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Ng H Y, et al.

and growth of vascular channels. For instance, oxygen that even though tunica intima was not fabricated in
as we know is vital for cellular survival, regulation, this study, it is of importance to note that endothelial
differentiation and metabolism etc .[56] . In response cells do play a role in regulating final mechanical
to in-vivo hypoxia, our bodies activate and produce property of construct as endothelial cells are known
hypoxia-inducible factors (HIFs) that accumulates to secrete transforming growth factors that promotes
and subsequently activate expression of various genes tissue homeostasis and regulates extra-cellular matrix
[59]
and cascades. A key function of HIFs is the regulation degradation and production .
in expression of angiogenic genes that promote Laser-based techniques are also a common technique
vasculogenesis. An interesting published study applied used in self-assembly method as it allows specific
the vast knowledge of hypoxia and fabricated a hypoxia- deposition of individual cells. Early work had
inducible gelatin-ferulic acid hydrogel. It is reported that demonstrated that such technique allowed cells to be
8
the authors were able to specifically control the oxygen printed individually suspended in 10 cells/ml viscous
levels within the hydrogels, thus was able to promote in- hydrogel consisting of glycerol and sodium alginate.
vitro tubulogenesis of endothelial-colony-forming- cells In addition, laser-based techniques have the fastest
by activating HIFs with subsequent in-vivo promotion of printing speed amongst all the other techniques, thus
[57]
blood vessels recruitment and infiltration . minimizing unnecessary damage to the cells [60] . In this
With the emergence of bioprinting, it is expected that study, the authors demonstrated the ability of laser-based
pre-placement or directed cell deposition can aid in technique to bioprint two distinct concentric circles of
directing and controlling distribution and direction of human endothelial cells and showed that different cell
vascular channels. Droplet-based direct extrusion was types can be printed in close proximity to one another
also used to biofabricate construct with micro-channels with high cell concentration and according to desired
by the self-assembly method. Early works include pattern. Another study exploited the advantages of laser-
modification of a home-based thermal inkjet printer based techniques and fabricated branch-stem structures
(Hewlett Packard HP500). Droplets volume of 130 of HUVEC and human umbilical vein smooth muscle
picoliter (pL) of human micro-vascular endothelial cells cells (HUVSMC) [61] . By depositing cells in a branch-
(HMVECs) and fibrin were simultaneously deposited stem structure, initial results showed that HUVSMC
via 50 nozzles on the printer head. At the same time, tend to cluster together when deposited individually
fibrinogen, thrombin and calcium ions were used for whilst HUVEC can be directed and guided to form
[35]
rapid gelation of constructs after printing . Even though lumen networks within a day. However, pure HUVEC
there were no clear structural integrity after 14 days of lumens have limited stability which could be augmented
culture, results demonstrated that the printed endothelial by depositing HUVSMC alongside the HUVEC layer.
cells were able to proliferate and form a confluent lining Therefore, this study demonstrated that a certain level
along the fibrin scaffold after 21 days of culture with of artificial guidance, coupled with self-assembly of
distinct tubular integrity. cells, could be the way ahead for fabrication of vascular
A novel self-assembly method to biofabricate constructs.
autonomous vascular structures was reported using In-vivo studies of implanted cardiac patches into
a single-step process approach. A sheet of tissue was rats that underwent myocardial infarction showed
produced by culturing smooth muscle cells along with enhanced angiogenesis in the borders of infarction as
fibroblasts on a gelatin-coated tissue culture plate. After well as formation of functional vascular networks in
[62]
which, the sheet was rolled onto a tubular support thus the patch . Previously, it was found to be challenging
forming a vascular tubular construct with an internal to fabricate viable vascular constructs for implantation
smooth muscle layer and an external fibroblast layer as newly formed vessels tend to be immature and
which attempts to mimic the structure of tunica media unstable. In addition, it was reported that co-culturing
and tunica adventitia. In this study, it was reported that of HUVEC and mesenchymal stem cells (MSCs) could
such a self-assembly approach was able to generate enhance stability of newly formed vessels. With laser-
vascular constructs with significant mechanical based technique, HUVECs and MSCs could now be
properties based on the types of fibroblasts used. Self- printed in close proximity to each other in an intended
assembly approach allowed the smooth muscles cells design. The construct was then cultured for eight days
and fibroblasts to produce extra-cellular matrix which to allow production of extra-cellular matrix and self-
helps in determining mechanical properties of assembled assembly of cells before implantation. Post-implantation
vascular construct. For instance, constructs with dermal results showed that angiogenesis and functional neo-
fibroblast were shown to have denser and more compact vascularization were enhanced significantly in the
extra-cellular matrix, thus resulting in higher mechanical infarction area.
strength and burst pressure [58] . The authors mentioned A recent study by Kim BS, et al. demonstrated the

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