Chitosan based bioadhesives for biomedical applications: A review.

Due to the promising properties of chitosan for biomedical engineering applications like biodegradability, biocompatibility, and non-toxicity, it is one of the most interesting biopolymers in this field. Therefore, Chitosan and its derivatives have attracted great attention in vast variety of biomedical applications. In the current paper, different types of chitosan-based bioadhesives including passive and active and their different types of external stimuli response structure such as thermo, pH and Light responsive systems are discussed. Different bioadhesives mechanisms with chitosan as an adhesive agent or main polymer component and some examples were also presented. Chitosan based bioadhesives and their potential biomedical applications in drug delivery systems, suture less surgery, wound dressing and hemostatic are also discussed. The results confirmed wound healing, hemostatic and bioadhesion capabilities of the chitosan bioadhesives and its great potential for biomedical applications.

Life before Stonehenge: The hunter-gatherer occupation and environment of Blick Mead revealed by sedaDNA, pollen and spores.

The Neolithic and Bronze Age construction and habitation of the Stonehenge Landscape has been extensively explored in previous research. However, little is known about the scale of pre-Neolithic activity and the extent to which the later monumental complex occupied an 'empty' landscape. There has been a long-running debate as to whether the monumental archaeology of Stonehenge was created in an uninhabited forested landscape or whether it was constructed in an already partly open area of pre-existing significance to late Mesolithic hunter-gatherers. This is of significance to a global discussion about the relationship between incoming farmers and indigenous hunter-gatherer societies that is highly relevant to both Old and New World archaeology. Here we present the results of plant sedaDNA, palynological and geoarchaeological analysis at the Late hunter-gatherer site complex of Blick Mead at the junction of the drylands of Salisbury Plain and the floodplain of the River Avon, on the edge of the Stonehenge World Heritage Site. The findings are placed within a chronological framework built on OSL, radiocarbon and relative archaeological dating. We show that Blick Mead existed in a clearing in deciduous woodland, exploited by aurochsen, deer and hunter-gatherers for approximately 4000 years. Given its rich archaeology and longevity this strongly supports the arguments of continuity between the Late Mesolithic hunter-gatherers activity and Neolithic monument builders, and more specifically that this was a partially open environment important to both groups. This study also demonstrates that sediments from low-energy floodplains can provide suitable samples for successful environmental assaying using sedaDNA, provided they are supported by secure dating and complementary environmental proxies.

Mechanically robust, rapidly actuating, and biologically functionalized macroporous poly(N-isopropylacrylamide)/silk hybrid hydrogels.

A route toward mechanically robust, rapidly actuating, and biologically functionalized polymeric actuators using macroporous soft materials is described. The materials were prepared by combining silk protein and a synthetic polymer (poly(N-isopropylacrylamide) (PNIAPPm)) to form interpenetrating network materials and macroporous structures by freeze-drying, with hundreds of micrometer diameter pores and exploiting the features of both polymers related to dynamic materials and structures. The chemically cross-linked PNIPAAm networks provided stimuli-responsive features, while the silk interpenetrating network formed by inducing protein ß-sheet crystallinity in situ for physical cross-links provided material robustness, improved expansion force, and enzymatic degradability. The macroporous hybrid hydrogels showed enhanced thermal-responsive properties in comparison to pure PNIPAAm hydrogels, nonporous silk/PNIPAAm hybrid hydrogels, and previously reported macroporous PNIPAAm hydrogels. These new systems reach near equilibrium sizes in shrunken/swollen states in less than 1 min, with the structural features providing improved actuation rates and stable oscillatory properties due to the macroporous transport and the mechanically robust silk network. Confocal images of the hydrated hydrogels around the lower critical solution temperature (LCST) revealed macropores that could be used to track changes in the real time morphology upon thermal stimulus. The material system transformed from a macroporous to a nonporous structure upon enzymatic degradation. To extend the utility of the system, an affinity platform for a switchable or tunable system was developed by immobilizing biotin and avidin on the macropore surfaces.

Synthesis of chitosan iodoacetamides via carbodiimide coupling reaction: Effect of degree of substitution on the hemostatic properties.

Uncontrolled hemorrhage continues to be the leading cause of death from traumatic injuries both in the battlefield and in the civilian life. Chitosan is among the very few materials that have made the short list of military recommended field-deployable hemostatic dressings. However, the detailed mechanism of its action is still not fully understood. Moreover, in the cases when patients developed coagulopathy, the efficacy of the dressings rely solely on those mechanisms that work outside of the regular blood coagulation cascade. In addition to the well-known erythrocyte agglutination, we proposed to use the reactive N-iodoacetyl group on a new chitosan derivative to accelerate hemostasis. In this paper, we describe the synthesis of chitosan iodoacetamide (CI) with considerations of the stoichiometry among the reagents, the choice of solvent, the pH of the reaction medium, and the reaction time. The reaction was confirmed by FT-IR, 1H and 13C NMR, elemental analysis, iodine content analysis, and SEM-EDS. Water contact angle measurements and Erythrocyte Sedimentation Rate (ESR) method were used to evaluate the hemostatic potential of the newly synthesized CI as a function of their degree of substitution (DS). The range of DS was 5.9% to 27.8% for CI. The mid-range of DS gave the best results for the ESR. CIs exhibit favorable cytocompatibilities up to DS 18.7 compared to the generic unmodified chitosan. In general, the biocompatibility of chitosan iodoacetamide slightly declined with increasing the iodide content up to DS 21.5 owing to its affinity to SH groups of cells.

Multi-layer dressing made of laminated electrospun nanowebs and cellulose-based adhesive for comprehensive wound care.

In this work, multi-layer wound dressing was made of laminated layers of electrospun fibers supported by adhesive sheet. Graft copolymerization of methyl methacrylate (MMA) and 2-Ethyl-1-hexyl acrylate (EHA) onto carboxymethyl cellulose (CMC) was conducted to obtain an adhesive sheet with 1.52 (N/cm2) loop tack, 1.7 (N/cm) peel strength and 25 s shear strength. Diclofenac sodium, anti-inflammatory drug, was loaded to the adhesive sheet with encapsulation efficiency 73%. The contact layer to wound was made of synthesized anti-bleeding agents, chitosan iodoacetamide (CI) loaded into electrospun polyvinyl alcohol (PVA) fibers. It was fabricated from fiber diameter 300 nm by electrospinning of 5% wt/v of CI (D.S. 18.7%) mixed with 10% wt/v PVA, at 20 kV and 17 cm airgap. The second, pain-relief layer was fabricated by encapsulating up to 50% wt/wt of capsaicin into gelatin nanofibers (197 nm) crosslinked by glyoxal. The third, antimicrobial layer was fabricated from PVA electrospun fibers loaded with 2% wt/wt gentamicin. Biocompatibility test showed insignificant adverse effects of the fabricated layers on fibroblast cells. Animal test on rat showed accelerated wound healing from 21 to 7 days for the multi-layer dressing. Histopathological findings corroborated the intactness of the epidermis layer of the treated samples.

Chitosan nanoparticles: Polyphosphates cross-linking and protein delivery properties.

Nanoparticles from chitosan, in general, can be prepared through ionotropic gelation, physical crosslinking, with anionic polyphosphates. Tripolyphosphate (TPP) is widely used for such purpose especially in drug delivery applications. TPP is a small ion with a triple negative charge throughout the physiologically acceptable pH range. However, the stability of size and surface charge of the particles still challenging. In the current work, the utilization of Hexametaphosphate (HMP) instead of Tripolyphosphate (TPP) as a cross-linking agent is being investigated. HMP is hexavalent molecule in the neutral and slightly basic medium which offers more binding sites readily available for interaction with chitosan. It is thought that increasing the availability of the binding sites in the HMP molecule would result in stronger ionic complexation with chitosan cationic charges. Consequently, such stronger binding improves particles' stability and lead to average size reduction. A comparative study between chitosan/TPP and chitosan/HMP nanoparticles under different complexation conditions was conducted to investigate the effect of HMP on nanoparticles formation. Bovine Serum Albumin (BSA) was applied as a protein model drug to explore the drug loading efficiency, 96.3%, is higher than its TPP, 91.87%, counterparts. However, TPP cross-linked particles showed superior stability upon storage.

Chitosan based hydrogels and their applications for drug delivery in wound dressings: A review.

Advanced development of chitosan hydrogels has led to new drug delivery systems that can release their active ingredients in response to environmental stimuli. This review considers more recent investigation of chitosan hydrogel preparations and the application of these preparations for drug delivery in wound dressings. Applications and structural characteristics of different types of active ingredients, such as growth factors, nanoparticles, nanostructures, and drug loaded chitosan hydrogels are summarized.

Fabrication and characterization of bactericidal thiol-chitosan and chitosan iodoacetamide nanofibres.

Two chitosan derivatives, namely, thiol-chitosan (TCs) and chitosan iodoacetamide (CsIA) were newly synthesized by reacting Cs with thiglycolic acid (TGA) and iodoacetic acid (IA) respectively. After being crosslinked with glutraldehyde (GA), the two derivatives were submitted to FT-IR and H1 NMR analysis for identification and characterization of their chemical features. As TCs and CsIA are water soluble, their electrospun nanofibres mats from aqueous solutions could be crosslinked and achieved using polyvinyl alcohol/Chitosan blend (PVA/Cs) polymers. Morphological structures of the obtained nanofibres and their webs were studied via those of TCs and CsIA free systems. The data also indicate that the crosslinked PVA/Cs/CsIA is more thermally stable than the crosslinked PVA/Cs/TCs and crosslinked PVA/Cs respectively. It was proved that the electrospun fibers containing TCs or CsIA display a superior antibacterial activity against negative bacteria E. Coli with a minimum inhibitory concentration (MIC) of 400µg/ml. These effects are rather in confirmation with bacterial kinetics essays which were also carried out in current work. Of particular interest is that the antimicrobial properties of fibers containing small concentration of either TCs or CsIA are much superior than those obtained with neat Cs electrospun nanofibres used as reference. By and large the results advocate the fibers webs containing TCs or CsIA as excellent candidates for wound dressing applications.

Formulation and characterization of a plasma sterilized, pharmaceutical grade chitosan powder.

Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was micronized using cryo-ball and cryo-jet milling and subsequently sterilized with nitrogen plasma. Micronized chitosan was characterized by laser diffraction, scanning electron microscopy (SEM), conductometric titration, viscometry, loss on drying, FTIR, and limulus amebocyte lysate (LAL) assays. Cryo-jet milling produced mean particle size of 16.05µm, 44% smaller than cryo-ball milling. Cryomilled chitosan demonstrated increased hygroscopicity, but reduced molecular weight and degree of deacetylation (DD). SEM imaging showed highly irregular shapes. FTIR showed changes consistent with reduced DD and an unexplained shift at 1100cm(-1). Plasma treated chitosan was sterile with <2.5EU/g after low-pressure plasma and <1.3EU/g after atmospheric pressure plasma treatment. Plasma treatment decreased the reduced viscosity of chitosan flake and powder, with a greater effect on powder. In conclusion, pharmaceutical grade, sterile chitosan powder was produced with cryo-jet milling and plasma sterilization.

Effect of beta-sheet crystals on the thermal and rheological behavior of protein-based hydrogels derived from gelatin and silk fibroin.

Novel protein-based hydrogels have been prepared by blending gelatin (G) with amorphous Bombyx mori silk fibroin (SF) and subsequently promoting the formation of beta-sheet crystals in SF upon exposure to methanol or methanol/water solutions. Differential scanning calorimetry of the resultant hydrogels confirms the presence and thermoreversibility of the G helix-coil transition between ambient and body temperature at high G concentrations. At low G concentrations, this transition is shifted to higher temperatures and becomes progressively less pronounced. Complementary dynamic rheological measurements reveal solid-liquid cross-over at the G helix-coil transition temperature typically between 30 and 36 degrees C in blends prior to the formation of beta-sheet crystals. Introducing the beta-sheet conformation in SF stabilizes the hydrogel network and extends the solid-like behavior of the hydrogels to elevated temperatures beyond body temperature with as little as 10 wt.-% SF. The temperature-dependent elastic modulus across the G helix-coil transition is reversible, indicating that the conformational change in G can be used in stabilized G/SF hydrogels to induce thermally triggered encapsulant release.

Synthesis and antimicrobial activity of a water-soluble chitosan derivative with a fiber-reactive group.

A novel fiber-reactive chitosan derivative was synthesized in two steps from a chitosan of low molecular weight and low degree of acetylation. First, a water-soluble chitosan derivative, N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC), was prepared by introducing quaternary ammonium salt groups on the amino groups of chitosan. This derivative was further modified by introducing functional (acrylamidomethyl) groups, which can form covalent bonds with cellulose under alkaline conditions, on the primary alcohol groups (C-6) of the chitosan backbone. The fiber-reactive chitosan derivative, O-acrylamidomethyl-HTCC (NMA-HTCC), showed complete bacterial reduction within 20 min at the concentration of 10ppm, when contacted with Staphylococcus aureus and Escherichia coli (1.5-2.5 x 10(5) colony forming units per milliliter [CFU/mL]).

Antimicrobial wound dressing nanofiber mats from multicomponent (chitosan/silver-NPs/polyvinyl alcohol) systems.

Novel hybrid nanomaterials have been developed for antimicrobial applications. Here we introduce a green route to produce antibacterial nanofiber mats loaded with silver nanoparticles (Ag-NPs, 25 nm diameter) enveloped in chitosan after reduction with glucose. The nanofiber mats were obtained from colloidal dispersions of chitosan-based Ag-NPs blended with polyvinyl alcohol. Nanofibers (150 nm average diameter and narrow size distribution) were obtained by electrospinning and cross-linked with glutaraldhyde. The effect of crosslinking on the release of silver was studied by atomic absorption spectroscopy. Antimicrobial activity was studied by the viable cell-counting; mats loaded with silver and control samples (chitosan/PVA) with different degrees of cross-linking were compared for their effectiveness in reducing or halting the growth of aerobic bacteria. The results showed superior properties and synergistic antibacterial effects by combining chitosan with Ag-NPs.

Kinetic properties and role of bacterial chitin deacetylase in the bioconversion of chitin to chitosan.

Chitin is an extremely insoluble material with very limited industrial use; however it can be deacetylated to soluble chitosan which has a wide range of applications. The enzymatic deacetylation of various chitin samples was investigated using the bacterial chitin deacetylase (CDA), which was partially purified from Alcaligenes sp. ATCC 55938 growth medium and the kinetic parameters of the enzyme were determined. Also, the efficiency of biocatalyst recycling by immobilization technique was examined. CDA activity reached its maximum (0.419 U/ml) after 18 h of bacterial cultivation. When glycol chitin was used as a substrate, the optimum pH of the enzyme was estimated to be 6 after checking a pH range between 3 and 9, while the optimum temperature was found to be 35°C. Addition of acetate (100 mM) in the assay mixture resulted in 50% loss of enzyme activity. The Km value of the enzyme is 1.6 × 10(-4) µM and Vmax is 24.7 µM/min. The average activity of CDA was 0.38 U/ml for both of immobilized and freely suspended cells after 18 h of bacterial growth. Some related patents are also discussed here.

Chitosan-coated poly(vinyl alcohol) nanofibers for wound dressings.

A PVA nanofibrous matrix was prepared by electrospinning an aqueous 10 wt % PVA solution. The mean diameter of the PVA nanofibers electrospun from the PVA aqueous solution was 240 nm. The water resistance of the as-spun PVA nanofibrous matrix was improved by physically crosslinking the PVA nanofibers by heat treatment at 150 degrees C for 10 min, which were found to be the optimal heat treatment conditions determined from chemical and morphological considerations. In addition, the heat-treated PVA (H-PVA) nanofibrous matrix was coated with a chitosan solution to construct biomimetic nanofibrous wound dressings. The chitosan-coated PVA (C-PVA) nanofibrous matrix showed less hydrophilic and better tensile properties than the H-PVA nanofibrous matrix. The effect of the chitosan coating on open wound healing in a mouse was examined. The C-PVA and H-PVA nanofibrous matrices showed faster wound healing than the control. The histological examination and mechanical stability revealed the C-PVA nanofibrous matrix to be more effective as a wound-healing accelerator in the early stages of wound healing than the H-PVA nanofibrous matrix.

Effect of silk fibroin interpenetrating networks on swelling/deswelling kinetics and rheological properties of poly(N-isopropylacrylamide) hydrogels.

Novel protein/synthetic polymer hybrid interpenetrating polymer networks (IPNs) of poly(N-isopropylacrylamide) (PNIPAAm) with Bombyx mori silk fibroin (SF) have been prepared by using methanol to postinduce SF crystallization. Those IPNs having the beta sheet crystalline structure of SF show improved storage and loss moduli. The IPN hydrogels show the same volume phase transition temperature and NaCl concentration as pure PNIPAAm hydrogels. The PNIPAAm/SF IPNs keep the swelling kinetics of PNIPAAm, while showing increased deswelling kinetics. The IPNs with SF beta sheet structure should decrease the formation of the skin layer observed in conventional PNIPAAm hydrogels. Therefore, the proposed IPN hydrogels composed of protein/polymer provide fast deswelling rates as well as improved mechanical properties over pure PNIPAAm hydrogels. The effect of SF beta sheet networks on the IPNs copolymerized with acrylic acid (AAc) (P(NIPAAm-co-AAc)/SF IPNs) is compared with that on the PNIPAAm/SF IPNs, and the parameters controlling the deswelling kinetics of the IPNs are investigated. Three parameters, (1) the skin layer formation, (2) the restriction of SF beta sheet networks, and (3) the aggregation force of NIPAAm chains, are cooperatively involved in the deswelling process of IPN hydrogels according to the SF content and the presence of the AAc moiety.

Mixed protein blends composed of gelatin and Bombyx mori silk fibroin: effects of solvent-induced crystallization and composition.

Novel protein blends have been prepared by mixing gelatin (G) with Bombyx mori silk fibroin (SF) and using aqueous methanol (MeOH) to post-induce SF crystallization. When co-cast from solution, amorphous blends of these polymers appear homogeneous, as discerned from visual observation, microscopy, and Fourier-transform infrared (FTIR) spectroscopy. Upon subsequent exposure to aqueous MeOH, SF undergoes a conformational change from random coil to beta sheet. This transformation occurs in pure SF, as well as in each of the G/SF blends, according to X-ray diffractometry and thermal calorimetry. The influence of MeOH-induced SF crystallization on structure and property development has been ascertained in terms of preparation history and blend composition. Thermal gravimetric analysis reveals that the presence of beta sheets in SF and G/SF blends improves thermal stability, while extensional rheometry confirms that SF crystallization enhances the tensile properties of the blends. By preserving a support scaffold above the G helix-to-coil transition temperature, the formation of crystalline SF networks in G/SF blends can be used to stabilize G-based hydrogels for biomaterial and pharmaceutical purposes. The present study not only examines the properties of G/SF blends before and after SF crystallization, but also establishes the foundation for future research into thermally responsive G/SF bioconjugates.

Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning.

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.

Structural study of irregular amino acid sequences in the heavy chain of Bombyx mori silk fibroin.

Recently, genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known from X-ray diffraction studies that the beta-sheet crystalline structure (silk II) of fibroin is composed of hexaamino acid sequences of GAGAGS. However, in the heavy chain of B. mori silk fibroin, there are also present 11 irregular sequences, with about 31 amino acid residues (irregular GT approximately GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized and its 3-D solution structure was studied by high-resolution 2-D NMR techniques. The 3-D structure determined for this peptide shows that it makes a loop structure (distorted omega shape), which implies that the preceding backbone direction is changed by 180 degrees, i.e., reversed, by this sequence. This may facilitate the beta-sheet formation between the crystal-forming building blocks, GAGAGS/GY approximately GY sequences, in the fibroin heavy chain.

Swelling behavior and morphological evolution of mixed gelatin/silk fibroin hydrogels.

Mixed protein-based hydrogels have been prepared by blending gelatin (G) with amorphous Bombyx mori silk fibroin (SF) and promoting beta-crystallization of SF via subsequent exposure to methanol or methanol/water solutions. The introduction of beta crystals in SF serves to stabilize the hydrogel network and extend the solidlike behavior of these thermally responsive materials to elevated temperatures beyond the helix-->coil (h-->c) transition of G. In this work, we investigate the swelling and protein release kinetics of G/SF hydrogels varying in composition at temperatures below and above the G h-->c transition. At 20 degrees C, G and G-rich mixed hydrogels display evidence of moderate swelling with negligible mass loss in aqueous solution, resulting in porous polymer matrixes upon solvent removal according to electron microscopy. When the solution temperature is increased beyond the G h-->c transition to body temperature (37 degrees C), these gels exhibit much higher swelling with considerable mass loss due to dissolution and release of G. The extent to which these properties respond to temperature decreases systematically with increasing SF content. The unique temperature- and composition-dependent properties of G/SF hydrogels dictate the efficacy of these novel materials as stimuli-responsive delivery vehicles.

Dissolution of Bombyx mori silk fibroin in the calcium nitrate tetrahydrate-methanol system and aspects of wet spinning of fibroin solution.

There are still several problems associated with the spinning of dialyzed silk fibroin solutions. In this work some of these problems have been examined. The calcium nitrate tetrahydrate-methanol system was used to dissolve the silk fibroin. A compositional phase diagram was constructed at various concentrations of the solvent system. Regenerated fibroin powders from undialyzed fibroin solution in several coagulants showed different conformations. Regenerated powders from several coagulants except methanol and ethanol were resoluble in water. Atomic absorption analysis revealed that the calcium cations strongly interact with fibroin molecules in dialyzed fibroin solution, which may interfere with the regeneration of a strong fiber. Kinetic studies to determine the diffusion coefficient of methanol into dialyzed and concentrated fibroin solution were reported. The properties of both original and regenerated fibroin such as solubility in water and thermal behaviors using DSC were compared. Regenerated fibroin fiber was spun by the wet spinning method. An X-ray diffractogram showed that the regeneration process decreased the crystallinity of regenerated fibroin fiber. SEM images of the surface and cross section of the regenerated fibroin fibers were discussed.