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Bioengineering

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

Published: May 30th, 2016

DOI:

10.3791/54099

1O'Brien Institute Department, St Vincent's Institute of Medical Research, 2Department of Surgery, University of Melbourne, 3Faculty of Health Sciences, Australia Catholic University
* These authors contributed equally

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.

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.

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....

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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 he.......

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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.......

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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.......

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This work was supported by grant funding from NHMRC and Stafford Fox Medical Foundation. The authors acknowledge the surgical assistance of Sue McKay, Liliana Pepe, Anna Deftereos and Amanda Rixon of the Experimental Medical and Surgical Unit, St. Vincent's Hospital, Melbourne. Support is also provided by the Victorian State Government's Department of Innovation, Industry and Regional Development's Operational Infrastructure Support Program.

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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 5000 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

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