Bone fracture model

Closed right femur fractures were produced according to a published procedure (28). In brief, the mice were anesthetized with isoflurane (2–3%). After shaving fur and disinfecting with Betadine, a small incision was made at the knee lateral and parallel to the patellar tendon to dislocate the patella. To provide access to the intramedullary canal, we made a hole in the distal end of the femur and placed thin tungsten guide wire (0.125 mm diameter) into the femur. The mouse was secured in a materials testing machine using a custom 3-point testing fixture (Dynamight; Instron, Norwood, MA, USA). The femur was broken at the mid-diaphysis with a rapid-displacement controlled ramp (30 mm/s). To prevent excessive angulation of the femur, displacement ramp was limited to 1 mm above the resting platform. Immediately after fracture, the broken femur was stabilized with a 24-gauge stainless steel hollow rod (guided over the tungsten wire). In case of nonfracture due to hip dislocation (observed in Scx-mutant mice), a second attempt was made to realign and fracture the femur. If, after 3 attempts, a fracture was not obtained, the mouse was euthanized immediately. The guide wire was removed, the stainless steel rod was trimmed to the end of the femur, and the wound was sutured closed. To manage pain after surgery, we gave all mice 1 dose of buprenorphine (0.05 mg/kg, subcutaneously).