Author ORCID Identifier
Date Available
8-7-2024
Year of Publication
2023
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Engineering
Department/School/Program
Chemical and Materials Engineering
Advisor
Dr. Bradley J. Berron
Abstract
Volumetric muscle loss (VML) remains one of the few skeletal muscle injuries without a reliable and repeatable treatment. In large volume muscle injuries, muscle fibers as well as the surrounding connective tissue are damaged, preventing therapeutic muscle stem cells, called myogenic progenitor cells (MPCs), from reaching the injury site and initiating repair. There is a clinical need to rapidly fabricate in vitro muscle tissue constructs that mimic the native tissue organization, with aligned myotubes, for insertion and integration at the patient’s injury site. In this dissertation, we utilize the MPC’s natural propensity to close gaps across an injury site to guide alignment on collagen I, an extracellular matrix (ECM) protein substrate. Through the binding affinity of biotin and streptavidin molecules, we pre-position MPCs in temporary straight-line patterns separated with small gaps, mimicking small-scale muscle injuries. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps. Additionally, we have developed a novel technique to wash the substrate once it is seeded with cells using centrifugation to maximize the cells adhered in the desired patterned structures, while minimizing the number of cells adhered in the gaps between them. The patterning system we have designed allows for long-range myotube ordering, confirmed through the expression of myosin heavy chain (MyHC).
The next part of this dissertation expands our work on cell migration and adhesion to study the properties of 3 pairs of epithelial cancer cell lines (breast, endometrium, tongue) with varying metastatic potential. According to the epithelial-to-mesenchymal transition, cancer cells lose their cell-cell and cell-matrix adhesion junctions as they become more metastatic, contributing to increased detachment from the primary tumor and a migratory response into nearby tissue and vasculature. Current in vitro strategies for characterizing a cell’s metastatic potential heavily favor quantifying the presence of cell adhesion biomarkers; however, little attention is placed on quantitative analysis of metastatic function across well-known cancer cell lines. To place cell lines more accurately within the spectrum of metastasis, we deploy two functional metrics of cancer aggression (wound closure migration velocity and cell detachment from a culture surface) on each cell line. We demonstrate that there is not a relationship between cell migration velocity and metastatic potential. Additionally, we test cell adhesive function through a shear flow assay to confirm that cell lines with higher metastatic potential had greater detachment from a culture surface compared to low metastatic potential cell lines in homogenous populations. Normalizing the data to the highest value within each metric reveals that cell lines with low metastatic potential rank higher wound closure migration velocity while cell lines with high metastatic potential rank higher in detachment from a culture surface.
Finally, this dissertation concludes with a study initiated during the COVID-19 pandemic to help local Kentucky distillers transition between hand sanitizer and spirits production. Hand sanitizer introduces the bitter tasting compound denatonium benzoate to production equipment, requiring appropriate cleaning solutions for removal to prevent changes in spirit flavors. Materials compatibility with high proof ethanol dominant in hand sanitizer plays a large role in the effectiveness of the tested cleaning solutions. Metals and rigid polymers do not retain denatonium benzoate due to being compatible with high proof ethanol, so they can be cleaned easily using any of the cleaning solutions. However, elastomers permeate denatonium benzoate into and out of the material due to their poor compatibility with high proof ethanol. Ethanol proves to be a useful cleaner, but can cause irreversible damage to these materials over time.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2023.344
Funding Information
Lauren E. Mehanna Funding:
National Science Foundation Graduate Research Fellowship Program (GRFP), Grant No. 1839289, 2019-2022
Berron and Grady Lab Funding:
University of Kentucky, Igniting Research Collaborations (IRC) Grant, 2018-2020
National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health (NIH), Grant No. UL1TR001998, 2020-2021
University of Kentucky COVID-19 Unified Research Experts (CURE) Alliance, 2020-2021
National Science Foundation CAREER Award, Grant No. 2045853, 2019-2023
National Institute of Health (NIH), Grant No. P20GM130456 and R03DE029547, 2019-2023
Recommended Citation
Mehanna, Lauren E., "LEVERAGING CELL-SUBSTRATE ADHESION AND CELL MIGRATORY PROPERTIES IN SKELETAL MUSCLE CONSTRUCTS AND CANCER METASTASIS ASSAYS" (2023). Theses and Dissertations--Chemical and Materials Engineering. 153.
https://uknowledge.uky.edu/cme_etds/153
Included in
Biochemical and Biomolecular Engineering Commons, Biomaterials Commons, Molecular, Cellular, and Tissue Engineering Commons, Translational Medical Research Commons
