Fibronectin interactions with osteoblasts: Identification of a non–integrin‐mediated binding mechanism using a real‐time fluorescence binding assay

Fibronectin (Fn) is an extracellular matrix protein that interacts with specific integrins on the cell surface, initiating signal transduction processes that lead to a reorganization of the cytoskeleton and the assembly of focal adhesions. Cell surface proteoglycans or glycosaminoglycans (GAGs) such as heparan sulfate are also known to participate in the interaction of Fn with the cell surface by binding to two different heparin‐binding domains. The influence of Fn and GAGs on the spreading and differentiation of human osteoblasts was also previously described. In the current work, a method developed in our laboratory is established to evaluate the interaction between Fn and human osteoblasts and the influence of GAGs on such interactions. This technique makes use of fluoresceinphosphatidylethanolamine (FPE) such that when inserted into the lipidic bilayer, it acts as a fluorescent indicator of membrane interactions. The results indicate that the binding profile of Fn with the osteoblast cell surface is best represented by a hyperbolic single binding site model with a membrane affinity of 120 n M . Removal of cell surface heparan sulfate by treatment with heparitinase indicates that the cell surface moiety is directly involved in the binding process. Studies directed to assess the influence of heparin on the interaction of Fn with osteoblasts reveal that although it does not hamper Fn binding to the cell surface, it blocks the initial attachment to Fn‐coated surfaces, indicating that binding to the integrin receptor alone is not enough to promote cell attachment but that the participation of the cell‐surface GAGs is also a necessary condition. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 352–359, 2004