Name: an injectable and drug-loaded supramolecular hydrogel for local catheter injection into the pig heart
Uploaded: 2015-06-07T14:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart at JoVE.com

这一工作是由教育，文化和科学（重力计划024.001.035）部，荷兰科学研究组织（NWO），欧洲研究委员会（FP7 / 2007-2013）ERC资助协议和308045的范围内进行LSH TKI框架。这项研究的形式生物医学材料研究所的研究计划的项目P1.03 PENT的一部分，共同资助下，经济事务部荷兰。荷兰心脏学会（ -该项目由ICIN支持<a href="www.icin.nl">www.icin.nl</a> ）和"Wijnand M.双响炮斯蒂廷"。作者想感谢亨克扬森和里斯彼得斯的UPy，水凝胶和REMCO艺术合成提供了mRuby2。我们感谢伯特·梅杰，东铭博斯曼，罗克珊Kieltyka，斯泰恩克莱默，Joost的Sluijter，伊莫Hoefer说，和Frebus面包车斯洛赫特伦了许多有益的讨论和Marlijn扬森，乔伊斯维瑟，格雷斯克罗夫特和面包车的Martijn为Nieuwburg TE技术注释援助。

注:所有的体内实验按照指南的护理和使用实验动物用实验动物资源研究所进行。实验批准了乌得勒支大学的良药系的动物实验委员会，荷兰。 1.制剂的水凝胶的<ol><li>为了制备将1ml 10％（重量）的凝胶，通过在70℃下搅拌1小时用磁力搅拌器溶解在900微升的PBS pH值11.7的小瓶的UPy-水凝胶100毫克。之后冷却该粘稠溶液到室温。该溶液现在应该有一个pH约为9.0。此溶液可以储存数天。 </li><li>吸取药物或生物分子的被溶解在中性的PBS成粘稠溶液，并搅拌10分钟以达到均匀分布的适当的量。如果解决方案变得太粘，不久用热水温暖它。 </li><li>将1小时的解决方案下的紫外线灯消毒。 </li></ol>水凝胶2.分析<ol><li>该溶液的流变评估<ol><li>前加载的凝胶，装入25mm的板 - 板几何形状，在流变仪中，设置温度至20℃，并用水加载板，以防止在测量过程中的凝胶的蒸发。 </li><li>吸管加入300微升上保持在20℃的流变仪的溶液到25毫米板 - 板几何形状，并降低板，以获得0.5mm的间隙距离。 </li><li>记录剪切粘度作为剪切应力的函数在0.1至500Pa的每十10分。 </li></ol></li><li>凝胶的流变评估<ol><li>移液器将300μl溶液到板和吸管共4.2微升的1M HCl中的溶液不同的地方以诱导凝胶形成。 </li><li>降低板到0.5mm的间隙距离，并让凝胶固化约30分钟。在这种固化工序中，测量存储和损耗模量在低频和应变，例如分别为1弧度/秒和0.5％。 </li><li>后的凝胶固化（后大约30分钟），记录存储和损失模​​量作为频率（0.1-100弧度/秒）的函数，并随后为应变（0.1-1,000％）的功能。 </li></ol></li></ol> 3.侵蚀和释放实验<ol><li>转移100μl的含有药物或生物分子粘稠溶液到聚（对苯二甲酸乙二醇酯）的悬细胞培养插管24井板用孔径8.0微米。为了防止聚合物溶液的泄漏，而在液相中，覆盖所述插入物用Parafilm（ 图2A）的底部。 </li><li>随即吸管的1M的HCl在粘性溶液的顶部1.4微升降低pH至约7.0-7.2，并让该插入物内的凝胶固化约30分钟。 </li><li>来回取出封口膜米的插入，放置在一个24孔板的插入件和填充井用800微升pH 7.4的PBS。孵育板在37℃下缓慢摇动或振荡运动。为了防止蒸发溶剂，填充剩余空孔用PBS和密封24孔板用Parafilm（ 图2B）。 </li><li>定期刷新PBS和分析取出PBS用于发布UPy糜烂产品或药物/生物分子。 <ol><li>通过分别测量UV吸光度在265纳米或320纳米的量化UPy侵蚀产品或吡非尼酮。在以激发波长559 587后，纳米荧光蛋白mRuby2测量荧光发射。 </li><li>翻译测得的吸收/发射值浓度，通过预定的校准曲线。 <ol><li>制备校准曲线中溶解了一系列已知浓度在缓冲液中的分析物的和测量的UV吸光度或这些样品的荧光发射。使用线性函数来DET插值数据貂未知样品的浓度。对于非荧光蛋白用ELISA法检测6。 </li></ol></li></ol></li></ol>经由导管4局部注射<ol><li>感应心肌梗塞<ol><li>经过12小时禁食，水除外，稳重以其稳定的猪注射咪达唑仑0.4毫克/千克，氯胺酮10 mg / kg和阿托品0.014毫克/公斤肌肉注射。 </li><li>管理硫喷妥钠5mg / kg的静脉内以诱导麻醉和插管的猪用气管内管。以12 /分钟的速度，如果在运送动物到手术室​​需要执行气球通风。 </li><li>在抵达手术室立即启动机械正压通气氧合指数2 0.50 10毫升/公斤潮气量，并根据连续二氧化碳浓度监测仪12 / min的频率。使用对眼睛兽医药膏，以防止干燥。 </li><li>首先，连续intraven平衡麻醉的OU输注咪达唑仑0.5毫克/千克/小时的，舒芬太尼2.5微克/ kg /小时和泮库溴铵0.1毫克/千克/小时。为了确保适当的麻醉持续监测心电图，动脉血压，体温和二氧化碳图。 </li><li>静脉内注入4.3毫克/千克胺碘酮和放置心腔内除颤导管中使用静脉sheeth 18的右心室。 </li><li>阻塞左前降支（LAD）远端的第二对角支冠状动脉内球囊闭塞，90分钟，根据先前描述协议18。 </li></ol></li><li>机电测绘<ol><li>在心肌梗死后四周，规划映射过程。制备系统（ 图4）在cathlab为左心室的3D机电映射器（EMM）。有了这个系统可行，冬眠和心肌梗死可以识别而不透视指导。要构建这样一个EM-MAP收购SE在对左心室心内膜表面的多个位置点的通过使用一个超低磁场能量源和一个传感器尖导管19,20里斯。 </li><li>麻醉猪，以下方案步骤4.1.1-4.1.4。 </li><li>放在猪背上外部参考补丁。 </li><li>根据协议，18安全血管通路（股动脉）。 </li><li>获得双翼飞机左心室造影在25°右前斜位（RAO）和40°左前斜（LA）查看后，估计左室大小，给予75 U /公斤肝素。 </li><li>前进8法语映射（D或F曲线）导管透视引导下向降主动脉，主动脉弓和整个主动脉瓣进入左心室（LV）。 </li><li>导管的尖端定向到LV的顶点以获得第一数据，接着流出道，侧和后点，以形成一个三维的轮廓，限定ventri的边界CLE。 </li><li>获得后续点，直到所有的心内膜的段已被采样通过拖动映射导管通过心内膜，并依次获取所述尖端的位置而与心内膜21,22接触。 </li><li>定义目标区域，即，其中电活性（近）正常和机械运动障碍，所谓冬眠心肌（ 图6）。 </li></ol></li><li>心肌内注射<ol><li>由心肌内注射导管，其由27号针和一个8弗伦奇导管内的芯腔（ 图5A和B）的替换映射导管。为了提供特定量，装入一个容积渐变注射器与约2毫升水凝胶溶液，并将其放置到注射器泵。 </li><li>调整针延伸在0°和90°弯曲并放置0.1水凝胶溶液的毫升以填充针死空间。然后，将在主动脉瓣和进入目标区域喷射导管末端。 </li><li>满足在4.2.9中确定的目标区域内的注入位置以下条件:所述导管到LV壁（1）的垂直位置; （2）优良的循环稳定性（&lt;4毫米），为计算由EMM系统; （3）标的电压&gt; 6.9 mV的21。 <ol><li>推进针进入心肌，（4）将LV的室性早搏证实，并注入0.1-0.3毫升丸药水凝胶在约0.4-0.5毫升/分钟使用注射泵以恒定的速率。 6-10在不同位置漫地重复这一点。的组织的天然pH值中和该溶液注射后，在其上的水凝胶形成。 </li></ol></li></ol></li><li>牺牲<ol><li>程序后，入道放血牺牲动物。切下腔静脉和除去血液与抽吸设备。诱发心室连接brillation放置一个9伏电池上的顶点。 </li></ol></li></ol>

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The authors have nothing to disclose.

一个关键的挑战是获得一种溶液，其为可注射的，通过一个长导管，同时保持溶液与生物活性化合物相容。虽然pH值应该增加，以增加注入性，生物活性化合物，如生长因子，应小心处理易碎分子。我们监测该溶液的pH值密切使用pH计加入的水凝胶，以确认它是pH值为9.0添加任何生物活性成分之前后。最初，要结束与右pH值必须经过几轮调整PBS的起始pH值。此外，因为我们使用相对粘性溶液和一个细长的导管，一个大的压降的情况下（在0.5MPa的顺序，这取决于注入的速度）。因此，特别应注意在选择注射器和导管之间的正确连接。注射泵支架控喷射，通过手工应用这种力量是具有挑战性的。 对于VITRÓ实验，将该溶液中和，用HCl将溶液凝胶化，而​​在体内这是通过组织的自然pH值进行。因此，要添加的HCl适量，以防止pH值的过冲是重要的。这种酸的扩散可能是在水凝胶的体外实验的凝胶化的限制因素;然而， 在体内的液体将与中和组织中的高接触表面积，这很可能会导致更快和更均匀的凝胶相比滴加浓酸。此外，该凝胶开关与此温和过程快得多相比以前使用的方法（0.5小时对2小时）25。使用身体的自然pH值的材料性质的切换是非常有吸引力的，因为转换是迅速的，可逆的，不能发生在导管内并且是在体内全自动的。这些特性赋予优势如热SWITChâble的胶凝26，其中凝胶化在一个导管的由于温度变化的风险是存在，需要光诱导聚合，这是具有挑战性，因为有限的光穿透和自由基的形成27，或需要共同注射的聚合引发剂的凝胶的凝胶或accellerator 28。 成功释放从水凝胶的药物主要是依赖于药物的大小。如图所示，小分子药物立即释放，而超过1周模型蛋白的逐渐释放显示了这些水凝胶作为递送系统的生长因子的承诺。在一般情况下，水凝胶是作为输送工具，用于较大物体如蛋白质，外来体和细胞29,30更有前途。 三维机电映射和注射过程提供了关于各种心肌再生疗法，例如水凝胶临床验证的基于导管的递送方法。该ADDE这种技术相对于其它非手术递送技术的d值是治疗计划，使得能够区分正常，梗塞和冬眠心肌，并指导治疗中感兴趣的区域。这种方法关注所需的技术技能和耗时且昂贵的过程20的缺点。在心肌梗塞机电映射的呈现猪模型随后引导的心肌内注射与生物活性超分子UPy-水凝胶。其他组合具有再生疗法在体外试验和体内获得这一新兴领域取得更大的成功。此外，可注射和灭菌程序优化已被执行以成功地把这种方法以临床环境。

虽然急性心肌梗死的治疗有显著提高生存率，慢性缺血性心脏衰竭是人口老龄化的推进一个重大的公共卫生问题。大约有600万心脏衰竭患者在美国，估计增加25％患病率在2030年1,2。心肌组织的初步损失导致心肌重构，最终导致慢性心脏衰竭。除了心脏移植，对于本组患者没有真正的治疗。捐助者的心越来越缺点强调有必要制定新的可用疗法逆转重塑的过程。因此，对于未来治疗的一个目标是失去心肌的再生。 水凝胶是令人感兴趣的材料，因为它们的生物相容性的再生医学的领域中，以及它们与外部触发器3的灵敏度。注射水凝胶提供广告vantages超过其在微创外科手术4采用非注射水凝胶。这些可注射的水凝胶可以通过注射，因为在生理条件5内其switchability的应用，并且在原则上允许基于导管的注射方法6。不同的策略已被用于可注射的材料，从化学交联注射到物理交联由任一温度，pH和剪切变稀行为4,7,8后。虽然几个系统已通过注射器9,10显示易于注射，全导管兼容性尚未经常6中。 从超分子聚合物制备的水凝胶通过使用环境触发器11的非共价相互作用，可以方便地切换从凝胶到溶液状态，反之亦然形成。此外，低分子量的前体允许容易加工12,13 </sup&gt;。需要用于切换允许添加的各种生物活性组分，例如常常难于处理的生长因子的温和的条件。 基于聚（乙二醇）（PEG）在水中的超分子瞬时网络，端改性的脲基嘧啶酮（UPy）部分14已经与生物医学应用的组合所示的非共价相互作用的好处，并已被用作药物递送系统在心脏6和下肾包膜15。这些网络由UPy基团被烷基隔板形成疏水口袋从含水环境屏蔽的二聚化形成的。尿素氢键有利于后续的堆叠这些二聚体成纳米纤维。由于UPy-UPy二聚体的可逆相互作用，触发如pH和温度可以用来从溶液中切换到凝胶。使用合成的基序允许分子和凝胶特性由用于考试设计PLE调节长度的PEG链和烷基间隔14,16。 此外，一些生物活性组分可通过简单地混合注射前的超分子水凝胶溶液，用药物或生物活性物质，如分别生长因子或外来体，被并入。外来体是包含胞质衍生物小膜囊泡。它们是由许多细胞分泌，并参与细胞间通讯。从心肌祖细胞衍生的外来体被建议在心脏保护17发挥作用。 这里，我们描述制剂的协议，并在体内心肌注射这样的生物活性的超分子水凝胶的， 在体外实验中，描述了提供关于正手的指示的凝胶稳定性和药物释放，其允许凝胶的调谐和之前释放性能体内应用。

<table><tbody><tr><td>1 M HCl</td><td></td><td></td><td></td></tr><tr><td>1 M NaOH</td><td></td><td></td><td></td></tr><tr><td>Polystyrene 24-well plate</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>Amiodarone</td><td>Cordaron I.V. (Sanofini)</td><td></td><td></td></tr><tr><td>Anton Paar Physica MCR501</td><td>Anton Paar GmbH</td><td></td><td>Equipped with a parallel-plate geometry (25 mm)</td></tr><tr><td>Atropine</td><td>PCH</td><td></td><td></td></tr><tr><td>Balloon ventilator</td><td></td><td></td><td></td></tr><tr><td>Cary 50 Scan UV-Visible Spectrophotometer</td><td>Varian</td><td></td><td></td></tr><tr><td>Cary Eclipse Fluorescence Spectrophotometer</td><td>Varian</td><td></td><td></td></tr><tr><td>Defibrillation patches</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td>Biosolve</td><td>44705</td><td></td></tr><tr><td>Endotracheal tube</td><td>Covidien</td><td></td><td></td></tr><tr><td>Heparin</td><td></td><td></td><td></td></tr><tr><td>Ketamine</td><td>Narketan 10 V&amp;eacute;toquinol</td><td></td><td></td></tr><tr><td>Mapping catheter 115&amp;nbsp;cm</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>Midazolam</td><td>Actavis</td><td></td><td></td></tr><tr><td>MilliQ</td><td>MD Milipore MilliQ Integral Water Purification System</td><td></td><td></td></tr><tr><td>mRuby2</td><td></td><td></td><td></td></tr><tr><td>NaCl 0.9% 500&amp;nbsp;cc</td><td>Braun</td><td></td><td></td></tr><tr><td>NOGA guided Myostar injection catheter</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>NOGA-RefStar EFO-patch</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>Pancuronium bromide</td><td></td><td></td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>IKAA3801100</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P4417&amp;nbsp;</td><td></td></tr><tr><td>PET millicel</td><td>Millipore</td><td>PIEP12R48</td><td></td></tr><tr><td>Pirfenidone</td><td>Sigma Aldrich</td><td>P2116</td><td>Used from 100&amp;nbsp;mM stock in DMSO</td></tr><tr><td>Sodiumthiopental</td><td>Inresa</td><td></td><td></td></tr><tr><td>Sufentanil</td><td>Sufentanil-Hameln</td><td></td><td></td></tr><tr><td>Tegaderm</td><td></td><td></td><td></td></tr><tr><td>UPy-PEG10k</td><td></td><td></td><td></td></tr><tr><td>UV-Lamp</td><td></td><td></td><td></td></tr><tr><td>Vet ointment</td><td></td><td></td><td></td></tr><tr><td>Visipaque contrastfluid 100&amp;nbsp;cc</td><td></td><td></td><td></td></tr></tbody></table>

an injectable and drug-loaded supramolecular hydrogel for local catheter injection into the pig heart

丢失心肌的再生是由于慢性缺血性心脏衰竭的增加发生和有限访问供心的未来治疗的重要目标。治疗以恢复心脏的功能的例子包括由水凝胶局部递送药物和生物活性物质的。本文提出了一种方法引入到制定和注入载药凝胶非侵入性和侧面特有的进猪心脏使用长，灵活的导管。经由导管使用的3-D机电映射和注入允许心肌侧特异性治疗。以提供与该导管兼容的水凝胶，超分子水凝胶的使用，因为从凝胶到使用环境触发器溶液状态的方便的切换。在碱性pH本脲嘧啶酮改性的聚（乙二醇）作为牛顿流体可以容易地注入，但在生理pH的溶液迅速切换到凝胶。这些温和开关条件允许的生物活性的药物和生物活性物质，如生长因子和外来体中掺入作为我们在座中在体外和体内实验。 在体外实验上的凝胶稳定性和药物释放的正手击球指示，其允许凝胶的调谐和体内的后续申请之前释放性能得到。这种组合允许对凝胶所使用的生物活性化合物的最佳调谐和种，并且所述喷射系统。

从双方的溶液和凝胶中的振荡流变 ​​学测量获得的典型结果示于图1中 。对于注射通过一个长导管，具有低粘度的牛顿 ​​流体是理想的。测定粘度随剪切速率的功能，显示出在pH 8.5的溶液是剪切稀化，但在pH 9.0和9.5如由0.54和0.36帕的恒定粘度·秒的解决方案表现为牛顿流体，分别（ 图1A） 。中和的样品后，将样品显示由储能模量G&#39;，它比损耗模量G"，因此一个的tanδ= G"/ G&#39;大&lt;1（ 图1B）观察到类似固体的反应。凝胶获得其30分钟内最终强度。振荡流变 ​​测量显示出与G&#39;几乎不受角频率的典型实样反应uency和G&#39;&gt; G"为测量所有的频率（ 图1C）。 必不可少的用作药物递送系统是水凝胶随时间的侵蚀。超分子相互作用是固有的动态，并允许在体外凝胶的缓慢侵蚀。侵蚀和释放实验都在37℃下使用多孔以及插入（ 图2A和B）进行。通过调整亲水和疏水块14的长度，其侵蚀过一段数周，可以得到（ 图3A）的凝胶。凝胶侵蚀在2周内的25％与10％，在第一天的初始侵蚀，大概是由于水凝胶的初始膨胀。作为示例，小分子药物（吡非尼酮），以及一个模型荧光蛋白（mRuby2）的释放的同时释放了研究。荧光模型蛋白可以容易读出;然而， 在体外 </em&gt;释放实验也可以使用酶联免疫吸附定量6其他蛋白质来执行。该小分子药物被释放在一天之内，而更大的分子如蛋白质1周以上（ 图3B）被逐渐释放出来。拟合mRuby2的释放曲线高达60％的释放与半经验Korsmeyer-Peppas模型指示释放由于扩散（N = 0.44）23。由于没有一个在（适于）Korsmeyer-Peppas模型偏移表明没有突释本为mRuby2 24。由于数据点与吡非尼酮的释放低于60％的限定量的，无拟合在此释放曲线进行。 导管导航系统包括一个通信单元的控制台，工作站（图4），一个三角定位垫（产生低磁场）与外部基准的补丁，和两个导管，传感器尖映射和注射液的Ñ ​​导管（图5）。 后的后处理分析过滤不稳定点的LV三维心内膜重建实时更新与采集的每个新的数据点，并连续地对渐变色标（ 图6A）显示为单极性和双极性电压的电位。局部线性缩短（LLS）功能通过获得距离采样点和邻近点之间结束收缩和舒张末期的平均变化量化室壁运动。平均电压和LLS值计算为每个段和在极性地图。（图6B）显示。的异常或低电位单极（≤6毫伏）和受损的机械活动（LLS≤4％）的存在特征梗死区22。 <img alt="图1" src="/files/ftp_upload/52450/52450fig1highres.jpg"宽度="700"/&gt; 图1 :.的溶液和凝胶的流变评估。（A）的粘度随剪切速率的溶液在不同pH值的函数。用于样品在pH 8.5的剪切稀化被观察到，但在pH 9.0和样品9.5恒定粘度得到，显示出这些解决方案的牛顿特性。 （B）的凝胶固化，接着通过绘制正切δ作为时间的函数。 （C）频率扫描为2小时后固化中和样品。误差线显示3次独立测量的标准偏差，表示一个典型的实验误差。 <img alt="图2" src="/files/ftp_upload/52450/52450fig2highres.jpg" width="600" /> 图2:设置为降解和释放实验（A）中的聚（对苯二甲酸乙二醇酯）以及插入件覆盖的石蜡膜，以防止泄漏杜尔荷兰国际集团准备。 （B）的 24孔板的插入，用Parafilm包住，以防止溶剂的蒸发。 <img alt="图3" src="/files/ftp_upload/52450/52450fig3highres.jpg" width="700" /> 图3:水凝胶的侵蚀和释放（A）侵蚀随着时间的推移。凝胶为至少2周逐渐侵蚀是观察。 （B）中推出一个小分子药物和模型的蛋白质。而小分子被释放在一天之内，该模型蛋白质逐渐释放在一个星期没有显著爆发释放。该线示出了Korsmeyer-Peppas模型在释放的初始阶段的配合。 <img alt="图4" src="/files/ftp_upload/52450/52450fig4highres.jpg" width="600" /> 图4:导管导航系统。 </STRONG&gt;通讯单元控制台NOGA XP心脏导航系统。 <img alt="图5" src="/files/ftp_upload/52450/52450fig5highres.jpg" width="700" /> 图5:（一 ）内注射导管，注射器连接。 （ 二）详细介绍注射针。 <img alt="图6" src="/files/ftp_upload/52450/52450fig6highres.jpg" width="700" /> 图6:单极性电压和LLS地图 。 （A）单极地图，老挝视图（顶部）和公牛眼（下）。红色表示在心肌基地（正常）与电活动丧失后外侧单极性低电压值。蓝色表示正常心肌，而绿色和黄色表明减少生存能力。 （二）LLS地图，老挝视图（顶部）和公牛眼（下）。红色籼稻TES在后外侧壁运动不能，绿色和黄色指示降低室壁运动。映射点显示由白点。绘制的白线显示感兴趣的区域，其特征在于降低的单极电压和障壁运动。布朗点表示注射部位。

Watch this Scientific Journal Video about An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart at JoVE.com

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

基于脲基嘧啶酮的水凝胶超分子允许完全控制宏观凝胶特性和使用pH值溶胶 - 凝胶开关行为。在这里，我们提出了一个协议，通过直接局部递送相关领域中的猪心脏的导管输送系统制定和注射这种超分子水凝胶。

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

an-injectable-drug-loaded-supramolecular-hydrogel-for-local-catheter

Research

JoVE Journal

Bioengineering

17.3K 次观看.  Eindhoven University of Technology. 基于脲基嘧啶酮的水凝胶超分子允许完全控制宏观凝胶特性和使用pH值溶胶 - 凝胶开关行为。在这里，我们提出了一个协议，通过直接局部递送相关领域中的猪心脏的导管输送系统制定和注射这种超分子水凝胶。 

视频: An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

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

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 ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

医学

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

水凝胶构造和纤维蛋白基胶方法来提供治疗的小鼠心肌梗死模型。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D全心脏心肌组织分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

用于细胞和药物输送应用的可注射超分子-纳米粒子水凝胶

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

在缺血性心脏病的猪模型中部署3D打印冠状动脉狭窄植入物的新颖的皮下方法

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.