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.     

Correlation of chitosan's rheological properties and its ability to electrospin

Chitosan-based, defect-free nanofibers with average diameters ranging from 62 +/- 9 nm to 129 +/- 16 nm were fabricated via electrospinning blended solutions of chitosan and polyethylene oxide (PEO). Several solution parameters such as acetic acid concentration, polymer concentration, and polymer molecular weight were investigated to optimize fiber consistency and diameter. These parameters were evaluated using the rheological properties of the solutions as well as images produced by scanning electron microscopy (SEM) of the electrospun nanofibers. Generally, SEM imaging demonstrated that as total polymer concentration (chitosan + PEO) increased, the number of beads decreased, and as chitosan concentration increased, fiber diameter decreased. Chitosan-PEO solutions phase separate over time; as a result, blended solutions were able to be electrospun with the weakest electric field and the least amount of complications when solutions were electrospun within 24 h of initially being blended. The addition of NaCl stabilized these solutions and increased the time the blended solutions could be stored before electrospinning. Pure chitosan nanofibers with high degrees of deacetylation (about 80%) were unable to be produced. When attempting to electrospin highly deacetylated chitosan from aqueous acetic acid at concentrations above the entanglement concentration, the electric field was insufficient to overcome the combined effect of the surface tension and viscosity of the solution. Therefore, the degree of deacetylation is an extremely important parameter to consider when attempting to electrospin chitosan.     

Electrospun polyethylene oxide/cellulose nanocrystal composite nanofibrous mats with homogeneous and heterogeneous microstructures

An electrospinning process was successfully used to fabricate polyethylene oxide/cellulose nanocrystal (PEO/CNC) composite nanofibrous mats. Transition of homogeneous to heterogeneous microstructures was achieved by tailoring the concentration of PEO/CNC mixture in the solution from 5 to 7 wt %. Morphology investigation of the obtained nanofibers demonstrated that rod-shaped CNCs were well-dispersed in the as-spun nanofibers and highly aligned along the nanofiber long-axis. PEO/CNC nanofibers became more uniform and smaller in diameter with increased CNC-loading level. The heterogeneous composite mats were composed of rigid-flexible bimodal nanofibers. Results of structure characterization indicated that the incorporated CNCs interacted strongly with the PEO matrix through hydrogen bonding. Mechanical properties of both types of mats were effectively improved by using CNCs, with heterogeneous mats being stronger than their homogeneous counterparts for all compositions (0-20 wt % CNC contents). When a smaller diameter needle was used to form homogeneous mats, enhanced thermal and mechanical properties were obtained.     

Morphological and surface properties of electrospun chitosan nanofibers

Nonwoven fiber mats of chitosan with potential applications in air and water filtration were successfully made by electrospinning of chitosan and poly(ethyleneoxide) (PEO) blend solutions. Electrospinning of pure chitosan was hindered by its limited solubility in aqueous acids and high degree of inter- and intrachain hydrogen bonding. Nanometer-sized fibers with fiber diameter as low as 80 +/- 35 nm without bead defects were made by electrospinning high molecular weight chitosan/PEO (95:5) blends. Fiber formation was characterized by fiber shape and size and was found to be strongly governed by the polymer molecular weight, blend ratios, polymer concentration, choice of solvent, and degree of deacetylation of chitosan. Weight fractions of polymers in the electrospun nonwoven fibers mats were determined by thermal gravimetric analysis and were similar to ratio of polymers in the blend solution. Surface properties of fiber mats were determined by measuring the binding efficiency of toxic heavy metal ions like chromium, and they were found to be related with fiber composition and structure.     

Chitosan nanofibers from an easily electrospinnable UHMWPEO-doped chitosan solution system

Conversion of natural biopolymer chitosan into nanofibers through electrospinning has significant usefulness in various biomedical applications, in particular, for constructing a biomimetic and bioactive nanofibrous artificial extracellular matrix for engineering various tissues. Here, we show that introduction of an ultrahigh-molecular-weight poly(ethylene oxide) (UHMWPEO) into aqueous chitosan solutions remarkably enhances the formation of chitosan nanofibrous structure and leads to much lower loading of the water soluble fiber-forming aiding agent of PEO down to 5 wt % as compared to previous high PEO loadings in the electrospun chitosan nanofibers. The excellent electrospinnability of the current formulation renders electrospinning of natural biopolymer chitosan a robust process for large-scale production of practically applicable nanofibrous structures.     

Electrospun sodium alginate/poly(ethylene oxide) core-shell nanofibers scaffolds potential for tissue engineering applications

Core-shell structure nanofibers of sodium alginate/poly(ethylene oxide) were prepared via electrospinning their dispersions in water solution. The core-shell structure morphology of the obtained nanofibers was viewed under scanning electron microscope (SEM) and transmission electron microscope (TEM), and X-ray photoelectron spectroscopy (XPS) analysis was used to further quantify the chemical composition of the core-shell composite SA/PEO nanofibers surface in detail. Furthermore, one-step cross-linking method through being immersed in CaCl₂ solution was investigated to improve the anti-water property of the electrospun nanofibers mats in order to facilitate their practical applications as tissue engineering scaffolds, and the changes of the structural of nanofibers before and after cross-linking was characterized by Fourier transform infrared (FT-IR). Indirect cytotoxicity assessment indicated that SA/PEO nanofibers membrane was nontoxic to the fibroblasts cells, and cell culture suggested that SA/PEO nanofibers tended to promote fibroblasts cells attachment and proliferation. It was assumed that the nanofibers membrane of electrospun SA/PEO could be used for tissue engineering scaffolds.     

Influence of Surfactant Type and Concentration on Electrospinning of Chitosan–Poly(Ethylene Oxide) Blend Nanofibers

Electrospun blend nanofibers were fabricated from chitosan (1,000 kDa, 80% DDA) and poly(ethylene oxide) (PEO; 900 kDa) at a ratio of 3:1 dispersed in 50% and 90% acetic acid. The influence of surfactants on the production of electrospun nanofibers was investigated by adding nonionic polyoxyethylene glycol dodecyl ether (Brij 35), anionic sodium dodecyl sulfate, or cationic dodecyl trimethyl ammonium bromide below, at, and above their specific critical micellar concentration to the polymer blend solution. Viscosity, conductivity, and surface tension of polymer solutions, as well as morphology and composition, of nanofibers containing surfactants were determined. Pure chitosan did not form fibers and was instead deposited as beads. Addition of PEO and an increasing concentration of surfactants induced spinnability and yielded larger fibers with diameters ranging from 10 to 240 nm. Surfactants affected morphology yielding needle-like, smooth, or beaded fibers. Compositional analysis revealed that nanofibers consisted of both polymers and surfactants with concentration of the constituents in nanofibers differing from that in polymer solutions. Results suggest that surfactants may modulate polymer–polymer interactions thus influencing the morphology and composition of deposited nanostructures.     

Solution blowing of submicron-scale cellulose fibers

Solution blowing is an innovative process for spinning micro-/nano-fibers from polymer solutions using high-velocity gas flow as fiber forming driving force. Submicron-scale cellulose fibers were successfully solution blown by two improvement measures. First, cellulose solution was directly blown to fibers of 260-1900nm in diameter by raising the air temperature along the spinning line which was proved to accelerate the evaporation of solvent and fiber forming. Second, coaxial solution blowing technique was established with cellulose solution and polyethylene oxide (PEO) solution used as core and shell liquids, respectively. The core-shell structures of the fibers were examined by SEM and TEM. Cellulose fibers with diameter between 160nm and 960nm were further obtained after removing PEO shell. X-ray diffraction studies showed that the two kinds of submicron-scale cellulose fibers are mostly amorphous.     

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.     

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.     

Unique natural-protein hollow-nanofiber membranes produced by weaver ants for medical applications

We report the properties of unique natural-protein hollow-nanofiber membranes produced by weaver ants (Oecophylla smaragdina) and the potential of using the nanofiber membranes for medical applications. Although natural proteins such as silk and collagen have been used to produce electrospun nanofibers for medical applications, there are no reports on producing hollow nanofibers from proteins. Hollow nanofibers are expected to have unique properties such as high drug loading. Weaver ant larvae extrude proteins in the form of nanofibers that are hollow and the adult ants build the nests using the hollow nanofibers. It was found that the nanofiber membranes are composed of fibers with average diameters of 450 nm. The membranes have tensile strength of about 4 MPa, high elongation of about 31% and modulus of 31 MPa, better than any protein nanofiber membrane reported so far. The membranes withstand rigorous boiling in weak alkali, show good attachment and proliferation of osteoblasts and can load up to 4.7 times higher drugs compared to common silk. These features make ant nanofiber membranes unique and preferable for medical and biotechnology industries.     

Dehydration from alcohols by polyion complex cross-linked chitosan composite membranes during evapomeation

This study describes the dehydration of an ethanol/water azeotrope during evapomeation using polyion complex cross-linked chitosan composite (q-Chito-PEO acid polyion complex/PES composite) membranes, constructed from quaternized chitosan (q-Chito) and poly(ethylene oxydiglycolic acid) (PEO acid) on a porous poly(ether sulfone) (PES) support. Both the q-Chito/PES composite and the q-Chito-PEO acid polyion complex/PES composite membranes showed high water permselectivity for an ethanol/water azeotrope. Both the permeation rate and the water permselectivity of the q-Chito/PES composite membranes were enhanced by increasing the degree of quaternization of the chitosan molecule because the affinity of the q-Chito/PES composite membranes for water was increased by introducing a quaternized ammonium group into the chitosan molecule. q-Chito-PEO acid polyion complex/PES composite membranes prepared from an equimolar ratio of carboxylate groups in the PEO acid versus quaternized ammonium groups in the q-Chito showed the maximum separation factor for water permselectivity without lowering the permeation rate. With an increasing molecular weight of PEO acid, the separation factor for water permselectivity increased, but the permeation rate almost did not change. The mechanism responsible for the separation of an ethanol/water azeotrope through the q-Chito-PEO acid polyion complex/PES composite membranes was analyzed by the solution-diffusion model. The permeation rate, separation factor for water permselectivity, and evapomeation index of q-Chito-PEO acid 400 polyion complex/PES composite membrane with an equimolar ratio of carboxylate groups in PEO acid 400 and ammonium groups in q-Chito were 3.5 x 10(-1) kg/(m(2) hr), 6300, and 2205, respectively, and very high membrane performance. The separation factor for water permselectivity for aqueous solutions of n-propyl and isopropyl alcohol was also maximized at an equimolar ratio of carboxylate groups and ammonium groups and was greater than that for an ethanol/water azeotrope. The above results were discussed from the viewpoint of the physical and chemical structure of the q-Chito-PEO acid polyion complex/PES composite membranes and the permeants.     

Novel chitosan-based films cross-linked by genipin with improved physical properties

Novel cross-linked chitosan-based films were prepared using the solution casting technique. A naturally occurring and nontoxic cross-linking agent, genipin, was used to form the chitosan and chitosan/poly(ethylene oxide) (PEO) blend networks, where two types of PEO were used, one with a molecular weight of 20 000 g/mol (HPEO) and the other of 600 g/mol (LPEO). Genipin is used in traditional Chinese medicine and extracted from gardenia fruit. Importantly, it overcomes the problem of physiological toxicity inherent in the use of some common synthetic chemicals as cross-linking agents. The mechanical properties and the stability in water of cross-linked and un-crosslinked chitosan and chitosan/PEO blend films were investigated. It was shown that, compared to the transparent yellow, un-cross-linked chitosan/PEO blend films, the genipin-cross-linked chitosan-based film, blue in color, was more elastic, was more stable, and had better mechanical properties. Genipin-cross-linking produced chitosan networks that were insoluble in acidic and alkaline solutions but were able to swell in these aqueous media. The swelling characteristics of the films exhibit sensitivity to the environmental pH and temperature. The surface properties of the films were also examined by contact angle measurements using water and mixtures of water/ethanol. The results showed that, with the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and chitosan/PEO blends were more hydrophobic than un-crosslinked ones.     

Structure of the shell and tertiary membranes of eggs of softshell turtles (Trionyx spiniferus)

Eggs of the turtle Trionyx spiniferus are rigid, calcareous spheres averaging 2.5 cm in diameter. The eggshell is morphologically very similar to avian eggshells. The outer crystalline layer is composed of roughly columnar aggregates, or shell units, of calcium carbonate in the aragonite form. Each shell unit tapers to a somewhat conical tip at its base. Interior to the crystalline layer are two tertiary egg membranes: the outer shell membrane and the inner shell membrane. The outer shell membrane is firmly attached to the inner surface of the shell, and the two membranes are in contact except at the air cell, where the inner shell membrane separates from the outer shell membrane. Both membranes are multi-layered, with the inner shell membrane exhibiting a more fibrous structure than the outer shell membrane. Numerous pores are found in the eggshell, and these generally occur at the intersection of four or more shell units.     

Core/shell nanofibers with embedded liposomes as a drug delivery system

The broader application of liposomes in regenerative medicine is hampered by their short half-life and inefficient retention at the site of application. These disadvantages could be significantly reduced by their combination with nanofibers. We produced 2 different nanofiber-liposome systems in the present study, that is, liposomes blended within nanofibers and core/shell nanofibers with embedded liposomes. Herein, we demonstrate that blend electrospinning does not conserve intact liposomes. In contrast, coaxial electrospinning enables the incorporation of liposomes into nanofibers. We report polyvinyl alcohol-core/poly-ε-caprolactone-shell nanofibers with embedded liposomes and show that they preserve the enzymatic activity of encapsulated horseradish peroxidase. The potential of this system was also demonstrated by the enhancement of mesenchymal stem cell proliferation. In conclusion, intact liposomes incorporated into nanofibers by coaxial electrospinning are very promising as a drug delivery system.     

Core‐shell nanofibers: Integrating the bioactivity of gelatin and the mechanical property of polyvinyl alcohol

Coaxial electrospinning is used to fabricate nanofibers with gelatin in the shell and polyvinyl alcohol (PVA) in the core in order to derive mechanical strength from PVA and bioactivity from gelatin. At a 1:1 PVA/gelatin mass ratio, the core‐shell nanofiber scaffolds display a Young's modulus of 168.6 ± 36.5 MPa and a tensile strength of 5.42 ± 1.95 MPa, which are significantly higher than those of the scaffolds composed solely of gelatin or PVA. The Young's modulus and tensile strength of the core‐shell nanofibers are further improved by reducing the PVA/gelatin mass ratio from 1:1 to 1:3. The mechanical analysis of the core‐shell nanofibers suggests that the presence of the gelatin shell may improve the molecular alignment of the PVA core, transforming the semi‐crystalline, plastic PVA into a more crystallized, elastic PVA, and enhancing the mechanical properties of the core. Lastly, the PVA/gelatin core‐shell nanofibers possess cellular viability, proliferation, and adhesion similar to these of the gelatin nanofibers, and show significantly higher proliferation and adhesion than the PVA nanofibers. Taken together, the coaxial electrospinning of nanofibers with a core‐shell structure permits integration of the bioactivity of gelatin and the mechanical strength of PVA in single fibers. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 336–346, 2014.     

Determination of the parameters affecting electrospun chitosan fiber size distribution and morphology

The production of chitosan nanofiber mats by electrospinning presents serious difficulties due to the lack of suitable solvents and the strong influence of processing parameters on the fiber properties. Two are the main problems to be solved: to control the properties of the solution in order to obtain large area uniform fiber mats by having a stable flow rate and to avoid sparks during the process, damaging the fiber mats. In this work chitosan electrospun mats have been prepared form solutions of trifluoroacetic acid/dichloromethane mixtures, allowing solving the aforementioned problems. Mats with uniform fibers of submicron diameters without beads were obtained. Further, the influence of the different solution and process parameters on the mean fiber diameter and on the width of the distribution of the fiber sizes has been assessed. Solvent composition, needle diameter, applied voltage and traveling distance were the parameters considered in this study.     

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.     

Design and performance study of a novel immobilized hollow fiber membrane bioreactor

A dual-layer coaxial hollow fiber (DLHF) bioreactor for cell immobilization developed to overcome nutrients transport limitation is presented. Cells were contained in the annular space between two coaxial hollow fibers, and nutrients were supplied by a forced convective transport from the shell side through the annular space to the lumen side. With judicious selection of the membrane materials, a low operating transmembrane pressure of 50 kPa, and using E. coli as the model organism, a high cell density of 10(11) cells/mL annular space volume and a high cell viability of (up to 80%) were obtained.     

Die Mikro- und Ultrastruktur abgedeckter Hohlelemente und die Conellen des Ammoniten-Gehäuses

Hollow floored spines in the shell ofKosmoceras (Kosmoceras) spinosum (Sow.) and the hollow floored keel ofEleganticeras elegantulum (Young & Bird) have been studied with the scanning electron microscope. In both cases the shell wall is complete in so far as it consists of the outer prismatic layer, the nacreous layer and at least the distal zones of the inner prismatic layer. Both types of hollow shell elements are separated from the lumen of the whorl by a floor which is made up by the proximal zones of the inner prismatic layer. This explains why conellae occur, with preference, along the floors of hollow spines and keels. The origin of primary aragonitic conellae and of secondary calcitic conellae is discussed as well as their dependence on structural properties of the corresponding shell layer, which is the inner prismatic layer. An attempt is made to reconstruct the way of formation of the floored hollow spines and the floored hollow keels by the mantle epithelium.