Wnt Pathway Inhibitor Delays Fracture Healing by Targeting Ephrin B1 (EFNB1) in Osteoprogenitor Cells

Our study assessed the role of Wnt signaling inhibitor (SM04690) in fracture healing and the underlying mechanism. Sprague Dawley (SD) rats were used to establish a fracture model which was then separated into SM04690 group which received SM04690 (50 mg/kg) by intraperitoneal injection once a day for one week, and control group which received saline once a day. After rats were sacrificed, the fractured femurs were harvested to measure femoral strength by stress testing, bone density and volume by CT. Femurs were sliced for immunohistochemical staining. Mesenchymal stem cells (MSCs), endothelial cells, osteoprogenitor cells and osteoblasts were detected by flow cytometer and EFNB1 expression was detected by immunoblotting and PCR. In addition, MSCs were treated with SM04690 (5 uM), followed by detection of EFNB1 expression. SM04690 treatment significantly inhibited EFNB1 expression and reduced bone volume and callus volume as well as decreased ultimate load of bones. Immunohistochemical staining and flow cytometry analysis showed no difference of osteoclast numbers at the fracture site between two groups, but proportion of osteoclasts in the cartilage tissue of SM04690 group was significantly decreased. In addition, the number of osteoblasts, osteoprogenitor cells and endothelial cells was significantly decreased after treatment. Under the conditions favoring osteogenic differentiation, the production of minerals by osteogenic cells was significantly decreased along with upregulated TAZ phosphorylation and downregulated Osterix in SM04690 group. In conclusion, SM04690 delays fracture healing by inhibiting EpRunB1 in osteoprogenitor cells.