Comparison of Antimicrobial Effects of 445 and 970 nm Diode Laser Irradiation with Photodynamic Therapy and Triple Antibiotic Paste on Enterococcus faecalis in the Root Canal: an In Vitro Study

Abstract

Background and aim: Colonization of residual organisms in the root canal are major causes of root canal treatment failure. Therefore, the effective removal of organisms during root canal cleaning stages is of great importance. In this study, we aimed to investigate and compare the antimicrobial effects of several methods including high power laser irradiation, photodynamic therapy and triple antibiotic paste (TAP) on Enterococcus faecalis in the root canal.

Materials and methods: For the present laboratory study, 80 anterior single canal teeth were randomly divided into seven experimental groups (970 nm diode laser irradiation, 445 nm diode laser irradiation, 660 nm photodynamic therapy with doxycycline as a photosensitizer, TAP, 970 nm diode laser combined with TAP, 445 nm diode laser combined with TAP and sodium hypochlorite groups), each containing 10 teeth, and two positive and negative control groups, each containing five teeth. Afterwards, an E. faecalis suspension was prepared and injected into the root canals of all groups (except the negative control group), and after using the desired lasers or drugs and incubating for the necessary time, the grown colonies were counted and significance level of less than 0.05 was considered.

Results:Among seven experimental groups, in those that included triple antibiotic paste (TAP, 970 nm diode laser with TAP, and 445 nm diode laser with TAP groups), no bacteria grew, while the 970 nm and 445 nm diode laser groups had the highest bacterial growth. Statistically, all studied groups were effective, and the results showed a statistically significant difference between groups (P value < 0.05).

Conclusion: The findings of the present study show that all of the above-mentioned methods were effective enough for bacterial reduction. Triple antibiotic paste was proved to achieve complete bacterial elimination. Photodynamic therapy with doxycycline as a photosensitizer was shown to provide significant results of bacterial reduction and diode laser irradiation at both wavelengths (970 nm and 445 nm) were also reported to have antibacterial effect, although slightly lower than the previous methods.

Keywords:Enterococcus faecalis, triple antibiotic paste, 970 nm diode laser, 445 nm diode laser, photodynamic therapy, doxycycline.

INTRODUCTION

Persistence of organisms in the root canal system as well as their re-colonization and metabolic products are the main causes of failure in root canal therapy (1, 2). Root canal therapy aims to not only eliminate pathogenic organisms from the root canal but also shape it appropriately and obturate it with appropriate materials (3). In contaminated root canals, intra-canal medications are used for various aims such as complete removal of residual bacteria after instrumentation (mechanical removal of infected walls using hand instruments or rotary instruments), reduction of inflammation in the pulp and periapical tissues (4). Irrigants and antimicrobial agents during instrumentation effectively reduce the bacterial population in the root canal (5). However, due to the anatomical complexity of the root canal, complete disinfection cannot be achieved through instrumentation alone (6). A small number of remaining microorganisms can proliferate between sessions and often reach the same level as that found in the previous session (7). Intracanal medicament usage between treatment sessions could be an effective method to inhibit those bacteria (3). Among intra-canal medicaments used between sessions, antibiotic paste has been frequently used. For example, a combination of several antibiotics is employed to eliminate bacteria in necrotic teeth undergoing root regeneration therapy (8). Metronidazole, ciprofloxacin and minocycline have been suggested as useful disinfectants of the root canal system (9, 10). This combination is effective in penetrating dentinal tubules and disinfecting them (11) and can eliminate bacteria in the deep areas of the root canal system (12). Also, high-power laser radiation has been shown to have an antibacterial effect (13). Studies are being conducted to find the most suitable, efficient, safe and accessible laser wavelengths. Diode lasers (especially 810 nm, 980 nm and 940 nm diode lasers), have been recommended for root canal disinfection due to their optimal antibacterial effect and low cost compared to most lasers used in endodontics (14). A 445 nm diode laser has recently entered the market, and there are fewer studies on the impact of its radiation on bacteria. Photodynamic therapy is a therapeutic modality in which free radicals are produced by irradiating the photosensitizer-absorbing wavelength of laser light, leading to bacterial cell destruction (15). Some periodontal studies reported that doxycycline could be used as a photosensitizer to eliminate periodontal bacteria through photodynamic therapy (16), while others mention the effects of tetracycline oxidation products when exposed to light (17). In this regard, a combination of the antibacterial effect of doxycycline itself and its light-sensitive feature can be used. Therefore, the present study aimed to investigate and compare the antimicrobial effects of lasers with different wavelengths, triple antibiotic paste (TAP), their combination, photodynamic therapy and sodium hypochlorite irrigation on Enterococcus faecalis. The completion of this study enable us to state that the most desirable combination among the seven exlored groups can be chosen as an efficient method in endodontic treatments.

The onset of early childhood caries (ECC) can be prevented to some extent by employing fluoride varnish, such as sodium fluoride 5%, and fluoride-containing toothpaste. Nevertheless, for the management of cavities caused by ECCs, a non-invasive therapeutic approach known as atraumatic restorative treatment (ART) has been suggested. Atraumatic restorative treatment is a cost-effective and uncomplicated method that can be conducted outside traditional clinical settings or where suitable facilities are lacking (4).

MATERIALS AND METHODS

The present experimental laboratory study was conducted at the Faculty of Dentistry, Mazandaran University of Medical Sciences, Sari, Iran. It was approved by the Ethics Committee of Mazandaran University of Medical Sciences (ethics code IR.MAZUMS.REC.1402.341). For the current research, 80 single-rooted human teeth which had been extracted due to periodontal and orthodontic reasons were collected from dental clinics and offices.

The inclusion criterium was permanent single root teeth with single direct canal and intact, mature apex, without visible fracture line. Exclusion criteria comprised deciduous teeth, single-rooted teeth with multiple canals and teeth with severe root curvature, root decay or root resorption.

The tooth crown was cut with a diamond bur to obtain a root with a length of approximately 10 mm (16). The root canals were cleaned and shaped using rotary files (Denco, China) with single- length technique up to MAF#30. Irrigation was done with 5.25% sodium hypochlorite solution. Since the smear layer prevented contamination of dentinal tubules by E. faecalis, the teeth were irrigated with 17% EDTA for one minute, followed by saline and 2.5% sodium hypochlorite solution. The apex of the teeth was sealed with light-cured cement and the root surfaces were coated with two layers of nail polish. Then the tooth blocks were autoclaved at 121oC for 30 minutes. Afterwards, 80 tooth blocks were randomly divided into nine groups, 10 teeth were considered for each intervention group and five teeth were assigned to the positive and negative control groups. Then, teeth were inoculated with E. faecalis, except for those in the negative control group.

The standard bacterial strain of Entrococcus faecalis (ATCC 29212) (Pasteur Institute of Iran) was inoculated on a blood agar culture medium and incubated aerobically at 37 °C for 24 hours. Then, a loop of standard microbial culture (equivalent to 0.5 McFarland standard) containing 108 × 5/1 CFU/mL of E. faecalis was injected into the root canals of eight out of nine study groups (except the negative control group). The desired solution was well distributed throughout the root canal areas using a size 25 K-file and the root canals of all those teeth were infected with E. faecalis. Finally, the access cavity was covered with aluminum foil and infected teeth were incubated at a temperature of 37 oC for 24 hours under aerobic conditions with 10% CO2 using a candle jar (18-23).

In group 1 (high power 970 nm laser irradiation), canal was irradiated with 970 nm diode laser by using a 200 ìm fiber tip, CW and 1.5 W power in a circular motion from the canal depth to the canal orifice for five seconds (22).

In group 2 (high power 445 nm laser irradiation) we employed 445 nm laser irradiation (Siro- Laser Blue; Dentsply Sirona) in continuous wave mode with a power adjustment of 0.6 W for five seconds using a 200 ìm fiber tip (Easy Tip Endo; Dentsply Sirona). The procedure was performed in a circular motion from the canal depth to the canal orifice (20).

In group 3 (photodynamic therapy with doxycycline as photosensitizer), one tablet of 100 mg doxycycline was diluted in 10 mL of distilled water to obtain a concentration of 1%. The resulting solution was injected into the canal using a syringe. The needle of the syringe was replaced for each tooth to maintain sterile conditions. After drug delivery, 30 seconds of laser therapy was performed using a 660 nm wavelength diode laser (SiroLaser Blue; Dentsply Sirona) with an output power of 0.3 watts (16, 22).

In group 4 (triple antibiotic paste – TAP), ciprofloxacin 250 mg, metronidazole 500 mg and doxycycline 100 mg were crushed into a fine powder using a mortar; 500 mg of each drug was placed in a dapen container to achieve a weight ratio of 1:1:1. The resulting powder was mixed with 1 mL of 0.9% normal saline and the obtained paste was placed in the root canal using a size 30 k-file (24). The coronal part of the tooth was sealed with aluminum foil and incubated at 37 °C for 21 days (25).

In group 5 (TAP and high power 970 nm laser irradiation), the root canal was irradiated with 970 nm diode laser, using a 200 ìm fiber tip, CW and a power of 1.5 W in a circular motion from the canal depth to the canal orifice (5 s) (26). Then, TAP was placed in the root canal using a size 30 k-file (24). The coronal part of the tooth was sealed with aluminum foil and incubated at 37 °C for 21 days (25).

In group 6 (TAP and high power 445 nm laser irradiation), a 445 nm laser (SiroLaser Blue; Dentsply Sirona) was irradiated in continuous wave mode with a power adjustment of 0.6 W for five seconds, using a 200-μm fiber tip (EasyTip Endo; Dentsply Sirona) (20). Then, TAP was placed in the root canal using a size 30 k-file (24). The coronal part of the tooth was sealed with aluminum foil and incubated at 37 °C for 21 days (25).

In group 7 (NaOCl), teeth were irrigated with 1 mL of 5.25% sodium hypochlorite for two minutes (27).

The positive control group was infected with E. faecalis strain and no medication was administered, while in the negative control group only sterile brain heart infusion was used, without medication.

After the end of incubation, aluminum foil was removed and canals were washed with sterile normal saline solution. Then, canal walls underwent circumferential filling using a #30 K file and dentinal fragments were detached and suspended inside the canal. After that, a paper cone with appropriate size was placed in the canal, where it remained for 60 seconds so the contents inside the root canal, including liquid and dental fragments, were absorbed by paper cone. Then, paper cones were placed inside falcon tubes containing 500 μL of physiological serum. Afterwards, all collected samples were vortexed for at least five minutes. Then, 1.0 mL of the desired solutions were inoculated onto agar plates and incubated at 37 °C (28). Bacterial colonies were visually observed after 24 hours and counted per mL for each tooth as antimicrobial assessment. Finally, the colony counts obtained from each sample were compared with the control sample and any reduction in colony count was subjected to statistical analysis.

Indexes such as mean, standard deviation (SD) and median were used to describe variables. And Kruskal-Wallis and Mann-Whitney tests were used to compare the colony counts in different groups. Data analysis was performed using SPSS software version 22 and a significance level of less than 0.05 was considered.

RESULTS

Images related to bacterial cultures in each group are shown in Figure 1.

Descriptive information about colony count in different groups was shown in Table 1 and Figure 2.

Since the average colony count was not normally distributed, all counts were ranked instead of directly using the average colony count.

Due to the significant difference between groups, pairwise comparisons of the study groups were conducted to find significant differences.

When the colony count rank in the 970 nm laser group was compared with that of the other study groups, using the Mann-Whitney test, we noticed that all the other groups had a significant difference (P value <0.001), except for the 445 nm laser group (P value 0.255).

When the colony count rank in the 445 nm laser group was compared with that of the other study groups, using the Mann-Whitney test, we found that all the other groups had a significant difference (P value <0.001), except for the doxycycline + 660 nm photodynamic therapy group (P value 0.069).

When the colony count rank in the doxycycline + 660 nm photodynamic therapy group was compared with that of other groups, using the Mann-Whitney test, we saw that all groups had a significant difference (P value <0.001).

When the colony count rank in the TAP group was compared with that of the other groups, using the Mann-Whitney test, we found that only the positive control group had significant differences (P value <0.001).

When the colony count rank in the 970 nm laser + TAP group was compared with that of the other groups, using the Mann-Whitney test, we observed that only the positive control group had a significant difference (P value <0.001).

When the colony count rank in the 445 nm laser + TAP group was compared with that of the other groups, using the Mann-Whitney test, we found that only the positive control had a significant difference (P value <0.001).

When the colony count rank in the NaOCL group was compared with that of the other group, using the Mann-Whitney test, we noticed that only the positive control had a significant difference (P value <0.001).

Finally, when we compared the colony count rank between the positive and negative control groups by the Mann-Whitney test, we found a significant difference in the two groups.

DISCUSSION

Root canal system infection is a polymicrobial infection primarily caused by anaerobic bacteria, which can lead to pulp and periapical inflammation (29, 30). In chronic and persistent infections, various organisms are associated with intra- and extra-radicular infections (31). Enterococcus faecalis is a facultative anaerobic Gram-positive bacterium that stands out more than other organisms in cases of chronic periapical infections and unsuccessful root canal treatments (32, 33). This organism, with its dentinophilic nature, can stick to the dentin, penetrate up to 400 ²m into dentinal tubules, replicate and colonize in the root canal system, form a biofilm and remain in the same location for a long time (34, 35), and it can survive root canal treatment under harsh conditions and resist to root canal disinfectants (36, 37). The low prevalence of this microorganism in primary infections (4-40%) and its high prevalence in chronic and persistent infections (24-77%) (38) is one of the reasons why E. faecalis was also explored in the present study, similar to many previous studies.

Laser light radiation can eliminate non-pigment bacteria by increasing the temperature of water inside bacterial cells, while in pigmented bacteria, due to their high absorption in the pigmented components of bacteria, laser energy is selectively absorbed and released locally as heat, causing bacteria to be eliminated by increasing the temperature. Nowadays, lasers with different wavelengths, mainly diode lasers, are used for this purpose, and various results regarding bactericidal reduction have been reported (39-43). Recently, diode lasers with wavelengths in blue light range have also been introduced in dentistry. Blue light has been shown to be an effective antimicrobial agent with high intensity (44). With laser light penetration into dentin, the elimination of organisms in areas of the canal that are not accessible with conventional irrigation solutions, such as lateral or secondary canals and deep dentinal tubules, becomes possible (41, 45).

Photodynamic therapy is a therapeutic approach in which free radicals are generated by laser light irradiation at a wavelength absorbed by the photosensitizer, leading to the destruction of bacterial cells (46, 47). In various studies, different substances have been selected as light-sensitive substances and used in photodynamic therapy to eliminate Enterococcus faecalis (48). Photosensitizer substances can be natural or synthetic, hydrophilic or hydrophobic, or amphiphilic. Often, phenothiazine salts, including toluidine blue and methylene blue, are used as cationic dyes. Hematoporphyrin, cyanine, phthalocyanine, chlorine and curcumin are also utilized (49). In some periodontal studies, it has been observed that doxycycline could be used as a photosensitizer to eliminate periodontal bacteria through photodynamic therapy (17). Astuti et al used doxycycline-assisted photodynamic therapy in the gingival sulcus of mice with periodontitis and successfully eliminated Porphyromonas gingivalis bacteria, resulting in effective improvement (16). A similar improvement by using laser radiation and doxycycline administered in gingival sulcus of rats with induced periodontitis was achieved in a study conducted by Silveira et al (50). Also, numerous studies found that tetracycline underwent oxidation and produced reddish-colored products when exposed to light (17). In a study by Michael Hamblin, doxycycline and demeclocycline were reported to act as light-activated antibiotics by binding to bacteria and eliminating them through light irradiation, with the remaining antibiotic being capable of preventing bacterial growth. Since topical tetracycline is used clinically, activation by light can enhance its bactericidal properties (17). The results of the above-mentioned studies are consistent with our study, which describes the effect of photodynamic therapy with doxycycline on Enterococcus faecalis (17).

Sodium hypochlorite solution is used as an irrigating solution inside the root canal due to its antibacterial properties and reaction with organic compounds in the dental pulp, such as amino acids and fatty acids, to dissolve them. Its low viscosity allows it to easily penetrate into the root canal. Sodium hypochlorite solution has also the advantage of being widely available and inexpensive (51). However, its use has some disadvantages such as an unpleasant taste and high toxicity (52).

Sodium hypochlorite is a strong antimicrobial agent but has no residual activity (53). Sodium hypochlorite loses its activity right after use. On the other hand, the antimicrobial activity of triple antibiotic paste remains effective over time.

A study conducted by Janani et al in 2016 (54) investigated the antibacterial effects of photodynamic therapy (using toluidine chloride as a photosensitizer and a 635 nm laser) and 2.5% sodium hypochlorite on root canals infected with Enterococcus faecalis, using the real-time PCR technique. The authors found that both antimicrobial agents significantly reduced the bacterial count compared to the control group; also, there was a significant difference between laser photodynamic therapy and sodium hypochlorite, with the latter showing higher efficacy than photodynamic therapy. The findings of that study are consistent with ours.

We have also investigated the use of TAP, a substance consisting of three antibiotics: metronidazole, ciprofloxacin and minocycline. The triple combination has the highest efficacy in reducing resistant strains in the root canal system (55, 56). In the present study, doxycycline was used instead of minocycline. A study conducted by Leila Moradi et al compared the antimicrobial effects of TAP, PDT and 940 nm diode laser, and found that the highest reduction in biofilm thickness occurred in the TAP group, but the difference between groups was not significant (P > 0.05) (57). The greater antimicrobial effect of TAP was also found in our research. However, there was a significant difference in this comparison to the present study, which could be related to the type of photosensitizer (toluidine blue in Moradi's study) and light employed (LED in Moradi's study). Additionally, the exposure time of triple antibiotic paste used for bacterial growth inhibition was different in Moradi's study (seven days) and ours (57).

In a study conducted by Mohammad Sameh et al in 2023, it was shown that the new paste, consisting of levofloxacin–tinidazole–tigecycline, had the greatest inhibitory effect on microbial growth compared to other medications, in the following order of effectiveness: new paste + laser > old paste (metronidazole–minocycline–ciprofloxacin) + laser > laser alone (58).

In a study by Lakhani et al, the antibacterial effect of TAP was investigated after 10 days, seven days and one day of treatment. The authors reported that a longer duration of treatment (days) and contact time of the paste with bacteria led to an increase in antimicrobial effect (59). Ghabraei’s study demonstrated that application of TAP for seven days could eliminate Enterococcus faecalis bacteria up to a depth of 400 ìm in dentinal tubules (60). Mandal et al investigated the effect of TAP using different carriers on Enterococcus faecalis for 21 days in their research (24).

A study conducted by Asnaashari et al in 2019 compared the antimicrobial effects of TAP, calcium hydroxide paste and photodynamic therapy. The authors reported a reduction in bacterial count in the root canal by 99.9% with regular TAP, by 98.8% with photodynamic therapy and 94.13% with calcium hydroxide (61). Their findings were consistent with ours, and the percentage of effectiveness for TAP and photodynamic therapy was very close to that found by us.

Finally, considering the positive and negative points mentioned for each group, it is not possible to definitively determine the superiority of one group over the others; however, TAP can be a suitable choice due to several features such as increasing antimicrobial effect over time, acceptable depth of penetration and powerful antimicrobial effect. So, environmental situations, patient's condition and diagnosis, preferences of the dentist and the availability of materials and equipment are important factors in choosing the best antibacterial method.

CONCLUSION

Results show that all of the methods used in the present study were effective enough for bacterial reduction. The use of triple antibiotic paste was proved to achieve complete bacterial elimination. Photodynamic therapy with doxycycline as a photosensitizer was shown to provide significant results of bacterial reduction and diode laser irradiation at both wavelengths used by us (970 nm and 445 nm) were also reported to have antibacterial effect, although slightly lower than the previous methods.

Financial support: Funding was provided by the Research and Technology Vice-President of Mazandaran University of Medical Sciences, Iran.

FIGURE 1.

A) Culture image related to the positive control group; B) 980 nm laser group; C) 445 nm laser group; D) photodynamic therapy group; E) sodium hypochlorite group; F) triple antibiotic paste; G) 970 nm laser group and antibiotic paste; and H) 445 nm laser group and antibiotic paste

TABLE 1.

Descriptive information on colony count rank in different study groups

FIGURE 2.

Average colony count rank in different study groups