## 4 Discussion
faecalis_ develops strong resistance to extremely harsh environments, including a highly alkaline pH, nutritional deficiency and many current clinical intra-canal medications, and it is resistant to most root canal disinfectants [35; 43]. Therefore, the continuous study of _E. faecalis_ and its elimination from the dental apparatus can define the future of the endodontic specialty and this resistance has made the control of _E. faecalis_ infection a great challenge for dentists worldwide. Moreover, it is widely used in endodontic disinfecting agents in vitro testing, as well as in the only study that has tested 3 MIX-C in vitro [26; 44; 45]. The dentin infection model was selected due to the need to assess the antibacterial effect on a heavy infection in the dentin tubules being more related to clinical reality [33]. The CLSM analysis, combined with viability staining, was proposed as a reliable method to assess the formation of a bacterial biofilm in the dental tubules after incubation [46]. Moreover, the quantitative analysis of bacterial viability after the disinfection was achieved through CLSM, which has been previously described as an accurate method to visualize the remaining bacteria in the dental tubules [33; 34]. The division of the samples with a scalpel on the previously prepared grooves is still proposed to obtain a clear and flat surface without artifacts and debris for the CLSM analysis [46]. Three weeks' aerobic incubation and centrifugation were used to ensure an adequate penetration of bacteria into the dental tubules [24; 33; 34]. The mean depth of action is a surrogate marker of the penetration ability of the tested antibiotic mixture into the dental tubules. For all groups, this parameter was comparable with existing reports in the literature [47]. The ratio of red fluorescence was slightly higher for the TAP groups compared with the DAP specimens, probably due to the higher antimicrobial action of TAPs [24; 26]. These values cannot be easily compared to previous studies due to the variety of infection protocols and solutions that were tested, which may influence the penetration ability and depth of action [24; 33]. Moreover, the absolute ratio of red fluorescence could be influenced by the threshold scale used for the green channel, due to the slight green fluorescence of the natural dentin structure observed with CLSM. However, the positive control specimens showed bright fluorescent staining, whereas negative samples did not stain with either dye. Interestingly, enhanced antimicrobial effects were reported when the antibiotic mixtures were conveyed with HA, possibly due to its well-described bacteriostatic effect [30; 48; 49], even if MG also displayed slight antibacterial properties [50]. HA displays unique structural and physiological functions within tissues, including extracellular and intracellular interactions, and growth factor modulation [51]. A recent study described a positive correlation between the HA scaffold and SCAP survival during REPs [52]. Moreover, HA-conveyed DAPs and TAPs appear more fluid and applicable inside the root canal. In conclusion, within the limitations of this in vitro study, the 3-MIXC mixed with the HA appeared to be a possible alternative for root canal disinfection during REPs. Both the antibacterial efficacy and the depth of action of the 3-MIXC are comparable to the traditional TAPs used for dental pulp regeneration, without the risk of tooth discoloration, and the use of HA to convey the TAPs could enhance the antibacterial properties. Conceptualization, D.P. and A.C.; Methodology, N.M., M.A., J.R., P.C., A.L., D.P. and S.A.; Software and Validation, M.A. and J.R.; Investigation, N.M., M.A., J.R., D.P. and G.B. ; Resources, M.A., D.P., E.G. and E.B; Writing--Original Draft Preparation, N.M., M.A. and J.R.; Review & Editing, N.M., M.A., J.R, S.A. and A.C.; Visualization, N.M., M.A., J.R., P.C., A.L., D.P., E.G., and G.B. ; Supervision, S.A., A.C. and E.B. All authors have read and agreed to the published version of the manuscript.