Page 123 - IJB-9-5

International Journal of Bioprinting DLP-printed scaffold for bone regeneration

2.4.7. Capacity of iron ion adsorption 1 mL of cell suspension (5 × 10 cells) was added. BMSCs
4
The different hydrogels were immersed in 10 mL of FeCl were seeded on different scaffolds and cultured in medium
3
and placed on an incubator shaker for 6 h. Afterward, for 48 h. The cells were incubated with live/dead dye for
the adsorbent was removed, and the biosorption was 15 min, and then observed under a confocal microscope
calculated to analyze the difference in adsorption capacity (Olympus, FV3000, Japan), where green represented living
by inductive coupled plasma (ICP) emission spectrometer cells and red represented dead cells.
of the different scaffolds.
2.7. Effect of scaffold on the chondrogenic induction
2.5. Cell extraction and culture in vitro
The BMSCs and human umbilical vein endothelial cells The experiments in this section were divided into three
(HUVECs) used in this experiment were both from the groups: control, GelMA, and GelMA/3%PMAA groups.
Orthopedic Laboratory of the PLA General Hospital (the All experiments were repeated three times.
ethical number: 2022-x18-51). The original generation
of BMSCs was expanded to the third generation with 2.7.1. Immunofluorescence staining
medium containing α-MEM, fetal bovine serum (FBS, After co-culture of BMSCs with different scaffolds in
10%), and penicillin–streptomycin (1%) for experiments. osteogenic induction medium for 5 days, the cells were
The HUVECs were cultured with medium containing high fixed with 4% paraformaldehyde and permeabilized with
glucose-Dulbecco’s Modified Eagle Medium (H-DMEM), 0.1% Triton-X100. Cells were then incubated with primary
FBS (10%), and penicillin–streptomycin (1%). Both types antibody overnight and secondary antibody for 1 h. Nuclei
of cells were cultured in this medium—refreshed every were then stained with DAPI and washed with PBS three
2 days—in a 37°C and 5% CO environment. times before observation under a confocal microscope.
2
2.6. Cell viability 2.7.2. Quantitative real-time PCR
2.6.1. Extracts of different scaffolds The scaffolds were immersed in the medium for half an hour
5
The scaffolds were soaked in medium for 48 h at 37°C. For and then removed from the medium. A total of 10 cells were
different scaffolds, 100% extracts were prepared according seeded on the surface of the scaffolds and cultured for 24 h in
to the standard of 1.25 cm /mL and diluted to different normal medium, which was then replaced with chondrogenic
2
concentrations of 75%, 50%, and 25%. induction medium (α-MEM, 10% FBS, 2 mg/L insulin, 3
mg/L transferrin, 1 mM pyruvic acid, 10 µg/L TGF-β1, and
2.6.2. CCK-8 100 nM dexamethasone). After 5 days and 10 days of culture,
BMSCs were cultured in extracts with different total RNA was extracted from the cells for real-time PCR
concentrations of different scaffolds for 1–5 days. After the using Trizol reagent (Servicebio, G3013).
color deepened for 1 h with the addition of CCK-8 (10%),
the cell viability was analyzed using a microplate reader 2.7.3. Western blot
(Thermo Fisher, Waltham, MA, USA). Cells were inoculated onto scaffolds and cultured
with chondrogenic induction medium for 5 days
2.6.3. Live/dead staining of cells cultured with extracts before total cellular protein was extracted using Radio
BMSCs were cultured for 48 h in the extracts at the optimal Immunoprecipitation Assay (RIPA) lysate (Servicebio,
concentration obtained via the CCK-8 assay. The cells G2002), and protein concentration was then determined
were incubated with live/dead dye for 15 min, and then using the BCA protein concentration kit (Servicebio,
observed under a fluorescence microscope (Ni-U, Nikon, G2026). After passing through the process of sodium
Tokyo, Japan), where green represented living cells and red dodecyl-sulfate polyacrylamide gel electrophoresis
represented dead cells. (SDS-PAGE), membrane transfer, immunoblotting, and
chemiluminescence detection, the signal intensity was
2.6.4. Phalloidin staining of cells cultured with extracts measured using ImageJ software.
BMSCs cultured for 48 h in the extracts were fixed in 4%
paraformaldehyde solution for 30 min. After three washes 2.8. Effect of scaffold on the blood vessel
with PBS, the cells were stained with phalloidin for 30 min regeneration in vitro
and DAPI for 5 min. The morphology of the cytoskeleton The experiments in this section were divided into three
was observed with a confocal microscope (Olympus, groups: control, GelMA, and GelMA/3%PMAA groups.
FV3000, Japan). All experiments were repeated three times.
2.6.5. Live/dead staining of cells cultured on scaffolds 2.8.1. Migration experiment using transwell assay
Sterile scaffolds were soaked in medium for 15 min in The effect of different scaffolds on the migration of
24-well plates; following their removal from the medium, HUVECs through transmembrane was observed using

Volume 9 Issue 5 (2023) 115 https://doi.org/10.18063/ijb.754