Dual-species biofilm of Streptococcus mutans and Candida albicans produces subsurface caries lesions on bovine enamel.

OBJECTIVES: To develop a protocol for forming subsurface caries lesions on bovine enamel by dual-species biofilms of Streptococcus mutans and Candida albicans in vitro. DESIGN: Biofilms were grown on bovine enamel specimens in artificial saliva (AS) for seven days. After 24 h of formation, the AS was supplemented or not with fluoride (F) using sodium fluoride (0.005 or 0.008 ppm F), and the biofilms were exposed or not to a 20 % sucrose solution (reproducing a cariogenic challenge) once/day. On the seventh day, the biofilms were harvested and had their extracellular polysaccharides (EPS) and inorganic components analyzed. The specimens were subjected to computed X-ray microtomography analysis to determine their mineral concentration. Data were compared using two-way analyses of variance, followed by Fisher's LSD or Student-Newman-Keuls tests (p < 0.05). RESULTS: Biofilms exposed to the cariogenic challenge had significantly higher EPS concentrations than those not exposed, regardless of the presence of F. For biofilms grown with 0.008 ppm F, those exposed to the cariogenic challenge had lower F levels than those not exposed. For biofilms exposed to the cariogenic challenge, those grown with 0.008 ppm F had lower lesion depths and integrated mineral loss, and higher outer layers than those grown without F. CONCLUSIONS: The dual biofilm model assessed was able to create subsurface caries lesions in bovine enamel in vitro, which was influenced by the presence of F in the culture medium and exposure to sucrose.

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.

In vitro effect of fluoride gels supplemented with nano-sized sodium trimetaphosphate on enamel erosive wear.

OBJECTIVE: To assess the protective effect of fluoride (F) gels supplemented with micrometric or nano-sized sodium trimetaphosphate (TMPmicro and TMPnano, respectively) against enamel erosion in vitro. METHODS: Bovine enamel blocks (n = 140) were selected according to their surface hardness, and randomly divided into seven groups (n = 20/group), according to the gels tested: Placebo (without F/TMP), 4,500 µg F/g (4500F), 9,000 µg F/g (9000F), 4500F plus 2.5 % TMPnano (2.5 % Nano), 4500F plus 5 % TMPnano (5 % Nano), 4500F plus 5 % TMPnano (Micro 5 %) and 12,300 µg F/g (Acid gel). Blocks were treated once during one minute with the gels, and submitted to erosive (ERO, n = 10/group) or erosive plus abrasive (ERO+ABR, n = 10/group) challenges 4 times/day, for 90 s for each challenge (under reciprocating agitation), during consecutive 5 days. Blocks were analyzed by profilometry, and by surface (SH) and cross-sectional hardness (∆KHN). Data were submitted to two-way ANOVA, and Fisher's LSD test (p < 0.05). RESULTS: For ERO, both TMPnano-containing gels promoted enamel wear significantly lower than Placebo and 4500F, reaching levels similar to both positive controls (9000F and acid gel); significantly lower softening was observed for enamel treated with 4500F+5 % Micro and 4500F+2.5 % Nano. Also, the lowest ∆KHN values were observed for 4500F+2.5 % TMPnano among the TMP-containing gels. For ERO+ABR, the lowest enamel wear was achieved by the use of 4500F+5 % Nano among all gels, including both positive controls; lower softening was observed for Placebo and 9000F groups. CONCLUSION: The addition of 5 % nano-sized TMP to a low-fluoride gel produced superior protective effects for enamel under both challenges conditions, when compared with micrometric TMP, reaching values similar to or superior than both positive controls, respectively for ERO and ERO+ABR. CLINICAL SIGNIFICANCE: The supplementation of low-F gels with TMP was shown to significantly improve their effects on enamel erosive wear, and the use of nano-sized TMP further enhances this protective action.

Amount of Dentifrice and Fluoride Concentration Affect the pH and Inorganic Composition of Dual-Species Biofilms of Streptococcus mutans and Candida albicans.

This work assessed the influence of the amount of dentifrice and fluoride (F) concentration in the product on the pH and inorganic components of Streptococcus mutans and Candida albicans dual-species biofilms. The biofilms were treated with suspensions of fluoride dentifrices containing 550 or 1100 ppm of F (550 F or 1100 F, respectively) administered at comparable intensities: (i-1) 550 F/0.08 g or 1100 F/0.04 g; (i-2) 550 F/0.16 g or 1100 F/0.08 g; and (i-3) 550 F/0.32 g or 1100 F/0.16 g. A placebo dentifrice (without NaF, 0.32 g) was used as a negative control. After the last treatment, the biofilm pH was measured and the F, calcium (Ca), and phosphorus (P) concentrations were determined. Data were subjected to an ANOVA/Kruskal-Wallis test, and a Student-Newman-Keuls test. The highest biofilm pH and F concentrations (biomass and fluid) were observed for 1100 F at i-3. Overall, 1100 F resulted in F levels similar to 550 F for i-1 and i-2. In addition, 550 F applied at i-2 and i-3 led to higher F in the biomass/fluid compared to 1100 F applied at i-1 and i-2, respectively. In biomass, the lowest Ca concentrations were observed for 1100 F at i-3. The conclusion drawn is that the treatment intensity holds greater significance as a parameter compared to the concentration of F or the amount of dentifrice when considered individually.

Dual nanocarrier of chlorhexidine and fluconazole: Physicochemical characterization and effects on microcosm biofilms and oral keratinocytes.

OBJECTIVES: This study assembled and characterized a dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluated its antibiofilm and cytotoxic effects. METHODS: CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS) and characterized by physical-chemical analyses. Biofilms from human saliva supplemented with Candida species were grown (72 h) on glass discs and treated (24 h) with IONPs-CS carrying CHX (at 39, 78, or 156 µg/mL) and FLZ (at 156, 312, or 624 µg/mL) in three growing associations. IONPs and CS alone, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, microbiological analyses were performed. The viability of human oral keratinocytes (NOKsi lineage) was also determined (MTT reduction assay). Data were submitted to ANOVA or Kruskal-Wallis, followed by Fisher's LSD or Tukey's tests (α=0.05). RESULTS: Nanocarriers with spherical-like shape and diameter around 6 nm were assembled, without compromising the crystalline property and stability of IONPs. Nanocarrier at the highest concentrations was the most effective in reducing colony-forming units of Streptococcus mutans, Lactobacillus spp., Candida albicans, and Candida glabrata. The other carriers and CHX156-FLZ624 showed similar antibiofilm effects, and significantly reduced lactic acid production (p<0.001). Also, a dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier. IONPs-CS-CHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations ≥7.8 and 31.25 µg/mL, respectively (p<0.05). CONCLUSION: The nanotherapy developed outperformed the effect of the combination CHX-FLZ on microcosm biofilms, without increasing the cytotoxic effect of the antimicrobials administered. CLINICAL SIGNIFICANCE: The dual nanocarrier is a promising topically-applied therapy for the management of oral candidiasis considering that its higher antibiofilm effects allow the use of lower concentrations of antimicrobials than those found in commercial products.

Fluoride varnishes supplemented with nano-sized sodium trimetaphosphate reduce enamel erosive wear in vitro.

OBJECTIVE: To evaluate the effect of fluoride (F) varnishes with sodium trimetaphosphate (TMP) on erosive tooth wear (ETW) in vitro. METHODS: Enamel blocks (n = 100) were divided into 5 experimental groups (n = 20/group): Placebo (Pla - without F/TMP); 5 % NaF (NaF); 5 % NaF + 5 % micrometric TMP (NaF+5 %MICRO); 5 % NaF + 2.5 % nano-sized TMP (NaF+2.5 %NANO), and 5 % NaF + 5 % nano-sized TMP (NaF+5 %NANO). Blocks received a single varnish application (6 h contact), and were submitted to 4 daily erosive challenges (ERO, 0.05 M citric acid, pH 3.2, 90 s, under agitation), for 5 days. After ERO, half of the blocks (n = 10/group) were subjected to brushing abrasion (ERO+ABR). Profilometry, surface hardness (SH), and cross-sectional hardness (ΔKHN) were determined. The data were submitted to 2-way ANOVA and Fisher's LSD test (p < 0.05). RESULTS: Enamel wear was significantly lower for ERO compared with ERO+ABR for all varnishes tested (p < 0.001), following the pattern NaF+5 %NANO < NaF+5 %MICRO < NaF < NaF+2.5 %NANO < Pla (both for ERO and ERO+ABR). The highest SH loss was observed for Pla and the lowest for NaF (ERO) and NaF+2.5 %NANO (ERO+ABR), without significant differences among NaF+2.5 %NANO, NaF, and NaF+5 %MICRO. The highest ΔKHN values were observed for NaF+5 %MICRO and NaF+5 %NANO at 5-30 µm, with less marked differences among the groups at 30-70 µm (ERO and ERO+ABR). CONCLUSIONS: The addition of TMP to F varnishes significantly improves protection against ETW in vitro. The use of 5 % nano-sized TMP further enhances such effects. CLINICAL SIGNIFICANCE: F varnishes containing TMP can reduce enamel loss caused by ERO or ERO+ABR.

Evaluation of Solutions Containing Fluoride, Sodium Trimetaphosphate, Xylitol, and Erythritol, Alone or in Different Associations, on Dual-Species Biofilms.

Although the association of polyols/polyphosphates/fluoride has been demonstrated to promote remarkable effects on dental enamel, little is known on their combined effects on biofilms. This study assessed the effects of solutions containing fluoride/sodium trimetaphosphate (TMP)/xylitol/erythritol on dual-species biofilms of Streptococcus mutans and Candida albicans. Biofilms were grown in the continuous presence of these actives alone or in different associations. Quantification of viable plate counts, metabolic activity, biofilm biomass, and extracellular matrix components were evaluated. Overall, fluoride and TMP were the main actives that significantly influenced most of the variables analyzed, with a synergistic effect between them for S. mutans CFUs, biofilm biomass, and protein content of the extracellular matrix (p < 0.05). A similar trend was observed for biofilm metabolic activity and carbohydrate concentrations of the extracellular matrix, although without statistical significance. Regarding the polyols, despite their modest effects on most of the parameters analyzed when administered alone, their co-administration with fluoride and TMP led to a greater reduction in S. mutans CFUs and biofilm biomass compared with fluoride alone at the same concentration. It can be concluded that fluoride and TMP act synergistically on important biofilm parameters, and their co-administration with xylitol/erythritol significantly impacts S. mutans CFUs and biomass reduction.

Antimicrobial Effect of Low-Fluoride Toothpastes Containing Polyphosphate and Polyols: An In Vitro Assessment of Inhibition Zones.

This study evaluated the antimicrobial effect of toothpastes containing 200 ppm fluoride (200F), xylitol (X, 16%), erythritol (E, 4%), and sodium trimetaphosphate (TMP, 0.25%), alone or in different associations, against Streptococcus mutans (SM), Lactobacillus casei (LC), Actinomyces israelii (AI), and Candida albicans (CA). Suspensions of the micro-organisms were added to a BHI Agar medium. Five wells were made on each plate to receive toothpaste suspensions at different dilutions. Toothpastes containing no actives (placebo) or 1100 ppm F (1100F) were used as negative and positive controls. Two-way ANOVA and Tukey's HDS test were used (p < 0.05). For SM, the largest halo was for 200F+TMP at all dilutions, followed by the 200F+X+E toothpaste (p < 0.001). For LC, the overall trend showed that the polyols effectively inhibited microbial growth, and the association with the other compounds enhanced such effects (p < 0.001). For AI, a less-defined trend was observed. For CA, the experimental toothpaste (200F+X+E+TMP) was consistently more effective than the other treatments, followed by 200F+X+E (p < 0.001). The association of polyols and TMP in a low-fluoride toothpaste effectively reduced the growth of cariogenic micro-organisms (SM, CA, and LC), suggesting that this formulation could be an interesting alternative for children due to its low fluoride content.

Effect of fluoride gels with nano-sized sodium trimetaphosphate on the in vitro remineralization of caries lesions.

OBJECTIVE: To evaluate the effects of fluoride (F) gels supplemented with micrometric or nano-sized sodium trimetaphosphate (TMPmicro and TMPnano, respectively) on the in vitro remineralization of caries-like lesions. METHODOLOGY: Bovine enamel subsurface lesions (n=168) were selected according to their surface hardness (SH) and randomly divided into seven groups (n=24/group): Placebo (without F/TMP), 4,500 ppm F (4500F), 4500F + 2.5% TMPnano (2.5% Nano), 4500F + 5% TMPnano (5% Nano), 4500F + 5% TMPmicro (5% Micro), 9,000 ppm F (9000F), and 12,300 ppm F (Acid gel). The gels were applied in a thin layer for one minute. Half of the blocks were subjected to pH cycling for six days, whereas the remaining specimens were used for loosely- (calcium fluoride; CaF2) and firmly-bound (fluorapatite; FA) fluoride analysis. The percentage of surface hardness recovery (%SHR), area of subsurface lesion (ΔKHN), CaF2, FA, calcium (Ca), and phosphorus (P) on/in enamel were determined. Data (log10-transformed) were subjected to ANOVA and the Student-Newman-Keuls' test (p<0.05). RESULTS: We observed a dose-response relation between F concentrations in the gels without TMP for %SHR and ΔKHN. The 2.5% Nano and 5% Micro reached similar %SHR when compared with 9000F and Acid gels. For ΔKHN, Placebo and 5% Nano gels had the highest values, and 5% Micro, 2.5% Nano, 9000F, and Acid gels, the lowest. All groups had similar retained CaF2 values, except for Placebo and Acid gel. We verified observed an increase in Ca concentrations in nano-sized TMP groups. Regarding P, TMP groups showed similar formation and retention to 9000F and Acid. CONCLUSION: Adding 2.5% nano-sized or 5% micrometric TMP to low-fluoride gels lead to enhanced in vitro remineralization of artificial caries lesions.

Are the counts of Streptococcus mutans and Staphylococcus aureus changed in complete denture wearers carrying denture stomatitis? A systematic review with meta-analyses.

STATEMENT OF PROBLEM: Despite the importance of Candida spp. on the etiology of denture stomatitis (DS), information on the role of the bacterial component is still scarce. PURPOSE: The purpose of this systematic review was to evaluate whether the counts of Staphylococcus aureus and Streptococcus mutans were changed in complete denture wearers diagnosed with Candida-associated DS. MATERIAL AND METHODS: The literature search was performed in 8 databases and by hand searching. The risk of bias was assessed according to the Newcastle-Ottawa qualifier. Meta-analyses were performed considering the microorganism evaluated (S. aureus or S. mutans) and the collection area (mucosa or dentures). The certainty of evidence was assessed according to the grading of recommendations assessment, development and evaluations (GRADE) criteria. RESULTS: Participants with DS presented higher counts of S. aureus in the mucosa compared with those from the control group (OR, 3.16 [1.62, 6.15]; P<.001). No significant difference between the groups was observed for samples collected from dentures (OR, 0.73 [0.50, 1.07]; P=.110). Conversely, participants without DS presented higher counts of S. mutans both in the mucosa (OR, 0.19 [0.06, 0.63]; P=.006) and dentures (OR, 0.64 [0.41, 1.0]; P=.050). CONCLUSIONS: Microbial counts in participants with DS changed as a function of the type of microorganism and collection site. The certainty of evidence ranged from very low to low. The findings reinforce the fact that bacteria also play a relevant role in DS and should be more extensively studied. Such information may be useful to guide further therapies to prevent or control DS.

The Activity of Calcium Glycerophosphate and Fluoride against Cariogenic Biofilms of Streptococcus mutans and Candida albicans Formed In Vitro.

This study evaluated the effects of calcium glycerophosphate (CaGP), with or without fluoride (F), on dual-species biofilms of Streptococcus mutans and Candida albicans. The biofilms were treated three times with 0.125, 0.25, and 0.5% CaGP solutions, with or without 500 ppm F (NaF). Additionally, 500 and 1100 ppm F-solutions and artificial saliva served as controls. After the final treatment, the microbial viability and biofilm structure, metabolic activity, total biomass production, and the composition of the extracellular matrix composition were analyzed. Regardless of the presence of F, 0.25 and 0.5% CaGP promoted a higher biomass production and metabolic activity increase than the controls (p < 0.05). F-free CaGP solutions reduced bacterial cell population significantly more than the 500 ppm F group or the negative control (p < 0.05). All the groups reduced the proteins, and 0.5% CaGP combined with F led to the highest reduction in the carbohydrate and nucleic acids content of the extracellular matrix (p < 0.05). It can be concluded that CaGP alone affected the number of bacterial cells and, when combined with F, reduced its production of biomass, metabolic activity, and the expression of the extracellular matrix components.

Effect of fluoride gels with nano-sized sodium trimetaphosphate on the in vitro remineralization of caries lesions

Abstract Objective To evaluate the effects of fluoride (F) gels supplemented with micrometric or nano-sized sodium trimetaphosphate (TMPmicro and TMPnano, respectively) on the in vitro remineralization of caries-like lesions. Methodology Bovine enamel subsurface lesions (n=168) were selected according to their surface hardness (SH) and randomly divided into seven groups (n=24/group): Placebo (without F/TMP), 4,500 ppm F (4500F), 4500F + 2.5% TMPnano (2.5% Nano), 4500F + 5% TMPnano (5% Nano), 4500F + 5% TMPmicro (5% Micro), 9,000 ppm F (9000F), and 12,300 ppm F (Acid gel). The gels were applied in a thin layer for one minute. Half of the blocks were subjected to pH cycling for six days, whereas the remaining specimens were used for loosely- (calcium fluoride; CaF2) and firmly-bound (fluorapatite; FA) fluoride analysis. The percentage of surface hardness recovery (%SHR), area of subsurface lesion (ΔKHN), CaF2, FA, calcium (Ca), and phosphorus (P) on/in enamel were determined. Data (log10-transformed) were subjected to ANOVA and the Student-Newman-Keuls' test (p<0.05). Results We observed a dose-response relation between F concentrations in the gels without TMP for %SHR and ΔKHN. The 2.5% Nano and 5% Micro reached similar %SHR when compared with 9000F and Acid gels. For ΔKHN, Placebo and 5% Nano gels had the highest values, and 5% Micro, 2.5% Nano, 9000F, and Acid gels, the lowest. All groups had similar retained CaF2 values, except for Placebo and Acid gel. We verified observed an increase in Ca concentrations in nano-sized TMP groups. Regarding P, TMP groups showed similar formation and retention to 9000F and Acid. Conclusion Adding 2.5% nano-sized or 5% micrometric TMP to low-fluoride gels lead to enhanced in vitro remineralization of artificial caries lesions.

Buffering Capacity and Effects of Sodium Hexametaphosphate Nanoparticles and Fluoride on the Inorganic Components of Cariogenic-Related Biofilms In Vitro.

Despite the remarkable effects of sodium hexametaphosphate nanoparticles (HMPnano) on dental enamel de-/re-mineralization processes, information on the effects of these nanoparticles on biofilms is scarce. This study assessed the effects of HMPnano, with or without fluoride (F), on the inorganic components and pH of Streptococcus mutans and Candida albicans dual-species biofilms. Solutions containing conventional/micro-sized HMP (HMPmicro) or HMPnano were prepared at 0.5% and 1%, with or without 1100 ppm F. A 1100 ppm F solution and pure artificial saliva were tested as positive and negative controls, respectively. The biofilms were treated three times and had their pH analyzed, and the concentrations of F, calcium, phosphorus, and HMP in the biofilm biomass and fluid were determined. In another set of experiments, after the last treatment, the biofilms were exposed to a 20% sucrose solution, and the biofilm pH and inorganic components were evaluated. The 1% HMPnano solution with F led to the highest biofilm pH, even after exposure to sucrose. The 1% HMPnano solution without F led to significantly higher phosphorus concentrations in comparison to all other groups. It can be concluded that 1% HMPnano and F influenced the biofilm pH, besides affecting most of the inorganic components of the dual-species biofilms.

Effects of Sodium Hexametaphosphate and Fluoride on the pH and Inorganic Components of Streptococcus mutans and Candida albicans Biofilm after Sucrose Exposure

In order to improve the anticaries effects of fluoridated products, the supplementation of these products has been considered a promising alternative for caries control. This study evaluated the effects of sodium hexametaphosphate (HMP) and/or fluoride (F) on the inorganic components and pH of Streptococcus mutans and Candida albicans dual-species biofilms. The biofilms were treated 72, 78, and 96 h after the beginning of their formation with 0.25, 0.5, or 1% HMP-containing solutions with or without F (500 ppm, as sodium fluoride). F-containing solutions (500 ppm and 1100 ppm) and artificial saliva were used as controls. The biofilms were exposed to a 20% sucrose solution after the third treatment. Along with the biofilm pH, the concentrations of F, calcium, phosphorus (P), and HMP were determined. HMP, combined with F, increased F levels and decreased P levels in the biofilm fluid compared to that of the solution with 500 ppm F. Exposure to sucrose decreased the concentrations of all ions in the biomass, except for HMP; 1% HMP, combined with F, promoted the highest pH. It can be concluded that HMP affected the inorganic composition of the biofilm and exerted a buffering effect on the biofilm pH.

Effects of sodium hexametaphosphate microparticles or nanoparticles on the growth of saliva-derived microcosm biofilms.

OBJECTIVES: This study evaluated the effects of sodium hexametaphosphate microparticles (HMPmicro) or nanoparticles (HMPnano) on the growth of saliva-derived microcosm biofilms MATERIALS AND METHODS: Saliva-derived biofilms were formed on glass coverslips for 24 h. Thereafter, Streptococcus mutans (C180-2) was incorporated or not into the biofilms. From that time point onwards, solutions containing 0.2% HMPmicro or HMPnano, combined or not with 220 ppm F, were constantly present in the culture medium. In addition, 220 ppm F alone (220F) and McBain medium without any compound were also tested as positive and negative controls (CTL), respectively. After 96 h, the biofilms were plated on anaerobic blood agar or sucrose agar bacitracin for total and S. mutans CFU-counting, respectively. Biofilms' lactic acid production was analysed spectrophotometrically. Data were submitted to ANOVA or Kruskal-Wallis' tests, followed by Student-Newman-Keuls' test (p<0.05; n=12). RESULTS: HMPmicro or HMPnano led to significantly lower lactic acid production, and significant reductions in total CFU-counting in microcosm biofilms, supplemented or not with S. mutans, in comparison to both controls, with significant differences between 220F and CTL. No significant differences were observed among the groups treated with HMPmicro or HMPnano (with or without F). The same trend was seen for S. mutans CFU-counting, in biofilms supplemented with S. mutans. CONCLUSIONS: HMP significantly reduced total and S. mutans CFU counts, as well as lactic acid production by saliva-derived microcosm biofilms. CLINICAL RELEVANCE: These findings in saliva-derived microcosm biofilms suggest that HMP stands as a promising alternative for the control of cariogenic biofilms.

Effects of nano-sized sodium hexametaphosphate on the viability, metabolism, matrix composition, and structure of dual-species biofilms of Streptococcus mutans and Candida albicans.

This study evaluated the effects of micrometric or nano-sized sodium hexametaphosphate (HMPnano), combined or not with fluoride (NaF, 1100 ppm), on dual-species biofilms of Streptococcus mutans and Candida albicans. Biofilms were treated with solutions containing the polyphosphates at 0.5% or 1.0%, with/without fluoride (F), in addition to positive and negative controls. Biofilms were analysed by colony-forming units (CFU) counting, metabolic activity, production of biomass, composition of extracellular matrix, and structure. 1% HMPnano + F led to the lowest S. mutans CFU, while C. albicans CFU counts were not affected by any solution. 1% HMPnano led to the lowest metabolic activity, except for 1% HMPnano + F. All solutions promoted reductions in biofilm biomass compared to controls. Also, 1% HMPnano + F promoted the lowest concentrations of carbohydrates in the biofilm matrix, besides substantially affecting biofilms' structure. In conclusion, HMPnano and F promoted higher antibiofilm effects compared with its micrometric counterpart for most of the parameters assessed.

Activity of Sodium Trimetaphosphate Nanoparticles on Cariogenic-Related Biofilms In Vitro.

In light of the promising effect of sodium trimetaphosphate nanoparticles (TMPn) on dental enamel, in addition to the scarce evidence of the effects of these nanoparticles on biofilms, this study evaluated the activity of TMPn with/without fluoride (F) on the pH, inorganic composition and extracellular matrix (ECM) components of dual-species biofilms of Streptococcus mutans and Candida albicans. The biofilms were cultivated in artificial saliva in microtiter plates and treated with solutions containing 1% or 3% conventional/microparticulate TMP (TMPm) or TMPn, with or without F. After the last treatment, the protein and carbohydrate content of the ECM was analyzed, and the pH and F, calcium (Ca), phosphorus (P), and TMP concentrations of the biofilms were determined. In another set of experiments, after the last treatment, the biofilms were exposed to a 20% sucrose solution, and their matrix composition, pH, and inorganic component contents were evaluated. 3% TMPn/F significantly reduced ECM carbohydrate and increased biofilm pH (after sucrose exposure) than other treatments. Also, it significantly increased P and F levels before sucrose exposure in comparison to 3% TMPm/F. In conclusion, 3% TMPn/F affected the biofilm ECM and pH, besides influencing inorganic biofilm composition by increasing P and F levels in the biofilm fluid.

Efeitos de nanopartículas de hexametafosfato de sódio, associadas ou não ao fluoreto, em biofilmes mistos de Streptococcus mutans e Candida albicans e em biofilmes microcosmos / Effects of sodium hexametaphosphate nanoparticles, combined or not with fluoride, on dual-species biofilms of Streptococcus mutans and Candida albicans and on microcosm biofilms

O presente estudo avaliou os efeitos de nanopartículas de hexametafosfato de sódio (HMPnano), associadas ou não ao fluoreto (F), na composição orgânica (Subprojeto 1- S1) e inorgânica (Subprojeto 2-S2) de biofilmes mistos de Streptococcus mutans e Candida albicans formados in vitro; e na viabilidade celular e atividade metabólica de biofilmes microcosmos derivados de saliva (Subprojeto 3-S3). Em S1 e S2, soluções de HMPnano ou HMP microparticulado (HMPmicro) foram preparadas a 0,5% ou 1%, com ou sem F (1100 ppm F, NaF), além de 1100 ppm F (controle positivo) e saliva artificial (controle negativo). S. mutans e C. albicans foram cultivados em saliva artificial. Os biofilmes foram formados no fundo de poços de placas de microtitulação e tratados 72, 78 e 96 horas após o início da formação, por 1 minuto. Em S1, após o último tratamento, realizou-se análises de quantificação das unidades formadoras de colônias (UFCs), produção de biomassa total, atividade metabólica, além da composição da matriz extracelular dos biofilmes e avaliação estrutural. Observou-se que 1% de HMPnano combinado ao F levou aos menores UFCs de S. mutans, bem como às menores concentrações de carboidratos da matriz extracelular dos biofilmes, além de afetar substancialmente a sua estrutura. Em S2, após o último tratamento, avaliou-se o pH e a composição inorgânica dos biofilmes (análise das concentrações de F, cálcio (Ca) e fósforo (P)), antes e após exposição a sacarose. Soluções contendo 1% HMPnano combinado ao F promoveram os maiores valores de pH dos biofilmes, mesmo após exposição à sacarose. Além disso, 1% HMPnano promoveu maiores concentrações de P, enquanto que o HMP (micro/nano, com/sem F) levou a concentrações inexpressivas de Ca no fluido do biofilme. Em S3, os efeitos do HMPmicro ou HMPnano, sozinhos ou associados ao F, foram avaliados em biofilmes microcosmos derivados de saliva. Os biofilmes foram formados sobre discos de vidro por 24 h e, em seguida, S. mutans (C180- 2) foi incorporado ou não aos biofilmes. A partir deste momento, os mesmos ativos avaliados em S1/S2 foram adicionados ao meio de cultura, a 20% das concentrações utilizadas nesses subprojetos. Após 96 h de formação, foram determinadas as UFCs totais e de S. mutans, e avaliada a produção de ácido láctico pelos biofilmes. Todos os meios de cultura contendo contendo HMP levaram às menores concentrações de ácido láctico e às maiores reduções de UFCs totais e de S. mutans dos biofilmes, sem influência do tamanho da partícula de HMP, associação com F ou adição de S. mutans. Conclui-se que o HMPnano a 1%, associado a 1100 ppm F, promoveu uma diminuição substancial no metabolismo de biofilmes mistos de S. mutans e C. albicans, e da viabilidade de S. mutans. Esta combinação também levou a valores de pH mais próximos do neutro, além de afetar a composição inorgânica destes biofilmes. Para biofilmes microcosmos, o HMP promoveu a diminuição da viabilidade microbiana e acidogenicidade, sem influência, entretanto, do tamanho da partícula de HMP e da presença de F(AU)

This study evaluated the effects of sodium hexametaphosphate nanoparticles (HMPnano), combined or not with fluoride (F), on the organic (Subproject 1-S1) and inorganic (Subproject 2-S2) compositions of dual-species biofilms of Streptococcus mutans and Candida albicans formed in vitro; and on the cell viability and metabolic activity of saliva-derived microcosms biofilms (Subproject 3-S3). In S1 and S2, solutions containing HMPnano or conventional/micrometric HMP (HMPmicro) were prepared at 0.5 or 1%, combined or not with F (1,100 ppm F, as NaF). Also, a solution containing 1,100 ppm F and pure artificial saliva were tested as positive and negative controls, respectively. S. mutans and C. albicans strains were cultivated in artificial saliva. The biofilms were formed in well plates, and treated with the test solutions at 72, 78 and 96 from the beginning of the biofilm formation, for 1 minute. In S1, after the last treatment, the number of the colony-forming units (CFUs), production of total biomass, metabolic activity, composition of the extracellular matrix, and the structure of the biofilms were determined. HMP at 1% combined with F led to the lowest S. mutans CFUs and lowest concentration of carbohydrates from the extracellular matrix of the biofilms, besides substantially affecting biofilm's structure. In S2, after the last treatment, the pH and the inorganic composition of the biofilms (analysis of F, calcium (Ca), and phosphorus (P) concentrations), prior to and after sucrose exposure, were evaluated. Solutions containing 1% HMPnano combined with F led to the highest biofilm pH, even after exposure to sucrose. In addition, 1% HMPnano promoted the highest P concentrations, while HMP (micro/nano, with/without F) led to inexpressive Ca levels in the biofilm fluid. In S3, the effects of HMPmicro or HMPnano, alone or associated with F, were evaluated in salivaderived microcosm biofilms, which were formed during 24 h attached to glass coverslips. Thereafter, S. mutans (C180-2) was incorporated to the biofilms. From that timepoint onwards, the same actives analyzed in S1/S2 were added to the culture medium at 20% of the concentrations used in those subprojects. After 96 h of formation, total and S. mutans CFU-counting were determined, and the production of lactic acid was assessed. All HMP-containing culture media led to the lowest lactic acid concentrations, and to the highest reductions in total and S. mutans CFU counts, with no significant influence of HMP's particle size, association with F or the addition of S. mutans. In summary, it was concluded from S1 and S2 that 1% HMPnano combined with 1,100 ppm F substantially reduced the metabolism of S. mutans and C. albicans dual-species biofilms, besides reducing the viability of S. mutans. Also, this combination led to biofilm pH values closer to neutral ones, besides affecting their inorganic composition. From S3, HMP promoted reductions in the microbial viability and acidogenicity, without the influence, however, of HMP's particle size or the presence of F(AU)

Effect of sodium hexametaphosphate and fluoride on dual-species biofilms of Candida albicans and Streptococcus mutans.

This study evaluated the effect of sodium hexametaphosphate (HMP), administered alone or in combination with fluoride (F), on dual-species biofilms of Streptococcus mutans and Candida albicans. Biofilms were treated with HMP solutions at 0.25%, 0.5% and 1%, alone or combined with F (0.05%), and compared by evaluating their structure and quantifying the colony-forming units (CFUs), metabolic activity, production of biomass and extracellular matrix components. All HMP-containing solutions were capable of reducing metabolic activity, the biofilm biomass, and the extracellular matrix components. Furthermore, the treatment with 1% HMP/F significantly reduced the CFUs of S. mutans, although it showed no effect on the CFUs of C. albicans, in the dual-species biofilms. In general, the combination of HMP and F influenced all the parameters analyzed from dual-species biofilms, except the CFUs of C. albicans.

Calcium glycerophosphate and fluoride affect the pH and inorganic composition of dual-species biofilms of Streptococcus mutans and Candida albicans.

OBJECTIVES: This study evaluated the influence of calcium glycerophosphate (CaGP), combined with or without fluoride (F), on the pH and concentrations of F, Ca, and P of dual-species biofilms of Streptococcus mutans and Candida albicans, with or without exposure to sucrose. METHODS: The biofilms (n = 9) received three treatments (72, 78, and 96 h after the start of their formation) at three CaGP concentrations (0.125, 0.25, or 0.5%), with or without F at 500 ppm (as NaF). Solutions containing 500 and 1100 ppm F and artificial saliva were also tested as controls. Biofilm pH was measured, and the concentrations of F, Ca, P, and CaGP were determined (solid and fluid phases). In a parallel experiment, after the third treatment, the treated biofilms were exposed to a sucrose solution, and the pH of the medium, F, Ca, P, and CaGP was determined. Data were subjected to two-way ANOVA, followed by Fisher's LSD test (p < 0.05). RESULTS: Treatment with CaGP and 500 ppm F led to the highest pH values and F and Ca concentrations in the biofilm biomass, both with and without sucrose exposure. CaGP without F led to higher Ca and P concentrations in the biofilm fluid. CONCLUSIONS: CaGP increased F, Ca, and P concentrations in the biofilm, and its presence promoted an increase in the pH of the medium, even after exposure to sucrose. CLINICAL SIGNIFICANCE: The present results elucidate the mechanism by which CaGP and F act on biofilms, further interfering with dental caries dynamics.