In vitro evaluation by quantitative real-time PCR and culturing of the effectiveness of disinfection of multispecies biofilms in root canals by two irrigation systems

Duo Zhang, Ya Shen, César de la Fuente-Núñez, Markus Haapasalo

The purpose of this in vitro study was by using quantitative real-time PCR and culturing to determine the effectiveness of two irrigation and cleaning systems in removing multispecies oral biofilms from root canals. Twenty extracted human molars were instrumented to size #15/.02 and then cleaned with the GentleWave (GW) System. The teeth were autoclaved to provide the same sterile baseline. The molars were filled with mixed plaque suspended in BHI and centrifuged to inoculate the biofilms. After 2 weeks of incubation, the teeth were randomly divided into two treatment groups. In GW group (26 canals), the teeth were further instrumented to size #15/04, and in PiezoFlow (PF) group (30 canals) to #35/.04. The teeth were then cleaned either with GW System or ProUltra PiezoFlow Active Ultrasonic System using 3% sodium hypochlorite NaOCl, 8% EDTA, and sterile water as irrigants. Samples (S1, S2, and S3) for bacterial cultures were taken from 13 canals before and after instrumentation and after final cleaning. Quantitative real-time PCR was performed from all 56 canals, and universal bacterial, one genus, and one species-specific primers were used to determine the presence of microorganisms in samples from root canals before and after instrumentation and after final cleaning. Statistical analyses were performed using the Mann-Whitney U test with the significance level set at P < 0.05. Bacterial culturing from the canal samples revealed strong reduction of bacteria from S1 to S2 in both groups after instrumentation and irrigation with water only. No growth was detected in any of the S3 samples after cleaning in either group. A highly significant reduction in bacterial DNA was recorded by qPCR for both groups (P < 0.001). GW System showed more constant and a significantly higher reduction of total microbial DNA (P = 0.007), Enterococcus faecalis DNA (P = 0.011) and Streptococcus spp. DNA (P = 0.029) than the Ultrasonic System. The amount of residual microbial DNA calculated as an average of residual DNA in each individual canal in PF group was 1.99% and in GW group 0.09%. While both systems demonstrated a highly effective reduction of intracanal bacterial DNA, the final total amount and variation in the number of residual bacterial DNA was significantly smaller in the GW group. Elimination of microbes from the infected root canal system is regarded as the key for long-term clinical success. While both GentleWave and Ultrasonic Systems used with NaOCl and EDTA demonstrated a highly effective reduction of intracanal bacterial DNA; GW produced higher reduction and better predictability.