Search

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.

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.

The present manuscript deals with the setup and evaluation of two analytical methods, namely Circular Dichroism (CD) and 23Na longitudinal relaxation, to determine the composition of binary mixtures of the sodium salts of alginate and hyaluronan in solution. The quantitative results obtained were confirmed by 1H NMR, and the developed methods were applied to the determination of the composition and polymer release of mixed hydrogels. Although focused on the specific case of binary mixtures of alginate and hyaluronan, the approach drawn in the present manuscript can find applications in different binary systems composed of natural or synthetic non-interacting polymers.

Alginate/hydroxyapatite composite scaffolds were developed using a novel production design. Hydroxyapatite (HAp) was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid hydrolyzing d-gluconic acid delta-lactone (GDL) for the direct release of calcium ions from HAp. Hydrogels were then freeze-casted to produce a three-dimensional isotropic porous network. Scanning electron microscopy (SEM) observations, confocal laser scanning microscopy (CLSM) and microcomputed tomography (micro-CT) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 and 300 microm and over 88% porosity. Proliferation assay and SEM observations demonstrated that human osteosarcoma cell lines were able to proliferate, maintain osteoblast-like phenotype and massively colonize the scaffold structure. Overall, these combined results indicate that the novel alginate based composites efficiently support the adhesion and proliferation of cells showing at the same time adequate structural and physical-chemical properties for being used as scaffolds in bone tissue engineering strategies.

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).

Polyuronates such as pectate and alginate are very well-known examples of biological polyelectrolytes undergoing, upon addition of divalent cations, an interchain association that acts as the junction of an eventually formed stable hydrogel. In the present paper, a thermodynamic model based on the counterion condensation theory has been developed to account for this cation-induced chain pairing of negatively charged polyelectrolytes. The strong interactions between cross-linking ions and uronate moieties in the specific binding site have been described in terms of chemical bonding, with complete charge annihilation between the two species. The chain-pairing process is depicted as progressively increasing with the concentration of cross-linking counterions and is thermodynamically defined by the fraction of each species. On these bases, the total Gibbs energy of the system has been expressed as the sum of the contributions of the Gibbs energy of the (single) chain stretches and of the (associated) dimers, weighted by their respective fractions 1 - theta and theta. In addition, the model assumes that the condensed divalent counterions exhibit an affinity free-energy for the chain, G(C)(aff,0), and the junction, G(D)(aff,0), respectively. Moreover, a specific Gibbs energy of chemical bonding, G(bond,0), has been introduced as the driving force for the formation of dimers. The model provides the mathematical formalism for calculating the fraction, theta, of chain dimers formed and the amount of ions condensed and bound onto the polyelectrolyte when two different types of counterions (of equal or different valence) are present. The effect of the parameter G(bond,0) has been investigated and, in particular, its difference from G(C,D)(aff,0) was found to be crucial in determining the distribution of the ions into territorial condensation and chemical bonding, respectively. Finally, the effect of the variation of the molar ratio between cross-linking ions and uronic groups in the specific binding sites, sigma0, was evaluated. In particular, a remarkable decrease in the amount of condensed counterions has been pointed out in the case of sigma0 = 1/3, with respect to the value of sigma0 = 1/4, characterizing the traditional "egg-box" structure, as a result of the drop of the charge density of the polyelectrolyte induced by complete charge annihilation.

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.

The long-term stability and success of orthopedic implants depend on the osseointegration process, which is strongly influenced by the biomaterial surface. A promising approach to enhance implant integration involves the modification of the surface of the implant by means of polymers that mimic the natural components of the extracellular matrix, for example, polysaccharides. In this study, methacrylate thermosets (bisphenol A glycidylmethacrylate/triethyleneglycol dimethacrylate), a widely used composition for orthopedic and dental applications, have been coated by electrostatic deposition of a bioactive chitosan-derivative. This polysaccharide was shown to induce osteoblasts aggregation in vitro, to stimulate cell proliferation and to enhance alkaline phosphatase activity. The coating deposition was studied by analyzing the effect of pH and ionic strength on the grafting of the polysaccharide. Contact angle studies show that the functionalized material displays a higher hydrophilic character owing to the increase of surface polar groups. The mechanical properties of the coating were evaluated by nanoindentation studies which point to higher values of indentation hardness and modulus (E) of the polysaccharide surface layer, while the influence of cyclic stress on the construct was assessed by fatigue tests. Finally, in vivo tests in minipigs showed that the polysaccharide-based implant showed a good biocompatibility and an ability for osseointegration at least similar to that of the titanium Ti6Al4V alloy with roughened surface.

The development of biomaterials endowed with bioactive features relies on a simultaneous insight into a proper terminal sterilization process. FDA recommendations on sterility of biomaterials are very strict: a sterility assurance level (SAL) of 10(-6) must be guaranteed for biomaterials to be used in human implants. In the present work, we have explored the potential of supercritical CO(2) (scCO(2)) in the presence of H(2)O(2) as a low-temperature sterilization process for thermoset materials and their bioactive surfaces. Different conditions allowing for terminal sterilization have been screened and a treatment time-amount of H(2)O(2) relationship proposed. The selected terminal sterilization conditions did not notably modify the mechanical properties of the thermoset nor of their fiber-reinforced composites. This was confirmed by μCT analyses performed prior to and after the treatment. On the contrary, terminal sterilization in the presence of H(2)O(2) induced a slight decrease in the surface hardness. The treatment of the thermoset material with scCO(2) led to a reduction in the residual unreacted monomers content, as determined by means of high performance liquid chromatography (HPLC) analyses. Finally, it was found that a thermoset coated with a polysaccharide layer containing silver nanoparticles maintained a very high antimicrobial efficacy even after the scCO(2)-based terminal sterilization.

The present contribution deals with the preparation and characterization of ternary mixtures of polysaccharides with potential applications in the field of tissue engineering. Two natural polyanions, i.e., alginate and hyaluronic acid, and a polycation, a lactose-modified chitosan (chitlac), were mixed in dilute conditions. The miscibility between the three components was explored in the presence of different amounts of supporting simple salt. These analyses allowed to identify the experimental conditions avoiding polymer phase, separation and leading to either solution of independent polymers or soluble nonstoichiometric interpolyelectrolyte complexes. The characterization of the interpolyelectrolyte complexes was tackled by means of viscometry, light scattering, fluorescence quenching, and energy transfer. The electrostatic interactions taking place among the different polyelectrolytes led to synergistic effects on the viscosity of the polymer mixtures which strongly depend on the ionic strength. It has been found that, starting from binary soluble complexes of alginate and chitlac, the addition of hyaluronan led to the dissolution of the complexes. At variance, the addition of alginate to a phase-separated binary mixture of hyaluronan and chitlac led to the formation of soluble complexes composed of all three polysaccharides and, eventually, to their dissolution. In addition, the results showed that at low ionic strength the overall properties of the ternary mixtures depend on their order of mixing.

The structure of calcium-saturated alginate hydrogels has been studied by combining rheological determinations and relaxometry measurements. The mechanical spectroscopy analyses performed on alginate gel cylinders at different polysaccharide concentration allowed estimating their main structural features such as the average mesh size. The calculation was based on the introduction of a front factor in the classical rubber elasticity approach which was correlated to the average length of the Guluronic acid blocks along the polysaccharide chain. Transverse relaxation time (T(2)) determinations performed on the cylinders revealed the presence of two relaxation rates of the water entrapped within the hydrogel network. The cross-correlation of the latter data with the rheological measurements allowed estimating the mesh size distribution of the hydrogel network. The results obtained for the hydrogel cylinders were found to be consistent with the relaxometric analysis performed on the alginate microbeads where, however, only one type of water bound into the network structure was detected. A good correlation was found in the average mesh size determined by means of relaxometric measurements on alginate microbeads and by a statistical analysis performed on TEM micrographs. Finally, the addition of a solution containing calcium ions allowed investigating further the different water relaxation modes within alginate hydrogels.

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.

Three derivatives of the biocompatible polymer poly(styrene-co-maleic anhydride) (SMA) were obtained with 1-amino-1-deoxy-beta-D-galactose, 1-amino-1-deoxy-beta-D-glucose, and 1-amino-1-deoxy-beta-D-lactose, respectively. The amino sugars were chemically conjugated via formation of an amide bond between the anomeric amino group of the sugar residue and the anhydride of the copolymer, giving the corresponding glycoconjugate derivatives. Colorimetric assay of the unreacted amino groups and elemental analysis were used to determine the degree of substitution. About 56%, 54%, and 94% of the available anhydride groups reacted to give galactosyl-amide (SMA-Gal), glucosyl-amide (SMA-Gluc), and lactosyl-amide (SMA-Lac) branched polymers, respectively. The synthesized glycopolymers were characterized by Fourier transform infrared spectroscopy, gel permeation chromatography, circular dichroism, and UV and fluorescence spectroscopy. The release of glucosylamine from the glucosyl-amide branched polymer, by basic hydrolysis, was monitored by high-performance anion-exchange chromatography and by capillary electrophoresis, providing for an additional check of the degree of substitution of this specific polymer derivative. Biological activity tests showed that both SMA-Gal and SMA-Lac allow adhesion of HepG2 hepatic cells about five times larger than that of hydrolyzed, underivatized SMA.