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| ORIGINAL ARTICLE |
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| Year : 2022 | Volume
: 12
| Issue : 2 | Page : 138-143 |
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The combined occluding effect of sodium fluoride varnish and erbium: Yttrium-aluminium-garnet laser irradiation on dentinal tubules − CLSM and SEM study
Sahil Kawle1, Harshraj Gawande1, Jaiti Gupte1, Apurva Jadhav1, Swati Raut2, Sonal Sinha3
1 Deparments of Conservative Dentistry and Endodontics, Dr G.D Pol Foundation's YMT Dental College and Hospital Kharghar, Navi Mumbai, India
2 Krishna Institute of Medical Sciences “Deemed to be University”, Karad, Maharashtra, India
3 B.R. Ambedkar Institute of Dental Sciences and Hospital, Patna, Bihar, India
| Date of Submission | 16-Sep-2021 |
| Date of Decision | 22-Nov-2021 |
| Date of Acceptance | 02-Dec-2021 |
| Date of Web Publication | 12-Feb-2022 |
Correspondence Address:
Sahil Kawle
Dr. G. D Pol Foundation Y.M.T Dental College and Hospital, Sector 4, Kharghar, Navi Mumbai - 410 210, Maharashtra
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/aihb.aihb_136_21
Introduction: Dentin hypersensitivity (DH) is one of the most common problems, but to date, no single agent or form of treatment has been found effective. Lasers have promising potential for the treatment of DH. This in vitro study aims to compare and evaluate the desensitising ability of a combination of sodium fluoride varnish (NaF) and Erbium: yttrium-aluminium-garnet (Er: YAG) laser by assessing tubule occlusion and depth of penetration of the agent in dentinal tubules through Scanning electron microscope (SEM) and Confocal laser scanning microscope (CLSM), respectively. Materials and Methods: Forty-five extracted maxillary premolar teeth were included in this study. A 3-mm-thick horizontal slice was prepared from the cementoenamel junction of each tooth. Each slice was transversely separated into two halves as buccal and lingual samples. Ninety samples were obtained, and these samples were randomly divided into six equal groups. Groups A, B, C, and D were used for the evaluation of tubule occlusion with SEM Groups E and F were evaluated for dentin permeability with CLSM. Results: Intergroup comparisons showed statistically significant differences among groups, and it was observed that the combined use of Sodium fluoride and Er: YAG Laser showed better occluding (Group D) and depth penetration (Group F) effect when analysed using SEM and CLSM, respectively. Conclusion: It can be concluded that a combination of fluoride varnish and lasers offers a significant role in dentinal hypersensitivity.
Keywords: Confocal laser scanning microscopy, dentin hypersensitivity, Erbium: yttrium-aluminium-garnet laser, fluoride, varnish
How to cite this article:
Kawle S, Gawande H, Gupte J, Jadhav A, Raut S, Sinha S. The combined occluding effect of sodium fluoride varnish and erbium: Yttrium-aluminium-garnet laser irradiation on dentinal tubules − CLSM and SEM study. Adv Hum Biol 2022;12:138-43 |
How to cite this URL:
Kawle S, Gawande H, Gupte J, Jadhav A, Raut S, Sinha S. The combined occluding effect of sodium fluoride varnish and erbium: Yttrium-aluminium-garnet laser irradiation on dentinal tubules − CLSM and SEM study. Adv Hum Biol [serial online] 2022 [cited 2023 Mar 30];12:138-43. Available from: https://www.aihbonline.com/text.asp?2022/12/2/138/337700 |
| Introduction | |  |
Dentin hypersensitivity (DH) is one of the common problems and is quite challenging to manage in the dental profession. It is characterised by short, sharp pain arising from exposed dentin in response to stimuli, typically thermal, evaporative, tactile, osmotic, or chemical, and which cannot be ascribed to other forms of dental defect or pathology.[1],[2]
The most common reason for hypersensitivity is the exposure of cervical cementum/dentin surfaces, which is seen in conditions such as abrasion, erosion, attrition and gingival recession following scaling and root planing. The pain in DH is caused by various stimuli, like electrical (pulp testers), thermal (cold-warm), mechanical – tactile (probe-scaling), osmotic (hypertonic solutions – sugars), evaporation (air blast – air-jet stimulator) or chemical stimuli (acids) cause fluid movements within the dentin tubules.[3]
'Hydrodynamic theory' explains the movements of fluid within exposed dentinal tubules, which may be responsible for stimulating the pulpal mechanoreceptors.[1] Exposure of dentin isn't sufficient to cause hypersensitivity; it is equally important that the corresponding dentinal tubules must be open to allow for fluid diffusion, which successively stimulates nerve fibers to elicit painful nerve stimuli in the pulp by activating mechanoreceptors of nerves situated at the inner ends of the tubules or in the outer layers of the pulp.[3]
A variety of chemical and physical agents have been used for the treatment of DH like anti-inflammatory agents (corticosteroids); protein precipitants (formaldehyde, silver nitrate and strontium chloride hexahydrate); tubule occluding agents (calcium hydroxide, potassium nitrate and sodium fluoride); and tubule sealants (resins and adhesives). Currently, the actions of desensitising agents are understood in two ways: (1) Blocking dentinal fluid movement by occluding the tubules and (2) blocking pulpal nerve activity by changing the excitability of the sensory nerves.[1]
One of the important ways to manage hypersensitivity is dentinal tubule occlusion. It may occur naturally through saliva or various therapeutic agents. Fluoride is used clinically as a mouth rinse, toothpaste, desensitising agent or as a topical agent in the form of gels or Varnish. Fluoride varnishes show comparatively acceptable efficacy to control dentine hypersensitivity compared to other measures.[4]
The combination of fluoride with laser technology is promising to control tooth hypersensitivity. Thus, the aim of this in vitro study was to evaluate and compare the combined occluding effect of sodium fluoride varnish and Erbium: yttrium-aluminium-garnet (Er: YAG) laser irradiation on dentinal tubules through scanning electron microscope and confocal laser scanning microscopy.
| Materials and Methods | | |
This study was conducted in the institute after obtaining the clearance from the Institutional Ethics Committee. The sample size was determined using the following formula:
where,
S1 = 0.20 Standard deviation in the 1st group; S2 = 0.25 Standard deviation in the 2nd group
The mean difference between the 1st and 2nd sample = d = 0.2974 Effect Size = 1.32177777777778
Alpha Error (%) = 5 Power (%) =95
Z1−α/2 = Z-value for α level = 1.96 and Z1−β = Z-value for β level = 1.682.
The final sample size was 15 samples per group.
Sample preparation
Freshly extracted maxillary premolar teeth were collected and stored in 4°C distilled water with thymol until further processing. Non-carious teeth with completely formed apices were included. Carious, fractured and restored teeth were excluded.
Forty-five extracted premolars were included in the study. Before sample preparation, soft tissue and debris were removed using a scaler and a low-speed handpiece with non-fluoride pumice powder. A 3-mm-thick horizontal slice was prepared from the cementoenamel junction of each tooth using a slow-speed diamond saw. Each slice was transversely split into two halves as buccal and lingual. The cementum layer from each slice was removed with a high-speed bur to expose the dentinal tubule orifices. Smear layers on the dentinal tubule orifices were removed by 1% citric acid application for 5 min, and then samples were washed with distilled water.
Ninety samples were thus obtained in this way, and these samples were randomly divided into six equal groups.
- Group A − Control group
- Group B − Application of sodium fluoride varnish only
- Group C − Application of Er: YAG Laser
- Group D – Combined application of sodium fluoride and Er: YAG laser
- Group E − Application of sodium fluoride varnish only
- Group F − Combined application of sodium fluoride varnish and Er: YAG laser.
Group A to D was analysed for dentinal tubules occlusion by scanning electron microscopy (SEM). Group E and Group F were analysed for analysis of the depth of penetration using the confocal laser scanning electron microscope (CLSM).
Erbium: Yttrium-aluminium-garnet laser irradiation
Samples of Groups C, D and F (following sodium fluoride varnish application), were irradiated with an Er: YAG laser (fotona fedelis III) with a straight quartz round tip of 600-micrometre diameter at an energy level of 60 millijoules/pulse and a repetition rate of 30 Hertz, without water irrigation. The laser beam was swept in a mesiodistal fashion for 10 seconds, with the beam directed perpendicularly to the dentine surface in non-contact mode at a distance of 3–4 mm.
Dentin analysis via confocal laser scanning microscopy
Samples of groups E and F were labelled with rhodamine B isothiocyanate dye (0.1% by weight) to determine the effect of different methods over the penetration depth of the potassium fluoride varnish. CLSM was used in fluorescent mode to obtain images. A Helium-neon laser was used as the light source.
Dentin analysis through scanning electron microscopy
Samples of Groups A, B, C, and D were washed with deionised water and gently dried with air by using a three-in-one syringe. The samples were coated with a thin layer of gold (20–30 nanometre) under vacuum in an ion-sputtering device to increase conductivity. The following SEM images were recorded. Image analysis was performed at ×2000 to determine the diameter of the open tubules.
Statistical analysis
The scores thus obtained were tabulated and statistically analysed by Kolmogorov−Smirnov test, analysis of variance (ANOVA) followed by Turkey's post hoc procedures, and independent tests were applied.
| Results | | |
The diameter of the tubule was assessed in the first four (A-D) groups [Figure 1], and the depth of penetration was assessed in Groups E and F [Figure 2]. Data obtained were tested for normality using the Kolmogorov − Smirnov test. P value for Group A, B, C and D is 0.6510; 0.2424; 0.8070 and 0.9480, respectively, and the values for Group E and F are 0.9980 and 0.9460, respectively. The diameter of the tubule and depth of penetration of die in dentinal tubules scores follows a normal distribution. Therefore, the parametric one-way ANOVA followed by Turkey's post hoc procedure and Independent sample t-test was done to assess the diameter of tubules and the depth of penetration of the dye, respectively. | Figure 1: Histological section of scanning electron microscopy (SEM) images of various groups. (a) Group A (Control). (b) Group B (NaF Varnish). (c) Group C (Erbium: yttrium-aluminium-garnet Laser). (d): Group D (NaF Varnish + Erbium: yttrium-aluminium-garnet Laser)
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 | Figure 2: Histological section of confocal laser scanning microscopy images. (e) Group E (NaF Varnish). (f) Group F (NaF Varnish + Erbium: yttrium-aluminium-garnet Laser)
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There was a statistically significant difference in the mean diameters of the groups (P 0.001) [Table 1]. Turkey's post hoc analysis revealed that Group A showed the highest tubule diameter followed by Groups B, C, and D [Table 2]. | Table 1: Summary of tubules diameter scores in four study groups (A, B, C and D) by one-way analysis of variance test
Click here to view |
 | Table 2: Pair wise comparisons of four study groups (A, B, C and D) with respect to mean tubules diameter scores by Turkey's multiple posthoc procedures
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The comparison between Groups E and F with respect to the mean depth of penetration of die in dentinal tubules scores was performed by independent t-test. The mean value for groups E and F is 35.07 and 20.33, respectively, and the standard of deviation is 3.52 and 4.24, respectively. The P value is 0.001, which is significant. Group E shows the highest depth of penetration of die in dentinal tubules than group F [Table 3]. | Table 3: Comparison of two study groups (E and F) to assess the mean depth of penetration of die in dentinal tubules scores by independent t-test
Click here to view |
The results of the current study show that combined application of sodium fluoride and Er: YAG laser on dentinal tubules was the best treatment option for dentinal hypersensitivity than the application of sodium fluoride varnish and Er: YAG laser alone.
| Discussion | | |
Dentine hypersensitivity (DH) is well explained by 'hydrodynamic theory' which was first described within the early twentieth century by Gysi[5] and was later studied and explained more thoroughly by Brannstrom It is 'hydrodynamic theory' which explains that it is the movement of tubule fluid, which provokes the pain sensation.[6],[7] The concept of tubule occlusion as a method of reducing DH could be a logical conclusion from the hydrodynamic hypothesis. It's been shown that sensitive teeth have a much greater number of open tubules per unit area of exposed dentin as about double the typical tubules' diameter compared to non-sensitive teeth.[3]
Tubule occlusion may occur naturally through normal remineralisation and sclerotic processes on the dentin surface by the conventional saliva content and function. Therapeutic interventions include direct sealing of the tubules by dentin bonding agents and derivates, use of depolarising agents, or different fluoride measures starting from toothpaste, fluoride rinsing solutions, professional application with fluoride varnish and fluoride gel application in trays, or fluoride rinsing solutions. Fluoride mainly acts by its chemical ability to cut back and block fluid movements within the dentin tubules through the formation of calcium–phosphorous precipitates as well as calcium fluoride (CaF2) and fluorapatite (FAp)[8],[9]
Lasers may either act by occluding effect on dentinal tubules or a desensitisation effect by reducing the pulpal nerve's absolute pain threshold. The effect of Laser depends on various parameters like the amount of energy applied to a given surface include power level (Watt), exposure time (seconds), energy density (Joule/cm2), distance from the surface, and also the angle between the target tissue and the tip. The most important advantage of Laser is the immediate effect after one treatment session.[10]
Recently, uses of various lasers, including He-Ne, CO2, Nd: YAG, Er: YAG, and 810-nm diode lasers, are presented as a potential possible alternative strategy for the treatment of hypersensitivity. A study by Gelskey et al. and Lan et al. showed less cure rate for treating DH using Nd: YAG laser is not very satisfactory.[11]
In 1974, Er: YAG, as a solid laser with a wavelength of 2940 nanometres, was created by Birang R et al. in 2002 used this Laser to treat DH in three patients.[12] Er: YAG Laser is considered as a gold standard for the treatment of hypersensitivity because it' has the capacity to obliterate the dentinal tubules by melting and resolidifying dentin without pulp injuries or crack.
Irradiated dentin, when used with an adequate protocol, has a depth of sealing of 4 micrometres within the dentinal tubules, which in turn has shown direct improvement of dentinal hypersensitivity. The Er: YAG laser has an added analgesic effect when compared with the other high-power lasers. This probably occurred because the irradiation might have temporarily altered the final part of the sensory axons and blocked both the C and Aβ fibres, preventing the patient from feeling pain.[13] Thus Er: YAG laser was chosen in the present study to treat dentinal hypersensitivity.
The fluoridated dentinal tubule-occluding agent utilised in treating hypersensitivity leads to immediate relief from DH. However, the agent used to block the dentinal tubules was incapable of binding to the walls of dentinal tubules and thus was kept in place only by mechanical means only. Therefore, this fluoridated agent may well be dissolved by vitamin C solution or worn away by the action of an electric toothbrush and may eventually become ineffective. Thus the stability of the fluoridated dentinal tubule-occluding agent isn't definitive, and its long-term effects may decrease with time. Therefore a combination of Laser with fluoridated Varnish is used in the current study.[14]
Although low-level lasers and fluoride varnishes present distinct modes of action, both treatments, when combined, provided significant overall relief in dentine hypersensitivity (particularly for the sharpest pains) and showed similar performance.[15]
The confocal laser scanning microscope (CLSM) is an optical microscope that has a laser light as a light source and an electronic system that helps with image processing. CLSM could be a system that tracks fluorescently labelled agents throughout biological specimens, which enables the researchers to watch the penetration of Varnish beneath the hard tissues without any destruction of samples. CLSM was thus chosen for the current study to evaluate the degree of dye penetration.[16]
In the present study, group D (NaF Varnish + Er: YAG Laser) showed the least tubular diameter, followed by Group E (Er: YAG laser) than Group B (NaF Varnish). A study was done by Ipci, et al. where two lasers CO2 and Er: YAG lasers, were utilised in addition to NaF gel application, Lasers together with NaF gel appear to show better efficacy compared to either treatment modality alone.[17] Lan and et al. also showed similar results, in which SEM analysis of dentinal tubules showed that NaF varnish in comparison with NaF varnish and Nd: YAG laser-irradiated samples even after being treated with Electronic tooth brushing, samples showed 90% of dentinal tubules still remained occluded than those in which only NaF was applied.[11]
Tosun et al. showed similar results in which Clinpro sealant was used along with Nd: YAG laser and it was observed that the combination showed better occlusion and thus better reduction in dentine hypersensitivity.[18] In a study done by Watanabe et al., the ratio of the blockade of dentinal tubules and reduction of its diameter after laser irradiation was 16%–61% compared to the control.[19]
This study is an in vitro study and contributes as the base to carry out more in vivo and randomised controlled trials. However, it might be recommended that one needs to assess dentine hypersensitivity with various clinical parameter's which include both objective and subjective parameters.
Praveen et al. conducted a study on Comparative evaluation of a low-level laser and topical desensitising agent for treating dentinal hypersensitivity, and they concluded that low-level Laser and Gluma topical desensitiser showed a similar immediate decrease in DH. Low-level Laser showed improved results at their study time intervals compared to the topical agent.[20]
Kunam et al. conducted a study on dentinal tubule occlusion, and depth of penetration of nano-hydroxyapatite derived from chicken eggshell powder with and without the addition of 2% sodium fluoride using a scanning electron microscope and confocal laser scanning microscope, and they concluded that The combination of nano-hydroxyapatite and 2% sodium fluoride was the most effective in occluding dentinal tubule.[21] Tubule occlusion may occur naturally through normal remineralisation and sclerotic processes on the dentin surface by the conventional saliva content and function. Therapeutic interventions include direct sealing of the tubules by dentin bonding agents and derivates, use of depolarising agents or different fluoride measures starting from toothpaste, fluoride rinsing solutions, professional application with fluoride varnish and fluoride gel application in trays, or fluoride rinsing solutions. Fluoride mainly acts by its chemical ability to cut back and block fluid movements within the dentin tubules through the formation of calcium–phosphorous precipitates as well as CaF2 and FAp.[8],[9]
Lasers may either act by occluding effect on dentinal tubules or a desensitisation effect by reducing the pulpal nerve's absolute pain threshold. The effect of Laser depends on various parameter's like the amount of energy applied to a given surface include power level (Watt), exposure time (seconds), energy density (Joule/cm2), distance from the surface and also the angle between the target tissue and the tip. The most important advantage of the Laser is the immediate effect after one treatment session.[10]
Recently, uses of various lasers, including He-Ne, CO2, Nd: YAG, Er: YAG and 810-nm diode lasers are presented as a potential possible alternative strategy for the treatment of hypersensitivity. A study by Gelskey et al. and Lan et al. showed less cure rate for treating DH using Nd: YAG laser isn't very satisfactory.[11]
In 1974, Er: YAG as a solid Laser with a wavelength of 2,940 nanometres, was created by Birang R et al. in 2002, used this Laser to treat DH in three patients.[12] Er: YAG Laser is considered as a gold standard for the treatment of hypersensitivity because it's has the capacity to obliterate the dentinal tubules by melting and resolidifying dentin without pulp injuries or crack.
Irradiated dentin, when used with an adequate protocol, has a depth of sealing of 4 micrometres within the dentinal tubules, which in turn has shown direct improvement of dentinal hypersensitivity. The Er: YAG laser has an added analgesic effect when compared with the other high-power lasers. This probably occurred because the irradiation might have temporarily altered the final part of the sensory axons and blocked both the C and Aβ fibres, preventing the patient from feeling pain.[13] Thus Er: YAG laser was chosen in the present study to treat dentinal hypersensitivity.
The fluoridated dentinal tubule-occluding agent utilised in treating hypersensitivity leads to immediate relief from DH. However, the agent used to block the dentinal tubules was incapable of binding to the walls of dentinal tubules and thus was kept in place only by mechanical means only. Therefore, this fluoridated agent may well be dissolved by vitamin C solution or worn away by the action of an electric toothbrush and may eventually become ineffective. Thus the stability of the fluoridated dentinal tubule-occluding agent isn't definitive, and its long-term effects may decrease with time. Therefore the combination of Laser with fluoridated Varnish is used in the current study.[14]
Although low-level lasers and fluoride varnishes present distinct modes of action, both treatments, when combined, provided significant overall relief in dentine hypersensitivity (particularly for the sharpest pains) and showed similar performance.[15]
The confocal laser scanning microscope (CLSM) is an optical microscope that has a laser light as a light source and an electronic system that helps with image processing. CLSM could be a system that tracks fluorescently labelled agents throughout biological specimens, which enables the researchers to watch the penetration of varnish beneath the hard tissues without any destruction of samples. CLSM was thus chosen for the current study to evaluate the degree of dye penetration.[16]
In the present study, group D (NaF Varnish + Er: YAG Laser) showed the least tubular diameter, followed by Group E (Er: YAG laser) than Group B (NaF varnish). In a study done by Ipci, et al. where two lasers CO2 and Er: YAG lasers, were utilised in addition to NaF gel application, lasers together with NaF gel appear to show better efficacy compared to either treatment modality alone.[17] Lan and et al. also showed similar results, in which SEM analysis of dentinal tubules showed that NaF varnish in comparison with NaF varnish and Nd: YAG Laser irradiated samples even after being treated with Electronic tooth brushing, samples showed 90% of dentinal tubules still remained occluded than those in which only NaF was applied.[11]
Tosun et al. showed similar results in which Clinpro sealant was used along with Nd: YAG laser, and it was observed that combination showed better occlusion and thus better reduction in dentine hypersensitivity.[18] A study done by Watanabe et al., the ratio of blockade of dentinal tubules and reduction of its diameter after laser irradiation was 16%–61% compared to the control.[19]
This study is an in vitro study and contributes as the base to carry out more in vivo and randomised controlled trials. However, it might be recommended that one needs to assess dentine hypersensitivity with various clinical parameter's which include both objective and subjective parameters.
Praveen et al. conducted a study on comparative evaluation of a low-level laser and topical desensitizing agent for treating dentinal hypersensitivity, and they concluded that low-level Laser and Gluma topical desensitiser showed a similar immediate decrease in DH. Low-level Laser showed improved results at their study time intervals compared to the topical agent.[20]
Kunam et al. conducted a study on dentinal tubule occlusion and depth of penetration of nano-hydroxyapatite derived from chicken eggshell powder with and without the addition of 2% sodium fluoride using a scanning electron microscope and confocal laser scanning microscope, and they concluded that The combination of nano-hydroxyapatite and 2% sodium fluoride was the most effective in occluding dentinal tubule.[21]
| Conclusion | | |
Within the limitations of this in vitro study, it can be concluded that all the treatment modality for reducing dentinal hypersensitivity was effective for the occlusion and narrowing of dentinal tubules. However, more prominent occlusion was observed in the combined treatment of Er: YAG laser and sodium fluoride varnish on dentinal hypersensitivity. Therefore, whether used alone or in the combination of Er: YAG laser and sodium fluoride varnish, Er: YAG laser irradiation is effective for the treatment of DH.
Acknowledgements
We would like to thank the faculty and staff of the Department of Biosciences and Bioengineering, Indian Institute of Technology, Powai, for their assistance, support, and cooperation and for letting us carry out this research at their esteemed institution.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 1], [Table 2], [Table 3]
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