In-vitro model of scardovia wiggsiae biofilm formation and effect of nicotine

Abstract Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

Resumo Recentemente, foi relatado que Scardovia wiggsiae está fortemente associado à formação de cáries. Este estudo teve como objetivo estabelecer um modelo in vitro de biofilme de S. wiggsiae e investigar o efeito da nicotina no crescimento de unidades formadoras de colônias (UFC) de S. wiggsiae. O biofilme de S. wiggsiae foi cultivado durante a noite usando caldo de infusão de cérebro-coração (BHI) suplementado com 5 g de extrato de levedura / L (BHI-YE). A cultura noturna foi usada como um inóculo para cultivar biofilme de S. wiggsiae em amostras padronizadas de esmalte e dentina. As amostras foram incubadas com diferentes concentrações de nicotina (0, 0,5, 1, 2, 4, 8, 16 e 32 mg/mL) por 3 dias. Os biofilmes dissociados foram diluídos, semeados em espiral em placas de ágar sangue e incubados por 24 h. UFC/mL foram quantificados usando um contador de colônias automatizado. Uma ANOVA de duas vias foi usada para comparar o efeito de diferentes concentrações de nicotina em UFCs de S. wiggsiae. Este estudo demonstrou que o biofilme de S. wiggsiae pode ser iniciado e formado in vitro. UFCs aumentadas foram observadas com 0,5-4 mg/mL e 0,5-8 mg/mL de nicotina usando substratos de esmalte e dentina, respectivamente. A concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) de nicotina foram determinadas, respectivamente, como 16 e 32 mg/mL. S. wiggsiae formou maior biofilme no esmalte do que espécimes de dentina em resposta ao estímulo de nicotina. Este estudo demonstrou o efeito negativo do tabagismo no aumento do biofilme de S. wiggsiae. O estabelecimento do biofilme de S. wiggsiae in vitro pode permitir que os pesquisadores no futuro tenham uma melhor compreensão da patogênese da cárie e da interação bacteriana.

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. Objective To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. Methodology Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. Results The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). Conclusion Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

Caries lesion remineralization with fluoride toothpastes and chlorhexidine - effects of application timing and toothpaste surfactant

Abstract Habitual toothbrushing with fluoridated toothpaste followed by rinsing with antibacterial mouthwashes is a method to maintain good oral hygiene and to diminish the occurrence and severity of dental caries and periodontal disease. However, our understanding of how antimicrobial agents in mouthwashes affect fluoride-mediated caries lesion remineralization is still poor. Objective: The objectives of this in vitro study were a) to determine the effects of the waiting period of chlorhexidine (CHX) rinsing after fluoride toothpaste use and b) to further determine the effect of the type of toothpaste surfactant [sodium dodecyl sulfate (SDS) or cocamidopropyl betaine (CAPB)] on caries lesion remineralization associated with CHX rinsing. Material and Methods: Caries lesions were formed in bovine enamel specimens and assigned to 10 treatment groups (n=18) based on Vickers surface microhardness (VHN). Lesions were then pH-cycled for 10 days with daily regimen comprised of twice daily toothpaste slurry treatments (1150 ppm fluoride, with SDS or CAPB), followed by CHX solution treatments [0, 15, 30 or 60 minutes following slurry treatment or no CHX treatment (negative control)]. VHN was measured again and the extent of lesion remineralization calculated (∆VHN). Results: ∆VHN with SDS-toothpaste was significantly lower than with CAPB-toothpaste, indicating more remineralization for the CAPB-toothpaste. ∆VHN with 0-minute waiting time was significantly lower than with 30-minute waiting time and with negative control. Conclusions: The absence of CHX as an adjunct to fluoride toothpastes led to greater remineralization of enamel lesions compared with the immediate use of CHX treatment for both SDS- and CAPB-toothpastes. CAPB-toothpastes indicated significantly greater remineralization than SDS-toothpastes, and can be suggested for patients at high risk of caries. A 30-minute waiting time for CHX treatment is recommended after brushing.