Non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices for potential biotechnological applications

This study reports a novel biopolymeric matrix fabricated by chemically cross-linking poly (vinyl alcohol) with silk sericin protein obtained from cocoons of the tropical tasar silkworm Antheraea mylitta. Glutaraldehyde was used as a cross-linking agent with hydrochloric acid acting as an initiator. The matrices were biophysically characterized and the cytocompatibility of the matrices was evaluated for their suitability as biomaterials. The surface morphology was assessed using atomic force microscopy while the changes taking place after cross-linking were confirmed by Fourier transform infrared spectroscopy. The enhanced thermal stability of the constructs was assessed by thermogravimetric and differential scanning calorimetry. Fourier transform infrared spectroscopy analysis showed that sericin was chemically cross-linked with poly (vinyl alcohol) using glutaraldehyde. Silk sericin protein demonstrated a favorable effect on animal cell culture by successfully improving the adhering and spreading of cells on the poorly adhering surface of poly (vinyl alcohol). Confocal microscopy revealed cell spreading and actin filament development in sericin/poly (vinyl alcohol) hydrogel matrices. These findings prove the potential of non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices to be used as biocompatible and biopolymeric material for tissue-engineering and biotechnological applications.     

Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts

The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta cocoon has been assessed and compared with that of the mulberry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive control for the experiment. Our results showed that the sericin obtained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-incubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase; lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant.     

广西桑蚕茧丝质量与性能指标的测定分析

本研究对广西桑蚕茧的茧丝质量与性能指标进行了测定分析.结果表明,广西桑蚕茧外形略小,茧层丝胶含量、茧层易溶丝胶含量、丝胶溶解性以及茧丝纤度等主要指标均与江浙夏秋用桑蚕茧有明显差异.桑蚕茧的茧层丝胶含量、易溶丝胶含量随庄口、季节不同而有显著差异;春季桂南地区的蚕茧茧层丝胶含量高于桂西北地区,秋季刚好相反.茧层易溶丝胶含量在11.6%～14.4%之间,约占丝胶总含量的41%～45%,茧层易溶丝胶含量和解舒率呈显著的正相关关系,即茧层易溶丝胶含量越多,蚕茧解舒越好.庄口和季节是影响桑蚕茧层易溶丝胶含量高低与蚕茧解舒优劣的重要因素.广西桑蚕茧层丝胶对煮茧温度更敏感,茧丝纤度粗细变化开差大.因此,我们认为目前困扰广西桑蚕茧加工技术突破的原因之一是缫丝工艺技术,采用传统缫丝工艺技术难以正常发挥广西桑蚕茧的茧丝优势与特性.     

Strain Rate and Anisotropic Microstructure Dependent Mechanical Behaviors of Silkworm Cocoon Shells

Silkworm cocoons are multi-layered composite structures comprised of high strength silk fiber and sericin, and their mechanical properties have been naturally selected to protect pupas during metamorphosis from various types of external attacks. The present study attempts to gain a comprehensive understanding of the mechanical properties of cocoon shell materials from wild silkworm species Antheraea pernyi under dynamic loading rates. Five dynamic strain rates from 0.00625 s^-1 to 12.5 s^-1 are tested to show the strain rate sensitivity of the cocoon shell material. In the meantime, the anisotropy of the cocoon shell is considered and the cocoon shell specimens are cut along 0°, 45° and 90° orientation to the short axis of cocoons. Typical mechanical properties including Young’s modulus, yield strength, ultimate strength and ultimate strain are extracted and analyzed from the stress-strain curves. Furthermore, the fracture morphologies of the cocoon shell specimens are observed under scanning electron microscopy to help understand the relationship between the mechanical properties and the microstructures of the cocoon material. A discussion on the dynamic strain rate effect on the mechanical properties of cocoon shell material is followed by fitting our experimental results to two previous models, and the effect could be well explained. We also compare natural and dried cocoon materials for the dynamic strain rate effect and interestingly the dried cocoon shells show better overall mechanical properties. This study provides a different perspective on the mechanical properties of cocoon material as a composite material, and provides some insight for bio-inspired engineering materials.     

Applications of natural silk protein sericin in biomaterials

Silk sericin is a natural macromolecular protein derived from silkworm Bombyx mori. During the various stages of producing raw silk and textile, sericin can be recovered for other uses. Also, sericin recovery reduces the environmental impact of silk manufacture. Sericin protein is useful because of its properties. The protein resists oxidation, is antibacterial, UV resistant, and absorbs and releases moisture easily. Sericin protein can be cross-linked, copolymerized, and blended with other macromolecular materials, especially artificial polymers, to produce materials with improved properties. The protein is also used as an improving reagent or a coating material for natural and artificial fibers, fabrics, and articles. The materials modified with sericin and sericin composites are useful as degradable biomaterials, biomedical materials, polymers for forming articles, functional membranes, fibers, and fabrics.     

Proteomic analysis of sericin in <Emphasis Type="Italic">Bombyx mori</Emphasis> cocoons

Cocoon sericin plays an important role in the reeling of silk and serves as a valuable biomaterial in the field of biomedicine, skincare, and food industries; however, knowledge about cocoon sericin proteins has been limited. For a comprehensive study on sericin, cocoons of eight varieties of silkworm of different geographic origin and with varied cocoon color were analyzed utilizing proteomics and bioinformatics approaches. The electrophoresis pattern demonstrated some common protein bands for all silkworm varieties and distinctive protein bands for some of those examined in the present study. The Ser2 protein, a new Ser3 protein, and four other novel sericin proteins were identified in cocoons for the first time. Products of both Ser1 and Ser3 genes appear to be ubiquitous in the cocoon shell of Bombyx mori. In addition, cocoons with especially high-reelability produced by the mutant strain B84 had an unique protein product of the Ser2 gene, indicating that the protein may play an important role in cocoon reelability. A series of sequence conflicts and post-translational modifications (PTMs) were also revealed in sericin proteins. Lipid modifications of sericin proteins, which promote waterproofing of the cocoon shell, were observed. Further, hydroxylation was identified, which provided evidence for intermolecular bonds among neighboring molecules of sericin as found in collagens. The sericin proteome data obtained from this study illuminated the molecular complexity of cocoon sericin and contributed to our understanding of the properties of sericin in filature and biomaterials.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

Isolation of Three Main Sericin Components from the Cocoon of the Silkworm,Bombyx mori

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.     

Isolation of three main sericin components from the cocoon of the silkworm,Bombyx mori

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials.

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

Recombinant DNA production of spider silk proteins

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.     

Transgenic silkworms that weave recombinant proteins into silk cocoons

As a result of breeding for more than 4,000 years, the silkworm, Bombyx mori, has acquired the ability to synthesize bulk amounts of silk proteins in its silk glands. To utilize this capacity for mass production of useful proteins, transgenic silkworms were generated that synthesized recombinant proteins in the silk gland and secreted them into the silk cocoon. The silk gland is classified into two main regions: the posterior (PSG) and the middle silk gland (MSG). By controlling the expressed regions of the recombinant protein gene in the silk gland, we were able to control the localization of the synthesized protein in the silk thread. Expression in the PSG or MSG led to localization in the insoluble fibroin core or hydrophilic outer sericin layer, respectively. This review focuses on the expression of recombinant protein in the MSG of transgenic silkworms. The recombinant protein secreted in the sericin layer is extractable from the cocoon with only a small amount of endogenous silk protein contamination by soaking the cocoon in mild aqueous solutions. The possibility of utilizing transgenic silkworms as a valuable tool for the mass production of therapeutic and industrially relevant recombinant proteins is discussed.     

Preparation of elastic silk sericin hydrogel

This paper reports a preparation method for silk sericin hydrogel using the Sericin-hope silkworm, whose cocoons consist almost exclusively of sericin. Sericin solution, prepared from Sericin-hope cocoons, contains intact sericin and forms elastic hydrogels with the addition of ethanol. The sericin hydrogel can be prepared without crosslinking by chemicals or irradiation and might be usable as a naturally occurring biomaterial.     

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

Non-bioengineered silk gland fibroin protein: characterization and evaluation of matrices for potential tissue engineering applications

The possibility of using wild non-mulberry silk protein as a biopolymer remains unexplored compared to domesticated mulberry silk protein. One of the main reasons for this was for not having any suitable method of extraction of silk protein fibroin from cocoons and silk glands. In this study non-bioengineered non-mulberry silk gland fibroin protein from tropical tasar silkworm Antheraea mylitta, is regenerated and characterized using 1% (w/v) sodium dodecyl sulfate (SDS). The new technique is important and unique because it uses a mild surfactant for fibroin dissolution and is advantageous over other previous reported techniques using chaotropic salts. Fabricated fibroin films are smooth as confirmed by atomic force microscopy. Circular dichroism spectrometry along with Fourier transformed infrared spectroscopy and X-ray diffraction reveal random coil/alpha-helix conformations in regenerated fibroin which transform to beta-sheets, resulting in crystalline structure and protein insolubility through ethanol treatment. Differential scanning calorimetry shows an increase in glass transition (Tg) temperature and enhanced degradation temperature on alcohol treatment. Enhanced cell attachment and viability of AH927 feline fibroblasts were observed on fibroin matrices. Higher mechanical strength along with controllable water stability of regenerated gland fibroin films make non-mulberry Indian tropical tasar silk gland fibroin protein a promising biomaterial for tissue engineering applications.     

Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma

Sericin, a constituent of the silkworm cocoon, was added to the culture of four mammalian cell lines: murine hybridoma 2E3-O,human hepatoblastoma HepG2, human epithelial HeLa and human embryonal kidney 293 cells. The proliferation of all cell lineswas accelerated in the presence of sericin. The hybridoma cellline was further studied. The 2E3-O cell line was so well adapted to serum-free medium that both the proliferation rate and maximum cell density in serum-free ASF103 medium were higher than in RPMI medium supplemented with all lots of FBS tested, and this proliferation was stimulated by the addition of sericin in a dose-dependent manner. Stimulation was observed at sericin concentrations from 0.01 to 0.1 %, although 1% sericin was severely harmful to the culture. In comparison with bovine serum albumin (BSA), a widely used supplement in serum-free medium, sericin had an equivalent effect on the proliferation of the hybridomas and sericin additively stimulated the proliferation with BSA. Although heat easily denatures and inactivates most proteins, the activity of sericin was not affected by autoclaving. In a similar manner to the silkworm-derived sericin, recombinant sericin synthesized in E. coli also stimulated the hybridoma proliferation, irrespective of whether it was autoclaved or filtered. Since BSA is obtained from bovine serum and the risk of infections such as bovine spongiform encephalopathy cannot be eradicated, sericin derived from insects could be a preferable culture medium supplement for stimulating the proliferation of mammalian cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biocompatible materials from regenerated silk for tissue engineering and medicinal therapy

The present review is devoted to the application of biomaterials from regenerated silk for designing tissue-engineered constructs—the basis for hybrid organs and tissues. Fibroin, the main structural protein of silkworm silk, can be used to design artificial cartilages, bone tissue fragments, blood vessels, as well as to regenerate nervous tissue. Fibroin capsules containing bioactive compounds are successfully applicable in medicinal therapy, such as controlled drug delivery in cancer treatment. Apart from fibroin, tissue engineering can successfully be based on biopolymer spidroin, a spider net protein, which is also a biocompatible material with valuable mechanical properties.     

Fabrication of silk sericin nanofibers from a silk sericin-hope cocoon with electrospinning method

In this study, silk sericin nanofibers from sericin hope-silkworm, whose cocoons consist almost exclusively of sericin were successfully prepared by electrospinning method. Scanning electron microscopy (SEM) was used to observe the morphology of the fibers. The effect of spinning conditions, including the concentration of sericin cocoon solution, acceleration voltage, spinning distance and flow rate on the fiber morphologies and the size distribution of sericin nanofibers were examined. The structure and physical properties were also observed by Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). The optimum conditions for producing finely thinner fibrous sericin nanofibers without beads were the concentration of sericin solution above 6-8 wt%, acceleration voltage ranging from 25 to 32 kV, spinning distance above 9 cm, and flow rate above 0.06 cm min(-1). The mean diameter of as spun sericin fibers varied from 114 to 430 nm at the different spinning conditions. In the as-spun fibers, silk sericin was present in a random coil conformation, while after methanol treatment, the molecular structure of silk sericin was transformed into a β-sheet containing structure. Sericin hope nanofiber demonstrated thermal degradation at lower temperature than the sericin hope cocoon, which probably due to the randomly coiled rich structure of the sericin hope nanofiber.     

Effect of polyamines on mechanical and structural properties of Bombyx mori silk

Silkworm, Bombyx mori (B. mori) belongs to the Lepidoptera family. The silk produced from this insect, mulberry silk, gained lot of importance as a fabric. Silk is being exploited as a biomaterial due to its surprising strength and biocompatibility. Polyamines (PA) are important cell growth regulators. In the present work the effect of treatment of polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm) on the quantity and quality of silk produced was assessed. Results showed that exogenous feeding of Spd at a concentration of 50 µM increased fiber length significantly. Analysis by Fourier transform infrared (FTIR) on the properties of silk obtained from Spd treated silkworms revealed an increase in percentage of absorption with no difference in peak positions of amide I and amide III groups. Scanning electron microscopy (SEM) revealed an increase in diameter of silk. Further, analysis at molecular level showed an increase in fibroin expression in Spd treated silk glands. However, the Spd treatment showed no significant difference with respect to fibroin to sericin ratio per unit weight of cocoon, silk tenacity, and percent elongation. Thus, the present results show that polyamine treatment would influence silk quality at structural, mechanical, and molecular level in the Bombyx mori, which can be exploited in silk biomaterial production.     

Facts and myths of antibacterial properties of silk

Silk cocoons provide protection to silkworm from biotic and abiotic hazards during the immobile pupal phase of the lifecycle of silkworms. Protection is particularly important for the wild silk cocoons reared in an open and harsh environment. To understand whether some of the cocoon components resist growth of microorganisms, in vitro studies were performed using gram negative bacteria Escherichia coli (E. coli) to investigate antibacterial properties of silk fiber, silk gum, and calcium oxalate crystals embedded inside some cocoons. The results show that the previously reported antibacterial properties of silk cocoons are actually due to residues of chemicals used to isolate/purify cocoon elements, and properly isolated silk fiber, gum, and embedded crystals free from such residues do not have inherent resistance to E. coli. This study removes the uncertainty created by previous studies over the presence of antibacterial properties of silk cocoons, particularly the silk gum and sericin. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 237–245, 2014.