ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2412 |
Comparative Efficacy of Different Remineralizing Agents and Iontophoresis-enhanced Remineralization on Surface Microhardness: An In Vitro Study
1Gupta Dental Professionals, Jammu, Jammu and Kashmir, India
2–5Department of Conservative Dentistry and Endodontics, Maharishi Markandeshwar College of Dental Sciences and Research, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
6Department of Prosthodontics, Maharishi Markandeshwar College of Dental Sciences and Research, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
Corresponding Author: Swati Chhabra, Department of Conservative Dentistry and Endodontics, Maharishi Markandeshwar College of Dental Sciences and Research, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India, Phone: +91 9034743780, e-mail: endoswatichhabra@gmail.com
Received: 05 March 2023; Accepted: 05 April 2024; Published on: 17 May 2024
ABSTRACT
Aim: To compare the efficacy of different remineralizing agents and to evaluate the beneficial role of iontophoresis along with remineralizing agents.
Materials and methods: A total of 120 specimens were divided into four groups—control group (n = 15), demineralized group (n = 15), remineralized group (n = 45), and remineralized + iontophoresis group (n = 45). The remineralized group was further divided into subgroup A = casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (n = 15), subgroup B = 8% grape seed extract (GSE) (n = 15), and subgroup C = NovaMin (n = 15). The remineralized + iontophoresis group was also divided into three subgroups—subgroup A1 = CPP-ACP + iontophoresis (n = 15), subgroup B1 = GSE + iontophoresis (n = 15), and subgroup C1 = NovaMin + iontophoresis (n = 15). Vickers microhardness test was used to measure the changes in the hardness of enamel, which occurred in three phases (i.e., control, after demineralization, and after treatment). Data was recorded and statistically analyzed.
Results: The mean microhardness for the control group was highest (359.72 kg/mm2 ± 15.3), while the demineralized group had the lowest value (294.2 kg/mm2 ± 16.3), which was statistically significant. Among the remineralizing agents, results were nonsignificant; however, CPP-ACP (330.4 ± 11.3 kg/mm2) had shown the highest mean microhardness values. On intragroup comparison, data was statistically significant for each group, with an increase in microhardness values in the remineralized + iontophoresis group compared to using remineralizing agent alone.
Conclusion: Mean microhardness values in descending order—control group > remineralized + iontophoresis group > remineralized group > demineralized group. CPP-ACP application, along with iontophoresis, had superior remineralizing efficiency compared to other groups.
Clinical significance: A pivotal goal of modern dentistry is to manage the noncavitated lesions noninvasively through the remineralization process in order to prevent disease progression and to improve esthetics, strength, and function. Iontophoresis has been shown to effectively accelerate the remineralization speed by driving the remineralizing agents deep into the subsurface enamel lesion in less time, leading to optimal enamel surface hardness.
How to cite this article: Gupta A, Kukreja N, Thakur A, et al. Comparative Efficacy of Different Remineralizing Agents and Iontophoresis-enhanced Remineralization on Surface Microhardness: An In Vitro Study. World J Dent 2024;15(4):331–335.
Source of support: Nil
Conflict of interest: None
Keywords: Casein phosphopeptide-amorphous calcium phosphate, Grape seed extract, Iontophoresis, NovaMin, Vicker’s hardness
INTRODUCTION
Dental caries, the most prevalent disease of mankind, is a multifactorial disease that occurs through a complex interaction between acid-producing bacteria, fermentable carbohydrates, and many host factors, including teeth and saliva overtime.1 It is a dynamic process characterized by alternating periods of demineralization and remineralization. A decrease in oral pH is due to the organic acids formed by the action of bacteria (in plaque) in the presence of dietary carbohydrates, which cause demineralization of the tooth as the oral environment becomes undersaturated with mineral ions compared to tooth’s mineral content but as the pH rises, the mineral gets deposited back to the tooth structure.2,3 As the pH drops below the critical pH of 5.5, decalcification occurs, which is visualized clinically as a white spot enamel lesion.4
Managing the noncavitated caries lesions noninvasively through remineralization highlights the concept of “prevention is better than cure” rather than simply “drill and fill.”5 Dental remineralization is basically a process of driving the lost minerals from the surrounding environment (saliva or biofilm) into the partially demineralized tooth structures. Remineralization can occur naturally or be induced by various therapies. Numerous remineralizing agents have been proven to be efficacious in preventing and reversing enamel subsurface lesions. The nonfluoride remineralizing systems can be categorized into (A) biomimetic systems that involve nanohydroxyapatite, leucine-rich amelogenin peptides, P11-4 peptides, electrically accelerated and enhanced remineralization and polyamidodentrimers and (B) fluoride boosters—the techniques that repair initial caries by enhancing fluoride efficacy.
Various fluoride boosters include calcium-phosphate systems [like casein phosphopeptide-amorphous calcium phosphate (CPP-ACP); NovaMin, enamelon], polyphosphate systems (sodium trimetaphosphate, calcium glycerophosphate, and sodium hexametaphosphate) and natural products [grape seed extract (GSE), hesperidin].6
Casein phosphopeptide-amorphous calcium phosphate with an increase in pH, the level of bound ACP increases, which stabilizes the free calcium and phosphate and thus delivers an anticalculus and anticaries action, thereby reducing enamel demineralization and promoting its remineralization.7
Proanthocyanidin present in GSE strengthens collagen-based tissues by increasing collagen synthesis and crosslinks and also by accelerating the conversion of soluble collagen to insoluble.8
NovaMin releases sodium ions in the oral cavity, which are then replaced with the hydrogen in the tooth to liberate calcium and phosphate ions, resulting in an accelerated pH increase.9
Remineralization using iontophoresis is one of the ideal ways of practicing noninvasive methods as it desires not only to save the tooth but also to strengthen it. The simple theory of iontophoresis suggests that charged particles (also anomalously uncharged particles) will move as far as possible in relation to the opposing polarity electrode until the electric field reduces.10
Therefore, the best strategy for caries management is to focus on methods of improving the remineralization process with the aid of remineralizing agents. However, as the literature evidence is limited and only a few studies have made the correlation between the enamel hardness and enamel mineral content Therefore, the purpose of this in vitro study was to compare the effectiveness of three different remineralizing agents (CPP-ACP, NovaMin, GSE) and iontophoresis-enhanced remineralization by using enamel microhardness test. The null hypothesis was that there would be no statistical difference in the mineral composition and microhardness between the tested materials; also, the microsurface of all the specimens would be the same.
MATERIALS AND METHODS
This in vitro study was conducted in 2022 at the Department Conservative and Endodontics, Maharishi Markandeshwar College of Dental Sciences and Research, Maharishi Markandeshwar (Deemed to be University), Ambala, Haryana, India. The research protocol was permitted by the Institutional Ethics Committee (IES/1580).
A total of 60 human premolar teeth extracted for orthodontic reasons, caries-free, unrestored, and without developmental defects, were selected for the study.
Preparation of Samples
Samples (n = 60) were cleaned ultrasonically and stored in 0.1% thymol (antifungal agent) for 24 hours prior to the start of the study and then in distilled water for storage till use. Samples were sectioned buccolingually (horizontally) into two equal halves, resulting in 120 specimens (n = 120). The surface of the test specimens was polished using 400-, 600-, and 1200-grit abrasive papers and water-based polishing cream using a slow-speed handpiece to increase the accuracy of microhardness measurements. Specimens were embedded in cold cure resin in cylindrical molds with the sectioned surface facing outward and stored in artificial saliva.
Grouping of Samples
A total of 120 specimens were divided into the following groups (Fig. 1):
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Control group: Without demineralization (n = 15).
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Demineralized group: Demineralized specimen (n = 15).
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Remineralized group: (n = 45).
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Subgroup A: CPP-ACP (GC Tooth Mousse paste) (n = 15).
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Subgroup B: GSE (n = 15).
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Subgroup C: NovaMin (Sensodyne–repair and protect) (n = 15).
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Remineralized group + iontophoresis: (n = 45).
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Subgroup A1: CPP-ACP + iontophoresis (n = 15).
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Subgroup B1: GSE + iontophoresis (n = 15).
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Subgroup C1: NovaMin + iontophoresis(n = 15).
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Fig. 1: Grouping of samples used in the study
Control Group
Specimens in the control group (n = 15) were washed with deionized water and kept in artificial saliva for 14 days. Baseline surface microhardness was determined for specimens in the control group using a Vickers microhardness tester.
Demineralization
To induce demineralization, 105 specimens were immersed in the demineralizing solution (2.2 mm of CaCl2·2H2O, 2.2 mM NaH2PO4·7H2O, 0.05 M C3H6O3, and 0.2 ppm fluoride, pH = 4.5) and incubated at 37°C for 96 hours. Demineralized specimens were then stored in artificial saliva solution, which was changed regularly after 24 hours. Out of 105 demineralized specimens, 15 specimens were randomly selected and designated as a demineralized group, which were tested for surface microhardness after 14 days.
The remaining 90 specimens were further divided equally into remineralized and remineralized + iontophoresis groups.
Remineralized Group
Subgroup A, B, and C (n = 15 each) were subjected to the respective remineralizing agents [GC Tooth Mousse paste containing CPP-ACP, GSE gel (proanthocyanidin, gallic acid, epicatechin, catechin, sodium hydroxide, sodium carboxymethyl cellulose, and distilled water), NovaMin containing Sensodyne toothpaste] for a period of 3 minutes, twice a day, for 14 days with the help of an applicator tip. The specimens were stored at room temperature in artificial saliva solution after washing with deionized water and tested for microhardness.
Remineralized Group + Iontophoresis
Specimens of test group A, B1, and C1 (n = 15 each) were subjected to respective remineralizing agents, which were applied to the lesions as a paste (ACP-CPP and NovaMin) and gel (GSE) formulation filled in a small plastic tube to serve as a reservoir of mineral material. An iontophoretic electric field was created by placing the suitable metal electrode into the remineralizing agent reservoir with the opposing polarity electrode placed in a Petri dish filled with sodium agarose gel to accelerate the movement of agents into the lesion (electrically shortest path to the opposing electrode). The electric power supply was switched on with the input voltage of 230V at 50 Hz frequency and a maximum current of 5 mA for 30 minutes, which accelerated the movement of remineralizing agent toward the opposite polarity electrode, that is, away from the electrode of similar polarity. Apparently, the specimens were then tested for microhardness.
Vickers Microhardness Testing
Test specimens were placed and stabilized on the tester stage. Two pyramid-shaped indentations were made at a 20 µm distance, making two testing sites per specimen with a rate of 500 gm (4.9 N) load for 20 seconds. From these two indentations, the average (mean) microhardness of specimens was determined.
Statistical Methods
The surface microhardness was compared among different study groups using one-way analysis of variance (ANOVA) Tukey’s post hoc tests, and the Wilcoxon rank test. Data was analyzed using Statistical Package for the Social Sciences (SPSS) 21 version (SPSS Inc., Chicago, Illinois, United States of America). The level of significance is set at p ≤ 0.05.
RESULTS
The statistical parameters were described in terms of mean and standard deviation for each group, as depicted in Table 1. The mean microhardness for the control group was the highest (359.72 kg/mm2 ± 15.3), while the demineralized group had the lowest value (294.2 kg/mm2 ± 16.3), which was statistically significant. On intragroup comparison, data was statistically significant for each group; there is an increase in microhardness values in remineralized + iontophoresis (CPP-ACP—350.13 kg/mm2, GSE—328.77 kg/mm2, and NovaMin 343.44 kg/mm) group compared to remineralizing agent alone (Table 2). On intergroup comparison, results were nonsignificant; however, subgroup A (CPP-ACP) had shown the highest microhardness value (330.4 ± 11.3 kg/mm2) after 14 days of treatment, followed by subgroup C (NovaMin 323.9 ± 6.7 kg/mm2) and subgroup B (GSE 315.4 ± 9.2 kg/mm2).
Group A (CPP-ACP) |
Group B (GSE) |
Group C (NovaMin) |
Group A vs B | Group A vs C | Group B vs C | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Mean | Standard deviation (SD) | Mean | SD | Mean | SD | F | p-value | p-value | p-value | p-value | |
Control | 359.42 | 17.29 | 358.74 | 20.00 | 361.00 | 14.50 | 0.044 | 0.957 | 0.996 | 0.978 | 0.955 |
Demineralized | 291.86 | 17.20 | 296.40 | 15.77 | 298.06 | 11.96 | 0.449 | 0.643 | 0.782 | 0.635 | 0.967 |
Remineralized | 330.43 | 12.45 | 315.44 | 9.71 | 323.42 | 7.06 | 5.644 | 0.009 | 0.006 | 0.276 | 0.193 |
Remineralized + iontophoresis | 350.13 | 18.38 | 328.77 | 11.16 | 343.44 | 10.85 | 6.175 | 0.006 | 0.005 | 0.537 | 0.064 |
Groups | Subgroups | Mean | SD | Standard error mean | t | p-value | Mean difference | SD difference |
---|---|---|---|---|---|---|---|---|
Group A (CPP-ACP) | Remineralized | 330.43 | 12.45 | 3.94 | −7.578 | 0.001 | −19.70 | 8.22 |
Remineralized + iontophoresis | 350.13 | 18.38 | 5.81 | |||||
Group B (GSE) | Remineralized | 315.44 | 9.71 | 3.07 | −9.901 | 0.001 | −13.33 | 4.26 |
Remineralized + iontophoresis | 328.77 | 11.16 | 3.53 | |||||
Group C (NovaMin) | Remineralized | 323.42 | 7.06 | 2.23 | −8.511 | 0.001 | −20.02 | 7.44 |
Remineralized + iontophoresis | 343.44 | 10.85 | 3.43 |
DISCUSSION
Vickers’s microhardness testing was used as it is a simple, fast, reliable, and least destructive method to determine demineralization and remineralization, and assess the hardness of materials within the range of microhardness test loads (typically 1–1000 gm). Pyramid-shaped indent obtained in Vickers microhardness testing is quite accurate to measure and to detect visually and digitally, whereas the rhomboid-shaped indent observed with Knoop hardness testing with opposing surfaces parallel to each other makes it difficult to detect errors.11,12
The specimens were stored in a demineralizing solution to produce artificial white spot lesions following the Amaechi protocol. In the demineralizing solution, calcium and phosphate concentration was kept at a 50% saturation level so as to cause dissolution of subsurface enamel only, and fluoride was added to prevent demineralization at the surface by forming fluorapatite, thus simulating the naturally occurring early enamel caries with intact surface layer.3 In order to standardize or to reduce the variability of conditions, artificial saliva was used and renewed daily to prevent any bacterial contamination and to provide new ions for remineralization. The remineralization potential of the agents was evaluated on the 14th day, as it takes a minimum of 14 days for any toothpaste/gel to express their desired therapeutic effect.
The control group showed the highest mean surface microhardness values, followed by remineralized + iontophoresis group, then by the remineralized group, and least by the demineralized group. The demineralized group showed the lowest mean surface microhardness as the tooth enamel subjected to demineralization undergoes significant loss of surface hardness due to loss of minerals when compared to that of intact tooth structure. The results of this study were in agreement with the studies conducted by Thakur et al. and Rai et al., who concluded that the application of remineralizing paste proved potent in improving remineralization in the demineralized enamel surface.13,14
After the 30-minute application of remineralizing agent along with iontophoresis, the mean microhardness of specimens was higher compared to the specimens subjected to remineralizing agent alone. The result is in concurrence with the studies done by Wilson et al., Zhang et al., and Yanko.15-17 Iontophoresis has been shown to effectively accelerate the mineralization speed in less time, almost 15 times more than that occurred by simple diffusion.16 Also, it effectively enhances the penetration of remineralizing agents deep into the lesion by spontaneous deposition of minerals (calcium, phosphate, and fluoride) onto crystallite remnants.18 Iontophoresis significantly enhanced the remineralization kinetics of the remineralizing agent, which is otherwise a slow and time-consuming process.16
Among the remineralizing agents used in the study, CPP-ACP showed a maximum increase in microhardness value followed by subgroups C and B in both remineralized and remineralized + iontophoresis groups. CPP-ACP, due to the presence of a supersaturation state of calcium and phosphate ions in close approximation to enamel, has the capability to remineralize the deeper areas of the lesion because of its small size (1.5 nm radius of nanocomplex).19,20 NovaMin has the disadvantage of disintegrating as soon as it comes in contact with saliva or any body fluids and loses some of its efficacy by the time it reaches the tooth surface.12 The action of GSE is restricted to the surface layer only because of the presence of high molecular weight proanthocyanidins in GSE that prevents its penetration deep into the underlying layers, resulting in porous underneath structure, and it interacts majorly with the organic portion through proanthocyanidins-collagen interactions thus more effective in dentin remineralization than enamel surface remineralization.21,22
Casein phosphopeptide-amorphous calcium phosphate forms nanocomplexes of calcium and phosphate ions, preventing the growth of the nanoclusters to this critical size required for nucleation and phase transformation, due to which movement of ions can be accelerated when exposed to a few mill amperes of electric current whereas GSE shows restricted penetration of ions deeper into the lesion due to its high molecular weight.23,21 CPP-ACP also behaves as a reservoir of the neutral calcium phosphate ion species (CaHPO40). Under low pH conditions in the oral cavity (produced by plaque bacteria), CPP-ACP buffers the plaque pH by dissociating to calcium, phosphate, and neutral ions. The diffusion of these ions into the subsurface lesion is the rate-limiting step in subsurface lesion remineralization when remineralization occurs naturally.
Though a wide variety of excellent new materials and treatment modalities have resulted in remarkable improvement in remineralization phenomenon iontophoresis has emerged to be a breakthrough to ease the process by boosting tooth’s natural repair process. In this study, all three remineralizing agents could significantly remineralize the artificial enamel caries, but complete remineralization did not occur for any of the remineralizing agents tested. The study concluded that microhardness values at baseline change after demineralization, after the application of remineralizing agent, and after the application of remineralizing agent along with iontophoresis in the order as control group > remineralized+ iontophoresis group > remineralized group > demineralized group.
The limitation of the study was the short duration of the experiment and the inability to fully reflect the in vivo conditions; thus the effect of masticatory forces and chewing on the retention of the remineralizing agents was not evaluated, and the results may vary in in vivo conditions depending upon the influence of saliva and oral microbial flora. Further investigations should be undertaken with an increased number of samples in order to evaluate the long-term effect of the application of these agents.
CONCLUSION
The results showed that CPP-ACP has a better remineralizing potential among the three agents and, when used along with iontophoresis, has been shown to impart remineralization within the subsurface enamel lesion over a shorter duration of time. The utilization of the iontophoresis will provide improved remineralization of incipient caries and prove to be a better minimal invasive method.
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