Effect of senescent macrophages on delayed wound healing in an in vitro co-culture scratch assay with fibroblasts

Abstract

Purpose: Hard-to-heal wounds impair patients′ quality of life. Senescence-associated secretory phenotype (SASP) factors from macrophages in wound exudates may contribute to delayed healing, but their interaction with fibroblasts remains unclear. This study examined whether senescent macrophages impair fibroblast wound healing using an in vitro co-culture scratch assay. Methods: Mouse bone marrow-derived macrophages (BMDMs) were treated with 750μM hydrogen peroxide (H₂O₂) to induce cellular senescence. Senescence in H₂O₂-treated BMDMs was evaluated by RT-PCR analysis of senescence-related genes and SA-β-Gal staining. Protein levels of SASP-related factors in the supernatants of senescent BMDMs were quantified by ELISA. H₂O₂-treated BMDMs were co-cultured with fibroblasts using an insert-based system, and fibroblast wound healing and gene expression were evaluated. Results: H₂O₂-treated BMDMs showed increased expression of Cdkn1a, Serpine1, Cxcl2, and Sod1 and a higher proportion of SA-β-Gal-positive cells. Protein levels of TNF-α and PAI-1 were significantly elevated. Fibroblast wound healing was significantly delayed at 24-72 h. Fibroblasts co-cultured with H₂O₂-treated BMDMs exhibited increased expression of Tnf, Il1b, and Sod1. Conclusion: Senescent macrophages impaired fibroblast wound healing, suggesting that macrophage-derived SASP factors may contribute to delayed wound repair.