这一工作是由美国陆军格兰特（W81XWH-08-1-0701）和卡尔基金会医院的院士。

程序符合IACUC协议号13302完全兼容，并已通过动物资源的划分。 1.仪器准备<ol><li>高压灭菌之前用来在外科手术，以保证无菌的所有非一次性的仪器。 </li><li>消毒与用途之间玻璃珠灭菌多次使用在会话仪器。 </li></ol> 2.水凝胶制剂<ol><li>解冻板干细胞至少24小时之前创建的水凝胶结构的。 </li><li>制备贴剂前一天已在全部细节在以前的出版物8,18所述的外科手术用立体光刻设备（或类似的仪器）。 </li><li>为了创建该水凝胶，首先生成一个计算机辅助设计（CAD）模型将所需的修补和出口设计stereolit​​hographic格式（STL）。 </li><li>创建由dissolvin预凝胶溶液克聚（乙二醇）二甲基丙烯酸酯（PEGDMA）在无菌的1×PBS，得到20％w / v的溶液。同时，溶解的光引发剂1- [4-（2-羟基 - 乙氧基） - 苯基] -2-羟基-2-甲基-1-丙烷-1-酮在DMSO中。添加光引发剂到PEGDMA溶液在最终瓦特/ V的0.5％。直接交联之前，加入100 ml细胞所需的密度。注：细胞密度在2.0×10 5 -2.0×10 6个细胞/ ml，推荐。 </li><li>吸取在该SLA平台中心的预凝胶溶液成菜并运行该SLA软件与以前上传的STL格式设计。 </li><li>除去从装置的补丁和孵化的水凝胶的O / N的Dulbecco改良的Eagle培养基（DMEM），补充有5％胎牛血清（FBS），100U / ml青霉素，和100毫克/毫升链霉素，以允许细胞以调节到贴片内的环境。 </li></ol> 3.动物制备及口服intubati上<ol><li>评估之前，过程中的每个鼠标，以保证动物健康状况良好，因为从他们的美容习惯和相互作用决定与他们的笼子队友。注意：女8-10周龄的C57BL / 6J小鼠用于这个过程，但是，任何小鼠年龄或类型都可以使用。 </li><li>将动物进入麻醉室，并暴露于5％异氟烷以1升/分钟O 2的支持。监测麻醉通过脚趾捏反射的水平。 </li><li>一旦动物被正确麻醉，称重，并把他们放在插管立场。 </li><li>引向动物的胸腔的光源，并使用镊子缩回喉面和露出声带。小心地将声带之间的20G的静脉留置针，并顺利插入气管。通过胸腔的机械运动检测正确插入一旦导管被连接到小动物呼吸机。 </li><li>调整通风设置为th动物根据制造商的指导方针ë重量。 </li><li>一旦插管，放置在加热垫上仰卧位的动物，以防止体温过低。确保该加热垫不超过40℃。如果必要的话，将动物和加热垫之间的屏障，以尽量减少烧伤的可能性。 </li><li>通过使用一个脱毛霜除去毛发从手术部位。由优碘和75％乙醇的3交替磨砂消毒的部位，以确保适当的消毒。放置一个悬垂性，以暴露刚好手术部位，并放置兽医软膏对眼睛，以防止在操作过程中干。 </li></ol> 4.手术过程<ol><li>开胸是在动物的左侧进行的，因此，使一个皮肤切口约1cm到胸骨的左侧，在运行胸骨的长度。 </li><li>使用镊子的肌肉层与底层胸腔分开在两个步骤。观察Deline的阿婷线表示该动物的胸大肌肌肉。解除这种肌肉微微，它从底层腹外斜肌分离，向内缩回。然后，释放从在相同的方式的底层胸腔腹外斜肌肌肉，和缩回横向，提供第二，第三和第四肋的视野开阔图。 </li><li>执行第三和第四肋之间的开胸。轻轻抬起第四肋并使用cauterizer打开第三和第四肋骨之间的胸腔内。拉钩将进一步打开腔暴露心脏。 </li><li>使用镊子破裂心脏的心包薄。 </li><li>结扎左冠状动脉用8-0尼龙单丝缝合。放置缝线从心脏的顶点约4mm，左心房的底部前端的正下方。由心肌的热烫和苏图之后增加左心房的大小决定适当缝合放置再被追平。 </li></ol> 5.补丁安置和胶<ol><li>保持贴剂在37℃和5％CO 2的条件下直至使用。 </li><li>轻轻抬起用平铲端补丁，轻轻把它放在心脏的表面上，在梗塞的部位。 注意：为防止滑落左心室，这个补丁可以代替轻轻接触保持使用无论是压舌板或钳子尖举行。 </li><li> 1比纤维蛋白原凝血酶：通过混合4准备胶水。通过重复的移液混合溶液，直至其开始变稠，通常在1分钟内制备开始后。 </li><li>一旦溶液粘度到达期望的水平，迅速转移约10毫升至贴片表面。注：纤维蛋白胶的凝固时间迅速，对胶水的有效转移提供机会的一个小窗口。 </li></ol> 6.缝合<ol><l我&gt;用6-0尼龙单丝缝合线进行关闭所有缝线。 </li><li>用三到四个独立的间断缝合关闭肋层。 </li><li>肋间层的全封闭之前，插入PE-10套管插入切口撤离胸腔闭合后完成并重新建立正确的胸腔内的压力。 </li><li>关闭胸肌用三到四个独立的间断缝合。 </li><li>密封皮肤层用连续缝合。 </li><li>以下全封闭，附加1.0毫升注射器到所述套管的端部，并用它来撤离胸腔。 </li><li>应用组织粘合剂的切口部位加强缝合部位。 </li></ol> 7.手术后治疗<ol><li>给小鼠注射丁丙诺啡（0.05-1.0毫克/千克）和卡洛芬（2.2毫克/千克）经皮下之前立即恢复冠状动脉结扎和至少20分钟以下的。管理这两种药物在手术后6-8小时，然后给每日两次，三天来控制疼痛和痛苦。 </li><li>连续监测动物直至意识和每隔一小时的前4个小时以下手术。进行第二次检查手术（2-4小时后）的夜间管理止痛的第二剂量。 </li><li>第二天继手术后每天监测动物，直到动物出现稳定。 </li></ol>心功能及组织学分析8 <ol><li>对小鼠后4周的过程来确定继心肌梗塞损伤的程度进行echocardiographs。 </li><li>麻醉小鼠4-5％的异氟醚麻醉室吸入诱导麻醉范围内，然后2％经面罩异氟醚维持麻醉。 </li><li>清除成像区域用脱毛霜，并用一个小动物超声获得跳动的心脏的二维M型超声心动图扫描和记录功能参数ASSOciated它。 </li><li>安乐死的小鼠在二氧化碳室中，然后收集来自动物和地方的心在24小时的10％的福尔马林溶液中。 24小时后，将心转移到70％的乙醇，并保持在4℃直至组织处理。 </li><li>过程中的心，将它们嵌入石蜡块。 </li><li>切5毫米部分通过心脏的心室区域，开始在顶点，并在庭结束。 </li><li>染色切片与两个Masson三色和苏木精和使用标准协议伊红染色切片。 </li></ol>

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The authors declare that they have no competing financial interests.

用这种方法与鼠心肌梗死模型中，我们已经开发出一种系统，其最小化到与其它鼠心肌梗死技术相关的非心肌区域的损害。这些领域包括引起气管损伤，肌肉层的切割，并且肋的破损，以暴露胸腔。我们认为，这些变化改善整体手术结果由于采取保持尽可能多的主要结构的，包括肋和肌肉层，完整的外科手术过程中的照料。 应当指出...

心肌梗死（MI）被定义为血液的中断造成的主要冠状动脉的闭塞心脏的区域。从一个MI造成的损害是由于活心脏组织的重塑成非功能性疤痕组织，从而降低心脏的，或者更具体地说，左心室，击败适当的能力。这导致在血液的体积可以递送到身体与每次心跳，被称为每搏输出量，以及血液泵出心脏的与每次心跳的百分比，被称为射血分数6下降。这些以及其它缩水功能，增加了对心脏的其余部分的应变，以保持足够的功能。通常情况下，它会导致第二心脏发作，这种现象出现在个体7的约10％这增加了应变能变得如此严重。 虽然医疗实践已经发展到治疗心肌梗死的直接后果，没有任何的技术已经发展到停止，慢，或逆转组织重塑的负面影响。干然而细胞疗法已成为一个可能的途径为这样的处理，尽管它们有希望潜力，干细胞还没有被证明是成功的临床设置。一个理论为他们的缺点是无法保证的有益细胞保持在梗死足够长，以产生有利的结果5的部位。它已经显示，细胞的不超过24％被简单地注入到梗死部位存活和保持在损伤部位后1天交货2。一种可能的前景为解决这一问题的细胞保留的是开发出封装任一细胞或治疗剂，其可以被输送到受损部位的生物相容水凝胶系统。所选择的这个协议的水凝胶是聚（乙二醇）二甲基丙烯酸酯，由于其先前在电池封装公关使用ocedures然而，能够包封的任何水凝胶可以用于8。贴片直接向损伤部位的递送保证细胞与组织接触过在延长的时间周期，增加的时间长度，细胞可以提供有利的因素对底层心肌。 瓶颈的修补方法是秉承补丁到心脏表面的难度。许多团体通过各种技术，最普遍的是一个简单的缝合打结构建到心脏表面9,10已经克服了这一点。这已被证明是成功的数目，其中所述构建体是由硬的材料制成的情况下，但试图在水凝胶系统时，由于高的水浓度和接插构建体的细腻性失败。为了克服这个问题，我们已利用模仿血块形成的化学纤维蛋白胶外部粘合剂系统。纤维蛋白胶已用于许多医疗手术，INC泸硬脑膜眼泪，支气管瘘，和角膜移植，突出产品的生物相容性用作伤口的密封剂11-13。此外，血纤维蛋白已经被用于各种心脏目的，包括手术治疗左心室破裂和冠状动脉旁路手术，然而，其作为粘附胶用于心脏修补不常用14-17。的凝血酶和纤维蛋白原的结果在生物相容性胶的简单制剂，可以直接放置在一个外部心脏贴片的外侧，提供了一个可行的粘附系统，以确保补丁到心脏的相互作用。

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

在外科手术过程中，左冠状动脉的结扎可以由标热烫闭塞动脉的下游来鉴定。作为结婚前一个测试，缝合线可以简单地拧紧，以检查它是否在适当的位置。此外，由于在动脉导致在左心房的适当敲打的能力几乎瞬间降低闭塞，左心房将放大响应于血液系统中的回流。 M型超声心动图测量，作为早在2天后梗死表现出停止左室壁运动的，表明肌肉重建。从数据做出定性的计算显?...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

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

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 ...

a-hydrogel-construct-fibrin-based-glue-approach-to-deliver

Research

JoVE Journal

Medicine

8.6K 次观看.  University of Illinois at Urbana-Champaign. 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.