Effectiveness of the ozone application in two-visit indirect pulp therapy of permanent molars with deep carious lesion: a randomized clinical trial



The aim of this randomized, three-arm parallel, single-blinded clinical trial was to evaluate the clinical and microbiological effectiveness of the ozone application in two-visit indirect pulp therapy.

Materials and methods

One hundred five lower first molar teeth with deep caries lesion were included and randomly assigned three groups to apply the two-visit indirect pulp therapy. Treatment procedure was applied without any disinfectant (control), with 60-s 2% chlorhexidine digluconate (CHX) or 60-s ozone application. In four different stages (after initial excavation, ozone/CHX application before the temporary restoration, 4 months later immediately after removing temporary restoration, and final excavation), dentin humidity, consistency, and color properties were recorded to evaluate the clinical characteristics of the tissue, and standard dentin samples were collected for the microbiological analysis of mutans streptococci, lactobacilli, and the total number of colony-forming units. The data were analyzed by using Mann-Whitney U test for multiple comparisons.


The remaining dentin became harder, drier, and darker after 4 months in all groups. However, CHX and ozone application were statistically better than the control group (p < 0.05). There was a gradual decrease in the total number of microorganisms in all groups. While cavity disinfectant applications were improved the antibacterial efficacy (control, 79.11%; CHX, 98.39%; ozone, 93.33%), CHX application exhibited a greater significant reduction than both groups (p = 0.000).


The two-visit indirect pulp therapy yielded successful results for all study groups. However, CHX would be conveniently preferable due to improving the treatment success.

Clinical relevance

The two-visit indirect pulp therapy applied with cavity disinfectant is a proper alternative treatment procedure in deep carious lesions, instead of conventional technique.

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  1. 1.

    Bjorndal L, Reit C, Bruun G, Markvart M, Kjaeldgaard M, Nasman P, Thordrup M, Dige I, Nyvad B, Fransson H, Lager A, Ericson D, Petersson K, Olsson J, Santimano EM, Wennstrom A, Winkel P, Gluud C (2010) Treatment of deep caries lesions in adults: randomized clinical trials comparing stepwise vs. direct complete excavation, and direct pulp capping vs. partial pulpotomy. Eur J Oral Sci 118:290–297. https://doi.org/10.1111/j.1600-0722.2010.00731.x

    Article  PubMed  Google Scholar 

  2. 2.

    Magnusson BO, Sundell SO (1977) Stepwise excavation of deep carious lesions in primary molars. J Int Assoc Dent Child 8:36–40

    PubMed  Google Scholar 

  3. 3.

    Leksell E, Ridell K, Cvek M, Mejare I (1996) Pulp exposure after stepwise versus direct complete excavation of deep carious lesions in young posterior permanent teeth. Endod Dent Traumatol 12:192–196

    Article  Google Scholar 

  4. 4.

    Orhan AI, Oz FT, Orhan K (2010) Pulp exposure occurrence and outcomes after 1- or 2-visit indirect pulp therapy vs complete caries removal in primary and permanent molars. Pediatr Dent 32:347–355

    PubMed  Google Scholar 

  5. 5.

    Falster CA, Araujo FB, Straffon LH, Nor JE (2002) Indirect pulp treatment: in vivo outcomes of an adhesive resin system vs calcium hydroxide for protection of the dentin-pulp complex. Pediatr Dent 24:241–248

    PubMed  Google Scholar 

  6. 6.

    Fairbourn DR, Charbeneau GT, Loesche WJ (1980) Effect of improved Dycal and IRM on bacteria in deep carious lesions. J Am Dent Assoc 100:547–552

    Article  Google Scholar 

  7. 7.

    Leung RL, Loesche WJ, Charbeneau GT (1980) Effect of Dycal on bacteria in deep carious lesions. J Am Dent Assoc 100:193–197

    Article  Google Scholar 

  8. 8.

    Bjorndal L, Larsen T, Thylstrup A (1997) A clinical and microbiological study of deep carious lesions during stepwise excavation using long treatment intervals. Caries Res 31:411–417

    Article  Google Scholar 

  9. 9.

    Bjorndal L, Larsen T (2000) Changes in the cultivable flora in deep carious lesions following a stepwise excavation procedure. Caries Res 34:502–508 16631

    Article  Google Scholar 

  10. 10.

    Maltz M, de Oliveira EF, Fontanella V, Bianchi R (2002) A clinical, microbiologic, and radiographic study of deep caries lesions after incomplete caries removal. Quintessence Int 33:151–159

    PubMed  Google Scholar 

  11. 11.

    Pinto AS, de Araujo FB, Franzon R, Figueiredo MC, Henz S, Garcia-Godoy F, Maltz M (2006) Clinical and microbiological effect of calcium hydroxide protection in indirect pulp capping in primary teeth. Am J Dent 19:382–386

    PubMed  Google Scholar 

  12. 12.

    Orhan AI, Oz FT, Ozcelik B, Orhan K (2008) A clinical and microbiological comparative study of deep carious lesion treatment in deciduous and young permanent molars. Clin Oral Investig 12:369–378. https://doi.org/10.1007/s00784-008-0208-6

    Article  PubMed  Google Scholar 

  13. 13.

    Bjorndal L, Thylstrup A (1998) A practice-based study on stepwise excavation of deep carious lesions in permanent teeth: a 1-year follow-up study. Community Dent Oral Epidemiol 26:122–128

    Article  Google Scholar 

  14. 14.

    Polydorou O, Halili A, Wittmer A, Pelz K, Hahn P (2012) The antibacterial effect of gas ozone after 2 months of in vitro evaluation. Clin Oral Investig 16:545–550. https://doi.org/10.1007/s00784-011-0524-0

    Article  PubMed  Google Scholar 

  15. 15.

    Duque C, Negrini Tde C, Hebling J, Spolidorio DM (2005) Inhibitory activity of glass-ionomer cements on cariogenic bacteria. Oper Dent 30:636–640

    PubMed  Google Scholar 

  16. 16.

    Hori R, Kohno S, Hoshino E (1997) Bactericidal eradication from carious lesions of prepared abutments by an antibacterial temporary cement. J Prosthet Dent 77:348–352. https://doi.org/10.1016/S0022-3913(97)70157-4

    Article  PubMed  Google Scholar 

  17. 17.

    Kidd EA (1991) Role of chlorhexidine in the management of dental caries. Int Dent J 41:279–286

    PubMed  Google Scholar 

  18. 18.

    Azarpazhooh A, Limeback H (2008) The application of ozone in dentistry: a systematic review of literature. J Dent 36:104–116. https://doi.org/10.1016/j.jdent.2007.11.008

    Article  PubMed  Google Scholar 

  19. 19.

    Millar BJ, Hodson N (2007) Assessment of the safety of two ozone delivery devices. J Dent 35:195–200. https://doi.org/10.1016/j.jdent.2006.07.010

    Article  PubMed  Google Scholar 

  20. 20.

    Celiberti P, Pazera P, Lussi A (2006) The impact of ozone treatment on enamel physical properties. Am J Dent 19:67–72

    PubMed  Google Scholar 

  21. 21.

    Nagayoshi M, Kitamura C, Fukuizumi T, Nishihara T, Terashita M (2004) Antimicrobial effect of ozonated water on bacteria invading dentinal tubules. J Endod 30:778–781 00004770-200411000-00007

    Article  Google Scholar 

  22. 22.

    Kidd EA (2004) How ‘clean’ must a cavity be before restoration? Caries Res 38:305–313. https://doi.org/10.1159/000077770

    Article  PubMed  Google Scholar 

  23. 23.

    Bjorndal L, Kidd EA (2005) The treatment of deep dentine caries lesions. Dent Update 32:402–4, 407-10, 413

    Article  Google Scholar 

  24. 24.

    Schulz KF, Altman DG, Moher D, Group C (2010) CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. PLoS Med 7:e1000251. https://doi.org/10.1371/journal.pmed.1000251

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Bjorndal L, Demant S, Dabelsteen S (2014) Depth and activity of carious lesions as indicators for the regenerative potential of dental pulp after intervention. J Endod 40:S76–S81. https://doi.org/10.1016/j.joen.2014.01.016

    Article  PubMed  Google Scholar 

  26. 26.

    Kidd EA, Joyston-Bechal S, Beighton D (1993) Microbiological validation of assessments of caries activity during cavity preparation. Caries Res 27:402–408

    Article  Google Scholar 

  27. 27.

    Ricketts DN, Kidd EA, Innes N and Clarkson J (2006) Complete or ultraconservative removal of decayed tissue in unfilled teeth. Cochrane Database Syst Rev:CD003808. https://doi.org/10.1002/14651858

  28. 28.

    Cohen S, Berman LH, Blanco L, Bakland L, Kim JS (2006) A demographic analysis of vertical root fractures. J Endod 32:1160–1163. https://doi.org/10.1016/j.joen.2006.07.008

    Article  PubMed  Google Scholar 

  29. 29.

    Kidd E (2000) The cartwright prize. caries removal and the pulpo-dentinal complex. Dent Update 27:476–482

    Article  Google Scholar 

  30. 30.

    Tziafas D, Smith AJ, Lesot H (2000) Designing new treatment strategies in vital pulp therapy. J Dent 28:77–92 S0300-5712(99)00047-0

    Article  Google Scholar 

  31. 31.

    Borges FM, de Melo MA, Lima JP, Zanin IC, Rodrigues LK (2012) Antimicrobial effect of chlorhexidine digluconate in dentin: in vitro and in situ study. J Conserv Dent 15:22–26. https://doi.org/10.4103/0972-0707.92601

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Kollmuss M, Kist S, Obermeier K, Pelka AK, Hickel R, Huth KC (2014) Antimicrobial effect of gaseous and aqueous ozone on caries pathogen microorganisms grown in biofilms. Am J Dent 27:134–138

    PubMed  Google Scholar 

  33. 33.

    Bocci V (2004) Ozone as Janus: this controversial gas can be either toxic or medically useful. Mediat Inflamm 13:3–11. https://doi.org/10.1080/0962935062000197083

    Article  Google Scholar 

  34. 34.

    Safwat O, Elkateb M, Dowidar K, El Meligy O (2017) Clinical evaluation of ozone on dentinal lesions in young permanent molars using the stepwise excavation. J Clin Pediatr Dent 41:429–441. https://doi.org/10.17796/1053-4628-41.6.3

    Article  PubMed  Google Scholar 

  35. 35.

    Camp JH, Barrett EJ, Pulver F (2002) Pediatric endodontics: endodontic treatment for the primary and young, permanent dentition. In: Cohen S, Burns RC (eds) Book title. Mosby Inc, 8th edn St Louis

    Google Scholar 

  36. 36.

    Safwat O, Elkateb M, Dowidar K, Salam HA, El Meligy O (2018) Microbiological evaluation of ozone on dentinal lesions in young permanent molars using the stepwise excavation. J Clin Pediatr Dent 42:11–20. https://doi.org/10.17796/1053-4628-42.1.3

    Article  PubMed  Google Scholar 

  37. 37.

    Krunic J, Stojanovic N, Dukic L, Roganovic J, Popovic B, Simic I, Stojic D (2018) Clinical antibacterial effectiveness and biocompatibility of gaseous ozone after incomplete caries removal. Clin Oral Investig. https://doi.org/10.1007/s00784-018-2495-x

  38. 38.

    Kapdan A, Oztas N, Sumer Z (2013) Comparing the antibacterial activity of gaseous ozone and chlorhexidine solution on a tooth cavity model. J Clin Exp Dent 5:e133–e137. https://doi.org/10.4317/jced.51130

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Hauser-Gerspach I, Pfaffli-Savtchenko V, Dahnhardt JE, Meyer J, Lussi A (2009) Comparison of the immediate effects of gaseous ozone and chlorhexidine gel on bacteria in cavitated carious lesions in children in vivo. Clin Oral Investig 13:287–291. https://doi.org/10.1007/s00784-008-0234-4

    Article  PubMed  Google Scholar 

  40. 40.

    Bocci VA (2006) Scientific and medical aspects of ozone therapy. State of the art. Arch Med Res 37:425–435. https://doi.org/10.1016/j.arcmed.2005.08.006

    Article  PubMed  Google Scholar 

  41. 41.

    Polydorou O, Pelz K, Hahn P (2006) Antibacterial effect of an ozone device and its comparison with two dentin-bonding systems. Eur J Oral Sci 114:349–353. https://doi.org/10.1111/j.1600-0722.2006.00363.x

    Article  PubMed  Google Scholar 

  42. 42.

    Fagrell TG, Dietz W, Lingstrom P, Steiniger F, Noren JG (2008) Effect of ozone treatment on different cariogenic microorganisms in vitro. Swed Dent J 32:139–147

    PubMed  Google Scholar 

  43. 43.

    Baysan A, Beighton D (2007) Assessment of the ozone-mediated killing of bacteria in infected dentine associated with non-cavitated occlusal carious lesions. Caries Res 41:337–341. https://doi.org/10.1159/000104790

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Johansson E, Claesson R, van Dijken JW (2009) Antibacterial effect of ozone on cariogenic bacterial species. J Dent 37:449–453. https://doi.org/10.1016/j.jdent.2009.02.004

    Article  PubMed  Google Scholar 

  45. 45.

    Ersin NK, Uzel A, Aykut A, Candan U, Eronat C (2006) Inhibition of cultivable bacteria by chlorhexidine treatment of dentin lesions treated with the ART technique. Caries Res 40:172–177. https://doi.org/10.1159/000091120

    Article  PubMed  Google Scholar 

  46. 46.

    Huang GT (2008) A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. J Dent 36:379–386. https://doi.org/10.1016/j.jdent.2008.03.002

    Article  PubMed  Google Scholar 

  47. 47.

    Pereira JC, Stanley HR (1981) Pulp capping: influence of the exposure site on pulp healing--histologic and radiographic study in dogs' pulp. J Endod 7:213–223. https://doi.org/10.1016/S0099-2399(81)80178-1

    Article  PubMed  Google Scholar 

  48. 48.

    Camp JH (1984) Pulp therapy for primary and young permanent teeth. Dent Clin N Am 28:651–668

    PubMed  Google Scholar 

  49. 49.

    Yoshiyama M, Doi J, Nishitani Y, Itota T, Tay FR, Carvalho RM, Pashley DH (2004) Bonding ability of adhesive resins to caries-affected and caries-infected dentin. J Appl Oral Sci 12:171–176

    Article  Google Scholar 

  50. 50.

    Hayashi M, Fujitani M, Yamaki C, Momoi Y (2011) Ways of enhancing pulp preservation by stepwise excavation-a systematic review. J Dent 39:95–107. https://doi.org/10.1016/j.jdent.2010.10.012

    Article  PubMed  Google Scholar 

  51. 51.

    Hevinga MA, Opdam NJ, Frencken JE, Truin GJ, Huysmans MCDNJM (2010) Does incomplete caries removal reduce strength of restored teeth? J Dent Res 89:1270–1275. https://doi.org/10.1177/0022034510377790

    Article  PubMed  Google Scholar 

  52. 52.

    Schwendicke F, Meyer-Lueckel H, Dorfer C, Paris S (2013) Failure of incompletely excavated teeth-a systematic review. J Dent 41:569–580. https://doi.org/10.1016/j.jdent.2013.05.004

    Article  PubMed  Google Scholar 

  53. 53.

    Gopikrishna V, Pradeep G, Venkateshbabu N (2009) Assessment of pulp vitality: a review. Int J Paediatr Dent 19:3–15. https://doi.org/10.1111/j.1365-263X.2008.00955.x

    Article  PubMed  Google Scholar 

  54. 54.

    Hoefler V, Nagaoka H, Miller CS (2016) Long-term survival and vitality outcomes of permanent teeth following deep caries treatment with step-wise and partial-caries-removal: a systematic review. J Dent 54:25–32. https://doi.org/10.1016/j.jdent.2016.09.009

    Article  PubMed  Google Scholar 

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This project was supported by Turkish Scientific and Technical Research Council (TUBITAK). Project No: 213S004.

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Correspondence to Merve Akcay.

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Durmus, N., Tok, Y.T., Kaya, S. et al. Effectiveness of the ozone application in two-visit indirect pulp therapy of permanent molars with deep carious lesion: a randomized clinical trial. Clin Oral Invest 23, 3789–3799 (2019). https://doi.org/10.1007/s00784-019-02808-0

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  • Chlorhexidine digluconate
  • Deep caries
  • Ozone
  • Stepwise excavation