*2.5. Histological Observation*

In order to further study the effects of the hydrogel on the regeneration of wound epidermis and dermis, skins of animals in each group were sampled on days 3, 7, 14, and 21, respectively, followed by hematoxylin and eosin (HE) staining to investigate wound tissue reepithelialization. As shown in Figure 6, on day 3 after scalding, epidermal layers in each group were seriously damaged, vesicles and inflammatory cell infiltration were visible in dermal layers, and dermal interstitium became loose. However, as compared with the blank group, inflammatory cell infiltration was milder in the GT and CS-GT groups. On day 7 after scalding, edema and many inflammatory cells were still present in the blank and matrix groups, and fewer inflammatory cells were observed in the

positive control group. In contrast, no epithelial regeneration was observed in all three groups. The inflammatory cell infiltration in the GT group was the mildest, whereas that in the CS-GT group was worse. In both groups, epithelial regeneration was observed. On day 14 after scalding, in addition to mild inflammatory cells, hyperkeratosis was also observed in the blank, matrix, and positive groups, while in the GT and CS-GT groups, complete epidermal regeneration was observed. On day 21 after scalding, scabs did not fall off completely, particularly in the blank group, and epidermal regeneration and angiogenesis, to some extent, were observed in other groups, especially in the CS-GT group, where connective tissue recombination was the best and previously unordered tight structure became more regular. In general, the skin healing process in the CS-GT hydrogel group was basically completed on day 21. The animals in CS-GT group had the thickest dermis, dense dermal mesenchyme, and spindle-shaped fibroblasts, indicative of optimal recovery.

**Figure 6.** Hematoxylin and eosin (HE) staining images showing pathological changes of skin in various groups (200×).

The final tissue remodeling phase of trauma repair is tissue maturation and extracellular matrix deposition, mainly manifested as a gradual increase and recombination of collagen fibers [38]. Masson staining is classical staining of collagen fibers; in this study, skins at original scald sites in each group were sampled on day 21 for Masson staining to check collagen content wherein. As shown in Figure 7, compared with normal skin, the skin in the blank and matrix groups had fewer stained collagen fibers, which were sparsely and unorderly arranged. In contrast, in the positive control, GT, and CS-GT groups, the content of stained collagen in the skin increased significantly. Additionally, in the case of the CS-GT group, collagen fibers in the skin were orderly arranged with more uniform density.

**Figure 7.** Masson staining on Day 21 postinjury (200×).

In addition, Image-pro plus software was used to quantitate the collagen fiber content (expressed as mean optical density) in a Masson plot. As shown in Figure 8, the collagen fiber contents of skin in the matrix group were comparable to those in the blank group, indicating that the PVP matrix has no significant impact on collagen fibrogenesis in the scald-skin healing process. In CS-GT group, the collagen fiber content in the skin was close to the level in normal skin, which was significantly different from those in the blank group (*p* < 0.05). The results of the positive control and GT groups were intermediate between those of the blank group and normal skin. Therefore, it can be found that the scald-skin healing effect in the CS-GT group was superior and the efficacy was most significant.

**Figure 8.** Mean density values of collagen in scald wound in various groups (mean ± SD, *n* = 4). Note: versus blank control group, \* *p* < 0.05.

#### *2.6. TP and HYP Contents in Granulation Tissue*

After a skin trauma, protein in granulation tissue will be substantially hydrolyzed, enabling the growth of a grea<sup>t</sup> number of bacteria at the wound, which will affect the epidermal formation and further wound healing rate. TP is the content of all proteins during the growth of granulation tissue on the wound surface. As shown in Figure 9, the content of TP in skin wound granulation tissue in each group increased gradually over time. Throughout the study cycle, the TP level in the skin in the blank group was basically consistent with that in the matrix group, without significant difference, indicating that PVP has no significant impact on TP content in tissue. On day 3 after scalding, compared with the TP content in the blank group, the TP contents in the positive control, GT, and CS-GT groups increased to some extent, among which the GT group had the highest TP content. On day 7, there was a trend similar to that on day 3. However, for the CS-GT hydrogel group relative to that in the blank group, the increase in TP content was significantly different (*p* < 0.05). From days 14 to 21, variations in TP content in the blank group were smaller, whereas an increase in TP in other groups was significant. On day 21, the TP content in CS-GT group was the highest with a significant difference from that in the blank group (*p* < 0.01). In summary, compared with GT alone, CS-GT was more capable of accelerating protein synthesis in wound granulation tissue.

**Figure 9.** Total protein (TP) contents of wounds in various groups (mean±SD, *<sup>n</sup>*=4). Note: versus blank control group, \* *p* < 0.05, \*\* *p* < 0.01.

During wound healing, connective tissue mainly consists of fibroblasts and collagen, and collagen content has an immediate impact on the wound repair process. HYP is a precursor amino acid of collagen synthesis and the HYP content of granulation tissue represents the collagen level; therefore, a study on HYP content enables further reflection of wound healing [39]. Based on variations in HYP content in various groups (Figure 10), on day 3 after scalding, as compared with normal skin, scalding led to a significant decrease in HYP content in skin wound tissue. From days 3 to 21, HYP content in wound tissue in each group increased gradually, among which, HYP content in CS-GT group at various time intervals was higher than those in the blank, matrix, and positive groups. The HYP content in the CS-GT group on days 7 and 21 exhibited significant di fferences from that in the blank group (*p* < 0.05). Additionally, from day 7 on, HYP contents in both the GT and CS-GT groups were close to that in normal skin. The above findings indicate that CS-GT hydrogel is able to e ffectively promote HYP synthesis in traumatic tissue and accelerate collagen synthesis and accumulation so as to provide a material basis for expediting wound healing.

**Figure 10.** Hydroxyproline (HYP) contents of wounds in various groups (mean ± SD, *n* = 4). Note: versus blank control group, \* *p* < 0.05.

#### *2.7. Expression of Proinflammatory Factors*

After skin damage, the damaged tissue will undergo a slow-onset persistent inflammatory response focusing on the release of pro-inflammatory factors TNFα and IL-6, among which, TNFα is capable of stimulating vascular endothelial cells to regulate cell metabolism within an organism [40], while IL-6 has an immediate impact on the growth of fibroblast and endothelial cells [41]. However, the persistent presence of excessively high levels of TNFα and IL-6 in tissues would enhance the toxicity of inflammatory cells and induction capacity of related inflammatory mediators, and eventually, leading to damage and necrosis of more tissue cells [42].

Based on the levels of TNFα and IL-6 in wound tissue in various groups (Figure 11), throughout wound healing, the levels of TNFα and IL-6 in skin tissue in the blank group did not di ffer significantly from those in matrix group, indicating that PVP matrix has no significant impact on the inflammatory response of wound tissue. From days 3 to 21 after scalding, the IL-6 levels in wound tissue in various groups tended largely to decrease gradually over time and were all higher than the level (Normal) in normal skin. In particular, in the early phase (days 3 and 7), the IL-6 levels in the skin in the GT and CS-GT groups decreased significantly, suggesting that the test article has a good inhibitory e ffect against early inflammation. On day 21, only during the study cycle, the TNFα level in the CS-GT group was significantly lower than that in the blank group and close to the level in normal skin, and such levels in all the other groups did not di ffer significantly from the level in the blank group. Therefore, CS-GT hydrogel is superior to that of positive control, and GT can alleviate the local inflammatory response of a wound to accelerate scald wound healing.

**Figure 11.** Levels of TNF-α (**a**) and IL-6 (**b**) in wounds in various groups (mean ± SD, *n* = 4). Note: versus blank control group, \* *p* < 0.05.

#### **3. Materials and Methods**
