Subject Allocation and Randomization Schedule

Once the subject was considered high risk and accepted into the study, all the necessary consent and HIPAA forms were reviewed and signed. A stratified block randomization schedule was used to assign subjects to intervention or control arms in a parallel group design with a 1:1 allocation. A permuted block design (Table 3) developed

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with a computer random number generator ensured that equal numbers of subjects (n=12) were assigned to each arm. This is critical in trials with small numbers of subjects. The purpose of randomization was to balance the arms as much as possible with respect to known and unknown prognostic factors for dental caries. Within blocks, stratification ensured that both groups were balanced on salivary pH (low versus high pH), an

important characteristic influencing dental caries risk. The salivary pH was measured by placing pHion Diagnostic pH Test Strips (pHion Balance, Scottsdale, AZ) in the buccal vestibule for 15 seconds. The chairside pH was determined by color transition of the strips and read as numerical scores in increments of 0.25. Those subjects 6.5 and above were considered low risk and those below were considered as high risk. Overall, this design optimized conditions to test the efficacy of the CariFree intervention on risk of dental caries.

The intervention group received the CariFree Treatment products to be used for 3 months as per manufacturer recommendations and the control group continued oral hygiene practices. Both protocols are described in detail in the following sections.

4.2.1 CariFree Treatment Protocol

The 12 subjects in the intervention group were provided with a treatment kit along with verbal and written instructions. The protocol is as follows:

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4.2.1.1 CariFree Treatment rinse

This rinse is used in the first 30 day of starting treatment. The rinse consists of sodium fluoride 0.05%, water, xylitol, menthol, natural flavors, sodium benzoate,

poloxomer 407, sodium hydroxide, sodium hypochlorite. Instructions include using rinse for two times daily for 30 days. Instructions- Mix 5mL from bottle A with 5ml from bottle B and rinse with solution for 1 minute and spit out. Wait 30 minutes before eating or drinking.

4.2.1.2 CariFree Maintenance rinse

The maintenance rinse is used after the 30 day use of the treatment rinse. It contains sodium fluoride 0.05%, menthol, natural flavors, polysorbate 20, potassium sorbate, sodium benzoate, sodium bicarbonate, water, xylitol. It must be used twice daily after brushing and flossing for 60 days. Instructions- Rinse with 10mL of the solution for 1 minute and spit out. Wait 30 minutes before eating or drinking.

4.2.1.3 CarieFree Oral neutralizer gel

The gel contains glycerin, hydrogenated starch hydrolysate, hydroxethyl

cellulose, menthol, natural flavors, polysorbate 20, potassium sorbate, sodium benzoate, sodium bicarbonate, sodium hydroxide, sodium lauryl sulfate, water, xylitol. Instructions- Use twice daily as a toothpaste supplement for 90 days.

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4.2.1.4 CariFree Boost

Boost is an oral spray with a pH close to neutral. It contains purified water, xylitol, glycerin, sodium benzoate, calcium hydroxide, natural flavors, and natural colors. Instructions-Use 2-3 sprays in mouth as often as needed to relieve dry mouth and

neutralize acids. Between meals and before bedtime recommended.

4.2.1.5 CariFree Xylitol gum

A small sample (6 parts) of chewing gum was included. This did not constitute the center of the intervention treatment, but was used more to introduce patients to a xylitol based chewing gum. It contains xylitol, gum base, natural flavors, glycerin, gum arabic, soy lecithin, calcium acetate, beeswax. Instructions- Chew 2 pieces, 3-5 times daily. Recommended after meals or when dry mouth/bad breath occurs.

4.2.2 Control group

The 12 subjects in the control group were asked to follow conventional oral hygiene practices for 90 days. This constitutes brushing 2 times daily with an over the counter fluoridated tooth paste and flossing once daily.

Subjects in both the intervention (n=12) and control group (n=12) had sample collection at baseline (visit 1) for their ATP bioluminescence scores and stimulated saliva. The measurements were repeated at day 30 (visit 2), and day 90 (visit 3) from the baseline records.

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4.2.3 Stimulated Saliva Collection

For the saliva collection, the participants were given a paraffin wax tablet and instructed to chew on it for 5 minutes and saliva was collected and expelled into a sterile calibrated collection container up to the 5 minute mark. All samples were collected at the same time of the day for each subject. The sample was coded with the unique participant identifier and transported on ice to the laboratory for determination of buffering capacity, the mutans streptococci (MS) and lactobacilli (LB) counts. The saliva was diluted four- fold in 0.005N HCl and the final pH determined after ten minutes. The pH values of 4.0 or less were considered high risk, 4.1 to 4.9 intermediate risk and 5.0 or greater were considered normal. Serial 10-fold dilutions of saliva were done and quantitatively plated (Spiral PlaterTM _{model DU2; Spiral Systems Inc.) to Difco}TM _{Mitis Salivarius Agar} supplemented with Chapman Tellurite solution and with bacitracin for the selective enumeration of MS and to BBLTM

LBS Agar for the selective isolation and enumeration of Lactobacilli (Figure 6). Counts were performed after 48 hours on a ProtoCOL RGB model no. 90000 (Microbiological International Inc.). The limit of detection for bacterial counts was 204 CFU/mL of saliva. MS counts less than 104 CFU/mL and for LB less than 103 CFU/mL were considered normally healthy. Total saliva and flow rates were also recorded. All samples were destroyed after testing.

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