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International Journal of Bioprinting The biological properties of WE43 scaffolds via the oxidative heat strategy

BMSCs cultured in the OHS group was between that expression of RUNX2 promotes cell proliferation. CCK-
of the blank control group and of the APS group, which 8 data showed that BMSC proliferation was greater in
suggested that oxidation heat treatment could improve the the OHS group than in the control group, which was
biocompatibility. related to the promotion of RUNX2 expression. From the
microscopy images of Alizarin Red staining, in addition
ALP is an osteogenic molecule expressed by BMSCs to having more mineralized nodules than the control
and osteoblasts and is highly expressed in mineralized group, the APS and OHS groups also had a greater cell
bone tissue . It has been reported that the expression density than the control group, which further shows that
[28]
level of ALP in BMSCs cultured in vitro can be used to WE43 magnesium alloy could promote the proliferation
predict their osteogenic ability in vivo . By measuring of BMSCs to indirectly promote osteogenesis. In
[29]
ALP activity, the ALP expression of both the APS and addition, the SP7 protein, or osterix, is a protein that acts
OHS groups was greater than that of the control group. as a transcription factor to stimulate the differentiation
In terms of the late phase of osteogenesis, this study of BMSCs into osteoblasts and ultimately osteocytes [36] .
used Alizarin Red staining, which can stain mineralized This transcription factor is often utilized as a marker of
nodules red. Since mineralization is the result of late osteoblast development since it acts in the transcriptional
osteogenesis, Alizarin Red staining mainly reflects the level cascade after RUNX2. Mouse embryos with the SP7 gene
of late osteogenesis . In Alizarin Red mineralized nodule knocked out have no bone formation [37] . BMP2 serves a
[30]
staining, the OHS group also showed the best osteogenic crucial function in osteogenic differentiation induction.
properties. In the ALP test, the ability of the APS group Previous literature has shown that RUNX2 is stabilized by
to induce osteogenic differentiation was similar to that BMP2-activated ERK/MAP kinase to promote osteoblast
of the blank group, while in the Alizarin Red staining differentiation [38] . Increased SP7 and BMP2 expression
experiment, the APS group had a much greater density compared to the control group was demonstrated by
of mineralized nodules than the blank group. Combined Western blotting in the APS and OHS groups, suggesting
with the results of the BCA kit, this may be due to the that WE43 magnesium alloy could promote osteogenesis
magnesium ions promoting cell proliferation and protein through SP7 and BMP2. This study mainly focused
expression. on the effect of magnesium ion on the differentiation

The RUNX2 protein is a transcription factor closely of BMSCs in vitro. In fact, magnesium ion promotes
related to osteoblast differentiation. Expression of this osteogenesis through additional mechanisms in vivo. For
gene is induced more strongly in preosteoblasts and example, magnesium ions can promote the expression
immature osteoblasts than in mature osteoblasts . It of calcitonin gene-related polypeptide-α in periosteal
[31]
has been shown in the literature that magnesium salts neurons, thereby promoting osteoblast differentiation [39] .
can promote the expression of RUNX2 in murine BMSCs Therefore, it is also expected that the scaffold has better
and promote their differentiation into osteoblasts. Using osteogenesis in vivo.
0 mM and 20 mM magnesium ion medium, it was found
that the cells cultured with 20 mM magnesium ion were 5. Conclusion
more sensitive to RUNX2 than the cells cultured in 0 mM
magnesium . In this experiment, magnesium alloy bone In summary, we demonstrate an oxidation heat-treated
[32]
implants were used, and it was intuitively observed that WE43 magnesium alloy made by additive manufacturing,
magnesium-rare earth alloys stimulated the expression of which possessed good biocompatibility, biodegradability,
molecules involved in osteogenesis in vitro. Among them, and superior osteogenic properties for clinical bone defect
the concentration of magnesium ion in the APS group was repair. The cytotoxicity of both APSs and OHSs was low
higher, approximately 17.13 mM, than that in the OHS and related to the induction of apoptosis. BMSCs are
group, which was approximately 3.53 mM, similar to the more likely to adhere and grow on OHSs. The osteogenic
results of culturing cells with magnesium salts. activities of the APSs and OHSs were better than those of
the control group. During the culture process, the ALP
As a transcription factor, RUNX2 is also closely activity of the APS and OHS groups was higher than that
related to the regulation of the cell cycle. In mitosis, of the control. Compared to the control group, the scaffold
there is a correlation between RUNX2 and cyclin B1 [33] . groups expressed more of the upstream proteins RUNX2,
RUNX2 expression in the MC3T3-E1 osteoblast cell SP7, and BMP2 and the downstream protein COL1.
line was very high during the G1 phase of mitosis and Thus, the scaffolds have the potential for future clinical
relatively low during the S, G2, and M phases [34] . In applications in orthopedic implants, but further animal
addition, knockdown of RUNX2 also produces G1- experiments and clinical studies are still needed to verify
related antiproliferative effects [35] . In conclusion, the their efficacy in the future.

Volume 9 Issue 3 (2023) 101 https://doi.org/10.18063/ijb.686