EFFECTIVENESS OF CHITOSAN-FLAVONOIDS (PROPOLIS EXTRACT) NANOFORMULATIONS AGAINST ENTEROCOCCUS FAECALIS BIOFILM IN THE ROOT CANAL: EX VIVO STUDY

Abstract

Background The complexity and morphological variation of root canal system along with the nature of biofilm make root canal disinfection extremely challenging. Bacteria exhibit increased virulent phenotypes when growing inside biofilms than in planktonic forms. Within a biofilm, wide variety of bacteria are found forming a multi-species community however, Enterococcus faecalis is one of the most persistent organisms that causes intra-radicular infections. Therefore, the outcome of endodontic treatment depends on controlling the microbial biofilm by instrumentation and disinfecting the root canals using various intracanal medicaments and endodontic irrigants. Rationale A number of antibiotics and surfactants are widely used in the treatment of biofilms and the current trend is towards identification of natural products in disinfection. Propolis, a natural product has been proven to have high antibacterial property due to presence of flavonoids. Similarly, chitosan due to its low toxicity and bio-adhesive properties and the use of nanoparticles due to their antibacterial activity have also gained popularity. Therefore, nanoformulations with natural products like chitosan-propolis extract may offer a broader potential for effective therapy. Hence, the aim of this research was to determine the antibacterial effectiveness of chitosan-propolis nanoparticles as intracanal medicaments and propolis nanoparticles as endodontic irrigants against E. faecalis biofilm in root canal therapy. Materials and Methods The antibacterial effectiveness of chitosan propolis nanoparticles intracanal medicament and propolis nanoparticles as endodontic irrigant was tested against E. faecalis in human tooth model. For testing the effectiveness of intracanal medicament, 240 extracted human maxillary anterior teeth were sectioned below the cemento-enamel junction and the apical third of the root to obtain 6 mm of the middle third of the root. Thereafter, the root canal was enlarged to an internal diameter of 0.9 mm using Pesso Reamer size No.2, irrigated and autoclaved. The specimens were inoculated with E. faecalis for a period of 21 days to create a biofilm. Following this, specimens were randomly divided into eight groups having 30 dentinal blocks in each group including group I: saline, group II: chitosan, group III: propolis 100 μg/ml (P100), group IV: propolis 250 μg/ml (P250), group V: chitosanpropolis nanoparticles 100 μg/ml (CPN100), group VI: chitosan-propolis nanoparticle 250 μg/ml, group (CPN250) VII: calcium hydroxide (CH) and group VIII: 2% chlorhexidine gel (CHX). Dentine shavings were collected at 200 and 400 μm of depth, and total number of colony forming units (CFUs) were determined at the end of day one, three, and seven. Additionally, scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) were used for qualitative assessment of antibacterial effectiveness. The effectiveness of CPNs were also evaluated against E. faecalis isolated obtained from patients having failed root canal treatment. To evaluate antibacterial effectiveness of propolis nanoparticles as an endodontic irrigant, a similar methodology as described for intracanal medicament testing was followed to obtain 210 dentine blocks to test the effectiveness of propolis nanoparticles as endodontic irrigant. The dentine blocks were randomly divided into seven groups having 30 blocks in each group including group I: saline, group II: propolis 100 μg/ml (P100), group III: propolis 300 μg/ml (P300), group IV: propolis nanoparticle 100 μg/ml (PN100), group V: propolis nanoparticle 300 μg/ml (PN300), group VI: 6% sodium hypochlorite (NaOCl),group VII: 2% chlorhexidine (CHX). Dentine shavings were collected at 200 and 400 μm of depth and total numbers of CFUs were determined at the end of one minute, five, and ten minutes. The qualitative assessment of antibacterial effectiveness was conducted using SEM and CLSM. The effectiveness of PNs were also evaluated against E. faecalis isolated obtained from patients having failed root canal treatment. The antibacterial effectiveness was statistically tested by comparing mean differences in reduction of CFUs between all groups using non-parametric Kruskal Wallis and Mann- Whitney tests with probability value set at p < 0.05. Results On statistical analysis, the mean reduction in CFUs was found to be significant (p<0.05) in all the groups when compared to the saline group at all times and all depths. On day one and day three, at 200 and 400 μm depths of the dentinal tubules,CPN250 showed significant mean reduction of CFUs (p<0.05) when compared to all other groups. Conclusion CPN250 was the most effective in reducing E. faecalis colonies on day one and three at 200 and 400 μm dentinal tubule depths, while at day seven, CPN250 was equally effective as CPN100 and 2% CHX. Therefore, CPN250 can be proposed as a potential intra-canal medicament to be used in future. PN300 was equally effective as 6% NaOCl and 2% CHX in reducing E. faecalis colonies after one, five and ten minutes at 200 and 400 μm of depths. These findings suggested PN300 can be considered as an alternate endodontic irrigant. Key words Dentinal tubule disinfection, Enterococcus faecalis, intracanal medicaments, chitosanpropolis nanoparticles, endodontic irrigants, propolis nanoparticles