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

The overall goal of this procedure is to provide a reproducible protocol for the surgical implantation of vascular grafts and novel instrumentation into the carotid artery. This is accomplished by first excising, a section of native carotid artery in a sheep. The second step is to suture in the vascular graft referred to here as the tissue engineered vessel or TEV using simple interrupted stitches and to establish anastomosis.

Next, a flow probe catheter. And so metric crystals are secured onto the carotid artery and vascular graft. Ultimately, after implantation, this procedure allows for diameter changes, blood flow, and arterial pressures to be recorded in vivo for the length of the experiment.

From these measurements, dynamic compliance, pulse wave velocity, and other hemodynamic parameters can be monitored to follow in vivo remodeling. The main advantage of this technique is that it provides an optimized, detailed vascular graft implantation protocol that can become standard practice, thereby minimizing surgical variability and providing a means to study in vivo remodeling. In general, individuals new to this method may struggle because they may not be familiar with the details of the optimized implantation procedure and the importance of monitoring vascular graft, adaptation, and remodeling in vivo.

This method can help answer key questions in the field of vascular graft development and biology, such as optimal vascular tissue engineering conditions and vascular graft remodeling and disease. The implications of this technique extend toward therapy of vascular replacement because it provides a procedure to better study the development of vascular grafts for clinical applications. Through this method, we can provide insights into the study of whole organ tissue engineering and vascular remodeling and disease such as hypertension or even whole organ transplantation.

We first had this idea when we used an ex vivo arteriovenous shunt model to assess optimal conditions for development of a tissue engineered vascular graft. Visual demonstration of this method is essential as the placement of sutures and instrumentation is critical for patency and successful data acquisition. To begin this procedure, induce the sheep for anesthesia and prepare the sheep for surgery.

As described in the text protocol, make an approximate 12 centimeter incision lengthwise over the ventral midline neck using electrocautery. Then, then isolate the left and right carotid arteries by removing connective tissue. Using a blunt dissection technique, 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.

Use an eight centimeter 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 to approximately 1.5 centimeters wide. Reach under the sterile drape and make a 1.5 centimeter incision through the skin over the tips of the hemostat with a sterile number 11 blade.

Pass the tigon tube containing all wiring and tubing through the subcutaneous tunnel. Hold the wires and tubing above the sterile drape. Then reach under the drape to remove the outer tigon 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. After placing the doppler flow probes and administering heparin as described in the text protocol, clamp the carotid artery using non crushing vascular clamps and excise a portion approximately four centimeters in length.

Suture the tissue engineered vessel or TEV in place using simple interrupted stitches with 7.0 proline F alloy. Double armed monofilament suture. First anchor four points of the TEV to the native artery by placing two opposing stitches on both proximal and distal ends.

Then add five to six more sutures on the superficial side of both the proximal and distal ends. To begin the anastomosis simultaneously, rotate the vascular clamps 180 degrees, reestablish tension on the anchoring sutures, and add five to six additional interrupted sutures to proximal and distal ends on the rotated side of the TEV. 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.

Next, suture one millimeter ultrasonic crystals to opposing sides of the TEV. Using 7.0, proline catheterize the artery using a modified 18 gauge catheter with a Teflon woven plaque. Suture the plaque to the arterial wall with 5.0 at theon to control any bleeding.

Use cyclone to adhere the micro bore tubing to the catheter that has been flushed with saline. Next, secure implanted lines and wires to nearby musculature using 2.0 silk and a taper needle. To do this position the vascular flow probe wire parallel to the vessel with the probe coddle and the wire extending cran, 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. Then suture the crystal wires and arterial catheter line to the lateral musculature, allowing for approximately 1.5 centimeters of slack similar to previous steps for securing the flow probe group all wires and lines together and anchor them to the musculature just before exiting out through the subcutaneous tunnel. Similar to previous S, close the surgical site with a 2.0 Vicryl suture in layers using a running suture pattern on the fascia and subdermal and a running mattress stitch on the skin.

Close the 1.5 centimeter incision at the dorsal neck around the exteriorized wires and lines using 2.0 Vicryl and a cutting needle. Finally, place flow probe wires, catheter lines, and ultrasonic crystal wires into a pouch that is securely sutured to the skin of the sheep. See the text protocol for the sheep postoperative care Place sheep into a mobile cart to ensure proper restraint.

This allows the sheep to remain calm and conscious without compromising the hardware. Remove all wires and lines from the pouch and connect them to the monitoring devices. Connect the flow.

Probe to a flow meter, the one millimeter ultrasonic crystals to A-T-R-B-U-S-B box and the catheter lines to pressure transducers. Calibrate the flow probes and pressure transducers. Prior to data acquisition.

Utilize an oscilloscope to fine tune the SOE crystal measurement. According to the manufacturer's protocol, record the data using computer software here. The top half in white color corresponds to the implanted TEV while the bottom half in red color corresponds to the sham or native artery for both the TEV and sham.

The diameter flow rate and arterial blood pressure are recorded. Live after recording for at least one minute with no disturbances. Export this data for more detailed analysis.

Then disconnect all wires and place them back into the pouch sutured on the neck of the sheep.Shown. Here is the in vivo live recording of the diameter flow rate and arterial blood pressure of the TEV, which may be recorded for the duration of the experiment. Optimizing the implantation of vascular grafts.

Using this surgical technique allows researchers to rule out surgical error as a cause of graft failure Once mastered, this technique can be done in as little as two and a half to three hours. If one TEV is being implanted or as long as six and a half hours. If a TEV autologous sham and instrumentation are to also be implanted While attempting this procedure, it's important to remember to maintain a proper level of anticoagulation.

Use vasodilatory agents an optimized precision in the anastomotic suture placement Following this procedure. Other methods such as immuno staining can be performed in order to answer additional questions such as cell migration and cell differentiation. After watching this video, you should have a good understanding of how to implant tissue engineered vessels into the carotid artery of a sheep, as well as how to monitor arterial blood flow, blood pressure, and changes in vascular graft, diameter in vivo.

Don't forget that working with sheep and other large animals for the use in an experimental setting need to follow species specific precautions. If you plan on working with these large animals for your own studies, be sure you are properly trained to handle these animals in a safe and ethical manner. Thanks for watching our video.