Characterisation of mineral loss as a function of depth using confocal laser scanning microscopy to study erosive lesions in enamel: A novel non-destructive image processing model

Abstract

Objective: The aim was to develop a novel image processing protocol for confocal laser scanning microscopy (CLSM) to study mineral distribution within erosive lesions as a function of depth.

Methods: Polished bovine enamel samples (n = 80) were divided into groups (8/group) with similar mean surface microhardness (SMH) values. Samples underwent erosion (1 % citric acid pH3.8) for 1,5,10,15, or 30 min, with or without stirring giving 10 treatment groups in a 2*5 factorial design. SMH was used to measure erosive softening. Profilometry was used to measure bulk tissue loss. Samples were then stained with rhodamine-B (0.1 mM, 24 h) and imaged using CLSM. Image processing was used to measure fluorescence volume (FV) as a function of depth for each image. The data from reference images were subtracted from post-erosive data to determine changes in fluorescent volume (ΔFV) as a function of depth. 2-way ANOVA and linear regression analysis were used where applicable.

Results: Surface softening and bulk tissue loss increased with acid erosion duration with or without stirring. Stirring significantly increased net softening at each time point; specimens underwent significantly more bulk tissue loss (P < 0.05). CLSM showed the erosive lesion deepened as exposure to acid increased, and that at the near surface (0-10 μm) FV and ΔFV increased rapidly for stirred solutions. The increase in pore space translated to a softer surface as measured by SMH.

Conclusion: This novel non-destructive method allows concurrent quantification of dental erosion by mineral loss as a function of depth, and qualitative characterisation of microstructural changes during early erosion.

Keywords: Confocal; Enamel; Erosion; Porosity.