INTRODUCTION:

Dentine hypersensitivity (DH) is reported as a sharp and short, localized, and rapidly spreading pain affected by thermal, evaporative, tactile, osmotic, or chemical stimuli affecting the comfort of oral function¹. Borges (2012) reports that about 8% -30% of the adult population shows teeth with DH, especially at 20 to 30 years. The teeth most commonly infected with DH. are first premolars with the specifications in the cervical area of the tooth's buccal surface².

Pathogenesis of DH occurs due to abrasion, erosion, and the fraction of dentine coated with enamel in the coronal and cementum at the root resulting in the opening of dentinal tubules and pain³.

Another report mentioned gingival recession, periodontal treatment, or a combination of both occurrences could trigger DH⁴. The mechanism of the onset of pain in dentine can be subjective and not specific. Many theories are introduced to explain the mechanism and characterize dentine hypersensitivity. However, the hydrodynamic theory mentioned by Brannstrom is the most widely accepted⁵. The hydrodynamic theory assumes that dentine's exposed surfaces cause stimulation from the outside, thus causing the movement of tubular fluid, which activates nerve mechanoreceptors, causing pain and discomfort⁶.

Dentine Hypersensitivity is treated by closing the dentinal tubules or stopping peripheral pulp nerve activity from preventing external stimulation that triggers pain. The treatments are achievable at home and or in the office⁷. Home treatment includes desensitizing toothpaste, while in-office therapies include applying desensitization materials such as fluoride, potassium nitrate, calcium phosphates, and oxalate. Calcium oxalate has been recommended as a desensitizing toothpaste agent in treating DH. with the formation of calcium oxalate precipitation in the dentinal tubules. This treatment effectively removes hypersensitivity dentine initially but is short-lived because of the dissolution/erosion of calcium oxalate⁸.

The use of desensitizing material is expected to have a long-term effect by maintaining quality in dentine's intra-tubular closure. However, desensitizing material has a short-term impact after being stimulated. Lynch (2012) states that sodium fluoride can close the dentinal tubules through the crystallization mechanism, and the flow of fluid to the pulp is reduced, thereby reducing DH⁹. Meanwhile, the varnish is also used for DH treatment by covering exposed dentine. But the effect is only short-term and unrecommended for long-term application¹. Kundapur (2011) reported that the use of potassium nitrate in the desensitization process showed the erosion of all potassium nitrate after rinsing using distilled water¹⁰. Also, 28% silver nitrate produces protein precipitation in the tubules, reducing hypersensitive dentine, but silver nitrate has a disadvantage that can cause discoloration of teeth¹¹.

Several materials have been proposed to treat hypersensitive dentine, but the application is still less efficient, and the development of new materials related to desensitization is still needed. Some researchers have started to develop natural materials, including chitosan. Chitosan is a deacetylated chitin N derivative with a positive charge and polymers¹². Based on findings from previous studies, the purpose of combining chitosan with silver nitrate is expected to have a better effect on desensitization in dentine hypersensitivity.

MATERIAL AND METHODS:

This research has been approved as research ethics from the Ethics Commission of the Faculty of Medicine, Universitas Sumatera Utara, Indonesia, No. 207/TGL/KEPK FKUSU-RSUP HAM/2019. The research material consisted of nano chitosan-silver nitrate gel obtained at the chemical laboratory, Faculty of Mathematics and Natural Sciences, University of North Sumatra, Indonesia. Besides, it also used varnish gel (5% sodium fluoride and tri-calcium phosphate) ® and 24 third molars.

Dental Sample Preparation:

Hank's Balanced Salt Solution (HBSS) stores 24 odontectomy third molars. All samples were cut at the crown, and the radicular was removed 1 mm from the Cementum enamel junction (CEJ) using a wheel disc. The buccal dentin surface of the sample is sharpened using silicon carbide paper (600-2000 grits) on the polish machine to expose the dentin's surface, then placed into the distillation liquid in the ultrasonic bath for 10 min to release the remaining abrasive polish. Then the sample was etched with 6% citric acid for 1 minute to expose the dentine tubules and then regained into the distillation liquid in the ultrasonic bath for 30 min to remove the residual smear layer. Samples were divided into six groups, and each group was divided by incubation time of 24 h and 48 h. Its Group 1 (8 teeth only immersed in artificial saliva), Group 2 (8 teeth applied with nano chitosan-silver nitrate gel), and Group 3 (8 teeth used with varnish gel).

SEM-EDX Tubulus Dentine Assay:

The tubules dentine closing was evaluated by Scanning Electron Microscope (TM3000, Hitachi High-Tech. Corp, Jepang) while examining Energy Dispersive X-ray (SwiftEd3000, Hitachi High-Tech. Corp, Japan) to determine the composition of chemical elements on the surface of the dentin¹³. The image was taken three times at different points to calibrate the covering area's profile in the dentinal tubules. The microstructure image of each sample group was captured using SEM. The sample is placed in the center of the chamber. The height of the sample is according to standard calibration. Then, the SEM tool was turned on with a power of 20 kV. The sample is shifted slowly to get the area to be captured on the SEM screen. Brightness, contrast, and focus are adjusted until the best picture is shown. Photos were captured with a magnification of 1500K and 2000K. Sample testing using EDX is carried out with the following procedure: Determined the area to be analyzed. The data were obtained using an EDX scanner within 1 sec. The results are displayed on the screen. The EDX database software confirmed the type and number of elements of the scanned area.

Ionic Assay:

The ionic analysis included a control sample of conductivity and dissolved oxygen groups (artificial saliva), chitosan-silver nitrate nano gel, and gel varnish. Each group's conductivity checks were carried out using a Conductivity meter (CyberScan CON 410, Eutech Instruments Pte Ltd, Singapore)¹⁴. The conductivity meter system comprises two electrodes connected to a voltage source and an ampere meter. The electrode is set to give a certain distance between the electrodes (usually 1 cm). These two electrodes are immersed in the sample solution during the measurement process, and the voltage is applied to a certain magnitude. The value of the electric current processed in an ampere meter is then used to calculate the solution's electrical conductivity value. Dissolved oxygen from each group was carried out using a Dissolved Oxygen (DO) meter (CyberScan DO 300, Eutech Instruments Pte Ltd, Singapore). The working principle of DO refers to conductivity checks, using an oxygen probe consisting of a cathode and anode immersed in a test solution. The test is carried out three times to obtain the optimal value of the two ionic tests.

Viscocity Assay:

The Schott Ostwald viscometer was used to measure the viscosity of the test material. The density of the pycnometer was measured beforehand to obtain the viscosity value, namely by weighing the empty pycnometer and the lid using analytical scales. The tube's test material was then taken using a 5 mL pipette, placed into a pycnometer and closed, then weighed using an analytical scale (Ohaus, max cap 210gr), and assessed for three repetitions. Acquisition of data is recorded on the provided sheet. The density of saliva is calculated using the following formula: ρ = m/v, ρ = density of saliva (gr/mL), m = weight of filled pycnometer-weight of empty dry pycnometer (gr), and v = volume of pycnometer (mL)¹⁵.

After obtaining the results from the density measurement, the saliva's viscosity was calculated using the Ostwald viscometer. Placed water into the viscometer, then sucked the test material using a suction ball (rubber ball) through the capillary tube until it crossed line A. When the water is exactly on line A, start the stopwatch, let the water flow, and how long it takes to reach line B. Then, remove the water and insert the test material in the same way. The assessment was carried out three times. The formula for calculating viscosity is: η1/η2 = ρ1 t1/ρ2 t2. η1 = Viscosity of water (0.008937 N seconds/m2), η2 = Viscosity of salivary samples (N seconds/m2) ρ1 = Density of water (gr/ cm3), ρ2 = density of salivary samples (gr/cm3), t1 = time of flow water (seconds), and t2 = flow time of saliva sample (seconds)

Data analysis:

Kruskal-Wallis analyzed data for dentinal tubular closure. The significant difference between each treatment group in dentinal tubular closure based on p<0.05 is significant.

results and DISCUSSION:

Fig 1 shows the SEM image of the dentinal tubules. In the untreated group, A (24 h) and group D (48 h) showed that the dentinal tubules' surface was still exposed, which showed that the dentinal tubule surface was intact. Meanwhile, in group B (24 h) and group E (48 h) in the chitosan treatment group, the tubule surface showed a closure starting 24 h to 48 hours. Likewise, the commercial gel varnish group, group C (24 hours) and group F (48 h), showed almost even dentinal tubules coverage.

Fig.1: SEM profiles of dentinal tubules. Group control (A 24 h, group D 48 h), Group nano chitosan-silver nitrate gel (B: 24 h and E: 48 h), and gel varnish group of 5% sodium fluoride -tricalcium phosphate (C: 24 h and F: 48 h). 2000 x Magnification

Fig. 2: Graph of dentinal tubules closure scores in teeth immersed in Chitosan-silver nitrate and gel varnish (5% sodium fluoride -Tricalcium Phosphate). There are differences in the assessment of dentinal tubule closure by observers 1 and 2. Observer 1, there is no difference in dentinal tubule closure after immersion in the test material. Otherwise, observer 2 shows dental samples immersed in Chitosan-silver nitrate indicate a higher effect on the dentinal tubule closure.

Fig. 2 illustrates the quantitative analysis of dentinal tubular closure scores from SEM images performed by two observers. Although not significantly different from varnish gel (p> 0.05), chitosan-silver nitrate at 24 hours showed the best dentinal tubule closure results. The Mann-Whitney test showed no significant difference in the dentinal tubular closure score between the first and second observers (p = 1,000> 0.05). In general, based on the Kruskal-Wallis analysis, there was a difference in the two observers' scoring (p<0.05; 0.011). Besides, there was no significant difference between the 24 and 48 hours incubation time (p> 0.05).

Figure 3 shows that chitosan gel's viscosity value is higher than gel varnish, especially at the 24-hour incubation time, while at 48 hours, it has the same value. This viscosity value becomes a reference for entering the dentinal tubules while interacting with the dentinal tubule's chemical components. This value has linearity with the dentinal tubule's closure, as shown in Figures 1 and 2.

Fig. 3: The viscosity value of the test material. The chitosan-silver nitrate nano gel group's viscosity showed a better value than the viscosity of the gel varnish (5% sodium fluoride -tricalcium phosphate) at 24 hours. Meanwhile, the saliva medium is lower than the two test materials. Bar (viscosity value) Error bar (standard error).

Table 1 shows that chitosan and commercial gel varnish substantially affect the dentin surface's chemical elements. This activity is related to interacting with the dentinal tubule's chemical elements in forming bonds to initiate and increase the number of chemical elements during the dentinal tubule's closure process. Indications that the dentinal tubules' chemical closure activity increase oxygen, phosphate, and calcium and decrease elemental carbon during the reactivity process.

Table 2 shows that chitosan has higher conductivity and oxygen solubility values than gel varnish. The test material's conductivity and oxygen dissolve values strongly influence the dentinal tubules' closure on dentin hypersensitivity. Based on this ionic value, chitosan shows stable properties in the dentinal tubules ionization process. The percentage of the ionic conductivity value is higher than the solubility value of oxygen.

Table 1: Profiles of chemical elements on the dentin surface of a tooth

Elements	Weight % (24 h)			Weight % (48 h)
Elements	No Treatment	Chitosan-Silver nitrate	gel varnish*	No Treatment	Chitosan-Silver nitrate	gel varnish
Carbon	55.551	35.154	18.217	0.000	0.000	0.000
Oxygen	36.673	38.285	44.558	0.000	52.101	50.213
Fluorine	0.000	4.636	0.000	76.965	0.000	0.000
Sodium	0.286	0.000	0.585	0.000	0.600	0.000
Magnesium	0.294	0.000	1.177	0.001	0.000	2.896
Phosphorus	0.049	5.623	11.871	0.026	15.911	22.067
Potassium	0.664	1.870	0.523	0.000	0.000	1.289
Calcium	6.482	14.432	23.069	23.009	31.389	23.535

*gel varnish (5% sodium fluoride -tricalcium phosphate)

Table.2. Conductivity and dissolved oxygen values assessment of material assay

Groups	Conductivity (µS)				Dissolve Oxygen (Ppm)
	24 h		48 h		24 h		48 h
	Value	Freq	Value	Freq	Value	Freq	Value	Freq
No Treatment (Control)	0,7425	13%	426,25	33%	0,01	25%	0,06	35%
Chitosan-Silver nitrate	2,9775	51%	477,5	37%	0,02	50%	0,05	29%
Gel varnish	2,155	37%	402,75	31%	0,01	25%	0,06	35%

This study evaluated the role of chitosan-silver nitrate nano gel in the desensitization process of DH. This in-vitro modeling considers several indicators on DH starting with exposing the dentinal tubules, applying the test material, and observing the effect on the dentinal tubules' closure. The success of closure of the dentinal tubules as an indicator of DH prevention was assessed based on SEM images of dentinal tubules, an increase in calcium and phosphor, and ionic properties and viscosity of the test material. Tosun (2016) reports that test materials in the DH treatment reduce the number or diameter of dentinal tubules either by sealing through a coating mechanism or through coagulation¹⁶. Thus, desensitizing agents are expected to increase the dentinal tubules' closure, thereby reducing the movement of the dentine fluid and being resistant to the acidic environment of the oral cavity.¹

Fig1 reports that nano chitosan-silver nitrate gel can better close dentine tubules than varnish gel (5% sodium fluoride-tricalcium phosphate), even though it is incomplete due to the detection of some exposed tubules, especially at 24 hours. Camilotti (2012) reports that not all DH treatment products are ideal for covering dentinal tubules¹⁷. The parameters set by Grossman (1934) DH treatment products are non-irritating pulp or painless, easily applied, long-term effect, do not lead to discoloration and stain on teeth, non-irritating to soft tissue or periodontal ligament, and low cost¹⁸. Based on the research results, chitosan as a natural material, besides closing the dentinal tubules, can maintain pH balance, thereby preventing the development of bacteria and accelerating absorption through anabolic and catabolic reactions between chitin and dentin surface reactors¹⁹.

The results of Fig 2 are in line with Fig 1, where the nano chitosan-silver nitrate gel has better closure power of the dentinal tubules than the varnish gel. The phenomenon mentioned above is believed that the chemical bond between chitosan and silver nitrate has a better adhesion power. This bonding power causes the two compounds to have good reactive properties with the mass of dentinal tubules. Yadav (2015) reported that silver nitrate is used as a desensitizing agent by coagulation action or destroying dentin protoplasm.²⁰ The results of Fig 2 are in line with Fig 1, where the nano chitosan-silver nitrate gel has better closure power of the dentinal tubules than the varnish gel. The phenomenon mentioned above is believed that the chemical bond between chitosan and silver nitrate has a better adhesion power. This bonding power causes the two compounds to have good reactive properties with the mass of dentinal tubules.

Besides being stable under the influence of light, silver nitrate/nitric acid also has a double bond constituent with the chitin group to facilitate the two compounds' interaction. Thus, the dentin's surface, desensitized with nano chitosan-silver nitrate gel, reacts with chloride ions originating from the dentine liquid to form a very homogeneous and thin white precipitation covering all dentinal tubules²¹. Meanwhile, this white residue, silver chloride crystals, originates from dentin, namely tubular fluid²². In addition, chitosan in silver nitrate can increase the rate of hydroxyapatite coatings because chitosan can be absorbed into hydroxyapatite particles, increasing the stability of hydroxyapatite particles while increasing particle load to speed up the coating process²³. Moreover, the nanometre-scale particle size of chitosan also increases the surface area by hundreds of times to increase the effectiveness of chitosan in binding other chemical groups²⁴. It is in line with Hutapea et al.'s (2015) research, which reported that toothpaste containing high molecular chitosan with a concentration of 0.5% was effective in covering dentinal tubules in a tightly closed dentinal tubule pattern with homogeneous crystallization, smooth, and the joined crystals resembled continuous film layer²⁵.

This study's findings also reported that the dentin surface treated with a commercial gel test material (gel varnish) resulted in a less dense closure of the dentinal tubules in coating the dentin surface. The layer formed is hexagonal crystals, and there is a visible plug on the dentin surface²⁶. Whereas in this study, the presence of a thin film of a commercial gel showed reactive fluoride ions, forming salts rapidly, resulting in a layer that blocked exposed dentinal tubules. Additional Tri-Calcium Phosphate (TCP) resulted in a more homogeneous change in the dentin surface²⁷.

Table 1 shows the better impact of chitosan nano gel and gel varnish to increase the chemical elements on the dentin surface. It indicates the dentin tubules' closure activity by finding an increase in oxygen, phosphate, and calcium and a decrease in elemental carbon during the reactivity process. Arrais (2004) reported that the desensitizing agent could react either by closing the dentinal tubules or changing the dentinal tubules' contents through the precipitation of protein and calcium crystals around or into the dentinal tubules. Thus the deposition of this dental mineral is expected to stop dentin hypersensitivity²⁸. Another assumption is that dentin produces a relatively high organic matrix such as carbon and oxygen and reduced minerals such as sodium, magnesium, phosphorus, and potassium due to initial demineralization caused by citric acid²⁹.

As a comparison, Jusman (2014) reported that carbon and oxygen elements could also be found in samples coated with aluminum or gold³⁰. This occurs due to the penetration of electrons into the dentin sample and is unavoidable because it is necessary to produce a conductive source³¹. The results also showed that the untreated samples with 48 hours incubation showed no elemental oxygen. Loss of oxygen may occur due to phosphate loss from minerals such as apatite, which dissolves easily with citric acid³². The results also reported that chitosan nano gel could increase calcium levels on the dentin surface to reduce tooth sensitivity by reducing fluid flow in the tubules and pain from exogenous stimuli. Thus it can be explained that the chitosan nano gel can play a role in the desensitization process of dentin hypersensitivity.

Table 2 states that chitosan's conductivity and oxygen solubility values are higher than the gel varnish. The test material's conductivity and Dissolved Oxygen (DO) values strongly affect the dentinal tubules' closure on dentin hypersensitivity. Chitosan shows relatively stable material properties in the dentinal tubule ionization process based on the ionic value. The percentage of the ionic conductivity value is higher than the solubility value of oxygen. The bigger the DO value of an ingredient, the better the material's quality becomes. The dissolved oxygen concentration in the material varies depending on the temperature and the easily oxidized elements. In the case of increasing temperatures, the solubility will decrease. The higher the temperature, the lower that oxygen level needed becomes. The higher the oxygen content required, the lower that alkalinity becomes^33,34. In the dentinal tubules' closure, chitosan uses relatively large amounts of oxygen to balance solubility and reactivity with calcium dentin, calcium ion bonds, and other minerals³⁵. Dissolved oxygen is needed by material for the diffusion process on the surface of the dentin. The dissolved oxygen value shows the amount of oxygen (O₂) available in a material³⁶.

In addition, Kaur (2016) reports that the conductivity value is needed to strengthen the interaction and reactivity between molecules during catabolism and anabolism³⁷. Furthermore, Rusydi (2018) notes the conductivity value as a reference for the number of ions and the concentration of dissolved solids (Total Dissolved Solid/TDS)³⁸. The ion concentration in the solution is linearly proportional to the electrical conductivity—the more dissolved mineral ions, the greater the ability of the solution to conduct electricity³⁹. Based on the results, the dentinal tubules' closure frequency is linear with the required conductivity and oxygen solubility values.

A number of these test materials were examined for viscosity as a reference during the dentinal tubules' closure activity. Figure 3 reports that chitosan gel's viscosity is higher than that of varnish gel, especially at the 24-hour incubation time, whereas at 48 hours, it has the same value. It is related to chitosan, which dissolves in organic solvents, such as HCl, HNO₃, 0.5% H₂PO₄, and 1% CH₃COOH, but it is insoluble in solid bases and H₂SO₄. Under acidic conditions, the amino group (-NH₂) will capture H⁺ from the environment. Thus the amino group is protonated to -NH₃. This -NH₃ group will cause chitosan to act as salt, thereby dissolving in water⁴⁰. Besides, the positive charge -NH³⁺ can be utilized in the adsorption of anionic substances (negatively charged). Adsorption of cations uses the lone pair electrons on the -OH and -NH₂ groups⁴¹.Therefore, the viscosity value of chitosan correlates with absorption during the dentinal tubule closure process.

Based on the viscosity analysis, it can be explained that the significant viscosity value after the application of the test material in the chitosan-silver nitrate nano gel group at the incubation time of 24 and 48 hours is relatively slightly higher, where the viscosity value of the test material is related to the conductivity and dissolved oxygen values. In this study, although the nano chitosan-silver nitrate gel's viscosity value experienced a slightly higher value, the dissolved oxygen value was still relatively high. Thus the ability of the material to conduct electricity also increased. It decreases the exposed dentin's oxygen element, causing the desensitizing material to penetrate the dentinal tubules rapidly. This penetration prevents fluid flow and transmits ions into the tubule, thereby reducing the pain in hypersensitive dentin⁴².

CONCLUSION:

Nano gel chitosan-silver nitrate within 24 hours incubation gives the best results in closing the dentinal tubules with a dense tubular closure pattern, homogeneous and smooth compared to gel varnish and capable of increasing the formation of calcium on the dentin surface while increasing conductivity and oxygen solubility, which supports accelerated closure of the dentinal tubules.

ACKNOWLEDGMENT:

Thank you to the Corrosion Laboratory, Faculty of Engineering, Veterinary Research Laboratory Faculty, Syiah Kuala University, Darussalam Banda Aceh, Indonesia.

CONFLICT OF INTEREST:

We declare no conflict of interest.

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Received on 22.04.2021 Modified on 04.11.2021

Research J. Pharm. and Tech. 2022; 15(8):3511-3517.

DOI: 10.52711/0974-360X.2022.00589