Cytotoxic and antibiofilm properties of antibiotic-loaded thermoresponsive hydrogels for root canal therapy

Dental infections can disrupt root development in immature permanent teeth, making traditional endodontic treatment challenging. Apexogenesis, a regenerative approach that promotes natural root development, offers a potential solution. However, issues related to disinfection and material biocompatibility still remain. The objective of this study was to evaluate the synergistic antimicrobial and antibiofilm properties of double and triple antibiotic combinations against common oral pathogens, and to incorporate the most effective combination into a thermosensitive hydrogel, to develop an alternative intracanal medication. Antibiotics were tested alone and in combination in planktonic and biofilm conditions of oral bacteria and Candida albicans. The antibiotic combinations with potential antimicrobial synergy were tested on Enterococcus faecalis biofilms in radicular dentin by confocal microscopy. Metronidazole (ME), ciprofloxacin (CI), and fosfomycin (FO) were incorporated into poly(N-vinylcaprolactam) (PNVCL) hydrogels, and their antibiofilm activity was compared to PNVCL hydrogels containing chlorhexidine (CHX) or calcium hydroxide (CH). The cytotoxicity of the hydrogels was assessed on MDPC-23 odontoblast-like cells using metiltetrazolium assays. A statistical analysis was performed using ANOVA followed by Tukey’s test (p < 0.05). The combination of ME + CI + FO showed superior antibiofilm effects in mono- and dual-species biofilms and on biofilms inside dentinal tubules, comparable to CHX. PNVCL hydrogels with ME + CI + FO significantly reduced E. faecalis biofilms in dentinal tubules, exhibiting a higher efficacy than PNVCL + CH. Cytotoxicity tests revealed minimal effects on cell viability for both PNVCL hydrogels with and without antibiotics. In conclusion, ME + CI + FO showed potent antimicrobial synergy and, when loaded in thermosensitive PNVCL hydrogel, demonstrated significant antibiofilm activity and low cytotoxicity. These findings emphasize the potential of this formulation as an effective and biocompatible endodontic medication, especially for the treatment of immature permanent teeth.