This protocol was developed for research supported by the Natural Sciences and Engineering Research Council of Canada (grant RGPIN 401963).

下面的协议在荷兰Bloorview儿童康复医院是经研究伦理委员会。 1.电机校准<ol><li>单片机电路板连接到使用USB端口的计算机。 </li><li>使用原装单片机软件，上传自定义脚本，"Motor_and_AccelerometerTest.ino"使用通过点击"上传"图标，在盘旋右箭头表示USB连接的电路板。 <ol><li>确保该振动电平被设置为零，以把使用"analogWrite"命令，在关闭位置的马达。该代码应改为"analogWrite（vibe1,0）;"。 </li><li>在微控制器的代码，指定脉冲宽度调制（PWM）输出引脚相应于通过初始化"vibe1"可变兴趣电机。 注意:PWM信号生成从由微控制器产生的数字信号近似模拟输出。该引脚labeleÐ数字上的物理单片机电路板。例如，如果电机被连接到PWM输出引脚"3"，则确保了"INT vibe1 = 3;"在代码被指定。 </li></ol></li><li>三轴加速计的z轴输出连接到微控制器板的模拟输入端口之一，以及连接正极和分别地从加速度计到5V和接地（GND）端口的微控制器板的引线。 </li><li>安装加速度计到振动电机，确保其z轴是垂直于马达的平表面， 如图2中 ，并放置在电机上的硬表面。 </li><li>打开在数据采集软件的"Motor_Calibration.vi"文件，并通过USB端口连接微控制器的计算机。 </li><li>使用提供的字段中，指定用于微控制器输入串行端口，使用下拉菜单，以及采样率，和num样品的误码率，收集。注意:500赫兹的采样速率是标准为这些实验，以防止加速度数据的走样，和1000的样品通常记录。 </li><li>使用"Motor_and_Accelerometer.ino"代码，指定供应到振动马达的PWM脉冲的所需占空比，再次使用"analogWrite"命令，并且该程序重新下载到微控制器板控制电机（参见步骤1.1 ）。例如，要设置脉冲100的数目，代码应为"analogWrite（vibe1,100）;"。 表1列出了在PWM值及其对应的占空比。 </li><li>了"MotorCalibration.vi"接口上使用快速傅立叶变换（FFT）显示，查明的最大峰并记录对应的振动频率的值（从横轴）。 </li><li>重复步骤1.7-1.8，调整PWM水平，直到达到所希望的频率，记录每个PWM频率PAI河例如，如果指定一个100赫兹的频率，执行步骤1.7-1.8直到发生在横轴上的100赫兹标记的最大峰。 注:有关本协议中使用的振动电机，振动的目标应在于在60-400赫兹范围在文献中描述5,10,13皮肤更好地匹配机械性的响应频率。 </li><li>重复步骤每个电机1.2.2到1.8，手动记录与电子表格或铅笔和纸每个电机的PWM频率的关系。 </li><li>打开"Experiment_1.vi"文件。对于每一个电机，下拉菜单频率右击并选择"属性"。在"编辑项目"选项卡中，使用表格，进入1.8-1.9的步骤来确定所需的频率和相应的PWM水平。选择"确定"退出。 </li><li>为系统设计软件的每个虚拟接口（VI）的文件重复步骤1.11的测试过程中使用（ 例如，"Experiment_2.vi"，"Experiment_3.vi" 等 ）。 </li></ol><img alt="图2" src="/files/ftp_upload/54223/54223fig2.jpg" /> 图2. 加速度安装到电机该三轴加速计（绿色）被安装到其z轴正交的马达用于校准的平坦表面的硬币电机。每个电机采用不同占空比激活，相应的振动频率是由加速度记录。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54223/54223fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.放置汽车<ol><li>一旦所有电机都被校准（第2节），将它们安装到大腿。 <ol><li>为了实现本手稿描述的结果，将一台电机上的每个前侧，后侧，内侧，和大腿的侧表面的，大约中途是吐温大粗隆和股骨外侧髁（或肢体的膝上截肢患者的远端）。 注意:每个电机的具体位置可以变化，这取决于所感兴趣的研究问题和地区，并且可以通过解剖和生理因素，如在皮肤机械性的类型和空间分布的影响。 </li></ol></li><li>直接连接马达用双面胶带在皮肤上。 注:刮围绕每个马达的区域是没有必要的，但可提高其对皮肤的粘附（ 图3）。对于其中的衣物，假体衬套，或对用户感知一些其它材料的影响感兴趣的应用程序，将在所述材料的顶部的电机，而不是抵靠皮肤。 </li></ol><img alt="图3" src="/files/ftp_upload/54223/54223fig3.jpg" /> 图3.测试平台的实验。自定义测试平台的建立是为了房子Ť他单片机电路板和按钮。汽车可以直接贴在皮肤上（如图所示），或与电机和皮肤之间的假体衬垫。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54223/54223fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.实验1:应用刺激和记录反应时间<ol><li>重新刷新微控制器板固件，以便通过与控制器的附带的软件包，打开"LVIFA_Base.pde"的文件，并重复步骤1.1数据采集软件董事会的控制权，取代"Motor_and_Accelerometer_Test.ino"与" LVIFA_Base.pde"脚本。 </li><li>直接连接的按钮来使用串行到USB连接电脑的USB端口中的一个。确保所有需要的驱动程序。 </li><li>打开"Experiment_1.vi"界面。 </li><li>指定串行端口，用于单片机电路板连接到电机，和按钮从标有"电机输入"和"绿色按钮"，分别下拉菜单选择相应的串行端口标识号。确定使用计算机的操作系统设备管理器实用程序的串行端口标识号。 </li><li>选择该文件以记录结果并启动程序。 </li><li>选择电机和频率通过从下拉菜单中的"Experiment_1.vi"接口每个马达选择被激活。有参与者按下按钮以在其上感觉到的振动时被施加反馈的腿。该按钮被按下后，确认通过注意到时钟已经停止计数在数据采集软件界面的响应，并通过从下拉菜单中选择一组新的频率重置为下一个试验的电机。 </li><li>一旦实验结束后，使用DRopdown菜单，所有机动频率恢复到零位，然后选择"停止程序"按钮以​​终止于电机控制板上的连接。 </li></ol> 4.实验2:刺激之间区分注意:这个实验可以完全独立于实验1.单个或多台电机可以用来进行。马达的具体位置可以根据不同的应用和研究问题而有所不同。 <ol><li>使用串行USB接口第二按钮连接到另一个USB端口。 </li><li>打开"Experiment_2.vi"的数据采集接口。 </li><li>将电机在具体的研究应用程序所需的位置和配置。例如，将单个马达上大腿，大转子和股骨外侧髁（或肢体被截肢者的前端）之间的中间，在每个前，后，外侧的，和大腿内侧表面到电子xamine在每个腿10,11的这些部分的频率灵敏度。 </li><li>指定用于微控制器板，并使用相同的方法步骤3.4既个按键的串行端口。一定要注意哪些按钮被分配到每一个端口。 </li><li>选择要激活的特定的电机和通过点击在软件接口"电机"的图标的频率的所需序列。例如，假设三个频率正在测试，如140赫兹，180赫兹，220赫兹。一系列测试可能是（1）180赫兹后跟140赫兹，（2）220赫兹后跟140赫兹，及（3）180赫兹后跟220赫兹。 </li><li>输入延迟开始时间和持续时间的刺激。 1.5秒是典型的两延迟和刺激次。 </li><li>启动该程序。 注:后在步骤4.6中指定的延迟，程序将激活与步骤4.5选择成对频率的序列电机（多个）。例如，如果180赫兹与220赫兹是本身在步骤4.5 lected，相应的马达将首先在180赫兹的指定的持续时间切换到220赫兹之前振动。 </li><li>有参与者按一项所述的两个按钮来选择第二感知频率是否比第更高或更低。响应将自动被程序记录下来。 </li></ol>

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The authors have nothing to disclose.

该协议的目的是提供一种用于在振动触觉的ASF应用评估刺激参数提供框架。具体地讲，它检查振动的频率，振幅，位置和序列的用户感觉运动响应的影响。这个框架可以在建立和扩展，其中包含另外的或可选择的类型可能更临床相关，如弯曲的联合，或从一条腿移重量到另一个用户应答。这些类型的变化将需要稍微不同的硬件配置，即置换设备的按钮的如惯性测量单元（IMU产品）或压力传感器，以及伴随的变化的虚拟接口。类似地，尽管这里介绍的协议要求参与者处于就座位置，只有小的硬件修改是必要的，以使过渡更临床相关的姿势，如站立平衡或沃尔王试验。 在这两个实验中，按钮（多个）可以与手，腿，脚被压，或通过一些其它手段，这取决于具体的研究问题和所希望的响应。此外，采用该基本协议额外的研究可用于探索不同的反馈编码策略，位置和掺入的影响到新的或现有的假肢。例如，实施震动触觉反馈到下肢假肢时，它可能是有趣的检查假肢插座和衬垫上的刺激使用者敏感度的影响。虽然在本手稿详述的协议所需要的振动电机（通过接口）的半手动激活，它们可以很容易地修改，以便使马达的激活以响应来自外部传感器动力学或运动的测量。使用的测量设备，例如惯性平台，角度计，压力传感器等 ，以代替按钮的，实验可以是conducted考察更多的生理有关用户的响应时间和准确性提供振动触觉反馈。这种类型的研究将采用类似的协议，以那些在实验1和2的说明，但将需要额外的控制系统传感器的输入转换成发送到振动马达的指令，以及变更到数据采集软件接口新硬件的变化。 实施生理学相关的响应的一个例子是用测角器来测量在膝角度的变化来取代按钮。对于这种类型的实验中，测角器将被安装在膝关节的侧表面上，而不是按压按钮，参与者将被指示在其膝盖弯曲到预定义膝角度（ 例如，90度）时电机振动的看法。用户的反应时间则定义为施加刺激并且当关节一个之间所经过的时间GLE沉降或接近期望的值（ 例如，90°±10°）。运动精度，也可以通过计算目标和实现角度之间的百分比误差进行评价。 在过去的十年中，许多研究已经探索在各种生物力学应用中使用的振动触觉反馈，包括其疗效作为改善步态和平衡14,15一个训练工具。这些研究大部分都集中在生物反馈的临床意义，研究具体的运动或运动参数的任何变化当施加振动触觉反馈。因此，大多数的协议选择一个组的刺激参数，很少检查用户灵敏度振动的位置，振幅，或图案。这里介绍的协议作为朝向理解用户感知的第一步骤，以振动触觉，应该在评估这些刺激对特定临床效果进行刺激iCal的条件。额外的工作，例如由Goodworth 等 7,16，探讨各种编码策略为翻译感觉信息到振动刺激，和Crea 等人的12，其评价用户在振动模式的改变的敏感性，补充这些实验，以提供如何更全面地理解，以优化对特定的生物力学应用振动触觉的反馈。 应当指出，这个实验系统的一个基本的限制，与在文献5,6报道的其他系统，在于所述振动电机，其中一对振动频率和幅度。即，在振动频率的增加或减少，伴随有幅度正比变化。这两个参数的分离需要不同类型的电动机，例如线性共振致动器，以及更先进的电动机驱动器的更复杂的马达供电。 ADDIT有理更新到现有的接口将需要以适应新的硬件和附加振幅参数。 校准过程是这些实验的成功执行关键的，并应在随后的实验中使用的各电动机独立地执行。而占空比频率关系应该是名义上相同类型相同电机，在电机结构小的差异，可能会导致非平凡的变化，从而导致频率。例如，当开发此过程中，一个180赫兹目标频率，使用范围从103-143为不同的马达的PWM值来实现的。

人工感觉反馈（ASF）可以被定义为提供实时生物信息对个人，往往补偿受损本体或其他感官机构的做法。 ASF已经受伤或残疾人康复领域长期被用来帮助身体机能和运动1方面恢复- 3，允许个人控制曾经是自主神经系统4的无意识反应的物理过程。 ASF，生物力学生物反馈的子类，用外部传感器来测量与平衡或步态运动学参数，并通过某种应用的刺激传达这一信息到个人。一种日益流行的方法的生物力学反馈采用小振动马达，或病理因素，放置在身体的不同部位，以提供空间以及时间上的反馈。以前的文献已经呈现promising结果支持的应用程序与下肢截肢，前庭障碍的人使用振动触觉反馈，和老化有关的平衡5的损失- 9。 控制一个人的感知和响应特定的刺激机制的深入理解是必要的通知有效实施针对不同的应用ASF系统。为振动触觉的反馈，这些机制之间主要是本体和感觉运动响应，特别是对施加振动用户的灵敏度和执行所需的反应所需的时间。通过振动刺激传达任何感觉信息必须被编码为振动频率的特定组合，幅度，位置和序列。因此，震动触觉ASF系统的设计应该选择的参数组合，以最大限度地刺激用户的感知和解释，如还有的及时性和所得到的马达响应的准确性。该协议的目的是提供一种从其中计算的响应时间和响应精度的各种振动刺激通知ASF系统设计为具有不同的感官受损人群使用的平台。 此处所描述的方法建立在以前的研究探索的触觉和震动触觉反馈3,5,6-人类感知，并用于沿两个以前的研究-10,11-用途被开发。后两种研究采用该协议来检查在下肢截肢的准确性和用户响应及时振动频率和位置的影响，显示出这两个参数显著影响结果的措施，而且有高度的响应精度的可实现。这些结果可以被用于通知病理因素的理想放置在今后的研究和振动触觉的ASF系统的临床应用。其他最近的工作由Crea 等人的12检查用户灵敏度行走期间施加到大腿中的振动模式的变化，使用口头响应来表示感知的改变振动模式，而不是电动机的响应。虽然这些口头响应可以被用于测量检测精度，他们不考虑错误和延迟可能存在在电动机控制过程。 用于以下实验的主要设置包括许多个连接到脉冲宽度调制的微控制器板的输出管脚振动马达中的。该板，反过来，通过通用串行总线（USB）连接到一台计算机上运行市售系统设计软件控制。电机需要额外的放大电路，以确保足够的电压和电流在很宽范围的振动频率的供给。一个例子放大器电路示于图1。的双极型结型晶体管（BJR）在图中可以用小的金属氧化物半导体场效应晶体管（MOSFET）为更有效的操作和更小的尺寸来代替。类似地，整个放大电路可以由一个关闭的，现成的触觉电机驱动器被替换，以提供额外的控制和减小的尺寸。每个电机都需要自己的电路，和使用本文中列出的设备，最多十个马达可以由单个微板来控制。 <img alt="图1" src="/files/ftp_upload/54223/54223fig1.jpg" /> 图1.电机配线。（A）中的放大电路，用于单个振动电机被示出。各电动机需要一个单独的电路和必须连接到微控制器上的一个独特的PWM输出端口。与V DD这里表示由微控制器板提供的3.3伏电源，并且当零电压是应用R2用作下拉电阻，以确保该晶体管开关的电阻保持打开撒了谎。 （B）的两个电机的物理线路的一个例子。尽管示出了八个单独放大电路，只有两个被连接到振动电机。在这个协议中，R1 = 4.7kΩ和R2 = 100kΩ的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54223/54223fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibrating Pager Motors</td>
			<td style="width:107px;">Precision Microdrives</td>
			<td style="width:119px;">Model 310-101</td>
			<td style="width:407px;">Coin eccentric rotating mass motors.&#160; As many as necessary to test all locations and interactions of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tri-axis Accelerometer</td>
			<td style="width:107px;">Dimension Engineering</td>
			<td style="width:119px;">ADXL 335</td>
			<td style="width:407px;">Advanced analog accelerometer. 500Hz bandwidth, 3.5-15V input.&#160; Designed for motion, tilt, and slope measurement, as well as vibration and shock sensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arduino Uno</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">DEV-11021</td>
			<td style="width:407px;">Microcontroller board for communicating with the tri-axis accelerometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arduion Mega 2560</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">DEV-11061</td>
			<td style="width:407px;">Microcontroller board for interfacing with the vibration motors.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabVIEW</td>
			<td style="width:107px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:407px;">Data acquisition software used to control motors and display accelerometer signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arduino IDE Software</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">v. 1.6.5</td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Push-Button</td>
			<td style="width:107px;">Bridges</td>
			<td style="width:119px;">Buddy Button</td>
			<td style="width:407px;">Wired switch featuring a 2.5in/6.35cm activation surface that provides an auditory click and tactile feedback.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optional:</td>
			<td style="width:107px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dedicated haptic motor driver</td>
			<td style="width:107px;">Texas Instruments</td>
			<td>DRV2605L</td>
			<td style="width:407px;">Can be used to replace the entire amplification circuit described in Step 1.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Flexible wearable goniometer</td>
			<td style="width:107px;">Biometrics Ltd.</td>
			<td style="width:119px;">SG110</td>
			<td style="width:407px;">Twin axis flexible goniometers to measure angles in up to two planes of movement that can be used in lieu of the push button to measure joint movement in response to stimuli. 
			www.biometricsltd.com/gonio.htm</td>
		</tr>
	</tbody>
</table>

a method for evaluating timeliness and accuracy of volitional motor responses to vibrotactile stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

图4示出的校准曲线识别为单个马达的180赫兹的振动频率的PWM值。以50％的占空比开始时，PWM值被重复，直到主频率尖峰发生在180赫兹。成功校准，试验应显示在主振动频率明显的尖峰。加速度计到电机，或者电机的支撑表面的差固定，可能会导致不明确的尖峰更弥漫的FFT。在这种情况下，支架已经调整后，以确保更好的连接的校准试验应该重复。 图5A示出记录实验1三个的振动频率，140赫兹，180赫兹，220赫兹，施加到大腿的前表面十身强力壮参与者和三个被截肢者10刺激和按钮响应之间的反应时间。重复测量的方差分析（ANOVA）和使用Bonferroni校正图基事后分析被用来确定各频率的具体效果。这些数据显示的数据在身强力壮的人口每个频率相对紧张的蔓延，以及显著频率的影响。用于对振动频率的区分反应时间示于图5B中 ，并且可以使用相同的步骤，在单一频率的测试来分析。类似的分析可以进行，以确定电动机的位置，反应机制的影响（ 例如 ，按下按钮以手或脚），或其它的测试条件。 <img alt="图4" src="/files/ftp_upload/54223/54223fig4.jpg" /> 图4.典型的校准曲线。在快速傅立叶结果变换的加速度数据的示为单个马达进行校准。四项试验进行了识别PWM水平correspon宝顶至180Hz振动（蓝色实线）。需要注意的是振动的不同频率之间变化。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54223/54223fig4large.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/54223/54223fig5.jpg" /> 图5.反应时间代表结果。（A）响应时间为个别频率显示。该线路连接的数据显示为健全的参与者（平均值±SD）中的数据，而各个数据点所代表的3个人一起经股截肢。反应时间频率显著下降。 &#39;*&#39;表示从140赫兹的反应时间都在显着差异p &lt;0.05显著差异，和"＃"从180赫兹频率的差。 （B）响应时间对区分频率s的绘制两个身强力壮的个人和那些与经股截肢。注意，在每一对中的数据的传播比为单频数据更大，这表明更可变的结果。这个数字已经从最初由Sharma等 10公布的数据修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54223/54223fig5large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> PWM值（脉冲） </td><td> 64 </td><td> 127 </td><td> 191 </td><td> 255 </td></tr><tr><td> 占空比 （％） </td><td> 25 </td><td> 50 </td><td> 75 </td><td> 100 </td></tr></tbody></table> 表1. PWM值和相应的占空比。样品PWM水平和相应的占空比所示。 ŧ他0-255范围为PWM值指定（在255）可为其中的信号是在每个脉冲的字节数。

Watch this Scientific Journal Video about A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli at JoVE.com

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

本文介绍用于施加振动触觉刺激对人参与者的大腿，并测量参与者的用于刺激的位置和频率的不同组合意志响应的精度和反应时间的技术。

一种意志的运动反应的及时性和准确性，以振动触觉刺激评价方法

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

a-method-for-evaluating-timeliness-accuracy-volitional-motor

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.2K 次观看.  Holland Bloorview Kids Rehabilitation Hospital. 本文介绍用于施加振动触觉刺激对人参与者的大腿，并测量参与者的用于刺激的位置和频率的不同组合意志响应的精度和反应时间的技术。 

视频: A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

Haptic/Graphic Rehabilitation: Integrating a Robot into a Virtual Environment Library and Applying it to Stroke Therapy

Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other forms of biofeedback. Previous human-robot interactive systems have required different software commands to be implemented for each robot leading to unnecessary developmental overhead time each time a new system becomes available. For example, when a haptic/graphic virtual reality environment has been coded for one specific robot to provide haptic feedback, that specific robot would not be able to be traded for another robot without recoding the program. However, recent efforts in the open source community have proposed a wrapper class approach that can elicit nearly identical responses regardless of the robot used. The result can lead researchers across the globe to perform similar experiments using shared code. Therefore modular "switching out"of one robot for another would not affect development time. In this paper, we outline the successful creation and implementation of a wrapper class for one robot into the open-source H3DAPI, which integrates the software commands most commonly used by all robots.

Recently, a vast amount of prospects have come available for human-robot interactive systems. In this paper we outline the integration of a new robotic device with open source software that can rapidly make possible a library of interactive functionality. We then outline a clinical application for a neurorehabilitation application.

触觉/图形康复:整合成一个虚拟的环境下图书馆的机器人，它应用于中风治疗

Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other ...

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 ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

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. ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Evaluating Primary Blast Effects In Vitro

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions,&#160;whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Evaluating Primary Blast Effects In Vitro

Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability.

评估主要爆炸效果体外

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...