Structure of the spinning apparatus of a wild silkworm Samia cynthia ricini and molecular dynamics calculation on the structural change of the silk fibroin

Silkworms have been developed over thousands years to optimize folding and crystallization of fibroin under highly controlled conditions which have resulted in their efficient fiber formation. In this paper, we reconstructed the three-dimensional architecture of the spinneret of a wild silkworm Samia cynthia ricini from approximately 1000 optical micrographs of the semi-thin cross sections. The chitin plates and muscles were observed in the silk press part together with large change in the diameter of the spinneret lumen at the press part by large shear stress. This is similar to the case of the spinneret of Bombyx mori silkworm, indicating that the structural change in the silk fibroin of S.c. ricini silkworm occurs exclusively at the silk press part due to large shear stresses. Molecular dynamics (MD) calculations were then performed to study the structural change that occurs in the crystalline region of S.c. ricini silk fibroin under shear stress. Namely, using the peptide AGGAGG(A)₁₂GGAGAG as a model of the crystalline part of the silk fibroin under different shear stresses in the presence of water molecules and followed by molecular mechanics (MM) calculation after removal of water molecules. The simulation indicates that the Ala residues in the model peptides adopt a predominantly β-sheet structure under shear stresses of above 1.0 GPa.     

Flow analysis of aqueous solution of silk fibroin in the spinneret of Bombyx mori silkworm by combination of viscosity measurement and finite element method calculation

Bombyx mori silk fibers possess outstanding mechanical properties in spite of being spun at room temperature and from the aqueous solution. Therefore, the mechanism of the structural transition has been studied with great attention, but still not be well understood. In this study the flow simulation of the silk fibroin aqueous solution using finite element method was performed on the basis of both the relationship between the viscosity and shear rate of the silk fibroin solution prepared from the silk gland, and the detailed structure of the spinneret including silk press part of the silkworm obtained from the optical micrographs. The viscosity of the silk fibroin solution decreased with power-law till the shear rate, about 1.5 s⁻¹ with increasing shear rate. Then the viscosity increased reversely which is speculated due to the fiber formation as a result of aggregation of the molecules. In the flow simulation analysis, the initiation site of the fiber formation was calculated by regulating the extrusion pressure. The fiber formation occurs in 550 μm from the spigot at 1 MPa and in 600 μm from the spigot at 50 MPa. The extrusion pressure in the range from 1 MPa to 50 MPa induces the fiber formation in the stiff plates (550-600 μm from the spigot), that is, the silk press part in the spinneret.     

Regeneration of Bombyx mori silk by electrospinning. Part 2. Process optimization and empirical modeling using response surface methodology

The response surface methodology was used to model and optimize the electrospinning parameters for the spinning of regenerated nanoscale silk fibers from domestic silkworm, Bombyx mori. Electric field and silk concentrations were chosen as variables to control fiber diameter at different spinning distances. Fiber diameter was correlated to these variables by using a second order polynomial function. The predicted fiber diameters were in agreement with the experimental results. Response surfaces were constructed to identify the processing window suitable for producing nanoscale fibers.     

Silk Spinning in Silkworms and Spiders

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.     

Development of silk-like materials based on Bombyx mori and Nephila clavipes dragline silk fibroins

In order to develop new silk-like materials in the form of fiber and non-woven nano-fiber, this study synthesized a new silk-like protein by selecting the sequence from the crystalline region of Bombyx mori silk fibroin, (GAGSGA)₆, to imitate the processing condition of the silk fibroin with the combination of the sequence YGGLGSQGAGRG, the hydrophilic motif of spider dragline silk which was considered as the origin of supercontraction of spider dragline silk (Yang Z, et al. J Am Chem Soc 2000;122: 9019-25). The CD pattern of the silk-like protein in hexafluoroacetone (HFA) indicates that it takes helical structure. The solid-state structures of the silk-like protein before and after methanol treatment were determined to be random coil and Silk II structure (mainly β-sheet structure), respectively, using ¹³C CP/MAS NMR. The ¹³C CP/MAS NMR spectra of the protein after methanol treatment in hydrated state showed that the random coil peaks from Ala Cβ and Ser Cβ carbons become sharper compared with the corresponding peaks in the dry state. The 2D-WISE spectra in the hydrated state also showed increase of the narrow components for these carbons. Thus, water molecules are relatively easy to access at the random coil regions of the protein. The fiber formation of the silk-like protein was possible with wet-spinning or electrospinning methods using HFA as dope solvent and methanol as coagulation solvent.     

Structure for electro‐spun silk fibroin nanofibers

Electro‐spinning was made using silk fibroin and it was found that silk fibroin nanofibers of partially oriented amorphous structure are producible using HFIP(1,1,1,3,3,3 hexafluoro‐2‐propanol) as a solvent for fibroin via the electro‐spinning setup equipped with parallel electrodes as a collector. Transformation from amorphous to silk I of a highly contracted beta‐turn form or amorphous to silk II of a regular array of antiparallel beta‐sheets occurred preferentially via the treatment with water vapor or ethanol, respectively. In addition the c‐axis of crystallites was oriented parallel to the fiber axis. When the electro‐spinning was made using a dish‐type collector filled with ethanol, a peculiar web texture was obtained. Such a web texture seems to be brought about by the shrinkage of fiber due to the crystallization of fibroin and/or surface tension of ethanol droplets formed between the fibers. In this spinning setup, the c‐axis of crystallites was also oriented parallel to the fiber axis. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.     

从丝素水溶液中再生的丝纤维的结构与性能

通过使用表面皿直接拉伸、毛细管重力纺丝和人工拉伸3种不同的成丝方法,从高浓度再生丝素水溶液中制得了丝纤维。用偏光显微镜观察了丝纤维的取向,用拉曼光谱仪和Instron拉力仪表征了丝纤维的结构和力学性能。结果发现,经毛细管剪切流动后再拉伸有利于再生丝性能的提高,所得的丝有较好的取向和较多的β折叠结构,力学性能也相对较好。剪切在丝纤维的成形过程中起重要的作用。     

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.     

The Role of Filippi’s Glands in the Silk Moths Cocoon Construction

Filippi’s glands (FGs), formerly also called Lyonet’s glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.     

Exploring the backbone dynamics of native spider silk proteins in Black Widow silk glands with solution-state NMR spectroscopy

Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, understanding how a spider converts the gel-like, aqueous protein spinning dope within the major ampullate (MA) gland into a super fiber is of great importance for developing future biomaterials based on spider silk. In this work, the initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy. ¹⁵N relaxation parameters, T₁, T₂ and ¹⁵N-{¹H} steady-state NOE were measured for twelve backbone environments at two spectrometer frequencies, 500 and 800 MHz. The NMR relaxation parameters extracted for all twelve environments are consistent with MA silk protein backbone dynamics on the fast sub-nanosecond timescale. Therefore, it is concluded that the repetitive core of spider MA proteins are in an unfolded, highly flexible state in the MA gland.     

Preparation of non-woven nanofibers of Bombyx mori silk, Samia cynthia ricini silk and recombinant hybrid silk with electrospinning method

Electrospinning is a good method to obtain nanoscale fibers from polymer solutions. In this paper, we successfully prepared non-woven nanofibers of Bombyx mori and Samia cynthia ricini silk fibroins, and of the recombinant hybrid fiber involving the crystalline domain of B. mori silk and non-crystalline domain of S. c. ricini silk from hexafluoroacetone (HFA) solution using electrospinning method. ¹³C cross polarization/magic angle spinning NMR spectroscopy was used to monitor the structural change of silk fibroins together with the detection of the residual HFA during the process of the fiber formation. Scanning electronic microscope was used to determine the diameters and their distributions of the fibers.     

Ultra-microindentation at the surface of silk membranes

Ultra-microindentation was used to measure the microhardness and modulus of silk (Bombyx mori) membranes, cast from 20 to 80 °C. The microhardness and modulus were determined from the loading/unloading curves. The membranes exhibit microhardness of about 400 MPa which is larger than the values for most common synthetic polymers (50-300 MPa) implying a greater scratch resistance. The moduli are of the order of those measured by the other means for B. mori silk membranes (5 GPa), and fibers (7-11 GPa). There is some correlation between microhardness and the dimensions of the grains/nanofibrils, but none with surface roughness. The results extend the range of an empirical correlation between microhardness and modulus. The present data together with previous data from other polymers fit the equation, H=0.55E0.74, with a correlation coefficient of 0.94. Finally, it is shown that elastic recovery of the silk membranes is an increasing function of the maximum load applied.     

Silks as ancient models for modern polymers

Silks have a great potential as sustainable, ecologically benign commercial polymers. Here we discuss this fascinating bio-material by merging the biologist's with the polymer scientist's views i.e. combine insights into the characterisation and understanding of evolved structure, property and function in natural silk proteins with the broad scope of applied disciplines ranging from molecular modelling to rheology and mechanical testing. We conclude that silk cannot be defined simply by only its origin or material composition but any meaningful designation must include the key feature of formation by extrusion spinning. We further conclude that silk ‘spinning’ largely depends on a highly specific denaturation process dependent on competing molecular-level interactions of hydrogen bonding between water and main chain amide groups in the silk protein chains. Finally we conclude that silks have a bright future not only as archetype models to guide our understanding of highly adapted and energy efficient bio-polymers but also as prototype models to guide the design of totally novel polymer systems.     

Structure development and properties of high-speed melt spun poly(butylene terephthalate)/poly(butylene adipate-co-terephthalate) bicomponent fibers

Ultra-high-speed bicomponent spinning of poly(butylene terephthalate) (PBT) as sheath and biodegradable poly(butylene adipate-co-terephthalate) (PBAT) as core was accomplished with the take-up velocity up to 10 km/min. The structure development of the individual component and the properties of PBT/PBAT fibers were investigated through the measurements on differential scanning calorimetry, wide-angle X-ray diffraction, birefringence and tensile test. Due to the mutual interaction between two polymer-melts along the spinline, the processability of both components in PBT/PBAT bicomponent spinning was improved compared with those of corresponding single component spinnings. Furthermore, in PBT/PBAT fibers, the structure development of PBT component was found to be greatly enhanced, which led to the improvement in its thermal and mechanical properties; whereas the structure development of PBAT component was significantly suppressed, in which nearly non-oriented structure was observed in both crystalline and amorphous phases.     

Molecular order in spider major ampullate silk (dragline): Effects of spinning rate and post‐spin drawing

Optical birefringence measurements are used to characterize how the molecular order of spider (Nephila clavipes) major ampullate silk is affected by linear spinning rate, by the extent of post‐spin drawing, and by post‐spin drawing rate. Results are interpreted qualitatively in terms of a simple microstructural model, in which birefringence depends on both the overall degree of molecular orientation and the extent to which crystalline regions are present. In contrast to the behavior of conventional, synthetic polymers, birefringence is found to be an unreliable predictor of tensile stiffness: microstructural changes that lead to increased birefringence may leave stiffness unchanged or, in some cases, lower than before. It is unlikely that economic processing of silk‐like polymers into fiber that exhibits biomimetic tensile properties can be achieved with spinning followed by drawing, or with a single spinning step. Instead, spinning followed by thermochemical treatment under load may be needed to obtain the critical combination of molecular orientation and crystallinity in commercially satisfactory time scales. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 895–903, 1999     

Differences in regenerated silk fibroin prepared with different solvent systems: From structures to conformational changes

Dissolution is an important part of silk fibroin (SF) reprocessing, and it is the only way to process it into films, gels, porous scaffold materials, and electrostatic spinning silk fibers. There are a variety of dissolution systems used to dissolve SF. However, few studies have focused on the differences between these different solvent systems. The dissolution of SF with different solvent systems was investigated in this study. Regenerated silk fibroin (RSF) solutions and films were characterized by dynamic light scattering, Fourier transform infrared spectroscopy, differential scanning calorimetry, X‐ray diffraction, and scanning electron microscopy. The results show that the RSF film structures changed with the solvent system, especially LiBr–H₂O. The characterization proved that the random coil did not change into a β‐sheet structure during film formation, and this indicated that its crystal structure and thermal stability was different from others. Interestingly, the differences in the morphologies of all of the RSF films prepared with different solvents were outstanding. Because the mechanism and force of the ion in the solvent systems were different, the SF molecule was hydrolyzed differently in individual solvent systems and produced different hydrolyzed SF molecular chains. These chains had different self‐assembly processes and would lead to RSF products with different microstructures and properties. This suggests that a suitable solvent should be chosen for different uses. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41959.     

Natural and unnatural silks

Natural silk is an important biopolymer with huge potential as it combines superb mechanical properties with environmentally sensitive production methods. Native silk dope taken straight from the gland can easily and without chemical assistance be drawn into strong fibres. Artificial silk fibres, on the other hand, rely on spinning dopes typically ‘reconstituted’ from natural silk fibres by strong chaotropic agents. Such fibres do not form readily, and often require chemical post-spin treatment for stabilisation. In addition these fibres tend to be brittle, and so far have been unable to match native fibres. Here we present novel rheometric data to argue that native and reconstituted silkworm silk dope differ in kind, not just in degree. While native silks behave like typical molten polymers, reconstituted silks do not. We conclude that rheology provides a powerful tool in the quest to learn from the Nature's polymer fibre technology.     

Unfolding the multi-length scale domain structure of silk fibroin protein

Multi-scale force spectroscopy was applied to measure unfolding properties and the internal domain structure of Bombyx mori silk fibroin. We demonstrated that the complex multi-domain sequence and block design in this protein can be directly related to multi-stage unfolding behavior of the specific regions through the use of force extension measurements. These new findings suggest relationships between polymer block chemistry and mechanical features, as origins of the remarkable mechanical properties of native silk fibers in general. We observed multiple consequential unfolding of hydrophilic and hydrophobic domains with characteristics that can be directly related to known molecular dimensions of the protein backbones. Future screening and selection approaches can be envisioned to exploit this approach to optimize specific material functional features for both biopolymers and synthetic polymers in relation to sequence chemistry, a capability not currently available.     

Optimized production of a high-performance hybrid biomaterial: biomineralized spider silk for bone tissue engineering

Silks have been widely used as biomaterials due to their biocompatibility, biodegradability, and excellent mechanical properties. In the present work, native spider silk was used as organic template for controlled nucleation of hydroxyapatite (HA) nano-coating that can act as biomimetic interface. Different bio-inspired neutralization methods and process parameters were evaluated to optimize the silk functionalization. The morphology and chemical composition were investigated by scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analysis and mechanical properties were studied through tensile tests. Results showed that the optimized protocol enabled a controlled and homogeneous nucleation of apatite nano-crystals while maintaining silk mechanical performances after mineralization. This study reports the optimization of a simple and effective bio-inspired nucleation process for precise nucleation of HA onto spider silk templates, suitable to develop high-performance hybrid interfaces for bone tissue engineering. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48739.