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Journal of Indian Society of Pedodontics and Preventive Dentistry Official publication of Indian Society of Pedodontics and Preventive Dentistry
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Year : 2022  |  Volume : 40  |  Issue : 3  |  Page : 324-329

Effect of non-thermal atmospheric pressure plasma and ErCr:YSGG LASER activation of three fluoride varnishes on surface re-mineralization of enamel: A SEM-EDX analysis

Department of Paediatric and Preventive Dentistry, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed-to-be University, Karad, Maharashtra, India

Date of Submission08-Mar-2022
Date of Decision24-Aug-2022
Date of Acceptance30-Aug-2022
Date of Web Publication18-Oct-2022

Correspondence Address:
Shreya Arun Bapat
Department of Paediatric and Preventive Dentistry, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed-to-be University, Karad - 415 110, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jisppd.jisppd_113_22

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Background: Dental remineralization is the process of transporting minerals from the surrounding environment (i.e., saliva and biofilm) into partially demineralized tooth structures. Remineralization can be induced by professional therapies such as fluoride-based treatments that have the highest level of supporting evidence. High-intensity LASER and nonthermal atmospheric pressure plasma therapy have been known to increase the resistance of enamel to demineralization by surface modification. Aim: The aim of the study was to evaluate and compare the surface remineralization of enamel using ErCr:YSGG LASER and nonthermal atmospheric pressure plasma (NTP) activation with three different fluoride varnishes. Methodology: Sixty-eight extracted premolars were used which were sectioned mesiodistally to obtain 135 specimens and artificial caries were induced on the experimental surface. They were then randomly divided into three groups (n = 45): MI Varnish (GC Japan), Vanish Varnish (3M ESPE), and Embrace Varnish (Pulpdent). After varnish application, these groups were further divided into three subgroups based on the activation therapy used. Fifteen samples from each group were treated with ErCr:YSGG LASER, 15 samples with NTP, and 15 samples were the control that did not undergo activation. After 9 days of pH cycling, the mean ion concentration of the surface calcium and phosphate ions was recorded using FEG-SEM and EDX analysis. The data were statistically analyzed. Results: One-way ANOVA and post hoc Tukey test accepting P < 0.05 were performed for comparisons between all analyses groups. Vanish Varnish showed a higher Ca/P ratio in LASER, NTP, and control subgroups, followed by MI Varnish and Embrace Varnish. ErCr:YSGG LASER therapy showed an improved Ca/P ratio in all varnishes than NTP therapy and control. Conclusion: ErCr:YSGG LASER therapy showed positive effects toward improving the Ca/P, followed by NTP therapy as compared to control in all three varnishes indicating their role in enhancing the effects of remineralization. Vanish Varnish showed a higher Ca/P ratio indicating better remineralization post activation.

Keywords: Caries, fluoride, LASER, nonthermal atmospheric pressure plasma, remineralization

How to cite this article:
Bapat SA, Shashikiran N D, Gugawad S, Gaonkar N, Taur S, Hadakar S, Chaudhari P. Effect of non-thermal atmospheric pressure plasma and ErCr:YSGG LASER activation of three fluoride varnishes on surface re-mineralization of enamel: A SEM-EDX analysis. J Indian Soc Pedod Prev Dent 2022;40:324-9

How to cite this URL:
Bapat SA, Shashikiran N D, Gugawad S, Gaonkar N, Taur S, Hadakar S, Chaudhari P. Effect of non-thermal atmospheric pressure plasma and ErCr:YSGG LASER activation of three fluoride varnishes on surface re-mineralization of enamel: A SEM-EDX analysis. J Indian Soc Pedod Prev Dent [serial online] 2022 [cited 2022 Nov 29];40:324-9. Available from: http://www.jisppd.com/text.asp?2022/40/3/324/358805

   Introduction Top

Dental caries is a transmissible bacterial disease process caused by acids from the bacterial metabolism diffusing into enamel and dentin and dissolving the mineral. The caries process is a continuum resulting from many cycles of demineralization and remineralization. Remineralization is the natural repair process for noncavitated lesions and relies on calcium and phosphate ions assisted by fluoride to rebuild a new surface on existing crystal remnants in subsurface lesions remaining after demineralization. These remineralized crystals are acid resistant, being much less soluble than the original mineral.[1]

The use of fluoride varnishes is considered appropriate for at-risk tooth surfaces in caries susceptible individuals and for moderate and high caries prevalence child populations in community-based preventive programs. The active ingredient of fluoride varnish is usually 5% sodium fluoride (22,600 ppm fluoride). To enhance the remineralization potential of the varnishes, adding agents such as CPP-ACP, tricalcium phosphate, xylitol, nanohydroxyapatite, dicalcium phosphate dehydrate, and bioactive glass which yields additional remineralizing ions are proposed. Intensive application regimens come with its drawbacks of supplementary expenditure, added product usage, and increased number of visits for the dentist and the patient. To overcome these drawbacks, the activation of the varnish in a single sitting is gaining importance. The procedure emphasizes increasing the potency of the varnish as well as enamel surface modification for deeper remineralization. Various means of activation practiced include heat application, LASER irradiation, and more recently, use of nonthermal atmospheric plasma.

High-intensity LASER is known to increase the resistance of enamel to demineralization by surface modification. For caries preventive treatment, LASER irradiation is not intended to ablate the surface but only to change the morphological/chemical composition of enamel instead. This change is known to alter the ability of the enamel to remineralize. The recent enormous progress in the understanding of plasma physics and the development of plasma jet has attracted focus on the application of plasma in medicine and dentistry alike. It has been used widely in the medical field ranging from the treatment of cancer to sterilization. However, its dental application, particularly in the process of remineralization of enamel requires wider and more comprehensive research. According to the limited results of a study by Kim et al., fluoride application with nonthermal atmospheric pressure plasma (NTP) was considerably more effective than fluoride application without NTP in terms of uptake and retention of fluoride to the enamel and resistance to demineralization.[2] In light of these findings, the present study was undertaken with the aim of evaluating and comparing the effect of ErCr:YSGG LASER and NTP activation on surface remineralization of enamel using three different fluoride varnishes.

   Methodology Top

The teeth samples selected for the study included 68 human premolars that were extracted for orthodontic purposes in patients of the age group of 12–15 years. Teeth with morphologically intact enamel surface not having white spots, cracks, caries, restorations, and hypocalcifications were included in the study. After extraction, the teeth were cleaned to remove blood or any tissue debris and were stored in 0.1% wt/vol thymol solution which acted as an antifungal storage media. The teeth then were decoronated at cementoenamel junction using a diamond disk mounted on a straight handpiece.

The 68 decoronated samples thus obtained were further sliced mesiodistally with the disk and water coolant jet to obtain two enamel surfaces. The buccal and palatal or lingual halves were used as two individual specimens in further procedures.

Thus, 136 specimens were obtained from 68 premolars, respectively, and 1 specimen was discarded to divide the specimens into three groups equally. Specimens thus obtained were mounted in autopolymerizing acrylic resin disk. The dimensions of the disk were standardized to a diameter of 1.2 cm and a height of 0.6 cm.

After mounting the specimens, a label of 3 mm × 4 mm was placed on the center of the tooth surface. The rest of the exposed enamel around the label was covered with nail varnish. After adequate drying of the nail varnish, the label was peeled off the tooth surface to obtain an enamel window of 3 mm × 4 mm which was used further in the study.

The exposed enamel surface was artificially demineralized to induce white spot lesions for the study. To induce these lesions, all the samples were placed in a Modified Ten Cate's demineralizing solution at 37°C for 96 h.[3] After 96 h of demineralization, the samples were removed and washed with distilled water. One hundred and thirty-five samples, after demineralization, were divided randomly into three groups of 45 samples. The groups were labeled as:

  • Group A: MI Varnish (5% NaF + CPP-ACP)
  • Group B: 3M Vanish Varnish (5% NaF + fTCP)
  • Group C: Pulpdent Embrace Varnish (5% NaF + cXp).

Each group (A, B, and C) was further divided into three subgroups. Each subgroup had 15 samples that were randomly selected from the group. The subgroups were based on the activation systems that were used. The subgroups were labeled as:

  • Subgroup a: ErCr:YSGG LASER
  • Subgroup b: Nonthermal atmospheric pressure plasma (NTP)
  • Subgroup c: No activation (control).

All three varnishes were applied using the applicator brush provided with each product, respectively, following the manufacturer's instructions. The samples in subgroup “a” were activated using ErCr:YSGG LASER Biolase Waterlase Turbo MD with 2790-μm wavelength, wherein the output power of 0.75W was maintained with a pulse rate of 15 pps. The activation was carried out for 10 s from a distance of 10 mm from the surface of the sample in noncontact mode and perpendicular to the tooth surface with a swiping motion.

Similar to the LASER group, the samples were activated after the varnish application using an NTP pen. The NTP groups were subjected to activation by plasma device with aluminum electrodes at 230 V AC. Helium was used as the propellant gas, and the gas flow rate was maintained at 100–200 ml/min for 10 s per sample, and the activation was carried out in noncontact mode from a distance of 10 mm. In the control group, after varnish application, the samples were stored in nonionic artificial saliva (Wet Mouth Liquid, ICPA Health products). The samples did not undergo any activation. Twenty-four h after storage in artificial saliva, the varnish was peeled off the surface using the periodontal curette.

To simulate the oral environment after varnish application and activation, all the samples were subjected to a pH cycling process. For 9 days and nights, a pH cycling scheme was performed with 6 h of demineralization, followed by 17.5 h of remineralization at 37°C. The demineralization solution contained 2.0 mmol/L calcium, 2.0 mmol/L phosphate, and 0.075 mol/L acetic acid at pH 4.3. The remineralization solution was supersaturated with calcium phosphate (calcium = 1.5 mmol/L and phosphate = 0.9 mmol/L), with potassium chloride at 150 mmol/L and cacodylate buffer to pH 7.0 (20 mmol/L).[4] An intermediate wash of samples with deionized water was performed before changing the solution. Both the solutions were changed daily. The enamel surface was viewed microscopically for surface modifications and crystal growth under a FEG-SEM (FEI Quanta 200 3D). EDX analysis was carried out on the enamel surface to obtain the Ca/P ratio. The ionic ratio obtained was compared statistically.

   Results Top

FEG-SEM imaging of the samples was done to visually assess the crystal growth and remineralization on the experimental surface, and the samples were checked for the ratio of calcium and phosphate ions on the treated enamel surface using EDX analysis. FEG-SEM images were obtained, and EDX analysis was done on three standardized points within the experimental enamel window. The mean ion concentration of the surface calcium and phosphate ions was recorded. The elements quantified were Ca (weight %) and P (weight %). Using the Ca and P values obtained, Ca/P stoichiometric ratios were calculated using the following formula: Ca (mol)/P (mol) % = (Ca [weight %]/40.08 [g/mol])/(P [weight %]/30.97 [g/mol]), the molecular masses of Ca and P being 40.08 and 30.97, respectively. The values obtained were tabulated in Microsoft Excel 2016 and analyzed using one-way ANOVA and post hoc Tukey test. All the statistical tests were carried out using the IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp. One-way ANOVA and post hoc Tukey test accepting P < 0.05 were performed for the comparisons between all analysis groups.

MI Varnish group [Figure 1] showed the formation of crystals on the experimental surface. (a) enamel surface modification after LASER treatment and the formation of calcium fluoride globule can be seen. Vanish Varnish group images (d, e, and f) show distinct layers of crystallization, and a calcium fluoride globule can be seen in image (e). Embrace Varnish group (g, h, and i) show intermittent zone of remineralization and the layer of remineralization appears to be discontinuous.
Figure 1: FE-SEM images of the enamel surface for the evaluation of surface modification. (a) LASER + MI Varnish; (b) NTP + MI Varnish; (c) control group in MI Varnish; (d) LASER + Vanish Varnish; (e) NTP + Vanish Varnish; (f) Control group in Vanish Varnish; (g) LASER + Embrace Varnish; (h) NTP + Embrace Varnish; (i) Control group in Embrace Varnish

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A comparison of Ca/P ratio within each varnish showed that Vanish Varnish showed a significantly higher Ca/P ratio on the enamel surface in all three therapy subgroups followed by MI Varnish, and least Ca/P ratio was seen with Embrace Varnish.

The intragroup comparison of three activation therapies within each varnish was done using a one-way ANOVA test [Graph 1].

In the intra-group comparison for MI Varnish, the Ca/P ratio of LASER subgroup (1.47 ± 0.16) was significantly higher than plasma (1.25 ± 0.14) and control (1.03 ± 0.14) subgroup (P = 0.002). For Vanish Varnish, the highest Ca/P ratio was seen in the LASER subgroup (1.77 ± 0.14) followed by plasma (1.46 ± 0.10), and the least Ca/P ratio was seen in the control subgroup (1.12 ± 0.07). The difference between these subgroups was found to be statistically significant (P = 0.001). In the Embrace Varnish group, the difference was statistically significant with the highest Ca/P ratio seen in the LASER subgroup (1.02±0.12), followed by plasma (0.84 ± 0.11) and control (0.72 ± 0.17).

Post hoc analysis was conducted for an exact pairwise comparison of Ca/P ratio of activation therapies within each varnish [Graph 2].

A pairwise comparison of subgroups in MI Varnish showed that there was a statistically significant difference in the LASER and control subgroup (P = 0.001). However, the comparison of control and plasma subgroup (P = 0.095) and LASER and plasma subgroup (P = 0.085) showed a statistically insignificant difference.

The pairwise comparison of subgroups in Vanish Varnish showed a statistically significant difference in the comparison of LASER and plasma subgroup (0.002), LASER and control subgroup (P = 0.001), and plasma and control subgroup (P = 0.001).

A pairwise comparison of subgroups in Embrace Varnish showed that there was a statistically significant difference in the LASER and control subgroups (P = 0.012). However, the comparison of control and plasma subgroup (P = 0.354) and LASER and plasma subgroup (P = 0.151) showed a statistically insignificant difference.

   Discussion Top

White-spot lesions are the first clinical presentation of caries. The critical balance of remineralization and demineralization is maintained in the oral cavity by the salivary ions and substrate ions. These localized areas of demineralization are a consequence of disturbed equilibrium in the ion exchange of enamel. When the equilibrium shifts toward the acidic conditions, a greater number of ions leach out of the enamel as compared to normal. This results in the dissolution of the hydroxyapatite crystals and weakens the enamel. The refractive index of the demineralized areas changes and the lesions appear chalky white under natural light. They are an esthetic concern and have the potential to progress into carious lesions. The advantage, however, is that this stage of carious process is reversible. If the ion equilibrium is regained at this stage, the caries progression stops and the cavitation of the lesion is prevented. Hence, early detection and interception are of utmost importance in treating white spot lesions.

In vitro demineralization and remineralization can be assessed using various methods. It is wise to measure the changes in the mineral content of the carious lesions quantitatively to provide more promising results in the remineralization process. One of the most common techniques with high accuracy is FEG-SEM with EDX attachment. It is a microanalytical technique that is employed to estimate quantitatively the amounts of minerals in a given tooth sample. FEG-SEM gives the topographical pictures and is used to assess the surface changes seen on enamel. EDX gives quantification of various elements such as calcium, phosphorus, fluoride, magnesium, and sodium in both atomic and weight percentages. In the present study, EDX was used as an elemental analytic method for both Ca and P since the main components of enamel hydroxyapatite are Ca and P. This method permits fast and quantitative microanalysis estimating the number of minerals in a given tooth sample in a nondestructive manner.[5] FE-SEM and EDX were chosen to qualitatively measure the remineralization in our study due to its accuracy, relevancy in terms of ion concentration measurement, and imaging potential to view the modification of enamel after the therapy.

In the present study, all the samples showed the presence of both the ions on the experimental surface which indicated the remineralization of the artificially induced WSLs. The presence of these ions is in accordance with the findings of De Carvalho Filho et al. and Hicks et al. who showed that fluoride application causes the chemical changes in the main component of the enamel hydroxyapatite as calcium fluoride deposition thus inducing chemical stability and remineralization.[6],[7] Vanish Varnish contains functionalized tricalcium phosphate as it is an active ingredient. When tricalcium phosphate in the varnish comes into contact with the tooth surface and is moistened by saliva, the protective barrier breaks down, making calcium and phosphate ions available on the tooth surface in free forms indicating a higher ratio in this group.[8] The results of our study show that the calcium and phosphate ratio on the enamel surface of the samples was the highest in the samples treated with Vanish Varnish irrespective of the activation used. The higher ratio of surface calcium and phosphate ions in our study could be attributed to the fact that 3M Vanish Varnish contains calcium and phosphate ions as its chemically active ingredient.

When comparing the MI Varnish with the Embrace Varnish in the present study, the MI Varnish-treated specimens had a significantly higher Ca/P ratio. The results are consistent with the data presented by Savas et al. who reported that the Ca/P ratio was significantly higher in the remineralization phase of MI Varnish as compared to those of the demineralized surfaces in bovine incisor teeth.[9] This may be due to calcium and phosphate ions being available in the high concentrations in the composition of MI as compared to Embrace Varnish indicating better remineralization capacity.[10]

The activation by LASER showed a significantly superior ratio of calcium and phosphate ions in all three varnish groups as compared to NTP therapy and control in the present study. The ErCr:YSGG power used in the present study was 0.75 W, as studies have revealed that 0.75 W, which is a subablative power, gave the best results toward acid resistance enhancement.[11],[12],[13] Our study showed the highest Ca/P ratio in ErCr:YSGG LASER therapy group and is in accordance with the study done by El Mansy et al. who found the highest resistance to demineralization process owing to higher Ca/P ratio in samples treated with ErCr:YSGG LASER.[5] ErCr:YSGG LASER emitted at subablative wavelength of 2780 is well absorbed by water and hydroxyl radical in the hydroxyapatite thus potentially improving enamel acid resistance and preventing mineral loss by inducing chemical and morphological changes in enamel without an excessive increase of heat.[14]

The FE-SEM images of LASER subgroup of Vanish Varnish show calcium fluoride globule formation on the treated surface. This may be due to the clearly visible morphological changes on the enamel surface that occurred after laser application. As a result of the morphological surface roughness, more hydroxyapatite crystals reacted with the varnish and ions from pH cycling solutions, thus increasing the formation and retention of CaF2 globules.[15] The surface of samples of MI Varnish treated with LASER shows uniform crystal growth [Figure 1]. The images of Embrace Varnish show intermittent areas of remineralization. The formation of crystal layer is not uniform [Figure 1]. The samples of three varnishes treated with NTP show uniform layers of remineralization. The enamel surface does not appear to be structurally modified. This is in accordance with the findings of Lehmann et al. who found that no measurable surface modification of enamel was seen after plasma treatment when compared to polished enamel.[16]

   Conclusion Top

Overall, Vanish Varnish showed a higher Ca/P ratio in LASER, NTP, and control subgroups among the three varnishes studied. ErCr:YSGG LASER therapy group showed an increased Ca/P ratio, followed by NTP therapy group. While NTP therapy showed increased in Ca/P ratio as compared to control in all three varnishes used indicating its role in enhancing remineralization. The paucity of literature in exploring the full scope of NTP as an emerging tool paves the way for clinically oriented research in this direction.

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   References Top

Featherstone JD. Dental caries: A dynamic disease process. Aust Dent J 2008;53:286-91.  Back to cited text no. 1
Kim YM, Lee HY, Lee HJ, Kim JB, Kim S, Joo JY, et al. Retention improvement in fluoride application with cold atmospheric plasma. J Dent Res 2018;97:179-83.  Back to cited text no. 2
Shaik ZA, Rambabu T, Sajjan G, Varma M, Satish K, Raju VB, et al. Quantitative analysis of remineralization of artificial carious lesions with commercially available newer remineralizing agents using SEM-EDX – In vitro study. J Clin Diagn Res 2017;11:C20-3.  Back to cited text no. 3
Malik A, Parmar G, Bansal P, Bhattacharya A, Joshi N. Effect of laser and fluoride application for prevention of dental caries: A polarized microscope analysis. J Dent Lasers 2015;9:11-15.  Back to cited text no. 4
  [Full text]  
El Mansy MM, Gheith M, El Yazeed AM, Farag DB. Influence of Er, Cr:YSGG (2780 nm) and nanosecond Nd: YAG laser (1064 nm) irradiation on enamel acid resistance: Morphological and elemental analysis. Open Access Maced J Med Sci 2019;7:1828-33.  Back to cited text no. 5
De Carvalho Filho AC, Sanches RP, Martin AA, Do Espírito Santo AM, Soares LE. Energy dispersive X-ray spectrometry study of the protective effects of fluoride varnish and gel on enamel erosion. Microsc Res Tech 2011;74:839-44.  Back to cited text no. 6
Hicks J, Garcia-Godoy F, Flaitz C. Biological factors in dental caries: Role of remineralization and fluoride in the dynamic process of demineralization and remineralization (part 3). J Clin Pediatr Dent 2004;28:203-14.  Back to cited text no. 7
Robert LK, Allen CM, Emily RW, Katherine EF, Christabel XF. In vitro evaluation of eroded enamel treated with fluoride and a prospective tricalcium phosphate agent. J Dent Oral Hyg 2009;1:52-8.  Back to cited text no. 8
Savas S, Kavrìk F, Kucukyìlmaz E. Evaluation of the remineralization capacity of CPP-ACP containing fluoride varnish by different quantitative methods. J Appl Oral Sci 2016;24:198-203.  Back to cited text no. 9
Salman NR, El-Tekeya MM, Bakry NS, Soliman S. Remineralization effect of fluoride varnish containing casein phosphopeptide amorphous calcium phosphate on caries-like lesions in primary teeth (in vitro study). Alex Dent J 2019;44:13-16.  Back to cited text no. 10
Kaur T, Tripathi T, Rai P, Kanase A. SEM evaluation of enamel surface changes and enamel microhardness around orthodontic brackets after application of CO2 LASER, Er, Cr:YSGG laser and fluoride varnish: An in vivo study. J Clin Diagn Res 2017;11:C59-63.  Back to cited text no. 11
de Oliveira RM, de Souza VM, Esteves CM, de Oliveira Lima-Arsati YB, Cassoni A, Rodrigues JA, et al. Er, Cr:YSGG laser energy delivery: Pulse and power effects on enamel surface and erosive resistance. Photomed Laser Surg 2017;35:639-46.  Back to cited text no. 12
de Freitas PM, Rapozo-Hilo M, Eduardo Cde P, Featherstone JD. In vitro evaluation of erbium, chromium: Yttrium-scandium-gallium-garnet laser-treated enamel demineralization. Lasers Med Sci 2010;25:165-70.  Back to cited text no. 13
Scatolin RS, Colucci V, Lepri TP, Alexandria AK, Maia LC, Galo R, et al. Er: YAG laser irradiation to control the progression of enamel erosion: An in situ study. Lasers Med Sci 2015;30:1465-73.  Back to cited text no. 14
Zamataro CB, Ana PA, Benetti C, Zezell DM. Influence of Er, Cr: YSGG laser on CaF2 -like products formation because of professional acidulated fluoride or to domestic dentifrice application. Microsc Res Tech 2013;76:704-13.  Back to cited text no. 15
Lehmann A, Rueppell A, Schindler A, Zylla IM, Seifert HJ, Nothdurft F, et al. Modification of enamel and dentin surfaces by non-thermal atmospheric plasma. Plasma Process Polym 2013;10:262-70.  Back to cited text no. 16


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