Date of Award

January 2015

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Christopher K. Breuer

Second Advisor

Edward L. Snyder

Subject Area(s)

Medicine, Biomedical engineering, Biology

Abstract

Objective: Tissue engineered vascular grafts (TEVGs) are useful in the surgical treatment of congenital heart defects. The development of TEVGs requires seeding of scaffolds composed of biodegradable polymers with bone marrow-derived mononuclear cells (BM-MNCs). The most common method used to isolate BM-MNCs involves density centrifugation in Ficoll. This process requires an International Organization for Standardization (ISO) class 7 clean room, is labor intensive, time intensive, and susceptible to operator variability. A recently developed filtration-based method for BM-MNC isolation uses a closed, sterile, and disposable system that removes the need for a clean room, decreases processing time, and is operator-independent. This study compared the efficacy of each method of BM-MNC isolation by evaluating the viability of cells recovered using each method, and by assessing the biologic and structure equivalence between neo-vessels created from scaffolds seeded using cells isolated by either method.

Methods: BM-MNCs were isolated from the bone marrow of immunocompetent syngeneic C57BL/6 wild type mice by either density centrifugation in Ficoll or using a filter-based method. The cells were seeded onto scaffolds fabricated from a polyglycolic acid (PGA) mesh coated with a 50:50 copolymer sealant of poly-L-lactide-co-ε-caprolactone. Seeded scaffolds were incubated overnight and then implanted as inferior vena cava (IVC) interposition grafts in 10-week-old wild type mice (n = 23 for each group). Grafts were explanted at 2 weeks post-implantation for analysis.

Results: Significantly greater total (filter: 44.3 ± 12.6x106 cells/mouse versus density centrifugation: 24.8 ± 8.8x106 cells/mouse, p=0.02) and viable (filter: 32.8 ± 6.7x106 cells/mouse versus density centrifugation: 20.6 ± 8.7x106 cells/mouse, p=0.04) BM-MNCs were isolated using filtration versus density centrifugation-based isolation. There was no significant difference in graft patency (filter: 78% patency versus density centrifugation: 87% patency, p=0.7), luminal diameter (filter: 633 ± 131 μm versus density centrifugation: 620 ± 82.9 μm, p=0.72) or neointimal thickness (filter: 37.9 ± 11.2 μm versus density centrifugation: 37.9 ± 7.8 μm, p=0.99) between groups at explantation. There was also no significant difference in quantitative macrophage infiltration between the two methods at explantation (filter: 1887 ± 907.7 cells/mm2 versus density centrifugation: 2041 ± 1078 cells/mm2, p=0.59).

Conclusion: BM-MNCs isolated using density centrifugation or the filter-based method were biologically equivalent and TEVGs formed from scaffolds seeded by each method were structurally similar when examined up to 14-days post-implantation in our in vivo murine model.

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