Materials Science and Engineering C 49 (2015) 463–471

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Incorporation of poly(ethylene glycol) grafted cellulose nanocrystals in poly(lactic acid) electrospun nanocomposite fibers as potential scaffolds for bone tissue engineering Chunmei Zhang a,b,c, Max R. Salick d, Travis M. Cordie e, Tom Ellingham b, Yi Dan a,⁎, Lih-Sheng Turng b,⁎ a

State Key Laboratory of Polymer Materials Engineering of China, Polymer Research Institute of Sichuan University, Chengdu 610065, China Department of Mechanical Engineering, University of Wisconsin–Madison, Madison, WI 53706, USA c Department of Chemistry and Materials Engineering, Guiyang University, guiyang 550005, China d Department of Engineering Physics, University of Wisconsin–Madison, Madison, WI 53706, USA e Department of Biomedical Engineering, University of Wisconsin–Madison, Madison, WI 53706, USA b

a r t i c l e

i n f o

Article history: Received 22 September 2014 Received in revised form 9 November 2014 Accepted 6 January 2015 Available online 8 January 2015 Keywords: Poly(lactic acid) Cellulose nanocrystals Poly(ethylene glycol) Interfacial adhesion Mechanical properties

a b s t r a c t Poly(ethylene glycol) (PEG)-grafted cellulose nanocrystals (CNCs) were successfully synthesized and incorporated into poly(lactic acid) (PLA) as a reinforcing filler to produce nanocomposite scaffolds consisting of CNC-g-PEG and PLA using an electrospinning technique. Morphological, thermal, mechanical, and wettability properties as well as preliminary biocompatibility using human mesenchymal stem cells (hMSCs) of PLA/CNC and PLA/CNCg-PEG nanocomposite scaffolds were characterized and compared. The average diameter of the electrospun nanofibers decreased with increased filler loading level, due to the increased conductivity of the electrospun solutions. DSC results showed that both the glass transition temperature and cold crystallization temperature decreased progressively with higher CNC-g-PEG loading level, suggesting that improved interfacial adhesion between CNCs and PLA was achieved by grafting PEG onto the CNCs. Wettability of the electrospun nanofibers was not affected with the addition of CNCs or CNC-g-PEG and indicating that the fillers tended to stay inside of the fiber matrix under electrical field. The tensile strength of the composite fiber mats was effectively improved by the addition of up to 5% CNC-g-PEG up to 5 wt.%. In addition, the cell culture results showed that PLA/CNC-gPEG composite nanofibers exhibited improved biocompatibility to hMSCs, which revealed the potential application of this nanocomposite as the scaffolds in bone tissue engineering. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cellulose is the world's most abundant renewable polymer resource and has been used as an engineering material for thousands of years [1]. By extracting cellulose at the nanoscale, the majority of the defects associated with its hierarchical structure can be removed, and a new generation of material–cellulose nanoparticles can be obtained, which is an ideal material on which to base the new biopolymer nanocomposite industry. Crystalline cellulose has a greater axial elastic modulus than Kevlar and its mechanical properties are within the range of other reinforcement materials [2,3]. The preparation of reinforced polymer materials with cellulose nanoparticles has seen rapid advances and considerable interest in the last decade owing to its renewable nature, high mechanical properties, and low density, as well as its availability and the diversity of its sources. Cellulose nanocrystals (CNCs) are defect-free, rod-like crystalline residues obtained when cellulose is subjected to acid hydrolysis. CNCs have attracted great attention due to ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Dan), [email protected] (L.-S. Turng).

http://dx.doi.org/10.1016/j.msec.2015.01.024 0928-4931/© 2015 Elsevier B.V. All rights reserved.

their high aspect ratio (3 to 5 nm wide, 50 to 500 nm in length) and high crystallinity (54 to 88%). It has been reported that CNCs can be aligned under high electrostatic fields [4]. During the electrospinning process of polymer/CNC nanofibers, CNCs orient along the fiber axis, which endows electrospun nanofibers with significantly enhanced axial strength [5,6]. Poly(lactic acid) (PLA) has been historically employed in the biomedical and tissue engineering fields in applications such as resorbable sutures, antibiotic release materials, and degradable implants due to its bioresorbable and biocompatible properties [7]. PLA-based cellulose nanocrystal composites have been extensively investigated in recent years to develop the next generation of lightweight and high performance materials for biomedical applications [8–11]. Among different bio-fabrication methods, electrospinning has been widely used to produce porous PLA-based scaffolds for tissue engineering applications due to its simple setup and its ability to generate fibers with diameters ranging from 50 nm to a few micrometers and a fiber mat with high interconnectivity and high surface areas that resembles natural extracellular matrix (ECM) [12–15]. It has been found that the physiological characteristics of the scaffolds such as mechanical properties can be

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tailored to control the cell behavior. However, the poor compatibility related to the interfacial adhesion between the hydrophilic CNCs and the hydrophobic PLA matrix hindered the nanoparticles from dispersing well in the matrix. Therefore, it has become increasingly important to improve the dispersion of CNCs in the PLA matrix to enhance the mechanical properties of such composites and improve the commercial viability of such products as scaffolds in tissue engineering. To overcome this challenge, surface modification of cellulose by partial substitution of hydroxyl groups with other functional groups seems to be an effective strategy. From the literature review, methods of surface modification of cellulose nanocrystals can generally be categorized into three distinct groups: (1) substitution of surface hydroxyl groups with small molecules [16], (2) polymer surface modification with different coupling agents [17,18], and (3) polymer surface modification with radical polymerization [19–21]. To facilitate the dispersal of CNCs in hydrophobic systems, as well as to achieve steric colloidal stabilization of CNCs, Grey et al. reported the method of surface grafting of cellulose nanocrystals with poly(ethylene glycol) (PEG) [18]. PEG is water soluble and also can be dissolved in hydrophobic solvents such as chloroform. By grafting the surface hydroxyl groups of CNCs with PEG, the polymer chains extend into the surrounding aqueous medium like “polymer brushes,” thus hindering direct contact between the nanoparticles and therefore inhibiting coagulation of the suspension. The monofunctionalized PEG with an epoxide end group can react with the hydroxyl groups on the surface of the CNCs in alkali aqueous media. Nucleophilic attack by surface hydroxyl groups under strongly alkaline reaction conditions opens the epoxide ring to form a covalent ether linkage between the CNCs and PEG chains. While CNC-g-PEG has been synthesized and reported in the literature [18], to the best of our knowledge, this modified form of CNC has not been incorporated into PLA-based composites to improve the compatibility between CNC and PLA and to enhance the mechanical properties of the resulting nanocomposite fibers. In addition, the PEG with an epoxy end group used in the previous study had a molecular weight of 2.086 kDa. In this study, the hydroxyl groups present on the surface of the cellulose nanocrystals were partially substituted with PEG. The PEG epoxide (PEG-EP) was synthesized from poly(ethylene glycol) hydroxyl (PEG-OH). PEG (Mn = 5000) was then grafted onto the CNCs. By grafting higher molecular weight of PEG onto CNCs, we have achieved longer “polymer brushes” on the surface of CNC. It is hypothesized that the grafting of CNCs with PEG will improve the dispersion of CNCs in organic solvents. It is further hypothesized that the addition of CNC-g-PEG will promote miscibility and interfacial adhesion with the PLA matrix, ultimately enhancing the mechanical performance of the resultant composites. The successful monofunctionalization of PEG-OH with an epoxide end group, and the further grafting onto CNCs, was confirmed by proton nuclear magnetic resonance (1H NMR) spectra and Fourier transform infrared spectroscopy (FTIR). Bionanocomposite nanofibers consisting of CNC-g-PEG and PLA were fabricated using the electrospinning technique. The composite nanofibers were characterized using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), water contact angle (WCA) testing, and tensile measurements. The formation of our bones involves the progeny of human mesenchymal stem cells (hMSCs) which can be differentiated into osteoblast-like cells [22]. Preliminary biocompatibility assay of PLA/CNC-g-PEG nanocomposite scaffold was investigated with hMSCs to explore its potential application in bone tissue engineering. 2. Experimental setup 2.1. Preparation of cellulose nanocrystals (CNCs) Commercially available dissolving pulp dry lap made from southern pine was used as the starting material for producing cellulose nanocrystals (CNCs). Strips of the dissolving pulp were reacted with

64% sulfuric acid at 45 °C for approximately 1.5 h under a nitrogen blanket with constant stirring. The hydrolysis reaction was terminated by diluting with water to approximately a 10-fold volume of the initial suspension. The CNCs were neutralized by adding an aqueous sodium hydroxide solution of about 5% concentration. At this point, the CNCs were not colloidal and settled because of the high salt concentration. Continued dilution and decanting were carried out until the sodium sulfate concentration was about 1%. The sodium sulfate and other salts were removed by ultrafiltration in a tubular ultrafiltration unit. Fresh water was added to the suspension during ultrafiltration to keep the CNC concentration at approximately 1%. After essentially all of the salt was removed, the CNC suspension was concentrated in the same ultrafiltration unit by no longer adding fresh water. The final concentration of the CNCs was approximately 10% by weight. 2.2. Synthesis of poly(ethylene glycol) epoxide (PEG-EP) Poly(ethylene glycol) epoxide (PEG-EP) with an epoxy end group was synthesized from poly(ethylene glycol) hydroxyl (PEG-OH) in two steps following the procedure described in the literature [23]. First, 10 g of PEG-OH (Mn 5000) and 0.05 g sodium were stirred in 80 ml toluene at 100 °C for 8 h under nitrogen flow and reflux water. The solution was then cooled down to 40 °C and 0.3 g epibromohydrin was added and further stirred for 12 h. PEG-EP was obtained after the solution was filtered, washed twice with diethyl ether, and freeze dried. 2.3. Poly(ethylene glycol) (PEG) grafting reaction The grafting reaction of poly(ethylene glycol) epoxide onto cellulose nanocrystals was performed as follows. Briefly, NaOH aqueous solution (0.62 g in 12 ml deionized water) was added dropwise to a diluted CNC suspension (30 g, 2.22% w/w) to give a total concentration of 0.37 mol/l NaOH. The mixture was stirred for 30 min before 1.03 g of PEG-EP was added. The mixture was heated to 65 °C and stirred for 6.5 h followed by exhaustive dialysis against DI water (dialyzed for 1 week in a 5 l container; water was changed 5 times a day) to remove any NaOH and unreacted PEG. Poly(ethylene glycol) grafted cellulose nanocrystal (CNC-g-PEG) powder was obtained after the dialyzed solution was freeze dried. 2.4. Fabrication of electrospun nanofibers Poly(lactic acid) (PLA, 2002D, D content of 4.25 wt.%, melt flow index of 7 g/10 min, density of 1.24 g/cm3) was purchased from NatureWorks, LLC (Minnetonka, MN, United States). N,N′dimethylformamide (DMF) and chloroform were purchased from Sigma-Aldrich Inc. (St. Louis, MO, United States). PLA was dissolved in 7 ml of chloroform with magnetic stirring for 3 h at room temperature. CNC-g-PEG was added to 3 ml of DMF and ultrasonic treated for 10 min. The two solutions were mixed together and further stirred for 6 h before electrospinning. The weight concentration of PLA in the mixture was fixed at 10%, and the loading levels of CNC-g-PEG by weight of PLA were 1, 5, and 10%. Pure CNCs were also added to PLA following the same procedure at a 1 and 5% content because the dispersion of CNCs in electrospun PLA nanofibers weakens above a 5% CNC loading level [12]. The nomenclature of the samples was designated as PLA/CNC (x%) and PLA/CNC-g-PEG (x%), where x was the weight percent of the fillers in relation to PLA. The PLA solution, and PLA/CNC and PLA/CNCg-PEG suspensions prepared, were separately loaded into a 5 ml plastic syringe with an 18 gauge blunt stainless steel needle and then mounted on a syringe pump. The flow rate of the suspension was controlled at 0.5 ml/h. The fibers were electrospun onto a piece of aluminum foil within a 20 kV electric field at a 15 cm needle tip-to-collector distance. The obtained nanofibers were dried under vacuum at 80 °C for 24 h and then stored in a desiccator prior to characterization.

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2.5. Transmission electron microscopy (TEM) imaging of cellulose nanocrystals For TEM imaging, a diluted CNCs suspension was deposited to a glowdischarged copper grid with formvar and carbon film (400 mesh). The droplet was maintained on the grid for 2 min, and then rinsed thoroughly using a 2% aqueous uranyl acetate stain followed by blotting. Samples were imaged using a Philips CM-100 TEM (Philips/FEI Corporation, Eindhoven, Holland) which operated at 100 kV, spot 3200 μm condenser aperture, and 70 μm objective aperture. The images were captured and recorded using a SIA L3C 4 Mpix CCD camera (Scientific Instruments and Application, Duluth, GA, USA). 2.6. Chemical structure analysis Proton nuclear magnetic resonance (1H NMR) spectra of PEG-OH and PEG-EP were recorded on a Varian Mercury 300 MHz spectrometer using deuterated dimethyl sulfoxide (DMSO) as the solvent. Tetramethylsilane (TMS) was used as the internal reference. Fourier transform infrared (FTIR) analysis of PEG-EP, CNCs, and CNC-g-PEG were performed by a Bruker Tensor 27 instrument with attenuated total reflectance mode in a frequency range of 4000 cm−1 to 400 cm−1. 2.7. Characterization of electrospinning solutions The electrical conductivity of the prepared electrospinning solutions was measured at room temperature using a Fisher Scientific accumet AP85 PH/conductivity meter. The meter was calibrated to the 0.0–19.9 μS range prior to use. The complex viscosity of the solutions was measured by AR 2000ex rheometer (TA Instruments, New Castle, DE, USA). A 25-mm parallel-plate geometry was used and all tests were performed at 25 °C. Oscillatory frequency sweep tests were performed at a constant stress of 1 Pa with an increase of angular frequency from 0.1 to 100 rad/s. 2.8. Morphological observation of the nanofibers The morphology of the electrospun nanofibers was observed with a LEO 1530 scanning electron microscope (SEM) with an accelerating voltage of 3 kV. Before SEM observation, the fiber mats were sputter coated with gold. The fiber diameters were determined by analysis of the SEM images using ImageJ 1.47 software. 2.9. Differential scanning calorimetry (DSC) A differential scanning calorimeter Q20 (TA Instruments) was used to study the thermal properties of the nanofibers. Prior to testing, the specimens were dried at 60 °C for 2 h. Samples of 6 to 10 mg were

Fig. 1. TEM image of cellulose nanocrystals (CNCs).

Fig. 2. 1H NMR spectra of PEG hydroxyl and PEG epoxide.

placed in aluminum sample pans and heated from room temperature to 180 °C, held for 3 min to remove any prior thermal history, and then cooled to − 20 °C. The samples were then reheated to 200 °C. The heating and cooling rates were both 10 °C/min. The heat flow of the second heating was recorded and compared. 2.10. Wettability The wettability of the electrospun fibers was characterized with a Dataphysics OCA 15 optical contact angle measuring system using the sessile drop method. Four microliters of deionized water was dropped on the fiber mats and images of the water on the surface at a time of 3 min were taken and measured. At least 5 spots of each composite nanofiber mat were tested and the average value was taken. 2.11. Tensile tests The tensile behaviors of the nanofibers were tested by an Instron 5967 universal testing machine with a load cell of 250 N. The experiments were performed at room temperature (25 °C) and atmospheric conditions (relative humidity of 20 ± 5%), with a crosshead speed of 5 mm/min. The samples were prepared by cutting strips of nanofiber mats 60 mm long by 10 mm wide. The distance between the jaws was 20 mm, whereas the thickness was measured (around 0.6 mm) before each measurement was taken. Ten samples were tested

Fig. 3. FTIR spectra of PEG epoxide, CNCs, and CNC-g-PEG.

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for each material and the average values and standard deviations were reported.

4–6 days once the cells reached 90% confluency. The mounted scaffolds were placed in 24-well tissue culture polystyrene (TCPS) culture plates (BD Falcon). The scaffolds were allowed to soak in hMSC medium for 48 h before cells were seeded to allow for sufficient protein attachment. hMSCs were seeded onto the scaffolds at a density of 50,000 cells per 24 well and cultured for 14 days. All samples were fed every other day with 1.0 ml per well of mesenchymal stem cell media, which was composed of 90% DMEM–low glucose (Life Technologies; 11885-076), 10% fetal bovine serum (Life Technologies; 26140-079), 1% non-essential amino acids (Life Technologies; 11140-050), 1% L-glutamine (Life Technologies; 25030081), 1% penicillin–streptomycin (Life Technologies; 15140148). Live/dead assays were conducted using a viability/cytotoxicity kit (Life Technologies). This kit utilizes Calcein-AM to label living cells by utilizing the esterase activity within the cytoplasm of living cells, while simultaneously labeling dead cell nuclei with red fluorescence using ethidium homodimer-1. Stained cells were imaged using a Nikon Eclipse Ti Microscope with an attached Photometrics CoolSNAP HQ2 camera. Nis-D Elements Advanced Research v.3.22 software was used for image analysis. To observe the cell spreading on scaffolds, the cells were fixed on scaffolds for SME imaging after live/dead assays. Samples were rinsed twice with Hanks' balanced salt solution (HBSS; Thermo Scientific) and immersed in 4% paraformaldehyde solution in HyClone HyPure molecular biology grade water (Thermo Scientific) for 30 min. Then the cells were stepwise dehydrated by ethanol aqueous solutions with 50%, 80%, 90%, and 100% of ethanol. Finally, the scaffolds with cells fixed were dried overnight at room temperature under vacuum before SEM imaging. Cell proliferation assays were conducted and cell numbers were collected using a flow cytometry (BD C6 flow cytometer). Dead cells were excluded from the assay using a 7-AAD stain (BD). Cells were removed from the membranes and tissue culture plastic using 300 μl of TrypLE Express (Life Technologies) for 8 min, then 300 μl of 10% FBS + PBS solution was used to deactivate the TrypLE, finally the cell solution was filtered and analyzed for total cell number.

2.12. Cell culture of hMSCs on scaffolds

3. Results and discussion

Scaffolds of PLA, PLA/CNC and PLA/CNC-g-PEG nanofibers were directly electrospun onto stainless steel washers for the sake of cell culture procedure. Human mesenchymal stem cells (hMSCs) were maintained in T75 flasks and were fed every other day and split every

3.1. Morphology of cellulose nanocrystals

Fig. 4. (a) Difference in stability of CNCs and CNC-g-PEG suspensions in DMF and (b) complex viscosity of as-prepared PLA, PLA/CNC, and PLA/CNC-g-PEG electrospinning suspensions.

The TEM image in Fig. 1 shows the size and size distribution of a dilute suspension of cellulose nanocrystals extracted from southern pine

Fig. 5. SEM micrographs of electrospun nanofibers: (a) PLA, (b) PLA/CNC (1%), (c) PLA/CNC (5%), (d) PLA/CNC-g-PEG (1%), (e) PLA/CNC-g-PEG (5%), and (f) PLA/CNC-g-PEG (10%).

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pulp. From the image it can be seen that the CNCs were well separated and showed no aggregation. The CNCs appeared as slender rods and had a broad distribution in length, ranging from 30 to 100 nm, as obtained using digital image analysis. The diameters fell between 6 and 10 nm. The average length and width were estimated to be 77 ± 21 and 9 ± 2 nm, respectively. The average aspect ratios (L/D) of the CNCs were around 8.5. 3.2. Characterization of PEG epoxide and its grafting onto CNCs Proton NMR spectra of the PEG epoxide and PEG hydroxyl in DMSO are shown in Fig. 2. Both PEG-EP and PEG-OH showed the large backbone (CH2CH2O, b) of PEG absorbance centered at 3.49 ppm and methoxy terminal group (a) at 3.28 ppm. The new peaks in the spectrum of PEG-EP indicate the characteristic epoxide absorbance at (f) 2.52 ppm, (f) 2.69 ppm, and (e) 3.07 ppm. This proved the successful synthesis of PEG with an epoxy end group from PEG hydroxyl. The spectrum of PEG-OH showed a clean triplet at (c) 4.53 ppm for the hydroxyl protons (OH) at the polymer terminus. No hydroxyl absorbance was observed at 4.53 ppm in the spectrum of PEG-EP, thus indicating complete conversion of hydroxyl groups to glycidyl ethers [24]. Comparison of the backbone areas to the epoxy ring areas gave greater than 100% conversion, which may have been due to a side reaction proceeding via a glycerol monoether of PEG that reacted with more than one mole of epibromohydrin [25]. This side reaction would be favorable for the grafting of PEG onto CNCs since more epoxy end groups are present on PEG. CNCs reacted with the synthesized PEG epoxide in the alkaline solution. After 1 week of dialysis, the reaction gave 1.23 g (133.7 g of a 0.92% w/w suspension in water) of PEG-grafted CNCs, corresponding to a yield of 72% based on the completed reaction of the CNCs starting material (0.67 g cellulose content) with the PEG epoxide (1.03 g). Unreacted PEG would have been removed by the extensive dialysis, so at least 0.56 g (=1.23 g − 0.67 g) of PEG must have been covalently bound to the CNCs. This is a minimum value since background hydrolysis of the nanocrystals cannot be excluded under the alkaline reaction conditions. Fig. 3 presents the FTIR spectra of PEG epoxide, ungrafted CNCs, and PEG-grafted CNCs. The spectrum of PEG-EP showed the characteristic absorption bands of the \CH2 and \CH3 framework stretching at 2883.5 cm−1, bending \CH2 and \CH3 at 1467.8 cm−1, and 1342.4 cm− 1, respectively, and \C\O\C\ asymmetrical and symmetrical stretching at 1103.3 cm−1 and 960.5 cm−1, respectively. The presence of an epoxide end group was also proved by the strong \CH\ cyclic epoxy absorption situated at 842.9 cm−1. From the spectrum of cellulose nanocrystals, it can be observed that the hydrogen bonded \OH stretching was located at 3340.7 cm− 1, the C\H stretching at 2891.3 cm− 1, the \CH2 bending situated at 1427.3 cm−1, and the C\H bending at 1369.4 cm−1, which represents characteristic peaks of cellulose nanocrystals. The peak at 1057.0 cm−1 belonged to the C\O stretching. In addition, the peak at 1653.0 cm−1 was related to the \OH bending of adsorbed water, because it was too difficult to completely extract the water adsorbed in the cellulose nanocrystals. The spectrum of CNC-g-PEG not only displayed all of the characteristic peaks of cellulose nanocrystals, but also showed the absorption bands of poly(ethylene glycol), such as \CH2 and \CH3 bending at 1467.8 cm− 1 and 1342.4 cm− 1, \C\O\C\ asymmetrical and symmetrical stretching at 1103.3 cm−1 and 960.5 cm−1, and \CH\ cyclic epoxy absorption at 842.9 cm−1. This indicates the success of poly(ethylene glycol) grafted onto the surface of cellulose nanocrystals. The resulting CNC-g-PEG aqueous suspension was freeze dried and dispersed in DMF for the fabrication of electrospinning solution. After ultrasonic treatment for 10 min, the CNC-g-PEG suspensions were stable for several days, and no precipitation of the colloidal nanoparticles was observed. This differed drastically from the unmodified CNC suspension, where the particles settled within several minutes, as shown

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Table 1 Conductivity of electrospinning PLA, PLA/CNC, and PLA/CNC-g-PEG suspensions, diameters, and water contact angles of the electrospun nanofiber mats. Samples

σ (μS·cm−1)

Diameter (nm)

θ (°)

PLA PLA/CNC (1%) PLA/CNC (5%) PLA/CNC-g-PEG (1%) PLA/CNC-g-PEG (5%) PLA/CNC-g-PEG (10%)

0.62 0.87 0.98 0.64 0.67 0.85

902 ± 153 637 ± 65 575 ± 84 800 ± 144 696 ± 125 664 ± 58

126.3 124.9 125.4 124.0 126.1 125.8

in Fig. 4(a). Thus, functionalizing the surface of CNCs with PEG chains achieved a significant improvement in stabilization for cellulose nanocrystals in DMF. 3.3. Morphology of the electrospun nanofibers Fig. 5 shows the SEM photographs of electrospun PLA, PLA/CNC, and PLA/CNC-g-PEG composite nanofibers. All of the fibers were randomly oriented and homogeneous without visible beads. The calculated average diameters and diameter deviations of as-spun fibers are displayed in Table 1. Compared with pure PLA, the PLA/CNC composite nanofibers exhibited much smaller diameters and were more evenly dispersed. Furthermore, the diameters decreased with increased CNCs content, which was similar to what was reported in the literature [12]. As shown, the average diameter of the PLA fibers was about 902 nm, and the diameters greatly decreased to 637 and 575 nm with 1% and 5% CNC loading levels, respectively. From Table 1, it can be seen that PLA/ CNC-g-PEG composite nanofibers also showed decreased diameters and distribution with increased CNC-g-PEG. But the average diameters of PLA/CNC-g-PEG composites were bigger than that of PLA/CNC composites. Even with 10% CNC-g-PEG, the composite nanofibers showed an average diameter of 664 nm, which was larger than that of PLA/ CNC (1%). Generally, when using the same polymer concentration and solvent for the electrospinning solutions containing nanoparticles, the diameter of the electrospun nanofibers is related to the electrical conductivity and viscosity of the solutions [26]. The effect of the electrical conductivity is opposite to the effect of viscosity in controlling the diameter of the electrospun fibers. Thus, increased electrical conductivity usually results in smaller electrospun nanofibers, while an increase in viscosity tends to produce larger fiber diameters. The complex viscosity and electrical conductivity of PLA, PLA/CNC, and PLA/CNC-g-PEG electrospinning solutions were measured, and the results are presented in Fig. 4(b) and Table 1, respectively. From Fig. 4(b) it is noticed that the addition of CNCs and CNC-g-PEG didn't have much impact on the viscosity of PLA composite suspensions compared with pure PLA solution. The reason can be addressed that in PLA/CNC composite system, the interaction between PLA and CNCs was weak and the movement of PLA chains in the solution was not influenced by CNCs within 5% filler loading level, while in PLA/CNC-g-PEG composite system, the grafted low-molecular PEG chains on CNCs acted as plasticizer in PLA suspension, so the viscosity was not increased even with 10% CNC-g-PEG. Since the viscosity of PLA, PLA/CNC and PLA/CNC-g-PEG composite suspensions are similar Table 2 Summary of the DSC data of electrospun PLA, PLA/CNC, and PLA/CNC-g-PEG composite nanofibers. Samples

Tg (°C)

Tcc (°C)

Tm (°C)

ΔHcc (J/g)

ΔHm (J/g)

PLA PLA/CNC (1%) PLA/CNC (5%) PLA/CNC-g-PEG (1%) PLA/CNC-g-PEG (5%) PLA/CNC-g-PEG (10%)

61.7 62.4 62.5 61.6 58.5 54.8

134.5 134.7 134.9 131.5 120.0 113.1

155.2 154.8 155.1 155.4 154.2 149.2, 155.6

2.5 1.6 2.4 12.4 26.4 26.1

2.8 1.9 3.5 13.2 26.5 26.9

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Fig. 6. DSC curves of electrospun PLA, PLA/CNC, and PLA/CNC-g-PEG composite nanofibers.

to one another, it is anticipated that the diameter difference of these electrospun nanofibers is mainly influenced by the electrical conductivity of the solutions. As shown in Table 1, the electrical conductivity (σ) of PLA solution was 0.62 μS·cm−1. With addition of CNCs, the σ value increased to 0.87 and 0.98 μS·cm−1 at 1% and 5% CNC loading levels, respectively. The electrical conductivity of the PLA/CNC-g-PEG solutions was also higher than that of PLA (but lower than that of PLA/CNC suspensions), and increased with higher CNC-g-PEG loading levels. The σ values for PLA/CNC-g-PEG (1%), PLA/CNC-g-PEG (5%), and PLA/CNC-gPEG (10%) were 0.64, 0.67, and 0.85, respectively. This trend is consistent with the results of the electrospun fiber diameters, that is, the higher the electrical conductivity, the smaller the nanofibers. This is because the increased electrical conductivity of the electrospinning solution results in a higher charge density on the surface of the ejected jet during spinning, and promotes the formation of higher elongation forces to drive the jet under the electrical field. The enhanced electrostatic forces facilitate the jet to split into smaller and more spindle-like shapes. Hence, the diameters of the fibers formed become substantially smaller [27]. The increased conductivity of the PLA/CNC suspensions was due to the negatively charged CNCs from the sulfate ester groups on its surface, which were produced during the sulfuric acid hydrolysis process. When

Fig. 8. Typical stress–strain curves of electrospun PLA, PLA/CNC, and PLA/CNC-g-PEG composite fiber mats.

the CNCs were subjected to grafting with PEG, the sulfate ester groups were not stable under the alkali condition and were replaced with hydroxyl groups that further reacted with PEG epoxide. This explains the lower conductivity of the PLA/CNC-g-PEG suspension as compared to the PLA/CNC suspension. 3.4. Thermal properties (differential scanning calorimetry) The thermal properties of electrospun PLA, PLA/CNC, and PLA/CNCg-PEG composite nanofibers based on DSC measurements are shown in Fig. 6. The glass transition temperature (Tg), cold crystallization temperature (Tcc), melting temperature (Tm), cold crystallization enthalpy (ΔHcc), and melting enthalpy (ΔHm) are summarized in Table 2. From the data available, it is evident that the addition of PEG-grafted CNCs decreased the glass transition temperature of PLA, while there was no major change in Tg for the PLA/CNC composite fibers. Pure PLA electrospun nanofibers had a Tg of 61.7 °C. By adding 5% CNC-g-PEG, the Tg of the composite nanofibers decreased to 58.5 °C, and further decreased to 54.8 °C at a 10% CNC-g-PEG loading level. This is because the grafted PEG chains were compatible with PLA; the low molecular PEG chains presented as plasticizers in the PLA matrix and improved the mobility of the PLA chain segments. Since PEG was covalently bound to the

Fig. 7. Images of water contact angle test of the electrospun fibers: (a) PLA, (b) PLA/CNC (1%), (c) PLA/CNC (5%), (d) PLA/CNC-g-PEG (1%), (e) PLA/CNC-g-PEG (5%), and (f) PLA/CNC-g-PEG (10%).

C. Zhang et al. / Materials Science and Engineering C 49 (2015) 463–471 Table 3 Tensile properties of electrospun PLA, PLA/CNC, and PLA/CNC-g-PEG composite fiber mats. Samples

σmax (MPa)

εb (%)

PLA PLA/CNC (1%) PLA/CNC (5%) PLA/CNC-g-PEG (1%) PLA/CNC-g-PEG (5%) PLA/CNC-g-PEG (10%)

2.8 ± 0.4 2.8 ± 0.5 2.3 ± 0.5 3.5 ± 0.2 4.7 ± 0.3 2.8 ± 0.3

95 ± 14 85 ± 10 71 ± 11 95 ± 12 106 ± 9 91 ± 10

CNCs, CNC-g-PEG could achieve better dispersion in the PLA than the ungrafted CNCs. This result also proved that the fillers were indeed incorporated in the electrospun fibers rather than being excluded during the electrospinning process. It was also observed that the addition of CNC-g-PEG prompted the cold crystallization of PLA, and with the addition of more CNC-g-PEG, cold crystallization occurred at a lower

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temperature. Thus, CNC-g-PEG dispersion affected the nucleation and growth geometry of the polymer crystals. Mukherjee et al. observed similar but much less obvious trends in their cellulose nanocrystal reinforced PLA composites, where they ascribed the reduced cold crystallization temperature to improved dispersion of the fillers. The greatly reduced cold crystallization of PLA/CNC-g-PEG composites indicated enhanced dispersion of cellulose nanocrystals in the PLA matrix by grafting with PEG [28]. It should be noted that there are two melting peaks for the PLA/CNCg-PEG (10%) composite nanofiber. The peak at the lower temperature is the melting of less perfect crystals that formed due to the improved cold crystallization. The area of the cold crystallization exotherm is quite similar to that of the melting endotherm, which suggests that the PLA component in each sample after cooling from the melt was almost amorphous. This is because the PLA used in this study had a D content of 4.25% and was very difficult to crystallize from the melt.

Fig. 9. Fluorescence micrographs of stained cells showing live (green) and dead (red) cells and corresponding SEM images of fixed cells on (a, a′ and a″) PLA, (b, b′ and b″) PLA/CNC (5%), (c, c′ and c″) PLA/CNC-g-PEG (5%) and (d, d′ and d″) PLA/CNC-g-PEG (10%) scaffolds after 14 days of culture. (a″ to d″ are the SEM images of fixed cells with larger magnification). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.5. Wettability It has been suggested that improved hydrophilicity is favorable for cell proliferation in materials intended for tissue engineering [29]. As such, the water contact angle in relation to the wettability of the electrospun fiber mats was measured. It was expected that the addition of CNCs and CNC-g-PEG would improve the hydrophilicity of the electrospun composite nanofibers by decreasing the water contact angle due to the hydrophilic nature of CNCs and PEG. The images of a drop of water on the surface of the fiber mats at a time point of 3 min are shown in Fig. 7, and the measured angles (θ) are presented in Table 1. It is clear that the contact angle for all of the nanofiber mats was more than 90°, which means that all of the materials are hydrophobic. The values didn't decrease with the addition of CNCs or CNC-g-PEG. Vicens et al. [13] also found the contact angle of PLA/CNC nanocomposites was not improved by incorporating CNCs, and PLA/CNC (11:1) saw even higher values than that of PLA. The authors explained that the roughness of the sample was a limiting factor for water penetration; that could be the case for their sample PLA/CNC (11:1) as well. It is possible that during the electrospinning process, CNCs and CNC-g-PEG would end up staying inside of the nanofibers rather than on the surface under the electrical field, resulting in negligible effect on the hydrophilicity of the electrospun composite nanofibers. 3.6. Mechanical properties Fig. 8 shows the typical tensile stress–strain curves of PLA, PLA/CNC, and PLA/CNC-g-PEG electrospun nanofibers, and their maximum tensile stress (σmax) and elongation-at-break (εb) are summarized in Table 3. The addition of CNCs didn't achieve an improvement in tensile stress, but resulted in decreased ductility with lower elongation-at-break. This can be ascribed to two factors: one is the poor dispersion of CNCs in DMF and further in PLA matrix; the other is the poor interfacial adhesion between CNCs and PLA. However, the composite fiber mats exhibited higher values of stress when CNC-g-PEG was added at 1% and 5% loading levels, indicating a reinforcing effect of the filler. What's more interesting is that the ductility of the fiber was retained and even improved a little with the addition of 5% CNC-g-PEG. Many factors, including the composition and structure of individual fibers, as well as the interaction among fibers, could affect the mechanical properties of the electrospun nanofibers. Zhou et al. [14] concluded that the dispersion, orientation, and interfacial adhesion of nanoparticles within polymer matrices had the most reinforcement effects associated with a mechanical percolation. The reinforcement of the PLA/CNC-g-PEG system could be due to the grafting of PEG onto the CNCs, which enabled the good dispersion of the filler in the PLA matrix, with the PEG chains playing the

Fig. 10. Proliferation cell counts of hMSCs on scaffolds after 14 days of culture.

role of interfacial binder between the CNCs and the PLA, which enabled the reinforcing tendency of the strong filler to be effective. The improved ductility was attributed to the plasticizing effect of the low molecular PEG chains. It was noted that the σmax value of PLA/CNC-g-PEG (10%) didn't achieve further enhancement and was comparable to that of the pure PLA nanofiber mats. It seems that the percolation threshold of CNC-g-PEG reinforcing PLA was achieved at around 5%. The further addition of filler was not favorable for the mechanical strength of the composite fiber mats due to the low strength of the PEG molecules. 3.7. Biocompatibility Preliminary biocompatibility assays were conducted to determine whether these materials have the potential bone tissue applications. Fig. 9(a to d) shows the fluorescence images of hMSCs on PLA, PLA/ CNC (5%), PLA/CNC-g-PEG (5% and 10%) nanofibrous scaffolds after 14 days of culture. From the images we can see that hMSCs could attach and grow well on the scaffolds, live cells (stained green) on all scaffolds showed spindle-like morphology and only a few dead cells (stained red) were observed, indicating that the addition of CNC and CNC-gPEG didn't compromise the good biocompatibility of PLA. It is obvious that there are more live cells on PLA/CNC-g-PEG (5%) nanofibrous scaffold than pure PLA, which is attributed to the decreased diameter of nanofibers that promoted cell-matrix interactions by providing more binding sites for cell adhesion, and also the improved mechanical strength that provided sufficient biomechanical support for cell growth. The morphology of the fixed cells on the scaffolds presented in Fig. 9(a′ to d′) showed that the cells spread out well on the scaffolds after 14 days of incubation, especially for the PLA/CNC-g-PEG (5%) composite nanofiber, which revealed that the hMSCs interacted and integrated well with the nanofibrous scaffolds. hMSC proliferation on PLA, PLA/ CNC (5%), and PLA/CNC-g-PEG (5% and 10%) nanofibrous scaffolds was analyzed by flow cytometry. As shown in Fig. 10, PLA/CNC-g-PEG (5%) scaffold also had more cell count than pure PLA after 14 days' incubation. In conclusion, the cell viability and proliferation results suggested that the biocompatibility of PLA was retained with the addition of CNCs and CNC-g-PEG. With improved tensile strength and decreased diameter of fibrous nanofibers, the scaffold exhibited better cellular compatibility as of PLA/CNC-g-PEG (5%) composite, which should have potential applications in bone tissue engineering. 4. Conclusions Reactive PEG with an epoxy end group was synthesized and successfully grafted onto CNCs, as proved by 1H NMR and FTIR results. PEG grafted CNCs had a better dispersion in DMF than unmodified CNCs. CNCs and CNC-g-PEGs were separately incorporated into a PLA matrix using the electrospinning technique. Morphological observation indicated that the diameters for both PLA/CNC and PLA/CNC-g-PEG composite nanofibers decreased with higher filler loading, and PLA/CNC nanofibers became smaller than PLA/CNC-g-PEG due to the higher conductivity of the electrospun solutions. Enhanced dispersion of cellulose nanocrystals in the PLA matrix was achieved by grafting with PEG. Based on the DSC results, the addition of CNC-g-PEG decreased the glass transition temperature and accelerated the cold crystallization of the nanocomposites. Wettability results from water contact angle measurements showed that both PLA/CNC and PLA/CNC-g-PEG composite fiber mats exhibited similar hydrophobic behavior, indicating that the filler could end up staying inside of the nanofibers rather than on the surface under the electrical field. Tensile tests showed that the addition of CNCs didn't achieve an improvement in tensile stress due to their poor dispersion in the PLA and the poor interfacial adhesion between CNCs and PLA. However, the addition of CNC-g-PEG effectively improved the strength of the PLA/CNC-g-PEG composite fiber mats, but 5% loading seemed to be the optimal content level. Moreover, PLA/

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CNC and PLA/CNC-g-PEG composite nanofibrous scaffolds were nontoxic to hMSCs and capable of supporting cell attachment and growth. PLA/CNC-g-PEG (5%) scaffold with enhanced mechanical strength and decreased diameter of nanofibers exhibited improved cell viability and proliferation cell count, which shows promises for its potential applications in bone tissue engineering. Acknowledgments The financial support of the United States Department of Agriculture National Institute of Food and Agriculture Award (No. 2011-6700920056) is gratefully acknowledged in this research. The authors would also like to acknowledge the financial support of the Wisconsin Institute for Discovery (WID) and the China Scholarship Council (CSC). References [1] Y. Habibi, L.A. Lucia, O.J. Rojas, Cellulose nanocrystals: chemistry, self-assembly, and applications, Chem. Rev. 110 (2010) 3479–3500. [2] R.J. Moon, A. Martini, J. Nairn, J. Simonsen, J. Youngblood, Cellulose nanomaterials review: structure, properties and nanocomposites, Chem. Soc. Rev. 40 (2011) 3941–3994. [3] N. Lin, J. Huang, A. Dufresne, Preparation, properties and applications of polysaccharide nanocrystals in advanced functional nanomaterials: a review, Nanoscale 4 (2012) 3274–3294. [4] Y. Habibi, T. Heim, R. Douillard, AC electric field-assisted assembly and alignment of cellulose nanocrystals, J. Polym. Sci. Polym. Phys. 46 (2008) 1430–1436. [5] H. Dong, K.E. Strawhecker, J.F. Snyder, J.A. Orlicki, R.S. Reiner, A.W. Rudie, Cellulose nanocrystals as a reinforcing material for electrospun poly(methyl methacrylate) fibers: formation, properties and nanomechanical characterization, Carbohydr. Polym. 87 (2012) 2488–2495. [6] R.T. Olsson, R.H. Kramer, A. Lopez-Rubio, S. Torres-Giner, M.J. Ocio, J.M. Lagaron, Extraction of microfibrils from bacterial cellulose networks for electrospinning of anisotropic biohybrid fiber yarns, Macromolecules 43 (2010) 4201–4209. [7] R.M. Rasal, A.V. Janorkar, D.E. Hirt, Poly(lactic acid) modifications, Prog. Polym. Sci. 35 (2010) 338–356. [8] M. Jonoobi, J. Harun, A.P. Mathew, K. Oksman, Mechanical properties of cellulose nanofiber (CNF) reinforced polylactic acid (PLA) prepared by twin screw extrusion, Compos. Sci. Technol. 70 (2010) 1742–1747. [9] A.H. Pei, Q. Zhou, L.A. Berglund, Functionalized cellulose nanocrystals as biobased nucleation agents in poly(L-lactide) (PLLA) — crystallization and mechanical property effects, Compos. Sci. Technol. 70 (2010) 815–821. [10] L. Petersson, I. Kvien, K. Oksman, Structure and thermal properties of poly(lactic acid)/cellulose whiskers nanocomposite materials, Compos. Sci. Technol. 67 (2007) 2535–2544. [11] C.H. Xiang, Y.L. Joo, M.W. Frey, Nanocomposite fibers electrospun from poly(lactic acid)/cellulose nanocrystals, J. Biobased Mater. Bioenergy 3 (2009) 147–155.

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