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 Mater. Res. Express 4 (2017) 105404 https://doi.org/10.1088/2053-1591/aa8e07

PAPER

Dissolution and regeneration of non-mulberry Eriogyna Pyretorum

silk fibroin
RECEIVED
14 July 2017
RE VISED
13 September 2017
ACCEP TED FOR PUBLICATION
Yuhang Guo1, Xiufang Li2, Qiang Zhang1, Shuqin Yan1 and Renchuan You1
1
21 September 2017 State Key Laboratory of New Textile Materials and Advanced Processing Technology, College of Textile Science and Engineering, Wuhan
PUBLISHED
Textile University, Wuhan 430200, People’s Republic of China
2
10 October 2017 Hubei Key Laboratory of Advanced Textile Materials and Application, College of Materials Science and Engineering, Wuhan Textile
University, Wuhan 430200, People’s Republic of China
E-mail: yourenchuan1182@126.com (R You)

Keywords: non-mulberry silks, regeneration, films, sol–gel transition, biomaterials

Abstract
Protein-based materials have been actively pursued as biomaterials because of their nontoxicity,
biocompatibility and biodegradability. In this work, we demonstrated the potential of Eriogyna
pyretorum silk fibroin (ESF), a non-mulberry silk protein, as biomaterials. The degummed ESF fibers
could be dissolved completely by Ca(NO3)2/H2O/C2H5OH solution to produce regenerated ESF. The
solubility was strongly dependent on the addition of C2H5OH, heating temperature and dissolving
time. α-helix and random coil are main molecular conformation in aqueous ESF solution. The
sol–gel transition behavior of regenerated ESF was also studied, indicating that the conformational
transition of regenerated ESF from random coil/α-helix to β-sheet during gelation. Especially, ESF
showed more rapid gelation than mulberry silk fibroin (BSF). Consequently, the gelation rate of BSF
could be controlled ranging from tens of minutes to days by changing the ESF ratio, providing useful
options for the fabrication of silk hydrogels. Water-stable regenerated ESF film could be achieved
by using aqueous ethanol to induce structural transition. Tensile tests showed that the ESF films
have a dry strength of approximate 31.0 MPa and a wet strength of approximate 3.3 MPa. This study
provides new opportunities as an alternative natural protein material for biomedical applications.

1. Introduction

The demand for innovative biomaterials is constantly increasing with recent advancements in the field of tissue
engineering. Protein-based materials hold great promise for biomaterials because of their intrinsic and unique
properties, including nontoxicity, biocompatibility and biodegradability [1]. Silks are naturally occurring
protein polymers that have been used clinically as sutures for centuries [2]. Silk is composed of a filament core
protein, termed fibroin, and a glue-like coating consisting of sericin proteins [3]. Silk fibroin has been increasingly
studied for new biomedical applications, because of outstanding biocompatibility, tunable degradability, and
low inflammatory response in vivo [4–7]. The silks produced by silkworms can be classified into two types—
mulberry and non-mulberry. In recent years, mulberry silk fibroin from domesticated Bombyx mori silkworm
(Bombycidae family) has been well explored and is now widely used as biomaterials involving drug delivery, tissue
engineering, and implantable devices [7, 8]. Non-mulberry silkworms are wild heterogeneous in nature and have
a wide distribution throughout the world. Non-mulberry silkworms like tasar, muga, eri, fagaria (belonging to
Saturniidae family) and shashe (Lasiocampidae family) produces a variety of silks [9]. However, limited study is
available on the potential of non-mulberry silks as biomaterials.
In previous studies, silks from non-mulberry silkworms namely Antheraea pernyi (Tussah), Antheraea mylitta
(Tropical Tasar), Antheraea assama (Muga), and Samia/Philosamia ricini (Eri) have been also demonstrated as
good source of raw materials to produce protein-based biomaterials for biomedical applications [9–15]. For
instance, non-mulberry silk fibroin (A. mylitta) enhanced differentiation of human mesenchymal stem cells into
osteocytes, suggest the potential of A. mylitta silk fibroin for bone-tissue engineering applications [15]. Mulberry
and non-mulberry silks are structurally and functionally distinguishable. B. mori silk fibroin (BSF) is composed
of 6 heavy-chains, 6 light-chains and a P25 glycoprotein, while non-mulberry SF lacks both an L-chain and the

© 2017 IOP Publishing Ltd

Mater. Res. Express 4 (2017) 105404 Y Guo et al

Table 1.  The solubility of E. pyretorum silk fibroin fibers.

Group Ca(NO3)2:H2O:C2H5OH (molar ratio) Temperature Time Solubility

A 1:4:0 100 °C 4h   √  

B 1:4:1.5 100 °C 2h   √  
C 1:4:1.5 100 °C 4h   √  √
D 1:4:1.5 90 °C 4h   ×  
E 1:6:1.5 100 °C 4h   ×  

  ×  Insoluble, √  Partially soluble, √√ Completely soluble.

P25 protein [9]. Moreover, the poly(–Ala–) β-sheets in non-mulberry silks impart a higher binding energy than
do poly(–Gly–Ala–) β-sheets, which make non-mulberry silk fibroin more resistant to dissolution and regenera-
tion in salt solvents in comparison to mulberry silk fibroin [9, 16]. Arginyl-glycyl-aspartic acid (RGD) tripeptide
sequences have been found in most non-mulberry silk fibroins, and as a result has been shown to provide much
stronger cell adhesion than BSF [9, 17]. A comparative study using mulberry (B. mori) and non-mulberry (A.
mylitta) scaffolds for osteochondral regeneration showed that silk fibroin scaffolds of A. mylitta are more chon-
droinductive while those of B. mori are more osteoinductive when compared mulberry silk fibroin [18], imply-
ing the potential of different kinds of silk proteins toward tissue-specific goals.
Eriogyna pyretorum (Saturnia) silk is an ancient non-mulberry silk variety. This E. pyretorum silkworm spe-
cies is mainly distributed in China, India, Burma and Vietnam. E. pyretorum silks are often made into high quality
fish lines, and have been used as sutures for thousands of years. However, we expect non-mulberry E. pyretorum
silks fibroin (ESF) to possess potential as a biomaterial. In this work, the feasibility of utilization of the silk fibroin
from non-mulberry E. pyretorum silks is investigated for potential biomaterial applications. The sericin could be
removed from E. pyretorum silk fibers after boiling in sodium carbonate solution. The degummed silk fibers were
dissolved by Ca(NO3)2/H2O/C2H5OH solvent system to obtain regenerated solution, and the molecular confor-
mation and sol–gel transition behavior of regenerated ESF were studied.

2.  Materials and method

2.1.  Degumming of silk fibers

Clean cocoon shells were dried at 60 °C and then weighted. The dried cocoon shells were degummed for different
times (30 min for each time) in 0.25% Na2CO3 solution at 98–100 °C. After the degumming process, the cocoons
were rinsed thoroughly with deionized water. The degummed silks were dried at 60 °C and then weighted. This
procedure was performed in triplicate, and the degumming ratio was expressed as the percentage of weight loss
relative to the initial dry cocoon weight.

2.2.  Scanning electron microscope

The surface morphology of E. pyretorum cocoon silks and sericin-extracted ESF fibers were observed by a
scanning electron microscope (SEM; Hitachi S-4800, Japan) after gold sputtering.

2.3.  Dissolution of ESF fibers

To determine the optimum conditions for the dissolution of ESF fibers, varied dissolution conditions, including
the addition of ethanol, treatment temperature, and heating time were performed. Table 1 lists the dissolution
conditions, as well as the solubility of ESF. The dissolved solution was dialyzed against deionized water for 3 d
with a 9–12 kDa molecular weight cut off dialysis tube. The purified ESF solution was centrifuged at 10 000 rpm
for 3 min to remove aggregates and debris, and then stored at 4 °C before use. The ESF solution were frozen
at  −20 °C for 6 h, followed by lyophilization for 48 h to obtain solid sponge.

2.4.  Circular dichroic spectroscopy

To detect the molecular conformation of regenerated ESF in aqueous solution, the secondary structure of aqueous
ESF solution were analyzed with circular dichroism (CD) spectra. The lyophilized ESF sponge was re-dissolved
in ultra-pure water to generate aqueous ESF solution. CD spectra were recorded on a spectrophotometer (J-815,
JASCO) equipped with a Peltier temperature controller, and the spectra from 250 to 190 nm were collected at a
scanning speed of 100 nm·min−1 at 25 °C. The data was recorded as mean residue ellipticity (deg·cm2·dmol−1).

2.5.  Preparation and characteristics of regenerated SF films

ESF films were prepared using regenerated ESF solution cast upon polystyrene plate, and the thickness of the
films was regulated by the ratio between solution volume and the surface area of plate. After casting, the films
were allowed to dry for 24 h at 16–20 °C in a fume hood. After drying, the films were peeled off and immersed

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Mater. Res. Express 4 (2017) 105404 Y Guo et al

Figure 1.  (A) and (B) The cocoon of wild E. pyretorum silkworm, (C) degummed silk fibers.

in 75% ethanol for 2 h. The secondary structure of the regenerated ESF films was analyzed by attenuated total
reflectance-fourier transform infrared spectroscopy (ATR-FTIR) on a VERTEX 70 spectrometer (Bruker,
Germany).
To investigate the mechanical properties of EFS film, BSF film was used as a control group. BSF solution was
extracted from B. mori silks according to previously reported methods [7]. Regenerated BSF film was prepared
in accordance with the abovementioned conditions. The mechanical properties of the SF films were measured
using an Instron 5967 mechanical testing instrument at 25 °C and 50  ±  5% RH for dry state and for wet films
after water soaking. The films were cut as uniform strips with a size of 50 mm  ×  10 mm (n  =  5 for each sample).
The gauge length and the drawing speed were preset as 30 mm and 20 mm min−1, severally.

2.6.  Gelation behavior of aqueous ESF solution

To determine the gelation behavior of ESF in aqueous solution, the sol–gel transition was monitored optically,
as previously [19]. The reported lyophilized ESF sponge was dissolved in ultra-pure water to obtain ESF solution
with varying concentrations. 0.5 ml of ESF solution was gently pipetted into each well of 24-well plates. The
regenerated BSF solution was used as a control. Regenerated BSF solution was prepared following the procedure
described previously [7]. To further investigate the gelation behavior of ESB/BSF blend, the blended SF solutions
(4%) with the desired blending ratios of BSF to ESF as 75/25, 50/50 and 25/75 were prepared. The plates were
incubated in a Synergy HT Multi-Mode Microplate Reader (Bio-Tek Instruments, USA) at a temperature of
37 °C. To monitor the gelation process, turbidity changes of silk fibroin were measured by the values of optical
density (OD) at 550 nm at predetermined time.
Regenerated ESF solution and hydrogel was frozen at  −40 °C, and then freeze-dried for 48 h for FTIR analy-
sis. The lyophilized sponges were cut into powder and then prepared in KBr pellets. FTIR spectra were collected
using a VERTEX 70 spectrometer.

3.  Result and discussion

3.1.  Silk degumming

The cocoon silk of wild E. pyretorum silkworm is a deep yellow silk fiber (figure 1(A)). The exogenous impurity
had been cleared before degumming (figure 1(B)). The filaments core protein of silkworm silks are surrounded
by a glue-like coating consisting of sericin proteins. In addition, non-protein component such as calcium oxalate
crystals are usually observed on the surface of undegummed non-mulberry silks [20]. As shown in figure 2(a),
the surfaces of E. pyretorum silks are covered by a large of calcium oxalate crystals at micrometer size. The removal
of the sericin and impurities is required for biomedical applications of ESF. Sericin is swollen and dissolved
in hot water, especially in alkaline hot water. The hot Na2CO3 solution is the frequently-used method for silk
degumming in the laboratory. Non-mulberry A. pernyi silks have been successfully degummed using a 0.25%
Na2CO3 solution [13, 16, 17]. In this work, the degumming ratio was about 18% after three times of boiling in
Na2CO3 solution (figure 1(C)), and increased times had no further effect on the degumming ratio, indicating
that the sericin had been almost removed from the fibroin fibers. The surfaces of degummed silk fibers are greatly
smooth, which show that only very shallow longitudinal striations attributable to the fibrillar fibroin structure
(figure 2(D)). SEM results indicated that sericin and impurities have been successfully removed from filaments,
and suggested that the degumming conditions did not destroy the microstructure of fibroin fibers.

3.2.  Effect of dissolution conditions on the solubility

Natural silk fibers must be regenerated into solution and then processed into desired material forms, such as films,
porous scaffolds and hydrogel for various biomedical applications. However, the uniquely intrinsic characteristic
makes non-mulberry silks very resistant to dissolution in salt solvents. Although there are many aqueous salt

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Mater. Res. Express 4 (2017) 105404 Y Guo et al

Figure 2.  (A) Surface morphology of E. pyretorum silks. (a) Micrometer-sized calcium oxalate crystals on the surface of
undegummed silk fiber. (B) Nanofibrillar fibroin structure in the defect of undegummed silk fiber. (C) Surface morphology of
degummed ESF fibers. (D) Nanofibrillar morphology of degummed ESF fibers. Scale bars: (A) and (C) 25 µm, (a) and (D) 2 µm, (B)
1 µm.

solvents for the dissolution of mulberry silk fibroin, rather few salts have been reported for non-mulberry silks
[21]. Here, the solubility of ESF fibers in Ca(NO3)2:H2O:C2H5OH solution was investigated. Table 1 shows the
solubility of ESF fibers under varied dissolution conditions. In the dissolution of ESF fibers, the solubility is
strongly dependent of the addition of ethanol, heating temperature, and the concentration of the calcium nitrate
solution. Although other non-mulberry silk variety, such as A. pernyi, could be dissolved completely by heated
Ca(NO3)2, ESF fibers showed only partial dissolution in molten Ca(NO3)2·4H2O solution at 100 °C after 4 h
heating. However, the addition of ethanol remarkably enhanced the dissolution of silk fibers. ESF fibers have
been almost dissolved after 2 h heating, and a more clear solution was achieved after 4 h heating. This may caused
by active molecular mobility and increased ions permeation when heating temperature is higher than the boiling
point of ethanol, but the mechanism for the dissolution of silk fibroin has yet to be identified. Furthermore,
we found that the solvent concentration and heating temperature play important roles in the solubility of ESF
fibers. Silk fiber was completely undissolved when the molar ratio of solvent was changed from 1:4:1.5 to 1:6:1.5.
Moreover, at least 100 °C temperature is required for complete dissolution, because silk fiber was nearly insoluble
at 90 °C. Therefore, we conclude that the preferable dissolution conditions of ESF fibers are the molar ratio of
Ca(NO3)2/H2O/C2H5OH of 1:4:1.5 at 100 °C for 4 h, which was used throughout the remaining experiments.

3.3.  Molecular conformation of regenerated ESF

As shown in figure 3, the molecular conformation of regenerated aqueous ESF solution was detected by CD
spectrum. The strong positive peak at 191 and 222 nm as well as weak negative peak at round 208 nm show that the
molecular conformations of regenerated ESF in water mainly are α-helix and random coil structure. Moreover,
the positive peak at 197 nm and weak peak at around 215 nm could be observed, suggesting that the existence of a
little β-sheet structure. CD spectroscopy analysis suggested that the different secondary structure co-exist in the
aqueous ESF solution, and α-helix and random coil structure should be main molecular conformation.

3.4. Sol–gel transition of regenerated ESF

There was a rapid increase in viscosity and subsequent conversion to a gel state when regenerated SF solution
was stored for a certain time [13, 22]. Therefore, the sol–gel transition behavior of regenerated ESF gelation
should be investigated. Gelation of silk fibroin is accompanied by a structural transition from a random coil to β-
sheets. The β-sheets then assemble into a physically cross-linked gel network [19, 23]. FTIR results demonstrated
the structural transition of ESF from α-helix/random coil to β-sheet after gelation. As shown in figure 4, the

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Mater. Res. Express 4 (2017) 105404 Y Guo et al

Figrue 3.  Circular dichroism spectrum of regenerated ESF solution.

Figure 4.  FTIR spectra of regenerated ESF: (a) lyophilized sponge, (b) lyophilized hydrogel.

lyophilized ESF sponge showed the obvious peaks at 1654 cm−1 (α-helix, amide I), 1540 cm−1 (α-helix, amide
II) and 1241 cm−1 (random coil, amide III), indicating that α-helix and random coil are the most typical
conformation in lyophilized ESF sponge. After gelation, the adsorption peaks shifted to 1631 cm−1 (β-sheet,
amide I), 1517 cm−1 (β-sheet, amide II) and 1227 cm−1 (β-sheet, amide III), respectively, indicating structural
transition from α-helix/random coil to β-sheet. Moreover, a weak peak at 1692 cm−1, which may attributed to
β-turn structure [24], can be observed (figure 4(b)).
The chemical structure of non-mulberry silk is remarkably different from that of BSF. For example, the hydro-
phobic sequences are mainly (GAGAGS)n for BSF and polyalanine for non-mulberry silk, and they are also chief
components of β-sheets in the corresponding silks [25]. The differences in the primary structure would lead to
different sol–gel transition behavior. For BSF solution with concentrations from 0.6% to 15% (w/v), the gela-
tion time was usually days to weeks at room temperature or 37 °C [22]. However, the gelation of ESF was much
more rapid than that of BSF and more sensitive to the effect of environmental temperature (figure 5). At lower
temperature, the gelation rate of ESF solution is slow. It was found that the 2% ESF solution could be maintained
for weeks without gelation at 4 °C. When the environmental temperature was increased above 30 °C, the gela-
tion rate for ESF solution remarkably increased. Figure 6 illustrates the gelation kinetics at 37 °C. The ­complete

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Mater. Res. Express 4 (2017) 105404 Y Guo et al

Figrue 5.  In situ observation of ESF gelation at different temperature.

Figrue 6.  Gelation kinetics of regenerated silk fibroin solutions.

g­ elation of 4% ESF solution needed only about 16 min, which is far faster than that of BSF solution (about 7 d,
data not shown). Moreover, ESF solution with concentrations from 2% to 4% showed approximate gelation time
with 4% ESF solution, suggesting that the effect of concentration is slight for ESF gelation.
Rapid gelation is useful for the biomedical applications of hydrogel. However, the clinical application of BSF
hydrogel is greatly limited because its gelation time is too long. We demonstrated that the gelation of ESF was
much more rapid than that of BSF. Therefore, we expected to control the gelation behavior by blending ESF
with BSF. After blending processing, the gelation rate can be reduced from days to tens of minutes by gradually
increase ESF ratio (figure 6), provide useful options for the fabrication of silk hydrogels. It is expected that the SF
hydrogel with controlled gelation time will be used as an injectable hydrogel for tissue engineering.

3.5.  Structural characteristics of regenerated ESF film

ATR-FTIR result showed the secondary structure of regenerated ESF film. As shown in figure 7(a), the structure
of ESF in the film was predominantly random coil structure, which is different from the α-helix-predominated
structure in lyophilized ESF, suggesting the structural changes during slow air drying. This amorphous
structure makes ESF films soluble in water. The ESF film was treated with 75% ethanol solution for 2 h to induce
crystallization of silk fibroin. The structural changes after ethanol treatment were observed by ATR-FTIR. After
ethanol treatment, the structure changed to predominantly β-sheet figure 7(b), and a weak peak at 1692 cm−1 (β-
turn) can be observed. The formation of insoluble β-sheets promotes the stability of ESF film in water, imparting
the feasibility of regenerated ESF-based materials for biomaterials.

3.6.  Mechanical properties of regenerated ESF films

In regeneration processing, severe dissolution conditions may result in excessive reduction of molecular weight,
which will strongly affect material forming ability. In this study, the regenerated ESF solution can be fabricated
into films. Furthermore, tensile test was carried out to evaluate the mechanical properties of regenerated ESF
films, both in dry and wet states. Regenerated BSF has been fabricated into biomaterials with various forms,
such as film, particles, fibers and porous scaffolds. Therefore, the traditionally prepared regenerated BSF film
was used as a control. The stress–strain curves of films are shown in figure 8. In dry state, the ESF films showed
the similar tensile strength and breaking elongation with the BSF films. The BSF films showed tensile strength of

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Mater. Res. Express 4 (2017) 105404 Y Guo et al

Figure 7.  ATR-FTIR spectra of regenerated ESF films: (a) without ethanol treatment, (b) after ethanol treatment.

Figure 8.  Tensile properties of regenerated SF films.

37.5  ±  3.4 MPa and breaking elongation of 1.01  ±  0.07%. Similarly, the tensile strength and break elongation
and of the ESF films are 31.0  ±  2.8 MPa and 1.26  ±  0.27%, respectively. After ethanol treatment, SF films became
water-insoluble. In wet state, the strength values of SF films reduced, but the flexibility remarkably increased.
The tensile strength of the BSF films reduced to 6.9  ±  0.9 MPa, whereas the break elongation increased to
32.9  ±  1.8%. Similarly, the tensile strength of the ESF films reduced to 3.3  ±  0.3 MPa, and the break elongation
increased to 12.9  ±  2.3%.

4. Conclusion

In this study, we have investigated Ca(NO3)2/C2H5OH/H2O mixtures as a solvent for the dissolution and
regeneration of non-mulberry ESF. The ESF fibers could be dissolved completely to produce regenerated ESF
solution, and random coil and α-helix are main conformations in aqueous ESF solution. The sol–gel transition
behavior of regenerated ESF showed that the conformational transition from random coil/α-helix to β-sheet.
Interestingly, ESF showed strikingly rapid gelation than mulberry silk fibroin. Consequently, the gelation rate
of BSF could be controlled ranging from tens of minutes to days by changing the ESF ratio. Water-stable ESF
film can be obtained using aqueous ethanol to induce crystallization of silk fibroin. Mechanical tests of ESF
films indicated that regenerated ESF solution should be feasible for biomaterial material fabrication with other
desired forms, such as nanofibers and porous scaffolds. This study confirms the fundamental properties of this

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non-mulberry silk as a potential biomaterial, showing that this regenerated fibroin could also be used as a new
prospective natural biomaterial for biomedical field. Further work relating to the processing of the regenerated
ESF into fibers and sponges as well as the cytocompatibility is currently underway.

Acknowledgments

This work was supported by the Natural Science Foundation of Hubei Province (2016CFB260) and the National
Nature Science Foundation of China (31600774, 51403163).

ORCID iDs

Renchuan You https://orcid.org/0000-0002-5984-0093

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