Advanced Bioink for 3D Bioprinting of Complex Free-Standing Structures with High Stiffness

Abstract

One of the challenges in 3D-bioprinting is the realization of complex, volumetrically defined structures, that are also anatomically accurate and relevant. Towards this end, in this study we report the development and validation of a carboxylated agarose (CA)-based bioink that is amenable to 3D printing of free-standing structures with high stiffness at physiological temperature using microextrusion printing without the need for a fugitive phase or post-processing or support material (FRESH). By blending CA with negligible amounts of native agarose (NA) a bioink formulation (CANA) which is suitable for printing with nozzles of varying internal diameters under ideal pneumatic pressure was developed. The ability of the CANA ink to exhibit reproducible sol-gel transition at physiological temperature of 37 °C was established through rigorous characterization of the thermal behavior, and rheological properties. Using a customized bioprinter equipped with temperature-controlled nozzle and print bed, high-aspect ratio objects possessing anatomically-relevant curvature and architecture have been printed with high print reproducibility and dimension fidelity. Objects printed with CANA bioink were found to be structurally stable over a wide temperature range of 4 °C to 37 °C, and exhibited robust layer-to-layer bonding and integration, with evenly stratified structures, and a porous interior that is conducive to fluid transport. This exceptional layer-to-layer fusion (bonding) afforded by the CANA bioink during the print obviated the need for post-processing to stabilize printed structures. As a result, this novel CANA bioink is capable of yielding large (5–10 mm tall) free-standing objects ranging from simple tall cylinders, hemispheres, bifurcated ‘Y’-shaped and ‘S’-shaped hollow tubes, and cylinders with compartments without the need for support and/or a fugitive phase. Studies with human nasal chondrocytes showed that the CANA bioink is amenable to the incorporation of high density of cells (30 million/mL) without impact on printability. Furthermore, printed cells showed high viability and underwent mitosis which is necessary for promoting remodeling processes. The ability to print complex structures with high cell densities, combined with excellent cell and tissue biocompatibility of CA bodes well for the exploitation of CANA bioinks as a versatile 3D-bioprinting platform for the clinical translation of regenerative paradigms.

Extrusion-Based 3D Bioprinting of Gradients of Stiffness, Cell Density, and Immobilized Peptide Using Thermogelling Hydrogels

“…The CA bioink was formulated in PBS and the cells suspended in phenol red supplemented media, thus allowing for the visual tracking of the printed gradient ( Figure 4 B). As previously reported on drop-on-demand, 22 and extrusion bioprinting, 21 incorporation of cells within the ink was possible ( Figure 4 C and D). To quantify the cellular gradient, immediately following printing, cells were stained with Hoechst dye (live cells stain blue) and imaged using a fluorescent microscope ( Figure 4 E and F).…”

Natural Biomaterials and Their Use as Bioinks for Printing Tissues

“…Carboxylated agarose is a derivative of native agarose with carboxylic acid groups on the polysaccharide backbone. Changing the degree of carboxylation causes an α-helix to β-sheet switch in secondary structure, allowing mechanical properties of the bioink to be modified without affecting the concentration [ 55 , 56 ]. Forget et al bioprinted carboxylated agarose with human MSCs and achieved a 95% cell survival rate [ 57 ].…”

“…Forget et al bioprinted carboxylated agarose with human MSCs and achieved a 95% cell survival rate [ 57 ]. Gu et al also created a carboxylated agarose-based bioink that had a high cell survival rate and was stiff enough to form 5–10 mm tall structures of various shapes without requiring extra support material [ 56 ]. Thus, agarose is a commonly used material for 3D printing applications.…”

Current Trends on Protein Driven Bioinks for 3D Printing

“…This procedure implies setting a slightly higher temperature to obtain physiological temperature at the nozzle tip or performed using room temperature (20–25 °C). Protein-based bioinks incorporating polysaccharides such as alginate [ 93 ] and agarose [ 94 ] can be printed by this process [ 3 ]. In this particular cases the temperature-induced gelation is faster than the Ca 2+ induced gelation, which can improve the initial stability of the printed construct [ 95 , 96 ].…”