Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

The cell-dependent polymerization of intercellular fibronectin fibrils can stimulate cells to self-assemble into multicellular structures. The local physical cues that support fibronectin-mediated cellular self-assembly are largely unknown. Here, fibronectin matrix analogs were used as synthetic adhesive substrates to model cell-matrix fibronectin fibrils having different integrin-binding specificity, affinity, and/or density. We utilized this model to quantitatively assess the relationship between adhesive forces derived from cell-substrate interactions and the ability of fibronectin fibril assembly to induce cellular self-assembly. Results indicate that the strength of initial, rather than mature, cell-substrate attachments correlates with the ability of substrates to support fibronectin-mediated cellular self-assembly. The cellular response to soluble fibronectin was bimodal and independent of the integrin-binding specificity of the substrate; increasing soluble fibronectin levels above a critical threshold increased aggregate cohesion on permissive substrates. Once aggregates formed, continuous fibronectin polymerization was necessary to maintain cohesion. During self-assembly, soluble fibronectin decreased cell-substrate adhesion strength and induced aggregate cohesion via a Rho-dependent mechanism, suggesting that the balance of contractile forces derived from fibronectin fibrils within cell-cell versus cell-substrate adhesions controls self-assembly and aggregate cohesion. Thus, initial cell-substrate attachment strength may provide a quantitative basis with which to build predictive models of fibronectin-mediated microtissue fabrication on a variety of substrates. Statement of significance Cellular self-assembly is a process by which cells and extracellular matrix (ECM) proteins spontaneously organize into three-dimensional (3D) tissues in the absence of external forces. Cellular self-assembly can be initiated in vitro, and represents a potential tool for tissue engineers to organize cells into modular building blocks for artificial tissue fabrication. Fibronectin is an ECM protein that plays a key role in tissue formation during embryonic development. Additionally, the cell-mediated process of converting soluble fibronectin into insoluble, ECM-associated fibrils has been shown to initiate cellular self-assembly in vitro. In this study, we examine the relationship between the strength of cell-substrate adhesions and the ability of fibronectin fibril assembly to induce cellular self-assembly. Our results indicate that substrate composition and density play cooperative roles with cell-mediated fibronectin matrix assembly to control the transition of cells from 2D monolayers into 3D multicellular aggregates. Results of this study provide a quantitative approach to build predictive models of cellular self-assembly, as well as a simple cell-culture platform to produce biomimetic units for modular tissue engineering.