Search

Developing synthetic materials able to mimic micro- and macrorheological properties of natural networks opens up to novel applications and concepts in materials science. The present contribution describes an active network based on a semi-synthetic polymer, a lactitol-bearing chitosan derivative (Chitlac), and a transient inorganic cross-linker, boric acid. Due to the many and diverse anchoring points for boric acid on the flanking groups of Chitlac, the cross-links constantly break and reform in a highly dynamic fashion. The consequence is a network with unusual non-equilibrium and mechanical properties closely resembling the rheological behavior of natural three-dimensional arrangements and of cytoskeleton. Concepts like network nucleation, reorganization and disassembly are declined in terms of amount of the cross-linker, which acts as a putative motor for remodeling of the network upon application of energy. The out-of-equilibrium and non-linear behavior render the semi-synthetic system of great interest for tissue engineering and for developing in-vitro mimics of natural active matrices.

The present paper explores the effect of boric acid on Chitlac, a lactose-modified chitosan which had previously shown interesting biological and physical-chemical features. The herewith-reported experimental evidences demonstrated that boric acid binds to Chitlac, producing conformational and association effects on the chitosan derivative. The thermodynamics of boric acid binding to Chitlac was explored by means of 11B NMR, circular dichroism (CD), and UV–vis spectroscopy, while macromolecular effects were investigated by means of viscometry and dynamic light scattering (DLS). The experimental results revealed a chain–chain association when limited amounts of boric acid were added to Chitlac. However, upon exceeding a critical boric acid limit dependent on the polysaccharide concentration, the soluble aggregates disentangle. The rheological behavior of Chitlac upon treatment with boric acid was explored showing a dilatant behavior in conditions of steady flow. An uncommonly high dependence in the scaling law between the zero-shear viscosity and the concentration of Chitlac was found, i.e., η0 ∝ CCTL5.8, pointing to interesting potential implications of the present system in biomaterials development.

Alginate-based membranes containing hyaluronic acid (HA) were manufactured by freeze-drying calcium-reticulated hydrogels. The study of the distribution of the two macromolecules within the hydrogel enabled to highlight a polymer demixing mechanism that tends to segregate HA in the external parts of the constructs. Resistance and pliability of the membranes were tuned, while release and degradation studies enabled to quantify the diffusion of both polysaccharides in physiological solution and to measure the viable lifetime of the membranes. Biological studies in vitro proved that the liquid extracts from the HA-containing membranes stimulate wound healing and that fibroblasts are able to colonize the membranes. Overall, such novel alginate-HA membranes represent a promising solution for several medical needs, in particular for wound treatment, giving the possibility to provide an in situ administration of HA from a resorbable device.

We report on a controlled process allowing for the gelation of a diol-rich chitosan-derivative named CTL (lactose-modified chitosan) in the presence of boric acid as the cross-linker. A two-step approach is described, namely (i) the mixing of CTL and boric acid at pH = 5, a condition where the inorganic component is mildly reactive; (ii) the addition of sodium bicarbonate (NaHCO3) as a trigger, allowing for the gradual and slow pH increase. The goal was to convert gradually the almost inert neutral boric acid into the much more reactive borate anion, the latter promoting the formation of borate esters with CTL diols. Gelling kinetics as well as mechanical behavior at small and large deformations was investigated by rheometry. CTL-boric acid gels behaved essentially as transient networks, hence continuously assembling and dissociating in a highly dynamic fashion. The present gelling mechanism preserves the strain-hardening behavior in the nonlinear region of stress-strain response, corroborating the already suggested potential applications of such gels as mimics of biological soft tissues.

The aim of the present contribution is twofold as it reports (i) on the role played by chitosan acetylation degree for the stability of nanoparticles (NPs) formed with hyaluronan and (ii) on the effect of the interaction of such NPs with immune cells. Chitosans with similar viscosity-average molecular weight, Mv, (i.e., 200 000) and different fractions of acetylated units (F A ) together with low-molecular-weight hyaluronan were chosen for developing a select library of formulations via electrostatic complex coacervation. The resulting NPs were analyzed in terms of size, polydispersity, surface charge, and stability in physiological-mimicked media by dynamic light scattering. Only medium acetylated chitosan (F A = 0.16) guaranteed the stability of NPs. To explore the effect of NPs interaction with immune cells, the release of proinflammatory cytokines and the reactive oxygen species production by human macrophages and neutrophils, respectively, were evaluated. Strikingly, a structure-function relationship emerged, showing that NPs made of chitosans with F A = 0.02, 0.25, 0.46, and 0.63 manifested a proinflammatory activity, linked to the instability of the system. Conversely, NPs made of chitosan with F A = 0.16 neither modified the functional response of macrophages nor that of neutrophils. Of note, such NPs were found to possess additional properties potentially advantageous in applications such as delivery of therapeutics to target inflamed sites: (i) they are devoid of cytotoxic effects, (ii) they avoid engulfment during the early stage of interaction with macrophages, and (iii) they are muco-adhesive, thereby providing for site-specificity and long-residence effects

A broad library of chitosans was produced varying the molecular weight and the fraction of acetylated units, FA. The produced chitosans were used for the formation of wall-to-wall cylindrical gels through a controlled external gelation using tripolyphosphate (TPP) as cross-linker. The resulting gels were analyzed by rheometry. Viscosity average degree of polymerization ( D P v ¯ ) > 152 was shown to be required for the formation of stable gels. Both gel stiffness and gel rupture strength were proportional to the molecular weight regardless of the applied deformation. Increasing acetylation produced a marked reduction of the shear modulus, but, in parallel, switched the networks from rigid and brittle to weak and elastic. Intriguingly, gels made of chitosan with FA = 0.37 displayed notable elasticity, i.e. up to 90% of applied strain falls into linear regime. These findings suggest that the frequency of glucosamine (D unit) and N-acetyl-glucosamine (A unit) contribute to a subtle structure-property relationship in chitosan-TPP gels.

Chronic non-healing wounds are a clinically important problem in terms of number of patients and costs. Wound dressings such as hydrogels, hydrocolloids, polyurethane films and foams are commonly used to manage these wounds since they tend to maintain a moist environment which is shown to accelerate re-epithelialization. The use of antibacterial compounds is important in the management of wound infections. A novel wound-dressing material based on a blended matrix of the polysaccharides alginate, hyaluronic acid and Chitlac-silver nanoparticles is here proposed and its application for wound healing is examined. The manufacturing approach to obtain membranes is based on gelling, foaming and freeze-casting of alginate, hyaluronic acid and Chitlac-silver nanoparticles mixtures using calcium ions as the cross-linking agent. Comprehensive evaluations of the morphology, swelling kinetics, permeability, mechanical characteristics, cytotoxicity, capability to inhibit metalloproteinases and of antibacterial property were conducted. Biological in vitro studies demonstrated that hyaluronic acid released by the membrane is able to stimulate the wound healing meanwhile the metal silver exploits an efficient antibacterial activity against both planktonic bacteria and biofilms. Overall, the experimental data evidence that the studied material could be used as antibacterial wound dressing for wound healing promotion.

Ionic chitosan gels fabricated using multivalent anions, tripolyphosphate (TPP) or pyrophosphate (PPi), respectively, have been investigated as potential biomaterials to be used in tissue engineering. Starting from the hypothesis that the polymer mesh texture at the microscale affects the final performance of the resulting materials, an innovative image analysis approach is presented in the first part of the article, which is aimed at deriving quantitative information from transmission electron microscopy images. The image analysis of the (more extended) central area of the gel networks revealed differences between both the cross-linking densities and pore size distributions of the two systems, the TPP gels showing a higher connectivity. Chitosan-TPP gels showed a limited degradation in simulated physiological media up to 6 weeks, reasonably ascribed to the texture of the (more extended) central area of the gels, whereas PPi counterparts degraded almost immediately. The release profiles and the calculation of diffusion coefficients for bovine serum albumin and cytochrome c, herein used as model payloads, indicated a different release behavior depending on the polymer network homogeneity/inhomogeneity and molecular weight of loaded molecules. This finding was ascribed to the marked inhomogeneity of the PPi gels (at variance with the TPP ones), which had been demonstrated in our previous work. Finally, thorough in vitro studies demonstrated good biocompatibility of both chitosan gels, and because of this feature, they can be used as suitable scaffolds for cellular colonization and metabolic activity.

The effect of transient cross-links has been explored on a lactose-modified chitosan, which previously had shown interesting biological features. The presence of galactose side chains and of the polyol spacer resulted particularly appealing for the reticulation by borate ions. The interaction between chitlac and borax was investigated by means of 11B NMR while rheology pointed to a marked non-linear behavior depending on the amount of borax added to the system. The presence of limited amount of cross-linking ion led to dilatant behavior when the steady flow curve was measured. In addition, strain stiffening was noticed on elastic response upon exceeding a critical stress, indicating a transient nature in the formation of the cross-links. The non-linear response of chitlac in the presence of borax compared surprisingly well with the one showed by proteins composing the natural ECM pointing at a possible role of mechanotransduction in the biological significance of the modified chitosan.

Complex coacervation of two oppositely charged polysaccharides, namely a lactose-modified chitosan (CTL) and hyaluronan (HA), was investigated in this study. Coacervates of the two polysaccharides were prepared by drop-by-drop injection of HA into CTL. Transmittance and dynamic light scattering (DLS) measurements in combination with TEM analyses demonstrated the formation of spheroidal colloids in the nano-/microsize range showing good homogeneity. Strikingly, the presence of 150 mM supporting NaCl did not hamper the colloid formation. Stability studies on selected formulations demonstrated that HA/CTL coacervates were stable up to 3 weeks at 37 °C and behaved as pH-responsive colloids since transition from entangled to disentangled chains was attained for a proper pH range. The possibility of freeze-drying the coacervates for storage purposes and the ability of encapsulating selected payloads were investigated as well, for two values of the fraction of the lactitol side-chain substitution (FL). Finally, biological tests using human neutrophils were undertaken at acidic pH value (pH = 6.0): under such experimental conditions, akin to those frequently occurring in the inflammatory microenvironment, coacervates scavenged reactive oxygen species (ROS) generated by these cells in basal conditions. Given the well documented bioactivity of CTL with respect to chitosan toward cartilage regeneration, these findings point to a possible application of HA/CTL-based colloids as scavenging and bioactive carriers for the delivery of therapeutic molecules at confined inflamed sites such as knee joints.

Chitosan macro- and micro/nano-gels have gained increasing attention in recent years, especially in the biomedical field, given the well-documented low toxicity, degradability, and non-immunogenicity of this unique biopolymer. In this review we aim at recapitulating the recent gelling concepts for developing chitosan-based physical gels. Specifically, we describe how nowadays it is relatively simple to prepare networks endowed with different sizes and shapes simply by exploiting physical interactions, namely (i) hydrophobic effects and hydrogen bonds—mostly governed by chitosan chemical composition—and (ii) electrostatic interactions, mainly ensured by physical/chemical chitosan features, such as the degree of acetylation and molecular weight, and external parameters, such as pH and ionic strength. Particular emphasis is dedicated to potential applications of this set of materials, especially in tissue engineering and drug delivery sectors. Lastly, we report on chitosan derivatives and their ability to form gels. Additionally, we discuss the recent findings on a lactose-modified chitosan named Chitlac, which has proved to form attractive gels both at the macro- and at the nano-scale.

The present contribution aims at describing the fabrication of coacervates in the nano-size range starting from a 1-deoxylactit-1-yl chitosan (in this manuscript termed as CTL60) and the multivalent anion tripolyphosphate (TPP). Colloidal coacervates have been obtained for precise values of the molar ratio of TPP to CTL60 repeating unit. Coacervation is ensured only at pH 4.5 and not at 7.4, thus demonstrating the key role of electrostatic interactions in the stabilization of the coacervates. At a variance with chitosan, CTL60 favors the formation of highly homogeneous coacervates with very low values of the polydispersity index (PDI). Moreover, CTL60 coacervates can be freeze-dried without any cryoprotectant, they can host a model molecule and are stable up to three weeks at 4 °C. Conversely, such coacervates dissolve upon increasing pH and ionic strength. By considering the bioactive polycation CTL60, the present system can be suggested as a first step in the development of innovative biologically-active nano-carriers to be used as drug delivery systems

Polysaccharide networks, in the form of hydrogels and dried membranes based on chitosan and on the cross-linker tripolyphosphate (TPP), were developed using a novel approach. TPP was incorporated into chitosan by slow diffusion to favor a controlled gelation. By varying chitosan, TPP, and NaCl concentration, transition from inhomogeneous to homogeneous systems was achieved. Rheology and uniaxial compression tests enabled to identify the best performing hydrogel composition with respect to mechanical properties. FTIR, (31)P NMR, and spectrophotometric methods were used to investigate the interaction chitosan-TPP, the kinetics of phosphates diffusion during the dialysis and the amount of TPP in the hydrogel. A freeze-drying procedure enabled the preparation of soft pliable membranes. The lactate dehydrogenase assay demonstrated the biocompatibility of the membranes toward fibroblasts. Overall, we devised a novel approach to prepare homogeneous macroscopic chitosan/TPP-based biomaterials with tunable mechanical properties and good biocompatibility that show good potential as novel polysaccharide derivatives.

In this study nano-composite scaffolds to be used as bone grafts have been endowed with antibacterial properties owing to the presence of silver nanoparticles. The alginate/hydroxyapatite composite scaffolds were prepared by internal gelation followed by a freeze-drying procedure to obtain a porous structure. The nanoparticles were prepared in presence of a lactose modified-chitosan and this colloidal solution was adsorbed on the scaffolds by exploiting electrostatic interactions. The adsorption and release of the silver from the composite scaffold was measured by ICP-AES and spectrofluorimetry measurements. Micro-computed tomography analysis of the scaffolds showed a homogeneous porous structure with average pore sizes of 341.5 μm and porosity of 80 %. In vitro biological tests (MTS and killing kinetics assays) demonstrated that silver does not affect the ability of the scaffolds to promote osteoblasts proliferation and that at the same time it exerts a strong bactericidal effect against both Gram+ and Gram- bacterial strains. Overall, the combined results indicate that these biocompatible antimicrobial scaffolds possess ideal characteristics for tissue engineering applications.

Sterilization methods such as gamma-irradiation, steam sterilization and ethylene oxide gas treatment can have negative effects on molecular structure and properties of polysaccharide-based biomaterials. In this perspective, the use of supercritical carbon dioxide (scCO(2)) has been proposed as an alternative method for biomaterial sterilization. In this work, chemical, mechanical and biological properties of polysaccharidic membranes for surgical applications were investigated after sterilization by scCO(2). Four sets of sterilizing conditions were considered and SEC analyses were performed in order to identify the one with lower impact on the polysaccharidic matrix of membranes (alginate). Mechanical tests showed that the resistance of membranes was slightly affected after sterilization. Biological analyses proved the biocompatibility of the sterilized membranes both in vitro and in a preliminary in vivo test. Overall, this study points out that this sterilization technique can be successfully employed to achieve an effective and safe sterilization of polysaccharidic membranes for surgical use. (C) 2017 Elsevier Ltd. All rights reserved.

Tissue damage caused by excessive amounts of neutrophil-derived reactive oxygen species (ROS) occurs in many inflammatory diseases. Butyrate is a short-chain fatty acid (SCFA) with known anti-inflammatory properties, able to modulate several neutrophil functions. Evidence is provided here that butyrate inhibits neutrophil ROS release in a dose and time-dependent fashion. Given the short half-life of butyrate, chitosan/hyaluronan nanoparticles are next designed and developed as controlled release carriers able to provide cells with a long-lasting supply of this SCFA. Notably, while the inhibition of neutrophil ROS production by free butyrate declines over time, that of butyrate-loaded chitosan/hyaluronan nanoparticles (B-NPs) is sustained. Additional valuable features of these nanoparticles are inherent ROS scavenger activity, resistance to cell internalization, and mucoadhesiveness. B-NPs appear as promising tools to limit ROS-dependent tissue injury during inflammation. Particularly, by virtue of their mucoadhesiveness, B-NPs administered by enema can be effective in the treatment of inflammatory bowel diseases.

Iodinated contrast media (ICM) are persistent and ubiquitous water pollutants. Because of their high water solubility and biochemical stability, their phase-separation and recovery from the aquatic environment is very difficult. Here, borate was chosen as a complexing agent of the two diagnostic aids iomeprol and iopamidol in order to provide them with a negative charge and to fix the resulting adducts on Dowex 1X4 ion exchangers. A systematic characterization study of the complex by means of capillary zone electrophoresis and 11B NMR revealed that iomeprol and iopamidol interact with borate anions in aqueous solutions giving a 1:1 single-charged adduct and that the association constant at 25 °C for both contrast agents is highest at pH 10.5. These findings allowed the proper calibration of experimental parameters for further batch adsorption–desorption trials, where the two ICM were shown to be almost completely removed from the water phase and released from the solid sorbents in mild conditions, enabling the recovery of functional resin.

Ionotropic gelation of chitosan by means of opposite charged ions represents an efficient alternative to covalent reticulation because of milder condition of use and, in general, higher biocompatibility of the resulting systems. In this work 90° light scattering (turbidimetry), circular dichroism (CD) and 1H NMR measurements have been performed to study the interactions between the biopolymer and ionic cross-linkers tripolyphosphate (TPP) and pyrophosphate (PPi) in dilute solutions. Thereafter, a dialysis-based technique was exploited to fabricate tridimensional chitosan hydrogels based on both polyanions. Resulting matrices showed a different mechanical behavior because of their peculiar mesh-texture at micro/nano-scale: in the present contribution we demonstrate that TPP and PPi favor the formation of homogeneous and inhomogeneous systems, respectively. The different texture of networks could be exploited in future for the preparation of systems for the controlled release of molecules.

In this study we present a novel silver complex of hyaluronan-lipoate (SHLS12) in a gel-state form. NMR analysis, conductometry and elemental analysis demonstrated stable non-covalent interactions between silver ions and the polysaccharide-lipoate backbone, whereas rheological investigations confirmed its gel-like physical-chemical behavior. Biological studies showed the ability of SHLS12 to exert a straightforward activity against different bacterial strains grown in sessile/planktonic state. The biocompatibility was also proved toward two eukaryotic cell lines. By considering both its ability to preserve antibacterial properties when exposed to the serum protein BSA and its low susceptibility to be degraded by hyaluronidase enzyme, this novel complex may be considered as a promising biomaterial for future in vivo applications.

In this work, hydrogen peroxide (H2O2) was employed to improve the adhesion of an alginate-based membrane to intestinal serosa: mechanical adhesion test and SEC-MALLS studies were performed to investigate the contribution of H2O2-induced modifications of both tissue and membrane to the adhesion enhancement. Adhesion tests confirmed that the formation of an adhesive gelatin layer on serosa plays a major role in increasing adhesion forces, while SEC-MALLS studies revealed a slight decrease of alginate molecular weight. However, the latter chain degradation does not seem to significantly impair the adhesion strength. Overall, this study describes a possible strategy to create adhesive interfaces between biomaterials and collagen-containing tissues.

Treatment of non-healing wounds represents hitherto a severe dilemma because of their failure to heal caused by repeated tissue insults, bacteria contamination and altered physiological condition. This leads to face huge costs for the healthcare worldwide. To this end, the development of innovative biomaterials capable of preventing bacterial infection, of draining exudates and of favoring wound healing is very challenging. In this study, we exploit a novel technique based on the slow diffusion of tripolyphosphate for the preparation of macroscopic chitosan hydrogels to obtain soft pliable membranes which include antimicrobial silver nanoparticles (AgNPs) stabilized by a lactose-modified chitosan (Chitlac). UV-Vis and TEM analyses demonstrated the time stability and the uniform distribution of AgNPs in the gelling mixture, while swelling studies indicated the hydrophilic behavior of membrane. A thorough investigation on bactericidal properties of the material pointed out the synergistic activity of chitosan and AgNPs to reduce the growth of S. aureus, E. coli, S. epidermidis, P. aeruginosa strains and to break apart mature biofilms. Finally, biocompatibility assays on keratinocytes and fibroblasts did not prove any harmful effects on the viability of cells. This novel technique enables the production of bioactive membranes with great potential for the treatment of non-healing wounds.

The theoretical model devised in the previous paper (Donati, I.; Benegas, J. C.; Cesàro, A.; Paoletti, S. Biomacromolecules 2006, 7 (5), 1587-1596) for the description of ion-induced chain aggregation is here applied to the case of chain dimerization of poly(galacturonate) in the presence of calcium ions. Particular attention has been directed toward the initial stage of dimer formation [i.e., in the low regime of calcium-to-polymer ratio (Rj)]. Circular dichroism (CD) data allowed evaluation of the fraction, theta, of calcium ions bound within chain dimers according to the "egg-box"-model. The theoretical model was able to reproduce satisfactorily the total molar enthalpy variation experimentally determined; the contributions of affinity (specificity in territorial condensation) and chemical bonding of calcium counterions to the thermodynamic properties of the system (i.e., enthalpy and entropy) were calculated. The intrinsic molar enthalpy of bonding, DeltaH(bond,0), displayed a peculiar sigmoid dependence on Rj. In particular, its decrease toward more negative values was interpreted as stemming from a (cooperative) calcium-induced conformational change that accompanies pectate chain pairing upon junction formation. The calculated pKin of instability of the Ca-(GalA-)4 complex was 10.80, in very good agreement with the corresponding value reported for the Ca-EDTA complex (i.e., 10.96). Significant contributions to the complex stability were the enthalpy of ion pairing (DeltaH(ionpairing,bond) = -5.1 kcal (mol calcium)-1, in good agreement with the value reported for calcium-EDTA: approximately -5.4 kcal (mol calcium)-1), and the entropy of desolvation (DeltaS(desolv,bond) = 43.7 cal mol-1 K-1, well within the range of values reported for calcium-EDTA: 42-57 cal mol-1 K-1).

Evidences for the involvement of the Galectin-1 in the interaction of pig chondrocytes with a lactose-modified chitosan, namely Chitlac, are reported. The Chitlac glycopolymer has been shown to promote pig chondrocyte aggregation and to induce extracellular matrix production. Highly pure Galectin-1 was obtained from pig spleen by affinity chromatography and its identity was determined by ion spray mass spectrometry analysis of tryptic peptide fragments obtained after in-gel digestion. The complete sequence of pig Galectin-1 CDS was obtained by screening a pig EST database using human Galectin-1 sequence as template. The Galectin-1 cDNA was cloned into a pGEX-4T-1 expression vector and the recombinant protein was purified, characterized and used to produce a rabbit anti-serum. Recombinant Galectin-1 interacts in a dose-dependent manner with Chitlac as determined with ELISA assay. Expression level of galectin-1 gene, quantified by real-time PCR, was significantly higher in chondrocytes cultivated on Chitlac. In the same way, the presence of Chitlac stimulates secretion of Galectin-1 in culture medium that, by immunohistochemical analysis, revealed to be clustered on the surface of Chitlac-induced aggregates. These data indicate the role of Galectin-1 as a bridging agent between Chitlac and chondrocyte aggregates.

Objectives The aim of this study was to determine the in vitro microbiological performances of a lactose-modified chitosan (Chitlac) coating inside which silver nanoparticles were embedded (Chitlac-nAg) for BisGMA/TEGDMA methacrylic specimens. Methods Different concentrations of nAg inside Chitlac coating were tested (1 mM, 2 mM, 5 mM). Specimen surface was analyzed by means of field-emission scanning electron microscopy (FEISEM) and energy-dispersive X-ray spectroscopy (EDS). A 48 h monospecific Streptococcus mutans biofilm was developed over the specimen surfaces using a modified drip-flow bioreactor; adherent viable biomass was assessed by MTT test and biofilm was imaged by confocal laser-scanning microscopy (CLSM). Results The presence of finely dispersed nanoparticles inside the Chitlac coating was confirmed by FEISEM and EDS analysis. All nanoparticles were embedded in the Chitlac coating layer. Chitlac-nAg coatings were able to significantly decrease biofilm formation depending on the nAg concentration, reaching a −80% viable biomass decrease when the 5 mM nAg-Chitlac group was confronted to non-coated control specimens. CLSM analysis did not provide evidence of a contact-killing activity, however the antibacterial Chitlac-nAg coating was able to alter biofilm morphology preventing the development of mature biofilm structures. Conclusions The microbiological model applied in this study helped in assessing the antibacterial properties of a coating designed for methacrylate surfaces. Clinical significance A microbiological model based on a bioreactor-grown biofilm is useful for preliminary in vitro tests of dental materials. In translational terms, an antibacterial nanocomposite coating based on Chitlac-nAg and designed to be applied to methacrylic surfaces may be a promising way to obtain dental materials able to actively prevent secondary caries.

Light scattering (LS) investigations have additionally confirmed the intramolecular nature of the fundamental ordered conformation (single helix) of κ-carrageenan in aqueous 0.1 M NaI. The partial replacement of sodium with cesium ions induces a (thermoreversible) intermolecular association of the polysaccharide in the ordered conformation. The association-inducing effect can be observed already at the lowest investigated concentration of the cation. We found no significant change of the thermodynamic properties related to an alleged composition threshold value in the range of the cesium equivalent fraction, X Cs , from 0.12 to 0.40. The extent and the rate of the association process strongly increase with the concentration of cesium. Equilibrium values for the association constant could be obtained except for the case of X Cs = 0.40, where the process is too massive to allow long-time LS investigation. In the latter condition, some additional stiffening of the chain was noticed.

The synthesis of some glycosylamines (1-amino-1-deoxy D-glucose, 1-amino-1-deoxy-D-galactose and 1-amino-1-deoxylactose) was carried out by treatment of the corresponding reducing sugars with ammonium hydrogencarbonate in concentrated ammonia. The reaction mixture was first analyzed by capillary electrophoresis with indirect absorbance detection and high performance anion-exchange chromatography with pulsed amperometric detection. Beside glycosylcarbamate, a known reaction by-product, fructose and lactulose were detected during the synthesis of 1-amino-1-deoxyglucose and 1-amino-1-deoxylactose, respectively. Quantification of glycosylamines was carried out by micellar electrokinetic chromatography with UV detection of their 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives; lactulosylamine was thus detected in the synthesis mixture of 1-amino-1-deoxylactose. The Fmocglycosylamines were easily purified from the other components of the crude synthesis mixtures.

The temperature-induced helix-to-coil conformational transition accompanying the thermoreversible gelation of syndiotactic poly(methyl methacrylate) in toluene was studied by rheology and FTIR and 1H NMR spectroscopy. Both rheological and FTIR measurements, carried out dynamically as a function of temperature, revealed a marked hysteresis between the heating and the cooling cycles. It was not possible to assess a quantitative difference between the values of the temperature of onset of the physical variations between the rheological and the spectroscopic experiments. It is then possible to conclude that a conformational change of the backbone (from the evolution of the band at 860 cm-1) is the molecular basis of the macroscopic formation of the gel. Spin−spin relaxation time, or T2, measurements were used in the 1H NMR study. The data obtained from the relaxation behavior of the α-methyl group, indicated a fraction of 0.28 for the chain repeating units incorporated in highly cooperative helical stretches,...

We examined the effect as donors of three aryl β-D-galactosides (i.e. p-nitrophenyl β-D-galactopyranoside, o-nitrophenyl β-D-galactopyranoside and phenyl β-D-galacto-pyranoside) on the regioselectivity and the yield of the synthesis of N-acetyllactosamine obtained from the transglycosylation reaction catalyzed by a crude preparation of β-D-galactosidase from Bacillus circulans at 25 °C, 37 °C and 55 °C, respectively. Using p-nitrophenyl β-D-galactopyranoside the reaction results were fully regiospecific at all the temperatures considered: the maximum molar yield (74%) was obtained at an incubation temperature of 55 °C. Using o-nitrophenyl β-D-galactopyranoside as the donor the reaction was still highly regioselective and the maximum molar yield (50%) was achieved at an incubation temperature also of 55 °C. Using phenyl β-D-galactopyranoside transglycolytic products appear only at an incubation temperature of 55 °C but at very low molar yield (about 14%) and lower regioselectivity.

The synthesis of the disaccharide 6-O-beta-D-xylopyranosyl-2-acetamido-2-deoxy-D-glucopyranose (N-acetylprimeverosamine), structurally related to the natural disaccharide 6-O-beta-D-xylopyranosyl-D-glycopyranose (primeverose), was obtained via a transglycosylation reaction catalyzed by a crude preparation of beta-D-xylosidase from Aspergillus niger, using p-nitrophenyl beta-D-xylopyranoside as the donor and 2-acetamido-2-deoxy-D-glucopyranose as the acceptor. The yield of the reaction was 36% on a molar basis with respect to the donor. The chemical identity of the product was assessed by HPLC, ionspray mass spectrometry and NMR spectroscopy.

The reaction of hyaluronic acid benzyl ester derivative (1) with trimethyl orthoacetate in the presence of an acid catalyst in N,N-dimethylformamide gave the expected 4,6-orthoester 2, along with its hydrolysed products the 6-O- (3) and the 4-O- (4) acetate hyaluronic acid benzyl esters as minor components. Acid hydrolysis of 2 followed by O-4→O-6 acetyl migration using t-butylamine afforded a mixture which contained the 6-acetate 3 as the major and the 4-acetate 4 as the minor compound. Synthesis of a mixture of 2′,3′,6- (6) and 2′,3′,4- (7) tri-acetates was achieved by peracetylation of 2 followed by treatment with aqueous acetic acid. Compound 1 on treatment with 2,2-dimethoxypropane in dimethyl sulfoxide in the presence of p-toluenesulfonic acid afforded the corresponding 4,6-O-isopropylidene derivative 8. The 2′,3′-di-O-acetate 10 was obtained from 8 by conventional acetylation followed by de-acetylation using trifluoroacetic acid. Acetylation of 1 with acetic anhydride in a mixture of N,N-dimethylformamide and pyridine gave the expected 2′,3′,4,6-tetraacetate derivative 11. In order to facilitate the NMR assignments of the acetate groups in 3–11, the 2′,3′-diacetate-4,6-di(trideuterioacetate) 12 and the 2′,3′-di(trideuterioacetate)-4,6-diacetate 13 were prepared. The effect of substituents on conformational changes around the glycosidic linkages has been investigated.

A transglycosylation reaction, carried out in an aqueous medium and catalyzed by a crude preparation of alpha-fucosidase (E.C. 3.2.1.51) from Aspergillus niger, allowed for the regiospecific synthesis of the disaccharide 3-O-alpha-L-fucopyranosyl-2-acetamido-2-deoxy-D-glucopyranose using p-nitrophenyl-alpha-L-fucopyranose as the donor and 2-acetamido-2-deoxy-D-glucopyranose as the acceptor. The chemical identity of the product obtained was demonstrated by HPLC, ion spray mass spectrometry and NMR spectroscopy. Further transglycosylation using a beta-galactosidase (E.C. 3.2.1.23) yielded the branched oligosaccharide 2-acetamido-2-deoxy-3-O-(alpha-L-fucopyranosyl)-4-O-(beta-D-galactopyran osyl)-D-glucopyranose, i.e., the Lewis(x) antigen.

Chitlac is a biocompatible modified polysaccharide composed of a chitosan backbone to which lactitol moieties have been chemically inserted via a reductive N-alkylation reaction with lactose. The physical-chemical and biological properties of Chitlac that have been already reported in the literature suggest a high accessibility of terminal galactose in the lactitol side chain. This finding may account for its biocompatibility which makes it extremely interesting for the production of biomaterials. The average structure and the dynamics of the side chains of Chitlac have been studied by means of NMR (nuclear Overhauser effect and nuclear relaxation) and molecular dynamics to ascertain this hypothesis. A complete assignment of the (1)H and (13)C NMR signals of the modified polysaccharide has been accomplished together with the determination of the apparent pKa values of the primary and secondary amines (6.69 and 5.87, respectively). NMR and MD indicated a high mobility of Chitlac side chains with comparable average internuclear distances between the two techniques. It was found that the highly flexible lactitol side chain in Chitlac can adopt two distinct conformations differing in the orientation with respect to the polysaccharide chain: a folded conformation, with the galactose ring parallel to the main chain, and an extended conformation, where the lactitol points away from the chitosan backbone. In both cases, the side chain resulted to be highly hydrated and fully immersed in the solvent.

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1H and 31P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1H and 31P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1H and 31P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3JPH and 4JPH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90 degrees Gaussian pulse on the 31P channel was introduced to increase the resolution in the F1-domain and make the method even faster. The procedure was then applied to unambiguously assign the ID 31P and 1H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31P and 1H methods lead to different results is discussed, and the use of 1H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31P spectrum is proposed.

Within this study we analyzed the degradation resistance of Chitlac both in solution and as a coating film in aqueous media upon exposure to the enzymatic attack of lysozyme or to the oxidant effect of H2O2. Possible coating degradation in such conditions was evaluated by measuring the amount of both polysaccharide and silver released in solution.

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are biomaterials commonly employed for orthopedic and dental applications; for both these fields, bacterial adhesion to the surface of the implant represents a major issue for the outcome of the surgical procedures. In this study, the antimicrobial properties of a nanocomposite coating formed by polysaccharide 1-deoxylactit-1-yl chitosan (Chitlac) and silver nanoparticles (nAg) on methacrylate thermosets were studied. The Chitlac–nAg system showed good anti-bacterial and anti-biofilm activity although its biocidal properties can be moderately, albeit significantly, inhibited by serum proteins. In vitro studies on the silver release kinetic in physiological conditions showed a steady metal release associated with a gradual loss of antimicrobial activity. However, after 3 weeks there was still effective protection against bacterial colonization which could be accounted for by the residual silver. This time-span could be considered adequate to confer short-term protection from early peri-implant infections. Preliminary in vivo tests in a mini-pig animal model showed good biological compatibility of Chitlac–nAg-coated materials when implanted in bony tissue. The comparison was made with implants of titanium Ti6Al4V alloy and with a Chitlac-coated thermoset. Bone healing patterns and biocompatibility parameters observed for nAg-treated material were comparable with those observed for control implants.

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are receiving increasing attention as biomaterials for dental and orthopedic applications; for both these fields, bacterial adhesion to the surface of the implant represents a major issue for the outcome of the surgical procedure. Moreover, the biological behaviour of these materials is influenced by their ability to establish proper interactions between their surface and the eukaryotic cells of the surrounding tissues, which is important for good implant integration. The aim of this work was to develop an antimicrobial non-cytotoxic coating for methacrylic thermosets by means of a nanocomposite material based on a lactose-modified chitosan and antibacterial silver nanoparticles. The coating was characterized by UV-vis spectrophotometry, optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In vitro tests were employed for a biological characterization of the material: antimicrobial efficacy tests were carried out with both Gram+ and Gram- strains. Osteoblast-like cell-lines, primary human fibroblasts and adipose-derived stem cells, were used for LDH cytotoxicity assays and Alamar blue cell proliferation assays. Cell morphology and distribution were evaluated by SEM and confocal laser scanning microscopy. In vitro results showed that the nanocomposite coating is effective in killing both bacterial strains and that this material does not exert any significant cytotoxic effect towards tested cells, which are able to firmly attach and proliferate on the surface of the coating. Such biocompatible antimicrobial polymeric films containing silver nanoparticles may have good potential for surface modification of medical devices, especially for prosthetic applications in orthopedics and dentistry.

In this study, we took advantage of the strong interaction between the antimicrobial peptide LL-37 and anionic polysaccharides, such as alginate, to design and evaluate a new biomaterial with putative antibacterial properties. To begin with, we have investigated the effect of different biocompatible polysaccharides on both the cytotoxicity and the antimicrobial activity of LL-37, a powerful endogenous antimicrobial peptide of human origin, whose use in therapy has been hampered by its toxicity to host cells. Interactions of the peptide with polysaccharides were evaluated by circular dichroism analyses, which revealed a different capacity of the polymers to induce the active helical conformation in the peptide. Among the polysaccharides tested, sodium alginate was the only one that significantly reduced the toxicity of LL-37 toward mammalian cells. A sodium alginate/LL-37 preparation was then tested on four bacterial strains. The Gram-negative bacteria resulted susceptible to the mixture, while the growth of the Gram-positive ones was poorly affected and only at the highest peptide concentration tested. Following the positive results with Gram-negative species, the alginate/LL-37 binary system was used for the preparation of calcium alginate beads, which were tested for peptide release.

In this work, the morphological characteristics of a calcium alginate gel and a binary (gel) mixture composed of (calcium) alginate and lactose-modified chitosan (chitlac) are evaluated and compared to quantify the differences between the two three-dimensional (3D) structures. A set of textural descriptors based on histogram analysis as well as on gray level co-occurrence matrix and on fractal dimension is extracted from transmission electron microscopy micrographs to describe the morphological differences that the images present. The obtained results reveal significant quantitative morphological differences between the calcium alginate gel and the binary gel mixture that were already inferred from rheological experiments, so as to provide a structural basis for developing new encapsulation systems based on such mixed polymer gels. Microsc. Res. Tech. 74:58-66, 2011. (C) 2010 Wiley-Liss, Inc.

Capillary electrophoresis is a consolidated analytical approach for the structural characterization of polysaccharide mono- and oligomer constituents, as demonstrated in this chapter, which surveys several applications of this technique on chemically and enzymatically degraded polysaccharides, covering the last 10 to 12 years. Capillary electrophoresis is also demonstrated to be highly reliable for determination of polysaccharides in biological samples, as it analyzes quite complex matrices even without any pretreatment, a distinctive feature with respect to other separative strategies. The versatility of this technique is clearly demonstrated by its potential in evaluating macromolecular features of polysaccharides, such as size (molecular weight, chain length), chain rigidity, charge density, and chemical modifications.

The solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae SK1, SK1-CPS, were investigated by various methods. The SK1-CPS repeating unit is a branched pentasaccharide containing one glucuronic acid as single unit side chain; acetyl groups are present as non-carbohydrate substituents on the uronic acid residue in non-stoichiometric amounts. Chiro-optical, potentiometric, viscometric and rheological measurements have been performed in order to characterize the conformational behaviour of the polymer in water and in aqueous salt solutions. Under the investigated experimental conditions, changes of temperature, ionic strength and pH were shown not to induce any cooperative conformational transition. All the results obtained suggest that the solution conformation of SK1-CPS is a random coil with a certain degree of chain flexibility. The removal of the acetyl substituents apparently does not modify the overall conclusions drawn for the native polymer, except for an incipient tendency to aggregation revealed for high salt conditions.

Lipid extracts of biopsy samples from normal and non-alcohol-induced fatty human liver were studied by 1H-NMR at 200 MHz. Spectra of the lipid extracts from 10 mg samples were obtained in 6 min with routine acquisition parameters and allowed the calculation of the phosphatidylcholine to total fatty acyl chain ratio, the cholesterol to total fatty acyl chain ratio, the average fatty acyl chain length, the unsaturation ratio and the acylated glycerol to total fatty acyl chain ratio. The data suggest that lipids with a higher ratio of de novo synthesized fatty acyl chains are stored in non-alcohol-induced fatty liver. NMR lipid analysis appears to be a reliable method for the rapid assessment of hepatic lipid composition on bioptic specimens.

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets and composites are well-known examples of biomaterials for dental applications that are receiving growing interest for orthopedic applications. While mechanical bulk properties are guaranteed by the presence of reinforcing fibers, in vitro and in vivo performances of these materials are ultimately driven by their ability to establish proper interactions between their surface and the surrounding tissues. Hence, the development of novel chemical processes enabling the introduction of bioactive molecules on the surface of these methacrylate-based thermosets is of particular interest. In the present work, we have devised a chemical strategy to expose carboxylic groups on the surface of the BisGMA/TEGDMA thermoset. The presence of negative charges was confirmed by Fourier transform infrared-attenuated total reflectance and by UV-vis spectrophotometry. Bulk mechanical properties and surface morphology of the thermoset were only slightly affected upon chemical functionalization. The activated material was further refined by the deposition of a lactose-modified chitosan (chitlac) driven by strong electrostatic interactions. The presence of the bioactive polysaccharide was confirmed by fluorescence spectroscopy and by confocal laser scanning microscopy measurements. Scratch tests were performed to evaluate the mechanical behavior of the coating. Finally, in vitro tests revealed that the presence of chitlac led to a slight enhancement of cell proliferation with respect to the unmodified BisGMA/TEGDMA thermoset. This effect was more pronounced when chitlac decorated with an arginine-glycine-aspartic acid (RGD) peptide was used in the preparation of the coating. In the latter case, the in vitro performance of the coated BisGMA/TEGDMA thermoset became comparable with that of clinically used roughened titanium.

The 1 H- and 13 C-NMR spectra of the ethyl and benzyl esters and the tetrabutylammonium and tetraethylammonium salts of hyaluronic acid { 2)-β- d -Glc p A-(1 → 3)-β- d -Glc p NAc-(1 n } in Me 2 SO- d 6 have been assigned using 1D and 2D techniques. The chemical shifts of the resonance of GlcNAc C-3 suggest that the relative orientations of the monosaccharides at the (1 → 3) linkage in the esters and salts are different. Small differences in the chemical shifts of the resonance GlcA C-4 suggest only a slight conformational variation around the (1 → 4) linkage. The 13 C-NMR data also suggest similarities in conformation between the esters in Me 2 SO- d 6 and the salts in water. The chemical shifts of the 1 H resonances for NH and OH groups and their temperature dependence for the esters and salts in Me 2 SO reveal markedly stronger inter-residue hydrogen bonds between the carboxyl and NH groups and between HO-4 of GlcA and O-5 of GlcNAc for the salts. The 3 J 2,NH values indicate a slightly different orientation for the acetamido group. For solutions in Me 2 SO, the higher segmental flexibility of the esters is supported by the line widths, whereas the reduced viscosity for the tetrabutylammonium salt showed a sigmoidal concentration dependence and suggests association of chains which could contribute to the segmental rigidity. The linear concentration dependence for the benzyl ester suggests a higher overall flexibility without chain association.

Alditol bearing chitosans have shown the ability to reduce silver ions in mild conditions and without addition of exogenous reducing agents. The ion reduction induces the formation of a lactone moiety on the polysaccharide (Fetizon reaction) without causing C-C bond cleavage on the polyol. The close and multivalent arrangement of the endogenous reducing agent (alditols) on the polysaccharide backbone resulted in the formation of silver nanoparticles (phi < 10 nm), which induced a considerable SERS effect and led to hydrogel formation.

In this work we study (i) the formation and stabilization of silver nanoparticles in a bioactive chitosan-derived polysaccharide solution, (ii) the antimicrobial properties, either in solution or in 3D hydrogel structures, obtained by mixtures with the polysaccharide alginate, and (iii) the cytotoxicity of the latter nanocomposite materials on different eukaryotic cell lines. Antimicrobial results show that these nanocomposite systems display a very effective bactericidal activity toward both Gram+ and Gram- bacteria. However, the hydrogel does not show any cytotoxic effect toward three different eukaryotic cell lines. This is due to the fact that the nanoparticles, immobilized in the gel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while they can not be uptaken and internalized by eukaryotic cells. This novel finding could advantageously contribute to responding to the growing concerns on the toxicity of nanoparticles and facilitate the use of silver-biopolymer composites in the preparation of biomaterials.

The present chapter deals with the description of the main characteristics of alginate as a material. Sources and the chemical structure of the polysaccharide are discussed. Particular attention is devoted to the definition of the comonomer sequence and its determination together with chemical and biochemical modifications of alginate. The stability of alginate molecules is discussed from the point of view of the effect on the polymer molecular weight. The characteristics of the polysaccharide in solution are tackled looking both at the equilibrium properties (thermodynamic properties) and at the nonequilibrium ones (viscosity in dilute and semidilute solution). The second part of this contribution is focused on the analyses of the ion-binding properties of alginate towards divalent ions. A general description of the “egg-box” model is provided together with a brief overview of recent findings on this topic. The mechanism of hydrogel formation and the description of the mechanical properties of alginate gels in small and large deformation ranges are addressed.

Enzymatic degradation of standard κ-carrageenan and the low-gelling hybrid κ-/μ-carrageenan were conducted using recombinant Pseudoalteromonas carrageenovora κ-carrageenase. The initial velocity of the enzyme was determined as a function of varying Tris or NaI concentrations and at constant 200 mM cosolutes concentration, adjusting NaI and Tris concentrations accordingly. In both cases, we observed strong inhibition of the enzyme with increasing amounts of iodide. The characterization of the κ- and κ-/μ-carrageenan ordering by optical rotation and the visualization of iodide binding on carrageenan by (127)I NMR revealed that inhibition was not caused by the disordered-ordered transition of carrageenan in NaI, but by iodide binding. These results were confirmed by analysis of the degradation products by gel permeation chromatography. Degradation of carrageenan in the disordered state led to a rapid decrease in molecular mass and the production of all possible neo-κ-carrabiose oligomers. In the ordered conformation, the degradation kinetics, the decrease of average molecular weight, and the chain population distribution of degradation products varied with iodide concentration. These observations were interpreted to be the result of increasing amounts of bound iodide on carrageenan helices that, in turn, impede enzyme catalysis. Based on these results, we propose a single-helix ordered conformation state for κ-carrageenan and reject the previously advocated double-helix model.

A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.

Osmotic coefficients, phi, and apparent molar volumes, PhiV, of aqueous solutions of isotactic, syndiotactic, and atactic poly(methacrylic acid), i-PMA, s-PMA, and a-PMA, respectively, were measured at 298 K in dependence on polymer concentration and on degree of neutralization, alphaN, of carboxyl groups. The solutions of i-PMA have lower phi values than those of a-PMA and s-PMA in the whole region of alphaN. Molecular dynamics simulation studies of the isotactic and the syndiotactic PMA 101-mer have shown that lower phi is a consequence of a shorter distance between charges, which leads to a greater charge density of the isotactic polymer and thus to stronger binding of counterions. The experimental phi data were analyzed using a cylindrical cell model. Good agreement between theory and experiment was achieved when charges on the polyion were distributed periodically along the z-axis of the polyion in accordance with the simulation results. The alphaN dependence of the PhiV data pointed out that all PMA isomers bind an appreciable amount of water in the elementary dissociation process (electrostriction). For a- and s-PMA, the PhiV values decrease linearly with increasing alphaN, whereas they show a marked nonlinear dependence in the case of i-PMA for alphaN < 0.6. The latter finding was ascribed to a very high intermolecular association tendency of the isotactic polymer. This association tendency of PMA chains was confirmed by light scattering measurements. It is present in both i- and a-PMA solutions but is much more pronounced in the i-PMA case.