Effect of Korean Bombyx mori Variety on Electro ... - Springer Link

Fibers and Polymers 2015, Vol.16, No.9, 1935-1940 DOI 10.1007/s12221-015-5472-x

ISSN 1229-9197 (print version) ISSN 1875-0052 (electronic version)

Effect of Korean Bombyx mori Variety on Electro-spinning Performance of Regenerated Silk Fibroin Bo Kyung Park and In Chul Um* Department of Bio-fibers and Materials Science, Kyungpook National University, Daegu 41566, Korea (Received June 20, 2015; Revised August 3, 2015; Accepted August 4, 2015) Abstract: The electro-spinning of silk has attracted the attention of researchers in biomedical fields because of 1) the unique properties of silk, including its biocompatibility, and 2) the simplicity of electro-spinning for nanoweb fabrication. Although there are hundreds of varieties of Bombyx mori, the effect of silkworm variety has not been investigated in the study of silk electro-spinning. As an effort to develop electro-spun silk with diverse performances, the effect of Korean silkworm variety on the electro-spinning of silk fibroin (SF) was examined in this study. The regenerated SF solutions showed different viscosities depending on the silkworm variety, which resulted in differences in the maximum electro-spinning rates and diameters of electro-spun SF fibers. However, the crystallization of SF during electro-spinning and the water content of the electro-spun SF webs remained the same between different silkworm varieties. Keywords: Silkworm variety, Silk fibroin, Electro-spinning

on the silkworm variety [15]. Electro-spinning is a simple and popular way of producing porous webs with nano-sized fibers, which can be applied as tissue engineering scaffolds [1-3] and nerve guidance conduits [8]. Researchers have studied electro-spun silk webs with various properties by controlling the degumming conditions [16-18] and the molecular weight (MW) [19] of regenerated SF. However, the effect of silkworm variety on the electrospinning of SF has not been considered to date. In this study, as a subsequent research on silkworm variety, the regenerated SF prepared from different Korean varieties of Bombyx mori were electro-spun, and the electro-spinning performance of SF was examined to investigate the effect of silkworm variety on electro-spinning.

Introduction Silk has been used as an excellent textile material for a long time. Recently, silk has attracted the attention of researchers because of its use in non-textile applications including tissue engineering scaffolds [1-3], burn dressings [4], wound dressings [5], artificial ear drums [6], membranes for guided bone regeneration [7], nerve guidance conduits [8], and bone substitutes [9]. It is revealed that silk possesses good blood compatibility [10-11] and cyto-compatibility [12], triggers minimal inflammatory reactions in the body [13], and is biodegradable [14]. These properties contribute to the diverse applications of silk. Silk is produced in nature, i.e. by silkworms. Silkworms can be classified as domestic (i.e. Bombyx mori) or wild silkworms. In South Korea, Bombyx mori has been mainly raised for silk production, and there are several hundred varieties of the Bombyx mori in South Korea. Silk produced from different silkworm varieties display diverse properties and performances. However, several Bombyx mori varieties have been selected and raised based on their performance, including the reelability of the cocoon, the weight of the silk cocoon, and the mechanical properties of the final silk yarn. The silkworm varieties are optimized to obtain the best quality of silk textile. The main problem in the previous optimization of silkworm variety is that some varieties of the Bombyx mori have been selected based on their textile performance only. The selected silk from the limited silkworm varieties is also used in nontextile applications for biomedical and cosmetic purposes. In previous studies, the structure and properties of silk cocoon and silk fibroin (SF) prepared from different silkworm varieties were examined. As a result, it was reported that the structure and properties of silk cocoon and SF vary depending

Experimental Preparation of Regenerated SF Four different original Korean Bombyx mori varieties were grown at Kyungpook Sericulture and Insect Research Center, and a hybrid Korean Bombyx mori variety (Baekokjam) was grown at the Yeongdeok Taeyang Farm in South Korea. Five silk cocoon samples were produced in total from the five silkworm varieties. The method to prepare regenerated SF has been reported elsewhere [15,20]. Briefly, the Bombyx 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 oC for 3 min. The liquor ratio was 1:20. The regenerated aqueous

*Corresponding author: [email protected] 1935

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Fibers and Polymers 2015, Vol.16, No.9

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 4 days at room temperature. The regenerated SF solutions were dried to obtain the regenerated SF powder. Electro-spinning of Regenerated SF Solution The method of electro-spinning regenerated SF solution has been introduced elsewhere [16,19]. The regenerated silk powder was dissolved in formic acid (98 %) and filtered twice by non-woven membranes to prepare an 11 % solution of regenerated SF for electro-spinning. The solution was loaded into a plastic syringe with a 21-gauge stainless steel needle (inner diameter=0.495 mm) at the tip. Electrospinning was performed at an applied voltage of 20 kV and a tip-to-collector distance of 19 cm. Measurement and Characterization The maximum electro-spinning rate of the dope solution was determined under a constant voltage and tip-to-collector distance [17]. The feed rate of the dope solution was controlled using a syringe pump (KDS100, KDScientific, USA) and was increased until the electrified jet was stable (e.g. Taylor cone stability and continuous spinning without dripping). The quality of the electro-spun fibers with the maximum spinning rate was confirmed by scanning electron microscopy (SEM). An 11 % (w/w) regenerated SF formic acid solution was used for the rheological measurements. The shear viscosity was measured by a rheometer (MARS III, Hakke, Germany) using a cone and plate geometry with a shear rate of 0.1100 s-1 at 25 oC. The radius and angle of the cone were 60 mm and 1 o, respectively. The silk nanoweb was gold-coated for SEM (FE-SEM, S570, Hitachi, Japan). The mean fiber diameter was determined by counting 100 fibers from the SEM images. A Fourier transform infrared (FTIR, Nicolet 380, Thermo Fisher Scientific, USA) spectrometer in the attenuated total reflection (ATR) mode was used to examine the molecular conformation and the crystallinity index of the electro-spun silk web. The crystallinity index was calculated from the FTIR spectrum as the intensity ratio of 1260 cm-1 and 1235 cm-1 using the following equation [21,22]. A1260cm–1 - × 100 Crystallinity index (%) = -----------------A1235cm–1

where, A1235cm–1 : Absorbance at 1235 cm-1 A1260cm–1 : Absorbance at 1260 cm-1 Electrospun SF webs were conditioned at 20 oC and 65 % relative humidity (R. H.) for more than 24 h, and the weight of SF webs was measured using a moisture analyzer (XM60, Precisa, Switzerland). Additionally, the dry weight of SF webs was obtained using the same moisture analyzer. The water content was calculated by the following equation:

Bo Kyung Park and In Chul Um

Water content (%) = (W2 − W1)/W1 × 100 where, W1: dry weight of the electrospun SF web W2: weight of the electrospun SF web under standard conditions (20 oC and 65 % R.H.) W1 and W2 of five electrospun SF web were measured for each silkworm variety sample and the average moisture content was obtained by averaging the five measurements.

Results and Discussion Rheological Properties of Regenerated SF Solution The rheological properties of regenerated SF solutions have been studied because they determine the wet- and electro-spinning performances of SF solutions [18,21-23]. The steady state flow of regenerated SF solutions of different silkworm varieties was measured, and the results are shown in Figure 1. 11 % Geumwangju and N74 SF solutions showed the highest viscosities among the samples, while Wonwon 126 showed the lowest viscosity. The differences in the viscosity of SF solutions between silkworm varieties are due to the different MWs of SF, as revealed in a previous study [15]. That is, Geumwangju and N74 have higher MWs than the other varieties, and Wonwon 126 has the lowest MW among the samples. Effect of Silkworm Variety on the Electro-spinning Performance Table 1 shows the SEM images of electro-spun regenerated SF fibers from different silkworm varieties. Regardless of silkworm variety, the 11 % regenerated SF solutions showed good electro-spinning performance, displaying good fibers without bead. Cho et al. reported that the critical viscosity range of SF solution for bead formation is 0.15-0.16 Pa·s, and fibers are formed above this range [18]. Considering that

Figure 1. Steady state flow of 11 % (w/w) regenerated SF formic acid solution prepared from different silkworm varieties.

Effect of Bombyx mori Variety on Electro-spinning of Silk


Table 1. SEM images of electro-spun regenerated SF fibers prepared from different silkworm varieties and at different feed rates Feed rate Silkworm varieties

Geumgwangju

N74

Baekokjam

Imbakgalwon

Wonwon126

0.3 ml/h

Maximum electro-spinning rate (ml/h)

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Figure 2. Effect of solution viscosity (at 1 s-1) on the maximum electro-spinning rate of regenerated SF solutions prepared from different silkworm varieties.

all SF solutions have viscosities higher than 0.2 Pa·s (Figure 1), the electro-spun morphologies of SF in Table 1 are consistent with the previous report. The rate of electro-spinning is intimately related to the mass production of electro-spun fibers, and the maximum electro-spinning rate is an important issue in the industrialization of electro-spun fibers [17]. The maximum electrospinning rate was measured for SF solutions from different silkworm varieties, and the results are displayed in Figure 2. In Figure 2, Geumgwangju showed the highest maximum electro-spinning rate (1.4 ml/h) and Wonwon 126 displayed the lowest rate (0.6 ml/h). On the whole, as the solution viscosity of SF at 1 s-1 was increased, the maximum electrospinning rate was increased. Yoon et al. studied the maximum electro-spinning rate of silk solutions with different residual sericin contents and reported that two factors determined the maximum electro-spinning rate: 1) solution viscosity and 2) residual sericin content [17]. In addition, Kim and Um reported that the maximum electro-spinning rate of SF solutions produced by different degumming methods was determined by solution viscosity [18]. Therefore, the positive relationship between solution viscosity and maximum electrospinning rate is consistent with the previous studies. When comparing the morphologies of SF that was electrospun at the maximum electro-spinning rate to that at a feed rate of 0.3 ml/h, a significant difference was not observed between the feed rates regardless of silkworm variety. It was reported previously that the electro-spun morphology was not affected by feed rate [17], and the result in the present study reconfirms this observation. Figure 3 shows the relationship between the mean diameter of electro-spun SF fibers and solution viscosity. As seen in the figure, as the viscosity of the SF solution increased, the fiber diameter also increased. The positive relationship


Figure 3. Effect of solution viscosity (at 1 s-1) on the mean diameter of electro-spun regenerated SF fibers prepared from different silkworm varieties (flow rate was 0.3 ml/h).

Figure 4. Effect of solution viscosity (at 1 s-1) on the mean diameter of electro-spun regenerated SF fibers from different silkworm varieties (flow rate was the maximum electro-spinning rate for each sample).

between mean diameter and viscosity has been reported in many previous studies [16,18,23-25], and the results obtained in this study are consistent with those in the previous reports. The positive relationship between solution viscosity and fiber diameter is also shown in the SF fibers that were electro-spun at the maximum electro-spinning rate, as seen in Figure 4. This indicates that the electro-spinning rate almost did not affect the mean diameter of the electro-spun fibers, which is consistent with a previous report [17]. Effect of Silkworm Variety on the Structure and Properties of Electro-spun SF Webs The molecular conformation of silk has been studied using

Effect of Bombyx mori Variety on Electro-spinning of Silk

Figure 5. FTIR spectra of electro-spun SF webs prepared from different silkworm varieties; (a) Geumgwangju, (b) N74, (c) Baekokjam, (d) Imbakgalwon, and (e) Wonwon126.

FTIR spectroscopy because it affects the properties of silk [21,22,26,27]. Figure 5 presents the FTIR spectra of electrospun SF webs from different silkworm varieties. The IR absorption peaks at 1650 and 1235 cm-1 were exhibited in the amide I and III band, respectively, regardless of silkworm variety. These peaks are attributed to the random coil conformation of silk. On the other hand, an IR absorption peak at 1515 cm-1 and a shoulder at 1540 cm-1 were shown in the amide II band. These peaks correspond to the β-sheet conformation and random coil conformation, respectively. These IR results imply that the β-sheet and random coil conformations co-exist in electro-spun silk web. Previous studies reported that the random coil conformation of SF exists in formic acid solutions [28], and it is partially transferred into a β-sheet conformation during electro-spinning [17]. Considering that all electro-spun SF webs showed almost the same FTIR spectra regardless of silkworm variety, it is thought that silkworm variety does not influence the molecular conformation of the electro-spun SF webs. The crystallinity index of the electro-spun SF webs does not change with silkworm variety, reconfirming that the silkworm variety does not affect the crystallization of the SF molecules during the electro-spinning process (Figure 6). Figure 7 shows the water content of SF webs electro-spun from different silkworm varieties. Although Imbakgalwon displayed a slightly higher water content than other electrospun SF web samples did, there was no significant difference among the silkworm varieties, considering the error range of the measurement. The similar water contents of the electrospun SF webs might be due to the similar amino acid composition of SF from the different silkworm varieties. That is, SF is a protein polymer consisting of various amino acids. The amino acid is either hydrophilic or hydrophobic


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Figure 6. Crystallinity index of electro-spun SF webs prepared from different silkworm varieties.

Figure 7. Water content of electro-spun SF webs prepared from different silkworm varieties.

depending on the side group. Therefore, if the amino acid composition of SF is different depending on silkworm variety, it may result in differences in hydrophilicity and water content in the electro-spun SF webs. However, the amino acid compositions of the SF used in this study are very similar between the silkworm varieties, as reported in a previous study [15], resulting in similar water content in the electro-spun SF webs.

Conclusion In this study, regenerated SF solutions prepared from different Korean silkworm varieties were electro-spun and their electro-spinning performance was examined. The molecular conformation and water content of the resultant SF webs were also investigated. On the whole, depending on the silkworm variety, different solution viscosities were

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obtained in the regenerated SF because of the different MWs of regenerated SF from the silkworm varieties. Other electrospinning performances including the electro-spinnability, the maximum electro-spinning rate, and the diameter of the electro-spun fibers were determined by the solution viscosities of the regenerated SFs from diverse silkworm varieties, rather than by other characteristics of silkworm variety. Although SF can be obtained from various forms of silks, the silkworm variety has not been used to control the electrospinning performance of SF and to obtain electro-spun SF webs with different properties. The results in this study indicate that the silkworm variety can be utilized to control solution viscosity and various electro-spinning performances, including the maximum electro-spinning rate and the mean diameter of the electro-spun fibers, without changing the molecular conformation and water content of SF. Considering that the electro-spinning performance of SF solutions from different silkworm varieties is dependent on the MW of the regenerated SF, the silkworm varieties can be utilized to obtain SF forms with different performances and to increase the possibility of industrialization of SF in applications in the biomedical and cosmetic fields.


8.

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10. 11. 12. 13. 14. 15. 16.

Acknowledgements 17.

This study was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012042016 and NRF2014R1A1A2056892).

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