*4.6. Macroscopic Evaluation*

Appetite and general health conditions were monitored daily, and body weight was measured every other day. For wound area study, the wound beds were photographed with a digital camera and a ruler at the specific time points (0 d, 3 d, 5 d, 7 d, 9 d, and 11 d). Wound images were used to calculate the wound area by the NIH/Image J software. The following equation was used to measure the rate of wound closure,

$$\mathbf{y}(\mathbf{n}) = \frac{\mathbf{A}\_0 - \mathbf{X}\_{\mathbf{n}}}{\mathbf{A}\_0} \cdot 100\% \tag{1}$$

where y (n): the rate of wound closure (%); A0: wound area at day 0; Xn: wound area at day (n). Wound healing curves were constructed by Graph Pad Prism 5.

#### *4.7. Sample Collection and Histological Analysis*

To examine re-epithelialization, granulation tissue, vessel counts, and collagen content of the wounds, three mice from each group were euthanized at 3, 5, 7, and 10 days after treatment. The entire wound and adjacent wound were harvested down to the fascia, and then bisected through the center of the lesion to ge<sup>t</sup> the largest diameter of the wound, one-part tissue was stored in liquid nitrogen to detect the expression of relative factors and another part was fixed in formalin solution (4%) for histological evaluation of wound healing. The fixed tissue samples were routinely processed and embedded in para ffin, 4 μm sections of middle wound bed were stained with hematoxylin and eosin (HE), CD31 antibody and Masson Trichrome. The positive area of CD31 and collagen deposition were measured by NIH/Image J software (https://imagej.net/NIH\_Image).

## *4.8. RNA Extraction and qRT-PCR*

Total RNA was extracted frozen tissues of wound healing models by using Trizol reagen<sup>t</sup> (Takara, Japan) according to the manufacturer's instructions. The purity and concentration of total RNA were determined by UV Spectrophotometer (Eppendorf, Hamburg, Germany) and 1% agarose gel electrophoresis. The first-stand cDNA was synthesized by the HiScript ®II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) following the manufacturer's instructions. The primers of the quantitative real-time PCR (qRT-PCR) was designed by Prim5 software (Table 2). The qRT-PCR was used to detect relative expression of b-FGF, PDGF-BB, TGF-β, VEGF, and MMP-2 by an ABI 7500 real-time PCR detection system (ABI, Carlsbad, CA, USA), and the relative expression of these genes were normalized by an internal control (β-actin). The qRT-PCR final reaction volume was 20 μL which was added according to the instructions, and under the following conditions: 2 min of pre-denaturation at 95 ◦C, followed by the 30 cycles for 15 s, 59–60 ◦C for 15 s, and 72 ◦C for 30 s, and each reaction was performed in triplicate. Relative expression of genes was calculated using the 2−ΔΔCT method.


