Versatility of hydrogelation by dual-enzymatic reactions with oxidases and peroxidase

Hydrogelation of aqueous solution of biocompatible macromolecules by mild chemical reaction is promising to be a universal technique in the field of biomaterials. This work demonstrates that the hydrogelation is achieved through dual enzymatic reactions using various kinds of oxidases (L-amino acid oxidase (L-AAOx), choline oxidase (ChOx), and galactose oxidase (GalOx)) in combination with horseradish peroxidase (HRP). The dual-enzymatic mechanism is confirmed from gelation experiments using varying amounts of enzymes or substrates. The results obtained in dynamic viscoelastic measurements indicate the formation of stable chemical gel networks and the oxidase-dependent elastic moduli of the gels. The viabilities of fibroblasts seeded on the gels formed by ChOx and GalOx are around 90% after 24 h of culture, whereas the viability on the gel formed by L-AAOx is less than 20%. This work shows the versatility of the hydrogelation mediated by cascade reactions of oxidases producing H2O2 and subsequent consumption of H2O2 by HRP and the importance of the careful selection of oxidases considering the potential H2O2 generation by remaining oxidases in resultant hydrogels.