Ultrastructure and nanomechanical properties of aggrecan from native cartilage and engineered tissue

Abstract

Electrostatic interactions associated with aggrecan, one of the major components of the cartilage extracellular matrix, are responsible for ~50% of the equilibrium compressive elastic modulus of the tissue. The bottle-brush-shaped aggrecan consists of a core protein to which ~100 sulfated glycosaminoglycan (sGAG) chains are attached. Loss of sGAG is one early events in the pathogenesis of osteoarthritis and the resulting degradation of cartilage is irreversible due to its limited capacity for self-repair. Tissue engineering is one of the techniques which holds great potential for cartilage repair. In order to achieve successful repair, a clear understanding of native and engineered cartilage aggrecan is essential. With atomic force microscopy and high resolution force microscopy, the structure of aggrecan single molecules and the nanomechanical properties of an end-grafted aggrecan monolayer were quantified. Adult human aggrecan showed significantly shorter GAG chains and core proteins as well as lower molecular stiffness compared to that of newborn aggrecan. After enzymatic digestion of chondroitin sulfate (CS) GAGs, keratan sulfate GAG chains were visualized near the N-terminal domain of a less extended core protein. Direct visualization of aggrecan aggregates confirmed the structure of the constituent hyaluronic acid, aggrecan G1 domain, and link protein. Increased flexibility of the core protein was found near the G1 domain, which may facilitate the aggregate self-assembly process. Aggregated and non-aggregated aggrecan both showed remarked flexibility (i.e., decreased extension ratio) when the aggrecan areal density increased. These findings on intra- and inter-molecular structure provide insights into the structure-property relationships of aggrecan in vivo. Aggrecan produced by animal-matched bone marrow stromal cells (BMSCs) and chondrocytes seeded in peptide hydrogel were evaluated for their age-associated structure and nanomechanical properties. Independent of age, BMSCs produced longer core proteins and GAG chains than the chondrocytes, suggesting that the BMSC-produced aggrecan was characteristic of that from young cartilage. Comparison of the adult BMSC-produced aggrecan with adult cartilage-extracted aggrecan revealed that adult BMSC-aggrecan has a phenotype characteristic of young growth cartilage: primarily full-length aggrecan core, longer GAG chains and a higher content of chondroitin-4-sulfate in the CS-GAG chains, the latter identified via fluorescence assisted carbohydrate electrophoresis. The nanomechanical stiffness of BMSC-aggrecan was demonstrably greater than that of cartilage-aggrecan at the same total sGAG (fixed charge) density. These results support the use of adult BMSCs for cell-based cartilage repair.