This method can help answer key questions in the field of bone biology, such as which interventions promote fracture healing. The main advantage of this technique is that it provides a simple method to derive parameters to generate consistent fractures. To locate the region for the fracture, obtain radiographs of the limb femur or tibia to be fractured in a representative sample of five euthanized animals.
Tibia images are shown here. Mark the desired location of the fracture on the radiograph of the limb to be fractured. Measure from the calcaneal tibial joint to the level of the marked fracture.
Calculate the mean fracture length for all trial specimens. On the adjustable fracture device, measure the distance from the outside surface of one support anvil to the center of guillotine impact. Subtract the center of guillotine impact from the fracture length to calculate the fracture positioning jig depth, or JD.Machine or 3D print a U-shaped channel with a height and width equal to the anvil and a depth equal to the JD.Position the specimen in the fracture apparatus in the prone position for femur fractures, or in the supine position for tibia fractures.
Press the dorsum of the foot against the end of the fracture positioning jig. Manually depress the guillotine until the limb fractures. Obtain a radiograph of the fractured limb to confirm the jig size and fracture location.
To determine pin length, measure the limb length from the tibial plateau to the level of the posterior malleolus for tibia fractures. To determine pin width, measure the minimum medullary diameter in the fractured limb. Select a needle with a gauge approximately equivalent to the medullary diameter and a length more than 1.5 times pin length.
An approximate pin size for a 14-week old C57 black six mouse is 27 gauge, one and a quarter inch, and 22 gauge, one and a half inch for tibia and femur, respectively. Machine or 3D print a gauge with a length equal to pin length minus the needle length. One end should have an overhang to rest against the hub of the needle and the other should indicate where the pin should be cut.
Use an electric clipper or depilatory cream to remove hair from the legs of non-fracture trial specimens from mid-tibia to mid-femur, exposing the knee joint. To pin the tibia, insert the needle percutaneously, lateral to the patellar ligament. Retract the patellar ligament medially and align the tip of the needle to the axis of the tibia.
Using a reaming motion, gently breach the tibial plateau and guide the needle down the medullary cavity. Next, use the gauge and ream until the exposed needle is equal to the gauge length. Retract the needle approximately three millimeters, to provide enough room to cut the needle at the level indicated by the gauge.
Crimp 3 millimeters of the distal end of the pin using a pin cutter, and then cut the pin at the level of the gauge. Sync the pin to the articular surface using a rod with a diameter 1.5 times larger than the diameter of the needle. Obtain radiographs to confirm the needle extends the length of the medullary canal of the limb and does not protrude from the proximal or distal end.
To determine impact depth, measure the diameter of the cortex at the level of the desired fracture on the radiograph. Position a pinned trial specimen in the fracture device using the fracture positioning jig. Rest the impact ram on the uninjured limb.
Do not allow the ram to drop. The bone should remain intact during this optimization step. Apply enough downward force on the ram to compress soft tissue, but not fracture the bone.
Adjust the impact depth to 75 times cortical diameter to account for the soft tissue. Set the drop height to two centimeters. Position the ram in its starting position by connecting it to the activated electromagnet.
Position a trial limb in the fracture apparatus. Press the dorsum of the foot against the fracture positioning jig. Briefly depress the foot switch to release the ram and then reset it to its starting position.
Radiograph the impacted trial limb and inspect for any evidence of a fracture. This can be subtle when using low velocities with a controlled impact depth. If no fracture is generated, increase the drop height by two centimeters.
If a fracture is generated, record the drop height and multiply it by 1.1. This is the new drop height. Use the new drop height to fracture the next trial limb.
Continue the procedure until all trial samples are fractured. Record the final drop height and all parameters from the optimization. Record the trial specimen's age, sex, genotype, and weight.
Using an adjustable fracture device and optimized parameters significantly improved the generation of simple transverse fractures. The pre-optimization group only generated a simple transverse fracture in 27 out of 58 samples, or 46.55%of the time. Whereas, the post-optimization group exhibited simple transverse fractures 98.28%of the time.
After its development, this technique increased the rigor and the reproducibility of animal models in fracture generation studies. Following the procedure, other techniques, such as micro-CT and histology can be used to answer additional fracture morphology questions.
