Vertical Extrusion Cryo(bio)printing for Anisotropic Tissue Manufacturing

Abstract

extrusion bioprinting is mainly used for constructing volumetric structures in a layer-wise manner. [5] Although the layerby-layer bioprinting method is functional in majority of the cases, [6] there are limitations associated with creating anisotropic tissues, such as muscle fibers [7,8] and nerve fibers [9] that heavily rely on cellular alignment for their physiologies. Therefore, developing a versatile strategy that allows convenient 3D bioprinting synergized with simultaneous generation of structural anisotropy is essential for these applications.Numerous studies have shown that porous hydrogel scaffolds can potentially enhance cell spreading and proliferation. [10,11] In particular, ice-templating, one of the most widely utilized techniques for the fabrication of materials with anisotropic microchannels, allows control over pore morphologies by controlling directional ice formation in a suspension of solute(s). [12][13][14][15] During the freezing process, ice crystals form and propagate through a set direction within the biomaterial solution. When the construct cross-links and thaws, the melted ice crystals form interconnected anisotropic microchannels within the scaffold. Importantly, previous studies have clearly demonstrated that the presence of anisotropic microchannels enhances Due to the poor mechanical properties of many hydrogel bioinks, conventional 3D extrusion bioprinting is usually conducted based on the X-Y plane, where the deposited layers are stacked in the Z-direction with or without the support of prior layers. Herein, a technique is reported, taking advantage of a cryoprotective bioink to enable direct extrusion bioprinting in the vertical direction in the presence of cells, using a freezing plate with precise temperature control. Of interest, vertical 3D cryo-bioprinting concurrently allows the user to create freestanding filamentous constructs containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performances. Skeletal myoblasts within the 3D-cryo-bioprinted hydrogel constructs show enhanced cell viability, spreading, and alignment, compared to the same cells in the standard hydrogel constructs. This method is further extended to a multimaterial format, finding potential applications in interface tissue engineering, such as creation of the muscle-tendon unit and the muscle-microvascular unit. The unique vertical 3D cryo-bioprinting technique presented here suggests improvements in robustness and versatility to engineer certain tissue types especially those anisotropic in nature, and may extend broad utilities in tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.

High Precision 3D Printing for Micro to Nano Scale Biomedical and Electronic Devices

“…Bio-ink is used as the printing material which contains cells, nutrients and growth factors that are critical for determining resolution and biological properties in the 3D printing of blood vessels [199]. Hydrogel precursors are a common constituent of the bio-ink as it mimics the extracellular matrix, and therefore provides a suitable microenvironment for cell proliferation [199,200]. The fabricated vessels must portray appropriate mechanical properties, remodeling capability and anti-thrombogenicity to achieve successful integration with the surrounding biologically developed vessels [199].…”

Recent Advances in Macroporous Hydrogels for Cell Behavior and Tissue Engineering

“…When polymers are cross-linked and melted, the removed ice crystals form interconnected anisotropic macro-channels within cryogels [ 119 , 120 , 121 , 122 ]. Anisotropic cryogels supported better cell migration and tissue infiltration than cryogels with randomly distributed pores [ 31 , 123 ]. Interestingly, Jiang et al used ice crystals to grow from the periphery to the center of the hydrogel to form a radial topology [ 124 ].…”

“…For example, cryoprotective bioinks containing skeletal muscle cells were vertically extruded printed onto cryoplates to achieve macroscopic filamentous structures and microscopic gradient axial channels ( Figure 8 ). Directional freezing overcomes the disadvantage of poor mechanical properties of bioinks in 3D printing, enabling the directional arrangement of cells at different levels [ 123 ]. Notably, although topology has a positive effect on cell adhesion and alignment, the addition of adhesion molecules is still necessary to enhance cell–macroporous hydrogel interactions [ 235 , 237 , 238 ].…”

“…( F ) Vertical 3D cryo-bioprinting for fabricating the muscle-tendon unit. Reprinted with permission from [ 123 ], Copyright 2021, Wiley.…”

Recent Progresses in Development of Biosensors for Thrombin Detection

“…Moreover, many 3D printing technologies have been recently utilized to construct biosensors or some parts of them due to the benefits of these approaches over conventional ones such as end-user customization, great functionality and adaptability, and quick prototyping. This technology has also quickly been developed in a wide range of biological and biomedical fields including organ on a-chip platforms, bioelectronics [ 17 , 18 ], microfluidic devices [ 19 ], and tissue-engineered implants [ 20 ].…”