Effect of myricetin on odontoblast-like cells and its potential to preserve resin-dentin Bonds.

Stabilization of the resin-dentin interface to increase the durability of adhesive dental restorations is a challenging task. The use of naturally occurring collagen crosslinking agents has been proposed to prevent degradation of the hybrid layer. Myricetin (MYR) is a flavonoid with a wide variety of beneficial effects and it has been used for the treatment of different systemic pathologies. The chemical structure of MYR makes it a powerful antioxidant, an inhibitor of matrix metalloproteinase (MMP) activity, and a collagen cross-linker. This study presents MYR as a novel treatment in operative dentistry to stabilize the resin-dentin interface by inhibiting MMPs and crosslinking the collagen. Viability tests carried out using a resazurin assay showed that MYR had no cytotoxic effects on human odontoblast-like cells and the phenotype was preserved. Fluorometric MMP activity assay and fluorescence microscopy revealed that the MMPs in the demineralized dentin were effectively inhibited by the application of MYR (600 µM for 120 s). A microtensile bond strength test was performed immediately and after six months of storage. The bond strength to dentin was not affected by MYR and was preserved over time. Demineralized dentin beams were evaluated to determine the dentin biomodification using microtensile strength and elastic modulus assays. MYR improved the biomechanical behavior of the demineralized dentin similarly to glutaraldehyde, a recognized crosslinking agent. These findings indicated that MYR acts as an MMP inhibitor, collagen cross-linker, and preserver of the bond strength. Furthermore, MYR is an ethanol-soluble molecule with a lower molecular weight than the other polyphenols; hence, it can be applied for a short time and diffuses deeply through the dentin without any associated cytotoxicity. This molecule has beneficial effects on the biological and mechanical behavior of the resin-dentin interface and may be used to effectively stabilize the hybrid layer in a clinical setting.

Polymodal Activation and Desensitization of TRPV1 Receptor in Human Odontoblasts-Like Cells with Eugenol.

Dentinal hypersensitivity is a frequent reason for dental consultation, and its pathophysiology has not been fully clarified. Previous findings have made it possible to establish a relationship between the cellular sensory capacity and the activation of the polymodal transient receptor potential vanilloid 1 (TRPV1), which is responsible for the nociceptive response and whose desensitization could cause analgesia. Thus, the objective of this study was to determine the expression, localization, and functional activity of TRPV1 in human odontoblasts-like-cells (hOLCs) and the effect of eugenol (EUG) on its activation and desensitization. Human dental pulp stem cells (hDPSCs) were obtained from third molars and were characterized using flow cytometry, and their differentiation potential toward the osteoblastic, chondrogenic, and adipogenic lineages was investigated. Subsequently, the hDPSCs underwent odontogenic differentiation for 7, 14, and 21 days, and their phenotype (odontogenic markers dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP)) was evaluated using immunofluorescence. The TRPV1 gene expression in hOLCs was estimated using RT-qPCR, and its localization was analyzed using immunofluorescence. Half-maximal effective concentration (EC50) from both eugenol (EUG) and capsaicin (CAP) was determined; in addition, receptor activation was evaluated against chemical, thermal, and pH stimuli. For the statistical analysis, a one-way ANOVA with a Tukey post hoc test (p < 0.05) was used. After establishing the in vitro model of hOLCs and the membrane location of TRPV1, its chemical activation with EUG and CAP was demonstrated, as well as its thermal activation at ≥ 43°C and with an acidic (<6) or basic pH (between 9 and 12). Receptor desensitization was achieved after 20 min of exposure to two concentrations of EUG (603.5 and 1000 µM). These findings represent a stepping-stone for the construction of a pulp pain study model oriented toward a therapeutic alternative for the treatment of dentinal hypersensitivity.

Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation.

Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC) were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-ß1), and OLC expanded after trypsinization (EXP-21) were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry.