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 50:50 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.

Figures

FIG. 1.

Comparison of isolated bone marrow-derived mononuclear cells (BM-MNCs) and cell attachment after seeding. (A) Total and viable cell count of BM-MNCs was performed by trypan blue staining with manual cell count. The filter method isolated significantly more BM-MNCs in both total and viable cells. Data were evaluated by Student t test. (B) Cell attachment on the graft was evaluated by PicoGreen DNA quantification after overnight incubation. The number of attached cells in the filter group was higher than that in the Ficoll group. Data were evaluated by Welch's t test.

FIG. 2.

Serial monitoring of graft patency and luminal diameter by ultrasonography. (A) Representative images of ultrasound. The graft luminal diameter was determined sonographically and patency was determined by measuring flow velocity proximal and distal to the graft. (B) Serial monitoring by ultrasound demonstrated no difference in graft patency and luminal diameter between groups at each time point. Color images available online at www.liebertpub.com/tec

FIG. 3.

Comparison of quantitative morphometric analysis of tissue-engineered vascular grafts (TEVG). (A) Representative scanning electron microscope images of scaffolds before cell seeding. (B) Representative hematoxylin and eosin (HE) staining images of patent and occluded TEVG. (C) The adventitia, media, and intima were manually identified and measured on HE staining, and patent was defined as greater than 50% in luminal diameter compared to graft at time of implantation. There was no statistical difference in patency of grafts at 2 weeks between the groups. Data were evaluated by Fisher's exact test. (D) Neither luminal diameter nor neointimal thickness differed between groups. Data were evaluated by Welch's t test. Color images available online at www.liebertpub.com/tec

FIG. 4.

Histological analysis of TEVG at 14 days after implantation. Histologic examination showed cellular infiltration into the TEVG, and robust deposition of collagen throughout the TEVG in both experimental groups, although there was no evidence of elastin. von Kossa stain revealed no instances of calcification in either group. Poly(glycolic acid) fibers, which may appear as vacuoles or capillaries, still existed in the TEVG and caused nonspecific staining of both von Kossa and Alcian blue stains. Arrows indicate representative remaining fibers on HE staining. Boxed area highlights the region of TEVG section corresponding to high-magnification sections in each group. Color images available online at www.liebertpub.com/tec

FIG. 5.

Immunohistological analysis of TEVG at 14 days after implantation. (Left) Smooth muscle cells were shown mainly in neointima of both groups. (Middle) Endothelialization of the luminal surface was found to line the luminal surface of the TEVG by anti-von Willebrand factor (vWF) antibody staining. (Right) Matrix metalloproteinase (MMP)-2 was positive around the remaining fibers in both groups. There was no difference in these results between the two groups. Color images available online at www.liebertpub.com/tec

FIG. 6.

Macrophage infiltration into TEVG evaluated by histological assessment and gene expression. (A) Representative images of immunohistochemical staining of F4/80 (entire macrophage), iNOS antibody (M1 macrophage), and CD206 (M2 macrophage). (B) The number of macrophages in TEVG, defined by F4/80-positive cells, at 14 days after implantation showed no difference between groups. Data were evaluated by the Mann–Whitney test. (C), Gene expression was analyzed by reverse transcription quantitative polymerase chain reaction using the ΔΔCT method. Gene expression of monocyte/macrophage marker (CD11b) demonstrated that there was no difference between the two experimental groups in each time point “before seeding” and “14 days after implantation” Furthermore, there was no difference in gene expression of macrophage phenotype (M1: iNOS and M2: CD206) between groups at 14-day time point. Data were evaluated by Student's t test. iNOS, anti-inducible nitric oxide synthase. Color images available online at www.liebertpub.com/tec