A randomized, controlled, clinical trial was designed to evaluate the antibacterial effect of ozone application in two-visit indirect pulp therapy. The sample size for this study was determined 35 for each group (%82 power, 0.50 significance level). This study conducted with 49 female and 56 male patients aged between 6-13 years old (average 9.72). The entire patient had deep caries lesions with pulp perforation risk at one of the lower first permanent molars.

The vitality of teeth were tested by cold thermal test, necessary clinical and radiographic examinations were performed, then the teeth were randomly divided into three treatment groups as: two-visit indirect pulp therapy without any disinfectant (control group), two-visit indirect pulp therapy with chlorhexidine digluconate application (positive control group), and two-visit indirect pulp therapy with gaseous ozone application (experimental group). Treatment procedure started with the local anesthesia and isolation of the tooth with rubber-dam. The peripheral demineralized dentin and the superficial necrotic dentin were then completely removed, and left some caries at the central part. It was decided to leave potentially remineralizable affected dentin by hand sensitivity. In control group, the remaining caries dentin was covered with calcium hydroxide base material and cavity sealed with glass ionomer cement temporarily. In positive control group, following the excavation, 2% chlorhexidine digluconate was applied to the cavity for 60 seconds using a brush. According to the manufacturer instructions, puddled solution was removed with a new brush without dry to leave site moist. In experimental group, following the excavation, the cavity was exposed to gaseous ozone for 60 seconds, with an ozone delivery system. According the manufacturers, HealOzone produces ozone at a fixed concentration of 2,100 ppm. Ozone production is stopped if the airtight seal over the tooth is broken during treatment, therefore, silicon caps were selected according to the size of each tooth. After the disinfectant applications, procedure was completed as aforementioned in the control group for these two groups. After four months, the clinical and radiographic examinations were repeated in order to assess pathological conditions of the teeth before removed the temporary glass ionomer cement and calcium hydroxide base material. Subsequent to this procedure, remaining demineralized dentin was completely removed in all groups. Floor of the cavity was then covered with calcium hydroxide base material and glass ionomer cement as liner. Final restorations were performed with 2-stage self etch bond and composite.

For the clinical analyses, the consistency, colour and humidity of the dentine were recorded at three times: after initial excavation at the first stage (T0), at the end of four months period, immediately after removing the calcium hydroxide base at second stage (T2) and after final excavation at second stage (T3). The consistency of dentin was determined by probing and classified as; very soft, soft, medium hard and hard \[8\]. The colour of dentin was classified as, light yellow, yellow, light brown, dark brown/black. In addition, dentine was probed and if moisture leakage was occurred, it was classified as wet or if did not it would be classified as dry.

For the microbiological analyses, dentine samples were taken at four different times: from the remain dentine after initial excavation (T0), immediately after ozone/chlorhexidine application (T1), at the end of four months period immediately after removing the calcium hydroxide base material (T2) and after final excavation (T3). T1 stage did not exist in control group, because in this group no disinfectant was used. Clinical photographs were taken to ensure that all samples were collected from the same site in all following stages. The dentine samples were collected enough to fill the slots of a sterile No.16 round carbide bur (SS White Burs, Inc., New Jersey, USA) and the samples were placed into 1 ml thioglucolate medium and transported the microbiology laboratory in 2 hours to be analyzed for the mutans streptococci, lactobacilli and the total number of colony forming units (CFU).

The samples were homogenized on a vortex mixer for 15 seconds under laboratory conditions. Mitis Salivarius agar as the selective medium for Streptoccus mutans, Rogossa agar for Lactobacilli and Brain Hearth agar supplemented %5 blood for the total numbers of CFU were used. Samples were incubated anaerobically for 48 hours and the colonies were counted.