Biomacromolecules 2004, 5, 786-792
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Structure and Properties of Silk Hydrogels Ung-Jin Kim,† Jaehyung Park,† Chunmei Li,† Hyoung-Joon Jin,†,‡ Regina Valluzzi,† and David L. Kaplan*,† Departments of Biomedical Engineering, Chemical and Biological Engineering, and Bioengineering Center, Tufts University, Medford, Massachusetts 02155, and Department of Polymer Science and Engineering, Inha University, Incheon 402-751, South Korea Received December 24, 2003; Revised Manuscript Received February 10, 2004
Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca2+, pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca2+, and addition of PEO. No change of gelation time was observed with the addition of K+. Upon gelation, a random coil structure of the silk fibroin was transformed into a β-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca2+ exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the solgel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials. Introduction Silks are protein polymers that are spun into fibers by silkworms and spiders.1 Silk proteins are usually produced within specialized glands after biosynthesis by epithelial cells, followed by secretion into the lumen of these glands where the proteins are stored prior to being spun into fibers.2 The most extensively characterized silks are from the domesticated silkworm (Bombyx mori) and from some spiders (Nephila claVipes and Araneus diadematus). Silkworm silk has been used commercially as biomedical sutures for decades and in textile production for centuries. Silkworm silk from B. mori consists primarily of two protein components. Fibroin is the structural protein of silk fibers and sericin is the water-soluble glue that binds the fibroin fibers together.3 The silk fibroin molecule consists of heavy and light chain polypeptides of ∼350 kDa and ∼25 kDa, respectively, connected by a disulfide link.4,5 The fibroin is a protein dominated in composition by the amino acids glycine, alanine, and serine, which form antiparallel β sheets in the spun fibers.6-8 Fibrous proteins such as silks exhibit impressive mechanical properties as well as biocompatibility; thus, these proteins are under study for biomaterials and scaffolds for tissue engineering.2,9-15 One important material option for bioma* Please address all correspondence to: David Kaplan, Department of Biomedical Engineering, 4 Colby Street, Medford, MA 02155, U.S.A. Phone: 617-627-3251. Fax: 617-627-3231. E-mail:
[email protected]. † Tufts University. ‡ Inha University.
terials is the formation of hydrogels, as has been extensively studied for a variety of polymers such as alginates, chitosan, and collagen.16-21 Silk fibroin solutions at concentrations e5 wt % have been previously studied with respect to hydrogel formation.22-24 The sol-gel transition depended on the concentration of the protein, temperature, and pH. Random coil to β-sheet structural transitions were noted during the process of hydrogelation. In the present study, we have studied in detail the environmental factors that influence silk fibroin sol-gel transitions. Most importantly, our ability to use osmotic stress to generate highly concentrated fibroin aqueous solutions provided the opportunity to explore these sol-gel transitions. Fibroin concentration in aqueous solution was varied by osmotic stress to avoid sudden “shocks” to the local chemical composition of the solutions as well as to achieve very high concentrations of fibroin in water to mimic the native processing of silk.25 The gelation rate of regenerated silk fibroin aqueous solutions was studied with varying concentrations of fibroin, poly(ethylene oxide) (PEO), Ca2+, K+, temperature, and pH. The salts and pH were selected as variables for study on the basis of their presence and changes in concentration in different regions of the storage glands of insects and spiders where silk proteins are processed.3,26 Temperature was selected for study as an artificial in vitro control of the process on the basis of potential impact on chain-chain interactions, as is well documented for synthetic polymers.18 PEO was selected as an internal control of osmotic effects, in combination with the osmotic stress
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induced via dialysis with poly(ethylene glycol) (PEG), to control water content.25,27 Morphological and structural changes in the gels obtained after the sol-gel transition were used to compare features to the environmental variables studied. The mechanical compressive properties of the hydrogels formed under these various conditions were also assessed. The correlations between the structures, morphology, and mechanical properties provide new insight into the role of environmental variables on the sol-gel transitions of silk fibroin proteins in aqueous solution.25 This insight also provides road maps to optimize silk structure, morphology, and function for these gels, depending on the desired material features. Experimental Section Preparation of the Silk Fibroin Aqueous Solution. Cocoons of B. mori, kindly provided by M. Tsukada (Institute of Sericulture, Tsukuba, Japan) and M. Goldsmith (University of Rhode Island), were boiled for 20 min in an aqueous solution of 0.02 M Na2CO3 and then rinsed thoroughly with distilled water to extract the gluelike sericin proteins and wax. The extracted silk fibroin was then dissolved in a 9.3 M LiBr solution at 60 °C for 4 h, yielding a 20% (w/v) solution. This solution was dialyzed in distilled water using a Slide-a-Lyzer dialysis cassette (molecular weight cutoff, MWCO, 3500, Pierce) for 2 days. The final concentration of the silk fibroin aqueous solution was about 8 wt %, which was determined by weighing the remaining solid after drying. The silk fibroin film prepared from the 8 wt % solutions was evaluated to verify the removal of the Li+ ion by X-ray photoelectron spectoscopy; no residual Li+ ion was detected. Preparation of the Concentrated Silk Fibroin Solution by Osmotic Stress. Silk fibroin aqueous solution (8 wt %, 10 mL) was dialyzed against a 10-25 wt % PEG (10 000 g/mol) solution at room temperature by using Slide-a-Lyzer dialysis cassettes (MWCO 3500). The volume ratio of PEG to the silk fibroin solution was 100:1. By osmotic stress, water molecules in the silk fibroin solution moved into the PEG solution through the dialysis membrane.28 After the required time, the concentrated silk fibroin solution was slowly collected by syringe to avoid excessive shearing and the concentration was determined. Silk fibroin aqueous solutions with a concentration less than 8 wt % were prepared by diluting the 8 wt % solutions with distilled water. All solutions were stored at 7 °C before use. Sol-Gel Transitions. A total of 0.5 mL of silk fibroin aqueous solution was placed in 2.5-mL flat-bottomed vials (diameter: 10 mm). The vials were sealed and kept at room temperature, 37, and 60 °C. Gelation time was determined when the sample had developed an opaque white color and did not fall from an inverted vial within 30 s. To investigate the effect of ions and ion concentration on the process, CaCl2 or KCl solutions were added into the silk fibroin aqueous solution to generate a final salt concentration of 2.5-30 mM. The pH of the silk fibroin solution was adjusted with HCl or NaOH solution. For the preparation of a silk fibroinPEO (900 000 g/mol) solution, the required amount of PEO solution (5 wt %) was added to a silk fibroin solution with
Figure 1. Concentration of silk fibroin solution (filled symbols) and gel (open symbols) prepared by dialysis against PEG solutions (circle, 25 wt %; square, 15 wt %; triangle, 10 wt %) at room temperature. Values are average ( standard derivation of three samples.
mild stirring for 5 min. The blend ratios of silk fibroinPEO were 100/0, 95/5, 90/10, 80/20, and 70/30 (w/w). Wide-Angle X-ray Scattering. X-ray profiles were recorded for freeze-dried silk fibroin solutions and hydrogels using a Brucker D8 X-ray diffractometer at 40 kV and 20 mA, with Ni-filtered Cu KR radiation. Scanning Electron Microscopy (SEM). Silk fibroin solutions and hydrogels were frozen at -80 °C and then lyophilized. The samples were fractured in liquid nitrogen and examined using a LEO Gemini 982 field emission gun SEM. To check for artifactual morphological changes due to freeze-drying, an alternative preparation used Karnovsky’s fixative at room temperature for 4 h. Hydrogels with and without fixative treatment showed little morphological change upon freeze-drying. Pore size was obtained by using ImageJ software developed at the U.S. National Institutes of Health. Mechanical Properties. Compression tests of hydrogels were performed on an Instron 8511 equipped with a 2.5-kN load cell at room temperature. A crosshead speed was 10 mm/min. The cross section of samples was 12 mm in diameter and 5 mm in height. The compression test was achieved conventionally as an open-sided method. The compression limit was 98% strain to protect the load cell. Five samples were evaluated for each composition. Results Concentrated Silk Fibroin Solutions. The silk fibroin aqueous solution with an initial concentration of 8 wt % was dialyzed against a 10-25 wt % PEG solution at room temperature. The silk fibroin aqueous solution was concentrated over time by osmotic stress, and concentrations of about 21 wt % were obtained after 9 h of dialysis against the 25 wt % PEG solution (Figure 1). Longer dialysis times were required to generate higher concentrations of silk fibroin aqueous solution, when lower concentrations of PEG solutions were used. Silk fibroin gels, 23-33 wt %, were spontaneously generated in the dialysis cassettes during the concentration process. These gels were transparent even after drying at room temperature and at 60 °C. Gelation of the Silk Fibroin Aqueous Solution. The influence of temperature, Ca2+ and K+ concentrations, pH,
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Figure 2. Gelation time of silk fibroin aqueous solutions at various temperatures (pH 6.5-6.8, without ions). Values are average ( standard derivation of seven samples.
and PEO concentration was investigated on the gelation of silk fibroin aqueous solutions. Figure 2 illustrates the gelation time of the silk fibroin aqueous solution (pH 6.5-6.8) at various temperatures. The gelation time of the silk fibroin aqueous solution decreased with increase in the fibroin content and temperature. Concurrently, a conformational change from a random coil to a β-sheet structure was observed and the formation of a β-sheet structure in the hydrogels was confirmed by X-ray diffraction as described later. Figure 3 shows the gelation time of the silk fibroin aqueous solution with different Ca2+ and K+ concentrations. The pHs of silk fibroin solutions with Ca2+ and K+ ions were 5.6-5.9 and 6.2-6.4, respectively. Ca2+ resulted in shorter gelation times, whereas there was no change in gelation time with the addition of K+ at any temperature. These results with regenerated silkworm fibroin differ from prior studies in which K+ ions added to solutions of spider silk influenced aggregation and precipitation of the protein, whereas there was no rheological change after addition of Ca2+ ions.26 Figure 4 shows the gelation time of the silk fibroin aqueous solution (4 wt %) at different pHs. The gelation time decreased significantly with a decrease in pH. This behavior is similar to that observed for the silk from the spider, A. diadematus, which gels at pH 5.5 but behaves as a viscous liquid at pH 7.4.29 Figure 5 shows the gelation time of the silk fibroin aqueous solution (4 wt %) with different PEO contents. By adding PEO solution, the pH decreased slightly to the range 6.1-6.4. The gelation time was significantly reduced with the addition of only 5% PEO, whereas there
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was no difference in gelation time when the concentration was above 5%. Structural Analysis of Hydrogels. Structural changes in the silk fibroin were determined by X-ray diffraction. Figure 6 shows X-ray profiles of freeze-dried silk fibroin solutions and hydrogels prepared from silk fibroin aqueous solutions. When silk fibroin solutions were frozen at a low temperature, below the glass transition (-34 to -20 °C), the structure was not significantly changed.30 The freeze-dried silk fibroin samples exhibited a broad peak at around 20° regardless of the silk fibroin concentration, indicating an amorphous structure. Silk fibroin in aqueous solution at neutral pH exhibited a random coil conformation.3,22 All hydrogels prepared from silk fibroin solutions showed a distinct peak at 20.6° and two minor peaks at around 9 and 24°. These peaks were almost the same as those of the β-sheet crystalline structure of silk fibroin.22,31 These peaks indicate β-crystalline spacing distances of 9.7, 4.3, and 3.7 Å corresponding to 9, 20.6, and 24°, respectively. From the results of X-ray diffraction, the gelation of silk fibroin solutions induced a conformational transition from random coil to β sheet as previously reported.22-24 Morphology of Freeze-Dried Hydrogels. Morphological features of silk fibroin solutions and hydrogels were observed by SEM after freeze-drying at -80 °C. Freeze-dried silk fibroin solutions of 4-12 wt % showed leaflike morphologies (Figure 7a; 8 wt %). Freeze-dried silk fibroin solutions of 16 wt % (Figure 7b) and 20 wt % exhibited network and spongelike structures with pore sizes of 5.0 ( 4.2 µm and 4.7 ( 4.0 µm, respectively. Figure 7c-h shows SEM images of freeze-dried hydrogels prepared from silk fibroin aqueous solutions at different temperatures. Freeze-dried hydrogels prepared from the 4 wt % silk fibroin solution showed leaflike morphologies and interconnected pores regardless of temperature (Figure 8a), and at higher fibroin concentrations than 4 wt %, spongelike structures were observed. The pore sizes of freeze-dried hydrogels (4 wt %, pore sizes of freeze-dried hydrogels with Ca2+ were larger than those of freeze-dried hydrogels prepared from silk fibroin aqueous solutions without Ca2+ ions. Interestingly, the pore size was larger in freeze-dried hydrogels with the same silk fibroin concentration with an increase in Ca2+ concentrations (Figure 8d,e). In contrast to freeze-dried hydrogels with Ca2+, pore sizes of freeze-dried hydrogels with K+ showed sizes similar to those of freeze-dried hydrogels prepared from the silk fibroin aqueous solutions (Figure 8f, see Figure 7e). These results imply that Ca2+ was more effective in inducing interactions among the silk fibroin chains than K+. This result
Gelation occurs because of the formation of inter- and intramolecular interactions among the protein chains, including hydrophobic interactions and hydrogen bonds.22-24 With an increase in fibroin content and temperature, interactions among the fibroin chains increases. Silk fibroin molecules are thereby able to interact more readily, leading to physical cross-links. The concentration of the Ca2+ ion in the silkworm (B. mori) increases from 5 to 15 mM as silk progresses toward the spinneret, while the K+ ion is present at 5-8 mM.3 Several calcium salts are known to dissolve silk fibroin because of strong interactions with the fibroin.32,33 Rheological measurements of dilute solutions of silk fibroin from B. mori revealed that the protein chains tend to form clusters by ionic interaction between COO- ions of amino acid side chains in the fibroin and divalent ions such as Ca2+ or Mg2+.34 Through these interactions, the pH of silk fibroin solutions with Ca2+ ions was significantly lower than that of silk fibroin solutions in the absence of these ions, whereas the addition of monovalent ions such as K+ showed only a slight decrease of pH. With lower pH, repulsion among silk fibroin molecules decreases and interactions among the chains is easier, resulting in stronger potential for the formation of a β-sheet structure through hydrophobic interac-
Figure 6. X-ray profiles of (a) freeze-dried silk fibroin solutions and (b) hydrogels prepared from the silk fibroin aqueous solution at 60 °C.
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Figure 7. SEM images for the freeze-dried silk fibroin solutions (a and b) and hydrogels prepared at various temperatures (room temperature, c and d; 37 °C, e and f; 60 °C, g and h). Fibroin concentration: 8 wt % (upper panels) and 16 wt % (lower panels).
Figure 8. SEM images for freeze-dried hydrogels prepared with various ion concentrations at 37 °C (a, 4 wt % without ion; b, 4 wt % with 5 mM Ca2+; c, 4 wt % with 10 mM K+; d, 8 wt % with 5 mM Ca2+; e, 8 wt % with 30 mM Ca2+; f, 8 wt % with 10 mM K+).
Figure 9. Compressive strength (a), compressive modulus (b), and strain at failure (c) of hydrogels prepared from silk fibroin aqueous solutions at various temperatures. Asterisks: the hydrogel prepared at 60 °C with the silk fibroin concentration of 16 wt % was not crushed under the conditions used in the study. Values are average ( standard derivation of five samples.
tions. A pH near the isoelectric point (pI ) 3.8-3.9)22,24 of silk fibroin accelerated the sol-gel transition of silk fibroin aqueous solutions in a fashion similar to that of other proteins that aggregate near their isoelectric points. These outcomes may reflect subtle differences in how different silk proteins from different organisms utilize physiologically relevant ions to facilitate sol-gel transitions.
Divalent ions may induce aggregation of silk fibroin molecules by ionic interactions with negatively charged amino acids present particularly near the chain ends of the heavy chain fibroin. The lack of response to different concentrations of Ca2+ may suggest a broad window of response physiologically or perhaps a role for combinations of ions to fully control this process in vivo or in vitro.
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Additional studies will be required to elucidate these relationships, particularly when considered in concert with observations on domain mapping of silks related to processing environments.35 The movement of water from the silk fibroin molecules to the hydrophilic PEO facilitates inter- and intramolecular interactions among the protein molecules and the subsequent formation of the β-sheet structure. This transition is evident with silk on the basis of our recent mechanistic understanding of the process.25 These transitions can be induced by direct addition of PEO into the fibroin aqueous solutions or via separation from the aqueous solutions across a dialysis membrane (with PEG). Thus, direct contact between the protein and the PEO is not required, only the facilitation of water transport from the protein to the PEO/PEG to drive the sol-gel transition. Conclusions From the primary sequence, the silkworm silk fibroin heavy chain is composed of seven internal hydrophobic blocks and seven much smaller internal hydrophilic blocks, with two large hydrophilic blocks at the chain ends.5,25 The percentage of hydrophobic residues in silk fibroin is 79%,36 and the repetitive sequence in hydrophobic residues consists of GAGAGS peptides that dominate the β-sheet structure forming crystalline regions in silk fibroin fibers and films.37 The formation of these β sheets results in insolubility in water.38 Hydrophobic regions of silk fibroin in aqueous solution assemble physically by hydrophobic interactions and eventually organize into hydrogels.25 Silk fibroin, Ca2+, and PEO concentrations; temperature; and pH affected the gelation of the silk fibroin aqueous solutions. With increase in the fibroin content and temperature, physical cross-linking among silk fibroin molecules formed more easily. Ca2+ ions accelerated these interactions, presumably through the hydrophilic blocks at the chain ends. The decrease in pH and the addition of a hydrophilic polymer decreased repulsion between silk fibroin molecules and promoted the desorption of water from the protein, resulting in shorter gelation times. Upon gelling, a conformational transition from random coil to β-sheet structure was induced and promoted the insolubility and stability of silk fibroin hydrogels in water. Silk fibroin hydrogels had network and spongelike structures. The pore size was smaller with increased silk fibroin concentration and gelation temperature. Freeze-dried hydrogels showed larger pore sizes with increases in Ca2+ concentrations than freeze-dried hydrogels prepared from silk fibroin aqueous solutions at the same fibroin content. The compressive strength and modulus of hydrogels prepared from the silk fibroin aqueous solution without ions increased with increase in the protein concentration and gelation temperature. Hydrogels from natural polymers, such as collagen, hyaluronate, fibrin, alginate and chotosan, have found numerous applications in tissue engineering as well as in drug delivery. However, they generally offer a limited range of mechanical properties.17 In contrast, silk fibroin provides an important set of material options in the fields of controlled release, biomaterials, and scaffolds for tissue engineering
because of combination with impressive mechanical properties, biocompatibility, biodegradability, and cell interaction.2,9-15 With more complete mapping of the influence of solution variables on structure, morphological transitions, and functional properties, we anticipate new options for the biomimetic processing of silk fibroin into new materials. Acknowledgment. We thank the NIH (R01EB003210) and the Air Force Office of Scientific Research for support of this program. References and Notes (1) Kaplan, D. L.; Adams, W. W.; Farmer, B.; Viney, C. Silk: biology, structure, properties and genetics. In Silk polymers: materials science and biotechnology; Kaplan, D. L., Adams, W. W., Farmer, B., Viney, C., Eds.; ACS Symposium Series 544; American Chemical Society: Washington, DC, 1994; p 2. (2) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Biomaterials 2003, 24, 401416. (3) Magoshi, J.; Magoshi, Y.; Becker, M. A.; Nakamura, S. In Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press: NewYork, 1996; Vol. 1, p 667. (4) Tanaka, K.; Kajiyama, N.; Ishikura, K.; Waga, S.; Kikuchi, A.; Ohtomo, K.; Takagi, T.; Mizuno, S. BBA Protein Struct. M. 1999, 1432, 92-103. (5) Zhou, C. Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z. G. Nucleic Acids Res. 2000, 28, 2413-2419. (6) 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. (7) He, S. J.; Valluzzi, R.; Gido, S. P. Int. J. Biol. Macromol. 1999, 24, 187-195. (8) Asakura, T.; Yao, J.; Yamane, T.; Umemura, K.; Ulrich, A. S. J. Am. Chem. Soc. 2002, 124, 8794-8795. (9) Cappello, J.; Crissman, J. W.; Crissman, M.; Ferrari, F. A.; Textor, G.; Wallis, O.; Whitledge, J. R.; Zhou, X.; Burman, D.; Aukerman, L.; Stedronsky, E. R. J. Controlled Release 1998, 53, 105-117. (10) Foo, C. W. P.; Kaplan, D. L. AdV. Drug DeliVery ReV. 2002, 54, 1131-1143. (11) Dinerman, A. A.; Cappello, J.; Ghandehari, H.; Hoag, S. W. J. Controlled Release 2002, 82, 277-287. (12) Megeed, Z.; Cappello, J.; Ghandehari, H. AdV. Drug DeliVery ReV. 2002, 54, 1075-1091. (13) Petrini, P.; Parolari, C.; Tanzi, M. C. J. Mater. Sci.: Mater. Med. 2001, 12, 849-853. (14) Altman, G. H.; Horan, R. L.; Lu, H. H.; Moreau, J.; Martin, I.; Richmond, J. C.; Kaplan, D. L. Biomaterials 2002, 23, 4131-4141. (15) Panilaitis, B.; Altman, G. H.; Chen, J. S.; Jin, H. J.; Karageorgiou, V.; Kaplan, D. L. Biomaterials 2003, 24, 3079-3085. (16) Knight, D. P.; Nash, L.; Hu, X. W.; Haffegee, J.; Ho, M. W. J. Biomed. Mater. Res. 1998, 41, 185-191. (17) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869-1879. (18) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliVery ReV. 2002, 54, 37-51. (19) Hoffman, A. S. AdV. Drug DeliVery ReV. 2002, 43, 3-12. (20) Shin, H.; Jo, S.; Mikos, A. G. Biomaterials 2003, 24, 4353-4364. (21) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337-4351. (22) Ayub, Z. H.; Arai, M.; Hirabayashi, K. Biosci. Biotechnol. Biochem. 1993, 57, 1910-1912. (23) Hanawa, T.; Watanabe, A.; Tsuchiya, T.; Ikoma, R.; Hidaka, M.; Sugihara, M. Chem. Pharm. Bull. 1995, 43, 284-288. (24) Kang, G. D.; Nahm, J. H.; Park, J. S.; Moon, J. Y.; Cho, C. S.; Yeo, J. H. Marcromol. Rapid Commun. 2000, 21, 788-791. (25) Jin, H. J.; Kaplan, D. L. Nature 2003, 424, 1057-1061. (26) Chen, X.; Knight, D. P.; Vollrath, F. Biomacromolecules 2002, 3, 644-648. (27) Jin, H. J.; Fridrikh, S. V.; Rutledge, G. C.; Kaplan, D. L. Biomacromolecules 2002, 3, 1233-1239. (28) Parsegian, V. A.; Rand, R. P.; Fuller, N. L.; Rau, D. C. In Methods in Enzymology; Packer, L., Ed.; Academic Press: 1986; Vol. 127, p 400. (29) Vollrath, F.; Knight, D. P.; Hu, X. W. Proc. R. Soc. London, Ser. B 1998, 265, 817-820.
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(30) Li, M.; Lu, S.; Wu, Z.; Yan, H.; Mo, J.; Wang, L. J. J. Appl. Polym. Sci. 2001, 79, 2185-2191. (31) Asakura, T.; Kuzuhara, A.; Tabeta, R.; Saito, H. Macromolecules 1985, 18, 1841-1845. (32) Ajisawa, A. J. Nippon Sanshigaku Zasshi 1998, 67, 91-94. (33) Ha, S. W.; Park, Y. H.; Hudson, S. M. Biomacromolecules 2003, 4, 488-496. (34) Ochi, A.; Hossain, K. S.; Magoshi, J.; Memoto, N. Biomacromolecules 2002, 3, 1187-1196.
Kim et al. (35) Bini, E.; Knight, D. P.; Kaplan, D. L. J. Mol. Biol. 2004, 335, 2740. (36) Braun, F. N.; Viney, C. Int. J. Biol. Macromol. 2003, 32, 59-65. (37) Mita, K.; Icimura, S.: James, T. C. J. Mol. EVol. 1994, 38, 583592. (38) Valluzzi, R.; Szela, S.; Avtges, P.; Kirschner, D.; Kaplan, D. L. J. Phys. Chem. B 1999, 103, 11382-11392.
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