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International Journal of Bioprinting 3D-printed Mg scaffolds promote bone defect repair

Figure 4. Osteoclast formation and bone resorption of BMMs cultured in the extract of magnesium (Mg) alloy scaffolds and stimulated with RANKL
(100 ng/mL). (A) Representative images of TRAP-positive osteoclasts. (B) Quantification of the number (left) and the average area (right) of TRAP-
positive multinucleated osteoclasts. (C) Representative images of F-actin ring formation in osteoclasts. (D) Quantification of the average number of F-actin
ring; (E) Representative SEM images of bone resorption. (F) Quantification of resorbed bone slice area. *P < 0.05 vs. Control group; P < 0.05 vs. Mg group.
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slightly lower than that in the control group; however, ZA groups was significantly higher than that in the control
the difference was not statistically significant. On the 1st group (Figure 3D), indicating that the environment of
3rd, 5th, and 7th days, the OD value of the Mg group rapid degradation of the Mg alloy in the Mg group was
was significantly lower than that of the control group, not conducive to osteogenic differentiation of rBMSCs. In
indicating that the alkaline environment and excessive contrast, the degradation products produced in the Mg/
concentration of Mg ions after the rapid degradation of the Sc and Mg/Sc/ZA groups were beneficial for osteogenic
Mg alloy slowed down the proliferation rate of rBMSCs. differentiation of rBMSCs because of the slow degradation
Under the protection of the ceramic coating converted by of the Mg alloy. In addition, there was no significant
polysilazane, the degradation rate of the Mg alloy substrate difference between the Mg/Sc and Mg/Sc/ZA groups.
of the Mg/Sc and Mg/Sc/ZA groups decreased, and the Therefore, the addition of ZA had no significant effect
physical and chemical properties of the culture medium on early osteogenic differentiation of rBMSCs. The semi-
minimally changed; therefore, the cell proliferation of the quantitative results for the mineralized nodules were
Mg/Sc and Mg/Sc/ZA groups was not significantly affected. similar to those for ALP activity (Figure 3E). As shown
in Figure 3F, the expression of ALPL, SPP1, RUNX2, SP7,
3.4. Osteogenic differentiation COL1A1, and BGLAP related to bone differentiation in
The effect of the 3D-printed Mg alloy scaffold extracts on the Mg/Sc and Mg/Sc/ZA groups was significantly higher
the osteogenic differentiation ability of rBMSCs is shown than that in the control and Mg groups, further confirming
in Figure 3. Figure 3B and C shows that the degree of ALP that the Mg/Sc/ZA extracts can promote osteogenic
and Alizarin Red staining in the Mg group was significantly differentiation in vitro.
weaker than that in the control group, whereas those in the
Mg/Sc and Mg/Sc/ZA groups were significantly stronger 3.5. Osteoclastogenesis, bone resorption, and
than those in the control group. The relative activity of intracellular reactive oxygen species
ALP in the Mg group was significantly lower than that in TRAP staining results are shown in Figure 4A.
the control group, whereas that in the Mg/Sc and Mg/Sc/ Quantitative statistics showed that the area and number

Volume 9 Issue 5 (2023) 409 https://doi.org/10.18063/ijb.769