*3.2. Experimental Materials*

Sixteen New Zealand rabbits (half male and half female, conventional grade) weighing 2000 ± 20 g before modeling were provided by Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Guangdong) 2014-0035, and the laboratory animal quality certificate number is 44411000004907. The rabbits were individually housed with free access to water and food under the following conditions: 25 ◦C temperature, 40–70% humidity, and 12 h light/12 h dark cycles. Experimental study on scald repair of CS-GT hydrogels after one week of adaptive breeding. Animal experiments were undertaken according to guidelines set by the Experimental Animal Center of Guangdong Ocean University (Guangdong, China) for the care and use of laboratory animals (SYXK (Yue) 2014-0053). This study was approved by the Animal Ethics Committee of Guangdong Ocean University.

#### *3.3. Preparation of CS-GT Hydrogel*

To fabricate CS-GT hydrogel, 8 g of PVP was firstly dissolved in 32 g of distilled water, which was then mixed with 20 g CS-GT aqueous solution containing 5 g of CS-GT [31] and 40 g glycerin under magnetic stirring till a homogeneous solution was obtained. GT hydrogel was prepared using the same method, where 5 g GT was introduced to replace the 5 g CS-GT. For comparison, the matrix was also prepared based on the same procedures without CS-GT and GT loaded.

#### *3.4. In Vitro Antibacterial Assay*

As *S. aureus* and *P. aeruginosa* often exist in patient exudate and are important pathogenic strains causing infections in burn/scald patients [43]. These two strains were chosen in this study as indicator microorganisms, followed by antimicrobial assay of CS-GT via diameter of zone of inhibition [44] according to the following procedure: 100 μL of bacterial suspension (1 × 10<sup>8</sup> CFU/mL) was evenly plated onto NB agar plate, and a sterile paper disc (6.0 mm in diameter, sterilized by autoclaving) was placed on the agar surface, onto which a test sample was then added dropwise. After the sample was incubated at 37 ◦C for 24 h, the diameter of the zone of inhibition was determined. The solvent of 1% HAc, CS-GT, and GT was distilled water, while the solvent of CS was 1% HAc. The concentration of all samples was 1 mg/mL. GT's graft rate was 22.1%, as reported in our previous research [31].

#### *3.5. In Vitro Cytotoxicity Study*

In this study, MTT assay was used to determine the e ffect of CS-GT on the viability of human skin fibroblasts (L929) so as to evaluate it in vitro cytotoxicity. A normal control group (Control) and test groups of CS-GT at various concentrations (100, 200, 300, 400, 600, 800, 1000, and 1200 μg/mL, respectively) were set up. Cells in good growth state were selected, and after adjustment of cell concentration, they were seeded onto a 96-well culture plate at a plating density of 5 × 10<sup>3</sup> cells/well (100 μL per well, 6 replicate wells per group); then, the plate was put into a cell incubator (37 ◦C, 5% CO2) and incubated for 24 h. After the culture medium was pipetted o ff, blank medium and DMEM medium for CS-GT at varied concentrations were added to the wells (100 μL/well), respectively, and the plate was put into the incubator again for further culturing. Cell viability assay was performed for each group by MTT after every 1 day of culture for 3 consecutive days to study the e ffects of CS-GT in varying action time on L929 viability [45]. The absorbance at 570 nm was measured by an RT-2100C microplate reader.

Cell viability was calculated using the following formula:

$$\text{Cell viability} (\text{\textquotedblleft} \text{\textquotedblright}) = \frac{OD\_{\text{test\textquotedblright}} - OD\_{\text{blank\textquotedblleft}\text{\textquotedblright}}}{OD\_{\text{control\textquotedblright}} - OD\_{\text{blank\textquotedblright}}} \times 100\%$$

In addition, in order to have a more straightforward observation of the compatibility of the test sample with L929 cells, the cells in this study were stained using a Calcein-AM/PI Double Stain Kit to observe cell viability state [46]. Cell suspension was prepared with 1× assay bu ffer with density ranging from (1 × 105) to (1 × 106) cells/mL. Then, such cell suspension was divided into two aliquots, into which CS-GT (100 μg/mL, 50 μL) and GT (100 μg/mL, 50 μL) were added, respectively, followed by incubation for 24, 48, and 72 h, respectively. Then, 100 μL of staining working solution was added into 200 μL of each part of cell suspension, mixed well, incubated at 37 ◦C for 15 min, and stained; then, cell morphology and staining status were observed using an inverted fluorescence microscope.

## *3.6. Hemolysis Assay*

According to the literature [36], the hemolysis rate of CS-GT was determined as follows: 3 mL of rat blood was centrifuged at 2000 rpm for 15 min to isolate RBCs from serum, and then the above RBC sediment was washed with 1× PB bu ffer three times until a clear supernatant was observed. Next, the RBC sediment was diluted with PBS to a concentration of 2% (*v*/*v*) for later use, while RBCs incubated with deionized water and PBS were used as the positive and negative controls, respectively. GT and CS-GT solutions at various concentrations (100, 200, 400, 800, 1600, and 3200 μg/mL) were added into the above 2% RBC suspension, respectively. RBS suspension samples prepared as above were incubated at 37 ◦C for 1 h and then centrifuged at 2000 rpm for 15 min; the image of each centrifuged

sample was captured with a digital camera, and absorbance of each supernatant was measured at 540 nm with a plate reader. The hemolysis rates were calculated using the following formula:

$$H\% = \frac{H\_1 - H\_0}{H\_{100} - H\_0} \times 100\%$$

where *H0*, *H1*, and *H100* are absorbance values of the negative control (PBS), test sample, and positive control (H2O), respectively.

In addition, GT-treated RBCs and CS-GT-treated RBCs at a concentration of 800 μg/mL were microscopically examined as follows for any morphological change: After incubation at 37 ◦C for 1 h, the RBC solutions were centrifuged at 20,000 rpm for 20 min. The supernatants were photographed using a digital camera. The collected RBC pellets were diluted in PBS, dispensed onto clean glass slides, covered with a coverslip, and then photographed by using a microscope equipped with a digital camera.

#### *3.7. In Vivo Animal Test*

Twenty-four hours before the experiment, each New Zealand rabbit was dehaired using 10% sodium sulfide, then, the dehaired area was immediately cleaned with lukewarm water and the rabbits housed in a single cage. Briefly, 3% pentobarbital sodium (30 mg/kg) was injected via ear vein to anesthetize the New Zealand rabbit and 75% ethanol was used to disinfect the skin of dorsal hair loss area, then, 5 scald wounds of 1.5 cm<sup>2</sup> in area were created laterally at points 2 cm away from the dorsal midline of the rabbit by using a scalding apparatus. The scalding conditions were described as follows: probe temperature, 100 ◦C; working pressure, 1000 g; probe-to-skin contact time, 6 s. Each animal was self-controlled, and 5 wounds of each rabbit correspond to the blank group, matrix group, positive control group, GT group, and CS-GT group, respectively, as shown in Figure 12.

**Figure 12.** Correspondence of groups to scald wounds.

After modeling, scald wounds exhibited typical scald features: pale opalescent appearance and dry surface. In the case of the blank group, no treatment was performed after successful modeling. In the case of matrix group, the scald was daily treated by applying PVP hydrogel, once in the morning and once in the evening (about 1 g/dose) for 21 consecutive days starting on day 1 after modeling. In the cases of the positive control, GT, and CS-GT groups, moist exposed burn repair ointment (MEBO), GT hydrogel, and CS-GT hydrogel were applied, respectively, in the same manner as in the case of the matrix group. On the day of modeling and upon dressing change each time, size, color, exudate, scab formation, and scab falloff of each wound were inspected, photographed, and documented. On days 3, 7, 14, and 21, variations in wound area were determined as follows to evaluate wound healing: wound closure was monitored by imaging with a digital camera and a scale bar, and wound

area was quantitated by Photoshop so that pixel area was calculated from wound contour plotted by a blinded observer. Then, wound healing rate *M* was calculated using the following formula:

$$M\% = \frac{M\_0 - M\_1}{M\_0} \times 100\%$$

where *M0* is the initial wound area, and *M1* is wound area at a time point.
