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3D-Printed β-TCP scaffolds promote Osteogenic Differentiation of BMSCs in an m6A-Dependent Manner
A

B C D E

F G H

Figure 2. Osteogenesis effects of β-TCP on BMSCs. (A) ALP staining of BMSCs in Ctrl, 1/32, 1/64, and 1/128 groups. Top-right corner
shows macroscopic pictures. Scale bars = 50 μm. (B) Quantitative results of (A). The mRNA expression levels of COL I (C), OPN (D),
BSP (E), and RUNX2 (F) in Ctrl, 1/32, 1/64, and 1/128 dilution groups are illustrated in bar charts. The mRNA expression levels were
determined by qRT-PCR. (G) The protein expression level of RUNX2 in Ctrl, 1/32, 1/64, and 1/128 dilution groups by Western blot. (H)
The quantitative result of (G). *P < 0.05; **P < 0.01.

(COL I) and OPN, which represent late osteogenesis, cytotoxicity and osteogenic differentiation assay results,
in the β-TCP extract groups, were slightly lower than we chose 1/128 dilutions of β-TCP extract for further
that of the Ctrl group, except for the 1/128 dilution of experiments.
the β-TCP extract. Meanwhile, the levels of RUNX2 and
BSP, which represent early osteogenesis, increased. Since 3.3. Expression level of m6A-related enzymes
RUNX2 was the key translation factor of osteogenesis, changed after β-TCP treatment
we further tested the expression of RUNX2 protein by
Western blotting. The 1/64 and 1/128 dilutions of β-TCP To study the effects of β-TCP on m6A-related enzymes
extract showed higher expression of RUNX2 protein than such as “writers” and “erasers,” we detected the mRNA
the Ctrl (Figure 2G and H). These results demonstrated and protein levels of several m6A-related enzymes.
the excellent osteoinductivity of β-TCP. Considering the The results of the qRT-PCR analysis indicated that the

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