## 1 Introduction  
Very wide canals, open apexes and thin dentinal walls characterize immature permanent teeth. When pulp necrosis occurs as a result of caries or trauma, root development is arrested. Therefore, root canal debridement and obturation present several clinical difficulties, and teeth are more prone to fracture [1; 2]. Apexification protocols with calcium hydroxide or mineral trioxide aggregate (MTA) are effective, but they do not consider the development of the root canal walls [3; 4]. Therefore, these teeth remain more prone to root fracture and present a lower long-term prognosis [4; 5]. Regenerative endodontic procedures (REPs) have been proposed as an alternative treatment approach [6; 7; 8]. Replacing cells in the pulpo-dentin complex is advantageous due to the potential for promoting root development and strengthening of the dentinal walls [9]. However, a complete disinfection of the canal space, without mechanical instrumentation, is mandatory to prepare for the ingrowth of vital tissue [10]. Contemporary guidelines suggest the use of calcium hydroxide or different antibiotic mixtures for the disinfection of the root canal during regenerative procedures [11]. Several in vitro and in vivo studies have described the efficacy of the combination of ciprofloxacin, metronidazolezole and minocycline (3-MIX), against several endodontic pathogens [12; 13; 14; 15]. However, this topical triple antibiotic paste (TAP) may result in substantial tooth discoloration, primarily due to the chelating effect of the minocycline against the calcium ions [16; 17; 18]. Additionally, the bonding agents applied in the pulp chamber may be partially ineffective against root and crown staining [19; 20]. For this reason, calcium hydroxide and a double antibiotic paste (DAP) were preferably proposed [21]. However, calcium hydroxide could present a weakening effect on the thin dentine walls [22]. Moreover, the alternative use of a DAP containing ciprofloxacin and metronidazolezole and without minocycline (2-MIX) has proven less effective at the same concentration [23; 24]. A recent study [25] reported that between 1 and 5 mg/mL DAP provided a significant, direct antibacterial effect against bacterial biofilm obtained from immature teeth with necrotic pulps, but only 5 mg/mL DAP showed substantial residual antibacterial effects against bacterial biofilms. Therefore, the recovery of a novel disinfecting solution is current in the emergent pulp regeneration field. Previous studies have reported that clindamycin or cefcolor-modified TAPs have demonstrated favorable outcomes when they were used during regenerative endodontic treatments [17]. However, an alternative antibiotic combination, composed of ciprofloxacin, metronidazolezole and clarithromycin (3-MIXC), has demonstrated high antimicrobial properties with no discoloration effects in vitro [26]. Clarithromycin is an advanced-generation macrolide with a wide spectrum of activity and excellent inflammatory-dependent activity, both intra and extracellular [27; 28; 29].  
In addition, the application of hyaluronic acid (HA) has been described in a variety of different uses in several medical settings [30]. HA is a biodegradable polymer, consisting of N-acetyl-D-glucosamine and beta-glucoronic acid. The gel is well-tolerated, safe and effective. The viscoelastic nature of HA and its biocompatibility and non-immunogenicity has led to its use in a large number of clinical applications, such as in controlled-release and targeted drug delivery systems. However, most studies are still only in the in vitro experimental stage and no data are available about the use of HA in place of the traditional macrogol gel (MG) as a vehicle for antibiotic mixtures in REPs [31].  
The aim of this study was to evaluate the antibacterial efficacy and depth of action of the novel 3-MIXC compared with the 3-MIX and 2-MIX antibiotic mixtures, vehicled with HA or MG, using confocal laser scanning microscopy (CLSM).