Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding

Abstract

Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH₃, OH, COOH, NH₂) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on focal adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity—α₅β₁ integrin binding affinity followed the pattern OH>NH₂COOH>CH₃, while integrin α_Vβ₃ displayed the relationship COOH>NH₂≫OHCH₃. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix adhesions as well as focal adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, α-actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH₂ and negatively charged COOH surfaces exhibited intermediate levels of recruitment of focal adhesion components, while the hydrophobic CH₃ substrate displayed the lowest levels. These patterns in focal adhesion assembly correlated well with integrin α₅β₁ binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in focal adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.

Introduction

Cell adhesion to synthetic surfaces is crucial to many biomedical and biotechnological applications [1], [2], [3], [4], [5], [6]. In addition to anchoring cells, adhesive interactions activate various intracellular signaling pathways that direct cell viability, proliferation, and differentiation [7], [8]. In many instances, cell adhesion to biomaterial surfaces is mediated by a layer of adsorbed proteins, such as immunoglobulins, vitronectin, fibrinogen and fibronectin (FN) [9], [10]. Numerous studies have shown that the type, quantity and activity of adsorbed proteins are influenced by the underlying substrate properties, including chemistry and hydrophobicity [9], [11], [12], [13], [14], [15], [16]. These substrate-dependent differences in protein adsorption have profound effects on cellular activities, including integrin receptor binding and subsequent cell adhesive events [17], [18], [19], [20], [21], [22], [23]. Several studies have demonstrated diverse cellular responses to substrates with different surface chemistries. For instance, Allen et al. showed differential gene expression for several cell types on surfaces of varying hydrophobicity [24]. Similarly, Brodbeck and colleagues demonstrated increased in vivo apoptosis and reduced foreign body giant cell formation on hydrophilic and anionic surfaces compared to hydrophobic and cationic substrates [25]. While these studies highlight the importance of biomaterial surface properties in modulating cellular behaviors, the underlying mechanisms responsible for generating dissimilar cell responses among different substrates remain poorly understood.

Using model substrates with well-controlled surface properties, we recently reported that surface chemistry alters the adsorption kinetics and structure of adsorbed FN [26]. Furthermore, we demonstrated that surface chemistry modifies the functional presentation of the major integrin binding domain of FN, alters integrin binding, and potentiates cell adhesion strength [23]. In the present work, we analyzed the effects of surface chemistry on focal adhesion assembly and signaling. Focal adhesions are specialized adhesive complexes containing structural and signaling molecules that regulate cell migration, survival, cell cycle progression, and differentiation [27], [28]. We demonstrate that surface chemistry modulates focal adhesion composition and signaling. These findings provide a potential mechanism for the diverse cellular responses to biomaterial surfaces and offer design criteria for the engineering of surfaces that elicit specific cellular responses.