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International Journal of Bioprinting G40T60@WNT5A promotes osteoblast differentiation

days of cultivation, fresh medium containing 10% CCK8 injection of equal volume; (ii) G40T60 group, in which a
(CA1210, Solarbio) was added to the 48-well plate. The scaffold was applied to each animal; (iii) WNT5A group,
plate was then incubated under light-shielding conditions in which an animal received only an injection of WNT5A
for 1 h. Subsequently, the absorbance of the supernatant in a concentration equal to that of G40T60@WNT5A; (iv)
culture medium in a 96-well plate (100 μl per well) was G40T60@WNT5A group, in which G40T60@WNT5A
measured at 450 nm using a microplate spectrophotometer scaffold was transplanted to the defect site of each animal.
(Bio-Tek, UK). 55 The final concentration of WNT5A is 500 μg/ml. After
2 weeks of treatment, the rats were euthanized. The skin
2.18. CTO&BD rat model construction over the original surgical incision on the left thigh was
Twenty-four Sprague-Dawley rats (male, 7 weeks old, cut open to expose the muscle and fascial tissues, and the
weighing 250–300 g) were purchased from Beijing Vital induced membrane formed on the surface of the scaffold
River Laboratory Animal Technology Co., Ltd. (strain was removed.
code: 101, Beijing, China). The rats were housed in an SPF-
level animal laboratory maintained at a humidity of 60% 2.19. Induction of osteoblast differentiation and
to 65% and a temperature of 22 to 25°C. They were given identification of osteoblasts
ad libitum food and water under a 12-h light/dark cycle. BMSCs were cultivated with induced membrane. An
After a week of adaptation feeding, the health status of osteogenic induction medium comprising of 10 mM
the rats was observed before the experiment commenced. β-glycerophosphate, 100 nM dexamethasone, and 50
All animal experiments have been approved by the mg/ml ascorbic acid-2-phosphate in the BMSCs culture
Animal Ethics Committee of the Third Hospital of Hebei medium was prepared. The culture medium should be
Medical University (No. KSD2022-033-1), and all animal replaced every 3 days during the induction of BMSCs
experiments in this study comply with the local principles osteogenic differentiation. Cells were obtained after
for the management and use of experimental animals. using GelMA lysis reagent kit (EFL-GM-LS-001, Suzhou,
China) and trypsin (R001100, Thermo Fisher Scientific) to
Under general anesthesia, the eccentric exercise of the degrade the hydrogel.
triceps surae in the rat’s leg was performed 3 times a week
for 1 h each time (30 stimuli per minute) to induce Achilles 2.19.1. Alkaline phosphatase staining
tendinitis and tendon disorders, which are equivalent to Alkaline phosphatase (ALP) staining was performed using
tendinopathy. After 3 weeks, a longitudinal incision was an ALP staining kit (40749ES60, Yeasen, China), following
made on the lateral side of the rat’s left thigh skin, followed the manufacturer’s instructions for detection. Fixed with
by dissection of the skin and subcutaneous tissue, blunt 4% paraformaldehyde for 7 days, the BMSCs were washed
separation of fascia and muscle, and exposure of the with PBS and stained for 30 min. Stained cells were imaged
femur. A 6-hole steel plate was placed on the outer side using a microscope (IX73, OLYMPUS, Japan). Meanwhile,
of the femur, and one screw was inserted into the distal we evaluated the ALP activity of the cell lysates of BMSCs
and proximal ends after adjusting the position. We found using ALP activity assay kit (MAK411, Sigma-Aldrich).
that the length of the bone section in the steel plate was 0.5 After being incubated with p-nitrophenyl phosphate
cm using a sterile stainless steel ruler. The steel plate was solution, the absorbance of the cell lysates was measured
secured with toothed forceps at the proximal and distal at 520 nm using a microplate spectrophotometer (Bio-
ends and partially cut through with an electric oscillating Tek, Thermo Fisher Scientific, USA) to determine the
saw (leaving approximately 1/5 of the bone closest to the ALP activity.
steel plate). Then, a diamond saw blade was used to cut 2.19.2. Alizarin red S staining
through the remaining bone in the proximal and distal The calcium deposited in the BMSCs induced for 21 days
regions of the bone section. The bone section was removed, was stained with Alizarin red S (ARS) solution. BMSCs
and a pre-prepared G40T60@WNT5A scaffold was placed were fixed with 4% paraformaldehyde and stained with
in the bone defect area.
ARS solution (PH 1354, PHYGENE, China) at room
The G40T60@WNT5A scaffold was secured with 2-0 temperature for 30 min, followed by PBS washing. The
sutures to prevent displacement. In the control group, stained cells were then imaged using an inverted microscope
WNT5A, in a concentration equal to that of G40T60@ (IX73, OLYMPUS). The calcium deposits stained with 10%
WNT5A, was applied to the corresponding defect area cetylpyridinium chloride (CPC, C0732, Sigma-Aldrich)
to build a CTO&BD model. The rats were randomly were dissolved in a microplate spectrophotometer at 562
56
divided into four groups, each consisting of six rats: nm to measure the absorbance, which helps gauge the
(i) PBS group, in which an animal received only a PBS degree of mineralization. 55

Volume 10 Issue 2 (2024) 233 doi: 10.36922/ijb.1461