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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

1. Introduction mechanosensitive cell sources as functionalized agents
to actively counteract microgravity-induced bone loss.
Bone loss in microgravity remains a significant challenge Recent research has developed microgravity-compatible
for astronauts, with bone mineral density (BMD) of the bioprinted constructs but relied on passive scaffold designs
lumbar spine and hip joint decreasing by 1–1.5% per that lacked innate osteoinductive signaling.
20
month, mainly due to suppressed osteoblast activity
1–3
and increased osteoclast-based osteolysis. Current Herein, we present two key innovations: (i) bioprinting
4,5
countermeasures, including physical exercise, nutritional of functionalized osteocytes and (ii) a 3D biomimetic
supplementation, and pharmacological protection, weightless biomicroenvironment (3D-BWBM) as a
have limited effectiveness, potential risks, and side bionic microgravity platform. We designed Wnt7b-
6
effects. A promising alternative is to target osteocytes, expressing osteocytes (MLO-Y4) as a bioactive cell source
the mechanosensory cells in the skeleton that regulate to bypass sclerostin-mediated Wnt inhibition. Unlike
osteoblast/osteoclast activity through Wnt signaling. previous studies using undifferentiated cells, Wnt7b-
7,8
However, the loss of mechanical loads, such as prolonged activated osteocytes promoted osteogenic differentiation
bed rest, paralysis, or space flight, upregulates osteocyte of undifferentiated cells via the non-canonical Wnt-PKCδ
9
sclerostin (gene: Sost), which blocks the binding of Wnt pathway while inhibiting adipogenic differentiation—a
ligand to its coreceptor Lrp5/6. This, in turn, inhibits the dual mechanism that is not possible with conventional
Wnt/β-catenin canonical signaling pathway, leading to a scaffolds or growth factors. Likewise, by combining a 3D
10
decrease in osteoblast differentiation. 11,12 bioprinted polycaprolactone (PCL)-gelatin methacryloyl
(GelMA) scaffold with NASA’s rotating cell culture
Wnt7b, a ligand that activates the non-canonical system (RCCS™), we have created 3D-BWBM that goes
Wnt pathway, has emerged as a potential solution. In beyond traditional 2D RCCS cultures. Our system
™
contrast to Wnt/β-catenin signaling, Wnt7b-PKCδ features multi-scale tunnels (500 μm) for enhanced
is sclerostin independent and promotes osteogenesis nutrient/metabolite transport. Intercellular crosstalk
in mice. 13,14 We recently observed that mice with is maintained by printing osteocyte-ST2 co-cultures
osteocyte-specific Wnt/β-catenin activation (daβcat ) for long-term osteogenic function under simulated
Ot
exhibit elevated Wnt7b expression (Figure 1A) and are microgravity conditions.
protected from weightlessness-induced bone loss. This
led us to hypothesize that osteocytic Wnt7b creates a This study combines, for the first time, mechanosensitive
microenvironment conducive to osteogenesis even under cell customization with a 3D-bioprinted exoskeleton for
microgravity conditions. microgravity applications, providing a scalable solution
for disuse osteoporosis. By expanding the scope of
The cost of conducting research under actual bioprinting to realize biological functions in extreme
microgravity conditions is high, resulting in limited environments, this study establishes a critical link between
research opportunities. Various types of bone tissue biomanufacturing and space medicine.
engineering (BTE) scaffolds are widely used in
microgravity environments. Although scaffolds may 2. Materials and methods
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provide temporary physical support for cell attachment,
proliferation, and differentiation, the microgravity 2.1. Materials
Puromycin (10 mg/mL stock solution), Rottlerin (PKCδ
environment confers a unique mechanical environment
with profound effects on cell fate. Therefore, cell culture inhibitor, dissolved in dimethyl sulfoxide [DMSO], stored
16
in microgravity environments could inspire greater at 10 mM), rapamycin (mTORC inhibitor, dissolved in
potential for scaffold-based BTE. The use of biomaterials DMSO, stored at 50 mM), and iCRT-14 (Wnt canonical
combined with a simulated weightlessness device for signaling inhibitor, dissolved in DMSO, stored at 50 mM)
in vitro 3D culture provides a platform that closely were purchased from MedChemExpress (China). The
mimics in vivo conditions, thereby supporting tissue and BCIP/NBT Alkaline Phosphatase Color Development kit,
cell regeneration. 17,18 modified Oil Red O staining kit, calcein/PI cell viability/
cytotoxicity assay kit, alkaline phosphatase (AP) assay
Recent advances in 3D bioprinting have enabled the kit, phenylmethanesulfonyl fluoride (PMSF), RIPA lysis
fabrication of bone-mimicking scaffolds with customized buffer, the nuclear and cytoplasmic protein extraction kit,
mechanical and biochemical properties. However, most phosphatase inhibitors, and DAPI staining solutions were
19
studies have focused on structural optimization (e.g., obtained from Beyotime Biotechnology (China). TRizol
porosity, stiffness) or general effector cell (e.g., mesenchymal reagent, Evo M-MLV reverse transcription premix kit, and
stem cells, osteoblasts) encapsulation, without utilizing SYBR Green Premix Pro Taq HS qPCR kit were obtained

Volume 11 Issue 4 (2025) 427 doi: 10.36922/IJB025240238