Name: a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.
Uploaded: 2015-06-14T14:00:00.000+00:00
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Description: 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

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。

<table><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 - 0.4 mm Tips Angled</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>Agricola Retractor - 3.5 cm Spread</td><td>Fine Science Tools</td><td>17005-04</td><td></td></tr><tr><td>Spring Scissors - 2.5 mm Blades Straight</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14 cm</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

8.7K 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.

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

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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering 

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented.&#160;Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area.&#160;LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10%&#160;post-operative mortality.&#160;

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

基于细胞治疗心脏衰竭大鼠：双开胸心肌梗死和细胞与生物基质植入心外膜

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

发育生物学

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

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

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 ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

法援署结扎：有心肌梗死的小鼠模型

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

生物学

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

医学

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 ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.