Name: preparation of thermoresponsive nanostructured surfaces for tissue engineering
Uploaded: 2016-03-01T14:00:00.000+00:00
Duration: 12 min 22 s
Description: Watch this Scientific Journal Video about Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering at JoVE.com

This study was financially supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Program's Project for Developing Innovation Systems "Cell Sheet Tissue Engineering Center (CSTEC)" of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

1.聚苯乙烯块聚（N -isopropylacrylamide）通过两步可逆加成断裂链转移（RAFT）自由基聚合合成<ol><li>溶解苯乙烯（153.6毫摩尔），4-氰基-4-（ethylsulfanylthiocarbonyl）sulfanylpentanoic酸（ECT; 0.2毫摩尔）和4,4&#39;-偶氮二（4-氰基戊酸）（ACVA; 0.04毫摩尔）在40ml 1， 4-二恶烷。冻结真空下在液氮中的溶液15-20分钟以除去反应性物质，并逐步在RT解冻。确保该解决方案完全解冻并重复这一冷冻泵解冻循环脱气三次。 </li><li>得到的聚苯乙烯（PST）（分子量:13500），通过聚合一大分子RAFT试剂在70℃的油浴中15小时。 </li><li>沉淀聚苯乙烯大分子RAFT试剂用800毫升乙醚，并在真空干燥。 </li><li>溶解在4ml的1,4-二恶烷的IPAAm单体（4.32毫摩尔），聚苯乙烯大分子RAFT试剂（0.022毫摩尔），和ACVA（0.004毫摩尔）。 </li><li>去掉如在步骤1.1中提到在由冷冻 - 抽吸 - 解冻脱气循环溶液中的氧气。 </li><li>在70℃下进行聚合为在脱气处理后的油浴15小时。获得合成的St-IPAAm分子（MW:32800）中相同的方式作为聚苯乙烯大分子RAFT试剂。 </li></ol> 2.硅烷化修饰的疏水玻璃基板的制备<ol><li>洗涤玻璃基板（24毫米×50毫米）与过量的丙酮和乙醇超声的为5分钟，除去表面的污染物。 </li><li>干燥烘箱中在基片在65℃下30分钟。然后用氧等离子体（400瓦特，3分钟）在RT以激活基片的表面上。 </li><li>浸入基板中含有1％己基三甲过夜在RT silanize基板甲苯。 </li><li>洗硅烷化基板在甲苯和丙酮中浸泡30分钟以除去未反应试剂。 </li><li>在110℃下进行2小时退火衬底以彻底固定在Surface。 </li><li>削减玻璃刀硅烷化衬底25毫米×24毫米，以适应细胞培养皿（碟尺寸:φ35毫米）。 </li></ol> 3.朗缪尔电影和电影调入表面的制备<ol><li>放置朗缪尔膜仪器在机柜防止尘埃积聚。 </li><li>洗朗缪尔槽（尺寸:580毫米×145毫米）中，用蒸馏水和乙醇以除去污染物的障碍。 </li><li>通过用絮的毛巾擦干槽和障碍。然后填充约1.1毫升蒸馏水槽，并设置在槽两侧的障碍。请注意，应在不在下面的步骤溢出从3.5至3.13中加入蒸馏水。 </li><li>热的铂的Wilhelmy板（周长:39.24毫米），用于与一个气体燃烧器监测的表面张力，直到板变红，然后用蒸馏水洗涤以除去污染物。暂停威廉米悬片上附接至电线表面压力测量仪。 </li><li>根据制造商的协议零表面压力测量仪。通过在槽的两侧的障碍压缩上槽的空气-水界面直到接口达到大约50厘米2没有聚合物的任何液滴。 </li><li>抽吸小的污染物，直到表面压力几乎为0达因/厘米。 </li><li>重新定位在两侧的障碍，并加入蒸馏水，以补偿的蒸馏水从步骤3.6的减少。 </li><li>溶解5毫克合成的St-IPAAm分子在5毫升氯仿中显影溶液。 注意:二氯甲烷或甲苯，也可以用作溶剂。 </li><li>轻轻放下圣IPAAm的27微升溶于氯仿到使用微量或微量低谷。 </li><li>等待5分钟后，以允许氯仿完全蒸发，水平移动两个壁垒以压缩的St-IPAAm molecu勒在界面。保持在0.5毫米/秒，直到50达到厘米2目标区域的障碍压缩率。 注:一种快速的压缩率会导致在朗缪尔薄膜缺陷。 </li><li>测量表面压力具有根据制造商的协议压缩过程中附着于表面压力测量仪器的铂的Wilhelmy板（π）-A 米等温线。 </li><li>到达目标区域的大小后，保持表面5分钟，以使圣IPAAm分子放松;该分子不压缩后立即达到平衡。 </li><li>使用传送装置5分钟以有力吸附膜转移的Langmuir膜疏水改性的玻璃基板。固定在装置上平行疏水玻璃基板。将设备连接到对准平台和垂直移动。 </li><li>在一个desiccato传送装置和干燥1天水平提起衬底河</li></ol> 4.培养细胞和朗缪尔电影转印表面优化细胞粘附和支队<ol><li>至在37℃，在5％的组织培养聚苯乙烯（TCPS）用含有10％胎的Dulbecco改良的Eagle培养基（DMEM）制备细胞悬浮液，培养的牛颈动脉内皮细胞（牛主动脉内皮）至三分之一合流的 CO 2和95％空气牛血清（FBS）和100单位/毫升青霉素。 </li><li>达到汇合后，在37℃下在5％CO 2和95％空气治疗的BAECs用3ml的0.25％胰蛋白酶-EDTA的3分钟。 </li><li>通过加入10毫升含有10％FBS的所述DMEM中停用胰蛋白酶-EDTA，并收集细胞悬液至50ml锥形管中。 </li><li>离心，在120×g离心5分钟，吸出上清液。重悬用10ml的DMEM中的细胞。 </li><li>将在紫外线照射下的圣IPAAm表面干净的长椅上消毒5分钟。 </li><li>种子回收CE在圣IPAAm LLS表面以1.0×10 4个细胞/ cm 2通过一次性血细胞计数器计数的浓度，并通过在37℃下装有的培养箱，用5％ 的 CO 2和95％的显微镜观察表面上的细胞空气。 注:装备净化台紫外线消毒的圣IPAAm表面。 </li><li>通过用放大10倍相差显微镜，在37℃大约24.5小时粘附的BAECs的记录时间推移的图像。后BAEC粘附，在20℃下从圣IPAAm表面的BAECs的记录脱离大约3.5小时。 </li></ol>在朗缪尔电影调入表面5.细胞板材加工<ol><li>以相同的方式用于培养牛主动脉内皮第4节中叙述。 </li><li>种子上的St-IPAAm表面共1.0×10 5个细胞/ cm 2的和在5％CO 2在37℃下孵育3天。汇合的BAECs在20℃自发分离。</li>
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All authors contributed equally to writing the manuscript and have approved the final version. The authors declare that they have no competing financial interests.

温度响应性表面由朗缪尔 - 谢弗方法制造，并为细胞粘附/拆卸和细胞片恢复表面性能进行了优化。当使用表面的制造这种方法，几个步骤是关键的。圣-IPAAm分子的分子的组合物具有在表面结构和有很大影响的表面的稳定性，并且通过扩展，对细胞粘附和脱离。特别是，圣IPAAm分子应具有窄的分子量分布。在我们的方法中，两次与不同PIPAAm链长的St-IPAAm分子通过RAFT聚合合成，使分子量和分子量分布的控制。 应采取措施，以防止空气 - 水界面的污染的圣IPAAm表面的制备过程中，以避免在纳米结构的缺陷。下探聚合物分子到界面上，contaminan之前TS应该吸气，直到表面压力达到约0达因/厘米。污染往往积累围绕槽和威廉米悬片的边缘。因为威廉米悬板具有在其表面上的一些污染，它被退火。当使用纸的Wilhelmy板，在界面处的抽吸步骤应至少重复两次。该πA M等温线也取决于污染的在空气-水界面的存在。我们建议的等温线的曲线膜的制造之前得到一次以上。因为疏水性修饰的玻璃基板的污染，也可能发生，衬底应与新鲜空气或氮气吹入以去除污染。 始终如一地制造的Langmuir膜转印表面（圣IPAAm表面），聚合物的疏水性链段是重要的，因为朗缪尔膜与疏水性修饰的玻璃I之间的疏水相互作用<html> S中的反应的驱动力。在这项研究中，由于嵌段共聚物由聚苯乙烯，这是强疏水性的，并在室温下亲水PIPAAm的，被转印膜稳定地，即使在水或细胞培养条件附着到基底上。这表明，疏水性链段中制造一个健壮的St-IPAAm表面发挥重要的作用。 为了控制上的温度响应表面的细胞粘附和脱离，两者的分子组成和密度的精确控制是重要的。在此朗缪尔-谢弗方法中，通过下降分子的给定量和空气-水界面的目标区域合成的聚合物的每个分子的面积（A M）可以通过压缩进行控制。滴加圣IPAAm分子的A M可以在理论上由下列公式计算: D / 53465 / 53465eq1.jpg"/&gt; 其中A是接口，M W为面积 是圣IPAAms的分子量，c是氯仿溶液的浓度，N A是阿伏伽德罗数，V是聚合物溶液的体积减少。纳米海岛结构加工后的St-IPAAm面观察，因为两亲聚合物形成空气-水界面自组织结构，因为在以往的研究描述了13-17。纳米结构的尺寸和量通过A M和St-IPAAms的分子量控制。 细胞粘附和脱离上通过各种方法，包括电子束照射，表面引发RAFT聚合和旋涂19-27制造PIPAAm涂层表面进行评价。因为密度，分子量，和PIPAAm结构影响CEL升粘附和脱离，用于制造PIPAAm涂覆表面精确的技术是重要的。在此朗缪尔 - 谢弗方法中，如上所述密度，分子量和表面的纳米结构可以控制。圣-IPAAm表面上的细胞粘附和脱离者急剧各A M和St-IPAAm分子组合物的影响，和圣IPAAm的一些条件面为细胞粘附和脱离可以被进一步优化。这些结果表明，该方法可以控制的St-IPAAm表面和细胞之间的相互作用。 为再现地恢复完整的细胞片，两亲性聚合物的设计是最重要的。当仅亲水性聚合物是由朗缪尔 - 谢弗方法的表面上制成，涂覆聚合物容易被蒸馏水或培养基冲洗掉。这个结果表明，弱疏水聚合物不应被用于在此甲基再现地回收细胞片OD因为聚合物改性，表面是在水环境不稳定。在我们的方法中，两亲聚合物的亲水性链段使表面上健壮制造不受水溶液冲洗掉。另外，由于在朗缪尔膜在这项研究中平行转移到玻璃基板上，观察到的表面上的纳米级的海岛结构。虽然朗缪尔膜的上基底侧的结构是公知的是，从不同的顶侧，该结构固定由一个平行转移。 细胞疗法和再生医学一直专注于医治病人谁不响应其他治疗的可能性。我们的实验室已使用具有在组织工程应用潜力上制造温度响应培养表面细胞片工程开发的原始的，独特的自由支架组织工程方法。与细胞片几个临床试验是alreaDY正在进行和已经证明成功的结果为多种类型的组织29-31。虽然牛主动脉内皮被用作细胞来源以制造细胞片层，其它细胞来源，包括干细胞，可用于通过优化的米和组合的St-IPAAm分子的分子的组合物来制造上的圣IPAAm表面片。这种非传统的技术将不仅有助于容易生产纳米结构表面，而且还用于在再生医学使用基本细胞培养物的策略。

纳米结构表面最近吸引了大量关注，因为它们的各种潜在应用，包括图案化，细胞培养，清洗，和表面交换。例如，通过荷叶和其它响应表面的纳米结构启发超疏水表面是能够反应以外部刺激1-4。 朗缪尔膜是最广泛研究的聚合物涂层中的一个。朗缪尔膜通过滴两性分子到空气-水界面5-8形成。该膜然后可通过物理或化学吸附被转移到一个固体表面上，并且可以使用垂直和水平转移方法9-12被控制在固体表面上的分子的构象。朗缪尔膜的密度可以通过压缩的空气 - 水界面精确地调节。最近，这种方法也被证明有效的用于制造纳米尺度的海岛structur利用两亲性嵌段共聚物上课。纳米结构被假定为是由疏水性链段的芯和亲水性链段13-17的壳的。此外，在表面上的纳米结构的数量是通过控制在界面上的嵌段共聚物的每个分子的面积（A M）调节。 我们专注于一个原始，独特的无支架组织工程方法，细胞片工程，使用温度响应培养表面。所开发的技术已经应用到再生疗法为各种器官18。温度响应培养表面是由接枝聚（N- -isopropylacrylamide）（PIPAAm），温度响应分子到表面19-27制成。 PIPAAm及其共聚物表现出的低临界溶液温度（LCST）在水性介质中在接近32℃的温度。培养表面还表现出一个温度响应alternati<html>关于疏水性和亲水性之间。在37℃时，PIPAAm接枝的表面变得疏水的，细胞容易附着和增殖的表面上，以及对现有的组织培养聚苯乙烯。当温度降低至20℃时，表面变得亲水，和细胞表面的自发分离。因此，表面上培养的汇合细胞可被收获作​​为通过改变温度完整片。这些细胞粘附和剥离性能也由朗缪尔膜包衣对实验室演示26,27制成的表面显示。聚苯乙烯（P（ST））和PIPAAm（ST-IPAAm）构成的嵌段共聚物的朗缪尔膜制成。与特定的A M的朗缪尔膜可以水平地转移到疏水改性的玻璃基板。此外，对细胞粘附和脱离由响应于温度的准备好的表面进行评价。 _content"&gt;这里，我们描述协议热 - 反应的两亲嵌段共聚物的玻璃基板上构成的纳米结构的Langmuir膜的制造。我们的方法可以用于在表面科学的各个领域的有机纳米膜提供有效的制造技术，并可以有利于更在有效控制细胞粘附和从表面自发支队。

<table><tbody><tr><td>&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide</td><td>Kohjin</td><td></td><td>No catalog number</td></tr><tr><td>Azobis(4-cyanovaleric acid)</td><td>Wako Pure Chemicals</td><td>016-19332</td><td></td></tr><tr><td>Styrene</td><td>Sigma-Aldrich</td><td>S4972</td><td></td></tr><tr><td>1,3,5-trioxane</td><td>Sigma-Aldrich</td><td>T81108</td><td></td></tr><tr><td>1,4-Dioxane</td><td>Wako Pure Chemicals</td><td>045-24491</td><td></td></tr><tr><td>DMEM</td><td>Sigma&amp;nbsp;</td><td>D6429</td><td></td></tr><tr><td>PBS</td><td>Nakarai</td><td>11482-15</td><td></td></tr><tr><td>Streptomycin</td><td>GIBCO BRL</td><td>15140-163</td><td></td></tr><tr><td>Penicillin</td><td>GIBCO BRL</td><td>15140-122</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4174</td><td></td></tr><tr><td>FBS</td><td>Japan Bioserum</td><td>JBS-11501</td><td></td></tr><tr><td>BAECs</td><td>Health Science Reserch Resources Bank</td><td>JCRB0099</td><td></td></tr><tr><td>Cover Glasses</td><td>Matsunami Glass Industry</td><td>C024501</td><td></td></tr><tr><td>AFM NanoScope V</td><td>Veeco</td><td></td><td></td></tr><tr><td>&lt;sup&gt;1&lt;/sup&gt;H NMR INOVA 400</td><td>Varian, Palo Alto</td><td></td><td></td></tr><tr><td>ATR/FT-IR NICOLET 6700</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>GPC HLC-8320GPC</td><td>Tosoh</td><td></td><td></td></tr><tr><td>TSKgel Super AW2500, AW3000, AW4000</td><td>Tosoh</td><td></td><td></td></tr><tr><td>Langmuir-Blodgett Deposition Troughs&amp;nbsp;</td><td>KSV Instruments</td><td>KN 2002</td><td>KSV NIWA Midium trough</td></tr><tr><td>Nikon ECLIPSE TE2000-U</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

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.

聚苯乙烯和聚（N- -isopropylacrylamide）（圣IPAAms）具有特定分子量的构成的嵌段共聚物通过RAFT自由基聚合来合成。 ECT被作为Moad 等人 28所述制备作为链转移剂。不同PIPAAm链长的二圣IPAAm分子合成，并且所得到的嵌段聚合物进行表征通过1 H核磁共振（NMR）和凝胶渗透色谱（GPC）。圣IPAAms的分子量分别为32800和67900，具有窄分子量分布（1.31和1.50）。聚苯乙烯大分子RAFT试剂和PIPAAm的单体转化率被发现是17.4％和85.0％以上。合成的圣IPAAms被命名为圣IPAAm170和圣IPAAm480分别。 每个分子不同区域（A M），朗缪尔电影以是捏造滴加溶解在氯仿溶液（ 图1A）的St-IPAAm分子空气-水界面。滴加圣IPAAm分子之后，空气-水界面上的表面压力固定为0达因/厘米，不断增加的屏障（ 图1B）在压缩过程中关闭的接口。在形成的空气-水界面上的Langmuir膜的任何缺陷可以从该πA M等温线来推断。所述制备的St-IPAAm朗缪尔膜转印到疏水改性的玻璃基板（圣IPAAm面）（ 图1C）。膜转印表面用原子力显微镜（AFM）进行评价。 图2示出了圣IPAAm170和St-IPAAm480表面的原子力显微镜地形图（1×1微米）。观察的St-IPAAm表面上的纳米结构，而这种结构分别在裸疏水基材上很少观察到。纳米结构的尺寸和形状的强烈dependenT于A M和圣IPAAm组成。 AFM地形图像证实，朗缪尔膜可以均匀地转印到疏水性修饰玻璃基板和，表面形貌可以由分子组成和A M进行控制。 圣-IPAAm朗缪尔膜的稳定性是由衰减全反射评估叶变换红外分光镜（ATR / FT-IR）。 PIPAAms的疏水玻璃基板上的量可以通过从千厘米峰强度比-1从玻璃（的Si-O）1650 -1从PIPAAm（C = O）衍生衍生获得的校准线来估计。校准线，从一系列已知量的疏水性玻璃PIPAAm铸造的计算。被发现的St-IPAAm170（10纳米2 /分子）和St-IPAAm480在衬底上（40纳米2 /分子）的PIPAAm的量为0.87微克/ cm 2，并且分别0.63微克/厘米2。 PIPAAm的表面上，用蒸馏水洗涤后的量几乎相同的表面上，而不洗涤。这些结果表明，所制作的圣IPAAm表面的水分条件是稳定的。 接下来，我们检查了附着力和圣IPAAm表面上的牛颈动脉内皮细胞（牛主动脉内皮）的脱离。在40纳米2 /分子的圣IPAAm480表面上粘附和分离的BAECs的延时拍摄显示在动画图1。粘附的BAECs在37℃来自圣IPAAm170和圣IPAAm480降低后迅速分离温度至20℃。上的St-IPAAm170和St-IPAAm480粘附细胞在37℃的数目为0.6×10 4个细胞/ cm 2和0.9×10 4个细胞/ cm 2，分别表示贴壁细胞的数量S为被圣IPAAm表面的组成进行调制。细胞片层也可通过分离来自这些的St-IPAAm表面汇合培养的牛主动脉内皮回收。将回收的细胞片可视化为二维单元结构（ 图3）。后在圣IPAAm170和St-IPAAm480培养3天达到汇合的细胞表面，在37℃，细胞片减少37℃的温度至20℃后迅速恢复。分子量和St-IPAAms对细胞片恢复A M的效果概括在表1中 。细胞片的生存能力通过台盼蓝染色的BAECs片的分离之后进行评价。用胰蛋白酶-EDTA在37ºC在玻璃基板上处理的细胞用作对照。上的St-IPAAm170，圣IPAAm480和疏水玻璃基板作为控制死细胞比率计算为约11.0％，7.7％，和11.7％之间。这些结果表明，策LL存活率几乎不受温度降低的影响。 <img alt="图1" src="/files/ftp_upload/53465/53465fig1.jpg" /> 图1:一个温敏的Langmuir膜转印表面的制备 （A）中的聚苯乙烯- 嵌段 -聚（N- -isopropylacrylamide）（圣IPAAm）的氯仿溶液轻轻滴到空气-水界面。压缩过程中测得的表面压力，以检测在朗缪尔膜的任何缺陷。 （B）的两个障碍被用来压缩所述接口上的圣IPAAm分子直至50厘米达到2目标区域。 （℃）压缩之后，经疏水改性的覆盖玻璃基片水平放置在接口上与对准平台5分钟。将基板水平地提升并干燥1天。这个数字已经从萨克出版了略作修改UMA 等人 27 <img alt="图2" src="/files/ftp_upload/53465/53465fig2.jpg" /> 图 2: 朗缪尔电影传送面（ST-IPAAm面）的原子力显微镜（AFM）地形图像（1×1微米）左面板:ST-IPAAm170与10纳米2 /分子的A M纳米结构表面。右面板:ST-IPAAm480与40纳米2 /分子的A M纳米结构表面。在用磷酸盐掺杂的硅悬臂用的3 N / m的弹簧常数和70-90千赫共振频率轻敲模式得到的AFM图像。 <img alt="图3" src="/files/ftp_upload/53465/53465fig3.jpg" /> 图 3: 圣IPAAm480朗缪尔膜转印表面上的回收细胞片和40nm的A M的宏观图像 <s高达&gt; 2 /分子。牛颈动脉内皮细胞（牛主动脉内皮）接种于圣IPAAm480表面，并在37℃孵育3天。达到汇合后，牛主动脉内皮自发脱离在20℃，细胞片回收后大约30分钟。插图图像示出了由相差显微镜（10X）观察到的表面上的confluently培养牛主动脉内皮。 <img alt="电影1" src="/files/ftp_upload/53465/53465movie1.jpg" /> <a href="https://www.jove.com/files/ftp_upload/53465/movie1.mp4">动画图1: <a href="https://www.jove.com/files/ftp_upload/53465/movie1.mp4">（</a></a> <a href="https://www.jove.com/files/ftp_upload/53465/movie1.mp4">右键点击下载）</a>牛主动脉内皮的粘附和支队与圣IPAAm480和40纳米2 /分子的上午朗缪尔膜转印表面上时间推移照片。该图像是在2分钟间隔收集，收集的图像被提供作为电影在960x速度。该在电影中比例尺为50微米。 <table fo:keep-together.within-page="1"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td colspan="3">圣IPAAm170 </td><td colspan="3">圣IPAAm480 </td></tr><tr height="23"><td> 3 [纳米2 /分子] </td><td> 10纳米2 /分子] </td><td> 40纳米2 /分子] </td><td> 3 [纳米2 /分子] </td><td> 10纳米2 /分子] </td><td> 40纳米2 /分子] </td></tr><tr><td>附着力弱</td><td>好</td><td>很难恢复</td><td>附着力弱</td><td>附着力弱</td><td>好</td></tr></tbody></table> 表1: 用各种密度和PIPAAm链长朗缪尔膜转印表面的牛颈动脉内皮细胞（BAEC）片恢复 "好"代表完好策LL表恢复。 "几乎恢复"是指该细胞可以是confluently培养和培养的细胞，即使在温度降低后显示出从表面很少或没有脱离。 "弱粘附"是指细胞不培养，在37℃至铺满3天。

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Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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 ...

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8.2K 次观看.  Tokyo University of Science. 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.

视频: Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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 ...

科研

生物工程

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

免疫与感染

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

温敏纳米结构表面的组织工程准备

摘要

探索更多视频

此视频中的章节

毛细力光刻技术在心脏组织工程

组织工程：建设多细胞三维支架的层状细胞片交货

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

自动点胶机器人技术在细胞表面和指导生物印刷

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

温敏纳米结构表面的组织工程准备

摘要

探索更多视频

此视频中的章节

毛细力光刻技术在心脏组织工程

组织工程：建设多细胞三维支架的层状细胞片交货

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

自动点胶机器人技术在细胞表面和指导生物印刷

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生