Influence of irradiated dentin, biofilm and different artificial saliva formulations on root dentin demineralization.

The aim of this study was evaluated the influence of radiation as well as of new formulations of artificial saliva on the development of root dentin lesions. Bovine root samples were divided into: irradiated (70 Gy) dentin or not; the type of biofilm (from irradiated patient-experimental or non-irradiated patient-control) and the type of artificial saliva (for the condition irradiated dentin/biofilm from irradiated patient): Control Artificial Saliva (inorganic); Control Saliva + 1 mg/ml hemoglobin; Control Saliva +0.1 mg/ml cystatin; Control Saliva + hemoglobin + cystatin; Bioextra (positive control) and deionized water (DiW, negative control) (n = 12/group). Biofilm was produced using human biofilm and McBain saliva (0.2 % of sucrose, 37o C and 5 % CO2); the treatments were done 1x/day, for 5 days. Colony-forming units (CFU) counting was performed; demineralization was quantified by transversal microradiography. Two-way ANOVA/Bonferroni or Sidak test for the comparison between biofilm x dentin and ANOVA/Tukey or Kruskal-Wallis/Dunn for comparing artificial saliva were done (p < 0.05). The type of biofilm had no influence on CFU and demineralization. Sound dentin under control biofilm presented the lowest Lactobacillus ssp. and Streptococcus mutans CFU and the lowest mean mineral loss (R) (25.6 ± 2.2; 23.7 ± 2.9 %) compared to irradiated dentin (26.1 ± 2.8; 28.1 ± 3.3, p < 0.004) for both types of biofilms (experimental and control, respectively). Bioextra was the only artificial saliva that reduced R (10.8 ± 2.5 %) and Lesion Depth (LD) (35 ± 15 µm) compared to DiW (17.3 ± 3.3 %, 81 ± 18 µm, p < 0.0001). Irradiation has impact on caries development; the experimental saliva were unable to reduce its occurrence.

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.

Salivary proteomic profile of response to different resistance training protocols: A case report.

Resistance training (RT) with blood flow restriction (BFR) or high intensity (HI) are effective to increase muscle mass. To understand this effect, techniques known as "omics" are used to identify possible biomarkers. This study analyzed the salivary proteomic profile of healthy individuals trained before and after two RT protocols both designed with eight exercises for upper- and lower-limbs, one performed at low percentage of one-maximum repetition (%1RM) with BFR technique, and other at high %1RM (HI) without BRF technique. Four healthy males between 18 and 28 years participated in the study. Stimulated saliva was collected before (BBFR/BHI) and immediately after (ABFR/AHI) the two RT protocols. All protein-related processing was performed using label-free proteomic. The difference in expression between groups was expressed as p < .05 for downregulated proteins and 1-p > .95 for upregulated proteins. There was difference in salivary flow between ABFR and BBFR (p = .005). For HI, 87 proteins were found after the practice and 119 before. Three hemoglobin isoforms were increased in AHI compared with BHI. In the BFR comparison, 105 proteins were identified after (ABFR) and 70 before (BBFR). Among those increased ABFR, we highlight five hemoglobin isoforms and Deleted in malignant brain tumors 1 protein. Between ABFR and AHI, 17 isoforms of histones, Transaldolase, Transketolase, Glyceraldehyde-3-phosphate dehydrogenase, and Antileukoproteinase were decreased ABFR. For HI, there was an increase in proteins related to oxidative stress and metabolism of the musculoskeletal system, compared with BFR. HI seems to induce higher anabolic signaling to muscle mass increase and antiatherosclerotic effects.

Bleaching as a complement to fluoride-enhanced remineralization or resin infiltration in masking white spot lesions

Abstract There are many suitable strategies for addressing caries, which is an ongoing worldwide problem. Although white spot lesions (WSLs) can be either remineralized naturally or treated with non- or micro-invasive strategies, their whitish and opaque appearance may persist. Objective To evaluate the effects of tooth bleaching as a complement to fluoride-enhanced remineralization or resin infiltration in masking WSLs, as well as in enamel surface roughness relative to that of the adjacent enamel. Methodology Flattened rectangular bovine enamel fragments (6×3×~2.9 mm length, width and thickness) were divided into six groups (L/N, F/N, F.BL/BL, I/N, I.BL/BL, N/N; n=15). Treatments applied to the 3×3 mm left half included: L (Lesion) - WSL simulation with 50 mM acetate buffer, 96 hours, 37ºC; F (Fluoride) - WSL treatment with 2% NaF neutral gel, 1x/week, 8 weeks; I (Infiltration) - WSL treatment with H3PO4 37%/10 s; Icon®-Dry/30 s; Icon®-Infiltrant/3 min+1 min; N (Nothing) - sound enamel/control. Treatments applied to both halves after F and I included: BL (Bleaching) - Opalescence Boost 40%, 3×/20 min each; N (Nothing) - control. The differences in color (ΔE00, ΔL, Δa, Δb) and surface roughness (ΔRa) between the left and right halves were measured. Kruskal-Wallis/post-hoc tests were applied to ΔE00, ΔL, Δa and ΔRa, and 1-way ANOVA/Tukey tests to Δb (α=0.05). Results The factor under study significantly influenced ΔE00 (p=0.0001), ΔL (p=0.0024), Δb (p=0.0015), and ΔRa (p<0.001), but not Δa (p=0.1592). Both fluoride-enhanced remineralization and resin infiltration were able to mask WSL, regardless of subsequent bleaching. However, when bleaching was performed, ΔE00 median values did not exceed the acceptability threshold for color difference. Only resin infiltration reduced ΔRa between WSL and the adjacent enamel. Conclusions Both remineralization and infiltration, particularly if complemented by bleaching, fostered satisfactory esthetic results. Only infiltration without bleaching led to really good results in surface roughness.

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

Abstract 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.

Proteomic analysis of stimulated saliva in gastroesophageal reflux disease patients with and without erosive tooth wear: Observational study.

OBJECTIVE: To evaluate the difference in the proteomic profile of stimulated saliva in patients with gastroesophageal reflux disease (GERD) with (GE) and without (GNE) erosive tooth wear (ETW), regarding both human and bacterial proteins. METHODS: Stimulated saliva (SS) was collected from 16 patients (8/group). Samples were centrifuged at 4.500 g for 15 min under refrigeration to remove all debris. The supernatant from each saliva sample was taken and frozen at -80 °C. After extracting the proteins, they were submitted to reverse phase liquid chromatography and mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification was performed using Protein Lynx Global Service (PLGS) software (p < 0.05) for human and bacterial proteins. RESULTS: In total, 67 human proteins were common for GNE and GE groups. GNE group presented, compared to GE group, increase in proteins that confer antimicrobial and acid resistant properties, such as cystatins, histatin and immunoglobulins. However, GNE group had a marked decrease in subunits of hemoglobin (α, ß and delta). Regarding bacterial proteins, for SS, 7 and 10 unique proteins were identified in the GE and GNE groups, respectively. They are related to protein synthesis and energy metabolism and interact with human proteins typically found in saliva and supramolecular complexes of the acquired pellicle. CONCLUSIONS: Our data indicate that the stimulation of the salivary flow increases acid resistant and antimicrobial proteins in saliva, which might protect against ETW. CLINICAL SIGNIFICANCE: This pioneer study showed important differences in the human and bacterial proteome of SS in patients with GERD with or without ETW.

Purification and Biological Properties of Raniseptins-3 and -6, Two Antimicrobial Peptides from Boana raniceps (Cope, 1862) Skin Secretion.

The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans' skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Effect of anaerobic or/and microaerophilic atmosphere on microcosm biofilm formation and tooth demineralization

Abstract Microcosm biofilms can reproduce the complexity of a dental biofilm. However, different forms of cultivation have been used. The impact of the culture atmosphere on the development of microcosm biofilms and their potential to cause tooth demineralization has not yet been deeply studied. Objective This study analyzes the effects of three experimental cultivation models (microaerophile vs. anaerobiosis vs. experimental mixed) on the colony-forming units (CFU) of the cariogenic microorganisms and tooth demineralization. Methodology 90 bovine enamel and 90 dentin specimens were distributed into different atmospheres: 1) microaerophilia (5 days, 5% CO2); 2) anaerobiosis (5 days, jar); 3) mixed (2 days microaerophilia and 3 days anaerobiosis), which were treated with 0.12% chlorhexidine (positive control - CHX) or Phosphate-Buffered Saline (negative control - PBS) (n=15). Human saliva and McBain's saliva containing 0.2% sucrose were used for microcosm biofilm formation, for 5 days. From the second day to the end of the experiment, the specimens were treated with CHX or PBS (1x1 min/day). Colony-forming units (CFU) were counted, and tooth demineralization was analyzed using transverse microradiography (TMR). Data were subjected to two-way ANOVA and Tukey's or Sidak's test (p<0.05). Results CHX was able to reduce total microorganism's CFU compared to PBS (differences of 0.3-1.48 log10 CFU/mL), except for anaerobiosis and microaerophilia in enamel and dentin biofilm, respectively. In the case of dentin, no effect of CHX on Lactobacillus spp. was observed. CHX significantly reduced enamel demineralization compared to PBS (78% and 22% reductions for enamel and dentin, respectively). Enamel mineral loss did not differ when compared with the other atmospheres; however, the enamel lesion depth was greater under anaerobiosis. Dentin mineral loss was lower under anaerobiosis when compared with the other atmospheres. Conclusion The type of atmosphere has, in general, little influence on the cariogenic ability of the microcosm biofilm.

Effect of experimental and commercial artificial saliva formulations on the activity and viability of microcosm biofilm and on enamel demineralization for irradiated patients with head and neck cancer (HNC).

The effect of different artificial saliva formulations on biofilm activity and viability, and on enamel demineralization for head and neck cancer (HNC) patients was evaluated. Irradiated enamel samples were treated (1 min) with BioXtra® or with experimental formulations containing carboxymethylcellulose plus inorganic constituents alone (AS) or containing 0.1 mg mL-1 CaneCPI-5 (AS + Cane), 1.0 mg mL-1 hemoglobin (AS + Hb) or combination of both (AS + Cane + Hb). Phosphate-buffered-saline and chlorhexidine (0.12%) were negative and positive control, respectively. Biofilm was produced from the saliva of five male HNC patients, under 0.2% sucrose exposure for 5 days, and daily treated with the formulations (1 min). No significant effects were observed for the different experimental treatments. BioXtra® significantly reduced lactobacilli, demonstrating antibacterial potential for this group. Chlorhexidine was an effective treatment to significantly reduce all parameters, being an important antimicrobial and anticaries agent. Future in vitro studies must be performed using a new approach for the design of the experimental formulations.

S-PRG-based toothpastes compared to NaF toothpaste and NaF varnish on dentin permeability in vitro

Abstract Objectives To analyze the effect of 5 toothpastes containing different percentages of S-PRG fillers compared to NaF toothpaste and NaF varnish on the dentin hydraulic conductance (Lp). Methodology Dentin disks (1.0±0.2 mm thickness) were cut from third molars, and their Lp values were evaluated using Flodec. The specimens were allocated into 7 groups (n=8). The minimum (smear layer) and the maximum (after acid etching) Lp values were recorded. Lp was also assessed after treatment with either a 0wt.%, 1wt.%, 5wt.%, 20wt.%, or 30wt.% S-PRG toothpaste, a NaF toothpaste, or a NaF varnish. Toothpastes were applied by brushing for 15 s, allowing it to settle for 1 min, and rinsing with deionized water. The NaF varnish was applied for 4 min and was removed with a probe. Specimens were exposed to citric acid (6%, pH 2.1, 1 min) and their final Lp was recorded. The pH of all products was recorded (n=3) and specimens from each group were analyzed by Laser Scanning Confocal Microscopy (LSCM). Data were subjected to 2-way repeated measures ANOVA and post-hoc Bonferroni (a=0.05). Results The highest Lp reduction was noticed for the 5wt.% S-PRG toothpaste, NaF toothpaste, and NaF varnish. However, the toothpastes containing 5wt.%, 20wt.%, and 30wt.% of S-PRG were similar to all toothpastes but differed from the NaF varnish. After erosion, all groups retrieved their maximum Lp values, except for the NaF varnish. The LSCM evidenced deposits on the surface of specimens treated with 5%, 20%, and 30% S-PRG-based toothpastes and NaF toothpaste. Even more deposits were observed for the NaF varnish. After the erosive challenge, the deposits were diminished in all groups. Conclusion Toothpastes containing 5wt.%, 20wt.%, and 30wt.% of S-PRG fillers behaved similarly to a conventional NaF toothpaste, even after an erosive challenge. The NaF varnish promoted better reduction of the Lp, but its effect was also diminished after erosion.

Characterization of white spot lesions formed on human enamel under microcosm biofilm for different experimental periods

Abstract The initial characteristics of white spot lesion (WSLs), such as the degree of integrated mineral loss (ΔZ), depth and pattern of mineral distribution, have an impact on further demineralization and remineralization. However, these lesion parameters have not been evaluated in WSLs produced from microcosm biofilms. Objective: This study characterized artificial white spot lesions produced on human enamel under microcosm biofilm for different experimental periods. Methodology: In total, 100 human enamel specimens (4x4mm) were assigned to 5 distinct groups (n=20/group) differing according to the period of biofilm formation (2, 4, 6, 8 or 10 days). Microcosm biofilm was produced on the specimens from a mixture of human and McBain saliva at the first 8h. Enamel samples were then exposed to McBain saliva containing 0.2% sucrose. WSLs formed were characterized by quantitative light-induced fluorescence (QLF) and transverse microradiography (TMR). Data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p<0.05). Results: A clear time-response pattern was observed for both analyses, but TMR was able to better discriminate among the lesions. Regarding QLF analysis, median (95%CI; %) changes in fluorescence ∆Z were -7.74(-7.74:-6.45)a, -8.52(-8.75:-8.00)ab, -9.17(-10.00:-8.71)bc, -9.58(-10.53:-8.99)bc and -10.01(-11.44:-9.72)c for 2, 4, 6, 8, and 10 days, respectively. For TMR, median (95%CI; vol%.µm) ∆Z were 1410(1299-1479)a, 2420(2327-2604)ab, 2775(2573-2899)bc, 3305(3192-3406)cd and 4330(3972-4465)d, whereas mean (SD; µm) lesion depth were 53.7(12.3)a, 71.4(12.0)a, 103.8(24.8)b, 130.5(27.2)bc, 167.2(39.3)c for 2, 4, 6, 8 and 10 days, respectively. Conclusion: The progression of WSLs formed on human enamel under microcosm biofilm can be characterized over 2-10 days, both by QLF and TMR analyses, although the latter provides better discrimination among the lesions.

Effect of fluoride, chlorhexidine or Nd:YAG on the progression of root dentin demineralization after removal of the demineralized organic matrix

Abstract Quantification of collagen degradation is an important parameter to evaluate dentin caries for preventive aid. Objectives: Evaluate preventive methods against root collagen degradation by the hydroxyproline assay (HYP) and microradiography technique (MRT). Methodology: Five bovine root dentin blocks were obtained and subjected to an artificial demineralization process by acetate buffer (pH 5) to induce carious lesion formation. Samples were subjected to the following therapeutic treatments: 1) 0.12% chlorhexidine for 1 min, 2) 2% fluoride for 1 min, 3) Nd:YAG Laser (400 μm diameter optical fiber, 10 Hz frequency, 60 mJ/pulse energy, 48 J/cm2 energy density, in noncontact mode for 10 s), 4) deionized water (control) for 1 min, 5) MRT control group (without treatment and removal of collagen). Samples were exposed to degradation by a collagenase enzyme for five days. The enzyme solution was collected, by colorimetry in a spectrophotometer, from the collagen matrix for the hydroxyproline release analysis. The same samples were subjected to an additional two days of demineralization to induce the progression of mineral loss. Samples were analyzed by MRT for the visualization of their degraded areas (estimation of lesion depth and mineral loss). ANOVA was applied to compare hydroxyproline release rates. MRT data were subjected to the Kruskal-Wallis test, followed by the Dunn's test. Comparisons between the initial five-day and the subsequent two-day demineralization processes were performed by repeated t-test or Wilcoxon (p<0.05) measurements. Results: The amount of HYP released from the dentin samples failed to show significant differences among the groups (p=0.09). Fluoride and chlorhexidine were able to interact with the samples, reducing the progression of dentin caries after removal of the demineralized organic matrix. CHX was the only treatment able to show significant lower lesion depth than the negative control. Conclusion: Chlorhexidine and fluoride were effective in reducing root caries progression.

Brazilian Theraphosidae: a toxicological point of view.

The Theraphosidae family includes the largest number of species of the Mygalomorphae infraorder, with hundreds of species currently catalogued. However, there is a huge lack on physiologic and even ecologic information available, especially in Brazil, which is the most biodiverse country in the world. Over the years, spiders have been presented as a source of multiple biologically active compounds with basic roles, such as primary defense against pathogenic microorganisms or modulation of metabolic pathways and as specialized hunters. Spider venoms also evolved in order to enable the capture of prey by interaction with a diversity of molecular targets of interest, raising their pharmaceutical potential for the development of new drugs. Among the activities found in compounds isolated from venoms and hemocytes of Brazilian Theraphosidae there are antimicrobial, antifungal, antiparasitic and antitumoral, as well as properties related to proteinase action and neuromuscular blockage modulated by ionic voltage-gated channel interaction. These characteristics are present in different species from multiple genera, which is strong evidence of the important role in spider survival. The present review aims to compile the main results of studies from the last decades on Brazilian Theraphosidae with special focus on results obtained with the crude venom or compounds isolated from both venom and hemocytes, and their physiological and chemical characterization.

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.

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.

Antimicrobial and anti-caries effects of a novel cystatin from sugarcane on saliva-derived multi-species biofilms.

This study evaluated the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of a new cystatin derived from sugarcane (CaneCPI-5). Microcosm biofilm was produced on bovine enamel specimens (4 x 4 mm; n=48) from a mixture of human saliva and McBain saliva at the first 8 h. From this moment until the end of the experiment, the enamel specimens were exposed to lsaMcBain saliva containing 0.2% sucrose and, once a day, they were treated with the test solutions for 1 min. This treatment was performed for 5 days. The solutions evaluated were: PBS (negative control), 0.12% chlorhexidine (positive control), 0.1 mg/ml CaneCPI-5 and 1.0 mg/ml CaneCPI-5. The biofilm viability was determined by fluorescence using confocal microscopy and the enamel demineralization was quantified using transverse microradiography (TMR). The data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests for biofilm and enamel, respectively (p<0.05). With respect to the antimicrobial effect, all treatment solutions significantly reduced the biofilm viability compared with PBS. The best antimicrobial effect was found for 1.0 mg/ml CaneCPI-5 (82.37±10.01% dead bacteria) that significantly differed from 0.12% chlorhexidine (73.13±15.07% dead bacteria). For the anti-caries effect, only 0.12% chlorhexidine (ΔZ: 2610, 1683-4343) performed significantly better than PBS (ΔZ: 8030, 7213-9115), but 0.12% chlorhexidine did not significantly differ from 0.1 mg/ml Cane-CPI-5. Under this experimental model, CaneCPI-5 significantly reduced the biofilm viability, but this effect was not reflected on its anti-caries potential.