Preparation of acid-resistant macroporous chitosan sponges using trans-2-hexenal as a chemical heterobifunctional cross-linker.

The aim of this study was to apply trans-2-hexenal (HX) as a chemical cross-linker to obtain chitosan sponges easily. For that, 4% (w/v) chitosan dissolved in 1% acetic acid solution was gelled with HX ranging from 3.125 to 25% (w/w). Rheological measurements at 23 °C showed a decrease in gelation time and an increase in the strength of the hydrogels as the concentration of HX increased. Spectroscopic analysis revealed the formation of imine bonds between the aldehyde and primary amino groups of chitosan and the presence of the ethylenic group of HX for concentrations >3.125%. Lyophilised hydrogels presented a macroporous sponge-like structure with rounded pores and high water holding capacity, moreover, they maintained their structural integrity in acidic aqueous solutions. Hydrolysis experiments and elemental analysis confirmed that for HX concentrations greater than 3.125%, some molecules were linked to chitosan only forming reversible conjugated imines, which gave rise to an increase in the hydrophobicity of the sponges, and a decrease in their thermal stability and compressive stress. Cytotoxic studies shown sponges formulated with 3.125% HX were biocompatible with HaCat cells. Spectroscopic and experimental results suggest that trans-2-hexenal behaves as a chemical cross-linker of chitosan, likely link-ing chains through the formation of imines and aza-Michael adducts.