Immediate and Sustained Root Caries Prevention of Fluoride Varnish Combined with Toothpastes.

The aim of this study was to determine the immediate and sustained effect of a fluoride varnish and its combinations with toothpastes in preventing root caries development using a salivary microcosm in vitro model. Human root dentin specimens (n = 150) were randomly divided into 5 experimental protocols (n = 30): (1) Fluoride Varnish (V); (2) V followed by Paste One (V + PO); (3) V followed by Paste Plus (V + PP); (4) V followed by PO and PP (V + PO + PP); and (5) No treatment (control). One varnish layer was applied on the specimens (except for the control group) and kept for 18 h. Then, the varnish was removed and toothpaste treatments were initiated according to experimental groups. For the short-term incubation model (n = 15), the specimens were also immediately subjected to 7-day cariogenic challenge. For that, human saliva was used as bacterial inoculum and McBain artificial saliva containing 2% sucrose as growth medium. The other half of the specimens (n = 15) were used to study the varnish's sustained effect by long-term incubation (8 weeks) before cariogenic challenge. The protocols' anti-caries properties were evaluated by dentin porosity (rhodamine intensity; RI) and mineral density, while their anti-biofilm effects were evaluated using biofilm's biomass and viability assays. For short- and long-term incubation models, all experimental regimens resulted in statistically significant decreases (p < 0.05) in the RI (up to 180 µm and 120 µm, respectively) as well as higher mineral density compared to No treatment (p < 0.001). V + PO + PP and/or V + PO resulted in statistically lower RI compared to V for some depths (p < 0.05) in both models. There were changes in RI and mineral density within groups over time. All experimental treatments exhibited anti-biofilm effects. All prevention protocols exhibited immediate and sustained anti-caries effect against root caries development. The combination of a fluoride varnish with PO resulted in superior additional anti-caries effects.

In vitro anti-erosive property of a mint containing bioactive ingredients.

PURPOSE: To evaluate the in vitro protective effect of a mint formulation containing (-)-epigallocatechin-3-gallate (EGCg-mint) on root dentin exposed to a highly erosive environment in the presence and absence of proteolytic challenge. METHODS: Root dentin specimens were subjected to an erosion-remineralization cycling model (6×/day; 5 days) that included 5-minute immersion in 1% citric acid and 60-minute immersion in remineralization solution (RS). At the remineralization half-time, the specimens were treated (n= 20) with EGCg-mint, RS (negative control) or sodium fluoride (1,000 ppm of NaF; positive control). Half of the specimens were kept overnight in RS (pH cycling) and the other half in RS with Clostridium histolyticum collagenase (pH-proteolytic cycling). Erosion depth was measured using optical profilometry and data analyzed by two-way ANOVA and Tukey tests (α= 0.05). RESULTS: Under pH-cycling, NaF resulted in statistically lower erosion depth compared to EGCg-mint (P= 0.020) and RS (P= 0.005). Under pH-proteolytic cycling, EGCg-mint and NaF significantly decreased the tissue loss (erosion depth, P< 0.001) compared to the RS. The EGCg-mint exhibited an anti-erosion property on root dentin under a proteolytic challenge. NaF presented an anti-erosion property regardless of the erosive cycling model. CLINICAL SIGNIFICANCE: The anti-erosive action of an over-the-counter mint, containing active ingredients, including epigallocatechin-3-gallate, is likely by the protective mechanisms of the dentin extracellular matrix.

In vitro Study of the Role of Human Neutrophil Enzymes on Root Caries Progression.

The role of the host immune system in caries progression is mainly speculative, and it is believed that it entails the enzymatic degradation of the dentin organic matrix. The aim of this study was to evaluate the proteolytic effect of human neutrophil enzymes on root caries progression. For this, specimens of bovine root dentin were divided into 4 groups (n = 30): caries (C), caries + neutrophils (C + N), no caries (Control), and no caries + neutrophils (Control + N). Streptococcus mutans biofilm (105 CFU/mL) was grown on the root surface to artificially induce root carious lesions (C and C + N groups). Specimens were then exposed to neutrophils (5 × 106 cells/mL) for 48 h (C + N and Control + N groups). Caries development and neutrophil exposures were repeated a 2nd and 3rd time. Caries depth (CD) and dentin demineralization (DD) were assessed by infiltration of rhodamine B using fluorescence microscopy. Collagen fibril ultrastructure was characterized under a polarized microscope with Picrosirius red staining. There were no significant differences (p > 0.05) in CD and DD between the C and C + N groups for 1, 2, and 3 caries-neutrophil exposures. Immature collagen was significantly less present in the carious groups (C, p = 0.003; C + N, p = 0.01) than in the noncarious groups in the most superficial 200 µm. We thus concluded that human neutrophil enzymes did not influence short-term root caries progression, and immature collagen fibrils were more susceptible to degradation during S. mutans-induced root caries progression.

On the bulk biomechanical behavior of densely cross-linked dentin matrix: The role of induced-glycation, regional dentin sites and chemical inhibitor.

Collagen glycation takes place under physiological conditions during chronological aging, leading to the formation of advanced glycation end-products (AGEs). AGEs accumulation induces non-enzymatic collagen cross-links increasing tissue stiffness and impairing function. Here, we focused on determining the cumulative effect of induced glycation on the mechanical behavior of highly collagen cross-linked dentin matrices and assess the topical inhibition potential of aminoguanidine. Bulk mechanical characterization suggests that early glycation cross-links significantly increase the tensile strength and stiffness of the dentin matrix and promote a brittle failure response. Histologically, glycation yielded a more mature type I collagen in a densely packed collagen matrix. The time-dependent effect of glycation indicates cumulative damage of dentin matrices that is partially inhibited by aminoguanidine. The regional dentin sites were differently affected by induced-glycation, revealing the crown dentin to be mechanically more affected by the glycation protocol. These findings in human dentin set the foundation for the proposed in vitro ribose-induced glycation model, which produces an early matrix stiffening mechanism by reducing tissue viscoelasticity and can be partially inhibited by topical aminoguanidine.

Regional contribution of proteoglycans to the fracture toughness of the dentin extracellular matrix.

This study investigated the contribution of small leucine rich proteoglycans (SLRPs) to the fracture toughness of the dentin extracellular matrix (ECM) by enzymatically-assisted selective removal of glycosaminoglycan chains (GAGs) and proteoglycans (PGs) core protein. We adapted the Mode III trouser tear test to evaluate the energy required to tear the dentin ECM. Trouser-shaped dentin specimens from crown and root were demineralized. Depletion of GAGs and PGs followed enzymatic digestion using chondroitinase ABC (c-ABC) and matrix metalloproteinase 3 (MMP-3), respectively. The legs from specimen were stretched under tensile force and the load at tear propagation was determined to calculate the tear energy (T, kJ/m2). SLRPs decorin and biglycan were visualized by immunohistochemistry and ECM tear pattern was analyzed in SEM. Results showed T of crown ECM was not affected by PGs/GAGs depletion (p = 0.799), whereas the removal of PGs significantly reduced T in root dentin ECM (p = 0.001). Root dentin ECM exhibited higher T than crown (p < 0.03), however no regional difference are present after PG depletion (p = 0.480). Immunohistochemistry confirmed removal of GAGs and PGs. SEM images showed structural modifications after PGs/GAGs removal such as enlargement of dentinal tubules, increased interfibrillar spaces and presence of untwisted fibrils with increased diameter. Findings indicate that the capacity of the PGs to unfold and untwist contribute to the dentin ECM resistance to tear, possibly influencing crack growth propagation. The regional differences are likely an evolutionary design to increase tooth survival, that undergoes repetitive mechanical loading and load stress dissipation over a lifetime of an individual.

Investigation of five α-hydroxy acids for enamel and dentin etching: Demineralization depth, resin adhesion and dentin enzymatic activity.

OBJECTIVES: Surface conditioning of enamel and dentin is a key step during adhesive restorative procedures and strategies. The aim of this study was to investigate the effectiveness of five α-hydroxy-acids (AHAs) as enamel and dentin surface etchants. METHODS: Enamel and dentin specimens were prepared from human molars to determine the depth of demineralization by optical profilometry (Δz), the resin bond strength to enamel and dentin (µTBS), the micro-permeability of dentin-resin interfaces, and the gelatinolytic activity of dentin matrix induced by AHAs [glycolic (GA), lactic (LA), citric (CA), malic (MI) and tartaric (TA)] and controls [phosphoric (PA) and maleic (MA)]. All acids were prepared at 35% concentration. Adhesion studies employed Adper Single Bond Plus bonding system. Data were individually processed and analyzed by ANOVA, post-hoc tests and Pearson correlations (α = 0.05). RESULTS: AHA exhibited statistically lower depth of demineralization of enamel and dentin (average 4 fold) than controls (p < 0.001). In enamel, MA and PA etching resulted in higher µTBS than AHA groups (p < 0.001). In dentin, GA, TA, CI and LA etching resulted in statistically similar µTBS than PA (p < 0.05). The hybrid-layer (HL) thickness and interfacial micro-permeability intensity were statistically lower for AHA groups (p < 0.05). A significant positive correlation was observed between the intensity of micro-permeability and the thickness of HL (p < 0.05). AHA etchants elicited lower dentin enzymatic activity than controls (p < 0.05). SIGNIFICANCE: AHAs effectively etched enamel and dentin surfaces. In particular, GA and TA resulted in suitable µTBS and sealing ability as well as induced less gelatinolytic activity in dentin than PA and MA.

Remineralization Potential of Mints Containing Bioactive Agents in Artificially Induced Root Caries.

This study investigated the remineralization effect of experimental mint formulations containing bioactive agents (xylitol; green tea extract, GT; and amorphous calcium phosphate, ACP) in the progression of artificially induced root caries. Root caries lesions were induced by demineralization solution (pH 4.6; 96 h; 37°C). The lesions were treated with mint A, mint B, mint C, xylitol, GT, ACP, or remineralization solution (RS; negative control). Specimens were pH-cycled through treatments (5×/day; 3 min) and 6 cycles of acidic (pH 5.0; 30 min) and neutral (pH 7.0; 10 min) buffers for 8 days. Bacterial collagenase (Clostridium histolyticum) was used overnight to simulate proteolytic challenge. Caries depth and porosity as well as mineral density were estimated using fluorescence microscopy (n = 15) and microcomputed tomography (n = 6). Analysis of variance (ANOVA, α = 0.05) showed no statistically significant difference in caries depth among all groups (p = 0.172). The highest fluorescence intensity decrease was observed for GT followed by mint C, with no significant difference between them (p = 0.868). There were significant differences among GT and mints A, B, and C when compared to RS (p < 0.001). No statistically significant differences in fluorescence intensity were observed among ACP, xylitol, and RS (p > 0.05). The mineral density of the lesions in GT, mints A, B, and C, and ACP was statistically similar (p > 0.05) and significantly higher than that in RS (p < 0.05). No significant difference was observed between xylitol and RS (p = 0.728). The experimental mints showed remineralization action on artificial root caries, and GT was found to be the main active ingredient in the investigated formulations.

New Preventive Approaches Part II: Role of Dentin Biomodifiers in Caries Progression.

Dental caries is the most prevalent infectious chronic disease in children and adults. With a globally aging population, new demands in the management of dental caries are awakened by the rampant increase in the incidence of dental root caries. Like crown caries, root caries requires bacterial driven tissue demineralization followed by the degradation of the extracellular dentin matrix. Due to the complex composition and ultrastructure, preventive strategies targeting the mineral phase of dentin are insufficient for managing the prevention and progression of root caries. However, the composition and ultrastructure of dentin has inspired novel strategies for the effective management of highly susceptible root surfaces. Specifically, the complex and dynamic dentin extracellular matrix (ECM). The ECM of mature dentin contains a robust type I collagen fibrils scaffold, and carefully distributed non-collagenous components, such as proteoglycans, phosphoproteins, and proteases. In this chapter, we will review the experimental strategies of potential clinical impact to prevent root caries progression by site modifications of the mature extracellular dentin matrix. This approach, termed dentin biomodification, encompasses bioinspired strategies to locally enhance the biological and biomechanical characteristics of the tissue by mimicking natural processes. Here, synthetic and biosynthetic compounds can decrease the biodegradability of the dentin ECM and provide mechanical enhancement of dentin. The resulting effect is the maintenance of the dentin ECM to halt root caries progression and possibly mediate effective remineralization of the caries affected root dentin.

Effect of surface roughness on the hydrophobicity of a denture-base acrylic resin and Candida albicans colonization.

AIM: The aim of the present study was to evaluate the effect of surface roughness (roughness average [Ra] µm) on the hydrophobicity of a denture-base acrylic resin and the initial adherence and biofilm formation of Candida albicans (C. albicans). METHODS: Disk-shaped specimens were divided into six groups: Ra 0.05, Ra 0.2, Ra 0.4, Ra 0.8, Ra 1.5, and Ra 3.0. Water contact angles (WCA) were measured, and the specimens incubated with C. albicans for 90 min (initial adherence, n = 108) or 48 h (biofilm formation, n = 108). Adhered and biofilm cells were evaluated by c.f.u./mL and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and the correlation between the two methods was evaluated. The surface of the specimens and cells (adhered and biofilm) were also analyzed by scanning electron microscopy (SEM). RESULTS: Groups Ra 0.05 and 3.0 exhibited the lowest (~75°) and the highest (~100°) WCA mean values, respectively. For both initial adherence and biofilm formation, no statistically-significant differences were observed among all groups, as determined by c.f.u./mL and XTT. A positive correlation between these two methods was found. SEM analysis showed the presence of scratches and valleys on the acrylic specimens and densely-packed yeast cells covering the entire surface. CONCLUSIONS: Roughness significantly increased hydrophobicity (WCA), but had no effect on the number and metabolic activity of adherent and biofilm cells of C. albicans.

Effect of human whole saliva on the in vitro adhesion of Candida albicans to a denture base acrylic resin: a focus on collection and preparation of saliva samples.

OBJECTIVE: In studies on Candida albicans adhesion to surfaces, diverse protocols have been used for collection and preparation of saliva samples. Thus, this study investigated whether variations in the centrifugation parameters and number of donors of saliva would influence the adhesion of C. albicans to a denture base resin. METHODS: Resin acrylic samples (n = 72) were made and then divided into four groups: (a) control - specimens were left without preconditioning in saliva; (b) three experimental groups, in which the specimens were preconditioned with saliva collected from 15 volunteers and centrifuged at 12 000 g for 5 min (G1 ); from 15 volunteers and centrifuged at 18 000 g for 30 min (G2 ); and from one volunteer and centrifuged at 12 000 g for 5 min (G3 ). Candida adhesion was evaluated by both the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction method and crystal violet staining. Data were analyzed by one-way analyses of variance (P = 0.05). RESULTS: For XTT reduction assay, groups G2 , G3 , and control were not significantly different, whereas group G1 showed significantly higher absorbance value than control. For crystal violet staining there were no significant differences among all groups. CONCLUSION: Variations in the centrifugation parameters and number of donors of saliva may influence C. albicans adhesion to denture base resins.