Cytotoxicity of Root Canal Sealers and Potential Clinical Implications: A Comprehensive Systematic Review of In Vitro Studies

<b>Background</b>: Root canal sealers may come into direct contact with periapical tissues, particularly in cases of apical extrusion, potentially influencing periapical healing and treatment outcomes. Cytotoxicity assessment represents a clinically relevant parameter when selecting endodontic sealers. However, evidence derived from in vitro studies remains heterogeneous and challenging to interpret from a clinical perspective. Therefore, the aim of this systematic review was to critically evaluate the in vitro cytotoxicity of all root canal sealers that have been commercially marketed over the years, excluding experimental materials, and to contextualize the findings in relation to clinically relevant experimental conditions. <b>Methods</b>: This systematic review was conducted according to PRISMA guidelines and preregistered on the Open Science Framework. PubMed, Scopus, and the Cochrane Library were searched up to 30 November 2025. In vitro studies evaluating the cytotoxicity of commercially available root canal sealers using validated cell viability or proliferation assays were included. Data extraction focused on sealer composition, setting condition, extraction protocols, exposure parameters, and cytotoxic outcomes. Due to marked methodological heterogeneity, a qualitative synthesis was performed. <b>Results</b>: Ninety-eight in vitro studies were included. All categories of root canal sealers demonstrated some degree of cytotoxicity, particularly when tested in freshly mixed conditions, at higher extract concentrations, or after prolonged exposure. Bioactive and calcium silicate-based sealers generally showed a more favorable cytotoxicity profile compared with conventional materials, especially after complete setting and at diluted concentrations, although cytotoxic effects were reported under specific experimental conditions. Resin-based sealers, including AH Plus, exhibited condition-dependent cytotoxicity, while zinc oxide-eugenol and glass ionomer sealers tended to display higher cytotoxic potential. <b>Conclusions</b>: In vitro cytotoxicity of root canal sealers varies according to material composition and experimental conditions. Bioactive sealers generally exhibit a more favorable biological profile, which may be clinically relevant in situations involving sealer extrusion or prolonged tissue contact. Standardized testing protocols and further translational studies are required to support evidence-based clinical material selection.