Antimicrobial electrospun silk fibroin mats with silver nanoparticles for wound dressing application

In this report, silk fibroin (SF) mats coated with silver nanoparticles (AgNPs) were manufactured as a prototypic wound dressing and evaluated for antimicrobial properties. SF was extracted from cocoons of Thai silkworms Bombyx mori (variant Nangnoi Si Sa Ket) and fabricated into nonwoven mats by electrospinning. In a one-step synthesis method, colloidal AgNPs were prepared from silver nitrate by gamma irradiation and inspected by transmission electron microscopy. Using the in vitro disc diffusion and growth-inhibition assays, AgNP-coated SF mats effectively inhibited the growth of Staphyllococus aureus and Pseudomonas aeruginosa when the coating solution containing colloidal AgNPs was 4 mM, or equivalent to 50.4 ng/cm² of adsorbed AgNPs. Based on these results, the AgNP-coated SF mats can potentially be used as antimicrobial wound dressings.     

Crosslinking reaction of phenolic side chains in silk fibroin by tyrosinase

Tyrosinase oxidizes the tyrosyl residues in silk fibroin (SF) with oxygen, resulting in the production ofo-quinone residues. Subsequently, the inter- or intramolecular crosslinks are formed by reaction with amino groups in through nonenzymatic process. The measurement of oxygen consumption proved that the tyrosyl residues in SF were mostly oxidized to quinone residues by tyrosinase. The reaction mechanisms were proposed in this study and the crosslinking reaction ofo-quinone residues and the enzymatic oxidation of tyrosyl residues could be confirmed by the measurements of UV,¹H-NMR and GFC.     

Silk grafting with chitosan and crosslinking agents

Chitosan grafting onto silk was tested with three crosslinking agents: trifunctional epoxy resin Araldite DY-T, PEG400 dimethacrylate, and glutaraldehyde in acetic as well as in tartaric acid solutions. Operating conditions were studied to obtain a significant silk weighting with satisfactory graft yields. With the epoxy crosslinker the weight gain was in the range from 1.8 to 8.8 % with graft yield from 8 to 23 %. In the case of PEG400DMA weight gain was 8–12 % with 22–24 % graft yield. With glutaraldehyde in tartaric acid solution a maximum weight gain of 8.4 % with 27.6 % graft yield was obtained. Results of determination of primary amino groups on the grafted silk showed that with epoxy and glutaraldehyde, unlike PEG400DMA, the amino groups of chitosan were only partially involved in crosslinking. Results of DSC analyses suggested that the modification of fibroin structure in chitosan-grafted silk was stronger with glutaraldehyde than with epoxy or dimethacrylate. FTIR-ATR spectra of grafted fibers were found very similar to that of control silk with an additional weak peak ascribable to chitosan in 1180–1080 cm⁻¹ range. Surface investigation through AFM showed clear morphology differences between chitosan-grafted silk with epoxy or dimethacrylate and that crosslinked with glutaraldehyde; the latter appears uneven and scale-like, the others slightly rougher than the original silk.     

Preparation and characterization of blended <Emphasis Type="Italic">Bombyx mori</Emphasis> silk fibroin scaffolds

The aim of this study was to compare physical, mechanical and biological properties of 3-dimensional scaffolds prepared from Bombyx mori silk fibroin (SF), fibroin blended with collagen (SF/C), and fibroin blended with gelatin (SF/G) using a freeze-drying technique. The prepared scaffolds were sponge-like structure that exhibited homogeneous porosity with highly interconnected pores. Average pore size of these scaffolds ranged from 65–147 μm. All biodegradable scaffolds were capable of water absorption of 90 %. The degradation behavior of these scaffolds could be controlled by varying the amount of blended polymer. The SF/C and SF/G scaffolds showed higher compressive modulus than that of SF scaffolds which could be attributed to the thicker pore wall observed in the blended constructs. The less crystalline SF structure was observed in SF/G scaffolds as compared to SF/C scaffolds. Thus, the highest compressive modulus was observed on SF/C matrix. To investigate the feasibility of the scaffolds for cartilage tissue engineering application, rat articular chondrocytes were seeded onto the scaffolds. The MTT assay demonstrated that blending collagen or gelatin into SF sponge facilitated cell attachment and proliferation better than SF scaffolds. The blended SF scaffolds possessed superior physical, mechanical and biological properties in comparison to SF scaffolds and showed high potential for application in cartilage tissue engineering.     

Surface modification of silk fibroin nanofibrous mat with dextran for wound dressing

In this study, we examined the effects of a dextran-modified silk fibroin nanofibrous mat (D-SFNM) on wound healing. To increase the hydrophilicity of silk fibroin (SF), the SF nanofibrous mat (SFNM) was modified with oxidized dextran. The D-SFNM absorbed water faster than the SFNM, and the swelling ratio was increased by approximately 80 % compared with the SFNM. An in vitro cell (NIH3T3) test revealed that fewer cells attached to the D-SFNM than the SFNM, but the proliferation of cells was not significantly affected by the presence of dextran. An in vivo wound healing test with mice indicated that the D-SFNM resulted in a good wound recovery effect similar to a commercial wound dressing material. The increased hydrophilicity of the D-SFNM might balance the moist environment at the wound site, which improves the wound healing compared with the SFNM.     

Metal ion adsorbability of electrospun wool keratose/silk fibroin blend nanofiber mats

In this study, electrospun wool keratose (WK)/silk fibroin (SF) blend nanofiber was prepared and evaluated as a heavy metal ion adsorbent which can be used in water purification field. The WK, which was a soluble fraction of oxidized wool keratin fiber, was blended with SF in formic acid. The electrospinnability was greatly improved with an increase of SF content. The structure and properties of WK/SF blend nanofibers were investigated by SEM, FTIR, DMTA and tensile test. Among various WK/SF blend ratios, 50/50 blend nanofiber showed an excellent mechanical property. It might be due to some physical interaction between SF and WK molecules although FTIR result did not show any evidence of molecular miscibility. As a result of metal ion adsorption test, WK/SF blend nanofiber mats exhibited high Cu²⁺ adsorption capacity compared with ordinary wool sliver at pH 8.5. It might be due to large specific surface area of nanofiber mat as well as numerous functional groups of WK. Consequently, the WK/SF blend nanofiber mats can be a promising candidate as metal ion adsorption filter.     

Effect of sericin blending on molecular orientation of regenerated silk fiber

The use of regenerated silk fiber is limited due to its inferior mechanical properties in spite of high potential in a wide variety of applications. Many studies have been conducted in order to improve the mechanical properties of the regenerated silk materials, but no one has so far suggested an obvious solution. Meanwhile, some reports showed evidence that structural development of silk protein can be manipulated by physical interactions between silk fibroin (SF) and silk sericin (SS) during the regeneration process, especially in recrystallization process of SF. Such a hypothesis suggests a promising clue to enhance the mechanical properties of silk-based materials. Therefore, in this study, we tried to elucidate how SS can promote developing the molecular chain orientation of SF, resulting in an improvement of mechanical properties of regenerated silk fiber during spinning process. The tensile properties of the regenerated silk fiber were significantly improved compared to those of pure SF fiber when a proper amount of SS was blend with SF; both tenacity and breaking elongation increased by approximately 30 % and 70 % at three fold draw ratio, respectively. Quantitative analysis of X-ray diffraction and Herman’s orientation coefficient confirmed that such an improvement of tensile property was mainly caused by an increase of molecular orientation induced by sericin during the drawing process.     

Enhancement biocompatibility of bacterial cellulose membrane via laccase/TEMPO mediated grafting of silk fibroins

Silk fibroin (SF) has the characteristic of moisture penetrability and biocompatibility. To enhance the biocompatibility of bacterial cellulose (BC), silk fibroin is grafted onto BC membrane using laccase and 2,2′,6,6′-tetramethylpiperidine-N-oxyl (TEMPO). As the model compound of BC, cellobiose is incubated with laccase/TEMPO for disclosing the mechanism of enzymatic oxidation. The structure and property of the composite membranes of SF/BC are investigated by means of FTIR, XPS, DSC, and biocompatibility analysis. The results indicate that cellobiose might react with hexamethylenediamine and form Schiff bases. The concentration of amino group in SF solution noticeably decreased after laccase/TEMPO oxidation, indicating the occurrence of self-crosslinking of SF. After enzymatic grafting of SF, the content of atomic nitrogen on BC surface was increased compared to that of the control. Meanwhile, the composite membrane of SF/BC exhibits more satisfactory biocompatibility compared to BC, and it has potential applications in biomedical fields.     

Key Characteristics of a Novel Silk Yarn from Fresh Cocoons

We studied the key characteristics of a novel silk yarn reeled from fresh cocoons. Compared with traditional silk yarn, this novel silk yarn displayed better mechanical properties, especially in terms of a higher breaking stress and toughness, and exhibited a different surface morphology. A cross-sectional observation and the sericin content results illustrated that different sericin coatings on the silk yarn reeled from fresh cocoons surface did not improve the mechanical properties. The degumming and tensile testing analysis indicated that degummed silk fibroin of novel silk yarn is able to resist deformation and fracture better than silk fibroin of traditional silk yarn. The FTIR results revealed that the selected techniques is an important contributor to the silk fibroin mechanical properties, because novel technique brought higher percentage beta-sheet structures in novel silk yarn fibroin than traditional silk yarn. The new technique that using novel silk yarn has improved its mechanical properties and it is expected that the silk yarn with superior mechanical properties could be used in fabrics transistors, electrodes and reinforced biomaterials.     

Relationships between antithrombogenicity and surface free energy of regenerated silk fibroin films

Silk fibroin (SF) was dissolved in calcium chloride/ethanol/water mixture (1/2/8 in mole ratio) at 70°C for 4 h. The dissolved silk fibroin was regenerated by casting the dialyzed solution into the films. The films were treated with 50% aqueous solution of methanol for different times, and their antithrombogenicity was evaluated byin vitro andin vivo tests.In vivo blood tests were made by a method of peripheral vein indwelling suture. It was found that the silk fibroin had a good anti-thrombogenicity and an absorbability even though the polymer showed foreign body reaction. Finally, the blood compatibilty of silk fibroin films which were subjected to structural change by the methanol treatment, was examined in connection with their interfacial surface energy, and a correlation between these properties was found to be present.     

A new consolidation system for aged silk fabrics: Interaction between ethylene glycol diglycidyl ether,silk Fibroin and Artificial Aged Silk Fabrics

Consolidation of fragile historic silks is of great importance for further displays and researches. An effective and convenient method to consolidate aged silk fabric has been proposed by using a silk fibroin (SF)/ethylene glycol diglycidyl ether (EGDE) consolidation system. Artificial aged silk fabrics treated with SF/EGDE show great improvement in mechanical properties. The chemical reaction between EGDE and silk fabrics has been proved in previous paper. And in this paper, mechanical test, field emission scanning electron microscopy (FESEM), Fourier transform infrared spectrum (FTIR) test and amino acid analysis (AAA) were applied to illustrate the interactions between SF and silk fabric, EGDE and SF. Results show that SF takes part in the consolidation in the form of adhesions on the surface of silk fibers. The chemical reactions and film adhesion are both responsible for the improvements of mechanical properties in the consolidation.     

The effect of casting solvent on the structural characteristics and miscibility of regenerated silk fibroin/Poly(vinyl alcohol) blends

Regenerated silk fibroin(SF)/Poly(vinyl alcohol)[PVA] blend films were prepared using different casting solvents, water and formic acid, to elucidate the effect of casting solvent on the structure and miscibility of SF/PVA blends. FTIR and XRD measurement suggested thatβ-sheet conformation of SF was not changed by addition of PVA in case of formic acid casting and the casting solvent determined the crystallized component of SF/PVA, leading to a different trend in the overall crystallinity between the two blends. The casting solvent had a dominant role in deciding phase behavior and molecular miscibility of blend films. SEM observation and DMTA measurement elucidated that water solvent produced phase-separated blend films while formic acid yielded one phase blend films with partial miscibility in molecular level indicating that the miscibility of SF blend can be improved by choosing a proper co-solvent.     

Electrospinning of <Emphasis Type="Italic">Bombyx mori</Emphasis> silk fibroin nanofiber mats reinforced by cellulose nanowhiskers

Cellulose nanowhisker (CNW) reinforced electrospun Bombyx mori silk fibroin (SF) nanofibers were fabricated. The morphology, structure, and mechanical properties of nanofibers were investigated by FE-SEM, TEM, FTIR, and tensile testing. It was found that the nanofiber size decreased obviously from 250 nm in the unreinforced mat to 77–160 nm in the CNW reinforced mats depending on the CNW content due to the increased conductivity of spinning dope. In the reinforced mats, the CNWs were embedded in the SF matrix separated from each other, and aligned along the fiber axis. There was a positive correlation between the CNW content and the tensile strength and Young’s modulus of reinforced mats. However the strain at break dropped gradually with the increase of CNW. When the CNW content was 2 w/w%, the tensile strength and Young’s modulus of reinforced SF nanofiber mats were about 2 times higher than those of unreinforced mat.     

Correlating the fineness and residual gum content of degummed hemp fibres

It is well known residual gum exists in degummed or retted hemp fibres. Gum removal results in improvement in fibre fineness and the properties of the resultant hemp yarns. However, it is not known what correlation if any exists between the residual gum content in retted hemp fibres and the fibre fineness, described in terms of fibre width in this paper. This study examined the mean width and coefficient of variation (CV) of fibre width of seventeen chemically retted hemp samples with reference to residual gum content. The mean and CV of fibre width were obtained from an Optical fibre diameter analyser (OFDA 100). The linear regression analysis results show that the mean fibre width is directly proportional to the residual gum content. A slightly weaker linear correlation also exists between the coefficient of variation of fibre width and the residual gum content. The strong linear co-relation between the mean of fibre width and the residual gum content is a significant outcome, since testing for fibre width using the OFDA is a much simpler and quicker process than testing the residual gum content. Scanning Electron Microscopy (SEM) reinforces the OFDA findings. SEM micrographs show a flat ribbon like fibre cross-section hence the term “fibre width” is used instead of fibre diameter. Spectral differences in the untreated dry decorticated skin samples and chemically treated and subsequently carded samples indicate delignification. The peaks at 1370 cm⁻¹, 1325 cm⁻¹, 1733 cm⁻¹, and 1600 cm⁻¹ attributed to lignin in the untreated samples are missing from the spectra of the treated samples. The spectra of the treated samples are more amine-dominated with some of the OH character lost.     

An adsorption study of alum-morin dyeing onto silk yarn

Silk yarn was dyed with morin (2′,3,4′,5,7-pentahydroxyflavone) by using alum as mordant. In order to optimize the process, three methods of dyeing involving: pre-mordanting, simultaneous mordanting, and post-mordanting were assessed and compared with a mordant-free process. The adsorption of alum-morin dye onto silk fibers indicated that the adsorption capacities were significantly affected by pH, the initial dye concentration, and temperature. The initial dye adsorption rates of alum-morin dye on silk before equilibrium was reached increased with higher dyeing temperatures. The pseudo second-order kinetic model was indicated for alum-morin dyeing (simultaneous mordanting) of silk at pH 4.0 with an activation energy (E _a ) of 45.26 kJ/mol. The value of the enthalpy of activation (ΔH ^#) for alum-morin dyeing on silk at pH 4.0 was −31.29 kJ/mol. Also, the free energy (ΔG ^o) and entropy changes (ΔS ^o) for alum-morin dyeing on silk were −17.73 kJ/mol and −45.7 J/molK, respectively, consistent with a spontaneous and exothermic adsorption process.     

Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme

Silk fibroin (SF) nanofibers were prepared by electrospinning and their application as an enzyme immobilization support was attempted. By varying the concentration of SF dope solution the diameter of SF nanofiber was controlled. The SF nanofiber web had high capacity of enzyme loading, which reached to 5.6 wt%. The activity of immobilizedα-chymotrypsin (CT) on SF nanofiber was 8 times higher than that on silk fiber and it increased as the fiber diameter decreased. Sample SF8 (ca. 205 nm fiber diameter) has excellent stability at 25°C by retaining more than 90 % of initial activity after 24 hours, while sample SF11 (ca. 320 nm fiber diameter) shows higher stability in ethanol, retaining more than 45% of initial activity. The formation of multipoint attachment between enzyme and support might increase the stability of enzyme. From these results, it is expected that the electrospun SF nanofibers can be used as an excellent support for enzyme immobilization.     

Fabrication of silk fibroin based three dimensional scaffolds for tissue engineering

Herein we report successful synthesis of silk fibroin (SF) three dimensional scaffolds (SF 3D-scaffold) from SF sponge and SF nanofibers. Both the nanofibers and sponge were prepared from Bombyx mori fibroin. The SF 3D-scaffold was prepared by electrospinning the fibroin nanofibers over the sponge. Surface morphology was determined by scanning electron microscopy (SEM), while nanofiber diameter and pore size were measured using imageJ software. Effect of spinning time on the pore size and cell adhesion was determined. Average diameter of the SF nanofibers was measured to be 320 nm and pore size was found to reduce with increasing spinning time, such that, for 1 h spinning time pore size was 231 µm and the same for 3.5 h was 4.1 µm. However, the number of pores increased with spinning time. The results confirmed adhesion of MC3T3-E1 cells on the SF sponge, SF nanofibers and SF three dimensional scaffolds. Higher cell adhesion was found on the three dimensional scaffold in comparison to the nanofibers and sponge, possibly due to highly porous structure with very small and numerous pores in the resultant composite; hence more cell adhesion sites. The cell adhesion result confirmed biocompatibility of the SF 3D-scaffold and hence its suitability for applications in tissue engineering.     

Structural and physical properties ofAntheraea pernyi silk fibroin fiber treated with I2-KI aqueous solution

Silk fibroin (SF) fiber from theAntheraea pernyi silkworm was treated with a 1.23 N iodine-potassium iodide (I₂-KI) aqueous solution, and the structure and physical properties were investigated to clarify the effects of the iodine treatment. The noticeably high weight gain value of SF fiber, about 25 wt% was attributed to the absorption of polyiodide ions in the form of I₃ ^? and I₅ ^?. Fourier transform infrared spectroscopy and X-ray diffraction measurements suggested that polyiodide ions mainly entered the amorphous region. In addition, a new sharp reflection on the meridional direction, corresponding to a period of 7.0 Å, was observed and indicated the possibility of the formation of mesophase structure ofβ-conformation chains. Dynamic viscoelastic measurements showed that the damping tanδ peak at 270°C gradually shifted to lower temperature in the iodinated SF fibers, suggesting an enhancement of the molecular motion of the fibroin chains induced by the presence of polyiodide ions. With heating above 254 °C, the iodine component introduced intermolecular cross-linking of SF, and the melt flow of the sample was inhibited. The thermal decomposition stability of fibroin molecules was greatly enhanced by iodine treatment.     

Growth of primary hippocampal neurons on multichannel silk fibroin scaffold

Particular attention has been given to axonal outgrowth of neurons to understand how topographical surface cues influence attachment and subsequent directional migration and growth. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional freeze-drying processes. The average pore diameter, the porosity, and pore density of the scaffold are 120 µm, 88 %, and 203 mm^?2, respectively. Further, hippocampal neurons were seeded onto the scaffold and the hippocampal neurons morphology was investigated. Cell-cell networks and cell-matrix interactions had been established by newly formed axons and the diversity of neurons was much higher after culturing 7 days. The neurons expressed β-III-tubulin and nerve filament, while glial fibrillary acidic protein immunofluorescence was barely above background. These results indicated that the SF scaffolds with uniaxial multichannels could be guided axons of neurons spread along the channels. SF scaffolds with oriented pores have a potential for nerve tissue regeneration.     

Fabrication of silk fibroin/eggshell nanofiber membranes for facemasks

We report our study on fabrication of soluble eggshell membrane (SESM) and silk fibroin (SF) nanofibers composite (SF/SESM) for facemasks by electrospinning. Biocompatibility of the SF and SESM, determined from hydrophilicity results, is exploited in SF/SESM nanocomposite for facemask application. The SF/SESM nanocomposites were prepared in different ratios of SF and SESM. The samples were characterized by scanning electron microscopy (SEM), FTIR and water droplet adsorption tests conducted via water contact angle (WCA) and water droplet diffusion. The results revealed that addition of SESM has insignificant effect on the electrospinnability of SF nanofibers in the studied ratios. The SEM results depicted regular morphology of the nanofibers except increase in nanofiber diameter with addition of SESM. The FTIR results confirmed respective peaks of SF and SESM in SF/SESM nanocomposite. WCA of the nanofibers decreased with addition of SESM such that for SF/SESM30, 30 % SESM, it reduced to 0 ° from 101 ° for pure SF nanofibers. The research results demonstrate SF/SESM30 nanocomposite as optimum ratio of SF and SESM for facemasks and other biomedical applications.