3D PRINTING AND PATTERNING VASCULATURE IN ENGINEERED TISSUES - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 178

grooves not only provide physical guidance for the sprouting capillaries, but also influence the local
concentration of autocrine growth factors released from the ECs actively participating in angiogenesis.
Organotypic models were also fabricated to investigate angiogenic sprouting and neovessel formation
within a 3D ECM by flowing combinations of factors next to an endothelium-lined channel ( Nguyen
et al ., 2013 ). In this approach, a 3D microfluidic device with two parallel channels was fabricated by
casting collagen into a PDMS mold with needles placed across the casting chamber, allowing the colla-
gen to polymerize, and then removing the needles to result in hollow cylindrical channels in the matrix.
After ECs were seeded into one of the channels, the second channel was perfused with angiogenic
factors to establish a gradient across the collagen matrix to the endothelium. The authors observed that
when a complex combination of proangiogenic factors was perfused into the second channel, more
substantial multicellular sprout-like structures were formed which mirror major steps of in vivo angio-
genesis ( Figure 8.3 B). After 1 week of culture, tip cells breached the source channel and resulted in a
connection between the two parallel channels. Perfusion studies showed that beads flowed through the
neovessels without leakage into the interstitial space, indicating fully developed, continuously endo-
thelialized lumens. Additionally, the authors observed maturation of secondary branches with their own
new tip cells in the fabricated organotypic model.
In addition to observing angiogenesis and vasculogenesis in vitro , some efforts demonstrating these
processes in vivo have emerged. One such technique, laser-induced forward transfer, involves transfer
of material from a donor slide, covered with a laser-absorbing gold layer, onto a collector slide by
pulsing the laser. Transfer of material to the collector slide is achieved through generation of high gas
pressure, which propels material on the donor slide toward the lower collector slide. This technique was
used by Gaebel et al. to create specific vascular patterns consisting of different cell types for cardiac
regeneration ( Gaebel et al., 2011 ). In addition to the gold layer, the donor slide also contained a layer of
cell-containing material to be transferred while the collector slide contained a polyester urethane urea
patch immersed in Matrigel ® . A cardiac patch composed of polyester urethane urea was spatially pat-
terned with ECs and mesenchymal stem cells (MSCs) in vitro and then transplanted to infarcted zone
of rat hearts. After 8 weeks of implantation, the authors observed increased vessel formation and func-
tional improvement of infarcted hearts of transplanted implants. Additionally, the authors mentioned
that histological analysis revealed integration of the implanted patch with the host myocardium. Tissue
constructs fabricated by ink-jet printing technology were also implanted into mice and then analyzed
for vasculature formation by using MRI ( Xu et al., 2008 ). In this approach, the print head consisted
of calcium chloride and cells, while the chamber consisted of sodium alginate and collagen solution
mixture. Upon ejection of the cell-calcium chloride drops, gelation occurred rapidly, resulting in con-
struction of 3D structures. After implanting the printed tissue constructs, the authors used MRI with
contrast perfusion to image the vasculature formation in vivo and performed histological analysis to
determine host cell invasion into the transplanted construct. The authors observed a vascular network in
the superficial area of the constructs printed with cells, which express EC-specific marker vWF, while
few vascular networks were found in the cell-free implant.
Interestingly, Skardal et al. (2012) also printed patches directly onto the wound site to accelerate
the healing process of large skin wounds. In this study, bioprinting technology was used to deposit
fibrin/collagen gels, with either MSCs or amniotic-fluid-derived stem (AFS) cells, directly onto the
defect site of mice. The printed patch provided full coverage over the wound area and evidence of
re-epithelialization was observed by histological analysis ( Figure 8.3 E). Compared to a gel-only treat-
ment, treatment with MSC and AFS resulted in thicker regenerating tissue with a greater number of