Osteoporotic fractures are very common and represent an enormous unmet medical need. Our group has previously reported that addition of rTNF to the fracture site promotes fracture healing in C57/BL6 mice (Glass et al. PNAS 2011). Using a murine fracture model of endochondral healing, we observed that local addition of rTNF only accelerates fracture repair if administered within the first 24 h following injury. The optimal therapeutic dose is 1 ng. TNF is first expressed by neutrophils in the first 72 h followed by F4/80+ monocytes/macrophages. Furthermore, downregulation of early inflammation using anti-TNF or rIL10 impaired fracture healing. To quantify the recruitment of innate immune cells, we used a murine air-pouch model whereby murine fracture supernatants +/− rTNF was injected. Fracture supernatants are generated by incubating fracture fragments in media overnight to capture the local cytokine environment of the fracture site. We found that addition of rTNF promoted neutrophil recruitment, which in turn promoted recruitment of monocytes/macrophages by CCL2 production. Macrophages have previously been reported to be critical in bone repair (Alexander et al. JBMR 2011). Furthermore, neutrophil depletion using anti-Ly6G antibody and inhibition of the chemokine receptor for CCL2, CCR2, led to significantly impaired fracture healing. Finally, local treatment with rTNF enhanced fracture repair in ovariectomy-induced osteoporotic mice by 40%. Thus, we have shown that TNF is a key upstream inflammatory mediator in fracture repair. Mechanistically, addition of rTNF upregulates CCL2 production and monocyte recruitment. Our findings provide evidence that the innate immune response represents a viable therapeutic target in the enhancement of fracture healing.
17 May 2014 - 20 May 2014