Interaction of epigallocatechin-gallate and chlorhexidine with Streptococcus mutans stimulated odontoblast-like cells: Cytotoxicity, Interleukin-1ß and co-species proteomic analyses.

OBJECTIVES: The dentin therapeutic agent chlorhexidine has inflammatory and cytotoxic characteristics urging investigation of alternatives like the natural compound epigallocatechin-gallate. The aim is to verify the effect of epigallocatechin-gallate and chlorhexidine on viability, interleukin-1ß (IL-1ß) and differential protein expression of MDPC-23 odontoblast-like cells stimulated by Streptococcus mutans. DESIGN: Cells were stimulated with heat-killed S. mutans at multiplicity of infection (MOI) of 100-1000 and subsequently treated with 100-1 µM of epigallocatechin-gallate. Cells with no treatment or chlorhexidine were controls. Combined stimulated/treated cells were tested for cytotoxicity (Alamar-Blue, N = 3, n = 3), total protein (N = 3, n = 3), IL-1ß (ELISA, N = 3, n = 3), and differential protein expression by liquid chromatography-tandem mass spectrometry (LC-MS/MS, n = 2). RESULTS: Cells stimulated at MOI 100/1000 and treated with 10 µM epigallocatechin-gallate and chlorhexidine did not present cytotoxicity. IL-1ß significantly increased in both un-stimulated and stimulated chlorhexidine 10 µM groups when compared to un-treated control (p < 0.05). MOI 100 chlorhexidine 10 µM group significantly increased IL-1ß compared to un-stimulated chlorhexidine 10 µM and epigallocatechin-gallate 10 µM groups, as well as to MOI 100 epigallocatechin-gallate 10 µM group (p < 0.05). LC-MS/MS revealed S. mutans and mammalian proteins, with tooth-specific proteins exhibiting different abundance levels, depending on the tested condition. CONCLUSIONS: Odontoblast-like cells stimulated with S. mutans at different MOI combined with epigallocatechin-gallate treatment did not cause cytotoxicity. S. mutans stimulation combined with chlorhexidine 100 µM treatment decreased cell viability, while treatment with chlorhexidine 10 µM concentration significantly increased IL-1ß. S. mutans stimulation and treatment of cells resulted in varied protein expression.

Profiling cytokine levels in chlorhexidine and EGCG-treated odontoblast-like cells.

OBJECTIVE: To screen the effect of two compounds, chlorhexidine diacetate (CHX) and epigallocatechin-gallate (EGCG), on the levels of cytokines produced by odontoblast-like cells (MDPC-23). METHODS: Cells were seeded at 24h and 48h with serial dilution of the compounds to determine cell metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n=3). Cells with no compound treatment were used as control (Ctr). For the highest equal non-cytotoxic compound dilution tested at 48h cell treatment, total protein concentration was measured using a Pierce bicinchoninic acid (BCA) assay (n=3), and expression of 23 cytokines was analyzed using the Bio-Plex cytokine assay (n=2). Data were analyzed by one-way ANOVA and Tukey's test (α=5%). RESULTS: The MTT assay revealed that at 24h and 48h, CHX and EGCG did not reduce cell metabolic activity at concentrations of 2.5-20µM (CHX) and 2.5-160µM (EGCG), respectively (p>0.05). At 48h, total protein levels were consistent across all groups for 20µM compound dilution (Ctr: 1.04mg/mL; CHX: 0.98mg/mL; and EGCG: 1.06mg/mL). At 20µM dilution, both CHX and EGCG significantly increased the secretion of IL-1ß, IL-10, IL-12, KC, MIP-1α, IFN-γ and IL-6 (p<0.05). Treatment with CHX significantly increased secretion of IL-4 and RANTES (p<0.05). TREATMENT: with EGCG significantly increased Eotaxin secretion (p<0.05). Both CHX and EGCG significantly decreased secretion of IL-17 (p<0.05). GM-CSF and TNF-α did not present significant change in secretion after treatment with either CHX or EGCG (p>0.05). SIGNIFICANCE: Both CHX and EGCG modulate secretion of various inflammatory and anti-inflammatory mediators in odontoblastic cells.

Anthocyanin incorporated dental copolymer: bacterial growth inhibition, mechanical properties, and compound release rates and stability by (1)h NMR.

Objective. To evaluate bacterial growth inhibition, mechanical properties, and compound release rate and stability of copolymers incorporated with anthocyanin (ACY; Vaccinium macrocarpon). Methods. Resin samples were prepared (Bis-GMA/TEGDMA at 70/30 mol%) and incorporated with 2 w/w% of either ACY or chlorhexidine (CHX), except for the control group. Samples were individually immersed in a bacterial culture (Streptococcus mutans) for 24 h. Cell viability (n = 3) was assessed by counting the number of colony forming units on replica agar plates. Flexural strength (FS) and elastic modulus (E) were tested on a universal testing machine (n = 8). Compound release and chemical stability were evaluated by UV spectrophotometry and (1)H NMR (n = 3). Data were analyzed by one-way ANOVA and Tukey's test ( α = 0.05). Results. Both compounds inhibited S. mutans growth, with CHX being most effective (P < 0.05). Control resin had the lowest FS and E values, followed by ACY and CHX, with statistical difference between control and CHX groups for both mechanical properties (P < 0.05). The 24 h compound release rates were ACY: 1.33 µg/mL and CHX: 1.92 µg/mL. (1)H NMR spectra suggests that both compounds remained stable after being released in water. Conclusion. The present findings indicate that anthocyanins might be used as a natural antibacterial agent in resin based materials.