"Four-dimensional (4D) bioprinting (i.e., fabrication via additive manufacturing of scaffolds characterized by a programmed change, over time, under a predefined stimulus ) can be exploited to produce active scaffolds that can modify their shapes upon desired stimulation, thus potentially recapitulating biological processes such as morphogenesis.
In this study, we exploited the 4D bioprinting approach to design and fabricate an innovative smart scaffold for in vitro modeling the development of the neural tube (NT), the structure from which the central nervous system stems in the embryo, with the final aim to guide stem cells towards neural differentiation. The smart scaffold is able to self-fold in time, mimicking the neural plate folding to create a hollow tube, namely the NT .
The requested behavior is achieved exploiting the differential swelling properties of bilayer films . Indeed, the different volumetric swelling of the two layers, when dipped in water, creates a deformation mismatch in the film that leads the folding of the film itself. In this study, the two layers were made of the same bulk material (i.e., gelatin crosslinked using (3-Glycidoxypropyl)methyldiethoxysilane, GPTMS-GEL), thus guaranteeing a chemical bond between the layers and avoiding delamination. The swelling behavior of the layers was tuned through the modification of the GPTMS and GEL concentrations.
GPTMS-GEL-1 monolayer films, with the higher volumetric swelling, were fabricated by solvent casting. Then, lines of GPTMS-GEL-2, with lower volumetric swelling, were deposited on the GPTMS-GEL-1 film by Extrusion-Based Bioprinting. The presence of precisely oriented lines (as second layer of the bilayer film) provides a constrain and, as a consequence, a complete control over the film folding direction.
When dipped in water the film self-folds, maintaining its shape in time and, as expected, the orientation of the folding depends on the printed line direction.
Cellular tests have been performed to verify the properties of the smart scaffold, using human induced pluripotent stem cells (iPSCs) directed toward neural progenitor fate via Dual SMAD inhibition. iPSC-derived neural progenitor population uniquely recapitulates in vitro the onset of the founder population of the developing NT. Indeed, the simultaneous application of these cellular and bio-engineering technologies will provide a platform to assess complex phenomena such as NT folding and cellular polarization in a dynamic 4D environment. This pioneering platform will provide an innovative standpoint to unravel neural tube defects and their clinical impact.
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 Kim, S. H., et al. (2020) Biomaterials, 260,120281"