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

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). 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). 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). 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.