Cytotoxicity and Senescence-Associated Responses Induced by GuttaFlow Bioseal in Human Gingival Fibroblasts
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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 mm×2 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 β-galactosidase (SA-β-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-β-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.
Abstract
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 mm×2 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 β-galactosidase (SA-β-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-β-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.