Anti-bacterial activity of applying chitosan and propolis dressing against Enterococcus faecalis in primary teeth: in vitro study

Introduction

Primary teeth with pulpal and periapical problems should be retained until their normal time of physiologic exfoliation to reserve chewing, pronunciation, aesthetics, and eruption of permanent teeth [1, 2]. Therefore, preserving primary teeth by performing endodontic treatment is necessary [3]. This procedure is considered a major thera¬peutic challenge in pediatric dentistry [4] because of internal complex nature of the root canal system in primary teeth that makes it difficult to achieve proper disinfection by bio-mechanical preparation of root canal [1].
One of the main keys in successful root canal treatment is disinfecting the root canal system prior to application of final filling material [5]. Although most bacterial species are eliminated after mechanical instrumentation and irrigation of canals, some of them may stay active leading to undesirable complications of the treated root canals, including periapical or root bifurcation lesions, root resorption, and alveolar abscess [2, 6, 7]. However, these microbial organisms may persist even after bio-mechanical preparation; hence, the use of intra-canal medicaments is necessary [8].
Most commonly used materials are calcium hydro¬xide Ca(OH)2-based materials [5]. The anti-microbial activity of Ca(OH)2 is related to a release of these hydro¬xyl ions, damaging cytoplasmic membranes and DNA of microbial cells [9].
Chitosan is a natural bio-material, derived mainly from chitin through the process of deacetylation. The main source of chitin is the exo-skeleton of shrimp and crab.
Chitosan has been recognized as an anti-microbial agent, with a broad anti-bacterial spectrum covering Gram-negative and Gram-positive bacteria as well as fungi [10-12]. In addition, it has bio-compatibility, non-toxicity, anti-inflammatory, and wound healing properties [11]. Therefore, chitosan has been used in a bio-dental application [12].
Several studies have shown the efficacy of chitosan in removing the smear layer from the root canal [13]. Moreover, chitosan was found superior to EDTA 17% in removing smear layer in the apical third of root canal [14]. The effectiveness of chitosan in reducing bacterial counts of Enterococcus faecalis when used as a root canal irrigation in permanent teeth, was also evaluated [15], and a significant increase in this efficacy when it was combined with chlorhexidine was observed.
Propolis or bee glue is a natural product, collected from plant by honeybees. It has many biologic activities, including anti-bacterial, anti-inflammatory, anti-oxidant, and healing properties [16, 17]. The main chemical composition of propolis are phenolics and flavonoids; they present anti-oxidant, anti-bacterial, anti-fungal, anti-viral, and anti-inflammatory proprieties [18].
Due to its’ anti-bacterial properties, propolis has been used in a wide range of medical and dental applications as root canal irrigation [19], in direct and indirect pulp capping [18], reduction of dentin hypersensitivity [17], and in preventing dental caries [20], in addition to a storage media for avulsed teeth [21].
Several studies have investigated the microflora in root canal infections. Enterococcus faecalis are a selective anaerobic Gram-positive cocci [7]. They are the most common species that has been detected in the root canal [22, 23], and their ability to invade dentinal tubules may be attributed to their high resistance to alkalinity [7].
Chitosan has been suggested for root canal irrigation in both primary and permanent teeth by several in vitro studies [13, 15, 24]. On the other hand, propolis has shown various anti-bacterial activity against Enterococcus faecalis [25, 26]. However, the combination of chitosan and propolis was not assessed as dressing materials in primary teeth.

Objectives

This study was designed to evaluate the synergistic efficacy of the combination of chitosan and propolis against Enterococcus faecalis in primary root canal as intra-canal medicament.

Material and methods

Study sample

Study sample included 72 primary second molars extracted for orthodontic reasons, with two third of their roots staying undamaged without any physiological or pathological resorption, and they were not subjected to any treatment before extraction. Teeth were stored in sterile saline until using in the study. Samples were distributed randomly into 3 groups, including chitosan + propolis, calcium hydroxide, and saline.

Chitosan and propolis dressing preparation

Chitosan (≥ 75% deacetylated) was acquired from Sigma-Aldrich Chemicals, Germany, and propolis was obtained from Sakka Amini Company, Syria.
2 g of chitosan was dissolved in 100 ml of acetic acid (2%), with continuous stirring for 24 hours to form chitosan 2% [27, 28]. 5 g of crude propolis was dissolved in ethyl alcohol (80%) in a ratio of 1 : 15, and then the solution was evaporated under reduced pressure, using a rotary evaporator (at 40-45°C); it was then filtered using filtering paper (Whatman paper) [29]. 1.5 g of ethanol extract of propolis (EEP) was mixed with the resulting 2% chitosan solution (CH) using constant stirring. 1 g of sodium alginate 1% was added until reached the required thickness. The mixture was steamed on Petri dishes in an oven at a temperature of 37°C until all water evaporation.

Teeth samples preparation

Teeth were cleaned with a gentle curettage to remove residual periodontal ligament, and scaled to remove any calculus. An access opening was prepared using a high-speed diamond bur, with working length 1 mm shorter than the root apex, and the root canals were prepared to size 30 K-File. 10 ml of a 2.5% sodium hypochlorite solution was used to irrigate the root canals during canal preparation. Apical foramen was sealed with flowable composite resin, and the teeth were placed in an acrylic resin block to allow handling the teeth during the experiment. Teeth were sterilized using moist heat at 121°C for 30 minutes, using a pressure of 15 pounds (lb.) [30].

Preparing bacterial suspension

Enterococcus faecalis was isolated clinically from the necrotic pulps of patients treated in the Department of Pediatric Dentistry, Damascus University, and the phenotype was determined using BD PHOENIX™ M100 (BD Company, USA). Enterococcus faecalis was cultured on Petri dishes with nutrient medium (Mueller-Hinton agar) at 37°C for 24 hours, in a 5% CO2 incubator. Bacterial suspension was formed and adjusted to 4.5 × 108 colony-forming units per ml (No. 1.5 McFarland Standard), using a BD PhoenixSpec™ nephelometer (BD Company, USA). 50 μl of bacterial suspension 4.5 × 108 (CFU/ml) was applied into the prepared root canals using a sterile micropipette [30]. The suspension was activated into the canal using a sterile file size 20 for the same period for each sample. All samples were incubated at 37°C for 24 hours under aerobic conditions [30].

Experimental procedure

Samples were divided randomly into 3 groups to apply the tested materials. Randomization was performed using an online service (www.randomization.com).
Group A: CH + EEP (n = 24): 2% chitosan + 10% ethanol propolis extract dressing was applied using a sterile syringe with disposable plastic needles to inject the dressing into the root canal.
Group B: Positive control group Ca(OH)2 (n = 24): calcium hydroxide dressing was applied using lentulo spirals at low speed after mixed with sterile saline solution in a volume ratio of 1 : 1.
Group C: Negative control group (n = 24): samples without any dressing in root canals application.
Each canal was filled with its’ dressing before sealing the access cavity with glass ionomer cement (Fuji IX GP, GC Corporation, Tokyo, Japan). Finally, specimens were incubated at 37°C/CO2 in 5% environment.

Evaluation of anti-bacterial effect

The bacterial colonies were counted after 24 hours, 72 hours, and 7 days of dressing application, as the study groups were divided into 3 sub-groups according to the three periods studied:
• 24 hours (n = 24): (A1 = 8, B1 = 8, C1 = 8),
• 72 hours (n = 24): (A2 = 8, B2 = 8, C2 = 8),
• 7 days (n = 24): (A3 = 8, B3 = 8, C3 = 8).
All the root canals were rinsed with 1 ml of sterile saline, and the solution was left inside the canals with a peri-pheral filing using a sterile H file for a minute. Then, a paper point was inserted over the entire length of the canal for 60 seconds to absorb the content of the canal, whereas the saline solution acted as a medium to transfer the bacteria to agar plate. The paper point was transferred to a sterile tube containing 2 ml of sterile saline, and the swab was repeated three times for each root canal to get a true microbial reality of the canal. The tubes, which contained the paper points were shaken using a vortex device for one minute to ensure homogeneity of the solution. Then, 20 μl of the solution was taken by a sterile micro¬pipette and inoculated on blood agar plates, and these plates were incubated with the same conditions as before for 24 hours (Figures 1-3). Bacterial colonies in colony forming units/ml (CFU/ml) were calculated, and were converted into logarithmic numbers to facilitate statistical analysis [30-32].

Results

The distribution of bacterial counts after disinfection procedures is presented in Table 1 and Figure 4. Differences between groups were analyzed using one-way ANOVA test. P-value less than 0.05 was considered statistically significant, and Bonferroni test was used to analyze differences between binary groups (Table 2). Wilcoxon test was used to evaluate the bilateral differences in the mean bacterial colony units between two stages before and after 24 hours, 72 hours, and 7 days of dressing application (Table 3). Statistical calculations were performed with SPSS Statistics for Windows software. As expected, the negative control group revealed the highest number of bacteria (mean, 3.927 ± 0.936). Chitosan + propolis exhibited the highest anti-bacterial effect (1.997 ± 0.515) after 24 hours of applying the dressing material, with statistically significant difference (p < 0.05) compared to calcium hydroxide group and saline group (3.408 ± 0.289 and 3.927 ± 0.936, respectively). Calcium hydroxide showed higher anti-bacterial effect than saline, with no statistically significant difference (p > 0.05). After 72 hours and 1 week, chitosan + propolis and calcium hydroxide demonstrated similar anti-bacterial effects, with no statistically significant difference (p > 0.05). Both showed statistically significant difference compared to saline (p < 0.05) (Table 2).
Table 1 and Figure 5 showed the average percentage of the bacterial decrease of each dressing in study groups.

Discussion

An ideal intra-canal medicament should exhibit maximum anti-bacterial and minimum toxic effects. Nowadays, calcium hydroxide is the most commonly used intra-canal medicament in endodontic practice. Moreover, it is clear that the anti-bacterial effect of calcium hydroxide is related to a release of hydroxyl ions in aqueous environment, which in turn affect the bacterial cells. However, there are bacterial strains that are resistant to calcium hydroxide alkalinity. Enterococcus faecalis is the most common bacteria detected in secondary infections, even in primary infections in permanent [7] and primary teeth [23, 33].
The anti-bacterial efficacy against Enterococcus faeca¬lis was preferred in this study as long as it was the most persistent bacteria strains of infected root canal; therefore, if the studied materials were effective against this type of bacteria, it would be effective against other species of bacteria in the root canal. The present study attempted to determine the effective duration of all intra-canal medications tested, as they were verified in three different time periods since the anti-bacterial effect is time-dependent, which is considered beneficial in clinical practice of root canal disinfection during endodontic treatment [34, 35].
The results showed reduction in the bacterial count after applying the dressings compared to the bacterial count before, with statistically significant difference in both study groups chitosan + propolis and Ca(OH)2 as well as during the three time periods studied.
Chitosan and propolis dressing showed the highest anti-bacterial efficacy 24 hours after applying the dressing. The anti-bacterial activity of chitosan is due to inter¬action of positively charged amino group in chitosan (exhibiting a polycationic nature), with negative charges of the bacterial cell wall resulting in a leakage of intra-cellular components that impart bacterio-static activity [10, 12].
Propolis also affects the bacterial cell wall, causing structural and functional damage [36]. Ethanolic extracts of propolis showed high anti-bacterial activity against anaerobic bacteria, including Enterococcus faecalis [25] and anti-bacterial efficacy of propolis extracts due to the presence of flavonoids content [37]. There is a high concentration of artepillin C in ethanolic extracts of pro¬polis [38], which is one of the numerous phenolic compounds found in propolis. It has a bacterio-static activity with membrane bulging, in addition to cinnamic acid and pinocembrin, which present similar anti-microbial activity against Enterococcus faecalis [37].
After 72 hours and 1 week, calcium hydroxide showed high anti-bacterial effect similarly to chitosan and pro-polis medicament.
This may be attributed to the fact that the anti-microbial efficacy of the calcium hydroxide dressing depends mainly on releasing hydroxyl ions and raising the level of pH, as studies reported that calcium hydroxide shows pH variation ranging from 8.1 to 11.8 after 72 hours of dressing application [39]. Bystrom et al. reported that Enterococcus faecalis is a resistant bacterium in root canals, surviving at pH 11.5. Therefore, a high pH level is desired to eliminate the bacteria [40].
These results are in accordance with Parolia et al. study, where calcium hydroxide was found to be less effective than chitosan and propolis nano-particle and 2% chlorhexidine on day one and day three. However, it was as effective as chitosan and propolis nano-particle on seventh day of application [35].
On the other hand, significant reduction in the number of bacterial colonies was found in the group A (chitosan + propolis) on the first, third, and seventh day after medicament application compared to the base counts of bacterial colonies, where the effectiveness of chitosan and propolis was highest one week after application, with significant differences compared to its’ effectiveness 24 and 72 hours after application. This is due to the fact that chitosan acts as drug delivery that indicate continuous active release of components carried by chitosan even after prolonged times, therefore it is possible that chitosan resulted in a continuous release of propolis throughout the period of dressing application, which resulted in increased anti-bacterial efficacy. In a study by Balata et al., chitosan chips containing propolis extract showed an initial release of 41.7% of loaded propolis, followed by continuous release up to 7 days [41].
Del Carpio-Perochena et al. also found that combining chitosan with calcium hydroxide dressing has increased chitosan anti-bacterial activity in short- and long-term exposure (day 7 and 14), and restrained bacterial re-colonization in root canal after endodontic treatment [42]. However, the bacterial inoculation in the present study was performed at a concentration of 4.5 × 108 cell/ml, which was stronger than that used by Del Carpio-Perochena et al.
Cationic nature of chitosan, which react with negative charge of bacteria cells wall, bio-compatibility, compatibility to blend with other materials, and the potential use of chitosan as oral delivery drug, are the main factors to explain the findings mentioned previously. However, further studies are necessary to validate the anti-bacterial behavior of EEP and chitosan in in-vivo conditions.

Conclusions

Our study showed that the combined application of chitosan and ethanol extract of propolis had a potential anti-bacterial activity against Enterococcus faecalis in primary teeth as intra-canal medication.

Conflict of interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References