A let-7a-5p-engineered extracellular vesicle-laden bioactive scaffold promotes immunomodulation and regeneration of segmental tracheal defects

Segmental tracheal defects resulting from trauma, tumor resection, or congenital malformations pose significant challenges for reconstruction. Persistent post-injury inflammation, inadequate neovascularization, and delayed epithelial restoration contribute to the formation of granulation tissue and subsequent graft failure. In this study, we developed a bioactive tracheal scaffold for sustained local delivery of let-7a-5p-engineered extracellular vesicles (let-7a-5p-EVs) to modulate the regenerative microenvironment during tracheal repair. The scaffold was constructed by integrating a mechanically optimized polycaprolactone (PCL) framework with a 20% Pluronic F127 hydrogel, which served as a local reservoir for EV retention and sustained release. Let-7a-5p-EVs were generated from lentivirally engineered human gingiva-derived mesenchymal stem cells and showed typical EV morphology, representative EV marker expression, and efficient uptake by airway epithelial cells. In vitro, let-7a-5p-EVs enhanced airway epithelial cell migration and shifted lipopolysaccharide (LPS)-induced macrophages from a pro-inflammatory M1-like phenotype toward a reparative M2-like phenotype, accompanied by reduced inflammatory cytokine expression. In a rabbit segmental tracheal defect model, the let-7a-5p-EV-laden scaffold enhanced neovascularization, mitigated local inflammatory responses, promoted mucociliary epithelial reconstruction, and maintained airway patency. The let-7a-5p-EV scaffold improved biomechanical recovery, and increased survival rates. Transcriptomic profiling and molecular validation indicated that these effects were associated with the attenuation of nuclear factor kappa B (NF-κB)-associated inflammatory activation, as evidenced by reduced p65 phosphorylation and nuclear localization. These findings demonstrate that sustained local delivery of engineered EVs from a mechanically competent bioactive scaffold provides an effective immunomodulatory strategy for the regenerative repair of segmental tracheal defects and offers translational potential for functional airway reconstruction.