Characterisation of mineral loss as a function of depth using confocal laser scanning microscopy to study erosive...

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.

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.

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.

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.