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Updated: May 5, 2026

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Published on: March 15, 2018
[RANKL and OPG expression in bone marrow-derived mesenchymal stem cells of ovariectomied promotes osteoclast
Bingyi Shao1, Yang Yu, Xiaohui Fu
1Department of Endodontics, The Affiliated Hospital of Stomatology, The Stomatology College The Chongqing Medical University, Chongqing 400015, China.
Objective:
To investigate the expression of receptor activator of nuclear factor-kappa B lig (RANKL), osteoprotegerin (OPG) in bone marrow mesenchymal stem cells (BMSCs) and its impact on osteoclast formation and function in the ovariectomied mice.
Methods:
An animal model of osteoporosis was established by ovariectomy (OVX bilateral ovarian resection) in 8-week-old healthy female mice. The sham group was the 8-week-old healthy female mice with bilateral resection. Macrophages from mice were inducted by M-CSF and RANKL, and co-cultured with the BMSCs collected from mice in the OVX group and sham group, respectively. The osteoclast numbers of the two groups were compared by TRAP staining. The resorption pits were measured by toluidine blue staining. The level of RANKL/OPG expression was detected by RT-PCR and Western blotting.
Results:
TRAP assay and toluidine blue staining showed that the numbers of osteoclasts and resorption pits in OVX group were more than that in the sham group. The expression of RANKL in BMSC was lower in sham group than that of the OVX group. On the contrary, the expression of OPG in BMSCs was higher in the shame group than that of the OVX group.
Conclusion:
Expression of RANKL/OPG are regulated by estrogen in BMSCs. RANKL and OPG expression promotes osteoclast development and enhances its function under the condition of estrogen deficiency.
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