Chitosan-anthracene hydrogels as controlled stiffening networks

In this study, we report the synthesis of single and dual-crosslinked anthracene-functional chitosan-based hydrogels in the absence of toxic initiators. Single crosslinking was achieved through dimerization of anthracene, whereas dual-crosslinked hydrogel was formed through dimerization of anthracene and free radical photopolymerization of methacrylated-chitosan in the presence of non-toxic initiator riboflavin, a well-known vitamin B2. Both single and dual-crosslinked hydrogels were found to be elastic, as was determined through rheological analysis. We observed that the dual-crosslinked hydrogels exhibited higher Young's modulus than the single-crosslinked hydrogels, where the modulus for single and dual-crosslinked hydrogels were measured as 9.2 +/- 1.0 kPa and 26 +/- 2.8 kPa, respectively resulting in significantly high volume of cells in dual-crosslinked hydrogel (2.2 x 107 mu m3) compared to single-crosslinked (4.9 x 106 mu m3). Furthermore, we investigated the cytotoxicity of both hydrogels towards 3T3-J2 fibroblast cells through CellTiter-Glo assay. Finally, immunofluorescence staining was carried out to evaluate the impact of hydrogel modulus on cell morphology. This study comprehensively presents functionalization of chitosan with anthracene, uses nontoxic initiator riboflavin, modulates the degree of crosslinking through dimerization of anthracene and free radical photopolymerization, and further modulates cell behavior through the alterations of hydrogel properties.