Fenghua Zhao

The extracellular matrix (ECM) is a complex and dynamic environment that regulates cellular behavior, tissue architecture, and disease progression through its biochemical and biomechanical properties. As a dynamic and tissue-specific scaffold, the ECM not only provides physical support but also influences vital processes such as cell differentiation, migration, and matrix remodeling. In fibrotic diseases and wound healing, changes in ECM mechanics are closely linked to pathological outcomes.

This thesis focuses on the development and application of organ-specific ECM hydrogels as advanced 3D culture systems that accurately mimic the native tissue environment. By tailoring the mechanical properties and composition of ECM hydrogels, we investigated their effects on endothelial cells, fibroblasts, and mesenchymal stem cells in the context of vascularization, scarring, and fibrosis. Through a series of experimental models, this work provides mechanistic insights into how ECM stiffness influences cellular responses, particularly ECM remodeling.

Chapter 2 introduces the development and application of organ-derived ECM hydrogels, emphasizing their biochemical and biomechanical capabilities for regulating cell behavior. Unlike 2D systems, these 3D hydrogels better replicate native tissue mechanics and support the continuous monitoring of ECM dynamics as carried out by the encapsulated cells. The mechanical properties of ECM hydrogels can be tuned to mimic both healthy and fibrotic conditions, making them powerful tools for studying cell–ECM interactions.

In Chapter 3, we investigated the mechanical differences of ECM hydrogels derived from skin, lung, and a hybrid mixture (1:1 mix), and their influence on vascular network formation (VNF) and ECM remodeling by endothelial cells. Physical characterization revealed distinct differences among the hydrogels in stiffness, viscosity, fiber diameter, pore size, and fiber density. Upon cell culture, skin-derived ECM hydrogels promoted VNF most efficiently compared to lung and hybrid hydrogels. Skin ECM hydrogels induced higher secretion of matrix metalloproteinase 1 (MMP1), while hybrid hydrogels were associated with increased MMP9 secretion and greater deposition of fibronectin and collagen IV. Principal component analysis highlighted that mechanical properties (e.g., stiffness, stress relaxation) exerted a more significant influence on VNF than biochemical composition. The study highlighted that the specific properties of ECM from different organs impact their ability to support VNF.

In Chapter 4, we explored how fibroblasts modulate the matrix to support angiogenesis in co-cultures of endothelial cells and fibroblasts within skin-ECM hydrogels. Fibroblasts increased hydrogel stiffness, fiber diameter, and porosity while promoting the deposition of fibronectin and fibulin-1 near endothelial cells. These findings suggest that fibroblasts can dynamically cooperate with endothelial cells and alter the ECM microenvironment to promote vascularization.

In Chapter 5, we aimed to modify the mechanical properties of lung-specific ECM hydrogels to mimic healthy and fibrotic environments. ECM hydrogels crosslinked with ruthenium (Ru) exhibited increased stiffness and decreased stress relaxation without alterations in composition. Analysis of fiber structures showed that crosslinked hydrogels featured higher fiber density and shorter average fiber length. When fibroblasts were cultured on these hydrogels, they exhibited myofibroblast differentiation, characterized by increased expression of α-smooth muscle actin and morphological changes. This system provided an in vitro model to study the mechanical effects of ECM during the progression of lung fibrosis.

In Chapter 6, we extended the Ru-crosslinking strategy to skin-derived ECM hydrogels. Using a custom-made mold and diffusion of ruthenium into skin-ECM hydrogels, we generated ECM hydrogels with a stiffness gradient that mimics the transition from normal skin to scar tissue in vivo. Along this gradient, higher-stiffness regions exhibited denser collagen and slower stress relaxation, requiring an additional Maxwell element during stress-relaxation curve fitting. Fibroblasts responded to this gradient by adjusting their orientation, ECM remodeling behavior, and local stiffness. 3D encapsulated fibroblasts oriented at around 45° in regions below 120 kPa, while they first reduced stiffness before reorienting in areas above 120 kPa. Beyond phenotype changes, fibroblasts also remodeled their surrounding ECM in a stiffness-dependent manner. In high-stiffness areas, fibroblasts created collagen-poor voids around cells, and formatted interlaced collagen bundles in low-stiffness areas. Overall, these results showed that fibroblasts actively remodeled their surroundings to optimize their mechanical and topographical environment.

Chapter 7 explored how ECM stiffness shaped matrix remodeling by umbilical cord-derived mesenchymal stem cells (UC-MSCs) within skin ECM hydrogels. The results showed that UC-MSCs exhibited stiffness-dependent behavior: in soft hydrogels (1.2 kPa, simulating a low-stiffness baseline), they caused contraction-driven stiffening; in medium stiffness (3.4kPa, mimicking normal skin), cells adopted a spindle-shaped morphology and maintained ECM integrity; while in stiff hydrogels (17.7 kPa, resembling scar tissue), cells remained more rounded and promoted ECM degradation, resulting in localized hole formation and a reduction in stiffness. Mechanistically, the mechanosensitive ion channel Piezo1 was identified as the regulator of these responses as Piezo1 inhibition altered remodeling outcomes-mitigating contraction in soft hydrogels and reducing ECM degradation in stiff hydrogels. Interestingly, while MMP2 and MMP14 expression was stiffness-dependent, it was unaffected by Piezo1 inhibition, suggesting the involvement of additional mechanotransduction pathways. Overall, this study underscored Piezo1’s role in mediating stiffness-dependent ECM remodeling by UC-MSCs.

In conclusion, this thesis advances our understanding of how mechanics of ECM influence cellular behavior and remodeling. It introduces versatile in vitro models that replicate fibrotic microenvironments, highlights key mechanotransduction pathways like Piezo1, and provides a foundation for the future identification of potential targets for development of antifibrotic therapies and regenerative medicine strategies.