## 4 Discussion  
Following endodontic surgery, bacteria may still invade a retrofilled root canal and deep into the den retinal tubules [1; 17], potentially leading to an inflammatory reaction when bacterial by-products invade the surrounding periradicular tissues [18]. These bacteria form biofilms, which are complex ecological communities that use diverse mechanisms to secure themselves against a harsh environment, including the immune system and antibacterial agents [19]. Gram-positive facultative anaerobes are the most commonly detected bacteria in endodontically treated teeth presenting with persistent endodontic infections [2]. Among them, _E. faecalis_ plays a major role in bacterial biofilm invasion and is considered a suitable model for assessing root canal bacterial invasion [2; 8]. Many microscopic, microbiological, and histological experimental models have been used to assess bacterial invasion within endodontically treated root canals [20; 21]. The most common traditional experimental model is the two-chamber bacterial leakage model, which is used to assess the diffusion of bacteria from an upper chamber and over the retrograde filling to a suspension placed in the lower chamber. However, it has been reported that this traditional model suffers from many inherent limitations, such as a lack of proper control groups and histological evaluation, and that its reliability is compromised [22; 23; 24]. Additional ex vivo models to simulate and assess bacterial invasion, such as the traditional dye-penetration model [25] or the high-pressure replica technique used to assess the pore morphologies and apical leakage of sealers in retrofilled teeth [26], are restricted because in these traditional models indirect evaluations are used, incapable of demonstrating the real routes of bacterial invasion, and because of the absence of proper histological controls in these models [22]. Furthermore, under the adverse conditions of a filled root canal, bacteria can increase their stress tolerance by entering a dormant state where they are viable but not culturable [5; 27], thus challenging the efficacy of traditional culture-based studies [5; 19; 27]. The authors of a recent study established a novel and reliable experimental model that enabled them to trace and quantify biofilm invasion into the den retinal tubules [2]. With this CLSM-based model, the actual routes of bacterial invasion are tracked histologically, and negative and positive histological controls are used to verify the suitability of the experimental model. Furthermore, the true viability of the proliferating bacteria, even when they enter a dormant, not-culturable state, can be assessed [5; 19; 27]. As a result, using CLSM together with live/dead bacterial staining techniques offers information regarding both the magnitude of the dentin infection and the vitality of the proliferating bacteria within the infected tubuli in situ [2; 8], thus enabling a better understanding of the microbiological-pathological course after endodontic surgical procedures [2]. In the current study, we used this CLSM-based experimental model to assess the invasion of bacterial biofilms into the den retinal tubules. Furthermore, bacterial invasion was assessed in the presence of fluorescently labeled retrograde filling materials. The use of CLSM to assess the penetration of the retrograde filling materials into the tubuli [6; 28], while assessing the depth of the bacterial invasion into the tubuli and their viability, provides a comprehensive view of the microbial-pathological process following surgery. Bacterial fluorescence was not detected in the negative control group, while fluorescence was detected in the positive control group specimens, confirming the reliability of the experimental model. The rationale of a retrograde filling material is to inhibit bacterial biofilm growth and the outflow of bacterial byproducts and toxins into the surrounding periradicular tissues [29].