Development of tissue engineered blood vessels using cell-seeded acellular porcine arterial scaffolds

The patency rate of small diameter synthetic bypass grafts remains poor. The aim of this study was to develop a biocompatible, acellular, arterial scaffold, assess the scaffolds regenerative capacity using ovine vascular cells and begin preliminary studies of a vascular tissue bioreactor for the development of a tissue engineered graft for peripheral and coronary arterial bypass. Porcine carotid arteries were decellularised using a protocol developed at the University of Leeds. Arteries were incubated sequentially in disodium ethylenediaminetetraacetic acid, hypotonic solution, sodium dodecyl sulphate [0.1% w/v], DNAse and RNAse, hypertonic solution and 0.1% (v/v) peracetic acid. To ensure decellularisation, representative arterial histological sections were stained using haematoxylin and eosin and 4’,6-diamidino-2-phenylindole to confirm removal of cells and cell nuclei. The total DNA content of treated arteries was also determined. Biocompatibility of the acellular scaffolds was assessed using contact and extract cytotoxicity assays using both primary cells (porcine and ovine endothelial cells and smooth muscle cells) and two distinct cell lines (murine 3T3 and BHK cells). Ovine endothelial cells were harvested from the femoral arteries of sheep following digestion with collagenase. Ovine smooth muscle cells were isolated from ovine arterial explant cultures. To determine correct cell phenotype, immuno-staining was performed using a variety of primary antibodies to vascular cell markers by indirect immunofluorescence. Ovine vascular cells were then seeded onto the luminal surface of the decellularised vessels in both a two-dimensional and three-dimensional manner. A cell viability assay (Live / Dead Stain ®) was performed to confirm the viability of seeded cells. A vascular bioreactor was successfully assembled and preliminary sterility runs were performed in preparation for future scaffold pre-conditioning. The decellularisation protocol resulted in porcine carotid arteries that were free from cells with >90% of the total DNA being removed. The decellularised porcine carotid artery was not cytotoxic to any test cells. Indirect immunofluorescence performed on the harvested cells confirmed correct ovine endothelial cell and smooth muscle cell phenotype after cell isolation using magnetic bead separation. Ovine vascular cells were successfully seeded onto the luminal matrix of decellularised arteries in both two-dimensional and three-dimensional experiments. Seeded cells were viable at 48 hours post incubation. A vascular bioreactor was successfully assembled and was kept free from macroscopic microbial contamination for a maximum of fourteen days. In conclusion, porcine carotid arteries were successfully decellularised using an established decellularisation protocol. The remaining acellular scaffolds demonstrated capability in allowing the attachment and proliferation of xenogeneic vascular cells onto the scaffold surface. Further work developing a vascular bioreactor in order assess the functionality and ongoing cell viability of the seeded scaffold will be needed to assess the efficacy of decellularised porcine carotid artery as a viable conduit for arterial bypass