Cariogenic Potential of Human and Bovine Milk on Enamel Demineralization.

The higher cariogenicity of human milk when compared with bovine milk is still a debatable subject. Therefore, we evaluated the effect of human or bovine milk exposure on biofilm composition and enamel demineralization using a validated cariogenic biofilm model. Streptococcus mutans UA159 biofilms (n = 8) were grown on human saliva-coated bovine enamel slabs of known surface hardness. The biofilms were exposed 8×/day to 0.9% NaCl (negative control), human milk, bovine milk, 7.0% lactose (active human milk control), 4.5% lactose (active bovine milk control), or 10% sucrose (positive control). The culture medium was changed twice daily, and the pH was analyzed as an indicator of biofilm acidogenicity. After 120 h of growth, biofilms were harvested to evaluate viable cells, and soluble and insoluble extracellular polysaccharides (EPS). Enamel demineralization was assessed by the percentage of surface hardness loss (%SHL). Data were analyzed by one-way ANOVA/Tukey's test (α = 5%). In terms of %SHL, negative control (7.7 ± 3.1), human milk control (13.3 ± 7.5), bovine milk control (15.3 ± 8.2), human milk (7.5 ± 5.0), and bovine milk (8.7 ± 6.3) did not differ among them (p > 0.05) but differed (p < 0.05) from sucrose (55.1 ± 5.4). The findings of enamel demineralization (%SHL) were statistically supported by the data of biofilm acidogenicity, bacterial counts and EPS biofilm composition. This experimental study suggests that human and bovine milk have low cariogenic potential to provoke caries lesions in enamel.

UV-photofunctionalization of a biomimetic coating for dental implants application.

Photofunctionalization mediated by ultraviolet (UV) rays changes the physico-chemical characteristics of titanium (Ti) and improves the biological activity of dental implants. However, the role of UV-mediated photofunctionalization of biofunctional Ti surfaces on the antimicrobial and photocatalytic activity remains unknown and was investigated in this study. Commercially pure titanium (cpTi) discs were divided into four groups: (1) machined samples without UV light application [cpTi UV-]; (2) plasma electrolytic oxidation (PEO) treated samples without UV light application [PEO UV-]; (3) machined samples with UV light application [cpTi UV+]; and (4) PEO-treated samples with UV light application [PEO UV+]. The surfaces were characterized according to their morphology, roughness, crystalline phase, chemical composition and wettability. The photocatalytic activity and proteins adsorption were measured. For the microbiological assay, Streptococcus sanguinis was grown on the disc surfaces for 1 h and 6 h, and the colony forming units and bacterial organization were evaluated. In addition, to confirm the non-cytotoxic effect of PEO UV +, human gingival fibroblast (HGF) cells were cultured in a monolayer onto each material surface and the cells viability and proliferation evaluated by a fluorescent cell staining method. PEO treatment increased the Ti surface roughness and wettability (p < 0.05). Photofunctionalization reduced the hydrocarbon concentration and enhanced human blood plasma proteins and albumin adsorption mainly for the PEO-treated surface (p < 0.05). PEO UV+ also maintained higher wettability values for a longer period and provided microbial reduction at 1 h of bacterial adhesion (p = 0.012 vs. PEO UV-). Photofunctionalization did not increase the photocatalytic activity of Ti (p > 0.05). Confocal microscopy analyses demonstrated that PEO UV+ had no cell damage effect on HGF cells growth even after 24 h of incubation. The photofunctionalization of a biofunctional PEO coating seems to be a promising alternative for dental implants as it increases blood plasma proteins adsorption, reduces initial bacterial adhesion and presents no cytotoxicity effect.

Systemic Effects (Risks) of Water Fluoridation.

Since the 1950s, the benefits and risks of fluoridated water use have been debated worldwide. In the past, it was considered that the systemically ingested fluoride would exert its primary preventive effect after being incorporated into the enamel as fluorapatite, making the enamel more resistant to the caries process; however, it is now recognized that the main effect of water fluoridation is local and post eruptive. On the other hand, irrespective of the caries decline reported worldwide, the anticaries benefit of water fluoridation continues to be observed even in developed countries. Regarding the risks, water fluoridation is considered an acceptable community-based method for fluoride delivery, because the risk of developing dental fluorosis lesions due to the ingestion of fluoride during the enamel formation period has been deemed acceptable when contrasted to the anticaries benefits of fluoride. However, the use of fluoride in water to control caries has created a controversy due to data associating water fluoridation as the cause of some systemic diseases. Therefore, the aim of this descriptive review was to discuss the systemic effects (risks) of water fluoridation use.

Systemic effects (risks) of water fluoridation

Abstract Since the 1950s, the benefits and risks of fluoridated water use have been debated worldwide. In the past, it was considered that the systemically ingested fluoride would exert its primary preventive effect after being incorporated into the enamel as fluorapatite, making the enamel more resistant to the caries process; however, it is now recognized that the main effect of water fluoridation is local and post eruptive. On the other hand, irrespective of the caries decline reported worldwide, the anticaries benefit of water fluoridation continues to be observed even in developed countries. Regarding the risks, water fluoridation is considered an acceptable community-based method for fluoride delivery, because the risk of developing dental fluorosis lesions due to the ingestion of fluoride during the enamel formation period has been deemed acceptable when contrasted to the anticaries benefits of fluoride. However, the use of fluoride in water to control caries has created a controversy due to data associating water fluoridation as the cause of some systemic diseases. Therefore, the aim of this descriptive review was to discuss the systemic effects (risks) of water fluoridation use.

Resumo Desde 1950, os benefícios e riscos do uso da água fluoretada têm sido debatidos mundialmente. No passado, foi considerado que o fluoreto ingerido sistemicamente exerceria seu efeito preventivo de cárie pela sua incorporação ao esmalte como fluorapatita, tornando o esmalte mais resistente ao processo de desenvolvimento de lesões de cárie; entretanto, é atualmente reconhecido que o principal efeito da fluoretação da água é local e pós-eruptivo. Por outro lado, independente do declínio de cárie que tem sido observado mundialmente, o benefício anticárie da fluoretação da água continua sendo observado mesmo em países desenvolvidos. Com relação aos riscos, fluoretação da água é considerada uma estratégia aceitável de saúde pública para uso de fluoreto, porque o risco de desenvolvimento de fluorose dental devido à ingestão de fluoreto durante o período de formação do esmalte foi considerado aceitável quando comparado aos benefícios anticárie do flúor. Entretanto, o uso de fluoreto na água para controlar cárie tem gerado controvérsias devido a dados de pesquisa que associam fluoretação da água como causa de algumas doenças sistêmicas. Assim, o objetivo dessa revisão descritiva foi discutir os efeitos sistêmicos (riscos) da fluoretação da água..

Effect of Chitosan Dispersion and Microparticles on Older Streptococcus mutans Biofilms.

(1) Background: The effectiveness of chitosan to improve the action of antimicrobial compounds against planktonic bacteria and young biofilms has been widely investigated in Dentistry, where the biofilm lifecycle is a determining factor for the success of antibacterial treatment. In the present study, mature Streptococcus mutans biofilms were treated with chitosan dispersion (CD) or chitosan microparticles (CM). (2) Methods: CD at 0.25% and 1% were characterized by texture analysis, while CD at 2% was spray-dried to form CM, which were characterized with respect to particle size distribution, zeta potential, and morphology. After determining the minimum inhibitory and bactericidal concentrations, S. mutans biofilms were grown on glass slides exposed 8×/day to 10% sucrose and 2×/day to CD or CM at 0.25% and 1%. Biofilm viability and acidogenicity were determined, using appropriate control groups for each experiment. (3) Results: CD had high viscosity and CM were spherical, with narrow size distribution and positive zeta potential. CM affected bacterial viability and acidogenicity in mature S. mutans biofilms more strongly than CD, especially at 1%. (4) Conclusions: Both chitosan forms exerted antimicrobial effect against mature S. mutans biofilms. CM at 1% can reduce bacterial viability and acidogenicity more effectively than CD at 1%, and thereby be more effective to control the growth of mature biofilms in vitro.

Visible-Light-Induced Photocatalytic and Antibacterial Activity of TiO2 Codoped with Nitrogen and Bismuth: New Perspectives to Control Implant-Biofilm-Related Diseases.

Biofilm-associated diseases are one of the main causes of implant failure. Currently, the development of implant surface treatment goes beyond the osseointegration process and focuses on the creation of surfaces with antimicrobial action and with the possibility to be re-activated (i.e., light source activation). Titanium dioxide (TiO2), an excellent photocatalyst used for photocatalytic antibacterial applications, could be a great alternative, but its efficiency is limited to the ultraviolet (UV) range of the electromagnetic spectrum. Since UV radiation has carcinogenic potential, we created a functional TiO2 coating codoped with nitrogen and bismuth via the plasma electrolytic oxidation (PEO) of titanium to achieve an antibacterial effect under visible light with re-activation potential. A complex surface topography was demonstrated by scanning electron microscopy and three-dimensional confocal laser scanning microscopy. Additionally, PEO-treated surfaces showed greater hydrophilicity and albumin adsorption compared to control, untreated titanium. Bismuth incorporation shifted the band gap of TiO2 to the visible region and facilitated higher degradation of methyl orange (MO) in the dark, with a greater reduction in the concentration of MO after visible-light irradiation even after 72 h of aging. These results were consistent with the in vitro antibacterial effect, where samples with nitrogen and bismuth in their composition showed the greatest bacterial reduction after 24 h of dual-species biofilm formation ( Streptococcus sanguinis and Actinomyces naeslundii) in darkness with a superior effect at 30 min of visible-light irradiation. In addition, such a coating presents reusable photocatalytic potential and good biocompatibility by presenting a noncytotoxicity effect on human gingival fibroblast cells. Therefore, nitrogen and bismuth incorporation into TiO2 via PEO can be considered a promising alternative for dental implant application with antibacterial properties in darkness, with a stronger effect after visible-light application.

Protocols to Study Dental Caries In Vitro: Microbial Caries Models.

Caries lesions result from the interaction between dental biofilm and sugars. Since the biofilm is an important component in the etiology of the disease, biofilm models have been developed to study the cariogenicity of dietary sugars, as well as the anticaries effect of substances. Two of such models, termed as "static" or "continuous flow," are described in details here together with their advantages, limitations, and applications.

Effect of sucrose on biofilm formed in situ on titanium material.

BACKGROUND: Because sucrose may change the composition of biofilms formed on dental surfaces, the aim of this study was to evaluate in situ the effect of this dietary sugar on biofilm formation on titanium surface. METHODS: In this blind, crossover, in situ study, 10 volunteers wore, in 3 phases of 7 days each, a palatal appliance containing titanium specimens. In each phase, the specimens were treated extraorally with 20% sucrose solution at a frequency of 4 or 8 times per day. As control, no treatment was rendered (0×). At the end of each phase, the biofilms were collected for biochemical analysis of biofilm wet weight (biomass), protein concentration, soluble (S-EPS), and insoluble (I-EPS) extracellular polysaccharides and intracellular polysaccharides (IPS), and for microbiologic analysis by checkerboard DNA-DNA hybridization (for levels and proportions of 40 bacterial species). Biochemical data were analyzed by linear regression and microbiological findings by Friedman and Dunn tests (α = .05). RESULTS: A positive significant linear relationship was found among sucrose exposure (0×, 4×, and 8×) and biomass, S-EPS, I-EPS and IPS (p < 0.05). The biofilms treated with sucrose (4× and/or 8×) presented higher mean total levels of the 40 bacterial species evaluated, higher proportions of red complex species and lower proportions of the host-compatible green complex species, in comparison with the control group (p < 0.05). CONCLUSION: The findings of the present study suggest that daily sucrose exposure has a harmful effect on the composition of biofilms formed on titanium surfaces.

Candida albicans Increases Dentine Demineralization Provoked by Streptococcus mutans Biofilm.

Streptococcus mutans are considered the most cariogenic bacteria, but it has been suggested that Candida albicans could increase their cariogenicity. However, the effect of this dual-species microorganisms' combination on dentine caries has not been experimentally evaluated. Biofilms of C. albicans, S. mutans and C. albicans + S. mutans (n = 12/biofilm) were grown in ultra-filtered tryptone yeast extract broth culture medium for 96 h on root dentine slabs of known surface hardness and exposed 8 times per day for 3 min to 10% sucrose. The medium was changed 2 times per day (after the 8 cariogenic challenges and after the overnight period of famine), and aliquots were analyzed to determinate the pH (indicator of biofilm acidogenicity). After 96 h, the biofilms were collected to determine the wet weight, colony-forming units, and the amounts of extracellular polysaccharides (soluble and insoluble). Dentine demineralization was assessed by surface hardness loss (% SHL). The architecture of the biofilms was analyzed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Data were analyzed by ANOVA followed by Tukey's test (α = 0.05). The dual-species C. albicans + S. mutans biofilm provoked higher % SHL on dentine (p < 0.05) than the S. mutans and C. albicans biofilm. This was supported by the results of biofilm acidogenicity and the amounts of soluble (6.4 ± 2.14 vs. 4.0 ± 0.94 and 1.9 ± 0.97, respectively) and insoluble extracellular polysaccharides (24.9 ± 9.22 vs. 18.9 ± 5.92 and 0.7 ± 0.48, respectively) (p < 0.05). The C. albicans biofilm alone presented low cariogenicity. The images by CLSM and TEM, respectively, suggest that the C. albicans + S. mutans biofilm is more voluminous than the S. mutans biofilm, and S. mutans cells interact with C. albicans throughout polysaccharides from the biofilm matrix. These findings show that C. albicans enhances the cariogenic potential of the S. mutans biofilm, increasing dentine demineralization.

Antibacterial photocatalytic activity of different crystalline TiO2 phases in oral multispecies biofilm.

OBJECTIVE: Titanium dioxide (TiO2) incorporation in biomaterials is a promising technology due to its photocatalytic and antibacterial activities. However, the antibacterial potential of different TiO2 crystalline structures on a multispecies oral biofilm remains unknown. We hypothesized that the different crystalline TiO2 phases present different photocatalytic and antibacterial activities. METHODS: Three crystalline TiO2 films were deposited by magnetron sputtering on commercially pure titanium (cpTi), in order to obtain four groups: (1) machined cpTi (control); (2) A-TiO2 (anatase); (3) M-TiO2 (mixture of anatase and rutile); (4) R-TiO2 (rutile). The morphology, crystalline phase, chemical composition, hardness, elastic modulus and surface free energy of the surfaces were evaluated. The photocatalytic potential was assessed by methylene blue degradation assay. The antibacterial activity was evaluated on relevant oral bacteria, by using a multispecies biofilm (Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum) formed on the treated titanium surfaces (16.5h) followed by UV-A light exposure (1h) to generate reactive oxygen species production. RESULTS: All TiO2 films presented around 300nm thickness and improved the hardness and elastic modulus of cpTi surfaces (p<0.05). A-TiO2 and M-TiO2 films presented superior photocatalytic activity than R-TiO2 (p<0.05). M-TiO2 revealed the greatest antibacterial activity followed by A-TiO2 (≈99.9% and 99% of bacterial reduction, respectively) (p<0.001 vs. control). R-TiO2 had no antibacterial activity (p>0.05 vs. control). SIGNIFICANCE: This study brings new insights on the development of extra oral protocols for the photocatalytic activity of TiO2 in oral biofilm-associated disease. Anatase and mixture-TiO2 showed antibacterial activity on this oral bacterial biofilm, being promising surface coatings for dental implant components.

Three-species biofilm model onto plasma-treated titanium implant surface.

In this study, titanium (Ti) was modified with biofunctional and novel surface by micro-arc oxidation (MAO) and glow discharge plasma (GDP) and we tested the development of a three-species periodontopatogenic biofilm onto the treated commercially-pure titanium (cpTi) surfaces. Machined and sandblasted surfaces were used as control group. Several techniques for surface characterizations and monoculture on bone tissue cells were performed. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum was developed onto cpTi discs for 16.5h (early biofilm) and 64.5h (mature biofilm). The number of viable microorganisms and the composition of the extracellular matrix (proteins and carbohydrates) were determined. The biofilm organization was analyzed by scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM). In addition, MC3T3-E1 cells were cultured on the Ti surfaces and cell proliferation (MTT) and morphology (SEM) were assessed. MAO treatment produced oxide films rich in calcium and phosphorus with a volcano appearance while GDP treatment produced silicon-based smooth thin-film. Plasma treatments were able to increase the wettability of cpTi (p<0.05). An increase of surface roughness (p<0.05) and formation of anatase and rutile structures was noted after MAO treatment. GDP had the greatest surface free energy (p<0.05) while maintaining the surface roughness compared to the machined control (p>0.05). Plasma treatment did not affect the viable microorganisms counts, but the counts of F. nucleatum was lower for MAO treatment at early biofilm phase. Biofilm extracellular matrix was similar among the groups, excepted for GDP that presented the lowest protein content. Moreover, cell proliferation was not significantly affected by the experimental, except for MAO at 6days that resulted in an increased cell proliferative. Together, these findings indicate that plasma treatments are a viable and promising technology to treat bone-integrated dental implants as the new surfaces displayed improved mechanical and biological properties with no increase in biofilm proliferation.

Production of a biofunctional titanium surface using plasma electrolytic oxidation and glow-discharge plasma for biomedical applications.

In this study, the authors tested the hypotheses that plasma electrolytic oxidation (PEO) and glow-discharge plasma (GDP) would improve the electrochemical, physical, chemical, and mechanical properties of commercially pure titanium (cpTi), and that blood protein adsorption on plasma-treated surfaces would increase. Machined and sandblasted surfaces were used as controls. Standard electrochemical tests were conducted in artificial saliva (pHs of 3.0, 6.5, and 9.0) and simulated body fluid. Surfaces were characterized by scanning electron microscopy, energy-dispersive spectroscopy, x-ray photoelectron spectroscopy, atomic force microscopy, x-ray diffraction, profilometry, Vickers microhardness, and surface energy. For biological assay, the adsorption of blood serum proteins (i.e., albumin, fibrinogen, and fibronectin) was tested. Higher values of polarization resistance and lower values of capacitance were noted for the PEO and GDP groups (p < 0.05). Acidic artificial saliva reduced the corrosion resistance of cpTi (p < 0.05). PEO and GDP treatments improved the surface properties by enrichment of the surface chemistry with bioactive elements and increased surface energy. PEO produced a porous oxide layer (5-µm thickness), while GDP created a very thin oxide layer (0.76-µm thickness). For the PEO group, the authors noted rutile and anatase crystalline structures that may be responsible for the corrosion barrier improvement and increased microhardness values. Plasma treatments were able to enhance the surface properties and electrochemical stability of titanium, while increasing protein adsorption levels.

The role of nicotine, cotinine and caffeine on the electrochemical behavior and bacterial colonization to cp-Ti.

Although smoking promotes deleterious effect to bone healing, there is a lack of study investigating its role on the implant structure and biofilm growth. We hypothesized that nicotine, cotinine and caffeine would impair the corrosion resistance of commercially-pure titanium (cp-Ti) and would enhance Streptococcus sanguinis biofilm growth. Neither the smoking products nor the caffeine affected the corrosion tendency (P>.05) and the oxide layer resistance (P=.762) of cp-Ti. Lower capacitance values were noted in the presence of nicotine (P=.001) and cotinine (P=.0006). SEM showed no pitting corrosion, and the EDS spectra did not differ among groups. Nicotine (300µg/mL) induced higher surface roughness (P=.03) and greater surface change of cp-Ti. Nicotine at 3µg/mL, and cotinine at 0.3 and 3µg/mL increased the number of viable cells (P<.05). Biofilm exposed to nicotine (0.3, 3 and 30µg/mL) (P=.025, .030, .040, respectively) and cotinine (3 and 30µg/mL) (P=.027, .049, respectively) enhanced carbohydrate content. Biofilm biomass and protein content were similar among groups (P>.05). These findings suggest a greater biofilm accumulation in smokers, a risk factor that may lead to peri-implantitis.