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

Summary

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.

Abstract

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.

Introduction

Fabricating functional vascularized tissue using a tissue engineering approach is an emerging paradigm in regenerative medicine.^1,2 Many approaches to engineer new and healthy tissue for the replacement of injured tissue or defective organs have been developed,^3-6 experimentally in small animal models with promising clinical potential.^7,8 However, vascularization remains one of the great challenges for tissue engineering limiting its potential to grow tissues of clinically relevant size.⁹

Current approaches to vascularize tissue follow either an extrinsic pathway where new vessels grow from the re....

Protocol

The protocols described here have been approved by the Animal Ethics Committee of St. Vincent's Hospital Melbourne, Australia, and were conducted under strict adherence to the Australian National Health and Medical Research Council Guidelines.

NOTE: Two chamber protocols are described below. The two different models and their specific chamber designs are illustrated in Figure 1. Chamber (1) is made of polycarbonate (for rat arteriovenous loop chamber model). It is cylindrical with an internal diameter 13 mm and height 4 mm. A window at one point in the wall allows unimpeded access for the pedicle. In the second model (for rat flow-through pedicle chamber model)....

Results

The microsurgical creation of tissue engineering chambers was performed as described in the protocol above. Tissues generated inside the chambers can be examined histologically as describe in protocol step 3. Various tissue types have been successfully engineered using the in vivo vascularized chamber (Figure 2). These include cardiac tissue with neonatal rat cardiomyocytes (Figure 2A), muscle tissue with rat skeletal myoblasts (Figure 2B.......

Discussion

Engineering of the microcirculation is currently being investigated essentially through two approaches. The first involves developing a highly interconnected vascular network within the construct in vitro so that when implanted, capillaries from the host vascular bed connect with those of the transplanted construct through a process called inosculation, thus ensuring the delivery of nutrients not only to the periphery but also to the core.^21,32,33 This is called pre-vascularization. The second approac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 15 Blade Scalpel	Braun	BB515
1 Toothed Adson Forceps	Braun	BD527R
1 Needle Holder	Braun	BM201R
1 Bipolar Coagulator	Braun	US335
1 Micro Needle Holder B-15-8.3	S & T	00763
1 Micro Dilator Forceps D-5a.2	S & T	00125
1 Micro Jeweler's Forceps JF-5	S & T	00108
1 Micro Scissors - Straight SAS-11	S & T	00098
1 Micro Scissors - Curved SDC-11	S & T	00090
2 Single Clamps B-3	S & T	00400
2 10/0 nylon suture	S & T	03199
1 6/0 nylon suture	Braun	G2095469
2 4/0 Silk Sutures	Braun	C0760145
Xilocaine 1%	Dealmed	150733	10 mg/ml
Heparin Sodium	Dealmed	272301	5,000 UI/ml
Ringer Lactate	Baxter	JB2323	500 ml
1 dome-shaped tissue engineering chamber	custom made
1 flow-through chamber	custom made
Lectin I, Griffonia Simplicifolia	Vector Laboratories	B-1105	1.67 μg/ml
Troponin T antibody	Abcam	Ab8295	4 μg/ml
Human-specific Ku80 antibody	Abcam	Ab80592	0.06 μg/ml
Desmin antibody	Dako	M0760	2.55 μg/ml
Cell Tracker CM-DiI dye	Thermo Fisher Scientific	C-7000	3 mg/106 cells
Lethabarb (sodium pentobarbitone)	Virbac Animal Health	LETHA450	325 mg/ml
Heat pad flexible	Redzone Heating	RZ/Medium