Background
Multinucleated osteoclasts responsible for bone resorption are derived from hemopoietic cells of monocyte-macrophage lineages. Osteoclast progenitors proliferate and differentiate into osteoclasts by a cell-to-cell contact with osteoblast/stromal cells. There are many model systems for studying osteoclasts formation: 1) culture system of monocytic cell line, 2) primary culture system of monocyte-macrophage, 3) bone marrow culture system, 4) coculture system of bone marrow or spleen cells with osteoblastic or stromal cells. To develop more efficient system for osteoclast assay, we have performed coculture system using osteoblastic cells and osteoclast progenitors from subcultured mouse calvarial cells.
Methods
ICR mice at age of 6~8 weeks were dissected, and the calvariae were removed the periostium and soft tissue. The calvariae were minced into small pieces and then digested in α-MEM containing bacterial collagenase (1 mg/ml) for 2 hours. The bone cells liberated by digestion were harvested by centrifugation and then cultured with bone fragments in α-MEM containing 10% fetal bovine serum for 10 days. After these cells were cultured for 6 days in the presence of 1α,25-(OH)2D3, IL-1β, TNF-α, macrophage inflammatory protein-1α, PGE2, respectively. Tartrate resistant acid phosphatase (TRAP) staining, alkaline phosphatase staining, RT-PCR of calcitonin receptor (CTR) were performed for the characterization of osteoclasts and osteoblasts.
Results
The subcultured calvarial cells (1×105 cells/ml) primed with 1α,25-(OH)2D3 formed the high number of TRAP-positive multinucleated cells, and multinucleated cells expressed the CTR mRNA. Osteoblastic cells were located in the near of TRAP-positive multinucleated cells. All osteotropic factors increased the number of TRAP-positive multinucleated cells. The efficiency in term of number of osteoclasts formed in this coculture system was higher than those of the mouse bone marrow culture system.
Conclusion
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