## 4 Discussion
Here, oxygen release was monitored using an oxygen meter, which showed that the release of oxygen was at a maximum 24 h and subsequently gradually decreased up until 72 h. These data prove that a double layer matrix allows for the controlled release of oxygen. During the normal wound healing process, the extracellular superoxide dismutase (SOD) metabolites, such as H\({}_{2}\)O\({}_{2}\) and superoxide (O\({}^{2-}\)), play roles as cell signaling messengers that switch on the key processes of wound healing, such as cell motility, cytokine action, and angiogenesis. Several studies have reported that angiogenesis and wound healing respond differently with respect to H\({}_{2}\)O\({}_{2}\) concentration [28; 29]. At 10 nM (0.003%) H\({}_{2}\)O\({}_{2}\), both angiogenesis and wound healing are facilitated, while at 166 mM (0.5%), wound healing is delayed. Another study has reported that theadhesion and migration of cells occurs at H\({}_{2}\)O\({}_{2}\) concentration up to 100 \(\upmu\)M, with 20 \(\upmu\)M (0.0006%) being the most effective, suggesting the effects varying depending on the H\({}_{2}\)O\({}_{2}\) concentration. This is because, at low H\({}_{2}\)O\({}_{2}\) concentrations, MAP kinase activation leads to the increased production of VEGF and MMP-9, which are factors that induce cell proliferation and facilitate the production of MMP-1, a growth factor that activates cell migration [30]. H\({}_{2}\)O\({}_{2}\), derived from the action of extracellular SOD, plays a crucial role in facilitating the phosphorylation of the VEGFR thereby enhancing cellular growth and differentiation through the proliferation and migration of endothelial cells, leading to an acceleration of angiogenesis in the body. It is true that an excessive amount of reactive oxygen species such as H\({}_{2}\)O\({}_{2}\) is toxic to the cells; however, if provided at an appropriate concentration in the place required, H\({}_{2}\)O\({}_{2}\) can promote cellular growth and differentiation [31]. In this study, the amount of H\({}_{2}\)O\({}_{2}\) released from the newly developed ORHS and the amount of SOD generated were compared. As the data showed, the H\({}_{2}\)O\({}_{2}\) concentration in the ORMs was 6.6 \(\upmu\)M, whereas that in the ORHS was 5.8 \(\upmu\)M, an extremely small amount. These data provide indirect evidence that 0-100 \(\upmu\)M H\({}_{2}\)O\({}_{2}\) can enhance cell adhesion and migration to activate the growth factors, leading to accelerated angiogenesis. The oxygen generated through the decomposition of H\({}_{2}\)O\({}_{2}\) is a key molecule in cellular metabolism and plays a crucial role in most wound healing processes. Although diffusion allows for an oxygen supply at 40-200 \(\upmu\)m from capillaries or vascular endothelial cell membranes [32], when the diameter of the tissue to be transplanted exceeds 0.75-1.5 cm [33], the consequent limited diffusion makes the supply of oxygen and nutrients difficult, resulting in possible tissue necrosis [8; 34]. Thus, in the present study, the size of the ORMs was restricted to be 250 \(\upmu\)m and the ORHS was designed to fall within 1.0 cm, so that oxygen diffusion could be maintained. The structure that allows oxygen diffusion was observed using a scanning electron microscope and a porosimeter. This structure was based on dressings with a large surface area that induce interactions between cells and pore structures. These data lend support to there being stable oxygen diffusion during cell maintenance and regeneration, in concordance with previous studies. As shown, the ORHS developed in this study using a biocompatible macromolecular scaffold, could maintain the wetness around the wounded area and induce the secretion of various growth factors (TGF-\(\upalpha\), VEGF, FGF, EGF, MMP-2, MMP-9, etc.) and cytokines (IL-1 \(\upalpha\), etc.) from the skin cells, the result of which improves wound healing [35]. The ORHS creates an appropriate environment around the wounded area for healing and the stimulation of angiogenesis, while enhancing the fibroblast proliferation.