Fabrication and Characterization of Biomaterial Film from Gland Silkof Muga and Eri Silkworms

Saranga Dutta,1 Bijit Talukdar,1 Rupjyoti Bharali,2 Rangam Rajkhowa,3 Dipali Devi11 Seri-biotech Laboratory, Life Sciences Division, Institute of Advanced Study in Science and Technology, Paschim Boragaon,

Guwahati 781035, Assam, India

2 Department of Biotechnology, Gauhati University, Guwahati 781014, Assam, India

3 Australian Future Fibres Research and Innovation Centre, Deakin University, Waurn Ponds, Victoria 3217, Australia

Received 4 May 2012; revised 5 September 2012; accepted 3 October 2012

Published online 10 October 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22168

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

Silks belong to a group of high-molecular-weight

organic polymers characterized by repetitive hydro-

phobic and hydrophilic peptide sequences.1 Silk

fibers produced by silkworms are nature’s most

highly engineered structured material and have a

good combination of strength and toughness that cannot be

reproduced by artificial means.2 Silk proteins are usually pro-

duced within specialized glands after biosynthesis in epithe-

lial cells, followed by secretion into the lumen of these glands

where the proteins are stored before spinning into fibers.3

Silkworm silk has been used commercially in textile produc-

tion for centuries and as biomedical sutures for decades. The

most extensively studied silk are Mulberry silk, produced by

Bombyx mori, a domesticated silkworm and has been studied

for use in surgery and tissue engineering.

In general, silk cocoons are composed of a fibrous protein

fibroin core (72–81%) and a surrounding glue protein, seri-

cin (19–28%). Fibroin is the major structural protein

secreted from the posterior silk gland (PSG), and sericin is

Fabrication and Characterization of Biomaterial Film from Gland Silkof Muga and Eri Silkworms

Correspondence to: Dipali Devi; e-mail: dipali.devi@gmail.com

ABSTRACT:

This study discusses the possibilities of liquid silk (Silk

gland silk) of Muga and Eri silk, the indigenous non

mulberry silkworms of North Eastern region of India, as

potential biomaterials. Silk protein fibroin of Bombyx

mori, commonly known as mulberry silkworm, has been

extensively studied as a versatile biomaterial. As

properties of different silk-based biomaterials vary

significantly, it is important to characterize the non

mulberry silkworms also in this aspect. Fibroin was

extracted from the posterior silk gland of full grown fifth

instars larvae, and 2D film was fabricated using standard

methods. The films were characterized using SEM,

Dynamic contact angle test, FTIR, XRD, DSC, and TGA

and compared with respective silk fibers. SEM images of

films reveal presence of some globules and filamentous

structure. Films of both the silkworms were found to be

amorphous with random coil conformation, hydrophobic

in nature, and resistant to organic solvents. Non

mulberry silk films had higher thermal resistance than

mulberry silk. Fibers were thermally more stable than the

films. This study provides insight into the new arena of

research in application of liquid silk of non mulberry

silkworms as biomaterials. # 2012 Wiley Periodicals, Inc.

Biopolymers 99: 326–333, 2013.

Keywords: fibroin; liquid silk; films; biomaterials; non

mulberry silkworm

Contract grant sponsor: Department of Biotechnology, Govt. of India

Contract grant number: BT/102/NE/ TBP/ 2010VVC 2012 Wiley Periodicals, Inc.

326 Biopolymers Volume 99 / Number 5

synthesized in the middle silk gland (MSG) of the mature

silkworm larvae. During spinning, the fibroin from PSG gets

coated with sericin from the MSG and comes out of the spin-

neret to form the fiber.4

The major biomedical applications of silk revolve around

fibroin, which is a homogenous protein with a molecular

mass of around 370 kDa.5 Sericin is a soluble glycoprotein6

and has a group of proteins ranging from 20 to 400 kDa.7,8

Silk fibroin from silkworms has been explored as a versatile

protein biomaterial for the formation of films, fibers, micro-

spheres, and porous scaffolds for various biomedical applica-

tions because of its biocompatibility, slow degradability, and

robust mechanical properties.9–11 These silk-based biomateri-

als have interesting mechanical, morphological, and structural

properties that fulfill important niches in biomaterial applica-

tions. The B. mori fibroin has been reported to be utilized for

a wide range of tissue engineering applications including

osteoblast, fibroblast, hepatocyte, and keratinocyte adherence

and growth in vitro and as an alternative to collagen in sur-

gery, mainly in the form of sutures.12Silk from Saturniidae

families is used in the textile industry and, therefore, offers a

good source for producing silk-based biomaterials for

advanced applications. They are commonly referred to as

non-mulberry silk. Non-Mulberry silk from Saturniidae fam-

ily has higher components of amino acids with polar, bulky,

basic, and hydrophilic side chains when compared with the

domestic Mulberry silk B.mori.5–9 Studies suggest that the fi-

broin from Non-mulberry silk is more bioactive than

B.mori.10,11 Previous studies on fibroin from tasar silk of A.

mylitta and A. pernyi13,14 showed that they have better cell

attachment and growth than B. mori silks. This calls for fur-

ther understanding of different non-mulberry silk species to

further explore their biomedical applications. However

among non-mulberry silk, only tasar silk has been studied to

a certain extent and other varieties remain largely unexplored.

Antheraea assamensis (muga silkworm) and Samia cynthia

ricini (eri silkworm) are nondiapausing silkworm species. In

other words, they survive without any physiological state of

dormancy in response to adverse environmental conditions.

These silkworm species are commonly known as non-mul-

berry silk, found in the North Eastern states of India, namely,

Assam, Meghalaya, and Mizoram states and especially con-

fined to Brahmaputra valley of Assam, India.15,16 Muga silk

is highly durable and so has high demands in the national

and international market.17 Muga silk has better mechanical

properties than other commercial silks (Tenacity-3.47 g/den,

Toughness-0.90 g/den, and Youngs Modulus-63.64 g/den),18

which imparts a wide range of use as a textile material. Eri is

a domesticated insect species produces the eri silk fiber hav-

ing good thermal insulation properties. High production

rate and low cost of eri silk is another advantage for exploit-

ing this fiber as a biomaterial.

This article reports the fabrication and characterization of

fibroin films from the liquid silk of these two non mulberry

silkworms which may be used as potential biomaterial, owing

to good mechanical (muga) and thermal properties (eri).

MATERIALS AND METHODS

MaterialsTwo indigenous species of non mulberry silkworm viz A. assamensis

and S. cynthia ricini were utilized in this study. Samples are

marked as

A. assamensis (muga) fiber-A and film-A1

S. cynthia ricini (eri) fiber-B and film-B1

Matured fifth instar larvae and cocoons were used for film and

fiber, respectively, from the rearing house of our institute. The fine

chemicals used in this study were obtained from Sigma Aldrich and

Merck. Double distilled water (Milipore, USA) was used throughout

the experiment.

Extraction of Native Silk Proteins from

the Silk GlandsExtraction of native liquid silk protein fibroin was done according

to Shimizu et al.19 and Putthanarat et al.20 The posterior part of

paired silk gland were dissected out and washed carefully without

any stress in double distilled water to remove any residual sericin.

Immediately, they were cut into small pieces and put into petri

plates containing small amount of cold distilled water without using

any shearing force. After several minute, the swollen glandular tissue

were removed from the petri plates, and the silk proteins were

shaken very gently overnight in the cold (2–38C). By keeping the fi-broin solutions at low temperature overnight, it becomes a colorless

viscous solution in water21 and prevents formation of gel. The

extracts were centrifuged for 60 min at 10,000g, and the supernatant

was collected by decantation.

Preparation of FilmsThe films were prepared by the method described by Putthanarat

et al.20 with slight modification. Briefly, the collected supernatant

was cast on a polyethylene plate as substrate, at 258C and 65% rela-

tive humidity and air-dried under a laminar flow for 12–14 h. The

protein films were peeled off and dried in desiccators for 12 h.

Degumming of FibersFibers were studied for comparison. Cocoons of muga and eri were

boiled in an aqueous solution of 0.02M Na2CO3 for 1 h to remove

sericin, washed thoroughly in distilled water, and dried for 1 h

at 508C.

Surface CharacterizationScanning Electron Microscopy. Scanning electron microscopy

images of silk fibroin films and fiber were obtained after gold

Biomaterial Film from Liquid Silk of Muga and Eri Silk 327

Biopolymers

sputtering using a JEOL JSM-5800 scanning electron microscope

with incident electron beam energy of 15 kV.

Dynamic Contact Angle Test. The advancing and receding con-

tact angles of fibroin films were determined using the Dynamic Wil-

helmy method (DCAT 11, Dataphysics) in water as well as in n-hep-

tane, to study the comparative solubility.

Physicochemical CharacterizationFourier Transform Infrared Spectroscopy. Fourier transform

infrared (FTIR) analyses of the samples were carried out using a

Bruker, vector 22 FTIR spectrometer. To avoid the effect of mois-

ture, the samples were dried overnight in desiccators. The IR spectra

were obtained in the spectral region of 400–4000 cm21.

X-Ray Diffraction. Wide-angle X-ray diffraction (XRD) patterns

of the samples were measured by an X-ray diffractometer (PANalyt-

ical, X’PertPRO PW3040/60) using CuKa radiation (k 5 1.54 A) in

the 2h range of 5–408 at 40 kV, 40 mA.

Differential Scanning Calorimetry. The thermal properties of

the samples were measured in a Perkin Elmer, DSC 6000, USA

under a dry nitrogen gas flow of 50 ml min21. The samples were

heated at 58C min21 from 30 to 3508C.

Thermogravimetric Analyses. The percentage weight loss of the

samples were determined by thermogravimetric measurements

(TGA), using a TGA 4000 (Perkin Elmer, USA) system. Samples

were heated from 30 to 4008C with a step increase of 28C min21

under an inert nitrogen atmosphere.

RESULTS

Surface CharacterizationScanning Electron Microscopy. The architecture of fibroin

fibers and films were studied by scanning electron micros-

copy and depicted as Figure 1. The surface structure of

degummed sample A was found to have stripe-like struc-

tures with deep grooves. The surface of the samples B and C

were smooth with absence of grooves. Wavy surfaces were

seen in A1 and B1 with some spots which on magnification

appears as globular structure (Figure 1D). In some cases,

the globular structures looks like microfibril (Figure 1E). In

case of B. mori film (C1), the surface was found to be

smooth; however, presence of some globular structures was

observed.

Dynamic Contact Angle Test. The wetting properties and

solubility of fibroin film surfaces were determined by

dynamic contact angle measurements in water and organic

solvent n-heptane. The data are shown in the Table I

(P\ 0.05).

Physicochemical Characterization of

Silk Fibroin FilmsFTIR Analysis of Fibroin Films. FTIR spectra of muga and

eri fibers and films are shown in Figure 2. FTIR spectra of A

showed presence of strong peaks at 1620 cm21 (amide I),

1518 cm21 (amide II), and 1234 cm21 (amide III), and sam-

ple B showed distinct peaks at 1620 cm21 (amide I), 1520

cm21 (amide II), and 1236 cm21 (amide III). The spectra of

A1 showed major peaks at 1643 cm21 (amide I), 1540 cm21

(amide II), and 1240 cm21 (amide III), whereas sample B1

showed major peaks at 1650 cm21 (amide I), 1546 cm21

(amide II), and 1240 cm21 (amide III).

X- Ray Diffraction Curves. Figure 3 shows the XRD data of

all the samples. Sample A showed major peaks at 16.78, 20.28(Silk II) and B showed peaks at 16.78, 20.18 (Silk II). In case

of A1 and B1, the main peak was found around 11.6 (Silk I)

with additional peaks at 22.318 (Silk II).

Differential Scanning Calorimetry. The differential scan-

ning calorimetry (DSC) results of muga and eri fibers and

films along with B. mori data are presented in Figure 4. Sam-

ple A showed an endothermic peak at 65.58C and a degrada-

tion peak at about 3648C. An endothermic peak at 77.58Cwas found in B and the degradation peak at 356.48C. In the

case of A1, the first endothermic peak was found at 938C, anendo-exo transition around the temperature range of 220–

2268C and final degradation at 3508C. In B1, the first endo-

thermic peak and the degradation peak were same as A1. The

endo-exo transition was found around 208–2238C. In case of

B. mori fiber, the first endothermic peak was evident around

648C and the degradation peak at 2678C. However in films,

the first peak shifted to 748C and the second to 2648C.

Thermogravimetric Analyses. TGA analyses of the samples

are depicted in Figure 5. In fibers (A and B), the first weight

loss was found around 96.48C, and the second weight loss

was evident around 245.7–386.28C. On the contrary, the

films showed the first weight loss around 138.58C, and the

second weight loss in the temperature range of 235.4–

377.38C.

DISCUSSIONThe silk fiber proteins are synthesized in the silk gland cells

of the silkworm and are formed by stretching of the liquid

silk through the movement of the head of the silkworm.

Therefore, the morphological structures are genetical charac-

ters, hence species specific. The fine external structure of

the silk fibers observed in A and B samples reflects the

328 Dutta et al.

Biopolymers

complicated and refined natural appearance which is respon-

sible for some special features like luster and color.22 In the

case of eri silk fiber (B), the external structure is close to B.

mori fibers (C) with smooth surface. However in case of

muga silks (A), stripe-like structures with deep grooves was

observed running along the length of the fibril (Figure 1A).

Similar striations were reported for the wild silkworm

Antheraea yamamai by Matsumura.23 These striations may

account for the uneven structure of the A. assamensis silk.

When thin films were prepared from the liquid silk, some

morphological differences were observed, and aggregation of

secondarily accumulated microfibril was observed (Figures

1A1 and 1B1). At the same time, aggregation of ball-like

globular particles of fibroin and formation of fibril of rela-

tively smaller size were observed (Figures 1D and 1E). How-

ever, B. mori film surface looked smooth and even than the

other films. Globular fibroin particles of 2 lm diameter and

filamentous structures have been seen in all the samples. The

FIGURE 1 SEM images of fibers and films. A: A. assamensis fiber, A1: A. Assamensis film, B: S.

ricini fiber, B1: S. ricini film, C: B. mori fiber, C1: B. mori film, D: globular structure, and E: filamen-

tous structure of films.

Biomaterial Film from Liquid Silk of Muga and Eri Silk 329

Biopolymers

globules and filamentous structures are due to the less

ordered random coil conformations of the films.24 Dynamic

contact angle tests showed that the film surfaces were hydro-

phobic in nature and immiscible in organic solvents like

n-heptane. The contact angles of muga and eri films were

found to be more than that of B. mori films prepared from

gland fibroin solutions. Previous reports on B. mori films

also showed lower contact angles compared to other fibroin

films.25,26 This property of the fibroin film may be advanta-

geous for application of pharmacological agents to the scaf-

fold during fabrication in solvent casting/particulate leaching

procedures in organic solvents.27FTIR spectroscopy is useful

in structural analysis of silk fibroin, because the position and

intensity of the amide bands are sensitive to the molecular

conformation.28–30 Amide I absorption primarily represents

the C¼¼O stretching vibrations of the amide group. The am-

ide C¼¼O bonds involved in different secondary structures

absorbs infrared radiation of different wave numbers. This

occurs because the frequency of vibration of C¼¼O in the

protein backbone changes due to the influence of hydrogen

bonds between N��H and C¼¼O, which are dependant on

conformations adopted by protein chains.31Therefore, the

major secondary structure contained in the protein can be

estimated from the amide I peak position. In the spectra of A

and B, the amide I peak was found at 1620 cm21 assigned to

b-sheet conformation by Chen et al.32 Additionally, the am-

ide II and III peaks for sample A at 1518 cm21 and 1235

cm21, respectively, have been assigned to b-sheet secondarystructures by Freddi et al.29 In case of B, the amide II peak

shifts to 1520 cm21 representing crystalline b-sheet confor-mation along with the peak at 1235 cm21. Earlier report

reveals that in muga fiber both a-helical and b-sheet confor-mation are present and interconversion occurs in them due

to stress.18 The films from gland proteins (A1 and B1)

showed highly amorphous structures. The peak at 1643

cm21 of A1 has been assigned to random coils/extended

chain conformations by Tretinnikov and Tamada33 and Tera-

moto and Miyazawa,34 and the 1650 cm21 peak of B1 to ran-

dom coil conformation. Arondo et al.35 has assigned the

1650 cm21 peak to a-helical structures. Additional peaks at1540 cm21 (Amide II) for A1 which is due to the out of

phase combination of the N��H in plane bend and the

C��N stretching can be assigned to random coil conforma-

tions36,37 and the peak at 1546 cm21 in B1 may be due to the

alpha helical conformation.38 The amide III band (C��H

stretching, N��H in-plane bending) at 1240 cm21 for A1

and B1 has been attributed to random coil conformations in

the films.36,37 Therefore, the films from muga (A1) and eri

(B1) are composed mainly of random coiled conformation

and a-helical structures of proteins. It was reported that a-phases are present in the liquid silk extracted from the silk

gland of S. cynthia ricini39,40 and random coil conformation

in regenerated and gland protein B. mori fibroin films.26 For

subsequent use of these films as biomaterials, a more ordered

crystalline structure is required which provides stability

to the films,26 and this can be achieved by treatment with

Table I Dyanamic Contact Angle of Fibroin Films in Two

Different Solvents—Water and n-Heptane

Type of film Solvent Mean Contact Anglea

A1 Water 77.5246 6.88

A1 n-heptane 84.0466 0.107

B1 Water 75.4316 4.56

B1 n-heptane 83.1246 0.97

B. mori Water 71.56 6 0.65

B. mori n-heptane 75.2 6 1.20

a Average 6 standard deviation (n 5 3).

FIGURE 2 FTIR spectra of A, A1, B, and B1 in the spectral range of 400–4000 cm21.

330 Dutta et al.

Biopolymers

methanol which is known to induce formation of crystalline

beta-sheets.41–43

Three types of structures are proposed for silk viz. Silk I,

Silk II, and Silk III. Silk I refers to the water soluble structure

existing within the silkworm gland before spinning. Silk II is

the insoluble extended beta-sheet conformation which forms

after spinning of silk fibers. Silk III is an unstable structure

observed at the water–air interface.44–46 Pure silk fibroin film

has a characteristic peak at 2h � 12.2847 which indicates Silk

I structure. In this study, this peak was evident around 11.68in the muga and eri film, confirming Silk I structure. The

degummed fibers (A and B) showed typical Silk II peaks at

16.78 and 20.28 denoting crystalline beta-sheet structures.48

The thermal properties were studied by DSC and TGA.

The DSC study reveals that the first endothermic peak

(Figure 5) below 1008C in A1 and B1 and C1 is due to the

evaporation of bound water. Two major endotherm-exo-

therm transitions above the glass transition temperature Tg

(190–2008C)49 reported for fibroin, were seen in the temper-

ature range of 220–2268C in A1 and 208–2238C in B1. These

transitions are due to the conversion of the amorphous films

to crystallized ones during the scans.50–52 The glass transition

and subsequent crystallization provide clear evidence of large

amount of amorphous structure in the films. After crystalli-

zation, A1 and B1 started to degrade around temperature

3508C. On the contrary, C1 degraded at a much lower tem-

perature of 2648C indicating lower thermal stability. On the

other hand in A, B, and C, the first endothermic peak was

around 65.58C, 77.58C, and 648C, respectively, which are due

to loss of moisture. The films contained higher amount of

bound water than the fibers because of their higher amount

of amorphous region as moisture can bind to silk through

FIGURE 4 DSC curves of fibers and films. A: A. assamensis fiber, A1: A. Assamensis film, B: S.

ricini fiber, B1: S. ricini film, C: B. mori fiber, C1: B. mori film.

FIGURE 3 X-ray diffraction curves of A, A1, B, and B1 in the 2h range of 10–408.

Biomaterial Film from Liquid Silk of Muga and Eri Silk 331

Biopolymers

hydrogen bonds in the amorphous region. Two minor and

broad endothermic transitions appeared above the glass tran-

sition temperature at 230 and 3008C in A and B which can

be related to the degradation of amorphous silk,49 and the

major endothermic peaks at 3648C for A, 356.48C for B, and

2678C for C are attributed to the thermal degradation of silk

fibers with beta-sheets.53,54 It is noteworthy that the films

decomposed at a lower temperature than the fibers because

of its amorphous nature and crystals that form during

the thermal transition are not as strong as those available

in a fiber.

The DSC and TGA data compliment each other. Silk

fibroin cast from aqueous solutions contains bound water

even after they are dried.26 The initial weight loss in the films

around 138.58C is due to the loss of moisture. The second

weight loss took place in the range of 235–3778C associated

with the breakdown of fibroin. In the fibers, the initial weight

loss was around 968C due to less water containment than the

films. The second weight loss took place in the range of 245–

3868C because of the highly ordered crystalline structure of

the fibers. The amorphous sample showed faster degradation

curve as supported by DSC results.

CONCLUSIONSThis report gives an insight into the structure and properties

of fibers and films from two wild silkworms from a very

remote corner of India, which may be helpful in exploiting

them as potential biomaterials. Films from this two species

are thermally more stable than the B. mori films, and the sur-

face wetting property shows that surface of muga and eri

films are more hydrophobic in water and unaffected by

organic solvent compared to B. mori film surfaces. Therefore,

the silk gland two dimensional films may be considered as

potential biomaterial by virtue of its good water stability and

thermal properties which are essential for biomedical

applications. Additionally, the film obtained from liquid silk

will be devoid of any chemicals; hence, it will be nontoxic

and the practice of removing the chemicals will be dimin-

ished. However, further studies are warranted for monitoring

a range of biological properties such as cell supporting,

biocompatibility, and biodegradability to understand their

biomedical applications.

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Reviewing Editor: Kenneth J. Breslauer

Biomaterial Film from Liquid Silk of Muga and Eri Silk 333

Biopolymers