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. 2025 Sep 26;25:1458. doi: 10.1186/s12903-025-06763-z

Evaluation of the effectiveness of different remineralization agents on primary and permanent teeth exposed to acidic liquids: a randomized controlled in vitro study

Kübra Bilge 1,✉, İrem İpek 2, Büşra Karaagac Eskibaglar 2
PMCID: PMC12465249  PMID: 41013545

Abstract

Background

The aim of this study is to evaluate the effectiveness of different remineralization agents applied to primary and permanent teeth that were demineralized in acidic environments with varying pH levels, using scanning electron microscopy (SEM) and microhardness measurements.

Methods

In this study, 80 primary second molars and 80 permanent premolars were used and demineralization procedure was applied by keeping in gastric acid (pH = 1.2) and kefir (pH = 4.5) for 72 h. Then, samples were randomly divided into groups (n = 10) to be applied with different remineralization agents (Curodont Protect, MI Paste Plus, Tooth Mousse) for 14 days. Samples in the control group were kept in artificial saliva for 14 days. Microhardness measurements were made at the beginning, after the demineralization procedure and after the remineralization procedure from all groups and SEM analyses were performed on one sample from each group. Since the assumptions of parametric test were met in the evaluation of the data (Kolmogorov-Smirnov), two-way ANOVA, followed by the Tukey post hoc test. p < 0.05 was accepted as the significance value.

Results

The lowest microhardness values and the most eroded areas in SEM images were observed in primary teeth. When acidic liquids were evaluated, microhardness values decreased more in those immersed in gastric acid than at the initial. After the application of remineralization agents, the microhardness values of primary and permanent teeth increased the most in the Curodont Protect group and the least in the control group. In SEM images, the most eroded areas were observed in the control group in which no remineralization agent was applied after immersed in gastric acid in primary teeth.

Conclusion

Gastric acid is a stronger acid compared to kefir, which may explain its more erosive effect on both primary and permanent teeth. Curodont Protect, a biomimetic peptide-based product, can be preferred primarily in clinical use for erosive enamel treatment because it shows an effective remineralization ability on eroded enamel.

Keywords: Remineralization agents, Primary and permanent teeth, SEM, Microhardness

Introduction

Various factors can cause erosion of the enamel surface, the hardest tissue in the human body. Compared to dental caries, which is a microbial disease of the hard tissues of the tooth, dental erosion occurs through chemical reactions without microbial participation. Therefore, even in patients with good oral hygiene, it can occur without caries, leading to exposure of the dentin, which can cause sensitivity and discomfort. Although a critical pH value is necessary for the development of caries, enamel erosion caused by exposure to acidic beverages or substances can occur over a wider pH spectrum, making it a more complex process [1, 2].

There were many factors that caused the erosion that occurred on the enamel. Both external factors, such as acidic foods and drinks, and internal factors, such as stomach contents reaching the oral cavity due to reflux, regurgitation, or vomiting, can contribute to enamel erosion [3, 4]. Gastroesophageal reflux disease (GERD) is a disorder with an increasing prevalence of 10–30% in the world population and is a disorder that occurs when stomach contents reflux into the esophagus, oropharynx, or airway [5, 6]. GERD has a complex and multifactorial etiology. Some physiological changes such as changes in esophageal sphincter relaxation, gastric emptying, peristaltic functions, genetic predisposition, salivation, gastric acid secretion, and increased intra-abdominal pressure may predispose to GERD [7]. Gastric juice has a much higher potential to cause tooth erosion in both enamel and dentin than common extrinsic acids. When gastric juice entering the oral cavity as a result of GERD suppresses the protective effects of saliva, the pellicle on tooth surfaces is lost and teeth become susceptible to erosion [8, 9].

In recent years, dietary habits have gradually changed due to changes in lifestyle and increased availability of dietary acids. Consumption of acidic beverages such as cola, energy drinks, orange juice and kefir has become the most common cause of diet-related enamel erosion [10]. Saliva plays an important role in neutralizing oral acidity due to its buffer system of bicarbonate and ions. However, the frequency of acidic challenges can exceed the regulatory and repairing capacity of saliva, resulting in mineral loss on the tooth surface [11]. Kefir contains granules containing a specific mixture of symbiotic microflora, including lactic acid bacteria, acetic acid bacteria, and yeast cells, surrounded by a matrix of casein, complex sugars, and polysaccharides. Thanks to this content, kefir is a fermented product with mild acidity and a natural carbonation mechanism [12].

Early diagnosis of enamel lesions is crucial for implementing preventive measures in both children and adults [13]. Gastroesophageal reflux, a common condition seen across all age groups, is a disease characterized by the frequent return of stomach contents to the oropharynx [14]. Due to dietary habits and/or diseases like these, the oral cavity may become acidic, initiating the demineralization process on the enamel surface. To slow down this process, repair through remineralization is necessary [15]. Saliva plays a protective role against erosive factors due to its buffering mechanism against pH changes, its ability to form a protective layer on the enamel surface, its diluting effect, and its promotion of remineralization by supplying supersaturated minerals [16, 17]. In addition to saliva, which acts as a natural defense against dental erosion, remineralization agents produced by various companies are also used in treatment.

Among the biomimetic agents, self-assembling peptides have a high affinity for calcium. Additionally, their ability to diffuse into the micro-porosities beneath the enamel surface to promote remineralization has recently brought these agents into the spotlight [18]. The self-assembling peptide P11-4 has a three-dimensional scaffold structure. Its main mechanism is to promote biomimetic mineralization by enhancing hydroxyapatite formation [19]. Specifically, at a pH below 7.5, P11-4 forms ß-sheet and ribbon-like structures to facilitate remineralization [20].

Casein phosphopeptide (CPP) is a bioactive phosphoprotein derived from milk, which acts as a carrier for calcium and phosphate ions. It can deliver these ions to the tooth surface and maintain the concentrations of amorphous calcium phosphate and fluoride ions in saliva at acidic pH levels, thereby promoting more effective remineralization [21]. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is believed to prevent demineralization, enhance remineralization, or do both. Due to its remineralization potential, CPP-ACP is used in treating dentin sensitivity, dental erosion, and halting white spot lesions [22–24]. The effectiveness of fluoride in remineralization has also been known for many years. The combination of CPP-ACP and fluoride is thought to enhance remineralization through a synergistic effect [25].

Although there are many studies evaluating the effectiveness of different remineralization agents after application on artificial caries lesions [26] and bleached enamel surfaces [27], there is no study evaluating the effectiveness of different remineralization agents after acidic exposure on both primary and permanent teeth. The null hypothesis of this study was that there would be no significant difference between the effectiveness of the different remineralization agents in primary and permanent teeth after acidic challenges.

Material- method

Sample Preparation

This study was approved by the Firat University Non-Interventional Research Ethics Committee (2024/15) and was conducted in accordance with the Declaration of Helsinki. For the extracted teeth used in this study, informed consent forms were obtained from the patients. A similar previous study was taken as a reference [28] in the sample size calculation and was determined by performing power analysis with the G-Power program (Version 3.1.9.4, Heinrich Heine University, Düsseldorf, Germany). The sample size was determined by power analysis with the G-Power programme (Version 3.1.9.4, Heinrich Heine University, Düsseldorf, Germany). In this study, when α = 0.05, β = 0.10, and (1 − β) = 0.90, it was decided to take n = 10 samples for each group and the total number of samples to be N = 160 and the power of the test p = 0.8961640.

Following the sample size calculation, 160 teeth without caries, cracks, fractures, or any defects were selected for this study, consisting of 80 primary second molars and 80 permanent premolars. Each selected tooth underwent thorough ultrasonic cleaning and was polished using pumice with a polishing brush/rubber cup. To limit the study area, after the samples were air-dried, a 3 × 3 mm window was placed in the middle of the coronal buccal surface of each tooth. The remaining area was painted with nail polish and left to dry. The samples were kept in deionized water containing 0.1% thymol to prevent dehydration until the experimental stage, and the water was renewed at regular intervals. This solution was preferred because it was reported that there was no change in the microhardness of teeth kept in a solution containing thymol for a long time [29]. Then, 80 primary teeth were divided into two groups of 40 samples to be immersed in gastric acid and kefir, permanent teeth were divided into groups of 40 samples to be immersed in kefir and gastric acid. A schematic representation of this study design is presented in Fig. 1.

Fig. 1.

Fig. 1

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Schematic representation of study design

Demineralization procedure

In this study, gastric acid and kefir were used for the demineralization procedure (Table 1). The pH of the liquids was measured using a pH meter (pH-2005, JP Selecta, Spain). The pH measurements were taken using a pH meter calibrated between pH 2–12, with a pH probe placed in the prepared solutions until stable values were achieved. The prepared samples were then immersed in the liquids at 100% humidity and 37 °C for 72 h, with the liquids being replaced daily [30]. At the end of the 72 h, the samples were thoroughly rinsed with deionized water and left for a 24-hour washing period [31].

Table 1.

Acidic liquids used in the study

Liquids Manufacturer Contents pH
Artificial gastric acid Elazığ, Türkiye Hydrochloric acid (HCl) 0.06 M (0.113% solution deionized water) 1.2
Kefir Altınkılıç, İstanbul, Türkiye Pasteurized Cow Milk, Kefir Yeast, Kefir Culture 4.5
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Remineralization procedure

After demineralization, samples were subdivided according to the remineralization procedure (n = 10). According to the manufacturer’s instructions:

  • Curodont Protect (P11-4) was applied using a fine-tipped disposable microbrush at a thickness of approximately 1 mm for 2 min, twice a week, for 14 days.

  • MI Paste Plus (CPP-ACP with F) was applied using a fine-tipped disposable microbrush at a thickness of approximately 1 mm for 3 min, twice daily, for 14 days.

  • Tooth Mousse (CPP-ACP) was applied using a fine-tipped disposable microbrush at a thickness of approximately 1 mm for 3 min, twice daily, for 14 days.

  • Artificial saliva: The samples in this group were kept in artificial saliva, which was replaced daily for 14 days.

The technical profiles of the remineralization agents used in the study are shown in Table 2.

Table 2.

Chemical composition of the tested materials

Material Manufacturer Contents
Curodont Protect (P11-4) Credentis, Windisch, Switzerland 900 ppm fluoride (sodiummonofluorophosphate), 0.1% dicalciumphosphate, 0.028% calciumglycerophosphate, self-assembling peptide P11-4 (1,000 ppm)
MI Paste Plus (CPP-ACP with F) GC Europe N.V Leuven, Belgium 900 ppm Fluor, glycerol, propylene glycol,CPP-ACP, D-sorbitol, colloidal silica,sodium carboxymethyl cellulose, titaniumdioxide, xylitol, phosphoric acid, sodiumsaccharin, zinc oxide, magnesium oxide,ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, flavor, pure wate
Tooth Mousse (CPP-ACP) GC Europe N.V Leuven, Belgium Glycerol, propylene glycol, CPP-ACP, D-sorbitol, colloidal silica, sodiumcarboxymethyl cellulose, titaniumdioxide, xylitol, phosphoric acid, sodium saccharin, zinc oxide,magnesium oxide, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, flavor, pure water
Artifical saliva Elazığ, Türkiye 1.160 g/l sodium chloride, 0.600 g/l calcium chloride, 0.600 g/l potassium phosphate, 1.491 g/l potassium chloride, 0.050 g/l sodium flouride, trace of sodium hydroxide
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Microhardness tests

The microhardness values of the teeth were determined at three stages: initially, after the demineralization procedure, and after the remineralization procedure. For Vickers microhardness (VHN) analysis, a microhardness tester (Shimadzu, Tokyo, Japan) was used, applying a pyramid-shaped diamond indenter with a vertical static load of 100 g for 5 s. The diagonals of the rhombus-shaped indentation formed on the sample surface were measured under a ×40 magnification eyepiece. Measurements were taken from three different random points, 100 μm apart from each other, on each test sample. The arithmetic mean of these three measurements was recorded as the Vickers microhardness value.

SEM analyses

SEM photographs were taken from randomly one selected samples from each group at initial, after the demineralization procedure, and after the remineralization procedure. Since SEM analysis would be performed initially, and after both demineralization and remineralization procedures, the samples were not coated with gold. To enhance conductivity, the samples were wrapped with carbon tape and placed on the platform of the SEM device (Zeiss EVO MA10, Zeiss, Germany). Representative images from the most characteristic areas of the samples were obtained at 20 kV electric voltage and magnifications of ×1kx, ×2.5kx, and ×5kx, ×10kx before demineralization, after demineralization, and after remineralization.

Statistical analysis

The data obtained from the present study were evaluated with the SPSS 22.0 (Statistical Package for Social Science Version: 22) program. Since the assumptions of parametric test were met in the evaluation of the data (Kolmogorov-Smirnov), two-way ANOVA, followed by the Tukey post hoc test were used. p < 0.05 was accepted as the significance value.

Results

Microhardness values

Microhardness values ​​of primary and permanent teeth are given in Tables 3 and 4.When microhardness values ​​were compared, the initial values ​​of primary teeth were found to be lower than those of permanent teeth. The decrease in microhardness values ​​after immersed in gastric acid in both primary and permanent teeth was significantly greater than after immersed in kefir. In addition, microhardness values ​​increased after remineralization in both primary and permanent teeth. When evaluated in terms of remineralization agents, the highest increase was observed in the Curodont Protect group. When evaluated according to the microhardness parameter, the control groups had the lowest remineralization potential.

Table 3.

Microhardness values ​​of primary teeth

Initial Kefir Curodont
Protect 		MI Paste Plus Tooth Mousse Artificial saliva P value
280.11±15.37A 243.87±62.12B,a 282.45±32.98A,a 270.31±67.21C,a 263.45±42.14C,a 260.56±61.23C,a <0.05*
Initial Gastric acid Curodont Protect MI Paste Plus Tooth Mousse Artificial saliva
280.11±15.37A 217.42±34.65B,b 273.33±45.05A,a 270.54±28.76A,a 258.22±31.09C,a 223.69±56.70B,b <0.05*
p value <0.05*
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Note: Different capital letters indicate significance horizontally, and different lower case letters indicate significance vertically

Table 4.

Microhardness values of permanent teeth

Initial Kefir Curodont Protect MI Paste Plus Tooth Mousse Artificial saliva p value
334.76±34.91A 275.12±43.56 B,a 340.18±23.87A,a 315.54±78.44C,a 310.44±62.13C,a 287.30±37.01D,a <0.05*
Initial Gastric acid Curodont Protect MI Paste Plus Tooth Mousse Artificial saliva
334.76±34.91A 253.45±55.82 B,b 328.98±45.21A,a 320.66±62.49A,a 307.31±43.22C,a 267.19±38.72B,a <0.05*
p value <0.05*
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Note:Different capital letters indicate significance horizontally, and different lower case letters indicate significance vertically

SEM analyses

SEM images of permanent teeth at the initial, post-demineralization and post-remineralization are shown in Fig. 2, and SEM images of primary teeth are shown in Fig. 3. However, due to the large number of images, images of the groups with the strongest and weakest demineralization and remineralization efficiency from each group were included. In SEM analyses, an increase in enamel surface irregularities was observed in images obtained after demineralization compared to the initial state, while surface irregularities decreased after remineralization. The most pronounced erosive areas were detected in the groups immersed in gastric acid. While the remineralization in erosive areas was lowest in the control groups immersed in artificial saliva after demineralization, the groups with the highest remineralization were the Curodont Protect groups.

Fig. 2.

Fig. 2

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SEM images of permanent teeth (a: inital, a’: after immersed in kefir,a’’: after applying Tooth Mousse, b: initial, b’: after immersed in gastric acid, b’’: after applying Curodont Protect, c: initial, c’: after immersed in kefir, c’’: after immersed in artificial saliva, d:initial, d’: after immersed in gastric acid, d’’: after applying MI Paste Plus)

Fig. 3.

Fig. 3

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SEM images of primary teeth (a: inital, a’: after immersed in kefir,a’’:after immersed in artificial saliva, b: initial, b’: after immersed in gastric acid, b’’:after applying Curodont Protect, c: initial, c’: after immersed in kefir, c’’:after applying MI Paste Plus, d:initial, d’:after immersed in gastric acid, d’’:after applying Tooth Mousse)

Discussion

According to the data of current study, it was found that the demineralization caused by different acidic liquids and the remineralization abilities of various remineralization agents differed when analyzed using SEM images and microhardness values. As a result, the null hypothesis was rejected.

Saliva in the oral cavity contains various ions and minerals that can aid in the remineralization process of enamel defects caused by erosion. When erosion occurs due to the content of saliva, it plays a remineralizing role when the mineral concentration exceeds the solubility limit [32]. Baltaci et al. [28] evaluated the efficacy of different remineralization agents (P11-4, CPP-ACPF, and fluoride varnish) on primary teeth after acidic erosion, using artificial saliva as the control group. In all groups, higher microhardness values were obtained compared to the control group. Similarly, in current study, microhardness values decreased after immersion in acidic liquids across all groups, but even in the groups that were placed in artificial saliva without any remineralization agents, improvements were observed in the microhardness values and SEM images of erosive areas. This may be attributed to the ions and minerals present in artificial saliva, which partially filled the erosive areas and improved the physical properties of the teeth.

In their study, Behl et al. [33] induced demineralization by immersing permanent teeth in cola (pH = 2.74) and aimed to promote remineralization by applying CPP-ACPF, P11-4, and Biomin F paste. Using microhardness as the parameter to evaluate remineralization agents, they found the highest remineralization potential and microhardness values in the P11-4-containing Curodont Protect group. Sindhura et al. [34] evaluated the effectiveness of the self-assembling peptide P11-4 in artificially induced enamel lesions, using CPP-ACP as the control group. They reported that P11-4 showed superior remineralization compared to CPP-ACP more uniform mineral deposition in initial lesions. Özdemir et al. [35] assessed the effects of remineralization agents after secondary caries in primary teeth and found that P11-4 had a high remineralization effect. In a study by Habashy et al. [36], different remineralization agents (fluoride varnish, CPP-ACPF, and P11-4) were applied to artificial carious lesions in primary teeth, and their efficacy was evaluated using SEM-EDX. They observed the highest percentage increase in the Ca/P ratio in the P11-4 group. In current study, when SEM images were examined in both primary and permanent teeth, the surface topography improved after remineralization, while more regular areas were observed in the P11-4 group compared to the others. The high remineralization efficacy of P11-4 may be explained by its ability to self-assemble into fibers and form a biomatrix in demineralized areas, promoting hydroxyapatite formation on the surface [37]. This is likely due to Curodont Protect’s ability to mimic the physiological function of hydroxyapatite crystals with its small self-assembling molecules. By forming a three-dimensional fibrillar scaffold and spreading into sub-surface microporosities, this agent may reduce porous areas caused by demineralization, leading to increases in microhardness values.

Jayarajan et al. [38] evaluated the remineralization efficacy of CPP-ACP and CPP-ACPF on enamel using DIAGNOdent and SEM, finding that CPP-ACPF had higher efficacy. In a study by Rallan et al. [39], which assessed the effectiveness of various remineralization agents on primary teeth, they reported that CPP-ACPF showed greater remineralization potential than CPP-ACP. Thierens et al. [40] evaluated the remineralization efficacy of CPP-ACP and CPP-ACPF on early carious lesions, finding no difference in mineral content and the area of remineralization. This discrepancy could be attributed to the pH cycling between 7 and 6 in their study. CPP-ACPF solutions may perform better than CPP-ACP solutions when the pH drops to 5.5, 5.0, and 4.5, due to the presence of fluoride [41]. In current study, the higher remineralization efficiency of CPP-ACPF compared to CPP-ACP in the gastric acid groups, which is a more acidic liquid, can be explained by this factor. Additionally, the stronger remineralization potential of CPP-ACPF, which is composed of Ca and PO4-based complexes, may be due to its ability to provide all the necessary elements for remineralization to occur on the tooth surface. The formation of fluorapatite may be promoted in the presence of sufficient amounts of Ca and PO4 ions, with the inclusion of F ions, resulting in stronger remineralization [42].

Agrawal et al. [43] compared the microhardness values of primary and permanent teeth after inducing demineralization by immersing them in a strong acid and applying different remineralization agents. They found that the initial and post-demineralization values in primary teeth were lower than in permanent teeth. Haghgou et al. [44] evaluated the microhardness of primary and permanent teeth after immersion in acidic beverages and found a greater loss of microhardness in primary teeth. In current study, similar results were observed when the surface properties were evaluated using SEM; higher erosion areas were seen in primary teeth compared to permanent teeth in all acidic liquids. Additionally, after demineralization, the microhardness values in primary teeth were found to be lower than those in permanent teeth. Dental erosion progresses more rapidly in primary dentition than in permanent dentition due to lower crystallite organization, lower mineralization levels, higher water content, and increased permeability [45]. Moreover, the pellicle, which serves as a protective factor against dental erosion, is smaller in primary teeth, has lower protein content, and absorbs proteins more slowly than in permanent teeth [46]. As a result, material loss occurs more easily on the enamel surface of primary teeth eroded by acids. This difference may be due to factors such as enamel thickness and mineralization differences between primary and permanent teeth.

Memarpour et al. [47] evaluated the efficacy of different remineralization agents (P11-4, CPP-ACP, fluoride paste) on early carious lesions and reported that microhardness values significantly decreased after carious lesion formation, and increased in all groups following remineralization. Tabatabaei et al. [48] evaluated the effect of applying different remineralization agents after the use of iron preparations on microhardness and mineral concentration, and found higher microhardness values in the CPP-ACPF group compared to the CPP-ACP group. CPP-ACP is an amorphous compound that binds tightly to the enamel surface. The presence of fluoride in CPP-ACPF may result in the formation of more stable compounds, leading to higher microhardness values. The combination of both CPP-ACP and fluoride may promote the formation of stabilized amorphous calcium fluoride phosphate, which leads to greater fluoride incorporation and the presence of more calcium and phosphate on the enamel surface, resulting in higher microhardness values. Kamal et al. [49] evaluated the remineralization efficacy of P11-4, CPP-ACPF, and fluoride varnish after creating artificial carious lesions and selected artificial saliva as the control group. Similarly, in current study, the highest microhardness values ​​were found in the P11-4 group and the lowest values ​​in the control group after remineralization. P11-4 transitions from a low-viscosity isotropic liquid to an elastomeric nematic gel at pH < 7.4, with the anionic groups of P11-4 side chains attracting calcium ions to activate the precipitation of new hydroxyapatite, thereby promoting mineral deposition [50]. This may explain the higher microhardness values observed in this group.

Conclusion

The results of this study suggest that biomimetic agents, especially P11-4-based Curodont Protect, may provide effective remineralization in cases of dental erosion, particularly due to gastric acid exposure. Clinicians should consider incorporating such agents as part of minimally invasive treatment strategies in pediatric and adult patients with early enamel erosion. The limitations of this study are that it does not fully reflect the conditions of the oral environment and that individual-specific factors (such as the standardized composition of artificial saliva) were not taken into account. Although the results obtained from the experiments were favorable, they were carried out only in a controlled environment.

Future studies should focus on in vivo evaluations that include longer observation periods and more clinically relevant protocols. Further research may also explore the combined use of remineralization agents or their effectiveness under dynamic pH cycling conditions. Comparative studies including different age groups, salivary flow rates, or the presence of biofilm could provide more comprehensive insights. In addition, investigating the long-term stability of the remineralized enamel under mechanical forces would be valuable.

Acknowledgments

None.

Abbreviations

SEM

Scanning Electron Microscopy

GERD

Gastroesophageal reflux disease

CPP-ACP

Casein phosphopeptide-amorphous calcium phosphate

VHN

Vickers microhardness

Authors’ contributions

Design, planning and execution of the study: KB, İİ, BKE Data collection: İİ, KB Data analysis and interpretation: İİ Literature review: KB, İİ, BKE Writing the article: KB, İİ Critical review: KB, İİ, BKE.

Funding

No external funding was applied to this study.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was approved by the Firat University Non-Interventional Research Ethics Committee (2024/15) and was conducted in accordance with the Declaration of Helsinki. For the extracted teeth used in this study, informed consent forms were obtained from the patients.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

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