Results

PCR Array: The expression of MCP-1 and MCP-3 had increased on day 2 than 0 or 7 in the rib fracture healing. Immunohistochemistry Staining: To verify the localization of MCP-1 expression, we examined the Wild type (WT)-mouse rib fracture healing. We observed high expression of MCP-1 and MCP-3 at the periosteum and the endosteum on post-fracture day 3. In vivo Antagonist Study: To elucidate whether MCP-1/CCR2 axis is involved during the early phase of fracture healing, we continuously administered RS102895, CCR2 antagonist, before or after rib fracture. Micro-CT analysis showed delayed fracture healing in the before-group compared with both the control and after-group. On day 21, the hard callus volume in the before-group was significantly smaller than that in the control-group. Histological analysis showed that fractures in both the control and the after-groups were healed by day 21. In contrast, less of cartilage in the callus was observed in the before-group on day 7. Gain of Function: To examine the roles of MCP-1 at the periosteum and the endosteum during the fracture healing, we created a segmental bone graft exchanging model. The bone grafts were transplanted from MCP-1^−/− mice to another MCP-1^−/− mice (KO-to-KO). Micro-CT analysis showed that KO-to-KO transplantation led to the delay of fracture healing on day 21. Next, we created exchanging-bone graft models between MCP-1^−/− and WT mice, in which a segmental bone derived from a WT mouse was transplanted into a host MCP-1^−/− mouse (WT-to-KO). In contrast to KO-to-KO bone graft transplantation, the transplantation of WT-derived graft into host KO mouse resulted in a significant increase of new bone formation on day 21. Histological analysis revealed that marked and localised induction of MCP-1 expression in the periosteum and the endosteum around the WT-derived graft was observed in the host MCP-1^−/− mouse. Loss of Function: To validate whether MCP-1 is a crucial chemokine for fracture healing, we created WT-to-WT and KO-to-WT bone graft models. When WT-donor graft was transplanted into WT-host, abundant new bone formation was observed around a WT-derived graft on day 21. In contrast, transplantation of KO-derived graft into WT-host resulted in a marked reduction of periosteal bone formation on a donor graft.