In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested

Review of nanomaterials in dentistry: interactions with the oral microenvironment, clinical applications, hazards, and benefits.

Interest in the use of engineered nanomaterials (ENMs) as either nanomedicines or dental materials/devices in clinical dentistry is growing. This review aims to detail the ultrafine structure, chemical composition, and reactivity of dental tissues in the context of interactions with ENMs, including the saliva, pellicle layer, and oral biofilm; then describes the applications of ENMs in dentistry in context with beneficial clinical outcomes versus potential risks. The flow rate and quality of saliva are likely to influence the behavior of ENMs in the oral cavity, but how the protein corona formed on the ENMs will alter bioavailability, or interact with the structure and proteins of the pellicle layer, as well as microbes in the biofilm, remains unclear. The tooth enamel is a dense crystalline structure that is likely to act as a barrier to ENM penetration, but underlying dentinal tubules are not. Consequently, ENMs may be used to strengthen dentine or regenerate pulp tissue. ENMs have dental applications as antibacterials for infection control, as nanofillers to improve the mechanical and bioactive properties of restoration materials, and as novel coatings on dental implants. Dentifrices and some related personal care products are already available for oral health applications. Overall, the clinical benefits generally outweigh the hazards of using ENMs in the oral cavity, and the latter should not prevent the responsible innovation of nanotechnology in dentistry. However, the clinical safety regulations for dental materials have not been specifically updated for ENMs, and some guidance on occupational health for practitioners is also needed. Knowledge gaps for future research include the formation of protein corona in the oral cavity, ENM diffusion through clinically relevant biofilms, and mechanistic investigations on how ENMs strengthen the tooth structure

The susceptibility of Streptococcus mutans to antibacterial photodynamic therapy: a comparison of two different photosensitizers and light sources

Streptococcus mutans is the main etiological agent for dental caries. Recently, photodynamic therapy (PDT) has been introduced as a new modality in bacterial decontamination. OBJECTIVE: This in vitro study was carried out to evaluate the susceptibility of Streptococcus mutans to antibacterial PDT using two different photosensitizers and light sources. MATERIAL AND METHODS: Standard suspensions of S. mutans were exposed to laser light at 662 nm and Radachlorin(®) or LED 630 nm in combination with Toluidine blue O (TBO). Radiation-only groups, photosensitizer alone, and groups with no treatment were used as controls. Bacterial suspension from each treatment was subcultured onto the surface of Mueller-Hinton agar plates and bacterial growth was assessed. The results were analyzed by analysis of variance and Tukey test (p<0.05). RESULTS: PDT with TBO and Radachlorin(®) significantly reduced S. mutans viability, whereas no difference was observed between two groups of PDT. In the groups treated just with the photosensitizer or irradiated alone, no significant reduction of S. mutans colonies was observed. CONCLUSION: S. mutans colonies were susceptible to either 662 nm laser or LED light in the presence of Radachlorin(®) and TBO respectively with no priority

Effects of low-dose doxycycline on cytokine secretion in human monocytes stimulated with Aggregatibacter actinomycetemcomitans

Doxycycline is an antibiotic used in the treatment of a variety of inflammatory conditions, including periodontitis. Apart from its antimicrobial properties, this drug also has independent anti-inflammatory effects at sub-antimicrobial doses. The present study aimed to investigate the effects of low-doses of doxycycline (LDD) on cytokine production by human monocytic cells challenged with the periodontal pathogen Aggregatibacter actinomycetemcomitans, for up to 6h. The simultaneous regulation of 12 cytokines were measured by a Human Cytokine Array Kit. To validate the array findings, selected cytokines were also measured by enzyme-linked immunosorbant assay (ELISA). A. actinomycetemcomitans stimulated the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6 and IL-8 by the cells after 6h of challenge, and doxycycline significantly inhibited this effect. The kinetics of this regulation demonstrated an early (within 2h) and significant (P<0.05) inhibition of pro-inflammatory cytokines, with a mild (0.5-fold) up-regulation of the anti-inflammatory cytokine IL-10. The results indicate that LDD acts as an anti-inflammatory agent in human monocytic cells stimulated with A. actinomycetemcomitans. This model provides clear evidence that some of the clinically proven benefits of LDD may be related to its ability to regulate inflammatory mediator release by monocytic cells. This property may contribute to the clinically proven benefits of this antibiotic as an adjunctive treatment for periodontitis

Nanomaterials as antimicrobials

The chapter considered the use of nano-particulate metals and metal oxides as antimicrobial agents that have the potential to provide an additional functionality to polymeric materials.Non peer reviewe