Abstract
Impaired wound healing in aged and diabetic wounds involves complex cellular dysregulation that hinders tissue repair. Using single-cell RNA sequencing (scRNA-seq) and validation techniques, we investigated impaired wound healing to identify whether there were significant changes linked to each condition. Comparative mucosal wound analysis revealed distinct differences between diabetic and normoglycemic (NG)-aged mice, which had an impact on connective tissue formation and epithelial closure. Wounds in NG-aged mice exhibited prolonged granulation tissue and upregulation of genes linked to chemotaxis, cell migration, neutrophil degranulation, and antimicrobial defense pathways compared to the diabetic wounds. In comparison to healing in young animals, wounds in NG-aged mice had a shift in fibroblast subtypes with fewer matrix-producing myofibroblasts and increased inflammatory fibroblasts. Furthermore, wounds in NG-aged mice versus wounds in diabetic mice had an upregulation of lytic enzymes, with striking differences in cathepsin-expressing fibroblasts. Since diabetic wounds healed more slowly than wounds in NG-aged mice, the results suggest that the upregulation of lytic enzymes that characterized diabetic wounds is particularly damaging to healing. In addition to the transcriptional differences, pseudotime analysis revealed that fibroblasts in wounds from diabetic mice progressed towards a protease-enriched state, while those in aged mice shifted towards an inflammatory phenotype. This is the first study to directly compare aged and diabetic healing at the single-cell level and provides distinct molecular mechanisms that may allow more precise therapeutic targets to improve healing in aged and diabetic wounds.