Dissolution of Bombyx mori Silk Fibroin in the Calcium Nitrate

Biomacromolecules 2003, 4, 488-496

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Dissolution of Bombyx mori Silk Fibroin in the Calcium Nitrate Tetrahydrate-Methanol System and Aspects of Wet Spinning of Fibroin Solution Sung-Won Ha,† Young H. Park,‡ and Samuel M. Hudson*,† Fiber and Polymer Science Program, North Carolina State University, Raleigh, North Carolina 27695-8301, and Department of Natural Fiber Science, Seoul National University, Suwon 441-744, Korea Received June 17, 2002; Revised Manuscript Received January 17, 2003

There are still several problems associated with the spinning of dialyzed silk fibroin solutions. In this work some of these problems have been examined. The calcium nitrate tetrahydrate-methanol system was used to dissolve the silk fibroin. A compositional phase diagram was constructed at various concentrations of the solvent system. Regenerated fibroin powders from undialyzed fibroin solution in several coagulants showed different conformations. Regenerated powders from several coagulants except methanol and ethanol were resoluble in water. Atomic absorption analysis revealed that the calcium cations strongly interact with fibroin molecules in dialyzed fibroin solution, which may interfere with the regeneration of a strong fiber. Kinetic studies to determine the diffusion coefficient of methanol into dialyzed and concentrated fibroin solution were reported. The properties of both original and regenerated fibroin such as solubility in water and thermal behaviors using DSC were compared. Regenerated fibroin fiber was spun by the wet spinning method. An X-ray diffractogram showed that the regeneration process decreased the crystallinity of regenerated fibroin fiber. SEM images of the surface and cross section of the regenerated fibroin fibers were discussed. Introduction In the past few years, there have been increasing interests in the use of silk fibroin in biomedical applications1 and biotechnological fields.2-4 The reasons for this increased interest are its useful mechanical properties combined with flexibility, tissue compatibility, and high oxygen permeability in the wet condition.5,6 To utilize this material, it needs to be regenerated into desirable forms such as powder, film, fiber, or nonwoven sheets. Bombyx mori silk fibroin dissolves in neutral salt-alcohol systems without degradation of its molecular weight.7 The lithium bromide- or lithium thiocyanate-ethanol system8,9 and hexafluoroisopropyl alcohol (HFIP)10 have been widely used to dissolve silk fibroin. Mathur et al.11 studied the dissolution behavior of B. mori silk fibroin using the calcium nitrate-methanol [Ca(NO3)2-MeOH] system, and regenerated thin films were prepared from this solution and characterized by several analytical methods. This paper reports a further study of B. mori silk fibroin solution using the calcium nitrate-methanol system. The dissolution of silk fibroin as related to the roles of salt, alcohol, and water and coagulation of the fibroin solution are discussed. Several factors and problems relating to the regeneration of fibroin fibers were examined. In the case of salt-alcohol systems, the concentration of salt remaining after dialysis has not been systematically studied. Here we * To whom correspondence may be addressed. E-mail: shudson@ unity.ncsu.edu. † North Carolina State University. ‡ Seoul National University.

report that appreciable quantities of calcium remain bound to the fibroin even after 96 h of dialysis. The calcium concentration in the dialyzed fibroin solution was measured using atomic absorption spectrometry. The diffusion coefficient of the coagulant, methanol, into fibroin dope solution was calculated using the Stokes-Einstein equation. The properties of the as-spun fiber were characterized by X-ray diffraction, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). This information is useful for the design of a fiber spinning machine for silk fibroin. Experimental Section Materials. Grade 5A raw silk with an average denier of 20.86 produced in Brazil by the Fiaç aò de Seda Bratac S.A. was used for this research. Raw silk was degummed using 0.25% w/v sodium lauryl sulfate and 0.25% w/v sodium carbonate in boiling water for 1 h [bath ratio of silk to bath was 1:100 w/v (g/mL)]. After the degumming, fibroin was washed in boiling water for 1 h to remove remaining sericin and surfactants and then washed again with distilled water. Chemicals for dissolving fibroin, such as calcium nitrate and methanol, were purchased from Fisher Scientific as reagent grades and used without further purification. Preparation of Solvent System. Calcium nitrate tetrahydrate [Ca(NO3)2‚4H2O] was dissolved in methanol at room temperature with adequate stirring for 1 h. Anhydrous calcium nitrate [Ca(NO3)2], prepared by baking tetrahydrate calcium nitrate in a vacuum oven to constant weight, was also dissolved in methanol. To examine the effect of water for the dissolution of silk fibroin, defined amounts of water

10.1021/bm0255948 CCC: $25.00 © 2003 American Chemical Society Published on Web 03/15/2003

Dissolution of B. mori Silk Fibroin

were added to the anhydrous calcium nitrate to prepare solutions ranging from calcium nitrate monohydrate to calcium nitrate trihydrate. To prepare the calcium nitrate penta- to decahydrate, tetrahydrate calcium nitrate was used. Then, methanol was added to obtain a concentration range between 40% w/w and 95% w/w Ca(NO3)2‚nH2O (n is the hydration number). Preparation of Fibroin Solution. The prepared calcium nitrate-methanol solutions with various hydration numbers and concentrations ranging from 40% w/w to 95% w/w were added to 0.5 g increments of fibroin to yield fibroin concentrations of 5%, 10%, 15%, and 20% w/v. The appearances of silk fibroin in these solutions were recorded to construct a compositional phase diagram. The optimum composition for dissolving silk fibroin in this solvent system was the molar ratio of calcium:water:methanol of 1:4:2, which is used throughout the remaining experiments. Powder Preparation and Characterization by FTIR. Fibroin solution was extruded into several coagulants (methanol, acetone, and isopropyl alcohol) using a hypodermic syringe and left in the coagulants for 6 h to give enough time for solidification. Completely precipitated fibroin materials were then dried in the vacuum oven and ground in the form of fine powders. FTIR spectra of degummed silk fiber cut into small pieces and powdered fibroin were obtained using a Nicolet 510P FTIR spectrophotometer. All samples were prepared as KBr pellets. Nitrogen gas was purged into the sample chamber to avoid the complication of water peaks from interfering with the amide I peak. Sixty-four repeated scans from 4000 to 400 cm-1 were averaged. Dialysis and Concentration. A 20% w/v fibroin solution (10 mL) was dialyzed using a regenerated cellulose membrane [molecular weight cutoff (MWCO) 6000-8000] against deionized water (1 L) for 4 days to reduce the salt concentration in the fibroin solution. The deionized water was replaced to a fresh one every 24 h. Dialyzed fibroin solution was dewatered in a centrifugal filter (MWCO 30000; Spectra/Por model, supplied by Spectrum Industries, Inc.) at a constant speed of 4000 rpm for 12 h. Atomic Absorption Analysis. A Perkin-Elmer atomic absorption spectrometer, model AAnalyst 300, was used to examine the remaining calcium concentration in the dialyzed fibroin solution. To increase calcium sensitivity, 5% v/v of 10% w/v aqueous lanthanum chloride (LaCl3) solution was added to the dialyzed fibroin solution.12 A monochromatic radiation with the wavelength of 422.7 nm was chosen to detect the calcium concentration in the samples. The width of the slit was 0.7 nm. Standard solutions containing 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 ppm of calcium were prepared for the calibration. Fibroin solutions and solvents [Ca(NO3)2‚4H2O-MeOH; molar ratio of Ca2+:H2O: MeOH ) 1:4:2] dialyzed for 1-4 days were prepared as samples. To obtain a clear solution, concentrated sulfuric acid (40% v/v of each sample) was added. Tracemetal grade sulfuric acid used in this study supplied by Fisher Scientific contains less than 0.5 ppb of calcium. Coagulation Rate. To determine the coagulation rate, a glass capillary of inside diameter 1 mm and an optical

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Figure 1. Schematic of the coagulation rate experiment using capillaries.

Figure 2. Schematic of a benchtop scale extrusion apparatus for the wet spinning of fibers: A, nitrogen gas cylinder; B, polymer solution in the spin cell; C, coagulation bath; D, wash bath; E, draw rolls; F, take-up roll.

microscope were used. The apparatus illustrated in Figure 1 was used to determine the rate of diffusion of coagulant into fibroin dope. As methanol diffuses into the capillary, fibroin precipitates. The moving boundary was observed by an optical microscope equipped with an eyepiece graticule, and the boundary distances were recorded with time. Film Preparation. Dialyzed fibroin solution was cast on Plexiglass to prepare fibroin films. As-cast films were treated with methanol, ethanol, isopropyl alcohol, tert-butyl alcohol, and acetone by soaking them for 10 min. Their thermal properties using DSC and physical properties such as brittleness and resolubility in water were compared. Wet Spinning. Dialyzed and concentrated fibroin solution was extruded using a wet spinning apparatus (shown in Figure 2) with different diameter-sized spinnerets [diameters of 0.254, 0.381, and 0.508 mm; nozzle lengths/nozzle diameters (l/D) were all 2]. The pressure from the nitrogen gas cylinder was 50 psi. Pure methanol was used as a coagulant. Solidified fibroin filament in the coagulation bath was passed through the wash bath (containing water; room temperature) and then wound at a constant speed of 35 m/min. Differential Scanning Calorimetry. A Perkin-Elmer DSC 7 differential scanning calorimeter was used to study the thermal properties of the regenerated fibroin films and fiber. The measurements were run in the range between 25 and 300 °C at a heating rate of 20 °C/min. X-ray Diffraction. The X-ray scans of raw silk, degummed silk, and regenerated fibroin fiber (dry and wet) were performed with a Seimens type F X-ray diffractometer. The X-ray source was Ni-filtered Cu KR radiation (30 kV, 20 mA). The samples were mounted on aluminum frames and scanned from 5° to 50° (2θ) at a speed of 1.0°/min.

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Ha et al. Table 1. Water Solubility of Original and Regenerated Fibroin Materials fibroin materials original powder

film

Figure 3. Effect of hydration number in the Ca(NO3)2-MeOH-H2O solvent system for the dissolution of silk fibroin.

Scanning Electron Microscopy. Regenerated fibroin fibers were examined by the Hitachi S-3200N scanning electron microscope (SEM). To obtain fine images, the fiber samples were coated with gold for 2 min. Their surface, cross-sectional, and fracture images were captured. Results and Discussion Compositional Phase Diagram. Figure 3 shows the difference in dissolution of silk fibroin in the calcium nitrate-methanol solvent system with different hydration numbers of calcium nitrate. In contrast to Mathur’s report,11 the anhydrous calcium nitrate-methanol system [75% w/w Ca(NO3)2-MeOH] did not dissolve the fibroin at all while 75% w/w Ca(NO3)2‚4H2O-MeOH (calcium nitrate tetrahydrate-methanol) dissolved the fibroin completely. The most critical factors for the fibroin dissolution are the molar ratios of calcium nitrate, water, and methanol (1:4:2). The optimum ratio of 1:4:2 is also independent of the fibroin concentration up to 25% w/v fibroin. Fibroin concentrations of 5%, 10%, 15%, and 20% w/v showed similar dissolution behaviors (solubility) in various solvent concentrations. At least three water molecules per one calcium ion are needed to begin dissolution of silk fibroin. Figure 4 is the phase diagram for the dissolution of silk fibroin in the Ca(NO3)2‚ nH2O-MeOH system with various solvent concentration ranges. As shown in Figure 5, the molar ratio of methanol to water is the critical factor for dissolution when the concentration of calcium ion is considered to be 1 mol. However, the mechanism for the dissolution of silk fibroin is still uncertain. Dissolution of Fibroin in Ca(NO3)2‚4H2O-MeOH. In a related system, Aoki13 proposed that the oxygen atoms of the carbonyl groups in the amide bonds of Nylon6 make coordination bonds with calcium ions. Similar to the case of Nylon6 in CaCl2‚2H2O-MeOH solution, we assume here that the oxygen atoms of the carbonyl groups in the peptide bonds of fibroin also make coordination bonds with calcium ions. However, the dissolution mechanisms for both cases would not be the same since the optimum fibroin-calcium nitrate-methanol system requires four water molecules per one calcium ion for the complete dissolution. It seems that the water molecules act either as a swelling agent for the silk fibroin or as components of the coordination bonds.

fiber a

preparation method

water solubilitya

raw degummed regenerated in MeOH regenerated in EtOH regenerated in isopropyl alcohol regenerated in acetone treated in MeOH treated in EtOH treated in isopropyl alcohol treated in tert-butyl alcohol treated in acetone coagulated in MeOH

I I I I S S I I S S S I

S, soluble; I, insoluble.

The dissolution process is a physical phenomenon. Dissolution requires the proper ratios of calcium ions and ligands, which is also dependent on the nature of the calcium counterion. These solvents are described as salt solutions with incomplete ionic solvation sheaths.14 Dissolution of the fibroin occurs because it completes the solvation sheath of the ions. If the binding of the solvent ligands and counterions to the calcium is too strong, dissolution will not occur. Also if the solvation sheath is complete, dissolution will not occur. Water Solubility of Regenerated Fibroin Mateials. Water solubility of regenerated fibroin materials is shown in Table 1. Water-soluble fibroin materials dissolved as soon as they contacted water. Water solubility indicates that the regenerating coagulants affect the crystallinity and conformation of the fibroin. Characterization of Regenerated Fibroin Powder by FTIR. Figure 6 shows the FTIR spectra of the degummed silk fiber (fibroin) and regenerated fibroin powders in methanol, isopropyl alcohol, and acetone. The silk fiber (a) and the regenerated fibroin powder in methanol (b) show similar spectra: amide I (CdO stretching around 1645 cm-1) and amide II (secondary NH bending around 1515 cm-1) bands, which represent the β-sheet and R-form (silk I) or random coil conformation of fibroin, respectively.15 The β-sheet conformation could be confirmed by amide III bands (C-C-N bending) appearing around 1230 cm-1.16 Only the original silk showed the carbonyl stretching band at 1704 cm-1. However, this band usually disappears when silk fibroin is dissolved and regenerated. Even though the regenerated fibroin powder in methanol has amide I and amide III bands in FTIR spectra, its conformation is different from that of the original silk, possibly having more R-form (silk I) or random coil conformation. The crystallinity of the regenerated fibroin in methanol would be very low, as confirmed by the weak intensity of the amide III band around 1230 cm-1. Another difference in the FTIR spectra of (a) and (b) is the CH3 deformation bands. While the original silk shows a sharp peak at 1442 cm-1 (antisymmetric), the regenerated fibroin powder in methanol shows a weak peak at 1384 cm-1 (symmetric). This indicates the conformational change of the methyl group in the alanine residues, which is critical for



Figure 4. Dissolution phase diagram for fibroin solution in various solvent concentration ranges of Ca(NO3)2‚nH2O-MeOH. A 10% w/v fibroin concentration was used. The insoluble region of calcium nitrate is shown by a dotted line in the region nearest pure calcium nitrate.

Figure 5. Molar ratio of MeOH to H2O required for dissolution of silk fibroin when [Ca2+] is 1 mol.

the β-sheet conformation of silk fibroin.17 This may have caused a change in the crystalline structure between them. Notable differences occurred in the FTIR spectra of the regenerated fibroin powders both in isopropyl alcohol and in acetone. Both of them show a strong amide I band around 1658 cm-1, which can be assigned to the random coil conformation of fibroin, and show several weak peaks for the β-sheet conformation. In addition, the strong peaks around 1300-1400 cm-1 indicate that a considerable amount of calcium nitrate remains in these samples. Water resolubility of these samples may be explained by their conformational nature and the residual calcium.

Figure 6. FTIR spectra of degummed silk fiber (a), regenerated fibroin powders treated with MeOH (b), isopropyl alcohol (c), and acetone (d).

Dialysis and Concentration. The dialyzed fibroin solution has a unique characteristic. It is very sensitive to shear stress. The fibroin can be precipitated simply by scrubbing a drop of solution on a glass slide. This result is in accordance with the results of Iizuka8 and Yamamura et al.18 on shear-induced crystallization. In addition, the dialyzed fibroin solution seems to be somewhat unstable, in that turbidity increases with time in the absence of shear. This result is very similar

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Ha et al. Table 2. Experimental Data of Boundary Distance of Several Coagulants for 60 min at 25 °C

MeOH EtOH isopropyl alcohol tert-butyl alcohol acetone

van der Waals volume (cm3/mol)

boundary distance () (mm)

ln V

ln 2/t

21.67 31.90 42.10 52.35 39.04

2.66 1.60 1.30 0.79 1.17

3.0759 3.4626 3.7400 3.9580 3.6646

-6.23 -7.25 -7.66 -8.66 -7.87

Figure 7. Calcium concentrations either in the dialyzed fibroin solution or in the solvent [Ca(NO3)2‚4H2O-MeOH; [Ca]:[H2O]:[MeOH] ) 1:4:2] with time.

to Magoshi’s experiment using aqueous silk from the posterior part of the middle division of the silk gland of the silkworm.19 He reported that evaporation increased the fibroin concentration and caused precipitation. However, the fibroin solution we dialyzed for 4 days and transferred to a tightly sealed container also precipitated within 10 days. This may result from spontaneous nucleation of the β-form.20 However, a fibroin solution dialyzed for 1 day required more than 1 month for complete precipitation in a tightly sealed container. The remaining calcium ions may play an important role in maintaining the stability of the dialyzed fibroin solution. After 4 days of dialysis, the fibroin solution was concentrated by centrifugation since it did not have enough viscosity for a spinning dope solution. Atomic Absorption Analysis. Figure 7 shows the calcium concentration of both dialyzed fibroin solution and dialyzed solvent. Even though both samples started with the same concentration of calcium nitrate (6.1 g/10 mL), dialyzed fibroin solution contained more calcium ions after 4 days of dialysis, indicating that the calcium ions are still interacting with fibroin molecules in the dialyzed fibroin solution. Diffusion Coefficient of Methanol into Dialyzed and Concentrated Fibroin Solution. The Stokes-Einstein equation was used to calculate the diffusion coefficient of methanol into dialyzed and concentrated fibroin solution: D)

kBT 6πηR0

where D is the diffusion coefficient, kB is Boltzman’s constant, T is temperature, η is the viscosity of solvent, and R0 is the radius of the solute molecule. This equation can be applied to a small and rigid sphere solute diffusing into a solvent. Liu et al.21 using cellulose solution and Knaul and Creber22 using chitosan solution developed an equation to examine the reliability of the Stokes-Einstein equation for the coagulant and polymer solution system: ln

2 B ) A - ln V t 3

where is the boundary distance, t is time, V is the molecular volume of the coagulant, and A and B are adjustable

Figure 8. Dependence of the diffusion rate at 60 min on the molecular volume of several different coagulants at 25 °C. Table 3. Viscosity of Fibroin Dope Solution at Low Shear Rate at 25 °C rpm

torque (%)

viscosity (cP)

shear rate (1/s)

shear stress (dyn/cm2)

0.03 0.05 0.07 0.09 0.10 0.20 0.30

12.3 20.2 27.5 34.6 37.1 64.7 86.6

37776 37223 36197 35421 34183 29806 26597

0.06 0.10 0.14 0.18 0.20 0.40 0.60

22.7 37.2 50.7 63.8 68.4 119.2 159.6

constants. When ln 2/t and ln V show a linear regression for several different penetrant molecules each with different volume, the Stokes-Einstein equation can be applied. Several coagulants (methanol, ethanol, isopropyl alcohol, tert-butyl alcohol, and acetone) were examined to confirm the eligibility of the Stokes-Einstein equation for the fibroin solution and methanol system. Table 2 shows the experimental data, and Figure 8 shows the linear relationship between ln 2/t and ln V. To calculate the diffusion coefficient of methanol into dialyzed and concentrated fibroin solution, a Brookfield cone and plate viscometer, model LVDV-II+, was used to obtain the viscosity of the dialyzed and concentrated fibroin solution (Table 3). The viscosity of the fibroin solution at 25 °C and at zero shear rate obtained by extrapolation of the data was 39044 cP (Figure 9) since the viscosity of the polymer solution is linearly proportional to the shear rate at the low shear rate region of less than 100 s-1. The radius of methanol (R0 ) 2.05 × 10-8 cm) was calculated from the van der Waals volume of methanol with the assumption that metha-


Figure 9. Linear dependence of viscosity at low shear rate for the fibroin solution.

nol is a spherical molecule. The calculated diffusion coefficient of methanol into dialyzed and concentrated fibroin solution was 0.276 × 10-11 cm2/s. The concentration of fibroin in the dope solution, however, varies greatly with the centrifugation condition. The diffusion coefficient mentioned above is only for this sample. The possible viscosity of fibroin dope solution is in the range between 10000 and 40000 cP (fibroin concentration is about 20-30% w/v). Therefore, the diffusion coefficient of methanol into dialyzed and concentrated fibroin solution varies from 0.266 × 10-11 to 1.064 × 10-11 cm2/s according to the Stokes-Einstein equation. Figure 10 shows the time-dependent relationship of the moving boundary of precipitated fibroin in the capillary. The boundary distance is linearly proportional to the square root of time. Wet Spinning. The spinnerets with nozzle diameters of 0.254, 0.381, and 0.508 mm were used to extrude the dialyzed and concentrated fibroin solution into the coagulation bath containing 100% methanol. When the spinneret with a nozzle size of 0.254 mm was used, however, as soon as the pressure increased, the fibroin dope solution was precipitated and plugged the nozzle. This is probably as a result of shear-induced crystallization. In the case of 0.508 mm nozzle, the dope solution spontaneously drained into the coagulation bath without pressure. Therefore, the 0.381 mm


nozzle was used for the spinning. During the coagulation, a change in refractive index was observed at the interface between the methanol and the dope. This is thought to arise from diffusion of the salt and water from the dope into the coagulation bath. Even though the as-spun fiber was elastic in the wet state (about 50% elongation), as soon as the water evaporated, the dried fiber became brittle (less than 1.5% elongation). This brittleness resulted in unreliable tensile data. This brittle characteristic was the same as the fibroin film treated with methanol. Differential Scanning Calorimetry. Figure 11 shows DSC curves for several fibroin materials in film and fiber form. Only the original silk shows an endotherm at 100 °C, which is considered to be evaporation of water. However, the endotherms around 100 °C (105-125 °C) of all the other materials have been shifted up. Since the calcium ions in the regenerated materials were not completely removed, the elevation of the boiling point of water may have taken place. Only regenerated fibroin film (untreated), which is in an amorphous state, shows a glass transition (Tg) peak at about 181 °C and an exotherm at 227 °C. This result is in accordance with the reports of Mathur et al.11 and Magoshi et al.23,24 The Tg peak at 181 °C indicates the segmental motion of loose fibroin molecules, which mainly have a random coil conformation and small amount of R-form crystals. The exotherm at 227 °C indicates the crystallization of the fibroin random coils to β-form crystals. Since the original silk and the regenerated fibroin materials treated with methanol both have β-sheet conformations, they showed neither the Tg peaks nor the exotherms. The decomposition peaks of all the regenerated fibroin materials (endotherms around 290 °C) were shifted down compared with the original silk fiber, indicating the lower thermal stability of regenerated samples. This may be due to lower crystallinity as well as molecular weight decrease during the degumming process25 of the regenerated fibroin materials compared with the original silk fiber. Wide-Angle X-ray Diffraction. Figure 12 shows the X-ray diffractogram of raw silk, degummed silk, and regenerated fibroin fiber (dry and wet). The original silk

Figure 10. Boundary movement in the coagulation process of fibroin solution. The concentration of fibroin is 20-30% w/v. Panels: (a) boundary distance vs time; (b) boundary distance vs time1/2.

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Figure 11. DSC curves for several fibroin materials: solid line, original silk fiber; dotted line, regenerated fibroin film (untreated); dashed line, regenerated fibroin film (MeOH treated); dashed and dotted line, regenerated fibroin fiber.

Figure 12. Wide-angle X-ray diffractograms of several silk materials.

fibers (raw and degummed) show a strong peak around 2θ ) 21° with a shoulder peak around 28° derived from the β-sheet crystal structure. On the other hand, a broad peak around 2θ ) 21° was obtained for the regenerated silk fiber (dry and wet). This result indicates that the crystalline region has been affected by the regeneration process. The size and perfection of the β-sheet crystal structure as well as the

degree of crystallinity are lower for the regenerated fiber compared with the original one. Even though the crystallinity for the regenerated fibroin materials in methanol is less than that of the native silk, it is expected that the regenerated fibroin fiber shows the X-ray diffractogram of the β-crystal structure similar to the original silk since the regenerated fiber is prepared with a methanol coagulation bath. One reason for the lower crystallinity of the regenerated fibroin fiber compared with the native silk protein is because the fibroin dope solution contacts the coagulant, methanol, at most for 5 s during the wet spinning, and therefore the crystallites may not have had enough time to be well developed. In addition, the lack of the shear-induced crystallization due to the large spinneret diameter and low take-up speed may be another reason for it. The regenerated fibroin fiber is very elastic in the wet state (about 50% elongation at break). The X-ray diffractogram in Figure 12 shows the greatly decreased intensity of the regenerated fibroin fiber in the wet state. Water molecules diffused into the amorphous region (free volume) of the regenerated fibroin fiber and disrupted its crystalline structure by forming new hydrogen bonds with the fibroin molecules. Therefore, this material shows an amorphous X-ray diffraction pattern with very low crystallinity.



Figure 13. Surface images of regenerated fibroin fiber (1): fiber end; (b) fiber surface.

Figure 14. Fracture images of the regenerated fibroin fiber (counter cross sections of a tensile broken fiber): (a) fiber fracture 1; (b) fiber fracture 2.

Scanning Electron Microscopy. The SEM images of the surfaces and fractures of the individual regenerated fibroin fiber are shown in Figures 13-15. The SEM images of the fiber surface and cross section are very similar to those of the previous study of genetically engineered silk/elastin-like protein (SELP) fibers by Martin et al.26 As shown in Figure 13, the fiber has horizontal lines on its surface along the fiber axis. They are most likely caused by a skin-core effect in the fiber structure, as it coagulates from solution. The regenerated fibroin fiber is very brittle. It cannot stand even 90° bending. The tenacities are only a small fraction of a gram per denier. As shown in the fracture images, the breakage started at the ductile part of the fiber, and then it propagated as catastrophic brittle failure.27 However, an elastic recoiling phenomenon, which can occur after the tensile break of ductile fibers such as for cross-linked chitosan fiber,28 did not occur. It is likely that the 100% methanol coagulation bath also causes internal voids in the fiber which contributes to the brittle behavior.

Conclusion To regenerate silk fibroin, it must be dissolved. In this paper, the dissolution condition of silk fibroin using the calcium nitrate tetrahydrate-methanol system was elucidated. Water molecules, which may be a component of the coordination bond and act as a swelling agent, are undoubtedly needed for the dissolution of silk fibroin. Calcium ions and methanol also play important roles in the construction of the coordination complex. The absence of a Tg peak in the DSC curve for the regenerated fibroin fiber in methanol indicates that it has a small amount of amorphous regions. The absence of an exotherm from the regenerated fibroin fiber in the DSC curve indicates that it already has β-form crystals, whose melting temperature is higher than the decomposition temperature. Even though both the regenerated fibroin film treated with methanol and the regenerated fibroin fiber have β-form crystals, their intermolecular interactions (hydrogen bonding) would be less than those of the original silk fiber. This is

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Figure 15. High-magnification fracture images of the regenerated fibroin fiber: (a) brittle part; (b) ductile part.

indicated by the X-ray diffractogram which shows the lowered peak intensity and peak broadening for the regenerated fibroin fiber. This indicates that it has smaller crystals (undeveloped β-form crystals, mostly two-dimensional β-sheets) than the original silk fiber due to low shear stress during the regeneration process. The diffusion coefficient of methanol into dialyzed and concentrated fibroin solution is relatively small. The combination of the low diffusion coefficient and the high throughput during the wet spinning process led to brittle tensile behavior. The key to the regeneration of silk fibroin seems to be the control of the crystalline structure of the regenerated fibroin materials. The regenerated fibroin fiber in the wet state has not brittle but elastic characteristics. This tells us that there may be a promising effect for the use of plasticizers that can remain inside the regenerated fibroin materials for a relatively long time (time of use). In addition, sufficient drawing during the spinning process would give the regenerated fibroin fiber a molecular orientation along the fiber axis resulting in three-dimensional β-form crystals in the regenerated fibroin fibers. Acknowledgment. We are pleased to acknowledge the ARO-MURI program (Grant DAAH 04-96-1-0018) for funding this work. We thank Professor Y. J. Lim for providing us the raw silk as a gift and Mr. Teruyuki Yamada for some useful ideas. We are grateful to Dr. Keith Beck for assistance with the atomic absorption analysis. References and Notes (1) Sofia, S.; McCarthy, M. B.; Gronowicz, G.; Kaplan, D. L. Biomed. Mat. Res. 2001, 54, 139-148. (2) Bunning, T. J.; Jiang, H.; Adams, W.; Crane, R. L.; Farmer, B.; Kaplan, D. Silk Polymers. Materials Science and Biotechnology; ACS Symposium Series 544; American Chemical Society: Washington, DC, 1994; pp 353-358. (3) Liu, Y.; Zhang, X.; Liu, H.; Yu, T.; Deng, J. J. Biotechnol. 1996, 46, 131-138. (4) Tsukada, M.; Freddi, G.; Minoura, N.; Allara, G. J. Appl. Polym. Sci. 1994, 54, 507-514.

(5) Minoura, N.; Tsukada, M.; Nagura, M. Polymer 1990, 31, 265269. (6) Lazaris, A.; Arcidiacono, S.; Huang, Y.; Zhou, J.; Duguay, F.; Chretien, N.; Welsh, E. A.; Soares, J. W.; Karatzas, C. N. Science 2002, 295, 472-476. (7) Freddi, G.; Pessina, G.; Tsukada, M. Int. J. Biol. Macromol. 1999, 24, 251-263. (8) Iizuka, E. J. Polym. Sci., Polym. Symp. 1985, 41, 173-185. (9) Tsukada, M.; Freddi, G.; Gotoh, Y.; Kasai, N. J. Polym. Sci., Part B: Polym. Phys. 1994, 32, 1407-1412. (10) Trabbic, K. A.; Yager, P. Macromolecules 1998, 31, 462-471. (11) Mathur, A. B.; Tonelli, A. E.; Rathke, T.; Hudson, S. M. Biopolymers 1997, 42, 61-74. (12) Atomic Absorption Spectroscopy Analytical Methods; The PerkinElmer Co., 1996. (13) Nakajima, A.; Tanaami, K. Polym. J. 1973, 5, 248-254. (14) Gordon, J. E. The Organic Chemistry of Electrolyte Solutions; John Wiley & Sons: New York, 1975; p 2. (15) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712-719. (16) Brame, E. G., Grassilli, J. G., Eds. Infrared and Raman Spectroscopy; Dekker: New York, 1977. (17) Liivak, O.; Blye, A.; Shah, N.; Jelinsky, L. W. Macromolecules 1998, 31, 2947-2951. (18) Yamamura, K.; Okumura, Y.; Ozaki, A.; Matsuzawa, S. J. Polym. Sci., Polym. Symp. 1985, 41, 205-220. (19) Magoshi, J. Polymer 1977, 18, 643-646. (20) Li, G.; Zhou, P.; Shao, Z.; Xie, X.; Chen, X.; Wang, H.; Chunyu, L.; Yu, T. Eur. J. Biochem. 2001, 286, 6600-6606. (21) Liu, C. K.; Cuculo, J. A.; Smith, B. J. Polym. Sci., Part B: Polym. Phys. 1989, 27, 2493-2511. (22) Knaul, J. Z.; Creber, K. A. M. J. Appl. Polym. Sci. 1997, 66, 117127. (23) Magoshi, J.; Nakamura, S. J. Appl. Polym. Sci. 1975, 19, 1013-1015. (24) Magoshi, J.; Magoshi, Y.; Nakamura, S.; Kasai, N.; Kakudo, M. J. Polym. Sci., Part B: Polym. Phys. 1977, 15, 1675-1683. (25) Yamada, H.; Nakao, H.; Takasu, Y.; Tsubouchi, K. Mater. Sci. Eng., C 2001, 14, 41-46. (26) Martin, D. C.; Jiang, T.; Buchko, C. J. Protein based materials; Birkhaüser: Boston, 1997; pp 339-371. (27) Hearle, J. W. S., et al. Fiber Failure and Wear of Materials: an Atlas of Fracture, Fatigue, and Durability; Halsted Press: New York, 1989. (28) Wei, Y. C.; Hudson, S. M.; Mayer, J. M.; Kaplan, D. L. J. Polym. Sci., Part B: Polym. Phys. 1992, 30, 2187-2193.

BM0255948

Dissolution of Bombyx mori Silk Fibroin in the Calcium Nitrate

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