Candidate Biomarkers for Hard‐to‐Heal Wounds Revealed by Single‐Cell <scp>RNA</scp> Sequencing of Wound Fluid in Murine Wound Models

Wound healing is often hindered by hyperglycemia, chronic inflammation and ageing. Despite extensive research on the pathophysiology of hard-to-heal wounds, wound healing remains complex and poses challenges in treatment and management. Current wound treatments and care mostly target a single pathology, such as infection, while most hard-to-heal wounds are multifactorial. Therefore, exploring the factors that do not rely on a single pathology is crucial to fill the gap in current wound management. Despite containing more comprehensive information than commonly used wound tissue samples, cells in the wound fluid have not drawn much attention because of collection difficulties. This study aimed to use single-cell RNA sequencing (scRNA-seq) of cells from wound fluid to identify specific biomarkers for hard-to-heal wounds, with the hypothesis that common biomarkers among various wound models can be potentially applied to complex hard-to-heal wounds in clinical settings. Three representative delayed wound models, aged, diabetic and lipopolysaccharide-induced inflammatory wound models, were compared with normal young mice to explore commonly shared genes that exist in different pathological delayed wound healing models. The shared upregulation of cell cycle and cellular senescence-related genes such as Rpl11, Rpl26, Rps3, Rps15, Rps 20, Rps26, Ccl2, Cdk2ap2 and Ccnd3 and the downregulation of immune response regulation genes such as Tnfaip3, Junb, Il1r2, Plaur, Il1rn, Il1a, Cxcl2, Cd14, S100a8 and S100a9 in all delayed healing wound models were found in most immune cell subgroups, especially the macrophage subgroup. The results of this study suggested cellular senescence of cells in wound fluid could be related to hard-to-heal wounds.