Quantitative investigation of integrin-mediated cellular adhesion: Role of non-specific, protein and peptide-mediated forces

Adhesion is the first step in a cascade of events through which cellular interaction with a material surface occurs. It is a complex phenomenon which is mediated by a combination of many overlapping interactions due to non-specific forces arising from the substrate’s physicochemical characteristics and to specific forces through binding events between physisorbed or chemisorbed adhesive proteins, peptides, and other bioactive factors on the material surface and receptors on the cell surface. Furthermore, these events occur concurrently at different rates. Quantitative studies of the contribution of each of these processes to the initial stages of adhesion allow for tailoring of the material surface properties to optimize cell-surface interactions, as well as elucidating the molecular mechanism of the receptor-ligand interactions which dictate cell function.

The principle goals of this research to essentially develop criteria for immobilization strategies for biomolecules so that optimization of the ligand “presentation” on the surface can be achieved for biomaterials/tissue engineering as well as biotechnology/nanotechnology applications. In order to achieve this, we’ve first developed a model system of materials (self-assembled monolayers – SAMs), ligands (designed RGD-containing peptides and proteins), cells (expressing only specific integrins of interest), and assays (spinning disc assay, biochemical crosslinking) to be able to study quantitatively the non-specific and specific contributions to the RGD-integrin binding event. We’re currently extending this system to study peptide-mediated integrin binding and its effects on the adhesion and downstream signaling of MC3T3-E1 osteoblasts as they’re “induced” into forming bone tissue.

In situ imaging of adsorbed fibronectin structure: Correlation between nano-scale structure and function

In conjunction with the quantitative studies of cellular adhesion (link to first page), we’re also interested in the nano-scale structure of and the cellular receptor’s interaction with bioactive substrates with immobilized peptides and proteins. By combining techniques such as ellipsometry and atomic force microscopy (AFM) in addition to cellular assays, much insight of this interface at the molecular-level can be obtained. For instance, we’ve imaged small groups of proteins in both “dry” and “in situ” (i.e. hydrated) to be able to determine the native structure of adsorbed proteins on these surfaces (see below) as a function of material surface properties. By using modified-tip techniques, we can not only probe the physical structure but also the functional interaction of the surface ligands with receptors (piconewton levels of forces). We’re interested in modeling this data to increase the current fundamental understanding of the receptor-ligand interactions, which is the primary mechanism by which biospecific function is triggered by bioactive substrates in the fields of biotechnology and biomaterials/tissue engineering.

Figure (above). The structure of sub-monolayer fibronectin clusters on bare silicon oxide detected by “dry” (left) and “in situ” (right) atomic force microscopy (AFM). The loss of the native structure of the protein is evident due to the drying process. (z-scale is 10 nm/div) Future in situ studies with modified tips can detect the “functional structure” of these cell adhesion proteins as well probe the strength of the ligand-receptor interaction.

This research was performed in collaboration with: (applies to both)

(Note for Ranjan: We may want to centralize the collaborators for the biomaterials section rather than project by project since many of them overlap)

1. Department of Bioengineering (PENN) – Dr. Paul Ducheyne
2. Department of Microbiology (PENN) – Dr. David Boettiger
3. Department of Biochemistry and Molecular Biophysics (PENN) – Dr. William F. DeGrado
4. Department of Orthopedic Surgery (Thomas Jefferson University) – Dr. Irving Shapiro

Publications:

1. Lee Mark H.; Ducheyne, P.; Morris Ronit; Boettiger David; Composto Russell J. “The effect of substrate properties on a5b1 integrin-mediated cellular adhesion to fibronectin.” Langmuir 2004 (submitted).
2. Lee, Mark H.; Brass, David A.; Morris, Ronit M.; Composto, Russell J.; Ducheyne, Paul. “Effect of non-specific interactions on cellular adhesion using model surfaces.” Biomaterials 2004 (in press).
3. Lee Mark H.; Morris Ronit; Shapiro Irving; Boettiger David; Composto Russell J. “Non-specific effects of self-assembled monolayer substrates on RGD peptide-mediated cell adhesion.” Transactions of the Society for Biomaterials 2003.
4. Lee Mark H.; Adams, Christopher J.; Composto, Russell J.; DeGrado, William F.; Ducheyne, Paul. Effect of RGD peptide design on the adhesion, differentiation and mineralization of MC3T3-E1 cells. Journal of Bone and Mineral Research 2005 (in preparation).

Selected References:

[1] Ulman A., An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self Assembly. (Academic Press: Boston, 1991).

[2] Plueddemann, E. P., Silane Coupling Agents. (Plenum Press: New York, 1982).

[3] Adamson A.W., Physical Chemistry of Surfaces. (John Wiley and Sons: New York, 1990).

[4] Israelachvili, J. N., Intermolecular and Surface Forces. (Academic Press: London, 1991).

[5] Hynes, R.O., “Integrins: Versatility, Modulation, and Signaling in Cell Adhesion,” Cell 69 (1992): 11–25.

[6] Rouslahti, E., Pierschbacher, M.D., “New perspectives in cell adhesion: RGD and integrins,” Science 23 (1994), 491-497.

[6] Mrksich M, Whitesides GM, “Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells,” Annu Rev Biophys Biomol Struct 25 (1996):55-78.

[7] Garcia, A. J., Ducheyne, P., Boettiger, D., “Quantification of cell adhesion using a spinning disc device and application to surface-reactive materials,” Biomaterials 18 (1997), 1091-1098.

[8] Massia, S. P., Hubbell, J. A., Anal. Biochem 187 (1990): 292.