Name: the arteriovenous (av) loop in a small animal model to study angiogenesis and vascularized tissue engineering
Uploaded: 2016-11-02T14:00:00
Description: 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

We would like to thank the following institutions for supporting our AV loop research: Staedtler Stiftung, Dr. Fritz Erler Fonds, Else Kr&#246;ner Fesenius Stiftung, Baxter Healthcare GmbH, DFG, IZKF/ELAN/EFI/Office for Gender and Diversity, the Forschungsstiftung Medizin, Friedrich-Alexander University of Erlangen-N&#252;rnberg (FAU), AO Foundation, Manfred Roth Stiftung, Xue Hong, Hans Georg Geis Foundation, Deutscher Akademischer Austauschdienst (DAAD), Germany, and the Ministry of Higher Education and Scientific Research, Iraq. We would like to thank Stefan Fleischer, Marina Milde, Katrin K&#246;hn and Ilse Arnold-Herberth for their excellent technical support.

The Animal Care Committee of the Friedrich-Alexander University of Erlangen-N&#252;rnberg (FAU) and the Government of Middle Franconia, Germany, approved all the experiments. For the experiments, male Lewis rats with a body weight of 300 - 350 g were used.
1. The Arteriovenous Loop Model in the Rat
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	<li>Implantation Procedure (Figure 2)
	<ol>
		<li>For anesthesia use a special plastic box that is connected via tube to the isoflurane vaporizer and closed by a lid. Turn on supply gas...

For over a decade, we have successfully used the arteriovenous (AV) loop for tissue engineering purposes and studying angiogenesis in vivo in the small animal model. We could demonstrate that this microsurgical model is very well suited for engineering different tissues and that it can also be used for angiogenesis or antiangiogenesis studies.
Significance of the Technique with Respect to Existing/Alternative Methods 
Engineered tissues or organs require a functi...

Most tissues and organs in the human body are dependent on a functional blood vessel network that supplies nutrients, exchanges gases and removes waste products. Malfunction of this system caused by local or systemic vascular problems can lead to a multitude of severe diseases. Moreover, in research areas such as tissue engineering or regenerative medicine, a functional blood vessel network within artificially generated tissues or transplanted organs is indispensable for successful clinical application.
For decades researchers have been investigating the exact mechanisms involved in the growing vasculature to gain deeper insight into pathological situations in order to find novel therapeutic interventions and provide better prevention of vascular disorders. In the first step, basic processes such as cell-cell interactions or the effect of molecules on cells of the vascular system are usually investigated by in vitro 2D or 3D experiments. Traditional 2D models are easy to perform, are well established and have contributed greatly to a better understanding of these processes. For the first time in 1980, Folkman et al. reported in vitro angiogenesis seeding of capillary endothelial cells on gelatin coated plates1. This immediately gave way to publication of a multitude of further 2D angiogenesis experiments on endothelial cell tube formation assay2, migration assay3 and the co-culturing of different cell types4, as well as others. These assays are still used today and accepted as standard in vitro methods.
However, this experimental setup is not always appropriate for the study of in vivo cell behavior since most cell types require a 3D environment to form relevant physiological tissue structures5. It could be shown that the architecture of the 3D matrix is decisive for capillary morphogenesis6 and that cell-extra cellular matrix (ECM) interactions and 3D culture conditions regulate important factors involved in tumor angiogenesis7. The 3D matrix provides complex mechanical inputs, can bind effector proteins and establish tissue-scale solute concentration gradients. Moreover, it is considered necessary in order to imitate in vivo morphogenetic and remodeling steps in complex tissues5. In these systems, both angiogenesis and vasculogenesis can be studied. While angiogenesis describes the sprouting of capillaries from preexisting blood vessels8, vasculogenesis refers to the de novo formation of blood vessels through endothelial cells or their progenitors9,10. Maturation of vessels is described in a process called &#39;arteriogenesis&#39; via recruitment of smooth muscle cells11. A typical angiogenic in vitro model is the sprouting of endothelial cells from existing monolayers seeded as a monolayer on gel surfaces, on surface of microspheres embedded within a gel or by building endothelial cell spheroids12. In vasculogenic models single endothelial cells are entrapped in a 3D gel. They interact with adjacent endothelial cells to form vascular structures and networks de novo, typically in combination with supportive cells12.
However, even complex 3D in vitro models cannot mimic in vivo settings completely given the multitude of cell-cell and cell-ECM interactions13. Substances with high in vitro activity do not automatically show the same effects in vivo and vice versa14. For a comprehensive analysis of vascularization processes there is an urgent need to develop in vivo models that better simulate the situation in the body. A large range of in vivo angiogenesis assays are described in the literature, including the chick chorioallantoic membrane assay (CAM), the zebrafish model, the corneal angiogenesis assay, the dorsal air sac model, the dorsal skinfold chamber, the subcutaneous tumor models14. However, these assays are often associated with limitations, such as rapid morphological changes, problems in distinguishing new capillaries from already existing ones in the CAM assay, or the limited space in the corneal angiogenesis assay15. Furthermore, non-mammalian systems are used (e.g., the zebrafish model16), which leads to problems in xenotransplantation17. In the subcutaneous tumor model, angiogenesis originating only from the tumor itself cannot be analyzed since the adjacent tissue greatly contributes to the vascularization process. Moreover, the surrounding tissue can have a decisive role in shaping the tumor microenvironment18.
Not only for studying angiogenesis or vasculogenesis is there a strong need for a standardized and well-characterized in vivo model but also for studying different vascularization strategies in tissue engineering and regenerative medicine. Today, the generation of complex artificial organs or tissues is possible both in vitro and in vivo. 3D bioprinting provides an on-demand fabrication technique for generating complex 3D functional living tissues19. Furthermore, bioreactors can be used for generating tissues20 or even the own body can be used as bioreactor21. However, the main barrier to successful application of artificially generated tissues is the lack of vascularization within the engineered constructs. Immediate connection to the host&#39;s vasculature after transplantation is a major prerequisite for survival, especially in the case of large-scale artificial tissues or organs.
Different in vitro or in vivo prevascularization strategies were developed to establish a functional microvasculature in constructs prior to implantation22. The implantation of a scaffold with in vitro preformed engineered capillaries onto the dorsal skin of mice led to rapid anastomosis of the mice vasculature within a day23. In contrast, a spheroid co-culture consisting of human mesenchymal stem cells and human umbilical vein endothelial cells assembled into a three-dimensional prevascular network developed further after in vivo implantation. However, anastomosis with the host vasculature was limited24. Above all, in poorly vascularized defects, such as necrotic or irradiated areas, this so-called extrinsic vascularization &#8212; the ingrowth of vessels from the surrounding area into the scaffold &#8212; often fails. Intrinsic vascularization, on the other hand, is based on a vascular axis as a source of new capillaries sprouting into the scaffold25. Using the axial vascularization approach, the engineered tissue can be transplanted with its vascular axis and connected to local vessels at the recipient site. Immediately after transplantation, the tissue is adequately supported by oxygen and nutrients, which creates the right conditions for optimal integration.
Due to the limited availability of models for investigating in vivo angiogenesis and in recognition of the growing importance of generating axially vascularized tissue, we developed the microsurgical approach of Erol and Spira further to generate an arteriovenous (AV) loop in the animal model26. The use of a completely closed implantation chamber makes this method very well suited to study blood vessel formation under &#34;controlled&#34;, well characterized in vivo conditions (Figure 1). This model is not only useful for the investigation of angiogenesis but is also optimally suited for the axial vascularization of scaffolds for tissue engineering purposes.

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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.

Tissue Engineering 
For bone tissue engineering purposes, a number of different bone substitutes were implanted in the small animal rat AV loop model27,28,33,34. Vascularization could perfectly be demonstrated by 3D micro-computed tomography (micro-CT) (Figure 3A). Vascularization of a processed bovine cancellous bone (PBCB) matrix was significantly higher in the loop group compared to the group without vascularization. A constantly growi...

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

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

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 ...

The overall goal of this microsurgical approach is to generate an arteriovenous loop for engineering axioli, vascularized, and translatable tissues, and analyzing vascularization in vivo and in isolated and well characterized environment. With this method, we can use our own bodies as living bio reactors for the generation of different kinds of axiolly vascularized tissue tailored to the individual patient's requirements. The main advantage of this technique is that the generated tissue can be translated with its vascular access and connected to local vessels at the recipient's side for immediate supplying with oxygen and nutrients.In addition to tissue engineering studies, this method can provide insight into the angiostenosis processes in a living organism. It can also be applied to pathological situations, such as cancer angiogenesis. Generally, individuals new to this method will struggle because of the high complexity of the surgery, such as the micro anastomosis of the submillimeter vessel.We first had the idea for this method when we were confronted with clinical cases with large tissue defects, which required the transplantation of atholo vascularized tissue creating a significant donor side mobility. After confirming proper anesthesia, weigh the rat to calculate the drug doses. Then, apply eye ointment to prevent dryness while under anesthesia.After administrating pain medications and antibiotics, place the rat over a heating pad and administer continuous isoflurane. Then, reconfirm the level of anesthesia using a toe pinch. If possible, also use pulse oximetry to check the rat's anesthesia level.Proceed by shaving the inner sides of the hind limbs. And then disinfecting the surgical area with alternative scrubs of betadine and alcohol. Then, spread the hind limbs and secure them with adhesive tape.Finally, position the rat under a surgical microscope and drape it. Proceed with the surgery using steril procedures. Open the skin in the middle of the left thigh with a longitudinal incision from the upper knee to the groin using a scalpel.Next, using dissecting scissors and micro forceps, cut the subcutaneous tissue and fascia in layers that are about three centimeters long. Stop when the femoral vascular bundle is exposed from the pelvic artery in the groin to the knee. Next, using admin tissue scissors and micro forceps separate the vessels and remove the admin tissue.As needed, coagulate the side branches using electric coagulation. Once completed, cover the operated field with a damp compress and repeat the procedure on the right side of the rat. Now, with the first procedure completed symmetrically, commence with harvesting the venus graft.First, ligate the right femoral vein using electric coagulation on the proximal and distal ends to obtain a graft of about one to 1.5 centimeters. Next, remove the venus graft with micro forceps. For dilating the opening of the venus graft, use the vessel dilator.Then, flush the venus graft with a haperin solution using an irrigation cannula. Transfer the fleshed graft to the left thigh and cover the field at the right side with a damp compress. Next, ligate the femoral vein on the left side proximately at the inguinal region with a mico vessel clamp.Then coagulate the femoral vein distally at the upper knee before the branching. For the anastomosis, connect the proximal end of the venus graft with the proximal end of the vein using an end to end technique with an 11-0 suture. Begin with placing the first two sutures at the 12 o'clock and the six o'clock positions.Then, put in two to three more sutures between these points on the front side. And then put two to three more sutures into the back side. The anastomosis of the loop vessels is the most critical step of the protocol.It is important to make sure that the sutures are performed as accurately as possible to reduce the risk of and closure of the loop vessels. Next, ligate the femoral artery with a micro vessel clamp and use electronic coagulation, similarly to ligating the femoral vein. Before proceeding, make sure the looped vessels are not twisted.Then, anastomose the distal end of the venus graft with the proximal end of the artery, using the same suture technique as demonstrated previously. After the second anastomosis, administer 25 iu of heparan intravenously. Now, double check that loop vessels are not twisted and then remove the clamps.Observe the loop for about five minutes, checking for leakage and patency. If there is patency, the loop will expand and the pulse of the artery will be visible. During the observation, dripple papaverine on the vessels to prevent vascular spasms.Next, pre-fill the implantation chamber with the first half of the matrix mixture, about half a millileter. Then, embed the loop into the implantation chamber. Close the chamber entrance with two clots of fibrin and fill the chamber with the second half of the matrix mixture, for a total volume of one millileter.Now, prefix the chamber on the thigh, seal the chamber with its lid, and secure the lid with a nonabsorbable 6-0 suture. Then, secure the implantation chamber to the thigh with an additional nonabsorbable 6-0 suture. Stop any bleeding using electric coagulation.Finally, close the skin with absorbable 4-0 sutures and cover the wound with aluminum spray. For bone tissue engineering purposes, a number of different bone substitutes were implanted in the small animal rat A-V loop model. Vascularization was observed using 3D micro computer tomography.Pre-vascularization of a processed bovine cancellus bone matrix for six weeks led to superior survival of the injected osteoblasts. Loops were harvested, sectioned, and stained at various time points post implantation. For example, H and E staining showed that use of a beta tricalcium phosphate hydroxyapatite bone substituent with mesenchymal stem cells resulted in many vessel connections within six weeks.Once mastered, this technique can be done in five to six hours, if it is performed properly. Following this procedure, a large variety of different The various cells factors or anti can be implanted to study a wide area of conditions. This technique also works in a large animal sheep venus loop model.So it should allow researchers to generate vascularized tissues in large clinically relevant amounts. This model may provide the opportunity to study vasularization under pathological conditions. For example, the influences of various growth factors and cells during tumor vessel network formation could be investigated

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