Airway mucosa-derived extracellular matrix bioink for 3D bioprinting of functional airway tissue

Abstract Airway epithelial dysfunction is a hallmark of chronic airway diseases; however, current clinical interventions fail to restore functional airway mucosa. We aimed to develop a bioink derived from airway mucosa-derived decellularized extracellular matrix (MudECM) and evaluate its potential for airway tissue engineering through in vitro and in vivo studies. Airway mucosal tissue was decellularized to preserve tissue-specific extracellular matrix components and processed into a thermo-responsive bioink. The biochemical composition, rheological properties, and printability of the bioink were characterized. Human tracheal epithelial cells (HTEpCs) were cultured on MudECM hydrogels under air–liquid interface conditions to assess epithelial differentiation and mucociliary function. A bilayered airway construct was fabricated by 3D bioprinting using MudECM-based bioinks and evaluated for epithelial–stromal organization in vitro . Functional regeneration was tested in a rat tracheal defect model by implanting 3D-printed MudECM scaffolds. MudECM retained collagen and glycosaminoglycans while achieving >98% decellularization. The bioink exhibited shear-thinning and thermal gelation properties suitable for bioprinting. HTEpCs cultured on MudECM hydrogels in vitro showed enhanced mucociliary differentiation, tight junction formation, goblet cell development, and directional mucus transport, compared to collagen controls. Bioprinted bilayered airway constructs supported fibroblast viability, growth factor expression, and epithelial differentiation with upregulation of Trp63, FoxJ1, and mucin 5AC. Implanting MudECM scaffolds into tracheal defects in vivo resulted in complete re-epithelialization, restoration of ciliary beating, and improved mucociliary clearance. By contrast, collagen controls showed only partial regeneration. Thus, MudECM bioink provides tissue-specific biochemical and mechanical cues that promote mucociliary epithelial regeneration. By enabling structural and functional restoration in vitro and in vivo , this bioink represents a clinically translatable biomaterial for airway reconstruction. Beyond regenerative graft fabrication, it offers a robust platform for disease modeling and drug testing in airway research.