## 4 Discussion  
REPs aim to regenerate the functional pulp tissue in necrotic immature teeth [6]. Although the histologic findings following REPs are variable, the complete elimination of bacteria from the canal system is mandatory to promote the ingrowth of any vital tissue [3,8,9]. A remarkable number of microorganisms are able to invade the root canal space, forming a biofilm and infiltrating the dentimal walls [35]. Therefore, REPs require a high-level, wide-spectrum disinfection, deep into the dentimal tubules, that does not disrupt the remaining dentin, or negatively affect stem cell viability [12,13,17,24]. The American Association of Endodontists' Regenerative Committee has established a recent protocol which includes the use of either calcium hydroxide or DAP and TAP [11]. Therefore, the use of effective antibiotic mixtures is a current subject of interest [36]. Due to the complexity of infection, a combination of different antibiotics is recommended to overcome root canal microbial flora [14,15,37]. Previous studies have assessed the antibacterial efficacy of the 3-MIX deep in the dentimal tubules of immature teeth [12,37,38]. One negative characteristic of the 3-MIX is the persistent tooth discoloration that may occur due to the presence of minocycline [19,23]. However, root staining was reported even when minocycline was replaced with doxycycline [18]. A further drawback is TAP toxicity, with concentrations above 1 mg/mL deemed to be toxic for stem cells from the apical papilla (SCAP) in vitro [24]. Previous studies have suggested that TAP toxicity may occur at even lower concentrations [38]. Based on current data, regenerative protocols using DAP or TAP at a concentration of 0.1 mg/mL are recommended [11], despite their variable efficacy [24,38,39,40,41]. A study from [PERSON] et al. demonstrated that TAP-containing clarithromycin showed high efficacy against cultures of _E. faecalis_, without any staining effect in vitro [26]. The objective of the cited study was to evaluate the incidence of potential discoloration of 3-MIXC and 3-MICF (a mixture of ciprofloxacin, metronidazoleazole and fosfomycine) as possible alternatives to minocycline-based TAPs. The root canals were randomly divided into five groups and brought into contact with the different antibiotic mixtures for 3 weeks. Within the study conditions, the discoloration effect was avoided with the use of clarithromycin instead of minocycline in TAPs and a dark green-brown shade appeared only for the 3-MIX samples containing minocycline. In the other groups and in the control group, the root stain remained unchanged. Moreover, clarithromycin showed a higher ability to kill the endodontic pathogens in vitro compared to fosfomycine. Clarithromycin is an advanced generation macrolide with a wide spectrum of activity and excellent inflammatory-dependent activity, both intra and extracellular [27,28,29]. Moreover, it has been shown to exhibit anti-inflammatory properties and the associated side effects are typically mild [26,42]. In the present study, the root samples were infected with _E. faecalis_, a Gram-positive facultative anaerbe, in order to test the antibacterial efficacy and depth of action of the antibiotic mixtures on dentin. As suggested by previous epidemiological studies, _E. faecalis_ is commonly present in persistent endodontic infections, with a prevalence of 24% to 77%. It is able to compete with other microorganisms, invade the dentimal tubules and resist nutritional deprivation. It is believed to be a major cause for the failure of root canal treatment. It can penetrate the dentimal tubules and it is able to create biofilm [35]. Figure 3: Confocal laser scanning microscopy composite reconstructions of dentimal tubules of positive (**C+**) and negative (**C-**) controls. **D**, dentin; **P**, pulpal side. The scale length is 300 \(\upmu\)m.  
_E.