Page 329 - IJB-10-6

International Journal of Bioprinting 3D-printed PPDO/GO stents for CHD treatment

2.6. Surface morphology analysis of PPDO/GO films supplemented with 1% penicillin-streptomycin (HyClone,
and sliding-lock stents USA) and 10% fetal bovine serum (Gibco, USA), before
incubation in a humidified incubator with 5% CO at 37°C.
The surface morphology of PPDO/GO films and 2
sliding-lock stents was analyzed by SEM using a RISE- The culture medium was refreshed every two days. PPDO/
GO stents were cut into small pieces (1 × 1 cm), washed
MAGNA system (TESCAN, Czech Republic) at an with 75% ethanol solution and deionized water, and
accelerating voltage of 5 kV. The surfaces were sputtered sterilized by ultraviolet radiation. After sterilization, the
with gold before observation.
samples were placed into 12-well culture plates, immersed
2.7. In vitro evaluation of hemocompatibility in DMEM, and incubated for 2 h.
2.7.1. Hemolysis test 2.8.2. Cell adhesion and proliferation
Fresh Sprague–Dawley (SD) rats blood was collected The HUVECs were seeded on the samples in 12-well
4
in heparinized test tubes. Red blood cells (RBCs) were culture plates at a density of 2 × 10 cells/mL and incubated
obtained by centrifuging the anticoagulated blood at 4000 in the culture medium. After incubation for 1, 3, 5, and
r/min for 5 min and then removing the supernatant plasma. 7 days, cell viability and proliferation were evaluated by
The 2% RBC solution was prepared by dispersing the RBCs a Cell Counting Kit-8 (CCK-8) assay according to the
in normal saline. PPDO/GO stents were cut into small manufacturer’s protocol. The culture medium was removed,
pieces and incubated with 2 mL of the 2% RBC solution and the samples were washed with PBS. The samples were
at 37°C for 2 h. After the removal of stents, the suspension then transferred to a 12-well plate, and CCK-8 reagent
was centrifuged at 4000 r/min for 5 min. For positive and (Beyotime, China) was added to each well and incubated
negative controls, the 2% RBC solution was incubated for 2 h at 37°C. The absorbance of the supernatant was
with deionized water and normal saline, respectively. measured using a microplate reader at 450 nm.
The absorbance of the supernatant was measured at 545 2.8.3. Cell morphology
nm using a microplate reader. The hemolysis rate was The HUVECs were seeded on the samples in 12-well culture
calculated using the following equation: plates at a density of 2 × 10 cells/mL and incubated in the
4
culture medium. After incubation for 1, 3, 5, and 7 days, the
OD − OD samples were washed with PBS, and the adhered HUVECs
Hemolysis % () = s n ×100 % (III)
OD − OD were fixed with 4% paraformaldehyde (Beyotime, China).
p
n
The HUVECs were permeabilized using 0.1% Triton X-100
(Beyotime, China). Actin filaments were stained with
where OD is the absorbance of the sample, OD is
s
n
the absorbance of the negative control, and OD is the rhodamine-conjugated phalloidin (Solarbio, China), and
cell nuclei were stained by 4,6-diamidino-2-phenylindole
p
absorbance of the positive control.
(DAPI; Vector Laboratories, USA). Afterwards, the samples
2.7.2. Platelet adhesion test were observed using laser confocal scanning microscopy
Platelet-rich plasma was prepared by centrifuging the (LCSM; Leica, Germany) to evaluate the cell morphology
anticoagulated fresh rat blood at 1000 r/min for 10 min. on the PPDO/GO stents.
PPDO/GO stents were cut into small pieces and incubated 2.9. In vivo evaluation
with 1 mL platelet-rich plasma for 2 h at 37°C. The All procedures were approved by the Animal Ethics
samples were rinsed with phosphate-buffered saline (PBS; Committee and performed in strict accordance with their
BasalMedia, China) three times and immersed in 2.5% regulations. 3D-printed PPDO/GO filaments with a length
glutaraldehyde solution (Solarbio, China) for at least 6 h to of 15 mm and a diameter of 0.1 mm were implanted in
fix the adhered platelets. The samples were then dehydrated the abdominal aortas of SD rats to evaluate the effect of
by gradient ethanol solutions (50%, 60%, 70%, 80%, 90%, PPDO/GO materials on in vivo endothelialization. After
and 100%) and dried at room temperature. Thereafter, the implantation for 2 weeks, the vessels containing embedded
samples were sprayed with gold and observed by SEM. filaments were harvested and cut longitudinally to expose

2.8. In vitro evaluation of cell compatibility the tissue on the surface of the filaments. The samples were
fixed by 2.5% glutaraldehyde solution (Solarbio, China) for
2.8.1. Cell culture at least 6 h and dehydrated using gradient ethanol solutions
Human umbilical vein endothelial cells (HUVECs) were (50%, 60%, 70%, 80%, 90%, and 100%). After drying at
sourced from the Type Culture Collection of the Chinese room temperature, the samples were sprayed with gold on
Academy of Sciences (China) and cultured in Dulbecco’s the surface and observed by SEM. After implantation for
Modified Eagle Medium (DMEM; Gibco, USA), 4 weeks, the PPDO and PPDO/GO filaments, along with

Volume 10 Issue 6 (2024) 321 doi: 10.36922/ijb.4530