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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

pathophysiological features of weightlessness-induced osteogenesis while suppressing adipogenesis—may be
bone loss. particularly well-suited for preventing the skewed lineage
Osteocytic Wnt7b represents a novel POME commitment characteristic of weightlessness-induced
acting through the Wnt noncanonical PKCδ pathway, bone loss.
distinguishing it from our previous reports on osteogenic Interestingly, unlike Wnt canonical signaling,
microenvironments, such as osteocyte-activated canonical osteocytic Wnt7b did not significantly alter the expression
Wnt signaling, 25,30,31 decellularized matrix, Dll4, of osteoclast regulators (Rankl, Opg, and Rankl/Opg ratio)
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and BMP7, all of which operate through different under static culture. However, microgravity itself robustly
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mechanisms. Importantly, Wnt7b-PKCδ signaling bypasses increased the Rankl/Opg ratio. Intriguingly, osteocytic
sclerostin-mediated inhibition of the canonical Wnt Wnt7b further elevated this ratio to control Y4-GFP
pathway, a major limitation of current anabolic strategies under microgravity (Figure S5, Supporting Information),
like Romosozumab (sclerostin antibody) and potentially suggesting that Wnt7b may paradoxically enhance
Teriparatide (parathyroid hormone, PTH1-34). While osteoclast differentiation in disuse conditions. While
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the anabolic mechanism of abaloparatide (parathyroid counterintuitive for an anabolic process, enhanced bone
hormone-related protein, PTHrP) remains unclear, resorption could potentially contribute to bone remodeling
our findings highlight Wnt noncanonical signaling— by recruiting mesenchymal stem cells to resorption sites,
particularly via osteocytic Wnt7b-PKCδ—as a previously ultimately promoting subsequent bone formation. The
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unexplored and highly promising target for counteracting net effect in vivo warrants further investigation.
disuse osteoporosis. Currently, no therapies specifically Current countermeasures against spaceflight bone loss,
target Wnt noncanonical signaling for weightlessness- including exercise, nutrition, and drugs (e.g., PTH ),
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induced bone loss, nor utilize osteocytes engineered with face significant challenges in terms of their efficacy and
this pathway as functional cells within 3D constructs to practicality in microgravity. Antibodies targeting sclerostin
promote osteogenesis under microgravity.
displayed promise but can only partially mitigate bone loss
While our data robustly support Wnt7b’s protective role in partially weight-bearing mice. Crucially, sclerostin
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against microgravity-impaired osteogenesis via sclerostin- production persists during prolonged spaceflight or bed
independent PKCδ activation, definitive proof of causality rest, raising concerns about the long-term efficacy and
necessitates further in vivo studies. Conditional knockout potential side effects of continuous antibody administration.
of Wnt7b specifically in the osteocytes of daβcat mice, Clinically effective PTH exhibits region-specific and load-
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or its neutralization via antibodies, will establish whether dependent responses under weightlessness conditions,
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Wnt7b is indispensable for the observed bone-sparing and an ultra-high dose (80 μg/kg/day) of PTH was required
effects. We are currently designing these experiments for protection in one study, posing osteosarcoma risks.
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using tail-suspended mouse models. Additionally, testing PTHrP promotes bone formation but may not directly drive
if osteocyte-specific Wnt7b overexpression alone (without osteogenic differentiation of progenitor cells. Therefore,
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concomitant β-catenin activation) replicates the protective novel anabolic strategies are urgently needed. Wnt7b,
phenotype in wild-type mice will further isolate its intrinsic acting independently of Sost/sclerostin and effectively
therapeutic potential. promoting osteogenesis even in its presence, emerges as a
Both Wnt/β-catenin and noncanonical pathways highly promising candidate for protecting weightlessness-
(especially the Wnt-Ca pathway) generally promote induced bone loss.
2+
osteoblastogenesis while inhibiting adipogenesis. PKC The 3D-BWBM system represents a significant
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isoforms exhibit complex, sometimes opposing, roles in technological innovation, integrating our in-house PCI3D
adipogenesis. 45–47 Fleming et al. demonstrated that PKC-α, bioprinting with the established RCCS bioreactor.
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TM
-δ, and -μ inhibit adipogenesis, while PKC-ε promotes it. This system enables rapid assessment of potential bone
Our results demonstrate that osteocytic Wnt7b inhibits homeostasis regulators under simulated microgravity
adipogenesis by downregulating the transcription of the within a biomimetic 3D microenvironment. Current
adipogenic transcription factors Pparg and Cebpa through microgravity tissue engineering models, whether 2D or
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PKCδ signaling under 2D and simulated 3D conditions. 3D, 56–59 often lack the interconnected tunnel structures
Although microgravity typically enhances adipogenesis essential for efficient nutrient/oxygen/metabolite
in human mesenchymal stem cells, the expression of exchange, limiting cell viability, and function deep within
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adipogenic factors tended to decrease in our 3D-BWBM constructs. Unlike conventional BTE scaffolds, where
system containing Wnt7b osteocytes. This suggests that cell activity is often confined to surfaces, our PCI3D
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the dual function of the Wnt7b-PKCδ axis—of promoting module features precisely printed, uniform tunnels

Volume 11 Issue 4 (2025) 441 doi: 10.36922/IJB025240238