4D printing and in vitro studies of bi-active bi-layer self-forming biocompatible tubular structures for advanced vascular tissue engineering

The fabrication of functional vascular grafts remains a significant challenge in tissue engineering, demanding sophisticated bioprinting techniques to create biomimetic constructs with clinical potential. This study describes a novel 4D printing method for creating small-diameter (500-1000 μm), vascular grafts using bilayered hydrogel scaffolds made of gelatin methacryloyl (GelMA) and decellularized extracellular matrix (dCEM), and methacryloyl-modified dCEM (dCEM-MA), printed with an extrusion-based 3D bioprinter and photo-crosslinked. Timoshenko's beam theory was used to model the curvature and predict tube diameters. The bi-active GelMA/dCEM-MA system showed faster morphing and improved stability compared to the active-passive GelMA/dCEM system. While simultaneous crosslinking led to interlayer adhesion, the crosslinking gradient provides precise spatial control over morphing, allowing for the creation of intima-like (dCEM/dCEM-MA) and adventitia-like (GelMA) layers. Biocompatibility was observed to be good in both bilayers. Compared to coaxial and single-layered bioprinted grafts, bilayered scaffolds provide better stability and simpler fabrication, removing the need for intricate coaxial systems while closely mimicking blood vascular design, and could leverage differential swelling for dynamic self-folding tube formation. • Developing a bi-active bilayer design of GelMA/dECM-MA scaffolds with controlled self-folding into tubular structures (500–1000 μm) • Presenting a biomimetic vascular architecture closely mimicking native blood arteries in structure and function • Demonstrated faster morphing, improved stability, and high cell viability compared to active–passive bilayer arrangements • Using extrusion-based 3D printing for precise, reproducible fabrication of dynamic vascular grafts.