Rheological Properties of Native Silk Fibroins from ... - ACS Publications

Biomacromolecules 2009, 10, 929–935

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Rheological Properties of Native Silk Fibroins from Domestic and Wild Silkworms, and Flow Analysis in Each Spinneret by a Finite Element Method Motoaki Moriya,† Frederico Roschzttardtz,‡ Yusuke Nakahara,§ Hitoshi Saito,| Yuichi Masubuchi,⊥ and Tetsuo Asakura*,# Department of Applied Chemistry, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan, IRC, School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, United Kingdom, PLAMEDIA Co. Inc., Honcho, Nakano-ku, Tokyo 164-0012, Japan, Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan, Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan, and Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan Received December 11, 2008; Revised Manuscript Received January 18, 2009

Silkworms can produce strong and tough fibers at room temperature and from an aqueous solution. Therefore, it seems useful to study the mechanism of fiber formation by silkworms for development of synthetic polymers with excellent mechanical properties. The rheological behaviors of native silk dopes stored in the silk glands of Bombyx mori and Samia cynthia ricini were clarified, and flow simulations of the dopes in each spinneret were performed with a Finite Element Method. Dynamic viscoelastic measurements revealed that silk fibroin stored in silk glands forms a transient network at room temperature, and that the molecular weight for the network node corresponds to the molecular weight of a heterodimer of H-chain and L-chain (B. mori) and a homodimer of H-chains (S. c. ricini), respectively. Also, each dope exhibited zero-shear viscosity and then shear thinning like polymer melts. In addition, shear thickening due to flow-induced crystallization was observed. The critical shear rate for crystallization of B. mori dopes was smaller than that of S. c. ricini dopes. From the flow simulation, it is suggested that domestic and wild silkworms are able to crystallize the dopes in the stiff plate region by controlling shear rate using the same magnitude of extrusion pressure despite differences in rheological properties.

Introduction The silk fibroin fibers produced from silkworms at room temperature and from an aqueous solution are strong and stiff fibers, whereas synthetic materials with comparable properties must be processed at higher temperatures and from less benign solvents.1,2 The properties of natural silk fibers are the consequence of molecular aggregation and flow properties of fibroin as well as of the chemical composition.3 Thus, rheological properties such as dynamic viscoelasticity and steady flow viscosity behaviors of silk fibroin from the silk glands of domestic Bombyx mori (B. mori) silkworms have been studied with great attention.4-8 B. mori dopes exhibit unique responses to fluidic force due to flow-induced crystallization.9-12 Therefore, the flow history should be investigated in the spinneret where silk fibers form from liquid silks to clarify the dope crystallization process.13,14 Because the flow field is affected by the duct shape, we have previously examined the shape of the spinneret using light microscopy15,16 and X-ray radioscopy.17 It is expected that dope crystallization occurs in the silk press part of the spinneret where stiff plates exist and the cross-sectional area abruptly narrows * To whom correspondence should be addressed. Tel./Fax: +81-42-3837733. E-mail: [email protected]. † Department of Applied Chemistry, Tokyo University of Agriculture and Technology. ‡ University of Leeds. § PLAMEDIA Co. Inc. | Kyoto Institute of Technology. ⊥ Kyoto University. # Department of Biotechnology, Tokyo University of Agriculture and Technology.

for B. mori silkworms. In addition, shear rate profiles in the B. mori spinneret were obtained by a Finite Element Method (FEM) calculation, and the relationship between extrusion pressure and dope crystallization in the silk press was discussed.12 FEM simulations have also been utilized for silk glands of domestic silkworms and spiders.18 Flow simulations for wild silkworms such as Samia cynthia ricini (S. c. ricini) and comparison with B. mori may provide us with beneficial information on silkworms’ spinning processes. The present study aims to clarify the spinning process of B. mori and S. c. ricini silkworms by rheological measurements and FEM calculation because different duct shape is expected for both silkworms and the rheological behavior will be different. Actually, the dynamic viscoelastic and shear flow properties for wild silkworm dopes have never been reported although comparisons of thermal properties by DSC19,20 and structures by solid-state NMR21 for B. mori and wild silkworms, for example, S. c. ricini and Antheraea pernyi (A. pernyi) have been reported. In addition, we made new observations on the rheological properties of B. mori dope to compare with those of S. c. ricini dopes under the same conditions, that is, temperature, time, and environment.

Experimental Section Native Silk Dopes Preparation. B. mori silkworm larvae were raised on fresh mulberry leaves in our laboratory. Also S. c. ricini silkworm (wild silkworm) larvae were reared on artificial diet; Silk Mate L4 M (Nosan Co. Inc., Japan). The silk glands of B. mori were pulled out from an anesthetized 7-day-old fifth instar larva and the middle division

10.1021/bm801442g CCC: $40.75  2009 American Chemical Society Published on Web 03/24/2009

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was cut off from other divisions by a pair of scissors. For wild silkworms, the middle and posterior silk glands were obtained. The tube-shaped samples were immersed in distilled water. The thin epithelial surfaces as well as hydrophilic sericin layers were then gently removed by tweezers.4 To eliminate possibility of the effects of shearing upon sample loading to the rheometer, the method in the literatures was employed.5,6 Rheological Measurements. All rheological measurements were run on a stress-controlled rheometer, Physica MCR-301 (AntonPaar Co. Inc.), with a solvent trap and a temperature regulator system to prevent the sample drying out. For both native silk samples a cone-and-parallel measuring geometry with 2° angle and 25 mm diameter was used.6,8 The temperature dependency of storage modulus G′(T) was measured by increasing the temperature from 5 to 65 °C at a rate of 1 °C/min and then cooling to 5 °C at the same rate under isochronal condition at ω ) 1.0 rad/s. The storage and loss moduli, G′(ω) and G′′(ω), were measured as a function of frequency ω in the range of 102-10-1 rad/s at three temperatures (5, 15, and 25 °C). Shear viscosity η(γ˙ ) was then measured as a function of shear rate γ˙ in the range of 10-2 and 10 s-1 for B. mori silk dope and 10-2 and 102 s-1 for S. c. ricini silk dope.6,12 Additionally, the shear viscosity η(γ˙ ) was measured by increasing shear rate in the range of 10-1 and 10 s-1 (B. mori) and 10-1 and 102 s-1 (S. c. ricini) and by shearing dopes for 30 s at each shear rate. Model Construction for Calculation. A three-dimensional structural model of the channel in both spinneret for the FEM calculation was constructed from sliced optical micrographs obtained previously on histological sections16 at intervals of 10 µm using a set of computer software, TRI/3D-SRFII and TRI/3D-BON (RATOC system engineering Co. Inc., Japan), and converted into triangular lattice surface elements.12 The duct model in the spinneret was then filled with tetrahedra lattice elements using the program, FEPartner (PLAMEDIA Co. Inc., Japan). Numerical Flow Simulation. FEM simulations were performed using the program, SUNDY TETRA (PLAMEDIA Co. Inc., Japan). The flow profile in the duct at steady state under a given pressure difference ∆P between inlet and outlet was calculated by the solving incompressible Navier-Stokes equation with the some assumptions:12,18 (i) The temperature is homogeneous and set to be constant at 25 °C. (ii) The fluid is purely viscous with non-Newtonian shear viscosity. We note that dopes are the viscoelastic fluid5,6 and this effect may influence results. (iii) The spinneret wall is specified to have a no slip boundary condition. Silkworms, however, are known to secrete another protein, sericin, around their silk secretions and secrin may act as a lubricating layer7,13,22 reducing the pressure drop required for flow.23 Also, rheological properties of sericin solutions have never been reported and this effect is not able to be considered. (iv) The rheological properties of dopes correspond to those of simulations. It has been known that, in vivo, pH5,24-26 and metallic ions4,27 in dopes are regulated and these influence rheological properties. (v) The water extraction in vivo is ignored, which affects the protein concentration and may change the flow behavior of the dopes. (vi) The lumen in the spinneret is completely filled with fibroin solutions. The assumption about the initial conditions, that is, when the silkworm starts to spin at which time the duct of the spinneret may be completely filled with concentrated proteins, may not be correct for equilibrium conditions during silk extrusion according to histological observations.15,16 However, these detailed factors described above are not able to be contained into calculation owing to current computer performance, namely calculation cannot converge. The density13 of the fluid was fixed at F ) 1.36 g/cm3 for B. mori. The density of the S. c. ricini dope was taken to be the same as that of B. mori dope. The examined pressure difference ∆P was from 1 to 100 M Pa.12

Results and Discussion Sol-Gel Transition of Dopes at the Critical Temperature. Figure 1 shows the temperature dependence of the storage modulus G′ from two samples of both silkworm

Moriya et al.

Figure 1. Temperature change in the storage modulus G′ for (a) B. mori dopes (square) and (b) S. c. ricini dopes (square). Results of two independent runs are shown.

dopes. The behavior observed from the storage modulus G′(T) is similar and consistent for both silkworm dopes evaluated here. With increasing temperatures, there is a small decrease in values of G′ followed by an abrupt increase in modulus values at a critical temperature TC. For B. mori dopes the critical temperature TC was evaluated as 38 ( 1 °C and 32 ( 1 °C in the case of S. c. ricini dopes. Note that the values obtained for B. mori are consistent with earlier results reported by Holland et al.6 In both dopes, the sharp increase of modulus is followed by a region where the temperature dependency is much smaller, leading to two visible regions with very distinct slopes. The first region characterized by the lager slope, just after the critical temperature, is related to the high kinetic process of the network formation due the phase transition from silk I28 to silk II.29 The second region observed here and characterized by the smaller gradient depicts a low kinetic process similar to those, observed in crystalline systems and consistent with an aging process of the phase transition.30,31 To clarify the detailed transition process to silk II, however, measurements combined with other methods for example SAXS/WAXS32 is required and it is out of scope of this study. During the cooling process we observe a nonreversible gel. Both dopes maintain their condition of strong gel after cooling. Interestingly, this irreversible process is coupled with whitening of the gel system, which confirms that the structure involved in the gel network has large scale domains, at least in the order of spectrum of the visible light. Based on the preceding results, in the following section rheological tests were performed below the critical temperature evaluated for the sol-gel transition. Therefore all tests were performed at temperatures below 25 °C ensuring a solution state condition. Dynamic Viscoelastic Behaviors of Dopes. G′(ω) and G′′(ω) at three temperatures are shown in Figure 2. For B. mori dopes, G′ has a pseudoplateau modulus at the high angular frequency end accompanied by a broad maximum of G′′ in the intermediate angular frequency range. In contrast, for S. c. ricini native dope, it appears that the plateau of G′ is observed at lower frequency

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Figure 4. Master curves of each dope at 25 °C, obtained by time-temperature superposition.

time of transient cross-links in a pseudonetwork structure formed by fibroin molecules and metallic ions. Let us assume that the elasticity is purely generated by entropy and the pseudoplateau observed in G′ corresponds to the elasticity of the pseudonetwork. Then a characteristic molecular weight for the network node (or the strand), Mx, can be obtained by,33

Mx ) Figure 2. Dynamic viscoelastic behaviors of (a) B. mori dopes and (b) S. c. ricini dopes at three temperatures; 5 °C (n ) 5 and 4 for B. mori dopes and S. c. ricini dopes, respectively), 15 °C (n ) 5 and 5), and 25 °C (n ) 8 and 9). Values are averages.

Figure 3. Temperature dependencies of horizontal shift factor of both dopes (Tref ) 25 °C).

and the maximum of G′′ was not observed. Time-temperature superposition33 held well for the viscoelastic data and shift factor aT was calculated from each crossover point ωc of G′(ω) and G′′(ω). Figure 3 shows horizontal a shift factor as a function of each temperature, and Figure 4 shows the master curve of G′(ω) and G′′(ω) at 25 °C. From the crossover frequency, we may estimate the relaxation time as τB ) 1.2 s for B. mori and τS ) 3.1 × 10-2 s for S. c. ricini. Note that the obtained viscoelastic behaviors for B. mori are consistent with earlier results reported by several groups.4-6 The difference in the viscoelasticity is due to the structure difference of fibroin molecules in each dope. It has been previously suggested that the elasticity of dope solution may originate from the structure formed by fibroin molecules and divalent metallic ions with ionic bonding.4 The relaxation time obtained above may exhibit the breakage and recombination

FRT GN

(1)

where F is the density of the solution, R is the gas constant, T is temperature, and GN is the plateau modulus. In the case of B. mori dope, with F ) 1.36 g/cm3 and GN ) 104 Pa, we obtain Mx ) 3.4 × 105 g/mol. This molecular weight intriguingly accords with the molecular weight of one heavy (H-) chain fibroin molecule (3.5 × 105 g/mol) or the molecular weight of a heterodimer of a H- and light (L-) chain linked by a disulfide bond (3.8 × 105 g/mol).34,35 On the other hand, it has been reported that a large protein complex with molecular weight of 2.3 × 106 g/mol is designated as an elementary unit (EU) of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, which is a glycoprotein of around 3.0 × 104 g/mol36 for B. mori silk fibroins. However, because the large complex is used for intracellular storage36 and is not present in lumen of the glands, the molecular weight calculated above may differ from the molecular weight of large complex EU. Because the plateau value of G′ is similar for S. c. ricini, the characteristic molecular weight of the pseudonetwork is close to that of B. mori The molecular weight is also consistent with the molecular weight of a pair of H-chain of S. c. ricini fibroin molecules (1.6 × 105 g/mol each) or the molecular weight of H-chain homodimer linked by a single disulfide bond (3.2 × 105 g/mol in total).37 Again, the detail of the rheologically observed network structure is unknown, though the difference in the characteristic time may be induced by the structure of the network and the mechanism of the relaxation. Flow Properties of Dopes and Flow Induced Crystallization. Figure 5 shows flow curves of B. mori and S. c. ricini dopes. Each native silk dopes exhibited an apparent zero shear viscosity η0 and in the high shear rate regime it showed shear thinning behavior. Note that the observed flow curve for the B. mori dope is consistent with earlier studies. In ordertoobtainarelaxationtimeundershearing,theCarreau-Yasuda equation,38 which can describe the viscosity behavior of a nonNewtonian fluid well was used

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η ) η0(1 + (τγ˙ )a)

n-1 a

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(2)

where τ is the relaxation time and a and n are fitting parameters. The obtained relaxation times (τB ) 33 s and τS ) 0.25 s) are different from those discussed in Figure 3. For B. mori dopes, the discrepancy in the characteristic time has been observed in measurements of Holland et al.6 and Terry et al.5 Although two characteristic relaxation times may show different relaxation phenomena in the linear and nonlinear viscoelastic regimes, the details are unknown. It has been known that spinning dopes of B. mori exhibit the repeated β-turn structure28 in the main sequence domain and those of S. c. ricini exhibit R-helical structure39 in main polyalanine domain. The difference of these structures regarding intra- and intermolecular hydrogen bondings could influence the discrepancy between shear viscosities. It has been reported that over shear rate 2 s-1, instabilities of B. mori dopes and spider dopes are observed by viscosity measurements of Holland et al.6 Also it has been known that shear history influences the flow curves from viscosity measurements of regenerated silk fibroin solutions.40 Thus, we provided dopes with different shear history to observe flow properties influenced by the instabilities. While both dopes were subjected to shearing for 30 s at each shear rate (γ˙ > 0.1 s-1), we measured the viscosity of solutions showing dope crystallization behavior to generate data for a simulation. Figure 6 shows the viscosity, including the dope transition process. The shear rate where the viscosity abruptly increases in the flow curve is defined as the critical shear rate γ˙ c for the crystallization (Figure 6) and the critical shear rates are listed in Table 1. This shear rate is slightly different from that measured in our previous study.12 The difference may be induced by both a difference in the measurement time when the dope underwent shearing and the geometry of the jig. The critical shear rate of B. mori (4.0 s-1) corresponds to the shear rate range where dope turned into white solid in Holland et al.’s measurements.6 The critical shear rate of S. c. ricini dope is much higher than that of B. mori dope (Table 1). It is suggested that B. mori dope is more sensitive to shear deformation than S. c. ricini dope in vitro. Note that in vivo pH5,26 and ion gradients4 influence the dopes/dopes’ properties. In addition, flow curves in Figure 5 were plotted also in Figure 6 for comparison. It is observed that slopes of curves decreased around 1 s-1 for B. mori and around 10 s-1 for S. c. ricini. The change of slope shows a decrease of degree

Figure 5. Flow curves of each dope at 25 °C. Black and white symbols correspond to B. mori dopes (n ) 4) and S. c. ricini dopes (n ) 8), respectively, and values are average. Dotted lines represent the Carreau-Yasuda fit from eq 2 to experimental data.

Figure 6. Flow-induced crystallization behaviors of each dope. Black and white symbols show viscosity of B. mori dopes (n ) 3) and S. c. ricini dopes (n ) 3), respectively, and values are average. Dotted lines show the flow curve in Figure 4.

of shear thinning. The difference may suggest that molecular alignment and/or nucleus formation before crystallization have an effect on the degree of shear thinning. For the flow simulation discussed later, a mathematical description of the measured flow curve was constructed. According to the power-law expression of the viscosity (eq 3)33

η ) aγ˙ n

(3)

where a and n are fitting parameters. The best fit a and n are listed in Table 1. To escape from numerical difficulty the viscosity was assumed to be constant below 0.1 s-1 and above 10 s-1 in the case of B. mori dope. In the case of S. c. ricini dope, the viscosity below 5 s-1 was assumed to be constant with a zero-shear viscosity from our measurements and the viscosity was assumed to be constant above 2 × 104 s-1. Structures of Each Flow Channel and Stiff Plate. Figure 7a shows a three-dimensional reconstruction of S. c. ricini silkworms’ flow channel and stiff plates obtained from crosssections, and Figure 7b shows optical micrographs of selected cross-sections of the spinning apparatus. The cross-sectional area of the lumen in the spinneret was obtained as a function of the distance from the spigot (Figure 7c). There were locations where the value of the cross-sectional area drastically decreases in both lumens. For B. mori, it decreased by 2000 µm2 at 600 µm from the spigot, while for S. c. ricini, it decreased by 1000 µm2 at 600 µm from the spigot (Figure 7a, (1) and (2)). This difference may be induced by size and shape of stiff plates of each silkworm. B. mori silkworms have a large ventral plate and a narrow but much longer dorsal plate.12,15 In contrast, S. c. ricini silkworms have only a strange shaped and longer dorsal plate (Figure 7a and b). As we already suggested,15 the silk press in the spinneret is larger than other parts for both silkworms. The muscles in the silk press are also related to the large shearing of the dope as well as the stiff plate. Also it has been suggested that characteristics of native silk fibroin appear depending on the location of the stiff plates. One of the examples is change in the birefringence corresponding to the change of molecular orientation and packing density. The birefringence of silk dope in B. mori silkworm’s spinneret abruptly increases near a boundary between the silk press and the common tube part.14 (There have been no reports about birefringence in the nascent silk of S. c. ricini.) It may be valid that the native dope starts to crystallize near the onset of the narrow flow channel in the



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Table 1. Critical Shear Rate and Parameters of Power-Lawa Before crystallization Fitting parameters

B. mori dope S. c. ricini dope a

Critical shear rate for crystallization -1

After crystallization Fitting parameters

a

n

γ˙ C [s ]

a

n

4.6×103 2.6×102

-0.38 -0.27

4.0 40

8.6×102 6.9

0.78 0.73

a and n show parameters in eq 3. These parameters were determined from the averaged value of three measurements shown in Figure 6.

Figure 7. (a) Three-dimensional structure of flow channels of S. c. ricini’s spinneret and stiff plates. (b) The selected histological sections obtained by optical microscopy. Arrows correspond to the positions along the duct. (c) Changes in the cross-sectional area (µm2) of the flow channel in the spinneret as a function of the distance from the spigot. For comparison, the cross-sectional area of B. mori’s flow channel is also shown. Abbreviated symbols in (b) show l.s. ) lumen of the spinneret and s.p. ) stiff plate.

silk press where the stiff plates and muscles are. We therefore carried out a flow calculation with a combination of the flow curve obtained from the measurements described above and a model of the flow channel, as described in the next section. Flow Simulation of Native Dopes in Each Spinneret. As described above, to discuss the crystallization of dopes in the duct it is important to focus attention in the simulation on the change in the shear rate at the location of the stiff plates. Silkworms’ spinning processes rely on the extrusion process and make use of the complicated structure of the flow channel.6 Therefore, it is essential to evaluate shear rate profile, accumulated shear strain and extrusion pressure to develop a better understanding of the spinning mechanism. As an example of the calculation, the profiles of shear rate and accumulated shear strain at 30 MPa are shown in Figure 8. The start of the crystallization (fiber formation) was defined by the critical shear rate obtained by present measurements. The initiation site of the crystallization is indicated by a circle in Figure 8. The dopes

exhibited different shear rate and extensional strain rate profiles. For B. mori, the dopes initially had a very low shear rate, which is at least one order smaller than the critical shear rate for dope crystallization, which then increased sharply to exceed the critical shear rate. In the case of S. c. ricini, the shear rate increased gradually toward the critical shear rate. These profiles might be correlated with the dope crystallization process in vivo and the characters of the fibers. In Table 2, the results of simulations at each regulated pressure are summarized. It is expected that B. mori native silk dope is subjected to large shearing in the location of the stiff plates in the silk press. The value of the shear rate in the spinneret changes depending on the extrusion pressure. The crystallization of native silk dope arises in the region of the stiff plates (550-600 µm from the B. mori spigot) in the pressure range of 10-80 MPa (Table 2). S. c. ricini silkworms also have a stiff plate and it is expected that S. c. ricini native silk dope undergoes a large shear force in the location of the

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from exposure to the flow field up to the initial site of crystallization using eq 4

γc )

∑ γ˙ ave,i × V∆z ave,i

(4)

i

where γ˙ ave,i is the average shear rate between adjacent nodes, Vi is the average velocity between adjacent nodes and ∆z is the distance between adjacent nodes on the near center line. Accumulated shear strain profiles are shown in Figure 8, and the results of accumulated shear strain under each extrusion pressure are summarized in Table 2. It is suggested that despite an order of magnitude difference of viscosity, each accumulated shear strain exhibited the same magnitudes. However, the calculated shear strain until crystallization was much smaller than the experimental strain. It is suggested that the dominant factor in dope crystallization is not the accumulated shear strain but the shear rate. Our simulation demonstrated that the two types of silkworm spin fibers in the location of the stiff plates at similar extrusion pressure. However these estimated extrusion pressures do not match the in vivo pressure. As described, our calculation includes many assumptions ((i)-(vi) in Experimental Section) due to the limitations of numerical simulation, which may give rise to the disagreement. If a flow simulation containing these effects is preformed, the results will provide us with more beneficial information for understanding the spinning mechanism, and these are issues in the future. Figure 8. Shear rate (solid line) and accumulated shear strain (dotted line) profiles in the spinneret at 30 MPa. (a) and (b) show simulations for B. mori and S. c. ricini, respectively. The shear rate along the central line is plotted against the distance from the spigot and shown below. The red dotted line and circle show the critical shear rate, i.e., γ˙ c.B ) 4.0 s-1 and γ˙ c.S ) 40 s-1 and the initiation site of fiber formation. Table 2. Information for Dope Crystallization in the Stiff Plate Region at Each Extrusion Pressurea extrusion pressure, ∆P [MPa] B. mori

S. c. ricini

10 15 30 50 80 10 15 30 50 70

flow volume, Q [mm3/s] 7.8 1.1 2.2 3.6 5.6 1.7 2.2 3.3 4.4 5.4

× × × × × × × × × ×

10-6 10-5 10-5 10-5 10-5 10-5 10-5 10-5 10-5 10-5

initiation accumulated site of strain for crystallization, crystallization, zc[µm] γc 550 580 590 590 600 500 560 590 590 600

6.9 3.2 3.1 3.0 3.1 10 4.8 3.4 3.2 2.6

a It is defined that stiff plate regions in each spinneret are at 550-600 µm from the spigot. The results for 1 and 100 MPa are not listed here because the dope crystallization did not occur in the silk press part.

stiff plate. It is assumed that the crystallization starts within 50 µm of the onset of the stiff plate region (550-600 µm from the S. c. ricini spigot). The crystallization of S. c. ricini native dope arises at 550-600 µm in the pressure range of 10-70 MPa (Table 2). It is suggested that the two types of silkworm are able to crystallize the dopes in the location of the stiff plate in the same pressure magnitudes. It is noted that the obtained pressure drop is consistent with the other FEM study.18 To discuss the effect of accumulated shear strain on the dope crystallization, we estimated the accumulated shear strain γc

Conclusion The temperature dependencies of storage modulus G′ were examined for B. mori and S. c. ricini dopes from silk glands. These tests revealed the critical temperature of sol-gel transition of dopes as 38 ( 1 °C (B. mori) and 32 ( 1 °C (S. c. ricini). The dynamic viscoelastic behaviors of dopes were measured, and the characteristic crossover points of the two moduli and a plateau value of G′ were observed. A characteristic molecular weight for the network node (or the strand), Mx, was estimated, and the molecular weight corresponded to a heterodimer of H-chain and L-chain (B. mori) and a homodimer of H-chain (S. c. ricini). Also, the characteristic relaxation times were obtained from the crossover point but these relaxation times were different from the relaxation times obtained from the flow curve. The discrepancies between these relaxation times are unclear. Additionally, we characterized the shear viscosity including flow-induced crystallization behaviors. The critical shear rate where the viscosity abruptly increased due to crystallization of S. c. ricini dope was larger than that of B. mori. From these rheological measurements, it is speculated that B. mori fibroin networks have a large sensitivity to shearing and S. c. ricini fibroin networks have a large sensitivity to temperature from ex vivo viscoelastic measurements. The flow fields in B. mori and S. c. ricini silkworms’ spinnerets were investigated by FEM calculations. Our simulation demonstrated that the two types of silkworm are able to crystallize the dopes in the location of the stiff plate at the same magnitude of extrusion pressure despite different viscosity behaviors. From estimation of accumulated shear strain, it is suggested that magnitude of shear rate mainly contributes to the transition from silk I to silk II. Acknowledgment. We acknowledge support from Promotion of Basic Research Activities for Innovative Biosciences, Japan.


We also acknowledge Prof. Mike Williamson at University of Sheffield for many suggestions and comments. M.M. thanks Dr. Nobuhito Nango (Ratoc System Engineering Co., Inc., Japan) for useful advice for producing three-dimensional reconstructions.

References and Notes (1) Asakura, T.; Kaplan, D. L. Silk Production and Processing. In Encyclopedia of Agricultural Science, Arutzen, C. J., Ed.; Academic Press: New York, 1994; Vol. 4, pp 1-11. (2) Shao, Z. Z.; Vollrath, F. Nature 2002, 418 (6899), 741–741. (3) Magoshi, J.; Magoshi, Y. Sen’i Gakkaishi 2007, 63 (9), P244-P252. (4) Ochi, A.; Hossain, K. S.; Magoshi, J.; Nemoto, N. Biomacromolecules 2002, 3 (6), 1187–1196. (5) Terry, A. E.; Knight, D. P.; Porter, D.; Vollrath, F. Biomacromolecules 2004, 5 (3), 768–772. (6) Holland, C.; Terry, A. E.; Porter, D.; Vollrath, F. Nat. Mater. 2006, 5 (11), 870–874. (7) Kojic, N.; Bico, J.; Clasen, C.; McKinley, G. H. J. Exp. Biol. 2006, 209 (21), 4355–4362. (8) Holland, C.; Terry, A. E.; Porter, D.; Vollrath, F. Polymer 2007, 48 (12), 3388. (9) Iizuka, E. Biorheology 1966, 3 (3), 141–152. (10) Yamaura, K.; Okumura, Y.; Matsuzawa, S. J. Macromol. Sci., Part B: Phys. 1982, B21 (1), 49–69. (11) Iizuka, E. Experientia 1983, 39 (5), 449–454. (12) Moriya, M.; Ohgo, K.; Masubuchi, Y.; Asakura, T. Polymer 2008, 49 (4), 952–956. (13) Kataoka, K.; Uematsu, I. Kobunshi Ronbunshu 1977, 34 (1), 37–41. (14) Kataoka, K.; Uematsu, I. Kobunshi Ronbunshu 1977, 34 (6), 457– 464. (15) Asakura, T.; Umemura, K.; Nakazawa, Y.; Hirose, H.; Higham, J.; Knight, D. Biomacromolecules 2007, 8 (1), 175–181. (16) Asakura, T.; Yao, J. M.; Yang, M. Y.; Zhu, Z. H.; Hirose, H. Polymer 2007, 48 (7), 2064–2070. (17) Moriya, M.; Ohgo, K.; Masubuchi, Y.; Knight, D. P.; Asakura, T. Polymer 2008, 49 (26), 5665–5669. (18) Breslauer, D. N.; Lee, L. P.; Muller, S. J. Biomacromolecules 2009, 10 (1), 49–57. (19) Magoshi, J.; Magoshi, Y.; Becker, M. A.; Kato, M.; Han, Z.; Tanaka, T.; Inoue, S.; Nakamura, S. Thermochim. Acta 2000, 352, 165–169.


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(20) Tanaka, T.; Magoshi, J.; Magoshi, Y.; Inoue, S.; Kobayashi, M.; Tsuda, H.; Becker, M. A.; Nakamura, S. J. Therm. Anal. Calorim. 2002, 70 (3), 825–832. (21) Asakura, T.; Nakazawa, Y. Kobunshi Ronbunshu 2006, 63 (11), 707– 719. (22) Magoshi, J.; Magoshi, Y.; Nakamura, S. Sen’i Gakkaishi 1997, 53 (3), P87-P97. (23) Vollrath, F.; Knight, D. P. Nature 2001, 410 (6828), 541–548. (24) Chen, X.; Knight, D. P.; Vollrath, F. Biomacromolecules 2002, 3 (4), 644–648. (25) Foo, C. W. P.; Bini, E.; Hensman, J.; Knight, D. P.; Lewis, R. V.; Kaplan, D. L. Appl. Phys. A: Mater. Sci. Process. 2006, 82 (2), 223– 233. (26) Matsumoto, A.; Lindsay, A.; Abedian, B.; Kaplan, D. L. Macromol. Biosci. 2008, 8 (11), 1006–1018. (27) Ochi, A.; Nemoto, N.; Magoshi, J.; Ohyama, E.; Hossain, K. S. J. Soc. Rheol. Jpn. 2002, 30 (5), 289–294. (28) Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. J. Mol. Biol. 2001, 306 (2), 291–305. (29) Asakura, T.; Yao, J. M.; Yamane, T.; Umemura, K.; Ultrich, A. S. J. Am. Chem. Soc. 2002, 124 (30), 8794–8795. (30) Tosh, S. M.; Marangoni, A. G.; Hallett, F. R.; Britt, I. J. Food Hydrocolloids 2003, 17 (4), 503–513. (31) da Silva, J. A. L.; Coutinho, J. A. P. Rheol. Acta 2004, 43 (5), 433– 441. (32) Martel, A.; Burghammer, M.; Davies, R. J.; Di Cola, E.; Vendrely, C.; Riekel, C. J. Am. Chem. Soc. 2008, 130 (50), 17070–17074. (33) Ferry, J. D. Viscoelastic Properties of Polymers; Wiley & Sons: New York, 1980. (34) Yamaguchi, K.; Kikuchi, Y.; Takagi, T.; Kikuchi, A.; Oyama, F.; Shimura, K.; Mizuno, S. J. Mol. Biol. 1989, 210 (1), 127–139. (35) Tanaka, K.; Inoue, S.; Mizuno, S. Insect Biochem. Mol. Biol. 1999, 29 (3), 269–276. (36) Inoue, S.; Tanaka, K.; Arisaka, F.; Kimura, S.; Ohtomo, K.; Mizuno, S. J. Biol. Chem. 2000, 275 (51), 40517–40528. (37) Inoue, S.; Tsuda, H.; Tanaka, T.; Kobayashi, M.; Magoshi, Y.; Magoshi, J. Nano Lett. 2003, 3 (10), 1329–1332. (38) Bird, R. B.; Armstrong, R. C.; Hassager, O. Dynamics of Polymeric Liquids. Fluid Mechanics, 2nd ed.; Wiley Interscience: New York, 1987; Vol. 1. (39) Nakazawa, Y.; Asakura, T. J. Am. Chem. Soc. 2003, 125 (24), 7230– 7237. (40) Chen, X.; Knight, D. P.; Shao, Z. Z.; Vollrath, F. Polymer 2001, 42 (25), 9969–9974.

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