We would like to thank the funding from Medical Research Council and Action Medical Research, which provided MG with a clinical fellowship, GN 2339, to conduct this work.

该协议遵循的关于禁止使用，储存和处置人体组织我们机构的人类研究伦理委员会的指导方针道德准则。人体组织样品可以从已同意为研究目的与相关的伦理批准尸体被切除。样品还可以从接受手术治疗的，具有相关的伦理批准同意患者丢弃的组织。 1.皮肤的制备<ol><li>通过手动解剖掉脂肪组织并使用手术刀刀片和镊子真皮深层的薄层制作标本。这个步骤是重要的，以确保样品14之间的一致性。 </li><li>所得刃厚皮片材切成标准化的样本大小（如 1厘米*5厘米样本）。确定基于所述检测装置的尺寸的试样尺寸。如果组织工程构建物也被测试，该specimeÑ尺寸应该适合感兴趣14的材料。手术刀刀片在适当的尖锐件箱处理。 </li><li>以使力学计算完成后，测量皮肤的厚度之前和机械测试之后使用电子卡尺进行测试。 </li></ol> 2.拉伸测试注意:所有的材料试验机应根据制造商的测试之前的准则进行校准。 <ol><li>试验皮肤样品在单轴拉伸，在室温（22℃）14使用材料试验机（ 图2A）。 </li><li>定向在所有样品（ 例如，垂直或在符合兰格线（绘制在地图上的人体的拓扑线和参照在真皮胶原纤维）的自然取向）14相同的方向上的皮肤样品。 </li><li>固定两个夹具之间的试样（共mmercial夹具），一个固定在98.07 N加载细胞，另一个不可移动基板14。在单轴拉伸测试夹具之间所产生的面积应为1厘米×4厘米（ 图2）。 注:商业夹具用来避免不均匀的把持和损坏测试前的样品。样品被固定在"用手指拧紧"密封性。 </li><li>盖两边用凡士林样品面积（在装置放置后），以防止样品脱水。 </li><li>程序中的拉伸负荷和放松的检验制度进入软件的操作列表如下:空载|零位|查找联系人（拉伸载荷）|等待（放松）。 </li><li>先从软件程序测试。荷载作用下的张力样品29.42ñ1毫米/秒。用率和负载不会引起皮肤的失败（ 例如，29.42Ñ在1毫米/秒）。 </li><li>达到29.42 N-二负载后，允许组织放松1.5小时，一个时间点在其中有在松弛行为变化最小，由计算机软件14控制。 注意:位移在松弛阶段，而不是负载保持恒定。 </li><li>计算弹性和粘弹性性质按上述分析部的准则。调查了机械性能将代表裂层皮肤成分（表皮和真皮）14的平均性质。 注意:没有定义的皮重负载的，因为它是从当变形发生时，因此，仅包含这些数据点的原始数据清楚。 </li></ol> 3.软骨的制备<ol><li>删除使用手术刀刀片和钳子15，16中的软骨样本的皮肤和筋膜。 </li><li>划分软骨标本成使用解剖刀刀片和镊子的标准化样品的大小（ 例如，1.5厘米的块）。对于所有的样品，使用semicircu拉尔形压头（ 图2B），其具有的直径与厚度比软骨样本的大小大至少8倍。这个比率可确保压头不会受到从标本制备15的任何边缘效应。手术刀刀片在适当的尖锐件箱处理。 </li><li>以使力学计算完成后，测量之前和之后的机械试验15，16利用电子卡尺加载的软骨的厚度。 </li></ol> 4.压缩压痕测试<ol><li>压缩在室温水合环境中使用的材料试验机的软骨样本。覆盖之前和压缩测试期间的磷酸盐缓冲盐水（PBS）的软骨样本以确保样品水合。 注:PBS不完全的生理环境相匹配，但它允许两种材料和组织是玉米同样相比15，16。 </li><li>定向的软骨样品所以表面是垂直于所述压头。这允许压缩是单轴，并限制任何剪切载荷15。 </li><li>程序中的压缩载荷和放松的检验制度进入软件的操作列表如下:空载|零位|查找联系人（压缩载荷）|等待（放松）。 </li><li>使用的软件程序开始测试。负载下压缩到2.94 n个样品以1毫米/秒15,16。 注意:这被确定为一个非破坏性的负荷是足够灵敏的，以确定软骨15的弹性和粘弹性。 </li><li>达到2.94-N限制后，使软骨到15分钟放松，一个时间点在其中有在松弛行为变化最小，使用计算机软件15,16。 注: 图图2C-D显示一组典型的为人体组织标本的压缩和拉伸试验。然后相同的协议可应用到合成生物材料的生物力学特性匹配被分析的天然组织。例如， 图2E-F展示了紧密匹配的合成材料的生物力学特性的人体组织的压缩和拉伸试验。 </li></ol> 5.计算杨氏弹性模量为压痕和拉伸试验<ol><li>收集的原始数据，包括从材料试验设备14-16时间（s），位移（毫米）和负荷（N）。 </li><li>计算使用图3中所示的式的应力（兆帕）和应变（％）。 注意:如果压缩测试期间使用的半球形压头，由截面积除以力可以提供标称（平均）应力，但不是峰值应力。 </li><li>使用线性散点图绘制应力兆帕（y轴）对应变（x轴）。确定线性曲线拟合。线性曲线拟合是等于y = mx + b中与各自的R值。 注:包括所有的数据点，实现最低R值&gt; 0.98。 m值是斜率，其对应于应力的过度应变模量，这表明在兆帕压电阻或耐张力（ 即，杨氏模量）。如果R值不&gt; 0.98，那么表征线性粘弹行为的假设是无效的。 </li><li>以识别在其中因暴露于变形的流体流动达到平衡，应力随时间过去200的机械试验，并在试验结束时的最终应力水平的比例来计算的粘弹性性质。 注:随着时间，紧张程度会降低（放松），为流体流动达到平衡17,18。快速应力松弛反应indicat上课，这是困难的样品17，18内保持高的应力。 </li></ol> 6.放松属性<ol><li>在兆帕（y轴）对上一个线性散点图以s（x轴）的时间曲线图的应力。 </li><li>确定的线性曲线拟合来计算松弛率。线性曲线拟合等于Y = mx + b中与最后200秒的相应数值。 m值是松弛的速率。 </li><li>包括所有的数据点，以获得最小R值&gt; 0.98。在用于皮肤1.5小时，对于软骨15分钟的最终应力（兆帕）是最后的绝对松弛值。 </li></ol>

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The authors have nothing to declare.

一些拉伸和压痕协议已经公布的表征人体软组织。我们已经提供了另一种方法，其目的是更诊断和非破坏性。在这个协议中进行机械测试的样品通过负载，而不是通过位移的限制，因为换能器是比位移加载更加敏感。因此，该实验的复制品可以横跨组织和合成材料更精确。使用这种技术，我们已经证明了皮肤组织和软骨组织分析的压痕协议评估拉伸协议。这两个协议都容易和简单的实现，并可能被视为人体软组织和组织工程结构的表征。 一项所述的方法的重要步骤，以获得适合于分析的应力松弛曲线是确保样品不测试期间打滑。充分固定是requir版，但这必须要针对在试样上产生的任何应力，并确保压头垂直于表面，以防止任何剪切负荷平衡。至关重要的是，该组织的组合物，以及尺寸和形状的样品相似。对于软骨，至关重要的是使用重复的解剖协议和样品尺寸。对于皮肤样品，至关重要的是要除去所有的皮下组织，以便获得可重复的样品。同样重要的是，以确保所有样品中，样品的条件是相同的，包括水合，室温，解冻过程中，如果合适的。 有所呈现的协议一定的局限性。有研究表明，皮肤和软骨变形特性取决于试样取向13。皮肤被认为是各向异性的，早在19 世纪 ，随着兰格证明于1861年，皮肤具有天然的线张力的称为兰格线4。因此，表征皮肤样品时，它向定向的所有样品平行或垂直于朗格行，以避免引入的方法偏压4是重要的。软骨还显示各向异性特性，并包含Hultkrantz线，这是相当于兰格线，所以软骨可以根据在它被加载12,19的方向变形不同。因此，重要的是要增加样本大小，以允许软骨在不同方向的检测。作为组织的生物力学性能还与年龄和性别不同，研究应该具有代表性患者队列中进行，以保持有效性临床设置。此外，一些机械协议主张预处理，其中所述组织经受循环加载，以确保组织是在随后的机械测试20的稳定状态。然而，p的确切机制再生是不清楚，以产生一致的和可重复的响应所需要的循环的确切数目在不同的研究20而变化。研究者应该考虑是否要包括评估执行特定的生物力学测试20的原因后预处理。 皮肤是一个复杂的，多层次的材料，分为三个主要层:表皮，真皮，和皮下组织4。 皮肤组织的机械性质最近已使用体内评估4评价。 然而，拉伸试验的协议可以利用来理解切除皮肤4的皮肤生物力学。这样的测试可以提供信息，以应力-应变关系进行建模，由于边界条件可以定义4。通常， 在体外试验制度使用高应变的材料表征故障，而在体内系统中使用低应变范围4。当张力切除皮肤的生物力学比较值，有不同的研究之间有很大的变化，从2.9-150兆帕4。主体之间的巨大差异，预计由于自然变异生物，但在协议制度的差异也可以加剧这种自然的生物学差异。例如，在协议之间加载率的差异会导致变型中，作为更大的负荷率造成的流体流出时间较少，造成了更高的刚度。皮肤组织的制备中，切除和处理协议也将导致机械性能4的差异。这个协议证明测试皮肤为研究人员提供以表征皮肤组织的另一种方法。它提供了一些优点，包括识别一个机械测试皮肤组织的弹性和粘弹性性质的能力，允许的皮肤更好地理解在很短的时间量。此外，相同的测试可应用于组织工程替代来制造具有类似生物力学性质如天然皮肤构造。 压痕试验相比，了解软骨21的生物力学密闭压缩测试提供了一个有吸引力的选择。压痕具有保护软骨的生理结构的能力，从而提供了模仿那些临床环境的值。使用压痕，它也有可能测试软骨同时仍然附连到下面的骨骼。压痕也允许作为体内软骨的生理测试。当两个软骨表面彼此接近时，由于接触面积下的水周围接触"隆起"的区域中的边缘被压缩变形发生17,21后横向移位。软骨缩进必须与在被进行具有比软骨样本更小的半径D输入，以允许类似鼓胀。压头的大小也应至少8倍的样本大小，以确保就好像它是一个不确定的样品22的部分的软骨反应。使用压头比样品直径的半径消除存在于试样创建任何边缘效应小得多。另外，压痕避免了通过测试由样品提取损坏软骨缺损可能的实验误差。压痕也不涉及深样品制备，如密闭压缩，允许小的，薄件软骨待测试17,21。另外，压痕的非破坏性方法意味着它在临床环境的潜在应用为确认和验证的研究后的诊断工具已被执行。 有齿痕，用户必须确保approp关键假设riate结果。在缩进装载一个关键边界条件要求压头和软骨表面之间的恒定接触（ 即，该表面不会变形从压头远）23，24。压痕载荷还包括假定的边界条件，即软骨表面与压头之间的接触是无损（ 即，压头是在与表面接触，但通过表面不走;软骨表面不应下失败压头）25 - 26。有研究表明，这个边界条件，可以通过使用印油墨，当施加到软骨表面25，26，这将弄脏损坏的区域进行验证。进一步的边界条件假定压头压缩垂直于样品表面的软骨。压缩的垂直方向是一个重要的边界COND银行足球比赛，因为在一个角度压缩，特别是如果使用循环加载，可能引起滑移，这可能导致剪切元件和改变机械加载。这个条件可以保证通过仔细的测试设备设置。 汇总协议已经针对感兴趣的软组织被优化后，为研究人员寻找到感兴趣的组织的动态测试将是有用的。标本适当的循环载荷应模仿正常的生理极限和行为，如模仿行走或其他重复动作27。总之，本报告展示了简单的机械测试协议，以评估人体组织。实施这些协议将提供有关组织的生物力学特性的关键信息，使组织工程结构，以更好地模仿天然组织。

患者越来越等待各种器官移植治疗失败或受伤的器官。然而，与合适的供体器官的短缺，再生医学的目标是创建患者终末期器官衰竭替代解决方案。再生医学的目的是满足这种临床需要通过工程材料充当组织代用品，包括软组织，例如软骨和皮肤。要创造一个成功的材料，以恢复受损的组织，更换材料应模仿它要更换1-2本地组织的属性。一旦手术植入，该材料将需要提供解剖学形状的组织缺陷，因此，该材料的机械性能是至关重要的1。例如，在更换耳廓软骨的材料应当具有适当的机械性能，以防止压缩由覆盖皮肤2。同样，材料以取代鼻车tilage将需要有足够的机械性能，以防止呼吸3期间塌陷。然而，尽管制造用于注入材料时，机械性能的重要性，没有证据集中于表征不同人体组织的机械性能。 机械测试机制可用于建立压力，拉力，弯曲，或一个组织的剪切性能。皮肤是一个高度各向异性，粘弹性，几乎不可压缩材料4-9。通常切皮肤使用单轴拉伸的方法，其中皮肤的一个适当形状的条带在两端夹住并拉伸而负荷和延伸记录4-9的测试。 由于所有的软组织的主要成分为间质水，软骨的机械响应强烈相关流体穿过组织10-11的流动。软组织，如软骨ħAVE使用压缩试验传统上进行测试。在压缩测试的方法是相当多样，以密闭，潜水和缩进是最普遍的（ 图1）。内密闭压缩，软骨样品放置在一个不透流体填充井并装载通过多孔板。由于阱是无孔的，流过的软骨是在垂直方向12-13。在无侧限压缩，软骨，使用的非多孔板到非多孔室加载，迫使流体流为主要径向12-13。缩进是用于评价软骨12-13的生物力学特性的最常用的方法。它由一个压头，比试样表面较小被测试的，即放倒到样品。压痕有超过压缩的其他方法，包括事实可以原位执行的压痕，enabli许多优点纳克测试更加生理（ 图1）12-13。 理解的组织的压缩和拉伸性能，杨氏弹性模量，通常通过分析应力-应变曲线的线性部分，指示压缩或拉伸的弹性阻力计算，而不管试件尺寸12。两个拉伸和压缩试验制度可以根据负载或变形施加并且两个这样的参数的速率而变化。目前，有许多不同的测试协议，以评估组织力学，这使得它非常难以解释或从不同研究6-13比较结果。此外，许多机械方法目前集中在通过检体检测到破坏表征的组织的机械性能。 我们的目标是展示一个压痕和拉伸协议，为人类提供直接的，非破坏性的比较软组织和组织工程化构建体。 我们表明，限制了机械测试压力，但仍获得一个年轻的公司在压缩和拉伸弹性模量的方法。样品被强调无论是在拉伸或压缩到一定值，并且一旦选择了应力值已达到，样品被允许放松，同时所有的数据被记录。此方法同时捕捉相同的测试中的组织，其可以直接应用到合成材料的粘弹性和松弛性能。我们已经使用了缩进协议 评估人体软组织，包括皮肤和软骨14-16。软骨是用压痕试验评估，皮肤是用拉伸试验14-16评估。研究人员旨在工程师具有相似属性的人的软组织材料，可以考虑实施这些协议。

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:143px;">Digitial Vernier Calipers</td>
			<td style="width:95px;">Machine Mart</td>
			<td style="width:104px;">40218046</td>
			<td style="width:427px;">Digitial vernier caliper is used to measure sample thickness.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:143px;">Water Bath&#160;</td>
			<td style="width:95px;">Cole Parmer</td>
			<td style="width:104px;">UY-12504-94</td>
			<td style="width:427px;">StableTemp Digital Water Bath Flask Holder used to defrost tissues samples if they are frozen.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:143px;">Mach-1 Material Testing Machine</td>
			<td>Biomomentum&#160;</td>
			<td style="width:104px;">V500c</td>
			<td style="width:427px;">Mechanical Testing Machine used to test the mechancial properties of the tissues.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:143px;">Scalpel Blade&#160;</td>
			<td style="width:95px;">VWR</td>
			<td style="width:104px;">233-5335</td>
			<td style="width:427px;">Scalpel blades using to cut and dissect the tissues.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:143px;">Forceps&#160;</td>
			<td style="width:95px;">VWR</td>
			<td style="width:104px;">470007-554</td>
			<td style="width:427px;">Forceps used to dissect the tissues.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:143px;">Phosphate Buffered Saline (PBS) pH 7.2</td>
			<td style="width:95px;">Life Technologies&#160;</td>
			<td style="width:104px;">20012019</td>
			<td style="width:427px;">PBS is used to hydate the tissue samples&#160;</td>
		</tr>
	</tbody>
</table>

biomechanical characterization of human soft tissues using indentation and tensile testing

再生医学的目的是工程师的材料来代替或恢复受损或患病的器官。这样的材料应该模仿他们的目标，以取代人体组织的机械性质;以提供所需的解剖学形状，材料必须能够承受的机械力时在缺损部位植入他们将遇到。虽然组织工程支架的机械性能是非常重要的，即经历恢复与工程材料许多人类组织没有得到充分的生物力学特征。几个压缩和拉伸协议报告用于评估材料，但有大的变化是困难的比较研究之间的结果。研究进一步复杂化的是机械测试的往往破坏性质。虽然组织失败的理解是重要的，同样重要的是具有下更生理学弹性和粘弹性特性的知识ogical负载条件。 此报告的目的是提供一种微创破坏性协议来评价人体软组织的压缩和拉伸性能。作为这种技术的例子中，皮肤的拉力试验和软骨的压缩测试中描述。这些协议也可以直接应用到合成材料，以确保机械性质类似于天然组织。协议评估人类天然组织的机械性能将允许一个基准由创建合适的组织工程化的替代品。

图4和5提供通过压痕和拉伸试验得到的数据的例子。 图4展示了人体软骨压痕试验后得到的典型值。 图4A是压痕试验之后获得的典型菌株抗应力曲线图的一个例子。以获得杨氏模量，所有的值都包括直到行曲线拟合具有0.98（ 图4B）的最小R值。 m值是杨氏模量在兆帕的指示符;例如，在该数据中，软骨具有1.76兆帕的模量。 图4C示出了应力对时间的典型曲线图，以评估软骨的松弛性能。松弛的速率从最后一个200秒计算的。同样，要获得松弛的速率，使用了线曲线拟合以MPa的m值的。例如，在该数据中，软骨具有的速度8.78×10 -6兆帕/秒（ 图4D）的放松。松弛的绝对最后一级是应力的以MPa最终点。例如，在该数据集，松弛的绝对最终水平将是0.028兆帕（ 图4D）。 图5显示了如何拉力试验评估后，皮肤组织的粘弹性。该分析作为每个压缩试验进行。 图5A展示了从拉伸试验协议获得的典型菌株抗应力图解。以获得杨氏拉伸模量，所有值都包含直到行曲线拟合具有0.98（ 图5B）的最小R值。 m值是杨氏模量在兆帕的指示符;例如，在该数据，对皮肤有0.62兆帕的模量。 图5C示出了相对于时间的压力的典型曲线图Ò评价松弛性能˚F皮肤。松弛的速率从最后一个200秒计算的。同样，要获得松弛的速率，使用了线曲线拟合以MPa的m值的。例如，在该数据，对皮肤有3.1×10 -5兆帕/秒（ 图5D）松弛的速率。松弛的绝对最后一级是应力的以MPa最终点。例如，在该数据组中，电平将是0.64兆帕（ 图5D）。然后在同样的分析可用于分析在压缩和拉伸测试生物材料到其生物力学特性匹配天然组织。 <img alt="图1" src="/files/ftp_upload/54872/54872fig1.jpg" /> 图1: 示意图来说明不同的压缩方法。 A.压痕测试。负载被施加到使用非多孔压头软骨的一个小区域。 B.限制ð压缩。软骨样本被放置在一个不透流体填充好。软骨然后通过多孔板加载。由于阱是不透水，流过软骨是仅在垂直方向上。 C.无侧限抗压强度。软骨是使用非多孔板到非多孔室加载，迫使流体流是主要径向的。 <img alt="图2" src="/files/ftp_upload/54872/54872fig2.jpg" /> 图 2: 力学试验机的设置。 A.试验机的插图。 B点 。用于压缩试验分析压头的插图。使用压缩压痕试验正在分析C.软骨。 D.皮肤组织被下拉力试验分析。合成生物材料E.拉伸试验。 F。 </stronG&gt; A合成生物材料的压缩试验。 <img alt="图3" src="/files/ftp_upload/54872/54872fig3.jpg" /> 图3: 用于计算的组织或组织工程构建物的压缩和拉伸的机械性能公式。 （N），用于计算力式中，应力（兆帕），和应变（％）。 <img alt="图4" src="/files/ftp_upload/54872/54872fig4.jpg" /> 图4: 人软骨的压缩分析的实施例。 A.应力与应变分析。 B.行曲线拟合方程的m个值是杨氏弹性模量在兆帕。 C.应力-时间分析来证明松弛性能。 D.行曲线拟合方程的m个值表示松弛率。最后一个bsolute速率是图表上的最后一个点。 <img alt="图5" src="/files/ftp_upload/54872/54872fig5.jpg" /> 图5: 人 的 皮肤拉伸分析的例子。 A.应力与应变分析。 B.行曲线拟合方程的m个值是杨氏弹性模量在兆帕。 C.应力-时间分析来证明松弛性能。行曲线拟合方程的D的m值的等同于松弛率。最终的绝对速率是图表上的最后一个点。

Watch this Scientific Journal Video about Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing at JoVE.com

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

人体软组织的生物力学特性采用压痕和拉伸试验

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

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31.2K 次观看.  University College London (UCL). Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

视频: Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

科研

生物工程

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, ...

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

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

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

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

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

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

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

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

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

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

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

新生儿小猪体内胸腺菌的活体力学试验方法

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

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

鼠头的生物力学试验

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

利用软光刻改变根面微结构的仿生复制

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

使用压痕和磁共振弹性成像对软组织模拟明胶模型进行粘弹性表征

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

基于原子力显微镜的人关节软骨外植体微压痕研究的实际问题

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...