Fibroin Fibers Research Articles

Domain-Driven Binding of Fibrin(Ogen) onto Silk Fibroin Biomaterials

Studies have demonstrated that serum protein adsorption onto silk fibroin-based biomaterials dramatically changes when the conformation of this natural polymer is rearranged by engineering procedures. In the present study, attention was paid to the binding of fibrin(ogen) to fibroin fibers and regenerated films. The fibroin specimens were incubated either in human plasma or in a fibrinogen solution to which thrombinwas added to activate the polymerization of the precursor into the final product, fibrin. The experiments were carried out in the presence and absence of calcium to investigate the role of calcium-dependent enzymes in the binding process. The two types of samples were analyzed by SEM, the micrographs showed completely different interactions with fibrinogen. Films did not show any visible fibrin polymerization, whereas the fibers were bound to the fibrin bundles by calcium-independent mechanisms.

Serum Protein Absorption on Silk Fibroin Fibers and Films: Surface Opsonization and Binding Strength

Fibroin, the core of the silk filament protein, has been proposed as a biomaterial for different biomedical applications since it can be engineered as a thread, fabric or film. Infrared spectroscopy suggests that the dissolution of the fibroin filaments and subsequent casting of the fibroin solution into films followed by treatment with methanol alters the protein structure leading to an increase in amorphous domains. The adsorption ofserum proteins on fabrics and films showed different hydrophobic binding strengths for the two materials with the protein binding being greatest for the more hydrophobic fibroin fibers. Differences between the materials were also observed in the adsorption of key immunoproteins. Although the C3 fragment of the complement system was adsorbed on both the surfaces, it appeared to be activated preferentially on the fibroin films and not on the fibroin fibers, whereas the Bb and C1q factors were only significantly present on the fibroin fabric. IgG appeared to be adsorbed, although to different extents, on both types of fibroin substrates. These results suggest that the biocompatibility of the silk fibroin may be affected by changes in protein structure induced by processing the material.

Subject index of volume 66

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.

Natural protein fibers

AbstractAlpha keratin fibers (hairs, wools, quills, and other mammalian appendages) together with fibroin fibers such as silks and spiders webs are all highly extensible fibrous proteins for which the mechanical properties are of primary importance both to the animal from which they originate and their ultimate application by man. Similarly, the collagens are highly inextensible fibrous proteins, which form the major component of mammalian skin and connecting structures such as tendons. All these fibrous proteins are biological polymers of polypeptide chains for which the mechanical and allied physical properties, such as water absorption, relate to both their macrostructure and their molecular and near‐molecular structure. Because of both their commercial application and their relatively complex structure at the molecular and near‐molecular level, interpretation of the physical properties of α‐keratin fibers represents the main component of this presentation. The mechanical properties of α‐keratin fibers are primarily related to the two components of the elongated cortical cells, the highly ordered intermediate filaments (microfibrils) which contain the α‐helices, and the matrix in which the intermediate filaments are embedded. The matrix consists of globular proteins plus water, the content of the latter being dependent on the fibers environment. The Extended Two‐Phase Model (ETPM) has been developed and results in a detailed coverage of the bulk mechanical properties of α‐helical fibers in terms of their known molecular and near‐molecular structure. The inextensible protein fibers, the collagens and fibroins, are also briefly discussed in terms of the relationship between mechanical properties and the structure of these fibers. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 489–507, 2002

Production of fine powder from silk by radiation

Silk fine powder was prepared directly from silk fibers irradiated with an accelerated electron beam (EB). Irradiated silk fibers were well pulverized only by physical crushing using a ball mill without any chemical pretreatment. Silk fibroin fibers were irradiated at ambient temperature in the dose range of 250–1 000 kGy. Although unirradiated silk fibers were not pulverized at all, irradiated fibers were easily pulverized and showed a high conversion efficiency from fiber to powder at high irradiation doses. The presence of oxygen in the irradiation atmosphere enhanced pulverization of the silk fibers. The electron microscopic observation showed that the minimum particle size of silk powder obtained from fibers irradiated at 1 000 kGy in oxygen was less than 10 μm. It was found that fibroin powder obtained in this work dissolved remarkably in water, thought unirradiated fibroin fibers were insoluble even in hot water. The soluble fraction was about 60% of fibroin powder for 1 000 kGy irradiation in oxygen.

Structure and dynamics of silk fibroin studied with 13C, 15N and 2H solid state NMR

Abstract[1‐13C]Gly, L‐[1‐13C]Ala, [15N]Gly, L‐[15N]Ala, [2,2‐2H2]Gly, L‐[3,3‐2H2]Ser and [3,3,3‐2H3]Ala labeled silk fibroin fibers from Bombyx mori and Samia cynthia ricini silkworms were prepared in order to analyze structure of backbone and dynamics of side chain. The torsion angles ϕ and Ψ were determined from the angular dependent 13C and 15N solid state NMR spectra for uniaxially oriented fiber samples. In addition, the characteristic side chain dynamics of Ser residue determined from solid state 2H NMR measurements was compared with those of Ala and Gly residues.

Structure of Alanine and Glycine Residues of Samia cynthia ricini Silk Fibers Studied with Solid-State 15N and 13C NMR

The structure of silk fibroin fiber from a wild silkworm, Samia cynthia ricini, whose amino acid sequence is similar to the spider (major ampullate) silk, was determined with solid-state NMR. The 15N and 13C labelings for the dominant amino acids, alanine and glycine, residues of S.c.ricini silk fibers, were performed by oral administration of [15N]alanine, [1-13C]alanine, [15N]glycine, or [1-13C]glycine to the fifth instar larvae of the silkworm, S.c.ricini. The blocks of the oriented silk fibers, stretched by about 10 times the original length of the sample, were prepared, and the 15N and 13C solid-state NMR were observed by changing the angles of the oriented silk fiber axis and the magnetic field. All of the oriented spectra of [15N]glycine silk fibroin fiber and [1-13C]glycine silk fibroin fiber were slightly broader than the corresponding spectra of alanine isotope-labeled silk fibers. The fraction of alanine residues in oriented domains in the silk fiber samples was 75%, and that of glycine residue...

Spectroscopic investigation of tertiary fold of staphylococcal protein A to explore its engineering application

Staphylococcal protein A is a cell wall constituent of most strains of Staphylococcus aureus, and it is characterized by its binding affinity to some immunological classes. A mutated low molecular weight type protein A (LPA; Mwt=27 kDa) which consists of the domains, E, D, A, B and 13 residues of the C-domain was prepared in this study. Since LPA does not possess a cell wall-bound region in contrast to wild-type protein A (WPA; Mwt=42 kDa), we have established a methodology of large scale purification of LPA without using any extracellular expression systems such as Escherichia coli. Using this relatively abundant protein, the immobilization of the LPA with silk fibroin of Bombyx mori was performed. Thermal stability of LPA immobilized with silk fibroin is higher than that of free LPA at high temperature judging from the immunoglobulin G (IgG)-binding affinity. However, the apparent value of its affinity decreased relative to that of immobilized WPA. These results indicate that structural information is essential to explore improvement of IgG-binding affinity of immobilized LPA. Therefore, secondary structure of free LPA was detected by its characteristic helical pattern in circular dichroism (CD) in aqueous solution. In addition to this, tertiary fold of four IgG-binding domains were investigated by two-dimensional 1H-NMR spectra. Four significantly high-field shifted cross-peaks attributed to methyl signals of alanine residues suggest that all four domains pack into a three helix bundle motif in solution. These structural data and properties of IgG-binding affinity suggest that spatial arrangement of four IgG-binding domains are packed into a compact globular molecular shape. This causes a certain active site of immobilized LPA to be buried in the silk fibroin fiber.

The preparation and applications of functional fibres from crab shell chitin

Novel chitin–silk fibroin fibres and chitin fibres were prepared by an environmental friendly wet-spinning method. Each aqueous solution of sodium chitin ( N-acetylchitosan) salt and its blends of silk fibroin in aqueous 14% sodium hydroxide was spun through a viscose-type spinneret into an aqueous 10% sulfuric acid solution saturated with ammonium sulfate (about 43%), and the corresponding white filament was obtained. The tenacity and elongation values of the chitin–silk fibroin filament decreased with an increase of fibroin content up to 33% by weight. A scanning electron microscopy analysis revealed that both the chitin filament and the chitin–silk fibroin (67:33, w/w) filament had vertical strips with faint scale structures on their surfaces. Some applications of these staple fibres were also reported.

Raman spectroscopic studies of silk fibroin fromBombyx mori

This study was focused on the conformational characterization of Bombyx mori silk fibroin in film, fiber and powder form by means of Fourier transform Raman spectroscopy. Native and regenerated silk fibroin films prepared by casting dilute silk fibroin solutions (<1%, w/v) display characteristic conformationally sensitive bands at 1660 cm-1 (amide I), in the range 1276–1244 cm-1 (a complex amide III region with multiple detectable maxima) and at 1107 and 938 cm-1. This spectral pattern can be related to a prevalently random coil conformation, with traces of α-helix. Liquid silk, prepared by casting the silk gland content (fibroin concentration 20–25%, w/v), shows almost the same wavenumbers in the amide I and III ranges, while differences appear below 1000 cm-1, where three bands at 952, 930 and 867 cm-1 increase in intensity. The spectral differences between films and liquid silk are discussed with a view to identifying possible markers for silk I structure, a crystalline modification of silk fibroin. The treatment of both native and regenerated films with 50% (v/v) methanol solution induces the conformational transition to a β-sheet structure, as demonstrated by the shift of amide I to 1665 cm-1 and the appearance of new maxima at 1262 and 1236 cm-1 (amide III) and at 1084 cm-1. When liquid silk is cast at above 50°C, the prevailing conformation taken by silk fibroin is β-sheet, whatever the rate of drying. By comparing the Raman spectra of silk fibroin fiber and powder, both having a β-sheet structure, a difference in the tyrosine doublet bands and in the amide I band can be observed. The value of the I853/I830 (Rtyr) intensity ratio increases in the powder while amide I shifts to lower wavenumbers, suggesting that the hydrogen bonds involving the tyrosil residues are weaker in the powder than in the fiber. © 1998 John Wiley & Sons, Ltd.

Structure of Bombyx mori Silk Fibroin Based on Solid-State NMR Orientational Constraints and Fiber Diffraction Unit Cell Parameters

Isotopic labeling of Bombyx mori silk fibroin was achieved biosynthetically using two approaches. First, labeled fibroin was achieved by feeding silk worms [13C1]Gly and [13C1]Ala along with an artificial diet. Second, in vitro production of [15N]Gly and [15N]Ala labeled B. mori silk fibroin was accomplished by culturing the posterior silk glands isolated from five-day-old silkworm larvae in the fifth instar stage. Orientation-dependent 15N and 13C solid-state NMR spectra of these isotope-labeled silk fibroin fibers were observed when the fiber axis was arranged at various angles between 0° and 90° to the magnetic field direction. Such data from the silk II structure was simulated on the basis of the chemical shift anisotropy to determine the Euler angles that relate the principal axis system (PAS) to the fiber axis coordinate system. The dipolar modulated 15N and 13C chemical shift powder pattern spectra of 13C−15N double-labeled silk fibroin model peptides were observed and simulated to determine the Eu...

Structure of uniaxially aligned 13C labeled silk fibroin fibers with solid state 13C-NMR

Carbon-13 isotopic labeling of B. mori silk fibroin was achieved biosynthetically with [1- 13C] glycine in order to determine the carbonyl bond orientation angle of glycine sites with the silk fibroin. Angular dependence of 13C solid state NMR spectra of uniaxially oriented silk fibroin fiber block sample due to the carbonyl 13C chemical shift anisotropy was simulated according to the chemical shift transformation with Euler angles, α F and β F , from principal axis system (PAS) to fiber axis system (FAS). The another Euler angles, α DCO and β DCO , for transformation from PAS to the molecular symmetry axis were determined from the [1- 13C] glycine sequence model compounds for the silk fibroin. By the combination of these Euler angles, the carbonyl bond orientation angle with respect to FAS of the [1- 13C] glycine sites of the silk fibroin was determined to be 90 ± 5°. This value is in agreement with the X-ray diffraction and our previous solid state NMR data of B. mori silk fibroin fiber (a typical β-pleated sheet) within experimental error.

Comparative Structural Characterization of Naturally- and Synthetically-Spun Fibers of Bombyx mori Fibroin

This investigation describes the comparative structural characterization of naturally- and synthetically-spun fibers of Bombyx mori fibroin. Wet spinning from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions was used to spin fibroin fibers with varying degrees of postspinning draw. Quantitative Raman spectroscopy showed that the secondary structure of fibroin changed dramatically from predominantly α-helical in HFIP solution to principally β-sheet in the undrawn synthetically-spun fiber. X-ray fiber diffraction showed that the undrawn fibers were highly crystalline (>50%) with little or no preferential crystalline alignment. The addition of a postspinning draw caused the polypeptide backbone and β-sheet crystals, which were formed largely by a methanol coagulation bath, to align with the fiber axis. The molecular and crystalline structures most similar to those of naturally-spun fibers were reproduced in synthetically-spun fibers with a minimum draw ratio of 2.5.

2H-Labeling of Silk Fibroin Fibers and Their Structural Characterization by Solid-State 2H NMR

Three kinds of 2H-labeled Bombyx mori silk fibroin samples (with [2,2-2H2]Gly, [3,3,3-2H3]Ala, or [2,3,5,6-2H4]Tyr) were obtained by oral administration of either the labeled amino acid or 2H2O to 5th instar larvae. The administration of 2H2O alone yielded a high degree of selective deuteration at the alanine methyl group, since the incorporation of 2H2O occurs between fumarate and malate in the tricarboxylic acid (TCA) cycle of the silk fibroin synthetic pathway. Uniaxially oriented silk fibers were prepared as samples for 2H-NMR spectroscopy. An analysis of the quadrupole echo line shape was carried out in order to determine the angle of the deuterium-labeled group relative to the fiber axis, i.e., of the Cα−2H bond vectors in glycine and of Cα−Cβ2H3 in alanine. With the fiber axis aligned parallel to the magnetic field, quadrupole splittings were obtained as 117.8 and 39.8 kHz for [2,2-2H2]Gly- and [3,3,3-2H3]Ala-labeled silk, respectively. These values are identical with those obtained from the 2H-NMR powder patterns of the unaligned samples, within experimental error. From the angular dependence of the quadrupole splittings, it was thus calculated that the Cα−2H bonds of glycine as well as the Cα−Cβ2H3 bond of alanine make an angle of approximately 90° relative to the fiber axis. These steric constraints were then used to evaluate the torsion angles, φ and ψ, for the glycine and alanine residues within the protein backbone. These data, determined independently by solid-state 2H NMR, thus verified and narrowed down the allowed region in the Ramachandran (φ, ψ) map obtained from previous solid-state 13C- and 15N-NMR studies.

Studies on regenerated protein fibers. III. Production of regenerated silk fibroin fiber by the self-dialyzing wet spinning method

The spinnable dope containing 20 wt % of silk fibroin dissolved in the MU solvent (e.g., LiBr · H2O = 40 wt %, and EtOH : H2O = 100 : 0 in volume) was spun into a coagulant and subsequently drawn, to give the following results. 1 Methanol, ethanol, and isopropanol were a predominant coagulant for the dope, and the coagulant containing 10% LiBr · H2O is best. 2 Spinnability depended upon the ratio of nozzle hole length to diameter (L/D) and was improved by increasing L/D from 4 to 10. Coagulativity was not sufficient at the SF concentration of 16%, but improved by raising it up to 20%. Drawability of the spun filaments in water was improved at a temperature above 60°C. 3 The drawn fiber showed excellent mechanical properties, especially tensile toughness when as-spun fiber was drawn in water at 61°C. An X-ray diffraction diagram of the drawn fibers indicated their crystalline axes were partially oriented along the fiber axis. © 1996 John Wiley & Sons, Inc.

Drawing of Silk Fibroin Fibers Treated with LiBr Aqueous Solution.

Mechanical and structural changes of silk fibers caused by stretch after treatment with LiBr aqueous solution were investigated. The mechanical properties of degummed silk depended upon the temperature of treatment with 8M LiBr aqueous solution. The strength and elongation increased with treatment temperature whereas the Young's modulus decreased. The X-ray diffraction pattern showed that the crystalline orientation was disturbed by the treatment at 80°C whereas the destruction of the crystallites did not occur. The stretch up to 160% was possible after the treatment at 80°C for 1h in 8M LiBr aqueous solution. The strength and Young's modulus increased while the elongation decreased. On the basis of the X-ray diffraction and SEM observations, the crystal orientation was found to become higher with increasing draw ratio.

Preparation and Antibacterial Activity of Silk Fibroin Fiber Containing Metal Complexes.

The preparation and the antibacterial activity of silk fibroin fibers containing metal complexes of Cu2+, Zn2+, Ni2+, Fe2+, and Ag+ were studied. The metalation was carried out in aqueous solutions of metal ions at low pH and in the solution of metal-ammine complexes at high pH using untreated silk fibroin fiber and fibers treated with a tannic acid aqueous solution. In solution of metal ions at low pH, unstable metal complexes were formed on the fiber without tannic acid, and exhibited no antibacterial activity owing to the low content of metal ions. From the metal-ammine complex solutions, very stable complexes were formed on the fiber but exhibited no activity owing to the high stability of biuret type complexes formed. On the other hand, metal complexes of Cu2+, Fe2+, Ag+ with tannic acid were formed on the fiber treated with tannic acid even at low pH. But the fixation of the complexes onto the fiber was not enough. Only Ag(I)-tannic acid complex on the fiber exhibited the antibacterial activity even in the low content. The sufficient metalation of the fiber containing tannic acid were under-gone in solutions of metal-ammine complexes of Cu2+, Zn2+, and Ni2+. These complexes on the fiber formed with tannic acid as ligands without peptide-nitrogen, and exhibited the strong antibacterial activity. Metalated silk fibroin fibers obtained from the metal-tannic acid complexes were effective, as one of the functional fibers, for the anti-bacterial fibers.

Changes in molecular aggregation structures of silk fibroin fibers treated with organic solvents

Changes in molecular aggregation structures of silk fibroin fibers treated with organic solvents

Physical and chemical properties of tussah silk fibroin films

AbstractPhysical and chemical structure, as well as thermal behavior of solution‐cast regenerated films, prepared from tussah (Antheraea pernyi) silk fibroin, were compared with those of solution‐cast native films, in order to ascertain whether treatment (degumming, dissolution) used for preparation affected their properties. Regenerated fibroin films exhibited a higher thermal stability than native ones, as shown by differential scanning calorimetry, thermomechanical analysis, and dynamic mechanical behavior. Glass transition temperature and other relevant thermal transitions of the regenerated silk specimen shifted to higher temperatures compared with those of native specimen. Molecular conformation and crystalline structure did not show significant differences between the two kinds of silk films. Amino acid composition and molecular weight, however, distribution changed markedly after dissolving tussah silk fibroin fiber in concentrated LiSCN in polypeptide size was the main features for the regenerated silk fibroin. © 1994 John Wiley &amp; Sons, Inc.

Changes in the fine structure of silk fibroin fibers following gamma irradiation

AbstractThe physicochemical changes of silk fibers irradiated with γ‐rays was studied in relation to the amount of absorbed does in the range 0–21 Mrad. The yellowing index (b/L) suddenly increased at low dose for both raw and degummed silk fibers. An equilibrium value was attained from 10 Mrad upward. The tensile properties were significantly affected by exposure to γ‐rays. Both strength and elongation at break decreased at almost the same rate and extent, attaining a final value that was one‐half of the untreated control. The birefringence and isotropic refractive index of exposed silk fibers decreased, the effect being larger in the low dose range, suggesting a decrease of crystallinity and molecular orientation. X‐ray diffraction curves, however, demonstrated that the crystalline structure remained unchanged even after exposure of the highest γ‐ray dose. The thermal behavior evaluated by DSC and TMA measurements showed that the γ‐irradiation induced a slight decrease of thermal stability in irradiated silk fibers, this effect being detectable only at 21 Mrad of the absorbed dose. The dynamic viscoelastic behavior suggested that the thermal movement of the fibroin molecules in the amorphous and crystalline regions increased with increasing absorbed dose, attributing to the physicochemical modifications induced by the ionizing radiations. © 1994 John Wiley &amp; Sons, Inc.