Development of a Self-Folding Engineered Trachea by 4D Bioprinting

<b>INTRODUCTION</b>: 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 with several advantages for tissue engineering applications: 1)recapitulating of biological dynamic processes, 2)easiness of cell seeding and achievement of 3D complex organization; 3)exerting of forces on the seeded cells. Here, we exploited the 4D bioprinting approach to design and develop an innovative smart scaffold able to fold in time when hydrated, to be used for trachea engineering. <b>MATERIALS AND METHODS</b>: The folding in time is achieved exploiting the differential swelling properties of bilayer films. This mismatch creates a deformation gradient in the film that drives the folding of the film. The two layers of the scaffold were made of the same bulk material, i.e., (3-Glycidoxypropyl) methyldiethoxysilane crosslinked gelatin, GPTMS-GEL), thus guaranteeing a chemical bond between the layers. The swelling behavior of the layers is tuned through the modification of the GPTMS and GEL concentrations. GPTMS-GEL-1 monolayer films (i.e., higher volumetric swelling, 15% w/v GEL in PBS with 92 µl/g of GPTMS) were fabricated by solvent casting. Then, 1 mm wide lines of GPTMS-GEL-2 (i.e., lower volumetric swelling, 5% w/v GEL in PBS with 368 µl/g of GPTMS), were deposited on the GPTMS-GEL-1 film by Extrusion-Based Bioprinting. The scaffold was then dried for 48 hours at room temperature. Scaffold folding behavior was tested in PBS 1X. Then, preliminary in vitro tests were performed. 10 mm x 5 mm rectangular scaffolds were fabricated as described above, sterilized by 1h of UV light and incubated in cell culture media for 2h. Then, 20k cells (see below) were seeded on each scaffold in its initial flat position. After 30 mins, 2 ml of cell culture media was added for each scaffold and changed every 3 days. Three different cell types were tested: human vocal folds fibroblasts and human lung endothelial cells, that mimic the internal epithelial and endothelial layer of the trachea, and human ear chondral progenitor cells that mimic the chondrocytes of the trachea cartilage rings. Cell proliferation via Alamar Blue Assay were evaluated for all the cell types and materials 24h, 72h and 5 days after the seeding. Finally, the ability of eCPCs to differentiate towards mature cartialge was evaluated. Briefly, cells were seeded as described before, and culture for 7 days to allow the complete coverage of the scaffold. Then, medium was switch to chondrogenic medium and cells were cultured for additional 21 days. Histological Assay and Real time PCR were performed to verify the chondral commitment of cells. Monolayer static scaffold made of the same materials were used as control. <b>RESULTS AND DISCUSSION</b>: In vitro tests showed the ability of cells to adhere on scaffolds in its flat position, maintain their adhesion during scaffold folding, that occurs 24h after seeding, and confirmed the biocompatibility of the GPTMS-GEL materials, that promotes viability, proliferation, and metabolic activity of all the tested cell types. Regarding the cartialge differentiation analysis, Alcian Blue histological analysis confirmed the deposition of cartilaginous matrix in the external layer of the scaffold. Moreover, an overexpression of SOX9, ACAN e COLII was observed on the 4D scaffold, when compared with static monolayer films. No differences arise in the expression of PRG4 and COLX, suggesting the establishment of a stable chondral phenotype on the 4D scaffolds. <b>CONCLUSION</b>: In this study, we exploited the 4D bioprinting approach to fabricate a self-folding bilayer scaffold for trachea engineering. The reported results represent an important milestone for 4D bioprinting, quantitatively showing that shape-morphing scaffolds in the millimeter scale can promote cells activity and differentiation.