Objectives: Collagen degradation is a crucial step in various dental pathologies. Cysteine cathepsins (CT) and matrix metalloproteinases (MMPs) are thought to play an important role in collagen degradation. This study evaluated the expression and localization of CTs and MMPs in dentin-pulp complex by confocal fluoresecnce microscopy.
Method: Freshly intact third molars were collected and carefully cleaned. Longitudinal and transversal slices (0.3 mm) were obtained from the crown segments of teeth, which were fixed with 2% formaldehyde (30 min). The teeth slices were incubated with primary antibodies (CTs, Sigma or MMPs, Santa Cruz) or with pre-immune serum (negative controls), diluted in assay buffer. After 2h-incubation, the slices were washed and re-incubated with the respective fluorescent secondary antibody, diluted in assay buffer. The expression/localization of CTs (CT-B and CT-L) and MMPs (MMP-2 and MMP-9) in labeled teeth slices and the localization of collagen were evaluated by multi-photon confocal microscopy LSM 510 (Carl Zeiss, Germany). Collagen images were obtained by secondary harmonic generation. 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 Holm-Sidak tests.
Results: The presence of the investigated proteases was observed in different compartments of the dentin-pulp complex. Higher expression of CT-B in comparison to CT-L; and MMP-9 in comparison to MMP-2 was clearly shown (p<0.05). Invariably, the presence and co-localization of proteases with collagen was more evident in predentin and in the half-potion of dentin that was bordering the predentin.
Conclusion: Co-expression of CTs and MMPs and their co-localization with collagen in human tooth indicate that these proteases may work in concert during the disruption of collagen in dental pathologies and in poorly resin-infiltrated hybrid layers.
Funding: FAPESP 07/54618-4 (P.I. Carrilho)