Solubility and rheological behavior of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide

As an important direct solvent for cellulose, N-methyl morpholine N-oxide (NMMO) is environmentally friendly, and potentially very economical. Silk fibroin (SF) (Bombyx mori) can also be dissolved directly in NMMO.H2O. However, it is unexpectedly difficult to obtain a silk fibroin solution with a concentration higher than 10wt.% in this way, and extensive degradation of silk fibroin occurs if the dissolution temperature is higher than 110 degrees C. On the other hand, it is found that regenerated silk fibroin (RSF) film is much easier to dissolve in NMMO.H2O than ordinary SF. The RSF in NMMO.H2O can be easily concentrated to a range from 10 to 25wt.%. The structural differences between the degummed silk fiber and the RSF film lead to this different solubility in NMMO.H2O. The rheological behavior of concentrated RSF/NMMO.H2O solutions were also investigated. Regenerated silk fiber was spun from this type of solution, and its strength can reach up to 3.07 cN/dtex.     

Morphology and structure of electrospun mats from regenerated silk fibroin aqueous solutions with adjusting pH

In this paper, regenerated silk fibroin (SF) aqueous solutions were adjusted to a pH of 6.9 by mimicing the condition in the posterior division of silkworm's gland and rheological behavior of solutions was investigated. The electrospinning technique was used to prepare fibers, and non-woven mats of regenerated B. mori silk fibroin were successfully obtained. The effects of electrospinning parameters on the morphology and diameter of regenerated silk fibers were investigated by orthogonal design. Statistical analysis showed that voltage, the concentration of regenerated SF solutions and the distance between tip and collection plate were the most dominant parameters to fiber morphology, diameter and diameter distribution, respectively. An optimal electrospinning condition was obtained in producing uniform cylindrical fibers with an average diameter of 1300nm. It was as follows: the concentration 30%, voltage 40kV, distance 20cm. The structure of electrospun mats was characterized by Raman spectroscopy (RS), wide-angle X-ray diffraction (WAXD) and modulated differential scanning calorimetry (MDSC). It was found that electrospun mats were predominantly random coil/silk I structure, and the transition to silk II (beta-sheet) rich structure should be further explored.     

Materials fabrication from Bombyx mori silk fibroin

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.     

Optical spectroscopy to investigate the structure of regenerated Bombyx mori silk fibroin in solution

Fluorescence and circular dichroism spectroscopy were used to monitor the conformational transition of regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions under different conditions. According to the analysis of fluorescence spectra using anilinonaphthalene-8-sulfonic acid magnesium salt (ANS) as an external probe, the destruction of the hydrophobic core prior to the secondary structure change suggests that this collapse may initiate the conformational transition from random coil to beta-sheet for RSF. The temperature dependence of the structural changes of RSF, detected by both fluorescence spectroscopy and circular dichroism, shows a reversible process upon heating and recooling, with the midpoint around 45 degrees C. The results also indicate that most of the tryptophan (Trp) residues contained in silk fibroin are concentrated on the surface of the unfolded protein. However, they will change their location in the highly ordered structure (e.g., becoming more homogeneous) with the conformational transition of silk fibroin. Moreover, our studies also suggest that the presence of water plays a crucial role during the structure changes of fibroin.     

X-ray photoelectron spectroscopic and Raman analysis of silk fibroin-Cu(II) films

There is evidence to suggest that Cu(II) is involved in the natural spinning process of a silkworm helping to convert the concentrated silk fibroin (SF) solution (or dope) into tough insoluble threads. To investigate the interaction between SF and Cu(II), a series of regenerated SF (RSF) films with different mass ratios of Cu(II) to SF were prepared. X-ray photoelectron spectroscopy (XPS) was employed to study the chemical interaction between Cu(II) and SF in these Cu(II)-RSF films. A significant change in the binding energy of Cu 2p(3/2) demonstrated that the chemical state of Cu(II) in the Cu(II)-RSF films was affected by the interaction between Cu(II) and SF. Moreover, chemical shifts of N 1s and O 1s of SF were also detected, implying that Cu(II) may coordinate with both N and O atoms in the SF. In addition, Raman spectra of the same series of Cu(II)-RSF films recorded the conformation transition of SF that may occur by the coordination of Cu(II) and SF macromolecular chains.     

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.     

Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.     

A study on the flow stability of regenerated silk fibroin aqueous solution

The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.     

Significantly reinforced composite fibers electrospun from silk fibroin/carbon nanotube aqueous solutions

Microcomposite fibers of regenerated silk fibroin (RSF) and multiwalled carbon nanotubes (MWNTs) were successfully prepared by an electrospinning process from aqueous solutions. A quiescent blended solution and a three-dimensional Raman image of the composite fibers showed that functionalized MWNTs (F-MWNTs) were well dispersed in the solutions and the RSF fibers, respectively. Raman spectra and wide-angle X-ray diffraction (WAXD) patterns of RSF/F-MWNT electrospun fibers indicated that the composite fibers had higher β-sheet content and crystallinity than the pure RSF electrospun fibers, respectively. The mechanical properties of the RSF electrospun fibers were improved drastically by incorporating F-MWNTs. Compared with the pure RSF electrospun fibers, the composite fibers with 1.0 wt % F-MWNTs exhibited a 2.8-fold increase in breaking strength, a 4.4-fold increase in Young's modulus, and a 2.1-fold increase in breaking energy. Cytotoxicity test preliminarily demonstrated that the electrospun fiber mats have good biocompatibility for tissue engineering scaffolds.     

Flexibility regeneration of silk fibroin in vitro

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were first prepared by slow drying process. Then, the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared with the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nanoassembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.     

Preparation and characterization of novel nanocomposite films formed from silk fibroin and nano-TiO2

This paper describes the synthesis and characterization of new regenerated silk fibroin (SF)/nano-TiO(2) composite films. The preparation method, based on the sol-gel technique using butyl titanate as oxide precursor, could avoid reagglomeration of the prepared nanoparticles. Samples were characterized mainly by X-ray diffraction (XRD), ultra-violet (UV) spectroscopy, atomic force microscopy (AFM), Fourier transform infrared (FT-IR) spectroscopy, and thermogravimetric analysis (TGA). The UV and AFM results indicated that TiO(2) nanoparticles could be well dispersed inside the SF film, and the size of TiO(2) was about 80nm. The XRD and FT-IR analysis implied that the formation of nano-TiO(2) particles may induce the conformational transition of silk fibroin to a typical Silk II structure partly with the increasing of crystallinity in the composite films. Compared to the pure SF films, the mechanical and thermal properties of composite films were improved, and the solubility in water was decreased due to the conformational transition of silk fibroin to Silk II structure.     

Production of scFv-conjugated affinity silk powder by transgenic silkworm technology

Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers.     

Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.     

家蚕不同茧色品种中cbp基因的结构和表达分析

类胡萝卜素结合蛋白(CBP)是唯一已被确认与家蚕黄色茧形成儡切相关的主要蛋白质。文章选择12个有色茧和白茧蚕品种, 调查了cbp基因结构、转录产物mRNA类型和丝腺类胡萝卜素紫外可见光吸收特征与其茧色的关系。结果表明: 黄色茧蚕品种含有2种或3种cbp基因结构, 同时转录具有CBP功能性的完整 mRNA和缺少第2外显子的mRNA; 绿茧品种间cbp基因结构存在差异, 转录缺少第2外显子的mRNA; 白茧蚕品种cbp基因只有1种结构, 转录缺少第2外显子的mRNA。文章在黄茧品种中新发现的cbp基因第1内含子序列可能具有茧色品种特异性, 家蚕黄茧品种丝腺的紫外可见吸收光谱特征显著区别于绿茧和白茧品种, cbp基因的结构和表达特征与家蚕茧色密切相关。     

Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 degrees C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented beta-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior.     

Vibrational 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly(alanine-glycine) as model for silk I Bombyx mori fibroin

This study focuses on the conformational characterization of poly(alanine-glycine) II (pAG II) as a model for a Bombyx mori fibroin silk I structure. Raman, IR, and 13C-cross-polarization/magic angle spinning NMR spectra of pAG II are discussed in comparison with those of the crystalline fraction of B. mori silk fibroin (chymotryptic precipitate, Cp) with a silk I (silk I-Cp) structure. The spectral data give evidence that silk I-Cp and the synthetic copolypeptide pAG II have similar conformations. Moreover, the spectral findings reveal that silk I-Cp is more crystalline than pAG II; consequently, the latter contains a larger amount of the random coil conformation. Differential scanning calorimetry measurements confirm this result. N-Deuteration experiments on pAG II allow us to attribute the Raman component at 1320 cm(-1) to the amide III mode of a beta-turn type II conformation, thus confirming the results of those who propose a repeated beta-turn type II structure for silk I. The analysis of the Raman spectra in the nuNH region confirms that the silk I structure is characterized by the presence of different types of H-bonding arrangements, in agreement with the above model.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.     

Conformation transition kinetics of Bombyx mori silk protein

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber.     

Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.     

Phosphorylation of silk fibroins improves the cytocompatibility of silk fibroin derived materials: A platform for the production of tuneable material

Silk fibroin demonstrates great biocompatibility and is suitable for many biomedical applications, including tissue engineering and regenerative medicine. Current research focuses on manipulating the physico‐chemical properties of fibroin, and examining the effect of this manipulation on firobin's biocompatibility. Regenerated silk fibroin was modified by in vitro enzymatic phosphorylation and cast into films. Films were produced by blending, at several ratios, the phosphorylated and un‐phosphorylated fibroin solutions. Fourier transform infra‐red spectroscopy was used to determine the specific P–OH vibration peak, confirming the phosphorylation of the regenerated silk fibroin solution. Differential scanning calorimetry showed that phosphorylation altered the intra‐ and inter‐molecular interactions. Further experiments demonstrated that phosphorylation can be used to tailor the hydrophylicity/hydrophobicity ratio as well as the crystalinity of silk fibroin films. Release profiling of a model drug was highly dependent on silk modification level. Cytotoxicity assays showed that exposure to lixiviates of phosphorylated films only slightly affected cellular metabolism and proliferation, although direct contact resulted in a strong direct correlation between phosphorylation level and cell proliferation. This new method for tuning silk biomaterials to obtain specific structural and biochemical features can be adapted for a wide range of applications. Phosphorylation of silk fibroins may be applied to improve the cytocompatibility of any silk‐based device that is considered to be in contact with live animals or human tissues.