A bilayer hydrogel enables spatiotemporal delivery of distinct hepcidin forms to reprogram macrophage responses for repair of infected bone defect

The treatment of infected bone defects remains challenging due to the dynamic coexistence of bacterial infection, dysregulated inflammation, and impaired tissue regeneration, which demands spatiotemporally coordinated immunomodulatory interventions. Here, we develop a bilayer self-assembling polypeptide hydrogel that enables the spatiotemporal delivery of distinct hepcidin forms to reprogram macrophage responses during different phases of infection and repair. The upper layer, consisting of a PC₁₀A-Hamp hydrogel loaded with hepcidin-overexpressing mesenchymal stem cells (PHMH), undergoes rapid degradation to provide early-stage antimicrobial immune activation, effectively eliminating bacteria, inhibiting biofilm formation, and scavenging excessive reactive oxygen species. In contrast, the lower layer, a PC₁₀A-RGD hydrogel loaded with hepcidin-engineered extracellular vesicles (PREH), enables sustained release to promote M2 macrophage polarization and support osteogenic and angiogenic regeneration. In vitro studies reveal that hepcidin enhances osteogenic differentiation of MSCs via activation of the p38/ERK pathway and promotes angiogenesis through the PI3K/AKT pathway. In vivo, the PHMH/PREH hydrogel demonstrates robust therapeutic efficacy in an infected bone defect model, achieving effective infection control and accelerated bone repair. Mechanistically, spatiotemporal hepcidin delivery orchestrates phase-adaptive macrophage reprogramming by maintaining M1 polarization during the infectious phase while driving Twist1-mediated M2 polarization during the resolution phase. Collectively, this bilayer hydrogel platform establishes a phase-adaptive immunomodulatory delivery strategy and offers a promising therapeutic approach for infected bone defect repair.