Formation of defined microporous 3D-structures starting from cross-linked hydrogels

A new and simple technique was developed to obtain polysaccharide (hyaluronane, alginate and carboxymethylcellulose) -based hydrogels with a defined porous morphology. The technique consists of stratifying a cross-linked hydrogel on a filter with known pore diameter. CO2 bubbles, produced by the addition of HCl to a porogen salt NaHCO3, are forced to pass through the filter, and they induce the hydrogel to assume a porous morphology. The presence and distribution of pores was confirmed by scanning-electron microscopy (SEM). A strict correspondence was found between the porosity of the filter and the pore diameter in the hydrogels. Water uptake measurements showed a decreased amount of water taken up by the porous hydrogels compared with the native hydrogels, due to a compacting of the material. An explanation of the porous material properties of Hyal hydrogel was given on the basis of FTIR spectra.

The role of Fibrinogen conformation on platelets activation.

Abstract Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces

Hyaluronic acid and sulfated hyaluronic acid in aqueous solution: effect of the sulfation on the protonation and complex formation with Cu2+ and Zn2+ ions

The behaviour of hyaluronic acid (Hyal) and sulfated hyaluronic acid (HyalS) in aqueous solution was investigated in terms of protonation and complex formation by thermodynamic and spectroscopic techniques. The presence of the scarcely protonable sulfate groups in the polymer chain provided the HyalS molecule with a more rigid structure in aqueous solution, allowing this polysaccharide to remain in the stretched conformation even after neutralisation of the carboxylate moieties. The presence of the sulfate groups also increases the basic character of the COO– group. Both Hyal and HyalS were able to form complexes with Cu2+ and Zn2+ ions in aqueous solution. The metal ion has a preferential binding site on these polysaccharides, providing a precise stoichiometry for the metal ion complex. The sulfate groups were not directly involved in the coordination of the metal ion, and the stoichiometry of the complex species is not affected by the presence of these groups. However, by increasing the negative charge on the polymer chain, the sulfate groups are responsible for the electrostatic attraction of the metal ion

Thermodynamic behavior of complexes with 5,6-membered condensed chelate rings. 1,4,8-Triazaoctane

The enthalpies of the reactions between NH2(CH2)2NH(CH2)3NH2 and Ni2+, Cu2+, and Zn2+ in 0.5M KNO3 at 25° were detd. by direct calorimetric titrations and previously reported free energy values were used to calc. the corresponding entropy values. Complexes formed contained a system of condensed 5- and 6-membered chelate rings and had higher heats of formation than corresponding complexes of NH2(CH2)2NH(CH2)2NH2 (5,5-ring size sequence) and NH2(CH2)3NH(CH2)3NH2 (6,6-ring size sequence.). Addn. of a 2nd mol. of ligand to the complex depended on steric factors

The role of fibronectin in cell adhesion to spiral patterned TiO2 nanoparticles

Spiral micropatterned surfaces of decreasing dimensions were produced by photo-immobilising a photoreactive hyaluronan (Hyal) derivative on TiO2 nanoparticles. The microstructured surfaces were characterised by both SEM and AFM analysis. The behaviour, of both endothelial cells (HCAEC) and tumoral mouse fibroblasts (NIH3T3) on the patterned surfaces was evaluated. HCAEC adhered only to the TiO2 nanoparticles avoiding the contact with Hyal. NIH3T3 adhered to and covered completely the TiO2 spiral and prolonging the culture time also the photo-immobilised Hyal. The role of fibronectin to mediate cell adhesion to the TiO2 pattern surfaces was evaluated by experiments with blocked fibronectin membrane receptors on both HCAEC and NIH3T3. The results showed the absence of any adhering cells. Thus, fibronectin seemed to be the only key protein in mediating cell adhesion to these TiO2 substrates

DEPENDENCE OF WATER UPTAKE AND MORPHOLOGY OF HYALURONAN AND ALGINATE BASED HYDROGELS ON pH AND DEGREE OF CROSSLINKING

Hydrogels of alginate and hyaluronan with 5%, 50% and 100% crosslinking, (percentage of polysaccharide carboxylate groups involved in crosslinking) are synthesized. Crosslinking and stoichiometry were varied by appropriate addition of chloromethylpyridine iodide (CMPI) to activate the carboxylic groups. Crosslinking percentage was determined by potentiometry, i.e., titrating the free carboxylate groups of the polysaccharide. Water uptake by the gels is studied in relation to crosslinking and pH. Hydrogen bonds in the three-dimensional network, as shown by FT-IR spectroscopy, are used to explain differences in water uptake and their dependence on pH. Scanning electron microscopy (SEM) analysis of the gels is performed at different pHs and the morphologies are compared with their chemical structures. Equilibrium water uptake of AA (gray) and HA (black) gels in relation to percentage crosslinking in 0.6 M NaCl

Heterotypic cell-cell interaction on micropatterned surfaces

Purpose. The aim of this paper was to study the influence of chemical and topographical signals on cell behaviour and to obtain a heterotypic cell-cell interaction on microstructured domains. Methods. The polysaccharide hyaluronic acid (Hyal) was photoimmobilised on glass surfaces in order to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterised by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The behaviour of Human Coronary Artery Endothelial Cells (HCAEC) and human tumoral dermal fibroblasts (C54) was investigated on these micropatterned surfaces by adhesion studies. Moreover heterotypic interaction among C54 and HCAEC adhered on patterned surfaces was evaluated by time-lapse video microscopy. Results. Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the centre to the edge of the sample. Neither HCAEC nor C54 adhered to the immobilised Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent cells depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain. Conclusions. A heterotypic cell-cell interaction occurred in the same chemical and topographic micro-domains but in narrow areas only. Moreover, the number of cells adhering to the glass domains and cell morphology depended on the dimensions of both adhesive areas and cell nuclei