Probing nanomechanics of aggrecan and the aggrecan-rich pericellular matrix of chondrocytes in cartilage

Abstract

The mechanical properties of articular cartilage are associated with the extracellular matrix network of type II collagen and the proteoglycan, aggrecan, which in combination provide the tensile, shear, and compressive stiffness of the tissue. While the collagen network mainly provides resistance to tensile and shear deformation, aggrecan enmeshed within this network contributes significantly to the tissue's compressive and shear properties under equilibrium as well as dynamic loading conditions. Aggrecan has a "bottle-brush" structure that includes -100 negatively charged chondroitin sulfate glycosaminoglycan (CS-GAG) chains attached covalently to a core protein. Electrostatic interactions between these GAGs contribute to the compressive and shear stiffness of the tissue. Variations in the structure of aggrecan and its GAG constituents are known to exist as a function of tissue age, disease, and species. Using atomic force microscopy (AFM), we directly visualized the nanometer scale structure of aggrecan deposited on a 2-D substrate, including the first high resolution imaging of individual GAG chains along the core protein. We also visualized and quantified the differences in structure between aggrecan obtained from fetal epiphyseal and mature nasal bovine cartilages.

(cont.) A combination of AFM, biochemical, and polymer statistical methodologies was used to better understand the dependence of aggrecan structure and stiffness on the properties of its constituent GAG chains. The fetal epiphyseal aggrecan had a denser GAG brush region and longer GAG chains, which correlated with a higher effective persistence length of fetal core protein compared to that of mature nasal aggrecan. The effect of increasing the concentration of aggrecan on the substrate resulted in a decrease in molecular extension, suggesting a flexible protein core backbone, which allowed aggrecan to entangle and interact with neighboring molecules. AFM imaging of the conformation of aggrecan that had been deposited on substrates from solutions of varying ionic strength (IS), from DI water to the hysiological IS of 0.1 M NaCl, allowed for direct visualization of the collapse of the molecule on the substrate at the highest IS, due to charge shielding of the CS-GAGs by by Na+ counter-ions. Lastly, the nanomechanical properties of cartilage cells (chondrocytes) and their aggrecan-collagen-rich pericellular matrix (PCM) were probed via AFM nanoindentation using both a sharp nano tip and a larger micro-colloidal tip to better understand the deformation of cells in cartilage.

(cont.) The properties of cells freshly isolated from cartilage tissue, devoid of PCM, were compared to that of cells isolated and then cultured for selected times in 3-D alginate gel to obtain cells surrounded by their newly developed PCM. Using Hertzian contact mechanics as well as finite element analyses, material properties were estimated from the AFM force-indentation curves measured with these cell preparations. We also studied the effects of culture conditions on the resulting PCM properties, comparing 10% fetal bovine serum vs. medium containing a combination of insulin growth factor-i (IGF-1) + osteogenic protein-i (OP-1). While both systems showed increases in matrix stiffness with time in culture between days 7 to 28, the IGF-1 + OP-1 combination resulted in a higher effective modulus for the cell-PCM composite. These AFM cell indentation studies were enabled by the use of microfabricated chips containing wells designed to immobilize the spherical chondrocytes during testing. Due to the nonconventional but known geometry of the microfabricated wells, finite element analysis was used to include the effects of the cell-well boundary conditions and tip geometries on the calculated cell-PCM material properties.

(cont.) Taken together, these studies examining cartilage mechanics at the molecular and cellular levels give insight into the intricate roles that proteoglycans and collagen play in governing tissue-level mechanical properties.