Date of Award

9-22-2010

Document Type

Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Christopher Breuer

Abstract

Significance: Each year approximately 10,000 children undergo operative repair of congenital cardiac anomalies. Long-term outcomes of these procedures, however, are limited by the lack of growth potential in the prosthetic vascular grafts currently used. Tissue engineered vascular grafts (TEVGs), created by seeding biodegradable polymer constructs with autologous cells, are attractive because of their ability to grow, repair, and remodel. As TEVGs evolve, there is a demand for non-invasive imaging modalities to evaluate graft structure and function. Aim: The aim of this project is to develop a novel label for mononuclear cells that would enable high definition Magnetic Resonance Imaging (MRI) analysis of tissue engineered vascular grafts. Methods: Human bone marrow mononuclear cells (hBM-MNCs) were incubated with a FITC labeled nanoparticulate dendrimer compound encapsulating a gadolinium core. Incubated cells were analyzed for label uptake and viability via fluorescent spectroscopy (480-520 nm) and FACS analysis (FITC and 7-AAD). PLLA/PCLA scaffolds seeded with labeled cells were imbedded in a 10 percent w/v gelatin suspension for MR evaluation. T1 and T2 images were acquired utilizing RARE and MSME sequences. Acquired images were evaluated qualitatively for increase in T1 and T2 signal. Results: Compound uptake by hBM-MNCs was proportional to compound concentration gradient and time of incubation. FACS analysis revealed a decrease in the size and granularity gated population and an increase in the apoptotic marker 7-AAD at higher concentrations (above 250 mcg/ml) demonstrating a decreasing 3 viability. Dendrimer compound embedded in gelatin displayed T1 signal intensity that correlated with increased compound concentrations, however labeled cells in DMEM suspension and labeled seeded cells on P(CL/LA) grafts displayed variable T1 intensities that were not correlative to compound concentration except when seeded cell numbers were increased to 10 x 106. Conculsion: The increased T1 signal intensity of label suspended in DMEM, coupled with high cell viability at lower concentration ranges, suggests gadolinium-dendrimer labeling is a viable model for non-invasive in vivo evaluation of tissue engineered conduits, however the lack of significant increases in T1 intensity of labeled cells suggest further investigations are needed to address intracellular compartmentalization and signal quenching. Clinically, with further modifications, this novel contrast agent can increase the ability to evaluate graft patency and morphology by clearly defining graft borders, providing non-user dependent reproducible images of high resolution as well potentially provide a mechanism in which to identify grafts at high risk for stenosis.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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