鼠头的生物力学试验
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 骨骼图像采集与3D骨骼模型构造在准备 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…
Representative Results
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。…
Discussion
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 …
Divulgations
The authors have nothing to disclose.
Acknowledgements
该研究得到了NIH/NIAMS（R01 AR055580，R01 AR057836）的支持。
Materials
| Agarose | Fisher Scientific | BP160-100 | Dissovle 1g in 100 ml ultrapure water to make 1% agarose |
| Bruker microCT | Bruker BioSpin Corp | Skyscan 1272 | Used by authors |
| ElectroForce | TA Instruments | 3200 | Testing platform |
| Ethanol 200 Proof | Fisher Scientific | A4094 | Dilute to 70% and use as suggested in protocol |
| Fixture to attach grips | Custom made | Used by authors | |
| Kimwipes | Kimberly-Clark | S-8115 | As suggested in protocol |
| MicroCT CT-Analyser (Ctan) | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
| MilliQ water (Ultrapure water) | Millipore Sigma | QGARD00R1 (or related purifier) | 100 ml |
| Meshmixer | Autodesk | http://www.meshmixer.com/ | Free engineering software used by authors to refine mesh |
| Objet EDEN 260VS | Stratasys LTD | Precision Prototyping | |
| Objet Studio | Stratasys LTD | Used by authors with 3D printer | |
| PBS – Phosphate-Buffered Saline | ThermoFisher Scientific | 10010031 | 2.5 L of 10% PBS |
| S&T Forceps | Fine Science Tools | 00108-11 | Used by authors |
| Scalpel Blade – #11 | Fine Science Tools | 10011-00 | Used by authors |
| Scalpel Handle – #3 | Fine Science Tools | 10003-12 | Used by authors |
| SkyScan 1272 | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
| Skyscan CT-Vox | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
| SkyScan NRecon | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
| SolidWorks CAD | Dassault Systèmes | SolidWorks Research Subsription | Solid modeling computer-aided design used by authors |
| SuperGlue | Loctite | 234790 | As suggested in protocol |
| Testing bath | Custom made | Used by authors | |
| Thin film grips | Custom made | Used by authors | |
| VeroWhitePlus | Stratasys LTD | NA | 3D printing material used by authors |
| WinTest | WinTest Software | Used by authors to collect data |
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Citer Cet Article
Kurtaliaj, I., Golman, M., Abraham, A. C., Thomopoulos, S. Biomechanical Testing of Murine Tendons. J. Vis. Exp. (152), e60280, doi:10.3791/60280 (2019).