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

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.

Do matrix metalloproteinase and cathepsin K inhibitors work synergistically to reduce dentin erosion?

OBJECTIVES: To evaluate the effects of matrix metalloproteinase (MMP) and cathepsin K (catK) inhibitors on resistance to dentin erosion. METHODOLOGY: A total of 96 dentin specimens (3×3×2 mm) were prepared and randomly assigned into four groups (n=24): deionized water (DW); 1 µM odanacatib (ODN, catK inhibitor); 1 mM 1,10-phenanthroline (PHEN, MMP inhibitor); and 1 µM odanacatib + 1 mM 1,10-phenanthroline (COM). Each group was further divided into two subgroups for the application of treatment solutions before (PRE) and after erosive challenges (POST). All specimens were subjected to four daily erosive challenges for 5 d. For each erosive challenge, the specimens in subgroup PRE were immersed in the respective solutions before cola drinks, while the specimens in subgroup POST were immersed in the respective solutions after cola drinks (the immersion duration was 5 min in both cases). All specimens were stored in artificial saliva at 37°C between erosive challenges. The erosive dentin loss (EDL) was measured by profilometry. The residual demineralized organic matrix (DOM) of specimens was removed using type VII collagenase and evaluated by profilometry. Both the EDL and thickness of the residual DOM were statistically analyzed by two-way analysis of variance (ANOVA) and Bonferroni's test (α=0.05). The surface topography and transverse sections of the specimens were observed using SEM. MMPs and catK were immunolabeled in the eroded dentin and in situ zymography was performed to evaluate the enzyme activity. RESULTS: Significantly lower EDL was found in the groups ODN, PHEN, and COM than in the control group (all p<0.05), while no significant difference in EDL was found among the groups ODN, PHEN, and COM (all p>0.05). The application sequence showed no significant effect on the EDL of the tested groups (p=0.310). A significantly thicker DOM was observed in the group ODN than in the control group regardless of the application sequence (both p<0.05). The treatment with ODN, PHEN, and COM inhibited the gelatinolytic activity by approximately 46.32%, 58.6%, and 74.56%, respectively. CONCLUSIONS: The inhibition of endogenous dentinal MMPs and catK increases the acid resistance of human dentin but without an apparent synergistic effect. The inhibition of MMPs and catK is equally effective either before or after the acid challenge.

Do matrix metalloproteinase and cathepsin K inhibitors work synergistically to reduce dentin erosion?

Abstract Objectives To evaluate the effects of matrix metalloproteinase (MMP) and cathepsin K (catK) inhibitors on resistance to dentin erosion. Methodology A total of 96 dentin specimens (3×3×2 mm) were prepared and randomly assigned into four groups (n=24): deionized water (DW); 1 µM odanacatib (ODN, catK inhibitor); 1 mM 1,10-phenanthroline (PHEN, MMP inhibitor); and 1 µM odanacatib + 1 mM 1,10-phenanthroline (COM). Each group was further divided into two subgroups for the application of treatment solutions before (PRE) and after erosive challenges (POST). All specimens were subjected to four daily erosive challenges for 5 d. For each erosive challenge, the specimens in subgroup PRE were immersed in the respective solutions before cola drinks, while the specimens in subgroup POST were immersed in the respective solutions after cola drinks (the immersion duration was 5 min in both cases). All specimens were stored in artificial saliva at 37°C between erosive challenges. The erosive dentin loss (EDL) was measured by profilometry. The residual demineralized organic matrix (DOM) of specimens was removed using type VII collagenase and evaluated by profilometry. Both the EDL and thickness of the residual DOM were statistically analyzed by two-way analysis of variance (ANOVA) and Bonferroni's test (α=0.05). The surface topography and transverse sections of the specimens were observed using SEM. MMPs and catK were immunolabeled in the eroded dentin and in situ zymography was performed to evaluate the enzyme activity. Results Significantly lower EDL was found in the groups ODN, PHEN, and COM than in the control group (all p<0.05), while no significant difference in EDL was found among the groups ODN, PHEN, and COM (all p>0.05). The application sequence showed no significant effect on the EDL of the tested groups (p=0.310). A significantly thicker DOM was observed in the group ODN than in the control group regardless of the application sequence (both p<0.05). The treatment with ODN, PHEN, and COM inhibited the gelatinolytic activity by approximately 46.32%, 58.6%, and 74.56%, respectively. Conclusions The inhibition of endogenous dentinal MMPs and catK increases the acid resistance of human dentin but without an apparent synergistic effect. The inhibition of MMPs and catK is equally effective either before or after the acid challenge.

Bonding to zirconia: Effects of translucency and cusp inclinations using an experimental methodology.

Purpose To investigate the effects of translucency and cusp inclination on the micro-shear bond strength (µSBS) of zirconia to self-adhesive resin cement (SARC).Methods Sixty U-shaped zirconia specimens with 1.0 mm thickness consisting of three planes (0°, 20°, and 30°) simulating different cusp inclinations were fabricated from zirconia blocks of high, moderate, and low translucency (n=20). SARC was bonded to the specimen and light-cured for 40 s. The µSBS was tested using a universal testing machine with a cross-head at a speed of 0.5 mm/min and fracture mode was observed using a stereomicroscope (×50). Two-way analysis of variance followed by a post hoc Tukey's honest significant difference test was used for the statistical analyses (α=0.05).Results Varied µSBS values were found in the test groups (7.1-14.9 MPa). Within the zirconia of the same translucency, the µSBS value of the plane 0° specimen was significantly higher than that of the 20° and 30° planes (all p<0.05). Within the zirconia of the same cusp inclination, the µSBS value of highly translucent zirconia was significantly higher than that of moderate and low translucent zirconia (all p<0.05). There was no significant interaction between cusp inclination and translucency (p=0.525). Among the fracture modes, 83.33% were adhesive failure, 11.11% mixed failure, and 5.56% were cohesive failure.Conclusions Translucency and cusp inclination significantly affect the µSBS of zirconia to SARC. The higher the translucency, the greater are the µSBS values; the higher the cusp inclination, the lower are the µSBS values.

In Vitro Cytotoxicity of Self-Adhesive Dual-Cured Resin Cement Polymerized Beneath Three Different Cusp Inclinations of Zirconia.

The aim of the present study was to evaluate the in vitro cytotoxicity of self-adhesive dual-cured resin cement (SADRC) polymerized beneath three different cusp inclinations of zirconia with different light curing time. A commercial SADRC (Multilink Speed) was polymerized beneath zirconia (ZrO2) with three different cusp inclinations (0°, 20°, and 30°) for 20 s or 40 s. After being stored in light-proof box for 24 h, the ZrO2-SADRC specimens were immersed in DMEM for 72 h and then we got the extract solution, cultured the human gingival fibroblasts (HGF, 8 × 103 per well) with 100% or 50% concentrations of the extract solution for 24 h, 72 h, and 120 h, respectively, and evaluated cytotoxicity of the polymerized SADRC with CCK-8 assay in optical density (OD) values, relative growth rates (RGR), and cytotoxicity grades. Statistical analysis was conducted using a two-way ANOVA followed by post hoc Student-Newman-Keuls test. The OD values varied from 0.8930 to 3.2920, the RGR varied from 33.93% to 98.68%, and the cytotoxicity grades varied from 0 to 2. There was significant difference in the OD values among the different cusp inclinations of zirconia (P < 0.001), and there was significant difference in the OD values between the different light curing times in some situations (P < 0.05). The cusp inclination of zirconia affects the in vitro cytotoxicity of SADRC. Prolonging the light curing time from 20 s to 40 s can reduce the in vitro cytotoxicity of SADRC when the cusp inclination of zirconia is smaller than 20°.

Effects of Cusp Inclination and Light-curing Time on Microshear Bond Strength of a Dual-cure, Self-adhesive Composite Cement to Zirconia.

PURPOSE: To evaluate the effect of cusp inclination and light-curing time on the microshear bond strength (µSBS) between zirconia and a dual-cure self-adhesive composite cement and zirconia. MATERIALS AND METHODS: Forty U-shaped specimens with angled surfaces, simulating different cusp inclinations (0 degrees, 20 degrees, and 30 degrees), were fabricated from zirconia blocks (Zenostar, Wieland). The specimens were randomly divided into two groups (n = 20) according to different light-curing times of the adhesive composite cements (20 s and 40 s). Three polyethylene tubes filled with a dual-curing self-adhesive composite cement (Multilink Speed, Ivoclar Vivadent) were bonded to the surfaces of each specimen with light-curing times of 20 s and 40 s. The µSBS was tested using a universal testing machine with a crosshead speed of 0.5 mm/min. Failure analysis was performed. Two-way ANOVA (cusp inclination and light-curing time) followed by two independent samples t-test were used for statistical analyses (α = 0.05). RESULTS: The composite cement showed various bond strengths to angled zirconia surfaces (7.07 ± 1.69 to 13.91 ± 3.11 MPa). The µSBS between composite cement and 0-degree zirconia surfaces was statistically higher than that of 20- and 30-degree zirconia surfaces. The composite cement light cured for 40 s showed nonsignificantly higher µSBS than did the composite cement cured for 20 s. Adhesive failure was the most common failure mode. CONCLUSIONS: Cusp inclination affected bond strength between the dual-curing self-adhesive composite cement and zirconia.