Biomechanical Study On The Mouse Model Of Ankle-subtalar Joint Complex Instability Induced Post-traumatic Osteoarthritis

Objective:The purpose of this study was to establish an animal model of anklesubtalar joint complex instability in mice,and to explore the relationship between anklesubtalar joint complex instability and post-traumatic osteoarthritis(PTOA).Methods:Twenty-one 6-week-old C57BL/6J male mice were selected,acclimated to the housing environment for two weeks,and trained for 5 days to familiarize them with balance beam and gait test experiments.When reaching 8 weeks of age,mice were randomly divided into three groups.In mice in the ligament transection group,the ligament around the ankle-subtalar joint complex of the right hindfoot was transected using a microscope to simulate acute injury of the ankle-subtalar joint complex,and to establish the animal model of ankle-subtalar joint complex instability in mice.In mice in the cervical ligament+anterior talofibular ligament group(CL+ATFL group),the cervical ligament and anterior talofibular ligament of the right hindfoot were transected.In mice in the cervical ligament+deltoid ligament group(CL+DL group),the cervical ligament and deltoid ligament of the right hindfoot were transected,and mice in the sham surgery group(SHAM group)the corresponding skin around the ankle-subtalar joint complex of the right hindfoot was transected without cutting any ligaments.After surgery,the skin incision was disinfected with Anerodine every day until the wound was healed.From 2 weeks to 12 weeks after surgery,mice in the three groups were trained in a running cage for 30 minutes every day with a rotary fatigue instrument to accelerate the formation of arthritis.All mice were tested on the balance beam before surgery and at 3 days,1 week,4 weeks,8 weeks,and 12 weeks after surgery.The time(seconds)and slipping times(Times)of mice passing through the balance beam were recorded and statistically analyzed.Gait tests were performed on all mice before and 12 weeks after surgery,and their stride length on the right was measured and recorded for further analysis.Balance beam and gait tests were used to evaluate the changes of self-movement in mice after an ankle-subtalar joint complex of ligament injury.Mice were euthanized 12 weeks after surgery,and the right hindfoot was fixed with 10%neutral formalin for 48 hours.Then,changes in the bone volume fraction of the ankle-subtalar joint complex and the formation of surrounding osteophytes of each specimen were analyzed by Micro-CT.The cartilage oligomeric matrix protein(COMP)in serum was measured by enzyme-linked immunosorbent assay(ELISA)to evaluate the progress of PTOA.Degeneration of the articular cartilage in the anklesubtalar joint complex was evaluated and analyzed by hematoxylin-eosin(H&E)and safranin O-fast green staining to evaluate the progress of PTOA.Immunohistochemical staining was used to observe the changes in type Ⅱ collagen with the progression of PTOA,to further verify the results of this study.Result:Compared with mice in the SHAM group,the balance beam and gait test in mice in the ligament transection groups were affected.In the behavioral test 12 weeks after surgery,the time used by the CL+ATFL group to pass the balance beam increased by 35.1%compared with that before surgery and increased by 27.7%compared with mice in the SHAM group,the number of slipes was 3.6 times that before surgery and 5.8 times that in mice in the SHAM group,and the average stride length on the right side was 7.2%smaller than that in SHAM group.The time it took mice in the CL+DL group to pass through the balance beam increased by 32.1%compared with that before surgery and increased by 28.5%compared with mice in the SHAM group,the number of slipes was 3 times that before surgery and 3.6 times that of mice in the SHAM group,and the average stride length of the right hindfoot was 5.6%smaller than that in SHAM group.Micro-CT analysis showed that the ankle bone volume fraction of the CL+ATFL group was 6%higher than that of mice in the SHAM group,and the ankle bone volume fraction of mice in the CL+DL group was 4.8%higher than that of mice in the SHAM group.The bone volume fraction of mice in the subtalar joint in CL+ATFL group was 6.7%higher than that of mice in the SHAM group,and the bone volume fraction of the subtalar joint of mice in the CL+DL group was 4.9%higher than that of mice in the SHAM group.The results of three-dimensional reconstruction showed that about 28.6%of the mice in the CL+DL group had talus dislocation,which destroyed the stability of the bone structure of the ankle-subtalar joint complex,and there was obvious osteophyte formation around the ankle-subtalar joint complex in the two groups with a severed ligament,and the joint surface was also coarser than that of mice in the SHAM group.Histopathological staining showed that the number of articular chondrocytes decreased,the cartilage layer was discontinuous,and that degradation of type Ⅱ collagen occurred in the ankle-subtalar joints complex of the two groups of mice with severed ligaments 12 weeks after surgery.Conclusions:The results of this study show that transection the CL+ATFL or CL+DL in mice will cause mechanical instability of ankle-subtalar joint complex.Some mice in the CL+DL group also present with talus dislocation due to ligament injury,and a destroyed stability of the bone structure of the ankle-subtalar joint complex.In addition,if there is no timely intervention treatment after joint instability,over time,the wear of articular cartilage will aggravate and lead to degenerative changes,which will affect the motor ability of mice.These results may provide a valuable reference for early ligament repair in the clinic.