Efficacy of diode laser and sonic agitation of Chlorhexidine and Silver-nanoparticles in infected root canals

Objective: To assess the efficacy of agitation of chlorohexidine (CHX) and Silver nanoparticles “AgNps” with 810nm diode laser or sonic endoactivator compared to side –vented needle on infected root canals with Enterococcus “E” Faecalis biofilms. Material and Methods: Sixty-five extracted human premolars with single oval canals were instrumented by protaper system up to F3. Biofilms of E. faecalis were generated based on a previously established protocol. Two teeth were used to check the biofilm formation, then the remaining Teeth were randomly divided into three equal experimental groups according to agitation techniques used: group 1 (810 nm diode laser with 1 watt) , group 2 (sonic endoactivator) and group 3 (Side vented needle). Each group was further divided into three equal subgroups according to the irrigant solution into; subgroup A: chlorohexidine, subgroup B: silver nanoparticles and subgroup C: distilled water: Confocal laser scanning microscopy “CLSM” was used to assess bacterial viability. Data were analyzed by appropriate statistical analyses with P = 0.05. Results: Regarding the activation method, all groups had a significantly high percentage of dead bacteria (P < 0.05). However, Laser was significantly the highest and Endoactivator the least (P < = 0.001). Diode laser agitation of AgNps irrigant showed the highest reduction percentage of bacteria (78.1%) with a significant difference with both CHX and water irrigation, Conclusion: Under the condition of the present study; results reinforced that laser activation is a useful adjunct, 810 nm diode laser agitation of AgNps or chlorhexidine was more effective in disinfection of oval root canals than endoactivator and side vented needle techniques. RESUMO Objetivo: Avaliar a eficácia da agitação de clorohexidina (CHX) e nanopartículas de prata (AgNps) , com laser de diodo de 810 nm ou endoativador sônico, em comparação à agulha de ventilação lateral, em canais radiculares infectados com biofilmes de Enterococcus “E”; Faecalis. Material e Métodos: Sessenta e cinco pré-molares humanos com um único canal oval, extraídos, foram instrumentados pelo sistema protaper até F3. Os biofilmes de E. faecalis foram gerados com base em um protocolo previamente estabelecido. Foram utilizados dois dentes para verificar a formação do biofilme, e os dentes restantes foram divididos aleatoriamente em três grupos experimentais iguais, de acordo com as técnicas de agitação utilizadas: grupo 1 (laser de diodo 810 nm com 1 watt), grupo 2 (endoativador sônico) e grupo 3 (Agulha com ventilação lateral). Cada grupo foi dividido em três subgrupos iguais, de acordo com a solução irrigante; subgrupo A: clorohexidina, subgrupo B: nanopartículas de prata e subgrupo C: água destilada: A microscopia confocal de varredura a laser foi usada para avaliar a viabilidade bacteriana. Os dados foram analisados por análises estatísticas apropriadas com P = 0,05. Resultados: Em relação ao método de ativação, todos osgrupos apresentaram percentual significativamente alto de bactérias mortas (P < 0.05). No entanto, para o laser foi significativamente o mais alto e, para oendoativador, o menos alto (P < = 0.001). A agitação com laser de diodo doirrigante AgNps apresentou a maior porcentagem de redução de bactérias (78,1%), com diferença significativa tanto para irrigação com CHX quanto comágua. Conclusão: Sob as condições do presente estudo; os resultadosreforçaram que a ativação a laser é um complemento útil, a agitação por laserde diodo de 810 nm de AgNps ou clorexidina foi mais eficaz na desinfecção dos canais radiculares ovais do que as técnicas de endoativador e agulha com ventilação lateral. O R I G I N A L A R T I C L E Efficacy of diode laser and sonic agitation of Chlorhexidine and Silver-nanoparticles in infected root canals Eficácia do laser de diodo e agitação sônica de clorexidina e nanopartículas de prata em canais radiculares infectados Latifa Mohamed ABDELGAWAD1, Niven ASMAIL1, Somia Abdel LATIF2, Ali Mohamed SAAFAN1 1 Medical Laser Applications Department National Institute of Laser Enhanced Sciences Cairo University Cairo Egypt. 2 Microbiology and Molecular Biology Department Faculty of Medicine Cairo University – Cairo Egypt. doi: 10.14295/bds.2020.v23i3.1989 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” Instituto de Ciência e Tecnologia Campus de São José dos Campos Ciência Odontológica Brasileira


INTRODUCTION
O ne of the main challenges facing the dentists in the endodontic steps is how to totally disinfect the root canal [1]. E faecalis is the most prominent microorganism involved in persistent infections after root canal therapy. E. faecalis has the ability to penetrate the dentinal tubules and cementum. It can survive in biofilm form at anatomical complexities of root canal system, over the foreign bodies like gutta-percha or other obturating materials extending into periapical tissues and can survive for prolonged periods under nutrientdepleted conditions [2].
Trying to overcome the challenge compulsory by the presence of biofilm and reach complete disinfection or significant bacterial reduction in the root canals, many irrigants have been indicated during endodontic treatment. Chlorhexidine gluconate (CHX) at 2% is one of the most commonly used irrigants and considered as an effective antimicrobial agent. It has many properties; broad-spectrum, substantively (extended outstanding activity) and a relative absence of toxicity that recommend it to be used as an endodontic irrigant [3,4].
Silver nanoparticles (AgNps) have gained popularity because of their unique ability to penetrate tissues, interact with bacteria, exhibit potent antimicrobial activity and biocompatibility [5].
The agitation of irrigating solutions with the laser has become common [6]. Researchers examined many laser systems to attain complete disinfection of root canal system and the adjacent dentinal tubules and yet there is still argument about which is the most powerful laser system in regards to providing a sterilized root canal system. The high bactericidal effect of diode laser has been reported in multiple studies [7]. Therefore, due to the bactericidal effect of diode laser and its low cost compared to other commonly used laser systems in endodontic, it can be used in agitation of the irrigation during the mechanical debridement procedures [8].
The Endoactivator System "EA" is a sonically driven irrigant activation system planned to produce dynamic fluid agitation inside the canal that has been shown to improve the effectiveness of irrigation better than the traditional syringe irrigation [9]. So the present study highlighted the effect of 810 nm diode laser and sonic agitation on two types of irrigants; [chlorohexidine (CHX) and silver nanoparticles (AgNps)] applied in root canals.

MATERIAL AND METHODS
This research was approved by the Ethics Committee of National Institute of Laser Enhanced Sciences (NILES), Cairo University, Egypt (CU/NILES/30/19). Also this study was done in the microbiology and molecular biology department, faculty of medicine, Cairo University.

Selection and Mechanical preparation of samples
Sixty -five extracted human premolars with single oval canal and fully formed apices were selected from hundred extracted teeth for periodontal reasons. Each root was digitally radiographed using RVG 6200 digital sensor (Carestream, Rochester, New York, United States) and measured by its software to select canals with ovality ratio more than 2.5. The crowns were sectioned such that roots were standardized to 12 + 2 mm and stored in labeled vials at 100% humidity.

Final irrigation and sterilization of the samples
Final irrigation was performed with 5 ml 15% Ethylenediaminetetraacetic acid "EDTA" (Endo-Solution, Cerkamed, Stalowa Wola, Polska) for 3 min and 5 ml NaOCl for 3 min to remove the smear layer, and then the canal was irrigated with 10 ml of physiological saline solution to remove the EDTA. Finally, the teeth were sterilized in the autoclave at 121°C for 30 min .

Inoculation and incubation of the teeth with E faecalis
Using a sterile micropipette, twenty microliters of E .faecalis suspension (matching McFarland's turbidity of tube no 0.5) was syringed into each root canal. Each inoculated root was kept in separate sterile test tube with caps in a rack. Then incubated at 37°C for 7 days, to allow the proliferation of microorganisms, and their further penetration into the dentinal tubules and formation of biofilms.

Classification of the samples:
Two teeth were used to check the biofilm formation, then the remaining teeth (63) were randomly divided into three equal experimental groups according to agitation techniques used: group 1 (810 nm diode laser with 1 watt), group 2 (sonic endoactivator) and group 3 (Side vented needle). Each group was further equally divided into three subgroups according to the irrigant solution into; subgroup A: chlorohexidine, subgroup B: silver nanoparticles and subgroup C: distilled water: The agitation mechanisms in the three groups were as a follow:

Group 1 (diode Laser)
Agitation was done with 810 nm Diode laser with continuous mode and output power 1 watt (Zolar lasers, Canada) which delivered into 200 um flexible plain endodontic fiber. The fiber was inserted parallel to root canal wall and used with helicoidally movement in apical-coronal directions. Agitation was done for 10 seconds to 1 ml irrigant. The sequence was repeated 5 times, giving 5 ml total volume and 50 sec total agitations.

Group 2 (Sonic endoactivator)
In this group agitation was done by Sonic endoactivator device (Dentsply, Tulsa) with red polymer tip # 25/0.04 at speed 10000 rpm. The samples in this group were irrigated with 5 ml of an irrigating solution, where each 1ml of the irrigant was followed by 10 seconds of sonic agitation [11].

Group 3 (side -vented needle)
Side vented needle delivered 5 ml irrigant during moving slowly up and down along canal length. All agitations were performed at 2 mm away from the working length.

Confocal laser scanning microscopy (CLSM) examination
Sixty -five roots were sectioned for detecting viable and nonviable bacteria on root canal walls. Roots were set in blocks of fast setting acrylic resin and two lines were drawn with a marker on the exposed part of the root, one buccal and one palatal for vertically sectioning into two halves, approximately parallel to the tooth axis, utilizing the microtome saw. A sample from each sectioned root was taken for scanning. Each sample was stained by both Acridine Braz Dent Sci 2020 Jul/Sep;23 (3) 4 orange (AO) and Propidium iodide (PI) dyes separately, just before the CLSM examination. Zeiss LSM 710 confocal microscope (Carl Zeiss, Germany) was used with 40 x objectives for scanning. Three random areas of the middle third of the root canal were scanned with a 2-mm step size by the CLSM. For each image the median intensity of green and red bacteria were calculated by the soft-ware (green for live and red for dead bacteria), this number was tabulated and statistically analyzed.

Data presentation and analysis
To identify the effect of activation protocols on bacterial reduction (dead cell %) in each irrigation protocol, one-way ANOVA was applied. Bonferroni's correction for multiple testing was used in one-way analysis of variance. P = 0.05 for the analyses. Twoway ANOVA was performed to weigh the effect of the irrigation protocol and activation method as the two independent variables on the outcome (percentage of dead cells. Data was analyzed using IBM SPSS advanced statistics (Statistical Package for Social Sciences), version 21 (SPSS Inc., Chicago, IL).

RESULTS
The data obtained from the CLSM are tabulated (Table I). Figure 1 shows a homogenous penetration of E. faecalis deep into the dentinal tubules of the root canal. figure 2 shows biofilm destruction within the root canal lumen for all groups. Regarding the activation method, all groups had a significantly high percentage of dead bacteria (P < 0.05). However, Laser was significantly the highest and Endoactivator the least (P <= 0.001).
Regarding the irrigant type; AgNps activated with diode laser had the highest percentage of dead bacteria (78.1%) followed by needle agitation (76.47%) then sonic (72.94%). Without significant difference (p > 0.05) In chlorhexidine subgroup, the high percentage of dead bacteria was in laser group (71.81%) followed by needle group (70.18%) then sonic group (68.99) Without significant difference (p > 0.05). while distilled water subgroup showed high percentage of bacterial reduction in laser group (71.46%) followed by sonic (62.7847%) then needle group (60.6%) with significant difference between laser and sonic and laser with needle (p < 0.05) while there was no significant difference between sonic and needle (p >0.05).   Table I percent of apparently dead bacterial cells in the biofilm within the dentinal tubules, assessed by confocal laser microscopy after three different activation techniques with three different irrigant.
(*) means significant between laser and sonic in H2O irrigant (#) means significant between laser and needle in H2O irrigant (a) Means significant CHX and AgNps within the same group (b) Means significant between CHX and H2O within the same group (c) Means significant between AgNps and H2o within the same group.

Group
Subgroup

DISCUSSION
The main goal of endodontic treatment is the complete disinfection of the root canal system; however, it is difficult due to complex anatomy of the root canal system and biofilm mediated infection. E. faecalis was chosen because it is generally believed that it is one of the most resistant microorganisms found in the infected root canals and endodontic treatment failures [9,10].
The clinical challenge to deliver irrigants into unprepared infected canal extensions; as well as the most apical infected segment recommended the use of automated agitation of irrigant in disinfection of root canal systems [7]. So the present study evaluated the antibacterial effect of activated agitation of diode laser, sonic and side-vented needle with three types of irrigants; Chlorhexidine, AgNps, and distilled water in infected oval root canal with E faecalis. Chlorhexidine (CHX) was selected in the present study as it has a broadspectrum antimicrobial effect and kills E. faecalis in the dentinal tubules. [9]. .as well as AgNps exhibit potential antibacterial activity and does not allow to develop resistance [11] Positively charged AgNps interact with the negatively charged bacterial cell walls, adhere, and penetrate into the bacterial cell leading to the loss of cell wall integrity and permeability [12][13][14][15][16][17].
Diode laser induced cavitation and side vented needle non laminar streaming probably provided better mechanical turbulence to penetrate infected dentin and effectively carry away the microorganisms than acoustic streaming by vibrating inserts of endoactivator [18][19][20].
In the present study the lasing protocol favored the disinfection of the root canal. Through limited studies existed specially on 810 nm diode laser; generally, diode agitation seems promising [6,18]. Agitation of AgNps by 810 nm diode laser I watt for 50 sec improved E. faecalis eradication compared to sonic or needle agitation. Using 10 sec continuous 810 nm diode laser which repeated five times for agitation of AgNps improved e faecalis eradication by 78.05% however inadequacy of diode laser to remove more percentage of bacteria may be due to using of low output power (1 watt) which is in accordance with other studies [21][22][23][24][25][26].
Sonic agitation produced bactericidal effect in the present study probably due to oscillating movement which allow hydrodynamic circulation of the irrigant. A reduced antibacterial efficiency of sonic compared with other techniques may be due to the greater displacement amplitude of the small vigorously vibrating polymer tip, also due To weakened. Currents, impeding microstreaming and irrigant activation in apical part of the root beyond the vibrating tip [27][28][29].
In the present study, laser activated agitation was more efficient than endoactivator Braz Dent Sci 2020 Jul/Sep;23 (3) 6 [18 ].Endoactivator in the present study was less efficient than needle agitation this may be due to vacuum and remove of irrigant before replenishing [30 ].Side -vented needle agitation came after laser and better than sonic as vertical up and down movements of the needle allowed distributing the localized high dynamic flow at the side exit along the oval canal allowed effective reflux of irrigant coronally as previously reported [ 30] .
The enhancing effect of 810 nm diode laser agitation in disinfection of oval canals can be complemented by a further study to evaluate other different protocols in canal disinfection with different irrigant and providing better bactericidal effect.

CONCLUSIONS
Under the condition of the present study; the results reinforced that laser activation is a useful adjunct, 810 nm diode laser agitation of AgNps and chlorhexidine was more effective in disinfection of oval root canals than endoactivator and side vented needle techniques.