Page 240 - IJB-10-2

International Journal of Bioprinting G40T60@WNT5A promotes osteoblast differentiation

the sample was heated for 3 min using a sputter coating (ab150165, Abcam, UK). After incubation at 4°C for 30
device (SCD 005; BAL-TEC, Liechtenstein) at a current of min, unbound antibodies were washed away with PBS.
30 mA. 52 The cells successfully targeted by the labeled antibodies
were analyzed by means of flow cytometry. To study the
2.13. Mechanical experiment differentiation of BMSCs into osteoblasts, adipocytes, and
The G40T60 bracket was placed on a load cell with a chondrocytes, an array of approaches including reagent
capacity of 500 N on a universal testing machine (EZ kit (PD-003/4/5, Procell), Alizarin red staining (ARS),
Test; SHIMADZU, Japan). The compressive strength of oil red O staining, and Alcian blue staining were used to
the bracket was measured at a crosshead speed of 5 mm/ observe the osteogenic, adipogenic, and chondrogenic
min. The compressive strength of five samples for each differentiation abilities of BMSCs. The above methods
proportion was determined for the measurements of mean confirmed the osteogenic, adipogenic, and chondrogenic
and standard deviation. 52 differentiation capacity of the BMSCs used in this study
2.14. Degradation experiment (Figure S1A in Supplementary File), and they showed high
β-TCP was incubated in PBS at 37°C for 4 weeks; the PBS expression of CD44 and CD90 but no expression of CD45
was replaced every 3 days. After incubation, the samples and CD34 (Figure S1B in Supplementary File), making
were dried using a freeze-dryer (TFD8503; Yixin Laboratory them suitable for use in the subsequent experiments.
Co., Ltd., China) for 14 or 28 days. The degradation value The rat umbilical vein endothelial cells (UVECs)
(W [%]) was calculated using Equation I: (catalog number: CP-R232, Procell, China) was cultured
d
in endothelial cell medium (ECM; 1001, Wegene, China)
W W supplemented with 5% fetal bovine serum, 1% v/v
W (%) before after 100 (I) penicillin/streptomycin, and 1% endothelial cell growth
d
W
before
supplement (ECGS; KGY1052, Kaiji, China). The cells were
where W before and W are the weights of the sample before incubated at 37°C in a 5% CO environment. UVECs from
after
2
and after degradation, respectively (n = 3). 52 the second to the fifth passages were used for subsequent
experiments to investigate their cellular functions. 53-55
2.15. Embedding WNT5A in a 3D-printed scaffold
A 3D bioprinting system was utilized to print the scaffold, 2.17. Testing of 3D scaffold cytotoxicity
with the following settings: speed = 300 mm/min, syringe 2.17.1. Cell adhesion and cell morphology detection
size = 21 G, bracket thickness = 500 µm, and spacing = 700 To investigate the cell viability and adhesion of BMSCs and
µm. After bioprinting, the sample was cross-linked at room UVECs on the scaffold, we used the LIVE/DEAD Viability/
temperature in 25% glutaraldehyde vapor for 24 h, resulting Cytotoxicity Assay Kit (Invitrogen, USA) for detection.
in a 3D-printed scaffold loaded with WNT5A (WNT5A The sample was washed once with PBS after removing the
final concentration of 500 μg/ml). Unreacted aldehyde culture medium. A dye solution containing 4 ml of calcein-
groups were capped with a 0.1 M glycine solution. 52 AM and 2 ml of homodimer-10 was added to each sample.
After being left for reaction at 37°C for 30 min, the scaffold
2.16. Cell culture and identification was washed with PBS. The live and dead cells on the
Bone marrow mesenchymal stem cells (BMSCs) from scaffold were observed under a fluorescence microscope
healthy rats (catalog number: CP-M131, Procell, China) (Eclipse Ti, Nikon, Japan), and the cell morphology on the
were cultured in α-MEM medium (catalog number: scaffold was examined using FE-SEM.
SH30265.01, HyClone, Thermo Fisher Scientific, USA)
supplemented with 15% fetal bovine serum (FBS; catalog In addition, the scaffolds cultivated for 4 and 7 days
number: 10091148, Thermo Fisher Scientific, USA) and were washed twice with PBS, for 5 min each time, and then
100 U/ml penicillin–streptomycin solution (catalog chemically fixed with a 2.5% glutaraldehyde solution. Then,
number: 10378016, Thermo Fisher Scientific, USA). The the cells were dehydrated in a gradient series of ethanol
culture was incubated at 37°C in a 5% CO environment. solutions (50%, 75%, 85%, 95%, and 100%), taking 10 min
2
The BMSCs identification process started with the for incubation in each ethanol solution. After dehydration,
the bracket was air-dried and coated with gold through a
preparation of a single cell suspension (1 × 10 /ml) after sputtering deposition for 3 min at 30 mA. Finally, the gold-
6
washing the cells with PBS. Various fluorescently labeled plated bracket was observed using SEM. 52
antibodies were added to different groups, including
CD44-FTITC (MA5-17522, Thermo Fisher Scientific), 2.17.2. CCK-8 cell proliferation assay
CD90-PE (ab33694, Abcam, UK), CD45-FITC (ab33916, BMSCs were seeded at a density of 1 × 10 cells/well in a 48-
4
Abcam, UK), CD34-PE (ab223930, Abcam, UK), and IgG well plate coated with scaffolds. On the 4th, 7th, and 10th

Volume 10 Issue 2 (2024) 232 doi: 10.36922/ijb.1461