Establishing a Diaphyseal Femur Fracture Model in Mice

The diaphyseal femur fracture model shown here is a simple and low cost murine model, suited for basic and preclinical studies of long-bone fracture healing. The open surgery technique does not require complex surgical instruments and fixation devices. It allows the visual inspection of the fracture being produced.

This model can be used for local transplantation and in vivo testing of therapeutic components, including stem cells, drugs and biomaterials that cannot be delivered by percutaneous, or systemic injection. The surgery protocol requires some knowledge of basic surgery techniques and of the mouse anatomy to perform the critical steps. The visual demonstration in this work can help even for those inexperienced with animal surgery.

Demonstrating the procedure will be Leonardo Muller and Bianca Frade, both master students from the Laboratory of Stem Cells and Bone Repair. After transferring the anesthetized animal to the surgical table, disinfect the area to be incised by rubbing the skin with a 10%povidone iodine sponge. Then dry the rubbed area with sterile gauze pads, wash with 70%ethanol and dry again with a sterile gauze pad.

Next, place the mouse in the right lateral decubitus position and drape the mouse, making only the incision region visible. Before surgery, place eyedrops in the mouse's eyes to avoid dryness and constantly check the mouse's breathing during the surgical procedure. Make a one centimeter cutaneous lateral parapatellar incision with a scalpel blade number 11, beginning at the level of the tibial tuberosity and extending to the level of the patella, and then for an equal distance towards the distal femur.

With blunt end scissors, dissect the subcutaneous fascia around the incision line to expose the fascia lata, the lateral vastus, and the femoral biceps muscles. With the scalpel blade number 11, make another incision in the fascia lata, similar to the one made in the skin, beginning at the level of the tibial tuberosity and running along the biceps femoris aponeurosis until the level of the distal femur to open the articular capsule and access the knee joint. Perform a medial luxation of the patella by placing the tip of a straight serrated precision tip tweezer under it and pushing it to the side together with the patellar and quadriceps ligaments, thus exposing the condyles of the femur.

Holding the femur with a serrated tip tweezer, flex the knee at 90 degrees and manually perforate the intramedullary canal of the femur through the intercondylar fossa with a 26-gauge hypodermic needle. Maintaining the knee flexed at 90 degrees, insert a segment of 1.0 centimeters, a 0.016 inch stainless steel rod wire through the opening into the medullary canal of the femur toward the great trochanter. Adjust the prevent distal extremity of the wire with a straight serrated tip tweezer to tightly fix it in the lateral condyle.

Next, separate the lateral vastus and femoral biceps muscles through blunt-end dissection with a serrated tip tweezer to access the distal diaphysis of the femur. Insert a dissecting scissor around the femur diaphysis in an angle of approximately 90 degrees, and gently perform a complete cortical osteotomy. Reposition the muscles and the patella by pushing the tip of a straight serrated precision tip tweezer over the condyle region.

Close the muscle fascia with a 6-0 reabsorbable suture, and then the skin using a 6-0 nylon suture, both in simple interrupted fashion. The radiographs of acceptable fracture patterns show transverse diaphyseal fractures in which the fracture lines are at a 90 degree angle to the axis of the bone, short oblique fractures in which the fracture line is less than 30 degrees relative to the axis of the bone, reducible fragmentary fractures, where a few small fragments of bone are seen, but the anatomical alignment of the bone remains. Representative radiographs of incorrectly placed wires are shown here where the wire is not inside the medullary canal of the proximal femur fragment, thus resulting in incorrect fixation of the fractured bone, and a case where the wire did not pass through any bone fragment and the fractured bone is completely unaligned.

Visible callus at the fracture site on day 14 and day 21 after surgery indicates the regenerative process of the model. Histological analysis shows the evaluation of the callus at the fracture site. The callus, initially on day seven, presents extensive areas of highline cartilage around the fracture line.

On day 14, ossification fronts are observed around the cartilage area, forming trabecular bone and cavities filled with reconstituted bone marrow. Finally, on day 21, the cartilage areas are almost completely replaced by trabecular bone, indicating successful bone bridging. The success of the model depends on the correct insertion of the wire.

Therefore, it's critical to keep the knee flexed at a 90 degree angle during this step. Additionally, micro-computed demography can be used to evaluate cell's evolution and the characteristics of the bone being formed in a tri-dimensional and quantitative scale.