Pure Chitosan Biomedical Textile Fibers from Mixtures of Low- and High-Molecular Weight Bidisperse Polymer Solutions: Processing and Understanding of Microstructure-Mechanical Properties' Relationship.

Natural polymers, as extracted from biomass, may exhibit large macromolecular polydispersity. We investigated the impact of low molar mass chitosan (LMW, DPw~115) on the properties of chitosan fibers obtained by wet spinning of chitosan solutions with bimodal distributions of molar masses. The fiber crystallinity index (CrI) was assessed by synchrotron X-ray diffraction and the mechanical properties were obtained by uniaxial tensile tests. The LMW chitosan showed to slightly increase the crystallinity index in films which were initially processed from the bimodal molar mass chitosan solutions, as a result of increased molecular mobility and possible crystal nucleating effects. Nevertheless, the CrI remained almost constant or slightly decreased in stretched fibers at increasing content of LMW chitosan in the bidisperse chitosan collodion. The ultimate mechanical properties of fibers were altered by the addition of LMW chitosan as a result of a decrease of entanglement density and chain orientation in the solid state. An increase of crystallinity might not be expected from LMW chitosan with a still relatively high degree of polymerization (DPw ≥ 115). Instead, different nucleation agents-either smaller molecules or nanoparticles-should be used to improve the mechanical properties of chitosan fibers for textile applications.

3-D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization.

Bone substitute fabrication is of interest to meet the worldwide incidence of bone disorders. Physical chitosan hydrogels with intertwined apatite particles were chosen to meet the bio-physical and mechanical properties required by a potential bone substitute. A set up for 3-D printing by robocasting was found adequate to fabricate scaffolds. Inks consisted of suspensions of calcium phosphate particles in chitosan acidic aqueous solution. The inks are shear-thinning and consist of a suspension of dispersed platelet aggregates of dicalcium phosphate dihydrate in a continuous chitosan phase. The rheological properties of the inks were studied, including their shear-thinning characteristics and yield stress. Scaffolds were printed in basic water/ethanol baths to induce transformation of chitosan-calcium phosphates suspension into physical hydrogel of chitosan mineralized with apatite. Scaffolds consisted of a chitosan polymeric matrix intertwined with poorly crystalline apatite particles. Results indicate that ink rheological properties could be tuned by controlling ink composition: in particular, more printable inks are obtained with higher chitosan concentration (0.19 mol·L-1).

Unraveling the Correlations between Conformation, Lubrication, and Chemical Stability of Bottlebrush Polymers at Interfaces.

In the present study, we monitored the conformation and chemical stability of a hydrophilic bottlebrush (BB) polymer in pure water and buffered saline solutions. We correlated these parameters to lubricating and wear protecting properties. Using the surface forces apparatus (SFA), we show that the BB polymer partially adsorbs on mica surfaces and extends half its contour length toward the aqueous media. This conformation gives rise to a strong repulsive interaction force when surfaces bearing BB polymer chains are pressed against each other. Analysis of these repulsive forces demonstrated that the adsorbed polymer chains could be described as end-attached elastic rods. After 2 months of aging at temperatures ranging from 4 to 37 °C, partial scission of the BB polymer's lateral chains was observed by gel permeation chromatography with a half-life time of the polymer of at least two years. The thickness of the BB polymer layer assessed by SFA appeared to quickly decrease with aging time and temperature, which was mainly caused by the adsorption to the substrate of the released lateral chains. The gradual loss of the BB polymer lateral chains did not significantly impact the tribological properties of the BB polymer solution nor its wear protection capacity. The friction coefficient between mica surfaces immersed in the BB polymer solution was µ = 0.031 ± 0.002, was independent of the aging conditions, and remained constant up to an applied pressure P = 0.2 to 0.25 MPa. Altogether, this study demonstrates that, besides the gradual loss of lateral chains, the BB polymer is still able to perform adequately as a lubricant and wear protecting agent over a time period suitable for in vivo administration.

Tuning the Hydrophilic/Hydrophobic Balance to Control the Structure of Chitosan Films and Their Protein Release Behavior.

The control over the crystallinity of chitosan and chitosan/ovalbumin films can be achieved via an appropriate balance of the hydrophilic/hydrophobic interactions during the film formation process, which then controls the release kinetics of ovalbumin. Chitosan films were prepared by solvent casting. The presence of the anhydrous allomorph can be viewed as a probe of the hydrophobic conditions at the neutralization step. The semicrystalline structure, the swelling behavior of the films, the protein/chitosan interactions, and the release behavior of the films were impacted by the DA and the film processing parameters. At low DAs, the chitosan films neutralized in the solid state corresponded to the most hydrophobic environment, inducing the crystallization of the anhydrous allomorph with and without protein. The most hydrophilic conditions, leading to the hydrated allomorph, corresponded to non-neutralized films for the highest DAs. For the non-neutralized chitosan acetate (amorphous) films, the swelling increased when the DA decreased, whereas for the neutralized chitosan films, the swelling decreased. The in vitro release of ovalbumin (model protein) from chitosan films was controlled by their swelling behavior. For fast swelling films (DA = 45%), a burst effect was observed. On the contrary, a lag time was evidenced for DA = 2.5% with a limited release of the protein. Furthermore, by blending chitosans (DA = 2.5% and 45%), the release behavior was improved by reducing the burst effect and the lag time. The secondary structure of ovalbumin was partially maintained in the solid state, and the ovalbumin was released under its native form.

Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation.

The formation of chitosan hydrogels without any external cross-linking agent was successfully achieved by inducing the gelation of a viscous chitosan solution with aqueous NaOH or gaseous NH3. The hydrogels produced from high molecular weight (Mw ≈ 640 000 g mol(-1)) and extensively deacetylated chitosan (DA ≈ 2.8%) at polymer concentrations above ∼2.0% exhibited improved mechanical properties due to the increase of the chain entanglements and intermolecular junctions. The results also show that the physicochemical and mechanical properties of chitosan hydrogels can be controlled by varying their polymer concentration and by controlling the gelation conditions, that is, by using different gelation routes. The biological evaluation of such hydrogels for regeneration of infarcted myocardium revealed that chitosan hydrogels prepared from 1.5% polymer solutions were perfectly incorporated onto the epicardial surface of the heart and presented partial degradation accompanied by mononuclear cell infiltration.

Enhancing radioprotection: A chitosan-based chelating polymer is a versatile radioprotective agent for prophylactic and therapeutic interventions against radionuclide contamination.

To mitigate the risk of radioactive isotope dissemination, the development of preventative and curative measures is of particular interest. For mass treatment, the developed solution must be easily administered, preferably orally, with effective, nontoxic decorporating properties against a wide range of radioactive isotopes. Currently, most orally administered chelation therapy products are quickly absorbed into the blood circulation, where chelation of the radioactive isotope is a race against time due to the short circulation half-life of the therapeutic. This report presents an alternative therapeutic approach by using a functionalized chitosan (chitosan@DOTAGA) with chelating properties that remains within the gastrointestinal tract and is eliminated in feces, that can protect against ingested radioactive isotopes. The polymer shows important in vitro chelation properties towards different metallic cations of importance, including (Cs(I), Ir(III), Th(IV), Tl(I), Sr(II), U(VI) and Co(II)), at different pH (from 1 to 7) representing the different environments in the gastrointestinal tract. An in vivo proof of concept is presented on a rodent model of uranium contamination following an oral administration of Chitosan@DOTAGA. The polymer partially prevents the accumulation of uranium within the kidneys (providing a protective effect) and completely prevents its uptake by the spleen.

Electrodeposition of a biopolymeric hydrogel: potential for one-step protein electroaddressing.

The electrodeposition of hydrogels provides a programmable means to assemble soft matter for various technological applications. We report an anodic method to deposit hydrogel films of the aminopolysaccharide chitosan. Evidence suggests the deposition mechanism involves the electrolysis of chloride to generate reactive chlorine species (e.g., HOCl) that partially oxidize chitosan to generate aldehydes that can couple covalently with amines (presumably through Schiff base linkages). Chitosan's anodic deposition is controllable spatially and temporally. Consistent with a covalent cross-linking mechanism, the deposited chitosan undergoes repeated swelling/deswelling in response to pH changes. Consistent with a covalent conjugation mechanism, proteins could be codeposited and retained within the chitosan film even after detergent washing. As a proof-of-concept, we electroaddressed glucose oxidase to a side-wall electrode of a microfabricated fluidic channel and demonstrated this enzyme could perform electrochemical biosensing functions. Thus, anodic chitosan deposition provides a reagentless, single-step method to electroaddress a stimuli-responsive and biofunctionalized hydrogel film.

Macrophage polarization in vitro and in vivo modified by contact with fragmented chitosan hydrogel.

We have previously shown that implantation of a fragmented chitosan hydrogel suspension (chitosan-FPHS) into a traumatic spinal cord lesion in adult rats led to significant axon regrowth and functional recovery, which was associated to a modulation of inflammation. Using an in vitro culture system, we show here that polarization of bone marrow-derived macrophages is indeed modified by direct contact with chitosan-FPHS. Reducing the degree of acetylation (DA) and raising the concentration of chitosan (Cp, from 1.5% to 3%), favors macrophage polarization toward anti-inflammatory subtypes. These latter also migrate and adhere efficiently on low, but not high DA chitosan-FPHS, both in vitro and in vivo, while inflammatory macrophages rarely invade a chitosan-FPHS implant in vivo, no matter the DA. Our in vitro model setup should prove a valuable tool for screening diverse biomaterial formulations and combinations thereof for their inflammatory potential prior to implantation in vivo.

Mechanisms involved during the ultrasonically induced depolymerization of chitosan: characterization and control.

The existence of two mechanisms involved in the ultrasonically induced depolymerization of chitosan is evidenced. The first leads to a rapid scission of polymer chains and a lowering of the polydispersity, and the second is responsible for obtaining short polymer chains and oligomers with a polydispersity increase. A systematic experimental study allowed us to identify and quantify the main parameters influencing the chain scission kinetics. Consequently, using a "master curve" approach, a general law of variation of the molecular weight during the depolymerization is proposed. This law can be used in various experimental conditions to easily control the production of chitosan chains of precise length and low polydispersity or a collection of chito-oligosaccharides (COS). Characterization of the latter by (1)H NMR and MALDI-TOF mass spectrometry shows their high purity and an unchanged primary structure.

Prilling and characterization of hydrogels and derived porous spheres from chitosan solutions with various organic acids.

This work emphazises the importance of the solubilizing conditions for the elaboration of chitosan hydrogel beads, which were produced using electromagnetic laminar jet breakup technology, resulting in dried porous beads by further freeze-drying. Paramaters such as the acid nature and concentration (acetic, formic, citric, lactic, maleic and malic, 0.1 to 0.5 mol·L-1), the chitosan concentration (2 to 5 wt%) and composition of the gelation bath (NaOH, with or without EtOH) were studied. Viscosity versus strain rate measurements were carried out on chitosan acidic solutions and the viscoelastic behaviour was studied on hydrogels. The solutions exhibiting the highest viscosities led to the stiffest macrohydrogels, as a result of chitosan carboxylate interactions. Specific surface areas of the freeze-dried beads were determined in the range from 12 to 107 m2·g-1. Their internal texture was observed by Scanning Electron Microscopy. Water uptake was also measured for further use in the field of water purification.

Cell and tissue responses at the interface with a chitosan hydrogel intended for vascular applications: in vitro and in vivo exploration.

AIMS: The need for small caliber vessels to treat cardiovascular diseases has grown. However, synthetic polymers perform poorly in small-diameter applications. Chitosan hydrogels can provide a novel biological scaffold for vascular engineering. The goal of this study was to explore host cell and tissue behavior at the interface with chitosan-based scaffolds in vitro and in vivo. METHODS AND RESULTS: in vitro, we assessed the ability of endothelial cells lining chitosan hydrogels to produce tissue factor (TF), thrombomodulin (TM) and nitric oxide. We showed that endothelial cells behave as a native endothelium since under stimulation, TF and TM expression increased and decreased, respectively. Endothelial cells seeded on chitosan produced nitric oxide, but no change was observed under stimulation. After in vivo subcutaneous implantation of chitosan hydrogels in rats, macrophage activation phenotypes, playing a crucial role in biomaterial/tissue, were explored by immunohistochemistry. Our results suggested a balance between pro- and anti-inflammatory signals since we observed an inflammatory response in favor of macrophage M2 phenotype. CONCLUSION: in vitro exploration of endothelial cell response at the interface with chitosan hydrogel showed a functional endothelium and in vivo exploration of tissue response revealed a biointegration of chitosan hydrogels.

Chitosan hydrogel micro-bio-devices with complex capillary patterns via reactive-diffusive self-assembly.

We present chitosan hydrogel microfluidic devices with self-assembled complex microcapillary patterns, conveniently formed by a diffusion-reaction process. These patterns in chitosan hydrogels are formed by a single-step procedure involving diffusion of a gelation agent into the polymer solution inside a microfluidic channel. By changing the channel geometry, it is demonstrated how to control capillary length, trajectory and branching. Diffusion of nanoparticles (NPs) in the capillary network is used as a model to effectively mimic the transport of nano-objects in vascularized tissues. Gold NPs diffusion is measured locally in the hydrogel chips, and during their two-step transport through the capillaries to the gel matrix and eventually to embedded cell clusters in the gel. In addition, the quantitative analyses reported in this study provide novel opportunities for theoretical investigation of capillary formation and propagation during diffusive gelation of biopolymers. STATEMENT OF SIGNIFICANCE: Hydrogel micropatterning is a challenging task, which is of interest in several biomedical applications. Creating the patterns through self assembly is highly beneficial, because of the accessible and practical preparation procedure. In this study, we introduced complex self-assembled capillary patterns in chitosan hydrogels using a microfluidic approach. To demonstrate the potential application of these capillary patterns, a vascularized hydrogel with microwells occupied by cells was produced, and the diffusion of gold nanoparticles travelling in the capillaries and diffusing in the gel were evaluated. This model mimics a simplified biological tissue, where nanomedicine has to travel through the vasculature, extravasate into and diffuse through the extracellular matrix and eventually reach targeted cells.

Chitosan Films for Microfluidic Studies of Single Bacteria and Perspectives for Antibiotic Susceptibility Testing.

Single-cell microfluidics is a powerful method to study bacteria and determine their susceptibility to antibiotic treatment. Glass treatment by adhesive molecules is a potential solution to immobilize bacterial cells and perform microscopy, but traditional cationic polymers such as polylysine deeply affect bacterial physiology. In this work, we chemically characterized a class of chitosan polymers for their biocompatibility when adsorbed to glass. Chitosan chains of known length and composition allowed growth of Escherichia coli cells without any deleterious effects on cell physiology. Combined with a machine learning approach, this method could measure the antibiotic susceptibility of a diversity of clinical strains in less than 1 h and with higher accuracy than current methods. Finally, chitosan polymers also supported growth of Klebsiella pneumoniae, another bacterial pathogen of clinical significance.IMPORTANCE Current microfluidic techniques are powerful to study bacteria and determine their response to antibiotic treatment, but they are currently limited by their complex manipulation. Chitosan films are fully biocompatible and could thus be a viable replacement for existing commercial devices that currently use polylysine. Thus, the low cost of chitosan slides and their simple implementation make them highly versatile for research as well as clinical use.

Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration.

OBJECTIVE: Regenerating a functional dental pulp in the pulpectomized root canal has been recently proposed as a novel therapeutic strategy in dentistry. To reach this goal, designing an appropriate scaffold able to prevent the growth of residual endodontic bacteria, while supporting dental pulp tissue neoformation, is needed. Our aim was to create an innovative cellularized fibrin hydrogel supplemented with chitosan to confer this hydrogel antibacterial property. METHODS: Several fibrin-chitosan formulations were first screened by rheological analyses, and the most appropriate for clinical use was then studied in terms of microstructure (by scanning electron microscopy), antimicrobial effect (analysis of Enterococcus fæcalis growth), dental pulp-mesenchymal stem/stromal cell (DP-MSC) viability and spreading after 7 days of culture (LiveDead® test), DP-MSC ultrastructure and extracellular matrix deposition (transmission electron microscopy), and DP-MSC proliferation and collagen production (RT-qPCR and immunohistochemistry). RESULTS: A formulation associating 10mg/mL fibrinogen and 0.5% (w/w), 40% degree of acetylation, medium molar mass chitosan was found to be relevant in order to forming a fibrin-chitosan hydrogel at cytocompatible pH (# 7.2). Comparative analysis of fibrin-alone and fibrin-chitosan hydrogels revealed a potent antibacterial effect of the chitosan in the fibrin network, and similar DP-MSC viability, fibroblast-like morphology, proliferation rate and type I/III collagen production capacity. SIGNIFICANCE: These results indicate that incorporating chitosan within a fibrin hydrogel would be beneficial to promote human DP tissue neoformation thanks to chitosan antibacterial effect and the absence of significant detrimental effect of chitosan on dental pulp cell morphology, viability, proliferation and collagenous matrix production.

Towards biocompatible vaccine delivery systems: interactions of colloidal PECs based on polysaccharides with HIV-1 p24 antigen.

This work reports on the interactions of a model protein (p24, the capside protein of HIV-1 virus) with colloids obtained from polyelectrolyte complexes (PECs) involving two polysaccharides: chitosan and dextran sulfate (DS). The PECs were elaborated by a one-shot addition of default amounts of one counterpart to the polymer in excess. Depending on the nature of the excess polyelectrolyte, the submicrometric colloid was either positively or negatively charged. HIV-1 capsid p24 protein was chosen as antigen, the ultrapure form, lipopolysaccharide-free (endotoxin-, vaccine grade) was used in most experiments, as the level of purity of the protein had a great impact on the immobilization process. p24 sorption kinetics, isotherms, and loading capacities were investigated for positively and negatively charged particles of chitosans and dextran sulfates differing in degrees of polymerization (DP) or acetylation (DA). Compared with the positive particles, negatively charged colloids had higher binding capacities, faster kinetics, and a better stability of the adsorbed p24. Capacities up to 600 mg x g(-1) (protein-colloid) were obtained, suggesting that the protein interacted within the shell of the particles. Small-angle X-rays scattering experiments confirmed this hypothesis. Finally, the immunogenicity of the p24-covered particles was assessed for vaccine purposes in mice. The antibody titers obtained with immobilized p24 was dose dependent and in the same range as for Freund's adjuvant, a gold standard for humoral responses.

Hyaluronan hydrogel: an appropriate three-dimensional model for evaluation of anticancer drug sensitivity.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. In order to investigate the sensitivity of cancer cells to antimitotic agents, we developed a cross-linked HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied three cell lines (SA87, NCI-H460 and H460M), from primary tumors and metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. Concurrently, we studied the growth of these cell lines in a usual monolayer culture system. In these two models, increasing concentrations of doxorubicin and 5-fluorouracil were evaluated for their ability to inhibit tumor cell growth and colony formation. Taken together, our data suggest that the cancer cells were more resistant in the three-dimensional model than in monolayer cell systems. The antimitotic drugs were efficient after 24h of treatment in the monolayer cultures, whereas they were significantly efficient only after one week of incubation in the HA hydrogels. Herein, we show that this cross-linked matrix provides a three-dimensional model particularly appropriate for investigating mechanisms involved in cancer cell line sensitivity to antimitotic drugs.

Physical chitosan microhydrogels as scaffolds for spinal cord injury restoration and axon regeneration.

Recovery from traumatic spinal cord injury (SCI) usually fails due to a cascade of cellular and molecular events that compromise neural tissue reconstitution by giving rise to glial scarring and cavity formation. We designed a scaffold material for SCI treatment containing only chitosan and water as fragmented physical hydrogel suspension (Chitosan-FPHS), with defined degree of acetylation (DA), polymer concentration, and mean fragment size. Implantation of Chitosan-FPHS alone into rat spinal cord immediately after a bilateral dorsal hemisection promoted reconstitution of spinal tissue and vasculature, and diminished fibrous glial scarring: with astrocyte processes primarily oriented towards the lesion, the border between lesion site and intact tissue became permissive for regrowth of numerous axons into, and for some even beyond the lesion site. Growing axons were myelinated or ensheathed by endogenous Schwann cells that migrated into the lesion site and whose survival was prolonged. Interestingly, Chitosan-FPHS also modulated the inflammatory response, and we suggest that this might contribute to tissue repair. Finally, this structural remodeling was associated with significant, long-lasting gain in locomotor function recovery. Because it effectively induces neural tissue repair, Chitosan-FPHS biomaterial may be a promising new approach to treat SCI, and a suitable substrate to combine with other strategies.

Synthesis of polymer latex particles decorated with organically-modified laponite clay platelets via emulsion polymerization.

We report a new route to colloidal nanocomposites consisting of polymer latex particles covered with Laponite clay nanoplatelets. These composite particles are prepared by seeded emulsion (co)polymerization of styrene and butyl acrylate from Laponite clay suspensions previously functionalized by ion exchange using either a free radical initiator: 2,2-azobis (2-methylpropionamidine) hydrochloride (AIBA) or a cationic vinyl monomer: 2-(methacryloyloxy) ethyl trimethyl ammonium chloride (MADQUAT). The successful intercalation of the cationic reactive molecules was confirmed by elemental analysis, FTIR, 13C solid-state NMR and WAXD. The organically-modified clays were dispersed into water with the help of tetrasodium pyrophosphate and an anionic surfactant. stable latexes, produced under different experimental conditions, were successfully obtained from the clay suspensions. Cryo-TEM images of the resulting latexes showed spherical composite particles with diameters in the 50-250 nm range with clay sheets located on their surface. This paper reports on the effect of the processing conditions on the particle morphology and latex stability, and describes the mechanism of formation of the nanocomposite particles.

Grafting of cellulose acetate with ionic liquids for biofuel purification by a membrane process: Influence of the cation.

A new strategy was developed for grafting ionic liquids (ILs) onto cellulose acetate in order to avoid IL extraction and improve its performance for ethyl tert-butyl ether (ETBE) biofuel purification by the pervaporation membrane process. This work extended the scope of IL-containing membranes to the challenging separation of organic liquid mixtures, in which these ILs were soluble. The ILs contained the same bromide anion and different cations with increasing polar feature. The membrane properties were strongly improved by IL grafting. Their analysis in terms of structure-property relationships revealed the influence of the IL content, chemical structure and chemical physical parameters α, ß, π* in the Kamlet-Taft polarity scale. The ammonium IL led to the best normalized flux of 0.182kg/m(2)h for a reference thickness of 5µm, a permeate ethanol content of 100% and an outstanding infinite separation factor for the azeotropic mixture EtOH/ETBE at 50°C.

Covalently-crosslinked mucin biopolymer hydrogels for sustained drug delivery.

The sustained delivery of both hydrophobic and hydrophilic drugs from hydrogels has remained a challenge requiring the design and scalable production of complex multifunctional synthetic polymers. Here, we demonstrate that mucin glycoproteins, the gel-forming constituents of native mucus, are suitable for assembly into robust hydrogels capable of facilitating the sustained release of hydrophobic and hydrophilic drugs. Covalently-crosslinked mucin hydrogels were generated via exposure of methacrylated mucin to ultraviolet light in the presence of a free radical photoinitiator. The hydrogels exhibited an elastic modulus similar to that of soft mammalian tissue and were sensitive to proteolytic degradation by pronase. Paclitaxel, a hydrophobic anti-cancer drug, and polymyxin B, a positively-charged hydrophilic antibacterial drug, were retained in the hydrogels and released linearly with time over seven days. After four weeks of drug release, the hydrogels continued to release sufficient amounts of active paclitaxel to reduce HeLa cell viability and sufficient amounts of active polymyxin B to prevent bacterial proliferation. Along with previously-established anti-inflammatory, anti-viral, and hydrocarbon-solubilizing properties of mucin, the results of this study establish mucin as a readily-available, chemically-versatile, naturally-biocompatible alternative to complex multifunctional synthetic polymers as building blocks in the design of biomaterials for sustained drug delivery.