Methods: Osteoblasts were isolated from mouse calvarial and long bone outgrowth after enzymatic removal of soft tissue and co-cultured for 10 days with osteoclast precursors obtained from bone marrow of both types of bone, spleen and blood. TRACP staining was performed to visualize and count TRACP-positive multinucleated cells (≥3 nuclei). The expression of osteoclastogenesis related genes, such as RANKL, OPG and TNF-α, was analyzed by qPCR. OPG was used to block RANKL/RANK pathway, and TNF-α antibody was used to block TNF-α pathway.
Results: Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. OPG gene expression was significantly higher expressed by long bone osteoblasts. The ratio of RANKL:OPG and TNF-α gene expression were significantly higher by calvaria osteoblasts. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no influence on osteoclastogenesis.
Conclusions: Our findings indicate that calvaria osteoblasts induce higher numbers of osteoclasts than osteoblasts obtained from long bone. This appears to be due to differences in the expression of RANKL and OPG, its ratio being higher in calvaria osteoblast cultures.