AGD - Academy of General Dentistry

One hundred twelve specimens from bovine incisors were divided into eight groups: Group 1 (treated with 10% strontium chloride gel), Group 2 (treated with 2% sodium fluoride gel), Group 3 (treated with 2% stannous fluoride gel), Group 4 (treated with 5% potassium nitrate gel), Group 5 (treated with 10% potassium nitrate gel), Group 6 (treated with 3% potassium oxalate gel), Group 7 (treated with hydroxyethylcellulose gel), and Group 8 (which received no treatment). Dentinal tubules were exposed after 0.5 mm of deep abrasion using a carbide bur and EDTA gel application. After each treatment, dentin permeability, tubule occlusion, and chemical elements on dentin were analyzed.

There was a significant difference among groups in dentin permeability (p < 0.05 ANOVA). Groups 4, 5, and 6 showed the lowest values, while Groups 1, 7, and 8 exhibited the highest. Groups 1, 2, 3, 7, and 8 showed open dentinal tubules, Groups 4 and 5 had partial tubule occlusion, and most of the tubules in Group 6 were obliterated. Energy-dispersive x-rays revealed similar chemical characteristics among the experimental agents used, with traces of strontium, fluoride, sodium, and potassium. Within the limits of the study, 3% potassium oxalate gel showed the best results in terms of dentin permeability and dentinal tubule occlusion.

Improvements in oral health can lead to a reduction in caries and support long-term tooth maintenance. However, improvements in oral health also may result in a higher risk of cervical dentin hypersensitivity.^1-6 Dentin hypersensitivity (DH) is neither a recent problem nor a rare one; nonetheless, this clinical condition remains poorly understood with no effective or permanent treatment available.^7-9

DH is clinically described as a painful response to thermic (hot or cold), chemical (acidic fruits, spicy foods, sugar, and salt), mechanical (brushing), evaporative, or osmotic stimuli applied to opened dentinal tubules, which cannot be ascribed to any other form of dental defect or pathology. DH may result from gingival recession, erosion, attrition, radicular abrasion, nonsurgical and surgical periodontal treatments, or improper brushing habits.^13-16

The exact mechanism of stimulus transmission across dentin is still unknown. The most widely accepted hypothesis about the influence of stimuli on nerve fibers is Brannstrom’s hydrodynamic theory, which states that fluid within the dentinal tubules moves in either an inward or an outward direction when stimuli are applied, stimulating the nerve endings at the pulp/dentin interface and resulting in the generation of pain impulses.^6,15,17,18

One method of treating DH is the remission of pain by dentinal tubule occlusion, which prevents stimuli from causing dentinal fluid movement.^11,19-22 Various treatment methods have been tested, including the application of desensitizing agents, anti-inflammatory substances, iontophoresis, and neodymium-doped yttrium aluminium garnet (Nd:YAG) and erbium:YAG (Er:YAG) lasers, as well as conventional treatments utilizing composite resins and dentin adhesives.^3,9,13,23-27

Desensitizing agents can act by either filling or changing the tubules’ contents via the precipitation of proteins and calcium crystals at the aperture of or inside the dentinal tubules.^3,7,14,17,24 However, the exact mechanism of action of desensitizing agents still is not understood.^6,11,15

Desensitizing agents have been tested frequently in in vitro studies.^{8,14,18-21,24,25} These research efforts complement clinical studies, which are difficult to execute since they focus on the subjective nature of pain.^{9,12,13,28,29} The results obtained in laboratory studies cannot be extrapolated completely for clinical practice, since they do not reproduce the variables that inherently affect the buccal cavity, such as saliva, microbiota, and eating habits. In addition, these studies do not consider the psychological components involved in DH.^15,30 Conversely, laboratory methods eliminate clinically uncontrollable variables (that is, the subjectivity of sensory responses).^5,6,17,22,27 This study sought to evaluate in vitro agents that are utilized in treating DH in terms of their effects on dentin permeability and dentinal tubule occlusion.

This study utilized 112 specimens obtained from bovine incisors, which were divided into eight groups (n = 14). Groups 1–7 were treated with desensitizing agents as shown in the table, which also lists the physical-chemical properties of the compounding gels. Group 8 samples received no treatment.

The crowns were removed from the teeth and the roots were sectioned longitudinally into two parts (in a mesiodistal direction). Each part was divided into thirds using a water-cooled diamond saw (Isomet 1000, Buehler Ltd.). The apical thirds were excluded, resulting in four samples per tooth (Fig. 1).

Dentinal tubules were exposed after abrasions 0.5 mm in depth were prepared on the sample surface using a carbide bur (No. 245, SS White Burs, Inc.) in a high-speed handpiece, with tap water used as a coolant. The samples provided a 4 mm x 4 mm area of exposed dentinal tubules, while their remaining surfaces were coated with a layer of epoxy resin and two layers of nail varnish (Fig. 1). Prior to the desensitizing application, the dentin samples were treated with a 24% EDTA gel (Biodinamica) on a tiny cotton pellet. This step was repeated every 30 seconds for three minutes to remove the smear layer and open the dentinal tubules.

The desensitizing agents were applied topically to the samples and kept in place for five minutes; after that, they were washed with 20 mL of distilled water for 15 seconds.

Ammonia silver nitrate solution (50% by weight) was used to analyze the dentin permeability of 64 dentin samples (eight per group). Following the desensitizing treatment, the samples were immersed in this freshly prepared solution in total darkness for two hours, followed by a thorough rising (using running distilled water) for five minutes. To promote silver ion precipitation, the stained samples were placed in a photo-developing solution for 16 hours under fluorescent light.

At that point, the samples were embedded in acrylic resin and sectioned longitudinally (in a buccolingual direction) using an Isomet 1000 water-cooled diamond saw. To remove sludge and dentin from the dentinal tubules, the specimens were cleaned ultrasonically for 10 minutes at 47°C. The samples were photographed (magnification 40x) and images were analyzed using Image Pro Plus Version 4.5.0.29 software (Media Cybernetics). The stored digital images were not enhanced and no transformation procedures were carried out. Each image was calibrated individually with a standard scale (in µm). Three measurements were taken of each image to determine the depth of silver nitrate infiltration; finally, the mean was calculated for each specimen. After testing the reproducibility of the data, all measurements were taken by the same examiner (CO).

The remaining 48 dentin samples (six per group) were evaluated by SEM. Following the desensitizing treatment, the specimens were washed with 20 mL of distilled water and cleaned ultrasonically for 10 minutes at 47°C. Dehydration was achieved to critical point dryness using a graded series of ethanol treatments (at 25%, 50%, 75%, 95%, and 100%, for 10 minutes each). Specimens were mounted on metal stubs, stored at 37°C for 24 hours, and stored in a vacuum silica-gel desiccator for 48 hours. To perform SEM analysis, the samples were sputter-coated with 25 nm of gold for 10 minutes.

Nine images from each sample (central area) were obtained (at both 500x and 3,000x magnification) by a scanning electron microscope (SSX 550, Shimadzu Corporation) operated at 20 Kv. The resulting photomicrographs were analyzed quantitatively, considering the dentin surface characteristics as well as the intertubular and peritubular dentin and smear layer deposits.

The 48 samples used for SEM analysis were examined by EDX microanalysis (SSX 550) to determine the presence of chemical elements in deposits found next to the dentinal tubules of each specimen. The spectrum was obtained at 20 Kv, with a spot size of 5 nm and a counting time of 300 seconds. EDX microanalysis provided qualitative information regarding the presence of fluorine, sodium, phosphorus, potassium, calcium, strontium, chlorine, tin, and nitrogen.

Intra-examiner reproducibility was assessed twice within 48 hours to check the reliability of the dentin permeability data using the intra-class correlation coefficient.

Comparisons among groups in terms of dentin permeability were tested by one-way ANOVA and the Student-Newman-Keuls post hoc test. The normality of the data distribution was confirmed using the Shapiro-Wilks test, while Levene’s test was used to determine the homogeneity of variance. An α value of ≤0.05 was used to indicate statistically significant differences among the groups. All analyses were performed using a software program (SPSS Version 11.5.1, SPSS Inc.). SEM evaluation was performed qualitatively.

Intra-examiner reliability was assessed by determining the intraclass correlation coefficient of agreement. The finding for dentin permeability measurements was 0.82 (adequate).

Chart 1 displays the mean (±SD) of dentin permeability in response to silver nitrate solution. There was a significant difference among the groups (p < 0.0001) using one-way ANOVA and the Student-Newman-Keuls post hoc test.

Qualitative analysis demonstrated the presence of opened and partially occluded dentinal tubules, peritubular and intertubular deposits, and the extent of the smear layer for Groups 1, 2, and 3 (Fig. 2). Most of the dentinal tubules in Groups 4 and 5 were partially obliterated by deposits on peritubular and intratubular dentin (Fig. 3). Most of the dentinal tubules in Group 6 were obliterated, with no smear layer detected on peritubular or intratubular dentin (Fig. 4). Groups 7 and 8 displayed more opened tubules and a moderate smear layer (Fig. 4).

EDX analysis revealed peaks for the primary chemical elements found in the experimental samples. Peaks were observed for sodium, phosphorus, calcium, chlorine, strontium, and nitrogen. In Group 3 (2% stannous fluoride), tin and fluorine were not identified, while Group 6 (3% potassium oxalate) showed only traces of potassium.

Dentinal tubule occlusion can contribute to the reduction of hypersensitivity.^14,17,19,20 This study sought to evaluate in vitro agents that are utilized in treating DH, as they can affect dentin permeability and dentinal tubule occlusion. Bovine incisors were used for this study based on indications that they provide an acceptable substitute for human dentin.²⁵

In the present study, strontium chloride resulted in high dentin permeability, with opened and partially obliterated dentin tubules visible through SEM analysis. These findings were similar to those found in the control groups (that is, those receiving hydroxyethylcellulose and those receiving no treatment). Greenhill and Pashley showed that strontium chloride has high permeability; however, strontium chloride demonstrated the capacity to occlude dentinal tubules and reduce dentin permeability when it was incorporated in dentifrices.^{14,17,19,22,24} These contrasting results may be attributed to the use of different formulations and the presence of abrasive agents, which could lead to alterations on the dentin surface. Kodaka et al used EDX analysis to identify silica (but not strontium chloride) on dentinal tubules.²⁴

Potassium nitrate (5% and 10%) yielded partially obliterated dentinal tubules and deposits on peritubular dentin. These agents had low dentin permeability in relation to Groups 1, 7, and 8. EDX analysis revealed traces of potassium and nitrogen, in keeping with the results of previous studies involving partial dentinal tubule occlusion.^3,25 Other studies have reported that potassium nitrate was unable to obliterate dentinal tubules or reduce dentin permeability.^17,21 These divergent results were due to differences in the methodology employed and in the concentration of potassium nitrate.

The potassium oxalate samples in Group 6 displayed lower dentin permeability and SEM analysis demonstrated a high number of obliterated dentinal tubules. EDX analysis did not show potassium; however, carbon and oxygen were observed. These elements react with the calcium in dentin to create calcium oxalate. Previous studies have shown that potassium oxalate has great potential for dentinal tubule occlusion and reduced dentin permeability.^7,8,17,18,25 In contrast, Gillam et al showed that potassium oxalate was unable to obliterate dentinal tubules.²⁰ It is possible that the formulation of potassium oxalate contributed to these conflicting results.

Two control groups (Group 7 and Group 8) were used in the present study. Group 7 samples were treated with hydroxyethylcellulose gel, which was considered a non-active or placebo agent, to assess the actual effect of a gel without the active components affecting dentinal tubule occlusion (and, consequently, dentin permeability). Hydroxyethylcellulose gel has been utilized frequently as a placebo in previous clinical studies.^12,13,28 Group 8 consisted of specimens that were not desensitized but did undergo the same preparatory measures.

Given the limitations of in vitro studies, the results obtained in this study should be interpreted cautiously. Despite the limitations of this research, the laboratory methods in DH studies control for variables that are difficult to standardize in clinical studies. Therefore, in vitro studies are necessary to elucidate the mechanisms involved and determine the most appropriate agent for treating DH in clinical practice.^{8,14,18,21,22,25}

Within the limits of this study, it is possible to conclude that none of the desensitizing agents utilized provided total dentinal tubule occlusion. Among all agents tested, dentin permeability was reduced most effectively by potassium oxalate, potassium nitrate, and stannous fluoride.

This work was supported by CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior), Brazil.

Dr. Oberg is a postgraduate student, Department of Dentistry, State University of Ponta Grossa, Parana, Brazil, where Drs. Pilatti and Santos are professors, Department of Dentistry; Dr. Farago is a professor, Department of Pharmaceutical Sciences; and Dr. Granado is a professor, Department of Materials Engineering. Dr. Pochapski is a postgraduate student, Department of Pharmacology, Anesthesiology, and Therapeutics, Piracicaba Dental School, University of Campinas, Sao Paulo, Brazil.

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