Collagen degrading enzymes localization in sound and caries affected dentin

Method: Longitudinal and transversal slices (200 µm) from freshly intact and carious third molars crows were fixed with 2% formaldehyde. The teeth slices were incubated with primary antibodies or with pre-immune serum (negative controls), diluted in assay buffer. Then, the slices were washed and re-incubated with the respective fluorescent secondary antibody diluted in assay buffer. Collagen images were obtained by secondary harmonic generation (SHG). The localization of CTs and MMPs in labeled teeth slices and the localization of collagen were evaluated by multi-photon confocal microscopy LSM 510 (Carl Zeiss, Germany). The acquired images were analyzed with LSM Image Browser software, which also measured quantitatively the co-localization ratio of proteases and collagen in predentin and dentin. Data were analyzed by ANOVA and Tukey’s post hoc tests (p < 0.05).

Result: MMPs and CTs were localized in different compartments of the sound and caries affected dentin-pulp complex. However, higher expression of some proteases was clearly shown in caries affected dentin when compared to sound dentin, especially for CT-B, CT-K and MMP-9 (p<0.05). Collagen images in caries affected dentin showed different pattern than sound dentin, with no or reduced SHG signal.

Conclusion: The higher localization of MMPs and CTs in caries affected dentin also suggests that these proteases may be responsible for the collagen degradation after demineralization by bacterial acids in caries lesions.