Biomaterial Films of Bombyx Mori Silk Fibroin with Poly(ethylene oxide)

Phase separation into controllable patterned microstructures was observed for Bombyx mori silkworm silk and poly(ethylene oxide) (PEO) (900000 g/mol) ...
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Biomacromolecules 2004, 5, 711-717

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Biomaterial Films of Bombyx Mori Silk Fibroin with Poly(ethylene oxide) Hyoung-Joon Jin,†,‡ Jaehyung Park,† Regina Valluzzi,† Peggy Cebe,§ and David L. Kaplan*,† Departments of Chemical & Biological Engineering, Biomedical Engineering, and Physics and Astronomy, Bioengineering Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155 Received August 31, 2003; Revised Manuscript Received December 8, 2003

Phase separation into controllable patterned microstructures was observed for Bombyx mori silkworm silk and poly(ethylene oxide) (PEO) (900 000 g/mol) blends cast from solution. The evolution of the microstructures with increasing PEO volume fraction is strikingly similar to the progression of phases and microstructures observed with surfactants. The chemically patterned materials obtained provide engineerable biomaterial surfaces with predictable microscale features which can be used to create topographically patterned or chemically functionalized biomaterials. Solution blending was used to incorporate water-soluble PEO into silk to enhance elasticity and hydrophilicity. The sizes of the globule fibroin phase ranged from 2.1 ( 0.5 to 18.2 ( 2.1 µm depending on the ratio of silk/PEO. Optical microscopy and SEM analysis confirmed the micro-phase separation between PEO and silk. Surface properties were determined by XPS and contact angle. Methanol can be used to control the conformational transition of silk fibroin to the insoluble β-sheet state. Subsequentially, the PEO can be easily extracted from the films with water to generate silk matrixes with definable porosity and enhanced surface roughness. These blend films formed from two biocompatible polymers provide potential new biomaterials for tissue engineering scaffolds. Introduction Silk is a natural fiber produced by the silkworm, Bombyx mori, which has been used traditionally in the form of threads in textiles for thousands of years. This silk contains a fibrous protein termed fibroin (both heavy and light chains) that form the thread core and glue-like proteins termed sericin that surround the fibroin fibers to cement them together. The fibroin is an insoluble protein containing up to 90% of the amino acids glycine, alanine, and serine leading to significant content of antiparallel β-pleated sheet formation in the fibers.1 Recent interest in the use of reprocessed silks such as fibroin in biotechnological materials and in biomedical applications originate from the unique mechanical properties of the silk fibers as well as their biodegradability and biocompatibility.2-6 Silk fibroin films cast from aqueous solution regenerated from fibers of cocoons undergo conformational transitions from random coil to the β structure. Thus, fibroin films in the dry state become brittle and unsuitable for practical use. These properties can be improved by blending with other natural or synthetic polymers. Silk blends have been extensively studied with respect to film formation.7 Blends with polyacrylamide,7 sodium alginate,8 cellulose,9,10 chitosan,11-13 poly(vinyl alcohol),14-17 acrylic polymers,18 poly(ethylene glycol) (PEG) (300 g/mol19 or 8000 g/mol20), poly(* To whom correspondence should be addressed. † Departments of Biomedical Engineering and Chemical & Biological Engineering. ‡ Present address: Inha University, Department of Polymer Science and Engineering, Incheon 402-751, South Korea. § Department of Physics and Astronomy.

caprolactone-co-D,L-lactide),21 and S-carboxymethyl keratin22 have been studied to improve the mechanical or thermal stability or membrane properties of silk films. Silk/syndiotactic-rich PVA blend films showed improved permeability to neutral salts.16 Thermal stability and tensile strength of silk were improved by blending with polyacrylamide.7 In the case of silk/cellulose blends, tensile strength and elongation at break were enhanced9 and porous cellulose membranes were formed by dissolving the silk in 10% NaOH.10 By blending with poly(-caprolactone-co-D,L-lactide), enhanced elastic behavior compared to pure silk was obtained.21 The surface properties of the silk film were improved by blending with S-carboxymethyl keratin based on reducing interfacial free energy between the blend films and water.22 Blending has included cosolvents such as water,7,8,14-17 a cuprammonium complex,10 formic acid,22 and acetic acid.12 In the present study, we sought to develop regenerated highly concentrated aqueous silk solutions (8 wt %) in blends with biocompatible polymers to improve silk film elasticity and to prepare materials for potential biomedical applications. Thick silk fibroin films (∼200 µm) were formed from concentrated silk solutions, since thin films (less than 50 µm) were brittle. PEO was selected for blending based on its aqueous solubility and known biocompatibility.23-27 By blending PEO with silk fibroin, a unique morphology was observed and could be controlled based on blend composition. Furthermore the PEO phase could be extracted from the films to generate silk with definable porosity and enhanced surface roughness. Processing was performed in water to enhance the potential biocompatibility of the silk

10.1021/bm0343287 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

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films by avoiding the use of organic solvents that can potentially pose problems when the processed materials are exposed to cells in vitro or in vivo. Experimental Section Materials. Cocoons of B. mori silkworm silk were kindly supplied by M. Tsukada, Institute of Sericulture, Tsukuba, Japan. PEO with an average molecular weight of 9 × 105 g/mol and PEG of 3400 g/mol were purchased from Aldrich (Milwaukee, WI) and used without further purification. Preparation of Regenerated B. mori Silk Fibroin Solutions. B. mori silk fibroin solutions were prepared as follows as a modification of our earlier procedure.4,28 Cocoons were boiled for 30 min in an aqueous solution of 0.02 M Na2CO3 and then rinsed thoroughly with water to extract the glue-like sericin proteins. The extracted silk was then dissolved in 9.3 M LiBr solution at room-temperature yielding a 20 wt % solution. This solution was dialyzed in water using Slide-a-Lyzer dialysis cassettes (Pierce, MWCO 3500) for 48 h. The final concentration of aqueous silk solution was 7.0-8.0 wt %, which was determined by weighing the remaining solid after drying. Preparation and Treatment of Blend Films. Various silk blends in water were prepared by adding 5 wt % PEO solutions into the silk aqueous solutions (Figure 1). The blending ratios of silk fibroin/PEO were 100/0, 95/5, 90/10, 80/20, 70/30, and 60/40 (wt %). The solutions were mildly stirred for 15 min at room temperature and then cast on polystyrene Petri dish surfaces for 24 h at room temperature in a hood. The films were then placed in a vacuum for another 24 h. Silk fibroin and blend films were immersed in a 90/10 (v/v) methanol/water solution for 30 min to induce an amorphous to β-sheet conformational transition of the silk fibroin.4 Characterization. The infrared spectra of silk fibroin and silk fibroin/PEO blend films were measured with a FT-IR (Bruker Equinox 55) spectrophotometer. These films were cast on a ZnS cell crystal surface directly from solution. Each spectrum for samples was acquired in transmittance mode by accumulation of 256 scans with a resolution of 4 cm-1 and a spectral range of 4000-400 cm-1. The contact angle, using Millipore purified water droplet, 17 MΩ, was measured to determine surface hydrophilicity. The water droplet, approximately 5 µL, was applied using a syringe and 22-guage needle, and the static contact angle was measured using a goniometer (Rame-Hart, Inc.). This analysis was performed after methanol treatment, and 5 replicates were averaged. Phase separation of blend films was observed by using a Nikon Eclipse E600 POL optical polarizing microscope before and after methanol treatment. Fractured surfaces of silk fibroin and silk fibroin/PEO blend films were imaged using a LEO Gemini 982 Field Emission Gun SEM. Fractured surfaces were obtained by splitting the film sample in liquid nitrogen. Sizes of globules and pores were measured from the SEM images of the blend film surfaces. A Surface Science Inc. model SSX-100 X-ray photoelectron spectrometer was used to analyze the surface

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of the silk films to estimate the surface density of silk versus PEO. Survey scans (spot 1000 µm, resolution 4, window 1000 eV) were performed using a flood gun (charge neutralizer) setting of 5 eV and nickel wire mesh held over the sample to prevent charging of the sample surface. Globule and pore sizes were measured from the electronic SEM images using Corel computer software. A total of 100 samples were averaged for each image. Individual globule surface morphology was observed by atomic force microscope (AFM). All imaging and lithography were performed in tapping mode on a Dimension 3100 Nanoscope III with rotated Tapping Mode etched silicon probes (RTESP) (Nanodevices, Stanta Barbara, CA). The RETSP probe cantilever length is 125 mm with a spring constant of 40 N/m developed to measure high-aspect ratio features. The images were obtained in phase-mode. Images were captured at the same time without digital filtering. Mechanical Properties of Films. The tensile properties of specimens (5 × 50 × 0.2 mm) were measured with a crosshead speed of 15 mm/min using an Instron tensile tester at ambient conditions after the methanol treatment. All samples were stored in a vacuum at room temperature before testing. The gauge length was set 30 mm, and an initial load cell of 100 kgf was applied. The tensile strength per crosssectional area (kg/mm2) and the ratio of the relative elongation to the initial film length at break (%) were determined from an observation of the stress-strain curves. Results and Discussion Phase Separation Behavior of Silk Fibroin/PEO Blends. PEG and PEO were initially selected for blending with silk fibroin to improve silk fibroin film properties with aqueous processability and biocompatibility as key criteria. Two molecular weights of PEG or PEO were studied for blending (3400 and 900 000 g/mol). Silk fibroin/PEG or PEO films were first prepared to identify concentrations of the components useful in materials processing. The films were cast from water solutions onto polystyrene Petri dishes in various ratios as described in the Experimental Section and dried overnight. In the case of silk and PEG (3400 g/mol) blends, the two components separated macroscopically into two phases during film formation throughout the range of compositions studied. Poorer quality films formed from all blend ratios except silk fibroin/PEG (98/2 wt %). Blends from silk fibroin/PEG were immersed in a 90/10(v/v) methanol/water solution for 30 min to convert the fibroin to the insoluble β-sheet structure. After this crystallization process, phase separation was more pronounced because the PEG phases became opaque while the silk fibroin phase was still transparent. Because of the evident phase separation and large domain sizes in the silk fibroin/PEG blend, further characterization was not considered. However, in the case of silk fibroin and PEO (900 000 g/mol) blends, no macroscopic phase separation occurred between the two components throughout the range of compositions studied, even after methanol treatment. Microscale phase separation in the silk fibroin/PEO blends was observed by optical microscopy

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Figure 1. Blend film processing steps. (A) Illustration of the film preparation process, (B) SEM images of film b (top) and film c (bottom), and (C) FT-IR data to show structural transitions of fibroin. a, As cast film. b, Methanol (90 wt %) treated film. c, PEO phase extracted film in water for 24 h at room temperature.

(Figure 2). After methanol treatment (Figure 2b), the size of the silk fibroin phase was reduced due to crystallization (β-sheet formation; Figure 1c) of the silk fibroin and dehydration. Unique globules of the silk fibroin phase were formed throughout the blend film except in the case of silk fibroin/PEO (98/2 w/w) blend before and after methanol treatment. FT-IR. Interactions between silk fibroin/PEO were assessed by FT-IR. As shown in Figure 1C, the structure of the silk fibroin in the films was predominantly random coil (1538 cm-1, amide II) or silk I (1658 cm-1, 1652 cm-1, amide I) before methanol treatment. The film was immersed

in 90/10 (v/v) methanol/water for 30 min to induce crystallization of fibroin. Structural changes after methanol treatment from random coil to predominantly β-sheet structure (1697 cm-1, 1627 cm-1, amide I; 1528 cm-1, amide II) were observed, mimicking the behavior typically seen with reprocessed silkworm silk. The amide I and amide II spectral bands for silk fibroin in the silk/PEO blend were similar to pure fibroin without PEO. Therefore, there was little evidence for a molecular level blend miscibility or secondary structural templating between the two polymers. Surface Properties of Silk Blend Films before and after Methanol Treatments. XPS was used to estimate the surface

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Table 1. High-Resolution XPS Results from the Silk Fibroin, PEO, and Silk Fibroin/PEO Blend Film Surfaces before and after Methanol Treatment O1s element

binding energy (eV)

Silk fibroin PEO02BMa PEO10BMa PEO20BMa PEO30BMa PEO40BMa PEO02AMb PEO10AMb PEO20AMb PEO30AMb PEO40AMb

530.3 530.9 530.9 531.1 530.8 530.5 530.7 531.1 530.5 530.7 531.1

N1s atom %

binding energy (eV)

24.6 24.2 29.0 25.3 25.1 24.6 23.8 23.7 29.3 18.3 27.0

398.4 398.5 398.6 398.9 398.6 398.4 398.4 398.5 398.4 398.4 398.5

C1s atom %

binding energy (eV)

atom %

N1s/C1

silk/PEO mol/mol

14.9 14.4 10.0 12.4 13.0 12.0 13.4 11.4 7.4 9.5 6.4

284.6 284.6 284.6 284.6 284.6 284.6 284.6 284.6 284.6 284.6 284.6

60.5 61.4 61.0 62.3 61.9 63.4 62.8 64.9 63.3 72.2 66.6

0.25 0.23 0.16 0.20 0.21 0.19 0.21 0.17 0.12 0.13 0.10

100/0 92/8 64/36 80/20 84/16 76/24 84/16 68/32 48/52 52/48 40/60

a PEO weight percent in blend with silk fibroin before methanol (BM) treatment. b PEO weight percent in blend with silk fibroin after methanol (AM) treatment.

Table 2. Contact Angle of Silk and Silk Fibroin/PEO Blend Films after Methanol Treatment (N ) 5, Average ( Standard Deviations)

a

sample

silk

PEO02AMa

PEO10AMa

PEO20AMa

PEO30AMa

PEO040AMa

angle(°)

81 ( 2

76 ( 2

72 ( 1

68 ( 1

68 ( 1

63 ( 2

PEO weight percent in blend with silk fibroin after methanol (AM) treatment.

Figure 2. Optical microscopy images of silk fibroin/PEO blend (80/20 wt %).

composition of the films. Table 1 shows the respective peak intensities of O1s, C1s, or N1s of silk fibroin and silk fibroin/ PEO blend films before and after methanol treatments. The ratios of N1s/C1s were used to estimate the composition of silk fibroin and PEO before and after methanol treatment from the surface of films. Based on these ratios, we can determine the blend film composition as shown in Table 1.

Before methanol treatment of silk fibroin/blend films, only the silk fibroin/PEO (90/10 wt %) blend film showed a higher PEO content on the film surface than the original blend composition. After methanol treatment, the surface PEO content of all blends increased. PEO migrates to the surface of the films during methanol treatment due to solubility of PEO in methanol and the lack of solubility of the fibroin. Since silk fibroin is relatively hydrophobic due to the amino acid content, β-sheet formation is induced by the methanol treatment and methanol compatible PEO on the film surface could be anticipated. The contact angle was used to characterize the surface of the films after methanol treatment as shown in Table 2. The hydrophilicity of surface increased with increased PEO content, confirming the XPS data. SEM and AFM. The fractured cross section side and surface morphologies of the silk fibroin and silk fibroin/PEO blend films were examined using high-resolution low voltage SEM after PEO extraction in water at room temperature for 24 h. In Figure 3, although the pure silk fibroin film exhibited a dense and uniform fractured surface, the fractured surfaces of all silk fibroin/PEO blends showed rough and reticulated morphologies due to the micro phase separation. In the case of the silk fibroin/PEO (98/02 wt %) blend, an evenly dispersed micro-size PEO phase was observed. Otherwise, the higher the PEO content in the films in the range of 1040 wt %, the sparser the film morphology based on the analysis of cross sections. The silk fibroin/PEO (90/10 wt %) blend showed the most dense structure between globules (Figure 3c) and the largest globule morphology from the fractured surfaces (Table 3). This blend generated the largest silk phases of all the blends studied. Therefore, by adding 10 wt % of PEO into the silk fibroin, the largest silk globule phases (15.4(3.4 µm diameter) and film pore sizes (6.6(2.0, inter-globule spaces) formed on the fractured surface. As the PEO portion increased, the silk phase separation size decreased as shown in Figure 3 and Table 3. In the blends

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Figure 3. Freeze fractured cross section images of silk fibroin and silk fibroin/PEO blend films imaged by SEM: (a) silk fibroin, (b) 98/2 wt %, (c) 90/10 wt %, (d) 80/20 wt %, (e) 70/30 wt %, and (f) 60/40 wt % (silk fibroin/PEO).

Figure 4. Surface images of silk fibroin/PEO (90/20 wt %) blend films imaged by SEM: (a) before methanol treatment, (b) after methanol treatment, and (c) after PEO extraction in water.

of 20-40 wt % PEO, well-distributed micro-size phase separation between silk fibroin and PEO was observed both

internally and on the surface of the films (Figure 4). The larger globule size was observed from the surface of the films

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Table 3. Silk Fibroin Globule Size and Pore Sizes (µM) of Silk Fibroin/PEO Blend Films after PEO Extraction in Water at Room Temperature (N ) 100, Average ( Standard Deviations) silk globules

PEO02a PEO10a PEO20a PEO30a PEO40a

Table 4. Mechanical Properties of Silk Fibroin and Silk Fibroin/ PEO Blend Films before and after Methanol (90%) Treatment (N ) 5, Average ( Standard Deviations)

pores

film surface

fracture urface

film surface

fracture surface

b 18.2 ( 2.1c (8.7 ( 2.5)d 13.1 ( 1.9 7.4 ( 1.5 3.1 ( 0.9

b 15.4 ( 3.4

1.9 ( 0.6 9.5 ( 2.1

2.5 ( 0.1 6.6 ( 2.0

7.3 ( 1.5 2.5 ( 0.9 2.1 ( 0.5

5.5 ( 1.2 4.5 ( 1.9 4.1 ( 1.1

2.5 ( 1.1 2.1 ( 1.5 2.0 ( 0.5

a PEO weight percent in silk/PEO blends. b Globule structure did not form at this composition. c Larger globule size. d Smaller globule size.

sample SILKBMa PEO02BMb PEO10BMb PEO20BMb PEO30BMb PEO40BMb SILKAMc PEO02AMd PEO10AMd PEO20AMd PEO30AMd PEO40AMd

tensile modulus tensile strength elongation at break (GPa) (MPa) (%) 3.9 ( 0.7 3.3 ( 0.6 3.2 ( 0.2 2.7 ( 0.3 2.3 ( 0.1 2.0 ( 0.03 3.5 ( 0.9 3.4 ( 0.1 3.2 ( 0.1 2.3 ( 0.2 2.1 ( 0.2 1.4 ( 0.2

47.2 ( 6.4 63.0 ( 8.7 42.5 ( 2.0 28.9 ( 2.8 29.5 ( 0.9 32.6 ( 3.4 58.8 ( 16.7 58.5 ( 6.5 43.3 ( 4.7 27.9 ( 3.0 29.2 ( 5.3 26.5 ( 2.3

1.9 ( 0.7 5.7 ( 2.0 2.7 ( 0.6 1.9 ( 0.7 6.2 ( 1.8 10.9 ( 4.5 2.1 ( 0.4 3.2 ( 1.0 2.6 ( 0.3 2.1 ( 0.2 4.9 ( 1.6 8.2 ( 1.3

a Pure silk film before methanol (BM) treatment. b PEO weight percent in blend with silk fibroin before methanol (BM) treatment. c Pure silk film after methanol (AM) treatment. d PEO weight percent in blend with silk fibroin after methanol (AM) treatment.

Figure 5. AFM images of silk fibroin/PEO (90/10 wt %) blends: (a) before methanol (90%) treatment, (b) after methanol treatment, and (c) after PEO extraction.

throughout all blend ratios than inside the film (Table 3). As the PEO content was increased, the pore size also decreased from 9.5 ( 2.1 to 4.1 ( 1.1 (Table 3) on the surface.

Optical microscopy images indicated similar phase separation and sizes of fibroin domains on the surface of blends of 10-40 wt % PEO before and after methanol treatment. The substructure was observed on the individual globules by AFM (Figure 5a). After methanol treatment, the surface of the globules was covered by PEO, which shows one phase by AFM (Figure 5b). After PEO extraction at room temperature in water, only silk fibroin was found on the surface of globules. A regular pattern was found on the surface by AFM (Figure 5). The pattern size spacing was in the range of 70-100 nm, which matched the size of similar morphological features on the fracture surface of native silk fibroin.29 Mechanical Properties of the Blend Films before and after Methanol Treatment. Tensile modulus, strength, and elongation of silk fibroin and silk fibroin/PEO blend films before and after methanol treatment are shown in Table 4. The pure silk film displayed typical behavior of brittle materials. To enhance mechanical properties of silk fibroin films, blending with PEO, a hydrophilic and biocompatible polymer, was used to impart flexibility while maintaining biodegradability.30 The addition of 2 wt % PEO to the silk

Figure 6. Optical microscopy and SEM images of Coomassie blue stained silk fibroin/PEO (80/20 wt %) blend film which was sliced and fractured mechanically after methanol treatment, PEO extraction in water and dried in a vacuum: (a) a slant slice from the film, (b) isolated bundle of silk fibroin globules, and (c) two different sizes of individual silk fibroin globules.

Films of B. mori Silk with Poly(ethylene oxide)

fibroin improved mechanical properties, whereas in the other blends, tensile modulus and strength decreased with increasing PEO content. This effect was likely due to phase separation between the two polymers, resulting in reduced interactions in the silk fibroin phase. Elongation at break increased up to 10.9% in silk fibroin/PEO (60/40 wt %) blends. Therefore, only toughness was slightly improved by addition of the PEO. Conclusions Solution blending of two biocompatible polymers was used to engineer novel biomaterials with highly regular patterned microstructures. Silk fibroin, a fibrous protein biopolymer, forms patterned domains resembling the lyotropic phases of many surfactants when combined with increasing volume fractions of PEO, also a biocompatible polymer. The blending and casting techniques used hinge on the availability of concentrated (8%) aqueous solutions of fibroin, prepared through a modified silk solubilization procedure. The microstructures obtained from the blends have uniform sized and shaped domains. These morphological features open up possibilities for porous biocompatible and biodegradable materials. Silk fibroin is biocompatible and biodegradable, therefore controlled membrane structure/porosity offers utility in scaffolds for tissue engineering. The aim of this study was to characterize new biomaterials based on silkworm silk fibroin/PEO blend films to improve film toughness while retaining biocompatibility. All processes were performed in aqueous environments with 8 wt % silk fibroin solution and PEO. By blending PEO with silk fibroin, the surface of the silk fibroin film was more hydrophilic, based on XPS and contact angle measurements. Interesting morphologies in the blends were observed in microscopically phase separated domains which depended on the ratio of PEO to silk. The sizes of silk fibroin globule phases were in the range of 2.1 ( 0.5-18.2 ( 2.1 µm depending on silk/PEO blend ratio. These phases produce predictable surface morphologies for bulk silk materials that will be of interest for biomaterial applications. Acknowledgment. We thank the NIH (R01 DE1340501), the NSF (DMR 0090384), and the DoD (Air Force) for support of this program. References and Notes (1) Asakura, T.; Kaplan, D. L. In Encylopedia of Agricultural Science; Arntzen, C. J., Ritter, E. M., Eds.; Academic Press: New York, 1994; Vol. 4, p 1.

Biomacromolecules, Vol. 5, No. 3, 2004 717 (2) Bunning, T. J.; Jiang, H.; Adams, W. W.; Crane, R. L.; Farmer, B.; Kaplan, D. L. In Silk Polymers-Material Science and Biotechnology; Kaplan, D. L., Adams, W. W., Farmer, B., Viney, C., Eds.; ACS Symposium Series 544: American Chemical Society: Washington, DC, 1993; p 353. (3) Kaplan, D. L.; Mello, C. M.; Arcidiacono, S.; Fossey, S.; Senecal, K.; Muller, W. In Protein-Based Materials; McGrath, K., Kaplan, D. L., Eds.; Birkhauser: Boston, 1997; p 103. (4) Sofia, S.; McCarthy, M. B.; Gronowicz, G.; Kaplan, D. L. J. Biomed. Mater. Res. 2001, 54, 139-148. (5) Shao, Z.; Vollrath, F. Nature 2002, 418, 741. (6) Altman, G. H.; Horan, R. L.; Lu, H. H.; Moreau, J.; Martin, I.; Richmond, J. C.; Kaplan, D. L. Biomaterials 2002, 23, 4131-4141. (7) Freddi, G.; Tsukada, M.; Beretta, S. J. Appl. Polym. Sci. 1999, 71, 1563-1571. (8) Liang, C. X.; Hirabayashi, K. J. Appl. Polym. Sci. 1992, 45, 19371943. (9) Freddi, G.; Romano, M.; Massafra, M. R.; Tsukada, M. J. Appl. Polym. Sci. 1995, 56, 1537-1545. (10) Yang, G.; Zhang, L.; Liu, Y. J. Membr. Sci. 2000, 177, 153-161. (11) Chen, X.; Li, W.; Yu, T. J. Polym. Sci. B: Polym. Phys. 1997, 35, 2293-2296. (12) Chen, X.; Li, W.; Shao, Z.; Zhong, W.; Yu, T. J. Appl. Polym. Sci. 1999, 73, 975-980. (13) Chen, X.; Li, W.; Zhong, W.; Lu, Y.; Yu, T. J. Appl. Polym. Sci. 1997, 65, 2257-2262. (14) Tanaka, T.; Suzuki, M.; Kuranuki, N.; Tanigami, T.; Yamaura, K. Polym. Int. 1997, 42, 107-111. (15) Tanaka, T.; Tanigami, T.; Yamaura, K. Polym. Int. 1998, 45, 175184. (16) Yamamura, K.; Kuranuki, N.; Suzuki, M.; Tanigami, T.; Matsuzawa, S. J. Appl. Polym. Sci. 1990, 41, 2409-2425. (17) Tsukada, M.; Freddi, G.; Crighton, J. S. J. Polym. Sci. B: Polym. Phys. 1994, 32, 243-248. (18) Sun, Y.; Shao, Z.; Ma, M.; Hu, P.; Liu, Y.; Yu, T. J. Appl. Polym. Sci. 1997, 65, 959-966. (19) Demura, M.; Asakura, T. J. Membr. Sci. 1991, 59, 39-52. (20) Kweon, H. Y.; Park, S. H.; Yeo, Y. W.; Lee, Y. W.; Cho, C. S. J. Appl. Polym. Sci. 2001, 80, 1848-1853. (21) Kesencl, K.; Motta, A.; Fambri, L.; Migliaresi, C. J. Biomater. Sci. Polym. Ed. 2001, 12, 337-351. (22) Lee, K. Y. Fibers Polym. 2001, 2, 71-74. (23) Kim, S. W. In Biomaterials Science-An introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, CA, 1996; p 297. (24) Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N. J. Biomed. Mater. Res. 2000, 51, 343-351. (25) Griffith, L. G. Acta Mater. 2000, 48, 263-277. (26) Elbert, D. L.; Hubbell, J. A. Ann. ReV. Mater. Sci. 1996, 26, 365394. (27) Desai, N. P.; Hubbell, J. A. Biomaterials 1991, 12, 144-153. (28) Jin, H.-J.; Fridrikh, S. V.; Rutledge, G. C.; Kaplan, D. L. Biomacromolecules 2002, 3, 1233-1239. (29) Poza, P.; Pe´rez-Rigueiro, J.; Elices, M.; LLorca, J. Eng. Fract. Mech. 2002, 69, 1035-1048. (30) Kim, K.-S.; Chin, I.-J.; Yoon, J. S.; Choi, H. J.; Lee, D. C.; Lee, K. H. J. Appl. Polym. Sci. 2001, 82, 3618-3626.

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