(L-442) In Vitro Testing of Tissue Engineered Small-Diameter Vascular Grafts for Diabetic Patients

Introduction::

Peripheral arterial disease (PAD) involves arterial narrowing or blockages that restrict blood flow to a patient's limbs. Current treatments for PAD include bypass surgery using the patient's arteries or veins as a graft. However, 1/3 of patients do not have adequate blood vessels, and diabetic patients exhibit chronic hyperglycemia, which leads to cross-linking and stiffening of the extracellular matrix, affecting both the natural arteries and the implanted grafts. Therefore, developing a tissue-engineered vascular graft for diabetic patients could improve the quality of life of patients with PAD.

This study aims to demonstrate the successful creation of living grafts using decellularized porcine carotid arteries seeded with human umbilical vein endothelial cells (HUVECs) to prevent the activation of the coagulation cascade and subsequent clotting and blockages upon implantation. HUVECs were seeded onto the lumen by infusion, and the arteries were placed into a sterile vascular bioreactor that mimics physiologically relevant pulsatile pressure and flow conditions of an artery. Initial Hhistology confirmed the presence of HUVECs on the lumen after two weeks in the bioreactor. While preliminary data are promising, future improvements in endothelialization techniques, successful seeding of vascular fibroblasts and smooth muscle cells, and testing in diabetic media are needed before preclinical testing.

Materials and Methods::

Fresh porcine carotid arteries were rinsed with sodium dodecyl sulfate to lyse cell membranes and remove all cells from the extracellular matrix (ECM). Complete decellularization was confirmed through DNA quantification and histological analysis. Cell removal significantly reduces the graft’s antigenicity while preserving the ECM. Arteries were then sterilely mounted and treated overnight in a solution of fibronectin and PBS, followed by an overnight treatment in a solution of Fetal Bovine Serum and Dulbecco's Modified Eagle Medium to improve cell adhesion. Arteries were then infused with 7 million Human Umbilical Vein Endothelial cells (HUVEC’s) over the course of 2 days. Immediately following infusion, they were placed onto a rotator overnight to allow even attachment of HUVEC’s to the lumen. A bioreactor to simulate physiologically relevant pulsatile pressures and flow conditions was then constructed and arteries were placed inside. The bioreactor was slowly brought up to its full flow level in 12-hour increments and ran for 2 weeks. Pulsatile pressures and flow were continuously monitored and maintained during this period. After 2 weeks, the arteries were removed and analyzed through histological staining to observe the presence of HUVEC’s.

Results, Conclusions, and Discussions::

Decellularized (see Figure 1) and fresh samples (see Figure 2) were histologically analyzed using H&E. The absence of cells within the extracellular matrix can be observed in Figure 1. Further validation of decellularization included DAPI nuclear staining, DNA quantification using spectrophotometry, and DNA gel electrophoresis. Following removal from the bioreactor, arteries were analyzed using DAPI (see Figure 3) and H&E histological staining. The presence of HUVEC’s can be seen around the lumen. This supports the claim that tissue engineered vascular grafts can be successfully endothelialized and that endothelialized acellular grafts can withstand physiological flow and pressures.

By proving successful endothelialization, there is improved likelihood that tissue engineered vascular grafts could be translational with utility in future clinical applications. Future research will look to reinfuse arteries with adventitial fibroblasts and smooth muscle cells alongside endothelial cells to help this study move towards in-vivo animal testing. The effects of diabetic conditions on tissue engineered vascular grafts long term also stands to be investigated.

Acknowledgements (Optional): : I would like to acknowledge Dr. Dan Simionescu, Dr. Agneta Simionescu, Dr. Juan Carlos Carillo Garcia, Alex Carter, Joshua Wingold, and all other members of the BTRL Lab for their support of this project.

References (Optional): :