± ± ± ± ± ±

212

172.3 261.7

355.3 301.7

221

264.0

306.8

215

264.5

307.3

212

280.4

323.6

a

16.6 ± 1.5 e 6.3 ± 1.3 d 19.5 ± 1.2 e 7.9 ± 1.6 d 26.3 ± 2.0 e 9.8 ± 1.2 d 33.6 ± 1.3 e 11.2 ± 1.4 d

143 0.69 0.46 1.32 0.67 1.66 0.81 1.87 1.21

± ± ± ± ± ± ± ±

0.06 0.07 0.09 0.07 0.06 0.08 0.07 0.06

3.3 2.0 2.8 1.6 3.2 1.9 2.6 1.7

0.3 0.4 0.4 0.3 0.3 0.4 0.4 0.3

a

Elastic modulus of CNCs was quoted from literature.13 bTi denoted the initial degradation temperature. cTd denoted the temperature at the maximum degradation rate. dMeasured in the direction along the fiber orientation. eMeasured in the direction perpendicular to the fiber orientation. (along the orientation of fibers) and width (perpendicular to the orientation of fibers), whereas, for tensile properties in the direction perpendicular to the orientation of fibers, nonwovens were cut by length (perpendicular to the orientation of fibers) and width (along the orientation of fibers). The samples have dimensions of 50 mm ×10 mm × d mm, where d was the thickness of the nonwovens. The thickness of each sample was approximately 0.04 mm. TGA was performed with a TG209 F1 instrument (NETZSCH Co., Germany) to measure the decomposition temperature of ECCNN. The samples (8−10 mg) were placed in the crucible and heated from 40 to 600 °C at a heating rate of 10 °C/min under a nitrogen atmosphere (nitrogen flow rate was 100 mL/min). hDFCs cultured on the surfaces of ECCNN were examined by SEM. Scaffolds (seeded with hDFCs, cultured for 3 days) were removed from the growth media, followed by gentle washing with 0.1 M sterile PBS. Cells were fixed on the surface of the scaffold with 2.5 wt % glutaraldehyde in 0.1 M sodium cacodylate at 4 °C for 4.5 h. Scaffolds were then dehydrated through a series of alcohol from 20 to 70 vol%, stained in 0.5 wt % uranyl acetate, followed by further dehydration in 90, 95, and 100 vol% alcohol. The final dehydration was followed by drying in a vacuum desiccator. Then the samples were sputter coated with gold before SEM imaging (S-4500; Hitachi, Japan) at an accelerating voltage of 20 kV. hDFCs grown inside the ECCNN were observed by CLSM. After 3 days culture, analysis media was aspirated and scaffolds were gently washed with cold PBS. Fixation of the cell−scaffold samples was carried out in freshly prepared 2 vol% paraformaldehyde for 20 min. Then the samples were washed in cold PBS three times, permeabilized in PBS containing 10 vol% FBS plus 0.5 vol% Triton-X 100 (Sigma Aldrich, St. Louis, MO, U.S.A.) for 20 min, and incubated in PBS containing 10 vol% FBS for 30 min. Thereafter, samples were stained

the manufacturer’s protocol, after that, MTT was removed gently followed by the addition of 1.5 mL of dimethylsulfoxide (DMSO). Plates were again incubated for 15 min at 37 °C with 5 vol% CO2. After the incubation period, the color development was measured by an enzyme-linked immunoabsorbent assay reader (ELISA Reader, BioRad) at 570 nm for the estimation of relative viability of the cells. Measurement and Characterization. The shape and dimension of CNCs were measured by atomic force microscope (AFM, Nanoscope Multimode and Explore, Veeco Instruments Inc., U.S.A.) in the noncontact mode. CNCs were spin-coated on a round mica sheet with a diameter of 10 mm. Polarized light microscope (PLM) images were obtained using an Olympus BX51 optical microscope equipped with two polarized light filters, a rotating stage, and a digital camera. Samples were viewed under white light conditions. The surface texture of ECCNN was observed on a field-emission scanning electron microscope (SEM, JEOL JSM-7500F, Japan) at an accelerating voltage of 5 kV. Fiber diameter distributions were measured from SEM images with the software Image J (at least 100 fibers were randomly selected from the images). The dispersion and orientation of CNCs in ECCNN were evaluated using transmission electron microscope (TEM, JEOL JEM-100CX, Japan) at 80 kV. Samples with a thickness of 100 nm for the TEM imaging were prepared by freezing and sectioning the electrospun nonwovens along the fiber alignment direction. Wide angle X-ray diffraction (WAXD) analysis was performed on a Philips Analytical X’Pert X-diffractometer (Philips Co., Netherlands) using Cu Kα radiation (λ = 0.1540 nm) at an accelerating voltage of 40 kV and a current of 40 mA. The data were collected from 2θ = 5−50° with a step interval of 0.03°. Tensile properties of ECCNN were measured by an Instron 5567 universal testing machine at a stretching rate of 1 mm/min. For tensile properties along fiber orientation, nonwovens were cut by length 621

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Figure 3. TEM micrograph of a single nanofiber in 20% ECCNN. Arrows point to CNCs. Scale bar: 100 nm for (a), 50 nm for (b). with Alexa-fluor 568 conjugated phalloidin for filamentous actin fluorescence (1:200) and cell nuclei were marked by 40,6-diamidino-2phenylindole (DAPI) staining for nuclei UV−visualization (1:2000) for 2 h. Subsequently, specimens were thoroughly washed with PBS, and rinsed with deionized water for 2 min. Finally, specimens were visualized using an Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) at 460 nm (emission) and the multitrack images were captured with a 60×/1.35 NA objective.

natively, the orientation of CNCs in this study was realized through the drawing forces from the rotating collector. Besides, the CNCs were also presented in a high strength electrostatic field during electrospinning. It was reasonable that the synergistic effect of mechanical forces and electrostatic field could induce efficient orientation of CNCs in cellulose nanofibers. The internal structural details of nanofibers could be revealed by TEM.63 As shown in Figure 3a,b, rod-like CNCs were well oriented along the nanofiber long axis. It is important to note that further dissolution of CNCs in the cellulose solutions can be largely prohibited. The AFM image of Figure S1b indicated the shape of CNCs was preserved when CNCs suspended in DMF was added into LiCl/DMAc (without cellulose as a solute) under the same condition for the preparation of cellulose/CNCs mixture. WAXD data (Figure S3) also suggested the crystallinity index (CrI) of treated CNCs (75.5%) just slightly decreased as compared with that of original CNCs (83.5%), possibly due to the surface swelling of CNCs. Consequently, ECCNN consisted of uniaxially aligned cellulose nanofibers with well oriented CNCs embedded was produced. The diagrammatic sketch of ECCNN structure was presented in Figure 1. PLM was utilized to evaluate the dispersion of CNCs in ECCNN. Figure 4 showed the PLM micrographs of ECCNN.

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RESULTS AND DISCUSSION Microstructure of ECCNN. A series of ECCNN mats were prepared by electrospinning of the spinning dopes with given compositions. The SEM images and the fiber diameter distributions for different samples (CNCs loading of 0, 5, 12.5, and 20%, in weight) were presented in Figure 2a, b, c, and d, respectively. Continuous nonwoven can be successfully fabricated even at a CNCs loading of 20%. However, when the CNCs loading increased to 25%, the spinning dope was no longer electrospinnable. With such a high loading of CNCs, the spinning dope on the tip of the needle would become highly unstable so that continuous fibers could not be obtained during electrospinning. To achieve better alignment of electrospun cellulose nanofibers, a high tangential speed (300 m/min) was applied on the rotating collector during electrospinning. From Figure 2, it was evident that most cellulose nanofibers aligned along the nanofiber long axis, suggesting a preferential alignment of nanofibers under the drawing force exerted by the rotating collector. As shown in Table 1, all samples had very close average fiber diameters ranging from 212 to 221 nm, but their diameter distributions were different. It was noticed that the fiber diameter distribution of ECCNN became narrower when the CNCs loading increased, indicative of the positive effect of CNCs on creating ECCNN with a more uniform morphology. It was noteworthy that there seemed to be several thick fibers with diameters of 500−1000 nm presented in the SEM images. These fibers were actually fiber bundles composed of two or more thinner fibers with diameters of about 200 nm (Figure S2). The electrospinning of cellulose required a water coagulation bath to solidify the electrospun fibers. When water was evaporated, the aligned electrospun fibers were likely to adhere with adjacent fibers due to capillary forces induced by water. Dispersion and Orientation of CNCs in ECCNN. TEM and PLM were used to visualize the dispersion as well as the orientation of CNCs in ECCNN. It has been reported that a high degree of orientation of CNCs could be achieved by mechanical forces,60 magnetic61 and electric fields.62 Alter-

Figure 4. PLM images of ECCNN: (a) 0% ECCNN, (b) 5% ECCNN, (c) 12.5% ECCNN, (d) 20% ECCNN. The arrows denote the long axis direction of electrospun cellulose nanofibers. Scale bar: 200 μm. 622

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cellulose nanofibers, the characteristic peak centered at 2θ = 21.9° for 0% ECCNN gradually shifted toward higher scanning angles (2θ = 22.6° for 20% ECCNN), since CNCs have their characteristic peak at 2θ = 22.7° for the [200] lattice plane of Cellulose I.65 It is noteworthy that the characteristic peak of Cellulose I was not clearly distinguishable even with the CNCs loading of 20%. This could be probably ascribed to the orientation of CNCs during electrospinning. However, WAXD patterns of 20% ECCNN with lower CNCs orientations (collected under a much lower rotating speed of the collector) still did not show Cellulose I peak (Figure S4). In a previous study, all-cellulose composite films with different ratios of Cellulose I and II were produced by partial dissolution of microcrystalline cellulose powder in LiCl/DMAc and subsequent film casting.20 When the Cellulose I proportion was 24%, the WAXD pattern of the composite appeared very similar to that of regenerated cellulose (with the highest peak intensity at an angle of 20.4°). By increasing the Cellulose I proportion in the composites, the peak at 22.7° became much sharper. Tensile Properties of ECCNN. Tensile properties of ECCNN were measured in directions both along and perpendicular to the fiber orientation. The stress−strain curves were presented in Figure 6 and the data was summarized in Table 1. For tensile properties along the fiber orientation (Figure 6a), by increasing the CNCs loading, tensile strength increased from 16.6 MPa (0% ECCNN) to 33.6 MPa (20% ECCNN) and elastic modulus increased from 0.69 GPa (0% ECCNN) to 1.87 GPa (20% ECCNN), enhanced by 101.7 and 171.6%, respectively. The hierarchical microstructure of nonwovens consisted of uniaxially aligned cellulose nanofibers with well dispersed and orientated CNCs as reinforcement was very much favorable for the long axial strength of cellulose nanofibers. Moreover, the intrinsic mutual compatibility could enable an intimate interfacial adhesion between CNCs and cellulose matrix, which enabled an effective stress transfer from cellulose matrix to the strong CNCs. However, owing to their high porosities, the ECCNN nonwovens still exhibited inferior tensile properties compared with other all-cellulose nanocomposites produced by film casting (126.9 MPa and 8.5 GPa for tensile strength and elastic modulus, respectively).23 Tensile properties in the direction perpendicular to orientation of fibers were also significantly enhanced with increased CNCs loading (Figure 6b). However, compared with those obtained along the fiber orientation, the mechanical properties in this direction were much lower. This could be ascribed to the fact that only a

There were almost no light regions in the sample of 0% ECCNN. That is, almost no crystal anisotropy could be identified, suggesting that 0% ECCNN was almost amorphous. This is in accordance with early discovery on cellulose nanofibers electrospun from their LiCl/DMAc or ionic liquid solutions.50,64 The PLM micrographs of the samples (5% ECCNN, 12.5% ECCNN, and 20% ECCNN) were shown in Figure 4b, c, and d, respectively. Distinct light domains along the nanofiber long axis could be observed in Figure 4b−d, which could be ascribed to the anisotropy properties of CNCs and the strong birefringence of CNCs under the polarized light. The light domain density increased as more CNCs were incorporated into ECCNN. Moreover, the light domains dispersed evenly in the PLM micrographs, indicative of good dispersion of CNCs in ECCNN. WAXD Analysis of ECCNN. Figure 5 showed typical WAXD patterns of ECCNN. All the samples exhibited similar

Figure 5. WAXD patterns of ECCNN.

WAXD profiles. As reported by Kim et al.50 and Xu et al.,64 the electrospun cellulose nanofibers regenerated from water were mostly amorphous with rather low crystallinity of the typical polymorph of Cellulose II. The WAXD pattern of 0% ECCNN had a characteristic peak at an angle of 21.9 o. This peak could be resulted from the overlapping between the amorphous cellulose peak (18.5°) and the Cellulose II peak (20.2° and 22.3°).50 As mentioned earlier, CNCs could largely maintain their crystallinity when their DMF suspension was mixed with LiCl/DMAc solution (Figure S3). By incorporating CNCs into

Figure 6. Stress−strain curves of ECCNN: (a) measured in the direction along the fiber orientation, (b) measured in the direction perpendicular to the fiber orientation. 623

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few fibers oriented in this direction, making it less effective for load bearing. TGA Thermograms of ECCNN. The TG and DTG curves of ECCNN samples and CNCs were presented in Figure 7, and

Figure 8. Cell viability and proliferation of hDFCs on ECCNN as evaluated by MTT assay. Initial cell density for seeding was 1 × 105 cells/mL.

between the positive control and the ECCNN group. The optical density (OD) value increased slowly with culture time. From this assay it could be concluded that ECCNN did not induce any toxic effects on the fibroblast cells (hDFCs), proving their potential to support cell proliferation and utility for tissue engineering applications. Morphology of hDFCs Cultured on ECCNN. The cell proliferation was also observed by SEM. As demonstrated in Figure 9a, after 3 days culture, cells clustered partly on the surface of ECCNN scaffold. On day 7, Figure 9b showed the confluent cells almost covered everywhere. Under higher magnification, spreading of hDFCs was observed on ECCNN scaffold on day 4, fibroblasts extended on the nanofibers of ECCNN (Figure 9c). The secreted extracellular matrix (ECM) could be seen in Figure 9d, which means ECCNN support hDFCs with a friendly growth environment for production of ECM. Furthermore, the cell proliferation was also confirmed by CLSM. In CLSM images, DAPI staining for nuclei UV− visualization (Blue) and Alexa-fluor 568 conjugated phalloidin for actin (Red), they showed similar results with SEM. On day 3, cells clustered on part of the scaffold. In the clustered part, cells inclined to interact with each other and proliferated orderly to form a confluent oriented structure (Figure 10a). However, for nonoriented scaffold, cells randomly scattered on the scaffold (Figure S5a), which was well in line with the SEM image (Figure S5b). A possible reason for this might be that aligned nanofibers retain fibroblast natural ECM structure which is more favorable for cell adhesion and proliferation than randomly aligned scaffold.68 On day 7, cells were highly confluent and grew along fiber alignment direction. Almost all cells were highly oriented. Both cell actin filament and cell nuclei stretched toward the same direction (Figure 10b). The aligned nanofibrous scaffold could mimic the ECM structure of periodontal fiber, for which ordered fibers were needed to share the occlusal force.69 CLSM could observe not only the surface of the ECCNN, but also the layers inside. Aided by Matlab software (MathWorks Co., U.S.A.), three-dimensional reconstruction of ECCNN cultured with cells for 7 days could be realized (Figure 10c). Red and blue fluorescence distributed among the ECCNN, which means cells could grow well in the entire ECCNN.

Figure 7. TG and DTG curves of ECCNN.

the data was summarized in Table 1. By incorporating CNCs, the thermal stability of ECCNN could be improved remarkably. The initial degradation temperature (Ti) and the maximum degradation temperatures (Td) of ECCNN increased with the increase of CNCs content. Ti and Td of 20% ECCNN were 280.4 and 323.6 °C, which were 18.7 and 21.9 °C higher than those of 0% ECCNN (261.7 °C for Ti and 301.7 for Td), respectively. The DTG peak temperature can be used as a measure of thermal stability. As shown in Figure 7, all peaks of ECCNN moved to higher temperatures after the addition of CNCs, indicating that CNCs improved the thermal stability of ECCNN. As is well recognized, the surface sulfate moieties of CNCs exhibited flame retardant properties and could act as a catalyst for dehydration and thermal degradation of CNCs.66 This would result in an increase of char residues, which functioned as an insulator and mass transport barrier to the volatile products generated during decomposition of the cellulose matrix. Growth and Proliferation of hDFCs on ECCNN Scaffolds by MTT. MTT was used for testing cell proliferation and this assay actually gives an idea about the toxicity of the material on the cells. The assay detects the reduction of MTT by mitochondrial dehydrogenase to blue formazan product, which reflects the normal function of mitochondria and hence the measurement of cytotoxicity and cell viability.67 From Figure 8, cell proliferation on the ECCNN scaffold was clear. The MTT activity was significantly decreased in negative control compared with the positive control and ECCNN group. Moreover, no significantly different changes were found 624

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Figure 9. SEM image of hDFCs cultured on ECCNN: (a) cultured for 3 days, (b) cultured for 7 days, (c) hDFCs spreads on the surface of ECCNN, (d) secreted ECM could be seen.

Figure 10. CLSM images of hDFCs loaded in ECCNN: (a) cultured for 3 days, (b) cultured for 7 days, (c) the three-dimensional view of ECCNN with cells. Scale bar: 50 μm.

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CONCLUSIONS

axis direction of electrospun nanofibers. PLM showed that CNCs were well dispersed in ECCNN. Due to the high stiffness of CNCs and strong interfacial bonding between CNCs and the regenerated cellulose matrix, remarkably improved tensile properties of ECCNN were achieved. By incorporating 20% loading of CNCs, tensile strength and elastic modulus of ECCNN in the fiber alignment direction increased by 101.7 and 171.6%, respectively. Moreover, the addition of CNCs could also enhance the thermal stability of ECCNN.

A series of ECCNN with different CNCs loadings were successfully fabricated via electrospinning. Morphology investigation from SEM images indicated that most of the obtained cellulose nanofibers were uniaxially aligned and had similar average fiber diameters ranging from 212 to 221 nm. More uniform morphology of electrospun cellulose nanofibers could be achieved with the incorporation of CNCs. TEM demonstrated that CNCs were well oriented along the long 625

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Cell culture experiments demonstrated ECCNN was very suitable for hDFCs attachment and proliferation in the entire scaffold and induce ordered hDFCs organization. For these unique features, we particularly envisage this robust scaffold to be applied in artificial blood vessels, which work against the high-shear stress condition under arterial blood flow. In addition, this study will also broaden the potential applications of all-cellulose nanocomposite nanofibers, such as filter membranes, protective clothing, and so on.

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ASSOCIATED CONTENT

S Supporting Information *

AFM images and WAXD patterns of CNCs before and after treatment. WAXD patterns of 20% ECCNN collected with different rotating speed on the collector. Magnified SEM images of 20% ECCNN. CLSM and SEM images for cells cultured on randomly oriented ECCNN scaffold. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (W.Z.); zhangximu1212@163. com (X.Z.). Tel.: (+086) 28-85460607 (W.Z.). Fax: (+086) 2885402465 (W.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Natural Science Foundation of China (51303112 and 51203105) and Young Scholar Fund of Sichuan University (2012SCU11074) for financial support of this work.

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