7 - Microbiology of Dentin Invasion

Abstract:

Purpose: To develop a method to detect collagen denaturation and cleavage and examine the spatial relationship between altered collagen and localization of bacteria and fungi within carious dentin.

Methods: Carious (n=3) and non-carious (n=3) primary teeth were collected from healthy children ages 2-10 years. Teeth were decalcified, paraffin-embedded, and histologically stained. Immunofluorescence staining with collagen hybridizing peptide and anti-collagen type 1 cleavage site antibody was used to identify collagen denaturation and cleavage, respectively. Streptococcus mutans (MS) and fungi were fluorescently labelled with an antibody against MS and wheat germ agglutinin, respectively and visualized with confocal microscopy. A dual Raman-confocal microscope enabled simultaneous localization of microbes and detection of altered collagen matrix, with spectral data analyzed using background-supervised non-negative matrix factorization.

Results: Immunofluorescence signals indicative of collagen denaturation, collagen cleavage, MS and fungi were detected in all carious teeth. No signals were detected for all four markers in non-carious teeth. In the carious lesion, collagen cleavage was concentrated along the outer dentin edge while collagen denaturation was predominantly localized to the inner dentin. Regions of collagen cleavage colocalized with areas colonized by both MS and fungi, while regions of collagen denaturation colocalized with regions colonized by fungi. Raman spectroscopy identified five distinct spectral signatures corresponding to bacteria, fungi, and collagen structural states.

Conclusions: This study establishes the feasibility of an integrated histologic and multimodal microscopic approach to detect collagen degradation in dental tissues and provides insight into how microbial activity contributes to dentinal extracellular matrix breakdown during caries progression.

Identify Supporting Agency and Grant Number: Research supported by the Paul L. Losche Fund.