Background The absence of hair follicles and other skin appendages regeneration is a difficult problem in the field of wound healing, especially cicatricial alopecia caused by scalp (burn) injury, which seriously affects the life and work of patients. Small molecule compounds can regulate cell proliferation and differentiation, promote the redifferentiation of mature cells into proliferative precursor cells or other cell lines, and also target the regulation of signaling pathways and metabolic processes, which is a safe and effective method to promote the in situ regeneration of skin and skin appendages.
Objective To explore the effect and mechanism of promoting hair follicle regeneration in mouse wound through the combination of small molecule compounds.
Methods Forty-five female C57BL/6 mice with a weight of (20 ± 5) g were selected in this study. The animal model of full-thickness skin defect on both sides of the back of mice was made with perforator and ophthalmic scissors. The left side of the wound was the drug side, and the right side of the wound was the control side. We prepared the Vaseline ointment containing the small molecules CHIR-99021, Purmorphamine, Tofacitinib, SJ000291942 and applied the ointment on the mouse skin wounds for 28 days. HE staining, Masson staining, and immunohistochemistry were performed to analyze the condition of wound healing and hair follicle regeneration.
Results There was no significant difference in the healing rate between the treatment group and the control group, suggesting that hair follicle regeneration ointment had no significant effect on the healing rate of the wound. HE staining showed that about 14 days after treatment of the wound with hair follicle regeneration ointment, hair germ cells were formed on the wound epidermis and dermal papilla cells were found in the treatment group. On the 28th day, a large number of follicle-like structures could be seen on the wound surface, which was confirmed by immunofluorescence staining to express characteristic markers of hair follicle cells (CK5, CK15, CK17 and Noggin). However, the number of hair follicles regenerated after applying ointment alone was very small, and there was a significant statistical difference in the number of new hair follicles between the treatment group and the control group (17 vs 4, P < 0.001). Masson staining showed that there was no significant difference in the density and arrangement of collagen fibers between the treatment group and the control group, suggesting that hair follicle regeneration ointment could induce hair follicle regeneration in fiber scar. Immunohistochemical staining showed that markers related to Wnt signaling pathway and sonic hedgehog (Shh) signaling pathway were obvious in the newly formed epidermal cells, hair germ cells, and newly formed dermal papilla cells on the drug side, suggesting that hair follicle regeneration ointment might promote hair follicle regeneration on the wound by activating Wnt and Shh signaling pathway.
Conclusion In this study, the self-made hair follicle regeneration ointment can promote hair follicle regeneration during wound healing by regulating the Wnt signaling pathway and Shh signaling pathway, which is expected to be transformed into a wound therapy drug for clinical application.
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