Comparison of biofilm models for producing artificial active white spot lesions.

OBJECTIVE: This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models. METHODOLOGY: In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001). RESULTS: In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols. CONCLUSION: The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

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.

The Synergistic Effect of High Intensity Focused Ultrasound on In-vitro Remineralization of Tooth Enamel by Calcium Phosphate Ion Clusters.

Background: Remineralization of dental enamel is an important intervention strategy for the treatment of demineralized lesions. Existing approaches have limitations such as failure to adequately reproduce both the ideal structural and mechanical properties of the native tooth. The ability of ultrasound to control and accelerate the crystallization processes has been widely reported. Therefore, a new approach was explored for in-vitro enamel remineralization involving the synergistic effect of high-intensity focused ultrasound (HIFU) coupled with calcium phosphate ion clusters (CPICs). Methods: The demineralized enamel was treated with CPICs, with or without subsequent HIFU exposure for different periods (2.5, 5, and 10 min). The specimens were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman spectroscopy. The surface hardness and crystallographic properties of the treated specimens were evaluated using Vickers microhardness testing and X-ray diffraction (XRD), respectively. Results: SEM revealed distinct, organized, and well-defined prismatic structures, showing clear evidence of remineralization in the combined CPIC/HIFU treatment groups. AFM further revealed a decrease in the surface roughness values with increasing HIFU exposure time up to 5 min, reflecting the obliteration of interprismatic spaces created during demineralization. The characteristic Raman band at 960 cm-1 associated with the inorganic phase of enamel dominated well in the HIFU-treated specimens. Importantly, microhardness testing further demonstrated that new mineral growth also recovered the mechanical properties of the enamel in the HIFU-exposed groups. Critical to our aspirations for developing this into a clinical process, these results were achieved in only 5 min. Conclusion: HIFU exposure can synergise and significantly accelerate in-vitro enamel remineralization process via calcium phosphate ion clusters. Therefore, this synergistic approach has the potential for use in future clinical interventions.

Comparative evaluation of the remineralizing potential of Salvadora persica and probiotic yogurt on incipient enamel lesions: An ex-vivo study.

BACKGROUND: Salvadora persica (miswak) is known to exert antibacterial, antifungal, antioxidant, and anticariogenic effects by elevating the pH of plaque after the consumption of sucrose. OBJECTIVES: The study aimed to compare the effectiveness of S. persica and probiotic yogurt in the remineralization of tooth enamel on artificially produced enamel lesions. MATERIAL AND METHODS: A total of 40 intact human premolars were collected and each tooth was sectioned longitudinally into 2 identical halves in a buccolingual direction. The buccal halves were selected for inclusion in this study, and standardized windows (5 mm × 3 mm) were isolated on the buccal surface of the enamel. The samples were incubated in a demineralizing solution at 37°C for 96 h. Subsequently, they were randomly selected for treatment with one of the experimental remineralizing solutions (S. persica or probiotic yogurt). After treatment, the samples were examined using scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and polarized light microscopy at baseline, after demineralization and after remineralization. RESULTS: The remineralizing effect of S. persica was found to be greater than that of probiotic yogurt. With regard to mineral content, S. persica exhibited the highest calcium and phosphorus levels among all groups. No significant differences were observed between the samples treated with S. persica and normal enamel. CONCLUSIONS: Salvadora persica extract has been demonstrated to effectively reduce the demineralization of enamel in experimental conditions. Furthermore, it has the potential to restore the mineral content to its original level.

Comparing Shear Bond Strength Between Pink Opaquer and Other Tooth-Colored Restorative Materials on Demineralized Dentin Treated with Silver Diammine Fluoride.

Purpose: The purposes of this study were to evaluate the effect of silver diammine fluoride (SDF) on the shear bond strength (SBS) of pink opaquer (PO) compared to resin-modified glass ionomer (RMGI) and conventional composite (COMP) on demineralized dentin, and also to investigate the mode of failure (MOF). Methods: Sixty extracted third molars were prepared, demineralized for 14 days, and divided into four groups: (1) COMP; (2) SDF+PO; (3) SDF+RMGI; and (4) SDF+COMP (restoration size: two by two mm). SBS, MOF, modified adhesive remnant index (MARI), and remnant adhesive volume (RAV) were evaluated using an Instron® machine, light microscopy, 3D digital scanner ( 3Shape©), and GeoMagic Wrap© software. Results: There was no significant difference in SBS (MPa) among the COMP mean??standard deviation (2.5±1.59), SDF+COMP (2.28±1.05), SDF+PO (3.31±2.63), and SDF+RMGI groups (3.74±2.34). There was no significant difference in MOF and MARI among the four groups (P>0.05). There was no significant difference in RAV (mm3) among the COMP (0.5±0.33), SDF+COMP (0.39±0.44), SDF+PO (0.42±0.38), and SDF+RMGI groups (0.42±0.38; P>0.05). A significant correlation existed between MOF and RAV (R equals 0.721; P<0.001). MOF, MARI, and RAV did not show any correlations with SBS (P>0.05). Conclusions: Silver diammine fluoride does not affect shear bond strength between carious dentinal surface and tooth color restorative materials. The amount of material left on the interface is not related to the amount of shear force needed to break the restoration.

Influence of different pre-treatments on the resin infiltration depth into enamel of teeth affected by molar-incisor hypomineralization (MIH).

OBJECTIVES: This in vitro pilot study aimed to evaluate whether different pre-treatments (demineralization, deproteinization, (chemo-)mechanical reduction of the surface layer) influence the penetration depth of a resin infiltrant into MIH-affected enamel compared to initial carious lesions. METHODS: Thirty extracted human permanent molars with non-cavitated initial carious lesions (n = 5) or MIH (n = 25) were chosen and randomly assigned to six experimental groups: IC: initial caries; M: MIH; MN: MIH, 5.25% sodium hypochlorite; MM: MIH, microabrasion; MA: MIH, air abrasion; MAN: MIH, air abrasion and 5.25% sodium hypochlorite. A modified indirect dual fluorescence staining method was adopted to assess the penetration depth (PD) of the resin infiltrant and the lesion depth (LD) by confocal laser scanning microscopy (CLSM). Exemplarily, scanning electron microscopic (SEM) images were captured. The relationship between group assignment and penetration/lesion depth was estimated using a linear mixed model incorporating the tooth as random effect (two observations/tooth). The significance level was set at p < 0.05. RESULTS: For MIH-affected molars, the mean PD (in µm; median, [minimum-maximum]) were M (178.2 [32.5-748.9]), MN (275.6 [105.3-1131.0]), MM (48.7 [0.0-334.4]), MA (287.7 [239.4-491.7]), and MAN (245.4 [76.1-313.5]). Despite the observed differences in PD between the groups, these could not be statistically verified (Bonferroni, p = 0.322). The percentage penetration was significantly higher for IC than for MIH groups (Bonferroni, p < 0.05). SIGNIFICANCE: Compared to IC, resin infiltration into MIH-affected enamel ist more variable. Different pre-treatments influence the resin penetration into developmentally hypomineralized enamel to a fluctuating level.

Enhanced anti-biofilm and anti-caries potential of arginine combined with calcium glycerophosphate and fluoride.

OBJECTIVE: The aim of this work was to evaluate the antibiofilm and anticaries properties of the association of arginine (Arg) with calcium glycerophosphate (CaGP) and fluoride (F). METHODS: An active attachment, polymicrobial biofilm model obtained from saliva and bovine teeth discs were used. After the initial biofilm growth period, the enamel discs were transferred to culture medium. The treatment solutions were added to the culture media to achieve the desired final concentration. The following groups were used: negative control (Control); F (110 ppm F); CaGP (0.05 %); Arg (0.8 %) and their associations (F + CaGP; Arg + F; Arg + CaGP; Arg +F + CaGP). The following analyses were carried out: bacterial viability (total bacteria, aciduric bacteria and mutans streptococci), pH assessment of the spent culture medium, dry weight quantification, evaluation of surface hardness loss (%SH) and subsurface mineral content. Normality and homoscedasticity were tested (Shapiro-Wilk and Levene's test) and the following tests were applied: two-way ANOVA (acidogenicity), Kruskall-Wallis (microbial viability) and one way ANOVA (dry weight, %SH, mineral content). RESULTS: The association Arg + F + CaGP resulted in the lowest surface hardness loss in tooth enamel (-10.9 ± 2.3 %; p < 0.05). Arg +F + CaGP exhibited highest values of subsurface mineral content (10.1 ± 2.9 gHAP/cm3) in comparison to Control and F (p < 0.05). In comparison to Control and F, Arg +F + CaGP promoted the highest reduction in aciduric bacteria and mutans streptococci (5.7 ± 0.4; 4.4 ± 0.5 logCFU/mL, p < 0.05). CONCLUSIONS: The Arg-F-Ca association demonstrated to be the most effective combination in protecting the loss of surface hardness and subsurface mineral content, in addition to controlling important virulence factors of the cariogenic biofilm. CLINICAL SIGNIFICANCE: Our findings provide evidence that the Arg-F-Ca association showed an additive effect, particularly concerning protection against enamel demineralization. The combination of these compounds may be a strategy for patients at high risk of caries.

Enamel remineralisation prospect of Moringa Oleifera hydrogel, eggshell hydrogel versus sodium fluoride varnish on artificially demineralised primary teeth: in vitro study.

PURPOSE: The purpose of the present in vitro study is to investigate and compare the remineralising potential of Moringa Oleifera extract, eggshell, and sodium fluoride varnish on microhardness of artificially demineralised enamel of primary teeth with biomimetic minimally invasive approach following the world paradigm shift towards natural products in paediatric dentistry. MATERIAL AND METHODS: Sample size included 44 primary molars. The mineral content and surface microhardness of all specimens were initially assessed using energy dispersive x-ray examination (EDX) and Vickers microhardness. The specimens were artificially demineralised for 96 h at a temperature of 37°C and then reassessed directly after demineralisation. The demineralised enamel specimens were randomly divided into four groups according to the remineralisation regimen utilised. Group 1: Artificial saliva (control); Group 2: Sodium fluoride varnish; Group 3: Eggshell hydrogel; and Group 4: Moringa Oleifera hydrogel. The specimens were stored for 8 days and then subsequently evaluated using EDX and microhardness assessment by Vickers microhardness test and scanning electron microscope (SEM).  Results: Regarding the microhardness test, there was a significant difference between the Moringa Oleifera group and Eggshell group compared to fluoride varnish (p < 0.05). Regarding EDX analysis, there was a statistically significant difference (p < 0.05) between Moringa Oleifera group and Eggshell group compared to fluoride varnish as the highest values were for Moringa Oleifera and Eggshell. On the other hand, there was no statistically significant difference (p > 0.05) between Moringa Oleifera and Eggshell in both the measurements. CONCLUSION: Moringa Oleifera and Eggshell might be considered as a biomimetic natural material capable of guiding enamel tissue remineralisation in early carious lesion of primary teeth. CLINICAL RELEVANCE: This research demonstrated the capability for early enamel caries to be remineralised using novel materials with a naturally counterpart implicated in biomineralisation as proved to be more effective than traditionally used fluoride varnish in primary teeth.

Acid challenge exacerbates activation of matrix metalloproteinases in permanent teeth undergoing radiotherapy.

The aim of this study was to investigate the effect of acid challenge on the activation of matrix metalloproteinases (MMPs) in the Dentinoenamel junction of primary and permanent teeth submitted to radiotherapy. For this purpose, a total of 178 dental fragments obtained from molars were used, and randomly divided into 2 groups (primary and permanent teeth) / 4 experimental subgroups (irradiated and non-irradiated, demineralized and non-demineralized). The fragments were exposed to radiation, with a dose fraction of 2 Gy, for 5 consecutive days, until a total dose of 60 Gy was reached, with a total of 30 cycles, for 6 weeks. To determine the activity of MMPs on the dentinoenamel junction (DEJ), in situ zymography assays on 0.6mm dental fragments were performed. To assess whether MMP activity would be impacted by an acidic environment, the fragments were placed in a demineralizing solution (pH of 4.8). The finding was that irradiation activated MMPs in DEJ and these effects were more evident in permanent when compared with primary teeth. When the effect of an acid challenge on MMPs activity was investigated, demineralization was observed not to increase MMPs activity in non-irradiated teeth, but it did increase MMPs activity in irradiated teeth. In conclusion, an acid challenge was found to exacerbate activation of MMPs in DEJ of permanent teeth submitted to irradiation, but not in primary teeth.

Inhibition of incipient caries lesion progression by different fluoridated varnishes.

The aim of this in vitro study was to evaluate the potential of different fluoridated varnishes to inhibit the progression of incipient caries lesions after cariogenic challenge. Seventy-five enamel specimens of bovine teeth were prepared and selected based on the initial surface microhardness (SMH). The specimens were first subjected to artificial demineralization (in buffer solution) after which SMH was re-analyzed (SM1). They were then randomly assigned to five experimental groups: 1- CONTROL (pH cycling), 2 - MI VAR (MI Varnish with RECALDENTTM - CPP-ACP), 3 - PROFL (Profluorid®), 4 - CLIN (ClinproTM White Varnish with TCP), and 5 - DUR (Duraphat®) (n=15). The varnishes were applied in a thin layer and the specimens were then subjected to pH cycling for eight days. The SMH and cross-sectional microhardness (CSMH) were then analyzed (SM2). The fluoride and calcium ion concentrations in the solution were analyzed by the indirect method and atomic absorption spectrophotometry, respectively. Data were statistically analyzed by Student's t-test, ANOVA/Tukey-Kramer, or Kruskall-Wallis/Dunn tests for individual comparisons (p˂0.05). All varnishes led to significantly higher surface and subsurface remineralization compared with the control group but did not differ from each other. The varnishes with the highest fluoride release were: PROFL and CLIN, followed by MI VAR and DUR. The varnishes with significantly higher release of calcium were: DUR, CLIN, and PROFL. In conclusion, all commercial fluoridated varnishes tested have good potential to inhibit the progression of demineralization, regardless of the ion release mechanisms.

Enamel Matrix Derivative, 58S5 Bioactive Glass, and Fluoride Varnish for Enamel Remineralization: A Multi-analysis Approach.

PURPOSE: This study aimed to evaluate the enamel remineralization efficacy of enamel matrix derivative (EMD), experimental bioactive glass (BAG), and fluoride varnish in vitro. METHODS AND MATERIALS: Artificial initial caries lesions were developed on fifty human enamel specimens using demineralization solution (pH 4.5, 37°C, 96 hours). Specimens were randomly assigned to five groups (n=10): I-5% NaF varnish (Enamelast), II-experimental 58S5 BAG+37% phosphoric acid (PA), III-EMD (Emdogain) + Ethylenediaminetetraacetic Acid (EDTA), IV-EMD+37% PA, V-Control (untreated). All remineralization agents were applied with pH cycling for seven days. The specimens were scanned by spectral domain optical coherence tomography (SD-OCT) at baseline, at demineralization, and after pH cycling. Lesion depths were measured using image analysis software (ImageJ). Lesions were evaluated using surface microhardness (SMH) and two fluorescence methods (FluoreCam and DIAGNOdent Pen [DDPen]). The data were statistically analyzed by Kruskal Wallis, Friedman, and Wilcoxon tests (α=0.05). RESULTS: According to SD-OCT results, fluoride varnish was found to be the most effective agent in reducing lesion depth (p=0.005). All agents increased the SMH values after pH cycling. No significant difference was found among fluoride varnish, BAG, and EMD+PA groups. These SMH values were significantly higher than EMD+EDTA and control groups (p<0.001). All groups showed lower DDPen scores compared with the control group (p<0.001), however, no significant difference was found among the remineralization agents. In FluoreCam assessment, size and intensity values of all treated groups showed improvement. However, there was no significant difference between the treatment groups in terms of FluoreCam size measurements (p=0.186). CONCLUSION: 58S5 BAG and EMD+PA have remineralization capacity as effective as fluoride varnish. EMD+PA showed better SMH and lesion intensity results than EMD+EDTA.

Efficacy of the Probiotic L. brevis in Counteracting the Demineralizing Process of the Tooth Enamel Surface: Results from an In Vitro Study.

BACKGROUND: Enamel plays an essential role in protecting the underlying layers of the human tooth; therefore, preserving it is vital. This experimental study aimed to evaluate the potential ability of L. brevis to counteract the action of a demineralizing agent on dental enamel morphology and mineral composition in vitro. METHODS: The sample consisted of 12 healthy human posterior teeth. The coronal portion of each tooth was subdivided into two equal parts longitudinally. The specimens were randomly divided into four groups: artificial saliva, L. brevis suspension, demineralizing agent (DA), and DA plus L. brevis. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used to evaluate the surface micromorphology and the mineral content, respectively. The statistical analysis was conducted using a one-way ANOVA, followed by Tukey's post hoc test. RESULTS: SEM analysis did not highlight significant changes in the enamel microstructure of L. brevis-treated specimens compared to the control. DA-induced damage to the enamel structure was drastically reduced when the specimens were contextually exposed to the probiotic. The treatment with DA substantially reduced the weight % of crucial enamel minerals, i.e., Ca and P. Notably, the probiotic was able to reverse the demineralization process, bringing Ca and P weight % back to basal levels, including the Ca/P ratio. CONCLUSIONS: The findings indicate that L. brevis is able to efficiently protect the dental enamel surface from the damage caused by DA and increase the enamel resistance to demineralization. Overall, L. brevis confirms its efficacy in preventing or counteracting the action of carious lesions through a novel mechanism that protects the tooth surface under a chemical challenge that mimics the caries process.

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.

Comparison of the effects of fluoride varnish containing silver nanoparticles and conventional fluoride varnish on the surface microhardness of tooth enamel.

BACKGROUND: Nano-silver fluoride (NSF) has been introduced to improve enamel lesions. The effective use of varnishes is important in the prevention of dental caries. OBJECTIVES: The study aimed to compare the effect of conventional sodium fluoride varnish with the same varnish containing 1% and 2% silver nanoparticles (AgNP) on the surface microhardness of enamel. MATERIAL AND METHODS: The baseline surface microhardness of 40 premolar teeth was measured using a Vickers microhardness tester. After immersing the samples in a demineralizing agent for 24 h, the microhardness was measured again. In group B, a layer of conventional fluoride varnish was applied to the tooth surfaces using a microbrush with soft bristles, following the manufacturer's instructions. Groups C and D were treated with 1% and 2% NSF varnishes, respectively, while group A received no varnish. Surface microhardness tests were conducted on all specimens, including those previously tested. RESULTS: The microhardness of the enamel surface increased significantly in all 3 test groups compared to the microhardness after demineralization (p < 0.05). CONCLUSIONS: Conventional fluoride varnish and fluoride varnishes containing 1% and 2% AgNP are equally effective in remineralizing initial caries.

Acquired enamel pellicle and biofilm engineering with a combination of acid-resistant proteins (CaneCPI-5, StN15, and Hemoglobin) for enhanced protection against dental caries - in vivo and in vitro investigations.

OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.

Microbial shift of oral biofilm associated with remineralization of root dentin lesions.

PURPOSE: To examine the relationship between remineralization of incipient root dentin lesions and the presence of polymicrobial biofilms, as well as examine changes in microbial composition. METHODS: Bovine root dentin disks used as specimens for biofilm formation, were cultured using saliva from a single donor. Amsterdam Active Attachment biofilm model was used to grow biofilms. The culture medium was McBain 2005 with 0.2% sucrose and 0.4 ppm F as sodium fluoride. After cultivation for 48 hours to achieve demineralization, a control group (n=10) was obtained and the other specimens were further cultured for 336 hours in two types of remineralization culture medium, with sucrose (S+) and without sucrose (S-), through continuous anaerobic incubation (10% CO2,10% H2, 80% N2). Then half of the specimens cultured in the S- medium were transferred to the S+ medium for an additional 48 hours resulting in three experimental groups S(+) (n=10), S(-) (n=10), and S(-)de (n=10), respectively. Experiment 1: Transverse microradiography (TMR) analysis - Immediately after respective culture treatments, integrated mineral loss (IML) and lesion depth (LD) in the dentin specimens were analyzed by TMR. Experiment 2: Microbiome analysis - Sequence data of the 16S rRNA gene of each sample was obtained using MiSeq, and partial base sequences were determined. Next-generation sequencing was performed to determine the taxonomic groups of fungi present in the biofilm samples. RESULTS: Experiment 1: In the control group, formation of dentin demineralization lesions by polymicrobial species biofilms was confirmed. The S(-) group showed significantly decreased IML and shallower LD compared to the control group. The S(-)de group showed a significant increase in IML and LD compared to the S(-) group. Experiment 2: There were statistically significant differences in microbiome between the control group and each of the three experimental groups, both at the genus and species levels. A significant difference in genus was observed between the S(-) group and the S(-)de group. CLINICAL SIGNIFICANCE: The confirmation of the possibility of microbial shift occurring during the remineralization process of root caries will lead to the development of new remineralization therapies.

The use of mouthwash containing trimetaphosphate as an adjunct therapy to fluoridated toothpaste reduces enamel demineralization.

INTRODUCTION: The decline in dental caries has been attributed to the widespread use of fluoride (F). Two forms of presentation are fluoridated toothpaste (FT) and mouthwash (MW), widely used by the population. MATERIALS AND METHODS: This study aimed to evaluate in vitro the effects of combining FT and MW, whether supplemented with sodium trimetaphosphate (TMP) or not, on dental enamel demineralization. Bovine enamel blocks (n = 60) were selected based on initial surface hardness (SHi) and divided into 5 experimental groups (n = 12 each): I) Placebo Toothpaste (without F/TMP); II) 1100 ppm F Toothpaste (FT); III) 1100F associated with a MW at 100 ppm F (FT + MW 100F); IV) 1100F associated with a MW at 225 ppm F (FT + MW 250F); and V) 1100F associated with a MW at 100 ppm F supplemented with 0.4 % TMP (FT + MW 100F-TMP). The blocks were treated twice a day, undergoing 5 pH cycles over 7 days. Thus, the percentage change in surface hardness (%SH), integrated subsurface hardness loss (ΔKHN), and the concentration of F, phosphorus (P), and calcium (Ca) in the enamel were determined. The data were submitted to ANOVA and Student-Newman-Keuls test (p < 0.001). RESULTS: The 1100F group was statistically inferior to the groups associated with MW for %SH, ΔKHN, and the concentration of P and Ca in the enamel (p < 0.001). Blocks treated with FT + MW 225F and FT + MW 100F-TMP showed significantly lower %SH compared to the other groups (p < 0.001). The FT + MW 100F - TMP group exhibited the lowest depth mineral loss (ΔKHN), and higher concentration de P in enamel (p < 0.001). CONCLUSION: The adjunct use of MW with FT produces a greater protective effect in inhibiting enamel demineralization, and the supplementation of TMP to the MW with 100F provides a superior effect compared to MW with 225F. CLINICAL SIGNIFICANCE: This combination of treatments could be regarded as one of several alternative fluoride supplements for subjects at elevated risk of caries.

Fluoride-amorphous calcium phosphate and biomimetic nano-hydroxyapatite for enamel remineralization: An in-vitro study of surface microhardness and composition.

AIM: Fluoride-Amorphous Calcium Phosphate and Biomimetic Nano-Hydroxyapatite for Enamel Remineralization; An In-Vitro Study of Surface Microhardness and Composition. MATERIAL AND METHODS: Ninety-six extracted human premolars with sound buccal surface were divided using a randomization computer-generating software into four groups; Group I (control) sound untreated enamel, Group II (demineralized) demineralized to create white spot lesions, Group III (biom-n-HA) demineralized and then treated with biomimetic nanohydroxyapatite cream, and Group IV (F-ACP) demineralized and then treated using Fluoride-Amorphous Calcium Phosphate varnish. Each group was divided into two subgroups; subgroup "A" evaluated for mineral content using energy dispersive x-ray spectroscopy (EDX) and for surface microhardness using the Vickers microhardness test and Subgroup "B" evaluated for white spot lesion depth using a polarized light microscope (PLM). RESULTS: The highest microhardness (VHN) was found in the (F-ACP) group (mean=428.61±54.43) and then in the (Biom-n-HA) group (mean=408.11±70.16) followed by the (Control) group (mean=402.13±53.40) with no significant difference between them and finally in the significantly different (Demineralized) group (mean=256.99±45.83). The weight percentage of Ca (30.29±1.04 and 33.44±1.07) and Ca/P ratio (1.87±0.06 and 2.03±0.05) were significantly different between Group III and Group IV respectively. PLM measurements in Group II (198.83µm), Group III (60.17µm), and Group IV (26.33µm) were significantly different. CONCLUSIONS: Both the (Biom-n-HA) cream and the (F-ACP) varnish showed promising results for enamel remineralization. The increased enamel surface microhardness was consistent with the mineral content and the changes in the birefringence.

Carbon dots combined with phytosphingosine inhibit acid-induced demineralization of hydroxyapatite in vitro.

OBJECTIVES: To study the effects of carbon dots (CDs), in combination with phytosphingosine (PHS), against acid-induced demineralization of hydroxyapatite in vitro. METHODS: CDs were generated from citric acid and urea by microwave heating. Transmission electron microscope (TEM), FT-IR, and fluorescence intensity were used to characterize the CDs. A hydroxyapatite (HAp) model was used to investigate the protective effects of CDs, PHS, and their combinations with and without a salivary pellicle against acid-induced demineralization in vitro. Ca2+ release as a parameter to evaluate the inhibition of demineralization was measured by capillary electrophoresis. The interactions between CDs, PHS, and HAp discs were investigated using a fluorescence detector. RESULTS: Uniform-sized CDs were synthesized, showing typical optical characteristics. CDs exhibited no inhibition of acid-induced demineralization in vitro, in contrast to PHS. Notably, a pre-coating of CDs increased the protective effects of PHS against acid-induced demineralization, which was not disturbed by the presence of a salivary pellicle and Tween 20. Scanning electron microscope (SEM) confirmed the binding and layers formed of both CDs and PHS to the HAp surfaces. Based on fluorescence spectra CDs binding to HAp seemed to be dependent on Ca2+ and PO43- interactions. CONCLUSIONS: CDs combined with PHS showed protective effects against acid-induced demineralization of HAp discs in vitro.

Effect of phosphoric acid containing polyvinylpyrrolidone as protective etchant for dentin bonding.

STATEMENT OF PROBLEM: Phosphoric acid is commonly used in dentistry as an etchant but can result in excessive demineralization of dentin, a major contributor to the instability of dentin-bonded restorations. Nevertheless, research on the development of etchants that can reduce acid damage is sparse. PURPOSE: The purpose of this in vitro study was to investigate the effects of polyvinylpyrrolidone-modified phosphoric acid on the dentin bonding of an etch-and-rinse adhesive. MATERIAL AND METHODS: Protective etchants were prepared by adding polyvinylpyrrolidone to 35% phosphoric acid aqueous solutions: the 3 concentrations were 0.5% (P0.5% group), 1% (P1% group), and 2% (P2% group) w/v. The treatment agent of the control group (C) was 35% phosphoric acid gel. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), microhardness, microtensile bonding strength (µTBS), nanoleakage, and in situ zymography were used to evaluate the appearance of the protective etchant on dentin bonding. The results were analyzed with a 1-way ANOVA test (α=.05). RESULTS: SEM showed no obviously exposed collagen fiber in the P1% and P2% groups. FTIR showed less demineralization of the dentin surface, and microhardness was higher after treatment with the protective etchant (P<.05). The µTBS of P1% (70 ±9.2 MPa) was the highest, and group C (44 ±5.8 MPa) was the lowest in all groups (P<.05). Moreover, there was weaker MMP activity in the P1% and P2% groups (P<.05). CONCLUSIONS: This study demonstrated that the protective etchant effectively reduced demineralization, enhanced bond strength, and reduced nanoleakage and enzyme activity within the hybrid layer.