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Ng H Y, et al.

for general metabolic and physiological functions. With be maintained for more than two weeks with cellular
respect to organs like liver whereby it is one of the most growth, proliferation and differentiation [47] . A more
perfused organs in the human body, vascularization recent novel direct extrusion bioprinting strategy include
became an important aspect of liver tissue engineering. using coaxial nozzle concurrently print hollow channel
In addition, it is reported that blood flow in liver exerts a vessel-like cellular micro-fluidic channels [48] . In this
certain level of shear stress on hepatocytes which causes study, a triple tube coaxial nozzle was set up with sodium
[44]
expression of several genes . alginate in the outer tube and calcium chloride in the
The most straightforward approach to perfusable inner tube. At the same time, the scaffold was bioprinted
scaffolds is to use extrusion-based technique to directly into a platform in a container filled with calcium
biofabricate a network of interconnected channels within chloride cross linking solution. By controlling the
the scaffold itself. Early works for extrusion-based concentration and rate of extrusion, this team was able
technique include using a homemade 3D bioprinter to to allow partial cross-linking in the inner walls of both
print bovine aortic endothelial cells in Type Ⅰ collagen alginates before allowing the whole extrusion to be
[45]
and human fibroblast in Pluronic-F127 . In this study, immersed into the solution for complete gelation. With
the researchers managed to generate spatially organized this technique, they were able to biofabricate scaffolds
constructs with customizable designs such as mimicking with hollow channels and subsequently demonstrated the
the structure of the left anterior descending artery of potential of coaxial nozzles in biofabricating large scale
a pig. A five layered bovine aortic endothelial cells in tissue constructs.
Type Ⅰ collagen bioink was biofabricated with the same However, it is not easy to maintain reproducibility
pattern as found in the angiogram, with maintenance of as visco-elasticity of hydrogels tend to vary over time
structural integrity after 35 days of culture. In general, it and between batches. Thus there is a need to fine
is reported that extrusion-based technique has the ability tune printing parameters after every session. A recent
to generate viable constructs by depositing various cell strategy for biofabrication of perfusable scaffolds has
types during one extrusion session, of which deposition been largely based on indirect extrusion such as using
of endothelial cells or micro-vessels fragments as one sacrificial inks [34] . Although indirect extrusion has its
of the bioink has the potential of promoting vascular own advantages in biofabricating well defined micro-
[46]
network formations with high viability . channels, it is found that these sacrificial inks tend
With advancement of technology and discovery be associated with cytotoxic by-products after post
of novel biomaterials, extrusion-based bioprinting dissolution treatment of sacrificial inks. For instance,
gradually branches out into direct and indirect extrusion. high concentrations of Pluronic-F127 is found to have
[49]
As mentioned, the difference between both techniques significant cytotoxic effects on cells . Regardless, we
is mainly on the usage of another sacrificial biomaterial can see increasing novel reports of indirect extrusion
as additional support. Early works for direct extrusion techniques with naturally derived or artificial sacrificial
include using a bottom-up layer-by-layer dual-nozzle inks. There are several criterions that the sacrificial ink
[2]
bioprinting approach with hepatocytes in gelatin/ must meet before it can be used with the main bioink .
alginate/chitosan (GAC) hydrogel and ADSCsin gelatin/ Firstly, the inks must be compatible with one another
alginate/fibrinogen (GAF) hydrogel. In this study, it is during printing under ambient condition. Secondly, the
clearly demonstrated that precise control of external cells in the bioink and its structural integrity must not be
parameters such as temperature and pressure is required altered or change during printing or removal of sacrificial
to maintain printability of hydrogel based largely on the ink. Inks that require harsh printing parameters or require
visco-elasticity behavior of the hydrogels. Therefore, harsh removal techniques are not suitable. A recent
with the above knowledge, this team used a digital study exploited the unique physical and shear-thinning
model to construct a circular biomimetic liver construct properties of Pluronic-F127 and different concentrations
with seven vascular channels within the construct. of cell laden GelMAto biofabricate 3D perfusable
Hepatocytes/GAC was used to construct the main scaffolds. A final four layered scaffold was produced in a
liver component surround the ADSC/GAF vascular layer-by-layer wise manner by co-printing different inks
channels. After which, the constructs undergo post- through 200μm nozzles, followed by photo-crosslinking
modifications with cross-linking agents such as thrombin of GelMA and removal of sacrificial Pluronic-F127. It
and calcium chloride solution to allow sol-gel transition was reported that high fidelity vascular channels with
for preservation of structural integrity. This pioneering customizable designs can be fabricated with such a
direct extrusion study demonstrated that complex hybrid method and these channels can be easily endothelized
constructs with distinct spatial organizations are possible and perfused with cell culture media. Even though the
and that with proper bioprinting, crosslinking methods various cells encapsulated in the GelMA hydrogel had
and differentiation induction, structural integrity can an initial decrease in viability (60–70% cell viability),

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