The Effects And Mechanisms In Bone Formation Process With The Activation Of Wnt/β-catenin Pathway

Objective To activate the Wnt/beta-catenin pathway with different methods, and investigate its effects and mechanisms on bone formation and bone healing.Methods 1. To produce rat femur fracture model, injecting the SB-415286 in fracture end. Using western blotting methods to investigate β-catenin, PCNA and BMP-2 protein expression in the callus; Immunohistochemical detection to study osteoblast proliferation and osteogenesis effect; Using the Micro-CT to measure callus bone mass and bone mineral density; At 3 weeks postoperatively, fracture specimens were performed biomechanical tests.2. Building lentivirus mi R-199 a gene expression vector, to infect MC3T3-E1 cells in vitro, in order to detect the infection efficiency. Injecting lentivirus vector in atrophic bone nonunion fracture end, adopting the method of real-time quantitative RT-PCR to detect the β-catenin gene expression in the callus. Using the histologic, immunohistochemical, Micro-CT, radiological methods to study the impact of lentivirus vector on atrophic bone nonunion healing.3. The osteoporosis models of rats were established by ovariotomy. Through the tail vein, lentivirus vector were injected. After operation, using the Micro-CT to detect right femoral neck bone microstructure and bone mass changes; Right femoral biomechanics changes were tested also.4. Building open right femoral fracture model of rats, and injecting Sclerostin monoclonal antibodies(Scl-Ab) postoperative, we observe its effects on fracture healing; the effect of complete femur was observed also.Results 1. SB-415286 promoted the β-catenin, PCNA and BMP-2 protein expression; promoted osteoblast proliferation and osteogenesis effect; improved the callus bone mass and bone mineral density; improve the biomechanical properties of callus and promoted the fracture healing.2. MC3T3-E1 cells were easy to be infected with lentivirus vector; mi R-199 a gene could be expressed in rats stablely; lentivirus vector injection enhanced healing of the atrophic bone nonunion.3. Lentivirus vector tail intravenous injection improved the right femoral neck bone microstructure of treatment group, increased bone mass; femoral biomechanics indexes of treatment group of were improved also.4. Scl-Ab treatment increased the expression of PCNA and BMP-2 in callus tissue; reduced the generation of cartilage; increased bone mass and biomechanical properties of callus tissue, promoted healing of the open fractures. In addition, Scl-Ab increased left intact femur anabolism also.Conclusion In conclusion, this study used a variety of methods to activate the Wnt/β-catenin signaling pathway, and studied the results of the fracture healing. Experiments showed that activation of Wnt/β-catenin signaling pathway can promote the healing of fracture. Considering the safety and effect, the use of sclerostin monoclonal antibody to antagonism sclerostin targets signals in Wnt/β-catenin signaling pathway, is a safe and effective method. Sclerostin antibodies can be used as a non-invasive treatment measures, for the treatment of open fractures.