Desenvolvimento de um gel enriquecido com trans-cinamaldeído para tratamento da periodontite apical: uma análise In vitro e In vivo

Abstract

Apical periodontitis (AP) is an inflammation that affects the periapical tissues associated with the colonization of bacteria from the oral microbiota, organized in biofilms, with the capacity to cause persistent infections, especially when Enterococcus faecalis is involved , making treatment with conventional intracanal medications difficult. In this sense, treatments with new approaches based on natural products have stood out in the search for effective antimicrobial activity, providing a sustainable and biocompatible alternative in the treatment of endodontic infections. Trans - cinnamaldehyde (TC), the main compound in cinnamon essential oil, has proven antibacterial potential. Therefore, the present thesis aimed to evaluate the effect of TC and a gel formulation enriched with TC (GelTC) on strains of E. faecalis isolated from raw foods. The potential of GelTC in an in vivo model of AP induced in rats was also investigated. The preparation of GelTC consists of a patent filing chapter. For the in vitro study , 5 strains (E1, E2, E3, E4 and E5) of E. faecalis from sushi were isolated, identified by Matrix Assisted Laser Desorption/Ionization (MALDI-TOF) and categorized according to the antibiotic resistance and biofilm formation capacity. The in vitro TC assays were performed by disk diffusion, agar diffusion per well, broth microdilution, biomass quantification by crystal violet (CV) and cell viability by methyl-thiazole-tetrazolium (MTT). After GelTC was formulated, its antibacterial efficiency was evaluated by per-well agar diffusion. In the in vivo study , 48 Wistar rats were used, divided into experimental groups: GelTC, calcium hydroxide (HC) as positive control and sick group (PA) that received only disease induction. To induce BP, the lower right molars were accessed and exposed to the oral cavity for 14 days. After the AP induction period, the conduits were prepared with manual files and irrigation with saline solution for subsequent insertion of intracanal medications, which were maintained for a period of 7 or 14 days. After 21 or 28 days of AP induction, the animals were euthanized, the hemimandibles were collected and x-rayed for later measurement of the area of the periapical lesions using ImageJ software. All assays were performed in triplicate with a significance level of p<0.05. Data from in vitro and in vivo assays were analyzed using GraphPadPrism 8.4.3 applying one-way analysis of variance (ANOVA) followed by Tukey's test. All E. faecalis isolates were sensitive to the antibiotics tested, with the exception of gentamicin (GEN) and imipenem (IMP). For GEN, the isolates showed low-level intrinsic resistance, while for IMP, all were classified as intermediate. Among the isolates, only strains E1, E3 and E5 are strong biofilm formers. The diameter halos of the zone of inhibition (DZI) of pure TC ranged from 26.33 to 39.33 mm, showing antibacterial potential against the strains tested. In microdilution, TC showed a minimum inhibitory concentration (MIC) ranging from 312.5 to 625 μg/mL, with a minimum bactericidal concentration (MBC) of 312.5 to 2500 μg/mL. The TC biofilm, CV and MTT assays were performed for strains E1, E3 and E5. Pure TC promoted a reduction in biomass and cell viability, especially for strains E1 and E5 (2 and 4 x MIC). Furthermore, the MIC of GelTC ranged from 1250 to 2500 μg/mL. In diffusion per well, TC 5% and GelTC 5% demonstrated efficient activity for all strains. GelTC 5% showed better results ranging between 17.33 and 30.67 mm of DZI, compared to TC 5% which varied between 17.67 and 20.33mm. In in vivo assays , morphometric differences were observed within 7 days of treatment between the PA, GelTC 5% and HC groups. However, after 14 days of treatment, there was a significant difference between the HC and GelTC 5% groups when compared to the PA. Furthermore, the 5% GelTC group, after 14 days of treatment, showed a statistically significant difference in relation to the HC group. It is concluded that TC and GelTC 5% showed in vitro antibacterial activity against E. faecalis isolates from food. GelTC 5% showed effective activity in the treatment of periapical lesions induced in rats, significantly reducing areas of bone resorption.