Cytotoxicity and Senescence-Associated Responses Induced by GuttaFlow Bioseal in Human Gingival Fibroblasts

Background: Root canal sealers play a crucial role in endodontic treatment by sealing the canal space, preventing microbial leakage, and supporting periapical tissue healing.Among these, bioceramic-based root canal sealers such as GuttaFlow Bioseal (GF-Bioseal) have been developed because of their favorable physicochemical properties and biocompatibility.However, most previous studies have reported low cytotoxicity, excellent sealing ability, and mineralization potential.This study evaluated the cytotoxicity and senescence-inducing potential of GF-Bioseal in human gingival fibroblasts (HGFs).Methods: GF-Bioseal specimens were prepared in standardized disk form (6 mm2 mm, 0.05 g/ml) according to ISO 10993-5, and extracts were obtained at varying dilutions.HGFs were cultured in three-dimensional conditions and treated with the extracts.Cell viability was measured using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay.Intracellular reactive oxygen species (ROS) levels were quantified using 2', 7'-dichlorofluorescein diacetate staining.Senescence was assessed by senescence-associated -galactosidase (SA--gal) staining and Western blotting of senescence-associated markers and stress-related proteins.Results: GF-Bioseal extracts reduced cell viability in a concentration-and time-dependent manner, but maintained 90% viability at 0.0125 g/ml for up to 72 hours.Cell adhesion was preserved with a fibroblast-like morphology, unlike AH Plus which reduced adhesion.GF-Bioseal increased intracellular ROS levels, elevated SA--gal-positive cells (40% at 72 hours), and upregulated inducible nitric oxide synthase (iNOS), p16, and p21.expression, whereas AH Plus increased cyclooxygenase-2 and iNOS levels.Conclusion: GF-Bioseal demonstrated relatively low acute cytotoxicity but induced oxidative stress and senescence-associated responses in oral fibroblasts.These findings highlight the need to evaluate both cytotoxicity and chronic cellular responses of assessing the biocompatibility of endodontic sealers.