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Electrospinning Bombyx mori Silk with Poly(ethylene oxide) Hyoung-Joon Jin,† Sergey V. Fridrikh,‡ Gregory C. Rutledge,‡ and David L. Kaplan*,† Department of Chemical & Biological Engineering, Bioengineering Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, and Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received May 30, 2002; Revised Manuscript Received July 25, 2002
Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900 000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein. Introduction Electrospinning for the formation of fine fibers has been actively explored recently for applications such as high-performance filters1,2 and biomaterial scaffolds for cell growth, vascular grafts, wound dressings, or tissue engineering.2-6 These applications benefit from the high surface area of the fibers. In this electrostatic technique, a strong electric field is generated between a polymer solution contained in a glass syringe with a capillary tip and a metallic collection screen. When the voltage reaches a critical value, the charge overcomes the surface tension of the deformed drop of suspended polymer solution formed on the tip of the syringe, and a jet is produced. The electrically charged jet thins under electrohydrodynamic forces,7 and under certain operating conditions undergoes a series of electrically induced bending instabilities during passage to the collection screen that results in extensive stretching.8-12 This stretching process is accompanied by the rapid evaporation of the solvent and results in a reduction in the diameter of the jet.10,13-16 The dry fibers accumulated on the surface of the collection screen form a nonwoven mesh of nanometer to micrometer diameter fibers even when operating with aqueous solutions at ambient temperature and pressure. The electrospinning process can be adjusted to control fiber diameter to some extent by varying the charge density and polymer solution concentration, while the duration of electrospinning controls the thickness of the deposited mesh.10-17 Protein fiber spinning in nature, such as for silkworm and spider silks, is based on the formation of concentrated † ‡
Tufts University. Massachusetts Institute of Technology.
solutions of metastable lyotropic phases that are then forced through small spinnerets into air.18 The fiber diameters produced in these natural spinning processes range from tens of micrometers in the case of silkworm silk to micrometers to submicrometers in the case of spider silks.18 The production of fibers from protein solutions has typically relied upon the use of wet or dry spinning processes.19,20 Electrospinning offers an alternative approach to protein fiber formation that can potentially generate very fine fibers. This would be a useful feature based on the potential role of these types of fibers in some applications such as biomaterials and tissue engineering.5,6,21 Electrospinning has been utilized to generate nanometer diameter fibers from type I collagen,5,6 recombinant elastin protein,21 and silklike protein.22-24 Zarkoob et al.25 have also reported that silkworm silk from Bombyx mori cocoons and spider dragline silk from Nephila claVipes silk can be electrospun into nanometer diameter fibers if first solubilized in hexafluoro-2-propanol (HFIP). Silk is a well described natural fiber produced by the silkworm, B. 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 forms the thread core and gluelike proteins termed sericin that surround the fibroin fibers to cement them together. The fibroin is a highly insoluble protein containing up to 90% of the amino acids glycine, alanine, and serine leading to antiparallel β-pleated sheet formation in the fibers.26 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 these fibers as well as their biocompatibility.18,27
10.1021/bm025581u CCC: $22.00 © 2002 American Chemical Society Published on Web 08/28/2002
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In the present study, we sought to develop silk electrospinning approaches that would address two major goals. First, we wanted to avoid problems encountered with conformational transitions of silkworm fibroin during solubilization and reprocessing from aqueous solution to generate new fibers and films. This problem derives from the formation of insoluble β-sheets. This process can result in embrittled materials that become difficult to utilize in subsequent studies. To overcome this problem we determined that blending would be a suitable option and we report on the incorporation of poly(ethylene oxide) (PEO) with silk in the electrospinning process. Second, we wanted to enhance the potential biocompatibility of the electrospun silk fibers by avoiding the use of organic solvents that can pose problems when the processed materials are exposed to cells in vitro or in vivo. Thus, we desired to develop an all-aqueous process for silk electrospinning in combination with PEO. PEO is well-documented as a biocompatible polymer29-31 and has been successfully blended with collagen in electrospinning.6,28
Jin et al.
Figure 1. Viscosities of silk/PEO blends in water.
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 (Aldrich) was used in blending. Preparation of Regenerated B. mori Silk Fibroin Solutions. B. mori silk fibroin was prepared as follows as a modification of our earlier procedure.32 Cocoons were boiled for 30 min in an aqueous solution of 0.02 M Na2CO3 and then rinsed thoroughly with water to extract the gluelike sericin proteins. The extracted silk was then dissolved in 9.3 M LiBr solution at 60 °C yielding a 20% (w/v) solution. This solution was dialyzed in water using a Slide-a-Lyzer dialysis cassette (Pierce, MWCO 2000). The final concentration of aqueous silk solution was 3.0-7.2 wt %, which was determined by weighing the remaining solid after drying. HFIP silk solution (1.5 wt %) was prepared by dissolving the lyophilized aqueous silk solution in HFIP. Preparation of Spinning Solutions. Silk/PEO blends in water were prepared by adding PEO (900 000 g/mol) directly into the silk aqueous solutions generating 4.8-8.8 wt % silk/ PEO solutions. Silk solutions in HFIP (1.5 wt %) and PEO (4.0 wt %) solution in water, respectively, were also prepared as controls for comparisons with the blends. Silk solutions in HFIP were prepared by dissolving the lyophilized silk fibroin in HFIP at room temperature. The viscosity and conductivity of the solutions were measured with a Couette viscometer (Bohlin V88), with a shear rate from 24.3 to 1216 per second, and a Cole-Parmer conductivity meter (19820) at room temperature, respectively. Electrospinning. Electrospinning was performed with a steel capillary tube with a 1.5 mm inside diameter tip mounted on an adjustable, electrically insulated stand as described earlier.13 The capillary tube was maintained at a high electric potential for electrospinning and mounted in the parallel plate geometry. The capillary tube was connected to a syringe filled with 10 mL of a silk/PEO blend or silk
Figure 2. Scanning electron micrograph of electrospun fibers (blend 5) (background) and sericin extracted native Bombyx mori silk fibers (foreground). (Scale bar ) 50 µm.)
solution. A constant volume flow rate was maintained using a syringe pump, set to keep the solution at the tip of the tube without dripping. The electric potential, solution flow rate, and the distance between the capillary tip and the collection screen were adjusted so that a stable jet was obtained. By variation of the distance between the capillary tip and the collection screen, either dry or wet fibers were collected on the screen. Scanning Electron Microscopy (SEM). Images of electrospun fibers were obtained with a LEO Gemini 982 field emission gun scanning electron microscope. Average fiber diameters were determined by measuring 50 fibers selected randomly from each electrospun blend. FT-IR. Infrared spectra were measured with an attenuated total reflectance Fourier transform (ATR-FTIR) (Bruker Equinox 55) spectrophotometer. Each spectrum was acquired in transmittance mode on a ZnSe ATR crystal cell by accumulation of 256 scans with a resolution of 4 cm-1 and a spectral range of 4000-600 cm-1. X-ray Photoelectron Spectroscopy (XPS). A Surface Science, Inc., model SSX-100 X-ray photoelectron spectrometer was used to analyze the surface of the silk films to estimate the surface density of protein versus PEO. Survey
Electrospinning Silk
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Figure 3. Scanning electron micrographs of electrospun fibers: (a) blend 1 (10 µm), (b) blend 2 (5 µm), (c) blend 3 (2 µm), (d) blend 4 (5 µm), (e) blend 5 (2 µm), (f) blend 6 (2 µm), and (g) blend 7 (20 µm). Numbers in parentheses are scale bars.
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Table 1. Characteristics of Silk, PEO, Silk/PEO Blend Solutions, Spinning Conditions, and Fiber Features
silk blend 1 blend 2 blend 3 blend 4 blend 5 blend 6 blend 7 PEO a
initial concn of
PEO ratio to silk
total concn
conductivity
applied field
av fiber diameter
silk solutions (%)
(PEO/silk)
(%)
(µS)
strength (kV/cm)
(nm) ((std dev)
7.2 7.2 7.2 6.3 6.0 5.3 4.1 3.0
1/3 1/4 1/4 1/3 1/3 1/2 2/3
7.2 8.8 (1.6a) 8.3 (1.1a) 7.3 (1.0a) 7.4 (1.4a) 6.6 (1.3a) 5.8 (1.7a) 4.8 (1.8a) 4.0
240.0 216.5 191.9 185.0 209.0 182.2 175.1 154.3 61.3
0.50 0.60 0.60 0.53 0.55 0.55 0.55 0.60
840 ( 80 740 ( 150 700 ( 100 730 ( 50 720 ( 100 850 ( 60 880 ( 50 410 ( 90
Weight percent of added PEO.
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. Results and Discussion Preparation of Silk/PEO Blend Solutions. Under shear conditions the amount of β-sheet precipitate formed from aqueous fibroin solutions is affected by the solution temperature, fibroin concentration, and stirrer rotation speed.33 Therefore, in the present study, all blend systems were gently mixed at low temperature, 4 °C in water. This procedure was used to maintain low shear in the concentrated fibroin solution to avoid premature induction of crystallization.34 Shear and elongation play important roles in the natural process of silk fiber formation.35 Despite these precautions, the silk fibroin solutions used in the present experiments exhibited the formation of some β-sheet precipitates36 even at low concentration, 3 wt % silk, during stirring at room temperature for blending. If the silk solutions were stored at room temperature, there was a rapid increase in viscosity and subsequent conversion to a gel state. The formation of the gel state when PEO was added into the solutions was much quicker than gel formation for the silk solutions. The gelation time for the silk solutions was usually 4-6 days at room temperature, which was dependent upon concentration, versus 6-18 h for the various blends with PEO. The blend solutions were frozen to reduce the induction of crystallization. However, crystallization of silk fibroin in the blend solutions nonetheless occurred due to fluctuations in solution temperature when returning the materials to ambient temperature. To overcome these difficulties, all blend solutions were mixed and stored at 4 °C before electrospinning. Properties of Silk/PEO Solutions. Aqueous silk solutions without PEO did not electrospin; no fibers were formed because the viscosity and surface tension of the solution were not high enough to maintain a stable drop at the end of the capillary tip. Higher concentrations of silk in water to increase the viscosity of the solution resulted in gel formation. A stable drop at the end of the capillary tip was achieved once the PEO was added to the silk solution at the ratios shown in Table 1. However, adding PEO at concentrations above 2 wt % led to the formation of opaque heterogeneous solutions. Therefore blending with PEO and silk was
confined to a maximum of 1.8 wt % PEO (Table 1). The viscosity of pure silk solution was much lower than the other solutions, even at 7.2% as shown (Figure 1). The jet breaks up into droplets as a result of surface tension in the case of low viscosity liquids.7,11 For high viscosity liquids the jet does not break up but travels to the grounded target. Therefore, jet breakup depends on viscosity so that lower viscosity solutions break up into droplets more readily.11,17,23 A small portion of PEO in the silk solution increased the viscosity of the blends, and the viscosity of silk/PEO blend solutions depended on the amount of PEO. The external electric field was an important process parameter during spinning (Table 1). High electric fields can cause hysteresis between the onset and disruption of a stable jet and low plate separations can be problematic for getting the submicrometer fibers formed by a fluid instability following a jet.13 The conductivities of silk and silk/PEO blend solutions were higher than thoes of pure PEO solutions at room temperature (Table 1). With their higher conductivity, the silk/PEO blend solutions exhibited stable jet behavior at lower field strength (less than 0.6 kV/cm). Fiber Formation and Morphology of Electrospun Silk/ PEO. The addition of PEO to silk solutions generated a viscosity and surface tension suitable for electrospinning. An aluminum foil was used as the collection screen. The distance between the tip and the collector was 20 cm, and flow rate of all fluids was 0.02-0.05 mL/min. Before all solutions were electrospun, Teflon was sprayed on the collection screen to facilitate removal of the mat. As the potential difference between the capillary tip and the aluminum foil counter electrode was gradually increased 10-12 kV (E ) 0.5-0.6 kV/cm), the drop at the end of the capillary tip elongated from a hemispherical shape into a cone shape, often referred to as a Taylor cone. The applied voltages resulted in a jet being initiated near the end of the capillary tip. Prior to deposition on the collector, the jet showed a fluid instability, the rapidly whipping jet, that leads to accelerated solidification of the fluid jet and the formation of submicrometer diameter solid fibers.8-13 The morphology and diameters of the electrospun fibers were examined using high-resolution low-voltage SEM. All silk/PEO blend solutions produced fine uniform fibers with less than 800 nm (100 average fiber diameters (Table 1). The fiber diameter is compared between sericin extracted natural silkworm silk and the electrospun fibers (blend 5) in Figure 2. The diameters of electrospun fibers were 40 times
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Table 2. XPS Results from the Surfaces of Electrospun Silk, PEO, and Silk/PEO Blends O1
N1s
element
binding energy (eV)
atom %
silka blend1 blend2 blend3 blend4 blend5 blend6 blend7 PEO
530.3 530.9 530.9 531.1 530.8 530.5 530.7 531.1 531.2
24.7 27.1 26.5 25.2 28.4 26.4 26.4 29.2 37.4
a
C1s
binding energy (eV)
atom %
398.4 398.5 398.6 398.9 398.6 398.4 398.4 398.5
18.9 15.3 15.0 16.4 13.8 15.8 14.2 10.1
binding energy (eV)
atom %
O1s/C1s
N1s/C1s
284.6 284.6 284.6 284.6 284.6 284.6 284.6 284.6 284.6
56.4 57.6 58.5 58.4 57.8 57.8 59.4 60.7 62.6
0.44 0.47 0.45 0.43 0.49 0.46 0.44 0.48 0.60
0.33 0.26 0.26 0.28 0.24 0.27 0.24 0.17
silk/PEO (mol/mol) 100/0 (100/0)b 79/21 (75/25) 79/21 (80/20) 85/15 (80/20) 73/27 (75/25) 82/18 (75/25) 73/27 (67/33) 51/49 (60/40) 0/100 (0/100)
For comparison, silk electrospun from HFIP (1.5 wt %). b In parentheses, blend composition (wt %) in solution.
smaller than the diameters of the native fibers after extraction of sericin. Although a systematic study to determine the optimal electrical field and concentration for these blend electrospun fibers with regard to fiber diameter was not completed, it appeared that the thickness of the electrospun blend fibers was relatively independent of the concentration of the silk and PEO dissolved in water electrospun under these conditions. The individual electrospun fibers appeared to be randomly distributed in the nonwoven mat. Micrographs of the electrospun fibers from a silk/PEO solution in water are shown in Figure 3. XPS was used to estimate the surface composition of the mats. Table 2 shows the respective peak intensities of O1s, C1s, or N1s of PEO, silk fibroin, and silk/PEO blends from electrospun mats. The ratios of N1s/C1s and O1s/C1s of the silk mat were 0.33 and 0.44, respectively. In the case of the silk/PEO mats, N1s/C1s decreased to 0.17 at minimum and O1s/C1s increased to 0.49 at maximum. On the basis of these ratios, we can estimate the fiber composition as shown in Table 2. The compositions reflect the solution composition used in the spinning process. Since silk is relatively hydrophobic, we anticipated a lower content of silk at the fiber surface where spin from water. However, this was not the case, perhaps due to the electrospinning process itself or the small diameters of the fibers to limit hydrophobic/ hydrophilic partitioning. Solvent Treatment of Electrospun Mats. The mat was contacted with 90/10 (v/v) methanol/water for 10 min to induce crystallization of silk. The structural changes after methanol treatment were observed by ATR-FTIR. As shown in Figure 4, the structure of the fibroin protein in the electrospun fibers was predominantly random coil or silk I. This amorphous structure promotes solubility in water.37,38 After methanol treatment, the structure changed to predominantly β-sheet (Figure 4), thus exhibiting behavior traditionally seen with reprocessed silkworm silk. This observation suggests that the electrospinning process preserves the ability of this protein to self-organize into its native structure. Figure 5 shows scanning electron micrographs of electrospun fibers after methanol treatment. After methanol treatment, the surface of fibers showed roughness apparently caused by phase separation between silk fibroin and PEO, possibly due to extraction of the PEO in the methanol.39 In the aqueous solution of the silk gland, fibroin has a random coil conformation. Usually, once this fibroin is spun into a cocoon
Figure 4. FTIR-ATR spectra of electrospun mat from silk/PEO (blend 5). Solid line is as spun; dotted line is after methanol/water (90/10 v/v) treatment.
fiber, the conformation of the fibroin dramatically changes to a semicrystalline structure, consisting of approximately 55% β-sheet crystallites dispersed throughout an amorphous or less-crystalline matrix.36 In the electrospun fibers from aqueous silk/PEO blend solutions, no β-sheet structure was apparent even though the chains were elongated during electrospinning. Further study of these transitions should provide additional options for control of solubility, mechanical properties, and likely biological responses to these nanometer diameter fibers. Conclusions Electrospinning fibers from B. mori fibroin was studied with a focus on blending with PEO and all aqueous processing. To improve the processibility of silk solutions for electrospinning, while maintaining biocompatibility, PEO with molecular weight of 900 000 was successfully blended with the aqueous solution of fibroin. Successful formation of electrospun mats with less than 1 µm diameter fiber was found, with the composition reflective of the solution concentrations. Traditional silk fibroin conformational transitions were induced with methanol treatment of the fibers, suggesting that native structural features of the silk were
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Figure 5. Scanning electron micrographs of electrospun fibers after methanol treatment: (a) blend 1 (2 µm), (b) blend 2 (5 µm), (c) blend 3 (2 µm), (d) blend 4 (5 µm), (e) blend 5 (5 µm), (f) blend 6 (2 µm), and (g) blend 7 (10 µm). Numbers in parentheses are scale bars.
Electrospinning Silk
preserved in the electrospinning process. These results open new directions in the fabrication of silk-based biomaterial scaffolds for biomedical applications. Acknowledgment. We thank the NIH (R01 DE1340501) and the DoD (Air Force) for support of this program and Regina Valluzzi (Tufts University) for valuable help with SEM and FT-IR characterization. References and Notes (1) Doshi, J.; Reneker, D. H. Electrospinning Process and Applications of Electrospun Fibers. J. Electrostat. 1995, 35, 151-160. (2) Bognitzki, M.; Czado, W.; Frese, T.; Schaper, A.; Hellwig, M.; Steinhart, M.; Greiner, A.; Wendorff, J. H. Nanostructured fibers via electrospinning. AdV. Mater. 2001, 13, 70-72. (3) Stitzel, J. D.; Pawlowski, K. J.; Bowlin, G. L.; Wnek, G. E.; Simpson, D. G.; Bowlin, G. L. Arterial smooth muscle cell proliferation on a novel biomimicking, biodegradable vascular graft scaffold. J. Biomater. Appl. 2001, 16, 22-33. (4) Boland, E. D.; Bowlin, G. L.; Simpson D. G.; Wnek, G. E. Electrospinning of tissue engineering scaffolds. Polym. Mater.: Sci. Eng. 2001, 85, 51-52. (5) Matthews, J. A.; Simpson, D. G.; Wnek, G. E.; Bowlin G. L. Electrospinning of Collagen Nanofibers. Biomacromolecules 2002, 3, 232-238. (6) Huang, L.; Nagapudi, K.; Apkarian, R. P.; Chaikof E. L. Engineered Collagen-PEO Nanofibers and Fabrics. J. Biomater. Sci., Polym. Ed. 2001, 12, 979-93. (7) Hohman, M. M.; Shin, M.; Rutledge, G. C.; Brenner, M. P. Electrospinning and electrically forced jets. II. Applications. Phys. Fluids 2001, 13, 2221-2236. (8) Baumgarten, P. K. J. Colloid Interface Sci. 1971, 36, 71. (9) Reneker, D. H.; Yarin, A. L.; Fong, H.; Koombhongse, S. Bending instability of electrically charged liquid jets of polymer solutions in electrospinning. J. Appl. Phys. 2000, 87, 4531-4547. (10) Shin Y. M.; Hohman M. M.; Brenner M. P.; Rutledge G. C. Electrospinning: A whipping fluid jet generates submicron polymer fibers. Appl. Phys. Lett. 2001, 78, 1149-1151. (11) Hohman, M. M.; Shin M.; Rutledge G.; Brenner M. P. Electrospinning and electrically forced jets. I. Stability theory. Phys. Fluids 2001, 13, 2201-2220. (12) Yarin, A. L.; Koombhongse, S.; Reneker, D. H. Bending instability in electrospinning of nanofibers. J. Appl. Phys. 2001, 89, 30183026. (13) Shin, Y. M.; Hohman, M. M.; Brenner M. P.; Rutledge G. C. Experimental Characterization of Electrospinning: the Electrically Forced Jet and Instabilities. Polymer 2001, 42 9955-9967. (14) Fong H.; Chun I.; Reneker D. H. Beaded nanofibers formed during electrospinning. Polymer 1999, 40, 4585-4592. (15) Jaeger R.; Scho¨nherr, H.; Vancso, G. J. Chain packing in electrospun poly(ethylene oxide) visualized by atomic force microscopy. Macromolecules 1996, 29, 7634-7636. (16) Deitzel J. M.; Kleinmeyer, J. D.; Hirvonen J. K.; Tan N. C. B. Controlled deposition of electrospun poly(ethylene oxide) fibers. Polymer 2001, 42, 8163-8170. (17) Deitzel J. M.; Kleinmeyer J.; Harris D.; Tan N. C. B. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 2001, 42, 261-272. (18) Bunning, T. J.; Jiang, H.; Adams, W. W.; Crane, R. L.; Farmer, B.; Kaplan, D.; Application of Silk. In Silk Polymers; Material Science and Biotechnology; Kaplan, D. L., Adams, W. W., Farmer, B., Viney, C., Eds.; ACS Symposium Series 544; Oxford University Press: Charlottesville, VA, 1993; pp 351-358. (19) Martin, D. C.; Tao, J.; Buchko, C. J. Processing and Characterization of Protein Polymers. In Protein-Based Materials; McGrath, K., Kaplan, D., Eds.; Birkhauser: Boston, MA, 1997; pp 339-370.
Biomacromolecules, Vol. 3, No. 6, 2002 1239 (20) Hudson, S. M. The Spinning of Silk-like Proteins into Fibers. In Protein-Based Materials; McGrath, K., Kaplan, D., Eds.; Birkhauser: Boston, MA, 1997; pp 313-337. (21) Huang, L.; McMillan, R. A.; Apkarian, R. P.; Pourdeyhimi, B.; Conticello, V. P.; Chaikof, E. L. Generation of synthetic elastinmimetic small diameter fibers and fiber networks. Macromolecules 2000, 33, 2989-2997. (22) Anderson, J. P.; Nilsson, S. C.; Rajachar, R. M.; Logan, R.; Weissman, N. A.; Martin, D. C. Biomolecular materials by design. In BioactiVe Silk-like Protein Polymer Films on Silicon DeVices; Alper, M., Bayley, H., Kaplan, D., Navia, M., Eds.; Materials Research Society: Pittsburgh, PA, 1994; Vol. 330, pp 171-177. (23) Buchko C. J.; Chen L. C.; Shen Y.; Martin D. C. Processing and microstructural characterization of porous biocompatible protein polymer thin films. Polymer 1999, 40, 7397-7407. (24) Buchko, C. J.; Kozloff K. M.; Martin D. C. Surface characterization of porous, biocompatible protein polymer thin films. Biomaterials 2001, 22, 1289-1300. (25) Zarkoob, S.; Reneker, D. H.; Eby, R. K.; Hudson, S. D.; Ertley, D.; Adams, W. W. Generation of synthetic elastin-mimetic small diameter fibers and fiber networks. Polym. Prepr. (Am. Chem. Soc., DiV. Polym. Chem.) 1998, 39, 244-245. (26) Asakura, T.; Kaplan, D. L. Silk Production and Processing. In Encylopedia of Agricultural Science; Arntzen, C. J., Ritter, E. M., Eds.; Academic Press: New York, 1994; Vol. 4, pp 1-11. (27) Kaplan, D. L.; Mello, C. M.; Arcidiacono, S.; Fossey, S.; Senecal, K.; Muller, W. Silk. In Protein-Based Materials; McGrath, K., Kaplan, D., Eds.; Birkhauser: Boston, MA, 1997; pp 103-131. (28) Huang, L.; Apkarian, R. P.; Chaikof E. L. High-Resolution Analysis of Engineered Type I Collagen Nanofibers by Electron Microscopy. Scanning 2001, 23, 372-75. (29) Kim, S. W. Nonthrombogenic Treatments and Stragegies. In Biomaterials Science: An introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: New York, 1996; pp 297-308. (30) Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N. Poly(ethylene glycol)coated biocompatible surfaces. J. Biomed. Mater. Res. 2000, 51, 343351. (31) Griffith, L. G. Polymeric Biomaterials, Acta Mater. 2000, 48, 263277. (32) Sofia, S.; McCarthy, M. B.; Gronowicz G.; Kaplan, D. L. Functionalized silk-based biomaterials for bone formation. J. Biomed. Mater. Res. 2001, 54, 139-148. (33) Trabbic, K. A.; Yager, P. Comparative Structural Characterization of Naturally- and Synthetically-Spun Fibers of Bombyx mori Fibroin. Macromolecules 1998, 31, 462-471. (34) Willcox, P. J.; Gido S. P.; Muller W.; Kaplan D. L. Evidence of a Cholesteric Liquid Crystalline Phase in Natural Silk Spinning Process. Macromolecules 1996, 29, 5106-5110. (35) Inoue, S.-I.; Magoshi J.; Tanaka, T.; Magoshi, Y.; Becker, M. Atomic Force Microscopy: Bombyx mori Silk Fibroin Molecules and Their Higher Order Structure. J. Polym. Sci., Part B: Polym. Phys. 2000, 38, 1436-1439. (36) Kobayashi, M.; Tanaka, T.; Inoue, S.; Tsuda, H.; Magoshi, J.; Magoshi, Y.; Becker, M. A. Rheological Behavior of Silk Fibroin Aqueous Solution: Gel-Sol Transition and Fiber Formation. Polym. Prepr. 2001, 42, 294-295. (37) Valluzzi, R.; Szela, S.; Avtges, P.; Kirschne, D.; Kaplan, D. Methionine Redox Controlled Crystallization of Biosynthetic Silk Spidroin. J. Phys. Chem. B 1999, 103, 11382-11392. (38) Wilson, D.; Valluzzi, R.; Kaplan, D. Conformational Transitions in Model Silk Peptides. Biophys. J. 2000, 78, 2690-2701. (39) Vandermiers, C.; Damman, P.; Dosiere, M. Static and quasielastic light scattering from solutions of poly(ethylene oxide in methanol). Polymer 1998, 39, 5627-5631.
BM025581U
