Effect of arginine-fluoride varnish on preventing enamel erosion by paediatric liquid medicaments.

BACKGROUND: The study objective was to examine the effect of arginine-sodium fluoride (Arg-NaF) varnish on preventing enamel erosion by acidic paediatric liquid medicaments (PLM). METHODS: The treatment groups were: 1) 2% Arg-NaF; 2) 4% Arg-NaF; 3) 8% Arg-NaF; 4) NaF; 5) MI (CPP-ACFP) varnishes; and 6) no varnish. The pH of PLM (paracetamol and chlorpheniramine) was measured at baseline and after immersing the Perspex® blocks coated with varnishes at 0 min, 30 min, 1 h, and 4 h. Seventy-two enamel specimens (n = 72) were randomly divided into 2 groups by PLM and further by treatment groups. Then, the specimens were pre-treated with varnishes and subjected to erosive cycles (5 min, 2×/day for 4 days) by PLM. After each erosive challenge, the specimens were stored in artificial saliva. At baseline and after 4 days, the specimens were assessed for surface roughness (Ra) using 2D-surface profilometric analysis (SPA) and atomic force microscopy (AFM). Additionally, the Ca/P ratio was determined using scanning electron microscopy with energy-dispersive X-ray spectroscopy. Paired samples dependent t-test, 1-way ANOVA and 2-way ANOVA with Bonferroni post-hoc tests were used to analyse data with the level of significance set at p < 0.05. RESULTS: The pH of PLM with 8% Arg-NaF was significantly higher than the other groups at 30 min and 4 h (p < 0.05). With paracetamol, no significant difference was observed between the baseline and post-erosive cycle measured enamel Ra (by SPA/AFM) and Ca/P ratio for all treatment groups (p > 0.05). The Ra determined by AFM, at the post-erosive cycle with chlorpheniramine, when treated with 4 and 8% Arg-NaF was significantly lower than the other groups (p < 0.05); except CPP-ACFP (p > 0.05). With the chlorpheniramine post-erosive cycle, the Ca/P ratio for 4, 8% Arg-NaF and CPP-ACFP treated specimens was significantly higher than the baseline Ca/P (p < 0.05). CONCLUSION: The 4%/8% Arg-NaF and MI varnish® application exhibit an enhanced preventive effect against low pH (pH < 3.0) PLM-mediated enamel erosive challenges compared to 5% NaF varnish.

The Influence of Surface Roughening and Polishing on Microbial Biofilm Development on Different Ceramic Materials.

PURPOSE: To test the hypothesis that surface roughening and polishing of ceramics have no effect on their surface roughness and biofilm adhesion. MATERIALS AND METHODS: Feldspathic ceramic Vitablocks™ TriLuxe forte (VTF), lithium disilicate glass IPS e.max Press™ (IPS) and zirconia reinforced lithium silicate Vita Suprinity™ (VS) ceramic blocks (n = 27 per group) were prepared from sintered CAD blocks using a water-cooled saw. They were further subdivided into 3 subgroups according to the surface treatment protocols (n = 9): as prepared, roughened and polished. The surface roughness of the ceramic blocks was measured using an electro-mechanical profilometer. The ceramic sections were inoculated with Streptococcus mutans and incubated for 48 hours to form a biofilm. The ceramic surfaces with the biofilms were analyzed using Confocal Laser Scanning Microscopy to calculate the percentage of live bacteria and substratum coverage by the biofilm, and further visualized using scanning electron microscopy. Statistical analysis was done with SPSS software using two-way ANOVA, followed by post hoc Bonferroni test to identify significant differences between the groups. The level of significance was set at p = 0.05. RESULTS: As prepared VTF showed significantly higher mean surface roughness values than as prepared IPS and VS. The mean percentage of live bacteria and biofilm coverage of the substrate were significantly higher in the roughened ceramic blocks than the as prepared and polished blocks for all three ceramic types (p < 0.05). Polished specimens of VS significantly lower percentage of biofilm coverage than the other groups (p < 0.05). CONCLUSIONS: This study sheds new light that adjustments of ceramic restorations prior to cementation increases the likelihood for formation and adhesion of microbial biofilms on the surface. Polished zirconia reinforced lithium disilicate ceramics demonstrated the lowest bacterial adhesion among the evaluated ceramics.

Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis.

INTRODUCTION: Stem cell-based dental pulp regeneration has been extensively studied, mainly focusing on exploiting dental stem cells' osteogenic and angiogenic potentials. Dental stem cells' neurogenic role is often overlooked. Stem cells from apical papilla (SCAPs), originating from the neural crest and capable of sphere formation, display potent neurogenic capacity. This study aimed to investigate the interactions of neuronally induced stem cells from apical papilla (iSCAP) spheres, SCAPs, and human umbilical vascular endothelial cells (HUVECs) on vasculogenesis and neurogenesis. METHODS: SCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small molecule neural induction medium, and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP sphere interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with ß-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. RESULTS: Neural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, ß-tubulin III, microtubule-associated protein 2, neuron-specific enolase, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared with iSCAPs. iSCAP sphere, HUVEC, and SCAP direct coculture significantly enhanced vessel formation along with up-regulated VEGF (P < .001) and multiple neural markers, such as Nestin (P < .01), microtubule-associated protein 2 (P < .001), S100 (P < .001), and NG2 (P < .001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and alpha-smooth muscle actin-positive and CD31-positive cells assembling for the vascular constitution. CONCLUSIONS: iSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.

Synbiotic-fluoride synergism on enamel remineralization, cytotoxicity and genotoxicity.

OBJECTIVE(S): The objectives of the present study were to examine the - a) enamel remineralization potential of synbiotic-fluoride (SF) therapy using a multi-species bacterial pH-cycling model; and b) cytotoxic and genotoxic effects of SF therapy extracts. MATERIALS AND METHODS: The SF therapy group comprised of 2% arginine (Arg), 0.2% NaF, and a probiotic Lactobacillus rhamnosus GG (LRG). The intervention groups studied were: 1) No treatment; 2) 2% Arg; 3) 0.2% NaF; 4) LRG; 5) 2% Arg+0.2% NaF; 6) 2% Arg+LRG; 7) 0.2% NaF+LRG; and 8) 2% Arg+0.2% NaF+LRG (SF therapy). The enamel remineralization potential of SF therapy was investigated under cariogenic biofilm challenge; while the cytotoxicity and genotoxicity of SF therapy extracts were examined on HGF-1 and Chinese hamster fibroblast V79, respectively. To determine the remineralization effect, the specimens were subjected to mineral density (MD) assessment using micro-CT, Ca/P molar ratio with SEM-EDX, and enamel fluoride uptake (EFU) estimates. The HGF-1 proliferation assessment was quantified using MTT/CCK-8 assays with qualitative analysis by nuclei staining Hoechst-based fluorescence imaging. The genotoxicity was determined by micronuclei formation test. RESULTS: Mineral gain and %remineralization derived from MD assessment for the SF therapy were significantly higher than the other groups (p<0.05). The %ΔCa/P for the SF and 2% Arg+0.2% NaF were significantly higher than the other groups (p<0.05). The SF and 2% Arg+0.2% NaF groups had the highest EFU compared to the other groups (p<0.05). No significant difference in the %viable HGF-1 cells were observed between the treatment interventions and no treatment group (p>0.05). Compared to the EMS-positive control, the micronuclei formation for all the intervention groups was significantly lower (p<0.05), with no significant difference among the treatment groups (p>0.05). CONCLUSION: The SF therapy enhanced enamel remineralization with no biocompatibility concerns. CLINICAL SIGNIFICANCE: With the enhanced enamel remineralization potential discerned in the present study, the SF therapy can be used as a promising caries-preventive agent targeted for high caries-risk individuals.

Enamel remineralization potential and antimicrobial effect of a fluoride varnish containing calcium strontium silicate.

OBJECTIVES: To investigate enamel remineralization and antimicrobial effect of sodium fluoride (NaF) varnish containing calcium strontium silicate (CSR). METHODS: CSR was synthesized by sol-gel process and incorporated in 5 % NaF varnish at three different concentrations (1 %, 2 %, and 4 % w/v). The treatment/control groups were: 1 % CSR+NaF, 2 % CSR+NaF, 4 % CSR+NaF, NaF, and no treatment. Strontium and fluoride release from the varnishes was evaluated. Sound enamel specimens (n = 6) were demineralized, varnish-treated, and subjected to remineralization cycle. Mineral density of enamel specimens was evaluated using micro-CT. Antimicrobial effect of the varnishes on Streptococcus mutans and Lactobacillus acidophilus biofilms was assessed using confocal laser scanning microscopy. The HGF-1 cytotoxicity of the varnishes was examined using CCK-8 assay. RESULTS: Both 2 % and 4 % CSR+NaF varnishes showed significantly higher F release and remineralization potential than NaF varnish (p < 0.05). Dead bacterial proportion of 4 % CSR+NaF varnish was significantly higher than NaF varnish (p < 0.05). The CFUs values of both S. mutans and L. acidophilus were significantly lower in 4 % CSR+NaF group than NaF group (p < 0.05). No significant difference in cell viability was observed among the groups (p > 0.05). CONCLUSIONS: Incorporation of 4 % CSR in a NaF varnish significantly enhanced its enamel remineralization and antimicrobial potential with no cytotoxic effect. CLINICAL SIGNIFICANCE: Dental caries is a major public health problem globally. The study highlights the great potential of CSR-doped NaF varnish as a novel anti-caries agent with synergistic remineralizing and antimicrobial properties to combat early enamel caries lesions in the general population.

The effect of synbiotic-fluoride therapy on multi-species biofilm.

OBJECTIVES: The study objective was to examine the effect of synbiotic-fluoride (SF) therapy within a multi-species cariogenic biofilm model system comprising of S. mutans, S. sanguinis, and S. gordonii. METHODS: The SF therapy was prepared using 2% L-arginine (Arg), 0.2% NaF and probiotic L. rhamnosus GG (LRG). The 8 treatment groups were: Group 1: No treatment, Group 2: 2% Arg, Group 3: 0.2% NaF, Group 4: LRG, Group 5: 2% Arg+0.2% NaF, Group 6: 2% Arg+LRG, Group 7: 0.2% NaF+LRG, and Group 8: SF therapy (2% Arg+0.2% NaF +LRG). Multi-species biofilm model over 96 h comprising Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii was utilized. The biofilms received cariogenic challenge and SF therapy 2 × /day. The extracellular matrix components were analyzed for carbohydrates, proteins, and extra-cellular DNA (eDNA). The live/dead cells were imaged and quantified using confocal microscopy. The viable/dead bacterial concentrations were estimated using propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). The gene expressions for gtfB, sagP, arcA, argG, and argH were measured using real-time reverse transcriptase qPCR. RESULTS: Carbohydrates and protein content with SF therapy were higher than non-LRG containing groups, while eDNA content was lower than other groups (p<0.05). Live bacterial proportions determined using confocal imaging with SF therapy were the lowest (p<0.05). The 2% Arg+LRG and SF therapy showed higher viable L. rhamnosus GG than 0.2% NaF+LRG (p<0.05). The dead S. mutans with SF therapy were higher than the other groups (p<0.05) with no difference from 2% Arg+0.2% NaF and 2% Arg+LRG (p>0.05). The SF therapy significantly downregulates gtfB and upregulates sagP, arcA, argG, argH gene expression (p<0.05). CONCLUSION: Synbiotic-fluoride therapy effectuates multi-fold changes in the multi-species biofilm matrix and cellular components leading to superior ecological homeostasis than its individual contents, prebiotics (arginine), probiotic (L. rhamnosus GG), and fluorides (NaF). CLINICAL SIGNIFICANCE: The ecological-based synbiotic-fluoride caries-preventive therapy aids in maintaining biofilm homeostasis to preempt/restore dysbiosis thereby sustaining dynamic-diverse health-associated microbial stability significant as a preventive regimen for high caries-risk patients.

Biofilm modulatory response of arginine-fluoride varnish on multi-species biofilm.

OBJECTIVES: To examine the biofilm modulatory effect of arginine (Arg)-fluoride (F) varnish on multi-species biofilms. METHODS: Experimental varnishes were prepared by incorporating L-Arg (1, 2, and 4%) in 5% NaF varnish, which served as the control. Multi-species biofilms comprising Streptococcus mutans, Streptococcus sanguinis and Streptococcus gordonii were grown on hydroxyapatite (HA) discs and treated with the Arg and F released from the experimental and control groups. The HA discs with treated biofilms were examined for biofilm composition. The biofilm thickness and live/dead counts were investigated using confocal microscopic imaging, while biofilm polysaccharides, proteins, and extracellular DNA (eDNA) were assessed spectrophotometrically. Bacterial composition in biofilms was analysed using viability real-time quantitative polymerase chain reaction (qPCR). The relative gene expression (RGE) was determined for gtfB, SMU.150, nlmD, arcA, and sagP. RESULTS: Both the 2 and 4% Arg-NaF groups reduced biofilm thickness, with the 4% Arg-NaF group showing a significantly greater proportion of dead bacteria, followed by 1 and 2% Arg-NaF (p < 0.001). All Arg-NaF groups significantly reduced the carbohydrate content of the biofilm, while the 4% Arg-NaF-treated biofilms demonstrated higher concentration of eDNA and proteins compared to the control NaF (p < 0.001). Expression of gtfB, SMU.150, and nlmD were significantly downregulated in 4% Arg-NaF-treated biofilms; while 2% Arg-NaF enhanced the expression of arcA. Both 2% Arg-NaF and 4% Arg-NaF significantly increased the expression of sagP. CONCLUSION: The incorporation of L-arginine (2%/4%) enhances the biofilm modulatory effect of 5% NaF varnish through released Arg and F. CLINICAL SIGNIFICANCE: The study results indicate that Arg-F varnish (at 2%/4% w/v. Arg) has the potential to modulate cariogenic biofilms in high-risk individuals.

Physicochemical Properties and Inductive Effect of Calcium Strontium Silicate on the Differentiation of Human Dental Pulp Stem Cells for Vital Pulp Therapies: An In Vitro Study.

The development of biomaterials that exhibit profound bioactivity and stimulate stem cell differentiation is imperative for the success and prognosis of vital pulp therapies. The objectives were to (1) synthesize calcium strontium silicate (CSR) ceramic through the sol−gel process (2) investigate its physicochemical properties, bioactivity, cytocompatibility, and its stimulatory effect on the differentiation of human dental pulp stem cells (HDPSC). Calcium silicate (CS) and calcium strontium silicate (CSR) were synthesized by the sol−gel method and characterized by x-ray diffraction (XRD). Setting time, compressive strength, and pH were measured. The in vitro apatite formation was evaluated by SEM-EDX and FTIR. The NIH/3T3 cell viability was assessed using an MTT assay. The differentiation of HDPSC was evaluated using alkaline phosphatase activity (ALP), and Alizarin red staining (ARS). Ion release of Ca, Sr, and Si was measured using inductive coupled plasma optical emission spectroscopy (ICP-OES). XRD showed the synthesis of (CaSrSiO4). The initial and final setting times were significantly shorter in CSR (5 ± 0.75 min, 29 ± 1.9 min) than in CS (8 ± 0.77 min, 31 ± 1.39 min), respectively (p < 0.05). No significant difference in compressive strength was found between CS and CSR (p > 0.05). CSR demonstrated higher apatite formation and cell viability than CS. The ALP activity was significantly higher in CSR 1.16 ± 0.12 than CS 0.92 ± 0.15 after 14 d of culture (p < 0.05). ARS showed higher mineralization in CSR than CS after 14 and 21 d culture times. CSR revealed enhanced differentiation of HDPSC, physicochemical properties, and bioactivity compared to CS.

The acid-resistance potential of arginine-fluoride varnish treated enamel.

The study objective was to examine the acid-resistance potential of enamel carious lesions treated with arginine (Arg)-sodium fluoride (NaF) varnishes using nano-mechanical testing and chemical mapping. L-arginine (at 1%, 2%, & 4%) was incorporated in 5% NaF varnish. The experimental/control groups were: 1% Arg-NaF, 2% Arg-NaF, 4% Arg-NaF, NaF, and no treatment. Enamel specimen blocks were subjected to incipient carious lesion formation. After treatment, the specimens underwent chemical pH-cycling for 8-days and acid challenge for 2 h. The specimens were characterised for surface nano-hardness (SNH) and calcium/phosphate content of the treated lesions to determine enamel solubility reduction (ESR). Post-acid challenge, X-ray diffraction crystallography (XRD), and energy dispersive X-ray spectrophotometry (EDX) were performed. The SNH for 2%/4% Arg-NaF demonstrated a higher resistance to acid challenge with significantly higher SNH recovery than NaF varnish (p<0.05). The ESR potential of 2%/4% Arg-NaF varnish was significantly higher than NaF varnish (p<0.05). The XRD crystalline phases demonstrated that 2%/4% Arg-NaF had intense hydroxyapatite peaks discerning its increased potential to resist demineralization than NaF varnish. The EDX results showed that 2%/4% Arg-NaF demonstrated Ca/P ratio closer to hydroxyapatite (~1.67) post-acid challenge. Incorporating 2%/4% L-arginine in a 5% NaF varnish enhances the acid-resistance potential of NaF varnish.

The effect of arginine-fluoride varnish on biochemical composition of multi-species biofilm.

OBJECTIVE: To examine the biochemical components of multi-species biofilm on the arginine (Arg)-sodium fluoride (NaF) varnish-treated enamel following bacterial pH-cycling. METHODS: l-arginine (at 1%, 2%, & 4% w/v.) was incorporated in a 5% NaF varnish. The experimental and control groups were: 1%, 2%, 4% Arg-NaF, NaF, and no treatment. Enamel blocks were prepared, acid-etched, varnish-treated and then subjected to 72 h bacterial pH-cycling in an oral biofilm reactor. The organic (carbohydrates, proteins, amyloids, and eDNA) and inorganic components (calcium, inorganic phosphate, F) were assayed for the obtained biofilm suspensions. The biofilms were stained for exopolysaccharides (EPS)/bacteria and the respective proportions of live/dead bacteria was determined using confocal imaging. RESULTS: The total carbohydrate content of the biofilm was the lowest for the 2% and 4% Arg-NaF (p < 0.05). Except for 2% Arg-NaF, the biofilm proteins for 4% Arg-NaF were significantly higher than the other groups (p < 0.05). The amyloids for Arg-NaF groups were significantly lower than the controls (p < 0.05). The eDNA for 4% Arg-NaF was significantly higher than the controls (p < 0.05). The 2% and 4% Arg-NaF-treated enamel had increased biofilm Pi and F compared to the NaF-treated enamel (p < 0.05). The proportion of biofilm EPS matrix to bacteria was significantly reduced in Arg-NaF groups compared to the controls (p < 0.05). The dead bacterial proportions of 4% Arg-NaF were significantly higher than the controls (p < 0.05). CONCLUSION: Higher concentrations (i.e. 2%/4%) of Arg in 5% NaF varnish have the potential to modulate the biochemical composition of the biofilm growing on the treated enamel.

Enamel remineralization potential of arginine-fluoride varnish in a multi-species bacterial pH-cycling model.

OBJECTIVES: To examine the enamel fluoride uptake and remineralization potential of arginine-fluoride (Arg-NaF) varnishes in a simulated clinical condition using a multi-species bacterial pH-cycling model. METHODS: L-Arginine (at 1 %, 2 %, and 4 % by wt.) was incorporated in a 5 % NaF varnish. Experimental and control groups were: 1 % Arg-NaF; 2 % Arg-NaF; 4 % Arg-NaF; NaF and no treatment. Artificial incipient caries-like lesions were formed on 30 enamel specimen blocks (n = 6). The specimens underwent multi-species bacterial pH-cycling in an artificial mouth system using oral biofilm reactor for 72 h after treatment. The specimens were evaluated for mineral density using micro-CT, Ca/P ratio with SEM-EDX, enamel fluoride uptake (EFU) and plaque fluoride uptake (PFU). RESULTS: Increasing concentrations of Arg in NaF varnish significantly increased the EFU of incipient caries-like lesions (p < 0.001). The PFU for 1 % Arg-NaF was significantly higher than 4 % Arg-NaF and the control NaF (p < 0.05). Post pH-cycling, Ca/P ratio with 1 %/2 % Arg-NaF was closest to hydroxyapatite (1.67). Mineral gain and % remineralization of 1 %/2 % Arg-NaF was significantly higher than the control NaF varnish (p < 0.05). CONCLUSION: The prebiotic L-arginine (at 1 %/2 % by wt.) in a 5 % NaF varnish enhanced the enamel fluoride uptake and remineralization potential of the conventional 5 % NaF varnish. CLINICAL SIGNIFICANCE: The Arg-NaF varnish addresses the limitations of fluorides on cariogenic biofilms. The Arg-NaF varnish appears a promising caries-preventive regimen that counters the pathogenic biofilms by Arg and promotes remineralization with fluorides. In high caries-risk patients, professional application of Arg-NaF varnish might aid to alleviate the global burden of dental caries.

A novel, doped calcium silicate bioceramic synthesized by sol-gel method: Investigation of setting time and biological properties.

The aim of the current study was to synthesize a fast-setting ion-doped calcium silicate bioceramic by the sol-gel method and to characterize its in vitro apatite-forming ability and cell viability. Calcium silicate (CS), doped calcium silicate with zinc and magnesium, with Ca/Zn molar ratios of 6.7:1 (DCS1), and 4.5:1 (DCS2), were synthesized by the sol-gel method. Matreva white MTA (WMTA, Matreva, CA, Egypt) was used as a control. The synthesized powders were characterized by x-ray diffraction. Setting time was measured using the Gilmore needle indentation technique. The in vitro apatite-forming ability of the materials was evaluated by scanning electron microscope and energy dispersive X-ray. NIH3T3-E1 cells viability was tested using MTT assay. The ion release of Ca, Si, Zn, and Mg was measured using inductive coupled plasma-optical emission spectroscopy (ICP-OES). One-way ANOVA was used to analyze setting time results. The Tukey's HSD post hoc test was used to establish significance (p < 0.001). For nonparametric data, the Kruskal-Wallis H test with Dunn's correction for post hoc comparison was used (p < 0.05). CS, DCS1, and DCS2 showed a significant decrease in setting time 33 ± 1.63 min, 28 ± 1.63 min, and 41.75 ± 2.87 min, respectively, compared to WMTA 91 ± 3.16 min (p < 0.001). DCS1 showed the highest apatite-forming ability and cell viability compared to the other groups. Ca and Si ions release decreased in both DCS1 and DCS2. The physical and biological properties of CS can be successfully improved by the sol-gel synthesis and ions doping. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:56-66, 2020.