Page 470 - IJB-10-1

International Journal of Bioprinting TPMS bone scaffold

and ethanol ultrasonic cleaning was performed for 30 2.4. Microscopic morphology observation and
s to remove the uncured resin. The support body was mechanical properties test of the scaffold
sintered at 1350°C for 3 h at a heating rate of 2°C/min A scanning electron microscope (SEM, JSM-IT500, JEOL,
to obtain a β-tricalcium phosphate biomimetic scaffold. Japan) was used to observe the β-surface morphology
After cleaning the support body with distilled water, the of the tricalcium phosphate biomimetic scaffold. The
scaffolds were placed in the autoclave, sterilized at 121°C β-tricalcium phosphate biomimetic scaffold was subjected
for 30 min, and then transferred to an oven to dry at 37°C to an axial compression fracture using a universal material
for 12 h. testing machine at a speed of 1 mm/min, and three
samples from each group were tested to obtain the average
2.2. I-PRF extraction and preparation mechanical strength.
After fixing the rabbit, the ear was disinfected with an
iodophor cotton ball prior to extracting blood from the 2.5. Evaluation of cell proliferation and cell adhesion
central artery of the rabbit ear with an arterial blood MC3T3 cells were inoculated at a density of 1 × 10 5
collection vessel. Approximately 40 mL of blood was cells/well on the scaffold in a 48-well plate and placed in
collected, transferred to a centrifuge tube, and incubated an incubator at 37°C with 5% CO . At each time point
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for 5 min in an ice bath. The tube was then quickly set in this experiment (1, 2, and 3 days), 50 μL CCK-8
transferred to the C-Tech centrifuge, selected the I-PRF solution was added to each well and incubated for 3 h,
mode, and centrifuged at 920 rpm for 7 min at room following which the CCK-8 solution in the 48-well plate
temperature. It was observed that the blood in the was transferred to the 96-well plate, and its absorbance
centrifuged tube was divided into two layers; the upper was measured at 450 nm using a microtiter plate reader.
layer contained the I-PRF, and the lower layer contained The experiment was repeated 3 times. After the cells were
the red blood cell; the I-PRF turned into light yellow gel cultured on the scaffolds for 24 h, the culture medium was
after 5–10 min at room temperature. The I-PRF gel was aspirated, samples were washed with phosphate-buffered
placed in an incubator at 37°C for 48 h and subjected to saline (PBS) 3 times, and 1 mL of Calcein-AM/PI mixed
subsequent experiments. All animal surgical procedures staining solution was added to each well and incubated in
were approved by the Institutional Animal Care and Use the dark at 37°C and 5% CO for 30 min. After washing
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Committee at Guangdong Medical University (Approval with PBS, the samples were visualized under the inverted
ID: GDY2204012). For the calculation of the drug release fluorescence microscope. The average total fluorescence
rate, 200 μg/mL methylene blue solution was prepared. intensity of the single hole of stents of each group was
Then, 500 μL Mix I-PRF was added to methylene blue calculated using ImageJ software.
solution, and the absorbance of the mix solution was read
at 664 nm at each time point (10 min, 30 min, 1 h, 2 h, 4 h, 2.6. Cell scratch test
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6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 3 days, 4 days, 5 days, 6 days, Two milliliters of 5 × 10 cells/mL cell suspension were
7 days, 8 days, 9 days, and 10 days). inoculated for each group in a 6-well plate for 24 h in a
culture chamber. Cells were washed twice with PBS, then
2.3. Preparation of TPMS bone scaffolds with culture medium was added to the blank control, and
different components scaffold medium extracts from different scaffold groups
To prepare the SDF-1-loaded TPMS bone scaffold (ST), were added to experimental groups. A scratch on the
500 μL SDF-1 solution with a concentration of 200 ng/mL cell monolayer was created using a pipette tip, and PBS
was transferred to the surface of the TPMS bone scaffold. was used to wash the created scratch. Two milliliters of
The solution was repeatedly aspirated and released on the the serum-free medium were added to each well. At this
scaffold to ensure full contact with the scaffold. To prepare point (0 h), the scratch was imaged under a microscope
the I-PRF-loaded TPMS bone scaffold (IT), 200 μL of 5% and then placed in the incubator for further cultivation.
I-PRF solution is placed on the surface of the TPMS bone After 24 h, the orifice plate was removed, and samples were
scaffold. To prepare TPMS scaffolds loaded with I-PRF and washed twice with PBS and imaged under the inverted
SDF-1 (SIT) 500 μL of 200 ng/mL SDF-1 solution and 200 phase contrast microscope. The Image J software was used
μL of I-PRF solution were mixed and placed on the surface to measure the distance between scratches to calculate the
of the TPMS bone scaffold. Repeated aspiration and release cell migration rate.
of the mix solution were used to ensure full contact with
the scaffold. The scaffold extraction solution was the liquid 2.7. Evaluation of osteogenic effect by performing
obtained by soaking the scaffold in a complete culture ALP and Alizarin red staining
medium for 48 h, which was used in the subsequent MC3T3 cells (passage 3) were inoculated on a 6-well plate
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experiments. at 2 × 10 cells/well. After 24 h, scaffold extracts from
different scaffold groups were added and cultured in an

Volume 10 Issue 1 (2024) 462 https://doi.org/10.36922/ijb.0153