Biological, antimicrobial, and apical tissue dissolution effects of ambroxol hydrochloride as an irrigant for regenerative endodontics: an in vitro study

This study evaluated, in vitro, the effects of ambroxol hydrochloride (ABX) and sodium hypochlorite (NaOCl), alone or in combination with EDTA and activation methods on cytotoxicity and metabolic activity of stem cells from the apical papilla (SCAPs), antimicrobial, antibiofilm, and apical tissue dissolution activity. Bovine dentin specimens were used to assess SCAP viability scores (Live/Dead assay) and metabolic activity (Alamar Blue assay) in a 3D culture model, and to evaluate biofilm disruption and intratubular bacterial reduction using dual-species biofilms of Enterococcus faecalis and Streptococcus mutans. Apical tissue dissolution was assessed using bovine muscle fragments. For all experiments, specimens were allocated according to the irrigation protocol (NaOCl, ABX, or their combinations with EDTA) and further subdivided by activation method: conventional irrigation (CI), ultrasonic activation (UA), or diode laser activation (LA). Data were analyzed with two-or three-way ANOVA and Tukey’s post hoc test (p<0.05). NaOCl and NaOCl+EDTA showed the highest cytotoxicity scores (p<0.05), while ABX-based protocols demonstrated lower cytotoxicity scores and preserved SCAP metabolism (p<0.05). NaOCl+EDTA promoted greater biofilm reduction under CI (p<0.05), with no significant differences between the activation methods (p>0.05). In intratubular analysis, NaOCl+EDTA achieved the greatest bacterial killing, especially in the apical and superficial dentin (p<0.05), with LA being superior to UA (p<0.05). Activation improved the antimicrobial effect of ABX in superficial dentin (p<0.05), whereas no statistically significant differences were observed in deep dentin (p>0.05). For tissue dissolution, NaOCl and NaOCl+EDTA promoted significantly greater apical tissue loss across all subdivisions (p<0.05). The toxicity of the irrigating solution was the main factor influencing cell viability and metabolism, while activation impacted antimicrobial outcomes depending on the irrigant used and the dentin region. NaOCl+EDTA showed the most potent antimicrobial effects, but with higher cytotoxicity and apical tissue dissolution. ABX-based protocols were more biocompatible and, when combined with EDTA and activation, provided satisfactory antimicrobial activity, supporting ABX as a promising candidate for regenerative protocols.