Biofabrication Under Fluorocarbon: A Novel Freeform Fabrication Technique to Generate High Aspect Ratio Tissue-Engineered Constructs

Abstract

Bioprinting is a recent development in tissue engineering, which applies rapid prototyping techniques to generate complex living tissues. Typically, cell-containing hydrogels are dispensed layer-by-layer according to a computer-generated three-dimensional model. The lack of mechanical stability of printed hydrogels hinders the fabrication of high aspect ratio constructs. Here we present submerged bioprinting, a novel technique for freeform fabrication of hydrogels in liquid fluorocarbon. The high buoyant density of fluorocarbons supports soft hydrogels by floating. Hydrogel constructs of up to 30-mm height were generated. Using 3% (w/v) agarose as the hydrogel and disposable syringe needles as nozzles, the printer produced features down to 570-μm diameter with a lateral dispensing accuracy of 89 μm. We printed thin-walled hydrogel cylinders measuring 4.8 mm in height, with an inner diameter of ∼2.9 mm and a minimal wall thickness of ∼650 μm. The technique was successfully applied in printing a model of an arterial bifurcation. We extruded under fluorocarbon, cellularized alginate tubes with 5-mm outer diameter and 3-cm length. Cells grew vigorously and formed clonal colonies within the 7-day culture period. Submerged bioprinting thus seems particularly suited to fabricate hollow structures with a high aspect ratio like vascular grafts for cardiovascular tissue engineering as well as branching or cantilever-like structures, obviating the need for a solid support beneath the overhanging protrusions.

Engineering alginate as bioink for bioprinting

“…Among the various liquid-dispensing systems, piston-driven deposition has recently received significant attention because it offers a significantly high fabrication speed and is capable of fabricating anatomically shaped, clinically relevant-sized constructs [29,30]. They require a bioink with a suitable density and viscosity as well as the capability to retain printing fidelity and high cell viability post-printing [31,32]. In this study, we used a custom-made, piston-driven deposition system, as a test bed to examine the printability of biodegradable alginates.…”

Mechanically Tunable Bioink for 3D Bioprinting of Human Cells

“…[8] However, the advantage of direct fabrication techniques, especially of 3D bioprinting, is its ability to generate constructs with spatially defined cell and material composition. Moreover, biomaterials that are conventionally used in cell culture such as alginate, [9] Pluronic, [10] gelatin, [11] nanocellulose, [12] self-assembling peptides, [13] and agarose [14] are highly advantageous for direct cell printing as they are soluble in water and hence can be formulated as a cell carrier. There has been extensive effort to build on and improve the properties of watersoluble polymers as bioinks.…”

A comprehensive review on droplet-based bioprinting: Past, present and future

“…A C C E P T E D Mechanical valve (plunger)-based DOD [89], TIJ [17]; Micro-valve [32]; PIJ [60] Vascular tissue [89], In-vitro and invivo evaluation of stem cells functionality and differentiation [17]; Liver tissue [32] [34]; 60 mg/ml fibrinogen and 40 NIH U/ml of cell-laden thrombin [92]; 20 U/ml of thrombin [35] ; 10 mg/ml of manually deposited fibrinogen and 20 IU/ml of bioprinted thrombin [54]; 50 U/ml of cellladen thrombin bioprinted over 60 mg/ml of fibrinogen substrate [52]; 10 mg/ml of fibrinogen and 3 U/ml of thrombin [130]; 0.1 mg/ml of fibrinogen plated glass slides incubated in 4 U/ml of thrombin for 2 h at 37 ° C [101] HMVECs (2 × 10 6 cells/ml) with thrombin [92]; HMVECs (1-8 × 10 6 cells/ml) [52]; NHLFs (2 × 10 6 cells/ml) and GFPtransfected HUVECs (1 × 10 6 cells/ml) with fibrinogen and thrombin [130] Micro-valve [34,35,92,1 30]; TIJ [52,54] VEGF delivery for NSCs [34]; HMVECs-laden fibrin interlayers (bioprinted cell-laden thrombin and pipetted fibrinogen) for skin graft fabrication [92]; Crosslinking of bioprinted chondrocytes/fibrinogen/collagen layers for cartilage tissue fabrication [35]; Fibrin interlayers for 3D neural sheet fabrication [54]; Human microvasculature engineering [52]; Angiogenic sprouting between two parallel vascular channels [130]; 5% w/v [33] hMSCs with BMP-2 or TGF-β1 [33] Micro-valve [33] Fibrocartilage tissue model [33] Biomimetic, biocompatible , enzymatically degradable, good mechanical strength Slow gelation, requires photoinitiator and UV-source which can harm cells Polyethylene glycol UV crosslinking: Functionalized with photocrosslinkable side groups such as methacryloyl chloride [56,98,99]...…”

“…16 melting agarose and 3% (w/v) low viscosity alginate [89]. The bioprinting process was performed under nontoxic fluorocarbon, which provided the necessary buoyancy forces needed to support the soft tissue structure.…”