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

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>

The authors have no conflicts of interest to declare.

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-rays pass through (and are attenuated by) varying amounts of bone tissue at each angular increment, this can lead to inaccuracy in the resulting data.
A second point for troubleshooting occurs in the reconstruction of scans. Depending on the program used, the user may define the density window for reconstruction, calculated from the attenuation coefficient values of the scan. This is a linear coefficient used to represent how much the X-ray beam is attenuated by an object5. Some programs obtain these densities and convert them to grayscale values ranging from 0 to 255 in a histogram. Two values will be shown in a histogram and are known as contrast limits10, which must be set appropriately to allow for recognition of bone versus non-bone voxels. The lower value will normally be set to a grey scale value of zero while the higher contrast value is recommended to be set to 10-20% of the maximum attenuation of the material of interest (bone)10. Errors may occur in analysis if this value is not set appropriately as portions of data could be cut off. Consequently, this value needs to be adjusted depending on the sample that was scanned. The best practice is to view the histogram on a logarithmic scale and select the upper limit as a number that is slightly larger than the end of the logarithmic tail to ensure all bone data are included10.
Additional points for troubleshooting occur during mechanical testing and analysis. During testing and viewing force-displacement curves after the test, samples that rolled may be spotted and should be removed from the dataset as discussed in the representative results. Moreover, caution should be taken regarding the orientation of the bone as there are assumptions governing the shape of the bone being tested. When using Euler-Bernoulli bending equations to calculate stress and strain, it is assumed the sample has a uniform cross section along its length3. Given that most bones do not have a uniform cross section, it is best to select the most uniform region of the bone to test (across the entire support span for three-point bending or between the loading points for four-point bending).
In a femur, testing in three-point bending at the midshaft is preferred. Because of the gentle curvature of the bone, one should test in the direction of the curvature to prevent local surface buckling (i.e., testing the femur with the anterior surface in tension). The tibia has a more variable cross-sectional shape, so the ideal region to test begins just proximal to the junction of the tibia and fibula. If the bone is placed with the medial surface in tension, the region of bone being tested is flat and has the least variability in radius and moment of inertia in the direction of bending. Caution should also be taken when interpreting the results of the bending tests due to the assumption that the material is isotropic, homogenous, and linearly elastic&#160;&#8211;&#160;each of these assumptions is violated to some degree when testing bone3. Bone&#39;s inability to fit these assumptions leads to results from bending tests that must be interpreted with caution. The properties that must be interpreted most carefully are those derived from the stress-strain curve past the yield point since by definition, passing yield violates the linear elastic assumption. While normalization to the bone&#39;s shape may be done, it is not recommended to attempt to normalize to animal body weight unless there are gross differences across groups. In this case, an analysis of covariance may be performed to compensate for these differences but general normalization to body weight should be avoided in most cases.
Despite the troubleshooting issues that may occur throughout this process, bending tests yield mechanical properties that can describe a bone&#39;s susceptibility to fracture. These tests are also relatively simple and quick to perform. Although absolute values from these tests might not always be completely valid, being able to detect relative differences between groups can be quite accurate for samples of various sizes and shapes. The mechanical properties obtained provide functional relevance in studies where differences in bone might be expected. Although monotonic failure tests are the most common and easily accessible mechanical test, other methods including fatigue life and fracture toughness may reveal additional mechanical properties of interest and could be considered.

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><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&amp;nbsp;&amp;nbsp;</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&amp;nbsp;</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.

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.
Once bending tests are complete, the force-displacement plots from each test should be examined to identify poor tests that may need to be removed from the dataset. An example of a problematic test is shown in Figure 4. The plot in Figure 4A shows the results of a correctly performed bending test. There is a clear linear region lacking a low-slope toe-in, a yield point, an ultimate point (maximum force), a drop in force as displacement extends past the maximum force, and a failure point. The curve is smooth with no abrupt changes in loading until after the ultimate point has been reached. Consequently, properties from this test may be easily identified and trusted. The plot shown in Figure 4B shows the result of a bending test with multiple concerning features. The abrupt changes in loading and the appearance of multiple peaks in the plot are major indicators of problems with this test. While minor peaks may occur in a proper test near the ultimate force, the magnitude and number of peaks in this plot suggest the bone may have rolled during the test. Whether observed and noted during the test or when examining the tests prior to analysis, the sample data should be investigated during post-testing analysis. If the data are indeed faulty or far outside of the range of other samples in the group, it would be ideal to not include this test in the final dataset. This is one reason for properly powering the experiment with priori power calculations. It might be possible to report only certain properties from a sample (in this case, preyield properties could be acceptable), but this is not ideal and should be clearly explained when reported.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65616/65616fig04.jpg" /> 
Figure 4: Force-displacement plots. (A) Ideal force-displacement plot. (B) Force-displacement plot resulting from a poor bending test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65616/65616fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
With all force-displacement plots examined and values normalized to stress-strain, the properties of interest may be identified and showcased in multiple ways. In Figure 5, resulting force-displacement and stress-strain plots for an entire study are shown. These are schematic representations whereby the force and displacement at the starting point (0,0), yield, ultimate point, and failure for each bone are determined and then, the force/stress and displacement/strain are averaged to provide an average plot for each group. The plots are not used for statistical analysis, but they can be used to showcase how overall behavior varies due to factors such as a treatment or disease state. The plots shown in Figure 5&#160;are from a study comparing control mice to those induced with a state of type 2 diabetes and chronic kidney disease (T2D-CKD). The right tibiae of these animals were tested to failure using four-point bending and analyzed to obtain the properties discussed in protocol section 8. From Figure 5, it is clear the T2D-CKD group had reduced mechanical properties, including strength and stiffness, at both the structural and tissue levels. These mice also appear to have reduced postyield properties, an indicator of brittleness. These plots should not be used to draw definitive conclusions from a study. Rather, they act as a visual representation and should be verified by performing a statistical analysis on all properties of interest.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65616/65616fig05.jpg" /> 
Figure 5: Force-displacement and stress-strain plots for an entire study. (A)&#160;Force-displacement plot for control animals and type 2 diabetes- and chronic kidney disease-induced animals. This plot resulted by averaging the yield force, displacement to yield, ultimate force, ultimate displacement, failure force, and total displacement for each group and plotting these means along with standard deviation. (B) Stress-strain for control animals and T2D-CKD animals. This plot resulted from averaging yield stress, strain to yield, ultimate stress, ultimate strain, failure stress, and total strain and plotting the resulting means along with standard deviation. Abbreviation: T2D-CKD = type 2 diabetes- and chronic kidney disease-induced animals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65616/65616fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The mechanical data and results from a two-tailed t-test are shown in Table I. Data are presented as mean &#177; standard deviation. A general examination suggests data within proper ranges and with expected levels of variability. Note that postyield properties tend to have the greatest variation and therefore, often require the largest sample sizes to detect meaningful differences. As the schematic curves in Figure 5&#160;suggested, there are significant decreases in almost all structural and tissue-level mechanical properties. From these data, it can be concluded that the induced disease state led to bones that are weaker, less stiff, and more susceptible to fracture due to loss of deformability and reduced toughness. Studies with more nuanced comparisons may not be as simple to interpret. An example of this may be if significant improvements are observed across structural-level mechanical properties but not tissue-level mechanical properties. In this case, the effects observed are likely driven by changes in the architecture of the bone (e.g., increased area, increased cortical thickness) rather than improvements in bone quality at the tissue level. For example, bone area increased due to the gain of woven bone, but tissue quality has decreased as an unorganized woven bone is now present rather than an organized lamellar bone. This could be supported by &#181;CT analysis in which statistically significant improvements in architecture may be observed. Contrastingly, there may be significant improvements in tissue-level mechanical properties with minimal/no improvements in structural-level mechanical properties. This high tissue quality could mask the pitfalls of smaller bones. Data interpretation may become further convoluted if changes are observed in preyield properties but not in postyield properties or vice versa. In the former case, a change in the bone&#39;s ability to resist deformation may be improved while its ability to tolerate damage is not. In each of these cases, the ability to reference architectural properties from &#181;CT analysis is highly beneficial and should be utilized (although describing that method is beyond the scope of this paper). Due to the complexity of the interpretation of these properties, presenting all properties in table or figure form (not just those properties that tend to be the easiest to interpret such as ultimate force, or that tell the story one is looking to tell) allows for a more complete representation of the mechanical impacts.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3"></td>
			<td>Control</td>
			<td>T2D-CKD</td>
			<td>P Value</td>
		</tr>
		<tr>
			<td colspan="3">Yield Force (N)</td>
			<td>19.7 &#177; 2.9</td>
			<td>15.2 &#177; 2.6</td>
			<td>0.0032**</td>
		</tr>
		<tr>
			<td colspan="3">Ultimate Force (N)</td>
			<td>22.8 &#177; 3</td>
			<td>17.6 &#177; 3.4</td>
			<td>0.0031**</td>
		</tr>
		<tr>
			<td colspan="3">Displacement to Yield (&#181;m)</td>
			<td>205 &#177; 17</td>
			<td>190 &#177; 21</td>
			<td>0.1039</td>
		</tr>
		<tr>
			<td colspan="3">Postyield Displacement (&#181;m)</td>
			<td>246 &#177; 235</td>
			<td>60 &#177; 51</td>
			<td>0.0435*</td>
		</tr>
		<tr>
			<td colspan="3">Total Displacment (&#181;m)</td>
			<td>451 &#177; 230</td>
			<td>249 &#177; 53</td>
			<td>0.0278*</td>
		</tr>
		<tr>
			<td colspan="3">Stiffness (N/mm)</td>
			<td>110 &#177; 10</td>
			<td>91 &#177; 13</td>
			<td>0.0037**</td>
		</tr>
		<tr>
			<td colspan="3">Work to Yield (mJ)</td>
			<td>2.16 &#177; 0.45</td>
			<td>1.54 &#177; 0.36</td>
			<td>0.0055**</td>
		</tr>
		<tr>
			<td colspan="3">Postyield Work (mJ)</td>
			<td>4.24 &#177; 3.01</td>
			<td>1.04 &#177; 0.9</td>
			<td>0.0109*</td>
		</tr>
		<tr>
			<td colspan="3">Total Work (mJ)</td>
			<td>6.4 &#177; 2.88</td>
			<td>2.58 &#177; 0.97</td>
			<td>0.0025**</td>
		</tr>
		<tr>
			<td colspan="3">Yield Stress (MPa)</td>
			<td>180 &#177; 20</td>
			<td>157 &#177; 25</td>
			<td>0.0504</td>
		</tr>
		<tr>
			<td colspan="3">Ultimate Stress (MPa)</td>
			<td>209 &#177; 26</td>
			<td>181 &#177; 27</td>
			<td>0.0434*</td>
		</tr>
		<tr>
			<td colspan="3">Strain to Yield (m&#603;)</td>
			<td>16.8 &#177; 2</td>
			<td>16.4 &#177; 1.5</td>
			<td>0.5771</td>
		</tr>
		<tr>
			<td colspan="3">Total Strain (m&#603;)</td>
			<td>36.6 &#177; 17.2</td>
			<td>21.5 &#177; 4.3</td>
			<td>0.0277*</td>
		</tr>
		<tr>
			<td colspan="3">Modulus (GPa)</td>
			<td>12.2 &#177; 1.1</td>
			<td>10.9 &#177; 1.1</td>
			<td>0.0171*</td>
		</tr>
		<tr>
			<td colspan="3">Resilience (MPa)</td>
			<td>1.62 &#177; 0.33</td>
			<td>1.38 &#177; 0.33</td>
			<td>0.1377</td>
		</tr>
		<tr>
			<td colspan="3">Toughness (MPa)</td>
			<td>4.85 &#177; 2.29</td>
			<td>2.26 &#177; 0.73</td>
			<td>0.0076**</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Results of mechanical testing and statistical analysis. Values shown as mean &#177; standard deviation. P values result from a two-tailed unpaired t-test. * P &#60; 0.05 and ** P &#60; 0.01. Abbreviation: T2D-CKD = type 2 diabetes- and chronic kidney disease-induced animals.

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

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

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.

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

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Video: Auteur in de schijnwerpers: verbetering van de nauwkeurigheid en reproduceerbaarheid bij buigtests voor het hele bot 

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Medicine

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Compression Testing of Mouse Vertebra for Determination of Mechanical Strength

Mouse Lumbar Vertebra Uniaxial Compression Testing with Embedding of the Loading Surface

There is increasing awareness that cortical and cancellous bone differ in regulating and responding to pharmaceutical therapies, hormone therapies, and other treatments for age-related bone loss. Three-point bending is a common method used to assess the influence of a treatment on the mid-diaphysis region of long bones, which is rich in cortical bone. Uniaxial compression testing of mouse vertebrae, though capable of assessing bones rich in cancellous bone, is less commonly performed due to technical challenges. Even less commonly performed is the pairing of three-point bending and compression testing to determine how a treatment may influence a long bone's mid-diaphysis region and a vertebral centrum similarly or differently. Here, we describe two procedures to make compression testing of mouse lumbar vertebrae a less challenging method to perform in parallel with three-point bending: first, a procedure to convert a three-point bending machine into a compression testing machine, and second, an embedding method for preparing a mouse lumbar vertebra loading surface.

Author Spotlight: Enhancing Small Animal Bone Compression Testing for Research

In this protocol, two approaches are described to make uniaxial compression testing of mouse lumbar vertebrae more attainable. First, the conversion of a three-point bending machine to a compression testing machine is described. Second, an embedding method for preparing the loading surface that uses bone cement is adapted for mouse lumbar vertebrae.

There is increasing awareness that cortical and cancellous bone differ in regulating and responding to pharmaceutical therapies, hormone therapies, and ...

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

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