财政支持是来自美国国立卫生研究院主任的新的创新奖（1DP2HL117750）提供给JMS来自美国国立卫生研究院生物力学在再生医学T32训练计划（2T32EB003392），以QJ从多德 - ICES奖学金和AWF。

1，制作母模使用光刻<ol><li>细胞外基质蛋白纳米纤维，nanofabrics和纳米结构是捏造的，首先设计使用计算机辅助设计（CAD）软件。然后这个CAD文件被传送到一个​​光掩模。光罩的类型将取决于功能的决议;用透明度为基础的光罩足够的特征尺寸降低到〜10微米。较小的特点&lt;10微米，需要在玻璃光掩膜铬。所有这里介绍的纳米纤维和纳米结构是使用一个透明的基于光掩模制​​造的，因而是纳米级的厚度，但不能横向尺寸。 注意：区分哪个光掩模的区域将成为暗是重要的（防止UV光通过），并且将是透明的（允许UV光穿过），因为这，随着光致抗蚀剂的类型（正或负） ，将决定主模的最终形貌。 </li><li>以开始主模具的制造中，通过将其放置在热板上设定为150℃下进行15分钟脱水4“硅晶片。 </li><li>围绕一个旋涂机的真空夹紧晶片。倾SU8-2015的负光致抗蚀剂到晶圆的中间，并继续在同心圆浇注直到大约三分之二晶片的覆盖。 注意：浇注时，尽量减少气泡的形成保持瓶SU8靠近晶片。 </li><li>编程spincoater如下： <ul><li>扩散周期：500 rpm的转速100转/秒，持续10秒的加速度。 </li><li>旋转循环：4,000 rpm的转速100转/秒，持续30秒的加速度。 </li></ul>注意：该旋转涂布配方会形成一个光致抗蚀剂层，它是约10微米厚。通过改变纺丝速度或SU8制剂，该厚度可以调整。 </li><li>软通过将其放置在热板上设定为95℃，3分钟烘焙晶片。 </li><li>通过与紫外线暴露晶圆光掩模为140毫焦耳/厘米2的总剂量。 注意：SU8是负光致抗蚀剂的区域，因此，其中UV光能够通过光掩模将保持显影后，成为在主模的凸起特征。 </li><li>曝光后烘烤的晶片通过将其放置在热板上设置到95℃持续4分钟。 </li><li>通过将其放置在SU8开发商为3分钟发展晶圆。 3分钟后，用异丙醇冲洗晶片。如果在白色膜的漂洗过程中产生的，该晶片没有发育完全，它应该被放回显影剂另外的30秒。用异丙醇再次冲洗。重复这个过程，直到一个白色的膜不异丙醇漂洗过程中形成。 </li><li>干燥晶片中的氮和地点在150mm的培养皿流以防止灰尘。 </li></ol> 2，制作的PDMS邮票<ol><li>通过结合弹性体基体，并在固化剂制备的PDMS预聚物10:1 w / w的比值。通常80克基和8g固化剂的使用，以确保有足够的PDMS以覆盖母模在1厘米厚的一层。 </li><li>混合脱泡用向心力搅拌机设置为以下的PDMS： <ul><li>混合：2,000 rpm离心2分钟</li><li>脱气：2,000 rpm离心2分钟。 </li></ul></li><li>如果混频器是不可用的用10ml的血清吸液管通过手混合的PDMS 10分钟。通过将其放置在真空干燥器中30分钟，以除去气泡脱气混合物。 </li><li>倒入足量的PDMS预聚物在母模（图案硅片），形成1厘米厚的一层。通过在65℃下烘烤4小时，或在室温下放置48小时固化的PDMS。 </li><li>一旦固化，包含模式的区域可切出，形成PDMS印章。从PDMS压模的背面区分特征侧，切出缺口的邮票上的背面的一个角的。 </li></ol> 3，微接触镨ECM模式的inting <ol><li>干净25mm直径的玻璃盖玻片上通过超声处理在95％乙醇中1小时，然后干燥在65℃的烘箱中。 </li><li>通过在10％的浓度（重量/体积，通常为1克配成10毫升）中的1 - 丁醇中溶解PIPAAm准备PIPAAm溶液。 </li><li>居中的玻璃盖玻片上spincoater和移液管200微升的PIPAAm溶液中的真空吸盘，使整个玻璃表面被覆盖。 </li><li>旋涂盖玻片在6000 rpm下离心1分钟。 </li><li>通过超声处理在50％乙醇清洗。PDMS压模30分钟，然后在氮气流中干燥。 注：干燥和随后的步骤应在生物安全柜内进行，以保持无菌的应用场合ECM的纳米结构将与细胞使用。 </li><li>用200μl的蛋白质溶液，在无菌蒸馏水中进行FN通常为50微克/毫升外套各PDMS的图案化表面的邮票。孵育1小时，在室温下进行。 注：TH被涂覆量为1.5 cm 2的PDMS压模，将需要取决于PDMS压模，用于对ECM蛋白质和在溶液中的细胞外基质蛋白的浓度的大小进行调整。 </li><li>在蒸馏水中洗涤。PDMS压模，以彻底在氮气流除去过量的蛋白质和干燥。 注：留在了邮票的任何水都会触发PIPAAm涂层的过早解散盖玻片上，影响正常的蛋白质转移。 </li><li>对于无菌生产，将PIPAAm涂盖玻片封闭的培养皿内，用灭菌的生物安全柜紫外线照射下，UV光45分钟就足够了。如果不需要无菌此步骤可以省略。 </li><li>通过将PDMS压模的特性侧与PIPAAm涂覆的盖玻片接触进行微接触印刷。如果需要，使用镊子轻轻敲打在邮票背面，以消除任何气泡，并确保均匀接触。 </li><li>经过5英里N，剥离PDMS印章从盖玻片。 </li><li>在此阶段，另外的ECM蛋白可被图案化，以创建更复杂的多组分结构。最多3个印刷已经核实这一过程的工作，更可能是可行的。 </li></ol> ECM纳米纤维和纳米结构4。释放<ol><li>将图案PIPAAm涂在盖​​玻片35毫米的培养皿中，并用相差显微镜检查图案逼真。取决于模式，CCD照相机，可能需要解决的图案的特征。荧光显微镜，也可用于检查所提供的ECM蛋白的荧光标记的图案。 </li><li>加入3毫升40℃的蒸馏水的培养皿中，并让水逐渐冷却。 </li><li>该PIPAAm层的溶解和ECM蛋白模式的释放可以用相差显微镜进行监测。如果应用程序不允许使用光学TEC的hniques，释放可以通过测量溶液的温度进行监测。通常，水被冷却至室温，远低于PIPAAm（32°C）的LCST，以确保对ECM蛋白的纳米结构已经被释放。 </li><li>释放后，纳米纤维，nanofabrics和其他纳米结构漂浮在水中。将它们用于进一步的应用，他们需要被操纵。的确切方法将取决于实验目的，并且可以包括以下步骤，例如固定到另一个表面上，用显微操作器系统移动或在水凝胶包埋。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

这里的新航法提出模仿细胞介导的​​基质组装，使之ECM蛋白纳米纤维和纳米结构工程与可调大小，组织和组成。虽然不完全相同的细胞产生的细胞外基质，新航造成细胞外基质蛋白纳米纤维20的经历可逆的折叠/内机械应变21展开，并能结合细胞20组成。这提供了构建的ECM蛋白的材料，概括ECM的体内发现了许多性能独特的能力。例如，细胞外基质的纳米纤维可以制造...

细胞外基质（ECM）的组织是由参与多种细胞过程，包括黏附，增殖，分化和凋亡1-3的物理和化学调控的多功能蛋白质。 ECM的合成，组装，并通过细胞组织组成的蛋白质纤维具有独特的成分，纤维尺寸，几何形状和相互关联的架构，随组织类型和发展阶段。最近的工作已经证明，细胞外基质可以提供指导性线索来指导细胞形成工程化组织4，这表明扼要的ECM中的组成和结构方面可能使仿生材料的开发用于组织工程和生物技术的应用。 许多制造方法已开发工程师的聚合物支架，可在组织中模仿的ECM的各方面。例如，静电纺丝和相位组合模式振动性也都表现出以形成纤维的多孔基质具有直径范围从几十微米下降到几十纳米5-7的能力。这两种技术也已经表明，纳米纤维的高度多孔的基质可支持细胞粘附和浸润到支架8。然而，这些方法都在能够建立可能的纤维的几何形状，取向和三维结构的限制。静电通常会产生与支架或者进行定向或随机高度一致的纤维，而相分离产生的支架与随机取向的纤维。也有对这些材料的限制，研究人员通常采用的合成聚合物，如聚（ε-己内酯）8和聚（乳酸-共-乙醇酸）9，即其后涂ECM蛋白，以促进细胞粘附。天然生物聚合物也被使用，包括I型胶原10，明胶11，纤维蛋白原12，脱乙酰壳多糖13，和丝14，但是表示在天然组织中发现的蛋白质的仅一小部分。大多数组织中含有ECM蛋白质和多糖，包括纤连蛋白（FN），层粘连蛋白（LN），IV型胶原和透明质酸，很难或不可能制造用现有的方法的纳米纤维的较大的环境。 为了应对这一挑战，我们一直致力于我们对模仿的方式细胞合成，组装和组织细胞外基质蛋白纤维在其周围的研究工作。而具体原纤维形成过程而变化为不同的细胞外基质蛋白，通常在ECM蛋白质分子的构象变化是由酶或受体介导的相互作用，它公开晦涩自组装站点触发。这里我们使用FN作为一个模型系统，以更好地了解纤维形成过程。简言之，FN同源二聚体结合到通过RGD氨基酸序列在第10的II型整合素受体在细胞表面我重复单元。一旦绑定，则通过整合肌动球蛋白收缩移开，然后展开FN二聚体，露出神秘的自组装基地。这些FN-FN的结合位点的曝光使FN二聚体装配成不溶性纤维权利于细胞表面15。在无细胞系统中的工作已经证明，隐蔽FN-FN的结合位点可以通过使用变性剂16或表面张力在空气-液体-固体界面17-19展开的被揭示。然而，通过这些技术产生的FN纤维被限制在特定的纤维的尺寸和几何形状，并且通常结合到一个表面。 在这里，我们描述了一种方法称为表面引发组装体（SIA）20是利用蛋白质表面的相互作用来创建独立的不溶性纳米纤维，nanofabrics（二维表）和一个或多个ECM蛋白组成（ 图1其它纳米结构克服了这些限制）。在这个Process，ECM蛋白质从溶液中一个紧凑的，球形构象吸附和部分变性（展开）上的图案化，疏水性聚二甲基硅氧烷（PDMS）的邮票。的ECM蛋白，然后在这种状态下转移到热响应聚（N-异丙基丙烯酰胺）（PIPAAm）表面通过微接触印刷22。当用40℃的水的水合的PIPAAm仍是固体，但是当冷却到32℃下其穿过其中​​它变成亲水性的低临界溶解温度（LCST），溶胀用水，然后溶解，释放出组装纳米结构的ECM销的表面上。新航方法提供了具有纳米级精度的尺寸控制。通过控制的关键参数，如组合物中，纤维的几何形状，和架构，它可以概括ECM的体内发现了许多属性和开发先进的支架用于组织工程和生物技术的应用。

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Poly(N-isopropylacrylamide) / PIPAAm</td>
			<td style="width:145px;">Polysciences</td>
			<td style="width:123px;">21458-10</td>
			<td style="width:167px;">40,000 Mw</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sylgard 184 Silicone kit (PDMS)</td>
			<td style="width:145px;">Dow Corning</td>
			<td style="width:123px;"></td>
			<td style="width:167px;">Mix 10 parts base with 1 part curing agent.&#160;</td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:216px;">Butanol</td>
			<td style="width:145px;"></td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fibronectin</td>
			<td style="width:145px;">BD biosciences</td>
			<td style="width:123px;">354008</td>
			<td style="width:167px;">Human, 1mg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Laminin</td>
			<td style="width:145px;">BD biosciences</td>
			<td style="width:123px;">354239</td>
			<td style="width:167px;">Ultrapure, mouse, 1mg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Negative Photoresist</td>
			<td style="width:145px;">Microchem</td>
			<td style="width:123px;">SU8-2015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">SU8 Developer</td>
			<td style="width:145px;">Microchem</td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:216px;">Sonicator Branson M3510</td>
			<td style="width:145px;">Branson Ultrasonic Corporation</td>
			<td style="width:123px;">CPN-952-318</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Thinky ARE-250 Mixer</td>
			<td style="width:145px;">Thinky Corporation</td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Spincoater</td>
			<td style="width:145px;">Specialty Coating Systems</td>
			<td style="width:123px;">G3P-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glass cover 25mm diameter, No 1.5</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:123px;">12-545-86</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

ecm protein nanofibers and nanostructures engineered using surface-initiated assembly

细胞外基质（ECM）在组织中被合成，并通过细胞组装以形成一个三维纤维状蛋白质与网络紧密调节纤维的直径，组成和组织。除了 ​​提供结构支撑，ECM的物理和化学性质在多种细胞过程，包括粘附，分化和凋亡中发挥重要作用， 在生物体内时，ECM是由蛋白质中暴露隐秘自组装（原纤维）位点组装。这个过程变化为不同的蛋白质，但纤连蛋白（FN）的原纤维是非常明确的，并作为细胞介导的​​ECM组件的模型系统。具体地，将细胞用对细胞膜整合素受体结合FN二聚体和肌动球蛋白生成的收缩力，以展开并暴露结合位点组装成不溶性纤维。该受体介导的过程使细胞从细胞聚集和组织的ECM来组织SCALES。在这里，我们提出了一个方法称为表面引发组装体（SIA），采用蛋白质表面的相互作用展开ECM蛋白和它们组装成不溶性的纤维，概括细胞介导的​​基质组装。首先，ECM蛋白吸附到疏水性聚二甲基硅氧烷（PDMS）表面，他们部分变性（折叠）和揭露隐秘结合结构域。未折叠的蛋白质，然后转移中良好定义的微和纳米图案通过微接触印刷到热响应聚（N-异丙基丙烯酰胺）（PIPAAm）表面。该PIPAAm的热引发溶解导致最终组装和不溶性的ECM蛋白的纳米纤维和纳米结构具有良好定义的几何形状的释放。复杂的架构是可以通过在用于微接触印刷的PDMS印章定义的工程模式。除了FN，新航过程中可以与层粘连蛋白，纤维蛋白原被使用和胶原蛋白I型和IV创建多组分的ECM nanostrucTures的。因此，SIA，可用于工程ECM蛋白质基材料，具有精确控制的蛋白质组合物，纤维的几何形状和脚手架架构中为了概括ECM的结构和组合物在体内 。

新航能工程的ECM蛋白纳米纤维与精确控制光纤的尺寸。为了证明这一点，FN纳米纤维为50×20微米平面尺寸的阵列，图案到PIPAAm涂盖玻片（ 图2A）。获释后，纤维收缩，因为他们是在一个固有的预应力对PIPAAm面（ 图2B）图案时。新生力量纳米纤维的分析表明它们是单分散的预发行以50.19的平均长度±0.49微米的19.98±0.17微米，宽度（ 图2C）。尽管收缩明显，FN纤维?...

Watch this Scientific Journal Video about ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly at JoVE.com

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

A到从单个或多个细胞外基质蛋白得到纳米纤维和纳米结构复杂的方法进行描述。此方法使用蛋白质表面的相互作用与可调的组成和架构，在各种组织工程和生物技术应用创造自由站立蛋白质为基础的材料。

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

ecm-protein-nanofibers-nanostructures-engineered-using-surface

Research

JoVE Journal

Bioengineering

12.4K 次观看.  Carnegie Mellon University.  A到从单个或多个细胞外基质蛋白得到纳米纤维和纳米结构复杂的方法进行描述。此方法使用蛋白质表面的相互作用与可调的组成和架构，在各种组织工程和生物技术应用创造自由站立蛋白质为基础的材料。 

视频: ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

为增强微型和纳米操纵利用电浆和光子晶体纳米结构

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

科研

生物工程

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

工程化血管肌瓣

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

将伽马改性肽核酸自组装成有机溶剂混合物中复杂的纳米结构

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

由去细胞化肺切片制备的工程肺组织

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...