As time progresses, the junction between dentin and restorative biomaterials deteriorates, facilitating the passage of saliva, tissue fluids, and bacteria between the filling or restorative substance and the dentin surface. The present investigation explored how long-term exposure to simulated human salivary/bacterial/blood esterases (SHSE) affects the development and survival of Enterococcus faecalis biofilms formed along the dentin interface when different endodontic materials are employed. Extracted human anterior teeth were mechanically prepared and obturated with gutta-percha in combination with one of four materials: a self-cured resin composite (Bisfil™ 2B, Bisco, Schaumburg, IL, USA) paired with either a self-etch adhesive (EasyBond) or a total-etch adhesive (Scotchbond™, 3M, Saint Paul, MN, USA), an epoxy resin-based sealer (AH Plus®, Dentsply Sirona, York, PA, USA), or a bioceramic sealer (EndoSequence® BC Sealer™, Brasseler USA, Savannah, GA, USA). Samples were then subjected to accelerated aging in SHSE or phosphate-buffered saline (PBS) for intervals of up to 360 days, after which stable E. faecalis biofilms were cultivated. The extent and vitality of bacterial growth within the interfacial zones were examined using confocal laser scanning microscopy coupled with live/dead viability staining. Data analysis was conducted using a three-way ANOVA with Scheffé’s post hoc comparisons. At baseline, all materials exhibited comparable biofilm penetration depths (p > 0.05). Prolonged aging led to significantly greater bacterial infiltration across all material types (p < 0.05). SHSE exposure intensified interfacial biofilm penetration for the TE, SE, and BC materials (p < 0.05), while the AH group remained unaffected. In unaged conditions, BC showed the smallest proportion of live bacterial cells, followed by AH, TE, and SE (p < 0.05). Overall, the proliferation and viability of interfacial E. faecalis biofilms were influenced by the type of biomaterial, the composition of the aging environment, and the duration of aging.
