Our research focuses on mechanical strength testing of small animal bones. Specifically, we aim to present an embedding method for compression testing of lumbar vertebrae. Applications for this research area include a better understanding of aging and dietary influences on vertebral bone mechanical properties.
Inherently, the small size of the vertebra is a challenge for compression testing, but the embedding method improves accessibility because the main preparation is to remove only the vertebral processes from the main body, but leaving other structures intact. The small size of mouse vertebra makes them challenging to prepare for compression testing. Our method provides a simpler means of preparing the loading surface for uniaxial compression testing.
Furthermore, we present the construction of compression testing machine from a three-point bending machine for easy conversion between the two. Our method allows researchers a simple means of pairing three-point bending and compression testing, which may allow researchers to better discern the effects of pharmacologic therapy, dietary dimension, or other treatments on a cortical bridge versus cancellous rich bone in a rodent model. We are exploring compression testing for other types of biological specimens.
For example, we are developing methods for testing mouse mandibles combined with other bone evaluation techniques. This will enable a more comprehensive approach to studying factors that influence bone properties such as aging and diet. To begin, unscrew the crosshead beam attached to the load sensor on the three-point bending machine.
Screw a self-aligning top platen onto the load sensor with threading identical to the crosshead beam. Turn on the machine at a constant lowering speed to begin compression testing. Using the threaded hex screws, secure the bottom platen to the two lower supports and tighten them until they are securely fastened.
Then to determine machine displacement, lower the top platen onto the bottom platen until light contact is made with no test material in between. Use digital data collection software to collect load and displacement measurements during the mechanical testing. Continue lowering the top platen onto the bottom platen at a constant speed until forces greater than that produced by all bone samples are reached.
Plot the data for system displacement versus applied load. Correct the recorded displacement for each data point from mouse vertebra samples obtained in subsequent steps. To begin, make a small incision on the dorsal midline of the mouse carcass near the base of the tail.
Extend the incision across each hind limb and gently pull to remove the pelt from the base of the tail toward the head of the animal. Cut the abdominal wall musculature away until the vertebral column becomes easily visible. Under a dissecting microscope, identify the two sacroiliac joints and locate the cranial end of the sacrum.
Using a razor blade or scalpel, make a precise cut to separate the last lumbar vertebra from the cranial end of the sacrum. Again, cut between the intervertebral space, remove L6 and L5 from the vertebral column and set aside L5 for analysis. Inspect the vertebra under a dissecting microscope and remove all soft tissues from the bone including the intervertebral disc using gauze pads and gently with forceps where necessary.
To begin, dissect the fifth lumbar vertebra isolated from the frozen mouse carcass. Using a diamond cutoff wheel attached to a rotary tool, cut each pedicle to remove the transverse and spinous processes. Gently sand the caudal end of the vertebra using fine 120-grit sandpaper to eliminate all intervertebral discs, soft tissue, and irregularities.
Then mark the sanded caudal end with a permanent marker for easy identification later. Prepare the PMMA bone cement according to the manufacturer's instructions. Apply a small amount of the semi-soft PMMA bone cement onto the upper side of the unmarked cranial end of the vertebra and rehydrate with saline.
While the PMMA is still semi-soft, position the vertebra on the bottom platen with the caudal side facing downwards. Now turn on the machine to engage the drive gears and gradually lower the top platen onto the vertebra and PMMA bone cement complex until it contacts the bone cement. Allow the PMMA bone cement to completely harden while the top platen gently presses down on it.
In real time, use digital software designed for data collection of mechanical testing to collect data for load and displacement from the sensors into a spreadsheet. After collecting baseline for five seconds, applied with a minimal preload force of less than 0.5 Newtons, gradually lower the top platinum onto the sample at a constant and predetermined lowering speed to initiate the compression test. Cease data collection once a significant reduction in load has been observed indicating material failure.
Correct each data point for system displacement using the equation obtained in previous steps. To create a load displacement curve with the load on the y-axis and the corrected specimen displacement on the x-axis, first create a table by clicking on Windows, then selecting New Table, followed by Do it. Copy the corrected displacement and load data from the raw data spreadsheet into the new table.
Then to generate a waveform representing the raw data, click Data and select XY Pair to Waveform. Select corrected displacement data for the X-Wave and load data for the Y-Wave. Ensure the correct data points are in the Number of Points box.
Name the waveform and click Make Waveform. After creating the waveform, visualize the load displacement curve by clicking on Windows and selecting New Graph. Place the waveform on the y-axis and calculate it on the x-axis to generate a graph.
Use the cursor tool to mark points or regions of interest on the graph for further analysis.
