Establishment Of Closed Fracture Model In Mouse And Study On The Effect Of Smurf1 Inhibitor On Fracture Healing

Fracture is a high-incidence traumatic disease,which seriously affects people’s daily life.The current clinical treatment methods mainly include bone transplantation,physical auxiliary therapy,systemic injection of parathyroid hormone,and local injection of human-derived recombinant BMP.Although the vast majority of fracture patients can heal after treatment,due to weakened blood supply at the fracture site,strong mobility of the fracture end,damage to the periosteum,and infection of the injured site,5%-10% of patients still fail to heal each year.BMPs are a class of acid glycoproteins isolated and purified from bone matrix that can efficiently induce the formation of bone and cartilage tissue.They play an important role in the process of embryogenesis,individual formation,and bone and cartilage repair.At present,human recombinant BMP-2(rh BMP-2)is the most effective growth factor for fracture treatment,spinal fusion and implant healing.However,due to the high cost of treatment,local injection is prone to adverse effects such as ectopic bone formation,natural bone resorption,soft tissue swelling,and osteolysis.Therefore,the FDA has not approved its application in children,pregnant women,and tumor-bearing patients.In view of the natural obstacles to the direct use of rh BMP-2 in clinical applications,the development of BMP pathway sensitizers as a new method to indirectly reduce the cost of rh BMP-2 treatment may occupy an important position in the future.As a negative regulator of bone,the ubiquitin ligase Smurf1 antagonizes the BMP signaling pathway to reduce its signaling activity.The preliminary laboratory established a new method based on computer simulation docking E3 antagonist screening,screened out small molecule compounds A01 and A17 that can inhibit Smurf1,and proved at the cellular level that Smurf1 is targeted to enhance the BMP signaling pathway as a drug target The feasibility.The purpose of this study is to do a more in-depth study of the previous results,and use the mouse fracture model to explore whether the Smurf1 inhibitors A01 and A17 can promote bone formation at the animal level.In this research,we invented a fracture modeling instrument for mouse bones,optimized the surgical plan of intramedullary nail fixation,and established the evaluation criteria for the recovery of mice.Using a new type of fracture modeling instrument to drop a weight of 120 g from a height of 200 mm can cause horizontal or oblique fractures to the femoral shaft of the mouse without damaging the skin and muscles of the mouse,and the success rate of fracture modeling is high;We use X-ray,Micro-CT and other clinical methods to examine the fracture healing of mice,and analyze the healing process through biological methods such as callus staining and m RNA detection of cartilage-specific gene expression.The results show that the healing method of mice is typical cartilage.Internal osteogenesis,this model can be used as a standard model for simulating human fracture healing.In terms of drug efficacy evaluation,we administered intragastric treatment to the fractured mice 3 days after the operation.At 28 days after the fracture,the three-point bending test was used to evaluate the biomechanical recovery status,which was used as the first criterion for drug efficacy evaluation.And at the landmark time points(14d,28d)after the fracture,the experimental mice were analyzed by Micro-CT and bone density scan to analyze the effects of Smurf1 inhibitors A01 and A17 in different stages of fracture healing.The results showed that the biomechanical strength and peak load of the fracture site of the mice in the medication group(A01,A17group)28 days after the fracture were higher;at 14 and 28 days after the fracture,the bone mineral density(BMD)of the mice in the medication group was higher Significantly higher than the control group;Micro-CT results also showed that the proportion of new bone formation in the drug group 14 days after fracture and the proportion of mineralized bone at 28 days after fracture were significantly higher than the control group.Therefore,the above results indicate that Smurf1 inhibitors A01 and A17 have a promoting effect on the biomechanical recovery of fractured mice.The reason may be due to the promotion of the formation of new bone and the deposition of mineralized bone during the healing process.Although there are countless therapies to promote fracture healing,drugs targeting ubiquitin-proteasomes and drugs targeting intracellular effector proteins have not been applied to the treatment of such diseases.Our experimental results are as follows: One program provides strong data to prove the feasibility of targeting the ubiquitin ligase Smurf1 to promote fracture healing.