Hematoxylin and eosin h e staining

All mice of the four groups were anesthetized using isoflurane (2-3% to induce anesthesia and 1.5-2% to maintain anesthesia) at day 14 post-operation, and wound tissues were harvested for histological analysis. The mice were then euthanized using CO₂ (injected into the box at a rate of 30% of the volume every min; the maximum flow rate did not exceed 0.5 kPa). The mice were considered dead when they maintained respiratory arrest, no motion, and pupil dilation for >5 min.
Wound samples were fixed in 4% paraformaldehyde at room temperature overnight and then dehydrated and embedded in paraffin. The samples were cut into 5-μm thick sections and mounted on slides for staining. Hematoxylin and eosin (H&E) staining (Beyotime Institute of Biotechnology) and Masson trichrome staining (Beyotime Institute of Biotechnology) were performed according to the manufacturers' instructions. The length of the scar and maturity of collagen were observed under a light microscope (BX21; Olympus Corporation).

Cells from their different groups were harvested, trypsinized for digestion, centrifuged, and resuspended in a culture medium. A cell count was performed, and cell density was adjusted to 1 × 10⁶ cells/mL, and 200 µL of the cell suspension was seeded into the upper chamber of a Transwell apparatus. The lower chamber was filled with 500 μL of DMEM culture medium containing 10% serum, and the setup was incubated for 24 hours. Following incubation, the Transwell chamber was removed, and cells were fixed with 4% paraformaldehyde for 15 minutes. Hematoxylin and eosin (HE) staining was then performed according to the manufacturer's instructions (Beyotime). Ten random fields of view were selected for cell counting under an inverted microscope.