Silk Gland Fibroin from Indian Muga Silkworm Antheraea assama as ...

Tissue Engineering and Regenerative Medicine, Vol. 10, No. 4, pp 1-11 (2013) DOI 10.1007/s13770-012-0008-6

｜Original Article｜

Silk Gland Fibroin from Indian Muga Silkworm Antheraea assama as Potential Biomaterial Subrata Kar1, Sarmistha Talukdar1, Shilpa Pal1, Sunita Nayak1, Pallavi Paranjape1 and S. C. Kundu1* 1

Department of Biotechnology,Indian Institute of Technology Kharagpur, West Bengal-721302, India (Received: December 2nd, 2012; Revision: April 7th, 2013; Accepted: April 7th, 2013)

Abstract : There is an increasing demand for new versatile biomaterials. Silk is reported by many researchers as an ideal biomaterial for different biomedical applications. Most of the studies are carried out on mulberry silk Bombyx mori, however silk from Indian non-mulberry silkworms are relatively unexplored. In this report we fabricate 2D matrices from the regenerated aqueous silk fibroin protein of the glands of non-mulberry Indian muga silkworm Antheraea assama (assamensis). Its biochemical, biophysical characteristics and its cytocompatibility for biomedical uses are evaluated. The properties of this muga gland fibroin are compared with silk gland fibroin from non-mulberry Indian tropical tasar silkworm Antheraea mylitta and with the fibroin from the cocoon of mulberry silkworm Bombyx mori. The gland fibroin of Antheraea assama is observed to consist of two polypeptides of approximately 250 kDa each linked by disulfide bond. Fourier transformed infrared spectroscopy and X-ray diffraction studies indicate random coil structure of dissolved fibroin solution. The alpha-helical structure in 2D films changed to beta-sheets upon ethanol treatment, imparting crystallinity and insolubility. The fibroin film is found to be the least hydrophilic, followed by B. mori and A. mylitta silk. Biocompatibility of the films from all three species is investigated through the cell attachment and spreading study of MG-63 human osteoblast-like cells. The cytocompatibility of non-mulberry fibroin matrices are comparable with that of standard tissue culture plates. The results indicate that the non-mulberry Indian muga silk gland fibroin is also suitable matrix as a natural biomaterial for tissue engineering applications. Key words: muga silkworm, fibroin, film, crystallinity, biomaterial

toughness and hardness to the fibre. Apart from its traditional use in the textile industry, mulberry silk has a huge role in biomedical applications,3,7,8 cosmetics9 and food industry.10 Non-mulberry muga silk is produced by Antheraea assama (assamensis), which is cultivated only in the North Eastern regions of India, particularly in the state of Assam. It feeds on the leaves of Machilus spp. The known superiority of this silk is in its durability and its glossy nature. The golden colour of the muga yarn as found in its original form is retained in the matrices because the fibres can neither be bleached nor dyed due to its low porosity.11 Earlier, fibroins of S. ricini have been compared with A. assama and B. mori12 and they are reported to be immunologically distinct from each other. The silk fibroin mainly from B. mori and also non-mulberry A. mylitta are widely used as a substrate for cell-culture in tissue engineering purposes 13-16 and for controlled drug delivery applications.17,18 Fabrication of blood compatible scaffolds having biomimetic architecture is also reported from fibroin derived from cocoons of A. assama.19 It is reported that RGD (arginine, glycine and aspartate) sequences are present in non-mulberry fibroin of A. mylitta,20 A. pernyi21

1. Introduction Silk is a proteinaceous secretion from diverse organisms of many arthropods to build structures external to their body, such as cocoons and webs.1 Among the silk producing organisms, spiders and silkworms have received special attention because of their tough mechanical properties and also for their wide range of applications.2-4 The silkworm species belong to the class Bombycidae (Bombyx mori) and Saturniidae of the order Lepidoptera.5 The non-mulberry silks which are mainly produced by species of the Antheraea and Attacini tribes of the family Saturniidae are tasar, muga, eri and fagaria.6 The silk obtained from silkworms consists of two kinds of protein: the core protein fibroin, which is hydrophobic and fibrous in nature and sericin, also known as the ‘glue protein’ which is hot water soluble glycoprotein that envelopes the inner fibroin imparting *Corresponding author Tel: +91-3222-283764; Fax: +91-3222-278433 e-mail: [email protected] (S. C. Kundu)

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Subrata Kar et al.

and A. yamamai.22 The RGD peptide sequence is partially responsible for cell attachment and proliferation.23 This makes the non-mulberry fibroin protein a potentially good biomaterial.1416,24,25 Sufficient amount of fibroin from the most widely used silk fibroin biomaterial can easily be obtained from Bombyx mori cocoons than from the glands using reported extraction and dissolution protocols.3 However, there is not much difference between the fibroins obtained from the silk gland as well as from the mulberry silk cocoons of B. mori.26 Fibroin from non-mulberry sources can also be extracted from the cocoons using such protocols, but the amount obtained is very low.14 The methodologies for dissolution of silk from nonmulberry cocoons to obtain higher yields of fibroin remain unsolved till today. Comparatively large amounts of fibroin can be obtained from the silk glands of non-mulberry silkworms. So far less work is carried out based on the cocoon fibres of non-mulberry silks as compared to B. mori due to the unavailability of suitable method to regenerate fibroin in aqueous solution from the fibres. For this reason the biomedical applications of silk fibroin from non-mulberry species including Antheraea assama are still limited. In the present work the silk fibroin is directly extracted from the silk gland, before the formation of the cocoon, using the presently available method for other non-mulberry species.24 In earlier work the cocoon mats (unwoven cocoon material) of Antheraea assama are used as scaffold material and not from the regenerated silk fibroin in aqueous solution.19,27 In the present study regenerated aqueous silk fibroin is used for fabrication of the matrices. The advantage of using the regenerated aqueous silk protein from the glands is the relative ease for fabricating different forms like 2-D films/ 3D matrices/ sponge scaffold /gels/nanoparticles for different biomedical applications in immediate future. Muga silk fibre (silk mat) produced by Antheraea assama is reported to be used as biomatrix for immobilization of cholesterol oxidase.28 The effect of different organic solvents on tensile strength of desericinized silk fibres produced by Antheraea assama is also studied.29 Very recently biophysical characterization of the films from silk gland of muga and eri silkworm is reported.30 Silk gland fibroins extracted from 5th instar larvae of muga silkworm is not yet utilized for cytocompatibilty test for the silk matrices for using as biomaterial. This gland fibroin will allow us to design various types of biomaterial matrices to be used for different biomedical applications. In this report we investigate the physico-chemical aspects of the non-bioengineered fibroin protein obtained from the silk gland of 5th instar larvae of Indian muga silkworm A. assama. Silk fibroin is characterized by SDS-PAGE, FTIR, XRD, SEM,

dynamic contact angle, and thermogravimetry. Simultaneously, for comparison, similar experiments are carried out with fibroin of the cocoon of mulberry B. mori and gland fibroin protein of non-mulberry A. mylitta. Biocompatibility of the silk gland fibroin from muga is carried out to establish it as biomaterial for different biomedical applications.

2. Materials and Methods 2.1 Materials Mature 5th instar larvae of muga silkworm were obtained from Khagrabari silk farm, Cochbehar district of West Bengal, a place close to the state of Assam. At the same time, mature 5th instar worms of tasar silkworm A. mylitta and cocoons of B. mori were obtained from nearby silk farms in West Midnapore district of West Bengal, India. Sodium dodecyl sulphate (SDS) (SRL, Mumbai, India), polyethylene glycol (PEG-6000) (SRL), cellulose dialysis tubes cut off 12 kDa (Pierce, USA), tissue culture grade polystyrene plastic flasks (Nunc, USA) and plates (Tarsons, India), cell culture grade chemicals including Modified eagle medium (MEM, Gibco BRL, USA), fetal bovine serum, trypsin-EDTA and penicillin-streptomycin antibiotics (Gibco BRL), Rhodamine-phalloidin and Hoechst 33342 (Molecular Probes, USA), Alamar Blue (Invitrogen, USA), MTT (Sigma-Aldrich, USA) were purchased for this experimentation. 2.2 Preparation of Regenerated Gland Fibroin Solution from Non-Mulberry Species and Cocoon Fibroin from Mulberry Species The fibroin from A. assama was extracted from the mature silk gland and compared with the fibroin of another non-mulberry silk species A. mylitta using same extraction procedure described by Mandal and Kundu24 and mulberry silk fibroin obtained from cocoon by most commonly used LiBr dissolution method.4 Mature larvae of A. assama and A. mylitta just prior to spinning were carefully dissected and the whole silk glands were taken out. After washing with deionised water the glands were carefully squeezed with forceps to extrude out the silk protein. This was followed by repeated washing to remove the water soluble sericin, and the remaining hydrophobic fibroin protein was collected. The water insoluble fibroin was dissolved according to the method described earlier.24 Briefly, the gland protein was dissolved in a solution containing 1% SDS, 10 mM Tris (pH 8), 5 mM EDTA under constant stirring for 30 mins at room temperature. Excess SDS was removed by dialysis against deionised water for 12 hrs with regular changes of water. B. mori fibroin can be largely extracted from the

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Silk Gland Fibroin from Indian Muga Silkworm Antheraea assama as Potential Biomaterial

cocoons hence the fibroin from the cocoon was extracted according to the established protocol.31 Fresh cocoons were cut into small pieces and degummed using 0.02 M sodium carbonate for 30 mins. The degummed fibres were washed several times with water and dried in the oven. The degummed fibre was then dissolved in 9.3 M lithium bromide at 55oC for 3 hrs. The Li+ ions in the solution were removed by dialysis against deionised water for 48 hrs with regular changes of water. Concentration of the dialysed protein solution was measured by gravimetric method. Films were casted on teflon coated boats using the 1% (w/v) fibroin solution and dried in laminar air flow followed by treatment of some films with 70% alcohol.

width at half maximum, and θ is the scattering angle.

2.3 Estimation of Molecular Weight Through (SDSPAGE) The dialyzed gland protein fibroin of A. assama was analyzed by 6% SDS-PAGE for estimating the molecular weight of the protein both in reducing and non-reducing conditions. The molecular weight of gland fibroin from A. mylitta20,24 and of B. mori fibroin32 was previously determined.

2.9 Dynamic Contact Angle Dynamic contact angle of the films (n = 3) was measured using a polar (distilled water) solvent and an organic solvent (nHexane) on SCAT (Data Physics InstrumentsQ13). Fibroin films were cut in a rectangular shape. The thickness and width of the immersing surface (sample) was recorded using digital digimetric callipers. All the experiments were conducted at a controlled temperature of 24oC.

2.7 Thermogravimetric Analysis (TGA) TGA of the films (5 mg) was carried out in Pyris Diamond TG-DTA to measure changes in the weight of the fibroin films with increasing temperature. The experiment was done in an alumina crucible under nitrogen atmosphere with a temperature range of 50o-650o with an increase of 10oC/min. 2.8 Differential Scanning Calorimetry (DSC) DSC studies of the films were done in Perkin-Elmer Diamond DSC in nitrogen atmosphere with temperature range of 50 to 400oC with an increase of 10oC/mins.

2.4 Scanning Electron Microscopy (SEM) SEM studies of fibroin films were performed in JEOL JSM 5800 at an operating voltage of 20 kV for the surface overview of untreated and alcohol treated fibroin films.

2.10 Cell Culture and Seeding 2.10.1 Maintenance of MG-63 Osteoblast-like Cell Line The cells were cultured in MEM medium with 10% FBS. The culture medium was replaced every 3 days. Confluent monolayers were washed with sterile phosphate-buffered saline (PBS) and split with the use of 0.05% trypsin/EDTA solution.

2.5 Fourier Transform Infrared (FTIR) Spectroscopy To understand the secondary structure of fibroin solution and films (untreated and alcohol treated), FTIR analysis was done from wave numbers 400 cm-1 to 4000 cm-1. Measurements were taken at a resolution of 4 cm-1 with 32 scans. For samples in aqueous solution form, the spectrum of water was subtracted. Analysis of the data was carried out using Origin 8.0 software.

2.10.2 Cell Attachment on Different Matrices MG-63 cell attachment kinetics was estimated by measuring the unattached cell concentration as a function of time after inoculation. MG-63 cell line was cultured in MEM medium with 10% FBS. The fibroin films were cast on tissue culture 6 well plates, air dried, sterilised by immersion in 70% ethanol for 30 mins and treated with UV for 20 mins. This was followed by washing the films in sterile phosphate-buffered saline (PBS). MG-63 cell line was cultured in MEM medium with 10% FBS. Cell attachment kinetics was estimated by measuring the unattached cell concentration as a function of time after inoculation. Cells were detached from the culture flasks with trypsin and resuspended in 2 mL of medium and seeded directly to the film already cast on 6 well tissue culture plates at a concentration of 1×105 cells/mL per well. Cells seeded in tissue culture plates were used as a control. Samples were incubated at 37o C in a humified 95% air, 5% CO2 atmosphere and maintained under the same conditions for 24

2.6 Wide Angle X-ray Diffraction (XRD) XRD study was carried out in Philips X’Pert using CuKα in the angle range 10o to 50o at 3o per minute increment target to determine the crystalline and amorphous nature of the film for understanding the secondary structural conformity of the alcohol untreated and treated fibroin film. The graphs were plotted using Origin 8.0 software. From the full width at half maximum intensity of the peaks the estimated crystallite size L was calculated using the Scherrer equation L = 0.9λ/ (B cosθ)

(1)

Where λ is the wavelength of radiation, B is the peak full

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hours. Observations were made after 0.5, 1, 2, 4, 6, 8, 10, 12 hrs of seeding. The number of cells present in the supernatant media was enumerated and deducted from the initial number of cells seeded. 2.10.3 Cell Spreading on Different Matrices MG-63 cells (1×105 cells/mL) were seeded on 6-well plates with or without fibroin coating of A. assama, A. mylitta, and B. mori. Cells were allowed to attach and spread. Observations were made after 0.5, 1, 2 and 4 hrs of seeding. The 2D image analysis was performed with Image J software program. At least ten microscopic fields per sample were randomly acquired in the central and peripheral regions. The following geometrical features were determined for each object element: (1) cell area (expressed in µm2), and (2) cell perimeter (µm). 2.10.4 Confocal Laser Scanning Fluorescence Microscopy Cell morphology of MG-63 osteoblast-like cells on A. assama, A. mylitta and B. mori fibroin coated plates and tissue culture plates were assessed using confocal microscopy. Cells were seeded on fibroin coated and uncoated films at a seeding density of 1×105 cells/mL. After 7 days of growth, the seeded films were fixed in 4% paraformaldehyde for 10 mins, followed by membrane permeabilisation in 0.2% Triton X-100 in PBS for 5 mins. Cells were then stained with rhodamine phalloidin(10 µg/mL) and Hoechst 33342 (5 µg/mL) for actin and nuclei visualization, respectively. Seeded fibroin coated and noncoated film cells were imaged using a confocal laser scanning microscope (CLSM, Olympus FV1000 attached with inverted microscope IX 81, Japan) equipped with Argon (488 nm) and He-Ne (534 nm) lasers. 2D multichannel image processing was carried out using FV 1000 Advance software version 4.1 (Olympus, Japan).

Figure 1. 6% SDS-PAGE gel showing bands of muga silk protein fibroin of A. assama; lane 1: non-reducing condition, lane 2: reducing condition, M: molecular weight marker.

Figure 2. SEM images of: A. assama fibroin films; (A) untreated, (B) 70% alcohol treated, B. mori fibroin films; (C) untreated, (D) 70% alcohol treated and A. mylitta fibroin films; (E) untreated, (F) 70% alcohol treated. Scale bar represents 10 µm.

2.11 Statistical Analysis All quantitative experiments were run in triplicate and the results are expressed as mean±standard deviation for n = 3, unless indicated otherwise. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA). Differences between experimental groups at a level of p ≤ 0.05 were considered as statistically significant and those at p ≤ 0.01 as highly significant.

3. Results and Discussion

tion as well as in the reducing condition. The estimated molecular weight of the non-reducing condition is approximately 500 kDa which is a homodimer of 250 kDa polypeptides. The A. mylitta silk gland protein fibroin is a dimer of approximately 197 kDa of total 395 kDa,20,24 while fibroin of B. mori is composed of two chains, one of 350 kDa and the other of 25 kDa.32

3.1 Gel Electrophoresis The SDS-PAGE analysis of A. assama protein fibroin is shown in Fig 1. The gel shows a single band in the non-reducing condi-

3.2 Scanning Electron Microscopy Surface analysis of the fibroin films prepared from three silkworm species (A. assama, A. mylitta and B. mori) are

4


Table 1. The FTIR data showing amide I region and their significance for fibroin solution, untreated and alcohol treated fibroin films of A. assama (Aa), A. mylitta (Am) and B. mori (Bm).

presented in Fig 2. Surface topography of alcohol untreated fibroin films of A. assama (Fig 2A), A. mylitta (Fig 2B) and B. mori (Fig 2C) observed by SEM images shows relatively flat and smooth morphology with no scratches. The surface of the treated silk fibroin films A. assama (Fig 2D), A. mylitta (Fig 2E) and B. mori (Fig 2F), as observed at higher magnifications revealed granular morphology with lateral granular sizes reaching a fraction of a micrometer. Ethanol treatment was carried out mainly for imparting crystallinity and insolubility to the films fabricated from the regenerated silk fibroin solution. The alcohol treatment induces crystallinity that produces a stretching force inducing rough surface formation.

Materials Fibroin solution

Aa

Am

Bm

1639 cm-1 1639 cm-1 1644 cm-1

Significance Random coil

Random coil Untreated 1660 cm-1 1660 cm-1 1655 cm-1 and alpha-helix (film) Treated (film)

3.3 Fourier Transform Infrared (FTIR) Spectroscopy The FTIR spectra pattern is used to interpret the secondary structural conformation of the silk fibroin. Out of many IR absorption bands of protein molecule, amide I (1600-1700 cm-1) due to C=O stretch vibration of protein chain, is very significant for understanding the protein secondary conformation. The FTIR spectra of muga silk in solution form and of treated and untreated films is shown in Fig 3. The regenerated fibroin solution of A. assama showed a peak at 1639 cm-1 in the amide I region (1600-1700 cm-1). This region signified that the solution was in random coil (silk I) conformation.33 A similar result was also observed for the fibroin solution prepared from A. mylitta and B. mori, showing absorption band at 1639 cm -1 and 1644 cm-1 respectively both signifying random coil structure. The untreated fibroin film of A. assama exhibited amide I absorption peak at 1660 cm-1 as seen in Fig 3 representing helical conformity. The untreated film from B. mori exhibited amide I peak at 1655 cm -1 , which represented the random coil conformation.26 The amide I absorption peak of A. assama fibroin film treated with 70 % alcohol was at 1625 cm -1 indicating β-sheet (silk II) structure.33 FTIR studies indicate that the random coil structure of the dissolved fibroin solution and the alpha-helical structure in 2D films changed to beta-sheets upon ethanol treatment, imparting crystallinity and insolubility to

1625 cm-1 1633 cm-1 1627 cm-1 β-sheet structure

Figure 4. XRD of 1% silk fibroin protein films (untreated and 70% alcohol treated) prepared from (A) A. assama, (B) A. mylitta and (C) B. mori.

the films. The amide I absorption band of each form of fibroin from the three silk species with their respective significance is represented in Table 1.

3.4 Wide Angle X-ray Diffraction (XRD) The X-ray diffractogram of fibroin film from A. assama was similar to the diffraction pattern of untreated A. pernyi film, representing β-helical conformation.34 After alcohol treatment, a new peak was observed at 16.67o along with the pre-existing peak, which signified the formation β-sheet (Fig 4A). The result supported the data obtained from the FTIR study.34 The presence of silk I structure in the A. assama fiboin film has also been confirmed.30 In the case of A. mylitta silk, a major peak was observed near 22.53o representing α-helix conformity and a peak near 28º (Fig 4B), which represented α form (silk I) conformity of the protein. After treatment with 70% ethanol a new peak was obtained at 17º representing the formation of the β-sheet structure34 and the peak near 28º was not visible. The B. mori fibroin showed a sharp peak at around 22° for both treated and untreated films (Fig 4C) signifying silk I structure.26 A

Figure 3. FTIR spectra of A. assama silk gland fibroin solution (1%), untreated and 70% ethanol treated films (1% fibroin).

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Table 2. Presentation of the results obtained from Scherrer analysis of the XRD data of untreated and alcohol treated fibroin films of A. assama (Aa), A. mylitta (Am) and B. mori (Bm). Parameters o

Aa

Am

represented in a summary Table 2.

3.5 Thermogravimetric Analysis (TGA) Fibroin films of the three different silkworm species showed a trend of weight loss with increase in temperature in the TGA plots (Fig 5). The mass loss of fibroin films in B. mori and A. assama showed a similar two -step breakdown. In the case of B. mori fibroin, the degradation of the untreated and alcohol treated films around 100oC was attributed to the loss of bound water. The degradation from 150oC to 298oC (Fig 5C) is due to the breakdown of the amino acid side chains. Final degradation started around 298oC with a dominant peak at around 302oC.36 This process continued up to 570oC resulting in the total breakdown of the protein. A. assama film showed a similar mass loss at around 140-150oC and continued up to 268oC in the case of untreated at 262oC in the case of treated film (Fig 5A). The next step in degradation started around 276oC in the case of the untreated film and 262oC for the treated film that continued up to 560-570oC. A. mylitta film showed a three -step degradation curve as also observed in A. pernyi (Fig 5B).21 The process of weight loss started at 140-150o C both for the untreated and treated films, and continued up to 268oC for the treated up to 244oC for the untreated film. The next step started in between 240 and 270oC and continued up to 320oC. The last step of degradation began from 320oC and ended around 542oC for the untreated film whereas the treated film degraded at around 620oC. The above process of degradation has been clearly shown in DTA curve given as an inset in Fig 5. Treated films prepared from B. mori and A. assama species did not show much difference in their degradation patterns. This may be due to the fact that the structural change had little effect on the thermal decomposition pattern. In the case of the A. mylitta film there was a sharp difference at the final degradation as the untreated film degraded at 542oC whereas the treated one degraded at 620o C showing an increase in stability. The degradation pattern of each of the alcohol treated and untreated

Bm

Untreated Treated Untreated Treated Untreated Treated

2θ/

22.53

22.45

22.55

22.57

22.32

22.42

d/Ao

3.94

3.95

3.93

3.93

3.977

3.96

FWHM/o

0.982

1.131

1.042

0.91

1.022

1.07

Crystal size Ao(L)

82.64

71.72

78.08

88.98

79.22

75.66

small peak at around 28o was also present in the untreated film, representing silk I conformity.26 The diffractograms of treated films showed a peak at around 14 o representing silk II structure,26 and a small peak at 17o. This signified that the silk II structure was induced upon alcohol treatment. It was not present in the untreated ones. The interplaner spacing (d) was calculated from the Braggs equation and crystal size and FWHM of each material was analysed by Scherrer equation as

Figure 5. TGA plots of the untreated and 70% alcohol treated fibroin films (A) A. assama, (B) A. mylitta and (C) B. mori.

Table 3. TGA data analysis indicating the temperature range on the degradation of A. assama (Aa), A. mylitta (Am) and B. mori (Bm) for untreated and alcohol treated fibroin films. Fibroin films

Aa

Am o

Untreated

Properties affected o

80-100 C

80-100 C

80-100 C

Loss of bound water

149-268oC

140-240oC

150-298oC

Disruption of side chain

276-560oC

240-320oC

298-570oC

Degradation of fibroin

320-542 C

-


80-100oC

80-100oC

Loss of bound water Disruption of side chain

o

80-100oC Alcohol treated

Bm o

o

150-262 C

140-268 C

150-298oC

262-570oC

270-320oC

298-570oC


-


-

o

o

320-620 C

6


an exotherm at 263ºC (Fig 6B). The endotherm at 234ºC corresponds to the breaking-up of the pre-existing bond while the exotherm signifies the formation of β-sheet structure molecules. This result represents the same nature as observed in the case of the A. pernyi fibroin.21 The treated fibroin films showed a single endotherm at 363oC. The major endotherms implied the degradation and disorientation of the ordered structure within the material.21 In the case of B. mori an endotherm was also observed at 284oC and 289oC for the untreated and treated films respectively (Fig 6C).37 The untreated film from B. mori showed an exothermic peak, near 214oC corresponding to the phase transition from the amorphous βhelical structure to a more crystalline β-sheet form. The thermal stability of the A. assama and A. mylitta gland fibroin is much more than that of the mulberry silk fibroin. This holds true even when compared with the untreated and alcohol treated fibroin protein obtained from non-mulberry silk gland and mulberry silk cocoon, respectively. This suggests a greater chemical stability of the non-mulberry gland fibroin.30

Figure 6. DSC thermograms of fibroin films (treated with 70% ethanol and untreated: A. assama (A), A. mylitta (B) and B. mori (C).

fibroin film with their respective significance is represented in a tabular form in Table 3.

3.7 Contact Angle The dynamic contact angle measurements of fibroin films in distilled water of the three species indicated that the hydrophilicity was highest in the A. mylitta film having the least mean contact angle (Table 4) followed by the B. mori and A. assama films. Previous studies of the dynamic contact angle measurement of B. mori fibroin film26 indicated the film to be hydrophobic. The water contact angle of A. mylitta gland fibroin film was previously studied24 were comparable with our results. The treated films behaved differently and provided erroneous results because of their repulsion to water when the film approached the solvent. The mean contact angle in nhexane for the untreated films showed that A. assama film had more solubility in the organic solvent than B. mori and A. mylitta. While the alcohol treated A. assama film was more soluble in the n-hexane than A. mylitta film. Moreover B. mori treated films showed the least solubility. The similar dynamic

3.6 Differential Scanning Calorimetry (DSC) DSC analysis was performed on the alcohol treated and untreated fibroin films from different species (Fig 6). The fibroin film of A. assama exhibited an endothermic peak at 371oC for the untreated and 361oC for the alcohol treated films representing the degradation and disorientation of the protein molecule (Fig 6a). This is supported by the recent work carried out on A. assama fibroin film.30 The untreated film showed an endothermic peak at 233.7oC, which represented the breakingup of the hydrogen bonds in α helical form. Treated films which had already attained β-sheet conformity did not show this endothermic peak. The commonly visible exothermic peak as observed in A. pernyi fibroin22 was not observed in the case of A. assama untreated fibroin film which signified that the heat treatment did not induce crystallization. A. mylitta untreated fibroin films showed two endotherms at 234ºC and 369ºC and

Table 4. Dynamic contact angle of untreated and treated fibroin films of A. assama (Aa), A. mylitta (Am) and B. mori (Bm) in water and n-hexane. Silk fibroin films Aa Am Bm

Advancing contact angle

Receding contact angle

Mean contact angle

Water

n-Hexane

Water

n-Hexane

Water

n-Hexane

84.25

94.70

96.18

94.45

90.21±2.4

94.50±1.63

Treated

-

20.70

-

11.92

-

16.00±2.65

Untreated

88.36

20.59

65.29

15.54

76.82±3.55

18.06±4.70

Untreated

Treated

-

96.67

-

75.10

-

86.78±5.06

Untreated

90.63

14.49

79.40

40.80

85.00±3.74

41.15±3.94

Treated

-

97.76

-

97.80

-

97.75±4.59

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Subrata Kar et al.

Figure 7. (A) Morphology of MG-63 cells grown on silk fibroin matrices after 7 days of culture; (a) A. assama (Aa) silk fibroin films, (b) B. mori (Bm) silk fibroin films, (c) A. mylitta (Am) silk fibroin films, and (d) tissue culture plate. (B) (Left) Time dependent attachment of cells on different 2D matrices (n = 3, p < 0.01), (Right) the matrix surface area covered by cells represented as mean±standard deviation of five fields studied for each type of matrices. (n = 10, * represent significant statistical difference, p < 0.05). The 2D image analysis was performed with Image J (NIH) software. Aa: A. assama silk fibroin, Bm: B. mori silk fibroin, Am: A. mylitta silk fibroin, TCP: tissue culture plate. Scale bars represent 250 µm. Cell spreading observed on TCP matrices were considered to be as the control group, depicting normal cell morphology. There was no significant difference in cell area and perimeter of the cells grown on TCP and Am matrices. Considering cell spreading on TCP to be 100%; cell area and perimeter of cells grown on Aa matrices were 72.7% and 70.8% respectively.

contact angle value both in water and n-hexane of A. assama fibroin was more than the silk fibroin from mulberry B. mori.30

in Fig 7B. Considering cell spreading on TCP is to be 100%; the cell area and perimeter of the cells grown on non-mulberry fibroin Am and Aa matrices were 72.7 % and 70.8% respectively. When plated onto artificial adhesive surfaces, the cells first attach themselves to the surface. The cells do not immediately attain their normal morphology. After ascertaining the cell adhesive property of the matrix, the signalling pathways to cell spreading is elicited. The early stages of cell spreading are biochemically and biophysically involved in regulating the cell functions.39 Many factors, mostly matrix properties, such as the presence of integrin binding sites, affect cell attachment and its spreading there after39 The cell morphology particularly the spreading of the cell, is an important indicator of healthy cells. Proper spreading (similar to the control matrix group) indicates that the supplied matrix may be cytocompatible. Either more or too little spreading of cells causes deformed cell morphology, which indicates the poor health of the cells.

3.8 Cell Attachment and Spreading So far no cell attachment, spreading, behaviour, morphology, viability and proliferation studies are carried out on gland fibroin of muga silk A. assama. Attachment of the cells to A. assama (Aa) fibroin films was similar to that of A. mylitta (Am) fibroin. The cells spread significantly more on TCP and A. mylitta fibroin than on B. mori fibroin. The cell spreading on A. assama fibroin is comparable to that on A. mylitta fibroin as shown in Fig 7A, a-d. The matrices of Aa and Am responded similarly to the control (TCP) in terms of cell adhesion properties. The cells (1×105) were initially seeded on all the matrices. It is considered that the cell attachment on TCP is to be 100%. The cell attachments were 95.4%, 94.9% and 52% on Aa, Am and Bm respectively after two hours only. After allowing the cells to attach for 4 hours no significant difference was observed in the cell attachment on TCP, Aa, Am or Bm matrices. There was no significant difference in cell area and perimeter of the cells grown on TCP and Am matrices as shown

3.9 Cell Viability and Proliferation The MTT results showed that the silk films obtained from all the three sources are biocompatible, and possess non-toxic

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Figure 8. (A) Cytocompatibilty tests of three different silk fibroin matrices by culturing MG-63 cells: (a) MTT assay and (b) Alamar blue assay. Each point represents mean from three independent experiments. The error bars denote standard deviation, n = 3, p < 0.05, * represent significant statistical difference. (B) Confocal laser scanning micrographs of MG-63 cells after 7 days of growth on the fibroin films of (a, e) A. assama fibroin, (b, f) B. mori fibroin, (c, g) A. mylitta fibroin and (d, h) tissue culture plate. (a-d) represent images taken at lower magnification and (e-h) represent images at higher magnification. The cells were stained with rhodamine-phalloidin for actin filaments (red) and Hoechst 33342 for nuclei (pseudo green). Scale bars represent 100 µm.

behaviour (Fig 8A, a). The tissue culture plate (TCP), A. mylitta and A. assama fibroin coated surfaces were found to be confluent within 5 days of incubation with 1×105 initial cell seeding density in 6-well tissue culture plates. The cells on B. mori reached confluency on day 7. The Alamar Blue assay was carried out on 0.5×105 cells seeded on fibroin coated and noncoated tissue culture plates. Maximum cell growth was obtained on day 7. The cell viability and proliferation of A. assama, and A. mylitta were comparable with that of TCP (Fig 8A, b). No significant differences were observed in the growth kinetics of the cells on the three matrices. The growth of cells on the non-mulberry fibroin and TCP was found to be significantly more than that on B. mori films. These results are in agreement with the earlier comparative studies conducted between nonmulberry and mulberry matrices.14,16 The cells showed a similar matrix response pattern irrespective of initial cell seeding density. The enhanced cell attachment, spreading and proliferation on non-mulberry fibroin matrices may be due to the presence of RGD sequences in the fibroin of A. mylitta, A. pernyi and A. yamamai.20,40,41 The results are in agreement with earlier works on non-mulberry fibroin.14,16,17,24,25 The cell culture results on A.

assama fibroin films indicate that the matrix is cytocompatible.

3.10 Cell Morphology under Confocal Microscopy MG-63 cells were cultured on three different types of fibroin films and TCP for 7 days. Cell morphology was observed after 7 days of culture (Fig 8B, a-h). Rhodamine -Phalloidin was used to stain actin microfilaments. The cells showed well developed actin filaments and distinct nuclei, indicating healthy cell morphology. Cell growth and morphology on A. assama and A. mylitta were comparable with TCP. The cells showed well spread and attached morphology on B. mori fibroin films.

4. Conclusions Silk gland fibroin films fabricated from Indian muga silkworm, Antherea assama display almost similar biophysical properties as those of mulberry and other non-mulberry silkworm species. This fibroin is also equally biocompatible and non-toxic. The cell response to the fibroin of A. assama and A. mylitta are comparable with that of the tissue culture plate. This may be due to the presence of RGD sequences in non-

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Bombyx mori, DNA Cell Biol, 23, 149, (2004). 13. K Inouye, M Kurokawa, S Nishikawa, et al., Use of Bombyx mori silk fibroin as a substratum for cultivation of animal cells, J Biochem Biophys Meth, 37, 159 (1998). 14. C Acharya, SK Ghosh, SC Kundu, Silk fibroin protein from mulberry and nonmulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion, J Mater Sci Mater Med, 19, 2827 (2008). 15. BB Mandal, SC Kundu, Non-bioengineered silk fibroin protein 3D scaffolds for potential biotechnological and tissue engineering applications, Macromol Biosci, 8, 807 (2008). 16. S Talukdar, M Mandal, DW Hutmacher, et al., Engineered silk fibroin protein 3D matrices for in vitro tumor model, Biomaterials, 32, 2149 (2011). 17. Y Wang, DD Rudym, A Walsh, et al., In vivo degradation of three-dimensional silk fibroin scaffolds, Biomaterials, 29, 3415 (2008). 18. BB Mandal, SC Kundu, Osteogenic and adipogenic differentiation of rat bone marrow cells on non-mulberry and mulberry silk gland fibroin 3D scaffolds, Biomaterials, 30, 5019 (2009). 19. N Kasoju, U Bora, Antheraea assama silk fibroin-based functional scaffold with enhanced blood compatibility for tissue engineering applications, Adv Eng Mater, 12, B139 (2010). 20. A Datta, AK Ghosh, SC Kundu, Differential expression of the fibroin gene in developmental stages of silkworm, Antheraea mylitta (Saturniidae), Comp Biochem Physiol B, 129, 197 (2001). 21. HY Kweon, IC Um, YH Park, Thermal behavior of regenerated Antheraea pernyi silk fibroin film treated with aqueous methanol, Polymer, 41, 7361 (2000). 22. Z Zheng, Y Wei, S Yan, et al., Preparation of regenerated Antheraea yamamai silk fibroin film and controlled-molecular conformation changes by aqueous ethanol treatment, J Appl Polym Sc, 116, 461 (2010). 23. CJ Wilson, RE Clegg, DI Leavesley, et al., Mediation of biomaterial–cell interactions by adsorbed proteins: A Review, Tissue Eng, 11, 1 (2005). 24. BB Mandal, SC Kundu, A novel method for dissolution and stabilisation of non mulberry silk gland protein fibroin using anionic surfactant sodium dodecyl sulphate, Biotechnol Bioeng, 99,1482 (2008). 25. S Talukdar, QT Nguyen, AC Chen, et al., Effect of initial cell seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage tissue engineering, Biomaterials, 32, 8927 (2011). 26. J Kundu, M Dewan, S Ghoshal, et al., Mulberry non-engineered silk gland protein vis-à-vis silk cocoon protein engineered by silkworms as biomaterial matrices, J Mater Sci Mater Med, 19, 2679 (2008). 27. N Kasoju, RR Bhonde, U Bora, Preparation and characterization of Antheraea assama silk fibroin based novel non-woven scaffold for tissue engineering applications, J Tissue Eng Regen Med, 7, 539 (2009). 28. U Saxena, P Goswami, Silk mat as bio-matrix for the immobilization of cholesterol oxidase, Appl Biochem Biotechnol, 162, 1122 (2010). 29. B Talukdar, M Saikia, PJHandique, et al., Effect of organic solvents on tensile strength of muga silk produced by Antheraea

mulberry silk. The results indicate that the muga fibroin proves to be an alternative natural biomaterial. Until a suitable method is available to dissolve the cocoons, silk glands cna be used for getting agueous fibroin solution as biomaterial of choice for tissue regeneraion and other biomedical applications. Acknowledgements: This work was supported by the Department of Biotechnology (NER programme) and IndoRussia Biotechnology Programme of the Department of Science and Technology, Govt. of India. We are grateful to Dr. Monoj K. Baidya (Deputy Director), Dr. Narayan Biswas, and Mr. Ram Kumar Saha of Central Silk Board, Coochbehar, and also Dr. Milli Banerjee and Mr. Mrinal Kanti Dey, Department of Textile (Sericulture division), West Bengal, India for providing us live 5th instar larvae of muga silkworms. We are thankful to the Sericulture Directorate, Midnapore, West Bengal, India for providing Indian Tropical tasar 5th instar larvae and fresh mulberry cocoons for our work.

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