Engineering Mechanically Dynamic Hyaluronic Acid Hydrogels to Investigate Stromal Cell Mechanosensitivity

The makeup and composition of the extracellular matrix (ECM), which supports tissues and organ structures, undergoes dynamic changes in response to tissue development, homeostasis, injury, and wound resolution. As such, there is growing appreciation towards understanding how changes to the ECM transmit to surrounding cells and affect subsequent cellular responses. This is of particular interest in the context of pulmonary fibrosis, a pathological disorder characterized by excessive ECM deposition by activated resident fibroblasts (myofibroblasts) following lung injury. The two current treatments are not curative, and more importantly, bear a 40% failure rate within one year of use due to extreme adverse side effects. The lack of effective therapies, coupled with the fact that fibrosis contributes to ~ 50% of deaths in developed countries, underscores a critical need to develop more effective treatments for this pathology. In vitro cell culture platforms provide a way to understand cell-ECM interactions responsible for fibrogenesis without extraneous biological processes and other cell types present in vivo. Specifically, hydrogel culture models closely mimic salient features of the native tissue microenvironment to study these interactions; however, many do not incorporate disease-relevant features like dynamic mechanics or stress relaxation, both of which have been found to be critical regulators of cell activation in lung fibrosis. Therefore, the objectives of this thesis were to design increasingly sophisticated mechanically dynamic hydrogels as models of normal and fibrotic lung tissue to investigate stromal cell mechanosensitivity and activation.

This thesis leverages various mechanically dynamic hyaluronic acid (HA)-based hydrogels as models of lung fibrosis to investigate how these cues regulate cell phenotype. Chapter 2 explores how standard culture practices of stromal cell expansion on tissue culture plastic (TCP) influence cell phenotype mechanosensitivity (e.g., spread area, elongation, focal adhesion formation and maturation) following in vitro culture on hydrogels modeling normal and fibrotic lung tissue. It also explores the influence of cell origin, either isolated directly from bone marrow (primary), or purchased from a cell line (immortalized). Chapter 3 utilizes in situ stiffening of the hydrogel to investigate how mechanical perturbations affect cell phenotype. This chapter also develops an appreciation for nuclear mechanosensing through chromatin condensation quantification. Chapter 4 discusses the development of a more complex mechanically dynamic hydrogel model incorporating viscoelasticity to assess similar cell behaviors as in Chapters 2 and 3, with the addition of another nuclear metric, an epigenetic event marked by DNA methylation. Lastly, Chapter 5 considers the design and synthesis of a mechanically cyclic hydrogel that models the tissue during homeostasis (compliant), injury (increased stiffness), and resolution (return to compliance) to better understand how prolonged mechanical dosing affects cell ‘mechanical memory’, a notion increasingly considered for its role in fibrogenesis. Collectively, this work presents the development of physiologically relevant hydrogel disease models to better understand how cell-ECM interactions regulate cell cytoskeletal and nuclear behaviors in lung fibrosis.