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 Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 1  |  Page : 18-23

Re-mineralization capability of two natural nano-plant extracts on early enamel caries using diode laser

Restorative and Dental Materials Department, National Research Centre, Giza, Egypt

Date of Submission26-Aug-2020
Date of Decision05-Apr-2021
Date of Acceptance26-Jul-2021
Date of Web Publication31-Dec-2021

Correspondence Address:
Lamiaa Mahmoud Moharam
Restorative and Dental Materials Department, National Research Centre, 33 El Bohouth Street (Former El Tahrir Street), Dokki, Giza
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJDS.IJDS_148_20

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Background: To investigate diode laser application and two natural nano-plant extracts effect on the re-mineralization and surface micro-hardness of early enamel carious lesions. Materials and Methods: Eighty enamel specimens were selected and prepared then divided into eight groups (10 specimens per group), according to diode laser irradiation, two nano-plant extracts (garlic and clove nano-plant extracts) and acidulated phosphate fluoride (APF) gel, that were applied to demineralized enamel surfaces as follows; Group A; garlic nano-plant extract, Group B; clove nano-plant extract, Group C; application of APF gel, Group D; garlic nano-plant extract + diode laser, Group E; clove nano-plant extract + diode laser, Group F; application of APF gel + diode laser, Group G; diode laser, Group H; early enamel caries (control). Specimens were examined for their content of Ca, P, and F ions and their surface Vickers micro-hardness. The tests used for the statistical analysis were one-way ANOVA followed by Tukey's (honestly significant difference) post hoc tests. Results: No statistically significant difference was detected between the tested groups for Ca and P content, while Group A recording the highest mean value. Group C recorded the highest F ion content values, while G and H groups recorded the least values. Group F recorded the highest significant micro-hardness values, while Group H recorded the least values. Conclusions: Garlic and clove nano-plant extracts may present a promising enamel re-mineralizing agents. Combination of both nano-extracts with diode laser application had a positive influence on enamel chemistry and micro-hardness.

Keywords: Chemical analysis, diode laser, early enamel caries, nano-plant extracts, surface micro-hardness

How to cite this article:
Moharam LM, Sadony DM. Re-mineralization capability of two natural nano-plant extracts on early enamel caries using diode laser. Indian J Dent Sci 2022;14:18-23

How to cite this URL:
Moharam LM, Sadony DM. Re-mineralization capability of two natural nano-plant extracts on early enamel caries using diode laser. Indian J Dent Sci [serial online] 2022 [cited 2022 May 22];14:18-23. Available from: http://www.ijds.in/text.asp?2022/14/1/18/334518

  Introduction Top

Dental caries is a common infectious disease that affects many patients of different ages around the globe. Nowadays, dental caries can be defined as alternative concurrent sequences of de- and re-mineralization. To prevent the development of initial enamel lesions, an ideal material releasing calcium and phosphate to the oral cavity is extremely essential.[1]

The clinical crown of the teeth is covered with a protective layer of enamel. The inorganic content of the enamel determines its hardness. When some minerals are absent from the tooth enamel, it might have a significant effect on the content of the others and may lead to an increased susceptibility to dental caries.[2]

Diode lasers have interesting features making them relatively prevalent amongst dentists, including small dimension and ease of application.[3] Generally, laser application alters physical properties, permeability, and morphology of enamel, thus preventing caries. It was concluded that laser irradiation had improved Ca/P ratio leading to caries inhibition. Combination of laser application with different re-mineralizing agents had a significant re-mineralizing effect on incipient caries.[4]

Searching for natural alternate tactics to reduce or eliminate dental caries is of prime importance nowadays. Therefore, several natural products were examined for their antibacterial efficacy. Some of them had efficiently shifted the de-/remineralization episodes. Yet, the exact mechanisms of their anti-caries property are still uncertain for many natural products.[5]

Garlic is a powerful natural antibiotic. It has a significant effect on many oral bacterial. It is rich in different vitamins, antioxidants, minerals, and flavonoids.[6] Clove consists mostly of phenolic compounds that are powerful antioxidants. It has antimicrobial, antiseptic, anesthetic, and anti-inflammatory effects.[7]

Therefore, the present study aimed to study the de-mineralization resistance of enamel following treatment with diode laser accompanied with a topical fluoride agent and two different natural nano-plant extracts.

The null hypothesis studied was that different tested nano-plant extracts of garlic and clove have no effect on either the enamel surface micro-hardness or the chemical analysis of early enamel carious lesions with and without diode laser irradiation.

  Materials and Methods Top

Selected materials

One commercial re-mineralizing agent; acidulated phosphate fluoride (APF gel) (Alpha-PRO®APF) and two experimentally prepared 5% concentration of nano-plant extracts of garlic and clove were tested. [Table 1] describes the material brand name, description, composition, and the manufacturer.
Table 1: Material, description, composition, and manufacturer

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Specimen grouping and study design

A total of 80 anterior bovine teeth were used in this study. The teeth were divided to eight groups (10 specimens each), regarding the applied re-mineralization procedures.

  • Group A – Garlic nano-plant extract application
  • Group B – Clove nano-plant extract application
  • Group C – APF gel application
  • Group D – Garlic nano-plant extract + Diode laser application
  • Group E – Application of clove nano-plant extract + Diode laser
  • Group F – Application of APF gel + Diode laser
  • Group G – Application of Diode laser
  • Group H – Early enamel carious lesions received no treatment (control).

R statistical package, version 2.15.2 (IBM Corp, Armonk, New York, USA). was used for sample size calculation (Copyright (C) 2012-R Foundation for Statistical Computing). Results of one-way ANOVA revealed that 10 samples for total sample size will be acceptable to perceive the mean difference between various groups tested, with 80% power and 5% two-sided significance level.

Nano-plant extracts preparation

Ultrasonic-solvent emulsification technique was used to prepare 5% concentration of garlic and clove essential oils, that were loaded with solid-lipid nanoparticles, according to Sjöström and Bergenståhl,[8] Siekmann and Westesen[9] and Asnawi et al.[10] Using 2.5% (w/w) soybean lecithin, 2.5% (w/w) Tween-80, 50-ml dichloromethane and 1% (w/w) stearic acid.

Selection and preparation of teeth

Eighty bovine anterior teeth were selected, and a scaler was used to scrap the teeth. Teeth were rinsed under running water to remove any debris. Then the teeth had their roots cut off using a double-side, low-speed cutting metal disc at 1-mm below enamel-cementum junction. A barbed broach was used to remove the pulp tissues from the pulp chambers, then pink was used to seal the pulp chambers. Enamel surfaces were wet grounded using 800–1000 grit silicon carbide abrasive papers.

Early enamel caries formation

An acid-resistant nail varnish (Super Stay Nail polish, Maybelline, NY, USA) was used to coat the facial surfaces of the teeth excluding a 3-mm × 3-mm window, located at the gingival 1/3 of teeth crowns.[11] The specimens were suspended in 20-ml of freshly prepared acidified buffered de-mineralizing solution at 37°C for 48-h. De-mineralizing solution composed of 2.2-mmol/L NaH2PO4, 2.2-mmol/L CaCl2, and 50-mM/L acetic acid of pH 4.5 adjusted with NaOH.[12]

Re-mineralization procedures

De-ionized water was used to rinse the prepared specimens then filter papers were used to dry them. The windows marked on the facial surfaces of the specimens were coated using the three re-mineralizing agents (APF gel-Alpha-PRO®, garlic and clove nano-plant extracts) with micro-brushes. APF gel was applied in 1–2 mm thick layer and left undisturbed[13] for 60-s according to manufacturers' recommendations. It was applied alone (Group C) or followed by an instant irradiation of diode laser (Group F). To remove any evident remnants of the gel, specimens were thoroughly washed with de-ionized water for 1-min. The nano-plant extracts of garlic and clove were applied on the facial surfaces of the assigned teeth and left undisturbed for 5-min[5] then washed thoroughly with de-ionized water for 1-min.

Diode laser application

Diode laser Gallium-Aluminum-Arsenide (Siro-Laser Advance Class III b, SIRONA Dental Company, Germany) of 2-W power and 970-nm wavelength was used in continuous mode for 15-s[14],[15] using a 220-μm fiber-optic conductor. The marked windows of the specimens (Group G) were directly irradiated using the laser beam or specimens were irradiated with diode laser instantly after treatment with the three re-mineralizing agents (Groups D, E, and F). Fiber-optic tip was positioned at a noncontact approach with 2-mm average distance,[16] using putty-molds to guarantee an equal distance between fiber-optic tip and enamel surface.[17] The optic fiber was moved longitudinally in a uniform way.[18] Through specimens rinsing with de-ionized water for 1-min to eradicate any re-mineralizing agent remnants was done. Specimens were kept in de-ionized water, in tight-seal poly-ethylene vessels at 37°C for 24-h before conducting the tests.

Assessment of the chemical analysis

Environmental Energy Dispersive X-ray Analysis (EDX, Quanta 250, FEI company, The Netherlands) was used for re-mineralization evaluation through Ca, P, and F content assessment. Specimens were positioned inside a chamber on aluminum stubs with their facial surfaces upwards. A compressed air was utilized to gently remove the water covering the specimen's surfaces. Wt% was used to determine Ca, P, and F ions contents.

Surface micro-hardness test

The treated specimens were set horizontally and centralized in chemical-cured acrylic resin blocks with their treated facial surfaces upwards using 4-cm diameter sectional Teflon molds. The acrylic blocks were left to harden for 1-h, then surface micro-hardness was calculated using Digital Vickers Micro-hardness Tester (NEXUS 4000TM, INNOVTEST, model 4503, The Netherlands) with 10-s dwell tine, ×20 magnification, and 200-g load.[19] Three random indentations were performed on the specimens marked windows. Calculations of the surface micro-hardness were evaluated using (Hardness-Course Brinell/Vickers/Rockwell, copyright IBS 2012, version 10.4.4) computer software.

Statistical analysis

For each group of each test standard deviation and mean values were determined. Kolmogorov–Smirnov and Shapiro–Wilk tests were utilized to check for data normality and the parametric (normal) distribution was displayed. To compare between more than two groups in nonrelated samples, One-way ANOVA followed by Tukey post hoc test was used. The level of significance was set at P ≤ 0.05. IBM® SPSS® Statistics Version 20 for Windows (IBM Corp, Armonk, New York, USA). was used to perform the statistical analysis.

  Results Top

Data of the assessment of the chemical analysis of the early enamel carious lesions using the three tested re-mineralizing agents in the presence or absence of diode laser irradiation is presented in [Table 2]. One-way ANOVA analysis for the Ca content (wt%) showed no statistically significant difference between all test groups (P = 0.577). Group A recorded the highest mean values, while Group H recorded the least mean values. P content (wt%) showed no statistically significant difference between the test groups (P = 0.821). Group A recorded the highest mean values, while Group C recorded the least mean values. F content (wt%) revealed a statistically significant difference between different test groups (P < 0.001). Group F and each of A, B, C, E, G, and H groups showed a statistically significant difference where P = 0.002, P = 0.001, P = 0.002, P = 0.023, P < 0.001 and P < 0.001 correspondingly. A statistically significant difference was found between Group C and each of A, B, D, E, G, and H groups (P < 0.001). Group C recorded the highest mean values, while the least mean value was found in G and H groups.
Table 2: Mean, standard deviation values of chemical analysis assessment of different groups

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Data of the surface micro-hardness of early enamel carious lesions using the three tested re-mineralizing agents in the presence or absence of diode laser irradiation is presented in [Table 3]. One-way ANOVA analysis displayed a statistically significant difference between all test groups (P < 0.001). Group A and each of C, D, F and H groups showed a statistically significant difference at P = 0.002, P = 0.028, P < 0.001 and P = 0.015 correspondingly. Group B and each of C, D and E presented a statistically significant difference at P = 0.001, P = 0.007 and P < 0.001 correspondingly. Group C and each of G and H groups revealed a statistically significant difference at P < 0.001. Group D and each of F, G and H groups showed a statistically significant difference at P = 0.008, P = 0.003 and P < 0.001 correspondingly.
Table 3: Mean, standard deviation values of surface micro-hardness of different groups

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Group F group showed the highest mean values, while Group H showed the least mean values.

  Discussion Top

New preventive modalities are highly required to overcome the spread of dental caries. Such modalities may include fluorides, antimicrobials, and herbal extracts, as relatively noninvasive and simple regimes to minimize the solubility of the protective enamel layer.

Lasers can successfully decrease the de-mineralization rate in enamel, through changing its crystallinity, permeability, and acid solubility.[20] However, their actual mechanism of action still uncertain and not fully understood even today.

A wide number of natural traditional therapeutic plants has been assessed for their role in the treatment or inhibition of various diseases of the oral cavity. The efficiency of different plant extracts against specific oral pathogens has been investigated. However, their capability to prevent dental plaque formation and development of dental caries still needs further investigations.[21]

The present study assessed the chemical analysis of early enamel carious lesions treated with a commercial fluoride agent (APF gel) and two experimentally prepared nano-plant extracts of garlic and clove with and without diode laser irradiation using environmental EDX as shown in [Table 2]. Ca content showed no statistically significant difference between all tested groups. This was in accordance with Pawlus et al.,[22] they concluded that Ca content in the tooth remains at relatively steady levels after the tooth is completely developed and over the lifetime. Ca is a significant chemical determining factor of quality of the enamel surface.[23]

On the other hand, the highest mean values were recorded for Group A (garlic nano-plant extract) and the lowest mean values were recorded for Group C (APF gel), this might be owed to the increased levels of calcium in the garlic extract while APF gel does not contain Ca in its components. This finding can be further supported by the nutritional composition of garlic, which indicated that every 100 g of garlic contain 181 mg of Ca.[24] Meanwhile, Group B (clove nano-plant extract) showed lower mean values than those of Group A, this might be explained by the nutritional composition of clove, which indicated that it has lesser content than garlic as every 100 g of clove contain 117 mg of Ca.[25]

The results of this study showed that diode laser application did not seem to significantly influence Ca content even when combined with garlic or clove nano-plant extracts. It was suggested that lasing of de-mineralized enamel might cause dissolution of Ca, P and F ions of the tooth which might be trapped in micro-pores and micro-fissures that were created due to evaporation of water and loss of the carbonate content.[26] Such results were in accordance with those of de Sant'anna et al.,[27] who studied changes at enamel chemistry following diode laser application. They stated that the enamel content of Ca was steady between the test groups upon the application of diode laser irradiation. Simmer and Fincham[28] had further explained such findings as they concluded that there will be a significant rise in the level of Ca activity up to 10th power within the equation of the product of enamel solubility. This might be owed to the existence of ten Ca ions in single unit of each hydroxyapatite crystal. It should be kept into consideration that the enamel solubility product significantly affects the enamel resistance to dental caries and different acidic challenges.

The results of P content showed a nonsignificant statistical difference between the different test groups with an insignificant decrease in its mean values decrease through all tested groups. Antunes et al.[29] agreed with such findings, they concluded that the drop in P content after laser application might be due to the heat generated during laser irradiation that might lead to a volatilization of the water content and creation of micro-spaces. Nonetheless, they applied different high power neodymium-doped yttrium aluminum garnet lasers. On the other hand, diode laser that was operated at low power was used in the present study.

Regarding F content, it showed a statistically significant difference between all test groups. A significantly higher mean values was recorded for groups treated with APF gel (Group C) and even higher for those irradiated with diode laser after APF gel application (Group F) than the rest of the test groups. This can be attributed to the increased fluoride concentration in APF gel that contains 12,300 ppm of fluoride according to its manufacturer and the absence of fluoride in the nutritional composition of both garlic and clove. Moreover, these findings might be due to the reduction of the acid-solubility of the enamel of the tested specimens as a result of laser treatment of their enamel surfaces. It was concluded that laser irradiation of partially de-mineralized enamel or incipient enamel caries could hinder the progress of dental caries.[30] In addition, laser irradiation along with a fluoride agent might increase the enamel fluoride uptake. Such findings agreed with those of Villalba-Moreno et al.,[31] and González-Rodríguez et al.,[18] their results presented a significant rise in enamel fluoride uptake of the test specimens subsequent to laser treatment. Some studies have concluded that combined fluoride application and laser irradiation improves the irradiated enamel uptake of fluoride and give much better results than laser treatment alone.[32],[33]

The outer enamel layer plays a critical role in the progression and initiation of caries process. Therefore, assessment of any alterations in such layer is of great concern. Vickers micro-hardness testing method has the advantage of being highly precise besides its quantitative measurement capability.[34]

This study investigated surface micro-hardness of early enamel carious lesions treated with APF gel and two experimentally prepared nano-plant extracts of garlic and clove with and without diode laser irradiation using Vickers micro-hardness test is shown in [Table 3]. Specimens treated with APF gel then irradiated with laser (Group F) recorded the highest mean value, while the least mean values were recorded for the control group (Group H). These results could be explained by high levels of fluoride concentration in APF gel that contains 12,300 ppm of fluoride according to its manufacturer. Similarly, this can be further explained by the significant role that fluoride plays in the process of re-mineralization of the tooth structure. According to Sh et al.,[35] fluoride has the capability to boost the re-mineralization procedure by increasing the crystal growth of the de-mineralized crystals of enamel. These findings were in accordance with Nammour et al.,[36] they concluded that the enamel surface micro-hardness has significantly increased following the application of two types of fluoride agents in comparison to the control group. Moreover, they also concluded that diode laser irradiation had a positive influence on the chemistry and surface micro-hardness of the de-mineralized enamel surface.

On the other hand, Lee et al.,[37] studied the effectiveness of three topical fluoride treatments on re-mineralization of de-mineralized enamel lesions and concluded that the three fluoride treatments had no significant differences in fluoride uptake.

Furthermore, it was concluded that diode laser irradiation of enamel surfaces increased the fluoride uptake providing an extra fortification to enamel surface from coexisting acidic attacks.[11] Hence, increasing the enamel surface micro-hardness. These findings were in accordance with de Magalhães et al.,[38] they concluded that the enamel microhardness had significantly improved when enamel was treated only with APF gel and photo-activated with a diode laser.

Garlic and clove nano-extracts showed a significantly higher values than the control or laser alone groups (Groups H and G respectively), either alone (Groups A and B respectively) or with laser irradiation (Groups D and E respectively). Due to the shortage of the studies that investigate the effect of natural nano-plant extracts on the surface microhardness of enamel, these findings might be owed to the calcium and phosphorus contents in garlic and clove nano-extracts.

Moreover, the application of laser has significantly increased the surface micro-hardness of enamel following the application of garlic and clove nano-extracts, which might be due to the capability of diode laser to ablate enamel surface which might have contributed to the increased surface micro-hardness of the treated specimens.

Finally, the null hypotheses studied in this study were not accepted as various tested re-mineralizing agents that were applied to the early enamel caries had various substantial consequences on enamel chemical assessment and surface micro-hardness.

  Conclusions Top

Due to the constraint of the current in vitro study; it could be concluded that garlic and clove nano-plant extracts may present potential dental re-mineralizing agents. Moreover, their re-mineralization capacity is enhanced by the action of diode laser irradiation.

Further research is compulsory on using natural plant extracts with diode laser application to improve re-mineralization and surface micro-hardness of enamel.

Ethical clearance

This study that was conducted at the National Research Centre in Egypt with Ethics Committee approval number 16/344.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3]


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