作者感谢支持由美国国立卫生研究院主任的新创新者奖（1DP2 OD006499-01至AA和1DP2 OD006460-01 AJE），和阿卜杜勒阿齐兹国王科技城（加州大学圣地亚哥分校在纳米医学的卓越中心）。我们要感谢杰西卡·穆尔女士为她的手稿上的批评意见。

1。合成荧光标记的丙烯酰-PEG-RGDS <ol><li>与羧甲基丙烯酰基-PEG-琥珀酰亚胺基酯（APEG供应链管理（SCM），PEG，分子量：3400克/摩尔）和NN二异丙基乙胺（DIPEA），1.2:1:2的摩尔比在二甲亚砜（DMSO）中在氩气下在室温下发生反应的RGDS肽过夜。 </li><li>确认结合基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF）。加入1μl的样本点的MALDI靶和干APEG-RGDS反应液。准备通用的MALDI基质的四氢呋喃（THF）和涡旋混合1分钟的饱和溶液。 〜1微升该溶液加入到相同的样品点。 APEG-SCM重复步骤进行比较。加载和分析。 APEG-RGDS的分子量应该是大于APEG-SCM（ 图2）。 </li><li>为共轭的荧光团，加入等摩尔量的的Alexa Fluor 350羧酸（succinimydyl酯），溶于在最小体积的DMSO中，从1.1 APEG-的RGDS反应溶液，并在室温下在氩气下反应过夜。 </li><li>透析（MW 3500大）与DI-H 2 O在4°C 1000:1的体积比为48小时，每天至少两次更换透析液，净化APEG-RGDS-350。 </li><li>冷冻干燥纯化的APEG-RGDS-350的LABCONCO自由贸易区的Plus或相当于冷冻干燥系统，并储存在-20°C </li></ol> 2。 RGDS-350层的二维模具和制造的二维PEG凝胶的制备与交流<ol><li>准备疏水性的载玻片上。将干净的玻璃在真空炉中的玻璃皿滑入。加热至80℃，进行30分钟至完全干燥的表面。将菜在通风橱中的幻灯片，并加入250μlSigmacote到每张幻灯片，轻轻摇晃30秒，整个表面涂层。彻底冲洗包被的载玻片，然后用100％甲醇通过在蒸馏水中洗涤两次5分钟，浸泡在执法机关T 10毫升。 </li><li>剪切硅氧烷间隔件（0.8mm厚），用10毫米活检冲头。 </li><li>高压灭菌的硅氧烷的衬垫和Sigmacote处理的载玻片。 </li><li>制订解决方案，为每个单独的微量离心管中的各自的层中混合，聚乙二醇二丙烯酸酯（PEGDA）前体（终浓度为15％）与不同量的碘克沙醇（在水中60％的原液），得到各种不同的最终浓度（ 例如 40％w / v的30％，20％和10％），补充的剩余体积用磷酸盐缓冲盐水（PBS），得到的梯度密度的解决方案。以类似的方式，交替层，混合APEG-RGDS-350（最终浓度为8毫摩尔）与碘克沙醇及PBS，得到各种浓度（ 例如 35％，25％，和15％）。 </li><li>添加光引发剂（2 - 羟基-4&#39;-（2 - 羟基乙氧基）-2 - 甲基苯丙酮，N-乙烯基吡咯烷酮的333mg/ml库存的）各种层（10μl的每个层溶液每毫升原液）的每个解决方案。光引发剂我S添加，以防止聚合，因为它是分层的凝胶在模具之前，对光线敏感。 </li><li>此协议的后续步骤将在生物安全柜中进行，以确保无菌。 </li><li>使用无菌1毫升注射器和0.2微米的过滤器过滤，消毒，每个解决方案。组装模具设定通过夹着隔板间Sigmacore处理载玻片和安全放置，如在图1中示出用夹子固定。 </li><li>铸造层状的凝胶，通过添加最密集的水溶液（ 如 40％的碘克沙醇PEGDA）第一，其次由密度较小的解决方案（ 例如 APEG-RGDS-350用35％的碘克沙醇）。重复备用层叠来实现，如在图1中所示的几个层的所需的组合物和密度。 </li><li>使用的是便携式UVR-9000灯照射3分钟365纳米的光模具。允许聚合凝胶固化5分钟。取下夹子，然后轻轻地抬起顶部玻璃幻灯片和模具的分层dGMP的凝胶会保持在幻灯片上。使用无菌抹刀，小心地将凝胶含有无菌PBS或培养基中用于洗涤，在50毫升的试管。 </li><li>在PBS中的聚合凝胶洗净1,000:1体积比，交换缓冲液每天至少两次以除去的密度调节剂，光引发剂，和未反应的聚合物。另外，PBS可以交换与细胞生长培养基中。存储dGMP的在PBS中的凝胶或生长培养基中，在步骤3中所述的细胞培养实验。 </li><li>交替层可视化，安排dGMP的凝胶（凝胶可用于细胞培养），沿着样品盘的VersaDoc凝胶单元上的标尺。公开的凝胶在350 nm处，曝光时间将取决于上的荧光基团的浓度。交替黑暗和蓝波段在dGMP的水凝胶中的证明形成独特的化学组合物（ 图3）的离散层。 </li></ol><ol><li>对于装有RGDS肽凝胶，使用粘附依赖性细胞，如C2C12细胞。 </li><li>入井的48孔细胞培养板中，使用在生物安全柜无菌细胞刮刀轻轻插入dGMP的凝胶（存储在PBS中）。 </li><li>预温生长培养基（Dulbecco改良的Eagle氏培养基或DMEM培养液，用10％体积/体积的胎牛血清和1％体积/体积的100倍的青霉素 - 链霉素溶液）及PBS在37℃下在水浴中设置</li><li> 60％汇合的板（10毫米）的C2C12细胞3次，用PBS洗净。吸干PBS和收获细胞，通过添加1毫升的0.25％胰蛋白酶-EDTA，在37℃下孵育2分钟。重悬在生长培养基中的细胞，计数细胞。种子dGMP的凝胶含有细胞培养C2C12细胞（20000个细胞/ cm 2）。在5％CO 2/95％相对湿度在37℃下孵育细胞。 4小时后，轻轻地交换介质，注意不要重新移动轻轻的贴壁细胞。 </li><li> 24小时后，C2C12成肌细胞附着的的RGDS含dGMP的凝胶层的上落射荧光相差显微镜（蔡司Axiovert 200）可以确认。 </li></ol>

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作者有没有利益冲突披露。

dGMP的是一个简单的策略，为编制多层凝胶，不依赖于昂贵的仪器。此协议可以使用其他的生物相容的材料，如胶原蛋白和透明质酸创建支架适于。生物活性的小分子物质，例如细胞的粘附促进RGDS肽，可以被拴到聚合物基体，以防止混合层与层之间的线索。蛋白质可被封装在不同的层中，而不需要进行化学缀合，因为他们，根据矩阵上的网目尺寸，是不容易扩散通过水凝胶10。在这里，我们用碘克沙醇（Nycoprep），惰性密度改性剂，已被用于活细胞的应用。也可用于其他密度调节剂，如蔗糖和葡萄糖。不同的稳定时间（T），可以精细调整产生光滑或尖锐的过渡，两层之间的接口（更长的稳定时间为平滑的过渡）&lt;SUP&gt; 10。例如，平滑的过渡之间dGMP的凝胶层，可以用于生成一个连续梯度的研究如趋化的细胞过程的生物提示。 密度调节剂的效果对凝胶刚度为15％aPEGda凝胶在图5中所示;作为PEGDA和碘克沙醇浓度的函数的刚度和孔隙度的一个更完整的表征是目前正在评估。虽然在这个例子中的PEGDA浓度是比较高的，我们观察到60％以上的弹性模量在30％的碘克沙醇凝胶相比，凝胶没有。凝胶刚度的变化可以进行调整，以通过调制的大单体浓度或交联密度。 我们还采用dGMP的技术来创建三维多层采用聚丙烯酰胺凝胶和PEG前体10。变的浓度或预聚物的交联度在允许结构变异支架，它可以被用来研究细胞的行为，如在3D的极性生长和迁移。 综上所述，dGMP的是，可应用于制作2D和3D的从生物相容性材料用于范围广泛的生物医学和基础研究应用各种支架的自适应性的技术。

<table border="1"><tbody><tr><td> 试剂或仪器 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>聚乙二醇succinimydyl羧甲基（α-PEG-SCM） </td><td>黑背</td><td> 120-64 </td></tr><tr><td> polyethelyene丙二醇二丙烯酸酯（PEGDA） </td><td> Dajac实验室</td><td> 9359 </td></tr><tr><td>精氨酸 - 甘氨酸 - 天门冬氨酸-丝氨酸（RGDS） </td><td>美国多肽</td><td> 49-01-4 </td></tr><tr><td> N，N -二异丙基乙胺（DIPEA） </td><td>西格玛</td><td> D125806 </td></tr><tr><td>二甲亚砜（DMSO） </td><td>西格玛</td><td> D2438 </td></tr><tr><td> N，N-二甲基甲酰胺（DMF）的</td><td>费舍尔</td><td> D119-4 </td></tr><tr><td>四氢呋喃（THF） </td><td>费舍尔</td><td> T397 </td></tr><tr><td>透析盒（3500达） </td><td> Thermo Scientific的</td><td> 66330 </td></tr><tr><td>的Alexa Fluor 350的羧酸succinimydyl酯</td><td> Life Technologies公司</td><td> A-10168 </td></tr><tr><td> Sigmacote </td><td>西格玛</td><td> SL2 </td></tr><tr><td>硅胶垫片</td><td>格兰杰</td><td> 1MWA4 </td></tr><tr><td>活检拳</td><td> Acuderm </td><td> P1025（10毫米） P850（8毫米） </td></tr><tr><td> Dulbecco氏磷酸盐缓冲盐水（DPBS） </td><td> Hyclone公司</td><td> SH30028 </td></tr><tr><td>克沙醇（NycoPrep） </td><td>费舍尔</td><td> NC9388846 </td></tr><tr><td> 2 - 羟基-4&#39;-（2 - 羟基乙氧基）-2 - 甲基苯</td><td>西格玛</td><td> 410896 </td></tr><tr><td> Dulbecco改良的Eagle培养基（DMEM） </td><td> Life Technologies公司</td><td> 11054 </td></tr><tr><td>胎牛血清</td><td> Life Technologies公司</td><td> 10082 </td></tr><tr><td>青霉素，链霉素</td><td> Life Technologies公司</td><td> 15140 </td></tr><tr><td> C2C12成肌细胞</td><td> ATCC </td><td> CRL-1772 </td></tr><tr><td> MALDI </td><td>布鲁克</td><td> N / A </td></tr><tr><td> UVR-9000 </td><td> Bayco </td><td> UVR-9000 </td></tr><tr><td> VersaDoc </td><td> Bio-Rad公司</td><td> N / A </td></tr></tbody></table>

density gradient multilayered polymerization (dgmp): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering

复杂的组织培养矩阵，其中的生物刺激（ 例如，生长因子，抑制剂，或小分子）或矩阵结构的类型和浓度（ 例如，组合物中，浓度，或刚度矩阵）随空间，将启用了广泛的调查关于这些变量如何影响细胞的分化，迁移等现象。创建层状矩阵的主要挑战是维持层接口的结构完整性没有单个组件从每个第1层中的扩散。目前的方法来实现这一点包括在光2-3，光刻4，顺序functionalization5，冷冻干燥6，微流体7，或离心分离8，其中有许多需要精密的 ​​仪器和技术技能。其他依靠连续的附件，这可能会导致个别层，9层分层</支持&gt;。 dGMP的克服了这些问题，通过使用惰性密度调节剂，如碘克沙醇，来创建层不同的密度10。由于密度调节剂可以混合使用任何预聚物或生物活性分子，dGMP的允许每个支架的层来进行定制。简单地改变的密度调节剂的浓度可以防止相邻的层的混合，同时它们保持水。随后的单步聚合产生结构连续多层的脚手架，其中每个层具有不同的化学和机械性能。密度调节剂可以容易地除去与无扰动各个层或它们的组成部分的充分漂洗。因此，这种技术是非常适合用于创建各种尺寸，形状和材料的水凝胶。 一种协议，用于制造一个2D - 聚乙二醇（PEG）的凝胶，其中的交替层包括RGDS-350，概述如下。我们使用PEG because它是具有生物相容性和惰性。 RGDS，细胞粘附肽11，是用来演示的生物提示的空间限制，和荧光团共轭的（的Alexa Fluor 350），使我们能够在视觉上区别各种层。此过程可适于其他材料（ 例如，胶原蛋白，透明质酸等），并可以扩展到制造三维凝胶与一些修改10。

MALDI-TOF分析证实RGDS肽丙烯酰基-PEG（ 图2）的共轭。凝胶成像显示RGDS-350（蓝色）层交替光聚合后（ 图3A）。正如图3A中所示，2D dGMP的凝胶尺寸可以变化的基础上的硅氧烷模具的直径（10毫米，左; 8毫米，右），并因此很容易定制的用于在多个检测的-在这种情况下，以适应48孔细胞培养板（ 图3B）。落射荧光和相衬显微镜C2C12细胞培养上dGMP的凝胶上显示选择性连接RGDS-350-含PEG层（ 图4），这表明细胞粘附肽（RGDS）的条块。 <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="图1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> 图1。分子weigHT MALDI-TOF比较APEG-SCM APEG-RGDS的RGDS肽结合后得到的分析。 <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> 图2。 dGMP的凝胶制作的示意图。梯度分层后，可以让它们来解决不同的时间段（T S）毕业的接口，然后通过光聚合。分层dGMP的凝胶可以容易地提取从模具中以便进一步使用。 <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="图3" /> 图3：A）光聚合后得到的二维多层凝胶成像使用350 nm和白色光通道的Versa文件凝胶文档单位。的灰度图像显示包含在白色的RGDS的交替层B）插入，dGMP的凝胶成48孔细胞培养皿。 <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="图4" /> 图4。C2C12成肌细胞生长在dGMP的凝胶（比例尺为50μm）的合并相衬和荧光显微镜图像。 <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="图5" /> 图5。碘克沙醇凝胶表面弹性的影响。原子力显微镜测量静态样品的交联PEG基板的使用以前建立的方法与2 NN力扳机12。 * p &lt;0.05和** P &lt;0.01。

Watch this Scientific Journal Video about Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering at JoVE.com

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

在这里，我们描述了一种独特的战略，创建连续不同的层之间的接口为组织工程的生物相容性，多层矩阵。这种支架可以提供一个理想的可定制的环境来调节细胞行为的各种生物，化学或机械的线索

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

density-gradient-multilayered-polymerization-dgmp-novel-technique-for

Research

JoVE Journal

Bioengineering

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

12.4K 次观看.  University of California, San Diego. 在这里，我们描述了一种独特的战略，创建连续不同的层之间的接口为组织工程的生物相容性，多层矩阵。这种支架可以提供一个理想的可定制的环境来调节细胞行为的各种生物，化学或机械的线索

视频: Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

科研

生物工程

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

娇嫩的组织操纵定制的软机器人气动设备手爪基于杆制造

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...