Hydrogel delivery of apoptotic neutrophils modulates macrophage angiogenic and inflammatory responses during wound healing

Effective wound healing requires coordinated angiogenesis and immune resolution. Biomaterial systems support tissue regeneration but typically lack intrinsic immunomodulatory signals needed to actively direct immune resolution and angiogenesis. In this study, localized delivery of apoptotic neutrophils (AN) encapsulated within Gelatin methacryloyl (GelMA) hydrogels was evaluated for its ability to enhance angiogenic responses and modulate inflammatory activity in vivo. In a murine splinted full-thickness skin wound model, AN-loaded GelMA significantly increased vessel length and CD31⁺ vascular area at 14 days post-wounding, while also enhancing cellular infiltration and hydrogel retention. Single-cell RNA sequencing (scRNA-seq) at day 5 post-wounding revealed that AN incorporation reshapes the early wound landscape, enriching macrophage states associated with tissue remodeling and increasing expression of efferocytosis-associated genes across myeloid populations while reducing inflammatory gene expression and altering chemokine expression. Intercellular communication analysis demonstrateds that AN-loaded GelMA shifts endothelial-myeloid signaling away from pro-inflammatory interactions toward pro-resolving signaling programs and enhances vascular crosstalk. In vitro assays confirmed modulation of macrophage paracrine activity, with reduced pro-inflammatory cytokine production, enhanced anti-inflammatory responses, and differential regulation of angiogenesis-associated gene expression. HUVEC vessel-like tube formation assay demonstrated that conditioned media from both GelMA- and AN-treated macrophages support endothelial network formation in vitro. Together, these findings demonstrate that combining GelMA hydrogel with AN-derived cues establishes a pro-resolving environment that reprograms macrophage states and coordinates vascular remodeling, highlighting a promising immunomodulatory biomaterial strategy for wound healing applications.