Establishment and Evaluation of a Rat Model of Medial Malleolar Fracture with Vascular Injury

Objective A stable animal model was needed to study bone non-union caused by insufficient blood supply, the main object of this paper is to develop a medial malleolar fracture model with controllable arterial vascular injuries in rats for revealing the biochemical mechanism of non-union by insufficient blood supply. Methods A total of 18 rats were randomly divided into three equal groups: the Sham group, the Fracture group, and the Fracture + Vascular group. The animals were subjected to unilateral medial malleolar bone fracture and vascular injury using customized molding equipment. The fracture site was scanned by micro-CT, and vascular injury was evaluated by laser Doppler flowmetry (LDF) 24 h after modeling. Histological examination (HE), alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry and immunofluorescence were conducted on the medial malleolar fracture tissues of three rats randomly selected from each group 24 h after modeling. Subsequently, to further confirm the success of fracture modeling, the fracture sites of three other rats in each group underwent micro-CT scanning again 6 weeks after surgery. Results The results of a 24 h micro-CT showed that all rats used to create the fracture models showed controlled injury of the medial malleolus. The model was stable, and the satisfaction of the homemade equipment agreed with the expectation. LDF showed that the blood flow of rats in the Fracture + Vascular group decreased significantly 24 h after fracture injury, while collateral blood flow perfusion increased by 50% on average. The results of HE, ALP and TRAP staining in the medial malleolus showed that the number of osteoblasts (OBs) and osteoclasts (OCs) in the Fracture group increased significantly, but the number of OBs and OCs in the Fracture + Vascular group decreased sharply relative to the number in the Sham group 24 h later. Furthermore, immunohistochemistry and immunofluorescence results showed that the number of neovessels in the Fracture group was significantly increased, while the number of neovessels in the Fracture + Vascular group was significantly decreased, which was consistent with the above results. After 6 weeks of modeling, the micro-CT results showed that the fractures in the Fracture group had healed substantially, while those in the Fracture + Vascular group had not. Conclusion This study provided a reproducible and stable experimental animal model for medial malleolar fractures with arterial injury.