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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

time points, with expression levels further elevated at Day osteoblast differentiation of ST2 cells, as observed by
14 (Figure 5C). AP staining and activity assays, compared to Y4-GFP
controls (Figure 7A and B). The qPCR results revealed
We next evaluated the effect of Y4-Wnt7b on
mineralization within the PCI3D modules. Following that Y4-Wnt7b significantly upregulated the expression
of osteoblast marker genes Alpl, Ibsp, Col1a1, Bglap, and
7 days in growth medium, the modules were cultured Sp7 (Figure 7). These data confirm that osteocytic Wnt7b
in osteogenic medium containing ascorbic acid and continues to promote osteogenic differentiation of ST2
β-glycerophosphate disodium for an additional 14 cells under microgravity conditions, suggesting a strong
days. ARS staining demonstrated a marked increase in protective effect of Wnt7b against weightlessness-induced
mineralized nodule formation in the Y4-Wnt7b modules bone loss.
compared to Y4-GFP controls, with a corresponding
1.8-fold increase in calcium deposition (Figure 5D). However, osteocytic Wnt7b had the least effect on
This further confirmed the potent role of osteocytic adipogenesis compared with the Y4-GFP group, with
Wnt7b in driving osteogenic differentiation and matrix no significant difference in the expression of adipogenic
mineralization within the 3D environment. transcription factors Pparg, Cebpa, or Fabp4, all of
which tended to decrease under microgravity conditions
Furthermore, analysis of adipogenic potential revealed (Figure 7D).
that Y4-Wnt7b significantly downregulated the expression
of adipogenic transcription factors (Pparg, Cebpa, Fabp4) 4. Discussion
compared to controls (Figure 5E). This suggests that
Wnt7b may also inhibit adipogenic differentiation within Our study demonstrated that osteocyte-derived Wnt7b
the simulated in vivo environment provided by the effectively promotes osteogenic differentiation of
PCI3D modules. bone marrow stromal cells (ST2) and protects against
microgravity-induced impairment of osteogenesis. This
3.6. Osteocytic Wnt7b promotes osteoblast protective effect stems from Wnt7b’s unique ability to
differentiation independent of microgravity- activate the sclerostin-independent Wnt noncanonical
induced sclerostin production PKCδ signaling pathway. We further established a
We found that osteocytic Wnt7b significantly promotes functionalized 3D-bioprinted osteocyte niche (PCI3D
ST2 cell osteogenic differentiation and mineralization module) and integrated it with a simulated microgravity
through the Wnt noncanonical signaling pathway platform (3D-BWBM) to validate Wnt7b’s efficacy under
in both 2D and 3D cultures. To test whether this conditions mimicking spaceflight.
mechanism functions in weightless environments, we The impetus for this investigation arose from our
built the 3D-BWBM system that simulates a microgravity observation that elevated Wnt7b expression correlates
biological microenvironment using RCCS (Figure 6A). with preserved bone mass in osteocyte-specific β-catenin
TM
The system enables rapid examination of the effects of activated (daβcat ) mice subjected to tail suspension, a
Ot
simulated in vivo Wnt7b on osteoblast differentiation model of disuse osteoporosis and simulated microgravity.
within a 3D-printed scaffold. After 7 days of cultivation We mechanistically demonstrated that osteocytic
in HARV , MLO-Y4 cells expressed higher levels of Wnt7b potently induces osteoblast differentiation
TM
myocyte enhancer factor 2C (Mef2c), which can activate and mineralization in co-cultured ST2 cells, while
the expression of Sost, leading to sclerostin production. concurrently suppressing adipogenesis by downregulating
36
The qPCR results demonstrated that the mRNA levels key adipogenic transcription factors Pparg and Cebpa.
of both Sost and Mef2c were significantly upregulated Critically, the specific PKCδ inhibitor Rottlerin inhibited
(Figure 6B), indicating that the microgravity conditions Wnt7b-induced osteogenesis and reversed its inhibition
were successfully simulated in osteocytes. of adipogenesis in ST2 cells (Figure 7E), unequivocally
Mechanosensory osteocytes upregulate the expression establishing the PKCδ pathway as the central mediator of
of Sost/sclerostin under mechanical unloading 11,37,38 and Wnt7b’s dual function. This positions Wnt7b as a potent
downregulate it under mechanical loading, 11,37 respectively. intrinsic physiological osteogenic microenvironmental
Sclerostin is known to bind to Lrp5/6 and inhibit the Wnt factor (POME) and a compelling candidate for BTE
canonical signaling pathway, thereby suppressing bone applications.
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formation both in vitro and in vivo. We placed PCI3D Beyond sclerostin upregulation, our gene expression
modules into HARV and suspended them for 7 days. analysis within the 3D-BWBM system confirmed a
TM
Meanwhile, under microgravity conditions, osteocytic comprehensive microgravity response. We observed the
Wnt7b significantly maintained and even enhanced expected upregulation of Sost and its transcriptional

Volume 11 Issue 4 (2025) 437 doi: 10.36922/IJB025240238