The cantilever bending setup outlined in this protocol creates a reproducible approach for generating femoral neck fractures, which are more clinically relevant and can be utilized in murine studies to look at osteoporosis. This protocol creates a testing platform that improves reproducibility, leading to decreased coefficient of variation in outcome measures. Therefore, it minimizes the sample size needed for robust studies.
Begin by cutting tubing sections of length 1/2-inch to 1-inch using 1/4-inch by 1/4-inch square aluminum tubing. Label each aluminum segment with the sample IDs with an etching tool. Fill half of the tubing segments with putty and place them into a fixture to hold them upright.
Place the clean femurs flat on the benchtop so the anterior surface is facing up. Then, place the 3D-printed guide directly below the third trochanter, where the shaft diameter becomes consistent. Hold the proximal and distal ends with one hand.
Firmly press the femur onto the workbench, and using the other hand, place the 3D-printed guide on the midshaft of the femur to prevent the femur from rotating to the lateral or medial side. Then, place them in front of the corresponding aluminum segments. Fill the aluminum segments with bone cement until almost full, leaving a little room for displacement.
Place the femurs with guides in the correct aluminum segment and allow the bone cement to set. After the bone cement has hardened, place the samples in a Petri dish with room temperature PBS and allow them to rehydrate for two hours. Use a mechanical testing system to attach and calibrate a load cell with the resolution less than 1 Newton.
Attach a fixture with a square slot to firmly hold the aluminum segments with the samples. Attach set screws to the two sides of the holding fixture to firmly hold samples in place. Then, attach a loading platen into the actuator.
Place a stereo microscope on a surface directly in front of the MTS. Place light sources around the system if additional lighting is needed to see the setup through the microscope. In the MTS software, begin the creation of a new flexural protocol.
Ensure that the protocol will operate in displacement control. Set the loading rate of the protocol to 0.5 millimeters per second. If the software has a setting for soft keys, add the soft keys Balance and Zero Extension to the protocol.
Ensure that the software program will record the time in seconds, load in Newtons, and extension or displacement in millimeters at a minimum sampling rate of 100 hertz. Save the new protocol and return to the main screen of the software program to begin the testing of a new sample set. Obtain an X-ray image of the samples in the aluminum pots.
Multiple samples can be imaged at once. Ensure that the anterior view of samples is captured to allow for verification measurements of potting angle. Place an aluminum segment with sample into the holding fixture and tighten the set screws.
Lower the actuator until it is within a few millimeters of the femoral head. Use the stereo microscope to adjust the biaxial stage to align the position of the femoral head directly underneath the loading platen. Lock the biaxial stage in place.
In the MTS software, zero the position of the actuator and balance the load cell using the added soft keys Balance and Zero Extension. Then, begin the loading protocol. Depending on how much space was left between the loading platen and the sample, testing will only take 10 to 30 seconds.
After testing, capture another anterior X-ray of the sample. This will be used to discern and document the mode of fracture. The coefficients of variation, or COV, from measured flexural properties of mouse femoral necks are presented here.
COV represents the ratio of the standard deviation and mean of a data set, and its decrease indicates a tighter grouping of the individual data points around the mean. This protocol decreased the COV for maximum load compared to other publications performing similar testing. A representative force displacement curve displaying a 0.2%offset linear fit is used to derive the stiffness and yield point.
Selected outcome measures are plotted, displaying mean and standard deviation, including maximum load at failure, stiffness, maximum displacement at failure, and and work to failure. Asterisks indicate significant differences determined using a one-tailed unpaired t-test. Femoral necks for male mice were significantly stronger and stiffer than specimens from female mice.
In addition, the female femoral necks experienced more significant deformations and work to failure compared to specimens from male mice. This is consistent with the lower bone mineral density in females and underscores the sensitivity of the test to detect physiologically-relevant differences. Biomechanical outcome measures, including maximum load, stiffness, maximum displacement, and work to failure, were plotted against the potting angle and correlated using a simple linear regression for the male cohort, female cohort, and all samples grouped together.
Solid black lines show linear regression of group samples, and dotted lines indicate confidence intervals. Variability in the potting angle did not significantly affect the maximum load, maximum displacement, or failure work. However, as the potting angle increased, the stiffness increased.
The most important thing to remember while attempting this protocol is to keep the potting procedure and loading rates the same to better compare your results to previously published data. If using an alternative to bone cement, such as a bismuth alloy that can be softened to remove the sample after testing, samples can undergo other procedures, such as Raman spectroscopy, DEXA, or three-point bending, to elucidate additional mechanical, chemical, and structural properties.
