The overall goal of this procedure is to investigate the cellular and molecular mechanisms of neo tissue formation and stenosis development in tissue engineered vascular grafts. First biodegradable tubular scaffolds are assembled by coating polyglycolic acid nonwoven felt mesh with an epsilon cap lactone and L lactide copolymer. The next steps are to harvest bone marrow and isolate the mononuclear cells by density gradient centrifugation.
Then approximately 1 million cells are seeded on the scaffold and they are incubated overnight. The final step is to implant the seeded scaffold as an inferior vena cava interposition graft. Ultimately, results can show cell infiltration and extracellular matrix deposition in the tissue engineered vascular graft through immunohistochemistry.
The implications for this technique are to better understand what causes the formation of stenosis and tissue engineered vascular grafts, and then use this information to design a better tissue engineered vascular graft. From our clinical study, we learned that nearly a quarter of patients who received the tissue engineered conduit developed a narrowing. Our goal is to eliminate this problem.
Although this method can apply to the congenital heart disease, it also can be applied to the colon bypass surgery or the the condu for the alter venous feature. Generally, individuals new to this method were struggle because of the fine microsurgery techniques required during the vein osmosis. We first had the idea for this method when we were looking for a better way to characterize the new tissue formation and ways to prevent stenosis development in the tissue engineered vascular grafts used in our human clinical trial.
First start mixing up the epsilon cap lactone and L Lactide copolymer. This takes 60 to 90 minutes to fully dissolve. Meanwhile, take a sheet of polyglycolic acid from the freezer and cut several five by eight millimeter sections from it.
Next, cut the filter off of a 10 microliter pipette tip and insert a 19 gauge needle into its distal end. Using forceps, wrap a piece of polyglycolic acid felt around the needle. Then using a blunted 18 gauge needle, carefully push the felt into the distal end of the pipette tip around the 19 gauge needle.
When the copolymer solution is ready, load 40 microliters into the top of the pipette tip to saturate the polyglycolic acid felt with solution. Push out the air bubbles with a pipetter until all the air bubbles are removed. Transfer all the prepared grafts to a 50 milliliter tube with the needle heads down and put them at minus 80 degrees Celsius for 20 minutes.
Then lyophilize the grafts under vacuum for 24 hours. With the tubes lid open the next day, isolate the grafts from the needles. Cut both ends of the graft for a clean four to five millimeter section.
Then slip those grafts back around their needles so they retain their shape. Store the graft in a desiccate and the night before they are seated with cells. Store them under UV light in a biosafety hood from a euthanized mouse.
Collect all the femurs and tibia into a dish with 10 milliliters of RPMI cut both ends off each bone. And in a second dish, flush out the marrow by injecting RPMI from a 25 gauge needle through all the bones. Use a total of three milliliters of RPM.
I collect all the bone marrow in RPMI into a single 15 milliliter tube and rinse the dish clean with an additional two milliliters of RPMI to make a total volume of five milliliters. Count the cells in this collection. Next, load five milliliters of fial into a 15 milliliter centrifuge tube, and slowly fill it with five milliliters of bone marrow.
With RPMI then spin the tube for 30 minutes at 528 G with the break off and at 24 degrees Celsius. Collect the middle layer from the tube it contains the mononuclear cells. Dilute this layer one-to-one in PBS.
Spin down the cell solution and resuspend the pellet in five milliliters of PBS. Then repeat the spin. Dilute the cell pellet in about 200 microliters of RPMI and count the cells twice.
Use the average value to concentrate them to 1 million cells per 10 microliters of RPMI for the graft. Use a six to eight week old C 57 black six female. Weigh the mouse and apply ketoprofen as a pre-anesthesia analgesic.
Then anesthetize it with a lower quadrant injection. After confirming the anesthesia with a toe pinch, clip the abdominal hair, lubricate the eyes and position the mouse for surgery on a pad wrapped with a sterile poly lined towel. Wipe down the surgical area with three alternating scrubs of betadine and alcohol.
Then drape the mouse, leaving only the abdomen exposed. Begin the surgery with a midline laparotomy. Make an incision from below the xiphoid to the supra pubic region.
Next displace and wrap the intestines with a saline moistened gauze. Then perform a blunt dissection of the clear tissue from the infrarenal aorta and vena cva. Clamp the distal sides and the proximal sides of the aorta and vena cva.
Once clamped bluntly, separate the two vessels. Now proceed with the implantation. Now transect the vena cva and if needed, ligate the abdominal aortic branches with 10 knot monofilament.
Suture on tapered needles prior to the transect throughout the implantation. Frequently flush the graft with heparin solution to prevent thrombosis. The first two sutures on both sides of the graft are the most important steps of the entire process.
When they are not placed carefully, it is difficult to separate the front and back layers of the IVC. If the layers are not clearly identified, there's a higher chance of accidentally suturing them together. Trim off the extra graft material and secure the graft with stitches at both ends.
Add four or five stitches using 10 t. Suture along the front side of the graft. Flip it over and do the same along the backside with the implantation complete.
Remove the proximal clamp and control the hemorrhaging with an absorbable hemostat agent. When the hemorrhage is completely finished, remove the distal clamp and repeat the hemorrhage control. Make certain that blood is flowing through the graft before up the animal.
Using six au black poly acrylamide monofilament sutures. Close the skin in two layers. Now inject a half milliliter of saline subcutaneously to prevent dehydration.
Then transfer the mouse to a recovery cage with a warm pad When ambulatory transfer the mouse to its home cage and provided ibuprofen for 48 hours. An SEM of the scaffold shows that the internal diameter is about one millimeter and the wall thickness is about 0.17 millimeters. The porosity of the scaffold was 78.5%with a mean pore size of 45 microns.
Right after implantation, the tissue engineered vascular graft was viewed under a microscope. Two weeks later, it was viewed again. It's still held its form.
It is clear that the graft was degrading and gradually replaced with new tissue. During the two weeks cell seeding in fact improved graft patency by 25%the graft was removed for histological analysis. H and d staining revealed neo tissue throughout the scaffold, a stain with hearts and masson's.
Trireme showed elastin in the intimal layers and showed collagen in the medial layers. CD 31 staining revealed an endothelial cell lining in the inal layer. Alpha SMA staining showed that the graft was also populated with confluent smooth muscle.
Even macrophage infiltration was evident as seen by F four 80 staining. At the conclusion of this video, you should have a better understanding of how to manufacture a small scale tissue engineered vascular graft, including the methods for manufacturing a small scale biodegradable scaffold methods for isolating bone marrow derived mononuclear cells and seeding them onto the scaffold. And finally, we should demonstrate to you the methods for using microsurgical techniques to implant the tissue engineered vascular graft in the mirroring model.
Once mastered, this technique can be done in 30 to 45 minutes. Other methods like aortic interpretation graft implantation can be performed afterwards to investigate the development of tissue engineered vascular grafts for coronary artery bypass surgery. While attempting this procedure is important to remember to keep the surgical area moisturized and flush the graft frequently to prevent acute thrombosis, These techniques paved the way for the researchers are in the field of tissue engineering to explore formation of nail tissue and prevention of stenosis in tissue engineered vascular graft.
