Irrigation with Laser-Activated sodium hypochlorite: An antimicrobial alternative in endodontics

Abstract

Bacteria and their sub-products are the main cause of the occurrence and perpetuation of endodontic infection. However, only a few bacterial species are seen as responsible of persistent endodontic infections, among them Enterococcus faecalis is the most frequently isolated species. It has several virulence factors, such as aggregation substances, enterococcal surface protein (Esp), “endocarditis and biofilm-associated pili” (ebp) and cytolysin. Moreover, its high resistance to antibacterial agents is emhanced by its ability to form biofilm.

Due to the complex and unpredictable root canal morphology, the complete removal of smear layer and bacterial biofilm is difficult. Recently, laser-activated irrigation (LAI) has been introduced as an alternative to achieve a deeper cleaning and disinfection of the root canal system. Its mechanism of action is based on the generation of cavitation bubbles, through the absorption of laser energy by the irrigant. The most used lasers are from the Erbium family, Er, Cr: YSGG (2780nm) and Er: YAG (2980nm).

Sodium hypochlorite (NaOCl) is considered the "gold standard" of endodontic irrigators. It has a broad antibacterial spectrum and is capable of dissolving organic tissue. It is used in a range between 0.5% and 6%, varying its degree of effectiveness. Nevertheless, at high concentrations it is toxic, causing damage to endothelial cells and the periodontal ligament cells, which generates an acute inflammatory reaction and pain.

Hence, the aim of this thesis was to explore the bactericidal effect of low concentration of NaOCl activated by Er, Cr: YSGG laser-activated irrigation against E. faecalis biofilms in root canals in order to decipher if it may be similar to the one achieved by high concentrations of NaoCl.

IN IN VITRO ROOT CANAL MODEL: The main objective of the first stage of this thesis was to build a laboratory model to simulate the conditions inside a single-tooth root canal. The Pasteur pipettes were inoculated with E. faecalis ATCC 29212 for 24 hours bacterial colonization and the subsequent formation of biofilm in the proposed in vitro model was demonstrated by atomic force microscope.

The second essential point of this part was to determine antimicrobial capacity of Er,Cr:YSGG laser against E.faecalis. In addition, passive ultrasonic irrigation was also tested. Laser-activated irrigation demonstrated higher antimicrobial activity than passive ultrasonic irrigation.

The final stage of this first part consisted in the analysis and measurement of the nano-roughness by atomic force microscopy of the cells treated and its comparison with that of untreated cells.

EXTRACTED TEETH: This part was focused on the endodontic preparation of extracted human teeth. The root canals were instrumented by a crown-down / step-back technique using conventional sequence of 0.02 taper files up to the master # 55. The teeth were inoculated with E. faecalis ATCC 29212 for 10 days.

The following stage was focused on the study of the antibacterial action of low concentration of NaOCl activated by Er,Cr: YSGG laser in extracted human teeth. The antimicrobial effectiveness of laser-activated irrigation was compared with passive ultrasonic irrigation activation and conventional manual irrigation. Er,Cr:YSGG laser and 0.5% NaOCl showed a considerable synergistic action.

Finally, the last part was focused on the microscopic visualization of the samples, to complement the results of the microbiological count. The scanning electron microscope was used to determine the degree of effectiveness of bacterial biofilm and smear layer removal both on the dentin surface and inside the dentinal tubules. Additionally, the confocal scanning electron microscope was used to visualize the proportion of alive and dead bacteria after treatment.

The results obtained in this thesis show that laser-activated irrigation is a therapeutic alternative for infections caused by E. faecalis inside of root canals.

Las bacterias y sus subproductos son la principal causa de la infección endodóntica y su perpetuación. E. faecalis es la especie más frecuentemente aislada de los fracasos endodónticos. Su alta resistencia a los agentes antibacterianos viene incrementada por la capacidad para formar biofilms. Recientemente, el riego activado por láser (LAI) se ha propuesto como una alternativa para lograr una limpieza y desinfección más profunda del sistema de canales radiculares. Su mecanismo de acción se basa en la generación de burbujas de cavitación, a través de la absorción de energía láser por parte del irrigante. El objetivo de esta tesis fue explorar si el efecto bactericida de una baja concentración de NaOCl activado por el láser Er, Cr: YSGG, puede ser similar al alzanzado por altas concentraciones de NaOCl contra biofilms de E. faecalis en canales radiculares.

MODELO DE CANAL RADICULAR IN VITRO:

El objetivo principal de la primera etapa de esta tesis fue construir un modelo de laboratorio para simular las condiciones al interior de un canal radicular. Las pipetas Pasteur fueron inoculadas con E. faecalis ATCC 29212 durante 24 horas. Luego se determinó la capacidad antimicrobiana del láser Er, Cr: YSGG contra E.faecalis. La irrigación activada por láser demostró una mayor actividad antimicrobiana que la irrigación ultrasónica pasiva. Finalmente se realizó el análisis y la medición de la nano rugosidad mediante microscopía de fuerza atómica de las células tratadas y no tratadas.

DIENTES EXTRAÍDOS:

Los dientes fueron inoculados con E. faecalis ATCC 29212 durante 10 días. El láser Er, Cr: YSGG y 0,5% de NaOCl mostraron una acción sinérgica considerable. Finalmente, el microscopio electrónico de barrido se utilizó para determinar el grado de efectividad en la remoción de biofilms bacterianos y de barro dentinario. Además, se utilizó el microscopio electrónico de barrido confocal para visualizar la proporción de bacterias vivas y muertas después del tratamiento.

Los resultados obtenidos en esta tesis muestran que la irrigación activada por láser es una alternativa terapéutica para las infecciones causadas por E. faecalis dentro de los canales radiculares.