Influence of Hydrogen Peroxide-Mediated Cross-Linking and Degradation on Cell-Adhesive Gelatin Hydrogels

Hydrogen peroxide (H₂O₂) is widely used for the gelation of aqueous solutions of gelatin derivatives with phenolic hydroxyl groups (Gelatin-Ph) catalyzed by horseradish peroxidase (HRP). Apart from this, H₂O₂ is known to cause degradation/depolymerization of various polymers. Here, we prepared Gelatin-Ph hydrogels from solutions containing Gelatin-Ph and HRP by continuously supplying H₂O₂ from the gas phase and investigated the mechanical properties of resultant hydrogels and the behaviors of rat fibroblast and human adipose-derived stem cells on them. Young's modulus of the hydrogel obtained from 5 w/v % Gelatin-Ph and 1 and 5 U/mL HRP increased when the exposure time to air containing H₂O₂ (16 ppm) was extended from 15 to 30 min. However, further prolonging the exposure time to 60 min reduced Young's modulus to the same magnitude as for the hydrogels exposed to air containing H₂O₂ for 15 min. Interestingly, the cell length and aspect ratio of the cells continued to increase, as the exposure time was extended, without reflecting the decrease in Young's modulus. These results indicate that when preparing Gelatin-Ph hydrogels through HRP/H₂O₂-mediated gelation, it is necessary to consider the effect of the degradation of Gelatin-Ph caused by H₂O₂ on the mechanical properties of the resultant hydrogels and the behaviors of cells on them.