Effect of Thermal Annealing on Mechanical Properties ... - Springer Link

Fibers and Polymers 2014, Vol.15, No.7, 1406-1413

DOI 10.1007/s12221-014-1406-2

Effect of Thermal Annealing on Mechanical Properties of Polyelectrolyte Complex Nanofiber Membranes Zelong Wang, Ning Cai, Qin Dai, Chao Li, Dajun Hou, Xiaogang Luo, Yanan Xue, and Faquan Yu* Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China (Received October 11, 2013; Revised January 6, 2014; Accepted January 12, 2014) Abstract: Optimization of mechanical properties is required in the applications of tissue-engineered scaffolds. Thermal annealing strategy is proposed to improve the mechanical properties of polyelectrolyte complex nanofiber membranes. The effects of annealing on the structural and mechanical properties of electrospun chitosan-gelatin (CG) nanofiber membranes were investigated using tensile tests, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Tensile test results showed that annealing processing at 90 C produced 1.3-fold and 1.1-fold increase on Young’s modulus and tensile strength, respectively. By scanning electron microscopy (SEM) observation, it was found there was a formation of partial interfiber bonding when annealing temperature was elevated over the glass transition temperature (Tg) of CG nanofibers. FTIR results showed enhanced molecular interactions within fibers, suggesting that annealing treatment promoted the conjunction between chitosan and gelatin. In contrast, no detectable changes in crystallinity for CG nanofiber specimens were exhibited on XRD patterns following annealing treatment. In addition, thermal annealing induced the improvement in thermal stability, aqueous stability and swelling capacity. Therefore, annealing is proved to be an effective strategy for mechanical enhancement of polyelectrolyte complex nanofibrous scaffolds. The enhanced stiffness and strength is mainly attributed to the formation of interfiber bonding and strengthened molecular interactions between chitosan and gelatin. o

Keywords: Electrospinning, Polyelectrolyte complex, Nanofiber membranes, Thermal annealing, Mechanical properties

alignment, lack of cohesion between the fibers and high porosity, which becomes a hurdle in expanding their potential applications in tissue engineering [8]. Previously, several methods had been reported to improve mechanical performance of electrospun nanofiber membranes. These methods, so far, mainly fall into two categories according to their action mechanisms. Firstly, nano-sized materials, including carbon nanotubes, graphite oxide nanoplatelets and so forth, are employed for reinforcing the single nanofiber strength [9]. Secondly, based on the concerns about the potential biotoxicity of some nano-sized reinforcement agents, reinforcement agent-free strategy was proposed to fulfill the mechanical enhancement of nanofiber scaffold by polyelectrolyte complex of two oppositely charged polymers in our previous work [10]. In the aforementioned study, tensile modulus of chitosangelatin (CG) polyelectrolyte complexes nanofiber membranes are elevated over one hundred times compared with the neat chitosan or gelatin ones. Furthermore, it is found that thermal annealing treatment has an influence on the mechanical strength of CG nanofiber membranes, which enlightens us to optimize the mechanical properties of polyelectrolyte complexes nanofiber membranes further simply by annealing processing. Currently, there are no systematic studies on the effect of annealing on mechanical properties of nanofiber membranes of polyelectrolyte complexes. The aim of this study is to evaluate the physical properties of polyelectrolyte complexes nanofiber membranes before and after annealing treatment and elucidate the underlying mechanism. The structure and morphology of the nanofiber membranes were examined by Fourier transform infrared spectroscopy (FTIR), X-ray

Introduction Electrospinning biopolymer to generate micro to nanometer scale fibers has emerged as a prominent method for fabricating 3D scaffolds with tissue-like microstructures due to its advantages in emulating the nano-scaled geometry and topology of extracellular matrix structures [1]. Numerous natural and synthetic biopolymers have been successfully electrospun to form micro or nanofibers for tissue engineering and other related applications [2]. It is known that some criterions should be abided for designing an ideal scaffold, such as proper porosity and pore size, adequate mechanical properties, good biocompatibility, and appropriate biodegradability [3,4]. The importance of mechanical properties of tissue engineering scaffolds has been demonstrated on regenerations of tissues, including bone, cartilage, blood vessels, tendons and muscles [5]. Indeed, it has been found that the strength and deformability of nanofibers influence in vitro cell migration, proliferation and differentiation, along with cell morphology [6]. Moreover, the structural integrity and the sufficient mechanical strength of scaffolds are indispensible for maintaining the desired pattern prior to the formation of new tissue [7]. Therefore, the mechanical properties of nanofibrous scaffolds are of great importance for the intended application of the constituent material for tissue regeneration. Generally, mechanical properties of electrospun nanofiber scaffolds are not satisfactory due to the random fiber *Corresponding author: [email protected] 1406

Annealing Effect on the Mechanical Properties of Nanofibers

diffraction (XRD), and scanning electron microscopy (SEM). Thermal properties were studied by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The mechanical properties of the nanofiber membranes under different annealing temperatures were investigated through the tensile tests. Furthermore, the reinforcement mechanism of annealing on polyelectrolyte complexes nanofiber membranes was discussed.

Materials and Methods Materials Chitosan (Mw=100 kDa, degree of deacetylation=90 %, Mw/Mn=1.8) was obtained from Zhejiang Golden-Shell Biochemical, China. Gelatin was purchased from Sinopharm Chemical Reagent, China. Acetic acid was obtained from Tianjin Bodi Chemical Holding. All above were used without further purification. Phosphate buffered saline (PBS) was prepared in our laboratory. Preparation of Nanofiber Membranes The nanofiber membranes of chitosan (CS) and gelatin (GE) have been reported before. In brief, 10 % (w/w) chitosan solution and 20 % (w/w) gelatin solution were prepared by dissolving them respectively in 90 % acetic acid. Molar ratios of aminoglucoside units (chitosan) to carboxyl units (gelatin) was maintained at 50:50 in chitosangelatin (CG) specimens by blending given volumes of 10 % chitosan solution with 20 % gelatin solution. The chitosan-gelatin solutions were electrospun into nanofibers using an electrospinning system (Beijing Kangsente, China). The elecrtrospinning system is comprised of a syringe pump and a high voltage power supply generating positive DC voltage. A 10 ml syringe containing electrospinning solution was connected to a stainless steel needle with an inner diameter of 0.6 mm. The needle tip was set up horizontally. A vertical metal plate wrapped with aluminum foil was used to collect the electrospun nanofibers. The electrospinning parameters were fixed as follows: feeding rate, 1.0 ml/h; voltage, 15 kV; distance between needle tip and collector, 15 cm; humidity, 30-40 %; temperature, 25 oC. The as-spun CG nanofiber membranes were then peeled off from the aluminum foil for further characterization. The electrospinning precursor solutions were also cast onto clean glass plates and dried in open air at room temperature for 48 h. The obtained cast membranes were vacuum-dried for 24 h before characterization. Thermal Annealing Treatment Thermal annealing treatment of the CG membranes was carried out in a vacuum oven at four different annealing temperatures: 60 oC (CG60), 90 oC (CG90), 120 oC (CG120) and 150 oC (CG150). The samples were maintained in vacuum for 90 min and then cooled down to room temperature.

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Characterization The structure and morphology of the membranes were examined by SEM (JEOL, JSM-5510LV) with an accelerating voltage of 10 kV. The chemical structure of the membranes was analyzed with an attenuated total reflectance FTIR (Nicolet 6700). XRD analysis was performed with a Bruker D8 ADVANCE X-ray diffractometer at a voltage of 40 kV with Ni-filtered Cu Kα radiation. The 2θ scan data were collected from 5.0 o to 40.0 o at a scanning speed of 1.0 o/min. The thermal analysis was carried out through a DSC (Seiko Instruments, DSC6220). Five-milligram samples were sealed in an aluminum pan for the measurements. A heating rate of 10 oC/min was used under nitrogen flow. The thermal stability was studied with TGA using a thermogravimeter (Netzsch TG209), with a 10 mg sample placed in an aluminum pan and a heating rate of 10 oC/min employed under nitrogen flow. The thermal decomposition temperature (Td) is defined as a temperature of 10 wt% thermal weight loss. Mechanical Testing The mechanical properties of the membranes were measured with an Instron tensile testing machine using a 200 N load cell. Rectangular specimens were cut to 60×5 mm and extended at a constant crosshead speed of 20 mm/min with a 40 mm gauge length. Five specimens were tested to determine the mean value; error bars on all plots represent one standard deviation of the mean. The thickness of each specimen was obtained from the average of three measurements taken along the gauge length with a digital micrometer. Swelling Behavior To simulate the in vitro response of the nanofiber scaffolds to physiological conditions, the scaffolds were incubated in PBS medium at pH 7.4 and temperature of 37 oC for different periods of time. The specimens with 2×2 cm2 dimension were weighed (mi) and immersed in 10 ml of PBS (0.1 M, pH 7.4). At specified time, a set of aged specimens (n=3) was dabbed with filter paper and weighed (mw). The percentage of PBS uptake, or the swelling behavior, was calculated from the following equation: mw – m i - × 100 % Swelling ratio = ---------------mi Mass Loss Behavior In order to determine the mass loss due to the possible dissolution of the scaffolds, electrospun membranes were cut into a rectangular shape with dimensions of 20×20 mm. The specimens were placed in closed bottles containing 50 ml of PBS, and incubated in vitro at a temperature of 37 oC. At each time point, the recovered specimens were vacuumdried and weighted (md). The mass loss percentage of the samples was calculated with the following equations, based on the initial mass of each sample (mi) before incubation:

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mi – md - × 100 % Percentage mass loss = --------------mi For each time point, the tests were performed in triplicate. Data were averaged and standard deviations were determined.

Results and Discussion Morphology The morphology of both as-spun and annealing-treated nanofiber membranes was characterized through SEM observation. It is shown in Figure 1(a) that flawless nanofibers were formed. In our previous work [10], chitosan with wide molecular weight distribution was blended with gelatin to generate composite nanofibers. However, there are many broken fibers in the as-prepared nanofiber sample. To improve the quality of nanofiber, chitosan with narrow molecular weight distribution was adopted in this study. Moreover, operation parameters were optimized to prevent generation of the beads and fracture points intertwined in the nanofibers. By this way, present CG nanofiber membranes with high quality were acquired. It is exhibited in Figure 1 that the characteristic morphology of the CG nanofibers varies as a function of annealing temperature. Following annealing at 60 oC which is below the Tg (determined to be ~83 oC obtained from DSC result), the morphology of the CG nanofiber did not make evident change by comparison of Figure 1(a) with Figure 1(b). The merging of overlapping fibers (fiber-fiber bonding) cannot be found either. The lack of overt merging between overlapping fibers is characteristic of electrospun materials as the fibers

Figure 1. SEM micrographs of CG nanofibers untreated (a), and thermal annealing at 60 C (b) 90 C (c), 150 C (d). Scale bar for each image is 1 µm. o

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are almost completely dry when deposited onto the collector [11]. Lack of interfiber bonding can negatively affect the nanofiber membrane’s mechanical properties [12]. When annealing temperatures was elevated over the Tg of the CG nanofibers, extensive fusions between fibers were observed. As pointed out by the arrows in Figure 1(c)-(d), the sharp vertex formed by overlapped fibers (shown in Figure 1(a)(b)) becomes rounded. Therefore, a webbing structure with interfiber bonding was formed following annealing treatment. It is known that the motions of polymer chains was restraint below the Tg and the polymer chains become flexible and start to wiggle over the Tg [13]. During thermal annealing, high annealing temperature accelerates the interdiffusion of the polymer molecules at the contact points between fiber [14], inducing the in situ fiber-fiber fusions. Therefore, annealing treatment produces the webbing effect within the membrane, which is beneficial for the improvement in the mechanical properties of nanofiber membranes. Mechanical Properties of Annealing-treated Nanofiber Membranes Uniaxial tensile testing was employed to observe the influence of varying degrees of annealing on the mechanical response of as-prepared membranes. Representative tensile stress-strain curves of annealing-treated CG nanofiber membranes are shown in Figure 2. The nanofiber membranes annealed at 60 oC exhibit large failure strains. In addition, yielding region takes up a large portion of the stress-strain curve. However, brittle fracture behavior lack of yielding region is displayed on the CG membranes annealed at over Tg (90, 120 and 150 oC). Therefore, the elevation of annealing temperature induces the ductile-to-brittle transition for the nanofiber membranes. The Young’s modulus and tensile strength for the as-spun CG nanofiber membranes were 21.5±2.0 MPa and 1.8± 0.1 MPa, respectively. Following 1.5 h of annealing treatment at 60 oC, there is only 15 % and 8 % improvement on Young’s

Figure 2. Stress-strain curves of CG nanofiber membranes at different annealing temperature.


modulus and tensile strength of the nanofiber membranes, respectively. Comparatively, annealing processing at 90 oC produced 1.3-fold and 1.1-fold increase on Young’s modulus and tensile strength, respectively. With the rise of annealing temperature, Young’s modulus and tensile strength climb up till acquiring almost their highest values at 150 oC. In contrast to ascending trend of Young’s modulus and tensile strength, the elongation at break descends with the increase of annealing temperature. The energy to break, which characterize the toughness of CG nanofiber membranes, was determined by integrating the stress-strain curves in Figure 2. There is no singular trend of the energy to break with annealing temperature. Under the combinational influence of Young’s modulus and elongation at break, the energy to break reaches 0.33 MJ/m2 at 90 oC, which is the highest one among the five cases. Annealing is the heating and cooling process for materials, which is usually to alter physical and mechanical properties. It is known that influence of annealing on polymer is often via the change of crystallinity [15]. Annealing process may induce the increase in elastic modulus and tensile strength for undrawn materials [16]. In addition, the changes in mechanical properties of annealed polymers are tightly correlated with annealing temperature [17]. Usually, the changes are conspicuous in the region near the glass transition temperature [18]. Therefore, the improvement in mechanical performance may possibly result from increased crystallinity for our CG system. To examine the crystallinity of the annealing-treated nanofiber membranes, XRD observation was performed. Figure 3 shows the XRD patterns of CS, GE and the CG nanofiber membranes treated under different annealing conditions. Two crystal peaks that appeared at 2θ =11 o and 20 o was observed in the XRD patterns of chitosan. It is well accepted that the crystal peaks at 2θ of 11 o reflects the presence of crystal form I and the strongest reflection at 2θ of 20 o corresponds to crystal form II [19].

Figure 3. XRD patterns of nanofiber membranes at different annealing temperature.


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The pattern of gelatin, as shown in Figure 3, had a very weak broad profile, indicating that gelatin is an amorphous material. If chitosan and gelatin was blended in an exclusively physical mixing mode and without any extra interactions, each component would maintain its original crystal region in the blends. Therefore, XRD patterns of chitosan-gelatin should be the superposition of the pattern of chitosan and gelatin in blending ratio. However, the diffraction peak at 2θ =11 o for chitosan shifted to 13.5 o for the chitosan-gelatin. In addition, diffraction peak for chitosan at 2θ=20 o disappeared on the chitosan-gelatin, demonstrating the decreased crystallinity of the chitosan-gelatin compared to chitosan. This change in crystallinity can be explained by the breaking of the hydrogen bonding in the chitosan molecules because of the formation of polyelectrolyte complex after addition of gelatin [20]. The deformation of crystal structure of polyelectrolyte complex have also been discussed and reported by other researchers [21]. In addition, almost identical diffraction patterns are shown among all the CG nanofiber membranes, indicating annealing treatment has almost no effect on the crystallinity of CG nanofiber membranes. So the conspicuous difference of mechanical properties on unannealed and annealed CG nanofiber membranes could not be attributed to the possible change in crystallinity induced by annealing treatment. There is lack of cohesions of nanofibers within the electrospun membranes, which may lead to delaminating when strained [22]. Introduction of interfiber bonding through heat treatment or cross linking within nanofiber membrane is a commonly reported strategy for promoting mechanical properties of electrospun materials, including PLA and PCL nanofiber membranes [23,24]. Therefore, the better mechanical performance shown on annealed CG nanofiber membranes may also stem from the interfiber bonding which are newly formed during heat treatment process. This structure was just observed in our morphology study (Figure 1). With the increase of annealing temperature, the tensile strength and modulus increased gradually as the interfiber junctions became more extensively bonded. The annealed samples are more rigid and brittle than the unannealed samples. Moreover, the annealing process may promote the conjugation of the oppositely charged polymers, leading to the mechanical enhancement of CG nanofiber membranes. To clarify this issue, we studied the effect of annealing treatment on the tensile strength and modulus of cast CG membranes in which the effect of interfiber bonding has been completely avoided. As depicted in Figure 4(a) and (b), annealing treatment at different temperature is beneficial for the improvement of mechanical performance of both electrospun and cast CG membranes. Further, the percentage increase of tensile strength or modulus is higher in electrospun membranes than that in corresponding cast membranes under the same treatment conditions. Obviously, the difference can mainly be attributed to the existence of interfiber bonding effect in electrospun samples accompanied by annealing treatment.

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Figure 4. The percentage increase of tensile strength (a) and modulus (b) of the electrospun and cast membranes due to annealing treatment.

Figure 5. FTIR spectrum of nanofiber membranes at different annealing temperature (a), and illustration of the intermolecular bonding of CG (b).

FTIR analysis was conducted to acquire the information about molecular interactions between chitosan and gelatin stimulated by the annealing treatment. Figure 5(a) shows the FTIR spectra of CS, GE and the CG nanofiber membranes treated under different annealing conditions. As shown in Figure 5(a), the spectra of all the samples exhibited a broad band at ~3300 cm-1 and a weak peak at 2900 cm-1, which are assigned to the groups of -OH and -CH2-, respectively [25]. For chitosan, the appearance of the band at 1650 cm-1 and a peak at 1594 cm-1 confirms the existence of C=O groups of acetylated amide and amine groups [26]. An absorbance peak at 1065 cm-1 corresponds to the characteristic -C-O-Cbonds [27]. This peak was prominent in pure chitosan as compared to those in the other samples. In the spectra of gelatin sample, the bands at 1640 cm-1 and 1540 cm-1 corresponded to the -CO-NH- stretching of amide I, and -NH2 stretching of amide II, respectively [28]. The chemical composition of all CG samples consists of the same functional groups, which give rise to the characteristic FTIR bands at 1640 cm-1 and 1540 cm-1. Despite of high similarity of CG FTIR spectrum, significant difference in the intensity ratio of the amide I region (1640 cm-1) to the amide II region (1540 cm-1) can still be found

for all CG samples. As shown in Figure 5(a), the intensity ratio value gradually increases from 1.02 (CG) to 1.39 (CG150) with increasing annealing temperature. The argument of the ratio is just the reflection of intensified molecular interactions induced by annealing treatment. Although electrospun solution was prepared according to the principle of maintaining net charge of zero, the two kinds of oppositely charged units is not paired perfectly [29]. During annealing process, thermodynamic movement of chain segment is expected to be intensified under high temperature [30], which is beneficial for the bonding of unpaired units. Besides promoting the complexation between chitosan and gelatin, heat treatment may produce the formation of covalent bonds. It has been reported that there are some degree of amidization between the ammonium (-NH3+) ions of the chitosan and the carboxylate (-COO−) ions of the gelatin in CG due to the partial conversion of electrostatic bonds into chemical bonds through condensation reaction [21,31]. Therefore, thermal processing may initiate the formation of covalent amide bonds in CG system, which is schematically shown in Figure 5(b). The intensified molecular interactions resulting from the formation of both polyelectrolyte complex and covalent bonding promote mechanical enhance-


ment of CG nanofiber membranes. All in all, the interfiber bonding (webbing effect) combined with intermolecular bonding (electrostatic interaction and covalent bonds) makes contribution of the elevated tensile strength and modulus of the annealed polyelectrolyte complexes nanofiber membranes. Thermal Properties of Annealing-treated Nanofiber Membranes The thermal annealing treatment may bring about the alteration of thermal properties of the CG, which was characterized by DSC. The DSC thermograms of as-spun and annealed samples under different conditions are shown in Figure 6(a). The endothermic peak for CG was visible at temperature of about 83 oC (Tg), which is in turn associated with the amount of hydrogen-bound water in the CG [32]. The peak position shifted to high temperature with increasing annealing temperatures up to 120 oC. The Tg values are summarized in Table 1. Compared with the as-spun CG nanofiber membranes, the annealed nanofiber membranes possesses higher Tg, which implies that the nanofiber membranes became steadier following annealing process. However, there is a slight drop of Tg when annealing temperature was set at 150 oC. Thermal treatment would assumedly induce


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partial decomposition of chitosan and gelatin. When annealing temperature is high enough (>120 oC), the decomposition effect offset the favorable CG conjunction induced by annealing treatment, leading to the decrease of Tg. In addition, no secondary peak was observed in any CG sample, suggesting good miscibility of all the blend samples and the absence of phase separation. The good miscibility is related to the intermolecular interactions between the two oppositely charged polyelectrolytes [33]. The thermal stability of the nanofiber membranes was studied by TGA. The TGA profiles of as-spun and annealed CG nanofiber membranes are shown in Figure 6(b). It showed that the initial decomposition temperature (Td) of unannealed CG was 155 oC. Comparatively, the initial decomTable 1. Thermal properties of CG nanofiber membranes at different annealing temperature Annealing Untreated temperature ( C) Glass transition 83 temperature Tg ( C) Decomposition 155 temperature Td ( C)

60

90

120

150

o

85

87

87

83

o

168

201

246

253

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Figure 6. DSC thermograms (a), and TGA profiles (b) of CG nanofiber membranes at different annealing temperature.

Figure 7. Swelling behavior (a), and mass loss behavior (b) of CG nanofiber membranes at different annealing temperature in PBS (0.1 M, pH 7.4).

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position temperatures of the thermal annealed nanofibers was 13-98 oC higher than that of the unannealed ones. Behavior of Swelling and Mass Loss The capacity to swelling is a characteristic property of bodies consisting of polymers. As shown in Figure 7(a), all samples possess the rapid and significant swelling capacity. The compact structure of the nanofiber membranes with nanopores favors swelling, leading to the rapid and significant swelling behavior. Comparatively, the samples annealed at higher temperature possess lower swelling capacity. The unannealed sample has the highest swelling ratio. It has been reported that enhanced molecular interactions usually reduce swelling capacity due to restricting the mobility and hydration of the macromolecular chain [34]. Therefore, the decreased swelling ratio with increasing annealing temperatures confirms the enhancing effect of annealing treatment on CG nanofiber membranes. The mass loss behavior was compared between the unannealed and the annealed CG nanofiber membranes, as shown in Figure 7(b). Analysis of the results shows that the mass loss of the unannealed CG nanofiber membranes was bigger than that of the annealed ones. The rate of mass loss (slope of the curves) decreases gradually with the rise of annealing temperature, indicating that aqueous stability of CG nanofiber membranes can be tailored by adjusting annealing temperature. The improved aqueous stability can be thankful to the formation of intensified molecular interactions due to annealing treatment, which is consistent with the swelling capacity results of CG nanofiber membranes.

Conclusion The effects of annealing treatment on the structural and mechanical properties of electrospun polyelectrolyte complex nanofiber membranes were studied. Significant improvement of mechanical properties of CG nanofiber membranes was achieved when annealing temperature is elevated above the Tg of CG. Annealing processing at 90 oC produced 1.3-fold and 1.1-fold increase on Young’s modulus and tensile strength, respectively, comparing with the unannealed nanofiber membranes. In addition, the energy to break of annealed CG specimens reaches its maximum value at annealing temperature of 90 oC. By SEM, XRD and FTIR analysis, it is found that both interfiber bonding and intermolecular bonding make contribution to the mechanical enhancement following annealing treatment. Therefore, thermal annealing treatment may be employed as an effective strategy for enhancing mechanical properties of polyelectrolyte complex nanofiber membranes/ scaffolds.

Acknowledgments This research was supported by the National Natural

Zelong Wang et al.

Science Foundation of China (Grant No. 21071114), as well as by the Excellent Program of Activity of Science and Technology for Overseas-Returned Scientists founded by the Ministry of Human Resources and Social Security of the People’s Republic of China, by the Program for Innovative Research Teams of Hubei Provincial Department of Education, China, by the project sponsored by the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry, and by Graduate Innovative Fund of Wuhan Institute of Technology (No. CX2013010).

References 1. B. M. Baker, R. P. Shah, A. M. Silverstein, J. L. Esterhai, J. A. Burdick, and R. L. Mauck, Proc. Natl. Acad. Sci. USA, 109, 14176 (2012). 2. C. I. Su, T. C. Lai, C. H. Lu, Y. S. Liu, and S. P. Wu, Fiber. Polym., 14, 542 (2013). 3. M. E. Gomes, A. Ribeiro, P. Malafaya, R. Reis, and A. Cunha, Biomaterials, 22, 883 (2001). 4. M. E. Gomes, J. Godinho, D. Tchalamov, A. Cunha, and R. Reis, Mater. Sci. Eng. C, 20, 19 (2002). 5. L. Ghasemi-Mobarakeh, M. P. Prabhakaran, P. Balasubramanian, G. Jin, A. Valipouri, and S. Ramakrishna, J. Nanosci. Nanotechnol., 13, 4656 (2013). 6. F. Croisier, A. S. Duwez, C. Jérôme, A. Léonard, K. Van der Werf, P. Dijkstra, and M. Bennink, Acta Biomater., 8, 218 (2012). 7. K. Leong, C. Cheah, and C. Chua, Biomaterials, 24, 2363 (2003). 8. Z. M. Huang, Y. Z. Zhang, M. Kotaki, and S. Ramakrishna, Compos. Sci. Technol., 63, 2223 (2003). 9. Z. Wang, N. Cai, D. Zhao, J. Xu, Q. Dai, Y. Xue, X. Luo, Y. Yang, and F. Yu, Polym. Compos., 34, 1735 (2013). 10. J. Xu, N. Cai, W. Xu, Y. Xue, Z. Wang, Q. Dai, and F. Yu, Nanotechnology, 24, 025701 (2013). 11. J. Deitzel, J. Kleinmeyer, J. Hirvonen, and N. Beck Tan, Polymer, 42, 8163 (2001). 12. Y. You, S. W. Lee, S. J. Lee, and W. H. Park, Mater. Lett., 60, 1331 (2006). 13. M. Sadrjahani and S. H. Ravandi, Fiber. Polym., 14, 1276 (2013). 14. J. Klein, Science, 250, 640 (1990). 15. C. Gao, H. Ma, X. Liu, L. Yu, L. Chen, H. Liu, X. Li, and G. P. Simon, Polym. Eng. Sci., 53, 976 (2012). 16. B. Babatope and D. H. Isaac, Polymer, 33, 1664 (1992). 17. Y. Srithep, P. Nealey, and L. S. Turng, Polym. Eng. Sci., 53, 580 (2012). 18. L. Huang, S. S. Manickam, and J. R. McCutcheon, J. Membrane Sci., 436, 213 (2013). 19. R. J. Samuels, J. Polym. Sci. B, 19, 1081 (1981). 20. B. Smitha, S. Sridhar, and A. Khan, Macromolecules, 37, 2233 (2004). 21. S. Haider, S. Y. Park, and S. H. Lee, Soft Matter., 4, 485


(2008). 22. S. Ramaswamy, L. I. Clarke, and R. E. Gorga, Polymer, 52, 3183 (2011). 23. S. J. Lee, S. H. Oh, J. Liu, S. Soker, A. Atala, and J. J. Yoo, Biomaterials, 29, 1422 (2008). 24. L. Li, R. Hashaikeh and H. A. Arafat, J. Membrane Sci., 436, 57 (2013). 25. Z. Liu, X. Ge, Y. Lu, S. Dong, Y. Zhao, and M. Zeng, Food Hydrocolloids, 26, 311 (2012). 26. S. Haider, W. A. Al Masry, N. Bukhari, and M. Javid, Polym. Eng. Sci., 50, 1887 (2010). 27. J. Zawadzki and H. Kaczmarek, Carbohyd. Polym., 80, 394 (2010).


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28. T. Jiang, Z. Zhang, Y. Zhou, Y. Liu, Z. Wang, H. Tong, X. Shen, and Y. Wang, Biomacromolecules, 11, 1254 (2010). 29. M. M. Daly and D. Knorr, Biotechnol. Progr., 4, 76 (1988). 30. T. Hesketh, J. Van Bogart, and S. Cooper, Polym. Eng. Sci., 20, 190 (1980). 31. G. Zhumadilova, A. Gazizov, L. Bimendina, and S. Kudaibergenov, Polymer, 42, 2985 (2001). 32. S. Balaji, R. Kumar, R. Sripriya, P. Kakkar, D. V. Ramesh, P. Reddy, and P. Sehgal, Mater. Sci. Eng. C, 32, 975 (2012). 33. X. Li, H. Xie, J. Lin, W. Xie, and X. Ma, Polym. Degrad. Stabil., 94, 1 (2009). 34. I. I. Muhamad, L. S. Fen, N. H. Hui, and N. A. Mustapha, Carbohyd. Polym., 83, 1207 (2011).