Association of Nd:YAG laser and calcium-phosphate desensitizing pastes on dentin permeability and tubule occlusion

Abstract Objective To evaluate the efficacy of Nd:YAG laser associated with calcium-phosphate desensitizing pastes on dentin permeability and tubule occlusion after erosive/abrasive challenges. Methodology Dentin specimens were exposed to 17% ethylene diamine tetra-acetic acid (EDTA) solution for 5 min and randomly allocated into five groups: G1, control (no treatment); G2, Nd:YAG laser (1 W, 10 Hz, 100 mJ, 85 J/cm2); G3, Laser + TeethmateTM Desensitizer; G4, Laser + Desensibilize Nano P; and G5, Laser+Nupro®. Specimens underwent a 5-day erosion-abrasion cycling. Hydraulic conductance was measured post-EDTA, post-treatment, and post-cycling. Post-treatment and post-cycling permeability (%Lp) was calculated based on post-EDTA measurements, considered 100%. Open dentin tubules (ODT) were calculated at the abovementioned experimental moments using scanning electron microscopy and ImageJ software (n=10). Data were analyzed using two-way repeated measures ANOVA and Tukey’s test (α=0.05). Results G1 presented the highest %Lp post-treatment of all groups (p<0.05), without significantly differences among them. At post-cycling, %Lp significantly decreased in G1, showed no significant differences from post-treatment in G3 and G4, and increased in G2 and G5, without significant differences from G1 (p>0.05). We found no significant differences in ODT among groups (p>0.05) post-EDTA. At post-treatment, treated groups did not differ from each other, but presented lower ODT than G1 (p<0.001). As for post-cycling, we verified no differences among groups (p>0.05), although ODT was significantly lower for all groups when compared to post-EDTA values (p<0.001). Conclusion All treatments effectively reduced dentin permeability and promoted tubule occlusion after application. Combining Nd YAG laser with calcium-phosphate pastes did not improve the laser effect. After erosive-abrasive challenges, treatments presented no differences when compared to the control.


Introduction
Dentin hypersensitivity (DH) is a common painful condition defined as a short and acute pain resulting from exposed dentin in response to external stimuli, such as thermal, osmotic, tactile, chemical, or evaporative, and unrelated to any other dental disease or defect. 1 DH prevalence may vary widely among studies, but can be as high as 89.1%. 2 Studies have demonstrated that sensitive teeth have more exposed dentin tubules and with larger diameter than those of non-sensitive teeth, so that the patency of dentine tubules is an important and necessary condition for DH to occur. 3 Formulated by Brännström and Aström 4 in 1972, the hydrodynamic theory postulates that nociceptors located at the pulp-dentin interface can be activated by stimuli-induced changes in dentin fluid flow. Based on this theory, occluding dentinal tubules and reducing dentin permeability are deemed a rational approach to avoid or disable triggers that cause pain.
Many are the desensitizers available for treating DH, each with a different mechanism of action and effectiveness. However, no consensus has been reached about the best treatment for this condition.
Fluoride, oxalates, strontium, calcium/phosphate formulations, and lasers are some of the agents used to manage DH. 5 For being able to occlude dentinal tubules by mineral deposition, calcium and phosphatebased products are promising options. 6 Although efficiently used to promote immediate relief, 7 some of these agents lack the necessary ability to resist erosive-abrasive challenges. 8 Associating treatments has shown to reach superior results in tubule occlusion, such as the use of high-power lasers along with desensitizing agents. 9 However, their effectiveness has not always been shown to be greater in the long-term when compared to a single treatment. 10 Among high-power lasers, Nd:YAG has been widely used to treat DH. 11 Nd:YAG laser features thermal effects by melting and re-solidifying dentin surface, obliterating or narrowing dentinal tubules and forming a glazed surface. 12 When compared to other lasers, 13 Nd:YAG presents superior effects on reducing pain and obliterating tubules by promoting homogeneous melting 12 . This laser was considered safe, resulting in no pulp damage or tissue cracks and craters. 14 Manual dentin irradiation may result in areas without morphological changes and with open tubules, once the irradiation may not cover the whole surface equally. 15 Considering that irradiation is always performed manually in the daily practice, applying another occluding agent post-irradiation may help to improve tubule occlusion, reducing dentin permeability.
This in vitro study aimed to evaluate the efficacy of Nd:YAG laser associated with three calcium and phosphate-based desensitizing agents -Tetracalcium phosphate and Dicalcium phosphate anhydrous (Teethmate TM Desensitizer); Nanohydroxyapatite (Desensibilize Nano P); and sodium calcium phosphosilicate (Nupro ® paste -NovaMin ® ) -in occluding dentinal tubules and reducing dentin permeability immediately after application and after erosive-abrasive challenges. Our null hypotheses were: 1. Groups would present no differences in dentin permeability and tubule occlusion after treatment; and 2. Groups would present no differences in dentin permeability and tubule occlusion after erosiveabrasive cycling.

Methodology
Based on a completely randomized design, this study included two experimental factors: desensitizing treatment and experimental moment. The desensitizing treatment factor comprised five groups ( Figure 1)

Dentin hypersensitivity simulation
To open dentin tubules and simulate a hypersensitive condition, all specimens were immersed in 17% ethylene diamine tetra-acetic acid (EDTA) solution (pH 7.4) for 5 min, 7 rinsed with distilled water, and stored in relative humidity at 4°C. Dentin permeability and tubule counting obtained after this procedure were considered as baseline values. Then, the specimens were randomly allocated into different groups (n = 10 for dentin permeability and n=10 for ESEM evaluation).

Application of the treatments
Specimens were taken off the relative humidity environment and gently wiped with a soft absorbent paper to remove excess water from the surface.  Apply the paste using rubber cup at low speed. Let the product in contact for 60 seconds and rinse were repeated three times for each specimen, and the average was calculated. Results were converted into flow volume (µl mim 1 ) and then into hydraulic conductance Lp (mim -1 cm 2 cmH 2 O -1 ). 17 The measured hydraulic conductance considered the flow volume previously calculated, the area through which the water passed in the specimen dentin disc (0.058 cm 2 ), and the system hydrostatic pressure (10 psi

Statistical analyses
Data on %Lp and ESEM were analyzed for normal distribution using the Shapiro-Wilk test and for homoscedasticity using the Brown-Forsythe test.
After verifying data normality and homoscedasticity, the experimental groups were compared with twoway repeated measures ANOVA and Tukey's test.
Significance level was set at 5%. All statistical analyses were performed with SigmaPlot 13 software (Systat Software Inc., USA).

Dentin permeability
We found no significant differences for dentin permeability among desensitizing treatments    Figure 4 shows all representative micrographs.

Qualitative evaluation
At post-EDTA, all groups showed the dentin surfaces without smear-layer and with a high number of ODT.
Post-treatment, the control group (G1) maintained the same characteristics, presenting opened and exposed dentin tubules. G2 specimens showed dentin melting areas and an irregular surface with obliterated and narrowed tubules, although areas with opened tubules were also present. G3 surface presented the formation of a crystal-like layer, with a wide range of crystals diameters. In areas without crystal coverage, we observed opened and narrowed tubules and dentin melting areas. G4 specimens' surfaces showed dentin melting areas (indicated by M in Figure 4) associated with small crystal-like deposits (indicated by C in Figure   4), besides areas with opened tubules, unreached by the treatment. These crystal-like deposits seemed smaller than those found in G3 and G5. Moreover, many surface areas showed a large number of visible tubules amidst the crystal layer. G5 presented the formation of a heterogeneous crystal-like layer with completely or partially occluded tubules. In areas with large crystals, opened tubules could be seen through these mineral deposits.
Post-cycling, G1 presented a smear layer-covered surface with most dentin tubules obliterated or narrowed. G2 specimens showed some surface cracks and open dentin tubules, although apparently small in diameter than those observed post-EDTA, as well as

Discussion
Considering the pain mechanism described by the hydrodynamic theory, 4 the literature tends to agree that reducing the number of open dentin tubules may minimize pain and treat dentin hypersensitivity (DH). 20 Our study employed a model capable of evaluating dentin permeability and tubule occlusion, thus suitable for assessing the efficacy of the treatments tested. For simulating pulpal pressure and expressing conductance is an effective method to evaluate dentin permeability. In turn, environmental scanning electron microscopy (ESEM) offers information on the dentin surface topography, allowing us to assess its morphology and the magnitude of tubular occlusion.
This method requires no sample preparation for analyzing dentin permeability, so that the same specimen could be measured at different time-points.

Data on dentin permeability and tubule occlusion
showed similar outcomes in our study.
We assessed the efficacy of Nd:YAG laser associated with calcium phosphate-based pastes in reducing dentin permeability and promoting tubule occlusion after treatment. Our results show that all treatments achieved the desired goal, promoting lower dentin permeability and tubules occlusion when compared with post-EDTA (baseline), when dentin permeability was considered 100%. Immediately after application, all treatments showed lower ODT and %LP than the control group. These findings indicate that our first study null hypothesis was rejected. We also found no significant differences among treatments, suggesting they were equally effective. Considering that, the laser effect was not improved in association with treatments. However, that was not the outcome observed. Although all specimens treated with Nd:YAG laser still presented some opened tubules in unirradiated areas, laser treatment might have been sufficiently efficient so that no additional effect was achieved by associating treatments. We could also assume that, differently from varnishes or more fluid desensitizers, pastes consistency precluded a greater surface coverage. 19 We set the Nd:YAG power at 100 mJ, 1 W, 10 Hz, ≈85 J/cm 2 , which, according to previous findings, has proved to be safe and efficient for dentin occlusion 10 .
Recently discovered and little tested, 21 teethmate desensitizer (TD) is a mildly alkaline material that contains a tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powder that transforms into hydroxyapatite (HA) when exposed to water or an aqueous solution, such as saliva. 21 An interesting feature of TD is that TTCP/DCPA dissolution supplies a supersaturated calcium and phosphate ion solution, favoring HA continued precipitation within the oral environment. 22 Calcium phosphate precipitation seems to occlude dentin tubules at 10-15 µm depth, and fluoride helps transforming DCPA into hydroxyapatite. 23 In our study, G3 revealed a heterogeneous crystal-layer deposit on dentin surface, melted dentin areas, and nearly no open tubules apparent in post-treatment ESEM, as well as some mineral crystals within dentin tubules. TD and Nd:YAG laser association showed to effectively narrow and occlude dentin tubules after treatment, corroborating previous studies addressing TD potential (when used alone) in reducing dentin permeability. 21,24 Nano-hydroxyapatite (N-HA) -the desensitizing agent present in Desensibilize Nano P (DNP) pasteis very similar to the dental apatite 25 and considered one of the most biocompatible materials, 26 so that it has been employed in bone regeneration and tooth remineralization. 26 When applied over dentin, N-HA effectively reduces dentin permeability by promoting remineralization and dentinal tubules occlusion. 27 In our study, Nd:YAG laser and DNP association (G4) showed a significant reduction in %Lp and ODT values after treatment, significantly lower than control. G4 micrographs exhibited areas of melting interleaved with crystal-like deposits, where dentin tubules were not apparent. However, we observed opened dentin tubules in several other areas, indicating that treatments were unable to cover the surface homogeneously. Despite that, we verified the presence of crystals within some dentin tubules, indicating that N-HA may have narrowed them (arrows in Figure 4).
Dentin permeability results are compatible with ODT counting and ESEM qualitative evaluation.

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Nupro ® prophylaxis paste (NU) contains 15% Novamin ® (the commercial name of calcium sodium phosphosilicate -CSP). In aqueous environment, CSP releases calcium and phosphate ions that react to form hydroxycarbonate apatite (HCA), a product chemically similar to dental HA whose deposition forms a crystal layer capable of occluding dentin tubules. 28 At posttreatment ESEM analyses, G5 images showed most of the dentin surface to be covered by a crystal-like layer; the few tubules apparent were also partially occluded.
We did not observe melting areas, suggesting that HCA paste deposition overlaid laser irradiation effects. This could be deemed as a good effect of the treatment association, as untreated areas were widely sparse. We found no differences for %Lp and ODT counting between G5 and the other treatment groups, corroborating previous studies that also tested Nd:YAG laser and CSP paste association. 5,29 Ideally, DH treatment should provide long-term In G2, %Lp values increased in post-cycling when compared to post-treatment, but we verified no differences regarding ODT for these two experimental moments, and its micrographs showed narrowed dentin tubules and some crystals scattered over the surface. The sealing depth of Nd:YAG laser in dentin tubules was nearly 4 µm. 12 The erosive-abrasive cycling model used in our study was adapted from Machado et al. 7 (2019) and attempted to simulate a high dietary intake of acid 4 times a day, with 2 abrasive challenges, causing a dentin surface loss greater than 10µm. 31 Thus, the erosive-abrasive cycling removed nearly all laser-treated surface, partially re-opening dentin tubules and causing %Lp values to increase as the tubules were re-exposed.
Post-cycling ODT counting was not significantly different from post-treatment. This may be explained by the melting residual effect or by the deposition of toothpaste abrasives, which created a smear layer that was not firmly attached to the dentin surface. that Nd:YAG laser associated with DNP can partially resist to the erosive-abrasive cycling. N-HA ability to resist to erosive cycling has been already described in the literature. 35 Given that occluded dentin tubules are directly related to lower dentin permeability, %Lp results corroborate ESEM observations. G3 and G4 were the only two groups to show no significant differences for %Lp and ODT post-cycling in relation to post-treatment.
As for G5, post-cycling %LP significantly increased compared to post-treatment, but without significant differences when compared to G1. Conversely, postcycling ODT counting did no differ from post-treatment, and ESEM images showed narrowed tubules and some smear on dentin surface. These findings allow us to infer that, regardless of the partial occlusion observed on dentin tubules, this effect failed in preventing post-cycling increase in dentin permeability. We could hypothesize that, although some dentin tubules were occluded, crystals and precipitates were not strongly attached to the dentin surface, being removed by the pressurized water flow during the dentin permeability experiment. The results for Nd:YAG laser and NU paste association corroborate those reported by previous studies. 19 Both analytical methods evaluated but a small area, which may be considered a limitation of this study given that it could lead to an underestimation or overestimation of the treatments effect. 18 When compared to baseline results (post-EDTA), all treatments presented significantly lower %Lp and ODT values at post-cycling. All treatments were administered according with the manufacturer's instructions to cover the complete dentin surface.
However, ESEM observations showed treatments to obtain a heterogeneous distribution on the dentin surface for all groups, evincing the difficulty in treating areas with exposed dentin. The treatments used in this study may present other agents capable of acting on DH, such as the potassium nitrate from Desensibilize Nano P 36 or the analgesic effect of Nd:YAG irradiation, which could contribute in relieving pain. 33 As we opted by not including groups treated with pastes only in the experiment, we cannot judge whether the melting promoted by laser could have negatively influenced pastes effect. All these aspects should be considered when extrapolating this study results to the clinical scenario.

Conclusions
The association between Nd:YAG laser and calcium and phosphate-based pastes effectively reduced dentin permeability and the number of open dentin tubules immediately after application. However, the values recorded post-treatment did not differ from the control group after 5 days of erosive-abrasive cycling.

Conflit of interest
All authors report no conflicts of interest in this work.