Straightforward determination of the degree of N-acetylation of chitosan by means of first-derivative UV spectrophotometry

First-derivative UV spectrophotometry is shown to be a reliable method for the determination of the degree of N-acetylation of chitosan samples. A mathematical expression is derived that allows to determine the DA directly from the mass concentration of a chitosan solution and the first derivative of its UV spectrum at 202 nm, thus eliminating the need for empiric correction curves for highly deacetylated samples. A procedure is proposed for the accurate mass determination of the hygroscopic chitosan. The proposed approach facilitates the routine determination of the DA, especially when using potent multiwell microplate readers, which allow hundreds of samples to be measured in just a few minutes

Poly(N-Isopropylacrylamide) surface-grafted ghitosan membranes as a new substrate for cell sheet engineering and manipulation

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosanmembranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance (1H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously describedmethodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.Contract grant sponsor: European NoE EXPERTISSUESContract grant number: NMP3-CT-2004-500283Contract grant sponsor: European UnionContract grant number: NMP3-CT-2003-50575

Transport of small anionic and neutral solutes through chitosan membranes: Dependence on cross-linking and chelation of divalent cations

Chitosan membranes were prepared by solvent casting and cross-linked with glutaraldehyde at several ratios under homogeneous conditions. The cross-linking degree, varying from 0 to 20%, is defined as the ratio between the total aldehyde groups and the amine groups of chitosan. Permeability experiments were conducted using a side-by-side diffusion cell to determine the flux of small molecules of similar size but with different chemical moieties, either ionized (benzoic acid, salicylic acid, and phthalic acid) or neutral (2-phenylethanol) at physiological pH. The permeability of the different model molecules revealed to be dependent on the affinity of those structurally similar molecules to chitosan. The permeability of the salicylate anion was significantly enhanced by the presence of metal cations commonly present in biological fluids, such as calcium and magnesium, but remained unchanged for the neutral 2-phenylethanol. This effect could be explained by the chelation of metal cations on the amine groups of chitosan, which increased the partition coefficient. The cross-linking degree was also correlated with the permeability and partition coefficient. The change in the permeation properties of chitosan to anionic solutes in the presence of these metallic cations is an important result and should be taken into consideration when trying to make in vitro predictions of the drug release from chitosan-based controlled release systems

Myoconductive and osteoinductive free-standing polysaccharide membranes

Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well-defined ∼50 μm thick polysaccharide membranes that could be easily manipulated, were mechanically resistant, and would enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via Fourier transform infrared spectroscopy and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over 1 month. The myogenic and osteogenic potentials of the membranes in vitro were assessed using BMP-2-responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded into and subsequently released from the membranes, depending on the crosslinking degree and the initial BMP-2 concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration.This work was financially supported by the Foundation for Science and Technology (FCT) through the scholarship SFRH/BPD/96797/2013, Fundo Social Europeu (FSE), and Programa Diferencial de Potencial Human (POPH) granted to Sofia G. Caridade. C.M. is indebted to the Association Francaise contre les Myopathies for financial support via a post-doctoral fellowship (AFM project 16673). J.A. acknowledges the Whitaker International Fellows and Scholars Program for support via a post-doctoral fellowship. This work was supported by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370 to C.P.) and by the AFM (grant Microtiss, 16530). We thank Isabelle Paintrand for her technical help with the confocal apparatus

Free-standing polyelectrolyte membranes made of chitosan and alginate

Free-standing films have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we prepared free-standing membranes by the layer-by-layer assembly of chitosan and alginate, two widely used biomaterials. Our aim was to produce a thick membrane and to study the permeation of model drugs and the adhesion of muscle cells. We first defined the optimal growth conditions in terms of pH and alginate concentration. The membranes could be easily detached from polystyrene or polypropylene substrate without any postprocessing step. The dry thickness was varied over a large range from 4 to 35 μm. A 2-fold swelling was observed by confocal microscopy when they were immersed in PBS. In addition, we quantified the permeation of model drugs (fluorescent dextrans) through the free-standing membrane, which depended on the dextran molecular weight. Finally, we showed that myoblast cells exhibited a preferential adhesion on the alginate-ending membrane as compared to the chitosan-ending membrane or to the substrate side.This work was financially supported by Foundation for Science and Technology (FCT) through the Scholarship SFRH/BD/64601/2009 granted to S.G.C. C.M. is indebted to Grenoble INP for financial support via a postdoctoral fellowship. This work was supported by the European Commission (FP7 Program) via a European Research Council starting grant (BIOMIM, GA 259370 to C.P.). C.P. is also grateful to Institut Universitaire de France and to Grenoble Institute of Technology for financial support. We thank Isabelle Paintrand for her technical help with the confocal apparatus and Patrick Chaudouet for his help with SEM imaging

Facilitating or disturbing? An investigation about the effects of auditory frequencies on prefrontal cortex activation and postural sway

Auditory stimulation activates brain areas associated with higher cognitive processes, like the prefrontal cortex (PFC), and plays a role in postural control regulation. However, the effects of specific frequency stimuli on upright posture maintenance and PFC activation patterns remain unknown. Therefore, the study aims at filling this gap. Twenty healthy adults performed static double- and single-leg stance tasks of 60s each under four auditory conditions: 500, 1000, 1500, and 2000 Hz, binaurally delivered through headphones, and in quiet condition. Functional near-infrared spectroscopy was used to measure PFC activation through changes in oxygenated hemoglobin concentration, while an inertial sensor (sealed at the L5 vertebra level) quantified postural sway parameters. Perceived discomfort and pleasantness were rated through a 0-100 visual analogue scale (VAS). Results showed that in both motor tasks, different PFC activation patterns were displayed at the different auditory frequencies and the postural performance worsened with auditory stimuli, compared to quiet conditions. VAS results showed that higher frequencies were considered more discomfortable than lower ones. Present data prove that specific sound frequencies play a significant role in cognitive resources recruitment and in the regulation of postural control. Furthermore, it supports the importance of exploring the relationship among tones, cortical activity, and posture, also considering possible applications with neurological populations and people with hearing dysfunctions

Chitosan Modification and Pharmaceutical/Biomedical Applications

Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Our recent efforts focused on the chemical and biological modification of chitosan in order to increase its solubility in aqueous solutions and absorbability in the in vivo system, thus for a better use of chitosan. This review summarizes chitosan modification and its pharmaceutical/biomedical applications based on our achievements as well as the domestic and overseas developments: (1) enzymatic preparation of low molecular weight chitosans/chitooligosaccharides with their hypocholesterolemic and immuno-modulating effects; (2) the effects of chitin, chitosan and their derivatives on blood hemostasis; and (3) synthesis of a non-toxic ion ligand—D-Glucosaminic acid from Oxidation of D-Glucosamine for cancer and diabetes therapy

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, Mw and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a "rolling ball" viscometer, respectively. The solution conformation of chitosan was then estimated from:(a)the Mark-Houwink-Kuhn-Sakurada (MHKS) power law relationship [η] = kMwa and(b)the persistence length, Lp calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464-2475]. Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (Lp = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales-van Holde ratio, the translational frictional ratio and sedimentation conformation zoning. © 2008 Elsevier Ltd. All rights reserved