Enterococcus faecalis biofilm removal efficacy, cytotoxicity and alkaline phosphatase activity on stem cells after the application of conventional medicaments and novel hydrogels used in regenerative endodontics

Background: To compare the antibacterial activity of conventional intracanal medicaments used in regenerative endodontic treatment (TAP, DAP, Ca(OH)₂, CHX gel) with novel hydrogel-based formulations (chitosan, CHX, hyaluronic acid, metronidazole, and diclofenac hydrogels) against Enterococcus faecalis biofilms, and to assess their cytotoxic effects on dental pulp stem cells (DPSCs). Methods: Antibacterial activity was evaluated using an E. faecalis biofilm model in single-rooted incisors. For cytotoxicity testing, dentine discs were treated with the medicaments and seeded with DPSCs. Cell viability was assessed using the WST-1 assay, and mineralization-related responses were evaluated by alkaline phosphatase (ALP) activity. Data were analyzed using the Kruskal–Wallis test with Bonferroni-adjusted post-hoc comparisons (p < 0.05). Results: TAP, DAP, diclofenac hydrogel, and CHX hydrogel completely eliminated the E. faecalis biofilm in the CFU analysis, whereas CHX gel reduced the biofilm but did not achieve full eradication. In contrast, Ca(OH)₂, chitosan, HA, and MTR hydrogels exhibited limited antibacterial effects. Ca(OH)₂ and chitosan hydrogel preserved the highest levels of cell viability, while DAP, TAP, HA, MTR, and diclofenac hydrogels caused moderate reductions; CHX gel and CHX hydrogel showed the lowest survival. ALP activity was maintained in the Ca(OH)₂, chitosan, HA, MTR, and diclofenac hydrogels but decreased in the CHX based groups (all relevant comparisons, p < 0.05). Conclusions: Hydrogel-based systems demonstrated a favorable balance between biofilm control and stem-cell biocompatibility. The diclofenac-loaded hydrogel achieved TAP/DAP-level biofilm reduction while preserving cell viability, supporting its potential as an antibiotic-free disinfection alternative. CHX hydrogel showed higher antibacterial performance than CHX gel, whereas hyaluronic acid hydrogel maintained high biocompatibility and demonstrated applicability as a biofunctional carrier system.