Protein-Containing Enamel Matrix Layer Bridging the Dentin-Enamel Junction

Objective: To analyze the distribution and quantity of matrix proteins localized to the dentin-enamel junction (DEJ) of mature human molar teeth.  Method: Healthy erupted human third molar extracted teeth were stored for 3-60 days in phosphate buffered saline prior to processing.  Bucco-lingual sections of the crowns were evaluated using scanning electron microscopy (SEM), with sections subjected to essential steps of etching, rinsing and air drying, with no fixation and no controlled dehydration, or, to a full sequence of required steps including final treatment with CPD or HMDS. Five replicate confocal Raman spectra were acquired from each tooth section (n=4 teeth) in two separate cuspal enamel regions, within 50 µm and 400 µm away from the DEJ.  Spectra were acquired from sections before and after etching with 4% EDTA, and following subsequent bleach treatment.  Representative spectra for the two regions of each tooth were obtained by averaging individual scans which were then subjected to baseline adjustment and normalization based on a hydroxyapatite phosphate signal at 430 cm^-1.  Result:  With SEM images, etching revealed that mature enamel contained an organic layer whose shape resembled the sheath regions of the enamel rods predominantly at and near the DEJ.  Following bleach treatment, the majority of this organic layer appeared to be removed in SEM images.  Confocal Raman spectroscopy confirmed this layer contained protein, as evidenced by a peak at ~2950 cm^-1 characteristic of bone proteins with protein/mineral ratios significantly higher (p<0.05) near the DEJ.  Following bleach treatment, the protein/mineral ratios near the DEJ were significantly reduced (p<0.05). Conclusion: We have identified an organic matrix layer in adult enamel that is morphologically distinct from enamel “tufts”.  We hypothesize that this protein-containing matrix layer bridging the DEJ could provide an important biomechanical linkage between enamel and dentin in the adult tooth.