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><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

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。

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

作者聚焦：提高全骨弯曲测试的准确性和可重复性 

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。

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

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

741 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。

视频: 作者聚焦：提高全骨弯曲测试的准确性和可重复性 

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.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

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

科研

生物工程

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.

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用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, ...

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.

组织工程生物反应器的双轴机械装载设计

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.

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

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.

鼠头的生物力学试验

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.

鼠股颈悬臂弯曲

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.

用膝关节关节仪评价大鼠膝关节屈曲收缩的特定组织贡献

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 ...

医学

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.

鼠类闭合稳定骨折的断裂装置设计与协议优化

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.

开发Wistar白化大鼠股骨截骨模型的协议

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

用于测定机械强度的小鼠椎骨压缩试验

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.

作者聚焦:增强小动物骨压缩检测用于研究

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.

嵌入加载面的小鼠腰椎单轴压缩测试

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