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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

overexpressing osteocytes in this study), with the conditioned medium exhibited enhanced osteogenic
R-approved microgravity-simulation system, RCCS . The differentiation-promoting activity. 29
TM
3D-BWBM was set up in a CO incubator. The prepared osteocyte cell lines were used as
2
In order to rapidly evaluate the effect of osteocytic functional cells and co-cultured with effector cells,
Wnt7b on osteogenic differentiation under microgravity in specifically bone marrow mesenchymal cells ST2, for
a 3D environment closely resembling in vivo conditions, 26,27 3 days (Figure 1C), followed by an assessment of their
we printed the PCI3D module and cut it into small osteogenic differentiation activity. The co-culture cell
modules about 5 × 5 mm with optional compartments, ratio of MLO-Y4 to ST2 at 1:4 was selected (Figure
2
allowing for installation within the high-aspect rotating S1, Supporting Information), consistent with previous
vessel (HARV ) of the RCCS . The HARV was filled reports indicating that osteogenic factors from osteocytes
TM
TM
TM
with growth medium, carefully degassed to remove promote ST2 osteogenic differentiation through activation
air bubbles, sealed with a plug at the filling port, and of the canonical Wnt signaling pathway. 25,30,31 Osteogenic
subsequently mounted onto the rotating base. To ensure differentiation, assessed by AP staining, displayed
clear experimental effects, prevent cell dislodgement, and significantly increased staining intensity in the co-culture
maintain the module suspended at the center of the vessel, of Wnt3a-treated MLO-Y4 and Y4-Wnt7b groups, but
a rotational speed of 20–30 rpm/min was optimized for no significant increase was observed in the Y4-Wnt5a
this experiment to simulate microgravity and low shear co-culture group (Figure 1D). The Y4-GFP co-culture
stress conditions. Finally, RCCS was placed in a CO group exhibited no significant difference compared to the
TM
28
2
incubator to culture the modules for 7 days, during which MLO-Y4 co-culture group treated with the Wnt3a control
the medium was changed once. medium (L-ctrl). Consistent with the staining results, Y4-
Wnt7b and Y4-Wnt3a significantly promoted osteogenic
2.16. Statistical analysis differentiation, with robust increases in AP biochemical
GraphPad Prism 8.0.1 software was used for statistical activity compared to their respective controls, Y4-GFP and
analysis. Each experiment was repeated three times. Data Y4-L-ctrl (Figure 1E).
were expressed as mean ± standard deviation. Differences
in data between two groups were analyzed by t-test; However, given that the osteogenic activity of Wnt3a
differences between multiple groups were analyzed by can be inhibited by the Wnt/β-catenin antagonist ICG-
32
one-way analysis of variance (ANOVA); and differences 001 and that sclerostin produced by microgravity also
between groups with two or more independent variables inhibits the canonical Wnt signaling pathway, we focused
were analyzed by two-way ANOVA. A p-value less than on Wnt7b as a potential strategy to bypass sclerostin
0.05 indicates statistical significance. inhibition under microgravity conditions. Furthermore,
RT-qPCR analysis revealed that the Y4-Wnt7b co-
3. Results culture group significantly upregulated the expression of
osteoblast marker genes (Alpl, Col1a1, Ibsp, and Bglap)
3.1. Osteocytic Wnt7b induces osteogenic in ST2 cells (Figure 1F). To verify whether the osteocyte-
differentiation of ST2 cells mediated osteogenic differentiation of ST2 cells in the
We observed that mice with osteocyte-specific Wnt/β- co-culture system was exclusively derived from ST2 cells,
catenin activation (daβcatOt) exhibited resistance to the we then examined the AP expression of Y4-GFP and Y4-
induced bone loss in a tail suspension model simulating Wnt7b cells, respectively. The AP staining results showed
weightlessness. To investigate this phenomenon, we that the intensity of AP staining of Y4-Wnt7b itself and
analyzed the expression of Wnt ligands in the long bones Y4-GFP cells was significantly weaker than that in the
of daβcatOt mice and their cohort control mice (WT). ST2 cells cocultured with L-ctrl medium-treated MLO-Y4
We found that the expression levels of Wnt3a, Wnt5a, and and Y4-GFP (Figure S2A, Supporting Information). Also,
Wnt7b were positively correlated with bone loss and bone the mRNA levels of osteoblast marker genes Alpl, Ibsp,
protection in the microgravity environment (Figure 1A). and Bglap were comparable to those of Y4-GFP cells in
To determine whether these three Wnt ligands possess Y4-Wnt7b cells (Figure S2B, Supporting Information),
a protective function against microgravity-induced suggesting that Wnt7b hardly altered the expression of
bone loss in osteocytes, we established stable osteocyte osteoblast marker genes in MLO-Y4 cells. Finally, we
cell line MLO-Y4 expressing Wnt5a, Wnt7b, and GFP assessed the effect of osteocyte Wnt7b on mineralization.
(control), namely Y4-Wnt5a, Y4-Wnt7b, and Y4-GFP, ARS staining demonstrated that the mineralized nodules
respectively. RT-qPCR analysis revealed that Y4-Wnt5a formed in the Y4-Wnt7b co-culture group were larger,
and Y4-Wnt7b overexpressed their respective Wnt5a and denser, and had 1.4 times more calcium deposition than
Wnt7b mRNAs (Figure 1B). MLO-Y4 treated with Wnt3a- those in the Y4-GFP co-culture group (Figure 1G).

Volume 11 Issue 4 (2025) 432 doi: 10.36922/IJB025240238