Molecular hydrogen promotes wound healing by inducing early epidermal stem cell proliferation and extracellular matrix deposition.

Abstract

Background: Despite progress in developing wound care strategies, there is currently no treatment that promotes the self-tissue repair capabilities. H₂ has been shown to effectively protect cells and tissues from oxidative and inflammatory damage. While comprehensive effects and how H₂ functions in wound healing remains unknown, especially for the link between H₂ and extracellular matrix (ECM) deposition and epidermal stem cells (EpSCs) activation.

Methods: Here, we established a cutaneous aseptic wound model and applied a high concentration of H₂ (66% H₂) in a treatment chamber. Molecular mechanisms and the effects of healing were evaluated by gene functional enrichment analysis, digital spatial profiler analysis, blood perfusion/oxygen detection assay, in vitro tube formation assay, enzyme-linked immunosorbent assay, immunofluorescent staining, non-targeted metabonomic analysis, flow cytometry, transmission electron microscope, and live-cell imaging.

Results: We revealed that a high concentration of H₂ (66% H₂) greatly increased the healing rate (3 times higher than the control group) on day 11 post-wounding. The effect was not dependent on O₂ or anti-reactive oxygen species functions. Histological and cellular experiments proved the fast re-epithelialization in the H₂ group. ECM components early (3 days post-wounding) deposition were found in the H₂ group of the proximal wound, especially for the dermal col-I, epidermal col-III, and dermis-epidermis-junction col-XVII. H₂ accelerated early autologous EpSCs proliferation (1-2 days in advance) and then differentiation into myoepithelial cells. These epidermal myoepithelial cells could further contribute to ECM deposition. Other beneficial outcomes include sustained moist healing, greater vascularization, less T-helper-1 and T-helper-17 cell-related systemic inflammation, and better tissue remodelling.

Conclusion: We have discovered a novel pattern of wound healing induced by molecular hydrogen treatment. This is the first time to reveal the direct link between H₂ and ECM deposition and EpSCs activation. These H₂-induced multiple advantages in healing may be related to the enhancement of cell viability in various cells and the maintenance of mitochondrial functions at a basic level in the biological processes of life.