Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Summary

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Abstract

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Introduction

The primary focus for the development of TEVs has been to provide a substitute for autologous graft replacement when autologous vessels are not available and to limit donor sight morbidity. For example, the number of coronary artery bypass surgeries per year has exceeded 350,000 in the USA, and the ideal source of suitable grafts remains the left internal mammary artery, left anterior descending coronary artery and saphenous vein¹. Since many individuals who suffer from vascular diseases may not have suitable arteries and veins for autologous graft replacement, the development of TEVs has thus become an intense field of research for decades^1-6. W....

Protocol

NOTE: This protocol has been approved by the Animal Care and Use Committee at the State University of New York at Buffalo.

1. Pre-surgical Preparation

1. Use sheep (Dorset cross, female, approximately 1-3 years old with a weight of 40-60 kg) for the following study. Administer cyclosporine A (200 mg/day), aspirin (975 mg/day), and coumadin (20-30 mg/day) by mouth, starting 3 days before surgery and continue for the duration of all studies.
2. Ensure the sheep has fasted 12 hr before surgery (normal feed = 1.8 kg hay and 0.4 kg grain).
3. Sterilize all surgical supplies using a steam autoclave at 121 °C and 15 psi....

Results

More than 30 sheep have undergone the surgical technique described in this report for the implantation of TEVs (in press) ⁹. A table summarizing the most recent sheep operations after protocol optimization are shown in Table 2. All sheep recovered after TEV implantation with no life threatening complications. In some animals, fibrosis was observed in native artery near the tip of the indwelling arterial catheter. A significant increase of inflammation with the presence of added instrumentation.......

Discussion

The purpose of this report is to provide a reliable and reproducible procedure to implant TEVs of interest in the ovine carotid artery. The native carotid arteries of the animals used in this model were 0.5-0.75 mm in thickness and 4.5-5 mm in outside diameter. The surgical technique described here has been successful for implanting TEVs of varying geometries measuring 0.25-1 mm in thickness, 4-5 mm outside diameter and 4 cm in length with great success proving effective up to 3 months duration, the intended end point. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pressure Transducer	Becton Dickinson	P23XL-1	Quantity: 1+ (1 for each artery). Used with water-filled diaphragm domes.
Amplifier and transducer box	Gould	5900 Signal Conditioner Cage	Quantity: 1. Two transducers and amplifiers should be included in cage. While this specific unit may be discontinued, other commercially available pressure transducers with a BNC/analog output will communicate with the Sonometrics equipment.
T403 Console with TS420 perivascular flowmeter module (x2)	Transonic Systems	T403 module and TS420 (x2)	Quantity: 1. Flow probes measuring flow through each of the carotid arteries will connect to each of the TS420 units.
Digital ultrasonic measurement unit	Sonometrics	TR-USB	Quantity: 1
Flow Probe Precision S-Series 4 mm	Transonic Systems Inc.	MC4PSS-LS-WC100-CM4B-GA	Quantity: 2
1 mm Sonometrics Crystals	Sonometrics Systems	1R-38S-20-NC-SH	Quantity: 2-4 (2 for each artery)
Catheter for implantation	BD (Becton Dickinson)	381447	Quantity: 1+ (1 for each artery). Catheter is cut and secured to microbore tubing, stylette is utilized for insertion.
Tygon Microbore Tubing	Norton Performance Plastics	(AAQ04127) Formulation S-54-HL	Cut to length for an extension set
Luer Stub Adapter	BD (Becton Dickinson)	427564 (20 gauge)	Quantity: 1+ (1 for each arterial catheter)
Surflo Injection Plug	Terumo	SR-IP2	Quantity: 1+ (1 for each arterial catheter)
Meadox	PTFE (Teflon) Felt	19306	Cut to size. The PTFE felt used in our studies was discontinued. However, comparable companies such as “Surgical Mesh” offer products which are equivalent.