The extracellular matrix (ECM) plays an important role in many crucial cellular processes such as gene expression, cancer progression and cell differentiation. Cells are able to sense the mechanical properties of their extracellular environment and adjust their gene expression accordingly. The ECM regulates numerous cell behaviors including cells adhesion, spreading, migration, proliferation, and death. Fibronectin, a key glycoprotein of the ECM, is critical in the early wound healing process, as it can stop bleeding and protect the tissue, facilitate cell motility and proliferation, and guide the remodeling of new ECM. Subsequently, collagen is also deposited at the wound. The resulting mature collagen-fibronectin ECM keeps the wound sterile and induces the wound closure. Three different systems of collagen-fibronectin three-dimensional (3D) scaffolds were developed for this thesis. First, a collagen porous scaffold was originally fabricated using ice templating techniques and then coated with a layer of fibronectin, whose conformation was controlled via incubation temperature and monitored using Fӧrster resonance energy transfer (FRET) spectroscopy. This platform was next utilized to investigate the effect of ECM structure and conformation on cellular invasion and viability. Second, fibronectin was assembled into 3D fibrillar matrix within the above-mentioned porous collagen scaffold via shearing methods, which provides a promising platform for further investigation of cell-ECM interactions. Third, collagen fibrillar scaffolds with tunable microarchitecture and cell content were generated via warm/cold casting techniques, which resulted in ECM platforms that closely mimic the wound physiological environment. Collectively, our study suggests that by tuning collagen (porous vs. fibrillar) structure and fibronectin molecular conformation, we were able to control cell behaviors including cell adhesion, migration and proliferation, and thus, potentially facilitate the wound healing process. These 3D ECM-mimicking platforms offer precise control of protein microarchitecture and conformation over large volumes, i.e., for long-term cell culture, and therefore have potential applications in tissue engineering as well as regenerative medicine.