Dentin proteoglycans: an immunocytochemical FEISEM study

Dentin proteoglycans are fundamental constituents of the dentin matrix and are distributed ubiquitously both in dentin and cement. They have several important functional properties; in particular, they have a fundamental role in the maintenance and the correct stabilization of collagen fibers. The use of phosphoric acid on dentin, as proposed in most common dental adhesive systems to establish a reliable bond, may affect the molecular structure of proteoglycans. The aim of this study was to evaluate, after the application of EDTA or phosphoric acid on dentin, the dentin proteoglycans with an immunocytochemical approach with high resolution SEM. For this purpose, dentin disks obtained from recently extracted human molars were etched with a 35% water solution of phosphoric acid for 15 s, 30 s, and 60 s. Control specimens were conditioned with EDTA. Specimens were immunolabeled with a monoclonal antibody antichondroitin sulfate and visualized with a gold-conjugated secondary antibody. Conditioning dentin with EDTA resulted in a distinct labeling of the proteoglycans, as visualized on branching fibrillar structures in the order of 10-20 nm. The use of 35% phosphoric acid on dentin revealed a coagulation of proteoglycans after etching for 15 s while a very low labeling signal was detectable after 30 s. No labeling was obtained after etching dentin with phosphoric acid for 60 s. These results suggest that the use of 35% phosphoric acid on dentin is able to produce significant structural modifications of the dentin proteoglycans even after short application times. Additionally, when applied on the dentin surface for more than 30 s, phosphoric acid produces a dramatic decrease in proteoglycans' antigenicity, probably due to structural modifications of the three-dimensional conformation of these molecules.