Poly(vinyl alcohol

Dec 8, 2007 - Department of Microbiology, Peking University Health Science Center, Beijing, 100083, P. R. China. Received August 12, 2007; Revised ...
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Biomacromolecules 2008, 9, 349–354

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Electrospun Water-Soluble Carboxyethyl Chitosan/Poly(vinyl alcohol) Nanofibrous Membrane as Potential Wound Dressing for Skin Regeneration Yingshan Zhou,† Dongzhi Yang,‡ Xiangmei Chen,§ Qiang Xu,§ Fengmin Lu,§ and Jun Nie*,†,‡ Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan, 430072, P. R. China, State Key Laboratory of Chemical Resource Engineering and College of Material Science and Engineering, Beijing University of Chemical Technology, Beijing, 100029, P. R. China, and Department of Microbiology, Peking University Health Science Center, Beijing, 100083, P. R. China Received August 12, 2007; Revised Manuscript Received October 30, 2007

Biocompatible carboxyethyl chitosan/poly(vinyl alcohol) (CECS/PVA) nanofibers were successfully prepared by electrospinning of aqueous CECS/PVA solution. The composite nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). SEM images showed that the morphology and diameter of the nanofibers were mainly affected by the weight ratio of CECS/PVA. XRD and DSC demonstrated that there was strong intermolecular hydrogen bonding between the molecules of CECS and PVA. The crystalline microstructure of the electrospun fibers was not well developed. The potential use of the CECS/PVA electrospun fiber mats as scaffolding materials for skin regeneration was evaluated in vitro using mouse fibroblasts (L929) as reference cell lines. Indirect cytotoxicity assessment of the fiber mats indicated that the CECS/PVA electrospun mat was nontoxic to the L929 cell. Cell culture results showed that fibrous mats were good in promoting the cell attachment and proliferation. This novel electrospun matrix would be used as potential wound dressing for skin regeneration.

1. Introduction Skin is a relatively soft tissue, covering the entire external surface and forming about 8% of the total body mass.1 Every year, millions of people need skin grafts due to dermal wounds caused by fire, heat, electricity, chemicals, ultraviolet, nuclear energy, or diseases. In the case of wounds that extend entirely through the dermis, such as full-thickness burns or deep ulcers, many skin substitutes such as xenografts, allografts, and autografts have been employed for wound healing. However, these approaches have disadvantages, which are high cost, the limited availability of skin grafts in severely burned patients, and problems of disease transmission and immune response.2–4 One strategy for dealing with serious skin damage is to develop tissue engineered skin substitutes. Nanofiber matrices have shown tremendous promise as tissue engineering scaffolds for skin substitutes. The advantages of a scaffold composed of ultrafine, continuous fibers are oxygen-permeable high porosity, variable pore-size distribution, high surface to volume ratio, and most importantly, morphological similarity to natural extracellular matrix (ECM) in skin, which promote cell adhesion migration and proliferation.5–7 A number of techniques, such as phase separation,6 self-assembly,8 and electrospinning5 have been developed to fabricate nanofibrous scaffolds with unique properties. * Corresponding author. E-mail: [email protected]. Fax: +861064421310. † Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University. ‡ State Key Laboratory of Chemical Resource Engineering and College of Material Science and Engineering, Beijing University of Chemical Technology. § Department of Microbiology, Peking University Health Science Center.

Among these techniques, electrospinning technology has become popular for the fabrication of tissue engineering scaffolds in recent years because it is a simple, rapid, efficient, and inexpensive method for producing nanofibers by applying a high voltage to electrically charge liquid. When the electric force on induced charges on the polymer liquid overcomes surface tension, a thin polymer jet is ejected. The charged jet is elongated and accelerated by the electric field, undergoes a variety of instabilities, dries, and is deposited on a substrate as a random nanofiber mat. For proper biomaterials in wound dressing, a number of natural and synthetic polymers, including chitosan,9 collagen,10 and poly(lactide-coglycolide), are currently being employed as tissue scaffolds for skin reconstruction. Chitosan has excellent biological properties such as biodegradability, biocompatibility, and antibacterial and woundhealing activity.11–13 Additionally, chitosan could achieve hemostasis and promote normal tissue regeneration.14 For these reasons, chitosan has been considered to be one of the most promising biomacromolecules for tissue engineered scaffolds and wound dressing. Recently, chitosan-based nanofibers have been successfully electrospun from chitosan solutions blended with poly(ethylene oxide) (PEO),15 poly(vinyl alcohol) (PVA),16,17 or silk fibroin.18 Also, electrospinning of homogeneous chitosan19,20 or chitosan derivatives21 has been also reported. However, some organic solvents or organic acids solvents such as 1,1,1,3,3,3-hexafluoro2-propanol (HFIP), chloroform, trifluoroacetic acid (TFA), acrylic acid, and acetic acid must be employed in the fabrication of these chitosan nanofibers. The trace toxic organic solvent or acid in electrospun products is harmful when it is applied to wounded human skin or tissue.

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To overcome these problems, in this research, the watersoluble N-carboxyethyl chitosan (CECS) was synthesized by Michael addition reaction of chitosan and acrylic acid. Because electrospinning from aqueous solution of CECS was unsuccessful either by regulating the viscosity or the conductivity of the solution, PVA was selected as the polymer additive to produce electrospun nanofibrous mat because of its good fiberforming, biocompatibility, and chemical resistance properties.22,23 Here, water is an ideal solvent for both fabrication processes and biomedical applications of CECS and PVA, and electrospinning carried out in neutral pH could not only avoid the trace presence of the toxic solvent in the produced fibers but also result in the fabrication of functional fibrous biomedical products containing water-soluble drugs. The effect of viscosity and conductivity of the solution on fiber formation and on the morphology of CECS/PVA bicomponent fiber was studied. The potential use of this as-spun fiber mat as a scaffolding material for skin was evaluated in vitro with mouse fibroblasts (L929). The indirect cytotoxicity, cell attachment, and cell proliferation were investigated as well.

2. Experimental Section 2.1. Materials. Chitosan (MW ) 1.2 × 105, degree of deacetylation ) 82.5%) was purchased from Zhejiang Golden-Shell Biochemical Co., Ltd., China. PVA (degree of polymerization ) 3500; 88% hydrolyzed) was obtained from Kuraray Co., Ltd., Japan. Acrylic acid was from Beijing Chemical Reagents Company, distilled under reduced pressure in the presence of hydroquinone, and stored at 4 °C until use. Mouse fibroblasts (L929) were obtained from Department of Microbiology, Peking University Health Science Center. 2.2. Synthesis of Water-Soluble CECS. N-Carboxyethyl chitosan was synthesized according to the method established by Sashiwa et al.24 Briefly, 2.0 g of chitosan (corresponding to 10 mmol NH2) was dissolved in 100 mL of water containing 1.40 mL of acrylic acid at 50 °C under constant stirring for 2 day. Thereafter, 1 N aqueous NaOH was added to the reaction mixture to adjust the pH to 10–12 in order to convert the carboxylic acid to its sodium salt. The mixture was precipitated by acetone and then dialyzed (membrane molecular weight cutoff 12 000 g µmol-1) against water for 2 days and lyophilized to obtain pure CECS. 1H NMR spectrum was recorded on a Bruker AMX 600 M NMR instrument. 2.3. Preparation of Polymer Solutions. A 9% (w/v) PVA solution was prepared by dissolution of 9.0 g PVA in 100 mL distilled water at 90 °C with vigorous stirring for a period about 4 h. CECS (6.0 g) was dissolved at a concentration of 6% (w/v) in 100 mL of distilled water. The PVA solution was mixed with the CECS solution at CECS/PVA weight ratios 100/0, 80/20, 60/40, 50/50, 30/70, 20/80, 10/90, and 0/100. The shear viscosities of the solutions of different CECS/PVA ratios were measured in a shear rate of 344 s-1 using a rotational viscometer (NDJ-79, Shanghai Jichang Geology Instrument Co. Ltd., China) equipped with coaxial cylinders, and the conductivities of the blend solutions were measured by electric conductivity meter (DDB-6200, Shanghai Rex Xinjing Instrument Co. Ltd., China). 2.4. Preparation of Fiber Mats. The electrospinning was performed at room temperature. The above mixed solution was placed into a plastic syringe (5 mL) with a metal capillary having an inner diameter of 0.57 mm. The positive electrode of a high voltage power supply (BMEI Co., Ltd., China) was connected to the metal capillary by copper wires. The voltage was 25 kV, and tip-to-collector distance was fixed at 12 cm. A grounded aluminum foil was used as the collector, and the nanofibrous nonwoven mats were collected on the surface of aluminum foil and dried at room temperature in vacuum for 24 h. 2.5. Cross-linking of the Electrospun Membranes. The crosslinking process was carried out according to the method established by Zhang et al.25 Briefly, in a sealed desiccator containing 10 mL of aqueous glutaraldehyde solution, the membranes were placed on a holed

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Figure 1. 1H NMR spectra of CECS and CS with CD3COOD/D2O as solvent.

ceramic shelf in the desiccator and were cross-linked in the glutaraldehyde vapor at room temperature for 2 days. After cross-linking, the samples were immersed in deionized water for 1 day by changing the water every 2 h. Finally, the cross-linked mats were dried at 60 °C in vacuum for 24 h. 2.6. Characterizations. 2.6.1. Scanning Electron Microscopy (SEM). The morphology and diameter of nanofibrous mats were determined by scanning electron microscopy (SEM) (S-450, Hitachi Ltd., Japan) at an accelerating voltage of 20 kV. The diameters of nanofibers were measured by using image analyzer. Fifty fibers were statistic in image. 2.6.2. Differential Scanning Calorimetry (DSC). The thermal analysis of the electrospinning fibers was studied by a DSC 204 F1 thermal analysis system (Netzsch, Germany). Samples sealed in aluminum pans were heated from room temperature to 220 °C at a heating rate of 10 °C/min under 50 mL/min of nitrogen flow. 2.6.3. X-Ray Diffraction (XRD). The XRD patterns of the electrospinning fibers were performed via X-ray diffractometer (Rigaku, Damax 2500) with Cu KR characteristic radiation (wavelength λ ) 0.154 nm at 40 kV, 50 mA, and scan speed of 1°/min in the 2θ range of 5–90°). 2.6.4. Methylthiazolydiphenyl-tetrazolium Bromide (MTT) Assay. The cytotoxicity of the electrospinning fibers and films was evaluated based on a procedure adapted from the ISO10993-5 standard test method. Mouse fibroblast (L929) was cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, together with 1.0% penicillin-streptomycin and 1.2% glutamine. Culture was maintained at 37 °C in a wet atmosphere containing 5% CO2. When the cells reached 80% confluence, they were trypsinized with 0.25% trypsin containing 1 mM ethylenediamine tetraacetic acid (EDTA). The viabilities of cells were determined by the MTT (3-[4, -dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; thiazolyl blue) assay. MTT reagent is a yellow substance that produces a dark-blue formazan product when incubated with viable cells. Therefore, the level of the reduction of MTT into formazan can reflect the level of cell metabolism. For the MTT assay, the film and the prepared as-spun membranes (∼0.3 mm thickness) cross-linked with glutaraldehyde were sterilized with highly compressed steam for 15 min and placed in wells of a 24-well culture plate, respectively. The samples were then incubated in 1 mL of RPMI1640 medium at 37 °C for 24 h. The extraction ratio was 6 cm2/mL. At the end of this period, the membranes were removed and the so-called extracts were obtained and further were diluted to obtain extraction medium samples with concentration of 10, 25, 50, and 100% (relative to original extracts). L929 cells were seeded in wells of a 96-well plate at a density of 103 cells per well. After incubation for another 24 h, the culture medium was removed and replaced with the as-prepared extraction medium and incubated for another 24 h, then 100 µL of MTT solution was added to each well. After 3 h incubation at 37 °C, 200 µL of dimethyl sulfoxide was added to dissolve the

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Figure 2. Effect of the composition of spinning solution on the morphology of CECS/PVA nanofiber. CECS/PVA weight ratio: (a) 80/20; (b) 60/40; (c) 50/50; (d) 40/60; (e) 30/70; (f) 20/80; (g) 10/90.

Figure 3. Diameter distribution of nanofibers for different composition. CECS/PVA weight ratio: (a) 60/40; (b) 50/50; (c) 40/60; (d) 30/70; (e) 20/80; (f) 10/90.

formazan crystals. The dissolved solution was swirled homogeneously for about 10 min by the shaker. The optical density of the formazan solution was detected by an ELISA reader (Multiscan MK3, Labsystem Co. Finland) at 570 nm. For reference purposes, cells were seeded to medium containing 0.64% phenol (positive control) and a fresh culture medium (negative control) under the same seeding conditions, respectively. 2.6.5. Cell Culture and Adhesion. The cross-linked nanofibrous mats were fixed on glass cover slips using copper tapes. The samples were then sterilized and extensively washed three times with sterile PBS prior to transfer to individual 24-well tissue culture plates. Aliquots (1 mL) of mouse fibroblasts (L929) suspension with 1.5 × 104 cell/mL were seeded on the sample membranes. After 48 h of culture, cellular constructs were harvested, rinsed twice with PBS to remove nonadherent cells, and subsequently fixed with 3.0% glutaraldehyde at 4 °C for 4 h. After that, the samples were dehydrated through a series of graded ethanol solutions and air-dried overnight. Dry samples were sputtered with gold for observation of cell morphology on the surface of the scaffolds by SEM. 2.6.6. Statistical Analysis. Results are depicted as mean ( standard deviation. Significance between the mean values was calculated using

ANOVA one-way analysis (Origin7.0 SRO, Northampton, MA). Probability values P < 0.05 were considered as significant (n ) 6).

3. Results and Discussion 3.1. Synthesis of Water-Soluble CECS. A water-soluble N-carboxyethyl chitosan was synthesized by a Michael addition reaction of chitosan with acrylic acid. The 1H NMR spectra of CECS and CS are shown in Figure 1. The chemical shift at 2.01 ppm (peak 1) is attributed to the methyl protons in the acetamide groups of chitosan. The occurrence of the two new peaks at 2.6 and 3.4 ppm (peak 2 and peak 4) indicate the presence of methylene protons, which suggest the successful grafting of acrylic acid to the amine groups of chitosan by the Michael addition reaction. The degree of substitution (DS) was calculated by comparison of the peak area at δ ) 2.6 of the -CH2- proton and that of δ ) 2.0 of the NHAc proton. The DS of synthesized CECS was 0.18, which meant that 21% of the NH2 reacted with acrylic acid. 3.2. Electrospinning of Aqueous CECS/PVA Solution. Our preliminary attempts for electrospinning of solutions of CECS

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Figure 4. Viscosity and conductivity of the CECS/PVA blend aqueous solutions.

Figure 7. Morphology of CECS/PVA ) 50/50 cross-linked membrane after immersion in water for 48 h.

Figure 5. XRD patterns of CECS, PVA, and the nanofibrous membranes with different CECS/PVA weight ratio: (a) CECS; (b) 50/ 50; (c) 40/60; (d) 30/70; (e) 20/80; (f) 10/90; (g) PVA.

Figure 8. Cytotoxicity test of cross-linked CECS membrane, crosslinked CECS/PVA membrane, and cross-linked CECS/PVA nanofiber with positive and negative controls (p < 0.05) *p < 0.05 when compared to the negative control of indirect cytotoxicity. The data represented mean and standard deviations of six samples.

Figure 6. DSC curves of CECS, PVA, and the nanofibrous membranes with different CECS/PVA weight ratio: (a) CECS; (b) 50/50; (c) 40/60; (d) 30/70; (e) 20/80; (f) 10/90; (g) PVA.

in distilled water over a broad concentration range, by regulating the viscosity or the conductivity of the solution, failed to obtain ultrafine fibers. Formation of “tailed” micro- and nanoparticles was observed, which meant that CECS itself was very difficult to electrospun. To obtain a nanofibrous mat, PVA was selected as a suitable nonionogenic partner for the preparation of CECS containing nanofibers. The morphology of electrospun nanofibers (presence or absence of defects, shape of the fibers, and the defects) and the

fiber diameters were strongly influenced by the composition of the spinning solution.26,27 SEM micrographs of the nanofibers obtained at different weight ratios are shown in Figure 2. It could be found that, when CECS/PVA ) 80/20, the jet was not stable, and a bead-on-string morphology with several big beads was obtained, as shown in Figure 2a. With an increase of PVA content, the number of spindles among the fibers decreased and the fiber formation ability improved (CECS/PVA ) 60/40, Figure 2b). As the CECS/PVA ratio reached up to 50/50, the bead-on-string morphology disappeared and smooth, homogeneous fibers were produced (Figure 2c). When the content of PVA increased above 50%, the electrospinning process became increasing fluent and the fiber diameter became larger (Figure 2e,f,g). Correspondingly, the diameter distribution of the nanofibrous mats is presented in Figure 3. As the CECS content in the blends decreased from 60/40 to 10/90, the average diameter of the blend nanofibers gradually increased from 131 to 456 nm and the distribution became slightly broader. The conductivities of the CECS/PVA mixed solutions increased from 0.95 to 3.14 mS · cm-1 with increasing of CECS content (Figure 4). Because CECS is a polyelectrolyte and PVA is nonionogenic polymer, an increase in CECS content in the blend solution could lead to an increase in the conductivity of the solution. However, it was found that viscosities of the mixed aqueous solutions reduced greatly with increasing CECS content and the viscosity changed from 751 mPa · s (CECS/PVA ) 10/ 90) to 227 mPa · s (CECS/PVA ) 80/20), as shown in Figure

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Figure 9. SEM images of L929 cell seeded on nanofibrous membrane of CECS/PVA (50/50) after 48 h culture.

4. Previous studies have shown that, during the smooth fiber process, the relatively higher conductivity and viscosity are both favorable factors for improving electrospinnability.28,29 Higher viscosity polymer solutions usually exhibit longer stress relaxation times, which could prevent the fracturing of ejected jets during electrospinning.30 Meanwhile, the high conductivity could enhance the electric force, which helps to strengthen the whipping instability and to improve the formation of smooth fibers.30 However, conventional CS solution formulations displayed electrical conductivities that were unacceptably large for electrospinning, where the liquids underwent deep atomization and broke into polydisperse electrosprays.31 In this work, when CECS/PVA ) 80/20, the blend solution could not be electrospun because of too low viscosity and too high conductivity. With an increase in the PVA content, the conductivities of the solutions gradually fell, whereas the viscosities of the solutions increased high enough to enhance the molecular entanglement necessary for fiber formation, which made the blend solutions electrospun. 3.3. X-Ray Diffraction (XRD) Spectra. Figure 5 presents XRD patterns of CECS/PVA blend nanofibers. For the pure PVA powder, there were three typical peaks at 2θ ) 10.7°, 2θ ) 19.3°, and 2θ ) 22.5° (Figure 5g). The diffraction model of CECS powder showed one typical peak at 2θ ) 19.9° (Figure 5a). If there were no or weak interaction between CECS and PVA molecules in the blend fibers, each component would show its own crystal region in the blend fibers and XRD patterns could be expressed as simple mixed patterns of CECS and PVA with the same ratio as those for mechanical blending. However, the peak of 2θ ) 10.7° of PVA disappeared with incorporation of CECS. These evidence indicated that strong interaction occurred between CECS and PVA molecule in the blend fibers. Moreover, compared with the powders, CECS/PVA blend nanofibers showed a relatively obtuse and broad peak around 19.4°. This phenomenon confirmed that electrospinning retarded the crystallization process of CECS/PVA blend, which did not lead to the development of the crystalline microstructure of electrospun fibers. Similar results of electrospinning were reported.32,33 The

reason is that, during the electrospinning process, the stretched molecular chains of the fiber solidified rapidly at high elongation rates, which significantly hindered the formation of crystals. 3.4. Differential Scanning Calorimetry (DSC). Figure 6 shows the DSC thermograms of CECS, PVA, and the electrospinning fibers of CECS/PVA blends. The pure PVA powder showed a relatively large and sharp endothermic curve with a peak at 194 °C. However, the endothermic curve of CECS/ PVA blend fibers became obtuse and broad, and the peak shifted toward lower temperatures. This indicated that the crystalline microstructure of electrospun fibers did not develop well. This was because the majority of the chains were in the noncrystalline state due to the rapid solidification process of stretched chains during electrospinning. 3.5. Stability in Water of Cross-linked Electrospun Membranes. CECS and PVA are water-soluble biopolymers, and they should be cross-linked to be water-resistant before use as wound dressings. In the present study, after the CECS/PVA (50/ 50) cross-linked fibers had been immersed in water for 48 h, the morphology hardly changed, as shown in Figure 7. No residual glutaraldehyde was detected by an ultraviolet spectrophotometer (U-3010, Hitachi, Tokyo, Japan) from waterimmersed nanofibers, which favored the cross-linked as-spun mats as biomedical materials. 3.6. MTT Assay. An ideal wound dressing should not release toxic products or produce adverse reactions, which could be evaluated through in vitro cytotoxic tests. In the evaluation, mouse connective tissue, fibroblast-like cells (L929) were used as reference. Figure 8 shows the absorbance illustrating the viability of L929 cells that were cultured with the extraction medium from various types of specimens. It could be seen that no statistically significant differences were observed in the cell activity of L929 culture for 48 h in the presence of CECS/PVA electrospinning mat extracts in comparison with control, independent of the dilution used, although the average absorbance values were lower than that of the control condition. However, when a 10% diluted cross-linked CECS membrane or crosslinked CECS/PVA membrane extract was used, statistically

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significant differences (p < 0.05) were observed in the cell activity in comparison with control, but the viability of the cell still reached 89% of that of the negative control. This indicates that the cross-linked CECS membrane or cross-linked CECS/ PVA membrane were less toxic to L929 cells. A similar result was observed in the cross-linked CECS/PVA membrane when 100% extract was used. The obtained results clearly suggest that electrospun mats of CECS/PVA are nontoxic to L929 cells and are good candidates to be used as wound dressing. 3.7. Cell Adhesion and Morphology. The cross-linked nanofibrous membrane (CECS/PVA ) 50/50) was used for cell adhesion, spreading, and interaction study. Figure 9 shows SEM images of L929 grown on the cross-linked CECS/PVA nanofibers after 48 h cell culture. It could be found that, L929 cells appeared to adhere well and exhibited a normal morphology on the surface, which was due to the large surface area available for cell attachment. It could be clearly seen from Figure 9b,c,d that cells are attached to the surfaces by discrete filopodia and exhibit short and numerous microvilli on their surfaces. It was interesting to see that microvilli of L929 cells tended to attach and grow along the polymer nanofibers.

4. Conclusions The best biomaterials for wound dressing should be biocompatible and promote the growth of dermis and epidermis layers. In this study, the biocompatible CECS/PVA nanofibers were successfully prepared by electrospinning of aqueous CECS/PVA with different weight ratios. Indirect cytotoxicity assessment of electrospun CECS/PVA fiber mats with mouse fibroblasts (L929) indicated that the material showed no cytotoxicity toward growth of L929 cell and had good in vitro biocompatibility. Cell culture suggested that the fibrous mats did well in promoting cell attachment and proliferation. These novel electrospun matrices have the potential to be used as materials for wound dressing for skin regeneration. Acknowledgment. This research was supported by Open Funding from State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology.

References and Notes (1) Chong, E. J.; Phan, T. T.; Lim, I. J.; Zhang, Y. Z.; Bay, B. H.; Ramakrishna, S.; Lim, C. T. Acta Biomater. 2007, 3, 321–330. (2) Sheila, M. Nature 2007, 445, 874–880. (3) Seal, B. L.; Otero, T. C.; Panitch, A. Mater. Sci. Eng., R 2001, 34, 147–230.

Zhou et al. (4) Marler, J. J.; Upton, J.; Langer, R.; Vacanti, J. P. AdV. Drug DeliVery ReV. 1998, 33, 165–182. (5) Li, W. J.; Laurencin, C. T.; Caterson, E. J.; Tuan, R. S.; Ko, F. K. J. Biomed. Mater. Res. 2002, 60, 613–621. (6) Smith, L. A.; Ma, P. X. Colloids. Surf., B 2004, 39, 125–131. (7) Yang, F.; Murugan, R.; Wang, S.; Ramakrishna, S. Biomaterials 2005, 26, 2603–2610. (8) Whitesides, G. M.; Boncheva, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4769–4774. (9) Ma, L.; Gao, C. Y.; Mao, Z. W.; Zhou, J.; Shen, J. C.; Hu, X. Q.; Han, C. Biomaterials 2003, 24, 4833–4841. (10) Chen, G. P.; Sato, T; Ohgushi, H; Ushida, T.; Tateishi, T.; Tanaka, J. Biomaterials 2005, 26, 2559–2566. (11) Khor, E.; Lim, L. Y. Biomaterials 2003, 24, 2339–2349. (12) No, H. K.; Park, N. Y.; Lee, S. H.; Meyers, S. P. Int. J. Food Microbiol. 2002, 74, 65–72. (13) Ueno, H.; Mori, T.; Fujinaga, T. AdV. Drug DeliVery ReV. 2001, 52, 105–115. (14) Malette, W. G.; Quigley, H. J. U.S. Patent 4,532,134, 1985. (15) Bhattarai, N.; Edmondson, D.; Veiseh, O.; Matsen, F. A.; Zhang, M. Q. Biomaterials 2005, 26, 6176–6184. (16) Jia, Y. T.; Gong, J.; Gu, X. H.; Kim, H. Y.; Dong, J. Carbohydr. Polym. 2007, 67, 403–409. (17) Zhou, Y. S.; Yang, D. Z.; Nie, J. J. Appl. Polym. Sci. 2006, 102, 5692– 5697. (18) Park, W. H.; Jeong, L.; Yoo, D.; Hudson, S. Polymer 2004, 45, 7151– 7157. (19) Ohkawa, K.; Cha, D.; Kim, H.; Nishida, A.; Yamamoto, H. Macromol. Rapid Commun. 2004, 25, 1600–1605. (20) Geng, X. Y.; Kwon, O. H.; Jang, J. Biomaterials 2005, 26, 5427– 5432. (21) Neamnark, A.; Rujiravanit, R.; Supaphol, P. Carbohydr. Polym. 2006, 66, 298–305. (22) Gimenez, V.; Mantecon, A.; Cadiz, V. J. Polym. Sci., Part A: Polym. Chem. 1996, 34, 925–934. (23) Krumora, M.; Lopez, D.; Benarente, R.; Mijangos, C.; Perena, J. M. Polymer 2000, 41, 9265–9272. (24) Sashiwa, H.; Yamamori, N.; Ichinose, Y.; Sunamoto, J.; Aiba, S. Macromol. Biosci. 2003, 3, 231–233. (25) Zhang, Y. Z.; Venugopal, J.; Huang, Z. M.; Lim, C. T.; Ramakrishna, S. Polymer 2006, 47, 2911–2917. (26) Zhang, C.; Yuan, X.; Wu, L.; Han, Y.; Sheng, J. Eur. Polym. J. 2005, 41, 423–432. (27) Spasova, M.; Manolova, N.; Paneva, D.; Rashkov, I. e-Polym. 2004, 056, 1–12. (28) Fong, H.; Chun, I.; Reneker, D. H. Polymer 1999, 40, 4585–4592. (29) Theron, S. A.; Zussman, E.; Yarin, A. L. Polymer 2004, 45, 2017– 2030. (30) Li, J. X.; He, A. H.; Zheng, J. F.; Han, C. C. Biomacromolecules 2006, 7, 2243–2247. (31) Subramanian, A.; Vu, D.; Larsen, G. F.; Lin, H. Y. J. Biomater. Sci., Polym. Ed. 2005, 16, 861–873. (32) Deitzel, J. M.; Kleinmeyer, J. D.; Harris, D.; Beck Tan, N. C. Polymer 2001, 42, 261–272. (33) Zong, X. H.; Kim, K. S.; Fang, D. F.; Ran, S. F.; Hsiao, B. S.; Chu, B. J. Polymer 2002, 43, 4403–4412.

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