Bombyx Mori Silk Fibroin Fiber Research Articles

Fine Structural Analysis of Degummed Fibroin Fibers Reveals Its Superior Mechanical Capabilities.

Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of functionalization and reprocessing into various material formats. The properties of these fibers are typically affected by the intense thermal treatments needed to remove the sericin gum coating layer. Additionally, their mechanical characteristics are often misinterpreted by assuming the asymmetrical cross-sectional area (CSA) as a perfect circle. The thermal treatments impact not only the mechanics of the degummed fibroin fibers, but also the structural configuration of the resolubilized protein, thereby limiting the performance of the resulting silk-based materials. To mitigate these limitations, we explored varying alkali conditions at low temperatures for surface treatment, effectively removing the sericin gum layer while preserving the molecular structure of the fibroin protein, thus, maintaining the hierarchical integrity of the exposed fibroin microfiber core. The precise determination of the initial CSA of the asymmetrical silk fibers led to a comprehensive analysis of their mechanical properties. Our findings indicate that the alkali surface treatment raised the Young's modulus and tensile strength, by increasing the extent of the fibers' crystallinity, by approximately 40 % and 50 %, respectively, without compromising their strain. Furthermore, we have shown that this treatment facilitated further production of high-purity soluble silk protein with rheological and self-assembly characteristics comparable to those of native silk feedstock, initially stored in the animal's silk gland. The developed approaches benefits both the development of silk-based materials with tailored properties and the proper mechanical characterization of asymmetrical fibrous biological materials made of natural building blocks.

Silk Fibroin Based Formulations as Potential Hemostatic Agents.

Effective hemorrhage control is indispensable for life-threatening emergencies in defense fields and civilian trauma. During major injuries, hemostatic agents are applied externally to mimic and accelerate the natural hemostasis process. Commercially available topical hemostatic agents are associated with several limitations, e.g., burning sensation, necrosis, futile in severe injuries, and high costs of the products. In the present study, we developed silk fibroin fiber-based formulations and evaluated their use as a cost-effective potential hemostatic agent with shortened clotting time. Silk fiber-based powder was produced following the alkaline hydrolysis process, wherein Bombyx mori silk fibroin fibers were treated with sodium hydroxide (NaOH) solution that randomly chopped the silk microfibers. Physicochemical reaction parameters, e.g., reaction temperature, molarity of NaOH solution, and incubation time, were optimized to achieve the maximum yield of microfibers. The surface properties of alkaline hydrolyzed silk microfibers (AHSMf) were analyzed by field emission scanning electron microscopy and energy dispersive X-ray studies. The water uptake capacity of AHSMf and the change in pH and temperature (∼30 °C) during blood clotting were analyzed. Further, the hemostatic potential of AHSMf was evaluated by an in vitro whole blood clotting assay using both goat and human blood. The in vitro studies demonstrated a reduced blood clotting time (CT = 20-30 s), prothrombin time (PT = ∼27%), and activated partial thromboplastin time (APTT = ∼14%) in the presence of AHSMf when compared to silk hydrogel powder (devoid of NaOH). Thus, the developed AHSMf could be a promising material to serve as a potential hemostatic agent.

Characterization of water in hydrated Bombyx mori silk fibroin fiber and films by 2H NMR relaxation and 13C solid state NMR.

The mechanical properties of Bombyx mori silk fibroin (SF) change remarkably upon hydration. However, the microscopic interaction between SF and water is not currently well understood on a molecular level. We were able to identify four distinct components in the relaxation times for water in SF fiber by 2H solution NMR relaxation and exchange measurements. In addition, the effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13C selectively labeled SF, respectively, could be determined independently. Thus, our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials.

Stability toward alkaline hydrolysis ofB.morisilk fibroin grafted with methacrylamide

Bombyx mori silk fibroin fibers were grafted with methacrylamide (MAA) and characterized by Raman and infrared (IR) vibrational spectroscopy before and after hydrolysis in NaOH 5% to elucidate the possible interactions between the two components and the stability of the fibers toward alkaline hydrolysis. Upon grafting, the fibers underwent conformational rearrangements toward a more unordered state and lost orientation at weight gains higher than 60%. Vibrational spectroscopy disclosed the occurrence of intermolecular interactions (mainly hydrogen bonds) between B. mori silk fibroin and polyMAA in the grafted fibers, and the formation of covalent bonds has been explored. These strong interactions made the grafted fibers as a whole more stable toward alkaline hydrolysis because they prevented the solubilization of the polymer upon hydrolysis and made slower the transformation of its CONH2 groups into COOH and COO− groups. Upon hydrolysis, silk fibroin underwent an enrichment in the β-sheet crystalline domains, because of the preferential removal of the unordered domains, which were more prone to the OH− attack. IR and Raman spectroscopy proved valid techniques to investigate the degradation mechanism and kinetics of grafted silk fibroin fibers and so for designing high-performing silk-based materials. The A731/A1004 Raman intensity ratio was proposed to spectroscopically evaluate the composition of the grafted samples; its value was found to linearly increase with weight gain (R2 = 0.998), envisaging the possibility of using Raman spectroscopy as a routine analytical technique for qualitative and quantitative characterization of grafted industrial samples. Copyright © 2016 John Wiley & Sons, Ltd.

Control of Olfactory Ensheathing Cell Behaviors by Electrospun Silk Fibroin Fibers

Olfactory ensheathing cells (OECs), the only glial cell type that normally penetrates the transition zone between the peripheral and central nervous systems, are one of the most promising candidates for cell transplantation in repairing spinal cord injury (SCI). However, we must manipulate and regulate OECs' behavior to make these cells effective in cell transplantation. In the present study, we assessed the response of rat OECs to different variants of nanofibrous silk fibroin mats with regard to cell morphology, adhesion, proliferation, and migration and the related gene and protein expression. Results showed that OECs adhere and spread more easily on Tussah silk fibroin (TSF) fibers than Bombyx mori silk fibroin fibers, resulting in a higher rate of cell proliferation and gene and protein expression, examined by RT-PCR and ELISA. In addition, the morphology of OECs on microfibers is mainly polygonal with short protrusions, whereas the OECs on nanofibers exhibit a bipolar shape with long protrusions that align along the fibers, especially when aligned fibers are employed. Moreover, OECs on silk fibroin nanofibers migrate more efficiently than those on poly-L-lysine (PLL). Based on the experimental results, the morphology, adhesion, spread, gene and protein expression, and migration of OECs could be modulated and regulated by adjusting the contents and structure of silk fibroin nanofibers, shedding light on the control of OECs' behavior in nerve tissue engineering and thus the future therapeutic intervention for nerve repair after injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Graphitization behavior of iodine-treated Bombyx mori silk fibroin fiber

In order to investigate the graphitization behavior of Bombyx mori silk fibroin (SF) fibers, structural inspection of carbonized SF fibers treated with iodine vapor was studied at different temperatures, from 800 to 1400 °C. Wide angle X-ray diffraction measurement suggested that both untreated and iodinated SF fibers exhibited amorphous structure carbonized to 1200 °C. After carbonization at 1400 °C for 12 h, a graphite-like structure was obtained. Raman spectroscopy and transmission electron microscopic observation showed that the graphite layers of SF fibers became more ordered after iodine treatment. The carbon yield obtained at higher temperature (1400 °C) after iodine treatment was higher, ca. 28 wt%, than that of untreated SF.

Surface Morphology and Properties of Bombyx mori Silk Fibroin Fiber Treated with I 2-KI Aqueous Solution

The surface morphology, thermal and mechanical properties of Bombyx mori silk fibroin (SF) fiber treated with a 1.23 N iodine-potassium iodide (I2-KI) aqueous solution were investigated using scanning electron microscopy (SEM), atomic force microscopy (AFM), differential scanning calorimetry, thermogravimetric analysis, and tensile measurements to clarify the effects of the iodine treatment. SEM and AFM analyses indicated that the SF fiber surface became rougher by the absorption of polyiodide ions. The mechanical properties of iodinated SF showed an increase in Young’s modulus, and strain remained constant although ultimate tensile strength slightly decreased. The thermal stability of SF molecules was greatly enhanced by iodine treatment. Iodinated SF fibers should be an attractive candidate for biomedical applications such as for producing antimicrobial filters, iodine containing wound-healing anion exchange fibers, etc.

Effects of Ultrasonic and Salt-shrinking Treatments on Structure and Physical Properties of Bombyx mori Silk Fibroin Fiber

Effects of Ultrasonic and Salt-shrinking Treatments on Structure and Physical Properties of Bombyx mori Silk Fibroin Fiber

Preparation and characterization of regenerated Bombyx mori silk fibroin fiber containing recombinant cell‐adhesive proteins; Nonwoven fiber and monofilament

AbstractTo improve the cell‐adhesive character of Bombyx mori silk fibroin fiber, the regenerated B. mori silk fibroin fibers with recombinant cell‐adhesive proteins were prepared. The recombinant proteins were originally designed in our laboratory and expressed in Eschericchia coli; one is the protein with the primary structure of (TGRGDSPAS)8 which was designed based on the cell adhesive sequence in fibronectin and the other is (GERGDLGPQGIAGQRGVVGERGERGERGAS)8GPPGPCC GGG originated from the cell adhesive sequences in collagen. The nonwoven silk fibroin nano‐fibers including these recombinant proteins were prepared by electrospinning method from the hexafluoroisopropanol (HFIP) solutions of mixture of B. mori silk fibroin and the recombinant proteins. The cell‐adhesive characters of such nonwoven silk fibers were much improved compared with that of the nonwoven silk fibroin fibers without the recombinant proteins. In addition, the degradation rates of the nonwoven fibers were accelerated by mixing of the recombinant proteins into silk fibroin. Here, we also prepared the regenerated silk fibroin monofilaments including the recombinant proteins by wet spinning from the HFIP dope solution. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Preparation and characterization of regenerated Bombyx mori silk fibroin fiber with high strength

Regenerated Bombyx mori silk fibers were spun from hexafluoro-iso- propanol solution of silk fibroin sponge in methanol used as a coagulant solvent and then elongated in water. The stress-strain curves of the regenerated fibers changed dramatically depending on the draw ratio and the structure was studied by 13 C CP/MAS NMR and X-ray diffraction meth- ods. The patterns of 13C CP/MAS NMR spectra of two regenerated fibers with different draw ratios (1× and 3×) and native silk fiber are all β-sheet structure although the fraction of random coil/distorted β-turn decreases in the order of 1×, 3× and native fiber gradually. On the other hand, azimuthal scans of their X-ray fiber patterns changed remarkably with increasing the draw ratio. This indicates that long-range orientation of the fibroin chain changes remarkably during the drawing process, but the short-range local structure does not change significantly. Regenerated silk fiber with a draw ratio of 3× is a fiber with high strength which is comparable with that of natural silk fiber. The regenerated fiber is also more degradable than natural silk fiber in enzyme solution in vitro.

Structural Analysis of Bombyx mori Silk Fibroin Peptides with Formic Acid Treatment Using High-Resolution Solid-State 13C NMR Spectroscopy

Bombyx mori silk fibroin fiber is a fibrous protein produced by the silkworm at room temperature and from an aqueous solution whose primary structure is highly repetitive. In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using high-resolution solid-state 13C NMR. We establish that the Ser residue bearing a short polar side chain has the ability to stabilize the conformation formed in the model peptides due to its ability to form intermolecular hydrogen bonds involving its hydroxyl group as a donor and the carbonyl groups of other residues as acceptors. On the other hand, insertion of Tyr residue in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Moreover, the environmental effect was investigated by mixing the native Cp fraction with the model peptides, showing that there is no significant structural difference on the Ser-containing peptides, while structural transformation was observed on the peptides containing the GAAS unit. This may be attributed to the fact that the Cp fraction promotes the formation of an antiparallel beta-sheet in the Ala-Ala unit. Such periodically disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical not only for facilitating the conformational transformation from silk I to silk II structural form but also for having some correlation with the unique properties of the silk materials.

Structural changes and dyeability of silk fibroin fiber following shrinkage in neutral salt solution

AbstractThe fine structural changes of Bombyx mori silk fibroin fibers, induced by shrinking with concentrated calcium chloride aqueous solution at elevated temperature were investigated as a function of shrinking rate. Tensile strength decreased and elongation at break increased in the shrinkage range 13–67%, the shape of the stress–strain curve changing from rubberlike to brittle at high shrinkage values (70–90%). The birefringence gradually decreased over the entire shrinking range examined, the curve becoming steep as the shrinkage exceeded about 67%. The behavior of isotropic refractive index (niso) closely resembled that of birefringence (Δn) in the shrinking range 13–67%. Beyond shrinkage of 67%, the niso showed a tendency to increase, especially for the sample with 80% shrinkage. Dichroism measurements showed that the molecular orientation within the amorphous regions decreased sharply at the beginning of the shrinking treatment, within the range 0–13%, then attained a saturation at about 55%. The position and intensity of the major X‐ray diffraction peak at 20.5° remained essentially unchanged regardless of the shrinking treatment. The results of dyeing behavior showed that the saturation value attained by shrunk silk fibers was significantly larger than that of the untreated control sample. Both standard affinity and the heat of dyeing increased slightly for the shrunk silk fibroin fiber, suggesting that a larger number of reactive sites became available for the interaction between dye molecules and fibroin chains. A schematic model is proposed for explaining the relation between structural changes and enhanced dyeability of the silk fibers following shrinkage in neutral salt solution. © 1994 John Wiley & Sons, Inc.