Electrospinning of Chitin and Chitosan Nanofibres
C. K. S. Pillai and Chandra P. Sharma*
Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Thirunal Institute for Medical Sciences & Technology, Poojappura, Thiruvananthapuram 695 012 INDIA
* Corresponding author sharmacp@sctimst.ac.in
Received 5 February 2009; Accepted 25 February 2009; published online 17 March 2009
The electrospinning technique is a versatile method to spin polymers into continuous fibers with diameters ranging from a few micrometers to a few nanometers. Electrospinning creates seemingly endless ultra fine fibers that collect in a random pattern. These nanofibers can form non-woven textile mats, oriented fibrous bundles and even three-dimensional structured scaffolds, all with large surface areas and high porosity. It is, thus, the most extensively used fabrication method that offers vast opportunities for control of the morphology of the electrospun fibers. Due to their intrinsic features, polymeric nanofibers are attractive for biomedical and biotechnological applications such as tissue engineering, nanocomposites for dental application, controlled drug delivery, medical implants, wound dressings, biosensors and filtration. The applications of chitin and chitosan (CS) nanofibers in these areas are reviewed in this paper. Because of the inherent biodegradability, biofunctionality and biocompatibility of the biopolymer, electrospun chitin and CS fibers have special advantages whereby properties such as cytocompatibility, tissue responses etc. could be controlled in critical applications.
© Society for Biomaterials and Artificial Organs (India), 20090205-21.
Electrospinning of nanofibers based on natural polymers have recently emerged as having enormous possibilities for better utilization of bio-based materials [17]. This paper focuses its attention to discuss this highly promising area on electrospinning of nanofibers from chitin and CS. Chitin and CS fibers are unique as they carry an acetamido/amino functionality that impart many biological properties. CS fiber is unlike other fibers is unique: it carries a positive ionic charge, it has remarkable affinity to proteins, it can be functionalized and it is renewable [9,18-22].
Although chitin fibers could be made into textile materials [23, 24], its application as sutures is remarkable because it can accelerate wound healing. [3,6,8,15,23,25-28, 28a]. CS-poly(L- glutamic acid) and CS-polyacrylic acid fiber are finding potential industrial applications due to their enhanced tensile strength and environmental biodegradability [29]. One study Introduction
Chitin and CS are polysaccharides having excellent biocompatibility and admirable biodegradability with versatile biological activities such as antimicrobial activity, low immunogenicity and low toxicity [1-5]. Coupled with the possibility of preparing a variety of chemically or enzymatically modified products and processes, these biopolymers having the rare amino functionality and two hydroxyl groups for chemical modifications are potential materials in a variety of applications in biomedical, biotechnological and pharmaceutical areas [6-8]. Despite its huge annual production and easy availability, chitin still remains an under utilized resource primarily because of its intractable molecular structure.
Chitin and CS fibers have attracted much attention due to their highly promising applications in textile materials, sutures and as scaffold materials for tissue engineering [9-16].
reports that chitin fibres have comparable properties to those of collagen and lactide fibres [30]. Rathke and Hudson [11] pointed out that chitin’s microfibrillar structure indicated its potential as fiber- and film-former, but as chitin was found to be insoluble in common organic solvents, the N-deacetylated derivative of chitin, CS, was developed. The polymeric linear chain structure of chitin is expected to give rise to fibre formation and film forming ability similar to those of cellulose [11]. Thus, the presence of the micro fibrils of chitin with diameters from 2.5 to 2.8 nm which are usually embedded in a protein matrix indicates that chitin can be spun into fibres [31, 32]. The polyamide-type structure should be broken up to enable solubilisation of chitin into a solvent [33]. This requires either melting or dissolution in appropriate solvents.
Melt spinning is ruled out as chitin decomposes prior to melting. There have been many attempts at dissolution of chitin and spinning of chitin and CS into fibre form. Variuos methods have been developed to produce chitin and CS fibres whose properties and applications are covered in a number of reviews [9-12]. The electrospinning technique is a highly versatile method to process solutions or melts, mainly of polymers, into continuous fibers with diameters ranging from a few micrometers to a few nanometers [34-36, 36a]. Considering the potential applications of chitin and CS fibres, it appears that a consolidation of the data relating to the electrospinning technology and its applications in chitin and CS polymers seems to be worthwhile.
The electrospinning process
The electrospinning method of producing non- woven nanofibrous mats [37] is attractive due mainly to its cost effectiveness, reproducibility and simplicity. It is a process by which polymer nanofibers can be produced using an electrostatically driven jet of polymer solution (or polymer melt). The method produces fibers 103 times larger specific surface to volume ratios, increased flexibility in surface functionalities, improved mechanical performances, and smaller pores than fibers produced using traditional methods. One reason for the upsurge in nanofibers fabrication research in the 1990s, was due to new found interest in producing polymeric nanofibers
Figure 1: Electro spinning apparaturs: (a) The schematic setup for manufac-turing nonwovens by electrospinning 1 - high voltage source, 2 - polymer container, 3 - tip of metal tube, 4 - droplet of polymer solution in the conical shape known as Taylor cone, 5 - rectilinear part of jet, 6 - electrically-driven bending instability of the jet (insta-bility region), 7 - collector a-lower electrode (grounded); b) photograph of the main parts of the apparatus, front view; c) photograph of the electrically- driven bending instability of the jet.
(Reproduced from reference [40] with permission from Text. Fibres Eastern Eur.
Poland)
under laboratory conditions. These nanofibers form non-woven textile mats, oriented fibrous bundles and even three-dimensional structured scaffolds, all with large surface areas and high porosity [38]. Applying a strong electric potential on a polymer solution or melt produces nanoscale fibers. Polymer solutions of sufficient viscosity are metered through a capillary and placed under high electric potential in the range of 10–30 kV. The large potential causes a droplet of solution to accelerate towards a grounded or oppositely charged collector. The solvent gets evaporated quickly from the polymer ‘jet’, reducing the jet diameter and increasing the charge density on the surface. The dry polymer fibers are deposited on the collector on the grounded target. The necessary components of an electrospinning apparatus include a high power voltage supply, a capillary tube with a needle or pipette, and a collector that is usually composed of a conducting material [35, 39]. Electrospinning is influenced by a variety of process parameters such as the operating voltage, the tip-to-target distance, the temperature, the pressure, and the flow rate [39a, 39b]. Of these parameters, the applied voltage is the most important one since it determines the degree of electrostatic interaction forces that induce the expulsion of a polymer jet [39]. The schematic setup for electrospinning is given in Fig. 1 [40].
Electrospinning creates seemingly endless ultra fine fibers that collect in a random pattern.
Fibrous mats produced could be used in filter media, catalysis, protective clothing, cosmetics, sensors, nanocomposites (dental application), controlled drug delivery, medical implants, wound dressings, biosensors and tissue engineering. More specifically, because of the biofunctionality and biocompatibility of the biopolymer, electrospun CS could be used to improve tissue engineering scaffolds by increasing their cytocompatibility while also mimicking the native extracellular matrix [37,41,42].
Electrospinning of chitin and CS solutions Previous research has shown that it is difficult to form nanofibers from pure CS solutions using electrospinning because CS does not give solutions with a sufficiently high polymer concentration but a viscosity that is low enough
to support electrospinning. Nevertheless, several research groups have succeeded in the preparation of CS based composite fibers by blending it with other polymers such as polyethylene oxide (PEO) [43-47,47a], or polyvinyl alcohol (PVA) [48-53]. Fabrication and characterization of poly (vinyl alcohol)/ CS blend nanofibers produced by electrospinning method [54] in acetic, acrylic, or other acids [55]. In this case, the excellent fiber forming properties of the co-spinning agent are utilized. CS has also been successfully blended with other natural biopolymers such as collagen that are more easily electrospun [56,57]
Application of electrospinning [58] to CS fibres and was first applied by Ohkawa et al.[59]. They prepared homogenous non-woven fabrics of nano-scaled fibers successfully with the samples of Mw 158 and 180× 10-4 having the average fiber diameters of 83, and 60 nm respectively [60].
Westbroek and group have shown that parameters such as type of solvent, pH, concentration of CS viscosity, charge density, applied voltage, solution flow rate, distance from nozzle tip to collector surface and time play a role in the characteristics of the obtained nanofibrous structures [61]. They provide a description of the setup for electrospinning and the method of production of fibres in detail. At longer production time, nano fibres spun from a 3% CS in 90% acetic acid solution split and form short side arms on the main fibre possibly due to distortion of the electrical field during fibre deposition.
Blasin´ska et al. produced smooth dibutyryl chitin fibers by means of the electrospinning classical method. [40]. This allowed preparation of fibres of transverse dimensions of below 0.4 ìm to be obtained. The supermolecular structure of fibres manufactured by means of the elec-trospinning technique did not differ significantly from the supermolecular structure of those fibres obtained using the classical wet spinning technique. Du et al. employed electrospinning method to produce cellulose/
CS hybrid nanofibers by electrospinning of their ester derivatives, cellulose acetate (CA) and dibutyryl chitin (DBC), followed by alkaline hydrolysis. [62]
Klossner et al. fabricated CS-based, defect-free nanofibers with average diameters ranging from 62 ± 9 nm to 129 ± 16 nm via electrospinning blended solutions of CS and polyethylene oxide PEO [63]. They demonstrated using SEM imaging that as total polymer concentration (CS + PEO) increased, the number of beads decreased, and as CS concentration increased, fiber diameter decreased. As CS-PEO solutions phase separate over time, the solutions were stabilized using NaCl. They also showed that the degree of deacetylation was an extremely important parameter to consider when attempting to electrospun CS [64]. Vodran et al.
studied the tensile and compressive mechanical properties of eletrospun CS- poly(ethylene oxide) nanofiber mats cross linked with glutaraldehyde (GA) for various time periods [64]. The mechanical studies confirmed that the GA vapor cross linking increased the stiffness and decreased the ductility of the electrospun mats. Increased exposure time to cross linking led to changes in the mat surface color and resistance to dissolution. Scanning electron microscopy fiber counts verified that exposure to GA vapor cross linking increased the average fiber diameter.
In another development, introduction of a dry- jet-stretching step could improve the mechanical properties of the CS fibers substantially (Young’s modulus of 82 g/d and tenacity of 2 g/d) [65].
Bead formation was found to occur during electrospinning and could be controlled by controlling the molecular weight of CS and the solvent used for spinning [66,67]. Viscosity is thus a key parameter in electrospinning because it is related to the extent of the polymer molecule chain entanglement within the solution (Ramakrishna, 2005) Blended fibres of hexanoyl CS/polylactide blend fibres were prepared without the presence of beads by electrospinning from solutions in chloroform with the H-CS solution content of less than or equal to 50% (w/w). Fig. 2 shows no beads at lower CS to PVP ratio [67]. In another, bicomponent system consisting of poly(vinyl alcohol) (PVA, Mw = 124-186 kDa) and 82.5%
deacetylated CS (Mv = 1600 kDa) in 2% (v/v) aqueous acetic acid, fewer beaded structures and more efficient fiber formation were observed on electrospinning with increasing PVA contents. The improved uniform distribution of CS and PVA in the bicomponent fibers was attributed to better mixing mostly due to the reduced molecular weight and to the increased deacetylation of the CS [54]. On replacing CS by N-carboxyethylCS (CECh), it was observed that the elctrospinning of CECh-containing nanofibers was enabled by the ability of PVA to form an elastically deformable entanglement network based on hydrogen bonds. The average diameters of the bicomponent fibers were in the range 100–420 nm [68,69].
Fig. 2. SEM images (magnification 500×, scale bar = 50 ìm) of as-spun products from blend solutions of H-CS (10% (w/v) in chloroform) and PLA (24% (w/v) in chloroform) at blend compositions (w/w): (a) 20 : 80 and (b) 80 : 20, respectively. The applied electrostatic field strength was 16 kV/15 cm. (Reproduced from reference [67] with permission from BRILL, Netherlands).
Ultra fine fibers could be generated by controlling the addition of PEO in 2:1 or 1:1 mass ratios of CS to PEO from 4-6 wt% CS/PEO solutions [44]. Fig 3 demonstrates that increasing the concentration of PEG reduces bead formation and enhances nanofiber formation [43]. It was also shown that addition of PEO brings about additive effects in enhancing the formation of a fibrous structure [70]. A scanning electronic microscopic study showed that electrospun CS fiber mats were indeed aligned and there was a slight cross- linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on electrospun CS
mats indicated the potential to be processed into three-dimensional scaffolds for cartilage tissue repair [46].FT-IR, XRD, and DSC studies demonstrated that there were strong intermolecular hydrogen bonds between the molecules of CS and PVA in the PVA/CS blend nanofibrous membranes [55]. SEM images showed that the morphology and diameter of the nanofibers were mainly affected by concentration of the blend solution and weight ratio of the blend, respectively [53].
Morphology of the electrospun fibers Electrospinning produces continuous fibers with a diameter in the range from 3 nm to several Figure 3. Scanning electron micrographs of electrospun CS/PEO obtained from their solutions in 1 : 1 mass ratio with 2300 kDa PEO at different concentrations (original magnification 104£): (a) 2 wt%;(b) 6 wt%.(Reproduced from reference [43] with permission from BRILL, Netherlands).
Figure 4. (A) Fibers produced by wet spinning of PHBV. (B) Fibers produced by electrospinning of PHBV. (Reproduced from reference 42 with permission from BRILL, Netherlands).
micrometers whereas other methods such as self-assembly, template synthesis and phase separation produce fibers only a few micrometers long with diameters ranging from 500 nm up to a few micrometers. Thus, electrospinning is the most extensively used fabrication method that offers vast opportunities for control of the morphology of the electrospun fibers. Nanofibers aligned for certain applications can be obtained by using patterned electrodes [71], conductive substrates separated by a nonconductive gap [72], disc collectors [73] or other methods as outlined by Teo and Ramakrishna [74,75]. Several parameters are known to affect the morphology of the fibers [35]. The molecular weight and molecular weight distribution, Tg, viscosity and viscoelastic properties are important parameters that effect the morphology of the spun fibres. Figure 4 gives a comparison of electron spun fibers with that of wet spun fibers of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) films [42]. The uniformity and the nanoscale dimension of the electropsun fibers could be easily discernable from the figures [42,76].
Due to their intrinsic features polymeric nanofibers are attractive for biomedical and biotechnological applications such as tissue engineering, nanocomposites for dental application, controlled drug delivery, medical implants, wound dressings, biosensors and filtration are among the most intensively studied areas [39a, 42, 76a]. A few examples are discussed in the present review taking chitin and CS as candidate materials.
Scaffolds for tissue engineering
Polymeric nanofibers are of great interest because they can serve as tissue-engineering scaffolds and their dimension and properties arising from their structures make them interesting materials. Human cells are known to attach, grow and organize well on fibers with diameters smaller than that of cells [39a, 42, 76a, 77]. So, nanofiber based tissue engineering research is being done on a number of areas such as cartilage, nerve, bone, skin, skeletal muscle and blood vessels.
Polylactic acid (PLLA), poly(lactic acid-co- glycolic acid) (PLGA), poly(å-caprolactone)
(PCL), polyhydroxy butarate-valerate (PHBV) and a variety of their blends have been used in the fabrication of scaffolds made from nanofibers [77-82, 82a, 82b]. PLGA-based scaffolds prepared through electro spinning have been shown to provide both guidance and flexibility to cardiac myocytes [83]. Genov et al.
showed that a self-assembled peptide nanofiber scaffold was used to create a basement membrane [84].
The design of biomimetic nanostructured scaffolds for tissue engineering, in general, involves mimicking the structure and biological function of native extra cellular matrix (ECM) proteins, which provide mechanical support and regulate cell activities [84a]. It should also provide a good environment for the cells so that they can easily attach, proliferate and differentiate [85]. These structures have morphologies and fiber diameters in a range comparable with those found in the extra cellular matrix of human tissues. Therefore, nanofibrous scaffolds are intended to provide improved environments for cell attachment, migration, proliferation and differentiation when compared with traditional scaffolds. In addition, the process versatility and the highly specific surface area of nanofiber meshes may facilitate their use as local drug-release systems [86]. The natural ECMs in the body are mainly composed of collagen and proteoglycans whose compositions depend on tissue types. To establish an ideal scaffold mimicking the natural ECM, nanofibrous structure composed of collagen and glycosaminoglycans (GAGs) (main component of proteoglycans), such as condroitin sulfates and hyaluronic acid, is desirable. Alternatively, the combination of natural polymers (fibrous proteins and polysaccharides), which could enhance biological interactions with cells and speed up tissue regeneration, should be considered as substitutes of ECM proteins. However, utility of collagen and GAGs has been limited due to extremely high price and poor mechanical properties. The similarity of structural characteristics of chitin to GAGs, such as condroitin sulfates and hyaluronic acid, in the ECM is advantageous for utilizing chitin and CS for preparing biomimetic nanostructured scaffolds. Chen et al. prepared collagen-CS complex nanofibers by electrospinning with a
view to mimicking the native ECM for tissue engineering and to develop functional biomaterials [87]. It was found that the diameter of the spun fibers became thick with the concentration of the solution increasing and became fine with the ratio of the CS/collagen increasing.
In the design of CS based scaffolds, CS derivatives with grafted L-lactide oligomers with controlled side chain length would be advantageous [88]. This allows the manipulation of the biodegradation rate and hydrophilicity of the tissue engineering scaffold material. This general synthetic route renders functionalized CS soluble in a broad range of organic solvents, facilitating formation of ultrafine fibers via electrospinning. A biocompatible and biodegradable ultra fine fibrillar scaffold material was prepared by grafting L-lactide oligomers via ring opening polymerization on to CS [89]. Cytotoxicity tests using fibroblasts (L929 cell line) performed on electrospun L-lactide modified CS fibers showed that the specimen with the highest molar ratio of L-lactide (1:24) investigated in this study is the most promising material for tissue engineering purposes, while less stable formulations might still find application in drug delivery vehicles.
Wan et al. used an electro wet spinning method to fabricate scaffolds from poly(CS-g-dl-lactic acid) (PCLA) copolymers with improved tensile strength and modulus [90]. The diameter of fibers in different scaffolds could vary from about 100 nm to around 3 ìm. The scaffolds exhibited various pore sizes ranging from about 1 ìm to less than 30 ìm and different porosities up to 80%. The data collected from in vitro rabbit- fibroblast/scaffold culture showed that there were no substantial differences in the viability, density and distribution of cultured fibroblasts between PCLA scaffolds and pure CS scaffolds.
In another work, Mo et al. fabricated poly(L-lactic- co-caprolactone) [P(LLA-CL)] nanofibers and collagen-CS complex nanofibers by electrospinning [91]. Results of the experiments showed that the mechanical properties of the collagen-CS complex nanofibers varied with the collagen content in the complex. It was also found that the biodegradability of P(LLA-CL) nanofibers was
faster than its membrane and that smooth muscle cells (SMC) grow faster on collagen nanofibers than on P(LLA-CL) nanofibers. PLLA and CS membranes wee also prepared by Chen et al. using the electrospinning technique [56]. When the osteoblastic cells are cultured with nano-fibrous membranes, the cell density was higher and the secretion of fibril was more significant compared with the cells cultured on dense films. The results indicated that the biocompatibility of PLLA and CS would be improved by changing their topography from smooth surface into nano-scaled structures.
Park et al. showed that chitin/silk fibroin (SF) blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds [85]. They showed that the nature of polymer plays an important role in determining the diameter of electrospun nanofibers.
Generally, the electrospinning of polymers with higher polarity provides nanofibrous products with smaller diameters. The average diameters of chitin/SF blend fibers increased from 340 to 920 nm, with the increase of SF content in blend compositions. This illustrated in Fig. 3(a) and (e) where the SEM of the nanofibers shows pure chitin having smaller average diameter (130 nm) and narrower distribution than pure SF nanofibers (1260 nm), although the viscosity (460 cP) of chitin solution was higher than that (221 cP) of SF solution. SF has a relatively hydrophobic character, compared with chitin.
Simultaneous electrospinning of chitin/SF solutions was also employed to fabricate biomimetic nanostructured bicomponent scaffolds. 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 [92].
Neamnark et al. employed electrospinning to fabricate ultra-fine fiber mats of hexanoyl CS (H-CS) for potential use as skin tissue scaffolds [93]. Indirect cytotoxicity evaluation of the fiber mats with mouse fibroblasts (L929) revealed
that the materials were non-toxic and did not release substances harmful to living cells. The potential for use of the fiber mats as skin tissue scaffolds was further assessed in terms of the attachment and the proliferation of human keratinocytes (HaCaT) and human foreskin fibroblasts (HFF) that were seeded or cultured on the scaffolds at different times. The results showed that the electrospun fibrous scaffolds could support the attachment and the proliferation of both types of cells, especially for HaCaT.
Duan et al. showed that the composite electrospun membranes of poly(lactide-co- glycolide) (PLGA) and CS/ poly(vinyl alcohol) (PVA) can positively mimic the structure of natural extracellular matrices and have the potential for application as three-dimensional tissue-engineering scaffolds [51, 94]
Poly(lactide-co-glycolide) (PLGA) and CS/
poly(vinyl alcohol) (PVA) were simultaneously electrospun from two different syringes and mixed on the rotating drum to prepare the nanofibrous composite membrane. The composite membrane was cross linked by glutaraldehyde vapor to maintain its mechanical properties and fiber morphology in wet stage.
The introduction of CS/PVA component changed the hydrophilic/hydrophobic balance and, thus, influenced degradation behavior and mechanical properties of the composite membranes during degradation. The human embryo skin fibroblasts cells (hESF)s seeded on each electrospun membrane could not only favorably attach and grow well on the composite membranes, but were also able to migrate and infiltrate the membranes.
Subramanium et al. generated CS mat composed of oriented sub-micron fibers using the ectrospinning technique [46, 95]. Scanning electronic microscope images showed the fibers in the electrospun CS mats were indeed aligned and there was a slight cross-linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on the electrospun mat was 69% of the cells on tissue-culture polystyrene (TCP control) after three days in culture, which was slightly higher than that on the cast films (63% of the TCP control).
A multi jet electrospinning method was employed by Mohammadi et al. to fabricate novel 3D nanofibrous hybrid scaffolds consisting of poly(å-caprolactone), poly(vinyl alcohol), and CS [96]. The influence of chemical, physical, and structural properties of the scaffolds on the differentiation of mesenchymal stem cells into osteoblasts, and the proliferation of the differentiated cells was investigated.
Osteogenically induced cultures revealed that cells were well-attached, penetrated into the construct and were uniformly distributed. The expression of early and late phenotypic markers of osteoblastic differentiation was upregulated in the constructs cultured in osteogenic medium.
Owida et al. prepared a sandwich structure nanofibre mesh to construct materials for leaflets of heart valve and blood vessel [97]. In the case of heart valve leaflet, the randomly oriented polyurethane nanofibres were prepared as the first layer, followed by gelatin- CS complex layer. Complex nanofibres were initially used to spin on the PU layer with cross orientation to mimic the fibrosa layer. A gelatin and CS complex was then spun onto the other side of PU nanofibre mesh to mimic the ventricularis layer. Smooth muscle cells adhered and flattened out onto the surface of the gelatin-CS complex as early as 1 day post seeding. There is great potential for this biosynthetic biocompatible nanofibrous
Figure 5. SEM image of electrospun one-step cross-linked CS fibers. (Reprinted from Ref.
[37] with permission from American Chemical Society).
material to be developed for various clinical applications.
Lou et al. performed electrospinning in an electric field of 0.6 kV/cm with a polyethylene oxide to CS ratio of 60:40 to prepare nanofibers with diameters that ranged from a few nanometers to several micrometers (approximately 30 nm) [47]. The potential use of the carboxyethyl CS/poly(vinyl alcohol) (CECS/
PVA) electrospun fiber mats as scaffolding materials for skin regeneration was evaluated by Zhou et al. in vitro using mouse fibroblasts (L929) as reference cell lines [48]. 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
The properties of the electrospun fibers could be improved by cross linking. Such an approach was adopted by Jin et al. to generate electrospun fibers of CS/polyvinyl alcohol (CS/
PVA) cross linked by the incorporation of photocrosslinking agent poly(ethyleneglycol)- 600- dimethacrylate (PEGDMA) and photoinitiator 2-hydroxy-l-[4-(2-hydroxyethoxy) phenyl]-2-methyl-l-propanone (HEPK) [98]. The results indicated that the water resistance of photocrosslinked CS-based nanofibers was improved obviously and the swelling ratio decreased with the increasing content of PEGDMA/HEPK. Cytotoxicity evaluation by 3-(4, 5-dimethylthia-zol-2-yl)-2, 5-diphenyltetrazolium bromide assay indicated that the photocrosslinked CS-based fiber membranes were nontoxic to L929 cells. Using cell culture SEM imaging the authors showed that cells which exhibited the spindle shape could grow properly on the surface of nanofibrous structure of the CS/PVA. In another work, Schiffman and Schauer developed a one step electro spinning technique to fabricate Schiff base cross-linked CS fibrous mats [37]. This method is 25 times faster and, therefore, more economical than a previously reported two-step vapor-cross- linking method. Fig.5 shows the structure of fibers fabricated by this method [37].
Earlier investigators used strong acidic solvents and blending with synthetic polymers has been used to achieve electrospun nanofibers containing CS. As an alternative approach, Ojha et al. used polyethylene oxide PEO a template to fabricate CS nanofibers by electrospinning in a core-sheath geometry, with the PEO sheath serving as a template for the CS core [99].
Solutions of 3 wt % CS (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, CS nanofibers were obtained. Electron microscopy confirmed nanofibers of 250 nm diameter with a clear core-sheath geometry before sheath removal, and CS nanofibers of 100 nm diameter after washing.
Nanofibers of ionogenic polymers are of great interest because of the peculiarities of the polyelectrolytes, and also because of the possibility of nanofiber modification on a subsequent step. Incorporation of PAAm into N-carboxyethylCS (CECh) allowed the preparation of fibers with average diameters 50 nm; the difficulties in cross linking the fibers focused the search to the preparation of nanofibrous CECh-based materials using PVA as the second component [68]. PVA is known to be a non-toxic, non-ionogenic and water soluble polymer. Therefore, the nanofibrous materials prepared by electrospinning of ECh/PVA aqueous solutions, dissolved when put in contact with water as can be seen from Figure 17. Cross linking by heating was adopted to stabilize the system, but after heating at 100 °C the fibre structure collapsed for the high PVA system whereas the ECh/PVA mat at low PVA content was promising. It is proposed that the CECh/PVA nanofibrous mats can find application as tissue engineering scaffolds.
Nural Tissue engineering
Bridging of nerve gaps after injury is a major problem in peripheral nerve regeneration.
Prabhakaran et al. fabricated polycaprolactone (PCL)/CS nanofibrous scaffolds and evaluated them in vitro using rat Schwann cells (RT4- D6P2T) for nerve tissue engineering. PCL, CS, and PCL/CS nanofibers with average fiber diameters of 630, 450, and 190 nm, respectively,
were fabricated using an electrospinning process [100]. Simple blending of PCL with CS proved an easy and efficient method for fabricating PCL/CS nanofibrous scaffolds, whose surface characteristics proved more hydrophilic than PCL nanofibers. Evaluation of mechanical properties showed that the Young’s modulus and strain at break of the electrospun PCL/CS nanofibers were better than those of the CS nanofibers. The authors could achieve 48% more cell proliferation on PCL/CS scaffolds than on PCL scaffolds after 8 days of culture in cell proliferation studies on these nanofibrous scaffolds using 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H- tetrazolium assay. PCL/CS scaffolds showed better cell proliferation than PCL scaffolds and maintained their characteristic cell morphology, with spreading bipolar elongations to the nanofibrous substrates.
Electrospun CS nano/microfiber mesh tubes with a DAc of 93% have been shown to exhibit sufficient mechanical properties to preserve tube space, provide a better scaffold for cell migration and attachment, and facilitate humoral permeation to enhance nerve regeneration [101]. Although the functional recovery of motor activity was delayed in each group, sensory function reemerged first in the isograft group followed by the group receiving nano/microfiber mesh tubes with a DAc of 93%.
The team could further fabricate with an electrospinning method a novel bilayered CS tube that comprises an outer layer of CS film and an inner layer of CS nonwoven nano/
microfiber mesh [102]. They introduced glycine spacers into the CYIGSR sequence, domain of laminin-1 that enhances Schwann cells migration and attachment, as well as neural outgrowth, resulting in the amino acid sequences CGGYIGSR and CGGGGGGYIGSR.
These peptides were covalently bound to the nano/microfiber mesh surface of the CS tube to examine the effects of peptide mobility on nerve regeneration. Scaffolds constructed from these bilayered CS tubes were grafted to bridge injured sciatic nerve. Isografting was performed as a control. These scaffolds were removed 5 and 10 weeks after implantation for histological analysis. Nerve regeneration into CS tubes, on which the CGGGGGGYIGSR peptide was immobilized, exhibited efficacy similar to that of
the isograft and represent a promising candidate for promoting peripheral nerve repair Bone tissue engineering
One of the most attractive areas in nano fibre application is bone tissue engineering [102a].
In an interesting study based on cell stain assay and SEM imaging, Bhatarai and his co-workers showed that CS nano fibres produced by electrospinnig exhibited cellular biocompatibility [45]. It was found that the nanofibrous structure promoted the attachment of human osteoblasts and chondrocytes and maintained characteristic cell morphology and viability throughout the period of study [40, 45] have investigated the use of PHBV and the effect of fiber thickness on cell behavior.
An in situ co-precipitation synthesis approach with an electrospinning process as employed by Zhang et al. to prepare a novel biomimetic nanocomposite nanofiber of hydroxyapatite/CS (HAp/CTS) [103]. A model HAp/CTS nanocomposite with the HAp mass ratio of 30 wt% was synthesized through the co- precipitation method so as to attain homogenous dispersion of the spindle-shaped HAp nanoparticles (ca. 100 × 30 nm) within the CS matrix in the presence of a small amount (10 wt%) of ultrahigh molecular weight poly(ethylene oxide) (UHMWPEO) as a fiber- forming facilitating additive (see Fig). Biological in vitro cell culture with human fetal osteoblast (hFOB) cells for up to 15 days demonstrated that the incorporation of HAp nanoparticles into CS nanofibrous scaffolds led to significant bone formation oriented outcomes compared to that of the pure electrospun CTS scaffolds.
Nie and Wang showed that DNA/CS nanoparticles encapsulated PLGA/HAp composite scaffold is promising for use in bone regeneration [104]. Poly (lactide-co-glycolide) (PLGA)/hydroxylapatite (HAp) composite scaffolds with different HAp contents (0%, 5%
and 10%) are fabricated by an electrospinning method and DNA is incorporated into the scaffolds in 3 ways (i.e. naked DNA, encapsulation of DNA/CS nanoparticles into scaffolds after fiber fabrication by dripping, and encapsulation of DNA/CS nanoparticles into scaffold by mixing with PLGA/HAp solution before fiber fabrication). Using SEM, XRD, DSC
and GC-MS, the authors showed that the scaffolds obtained were non-woven, nano- to micro-fibered membrane structures composed predominantly of PLGA with amorphous dispersion of HAp nanoparticles inside polymeric matrix. In vitro release tests were carried out on 9 different scaffolds to check the effects of HAp contents and the encapsulation ways of DNA on the release properties.
Investigations on the cell attachment ability, cell viability and DNA transfection efficiency by human marrow stem cells demonstrated that the addition of HAp nanoparticles increased the release rate of DNA for both naked and encapsulated DNA. Cell culture experiments showed that the scaffolds (hMSCs) with encapsulated DNA/CS nanoparticles had higher cell attachment, higher cell viablility and desirable transfection efficiency of DNA.
Nie et al. studied three different types of scaffolds, encapsulating bone morphogenetic protein-2 (BMP-2) plasmid, in terms of their performances in bone regeneration in nude mice [105]. The plasmid was loaded into fibrous matrices in three different ways: coating of naked DNA (Group A) or DNA/CS nanoparticles (Group B) onto scaffolds after fiber fabrication by dripping and encapsulation of DNA/CS nanoparticles into scaffold by mixing them with PLGA/DCM solution before fiber fabrication (Group C). Their individual performances were examined by soft X-ray observation, histological analysis and immunostaining of bone tissue.
In addition, the BMP-2 protein concentration and alkaline phosphatase (ALP) activity in serum were monitored. Examinations using by soft X- ray observation, histological analysis and immunostaining of bone tissue revealed that the bioactivity of BMP-2 plasmid released from all three kinds of scaffolds was well maintained;
this eventually helped improve the healing of segmental defects in vivo. The three kinds of scaffolds released DNA or DNA nanoparticles in different modes and their performances in bone healing were diverse.
A biocomposite of hydroxyapatite (HAp) with electrospun nanofibrous scaffolds was prepared Yang et al. by using CS/polyvinyl alcohol (CS/PVA) and N-carboxyethyl CS/PVA (CECS/PVA) electrospun membranes as organic matrix, and HAp formed in
supersaturated CaCl2 and KH2PO4 solution [106]. Their studies showed that addition of poly(acrylic acid) to the mineral solution and use of matrix with carboxylic acid groups promoted mineral growth and distribution of HAp. MTT tests and SEM imaging from mouse fibroblast (L929) cell culture revealed the attachment and growth of mouse fibroblast on the surface of biocomposite scaffold, and that the cell morphology and viability were satisfactory for the composite to be used.
Drug release
Delivery of drugs at the appropriate time, duration and site are among the most important points that have to be considered while designing controlled release systems. In meeting these requirements, polymeric nanofibers are emerging as important biomaterial vehicles for drug-delivery. Jin et al.
applied coaxial electrospinning to prepare core- shell structured CS (CS)/poly(vinyl alcohol) (PVA)-poly(propylene carbonate) (PPC) vehicles for encapsulation of drugs to deliver a biomolecular drug in a sustained fashion [107].
The authors could encapsulate hydroxyapatite into the core of composite fibers, which was characterized by Fourier transform infrared spectroscopy (FTIR).
Electrospun non-woven fabrics (ESNWs) were prepared by Ohkawa et al. by the electrospinning technique [36]. They discuss the issues such as (i) relationship between the chain conformation of PBLG in the pre-spun solution and the resulting post-spun morphology and mechanical property of PBLG- ESNWs, (ii) direct electrospinning of CS solution, preparation of the nanofibers, and post-spun treatment of the CS-ESNWs, with respect to the polymer chemical properties, and (iii) preparation of cellulose-ESNWs composed of the nano-scaled fibrous network and biomedical application as drug-carrier and releasing materials.
Nanofibrous mats of CS/poly(vinyl alcohol) (CS/
PVA) loaded with the antiseptic ofloxacin were prepared by Zhen et al. using the electrostatic spinning methods [108]. The hybrid fibers had diameters about 200 nm and there was good compatibility between CS, poly(vinyl alcohol)
and ofloxacin molecules in the blended nanofibrous mats.
Antimicrobial and wound healing applications The electrospinning technology opens up enormous possibilities for the preparation of chitin and CS mats for antimicrobial and wound healing applications. Spasova and coworkers propose that the CS nano-fibrous obtained by electrospun mats are promising for wound- healing applications as they could demonstrate the antibacterial activity of the photo-cross- linked electrospun mats against Staphylococcus aureus and Escherichia coli [109]. The fibres were prepared by electrospinning of quarternised CS solutions mixed with poly(vinyl alcohol) [109]. Their group also prepared successfully nanofibers of the polyampholyte (N-carboxyethylCS) by electrospinning adding a non-ionogenic water- soluble polymer poly(acrylamide) to the spinning solution. [110]. The electrospun mats dissolved when put in contact with water or water vapor. To render the nanofibers insoluble, experiments on their cross linking were performed by heat treatment. They could achieve the preparation of continuous defect-free fibers from quaternized CS derivative (QCh) by electrospinning of mixed aqueous solutions of QCh with poly(vinyl pyrrolidone) (PVP) [111]. On blending with poly(ethylene oxide), CS nanofibres could be produced with diameters in the range 40 - 290 nm by electrospinning of CS/poly(ethylene oxide) (PEO) blend aqueous
solutions. The diameters of the nanofibres were in the range 40 - 290 nm [46]. They prepared fibrous poly(L-lactide) (PLLA) and bicomponent PLLA/poly(ethylene glycol) mats by electrospinning and then were coated with CS [112]. Microbiological studies against Staphylococcus aureus revealed that the CS coating imparts antibacterial activity to the hybrid mats. The combined haemostatic and antibacterial activities render these novel materials suitable for wound-healing applications [109].
Figure 6 The effect of CS content on bacteria growth inhibition of PET/CS nanofiber mats: ~;
Staphylococcus aureus, ; Klebsiella pneumoniae. (Reproduced from reference 57 with permission from Wiley InterScience Inc.)
Figure 7 Degree of growth inhibition of PET, PET/chitin, and PET/CS nanofiber mats against Staphylococcus aureus (&) and Klebsiella pneumoniae. (Reproduced from reference 57 with permission from Wiley InterScience Inc.)
Figure 8 MTT reduction obtained from the NIH3T3 fibroblasts cultured on the nanofiber mats as a function of incubation time: (a) PET, (b) PET/chitin, (c) PET/CS. . (Reproduced from reference 57 with permission from Wiley InterScience Inc.)
Torres-Giner et al. studied the effect of a number of parameters (including solvent nature, polymer origin, molecular weight and spinning conditions) on the morphology to ascertain the antimicrobial properties of the generated biofibers of CS and to relate them to its chemical structure [113]. They reported a new route to generate CS based nanoporous structures starting from blends of CS and polylactic acid.
Nanofibers were produced by Gholipour et al.
from CS (Cs) in 2% acetic acid and poly vinyl alcohol (PVA) blend in DW in 10/90, 20/80, 25/
75, 50/50 mass ratio of Cs/PVA [114]. The response surface methodology was used to model and optimize the electrospinning parameters for the spinning of blend CS PVA nanofibers. Fiber diameter was correlated to these variables by using a second order polynomial function. The predicted fiber diameters were in agreement with the experimental results.
Jung et al employed the electrospinning method to introduce antibacterial activity and biocompatibility to the surface of poly (ethylene terephthalate) (PET) textiles [55]. The PET/CS nano-fibers were evenly deposited on to the surface, and the diameter of the nanofibers was in the range between 500 and 800 nm. The wettability of the PET nanofibers was significantly enhanced by the incorporation of CS. The antibacterial activity of the samples was evaluated utilizing the colony counting method against Staphylococcus aureus and Klebsiella pneumoniae. The results indicated that the PET/
CS nanofiber mats showed a significantly higher growth inhibition rate compared with the PET nanofiber control. In addition, the fibroblast cells adhered better to the PET/CS nanofibers than to the PET nanofibers mats, suggesting better tissue compatibility. Fig. 6 shows that the growth inhibition of bacteria gradually increases with an increase in CS content and it reaches a maximum value when the CS content is 10 wt
%.
Figure 7 shows the growth inhibition of the PET,PET/chitin, and PET/CS nanofiber mats against S. aureus and K. pneumoniae, respectively. The electrospun PET, which was blended with 10 wt % CS, exhibited a degree growth inhibition of more than 95% while PET alone showed a degree growth inhibition under 20%. The degree growth inhibition of 10 wt % of chitin blended with PET was around 60%, not as high as that of CS. Adding CS to PET textiles showed significant improvement of antibacterial activity, much higher than adding chitin, on both bacteria and gave high functionality of textiles.
Figure 8 shows the cell viability of fibroblasts that had been cultured for three days on nanofibrous mats. The cell viability on the PET/
CS was almost the same as that on PET/chitin, but it was higher than that of the PET control.
Figure 9. Water drop containing phenol red on the PET (a), PET/chitin (b), and PET/CS nanofiber (c) mats after 5 secs. (Reproduced from reference 57 with permission from Wiley InterScience Inc.)
The authors evaluated the surface wettability by placing a stained water drop on the nanofiber mat and observing the drop visually (Fig. 9). The water drop on the PET nanofiber mat maintained a spherical shape for a long time. However, a water drop on the PET/CS nanofiber mats spread immediately while water drop on PET/
chitin did slowly. Blending CS improved hydrophile property to PET surface much more than blending chitin. This result suggests that the PET nanofiber mat became more hydrophilic after the incorporation of CS into the PET fibers.
The electrospun CS/PVA membranes were shown to absorb higher water uptake indicating its potential applications in wound dressings.
Zhang et al. demonstrated this by preparing electrospun CS / PVS system from its solutions in 2% aqueous acetic acid by adding poly(vinyl alcohol) (PVA) as a “guest” polymer [49].
Analyses of scanning electron micrographs and transmission electron micrographs suggested that the CS/PVA ultra fine fibers were often obtained along with beads, and CS was located in the elctrospun fibers as well as in the beads.
Uniform CS/PVA fibers with an average diameter of 99±21 nm could be prepared from a 7% CS/
PVA solution in 40:60 mass ratio. Results of Fourier transform infrared spectroscopy and X- ray diffraction demonstrated that there were possible hydrogen bonds between CS and PVA molecules, which could weaken the strong interaction in CS itself and facilitate CS/PVA electrospinnability.
The biodegradability and cell behavior of electrospun chitin nano finbers were studied by Noh et al. [115]. The image analysis by scanning electron microscopy of the as-spun chitin nanofibers (Chi-N) and commercial chitin microfibers (Beschitin W®; Chi-M) showed that the average diameters of Chi-N and Chi-M were 163 nm and 8.77 ìm, respectively. During in vitro degradation for 15 days, the degradation rate of Chi-N was faster than that of Chi-M, likely due to higher surface area of Chi-N. Chi-N that was grafted into rat subcutaneous tissue had almost degraded within 28 days, and no inflammation could be seen on the nanofiber surfaces or in the surrounding tissues.
Cytocompatibility and cell behavior studies revealed relatively high cell attachment and spreading of all the cells tested were observed
on Chi-N in comparison to Chi-M, and Chi-N treated with type I collagen significantly promoted the cellular response. Chi-N, alone or with extracellular matrix proteins (particularly type I collagen), could turn out to be potential candidates for the cell attachment and spreading of normal human keratinocytes and fibroblasts. The authors suggest that this property of Chi-N might be particularly useful for wound healing and regeneration of oral mucosa and skin.
Nanocomposites & Hybrid nanofibres Preparation of hybrid nanomaterials, sensitive to changes in external magnetic field is of particular interest in the field of nanotechnology [116]. Duan et al. prepared hybrid nanofibrous membranes of poly(lactic-co-glycolic acid) (PLGA) and CS with different CS amounts (32.3, 62.7, and 86.5%) via a specially designed electrospinning setup [117]. They showed that that the CS amount in PLGA/CS membranes could be well controlled by adjusting the number of syringe for electrospinning of PLGA or CS, respectively. Mechanical properties improved on cross linking of CS. The cytocompatibility of hybrid PLGA/CS membranes was better than that of the electrospun PLGA membrane. The authors recommend that electrospun hybrid nanofibrous membranes of PLGA and CS appear to be promising for skin tissue engineering. In another work, hybrid nanofibers of silk with chitin and carbon nanotubes were prepared by elecrospinning and compared with fibers produced through wet spinning [118]. It was determined that carbon nanotubes offer the best potential for improving the thermal properties of silk.
Polymer-stabilized magnetic nanoparticles were obtained by Rashkov et al. using two biocompatible polyelectrolytes: N- carboxyethylCS (CECh) and poly(2-acrylamido- 2-methylpropanesulfonic acid) (PAMPS) [119].
The size of the particles (mean diameter 10 or 30 nm, respectively) and the stability of the dispersions could be effectively controlled depending on the polyelectrolyte nature.
Fabrication of nanocomposite magnetic fibers with mean diameter in the range 100-500 nm was achieved using electrospinning of the system CECh/ferrofluid/non-ionogenic polymer.
CS was as coating for to improve the surface compatibility of fibrous poly(L-lactide) (PLLA) and bicomponent PLLA/poly(ethylene glycol) mats prepared by electrospinning [112]. On contact with blood, the CS coating led to changes in erythrocyte shape and in their aggregation. The haemostatic activity of the mats increased with increasing CS content.
Other applications
The electrospinning process was employed by Xu and coworkers [50] to prepare stabilized CS nanofibrous membrane as support for enzyme immobilization. CS nanofibrous membrane was directly fabricated from a mixed solution of CS with poly(vinyl alcohol) (PVA) and then treated in a NaOH solution to remove PVA and stabilize the morphologies of CS nanofibrous membrane in aqueous media. CS can provide an optimal microenvironment for the immobilized enzyme to maintain relatively high biological activity and stability.
Mincheva et al. reported he preparation of polymer-stabilized magnetic nanoparticles using two biocompatible polyelectrolytes: N- carboxyethylCS (CECh) and poly(2-acrylamido- 2-methylpropanesulfonic acid) (PAMPS) by the application of electrospinning method. The size of the particles (mean diameter 10 or 30 nm, respectively) and the stability of the dispersions could be effectively controlled depending on the polyelectrolyte nature. Depending on the polyelectrolyte nature the magnetic nanoparticles existed in different magnetic states – superparamagnetic or intermediate state between superparamagnetic and ferrimagnetic one, as evidenced by the measurements of the magnetization and Mo¨ssbauer analyses [119].
Conventional ultra filtration (UF) or nanofiltration (NF) filters for water treatments based on porous membranes, typically manufactured by the phase immersion method usually exhibits only relatively low flux rate. [120]. Yoon et al.
demonstrated a new type of high flux UF/NF medium based on an electrospun nanofibrous scaffold (e.g. polyacrylonitrile, PAN) coupled with a thin top layer of hydrophilic, water-resistant, but water-permeable coating (e.g. CS) [120].
The interconnected porosity of the non-woven
nanofibrous scaffold can be controlled partially by varying the fiber diameter (from about 100 nm to a few micrometers) through the electrospinning processing. Addition of poly(ethyleneoxide) (PEO) to CS enhanced its solubility and enabled fiber formation during electrospinning. Thus, nanometer-sized fibers with fiber diameter as low as 80 ± 35 nm without bead defects were made by electrospinning high molecular weight CS/PEO (95:5) blends for applications in air and water filtration [121].
Desai et al. reported preparation of non-woven fibers without bead defects by electrospinning blend solutions of CS and polyacrylamide (PAAm) with blend ratios varying from 75 wt% to 90 wt% CS using a modified electrospinning unit wherein polymer solutions can be spun at temperatures greater than ambient up to 100
°C [122]. Zhang et al. showed that introduction of an ultrahigh-molecular-weight poly(ethylene oxide) (UHMWPEO) into aqueous CS solutions remarkably enhanced the formation of CS nanofibrous structure and led 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 CS nanofibers [123]. The excellent electrospinnability of the current formulation renders electrospinning of natural biopolymer CS a robust process for large-scale production of practically applicable nanofibrous structures.
Coaxial electrospinning is a new technique to fabricate continuous composite ultra fine fibers with core/shell structure, which has a broad application perspective in the biomedical field [124]. Ultra fine fibrous membranes of core/
shell poly(vinyl pyrrolidone)/poly(L-lactide-co- &
å caprolactone) (PVP/PLCL) produced by coaxial electrospinning showed the largest water absorption (501.3%) in phosphate buffer solution due to introduction of the PVP component and the core/shell fiber structure.
Results of tensile tests indicated that the electrospun PVP/PLCL membranes possessed higher tensile strength and elongation-at-break, and lower Young’s modulus than those of PLCL and CS membranes in both dry and wet states. Studies on cell adhesion, viability and morphology on the fibrous membranes showed that PVP/PLCL membranes could mimic the structure of natural extracellular matrices and positively promote
cell-cell and cell-matrix interactions because of hydrophilicity/hydrophobicity balance.
The miscibility of the fiber blends could be enhanced by polymers that can contribute to intermolecular interation. This was achieved by Chen et al. in preparing collagen-CS complex nanofibers by electrospinning [57]. It was found that the -OH group, the -NH2 group and the amide I, II and III characteristic absorption bands in FT-IR spectra of electrospun collagen and CS blends were shifted and modified with the difference of CS content in electrospun fibers.
CS, which is difficult to electrospin into nanofibers, could be easily electrospun into nanofibers with addition of a small portion of poly(L-lactic acid-co-å-caprolactone), P(LLA- CL). The fiber diameter depended on both the polymer concentration and the blend ratio of CS to P(LLA-CL). The average fiber diameter increased with increasing polymer concentration and decreasing the blend ratio of CS to P(LLA-CL) [125]. The porosity of CS/
P(LLA-CL) nanofiber mats increased with increasing the weight ratio of CS to P(LLA-CL), while both the tensile strength and the ultimate strain increased with increasing P(LLA-CL) ratio. Fibroblast cell growth on nanofiber mats were investigated with MTT assay and scanning electron microscope (SEM) observation. The highest cell proliferation was observed on the nanofiber mats when the weight ratio of CS to P(LLA-CL) was 1:2. As SEM images shown, fibroblast cells showed a polygonal shape on blend nanofiber mats and migrated into the nanofiber mats.
Electrospinning of aqueous solutions of carboxymethyl chitosan (CMCS) was facilitated with the addition of water-soluble polymers, including PEO, PAA, PAAm and PVA. [126]. The optimal fiber formation was observed at equal mass composition of O-CMCS (89 kDa at 0.36 DS) and PVA, producing nanofibers with an average diameter of 130 nm. Heat-induced esterification (at 140°C for 30 min) produced inter-molecular covalent cross-links within and among fibers, rendering the fibrous membrane water-insoluble. Membranes containing higher CMCS carboxyl to PVA hydroxyl ratio retained better fiber morphology upon extended water exposure, indicating more favorable inter-
molecular cross-links. The fibrous membranes generated with less substituted CMCS were more hydrophilic and retained a greater extent of the desirable amine functionality. Aqueous solutions of PEG-N-CS or PEG-N,O-CS alone could not be electrospun into fibers [127].
Fibrous structure intermixed with beads were observed during electrospinning of PEG-N,O- CS at 25% in DMF. However, ultra-fine fibers with diameters ranging from 40 nm to 360 nm and an average diameter of 162 nm were efficiently generated from electrospinning of 15% PEG-N,O-CS in 75/25 (v/v) THF/DMF co solvents with 0.5% Triton X-100TM. Soluble poly (vinyl alcohol) and soluble starch and polyvinyl pyrrolidone were used along with CS for the preparation of ultra fine fibers [53,128,129,130].
Zhen et al. used photo curable monomer triethylene glycol dimethylacrylate (TEGDMA) and photoinitiator 2-hydroxy-2-methyl-1- phenylpropan-1-one were introduced into the CS/PVA blend solution to increase the water- resistance of the fibers, which is a common shortcoming of water soluble polymer fiber [131]. De Vrieze et al. produced CS nanofibers by electrospinning from aqueous solutions containing solution 90% acetic acid.[61].
Solubility was greatly improved on cross linking with glutaraldehyde [132]. The as-spun medium molecular weight CS 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 CS 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 with average diameters between 20 and 100 nm have been prepared by Li et al. by electrospinning of 82.5% deacetylated CS (Mv = 1600 kDa) mixed with poly(vinyl alcohol) (PVA, Mw = 124-186 kDa) in 2% (v/v) aqueous acetic acid. Other cross linking agents such as
triethylene glycol dimethacrylate (TEGDMA) have also been used to make cross linked CS-based nanofibers via electrospinning technique with heat mediated chemical cross linking [133].
The results showed that, nanofibers exhibited a smooth surface and regular morphology, and tensile strength of nanofibers improved with increasing of TEGDMA content [56]. Matsuda et al. fabricated water-insoluble CS nanofiber sheets and tubes coated with CS-cast film by electrospinning by immersing the as-spun CS nanofiber sheets and tubes in 28% ammonium aqueous solution [134].
Another interesting development is the electrospinning of multiwalled carbon nanotubes (MWCNTs) grafted onto CS (CS) [135]. Raman spectra indicated that the electrospinning process did not severely alter the electron hybridization of carbon atoms within the nanotube framework. It was interesting to observe that these nanofibers showed a novel sheath-core structure; the outer and inner diameters of these sheath-core nanofibers were about 200 nm and 100 nm, respectively. The electrospun fibers’ web displayed faster electron transfer kinetics and better electrochemical properties than its cast film, which justified further applications in biological areas.
CS ahs also been sued to improve the surface properties of poly(acrylonitrile-co-acrylic acid) (PANCAA) based nanofibrous membranes produced by an electrospinning technique [136].
Platelet adhesion experiments indicated that the immobilization of CS on the PANCAA nanofibrous membranes was favourable for platelet adhesion. It appears that electrospinning has emerged as a versatile method to manufacture CS fibres [37-40, 137- 139]. There are great expectations for the use these technologies in the newly emerging area of nanomedicine that is aiming for disease diagnosis and treatment with unprecedented precision and efficacy by integrating nanotechnology with medicine [140, 141].
Conclusions and perspectives
During the last decade, enormous progress has been made in the area of electrospinning to produce polymeric ultra fine/nano scale fibres with diameters ranging from a few micrometers
to tens of nanometers. Using this simple and versatile technique, biopolymers such as chitin and CS have been successfully electrospun into nanofibers and their production variables and morphology have been established. The prospective biomedical applications of electrospun polymeric nanofibres include tissue engineering, wound dressings, medical prostheses, drug delivery systems etc.
Blending with other polymers gave a good option as they could reduce repulsive forces within the charged biopolymer solutions and allow fiber spinning.
However, there are a few significant challenges that need to be addressed for large-scale manufacturing of new products containing these biopolymer nanofibers. Methodology to fabricate reproducible, uniform nanofibers, which have particular morphologies, mechanical and chemical properties that are oriented for the demands of particular tasks need to be addressed. In tissue engineering as the cells grows in the direction of the nanofibers, further improvement in the alignment of the nanofibers can induce arrangement of cells in 2- and 3D architectures to achieve better cell proliferation, differentiation and functional longevity. The nanofibrous scaffold functions can be further improved with innovative development in electrospinning processes, such as two-component electrospinning and in-situ mixing electrospinning. Post modifications of electrospun membranes could be another effective means to render the electrospun scaffolds with controlled anisotropy and porosity. A silver lining in the area is report that there at least one product based on electrospun nanofibers with drug-release properties in a Phase III clinical trial, for wound dressing.
Electrospinning has been recognized as a feasible technique for the fabrication of continuous polymeric nanofibre yarns desired in the textile industry. Potential applications based on electrospun nanofibres as a new- generation material in the textile industry will be realized if suitable nanofibre yarns become available to textile processes like weaving, knitting and embroidery.
There is a growing interest in integrating nanotechnology with medicine, creating so- called nanomedicine aiming for disease diagnosis and treatment with unprecedented precision and efficacy. New products are being designed, or have been designed based on chitin, which present such innovative features, also in terms of their applications, as to have already influenced our current lifestyle. With the use of the newly developing technological platforms, it is possible to obtain thin nanostructured films organized as nets, capable of providing a huge surface that is available for interaction with the skin tissue and the external environment.
Acknowledgement
We are grateful to the Prof. K. Mohandas, Director, and Dr. G.S. Bhuvaneshwar, Head, BMT Wing of Sree Chitra Tirunal Institute for Medical Sciences & Technology for providing facilities and support for preparing this report. We are thankful to the laboratory staff and library staff for their assistance. Thanks are also due to Miss Minimol for rendering help in the preparation of this article. Thanks are also due to FADDS for support.
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