REFRESH-Reversible Embedded Bioprinting for Faster Reinforcement and Structuring of dECM Hydrogels.

Fabrication of complex, multi-layered tissue architecture using decellularized extracellular matrix (dECM)-based hydrogel ink is fundamentally limited by the biomaterial's inherent mechanical fragility and slow gelation kinetics, which severely compromise structural fidelity. Herein, we present REFRESH-Reversible Embedded Bioprinting for Faster Reinforcement and Structuring of dECM Hydrogels, a next-generation embedded bioprinting platform designed for high-fidelity fabrication of anatomically relevant tissue constructs using dECM hydrogels. At the core of this system is a custom-engineered polyethylene glycol (PEG)-gelatin microgel suspension bath that performs dual functions: it modulates the bath's rheological behavior, enhancing yield stress, shear-thinning, and self-healing properties necessary for precise deposition, and actively promotes in situ gelation of dECM hydrogel inks via hydrogen bonding and crowding-induced interactions. This enables faster filament stabilization, reduced structural fusion, and improved print fidelity compared to conventional thermal gelation. We recreated the zonal architecture of the trachea by co-printing cartilage- and trachealis muscle-derived dECM's encapsulating primary chondrocytes, fibroblasts, and subsequent epithelialization to form a biomimetic luminal surface. Furthermore, we fabricated functional trifurcated bronchial structures using lung-derived dECM that supported stromal-like mesenchymal behavior with contractile marker expression. The versatility of the REFRESH platform was further validated by the successful printing of a diverse array of tissue-specific dECM hydrogels beyond the airway system.