**1. Introduction**

Dentin hypersensitivity (DH) has become a more frequently encountered issue in diagnostic and therapeutic clinical situations. DH is identified by a typical short sharp pain on the exposed dentin because of the thermal, evaporative, tactile, osmotic, or chemical stimuli. The common clinical manifestations of DH include exposed dentin from abrasion, erosion or exposed root surface. Although the mechanism of pain transmission and sensitivity in dentin is still under speculation, the most accepted theory is the hydrodynamic mechanism of sensitivity. It is postulated that the sudden flow of tubular fluid within the tubules in the presence of irritating stimulus results in the activation of sensory nerve ending thereby inducing pain or sensitivity [**? ?** ]. The density and size of patent dentinal tubules are critical factors to the severity of DH in patients.

A standard treatment for DH has not ye<sup>t</sup> been established [**?** ]. Two main methods utilized for the treatment of DH are occlusion of the dentin tubules and interference with the sensitivity of the mechanoreceptor. A plethora of products have been developed with the aim to reduce the discomfort from DH. Dentin desensitizers have been introduced for DH treatment mainly through tubular occlusion [**????** ]. Calcium phosphate mineral-based desensitizers have attracted considerable interest in recent years thanks to their biocompatible properties yielding an effective dentinal tubule

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occlusion and reduction in dentin permeability. They also possess the possibility of mineral crystal growth in the oral environment [**???** ]. The composition of calcium and phosphate in a material may exhibit the ability to form hydroxyapatite (HA; Ca10(PO4)6(OH)2) as a final product. HA and its variations are the main mineral component in human tooth and their biocompatibility have rendered them as clinically practical choices [**?** ].

Dental adhesives have been utilized in the treatment of restoring teeth in combination with DH. The component in the bonding agen<sup>t</sup> provides the simultaneous conditioning and priming for the underlying dentin, resulting in superior bond strength and reduced DH [**? ?** ]. However, the sealing ability of adhesives on the dentin surface could be a ffected by an incomplete micro-emulsion polymerization of the hydrophilic primer and hydrophobic bonding [**?** ]. Therefore, in order to increase the e ffectiveness of treatment, it would be necessary to desensitize dentin exhibiting DH prior to the placement of a restoration. It was reported that one-step self-etching adhesives and dentin desensitizers could significantly relieve DH immediately and over a month after treatment [**?** ]. The usage of dentin desensitizer in the combination with the bonding agen<sup>t</sup> can alleviate the postoperative sensitivity with the composite restoration [**?** ]. Nevertheless, its e ffect on bonding performance using di fferent bonding systems remains to be evaluated, as the desensitized treated dentin may be unfavorable for bonding [**? ?** ]. The objective of this in vitro study was to evaluate the morphological characteristic of the new nanohydroxyapatite-based desensitizer with the proprietary composition of calcium, sodium, phosphate, and silica when applied on the tooth surface and the e ffect of this material on the bond strength. The null hypotheses were that the novel desensitizer could modify the morphological characteristic of the dentin of the extracted teeth and did not interfere with the bonding performance of the tested adhesives.

#### **2. Materials and Methods**

The extracted human teeth were used in this study in accordance with the guideline of the Ethics Committee of the University of Washington Human Subject Division and the Declaration of Helsinki, which allow the use of extracted deidentified human teeth. Forty molars were collected during treatment plan and patient care and stored at 4 ◦C in water with 0.02% thymol.

The occlusal cusps were removed by cutting perpendicularly to the long axis of the tooth 2–3 mm from the tip of the cusp to expose superficial midcoronal dentin using a low-speed water-cooled diamond saw (Isomet; Buehler, Lake Blu ff, IL, USA). The roots were then sectioned at the cement-enamel junction to obtain 2-mm-thick dentin slices. The occlusal side of each disc was polished under running water by 600-grit paper (3M, St. Paul, MN, USA) for 30 s in circular motion to create a standardized smear layer. The surfaces were observed under the digital stereomicroscope (MU1000, AmScope, Irvine, CA, USA) to eliminate the cracks, residual enamel, coarseness and pollutants.

The composition and application of desensitizers and adhesives according to the manufacturer's instruction were listed in Table **??**.




**Table 1.** *Cont.*

#### *2.1. Morphological Observation*

Ten dentin discs were pretreated with 0.5 M EDTA (pH 7.4) for 2 min to completely remove the smear layer and widely open the dentinal tubules before applying the deionized water. Half of the disc surfaces were subjected to the Predicta Bioactive Desensitizer (PBD, Parkell, Edgewood, NY, USA) whereas the other half served as control (no PBD). Five discs were immediately processed for SEM examination while the other five were stored for four weeks in simulated body fluid (SBF) for bioactivity test (specified by the ISO 23317:2014) (Figure **??**A).

**Figure 1.** Methodology: Dentin discs were produced from human molar for morphological observation (**A**) and microshear bond strength test (**B**). For (**A**): Ten dentin discs were pretreated with EDTA (pH 7.4) to remove the smear layer and open dentinal tubules before rinsing. Half of the disc surfaces were subjected to Predicta Bioactive Desensitizer (PBD) and the other half served as control (no PBD). Five discs were immediately processed for SEM examination while the other five were stored for four weeks in simulated body fluid (SBF) for bioactivity test (specified by the ISO 23317:2014). For (**B**): Thirty dentin discs were allocated into three groups (n = 10): Teethmate Desensitizer (TMD) treatment, PBD treatment and no desensitizer as control. The universal adhesive (PUB) in self-etch (SE) or with phosphoric acid etching (PA)) and two-step self-etch adhesive Clearfil SE Bond 2 (CSE) were used. Small cylinders of the hybrid composite were bonded on the dentin surface and the wire loop bond test was performed at a crosshead speed of 1 mm/min.

#### *2.2. Observation after Application of PBD*

The specimens were dried in a desiccator without heating for 24 h. The discs were then superficially scored using a diamond bur and delicately fractured by finger pressure revealing the cross-section of dentinal tubules without creating an additional smear layer. The boundary between applied and non-applied dentin surface was targeted for visualization under SEM (JEOL JSM-6010LA, Tokyo, Japan). After gold-sputter coating, the micromorphology was examined at magnifications of 1000×, 4000×, 5000×, and 10,000<sup>×</sup>. Furthermore, in order to anticipate the bioactivity of PBD-treated dentin in the oral environment, the dentin discs were further placed in dust-free SBF solution incubated at 37 ◦C for four weeks in a transparent bottle.

#### *2.3. Microshear Bond Strength Test*

Thirty dentin discs were randomly distributed into three groups (n = 10): Teethmate Desensitizer (TMD, Kuraray Noritake Dental Inc, Tokyo, Japan) treatment, Predicta Bioactive Desensitizer (PBD) treatment and no treatment (control). The universal adhesive PBOND (PUB, Parkell, Edgewood, NY, USA) in self-etch (SE) or with phosphoric acid etching (PA, Parkell Etching Gel, Parkell, Edgewood, NY, USA) and two-step self-etch adhesive Clearfil SE Bond 2 (CSE, Kuraray Noritake Dental) were used in the experiment of microshear bond strength test (MSBS). Figure **??** represented the method of the study.

Prior to the irradiation, three pieces of Tygon tubes (Saint-Gobain Performance Plastic, Nagano, Japan) with an internal diameter of 0.8 mm and 1.0 mm in height were fixed on the dentin surface of each slice. The adhesive was light-cured for 10 s at 600 mW/cm<sup>2</sup> (Yoshida Light Curing Unit, Japan). The hybrid composite Z100 (A2 Shade, 3M ESPE, St. Paul, MN, USA) was carefully placed inside the tubing lumens before irradiation for 40 s. After 1 h-storage at room temperature (25 ◦C), the tygon tubes were removed using a sharp blade to obtain the cylinders of resin with the size of 0.8 mm in diameter and 1.0 mm in height bonded to the dentin surface. The specimens were then stored in distilled water in an incubator at 37 ◦C.

After storage, an MSBS test was conducted. The dentin slice was attached to the testing apparatus (Mecmesin, Compact Force Gauge, CFG+, UK) with a thin layer of cyanoacrylate glue (Model Repair II Blue, Dentsply-Sankin, Tochigi, Japan) and tested in a microshear tester (Bisco, Inc., Schaumburg, IL, USA). A 0.2 mm-diameter steel wire was looped at the bottom of the resin cylinder, in contact with the lower half-circle of the cylinder and the dentin surface. The wire loop was maintained according to the shear stress orientation at the bonding interface. The force was applied at a crosshead speed of 1 mm/min until failure then the MSBS data were recorded (Figure **??**B).

#### *2.4. Evaluation of Failure Mode*

After the MSBS test, the mode of failure was determined for each specimen under the digital stereomicroscope. The fractured surfaces were classified as followed: adhesive failures between resin and dentin, mixed failures that were partially adhesive and partially cohesive, and cohesive failures that occurred entirely within the bonding resin or entirely in dentin.

#### *2.5. Statistical Analysis*

The distribution of data in each group was normal (Kolmogorov–Smirnov *p* > 0.05), parametric tests were performed. The MSBS values were statistically analyzed using two-way ANOVA with application mode and desensitizer treatment as factors. The statistical procedures were analyzed at 0.05 significance level using IBM SPSS Statistics 23 Software.
