Page 139 - IJB-9-5

International Journal of Bioprinting 3D printed PEEK scaffold mediates macrophages to affect osseointegration

filter, and CM of scaffolds with different pore sizes was were incubated in 40 mM Alizarin red S for 10 min after
finally obtained. being fixed with 4% paraformaldehyde for 30 min. The cell
mineralization was observed by using stereomicroscope
2.4.2. BMSCs growth and adhesion (SteREO, ZEISS, Germany). The optical density (OD) value
BMSCs were seeded at a density of 3 × 10 cells/well and was measured at 540 nm after the stain was solubilized
4
cultured in the CM for 48 h and then cultured for a further with 10% cetylpyridinium chloride.
3 and 7 days in osteogenic media (DMEM supplemented
with 10 M dexamethasone, 10 M β-Glycerophosphate, 2.5. Paracrine effect of macrophage on HUVECs
-7
-2
and 50 μg/mL ascorbic acid [Sigma, USA]) . Then, the 2.5.1. Scratch healing experiment
[28]
original medium was removed, and CCK-8 working HUVECs were seeded in 6-well plates at a density of 1 ×
solution (a mixture of 90% complete medium and 10% 10 cells/well and incubated overnight. When cells reached
5
CCK-8) was added and incubated at 37°C, 5% CO 90% confluency, a sterile 200-μL pipette tip was used to
2
for 30 min. One hundred microliter of the incubated make a cell-free scratch in the middle of the monolayer
culture solution was transferred to a 96-well plate, and a of HUVECs and rinsed with PBS to remove unadhered
microplate reader (BIO-RAD 680, USA) was used to test cells as well as cell fragments . Furthermore, after adding
[29]
the absorbance value (OD ) of the culture solution at different CMs, HUVECs were further cultured at 37°C and
450
450 nm. 5% CO for 12 and 24 h. After 0, 12, and 24 h of incubation,
2
The adhesion and distribution of BMSCs on the the scratches were photographed with a microscope, and
surface of PEEK scaffolds were observed by fluorescence then the scratch area was quantitatively analyzed using
microscope. After BMSCs seeding for 7 days, the cells were ImageJ software, and the scratch healing rate was calculated
fixed in 4% paraformaldehyde for 30 min. All samples using Equation I:
were rinsed several times with PBS and permeabilized At
with 0.5% Triton X-100. After that, cells were stained with Wound area % A0 100 % (I)
70 nM FITC-labeled rhodamine phalloidin (Solarbio,
China), washed with PBS after 30 min of incubation. The where At is the area of scratches at different time points,
fluorescence images of the constructs were observed with a and A0 is the area of scratches at 0 h.
confocal microscope (LSM880, Zeiss, Germany). 2.5.2. Expression of angiogenesis-related genes

2.4.3. Expression of osteogenesis-related genes HUVECs cells were seeded in 24-well plates at a density
5
BMSCs were seeded at a density of 3 × 10 cells/well and of 1 × 10 cells/well and cultured overnight, and then, the
4
cultured in the basal culture media or CM for 48 h and then cell culture medium was changed to a 1:1 mixed medium
cultured for a further 7 days in osteogenic media. After 7 of CM and complete medium. After 3 days of co-culture,
days of culture, the cells were collected and washed with the cells were collected and washed with PBS for 3 times.
PBS for 3 times. RT-PCR was used to detect the expression RT-PCR was used to detect the expression of angiogenesis-
of osteogenesis-related genes, and the detection steps were related genes, and the detection steps were the same as
the same as those in Section 2.3.4. The sequences of forward those in section 2.3.3. The sequences of forward and
and reverse primers of target genes (ALP, OCN, OPN, and reverse primers of target genes (Angiogenin, FGF, and
RUNX2) are listed in Table S2 (Supplementary File). SDF) are listed in Table S3 (Supplementary File).

2.4.4. ALP activity analysis 2.6. Animal experiment
BMSCs were seeded at a density of 3 × 10 cells/well and 2.6.1. Surgical procedure
4
cultured in the basal culture media or CM for 48 h and Ethical approval was obtained from the Experimental
then cultured for a further 7 and 14 days in osteogenic Animal Ethics Committee of Jinan University. The
media. According to the instructions of the kit, ALP assay tibial defect model animals used were New Zealand
kit (Solarbio, China) and BCA Protein assay kit (Solarbio, rabbits, which were purchased from Huadong Xinhua
China) were used to determine the ALP activity of BMSCs, Experimental Animal Farm. Then, the rabbits were
and three independent experiments were conducted. anesthetized with 3% sodium pentobarbital (1 mL/kg) and
xylazine hydrochloride (0.1 mL/kg), and the hind limbs
2.4.5. Alizarin red S staining of rabbits were cleaned and disinfected with iodophor
BMSCs were seeded at a density of 3 × 10 cells/well and and alcohol. Afterward, a defect of 8 mm in diameter
4
cultured in the basal culture media or CM for 48 h and and 3 mm in depth was made on the tibial plateau with a
then cultured for a further 21 days in osteogenic media. trephine with a diameter of 8 mm, and then the sterilized
After 21 days of culture, the supernatants were removed, 3D-printed PEEK scaffold was implanted into the defect.
and cells were rinsed with PBS for 3 times. Then, the cells Then, the wound was sutured layer by layer, and the rabbits

Volume 9 Issue 5 (2023) 131 https://doi.org/10.18063/ijb.755