The activity, distribution, and colocalization of cathepsin K and matrix metalloproteases in intact and eroded dentin

Abstract

Objectives: This study aimed to assess the activity, distribution, and colocalization of cathepsin K (catK) and matrix metalloproteases (MMPs) in both intact and eroded dentin in vitro.

Materials and methods: Eroded dentin was obtained by consecutive treatment with 5% citric acid (pH = 2.3) for 7 days, while intact dentin remained untreated. Pulverized dentin powder (1.0 g) was extracted from both intact and eroded dentin using 5 mL of 50 mM Tris-HCl buffer (0.2 g/1 mL, pH = 7.4) for 60 h to measure the activity of catK and MMPs spectrofluorometrically. In addition, three 200-μm-thick dentin slices were prepared from intact and eroded dentin for double-labeling immunofluorescence to evaluate the distribution and colocalization of catK and MMPs (MMP-2 and MMP-9). The distribution and colocalization of enzymes were analyzed using inverted confocal laser scanning microscopy (CLSM), with colocalization rates quantified using Leica Application Suite Advanced Fluorescent (LAS AF) software. One-way analysis of variance (ANOVA) was used to analyze the fluorescence data related to enzyme activity (α = 0.05).

Results: The activity of catK and MMPs was significantly increased in eroded dentin compared with intact dentin. After erosive attacks, catK, MMP-2, and MMP-9 were prominently localized in the eroded regions. The colocalization rates of catK with MMP-2 and MMP-9 were 13- and 26-fold higher in eroded dentin, respectively, than in intact dentin.

Conclusions: Erosive attacks amplified the activity of catK and MMPs in dentin while also altering their distribution patterns. Colocalization between catK and MMPs increased following erosive attacks.

Clinical relevance: CatK, MMP-2, and MMP-9 likely play synergistic roles in the pathophysiology of dentin erosion.

Keywords: Collagen; Fluorescence; Fluorescent antibody technique; Proteolytic enzymes; Tooth erosion.