|Year : 2022 | Volume
| Issue : 3 | Page : 330-337
Evaluation of three different remineralizing agents on artificially demineralized enamel lesions: Using scanning electron microscopy-energy dispersive X-ray and magic-angle spinning nuclear magnetic resonance - An in vitro study
Divya Vijay Mehta, Shakuntala B Siddaiah
Department of Pediatric and Preventive Dentistry, Rajarajeswari Dental College and Hospital, Rajiv Gandhi University, Bengaluru, Karnataka, India
|Date of Submission||13-Jun-2022|
|Date of Decision||07-Sep-2022|
|Date of Acceptance||13-Sep-2022|
|Date of Web Publication||18-Oct-2022|
Divya Vijay Mehta
Department of Pediatric and Preventive Dentistry, Rajarajeswari Dental College and Hospital, No. 14, Ramohalli Cross, Mysore Road, Kumbalgodu, Bengaluru - 560 074, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Demineralization can be arrested or reversed when remineralization agents are applied to incipient carious or noncavitated carious lesions. A large number of therapeutic agents, including nonfluoridated products, have been developed to promote enamel remineralization. This study aims to evaluate the efficacy of different remineralizing agents on artificially demineralized enamel lesions. Materials and Methods: The present in vitro study was conducted on 75 sound premolars divided into three groups of normal, demineralized (n = 15 each), and remineralized teeth (n = 45). The remineralized teeth were further subdivided into three groups (n = 15) as remineralized with 2% sodium fluoride (NaF), 2% NaF, and Psoralea corylifolia (bakuchi) and white mineral trioxide aggregate. Specimens of each group were treated with the above-mentioned remineralizing agents and then subjected to Vickers hardness number (VHN), scanning electron microscopy–energy dispersive X-ray (SEM-EDX), and magic-angle spinning nuclear magnetic resonance (MAS-NMR) 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, phosphorous, oxygen, and fluorine with remineralized with NaF + bakuchi. MAS-NMR spectra showed fluorine and phosphorous peak in a group with NaF + bakuchi indicative of the increase in remineralization. NaF + bakuchi showed effective results in VHN, SEM-EDX, and MAS-NMR with no antagonist interaction. Conclusion: Thus, P. Corylifolia presents an advantage in enhancing remineralization and inhibiting demineralization for early carious lesions and can be used as a herbal extract for effective reduction in pathogenic bacteria.
Keywords: Magic-angle spinning nuclear magnetic resonance, Psoralea corylifolia (bakuchi), remineralization, scanning electron microscopy–energy dispersive X-ray, sodium fluoride, Vickers hardness number
|How to cite this article:|
Mehta DV, Siddaiah SB. Evaluation of three different remineralizing agents on artificially demineralized enamel lesions: Using scanning electron microscopy-energy dispersive X-ray and magic-angle spinning nuclear magnetic resonance - An in vitro study. J Indian Soc Pedod Prev Dent 2022;40:330-7
|How to cite this URL:|
Mehta DV, Siddaiah SB. Evaluation of three different remineralizing agents on artificially demineralized enamel lesions: Using scanning electron microscopy-energy dispersive X-ray and magic-angle spinning nuclear magnetic resonance - An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2022 [cited 2022 Nov 29];40:330-7. Available from: http://www.jisppd.com/text.asp?2022/40/3/330/358836
| Introduction|| |
Dental caries is a disease process caused by an imbalance between demineralization and remineralization. Losing minerals from the tooth after an acidic encounter is called demineralization, whereas restoration of these minerals back to the tooth structure is termed remineralization.,
Saliva has the ability to remineralize along with its calcium, phosphate, and fluoride content.
Fluoride has been commonly used as a remineralization agent for preventing early enamel carious lesions. The main mechanism of action of fluoride can be attributed to its activity to inhibit bacteria growth.,
Medicinal plants have been found useful in the cure of several diseases including bacterial diseases owing to a rich source of antimicrobial agents and phytochemicals that have disease preventive or protective properties.
Psoralea corylifolia L (bakuchi), a traditional ayurvedic medicinal plant, was found to inhibit the growth of Streptococcus mutans under a range of sucrose concentrations, pH values, and in the presence of organic acids in a temperature-dependent manner. Thus, the aim is to incorporate P. corylifolia since it is a useful compound for the development of antibacterial agents against oral pathogens with great potential for use in mouthwash and denitrifies for preventing and treating dental caries.
Mineral trioxide aggregate (MTA) is a modified preparation of Portland cement, which is the basic ingredient of concrete and mortar that may never have been used as a dental material before the development of MTA.,
Tricalcium in MTA is a major component that is biocompatible and bioactive. MTA has high alkaline pH, antibacterial, biocompatibility, radiopacity, and good closure properties also has the capacity to stimulate cell differentiation or activation, which may contribute to hard tissue matrix formation and mineralization and induce reparative dentinogenesis, which involves complex cellular and molecular events, leading to hard tissue repair by newly differentiated odontoblast-like cells. It can remove calcium and hydroxyl ions that form hydroxyapatite crystals on dentin surfaces and also interact with dentin during intrafibrillar deposition.
The present study aimed to evaluate the three different remineralizing agents on artificially demineralized enamel lesions using Vickers hardness number (VHN), scanning electron microscopy–energy dispersive X-ray (SEM-EDX), and magic-angle spinning nuclear magnetic resonance (MAS-NMR).
VHN is a microhardness test used to quantitatively evaluate the tooth hardness before and after remineralization.
SEM-EDX analysis was used to evaluate the changes of enamel surface morphology and roughness at different stages of demineralization and remineralization.
MAS-NMR analysis was used to characterize the solid-phase precipitation of fluoride, calcium, and phosphorous ions on the enamel surface. It selectively probes the local environment of only the fluorine atoms within the enamel in crystalline, amorphous, or adsorbed forms and measures very low concentrations of fluoride in the order of 0.1% fluorine-19.
| Materials and Methods|| |
Source of Data: The present in vitro study was conducted on 75 sound premolar teeth freshly extracted for the purpose of orthodontic treatment, obtained from patients visiting Rajarajeswari Dental College and Hospital, Bengaluru. Premolars with intact buccal and lingual surfaces were included in the study, but the grossly decayed coronal structure, hypoplastic tooth or developmental defects, and teeth with fluorosis were excluded from the study.
Preparation of sample
The debris was removed, teeth were cleaned using an ultrasonic scaler and stored in distilled water, and used within 3 months of collection. The study was divided into three groups as GROUP I (n = 15) of normal samples, GROUP II (n = 15) of demineralized samples, and GROUP III (n = 45) of remineralized samples.
Preparation of sample for Vickers hardness number
Vickers hardness test was carried out using microhardness tester, FM800 equipped with optical system in the Department of Materials Engineering, Indian Institute of Science (IISC), Bengaluru. Each tooth crown was sectioned mesiodistally and embedded in acrylic resins, further polished and stored in distilled water at 37°C.
Preparation of sample for scanning electron microscopy–energy dispersive X-ray
This analysis was performed on model JEOL-SEM IT 300 at the Department of Advanced Facility for Microscopy and Microanalysis, IISC, Bengaluru. The other part of the sectioned tooth samples was sputter coated with conductive gold for SEM-EDX analysis.
Preparation of sample for magic-angle spinning nuclear magnetic resonance
This was conducted on Bruker 400MHz Machine in the Department of NMR Facility, IISC, Bengaluru. Tooth samples were dried and ground to a fine powder for solid-state magic-angle spinning nuclear magnetic resonance, under 400 MHz, 9.4T (Tesla) spectrometer (Jeol).
Preparation of demineralized solution
The solution contents are 2.2 mM calcium chloride (CaCl2), 2.2 mM potassium dihydrogen phosphate (KH2PO4), and 0.05M acetic acid, and pH was adjusted to 4.4 with 1M potassium hydroxide (KOH).
Preparation of remineralizing solution
The solution contents are 1.5 mM CaCl2, 0.9 mM sodium dihydrogen phosphate, and 0.15 M potassium chloride (KCL), and pH was 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 (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 grams per liter) – The artificial saliva contained 2.0 g of methyl p-hydroxybenzoate, 10.0 g of sodium carboxymethyl cellulose, 0.625 g of KCl, 0.059 g of MgCl2.6H2O, 0.166 g of CaCl2.2H2O, 0.804 g of K2HPO4, and 0.326 g of KH2PO4 per liter of solution. The pH of artificial saliva was adjusted to 6.75 with KOH.
The GROUP I samples (n = 15) were first tested under normal conditions for their hardness (VHN) at 50-g load force with a dwell time of 10 s. These were further subjected to SEM-EDX analysis, after which MAS-NMR was also performed.
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-g load force with a dwell time of 10 s to determine the hardness. SEM-EDX analysis was also performed to assess any loss of mineral content and the changes in surface structure and further subjected to MAS-NMR 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% sodium fluoride (NaF) –Solution was prepared by mixing 2 g 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 P. corylifolia L.(bakuchi) – The pure form of bakuchi seeds (which are easily available) was procured from an authentic Ayurvedic store (Amruth Kesari, Chickpet, Bengaluru). The seeds received from the store were sieved and sun-dried for 4 h, after which they were fine grounded into a powdered state and stored in an airtight container. The powdered seeds were mixed with NaF in a 1:2 ratio, i.e. 2 g bakuchi: 4 g NaF. Bakuchi was prepared by mixing 2 g of bakuchi in 200 ml of water and reducing it up to 100 ml of concentrate by boiling. Four g 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 min, followed by incubation in remineralizing solution for 5 min, then immersed in artificial saliva at 37°C for 24 h.
Each remineralizing experimental group and the remineralizing solution were freshly prepared daily. After 7 days, the remineralized tooth under GROUP III-A, III-B, and III-C was subjected to a microhardness test (VHN) at 50-g load force with a dwell time of 10 s to determine the hardness, and SEM-EDX analysis was also done for assessing the surface structure, and mineral content further MAS-NMR was also performed.
The results obtained were subjected to further statistical analysis.
The Statistical Package for the Social Sciences (SPSS) for Windows Version 22.0 (2013). Armonk, NY, USA: IBM Corp. was used to perform statistical analyses.
The descriptive analysis includes the expression of VHN and EDX values in terms of mean and standard deviation for each study group.
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|| |
[Table 1] demonstrates the mean results of VHN for the normal teeth group was 240.52 ± 58.97, for the demineralized group was 62.69 ± 14.66, remineralized with NaF group was 486.44 ± 27.65, and remineralized with NaF + bakuchi group was 547.65 ± 66.39 showing significantly highest VHN as compared to other groups and for remineralized with MTA group was 203.79 ± 22.63. This mean difference in the VHN between the five groups was statistically significant at P < 0.001. The SEM results are as per [Figure 1]. The EDX results are as per [Figure 2] giving the graphical analysis of the values.
|Figure 1: SEM images of (a) Normal (b) Demineralized (C) Remineralized with NaF (d) Remineralized with NaF (e)Remineralized with MTA. SEM = Scanning Electron Microscopy|
Click here to view
|Figure 2: Graphs showing the Mean EDX K-energy values for different elements (a) Phosphorous (b) Calcium (c) Potassium (d) Fluorine, between different groups arranged in descending order. EDX = Energy Dispersive X-ray|
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[Table 2] shows the test results demonstrate the mean EDX descriptive for other sparse elements present among different groups mentioned as EDX chlorine energy, EDX iron energy, EDX tungsten energy, EDX arsenic energy, EDX nitrogen energy, EDX mercury energy, EDX tantalum and barium, EDX titanium energy, EDX strontium, EDX rubidium, and EDX tungsten.
[Table 3] of the MAS-NMR spectra of the enamel surface were assessed in five states: normal teeth (sound), demineralized teeth, after remineralizing with NaF, remineralizing with NaF along with bakuchi, and remineralizing with MTA for phosphorous and fluorine.
| Discussion|| |
Demineralization occurs when pH drops due to the formation of organic acids due to the action of plague bacteria in the presence of dietary carbohydrates and remineralization of early carious lesions is one of the major advancements in this field, which retards the progression of a lesion causing its arrest and achieves lesion regression ideally.
Various materials used are fluorides with types of fluorides such as NaF, stannous fluoride, silver diamine fluoride, and varnishes are used at different concentrations, where fluoride or fluoride-releasing materials inhibit bacterial growth as well as reduce the solubility of enamel by forming fluorapatite- and/or fluoride-rich hydroxyapatite.
MTA and a few herbal agents are also used, and various means available, such as chewing gums (containing xylitol or tricalcium phosphate) and casein phosphopeptides-amorphous calcium phosphate casein phosphopeptides amorphous calcium phosphate (CPP-ACP) have been introduced over time; however, their use in daily practice has not been established. Moreover, the risk of ingestion and hence, toxicity has been a cause of concern in pediatric patients.,
The root of P. corylifolia has shown its effectiveness in dental caries. P. corylifolia seed extract (bakuchiol) was tested against some oral microorganisms in vitro and showed antimicrobial activity against various strains of bacteria.
Bakuchiol also showed antibacterial effects against other bacteria tested, including S. mutans, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Actinomyces viscosus, and Porphyromonas gingivalis. The antimicrobial activity was due to the presence of monoterpene phenol, responsible for the inhibition of cell growth of S. mutans in a concentration-dependent and temperature-dependent bactericidal effect on the growth of S. mutans was completely prevented by sterilizing concentration for 15 min ranging from 5 to 20 μg/ml.
MTA is a mechanical mixture of portland cement, bismuth oxide, and gypsum that promotes biomineralization as it can form hydroxyapatite crystals, and thus, calcium ions are released in the presence of phosphate-buffered saline.
MTA can release calcium ions; Sarkar et al. stated that the dominant calcium ions released from the MTA will react with phosphates in synthetic tissue fluids and form hydroxyapatite. Physicochemical analyses have revealed that MTA not only acts as a “calcium hydroxide-releasing” material but also interacts with phosphate-containing fluids to form apatite precipitates. Thus, with a selective caries removal method accompanied by the application of MTA material to deep carious lesions remineralization may occur.
In the present study, surface hardness was tested using VHN. This was done for all the groups, i.e. for normal, demineralized, and later remineralized teeth with NaF, NaF with bakuchi, and MTA. Since VHN is a relatively simple, rapid, noninvasive, and reproducible technique for assessing the changes of surface mechanical properties, the hardness of mineralized hard tissues is directly related to their mineral content. The present study revealed a VHN of normal teeth was 240.52 ± 58.97 and the demineralized group was 62.69 ± 14.66, which shows there is a loss of minerals from enamel (for example, in cases of caries or acid etching) that affects surface mechanical properties including hardness. The VHN value was found to be statistically significant highest for samples in experimental GROUP III-B remineralized with NaF + bakuchi group at 547.65 ± 66.39, followed next with samples in experimental GROUP III-A NaF group then by samples in experimental GROUP III-C MTA, respectively. This suggests that bakuchi with NaF has remineralizing effects on the tooth surface and there is no antagonistic interaction between the herbal extracts and NaF. A similar study was conducted by Hong et al. showed that NaF along with the P. corylifolia group showed a higher surface hardness number. Another study where microhardness of the surface and subsurface enamel was significantly reduced after the bleaching treatment and increased by the addition of fluoride and calcium to the bleached enamel which was assessed by Borges et al., using a Vickers micro durometer.
SEM-EDX is an efficient way to assess the changes in surface structure during the demineralization and in vitro remineralization process. It was used to determine calcium phosphorus and fluoride content in the normal tooth (sound), demineralized tooth, and remineralized enamel tooth under each experimental group.
In our study, the SEM image under the normal tooth group (GROUP I) was seen as a smooth homogenous flat surface with slightly discernible prism shadows. Demineralized tooth group (GROUP II) showed deep prismatic holes giving a typical honeycomb appearance and/or single or groups of focal holes without a protective layer. Based on observations, SEM images showed spherulite minerals formed after the application of bioactive material MTA.
The characterization of SEM for remineralized groups revealed that enamel surfaces were covered by a superficial layer of precipitated crystals to repair the erosive enamel, suggesting the remineralizing ability of these three treatments. These findings were consistent with microhardness test results. The study done by Kapoor et al. showed similar results of SEM where enamel prisms pits were visible as honeycomb appearance and after remineralization using dentifrice slurry, the mineral deposit and filling up prism cores were observed and honeycomb appearance was not visible.
Another study by Wang et al. remineralized casein phosphopeptide–amorphous calcium phosphate (CPP-ACP), arginine and calcium carbonate group, and calcium sodium phosposphosilicate groups and revealed that the demineralized tooth group showed a typical honeycomb appearance. Taha et al. assessed remineralization under SEM images after glass propulsion and noticed remineralizing surface had mineral precipitate-like deposits.
Further EDX analysis was conducted in all five groups revealing calcium and phosphorous content and was highest in experimental GROUP III-B (NaF + bakuchi) when compared to other groups. Fluorine content in EDX was higher in experimental GROUP III-A (remineralized with NaF), followed by experimental GROUP III-B (NaF + bakuchi). The presence of calcium, fluoride, and phosphorous in the remineralizing groups will cause effective remineralization due to the redeposition of these ions on the affected enamel surface crystals.
Test results in EDX analysis demonstrate a few of the trace elements such as chlorine, iron, tungsten, arsenic, nitrogen, mercury, tantalum and barium, titanium, strontium, rubidium, and tungsten were found in our study.
Similar results were seen with the study conducted by Wu et al., where the mineralized intertubular dentin seemed similar to the appearance of mineralized sound dentin of calcified collagen matrix. EDX showed that calcium, oxygen, fluorine, and phosphorus were the main elements comprising the intertubular dentin. The Ca/P ratio of the regenerated crystals was 1.70, which revealed that the crystals were similar to the HA of sound tooth enamel.
The MAS-NMR spectra show the formation of calcium fluoride and fish scale hydroxyapatite (Fs-Hap) as the main chemical species formed on the enamel samples that were demineralized with the addition of varying fluoride concentrations.
In our study, fluorine peak was more in remineralized experimental GROUP III-B with NaF + bakuchi peak and resonates at-128.201 ppm and phosphorous was stable with the peak resonating value of 3.983 with all three remineralizing groups.
The present study demonstrates that the addition of fluoride produces Fs-HAp as a major chemical species. There is overwhelming evidence by Fox et al. in 1983 and Lynch et al. in 2006 that low fluoride levels found in saliva can significantly reduce enamel demineralization and those found in plaque have the potential to remineralize, even at pH values typically regarded as demineralizing.,
Conversely, there was no difference between the NMR spectra obtained with fluorine for the demineralized sample (GROUP II) when compared to the spectra of remineralization with NaF (experimental GROUP III-A) and remineralized with NaF + bakuchi (experimental GROUP III-B). Further, we recommend re-evaluating these values under a larger sample size to correlate with the previous data.
| Conclusion|| |
Interestingly, in the present study, all three tests were indicative of the increase in remineralization under experimental GROUP III-B. Since P. corylifolia (bakuchi) extracts with NaF have the remineralizing effect on the tooth surface and no antagonist interactions can occur between P. corylifolia (herbal extract) and NaF also an herbal extract of P. corylifolia acts as a microbial agent which is safe and can reduce pathogenic bacteria effectively. The applied progressive research and insight on P. Corylifolia (bakuchi), when mixed with NaF has great potential for use in dentifrices and mouthwashes for preventing and treating dental caries.
My heartfelt gratitude to IISC, Bengaluru, India, for providing me laboratory infrastructure to execute my Research in their Department of Materials Engineering, Department of Advanced Facility for Microscopy and Microanalysis (AFMM), and Department of NMR Facility.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]