**4. Discussion**

Desensitizing agents occlude the dentinal tubules at the tubular orifice or within the dentinal tubules to prevent the fluid flow and therefore decrease the pain sensation by counteracting the hydrodynamic mechanism of DH. The effectiveness of various desensitizing agen<sup>t</sup> to mitigate the DH has been reported in previous studies. The clinical efficacy has been reported in the application on vital abutment teeth prepared to receive full coverage or porcelain fused to metal restoration [**? ?** ]. The pre-impression sealing of the dentin was recommended for tooth preparation on vital teeth to reduce the pressure transmitted to the pulp chamber during the crown cementation [**?** ].

In this study, immediately after the application of PBD, the untreated dentin showed patent dentinal tubules. At the area adjacent to the PBD-treated dentin, random mineral deposits were observed on the untreated dentin. It is assumed that these deposits have formed as a result of free PBD nanoparticles adjacent to the PBD-treated dentin (Figure **??**).

For evaluation of the mineral forming ability of PBD-treated dentin, SBF immersion proceeded for four weeks. The SBF was used to simulate the clinical situation where dentin interacts with body fluids, such as saliva from the oral environment and dentinal fluid and plasma from the pulpal side. The SBF is an acellular and protein-free simulated fluid with ion concentration nearly equal to those of human blood plasma. SBF was preferred over artificial saliva formulations for the bioactivity experiment in this study since the ISO test has standardized the SBF for bioactivity test and apatite formation ability, while there is not a comparable standard established for artificial saliva formulations. Due to the similarity between bone and dentin in terms of mineral composition and structure, the bioactivity test for bone implant devices would be applicable to a desensitizer material applied to dentin. The bioactive material is expected to form a layer of apatite on their surface [**?** ]. In this study, PBD demonstrated bioactivity as defined by the ISO 23317-2014 document. Considering the composition of SBF, the forming mineral in the ISO experiment is expected to be apatite.

The SEM micrographs confirmed that the dentin regions exposed for the experiment were rich in open dentin tubules, and tubular sealing was achieved immediately by PBD. The mineral growth matured through the SBF-storage period. Fine precipitates are observed inside the tubules close to the surface whereas the group without PBD, there were patent tubules. The principal mineral content in PBD (calcium and phosphate, as disclosed by the manufacturer) could stimulate the mineralization of the dentine tubules. During immersion in SBF, di fferent processes occur resulting in structural and chemical change at the surface of dentin: leaching, degradation, and precipitation. Calcium ions dissolve from the bioactive substance into the body fluid. The nucleation of hydroxyapatite is possible because the surrounding fluid is supersaturated with respect to hydroxyapatite due to the dissolution of the calcium ions. The process of formation and growth of the hydroxyapatite layer continues by the reactions of calcium, phosphate and hydroxide ions [**?** ]. In the oral cavity, the supersaturated calcium phosphate salts contained in the saliva could precipitate to form a less soluble compound HA continuously [**?** ].

The study compared the bond strength of adhesives to dentin treated with PBD and TMD. The main components of TMD are TTCP and DCPA, which are eventually converted to hydroxyapatite in an aqueous environment. This conversion is predicated upon the dissolution-precipitation reaction mechanism [**?** ]. TTCP and DCPA dissolve and supply Ca2+ and PO4 3−. The supersaturation of the apatite-contained solution prompted the precipitation of HA crystals. TMD has been investigated previously in vitro with a superior tubule occlusion rate compared to silver diamine fluoride and resin-containing oxalate [**???** ]. SEM images of TMD showed all tubules occluded with crystalline precipitates, with similar precipitation on the intertubular surface [**?** ]. The application of TMD occluded dentinal tubules and reduced dentin permeability by 92% regardless of the exposed collagen network [**?** ]. A comparable tubule occlusion was found in the novel PBD, therefore its sealing ability for treatment DH is promising.

As far as bonding performance is concerned, dentin desensitizers significantly decreased the bond strength of bonding agents. These findings are in alignment with the results of previous work [**? ? ?** ]. The application of desensitizers before the bonding system significantly reduced shear bond strengths to sound dentin. In the three-step bonding system, the protocol included acid etching, rinsing, and drying of dentin before applying the primer. Self-etching primers succeeded in eliminating the dentin-conditioning steps before priming. Nevertheless, the majority of self-etching primers have a milder acidity. Therefore, the lower bond strengths to sound dentin might be accounted for the desensitizer deposition blocking the dentin tubule orifices and intertubular di ffusion channels. The nanoparticles layer of the desensitizer dispersed on the dentin could hinder the adhesives to interact with the demineralized substrate, resulting in reducing the bond strength [**?** ].

The literature has reported that the dentin treated with mineral-based desensitizers did not show a reduction in the bond strength when treated with a two-step self-etching adhesive material [**?** ]. The reason for this phenomenon was explained by the interaction created between the desensitizer, the smear layer, and dentin. This was in contrast with the result in this present study where bond strength in PUB-SE mode after application of PBD and TMD was reduced compared to the control group.

The functional monomer in self-etch adhesives could contribute to the superficial micromechanical interlocking through hybridization of dentin substrate. The exposed HA crystal that remains around collagen is expected to be beneficial, as a substrate for chemical bonding. A mineral-rich substrate allow a more substantial chemical interaction with the functional monomer and protect the collagen against hydrolysis thus prevent from premature degradation [**? ?** ].

It was of interest to compare the bond strength characteristics of two products PBD and TMD in a combination of adhesives. The result indicated that bonding to PBD with PUB-PA and CSE is comparable to that to TMD. The bonding performance of the PBD-treated specimen was corroborated with SEM images obtained by the application of PUB on dentin. PUB with acid etching exhibited resin tag formation, which could explain higher values in groups bonded in PA mode. Nonetheless, it was reported that the sequence of application of etching and desensitizer a ffected the bonding performance [**? ?** ]. When dentin was acid-etched before the application of oxalate desensitizer [**?** ] or Novamin [**?** ], the bonding performance was not compromised. However, in this application sequence, the formation of resin tags might be a ffected by tubule occlusion with calcium-containing desensitizer pastes [**?** ]. The clinical decision of whether to etch dentin or not after desensitizer application should be made considering the potential increase in permeability of dentin following etching. The interaction of PBD and phosphoric acid etched dentin, as application of the acid prior to treatment with PBD and the universal bond, warrants further investigation.

Successful bonding to dentin may further reduce the e ffects of hydraulic pressure for the treatment of the post-operative DH [**? ?** ]. The use of the universal bonding system with acid phosphoric etching or the two-step self-etching is recommended following the usage of mineral-based desensitizer to achieve a high bond strength.

The study highlights the biomimetic remineralization shown by desensitizing agen<sup>t</sup> PBD. It could be of interest to compare investigate the characteristics of PBD in vivo. The present laboratory test could be to some extent a predictive tool for the clinical e fficacy of desensitizing materials, while the effect of adhesive systems on the survival rate of restorations following desensitizing treatment should be further studied.
