Our previous study, at light and electron microscopic level, (A.Oyarzún and E.Dreyer, J Dent Res, Vol 80 Special Issue,2001) indicated that intratubular glycosaminoglycans-proteoglycans complexes were embedded within the hybrid layer and were detected associated with the lamina limitans. Objective: The aim of this study was to examine by means of confocal laser scanning microscopy the structural interactions between intratubular proteoglycans and dentin adhesives in a total-etch wet-bonding technique. Methods: For that purpose, cervical root dentin segments (n=30) obtained from human teeth extracted for orthodontics reasons were used. The etched surfaces with 35% phosphoric acid gel were bonded using Scotchbond Multi-Purpose (3M Dental Products, St. Paul, MN, USA) according to the manufacturer's instructions. The primer solution was labeled with TRITC (1 mg/ml). Dentin segments were fixed in an ethanol-formaldehyde-acetic acid solution, demineralized in formic acid and embedded in paraffin. Sections of 20 um were treated with a mouse monoclonal antibody anti-chondroitin sulfate (clone CS-56) and then exposed to FITC conjugated anti-mouse IgG. Confocal optical sections were collected with a confocal laser scanning microscope LSM 410 Invert (Carl Zeiss;Germany). Results:The merged confocal images of immunolabeling and the adhesive system demonstrated that resin tags beneath the hybrid layer were only observed within lamina limitans in all optical sections. TRITC fluorescence was never detected between lamina limitans and the tubule walls composed by intertubular matrix. Moreover, colocalization of chondroitin sulfate epitopes with the adhesive was not observed. Conclusions: Our observations proved that lamina limitans, composed at least by chondroitin sulfates, provided a restricted pathway for adhesive penetration and determined the tag morphology. However,the lack of colocalization between both fluorescent labels suggest that morphological or chemical interactions between dentin proteoglycans and the adhesive system was not observed with this method. Supported by 3M, St. Paul MN, USA and DID grant ODO-013/2 University of Chile.

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