Remineralization of in vitro Root Caries after Exposure to Human Neutrophil Enzymes

Although there is a consensus that root caries (RC) progression depends on the degradation of the dentin extracellular matrix (dECM) by host proteolytic enzymes, source(s) of these enzymes are not completely known. Proteolytic enzymes derived from human neutrophils have been associated with ECM degradation and tissue damage in multiple systemic diseases. We hypothesized that neutrophil enzymes may prevent caries remineralization through dECM degradation. This study evaluated the potential of human neutrophil enzymes on compromising in vitro RC remineralization. For that, human root dentin specimens (~2×4×4 mm) were covered by nail polish except for a central area (~3×3 mm) where RC lesions were created by a chemical model (pH 4.6; 37°C and 5% CO2). After 4-day RC formation, half of each lesion was covered to keep the baseline lesion (initial lesion; IL). The other half was exposed to one of the following groups (n=10): Control (DPBS buffer; negative control; C); Neutrophils (N); Collagenase (100 μg/mL; positive control; Col) and incubated (37 °C and 5% CO2) for 3 weeks. Solutions were renewed 1×/week. Neutrophils were isolated from human peripheral blood by density gradient centrifugation. Following 3-week incubation, the second half of all specimens was 8-day pH-cycled and 3-min treated with sodium fluoride (5000 ppm; 2×/day) to induce remineralization (final lesion; FL). The porosity and depth of the RC lesions (n=10) were evaluated by rhodamine infiltration using confocal laser scanning microscopy and the final remineralization percentage (IL-FL) for each specimen was calculated. Additionally, RC specimens from different groups (n=3) were fully demineralized in EDTA for 3 weeks and 8 µm-histological sections were obtained, stained with picrosirius red staining and evaluated by polarized light microscope. Caries porosity and depth data were analyzed by Analysis of Variance (ANOVA) followed by Tukey test or T test at α=0.05 using SPSS 25 software. Results: There were statistically significant differences (p≤0.05) in porosity of the remaining RC between N (p=0.049) and Col (p=0.041) groups in comparison to C group, as showed by Analysis of Variance and Tukey. Statistically significant differences (p≤0.05) in porosity between the IL and FL within each group were detected by T test, except for N group. No statistically significant differences in depth of the remaining RC were observed among groups (p=0.832). Discontinuity of the dECM in RC exposed to neutrophils and collagenase was observed on histological sections. Human neutrophil enzymes compromise in vitro RC remineralization by dECM degradation. Funding: UIC College of Dentistry, Nakao Aging Population Funds; and KL2 (CATS) Scholars Program 2022 (NIH UL 1TR002003). IRB Protocol #: 2020-155. Although there is a consensus that root caries (RC) progression depends on the degradation of the dentin extracellular matrix (dECM) by host proteolytic enzymes, source(s) of these enzymes are not completely known. Proteolytic enzymes derived from human neutrophils have been associated with ECM degradation and tissue damage in multiple systemic diseases. We hypothesized that neutrophil enzymes may prevent caries remineralization through dECM degradation. This study evaluated the potential of human neutrophil enzymes on compromising in vitro RC remineralization. For that, human root dentin specimens (~2×4×4 mm) were covered by nail polish except for a central area (~3×3 mm) where RC lesions were created by a chemical model (pH 4.6; 37°C and 5% CO2). After 4-day RC formation, half of each lesion was covered to keep the baseline lesion (initial lesion; IL). The other half was exposed to one of the following groups (n=10): Control (DPBS buffer; negative control; C); Neutrophils (N); Collagenase (100 μg/mL; positive control; Col) and incubated (37 °C and 5% CO2) for 3 weeks. Solutions were renewed 1×/week. Neutrophils were isolated from human peripheral blood by density gradient centrifugation. Following 3-week incubation, the second half of all specimens was 8-day pH-cycled and 3-min treated with sodium fluoride (5000 ppm; 2×/day) to induce remineralization (final lesion; FL). The porosity and depth of the RC lesions (n=10) were evaluated by rhodamine infiltration using confocal laser scanning microscopy and the final remineralization percentage (IL-FL) for each specimen was calculated. Additionally, RC specimens from different groups (n=3) were fully demineralized in EDTA for 3 weeks and 8 µm-histological sections were obtained, stained with picrosirius red staining and evaluated by polarized light microscope. Caries porosity and depth data were analyzed by Analysis of Variance (ANOVA) followed by Tukey test or T test at α=0.05 using SPSS 25 software. Results: There were statistically significant differences (p≤0.05) in porosity of the remaining RC between N (p=0.049) and Col (p=0.041) groups in comparison to C group, as showed by Analysis of Variance and Tukey. Statistically significant differences (p≤0.05) in porosity between the IL and FL within each group were detected by T test, except for N group. No statistically significant differences in depth of the remaining RC were observed among groups (p=0.832). Discontinuity of the dECM in RC exposed to neutrophils and collagenase was observed on histological sections. Human neutrophil enzymes compromise in vitro RC remineralization by dECM degradation. Funding: UIC College of Dentistry, Nakao Aging Population Funds; and KL2 (CATS) Scholars Program 2022 (NIH UL 1TR002003). IRB Protocol #: 2020-155. Although there is a consensus that root caries (RC) progression depends on the degradation of the dentin extracellular matrix (dECM) by host proteolytic enzymes, source(s) of these enzymes are not completely known. Proteolytic enzymes derived from human neutrophils have been associated with ECM degradation and tissue damage in multiple systemic diseases. We hypothesized that neutrophil enzymes may prevent caries remineralization through dECM degradation. This study evaluated the potential of human neutrophil enzymes on compromising in vitro RC remineralization. For that, human root dentin specimens (~2×4×4 mm) were covered by nail polish except for a central area (~3×3 mm) where RC lesions were created by a chemical model (pH 4.6; 37°C and 5% CO2). After 4-day RC formation, half of each lesion was covered to keep the baseline lesion (initial lesion; IL). The other half was exposed to one of the following groups (n=10): Control (DPBS buffer; negative control; C); Neutrophils (N); Collagenase (100 μg/mL; positive control; Col) and incubated (37 °C and 5% CO2) for 3 weeks. Solutions were renewed 1×/week. Neutrophils were isolated from human peripheral blood by density gradient centrifugation. Following 3-week incubation, the second half of all specimens was 8-day pH-cycled and 3-min treated with sodium fluoride (5000 ppm; 2×/day) to induce remineralization (final lesion; FL). The porosity and depth of the RC lesions (n=10) were evaluated by rhodamine infiltration using confocal laser scanning microscopy and the final remineralization percentage (IL-FL) for each specimen was calculated. Additionally, RC specimens from different groups (n=3) were fully demineralized in EDTA for 3 weeks and 8 µm-histological sections were obtained, stained with picrosirius red staining and evaluated by polarized light microscope. Caries porosity and depth data were analyzed by Analysis of Variance (ANOVA) followed by Tukey test or T test at α=0.05 using SPSS 25 software. Results: There were statistically significant differences (p≤0.05) in porosity of the remaining RC between N (p=0.049) and Col (p=0.041) groups in comparison to C group, as showed by Analysis of Variance and Tukey. Statistically significant differences (p≤0.05) in porosity between the IL and FL within each group were detected by T test, except for N group. No statistically significant differences in depth of the remaining RC were observed among groups (p=0.832). Discontinuity of the dECM in RC exposed to neutrophils and collagenase was observed on histological sections. Human neutrophil enzymes compromise in vitro RC remineralization by dECM degradation. Funding: UIC College of Dentistry, Nakao Aging Population Funds; and KL2 (CATS) Scholars Program 2022 (NIH UL 1TR002003). IRB Protocol #: 2020-155.