Evaluation of cold plasma treatment and safety in disinfecting 21-day root canal enterococcus faecalis biofilm in vitro

Many studies have demonstrated that Enterococcus faecalis (E. faecalis) is the most important species responsible for persistent endodontic infection. E. faecalis biofilm often exhibits increased resistance to antimicrobial agents compared with planktonic bacteria. The aim of this study is to evaluate the effectiveness and safety of cold plasma therapy in disinfecting 21-day E. faecalis biofilms. Teeth with 21-day E. faecalis biofilm were treated with an argon/oxygen cold plasma for various treatment times and compared to those treated with Ca(OH)₂, 2% CHX gel and Ca(OH)₂/CHX gel for 7 days (positive controls). Negative control groups included normal saline treatment and Ar/O₂ gas treatment. Antimicrobial efficacy was determined by colony forming unit (CFU) method. Scanning Electron Microscopy (SEM) was employed to assess the morphological changes of E. faecalis biofilm by plasma. Confocal Laser Scanning Microscopy (CLSM) was used to confirm the viability of the biofilm after the plasma treatment. Microhardness and microroughness changes caused by plasma were tested with Vickers Hardness Tester and 3D Laser Scanning Microscope respectively. Energy Dispersive X-ray Spectroscopy (EDS) was used for elemental analysis of root canal dentin. The results indicated that a 12 min cold plasma treatment could inactivate E. faecalis 21-day biofilm completely. Atmospheric pressure cold plasma is an effective tool in endodontics for its strong biofilm sterilization effect and has an accepted safety for its low temperature without significantly affecting the microhardness and microroughness of tooth enamel if placed at proper working distance.