Remineralization potential of gum arabic versus casein phosphopeptide–amorphous calcium phosphate–fluoride for demineralized enamel in acidic challenges: In vitro study

Rasha R Hassan; Shereen H Ibrahim; Rawda H Abd ElAziz

doi:10.4103/jioh.jioh_258_21

ORIGINAL RESEARCH

Year : 2022 | Volume : 14 | Issue : 2 | Page : 195-202

Remineralization potential of gum arabic versus casein phosphopeptide–amorphous calcium phosphate–fluoride for demineralized enamel in acidic challenges: In vitro study

Rasha R Hassan, Shereen H Ibrahim, Rawda H Abd ElAziz
Department of Conservative Dentistry, Faculty of Dentistry, Cairo University, Cairo, Egypt

Date of Submission	22-Sep-2021
Date of Decision	11-Dec-2021
Date of Acceptance	05-Feb-2022
Date of Web Publication	26-Apr-2022

Correspondence Address:
Dr. Rawda H Abd ElAziz
Department of Conservative Dentistry, Faculty of Dentistry, Cairo University, 11 Al Saraya, St. Manial, Cairo 11553
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jioh.jioh_258_21

Abstract

Aim: To compare the remineralizing effect of gum arabic and casein phosphopeptide–amorphous calcium phosphate–fluoride (CPP–ACP–F). Materials and Methods: Twenty extracted sound human central incisors were selected and divided into four main groups (five specimens each) according to remineralization treatment: group 1, remineralization by gum arabic; group 2, MI Paste Plus CPP–ACP–F; group 3, gum arabic and CPP–ACP–F (MI Paste Plus); and group 4, artificial saliva. Surface microhardness analysis through Vickers hardness number and morphological evaluation using environmental scanning electron microscope were assessed initially to the sound enamel, demineralized enamel, and finally after 28 days of remineralization protocol and cycles of acidic challenges. Data were collected and statistically analyzed with one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test at the significance level P ≤ .05. Results: Intergroup comparisons showed that there was no significant difference between different groups regarding microhardness at baseline, after demineralization and remineralization (P > .05). However, intragroup comparisons showed that there was a significant difference between different time intervals in the gum arabic group and the MI paste group (P > .001). Conclusion: Both gum arabic and CPP–ACP–F were able to regain and maintain surface microhardness. CPP–ACP–F is a very potent remineralizing agent due to its biomimetic remineralization strategy featuring in the treatment of early enamel lesion. Gum arabic could be a promising remineralizing agent although it had a limited initial remineralizing potential. However, the painting on of CPP–ACP–F over the gum arabic varnish could not provide the desired synergistic remineralizing action.
Clinical Significance: Biomimetic remineralization of subsurface enamel lesion is the key to success in the management of early enamel lesion such as in CPP–ACP–F.

Keywords: Acidic Challenge, Biomimetic Remineralization, CPP–ACP–F, Enamel Remineralization, Gum Arabic, Laboratory Research

How to cite this article:
Hassan RR, Ibrahim SH, Abd ElAziz RH. Remineralization potential of gum arabic versus casein phosphopeptide–amorphous calcium phosphate–fluoride for demineralized enamel in acidic challenges: In vitro study. J Int Oral Health 2022;14:195-202

How to cite this URL:
Hassan RR, Ibrahim SH, Abd ElAziz RH. Remineralization potential of gum arabic versus casein phosphopeptide–amorphous calcium phosphate–fluoride for demineralized enamel in acidic challenges: In vitro study. J Int Oral Health [serial online] 2022 [cited 2023 Jun 7];14:195-202. Available from: https://www.jioh.org/text.asp?2022/14/2/195/344059

Introduction

The oral environment comprises continuous repetitive cycles of demineralization and remineralization that represent a major acidic challenge in front of the natural tooth structure. An important factor affecting the strength and hardness properties of a tooth structure is demineralization/remineralization ratio.^[1] As we know, dental enamel is one of the hardest materials of the human body.^[2] Dental caries occurs when enamel is lost due to the imbalance between the demineralization and the remineralization phases. Certainly, prevention can be accomplished when the remineralization phase is enhanced.^[3] Therefore, the best strategy for the prevention of caries is to focus on the methods of enhancing the remineralizing process with the help of different remineralizing agents.^[1]

Contemporarily, there is a variety of remineralizing agents available in the market such as fluoride, NovaMin, and casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) that helping in improving the remineralizing phase. CPP–ACP has shown to minimize demineralization and enhance the remineralization of enamel surface by controlling the bioavailability of ionic phosphate and calcium supersaturation in saliva to motivate the remineralization process.^[1]

Gum arabic is an extract from different African species of Acacia stems and branches. It consists of polysaccharides of high molecular weight and is rich in minerals such as calcium, magnesium, and potassium salts, which form arabinose, galactose, rhamnose, and glucuronic acid following hydrolysis.^[3] It is usually used as an additive for viscosity in food industries including gums, pastilles, and marshmallows. Several studies discussed the remineralization potential of gum arabic being possessed at a great concentration of calcium ions that would be able to substitute the lost minerals upon enamel demineralization and prevent any further demineralization to occur.^[3],[4] It was also reported that the Acacia arabica type of gum arabic inhibited early deposition of dental plaque, which is one of the causes of dental caries and gingivitis.^[4]

Therefore, the aim of this study was to compare the effect of remineralization potential of gum arabic versus casein phosphopeptide–amorphous calcium phosphate–F (CPP–ACP–F) on demineralized enamel subjected to acidic challenges. The null hypothesis tested was that there would be no difference in remineralizing potential between gum arabic and CPP–ACP–F.

Materials and Methods

Remineralizing agents

Two different materials were used in this study for a proposed remineralization protocol of demineralized enamel:

Specially prepared gum arabic varnish according to Onishi et al.^[3]

CPP–ACP–F: A commercially available MI Paste Plus (GC America Inc, Alsip, IL, USA).

Sample size calculation

Sample size was calculated with probability (power) 0.9, according to a previous study.^[5] We needed to study four experimental subjects and four control subjects to be able to reject the null hypothesis. To compensate for any loss of samples drop out, the sample size was increased to become by 20% (n = 5) for each group. The Type I error probability associated with this test of this null hypothesis is 0.5.

Specimen’s preparation

Twenty extracted sound human central incisors were selected for this study having coronal labial dimensions of 8 mm in width × 11 mm in length, which was checked by a digital caliper (Mitutoyo, Tokyo, Japan). The teeth were extracted either from diabetic patients or patients with compromised periodontal diseases according to the ethical regulations for manipulation of extracted teeth by the research ethics committee of the faculty. They were washed thoroughly under tap water and polished with fluoride-free polishing paste to remove any plaque or soft remnants; examined under a stereomicroscope to ensure that they were free from cracks, fractures, or any defects; and finally stored in the physiologic saline solution at room temperature till the beginning of the experiment.

Grouping of the specimen and study design

This in vitro study involved four main testing groups (five specimens each) according to the enamel surface treatment performed. Group 1 represents remineralization by gum arabic, group 2 represents remineralization by CPP–ACP–F, group 3 represents remineralization by both gum arabic and CPP–ACP–F, and group 4 represents the control group, artificial saliva. The selected teeth were randomly allocated in the different tested group. Within each group surface, microhardness and morphological evaluation using scanning electron microscope data were collected at three different circumstances, whereby a primary stage represented sound enamel, a secondary stage the demineralized enamel surface, and finally a tertiary stage after remineralization protocol and acidic challenges.

Specimen’s preparation

The selected teeth were embedded horizontally in chemically activated acrylic resin blocks from their lingual surfaces so that the tested labial enamel surfaces were looking outward and remained uncovered by resin. The labial surfaces were gently finished and polished with Sof-Lex disc (3M) with different abrasive sizes (medium, fine, and superfine) and mounted to a low-speed handpiece and copious amount of water irrigation to remove the aprismatic enamel.

Enamel demineralization procedures

All enamel initial microhardness values and analysis of the structural morphology were measured before the treatment as a primary stage. To induce demineralization of enamel, acid etching of enamel with 37.5% phosphoric acid was done for 90 s and then thoroughly rinsed with water and air-dried;^[6] the data were recorded as a secondary stage. This demineralization process using phosphoric acid would create microporosities with the removal of minimal amounts of mineral contents from the prismatic and interprismatic structures of enamel that would increase the enamel surface’s reactivity.

Remineralization protocols

Group 1: The gum arabic varnish was prepared in the National Research Centre, Cairo, Egypt. Material preparation was performed as follows. The gum arabic was dispersed in double distilled water in a concentration of 10 mg/mL as recommended by Onishi et al.^[3] using a magnetic stirrer bar under ambient temperature. This selected concentration when compared to a lower concentration has been proven to significantly inhibit the cell adherence of Streptococcus mutans and Streptococcus sobrinus. One coat of the prepared varnish was applied on the demineralized tested enamel surface with a varnish brush.

Group 2: A pea-sized amount of MI Paste Plus (CPP–ACP–F) was applied to the demineralized enamel surface using a varnish brush.

Group 3: One coat of gum arabic varnish was applied followed by the application of MI Paste Plus as an external source of calcium and phosphate.

Group 4: Specimens were soaked in artificial saliva at 37°C.

Demineralization and remineralization cycle

All specimens were subjected to demineralization and remineralization cycles according to Gao et al.^[7] The artificial saliva was prepared at the Laboratory of Biochemistry, Faculty of Pharmacy, Cairo University, Egypt. The artificial saliva was prepared by dissolving the following composition in water: 1.5 mmol/L CaCl₂, 0.9 mmol/L KH₂PO₄, 130 mmol/L KCl, 1.0 mmol/L NaN₃, and 20 mmol/L 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid. The pH 7.0 was adjusted with KOH (1 mmol/L). The prepared artificial saliva has close similarity to human saliva that can act as source of Ca and P ions in the remineralization cycle. For the demineralization cycle, a demineralizing solution of pH 4 was prepared by using 133 mmol/L NaCl and 50 mmol/L lactic acid. A demineralization cycle and a remineralization cycle were repeated daily for 28 consecutive days by immersing the specimens in 11.5 mL of artificial saliva for 23 h, and then they were immersed for 1 h in 11.5 mL of the demineralizing solution at 37°C. This immersion of 1-h duration in the demineralizing solution is nearly equivalent to the cumulative acidic challenge times of 24-h period inside the oral cavity.^[7]

Microhardness test procedure

Surface microhardness of the specimens (baseline, after demineralization, after 28 days of repeated cycles) was determined using a digital display Vickers microhardness tester (Shimadzu HMV-M Microhardness Tester; NewAge Testing Instruments Inc., Southampton, Pennsylvania) with a Vickers diamond indenter and a 20× objective lens. A load of 200 g was applied to the surface of the specimens for 15 s. Three indentations were made on the surface of each specimen and were equally placed over a circle and not closer than 0.5 mm to the adjacent indentations. The diagonals’ lengths of the indentations were measured by built-in scaled microscope, and Vickers values were converted into microhardness values.

Microhardness calculation: Microhardness was obtained using the following equation:

[INLINE 1]

where HV is Vickers hardness in kgf/mm², P is the load in kgf, and d is the length of the diagonals in millimeter.

Ultrastructural morphology analysis of enamel surface

The surface morphology of each tested group was examined using an environmental scanning electron microscope (ESEM) attached with an energy dispersive X-ray analyzer (ESEM–EDXA Unit; Main Defense Chemical Laboratory, Cairo, Egypt). For scanning electron microscopy (SEM) examination, the collected specimens were examined at 30 kV using the secondary electron large field detector (LFD) detector under the magnification (×4000) with a spot size 20 μm.

Statistical analysis

Numerical data were presented as mean and standard deviation values. Data were explored for normality by checking the data distribution, calculating the mean and median values, and using Kolmogorov–Smirnov and Shapiro–Wilk tests, and they showed parametric distribution. One-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test was used for intergroup comparisons, whereas one-way repeated measures ANOVA followed by comparison of main effects utilizing Bonferroni correction was used for intragroup comparisons. The significance level was set at P ≤ .05 within all tests. Statistical analysis was performed with IBM SPSS software program, version 26.0 for Windows.

Results

Microhardness results

Results of intergroup comparisons showed that there was no significant difference between different groups regarding microhardness at baseline, after demineralization and remineralization (P > .05). Results of intragroup comparisons showed that there was a significant difference between different time intervals in the gum arabic group and the MI paste group (P > .001). However, values recorded in the artificial saliva group and the gum arabic and MI paste group showed no significant difference between different time intervals (P > .05). Intergroup, intragroup, and post hoc pairwise comparisons are shown in [Table 1] and [Figure 1]A–C.

Table 1: Mean ± standard deviation (SD) of microhardness for different groups and time intervals

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Figure 1: (A) Bar chart showing average microhardness for different groups and time intervals. (B) Line chart showing average microhardness for different groups and time intervals. (C) Bar chart showing average percentage change (%) in microhardness for different groups

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There was a significant difference between different groups regarding microhardness percentage of change (%) between demineralization and remineralization (P < .001). The MI paste group (456.89 ± 48.94) showed the highest mean value followed by the artificial saliva group (184.15 ± 160.16) and gum arabic group (132.64 ± 65.81), whereas the gum arabic and MI paste group (66.40 ± 47.05) showed the lowest mean value. Post hoc pairwise comparisons showed that the MI paste group had a significantly higher mean value than all other groups (P < .001).

Elemental analysis of enamel surface after different treatment protocols

ESEM examination of sound enamel revealed smooth enamel surface and some microporosities were apparent [Figure 2], whereas SEM examination of demineralized enamel showed loss of uniform structure of enamel with uniform mineral depletion. Enamel rods and interprismatic structures showed etching-like appearance due to demineralization. Meanwhile, an increase in the enamel microporosities and pitting erosion showed the honeycomb appearance [Figure 3].

Figure 2: Environmental scanning electron microscope (ESEM) for sound enamel

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Figure 3: Environmental scanning electron microscope (ESEM) for demineralized enamel

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ESEM examination of demineralized enamel remineralized by gum arabic revealed the presence of surface irregularities with minimal mineral deposition. Pitting erosion and some microporosities were still present [Figure 4]. However, scanning electron micrograph of the demineralized enamel remineralized by MI Paste Plus (CPP–ACP–F) showed uniform mineral-rich layer deposition covering all etching-like patterns [Figure 5]. On the contrary, scanning electron micrograph of demineralized enamel remineralized by gum arabic and MI Paste Plus (CPP–ACP–F) revealed the deposition of nonuniform mineral-rich layer over the demineralized enamel surface with the presence of some surface irregularities and microporosities [Figure 6]. SEM examination of demineralized enamel remineralized by artificial saliva showed surface irregularities with minimal mineral deposition, and the presence of pitting and microporosities [Figure 7].

Figure 4: Environmental scanning electron microscope (ESEM) for remineralized specimen with gum arabic

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Figure 5: Environmental scanning electron microscope (ESEM) for remineralized specimen with MI Paste Plus

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Figure 6: Environmental scanning electron microscope (ESEM) of remineralized specimen with gum arabic and MI Paste Plus

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Figure 7: Environmental scanning electron microscope (ESEM) of remineralized specimen with artificial saliva

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Discussion

Natural tooth structure is continuously facing different challenges in the oral environment such as chemical, thermal, biological, and mechanical ones. The God-created hierarchy of the tooth structure provides it with the ability to withstand functioning in this atmosphere as long as there is a balance between different environmental factors. The unbalanced acidic challenge is a major factor that has a detrimental effect on the tooth structure.^[1] As the presence of preventative mechanism would be better than cure at this stage, several studies were conducted on the different remineralizing protocols that could be used for the prevention of tooth demineralization and enhance the remineralization if initial mineral loss occurs.^{[1],[2],[6],[8]} Here comes the importance of this study, which was carried out to compare the effect of remineralization potential of a natural product known as gum arabic (Acacia senegal) versus CPP–ACP–F on demineralized enamel subjected to acidic challenges.

Demineralization and remineralization of the tooth enamel could be assessed by several methods; one of these methods is surface microhardness assessment as their values change greatly upon mineral loss or gain.^[5],[8] Also the analysis of changes in the ultrastructural morphology is another relevant technique that could be performed by SEM where the morphological patterns of mineral loss and/or gain could be accurately visualized.^[8],[9] In this study, surface microhardness of the specimens (baseline, after demineralization, and after 28 days of repeated cycles) was determined. This reliable method assesses the resistance of the substance against plastic deformation from a standard load.^[10] Evaluation of the same specimen during the different stages of the study gives an accurate prediction of the amount of mineral changes during the whole study timelines, so intra- and intergroup comparison would be valuable. The scanning electron microscope was used as an adjunct qualitative exploratory tool to explore the changes that resulted from different studies’ stages from sound to demineralization ending to different remineralization protocols and acidic challenges.

In this study, microhardness assessment revealed that there was no statistically significant difference between different study groups at different stages of the study: baseline, after demineralization, and remineralization (P > .05). However, within each group, there was a statistically significant difference at different stages in the gum arabic group and MI paste group (P > .001). On the contrary, there was no statistically significant difference in the artificial saliva group and the gum arabic and MI paste group at different time intervals (P > .05). Regarding the percentage of change (%) between demineralization and remineralization, there was a significant difference between different groups (P < .001), where the MI paste group showed the highest mean value followed by the artificial saliva group and the gum arabic group, whereas the gum arabic and the MI paste group showed the lowest mean value.

These results could be interpreted and correlated to the ultrastructural morphological changes assessed by a scanning electron microscope that revealed that smooth enamel surface and some microporosities are apparent in sound enamel [Figure 2]. However, the SEM examination of demineralized enamel showed loss of uniform structure of enamel with uniform mineral depletion. Enamel rods and interprismatic structures appeared that may be due to demineralization giving etching-like appearance. In addition, increase in the enamel microporosities and pitting erosion gives honeycomb appearance [Figure 3]. Examination of demineralized enamel remineralized by gum arabic revealed the presence of surface irregularities with minimal mineral deposition. Pitting erosion and some microporosities are still present [Figure 4]. However, scanning electron micrograph of demineralized enamel remineralized by MI Paste Plus (CPP–ACP–F) showed uniform mineral-rich layer deposition covering all etching-like patterns [Figure 5]. On the contrary, micrograph of demineralized enamel remineralized by gum arabic and MI Paste Plus (CPP–ACP–F) revealed the deposition of nonuniform mineral-rich layer over the demineralized enamel surface with the presence of some surface irregularities and microporosities [Figure 6]. Examination of demineralized enamel remineralized by artificial saliva showed surface irregularities with minimal mineral deposition and presence of pitting and microporosities [Figure 7].

These results designate a greater remineralization potential of CPP–ACP–F compared to the other groups. This might be attributed to the powerful bioactive ingredients of CPP–ACP–F, using the nonclassical theory of biomimetic remineralization,^[11] whereby casein phosphopeptide is responsible for gathering the amorphous calcium phosphate particles to form a complex that was deposited on the demineralized enamel surface, diffuse into the body of the lesion, and also help this complex to remain stable upon facing acidic challenges.^{[12],[13],[14],[15],[16],[17]} The CPP–ACP–F provides a high level of calcium and phosphate ions that were visualized by SEM as a uniform mineral-rich layer deposition covering all etching-like patterns [Figure 5]. The presence of fluoride in CPP–ACP–F, known with its antibacterial properties and its classical pathway for remineralization to form fluoroapatite crystals, which are stable to acidic challenges,^[11] empowers the remineralizing potential of CPP–ACP–F, providing both surface and subsurface remineralization.^[1],[11] The prepared artificial saliva has close similarity to human saliva that can act as a source of Ca and P ions in remineralization cycle; however, it did not contain fluoride; hence, it had limited remineralizing potential as revealed by microhardness results of this study. This was confirmed by qualitative assessment of structural morphology using SEM that revealed the presence of surface irregularities with minimal mineral deposition and the presence of pitting and microporosities This was in accordance with several previous studies^{[11],[12],[13],[14]} where it was affirmed that for the treatment of early enamel lesions, nonclassical remineralization, whether used alone or in combination with fluoride, showed a positive outcome.^[11],[12] The remineralizing potential of CPP–ACP with fluoride was significantly better than that of CPP–ACP; thus, it can be used in preventing erosive tooth wear from acidic beverages.^[13] Remineralizing agents containing different calcium phosphate formulas and fluoride have increased remineralization potential compared to artificial saliva.^[14]

The result of this study regarding the gum arabic group and the gum arabic with MI Paste Plus group revealed that both groups were able to restore back effectively the lost minerals after demineralization, and this was in accordance with other research,^[3],[4] suggesting that gum arabic is a promising remineralizing agent.The percentage of change (%) between demineralization–remineralization revealed that there was no statistically significant difference among the following groups; the gum arabic group (132.64 ± 65.81) also, the gum arabic and MI Paste Plus group (66.40 ± 47.05) which showed the lowest mean value and the artificial saliva group (P < .001). This might designate the limited remineralizing potential of gum arabic. This might be attributed to its inherent polysaccharide nature of high molecular weight that allowed only for surface remineralization but was not able to remineralize subsurface lesion and hence was not able to resist the acidic challenges as shown by microhardness results and explored by SEM the deposition of nonuniform mineral-rich layer over the demineralized enamel surface with the presence of some surface irregularities and microporosities. This mode of action might be similar to that of fluoride. This might be reported in limited numbers of literature on gum arabic,^[3],[4],[9] as they suggested that gum arabic increases the remineralization of demineralized enamel vitro due to the mineral contents of calcium, potassium, and magnesium within its polysaccharide structure besides its antibacterial property due to the presence of cyanogenic glycosides and several different types of enzymes, such as oxidases, peroxidases, and pectinases.^[18],[19] It was suggested previously that addition of calcium and possibly phosphate to gum arabic in the remineralization solution as an extra source of minerals might enhance its remineralizing potential.^[4] However, in this study, the painting of MI Paste Plus over the gum arabic could not perform the required synergistic action and could not resist the acidic challenges. This might denote that the gum arabic varnish acted as a barrier against MI Paste Plus to perform its remineralizing action. The null hypothesis was accepted.

Conclusion

Although this study is in vitro type, all concerns were provided in the methodology and designing of every step in this study to simulate the oral environmental condition to attain results with a clinical significance. Considering the restrictions and limitations of this in vitro study, it could be settled that:

Both gum arabic and CPP–ACP–F were able to regain and maintain surface microhardness.

CPP–ACP–F is a very potent remineralizing agent with high resistance to acidic challenge due to its biomimetic remineralization strategy. It would be very successful in the treatment of early enamel lesion.

Gum arabic is a promising remineralizing agent, but it had a limited initial remineralizing potential; however, it could not face the acidic challenge; thus, the addition of other remineralizing agents to empower its action is strongly advised. Therefore, it could be used for the prevention of caries occurrence rather than the treatment of early enamel lesion.

Artificial saliva being deprived from fluoride had a limited remineralizing potential, although it gives indication that enriching the natural saliva with mineral through different remineralizing protocols is essential to enhance the caries preventive protocol.

The paint on of CPP–ACP–F over the gum arabic could not provide the desired synergistic remineralizing action.

Clinical significance

Remineralization of subsurface enamel lesion is the key to success in the management of early enamel lesion. A potent remineralizing agent might be characterized by taking the nonclassical pathway for biomimetic remineralization such as CPP–ACP–F. Restoring the minerals lost at the surface only is not enough as provided by fluoride varnish and gum arabic could not withstand the continuous acidic challenges of the oral environment.

Recommendation

Further in vitro studies are required with different formulations of gum arabic to reach the desired remineralizing potential by mixing it with other remineralizing agents and not painting on.

Acknowledgement

Not applicable.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Author contributions

RRH: Primary author, concept designs, data extraction, writing original draft, methodology, resources, conceptualization, validation, data curation, manuscript review, and guarantor.

RHA: Corresponding author, concept designs, data extraction, writing original draft, methodology, resources, conceptualization, validation, data curation, manuscript review, and guarantor.

SHI: Concept designs, data extraction, writing original draft, methodology, resources, conceptualization, validation, data curation, manuscript review, and guarantor.

Ethical policy and institutional review board statement

Not applicable.

Patient declaration of consent

Not applicable.

Data availability statement

Not applicable.

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