Knockout of TLR4 promotes fracture healing by activating Wnt/β-catenin signaling pathway

Objectives: The aim of this study was to investigate the effect of Toll like receptor 4 (TLR4) on fracture healing. Methods: The open tibial fracture models in TLR4 knockout (TLR4(-/-)) and wild type (WT) C57BL-6 J mice were established. The radiological examination, tartrate-resistant acid phosphatase (TRAP) staining, Micro-CT scan and biological torsion test were performed on 7, 14 and 21 days after operation. Enzyme Linked Immunosorbent Assay (ELISA) kit was used to detect the expression levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin 6 (IL-6). Western blotting was used to detect the expression of beta-catenin, Wingless-type MMTV integration site family, member 4 and 5B (Wnt4 and Wnt5B), proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) of the callus tissue obtained from mice. Results: TLR4 knockout promoted fracture healing, reduced the number of osteoclasts, increased bone callus volume (BV) and callus mineralized volume fraction (BV/TV%) (P < 0.05), increased the maximum torque and torsional stiffness of callus (P < 0.05), reduced TNF-alpha, IL-1 beta and IL-6 expression (P < 0.01), and increased the expression levels of beta-catenin, Wnt4, Wnt5B, PCNA and BMP-2 (P < 0.01). Conclusion: TLR4 knockout reduced inflammatory and promoted fracture healing by activating Wnt/beta-catenin signaling pathway.