Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

The current experimental challenges of bending tests include consistent placing of the bones in the same orientation, documenting the break progress of the bone during the test, and interpretation of complex results. Our protocol provides a guide not only for the testing procedure and data analysis for whole bone bending tests, but also for sample preparation and storage. And this is the first instance where the entire protocol has been presented.

Whole bone mechanical testing is a quick and simple method to obtain measures of bone fracture susceptibility when compared to other testing methods. After harvesting the femur and tibia from the euthanized mouse, obtain a 6-10 micrometer resolution Micro CT scan reconstructed for each sample to calculate fracture size geometry. Thaw all the bones for testing, and ensure to test all bones from the same experiment in one session.

Now locate a load cell with sensitivity and capacity suitable for the specimen. Considering the failure range of the specimen, select a load cell with 50%more capacity while maximizing sensitivity. Locate the loading and support span fixtures.

Install the load cell and fixtures by screwing the load cell onto the upper support of the tester, attaching the upper loading fixture to the load cell and mounting the lower fixture to the bottom support of the tester. Then select a support span length which will remain constant for all samples. Use the shortest bone in the sample set to determine the support span distance, and properly orient the bone between the fixtures.

To perform a three-point bending test of the femur, place the anterior surface of the bone against the support span, ensuring the position is within the diaphysis of the sample. Exclude the third trochanter on the proximal end and the transition point where the bone widens into the metaphysis and condyles on the distal end. Ensure the loading span is centered within the support span and contains a uniform region of the bone.

Then measure and record the support span distance. Once the support span has been set and recorded, bring the lower fixture close to the upper fixture with enough room between the two for a bone to be placed. Place the bone back into saline or rehydrated with a saline bolus.

Ensure all fixture parts are tight and free of movement. In the mechanical tester software, design a bending test profile with a slow displacement ramp rate to avoid viscoelastic effects to load the bone until it fails. After creating a testing profile, create distinct folders for every study group.

Select a properly thawed bone, then measure and record its full length using calipers. Load the bone sample onto the fixture in the proper orientation. Alter the file name to reflect the particulars of the sample being tested, and save each test in its corresponding folder.

Zero the load and activate the system's mover, ensuring it's not in load or displacement control. Carefully apply a slight preload to the bone to secure its position. Avoid compromising the sample.

Aim for a preload of about 0.25 newtons. Make sure the desired bone orientation is kept consistent before moving forward. Hydrate the sample by generously dowsing it with saline.

On the software, initiate the bending test by selecting Start or Run. It's crucial to observe the sample for the duration of the test accurately, noting down any occurrences of issues. Monitor closely as the bone starts to fracture.

Tests stop automatically when they reach their set limits. But if there's a failure, stop the test manually to avoid damaging the load cell. Once testing is complete, measure the length from the distal end to the break point using calipers and record it.