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
Summary
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。
Abstract
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.
Introduction
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'动脉' 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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.9% sodium chloride | Berlin-Chemie AG | 34592508 | |
| 11-0 Ethilon / polyamide 6/6 | Ethicon | EH7438G | |
| 4-0 Vicryl / polygalactin 910 | Ethicon | V392H | |
| 6-0 Prolene / polypropylene | Ethicon | 8695H | |
| aluminium spray | Pharma Partner Vertriebs-GmbH | 1020 | |
| antiseptics | BODE Chemie GmbH | ||
| Catheter | B Braun Meslungen AG | 4251612-02 | |
| contrast agent | Flowtech | MV-122 | |
| embutramide, mebezonium iodide, tetracaine hydrochloride injectable solution | Intervet International GmbH | ||
| encre de chine intense indian ink | Lefranc & Bourgeois | ||
| Enrofloxacin | Bayer AG | ||
| eye ointment | Bayer AG | ||
| Formalin 4 % | Carl Roth GmbH & Co. KG | P087.4 | |
| Heparin | Ratiopharm GmbH | ||
| isoflurane | Abbott Laboratories | 6055482 | |
| Lewis rat, male | Charles River Laboratories | ||
| Metamizol-Natrium | Ratiopharm GmbH | ||
| papaverine / Paveron N | Linden Arzneimittel-Vertrieb-GmbH | ||
| tramadol / Tramal | Grünenthal GmbH |
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