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International Journal of Bioprinting Precise fabrication of engineered vascular networks

2.6. Characterization scaffolds to investigate the intercellular connection and
The P/G hydrogels and PF-127 were measured by Fourier endothelialization function of the endothelial monolayer
transform infrared spectroscopy (FTIR) to determine formed on the inner surface of the engineered vasculature.
the presence of residual PF-127 in the prepared P/G Samples were washed with pre-warmed PBS and fixed
hydrogels. To confirm the surface characteristics’ changes in 4% paraformaldehyde (BL539A, Biosharp, China) for
of the P/G hydrogel at 25°C and 37°C, the contact angles 30 min at room temperature. Then, the samples were
were examined. The contact angle of the P/G hydrogel treated with 0.1% Triton X-100 (Triton X, Biosharp, China)
film at 25°C was measured directly by placing the film diluted in PBS for 30 min. Subsequently, the scaffolds were
on the platform of the contact angle meter (JC2000D1, blocked in 1% bovine serum albumin (BSA) in PBS for
POWEREACH, China). After shrinking in a 37°C water 1 h at room temperature. Upon washing thrice with PBS,
bath for 2 h, the P/G hydrogel film was placed on a heating the scaffolds were incubated in the diluted CD31 antibody
plate at the temperature of 37°C. The heating plate was (Abcam, USA) at 1:500 ratio, vinculin antibody (Abcam,
transferred to the platform of the contact angle meter USA) at 1:100 ratio, and VEGF antibody (Invitrogen,
to measure the contact angle of the P/G hydrogel film at USA) at 1:200 ratio in 1% BSA overnight at 4°C. After
37°C. The contact angles were captured immediately after washing thrice with PBS, the scaffolds were incubated
dropping the water on the hydrogel samples to avoid the with Alexa Fluor 488-conjugated goat anti-rabbit IgG H&L
absorption. The surface of the vasculature within scaffolds secondary antibody (1:500 dilution, Invitrogen, USA) in
fabricated by PF-127 and PF-127 + gelatin was observed 1% BSA for 2 h at room temperature in the dark. In the
by scanning electron microscopy (SEM, SIRION 200, following step, the scaffolds were stained with TRITC
USA). The samples were freeze-dried in liquid nitrogen Phalloidin (Maokang Biotechnology, China) in 1% BSA
and lyophilized, and the inner surface of the vasculature for 30 min. The scaffolds were then stained with Hoechst
was sputtered with gold for observation using SEM. 33342 (Solarbio Life Science, China) for 8 min after being
Compression modulus was measured with a mechanical washed with PBS thrice. The same protocol was used to
tester (BAIROE, China) for the control group (no vascular stain the attached HUVECs on the outer surface of the P/G
scaffold), group I (1 × 1 scaffold), group II (4 × 4 scaffold), hydrogel scaffolds. Finally, the images of scaffolds were
and group III (8 × 8 scaffold). captured using a confocal microscope (TCS SP8 STED 3X,
Leica, Germany).
2.7. Formation of endothelial monolayer
Hydrogel scaffolds with engineered vasculature were used 2.8. Interaction of HUVECs with OCs
in this section to study the endothelialization of HUVECs. P/G was chosen as the hydrogel concentration, and a 20-G
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P/G was used as the hydrogel concentration, and a 20-G needle was used. The wet spinning method was used to use
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needle was employed. Pattern 2 with six fibers was used sodium alginate as a tiny channel to communicate between
to fabricate the samples. Cells up to passage 6 were used endothelial cells and osteosarcoma cells. Cell viability of
in the following experiments. The fabricated samples were HUVECs and MG63 (Fuheng Biology, China) was first
immersed in 75% ethanol overnight under UV light, washed detected using Cell Counting Kit-8 (CCK-8) (Bimake,
with phosphate-buffered saline (PBS; Cytiva, USA) thrice, USA). The sterilized samples were washed three times with
shrank in the 37°C incubator for 30 min, and immersed in PBS, and 10 mL of Dulbecco’s Modified Eagle Medium
freshly prepared endothelial cell medium (ScienCell, USA) (DMEM; Gibco, Ireland) was added to the mixed hydrogel
at 37°C before use. After trypsinization, the cells were for 24 h to obtain the extracted solution of the hydrogel.
resuspended in endothelial cell medium at a concentration Diluted 100 μL of the extracted solution containing 0%,
of 2.5 × 10 cells/mL. Then, the cell suspension was slowly 25%, 50%, 75%, and 100% of the mixed hydrogel was added
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injected into the vasculature channels using a 1-mL to each well of a 96-well culture plate. Cell suspensions
sterilized syringe. After culturing in the incubator for of HUVECs and MG63 were then added to the 96-well
4 h, the samples were flipped upside down, and the cell plates (5000 cells/well), respectively. Cell viability was
suspension with the same concentration was injected into detected using CCK-8 after 24 h and 48 h of incubation at
the vasculature again to achieve a uniform and complete 37°C in 5% CO . MG63 and HUVECs were injected into
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cell seeding. Finally, the samples were statically cultured the channels according to the above method, immersed
with the medium changed every day and observed under a in DMEM medium, and cultured statically. The medium
microscope (Olympus, Japan). Fresh medium was injected was changed daily and observed under a microscope
into the channel gently to ensure the nutrient supply for (Olympus, Japan). After 5 days of culture, cytoskeletal and
cells. After culturing for 5 days, CD31 antibody, vinculin nuclear staining was performed on hydrogel scaffolds to
antibody, and vascular endothelial growth factor (VEGF) study the interaction between OCs and HUVECs. Samples
antibody immunostaining was performed on the hydrogel were washed with pre-warmed PBS and fixed with 4%

Volume 9 Issue 5 (2023) 38 https://doi.org/10.18063/ijb.749