Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

My research area is cardiovascular biomaterial, and regenerative medicine. We are trying to solve the problem of low patency of small-diameter vascular grafts.

The bio tubes developed recently are composed of polymer fiber skeletons and animal extracellular matrix, and are currently in phase three clinical trials.

Currently, the cell sources and the mechanism involved in vascular regeneration are not clear and require proper research studies to fairly understand these processes. I have fabricated the small diameter vascular grafts, modified with various bioactive using the electrospinning technique, which has improved their patency rates in diseased animal models. The technique is easy to operate and can improve their efficiency and the success rate.

In the future, our research group will focus on the mechanism of action of vascular stem cells in maintaining the vascular patency and the process of vascular regeneration.

[Narrator] To begin, load the polycaprolactone or PCL solution into a 10 milliliter syringe and position it with a 21 gauge stainless steel needle. Place a tungsten steel mandrel measuring 0.7 millimeters in diameter and 20 centimeters in length on the collection instrument. Fabricate nine groups of PCL vascular grafts using the electrospinning technique. Connect a high voltage power supply to the needle, then position a tungsten steel rod with an inner diameter of 0.7 millimeters at a fixed distance in front of the needle to serve as a receiving device. Using a one milliliter pipette, discard the medium from the culture and wash the cells with PBS. Now add two milliliters of fresh medium to the culture flask and gently scrape the surface with a cell scraper. Transfer the collected cells to a tube and centrifuge at 1000 G for five minutes at room temperature. Then resuspend five times 10 to the power of six cells in 100 microliters of complete medium. Next, place a PCL vascular graft measuring one centimeter in length in a 15 milliliter tube filled with DMEM. Centrifuge the tube at 4,000 G for five minutes. Then prepare a 10 centimeter Petri dish lined with a layer of filter paper. Place the moistened PCL vascular graft on the filter paper and roll it to remove excess medium. Now, using a P10 pipette, take 10 microliters of the cell suspension and inject the suspension into one end of the vascular graft. Rotate the vascular graft over the filter paper to ensure uniform distribution of the suspension. Place the macrophage loaded vascular graft in a 24 well plate containing one milliliter of complete medium. Incubate the plate for two hours in a cell culture incubator before implantation. Position the anesthetized mouse supine on the operating table. After making a midline incision, elevate the left salivary glands and excise the left cleidomastoid muscle to improve the surgical field of view. Then expose the left common carotid artery using micro tweezers. Now isolate the left carotid artery using micro forceps. Ligate the carotid artery in two locations at the mid portion using a 9-0 suture and transect the artery between the two ligatures using micro scissors. Pass the cuff through the arteries at each end. Then secure the artery and cuff together using arterial clips. Next, turn the artery outward to cover the cuff body and secure the artery to the cuff using a 9-0 suture with micro forceps. Carefully implant a vascular graft between the two ends of the carotid artery and slide the graft ends over the artery cuff. Then secure the grafts with 9-0 sutures. Next, remove the arterial clips at both ends before irrigating the implantation site with saline. Then observe distal arterial pulsation to assess the patency of the vascular graft. Finally, reposition the left salivary gland and close the surgical site using 6-0 sutures. Small diameter vascular grafts exhibited a uniform fiber distribution with irregular arrangement and the presence of pore structures. As the PCL concentration increased, both fiber diameter and pore size increased. Mechanical testing showed that all vascular grafts met the required mechanical standards. The maximum load and strain at break increased with higher PCL concentrations and greater vascular wall thickness, while the modulus of elasticity decreased. Macrophages were successfully implanted into vascular grafts using the perfusion adsorption method and their uniform distribution was confirmed by scanning electron microscopy. Cross-sectional imaging showed that the macrophages infiltrated from the lumen into the graft wall. After 30 days of in vivo implantation, the survival rate was 100% and two out of three vascular grafts remained patent with no aneurysms or fibrous encapsulation. Hematoxylin and eosin staining demonstrated substantial cellular infiltration and neo tissue formation within the graft lumen CD31 immunofluorescence staining confirmed endothelial tissue regeneration with intact endothelial cell coverage.