A chemically addressable polyurethane graft coordinates compartment-specific defense and regeneration for tracheal reconstruction

Reconstruction of segmental tracheal defects requires simultaneous restoration of luminal defense and load-bearing wall integrity, yet conventional grafts treat these requirements as spatially uniform. Here, we developed NG-PUNT, a compartmentalized bilayer electrospun graft that matches distinct bioactivities to the luminal and abluminal pathological environments. Pendant alkenes were introduced into polyurethane to create a chemically addressable platform, followed by photoinitiated thiol-ene functionalization with N-acetylcysteine (NAC) or glutathione (GSH). Sequential electrospinning generated an NAC-derived functionalized luminal layer and a GSH-derived cysteinyl-thioether-functionalized abluminal layer while preserving graft architecture and mechanics. Incubation with NAC-derived functionalized scaffolds altered mucus rheological properties. In separate assays, scaffold-conditioned preparations generated from NAC-derived functionalized scaffolds inhibited bacterial growth and biofilm accumulation and attenuated bronchial epithelial oxidative injury and apoptosis. Preparations generated from GSH-derived functionalized scaffolds attenuated macrophage oxidative and inflammatory responses and were associated with preservation of redox-responsive signaling and chondrogenic marker expression under oxidative stress. In a rabbit segmental tracheal-defect model, NG-PUNT improved survival and airway patency, reduced luminal bacterial burden and inflammation, supported repopulation of basal, ciliated and secretory epithelial phenotypes, and promoted COL II- and aggrecan-rich cartilage-matrix reconstruction. Transcriptomic profiling revealed coordinated antimicrobial, redox-responsive and chondrogenic signatures with suppression of inflammatory and chemokine-related programs. These findings support compartment-matched biofunctionalization as a strategy for coupling luminal defense with functional structural regeneration in tracheal grafts and hollow-organ regenerative implants.