## 4 Discussion
First, RONS may have been generated in the oral cavity during CAP treatment but were not evaluated. However, the survival rate of L-929 mouse fibroblasts subjected to up to 2 min of CAP treatment demonstrated no significant difference from the control group, as indicated in a previous study [42]. Further cytotoxicity studies should be conducted to support the clinical use of CAP. Second, the biofilms were formed on HAP disks rather than on actual enamel, which could have influenced initial biofilm adherence. Nevertheless, the HAP disk surfaces were prepared similarly to the process of specimen preparation using actual enamel [25], thereby minimizing the potential impact of the HAP disk surface characteristics on biofilm formation. Third, the CAP treatment duration was set to 2 min based on the unique characteristics of the plasma application, accounting for the 1 mm diameter jet projection onto the 7 mm disk and the output pressure of 0.02 MPa. Therefore, the area of immediate exposure was inherently limited due to the 1 mm diameter nozzle, resulting in non-uniform biofilm CAP exposure, unlike the CHX treatment, where the entire biofilm surface received simultaneous and uniform exposure. Consequently, while the CHX treatment, as per the manufacturer's instructions, only required 1 min due to its comprehensive coverage, the CAP treatment required prolonged exposure to ensure that the entirety of the biofilm surface received adequate treatment. However, the 2 min duration for CAP treatment does not correspond directly to the 1 min duration for the CHX treatment in terms of efficacy and exposure. Therefore, future studies should explore methods that could reduce the optimum CAP treatment duration, potentially through modifications to the nozzle length and enrichment of the working gas with oxygen, to optimize treatment efficiency. This would help in aligning the treatment times more closely and mitigating the issue of time as a confounding variable. Lastly, the differences in ease of use and cost-effectiveness could prominently differ between the CHX and CAP treatments. The current form of CAP application via a pipette could be less practical for everyday use and involve higher costs. However, this study aimed to lay the groundwork for future advancements in this technology. The ultimate goal is to eventually develop more user-friendly and cost-effective CAP devices that could offer viable alternatives or adjunctive therapies to existing treatments. These findings could highlight the potential for innovation in dental treatment methods and underscore the need for ongoing research to make emerging technologies more accessible and practical for patient use. Nevertheless, this novel study measured changes in oral biofilms using QLF-D camera over 6 days, which allowed for precise and non-invasive biofilm monitoring over time and provided detailed quantitative data on biofilm pathogenicity and antibacterial treatment responses. The antibacterial efficacy of CAP demonstrated using an oral microcosm biofilmmodel that closely simulates the oral environment presents another significant advantage, as this model closely mimics the natural environment of the oral cavity, providing a more accurate and realistic assessment of biofilm behavior and treatment efficacy compared with traditional models. By replicating the complex microbial community and environmental conditions of the oral cavity, the model presented in this study offers valuable insights into oral biofilm dynamics and their treatment responses.