Effect of different Bombyx mori silkworm varieties on the wet spinning of silk fibroin

Abstract

The wet spinning of silk solution has attracted researchers' attention because of 1) unique properties of silk as a biomedical material and 2) easy control of the structure and properties of the regenerated silk fiber. Recently, studies have reported that different silkworm varieties produce silk with differences in the molecular weight (MW) and other mechanical properties of the regenerated silk fibroin (SF) film. In this study, we look at the effect of different Bombyx mori varieties on the wet spinning of SF. Although five regenerated SFs from different silkworm varieties have different MWs and solution viscosity, the wet spinnability and post drawing performance of regenerated SFs were not different. This result is due to low variability in the MW of the regenerated SF samples from the different silkworm varieties. In addition, unlike regenerated SF films, the mechanical properties of wet spun regenerated SF filament were not affected by silkworm variety. This result suggests that the mechanical properties of wet spun SF filament are less affected by MW than those of SF film are.

Introduction

Silk has been an important textile material for a long time due to its excellent luster and touch. Additionally, owing to its compatibility with blood (Sakabe et al, 1989; Um et al., 2002) and excellent cyto-compatibility (Minoura et al., 1995), silk has many biomedical applications. Specifically, silk can successfully be used in artificial eardrums (Kim et al., 2010), as a membrane for guided bone regeneration (Song et al., 2011), in burn dressings (Lee et al., 2014), and as a nerve guidance conduit (Park et al., 2015).

Natural silk exists as a fiber form with structures and properties as determined by the silkworm. Technology to modify the structure and properties of natural silk fiber is limited. On the other hand, the structure and properties of wet spun silk filament can be changed by controlling the preparation conditions, including the molecular weight (MW) of silk fibroin (SF)(Cho et al., 2012; Chung and Um, 2014), the degumming conditions (Kim and Um, 2014), the coagulation conditions (Um et al., 2004a), and the post-drawing conditions (Um et al., 2004b). Therefore, there have been extensive studies examining the wet spinning of silk solutions (Trabbic and Yager, 1998; Ha et al., 2005).

There are many varieties of Bombyx mori, with more than 300 B. mori varieties in South Korea. Recently, Chung et al. (2015) studied the differences in structural characteristics and properties of silk produced by different B. mori varieties and reported that different varieties produce regenerated SF with different MW, solution viscosity, and mechanical properties.

In this study, five different B. mori varieties were used to prepare wet spun regenerated SF filaments. We looked at the effect of silkworm variety on the wet spinning performance of the SF solution as well as the structure and properties of the wet spun SF filament.

 

Materials and Methods

Preparation of regenerated SF

Four different B. mori varieties were grown at Kyungpook Sericulture and Insect Research Center and a hybrid B. mori variety (Baekokjam) was grown at Yeongdeok Taeyang Farm in South Korea. Five silk cocoon samples were produced from the five silkworm varieties.

The method used to degum silk cocoons has been reported elsewhere (Cho et al., 2012; Chung et al., 2015). Briefly, to prepare SF, the B. mori cocoons were degummed in a boiling aqueous solution containing sodium oleate (0.3% [w/v]) and sodium carbonate (0.2% [w/v]) for 1 h. The liquor ratio was 1:25. After the degumming process, the cocoons were rinsed thoroughly with purified water and dried. The purified water was obtained using a Water Purification System (RO50, Hana Science, South Korea) with a reverse osmosis membrane.

The degummed silk (SF) was dissolved in a ternary solvent containing CaCl2:H2O:EtOH (1:8:2 molar ratio) at 85℃ for 3 min. The liquor ratio was 1:20. The regenerated aqueous SF solutions were obtained by dialyzing the dissolved SF solutions in a cellulose tube (molecular weight cut off [MWCO]) = 12,000–14,000) against circulating purified water for 5 d at room temperature. The regenerated SF solutions were dried at 80℃ and then ground to obtain the regenerated SF powder.

Wet spinning of regenerated SF solution

The wet spinning method of SF solution has been reported elsewhere (Chung and Um, 2014). Briefly, the regenerated SFs were dissolved in a 98% formic acid solution to prepare 9% (w/w) SF formic acid dope solutions. The dope solutions were filtered through nonwoven fabric to remove the insoluble particles prior to wet spinning. The regenerated SF filaments were spun using a syringe and syringe pump by extruding the dope solution through a 21-gauge needle (inner diameter = 0.514 mm) into methanol as a coagulation bath. The flow rate of the fiber extrusion was controlled to 20 mL/h. The as-spun SF filaments were left to stand in the coagulant for 1 h and then in purified water for 20 h to allow the solvent (formic acid) to diffuse out completely from the filament prior to drying. Post-drawing of the as-spun SF filament was performed manually at a drawing speed of ca. 15 cm/s at 25℃ in air.

Measurement and characterization

A photograph of the wet spun silk filament in the coagulant was obtained using a digital camera (Canon PowerShot A2000 IS, Canon Inc., Japan). The maximum draw ratio was calculated from the ratio of the maximum drawn length of the fiber and length of the as-spun fiber. The drawing of the as-spun SF filament was performed manually with ca. 15 cm/s drawing speed at 25℃ in the air. The fiber lengths were measured at 20 different parts of the filament and the maximum draw ratio was determined from the mean.

The Fourier transform infrared (FTIR; Nicolet 380, Thermo Fisher Scientific, USA) spectra were obtained using the attenuated total reflectance (ATR) method. The crystallinity index was calculated as the intensity ratio of 1260 and 1230 cm−1 from the FTIR spectrum using the following equation (Chung and Um, 2014; Kim and Um, 2014).

To evaluate the mechanical properties of the regenerated SF filaments, stress-strain curves were obtained with a universal testing machine (UTM, OTT-003, Oriental TM, South Korea). The SF filaments tested were 70 mm in length. The tensile tests were performed under standard conditions with a 3 kgf load cell at an extension rate of 10 mm/min and a gauge length of 30 mm. All samples were preconditioned at the standard conditions for more than 1 d. Seven wet spun SF filaments produced by each silkworm variety were tested.

 

Results and Discussion

Wet spinning and post drawing performances of regenerated SF

Table 1 shows the wet spun feature of 9% regenerated SF solution from different silkworm varieties. Regardless of the silkworm variety, all regenerated SF solutions showed continuous fiber formation without bead formation, indicating the solutions has a good wet spinnability. This suggests that the silkworm variety almost does not affect the fiber formation at the condition used in this study.

Table 1.The effect of silkworm varieties on the wet spinnability of regenerated SF solution.

We believe all SF solutions show good fiber formation due to the viscosity of the SF dope solution. More than 80 mPa·s solution viscosity of SF solution results in continuous fiber formation without bead formation (Chung and Um, 2014). Furthermore, Chung et al. (2015) reported that the regenerated SF from the same five silkworm varieties used in this study showed 200 - 800 mPa·s solution viscosity due to differences in MW when the SF concentration was 10% (w/w) in formic acid. Therefore, considering the relationship between SF concentration and solution viscosity reported in the previous study (Chung and Um, 2014), it is expected that 9% SF solutions have more than 80 mPa·s. That is why all SF solutions show good fiber formation in table 1.

The molecular orientation of wet spun silk filament is very important because it strongly affects the mechanical properties (Um et al., 2004b). The molecular orientation can be increased by post drawing the regenerated silk fiber. Previous studies have examined the maximum draw ratio, which can be utilized to evaluate the post drawing ability of wet spun silk filament (Chung and Um, 2014; Kim and Um, 2014; Cho et al., 2012; Cho et al.,2010; Ko et al., 2009).

Fig. 1 shows the maximum draw ratio of wet spun SF filament from different silkworm varieties. Regardless of silkworm variety, all wet spun SF filaments displayed similar maximum draw ratios (4~4.5) indicating that the silkworm variety almost does not influence the post drawing ratio. Kim and Um (2014) reported that the maximum draw ratio of wet spun SF filament is strongly affected by MW and silk concentration. Therefore, the similar maximum draw ratio of SF filament from different silkworm varieties is likely because we used the same (9%) silk concentration for all samples and because there is not a significant difference in the MW between samples.

Fig. 1.Maximum draw ratio of wet spun regenerated SF filaments from different silkworm varieties.

Structure and properties of wet spun regenerated SF filaments

The chemical and physical properties of regenerated silk are affected by its molecular conformation. FTIR can be used to examine the molecular conformation of regenerated silk (Kim et al., 2013; Lee et al., 2013; Kim and Um, 2014; Chung and Um, 2014). Fig. 2 shows the FTIR spectra of regenerated SF filament from the five silkworm varieties tested. Strong IR absorption peaks at 1620 and 1510 cm−1 and a shoulder peak at 1260 cm−1 are seen for all regenerated SF filaments. These peaks are attributed to the β-sheet crystallite of SF. β-sheet crystallization takes place in the wet spinning of SF in formic acid solution with methanol as the coagulant because of the crystallization action of formic acid and methanol (Um et al., 2003). The crystallinity index of wet spun SF filament was calculated to examine the crystallization of SF quantitatively and the result is shown in Fig. 3. The wet spun SF filaments displayed similar crystallinity index (60~65%). This further confirms that silkworm variety almost does not affect the crystallization of SF during wet spinning process.

Fig. 2.FTIR spectra of wet spun regenerated SF filament (1X) from different silkworm varieties.

Fig. 3.Crystallinity index of wet spun regenerated SF filament (1X) from different silkworm varieties.

Previous studies have reported that there is a positive relationship between the crystallinity index of as-spun regenerated SF filament and the post drawing performance (i.e. maximum draw ratio) (Kim and Um, 2014; Chung and Um, 2014). Specifically, as the crystallinity index is increased, the maximum draw ratio of the as-spun SF filament increases because a more crystallized fiber can better resist the tensile force, which results in a higher draw ratio. Thus, our results of a similar crystallinity index and maximum draw ratio of wet spun SF filaments (Figs. 1 and 3) are congruent with previous findings.

Fig. 4 shows the tensile strength of regenerated SF filament from different silkworm varieties. Regardless of silkworm variety, the tensile strength of SF filaments was around 300 MPa. In the case of the elongation at break, the regenerated SF filament showed 20~30% (Fig. 5). However, considering the error range of result, the difference between the silkworm varieties is not significant. Initial Young’s modulus of wet spun SF filaments also showed a similar trend in strength and elongation. The wet spun SF filaments had similar Young’s modulus (9~10 GPa) regardless of silkworm variety (Fig. 6).

Fig. 4.Tensile strength of wet spun regenerated SF filament (4X) from different silkworm varieties.

Fig. 5.Elongation of wet spun regenerated SF filament (4X) from different silkworm varieties.

Fig. 6.Initial Young’s Modulus of wet spun regenerated SF filament (4X) from different silkworm varieties.

Chung et al. (2015) reported that silkworm variety strongly affects the mechanical properties of regenerated SF film Specifically, N74 (76.6 MPa) showed almost double the tensile strength of Wonjam 125 (38.7 MPa). Baekokjam (4.6%) showed much greater elongation than Wonjam 125 (2.5%). Considering the significant differences in mechanical properties of regenerated SF film between silkworm variety silk samples, it is interesting that the silkworm variety has little effect on the mechanical properties of wet spun regenerated SF filament.

The different fabrication characteristics of different silkworm varieties explain the differences in the mechanical properties of regenerated SF film and filament. In the case of cast film, the fabrication process is simple. Regenerated SF film is obtained after complete evaporation of the solvent (formic acid). Chung et al. (2015) reported that the mechanical properties of regenerated SF film from different silkworm varieties are mainly affected by the differences in the MW of the regenerated SF from the different silkworm varieties. For example, the higher tensile strength of N74 and the greater elongation of Baekokjam than Wonjam 125 are due to the higher MW of N74 and Baekokjam than Wonjam 125.

Wet spinning has a different fabrication process and thus the mechanical properties of the fiber are affected by additional factors: the uniformity as determined by wet spinnability and the molecular orientation by drawing. Although the regenerated SF has different MWs, the wet spinnability and post drawing performance of regenerated SF were not affected by the silkworm variety, likely because the MW difference among the silkworm variety SF samples was not high enough to result in a measurable difference. This finding is in contrast with previous studies (Chung and Um, 2014) in which they found that MW strongly affects wet spinnability and the post drawing of regenerated SF.

In this study, we examined the wet spinning of SF solutions from five different silkworm varieties. Unlike in regenerated SF film, silkworm variety almost does not affect the wet spinning of the regenerated SF solution nor the structure and properties of the resulting SF filament. This indicates it is difficult to obtain various properties of regenerated SF filament by controlling silkworm varieties, at least, in silkworm varieties used in this study. However, in other viewpoint, this also implies that the structure and properties of wet spun SF filament can be controlled uniformly regardless of silkworm variety. Considering there are more than 300 Bombyx mori varieties in South Korea, additional studies should be conducted to understand the characters of silkworm varieties.