Organ printing offers a great potential for the freeform layer-by-layer fabrication of three-dimensional (3D) living organs using cellular spheroids or bioinks as building blocks. Vascularization is often identified as a main technological barrier for building 3D organs. As such, the fabrication of 3D biological vascular trees is of great importance for the overall feasibility of the envisioned organ printing approach. In this study, vascular-like cellular structures are fabricated using a liquid support-based inkjet printing approach, which utilizes a calcium chloride solution as both a cross-linking agent and support material. This solution enables the freeform printing of spanning and overhang features by providing a buoyant force. A heuristic approach is implemented to compensate for the axially-varying deformation of horizontal tubular structures to achieve a uniform diameter along their axial directions. Vascular-like structures with both horizontal and vertical bifurcations have been successfully printed from sodium alginate only as well as mouse fibroblast-based alginate bioinks. The post-printing fibroblast cell viability of printed cellular tubes was found to be above 90% even after a 24 h incubation, considering the control effect.

Silk fibroin is a natural protein which has shown great promise for tissue engineering but is not printable due to slow gelation or harsh gelation conditions which are not cell-friendly. In this study, a two-step gelation process is proposed for the printing of silk fibroin, which utilizes alginate as a sacrificial hydrogel during an inkjetting-based process. A cell-laden blend of alginate with silk fibroin is utilized to achieve rapid gelation by calcium alginate formation during printing; it is followed by horseradish peroxidase (HRP) catalyzed covalent cross-linking of the fibroin protein at tyrosine residues after printing. This two-step gelation process successfully enables 3D bioprinting of well-defined cell-laden silk fibroin constructs suitable for long-term culture. The constructs remain intact after calcium chelation to liquefy the alginate component, demonstrating the formation of silk fibroin hydrogel. NIH 3T3 fibroblasts proliferate and spread through the hydrogel after printing. Increasing metabolic activity is observed for 5 weeks after printing, and histology shows dense cell populations in cultured constructs. The proposed two-step gelation technique is expected to enable 3D silk fibroin printing for various applications.

Three-dimensional (3D) bioprinting, as a freeform biomedical manufacturing approach, has been increasingly adopted for the fabrication of constructs analogous to living tissues. Generally, materials printed during 3D bioprinting are referred as bioinks, which may include living cells, extracellular matrix materials, cell media, and/or other additives. For 3D bioprinting to be an enabling tissue engineering approach, the bioink printability is a critical requirement as tissue constructs must be able to be printed and reproduce the complex micro-architecture of native tissues in vitro in sufficient resolution. The bioink printability is generally characterized in terms of the controllable formation of well-defined droplets/jets/filaments and/or the morphology and shape fidelity of deposited building blocks. This review presents a comprehensive overview of the studies of bioink printability during representative 3D bioprinting processes, including inkjet printing, laser printing, and micro-extrusion, with a focus on the understanding of the underlying physics during the formation of bioink-based features. A detailed discussion is conducted based on the typical time scales and dimensionless quantities for printability evaluation during bioprinting. For inkjet printing, the Z (the inverse of the Ohnesorge number), Weber, and capillary numbers have been employed for the construction of phase diagrams during the printing of Newtonian fluids, while the Weissenberg and Deborah numbers have been utilized during the printing of non-Newtonian bioinks. During laser printing of Newtonian solutions, the jettability can be characterized using the inverse of the Ohnesorge number, while Ohnesorge, elasto-capillary, and Weber numbers have been utilized to construct phase diagrams for typical non-Newtonian bioinks. For micro-extrusion, seven filament types have been identified including three types of well-defined filaments and four types of irregular filaments. During micro-extrusion, the Oldroyd number has been used to characterize the dimensions of the yielded areas of Herschel-Bulkley fluids. Non-ideal jetting behaviors are common during the droplet-based inkjet and laser printing processes due to the local nonuniformity and nonhomogeneity of cell-laden bioinks.

F re e fo rm V e rtic a l and H o rizo n ta l F a b ric a tio n of A lg in a te -B a s e d V a s c u la r-L ik e T u b u la r C onstructs U sing In k je ttin g

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