Significantly reinforced composite fibers electrospun from silk fibroin/carbon nanotube aqueous solutions

Microcomposite fibers of regenerated silk fibroin (RSF) and multiwalled carbon nanotubes (MWNTs) were successfully prepared by an electrospinning process from aqueous solutions. A quiescent blended solution and a three-dimensional Raman image of the composite fibers showed that functionalized MWNTs (F-MWNTs) were well dispersed in the solutions and the RSF fibers, respectively. Raman spectra and wide-angle X-ray diffraction (WAXD) patterns of RSF/F-MWNT electrospun fibers indicated that the composite fibers had higher β-sheet content and crystallinity than the pure RSF electrospun fibers, respectively. The mechanical properties of the RSF electrospun fibers were improved drastically by incorporating F-MWNTs. Compared with the pure RSF electrospun fibers, the composite fibers with 1.0 wt % F-MWNTs exhibited a 2.8-fold increase in breaking strength, a 4.4-fold increase in Young's modulus, and a 2.1-fold increase in breaking energy. Cytotoxicity test preliminarily demonstrated that the electrospun fiber mats have good biocompatibility for tissue engineering scaffolds.     

Electrospun PVA/HAp nanocomposite nanofibers: biomimetics of mineralized hard tissues at a lower level of complexity

Based on the biomimetic approaches the present work describes a straightforward technique to mimic not only the architecture (the morphology) but also the chemistry (the composition) of the lowest level of the hierarchical organization of bone. This technique uses an electrospinning (ES) process with polyvinyl alcohol (PVA) and hydroxyapatite (HAp) nanoparticles. To determine morphology, crystalline structures and thermal properties of the resulting electrospun fibers with the pure PVA and PVA/HAp nanocomposite (NC) before electrospinning various techniques were employed, including transmission electron microscopy (TEM), high-resolution TEM (HR-TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). In addition, FT-IR spectroscopy was carried out to analyze the complex structural changes upon undergoing electrospinning as well as interactions between HAp and PVA. The morphological and crystallographic investigations revealed that the rod-like HAp nanoparticles exhibit a nanoporous morphology and are embedded within the electrospun fibers. A large number of HAp nanorods are preferentially oriented parallel to the longitudinal direction of the electrospun PVA fibers, which closely resemble the naturally mineralized hard tissues of bones. Due to abundant OH groups present in PVA and HAp nanorods, they strongly interact via hydrogen bonding within the electrospun PVA/HAp NC fibers, which results in improved thermal properties. The unique physiochemical features of the electrospun PVA/HAp NC nanofibers prepared by the ES process will open up a wide variety of future applications related to hard tissue replacement and regeneration (bone and dentin), not limited to coating implants.     

Study on cast membranes and electrospun nanofibers made from keratin/fibroin blends

Keratin regenerated from wool and fibroin regenerated from silk were mixed in different proportions using formic acid as the common solvent. Both solutions were cast to obtain films and electrospun to produce nanofibers. Scanning electron microscopy investigation showed that, for all electrospun blends (except for 100% keratin where bead defects are present), the fiber diameter of the mats ranged from 900 (pure fibroin) to 160 nm (pure keratin). FTIR and DSC analysis showed that the secondary structure of the proteins was influenced by the blend ratios and the process used (casting or electrospinning). Prevalence of beta-sheet supramolecular structures was observed in the films, while proteins assembled in alpha-helix/random coil structures were observed in nanofibers. Higher solution viscosity, thinner filaments, and differences in the thermal and structural properties were observed for the 50/50 blend because of the enhanced interactions between the proteins.     

Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells

Electrospun nanocomposite scaffolds were fabricated by encapsulating multi-walled carbon nanotubes (MWNT) in poly (lactic acid) (PLA) nanofibers. Scanning electron microscopy (SEM) confirmed the fabrication of nanofibers, and transmission electron microscopy identified the alignment and dispersion of MWNT along the axis of the fibers. Tensile testing showed an increase in the tensile modulus for a MWNT loading of 0.25 wt% compared with electrospun nanofibrous mats without MWNT reinforcement. Conductivity measurements indicated that the confined geometry of the fibrous system requires only minute doping to obtain significant enhancements at 0.32 wt%. Adipose-derived human mesenchymal stem cells (hMSCs) were seeded on electrospun scaffolds containing 1 wt% MWNT and 0 wt% MWNT, to determine the efficacy of the scaffolds for cell growth, and the effect of MWNT on hMSC viability and proliferation over two weeks in culture. Staining for live and dead cells and DNA quantification indicated that the hMSCs were alive and proliferating through day 14. SEM images of hMSCs at 14 days showed morphological differences, with hMSCs on PLA well spread and hMSCs on PLA with 1% MWNT closely packed and longitudinally aligned.     

Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration

Biocompatible carboxyethyl chitosan/poly(vinyl alcohol) (CECS/PVA) nanofibers were successfully prepared by electrospinning of aqueous CECS/PVA solution. The composite nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). SEM images showed that the morphology and diameter of the nanofibers were mainly affected by the weight ratio of CECS/PVA. XRD and DSC demonstrated that there was strong intermolecular hydrogen bonding between the molecules of CECS and PVA. The crystalline microstructure of the electrospun fibers was not well developed. The potential use of the CECS/PVA electrospun fiber mats as scaffolding materials for skin regeneration was evaluated in vitro using mouse fibroblasts (L929) as reference cell lines. Indirect cytotoxicity assessment of the fiber mats indicated that the CECS/PVA electrospun mat was nontoxic to the L929 cell. Cell culture results showed that fibrous mats were good in promoting the cell attachment and proliferation. This novel electrospun matrix would be used as potential wound dressing for skin regeneration.     

Fabrication of silk sericin nanofibers from a silk sericin-hope cocoon with electrospinning method

In this study, silk sericin nanofibers from sericin hope-silkworm, whose cocoons consist almost exclusively of sericin were successfully prepared by electrospinning method. Scanning electron microscopy (SEM) was used to observe the morphology of the fibers. The effect of spinning conditions, including the concentration of sericin cocoon solution, acceleration voltage, spinning distance and flow rate on the fiber morphologies and the size distribution of sericin nanofibers were examined. The structure and physical properties were also observed by Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). The optimum conditions for producing finely thinner fibrous sericin nanofibers without beads were the concentration of sericin solution above 6-8 wt%, acceleration voltage ranging from 25 to 32 kV, spinning distance above 9 cm, and flow rate above 0.06 cm min(-1). The mean diameter of as spun sericin fibers varied from 114 to 430 nm at the different spinning conditions. In the as-spun fibers, silk sericin was present in a random coil conformation, while after methanol treatment, the molecular structure of silk sericin was transformed into a β-sheet containing structure. Sericin hope nanofiber demonstrated thermal degradation at lower temperature than the sericin hope cocoon, which probably due to the randomly coiled rich structure of the sericin hope nanofiber.     

Mechanisms and control of silk-based electrospinning

Silk fibroin (SF) nanofibers, formed through electrospinning, have attractive utility in regenerative medicine due to the biocompatibility, mechanical properties, and tailorable degradability. The mechanism of SF electrospun nanofiber formation was studied to gain new insight into the formation and control of nanofibers. SF electrospinning solutions with different nanostructures (nanospheres or nanofilaments) were prepared by controlling the drying process during the preparation of regenerated SF films. Compared to SF nanospheres in solution, SF nanofilaments had better spinnability with lower viscosity when the concentration of silk protein was below 10%, indicating a critical role for SF morphology, and in particular, nanostructures, for the formation of electrospun fibers. More interesting, the diameter of electrospun fibers gradually increased from 50 to 300 nm as the concentration of SF nanofilaments in the solution increased from 6 to 12%, implying size control by simply adjusting SF nanostructure and concentration. Aside from process parameters investigated in previous studies, such as SF concentration, viscosity, and electrical potential, the present mechanism emphasizes significant influence of SF nanostructure on spinnability and diameter control of SF electrospun fibers, providing a controllable option for the preparation of silk-based electrospun scaffolds for biomaterials, drug delivery, and tissue engineering needs.     

The pattern of cell wall deterioration in lignocellulose fibers throughout enzymatic cellulose hydrolysis

Cell wall deterioration throughout enzymatic hydrolysis of cellulosic biomass is greatly affected by the chemical composition and the ultrastructure of the fiber cell wall. The resulting pattern of cell wall deterioration will reveal information on cellulose activity throughout enzymatic hydrolysis. This study investigates the progression and morphological changes in lignocellulose fibers throughout enzymatic hydrolysis, using (transmission electron microscopy) TEM and field emission scanning electron microscopy (FE‐SEM). Softwood thermo‐mechanical pulp (STMP) and softwood bleached kraft pulp (SBKP), lignocellulose substrates containing almost all the original fiber composition, and with lignin and some hemicellulose removed, respectively, was compared for morphology changes throughout hydrolysis. The difference of conversion between STMP and SBKP after 48 h of enzymatic hydrolysis is 11 and 88%, respectively. TEM images revealed an even fiber cell wall cross section density, with uneven middle lamella coverage in STMP fibers. SKBP fibers exhibited some spaces between cell wall and lamella layers due to the removal of lignin and some hemicellulose. After 1 h hydrolysis in SBKP fibers, there were more changes in the fiber cross‐sectional area than after 10 h hydrolysis in STMP fibers. Cell wall degradation was uneven, and originated in accessible cellulose throughout the fiber cell wall. FE‐SEM images illustrated more morphology changes in SBKP fibers than STMP fibers. Enzymatic action of STMP fiber resulted in a smoother fiber surface, along with fiber peeling and the formation of ribbon‐disjunction layers. SBKP fibers exhibited structural changes such as fiber erosion, fiber cutting, and fiber splitting throughout enzymatic hydrolysis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Electrospun poly(lactic acid)/chitosan core-shell structure nanofibers from homogeneous solution

The core-shell structure nanofibers of poly(lactic acid)/chitosan with different weight ratios were successfully electrospun from homogeneous solution. The preparation process was more simple and effective than double-needle electrospinning. The nanofibers were obtained with chitosan in shell while poly(lactic acid) in core attributing to phase separation, which were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectrometer (EDS). The electrospun nanofibrous membrane was evaluated in vitro by using mouse fibroblasts (L929) as reference cell lines. Cell culture results indicated that these materials were good in promoting cell growth and attachment, thus they could be used for tissue engineering and wound healing dressing.     

The properties of natural silk fibers: deformation study and NMR data

Natural silk (Bombyx mori) fibers with low humidity (0.07 g H2O/g dried silk) after temperature influence were studied for mechanical longitudinal deformation. On the basis of the stress-strain curves, some estimates of tensile properties for silk fibers were obtained. It was found that the maximal tension (sigma(max) in tensile-linear field of deformation of silk fibers decreases with increasing fiber diameter. The results showed that the heating of fibers (100 degrees C) results in a diminishing of the sigma(max)-value. Scanning electron microscopy pictures for cross section and longitudinal fiber surface were obtained. Natural silk fibers were studied by the NMR relaxation method (free induction decay curves) and the second moments of NMR-line shape in silk samples were calculated. The intra- and intermolecular contributions into the second moment were analyzed. The results showed a strong interaction of water molecules with macromolecules and a low molecular mobility. Some characteristics of interactions between silk macromolecules and water molecules as well as the role of intermolecular links in the change of the structure-function properties of natural silk under the action of external factors are discussed.     

Complementary Effects of Two Growth Factors in Multifunctionalized Silk Nanofibers for Nerve Reconstruction

With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the ‘bands of Bungner’ found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.     

Ultrastructure of insect and spider cocoon silks

Despite much interest in the extraordinary mechanical properties of silks, the structure of native silk fibers is still not fully understood. In the present study, the morphology, topography, and organization of insect and spider cocoon silks were investigated using a range of imaging methods. Field emission scanning electron microscopy was used to observe transverse and longitude structures in silk fibers subjected to tensile fracturing, freeze fracturing, or polishing. In addition, ultrathin sections of silk brins embedded in resin were examined using transmission electron microscopy. Finally, dry silk brins were examined by confocal microscopy. The results confirmed the existence of well-oriented bundles of nanofibrils in all the silks examined and gave an indication of a hierarchical construction of the brin. Observed separation of the microfibrils in fractured brins suggests that the multifibrillar structure of the silk fiber contributes to toughness by allowing dissipation of energy in the controlled propagation of cracks.     

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.     

Preparation and mechanical properties of chitosan/carbon nanotubes composites

Biopolymer chitosan/multiwalled carbon nanotubes (MWNTs) nanocomposites have been successfully prepared by a simple solution-evaporation method. The morphology and mechanical properties of the chitosan/MWNTs nanocomposites have been characterized with field emission scanning electron microscopy (SEM), bright field transmission electron microscopy (TEM), optical microscopy (OM), wide-angle X-ray diffraction (XRD), and tensile as well as nanoindentation tests. The MWNTs were observed to be homogeneously dispersed throughout the chitosan matrix. When compared with neat chitosan, the mechanical properties, including the tensile modulus and strength, of the nanocomposites are greatly improved by about 93% and 99%, respectively, with incorporation of only 0.8 wt % of MWNTs into the chitosan matrix.     

Fabrication and characterization of electrospun chitosan nanofibers formed via templating with polyethylene oxide

Chitosan is an abundantly common, naturally occurring, polysaccharide biopolymer. Its biocompatible, biodegradable, and antimicrobial properties have led to significant research toward biological applications such as drug delivery, artificial tissue scaffolds for functional tissue engineering, and wound-healing dressings. For applications such as tissue scaffolding, formation of highly porous mats of nanometer-sized fibers, such as those fabricated via electrospinning, may be quite important. Previously, strong acidic solvents and blending with synthetic polymers have been used to achieve electrospun nanofibers containing chitosan. As an alternative approach, in this work, polyethylene oxide (PEO) has been used as a template to fabricate chitosan nanofibers by electrospinning in a core-sheath geometry, with the PEO sheath serving as a template for the chitosan core. Solutions of 3 wt % chitosan (in acetic acid) and 4 wt % PEO (in water) were found to have matching rheological properties that enabled efficient core-sheath fiber formation. After removing the PEO sheath by washing with deionized water, chitosan nanofibers were obtained. Electron microscopy confirmed nanofibers of approximately 250 nm diameter with a clear core-sheath geometry before sheath removal, and chitosan nanofibers of approximately 100 nm diameter after washing. The resultant fibers were characterized with IR spectroscopy and X-ray diffraction, and the mechanical and electrical properties were evaluated.     

Effect of loading rate on mechanical properties and fracture morphology of spider silk

Spider silks have been shown to have impressive mechanical properties. In order to assess the effect of extension rate, both quasi-static and high-rate tensile properties were determined for single fibers of major (MA) and minor (MI) ampullate single silk from the orb weaving spider Nephila clavipes . Low rate tests have been performed using a DMA Q800 at 10(-3) s(-1), while high rate analysis was done at 1700 s(-1) utilizing a miniature Kolsky bar apparatus. Rate effects exhibited by both respective silk types are addressed, and direct comparison of the tensile response between the two fibers is made. The fibers showed major increases in toughness at the high extension rate. Mechanical properties of these organic silks are contrasted to currently employed ballistic fibers and examination of fiber fracture mechanisms are probed via scanning electron microscope, revealing a globular rupture surface topography for both rate extremums.     

Inducing β-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of β-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers.     

Functionalizing electrospun fibers with biologically relevant macromolecules

The development of functionalized polymers that can elicit specific biological responses is of great interest in the biomedical community, as well as the development of methods to fabricate these biologically functionalized polymers. For example, the generation of fibrous matrices with biological properties and fiber diameters commensurate with those of the natural extracellular matrix (ECM) may permit the development of novel materials for use in wound healing or tissue engineering. The goal of this work is, therefore, to create a biologically active functionalized electrospun matrix to permit immobilization and long-term delivery of growth factors. In this work, poly(ethylene glycol) functionalized with low molecular weight heparin (PEG-LMWH) was fabricated into fibers for possible use in drug delivery, tissue engineering, or wound repair applications. Electrospinning was chosen to process the LMWH into fiber form due to the small fiber diameters and high degree of porosity that can be obtained relatively quickly and using small amounts of starting material. Both free LMWH and PEG-LMWH were investigated for their ability to be incorporated into electrospun fibers. Each of the samples were mixed with a carrier polymer consisting of either a 10 wt % poly(ethylene oxide) (PEO) or 45 wt % poly(lactide-co-glycolide) (PLGA). Field emission scanning electron microscopy (FESEM), energy-dispersive X-ray analysis (EDX), UV-vis spectroscopy, and multiphoton microscopy were used to characterize the electrospun matrices. The incorporation of heparin into the electrospun PEO and PLGA fibers did not affect the surface morphology or fiber diameters. The fibers produced had diameters ranging from approximately 100 to 400 nm. Toluidine blue assays of heparin suggest that it can be incorporated into an electrospun matrix at concentrations ranging from 3.5 to 85 mug per milligram of electrospun fibers. Multiphoton microscopy confirmed that incorporation of PEG-LMWH into the matrix permits retention of the heparin for at least 14 days. Improvements in the binding of basic fibroblast growth factor to the electrospun fibers were also observed for fibers functionalized with PEG-LMWH over those functionalized with LMWH alone. The combination of these results suggests the utility for producing electrospun fibers that are appropriately functionalized for use in biomaterials applications.     

Cross-linking chitosan nanofibers

In the present study, we have electrospun various grades of chitosan and cross-linked them using a novel method involving glutaraldehyde (GA) vapor, utilizing a Schiff base imine functionality. Chemical, structural, and mechanical analyses have been conducted by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and Kawabata microtensile testing, respectively. Additionally, the solubilities of the as-spun and cross-linked chitosan mats have been evaluated;solubility was greatly improved after cross-linking. SEM images displayed evidence that unfiltered low, medium, and high molecular weight chitosans, as well as practical-grade chitosan, can be electrospun into nanofibrous mats. The as-spun medium molecular weight chitosan nanofibers have a Young's modulus of 154.9 +/- 40.0 MPa and display a pseudo-yield point that arose due to the transition from the pulling of a fibrous mat with high cohesive strength to the sliding and elongation of fibers. As-spun mats were highly soluble in acidic and aqueous solutions. After cross-linking, the medium molecular weight fibers increased in diameter by an average of 161 nm, have a decreased Young's modulus of 150.8 +/- 43.6 MPa, and were insoluble in basic, acidic, and aqueous solutions. Though the extent to which GA penetrates into the chitosan fibers is currently unknown, it is evident that the cross-linking resulted in increased brittleness, a color change, and the restriction of fiber sliding that resulted in the loss of a pseudo-yield point.     

Nanofibers from blends of polyvinyl alcohol and polyhydroxy butyrate as potential scaffold material for tissue engineering of skin

Nanofibers were prepared by electrospinning from pure polyvinyl alcohol (PVA), polyhydroxybutyrate (PHB), and their blends. Miscibility and morphology of both polymers in the nanofiber blends were studied by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC), revealing that PVA and PHB were miscible with good compatibility. DSC also revealed suppression of crystallinity of PHB in the blend nanofibers with increasing proportion of PVA. The hydrolytic degradation of PHB was accelerated with increasing PVA fraction. Cell culture experiments with a human keratinocyte cell line (HaCaT) and dermal fibroblast on the electrospun PHB and PVA/PHB blend nanofibers showed maximum adhesion and proliferation on pure PHB. However, the addition of 5 wt % PVA to PHB inhibited growth of HaCaT cells but not of fibroblasts. On the contrary, adhesion and proliferation of HaCaT cells were promoted on PVA/PHB (50/50) fibers, which inhibited growth of fibroblasts.