The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

This method can help answer key questions in fracture healing research about the biological mechanisms of repair and the development of novel therapeutic strategies for promoting efficient bone healing. The main advantage of this technique is that it serves as minimally invasive procedure for generating standardized fractures to study bone fracture healing in mice. Demonstrating the surgical procedure will be Yong Li and Anuradha Valiya Kambrath, technicians from our laboratory.

After confirming the appropriate level of sedation by toe pinch, apply ointment to the eyes of an anesthetized 10-week old male mouse and remove the fur from the right hind limb. Starting at the center of the knee and making a circular sweep outward, disinfect the exposed skin with three sequential iodine-based and 70%ethanol scrubs, and place the mouse on a heating pad covered by a sterile surgical drape in the supine position. Flex the knee of the operative leg and use a scalpel blade to make a 1.5 centimeter incision centered over the knee joint.

Use forceps to laterally displace the patella, exposing the distal end of the femur and insert a 1.5 inch long, 25 gauge stainless steel hypodermic needle at the center of the trochlear groove in a retrograde direction down the length of the medullary canal and through the proximal end of the femur. Obtain an X-ray to ensure the proper placement of the pin. Then, pass a four-inch long, 36 gauge tungsten guidewire through the shaft of the needle into the hub at the distal femur and exiting the bevel on the dorsal side of the mouse.

Following a successful placement of the guidewire, gently pull the hub to carefully remove the needle while holding the limb and guidewire in place, and confirm the placement of the guidewire by X-ray. Now, place the mouse in the gravity dependent three-point bending device, positioning the femur horizontally across the two supporting points, such that the intertrochanteric and supracondylar regions of the femur rest on the support anvils and the lateral side of the limb is facing the loading point. The most critical step of this protocol is placement of the femur within the three-point bending apparatus, as the fracture geometry is dependent on the applied bending force and the positioning of the hind limb.

When the femur is in position, position a 391 gram weight, 34.6 centimeters above the impact disc of the device and drop the weight. Immediately, but carefully, remove the mouse from the device and confirm the fracture location by X-ray. Insert a piece of 24 gauge stainless steel hypodermic tubing over the guidewire to stabilize the fractured bone, and confirm the position of the steel rod and the stabilization of the fractured femur by X-ray.

Remove the guidewire and use wire cutters to remove any excess tubing at the distal end of the femur. Using forceps, apply a gentle downward force to bury the exposed tubing under the surface of the condyles, taking care not to dislocate the knee joint, and reposition the patella. Then, close the incision site with a 5-0 absorbable suture.

Once the surgeries have been completed, inject each mouse with up to 500 microliters of sterile saline, intraperitoneally, to aid in their postoperative recovery, and monitor the animals until their full recumbency. Weekly monitoring of the healing progression of the fracture callus up to 28 days following the surgery, reveals a prominent soft callus formed from mineralized cartilage by day 14 post-fracture, which is remodeled into a hard callus by day 21 post-fracture. Toluidine blue staining reveals the formation of a cartilage matrix at the fracture gap by day seven, that becomes aligned with the fracture gap by day 14, post-fracture.

Staining for Type one collagen expression illustrates the spatial organization and relative amounts of bone matrix that is present 14 days post-fracture. Micro-computed tomography analysis demonstrates a decrease in the callus volume of approximately 50%between days 14 and 28, post-fracture, indicating an effective remodeling of the callus. Before attempting this procedure, it is important to determine the weight and drop height, as they are dependent on the age, sex, and strain of the mice.

Radiographic imaging is commonly used to monitor the healing progression, while techniques such as immunohistochemistry and micro-CT can be used to answer questions about the mechanisms of bone regeneration and to assess the extent of the bone fracture healing. The development of this technique has provided a platform for the study of complex biological mechanisms of fracture healing and for exploring novel therapeutic strategies that accelerate bone tissue regeneration in mice.