我们非常感谢安雅·劳特巴赫（Lehrstuhl Biomaterialien）的，洛伦兹大全（桑德勒AG）和Armin BOECK的（B / S / H / G）的技术和科学支持。 SEM成像由约翰内斯·迪尔（Lehrstuhl Biomaterialien）。资金来自BMBF（01RB0710）。

1。纺丝原液的制备<ol><li>以冻干eADF4蛋白（C16）。 </li><li>通过使用高的精度为1毫升的反应容器中称取20毫克共eADF4（C16）。 </li><li>加入200μl的六氟异丙醇，并用封口膜密封的容器。 注意事项： 六氟异丙醇是高挥发性和有害的。因为它可以造成伤害下呼吸跟踪，工作的安全罩，吸管仔细，并加盖管。 </li><li>涡暂停1分钟，并进一步动摇它清除解决方案。为了确保整个量的蛋白质完全溶解，等待过晚。 </li></ol> 2。静电<ol><li>准备的静电纺丝装置（ 图1）：将过滤材料上的对置电极的顶部和预先设定的电压的两个电极（-22.5千伏）和反电极（2.5千伏）。将315微升/小时的体积流量。 注：W虽然静电，无毒HFIP将被蒸发。请确保您的静电纺丝装置连接到通风柜。 </li><li>以市售的20 G针，并用一只手研磨机研磨锐利尖端的剩余长度为30mm。将1 ml注射器的针。 注：A面的针尖是必需的，以便产生一个明确的泰勒锥。 </li><li>入注射器装入整个纺丝液（200微升）。叠加，用100μl的空气，纺丝原液，以允许完整的解决方案，在纺丝过程中被挤出。 注：为了避免针头堵塞，确保不存在任何颗粒（骨料或杂质）在纺丝原液。在通风橱！ </li><li>将填充注射器的电纺丝装置的注射器泵，并小心地按下活塞在注射器上的液滴，直到出现上面的针尖。锁定活塞。 </li><li>设置的距离针尖和反电极为8-20厘米之间。 </li><li>注射器泵启动和移除的针从开口（通常是干燥的）液滴。激活所有电设备的安全装置立即启动高压源，尽快出现一个新的液滴。静电纺丝原液，随后将开始。使用一个秒表来控制纺纱期限。 注意：为了避免晒出的解决方案，从而针的阻塞，有必要立即开始纺纱过程中取出后，将干燥的液滴。 </li><li>由于静电的重组蜘蛛丝蛋白依赖于湿度和温度，对单独的实验室条件下的一个适应的工艺参数可能是必要的（ 图2）。 注意：为了防止液滴干燥（ 图2B），使能以足够的流量。如果有一个低湿度性在周围气氛中，调整相对湿度或提高流速。降低电压，直至适当的泰勒锥时（ 图2A）。当不存在溶液的前端（ 图2C），提高的流率，并降低电压，直到微滴发生。然后调整电压，以建立定期和稳定的泰勒锥（ 图2A）。 </li><li>经过30秒/ 60秒/ 90秒的注射泵电关掉。为了避免水滴下降，等待10秒才关闭高电压源，以释放注射器中的残余压力。 </li><li>不同类型的过滤材料，如聚酰胺，聚丙烯和聚酯的非织造网格，以及用于比较的黑纸，可以执行步骤6至8。 </li><li>为了产生一个非织造网眼为后续的稳定性实验，用黑色的纸，而不是过滤材料，执行步骤5至7。静电5分钟后，停止在步骤8中所描述的过程。 </li></ol> 3。处理后的丝绸无纺布网格<ol><li>一个烤箱预热至60℃。 </li><li>将过滤器基板与eADF4（C16）垂直无纺网格使用封闭的玻璃容器中的最小距离为2厘米。该容器应该有两个开口，将用于随后引入乙醇和水。 注：在固定过滤材料时，确保渗透性实验所需的面积不被损坏夹具。 </li><li>连接两个60毫升的注射器，一个充满用乙醇，水满的，用硅胶管指向的后处理容器内底部（ 图3）。 注意：为了能够在处理后从容器中除去液体，将尽可能接近底部的管道的开口。 </li><li>将处理后的容器中的OVen，并加到60毫升乙醇中，挤出注射器。使用秒表来控制治疗时间。 </li><li>乙醇蒸汽处理90分钟后，用注射器从玻璃中除去乙醇，加60毫升水，从第二注射器。 </li><li>等待90分钟，然后取出水，然后关掉烤箱。为了避免通过冷凝的液滴，离开容器的烘箱中，直到它已完全冷却。 </li></ol> 4。蜘蛛丝无纺布网格分析<ol><li>准备蚕丝无纺网格黑纸或任何其他可移动的支持的稳定性测试。切出的纸板和两帧调整双粘性胶带。按一帧到丝绸织造网眼黑纸存放和使用手术刀切断多余的蚕丝纤维（随后SEM成像保持多余的纤维）。为了从纸张分离的非织造布，小心地取出帧。重复此步骤与第二帧（ 图4）。 </li><li>实用浸渍试验：剪下一块（1厘米2），后处理和非处理的丝绸织造网眼浸入去离子水。未处理的丝非织造网眼将立即溶解，而处理过的非织造网眼将是稳定的（ 图5）。浸渍后，晾干浸样品，并准备进行扫描电镜成像。 </li><li>傅里叶变换红外光谱（FTIR）计量和后续Fourier自去卷积（FSD）：为了获得信息后处理的非织造网格后的丝蛋白的结构变化，红外光谱可以应用于使用参数：透光率模式下，扫描800〜4000厘米-1，60的累积为每个谱的测量和平均光谱测定，每一个参考。消防处的数据进行定量分析，可以采用（ 图6和图7）。由此，曲线减少数据芳件1590和1705厘米-1，基线校正之间进行。本地最小二乘拟合计算峰值位置从以往的研究（1611，1619，1624，1630，1640，1650，1659，1666，1680，1691，1698厘米-1）22。 </li><li>扫描电镜成像：扫描电镜可以应用于调查纤维的直径和不同的过滤器基体上的丝纤维的形态上的纤维形态的分析后处理的影响（ 图8）。使用放大倍数为5000倍的为25,000 x为了得到足够详细的图像。 </li></ol> 5。透气性的测定<ol><li>放置一个合适的过滤材料一个Akustron透气性装置的测量面积。 注::如果你使用另一种类型的透气性设备，确保它符合DIN 53 887，DIN 53 120，ISO的要求9237和ASTM D 737-96标准。 </li><li>，运用Akustron的透气性设备（或任何其它5.1所描述的）。如果有必要计算范的数据[L /（M 2 X 秒）]。 </li><li>重复步骤1和2与样品的不同部分的至少10倍，计算算术运算的中间（ 图9）。 注意：测量透气性要求的设备的接触和非织造网眼。因此，认真处理的样本是必要的，以防止破裂的细腻丝绸织造网眼。 </li></ol> 6。过滤效率的测定<ol><li>使用合适的机器，压力控制和粒子计数器，如通用粒度仪（帕拉斯联系，卡尔斯鲁厄，德国）。 </li><li>将过滤器的样品中的移动设备和测量颗粒的滞留（ 图10）。气雾剂：迪-乙基己基sebacat（DEHS）;粒径0.3-3微米，持续时间：30秒;液体速度：2,350厘米/秒，空气流量：3,400 立方米 /小时。 <br/&gt; 注意：小心，不要触摸表面，防止破坏的非织造网格和避免任何污染处理样品。要确保创造足够的样品同等质量，性能测量。 </li></ol>

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新的过滤装置应允许在空气过滤在恒定或更高的过滤效率，降低整体能源消耗。在这里，这种装置使用蜘蛛丝制成的非织造布。由于其低的表面张力和高挥发性，HFIP中已被选择作为合适的溶剂静电纺丝过程。此外，丝水溶液已在以前的实验测试，但可产生任何纤维。在这里，使用的添加剂，为了降低表面张力的溶液的可纺性，从而改善将是至关重要的。最关键的步骤是调整的条件和所使用的材料浓度和溶剂的纺丝溶液，纺纱机的高度，电压和挤出速度。期间的性能，例如堵塞的前端，可以防止与水分以水蒸汽的形式供给针尖，但随后可能会扰乱静电设置任何种类的增加在敏感的过程和电场。基本工艺参数（浓度，电压，湿度，距离）单独决定进行独立的实验系列（数据未显示）。以考虑所有参数连续的泰勒锥和创建统一纤维的纺纱工艺的关键是要照顾。 该过滤器的效率是过滤材料的最重要的参数之一。此参数主要是由该过滤材料的结构的影响。织造布继承均匀的孔径和其后一致的透气性。这是至关重要的创建这些模板材料的均匀的非织造网眼上，以填充孔，并生成一个零缺陷滤波器的。示出了在我们的过滤器的过滤效率直接依赖于纺丝持续时间（丝蛋白），并且，因此，非织造网眼层的数量。单纤维之间的差距始终充满，使的保留f更小的颗粒。 在这项工作中，我们介绍了一个方法来产生蜘蛛丝无纺布网的一种新型过滤材料，过滤效率高。因此，这些过滤器的空气过滤系统，有希望的候选人，以供日后使用。

蜘蛛丝纤维由于其组合的强度和可扩展性，可以吸收更多的动能比大多数其他天然或合成纤维1。此外，与大多数合成的高分子材料，丝绸材料无毒，生物相容性，不会引起过敏性反应时并入2,3。公认的健康风险是可以预防的，用蜘蛛丝。这些特点使得蜘蛛丝非常有吸引力的各种医疗和技术的应用程序。由于蜘蛛不能养殖，由于他们的吃人的行为，已经开发了生物技术方法生产蜘蛛丝蛋白，无论是成本效率和足够的数量4。 该重组丝蛋白eADF4的（C16）已经设计根据自然顺序蛛diadematus素蛋白的4（ADF4）。图eADF4（C16）的分子量为48kDa 5，在各种溶剂中是可溶的（hexafluoroisopropanol异丙醇（HFIP）7 6，甲酸和水相缓冲液）8。 eADF4（C16），可以加工成不同的形态，如膜9 8，胶囊剂，颗粒10，11水凝胶，涂料7，纤维12和非织造的网格6。后者由于其化学稳定性，在过滤器的应用提供高电位。 在这里，我们提出了一个协议，以制造空气过滤器设备，包括非织造网眼的静电重组蜘蛛丝蛋白。静电纺丝或静电纺丝是一种技术，通常采用的制造聚合物纤维的直径在10纳米-10微米的13和非织造啮合的范围内，已被研究了过滤器应用程序14。静电纺丝在过去，已成功地应用于处理再生15以及重组产生的16蜘蛛丝蛋白质。通常情况下，高电电压（5-30千伏）被施加到注射器和8-20厘米的距离放置在反电极（0-20千伏）。强大的静电场诱导内充电解决方案的排斥力。如果被超过时，表面张力形成的泰勒锥，一个薄的射流从前端17,18爆发。形成后，发生弯曲的不稳定性，射流内造成进一步的拉伸作为溶剂的蒸发，形成了坚实的纤维。最后，该纤维随机地沉积作为非织造网眼19的对置电极上。类似直径和表面的拓扑结构（光滑，多孔的）纤维的性能主要依赖于溶液的参数，如浓度，粘度，表面自由能和溶剂的本征导电率和磁导率20。静电纺丝的eADF4（C16）在光滑的纤维直径从80-1,100纳米，这取决于在溶液中的蛋白质浓度溶解在六氟异丙醇的结果。eADF4（C16）从HFIP静电显示主要是α-螺旋二级结构的纤维是水溶性的6。为了稳定的丝纤维，β-折叠结构具有诱导随后用乙醇处理。在先前建立的后处理方法（21）相反，在本研究中eADF4（C16），非织造布已与乙醇蒸汽处理，以保持丝纤维的形态。使用傅里叶变换红外光谱（FTIR）和随后的Fourier自去卷积（FSD）的文献[22]中所描述的二级结构进行分析。 消防处是信号处理工具，它允许几个重叠的频段，包括红外光谱分辨率。由此，I区域中的广泛的模糊的条带，可以使用一个高通滤波器，接收与改进的峰值分辨率的解卷积的频谱变窄。 为了评估EFF器基板，并与丝无纺网格iciency，透气性测试采用根据标准方案的Akustron装置。使用一个帕拉斯通用的粒度仪测量沉积速率。

air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins

基于蛛diadematus素4（ADF4），重组蜘蛛丝蛋白eADF4的自然顺序（C16）已经设计好了。此高度重复的蛋白的分子量为48kDa，并在不同的溶剂中是可溶的（六氟异丙醇（HFIP），甲酸和水相缓冲液）。 eADF4（C16）加工成的形态，如片，胶囊，颗粒，水凝胶，涂料，纤维和无纺网格时各种技术应用提供了一个高电位。由于它们的化学稳定性和可控形态，后者可以被使用，以提高过滤材料。在这个协议中，我们提出了一个程序，提高效率不同的空气过滤装置，织造网格静电重组蜘蛛丝蛋白的沉积。静电eADF4（C16）在HFIP结果顺利纤维溶解。变异的蛋白质浓度（5-25％w / v的）的结果，在不同的纤维直径（80-1,100纳米）和因此，非织造网眼孔径。 从HFIP后处理eADF4（C16）静电是必要的，因为该蛋白显示在刚纺纤维的二级结构主要是α-螺旋的，因此纤维是水溶性的。随后用乙醇蒸汽诱导形成防水的，稳定的β-折叠结构，保持形态的纤​​维丝啮合。二级结构分析，采用傅里叶变换红外光谱（FTIR）和随后的Fourier自去卷积（FSD）。 加入真丝无纺布层之上的首要目标是改善现有的过​​滤器基板过滤效率。为了评价电持续时间的影响，并在过滤器上的效率的这样的非织造层的厚度，进行透气性测试与测量颗粒物沉积的组合。进行了实验，根据标准的协议。

静电从HFIP与浓度为10％w / v的重组蜘蛛丝的解决方案导致在光滑的纤维直径为80〜120nm的，使形成的非织造网格。与乙醇蒸汽处理后并没有导致明显的形态学变化，因此，作为一个适当的方式建立的丝绸织造后处理（ 图8）。结构变化，采用FT-IR和随后的FSD中我单的贡献峰（ 图6）频段进行分析检测。这可能是表明，治疗后导致在β-折叠结构中增加，而α-螺旋和无规线圈结构，内容的降低（ 图7）。实际上这个结果可以证明，通过浸渍后的处理过的无纺入水（ 图5）。即使一周后，不溶解的非织造网眼会发生。 纺杜配给关于丝非织造布中的应用在过滤材料由于基于电纺纤维密度的增加的压力降是最重要的参数。扩展纺丝持续时间达科较高数目的纤维层的结果以指数下降的透气性。这种效应可检测到的所有不同的过滤器之前和之后的后处理的衬底材料（ 图9）。同样地，丝含有亚微米颗粒的增加（ 图10）的过滤材料的过滤效率。虽然纺纱持续时间短（30秒）获得低通滤波器的效率，更高的纺纱持续时间（90秒）导致更高的效率。 <img alt="图1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50492/50492fig1highres.jpg" src="/files/ftp_upload/50492/50492fig1.jpg" /> 图1。高电压（0-30千伏）<html>小号应用与丝溶液填充到注射器中，并为8-20厘米的距离放置在反电极（0-20千伏）。这种设置导致强静电场，感应带电的溶液内的排斥力。如果被超过时，表面张力形成的泰勒锥，一个薄的射流从前端爆发。形成后，发生弯曲的不稳定性，射流内造成进一步的拉伸作为溶剂的蒸发，形成了坚实的纤维。最后，该纤维随机地沉积在非织造网眼形式的对置电极上。 <img alt="图2" src="/files/ftp_upload/50492/50492fig2.jpg" /> 一个普通的泰勒锥（A），（B）干液滴，液滴（C）的设定，不需图2。照片。 iles/ftp_upload/50492/50492fig3highres.jpg“SRC =”/ files/ftp_upload/50492/50492fig3.jpg“/&gt; 图3。原理程序在蒸汽后处理。在第一步骤中，腔室被填充有乙醇，并将样品在60℃下蒸90分钟。以减轻后续处理的非织造网格，乙醇去除纤维与水蒸汽蒸90分钟，在60°C <a href="https://www.jove.com/files/ftp_upload/50492/50492fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/50492/50492fig4.jpg" /> 图4。一个纸板框架连接的丝被用于后处理的非织造网的照片。 <img alt="图5" src="/files/ftp_upload/50492/50492fig5.jpg" /> 图5。Electrosp联合国和随后在干燥状态（A）和（B）水无纺布后处理。 <img alt="图6" fo:content-width="6in" fo:src="/files/ftp_upload/50492/50492fig6highres.jpg" src="/files/ftp_upload/50492/50492fig6.jpg" /> 图6。Fourier自去卷积的酰胺I带未处理的（A）和（B）的处理后的蜘蛛丝非织造网眼的吸收光谱。实线显示来自卷积后产生单贡献峰（虚线）的吸收带。分配各自的曲线是基于先前公布的数值从文献22。 <a href="https://www.jove.com/files/ftp_upload/50492/50492fig6large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图7" fo:conten<html>吨宽度=“5英寸”FO：SRC =“/ files/ftp_upload/50492/50492fig7highres.jpg”SRC =“/ files/ftp_upload/50492/50492fig7.jpg”/&gt; 图7。二级结构含量的非处理和处理后的eADF4（C16）的非织造网格。 <img alt="图8" fo:content-width="6in" fo:src="/files/ftp_upload/50492/50492fig8highres.jpg" src="/files/ftp_upload/50492/50492fig8.jpg" /> 图8的 SEM图像静电eADF4（C16）纤维对不同的过滤器基板：聚酰胺（PA），聚酯（PE），聚丙烯（PP）和纯eADF4（C16）纤维前（S1）和（S2）职位治疗用乙醇蒸汽。 <a href="https://www.jove.com/files/ftp_upload/50492/50492fig8large.jpg" target="_blank">点击这里查看大图</a> 。 / ftp_upload/50492/50492fig9.jpg的“/&gt; 图9。透气度测试之前，（A）和后处理后的（B）的丝绸无纺布与乙醇蒸汽啮合，增加纺丝时间导致更多个非织造层，其后降低的透气性。 <img alt="图10" fo:content-width="5in" fo:src="/files/ftp_upload/50492/50492fig10highres.jpg" src="/files/ftp_upload/50492/50492fig10.jpg" /> 图10。静电蜘蛛丝无纺网格聚酰胺纺丝的持续时间在不同的过滤材料，影响丝层的数量后，进行后处理，用乙醇，二乙基-己基- sebacat气溶胶的过滤效率。

Watch this Scientific Journal Video about Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins at JoVE.com

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

蜘蛛丝纤维显示出非凡的机械性能。工程<em&gt;蛛diadematus</em&gt;素4（eADF4）可以被加工成非织造网格，使用静电纺丝。在这里，的eADF4无纺网格用于提高性能的空气过滤装置。

空气过滤设备，包括非织造网格静电重组蜘蛛丝蛋白

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

air-filter-devices-including-nonwoven-meshes-electrospun-recombinant

Research

JoVE Journal

Bioengineering

16.0K 次观看.  University of Bayreuth. 蜘蛛丝纤维显示出非凡的机械性能。工程蛛diadematus素4（eADF4）可以被加工成非织造网格，使用静电纺丝。在这里，的eADF4无纺网格用于提高性能的空气过滤装置。

视频: Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

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 ...

科研

生物工程

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

制造棉分析仪器

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

热、压水固化重组蜘蛛丝的材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...