This study assessed a new antimicrobial paste formulation containing nitrofurantoin for regenerative endodontics against multispecies biofilms. Four groups of 11 single-root teeth each were tested: Negative control group (BHI + microorganisms); Hoshino 1 conventional tri-antibiotic paste group (1:1:1 ratio); Hoshino 5 group (5:5:5 ratio); Experimental group with nitrofurantoin, ciprofloxacin, and metronidazole. Minimum inhibitory concentrations of each antimicrobial were determined. A polymicrobial biofilm (E. faecalis, S. aureus, P. aeruginosa, C. albicans) was created in root canals, and pastes were inserted for 3 minutes with lentulo. After 7 days, samples were sonicated and spectrophotometrically analyzed. Microbiological assays, plasma membrane permeabilization, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) evaluated biofilm eradication. Statistical significance was set at p < 0.05. Spectrophotometry indicated Experimental group outperformed Hoshino 1 group and Negative control group (p<0.05), with results similar to Hoshino 5 group (p>0.05). Experimental group showed increased membrane damage compared to Hoshino 1 group and Hoshino 5 group. CLSM and SEM revealed fewer microorganisms and no residual biofilm in Experimental group. Substituting minocycline with nitrofurantoin in the paste effectively reduced microorganism counts in root canals system.
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