The structure of proteoglycan aggregate determined by atomic force microscopy

Abstract

Summary: Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.

Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

“…6E) is visible as a honeycomb‐like texture, which indicates an incomplete surface coverage. The estimation of the width of these visual structures (15–30 nm) does not match with simulated or experimental data (∼1 nm) for HA helices 38,41. These findings indicate a rather aggregated structure of multiple HA chains or helices.…”

“…When pHA or aHA was measured on NH 2 –Si, the layer thickness increased approximately 1 nm again. Compared with the molecular dimensions of HA, assuming a random coil, one comes to the conclusion that HA is attached side‐on in a single layer in these cases 38. A different behavior was observed in the case of tHA.…”

Biological Calcification

“…To achieve this, one of the substances most often used is colloidal iron (Fig. Under the electron microscope, the extended molecules of native proteoglycans appear as bottlebrush-like structures (i. e., they appear to consist of an axial filament with attached lateral chains) when aggregates are isolated and spread Morgelin et al 1994;Ng et al 2003;Yeh and Luo 2004). If this procedure is carried out at low pH (about 2.8), a satisfactory specificity is obtained, as also shown by the disappearance of iron-positive granules after protease or hyaluronidase digestion (Matukas et al 1967;Bonucci 1971a).…”

The Molecular Organization of Polymers of Cartilage in Health and Disease

“…It should be noted that the images of the ultrastructures of PG aggregates visualized by AFM are comparable with those using conventional electron microscopy. Thus, AFM represents an alternative approach for future analysis of PG aggregates and its assembly [123]. AFM does not exclusively represent a qualitative but also a quantitative tool: The direct measurement of GAG intermolecular interactions was recently performed by high-resolution force spectroscopy [124].…”

“…Finally, PGs were also investigated by AFM [123]: The images illustrated the ultrastructure of the aggregates, especially the individual PG and the core HA strand. In addition to the length and width of each molecule, the height of the individual PG aggregates could be directly measured.…”