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

Abstract

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.

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

“…The dynamic trend in the mechanical property of the hydrogel that shows an initial increase, which peaked at 30 min, followed by a reduction in the Young’s modulus in prolonged exposure time ( Figure 2 d) is consistent with our previous studies on Gelatin-Ph hydrogel and Gelatin-Ph/HA-Ph composite hydrogel [ 15 , 17 ]. The decreased Young’s moduli of Gelatin-Ph hydrogel in prolonged exposure to air containing 16 ppm H 2 O 2 could be a consequence of cross-linking inhibition due to HRP inactivation.…”

“…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 ).…”

“…A stiffer substrate allows actin polymerisation and assembly to occur and the cells to maintain the cytoskeletal tension, which results in the cell elongation [ 37 , 38 ]. In contrast, the cells cultured on the soft substrate cannot form F-actin bundles and stress fibres; thus, the cells appear in a round morphology [ 17 , 39 ].…”

Methanol extract of Myelophycus caespitosus ameliorates oxidative stress-induced cytotoxicity in C2C12 murine myoblasts via activation of heme oxygenase-1

“…Induction of cytotoxicity, including DNA damage and apoptosis, by oxidative stimuli is mostly accompanied by cell cycle arrest. Many previous studies have indicated that the cytotoxic effect of H 2 O 2 is closely related to cell cycle arrest at the G2/M phase (Ding et al, 2019;Mubarok et al, 2022). Similar results were also observed in this study using C2C12 myoblasts due to an increase of Cdk inhibitor p21, a negative regulator of cell cycle progression, and decreases of cyclin A and cyclin B1 required for G2 to M phase progression (Dai et al, 2019;Taylor & Stark, 2001).…”

Sterilization of Polymeric Implants: Challenges and Opportunities

“…Benefits of H 2 O 2 sterilization are similar to those of other chemical sterilants, including relatively low operating temperatures and better compatibility with heat sensitive polymers. , Compared to EtO gas, however, vaporized H 2 O 2 has less effective penetration of porous scaffolds ,,,, Furthermore, although deliberate H 2 O 2 incorporation the in polymeric scaffold may be leveraged for certain drug delivery or tissue engineering applications, the hydroxyl free radical generated during H 2 O 2 sterilization has the potential to oxidatively cross-link polymers, resulting in deleterious changes. ,, Studies characterizing the morphological and biochemical changes induced by H 2 O 2 sterilization in UHMWPE revealed changes similar to those induced by gamma irradiation, including embrittlement . H 2 O 2 sterilization has also been observed to induce mechanical and degradative property changes to degradable polymers such as PLA. ,, Lastly, it is of note that H 2 O 2 sterilization was found to be ineffective when paired with cellulose-based products due to the high tendency of cellulose to absorb H 2 O 2 , thereby reducing its effective concentration .…”