3D PRINTING AND PATTERNING VASCULATURE IN ENGINEERED TISSUES - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 173

layer-by-layer hydrogel photopatterning so that layers sufficiently adhere to each other and reduce
overcuring of printed layers, as well as swelling behavior and mechanical properties of the laser polym-
erized PEGDA hydrogels. The authors were also able to demonstrate control over spatial distribution of
cells in a multilayered structure with minimal mixing as well as high viability, proliferation, and spread-
ing of entrapped cells, indicating that this methodology can be applied to achieve high spatial control
to recapitulate complex 3D tissue microarchitecture. Tissue constructs with open channels can also be
obtained by using a light-based projection stereolithography system due to the high fabrication rate and
resolution of this technique in printing layer-by-layer ( Lin et al., 2013 ). However, solutions contain-
ing cells in the apparatus chamber tend to settle down over time during the printing process due to the
inherent higher density of the cells, resulting in constructs with nonhomogenous encapsulation of cells.
To address this issue, the authors included optimized concentration of Percoll to increase the density of
the precursor solution to match the density of the cells, making them neutrally buoyant. However, addi-
tion of Percoll resulted in some undesirable curing since the authors observed that fabrication of porous
structures resulted in partial pore occlusion. The authors also fabricated open channel scaffolds with
encapsulated cells and observed high cell viability and metabolic activity in the fabricated hydrogels,
even though the scaffolds did not contain any adhesive peptides.
Other methods for fabricating vessel-like tissue constructs involve dispensing cell containing mac-
rofilaments or spheroids and then allowing the cells to fuse into whole tissue constructs. One strategy
for direct-write bioprinting of a cell-laden ECM hydrogel dispensed prepolymerized cell laden GelMA
hydrogel fibers from glass capillary tubes ( Bertassoni, Cardoso, et al., 2014 ). The bioprinting process
involves aspirating the hydrogel precursor into a glass capillary by the upward movement of a metallic
piston, followed by photopolymerization of the precursor inside the capillary by exposure to UV light,
and then dispensing the hydrogel fiber as the metallic piston is pushed down against the cross-linked
gel. The authors demonstrated bioprinting of macroscale 3D cell-laden constructs by positioning hy-
drogel fibers in one plane and stacking a second layer of perpendicular fibers on the plane above. More
complex constructs were fabricated that contained GelMA hydrogel blocks with impregnated planar
and 3D bifurcating fiber networks to achieve tissue analogs that mimic vasculature. To fabricate con-
structs with hollow fibers, a sacrificial layer was dispensed during the printing process and layer re-
moved, resulting in microchannels within the fabricated construct. The authors demonstrated relatively
high cell viability and proliferation rates of encapsulated cells in the printed gels.
Additionally, other direct-write processes utilize a scaffold-free approach to circumvent some hur-
dles in tissue engineering approaches that utilize a biomaterial scaffold, such as a plethora of materials
to choose from, constructing viable tissues with high cell density, and potential uncontrollable scaffold
mechanical effects on cell behavior. Thus, utilization of a scaffold-free approach can avoid some of
these issues. Furthermore, a scaffold-free approach is inspired by the self-assembly process of cells
during early morphogenetic processes, in which individual cells organize into multicellular subunits
( Chang et al., 2013 ). To fabricate bioartificial vessel-like grafts using a scaffold-free approach, an
open-source printer, modified to hold microcapillary tubes for bioprinting of hydrogel macrofilaments,
was used to dispense cylindrical macrofilaments layer-by-layer ( Skardal et al., 2010 ). Culture of the
filaments resulted in discrete structures, as the filaments fused with each other to form a continuous
structure. With this approach, the resolution of the printed tissue construct was determined by the capil-
lary internal diameter, which was 500 m m. To better facilitate the fusion of dispensed cell-containing
materials, Jakab et al . utilized a scaffold-free approach by dispensing multicellular spheroids and
cylinders to allow cells to self-organize into functional living structures of prescribed shape ( Jakab