Kloxin, April M.Fibrosis is a common and frequently life-threatening condition characterized
by a buildup of stiff scar-like tissue that affects a variety of organs throughout the
body and impacts millions of people every year. Fibroblast activation is a key event in
fibrosis progression and is the subject of widespread study in vivo and in vitro. In
healthy tissues, fibroblasts are responsible for tissue maintenance through protein
secretion and matrix remodeling. In fibrotic tissues, fibroblasts are persistently
activated, meaning they respond to changes in the microenvironment including the
extracellular matrix (ECM) through overactive protein secretion and adoption of a
contractile phenotype, disrupting matrix architecture, and causing stiff scar-like tissue
to form. Fibrosis is understood to be the result of a feedback loop in which fibroblasts
activate and create a fibrotic environment, and signals from that environment, like
increased tissue stiffness, cause fibroblasts to persist in an activated state. If it were
possible to disrupt this maladaptive feedback loop, it may be possible to halt the
progression of fibrosis or even reverse its effects. ☐ Toward this goal, a variety of in vitro cell culture models have been developed
to study cell-matrix interactions that contribute to fibroblast activation. These models
are formed from a wide range of materials, use different methods of promoting
fibroblast adhesion and promoting integrin binding, and culture fibroblasts in different
cell culture geometries. Given this diversity, approaches are needed for comparison of
different integrin-binding motifs and culture geometries for further insights into the
drivers of fibroblast activation and improved designs of materials systems for studying
and regulating these processes. In this thesis, I first review different types of hydrogel
in vitro cell culture models that have been essential for understanding fibroblast
activation. I then present new strategies for probing fibroblast response to different
integrin binding motifs (whole proteins and integrin binding peptides) and different
cell culture geometries (2D, 2.5D, and 3D geometries). I also present work
investigating the use of a new boronic acid-based dynamic covalent material to enable
fibroblast co-culture with other cell types. Throughout this work, pulmonary
fibroblasts are used as a model fibroblast cell type and are of particular relevance for
insights into lung fibrosis. The overall goals of this work are to understand how cell
matrix interactions affect fibroblast activation and how the design of in vitro cell
culture models affects fibroblast phenotype to enable comparisons of results among
different models. ☐ First, for comparing fibroblast activation in response to different integrin
binding motifs, I developed a technique for conjugating whole proteins to step growth
hydrogel surfaces. This method enabled a side-by-side comparison of two protein
peptide pairs commonly used in hydrogel in vitro models and relevant to fibroblast
activation: collagen I and GFOGER, and fibronectin and PHSRN RGDS.
Interestingly, significant differences were observed between collagen I and GFOGER,
where the peptide promoted increased fibroblast activation and the formation of
clusters reminiscent of activated foci observed in fibrotic tissue. ☐ Next, towards bridging the gap between traditional 2D culture and
multidimensional in vivo microenvironments, I developed a technique for culturing
fibroblasts in a layered hydrogel geometry referred to as 2.5D cell culture. This
geometry mimics aspects of two common cell culture geometries, cells on top of
hydrogels (2D culture) and cells encapsulated within hydrogels (3D culture), by
allowing cells to initially spread without the requirement of matrix degradation while
also reducing asymmetric cell polarization. Cell activation (alpha smooth muscle actin
(αSMA) positive cells) was high and similar between the 2D and 2.5D geometry but
less in the 3D geometry. Further, gene expression levels of CDH11, ITGB1, and
Serpine 1, genes associated with cell-cell and cell-matrix interactions, were similar in
2D and 2.5D but lower in 3D culture. Interestingly, differences were observed in YAP
nuclear localization between fibroblasts cultured in 2D and 2.5D cell culture. These
results highlight the important role hydrogel geometry plays in directing fibroblast
activation and mechanotransduction. ☐ Given the complicated interplay between matrix modulus, cell confinement,
and cell polarization in multidimensional cell culture, new materials that allow for
dynamic cell-matrix and cell-cell interactions would be useful and complement more
traditional materials for 3D cell culture (e.g. cell-degradable, covalently-crosslinked
hydrogels). To achieve this, I established approaches for using boronic acid-based
hydrogels formed using dynamic covalent chemistry for 3D culture of fibroblasts and
other cells of interest in the lung microenvironment, such as disseminated tumor cells.
Good cell viability was observed in these materials over time, and the self-healing
nature of these hydrogels was utilized for the construction of more complex
geometries for dynamic co-culture of fibroblasts and breast cancer cells. ☐ Collectively, this work provides context for the interpretation of fibroblast
behavior in common in vitro cell culture models by demonstrating the effects of
integrin binding motifs and cell culture geometry on fibroblast activation. These
studies lay the groundwork for future investigations into the mechanisms by which
extracellular cues regulate fibroblast behavior, towards a better understanding
fibroblast activation in vivo. Additionally, dynamic-covalent materials, such as the
boronic acid-based hydrogels established here, could be useful for investigating the
role of network structure on fibroblast behavior in 3D cell culture.University of Delaware, Department of Chemical and Biomolecular EngineeringPh.D
