Clinical Oral Investigations

, Volume 23, Issue 2, pp 913–920 | Cite as

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 HaapasaloEmail author
Original Article



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.

Material and methods

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.

Clinical relevance

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.


Biofilm Disinfection GentleWave Quantitative real-time PCR Ultrasonic 



Supported by the Canadian Academy of Endodontics, by start-up funds provided by the Faculty of Dentistry, University of British Columbia, and by Canada Foundation for Innovation (CFI fund; project number 32623). CF-N acknowledges funding from the Ramon Areces Foundation. We thank prof. HsingChi von Bergmann, UBC, for valuable help in statistical analysis. We thank prof. Anil Kishen for providing the E. faecalis standard strain. One of the authors (MH) has commercial interest in one of the tested products.


This study was supported by the Canadian Academy of Endodontics, by start-up funds provided by the Faculty of Dentistry, University of British Columbia and by Canada Foundation for Innovation (CFI fund; project number 32623). CF-N acknowledges funding from the Ramon Areces Foundation.

Compliance with ethical standards

Conflict of interest

One of the authors (MH) consults to Sonendo Inc. (manufacturer of GW) and has economic interest in one of the products (GW) used in this study. The other authors declare that they have no conflict of interest.

Ethical approval

Ethics permission was obtained from the University of British Columbia Office of Research Services, Clinical Research Ethics Board (certificate number H15-02793).

Informed consent

Informed consent was obtained from the volunteer providing oral plaque in this study. According to the Clinical Research Ethics Board, no informed consent was required from patients whose teeth, extracted for nonrelated reasons, were used in this study.


  1. 1.
    Sjogren U, Figdor D, Persson S, Sundqvist G (1997) Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 30:297–306. CrossRefGoogle Scholar
  2. 2.
    Malentacca A, Uccioli U, Zangari D, Lajolo C, Fabiani C (2012a) Efficacy and safety of various active irrigation devices when used with either positive or negative pressure: an in vitro study. J Endod 38:1622–1626. CrossRefGoogle Scholar
  3. 3.
    Balic M, Lucic R, Mehadzic K, Bago I, Anic I, Jakovljevic S, Plecko V (2016) The efficacy of photon-initiated photoacoustic streaming and sonic-activated irrigation combined with QMiX solution or sodium hypochlorite against intracanal E. faecalis biofilm. Lasers Med Sci 31:335–342. CrossRefGoogle Scholar
  4. 4.
    Shon WJ (2016) Introducing the GentleWave System. Restor Dent Endod 41:235. CrossRefGoogle Scholar
  5. 5.
    Zandbiglari T, Davids H, Schäfer E (2006) Influence of instrument taper on the resistance to fracture of endodontically treated roots. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:126–131. CrossRefGoogle Scholar
  6. 6.
    Haapasalo M, Wang Z, Shen Y, Curtis A, Patel P, Khakpour M (2014) Tissue dissolution by a novel multisonic ultracleaning system and sodium hypochlorite. J Endod 40:1178–1181. CrossRefGoogle Scholar
  7. 7.
    Molina B, Glickman G, Vandrangi P, Khakpour M (2015) Evaluation of root canal debridement of human molars using the Gentle Wave System. J Endod 41:1701–1705. CrossRefGoogle Scholar
  8. 8.
    Ma J, Shen Y, Yang Y, Gao Y, Wan P, Gan Y, Patel P, Curtis A, Khakpour M, Haapasalo M (2015) In vitro study of calcium hydroxide removal from mandibular molar root canals. J Endod 41:553–558. CrossRefGoogle Scholar
  9. 9.
    Sigurdsson A, Le KT, Woo SM, Rassoulian SA, McLachlan K, Abbassi F, Garland RW (2016a) Six-month healing success rates after endodontic treatment using the novel GentleWave System: the pure prospective multi-center clinical study. J Clin Exp Dent 8:e290–e298. Google Scholar
  10. 10.
    Sigurdsson A, Garland RW, Le KT, Woo SM (2016b) 12-month healing rates after endodontic therapy using the novel GentleWave System: a prospective multicenter clinical study. J Endod 42:1040–1048. CrossRefGoogle Scholar
  11. 11.
    Chu FC, Tsang CS, Chow TW, Samaranayake LP (2005) Identification of cultivable microorganisms from primary endodontic infections with exposed and unexposed pulp space. J Endod 31:424–429. CrossRefGoogle Scholar
  12. 12.
    Le Goff A, Bunetel L, Mouton C, Bonnaure-Mallet M (1997) Evaluation of root canal bacteria and their antimicrobial susceptibility in teeth with necrotic pulp. Oral Microbiol Immunol 12:318–322. CrossRefGoogle Scholar
  13. 13.
    Siqueira JF Jr, Rocas IN (2005) Exploiting molecular methods to explore endodontic infections: part 1—current molecular technologies for microbiological diagnosis. J Endod 31:411–423. CrossRefGoogle Scholar
  14. 14.
    Rocas IN, Lima KC, Assuncao IV, Gomes PN, Bracks IV, Siqueira JE (2015) Advanced caries microbiota in teeth with irreversible pulpitis. J Endod 41:1450–1455. CrossRefGoogle Scholar
  15. 15.
    Antunes HS, Rocas IN, Alves FRE, Siqueira JE (2015) Total and specific bacterial levels in the apical root canal system of teeth with post-treatment apical periodontitis. J Endod 41:1037–1042. CrossRefGoogle Scholar
  16. 16.
    Rodrigues RCV, Antunes HS, Neves MAS, Siqueira JF, Rocas IN (2015a) Infection control in retreatment cases: in vivo antibacterial effects of 2 instrumentation systems. J Endod 41:1600–1605. CrossRefGoogle Scholar
  17. 17.
    Fouad AF, Barry J, Caimano M, Clawson M, Zhu Q, Carver R, Hazlett K, Radolf JD (2002) PCR-based identification of bacteria associated with endodontic infections. J Clin Microbiol 40:3223–3231. CrossRefGoogle Scholar
  18. 18.
    Kim SY, Shin Y, Lee CY, Jung IY (2013) In vivo quantitative evaluation of live and dead bacteria in root canal infection by using propidium monoazide with real-time PCR. J Endod 39:1359–1363. CrossRefGoogle Scholar
  19. 19.
    Dron-In S, Bassitta R, Schwaiger K, Bauer J, Bauer J, Hölzel CS (2015) Specific amplification of bacterial DNA by optimized so called universal bacterial primers in samples rich of plant DNA. J Microbiol Methods 113:50–56. CrossRefGoogle Scholar
  20. 20.
    Rocas IN, Siqueira JF Jr (2012) Characterization of microbiota of root canal-treated teeth with posttreatment disease. J Clin Microbiol 50:1721–1724. CrossRefGoogle Scholar
  21. 21.
    Shen Y, Stojicic S, Haapasalo M (2010) Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture. J Endod 36:1820–1823. CrossRefGoogle Scholar
  22. 22.
    Castelo-Baz P, Varela-Patino P, Cantatore G, Dominguez-Perez A, Ruiz-Pinon M, Miguens-Vila R, Martin-Biedma B (2016) In vitro comparison of passive and continuous ultrasonic irrigation in curved root canals. J Clin Exp Dent 8:e437–e441. Google Scholar
  23. 23.
    Tran KT, Torabinejad M, Shabahang S, Retamozo B, Aprecio RM, Chen JW (2013) Comparison of efficacy of pulverization and sterile paper point techniques for sampling root canals. J Endod 39:1057–1059. CrossRefGoogle Scholar
  24. 24.
    Stojicic S, Shen Y, Haapasalo M (2013) Effect of the source of biofilm bacteria, level of biofilm maturation, and type of disinfecting agent on the susceptibility of biofilm bacteria to antibacterial agents. J Endod 39:473–477. CrossRefGoogle Scholar
  25. 25.
    Alturaiki S, Lamphon H, Edrees H, Ahlquist M (2015) Efficacy of 3 different irrigation systems on removal of calcium hydroxide from the root canal: a scanning electron microscopic study. J Endod 41:97–101. CrossRefGoogle Scholar
  26. 26.
    Yücel AÇ, Gürel M, Güler E, Karabucak B (2013) Comparison of final irrigation techniques in removal of calcium hydroxide. Aust Endod J 39:116–121. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Duo Zhang
    • 1
  • Ya Shen
    • 1
  • César de la Fuente-Núñez
    • 2
    • 3
  • Markus Haapasalo
    • 1
    Email author
  1. 1.Division of Endodontics, Department of Oral Biological & Medical Sciences, Faculty of DentistryThe University of British ColumbiaVancouverCanada
  2. 2.Synthetic Biology Group, MIT Synthetic Biology Center, Research Laboratory of Electronics, Department of Biological Engineering, Department of Electrical Engineering and Computer ScienceMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Broad Institute of MIT and HarvardCambridgeUSA

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