我们感谢BBSRC的资助博士先生弗雷泽细则。

1。静电的随机和不结盟的纤维静电创建通过电位对接地收藏家绘制的聚合物溶液的细纤维网络。集热器可以在很多的形状，可以是静态的，更常见的旋转。溶剂蒸发解决方案之前到达集电极和喷流凝固成纤维。 每种聚合物需要其自己的一套条件，生产的纤维类型。的聚合物，溶剂的浓度，泵浦解决方案和集电极接地，两者之间的电位差之间的距离，速度旋转收集器，流速，温度和湿度都会影响静电。有许多研究，描述电参数的选择，以及如何将这些影响生产的支架（如纤维直径，形态和方向），5，6，7，8在这些实验中支架被剥离的基础上在我们以前的研究中选择的条件。2，9 下面的方法是合适的静电支架生 ​​产使用，如在图1所示的旋转收集器从共聚物，聚乳酸（PLA），聚ε-己内酯（PCL），聚羟基丁酸酯联合戊酸（PHBV的）。整个溶剂二氯甲烷（DCM）的使用。这里的方法生产的微型珠（"珍珠项链"形态）的PLGA微纤，PLA和PCL和PHBV的纳米纤维支架。 <ol><li>大衣抛光/闪亮的一面朝外，旋转芯棒收藏家铝箔。我们芯棒宽20厘米，直径10厘米。 </li><li>准备聚合物溶液，PLA，PCL和PHBV的10 wt％的解决方案作为一个在DCM。 PLGA是在DCM的20 wt％的解决方案。 </li><li>地方4注射器，5毫升注射器泵体积。注射器装到contain聚合物每5毫升，共20毫升。 </li><li>对于解放军，PCL和PHBV使用每注射器-1的40μLmin的流量，。 </li><li>对于共聚物的30μLmin-1％注射器使用的流量。 </li><li>对于解放军，PCL和PLGA使用了17厘米，从针尖到芯棒的工作距离。 </li><li>为PHBV的使用了10厘米，从针尖到芯棒的工作距离。 </li><li> 17000五（73030磷，Genvolt，别让英国）和electrospin收取注射器针头从铝箔涂芯棒到适当的距离。 </li><li>对于随机的纤维在200 rpm的旋转芯棒。 </li><li>对于对齐纤维在1000 rpm的旋转的芯棒。 </li><li>支架可以储存在干燥条件下的铝箔。建议储存在4℃中存在的干燥剂的密封容器中。支架在我们的经验在这些条件下保持稳定（我们不知道任何PU至少4个月（可能更长）blished支架的长期贮存条件）的研究。 </li></ol> 2。连续纺生产的复合支架连续纺丝提供了一个方法，结合不同材料的特性，以创建一个材料，具有最好的两个属性。 PHBV的生产而解放军或PCL纺生产低密度弹性纸平整，致密，脆表。这两种材料的支持细胞附着。连续纺丝这些材料在一个密集的细胞，防渗膜，是有弹性的结果。 <ol><li>设置为每第一节电钻机，与PHBV的纺纱条件。 </li><li>如上electrospin PHBV的。 </li><li>没有改变铝箔，electrospin第二聚合物，聚合物（17厘米如鼓针，17000伏，为解放军200 RPM）在最上层使用的参数和正常条件。这种添加剂的过程中建立了一个支架的双层生产双层。 </li&gt; </ol> 3。多层支架生产退火几层<ol><li>支架可以通过使用热退火多层。要做到这4张被放置在彼此顶部的PLGA，然后在60°C加热退火3小时。 </li><li>支架也可通过蒸汽退火退火。这里的PLGA 4张是放置在彼此顶部和暂停1小时2厘米以上的DCM池（10毫升）。这是在密封容器中在室温下进行。 </li></ol> 4。无菌生产和产后消毒，静电支架<ol><li>无菌脚手架生产，可以实现在无菌环境的洁净室环境内的层流罩静电。做要么无菌医疗级或在DCM孵化消毒聚合物聚合物可以使用。一旦解散，到无菌铝箔裹住为静电聚合物terilised芯棒。支架，然后无菌处理。无菌验证，在无抗生素的培养基中培育适当的时期样品的脚手架。 </li><li>乙醇消毒（这是使用实验，但不是一个公认的杀菌方法，可采取诊所）支架简要放置在70％V / V乙醇溶液，蒸馏水（15分钟）。对于实际的实验目的，这通常是足够的消毒，使他们能够与细胞培养相结合，成功支架。 </li><li>对于过氧乙酸消毒支架都沉浸在过氧乙酸（0.1％V / V磷酸盐缓冲液（PBS）），并在室温孵育3小时，塞利姆等9。 </li><li>伽玛3 kGy的剂量辐照灭菌支架使用铯源塞利姆等9。 </li></ol>5。支架的生物力学测试<ol><li>支架被切断成长方形5毫米×20毫米，使用微米的厚度，并放置到玻色Electroforce 3100的仪器测量。本机适用于0-22列印的力量了6mm和应力/应变曲线绘制负载与位移的位移。这使得计算的杨氏模量和弹性。 </li></ol> 6。 Visualising支架上的细胞和评估流脑生产细胞可以允许一见细胞在支架上，由于它们附着，迁移和增殖的重要荧光染料染色。邮政文化的支架上细胞的存在，可以决定由4&#39;,6-diamidino-2-苯基盐酸（DAPI）的细胞核染色。生产的ECM支架上的细胞，可以评估范围包括弹性，在这个例子中所示的ECM蛋白染色细胞。支架用于测定有播种前1.5厘米×1.5厘米到平方厚度至少0.2毫米，切。 在这些研究中，人类的皮肤成纤维细胞中使用，因为他们在软组织重建中发挥的作用，这是我们实验室的主要研究兴趣。 细胞是从从接受乳腺癌减少或腹壁（同意他们的组织被用于研究目的）的择期手术的患者的皮肤样​​本。组织收集和研究组织银行牌照12179下使用匿名。组织洗净，用PBS包含链霉素（0.1毫克/毫升）和青霉素（100国际单位/毫升）和两性霉素B（0.5微克/毫升）。组织标本培养在0.1％W / V胰蛋白酶和0.1％葡萄糖的PBS（12-18小时，4℃）。真皮层脱落，剁碎精心培养胶原酶10毫升（0.5％W / DMEM培养液中的V和10％FCS，37°C间为18小时）。离心导致细胞suspens离子为10分钟（400克），产生沉淀的细胞可以在DMEM培养和传代辅以胎牛血清（FCS中，10％V / V），链霉素（0.1毫克/毫升），青霉素（100 IU /毫升）和两性霉素B（0.5微克/毫升）。只有通过4-9成纤维细胞用于实验。 <ol><li>加入胰蛋白酶/ EDTA（5毫升，5毫克/毫升胰蛋白酶，2毫克/毫升EDTA生理盐水），5分钟的孵化在接种人类皮肤成纤维细胞，一旦融合中的T75（EasyFlask，NUNC，美国纽约） 37°C。悬浮离心10分钟（150克）。细胞悬浮于5毫升的DMEM（FCS（10％V / V），链霉素（0.1毫克/毫升），青霉素（100国际单位/毫升）和两性霉素B（0.5微克/毫升）的补充），并计算使用血球，浓度调整播种。细胞通常在50,000元细胞接种。 </li><li>如果需要，前播种支架上的细胞，细胞可以预先标记，用红色或绿色的CellTracker。在细胞S是3×5毫升PBS洗涤。 10毫米无血清，细胞，适当的，中期（10毫升）加入细胞孵育45分钟37°C。CellTracker的解决方案孵育后，细胞被冲洗3×5毫升PBS以下，它们接种到支架上。继支架表面，可在轴突ImageExpress显微镜（分子器件，桑尼维尔，美国），在570纳米波长λex成像 - 发射波长620纳米（CellTracker红色）和480 nm波长λex - 533 nm的发射波长（CellTracker绿色）。调查到支架的多光子共聚焦显微镜可用于细胞的渗透更深。这可以实现绕成最支架或无细胞200微米渗透。 </li><li>发布文化的样品在1毫升3.7％甲醛的PBS固定在37°C，20分钟，然后用3×1毫升PBS洗涤。 </li><li> 200μL弹性的主要抗体被添加到每个样品（5％V / PBS中至五，兔抗人αELAS锡，AbDserotec，基德灵顿，英国），在37°C孵育30分钟。 </li><li>样品3×1毫升PBS冲洗，然后在二级抗体溶液（0.5％V / V山羊抗兔IgG（下称"财委会"）:FITC标记）孵育30分钟，PBS中含1微克/毫升）的DAPI（。 </li><li>在此之后的样品3×1毫升PBS洗涤。 </li><li> DAPI和二次抗体染色样品，然后成像轴突ImageExpress荧光显微镜，365纳米波长λex - DAPI和480 nm的波长λex460 nm的发射波长 - 533 nm的发射波长为二级抗体。的DAPI污渍的原子核，并允许一见纤维细胞内的分布很爽快。 </li></ol> 7。支架遭受双向动态调节细胞检查动态调节成纤维细胞外基质生产的影响，我们开发了一个简单的概念证明型生物反应器探索。 <ol><li>组装气球和流调控仪器和准备系统，所以它可以很容易地放入无菌容器适用于细胞培养后，它是涂层。 </li><li>高压灭菌的仪器，包括气球（122℃1小时，220毫巴）。我们可以确认，气球生存而不影响其功能，通过反复膨胀和扣除他们的灭菌。 </li><li>在一个干净的房间，解开在一个位置的层流罩仪器是静电上。 </li><li>气球充气到所需的表面积（记得气球仍需要融入文化船只）与磷酸盐缓冲液和连接电气接地在设备中的点，这并不需要无菌的PBS（支管3路自来水）。 </li><li> electrospin到使用正常的纺纱条件的气球所需的聚合物，用10厘米的距离。让脚手架干燥1小时。 &#39;湿&#39;纤维&#39;粘&#39;，足以坚持冲浪ACE的气球而不随后分离。 </li><li>气球放入无菌容器和运输层流罩，适用于细胞培养。 </li><li>从船只及地方到无菌的表面（培养皿中）取出气球和多次（每20秒）吸取细胞悬浮液（1×10 6细胞的培养液5毫升），以试图分发到20分钟的涂层球囊细胞在其表面均匀。 </li><li>气球放入培养容器，并添加适当的细胞类型的预热的媒体。 </li><li>通胀仪器连接到注射泵（肯特科学，精灵此外，康涅狄格州，美国）和充气/放气的气球给双轴腹胀。一个计算机控制的注射泵，可以用来实现更复杂的腹胀制度。 </li></ol> 8。代表结果下面的数字代表的结果是可以预期的如果上面的方法也随之而来。 静电可以用来创建与随机和有序的架构（ 图1）支架，这是重复和纤维均匀。许多类型的聚合物可静电与可以有很大的不同，PHBV的解放军或PCL 图2所示的特点。静电能产生轻盈蓬松棚架或密集的细胞不透膜（ 见图3）。这里显示所有棚架，促进细胞附着和增殖。以前的工作表明，细胞可以通过这些支架迁移到至少500-600微米的深度对于解放军9，平均纤维直径为3μm; PHBV的，它是从5-20微米不等的珍珠为0.3μm; PCL的是3微米;共聚物，它是11微米。使用其他溶剂系统的其他研究报告，PHBV的无珠或聚合物珍珠可以作为纤维的静电。10,11 &lt;一类p =的"jove_content"&gt;如果需要较厚的支架可以采用蒸汽和热退火，退火层支架一起（ 见图4）。这些脚手架层不分层，层与层之间的交界处，它可以是非常困难的。 我们表明，双层膜可制成细胞A和B都可以在一个单独的膜培养无混杂， 如图5所示。在这里，我们展示利用人类皮肤成纤维细胞与两种不同的荧光细胞追踪染料着色。这种双层膜将是有益的培养细胞时，如硬骨或软骨组织，从设计，形成柔软的细胞分离的一方（通常增长较快）组织形成的另一侧，如腭裂修复或重建牙周手术12，13 我们尊重静电杀菌影响支架先前报道说"的脚手架和随后的细胞培养灭菌影响的方法。9这是在图6所示这表明过氧酸，γ射线辐照和乙醇对的纤维直径和极限拉伸强度和杨氏1的PLGA支架的弹性模量的影响。 γ射线辐照对纤维直径没有显着的效果，而过氧乙酸和乙醇，纤维直径减少约50％。每个灭菌方法与极限抗拉强度改变抗拉强度和弹性支架。这些支架上的细胞培养，进一步降低了最终的拉应力，但增加了弹性。 最后，静电支架上培养的细胞，动态双轴腹胀的效果测试方法。这个概念证明型方法表明，细胞保持在动态的可行腹胀，但也产生这些条件下的弹性增加的数额。这反差明显缺乏弹性时保持相同的脚手架上相同的细胞在静态条件下（ 见图7）。 <img src="/files/ftp_upload/4172/4172fig1.jpg" alt="图1"/> 图1显示一个卡通电钻机和随机和平行纤维，然后放置在彼此的纤维层的旋转。垂直纤维可以创建一套一致纤维电到铝箔，箔旋转90°，然后立即电第二套的对准这些顶级的纤维。 <img src="/files/ftp_upload/4172/4172fig2.jpg" alt="图2"/> 图2显示的形态（一）解放军（比例尺为100微米），（二）PHBV的（比例尺为100微米），（三）的PCL（比例尺是随机的静电垫<html> 100微米），（四）共聚物（比例尺为200微米）。需要注意的是解放军，PCL和PLGA是所有微纤统一支架。 PHBV的是纺纳米纤维与连接5-20微米大小的珠子作为"珍珠项链"。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4172/4172fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/4172/4172fig3.jpg" alt="图3"/> 图3。一个多层支架的生产。这里的支架最初纺PHBV的和然后使用PLA或PCL填充注射器。这些纺PHBV的支架上。图中显示的外观，这些多层支架（A）单PHBV的层，（B）一个PHBV的解放军双层截面，呈现出密集的纳米纤维，"珍珠项链"PHBV的层（左）和更开放的微纤解放军层（右）和（C）单解放军层。NK"&gt;点击这里查看大图。 <img src="/files/ftp_upload/4172/4172fig4.jpg" alt="图4"/> 图4。厚的支架，可以产生热退火和蒸汽退火。 （A）和（b）显示通过蒸汽退火解放军的支架部分，约150微米的初始纤维支架放在一起，二氯甲烷蒸汽是用来制造更厚的支架高达500微米。 （C）和（四）我们可以看到，脚手架层较厚的纤维层较薄的纤维由热退火薄和厚纤维层一起创建穿插组成。这种方法可以用来生产复杂的机械性能的支架。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4172/4172fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/4172/4172fig5.jpg" alt="图5"/> 连接<html>古雷5。外观上的双层支架的细胞。在所有情况下的细胞中存在是人类的皮肤成纤维细胞。 （一）静电解放军成纤维细胞的细胞是固定的，用DAPI染色。 （二）的DAPI染色PHBV的细胞。 （c）中成纤维细胞是一个重要的染料，CellTracker绿色预染，你可以看到他们的外观上解放军的双层侧。 （D）部分通过降低PHBV的表面上的解放军表面上的绿色染成纤维细胞的双层红色染的成纤维细胞。 （五）对PHBV的表面与CellTracker红色预染的成纤维细胞生长。重要的荧光染料的使用提供了一个方便的方法，寻找分布在细胞上的脚手架，而细胞仍然增长。人们可以经常使用这些染料至少7天。但是染料浓度变成稀释的细胞分裂。比例尺等于0.1毫米。 g6.jpg"ALT ="图6"/&gt; 图6。静电支架的生物力学性能得到了玻色Electroforce张力装置（一）。 （二）应力/应变曲线的PLGA支架消毒伽马射线照射，酒精，过氧乙酸，或无菌生产。从这样一个图表:之前，它打破极限拉伸应变和杨氏模量的纤维可承受的极限拉伸应力（悉尼），可以得到三个测量。后者给出了一个支架的弹性的迹象。 （三）上的PLGA微米纤维直径的影响，每个灭菌方法。每个灭菌方法降低抗拉强度。过氧酸和γ射线辐照降低的杨氏模量，给予更弹性的支架，酒精使脚手架特别脆。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4172/4172fig6large.jpg" target="_blank">点击这里查看大图</a> 。 <imgSRC ="/ files/ftp_upload/4172/4172fig7.jpg"ALT ="图7"/&gt; 图7。此图显示了使用一个简单的气球提供了一个双向的生物反应器支架（及内支架生 ​​长的细胞）可以受到时期双轴腹胀。 （一）泄了气的皮球到PHBV的静电纤维，已存入。在这个阶段，气球被部分覆盖纤维。 （B）气球完全涂上PHBV和PLA纤维。 （C）细胞悬液，反复洗涤后到气球。 （四）内放置一个气球，气球连接到一个注射器泵和PBS作为导体电解质的无菌媒体瓶轻轻吹气球对一个程序的时间表，并允许通缩。 （五）（六）从气球中删除显示细胞活力进行分析和实验结束在支架上的细胞活菌开发一个深蓝色的代谢INDicator 3 - （4,5 - 二甲基-2 - 基）-2,5 - 二苯基溴化。 （G）显示，培养细胞（蓝色）气球和双轴腹胀，发展弹性纤维（绿色，使用弹性蛋白特异性抗体染色），而在静态条件下培养相同的支架（高）相同的细胞可以忽略不计的弹性生产。比例尺等于0.025毫米。

<ol>
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J., Blackwood, K. A., Chapple, C. R., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+biodegradable+scaffolds+for+tissue+engineering+of+the+urethra.">Developing biodegradable scaffolds for tissue engineering of the urethra.</a> BJU Int. 107 (2), 296-302 (2010).</li><li>Tong, H. -. W., Wang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+into+the+influence+of+electrospinning+parameters+on+the+diameter+and+alignment+of+poly(hydroxybutyrate-co-hydroxyvalerate)+fibers.">An investigation into the influence of electrospinning parameters on the diameter and alignment of poly(hydroxybutyrate-co-hydroxyvalerate) fibers.</a> Journal of Applied Polymer Science. 120 (3), 1694-1706 (2011).</li><li>Tong, H. -. W., Wang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+poly(hydroxybutyrate-co-hydroxyvalerate)+fibrous+tissue+engineering+scaffolds+in+two+different+electric+fields.">Electrospinning of poly(hydroxybutyrate-co-hydroxyvalerate) fibrous tissue engineering scaffolds in two different electric fields.</a> Polymer Engineering &amp; Science. 51 (7), 1325-1338 (2011).</li><li>Retzepi, M., Donos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+Bone+Regeneration:+biological+principle+and+therapeutic+applications.">Guided Bone Regeneration: biological principle and therapeutic applications.</a> Clinical oral implants research. 21, 567-576 (2010).</li><li>Moreau, J., Caccamese, J., Coletti, D., Sauk, J., Fisher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+solutions+for+cleft+palates.">Tissue engineering solutions for cleft palates.</a> Journal of oral maxillofacial. 65, 2503-2511 (2007).</li><li>Yang, F., Both, S., Yang, X., Walboomers, X., Jansen, J. <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+an+electrospun+nano-apatite/PCL+composite+membrane+for+GTR/GBR+application.">Development of an electrospun nano-apatite/PCL composite membrane for GTR/GBR application.</a> Acta biomaterialia. 5, 3295-3304 (2009).</li><li>Yoshimoto, H., Shin, Y., Terai, H., Vacanti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biodegradable+nanofiber+scaffold+by+electrospinning+and+its+potential+for+bone+tissue+engineering.">A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering.</a> Biomaterials. 24, 2077-2082 (2003).</li><li>Telemeco, T., Ayres, C., Bowlin, G., Wnek, G., Boland, E., Cohen, N., Baumgarten, C., Mathews, J., Simpson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cellular+infiltration+into+tissue+engineering+scaffolds+composed+of+submicron+diameter+fibrils+produced+by+electrospinning.">Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning.</a> Acta biomaterialia. 1, 377-385 (2005).</li></ol>

没有利益冲突的声明。

静电是一个非常流行的技术，生产组织工程支架。，14，15，16，虽然它是相对简单的生产实验用静电支架基本技术是复杂和多方面的，有许多变数。有许多研究描述如何电参数确定支架的生产。在这项研究中的重点是在相当大的挑战"的后期制作，作出适当的结构和力学性能的支架，并鼓励在他们的细胞，使细胞外蛋白质，以实现组织适合植入人。 我们在这篇文章的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚乳酸 - 乙醇酸</td><td> Sigma Aldrich公司</td><td></td><td></td></tr><tr><td>聚乳酸</td><td> Sigma Aldrich公司</td><td> 81273 </td><td>固有粘度〜2.0dl /克</td></tr><tr><td>聚ε-己内酯</td><td> Sigma Aldrich公司</td><td></td><td></td></tr><tr><td>聚羟基丁酸酯-CO-羟基12:1 </td><td>古德费洛</td><td> 578-446-59 </td><td> PHB88/PHV12 </td></tr><tr><td>二氯甲烷</td><td> Sigma Aldrich公司或Fisher </td><td> 270997或D/1850/17 </td><td> &gt; 99.8％，包含50-150ppm的稳定戊烯</td></tr><tr><td> 50多彩色气球</td><td>威尔金森的五金百货有限公司</td><td> 0105790 </td><td></td></tr><tr><td>羊抗兔IgG（FC）:FITC标记</td><td> AbDserotec </td><td> STAR121F </td><td></td></tr><tr><td>兔抗人α弹性</td><td> AbDserotec </td><td> 4060-1060 </td><td></td></tr><tr><td>螺丝帽GL45 PP 2端口，PK / 2 </td><td>补充劳工计划</td><td> 1129750 </td><td></td></tr><tr><td> 4&#39;,6-Diamidino-2-苯基二胺</td><td> Sigma Aldrich公司</td><td> 32670 </td><td></td></tr><tr><td> CellTracker绿色CMFDA </td><td> Invitrogen公司</td><td> C7025 </td><td></td></tr><tr><td> CellTracker红色CMTX </td><td> Invitrogen公司</td><td> C34552 </td><td></td></tr></tbody></table>

postproduction processing of electrospun fibres for tissue engineering

静电是一种常用和通用的方法来产生3D组织工程支架材料（通常是可生物降解），1，2，3 在体内许多组织进行双向腹胀如皮肤，膀胱，盆底，甚至为儿童硬腭不同程度的增长。在生产支架为这些目的，有必要制定适当的生物力学性能的支架（是否没有或细胞与实现）和临床使用无菌。本文的重点是如何建立基本的静电参数（如静电有大量的文献），但对如何修改纺支架的后期制作，以使他们适合组织工程的目的 - 这里的厚度，力学性能和灭菌（需要临床使用）被认为是，我们还描述了如何可以培养细胞支架和双轴应变条件为特定应用。静电往往会产生薄片;静电收集变得与中空纤维涂上它变成不良导体，如纤维不再存款。因此，我们描述的方法来产生热或蒸汽退火增加支架的强度，但不一定是弹性的较厚结构。连续纺丝支架不同的聚合物，以实现复杂的支架也被描述。灭菌方法可以产生不利影响支架的强度和弹性。我们比较三种方法对静电聚乳酸 - 乙醇酸共聚物（PLGA）支架的生物力学性能的影响。 细胞外基质（ECM）在支架上的细胞蛋白质生产的支架和评估被描述成像。支架在体外培养细胞可以提高支架强度和弹性，但组织工程literatu再次表明，细胞往往不能产生相应的ECM静态条件下培养。很少有商业系统，使一个动态调节制度下的支架上的培养细胞-一个例子是，它可以用来对细胞在支架上的空调方案在媒体使用机械交手充满室举行的玻色Electroforce 3100 4预算控制在2维的失真细胞培养生物反应器的方法描述。我们表明，细胞可以诱导产生这些条件下的弹性。最后加工或无细胞培养支架的生物力学性能的评估描述。

Watch this Scientific Journal Video about Postproduction Processing of Electrospun Fibres for Tissue Engineering at JoVE.com

Postproduction Processing of Electrospun Fibres for Tissue Engineering

静电支架可以处理后生产组织工程的应用。在这里，我们描述复杂的支架（连续纺），为实现无菌（无菌生产或灭菌后生产）和较厚的支架（多分层使用热或蒸汽退火）为实现适当的生物力学性能，纺纱方法。

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 ...

postproduction-processing-of-electrospun-fibres-for-tissue-engineering

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

18.0K 次观看.  University of Sheffield. 静电支架可以处理后生产组织工程的应用。在这里，我们描述复杂的支架（连续纺），为实现无菌（无菌生产或灭菌后生产）和较厚的支架（多分层使用热或蒸汽退火）为实现适当的生物力学性能，纺纱方法。

视频: Postproduction Processing of Electrospun Fibres for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

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 ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

保利的静电纤维支架（甘油 - 十二双）工程神经组织从小鼠胚胎干细胞

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

静电纤维组织工程的后期处理

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