Date of Award

2019

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Michael J. Yost

Second Advisor

Robin Muise-Helmericks

Third Advisor

Carl Atkinson

Fourth Advisor

Stephen Fann

Fifth Advisor

Satish Nadig

Abstract

Despite a strong clinical demand for tissue replacement therapies, few tissue-engineered constructs (TECs) have attained FDA approval. Fewer still demonstrate long term viability of implanted cells, with root causes of failure of these devices identified as poor cell retention, poor vascularization, and inflammation following implantation. Focusing on the first two of these issues, we attempt to create a rapidly vascularizable TEC by optimizing a novel vascular implant model developed in our laboratory: the scaffold-free, prevascular endothelial-fibroblast construct (SPEC). The optimization process calls on a hybrid in vivo, in vitro, and in silico approach. We first developed an in vivo temporal model of TEC vascularization by comparing endothelial invasion, cord development, anastomosis, and vessel maturation dynamics of SPECs to avascular grafts such as fibroblast-only spheroids and silicone implants. While the existing microvessel architecture of the SPECs confers an advantage in anastomosis and endothelial infiltration of an implant in the first 12 hours post-implantation, poor lumen patency limits the rate of vessel development in the TECs. Perfusion is apparent at later time points (24-72 h) in both SPECs and fibroblast-only spheroids. Analysis of in vivo vascularization dynamics is augmented by a control flow simulation model which reveals that delayed vascular development coincides with poor accumulation of pro-angiogenic factors such as VEGF. Our in vivo observations drove corrections of our SPEC model, with efforts undertaken to improve lumen formation during the in vitro development period. These approaches include pre-dosing implants with pro-angiogenic factors such as VEGF, inducing endothelial cell realignments in a perfusion chamber, and incorporation of perivascular cells to improve patency of forming tubes. Recombinant human VEGF165 (rhVEGF165) dosing was most consistently associated with increased formation of endothelial-lined lumens, with a dose (ranging from 0-50 ng/mL) and time dependent increase in diameters of these lumens during SPEC formation. Finally, we generated computational models of SPEC formation in a rhVEGF165 field in order to combine our observations of endothelial clustering behavior, SPEC reorganization, and dose/time dependent cord hollowing behavior observed in vitro with existing stochastic models of tissue assembly and cell-cell interface optimization. Through careful control of model parameters, we generated a list of in silico simulations to enable optimization of vascularization response, ultimately resulting in a list of candidate treatments built on the backbone of VEGF pre-dosing. This candidate list can serve as a starting point for future experiments, with a goal of rapid and stable lumen formation and blood perfusion.

Rights

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