Tissue engineering aims to regenerate new biological tissue for replacing diseased or injured tissues. We propose a new approach to accelerate the deposition of cell-secreted matrix proteins into extracellular matrix fibrils. We examined whether dynamic substrates with nanoscale ligand features allowing for alpha5beta1 integrin recruiting, cellular tension generation, and alpha5beta1 integrin mobility would enhance fibronectin matrix assembly in a ligand model system that is routinely not sufficient for its induction. To this end, we developed biodynamic substrates consisting of cell adhesive fragment from the 9th and 10th type repeats of fibronectin (FNf ) functionalized to 100 nm prefabricated albumin nanoparticles (ANPs). FNf-ANPs modulated cellular spreading processes, promoting the development of stellate or dendritic morphologies. Concomitant with the spreading, FNf-ANPs rapidly recruited beta1 integrins to focal contacts and promoted the migration of beta1 integrins centripetally from the cell periphery toward the center. FNf-ANPs stimulated the deposition of secreted fibronectin into matrix fibrils; FNf, the key ligand alone, was not sufficient for fibronectin fibrillogenesis. When FNf-ANPs were displayed from "immobilized" substrates, abolishing any mobility of ligated beta1 integrins, fibronectin matrix assembly was abrogated, implicating the role of dynamic matrix display on matrix assembly. Receptor ligation of FNf-ANPs via noncontractile adhesions was not sufficient to stimulate fibrillogenesis, and Rho-kinase inhibitors abolished fibronectin matrix deposition. Our approach highlights the possibility of engineering integrin-based extracellular matrix assembly using nanotechnology, which may have implications for improved biomaterials for wound repair and basic understanding of matrix remodeling within pathogenesis and biomedicine.