Peroxidase-catalyzed microextrusion bioprinting of cell-laden hydrogel constructs in vaporized ppm-level hydrogen peroxide

Hydrogels were prepared by contacting air containing 10–50 ppm H 2 O 2 with an aqueous solution containing polymer(s) possessing phenolic hydroxyl (Ph) moieties (polymer-Ph) and horseradish peroxidase (HRP). In this system, HRP catalyzes cross-linking of the Ph moieties by consuming H 2 O 2 diffused from the air. The hydrogelation rate and mechanical properties of the resultant hydrogels can be tuned by controlling the H 2 O 2 concentration in air, the exposure time of the air containing H 2 O 2 to the solution containing polymer-Phs and HRP, and the HRP concentration. The shortest hydrogelation time of the solution stirred in air containing 16 ppm H 2 O 2 was 6 s. Based on these findings, this hydrogelation system was applied to microextrusion bioprinting, in which bioink containing polymer-Phs, HRP, and cells were extruded into air containing H 2 O 2 . The superior cytocompatibility of the bioprinting method was confirmed by more than 90% viability, migration, and the spreading of mouse fibroblast 10T1/2 cells enclosed in the bioprinted hydrogels composed of derivatives of hyaluronic acid and gelatin, both possessing Ph moieties. These results demonstrate the great potency of HRP-catalyzed hydrogelation consuming H 2 O 2 supplied in surrounding air for various biomedical applications, especially bioprinting.