Purpose: The purpose of this study was to investigate the clinical and microbiological effectiveness of the ozone application in stepwise excavation of primary molars.

Methods: This study was conducted in vivo conditions with 105 lower primary second molars that had deep caries lesions with the risk of pulpal exposure. The teeth were randomly divided into three groups: Conventional stepwise excavation without any disinfectant, 2% chlorhexidine digluconate(CHX) and ozone application. In four different stages (after; initial excavation, ozone/CHX application, four months, final excavation), dentine samples were collected for microbiological analysis of mutans streptococci, lactobacilli and total number of colony forming units. Clinical changes as dentine colour, humidity, consistency were recorded. The data were analysed by Mann-Whitney U, Friedman and chi-square test.

Informed consent including possible risks, discomforts and benefits about the procedure were obtained from all parents of the young patients.

The required sample size for this study was 35 in each subgroup (82 percent power, two-sided five percent significance level) for the detection of a significant difference. This study was conducted on 105 patients aged 6 to 10 years (59 girls, 46 boys) who attended the Pediatric Dentistry Department. None of the patients included in the study were medically compromised. Each patient had one mandibular second primary molar with deep carious lesion and pulp perforation risk. All teeth with deep carious lesions were examined clinically and radiographically.

The following inclusion criteria were used;

• Active carious lesion in deep dentine of primary second molars,
• Absence of clinical symptoms of irreversible pulpitis such as spontaneous pain or longtime pain against thermal and chemical stimuli,
• Absence of sensitivity to percussion or palpation, pathological mobility, swelling or fistula any discoloration except for carious lesion.
• Absence of any interradicular lesions, periapical radiolucency, pathological resorptions and pulpal calcifications.

A randomized, controlled, clinical trial was designed in a sample of 105 primary molars (35 teeth in each group) as three treatment groups: Step-wise excavation without any disinfectant (control group), step-wise excavation with CHX application (positive control group), and step-wise excavation with gaseous ozone application (experimental group).

All procedures were performed by one investigator. After mandibular anesthesia of the tooth, rubber dam isolation was provided. Carious enamel, lateral walls of the cavity and necrotic dentin was removed with carbide burs. Removal of carious dentin was performed with dentin excavators and low speed-burs, and left some caries at the central part. It was decided to leave potentially mineralizable affected dentin by hand sensitivity. Following the same procedures, the teeth were randomly divided into 3 groups (n=35):

Control Group: After partial removal of carious dentin, calcium hydroxide [Ca(OH)2] base material (Dycal; Dentsply International Milford, USA) was placed on the remaining carious dentin without applying any cavity disinfectants.

Positive Control Group: After partial removal of carious dentin, 2% CHX solution (Cavity Cleanser, Bisco, USA) was applied to the cavity for 60 s with an applicator brush for disinfection procedure. According to the manufacturer instructions, puddled solution was removed with a new brush without dry to leave site moist. Then, Ca(OH)2 base (Dycal; Dentsply) was placed on the remaining carious dentin.

Experimental Group: After partial removal of carious dentin, ozone gas (Heal ozone, KaVo Dental, Germany) with the concentration of 2100 ppm was applied to the cavity for 60 s for disinfection procedure. Ozone production stopped if the airtight seal over the tooth was broken during treatment, therefore, silicon caps were selected according to the size of each tooth. And Ca(OH)2 base was placed on the remaining carious dentin.

All the teeth were then temporarily sealed with glass ionomer cement and coating agent. After 4 months, radiographic and clinical examinations were repeated. Following rubber dam isolation and anesthesia, temporary fillings were removed. Removing of Ca(OH)2 on the remaining carious dentin was carefully performed with excavators. Subsequent to this procedure, remaining demineralized dentin was completely removed in all groups. Floor of the cavity was then covered with Ca(OH)2 base material (Dycal; Dentsply/Caulk) and glass ionomer lining. The cavities were then restored with composite resin after the 2-stages self-etch bond application.

Microbiological analysis:

During these procedures dentin samples were collected with a sterile carbide bur #14 from each group for microbiological analyses.

Clinical photograph was taken with dental explorers pointing 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.14 round carbide bur and the respective samples were placed into 1 ml thiogluconate medium then transported to 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 Streptococcus mutans, rogosa agar for Lactobacilli and Brain Hearth agar supplemented 5% blood for the total number of CFU were used. Tenfold dilution were plated onto agars and incubated anaerobically for 48 hours and the colonies were counted on the convenient dilution.

Clinical Evaluation For the clinical analysis, the colour, consistency and humidity of the dentine were also recorded during taking microbiological samples.

Color findings classifications were recorded according to following criteria: Light yellow, yellow, light brown, dark brown, black. Classification of dentin consistency was determined by probing and criteria were "very soft", " "soft", "moderate hard" and "hard". Dentin humidity was recorded as "wet" and "dry". Accordingly, the dentine was probed and if moisture leakage occurred, it was classified as wet or if it did not then it would be classified as dry.

Inclusion Criteria:

• Previously untreated, vital and asymptomatic lower primary molars with deep caries lesions considered likely to result in pulp exposure if they were treated by a single and terminal excavation
• Positive pulp sensibility tested by an electric pulp tester and cold stimulation,
• Mild discomfort from chemical and thermal stimuli,
• Cooperative children and parents willing to follow the instructions and report for follow-up.

Exclusion Criteria:

• Signs of irreversible pulpitis (spontaneous pain, prolonged pain response etc.)
• The presence of percussion or palpation sensitivity, pathological mobility, or infection symptoms like fistula or abscess or discoloration in the clinical examination,
• Absence of normal lamina dura and periodontal range, presence of lesion, internal or external resorption or calcification in or around the root in the radiological examination,
• Children with special health care needs.