Novel chitin and chitosan nanofibers in biomedical applications

Chitin and its deacetylated derivative, chitosan, are non-toxic, antibacterial, biodegradable and biocompatible biopolymers. Due to these properties, they are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressings, separation membranes and antibacterial coatings, stent coatings, and sensors. In the recent years, electrospinning has been found to be a novel technique to produce chitin and chitosan nanofibers. These nanofibers find novel applications in biomedical fields due to their high surface area and porosity. This article reviews the recent reports on the preparation, properties and biomedical applications of chitin and chitosan based nanofibers in detail.     

基于 AOPAN 纳米纤维膜的粘杆菌素发酵液后处理研究

使用静电纺丝再经胺肟化改性制备AOPAN纳米纤维膜,并基于AOPAN纳米纤维膜构建膜分离装置,用于粘杆菌素发酵液的后处理。研究中,对不同厚度的纳米纤维膜的渗透通量及分离性能进行比较,最终确定双层叠合的纳米纤维膜为分离膜,最适操作压力为0.14 MPa。在此条件下,分离膜的渗透通量为2.61 L/m2．min,蛋白质的截留率达到90％,色素等其他杂质也得到有效去除。     

Effect of deacetylation on wicking behavior of co-electrospun cellulose acetate/polyvinyl alcohol nanofibers blend

Cellulose acetate (CA) nanofibers webs deserve a special attention because of their very good water retention properties. CA nanofibers based biosensor in certain application come into contact with various liquids and requires high degree of wicking rate to transport liquid to its destination. Cellulose acetate (CA)/polyvinyl alcohol (PVA) blended nanofibers were prepared via co-electrospinning using double nozzle for jetting cellulose acetate and polyvinyl alcohol independently. The CA/PVA blend nanofibers webs were deacetylated in aqueous alkaline solution to convert CA in to regenerated cellulose and to remove PVA nanofibers from the raw web. The resultant nanofibers webs were characterized by wicking rate, water contact angle, SEM and FTIR analysis. The results revealed that by varying concentration of PVA solution enhances the wicking rate. Such a nanofibers web may be used in biosensor strip and other medical applications where the high wicking rates are desired.     

Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications

Chitin is a natural biopolymer have been used for several biomedical applications due to its biodegradability and biocompatibility. By using the calcium solvent system, chitin regenerated hydrogel (RG) was prepared by using -chitin. And also, the swelling hydrogel (SG) was prepared by using β-chitin with water. Then, both RG and SG were mixed with gelatin and N-acetyl-d-(+)-glucosamine (GlcNAc) at 120 °C for 2 h. The chitin/gelatin membranes with GlcNAc were also prepared by using RG and SG with GlcNAc. The prepared chitin/gelatin membranes with or without GlcNAc were characterized by mechanical, swelling, enzymatic degradation, thermal and growth of NIH/3T3 fibroblast cell studies. The stress and elongation of chitin/gelatin membrane with GlcNAc prepared from RG was showed higher than the chitin/gelatin membranes without GlcNAc. But, the chitin/gelatin membranes prepared from SG with GlcNAc was showed higher stress and elongation than the chitin/gelatin membranes without GlcNAc. It is due to the crosslinking effect of GlcNAc. The chitin/gelatin membranes prepared from SG showed higher swelling than the chitin/gelatin membranes prepared from RG. In contrast, the chitin/gelatin membranes prepared from RG showed higher degradation than the chitin/gelatin membranes prepared from SG. And also, these chitin/gelatin membranes are showing good growth of NIH/3T3 fibroblast cell. So these novel chitin/gelatin membranes are useful for tissue engineering applications.     

Preparation of chitin nanofibers from squid pen beta-chitin by simple mechanical treatment under acid conditions

A procedure for preparing individualized chitin nanofibers 3-4 nm in cross-sectional width and at least a few microns in length was developed. The key factors to prepare the chitin nanofibers with such high aspect ratios are as follows: (1) squid pen beta-chitin is used as the starting material and (2) ultrasonication of the beta-chitin in water at pH 3-4 and 0.1-0.3% consistency for a few minutes. Transparent and highly viscous dispersions of squid pen beta-chitin nanofibers in water can be obtained by this method. No N-deacetylation occurs on the chitin molecules during the nanofiber conversion procedure. Moreover, the original crystal structure of beta-chitin is maintained, although crystallinity index decreases from 0.51 to 0.37 as a result of the nanofiber conversion. Cationization of the C2 amino groups present on the crystallite surfaces of the squid pen beta-chitin under acid conditions is necessary for preparing the nanofibers.     

Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose

Never-dried and once-dried hardwood celluloses were oxidized by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated system, and highly crystalline and individualized cellulose nanofibers, dispersed in water, were prepared by mechanical treatment of the oxidized cellulose/water slurries. When carboxylate contents formed from the primary hydroxyl groups of the celluloses reached approximately 1.5 mmol/g, the oxidized cellulose/water slurries were mostly converted to transparent and highly viscous dispersions by mechanical treatment. Transmission electron microscopic observation showed that the dispersions consisted of individualized cellulose nanofibers 3-4 nm in width and a few microns in length. No intrinsic differences between never-dried and once-dried celluloses were found for preparing the dispersion, as long as carboxylate contents in the TEMPO-oxidized celluloses reached approximately 1.5 mmol/g. Changes in viscosity of the dispersions during the mechanical treatment corresponded with those in the dispersed states of the cellulose nanofibers in water.     

Biomimetic nanofibrous scaffolds: preparation and characterization of chitin/silk fibroin blend nanofibers

Electrospinning of chitin/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was investigated to fabricate a biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun chitin/SF blend nanofibers was investigated with a field emission scanning electron microscope (FE-SEM). The average diameters of chitin/SF blend fibers decreased from 920 to 340 nm, with the increase of chitin content in blend compositions. The miscibility of chitin/SF blend fibers was examined by solution viscosity measurement. The chitin and SF were immiscible in the as-spun nanofibrous structure. The dimensional stability of chitin/SF blend nanofibers, with or without water vapor after-treatment, was conducted by immersing in water. As-spun SF-rich blend nanofibrous matrices were lost their fibrous structure after the water immersion for 24 h, and then changed into membrane-like structure. On the contrary, nanofibrous structures of water vapor-treated SF-rich blends were almost maintained. To assay the cytocompatibility and cell behavior on the chitin/SF blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal keratinocyte and fibroblasts seeded on the scaffolds were studied. Our results indicate that chitin/SF blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds because it has both biomimetic three-dimensional structure and an excellent cell attachment and spreading for NHEK and NHEF.     

Chitin whiskers: an overview

Chitin is the second most abundant semicrystalline polysaccharide. Like cellulose, the amorphous domains of chitin can also be removed under certain conditions such as acidolysis to give rise to crystallites in nanoscale, which are the so-called chitin nanocrystals or chitin whiskers (CHWs). CHW together with other organic nanoparticles such as cellulose whisker (CW) and starch nanocrystal show many advantages over traditional inorganic nanoparticles such as easy availability, nontoxicity, biodegradability, low density, and easy modification. They have been widely used as substitutes for inorganic nanoparticles in reinforcing polymer nanocomposites. The research and development of CHW related areas are much slower than those of CW. However, CHWs are still of strategic importance in the resource scarcity periods because of their abundant availability and special properties. During the past decade, increasing studies have been done on preparation of CHWs and their application in reinforcing polymer nanocomposites. Some other applications such as being used as feedstock to prepare chitosan nanoscaffolds have also been investigated. This Article is to review the recent development on CHW related studies.     

Studies on chitin. 2. Preparation and properties of chitin membranes

A chitin membrane was prepared by a new procedure involving coagulation of the chitin solution in N,N-dimethyl acetamide, N-methyl 2-pyrrolidone and lithium chloride (DMA-NMP-LiCl) with 2-propanol. The solute permeability, water sorption and mechanical properties were compared with membranes prepared by two previously reported methods (coagulation of a formic acid and dichloroacetic acid (FA-DCA) solution of chitin with 2-propanol; and coagulation of a trichloroacetic acid and dichloroethane (TCA-DCE) solution of chitin with acetone). The permeability coefficients of the three chitin membranes were higher than a regenerated cellulose membrane (Cuprophane®). The membrane prepared from DMA-NMP-LiCl solution had a higher tensile strength (3·3 Mpa) in the wet state than the others. The membrane obtained from TCA-DCE solution absorbed more water (360%) and the membrane prepared from FA-DCA solution was relatively weak (1·8 MPa) in the wet state. It was suggested that 2-propanol was a favourable coagulant for membrane production. In addition, the effect of the origin of chitin on molecular weight and tensile properties of the membranes was studied.     

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.     

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

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

Core-shell structured PEO-chitosan nanofibers by coaxial electrospinning

Core-shell structured PEO-chitosan nanofibers have been produced using a coaxial electrospinning setup. PEO and chitosan solutions, both in an aqueous acetic acid solvent, were used as the inner (core) and outer (shell) layer, respectively. Uniform-sized defect-free nanofibers of 150-190 nm diameter were produced. In addition, hollow nanofibers could be obtained subsequent to PEO washing of the membranes. The core-shell nanostructure and existence of chitosan on the shell layer were confirmed by TEM images obtained before and after washing the PEO content with water. The presence of chitosan on the surface of the composite nanofibers was further supported by XPS studies. The chitosan and PEO compositions in the nanofibrous mats were determined by TGA analysis, which were similar to their ratio in the feed solutions. The local compositional homogeneity of the membranes and the efficiency of the washing step to remove PEO were also verified by FTIR. In addition, DSC and XRD were used to characterize the crystalline structure and morphology of the co-electrospun nonwoven mats. The prepared coaxial nanofibers (hollow and solid) have several potential applications due to the presence of chitosan on their outer surfaces.     

Biosynthesis of hetero-polysaccharides by Acetobacter xylinum - Synthesis and characterization of metal-ion adsorptive properties of partially carboxymethylated cellulose

Biological reconstruction of water-soluble carboxymethylated cellulose (CMC; D.S. =0.47) has been achieved by culturing Acetobacter xylinum in medium containing CMC and -glucose to give a novel hetero-polysaccharide having a carboxymethyl function. The novel extracellular polysaccharide, carboxymethylated-bacterial cellulose (CM-BC), had an ion exchange ability with enhanced specific adsorption for lead and uranyl ions compared to the original CMC and bacterial cellulose. The contribution of the hydroxy group at C-2 was confirmed by applying carboxymethylated chitin, which possesses acetamido group at C-2 of the glucose residue, as the carbon source of the incubation.     

"Nanocellulose" as a single nanofiber prepared from pellicle secreted by Gluconacetobacter xylinus using aqueous counter collision

This study attempted to prepare a single cellulose nanofiber, "nanocellulose", dispersed in water from 3D networks of nanofibers in microbial cellulose pellicle using aqueous counter collision (ACC), which allows biobased materials to be down-sized into nano-objects only using water jets without chemical modification. The nanocellulose thus prepared exhibited unique morphological properties. In particular, the width of the nanocellulose, which could be controlled as desired on nanoscales, was smaller than that of just secreted cellulose nanofiber, resulting in larger specific surface areas. Moreover, ACC treatment transformed cellulose I(α) crystalline phase into cellulose I(β) phase with the crystallinity kept >70%. In this way, ACC method depending on the treatment condition could provide the desired fiber width at the nanoscale and the different ratios of the two crystalline allomorphs between cellulose I(α) versus I(β), which thus opens further pathways into versatile applications as biodegradable single nanofibers.     

Preparation,characterization, bioactive and cell attachment studies of α-chitin/gelatin composite membranes

The chitin/gelatin composite membranes were prepared by mixing of chitin hydrogel with gelatin. The prepared composite membranes were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical, swelling, enzymatic degradation and thermal studies. The XRD pattern of the chitin/gelatin composite membranes showed almost the same pattern as α-chitin. The bioactivity studies of these chitin/gelatin membranes were carried out with the simulated body fluid solution (SBF) for 7, 14 and 21 days followed by the characterization with the scanning electron microscopy (SEM) and Energy Dispersive Spectrum (EDS) studies. The SEM and EDS studies confirmed the formation of calcium phosphate layer on the surface of chitin/gelatin membranes. Biocompatibility of the chitin/gelatin membrane was assessed using human MG-63 osteoblast-like cells. After 48 h of incubation, it was found that the cells had attached and completely covered the membrane surface. Thus, the prepared chitin/gelatin membranes are bioactive and are suitable for cell adhesion suggesting that these membranes can be used for tissue-engineering applications.     

Competition for polymers among heterotrophic bacteria, isolated from particles of the Equatorial Atlantic.

Three heterotrophic bacterial strains, isolated from organic particles of the upper water column of the Equatorial Atlantic, taken during a cruise on the R/V METEOR (1997), were investigated concerning their physiological and phylogenetic properties using classic microbiological and modern molecular-biological methods. All isolates are gram-negative rods able to use polymers such as cellulose, chitin or starch as sole carbon source. The phylogeny of these isolates was investigated by fluorescence in situ hybridization (FISH) and 16S rDNA sequencing. The three isolated strains belong to the Cytophaga/Flavobacteria, gamma-Proteobacteria (Marinobacter sp.), and alpha-Proteobacteria (Sulfitobacter pontiacus). In order to study succession during growth on polymers naturally occurring in marine habitats, FISH was used as a new approach to detect cells from different phylogenetic clusters in the course of a single growth experiment. Mixed cultures consisting of the isolated strains in equal amounts were incubated with cellulose, chitin or starch. Isolate 4301-10/2, a member of the gamma-Proteobacteria, dominated in mixed cultures growing on cellulose, chitin, or starch after only 10 days, with 55, 60, and 95%, respectively, of cells hybridizing with 4,6-diamidino-2-phenylindole (DAPI).     

Structure, morphology and thermal characteristics of banana nano fibers obtained by steam explosion

In this work, cellulose nanofibers were extracted from banana fibers via a steam explosion technique. The chemical composition, morphology and thermal properties of the nanofibers were characterized to investigate their suitability for use in bio-based composite material applications. Chemical characterization of the banana fibers confirmed that the cellulose content was increased from 64% to 95% due to the application of alkali and acid treatments. Assessment of fiber chemical composition before and after chemical treatment showed evidence for the removal of non-cellulosic constituents such as hemicelluloses and lignin that occurred during steam explosion, bleaching and acid treatments. Surface morphological studies using SEM and AFM revealed that there was a reduction in fiber diameter during steam explosion followed by acid treatments. Percentage yield and aspect ratio of the nanofiber obtained by this technique is found to be very high in comparison with other conventional methods. TGA and DSC results showed that the developed nanofibers exhibit enhanced thermal properties over the untreated fibers.     

Effects of nitrogen delivery on chitin nanofiber production during batch cultivation of the diatom <Emphasis Type="Italic">Cyclotella</Emphasis> sp. in a bubble column photobioreactor

The photosynthetic diatom Cyclotella sp. extrudes chitin nanofibers following cell division. This diatom requires silicon for cell wall biosynthesis and division, as well as nitrogen for biosynthesis of intracellular material and extracellular chitin, an N-acetyl glucosamine biopolymer. The initial nitrogen/silicon molar ratio was the critical parameter for assessing the limits of nitrogen delivery on cell number and chitin production during batch cultivation of Cyclotella in a bubble column photobioreactor under silicon-limited growth conditions, using nitrate as the nitrogen source. The peak rate of volumetric chitin production increased linearly, from 3.0 to 46 mg chitin L^?1 day^?1, with increasing N/Si ratio over the range studied (0.82 to 8.6 mol N mol^?1 Si). However, the cell number yield and the chitin yield per cell increased asymptotically with increasing N/Si ratio, achieving a final cell number yield of 5.3?×?10⁹?±?2.6?×?10⁸ cells mol^?1 Si and chitin yield of 28.7?±?1.2 mg chitin per 10⁹ cells (1.0 S.E.). An N/Si ratio of at least 4.0 mol N mol^?1 Si achieved 90% of the asymptotic chitin yield. This study has shown that scalable cultivation systems for maximizing chitin nanofiber production will require delivery of both silicon and optimal nitrogen under silicon-limiting growth conditions to promote cell division and subsequent chitin formation.     

Comparative protein analysis of the chitin metabolic pathway in extant organisms: A complex network approach

Chitin is a structural endogenous carbohydrate, which is a major component of fungal cell walls and arthropod exoskeletons. A renewable resource and the second most abundant polysaccharide in nature after cellulose, chitin is currently used for waste water clearing, cosmetics, medical, and veterinary applications. This work comprises data mining of protein sequences related to the chitin metabolic pathway of completely sequenced genomes of extant organisms pertaining to the three life domains, followed by meta-analysis using traditional sequence similarity comparison and complex network approaches. Complex networks involving proteins of the chitin metabolic pathway in extant organisms were constructed based on protein sequence similarity. Several usual network indices were estimated in order to obtain information on the topology of these networks, including those related to higher order neighborhood properties. Due to the assumed evolutionary character of the system, we also discuss issues related to modularity properties, with the concept of edge betweenness playing a particularly important role in our analysis. Complex network approach correctly identifies clusters of organisms that belong to phylogenetic groups without any a priori knowledge about the biological features of the investigated protein sequences. We envisage the prospect of using such a complex network approach as a high-throughput phylogenetic method.     

Viscoelastic evaluation of average length of cellulose nanofibers prepared by TEMPO-mediated oxidation

Dynamic viscoelasticity measurements were performed for aqueous dispersions of cellulose nanofibers prepared by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation and subsequent mechanical disintegration in water. The frequency dependence of the storage and loss moduli of 0.02% (w/v) dispersions of TEMPO-oxidized cellulose nanofibers in water showed terminal relaxation behavior at relatively lower angular frequencies. This strongly suggests that each cellulose nanofiber in the dispersion behaves as a semiflexible rod-like macromolecular chain or colloidal particle. Furthermore, a clear boundary was observed between the terminal relaxation and rubbery plateau regions. The longest viscoelastic relaxation time, τ, was estimated from the angular frequency, corresponding to the boundary point, and the average length of the cellulose nanofibers, L, was estimated using the equation τ = πη(s)L(3)/[18k(B)T ln(L/d)]. The equation gave a value of L = 2.2 μm, which was in good agreement with TEM observations.