Evaluation of Calcium Hydroxide Nanoparticles on the Anti-osteoclastic Activity and Antimicrobial Activity Against Endodontopathogenic Microorganisms in Mixed-species Biofilm Model

Abstract

Objective: To evaluate the efficacy of calcium hydroxide nanoparticles (Ca(OH)2 NPs) in eliminating mixed-species biofilms composed of E. faecalis, S. gordonii and C. albicans. both in an in vitro and an ex vivo tooth model and evaluate the inhibitory effect of Ca(OH)2 NPs on osteoclast activity, as opposed to conventional Ca(OH)2. Method: Firstly, antimicrobial activity of Ca(OH)2 NPs was determined by the Alamar Blue assay to find the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) was assessed by colony counting on agar. Then, mixed-species biofilm of E. faecalis, S. gordonii and C. albicans (1:1:1) were inoculated in root dentin blocks for 21 days. The teeth were then randomly treated with different medicaments (each n=10): (i) Ca(OH)2 NPs, (ii) conventional Ca(OH)2 (Metapaste®) (iii) chlorhexidine (iv) normal saline. After 7 days of medication, dentine chips from the root canal were collected and the viable cell count was determined using a colony-forming unit assessment. The morphological structures of each group were analysed by using scanning electron microscopy (SEM). Secondly, to evaluate the anti-osteoclastic activity. The osteoclast differentiation was evaluated by Tartrate-resistant acid phosphatase (TRAP) activity. RAW 264.7 cells third – fifth passage were cultured in triplicated with soluble receptor activator of nuclear factor‐κB ligand (RANKL) in different conditions: (i) media (control) (ii) Metapaste® (iii) Ca(OH)2 NPs for 7 days. TRAP-positive cells with more than three nuclei were defined as mature osteoclasts. Quantification of mature osteoclasts was performed in each whole well. The cytotoxic effect of medicaments was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: An in vitro assay revealed that the MBIC of Ca(OH)2 NPs and conventional Ca(OH)2 was observed at 1:128 of primary concentration. At a 1:16 ratio of primary concentration to the given product concentration, Ca(OH)2 NPs efficiently inhibited polymicrobial biofilms compared to chlorhexidine. Additionally, it was observed that the lowest concentration of Ca(OH)2 NPs was able to eliminate all viable biofilm, as confirmed on the agar plate at a dilution of 1:16, while the MBEC of Ca(OH)2 required at least 1:8 concentration, showing no growth. Ex vivo analysis revealed the lowest viable cell count was recorded with chlorhexidine, followed by Ca(OH)2 NPs, Ca(OH)2 and normal saline..The group treated with Ca(OH)2 NPs showed a significant reduction in viable cell count compared to the group treated with Ca(OH)2 (p < 0.05). However, this reduction was not statistically significant compared to the group treated with chlorhexidine. Disruption of biofilm structure and clumps of debris were observed in Ca(OH)2 NPs by SEM. Furthermore, Ca(OH)2 NPs exhibited an inhibitory effect on osteoclast differentiation, resulting in a reduction in the size and also number of osteoclasts however non-statistically significant. Additionally, MTT analysis revealed that the Ca(OH)2 NPs group did not exhibit cytotoxic effects on RAW 264.7 cell viability. Conclusions: Ca(OH)2 NPs had the potential for antimicrobial activity in disrupting and eliminating mixed-species biofilms composed of E. faecalis, S. gordonii and C. albicans in both in vitro and ex vivo. Additionally, Ca(OH)2 NPs inhibited osteoclast differentiation, thus advocating for their use to enhance the success of root canal treatment. Ca(OH)2 NPs had potential to be used as an alternative intracanal medicament in cases of external inflammatory root resorption with persistent root canal infection.