4-D printing of self-folding and cell-encapsulating 3-D microstructures as scaffolds for tissue-engineering applications.

Technology of Tissue-Engineering advanced rapidly in the last decade and motivated numerous studies in cell-engineering and biofabrication. Three-dimensional (3-D) tissue-engineering scaffolds play a critical role in this field, as the scaffolds provide the biomimetic microenvironments that could stimulate desired cell behaviors for regeneration. However, despite many achievements, the fabrication of 3-D scaffold remains challenging due to the difficulty of encapsulating cells in 3-D scaffolds, controlling cell-cell organization in 3-D, and being adapted by users unfamiliar with 3-D biofabrication. In this study, we circumvent these obstacles by creating a 4-D inkjet-printing platform. This platform produces micropatterns that self-fold into a 3-D scaffold. Seeding live cells uniformly onto the micropatterns before self-folding leads to cell-encapsulating 3-D scaffolds with layer-wise cell-cell organization. Photo-crosslinkable biomaterial-inks of distinct swelling rates were synthesized from gelatin, and the biomaterial-inks were patterned by a customized high-precision inkjet-printer into bilayer micropatterns that were capable of self-folding into 3-D microstructures. A mathematical model was developed to help design self-folding and to aid the understanding of the self-folding mechanism. Human umbilical vein endothelial cells (HUVECs) were embedded in self-folded microtubes to mimic microvessels. HUVECs in the microtube spread, proliferated, showed high cell viability, and engrafted on the microtube's inner wall mimicking the native endothelial cells. For physician and biologist end-users, this 4-D printing method provides an easy-to-use platform that supports standard 2-D cell-seeding protocol while enabling the users to customize 3-D cellularized scaffold as desired. This work demonstrated 4-D printing as a promising tool for tissue-engineering applications.