Arginine and sodium fluoride affect the microbial composition and reduce biofilm metabolism and enamel mineral loss in an oral microcosm model.

OBJECTIVE: To assess the effects of arginine, with or without sodium fluoride (NaF; 1,450 ppm), on saliva-derived microcosm biofilms and enamel demineralization. METHODS: Saliva-derived biofilms were grown on bovine enamel blocks in 0.2 % sucrose-containing modified McBain medium, according to six experimental groups: control (McBain 0.2 %); 2.5 % arginine; 8 % arginine; NaF; 2.5 % arginine with NaF; and 8 % arginine with NaF. After 5 days of growth, biofilm viability was assessed by colony-forming units counting, laser scanning confocal microscopy was used to determine biofilm vitality and extracellular polysaccharide (EPS) production, while biofilm metabolism was evaluated using the resazurin assay and lactic acid quantification. Demineralization was evaluated by measuring pH in the culture medium and calcium release. Data were analyzed by Kruskal-Wallis' and Dunn's tests (p < 0.05). RESULTS: 8 % arginine with NaF showed the strongest reduction in total streptococci and total microorganism counts, with no significant difference compared to arginine without NaF. Neither 2.5 % arginine alone nor NaF alone significantly reduced microbial counts compared to the control, although in combination, a reduction in all microbial groups was observed. Similar trends were found for biofilm vitality and EPS, and calcium released to the growth medium. CONCLUSIONS: 8 % Arginine, with or without NaF, exhibited the strongest antimicrobial activity and reduced enamel calcium loss. Also, NaF enhanced the effects of 2.5 % arginine, yielding similar results to 8 % arginine for most parameters analyzed. CLINICAL SIGNIFICANCE: The results provided further evidence on how arginine, with or without NaF, affects oral microcosm biofilms and enamel mineral loss.

Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization.

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60 s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500 ppm F, as NaF), (4) 0.025 mg/ml CaneCPI-5, and (5) 0.05 mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8 h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60 s) with the solutions for 5 days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.

In vitro effect of curcumin-mediated antimicrobial photodynamic therapy on fibroblasts: viability and cell signaling for apoptosis.

Although it was demonstrated that curcumin-mediated antimicrobial photodynamic therapy (aPDT) is effective for reducing the viability of microbial cells and the vitality of oral biofilms, the cytotoxicity of this therapeutic approach for host cells has not been yet elucidated. Hence, the aim of this study was to evaluate the cytotoxicity and apoptotic effects of curcumin-mediated aPDT on mouse fibroblasts. Cells were treated with 0.6 or 6 µmol.L-1 curcumin combined with 0.075 or 7.5 J.cm-2 LED at 455 nm. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while quantitative reverse transcriptase-PCR (qRT-PCR) was used to assess the expression of Bax, Bad, Bcl-2, VDAC-1, cytochrome C, and Fas-L genes for apoptosis. The differences between groups were detected by Kruskal-Wallis and post hoc Dunn's tests for MTT and CV assays and by ANOVA and post hoc Tukey test for qRT-PCR (P < 0.05). The effect of 0.6 µmol.L-1 curcumin plus 0.075 J.cm-2 LED (minimum parameter) did not differ statistically from control group; however, the combination of 0.6 µmol.L-1 curcumin plus 7.5 J.cm-2 LED reduced viable cells in 34%, while the combinations of 6 µmol.L-1 curcumin plus 0.075 and 7.5 J.cm-2 LED reduced viable cells in 47% and 99%, respectively. aPDT increased significantly the relative expression of Bax/Bcl-2, cytochrome C, VDAC-1, and Fas-L genes, without influence on the ratio Bad/Bcl-2. Therefore, curcumin-mediated aPDT activated Bcl-2 apoptosis signaling pathways in mouse fibroblasts regarding present conditions, reducing the viability of cells with the increase of curcumin concentrations and light energies.

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin.

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6 h day-1) to 1% sucrose for 4 days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student's-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48 h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.

Could a chelant improve the effect of curcumin-mediated photodynamic antimicrobial chemotherapy against dental intact biofilms?

To our knowledge, there is still no evidence in relation to the combination of curcumin with chelants to improve the effects of antimicrobial photodynamic therapy (aPDT) on complex dental caries biofilms. Therefore, the aim of this study was to evaluate the antimicrobial effect of curcumin-ethylenediaminetetraacetic acid (EDTA)-mediated aPDT on the vitality of intact biofilms of dentin caries microcosms. Biofilms were grown on glass slabs in McBain medium plus 1% sucrose in microaerophily at 37 °C for 5 days. Then, biofilms were treated with associations of 600 µmol L-1 curcumin combined or not with 1% EDTA and 37.5 or 75 J cm-2 LED (455 nm). The vitality was determined by a confocal laser scanning microscopy (CLSM) after staining biofilms with a mixture of 2.5 g L-1 fluorescein diacetate and 0.25 g L-1 ethidium bromide. Statistical analysis was conducted by Kruskal-Wallis and post hoc Dunn's test (P < 0.05). Three treatments were able to reduce the vitality of overall biofilms: curcumin + 75 J cm-2 LED, curcumin-EDTA + 37.5 J cm-2 LED, and curcumin-EDTA + 75 J cm-2 LED. Also, the vitality of inner layers of biofilms was significantly reduced only after the combination of aPDT with EDTA. Therefore, the association of curcumin and EDTA improved the antimicrobial effect of aPDT on dentin caries microcosms, considering the application of lower light densities and deeper layers of biofilms.

Could chlorhexidine be an adequate positive control for antimicrobial photodynamic therapy in- in vitro studies?

BACKGROUND: Chlorhexidine digluconate (CHX) is commonly applied as positive control of new antimicrobials, because it is considered the gold-standard for chemical plaque control. The aim of this study was to compare the effect of treatments with curcumin-mediated aPDT and CHX in relation to the viability of specific microorganism groups in two distinct times (immediately and 24 h later). METHODS: Dentin caries microcosms were grown on bovine dentin discs (37 °C, anaerobiosis) for 3 days in the Active Attachment Amsterdam Biofilm Model. The biofilms were treated with 300 µM curcumin and 75 J.cm-² LED, or 0.06% and 0.12% CHX. Then, total microorganisms, total streptococci, mutans streptococci, and total lactobacilli counts were determined. The statistical analysis was conducted by Kruskal-Wallis and post-hoc Dunn's tests (P < 0.05). RESULTS: Curcumin-mediated aPDT (C + L+), 0.06% and 0.12% CHX reduced mutans streptococci counts (0.19, 0.10 and 0.07 log10 respectively) in the immediate analysis. After 24 h, it was observed a re-growth of microorganisms treated by curcumin-mediated aPDT, whereas both CHX concentrations demonstrated a decrease of the viable microorganisms. CONCLUSION: This study confirmed the substantive effect of CHX and the immediate effect of aPDT. The use of a neutralizer solution was important to block the substantivity of CHX and permit its fair comparison with aPDT, allowing its use as a positive control in further studies.

Effect of methylene blue-mediated antimicrobial photodynamic therapy on dentin caries microcosms.

Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjuvant treatment of dental caries, although there are no well-defined protocols to its clinical application. This study aimed to evaluate the influence of aPDT on the viability of microorganisms, vitality of biofilms, and lactic acid production of dentin caries microcosms. Biofilms were grown on bovine dentin discs in anaerobic conditions at 37 °C for 5 days, inoculating infected carious dentin in modified McBain medium plus 1% sucrose. The biofilms were treated by the combination of deionized water or 100 mg L-1 methylene blue (MB) with 0, 37.5, or 75 J cm-2 LED at 630 nm. The counts of total microorganisms, total streptococci, mutans streptococci, and total lactobacilli were determined by colony-forming units (CFU). The vitality of microbial cells in intact biofilms was measured by confocal laser scanning microscope (CLSM). The lactic acid production was analyzed by enzymatic spectrophotometry at 340 nm. Statistical analysis was conducted by Kruskal-Wallis and post hoc Dunn's tests (P < 0.05). MB and 37.5 J cm-2 LED alone did not interfere in the viability of microorganisms, unlike 75 J cm-2 LED alone that decreased the total microorganism and lactobacillus counts. The combination of MB and 75 J cm-2 LED reduced the viability of all microorganisms and the vitality of intact biofilms. The production of lactic acid was statistically lower in all treatment groups in comparison with that of the control (no treatment), except for MB alone. Therefore, the MB-mediated aPDT was effective in controlling the viability, vitality and the acidogenicity of dentin caries microcosms.

The Diagnosis and Treatment of Multiple Factitious Oral Ulcers in a 6-Year-Old Boy.

Factitious ulcers are characterized by self-inflicted lesions with multifactorial origin. These lesions are frequently found in head, neck, and hands. This report shows a 6-year-old boy diagnosed with factitious oral ulcers that occurred after the self-biting of buccal vestibule and nail-scratching of gingival tissue. Clinically, a significant swelling was observed, hard on palpation, located at the right lower third of the face, next to the posterior area of the mandible. In the intraoral examination, ulcers at different healing stages were noted on the swelling area. During the anamnesis, the father reported a change in his familial structure that triggers psychological stress, providing the clues to the presumptive diagnosis of factitious oral ulcers. We prescribed the topical use of Gingilone® three times a day to control the local pain and inflammation. At 7-day follow-up, we noticed the reduction of extraoral swelling and the initial healing of the ulcers. The presumptive diagnosis was confirmed at 30-day follow-up, with the lasting remission of oral lesions. The treatments of factitious oral ulcers should be individually tailored for each patient, focused on a multidisciplinary approach, including psychotherapy and periodic clinical control. To the best of our knowledge, gaps of evidence lead to the lack of standardized clinical protocols on this issue.

Relação entre tempo de aleitamento materno, hábitos bucais deletérios e cárie dentária em bebês / The relationship between breastfeeding duration, deleterious oral habits, and dental caries in babies

A associação entre amamentação materna e cárie dentária é controversa. O presente estudo avaliou a experiência de cárie dentária entre bebês exclusivamente amamentados pelo seio materno ou pelo uso complementar de métodos artificiais auxiliares. Dados demográficos, valores do índice ceod, tempo de aleitamento materno e diagnóstico de hábitos bucais deletérios de sucção foram coletados dos prontuários clínicos-odontológicos de 299 bebês. Valores de P < 0,05 foram considerados para diferenças estatisticamente significantes. Os resultados demonstraram um significativo aumento da experiência de cárie dentária entre os bebês amamentados exclusivamente pelo seio materno. A média do tempo de aleitamento materno foi maior entre bebês com diagnóstico de cárie precoce da infância (CPI) (14,7±8,7 meses) quando comparado com bebês sem diagnóstico de CPI (8,3±6,8 meses) (P<0,001). Entretanto, bebês que desenvolveram hábitos bucais deletérios foram amamentados por menos tempo. O aleitamento materno deve ser estimulado, pois é capaz de prevenir infecções, mortalidade infantil, respiração bucal e hábitos bucais deletérios, além de favorecer o estabelecimento do vínculo emocional entre mãe e flho. Entretanto, o maior risco de lesões de cárie dentária deve determinar atenção especial da equipe de saúde da família em relação à manutenção das condições adequadas da saúde bucal dos bebês

The association between breastfeeding and dental caries is controversial. This study aimed to evaluate the experience of dental caries among exclusively breastfed infants and non-exclusively breastfed infants. Demographic data, dmft index scores, breastfeeding duration, and diagnosis of deleterious oral sucking habits were collected from 299 patient records of the Clinic of Babies of Cuiabá School of Dentistry. Statistical differences were considered to be signifcant with P values < 0.05. The results demonstrated a signifcant increase of experience of dental caries among exclusively breastfed infants. The mean of breastfeeding duration in infants with early childhood caries (ECC) diagnosis (14,7±8,7 months) was higher than those infants without ECC diagnosis (8,3±6,8 months) (P<0,001). The breastfeeding needs to be continuously estimulated because it prevents infections, infant mortality, oral breathing, deleterious oral sucking habits, besides favoring the maternal bonding. Higher risks of dental caries lesions among infants breastfed for longer, however, must lead family health teams to a special focus on the maintenance of good conditions of infants' oral health