## 4 Discussion  
Understanding the sealing ability of materials used in the repair of perforations and predicting the amount and direction of bacterial leakage are of paramount importance in addressing the pathologies related to perforations [25; 26]. CLSM could be considered a useful modality of choice, in addition to traditional standard electron microscopy and PCR-based techniques, to identify viable bacteria in dental tubules [16; 17; 24; 27]. Using CLSM together with the live/dead staining method allows us to assess the extent of contamination and the viability of bacteria in contaminated dental tubules [16; 17; 24]. To our knowledge, this is the first study to use CLSM to assess bacterial colonization in perforation sites of extracted human molar teeth repaired with three different materials. In contrast to previous reports using bacterial leakage models, our current study provides histological evidence for the actual routes of bacterial colonization. One positive and four negative histological controls were used to confirm the validity of the experimental model. After perforation occurs, the success of the treatment relies on suitably placed repair material designed to prevent communication between the root canal space, peri-radicular tissues, and oral environment [28; 29; 30; 31; 32]. MTA is a hydrophilic calcium silicate-based material and is compatible with moist conditions such as those found in perforation sites [33]. Additional favorable properties include biocompatibility, non-cytotoxicity, radiopacity, availability, and promotion of tissue regeneration [8; 34; 35; 36; 37], as well as induction of cementogenesis and osteogenesis [11; 12]. However, the material also has a number of disadvantages, such as the long setting time, difficulties in manipulation, and potential for tooth discoloration [38; 39; 40]. Recently, novel calcium silicate-based materials have been introduced in order to overcome these MTA shortcomings [41; 42]. Biodentine is a widely used bioactive material consisting of tricalcium silicate, calcium carbonate, zirconium oxide, and calcium chloride. Biodentine has been reported not only to have improved sealing ability and a short setting time [42; 43] but also to possess bioactivity and biomineralization properties [44; 45; 46; 47; 48]. Endocem MTA is another recently introduced fast-setting calcium silicate-based cement and is composed of calcium oxide, silicate oxide, aluminum oxide, and bismuth trioxide. Its fast-setting property depends on the presence of fine particles of pozzolan, which is a siliceous and/or aluminous material. Endocem MTA exhibits acceptable biocompatibility, induces reasonable mineralization, and has less discoloration potential than traditional MTA [41]. Most bacteria cannot survive at an alkaline pH. Accordingly, the growth of _Enterococcus faecalis_ can be suppressed at pH 10.5-11.0, and there was no survival reported above pH 11.5 [47]. Calcium silicate-based materials form a silicate gel at their surface when mixed with water, and any calcium hydroxide in the silicate gel releases hydroxyl ions into the environment, which increases the pH [48; 49]. The antibacterial effects of calcium silicate-based materials have generally been attributed to the resulting high pH levels, although this assumption is not totally approved in clinical situations. In the present study, higher numbers of dead bacteria than live bacteria were detected on the \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & \multicolumn{3}{c}{**Quantile**} & \multirow{2}{*}{**Min**} & \multirow{2}{*}{**Max**} & \multirow{2}{*}{**Median**} & \multirow{2}{*}{**Stdv**} & \multirow{2}{*}{**\(p\)-Value**} \\ \cline{2-2} \cline{6-8}  & **Q1** & & & & & & & \\ \hline MTA-Angelus & 862 & 987 & 1295 & 159 & 1790 & 987 & 455 & \(p\) = 0.092 \\ MTA CEM & 325 & 432 & 456 & 211 & 782 & 832 & 176 & \(p\) = 0.229 \\ Biodentine & 467 & 720 & 930 & 224 & 1641 & 720 & 289 & \(p\) = 0.079 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Bacterial Penetration depth (in um) in the different groups.