(567d) Engineering of Implantable, Bi-Layered Tissue-Engineered Blood Vessels from Adult Bone Marrow Stem Cells

We developed a novel method for isolating functional smooth muscle cells from ovine bone marrow (BM-SMCs) using tissue specific promoter. BM-SMCs exhibited the morphology of V-SMCs and expressed of smooth muscle á actin, calponin and myosin heavy chain as shown by immunofluorescence staining and western blot. The BM-SMCs could be easily and rapidly expanded for more than 10 passages with no signs terminal differentiation, providing nearly unlimited cell source for tissue engineered blood vessels. Furthermore we engineered blood vessels (BM-TEVs) using the BM-SMCs as the cell source and fibrin hydrogel as scaffold. BM-TEVs demonstrated significant mechanical strength and vasoreactivity to potassium chloride (KCl) and norepinephrine (NE) and most important, they were implantable into the jugular vein of an ovine animal model. The strength of BM-TEVs was further enhanced by engineering bi-layered tissues. Specifically, the inner layer of TEVs was composed of high concentration of fibrin hydrogel (10-30mg/ml) to provide mechanical strength and the outer layer consisted of BM-SMCs embedded into low concentration fibrin (2.5 mg/ml) to provide compaction and vascular reactivity. BM-SMCs in the inner layer of bi-layered BM-TEVs were distributed in the fibrin gel uniformly and aligned circumferentially around the silastic tube. Vasoreactivity measurement showed the bilayered TEVs retained high reactivity to KCl, independent of the concentrations of fibrin hydrogel in the inner layer. Notably, the strength of the bi-layered TEVs enhanced significantly. Specifically, burst pressure increased by more than 10-fold as compared to single layered TEVs, suggesting that these tissues may withstand implantation. Indeed, implantation into the jugular veins of eight-week old lambs showed that BM-TEV remained patent. At 5-8 weeks post-implantation, explanted BM-TEVs displayed a confluent endothelial monolayer, circumferential alignment of smooth cells in close proximity to the lumen and remarkable matrix remodeling. Specifically, BM-TEVs showed high levels of collagen and most importantly, elastin that was organized in well-defined fibers very similar to native veins. Our results demonstrate that BM-derived progenitor cells can be used to engineer implantable TEVs thus providing an unlimited supply of highly proliferative, autologous cells for cardiovascular tissue engineering.