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International Journal of Bioprinting Precise fabrication of engineered vascular networks

paraformaldehyde (BL539A, Biosharp, China) for 30 min the subcutaneous tissue and skin were sutured. Penicillin
at room temperature. Then, the samples were treated with sodium was routinely administered intramuscularly for 3
0.1% Triton X-100 (Triton X, Biosharp, China) diluted in consecutive days after surgery to prevent infection. Mice
PBS for 30 min. Subsequently, the samples were stained were housed and monitored in groups. The implanted
with TRITC Phalloidin (Maokang Biotechnology, China) stents and surrounding tissues were removed after 4 weeks
in 1% BSA for 30 min. The samples were then stained with and 8 weeks of in vivo implantation, respectively, for
Hoechst 33342 (Solarbio Life Science, China) for 10 min preliminary observation of blood vessel formation in and
and washed three times with PBS. Finally, images of the around the implanted stents. Samples were fixed in 4%
scaffolds were captured using a confocal microscope (TCS paraformaldehyde solution for 48 h and then paraffin-
SP8 STED 3X, Leica, Germany). embedded. Sections (5 μm) were stained with H&E and
Masson’s trichrome, and the number of vessels was counted
2.9. In vivo study under the microscope. The sections were stained with
This study was carried out following the recommendations fluorescent double-labeling (CD31, α-SMA) and finally
of the Animal Care and Experiment Committee of Shanghai scanned.
Ninth People’s Hospital, Shanghai Jiao Tong University
School of Medicine. The protocol was approved by the 2.10. Statistical analysis
Animal Care and Experiment Committee of Shanghai Ninth Each experiment was performed in triplicate. The count
People’s Hospital, Shanghai Jiao Tong University School of data are presented as mean ± standard deviation. All
Medicine (SH9H-2022-A11-1/SH9H-2022-A10-1). All quantitative data were analyzed using a one-way analysis
methods were performed following relevant guidelines and of variance (ANOVA) with Tukey’s significant difference
regulations. A total of 12 eight-week-old male C57BL/6 post-hoc test for multiple comparisons (OriginPro 2021
mice were selected and randomly divided into four groups: Learning Edition, USA). Values of *p < 0.05, **p < 0.01,
the control group (no vascular scaffold), group I (1 × 1 and ***p < 0.001 were considered statistically significant.
scaffold), group II (4 × 4 scaffold), and group III (8 × 8
scaffold). Mice were anesthetized during surgery, and 3. Results and discussion
three mice in each group were implanted subcutaneously
with hydrogel scaffold in the back. No signs of pain or 3.1. Swelling compensation for 3D-printed
discomfort were observed after surgery or throughout the vasculature
study. The mice were kept in groups and monitored for To investigate the potential of using the volume shrinkage
4 weeks. After 4 weeks of subcutaneous implantation, mice induced by the P/G hydrogel to compensate for the
from each group were euthanized to remove the implanted swelling of the sacrificial template, a customized 3D
scaffolds and surrounding tissues for initial observation printing platform based on an FDM printer and a syringe
of blood vessel formation in and around the implanted pump was used to print the sacrificial PF-127 (Figure 2A).
scaffolds. Samples were fixed with 4% paraformaldehyde Three needles of different sizes (19 G [I. D. 720 μm], 20 G
solution for 48 h and paraffin-embedded. Sections [I. D. 610 μm], and 21 G [I. D. 520 μm]) (Figure 2B) were
(5 μm) were stained with hematoxylin and eosin (H&E) used to print the pre-designed zigzag path (Figure 2C).
and Masson’s trichrome, and the number of vessels was First, different feed rates of the sacrificial PF-127 were set
counted under a microscope. Sections were fluorescently to study their effect on the structure of printed sacrificial
double-stained (CD31, α-SMA) to examine the formation templates. In this section, the inner diameter of the needle
of vasculature and finally scanned. In addition, 24 six- was set as the designed diameter of the fiber in the printed
[35]
week-old male Sprague-Dawley rats were selected and sacrificial templates. As shown in Figure 2D, an ideal
randomly divided into four groups: blank control group, zigzag structure could be printed when the feed rate was
group I (1×1 scaffold), group II (4×4 scaffold), and group set above 275 μL/min for a 19-G needle. The optimal feed
III (8×8 scaffold). All rats were injected intraperitoneally rates for 20- and 21-G needles to obtain the target structure
with anesthetic, the inguinal skin of the left leg of the rat are 225 and 175 μL/min, respectively. Microscopy images
was incised, the artery was stripped out, and the proximal of the printed sacrificial templates were captured, and
and distal points (distance about 8 mm) were ligated and the diameters of the template fibers were quantitatively
cut off after ligation. The blank control group was injected analyzed, as shown in Figure 2E and F. Template fibers
with about 1 mL of PBS, while the hydrogel of the test with a diameter of 750.6 ± 24.4 μm, which is close to the
group was implanted in the center of the ischemic vascular inner diameter of the 19-G needle, can be printed when the
resection area of the rat after surgery, and the severed feed rate is set at 275 μL/min. For 20- and 21-G needles,
end of the vessel was placed above and to the side of the the diameters of the template fibers were 638.4 ± 21.4 and
sample holder. The samples were fixed with sutures, and 536.1 ± 33.7 μm, respectively, with feed rates set at 225

Volume 9 Issue 5 (2023) 39 https://doi.org/10.18063/ijb.749