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Jiao, et al.
Dawley (SD) rats. The β-TCP was mixed with Pluronic 2.4. Cytotoxicity assay
F-127 in an aqueous solution to obtain a β-TCP ink, and After being incubated in the above-mentioned extract
the mixture was then stirred until it was homogeneous. with three replicates per group, BMSCs were seeded in
The ink was printed using a 22G needle, and the scaffolds 96-well plates at a density of 2.5×10 cells per well for
3
were sintered at 1100°C for 3 h. The sintered β-TCP cytotoxicity assay. Cell Counting Kit-8 (CCK-8) assay
scaffolds were observed by scanning electron microscopy was performed on days 3 and 7 to assess the cytotoxicity
(SEM, USA). The scaffolds were then used for animal of the β-TCP extract. The BMSCs were incubated in
experiments. complete medium with 10% CCK-8 reagent (Dojindo,

2.2. Cell culture CK04-05, Japan) for 2 h at 37°C. The absorbance of the
supernatant at 450 nm was measured using a microplate
Primary bone marrow mesenchymal stem cells were reader (Infinite M200 Pro, Tecan, Switzerland).
isolated after flushing the bone marrow of tibiae and
femurs from 1-week-old SD rats. The BMSCs were 2.5. ALP staining
cultured in α-minimum essential medium (α-MEM) ALP staining was conducted on the BMSCs after
(HyClone, SH30265.01, USA) supplemented with 10% incubation in the above extract for 7 days. BMSCs were
fetal bovine serum (FBS; Gibco, 10099-141, USA), 1% washed 3 times with phosphate-buffered saline (PBS)
penicillin/streptomycin (Gibco, 15140122, USA), and and fixed with 4% paraformaldehyde (Beyotime, P0099,
0.4% gentamicin (Sangon Biotech, Shanghai, China), China) for 15 min. After that, the BMSCs were washed
after which the cells were cultured at 37°C with 5% CO 3 times with PBS. An ALP kit (Hongqiao Lexiang, LRB-
2
and 95% humidity. The cell medium was changed every ALP, China) was used to perform ALP staining according
3–4 days and the BMSCs were passaged when they were to the manufacturer’s instructions. The results were
at nearly 80% confluence. analyzed by ImageJ.

2.3. Preparation of β-TCP extract and 2.6. RNA extraction and quantitative real-time
characterization measurement of β-TCP extract polymer chain reaction (PCR)

β-TCP powder was purchased from Kunshan Chinese After a culture period of 7 days, the total RNA from
Technology New Materials Co., Ltd (China). Two the BMSCs seeded on 6-well plates, at a density of
grams of the powder were incubated in 10 ml α-MEM 2*10^5 cells per well, was extracted using TRIzol
at 37°C for 24 h to prepare β-TCP extract (200 mg/ reagent (Invitrogen, 15596018, USA) following the
ml) [23-25] . Then, the mixture was centrifuged at 3000 manufacturer’s instructions. The concentration of
×g, and the supernatant was collected. The extract total RNA was measured using a NanoDrop ND-
was sterilized through 0.22 μm filter membranes 1000 Spectrophotometer (Thermo Fisher Scientific,
(Millipore, SLGPR33RB, USA) and stored at 4°C USA), and 1000 ng of the extracted RNA was reverse
until further use. Size distribution and zeta potential transcribed to cDNA using PrimeScript Master Mix
were detected by Nano Sizer and Zeta potential Tester (TaKaRa, RR036A, Japan). For the qRT-PCR reaction,
(Omni, USA). We diluted the β-TCP extract with 2× SYBR Green qPCR Master Mix (Low ROX)
α-MEM to concentrations of 1/32, 1/64, and 1/128. (Bimake, B21703, China) and Applied Biosystems
The concentrations of calcium (Ca) and phosphorus 7500 Real-Time PCR System (Applied Biosystems,
(P) ions in the three extracts with the concentrations of Foster City, CA, USA) were used. Glyceraldehyde-
1/32, 1/64, and 1/128 and α-MEM (control, ctrl) were 3-phosphate dehydrogenase (GAPDH) was used as
detected using an inductively coupled plasma atomic the quantitative control for normalization. The 2 −ΔΔCt
emission spectrometer (ICP-AES; avio500, USA). The method was used to calculate the relative mRNA
β-TCP extract at the three concentrations with α-MEM levels. The primers used in this study are listed in
was used for cell experiments and supplemented Table 1.
with 10% FBS, 1% penicillin/streptomycin, 0.4%
gentamicin, and osteogenic induction component (10 2.7. Prediction of Runx2 m6A methylation sites
mM β-glycerophosphate, 50 μM ascorbic acid, and The prediction of possible sites modified by m6A
10 nM dexamethasone). Cells in the control group was performed using SRAMP, a sequence-based m6A
were treated with α-MEM supplemented with 10% modification site predictor (www.cuilab.cn/sramp).
FBS, 1% penicillin/streptomycin, 0.4% gentamicin, The entire sequence of RUNX2 mRNA was imported
and osteogenic induction component (10 mM onto the server. Then, the possible Runx2 m6A
β-glycerophosphate, 50 μM ascorbic acid, and 10 nM methylation sites were exported from the online tool
dexamethasone). The treatment lasted for 7 days. automatically.

International Journal of Bioprinting (2022)–Volume 8, Issue 2 33