Abstract
Long-segment tracheal reconstruction remains a formidable clinical challenge due to failures of conventional tissue-engineered trachea (TET) grafts, stemming from inadequate vascularization, compromised cartilaginous framework, and epithelial deficiency. Transcriptomic profiling of conventional TETs revealed enriched hypoxia and inflammatory pathways, underscoring the need for robust vascular and epithelial strategies. Herein, a dual-pedicle biomimetic TET is developed to provide a stable cartilaginous framework for airway support, immediate blood supply for tissue perfusion, and functional epithelial lining for barrier protection. Autologous rabbit auricular skin is sutured into tubes for epithelial lining, while chondrocytes in photo-crosslinkable decellularized Wharton's jelly matrix hydrogels formed C-shaped constructs assembled into cartilage tubes. Both are pre-vascularized separately in cervical muscle for 4 weeks, then assembled with preserved unilateral pedicles, yielding integrated TETs with dual-pedicle vascular networks and enhanced biomechanical properties. Orthotopic tracheal transplantation of this construct into rabbits demonstrated sustained patency, minimal stenosis, and improved 28 day survival (60% vs 40% for single-pedicle controls). Immunofluorescence confirmed reduced bacterial infiltration, apoptosis, and inflammation (TLR4, MPO), with restored epithelial barriers (Claudin-1, β-defensin 1) and airway differentiation (cytokeratin 8 upregulation). Transcriptomics validated regenerative pathways, including oxidative phosphorylation and tight junctions. This dual-pedicle approach preempts pathological cascades, offering a promising paradigm for clinical tracheal regeneration.