Adaptive responses of the ileum of NOD mice to low-dose fluoride: A proteomic exploratory study.

Fluoride (F) has been employed worldwide to control dental caries. More recently, it has been suggested that the consumption of low doses of F in the drinking water may reduce blood glucose levels, introducing a new perspective for the use of F for the management of blood glucose. However, the exact mechanism by which F affects blood glucose levels remains largely unexplored. Given that the small gut plays a pivotal role in glucose homeostasis, the aim of this study was to investigate the proteomic changes induced by low doses of F in the ileum of female nonobese-diabetic (NOD) mice. Forty-two female NOD mice were divided into two groups based on the F concentration in their drinking water for 14 weeks: 0 (control) or 10 mgF/L. At the end of the experimental period, the ileum was collected for proteomic and Western blot analyses. Proteomic analysis indicated an increase in isoforms of actin, gastrotropin, several H2B histones, and enzymes involved in antioxidant processes, as well as a decrease in enzymes essential for energy metabolism. In summary, our data indicates an adaptive response of organism to preserve protein synthesis in the ileum, despite significant alterations in energy metabolism typically induced by F, therefore highlighting the safety of controlled fluoridation in water supplies.

Acquired Pellicle and Biofilm Engineering by Rinsing with Hemoglobin Solution.

INTRODUCTION: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the "acquired pellicle engineering" concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization. OBJECTIVE: Combining in vivo and in vitro proof-of-concept protocols, we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization. METHODS: In the in vivo studies, 10 volunteers, in 2 independent experiments, rinsed (10 mL,1 min) with deionized water-negative control or 1.0 mg/mL Hb. The AEP and biofilm formed along 2 or 3 h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA next-generation sequencing (NGS). In in vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with (1) phosphate-buffered solution (PBS), (2) 0.12% chlorhexidine, (3) 500 ppm NaF, (4) 1.0 mg/mL Hb, (5) 2.0 mg/mL Hb, and (6) 4.0 mg/mL Hb. The biofilm was cultivated for 5 days. Resazurin, colony forming units (CFU), and transversal microradiography were performed. RESULTS: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU, and enamel demineralization compared to PBS. CONCLUSION: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.

Proteomic profiles of the acquired enamel pellicle formed in vitro, in situ, or in vivo.

This study compared the protein profile of the acquired enamel pellicle (AEP) formed under three conditions: in vitro, in situ, and in vivo. Nine volunteers participated in all procedures. In the in vitro condition, the volunteers donated saliva, in which specimens were incubated to form the AEP. In the in situ condition, the volunteers used an oral device containing specimens where the AEP was formed. In the in vivo condition, the AEP was collected from the volunteers own teeth. All AEPs were formed for 120 min, collected and processed by mass spectrometry. Overall, a total of 321 proteins were identified, among which 37 proteins are commonly considered typical in the AEP. For each of the in vitro, in situ, and in vivo conditions, respectively, 66, 174, and 170 proteins were identified. For the in vitro condition, 17 pellicle-typical proteins were not identified. Furthermore, several proteins with important functions within the AEP presented differences in expression in the three conditions. The qualitative profile of the proteins, especially the typical ones, is different in the in vitro condition. In addition, there are important quantitative differences that may interfere when attempting to extrapolate in vitro results to an in situ and in vivo condition.

The effect of fluoride on the structure, function, and proteome of a renal epithelial cell monolayer.

High concentrations of fluoride in the body may cause toxic effects. Here, we investigated the effects of fluoride on the structure, function, and proteome of a cortical collecting duct epithelium in vitro. Kidney tubule cells (M-1) were chosen because the concentration of fluoride in the kidney is 4-5-fold higher than that in plasma. Mouse M-1 cell monolayers were incubated in fluoride-containing media, and the amiloride-sensitive short-circuit current and transepithelial resistance were measured. The Young's modulus of the epithelium was determined using atomic force microscopy, and the effect of fluoride on epithelial structure was assessed using scanning and transmission electron microscopy, and immunofluorescence. Differences in the expression of membrane proteins were evaluated using proteomics and bioinformatics. Fluoride exposure reduced both transepithelial Na+ transport and resistance. The IC50 for fluoride was ∼300 µM for both effects, and the half-times for the decays of ion transport and resistance were 8.4 h and 3.6 days, respectively. Fluoride treatment did not affect the sensitivity of Na+ transport to amiloride. The Young's modulus of the epithelium was also unaffected by fluoride; however, the functional effects of fluoride were accompanied by marked structural effects. Proteomic analysis revealed changes in expression of a number of proteins, and particularly mitochondrial proteins. Treatment with fluoride had profound effects on the structure, function and proteome of a model cortical collecting duct epithelium. Significantly, however, these effects were produced only at concentrations considerably higher than those likely to be encountered in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1455-1467, 2017.

Fluoride varnishes containing calcium glycerophosphate: fluoride uptake and the effect on in vitro enamel erosion.

OBJECTIVES: Calcium glycerophosphate (CaGP) was added to fluoride varnishes to analyze their preventive effect on initial enamel erosion and fluoride uptake: potassium hydroxide (KOH)-soluble and KOH-insoluble fluoride bound to enamel. MATERIALS AND METHODS: This study was carried out in two parts. Part 1: 108 enamel samples were randomly distributed into six varnish groups: base varnish (no active ingredients); Duraphat® (2.26%NaF); Duofluorid® (5.63%NaF/CaF2); experimental varnish 1 (1%CaGP/5.63 NaF/CaF2); experimental varnish 2 (5%CaGP/5.63%NaF/CaF2); and no varnish. Cyclic demineralization (90 s; citric acid, pH = 3.6) and remineralization (4 h) was made once a day, for 3 days. Change in surface microhardness (SMH) was measured. Part 2: 60 enamel samples were cut in half and received no varnish (control) or a layer of varnish: Duraphat®, Duofluorid®, experimental varnishes 1 and 2. Then, KOH-soluble and KOH-insoluble fluoride were analyzed using an electrode. RESULTS: After cyclic demineralization, SMH decreased in all samples, but Duraphat® caused less hardness loss. No difference was observed between varnishes containing CaGP and the other varnishes. Similar amounts of KOH-soluble and insoluble fluoride was found in experimental varnish 1 and Duofluorid®, while lower values were found for experimental varnish 2 and Duraphat®. CONCLUSION: The addition of CaGP to fluoride varnishes did not increase fluoride bound to enamel and did not enhance their protection against initial enamel erosion. CLINICAL RELEVANCE: We observe that the fluoride varnishes containing CaGP do not promote greater amounts of fluoride bound to enamel and that fluoride bound to enamel may not be closely related to erosion prevention.

Efficacy of TiF4 and NaF varnish and solution: a randomized in situ study on enamel erosive-abrasive wear.

OBJECTIVES: This in situ/ex vivo study analysed the anti-erosive/abrasive effect of TiF4 and NaF varnish and solution on enamel wear. MATERIALS AND METHODS: Twelve subjects took part in this study which was performed in three periods (phases) with the duration of 5 days each. Each two human enamel specimens per subject were pretreated with experimental NaF varnish or solution (phase A), experimental-TiF4 varnish or solution (phase B) and placebo varnish or untreated control (phase C). The specimens were worn in palatal appliances; one enamel specimen, from each treatment, was subjected to erosion (ERO; cola soft drink, 4 × 90 s/day), and the other specimen was subjected to erosion plus abrasion (ERO + ABR; tooth brushing, 2 × 10 s/day). The tooth wear was quantified by a contact profilometer (micrometre) and analysed using two-way repeated measures ANOVA and Bonferroni's test (n = 12 subjects, p < 0.05). RESULTS: All fluoride varnishes and solutions reduced the enamel wear (around 25 %) significantly compared to the control and placebo varnish. There were no significant differences among the fluoride formulations and between the conditions ERO and ERO + ABR. CONCLUSIONS: Therefore, it can be concluded that TiF4 has the same protective potential as NaF formulations to reduce human enamel wear under this experimental in situ model. CLINICAL SIGNIFICANCE: In vitro studies have indicated a better anti-erosive/abrasive effect of TiF4 compared to NaF varnish. The present in situ study does not support the previous findings. Therefore, any of the tested professional fluoride varnishes in principle could be able to partially reduce enamel wear.

Differential analysis of the dentin soluble proteomic.

OBJECTIVES: To perform a differential analysis of the dentin soluble proteomic and assess the effects of tissue health state and protocol for protein extraction. We hypothesized the dentin soluble proteomic varies according to the tissue physiopathological state (intact vs. caries-affected) and protocol used to extract its proteins. METHODS: Dentin from freshly extracted non-carious and carious teeth were randomly assigned for protein extraction using either guanidine-HCl/ethylenediaminetetraacetic acid (EDTA) or acetic acid. Protein extracts from intact and caries-affected dentin were processed and digested with trypsin for shotgun label-free proteomic analysis (nLC-ESI-MS/MS). Peptides identification was performed on a nanoACQUITY UPLC-Xevo Q-Tof MS system. Peptides identified with scores of confidence greater than 95% were included in the quantitative statistical analysis embedded in the PLGS software. Differences between experimental conditions were calculated using Student test-t with significance pre-set at α=0.05. RESULTS: A total of 158 human proteins were identified. Approximately one-sixth of proteins (24/158) were present in at least two different extracts. Conversely, the greatest number of proteins (134/158) was identified uniquely in only one of the extracts. Overall, a larger number of soluble proteins was retrieved from caries-affected than intact dentin (86/158). Likewise, a greater number of proteins was extracted by the guanidine-HCl/EDTA (106/158) in comparison to acetic acid protocol. Several proteins detected in dentin extracts, mainly those from caries-affected teeth, are biological and/or metabolically involved with tissue turnover/remodeling. CONCLUSION: The identity/abundance of soluble proteins retrieved from and remained in dentin noticeably depend on this tissue physiopathological state and protocol used to remove its minerals. CLINICAL SIGNIFICANCE: The present findings brought new insight into the proteomic phenotype of human dentin and may provide targets for the development of novel caries disease-prevention therapies.

In situ effect of sodium fluoride or titanium tetrafluoride varnish and solution on carious demineralization of enamel.

This study evaluated the effect of titanium tetrafluoride (TiF(4)) formulations on enamel carious demineralization in situ. Thirteen subjects took part in this cross-over, split-mouth, double-blind study performed in three phases of 14 d each. In each subject, two sound and two predemineralized specimens of bovine enamel were worn intra-orally and plaque accumulation was allowed. One sound and one predemineralized specimen in each subject was treated once with sodium fluoride (NaF) varnish or solution (Treatment A); TiF(4) varnish or solution (Treatment B); or placebo varnish or no treatment (Treatment C). The initially sound enamel specimens were exposed to severe cariogenic challenge (20% sucrose, eight times daily for 5 min each time), whereas the predemineralized specimens were not. Eleven subjects were able to finish all experimental phases. The enamel alterations were quantified by surface hardness and transversal microradiography. Demineralization of previously sound enamel was reduced by all test formulations except for the NaF solution, while both TiF(4) formulations were as effective as NaF varnish. For the predemineralized specimens, enamel surface hardness was increased only by TiF(4) formulations, while subsurface mineral remineralization could not be seen in any group. Within the experimental protocol, TiF(4) was able to decrease enamel demineralization to a similar degree as NaF varnish under severe cariogenic challenges, while only TiF(4) formulations remineralized the enamel surface.

Positive correlation between fluoride release and acid erosion of restorative glass-ionomer cements.

OBJECTIVE: The aim of this study was to determine whether there is a correlation between acid erosion and fluoride release of conventional glass ionomer cements. METHODS: Ten specimens for each material were prepared for fluoride release tests and five for acid erosion tests separately. After placed in pH cycling solution, concentration of fluoride was measured by a fluoride-ion selective electrode each day for 15 days. For the acid erosion test, specimens were immersed in a lactic acid solution and their depth measured with a spring-loaded dial gauge. The data were submitted to 3-way ANOVA, followed by Tukey's test (p<0.05) RESULTS: All materials showed ability to elute fluoride in the 15 day period of the test, with the same pattern of high fluoride release at the first 24h. Despite this, the amount of fluoride released was statistically different among the 18 groups, with the highest for Maxxion R and the lowest for Chemfil Rock (p>0.05). The highest acid erosion values were registered for Magic Glass, Ion Z, VitroFil and Maxxion R, which exceeded the maximum stipulated by the relevant ISO test (ISO 9917-1). A positive linear correlation (r2=0.4886) was found for both properties, i.e., higher fluoride release is related to higher acid erosion. SIGNIFICANCE: Acid erosion and fluoride release are related properties of GICs, though factors such as pH and P/L ratio lead to differences between actual values for individual brands of these materials.

Effect of chronic exercise on fluoride metabolism in fluorosis-susceptible mice exposed to high fluoride.

The present study investigated the effect of chronic exercise on fluoride (F) metabolism in fluorosis-susceptible mice exposed to high-F and explored the relationship between F concentrations in bone and plasma. Thirty male mice were randomised into three groups: Group I (No-F, No-Exercise), Group II (50 ppmF, No-Exercise), Group III (50 ppmF, Exercise). Body weight and physical performance of all mice were measured at baseline and end of experiment. F concentrations of plasma and bone were measured at the end of experiment. Mean plasma F concentration was significantly higher (p < 0.001) in Groups II and III compared with Group I. Mean bone F concentration was also significantly higher (p < 0.01) in Groups II and III compared with Group I. There was a significant correlation (p = 0.01, r = 0.54) between F concentration of plasma and bone. Mean body weight of Group I mice was significantly higher than Group II (p < 0.001) and Group III (p = 0.001) mice at the end of the experiment. This study, which provides the first data on the effect of chronic exercise on F metabolism in fluorosis-susceptible mice, suggests no effect of chronic exercise on F in plasma and bone. However, exposure to high-F resulted in lower body weight and exercise capacity in mice.

Consumption of bottled water by children in the city of Bauru, State of São paulo, Brazil -- a brief communication.

OBJECTIVES: To determine the consumption of bottled water by children in the city of Bauru, State of São Paulo, Brazil. METHODS: A stratified sample of the 17 areas established by the city plan was used to identify a total of 1,000 homes for visitation. Information was collected using a questionnaire concerning the type of water consumed and population demographics. RESULTS: Overall, around 30 percent of all residences used bottled water. Among all households where bottled water was consumed, about 26 percent had children residing. For those with children and bottled water, 81 percent reported to use bottled water for the preparation of the children's foods and beverages. CONCLUSIONS: An important percentage of children consume bottled water in the city of Bauru, State of São Paulo, Brazil. Considering previous studies showing that fluoride concentrations vary in bottled water, public health measures should be implemented in order to guarantee adequate levels of fluoride in commercialized water.

Relationship between daily fluoride intake from diet and the use of dentifrice and human plasma fluoride concentrations.

The literature contains reports of the relationship between the fluoride concentrations in drinking water and human plasma. None of these studies, however, documented individual levels of daily fluoride intake, which can vary considerably among individuals served by the same water supply. Furthermore, while water can be an important source of fluoride, other sources, especially fluoridated dentifrices, also contribute substantially. This 2-day study with five 25-35-year-old subjects in each of three communities (Bauru, 0.6-0.8 ppm F; Domelia, 0.7 ppm F; Floresta, 0.3 ppm F) determined plasma fluoride concentrations and fluoride intake from diet and the use of dentifrice which, together, approximate total daily fluoride intake. The purposes were to determine: (1) the extent to which plasma fluoride concentrations approached levels known to affect the quality and quantity of bone; (2) the relationship between fluoride intake and plasma concentrations. Plasma was collected at 4-h intervals starting at 0800 h and ending at 2000 h each day. Average fluoride intakes from diet and the use of dentifrice in the three communities ranged from 0.16 to 0.82 mg/day and from 0.29 to 3.16 mg/day, respectively. The overall average plasma concentrations in the three communities were 0.44, 0.45 and 0.54 micromol/l (P<0.005). They were directly related to intake from the use of dentifrice (P=0.030) and to total intake (P=0.033), but were not related to dietary intake (P=0.176). In conclusion, despite fluoride intake from various sources, the plasma fluoride concentrations of the study subjects remained at levels far below those associated with effects on bone production.

Effect of Sodium Fluoride on the endogenous MMP Activity of Dentin Matrices.

OBJECTIVES: This study evaluated the effect of incorporating increasing concentrations of sodium fluoride in incubation media, on the loss of dry mass and solubilization of collagen from demineralized dentin beams incubated for up to 7 days. The effect of fluoride on the inhibition of matrix-bound metalloproteinases (MMPs) was also measured. METHODS: Dentin beams were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass, the beams were divided into six groups (n=10) and incubated at 37°C either in buffered media containing sodium fluoride (NaF) at 75, 150, 300, 450, 600 ppm or in fluoride-free media (control) for seven days. Following incubation, dry mass was re-measured. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HYP) as an index of solubilization of collagen by endogenous dentin proteases. Increasing concentrations of fluoride were also evaluated for their ability to inhibit rhMMP-9. RESULTS: Addition of NaF to the incubation media produced a progressive significant reduction (p<0.05) in the loss of mass of dentin matrices, with all concentrations demonstrating significantly less mass loss than the control group. Significantly less HYP release from the dentin beams was found in the higher fluoride concentration groups, while fluoride concentrations of 75 and 150 ppm significantly reduced rhMMP-9 activity by 6.5% and 79.2%, respectively. CONCLUSIONS: The results of this study indicate that NaF inhibits matrix-bound MMPs and therefore may slow the degradation of dentin matrix by endogenous dentin MMPs.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

Fluctuations in public water fluoride level in Bauru, Brazil.

OBJECTIVES: The aim of this study was to monitor the quality of public water supply fluoridation in Bauru, in the state of São Paulo, Brazil. METHODS: Water samples collected three times a week during four weeks in 20 areas of distribution of water were supplied by the City Water Department of Bauru, São Paulo, Brazil. Fluoride was analyzed using the ion-specific electrode (Orion model 9409) after sample buffering with an equal volume of TISAB II. Data were compared to fluoride concentration reported by City Water Department. RESULTS: Fluoride content ranged between 0.01 ppm and 9.35 ppm (n = 240). There was great variation among the different areas of distribution of water, as showed by the Levene test (P < .001). Kruskal-Wallis test showed a statistically significant difference in mean fluoride concentration among different areas (P < .001). Mean fluoride concentration was less than the optimum concentration (0.8 ppm) in 89 percent of samples. The values reported by the City water Department were always higher than the analyzed fluoride concentrations. CONCLUSIONS: Based on the results from this study, more rigorous surveillance and monitoring of water fluoridation in Bauru is recommended.

Enamel fluorosis prevalence after a 7-year interruption in water fluoridation in Jaú, São Paulo, Brazil.

OBJECTIVES: This paper analyzes the impact of a 7-year interruption in water fluoridation on the prevalence of enamel fluorosis in Jaú, state of São Paulo, Brazil. METHODS: Fluorosis prevalence (TF index) was evaluated in permanent maxillary central incisors of children (9-14 years old) that were 36 (n=81; cohort -36), 27 (n=81; cohort -27), and 18 months old (+/-1 month; n=89; cohort -18) in October 1991, when the breakstarted, and 18months old(+/-1 month;n=70; cohort 18) after that date. Children brushed their teeth prior to examination, which was conducted under natural light by three calibrated examiners (agreement 87.8-93.8%, kappa 0.72-0.85). RESULTS: The fluorosis prevalence (TF> or =1) was 7.41 percent, 3.70 percent, 7.87 percent, and 18.57 percent, respectively, for cohorts -36, -27, -18, and 18. The difference between cohort 18 and the other groups was statistically significant (Kruskall-Wallis test, P=.05). CONCLUSIONS: These results suggest that the fluoridated water is not an important risk factor for enamel fluorosis, since the prevalence of enamel fluorosis was low in the cohorts -36, -27, and -18 when fluoridated water was used.

Acquired pellicle as a modulator for dental erosion.

Dental erosion is a multifactorial condition that can result in the loss of tooth structure and function, potentially increasing tooth sensitivity. The exposure of enamel to acids from non-bacterial sources is responsible for the progression of erosion. These erosive challenges are counteracted by the anti-erosive properties of the acquired pellicle (AP), an integument formed in vivo as a result of selective adsorption of salivary proteins on the tooth surface, containing also lipids and glycoproteins. This review provides an in-depth discussion regarding how the physical structure of the AP, along with its composition, contributes to AP anti-erosive properties. The physical properties that contribute to AP protective nature include pellicle thickness, maturation time, and site of development. The pellicle contains salivary proteins embedded within its structure that demonstrate anti-erosive properties; however, rather than individual proteins, protein-protein interactions play a fundamental role in the protective nature of the AP. In addition, dietary and synthetic proteins can modify the pellicle, enhancing its protective efficiency against dental erosion. The salivary composition of the AP and its corresponding protein-profile may be employed as a diagnostic tool, since it likely contains salivary biomarkers for oral diseases that initiate at the enamel surface, including dental erosion. Finally, by modifying the composition and structure of the AP, this protein integument has the potential to be used as a target-specific treatment option for oral diseases related to tooth demineralization.

The Influence of Small Quantities of Oxygen in the Structure, Microstructure, Hardness, Elasticity Modulus and Cytocompatibility of Ti-Zr Alloys for Dental Applications.

The mechanical properties of Ti alloys are changed significantly with the addition of interstitial elements, such as oxygen. Because oxygen is a strong stabilizer of the α phase and has an effect on hardening in a solid solution, it has aroused great interest in the biomedical area. In this paper, Ti-Zr alloys were subjected to a doping process with small amounts of oxygen. The influence of interstitial oxygen in the structure, microstructure and some selected mechanical properties of interest for use as biomaterial and biocompatibility of the alloys were analyzed. The results showed that in the range of 0.02 wt% to 0.04 wt%, oxygen has no influence on the structure, microstructure or biocompatibility of the studied alloys, but causes hardening of the alloys, increasing the values of the microhardness and causing variation in the elasticity modulus values.

Bone response to fluoride exposure is influenced by genetics.

Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue.

Impact of experimental nano-HAP pastes on bovine enamel and dentin submitted to a pH cycling model.

This in vitro study evaluated the preventive potential of experimental pastes containing 10% and 20% hydroxyapatite nanoparticles (Nano-HAP), with or without fluoride, on dental demineralization. Bovine enamel (n=15) and root dentin (n=15) specimens were divided into 9 groups according to their surface hardness: control (without treatment), 20 Nanop paste (20% HAP), 20 Nanop paste plus (20% HAP + 0.2% NaF), 10 Nanop paste (10% HAP), 10 Nanop paste plus (10% HAP + 0.2% NaF), placebo paste (without fluoride and HAP), fluoride paste (0.2% NaF), MI paste (CPP-ACP, casein phosphopeptide-amorphous calcium phosphate), and MI paste plus (CPP-ACP + 0.2% NaF). Both MI pastes were included as commercial control products containing calcium phosphate. The specimens were treated with the pastes twice a day (1 min), before and after demineralization. The specimens were subjected to a pH-cycling model (demineralization-6-8 h/ remineralization-16-18 h a day) for 7 days. The dental subsurface demineralization was analyzed using cross-sectional hardness (kgf/mm 2 , depth 10-220 µm). Data were tested using repeated-measures two-way ANOVA and Bonferroni's test (p<0.05). The only treatment able to reduce the loss of enamel and dentin subsurface hardness was fluoride paste (0.2% NaF), which differed significantly from the control at 30- and 50-µm depth (p<0.0001). The other treatments were not different from each other or compared with the control. The experimental Nanop pastes, regardless of the addition of fluoride, were unable to reduce dental demineralization in vitro.