## 4 Discussion
However, for Prime & Bond One Select, the comparison of all the study groups with the control in terms of adhesive layer thickness proved that there was no statistically significant difference between them. The present study reported the great variability of the adhesive layer thickness measurements, that may result from a low number of specimens and few measurement points usually used in SEM observations [30]. Also, faulty positioning of the samples or undefined reference points could influence the measurements of adhesive layer thickness [51]. The present study showed that the correlation between dentin shear bond strength and adhesive layer thickness produced by tested all-in-one adhesives was statistically insignificant. Previous study reported similar findings on the lack of positive correlation between these parameters [52; 53], depicting that it is the quality of the adhesive layer that might play a key role in dentin bond strength [51]. The increase in SBS of self-etch and universal adhesives observed in the present study could be related to an improvement of the resin-dentin interface quality, or the quality of the adhesive layer rather than to an increase in its thickness. This is in line with the findings of previous studies, which showed that some alterations of application procedures of all-in-one adhesives improved the quality of hybrid and/or adhesive layer and consequently led to an increase of SBS [25; 27; 50; 54]. For all tested all-in-one adhesives, except for Prime&Bond One Select, increased number of applications along with extended application time (groups 2A40 and 3A60) resulted in significant increase in SBS when compared to the controls. The limitations of this _in vitro_ study should be taken into consideration. Bond strength tests are generally a valuable tool aiming to rank dental adhesives according to their bond strength performance. However, they do not examine crucial factors influencing bond strength, which take place in clinical situation, such as pH, temperature changes, moisture or masticatory stress [55]. Some authors regard bond strength tests to be invalid in order to conclude the clinical results of dental adhesives [56]. Other studies proved a relationship between bond strength values and clinical performance of these adhesives [16]. Therefore, as long as the validity of this study to predict the behavior of tested adhesives in the oral cavity is questionable, it can provide valuable data to identify substrate variables and establish the most effective application protocol for each adhesive tested. This information is relevant especially in clinically demanding situations where maximum bond strength is required, e.g., fiber post cementation, direct pulp capping, or class V cavity. Considering the abovementioned results, the first part of the null hypothesis can be partially rejected; it is supported in case of Prime & Bond One Select and rejected in the case of Single Bond Universal, Xeno V, and Adper Easy One. Increasing the number of applications to two or three and extending the application time (40-60 s) significantly improved bond strength of the latter adhesives to dentin. Prime & Bond One Select showed no enhancement of bond strength to dentin in study groups. The second part of the null hypothesis, considering adhesive layer thickness after modified application, is rejected. The tested adhesives did not present increased thickness of the adhesive layer in the study groups. Furthermore, the third part of the null hypothesis is refuted. Improved bond strength of tested adhesives did not correspond with an increase of adhesive layer thickness.