Page 105 - IJB-9-3

International Journal of Bioprinting The biological properties of WE43 scaffolds via the oxidative heat strategy

5 min at 1000 rpm. The cells were centrifuged after being conditions were the same as those in the ALP activity
rinsed twice with precooled PBS, and the supernatant test. Tests were performed on day 21 of culture. The
was discarded after separation. Following the removal of media was removed, and PBS was used to rinse the cells
the supernatant, the cells were resuspended in 100 µL of twice. Then, the samples were fixed for 30 min in a 4%
binding buffer. The 10× binding buffer was a concentrate paraformaldehyde solution. The fixative was disposed, and
composed of 0.2 µm sterile-filtered 0.1 M HEPES (pH 7.4), after going through two rounds of cleaning with PBS, the
1.4 M NaCl, and 25 mM CaCl solution. The 10× buffer samples were stained with Alizarin Red.
2
was diluted to 1× with ultrapure water. Finally, 10 μL
of propidium iodide (PI) staining solution and 5 μL of 2.8. Western blot analysis of osteogenic markers
annexin V-FITC (Yeasen, China) were used to stain the By using Western blot analysis, we were able to evaluate
samples, and the samples were left at room temperature for the protein expression levels of COL-1, BMP2, RUNX2,
10–15 min away from light. Then, 400 μL of 1× binding and SP7. After separating identical amounts of protein
buffer was added, each solution was mixed well and stored (20 μg) using sodium dodecyl sulfate-polyacrylamide gel
on ice, and a flow cytometer (Beckman Coulter, USA) was electrophoresis (SDS-PAGE) at a concentration of 10%, the
used for measurement. separated proteins were then transferred to polyvinylidene
fluoride (PVDF) membranes. Two hours of blocking
2.5. Cell morphology in TBST with 50 g/L skim milk powder was followed by
Seeding densities of 1 × 10 cells/disk were used for the an overnight incubation at 4°C with primary antibodies.
4
BMSCs for up to 24 h of culture on WE43 and heat-treated The primary antibodies included rabbit anti-rat COL1,
WE43. After incubation, the samples were washed in anti-BMP2, anti-RUNX2, anti-SP7 monoclonal antibody
phosphate buffer, fixed in 2.5% glutaraldehyde, dehydrated (Abcam, UK), and anti-β-actin polyclonal antibody
in a series of ethanol concentrations (50, 70, 80, 90, 95, (Applygen, China). The membranes were subsequently
99, and 100%), subjected to critical point drying, and stained with a secondary antibody against rabbit IgG
then coated with gold by sputtering. Next, field emission (Invitrogen, USA) after three rounds of washing. The data
scanning electron microscopy (FESEM) was used to were obtained using an ECL detection kit, and to measure
examine the samples. the relative intensity of the protein bands, ImageJ software
was used.
2.6. ALP activity
Three groups were used in the experiment: the control 2.9. Statistical analysis
group, the APS group, and the OHS group. In 24-well The SPSS 26.0 statistical package was used for the data
plates, BMSCs were cultured at a density of 2 × 10 mL . analysis. The differences between pairs of groups were
-1
4
Dexamethasone, L-ascorbic acid, and β-glycerophosphate analyzed using t-tests for independent samples, while the
(Sigma-Aldrich, USA) were added to the total medium, differences involving three or more groups were analyzed
APS and OHS extract to reach concentrations of 8–10 M using one-way analysis of variance (ANOVA) followed by
dexamethasone, 5 mg/mL L-ascorbic acid, and 5 mM Tukey’s test.
β-glycerophosphate soon after incubation for a day, and
the medium was exchanged. After that, the frequency 3. Results
of changing the medium was the same as that of the
cytotoxicity test. On the 14th day, cells were lysed with 3.1. XRD
radioimmunoprecipitation assay buffer (Lablead, China), As shown in Figure 2, the main component of the as-
and the lysate was added to a 96-well plate, followed by printed WE43 is α magnesium; there are also some β phase,
p-nitrophenyl phosphate (pNpp). ALP can catalyze the and the composition of Y O is lower. In the composition
3
2
reaction of pNpp to generate p-nitrophenol, with the of oxidation heat-treated WE43, in addition to the main
strongest absorbance at a wavelength of 405 nm. To assess component of α magnesium, there are more metal oxides,
the ALP activity, a microplate reader was used to obtain including Y O and Nd O , and the peak value of Y O
2
3
2
3
3
2
the absorbance readings, and a standard curve was created is higher than that of the as-printed group. The β phase
using a standard. To determine the amount of total cellular of oxidation heat-treated WE43 was also significantly
protein, a BCA kit (Yeasen, China) was used. reduced.
2.7. Alizarin Red test 3.2. ICP-AES of the extracts
Three groups were used in the experiment: the control The Mg content in the APS extract was 17.13 ± 0.10 mM, and
group, the APS group, and the OHS group. After being the Mg content in the OHS extract was 3.53 ± 0.02 mM. Gd,
seeded in 25 cm cell culture flasks, BMSCs were cultured Nd, and Y in the diluents of the two diluents with a volume
2
for 24 h to allow them to adhere. Then, the culture of 20 mL did not reach the detection limit of ICP-AES.
Volume 9 Issue 3 (2023) 97 https://doi.org/10.18063/ijb.686