4D Biofabrication, a recently growing fabrication technique, is a 3D biofabrication method, which involves the automated production of 3D constructs that are dynamic and present a shape transformation capability [1] [2]. Although recent biofabrication methods have shown satisfactory results in the fabrication of vascular networks, their resolution is limited for the application of cells and the production of tubular constructs of small diameters is still a challenge [1] [3] [4]. Thus, 4D biofabrication involves the fabrication of hollow tube-like structures, which is promising for the fabrication of blood vessel substitutes of scalable diameters with high resolutions [1] [5].

Here, we introduce a biofabrication technique for the production of T-shaped vascular bifurcation substitutes using oxidized alginate crosslinked with gelatin ADA – Gel as a biomaterial ink. In which, alginate provides excellent rheological properties for 3D printing, whereas gelatin enhances cell adhesion and proliferation characteristics [4]. The formation of tube-like structures with diameters ranging from 2 – 15 mm were achieved by precisely controlling the printing parameters, rheological properties, and the crosslinking degree. Consequently, the 3D-printed T-shaped thin films (200 μm in thickness) showed automated self-transformation upon immersion in water with a final diameter of 3 mm and actuation time of 12 min. The perfusion of the T-shaped constructs with an aqueous solution simulating the blood flow through native blood vessels showed minimal leakages and tight tube formation, with a maximum flow velocity of 0.11 m/s.

Furthermore, human umbilical vein endothelial cells (HUVECs) were seeded on the plain T-shaped structures to accommodate the inner surface of the tube upon actuation. The fabricated structures showed outstanding adhesion and growth properties with cell viability of 98 % and proliferation of 35 % at day 7 of culture. The achieved diameters are comparable to native blood vessels, which is still a challenge in 3D biofabrication [4]. This technique will pave the way for the fabrication of vascular grafts of different geometries made of natural hydrogels with tunable properties.

[1] Ionov, L. (2018). 4D Biofabrication: Materials, Methods, and Applications. Advanced Healthcare Materials, 7(17), 1800412. https://doi.org/10.1002/adhm.201800412

[2] Zhou, W., Qiao, Z., Nazarzadeh Zare, E., Huang, J., Zheng, X., & Sun, X. et al. (2020). 4D-Printed Dynamic Materials in Biomedical Applications: Chemistry, Challenges, and Their Future Perspectives in the Clinical Sector. Journal Of Medicinal Chemistry, 63(15), 8003-8024. https://doi.org/10.1021/acs.jmedchem.9b02115

[3] Pashneh-Tala, S., MacNeil, S., & Claeyssens, F. (2016). The Tissue-Engineered Vascular Graft—Past, Present, and Future. Tissue Engineering Part B: Reviews, 22(1), 68-100. https://doi.org/10.1089/ten.teb.2015.0100

[4] Ruther, F., Distler, T., Boccaccini, A., & Detsch, R. (2018). Biofabrication of vessel-like structures with alginate di-aldehyde—gelatin (ADA-GEL) bioink. Journal Of Materials Science: Materials In Medicine, 30(1). https://doi.org/10.1007/s10856-018-6205-7

[5] Kirillova, A., Maxson, R., Stoychev, G., Gomillion, C., & Ionov, L. (2017). 4D Biofabrication Using Shape‐Morphing Hydrogels. Advanced Materials, 29(46), 1703443. https://doi.org/10.1002/adma.201703443