Evaluation of the Antimicrobial Efficacy of Cerium Oxide Nanoparticles and Chlorhexidine Gluconate Against Enterococcus faecalis in Root Canal Systems: Protocol for an In Vitro Study

Abstract

Background: Enterococcus faecalis is a facultative anaerobic bacterium frequently associated with persistent root canal infections and endodontic treatment failures. Its resistance is primarily attributed to its ability to form robust biofilms, survive under harsh conditions, and penetrate deep into dentinal tubules. Chlorhexidine gluconate (CHX), commonly used as an intracanal medicament, exhibits broad-spectrum antimicrobial activity; however, its limited ability to eliminate biofilms, potential cytotoxicity, and restricted dentinal penetration pose significant drawbacks. In recent years, nanotechnology has introduced promising alternatives such as cerium oxide nanoparticles (CeO₂-NPs), which exhibit unique redox properties, reactive oxygen species scavenging, and antimicrobial action due to their nanoscale size and surface chemistry.

Objective: This study aims to evaluate and compare the antimicrobial efficacy of 0.05% CeO₂-NPs, 2% CHX, and their combination against E. faecalis in root canal systems.

Methods: Forty-eight extracted human single-rooted premolars will be decoronated, instrumented, and sterilized. The root canals will be inoculated with E. faecalis and incubated for 21 days to allow biofilm formation. The specimens will then be divided into four groups (n=12 per group), each receiving one of the following treatments: (1) 0.05% CeO₂-NPs, (2) 2% CHX, (3) a combination of CeO₂-NPs and CHX, and (4) sterile water as the negative control. After the medicaments are applied for 5 days, microbial sampling will be conducted, and the remaining viable bacteria will be quantified using colony-forming unit (CFU) counts on selective agar.

Results: Ethics approval was obtained from the institutional ethics committee of Datta Meghe Institute of Higher Education and Research, Wardha (approval DMIHER(DU)/IEC/2025/546) in January 2025. As of January 2026, laboratory procedures, including specimen preparation, microbial inoculation, and data collection, have not yet commenced. Data collection is planned to begin in February 2026 and is expected to be completed by April 2026. Data analysis will follow immediately thereafter, with results anticipated to be submitted for publication in mid-2026. As this is an in vitro study, no participant recruitment, funding allocation, or clinical trial registration is applicable. It is expected that the combination of CeO₂-NPs and CHX will result in the greatest reduction in bacterial load, followed by CeO₂-NPs alone, CHX alone, and sterile water. These expected outcomes will be assessed based on comparative CFU counts for each treatment group.

Conclusions: This study aims to provide insight into the antimicrobial potential of CeO₂-NPs either alone or in combination with CHX against E. faecalis. If proven effective, CeO₂-NPs could represent a novel and biocompatible adjunct or alternative to conventional intracanal medicaments, contributing to improved disinfection protocols and better clinical outcomes in endodontic therapy.

International registered report identifier (irrid): PRR1-10.2196/77998.

Keywords: Enterococcus faecalis; biofilm; cerium oxide nanoparticles; chlorhexidine gluconate; intracanal medicament; nanoparticles; single-rooted premolar.