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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.

Structural properties of several cross-linked hyaluronan derivatives, obtained by scanning electron microscopy, monodimensional NMR microscopy and small angle X-ray scattering of synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid, used as a reference material. The experimental results, obtained in different media, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of denser polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover, the presence of polymer junction zones gave rise to the appearance of a well-defined correlation peak in the pattern of intensity of the scattered X-radiation.

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, 1H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5–1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Galactose moieties have been introduced on the uronic groups of alginates from different sources via an N-glycosidic bond, thus affecting the net charge on the polymer chain. The modified polymers have been analyzed by means of viscosity and of high-performance size-exclusion chromatography combined with refractive index multiple angle laser light scattering (HPSEC-RI-MALLS) measurements. The latter technique enabled us to determine the molecular weight of the modified polymers, proving that the synthetic procedure did not affect the chemical integrity of the chain. The intrinsic viscosity and the radius of gyration data showed that the hydrodynamic properties of the polymer chain varied with the degree and the pattern of substitution. In the presence of a relatively low galactose content (up to 19%), a decrease of the hydrodynamic dimensions of the coil was experienced, while on increasing the degree of substitution (especially on GG diads) a re-extension of the chain was discovered. Measurements of intrinsic viscosity at different values of the degree of dissociation have demonstrated that this effect cannot be solely explained by the reduction of the charge density of the polymer. Rather, it implies the occurrence of conformational changes of the chain that are specific to the chemical nature of the site of substitution. These data have been supported by the values of the persistence length of the natural and modified polymers obtained with the Doty-Benoit equation. The chiro-optical properties of the modified polymers studied by means of circular dichroism (CD) spectroscopy confirmed that conformational variations occurred to the polymeric chain upon introduction of galactose residues.

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.

The secondary structure of xanthan in solutions of relatively low salt concentration and at room temperature has been investigated using static light scattering experiments. Additional evidence has been found for a dimeric structure at 25 degrees C in 0.01 M NaCl. From the experimental z-average mean square (ms) radius of gyration, a value for the persistence length p has been estimated, taking explicitly into account the polydispersity of the three samples used, which has been established by gel permeation chromatography (GPC) measurements. The experimental particle scattering functions of the three samples are consistent with theoretical estimates for polydisperse systems with the same value of p = 65 +/- 10 nm and the molar mass per unit length for a dimeric structure. This secondary structure remains unaffected by the ionic strength in the 0.005-0.01 M range. Partial aggregation seems to occur at higher NaCl concentrations. Light scattering and GPC data show that heating the xanthan 0.01 M NaCl solutions to about 70 degrees C considerably reduces the Mw of the low molar mass sample (2.3 x 10(5) g.mol-1), contrary to what is observed for the high molar mass sample (1.8 x 10(6) g.mol-1). These experimental findings can be accounted for by a partial temperature-induced dissociation of the xanthan dimers according to an all-or-none mechanism.