Page 54 - IJB-9-5

International Journal of Bioprinting Precise fabrication of engineered vascular networks

surface of the vasculature after removing the sacrificial and OCs. P/G hydrogel was prepared as described
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PF-127. SEM images of the vasculature surface showed that previously. The prepared hydrogel extracts were added to
the vasculature fabricated by PF-127 was glossy (Figure S8 the cell culture medium at different concentrations (0%,
in Supplementary File), which may make it difficult for cells 25%, 50%, 75%, and 100%) and incubated with HUVECs
to attach to and grow on. Thus, in this experiment, 5% (w/v) and MG63 for 24 and 48 h, respectively. After 24 and 48 h
gelatin was added to PF-127 to change the morphology of incubation, the cell survival rate of each group was found
of the vasculature surface, as gelatin exhibits a granular to be not significantly different after assaying with the
structure in the sacrificial PF-127 hydrogel. SEM images of CCK-8 test (Figure S9 in Supplementary File), indicating
the vasculature fabricated by PF-127 + gelatin showed that that the P/G hydrogel extracts were non-toxic to the cells.
the vasculature surface has a honeycomb structure packed For the micro-channel, sodium alginate sacrificial fibers
with grooves (Figure S8 in Supplementary File). Cells tend were wet-spun in calcium chloride solution and orderly
to adhere to a rough surface more than to a smooth one . arranged on the crosslinked P/G hydrogel, as shown in
[40]
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Thus, the porous structure can benefit the attachment of Figure 7A. 34-G (I. D. 60 μm, O. D. 230 μm) needle was
cells. As shown in Figure S7 (Supplementary File), the cells used in this study. After that, PF-127 + gelatin sacrificial
could stably attach to the vasculature surface with spindle- fibers were printed on the sodium alginate sacrificial fibers
like morphologies during the culturing. Significant cell and the crosslinked P/G hydrogel to serve as macro-
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proliferation was discerned. Thus, PF-127 + gelatin was channel and cellular channel. PF-127 + gelatin and sodium
selected as the sacrificial material in this experiment. alginate were removed in ethylenediaminetetraacetic acid
For the formation of the endothelial monolayer, the disodium salt (EDTA-2Na) at 4°C after crosslinking by
growth of HUVECs inside the lumens was recorded every light. Subsequently, the hierarchical vasculature model
day (Figure 6B). The results showed that the HUVECs shrunk at 37°C for 2 h to prepare an in vitro model for
could distribute on the surface of the vasculature lumens HUVECs and OCs, as shown in Figure 7A. MG63 and
and predominately form spindle-like morphologies, which HUVECs were injected into the prepared in vitro model
further indicates the biocompatibility of the P/G hydrogel. shown in Figure 7B, and the growth status of both cells was
The number of cells significantly increased during observed.
the culturing. After 5 days of culturing, the HUVEC The results revealed that OCs could migrate into the
monolayer could be visualized in the microscopy images. micro-channels after culturing with HUVECs. It could
Immunofluorescence staining of CD31, vinculin, and be seen on confocal fluorescence imaging that MG63 and
VEGF of the formed endothelial monolayer was conducted HUVECs in the experimental group spread throughout
on day 5 to evaluate the endothelialization of the HUVECs the channels, as shown in Figure 8. The enlarged views of
attached to the inner surface of the vasculature lumen. As HUVECs and OCs in the experimental group can be seen
shown in Figure 6C, cells were interconnected and formed from Figure S10A (Supplementary File). In the control
the endothelial monolayer in the inner surface of the group, OCs could not migrate into the micro-channels
vasculature lumen. High expression of the CD31 marker and grew in a single channel when culturing without
demonstrated the attainment of endothelial function. As HUVECs, as shown in Figure 8 as well as Figure S10B
a pivotal protein marker in the cell–scaffolds interaction, and S10C (Supplementary File). However, HUVECs could
the appearance of the vinculin maker suggests that the migrate to other channels along the micro-channels when
firm adhesion of the HUVECs was achieved on the inner culturing without OCs, confirming that this engineered
surface of the vasculature lumen. Moreover, the enormous vasculature model has good biocompatibility and can
expression of VEGF for angiogenesis further exhibits the promote the growth of endothelial cells to promote blood
potential of the P/G hydrogel scaffolds with vasculature vessel formation. Thus, the engineered vasculature within
to support vascularization. Although PNIPAM has been the P/G hydrogel fabricated in this study can be a potential
reported to be cytotoxic to endothelial cells , we prepared model for drug screening and organ-on-a-chip.
[41]
a hydrogel scaffold by mixing GelMA with PNIPAM to
enhance the cell adhesion of P/G hydrogel since GelMA has 3.7. In vivo study
been widely used as a good cytocompatible material [42–44] . To evaluate the in vivo vessel formation ability of the P/G
From the above results, it can be seen that P/G hydrogels hydrogel, scaffolds with vasculature, scaffolds without
have good cytocompatibility and good expression of vasculatures for the control group, and scaffolds (1 × 1, 4 ×
relevant markers. 4, and 8 × 8) with a different number of vasculatures were
implanted subcutaneously in mice. The size of the scaffolds
3.6. Interaction between HUVECs and OCs in each group was 1 × 1 × 0.2 cm. The P/G hydrogel and
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In this section, the engineered vasculature was utilized as an 20-G needle were used in this experiment. After 4 weeks of
in vitro model to explore the interaction between HUVECs implantation, the scaffolds with the surrounding skin were

Volume 9 Issue 5 (2023) 46 https://doi.org/10.18063/ijb.749