Comparison of antibacterial activity of five root canal filling materials in primary dentition: an in vitro study

INTRODUCTION

Pulpectomy in primary dentition is a root canal treatment for irreversibly infected or necrotic pulp due to tooth decay or trauma, to avoid premature extractions in young children. It consists of debriding the root canal, chemical irrigation, and obturation with a resorb­able paste [1-3]. However, even when the primary tooth receives sufficient chemo-mechanical prep-aration, some of the infected bacteria still remain in the complicated and tortuous anatomy of the primary root canals, despite intense mechanical debridement and abundant irrigation, leading to secondary infection and failure of endodontically treated primary teeth [4, 5]. Therefore, a root canal filling material with optimal antibacterial properties might suppress the leftover bacteria that remain trapped, and increase the success rate [6, 7].
Zinc oxide eugenol (ZOE) in the form of powder and liquid, is the most commonly used root canal filling material for pulpectomy in the primary dentition [8-11]. Eugenol has antibacterial properties; it can denature proteins and impair bacterial metabolism and functioning [12]. However, ZOE is far from ideal. The material has a slow resorption rate, it can irritate the periapical tissues, cause necrosis of the bone and cementum, and lead to alteration of the underlying permanent tooth bud when extruded beyond the apex [9, 10, 13, 14].
Combinations of calcium hydroxide and iodoform (Ca(OH)2/iodoform), such as Calplus (Prevest DenPro, Jammu, India) in pre-mixed syringes, have been recently proposed, showing high clinical and radiographic success rate in clinical studies [15]. Calcium hydroxide has the advantage of eliminating residual mi-cro-organisms and preventing the infiltration of peri-apical exudates towards the root canals due to its alka-line pH [16]. Iodoform has been added because of its antibacterial effect, bio-compatibility, radiopacity, heal-ing properties, and its ability to be resorbed in case of extrusion outside the root canals within 8 weeks [6, 17]. Nevertheless, the main disadvantage of Ca(OH)2/iodoform pastes is an accelerated external resorption rate and a potential intra-canal resorption, which reduces the long-term prognosis of pulpectomy [6, 9].
In an attempt to benefit from the advantages of the above-mentioned materials, EndoflasTM paste (Sanlor Laboratories, Cali, Colombia) combining ZOE with calcium hydroxide and iodoform, has been suggested [9]. Due to its composition, EndoflasTM paste can easily resorb at the same physiological rate with the root. It also possesses a large antibacterial spectrum, demonstrating high success rates in clinical research [1, 9, 15].
In some recent studies, new combinations of exist­ing root canal filling materials for primary teeth are proposed. Zinc oxide eugenol has been combined with propolis, and the results are encouraging [1, 8-11]. Pro­polis is a natural resinous substance derived from plants with healing and anti-inflammatory properties, which can increase cell regeneration [10, 15]. In addition, it has antimicrobial properties, particularly against Gram-posi­tive bacteria, such as Streptococcus mutans [15, 18].
This antibacterial activity is linked to direct activity of bacteria on cellular metabolism, and to stimulation of immune system, leading to the activation of body’s natural defenses [18]. Considering the beneficial prop-erties of propolis, clinical trials have revealed clinical and radiographic successes of the mixture of zinc oxide and propolis (ZOP) as a root canal filling material in primary dentition [10, 11]. Another association of ZOE with sodium fluoride was also suggested. This could lead to the release of fluoride that can be beneficial to the germ of underlying tooth, and may allow a delay in the resorption of material, to coincide with that of the physiological root [9].
Infected root canals of deciduous teeth harbor a polymicrobial microbiome, which plays a role in the progression of pulpal and periapical infections. Higher prevalence of S. mutans and Enterococcus faecalis was revealed by molecular analysis of microbiota in the root canals of primary teeth with pulpal necrosis [19]. E. faecalis is an opportunistic bacterium that can develop resistance to antimicrobials during chemo-mechanical preparation, due to its capacity to create bio-films and ability to lower intra-cytoplasmic pH by operating a proton pump [19]. E. faecalis has a role in the recurrence of peri­radicular lesions in prima-ry teeth following endodontic therapy. Therefore, the choice of a root canal filling material that is effective against both strains of bacteria is crucial.
There is a lack of comparative studies on the antibacterial activity of different combinations of root canal filling materials in the primary dentition against S. mutans and E. faecalis.

OBJECTIVES

The aim of this study was to evaluate in vitro the anti­bacterial activity of five root canal filling materials for primary teeth pulpectomy against S. mutans and E. faecalis: ZOE, ZOE combined with fluoride, ZO with pro­polis, EndoflasTM, and Calplus. The null hypothesis was that there is no significant difference in the antibacterial activity between the five materials.

MATERIAL AND METHODS

After receiving approval from the Ethics Committee of Saint-Joseph University of Beirut (USJ) (ref. No. USJ-2023-178), comparative microbiological study eva­luating the antibacterial activity of ZOE, ZOE combined with fluoride, ZOP, EndoflasTM, and Calplus, was carried out at the Laboratory of Pathogens (LAP) of USJ.

SAMPLE SIZE

To determine the sample size, a power analysis for one-way ANOVA was conducted using G*Power soft-ware v. 3.1.9.7 for Windows (Heinrich Heine, Universitat Düsseldorf, Düsseldorf, Germany). Six groups of samples were necessary to achieve a power of 0.9. Alpha level was set at 0.05 and an effect size of 0.66 was calculated based on a previous study (Navit et al. [2]). Minimum sample size required was 8 samples per group. A sample size of 9 samples per group was used in this study.

EVALUATION OF ANTIBACTERIAL EFFICIENCY OF ROOT CANAL FILLING MATERIALS BY AGAR DIFFUSION METHOD

Antibacterial activity of root canal filling materials against S. mutans and E. faecalis was evaluated by agar medium disk diffusion method in Petri dishes [2].

PREPARATION OF CULTURE MEDIUM

PREPARATION OF S. MUTANS CULTURE MEDIUM

The culture medium for S. mutans was blood agar. In order to prepare them, a powder from Mueller- Hinton Agar (MHA, ref. DM170D, Mast, Bootle, Liver­pool, Merseyside, United Kingdom) was mixed with dis-tilled water, according to the recommendations of the manufacturer, in sufficient quantity; then, it was brought to boil and placed in autoclave (TMJ01-SACV-01, SCI FINETECH Co., Seoul, Korea), to a temperature of 121°C. Subsequently, after cooling to a temperature between 55° and 60° Celsius, 5% blood was added aseptically. After cooling, 20 ml of agar was placed in each Petri dish of 9 centimeters diameter.

PREPARATION OF E. FAECALIS CULTURE MEDIUM

The culture medium for E. faecalis was MHA. In order to prepare them, MHA powder was mixed with dis-tilled water, according to the recommendations of the manufacturer, in sufficient quantity. The mixture was then brought to boil, and placed in autoclave up to a temperature of 121°C. After cooling, 20 ml of agar was placed in each Petri dish.

PREPARATION AND SEEDING OF BACTERIAL STRAINS ON THEIR CULTURE MEDIUM

Three separate manipulations were carried out, with two bacterial strains of E. faecalis and S. mutans in-dependently inoculated on their culture medium at three days intervals. E. faecalis ATCC 29212 and S. mu-tans ATCC 25175 strains were obtained from the USJ Pathogens Laboratory, and cultured in Petri dishes for E. faecalis and blood agar for S. mutans, over the entire surface in three different directions in a homogeneous way, using a sterile cotton swab. Then, Petri dish containing E. faecalis was incubated for 24 hours in an aero-anaerobic atmosphere, and one containing S. mutans in an anae­robic container was incubated for 48 hours at 37°C, with a bag of 5% CO2 (lot No. 0325MJ-4, Mitsubishi Gas Chemical, Japan) in a laboratory oven (model 100-800, Memmert GmbH + Co. KG, Schwabach, Germany). Bacterial suspensions with a density of 0.5 McFarland (3 × 108 CFU/ml) (ref. No. 046457, Biosan Densitometer, Latvia) were prepared, and then inoculated into Petri dishes containing Mueller-Hinton’s medium for E. faecalis and blood agar for S. mutans.

PREPARATION OF WELLS IN PETRI DISHES CONTAINING BACTERIAL STRAINS AND ROOT CANAL FILLING MATERIALS

Six groups were tested in the current study.
Group 1: ZOE (lot No. 20071603, Prevest DenPro, Lewes, DE, United States).
Group 2: EndoflasTM (lot No. 149, Sanlor, Cali, Colombia).
For both groups 1 and 2, the mixture was prepared by gradually adding 0.40 g of powder to 250 μl of eugenol, until obtaining a cone after separation of the spatula from the glass plate.
Group 3: ZOE combined with fluoride; ZOE paste was mixed with 100 μl of 5% fluoride varnish (lot No. A63174, FluoroDose, Centrix Inc., Shelton, CT, United States).
Group 4: ZO combined with propolis (ZOP); 0.40 g of zinc oxide powder was mixed with 250 μl of pure and concentrated propolis (lot No. 33954, Officine Immortelle, Paris, France).
Group 5: Calplus (lot No. PK2223827, Prevest DenPro).
Group 6: Control group composed of saline solution.
Each material was placed in 3 wells for every mani­pulation for both bacterial strains (Figure 1).

MEASUREMENT OF ZONES OF INHIBITION

Diameters of clear zones around the wells indicating inhibition of bacterial growth were measured in mil-limeters (mm) using a caliper and a ruler graduated in millimeters. Absence of clear zones around the disc implied bacterial growth, and indicated absence of antibacterial activity of the root canal filling material.

EVALUATION OF ANTIBACTERIAL ACTIVITY’S KINETICS

In order to evaluate the kinetics of antibacterial activity of root canal filling materials against E. faecalis and S. mutans over time, the strains of E. faecalis and S. mutans were cultured for 24 hours and 48 hours, respec-tively, in Mueller-Hinton agar; then, they were placed in a liquid medium of 5 ml of brain heart infusion (BHI, ref. No. 64014, Bio-Rad, Marnes-la-Coquette, France) for 4 hours, to obtain young cultures in exponential growth phase. Subsequently, each root canal filling product was put into 12-well plates to which, 500 μl of BHI and 500 μl of bacterial growth were added, and then incubated at a temperature of 37°C under constant stirring (Thermo Scientific SHKE4450-1CE, Marietta, OH, United States). For E. faecalis, for each group, samples of 100 μl were taken after 2 hours and introduced into sterile Eppendorf tubes containing 900 μl of sterile distilled water, and serial dilutions from 100 to 104 in distilled water were carried out. 10 μl samples were taken after 24 h and added to 990 μl of sterile distilled water; serial dilutions of 100 to 109 in distilled water were performed.
As for S. mutans, 100 μl samples were taken after 2 hours and introduced into sterile Eppendorf tubes containing 100 μl of sterile distilled water. Serial dilutions of 100 to 104 in distilled water were carried out, followed by 100 μl samples after 24 hours added to 900 μl of sterile distilled water, with serial dilutions of 100 to 104 in distilled water.
The mixtures in Eppendorf tubes were agitated on a Mini Vortex Mixer (DragonLab MX-S, United States) between each dilution, and 100 μl from each tube were removed and placed in Petri dishes containing the MHA medium. Then, they were dispersed using rakes, and incubated at a temperature of 37°C for 24 hours in the laboratory oven, in an aero-anaerobic atmosphere for E. faecalis and anaerobic for S. mutans. Colony counting was performed after 24 hours manually to determine colony forming units per milliliter (CFU/ml). The procedure was triplicated.

STATISTICAL ANALYSIS

Data were analyzed using IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, United States). Means ± standard deviations, medians (inter­quartile ranges), and minimum and maximum values were computed and reported for quantitative variables. Shapiro-Wilk test was employed to evaluate the normality of distribution of quantitative variables. To compare zones of inhibition of S. mutans and E. faecalis between groups, Kruskal-Wallis test was used, followed by Mann-Whitney tests for multiple pair-wise compari­sons. To compare bacterial count within the same time point between groups, one-way ANOVA was utilized, followed by Tukey HSD post-hoc test for pair-wise comparisons. Finally, to compare bacterial count within each group between the two time points (2 h and 24 h), paired t tests were applied whenever possible. The level of significance was set at 5%, and all tests were two-sided.

RESULTS

STATISTICAL COMPARISON OF DIFFERENT MEASUREMENTS OF ZONES OF INHIBITION OBTAINED FOR THE DIFFERENT MATERIALS AGAINST S. MUTANS USING AGAR DIFFUSION IN PETRI DISHES

The measurements of zones of inhibition and counts showed different results for S. mutans among the groups (Figure 1). The highest average was observed for ZOE + fluoride (24.22 mm ± 0.44), followed by EndoflasTM (17.78 mm ± 0.44), ZOP and ZOE (16.56 mm ± 0.53 for both the groups), Calplus (9.22 mm ± 0.44), and finally saline solution (9.00 mm ± 0.00). No statistically sig-nificant difference was observed between ZOE + fluoride and EndoflasTM, and between ZOE and ZOP, ZOE and EndoflasTM, and ZOP and EndoflasTM. Additionally, no statistically significant difference was observed between Calplus and saline. However, statistically significant differences were observed between all other groups and saline as well as between all groups and Calplus (p < 0.001). The different uppercase superscript letters in Figure 1 indicate statistically significant differences between the groups.

STATISTICAL COMPARISON OF DIFFERENT S. MUTANS COUNT OBTAINED AT 2 H AND 24 H USING DIRECT CONTACT TEST IN THE DIFFERENT MATERIALS

As presented in Table 1, a statistically significant increase was observed at 2 hours (1.3333 × 102 CFU/ml ± 5.770) and 24 hours (5.8333 × 103 CFU/ml ± 5.7735 × 102) for the ZOE + fluoride group. At 2 hours, the highest number of bacteria was observed in the positive control group (1.6666 × 106 CFU/ml ± 1.6622 × 102), followed respectively by the Calplus group (4.6166 × 104 CFU/ml ± 9.1150 × 103), ZOE (1.2666 × 103 CFU/ml ± 5.6862 × 102), EndoflasTM (1.6000 × 102 CFU/ml ± 3.4640 × 101), ZOE + fluoride (1.3333 × 102 CFU/ml ± 5.770), and finally the ZOP group (7.3330 × 101 CFU/ml ± 2.0820 × 101). Moreover, statistically significant differences were observed between the positive control and all other groups (p < 0.001). Calplus showed a significantly higher number of CFU/ml than ZOE, ZOE + fluoride, ZOP, and EndoflasTM. No statistically significant difference was observed between ZOE, ZOP, EndoflasTM, and ZOE + fluoride.
As presented in Table 1, at 24 hours, the highest number of bacteria was observed in the positive control group (1.6666 × 106 CFU/ml ± 1.6622 × 102), followed respectively by the Calplus group (6.2333 × 104 CFU/ml ± 3.3080 × 104), ZOE (9.8333 × 103 CFU/ml ± 6.6395 × 103), ZOE + fluoride (5.8333 × 103 CFU/ml ± 5.7735 × 102), EndoflasTM (4.0000 × 103 CFU/ml ± 2.5980 × 103), and finally the ZOP group (1.6667 × 102 CFU/ml ± 2.8867 × 102). Statistically significant diffe­rences were found between the positive control and all other groups (p < 0.001). Calplus showed a significantly higher number of bacteria count than ZOE, ZOE + fluoride, ZOP, and EndoflasTM. However, no statistically significant difference was observed between ZOE, ZOP, EndoflasTM, and ZOE + fluoride. The different uppercase superscript letters in Table 1 indicate statistically significant differences between the groups within each time point, i.e., 2 hours and 24 hours.

STATISTICAL COMPARISON OF DIFFERENT MEASUREMENTS OF ZONES OF INHIBITION OBTAINED FOR THE DIFFERENT MATERIALS USED AGAINST E. FAECALIS WITH AGAR DIFFUSION IN PETRI DISHES

The measurements of zones of inhibition and counts showed different results for E. faecalis among the groups (Figure 2). The highest mean diameter of inhibition was observed for EndoflasTM (18.78 mm ± 0.67), followed by ZOP (13.22 mm ± 0.44), ZOE (13.22 mm ± 0.44), ZOE + fluoride (13.22 mm ± 0.44), Calplus (9.22 mm ± 0.44), and finally saline solution (9.22 mm ± 0.44). No statistically significant difference was not-ed between ZOE, ZOE + fluoride, and ZOP (p > 0.05). However, statistically significant differences were found between Calplus and all other groups, except for saline. Statistically significant differences were noted be-tween EndoflasTM and Calplus, ZOE, ZOP, and ZOE + fluoride (p < 0.001). The different uppercase superscript letters in Figure 2 indicate statistically significant differences between the groups.

STATISTICAL COMPARISON OF DIFFERENT E. FAECALIS COUNTS OBTAINED AT 2 H AND 24 H USING DIRECT CONTACT TEST WITH THE DIFFERENT MATERIALS

As presented in Table 2, at 2 hours, ZOE (7.3333 × 104 CFU/ml ± 9.6006 × 104), ZOE + fluoride (1.6000 × 104 CFU/ml ± 1.5000 × 103), EndoflasTM (1.3000 × 104 CFU/ml ± 5.7662 × 103), and ZOP (1.1333 × 104 CFU/ml ± 3.8837 × 103) demonstrated a significantly lower number of bacteria than Calplus (4.0750 × 106 CFU/ml ± 4.2500 × 105) and the positive control group (1.6666 × 106 CFU/ml ± 1.6622 × 102). No statistically significant difference was noted between ZOE, ZOE + fluoride, ZOP, and EndoflasTM. However, a significant difference was observed between positive control and Calplus. At 24 hours, ZOE, ZOE + fluoride, ZOP, and EndoflasTM killed all bacteria present in the medium (00.0000 CFU/ml ± 00.0000). As for Calplus (1.4975 × 108 CFU/ml ± 1.0740 × 107), no significant difference was observed with positive control (3.3867 × 1011 CFU/ml ± 2.5015 × 1011). The different uppercase superscript letters in Table 2 indicate statistically significant diffe­rences between the groups within each time point, i.e., 2 h and 24 h.

DISCUSSION

The complex root canal morphology of deciduous teeth makes effective cleaning by mechanical instru-mentation and chemical irrigation challenging and insufficient [10]. Moreover, the root canal obturation relies entirely on a resorbable filling paste; therefore, choosing a material with optimal antibacterial properties is crucial for a better prognosis. Among the materials available on the market, there is ZOE, calcium hydroxide and iodoform, and EndoflasTM, which is a combination of ZOE, calcium hydroxide, and iodoform. Since the drawbacks and limitations of each material are well-documented, the search for new materials is still ongoing.
In the current study, antibacterial properties were evaluated against two frequent intra-canal bacteria in canal infections, i.e., E. faecalis and S. mutans [2, 20-23].
E. faecalis plays a major role in the recurrence of lesions in primary teeth following endodontic therapy. Den-tal caries are the precursors of endodontic infections, and S. mutans is one of the main bacterial species asso-ciated with caries. Also, S. mutans is found in high numbers in chronic endodontic infections, including ab-scesses [20-22]. This was first carried out by the agar disc diffusion me­thod, and then by using a direct con-tact test [2, 12, 24].
The results of this study showed significant diffe­rences between the different groups regarding their an-tibacterial activity against E. faecalis and S. mutans (p < 0.05). Therefore, the null hypothesis was rejected.
For E. faecalis in the disc diffusion method, the best antibacterial activity was observed for EndoflasTM, followed by ZOP, ZOE, and ZOE + fluoride (having the same disc diameter), Calplus, and finally saline solution. Additionally, EndoflasTM demonstrated the best antibacterial activity in Navit et al. [2] and Ibrahim et al. [25]studies.
The composition of EndoflasTM may account for its potent antibacterial effects [26]. The eugenol compo-nent induces protein denaturation and disrupts bacterial metabolism [12]. Iodoform works by releasing io-dine, which can oxidize components and inactivate vital metabolic compounds, such as proteins, nucleotides, and fatty acids, leading to cell death [17]. Lastly, calcium hydroxide eradicates the remaining micro-organisms due to its high pH of 12, and helps prevent the infiltration of peri-apical exudates into the root canals [16]. Calplus and saline were not significantly dif-ferent (p > 0.05). By evaluating the kinetics at 2-time intervals, starting from 2 hours already, ZOE, ZOE + fluo-ride, EndoflasTM, and ZOP, presented a significantly higher antibacterial activity than Calplus, followed by the positive control group (p < 0.05). At 24 hours, ZOE, ZOE + fluoride, ZOP, and EndoflasTM killed all the bacteria present in the medium, while Calplus had no statistically significant antibacterial effect on E. faecalis. This result suggests that the addition of fluoride to ZOE brings only an extra cost to the material, since its absence presented the same antibacterial activity against E. faecalis.
Furthermore, the substitution of eugenol by propolis does not seem to affect the antibacterial properties of ZOE. This is in line with previous randomized clinical trial by Al-Ostwani et al. [11], where ZOP showed clin-ical and radiographic success after 6 months and 1 year post-operatively, similar to ZOE. However, ZOP demonstrated higher clinical and radiographic success than ZOE after 2 years follow-up, by 95% and 70%, respectively, in a study by RojaRamya [10]. The antibacterial effects of propolis are attributed to both its action on bacteria and its stimulation of the immune system, which activates the body’s natural defenses. It is particularly effective against Gram-positive bacteria, because in Gram-negative bacteria, the outer membrane and the production of hydrolytic enzymes break down the active components of propolis, thus diminishing its efficacy [18, 27]. Calplus was not effective against E. faecalis, and was the only material that did not show statistically signifi-cant antibacterial activity after 24 hours. Its action is therefore very limited and no longer effective after a few hours. These results are in agreement with previous study by Navit et al. [2], where Metapex, which is also a paste of calcium hydroxide combined with iodoform, showed minimal inhibitory antibacterial action com-pared with EndoflasTM, ZOE, and the combination of calcium hydroxide with chlorhexidine. The same tendency was confirmed in a study of El Hachem et al. [12], where Calplus had the least antibacterial activity with an elimination of 50% of bacterial colonies of E. faecalis when compared with ZOE (100%) and Bio-C Pulpecto (Angelus, Brazil) (75%). Calcium hydroxide in Calplus changes into calcium and hydroxyl ions in an aqueous solution, which increases alkalinity of the environment. However, its alkalinity decreases after 1 week, making it ineffective against E. faecalis [28]. The limited antibacterial effect can also explain why in recent guidelines; non-setting oil-based calcium hydroxide pastes containing iodoform are now being the preferred materials for teeth, and are expected to remain in the mouth for no longer than 18 months [6].
In this study, the substitution of eugenol with propolis in ZOE did not reduce its antibacterial efficacy. In addition, ZOP showed the same antibacterial efficacy as EndoflasTM, a product containing eugenol. Further-more, Calplus demonstrated a significantly higher number of S. mutans compared with other groups, which was confirmed by Pimenta et al. [29] and Hegde et al. [26], where calcium hydroxide had minimal or no anti-bacterial activity against S. mutans compared with that of ZOE and EndoflasTM. It is possible that the elevated pH of calcium hydroxide was neutralized by the buffers in the medium in vitro, which may also be present in vivo [2]. Its effect, when used alone with iodoform, is insufficient and requires the presence of zinc oxide eu-genol, as confirmed by the action of EndoflasTM.
In this study, the antibacterial effect of the filling materials was tested against E. faecalis and S. mutans, using direct contact tests and agar diffusions. However, dental infections involve bio-films, which are commu-nities of aggregated bacteria, coated in secreted micro- colonies, and adhering to inert or biological surface [30]. Several bacteria are involved in endodontic infec-tions, such as Lactobacillus, Streptococcus, Propionibacterium, and E. faecalis in recurrent periradicular le-sions after endodontic treatment or untreated canals [2, 19, 31-35]. E. faecalis is an anaerobic Gram-positive coccus that presents a high resistance to disinfection and antimicrobials, including chlorhexidine, sodium hypochlorite, and calcium hydroxide. It can penetrate far into the dentinal tubules, which also provides pro-tection against antiseptic measures [2, 33, 36]. It has also the ability to grow whether as a bio-film on the walls of root canals or alone. Finally, E. faecalis is able to retain in environments without sufficient nutri-ent supply, and with high alkaline pH of 11.5 [33].
In an attempt to search for new materials with limited side-effects, different combinations were tested. One of them is a triantibiotic mixture that contains ciprofloxacin, metronidazole, and minocycline; it is effec-tive against many micro-organisms, such as E. faecalis [37]. Moreover, several studies have shown 100% re-duction in E. faecalis growth using 2% chlorhexidine gel as an intra- canal medicament. On another note, aloe vera has shown anti-inflammatory, antibacterial, and hypoglycemic ef­fects, due to its vitamins, enzymes, minerals, amino acids, salicylic acid, lignin, and saponin [38]. Also, a 2.5% triclosan was tested, and incorporated into ZOE and EndoflasTM by Deepak et al. [39]. It increased the antibacterial effect of both bio-materials, with lasting an-ti­microbial activity. However, according to some studies, it may demonstrate secondary effects, such as chronic dermal toxicity and carcinogenic effect [40]. In Al-Quraine et al. [41] and Mukorera et al. [42] studies, EndoRez, a metha­crylate-based root canal sealer, was assessed against several micro-organisms, such as E. faecalis and Staphylo­coccus aureus, but showed minimal antibacterial activity.
The antibacterial efficacy of bio-materials was eva­luated in vitro against two bacteria, which surely are the main bacteria involved in endodontic infections, but there is still a large number of other bacteria with infectious potential that should be tested. This research showed that certain filling materials currently availa-ble on the market have strong antibacterial properties against E. faecalis and S. mutans, such as EndoflasTM and ZOE, while ZOP with comparable antibacterial effects and lower toxicity is still not readily available. In subsequent studies, it would therefore be interesting to evaluate its antibacterial properties against other bacteria involved in endodontic infections using different methods, such as confocal laser scanning microsco-py and transmission electron microscope [43, 44] as well as physico-chemical properties, such as pH, solu-bility, the rate of root resorption, adhesion to the canal walls, radiopacity, etc., to confirm the results.

CONCLUSIONS

The current study highlights the need for further research to identify an optimal root canal filling material for primary dentition. The addition of fluoride to ZOE did not demonstrate significant advantages. Replacing eugenol, known for its periapical toxicity, with propolis, a natural extract with comparable antibacterial effi-cacy against E. faecalis and S. mutans, shows potential. However, further studies investigating physi-co-chemical properties of these materials are required to optimize their clinical application, and to determine the most effective combination.

DISCLOSURES

1. Institutional review board statement: This study was approved by the Ethics Committee of the Saint- Joseph University of Beirut (ref. No. USJ-2023-178).
2. Assistance with the article: The authors would like to express their gratitude to the staff of LAP in USJ, for their administrative and technical support as well as for ATCC bacterial strains and microbiological sup-plements.
3. Financial support and sponsorship: None.
4. Conflicts of interest: The authors declare no potential conflicts of interest concerning the research, au-thorship, and/or publication of this article.

References