Date of Award

6-3-2009

Document Type

Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Christopher Breuer

Abstract

Purpose: Investigate vascular neotissue formation in vivo within human tissue-engineered arterial grafts (TEAG) implanted in an immunodeficient (SCID/bg) mouse model. Methods: Human aortic smooth muscle cells (hASMC) and endothelial cells (hAEC) were statically seeded onto porous biodegradable polymeric scaffolds. These two-cell tissue-engineered vascular grafts were implanted into immunodeficient female mice as aortic interposition grafts. Grafts were evaluated over a thirty-week time course to investigate their patency, structure, and histological appearance. Species-specific immunohistochemical stains were used to determine the cellular composition of developing TEAG. In order to understand seeded cell retention at early time points, human aortic smooth muscle cells (hASMC) were labeled with superparamagnetic iron oxide (SPIO) nanoparticles and grafts were imaged in vivo with spin and gradient echo imaging sequences. Setting: In vivo animal study. Subjects: 13 female C.B-17 SCID/bg mice. Interventions (if any): TEAG implanted as infra-renal abdominal aortic interposition grafts. Main Outcome Measures: Selective micro-computed tomography with intra-arterial contrast revealed graft patency and structure. Histological and immunohistochemical evaluations revealed cellularity and ECM composition. Species-specific immunohistochemistry determined the source of cells within the developing TEAG. Magnetic resonance images of SPIO-labeled grafts verified the presence of labeled hASMC at early time points (weeks 1-3). Results: All TEAG were patent without evidence of thrombosis or graft rupture over the 30-week time course. Histological and immunohistochemical analysis revealed a monolayer of vWF-positive cells lining the inner lumen, surrounded by concentric collagen-rich layers of α-SMA-positive cells. Species-specific immunohistochemical analysis demonstrated progressive loss of hAEC and repopulation of the luminal monolayer by mouse endothelial cells, but maintenance of the hASMC. Histological morphometry demonstrated evidence of remodeling with initial luminal narrowing followed by dilation. Seven of the 13 grafts included labeled hASMC, which were incorporated into tissue-engineered vascular grafts during in vivo development. Magnetic resonance imaging of the labeled vascular grafts over the first three weeks demonstrated that the hASMC persisted within the neo-media of the tissue-engineered vessels; this observation was confirmed by Prussian blue histology. Conclusions: Over thirty weeks, we see evidence that these grafts not only remain patent, but also develop into functional vascular neo-tissue histologically similar to that of native aorta. In part, their development includes integrating seeded human aortic smooth muscle cells, whose presence was confirmed with SPIO labeling and MRI, as well as Prussian blue histology. This chimeric animal model, having enabled determination of seeded cell retention through histology and MR labels, provides insight into cellular mechanisms underlying neotissue formation and is therefore a valuable tool for investigating TEAG development.

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