## 3 Results
### In Vivo Evaluation  
In wound group using ORHS, wound size was smaller than that of gauze group and alginate group, and surface scar was also improved. Especially after 3 days of injuries, the size of the wound covering the ORHS group decreased compared to the gauze group and the suture was maintained from 5 th to 7 th day (Figure 8). An immunohistochemical analysis of the wound tissue collected from each group on days 1 and 7 showed that the distribution of inflammatory cells (red) on day 1 became more clearly distinguishable in the 'with ORHS' group than in the control group or in the 'without ORHS' group, and a complete formation of the skin layer was observed on day 7 (Figure 9). Neutrophils were detected in the wound area in all three groups, although on day 7 there was a slight decrease only in the 'with ORHS' group. These data provide indirect proof that the release of oxygen prevents infection at the wounded area and consequently reduces the inflammatory reaction. The results of Masson's Trichrome  
Figure 6: Cell proliferation (CCK-8 assay) in the alginate group and oxygen-releasing hydrogel sponge (ORHS) group. Cell proliferation was observed for 7 days. No toxicity compared to the control was observed. Therefore, the ORHS did not affect cell viability. The assay was performed in hypoxia conditions (1% O\({}_{2}\), 5% CO\({}_{2}\), 94% N\({}_{2}\)) (n = 5).  
Figure 7: Scratch wound closure model using HaCaT cells; (**A**) Cells only, (**B**) alginate group, (**C**) oxygen-releasing hydrogel sponge (ORHS) group. Wound closure was significantly faster with ORHS. The ORHS group significantly increased epithelial keratinocyte cell migration.  
staining to examine collagen deposition showed that the healing wound had significantly increased numbers of fibroblasts(blue). The 'with ORHS' group, in particular, showed larger blue stained areas compared to the control, indicating that the release of oxygen allowed for the effective synthesis of collagen in the wounded area. Furthermore, the ORHS was found to improve the wound healing process based on the levels of gene expression of the angiogenesis factor VEGF. The 'with ORHS' group showed enhanced VEGF expression and a facilitation of the wound healing process was also observed.  
Figure 8: **(Left)** Gross morphology of wounds at different time points after surgery. (**Right**) Wound closures were observed on days 1, 3, 5, and 7 after wound generation via surgery; (A) full thickness skin defect group, (B) gauze group, (C) alginate group, (D) oxygen-releasing hydrogel sponge (ORHS)group.  
Figure 9: Histologic findings of the wounds at postoperative day 7 of the (**A**) full thickness skin defect group, (**B**) gauze group, (**C**) alginate group, and (**D**) oxygen-releasing hydrogel sponge (ORHS) group. Control groups showed that the dermis layer was uneven and remained patent area. The epidermal layer which was covered by new epithelial keratinocytes was also formed irregular. The alginate group (**C**) showed no patent area of dermis layer. But, epidermal layer was irregular. The ORHS group (**D**) had a uniform dermis layer and hair follicles were present in the wound area. The epidermal layer was regular as layer-by-layer. Control groups showed that dermis layer was uneven and remained patent area (H&E, \(\times 100\)). On the 7 th day, a mature arrangement of collagen fiber was shown in the alginate group (**C**), and ORHS group (**D**) (Tirchrom, \(\times 100\)). On the 7 th day, increased TGF-\(\beta 1\) expression was shown in (**D**) and increase VEGF expression was shown in (**D**) (Immuno-histochemical staining, \(\times 100\)).