Development of a 4D printing strategy to fabricate smart bioscaffolds

There is a constant need to improve materials and manufacturing techniques to fabricate substrates able to reproduce the suitable biological environment for in vitro testing. To date, conventional techniques have not been able to address this urgent prerequisite yet, due to the inability to properly mimic the biological stimuli. An approach based on the combination of tissue engineering and 4D printing could represent a more effective strategy. This method combines the manufacturing precision of 3D printing systems and the advanced functionality of materials to create complex biocompatible structures with high-resolution microstructure features. To this aim, we studied the feasibility of printing a bilayer scaffold made from a biocompatible ink, suitable for the cell culture, and a smart material for the folding of the printed structure. The two materials were printed by using a commercial 3D printer “Biobots”, with capabilities augmented through a customized multiple extruder in order to dispense fibers in a high controlled manner. GelMA-Alginate inks were optimized to print the first layer of the bioscaffold. Subsequent UV exposure showed to strongly influence the mechanical properties of the fabricated scaffold. The biocompatibility of the material was evaluated through cell culture experiments with 3T3 cell line, which successfully populated the substrate in 3 days from seeding. Concerning the second layer, the shrinking and printability of a thermo-sensitive polymer, PNIPAm, has been assessed as a potential candidate for the structure folding. Alginate-PNIPAm and Alginate-PNIPAm-GelMA samples exhibited a relevant shrinking when moved from room temperature to 37°C. Compression testing showed higher mechanical properties for samples at 37°C as compared to those kept at room temperature. Finally, as a proof of concept, GelMA-Alginate and Alginate-NIPAm bilayer ribbons were tested to evaluate the bending of the whole structure, which varied from 76° to 116° based on the thickness of layers. All samples were crosslinked by using a customized UV box. The study showed the feasibility of the proposed strategy and permitted a first collection of data as well as the development of essentials tools that will be useful for the future steps of the project.