In vitro reduction of enamel erosion by sugarcane-derived cystatin associated with sodium trimetaphosphate

Abstract

The objective of this in vitro study was to assess the efficacy of CaneCPI-5, either alone or in combination with various concentrations of sodium trimetaphosphate (TMP) in protecting against initial enamel erosion. A total of 135 bovine enamel specimens were prepared and categorized into nine groups (n/group=15) according to the following treatments: Deionized water; Commercial solution (Elmex Erosion Protection^TM); 0.1 mg/mL CaneCPI-5; 0.5% TMP; 1.0% TMP; 3.0% TMP; 0.1 mg/mL CaneCPI-5+0.5% TMP; 0.1 mg/mL CaneCPI-5+1.0%TMP; and 0.1 mg/mL CaneCPI-5+3.0%TMP. The specimens were treated with the respective solutions for 2 h, followed by acquired enamel pellicle formation for 2 h and exposure to 0.65% citric acid (CA) for 1 min. These procedures were repeated once a day for three consecutive days. Demineralization was assessed by the percentage change in surface hardness (%CSH) and calcium release into CA, analyzed by the Arsenazo III method. The data were evaluated using Kruskal-Wallis/Dunn's tests. Regarding %CSH, CaneCPI-5+3.0%TMP was the most effective treatment when compared to the CaneCPI-5 group alone. As for calcium release into CA, the CaneCPI-5+0.5% TMP and CaneCPI-5 groups (both with lower calcium release) did not significantly differ from the commercial solution. In conclusion, combination of CaneCPI-5 with TMP enhances the protective potential against initial enamel erosion in vitro.

Keywords:
Cystatins; Dental Pellicle; Saliva; Tooth Erosion

Introduction

Erosive tooth wear (ETW) consists of the progressive loss of mineralized tooth substance with dental erosion as the primary causal factor.¹,² The acids involved in this process are of non-bacterial origin² and can be either intrinsic³ or extrinsic.⁴ In the earliest stages of dental erosion, only surface softening occurs.⁵ With increased acid exposure, loss of volume and fine subsurface softening take place.⁵ ETW has a significant prevalence in the population,⁶ and there is a consensus that the severity of ETW increases with age.⁷ An individual's key protective factor against ETW is saliva, mainly because of its role in acquired enamel pellicle (AEP) formation.⁸

AEP is a bacteria-free film composed of salivary proteins, glycoproteins, and lipids that are adsorbed onto the dental surface, forming a highly organized and dynamic film.⁹ This protective layer functions as a mechanical barrier against acids on the dental surface, reducing demineralization.¹⁰ It is known that the pellicle's protective effectiveness is determined by its composition and physical properties, such as thickness.¹¹ Importantly, proteins in the basal layer of the AEP remain intact, even after prolonged erosive challenges.¹² Drawing on this concept, a new protection strategy against ETW was developed, based on "acquired pellicle engineering," which consists in reinforcing the basal layer of the AEP with acid-resistant proteins.¹³,¹⁴

Among these acid-resistant proteins, cystatin-B has been identified¹⁵ as a promising option against ETW. Nonetheless, because of the high cost of the human recombinant protein, it is considered commercially impractical. Therefore, our group identified and characterized a new sugarcane-derived cystatin (CaneCPI-5) that has a strong binding affinity for enamel.¹⁶ This protein demonstrated a reduction in enamel surface free energy, with a more negative polar component,¹⁷ which may influence AEP formation, owing to the change in protein composition. In vitro,¹⁶ in situ,¹⁸ and in vivo¹³,¹⁹ studies have demonstrated the high potential of this recombinant protein to protect dental enamel from ETW-causing acids.

The addition of sodium trimetaphosphate (TMP) to fluoride vehicles promotes a significant increase in their ability to favorably interfere in demineralization and remineralization of enamel induced by cariogenic and erosive challenges, even when associated with low-concentration fluoride varnishes.²⁰,²¹ Such effects are attributed to its high adsorption capacity to enamel, consequently reducing its solubility, in addition to having a strong tendency to form complexes with cations, due to the presence of a phosphate group in the molecular structure of TMP.²²,²³ The action of TMP is associated with: a) significant reduction in enamel mineral loss both in vitro and in vivo; b) reduced formation of extracellular polysaccharides in the dental biofilm formed in situ; c) increase in fluoride, calcium, and phosphate concentrations in enamel and dental biofilm; d) increase in the remineralization rate of previously demineralized dental enamel; and e) dental erosion reduction.²⁴–²⁷ This is associated with a decrease in the enamel surface free energy and its nonpolar component, coupled with an increase in the number of electron donor sites, which promotes greater adsorption of Ca²⁺ and PO₄^3- ions.²⁴ Despite the lack of data on the effects of TMP on the AEP, evidence indicates that the effects of this phosphate on enamel are related to changes in AEP composition, as a result of its high capacity of adsorption to enamel²²,²³ and alteration of the surface free energy of this substrate.²⁸

Thus, the association of CaneCPI-5 (organic component) with an inorganic component such as TMP may be an alternative to enhance the effect of protein against ETW, given the synergistic effect observed when CaneCPI-5 is combined with sodium fluoride (NaF).²⁹ Therefore, the aim of this study was to evaluate the effect of CaneCPI-5 associated or not with different concentrations of TMP on the protection against initial enamel erosion in vitro. The null hypothesis to be tested is that CaneCPI-5 associated or not with TMP would not have a protective effect against initial enamel erosion in vitro.

Methods

Ethical considerations

The protocol of this study was approved by the Institutional Human Research Ethics Committee (protocol No. 64568622.2.0000.5417) and by the Ethics Committee on the Use of Animals (protocol 009/2022)

CaneCPI-5 production

CaneCPI-5 was synthesized using the recombinant bacterial strain Escherichia coli Rosetta (DE3), as previously described.¹⁶,³⁰ Briefly, the bacterial strain was transformed with the plasmid pET28aCaneCPI-5. The expressed protein was purified from the soluble fraction of bacterial cultures induced by IPTG (Isopropyl-β—Dt-hiogalactoside), centrifuged, and sonicated. Purification was performed by affinity chromatography using columns containing Ni-NTA Superflow nickel resin (Qiagen).

Specimen preparation

The study by Santiago et al.¹⁶ was used for sample size calculation. A minimum detectable difference in %CSH of 27% was considered, with a standard deviation of 9%, a type α error of 5%, and a type β error of 20%. Therefore, 15 specimens were included.

One hundred and thirty five bovine enamel specimens (4 x 4 x 3 mm) were prepared from the buccal surface of bovine incisors. The crowns of the bovine teeth were individually mounted on acrylic plates (40 x 40 x 5 mm³) screwed into an ISOMET Low-Speed Saw precision cutting device (Buehler, Lake Bluff, USA). The specimens were cut with two double-sided diamond discs: XL 12205, "High concentration," 102 x 0.3 x 12.7 mm³ (Extec Corp., Ref: 12205, Enfield, USA) and a stainless-steel spacer (diameter: 7 cm, thickness: 4 mm, and central hole: 1.3 cm) between the discs.

After the samples were cut, they were polished. To achieve this, they were fixed with sticky Kota wax using a PKT instrument (Kota Ind., São Paulo, Brazil) and a lamp (JON, Ind. Bras., Juiz de Fora, Brazil) at the center of an acrylic disc (30 mm in diameter and 8 mm in thickness), with the enamel surface facing the disc, with the intention of initially planing the internal dentin. The assembly (disc/tooth) was mounted on a metallographic polisher (Arotec, São Paulo, Brazil) for dentin planing, allowing parallelism between the polished surfaces and the acrylic base to which the specimens were attached. A 320-grit silicon carbide sandpaper was then used under cooling with deionized water until the specimens reached a thickness of approximately 2.5 to 3 mm. The polisher was operated at a low speed for 30 s to 3 min until the desired thickness was achieved. Subsequently, the specimens were removed from the acrylic disc and the discs were cleaned with xylene (Merck, Darmstadt, Germany) to remove any residual wax adhered to them. Thereafter, the enamel specimens were fixed again with sticky wax at the center of the acrylic plate with the enamel surface exposed, for polishing, thus obtaining a surface that was flat and parallel to the base, essential for the tests. Again, the assembly was mounted on the polisher and the surface was initially worn down with a 600-grit silicon carbide sandpaper under cooling with deionized water for approximately 2 min (removal of approximately 150–200 μm of tissue), using two weights, at low speed. Polishing was performed with a 1200-grit silicon carbide sandpaper (Extec Corp., Enfield, USA) under cooling for 2-4 min, applying two weights, at high speed. This was followed by polishing with felt and diamond solution for 2 – 4 min, also applying two weights. The wear on the enamel surface was controlled using a micrometer to prevent excessive wear that could reach the dentin (region of non-interest in the present study). All specimens underwent this wear control, with measurements taken before and after polishing, and the difference between the initial and final data was employed to calculate the total wear value. In addition, some representative enamel specimens were examined under the microscope to confirm the absence of dentinal tubules, indicating that the specimens were within the dentin (region of non-interest in the present study). Thus, specimens that exceeded the parameters of enamel tissue removal and those in which the wear reached the dentin region were discarded. The specimens were cleaned in distilled water in an ultrasonic bath and finally stored at 4°C until the start of the experiments.

Human saliva collection

Nine volunteers (five men and four women) aged 20 to 38 years were selected. The following inclusion criteria were adopted: nonsmokers, individuals with no history of long-term medication use, nonpregnant women, individuals with no systemic diseases and with good oral health, individuals without active caries, individuals without periodontal disease, and those with normal salivary flow rates (stimulated saliva > 1.0 mL/min and unstimulated saliva > 0.3 mL/min). Volunteers provided stimulated saliva after chewing paraffin wax for 10 min, from 9:00 a.m. to 10:00 a.m, with samples stored on ice afterwards. Collected saliva was pooled and centrifuged for 20 min at 14,000 g and stored at 4°C.³¹ The supernatants were collected and stored at −80°C until the start of the experiment.

Treatment, incubation in human saliva, and acid challenge

Bovine enamel specimens were randomly distributed into nine groups (n = 15/group) according to the treatments: a) Deionized water (negative control); b) Commercial solution containing SnCl₂/NaF/AmF (800 ppm Sn⁺² + 500 ppm F^-, pH 4.5, Elmex Erosion Protection® – GABA International AG (positive control 1); c) 0.1 mg/mL CaneCPI-5 (positive control 2); d) 0.5% TMP; e) 1.0% TMP; f) 3.0% TMP; g) 0.1 mg/mL CaneCPI-5 + 0.5% TMP; h) 0.1 mg/mL CaneCPI-5 + 1.0% TMP; and i) 0.1 mg/mL CaneCPI-5 + 3.0% TMP. All treatments, except for the commercial solution, had the same consistency, color, and smell.

To ensure standardization of the exposed area, bovine enamel blocks were placed in 96-well plates with the enamel surface facing upwards, making sure that the entire enamel surface was immersed in the treatment solution. To prevent exposure of the lateral and bottom walls, these surfaces were protected with red nail varnish (Risqué®, São Paulo, Brazil).

Initially, the samples were individually treated with the respective solutions (250 μL, 2 h, 37°C, under constant agitation). The acquired pellicle was then individually formed by adding human saliva to each specimen (250 μL, 2 h, 37°C, under constant stirring). Subsequently, the specimens were individually subjected to the erosive challenge by immersion in 0.65% citric acid (pH 3.6, 1 mL, 1 min, 37°C, 70 rpm). Between each application (treatment, AEP formation, and acid challenge), the specimens were carefully washed with deionized water (10 s) and dried (5 s). These procedures were performed once a day for three consecutive days to simulate initial erosion.³² During brief interims and overnight, the specimens were stored in humidity chambers (4°C) (Figure 1).

Figure 1
In vitro experimental protocol and calcium analysis.

Percentage change in surface hardness

Measurements were performed using a Knoop diamond indenter with a load of 50 g and a dwell time of 15 s (HMV-2, Buehler, Lake Bluff, USA). Five indentations per specimen were made (spaced 150 μm apart on a defect-free surface) at the beginning of the experiment (SH_initial) and after the final erosive challenge (SH_final). The percentage change in surface hardness (%CSH) was calculated as a measure of enamel softening, according to the following equation: %CSH = [(SH_initial-SH_final)/SH_initial]x100 for statistical analyses.

Analysis of released calcium

The amount of calcium present in the citric acid after the erosive challenges was individually analyzed by the Arsenazo III colorimetric method (Sigma Aldrich, CAS 1668-00-4, Burlington, USA,).³³ The reagent was diluted with part of the sample or calcium standard, in a 1:1 ratio (50 μL of sample and 50 μL of reagent) and pipetted into 96-well plates. A calibration curve was constructed so that the absorbances remained within the linearity range of 0-1 using different concentrations of calcium. Absorbances were analyzed at 650 nm, at 25°C. The analyses were performed in triplicate on a microplate spectrophotometer (Biotek Instrumentals, Inc., Winooski, USA) (Figure 1).

Statistical analysis

GraphPad Prism software (version 6.0 for Windows; GraphPad Software Inc., La Jolla, USA) was used. Data were checked for normality (Kolmogorov-Smirnov test) and homogeneity (Bartlett test) and were analyzed by the Kruskal-Wallis test, followed by Dunn's test. Results are presented as median and interquartile range. The significance level was set at 5% (two-tailed).

Results

Change in surface hardness

After three consecutive days CaneCPI-5 and all combinations of Cane-CPI-5 + TMP significantly protected the enamel when compared to the negative control and did not show significant difference when compared to Elmex Erosion^TM. The isolated TMP groups did not provide a protective effect when compared to deionized water.

CaneCPI-5 + 3% TMP (26.75%; 1.82%) was the most effective treatment. It did not show significant differences when compared with Elmex (31.12%; 3.19%) or CaneCPI-5 + 1.0% TMP (29.00%; 6.20%), but performed significantly better than all the other groups, including CaneCPI-5 and TMP alone, regardless of the concentration (p < 0.05) (Figure 2). Table 1 shows the confidence interval values (95%CI).

Figure 2
Percentage change in surface hardness after treatments of bovine enamel specimens with 0.1 mg/mL CaneCPI-5 alone, TMP alone at different concentrations or their combination, followed by acquired enamel pellicle formation for 2 h and erosive challenges with 0.65% citric acid at pH 3.6 for 1 min.

Table 1
Effect size and confidence interval (CI) for change in surface hardness.

Groups	Minimum – Maximum (95%CI)
Deionized water	47.31–59.25
Commercial solution (Elmex Erosion ProtectionTM)	22.82–35.48
0.1 mg/mL CaneCPI-5	31.21–43.33
0.5% TMP	39.01–47.16
1.0% TMP	40.09–52.46
3.0% TMP	45.82–53.41
0.1 mg/mL CaneCPI-5+0.5% TMP	35.01–44.41
0.1 mg/mL CaneCPI-5+1.0% TMP	24.12–38.46
0.1 mg/mL CaneCPI-5+3.0% TMP	20.01–29.60

Calcium analysis by the Arsenazo III colorimetric method

The groups with lowest calcium release into the citric acid were Elmex (7.09 μM; 6.97μ), CaneCPI-5 alone (14.90 μM; 7.04 μM), and CaneCPI-5 + 0.5% TMP (22.80 μM; 4.35 μM). These groups did not show significant differences among themselves, but they released significantly lower amounts of calcium when compared to the negative control (38.70 μM; 9.16 μM). All the other experimental groups did not significantly differ from the negative control (Figure 3). Table 2 shows the confidence interval values (95%CI).

Figure 3
Calcium released into the citric acid (μM) by the Arsenazo III method. Bovine enamel specimens were treated with 0.1 mg/mL CaneCPI-5 alone, TMP alone at different concentrations or their combination, followed by acquired enamel pellicle formation for 2 h and erosive challenges with 0.65% citric acid at pH 3.6 for 1 min.

Table 2
Effect size and confidence interval (CI) for calcium analysis by the Arsenazo III colorimetric method.

Groups	Minimum – Maximum (95%CI)
Deionized water	0.0212–0.0784
Commercial solution (Elmex Erosion ProtectionTM)	0.0025–0.0221
0.1 mg/mL CaneCPI-5	0.0004–0.0415
0.5% TMP	0.0098–0.0549
1.0% TMP	0.0080–0.0646
3.0% TMP	0.0142–0.0560
0.1 mg/mL CaneCPI-5+0.5% TMP	0.0051–0.0540
0.1 mg/mL CaneCPI-5+1.0% TMP	0.0163–0.0476
0.1 mg/mL CaneCPI-5+3.0% TMP	0.0056–0.0377

Discussion

In recent decades, extensive research has explored ways to enhance the protective effect of active agents against erosive tooth wear. TMP, in particular, has been suggested as an enhancer of the effect of fluoride, whether associated with mouthwashes,²⁸ varnishes,³⁴ or toothpastes.³⁵ Considering the promising results of these studies, in which the addition of TMP to these fluoride-containing vehicles promoted a significant increase in the therapeutic potential of fluoride, in the present study, we proposed the addition of TMP to CaneCPI-5, a sugarcane-derived phytocystatin, which has shown good results against erosive demineralization.¹⁶ Our study corroborates the protective effect of the association between phosphate and protein, demonstrating that the association between CaneCPI-5 and TMP at the concentration of 3.0% yielded the best result . This association significantly decreased surface hardness loss compared to both CaneCPI-5 alone and TMP alone, regardless of the concentration. The absence of a significant effect of TMP alone was expected, as reported previously.³⁶ Consequently, the null hypothesis of the study was rejected.

To interpret calcium analysis results, it is crucial to consider various factors. The AEP can release calcium into an acidic environment.³⁷ This released calcium adds to the calcium released from hydroxyapatite. During treatments that involve acquired pellicle engineering, caution is advised when interpreting this response variable. This is because, during the engineering process, proteins with varying calcium-binding capabilities might replace the pellicle's initial proteins, affecting the outcomes across different groups. Notably, CaneCPI-5 + 0.5% TMP showed the most favorable outcome in calcium analysis, presenting intriguing results. Hence, the use of the Arsenazo III method in this analysis could have been influenced by the presence of calcium within the AEP. Therefore, given these considerations, the %CSH analysis might provide more reliable results.

Regarding the concentrations and particle sizes of the TMP employed in the present study, initial research shows no difference in the effectiveness of TMP associated with fluoride at the concentrations between 1.0% and 2.0%.³⁴ Therefore, concentrations of 0.5%, 1.0%, and 3.0% were used in this study. Additionally, although nanoparticulate TMP shows better results and can be used at lower concentrations,²⁸ in the present study, microparticulate TMP was employed, given that this is the first study to associate phosphate with protein. Furthermore, a previous study has shown that micrometric particles associated with fluoride toothpaste were more effective against dental erosion compared to groups that did not include phosphate.³⁸,³⁹ However, it should be noted that the hypothesis suggests smaller TMP particles may react more effectively with the enamel surface, potentially leading to a significant reduction of enamel erosion.²¹ Therefore, future studies should be conducted with nanometric particles.

Both TMP²²,²³ and CaneCPI-5¹⁶ have a high binding force to enamel and, once bound, they reduce the surface free energy of this substrate.¹⁶,²⁸ In the case of TMP, the reduction of surface free energy facilitates the diffusion of Ca⁺². CaneCPI-5 also increases the electron-donor sites on the enamel surface.¹⁶ This favors the adsorption of cationic species, such as Ca²⁺ and Ca(H₂PO₄) (calcium phosphate), as well as cationic acid-resistant salivary proteins, helping to explain the reduction in %CSH. The enhanced adsorption of Ca(H₂PO₄)₂ by CaneCPI-5 helps to explain why the association of protein with lower concentrations of TMP was less effective than the higher concentration (3%) (Figure 4).

Figure 4
Representation of the interaction of CaneCPI-5 and TMP with the tooth surface and their effects: increase in electron donor sites that favor the adsorption of salivary proteins and increase in the diffusion of calcium ions, respectively.

Although the in vitro protocol has been considered appropriate for preliminary investigations and for screening of new potential products,³² it does not accurately reflect the clinical setting. The main obstacle lies in the exposure time to protein solutions (2 h), required because of the unique combination of components. In dental practice, mouthwashes are specifically used for just 1–2 min. Furthermore, this study did not include a comprehensive assessment of erosive tooth wear because the effect of abrasion-related components of toothbrushing was not assessed. Therefore, additional studies are needed to evaluate the effect of protein solutions at time intervals that more closely reflect clinical practice. Moreover, the static model utilized in this study, in which the same saliva was in contact with the specimens for 2 h to form the AEP, contrasts with the clinical practice. In the clinical setting, saliva is continuously produced and removed, potentially impacting protein absorption and effectiveness of the protection system. Further studies evaluating the effect of abrasion and in vivo studies examining the impact of this combination of components in the AEP protein profile should be considered.

Conclusion

In conclusion, our study showed that combining the organic component, CaneCPI-5, with the inorganic component, TMP, effectively reduced initial enamel erosion in vitro, particularly in relation to the loss of surface hardness. To further explore the potential of CaneCPI-5 and TMP in preventive dentistry, future research should employ methodologies more closely aligned with the clinical setting. This includes in vivo study designs, shorter treatment durations, and the incorporation of abrasives, which are vital for facilitating the practical application of this combination.

Acknowledgments

This study was supported by FAPESP (2022/11297-3). The authors thank FAPESP for the concession of scholarships to Carolina Ruis Ferrari (2021/14161-2) and Karolyne Sayuri de Araujo Kitamoto (2022/11297-3).

References

• ¹ Lussi A, Carvalho TS. Erosive tooth wear: a multifactorial condition of growing concern and increasing knowledge. Monogr Oral Sci. 2014;25:1-15. https://doi.org/10.1159/000360380
  » https://doi.org/10.1159/000360380
• ² Schlueter N, Amaechi BT, Bartlett D, Buzalaf MA, Carvalho TS, Ganss C, et al. Terminology of erosive tooth wear: consensus report of a workshop organized by the ORCA and the Cariology Research Group of the IADR. Caries Res. 2020;54(1):2-6. https://doi.org/10.1159/000503308
  » https://doi.org/10.1159/000503308
• ³ Moazzez R, Bartlett D. Intrinsic causes of erosion. Monogr Oral Sci. 2014;25:180-96. https://doi.org/10.1159/000360369
  » https://doi.org/10.1159/000360369
• ⁴ Barbour ME, Lussi A. Erosion in relation to nutrition and the environment. Monogr Oral Sci. 2014;25:143-54. https://doi.org/10.1159/000359941
  » https://doi.org/10.1159/000359941
• ⁵ Eisenburger M, Shellis RP, Addy M. Scanning electron microscopy of softened enamel. Caries Res. 2004;38(1):67-74. https://doi.org/10.1159/000073923
  » https://doi.org/10.1159/000073923
• ⁶ Donovan T, Nguyen-Ngoc C, Abd Alraheam I, Irusa K. Contemporary diagnosis and management of dental erosion. J Esthet Restor Dent. 2021 Jan;33(1):78-87. https://doi.org/10.1111/jerd.12706
  » https://doi.org/10.1111/jerd.12706
• ⁷ Van't Spijker A, Rodriguez JM, Kreulen CM, Bronkhorst EM, Bartlett DW, Creugers NH. Prevalence of tooth wear in adults. Int J Prosthodont. 2009;22(1):35-42.
• ⁸ Buzalaf MA, Hannas AR, Kato MT. Saliva and dental erosion. J Appl Oral Sci. 2012;20(5):493-502. https://doi.org/10.1590/S1678-77572012000500001
  » https://doi.org/10.1590/S1678-77572012000500001
• ⁹ Dawes C, Jenkins GN, Tongue CH. The nomenclature of the integuments of the enamel surface of the teeth. Br Dent J. 1963;115:65-8.
• ¹⁰ Hara AT, Zero DT. The potential of saliva in protecting against dental erosion. Monogr Oral Sci. 2014;25:197-205. https://doi.org/10.1159/000360372
  » https://doi.org/10.1159/000360372
• ¹¹ Amaechi BT, Higham SM, Edgar WM, Milosevic A. Thickness of acquired salivary pellicle as a determinant of the sites of dental erosion. J Dent Res. 1999 Dec;78(12):1821-8. https://doi.org/10.1177/00220345990780120901
  » https://doi.org/10.1177/00220345990780120901
• ¹² Hannig M. Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period. Clin Oral Investig. 1999 Jun;3(2):88-95. https://doi.org/10.1007/s007840050084
  » https://doi.org/10.1007/s007840050084
• ¹³ Carvalho TS, Araújo TT, Ventura TM, Dionizio A, Câmara JV, Moraes SM, et al. Acquired pellicle protein-based engineering protects against erosive demineralization. J Dent. 2020 Nov;102:103478. https://doi.org/10.1016/j.jdent.2020.103478
  » https://doi.org/10.1016/j.jdent.2020.103478
• ¹⁴ Pelá VT, Buzalaf MA, Niemeyer SH, Baumann T, Henrique-Silva F, Toyama D, et al. Acquired pellicle engineering with proteins/peptides: mechanism of action on native human enamel surface. J Dent. 2021 Apr;107:103612. https://doi.org/10.1016/j.jdent.2021.103612
  » https://doi.org/10.1016/j.jdent.2021.103612
• ¹⁵ Delecrode TR, Siqueira WL, Zaidan FC, Bellini MR, Moffa EB, Mussi MC, et al. Identification of acid-resistant proteins in acquired enamel pellicle. J Dent. 2015 Dec;43(12):1470-5. https://doi.org/10.1016/j.jdent.2015.10.009
  » https://doi.org/10.1016/j.jdent.2015.10.009
• ¹⁶ Santiago AC, Khan ZN, Miguel MC, Gironda CC, Soares-Costa A, Pelá VT, et al. A new sugarcane cystatin strongly binds to dental enamel and reduces Erosion. J Dent Res. 2017 Aug;96(9):1051-7. https://doi.org/10.1177/0022034517712981
  » https://doi.org/10.1177/0022034517712981
• ¹⁷ Gironda CC, Pelá VT, Henrique-Silva F, Delbem AC, Pessan JP, Buzalaf MA. New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action. J Appl Oral Sci. 2022;29;30:e20210698. doi: 10.1590/1678-7757-2021-0698
  » https://doi.org/10.1590/1678-7757-2021-0698
• ¹⁸ Pelá VT, Lunardelli JG, Tokuhara CK, Gironda CC, Silva ND, Carvalho TS, et al. Safety and in situ antierosive effect of CaneCPI-5 on dental enamel. J Dent Res. 2021 Nov;100(12):1344-50. https://doi.org/10.1177/00220345211011590
  » https://doi.org/10.1177/00220345211011590
• ¹⁹ Pelá VT, Ventura TM, Taira EA, Thomassian LT, Brito L, Matuhara YE, et al. Use of reflectometer optipen to assess the preventive effect of a sugarcane cystatin on initial dental erosion in vivo. J Mech Behav Biomed Mater. 2023 May;141:105782. https://doi.org/10.1016/j.jmbbm.2023.105782
  » https://doi.org/10.1016/j.jmbbm.2023.105782
• ²⁰ Moretto MJ, Delbem AC, Manarelli MM, Pessan JP, Martinhon CC. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion: an in situ/ex vivo study. J Dent. 2013 Dec;41(12):1302-6. https://doi.org/10.1016/j.jdent.2013.09.008
  » https://doi.org/10.1016/j.jdent.2013.09.008
• ²¹ Danelon M, Pessan JP, Santos VR, Chiba EK, Garcia LS, Camargo ER, et al. Fluoride toothpastes containing micrometric or nano-sized sodium trimetaphosphate reduce enamel erosion in vitro. Acta Odontol Scand. 2018 Mar;76(2):119-24. https://doi.org/10.1080/00016357.2017.1388442
  » https://doi.org/10.1080/00016357.2017.1388442
• ²² Takeshita EM, Exterkate RA, Delbem AC, ten Cate JM. Evaluation of different fluoride concentrations supplemented with trimetaphosphate on enamel de- and remineralization in vitro. Caries Res. 2011;45(5):494-7. https://doi.org/10.1159/000331209
  » https://doi.org/10.1159/000331209
• ²³ Dijk JW, Borggreven JM, Driessens FC. The effect of some phosphates and a phosphonate on the electrochemical properties of bovine enamel. Arch Oral Biol. 1980;25(8-9):591-5. https://doi.org/10.1016/0003-9969(80)90072-2
  » https://doi.org/10.1016/0003-9969(80)90072-2
• ²⁴ Danelon M, Garcia LG, Pessan JP, Passarinho A, Camargo ER, Delbem AC. Effect of fluoride toothpaste containing nano-sized sodium hexametaphosphate on enamel remineralization: an in situ study. Caries Res. 2019;53(3):260-7. https://doi.org/10.1159/000491555
  » https://doi.org/10.1159/000491555
• ²⁵ Danelon M, Pessan JP, Souza Neto FN, Camargo ER, Delbem AC. Effect of toothpaste with nano-sized trimetaphosphate on dental caries: in situ study. J Dent. 2015 Jul;43(7):806-13. https://doi.org/10.1016/j.jdent.2015.04.010
  » https://doi.org/10.1016/j.jdent.2015.04.010
• ²⁶ Manarelli MM, Delbem AC, Lima TM, Castilho FC, Pessan JP. In vitro remineralizing effect of fluoride varnishes containing sodium trimetaphosphate. Caries Res. 2014;48(4):299-305. https://doi.org/10.1159/000356308
  » https://doi.org/10.1159/000356308
• ²⁷ Takeshita EM, Danelon M, Castro LP, Sassaki KT, Delbem AC. Effectiveness of a toothpaste with low fluoride content combined with trimetaphosphate on dental biofilm and enamel demineralization in situ. Caries Res. 2015;49(4):394-400. https://doi.org/10.1159/000381960
  » https://doi.org/10.1159/000381960
• ²⁸ Mancilla JOFC. Desenvolvimento de protocolo in vitro do processo erosivo inicial do esmalte e efeito de enxaguatório bucal fluoretado associado ao trimetafosfato nanoparticulado contra a erosão. Araçatub]: Faculdade de Odontologia de Araçatuba; 2018.
• ²⁹ Pelá VT, Niemeyer SH, Baumann T, Levy FM, Henrique-Silva F, Lussi A, et al. Acquired pellicle engineering using a combination of organic (sugarcane cystatin) and inorganic (sodium fluoride) components against dental erosion. Caries Res. 2022;56(2):138-45. https://doi.org/10.1159/000522490
  » https://doi.org/10.1159/000522490
• ³⁰ Soares-Costa A, Beltramini LM, Thiemann OH, Henrique-Silva F. A sugarcane cystatin: recombinant expression, purification, and antifungal activity. Biochem Biophys Res Commun. 2002 Sep;296(5):1194-9. https://doi.org/10.1016/S0006-291X(02)02046-6
  » https://doi.org/10.1016/S0006-291X(02)02046-6
• ³¹ Ventura TM, Ribeiro NR, Dionizio AS, Sabino IT, Buzalaf MA. Standardization of a protocol for shotgun proteomic analysis of saliva. J Appl Oral Sci. 2018;11;26:e20170561. doi: 10.1590/1678-7757-2017-0561
  » https://doi.org/10.1590/1678-7757-2017-0561
• ³² Cheaib Z, Lussi A. Impact of acquired enamel pellicle modification on initial dental erosion. Caries Res. 2011;45(2):107-12. https://doi.org/10.1159/000324803
  » https://doi.org/10.1159/000324803
• ³³ Vogel GL, Chow LC, Carey CM, Schumacher GE, Takagi S. Effect of a calcium prerinse on salivary fluoride after a 228-ppm fluoride rinse. Caries Res. 2006;40(2):178-80. https://doi.org/10.1159/000091121
  » https://doi.org/10.1159/000091121
• ³⁴ Danelon M, Pessan JP, Prado KM, Ramos JP, Emerenciano NG, Moretto MJ, et al. Protective effect of fluoride varnish containing trimetaphosphate against dentin erosion and erosion/abrasion: an in vitro study. Caries Res. 2020;54(3):292-6. https://doi.org/10.1159/000505179
  » https://doi.org/10.1159/000505179
• ³⁵ Toledo PT, Delbem AC, Cannon ML, Sakamoto AE, Pedrini D. The effect of toothpaste with reduced concentration of fluoride-containing sodium trimetaphosphate and polyols on initial enamel erosion. Clin Oral Investig. 2022 Dec;26(12):7243-52. https://doi.org/10.1007/s00784-022-04684-7
  » https://doi.org/10.1007/s00784-022-04684-7
• ³⁶ Manarelli MM, Pessan JP, Delbem AC, Amaral JG, Paiva MF, Barbour ME. Protective effect of phosphates and fluoride on the dissolution of hydroxyapatite and their interactions with saliva. Caries Res. 2017;51(2):96-101. https://doi.org/10.1159/000452716
  » https://doi.org/10.1159/000452716
• ³⁷ Jager DH, Vieira AM, Ligtenberg AJ, Bronkhorst E, Huysmans MC, Vissink A. Effect of salivary factors on the susceptibility of hydroxyapatite to early erosion. Caries Res. 2011;45(6):532-7. https://doi.org/10.1159/000331938
  » https://doi.org/10.1159/000331938
• ³⁸ Moretto MJ, Magalhães AC, Sassaki KT, Delbem AC, Martinhon CC. Effect of different fluoride concentrations of experimental dentifrices on enamel erosion and abrasion. Caries Res. 2010;44(2):135-40. https://doi.org/10.1159/000302902
  » https://doi.org/10.1159/000302902
• ³⁹ Cruz NV, Pessan JP, Manarelli MM, Souza MD, Delbem AC. In vitro effect of low-fluoride toothpastes containing sodium trimetaphosphate on enamel erosion. Arch Oral Biol. 2015 Sep;60(9):1231-6. https://doi.org/10.1016/j.archoralbio.2015.05.010
  » https://doi.org/10.1016/j.archoralbio.2015.05.010

Publication Dates

• Publication in this collection
  09 Dec 2024
• Date of issue
  2024

History

• Received
  22 Jan 2024
• Reviewed
  13 Sept 2024
• Accepted
  13 Aug 2024