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. While the engineering and optimization of novel TEVs have undergone many advancements, reporting on the surgical techniques employed to implant the TEVs themselves has not been a topic of such intense discussion. Rather, protocols regarding the implantation of TEVs into animal models are largely left up to research investigators.

The following manuscript demonstrates how to implant TEVs by utilizing an end-to-end anastomosis approach. This procedure was optimized by using a specific anastomotic suturing pattern, stabilizing suture technique, optimizing longitudinal tension and the addition of in vivo monitoring instrumentation. This method is contrasted with some of the many variations that have been previously used. Furthermore, this procedure describes how to acquire parameters such as arterial blood pressure, TEV diameter/compliance and flow rate through the TEV after surgery up until explantation. This data collection provides an indispensable analysis of the TEV while it is in the process of remodeling.

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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 for 30 min.
4. Place the following items in a paraformaldehyde desiccator 48 hr prior to surgery: 4 mm Doppler flow probes, 1 mm ultrasonic crystals, indwelling arterial catheters, extension tubing, and 42” Tygon Tubing.
   NOTE: A table of relevant medical equipment needed for the surgery and for sterilization is listed in Table 1. Pre-placement of the instrumentation into the Tygon tubing at this step will help save time during surgery.
   1. Label each end of the probes, catheters and crystals as left and right, if applicable.

2. Surgical Operation

1. Induce sheep for anesthesia with diazepam (0.5 mg/kg) and ketamine (4 mg/kg) intravenously (IV). Alternatively, use Telazol (4 mg/kg) IV.
   1. Perform orotracheal intubation with an 8.5-10.0 mm internal diameter cuffed endotracheal tube⁷.
   2. Administer inhalant anesthesia through a rebreathing circuit with a tidal volume ventilator (7-10 ml/kg) or a pressure-regulated ventilator (15-20 cm H₂O). Use a precision vaporizer to administer isoflurane or sevoflurane at an appropriate amount (3%-4% initially) to reach a medium to deep surgical anesthetic stage. The minimum alveolar concentration for sheep is 1.4% or 1.9% respectively⁸.
   3. Assess anesthetic depth by observing motor response to stimuli, palpebral reflex, eye position, and heart rate. Monitor blood oxygen saturation (95%-100%) using pulse oximetry, CO₂ concentration (45-55 mmHg) in expiratory gases using capnography and maintain body temperature during the procedure (38.5-39.5 °C) using an auto regulated warming blanket.
2. Shave wool from the entire neck of the sheep, and over one cephalic vein, using a #40 blade on standard clippers. Prepare the skin of both sites for surgery using 70% isopropyl alcohol and 7.5% betadine scrub saturated gauze. Begin with alcohol gauze to facilitate removal of skin oils. Alternate between betadine gauze and alcohol gauze three times.
3. Place sheep onto the operation table in dorsal recumbency on top of a warming blanket. Pass a medium sized orogastric tube to allow for passive expulsion of stomach contents. Extend the sheep’s neck and use supportive cushioning as needed to maintain placement.
   1. Perform a final aseptic scrub using 7.5% betadine soaked gauze and allow to sit for 5 min prior to surgery.
4. Administer IV fluids (Lactated Ringer’s Solution or 0.9% Saline) at 10 ml/kg/hr through an angiocath placed in the cephalic vein. Administer intraoperative antibiotics and analgesia: Penicillin G Procaine 6,600 U/kg intramuscularly (IM), Gentamicin 1.6 mg/kg IM, and Buprenorphine 0.005-0.01 mg IV or IM.
5. Make a ~12 cm incision lengthwise over the ventral midline neck using electro cautery. Isolate left and right carotid arteries (~6 cm) by removing connective tissue using a blunt dissection technique. Tie off and cauterize micro-vessels branching from the carotid arteries to minimize bleeding.
6. Maintain sterility by utilizing (a non-sterile) surgical nurse to assist with burrowing all wiring and tubing (flow probe, ultrasonic crystal wires and catheter tubing) in the subcutaneous layer of the skin. Use a blunted trocar, which exits through the surgically prepped incision at the dorsolateral neck.
   1. Reach under the sterile drape and turn the head of the sheep so that the side of the neck can be visualized under the drape.
   2. Use an 8 cm curved hemostat to tunnel through the subcutaneous space between the ventral midline neck incision and the side of the neck. Open and close the hemostat to bluntly dissect a space for the tubing ~1.5 cm wide. The tips of the hemostat should reside halfway between the head and shoulders, approximately 10 cm caudal to the right or left ear. Turn the hemostat so that the tips are pointing toward the superficial skin.
   3. Reach under the sterile drape and make a 1.5 cm incision through the skin, over the tips of the hemostat with a sterile #11 blade. Visualize the tips of the hemostat to confirm a clear exit through the skin.
   4. Pass the Tygon tube containing all wiring and tubing through the subcutaneous tunnel. Hold the wires and tubing above the sterile drape.
   5. Reach under the drape to remove the outer Tygon tubing from the neck, exposing the implanted wiring as it leaves the neck of the sheep. Pull individual lines out to minimize any slack in the subcutaneous space. Leave enough distance to properly attach the instrumentation to the artery.
7. Place 4 mm Doppler flow probes on both carotid arteries and attain an initial reading (Figure 1). Administer 100 U/kg of heparin IV 30 min before clamping the artery.
8. Continue heparin administration at 100 U/kg/hr until the end of surgery. Clamp the carotid artery using non-crushing vascular clamps and excise a portion (approximately 4 cm in length). The contralateral carotid flow rate will increase 50%-100% to maintain blood flow to the brain.
   NOTE: It is possible to limit longitudinal stretch by recoil of native vessel by removing shorter segments than being replaced and/or stretching the native artery with vascular clamps to shorten gap until full anastomotic procedure is completed. This will help limit the tension on individual holding sutures and the implanted graft.
9. Suture the TEV in place using simple interrupted stitches with 7-0 proline ethalloy double armed monofilament suture. If necessary, apply vascular smooth muscle relaxants such as Papaverine (15 mg/ml) or Nicardipine (1.25 mg/ml) topically to the native vasculature in order to prevent vasoconstriction which would hamper anastomotic suturing.
   NOTE: Begin placing sutures with approximately 1 mm spacing. This can greatly vary from case to case. The composition and TEV thickness will affect the effective distance between sutures. As the thickness of the native tissue or TEV decreases, it may be necessary to place the sutures closer together.
   1. First anchor four points of the TEV to the native artery by placing two opposing stitches on both proximal and distal ends (Figure 2-Di). Hold each anchor taught using hemostats.
      NOTE: Proximal and distal descriptions are in reference to direction of blood flow throughout the paper.
   2. Add 5-6 more sutures on the superficial side of both the proximal and distal ends to begin the anastomosis. (Figure 2-Pr). Simultaneously rotate the vascular clamps 180 degrees.
   3. Re-establish tension on the anchoring sutures. Add additional (5 to 6) interrupted sutures to proximal and distal ends on rotated side of the TEV.
10. Once the TEV is securely sutured in place, rotate it back to the original position and remove the vascular clamps one at a time, distal clamp first. Slight bleeding at the anastomosis sites is common. This may naturally resolve after several minutes of clamp release and reclamping or require the placement of additional sutures. Place the Doppler flow probe (Figure 3-Fl) back on native artery proximal to blood flow entering the TEV and monitor flow rate.
    NOTE: Expect the flow rates of the left and right carotid to equilibrate after approximately 15 min. If the flow rate on the carotid artery with implanted TEV steadily drops, it is possible the TEV is clotting. Other possible abnormalities regarding flow can be attributed to constriction of the native arteries proximal or distal to the TEV. If this occurs, the use of additional vascular smooth muscle relaxant may be applied, and the native vessel should return to a basal tone after 30-60 min following the closure of tissue over the graft site.
    1. If desired, excise the contralateral carotid artery and suture it back into place as a “Sham” control. This is more clinically relevant than leaving the right carotid artery alone and only attaching the flow probe, ultrasonic crystals, and catheter. If a sham control is wanted, perform this before continuing to step 2.11.
11. Suture 1 mm ultrasonic crystals (Figure 3-cr1 cr2) to opposing sides of the TEV using 7-0 Proline. Thread suture through the ultrasonic crystal head and stitch only to the superficial layer of the TEV.
12. Catheterize the artery using a modified 18 G catheter with a Teflon woven placket (Figure 3-Ca & Figure 4A). Place the catheter distal to the TEV on native arterial tissue.
    1. Suture the placket to the arterial wall with 5/0 Ethibond to control any bleeding. Use cyclohexanone to adhere the microbore tubing to the catheter that has been flushed with saline. Use the tubing as an extension line.
    2. Use a 20 G Luer Stub Adapter with a Surflo Injection Plug to seal the exteriorized end of tubing (Figure 4B). To maintain patency of the catheter, obtain the priming volume of the line and flush it with 10 ml of saline and then 5,000 U/ml of heparin sodium injection every 2-3 days.
13. Record the distance between the flow probe and the ultrasonic crystals, as well as the distance between the flow probe and the catheter. This will enable pulse wave velocity to be calculated in conjunction with software. If such calculations are not needed, do not implant a catheter.
14. Obtain an intraoperative reading if desired to ensure all implanted hardware is functional (see section 3).
15. Secure implanted lines and wires to nearby musculature using 2/0 Silk and a taper needle (Figure 3).
    1. Position the vascular flow probe wire parallel to the vessel, with the probe caudal and the wire extending cranially, and then making a “U-Turn” toward the lateral musculature. Secure the wire to adjacent musculature, using 2-0 silk on a taper needle at two locations, so that the wire or flow probe is not able to place any strain on the vessel. Be sure sutures are snug but do not over tighten and strangulate musculature (Figure 3).
    2. Suture the crystal wires and arterial catheter line to the lateral musculature, allowing for ~1.5 cm of slack, similar to previous steps for securing flow probe (Figure 3).
    3. Group all wires and lines together and anchor them to musculature just before exiting out through subcutaneous tunnel, similar to previous steps.
16. Close the surgical site with a 2-0 Vicryl suture in layers using a running suture pattern on facia and subdermal, running mattress stitch on the skin (facia, non-cutting needle; skin, cutting needle). Close the 1.5 cm incision at the dorsal neck around the exteriorized wires and lines using 2/0 Vicryl and a cutting needle.
17. Place flow probe wires, catheter lines, and ultrasonic crystal wires into a pouch (10 cm x 10 cm) that is securely sutured to the skin of the sheep (Figure 5 — after recovery).
18. Gradually wean the sheep off anesthesia and the tidal volume ventilator then extubate the sheep when spontaneous breathing is resumed. Remove the angiocath inserted into the cephalic vein and bandage if needed.
19. Bandage the neck using triple antibiotic ointment on the incisions, a Telfa pad, stretch roll gauze, and elasticon.
20. Administer post-operative analgesia: flunixin meglumine 2.2 mg/kg IM once during recovery, then 1.1 mg/kg IM once a day for two days, buprenorphine 0.005-0.01 mg IV or IM twice a day for one day.

3. In Vivo Monitoring

1. Place sheep into mobile cart to ensure proper restraint. This allows the sheep to remain calm and conscious without compromising the hardware. May need to acclimate sheep to cart 2 or 3 times for 30 min prior to obtaining instrumentation recordings.
2. Remove all wires and lines from pouch and connect to the monitoring devices. Connect flow probe to a Flowmeter, 1 mm ultrasonic crystals connected to TRB-USB box, and catheter lines to pressure transducers. A flow chart of this setup is provided (Figure 6).
3. Calibrate flow probes and pressure transducers prior to data acquisition.
   NOTE: Due to potential variability between software versions and differences in equipment used, calibrations and settings will vary from case to case.
4. Utilize an oscilloscope to fine tune the Sonometrics crystal measurement, according to manufacturer’s protocol.
5. Record data using computer software (Figure 7). Traces in the top half of Figure 7 in white color corresponds to the implanted TEV, while traces in the bottom half in red color corresponds to the Sham/Native. For both the TEV and Sham the flow rate (ml/min), arterial blood pressure (mm Hg) and diameter (mm) are recorded live.
6. Record for at least 1 min with no disturbances. Export this data for more detailed analysis. After the recording, disconnect all wires and place back into pouch sutured on the neck of the sheep.

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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...

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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...

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Materials

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.