Identification of a protein-containing enamel matrix layer which bridges with the dentine-enamel junction of adult human teeth

Abstract

Objective: To investigate the ultrastructure and chemical composition of the dentine-enamel junction and adjacent enamel of minimally processed third molar tooth sections.

Design: Undecalcified human third molar erupted teeth were sectioned and etched with 4% EDTA or 37% phosphoric acid prior to visualization by scanning electron microscopy. Confocal Raman spectroscopy was carried out at 50 μm and more than 400 μm away from the dentine-enamel junction before and after mild etching.

Results: A novel organic protein-containing enamel matrix layer was identified for the first time using scanning electron microscopy of etched bucco-lingual sections of crowns. This layer resembles a three-dimensional fibrous meshwork that is visually distinct from enamel "tufts". Previous studies have generally used harsher solvent conditions which likely removed this layer and precluded its prior characterization. The shape of the organic enamel layer generally reflected that of sheath regions of enamel rods and extended from the dentine-enamel junction about 100-400 μm into the cuspal enamel. This layer exhibited a Raman CH stretching peak at ∼2931 cm(-1) characteristic of proteins and this signal correlated directly with the presence and location of the matrix layer as identified by scanning electron microscopy.

Conclusions: The enamel protein layer was most prominent close to the dentine-enamel junction and was largely absent in cuspal enamel >400 μm away from the dentine enamel junction. We hypothesize that this protein containing matrix layer could provide an important biomechanical linkage between the enamel and the dentine-enamel junction and by extension, with the dentine, of the adult tooth (246 words).

Fig. 1. Identification of interphase organic matrix layer within enamel of mature teeth near the dentin enamel junction

a, a diagram illustrating shape and orientation of tooth sections used in this study. b, specimen processed according to “minimalist” protocol and observed without coating at 1 kV accelerating voltage. c, the microstructure of organic matrix of the “minimalist” specimen, observed on Au-Pd coated specimen. d, the microstructure of underlying enamel rods after removing of organic matrix from “minimalist” specimen with sodium hypochlorite. e, specimen processed according to “minimalist” protocol and backscattered electron image collected at 5 kV. f, specimen processed according to “minimalist” protocol and backscattered electron image collected at 15 kV. g, specimen processed according to “minimalist” protocol and backscattered electron image collected at 25 kV. Individual bars refer to microscopic scales for each image.

Fig. 2. Ultrastructural characteristics of the enamel interphase matrix layer

a, appearance of organic enamel matrix at fracture surface prefixed for 6 days, etched in EDTA for 90 min, fixed in glutaraldehyde, post fixed in OsO₄ and subjected to critical point drying. b, higher magnification view of enamel organic matrix at fracture surface after specimen prefixation for 6 days, etching in phosphoric acid for 45 sec, fixing in glutaraldehyde, and critical point drying. c, enamel hydroxyapatite crystals after etching in phosphoric acid for 45 sec, fixing in glutaraldehyde and drying (same magnification and same specimen as in 1B above). d, collagen network of dentin (same magnification, processing, and specimen as in 1C above) (bar=500 nm). Individual bars refer to microscopic scales for each image. KEY: E, enamel; OM, organic matrix; D, dentin

Fig. 3. Identification of sheath-like structures within the enamel interphase matrix

a, appearance of organic enamel matrix at flat surface of tooth section after etching in EDTA for 90 min, fixation in glutaraldehyde, post fixing in OsO₄ and critical point drying. b, interphase matrix at fracture surface after prefixation for 3 days, etching in phosphoric acid for 15 sec, fixation in glutaraldehyde, post fixation in OsO₄, and critical point drying. Asterisks refer to mineral crystals within sheath-like structures. c, higher magnification view of same specimen as in B. Asterisks refer to mineral crystals within sheath-like structures. d, appearance of region near the dentin-enamel junction at flat surface after prefixation for 6 days, etching in EDTA for 60 min, fixation in glutaraldehyde, and dehydration with HMDS. e, higher magnification view of same specimen as in D. f, appearance of region at fracture surface after prefixation for 6 days, etching in EDTA for 30 min, fixation in glutaraldehyde, post fixation in osmium tetroxide and critical point drying with built in magnetic stirrer turned on (jagged edge of dentin-enamel junction readily visible, arrows). g, higher magnification view of same specimen as in F (collagen bundles readily visible). Individual bars refer to microscopic scales for each image. KEY: E, enamel; OM, organic matrix; D, dentin; DEJ, dentin-enamel junction

Fig. 4. Confocal Raman spectroscopic analysis of the enamel interphase matrix

Data shown are representative of scans of sectioned teeth from four different individuals. A, baseline corrected Raman spectra of enamel at a location 50 μm (Inner) from the dentin-enamel junction. Spectra were recorded before and after sequential etching and bleaching steps. Five separate scans were made at different sites for each condition and averaged to generate the spectra shown. A higher resolution view of the region of interest between 2500 and 3200 cm⁻¹ is presented in B. KEY: NEt, non-etched; Et, etched; and, Et/B, etched and bleached. B, baseline corrected Raman scans of a sectioned tooth at two different locations, near the dentin-enamel junction (Inner) and at least 400 μm away from the dentin-enamel junction (400 μm). Spectra were recorded at these locations before and after etching and bleaching steps. Five separate scans were made at each enamel location for each condition and averaged to generate the curves shown. KEY: Inner, enamel 50 μm from the dentin-enamel junction; 400 μm, enamel at least 400 μm away from the dentin-enamel junction; NEt, non-etched; Et, etched; and, Et/B, etched and bleached.