Wet spinning of silk polymer. II. Effect of drawing on the structural characteristics and properties of filament

Regenerated silk fibroin (SF) filaments were prepared by the wet spinning technique. The rheological behavior of the SF dope solution prepared with formic acid was examined and the drawing effect on the structural characteristics and mechanical properties of SF filament was comparatively studied with those of natural silk fiber. SF dope exhibited shear thinning, but, as the dope concentration increased, the effect of shear thinning decreased, an indication that a higher concentration of dope solution will result in good spinnability. Wet-spun SF filaments exhibited a uniform and circular cross-sectional shape and dense morphology under SEM observation. X-ray diffraction (XRD) results revealed that the crystallinity of wet-spun regenerated filaments was hardly affected by the draw ratio, whereas the crystalline and amorphous orientation of regenerated SF filament showed different features depending on the drawing. The crystalline orientation of regenerated filaments increased with an increase of draw ratio and was lower than that of natural silk fiber. On the contrary, the amorphous orientation was constant throughout 1X-5X draw ratios, after an abrupt increase at 1X, and was higher than that of natural silk fiber. These differences in the orientation behaviors are attributed to the different spinning mechanisms involved. The tensile property was strongly dependent on the draw ratio. The breaking strength and elongation of the regenerated filament at 5X draw ratio were 2.2 g/day and 17%, respectively.     

A Novel Multifunctional Crosslinking PVA/CMCS Hydrogel Containing Cyclic Peptide Actinomycin X2 and PA@Fe with Excellent Antibacterial and Commendable Mechanical Properties

Bacterial infected environments and resulting bacterial infections have been threatening the human health globally. Due to increased bacterial resistance caused by improper and excessive use of antibiotics, antibacterial biomaterials are being developed as alternatives to antibiotics in some cases. Herein, an advanced multifunctional hydrogel with excellent antibacterial properties, enhanced mechanical properties, biocompatibility and self-healing performance, was designed through freezing-thawing method. This hydrogel network is composed of polyvinyl alcohol (PVA), carboxymethyl chitosan (CMCS), protocatechualdehyde (PA), ferric iron (Fe) and an antimicrobial cyclic peptide actinomycin X2 (Ac.X2). The double dynamic bonds among protocatechualdehyde (PA), ferric iron (Fe) and carboxymethyl chitosan containing coordinate bond (catechol-Fe) as well as dynamic Schiff base bonds and hydrogen bonds endowed the hydrogel with enhanced mechanical properties. Successful formation of hydrogel was confirmed through ATR-IR and XRD, and structural evaluation through SEM analysis, whereas mechanical properties were tested with electromechanical universal testing machine. The resulting PVA/CMCS/Ac.X2/PA@Fe (PCXPA) hydrogel has favorable biocompatibility and excellent broad-spectrum antimicrobial activity against both S. aureus (95.3 %) and E. coli (90.2 %) compared with free-soluble Ac.X2, which exhibited subpar performance against E. coli reported in our previous studies. This work provides a new insight on preparing multifunctional hydrogels containing antimicrobial peptides as antibacterial material.     

Chitosan-functionalized silk fibroin 3D scaffold for keratocyte culture

The goal of this study was to evaluate the potential suitability of an artificial membrane composed of silk fibroin (SF) functionalized by different ratios of chitosan (CS) as a substrate for the stroma of the cornea. Keratocytes were cultured on translucent membranes made of SF and CS with different ratios. The biophysical properties of the silk fibroin and chitosan (SF/CS) membrane were examined. The SF/CS showed tensile strengths that increased as the CS concentration increased, but the physical and mechanical properties of chitosan-functionalized silk fibroin scaffolds weakened significantly compared with those of native corneas. The resulting cell scaffolds were evaluated using western blot in addition to light and electron microscopy. The cell attachment and proliferation on the scaffold were similar to those on a plastic plate. Keratocytes cultured in serum on SF/CS exhibited stellate morphology along with a marked increase in the expression of keratocan compared with identical cultures on tissue culture plastics. The biocompatibility was tested by transplanting the acellular membrane into rabbit corneal stromal pockets. There was no inflammatory complication detected at any time point on the macroscopic level. Taken together, these results indicate that SF/CS holds promise as a substrate for corneal reconstruction.     

The effect of residual silk sericin on the structure and mechanical property of regenerated silk filament

In this study, we elucidated the effect of residual silk sericin (SS) on structure and mechanical properties of regenerated silk filament as well as on fiber formation. The dope viscosity markedly increased with increasing residual SS content in dope solution which was prepared by dissolving the silk protein in formic acid. As a result of FTIR, (13)C NMR, and XRD, a small amount of SS (9.6%) contained in the filament showed highest content of beta-sheet conformation and maximum crystallinity. It seems that the SS affects the structural change of SF up to a certain level by inducing the beta-transition easily. The tenacity of the filaments, containing 9.6-18.9% SS, was in the range of 2.1-2.4 gf/d, which was about 50% higher than the filament without SS (pure SF). Consequently, with the enhancement of spinnability in wet spinning process, the SS can play an important role for developing the crystalline structure of SF as well as for improving mechanical properties of the regenerated silk fiber.     

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 340 nm, 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 24 h, 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.     

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200_tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.     

Effect of chitin/silk fibroin nanofibrous bicomponent structures on interaction with human epidermal keratinocytes

To fabricate a biomimetic nanostructured bicomponent scaffolds, two types of chitin/silk fibroin (SF) nanofibrous scaffolds (blend scaffolds and hybrid scaffolds) were prepared by electrospinning or simultaneous electrospinning of chitin/SF solutions. The chitin/SF bicomponent scaffolds were after-treated with water vapor, and their nanofibrous structures were almost maintained. From the cytocompatibility and cell behavior on the chitin/SF blend or hybrid nanofibrous scaffolds, the hybrid matrix with 25% chitin and 75% SF as well as the chitin/SF blend nanofibers could be a potential candidate for tissue engineering scaffolds.     

Preparation and properties of alginate/carboxymethyl chitosan blend fibers

Alginate/carboxymethyl chitosan blend fibers, prepared by spinning their mixture solution through a viscose-type spinneret into a coagulating bath containing aqueous CaCl₂, were studied for structure and properties with the aid of infrared spectroscopy (IR), X-ray diffraction (XRD) and scanning electron micrography (SEM). The analyses indicated a good miscibility between alginate and carboxymethyl chitosan, because of the strong interaction from the intermolecular hydrogen bonds. The best values of the dry tensile strength and breaking elongation were obtained when carboxymethyl chitosan content was 30 and 10 wt%, respectively. The wet tensile strength and breaking elongation decreased with the increase of carboxymethyl chitosan content. Introduction of CM-chitosan in the blend fiber improved water-retention properties of blend fiber compared to pure alginate fiber. Antibacterial fibers, obtained by treating the fibres with aqueous solution of N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride and silver nitrate, respectively, exhibited good antibacterial activity to Staphylococcus aureus.     

Silk–PVA Hybrid Nanofibrous Scaffolds for Enhanced Primary Human Meniscal Cell Proliferation

In this study, silk fibroin nanofibrous scaffolds were developed to investigate the attachment and proliferation of primary human meniscal cells. Silk fibroin (SF)–polyvinyl alcohol (PVA) blended electrospun nanofibrous scaffolds with different blend ratios (2:1, 3:1, and 4:1) were prepared. Morphology of the scaffolds was characterized using atomic force microscopy (AFM). The hybrid nanofibrous mats were crosslinked using 25 % (v/v) glutaraldehyde vapor. In degradation study, the crosslinked nanofiber showed slow degradation of 20 % on weight after 35 days of incubation in simulated body fluid (SBF). The scaffolds were characterized with suitable techniques for its functional groups, porosity, and swelling ratio. Among the nanofibers, 3:1 SF:PVA blend showed uniform morphology and fiber diameter. The blended scaffolds had fluid uptake and swelling ratio of 80 % and 458 ± 21 %, respectively. Primary meniscal cells isolated from surgical debris after meniscectomy were subcultured and seeded onto these hybrid nanofibrous scaffolds. Meniscal cell attachment studies confirmed that 3:1 SF:PVA nanofibrous scaffolds supported better cell attachment and growth. The DNA and collagen content increased significantly with 3:1 SF:PVA. These results clearly indicate that a blend of SF:PVA at 3:1 ratio is suitable for meniscus cell proliferation when compared to pure SF-PVA nanofibers.     

Nanofibrous nonmulberry silk/PVA scaffold for osteoinduction and osseointegration

Poly‐vinyl alcohol and nonmulberry tasar silk fibroin of Antheraea mylitta are blended to fabricate nanofibrous scaffolds for bone regeneration. Nanofibrous matrices are prepared by electrospinning the equal volume ratio blends of silk fibroin (2 and 4 wt%) with poly‐vinyl alcohol solution (10 wt%) and designated as 2SF/PVA and 4SF/PVA, respectively with average nanofiber diameters of 177 ± 13 nm (2SF/PVA) and 193 ± 17 nm (4SF/PVA). Fourier transform infrared spectroscopy confirms retention of the secondary structure of fibroin in blends indicating the structural stability of neo‐matrix. Both thermal stability and contact angle of the blends decrease with increasing fibroin percentage. Conversely, fibroin imparts mechanical stability to the blends; greater tensile strength is observed with increasing fibroin concentration. Blended scaffolds are biodegradable and support well the neo‐bone matrix synthesis by human osteoblast like cells. The findings indicate the potentiality of nanofibrous scaffolds of nonmulberry fibroin as bone scaffolding material. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 271–284, 2015.     

Fabrication and evaluation of poly(epsilon-caprolactone)/silk fibroin blend nanofibrous scaffold

In this study we investigated the blend electrospinning of poly(?‐caprolactone) (PCL) and silk fibroin (SF) to improve the biodegradability and biocompatibility of PCL‐based nanofibrous scaffolds. Optimal conditions to fabricate PCL/SF (50/50) blend nanofiber were established for electrospinning using formic acid as a cosolvent and three‐dimensional (3D) PCL/SF blend nanofibrous scaffolds were prepared by a modified electrospinning process using methanol coagulation bath. The physical properties of 2D PCL/SF blend nanofiber mats and 3D highly porous blend nanofibrous scaffolds were measured and compared. To evaluate cytocompatibility of the 3D blend scaffolds as compared to 3D PCL nanofibrous scaffold, normal human dermal fibroblasts were cultured. It is concluded that biodegradability and cytocompatibility could be improved for the 3D highly porous PCL/SF (50/50) blend nanofibrous scaffold prepared by blending PCL with SF in electrospinning. In addition to the blending of PCL and SF, the 3D structure and high porosity of electrospun nanofiber assemblies may also be important factors for enhancing the performance of scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 265–275, 2012.     

Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D

In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze‐drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.     

Depleting deubiquitinating enzymes promotes apoptosis in glioma cell line via RNA binding proteins SF2/ASF1

USP5 and USP8 (Deubiquitinating enzyme) are highly overexpressed and more recognized as poor prognosis marker in various cancers. Depleting USP5 or USP8 to assess the synergism with proteasome inhibitor (Bortezomib) were measured. Furthermore, in present finding USP5 cooperates hnRNPA1 & USP8 cooperate SF2/ASF1, therefore gain in expression of either hnRNPA1 or SF2/ASF1 is sufficient to promote cell survival. On the other side, apoptosis markers were more pronounced in U87 or T98G cells devoid of either USP5 or USP8. However, apparent increase in SF2/ASF1 in absence of USP5, providing resistant factor is new. Antiapoptotic activity due to rise in SF2/ASF1 was validated after co-knock down of SF2/ASF1 in addition to USP5 induces more apoptosis comparing to individual knock down of USP5 or SF2/ASF1. This reveals SF2/ASF1 (RNA binding protein) delayed the apoptotic effect due to loss of USP5, lends ubiquitination of hnRNPA1. In presence of USP5, PI3 kinase inhibition promotes even more interaction between USP5 and hnRNPA1, thereby stabilizes hnRNPA1 in U87MG. In that way hnRNPA1 and SF2/ASF1 impart oncogenic activity. In conclusion, siRNA based strategy against USP5 is not enough to inhibit glioma, moreover targeting additionally SF2/ASF1 by knocking down USP8 is suitably more effective to deal with glioma tumour reoccurrence by indirectly targeting both SF2/ASF1 and hnRNPA1 oncogene.     

Collagen-based biomimetic nanofibrous scaffolds: preparation and characterization of collagen/silk fibroin bicomponent nanofibrous structures

Electrospinning of collagen (COL)/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol was investigated for fabrication of a biocompatible and biomimetic nanostructured scaffold for tissue engineering. The morphology of the electrospun COL/SF blend nanofibers was observed by scanning electron microscopy. The average diameters of COL/SF blend fibers ranged from 320 to 360 nm, irrespective of SF content in the blends. Both COL and SF components in the as-spun COL/SF blend matrices were stabilized by glutaraldehyde and water vapor, respectively, under the saturated glutaraldehyde aqueous solution at 25 degrees C. The glutaraldehyde vapor chemically stabilized the COL component via cross-linking, whereas the water vapor physically stabilized the SF component via crystallization to the beta-sheet structure. These structural changes of after-treated COL/SF blend matrices were examined using ATR-IR and CP/MAS (13)C NMR spectroscopy. To assay the cytocompatibility and cellular behavior of the COL/SF blend nanofibrous scaffolds, cell attachment and the spreading of normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) seeded on the scaffolds were studied. In addition, both morphological changes and cellular responses of COL/SF blend nanofibrous matrices were also compared with COL/SF hybrid nanofibrous matrices. Generally similar levels of cell attachment and spreading of NHEF were shown in the COL/SF blend nanofibrous matrix compared with those of the pure COL and pure SF matrices; the cellular responses of NHEK were, however, markedly decreased in the COL/SF blend nanofibrous matrix as compared to the pure matrices. In contrast, cell attachment and spreading of NHEK on the COL/SF hybrid nanofibrous matrix were significantly higher than that of the COL/SF blend nanofibrous matrix. Our results indicate that a COL/SF hybrid nanofibrous matrix may be a better candidate than a COL/SF blend nanofibrous matrix for biomedical applications such as wound dressing and scaffolds for tissue engineering.     

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (?-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to β-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.     

Solubility and rheological behavior of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide

As an important direct solvent for cellulose, N-methyl morpholine N-oxide (NMMO) is environmentally friendly, and potentially very economical. Silk fibroin (SF) (Bombyx mori) can also be dissolved directly in NMMO.H2O. However, it is unexpectedly difficult to obtain a silk fibroin solution with a concentration higher than 10wt.% in this way, and extensive degradation of silk fibroin occurs if the dissolution temperature is higher than 110 degrees C. On the other hand, it is found that regenerated silk fibroin (RSF) film is much easier to dissolve in NMMO.H2O than ordinary SF. The RSF in NMMO.H2O can be easily concentrated to a range from 10 to 25wt.%. The structural differences between the degummed silk fiber and the RSF film lead to this different solubility in NMMO.H2O. The rheological behavior of concentrated RSF/NMMO.H2O solutions were also investigated. Regenerated silk fiber was spun from this type of solution, and its strength can reach up to 3.07 cN/dtex.     

The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs)

Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine-serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.