In Vitro Evaluation of the Antibacterial Properties of Tea Tree Oil on Planktonic and Biofilm-Forming Streptococcus mutans.

Streptococcus mutans (S. mutans) is the principal etiologic agent in the occurrence of human dental caries and the formation of biofilms on the surface of teeth. Tea tree oil (TTO) has been demonstrated to exhibit a wide range of pharmacological actions that can effectively inhibit the activity of bacteria. In this context, we evaluated the in vitro antimicrobial effects of TTO on S. mutans both during planktonic growth and in biofilms compared with 0.2% CHX. We determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the microdilution method, the bacteriostatic rate using an MTT assay, and the antimicrobial time using a time-kill assay. Then, we explored the effects of TTO on acid production and cell integrity. Furthermore, the effects of TTO on the biomass and bacterial activity of S. mutans biofilms were studied. Finally, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to investigate the structure and activity of biofilms. The MIC and MBC values were 0.125% and 0.25%, and the bacterial inhibition rate was concentration dependent. TTO can effectively inhibit bacterial acid production and destroy the integrity of the cell membrane. Electron micrographs revealed a reduction in bacterial aggregation, inhibited biofilm formation, and reduced biofilm thickness. The effect of TTO was the same as that of 0.2% CHX at a specific concentration. In summary, we suggest that TTO is a potential anticariogenic agent that can be used against S. mutans.

Exogenous nitric oxide stimulates the odontogenic differentiation of rat dental pulp stem cells.

Nitric oxide (NO) is thought to play a pivotal regulatory role in dental pulp tissues under both physiological and pathological conditions. However, little is known about the NO functions in dental pulp stem cells (DPSCs). We examined the direct actions of a spontaneous NO gas-releasing donor, NOC-18, on the odontogenic capacity of rat DPSCs (rDPSCs). In the presence of NOC-18, rDPSCs were transformed into odontoblast-like cells with long cytoplasmic processes and a polarized nucleus. NOC-18 treatment increased alkaline phosphatase activity and enhanced dentin-like mineralized tissue formation and the expression levels of several odontoblast-specific genes, such as runt related factor 2, dentin matrix protein 1 and dentin sialophosphoprotein, in rDPSCs. In contrast, carboxy-PTIO, a NO scavenger, completely suppressed the odontogenic capacity of rDPSCs. This NO-promoted odontogenic differentiation was activated by tumor necrosis factor-NF-κB axis in rDPSCs. Further in vivo study demonstrated that NOC-18-application in a tooth cavity accelerated tertiary dentin formation, which was associated with early nitrotyrosine expression in the dental pulp tissues beneath the cavity. Taken together, the present findings indicate that exogenous NO directly induces the odontogenic capacity of rDPSCs, suggesting that NO donors might offer a novel host DPSC-targeting alternative to current pulp capping agents in endodontics.

Sequential expression of endothelial nitric oxide synthase, inducible nitric oxide synthase, and nitrotyrosine in odontoblasts and pulp cells during dentin repair after tooth preparation in rat molars.

Nitric oxide (NO) stimulates osteoblast differentiation, but whether NO contributes to odontoblast differentiation during dentin repair is unknown. By using reverse transcription/polymerase chain reaction and immunostaining, we investigated the gene expression and/or immunolocalization of endothelial NO synthase (eNOS), inducible NOS (iNOS), and nitrotyrosine (a biomarker for NO-derived peroxinitrite), and alkaline phosphatase (ALP) and osteocalcin (early and terminal differentiation markers of odontoblasts, respectively) in dental pulp tissue after rat tooth preparation. At the early stage (1-3 days) post-preparation, markedly increased expression of iNOS and nitrotyrosine was found in odontoblasts and pulp cells beneath the cavity, whereas eNOS expression was significantly decreased. ALP mRNA expression was significantly increased after 1 day but decreased after 3 days, whereas ALP activity was weak in the dentin-pulp interface under the cavity after 1 day but strong after 3 days. Osteocalcin mRNA expression was significantly increased at this stage. At 7 days post-preparation, tertiary dentin was formed under the cavity. All the molecules studied were expressed at control levels in odontoblasts/pulp cells beneath the cavity. These findings show that abundant NO is released from odontoblasts and pulp cells at an early stage after tooth preparation and indicate that, after tooth preparation, the up-regulation of iNOS and nitrotyrosine in odontoblasts is synchronized with increased cellular expression of ALP and osteocalcin. Therefore, the NO synthesized by iNOS after tooth preparation probably participates in regulating odontoblast differentiation during tertiary dentinogenesis.

Expression of osteocalcin and Jun D in the early period during reactionary dentin formation after tooth preparation in rat molars.

We examined, in rats, the expression of osteocalcin and Jun D in the early stage of reactionary dentin formation after tooth preparation and the accompanying morphological changes. Reverse transcription/polymerase chain reaction analysis revealed strong expression of osteocalcin mRNA in pulp tissue at 2 and 3 days post-preparation compared with that in control teeth. Light microscopy demonstrated that, at the dentin-pulp interface, damaged odontoblasts were detached from the dentin matrix immediately after preparation, with neutrophils lining the dental surface after 1 day. After 2-3 days, differentiated odontoblasts appeared at the interface. Reactionary dentin with tubular structures was formed under the cavity after 10 days. Immunoelectron microscopy showed that trace amounts of osteocalcin were expressed in odontoblasts at 2 days post-preparation, and abundant osteocalcin was found in the highly developed Golgi apparatus and granules at 3 days post-preparation. Osteocalcin was also found on type I collagen fibrils in newly formed predentin. The existing dentinal tubules were filled with osteocalcin-coated type I collagen fibrils. We observed, by immunohistochemistry, that Jun D was temporally expressed in the nuclei of the odontoblasts at 1 and 2 days post-preparation. However, no Jun D was found in the dental pulp cells at any other time or in control teeth. Thus, osteocalcin expression is correlated with reactionary dentin formation, and Jun D is associated with osteocalcin expression in odontoblasts. Osteocalcin may also serve as an obturator of the dentinal tubules to protect dental pulp vitality against external irritants after preparation.