Adsorbed layers of oriented fibronectin: A strategy to control cell–surface interactions Claudio Calonder, 1 * Howard W. T. Matthew, 1 Paul R. Van Tassel 2 1 Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202 2 Department of Chemical Engineering, Yale University, P.O. Box 208286, New Haven, Connecticut 06520-8286 Received 12 January 2005; revised 28 March 2005; accepted 29 March 2005 Published online 29 July 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30417 Abstract: Fibronectin (Fn) is a matrix protein known to induce cell attachment and spreading through its cell bind- ing site and related synergy sites. Fn-coated surfaces are therefore useful in tissue engineering and other cell contact- ing applications, but a problem with many immobilization strategies is a random distribution of molecular orientations. We sought to control Fn orientation, and thus enhance the availability of its cell binding site, by immobilizing Fn via a carboxymethyl dextran layer onto which are chemically at- tached monoclonal antibodies specific to a region near to Fn’s C terminus (and thus away from the cell binding site). Using optical waveguide lightmode spectroscopy, we show the presence of chemically coupled antibodies to yield a considerably denser and thicker Fn layer, consistent with a more vertically aligned protein. Human umbilical vein en- dothelial cells spread significantly faster, and in a more spherically symmetric way, on an oriented Fn layer (i.e., in the presence of immobilized monoclonal antibodies) as com- pared with a control Fn layer (i.e., in the absence of bound antibodies). However, we observe human umbilical vein endothelial cell spreading on the oriented Fn layer to be similar to that on a Fn layer in the absence of a carboxy- methyl dextran layer, suggesting that although orienting Fn is a promising strategy, coupling strategies using linkers other than dextran may be needed. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res 75A: 316 –323, 2005 Key words: fibronectin; dextran; human umbilical endothe- lial cell; optical waveguide lightmode spectroscopy; optical microscopy INTRODUCTION The incorporation of biomolecules onto or within synthetic materials offers the promise of new devices of high impact on the quality of human life. Substrates onto which a monolayer of protein molecules is im- mobilized are useful as biomaterials, biosensors, and biocatalysts. Just as the function of a biological mole- cule in solution depends critically on its structure, the function of a surface-immobilized protein monolayer heavily depends on its structural properties; these in- clude internal conformation, relative molecular orien- tation, and spatial homogeneity. Fibronectin (Fn) is an extracellular matrix protein known to promote cell attachment and spreading. 1 The mechanism is thought to involve attachment of transmembrane integrin receptors to Fn’s cell binding site, located on the 10th type III repeat module and containing the specific amino acid sequence RGD, as well as to synergy sites located on the 8th and 9th type III repeats. Thus, materials coated with Fn monolayers are promising as substrates for cell-based sensors or tissue engineering scaffolds. Indeed, a number of studies attest to the enhanced cell attachment and spreading of surfaces coated with Fn, compared with identical materials in the absence of Fn. 2–27 However, most current immobilization procedures result in a random distribution of protein orientations on the surface, leaving a significant fraction of binding sites inaccessible to cells. A material with a layer of surface- immobilized Fn, oriented with the cell binding site facing away from the surface and accessible to cells, may offer an enhanced degree of control over the cellular response. We present herein a strategy for placing Fn with a specific predominant orientation. Orientational con- trol is achieved through the preplacement of a mono- clonal antibody targeted to a region of Fn away from its cell binding site. Specifically, we consider a metal oxide substrate, coated with linear carboxymethyl dextran, onto which are chemically coupled monoclo- nal antibodies against a region near the C terminus of **Present address: Zeptosense AG, CH-4108 Witterswil, Switzerland Correspondence to: P. R. Van Tassel; e-mail: paul. vantassel@yale.edu Contract grant sponsor: National Institutes of Health; con- tract grant number: R01 EB000258 © 2005 Wiley Periodicals, Inc.