Cytocompatibility, Antibacterial, and Anti-Biofilm Efficacy of Grape Seed Extract and Quercetin Hydrogels Against a Mature Endodontic Biofilm Ex Vivo Model

<b>Background/Objectives</b>: To assess the cytocompatibility, antibacterial and anti-biofilm efficacy of grape seed extract (GSE) and quercetin hydrogels versus calcium hydroxide (CH) as intracanal medications (ICMs) against an endodontic ex vivo biofilm model. <b>Methods</b>: Single-rooted teeth (<i>n</i> = 50) were prepared and sterilized before being infected with <i>E. faecalis</i> to develop a mature biofilm. They were divided into five equal groups according to the ICM used: G1: medicated with CH paste, G2: medicated with GSE hydrogel, G3: medicated with quercetin hydrogel, G4: positive control group that was infected and not medicated, and G5: negative control group that was neither infected nor medicated. After 1 week, the ICM was removed, and the root canals were cultured to assess the antibacterial efficacy by counting the colony-forming units and the anti-biofilm efficacy by the crystal violet assay. Dead/live bacterial viability was assessed by CFLSM examination, while the cytocompatibility was assessed using the MTT assay. <b>Results</b>: CH had the best antibacterial efficacy, followed by GSE and quercetin hydrogels (<i>p</i> < 0.001). Regarding the anti-biofilm efficacy, GSE was superior, followed by quercetin and CH (<i>p</i> < 0.001). CFLSM examination showed CH and GSE hydrogel to be highly effective in comparison to the positive control (<i>p</i> < 0.0001), with no statistical difference between them (<i>p</i> > 0.05). CH showed significantly higher cell viability percentages using a 500 μg/mL, while quercetin and GSE started to show cell viability > 70% at concentrations of 125 μg/mL and 62.5 μg/mL. <b>Conclusions</b>: CH fulfilled the ideal requirements of ICM as being both antibacterial and non-cytotoxic compared to the other materials tested.