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International Journal of Bioprinting G40T60@WNT5A promotes osteoblast differentiation

2.20. Transwell, scratch assay, and tube 2.22. Double immunofluorescence
formation assay The cells were fixed with 4% paraformaldehyde at room
First, the migration of UVECs was assessed through temperature for 15 min, and then washed twice with PBS.
Transwell experiments. UVECs, co-cultured and The cells were permeabilized with 0.5% Triton X-100
trypsinized, were resuspended and then seeded on the (P0096, Beyotime, China) for 10 min. Then, the cells were
apical chamber of 8.0 μm Transwell inserts (BDFalcon™, incubated overnight at 4°C with primary antibodies: sheep
USA) at a density of 2 × 10 /insert. The induced membrane anti-OPN (ab11503, 1:200, Abcam, UK) and rabbit anti-
5
was placed in the sub-chamber. After 24 h, the cells were RUNX2 (#12556, 1:6400, CST, USA). After washing the
fixed with 4% paraformaldehyde for 30 min and then slides 3 times with PBS, the slides were incubated with Alexa
stained with 0.1% crystal violet. Imaging of migrating cells Fluor 488-conjugated secondary antibody (ab150129/
was performed using a microscope (IX73, OLYMPUS). ab150077, 1:200, Abcam, UK) for 1 h. Then, the slides were

Scratch assay was used to detect the migration of washed 3 times with PBS and stained with DAPI (10 μg/
UVECs. UVECs were seeded at a density of 2 × 10 cells/ ml; D3571, Thermo Fisher, USA) at room temperature for
5
well in a 12-well plate and allowed to grow until 100% 10 min. The slides were stored at 4°C and then observed
59
confluence. Scratch, measuring approximately 600 μm using a fluorescent microscope (IMT-2, Olympus).
in width, was made on monolayers after 3 h of nutrient 2.23. RT-qPCR
deprivation. Cells were incubated at 37°C and 5% CO with Total cellular RNA was extracted using Trizol (Catalog #:
2
different induced membranes (i.e., induced membranes 16096020, Invitrogen, USA). The purity and concentration
were soaked in serum-free culture medium for 72 h) for 12 of the obtained RNA were assessed by measuring the
h. Then, the cells were imaged using a microscope (IX73, absorbance of the solution at 260 and 280 nm by means
OLYMPUS). The relative closure degree of quantified of spectrophotometry. The A /A ratio of the sample
280
260
scratches was measured using ImageJ software. should be ≥1.8. To obtain cDNA, reverse transcription was
The tube-forming ability of UVECs was also assessed. performed using the Reverse Transcription Kit (Catalog
After being thawed overnight on ice, the UVECs in a number: 11483188001, Roche, Switzerland). Reaction
48-well plate were added with 250 μl of Matrigel (BD conditions were set as follows: 42°C for 15 min (reverse
Biosciences, USA) and incubated at 37°C for 30 min transcription reaction); 85°C for 5 s (reverse transcriptase
to allow gel formation. The UVECs were inoculated at inactivation reaction). The reverse-transcribed cDNA was
a concentration of 2.5 × 10 /cell in Matrigel and treated diluted to 50 ng/μl for subsequent fluorescence quantitative
4
with different elution media. Subsequently, the cells were polymerase chain reaction (PCR). PCR was performed
incubated at 37°C in 5% CO for 6 h. Then, the cells were using the LightCycler 480 SYBR Green I Master under the
2
imaged using a microscope (IX73, OLYMPUS), while following conditions: initial denaturation at 95°C for 10 min,
55
quantitative analysis was performed using ImageJ software. followed by denaturation at 95°C for 15 s, annealing at 60°C
for 20 s, and extension at 72°C for 20 s, for a total of 40 cycles.
2.21. Hematoxylin and eosin staining With Gapdh as the internal reference, 2-ΔΔCt method was
The extracted induced membrane was fixed with 4% employed. 2-ΔΔCt represents the fold change in expression
formaldehyde, and after 24 h of fixation, paraffin embedding of the target gene in the experimental group compared to the
and slicing were performed. The routine hematoxylin and control group, and the formula is given in Equation II:
60
eosin (H&E) staining was carried out. Briefly, after being
subjected to gradient alcohol dehydration, the slides were ΔΔCt = ΔCt experimental group − ΔCt control group (II)
stained in hematoxylin for 3 min, and then rinsed with tap
water. Next, 0.5% hydrochloric acid alcohol differentiation where ΔCt = Ct (target gene) − Ct (reference gene). The
solution was applied for 10 s, and then the slides were experiment was repeated 3 times. The primer sequences
rinsed with water. Counterstaining with bluing solution can be found in Table S1 (Supplementary File).
was performed for 10 min, and then the slides were stained
with eosin solution for 5 min. Subsequently, routine 2.24. Western blot
dehydration, application of coverslips, and ultimately Total protein was extracted from tissues or cells using
sealing with neutral gum were performed. The slides were efficient RIPA lysis buffer (C0481, Sigma-Aldrich, USA)
observed under an optical microscope (XP-330, Shanghai containing 1% proteinase inhibitor and 1% phosphatase
Bingyu Optical Instrument Co., Ltd., Shanghai, China). In inhibitor (ST019-5mg, Beyotime, Shanghai, China), in strict
particular, the cells, fibrous tissue, and microvessels in the adherence to the manufacturer’s instructions. After 15 min of
induced membrane were observed. 57,58 cracking at 4°C, the suspension was centrifuged at 15,000 r/

Volume 10 Issue 2 (2024) 234 doi: 10.36922/ijb.1461