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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

Therefore, we will conduct further studies on the p-MARCKS levels (Figure 2D). Functionally, 1 μM
force-sensitive and functional bone cell Wnt7b, which Rottlerin completely inhibited osteoblast differentiation
actively counteracts microgravity-induced impairment of in ST2 cells co-cultured with Y4-Wnt7b cells, as observed
osteoblast differentiation. in AP staining and its biochemical activity assays (Figure
2E and F), significant downregulation of osteoblast marker
3.2. Osteocytic Wnt7b activates the PKCδ pathway genes (Alpl, Col1a1, Ibsp) via qPCR (Figure 2G), and a
of Wnt noncanonical signaling to promote marked 1.4-fold reduction in osteocytic Wnt7b-induced
osteogenic differentiation mineralization by ARS staining (Figure 2H).
To determine whether osteocytic Wnt7b signals through
the canonical Wnt pathway, we assessed β-catenin Collectively, these results demonstrate that osteocytic
protein levels in Y4-Wnt7b cells, as β-catenin is a central Wnt7b promotes osteogenic differentiation of ST2 cells
mediator of canonical signaling. Western blot analysis of primarily through activation of the PKCδ-mediated
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cytoplasmic and nuclear fractions revealed no significant noncanonical Wnt signaling pathway, rather than through
difference in β-catenin levels compared to Y4-GFP controls canonical Wnt or mTORC1 signaling.
(Figure 2A). Furthermore, immunofluorescence staining 3.3. Osteocytic Wnt7b-PKCδ pathway inhibits
with an anti-β-catenin antibody demonstrated no adipogenic differentiation
observable translocation of β-catenin from the cytoplasm To investigate whether osteocytic Wnt7b inhibits
to the nucleus in Y4-Wnt7b cells relative to Y4-GFP cells adipogenic differentiation, we co-cultured Y4-Wnt7b
(Figure 2B). Moreover, at concentrations below 10 μM, and ST2 cells (1:4 ratio) in adipogenic medium for 7
iCRT-14 (the canonical Wnt signaling antagonist) did not days. While abundant lipid droplets formed in ST2 cells
attenuate Y4-Wnt7b-induced osteoblast differentiation of co-cultured with Y4-GFP controls, visualized by Oil Red
ST2 cells, as measured by AP staining and its biochemical O staining (Figure 3A), significantly fewer droplets were
activity assays (Figure S3A and B, Supporting Information). observed in the Y4-Wnt7b group. Furthermore, osteocytic
Similarly, iCRT-14 did not alter the expression of the Wnt7b downregulated the expression of key adipogenic
canonical Wnt target gene Axin2 compared to the DMSO transcription factors PPARγ (Pparg) and C/EBPα (Cebpa)
control (Figure S3C, Supporting Information). These (Figure 3B).
findings indicate that osteocytic Wnt7b does not activate
the canonical Wnt signaling pathway. Next, we investigated whether inhibition of PKCδ
signaling could restore the adipogenic differentiation of
Given our previous reports that Wnt7b activates ST2 cells by using Rottlerin in the co-culture system. After
noncanonical pathways involving mTORC1 and PKCδ in 7 days of adipogenic induction, lipid droplet accumulation
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osteoblasts to promote bone formation, we next investigated increased with Rottlerin concentration, largely reaching
these possibilities in our osteocyte model. Treatment with control (Y4-GFP) levels at 1 μM (Figure 3A). However, the
the mTORC1-specific inhibitor rapamycin in the co- lipid droplets were smaller and denser, which may be due
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culture of Y4-Wnt7b and ST2 cells for 3 days displayed no to the continued inhibition of Wnt7b-PKCδ signaling by
significant inhibition of AP activity. In contrast, rapamycin Rottlerin, thereby preventing the formation of larger lipid
effectively inhibited osteoblast differentiation induced droplets. Consistent with this observation, 2 μM Rottlerin
by Wnt3a-treated MLO-Y4 cells (Figure S4, Supporting restored Pparg and Cebpa expression to control levels
Information). This suggests that mTORC1 signaling is not (Figure 3B).
essential for osteocytic Wnt7b-mediated osteogenesis.
The above results indicated that Rottlerin reverses the
Finally, we tested the involvement of PKCδ signaling osteocytic Wnt7b-mediated inhibition of adipogenesis,
using its selective inhibitor Rottlerin. Firstly, we tested suggesting that the Wnt7b-PKCδ signaling axis in
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the effect of different concentrations of Rottlerin on osteocytes plays an important role in the negative
Wnt7b-induced osteogenic differentiation. AP staining regulation of adipogenesis.
revealed that treatment with 1 μM Rottlerin significantly
reduced the differentiation function of Y4-Wnt7b cells 3.4. Construction of Y4-Wnt 7b PCI3D modules
(Figure 2C). Furthermore, Rottlerin inhibited the We fabricated 3D scaffolds using our in-house developed
phosphorylation of the myristoylated alanine-rich C-kinase PCI3D system, simultaneously printing PCL frameworks
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substrate (MARCKS), which is a well-established marker of and cell-laden hydrogel bundles containing either Y4-
PKC activation. Western blot analysis demonstrated that GFP or Y4-Wnt7b cells mixed with ST2 cells at a 1:4
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Y4-Wnt7b cells exhibited increased levels of p-MARCKS ratio (Figure 4A). GelMA hydrogel served as the bioink
(Ser159/163) compared to controls. Treatment of Y4- for the cell bundles. Utilizing its temperature-dependent
Wnt7b cells with 1 μM Rottlerin significantly reduced viscosity, GelMA was uniformly extruded at 25°C using a

Volume 11 Issue 4 (2025) 433 doi: 10.36922/IJB025240238