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INTRODUCTION
Dentin hypersensitivity is characterized by pain caused by exposure of dentin due to opening of the dentinal tubules, and displacement of intratubular fluid. The pain caused by this inner fluid movement1can be exacerbated by thermal, tactile, or chemical stimuli2. Different treatment strategies have been proposed to minimize hypersensitivity. One approach is to apply tubular occlusion agents to the dentin surface as a mechanical barrier to hinder the movement of dentinal fluid1.
Adhesive systems promote the formation of a hybrid layer by micromechanical retention between dentinal collagen fibrils3. Fu et al.4 observed that adhesives effectively occluded dentinal tubules, thereby significantly reducing dentin permeability. Dentin permeability is reduced regardless of whether total-etching, self-etching or universal adhesive systems are used5. However, an adhesive system using a total-etching strategy -requiring prior acid etching-can open dentinal tubules, increasing their diameter, and immediately exacerbating pre-existing pain6. In addition, there is a discrepancy between the depth of demineralization from acid use and the penetration capacity of the adhesive due to its molecular size7. Self-etching adhesive systems eliminate the step of total removal of the smear layer, and reduce the clinical steps required.
Adding components to the adhesive systems enables deposition of products on the dentin surface or at the entrance of the dentinal tubules, thereby reducing dentin permeability and sensitivity. Chitosan has been evaluated for its interactive effect with dentin collagen to stabilize the dentin matrix 8, 9 . It is deposited on the surface and inside the dentinal tubules when applied as a rewetting agent, and it forms a calcium phosphate layer on the demineralized dentin layer10. This effect may reduce dentin permeability and hypersensitivity, regardless of the removal of the smear layer.
The addition of chitosan to total-etching and self-etching adhesive systems has been evaluated for its antimicrobial effect in concentrations of 0.12 to 5% 10, 11 , and to determine any interference regarding bond strength to dentin8, or possible improvement in the quality of adhesion 10 . However, none of the concentrations tested has been found to improve these properties. Chitosan added to an adhesive system may promote remineralization 10 , especially remineralization of deep cavities, thereby reducing permeability and pulp cytotoxicity 12 . It is therefore important to analyze the influence of chitosan on dentin permeability if it is added to a universal adhesive system used in total-etching or self-etching mode. Thus, the null hypotheses were that there is no difference regarding dentin permeability: H01) when chitosan is added to a universal adhesive system, regardless of whether total-etching or self-etching bonding strategy is used; and H02) when a universal adhesive system is used in total-etching or self-etching strategy.
MATERIALS AND METHOD
Adhesive system specifications and preparation
The materials used in the study are specified in Table 1. The adhesive system with added chitosan was prepared by weighing chitosan PA (low molecular weight Chitosan 448869) on a precision analytical balance (XPR10, Mettler-Toledo GmbH, Greifensee, Switzerland), and adding it to the single bond universal (3M ESPE, St. Paul, MN, USA) adhesive system at a concentration of 1%. Each solution was vortexed vigorously (Phoenix, AP-56, Araraquara, SP, Brazil) for 180 seconds, and stored in a hermetically closed flask, isolated from light and moisture to eliminate bubbles. The pH of the adhesive system, whether or not chitosan was added, was measured in triplicate with a microelectrode coupled to a pH meter (pH meter TEC-2/Tecnal/Piracicaba, SP, Brazil) ( Table 1 ).
Tooth selection and dentin disc preparation
The project was approved by the Research Ethics Committee of the Faculdade Sao Leopoldo Mandic (CAAE 26445019.7.0000.5374). Forty sound third molars, which had been extracted, stored, and frozen (for a maximum period of six months) were selected. After the teeth were cleaned with periodontal curettes and scalpel blades, they were sectioned using a precision metallographic cutter (Isomet 1000, Buehler, Springfield, VA, USA), with a high-concentration diamond disc (Buehler, 102mm x 0.3mm) under cooling, to obtain dentin discs. The crowns were sectioned perpendicular to the long axis of the tooth, 1.5 mm above the cementoenamel junction 13 , to obtain discs approximately 1.7-mm. thick (+0.1 mm), and samples with a completely enamel-free occlusal surface.
A small diamond bur marking was made to identify the occlusal surface. Next, both sides of the disc were sanded with 600-grit sandpaper (Lixa de Água, 3M, 3M do Brasil, Sumaré, SP, Brazil) to obtain specimens with uniform, smooth surfaces 1.5-mm thick. The thickness of each specimen was checked with a digital caliper (Mitutoyo Sul Americana LTDA, MIP/E - 103, Suzano, SP, Brazil). The discs were washed with water, and stored in J10 flasks with 5 mL of distilled water for 24 hours.
Both surfaces of the discs were submitted to acid-etching (Ultra-Etch 35%, Ultradent Products, South Jordan, UT, USA) for 15 seconds, to promote opening of the dentinal tubules, and measure the initial dentin permeability values. This served as the first measurement of hydraulic conductance, considered 100% maximum filtration. Then, the occlusal surfaces of the specimens were sanded with 600 grit sandpaper for 30 seconds to form a standardized smear layer.
The 40 dentin discs were randomly divided into four groups (n=10), according to the type of adhesive strategy (Total-Etch/TE or Self-Etch/SE), and addition (C) or non-addition of chitosan to the adhesive system:
-
TE
application of 35% phosphoric acid for 15 seconds, and application of a universal adhesive system
-
TEC
application of 35% phosphoric acid for 15 seconds, and application of a universal adhesive system containing 1% chitosan
-
SE
application of a universal adhesive system
-
SEC
application of a universal adhesive system containing 1% chitosan
The adhesive system (with or without chitosan) was applied according to the manufacturer’s instructions ( Table 1 ). When the total-etch strategy was used, phosphoric acid was applied for 15 seconds, followed by washing for 20 seconds, and drying with absorbent paper strips. Photoactivation was performed for 10 seconds using a photoactivating device (Valo Cordless, LED, Ultradent Products, South Jordan, UT, USA) with a power of 1,000mW/cm2. Next, the second (final) measurement of hydraulic conductance was performed.
Permeability Test
Permeability was evaluated with a device for measuring dentin permeability (THD, Odeme Dental Research, Luzerna, SC, Brazil) under pressure of 5 psi, equivalent to 351.54 cmH2O. The specimen was mounted in the filtration chamber, and the device adjusted so that water penetrated the dentinal tubules under pressure and was pushed upward toward the occlusal surface. This fluid movement was recorded according to the difference in the position of an air bubble in the glass capillary of the device. Three measurements were taken, the first after 4 minutes, and the next two at 3-minute intervals 14 . The measurements were performed with the digital caliper attached to the permeability measuring device. After the three measurements were taken, the system was depressurized, and the sample dismounted from the chamber. Each dentin disc was repositioned for the final measurement (after the treatments), and mounted in the filtration chamber using a standardized procedure, after which the system was closed and prepared to take a new measurement. The linear displacement of the liquid in the glass capillary for a preset time was used to calcúlate the amount of fluid that passed through the specimen by the following formula: Q = (ri2l)/t, where Q (pL/min-1) is the amount of fluid that passed through the specimen, l (cm) is the linear displacement in the glass capillary, t (min) is the time, and ri (cm2) is the inner radius of the glass capillary tube.
Hydraulic conductance (L) was calculated using another formula, considering water viscosity and constant specimen thickness: L = Q/(AP), where L is hydraulic conductance (pL cm-2 min-1 cmH2O-1), A (cm2) is the dentin surface area and P (cmH2O) is the pressure applied. The hydraulic conductance of each dentin disc was evaluated at two time points: initially (after the acid etching) and after the treatments. The percentage of dentin permeability was calculated using the following equation, and each tooth was its own control: L (%) = [(L1-L2) x100]/L1 15 , considering L as the permeability percentage, L1 as the hydraulic conductance after removal of the smear layer, and L2 as the hydraulic conductance after application of the treatments.
Analysis by Scanning Electrón Microscopy
To determine what material could penetrate deeply into the dentinal tubules, three treated discs were cut sagittally to observe penetration into the dentinal tubules of the adhesive options with or without chitosan. Another three discs from each group were not cut, to be used to evaluate the dentin surface for obliteration or non-obliteration of the dentinal tubules.
The interface of the cut disc sections was polished with sandpaper of decreasing abrasive grit (400, 600, 1200) (Imperial Wetordry, 3M, Sumaré, SP, Brazil). After abundant washing, the samples were demineralized for 30 seconds with hydrochloric acid (HCl), washed again, and subjected to a deproteinizing treatment with 1% sodium hypochlorite solution (Milton’s Solution) for 2 min, washed with distilled water for 15 seconds, and dried with absorbent paper 16 .
All specimens (sections and uncut discs) were coated in gold for 60 seconds, and examined by Scanning Electron Microscopy (SEM) (Jeol 5900LV, Jeol Ltd, Tokyo, Japan), at a voltage of15 Kv, with 1000x and 3000x magnification, respectively. The differences in the adhesive penetration observed in the images of the sections and the surface were evaluated qualitatively according to the groups, regarding obliteration or non-obliteration of the dentin surface from the treatments. The images were evaluated by a single examiner previously trained to perform micromorphological evaluations.
Statistical analysis
All the analyses were performed using the R program (R Core Team, 2021). Initially, descriptive and exploratory analyses of the permeability percentage data were performed. The exploratory analysis indicated that the data did not meet the assumptions of parametric analysis, and the Mann Whitney test was applied to compare the two bonding strategies to each other, and with and without chitosan. The significance level was set at 5%.
RESULTS
There was no significant difference in the permeability percentage between the groups with and without chitosan, either for the total-etching (p=0.8206) or the self-etching (p=0.5454) strategies ( Table 2 ). However, there was a significant difference in this percentage between the adhesive system strategies when chitosan was added. Dentin permeability was higher when the total-etching strategy was used, and lower when the self-etching strategy was used, based on the addition (p=0.0002) or non-addition (p=0.0126) of chitosan in the adhesive system ( Table 2 ).
Table 2 Median (mínimum and máximum value) of the dentin permeability percentage according to the type of strategy and presence of chitosan in the adhesive system
Micromorphological images of the dentin sections showed that adhesive penetration was deeper for the total-etching bonding strategy (h 10 gm) than for the self-etching strategy (h 1 to 3 gm) ( Fig. 1 ), regardless of the presence of chitosan. Application of the adhesive system provided a more intact surface [with fewer irregularities] in the total-etching strategy than in the self-etching strategy, whether with or without chitosan. In the latter application, the surfaces presented more holes and cracks ( Fig. 2 ).
Fig. 1 Images of the micromorphology of dentin sections (1000x magnification).The red arrows show the penetration depth of the adhesive system. Note that penetration in the total-etch strategy (TE and TEC) was greater, and penetration in the self-etch-ing strategy (SE and SEC) was lower. TE - Total-etch; TEC - Total-etch with chitosan; SE - Self-etching; SEC - Self-etching with chitosan.
Fig. 2 Images of dentin surface micromorphology (3000x magnification).A surface with greater uniformity and fewer pores or holes is observed with the total-etch strategy (TE and TEC). Use of the self-etching strategy (SE and SEC) caused more cracks and pores in the dentinal tubules due to greater dentin permeability. TE - Total-etch; TEC - Total-etch with chitosan; SE - Self-etching; SEC - Self-etching with chitosan.
DISCUSSION
There are various components that can be added to dental adhesives, and which could result in lower permeability because they contain particles which, when deposited at the base of dentinal tubules, can block fluid movement. This mechanism of action —explained by Brannstrom’s Theory of Hydrodynamics— promotes a reduction in dentin permeability 17 . Hindering the fluid movement in the dentin tubules helps reduce dentin sensitivity. Nevertheless, the addition of chitosan to the universal adhesive system did not influence dentin permeability, regardless of the adhesive strategy, thereby leading to the acceptance of the first null hypothesis of the present study.
Chitosan has an average molecular size of 0.5 gm 7 , which enables it to penetrate the dentinal tubules passively, even through the adhesives, considering that the adhesives have a diameter 4 times greater than the average size of chitosan. However, when chitosan is added to the adhesive system, some changes in its physicochemical properties occur 18 . Dacoreggio et al. 18 used a 0.5% concentration of chitosan in the adhesive without causing any changes in polydispersity or partióle size, but causing an impact on the colloidal stability of the solution. The higher concentration of chitosan used in the present study may therefore have influenced this stability by increasing the forces of attraction and repulsion between the particles, and causing a trend toward agglomeration. Further studies are needed to confirm this.
Chitosan may not have affected the micromorphological characteristics of the adhesive layer in the current study. Diolosá et al. 8 observed that chitosan was detected at a 100-pm depth, and explained this as a consequence of its low molecular weight (between 50 and 190 g/mol), compared to HEMA (130 g/mol) and BisGMA (512 g/mol). Dacoreggio et al. 18 observed a reduction in the number of tags in dentin, and lower bond strength, suggesting that this might be attributed to the presence of chitosan at the restorative interface. However, the higher concentration of chitosan added to the adhesive (Scotchbond Universal /3M ESPE St. Paul, MN, USA) in the present study may be one of the factors that prevented the hydraulic conductivity of the adhesive from decreasing. This outcome, together with the characteristics of polydispersity and particle distribution, should be investigated in future studies.
In the current study, dentin permeability was higher with the total-etching bonding strategy then with the self-etching strategy, leading to the rejection of the second null hypothesis. Acid etching promotes an increase in the hydraulic conductivity of dentin 13 , since the increase in the osmotic pressure exerted by the permeability measuring device promotes strong movement of dentinal fluid towards the occlusal surface, resulting from the removal of the smear layer and smear plugs 19 .
The opening of the dentinal tubules and the demineralization of the intertubular dentin allow greater penetration of the adhesive system 20 , resulting in a thicker adhesive layer. The micromorphological images of the dentin section in the current study confirm the greater penetration depth of the adhesive system achieved with the total-etching strategy ( h 10 pm). This is further corroborated by Wagner et al. 3 and Chen et al. 21 This greater penetration is attributed to the properties ofthe surface energy ofthe substrate, surface tension, and viscosity of the adhesive 22 . Moreover, the surface images show a more uniform layer with the total-etching than the self-etching strategy, which may be attributed to the thickness obtained by avoiding greater water permeation, achieved by using the adhesive system in the total-etch mode.
Maintaining the smear layer when using the self-etching strategy provides a thinner hybrid layer 3, 21 . However, the reduced permeability provided by not opening the dental tubules explains the lower dentin permeability observed for this strategy in this study. The micromorphological images of the surface after use of the self-etching strategy show cracks and fissures, which may have occurred due to the higher osmotic pressure exerted on the thinner adhesive layer. Sahin et al. 19 report that adhesives containing HEMA have greater ability to absorb water which remains trapped in the resin/dentin interface. Although universal adhesives promise easier technological processes, the benefits of the self-etching as opposed to the total-etching strategy include reducing the number of clinical steps, and lower dentin permeability. The addition of 1% chitosan did not interfere with permeability in either strategy, suggesting the need for further research into other concentrations and properties of the modified adhesive. Regarding clinical applicability, the results show that the total-etching strategy presented higher permeability than the self-etching strategy, and that the use of the self-etching strategy in Class V restorations might be better in clinical situations marked by greater hypersensitivity.
The addition of chitosan to the universal adhesive system did not affect dentin permeability. The self-etching bonding strategy led to lower dentin permeability.
