Effect of Rubusoside, a Natural Sucrose Substitute, on Streptococcus mutans Biofilm Cariogenic Potential and Virulence Gene Expression In Vitro.

Dental caries is a biofilm-mediated disease in which Streptococcus mutans is the main pathogenic microorganism, and its incidence is closely related to sucrose. Rubusoside is a natural nonnutritive sweetener isolated from Rubus suavissimus S. Lee. This study was designed to determine the effect of this sucrose substitute on the cariogenic properties and virulence gene expression of S. mutans biofilms. S. mutans was exposed to brain heart infusion (BHI) medium (as a control), 1% sucrose-supplemented medium, 1% rubusoside-supplemented medium, and 1% xylitol-supplemented medium. The growth curve of the biofilm was monitored by crystal violet staining, and the pH was measured every 24 h. After 5 days, the biofilms formed on the glass coverslips were recovered to determine the biomass (dry weight and total amount of soluble proteins), numbers of CFU, and amounts of intra- and extracellular polysaccharides. Biofilm structural imaging was performed using a scanning electron microscope (SEM). Virulence gene expression (gtfB, gtfC, gtfD, ftf, spaP, gbpB, ldh, atpF, vicR, and comD) was determined by reverse transcription-quantitative PCR. Growth in rubusoside resulted in lower levels of acid production than observed during growth in sucrose, xylitol, and the control, while it also reduced the level of biofilm accumulation and bacterial viability and even reduced the level of production of extracellular polysaccharides. By SEM, the levels of biofilm formation and extracellular matrix during growth in rubusoside were lower than these levels during growth in sucrose and xylitol. From the perspective of virulence genes, growth in rubusoside and xylitol significantly inhibited the expression of virulence genes compared with their levels of expression after growth in sucrose. Among these genes, gtfB, gtfC, gbpB, ldh, and comD downregulation was found with growth in rubusoside compared with their expression with growth in xylitol. Therefore, rubusoside appears to be less potentially cariogenic than sucrose and xylitol and may become an effective sucrose substitute for caries prevention. Further studies are needed to deepen these findings.IMPORTANCE Dental caries is a major public health challenge and places heavy biological, social, and financial burdens on individuals and health care systems. To palliate the deleterious effect of sucrose on the virulence factors of S. mutans, massive commercial efforts have been oriented toward developing products that may act as sucrose substitutes. Rubusoside, a natural sucrose substitute, is a plant extract with a high level of sweetness. Although some studies have shown that rubusoside does not produce acids or inhibit the growth of S. mutans, little attention has been paid to its effect on dental biofilm and the underlying mechanisms. Our study focuses on the effect of rubusoside on the formation and structure of biofilms and the expression of virulence genes. The results confirm that rubusoside can inhibit accumulation, bacterial viability, polysaccharide production by the biofilm, and related gene expression. These results provide further insight into the cariogenicity of S. mutans biofilms and demonstrate a new perspective for studying the impact of sucrose substitutes on caries.

Galla chinensis Compounds Remineralize Enamel Caries Lesions in a Rat Model.

OBJECTIVES: To investigate the effect of Galla chinensis chemical compounds on enamel caries remineralization in rats. METHODS: A total of 40 rats were inoculated with Streptococcus sobrinus 6715 and fed a cariogenic diet (Keyes 2000). The rats were randomly divided into 4 groups and treated topically twice a day with each of the following extracts (or control) for 5 weeks: distilled and deionized water (DDW, negative control); 1,000 ppm NaF (positive control); 4,000 ppm G. chinensis crude aqueous extract (GCE), or 4,000 ppm gallic acid (GA). After the experimental period, Keyes' caries diagnosis and scoring technique was applied as a preliminary evaluation on the molar teeth. For more accurate remineralization data, the residual enamel volume of the first molar and the mineral density (MD) of the enamel were further analyzed by micro-CT. RESULTS: The severity of molar enamel caries decreased in the following order of treatment groups: DDW > GA > GCE > NaF (p < 0.05). In addition, the residual first molar enamel volume and MD values increased in the order of DDW, GA, GCE and NaF treatment groups (p < 0.05). In most molars, remineralization layers were observed on the surface of lesions. CONCLUSION: G. chinensis compounds remineralize enamel caries lesions in a rat model.

Cariogenicity features of Streptococcus mutans in presence of rubusoside.

BACKGROUND: One promising way of reducing caries is by using sucrose substitutes in food. rubusoside is a prototype sweet substance isolated from the leaves of the plant Rubrus suavissimus S. Lee. (Rosaceae), and is rated sweeter than sucrose. The purpose of this study was to investigate the effects of rubusoside on Streptococcus mutans growth, acidogenicity, and adherence to glass in vitro. METHODS: The effects of rubusoside on the growth and glass surface adhering of Streptococcus mutans were investigated by measuring the optical density of the culture at 540 nm with a spectrophotometer. Rubusoside influence on Streptococcus mutans acidogenicity was determined by measuring the pH of the culture. Sucrose, glucose, maltose, fructose and xylitol were designed to compare with rubusoside. RESULTS: S. mutans growth in the rubusoside-treated group was significantly lower than that in the sucrose, glucose, maltose and fructose groups (p < 0.05) except for xylitol group (p > 0.05). Sucrose-treated S. mutans exhibited the highest adherence to glass, and rubusoside-treated S. mutans exhibited the lowest. S. mutans adherence to a glass surface and acidogenicity with sucrose were significantly reduced by rubusoside. CONCLUSIONS: Rubusoside may have some potential as a non-cariogenic, non-caloric sweetener.

Anti-carious Effects of Galla chinensis: A Systematic Review.

As a natural traditional Chinese medicine, Galla chinensis has been widely used since ancient times for its astringency, stypticity, detoxification, and antibacterial activity. Our group has concentrated on the research about its potential of being an applicable anti-caries agent. The crude extract and some other components purified from it show remarkable efficacy on anticaries, and the most likely mechanism is proposed through specific research. For the fact that crude drugs consist of numerous compounds, and their bioactivity is a result of synergistic effects and/or antagonistic effects of several compounds, it is difficult to clarify the exact mechanism and evaluate the safety and effectiveness of G. chinensis. This review article systematically summarizes previous findings from the following aspects: (1) inhibitory effect on oral bacteria; (2) the demineralization inhibition property; (3) the remineralization-enhancing property; and (4) stability and toxicity evaluation, and thus indicates the further research direction.

Effect of phosphate group on remineralization of early enamel caries regulated by amelogenin peptide.

OBJECTIVE: To show the effect of the phosphate group on the remineralization process of early enamel caries mediated by amelogenin peptide. METHODS: Freshly extracted, completed, and crack-free bovine teeth were used to create artificial early enamel caries, which were randomly divided into four groups: Group A: fluorination remineralized solution treatment group; Group B: pure remineralized solution treatment group. Group C: 100 g/ml recombinant Amelogenin peptide remineralized solution treatment group (with single phosphate group on N-terminus); Group D: 100 g/ml non-phosphorylated recombinant Amelogenin peptide remineralized solution treatment group (without single phosphate group on N-terminus). For 12 days, fresh remineralized solutions were replaced daily. Transverse microradiography (TMR) was used after remineralization to determine mineral loss and demineralization depth before and after each sample's remineralization. Each sample's depth of remineralization and mineral acquisition were then determined. RESULTS: The recombinant amelogenin peptide group significantly outperformed the non-phosphorylated amelogenin peptide group in terms of mineral acquisition and mineralization depth (P<0.05). CONCLUSIONS: The recombinant Amelogenin's solitary phosphate group at the N-terminus helps recombinant Amelogenin to encourage the remineralization process of early enamel caries.

Recombinant amelogenin peptide TRAP promoting remineralization of early enamel caries: An in vitro study.

Objective: To explore the regulatory effect of recombinant amelogenin peptide TRAP on the remineralization of early enamel carious lesions. Methods: Forty-eight bovine enamel blocks that prepared initial lesions in vitro were split at random into four groups for immersion treatment for 12 days: 1) remineralizing medium; 2) studied peptide 1 (consisting of the N- and C-termini of porcine amelogenin) + remineralizing medium; 3) studied peptide 2 (TRAP) + remineralizing medium; 4) fluoride + remineralizing medium. After demineralization and remineralization immersion, each specimen's mean mineral loss and lesion depth were measured using micro-computed tomography (micro-CT). The changes in lesion depth (∆LD) and mineral gain (∆Z) were computed following remineralization. The enamel samples were then cut into sections and examined with polarized light microscopy (PLM). The cross-section morphology was observed by scanning electron microscopy (SEM). The crystal phase was analyzed by an X-ray micro-diffractometer (XRD). The calcium-binding properties were determined using isothermal titration calorimetry (ITC). Results: Micro-CT analysis revealed a significant reduction in mineral loss in the four groups following the remineralization treatment (p < 0.05). The treatment with fluoride resulted in the greatest ∆Z and ∆LD, whereas the treatment with a remineralizing medium showed the least ∆Z and ∆LD among all groups. The ∆Z and ∆LD of the studied peptide 1 and studied peptide 2 groups were greater than those of the remineralizing medium group. However, there was no significant difference between the studied peptide 1 and studied peptide 2 groups (p > 0.05). All of the samples that the PLM analyzed had a thickening of the surface layer. A negative birefringent band changed in the lesion's body. The SEM displayed that minerals were formed in all four groups of samples. The XRD results indicated that the products of remineralization of the studied peptide were hydroxyapatite crystals (HA). ITC showed that there were two binding modes between the calcium and peptide TRAP. Conclusion: This study confirmed the potential of the recombinant amelogenin peptide TRAP as a key functional motif of amelogenin protein for enamel remineralization and provided a promising biomaterial for remineralization in initial enamel carious lesion treatment.

Suppression of puerarin on polymethylmethacrylate-induced lesion of peri-implant by inhibiting NF-κB activation in vitro and in vivo.

Puerarin (PR), a natural isoflavone isolated from Chinese traditional plant pueraria lobata, has attracted considerable attention due to its important biological and pharmacological activities. However, its effects on lesion of peri-implant and related mechanism of action are still not clear, which require further investigation. In this study, we evaluated the effects of PR on polymethylmethacrylate (PMMA)-induced lesion of peri-implant in vitro and in vivo, and explored its possible mechanism of action. Our results indicated that PR could inhibit PMMA-induced osteoclastogenesis in RAW264.7 cells with a dose-dependent manner in vitro and effectively down-regulate mRNA and protein expressions of matrix metalloprotein 9 (MMP-9), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and receptor activator of nuclear factor (NF)-κB (RANK), primarily via the suppression of NF-κB signaling. Furthermore, we found that PMMA induction could directly cause the phosphorylation of IκB and significantly promote the nuclear translocation of p65 in RAW264.7 cells. In other words, PR was able to dose-dependently attenuate the PMMA-induced nuclear translocation of p65 in RAW264.7 cells. In vivo, PR was observed to attenuate PMMA-induced osteoclastogenesis, osteolysis, mRNA expressions of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and RANK, as well as protein levels of MMP-9, TNF-α, IL-6, and p65 in a murine calvarial osteolysis model. These findings suggested that PR might be a potential therapeutic drug to lesion of peri-implant, and provided new insights for understanding its possible mechanism.

Remineralization Efficacy of an Amelogenin-Based Synthetic Peptide on Carious Lesions.

Objective: The aim of this study was to evaluate the remineralization efficacy of an amelogenin-based peptide on initial enamel carious lesions in vitro. Furthermore, we attempted to provide insights into the possible mechanism of the remineralization, including determining the calcium-binding properties of the peptide and its effects on calcium phosphate mineralization. Methods: The peptide comprising the N-terminus and the C-terminus of porcine amelogenin was synthesized by Synpeptide Co., Ltd. Fifty specimens were randomly assigned to five immersing treatment groups for 12 days: remineralizing medium only; 12.5 µg/mL peptide + remineralizing medium; 25 µg/mL peptide + remineralizing medium; 50 µg/mL peptide + remineralizing medium; fluoride + remineralizing medium. After immersion, mean mineral loss before and after remineralization of each specimen was determined using micro-CT. Mean mineral gain after remineralization was calculated. Calcium binding properties were measured by Isothermal titration calorimetry (ITC). TEM and Fourier transform-infrared were used to determine the effects of the peptide on calcium phosphate mineralization. Results: A significant decrease in mineral loss after remineralization process in all groups was observed (p < 0.05). Treatment in remineralizing medium resulted in the lowest mineral gain while the fluoridated treatment exhibited the highest mineral gain among all groups. Inclusion of synthetic peptide in the remineralizing medium exhibited a higher mineral gain and the gain of 50 µg/mL group was greater than that of the 25 µg/mL group. No significant difference in mineral gain was observed between the remineralizing medium only group and the 12.5 µg/mL peptide group (p > 0.05). ITC values showed that the Ca2+-binding affinity of the peptide is about 9.914 × 104M-1. Furthermore, the peptide was found to inhibit calcium phosphate precipitation and stabilize amorphous calcium phosphate formation for more than 2 h and finally transform into ordered hydroxyapatite crystals. Conclusion: Specific concentrations of the amelogenin-based synthetic peptide promoted in vitro remineralization, with higher concentrations exhibiting significantly greater remineralization. This study presented evidence suggesting that the peptide may act as a Ca2+carrier as well as a regulating factor. When the stabilizing calcium and phosphorus ions bind with the peptide they become biologically available for the remineralization of deeper carious lesions, while also regulated by the peptide to transform into ordered hydroxyapatite crystals.

[In vitro study of the effect of a lactoperoxidase-peroxidase-thiocyanate system with iodine on the cariogenicinity of streptococcus mutans].

OBJECTIVE: This study aimed to investigate the effect of a lactoperoxidase-peroxidase-thiocyanate (LPO-H2O-SCN-) system with different concentrations of iodine (I-) on Streptococcus mutans (S. mutans), particularly on various parameters, including growth, adhesion, glucosyltransferase (GTF) enzyme activity, and insoluble exopolysaccharide synthesis. METHODS: S. mutans ATCC 25175 was used as experimental species. Clonal formation unit (CFU) were counted to investigate the inhibitory effect on bacterial growth. The inhibition rate of bacterial adherence was calculated to analyze the effect on adhesion. Anthrone method was used to determine the content of insoluble exopolysaccharides and the amount of reducing saccharides. GTF activity and enzyme activity were then determined. RESULTS: The inhibitory ability of the LPO-H2O2-SCN- system with I- on the cariogenicinity of S. mutans was strengthened as I- concentration was increased. At I- concentration > or = 100 micromol x L(-1) the antibacterial effects were significantly increased compared with those of the control group (P < 0.05). At I- concentration > or = 1,000 micromol x L(-1), the antibacterial effects were significantly improved compared with those of the group with SCN-only (P < 0.05). At I- concentration > or = 100 micromol x L(-1), the inhibition rate of bacterial adherence was > 50%; insoluble exopolysaccharide synthesis and GTF enzyme activity were reduced (P < 0.05). CONCLUSION: The antibacterial effects of the LPO-H2O2-I- system were enhanced by adding I- to overcome the antagonistic effect of physiological SCN- concentration. LPO-H2O2-SCN- system with different concentrations of I- showed statistically significant inhibitory effects on growth, adhesion, insoluble exopolysaccharide synthesis, and GTF enzyme activity.

[The effect of coaggregation of A.viscosus and P.gingivalis on the adherence of A.viscosus to hydroxyapatite].

PURPOSE: To examine the effect of coaggregation of A.viscosus and P.gingivalis on the adherence of A.viscosus to saliva-coated hydroxyapatite(S-HA). METHODS: The suspensions of A.viscosus and P.gingivalis were rotated and dropped on cover slips to visualize the coaggregation under scanning electron microscope(SEM). Different concentrations of P.gingivalis were mixed with A.viscosus radiolabeled with 10 uCi of [methyl-(3)H] thymedine per milliliter, then rotated for periods up to 1.5 hours at room temperature and the numbers of absorbed A.viscosus cells were determined by liquid scintillation. A.viscosus alone was used as the control, and its adhesion percent was set for 100%. One-way ANOVA with SPSS10.07 software package was used to analyze the effect of P.gingivalis on adherence of A.viscosus. RESULTS: Several cells of P.gingivalis could aggregate on the surface of A.viscosus. The adherence of A.viscosus to S-HA was promoted slightly when the concentration of P.gingivalis was rather small, then further increase in concentration of P.gingivalis resulted in a steady decline in the number of adherent A.viscosus, and the difference was significant (P<0.05). CONCLUSION: These results show that the adherence of A.viscosus to S-HA could be influenced by coaggregation of P.gingivalis and A.viscosus.

[Effect of chemical compounds of Galla chinensis on enamel surface rehardening in vitro].

OBJECTIVE: To evaluate the effect of chemical compounds extracted from Galla chinensis on enamel surface rehardening in vitro. METHODS: Sixty bovine enamel blocks with early carious lesions were randomly divided into six groups: group1 treated NaF (positive control); group2 with GCE; group3 with GCE-B; group4 with GCE-B1; group5 with GCE-B2 and group6 with deionized water (negative control). The lesions were subjected to a pH-cycling regime for 12 days. Surface enamel microhardness was measured on the enamel blocks before and after demineralization. After pH-cycling, and the percentage of surface microhardness recovery (SMHR) was calculated. RESULTS: Obvious increase of the surface hardness of the enamel was observed in all the treatments except GCE-B2 and deionized water (P < 0.05). CONCLUSIONS: The present study demonstrates the potential of the three GCEs (GCE, GCE-B and GCE-B1) to effect net rehardening of artificial carious lesions under dynamic pH-cyclic conditions.