Distribution of SIBLING Proteins in Human Dentin: A Pilot Study

Objectives: The SIBLING protein family is a group of non-collagenous proteins involved in biomineralisation of dentin including dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1) and osteopontin (OPN). The aim of the pilot study is to examine and compare the immunohistochemical localization of these three proteins in human dentin and dental pulp.
Methods: Seven freshly extracted intact third molars of healthy young individuals (mean age 22.2) were enrolled in the study. All teeth were indicated for surgery due to limited jawbone space for eruption. Immediately after extraction the molars were fixed overnight in 10% buffered paraformaldehyde and reduced in size by trimming the enamel, superficial coronal dentin and the roots up to 2mm below the CEJ. The coronal dentin-pulp specimens were decalcified in a 3% hydrochloric acid (HCl) for 6 hours and dehydrated using graded ethanol and acetone, embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin. For immunohistochemistry, paraffin sections were dewaxed in xylene, rehydrated with distilled water, and then subjected to antigen retrieval (sodium citrate buffer, pH 6.0) and incubation with mouse monoclonal antibodies DMP1 (LFMb-31), DSPP (LFMb-21) and OPN (AKm2A1), Santa Cruz Biotechnology Inc., USA using Leica-Bond Max automated system (Leica Biosystems, Germany).
Results: All tissue sections were positively stained for DMP-1 including both the full length of dentin and the odontoblast cells layer. DSPP was positively immunolabeled mainly in odontoblasts in less than a half of the specimens. Immunohistochemical localization of OPN showed narrow spreading only in the deepest dentin layer and odontoblast cells in most of the sections. The predentin was negatively stained in all tested specimens confirming lack of mineralization.
Conclusions: The difference in the distribution of the SIBLING proteins supports the belief that these molecular species play different roles in dentinogenesis in the perspective of age-related physiological changes in pulp-dentin complex.