The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H2O2)-mediated cross-linking and degradation of the polymers by varying the exposure time to H2O2 contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young’s modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H2O2 exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H2O2-mediated degradation. These results indicate that H2O2-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.

Engineering skeletal muscle tissue in vitro is important to study the mechanism of myogenesis, which is crucial for regenerating muscle cells. The physicochemical properties of the cellular microenvironment are known to govern various cell behaviours. Yet, most studies utilised synthetic materials to model the extracellular matrix that suffers from cytotoxicity to the cells. We have previously reported that the physicochemical property of hydrogels obtained from horseradish peroxidase (HRP)-catalysed cross-linking could be controlled by a simple adjustment to the exposure time to air containing H2O2. In this study, we evaluated the influence of physicochemical properties dynamics in the gelatin possessing phenol groups (Gelatin-Ph) hydrogel to regulate the myogenesis in vitro. We controlled the Young’s modulus of the Gelatin-Ph hydrogel by tuning the air containing 16 ppm H2O2 exposure time for 15–60 min. Additionally, prolonged exposure to air containing H2O2 also induced Gelatin-Ph degradation. Myoblasts showed higher adhesion and myotube formation on stiff hydrogel (3.53 kPa) fabricated through 30 min of exposure to air containing H2O2 compared to those on softer hydrogel (0.77–2.79 kPa) fabricated through 15, 45, and 60 min of the exposure. These results demonstrate that the myogenesis can be tuned by changes in the physicochemical properties of Gelatin-Ph hydrogel mediated by H2O2.

No abstract

The study of the capillary-like network formation of human umbilical vein endothelial cells (HUVECs) in vitro is important for understanding the factors that promote or inhibit angiogenesis. Here, we report the behavior of HUVECs on the composite hydrogels containing hyaluronic acid (HA) and gelatin with different degrees of degradation, inducing the different physicochemical properties of the hydrogels. The hydrogels were obtained through horseradish peroxidase (HRP)-catalyzed hydrogelation consuming hydrogen peroxide (H2O2, 16 ppm) supplied from the air, and the degradation degree was tuned by altering the exposure time to the air. The HUVECs on the composite hydrogel with intermediate stiffness (1.2 kPa) obtained through 120 min of the exposure were more elongated than those on the soft (0.4 kPa) and the stiff (2.4 kPa) composite hydrogels obtained through 15 min and 60 min of the exposure, respectively. In addition, HUVECs formed a capillary-like network only on the stiff composite hydrogel although those on the hydrogels with comparable stiffness but containing gelatin alone or alginate instead of HA did not form the network. These results show that the HA/gelatin composite hydrogels obtained through the H2O2-mediated crosslinking and degradation could be a tool for studies using HUVECs to understand the promotion and inhibition of angiogenesis.