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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

Biological Technology, China). Membranes were blocked cells and 8 × 10 ST2 cells in 0.5 mL α-MEM was mixed
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with 5% bovine serum albumin (BSA) for 2 h, and then with an equal volume of GelMA/LAP solution, resulting in
incubated overnight at 4°C with primary antibodies: a final bioink containing 10% (w/v) GelMA, 0.25% (w/v)
β-catenin antibody (1:2000), Lamin B1 antibody (1:2000), LAP, and cells. The cell-laden bioink was loaded into a
Gapdh antibody (1:2000), MARCKS antibody (1:2000), cooled syringe and printed using a cell-printing nozzle.
or phospho-MARCKS (Ser159/163) (1:2000). After Simultaneously, PCL pellets were melted at 95°C
washing with PBST, membranes were incubated with for 30 min in a separate printing nozzle designed for
the secondary antibody goat anti-rabbit IgG H&L (HRP) thermoplastics. Melted PCL was printed at a speed of 2
(1:5000) for 1.5 h at room temperature. Protein bands mm/s to form a framework with a diameter of 400 µm
were visualized using enhanced chemiluminescence (ECL) and a spacing of 1100 µm. The cell-laden GelMA bioink
reagent (Abbkine Scientific, China) and imaged using an
ECL imager (CLINX Qinxiang Scientific, China). Band was printed alongside the PCL strands at a speed of 5
intensities were quantified using ImageJ and normalized to mm/s (strand diameter: 300 µm; spacing: 500 µm). After
loading controls. 13 printing each layer, the GelMA hydrogel was crosslinked
under 405 nm blue light for 10 s. Printing was performed
2.10. Immunofluorescence detection of β-catenin sequentially in three layers, with the PCL and cell-laden
nuclear translocation GelMA strands oriented perpendicularly between adjacent
Nuclear translocation of β-catenin was detected by layers. The fabricated PCI3D modules were transferred to
immunofluorescence, as described. Y4-GFP and Y4- 6-well plates containing growth medium and cultured at
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Wnt7b cells were cultured in a 24-well plate for 24 h. 37°C with 5% CO .
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Cells were fixed with 3.7% formaldehyde solution at room
temperature for 3 min, permeabilized with 0.25% Triton 2.13. Cell viability within PCI3D modules
X-100 for 30 min, and blocked with 1% BSA for 30 min. Cell viability within PCI3D modules was quantified at
Cells were then incubated overnight at 4 °C with β-catenin Days 1, 4, and 7 of culture using a Calcein/ Propidium
antibody (1:100). After washing three times with PBS, Iodide (PI) cell viability and cytotoxicity detection kit,
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cells were incubated with Cy3-labeled goat anti-rabbit IgG as described previously. Modules were washed with
secondary antibody (1:200) for 60 min in the dark. Nuclei PBS and incubated in staining solution (prepared by
were counterstained with DAPI solution (200 µL per well) diluting Calcein AM and PI stock solutions in assay buffer
for 3 min. Images were acquired using a fluorescence according to the kit instructions) for 30 min at 37 °C in
microscope (Leica, Germany). the dark. Calcein AM stains live cells green, while PI stains
dead cells red. Immediately after staining, the modules
2.11. Induction of adipogenic differentiation were imaged using an inverted fluorescence microscope
Adipogenic differentiation was induced as previously (Leica, Germany). Cell viability was quantified using
described. Briefly, Y4-GFP or Y4-Wnt7b cells were ImageJ software (64-bit, v1.46).
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cultured with ST2 cells at a 1:4 ratio in 12-well plates.
Upon reaching confluency (typically after 3 days), the 2.14. Cell proliferative activity within
growth medium was replaced with adipogenic induction PCI3D modules
medium (DMEM supplemented with 10% FBS, 0.5 µM Cell proliferation in PCI3D modules was evaluated on
hydrocortisone, 0.5 mM IBMX, and 60 µM indomethacin). Days 1, 4, and 7 using the CCK-8 assay kit, according to
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Cells were maintained in this medium for up to 7 days, the manufacturer’s instructions. The PCI3D module was
with half of the medium replaced every 3 days, until lipid first washed with PBS, then cut into four pieces and placed
droplets were observed microscopically. Adipogenesis in a 96-well plate. Subsequently, 90 μL of PBS and 10 μL of
was visualized for 30 min using the Oil Red O staining kit CCK-8 solution were added to each well and incubated for
according to the manufacturer’s instructions. Lipid droplets 2 h. The supernatant was then transferred to a new 96-well
were imaged using an inverted microscope (Nikon, Japan). plate, and the absorbance was measured at 450 nm using
the multi-function microplate reader (Thermo Fisher
2.12. PCI3D module preparation Scientific, USA).
The PCI3D modules were biofabricated using a dual-nozzle
3D bioprinter (Sunpbiotech, China), incorporating PCL 2.15. 3D-BWBM system
as structural support and cell-laden hydrogels containing The 3D-BWBM system was established to evaluate the
functional MLO-Y4 and ST2 cells, as previously detailed. protective effect of osteocytic Wnt7b against microgravity-
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Briefly, lyophilized GelMA was dissolved in α-MEM to impaired osteoblast differentiation. This system integrates
prepare a 20% (w/v) GelMA solution containing 0.5% the PCI3D system, which prints PCI3D modules
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(w/v) LAP. A cell suspension containing 2 × 10 MLO-Y4 containing the biological microenvironment (Wnt7b-
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Volume 11 Issue 4 (2025) 431 doi: 10.36922/IJB025240238