Name: biomechanical testing of murine tendons
Uploaded: 2019-10-15T14:30:00
Description: Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

该研究得到了NIH/NIAMS（R01 AR055580，R01 AR057836）的支持。

动物研究得到了哥伦比亚大学机构动物护理和使用委员会的批准。本研究中使用的小鼠具有 C57BL/6J 背景，是从杰克逊实验室（美国梅港巴港）购买的。他们被安置在无病原体屏障条件下，并向他们提供食物和水。
1. 开发用于夹持骨的定制贴合 3D 打印夹具
<ol><li>骨骼图像采集与3D骨骼模型构造<ol><li>在准备 3D 模型创建和 3D 骨夹持打印时，解剖感兴趣的骨骼;腐殖质和卡卡内乌...

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鼠群模型通常用于研究肌腱疾病，但其机械特性的表征在文献中具有挑战性且罕见。该协议的目的是描述一种对肌腱进行拉伸测试的时间高效和可重复的方法。新方法将测试样本所需的时间从数小时缩短到几分钟，并消除了以前方法中常见的重大夹持伪影。
该协议中描述的几个步骤对于生产有效的夹具至关重要，机械测试鼠超脊柱和跟腱。首先，步骤 1.1.4 是创建所需骨骼的 ...

肌腱疾病在老化、运动和活跃人群1、2、3中很常见和高度流行。在美国，每年有1640万结缔组织损伤报告，占所有与伤害有关的医生办公室就诊人数的30%， 8.最常见的受影响的部位包括旋转手铐，跟腱，和骨腱9。虽然已经探索了各种非手术和手术治疗，包括消炎药、康复和手术修复，但效果仍然很差，恢复功能有限，失败率高5 6.这些不良的临床结果促使基础和翻译研究寻求了解肌腱病，并开发新的治疗方法。
拉伸生物力学特性是定义肌腱功能的主要定量结果。因此，肌腱病变和治疗效果的实验室表征必须包括对肌腱拉伸特性的严格测试。许多研究描述了从动物模型（如大鼠、绵羊、狗和兔子10、11、12）中确定肌腱的生物力学特性的方法。然而，很少有研究测试过肌腱的生物力学特性，这主要是因为难以抓住小组织进行拉伸测试。由于鼠模型在机械研究肌腱病方面有许多优点，包括基因操作、广泛的试剂选择和低成本，因此需要开发准确、高效的生物力学方法来检测鼠组织。
为了正确测试肌腱的机械特性，必须有效地抓住组织，而不会在抓斗界面处打滑或被工器撕裂或生长板破裂。在许多情况下，特别是对于短肌腱，骨头被抓住一端，肌腱抓住另一端。骨骼通常通过嵌入环氧树脂13和聚甲基丙烯酸酯14、15等材料来固定。肌腱通常放置在两层沙纸之间，用氰丙烯酸酯粘附，并使用压缩夹（如果横截面平坦）或冷冻介质（如果横截面较大）15、16、17 固定.这些方法已应用于生物力学试验鼠肌腱，但由于标本体积小，生长板的符合性，从未出现过18种，因此出现了挑战。例如，鼠头的直径只有几毫米，因此很难抓住骨头。具体来说，对肌腱到骨骼样本的拉伸测试通常会导致生长板的失效，而不是在肌腱或肌腱上。同样，对跟腱进行生物力学测试也极具挑战性。虽然跟腱比其他肌腱大，但卡内肌很小，使得抓住这个骨头变得困难。骨骼可以去除，然后抓住两个肌腱末端;然而，这排除了肌腱到骨骼附件的测试。其他团体报告使用定制的夹具19，20，锚定在夹21，固定在自固化塑料水泥22或使用锥形槽22，但这些以前的方法仍然受到低重现性、高夹持故障率和繁琐的准备要求的限制。
目前的研究目标是开发一种准确而有效的方法，用于对肌腱进行拉伸生物力学测试，以上皮肌腱和跟腱为例。利用来自原生骨骼解剖、实体建模和增材制造的 3D 重建组合，开发了一种控制骨骼的新方法。这些夹具有效地固定了骨骼，防止了生长板故障，减少了试样制备时间，并增加了测试的可重复性。新方法很容易适应测试其他鼠肌腱以及大鼠和其他动物的肌腱。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose</td>
			<td >Fisher Scientific</td>
			<td >BP160-100</td>
			<td >Dissovle 1g in 100 ml ultrapure water to make 1% agarose&#160;</td>
		</tr>
		<tr >
			<td >Bruker microCT&#160;</td>
			<td >Bruker BioSpin Corp</td>
			<td >Skyscan 1272&#160;</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >ElectroForce&#160;</td>
			<td >TA Instruments</td>
			<td >3200</td>
			<td>Testing platform</td>
		</tr>
		<tr >
			<td >Ethanol 200 Proof</td>
			<td >Fisher Scientific</td>
			<td >A4094</td>
			<td>Dilute to 70% and use as suggested in protocol</td>
		</tr>
		<tr >
			<td >Fixture to attach grips</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Kimwipes</td>
			<td >Kimberly-Clark</td>
			<td >&#160;S-8115</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >MicroCT CT-Analyser (Ctan)</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >MilliQ water (Ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>100 ml&#160;</td>
		</tr>
		<tr >
			<td >Meshmixer</td>
			<td >Autodesk</td>
			<td >http://www.meshmixer.com/</td>
			<td >Free engineering software used by authors to refine mesh</td>
		</tr>
		<tr >
			<td >Objet EDEN 260VS&#160;</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Precision Prototyping</td>
		</tr>
		<tr >
			<td >Objet Studio</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Used by authors with 3D printer</td>
		</tr>
		<tr >
			<td >PBS - Phosphate-Buffered Saline</td>
			<td >ThermoFisher Scientific</td>
			<td >10010031</td>
			<td >2.5 L of 10% PBS&#160;</td>
		</tr>
		<tr >
			<td >S&#38;T Forceps</td>
			<td >Fine Science Tools</td>
			<td >00108-11</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Blade - #11</td>
			<td >Fine Science Tools</td>
			<td >10011-00</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Handle - #3</td>
			<td >Fine Science Tools</td>
			<td >10003-12</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >SkyScan 1272</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >Skyscan CT-Vox</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SkyScan NRecon</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SolidWorks CAD</td>
			<td >Dassault Syst&#232;mes</td>
			<td >SolidWorks Research Subsription</td>
			<td>Solid modeling computer-aided design used by authors</td>
		</tr>
		<tr >
			<td >SuperGlue</td>
			<td >Loctite</td>
			<td >234790</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >Testing bath</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Thin film grips&#160;</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >VeroWhitePlus</td>
			<td >Stratasys LTD</td>
			<td >NA</td>
			<td>3D printing material used by authors</td>
		</tr>
		<tr >
			<td >WinTest&#160;</td>
			<td >WinTest Software</td>
			<td ></td>
			<td>Used by authors to collect data</td>
		</tr>
	</tbody>
</table>

biomechanical testing of murine tendons

肌腱疾病是常见的，影响所有年龄的人，并经常使人衰弱。标准治疗，如消炎药、康复和手术修复，往往失败。为了定义肌腱功能并展示新疗法的疗效，必须准确确定动物模型中肌腱的机械特性。鼠种模型现在被广泛用于研究肌腱疾病和评估肌腱病的新疗法;然而，确定小鼠肌腱的机械特性一直具有挑战性。在这项研究中，为肌腱机械测试开发了一个新的系统，其中包括三维打印的固定装置，这些装置与腐殖质和钙化物的解剖完全匹配，分别用于机械地测试上皮肌腱和跟腱。这些夹具是使用原生骨解剖、固体建模和增材制造的 3D 重建来开发的。新方法消除了工件夹持故障（例如，生长板故障而不是肌腱故障），减少了总体测试时间，并增加了可重复性。此外，这种新方法很容易适应测试其他动物的其他肌腱和肌腱。

3D打印的固定装置用于测试8周大的鼠超脊柱和跟腱。所有经过机械测试的样品在检测时均失败，其特点是微CT扫描、目视检查和拉伸测试后的视频分析。图3对实验室中以前和当前超皮肌腱测试方法的一对一比较。在上一种方法28，29，30中，腐殖质骨被嵌入环氧树脂中，一个回形针被放置在腐殖质头部，以防止生长板断裂。...

Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

Biomechanical Testing of Murine Tendons

该协议描述了使用定制尺寸的3D打印夹具对肌腱进行高效和可重复的拉伸生物力学测试方法。

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, ...

该协议描述了一种高效且可重复的方法，通过可重复使用的定制 3D 打印夹具对小鼠肌腱进行生物力学测试。新方法将测试样品的时间从数小时缩短到数分钟，并消除了以前方法中常见的重大夹持工件。此方法不限于超性肌腱和跟腱。它可以很容易地适应测试其他小鼠肌腱和肌腱从较大的动物。为了确保该方法的可重复性和效率，此处未描述的夹具可能需要多个设计原型设计和验证周期。首先，对阿加罗斯凝胶中的整个骨骼进行微算断层扫描。将重建为横截面映像后，选择"文件打开"命令以打开文件数据集。打开对话框文件首选项并选择"高级"选项卡。使用自适应渲染算法构建提供更平滑表面细节的 3D 模型。使用 10 作为位置参数，该参数定义用于查找对象边框的邻点（以像素为单位）的距离。将容差最小化为 0.1 以减小文件大小。要指定感兴趣的体积，请手动选择要设置为所选 VOI 范围的顶部和底部的两个图像，然后移动到第二页"感兴趣区域"。手动选择单个横截面图像上感兴趣的区域。每 10 到 15 个横截面图像重复选择 ROI。接下来，移动到第三页，二进制选择。在"直方图"菜单上，单击"从数据集"以显示来自数据集所有图像的亮度直方图分布。此外，在直方图菜单上，单击"创建 3D 模型文件"菜单。以 STL 文件格式保存骨骼的 3D 模型。在 Meshmixer 软件中，导入网格并选择"全部编辑"。从"工具集"编辑中选择"生成"，然后从"工具集""减少目标"中选择"三角形预算"。减少三算并接受更改。通过选择 Export As.To 设计超平头肌腱腐殖质骨，使用实体建模计算机辅助设计程序创建腐殖质夹具的自定义拟合模型，以重新保存 STL 格式的新缩减文件。在实体建模程序中打开腐殖质骨骼的 STL 格式文件，然后以 SLDPRT 格式保存为零件文件。然后打开零件文件以手动创建三个与解剖学相关的平面，例如 sagittal、日冕和横向。单击"文件更新"以创建实体块组件零件。单击"文件更新"以创建包含两个组件的装配体模型，即实体块和右侧或左侧腐殖质骨骼。定义块内骨骼的方向，以确保肌腱和骨骼之间的角度为 180 度。确保整个骨骼体积适合块内。在"装配体"窗口中，选择块，然后从"装配体"工具栏中单击"编辑零部件"。单击"插入、特征、腔"。选择"均匀缩放"并输入 0% 作为在所有方向缩放的值。抑制骨骼零件并保存装配体作为零件。使用型腔部件打开气缸。创建草图，确保仅将 0.5 毫米留在腐殖质头部上方。从"特征"中选择"拉伸切割"。沿下垂平面切割组件，以创建两个对称的组件，适合骨骼的前部和后部。沿下垂平面切割装配零件，以创建两个对称部件，这些部件适合骨骼前部和后部。确保腐殖质头防止生长板故障，并定义紧密间隙，避免在测试期间将腐殖质与模型分离，这一点至关重要。按照手稿中所述继续，完成后部组件和前部组件。将两个组件另存为单独的零件文件。现在，按"插入图案"、"镜像"并选择"镜像"，为固定装置的每个组件创建对立面的 3D 镜像模型。选择前脸作为镜面平原。选择要镜像的零件。选择镜面并创建包含所有材质的草图。从"特征"中选择"拉伸切割"以移除原始零件并仅保留镜像零件。单击零件底部，用作平面以绘制草图。单击草图文本并添加 L 或 R.，这将在夹具底部添加蚀刻以区分左右两侧。以 STL 标准文件格式保存所有夹具部件，为 3D 打印做准备。解剖超性肌肉肌腱，腐殖质骨标本，首先将安乐死小鼠定位在易发位置，并在皮肤上从前爪肘上方向肩部切开。使用钳子小心地去除皮肤与钝解剖，使肩膀的肌肉是可见的。取出腐殖质周围的组织，直到骨骼暴露。用钳子握住腐殖质，小心地取出三叶虫和梯形肌肉，露出科拉科罗马拱门。识别交流接头，用手术刀刀片小心地将锁骨与锁骨分离。注意不要损坏超面肌腱及其骨质附件，使用手术刀刀片将其肩骨附件的肌肉移除。将腐殖质头部与软骨分离，并撕裂关节胶囊和炎素亚帽和肌腱。分离肘关节，将腐殖质与 ulna 和半径分开。分离腐殖质肌腱肌肉标本，清理腐殖质和腐殖质头上多余的软组织。解剖跟腱，钙质骨，将安乐死小鼠定位在容易的位置。注意不要损坏跟腱及其骨质附件，用钝解剖去除皮肤，使脚踝和膝盖关节周围的肌肉暴露。使用手术刀刀片，从跟腱-钙化物附件开始，小心地将胃毛肌从近近的附件中分离出来。小心地将钙化物与各种相邻的骨骼分离开来，分离出跟腱-钙化物标本并清除多余的软组织。要确定肌腱的横截面区域，请将骨倒置，将标本悬浮在充满阿加罗斯凝胶的冷冻管中，其中含有阿加罗斯凝胶中的骨骼以及肌腱和肌肉。使用微算断层扫描后，使用手术刀刀片轻轻去除肌腱上的肌肉。将骨骼插入 3D 打印夹具，并将其附加到测试网格。将肌腱插入并粘在折叠的薄纸巾之间，并使用薄膜手柄夹住它。将样品和夹具插入 37 摄氏度的 PBS 测试浴池中，并执行机械测试。在这项研究中，使用可重复使用的3D打印夹具来抓骨，标本制备时间从数小时缩短到几分钟。使用电流方法对超平肌肌腱进行拉伸测试的代表性负载变形曲线消除了生长板故障等主要夹持工事。超性肌腱和跟腱的机械特性表明，基于P值小于0.05的未配对T测试，性效果显著。微CT成功地测量了沿上肌腱长度沿跟腱长度的横截面区域。重要的是要记住，每个解剖站点都有有效抓取所需的特定设计标准。因此，每个夹具的设计应相应地进行调整。除了对故障的拉伸测试外，这些夹具还可用于循环加载测试，这些测试提供有关肌腱疲劳和粘骨特性的信息。由于许多原因，包括难以抓住这些小组织、繁琐而耗时的标本制备方法以及生长板断裂，在文献中很难确定肌腱的机械特性。该协议提供了一种时间高效且可重复的方法，用于测试小鼠肌腱，从而消除了伪人抓地力故障，并且将一天可测试的样本数量增加了两倍。

Research

JoVE Journal

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

科研

生物工程

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

小鼠颈动脉的力学性能测试：从新生儿到成人

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

测试床, 检查头盔的拟合和保留, 以及在模拟撞击时头颈部损伤的生物力学措施

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

新生儿小猪体内胸腺菌的活体力学试验方法

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

使用压力表谱对毛质生殖系统的双轴基底音和被动测试

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

The Quantification of Injectability by Mechanical Testing

机械测试的注入性量化

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

基于光学相干断层扫描的冠状动脉粥样硬化进展生物力学流固相互作用分析

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...