Anti-microbial and anti-inflammatory effect of isolated, combined and incorporated phenolic acids in chitosan hydrogels for endodontic application

Author(s)

Vanessa Rodrigues dos Santos

• Document type: Doctoral Thesis
• Press: Araçatuba. 2022-12-13.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araçatuba
• Defense date: 2022-10-24
• Advisor: Cristiane Duque; Antonio Hernandes Chaves Neto

Abstract

The objective of endodontic treatment is to maintain the integrity of the root, as well as to prevent or resolve periapical diseases, by eradicating microorganisms and their sources of nutrients from the root canal system. The complexity of root canal anatomy and multispecies biofilms increases the difficulty in eliminating microorganisms and controlling inflammation by conventional chemical-mechanical procedures, which justifies the use of intracanal medications. New compounds with broad antimicrobial effect and anti-inflammatory potential, such as phenolic acids, could be explored as active principles of intracanal medications. However, to increase the solubility, control the release and extend the biological effects of phenolic acids, it would be interesting to incorporate them into drug carriers such as chitosan hydrogels. This study was divided into two chapters with the following objectives: 1) to evaluate the antimicrobial, antibiofilm and anti-inflammatory activities and the cytotoxicity of cinnamic acid and its derivatives; 2) synthesize and characterize chemical and physico-mecanical properties of thermosensitive chitosan and poloxamer hydrogels containing phenolic acids and evaluate the effect of these hydrogels on multispecies biofilms and on the viability of macrophages and fibroblasts. In chapter 1, the antimicrobial activity of cinnamic acid (CI) and its derivatives coumaric acid (CO), caffeic acid (CA), ferulic acid (FE) and sinapic acid (SI) was evaluated by determining the minimum inhibitory and bactericidal concentration (MIC/MBC) and Fractional Inhibitory Concentration (FIC) on Enterococcus faecalis, Streptococcus mutans, Lactobacillus casei, Actinomyces israelii and Fusobacterium nucleatum. Phenolic acids were selected and their effect on bispecies and multispecies biofilms with the same standard or clinical strains were evaluated by bacterial counts, scanning electron microscopy and confocal microscopy. The viability of L929 fibroblasts and RAW 264.7 macrophages in the presence of these phenolic acids was evaluated by resazurin assays. In addition, mRNA levels of the pro-inflammatory markers TNF-α, IL-1β, iNOS and COX-2 were determined by quantitative TaqMan PCR after macrophage exposure to phenolic acids and lipopolysaccharide (LPS). In chapter 2, the synthesis and physical-mechanical characterization of chitosan-poloxamer (CPH) hydrogels containing phenolic acids were performed and their effects on multispecies biofilm and on the viability of macrophages and fibroblasts were evaluated. Data were statistically analyzed considering p<0.05. Cinnamic acid and caffeic acid showed an inhibitory and bactericidal effect against all bacterial species tested, with the lowest MIC and MBC values. However, no synergistic effect was observed between the compounds (FICI>0.5). Both compounds (at 5x the highest MIC) were effective in eliminating dual-species biofilms and significantly decreasing multispecies biofilms, especially cinnamic acid. Cinnamic acid caused minimal toxicity to both cell cultures at MIC concentrations and caffeic acid was not cytotoxic at concentrations below 0.125 mg/mL. Both compounds significantly reduced TNF-α, IL-1β, iNOS and COX-2, in a dose-dependent manner. CPH were characterized as thermoreversible and with adequate mechanical and bioadhesive properties. The effect of CPH+CA (77.8%) and CPH+CI (73.2%) hydrogels against multispecies biofilms was superior to CPH + calcium hydroxide (CH) (53.6%) and CPH + chlorhexidine (CHX) (39.9%). In general, CPH + CI caused less cytotoxicity when compared to CPH + CA, for both cell lines. In conclusion, cinnamic acid and caffeic acid showed bactericidal effect and against biofilms of bacteria associated with endodontic infections, causing minimal cytotoxicity. In addition, both compounds showed an anti-inflammatory effect, inhibiting the expression of pro-inflammatory markers in LPS-stimulated macrophages. The chitosan-poloxamer hydrogels were thermoreversible and presented adequate mechanical and bioadhesive properties for clinical application, and when combined specially with cinnamic acid, they promoted the reduction of multispecies biofilms formed in the dentinal tubules, causing low toxicity to fibroblasts and macrophages. (AU)

• FAPESP's process: 19/01949-0 - Antimicrobial and anti-inflammatory effect of phenolic acids alone, in combination and incorporated in chitosan hydrogels for endodontic application
• Grantee: Vanessa Rodrigues dos Santos
• Support Opportunities: Scholarships in Brazil - Doctorate