Comparison of the biological equivalence of two methods for isolating bone marrow mononuclear cells for fabricating tissue-engineered vascular grafts.

ABSTRACT

Our approach for fabricating tissue-engineered vascular grafts (TEVG), applied in the surgical management of congenital heart disease, is accomplished by seeding isolated bone marrow-derived mononuclear cells (BM-MNCs) onto biodegradable scaffolds. The current method used for isolation of BM-MNCs is density centrifugation in Ficoll. This is a time-consuming, labor-intensive, and operator-dependent method. We previously demonstrated that a simpler, faster, and operator-independent method for isolating BM-MNCs using a filter elution technique was feasible. In this study, we compare the use of each technique to determine if the BM-MNCs isolated by the filtration elution method are biologically equivalent to BM-MNCs isolated using density centrifugation. Scaffolds were constructed from a nonwoven poly(glycolic acid) fiber mesh coated with 5050 poly(l-lactide-co-ɛ-caprolactone) sealant. BM-MNCs were isolated from the bone marrow of syngeneic C57BL/6 mice by either density centrifugation with Ficoll or filtration (Ficoll vs. Filter), then statically seeded onto scaffolds, and incubated overnight. The TEVG were implanted in 10-week-old C57BL/6 mice (n=23 for each group) as inferior vena cava interposition grafts and explanted at 14 days for analysis. At 14 days after implantation, there were no significant differences in graft patency between groups (Ficoll 87% vs. Filter 78%, p=0.45). Morphometric analysis by hematoxylin and eosin staining showed no difference of graft luminal diameter or neointimal thickness between groups (luminal diameter, Ficoll 620.3±82.9 µm vs. Filter 633.3±131.0 µm, p=0.72; neointimal thickness, Ficoll 37.9±7.8 µm vs. Filter 37.9±11.2 µm, p=0.99). Histologic examination demonstrated similar degrees of cellular infiltration and extracellular matrix deposition, and endothelial cell coverage on the luminal surface, in either group. Macrophage infiltration showed no difference in the number of F4/80-positive cells or macrophage phenotypes between the two experimental groups (Ficoll 2041±1048 cells/mm(2) vs. Filter 1887±907.7 cells/mm(2), p=0.18). We confirmed the biological equivalence of BM-MNCs, isolated using either density centrifugation or filtration, for making TEVG.