ORIGINAL RESEARCH


https://doi.org/10.5005/jp-journals-10015-2235
World Journal of Dentistry
Volume 14 | Issue 5 | Year 2023

Effect of Three Different Remineralizing Agents on In Vitro Enamel Demineralization: Using Vickers Hardness Test and SEM-EDX


Shakuntala B Siddaiah1, Divya V Mehta2

1,2Department of Pediatric & Preventive Dentistry, RajaRajeswari Dental College & Hospital, Bengaluru, Karnataka, India

Corresponding Author: Divya V Mehta, Department of Pediatric & Preventive Dentistry, RajaRajeswari Dental College & Hospital, Bengaluru, Karnataka, India, Phone: +91 9900295444, e-mail: divyavmht@gmail.com

Received on: 01 April 2023; Accepted on: 02 May 2023; Published on: 02 August 2023

ABSTRACT

Aim: The present in vitro study aimed to evaluate and compare the remineralization potential of—2% sodium fluoride (NaF), 2% NaF along with Psoralea corylifolia L. (Leguminosae) (Bakuchi), and white mineral trioxide aggregate (MTA) using Vickers hardness number (VHN) and scanning electron microscopy–energy dispersive X-ray analyzer (SEM-EDX).

Materials and methods: The present in vitro study was conducted on 45 sound premolars, sectioned into two parts, further embedded in acrylic resins, polished, and divided into three groups of normal, demineralized, and remineralized teeth (n = 15). The remineralized teeth were further subdivided into three groups (n = 15) as remineralized with 2% NaF, 2% NaF, and Psoralea corylifolia L. (Bakuchi) and white MTA. Specimens of each group were treated with the above-mentioned remineralizing agents and then subjected to VHN and SEM-EDX for further evaluation.

Results: The test results showed significantly the highest VHN and the emission peak of elements under the EDX test, such as calcium (Ca), phosphorous (P), oxygen, and fluorine (F) with the group remineralized with NaF + Bakuchi (group III-B).

Conclusion: Sodium fluoride (NaF) + Bakuchi showed effective results in VHN and SEM-EDX with no antagonist interaction. Thus, Psoralea corylifolia L. has a remineralizing effect on the tooth and can be used as a herbal extract for effective reduction in pathogenic bacteria.

Clinical significance: Psoralea corylifolia L. ( Bakuchi), when mixed with NaF, has great potential for use in dentifrices and mouthwash and has revealed appreciable results on remineralization of artificially demineralized enamel lesions when compared to the MTA and NaF alone.

How to cite this article: Siddaiah SB, Mehta DV. Effect of Three Different Remineralizing Agents on In Vitro Enamel Demineralization: Using Vickers Hardness Test and SEM-EDX. World J Dent 2023;14(5):402–408.

Source of support: Nil

Conflict of interest: None

Keywords:Psoralea corylifolia L. (Bakuchi), Remineralization, Scanning electron microscopy–energy dispersive X-ray analyzer, Sodium fluoride, Vickers hardness number

INTRODUCTION

Dental caries is a disease of the hard tissues of the teeth caused by interactions between cariogenic bacteria in dental plaque and fermentable carbohydrates, mainly sugars.1

Demineralization of teeth by erosion is caused by frequent contact between teeth surface and acids resulting in the movement of hydroxyapatite (HAP) and diffusion of calcium (Ca) and phosphate ions toward the enamel surface–the process of remineralization restores these minerals again into HAP crystal lattice as a natural repair mechanism.1,3

Remineralization procedures are an ideal way of regeneration of lost tooth structure where a preventive approach of identification, conservation, and nonrestorative treatment of incipient caries saves both dental manpower, expense, and suffering for the patient.4

Small amounts of fluoride provide some degree of cariostatic effect, and a great number of fluoride-containing dental materials (e.g., glass ionomer-based materials, composites, adhesives, and varnishes) and oral care products (mouth rinse, toothpaste, topical gels), have been developed and commercialized so far. However, fluoride has a dose-response relationship, and overdose/overuse of fluoride toothpaste may lead to adverse effects like fluorosis.

The recent advances are based on biomimetic remineralizing materials, having the capability to create apatite crystals within the completely demineralized collagen fibers. Mineral trioxide aggregate (MTA) is an example that has high alkaline pH, antibacterial, biocompatibility, radiopacity, good closure properties, and can interact with dentine during intrafibrillar deposition.5

Natural products have been used as folk medicines for thousands of years and are promising sources of potential agents in the prevention of oral diseases, particularly plaque-related diseases such as dental caries.6

Psoralea corylifolia L. has a significant role in therapeutics in Ayurvedic medicines. The family Leguminosae is among the largest families of flowering plants, with 500 genera containing medicinal qualities, for example, Psoralea, Indigofera, Pongamia, and Alhagi.7

In India, due to the cold weather of the Himalayas, it is found as a wild weed, also seen in Mumbai, Bengal, Uttar Pradesh, Rajasthan, Karnataka, Bihar, and Deccan.7

Psoralea corylifolia L. is an erect herb with a height of 30–180 cm; it grows annually and gets mature in November. The plant yields for 5–7 years.7

Phytochemicals such as flavones, coumarins, monoterpenes, chalcones, lipids, resins, stigma steroids, and flavonoids are rich in Psoralea corylifolia L., where the bioactive compounds like coumarins, are concentrated in the seeds.7 The seed is the nucleus of every plant and tree, and it is pregnant with all qualitative aspects of health and healing with elemental harmony.

Psoralea corylifolia L. (Bakuchi), in traditional Chinese medicine, has been studied as an antimicrobial agent, is effective against Streptococcus mutans, and has shown remineralizing effects in combination with sodium fluoride (NaF).5 It has vasodilator, pigmentors, antitumor, antibacterial, cytotoxic, and antihelminthic properties and is locally used for alopecia, inflammation, leucoderma, leprosy, psoriasis, and eczema. Psoralea corylifolia L. (Bakuchi) seeds are a repository of the mineral patch, which contributes to bone and teeth health. A comprehensive herbal seed of this kind motivated me to venture into herbo-mineral preparation. Only a herb can link a microcosm and a macrocosm since it contains natural ingredients with three biological energies, namely potential energies, kinetic energy, and thermal energy.

Demineralization and remineralization affect the surface mechanical properties, including hardness which is directly related to its mineral content. Scanning electron microscopy–energy dispersive X-ray analyzer (SEM-EDX) is an efficient way to assess the changes in surface structure during demineralization and in vitro remineralization process.1

Very few studies have compared the remineralizing potential of NaF, MTA, and the herbal combination of Psoralea corylifolia L. (Bakuchi) and NaF. Hence the present study aims to evaluate the three different remineralizing agents on artificially demineralized enamel lesions by using Vickers hardness number (VHN) and SEM-EDX analysis. Consequently, the installation and application of graduated procedures for conventional treatment and remedies is the need for this study.

MATERIALS AND METHODS

Source of Data

In our study, 75 sound premolar teeth, freshly extracted for the purpose of orthodontic treatment, were obtained from the patients visiting RajaRajeswari Dental College and Hospital, Bengaluru.

Inclusion Criteria

Premolars with intact buccal and lingual surfaces were included.

Exclusion Criteria

Teeth with grossly decayed coronal structure, hypoplastic or developmental defects, or fluorosis were excluded.

Preparation of Sample

The debris was removed, and selected teeth were cleaned using an ultrasonic scaler and stored in distilled water, and used within 3 months.

This study included a total of three groups:

  • Group I (n = 15) = Normal samples

  • Group II (n = 15) = Demineralized samples

  • Group III (n = 45) = Remineralizing groups

Method of Preparation for VHN

Vickers hardness test was undertaken in microhardness tester FM800 equipped with an optical system in the Department of Materials Engineering, Indian Institute of Science (IISC), Bengaluru. For this, all teeth were cut using an angular diamond disk at the cement enamel junction. Each tooth crown was sectioned mesiodistally into two parts, and the sectioned buccal surface was embedded in acrylic resins and polished progressively using a 600-grit wet silicon-carbide paper for testing. All samples were stored in distilled water at 37°C.

Method of Preparation for SEM-EDX

Scanning electron microscopy–energy dispersive X-ray analysis (SEM-EDX) analysis was undertaken in the Department of Advanced Facility for Microscopy and Microanalysis, IISC, Bengaluru, on machine model JEOL-SEM IT 300. The other part of the sectioned tooth samples of each group were numbered and placed on carbon tape, and mounted over aluminum stubs (sample holder). This was then placed in the BAL-TEC SCD 500 sputter coating machine for gold sputtering on the sample surface. Next, the sample holder was placed in the JOEL SEM IT 300, which uses up to 30 kV tungsten source for SE/BSE imaging with EDS spectroscopy and cathodoluminescence (SEM-CL) for SEM-EDX analysis for evaluating any loss of mineral content and assess the changes in surface structure.

Method of Preparation of Demineralized Solution

The solution contents are 2.2 mM calcium chloride (CaCl2), 2.2 mM potassium dihydrogen phosphate (KH2PO4), and 0.05 M acetic acid, and the pH was adjusted to 4.4 with 1 M potassium hydroxide (KOH).

Method of Preparation of Remineralizing Solution

The solution contents are 1.5 mM CaCl2, 0.9 mM sodium dihydrogen phosphate, 0.15 M potassium chloride (KCL), and pH 7.0. This solution was approximated to the supersaturation of apatite minerals found in saliva.

Method of Preparation of Artificial Saliva

The artificial saliva used in this present study was prepared according to Macknight-Hane and Whitford’s (1992) formula. Sorbitol was not used because artificial saliva would be more viscous than normal saliva when sorbitol is mixed with sodium carboxymethyl cellulose (Levine et al.,1987).

Composition (in gm/L): The artificial saliva contained 2.0 gm of methyl-p-hydroxybenzoate; 10.0 gm of sodium carboxymethyl cellulose; 0.625 gm of KCl; 0.059 gm of magnesium chloride hexahydrate; 0.166 grams of CaCl dihydrate; 0.804 gm of K2HPO4, and 0.326 gm of KH2PO4 per liter of solution. The pH of artificial saliva was adjusted to 6.75 with KOH.

Methodology: Chronology of the Process

The group-I samples (n = 15) were first tested under normal conditions for their hardness (VHN) at 50 gm load force with a dwell time of 10 seconds. These were further subjected to SEM-EDX analysis.

The demineralizing solution was prepared, and the remaining 60 teeth were immersed into this freshly prepared demineralizing solution for 4 days to produce the carious lesion. Out of this, 15 teeth were selected as group II and subjected to a microhardness test (VHN) at 50 gm load force with a dwell time of 10 seconds to determine the hardness. SEM-EDX analysis was also done to assess any loss of mineral content and the changes in surface structure in this group-II demineralized teeth.

The remaining 45 demineralized teeth were then divided into remineralizing experimental groups for remineralization, as group III, which was further subdivided into the following subgroups:

  • Experimental group III-A (n = 15): Remineralized with 2% NaF; the solution was prepared by mixing 2 gm of NaF in 100 mL of distilled water, kept in a plastic bottle marked as group III-A, where the demineralized teeth were immersed.

  • Experimental group III-B (n = 15): Remineralized with 2% NaF and Psoralea corylifolia L. (Bakuchi)—the pure form of Bakuchi seeds (which are easily available) were procured from an authentic ayurvedic store (Amruth Kesar, Chikpet, Bengaluru). The seeds received from the store were sieved and sun-dried for 4 hours, after which they were fine-grounded into a powdered state and stored in an airtight container. Psoralea corylifolia L. is also available in various forms of ayurvedic marketed formulations in India and across the world; some of the brands are Safuf Bars, Zimad Kibrit, Svitrakaravati, Khadirarista, Algushadiyoga, Sarvangasundarigutika, Bhallatakawaleha, Maheshwaraghrita, Ayorajodilepa, Brihatsomarajitaila, Somarajighrita, Bawchichurna, etc.7

  • The powdered seeds were mixed with NaF in a 1:2 ratio, that is, 2 gm Bakuchi:4 gm NaF.

  • Bakuchi was prepared by mixing 2 gm of Bakuchi in 200 mL of water and reducing it up to 100 mL concentrate by boiling. A total of 4 gm of NaF was mixed in 100 mL of distilled water. Both the concentrates were mixed and marked as group III-B. The demineralized teeth were then immersed in this solution.

  • Experimental group III-C (n = 15): Remineralized with white MTA; the mixing of MTA was done as per the manufacturer’s instructions and marked as group III-C. The demineralized teeth were coated with MTA.

  • The demineralized teeth were treated with the three remineralizing experimental groups for 7 days, twice daily for 3 minutes, followed by incubation in remineralizing solution for 5 minutes, then immersed in artificial saliva at 37°C for 24 hours.

Each remineralizing experimental group and the remineralizing solution were freshly prepared daily. After 7 days, the remineralized teeth under groups III-A, III-B, and III-C were subjected to a microhardness test (VHN) at 50 gm load force with a dwell time of 10 seconds to determine the hardness and SEM-EDX analysis was also done for assessing the surface structure and mineral content.8

The results obtained were subjected to further statistical analysis (Flowchart 1).

Flowchart 1: Chronology of the process—Anatomy of the study

Statistical Analysis

Statistical Package for Social Sciences for Windows Version 22.0 Released 2013. Armonk, New York, United States of America: IBM Corp. was used to perform statistical analysis.

Descriptive Statistics

The descriptive analysis includes the expression of VHN and EDX values in terms of mean and standard deviation for each study group.

Inferential Statistics

The Kruskal–Wallis test was followed by Mann–Whitney’s post hoc test to compare the mean VHN and EDX values between different study groups. The level of significance was set at p < 0.05.

RESULTS

Vickers Hardness Test

The data in Table 1 demonstrates that the mean results of VHN for the normal teeth group were 547.65 ± 66.39 showing the highest VHN significantly as compared to other groups. Intercomparison results of mean difference in VHN between groups, where remineralized with NaF + Bakuchi group showed significantly highest VHN as compared to other groups at p < 0.001 and remineralized with NaF group at p = 0.008, respectively. This mean difference for VHN was statistically significant between the five groups at p < 0.001.

Table 1: Multiple comparisons of mean difference in VHN between groups using Mann–Whitney post hoc test
(I) Groups (J) Groups Mean Diff. (I−J) 95% confidence interval for the difference p-value
Lower Upper
Normal Demineralized 177.83 133.55 222.11 <0.001*
R + NaF −245.92 −290.20 −201.64 <0.001*
R (NaF + Bakuchi) −307.13 −351.41 −262.85 <0.001*
R + MTA 36.73 −7.55 81.01 0.04*
Demineralized R + NaF −423.75 −468.03 −379.47 <0.001*
R (NaF + Bakuchi) −484.96 −529.24 −440.68 <0.001*
R + MTA −141.10 −185.38 −96.82 <0.001*
R + NaF R (NaF + Bakuchi) −61.21 −105.49 −16.93 0.008 *
R + MTA 282.65 238.37 326.93 <0.001*
R (NaF + Bakuchi) R + MTA 343.86 299.58 388.14 <0.001*

*Statistically significant

Scanning Electron Microscope (SEM) Results

The scanning electron microscope results, as in Figures 1 and 2, show the significant changes observed for the various groups.

Fig. 1: Mean EDX P-Kα values between different groups (arranged in descending order)

Fig. 2: Mean EDX CA-Kα values between different groups (arranged in descending order)

Energy Dispersive X-ray Analysis (EDX) Results

The mean EDX K-energy (Kα) values for phosphorous (P-Kα), Ca-Kα, K-Kα, and fluorine (F-Kα) were observed from the EDX results between different groups arranged in descending order and represented in individual graphs in which, for each element, the mean difference in the EDX energy value between five groups was statistically significant at p ≤ 0.001. The result shows a clear advantage when remineralized with NaF + Bakuchi.

Figures 1 and 2 show that the remineralized group with NaF + Bakuchi was highest for P with Kα levels at 17.394 ± 2.842 and Ca with Kα levels at 42.981 ± 9.490, followed by the group that was remineralized only with NaF.

Similarly, Figure 3 shows that the remineralized group with NaF + Bakuchi was highest for K with K-α levels at 2.628 ± 1.035, followed by the group that was remineralized only with NaF.

Fig. 3: Mean EDX K-Kα values between different groups (arranged in descending order)

Figure 4 shows similar energy values for F; the remineralized group with only NaF showed energy levels at 23.777 ± 5.687, followed by the remineralized with NaF + Bakuchi group, which was at 13.469 ± 4.775 levels.

Fig. 4: Mean EDX F-Kα values between different groups (arranged in descending order)

DISCUSSION

Dental caries is the most common infectious disease in children. It is characterized by progressive demineralization and loss of dental hard tissues, owing to acids produced by the bacteria present in dental plaque.9

Demineralization occurs at or below a pH of 5.5, and this causes an imbalance in the phosphate content between the enamel surface and its environment; the HAP dissolves to provide more phosphate ions to restore the balance.10

Remineralization is novel noninvasive management of early carious lesions, which bridges the traditional gap between preventive and invasive dentistry.11

Fluoride, in combination with Ca and phosphate ions, creates a veneer of fluorapatite on the surface of existing enamel crystals, which not only acts as a replacement for minerals lost from the tooth structure but also is much less soluble than the original carbonated HAP.12

Medicinal plants, though, produce progressive recovery, but their therapeutic effect is miraculous, gaining popularity over the drugs because of the rapid increase in the rate of infections, antibiotic resistance in microorganisms, and side effects of synthetic antibiotics.6

The shape of the Psoralea corylifolia L. seed is elongated with a smooth surface. The seed is without hairs, compressed, and pitted closely. The color of the seed is dark brown. The seeds have no starch, have an oily texture, and have no endosperm (Krishnamurthi, 1969). The crop gets fully mature in 7–8 months. The seeds take time to mature, and therefore collection can be rendered 4–5 times from December to March (Sharma et al., 2000).7

Psoralea corylifolia L. seed extract has antimicrobial activity against numerous strains of bacteria in vitro due to the presence of monoterpene phenol which is responsible for the inhibition of cell growth of Streptococcus mutans. The biological effects of bakuchiol are anti-inflammatory effect, inhibition of mitochondria lipid peroxidation, stimulation of the immune system, and inhibition of DNA polymerase.8

The biological and physicochemical properties of MTA with respect to its ability to induce reparative dentinogenesis, which involves complex cellular and molecular events leading to hard-tissue repair by newly differentiated odontoblast-like cells.13

Microhardness tester and EDX were chosen to be tested in this study as they are indicative of chemical changes. Chemical changes indicate that the mineral phase was distorted or that a significant replacement of ions may have happened.14

The hardness of mineralized hard tissues is directly related to their mineral content. Loss of minerals from enamel affects surface mechanical properties, including hardness.4

In our present study, the result of VHN showed significantly highest for remineralized with NaF + Bakuchi group at 547.65 ± 66.39, and also, the test results for the demineralized group showed decreased values, that is, 62.69 ± 14.66. Statistically significant at p < 0.001 was seen in all five groups.

Hong et al. showed that fluoride, along with the Psoralea corylifolia L. group, showed a higher surface hardness number which is in accordance with our study. Psoralea corylifolia L. extracts have the potential to use as a third substance for the antimicrobial activity of fluoride dentifrice, and the herbal extracts may not decrease the remineralization effect of NaF in dentifrice.15 Thus NaF + Bakuchi group exhibited a significantly higher efficacy in enhancing remineralization.

Microhardness of the enamel was significantly reduced after the bleaching treatment and increased by the addition of fluoride and Ca, which was assessed by Borges et al. using a Vickers micro durometer.16

Scanning electron microscopy (SEM) observation was carried out using a scanning electron microscope to investigate morphological changes. In the present study, SEM with high-resolving power was used because of reported high sensitivity toward early reactions occurring at crystal level.1

The normal tooth group showed a homogenous flat and smooth surface, whereas the demineralized tooth revealed rough and porous voids of variable sizes distributed nonuniformly with irregular patterns of surface destruction under SEM image (Fig. 5).

Figs 5A and B: (A) Normal teeth (SEM image: 1000× magnification) appeared homogenous, flat, and smooth; (B) Demineralized teeth (SEM image: 1700× magnification) appeared rough and porous with voids of various sizes distributed nonuniformly and with irregular patterns of surface destruction (honeycomb appearance)

Scanning electron microscopy (SEM) images under all three remineralizing groups revealed a nonhomogeneous thin film with an amorphous-like precipitate scattered uniformly and remaining visible on the enamel surface (Fig. 6).

Figs 6A to C: )A) Remineralized tooth with NaF; (B) Remineralized tooth: NaF + Bakuchi; (C) Remineralized tooth + MTA; SEM images A, B, and C, above (1000× magnification) under all three remineralizing groups revealed amorphous precipitate-like deposit scattered unevenly on the surface or globular particles deposited uniformly on the enamel surface

Scanning electron microscopy (SEM) images after MTA application (experimental group III-C) showed spherulite minerals formed (Fig. 6C), and EDX showed there was an increase in Ca and fluoride suggestive of remineralization. Similar results were seen in the study conducted by Pratiwi et al.5

Mineral trioxide aggregate (MTA) mimics HAP in terms of composition and structure when placed on dentine with intra-fibrillar apatite deposition and also releases Ca ions during the setting reaction resulting in prolonged alkaline pH of 12.5, responsible for antibacterial and antifungal properties.5

Similar results of SEM where remineralization was conducted using dentifrice slurry were observed with the mineral deposit partially filling up prism cores with the study done by Kapoor et al.,1 Wang et al. remineralized with casein phosphopeptide–amorphous Ca phosphate, arginine and Ca carbonate group, and Ca sodium phosphosilicate groups and revealed that demineralized tooth group showed a typical honeycomb appearance.4

Energy dispersive X-ray analyzer (EDX) test was conducted on all the groups of tooth samples to identify the elemental compositions where the emission peak of elements such as Ca, P, oxygen, and F was observed under all five groups.

The highest value under each element was identified in group III B (NaF + Bakuchi) when compared to other groups.

As a mineral loss, or demineralization, occurs, Ca and phosphate are lost to the external environment. The EDX results in our study showed evident loss of Ca (27.088 ± 2.906) and phosphate (9.314 ± 4.168) in the demineralized samples (group II) when compared to the normal group samples (Ca, 33.239 ± 5.305; phosphate, 15.792 ± 2.707) (group I).

If mineral loss continues, eventually, it leads to cavitations by more extensive damage to crystal structure. In this study, the principle of remineralization is mainly the balancing of energy levels. Remineralization results if Ca and phosphate gradients are reversed and restored back into the tooth. Crystal regrowth, or new precipitation, will also occur as the pH rises along with fluoride, if present at the crystal surface, which promotes remineralization by dramatically speeding up the process. With this evidence, in our present study, the Ca (42.981 ± 9.490) and phosphate (17.394 ± 2.842) had significantly increased after remineralization with group III-B (NaF + Bakuchi) when compared to other groups. This explains the mechanism of remineralization of NaF along with Bakuchi and the qualitative aspects of the combination of Bakuchi along with fluoride, which is more productive in restoring the Ca and phosphate minerals and enhancing the potential of fluoride with no antagonist reaction.

Bakuchiol has a bactericidal effect on S. mutans with minimal inhibitory concentrations of 1–4 μg/mL also effective against adherent cells of S. mutans in the presence of sucrose and inhibits the reduction of pH in the broth.8 A study by Katsura et al. showed the bactericidal effect of Bakuchi was stable in the mouth though there is variation in temperature, sugar, pH, and organic acid.17

Psoralea corylifolia L. and its natural compounds (bavachin, corylifola, neobavaisoflavone, isobavachalcone, and bavachinin) had potential toxicity and showed potent inhibitory effects against human uridine diphosphate–glucuronosyltransferase, which is the main stimulant for Psoralea corylifolia L. related toxicity including hepatic injury and raised bilirubin level (Wang et al. 2015).7

Thus, the qualitative aspect of bakuchiol promotes it to be used as an antibacterial agent in mouthwash for preventing and developing dental caries.7,8 The biochemical effects of the various phytoconstituents in various parts of the plant possess multiple biological activities and has numerous medicinal application. Newer drugs can be made after extensive research on the bioactivity, mechanism of action, pharmacotherapeutic, toxicity, and clinical trials.

Psoralea corylifolia L. is an exclusive medicine as an anti-inflammatory, bone, and teeth rejuvenator, and for skin diseases; the utility of such a nectarious herb is up to the experience and discretion of the physician.

CONCLUSION

Psoralea corylifolia L. (Bakuchi), when mixed with NaF, has great potential for use in dentifrices and mouthwashes, for preventing and treating dental caries and has revealed appreciable results on remineralization of artificially demineralized enamel lesions when compared to the MTA and NaF alone.

Within the limitation of this study, research and development should be performed with different parts of the Psoralea corylifolia L. plant and its products for better economic and therapeutic utilization.

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