Antimicrobial efficacy of 2- hydroxyisocaproic acid (hica) towards endodontic pathogens and its cytotoxicity on dental pulp stem cells: an in vitro and ex vivo study.

Numerous intracanal medicaments are currently used in endodontic therapy. In regenerative endodontics, common medicaments are calcium hydroxide and antibiotic pastes; however, increasing antimicrobial resistance and limitations of current medicaments have led to research on alternative medicament. 2-hydroxyisocaproic acid (HICA) is a derivative of leucine, a normal component of human plasma that shows potential as an endodontic intracanal medication. Objective: This study aimed to determine the antimicrobial efficacy of HICA against two common endodontic pathogens. Enterococcus faecalis (E. faecalis) and Prevotella intermedia (P. intermedia), and assess its cytotoxicity towards dental pulp stem cells (DPSCs). Methods: The antimicrobial efficacy of HICA against both bacteria were determined using disk diffusion and broth microdilution methods. Triple antibiotic paste (TAP) containing minocycline, metronidazole, and ciprofloxacin (1:1:1 ratio) was used as a positive control, while sterile saline and sterile broth were used as a negative control for respective assays. Dentine block sampling was performed to investigate antimicrobial efficacy in an ex vivo experiment. A total of 66 extracted permanent teeth were infected with E. faecalis for 21 days. HICA at concentration of 16 mg/ml, TAP (1 mg/ml), and saline (negative control) were introduced into the canal and further incubated for seven days. Viable bacterial counts were used as indicators of bacterial growth. Scanning electron microscope (SEM) was used to observe changes in the morphology of E. faecalis in samples treated with HICA at MIC value and 1 mg/ml TAP at magnification of 1000X, 4000X, 10000X, 16000X and 30000X. The cytotoxicity of HICA on DPSCs was tested using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Results: In the disk diffusion assay, HICA displayed a greater inhibitory effect on P. intermedia at a lower concentration (128 mg/ml) compared to E. faecalis (256 mg/ml). At 256 mg/ml, HICA showed a zone of inhibition (ZOI) of 15.72±1.60 mm against E. faecalis and 30.74±0.71 mm for TAP at 4 mg/ml. Against P. intermedia, HICA at 128 mg/ml demonstrated a ZOI of 17.79±2.44 mm, while TAP at 0.25 mg/ml displayed a ZOI of 46.67±3.07 mm. The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) of HICA against E. faecalis were 8 mg/ml and 16 mg/ml, respectively. In contrast, the MIC of TAP was equal to the MBC at 0.25 mg/ml. When compared to P. intermedia, the MIC of HICA was equal to its MBC (4 mg/ml), while TAP concentrations were 0.078 and 0.156 pg/ml for MIC and MBC, respectively. These results indicate that P. intermedia is more susceptible to HICA and TAP than E. faecalis. TAP was found to be more potent than HICA, as it required a lower concentration for inhibition and bactericidal effects. Additionally, SEM analysis showed that HICA inhibited biofilm formation by E. faecalis at MIC value. HICA was also found to be non-cytotoxic at 1 mg/ml. Conclusion: Although TAP is more potent as an intracanal medicament, HICA shows potential as an alternative intracanal medicament to eradicate E. faecalis and P. intermedia in endodontic treatment.