这项工作得到了卫生部根据批RHD064968A的国家机构，部分由美国国家科学基金会根据批0931820，1149385格兰特，格兰特1361549部分支持，并在通过根据批H133G120165的国家残疾与康复研究的一部分。作者感谢林都鼎旺和杰拉尔德Hefferman在罗德岛的大学和迈克尔J.庵在庵矫正和假肢技术有限责任公司，为他们的伟大的建议，并在此研究的援助。

1，平台的供电采用经股假肢的神经控制的实现工程平台，在这项研究中，以实施和评估的动力人工腿神经控制开发的。硬件包括台式电脑采用2.8 GHz CPU和4 GB的RAM，一个多功能数据采集板与模拟 - 数字转换器（ADC）和数字 - 模拟转换器（DAC），一个电机控制器，数字I / O，以及一个典型的动力经股假体设计在我们的小组12。首先由ADC的数字化和流进入桌面PC进行信号处理的模拟传感器输入。该DAC被用于控制输出通过电机控制器来驱动直流电动机中的假体。数字I / O被用来使能/禁止电机控制器。该供电假体拴在台式PC和搭载一台24 V电源。 该软件在编程的广告适用于虚拟仪器在桌面PC上运行才有发展环境。开发环境是基于虚拟仪器技术，它有效地结合了用户自定义的软件和硬件来实现自定义的平台。通过使用图形框图的结构，不同的模函数的节点可以容易地和有效地实现和更新。为了演示平台的动力人工腿的在线控制功能，初步设计的假肢控制是在这个平台上实现。该控制系统包括一个神经控制器和一个内在的控制器。神经控制器包括我们基于神经肌肉机械融合，其中确认了用户的活动模式，以前的设计NMI的。神经控制器作为一个高层次的控制器，分层次的内在控制供电下肢假肢的控制连接。 续的体系结构在平台上ROL软件如图1所示的NMI包含两个部分：离线训练模块和在线测试模块。离线训练模块被设计用于收集训练数据和NMI建立分类器。所收集的多通道表面肌电信号和机械测量第一预处理和分割成连续的滑动窗口。在每个窗口中，特征的表征信号模式被提取，然后融合成一个特征向量。在每个窗口的特征向量被标记的训练数据收集过程中根据假体用户的演出活动活动模式（类）和相位指数和假体的状态。标记的特征向量，然后用来建立一个相依赖的模式分类器，它包含多个子分类器具有单独的阶段相关。所创建的分类保存，转移到后在线评估在线测试模块。&lt;/ P&gt; 在线测试模块来识别在线用户的运动意图，并在切换内在控制器的活动模式。多通道神经肌肉和机械测量，同时涌入的在线测试模块，并转化为特征向量。然后将特征向量送入这是已经建成的离线训练模块相依赖的分类。基于固有控制电流相位，则相应的子分类器被开启，并用于识别用户的意图。分级输出被进一步后处理和发送到固有控制器切换活动模式。 有限状态机（FSM）的阻抗控制器的供电人工腿的内在控制实施。产生的阻抗控制器所需力矩输出对膝盖关节。有限状态机按照调整后的关节阻抗表演活动的当前状态。对于运动活动（ 即水平地面行走和斜坡上升/下降），密克罗尼西亚包括对应于5个步态阶段五种状态：立场屈曲（STF），姿态扩展（STE），预摆（PSW），摆动屈伸（ SWF），并且摆动扩展（SWE）;静态站立，密克罗尼西亚包括两个阶段：负重（WB）和非负重（新巴）。状态之间的转换是由地面反作用力，膝关节位置触发。活动模式之间的转换是通过从在线测试模块的输出控制。对于上面所讨论的所有三个模块，图形用户界面（GUI）建成，这使得在实验室实验人员能够轻松地调整控制参数，监控系统性能，并进行评估实验。 2。实验装置<ol><li>表面肌电信号（sEMG的） 从截肢的大腿肌肉subjectR的表面肌电信号17，S残肢通过无线肌电采集系统记录。有针对性的大腿肌肉包括股直肌 （RF）， 股laeralis（VL），股内侧肌 （VM）， 股二头肌长 （BFL）， 缝匠肌 （SAR）， 半腱肌 （SEM）和收肌 （ADM）。值得注意的是，正是针对特定肌肉是没有必要的，因为8在NMI的用于模式识别算法搜索多个肌肉激活模式以识别活动模式。只要测得神经肌肉控制信息髋关节和膝关节的控制就足够了，肌电图记录之间的交叉谈判对NMI的性能影响不大。 <ol><li>让拍摄对象进行髋关节屈曲/伸展，髋关节内收/外展，并尝试弯曲/延长截肢膝关节时，他是在站立姿势。 </li><li>近似​​的，由肌肉触诊的确定电极安置的位置肌电图记录的ð检查。 </li><li>嵌入在电极中的新设计的肌电电极插座接口， 如图2中所示，两个主体的舒适和可靠的电极-皮肤接触。 </li></ol></li><li>从有源假体的机械测量 由6自由度称重传感器从假肢塔测量的机械地面反作用力和力矩融合与肌电信号识别拍摄对象的运动意图。此外，一个电位器仪表上的膝关节测量膝关节角度和一个编码器被连接到DC电动机来计算膝关节角速度。这些测量被用作内在的控制反馈信号。 <ol><li>挂载6度自由度称重传感器上的假肢塔。 </li><li>使X轴，Y轴，和称重传感器的Z轴与内外侧方向，前后方向，和假体的superoinferior方向，各自LY。 </li></ol></li><li>实验室环境设置 为了评估的动力人工腿被截肢者经股神经控制，一个超越障碍训练场始建于实验室， 如图3，测试环境包括5米直走道，一个4米长的坡道倾斜8°，并与坡道级平台的角度连接牢固。沿着坡道，扶手栏杆安装了以提高受试者的安全。另外，在天花板轨道系统具有防坠落安全带被设置成保护的主题从实验过程中掉落。 </li></ol> 3，实验协议这项研究是与机构审查委员会（IRB）在罗得岛大学的批准，并与招募受试者的知情同意下进行。一名男性单方面的经股截肢截肢（病因：外伤，年龄：57岁;截肢的持续时间：32年S）被吸收了这项研究。残肢（从坐骨结节测到残肢的前端）向非受损侧（从坐骨结节测到股骨上髁）的长度的长度之间的比率为51％。主体通过他的日常生活吸入悬浮插座穿的是微处理器控制的假肢膝关节。在实验前在这项研究中，这个问题收到由一名物理治疗师几次培训班，以便让被摄体来适应用电设备和校准每个活动模式所需的阻抗。 <ol><li>主题准备<ol><li>测量受试者的体重，身高，并记录他的性别和年龄。 </li><li>让拍摄对象放在了自己的短裤在一个私人制剂室。 </li><li>把大小安装防坠落安全带的主体，然后将其连接到天花板的铁路系统。 </li></ol></li><li>肌电图记录准备<ol><li>选出七个充满电的无线肌电传感器和打开它们。 </li><li>将肌电传感器插入定制的吸插座准备位置。写下传感器的顺序号并将其与肌电图的位置联系起来。 </li><li>清洁主体的异丙醇垫残肢的皮肤上。 </li><li>帮助主题在穿上吸插座，并确认插座牢固地连接到主体的残肢。 </li><li>打开实时肌电图的模拟数据流的软件。 </li><li>让拍摄对象进行髋关节屈曲/伸展，髋关节内收/外展，膝关节屈曲/伸展并检查肌电图信号来验证肌电电极接触和数据传输。 </li></ol></li><li>对齐和有源假腿的初始校准<ol><li>指示须留在站立姿势，同时举行一个辅助步行器。 </li><li>装上假肢供电到吸入插座一个金字塔ADAP器。调整一组旋转螺钉适配器上，直到假体的位置在几何上与插座对齐。这个过程是由假肢进行。 </li><li>让拍摄对象解除假体离地面和校准称重传感器上的假肢塔。 </li><li>指示主题实践走在不同的地形（ 例如 ，水平地面，斜坡上升和斜坡下降）穿着动力假肢时。这个过程一直持续到主题感到有信心在行走与用电设备并在每个活动的收益率表现一致步态。 </li></ol></li><li>训练数据集为训练分类器在NMI <ol><li>指导受试者站在一个预定义的行走路径的起始位置，如示于图3。 </li><li>打开电动假体和加载参数到本征控制器。 </li><li>运行一个培训数据收集COM计算机程序和设置的内在控制，通过点击图形用户界面（GUI）上的“站立”按钮站立模式。 </li><li>通过点击界面上的“开始录制”按钮，开始采集数据。指示须留在站立姿势5秒。 </li><li>指示须走过去的水平地面上他/她的自我选择舒适的步行速度;同时，点击前脚尖离被摄物体的领先腿的GUI中的“行走”按钮，设置的内在控制水平地面行走模式。 </li><li>当拍摄对象是接近斜坡上升的边缘，单击“斜坡上升”按钮，GUI上的脚趾离地的假腿踩着斜坡前和开关的内在控制斜坡上升模式。为了安全起见，让被摄体在斜坡上行走时用一只手栏杆。 </li><li>当物体在斜坡末端，点击“行走”按钮前再次假腿的脚跟罢工踏上级平台和开关假体的内在控制水平地面行走模式。 </li><li>在行走路径的末尾，指示须停止并保持站立姿势。与此同时，单击“常规”按钮，双支撑期前和切换的内在控制权交还给站立模式。 </li><li>经过大约5秒，单击“停止”按钮停止收集数据。标签收集到的数据为“训练数据集1”。 </li><li>重复上述步骤3.4.4-3.4.9当主体走在相反的路线回到起始位置;唯一的区别是开关的内在控制坡道下降模式时，须走在下降斜坡。 </li><li>重复3.4.4-3.4.10到10完整的训练数据集收集。检查所收集训练数据集的信号质量。 </li><li>让被摄体有日之后的休息时间E数据采集时段。 </li><li>通过离线训练模块（ 图1）在训练NMI模式识别分类器。使用收集到的肌电图和机械信号，活动模式（类）培训过程中标记和检测阶段建设相依赖的模式分类。自动保存分类器的参数后在线测试环节。 </li></ol></li><li>供电采用经假体的神经网络控制的在线测试<ol><li>指示须站在步行路径的起点。 </li><li>打开电动假体。加载训练的分类器，以在线测试模块和所述参数的特性的控制器。 </li><li>指示对象开始测试试验中站立姿势，然后连续过渡到水平地面行走，坡道行走，水平地面行走，并最终停止，并在行走路径的末尾完成这项试验。指示受在一个舒适的步伐进行每一项活动。允许试验之间的休息时间，避免过度疲劳。 </li><li>在每个测试试验中，显示假体电视监视器上的活动模式和膝关节角度读数。保存所有的测量和控制输出后评估的目的。 </li><li>重复步骤3.5.1-3.5.4到10齐全的检测试验已完成。 </li></ol></li></ol>

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没有利益冲突的声明。

工程平台，在这项研究中来轻松实现，优化，发展动力假肢的真实神经控制开发的。整个平台被设计在一个虚拟仪器的开发环境，并在台式机上实现的。控制软件是由几个独立的和可互换的模块，在其中每一个特定的功能被执行（ 即 NMI意图识别，和固有控制）。这种模块化设计的优点在于，每个功能块可以很容易地调试，修改和更新。此外，添加或删除功能，或改变模块之间的连接可以?...

本站下肢假肢已经获得了越来越多的关注在商业市场1,2和研究团体3-5。相较于传统的被动假肢，假肢机动关节有让下肢截肢者更有效地执行，穿无源器件时，是很难或不可能活动的优势。然而，目前，平滑无缝过渡的活动（ 例如 ，从水平地面步行到上楼梯）仍是动力假肢使用者一个具有挑战性的问题。这种困难主要是由于缺乏用户的人机界面，可以“阅读”用户的运动意图，并及时调整，以假体的控制参数，使用户能够无缝地切换活动模式。 为了应对这些挑战，各种方法在设计人机界面已经探索。根据其中NMI的肌电（EMG）的信号已显示出巨大的潜力，让直观的控制的电动下肢假肢。最近的两项研究6,7报道经股截肢者的膝盖缺失的监测过程中坐姿残余的肌肉记录的肌电信号拟提出的动议进行解码。金等 。5用于从残留柄的肌肉测量，以确定一个小腿截肢2运动模式（水平地面行走和下楼梯）肌电图信号。 Huang 等人 8提出了一种相依赖的肌电模式识别方法，可以识别7活动模式与约90％的准确度为展示两个经股截肢。为了更好的提高意图识别性能的基础上，神经肌肉机械融合一个NMI的目的是在我们组9和在线评估，对经股截肢者佩戴被动式义肢的意图识别10,11。这NMI能准确识别用户的预定活动及预测活动转变9，这是为动力人工腿神经控制可能有用。 我们所面临的当前问题是如何对我们的NMI融入假体控制系统，以使假体直观操作，保证了用户的安全性。开发真正neurally控制的人工腿需要一个灵活的平台，在实验室中，便于实施和假肢控制算法的优化。因此，本研究的目的是报告用于测试和优化假肢控制算法，在我们的实验室开发了一种灵活的工程平台。此外，新的实验装置和协议都用于评估患者的neurally控制动力经股的假肢下肢截肢安全和有效地。该平台和实验设计本研究提出可能有利于未来德韦讲习班中的真实neurally控制，供电的假腿。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Trigno Wireless EMG Sensors</td>
			<td style="width:221px;">Delsys, Inc.</td>
			<td style="width:143px;">7</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Trigno Wireless EMG Base Station</td>
			<td style="width:221px;">Delsys, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Multi-functional DAQ card (PCI-6259)</td>
			<td style="width:221px;">National Instruments, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Potentiometer (RDC503013A)</td>
			<td style="width:221px;">ALPS Electric CO., LTD</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Encoder (MR series)</td>
			<td style="width:221px;">Maxon Precision Motors, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motor controller (ADS50/10)&#160;</td>
			<td style="width:221px;">Maxon Precision Motors, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">24 V Power Supply (DPP480)</td>
			<td style="width:221px;">TDK-Lambda Americas, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">6 DOF Load Cell (Mini58)</td>
			<td style="width:221px;">ATI Industrial Automation</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Ceiling Rail System</td>
			<td style="width:221px;">RoMedic, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NI LabView 2011</td>
			<td style="width:221px;">National Instruments, Inc.</td>
			<td style="width:143px;">1</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering platform and experimental protocol for design and evaluation of a neurally-controlled powered transfemoral prosthesis

以使动力人工腿直观的操作，用户和假体之间的界面处，可以识别用户的运动的意图是需要的。一种新型的神经 - 机接口（NMI）的基础上在我们以前的研究开发神经肌肉机械融合已经表现出了很大的潜力，找准经股截肢者的预期变动。然而，该接口还没有被集成到一个有源假腿为真神经控制。本研究旨在汇报（1）一个灵活的平台来实现和优化供电下肢假肢神经控制和（2）的实验装置和协议，以评估患者的神经控制假肢下肢截肢。第一个基于PC和可视化编程环境在一个平台上开发，以实现假体的控制算法，包括NMI的训练算法，NMI在线检测算法，和内在的控制算法。为了演示功能这个平台，在此基础上研究神经肌肉的机械融合的NMI是分层与原型经股假体的内在控制一体化。一个病人有单侧经股截肢招募表演活动，如站立，电平的地面上行走，斜坡上升时，以评估我们实施的神经控制器，并在实验室连续坡道血统。一种新型实验装置和协议进行，以安全，高效地测试新的假肢控制开发的。概念证明的提交平台，实验装置和协议可以协助的neurally控制供电人工腿的未来发展和应用。

图4a显示七个通道表面肌电信号从主体的残肢大腿肌肉测量，当他进行髋关节屈/伸，如协议3.2.6中所述， 图4b显示肌电信号六步态周期在录制时须走过电平的地面上行走路径，协议3.3.4中。从图中可以看出，新设计的肌电电极插座接口可以提供表面肌电信号测量的质量好。 图5显示控制模式，检测阶段，并导致膝盖在一个代表性的在线检?...

Watch this Scientific Journal Video about Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis at JoVE.com

Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

神经 - 机接口（NMI）已开发，以确定用户的运动模式。这些NMI的是潜在的动力驱动的人工腿的神经控制是有用的，但没有得到充分展现。本文提出了（1）我们设计的工程平台，便于实施和神经控制的发展动力下肢假肢和（2）的实验装置和协议在实验室环境评估neurally控制的人工腿的患者下肢截肢安全和有效率。

工程平台和设计一个Neurally控制供电采用经假体与评价实验方案

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is ...

engineering-platform-experimental-protocol-for-design-evaluation

Research

JoVE Journal

Bioengineering

16.2K 次观看.  North Carolina State University & University of North Carolina at Chapel Hill. 神经 - 机接口（NMI）已开发，以确定用户的运动模式。这些NMI的是潜在的动力驱动的人工腿的神经控制是有用的，但没有得到充分展现。本文提出了（1）我们设计的工程平台，便于实施和神经控制的发展动力下肢假肢和（2）的实验装置和协议在实验室环境评估neurally控制的人工腿的患者下肢截肢安全和有效率。 

视频: Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

科研

生物工程

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

基于海胆大白鲨A接地采样：仿生为设计一个协议

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

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 Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

可重构 DNA 手风琴架的设计与合成

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...