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.     

Cellulose nanofiber-reinforced silk fibroin composite film with high transparency

We successfully prepared optically transparent silk fibroin-cellulose nanofiber (CN) composite films from solvent casting using a stable CN suspension in an aqueous silk fibroin solution. The transmittance of the silk fibroin composite films was observed by a UV-visible spectrophotometer. The secondary structural change of the silk fibroin caused by the incorporation of CNs was characterized using Fourier transform infrared spectroscopy. A tensile test was carried out to investigate the mechanical properties. The results showed that the composite film exhibited visible-light transmittance of 75 %, and its mechanical strength and Young’s modulus were increased by 44 % and 35 %, respectively, as compared to a neat silk fibroin film.     

Structure and properties of silk fibroin modified cotton

In this research, a novel cotton fiber with a silk fibroin (SF) coating was prepared by the oxidation of a cotton thread with sodium periodate and subsequent treatment in a solution of silk fibroin. The structures of both the oxidized cotton samples and the SF modified cotton samples were investigated by Fourier transform infrared (FT-IR) in combination with X-ray photoelectron spectroscopy (XPS) analysis. Other performances such as surface morphology and breaking strength were also studied. The results indicated that the weight of the oxidized cotton samples increased during SF treatment, while that of the un-oxidized cotton (pure cotton) samples reduced after SF treatment. Compared with the pure cotton samples, the oxidized cotton clearly showed a characteristic absorption band at 1730 cm⁻¹ due to the stretching vibration of the C=O double bond of the aldehyde group. After being treated with the SF solution, the oxidized cotton fiber showed a weakened characteristic absorption band at 1730 cm⁻¹ and a new absorption band at round 1540 cm⁻¹, suggesting the formation of C-N bond between aldehyde groups in the oxidized cotton and primary amines in the silk fibroin. The results were also confirmed by XPS analysis. Compared with the oxidized cotton samples, the SF treated cottons had relatively smooth surfaces, similar breaking strength, and the improved wrinkle recovery angles. The results in this research suggest that cotton based materials with protein coating can be achieved without using any other crosslinking agents by the method introduced.     

Chondrogenic differentiation of rat mesenchymal stem cells on silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds

Cartilage repair is a challenge in bone tissue reconstruction. In this study, silk fibroin (SF), chondroitin sulfate (CS) and hyaluronic acid (HA) were employed to fabricate scaffolds for tissue engineered cartilage by freeze drying technique. The secondary pores were formed in the main pores of SF/CS/HA scaffold which improved the pore connectivity and equilibrium swelling of the scaffold. Furthermore, rat bone marrow mesenchymal stem cells were seeded on the scaffolds to evaluate the cell adhesion and proliferation. Results of hematoxylin/eosin staining and cell counting kit-8 assay showed that the cells migration and differentiation of SF/CS/HA (80/15/5) scaffold were better than that of SF/CS/HA scaffolds with different ratios after 7 days culture. Moreover, immunohistochemistry and scanning electron microscope demonstrated that large amounts of collagen II and proteoglycans of the cells were expressed in the SF/CS/HA 3D scaffold, while the expression of collagen I was barely visible by immunohistochemistry. Abound of extracellular matrix was formed to morphologically round and distributed uniformly throughout the scaffolds. The 3D ternary scaffold could promote the cells chondrogenic differentiation without using any inductive agent and offer potential for cartilage tissue regeneration.     

Production and characterization of fibroin hydrogel using waste silk fibers

The use of natural resources, especially processing wastes, as low cost and environmentally friendly alternative aiming high value-added applications is a subject of broad interest. Since the Brazilian silk production annually generates a large amount of waste during the silk fibers processing, this work explores the preparation and characterization of silk fibroin hydrogels using spinning waste silk fibers from textile processing and the processed ones. Hydrogels were obtained directly by dialyzing silk fibroin solutions against frequent changes of water until the gelation point and then lyophilized and characterized in terms of their morphology, crystallinity, thermal resistance and secondary structure. X-ray diffraction analysis revealed the presence of β-sheet conformation related to sol-gel transition. FT-IR spectra indicated the coexistence of random coil (silk I) and β-sheet (silk II) structures, with predominance of β-sheet conformation for hydrogels from processed silk fibers. From thermogravimetric analysis the presence of β-sheet secondary conformation was demonstrated by a degradation peak around 292 °C for both hydrogels. Freeze-dried hydrogels presented sheet or leaf like morphology and no significant change was observed among the hydrogels from waste silk fibers and processed ones. These characteristics suggest that silk fibroin hydrogels prepared from spinning waste silk fibers and obtained directly by dialysis can be potential candidates for biomaterials application, such as drug delivery systems and for wound dressings.     

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.     

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.     

Antibacterial properties of silk fibroin/chitosan blend films loaded with plant extract

The silk fibroin (SF)/chitosan (CHI) blend films were prepared by dissolving them in formic acid. The morphology of the films was examined by scanning electron microscopy (SEM). The roughness of the membranes was determined by atomic force microscopy (AFM). These films were treated with the extracts of Pistacia terebinthus, Pistacia lentiscus, and Hypericum empetrifolium. Folin-Ciocalteu assay was used to determine the amount of total phenols adsorbed on these blend films. The antibacterial properties of films were tested using disc diffusion and turbidity measurement methods against Escherichia coli and Staphylococcus epidermidis. The release of adsorbed phenolics from the film surface resulted in antibacterial properties.     

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.     

Pentacene crystal formation on the surface of silk fibroin films

Silk fibroin (SF) has attracted interest as a gate dielectric due to its electrical insulation and high mobility in pentacene based organic thin film transistors (OTFTs). In this research, the surface energy of SF is controlled by water annealing, ethanol, and methanol solution treatments in order to study the effect of pentacene morphology on SF thin films with various treatments. For different crystallization methods, the crystal structures and surface energies of SF were investigated in detail by FT-IR and contact angle. Methanol treated SF thin film has a lower surface energy than the other two thin films. Topologies of pentacene on the SF thin films with various surface energies were obtained by atomic force microscopy (AFM). The AFM results showed that the smallest grain size of pentacene was that on methanol treated SF thin film which demonstrated that methanol treated SF thin film can be a proper candidate for a gate dielectric in OTFTs.     

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.     

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.     

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.     

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.     

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.     

Grafting of tyrosine-containing peptide onto silk fibroin membrane for improving enzymatic reactivity

During enzymatic modifications of silk fibroins, the accessibility of tyrosinases to the reactive sites was limited owing to the steric hindrance of tyrosine residues in the fibroin proteins. To improve the reactivity of silk fibroin, a tyrosine-containing peptide (TyrP) was covalently grafted onto the fibroin surfaces using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Enzymatic oxidation of the modified fibroins was subsequently carried out with a mushroom tyrosinase, followed by coupling of ε-polylysine (ε-PL) with the generated o-quinone residues of silk fibroins. The efficacy of grafting reaction was examined by means of SDS-PAGE and amine acid analysis. The results indicated EDC treatment might cause the direct self-crosslinks of silk fibroins and TyrP-bridged cross-links of fibroin molecules as well, which led to a noticeable increase in the molecular weight of fibroin proteins. TyrP-grafted fibroins displayed higher reactivity compared to the untreated, and more ε-PL was bonded to the fibroin surfaces when incubating with tyrosinase, resulting in improved wettability and mechanical property. The presented work offers an efficient alternative for the enzymatic modification of the fibroin-based materials with tyrosinase.     

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.     

Effects of environment parameters on sol-gel transition and dry-spinnability of regenerated silk fibroin aqueous solution

Protein concentration, pH, the types and concentrations of metallic ions, and extensional flow are thought to be important environment parameters affecting the natural spinning process. In this study, we investigate the effects of the types and concentrations of metallic ions (Ca²⁺, Mg²⁺, and K⁺ ions), pH, and silk fibroin concentration on the sol-gel transition and the rheological behavior of a regenerated silk fibroin (RSF) aqueous solution. The results show that with an increase in the silk fibroin concentration, the weak acidic RSF aqueous solutions containing Mg²⁺ or Ca²⁺ ions undergo a phase transition to a weak gel state. Moreover, the rheological characterization of RSF aqueous solutions shows a dramatic change, and their apparent viscosities increase by almost three orders of magnitude and approach the apparent viscosity of the native dope in the silkworm gland. By using conventional pressure equipment, we investigate the dry-spinnability of weak gels. Further, we observe that the as-spun fibers exhibit a smooth surface and have inferior mechanical properties. The structure of the as-spun fibers is predominantly in a random coil or Silk I conformation.     

Preparation and characterization of Ceftazidime loaded electrospun silk fibroin/gelatin mat for wound dressing

Fabrication of Ceftazidime (CTZ) loaded silk fibroin/gelatin (SF/GT) nanofibers (NFs) without the loss of structure and bioactivity of CTZ was demonstrated by electrospinning method. The structure, morphology and mechanical properties of the electrospun SF/GT nanofibrous mats were characterized using FT-IR, SEM and DSC. The drug release profile of different electrospun fibers was analyzed using spectrophotometric method, and also diffusion method was applied to assess the antibacterial effect of NFs. Cell viability was evaluated by MTT assay. The results show that the average diameter of drug loaded NFs at the optimum polymer to drug feeding ratio (10:1) was 276.55±35.8 nm, while increasing the feeding ratio to 1:1 increases the average diameter to 825.02±70.3 nm. FT-IR of drug loaded NFs was revealed that CTZ was successfully encapsulated into NFs while viability study approved cytocompatibility of SF/GT NFs. CTZ was released from NFs during 6 h, and formation of inhibition zone in diffusion test demonstrated the antibacterial effect of drug loaded NFs. Altogether, the CTZ loaded SF/GT NFs can improve the drug effectiveness particularly in the prevention of post-surgical adhesions and infections for wound dressing.