Localization And Quantification Of Advanced Glycation End-Products In Dentinal Caries

Objectives: Crosslinked collagen, also known as advanced glycation end-products (AGEs), may play a role in the progression of caries infection. To investigate the relationship between sound and caries-affected dentin, we used transmission electron microscopy (TEM), immunohistochemistry, fluorescence lifetime measurement, and Western blotting.
Methods: Sound and caries-affected human teeth were obtained as part of standard dental treatment, after obtaining informed consent. Each tooth was fixed in 4% paraformaldehyde, and was cut longitudinally into 1-mm sections using a low-speed diamond saw. (1) TEM: Non-decalcified teeth were embedded in EPON, sliced into thin sections and observed. (2) Immunohistochemistry: Teeth were decalcified for 2 weeks in 10% EDTA, and were then paraffin-embedded and sectioned with a microtome. Sections were stained with anti-AGE and anti-collagen antibodies. (3) Fluorescence lifetime measurement: Decalcified teeth were observed under a fluorescence microscope equipped with a time correlated single photon counting (TCPLC) system. (4) Western blotting: Non-decalcified teeth were separated into caries-affected and sound regions, and were homogenized with a bead crusher and ultra-sonic homogenizer. Each sample was pepsin-solubilized or treated with 3N HCl.
Results: (1) In caries-affected areas, the structure of collagenous fibers became amorphous, and the intensity of diffraction rings in caries-affected regions was low. (2) The localization of AGEs in caries-affected dentin was similar to areas showing bacterial invasion. (3) Fluorescence lifetime was shorter in the caries-affected area than the sound areas. (4) The accumulation of AGEs in caries-affected dentin was higher than in sound dentin treated with HCl. However, there were no significant differences when compared with pepsin-solubilized dentin.
Conclusions: The ultrastructure of type1 collagen and the total amount of AGEs varied markedly in the dentinal caries region. The increase in AGEs in caries-affected regions may influence the progression of dental caries.