Engineering Dynamic Extracellular Microenvironments: Temperature-Casted Collagen-Fibronectin Scaffolds with Controlled Structure, Mechanics, and Cell-Driven Re-Organisation

Abstract

Understanding how to engineer biomimetic scaffolds is critical for advancing tissue engineering, regenerative medicine, and disease modelling. This thesis investigates the design, fabrication, and characterization of three-dimensional (3D) extracellular matrix (ECM) scaffolds, focusing on collagen - fibronectin (ColFn) hydrogels that closely replicate native ECM environments.

The introductory chapters establish the fundamental principles of tissue engineering, emphasizing the role of collagen and fibronectin in ECM mechanics, cellular interactions, and mechanotransduction. Building upon this framework, the thesis integrates advanced confocal microscopy and oscillatory rheology to enable high-resolution, real-time imaging and precise mechanical analysis of ECM scaffolds.

A temperature-controlled fabrication technique was developed to modulate scaffold architecture and mechanical properties. Incorporating fibronectin significantly enhanced scaffold stability, fibre organization, and cellular engagement, supporting dynamic reorganization processes essential for tissue regeneration. The experimental work highlights how fibronectin modulates collagen fibre formation kinetics, increases ECM stiffness, and promotes cell adhesion and migration.

Live imaging and rheological analyses revealed that fibroblasts dynamically remodel their microenvironment, forming pericellular collagen - fibronectin micro-islands and aligning fibres along force axes. Importantly, a critical cell concentration was identified, beyond which collective cell generated forces induced a phase transition in the ECM. At low cell densities, cells contributed to local stiffening and retained more space to spread and interact with their surroundings. However, as cell density increased, mechanical stress and crowding altered ECM behaviour, transforming the structure from a continuous fibrous network into distinct, clustered islands of collagen and fibronectin. This structural shift was accompanied by a substantial increase in ECM stiffness, reflecting how cell density and mechanical feedback collectively govern matrix architecture and mechanical properties. While these observations parallel aspects of natural tissue remodelling, they are unique insights into the phase transition behaviour of engineered ECM under cellular stress.

Overall, this thesis provides a comprehensive framework for understanding scaffold design, ECM reorganization, and cellular interactions under physiologically relevant conditions. By integrating scaffold fabrication methods with mechanobiological insights, the findings contribute to the development of next-generation, bioactive materials for regenerative medicine and tissue engineering applications.