The work done to develop this protocol has been supported by the National Institutes of Health [AR072609].

All procedures described throughout this protocol that involved animals have been approved by the Indiana University School of Science Institutional Animal and Use Committee (IACUC) prior to the procedure. Animals described in the procedure were euthanized via CO2inhalation followed by cervical dislocation as a secondary means of euthanasia.
1. Harvest, storage, and thawing of bones
<ol>
	<li>Harvest and storage
	<ol>
		<li>Place the mouse ventral side up. Use a s...

Principles and Protocols of Mechanical Testing for Bone Fracture Susceptibility

<ol>
	<li>Vesper, E. O., Hammond, M. A., Allen, M. R., Wallace, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Even+with+rehydration,+preservation+in+ethanol+influences+the+mechanical+properties+of+bone+and+how+bone+responds+to+experimental+manipulation.">Even with rehydration, preservation in ethanol influences the mechanical properties of bone and how bone responds to experimental manipulation.</a> Bone. 97, 49-53 (2017).</li><li>Jepsen, K. J., Silva, M. J., Vashishth, D., Guo, X. E., van der Meulen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+biomechanical+mechanisms+in+mouse+models:+practical+guidelines+for+systematically+evaluating+phenotypic+changes+in+the+diaphyses+of+long+bones.">Establishing biomechanical mechanisms in mouse models: practical guidelines for systematically evaluating phenotypic changes in the diaphyses of long bones.</a> Journal of Bone and Mineral Research. 30 (6), 951-966 (2015).</li><li>Eds Burr, D. B., Allen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic and Applied Bone Biology. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> microCT SkyScan 1272 User Manual. , (2018).</li><li>Kim, Y., Brodt, M. D., Tang, S. Y., Silva, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+scanning+and+analysis+of+mouse+bones.">MicroCT for scanning and analysis of mouse bones.</a> Methods in Molecular Biology. 2230, 169-198 (2021).</li><li>Bouxsein, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> Journal of Bone and Mineral Research. 25 (7), 1468-1486 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Micro-CT specimen scanner, Centre for high-throughput phenogenomics. , (2023).</li><li>ASTM International. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard test method for flexural properties of unreinforced and reinforced plastics and electrical insulating materials by four-point bending. , (2020).</li><li>ASTM International. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard test methods for flexural properties of unreinforced and reinforced plastics and electrical insulating materials. , (2017).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruker+microCT+NRecon:+An+overview.">Bruker microCT NRecon: An overview.</a> , (2023).</li></ol>

Throughout the scanning and testing process, there are moments when troubleshooting and optimization are appropriate. The first of these occurs when scanning bones using &#181;CT. While many systems come with a holder in which one object may be held and scanned, custom holders can be fabricated to scan multiple bones at the same time. Scanning multiple bones can be an excellent point for optimization, but caution should be taken throughout the scanning and analysis process to ensure artifacts are not being induced. As X-...

Mechanical testing of bone is the primary method to extract functional information related to a bone&#39;s susceptibility to fracture. In preclinical studies, several testing modalities can be used but by far the most common is the bending of long bones. These tests are easy to perform and can be used on bones ranging in size from human to mouse. As mice are one of the most commonly studied animals in preclinical research, this protocol will focus on bending tests performed on the femora and tibiae of mice.
Prior to performing bending tests, bones must be properly harvested and stored. The most common storage methods have traditionally been freezing bones in saline-soaked gauze, freezing in saline alone, or dehydrating bones in ethanol 1. Bones stored in ethanol have been shown to have increased stiffness and elastic modulus and decreased deformation parameters versus those stored frozen1. Even rehydrating the bones prior to testing does not recover these properties back to normal levels 1. Storing submerged in saline could cause damage to the bone since pressure is exerted as the saline expands. In addition, a complete thaw of the solution would be required to remove the bones for microcomputed tomography (&#181;CT) scanning. Consequently, freezing freshly harvested bones in saline-soaked gauze has become the standard storage method and is recommended throughout this protocol.
Because the size and shape of a bone affect its bulk strength and many disease models significantly alter bone size and morphology, engineering principles are used to normalize away the effects of size to produce properties that estimate the behavior of the tissue2. This approach requires cross-sectional geometry of the failure location, which is most commonly acquired using &#181;CT to create scans of the bones prior to testing. &#181;CT is widely used due to its availability and high image resolution. Moreover, contributions of soft tissue are not included, and scanning does not require chemical fixation or other modifications to the bone3,4. In all forms of CT, an X-ray source is focused on an object while a detector on the other side of the object measures the resulting X-ray energy. This produces an X-ray shadow of the sample that can be converted into an image3,5. The object being scanned is rotated (or the X-ray source and detector are rotated around the sample), generating images that can be reconstructed into a three-dimensional data set representing the object5.
Scan resolution, or how close together two objects can be and still be resolved individually, is controlled by changing the nominal voxel size or the size of a pixel in the resulting image. It is generally accepted that objects must be at least two times the size of a single voxel to be identified3, but a higher ratio will allow for improved precision. Further, larger voxels are more prone to partial volume effects: when a single voxel contains tissues of varying densities, it is assigned the average of these densities, rather than the specific density of a single tissue, which may lead to an over- or under-estimation of tissue areas and mineral density3. While these issues can be mitigated by choosing smaller voxel sizes, using a higher resolution does not ensure the elimination of partial volume effects and may require longer scan times3. When scanning bones ex vivo, a voxel size of 6-10 &#181;m is generally recommended to accurately assess the trabecular architecture of mouse bones. A larger voxel size of 10-17 &#181;m can be used for cortical bone, although the smallest reasonable voxel size should be used. This protocol uses a 10 &#181;m voxel size, which is small enough to differentiate key trabecular properties and minimize partial volume effects without extensive scan time.
X-ray energy and energy filter settings must also be selected carefully, as the high mineral density and thickness of bone tissue greatly attenuates and alters the transmitted X-ray energy spectrum. It is generally assumed that because the emitted X-ray spectrum is equivalent to the spectrum that exits the object6, using low-energy X-rays on dense objects such as bone can lead to an artifact known as beam hardening7. A higher voltage of 50-70 kVp is recommended when scanning bone samples to reduce the incidence of these artifacts5. In addition, inserting an aluminum or copper energy filter creates a more concentrated energy beam, further minimizing artifacts4,7. A 0.5 mm aluminum filter will be used throughout this protocol.
Finally, the scan rotation step and rotation length (e.g., 180&#176;-360&#176;), together control the number of images captured, which determines the amount of noise in the final scan4. Averaging multiple frames in each step can reduce noise but may increase scan time4. This protocol uses a rotation step of 0.7 degrees and a frame averaging of 2.
One final note about scanning: hydroxyapatite calibration phantoms should be scanned using the same scan settings as the experimental bones to enable the conversion of attenuation coefficients to mineral density in g/cm35. This protocol uses phantoms of 0.25 g/cm3 and 0.75 g/cm3 of hydroxyapatite, although different phantoms are available. Note that some scanning systems use internal phantoms as part of daily system calibration.
Once scanning is complete, the angular projections are reconstructed into cross-sectional images of the object, typically using the manufacturer&#39;s accompanying software. Whatever system is used, it is important to ensure that the whole bone is captured in the reconstruction and that thresholding is set appropriately to allow for the recognition of bone versus non-bone. After reconstruction, it is critical to rotate all scans in three dimensions so that bones are oriented consistently and properly aligned with the transverse axis, again using the manufacturer&#39;s software.
Following rotation, regions of interest (ROI) for analysis may be selected based on whether cortical properties, trabecular properties, or fracture geometry for mechanical normalization are desired. For the latter, ROIs should be selected after testing by measuring the distance from the fracture site to one end of the bone and using voxel size to determine the corresponding slice location in the scan file. The selected region should be at least 100 &#181;m in length, with the fracture point at the approximate center of the ROI, to provide adequate estimation4.
With ROIs selected, two properties are needed for mechanical normalization (to calculate bending stress and strain): the maximum distance from the neutral bending axis to the surface where failure is initiated (assumed to be the surface loaded in tension, determined by the testing setup), and the area moment of inertia around the neutral axis, (also dependent on testing setup). This protocol recommends the use of a custom code to determine these values. For access to the code, contact the corresponding author directly or visit the lab website at&#160;https://bbml.et.iupui.edu/ for more information.
Once &#181;CT scanning has been completed, mechanical testing can begin. Bending tests can be performed in either four-point or three-point configurations. Four-point bending tests are preferred as they eliminate shear stress in the bone between loading points, allowing for pure bending to occur in this region3. The bone will then fracture due to tension, creating a failure that is more representative of the true bending properties of the bone3. However, the bone must be loaded in such a way as to deliver the same load at both loading points (this can be facilitated with a pivoting loading head). In three-point bending tests, there is a large change in shear stress where the load point meets the bone, which causes the bone to break at this point due to shear, not tension3. ASTM standards recommend materials undergoing bending should have a length-to-width ratio of 16:1, meaning the length of the support span should be 16 times larger than the width of the bone to minimize impacts of shear8,9. This is often impossible to achieve when testing small rodent bones, so the loading span is simply made as large as possible but with as small of a change in cross-sectional shape as possible. Moreover, when performing four-point bending, the ratio between the lengths of the lower and upper span should be ~3:18, which can usually be achieved in the tibia, but it is difficult in the shorter femur. In addition, the thinner cortical walls of femurs make them susceptible to ring-type deformation which changes the shape of the bone cross-section during the test (this can be accentuated in four-point tests as a greater force is required to induce the same bending moment compared to three-point bending). Therefore, three-point bending will be utilized for mouse femora while four-point bending will be used for tibiae throughout this protocol.
Finally, it is important to properly power the study for statistical analysis. A general recommendation for mechanical testing is to have a sample size of 10-12 bones per experimental group to be able to detect differences, as some mechanical properties, especially postyield parameters, can be highly variable. In some cases, this may mean starting with a higher animal sample size given attrition that could occur during the study. Sample size analysis using existing data should be completed prior to attempting a study.
There are numerous limitations and assumptions, but bending tests can provide quite accurate results, especially when relative differences between groups are of interest. These properties, together with the analysis of trabecular architecture and cortical morphology, can provide better insight into disease states and treatment regimens. If care is taken with those aspects of the experiment that are in our control (e.g., harvesting, storing, scanning, and testing), we can feel confident that accurate results have been generated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CTAn</td>
			<td>Bruker</td>
			<td>NA</td>
			<td>CT Scan Analysis Software</td>
		</tr>
		<tr>
			<td>DataViewer</td>
			<td>Bruker</td>
			<td>NA</td>
			<td>CT Scan Rotation Software</td>
		</tr>
		<tr>
			<td>Matrix Laboratory (MATLAB) 2023a</td>
			<td>MathWorks</td>
			<td>NA</td>
			<td>Coding platform used for data analysis</td>
		</tr>
		<tr>
			<td>NRecon</td>
			<td>Bruker</td>
			<td>NA</td>
			<td>CT Scan Reconstruction software</td>
		</tr>
		<tr>
			<td>SKYSCAN 1272-100 kV w/ 16 MP CCD detector, incl 3D Suite Software&#160;&#160;</td>
			<td>Micro Photonics Inc</td>
			<td>SKY-016814</td>
			<td>Micro-CT system that can non-destructively visualize up to 209 mPs in every virtual slice through an object&#160;</td>
		</tr>
	</tbody>
</table>

practical considerations for the design, execution, and interpretation of studies involving whole-bone bending tests of rodent bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

Author Spotlight: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests 

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.

Upon completion of CT scanning, most inadequate scans can be caught in reconstruction. Often, poor scans will have a high misalignment compensation that is a clear indicator of an error during the scan. However, errors may occur in other steps and could also lead to inaccurate data. These errors can often be spotted as the individual calculated architectural properties are examined. If values fall far out of the range of others in a group, the scan, ROI, and method of calculating the properties should be re-examined....

Watch this Scientific Journal Video about Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests at JoVE.com

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

practical-considerations-for-design-execution-interpretation-studies