CD11b+ Depletion of Bone Marrow Derived Macrophages Negatively Affects Bone Fracture Healing

Hematoma formation and inflammation occurs during the beginning stages of fracture repair, which require the presence of innate cells such as macrophages. Macrophages are subdivided into proinflammatory, M1, or anti-inflammatory, M2, phenotypes with different functions, cytokine profiles, and surface markers. In this study, in vitro and in vivo models were used to deplete M1 macrophages, using Mac-1 Sap conjugated antibody (Mac1SAP), to determine the effects on fracture healing. In vitro study, primary macrophages isolated from mice femoral bone marrow were harvested and differentiated into M1 macrophages (+LPS), M2 macrophages (+IL-4), or undifferentiated then treated with either vehicle or 10pM Mac1SAP. Primary macrophages and media were collected at days 2 and 5 post Mac1SAP treatment for flow cytometry and cytokine quantification. For the in vivo model, mice were treated with Mac1SAP prior to fracture. Bone marrow was then harvested from femurs on days 2 and 4 post- fracture for flow cytometry and cytokine quantification. MicroCT, trichrome staining, SAFO staining was used to evaluate the bone repair process. Immunohistochemistry was also performed using iNOS to identify M1 macrophages and Arg-1 to identify M2 macrophages within the fracture callus. FACS and IHC results demonstrated that Mac1SAP decreased the M1 macrophage population while protein multiplexing showed significant changes in cytokine expression profiles both in vitro and in vivo. MicroCT and histology data demonstrated that Mac1SAP treatment impaired bone healing. Overall, the data suggest that depletion of M1 macrophages by Mac1SAP treatment negatively affected the bone healing process by changing the inflammatory response