Real-time binding kinetic analyses of the interaction of the dietary stain orange II with dentin matrix

Objectives: Dietary stains can be adsorbed into the dentin of teeth. Using Orange II as a model dietary stain, this study investigated the strength of its interaction with the mineral and protein components of dentin matrix and how hydrogen peroxide (H2O2) treatment influences this interaction. Methods: Dentin slices were prepared from human teeth and were either deproteinized (5.6% sodium hypochlorite, 12 days), demineralised (0.5 M EDTA, 3 days) or left as intact control samples. Samples were stained with Orange II for 1-168 h, during which staining intensity was quantified by image analysis. Similarly, uptake of stain by deproteinized / demineralized samples treated with 10 or 30% H2O2 was investigated. Using surface plasmon resonance technology, real-time binding kinetics were determined assessing the interaction of Orange II with the dentin matrix protein constituents, collagen type I, biglycan, decorin, dentin sialoprotein and osteopontin. Results: Deproteinization of dentin matrix reduced the uptake of the Orange II compared to the intact control. Conversely, demineralization of dentin samples increased the uptake of the dye. Treatment of samples for 48 h with H2O2 reduced subsequent uptake of the Orange II. Real-time kinetic analysis indicated moderate strength of binding for Orange II with collagen type I, weak binding with decorin and biglycan and negligible binding with dentine sialoprotein and osteopontin. Conclusion: These results indicate a predominant role for collagen type I, which accounts for 90% of the organic protein matrix of teeth, for attracting dietary stains. Binding analyses indicate that the interaction is highly dissociable, and further binding is reduced following H2O2 treatment. Clinical significance: This study provides new information regarding adsorption of dietary stains into tooth dentin, suggesting that they are attracted and moderately bound to the collagen type I matrix. This study also contributes valuable information for discussion for considering the effect of H2O2 on bleaching teeth and its influence on subsequent uptake of dietary stains following whitening treatments.