美国国立卫生研究院（National Institutes of Health）[AR072609]支持制定该协议的工作。

骨折易感性机械测试的原则和方案

<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>

作者没有利益冲突需要声明。

在整个扫描和测试过程中，有时需要进行故障排除和优化。第一种发生在使用μCT扫描骨骼时。虽然许多系统都带有一个支架，可以在其中固定和扫描一个物体，但可以制造定制支架来同时扫描多个骨骼。扫描多块骨骼可能是一个很好的优化点，但在整个扫描和分析过程中应谨慎行事，以确保不会诱发伪影。当 X 射线以每个角度增量穿过（并被衰减）不同数量的骨组织时，这可能导致结果数据不准...

骨骼的机械测试是提取与骨骼骨折易感性相关的功能信息的主要方法。在临床前研究中，可以使用几种测试方式，但到目前为止，最常见的是长骨的弯曲。这些测试易于执行，可用于从人类到小鼠大小不等的骨骼。由于小鼠是临床前研究中最常研究的动物之一，因此该协议将侧重于对小鼠的股骨和胫骨进行的弯曲测试。
在进行弯曲测试之前，必须正确采集和储存骨头。传统上，最常见的储存方法是在盐水浸泡的纱布中冷冻骨头，单独在盐水中冷冻，或在乙醇中脱水骨头 1。与冷冻储存的骨头相比，储存在乙醇中的骨头已被证明具有更高的刚度和弹性模量，并降低了变形参数1。即使在测试前对骨骼进行补水也无法将这些特性恢复到正常水平 1.浸没在盐水中储存可能会对骨骼造成损伤，因为随着盐水的膨胀而施加压力。此外，需要完全解冻溶液才能取出骨头进行显微计算机断层扫描 （μCT） 扫描。因此，将新鲜收获的骨头冷冻在盐水浸泡的纱布中已成为标准储存方法，并在整个协议中推荐。
由于骨骼的大小和形状会影响其体积强度，并且许多疾病模型会显着改变骨骼的大小和形态，因此使用工程原理来规范化大小的影响，以产生估计组织行为的属性2.这种方法需要故障位置的横截面几何形状，最常使用μCT获取，以便在测试之前对骨骼进行扫描。μCT因其可用性和高图像分辨率而被广泛使用。此外，不包括软组织的贡献，扫描不需要对骨骼进行化学固定或其他修饰 3,4。在所有形式的 CT 中，X 射线源聚焦在物体上，而物体另一侧的探测器测量产生的 X 射线能量。这会产生样品的 X 射线阴影，可以转换为图像 3,5。被扫描的物体被旋转（或X射线源和探测器围绕样品旋转），产生图像，这些图像可以重建为代表该物体的三维数据集5。
扫描分辨率，即两个物体可以单独分辨的距离，以及仍然单独分辨的距离，是通过更改标称体素大小或生成图像中像素的大小来控制的。人们普遍认为，物体的大小必须至少是单个体素的两倍才能被识别3，但更高的比率将允许提高精度。此外，较大的体素更容易产生部分体积效应：当单个体素包含不同密度的组织时，它被分配了这些密度的平均值，而不是单个组织的特定密度，这可能导致高估或低估组织面积和矿物质密度3.虽然这些问题可以通过选择较小的体素尺寸来缓解，但使用更高的分辨率并不能确保消除部分体积效应，并且可能需要更长的扫描时间3.当体外扫描骨骼时，通常建议使用6-10μm的 体素大小，以准确评估小鼠骨骼的小梁结构。皮质骨可以使用较大的 10-17 μm 体素尺寸，但应使用最小的合理体素尺寸。该协议使用 10 μm 体素尺寸，该尺寸足够小，可以区分关键的小梁特性，并在没有大量扫描时间的情况下最大限度地减少部分体积效应。
还必须仔细选择 X 射线能量和能量滤光片设置，因为骨组织的高矿物质密度和厚度会大大衰减和改变透射的 X 射线能量谱。通常认为，由于发射的X射线光谱等同于离开物体6的光谱，因此在致密物体（如骨骼）上使用低能X射线会导致称为光束硬化7的伪影。扫描骨骼样本时，建议使用 50-70 kVp 的更高电压，以减少这些伪影的发生率5.此外，插入铝或铜能量滤波器可产生更集中的能量束，进一步减少伪影 4,7。在整个协议中将使用 0.5 毫米铝过滤器。
最后，扫描旋转步长和旋转长度（例如，180°-360°）共同控制捕获的图像数量，这决定了最终扫描中的噪声量4。在每个步骤中对多个帧进行平均可以减少噪声，但可能会增加扫描时间4.该协议使用 0.7 度的旋转步长和 2 的帧平均值。
关于扫描的最后一点：羟基磷灰石校准模型应使用与实验骨骼相同的扫描设置进行扫描，以便将衰减系数转换为以 g/cm35 为单位的矿物密度。该协议使用0.25 g / cm 3和0.75g / cm3的羟基磷灰石的模型，尽管可以使用不同的模型。请注意，某些扫描系统使用内部模型作为日常系统校准的一部分。
扫描完成后，通常使用制造商随附的软件，将角度投影重建为物体的横截面图像。无论使用哪种系统，重要的是要确保在重建中捕获整个骨骼，并适当设置阈值以允许识别骨骼与非骨骼。重建后，必须再次使用制造商的软件，在三维空间中旋转所有扫描，以便骨骼的方向一致并与横轴正确对齐。
旋转后，可以根据是否需要皮质特性、小梁特性或断裂几何形状来选择用于分析的感兴趣区域 （ROI） 进行机械归一化。对于后者，应在测试后通过测量从骨折部位到骨骼一端的距离并使用体素大小来确定扫描文件中相应的切片位置来选择 ROI。所选区域的长度应至少为 100 μm，断裂点位于 ROI 的近似中心，以提供充分的估计4。
选择 ROI 后，机械归一化需要两个属性（用于计算弯曲应力和应变）：从中性弯曲轴到开始失效的表面的最大距离（假设是受拉加载的表面，由测试设置确定）和围绕中性轴的惯性矩（也取决于测试设置）。此协议建议使用自定义代码来确定这些值。如需访问代码，请直接联系通讯作者或访问实验室网站 https://bbml.et.iupui.edu/ 了解更多信息。
μCT扫描完成后，即可开始机械测试。弯曲测试可以在四点或三点配置中进行。四点弯曲测试是首选，因为它们消除了加载点之间骨骼中的剪切应力，允许在该区域发生纯弯曲3.然后，骨头会因张力而断裂，从而产生更能代表骨头真实弯曲特性的故障3.然而，骨头的加载方式必须能够在两个加载点提供相同的负载（这可以通过旋转加载头来促进）。在三点弯曲试验中，荷载点与骨骼相交处的剪切应力变化很大，这导致骨骼此时因剪切而不是张力而断裂3.ASTM标准建议进行弯曲的材料应具有16：1的长宽比，这意味着支撑跨度的长度应比骨的宽度大16倍，以尽量减少剪切的影响8,9。在测试小型啮齿动物骨骼时，这通常是不可能的，因此只需将加载跨度做得尽可能大，但横截面形状的变化尽可能小。而且，在进行四点弯曲时，下跨和上跨长度的比例应为~3：18，这在胫骨中通常可以达到，但在较短的股骨中则很难。此外，股骨皮质壁较薄，使其容易受到环形变形的影响，从而在测试过程中改变骨横截面的形状（这在四点测试中可以强调，因为与三点弯曲相比，需要更大的力来诱导相同的弯矩）。因此，在整个协议中，三点弯曲将用于小鼠股骨，而四点弯曲将用于胫骨。
最后，重要的是要为统计分析的研究提供适当的动力。机械测试的一般建议是每个实验组的样本量为 10-12 块骨头，以便能够检测差异，因为某些机械性能，尤其是后屈服参数，可能变化很大。在某些情况下，这可能意味着考虑到研究期间可能发生的损耗，从更高的动物样本量开始。在尝试研究之前，应使用现有数据完成样本量分析。
有许多限制和假设，但弯曲测试可以提供相当准确的结果，特别是当组之间的相对差异是感兴趣的时。这些特性，加上对小梁结构和皮质形态的分析，可以更好地了解疾病状态和治疗方案。如果仔细处理我们控制的实验方面（例如，收获、储存、扫描和测试），我们可以确信已经产生了准确的结果。

<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

导致骨折的骨骼脆性是美国的公共卫生危机，每年导致 150 万例骨折和 180 亿美元的直接护理费用。了解骨病的潜在机制和对治疗的反应的能力不仅是需要的，而且是至关重要的。骨骼的力学测试是了解和量化骨骼对骨折的易感性的宝贵技术。虽然这种方法看起来很容易执行，但如果用户忽略管理假设和关键步骤，可能会得出不适当和不准确的结论。随着研究继续发表，滥用方法和对结果的错误解释，跨学科都观察到了这一点。该协议将作为与机械测试相关的原理以及这些技术应用的入门书 - 从考虑样本量到组织收获和储存，再到数据分析和解释。有了这些，就可以获得有关骨骼骨折易感性的宝贵信息，从而进一步了解学术研究和临床解决方案。

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.

作者聚焦：提高全骨弯曲测试的准确性和可重复性 

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

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.

完成 CT 扫描后，大多数不充分的扫描都可以在重建中被发现。通常，糟糕的扫描将具有较高的错位补偿，这是扫描过程中错误的明确指示。但是，其他步骤中可能会出现错误，也可能导致数据不准确。在检查各个计算的体系结构属性时，通常可以发现这些错误。如果值远远超出组中其他值的范围，则应重新检查扫描、ROI 和计算属性的方法。
弯曲测试完成后，应检查每个测试...

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

啮齿动物骨骼的机械测试是提取有关骨骼对骨折的易感性信息的宝贵方法。由于缺乏适当的实践理解，结果可能会被过度解释或缺乏有效性。该协议将作为指南，以确保准确执行机械测试以提供有效和功能数据。

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 ...

目前弯曲测试的实验挑战包括：将骨骼一致地放置在相同的方向，记录测试过程中骨骼的断裂过程，以及解释复杂的结果。我们的方案不仅为全骨弯曲测试的测试程序和数据分析提供了指南，还为样品制备和储存提供了指南。这是第一个提出整个协议的实例。与其他检测方法相比，全骨力学检测是一种快速简便的方法来测量骨折易感性。

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

Research

JoVE Journal

Bioengineering

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

574 次观看.  Indiana University-Purdue University Indianapolis. 目前弯曲测试的实验挑战包括：将骨骼一致地放置在相同的方向，记录测试过程中骨骼的断裂过程，以及解释复杂的结果。我们的方案不仅为全骨弯曲测试的测试程序和数据分析提供了指南，还为样品制备和储存提供了指南。这是第一个提出整个协议的实例。与其他检测方法相比，全骨力学检测是一种快速简便的方法来测量骨折易感性。