我们要感谢以下机构支持我们的AV循环的研究:施德楼基金会，弗里茨Erler博士全宗，否则克朗Fesenius基金会，百特医疗用品有限公司，东风集团，IZKF / ELAN / EFI /性别办公室和多样性的Forschungsstiftung MEDIZIN ，埃尔兰根 - 纽伦堡（FAU），AO基金会，曼弗雷德·罗斯基金会，薛虹，汉斯格奥尔格盖斯基金会，德意志学术Austauschdienst（DAAD），德国和高等教育部和科研，伊拉克的弗里德里希 - 亚历山大大学。我们要感谢斯特凡·弗莱舍，滨海米尔德，卡特琳·科恩和伊尔莎·阿诺德Herberth的出色的技术支持。

埃尔兰根 - 纽伦堡的弗里德里希 - 亚历山大大学的动物护理委员会（FAU）和中弗兰肯的政府，德国，批准所有的实验。对于实验，雄性Lewis大鼠体重为300 - 被用来350克。 1.动静脉循环模型大鼠<ol><li> 注入步骤（图2） <ol><li>麻醉使用通过管道连接到异氟醚蒸发和盖子封闭一种特殊的塑料盒。打开供给气体和0.8之间流量计 - 1.5升/分。 </li><li>放置在感应塑料盒大鼠和密封的顶部。打开异氟烷蒸发器至5％。 </li><li>麻醉诱导时仔细观察大鼠。 3后 - 4分钟，大鼠将被麻醉。 </li><li>确认正确的麻醉:翻正反射，眼睑反射消失，停药反射，角膜正反射消失。 </li><li>从包装盒中取出老鼠并检查其权重的计算药物。应用眼膏，以防止干燥，而在麻醉下。 </li><li>管理止痛药和抗生素（ 例如 ，7.5毫克/千克恩诺沙星皮下（SC），12.5毫克/千克曲马多和100mg / kg的安乃近两个静脉内（ⅳ））。操作期间管理重适于晶体溶液（ 例如 ，30毫升/千克SC）。 </li><li>放置老鼠上加温板，在37℃麻醉下用1背面- 经由掩模施用2％异氟醚吸入。 </li><li>监测麻醉适当，增加异氟烷如果麻醉水平太低（大鼠的运动，响应于疼痛，颚音调，无反射消失（见1.1.4。），心脏的速度增加）。要小心，不要过量异氟醚（角膜反射，高心脏率，氧饱和度下降的损失）。使用小动物一个特殊的脉搏血氧仪的检查血氧饱和度（95 - 100％）和心脏率 - 大鼠（250 450 /分）。在操作，实际对大鼠的监视温度 - 如果必要的（36 40℃），并调整加热板的温度。 </li><li>剃后肢的内侧用电动剃须刀和消毒用防腐剂的区域。传播后肢并用胶带固定。 </li><li>躺在手术显微镜下，大鼠和覆盖无菌悬垂的老鼠。确保整个操作过程在无菌条件下进行的。 </li><li>与从上部膝部到使用解剖刀（第10号）的腹股沟的纵向切口打开的左大腿中部皮肤。 </li><li>切的使用解剖剪刀和microforceps直到股骨维管束是从腹股沟在膝盖股骨动脉分叉骨盆动脉露出长度约3cm层皮下组织和筋膜。 </li><li>分离容器和使用外膜剪刀microforceps去除外膜。 </li><li>凝结的b侧牧场使用电凝。盖上湿布敷在经营领域。 </li><li> 1.1.14 - 作为左侧描述的，1.1.11打开右侧皮肤。 </li><li>为收获静脉移植物，结扎通过在近端和远端电凝右股静脉以1:1的距离 - 1.5厘米</li><li>删除与microforceps静脉移植物和冲洗用的冲洗套管肝素溶液（50IU / ml，在0.9％氯化钠溶液）的静脉移植物，并将其转移到左大腿。覆盖操作字段在右侧用湿压缩。 </li><li>结扎左侧股静脉近侧在用微血管钳腹股沟区域。支，在约2厘米的距离之前通过电凝远侧凝结股静脉，在上部膝部。 </li><li>用于吻合术的静脉移植物的近端与静脉的近端通过端 - 端吻合用11-0 sutur连接即使用约8间断缝合。与将前两个缝线在12点钟和6点钟位置上开始。然后，把在2到这些点之间3个缝合线的前侧，然后把2至3个缝线入背面。 </li><li>结扎在股静脉中描述的相同的方式，股动脉（1.1.18）。确保环路船舶不扭曲。吻合用作为用于股静脉中描述的动脉的近端静脉移植物的远端（步骤1.1.19）。 </li><li>辖25个国际单位的肝素静脉注射。请再次确认环路船舶不扭曲，取下夹子，并检查是否有泄漏和循环通畅约5分钟。 </li><li>运球罂粟碱（ 例如，4毫克/毫升）上的船只，以防止血管痉挛。如果有通畅，循环膨胀并可以观察到动脉的脉搏。 </li><li>与基质的前半部分（约500微升）预填注入室（例如&lt;/ em&gt;的，水凝胶或有或无的细胞的骨基质）。嵌入循环到注入室。 </li><li>与基质的后半部分注入室填充至1000微升的总体积。密封与所述室盖的腔室。 </li><li>固定在用非可吸收的6-0缝合大腿注入室。固定用非可吸收的6-0缝合室盖。停止与电凝止血可能。 </li><li>关闭可吸收缝线4-0皮肤。盖上喷铝伤口。 </li><li>管理抗生素和止痛药（ 例如，7.5毫克/千克恩诺沙星SC，经口（PO曲马多12.5毫克/千克）（通过饮水）为3 - 5天，之后根据对大鼠的行为）。 </li><li>关闭蒸发器关闭并允许大鼠呼吸供应气体，直到它开始唤醒。 </li><li>将大鼠在热支持一个盒子，仔细地观察它，直到完全康复。 </li><li>不要让大鼠未参加直到它已经恢复了足够的意识，以保持胸骨斜卧。 </li><li>不要大鼠回公司其他动物，直到完全康复。 </li></ol></li><li> 外植过程 <ol><li>短时或长期植入后执行外植（根据研究设计，为示例，请参阅参考资料27-32）。根据步骤1.1.1.-1.1.9麻醉大鼠。 </li><li>开用手术刀（第10号）的腹部皮肤，并用压缩一旁移动肠道。暴露腹主动脉和腔静脉尾用棉签。 </li><li>导管插入腹主动脉（ 例如 ，24号塑料套管）中，用解剖剪切腔静脉尾。 </li><li>冲洗用含100国际单位/毫升肝素直到泄漏流体是清楚0.9％氯化钠溶液的腹主动脉。灌流用30毫升灌流液大动脉（ 如造影剂或印度K）。 </li><li>在深度麻醉 - （0.3毫升/公斤0.1）由embutramide，mebezonium碘化物，盐酸丁卡因注射溶液的致死剂量的注射安乐死大鼠。 </li><li>用非可吸收的4-0缝线结扎腹主动脉和腔静脉尾。 </li><li>关闭1开放性伤口面积 - 2夹子。在4℃（灌注溶液的固化）储存24小时的大鼠。 </li><li>将在它的后面的老鼠。传播后肢并用胶带固定。 </li><li>用手术刀（第10号）的纵向切口打开腔上方皮肤。 </li><li>除去从室的结缔组织，并使用解剖剪刀和microforceps环路椎弓根。切断环椎弓根在从腔室口1厘米解剖剪刀的距离并取出室中。 </li><li>打开腔室的盖子，并小心地从钳腔室移除该构建和修复它在4％缓冲的福尔马林溶液，在室温24小时温度。之后使用3D微型计算机断层成像或石蜡包埋用于组织学分析30中的构建体。 </li></ol></li></ol>

The authors have nothing to disclose.

十多年来，我们已成功地用于动静脉（AV）用于组织工程目的， 在体内循环，研究血管生成的小动物模型。我们可以证明这种显微模型是非常适合于工程不同组织，它也可以用于血管发生或抗血管生成的研究。 关于到现有/替代方法的技术意义 工程组织或器官需要的功能血管网络来提供他们所需要移植到缺损部位41后，他们的生存和成功整合?...

大多数组织和器官在人体内是依赖供给营养物质，交换气体和除去废产物的官能血管网络。该系统所造成的局部或全身血管问题的故障可能导致严重的疾病，众说纷纭。此外，在研究领域，如组织工程和再生医学，人工产生的组织或器官移植中的功能性血管网络是成功的临床应用是必不可少的。 数十年的研究人员一直在调查参与生长脉管获得，以便找到新的治疗干预，并提供更好的预防血管疾病的更深入的了解病理情况的确切机制。在第一步骤中，如细胞-细胞相互作用或分子的血管系统的细胞的作用基本过程通过体外 2D或3D通常调查实验。传统的二维模型很容易执行，已经非常成熟，并已大大更好地理解这些过程做出了贡献。对于在1980年第一次，Folkman 等 。报道在明胶涂层板1毛细血管内皮细胞体外血管生成播种。这立即让位给对内皮细胞管形成测定2，迁移试验3和不同细胞类型4的共培养进一步2D血管生成的实验，以及其他众多的出版物。这些试验至今仍在使用，并接受体外方法标准中。 然而，该实验装置并不总是适合于体内细胞行为的研究，因为大多数的细胞类型需要3D环境，以形成相关的生理组织结构5。它可以表明3D矩阵的结构是决定性的毛细管morphogenesi第6和细胞-细胞外基质（ECM）的相互作用和三维培养条件调节参与肿瘤血管生成7的重要因素。的3D矩阵提供了复杂的机械输入，可以绑定效应蛋白，建立组织规模的溶质浓度梯度。此外，它是为了在体内形态发生和重塑步骤模仿在复杂的组织5认为是必要的。在这些系统中，无论是血管发生和血管生成，可以研究。而血管发生描述了从预先存在的血管8毛细血管的发芽，血管是指通过血管内皮细胞或它们的祖细胞9,10-血管的从头形成。血管成熟在一个名为&#39;动脉&#39; 通过平滑肌细胞11的招聘过程进行描述。 体外模型一个典型的血管生成是血管内皮细胞从现有的单层萌小号种植作为凝胶表面的单层，嵌入式凝胶微球内表面，或通过建立内皮细胞球体12。在血管模型单一内皮细胞被截留在三维凝胶。它们与相邻的内皮细胞相互作用以形成血管结构和网络从头，典型地结合支撑单元12。 然而，即使在体外模型体内设置不能模仿复杂的3D完全给定的细胞-细胞的大量和细胞-ECM相互作用的13。具有较高的体外活性物质不会自动显示在体内相同的效果， 反之亦然 14。对血管的综合分析处理，迫切需要在体内模型中 ，更好地模拟在体内的情况发展。大范围的体内血管生成测定法在文献中描述，包括鸡胚绒毛尿囊膜分析（CAM），斑马鱼模型，角膜血管生成测定，背侧空气囊模型中，背皮褶室中，皮下肿瘤模型14。然而，这些测定通常与局限性，如快速形态的变化，从在CAM测定中，或在角膜血管生成测定15在有限的空间已经存在的在识别新的毛细血管的问题相关联。此外，使用非哺乳动物系统（ 例如 ，斑马鱼模型16），这导致的问题中的异种移植17。在皮下肿瘤模型中，不能被分析血管生成只从肿瘤本身始发由于相邻组织大大有助于血管形成过程。此外，周围组织可具有在塑造肿瘤微环境18的一个决定性的作用。 不仅为研究血管生成或血管有强烈的NE编了一个标准化和良好的特点体内模型也为研究组织工程和再生医学的不同血管的策略。今天，复杂的人造器官或组织的产生是可能在体外和体内 。生物印刷3D为生成复杂的3D功能生活组织19按需制造技术。此外，可以用于产生组织20甚至自己的身体的生物反应器可以用作生物反应器21。然而，主要的障碍人工生成组织的成功应用是工程化构建体中缺乏血管形成。移植后宿主的脉管系统直接连接是生存的主要先决条件，特别是在大型的人工组织或器官的情况下。 在体外或体内 prevascularization策略不同分别DEVELOPED建立在结构功能性微血管植入22之前。 在体外预成形设计的毛细管的支架上的小鼠的背部皮肤的植入导致了小鼠脉管的快速吻合每天23内。与此相反，由组装成一个三维prevascular网络人类间质干细胞和人脐静脉内皮细胞的球状体共培养在体内植入后进一步发展。然而，吻合与宿主脉管限于24。首先，在血管不佳的缺陷，如坏死或照射区，这个所谓的外在血管 - 从周围区域进入支架血管的向内生长 - 经常失败。固有血管化，另一方面，是基于血管的轴线的新的毛细血管发芽到支架25的来源。使用轴向血管的方法中，工程组织可以与它的血管轴被移植，并连接到在接收站点本地船只。立即移植后，将组织充分的氧和营养物，它创建用于优化集成在合适的条件的支持。 由于模型研究体内血管生成和表彰产生轴向血管组织的重要性日益有限，我们开发埃罗尔和斯培拉的显微外科方法进一步产生在动物模型26动静脉（AV）循环。使用一个完全封闭的注入室使得这种方法非常适合研究根据"控制"，以及其特征在于在体内条件（ 图1）的血管形成。这种模式不仅对血管生成的调查有用的，但也最适合于支架用于组织ENGIN轴向血管eering目的。

<table><tbody><tr><td>0.9% sodium chloride</td><td>Berlin-Chemie AG</td><td>34592508</td><td></td></tr><tr><td>11-0 Ethilon / polyamide 6/6</td><td>Ethicon</td><td>EH7438G</td><td></td></tr><tr><td>4-0 Vicryl / polygalactin 910</td><td>Ethicon</td><td>V392H</td><td></td></tr><tr><td>6-0 Prolene / polypropylene</td><td>Ethicon</td><td>8695H</td><td></td></tr><tr><td>aluminium spray</td><td>Pharma Partner Vertriebs-GmbH</td><td>1020</td><td></td></tr><tr><td>antiseptics&amp;nbsp;</td><td>BODE Chemie GmbH&amp;nbsp;</td><td></td><td></td></tr><tr><td>Catheter&amp;nbsp;</td><td>B Braun Meslungen AG</td><td>4251612-02</td><td></td></tr><tr><td>contrast agent</td><td>Flowtech</td><td>&amp;nbsp;MV-122</td><td></td></tr><tr><td>embutramide, mebezonium iodide, tetracaine hydrochloride injectable solution&amp;nbsp;</td><td>Intervet International GmbH</td><td></td><td></td></tr><tr><td>encre de chine intense Indian ink</td><td>Lefranc &amp;amp; Bourgeois</td><td></td><td></td></tr><tr><td>Enrofloxacin</td><td>Bayer AG</td><td></td><td></td></tr><tr><td>eye ointment</td><td>Bayer AG</td><td></td><td></td></tr><tr><td>Formalin 4%</td><td>Carl Roth GmbH &amp;amp; Co. KG</td><td>P087.4</td><td></td></tr><tr><td>Heparin</td><td>Ratiopharm GmbH</td><td></td><td></td></tr><tr><td>isoflurane&amp;nbsp;</td><td>Abbott Laboratories</td><td>6055482</td><td></td></tr><tr><td>Lewis rat, male</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Metamizol-Natrium&amp;nbsp;</td><td>Ratiopharm GmbH</td><td></td><td></td></tr><tr><td>papaverine / Paveron N</td><td>Linden Arzneimittel-Vertrieb-GmbH</td><td></td><td></td></tr><tr><td>tramadol / Tramal</td><td>Gr&amp;uuml;nenthal GmbH</td><td></td><td></td></tr></tbody></table>

the arteriovenous (av) loop in a small animal model to study angiogenesis and vascularized tissue engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

组织工程 骨组织工程的目的，许多不同的骨替代，在小动物大鼠AV环模型27,28,33,34植入。血管可以完全由3D显微计算机断层摄影术（微CT）（ 图3A）来证明。经处理的牛松质骨（PBCB）矩阵的血管形成是环基中显著高于与没有血管的组。不断增长，maturating血管网8周以上的注入室之内的发展。 4和8周之间，观察到朝向构建体的中心的血管...

Watch this Scientific Journal Video about The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering at JoVE.com

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

我们描述了动静脉（AV）循环作为在一个孤立的和充分表征的环境在体内分析血管模型的生成一个显微方法。这种模式不仅对血管生成的调查是有用的，但也最适合用于工程轴向血管化和移植的组织。

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

the-arteriovenous-av-loop-small-animal-model-to-study-angiogenesis

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

12.5K 次观看.  University Hospital of Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg (FAU). 我们描述了动静脉（AV）循环作为在一个孤立的和充分表征的环境在体内分析血管模型的生成一个显微方法。这种模式不仅对血管生成的调查是有用的，但也最适合用于工程轴向血管化和移植的组织。 

视频: The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

科研

发育生物学

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

生物工程

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

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

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

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

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

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

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

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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

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

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

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

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

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

医学

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

摘要

探索更多视频

此视频中的章节

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

一个完整的皮肤缺损模型，以评估生物材料的血管化

手术技术组织工程血管移植和后续的植入

在组织工程由内在血管化

缩微和3D微血管大会

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

微流控装置中组织模型的 Perfusable 血管网

猪胫骨移植术后血管生成的动物模型

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

体外使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

摘要

探索更多视频

此视频中的章节

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

一个完整的皮肤缺损模型，以评估生物材料的血管化

手术技术组织工程血管移植和后续的植入

在组织工程由内在血管化

缩微和3D微血管大会

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

微流控装置中组织模型的 Perfusable 血管网

猪胫骨移植术后血管生成的动物模型

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

体外使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定