Wounds have a sizable impact on public health: 1 out of 50 Americans has a chronic wound at any given time and the cost of wound care exceeds $100 billion per year. Similarly, hypertrophic scars with excessive scar tissue greatly impair quality of life for millions of patients. However, therapeutic solutions for wound healing (WH) remain quite limited. Sex has received growing interest as a factor in wound healing, and some studies show higher rates of non-healing wounds in men. It has long been known that in male rodents testosterone (T) impairs wound healing via androgen receptor effects. Sex hormone (SH)-induced changes in wound repair have been studied as a possible clinical risk factor, but their impact is uncertain due to factors like illness that alter hormone levels. We developed clinically relevant new experimental models and showed that T limits wound healing. Further, we have proven that patients on T have impaired wound healing. A critical knowledge gap that remains unaddressed is the set of molecular mechanisms and pathways T acts through to change the wound healing response. We have identified new immunomodulatory effects of T that may mediate these effects. Conversely, estradiol (E2) may accelerate wound repair rates. Essential aspects of how E2 improves wound healing remain similarly unknown. We will address these gaps in understanding hormone modulation of WH with a program spanning three project directions, including overcoming the failure of current in vitro (petri dish) models to recreate in vivo wound cell behavior changes induced by sex hormones (Project 1: In Vitro System) reducing the need for animal experiments, and defining how testosterone modulates the evolution of the immune response during wound healing to impair closure and this induces a fibrotic or regenerative repair response (Project 2: Mechanisms of T Immunomodulation). Finally, we will expand our work to deeply profile the effects of estradiol on immune cell subtypes and the wound cytokine milieu (Project 3: Estradiol Wound Healing Mechanisms). This project will deploy innovative tools to probe SH-WH mechanisms, expanding scientific knowledge of how T and estradiol interact with sex chromosomes and the immune system to regulate wound healing biology. This may enable identification of new therapeutic targets that can alter only those dysfunctional wound healing pathways with high specificity (ie, without any hormone effects). This study seeks to transform current concepts of how hormones modulate wound repair and develop potential new treatment approaches for millions of patients with chronic wounds.