## 2 Materials and Methods
After the exposure, the specimens were rinsed with sterile saline solution to remove the antibiotic paste from the root canal and each cylindrical dentin block was fractured into 2 identical semi-cylindrical halves. The fracture was performed by making a longitudinal groove on the external root surface with a thin bur (Dentsply Sirona) and then by using a blade with a stable support, in order to ensure a very thin and flat surface. The outer surfaces of the semi-cylindrical halves were ground with 600-grit silicon carbide paper (ARC Abrasives Inc, Troy, OH) to achieve a standard thickness of 2 mm and to remove the root surface cement. The dentin specimens were then shaped with a water-cooled low-speed headpiece and a fine carbide bur at 300 rpm to fit each specimen into the inner wall of a filter tube with a 0.45 mm pore size (Pall Corporation, Ann Arbor, MI, USA). The refined specimens measured approximately \(4\times 4\times 2\) mm. The LIVE/DEAD (L/D) _Bac_Light Bacteria Viability Kit (TermoFisher Scientific, Waltham, MA, USA) was used to detect viable and dead bacteria using a 1:1 ratio of SYTO 9 (20/1) and propidium iodine (PI, 120/1) [34]. After staining with the L/D viability test solution, the specimens were positioned in 100 ul of saline solution in a multi-well and stored within a microscopy chamber in a dark room. The specimens were washed in sterile water for 1 min and were then vertically fractured--through the root canal--into 2 halves, as described above, to expose fresh and flat surfaces of longitudinally visible dentin canals for CLSM examination. The imaging was performed using a laser scanning confocal system (Fluouview 200, Olympus America, Melville, NY, USA), with the specimens mounted on an inverted microscope (model IX0, Olympus Optical Co. GMBH, Hamburg, Germany), illuminated by a krypton/argon laser (488 nm). Emission wavelengths of 505-550 nm (green, SYTO 9) and 650-750 nm (red, PI) were utilized to visualize SYTO 9 (live bacteria) and PI (dead bacteria), respectively. The images were taken using a 20\(\times\) objective with an additional zoom of 2\(\times\) and processed and analyzed with ImageJ software (NIH, Bethesda, MD, USA). Six samples were analyzed as positive controls to assess the formation of a bacterial biofilm and the bacterial viability (%) was calculated from the CLSM images. For the tested samples, the mean antibiotic depth of action was calculated from 10 separate measurements for each single image, adjusted for the red color channel. Data were recorded and differences were analyzed with one-way ANOVA and a post-hoc Bonferroni test (\(p<0.05\)). The ratio of red fluorescence to green-and-red fluorescence (indicating the proportion of dead cells for each group) was calculated from merged images and three-dimensional reconstructions. This measurement was considered to be a surrogate marker of bactericidal efficacy. Figure 1: Flow diagram of the study design. 2-MIX, ciprofloxacin and metronidazolezo; 3-MIX ciprofloxacin, metronidazolezo and minocycline; 3-MIX, ciprofloxacin, metronidazolezo and clarithromycin; HA, hyaluronic acid; MG, macrogol gel. The Kolmogorov-Smirnov test for normality was used to analyze the data distribution. The data were collected and the differences among the groups were analyzed by using [PERSON]'s post-hoc tests (\(p<0.05\)).