159. Differences In Age-related Regenerative Decline Using a Wound-Induced Hair Neogenesis (WIHN) Model

PURPOSE: The skin is our largest and first defense against the insults of the outside world, yet as we age, our ability to regenerate this critical barrier after injury greatly diminishes. By the 7th decade of life, the skin shows significant impairment in numerous regenerative processes, including re-epithelialization, revascularization, stem cell recruitment, cell migration, and cell proliferation. Consequently, in an aging population, there is an increase in the morbidity of chronic or recurring wounds and their associated complications. Moreover, chronic wound care has an annual cost of $10 billion in the US, with wound care in those aged 65+ accounting for the majority of this financial burden. While much is known about the signaling pathways and cellular dynamics involved in cutaneous wound healing, little is known about how these processes change with age. Understanding the molecular and cellular underpinnings of age-related regenerative decline is a crucial first step in the development of therapies to treat the increased prevalence of poorly healing cutaneous wounds in the elderly. METHODS: To elucidate aging’s role in regenerative capacity, we utilized a wound-induced hair neogenesis (WIHN) mouse model to quantify regeneration. WIHN is a phenomenon where adult mammals undergo embryonic levels of regeneration in large cutaneous wounds via the development of de novo hair follicles (HFs) and functional, non-scarred skin within the center of the re-epithelialized wound bed. C57BL/6J mice aged 4 weeks and 91 weeks were given full-thickness dorsal cutaneous wounds of 1.25 cm x 1.25 cm and 1.5 cm x 1.5 cm, respectively. Wound healing kinetics were tracked and regenerated skin was harvested at set timepoints for phenotypic and molecular analysis. De novo HFs within the healed wounds were visualized and quantified using confocal scanning laser microscopy. Histology was performed to detect morphological differences in skin structure and HFs between the two populations, which were characterized using a novel machine-learning system for quantitative three-dimensional histology termed CODA. Additionally, miRNA analysis, qPCR, immunostaining, and immunoblotting were conducted to elucidate molecular expression disparities between regenerating young and aged skin. RESULTS: Compared to aged mice, young mice exhibited faster wound closure during WIHN (p<0.0001) and had a 20-fold increase in regenerated HFs despite a comparable healed wound size (p<0.0001). Histology and immunostaining revealed the development of morphologically abnormal HFs in the aged group that, compared to young, had strikingly similar distributions of molecular markers such as cytokeratins (K1/K5/K10/K14), stem cell markers (Sox9) and differentiation markers (B-catenin, LEF-1). miRNA sequencing of regenerated wounds revealed that miR-133 had the greatest differential expression, with aged mice showing 4-fold higher expression. Young mice topically treated with miR-133 showed a 3.5-fold decrease in neogenic HFs (p<0.005). CONCLUSION: These findings highlight distinct skin regeneration and wound healing patterns between young and aged mice. There is an abundance of miR-133 in aged skin that may be impeding proper healing through alteration of key signaling pathways during regeneration, although the exact mechanism has yet to be elucidated. miR-133 may be considered a potential therapeutic target for improving cutaneous regeneration in frail/elderly individuals.