## 4 Discussion
[19] reported that double application and the placement of an additional hydrophobic resin layer had a negative impact on the \(\upmu\)-TBS of the enamel when using Clearfil SE Bond. On the other hand, [PERSON] et al. [8] found no significant difference in the SBS between the single and double-layer application methods when using Clearfil SE Bond. [PERSON] et al. [7] reported no significant change in the SBS between the single- and double-layer application methods with OptiBond XTR. In the present study, although CS did not show any significant differences between groups II and III in SBS regardless of etching mode, the other adhesivesshowed lower SBS values in group III than group II. Additionally, the proportion of adhesive failure was higher in all the adhesives in Group III when compared to those in Group II. Thus, it can be inferred that an excessively thick adhesive layer might be more prone to defects and voids leading to increased residual stress after polymerization [23]. This might have a negative impact on the mechanical-physical properties of the resin composite restoration when using the two-step adhesive system [24]. In Group I, although no significant differences were observed between the TC groups and the 24 h Group in CS, GU and OX presented with significantly higher SBS values in the TC groups in both etching modes. In particular, a drastic increase in SBS after TC was observed in GU when compared to those in the other adhesives. This might be attributed to a possible delay in the post-polymerization reaction in the adhesive layer in Group I when compared to those in Groups II and III. In the case of GU in Group I, the remnant water and solvent derived from the primer might inhibit the polymerization of the bonding agent during the early phase. Thus, the second null hypothesis that the trend in the durability of the enamel bond during the different application methods was similar among the tested two-step adhesive systems was rejected. Most of the application groups and adhesive systems demonstrated significantly higher SBS values in the ER mode than in the SE mode. This was in line with previous studies, which showed that phosphoric acid pre-etching before the application of a primer contributes to enhanced immediate and durable enamel bond performance [25; 26]. However, none of the adhesives in Group III after TC showed any significant differences in SBS between the ER and SE modes, apart from GU at TC 10,000. For all the adhesives in Group III, the proportion of adhesive failure tended to increase after TC, even in those subjected to the ER mode. This finding suggested that although a strong mechanical interlocking between the etched enamel substrate and the adhesive layer might have been achieved in Group III, the deterioration of the adhesive layer might have been accelerated by both thermal stress and hydrolytic degradation during TC. Additionally, the stress distribution might be different because of the different mechanical properties of each adhesive layer. In particular, the HEMA-, solvent-, and functional monomer-free bonding agent in GU is thought to create a hydrophobic adhesive layer, which might have higher mechanical properties than those formed in the other adhesives [15; 16]. This might explain why GU was the only adhesive to show a significantly higher SBS in the ER mode than that in the SE mode at TC 10,000 in Group III. Among the tested adhesive systems, the new-generation two-step adhesive GU, which utilizes a universal adhesive-derived primer, showed SBS values equal to or higher than the other conventional two-step adhesive systems, regardless of the application mode, etching mode, or bonded specimen storage period. In particular, GU in Group II (similar to the manufacturer's recommended bonding procedures) showed superior immediate and durable enamel bond performances to the other adhesives. Alternatively, GU did not demonstrate any significant differences in SBS under TC between Groups I and II.