A novel mouse model to study fracture healing of the proximal femur

Objectives The majority of fractures especially in elderly and osteoporotic patients occur in metaphyseal bone due to the susceptibility of trabecular bone to microstructural damage. These fractures are challenging to treat due to the difficult fracture fixation in the fragile osteoporotic bone and its poor healing capacity. Therefore, advanced strategies are needed to facilitate optimal bone healing of those fractures. However, adequate small animal models to study them are rare. Mice are increasingly used in fracture healing research, but metaphyseal fracture models in mice are challenging. Here we present a novel mouse model to study fracture healing of the proximal femur.

Methods All animal experiments were approved by the local animal welfare committee (IACUC, UCSF). 12 weeks old female C57BL/6J mice were anaesthesized using Ketamin hydrochloride and analgesized using Buprenex. A 24G needle was introduced retrograde into the right femur. For open femur osteotomy, femoral muscles were separated bluntly. The osteotomy was sawed between the third and the lesser trochanter using a 0.44 mm Gigli wire saw. After 14 and 21 days, mice were euthanized and fractured femurs were harvested and fixed in 4% PFA for microCT and histological analysis. Statistics: Student’s t-test. p < 0.05. n = 6.

Results and Conclusion microCT analysis revealed successful implementation of the osteotomy at the proximal femur in all animals. Callus bone volume ratio increased significantly between d14 and d21. Total callus volume decreased significantly between d14 and d21. Histomorphometric analysis of Safranin O-stained sections from the fractured femurs revealed that all fractures healed via endochondral ossification. All fracture calluses at d21 displayed less than 10% of cartilage tissue, indicating successful cartilage-to-bone transition between d14 and d21. TRAP staining showed high osteoclast abundance and activity at the rims of the fracture callus at d14 and throughout the whole fracture callus at d21 after fracture, further indicating that fracture callus remodelling has already started. Immunohistochemical staining for the late chondrocyte marker collagen X revealed high expression at the cartilage-to-bone transition zone of the fracture callus and in the trabecular bone in the peripheral callus at d14. At d21, collagen X was present only sparely in the trabecular bone in the fracture callus. The late osteogenic marker osteocalcin was stained in hypertrophic chondrocytes, osteoblasts, osteocytes and bone matrix both at d14 and d21 with a more abundant staining at d21.

In conclusion, we successfully combined an open osteotomy approach to the proximal femur with a closed approach for intramedullary stabilization. All fractures healed by endochondral ossification within 21 days. Our novel model provides a fast, reliable and inexpensive way to study metaphyseal fracture healing in mice. Future studies using osteoporotic mice might help to unravel molecular mechanisms of delayed osteoporotic fracture healing.

Stichwörter fracture healing; mouse model; osteoporosis

Publication History

Article published online:
15 October 2020