## 4 Discussion
In addition, the root-end filling is expected to entomb any remaining bacteria beneath the filling in a way that would eventually result in bacterial death [30]. However, studies found bacterial biofilm at the interfaces between the filling and the canals' dentin walls, and also deep in the den retinal tubules [7; 15]. In order to provide a comprehensive view and analysis of the relationships between the proliferating biofilm and the filling material, in the current study, different filling materials were fluorescently labeled in order to evaluateand understand the influence of the filling materials and their penetration depths into the tubuli on bacterial invasion within the tubuli. The same as in a previous study [2], in the current study, we found a pattern of bacterial invasion and filling penetration in which the bucco-lingual direction was preferred compared to the meso-distal direction, regardless of the type of filling material. This finding was also supported by previous investigations [2; 23; 24; 31]. The fact that both the fillings and the bacteria penetrated deeper in the bucco-lingual direction may be associated to an anatomical-physiological phenomenon, \"the butterfly effect\", which means a butterfly-like appearance observed on the cross sections of roots that are associated with higher sclerosis down the tubuli at the distal and mesial sides of the root canal [24]. This phenomenon is commonly detected in single-rooted human teeth in a broad range of ages [32; 33]. Similarly, in a study by [PERSON] et al. [24], histological observations revealed that the penetration of microorganisms might predominantly happen through tubular aspects of the dentin, whereas atubular or sclerotic dentin and the interfaces between dentin and sealer remained bacteria-tight [24]. Thus, it seems that while a two-chamber model is not suitable for the evaluation of bacterial leakage, it reliably reflects the invasion of bacteria into the tubuli [2; 23; 24]. The bacterial invasion depths into the dental tubules were variable, reaching as deep as 1480 um (with a mean of 167 um), which corroborates the findings of a previous study [2]. [PERSON] et al. [34] evaluated the viable endodontic bacteria in the root dentin of infected teeth presenting with apical periodontitis using culturing methods. They found that in the majority of the roots, bacteria were identified deep within the dentin near the cement. They also found that an anaerobic culturing method is more sensitive than histology in identifying these bacteria in the dentin. Within that scope, confocal microscopy seems to be a promising technique to assess bacterial penetration within the tubuli because it enables us to evaluate live and dead bacteria, as well as bacteria in dormant, not-culturable states [5; 19; 27]. Thus, CLSM is capable of assessing the actual level of bacterial invasion within the tubuli in situ [2]. In the current study, bacterial penetration into the dental tubules was significantly influenced by the type of filling material and its penetration depth into the dentinal tubules. For all materials, it was found that deeper filling penetration within the tubuli was significantly related with shallower bacterial penetration. This finding confirms that an appropriate retrograde filling material may inhibit bacterial penetration across the dentin into the periradicular tissues of teeth following root end resection and filling [35]. The fact that the Biodentine fillings penetrated less, and their associated bacterial penetration depths were deeper compared to the MTA and IRM groups (\(p<0.05\)), may suggest that the type of root end filling material and its ability to penetrate into the tubuli may play a significant part in the risk of bacterial invasion following endodontic surgery (Figure 4). Biodentine is a material based on calcium silicate that has several applications, including as a root-end filling material used in endodontic surgical procedures [36].