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International Journal of Bioprinting Osteocytic Wnt7b-PKCδ against microgravity

from Accurate Biotechnology (China). Lyophilized was assessed by AP staining after co-culturing MLO-Y4
GelMA and photo-crosslinking agent lithium phenyl- and ST2 at 1:2, 1:4, 1:6, and 1:8 ratios, respectively.
2,4,6-trimethylbenzoylphosphinate (LAP) were obtained
from Sunp Biotech (China). Hydrocortisone, 3-isobutyl- 2.3. Isolation of long bones of tail-suspended
1-methylxanthine (IBMX), indomethacin, PCL, and mice for RNA extraction
polybrene were obtained from Sigma (United States of Mice with dominantly active β-catenin in osteocytes
Ot
America [USA]). The quantitative polymerase chain (daβcat ) and wild-type control mice (WT) were generated
21
reaction (qPCR) primers were synthesized by Sangon as previously reported. Briefly, DMP1-8kb-Cre mice
Biotech (China). Alizarin Red S (ARS), β-glycerophosphate expressing Cre recombinase in osteocytes were crossed with
lox(ex3)/lox(ex3)
disodium salt solution, and l-ascorbic acid were obtained Catnb mice, in which exon 3 of the β-catenin gene
from Solarbao Biotechnology (China). The Cell Counting (encoding the degradation domain) is flanked by LoxP sites.
Kit-8 (CCK-8) was obtained from Yeasen Biotechnology All animal procedures were approved by the Institutional
(China). Phosphate-buffered saline (PBS) and SDS-PAGE Animal Care and Use Committee of Chongqing Medical
gels were obtained from Epizyme Biotech (China). University and conducted in accordance with relevant
guidelines (IACUC-CQMU-2024-0277).
Rabbit polyclonal anti-mouse β-catenin antibody, Ot
The 14-week-old daβcat and WT mice underwent
Lamin B1 polyclonal antibody, MARCKS polyclonal suspension for 14 days. Following euthanasia, tibiae
22
antibody, and phospho-MARCKS (Ser159/163) polyclonal and femurs were dissected, and soft tissue (skin, muscle)
antibody were purchased from Proteintech (China). Cy3-
labeled goat anti-rabbit IgG (H+L) was obtained from was meticulously removed. After clipping the femoral
Beyotime Biotechnology (China). Polyclonal anti-Gapdh epiphysis, the bone marrow was flushed three times with
α-MEM containing 1% FBS. The cleaned long bone shafts
antibody and secondary antibody goat anti-rabbit IgG were immediately snap-frozen in liquid nitrogen and
H&L (HRP) were obtained from ZENBIO (China).
stored in –80°C until RNA extraction.
Alpha modified eagle’s minimum essential medium
(α-MEM) and Dulbecco’s modified eagle (DMEM) 2.4. Stable transfection of MLO-Y4 with lentivirus
medium were purchased from Gibco (USA). The penicillin- MLO-Y4 cells overexpressing Wnt7b or Wnt5a were
streptomycin solution (100×) was obtained from Beyotime generated via stable lentiviral transduction, as previously
23
Biotechnology (China). Fetal bovine serum (FBS) was described. Lentiviruses expressing Wnt7b, Wnt5a,
obtained from Biological Industries (Israel). and GFP were purchased from GENECHEM (China).
MLO-Y4 cells were transfected at a multiplicity of infection
2.2. Cell lines and cell culture (MOI) of 100 in the presence of 7 μg/mL polybrene.
The osteocytic cell line (MLO-Y4) was provided by Transfection efficiency was assessed 48 h post-infection
Professor Lynda Bonewald, Indiana University School by GFP fluorescence intensity. Transfected cells were
of Medicine, and the ST2 cells were provided by Dr. selected and maintained in culture medium containing
Steve Teitelbaum, Washington University. Both cell lines 0.5 μg/mL puromycin for at least 1 week to eliminate
were cultured in α-MEM growth medium supplemented non-transfected cells.
with 10% FBS and 1% penicillin-streptomycin. Wnt3a-
expressing cells (Wnt3a) and their control cells (L-ctrl) 2.5. RNA extraction and quantitative real-time PCR
were purchased from the American Type Culture Total RNA was extracted from long bone shafts or cultured
Collection (ATCC, USA) and cultured in DMEM cells using TRIzol reagent according to the manufacturer’s
23
growth medium containing 10% FBS and 1% penicillin- instructions, as previously described. Complementary
streptomycin. All cells were cultured in an incubator with DNA (cDNA) was synthesized using the Evo M-MLV
reverse transcription premix kit, and qPCR was performed
5% CO at 37°C. using the SYBR Green Premix Pro Taq HS qPCR kit in
2
MLO-Y4 cells stably transfected with lentivirus the CFX Connect Real-Time PCR Detection System (Bio-
expressing green fluorescent protein (GFP) (control), Rad Laboratories, USA). All primer sequences are listed in
Wnt7b, and Wnt5a were labeled as Y4-GFP, Y4-Wnt7b, Table 1. All qPCR reactions were performed three times.
and Y4-Wnt5a, respectively. For co-culture experiments, The relative mRNA expression levels were calculated
Y4 (1 × 10 cells/well) and ST2 cells (4 × 10 cells/well) using the 2 -△CT method, with the housekeeping gene
4
4
were seeded together in 24-well plates and cultured for 3 glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as
days or as otherwise indicated. Osteogenic differentiation the reference gene.

Volume 11 Issue 4 (2025) 429 doi: 10.36922/IJB025240238