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Analysis of dental enamel remineralization: a systematic review of technique comparisons.

Graphical Abstract

1. Introduction

2. materials and methods, 2.1. protocol and registration, 2.2. search processing, 2.3. inclusion criteria, 2.4. data processing, 4. discussion, 4.1. casein-phosphopeptide-based studies, 4.2. tricalcium phosphate studies, 4.3. self-assembling peptide 11-4 studies, 4.4. nano-hydroxyapatite studies, 4.5. ozone therapy studies, 4.6. fluoride agent studies, 4.7. sealant agent studies, 5. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest, abbreviations.

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Malcangi, G.; Patano, A.; Morolla, R.; De Santis, M.; Piras, F.; Settanni, V.; Mancini, A.; Di Venere, D.; Inchingolo, F.; Inchingolo, A.D.; et al. Analysis of Dental Enamel Remineralization: A Systematic Review of Technique Comparisons. Bioengineering 2023 , 10 , 472. https://doi.org/10.3390/bioengineering10040472

Malcangi G, Patano A, Morolla R, De Santis M, Piras F, Settanni V, Mancini A, Di Venere D, Inchingolo F, Inchingolo AD, et al. Analysis of Dental Enamel Remineralization: A Systematic Review of Technique Comparisons. Bioengineering . 2023; 10(4):472. https://doi.org/10.3390/bioengineering10040472

Malcangi, Giuseppina, Assunta Patano, Roberta Morolla, Matteo De Santis, Fabio Piras, Vito Settanni, Antonio Mancini, Daniela Di Venere, Francesco Inchingolo, Alessio Danilo Inchingolo, and et al. 2023. "Analysis of Dental Enamel Remineralization: A Systematic Review of Technique Comparisons" Bioengineering 10, no. 4: 472. https://doi.org/10.3390/bioengineering10040472

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• Open access
• Published: 09 September 2024

Morphological study of remineralization of the eroded enamel lesions by tyrosine-rich amelogenin peptide

• Mingzhu Wen 1 ,
• Qinghua Bai 1 ,
• Yiwei Li 1 ,
• Yaru Li 1 ,
• Dandan Ma 1 &
• Jinpu Chu 1  

BMC Oral Health volume  24 , Article number:  1054 ( 2024 ) Cite this article

26 Accesses

Metrics details

Tyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix. It has been shown to promote remineralization of demineralized enamel in our previous study. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth on an enamel surface has not been reported. This study aimed to examine the effect of TRAP on the morphology of calcium phosphate crystals grown on early enamel erosion using a pH-cycling model.

Eroded lesions were produced in human premolars by 30-second immersion in 37% phosphoric acid. Forty-five samples of eroded human premolar enamel blocks were selected and randomly divided into 3 groups: deionized water (DDW, negative control); 100 µg/mL TRAP, and 2 ppm sodium fluoride (NaF, positive control group). For 14 days, the specimens were exposed to a pH-cycling model. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods, the surface morphology, calcium-phosphorus ratio, and enamel surface roughness were examined. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics.

After pH-cycling, compared to the two control groups, the surface of the eroded enamel of the peptide TRAP group shows a large number of new, densely arranged rod-like crystals, parallel to each other, regularly arranged, forming an ordered structure, with crystal morphology similar to that of natural enamel. The crystals are mostly hydroxyapatite (HA).

This study demonstrates that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals and promote the remineralization of enamel, providing a promising biomaterial for remineralization treatment of enamel lesions.

Peer Review reports

Introduction

The hardest tissue in the body, tooth enamel is a highly mineralized tissue that makes up the tooth’s outermost layer. The basic component of it is the enamel rod. The enamel rods consist of nano-fibrous hydroxyapatite (HA) crystals. When exposed to acidic conditions (pH < 5.5), hydroxyapatite crystals dissolve, enamel rods and internal rods are destroyed, and enamel is demineralized, leading to tooth erosion [ 1 ]. Dental erosion is the loss of dental hard tissue brought on by chemical processes without the presence of microbes. The etiology of this disease is usually acidic substances such as food, drink, and gastro-oesophageal reflux [ 2 ]. Dental erosion is a serious and common issue due to the rising popularity of acidic diets (especially drinks). Demineralized enamel cannot be fully repaired due to its non-regenerative nature. It must rely on a physicochemical procedure (remineralization) that uses inorganic components from solutions [ 3 ].

Now, methods including bioglass [ 4 ], chitosan [ 5 ], and fluoride [ 6 ] are now being used in the treatment of enamel erosion. Fluoride, in particular, is widely used in the treatment of enamel erosion. However, the newly precipitated apatite crystals on the surface of the enamel differ significantly in morphology and arrangement from the structure of natural enamel. Furthermore, dental and bone fluorosis caused by excessive fluoride intake cannot be ignored [ 7 ].

Enamel remineralization may now be safely encouraged by new biomaterials. Over the past 40 years, research has confirmed the function of amelogenin proteins in the mineralization of enamel. The interaction of matrix materials with amelogenin and fragments to mediate the synthesis of HA is continually being investigated [ 8 ].

The main enamel matrix protein, amelogenin, is necessary for optimal enamel development and is thought to be crucial for controlling the nucleation, growth, and morphology of enamel mineral phases. Amelogenin consists of a hydrophilic C-terminal domain, a hydrophobic central domain, and a tyrosine-rich N-terminal domain [ 9 ]. Studies have confirmed that the N- and C- termini domains are highly conserved and are crucial for the development of healthy enamel [ 10 ].

Full-length amelogenin was found to be less abundant during enamel development, mostly as an enzymatically cleaved polypeptide fragment [ 11 ]. Our previous study revealed that a recombinant amelogenin peptide (consisting of the C- and N- termini) induces remineralization of demineralized enamel. The peptide may serve as a calcium ion carrier and regulator for the formation of directly ordered crystals. This calcium carrier was associated with the S-16 monophosphate group (at the N- termini) in this peptide [ 12 ].

Tyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix that is the N-termini domain of amelogenin. TRAP contains all the self-colonizing ‘A-domains’, the only phosphate group, and an exogenous lectin binding motif ‘PYTSYGYEPMGGW ' of amelogenin [ 13 ].

Studies have shown that TRAP, like this peptide (consisting of the C- and N- termini), can encourage the remineralization of demineralized enamel [ 14 ]. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth at a nanoscale level on an enamel surface has not been reported.

According to previous studies, changes in surface morphology are evident during enamel demineralization and remineralization [ 15 ]. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods were used in this study to examine enamel surface morphology, calcium-phosphorus ratio and surface roughness. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics. Therefore, in this study, the effect of TRAP on the morphology and structure of crystal growth on eroded enamel surfaces was investigated, thus providing further evidence of the important role of TRAP in enamel remineralization.

Materials and methods

Peptide synthesis and characterization.

TRAP was synthesized by standard solid-phase peptide synthesis from Synpeptide Co., Ltd., (Nanjing, China) that consists of residues (MPLPPHPGHPGYINFSPYEVLTPLKWYQNMIRHPYTSYGYEPMGGW). High-performance liquid chromatography (RP-HPLC) was used to purify it, and mass spectrometry was used to characterize it. The lyophilized peptides are dissolved in 4 mg/ml microporous purified water and stored at 4℃. Centrifuge the peptide stock solution before use (10,900 x g, 4 °C, 20 min).

Sample preparation

Extracted human teeth were collected according to the guidelines approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (2021-KY-1050-002). Premolars extracted for orthodontic reasons were obtained from the Department of Dentistry of the First Affiliated Hospital of Zheng University in the age group of 18–30 years. After extraction, soft tissue debris was removed, examined for fissures, dysplasia, and white spot lesions, and teeth were sterilized by cleaning with 5% NaClO for 1 h. A vertical incision was made along the buccal crown of the sample to obtain an enamel block 3 × 3 × 2 mm in size. The surface was smoothed with water-cooled silicon carbide paper of 400, 600, 800, 1000, 1200, 1500, 2000, 2500, 3000, and 4000 grit (Buehler Ltd.). The buccal surface was ultrasonically cleaned for 10 min to remove impurities. Randomly selected 15 samples and obtained baseline surface roughness, EDXS data, and raw image data of enamel blocks using SEM, AFM, FT-IR, and XRD prior to the formation of acid eroding. After that, all enamel samples were kept at 4 °C in a deionized water solution.

Eroded enamel preparation

Apply a gel of 37% phosphoric acid to the buccal surface of enamel blocks for 30 s and wash the surface of enamel blocks thoroughly with deionized water (DDW) [ 6 ]. After naturally drying, a double layer of acid-resistant nail polish was applied to the rest of the region, except the buccal surface. In the same way as before acid eroding, randomly selected 15 samples and obtained the enamel’s surface roughness, EDXS data, and image data of enamel blocks using SEM, AFM, FT-IR, and XRD after the formation of acid eroding.

pH-cycling regime

Standard pH-cycling was performed according to a previously reported protocol [ 16 ]. Randomly selected 45 samples and divide the enamel blocks into 3 groups of 15 samples each. The groups were divided as follows: Group 1: DDW group (negative control group); Group 2: 100 µg/mL tyrosine-rich amelogenin peptide (TRAP); Group 3: 2 ppm NaF (positive control group). Three research groups had their enamel samples submerged in the treatment solution four times a day for 10 min each at 8:00, 9:00, 15:00, and 16:00. The acid challenge was carried out by immersion for two hours between 11:00 and 13:00 in a demineralization solution containing 2.2 mmol/L Ca(NO 3 ) 2 , 5 mmol/L NaN 3 , 50 mmol/L acetic acid, and 2.2 mmol/L KH 2 PO 4 (pH = 4.5). Samples were submerged in the remineralization solution for the remaining period, which included the following ingredients: 5 mmol/L NaN 3 , 20 mmol/L HEPES, 130 mmol/L KCl, 0.9 mmol/L KH 2 PO 4 , and 1.5 mmol/L CaCl 2 (pH = 7.0) [ 17 ]. In a tank that was sealed and continuously stirred with a low-speed magnetic stirring at 100 rpm while keeping the temperature at 37 °C, the cycle was repeated for 14 days. The samples were carefully rinsed with deionized water following each procedure. Prepare and replace fresh solutions daily. After pH-cycling for 14 days, washed well with deionized water and allowed to air dry naturally.

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDXS)

After pH-cycling remineralisation, five samples were randomly selected from each group to examine the surface crystal using a Scanning electron microscopy (SEM, Gemini 300 M, Zeiss, Germany). Using an Oxford Quorum SC7620 sputter coater at 10 mA, the samples were collected, adhered directly on the conductive adhesive, and coated with gold for 45 s. The surface morphology of the enamel mass was then photographed using the SEM at 3 kV of accelerating voltage. Energy spectrum mapping tests were carried out with a 15 kV EDXS detector (Smartedx detector, Germany) to The Ca and P ratios (At. %) in the samples after treatment with remineralizes were assessed. Data were calculated as mean and standard deviation and expressed as mean ± standard deviation. On the basis of these EDXS data, potential mineral phases were predicted. On the basis of these EDXS data, potential mineral phases were predicted.

Atomic force microscopy (AFM)

After pH-cycling remineralisation, five samples were randomly selected from each group to observe the surface morphology of the enamel using an atomic force microscope (Bruker Dimension Icon AFM, Germany) and to determine the surface roughness. The samples were fixed on slides and five loci were randomly selected for each sample and photographed in intermittent contact mode using an AFM instrument with a scanning area of 30 μm x 30 μm. NanoScope Analysis software was used to analyze the images and the origin software to plot them.

Fourier transform infrared spectroscopy (FT-IR)

After pH-cycling remineralisation, two samples were randomly selected from each group, and the surface crystals were analysed for composition using a Fourier transform infrared spectrometer (FT-IR, Nicolet iS10, USA). In a dry environment, the ATR accessory was placed in the optical path of the IR spectrometer, the air background was scanned, and the sample surface was pressed against the crystal surface of the ATR accessory, and then the infrared spectra of the sample were tested and captured in the wave number range of 2000 –500 cm − 1 , with a resolution of 4 cm − 1 , and the number of scans was 32.

X-ray diffraction (XRD)

After pH-cycling remineralisation, three samples were randomly selected from each group, and the mineral phase of the new crystals were analysed for composition using a X-ray diffraction analysis(Rigaku Ultima IV, Japan). By using CuKa (λ = 1.5418 Å) radiation at 40 mA and 40 kV, the samples were examined using XRD. Data were gathered over a 2Ɵ angle range of 20–70°at a scan rate of 350 s/step and in 0.02° steps. The chemical phases were indexed using the International Centre for Diffraction Data (ICDD, PDF-2 release 2004 version). Diffractograms were analyzed using MDI Jade v6.0 and plotted using origin software.

Statistical analysis

All data were statistically analyzed using SPSS 21.0 (IBM, Chicago, IL, USA) and plotted using origin software. The enamel surface roughness data before and after acid eroding and after remineralization were compared by t-test. After remineralization, statistical significance between groups was assessed using analysis of variance and Bonferroni correction. P  < 0.05 was used as the significance criterion.

SEM morphology

Figure  1 showed the surface morphology of the enamel samples. The surface was analyzed for elemental composition using EDXS. Newly deposited crystals were observed in all groups, but their morphology, growth direction, and distribution on the enamel surface differed after pH-cycling for 14 days. The intact enamel surface was flat and a few scratches were visible. EDXS investigation of the surface’s elemental composition showed a Ca/P ratio of 1.66 ± 0.06 (Fig.  1 A, B). The eroded enamel surface had an overall fish-scale appearance, with a preferential loss around the enamel prisms, protrusion of demineralized prismatic nuclei towards the original enamel surface, and discontinuity and fragmentation of crystals at the surface. The enamel surface was analyzed for elemental composition and content using EDXS and a Ca/P ratio of 1.61 ± 0.08 was obtained (Fig.  1 C, D). In all three groups, the enamel surface had a coating of precipitated crystals after pH-cycling remineralization. In contrast to the other groups, the surface of the enamel in the DDW group was characterized by large holes and pits, which were broadly visible as enamel prisms, with no obvious restoration of the interstitial enamel rods. Loose and disordered accumulations of fragmentary short rod-shaped crystal particles were visible on magnification (Fig.  1 E). The Ca/P ratio was determined by elemental analysis to be 1.61 ± 0.12, probably a mixed mineral composition of calcium-phosphate transition phases and HA (Fig.  1 F) [ 15 ]. The remineralized enamel surface of the TRAP group was relatively flat, the precipitates were connected in sheets and covered some of the pores and pit areas. The damaged enamel interstices were well repaired, and a few pits were visible. On magnification, many new, densely arranged rod-like crystals were visible on the surface, parallel to each other, regularly arranged and forming an ordered structure, closely mosaic with the enamel crystals at the base. It was difficult to distinguish between the natural enamel and the new crystals in terms of morphological and structural features, suggesting that the new crystal morphology is similar to that of the natural enamel crystals (Fig.  1 G). The Ca/P was 1.65 ± 0.23, close to the ideal HA ion ratio of 1.67 (Fig.  1 H) [ 15 ]. Fluoride addition increased the extent of enamel surface repair. The surface of the enamel was flat and the pits were largely invisible (Fig.  1 I). Magnification of the rougher areas reveals a looser arrangement of relatively regular columnar crystals. Magnification of the flat areas shows that the gaps between these columnar crystals were filled with a scattered distribution of short rod-shaped crystals. The NaF group had Ca/F of 11.40 ± 1.88 and Ca/P of 1.53 ± 0.06 (Fig.  1 J). According to an elemental investigation, the crystals were probably composed of fluorohydroxyapatite (FHA) and CaF 2 [ 18 ].

I SEM images and EDXS analyses of the surface of the enamel. Intact enamel: (A , B) . eroded enamel: (C , D) . DDW group: (E , F) . TRAP group: (G , H) . NaF group: (I , J)

AFM morphology

For the AFM images, the surface of the intact enamel was smooth, with some fine scratches visible. The 3D image showed some areas of roughness (Fig.  2 A, B). The roughness was 21.10 ± 8.19 nm. After acid eroding, the enamel surface exhibited a high degree of surface porosity. It showed the classic fish-scale surface morphology of demineralized enamel and the typical prismatic structure of enamel (Fig.  2 C, D). This change in morphological aspect was also manifested in the change in surface roughness, which increased significantly from 21.1 nm to 358.6 nm. After pH-cycling remineralization, all three groups had a deposit on the enamel surface. Damage was filled in and repaired, the height difference between the enamel and internal rods was reduced and the roughness was significantly reduced. The AFM 2D image of the DDW group showed a heterogeneous distribution of color on the enamel surface, indicating a highly undulating surface (Fig.  2 E). The AFM 3D image showed a phenomenon resembling a fish scale after acid eroding. The enamel surface was deposited with a layer of disordered minerals and was overall uneven and mountainous in appearance. The surface pores had been repaired to some extent, but their depth was still large (Fig.  2 F). The surface roughness was significantly lower (114 ± 21.13) compared to after acid eroding. On the basis of the SEM images, the AFM images also provide further evidence of the effect of TRAP on enamel remineralization. The AFM 2D image of the peptide TRAP group showed a more even distribution of color, indicating a flatter surface of the enamel (Fig.  2 G). The AFM 3D image showed a typical deposit layer on the enamel surface, with a large number of closely spaced protruding rod-shaped crystal structures. The enamel rods were slightly visible, but the pores were well-filled. The structure of the new crystals was similar to that of natural enamel. Both enamel rods and interstitial enamel rods were repaired (Fig.  2 H). The surface roughness increased to (76.8 ± 22.1). The AFM 2D and 3D images of the NaF group showed a flat surface. This demonstrated better repair of enamel rods and interstitial enamel rods (Fig.  2 I, J). The surface roughness increased to (73.72 ± 15.98).

I AFM images of the surface of the tooth enamel. Intact enamel: (A , B) . eroded enamel: (C , D) . DDW group: (E , F) . TRAP group: (G , H . NaF group: (I , J)

A comparison of the roughness values of the surface of intact, eroded, and remineralized enamels showed that the eroded enamel surface roughness increased significantly than the intact ( p  < 0.05) and the remineralized enamel surface roughness reduced significantly than the remineralized ( p  < 0.05). The differences between the two groups were statistically significant ( p  < 0.05), except for the TRAP and NaF groups ( p  > 0.05) (Fig.  3 ).

I SD bars labeled with different letters show a statistically significant difference, P  < 0.05

FT-IR Spectra and XRD Spectra

The FT-IR (Fig.  4 ) and XRD (Fig.  5 ) spectra show that the nascent mineral phases of the DDW and TRAP groups are mainly HA. The nascent mineral phases of the NaF group are mainly FAP. After pH-cycling, the FT-IR (Fig.  4 ) spectrum shows a clear PO 4 3− band (597.97 cm − 1 and 986.59 cm − 1 ) indicating the formation of HA. In addition, the results of the peptide TRAP group showed that amide I (1650 cm − 1 ) was detected. The results of the NaF group showed that a new wave peak (742 cm − 1 )was detected. This is due to the F − substituted OH. The XRD spectra of the nascent crystals after remineralization are shown in Fig.  5 , indicating the presence of apatite crystals. The diffraction peaks at 2Ɵ = 25.8°, 32.2° and 34.1° corresponding to the reflections of HA (002), (112), and (202) are observed in the HA diffraction bands for all sets of results. Since the XRD spectra did not reveal CaF 2 diffraction bands, combined with the results of the EDXS, we infer that the crystals of the NaF group are FHA.

I FT-IR images of the surface of the buccal surface of deionized water-treated demineralized enamel, TRAP-treated demineralized enamel, and sodium fluoride-treated demineralized enamel after 14 days of pH-cycling, intact enamel and eroded enamel

The pH-cycling model was used for the first time in this study to investigate the changes in surface morphology after the remineralization of eroded enamel by the peptide TRAP. The results showed that there were significant differences in the surface characteristics of the three groups of test samples and that the peptide TRAP had a lower remineralizing effect on eroded enamel than sodium fluoride and a higher impact than DDW. The peptide TRAP can promote the remineralization of enamel.

We used the pH-cycling model reported by White DJ et al. to investigate the remineralization effect of TRAP on eroded enamel surfaces in this study. The model consists of a demineralizing solution and a simple remineralizing solution soaked in enamel samples, and the specimens were subjected to cyclic demineralization and remineralization using an artificial mouth to simulate the physiological state of the mouth. Remineralization to repair surface damage to demineralized enamel in the presence of fluoride or TRAP, using appropriately modulating Ca 2+ /PO 4 3− ion concentrations [ 16 ]. As a large amount of saliva is required to perform this study in an artificial mouth, a remineralizing solution was primarily chosen to replace human saliva. Mayumi Iijima used a cation-selective membrane model to compare the types of surface crystals formed after remineralization of demineralized enamel at different F − concentrations. At low concentrations of F − (< 2 ppm), the newly formed crystals were mostly OCP or a mixture of OCP and HA. At a concentrations of 2 ppm, FHA can be formed. The FT-IR and XRD results are also consistent with their results in this study [ 6 ].

The effect of TRAP on the surface of enamel after being eroded was compared with that of DDW and NaF by SEM. TRAP and NaF clearly showed the potential for remineralization, with changes at the level of surface morphology, maintaining a rough and homogeneous appearance. The enamel crystals were discontinuous and broken at the surface after being eroded, which is consistent with the typical pattern of type 2 demineralization and was confirmed by the Ca/P ratio results [ 19 , 20 ]. After the pH-cycling, apatite coatings were formed on the eroded enamel surfaces. The enamel surface of the DDW group was seen to be porous with a haphazard, scattered distribution of loose banded crystalline coatings, in agreement with the results of Sami Dogan et al. [ 21 ]. The enamel surface may deposit calcium and phosphorus ions derived from supersaturated solutions. Still, they are too weakly bound to the enamel substrate and are easily dislodged during pH-cycling remineralization. The enamel surface of the polypeptide TRAP group shows a structure similar to that of natural enamel. The border between the restored enamel and the eroded enamel reflects the successful growth of a new HA layer, with the new needle-like crystals arranged in parallel and tightly packed, growing in a manner consistent with the orientation of the existing enamel crystals. This organized crystal bundle was also observed in the previously reported remineralization pattern of the recombinant amelogenin polypeptide [ 22 ]. Previous research has demonstrated that one of the most crucial properties the material should possess for enamel remineralization is a significant affinity with the substratum [ 23 ]. In a previous study, we found that TRAP has a good affinity for calcium ions and binds to them [ 14 ]. In our study, the FT-IR test results for the treated demineralized enamel samples in our investigation showed typical peaks of the TRAP peptide, showing that TRAP had been adsorbed on the enamel surface. As a result, we propose that TRAP may help remineralize enamel by interacting with Ca 2 +  to produce a TRAP-ACP complex that strongly binds the developing ACP to the enamel and subsequently transforms it into HA. The enamel surface of the NaF group was flat and formed a large number of relatively regular columnar crystals. This is mainly due to the fact that the addition of F - increases the crystalline properties of HAP and the structure of the HA molecule becomes more dense. This is similar to the results of Longjiang Ding et al. [ 15 ].

According to the AFM images (Fig.  3 ), after the pH-cycling, remineralization caused the deposition of crystalline material and a decrease in surface roughness, which greatly decreased the depth of the erosion cavities. PH-cycling resulted in a distinct surface layer on the surface of the TRAP and NaF groups enamel, whereas no surface layer was evident on the surface of the enamel treated with DDW only. The AFM images of the DDW group showed minimal morphological changes on the enamel surface compared to the eroded enamel surface, which still showed a porous fish scale structure. This is similar to the results of Lippert et al. [ 24 ]. Lesions in the TRAP and NaF groups were significantly shallower than in the DDW group. These results provide direct evidence that TRAP and NaF promote remineralization. The enamel surface roughness was similar for the TRAP group and NaF group after pH-cycling. However, combined with SEM and AFM topographic analysis, the enamel surface was flatter and had more tissue recovery in the NaF group. On the enamel surface of the polypeptide TRAP group, the crystals showed typical nano-HA microcrystals with a needle-like morphology, arranged in parallel and densely packed along the c-axis, forming the prototype of enamel prismatic bundles. The enamel prismatic crystals are regularly aligned in the same direction and grow in a manner consistent with the existing orientation of enamel crystals, successfully mimicking the texture of natural enamel. This may be related to the fact that TRAP contains the exogenous lectin-binding motif “PYTSYGYEPMGGW”, “A-domain” of amelogenin self-assembly, and the unique phosphate group of amelogenin. Combined lectin properties may have a function to determine the direction of the “nanosphere”, which will affect the orientation of the microcrystals [ 25 ]. TRAP contains all the “A-domain” of the amelogenin self-assembly. Additionally, in vitro and in vivo studies have shown that the “A-domain” is removed, which interferes with amelogenin self-assembly [ 26 , 27 ]. This suggests that TRAP can self-assemble to form ‘nanospheres’. When HA crystals adsorbed on particular surfaces, the acidic amino acids may have controlled how the crystals were oriented [ 28 ]. The phosphate group plays an important role in stabilizing ACP, inhibiting its premature precipitation, and regulating the formation of ordered hydroxyapatite crystals, as verified in results obtained by comparing naturally phosphorylated full-length (P173) and dephosphorylated P173 and LRAP (+ P, -CT) and LRAP (-P, -CT) [ 29 , 30 ].

Combined with the results of elemental analysis, the FT-IR (Fig.  4 ) and XRD (Fig.  5 ) Spectra indicate that the mineral phases of the DDW and TRAP groups are predominantly HA. The NaF group is predominantly FAP. The remineralized coating of the TRAP group contains protein and phosphate, as shown by FT-IR analysis. The peptide TRAP could adsorb on the enamel surface. By combining the findings from the AFM, SEM, and FTIR experiments, we now propose that TRAP can depend on the interaction of phosphate groups with Ca 2+ to form TRAP-ACP complexes and temporarily stabilize ACP, which can then encourage the deposition of phosphorus and calcium on the enamel surface and also enter the interior of the lesion through the pores of demineralized enamel lesions, eventually resulting in their transformation into hydroxyapatite crystals. As a result of the F - substituted OH, the original hydroxyl vibrational mode changes, and splits, so that a new peak appears at 742 cm − 1 in the NaF group.

I XRD images of the buccal surface of deionized water-treated demineralized enamel, TRAP-treated demineralized enamel, and sodium fluoride-treated demineralized enamel after 14 days of pH-cycling, intact enamel and eroded enamel

The XRD results show that the diffraction peak positions are essentially the same for all three sets of samples, indicating that the addition of TRAP and fluorine did not affect the mineral phase and that the crystals of HA remained essentially unchanged in their dense hexagonal structure. The increase in the spreading peak at 2Ɵ = 20–22° may indicate the presence of amorphous calcium phosphate in the TRAP group [ 31 ]. This spreading may also be the result of the presence of smaller apatite crystals. The XRD results for NaF partially shift the reflection peak to the right, a reflection pattern similar to that previously reported for fluorohydroxyapatite [ 32 ]. Because when a HA layer is developed on a layered OCP precursor in the presence of F ions, as in the structure described by Iijima et al., this shift may represent an overlap of fluorinated HA and OCP [ 6 ].

These results indicate that the TRAP peptide remineralized the eroded enamel more efficiently than DDW but less efficiently than NaF. However, in the TRAP peptide group, after the remineralization of eroded enamel, the crystals deposited on the surface of the enamel have the same morphology and growth direction as normal enamel.

Conclusions

This study demonstrates that the peptide TRAP can promote the remineralization of eroded enamel surfaces. TRAP binds to the enamel surface, temporarily stabilizes ACP, controls further crystallization of HA on the original enamel crystals, promotes the remineralization of eroded enamel, and restores the original enamel surface. It is suggested that it has significant potential for remineralization, and provides a promising biomaterial for the remineralization treatment of enamel lesions.

Data availability

Upon reasonable request, the corresponding author will provide the datasets used and/or analyzed in the current work.

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Our study was supported by the National Natural Science Foundation of China (No.U2004108) and the Henan Province’s Health Science and Technology Innovation Leading Talent Cultivation Program for Middle-aged and Young Professionals in 2023. (No. LJRC2023006). The funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to publish.

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MW and JC took part in the design of this study, and they both conducted the data analysis. MW and QB carried out the experiment and collected important background information. Yi L and Ya L provided assistance for data collection and analysis. DM and Ya L carried out a literature search, data collection, and manuscript editing. All authors reviewed the manuscript.

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Wen, M., Bai, Q., Li, Y. et al. Morphological study of remineralization of the eroded enamel lesions by tyrosine-rich amelogenin peptide. BMC Oral Health 24 , 1054 (2024). https://doi.org/10.1186/s12903-024-04777-7

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The use of fluoride for promoting oral health has always involved a balance between the protection against caries and the risk of fluorosis. Dental fluorosis, a developmental condition of enamel, results from excessive intake of fluoride during the tooth development period. Dental fluorosis is the most common adverse effect of fluoride use in prevention of dental caries. The public health importance of dental fluorosis lies in its role as the canary in the coalmine , that is, a population indicator of excessive fluoride exposure. Dental fluorosis is an important aspect of oral health because (a) scientific evidence has recently elevated dental fluorosis to prominence as the adverse outcome associated with fluoride use; (b) public opinion on the safety of fluoride use now routinely includes dental fluorosis as a concern; and (c) recommendations about the use of fluoride should be based on evidence of a risk-benefit trade-off between a preventive benefit against dental caries and a risk of having fluorosis. Research on various aspects of dental fluorosis is important to inform oral health policies.

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Dental Fluorosis: the Risk of Misdiagnosis—a Review

Fluoride Agents and Dental Caries

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Do, L.G., Ha, D.H. (2021). Dental Fluorosis: Epidemiological Aspects. In: Peres, M.A., Antunes, J.L.F., Watt, R.G. (eds) Oral Epidemiology. Textbooks in Contemporary Dentistry. Springer, Cham. https://doi.org/10.1007/978-3-030-50123-5_7

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• Published: 27 September 2013

The role of teeth in human evolution

• G. H. Sperber 1  

British Dental Journal volume  215 ,  pages 295–297 ( 2013 ) Cite this article

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Highlights that the chemical composition of teeth and calculus provide evidence of the dietary components of past and present populations.

Stresses the genetic and cultural relationships between generations of the hominin lineage can be elucidated from dental data.

A review of recent insights into palaeodiets provided by new dating techniques, spectroscopy and attritional wear of enamel in ancient and recent human fossils. Fossilised dental plaque reveals changing dietary content and varying oral microbiota between Neolithic and Industrial era populations. DNA analysis of ancient dental pulpal tissue provides evidence of contemporary hereditary relationships and gene flow of human populations.

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Windows into the past: recent scientific techniques in dental analysis

Dental calculus - oral health, forensic studies and archaeology: a review

Multipronged dental analyses reveal dietary differences in last foragers and first farmers at Grotta Continenza, central Italy (15,500–7000 BP)

The field of human evolution is not normally considered within the scope of dentistry, yet the very same organs upon which dentistry depends provide much of the essential evidence for human evolution. The changing anatomy of teeth over the eons reflects the changing diets dictated by the altering ambient environments in which modern mankind's predecessors survived.

That teeth are composed of the most durable tissues, enamel and dentine, ensures their postmortem survival long after all other tissues have decayed, or have been fossilised. In fact, dental enamel is a bioceramic fossilised tissue existing within us, having had its organic matrix nearly totally replaced by minerals, which, in essence, is the process of fossilisation.

It is paradoxical that the durable postmortem survival of teeth is most susceptible to decay during life, providing the dental profession its raison d'être .

The dentine component of teeth is that of ivory, surprising most people that their mouths are filled with ivory. Accordingly, the preparation of teeth for dental restorations makes dentists 'ivory carvers' on living subjects, but resembling artists sculpting ivory carvings. Finally, the pulp of teeth has become an invaluable resource of stem cells for regenerative medicine. 1 , 2 , 3

The preservation of the dental pulp postmortem by virtue of its entombment within hard tissues has provided the resource for DNA identification of Neanderthal genomes. 4

Teeth are unique among organs by allowing direct comparison between extant and fossilised specimens formed millions of years apart. Teeth, by their postmortem persistence, depict their genetically inherited patterns, and thus their evolutionary history, more accurately than any other organs.

The incremental deposition of enamel matrix during amelogenesis provides histological evidence of differing chronological deposition rates between apes, hominids and hominins. 5 Moreover, the different rates of body maturation between these different genera has been evinced from dental data. The rapid growth of Neanderthals compared to modern humans has been deduced from comparative incremental dental data. 6

Apart from the genetic evidence to be gleaned from dental morphology, significant information has been obtained about the nature of the diet, and thereby, indirectly, the cultural and evolutionary status of the propositi. 7 Fossilised dental calculus entrapping pollen phytoliths and silica bodies from plants reveal the composition of food eaten two million years ago. 8 Analysis of ancient calcified dental plaque revealed the dietary changes between the Neolithic and Industrial Revolutions. The changes in the oral microbiota and dietary shifts between Neolithic and Industrial era populations have been revealed by sequencing ancient calcified dental plaque ( Figs 1 , 2 ). The shift in microbial populations was concurrent with the new widespread availability of processed sugar and flour, leading to increased dental caries and gingivitis. 9 , 10

a) The right maxilla of sample 8,482 from the medieval cemetery of the UK population Jewbury, with a large deposit of buccal calculus over maxillary molar teeth; b) Another calculus specimen from the same population showing incremental growth lines. Magnification ×50 (SEM) c) Further calculus specimen containing a colony of unidentified rod-shaped bacteria. Magnification ×3,000 (SEM). (By kind permission of Dr K. Dobney and Nature Genetics )

(By kind permission of Dr Alan Cooper and The Scientist )

Further details of the diets of hominids and hominins have been extracted from dental enamel by microwear texture analysis 11 , 12 and by isotopic analysis of enamel, revealing fundamental dietary changes in the Pliocene and subsequently. 13 , 14 , 15 , 16 Using laser ablation mass spectrometry on dental enamel slivers allowed determination of dietary plant origins of isotopic 13 C on three million year-old Australopithecus bahrelghazali individuals. Comparison with contemporary dietary plant resources revealed an early and fundamental shift in hominin dietary intake, explaining the exploration of new habitats 17 and indicative of palaeoclimate change.

The advanced technology of electron spin resonance in combination with uranium series isotopic analysis and infrared/post infrared luminescence dating applied to a 500,000-year-old mandible from Serbia established the existence of a Middle Pleistocene hominin ancestral to both Neanderthals and modern humans ( Fig. 3 ). 18 , 19 , 20 DNA analyses of the teeth of 7,500-year-old and subsequent populations revealed the prehistoric gene-flow from Siberia in the complex human population history of North East Europe. 21

a) lateral view, b) medial view, c) occlusal view, d) from left to right: M1, M2, M3. Scale in centimetres. (By kind permission of Dr M. Roksandic and the Journal of Human Evolution )

DNA analysis of genetic identification of deceased individuals and their possible inherited relationship to living relatives is fundamental to forensic investigations. Insights into deceased historical figures and their identification by DNA analysis is being applied to the skeletal remains of the late King Richard III. 22 , 23 Detailed isotopic and radiological analyses of the oral lesions of the Neolithic Iceman, circa 3,300 years BCE, have revealed astonishing palaeopathological information on the life and death of the Iceman. 24

Dental caries undoubtedly existed at the dawn of human history, but was exceedingly rare among australopithecines and later hominins. 25 The earliest known direct evidence of a dental filling in a 6,500-year old Neolithic human tooth was reported by Bernardini et al . 26

The revelations of distant human ancestry revealed by teeth provide dentists, as potential recorders of our past, an expanded role in the practice of their profession. 27 While the speciality of palaeodontopathology already exists, the speciality of 'Palaeodentoanthropologists' could be added as an additional attribute to the profession's already large spectrum of expertise.

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Sperber, G. The role of teeth in human evolution. Br Dent J 215 , 295–297 (2013). https://doi.org/10.1038/sj.bdj.2013.878

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DOI : https://doi.org/10.1038/sj.bdj.2013.878

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The use of silver diamine fluoride to prevent/treat enamel carious lesions: a narrative review

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• 1 Restorative Dental Science, College of Dentistry, Imam Abdulrahman bin Faisal University, Dammam, Eastern Province, Saudi Arabia.
• PMID: 39210918
• PMCID: PMC11361260
• DOI: 10.7717/peerj.17897

This comprehensive literature review examines the use of silver diamine fluoride (SDF) for the prevention and treatment of enamel carious lesions. SDF has been approved by different international drug associations as a caries-preventing agent to be used on deep carious lesions (dentin). However, SDF can cause staining of exposed tooth structures. Furthermore, the effect of SDF on the bond of adhesives to the tooth structure is still being determined. This review explores various studies on the use of SDF to treat enamel carious lesions, highlighting its effectiveness and preventive action. The literature suggests that SDF inhibits bacterial growth, promotes remineralization, and does not negatively affect adhesive retentions. Potassium iodide (KI) or glutathione (GSH) can reduce staining and discoloration. However, the reviewed studies have limitations. Further research, including well-designed clinical trials, is necessary to validate the findings and evaluate the long-term implications of SDF treatment. Conclusion: Despite the above-mentioned limitations, SDF shows potential as a therapy for enamel caries prevention, remineralization, and use as an adjuvant to other dental treatments, warranting further investigation and the refinement of application methods.

Keywords: Enamel carious lesions; SDF; Silver diamine fluoride; Tooth staining.

©2024 AlSheikh.

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Conflict of interest statement

The author declares they have no competing interests.

Figure 1. The effects of fluoride, silver…

Figure 1. The effects of fluoride, silver nitrate, and silver diamine fluoride on bacteria and…

Figure 2. Scanning electron micrographs of the…

Figure 2. Scanning electron micrographs of the enamel surface morphology.

Representative scanning electron micrographs of…

Figure 3. Transmission electron microscopy data of…

Figure 3. Transmission electron microscopy data of experimental groups.

SDF + NaF group: (A) morphology,…

Figure 4. Polarization light micrograph images of…

Figure 4. Polarization light micrograph images of post-demineralization and remineralization.

Representative polarization light micrograph images…

Figure 5. Scanning electron micrographs of enamel…

Figure 5. Scanning electron micrographs of enamel surfaces.

Scanning electron micrographs of enamel surfaces after…

• Abdelaziz M, Yang V, Chang NN, Darling C, Fried W, Seto J, Fried D. Monitoring silver diamine fluoride application with optical coherence tomography and thermal imaging: an in vitro proof of concept study. Lasers in Surgery and Medicine. 2022;54:790–803. doi: 10.1002/lsm.23528. - DOI - PMC - PubMed
• Akyildiz M, Sönmez IS. Comparison of remineralising potential of nano silver fluoride, silver diamine fluoride and sodium fluoride varnish on artificial caries: an in vitro study. Oral Health & Preventive Dentistry. 2019;17:469–477. doi: 10.3290/j.ohpd.a42739. - DOI - PubMed
• Alcorn A, Al Dehailan L, Cook NB, Tang Q, Lippert F. Longitudinal in vitro effects of silver diamine fluoride on early enamel caries lesions. Operative Dentistry. 2022;47:309–319. doi: 10.2341/20-237-L. - DOI - PubMed
• American Dental Association . CDT 2017 Dental Procedures Codes. Chicago: American Dental Association Publishing; 2017.
• Baiju RM, Peter E, Varghese NO, Sivaram R. Oral health and quality of life: current concepts. Journal of Clinical and Diagnostic Research. 2017;11:ZE21–ZE26. doi: 10.7860/JCDR/2017/25866.10110. - DOI - PMC - PubMed

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Effect of carbonated water manufactured by a soda carbonator on etched or sealed enamel

Hyo-kyung ryu.

Department of Orthodontics, Wonkwang University School of Dentistry, Iksan, Korea.

Yong-do Kim

Sung-su heo, sang-cheol kim.

The purpose of this study was to determine the effects of carbonated water on etched or sealed enamel according to the carbonation level and the presence of calcium ions.

Carbonated water with different carbonation levels was manufactured by a soda carbonator. Seventy-five premolar teeth were randomly divided into a control group and 4 experimental groups in accordance with the carbonation level and the presence of calcium ions in the test solutions. After specimen preparation of the Unexposed, Etched, and Sealed enamel subgroups, all the specimens were submerged in each test solution for 15 minutes three times a day during 7 days. Microhardness tests on the Unexposed and Etched enamel subgroups were performed with 10 specimens from each group. Scanning electron microscopy (SEM) tests on the Unexposed, Etched, and Sealed enamel subgroups were performed with 5 specimens from each group. Microhardness changes in different groups were statistically compared using paired t -tests, the Wilcoxon signed rank test, and the Kruskal-Wallis test.

The microhardness changes were significantly different between the groups ( p = 0.000). The microhardness changes in all experimental groups except Group 3 (low-level carbonated water with calcium ions) were significantly greater than those in the Control group. SEM showed that etched areas of the specimen were affected by carbonated water and the magnitude of destruction varied between groups. Adhesive material was partially removed in groups exposed to carbonated water.

Conclusions

Carbonated water has negative effects on etched or sealed enamel, resulting in decreased microhardness and removal of the adhesive material.

INTRODUCTION

Carbonated water, colloquially called sparkling water, is an aqueous solution of carbon dioxide. 1 Some of these have additives, such as sweeteners or fruit flavors, but plain carbonated water is composed mostly of water and carbon dioxide, with no other additives. For this reason, carbonated water is considered as a healthier beverage than conventional soft drinks and its consumption is increasing. According to a statistical survey on the production of food and food additives conducted by the Ministry of Food and Drug Safety in Korea, 2 carbonated water sales in 2014 saw a 100.63% increase from those in 2013, as compared to an 8.32% increase in conventional soft drink sales within the same period. Despite the increase in consumption, the general public is not well-informed of the negative effects of carbonated water on dental health.

Erosion of enamel is defined as the physical result of dental hard tissue being chemically etched away from the tooth surface by acid. 3 Previous studies have shown that dietary habits accompanied by excessive consumption of acidic foods and beverages increase enamel erosion. 3 , 4 , 5 , 6 A healthy enamel surface is an important factor for a favorable prognosis during orthodontic treatment; since erosion of the enamel can occur due to increased consumption of acidic drinks, it can negatively affect the outcome of orthodontic treatment, especially on teeth that have been etched for orthodontic appliance bonding.

Not only conventional soft drinks, but also plain carbonated water is an acidic liquid. In previous studies, commercial carbonated waters showed a wide range of pH (pH 4.18-5.87) 7 , 8 and some showed a pH level that is below the critical level, pH 5.5, which is required for enamel demineralization. Since appliances that can carbonate water easily, such as soda carbonators, have become more common recently, carbonated water can easily be prepared and consumed at home. Consumers can manufacture carbonated water with varying degrees of carbonation, and a high degree of carbonation results in a high level of acidity, which can be harmful to teeth. Some oral health officials are aware of this problem and have suggested that the intake of carbonated water containing calcium ions is better than that of carbonated water without calcium ions.

Numerous studies 9 , 10 , 11 , 12 on the negative effects of conventional soft drinks on etched and sealed enamel have been conducted, and have shown that the drinks could cause erosion of etched enamel and dissolve adhesive material; however, studies that focus on the effects of plain carbonated water in this respect remain scarce. The purpose of this study was to determine the in vitro influences of carbonated water on etched or sealed enamel in accordance with carbonation level and the presence of calcium ions.

MATERIALS AND METHODS

Specimen preparation and treatment.

This in vitro study was approved by the institutional review board at the Dental Hospital of Wonkwang University (No. WKDIRB201610-01). Seventy-five human premolars, extracted from young adults (age range, 20–29 years) for orthodontic purposes, were used in this study. Teeth with caries, erosion and/or other damage, or restorations were excluded. These teeth were obtained from various private practices and from clinics at the Wonkwang University Dental Hospital (Iksan, Korea). All teeth were cleansed and stored in normal saline solution. The roots of the teeth were cut off with a water-cooled, low-speed diamond saw for ease of use. The teeth were cleansed with nonfluoridated pumice and a rubber cup, thoroughly washed, and then air-dried. The left side of the buccal surface of each tooth was incisogingivally covered with melted acid-resistant wax (Sticky Wax; Kerr Co., Orange, CA, USA), and after cooling, the area was isolated from the test solution or artificial saliva during the experiment ( Figure 1A and 1D ). This established the Unexposed enamel subgroups prior to immersion of the teeth in the test solutions, and allowed comparison of the exposed surfaces. The right side of the buccal surface, i.e., the unwaxed part of each tooth, was etched with 37% phosphoric acid (DB-Etching 37; Denbio, Gwangju, Korea) for 20 seconds, followed by cleansing for 20 seconds, and drying for 10 seconds ( Figure 1B ). When the white, chalky surface appeared, Transbond XT Light Cure Adhesive Primer (3M Unitek, Monrovia, CA, USA) was applied on the cervical half of the etched surface, and this was then cured with an light emitting diode (LED) curing light (Ortholux; 3M Unitek) for 20 seconds ( Figure 1C ). Thus, the occlusal part of the etched surface was designated as the Etched enamel subgroup of each tooth, and the cervical part of the etched surface was designated as the Sealed enamel subgroup of each tooth ( Figure 1D ).

A soda carbonator with a 3-level LED fizz indicator (Source Sparkling Water Maker, SodaStream, NJ, USA), which is a popular commercial apparatus for manufacturing carbonated water, was used in this study. The LED fizz indicator shows the 3 stages of carbonation levels, by changes in LED lights when the carbonated level reaches a certain range. This was used to prepare carbonated water with different carbonation levels according to the manufacturer's instructions.

The tooth specimens were then randomly divided into 5 groups, in accordance with the carbonation level and the presence of calcium ions in the test solutions (i.e., a control group and 4 experimental groups):

Control group: Ultrapure deionized water

Group 1: Low-level carbonated water

Group 2: High-level carbonated water

Group 3: Low-level carbonated water with calcium ions

Group 4: High-level carbonated water with calcium ions

The product name, manufacturing method, and pH value of each test solution are listed in Table 1 .

Values of pH are presented as mean ± standard deviation.

Control group, ultrapure deionized water; Group 1, low-level carbonated water; Group 2, high-level carbonated water; Group 3, low-level carbonated water with calcium ion; Group 4, high-level carbonated water with calcium ion.

Ultrapure deionized water used in Group 1–4 is the same product of the Control group. Ultrapure deionized water was stored at a temperature of 5℃ right before manufacturing test solutions. Calcium ion was added as calcium chloride in Groups 3 and 4. The pH of each test solution was measured electronically every time those were made (Orion 3-Star Benchtop pH Meter; Thermo Scientific, Waltham, MA, USA).

Over a period of 7 days, all the specimens were submerged in each test solution for 15 minutes, 3 times a day, with 2-hour intervals. When the teeth were not submerged in test solutions, they were stored in artificial saliva at a constant temperature of 37℃. Before being submerged in the test solution, all the specimens were washed with ultrapure deionized water. The artificial saliva was prepared with 0.4 g NaCl, 1.21 g KCl, 0.78 g NaH 2 PO 4 ·2H 2 O, 0.005 g Na 2 S·9H 2 O, 1 g CO(NH 2 ) 2 , and 1,000 mL of distilled and deionized water in the laboratory, as described previously. 4 Then, sodium hydroxide was added to this solution until the pH value was measured electrometrically as 6.75. 4 The test solutions and artificial saliva were remade and refreshed at the beginning of every experimental session.

After the immersion procedure, the specimens were washed with ultrapure deionized water and the wax was removed mechanically as a whole block without any effect on the enamel surface.

Measurement of microhardness

After the immersion procedure, 10 specimens from each group were cross-sectioned mesiodistally to divide the tooth into occlusal and cervical specimens. The cross-section was made at the level of the occlusal third ( Figure 1E ). Each occlusal specimen was embedded in acrylic resin, such that the cut surface was exposed for the microhardness test ( Figure 1F ). The surface of each specimen was ground to achieve a favorable flat enamel surface, using 600, 1,000, 1,200 grit silicon carbide abrasive paper sequentially, followed by polishing with 0.05-mm alumina slurry. All the specimens were kept in ultrapure deionized water prior to the microhardness test.

Microhardness tests on the Unexposed enamel subgroup and the Etched enamel subgroup were performed using a Vickers indentor attached to a microhardness tester (MXT-70 Microhardness tester; Matsuzawa, Tokyo, Japan). Microhardness tests were conducted under a 100-g load for 20 seconds dwell time. Three indentations per test were made on the outer enamel surfaces which were 10, 55, or 100 µm apart, making rows toward the dentin. Three tests were performed on each subgroup ( Figure 1F ). Three rows of indentations were made with a distance of at least 300 µm. The length and width of each indentation were measured and the Vickers hardness number was calculated from the obtained values by the indentation apparatus of a measuring microscope. The microhardness was determined using the average value of 3 indentations per test and 3 rows were assessed for each subgroup.

Observation of specimen surface

After the immersion procedure, 5 specimens from each group were cross-sectioned incisogingivally to divide the tooth into buccal and palatal specimens ( Figure 1G ). Each buccal specimen was kept in a desiccator containing CaCl 2 for 3 days, affixed to scanning electron microscopy (SEM) stubs, coated with platinum twice with a 108 Auto Sputter Coater (Cressington, Watford, UK), and was then examined with a JSM-6360 SEM (JEOL, Tokyo, Japan) at 15 kV, at different magnifications.

Statistical analysis

The microhardness of each Unexposed enamel subgroup was also compared ( Table 2 ). The Shapiro-Wilks test found a lack of normality of the distribution of the microhardness of each Unexposed enamel subgroup. Thus, the nonparametric Kruskal-Wallis test was used for analysis, and showed no significant difference ( p > 0.05).

Values are presented as mean ± standard deviation.

* p < 0.05 in Kruskal–Wallis test; † p < 0.05 in paired t -test; ‡ p < 0.05 in Wilcoxon signed rank test.

Descriptive statistics were calculated for the microhardness of each subgroup. Statistical analyses were performed using the Predictive Analytics Software (version 18.0; SPSS Inc., Chicago, USA).

To assess microhardness changes of each group after the exposure to test solutions, microhardness of the Unexposed enamel subgroup and the Etched enamel subgroup were compared ( Table 2 ). Difference values between subgroups in the Control group and Groups 1, 3, and 4 were normally distributed. Paired t -tests were used for the Control group, and Groups 1, 3, and 4, while the Wilcoxon signed rank test was used for Group 2. The nonparametric Kruskal-Wallis test and Mann-Whitney U -test with Bonferroni correction were used as post-hoc comparisons to compare difference values between subgroups ( Tables 3 and ​ and4 4 ).

a Significantly different from Control group; b significantly different from Group 1; c significantly different from Group 2; d significantly different from Group 3; e significantly different from Group 4.

* p < 0.05 in Kruskal-Wallis test.

* p < 0.005 in Mann-Whitney test with Bonferroni correction.

Microhardness change

Descriptive data and statistical comparisons of microhardness between the Unexposed enamel subgroup and Etched enamel subgroup in each group are given in Table 2 , and microhardness changes in specimens after exposure to test solutions are given in Tables 3 and ​ and4. 4 . The results showed statistically significant differences between groups ( p = 0.000).

All experimental groups, except Group 3, showed greater statistically significant change than the Control group. High-level carbonated water (Group 2, 90.18 ± 33.16) resulted in greater statistically significant changes than low-level carbonated water, without or with the presence of calcium ions (Group 1, 49.57 ± 26.13; Group 3, 32.60 ± 19.83). High-level carbonated water with calcium ions (Group 4, 71.33 ± 38.28) resulted in a greater statistically significant change than low-level carbonated water with calcium ions (Group 3, 32.60 ± 19.83). Low-level carbonated water with calcium ions (Group 3, 32.60 ± 19.83) showed lesser statistically significant change than low-level carbonated water without calcium ion (Group 1, 49.57 ± 26.13). There was no significant difference in microhardness changes between high-level carbonated water with calcium ions (Group 4, 71.33 ± 38.28) and low/high-level carbonated water without calcium ions (Group 1, 49.57 ± 26.13; Group 2, 90.18 ± 33.16).

Specimen surface change

The enamel surface of the Unexposed enamel subgroup in each group was observed. All groups showed a smooth enamel surface ( Figure 2 ).

Demineralized areas were observed in each group that had been etched and exposed to test solutions. The etched enamel surfaces in Groups 1–4 lost more tissue than the Control group ( Figure 3 ). Dissolution of the peripheral prism was observed in the Control group ( Figure 3A ). Apparent dissolution of the prism core and a slightly flattened honeycomb-like appearance were observed in Group 1 ( Figure 3B ), and a flattened and agglomerated honeycomb-like structure was observed in Group 2 ( Figure 3C ). Dissolution of the prism core and a well-defined honeycomb-like appearance were observed in Group 3 ( Figure 3D ), while a fuzzy and worn honeycomb-like structure was observed in Group 4 ( Figure 3E ).

Different amounts of adhesive primer removal were observed on the sealed enamel surfaces ( Figure 4 ). Intact adhesive primer was observed in the Control group ( Figure 4A ). Adhesive primer with a roughened surface remained in Group 1 ( Figure 4B ) and some adhesive primer was stripped off ( Figure 4C ) in Group 2. The border of the adhesive primer was partially removed in Group 3 ( Figure 4D ) and the border of the adhesive primer was irregular and some part of it had disappeared in Group 4 ( Figure 4E ).

The objective of this study was to determine the in-vitro effect of carbonated water on erosion of the etched enamel and degradation of the adhesive material in accordance with the carbonation level and the presence of calcium ions. This study revealed that carbonated water with different carbonation levels affects etched and sealed enamel to varying degrees. Higher carbonation levels show a higher tendency for enamel erosion, and addition of calcium alleviates this tendency ( Table 3 ).

In this study, the waxed part of the teeth was used as the Unexposed enamel subgroups for comparing conditions after etching and exposure to test solutions. Prior to the experiment, we conducted a pilot study, which showed that coating and mechanical removal of the wax had no effect on microhardness and the surface structure of the enamel.

We designed this study to reproduce the oral environment in vivo , including the immersion time, protocol, and storage medium in intervals, based on previous studies. 11 , 12 Since meal time was assumed to be 15 minutes, the teeth used in the experiment were submerged in test solutions for 15 minutes at a time and in artificial saliva between experimental sessions to simulate the oral environment. This procedure was repeated 3 times a day to simulate every meal.

This study had some limitations because of the in-vitro study design. We considered that the post-eruptive age of teeth could affect enamel surface structures and microhardness. 13 Thus, the premolars extracted from young adults were used in this study. Nonetheless, exposure level to acidic beverages or food before extraction and its impact on the teeth could not be estimated. Furthermore, we used artificial saliva at a constant temperature of 37℃ to simulate the oral environment. To exclude the possibility of remineralization effects by calcium ions in saliva and to focus on the role of calcium ions in the test solutions, we used artificial saliva that did not include calcium ions. 5 Previous studies have shown that saliva has protective effects against demineralization of teeth, not only due to its buffering capacity, but also by enhancing ion-induced remineralization. 6 , 14 Thus, in the oral environment in vivo , with normal saliva secretion and function, protective mechanisms against demineralization might have a stronger effect than the in-vitro environment. Additionally, the effects of biofilms on the reactivity of the enamel in the oral environment could decrease demineralization by the carbonated water. The effects of carbonated water on the etched and sealed enamel must be further studied in vivo to be verified in an oral environment.

To assess dental erosion, the microhardness test and SEM test were used. Microhardness tests directly assess the condition of a tooth surface and quantify dental erosion. 15 SEM tests, on the other hand, allow visual observation of the enamel surface change. These 2 test methods are complementary.

Microhardness of teeth showed significant differences and different features were observed in SEM images. The result from this study showed that the teeth exposed to carbonated water had more enamel erosion than those of the Control group. Other studies 4 , 11 , 12 have analyzed the effect of acidic drinks, such as soft drinks on the teeth, but they have not mainly dealt with the effect of carbonated water. The microhardness values of teeth exposed to acidic drink were significantly lower than those of the control group. 16

Erosion of teeth exposed to carbonated water (Groups 1–4) as observed by SEM was greater than that observed in a previous study. 11 In a previous study, commercial sparkling mineral water was used, whereas carbonated water made by a soda carbonator was used in this study. In our pilot study, we found that carbonation levels vary by products similar to previous findings 8 , 17 and a soda carbonator can manufacture water with higher levels of carbonation. Accordingly, the carbonated waters used in this study were predicted to result in a higher tendency for enamel erosion than commercial sparkling waters.

The critical pH of demineralization, defined as the highest pH at which saliva crosses the saturation line, has been established as pH 5.5. 18 When exposed to an acidic solution with a lower pH than this critical pH, enamel dissolution is initiated. We used manufactured carbonated water of two different carbonation levels, and their pH levels were lower than the critical pH, even at the lowest carbonation level. Furthermore, in this study, the teeth were intermittently exposed to carbonated waters and artificial saliva during 7 days, and demineralization occurred even during this short period. Since the orthodontic treatment period is estimated to be about 1.5 years or more, carbonated water can cause greater harmful effects.

A greater amount of erosion was seen in the high-level carbonated water group than in the low-level carbonated water group. This tendency was not influenced by the presence of calcium ions. This demonstrated that higher levels of carbonation have a more destructive effect on enamel surface. In this study, higher levels of carbonation resulted in water with a more acidic pH value. However, this result might have been overestimated because the study design did not include a positive control. Nevertheless, it could provisionally be concluded, based on similar results for potential of erosion after consumption of acidic drinks that have previously been reported. 11 , 16 , 19

In particular, the amount of erosion differed significantly between Group 1 and Group 3, whereas there was no significant difference between Group 2 and Group 4. These results reflect that calcium ions reduced erosion of the enamel, such that the microhardness was decreased in Group 3 compared to the Control group, and in Group 4 as compared to Group 1. The presence of calcium ions influenced the dissolution equilibria of the dental enamel. Nonetheless, addition of calcium ions seemed to have a limited effect in high-level carbonated water. This result corresponded with that of a previous study. 20 In terms of assessing the effect of the calcium ions in carbonated water, the calcium ion concentration was adjusted to 100 mg/L that resulted in an approximately 50% reduction in dissolution of hydroxyapatite in the previous study 20 ; this reflects the highest calcium ion concentration among commercial mineral waters. Additionally, if we used human saliva, the calcium ions influence of on reduction of enamel erosion could be decreased due to remineralization. Therefore, further studies with human saliva are needed to confirm the role of calcium ions in carbonated water.

In a previous study, 21 calcium ions were released into the carbonated water after bovine teeth were immersed in carbonated water. The concentration of calcium ions from the bovine teeth increased over time and continued, and moreover detection of the calcium ion was performed after several hours of immersion in carbonated water. In this study, the immersion time was shorter than that in the previous study, 21 and the test solutions were remade and refreshed at the beginning of every experimental session to minimize the effect of the calcium ions released from the teeth. In addition, calcium ions were added as calcium chloride, based on a previous study, 20 because sodium chloride has been reported to have a negligible influence on the potential of erosion.

A SEM test showed significant features associated with these erosive tendencies. Phosphoric acid etchant changed the enamel morphology of the Etched enamel subgroup in the Control group, and this showed a typical etching pattern, type 2, as a predominant pattern. 22 When the etched enamel surface was exposed to carbonated water, this well-defined pattern was collapsed by the destructive effect of the carbonated water. The level of influence varied according to the carbonation levels and the presence of calcium ions. These observations accorded with the results of the microhardness test.

In Sealed enamel subgroups, the adhesive primer was also affected by carbonated water. The adhesive primer was roughened and partially removed in Groups 1–4, and some parts of the etched surface were revealed. In this context, the revealed etched surface had a more important significance in practice, because of demineralization. In the Sealed enamel subgroup, calcium ions had little effect on the protective effect of the adhesive primer.

During orthodontic treatment with fixed appliances, frequent intake of carbonated water could increase the risk of erosion of enamel around the brackets. Erosion of enamel around the brackets, which had been etched for bonding, 23 , 24 could cause dental caries and decrease the retention of the appliances. Even though the etched enamel had been sealed with adhesive material, carbonated water could strip off the adhesive and reveal the etched enamel. Therefore, clinicians should instruct orthodontic patients that carbonated water has negative effects on teeth, especially those with fixed appliances.

Carbonated water has negative, destructive effects on teeth, and result in decreasing microhardness and removal of the adhesive material on etched or sealed enamel. Erosion occurred when the etched enamel of teeth was exposed to carbonated water, particularly in groups exposed to high-level carbonated water. Alleviation of this destructive effect is observed in groups exposed to carbonated water with calcium ion. Partial removal of the adhesive material on sealed enamel could be observed after exposure to carbonated water.

This paper was supported by Wonkwang University in 2017.

The authors report no commercial, proprietary, or financial interest in the products or companies described in this article.