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PubMed2005

Wet spinning of Bombyx mori silk fibroin dissolved in N-methyl morpholine N-oxide and properties of regenerated fibres.

Silk fibroin (SF) was dissolved in N-methyl morpholine N-oxide (NMMO) at a polymer concentration of 13% (w/w); thermal and rheological solution properties were characterized. The melting/crystallization behaviour of NMMO was influenced by SF presence. Melting of NMMO hydrate decreased to 71 degrees C and a cold crystallization peak appeared at 35 degrees C on heating. None crystallization occurred on cooling. Quenching at a temperature of 50 degrees C or higher did not induce any crystallization on heating. Viscosity of SF-NMMO solutions decreased as a function of temperature. At 75 degrees C, viscosity remained constant for 360 min. SF-NMMO dope was spun by using a lab-scale wet spinning line. The extruded filament was coagulated in an ethanol bath. Regenerated SF fibres were collected at different draw ratios and their morphological, physical, and mechanical properties were characterized. Fibre diameters ranged from 133 to 19mum, cross-section was regularly circular, and surface was generally smooth, with a very fine granular aspect. Birefringence increased with increasing the draw ratio, especially when take up and post-spinning draw were coupled. FT-IR spectra and DSC thermograms confirmed that SF fibres crystallized into Silk II structure. The IR crystallinity index did not change as a function of drawing. Regenerated SF fibres undrawn or drawn only during the coagulation step showed the mechanical behaviour typical of a brittle material. However, when both take up and post-spinning draw were applied, fibres displayed a ductile-stable behaviour. Typical values of the mechanical parameters of regenerated SF fibres were: E=8.7 GPa, sigma(b)=120 MPa and epsilon(b)=35%.

Animals↗

PubMed2006

Time-resolved structural investigation of regenerated silk fibroin nanofibers treated with solvent vapor.

Nonwoven matrices of silk fibroin (SF) nanofibers were prepared by electrospinning a regenerated SF solution, followed by treatment with solvent vapor including water, methanol, ethanol, and propanol. Structural changes of solvent vapor-treated SF nanofibers were investigated in a time-resolved manner using IR spectroscopy. Conformational transitions of SF nanofibers from random coil to beta-sheet forms were dependent on the type of solvent vapor used, and their transition rates were strongly influenced by treatment temperatures. Consistent with previous findings, methanol vapor treatment provided a fast and effective means by which to alter the secondary structure of SF nanofibers. However, treatment with water vapor, as compared to treatment with alcohol vapor, was also useful for inducing structural changes in SF nanofibers. As demonstrated in the present study, our approach of controlling secondary structure formation of proteins by solvent vapor treatment and monitoring real-time conformational changes may be useful for the design and tailoring of materials for biomedical applications.

1-Propanol↗

PubMed2006

Biomimetic nanofibrous scaffolds: preparation and characterization of chitin/silk fibroin blend nanofibers.

Electrospinning of chitin/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was investigated to fabricate a biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun chitin/SF blend nanofibers was investigated with a field emission scanning electron microscope (FE-SEM). The average diameters of chitin/SF blend fibers decreased from 920 to 340nm, with the increase of chitin content in blend compositions. The miscibility of chitin/SF blend fibers was examined by solution viscosity measurement. The chitin and SF were immiscible in the as-spun nanofibrous structure. The dimensional stability of chitin/SF blend nanofibers, with or without water vapor after-treatment, was conducted by immersing in water. As-spun SF-rich blend nanofibrous matrices were lost their fibrous structure after the water immersion for 24h, and then changed into membrane-like structure. On the contrary, nanofibrous structures of water vapor-treated SF-rich blends were almost maintained. To assay the cytocompatibility and cell behavior on the chitin/SF blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal keratinocyte and fibroblasts seeded on the scaffolds were studied. Our results indicate that chitin/SF blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds because it has both biomimetic three-dimensional structure and an excellent cell attachment and spreading for NHEK and NHEF.

Adolescent↗

PubMed2006

Effect of shearing on formation of silk fibers from regenerated Bombyx mori silk fibroin aqueous solution.

In this paper, the spinnable regenerated silk fibroin aqueous solution with high concentration was prepared and the regenerated silk fibers were obtained from the aqueous solution by two different spinning processes at ambient temperature. The orientation of these fibers was characterized by polarizing microscope. Their secondary structure was investigated by Raman spectroscopy and related mechanical properties were also measured. These data showed that shearing is an important step for increasing orientation and silk II (beta-sheet) structure, and the mechanical properties of the regenerated silk fibers can also be improved by shearing.

Animals↗

PubMed2006

Complex coacervation of silk fibroin and hyaluronic acid.

This study aimed to investigate the pH-induced complexation of silk fibroin (SF) and hyaluronic acid (HA). SF-HA complex coacervation was investigated by monitoring turbidity of the SF-HA system under slow acidification. Gravimetric analysis was performed to determine the yield of complex coacervation and viscosity of the system was measured to study the formation of the complexes at different pH values. The influences of total biopolymer concentration and biopolymer weight ratio on complex coacervation were examined during the analyses. Formation of the complexes was evidenced by the minimum viscosity and the maximum turbidity observed in the system. SF-HA complexes were formed within the pH-window of 2.5-3.5 regardless of the total biopolymer concentration or biopolymer ratio. Complex coacervation of SF-HA showed a reversible behavior and coacervation could be handled even in excess amounts of the biopolymers, which pointed out a non-stoichiometric complexation.

Animals↗

PubMed2004

Enzymatic grafting of chitosan onto Bombyx mori silk fibroin: kinetic and IR vibrational studies.

The potential for using tyrosinase to graft the polysaccharide chitosan (Ch) onto Bombyx mori silk fibroin (SF) was examined. FT-IR spectroscopy coupled to HPLC amino acid analysis showed that mushroom tyrosinase (MT) catalyses the oxidation of tyrosine (Tyr) of SF to electrophilic o-quinones. Kinetic studies showed that only a fraction of the Tyr residues available on the SF chain were oxidized. This result was interpreted in the light of the structure assumed by SF in aqueous solution: Tyr aromatic side chain groups buried into the folded hydrophobic portions of the chain were probably less accessible to MT for steric reasons. Using slightly acidic conditions (pH 6), it was possible to modify SF under homogeneous conditions. FT-IR spectroscopy provided evidence that Ch was grafted onto MT-oxidized SF: the o-quinones were found to undergo a subsequent non-enzymatic reaction with nucleophilic amino groups of Ch via Schiff-base and Michael addition mechanisms. Various factors, i.e. reaction time, pH, MT/SF ratio, were found to influence the grafting yield. The highest grafting yield was achieved at pH 7, i.e. more favorable to MT activity rather than to Ch solubility, suggesting that the determining step of the grafting reaction is the formation of o-quinones. The FT-IR spectroscopy revealed that grafting induced a beta-sheet --> random coil conformational transition.

Animals↗

PubMed2005

Biological efficacy of silk fibroin nanofiber membranes for guided bone regeneration.

The favorable biological properties of silk fibroin (SF) nanofiber membrane make it a good candidate for clinical applications as a device in bone and periodontal regenerative therapy. The purpose of this study is to evaluate the biocompatibility of the SF nanofiber membrane, and to examine its effect on bone regeneration in a rabbit calvarial model. To examine the biocompatibility of the electrospun SF membrane, we investigated cell proliferation, morphology, and differentiation. The bone regenerative efficacy of the membrane was evaluated in the calvarial defect of rabbits. The cell numbers and osteocalcin production labels were significantly increased in accordance with culture period. Cells had a stellate shape and broad cytoplasmic extensions on the membrane. The cells showed activity of ALPase that was comparable to culture dishes, and were calcified similarly to culture dishes. In in vivo tests, a complete bony union across the defects was observed after 8 weeks. At 12 weeks, the defect had completely healed with new bone. In conclusion, the SF nanofiber membrane was shown to possess good biocompatibility with enhanced bone regeneration and no evidence of any inflammatory reaction. These results strongly suggest that the SF membrane should be useful as a tool for guided bone regeneration.

3T3 Cells↗

PubMed2001

Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid.

Structural characteristics and thermal and solution properties of the regenerated silk fibroin (SF) prepared from formic acid (FU) were compared with those of SF from water (AU). According to the turbidity and shear viscosity measurement, SF formic acid solution was stable and transparent, no molecular aggregations occurred. The sample FU exhibited the beta-sheet structure, while AU random coil conformation using Fourier transform infrared (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry. The effects of methanol treatment on samples were also examined. According to the measurement of crystallinity (XRD) and crystallinity index (FTIR), the concept of long/short-range ordered structure formation was proposed. Long-range ordered crystallites are predominantly formed for methanol treated SF film while SF film cast from formic acid favors the formation of short-range ordered structure. The relaxation temperatures of SF films measured by dynamic thermomechanical analysis supported the above mechanism due to the sensitivity of relaxation temperature on the short-range order.

Animals↗

PubMed2002

Structure and properties of silk fibroin-poly(vinyl alcohol) gel.

A series of porous silk fibroin-poly(vinyl alcohol) blend gels were prepared from corresponding aqueous mixtures by freeze- or air-drying, and their structure and mechanical properties were examined. The air-dried gels had higher crystallinity and much greater strengths than the freeze-dried gels, and therefore are suitable for mechanical uses. The freeze-dried gels had characteristic porous structure potentially useful as cell culture substrate. Its structure could be systematically varied by changing freezing temperature and freeze-thaw pretreatments before drying.

Fibroins↗

PubMed2004

Preparation of lactose-silk fibroin conjugates and their application as a scaffold for hepatocyte attachment.

We prepared glycoconjugates (Lac-CY-SF) by the homogeneous chemical modification of solubilized silk fibroin (SF) with lactose bearing the galactose residue using cyanuric chloride (CY) as a coupling spacer and examined the usefulness of their application as a scaffold for hepatocyte attachment. The covalent immobilization of lactose into SF was assessed by several criteria including 1H-NMR measurements and reactions with lectins such as Ricinus communis agglutinin (RCA(120)) and fluorescein isothiocyanate-labeled RCA(120) (FITC-RCA(120)). The 1H-NMR spectrum of Lac-CY-SF showed new broad peaks attributed to methine and methylene protons of lactose, and by the integrated intensities of the peaks the weight ratio of lactose to SF in Lac-CY-SF was determined to be 0.20. Addition of RCA(120) to Lac-CY-SF solution caused an increase in the turbidity of the Lac-CY-SF solution. Incubation of Lac-CY-SF films with FITC-RCA(120) showed that the fluorescence emitted from the whole surface of Lac-CY-SF films. Furthermore, we examined the effect of the Lac-CY-SF conjugate-coating onto polystyrene culture dishes on the attachment of rat hepatocytes and their morphology. Attachment of hepatocytes onto the 0.1% (w/v) conjugate-coated dishes showed about an 8-fold increase as compared with that on uncoated dishes, being comparable to that on collagen-coated dishes, whereas the attachment on the SF-coated dishes was lower than that on uncoated dishes. Hepatocytes cultured onto the conjugate-coated dishes for 2.5 h showed smaller round-shaped morphology compared to those on collagen-coated dishes. After 2 days of culture in medium containing 100 nM insulin and 100 nM dexamethasone, hepatocytes on the conjugate-coated dishes formed monolayer islands with a slightly dispersed morphology, while hepatocytes cultured on collagen-coated dishes were uniformly spread flat. These results indicate that the Lac-CY-SF conjugates are useful as a scaffold for hepatocyte attachment, but the morphology of hepatocytes cultured on the conjugate-coated dishes is different from that on collagen-coated dishes.

Animals↗

PubMed1997

Synthesis of poly(ethylene glycol)-silk fibroin conjugates and surface interaction between L-929 cells and the conjugates.

Poly(ethylene glycol) (PEG)-silk fibroin (SF) conjugates (PEG2-SF) were prepared by the chemical modification of solubilized SF with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (actPEG2) in borate buffer at 37 degrees C. The IR spectra and DSC curves of PEG2-SF and SF suggested the introduction of PEG into SF by the modification and the beta-sheet structure of both SF and PEG2-SF induced by the treatment with methanol aqueous solutions. The content of the PEG component in PEG2-SF was evaluated to be 67% by weight from the melting enthalpy change of PEG observed on the DSC thermogram of PEG2-SF. Water content and contact angle measurements of SF before and after the modification indicated that the hydrophilicity of the PEG2-SF surface increased compared with that of SF. The attachment and growth of fibroblast cells (L-929) on the matrix of PEG2-SF were studied by a cell culture method. PEG2-SF exhibited very low cell attachment and growth, though SF exhibited high cell attachment and growth. The filopodium of the cells attached to PEG2-SF could not be found, and the cells aggregated to form masses in scanning electron microscopy images. These results could be explained in terms of the increased hydrophilicity of the PEG2-SF surface.

Animals↗

PubMed2001

Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy.

The ethanol-induced conformation transition of regenerated Bombyx mori silk fibroin membrane from a poorly defined to the well ordered state was monitored by time-resolved Fourier transform infrared spectroscopy (FTIR) for the first time. From the analysis of FTIR difference spectra, taken on time scales as short as 6 s and up to 1 h after addition of ethanol, intensity vs. time plots of an increasing band at 1618 cm(-1) were observed indicating formation of a beta-sheet coincident with the loss of intensity of a band at 1668 cm(-1) indicating decreases of random coil and/or silk I structure. Both infrared markers were fitted with identical biphasic exponential decay functions, however, there was a clear burst phase occurring prior to the onset of the observed transitions. The conformation transition process is indicated to either proceed sequentially through (at least) two intermediate states that contain different levels of beta-sheet structure or to have parallel pathways of initial beta-sheet formation followed by a slower 'perfection' phase. The first observed process forms in a burst phase a few seconds after mixing (or even faster), prior to the collection of the first spectrum at 6 s. The second observed process occurs with a time constant of approximately 0.5 min, the intermediate present at this stage then continues with a time constant of 5.5 min completing the observed formation of the beta-sheet. The conformation transition of this slower intermediate is not only indicated by an analysis of the kinetics of the random coil and beta-sheet-specific bands discussed above, it roughly coincides with the appearance of an additional infrared marker at 1695 cm(-1), which may be a marker for beta-sheet structure specific to the formation of the perfected structure. The conformation transition of this protein analyzed by infrared spectroscopy provides insight into a part of the fascinating process of cocoon formation in B. mori.

Animals↗

PubMedDate not supplied

Chemical modification of silk fibroin with cyanuric chloride-activated poly(ethylene glycol): analyses of reaction site by 1H-NMR spectroscopy and conformation of the conjugates.

Solubilized silk fibroin (SF) in 0.1 M borate buffer (pH 9.4) was modified with 2-O-[methoxy(polyethylene glycol)]-4,6-dichloro-s-triazine (actPEG1) at 4 degrees C. The weight of the modified SF (PEG1-SF) was at least 3.2 times that of the starting material SF. Amino acid analysis of PEG1-SF suggested that the nucleophilic epsilon-amino group of the lysine residue and the nucleophilic imidazole group of the histidine residue in SF reacted with actPEG1. The 1H-NMR spectrum of PEG1-SF showed a downfield shift of the aromatic protons of the tyrosine residue from the corresponding protons of SF. The 1H-NMR spectrum of the SF reacted with cyanuric fluoride (CyF), whose fluorine atoms are known to react with the phenolic hydroxyl group of the tyrosine residue, also showed the downfield shift. These results suggested that the reaction site of SF with actPEG1 was the phenolic hydroxyl group of the tyrosine residue in addition to the lysine and histidine residues. The conformation of PEG1-SF in a solid state was examined by means of IR and X-ray measurement. The IR spectrum of PEG1-SF revealed a change in secondary structure from random coil to beta-sheet due to the coexistence of PEG molecules. The X-ray diffraction pattern of PEG1-SF indicated that the PEG molecules covalently bonding to SF narrowed the spacing of the interchain periodicity and promoted the formation of the interchain beta-sheet.

Amino Acids↗

PubMed2004

Effect of pH and copper(II) on the conformation transitions of silk fibroin based on EPR, NMR, and Raman spectroscopy.

Much attention has been paid to the natural mechanism of silkworm spinning due to the impressive mechanical properties of the natural fibers. Our results in the present work show that the fractional changes of the conformational components in regenerated silk fibroin (SF) extracted from Bombyx mori fibers is remarkably pH- and Cu(II)-dependent as demonstrated by Cu(II) EPR, (13)C NMR, and Raman spectroscopy. Cu(II) coordination atoms in SF are changed from four nitrogens to two nitrogens and two oxygens as well as to one nitrogen and three oxygens when the pH is lowered from 8.0 to 4.0. The addition of a given amount of Cu(II) into a SF solution could induce efficiently the SF conformational fractional change from silk I, a soluble helical conformation, to silk II, an insoluble beta-sheet conformation. This behavior is strikingly similar to that seen in prion protein and amyloid beta-peptide. On the basis of the similarity in the relevant sequence in SF to the octapeptide PHGGGWGQ in PrP, we suggest that at basic and neutral pH polypeptide AHGGYSGY in SF may form a 1:1 complex with Cu(II) by coordination of imidazole N(pi) of His together with two deprotonated main-chain nitrogens from two glycine residues and one nitrogen or oxygen from serine. Such a type of coordination may make the interaction between two adjacent beta-form polypeptide chains more difficult, thereby leading to an amorphous structure. Under weakly acidic conditions, however, Cu(II)-amide linkages may be broken and Cu(II) may switch to bind two N(tau) from two histidines in adjacent peptide chains, forming an intermolecular His(N(tau))-Cu(II)-His(N(tau)) bridge. This type of coordination may induce beta-sheet formation and aggregation, leading to a crystalline structure.

Animals↗

PubMedDate not supplied

Porous 3-D scaffolds from regenerated silk fibroin.

Three fabrication techniques, freeze-drying, salt leaching and gas foaming, were used to form porous three-dimensional silk biomaterial matrixes. Matrixes were characterized for morphological and functional properties related to processing method and conditions. The porosity of the salt leached scaffolds varied between 84 and 98% with a compressive strength up to 175 +/- 3 KPa, and the gas foamed scaffolds had porosities of 87-97% and compressive strength up to 280 +/- 4 KPa. The freeze-dried scaffolds were prepared at different freezing temperatures (-80 and -20 degrees C) and subsequently treated with different concentrations (15 and 25%) and hydrophilicity alcohols. The porosity of these scaffolds was up to 99%, and the maximum compressive strength was 30 +/- 2 KPa. Changes in silk fibroin structure during processing to form the 3D matrixes were determined by FT-IR and XrD. The salt leached and gas foaming techniques produced scaffolds with a useful combination of high compressive strength, interconnected pores, and pore sizes greater than 100 microns in diameter. The results suggest that silk-based 3D matrixes can be formed for utility in biomaterial applications.

Animals↗

PubMedDate not supplied

Swelling behavior and morphological evolution of mixed gelatin/silk fibroin hydrogels.

Mixed protein-based hydrogels have been prepared by blending gelatin (G) with amorphous Bombyx mori silk fibroin (SF) and promoting beta-crystallization of SF via subsequent exposure to methanol or methanol/water solutions. The introduction of beta crystals in SF serves to stabilize the hydrogel network and extend the solidlike behavior of these thermally responsive materials to elevated temperatures beyond the helix-->coil (h-->c) transition of G. In this work, we investigate the swelling and protein release kinetics of G/SF hydrogels varying in composition at temperatures below and above the G h-->c transition. At 20 degrees C, G and G-rich mixed hydrogels display evidence of moderate swelling with negligible mass loss in aqueous solution, resulting in porous polymer matrixes upon solvent removal according to electron microscopy. When the solution temperature is increased beyond the G h-->c transition to body temperature (37 degrees C), these gels exhibit much higher swelling with considerable mass loss due to dissolution and release of G. The extent to which these properties respond to temperature decreases systematically with increasing SF content. The unique temperature- and composition-dependent properties of G/SF hydrogels dictate the efficacy of these novel materials as stimuli-responsive delivery vehicles.

Animals↗

PubMed2006

Mixed protein blends composed of gelatin and Bombyx mori silk fibroin: effects of solvent-induced crystallization and composition.

Novel protein blends have been prepared by mixing gelatin (G) with Bombyx mori silk fibroin (SF) and using aqueous methanol (MeOH) to post-induce SF crystallization. When co-cast from solution, amorphous blends of these polymers appear homogeneous, as discerned from visual observation, microscopy, and Fourier-transform infrared (FTIR) spectroscopy. Upon subsequent exposure to aqueous MeOH, SF undergoes a conformational change from random coil to beta sheet. This transformation occurs in pure SF, as well as in each of the G/SF blends, according to X-ray diffractometry and thermal calorimetry. The influence of MeOH-induced SF crystallization on structure and property development has been ascertained in terms of preparation history and blend composition. Thermal gravimetric analysis reveals that the presence of beta sheets in SF and G/SF blends improves thermal stability, while extensional rheometry confirms that SF crystallization enhances the tensile properties of the blends. By preserving a support scaffold above the G helix-to-coil transition temperature, the formation of crystalline SF networks in G/SF blends can be used to stabilize G-based hydrogels for biomaterial and pharmaceutical purposes. The present study not only examines the properties of G/SF blends before and after SF crystallization, but also establishes the foundation for future research into thermally responsive G/SF bioconjugates.

Animals↗

PubMed2006

In vitro study of the proteolytic degradation of Antheraea pernyi silk fibroin.

In this study, Antheraea pernyi silk fibroin (Ap-SF) films were incubated with Protease Type XXI from Streptomyces griseus, at 37 degrees C, to investigate the degradation behavior in an in vitro model system. The enzyme-resistant fractions of Ap-SF films and the soluble peptides formed by proteolytic degradation were collected at specified times, from 1 to 17 days, and analyzed by high performance liquid chromatography, differential scanning calorimetry, FT-Raman, and FT-IR spectroscopy. Proteolysis resulted in extensive weight loss and progressive fragmentation of films, especially at long degradation times. A range of soluble peptides was formed by proteolysis. By high performance-size exclusion chromatography it was found that their average molecular weight changed with the time of incubation. The chemical analysis of the enzyme-resistant fraction of Ap-SF films at different times of degradation indicated that the proteolytic attack preferentially occurred in the less ordered Gly rich sequences and that the contribution of the Ala rich crystalline regions to the composition of biodegraded films became progressively larger. Accordingly, DSC and spectroscopic results showed an enhancement of the crystalline character of the biodegraded films. From the behavior of the most important thermal transitions, it was deduced that the alpha-helix domains probably represent the most enzyme-resistant fraction. The in vitro approach used in the present study seems to be a valid tool for studying the rate and mechanism of degradation of Ap-SF films and of other biopolymers of potential biomedical utility.

Animals↗