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

Summary

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.

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

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 pathol....

Protocol

The Animal Care Committee of the Friedrich-Alexander University of Erlangen-Nürnberg (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

1. Implantation Procedure (Figure 2)
   1. 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 and flow meter between 0.8 - 1.5 L/min.
   2. Place the rat in the induction plastic box and seal the top. Turn on isoflurane vaporizer to 5%.

Results

Tissue Engineering
For bone tissue engineering purposes, a number of different bone substitutes were implanted in the small animal rat AV loop model^27,28,33,34. 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 growing.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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