Evaluation of wound healing

All animals survived and no complications related to the procedure, including infection, were observed. After treatment on days 3, 7, and 14, the wound areas were calculated in cm². The surface area on the first day after the wounds were prepared was deemed to be 100%, and subsequent changes were compared to that initial area.

On day 7 after wounding, rats from each group were sacrificed and tissue was collected from near the wound site and the wound itself. Tissues were fixed in a 10% formalin solution for hours and prepared for hematoxylin and eosin (H&E) staining to compare the status of re-epithelialization [9]. Re-epithelialization (%) was calculated by sum of each edge's epithelialized area, which was divided by total lengths of wound.

On day 14 after wounding, rats from each group were sacrificed and tissues were collected and stained (H&E stain kit, American MasterTech, Lodi, CA, USA) to assess angiogenesis in the newly forming skin tissue. Collagen deposition over the wound dressing was assessed as the level of inner skin deposition, and the thickness was evaluated by assessment of the full recovery of the impaired skin tissue (Masson's trichrome staining) [9]. Masson's trichome is a 3-color staining protocol used in histology. This method is used for the detection of collagen fibers in tissues such as skin, heart, etc. The collagen fibers will be stained blue and the nuclei will be stained black and the background is stained red. Tissues which stained with Masson's trichrome staining were evaluated its collagen density by using polarized light microscope with the aid of image analyzer software, Image J program (National Institutes of Health, Bethesda, MD, USA).

To evaluate the tissue invasion for exchanged dressings, the amount of residual DNA remaining in the dressings was measured on days 3, 7, 10, and 14. The total DNA was quantified with the CyQuant DNA kit according to the manufacturer's description (Molecular Probes, Eugene, OR, USA) using a fluorescent plate reader (emission, 520 nm; excitation, 480 nm) (Synergy H1, BioTek, Winooski, VT, USA). The standard curve for DNA analysis was generated with λ DNA provided with the CyQuant DNA kit.