4D Bioprinted Self‐Folding Scaffolds Enhance Cartilage Formation in the Engineering of Trachea

4D bioprinting is a cutting-edge approach for manufacturing active scaffolds able to shape-morph in a predefined way after the application of an environmental stimulus, thus enabling to mimic the dynamics of native tissues. In this study, we developed a self-folding gelatin-based bilayer scaffold for trachea engineering exploiting the 4D bioprinting approach. Starting from a 2D flat configuration, upon hydration, the scaffold automatically forms a closed tubular structure. An analytical model, based on Timoshenko's beam thermostats, was developed and validated to predict the radius of curvature of the scaffold. The 4D bioprinted structure was tested with airway fibroblast, lung endothelial cells and cartilage progenitor cells (CPCs) toward the development of a tissue engineered trachea. Cells were seeded on the scaffold in its initial flat configuration, maintained their position after the scaffold actuation and proliferated over or inside it. The ability of CPCs to differentiate towards mature cartilage was evaluated. Interestingly, real-time PCR revealed that differentiating CPCs on the 4D bioprinted scaffold promotes healthier cartilage formation, if compared with CPCs cultured on 2D static flat scaffold. Thus, CPCs can perceive scaffold folding and its final curvature and react to it, towards the formation of mature cartilage for the airway.