Introduction
Most cells are adherent and must attach to and spread on a surface in order to survive, proliferate and function. In tissue, this surface is the extracellular matrix (ECM), an insoluble scaffold formed by the assembly of several large proteins -- including fibronectin, the laminins and collagens and others -- that provide a wide range of biochemical and mechanical cues to cells. Studies of cellular processes in the laboratory routinely use protein-coated dishes to mimic the in vivo environment and to elucidate the functions of the matrix in regulating cellular processes. The adsorption of protein, however, is complicated and often proceeds with a lack of control over the orientation and conformation of proteins at the surface. As a result, it remains difficult to control the biological activities of proteins that are adsorbed to man-made materials, and in turn compromises the use of these substrates as models of the ECM.
This realization has motivated a significant effort over the past two decades to develop materials that present well-defined biological motifs for use as mimics of the ECM. Many of the approaches have used substrates modified with polymeric materials or monolayer chemistries that can be tailored with cell adhesion motifs. These important approaches have been reviewed elsewhere. The present review specifically focuses on the use of self-assembled monolayers of alkanethiolates on gold for this application. These surfaces are a recent addition to the strategies that are now used and offer a set of characteristics that make them well-suited to certain classes of problems in studies of cell-ECM interactions. These characteristics and early applications of the monolayers are described in the following pages.
Protein adsorption to materials
Most man-made materials, when placed in solutions containing proteins, are rapidly coated with an adsorbed layer of protein. This property is exploited in the routine preparation of substrates for cell culture. For example, polystyrene substrates are often treated with a solution of fibronectin prior to seeding with cells. The resulting protein film is heterogeneous in structure -- in that the proteins are presented in a range of orientations and denaturated states -- but still presents the cell-binding motifs at sufficient density and in a functional conformation to promote attachment, spreading and migration. In practice, the conditions for adsorbing protein are identified empirically and depend on the structures of the substrates in ways that remain poorly understood. One study, for example, used atomic force microscopy and surface plasmon resonance spectroscopy to characterize fibrinogen and fibronectin that had adsorbed to monolayers terminated either in methyl or carboxyl groups and found that the densities and conformations of the adsorbed proteins strongly depended on surface chemistry.
The tendency for proteins to adsorb non-specifically to materials has also interfered with efforts to use model surfaces that present defined cell-binding motifs. When the slide is placed in a suspension of cells, for example, proteins in the medium can rapidly adsorb to the surface, thereby physically preventing access to the immobilized ligands or introduce additional ligands that mediate cell attachment. The common approach to this problem has relied on treating the substrate with a solution of a "blocking" protein -- often bovine serum albumin -- prior to cell attachment with the expectation that "sticky" sites on the surface are passivated, now allowing the ligands to mediate cell adhesion. But the large fraction of the surface that is occupied by the blocking protein and the substantially larger size of the protein relative to the ligand make this approach ineffective in studies that require molecular control over the surface.
Monolayers for cell adhesion
This same approach has been validated for studying the roles of peptide ligands in cell adhesion and migration. In an early example, we had prepared monolayers that presented the peptide Gly-Arg-Gly-Asp-Ser against a background of tri(ethylene glycol) groups. This peptide is found in fibronectin and other ECM proteins and is a ligand for approximately one-half of the integrin family cell-surface receptors, which are an important class of receptors found on all cellular surfaces and that mediate the attachment of cells to ECM. We found that Swiss 3T3 fibroblasts attached and spread efficiently to the monolayer and immunostaining showed that the adherent cells assembled normal focal adhesion complexes -- a cluster of integrin receptors that forms strong attachments to the substrate and initiates adhesion signals -- and actin stress filaments. Control experiments established that the adhesion of cells could be blocked with a soluble RGD peptide and that cells failed to attach to a monolayer presenting a scrambled form of this peptide. Hence, this work validated the use of monolayers to control the ligand-receptor interactions that mediate the adhesion of cells and it also showed that the ligand RGD alone could support cell attachment, spreading and a proper organization of the cytoskeleton.
Studies of cell-ECM interactions
The application of monolayer substrates to understanding the roles of ligands in fibronectin illustrates the benefits of employing structurally well-defined substrates. Fibronectin is a primary ECM protein that is found in many tissues. This protein comprises more than 30 globular domains that include the type III domains important for cell adhesion. The RGD peptide is found within the 10th type III repeat and was among the earliest ligands discovered to mediate cell adhesion by interacting with integrin receptors. Recent work has implicated a peptide, having sequence PHSRN, residing in the 9th type III domain in the adhesion of cells. A series of studies by Yamada and Grant and coworkers led to the proposal that PHSRN is a synergy peptide that cooperates with RGD to enhance the attachment and spreading of cells but that fails to support cell attachment when present alone. Mardon suggested a mechanism wherein the RGD and PHSRN peptides in adjacent domains of fibronectin simultaneously interact with separate binding sites on opposite sides of the integrin receptor. This model was based on studies of cell adhesion to recombinant proteins having scrambled RGD and PHSRN sequences and on a crystal structure of a fragment of fibronectin, which shows that the two peptides are directed towards the same region of space and are separated by a distance that matches the width of the receptor. Yet studies that use protein-coated substrates to study cell adhesion are complicated by the inability to establish that peptide sequences in the proteins are available to interact with cellular receptors once the proteins are adsorbed to tissue culture plastic.
Monolayers erase this ambiguity and were used to revise the mechanistic understanding of the synergy peptide. First, Baby Hampster Kidney (BHK) cells attached with similar efficiency to monolayers presenting either the RGD or the PHSRN peptide, although cells failed to spread on the latter substrate. Second, the adhesion of cells could be inhibited by either peptide, i.e. either RGD or PHSRN could block the attachment of cells to a monolayer presenting RGD (and in the same sense, to a monolayer presenting PHSRN). These results require that the two peptides either share a binding site on the integrin receptor or bind to separate sites that are allosterically connected. In either case, these observations are inconsistent with the standing model invoking a cooperative two-point binding of peptides. Indeed, the ability to present defined ligands at controlled densities and in a regular environment (to ensure that all of the peptides are active) against an otherwise non-interacting background represents a powerful tool for establishing the function of ECM ligands and addressing the relationship between distinct ligands.
Dynamic substrates
The self-assembled monolayers have enabled models of the ECM that are dynamic, and that allow the activities of immobilized ligands to be switched on and off during the course of cell culture. The approach exploits the conductivity of the gold film that supports the monolayer and follows from extensive work that has shown that electroactive molecules that are tethered to the monolayer can be reduced or oxidized by applying electrical potentials to the gold film. By designing monolayers that incorporate molecular groups that undergo oxidation or reduction and subsequent reactions, it is possible to engineer surfaces that dynamically inactivate or activate ligands that interact with cell-surface receptors. In one example, the RGD peptide was immobilized to a monolayer by way of a tether that incorporated a propanate-benzoquinone fragment. The peptide ligand mediated the attachment and spreading of cells and was stable for several days. Application of a negative potential, however, resulted in reduction of the benzoquinone group and subsequent lactonization to release the peptide from the monolayer. As a result, the adherent cells assumed a rounded morphology and detached from the substrate. This example illustrates the molecular-level design of a monolayer that could selectively release ligands that were tethered by way of the redox-active moiety and in this case non-invasively release an adherent cell culture. We have also developed a tether that released immobilized ligand in response to a positive electrical potential. By combining these two strategies with microelectrode arrays, it is possible to prepare adherent cell cultures and selectively release sub-populations of cells at different times. Another notable approach to the development of dynamic substrates has used polymeric gels that undergo a thermally induced phase transition to release cells. The approach of using physical organic chemistry to design molecules or polymers that undergo redox-active reactions to manipulate the activities of ligands on a monolayer is general and can be applied to the preparation of dynamic substrates having a range of activities.
In another example, we developed a monolayer that could be electrically switched to permit the immobilization of cell-adhesive ligands]. A monolayer presenting the hydroquinone group against a background of tri(ethylene glycol) groups is inert and does not support the attachment of cells.
Summary and outlook
This review provides a perspective of work over the past decade that has developed self-assembled monolayers as model substrates for studies of cell adhesion. Several characteristics inherent to the monolayers make them well-suited for preparing mimics of the ECM. These points include:
Well-defined structure. The regular structure of the monolayers enables wide flexibility in tailoring the surface with ligands and other functional groups. Ligands can be presented with excellent control over their densities and in a uniform environment. This flexibility also enables the preparation of dynamic substrates that can manipulate the presentation of ligands.
Inert surfaces. Self-assembled monolayers that present oligo(ethylene glycol) groups are highly effective at preventing the non-specific adsorption of protein. These surfaces maintain this property in complex solutions, including serum-containing cell culture media and void the need for blocking.
Immobilization schemes. A full portfolio of immobilization chemistries that can be used to tether ligands to monolayers, and that provide for a defined orientation of the ligands and a rigorous control over the density, is available.
Analytical methods. Monolayers are compatible with multiple analytical methods used in characterizing biochips, including surface plasmon resonance spectroscopy, fluorescence imaging, radioisotope detection, and mass spectrometry patterning methods. The availability of several patterning methods -- and specifically the microcontact printing method -- provides routine access to substrates that can control the shapes, sizes and positions of cells. These substrates enable studies of cytoskeleton function in cells and cell-based technologies.
Proven performance. There are hundreds of publications that describe the use of monolayers in biological and bioanalytical applications. These systems are well-suited to experiments involving attached cell cultures and are used commercially in bioanalytical devices.
The ECM is complex and consequently experimental studies benefit from a range of methods and tools that bring insights to the structures and functions of the matrix. The self-assembled monolayers represent one important component of this toolbox and are significant because they offer a straightforward approach to prepare structurally well-defined mimics of the matrix. The tailored substrates are admittedly simple mimics of the matrix but they allow unambiguous studies of the roles that discrete motifs play in mediating cell adhesion and regulating downstream signaling processes. Early work with the monolayers has been important for addressing the roles of matrix ligands, understanding the relationships between cell shape and function, and enabling a class of dynamic substrates that can modulate, in real-time, the activities of immobilized ligands. Future work will see an increased use of the monolayers for current problems in cell adhesion and matrix biology.