Direct Measurement of Glycosaminoglycan Intermolecular Interactions via High-Resolution Force Spectroscopy

Abstract

Intermolecular repulsion forces between negatively charged glycosaminoglycan (CS−GAG) macromolecules are a major determinant of cartilage biomechanical properties. It is thought that the electrostatic component of the total intermolecular interaction is responsible for 50−75% of the equilibrium elastic modulus of cartilage in compression, while other forces (e.g., steric, hydration, van der Waals, etc.) may also play a role. To investigate these forces, radiolabeled CS−GAG polymer chains, with a fully extended contour length of 35 nm, were chemically end-grafted to a planar surface to form model biomimetic polyelectrolyte “brush” layers whose environment (e.g., ionic strength, pH) was varied to mimic physiological conditions. The total intersurface force (≤nN) between the CS−GAG brushes and chemically modified probe tips (SO3- and OH) was measured as a function of tip−substrate separation distance in aqueous solution using the technique of high-resolution force spectroscopy (HRFS). These experiments showed long-range, nonlinear, purely repulsive forces that decreased in magnitude and range with increasing ionic strength and decreasing pH. To estimate the contribution of the electrostatic component to the total intersurface force, the data were compared to a theoretical model of electrical double layer repulsion based on the Poisson−Boltzmann formulation. The CS−GAG brush layer was approximated as either a flat surface charge density or a smoothed volume of known fixed charge density and the probe tip was modeled as a smooth hemisphere of constant surface charge density. Modeling the CS−GAG brush as a volume charge yielded theoretical fits much closer to the experimental data and is a good first step toward deconvolution of the force components.