A Simple Method To Specifically Extract The Dentin-Enamel Junction Contents

Objectives: We have shown by immunofluorescent staining that type IV collagen is enriched within the dentin-enamel junction (DEJ) and that an adjacent enamel organic matrix (EOM) layer extends ~200 um into bulk phase enamel. We hypothesize that type IV collagen stabilizes the dentin-enamel interface. The goal was to determine if the structure of type IV collagen in the DEJ and EOM was different.
Methods: The EOM of adult human third molar crowns and young adult mouse molars was extracted with 0.5M EDTA; extracts were dialyzed against 5% acetic acid and lyophilized. Teeth were subsequently extracted with 8M urea/0.5% CHAPS or 8M urea/0.1% SDS (denaturing conditions). Extracts were subjected to Western blotting with reduction.
Results: EDTA enamel extracts from mouse and human molars were enriched in a ~25 kDa fragment of type IV collagen. Subsequent extraction of the residual DEJ-associated matrix with denaturing conditions yielded two high molecular weight type IV collagen chains (Mr>200 kDa) but no ~25 kDa fragment. Preliminary evidence supports a similar differential distribution for fragments and macromolecular forms of laminin and type VII collagen.
Conclusions: Mild decalcification/extraction readily removes the EOM layer and shows the EOM is enriched in fragments of type IV collagen and other extracellular matrix proteins. In contrast, denaturing conditions are required to release macromolecular type IV collagen. These results provide an explanation for why immunostaining of EDTA-treated tooth sections previously detected type IV collagen only in the DEJ and not the EOM. Specifically, ~25 kDa fragments in the EOM were likely removed by EDTA pretreatment, whereas tightly bound macromolecular type IV collagen at the DEJ was not. We hypothesize only macromolecular type IV collagen participates in stabilizing the dentin-enamel interface. In summary, this work provides a simplified method to specifically extract the contents of the dentin-enamel junction. Supported by UMKC SOD Summer Scholars program.