作者承认，从美国国家科学基金会资助CBET-0909678和资金从GW中心仿生学仿生与工程（COBRE）的支持。我们感谢同学，克里斯托弗Popma先生，李亚男Penna的女士，香农·卡拉汉女士，Shadman Hussain先生，穆罕默德·R. Najjari先生，和杰西卡Hinke女士的帮助在实验室和马蒂厄Barraja先生协助CAD图纸。

<ol>
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没有利益冲突的声明。

本文介绍的协议描述了采集中使用粒子图像测速技术（PIV）和相干结构检测方法，即，连续小波变换高保真的实验数据， <img alt="式（1）" src="https://neuron2-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/51288eq1.jpg" /> ，适合于涡和切主导的流动鉴定。从以理想化"IV型"断裂的存在生理流入实验数据的分析表明，与复杂的流体动力效应，如流动结构不对称性和变异在时空分布不能从简单的流体动力学理论预测的次要流结构。 有在此协议即执行四个关键步骤。，（ⅰ）的设计与实验室规模的支架模型​​的制造中，（ii）一种血液模拟工作流体的制备与血液和refrac运动粘度匹配弯曲的动脉模型略去指数，（ⅲ）的非侵入性的实验装置（2C-2D PIV）和（iv）用于识别动脉血流模式高级相干结构的检测方法。 Womersley号是涉及脉动流频率粘性效应7的无量纲参数。雷诺数涉及惯性力粘性流动的力量。迪恩数涉及在流中出现的通过弯曲管道惯性向心力和粘性力1,2。细节有关与Womersley和雷诺数生理波形的缩放在5 6给出。在这项研究中所用的流入波形是从典型的颈动脉流速测量重建的17-20健康的病人由Holdworth 等 15（平均）。导致弯曲动脉试验段的管道是足够长的，从而使流动得到充分的发展，使得脉动流赖斯在入口至弯曲动脉试验段系统蒸发散是相同的泵（ 图3a，3b和4a）。供给生理波形的重复性通过使流速和上游使用2C-2D PIV系统模型动脉散装速度的轴向PIV测量保证（参见图4b）。 从对上述临床并发症动脉血流动力学的流体力学刺激不为人所熟知。涉及支架和支架-骨折生理流动带来复杂性在体内和体外测量。这里介绍的协议可以被修改为包括在管道遵守研究动脉二次流结构下的非理想和更逼真流方案的影响。这样的实验会带来的测量和数据后处理更多的挑战。采用立体或断层-PIV技术，能够映射三维veloci的TY字段可以显著改善二次流结构的动力学的理解。 实验安排在于在近壁（模型动脉管腔）区和缺乏到支架植入区域内的血流的光接入缺乏分辨率的局限性。然而，这些因素制约介绍了协议的优雅扩展。对于支架的三维印刷中使用光学透明材料，现实和具体的患者动脉的几何形状将允许支架植入物和断裂，支架的血流动力学前所未有的访问。 本文提出的协议的扩展成果涉及"最佳"小波尺度的选择相干结构检测。步骤5.6.3 - 5.6.7是一个建议的解决方案，以"最好"小波尺度（或基函数）的相干结构检测的问题。研究人员发现，下面的步骤5.6.3 - 5.6.7决心D ALL大型相干结构，此外，检测到了前所未有的弯曲动脉模型试验未被发现的小尺度相干结构。作者建议参考。 34，35，其中香农熵用于评估在离散小波分组中的"最佳"的基础上变换（DWPT）算法朝向湍流实验检测相干结构。有关涉及连续小波方法的进一步信息变换算法，笔者建议参考。其中引用的5，6，35和引用。 在支架植入物和伴随气流扰动骨折的发生率导致具有复杂的，多尺度形态和不同大小的强度特性二次流结构。的方法，如在用相干结构检测特别组合粒子图像测速仪（PIV）的意义，小波变换允许多尺度，多强度分辨率secondar根据支架及支架断裂引起的流方案Ÿ流结构。本文中所呈现的协议铺平了道路调查医疗并发症如支架内再狭窄（ISR），支架内血栓形成和动脉瘤形成8,11 - 14由于二次流。此外，在纤芯区域中遇到的二次流涡旋模式将倾向于影响血源性粒子的运动和曝光时间，如血小板，敏化它们朝向血栓形成活化。应变主导近壁（流明）二次流结构将最终影响是密切相关的动脉粥样硬化，尤其是在动脉曲率壁剪切应力。 分析方法来预测二次流（涡流）结构复杂，需要在环形坐标Navier-Stokes方程和渐近理论，1 - 3，7实验和高阶分析方法相结合将促进新的见解弯曲动脉易于与支架植入和支架断裂相关的几个心血管疾病和并发症临床的血液动力学。

二次流结构是发生在内部流动的几何形状与弯曲，例如弯曲管道和渠道涡流模式。这些涡结构的出现是由于离心力的作用，不良压力梯度和流入特性的共同作用。在一般情况下，二次流结构出现在弯曲的管道作为下稳定流入对称迪安型涡流的平面的横截面，并振荡流入条件下对称迪安和莱恩型涡流。1 - 3二次流形态有很大的影响搏动搏动和多个谐波，生理条件的流入。相比于非生理（稳定振荡）流量1这些结构取得明显不同的大小强度，形状特征- 6在动脉粥样硬化病变的发展是由高频剪切振荡的存在，在地区的影响经历了低平均剪切27,28 </suP&gt;。二次流的结构可能影响疾病，如动脉粥样硬化和可能的进展，介导内皮反应由于通过改变壁剪切应力和血源性粒子的曝光时间脉动血流。 动脉粥样硬化的常见治疗，造成阻塞性病变动脉狭窄的并发症，是支架植入。支架骨折植入的支架导致进一步的并发症，如支架内再狭窄（ISR），支架内血栓形成和动脉瘤形成9的结构破坏- 13支架断裂被分为不同的失败"类型I至IV"，其中"IV型"特征的最高临床严重性和被定义为与支架的片段14的线性位移沿着支架支杆的完全横向断裂。在本研究中提出的协议描述了一种experimenta一个理想化的"IV型"支架在弯曲的动脉断裂模型下游二次流结构的可视化l的方法。 建议的协议有以下四个基本特征: 设计和实验室规模的支架模型的制造:支架的几何描述可以与一组自膨胀螺旋（弹簧或螺旋）的使用镍钛诺金属丝29（镍和钛的合金）相关的交织在一起。支架和其支柱直径的长度取决于动脉损伤临床植入5中遇到的长度尺度。支柱直径的参数变化和绕组（或沥青）的上升沿导致各种几何结构的支架。中选择了3D打印支架设计参数汇总如表1所示。 相匹配的血液模拟工作流体的制备用的血液动粘度和试验段的折射率:需要为了使非侵入速度测量到弯曲动脉测试部分光学连接。因此，牛顿血液-模仿工作流体与血管模型的折射率和理想情况下，一个动态粘度，匹配的人类血液被用来获得精确的血液流量测量16 - 18日，30日在此研究中使用的工作流体被报道由Deutsch 等人 。 （2006），其含有79％饱和的碘化钠水溶液（碘化钠），20％的纯甘油和1％的水（按体积计）16。 用于检测使用的双组分，二维粒子图像测速（2C-2D PIV）相干二次流结构的实验装置:实验设计成在下游的各种平面剖地点获取相平均二次流速度数据海峡的组合aight和实施弯曲支架部分的理想化"Ⅳ型"支架断裂5，6，9，14。使用粒子图像测速仪（PIV）技术有关获得次级流速场的协议步骤涉及一个PIV系统，其包括的激光（光片）的源，光学聚焦和照射流，一个特殊的交叉相关电荷耦合器件（CCD传感器或相机）和示踪粒子的区域，以通过一个很短的时间间隔内的光片（被照射Δt的见表4）31，32。 在该协议的步骤假设以下几点:首先，校准，实验，通过双帧，单次曝光的记录计算的图像的双组分，二维（2C-2D）PIV系统的设置。其次，2C-2D PIV系统由每个记录期间获得的两个图像帧之间执行互相关计算示踪粒子的平均位移。 BR的PIV规格和图像采集软件的IEF总结示于材料和设备表。三，操作激光所需的所有安全防范措施，根据主办机构提供的指导原则，随后由经过培训的实验室工作人员。作者建议参考文献。 31和32为在空气动力学，加氢和微流体动力学，相关峰值检测和位移估计，材料和示踪粒子的密度，并测量噪声和精度执行，功能和PIV技术的应用一个全面的理解。还要注意的是激光和摄像机可由PIV数据采集计算机（ 图3A）和数据处理软件来控制。 使用下面的协议说明中生成利用图2C-2D PIV相平均次级流速测量:数据采集和后处理用于相干结构检测 。后期处理<html>数据ING参与连贯二次流结构检测采用以下三种方法:连续小波变换， <img alt="式（1）" src="/files/ftp_upload/51288/51288eq1.jpg" /> 5，6，19 - 24，26。 作者指出的速度梯度张量本质上是一个3×3的矩阵， <img alt="公式（2）" src="/files/ftp_upload/51288/51288eq2.jpg" /> 。 协议提出获取二维实验测量（从图2C-2D PIV技术）的方法。因此，与速度梯度张量充分实验访问将无法达到使用这种方法。速度梯度张量对每一个像素<img alt="公式3" src="/files/ftp_upload/51288/51288eq3.jpg" /> PIV的图像<img alt="公式4" src="/files/ftp_upload/51288/51288eq4.jpg" />应该是一个2×2矩阵， <img alt="公式5" src="/files/ftp_upload/51288/51288eq5.jpg" /> 。 Z-元件涡quation 6"SRC ="/文件/ ftp_upload / 51288 / 51288eq6.jpg"/&gt;对于每个像素<img alt="公式7" src="/files/ftp_upload/51288/51288eq7.jpg" />使用速度梯度张量的反对称部分计算<img alt="式（8）" src="/files/ftp_upload/51288/51288eq8.jpg" /> 。其结果将是涡的2D阵列<img alt="公式9" src="/files/ftp_upload/51288/51288eq9.jpg" />可在等值线图被可视化。笔者强烈建议参考。 25雄辩的讨论，实验获得对提高涡耗散，应变率和连贯的结构检测知识的速度梯度张量。此外，作者不要试图探讨上述相干结构检测方法之间的相互关系，并提出参考。 23，24对这一主题进行全面的讨论。 在该协议中的步骤的焦点是二次流（涡流）的定量识别STructures（也称为相干结构）。相干结构检测即的三种方法， <img alt="10式" src="/files/ftp_upload/51288/51288eq10.jpg" />和小波变换的涡<img alt="11式" src="/files/ftp_upload/51288/51288eq11.jpg" />应用于速度场的数据对检测多尺度，多力理想化的"IV型"支架断裂的下游二次流结构的出现。 该 <img alt="12式" src="/files/ftp_upload/51288/51288eq12.jpg" /> 定义了一个涡流作为空间区域，其中涡张量的欧几里得范数支配该菌株19，23，速率的24 .The速度梯度矩阵分解成对称的（应变率）和非对称（旋转）的部件。应变率矩阵的特征值计算; <img alt="13式" src="/files/ftp_upload/51288/51288eq13.jpg" /> 。应变速率的规范然后计算; <im摹ALT ="14式"SRC ="/文件/ ftp_upload / 51288 / 51288eq14.jpg"/&gt;。涡从反对称部分计算的。拟涡能或z分量涡广场， <img alt="15式" src="/files/ftp_upload/51288/51288eq15.jpg" /> ）然后被计算。该<img alt="16式" src="/files/ftp_upload/51288/51288eq16.jpg" />终于计算; <img alt="17式" src="/files/ftp_upload/51288/51288eq17.jpg" /> 。整个集的等高线图<img alt="18式" src="/files/ftp_upload/51288/51288eq18.jpg" />同的异区<img alt="19式" src="/files/ftp_upload/51288/51288eq19.jpg" /> ，将表明二次流结构19。 该<img alt="20式" src="/files/ftp_upload/51288/51288eq20.jpg" /> ，也被称为"回旋强度"是由局部速度梯度张量的临界点分析进行的涡流识别方法和其相应的特征值20 - 24 <s仲&gt;。在每个像素中的速度梯度张量的本征值<img alt="21式" src="/files/ftp_upload/51288/51288eq21.jpg" />计算。特征值应该是这样的形式， <img alt="式（22）" src="/files/ftp_upload/51288/51288eq22.jpg" /> 。的等高线图<img alt="23式" src="/files/ftp_upload/51288/51288eq23.jpg" />同的异区<img alt="24式" src="/files/ftp_upload/51288/51288eq24.jpg" />将表明二次流结构20 - 22。 小波变换方法利用具有光滑的物理和光谱的空间分析功能（或波），是可以受理（或具有零均值），并具有有限<img alt="25式" src="/files/ftp_upload/51288/51288eq25.jpg" /> 5，6，26，通过进行卷积一个扩张或收缩与2D涡字段小波，小波变换的涡<img alt="26式" src="/files/ftp_upload/51288/51288eq26.jpg" />场是C产生具有广泛的规模和优势5，6，二维小波变换的涡场的26。熵相干结构omprising计算估计最优小波规模在所有的相干结构得到充分的解决。这个熵估计涉及一组概率<img alt="27式" src="/files/ftp_upload/51288/51288eq27.jpg" />对于每个像素<img alt="21式" src="/files/ftp_upload/51288/51288eq21.jpg" />这样<img alt="28式" src="/files/ftp_upload/51288/51288eq28.jpg" />中，归一化模平方与在单元m的像素相关联的涡中，n 5,6。在图6以图形方式的程序的步骤。放置在小波的选择的限制中详细参考呈现。 26.该协议的步骤介绍了使用二维Ricker子波相干结构检测的过程。对于使用该W的理由子波的旋涡模式匹配，提出在文献。其中引用5,6和相关引用。

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Acrylic tubes and sheet</td>
			<td style="width:155px;">McMaster-Carr Supply Company</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">Inlet and outlet pipes and, material of the curved artery test section</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Object24 Desktop 3D printer</td>
			<td style="width:155px;">Stratasys</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">Desktop rapid prototyping machine. http://www.stratasys.com/.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VeroWhitePlus Opaque material</td>
			<td style="width:155px;">Stratasys</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">Building material for Object24 Desktop 3D printer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fullcure 705</td>
			<td style="width:155px;">Stratasys</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">&#160;Non-toxic gel-like photopolymer Support material for Object24 Desktop 3D printer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ubbelhode viscometer</td>
			<td style="width:155px;">Cole Parmer</td>
			<td>YO-98934-12</td>
			<td style="width:208px;">Toward measurement of kinematic viscosity of the blood-analog fluid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VELP scientifica - ESP stirrer&#160;</td>
			<td style="width:155px;">VELP Scientifica</td>
			<td>F206A0179</td>
			<td style="width:208px;">Magnetic stirrer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ohaus Scout Pro SP 601&#160;</td>
			<td style="width:155px;">The Lab Depot</td>
			<td>SP4001</td>
			<td style="width:208px;">Weigh scale</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Refractometer</td>
			<td>Atago</td>
			<td style="width:119px;">PAL-RI</td>
			<td style="width:208px;">Toward measurement of refractive index of blood-analog fluid</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Beakers, pipettes, syringes and spatula</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">CLS710110,&#160; CLS10031L, CLS71015, CLS71011 Z193216</td>
			<td style="width:208px;">Toward handling materials required for blood-analog solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Iodide</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>383112-2.5KG&#160;</td>
			<td style="width:208px;">Crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>G5516-1L</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deionized Water</td>
			<td style="width:155px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium thiosulfate anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>72049-250G</td>
			<td style="width:208px;">Powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Recording medium</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">Imager Intense 10Hz</td>
			<td style="width:208px;">PIV Image acquisition CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PIV Illumination source</td>
			<td style="width:155px;">New Wave Research</td>
			<td style="width:119px;">Solo III-15</td>
			<td style="width:208px;">PIV Laser source, Nd:YAG laser, 532 nm, dual pulse 70 mJ/pulse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Imaging software</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">DaVis 7.2&#160;</td>
			<td style="width:208px;">PIV data acquisition and instrument control&#160;</td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:216px;">PIV Seeding material</td>
			<td style="width:155px;">Thermo-scientific&#160;&#160;</td>
			<td style="width:119px;">Flouro-Max</td>
			<td style="width:208px;">Red fluorescent polymer microspheres (&#8776; 7 &#181;m); Dry dyed polystyrene (DVB) fluorescent microspheres emit bright and distinct colors when illuminated by the light of shorter&#160; wavelengths than the emission wavelength.&#160;</td>
		</tr>
	</tbody>
</table>

experimental investigation of secondary flow structures downstream of a model type iv stent failure in a 180&#176; curved artery test section

动脉网络在人血管包括目前普遍存在血管复杂的几何形状（树枝，曲率和扭曲）的。二次流结构，由于离心力的作用，不良压力梯度和流入特性的共同作用发生弯曲动脉涡流模式。这样的流动形态由搏动和生理流入条件的多次谐波很大的影响，并且尺寸强度形特性变化很大相比非生理（稳定和振荡）流1 - 7。 二次流结构可能最终影响血液传播颗粒的壁面剪切应力和暴露时间对动脉粥样硬化，再狭窄，血小板的宣传和血栓形成4进展- 6，8 - 13因此，要检测和检定这些结构在实验室的能力。 -controlled条件precurs或者进一步的临床研究。 一个常见的​​手术治疗动脉粥样硬化是支架植入术，开辟了通畅血流狭窄动脉。但随之而来的气流扰动由于支架安装导致多尺度二次流形态4 - 6逐步高阶复杂如在相干不对称和损失可以通过随后的支架故障相-à-相那些未扰动流5下被诱导。这些支架的失败被列为基于故障因素和临床严重度14"类型I至IV"。 这项研究提出了预计将在弯曲动脉模型来完成横向支架断裂和骨折部位的线性位移（"IV型"）的复杂的二次流结构的实验研究的协议。实验方法包括粒子图像测速的实现（2C-2ðPIV）技术，与典型的颈动脉流入波形，折射率匹配的血液模拟工作流体相的平均测量15 - 18使用流的物理概念，临界点理论和新型达到二次流结构的定量化识别小波变换应用到实验数据PIV 5，6，19算法- 26。

在图7A-D呈现的结果的后处理的二次流的速度数据之后产生（参见图5，图6）从图3A所示2C-2D PIV系统获得的。供给到与理想化"Ⅳ型"支架断裂弯曲动脉试验段的流入条件是在图4B所示的颈动脉波形。我们以前的研究已经证明二次流结构的敏感性减速条件在各种脉动流入波形的一定范围Womersley数字内<img alt="式（55）" src="/files/ftp_upload/51288/51288eq55.jpg" /> 4 - 6因此，时间实例<img alt="56式" src="/files/ftp_upload/51288/51288eq56.jpg" />在图7A-D呈现的结果的，被选择以对应于颈动脉流入的收缩期减速W相aveform。不同大小强度形态特征的相干二次流结构在不同平面的横截面呈现<img alt="57式" src="/files/ftp_upload/51288/51288eq57.jpg" /> 如图7A-D。在弯曲动脉试验段出现大规模一致的二次流结构已被列为变形迪安，Lyne-和壁挂式（DLW）旋涡。通常情况下，DLW旋涡在收缩期加速阶段发展。在收缩期减速阶段，DLW结构体验的连贯性，不对称性非典型的损失，并在旋涡的位置，大小，实力和形态的变化。下面是在图7A-D呈现的结果的描述: 在 <img alt="58式" src="/files/ftp_upload/51288/51288eq58.jpg" /> 位置（图7A）:一对对称的，连贯的，变形狄恩涡流（D）中观察到<img alt="59式" src="/files/ftp_upload/51288/51288eq59.jpg" />场T / T = 0.23和0.27。这些D型涡流出现减速期间朝向外壁翻译。 C-领域本菌株和剪切为主的图案在t / T = 0.23，除了D型涡流。由于减速在t / T = 0.27，在D型涡和近壁应变为主的流场的强度降低的可能效果观察。除了D型多尺度二次流的形态在小波变换的涡字段被检测<img alt="60式" src="/files/ftp_upload/51288/51288eq60.jpg" />说明几个应变为主的旋涡图案的存在。 在 <img alt="61式" src="/files/ftp_upload/51288/51288eq61.jpg" /> 位置（图7B）:从在一对D型涡流的过渡<img alt="62式" src="/files/ftp_upload/51288/51288eq62.jpg"/&gt;的位置，三对对称的，连贯的，变形迪安，Lyne-和壁挂式涡流（DLW）在观察<img alt="59式" src="/files/ftp_upload/51288/51288eq59.jpg" />领域。如由漩优势的大小，L-和W-型涡流比D型涡高循环。流扰动从断裂支架在发出<img alt="62式" src="/files/ftp_upload/51288/51288eq62.jpg" />位置可能已经促成DLW涡流的形成。被观察为在L-中强度的降低和W型涡流减速的影响。有一个在与大型相干DLW结构的位置一致<img alt="63式" src="/files/ftp_upload/51288/51288eq63.jpg" />和<img alt="59式" src="/files/ftp_upload/51288/51288eq59.jpg" />领域。其他规模较小的二次流动形态的检测<img alt="63式"SRC ="/文件/ ftp_upload / 51288 / 51288eq63.jpg"/&gt;字段。 C-领域大多包括剪切为主的近壁区的暗示对称大规模DLW涡旋的存在。 在 <img alt="式（64）" src="/files/ftp_upload/51288/51288eq64.jpg" /> 位置（图7C）: <img alt="59式" src="/files/ftp_upload/51288/51288eq59.jpg" />在t字段/ T = 0.23表示L型涡流的损失和细长D-和W型涡旋的存在。在t / T = 0.27有漩涡状两个D-和W型涡流强度的损失。减速的作用是通过在所观察到的旋涡结构的不对称性指示<img alt="63式" src="/files/ftp_upload/51288/51288eq63.jpg" />领域。随着细长D型的存在涡流的小规模W型涡流大量观察。 Q-字段表示的近壁切为主的区域T中的存在<html>帽子是从暗示支架断裂引起的流动扰动增加了不稳定的。 在 <img alt="65式" src="/files/ftp_upload/51288/51288eq65.jpg" /> 位置（图7D）: <img alt="59式" src="/files/ftp_upload/51288/51288eq59.jpg" />在t / T字段= 0.23包括虚弱，DLW结构。由于流动减速的影响，这些DLW结构往往在t / T = 0.27进一步消耗。损失在近壁剪切流中的Q字段在时间两个实例观察。在t / T = 0.23， <img alt="63式" src="/files/ftp_upload/51288/51288eq63.jpg" />字段显示的D-旋涡更靠近内壁与多尺度W型涡流和周边应变为主的结构沿与相应的协议<img alt="59式" src="/files/ftp_upload/51288/51288eq59.jpg" />领域。51288eq63.jpg"/&gt;字段清楚地显示在时间而这两个实例中DLW结构的连贯性和不对称性亏损<img alt="式（66）" src="/files/ftp_upload/51288/51288eq66.jpg" />字段不捕获的现象。 该协议的成功执行后，广泛的推论 <img alt="67式" src="/files/ftp_upload/51288/51288eq67.jpg" />检测大型二次流结构及其变化的流动形态。 <img alt="式（68）" src="/files/ftp_upload/51288/51288eq68.jpg" />了在近壁区通常会遇到的高应变率的检测区域。连续小波变换算法检测与unthresholded一致大型二次流结构<img alt="67式" src="/files/ftp_upload/51288/51288eq67.jpg" /> 。二维Ricker子波的内核还解决了几个低循环，多尺度二次流morpholo<html>这是未被发现与吉斯<img alt="式（69）" src="/files/ftp_upload/51288/51288eq69.jpg" />和unthresholded <img alt="70式" src="/files/ftp_upload/51288/51288eq70.jpg" /> 。这三个指标的组合整体确定二次流涡和应变为主的结构。 <img alt="图1" src="/files/ftp_upload/51288/51288fig1.jpg" /> 图1. 设计，制造和安装的直线和曲线的支架。（A）使用左，右转动螺旋的组合直支架配置CAD模型。 （B）弯曲支架配置的CAD模型。用于支架的制造（C）的三维打印机。 （D）和（E）3D打印后直线和曲线的支架。安装在180°弯曲动脉测试部分（F）支架。://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/51288fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/51288/51288fig2.jpg" /> 图 2. 该粒子图像测速仪（PIV）系统的示意图以下系统组件被表示:1.钕YAG激光光学器件以产生一个由PIV-数据采集控制的激光片和2的CCD相机提供的电压 - 时间波形到泵和同步的计算机，3.泵仪器控制计算机将触发对PIV-数据采集计算机，4.产生生理流速可编程齿轮泵，5，一种闭环，具有实验测试部入口和出口管道，180°弯曲动脉试验段和用于血 - 模拟流体的贮存器。插图:不同平面的横截面，其中PIV测量可以是的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/51288fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51288/51288fig3.jpg" /> 图3. PIV-系统支架的安装位置的实验安排。（A）与各种系统组件的光学平台的PIV系统的安排。 （B）的重要方面180°弯曲动脉试验段示意图，在直线和曲线支架体现了"IV型"支架断裂和裂隙支架部分（D 空间 ）之间的间距的位置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/51288fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 180°弯曲动脉试验段的上游测量超过20的波形周期由可编程泵具有的特性的功能，如收缩峰值在时间t / T = 0.19产生 图4 的生理波形（A）流速（毫升/秒） 。 （B）流量波形与超过20波形周期测量的时间各种情况下的标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/51288fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/51288/51288fig5.jpg" /> 图5. 序列PIV测量，并在180°弯曲动脉试验段二次流结构的检测。（A </strong<html>通过由泵的仪器控制计算机产生的触发同步使用PIV技术二次流速场数据&gt;）生成。 （ 二 ）使用辅助流场数据由Q-和λCI治疗像素化图像（或矩阵）后处理程序-标准，和小波变换的涡（Ω"） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/51288fig5large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图6" src="/files/ftp_upload/51288/51288fig6.jpg" /> 在任意规模（ℓ），2D涡量场，香农与小波尺度（ℓ）熵的变化的一个例子2D-Ricker子波: 图6. 连续小波算法变换的代表动脉二次流结构检测方法插图。<html> COM /文件/ ftp_upload / 51288 / 51288fig6large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/51288/51288fig7.jpg" /> 图 7. 二次流结构中在45°180°弯曲动脉试验段，90°，135°和180°的平面的位置和时间的情况下，T / T = 0.23，0.27，在收缩期减速插图:示意图描绘测量位置，Q-，λ 词的比较-标准，和小波变换涡（Ω&#39;）在收缩减速期间每个平面的位置和实例数据字段，colorbars指示由Q-并且λ 词所获取的值的范围 - 标准，和小波变换的涡（Ω&#39;）数据及其解释。<html> PG"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"> 参数 </td><td> 公式没有。 </td><td colspan="2"> 初始值 </td><td> 支架型号类别 </td><td> 描述 </td></tr><tr><td colspan="2"> θ= 360 n 关闭 Ť </td><td> 1 </td><td colspan="2"> n 关闭 = 4 </td><td>直行;弯曲</td><td>在螺旋圈数 （正转 ） </td></tr><tr><td colspan="2"><img alt="表1式（1）" src="/files/ftp_upload/51288/51288tbl1.jpg" /></td><td> 2 </td><td colspan="2">每回合间距=22.225毫米</td><td>直行;弯曲</td><td>螺旋的螺距 （间距 ） </td></tr><tr><td colspan="2"><html>能1公式2"SRC ="/文件/ ftp_upload / 51288 / 51288tbl2.jpg"/&gt; </td><td> 3 </td><td colspan="2"> D =11.84毫米</td><td>直行</td><td>支架的公称通径 （D） </td></tr><tr><td colspan="2"><img alt="表1式3" src="/files/ftp_upload/51288/51288tbl3.jpg" /></td><td> 4 </td><td colspan="2"> D =11.84毫米</td><td>直行</td><td>支架的公称通径 （D） </td></tr><tr><td colspan="2"><img alt="表1式5" src="/files/ftp_upload/51288/51288tbl5.jpg" /></td><td>五</td><td colspan="2">每回合间距=22.225毫米</td><td>直行</td><td>螺旋的螺距 （ 间距 ） 直支架模型的长度（Z） </td></tr><tr><td colspan="2"><img alt="表1式6" src="/files/ftp_upload/51288/51288tbl6.jpg" /></td><td> 6 </td><td colspan="2"><html> l7.jpg"/&gt; </td><td>弯曲</td><td> 180°弯曲动脉模型半径 R 圆弧 </td></tr><tr><td colspan="2"> β=180吨</td><td> 7 </td><td colspan="2"> β= 45 </td><td>弯曲</td><td>角在曲率中心的弧形支架对着</td></tr><tr><td colspan="2"> ð 线 </td><td> - </td><td colspan="2"> Ð 线 =0.85毫米</td><td>直行;弯曲</td><td>支架支撑直径</td></tr><tr><td colspan="2">大号直 = Z </td><td> - </td><td colspan="2">大号直 =88.9毫米</td><td>直行</td><td>直支架模型长度</td></tr></tbody></table> 表1.左，右螺旋和，初始化参数值的参数方程。 lways"&gt; <tbody><tr><td> 化学溶剂和试剂 </td><td> 化学式 </td><td> 密度在20℃下 （克/厘米3） </td><td> 折射率 </td><td> 运动粘度 （米2 /秒）×10 -6 </td><td> 形成 </td><td> CAS登记号 </td></tr><tr><td>碘化钠</td><td>碘化钠</td><td> 3.67 </td><td> 1.7745 </td><td> - </td><td>晶</td><td> 7681-82-5 </td></tr><tr><td>甘油</td><td> C 3 H 8 O 3 </td><td> 1.262 </td><td> 1.4746 </td><td> ≈1115 一 </td><td>液体</td><td> 56-81-5 </td></tr><tr><td>去离子水</td><td> H 2Ø </td><td> 1 </td><td> 1.333 </td><td> 1.002 </td><td>液体</td><td> - </td></tr><tr><td>钠无水硫代</td><td> 的 Na 2 O 3 S 2 </td><td> 1.01 </td><td> - </td><td> - </td><td>粉末</td><td> 7772-98-7 </td></tr><tr><td colspan="7">由塞居尔和Oberstar 16日报道一个测量</td></tr></tbody></table> 表2说明在创造血-模拟溶液中使用的化学溶剂以及试剂。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> 公式没有。 </td><td> 描述 </td><td> 建议实验室设备 </td></tr><tr><td><img alt="表3公式100" src="/files/ftp_upload/51288/51288eq100.jpg" /></td><td> 8 </td><td>饱和碘化钠溶液（碘化钠）的密度是通过测量的这样的质量，计算lution和少量加至50毫升的烧杯中的体积。 </td><td> 1.烧杯（50ml）中 2.电子秤 3.毕业或体积吸管</td></tr><tr><td><img alt="表3公式101" src="/files/ftp_upload/51288/51288eq101.jpg" /></td><td> 9 </td><td>制备饱和碘化钠溶液的整批的体积</td><td> 1.烧杯用饱和碘化钠溶液（2,000毫升） 2.电子秤</td></tr><tr><td><img alt="表3公式102" src="/files/ftp_upload/51288/51288eq102.jpg" /></td><td> 10 </td><td>血液模拟解决方案的总成交量有望滴定溶液配制后</td><td> 1.烧杯用饱和的NaI溶液（2,000毫升）与甘油和去离子水混合。 2.电子秤</td></tr><tr><td><img alt="表3公式103" src="/files/ftp_upload/51288/51288eq103.jpg"/&gt; </td><td> 11 </td><td>甘油的总体积被添加到饱和碘化钠溶液</td><td> 1.烧杯用饱和碘化钠溶液（2,000毫升） 2.电子秤 3.烧杯（100毫升），以甘油转移到饱和的NaI溶液</td></tr><tr><td><img alt="表3公式104" src="/files/ftp_upload/51288/51288eq104.jpg" /></td><td> 12 </td><td> DI水的总体积被添加到饱和的NaI和甘油的溶液</td><td> 1.毕业或体积移液器去离子水转移到饱和碘化钠和甘油溶液</td></tr></tbody></table> 表3. 表百分比按体积计算为血-模拟溶液:79％的NaI，20％甘油和1％的去离子水。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>PIV系统规范 </td><td> 几何形状或特征值 </td><td> 描述 </td></tr><tr><td>流动几何</td><td>横截面平行光片通函</td><td>动脉弯曲试验段</td></tr><tr><td>最大平面内速度</td><td> 0.16米秒-1 </td><td>二次流速规模</td></tr><tr><td>图片大小</td><td>点¯x1376像素ÿ1,040像素</td><td> PIV相机CCD数组大小</td></tr><tr><td>激光脉冲之间的时间间隔（ΔT） </td><td> 600 - 3200微秒</td><td>投入PIV图像采集软件（DaVis软件7.2） </td></tr><tr><td>载体的最终数目</td><td>的X86，Y 65 </td><td>从PIV数据后处理输出（DaVis软件7.2） </td></tr></tbody></table> 表4双COMPON的规格耳鼻喉科，二维（2C-2D）PIV系统。 <img alt="补充1" src="/files/ftp_upload/51288/51288supplement1.jpg" /> 补充代码文件1. 创建一个面具。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/Supplemental_Code_File_1-creating_a_mask.docx" target="_blank">请点击此处下载此文件。</a> <img alt="补充2" src="/files/ftp_upload/51288/51288supplement2.jpg" /> 补充代码文件2.创建一个张贴处理例程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/Supplemental_Code_File_2-creating_a_post_processing_routine.docx" target="_blank">请点击此处下载此文件。</a> <img alt="补充3" src="/files/ftp_upload/51288/51288supplement3.jpg" /> 补充代码FILE 3:MATLAB代码 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51288/Supplemental_Code_File_3-MATLAB_Codes.docx" target="_blank">，请点击此处下载该文件。</a>

Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Ã‚Â° Curved Artery Test Section at JoVE.com

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

在180°弧形动脉试验段的二次流动结构下游的型号IV型支架失败的实验研究

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

experimental-investigation-secondary-flow-structures-downstream-model

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.5K 次观看.  The George Washington University. Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

视频: Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

表征多层鱼鳞（中<em&gt; Atractosteus锅铲</em&gt;）基于纳米压痕技术，X射线CT，红外光谱和SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...