Impact of Order and Disorder in RGD Nanopatterns on Cell Adhesion

Abstract

We herein present a novel platform of well-controlled ordered and disordered nanopatterns positioned with a cyclic peptide of arginine-glycine-aspartic acid (RGD) on a bioinert poly(ethylene glycol) background, to study whether the nanoscopic order of spatial patterning of the integrinspecific ligands influences osteoblast adhesion. This is the first time that the nanoscale order of RGD ligand patterns was varied quantitatively, and tested for its impact on the adhesion of tissue cells. Our findings reveal that integrin clustering and such adhesion induced by RGD ligands is dependent on the local order of ligand arrangement on a substrate when the global average ligand spacing is larger than 70 nm; i.e., cell adhesion is "turned off" by RGD nanopattern order and "turned on" by the RGD nanopattern disorder if operating at this range of inter-ligand spacing.Integrin plays a central role in the formation of focal adhesions (FAs), which anchor cells to the extracellular matrix (ECM). 1 High-affinity binding of the integrin transmembrane proteins to ECM ligands has been extensively exploited for tailoring artificial synthetic ECM systems. 2 Thus far, it has been reported that cell responses to the synthetic ECM depend to a large extent on multiple substrate features, such as its chemical composition, 3-6 geometry and topographical features, 7 ligand organization, 8,9 and even substrate stiffness. 10,11 In particular, the spatial organization of the integrin-specific peptide sequence of arginineglycine-aspartic acid (RGD) on non-fouling substrates has attracted much attention. This sequence, contained in many ECM proteins, can be recognized by all five aV integrins (αVβ1, αVβ3, αVβ5, αVβ6, αVβ8), two β1 integrins (α5β1, α8β1) and αIIbβ3. 12 Once ligated, the integrin receptors link the ECM to the cytoskeleton and integrate intracellular and extracellular events. Furthermore, it is known that cellular behaviors such as adhesion, migration, proliferation and differentiation, are quite sensitive to the bioactivity, tether length, interspacing and density of surface RGD ligands in artificial ECM materials. 13-21Recent developments in nanotechnology have given access to the nanoscale organization of RGD ligands in both inorganic and polymeric substrates mimicking ECMs. Research concerning randomly dispersed RGD ligands grafted onto polymeric materials suggested that *Corresponding authors: E-mail: E-mail: Spatz@mf.mpg.de (J.P. Spatz); E-mail: jdding1@fudan.edu.cn (J. Ding). Supporting Information Available: A detailed description of the experimental protocols for sample preparation and characterization is available free of charge via the Internet at http://pubs.acs.org. Nevertheless, there has been no report to date of studies comparing cellular responses to nanostructured surfaces characterized by ordered or disordered organization of biomolecules such as RGD ligands. Herein, we chose to examine this critical issue in cell-nanomaterial interactions by exploring osteoblast adhesion regulated by the nanoscale organ...

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