Validation of enamel erosion in vitro

Objectives

Many tools are available to quantify dental erosion, but each technique has its own inherent disadvantages. This study aims to validate the use of quantitative light-induced fluorescence (QLF) and non-contacting surface profilometry compared to the gold standard transverse microradiography (TMR) for the quantification of enamel erosion in vitro.

Methods

This was an in vitro laboratory based study. 60 bovine incisors were divided into 6 groups of 10. Each tooth's labial surface was completely varnished except for a window of enamel approximately 3 mm × 5 mm. Each was baseline imaged with QLF and non-contacting surface profilometry before being subjected to an erosive solution (pH 3.4) for up to 36 h. The lesions were imaged using non-contacting surface profilometry and QLF, sectioned and analysed with TMR. Correlation coefficients were calculated to assess the validity of the methods of measurement as compared to TMR.

Results

A range of lesion severities resulted. Mineral loss measured as ΔQ (QLF) and step height (profilometry), was recorded and confirmed by TMR. A correlation was found between ΔZ (TMR) and profilometry lesion depth of r = 0.648 (p < 0.001). A poorer correlation was found between ΔZ and ΔQ: r = 0.217 (p = 0.096).

Conclusions

Profilometry lesion depth and ΔZ correlated significantly. Both methods allow for quantification of erosive crater depth. QLF correlated poorly with ΔZ, but is useful for measuring subsurface loss of mineralisation. TMR is valuable but is destructive and can only be used in vitro. Currently only QLF can be used in vivo. Advances in these technologies may allow the development of non-destructive in vivo measurements of mineral loss, combining the positive features of each measurement method.     

Physical chemical effects of zinc on in vitro enamel demineralization

Objectives

Zinc salts are formulated into oral health products as antibacterial agents, yet their interaction with enamel is not clearly understood. The aim was to investigate the effect of zinc concentration [Zn²⁺] on the in vitro demineralization of enamel during exposure to caries-simulating conditions. Furthermore, the possible mechanism of zinc's action for reducing demineralization was determined.

Methods

Enamel blocks and synthetic hydroxyapatite (HAp) were demineralized in a range of zinc-containing acidic solutions (0–3565 ppm [Zn²⁺]) at pH 4.0 and 37 °C. Inductively coupled-plasma optical emission spectroscopy (ICP-OES) was used to measure ion release into solution. Enamel blocks were analysed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and HAp by X-ray diffraction (XRD) and neutron diffraction (ND).

Results

ICP-OES analysis of the acidic solutions showed a decrease in [Ca²⁺] and [PO₄³⁻] release with increasing [Zn²⁺]. FTIR revealed a α-hopeite (α-Zn₃(PO₄)₂.4H₂O)-like phase on the enamel surfaces at >107 ppm [Zn²⁺]. XRD and ND analysis confirmed a zinc-phosphate phase present alongside the HAp.

Conclusions

This study confirms that zinc reduces enamel demineralization. Under the conditions studied, zinc acts predominantly on enamel surfaces at PO₄³⁻ sites in the HAp lattice to possibly form an α-hopeite-like phase.

Clinical significance

These results have a significant implication on the understanding of the fundamental chemistry of zinc in toothpastes and demonstrate its therapeutic potential in preventing tooth mineral loss.     

Effect of sodium fluoride on oral biofilm microbiota and enamel demineralization

ObjectiveFluoride is widely used as an anti-caries agent, e.g. in toothpastes and mouth rinses. However, the nature of the anti-caries action is not entirely clear. Mechanisms suspected to explain the cariostatic effect include inhibitory effects on acid formation by bacteria, inhibition of extracellular polysaccharide (EPS) production, inhibition of enamel demineralization and enhancement of remineralizaton or combination thereof. The aim of this study was to examine with the supragingival Zurich in vitro biofilm model the effect of fluoride in NaF formulation, on the microbiota and on demineralization.MethodsBiofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar and Streptococcus sobrinus, were grown anaerobically on sintered hydroxyapatite or bovine enamel disks, exposed to 200, 400, and 1400 ppm of NaF, or 0.1% chlorhexidine (positive control). The biofilms were harvested after 64 h and CFUs were assessed for total bacteria. Demineralization of enamel disks was measured by quantitative light-induced fluorescence.ResultsNaF did not affect the bacterial numbers. No enamel mineral loss was observed at 1400 and 400 ppm of fluoride, whereas the pH of the surrounding medium was increased to 5.5 and 5.0, respectively, compared to the untreated control (pH 4.5 and mineral loss ΔF of −32%). At 1400 ppm NaF the biofilm’s EPS volume was also significantly reduced.ConclusionsAdministration of NaF completely prevented demineralization without affecting biofilm composition and growth. This protective effect may be attributed to the observed decrease in acid production or EPS volume, or to a shift in the de/remineralization balance.     

Effects of fluoride concentration on enamel demineralization kinetics in vitro

Objectives

The aim of the present study was to measure the effects of fluoride concentration on the real-time in vitro demineralization of enamel during exposure to caries-simulating conditions using Scanning Microradiography (SMR).

Methods

Enamel blocks obtained from non-carious human molars were fixed in SMR environmental cells, through which acidic solutions (0.1 M acetic acid, pH 4.0) were circulated for periods of 48 h. SMR was used to quantitatively measure continuous mineral mass loss. Subsequently, the effects of sequentially increasing fluoride concentration (0.1–4500 mg/L [F⁻]) in the acidic solutions were measured on the rate of enamel demineralization.

Results

The data shows a log-linear relationship between [F⁻] and reduction in demineralization up to 135 mg/L [F⁻]. Above 135 mg/L, no further significant decrease in demineralization occurred.

Conclusion

The optimum range of local fluoride concentration for reducing enamel demineralization was in the range 0.1–135 mg/L [F⁻] under the conditions studied.

Clinical significance

Relatively low [F⁻] can exhibit near-optimum protection. Increasing the fluoride concentrations above 135 mg/L may not necessarily give an increased cariostatic benefit. Improving the means of delivery of relatively low fluoride concentrations to the oral fluids through slow releasing mechanisms, such as the oral fluoride reservoirs, is the more appropriate way forward for sustaining long-term clinical efficacy.     

Association of amoxicillin use and molar incisor hypomineralization in piglets: Visual and mineral density evaluation

AimThe aim of the present study was to determine the prevalence of MIH both visually and quantitatively, and describes the range of mineral densities of enamel specimens from three groups of piglets where two groups were given different doses of amoxicillin in infancy.MethodsIn this blind randomized clinical study, 20 piglets were randomly divided into three groups. Group A received a standard dose (50 mg/kg/day) and Group B received a high dose (90 mg/kg/day) of amoxicillin in selected days of the month (20 working days) they were born. Group K did not receive any medication and served as control. Thirteen right mandibular permanent first molars (PFMs) were randomly collected from 3 groups of piglets at age 10 months for evaluation under X-ray micro-tomography. Tomographic data were obtained using a Skyscan 1174 compact micro-CT in the Department of Anatomy.ResultsPrevalence of MIH was 0% in all groups. MD values were quantified after enamel grey level (0–255) measurements on horizontal cross-sectional slices. After MD measurements, the effects of amoxicillin use on MIH are presented.ConclusionsWhile MIH is a multifactorial disturbance, the present study attempted to highlight the clinical findings of a possible relationship between amoxicillin use and MIH with the aid of X-ray micro-tomography.     

Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion: An in situ/ex vivo study

Objective

To evaluate the effect of fluoride (F) varnishes supplemented or not with sodium trimetaphosphate (TMP) on enamel erosive wear followed or not by abrasion in situ.

Methods

Ten volunteers were selected and randomly divided into four groups, according to the varnishes tested: placebo (no F or TMP), 5% NaF (positive control), 2.5% NaF and 2.5% NaF/5% TMP. Enamel blocks (n = 4) were mounted in palatal devices and received an application of each test varnish, following a double-blind, crossover protocol. After 6 h, varnishes were completely removed and the blocks were subjected to erosive challenges by ex vivo immersion in citric acid (5 min, 4×/dia, 5 days). Following, half of the blocks were subjected to abrasion by brushing with a placebo dentifrice slurry for 15 s. Enamel wear (μm), surface hardness (SH_f) and cross-sectional hardness (ΔKHN) were assessed after each experimental period. Results were analyzed by ANOVA, Student–Newman–Keuls's test and Pearson correlation coefficient (p < 0.05).

Results

The fluoride varnish supplemented with TMP promoted significantly lower wear and ΔKHN when compared to the other groups after erosive challenges, followed or not by abrasion (p < 0.05). As for (SH_f) the fluoride varnish supplemented with TMP promoted similar results to the 5% NaF product, being significantly higher than the remaining groups after erosive and erosive + abrasive challenges (p < 0.05).

Conclusion

TMP significantly enhanced the effects of F on enamel wear after erosive challenges, followed or not by abrasion.     

Effect of experimental mouthrinses containing the combination of NaF and TiF4 on enamel erosive wear in vitro

Objective

This study analysed the anti-erosive effect of experimental solutions containing TiF₄ and NaF.

Methods

Bovine enamel samples (n = 15) were treated with: (1) commercial solution with SnCl₂/NaF (Erosion Protection^®); (2) experimental solution with 0.0815% TiF₄; (3) 0.105% NaF; (4) 0.042% NaF + 0.049% TiF₄; (5) 0.063% NaF + 0.036% TiF₄ or (6) control. The samples were submitted to pH cycles and daily fluoride applications for seven days. The enamel wear was measured using a contact profilometer and analysed by ANOVA (p < 0.05).

Results

The best anti-erosive effect was found for experimental solution with 0.0815% TiF₄ (99% reduction in enamel wear), followed by SnCl₂/NaF (78%) and 0.049% TiF₄ + 0.042% NaF solution (41%).

Conclusions

The experimental solution containing a specific combination of TiF₄ + NaF has the ability to partially reduce enamel erosion.     

Light and electron microscopic study on the effect of phenylbutazone on developing mouse palatal epithelium in vitro

It is well-known that degeneration of the midline epithelial seam occurs after palatal fusion in normal mammalian embryos. Phenylbutazone, a non-steroidal anti-inflammatory drug inhibits epithelial cell death in both in vivo and in vitro experiments. Paired palatal processes from 13.5- and 14.5-day-old mouse embryos were organ-cultured in control and phenylbutazone-containing media for 72 h. The medial epithelial cells of the palatal processes were completely disrupted after 72 h in control cultures, but in treated expiants, no epithelial cell disruption took place, mitotic figures were seen in the epithelium and lysosomes did not appear in these epithelial cells. Phenylbutazone treatment seems to interfere with epithelial-mesenchymal interactions by inhibiting prostaglandin synthesis suggesting that these compounds are necessary for cell autolysis.     

Preventive effect of an iron varnish on bovine enamel erosion in vitro

Objective

The aim of this study was to evaluate, in vitro, the effect of an experimental varnish containing iron on the dissolution of bovine enamel by carbonated beverage.

Methods

Eighty specimens were randomly allocated to four groups (n = 20 per group), according to the following treatments: Fe varnish (FeV, 10 mmol/L Fe), F varnish (FV, 2.71% F), placebo varnish (PV) and control (not treated, NT). The varnishes were applied in a thin layer and removed after 6 h. Then, the samples were submitted to six cycles, alternating re- and demineralisation (only 1 day). Demineralisation was performed with the beverage Coca-Cola^® (10 min, 30 mL/block) and remineralisation with artificial saliva for 1 h. In order to determine the amount of enamel dissolved, the wear was analysed by profilometry. Data were analysed by ANOVA and Tukey's test (p < 0.05).

Results

The mean wear (±S.E.) was significantly lesser for the FeV (0.451 ± 0.018 μm) when compared to the other treatments. The FV caused significantly less wear (0.554 ± 0.022 μm) when compared to PV (0.991 ± 0.039 μm) and NT (1.014 ± 0.033), which did not significantly differ from each other.

Conclusions

The results suggest that the iron varnish can interfere with the dissolution of dental enamel in the presence of acidic beverages.     

Effect of rinsing with water immediately after neutral gel and foam fluoride topical application on enamel remineralization: An in situ study

Objective

The aim of this study was to evaluate, in situ, the effect of rinsing with water immediately after neutral fluoride foam application (Foam) or fluoride gel application (Gel).

Design

Ten volunteers wore acrylic palatal appliances containing 4 enamel blocks selected by surface hardness with artificial caries. Five experimental regimes of 3 days each were set according to treatment: placebo; Gel and Foam followed by no rinsing or consuming of liquids or solids for the next 30 min; Gel and Foam followed by immediately washing with water jet. After each phase, surface hardness was again measured for analysis of mineral gain, evaluated through percentage of surface hardness recovery (%SHR) and integrated loss of subsurface hardness (ΔKHN). The concentration of loosely bound fluoride (CaF2) and firmly bound fluoride (FA-like) formed and retained were also determined.

Results

Fluoride treatments produced greater remineralization (%SHR and ΔKHN) compared to placebo group (p < 0.05). There was no difference in the ability to promote remineralization and in the concentration of fluoride formed and retained, in each analysis, between Gel and Foam (p > 0.05).

Conclusion

The data suggest that rinsing with water immediately after topical application does not seem to have an influence on the ability of fluoride to promote remineralization.     

Fluoride uptake by powdered human enamel treated with prolonged acting fluoride pellets in vitro

A preparation for the prolonged release of fluoride for topical use was developed. The delayed release of the fluoride was achieved by dispersing NaF or CaF₂ in ethyl cellulose with or without a stearic acid matrix. Orthodontic plates were used as delivery systems. The kinetics of fluoride release was controlled by diffusion mechanisms. The major compound resulting from enamel treatment with prolonged acting fluoride is fluoridated apatite.     

Remineralisation of enamel with silver diamine fluoride and sodium fluoride

Objective

To evaluate the remineralising effect of the adjunctive application of 38% silver diamine fluoride (SDF) solution and 5% sodium fluoride (NaF) varnish on artificial enamel caries lesions.

MMP20 and KLK4 activation and inactivation interactions in vitro

Objective

Enamelysin (MMP20) and kallikrein 4 (KLK4) are believed to be necessary to clear proteins from the enamel matrix of developing teeth. MMP20 is expressed by secretory stage ameloblasts, while KLK4 is expressed from the transition stage throughout the maturation stage. The aim of this study is to investigate the activation of KLK4 by MMP20 and the inactivation of MMP20 by KLK4.

Design

Native pig MMP20 (pMMP20) and KLK4 (pKLK4) were isolated directly from enamel scrapings from developing molars. Recombinant human proKLK4 (rh-proKLK4) was activated by incubation with pMMP20 or recombinant human MMP20 (rhMMP20), and the resulting KLK4 activity was detected by zymography. Reaction products were isolated by reverse-phase high performance liquid chromatography (RP-HPLC), and their N-termini characterized by Edman degradation. The pMMP20 was incubated with pKLK4 under mildly acidic or under physiologic conditions, and enzyme activity was analyzed by zymography. The catalytic domain of rhMMP20 was incubated with pKLK4 or recombinant human KLK4 (rhKLK4) and the digestion products were characterized by zymography and Edman degradation.

Results

Both pMMP20 and rhMMP20 activated rh-proKLK4 by cleaving at the propeptide-enzyme junction used in vivo. The pMMP20 was inactivated by pKLK4 under physiologic conditions, but not under mildly acidic conditions. Both pKLK4 and rhKLK4 cleaved MMP20 principally at two sites in the catalytic domain of MMP20.

Conclusions

MMP20 activates proKLK4 and KLK4 inactivates MMP20 in vitro, and these actions are likely to occur during enamel formation in vivo.     

Effect of tricalcium silicate (Ca3SiO5) bioactive material on reducing enamel demineralization: An in vitro pH-cycling study

Objectives

The aim of this study was to investigate the effect of Ca₃SiO₅ on reducing enamel demineralization under pH-cycling conditions.

Methods

Forty bovine enamel samples were treated under four conditions: group 1, double distilled water (negative control); group 2, 1000 ppm F (as NaF, positive control); group 3, Ca₃SiO₅ slurry; and group 4, Ca₃SiO₅–F slurry (Ca₃SiO₅ with 1000 ppm F aq.). All the specimens were treated with treatment materials 4 times each day. Samples in groups 1 and 2 were soaked in test solutions for 2 min and samples in groups 3 and 4 were painted in treatment slurry for 2 min. At times between treatments, they were immersed in citric acid solution 3 times a day and 15 s each time. All the procedures were repeated for 7 days. Knoop microhardness, scanning electron microscopy (SEM), X-ray diffraction (XRD) and atom force microscopy (AFM) were used to examine samples.

Results

After treatment for 7 days, enamels in all the groups were significantly softened. The extents of microhardness reduction were 52.3%, 28.5%, 28.5% and 20.2% for groups 1, 2, 3 and 4, respectively. Samples in the negative control group showed a typical acid etched pattern while enamels in other groups were relatively compact. There was no significant difference between samples treated with Ca₃SiO₅ and F. The combination of Ca₃SiO₅ with F showed the best effect on reducing enamel demineralization.

Conclusions

Ca₃SiO₅ is an effective material against enamel demineralization alone but in combination with F a better anti-demineralization effect may be obtained.     

The effect of ozone on progression or regression of artificial caries-like enamel lesions in vitro

Objective

This study investigated the effect of ozone on the progression or regression of artificial caries-like lesions on enamel following pH cycling conditions in vitro.

Methods

A randomized, single blind, four legs design was used. 20 full thickness enamel slabs were allocated to each of the four groups which were: Fluoride free toothpaste (control); ozone alone; Reductant/Patient Kit alone and a combination of both ozone/Reductant/Patient Kit. Artificial lesions were created and subjected to the pH cycling regime for a 14 days period. Assessments were carried out before and after the pH cycling on the slabs using the microhardness testing and Quantitative Light-induced Fluorescence (QLF).

Results

Statistical significant difference were found in the percentage change of enamel microhardness before and after pH cycling between ozone/Reductant/Patient Kit group and all the other three groups of the study, as well as between Reductant/Patient Kit group and control. There was a statistical significant difference in the change of size and severity of the lesion (ΔQ) between all the three regimes tested and the control with a trend favouring ozone/Reductant/Patient Kit group.

Conclusions

In our model, it appeared that ozone treatment alone is not effective in protecting the enamel against demineralisation or promoting remineralisation, unless combined with the Reductant/Patient Kit, which contain high levels of fluoride.     

Effects of fluoride on locomotion of human blood leucocytes in vitro

The effect of NaF on the locomotion and chemotaxis of human blood neutrophils and monocytes was studied using two assays: the micropore filter assay and a time-lapse cinematographic assay in which the chemotaxis of cells in response to spores of Candida albicans was filmed. At high concentrations (> 10^?4 M), NaF inhibited locomotion of both cell types, but no inhibition of locomotion of either cell-type was seen in either assay using NaF at ? 10^?4 M, whether or not the cells were responding to a chemotactic source. This was so, even for monocytes incubated for 48 h in the presence of NaF. It is therefore improbable that fluoride, at levels added to drinking water or found in the body fluids of persons drinking fluoridated water, has any deleterious effect on the locomotor capacity of phagocytic cells or on their capacity to detect and home on to chemotactic sources.     

Effect of fluoride gels supplemented with sodium trimetaphosphate on enamel erosion and abrasion: In vitro study

Objective

This in vitro study aims to evaluate the effect of low fluoride (F) gel associate sodium trimetaphosphate (TMP) on erosion with or without abrasion.

Design

Enamel blocks (4 mm × 4 mm) selected through surface hardness (SH) is divided into five groups (n = 12): gel without F and TMP (placebo), gel containing 4500 ppm F (4500), gel containing 4500 ppm F plus TMP5% (4500 TMP5%), gel containing 9000 ppm F (9000), and gel containing 12,300 ppm F (acid gel). Those groups were additionally subdivided into conditions of erosion (Ero) and of erosion plus abrasion (Ero/Abra). The blocks have undergone a single application of gel on the first day of the study. The erosion challenge was produced by Sprite Zero^® for five minutes four times a day and abrasion was carried out by machine brushing for 15 s. After the challenges, the surface hardness (%SH), wear and cross-sectional hardness (ΔKHN) were analyzed. The data were analyzed using a 2-way ANOVA test followed by a Student-Newman–Keuls (p < 0.05).

Results

Lower values of %SH, wear and ΔKHN were observed for erosion challenge (p < 0.001). The %SH was lower in groups treated with fluoride gels, differing in the placebo (p < 0.05). With addition of TMP to the gel 4500, enamel wear was lower when compared with another groups (p < 0.05).

Conclusion

In vitro conditions, the 4500 5%TMP gel showed greatest effect against erosion and erosion/abrasion.     

The adsorption in vitro of purified salivary proteins on bovine dental enamel

The adsorption of proteins isolated from human submandibular saliva was studied on intact enamel surfaces and on enamel powder. All proteins adsorbed in a monolayer, in a single-step adsorption mechanism, suggesting that some of the molecules have a flexible (random coil) structure. The proteins did not release measurable amounts of ions from the adherent liquid layer during adsorption, in contrast to previous findings on the adsorption of several homopolypeptides (Juriaanse et al., J. Colloid Interface Sci.76, 212–220; 1980). Salivary phosphoproteins are more firmly bound to enamel surfaces than are non-phosphorylated proteins, which is due to an exchange reaction in which protein phosphate groups replace surface phosphates in the enamel mineral. In the model used for pellicle maturation, the enamelpellicle interface was slowly saturated with phosphoproteins which displaced non-phosphorylated proteins from the surface.     

Expression, localisation and synthesis of versican by the enamel organ of developing mouse molar tooth germ: An in vivo and in vitro study

Objective

Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ.

Design

To elucidate versican localisation in vivo, in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5–E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [³⁵S], followed by gel filtration, was performed to analyse secreted proteoglycans.

Results

Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2–3/4 of the [³⁵S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ.

Conclusions

The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.     

Synchrotron radiation microbeam X-ray fluorescence analysis of zinc concentration in remineralized enamel in situ

Objective

Remineralization is an indispensable phenomenon during the natural healing process of enamel decay. The incorporation of zinc (Zn) into enamel crystal could accelerate this remineralization. The present study was designed to investigate the concentration and distribution of Zn in remineralized enamel after gum chewing.

Methods

The experiment was performed at the Photon Factory. Synchrotron radiation was monochromatized and X-rays were focused into a small beam spot. The X-ray fluorescence (XRF) from the sample was detected with a silicon (Si) (lithium (Li)) detector. X-ray beam energy was tuned to detect Zn. The examined samples were small enamel fragments remineralized after chewing calcium phosphate-containing gum in situ. The incorporation of Zn atom into hydroxyapatite (OHAP), the main component of enamel, was measured using Zn K-edge extended X-ray absorption fine structure (EXAFS) with fluorescence mode at the SPring-8.

Results

A high concentration of Zn was detected in a superficial area 10-μm deep of the sectioned enamel after gum chewing. This concentration increased over that in the intact enamel. The atomic distance between Zn and O in the enamel was calculated using the EXAFS data. The analyzed atomic distances between Zn and O in two sections were 0.237 and 0.240 nm.

Conclusion

The present experiments suggest that Zn is effectively incorporated into remineralized enamel through the physiological processes of mineral deposition in the oral cavity through gum-chewing and that Zn substitution probably occurred at the calcium position in enamel hydroxyapatite.