Effect of osteoclasts on murine osteoblastic differentiation in early stage of co-culture

Interaction between osteoclasts and osteoblasts is known to play critical roles in bone formation. However, the biological significance of this interaction remains incompletely understood. This study aimed to investigate the early-stage effects of osteoclasts on the differentiation of murine osteoblasts. Osteoclasts were isolated from mouse bone marrow, and osteoblasts were harvested from neonatal mouse calvaria. Both cell types were co-cultured using a Millipore cell culture system. Expression of osteoblast marker genes was assessed using real-time polymerase chain reaction. Then changes in the protein expression profile of osteoblasts co-cultured with osteoclasts were examined using antibody array analysis and western blotting. The osteoblastogenic activities of cells under these conditions were also investigated using an in vitro assay. Osteoblasts and osteoclasts were successfully isolated, as confirmed by alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) staining, respectively. The expression of osteoblast specific marker genes, such as RUNX-2, ALP, and collagen-1a, was significantly increased. In addition, six proteins were found to be up-regulated and another six were down-regulated in osteoblasts cocultured with osteoclasts compared to expression levels in osteoblasts cultured alone. In particular, the expression levels of basic fibroblast growth factor and insulin-like growth factor-1 were remarkably altered. Indeed, osteoclasts grown in co-culture with osteoblasts significantly promoted osteoblastogenesis in vitro (P<0.005). In conclusion, osteoclasts may up-regulate osteoblastogenesis-related gene expression in osteoblasts and enhance osteoblastogenesis. Together, the results of this study indicate that osteoclasts play a critical role in the differentiation of osteoblasts during the early stage of co-culture.