1 Volume 24 2025 e257345 Short communication Braz J Oral Sci. 2025;24:e257345http://dx.doi.org/10.20396/bjos.v24i00.8677345 1 Faculdade de Odontologia de Piracicaba (FOP), Universidade Estadual de Campinas (UNICAMP), Piracicaba, São Paulo, Brazil. Corresponding author: Cinthia Pereira Machado Tabchoury E-mail: cinthia@fop.unicamp.br Faculdade de Odontologia de Piracicaba, UNICAMP Av. Limeira, 901, 13414-903, Piracicaba, São Paulo, Brazil Editor: Dr. Altair A. Del Bel Cury Received: July 28, 2024 Accepted: July 16, 2025 Histidine buffering of acidic fluoridated solutions increases CaF2-like formation on carious enamel Waldemir Francisco Vieira-Junior1 , Lenita Marangoni Lopes1 , Jaime Aparecido Cury1 , Cinthia Pereira Machado Tabchoury1* Aim: Since the reactivity of fluoride with enamel decreases when the pH of slightly acidic solutions is not maintained, we evaluated the effect of acidic fluoridated solutions buffered with histidine on the formation of loosely (CaF2-like) bound products on enamel. Methods: Demineralized enamel slabs were treated with 0.05% NaF solution at pH levels of 5.0, 5.5, 6.0, and 6.5, either buffered or not with histidine (n=12). CaF2- like product formation was determined in enamel, and the data were submitted to ANOVA, Tukey, and Dunnett’s tests. Results: Buffered fluoride solutions, regardless of pH, formed higher concentrations of CaF2-like products on enamel than the respective group without buffering (p<0.05). Conclusion: The findings show that the buffering increases CaF2-like formation on carious enamel, although a direct effect of histidine cannot be entirely ruled out. Keywords: Fluorides. Dental enamel. Dental caries. Buffers. Histidine. https://orcid.org/0000-0001-8226-3100 https://orcid.org/0000-0002-5565-5974 https://orcid.org/0000-0003-1046-5605 https://orcid.org/0000-0002-7660-5685 2 Vieira-Junior et al. Braz J Oral Sci. 2025;24:e257345 Introduction Fluoride is the most relevant anticaries ion1, and its effect is considered mainly local. One of the caries control mechanisms comes as fluoridated toothpastes, rinses, and professional application products promoting a chemical reactivity with the enamel and dentine surface1. During this reactivity, two fluoridated products are formed: one firmly bound as fluorapatite (FAp) and another loosely bound as a calcium fluoride-like product (CaF2-like). During the formation of both products (Figure 1)2, phosphate (PO4 3-) and hydroxyl ions (OH-) are released from the hydroxyapatite crystals into the environment (liquid/solid interface). As these two ions act as conjugate bases and accept protons (H+) from the medium, an increase in local pH is expected (reactions are represented in Figure 1)2. The formation of FAp is predominant at low fluoride concentrations (<10 μg F/mL) and occurs mainly by ionic exchange between OH- ions present in the mineral structure of enamel and fluoride present in the chemical solution2. When fluoride is present at high concentrations (>50 μg F/mL), its reaction with enamel involves enamel dissolution, the release of calcium ions, and the subsequent precipitation of CaF2-like material on the enamel surface3. Thus, it is considered that CaF2-like products have better anticaries properties than FAp4. Figure 1. Fluoride reaction with the enamel and relation with the pH of the solution (H+). Therefore, the maintenance of pH during fluoride reaction with enamel may be relevant mainly to the formation of CaF2-like products and commercial fluoride rinses present pH values from 4.23 up to 7.345. If the pH levels are not maintained and increase, the availability of calcium will decrease (Figure 1), diminishing the reaction potential of mineral by-products on enamel. Indeed, it was possible to demonstrate that the pH of fluoridated solutions increases after a reaction with enamel, either sound or with caries-like lesions. In addition, the solution that exhib- ited the smaller rise in pH during the treatment was the more reactive6. In this con- text, histidine would be a suitable buffer, as its pKa is close to 6, providing buffer- ing capacity within the pH range of 5 to 7. Thus, we aimed to evaluate whether the buffering of slightly acidic fluoride solutions with histidine would result in greater CaF2-like formation on enamel presenting caries-like lesions. 3 Vieira-Junior et al. Braz J Oral Sci. 2025;24:e257345 Material and Methods Experimental Design In this in vitro, operator-blind experiment, demineralized enamel slabs (n = 12/group) were randomized into the following treatments: 0.05% NaF solutions at pH 5.0, 5.5, 6.0, and 6.5 either buffered with 0.1 M histidine or not, but with their pH adjusted. As a control group, 0.1 M histidine solution without pH adjustment was used. His- tidine concentration was determined by a pilot study. The enamel slabs were sub- jected to the treatment groups, and loosely bound fluoride formed was determined in enamel as described further. Fluoride solutions preparation Fluoride solutions were prepared using NaF (Synth, Diadema, Brazil) at a concentra- tion of 226 ppm F. The buffered solutions were prepared with histidine (L-histidine, Dinâmica, Indaiatuba, Brazil) at a concentration of 0.1 M, and the pH of both the buff- ered and unbuffered solutions was adjusted with 0.1 M HCl or 0.1 M NaOH. The pH values of the solutions were adjusted using a pH-electrode coupled to a potentiome- ter, which was calibrated with standard buffers pH 4.0 and 7.0. Fluoride concentration in the solutions was checked as described further. Preparation of the demineralized enamel slabs Enamel slabs (4×4×2 mm) were obtained from sound bovine incisor teeth. The den- tine was flattened, and the enamel surface was flattened and polished. The den- tal slabs were then measured with a digital caliper, and the exposed enamel area (mm2) was determined. All surfaces of the slabs, except the enamel surface, were protected with acid-resistant nail varnish. Caries-like lesions were produced by immersing the dental slabs in a demineralizing solution (0.1 M acetate buffer pH 5.0, 1.28 mM Ca, 0.74 mM Pi, and 0.03 mg F/mL) at a proportion of 2 mL/mm2 enamel for 16 h at 37°C7. Reactivity tests The demineralized enamel slabs were individually immersed in the respective treat- ment solution at 2 mL/mm2 of exposed enamel surface under agitation (100 rpm) at room temperature for 10 min, using a validated protocol8. After the reaction, the pH of the solutions was again determined, and the variation of pH (ΔpH) was calculated considering ΔpH = pHinitial – pHfinal. Determination of CaF2-like product formed For the extraction of the CaF2-like product, the slabs were individually immersed in 0.5 mL of 1.0 M KOH solution at room temperature and gently agitated for 24 h9. The extract was then neutralized and buffered with 0.5 mL of TISAB II containing 1.0 M HCl10. Fluoride in the alkali extract was analyzed with an ion-specific electrode coupled to an ion analyzer as described below. 4 Vieira-Junior et al. Braz J Oral Sci. 2025;24:e257345 Fluoride Analysis Fluoride in the treatment solutions and fluoride in the alkali (CaF2-like) extract were determined with an ion-specific electrode (Orion 96-06) coupled to an ion analyzer (Orion EA-940; Orion Research) using the direct technique. Calibration curves with standard fluoride solutions (Orion 940907) were prepared and the data were plotted in an Excel spreadsheet (Microsoft). For the analysis of fluoride in the treatment solu- tions, standards ranging from 2.0 to 32.0 μg F/mL in TISAB II 50% (v/v) were used. For CaF2-like, standards containing from 0.125 to 16.0 μg F/mL in 0.5 M KOH and TISAB II (1 M HCl) 50% (v/v) were used. From the amount of fluoride in the alkali extract, the concentrations of CaF2-like in enamel were calculated and expressed in μg F/cm2. Statistical Analysis After exploratory analysis using SAS software (SAS Institute Inc., Cary, NC, USA, Release 9.1, 2003), the ΔpH was analyzed using Kruskal-Wallis, Dunn, and Mann-Whit- ney tests. The values of CaF2-like concentration were transformed by the square root. The data were subjected to analysis using the ANOVA test in 4 × 2 + 1 (pH × buffer + control) factorial design, followed by Tukey’s test for comparisons among groups and Dunnett’s test to compare the experimental groups with control, at a 5% level of significance. Results The pH of the treatment solutions measured after the reaction with the enamel slabs (Table 1) showed that histidine was effective in preventing pH changes in the solu- tions at pH 5.0, 5.5, and 6.0. For pH 5.5 and 6.0, the pH increased 0.12-0.13 units in the fluoridated solutions without histidine. When comparing the solutions with and without histidine, statistically significant differences were found in the groups at pH 5.0 and 6.0 (p<0.05). Table 1. Median (minimum; maximum) of ΔpH values according to the treatment with F solutions, buffered or not, at different pH values (n = 12). pH Without histidine With histidine 5.0 0.04 (-0.11; 0.08) Ab -0.14 (-0.18; -0.07) Bb 5.5 0.12 (0.02; 0.24) Aa -0.10 (-0.16; 0.02) Ab 6.0 0.13 (0.07; 0.25) Aa 0.01 (-0.03; 0.07) Ba 6.5 -0.07 (-0.19; 0.07) Ab -0.09 (-0.18; -0.02) Ab Medians followed by distinct letters (uppercase in rows and lowercase in columns) are different according to the Kruskal-Wallis, Dunn, and Mann-Whitney tests (p<0.05). Concerning CaF2-like formation (Table 2), the effects of the isolated factors, pH (p<0.0001), buffer (p<0.0001), and the interaction of the factors (p=0.0006) were sig- nificant. Dental slabs from all groups treated with buffered solutions showed higher CaF2-like concentrations than those without histidine (p<0.0001). In the groups with- out buffer, there was no significant difference between pH 5.0, 5.5, and 6.0 (p>0.05). 5 Vieira-Junior et al. Braz J Oral Sci. 2025;24:e257345 Considering the groups with buffer, the slabs treated with F solution at pH 5.0 had the highest concentration of CaF2-like, which statistically differed from all other groups (p<0.0001). All groups statistically differed from the control group (p<0.05). Table 2. Mean (standard deviation) of the CaF2-like (µg F/cm²) products found on enamel according to the treatments and the control (n=12/group). pH Without histidine With histidine 5.0 4.80 (1.23) Aab 14.36 (3.34) Ba# 5.5 4.90 (1.12) Aab 9.91 (2.20) Bb 6.0 5.20 (1.27) Aa 10.04 (1.33) Bb 6.5 3.59 (1.19) Ab 8.89 (1.20) Bb Control (histidine solution): mean = 0.21, standard deviation = 0.04. All groups differed from the control group according to Dunnett’s test (p<0.05). Distinct letters (uppercase in rows and lowercase in columns) indicate statistically significant differences (p<0.05). The # represents an outlier statistically identified in the group (n=11). Discussion This initial hypothesis that the buffering of 226 ppm F solutions slightly acidic (pH ≥ 5.0) would increase the formation of CaF2-like products on enamel was accepted. Histidine was chosen because the pKa of its imidazole group is 6.0, pro- viding good buffer capacity to prevent the pH increase that occurs when fluoride solutions at pH levels between 5.0 to 6.0 react with enamel6. Based on Table 1, the histidine buffer effectively maintained pH stability, while solutions with initial pH val- ues of 5.0, 5.5, and 6.0 exhibited a slight pH increase after exposure in the absence of histidine, as expected and shown in Figure 1. Consequently, an increased forma- tion of CaF2-like products was observed in all fluoridated groups containing histi- dine, especially at pH 5.0. The maintenance of lower pH values may have prevented chemical equilibrium from being reached11, sustaining hydroxyapatite dissolution and releasing Ca2+ for the formation of CaF2 products. The pH of the solutions played a crucial role during the reaction with the dental substrate, as H+ ions could potentially interfere with fluoride reactions. Specifically, for the solution with pH=5.0 (Table 2), the increase in fluoride concentrations could stem from either the easier diffusion of HF or from the dissolu- tion and recrystallization of enamel, particularly potentiated under low pH conditions2,3. Moreover, in enamel with caries-like lesions, the formation of CaF2-like compounds could serve as a local ion reservoir, increasing F bioavailability to the enamel1,4. Con- sidering the action mechanism of fluoride, under undersaturated conditions, fluoride ions will be released from CaF2-like products, thereby promoting remineralization or decreasing demineralization1,4,12. Previous studies have reported more effective treatments and an increase in the FAp and CaF2-like formations when products with acidic pH were evaluated, particularly with more concentrated fluoridated solutions, professional gels, or toothpastes13,14. In the present investigation, the most acidic solution had a pH of 5.0, and the concentration of fluoride in the solution was lower (226 ppm), thus, the increase in the formed fluoride products cannot be solely attributed to Ca2+ release induced by 6 Vieira-Junior et al. Braz J Oral Sci. 2025;24:e257345 the low pH. Corroboratively, an increase in CaF2-like products was observed for the histidine-NaF group at pH 6.5 (Table 2), suggesting a specific effect mediated by histidine. L-histidine may facilitate the nucleation and precipitation of hydroxyapatite15 due to its ability to chelate calcium ions or increase local concentrations of PO4 3- and Ca2+, which could then reprecipitate as CaF2-like compounds. Future investigations should explore other reactions occurring in the presence of histidine in fluoridated solutions, including its potential role in the formation of fluoride-enriched mineral complexes or the transport of fluoride into caries-like enamel. The limitations of the study include the use of an in vitro caries model that did not use biofilm or pH cycling, the non-determination of FAp products, and the inability to extrapolate the subclinical and clinical effects of increased reactivity of the fluoridated solutions. It is also imperative to consider that the effects observed in the present study may be related to the chemical structure of histidine rather than solely to its buffering capacity. In conclusion, the addition of histidine as a buffer to fluoridated solution has the potential to enhance fluoride reactivity on enamel with caries-like lesions. Data availability Data is available on demand from referees. Conflict of Interest The authors have no conflicts of interest to declare. Author Contribution: Waldemir Francisco Vieira-Junior: Data curation, investigation, methodology, origi- nal draft, writing, review, and editing. Lenita Marangoni Lopes: Data curation, investi- gation, methodology, review, and editing. Jaime Aparecido Cury: Conceptualization, data curation, investigation, methodology, writing, review, and editing. Cinthia Pereira Machado Tabchoury: Conceptualization, data curation, investigation, methodology, project administration, validation, original draft, writing, review, and editing. All authors actively participated in the discussion of the manuscript’s findings and have revised and approved the final version of the manuscript. References 1. Cury JA, Tenuta LM. How to maintain a cariostatic fluoride concentration in the oral environment. Adv Dent Res. 2008 Jul;20(1):13-6. doi: 10.1177/154407370802000104. 2. Brudevold F. Interaction of fluoride with human enamel. 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