Multifunctional hydrogel delivery of mesenchymal stem cell secretome suppresses neutrophil extracellular trap formation and promotes diabetic wound healing via PGE2/BMAL1 pathway.

Excessive formation of neutrophil extracellular traps (NETs) significantly impedes diabetic wound healing. Although conditioned medium of mesenchymal stem cells (MSC-CM) has shown therapeutic potential due to its anti-inflammatory, antioxidant, and immunosuppressive properties, its regulatory effects on NETs remain poorly understood. Herein, this study systematically investigates the impact of MSC-CM, particularly conditioned medium of hypoxia-conditioned MSCs (HCM), on NET formation and diabetic wound repair. Transcriptomic analysis and public database mining revealed that circadian clock dysfunction drives aberrant NET formation. Notably, HCM exhibited superior efficacy over conditioned medium of normoxia-conditioned MSCs (NCM) in suppressing reactive oxygen species (ROS) production and NET formation while accelerating diabetic wound healing. Mechanistically, HCM-derived prostaglandin E2 (PGE2) upregulated brain and muscle ARNT-Like protein 1 (BMAL1), a core circadian regulator, through PGE2 receptor 2 (EP2)-EP4 signaling, thereby reducing ROS accumulation and subsequent NET formation. To achieve non-invasive, on-demand delivery of HCM bioactive components (especially PGE2), we developed a multifunctional hydrogel composed of phenylboronic acid-grafted quaternized soy protein isolate, sodium alginate and HCM (BQSA-HCM). This hydrogel system features glucose/ROS responsiveness, self-healing capability, injectability and excellent biocompatibility, enabling controlled PGE2 release. Importantly, HCM incorporation endows the hydrogel with immunomodulatory properties. In vivo experiments demonstrated that BQSA-HCM hydrogel significantly enhanced BMAL1 expression, suppressed NET formation, and promoted diabetic wound healing. This study clarifies the critical role of BMAL1 in regulating NET formation, validates the therapeutic potential of HCM in targeting NET-related diabetic wounds, and presents an innovative controlled-release hydrogel platform for advanced diabetic wound therapy.