This protocol describes a reliable technique to perform and study femur fracture on adult mice. This technique is easy, fast and requires minimal tools to be performed. It allows for the characterization of different stages of fracture healing.
Individuals may initially struggle with properly aligning the fracture ends and creating consistent fractures between mice. Paying close attention to the site of fracture, the insertion of the pin and the closure of the wound will improve success of this surgery. To begin, prepare the animal by injecting 0.05 milligrams per kilogram of buprenorphine subcutaneously.
Using an electric trimmer shave a two by two centimeter square on both thighs corresponding to the location of the femur. Sterilize a shaved area using iodine and rinse with 70%ethanol solution. Make a five millimeter incision and expose the underlying fascia.
Expose the muscle covering the femur by cutting the fascia. Separate the muscle from the femur without damaging the tissue, then detach the muscle from the bone and secure the femur to make a cut. Make a transverse cut in the middle of the femur shaft.
Insert a guide needle through the distal section of the marrow cavity and push the thread across the knee joint. Then, remove the guide needle and perform a similar procedure from the proximal end. Leave the needle in the proximal end with its tip emerged above the skin.
Insert a stabilizing pin within the tip of the guide needle, pushing it so that it enters the proximal cavity as the guide needle is pulled out, then discard the guide needle. Align the distal end with the proximal end so that the thread is passed through the distal marrow cavity as it exits the knee joint. Pull the tip of the pin using a surgical clamp so that the proximal and distal sections are pulled together.
Then fold the ends of the surgical pin toward the fracture site to realign the fracture ends. Using wire cutters remove the plastic from the base of the needle. Twist the ends of the pin to avoid any internal tissue injury.
Finally, replace the muscles onto the femur and close the wound opening by pinching the skin together with clips. Perform a similar procedure without the femur fracture on the other leg and close the wound. Following the successful surgery, fluorescent imaging revealed various Messent-Kinal progenitors, such as Prx1-positive periosteal cells, which were found during the femur fracture repair process.
Alcian blue staining of the fractured femur revealed the formation of soft callus. Furthermore, the percentage of the callus or cartilage formation was also determined. By employing Micro-CT at approximately 28 days post fracture, the mineralization process was detected along with the callus gap and fracture gap.
It was also observed that if the fracture ends were not aligned properly or secured with a pin, there was a lack of callous formation on all or one side of the fracture end. When attempting this procedure remember to carefully align the two ends of the broken femur and stabilize them with the pin to ensure proper healing. After the surgery, femora can be collected for imaging and molecular analysis.
