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

Abstract

Hydrogen peroxide (H 2 O 2 ) 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, H 2 O 2 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 H 2 O 2 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 H 2 O 2 (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 H 2 O 2 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/H 2 O 2 -mediated gelation, it is necessary to consider the effect of the degradation of Gelatin-Ph caused by H 2 O 2 on the mechanical properties of the resultant hydrogels and the behaviors of cells on them.

Modulation of Cell-Cycle Progression by Hydrogen Peroxide-Mediated Cross-Linking and Degradation of Cell-Adhesive Hydrogels

“…Cell morphology analysis showed that while NMuMG/Fucci2 cells also had small and round morphology on the hydrogel obtained through 15 min of exposure to H 2 O 2 , cells cultured on the hydrogel obtained through 120 min of the exposure had an elongated shape, despite having almost similar stiffness (0.40 kPa) with hydrogel obtained through 15 min of the exposure (0.20 kPa) ( Figure 6 b–f). This phenomenon is similar to our previous report in human adipose-derived stem cells (hASCs) and rat fibroblast (3Y1) cells cultured on Gelatin–Ph hydrogel that showed elongated morphology on the hydrogel obtained through 60 min of exposure to H 2 O 2 despite having similar stiffness with hydrogel obtained through 15 min of the exposure [ 31 ]. This phenomenon could be attributed to other factors such as mesoscale topographical changes and mesh size [ 67 ].…”

“…In the applications of using H 2 O 2 for inducing HRP-mediated hydrogelation, no attention has been paid to the contradictory functions of H 2 O 2 for inducing the degradation of the polymers. Recently, we reported that the contradictory function of H 2 O 2 affects the mechanical property of hydrogels, which leads to changes in adhesion of the human adipose-derived stem cells (hASCs) and rat fibroblast cells [ 31 ]. However, the contradictory effect of H 2 O 2 on other cell behaviors, such as cell-cycle progression, has not been investigated.…”

Tuning Myogenesis by Controlling Gelatin Hydrogel Properties through Hydrogen Peroxide-Mediated Cross-Linking and Degradation

“…At 5.0% w / v , the Gelatin-Ph solution quickly forms hydrogel at room temperature, while at 1.0% w / v , the resultant hydrogels are too weak to handle for experiments. In addition, 1 U mL −1 HRP is used, since previous studies have reported an H 2 O 2 -mediated dynamic of the stiffening and softening of the hydrogels using a similar setup [ 15 , 17 ]. The scanning electron microscope (SEM) observation on the cross-section of the hydrogels showed a porous structure with a pore diameter of 28–42 µm ( Figure S2 ).…”

“…Recently, we have reported that hydrogen peroxide (H 2 O 2 ) could be used to control the physicochemical property of gelatin derivatives possessing phenolic hydroxyl moieties (Gelatin-Ph) hydrogel obtained from horseradish peroxidase (HRP)-catalysed cross-linking [ 15 ]. This system exploits the contradictory effect of H 2 O 2 that simultaneously induces the HRP-catalysed cross-linking as an electron donor while degrading the polymer as an oxidant ( Figure 1 a) [ 15 ]. The advantage of this system is that the mechanical property and molecular weight of the hydrogel can be controlled by a simple adjustment of the air containing H 2 O 2 exposure time.…”

“…The advantage of this system is that the mechanical property and molecular weight of the hydrogel can be controlled by a simple adjustment of the air containing H 2 O 2 exposure time. Using this system, the adhesion of stem cells and fibroblasts can be controlled [ 15 ]. However, there are no reports that study the effect of the dynamics of the physicochemical properties of the Gelatin-Ph hydrogel by H 2 O 2 to control the myogenesis.…”