*2.3. Hemolysis Assay*

Hemolysis assay can be used to evaluate material hemocompatibility as a hemolysis rate of ≤5% meets the hemolysis criterion for biomaterials [35]. Based on the hemolysis results of GT and CS-GT in Figure 4, the hemolysis rate of GT at a concentration of 100 μg/mL was lower than 5%, while the hemolysis rate of GT at a concentration of 200 μg/mL or above was higher than 5%. The hemolysis rate of CS-GT at a concentration of 800 μg/mL or below was lower than 5% (Figure 4a), indicating that CS-GT at the concentrations below 800 μg/mL has no hemolytic activity on red blood cells (RBCs) and is more hemocompatible than GT [36]. At the same concentrations, there was no significant difference in hemolysis rates between the two groups. As shown in Figure 4b, the content of cell-released hemoglobin increased with the increase of GT and CS-GT concentrations, and the higher the content of released hemoglobin, the stronger the hemolytic activity [37]. However, in general, at a concentration below 800 μg/mL, GT induced higher hemoglobin release than CS-GT, indicating that hemolytic activity of CS-GT was lower than that of GT. In addition, in order to further assess the hemolytic properties of GT and CS-GT, the morphology of RBCs treated with GT and CS-GT at 800 μg/mL was microscopically observed, and the results are shown in Figure 4c. After treatment with water, all RBCs were disrupted basically. Additionally, after treatment with PBS, RBCs were round-shaped and basically free of damage. As compared with PBS-treated RBCs, GT-treated RBCs exhibited more prominent damage and rupture in morphology, while only a minimal part of CS-GT-treated RBCs was damaged, without significant morphological variation in general. The above results indicate unanimously that the hemocompatibility of CS-GT is superior to GT.

**Figure 4.** (**a**) Hemolysis rates of RBCs treated with GT and CS-GT, respectively; (**b**,**<sup>c</sup>**) images of RBCs treated with GT and CS-GT, respectively (mean ± SD, *n* = 3).

#### *2.4. Wound Macroscopy and Healing Rates*

Based on information on scald wound healing in each group at various time points in Figure 5, the scald wounds appeared subcircular and edematous, and their surfaces were softened and blanched on the day of modeling (Day 1). Over time, scald areas in each group turned smaller gradually, while the wound healing rate tended to increase (Table 2). Throughout the healing cycle, blank group and matrix group did not differ significantly in macroscopic wound change, and no significant difference in wound healing rate was noted in either group, indicating that the PVP matrix has no significant impact on wound healing. On day 7, the edema on the wound surface in each group began to resolve; however, in the case of the blank group, such edema was associated with certain tissue fluid exudation and purulent substance secretion and inflammatory response around the wound was more significant. On day 14, the peripheral scab of each scald wound in each group began to fall <sup>o</sup>ff, and the wound surface was dry without tissue fluid exudation and purulent substance secretion. The wound turned smaller without a distinct boundary with surrounding normal tissue, whereas effects on wound shrinkage in the positive control group, GT group, and CS-GT group were superior to those in the blank group and matrix group to a varied extent. On day 21, in the positive control group and CS-GT group, scald wound scabs basically fell off; however, a swelling phenomenon occurred in the positive control group where the scabs fell <sup>o</sup>ff. In the cases of the blank group, matrix group, and GT group, a minor amount of scabs were still attached to wound cores and appeared pale red.

A wound healing rate of 100% is a criterion for trauma repair. As shown in Table 2, throughout the healing cycle, the blank group and matrix group had no significant difference in wound healing rate, indicating that the matrix has no prominent action on wound healing. On day 3 after scalding, wound healing rates in the blank group were all negative, possibly because blank samples had no anti-inflammatory effect after scald wound modeling. When various proinflammatory cytokines were triggered, such wounds started to ge<sup>t</sup> inflamed and edematous, resulting in a larger wound area. On day 3, compared with the blank group, the GT and CS-GT groups exhibited significant wound healing effects, suggesting that GT and CS-GT exhibited stronger antimicrobial effects in the early phase of wound healing to reduce infection probability and shorten the inflammation phase so that the proliferation phase came in advance. On day 7 after trauma, wound healing rates in the blank group were the lowest, although wound healing rates in the positive control, GT, and CS-GT groups did not differ from those in the blank group significantly, which increased to a varying extent. The wound healing effects in the GT and CS-GT groups were the best, which was thought to be closely associated with the strong antibacterial activity of GT and CS-GT. On day 14, wound healing rates in the CS-GT group were 89.18% ± 11.75%, significantly different (*p* < 0.05) from those in the blank group (55.88% ± 7.07%). On day 21, in CS-GT group, the healing rates reached 99.61% ± 0.23% (*p* < 0.01 vs. blank group), and wounds were basically intact, while in the blank group, the healing rate was only 75.45% ± 2.17% and scabs did not fall <sup>o</sup>ff, which were congruen<sup>t</sup> with the above macroscopic wound recovery. In the early phase of trauma, healing in the positive control group was slower than that in the CS-GT group. However, in the late phase of wound healing, the positive control sample had better trauma repair function, and wound healing rates on day 21 reached 94.98% ± 0.04%, only lower than those in CS-GT group. In contrast, GT had no trauma repair effect, and its healing effect in the late phase was inferior to that of CS-GT, with wound healing rates of only 87.50% ± 2.09% after 21 days of action. A synergic effect was observed to occur between CS and GT in the CS-GT group, and therefore in the late phase of trauma healing, the wound healing rates in the CS-GT group were significantly higher than those in the GT group.

**Figure 5.** Different phases of wound healing in New Zealand rabbits treated with hydrogels.

**Table 2.** Wound healing rates (%) at different times in each group of scald wounds (mean ± SD, *n* = 4). Note: versus blank control group, \* *p* < 0.05, \*\* *p* < 0.01.

