The overall goal of this surgical intervention is to transplant an electrospun vascular graft into a rat abdominal aorta, and evaluate the longterm performance of the graft. This method has been established by Professor Deling Kong and other colleagues in our group, and it can help answer key questions in the study of electrospun vascular grafts, such as the effect of structure and the surface properties of the graft on the vascular regeneration. The main advantage of this technique is that it has good repeatability and high animal survival rate.
Besides, the animal model is easy to manage at a low cost. This work will be conducted by our colleague, Doctor Wang. To begin, prepare the polymer solution by dissolving polycaprolactone in a 1-to-5 mixture of methanol and chloroform, and stirring it at room temperature for 12 hours.
Load the polymer solution into a 10 milliliter glass syringe, and equip it with a blunt 21 gauge needle. Then, place a stainless steel mandrel on the collection instrument, and make sure that it is properly grounded. Adjust the working distance from needle tip to collector to 17 centimeters for the 25%polycaprolactone solution.
Then, set the flow rate on the syringe pump to eight milliliters per hour. Next, set the voltage on the power supply to 11 kilovolts, and start the mandrel spinning at 40 RPMs in order to collect electrospun fibers for appropriate fiber distribution. With everything now set up, turn on the power supply and the syringe pump to begin collecting the electrospun fibers.
After 4.5 minutes, turn off the syringe pump, power supply, and the mandrel motor. Allow the graft to dry in the hood for one hour. Then, remove the graft from the mandrel using absolute alcohol, and place it in a vacuum overnight to remove the residual solvent.
Sterilize the graft with ethylene oxide, and allow it to dry in a sterile biological containment hood. Using standard techniques, obtain SEM images of the lumen of the grafts, and import them into ImageJ. Use the measurement tools to measure both the fiber diameter and average pore size in each image, to ensure consistency across samples.
To obtain information on the mechanical properties of the graft material, first cut a tubular scaffold into three millimeter sections using a razor blade. Then, use calipers to measure the thickness of the scaffolds. Next, load the scaffolds into the grips of a tensile testing machine, so that there is one millimeter between the grips.
Hold the scaffolds axially, at a rate of 10 millimeters per minute, until they rupture. Be sure to record both the tensile strength and ultimate elongation at break for the samples. Prior to surgery, fast male Sprague Dawley rats, weighing 240 to 270 grams, for 24 hours prior to the implantation surgery.
Anesthetize the rat and prepare it for surgery as described in the accompanying text protocol. Confirm adequate anesthetization by ensuring that the rat has relaxed muscles and steady breathing. Then, place the rat under the operating microscope in a supine position.
Using surgical scissors, perform a four to five centimeter long midline laparotomy incision, and expose the abdominal cavity. Carefully retract and wrap the intestines with gauze that has been moistened with saline solution. While looking through the operating microscope strip the abdominal aorta from the adjacent tissues.
Identify and ligate all of the small branches of the descending aorta, using 9-0 monofilament nylon sutures. Next, use vascular clamps to clamp up to a one centimeter isolated section of the aorta. Then, use a pair of micro scissors to transect the abdominal aorta between the clamps.
Flush the two ends of the aorta using heparinized saline solution to remove the residual blood. Peel off the adventitia of the clamped aorta using micro scissors. Then, place a one centimeter long section of the sterilized graft in the gap created by the removed section of aorta.
Using 9-0 monofilament nylon sutures, construct four anastomoses using a figure eight suture pattern at nine o'clock, three o'clock, 12 o'clock, and then six o'clock positions of the proximal side. Then, construct the other four anastomoses between each of the sutures in the proximal side. Place the graft vertically to show the equal interval among the eight stitches under the operating microscope.
After finishing the proximal suture, repeat the process on the distal side. Once the graft has been attached, remove the distal clamp to allow the blood to flow into the graft, then remove the proximal clamp. Using a sterile cotton ball, apply pressure to stop any observed bleeding at the suture points.
Press for about three minutes, until hemostasis occurs. Next, return the intestines into the abdominal cavity, and flush the abdominal cavity using warm physiological saline solution containing gentamicin. Then, sew up the abdominal wall using 3-0 nylon suture in the muscle and skin layers.
Use iodine on the incision after surgery only once. Return the rat to the company of others once it has fully recovered. The concentration of polycaprolactone in the electrospinning solution can affect both the fiber diameter, and the mechanical properties of the grafts.
The higher concentration 25%solution produces vascular grafts with thicker fibers and larger pores than the 10%solution. After three months in vivo, histological staining revealed a layer of new tissue formed on the lumen of the graft, and a significant amount of collagen deposition within the graft itself. This new tissue layer is lined with endothelial cells, below which can be found a layer of both smooth muscle cells and circumferentially aligned elastin.
After 12 months in vivo, the new tissue appeared well integrated, and the endothelial and smooth muscle layers remained intact. Once mastered, this technique can be completed in one to two hours, if it is performed appropriately. Following this procedure, other measures like color doppler ultrasound, can be performed in order to answer additional questions, like After watching this video, you should have a good understanding of how to fabricate the electrospun vascular grafts, and evaluate its in vivo performance in a rat abdominal artery replacement model.
