Acquired pellicle and biofilm engineering with CaneCPI-5: Insights from proteomic and microbiomics analysis.

OBJECTIVE: In this in vivo proof-of-concept study, acquired pellicle engineering was implemented to promote alterations in the protein composition of the acquired enamel pellicle (AEP) and the bacterial composition of the dental biofilm after treatment with Sugarcane cystatin (CaneCPI-5). DESIGN: After prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with the following solutions: 1) deionized water (H2O- negative control or 2) 0.1 mg/mL CaneCPI-5. The AEP and biofilm were formed along 2 or 3 h, respectively. The AEP was collected with electrode filter papers soaked in 3 % citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. The biofilm microbiome was collected with a dental curette. The DNA was extracted, amplified, and analyzed by 16S-rRNA Next Generation Sequencing (NGS). RESULTS: Treatment with CaneCPI-5 increased several proteins with antimicrobial, acid-resistance, affinity for hydroxyapatite, structural and calcium binding properties, such as Cysteine-rich-3 (6-fold-p = 0.03), Cystatin-B (5.5-fold-p < 0.01), Neutrophil-defensin 1 (4.7-fold-p < 0.01), Mucin (3.9-fold-p < 0.01), Immunoglobulin-heavy-constant (3.8-fold-p < 0.01) and Lactotransferrin (2.8-fold-p < 0.01). Microbiome revealed that several commensal bacteria had their abundance increased after rinsing with CaneCPI-5, such as Corynebacterium and Neisseria, while Streptococcus and Prevotella nigrescens were decreased. The results indicate the efficiency of CaneCPI-5 in promoting beneficial changes in the AEP and biofilm, making this phytocystatin a potential target for incorporation into dental products. CONCLUSION: Cane demonstrated the capability to alter the protein composition of the acquired enamel pellicle (AEP) and the initial colonizers of the biofilm, enhancing the presence of proteins and bacteria crucial for dental protection.

In vitro effect of TiF4/NaF solution on the development of radiation-induced dentin caries.

OBJECTIVE: To evaluate the protective effect of an experimental solution containing TiF4/NaF on the development of radiation-induced dentin caries lesions. METHODOLOGY: bovine root samples were irradiated (70Gy) and distributed as following (n=12/group): Commercial Saliva (BioXtra), NaF (500 ppm F-), TiF4 (500 ppm F), TiF4/NaF (TiF4: 300 ppm F-, NaF: 190 ppm F-), and Phosphate buffer solution (PBS, negative control). Biofilm was produced using biofilm from irradiated patients and McBain saliva (0.2% of sucrose, at 37oC and 5% CO2) for five days. The treatments were applied 1x/day. Colony-forming units (CFU) were counted and demineralization was quantified by transversal microradiography. The ANOVA/Tukey test was applied for all parameters. RESULTS: All treatments reduced CFU for total microorganisms. TiF4 reduced Lactobacillus sp. (7.04±0.26 log10 CFU/mL) and mutans streptococci (7.18±0.28) CFU the most, when compared to PBS (7.58±0.21 and 7.75±0.17) and followed by NaF (7.12±0.31 and 7.34±0.22) and TiF4/NaF (7.16±0.35 and 7.29± 0.29). TiF4 and Commercial saliva showed the lowest integrated mineral loss (ΔZ-vol%.mm) (1977±150 and 2062±243, respectively) when compared to PBS (4540±335), followed by NaF (2403±235) and TiF4/NaF (2340±200). Commercial saliva was the only to significantly reduce mineral loss (LD-µm) (111±25) compared to PBS (153±24).Mean mineral loss (R-vol%) decreased by 35.2% for TiF4 (18.2±3.3) when compared to PBS (28.1±2.9) Conclusion: TiF4/NaF has a comparable anti-cariogenic effect to TiF4 and Commercial saliva under the model in this study.

Protein-based engineering of the initial acquired enamel pellicle in vivo: Proteomic evaluation.

OBJECTIVE: To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. MATERIALS AND METHODS: In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10-5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. RESULTS: Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2-2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7-25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. CONCLUSION: Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. CLINICAL RELEVANCE: Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP.

Protective effect of titanium tetrafluoride and silver diamine fluoride on radiation-induced dentin caries in vitro.

This in vitro study evaluated the protective effect of titanium tetrafluoride (TiF4) varnish and silver diamine fluoride (SDF) solution on the radiation-induced dentin caries. Bovine root dentin samples were irradiated (70 Gy) and treated as follows: (6 h): 4% TiF4 varnish; 5.42% NaF varnish; 30% SDF solution; placebo varnish; or untreated (negative control). Microcosm biofilm was produced from human dental biofilm (from patients with head-neck cancer) mixed with McBain saliva for the first 8 h. After 16 h and from day 2 to day 5, McBain saliva (0.2% sucrose) was replaced daily (37 °C, 5% CO2) (biological triplicate). Demineralization was quantified by transverse microradiography (TMR), while biofilm was analyzed by using viability, colony-forming units (CFU) counting and lactic acid production assays. The data were statistically analyzed by ANOVA (p < 0.05). TiF4 and SDF were able to reduce mineral loss compared to placebo and the negative control. TiF4 and SDF significantly reduced the biofilm viability compared to negative control. TiF4 significantly reduced the CFU count of total microorganism, while only SDF affected total streptococci and mutans streptococci counts. The varnishes induced a reduction in lactic acid production compared to the negative control. TiF4 and SDF may be good alternatives to control the development of radiation-induced dentin caries.

A sugarcane cystatin (CaneCPI-5) alters microcosm biofilm formation and reduces dental caries.

The antimicrobial and anticaries effects of CaneCPI-5 were evaluated. Ninety bovine enamel samples were treated for 60 s with either phosphate-buffered-saline (PBS), 0.12% chlorhexidine (CHX), 0.05 mg ml-1 CaneCPI-5, 0.1 mg ml-1 CaneCPI-5 or 0.5 mg ml-1 CaneCPI-5. They were incubated with inoculum (human saliva + McBain's saliva) for the first 8 h. From then until the end of the experiment, the enamel was exposed to McBain saliva with sucrose and, once a day, for 5 days, they were treated with the solutions. At the end of the experimental period, resazurin and viable plate count assays were performed. Enamel demineralization was also measured. All concentrations of CaneCPI-5 and CHX significantly reduced the activity of biofilms compared with PBS. For viable plate counts, all treatments similarly reduced the lactobacilli and total streptococci; for the mutans streptococci, 0.05 mg ml-1 CaneCPI-5 performed better than CHX. All CaneCPI-5 concentrations significantly reduced the integrated mineral loss. This study represents the first step regarding the use of CaneCPI-5 within the concept of acquired enamel pellicle and biofilm engineering to prevent dental caries.

Acceptability and effect of TiF4 on dental caries: a randomized controlled clinical trial

Abstract: This randomized three-armed controlled clinical trial compared the effect of titanium tetrafluoride (TiF4) and sodium fluoride (NaF) varnishes on caries control in smooth surfaces of permanent dentition and children's acceptability. Sixty children (6-8 y/o) were randomly divided into TiF4 (2.45% F-), NaF (2.26% F-) or placebo (control) groups. Varnishes were applied on permanent teeth once a week for the first 4 weeks and after the 6th and 12th months of the study. The variables were as follows: International Caries Detection and Assessment System (ICDAS) scores, quantitative fluorescence changes, visual plaque index (VPI) and degree of acceptability. Two-way RM-ANOVA, ANOVA/Tukey and χ2 tests were performed (p < 0.05). No differences were found between the treatments with respect to ICDAS scores (p = 0.32). Only TiF4 reduced the mean fluorescence loss significantly at 18 months compared to the baseline (p = 0.003). TiF4 showed a lower percentage of new caries lesions by tooth surface than the placebo, while NaF did not induce such a change (p < 0.014). Regardless of the treatment, more than 95% of the participants reported being satisfied. For all groups, the VPI decreased significantly at 3 months compared to the baseline value (p < 0.001), with no differences between the treatments (p = 0.17). TiF4 had a similar ability to control caries lesions as NaF; however, only TiF4 differed from the placebo (p = 0.004). The acceptability of TiF4 varnish was similar to that of NaF varnish.

Effect of different citrus sweets on the development of enamel erosion in vitro

Abstract The increased consumption of citrus sweets can contribute to the development of erosive tooth wear (ETW). Objective This in vitro study evaluated the erosive potential of citrus sweets on bovine enamel samples regarding the quantification of wear. Methodology Ninety bovine crowns were prepared and samples were randomly distributed into 6 groups (n=15): 0.1% citric acid solution (pH 2.5); Coca-Cola ® Soft Drink (pH 2.6); Fini ® Diet (lactic and citric acid, pH 3.3); Fini ® Jelly Kisses (lactic and citric acid, pH 3.5); Fini ® Fruit Salad Bubblegum (maleic acid, pH 2.6); Fini ® Regaliz Acid Tubes (maleic and citric acid, pH 3.1). Sweets were dissolved in the proportion of 40 g/250 mL of deionized water. Enamel samples were submitted to erosive challenges for 7 days (4 daily acid immersion cycles for 90 s each). Enamel wear was measured using contact profilometry (μm), and data (median values [interquartile range]) were submitted to Kruskal-Wallis/Dunn's test (p<0.0001). Results All citrus sweets tested present a high erosive potential, Fini Diet ® (2.4 [1.2]) and Fini Regaliz Tubs ® (2.2 [0.5]) show the highest erosive potential, similar to 0.1% citric acid (2.3 [0.7]); Fini Regaliz Tubs ® is more erosive than Coca-Cola ® (1.4 [0.9]). Conclusion The evaluated citrus sweets have great erosive potential and play a key role in the development of ETW.

Antimicrobial and anti-caries effect of new glass ionomer cement on enamel under microcosm biofilm model

Abstract The occurrence of caries lesions adjacent to restorations is a serious problem in Dentistry. Therefore, new antimicrobial restorative materials could help to prevent recurrent carious lesions. This study evaluated the effect of a new glass ionomer cement (Ion Z) on the viability of a microcosm biofilm and on the development of enamel demineralization. Enamel samples were filled with the following materials (n=9): A) Ion-Z (FGM Ltda); B) Maxxion R (FGM Ltda); C) Ketac Fil Plus (3M ESPE) and D) no restoration (control). The samples were then exposed to human saliva mixed with McBain saliva (1:50) containing 0.2% sucrose for 14 days. The live and dead bacteria were quantified by fluorescence using a confocal laser-scanning microscope. The enamel demineralization was analyzed using transverse microradiography (TMR). The data were submitted to ANOVA/Tukey or Kruskal-Wallis/Dunn test (p<0.05). Ion Z induced a higher percentage of dead bacteria (60.96±12.0%) compared to the other groups (Maxxion R: 39.8±6.7%, Ketac Fil Plus: 43.7±9.71% and control 46.3±9.5%). All materials significantly reduced the average mineral loss compared to control (Ion-Z 25.0±4.2%vol, Maxxion R 23.4±8.0%vol, Ketac Fil Plus 30.7±7.7 and control 41.2±6.6%vol). Ion-Z was the only material able to significantly improve the mineral content at the surface layer (Zmax: 63.5±18.2%vol) compared to control (38.9±11.3%vol). Ion-Z shows antimicrobial potential, but its anti-caries effect was similar to the other materials, under this model.

Resumo A ocorrência de lesões de cárie adjacentes a restaurações é um sério problema na Odontologia. Portanto, novos materiais restauradores antimicrobianos poderiam ajudar a prevenir as lesões cariosas recorrentes. Este estudo avaliou o efeito de um novo cimento de ionômero de vidro (Ion Z) sobre a viabilidade de um biofilme microcosmo e o desenvolvimento da desmineralização do esmalte. Amostras de esmalte foram restauradas com os seguintes materiais (n=9): A) Ion-Z (FGM Ltda); B) Maxxion R (FGM Ltda); C) Ketac Fil Plus (3M ESPE) e D) sem restauração (controle). As amostras foram submetidas a uma mistura de saliva humana com saliva de McBain (1:50) contendo sacarose a 0,2% por 14 dias. As bactérias vivas e mortas foram quantificadas por fluorescência usando um microscópio confocal de varredura à laser. A desmineralização do esmalte foi analisada usando microradiografia transversal (TMR). Os dados foram submetidos aos testes ANOVA/Tukey ou Kruskal-Wallis/Dunn (p<0,05). O Ion Z induziu uma porcentagem mais elevada de bactérias mortas (60,96 ± 12,0%) comparado aos outros grupos (Maxxion R: 39,8 ± 6,7%, Ketac Fil Plus: 43,7 ± 9,71% e controle 46,3 ± 9,5%). Todos os materiais reduziram significativamente a perda mineral média em relação ao controle (Ion-Z 25,0 ± 4,2% vol, Maxxion R 23,4 ± 8,0% vol, Ketac Fil Plus 30,7 ± 7,7% vol e controle 41,2 ± 6,6% vol). O Ion-Z foi o único material capaz de melhorar significativamente o conteúdo mineral na camada superficial (Zmax: 63,5 ± 18,2% vol) em comparação com o controle (38,9 ± 11,3% vol). Ion-Z mostrou potencial antimicrobiano, mas seu efeito anti-cárie foi semelhante aos outros materiais, sob este modelo.

Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel

Abstract Titanium tetrafluoride (TiF4) is known for interacting with enamel reducing demineralization. However, no information is available about its potential antimicrobial effect. Objectives This study evaluated the antimicrobial and anti-caries potential of TiF4 varnish compared to NaF varnish, chlorhexidine gel (positive control), placebo varnish and untreated (negative controls) using a dental microcosm biofilm model. Material and Methods A microcosm biofilm was produced on bovine enamel previously treated with the varnishes, using inoculum from human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. All experiments were performed in biological triplicate (n=4/group in each experiment). Factors evaluated were: bacterial viability (% dead and live bacteria); CFU counting (log10 CFU/mL); and enamel demineralization (transverse microradiography - TMR). Data were analysed using ANOVA/Tukey's test or Kruskal-Wallis/Dunn's test (p<0.05). Results Only chlorhexidine significantly increased the number of dead bacteria (68.8±13.1% dead bacteria) compared to untreated control (48.9±16.1% dead bacteria). No treatment reduced the CFU counting (total microorganism and total streptococci) compared to the negative controls. Only TiF4 was able to reduce enamel demineralization (ΔZ 1110.7±803.2 vol% μm) compared to both negative controls (untreated: ΔZ 4455.3±1176.4 vol% μm). Conclusions TiF4 varnish has no relevant antimicrobial effect. Nevertheless, TiF4 varnish was effective in reducing enamel demineralization under this model.

Evaluation of fluoride release from experimental TiF4 and NaF varnishes in vitro

Fluoride varnishes play an important role in the prevention of dental caries, promoting the inhibition of demineralization and the increase of remineralization. Objective: This study aimed to analyze the amount of fluoride released into water and artificial saliva from experimental TiF4 and NaF varnishes, with different concentrations, for 12 h. Material and Methods: Fluoride varnishes were applied on acrylic blocks and then immersed in 10 ml of deionized water and artificial saliva in polystyrene bottles. The acrylic blocks were divided in seven groups (n=10): 1.55% TiF4 varnish (0.95% F, pH 1.0); 3.10% TiF4 varnish (1.90% F, pH 1.0); 3.10% and 4% TiF4 varnish (2.45% F, pH 1.0); 2.10% NaF varnish (0.95% F, pH 5.0); 4.20% NaF varnish (1.90% F, pH 5.0); 5.42% NaF varnish (2.45% F, pH 5.0) and control (no treatment, n=5). The fluoride release was analyzed after 1/2, 1, 3, 6, 9 and 12 h of exposure. The analysis was performed using an ion-specific electrode coupled to a potentiometer. Two-way ANOVA and Bonferroni's test were applied for the statistical analysis (p<0.05). Results: TiF4 varnishes released larger amounts of fluoride than NaF varnishes during the first 1/2 h, regardless of their concentration; 4% TiF4 varnish released more fluoride than NaF varnishes for the first 6 h. The peak of fluoride release occurred at 3 h. There was a better dose-response relationship among the varnishes exposed to water than to artificial saliva. Conclusions: The 3.10% and 4% TiF4 -based varnishes have greater ability to release fluoride into water and artificial saliva compared to NaF varnish; however, more studies must be conducted to elucidate the mechanism of action of TiF4 varnish on tooth surface. .

Dental erosion: an overview on definition, prevalence,diagnosis and therapy / Erosão dentária: uma visão sobre definição, prevalência, diagnóstico e terapia

Currently, it has been observed a significant increase in the prevalence of dental erosion as a consequence of frequent exposure to acids from foods, drinks and gastric juice. The aim of this review was to give some new insights about the definition and diagnosis of this condition, to clarify the causal factors and to show the preventive strategies and restorative therapy. Dental erosion is complex condition dependent on the interaction of chemical, biological and behavior factors. The diagnosis is generally performed by the analysis of the clinical appearance of the lesions in combination with the patient’s history. Some new technologies have been developed to help in early diagnosis and to quantify dental erosion in different phases. Preventive measures are established according to the causal factors, which may include the dietary intervention, modification of acidic drinks, and behavioral changes, or the modification of the tooth surface to increase its resistance against acidic attacks. The restorative treatment may range from minimally invasive therapies to multidisciplinary interventions. The clinicians should know how to detect the condition early, so that preventive measures can be applied before the lesions progress. Therapeutic strategies in high-risk patients should be as conservative as possible, involving multidisciplinary and preventive approaches with a periodic control for the success of the treatment.

Atualmente, tem-se observado um aumento significativo na prevalência de erosão dentária como consequência da exposição frequente aos ácidos oriundos de alimentos, bebidas e do suco gástrico. O objetivo desta revisão foi expor alguns novos direcionamentos sobre a definição e diagnóstico desta condição, esclarecer os fatores causais e apresentar as estratégias para a prevenção e o tratamento. A erosão dentária é uma condição complexa dependente da interação entre fatores químicos, biológicos e comportamentais. O diagnóstico é geralmente realizado por meio da análise da aparência clínica das lesões em combinação com a história do paciente. Novas tecnologias foram desenvolvidas para ajudar no diagnóstico precoce e para quantificar as diferentes fases da erosão dentária. As medidas preventivas são estabelecidas de acordo com os fatores causais que podem incluir a intervenção na dieta, modificação de bebidas ácidas, mudanças de comportamento, ou a modificação da superfície dentária com o objetivo de aumentar a sua resistência ao ataque ácido. O tratamento restaurador pode variar de terapias minimamente invasivas a intervenções multidisciplinares. Os clínicos devem saber como detectar a condição na sua fase inicial, para que medidas preventivas possam ser aplicadas antes da progressão da lesão. Estratégias terapêuticas em pacientes de alto risco devem ser as mais conservadoras possíveis, envolvendo abordagens multidisciplinares e preventivas com um controle periódico do paciente, para o sucesso do tratamento