这项工作是由国家科学基金会事业CBET - 0747615。作者想感谢博士。道格拉斯史密斯和大卫F.米尼为他们的辅导和支持。

1。轴突的拉伸生长生物反应器系统的概述<ol><li>已动用两种主要的方法，适用于神经元的实验部队。部队在第一种方法，适用于整个神经元（陆，Franze等人，2006年 ; Chetta 2010年，桂等 ;。林克韦斯特，刘等2010 ）。在第二种方法中，直接应用于部队是轴突生长锥的拉动。使用玻璃针，这后一种方法已用于模式形成新的轴突（贝尔纳尔，Pullarkat 等2007年;奥图尔，Lamoureux 等2008;。Lamoureux，海德曼等 2010），以确定伸长力阈值和回缩（Dennerll，Lamoureux 等 1989;郑Lamoureux 等人 ，1991年;。Lamoureux，郑等人1992年），并分析在轴突末端的神经递质集群（Siechen 2009年，羊等）。这种方法的一个独特的组织工程延伸，开发生产大脑神经构造使用一个自动化的轴突伸展增长（ASG），生物反应器系统（磐田， 布朗等人2006 年 。普菲斯特， 岩田等2006） 。最近，我们开发了一个实时微观助理秘书长（Loverde，Ozoka 等2011）研究的生物反应器系统的小型化版本。在这里，我们详细为期9天的协议（表1），我们目前使用的准备生物反应器和执行助理秘书长常规。 </li><li> 整体运作 ：助理秘书长的生物反应器系统是由三个主要部分组成，1）生物反应器与独立的车道（拉长井），神经元培养和延伸，2）室申请伸展的力量和3）步进电机自动化直线运动表驱动器控制器的软件来控制舒展增长（图1）。 </li><li>简言之，生物反应器的原型的制备是在一台机器使用的是立式铣床（布里奇波特，埃尔迈拉，纽约州）的店。生物相容性好，易于杀菌和耐用性，内部的生物反应器组件加工从3 / 8“聚醚醚酮（PEEK）。透明的聚碳酸酯盖使用，以便光镜和文化观赏，耐腐蚀316不锈钢螺钉和硬件的完整大会（麦克马斯特卡尔，艾姆赫斯特，IL）。 </li><li> 生物反应器室是由一个伸缩的框架，形成外部操纵（图2）3车道，一个可调节的牵引块，操纵细胞穿过小巷，和突起的牵引杆。神经元的文化是一次性ACLAR拖车牵引块（图2和3）支持的文化基板上镀。一次性盖玻片固定基板重视伸展框的底部，并跨越所有3个车道。镀神经元伸展之前，必须通过生长锥延伸到固定基板延长牵引基板轴突的进程。轴突的人口，桥梁基板也将随之发生拉伸牵引块的位移控制。 </li><li> 自动化直线运动表由步进电机（HT23 - 397，应用运动产品，沃森维尔，CA）和直线运动的表安装到聚甲醛对齐表（MIPS - 2 - 10 - 1.0MM，伺服系统，Monteville，新泽西州） 。生物反应器室是平行的直线运动表的表内就位。从生物反应器腔延长牵引杆固定在直线运动表，使用适配器。 </li><li> 步进电机驱动器控制器 （四2035年，应用运动产品）是使用包括四程序员软件编程控制操纵牵引基板。轴突伸展是一个循序渐进的方式，采取了一系列间隔停留时间（普菲斯特，2004年Iwata 等。普菲斯特，岩田等人 2006年）的小排量步骤的应用。这台计算机控制系统提供方案助理秘书长定制的配置文件的能力，和超过几天至数周不断的实验至关重要。 </li></ol> 2。生物反应器商会的制备<ol><li>准备一次性神经元文化的牵引和固定的文化基板： <ol><li>拖车文化基板削减8.5“× 11”张ACLAR薄膜（33C 2.0 MIL，结构探头，西切斯特，PA）。使用锋利的刀片切割基板约0.5 x 2.5厘米，或略高于车道的宽度至少有1 - 2mm的间隙，允许双方较短。 </li><li>轻轻砂使用罚款1200砂纸（麦克马斯特卡尔）两侧牵引文化基板的下1 / 3路。三鼎牵引基板，促进轴突牵引到盖玻片固定基板基板的生长。 </li><li>剪下5 × ACLAR7厘米件或使用1号玻璃盖玻片为固定基板（＃4865-1，脑研究实验室，牛顿，MA）。 </li><li>清洁日Ë文化基板用稀Alconox溶液，并彻底冲洗用纯化的DH 2 O </li><li>在70％乙醇30分钟的浸泡消毒的培养基质。允许基板无菌组织培养罩内空气干燥。 </li></ol></li><li>稀释Alconox和高压灭菌器生物反应器腔清洁与消毒高压灭菌器容器内。后高压灭菌，立即转移到无菌罩和风干。 </li><li>继续在无菌罩，胶水内的培养基质，生物反应器使用的硅室温（​​道康宁，麦克马斯特卡尔＃732）和无菌棉拭子（麦克马斯特卡尔）： <ol><li>在牵引块腿直立，胶水的牵引培养基质，在非打磨的部分牵引块腿。避免接触打磨文化表面。 </li><li>胶固定的文化底物的生物反应器室的底部。 </li><li>对粘基材的干棉签轻轻按下一个删除多余的胶水和气泡。 </li><li>由于硅室温滤醋酸是有毒的神经细胞，生物反应器是左干罩内，在紫外光下，2天前引入神经元文化的完整。 </li></ol></li><li>下拖带块双腿之间的打磨牵引基板和固定基板的提示，实现了2 - 3mm的重叠。关键注意必须支付的重叠的大小，如果过大，牵引基板提示可能转移的固定基板;减少轴突跨越重叠（图3）。 </li><li>定位在牵引块的起始位置，用生物反应器内收回的牵引杆。拧紧固定螺丝，以防止运动的牵引杆，前伸展增长。 </li></ol> 3。神经元文化<ol><li>池1ml含10μg/ mL的超高分子量聚- D -赖氨酸（猫＃354210，BD，贝德福德，马）在每个车道的衬底的界面面积的无血清培养基。允许的解决方案，坚持在室温下1小时不受干扰。冲洗轻轻地用最后的冲洗与文化传媒，卫生署2 O 3倍。移液应在后面的小巷，最远的衬底的界面。 </li><li>从E16大鼠小狗分离背根神经节植（病种付费）。使用立体显微镜，稀释成滴用皮氏培养皿中的植。收集到100μL移液器和板边缘打磨的牵引文化基板的3-4植。必须小心板植牵引基板使用媒体的一个小水坑的边缘。每巷文化传媒1ML是足够的限制外植体的运动的同时，为几个小时，以防止蒸发。媒体的提法：与B - 27 + 0.5mm的L -谷氨酰胺（Invitrogen公司，卡尔斯巴德，CA），1％FBS - HI（Hyclone公司，马萨诸塞州沃尔瑟姆），2.5G / L D -葡萄糖（G - 7528，Sigma公司，ST Neurobasal路易斯，密苏里州），20ng/mL神经生长因子（13290-010，Invitrogen公司）和20微米FDU 20μM的尿苷有丝分裂抑制剂（F - 0503，U - 3003，Sigma公司）。 </li><li>将盖子和转移为1小时，或直至细胞生物反应器的孵化器。 </li><li>填写与文化传媒，生物反应器，在最远的外植体，以避免脱落的点。孵育至少5天的生物反应器，而神经元延伸到固定基板轴突的进程。 </li></ol> 4。轴突伸展增长<ol><li>生物反应器的无菌罩内进行最后的准备程序： <ol><li>生物反应器内填充的水库与磷酸盐缓冲液加湿文化传媒商会和限制蒸发。在我们的系统，镂空围墙作为水库。另外，小培养皿盖子可放在文化车道之上的牵引块两侧。 </li><li>更换培养基和填写的行车线，以能力。移液应在后面的小巷，最远的衬底的界面。 </li></ol></li><li>座椅内的生物反应器的自动直线运动表。如果实验是一个孵化器内运行，它必须不加湿，由于自动化直线运动中的潜在的腐蚀。 </li><li>牵引杆适配器固定牵引杆。 </li><li>使用SI Programmer软件慢跑直线运动表的阶段，以配合牵引棒适配器，并拧紧到舞台。为方便起见，提前准备泗编程序列，以操纵的阶段，在特定的增量。 </li><li>松开固定螺丝上的生物反应器室，允许自由流动的牵引块。 </li><li>弹力是应用在一个循序渐进的方式，发起了一系列间隔停留时间（普菲斯特， 岩田等人，2006年）的小排量步骤。我们的范式开始到2微米步骤，每172秒，净1毫米舒展超过24小时。一天后，伸长率可憋足的位移增加或减少的停留时间（表2）。 </li><li>一旦启动了助理秘书长，媒体的变化通常不是必需的。然而，随着时间的推移，文化传媒普遍变为酸性和明显的是在颜色偏黄变化。如果需要进一步的实验是，旧媒体是永远不会完全耗尽，而只是部分地改变浮动轴突，以尽量减少创伤。 </li></ol> 5。代表性的成果： 轴突的进程可以接受的迅速和强大的伸展增长。最初，这个过程就开始一段慢伸展（≤1mm/day）小，罕见的位移组成。在第一个24小时的伸展运动，神经生长途径mechanotransduction时，即神经元开始除了轴突缸。在24小时连续助理秘书长，轴突显示一个更大和更频繁的的位移越来越宽容。在一般情况下，轴突可以承受1mm/day每12-24小时的伸长率（普菲斯特， 岩田等人2004 年 。普菲斯特，Bonislawski等2006年;。普菲斯特， 岩田等2006年）的增加。然而，伸长率增加太快，可能导致更快速的选择轴突的生长，但也将导致病理闭塞，导致断线。 伸缩轴突生长的倾向，形成捆绑，类似的架构分册。利用当前的协议，中央，舒展生长的轴突束部分没有文化基板的粘连。只有最初坚持，近端和远端伸展轴突生长段保持培养基质。因此，舒展生长轴突的中央部分自由浮动，并中断由于处理不当很敏感。 对于各种各样的原因，一些轴突不能生长在应用的延伸率。例如，一个背根神经节神经元有两个轴突的过程，这两者都经历舒展，未必能够将足够的蛋白质和应用的延伸率增长。轴突不能容纳适用于拉伸薄以下泊松效应。随后的拉伸会导致轴突，抑制大会闭塞，导致病理断线。然而，大部分的轴突，是能够接受助理秘书长成功的，只有很小的百分比轴突接受这个修剪过程。 <img alt="图1" src="/files/ftp_upload/2753/2753fig1.jpg" /> 图1。轴突伸展生长生物反应器系统。生物反应器（一）文化室及自动直线运动表，（二）步进电机驱动器控制器和硅的编程软件。 <img alt="图2" src="/files/ftp_upload/2753/2753fig2.jpg" /> 图2生物反应器的文化会所。这幅漫画描绘了从拆除盖顶部的生物反应器腔。牵引块的位置反映了轴突伸展生长的最后阶段。可以看出，在文化车道内束拉伸生长轴突。 <img alt="图3" src="/files/ftp_upload/2753/2753fig3.jpg" /> 图3。文化基板电镀接口。这幅漫画描绘的每个生物反应器从侧面看弄内的拖车机制的组成部分。 （上）的牵引和固定的培养基质的正确重叠。 （下）的牵引和固定基板过多的重叠，导致牵引基板的一角卷曲。 电影1。附件商会生物反应器培养基质。 <a href="http://www.jove.com/files/ftp_upload/2753/movie1.mov">点击这里观看视频</a> 电影2。背根神经节植到拖车基板的电镀<a href="http://www.jove.com/files/ftp_upload/2753/movie2.mov">。点击这里观看</a>视频 电影3。SiProgrammer用法。 <a href="http://www.jove.com/files/ftp_upload/2753/movie3.mov">点击这里观看视频</a> 电影4。生长锥扩展到固定基板。 <a href="http://www.jove.com/files/ftp_upload/2753/movie4.mov">点击这里观看视频</a> 电影5。轴突伸展生长。 <a href="http://www.jove.com/files/ftp_upload/2753/movie5.mov">点击这里观看视频</a> <table border="1"><tbody><tr><td> 日 </td><td> 步骤 </td></tr><tr><td> 1 </td><td>杀菌及烘干</td></tr><tr><td> 2 </td><td>涂胶及装配</td></tr><tr><td> 4 </td><td>涂料及神经文化电镀</td></tr><tr><td> 9 </td><td>拉伸生长开始</td></tr></tbody></table> 表1。实验附表。 <table border="1"><tbody><tr><td></td><td> 时间[HR] </td><td> 伸长率[毫米/天] </td><td> 停留时间[S] </td><td> 总长度[mm] </td><td> 共有拉伸时间[天] </td></tr><tr><td>预紧</td><td> 24 </td><td> 1 </td><td> 172.8 </td><td> 0 </td><td> 1 </td></tr><tr><td>伸展</td><td> 24 </td><td> 1 </td><td> 172.8 </td><td> 1 </td><td> 2 </td></tr><tr><td>伸展</td><td> 24 </td><td> 2 </td><td> 86.4 </td><td> 3 </td><td> 3 </td></tr><tr><td>伸展</td><td> 24 </td><td> 3 </td><td> 57.6 </td><td> 6 </td><td> 4 </td></tr><tr><td>伸展</td><td> 24 </td><td> 4 </td><td> 43.2 </td><td> 10 </td><td> 5 </td></tr><tr><td>伸展</td><td> 24 </td><td> 5 </td><td> 34.6 </td><td> 15 </td><td> 6 </td></tr></tbody></table> 表2。伸长率附表。所有的拉伸步骤2微米的位移（10步进电机步骤= 2微米长）。

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B., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc Natl Acad Sci U S A. 103, 17759-17764 (2006).</li><li>O'Toole, M., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Pfister, B. J., Bonislawski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-grown+axons+retain+the+ability+to+transmit+active+electrical+signals.">Stretch-grown axons retain the ability to transmit active electrical signals.</a> FEBS Lett. 580, 3525-3531 (2006).</li><li>Pfister, B. J., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+Engineering+Strategies+for+Peripheral+Nerve+Repair:+Surgical+Applications,+State+of+the+Art,+and+Future+Challenges.">Biomedical Engineering Strategies for Peripheral Nerve Repair: Surgical Applications, State of the Art, and Future Challenges.</a> Crit Rev Biomed Eng. 39, 81-124 (2011).</li><li>Pfister, B. J., Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Pfister, B. J., Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transplantable+nervous+tissue+constructs+comprised+of+stretch-grown+axons.">Development of transplantable nervous tissue constructs comprised of stretch-grown axons.</a> J Neurosci Methods. 153, 95-103 (2006).</li><li>Siechen, S., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+contributes+to+clustering+of+neurotransmitter+vesicles+at+presynaptic+terminals.">Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals.</a> Proc Natl Acad Sci U S A. 106, 12611-12616 (2009).</li><li>Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+extremes+and+exploitations.">Stretch growth of integrated axon tracts: extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Smith, D. H., Wolf, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+to+produce+sustained+growth+of+central+nervous+system+axons:+continuous+mechanical+tension.">A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension.</a> Tissue Eng. 7, 131-139 (2001).</li><li>Weiss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+patterns:+The+mechanics+of+nerve+growth.">Nerve patterns: The mechanics of nerve growth.</a> Growth, Third Growth Symposium. 5, 163-203 (1941).</li><li>Zheng, J., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+regulation+of+axonal+elongation+and+initiation.">Tensile regulation of axonal elongation and initiation.</a> J Neurosci. 11, 1117-1125 (1991).</li></ol>

神经细胞的体外机械伸长率是根据美国专利＃6264944和＃7429267的专利。

两个关键的步骤，应遵守在生物反应器的准备。首先，在衬底的界面是一个最佳的重叠，以确保轴突可以跨到固定基板。 ACLAR过于卷曲或以其他方式不完善，不应该使用（图3）。为了优化重叠，确认牵引基板打磨均匀，接触超过2 - 3mm长的接触面固定基板均匀。每次实验前，通过仔细调整的牵引腿的高度重叠，应优化。 二，同时提供用于连接的神经元，基材涂料维持相当sheering轴突（1990年海德曼和巴克斯鲍姆;普菲斯特，2004 年 Iwata等;。Loverde，Ozoka等2011）的生物反应器和收缩张力位移造成的力量。基板应当用无菌水冲洗彻底之前和之后赖氨酸涂层。涂料应适用于从刚解冻等份，并尽可能均匀地扩散。更重要的是，不应该被移到基板或以其他方式扰乱粘附期间。在随后的步骤，避免与基板接触，在电镀过程中，所有的解决方案，从尽头的小巷，远离衬底的界面和吸管。 每个生物反应器组件连接的公差可以体现在农闲的形式。在初期的助理秘书长，在系统的松弛明显自动化直线运动中的运动，没有运动的牵引块发生。臂可以每个实验的不同而有所差异，但在我们的经验通常小于1mm。出于这个原因，“预张力”1mm/day懈怠消除相，有一天，之前的助理秘书长的时间表，在此期间，从事生物反应器部件开始运动的牵引基板。 故障排除可能需要自动化直线运动表的位移步骤不匹配的牵引块前的紧张阶段后的位移是完整的。异步牵引块不准确的位移与“粘连”或摩擦产生静电内牵引硬件适配器弯曲。要防止出现这些问题，牵引块的运动应该由专人进行检查，组装后，顺利的接近毫不费力的运动。如果绑定时，牵引大会应事先释放试验。适配器足够的刚度也是必要的，以保证准确，同步位移牵引硬件黏附得不到克服。

<table border="1"><tbody><tr><td> 组件名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚醚醚酮（PEEK） </td><td>麦克马斯特卡尔，艾姆赫斯特，IL </td><td> 8504K69 </td><td>自定义作出反应器室</td></tr><tr><td>聚碳酸酯</td><td>麦克马斯特卡尔，艾姆赫斯特，IL </td><td> 8574K28 </td><td>定制生物反应器室盖</td></tr><tr><td>步进电机</td><td>应用运动产品，沃森维尔，加州</td><td> HT23 - 397 </td><td></td></tr><tr><td>直线运动表</td><td>新泽西州蒙特维尔，伺服系统， </td><td> MIPS - 2 - 10 - 1.0MM </td><td></td></tr><tr><td>步进电机驱动器控制器</td><td>应用运动产品，沃森维尔，加州</td><td> Si2035 </td><td></td></tr><tr><td> ACLAR </td><td>结构探针公司，西切斯特，PA </td><td> 1859年</td><td>拖车文化基板</td></tr><tr><td>第1盖玻片</td><td>脑研究实验室，牛顿，MA </td><td> 4865-1 </td><td>固定文化基板</td></tr><tr><td>硅室温</td><td>麦克马斯特卡尔，艾姆赫斯特，IL </td><td> 7587A37 </td><td></td></tr><tr><td>棉拭子</td><td>麦克马斯特卡尔，艾姆赫斯特，IL </td><td> 7074T62 </td><td></td></tr><tr><td>聚- D -赖氨酸</td><td> BD，贝德福德，马萨诸塞</td><td> 354210 </td><td></td></tr></tbody></table>

axon stretch growth: the mechanotransduction of neuronal growth

在突触前的胚胎发育，神经元突起穿过短的距离，以达到他们的目标，通过生长锥。随着时间的推移，神经somata脱离其轴突终端由于骨骼生长的扩大的有机体（魏斯1941 年，灰色，Hukkanen等1992年） 。 mechanotransduction诱导能够容纳不断伸长的轴突的神经细胞生长的辅助模式（布雷1984年，1994年海德曼和巴克斯鲍姆; 海德曼，Lamoureux等，1995;。普菲斯特 ，岩田等 2004年）。 可以想象轴突伸展增长（ASG）是在胚胎流程短到长神经和成年神经系统的特点脑白质大片成熟的核心因素。要研究在体外的助理秘书长，我们设计的生物反应器，适用于短轴突的神经元文化的进程（Ozoka等。Loverde，2011年）的紧张。在这里，我们详细介绍使用方法，我们准备的生物反应器和进行阿布沙耶夫。首先，在生物反应器的每个伸展巷，神经元是一个微操纵牵引基板镀后。接下来，神经元的再生轴突过程，通过生长锥的延长，到一个固定的衬底。最后，由牵引拉伸增长镀胞体坚持固定基板的轴突终端;扼要骨骼生长后，生长锥的延伸。 以前的工作表明，胚胎大鼠背根神经节神经元的助理秘书长是前所未有的增长率高达10mm/day的，深远的长度可达10cm，而同时增加轴突直径（ 史密斯 ，2001 年 Wolf等人。普菲斯特， Iwata 等人，2004年;普菲斯特2006年，Bonislawski等;。普菲斯特， 岩田等人，2006年; 2009年 ，史密斯）。这是在巨大反差再生生长锥延伸（机械性刺激的情况下）增长率平均1mm/day有限长度小于3cm的成功再生（1997年富和戈登。普菲斯特，Gordon等人2011）。因此，进一步研究助理秘书长，可能有助于揭示失调的增长限制在机械性刺激的情况下的再生机制。

Watch this Scientific Journal Video about Axon Stretch Growth: The Mechanotransduction of Neuronal Growth at JoVE.com

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

一个独特的组织工程方法开发拉长文化中的许多神经纤维扼要轴突伸展增长;一种神经系统发育，神经拉长结合扩大身体的增长。

轴突伸展生长：神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

axon-stretch-growth-the-mechanotransduction-of-neuronal-growth

Research

JoVE Journal

Bioengineering

16.7K 次观看.  New Jersey Institute of Technology. 一个独特的组织工程方法开发拉长文化中的许多神经纤维扼要轴突伸展增长;一种神经系统发育，神经拉长结合扩大身体的增长。

视频: Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

科研

生物工程

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

去耦力学传递线索的影响制备羟基聚丙烯酰胺水凝胶的

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

乳腺辐照有机体的生长与表征

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

在机械生物学研究的轨道摇床上的 6 井板中分段内皮细胞的生长

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...