Trial Outcomes & Findings for Fluoride Retention in the Mouth of Older Adults (NCT NCT04239872)

NCT ID: NCT04239872

Last Updated: 2023-01-31

Results Overview

Dental biofilm was collected at baseline (before mouthwash(es)), and the fluid phase was separated using centrifugation. Fluoride concentration in the dental biofilm fluid was measured using an ion-specific electrode adapted for microanalysis.

• Recruitment status: COMPLETED
• Study phase: PHASE1
• Target enrollment: 20 participants
• Primary outcome timeframe: Baseline
• Results posted on: 2023-01-31

Participant Flow

Participant milestones

Measure	Fluoride Mouthwash Alone, Then Calcium Mouthwash Before a Fluoride Mouthwash Participants with normal to dry mouth first tested a Fluoride mouthwash containing 226 ppm of fluoride, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours. After a washout period of at least 3 days, they then tested a Calcium mouthwash (150 millimoles per liter of calcium) immediately before the Fluoride mouthwash, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours.	Calcium Mouthwash Before a Fluoride Mouthwash, Then Fluoride Mouthwash Alone Participants with normal to dry mouth first tested a Calcium mouthwash (150 millimoles per liter of calcium) immediately before a Fluoride mouthwash containing 226 ppm of fluoride, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours. After a washout period of at least 3 days, they then tested the Fluoride mouthwash alone, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours.
Overall Study STARTED	11	9
Overall Study COMPLETED	11	8
Overall Study NOT COMPLETED	0	1

Reasons for withdrawal

Measure	Fluoride Mouthwash Alone, Then Calcium Mouthwash Before a Fluoride Mouthwash Participants with normal to dry mouth first tested a Fluoride mouthwash containing 226 ppm of fluoride, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours. After a washout period of at least 3 days, they then tested a Calcium mouthwash (150 millimoles per liter of calcium) immediately before the Fluoride mouthwash, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours.	Calcium Mouthwash Before a Fluoride Mouthwash, Then Fluoride Mouthwash Alone Participants with normal to dry mouth first tested a Calcium mouthwash (150 millimoles per liter of calcium) immediately before a Fluoride mouthwash containing 226 ppm of fluoride, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours. After a washout period of at least 3 days, they then tested the Fluoride mouthwash alone, and fluoride concentration in saliva and dental biofilm was assessed overtime for up to 2 hours.
Overall Study Lost to Follow-up	0	1

Baseline Characteristics

Fluoride Retention in the Mouth of Older Adults

Baseline characteristics by cohort

Measure	Participants With Normal Salivary Flow to Dry Mouth n=20 Participants Participants were randomly assigned to two arms, with varying sequence of treatment used, under a crossover design: Fluoride mouthwash alone, then Calcium mouthwash before a fluoride mouthwash; OR Calcium mouthwash before a fluoride mouthwash, then Fluoride mouthwash alone Fluoride mouthwash consisted of Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), used to rinse the mouth for 1 minute and expectorated. Calcium and fluoride mouthwash: Calcium lactate, at a concentration of 150 millimoles per liter, used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), used to rinse the mouth for 1 minute, and expectorated.
Age, Categorical <=18 years	0 Participants n=5 Participants
Age, Categorical Between 18 and 65 years	0 Participants n=5 Participants
Age, Categorical >=65 years	20 Participants n=5 Participants
Age, Continuous	70.9 years STANDARD_DEVIATION 4.6 • n=5 Participants
Sex: Female, Male Female	12 Participants n=5 Participants
Sex: Female, Male Male	8 Participants n=5 Participants
Ethnicity (NIH/OMB) Hispanic or Latino	0 Participants n=5 Participants
Ethnicity (NIH/OMB) Not Hispanic or Latino	18 Participants n=5 Participants
Ethnicity (NIH/OMB) Unknown or Not Reported	2 Participants n=5 Participants
Race (NIH/OMB) American Indian or Alaska Native	0 Participants n=5 Participants
Race (NIH/OMB) Asian	0 Participants n=5 Participants
Race (NIH/OMB) Native Hawaiian or Other Pacific Islander	0 Participants n=5 Participants
Race (NIH/OMB) Black or African American	1 Participants n=5 Participants
Race (NIH/OMB) White	18 Participants n=5 Participants
Race (NIH/OMB) More than one race	0 Participants n=5 Participants
Race (NIH/OMB) Unknown or Not Reported	1 Participants n=5 Participants
Region of Enrollment United States	20 participants n=5 Participants
Unstimulated salivary flow rate, mL/min	0.3 mL/min STANDARD_DEVIATION 0.33 • n=5 Participants
Stimulated salivary flow rate, mL/min	1.21 mL/min STANDARD_DEVIATION 0.87 • n=5 Participants

PRIMARY outcome

Timeframe: Baseline

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected at baseline (before mouthwash(es)), and the fluid phase was separated using centrifugation. Fluoride concentration in the dental biofilm fluid was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=13 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Fluid at Baseline	0.24 parts per million (ppm) of fluoride Interval 0.18 to 0.32	0.22 parts per million (ppm) of fluoride Interval 0.17 to 0.29

PRIMARY outcome

Timeframe: 15 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 15 minutes after mouthwash(es), and the fluid phase was separated using centrifugation. Fluoride concentration in the dental biofilm fluid was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Fluid at 15 Minutes	15.27 parts per million (ppm) of fluoride Interval 7.84 to 29.76	12.29 parts per million (ppm) of fluoride Interval 9.46 to 15.98

PRIMARY outcome

Timeframe: 60 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 60 minutes after mouthwash(es), and the fluid phase was separated using centrifugation. Fluoride concentration in the dental biofilm fluid was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=13 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=15 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Fluid at 60 Minutes	1.57 parts per million (ppm) of fluoride Interval 0.79 to 3.11	4.07 parts per million (ppm) of fluoride Interval 2.63 to 6.31

PRIMARY outcome

Timeframe: 120 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 120 minutes after mouthwash(es), and the fluid phase was separated using centrifugation. Fluoride concentration in the dental biofilm fluid was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=10 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Fluid at 120 Minutes	0.63 parts per million (ppm) of fluoride Interval 0.4 to 0.99	2.58 parts per million (ppm) of fluoride Interval 1.33 to 5.0

PRIMARY outcome

Timeframe: Baseline

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected at baseline (before mouthwash(es)). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of fluoride reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Fluoride concentration in the dental biofilm solids acid extract was measured using an ion-specific electrode adapted for microanalysis; protein concentration in the alkali extract was determined using the Lowry colorimetric method. Fluoride concentration in the biofilm was expressed as micrograms of fluoride/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Solids at Baseline	0.17 micrograms fluoride/milligram protein Interval 0.09 to 0.32	0.13 micrograms fluoride/milligram protein Interval 0.07 to 0.24

PRIMARY outcome

Timeframe: 15 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 15 minutes after mouthwash(es). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of fluoride reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Fluoride concentration in the dental biofilm solids acid extract was measured using an ion-specific electrode adapted for microanalysis; protein concentration in the alkali extract was determined using the Lowry colorimetric method. Fluoride concentration in the biofilm was expressed as micrograms of fluoride/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Solids at 15 Minutes	0.44 micrograms fluoride/milligram protein Interval 0.27 to 0.73	5.30 micrograms fluoride/milligram protein Interval 3.87 to 7.27

PRIMARY outcome

Timeframe: 60 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm will be collected 60 minutes after mouthwash(es). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of fluoride reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Fluoride concentration in the dental biofilm solids acid extract was measured using an ion-specific electrode adapted for microanalysis; protein concentration in the alkali extract was determined using the Lowry colorimetric method. Fluoride concentration in the biofilm was expressed as micrograms of fluoride/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=15 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Solids at 60 Minutes	0.23 micrograms fluoride/miligram protein Interval 0.1 to 0.53	5.98 micrograms fluoride/miligram protein Interval 4.36 to 8.21

PRIMARY outcome

Timeframe: 120 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 120 minutes after mouthwash(es). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of fluoride reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Fluoride concentration in the dental biofilm solids acid extract was measured using an ion-specific electrode adapted for microanalysis; protein concentration in the alkali extract was determined using the Lowry colorimetric method. Fluoride concentration in the biofilm was expressed as micrograms of fluoride/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=15 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Dental Biofilm Solids at 120 Minutes	0.18 micrograms fluoride/miligram protein Interval 0.08 to 0.4	4.84 micrograms fluoride/miligram protein Interval 3.61 to 6.49

PRIMARY outcome

Timeframe: Baseline, 15, 60 and 120 minutes after mouthwash(es)

Population: The area under the curve was calculated only for participants with all time points collected. This accounts for the smaller number of participants analyzed for this variable.

Fluoride concentration in dental biofilm fluid collected at baseline (before mouthwash(es)), 15, 60 and 120 minutes after mouthwash(es) was expressed as the area under the curve of fluoride concentration in dental biofilm fluid as a function of time (parts per million x min).

Outcome measures

Measure	Fluoride Mouthwash n=10 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=8 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Area Under the Curve of Fluoride Concentration in Dental Biofilm Fluid Overtime	841.6 parts per million x minutes Standard Deviation 543.2	764.4 parts per million x minutes Standard Deviation 234.1

PRIMARY outcome

Timeframe: Baseline, 15, 60 and 120 minutes after mouthwash

Population: The area under the curve was calculated only for participants with all time points collected. This accounts for the smaller number of participants analyzed for this variable.

Fluoride concentration in dental biofilm solids collected at baseline (before mouthwash(es)), 15, 60 and 120 minutes after mouthwash(es) was expressed as the area under the curve of fluoride concentration in dental biofilm solids as a function of time (micrograms of fluoride x minutes/ milligram of proteins).

Outcome measures

Measure	Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=10 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Area Under the Curve of Fluoride Concentration in Dental Biofilm Solids Overtime	37.85 microgram fluoride/milligram prot x min Interval 18.33 to 78.14	521.2 microgram fluoride/milligram prot x min Interval 376.7 to 721.2

SECONDARY outcome

Timeframe: Baseline

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at baseline (before mouthwash(es)), for 5 minutes. Fluoride concentration in unstimulated saliva was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=20 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Saliva at Baseline	0.14 parts per million (ppm) of fluoride Interval 0.09 to 0.21	0.11 parts per million (ppm) of fluoride Interval 0.09 to 0.13

SECONDARY outcome

Timeframe: 16-21 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected 16 minutes after mouthwash(es), for 5 minutes. Fluoride concentration in unstimulated saliva was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=20 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Saliva at 16 Minutes	4.60 parts per million (ppm) of fluoride Interval 2.7 to 7.81	20.36 parts per million (ppm) of fluoride Interval 15.08 to 27.48

SECONDARY outcome

Timeframe: 30-35 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected 30 minutes after mouthwash(es), for 5 minutes. Fluoride concentration in unstimulated saliva was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Saliva at 30 Minutes	2.18 parts per million (ppm) of fluoride Interval 1.21 to 3.9	17.05 parts per million (ppm) of fluoride Interval 14.54 to 19.99

SECONDARY outcome

Timeframe: 61-66 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected 61 minutes after mouthwash(es), for 5 minutes. Fluoride concentration in unstimulated saliva was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=20 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Saliva at 61 Minutes	1.07 parts per million (ppm) of fluoride Interval 0.67 to 1.72	9.98 parts per million (ppm) of fluoride Interval 6.89 to 14.5

SECONDARY outcome

Timeframe: 90-95 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected 90 minutes after mouthwash(es), for 5 minutes. Fluoride concentration in unstimulated saliva was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Saliva at 90 Minutes	0.80 parts per million (ppm) of fluoride Interval 0.49 to 1.32	7.66 parts per million (ppm) of fluoride Interval 5.61 to 10.5

SECONDARY outcome

Timeframe: 121-126 minutes after mouthwash(es)

Unstimulated saliva was collected 121 minutes after mouthwash(es), for 5 minutes. Fluoride concentration in unstimulated saliva was measured using an ion-specific electrode adapted for microanalysis.

Outcome measures

Measure	Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=20 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Fluoride Concentration in Saliva at 121 Minutes	0.46 parts per million (ppm) of fluoride Interval 0.3 to 0.69	3.71 parts per million (ppm) of fluoride Interval 2.18 to 6.3

SECONDARY outcome

Timeframe: Baseline

Population: Due to the small amount of biofilm collected from participants, resulting in a reduced biofilm fluid volume, there was not enough sample left for calcium measurement after fluoride concentration in the fluid was determined.

Dental biofilm will be collected at baseline (before mouthwash(es)), and the fluid phase will be separated using centrifugation. Calcium concentration in the dental biofilm fluid will be measured using an ion-specific electrode adapted for microanalysis, and will be expressed in millimolar.

Outcome measures

Outcome data not reported

SECONDARY outcome

Timeframe: 15 minutes after mouthwash(es)

Population: Due to the small amount of biofilm collected from participants, resulting in a reduced biofilm fluid volume, there was not enough sample left for calcium measurement after fluoride concentration in the fluid was determined.

Dental biofilm will be collected 15 minutes after mouthwash(es), and the fluid phase will be separated using centrifugation. Calcium concentration in the dental biofilm fluid will be measured using an ion-specific electrode adapted for microanalysis, and will be expressed in millimolar.

Outcome measures

Outcome data not reported

SECONDARY outcome

Timeframe: 60 minutes after mouthwash(es)

Population: Due to the small amount of biofilm collected from participants, resulting in a reduced biofilm fluid volume, there was not enough sample left for calcium measurement after fluoride concentration in the fluid was determined.

Dental biofilm will be collected 60 minutes after mouthwash(es), and the fluid phase will be separated using centrifugation. Calcium concentration in the dental biofilm fluid will be measured using an ion-specific electrode adapted for microanalysis, and will be expressed in millimolar.

Outcome measures

Outcome data not reported

SECONDARY outcome

Timeframe: 120 minutes after mouthwash(es)

Population: Due to the small amount of biofilm collected from participants, resulting in a reduced biofilm fluid volume, there was not enough sample left for calcium measurement after fluoride concentration in the fluid was determined.

Dental biofilm will be collected 120 minutes after mouthwash(es), and the fluid phase will be separated using centrifugation. Calcium concentration in the dental biofilm fluid will be measured using an ion-specific electrode adapted for microanalysis, and will be expressed in millimolar.

Outcome measures

Outcome data not reported

SECONDARY outcome

Timeframe: Baseline

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected at baseline (before mouthwash(es)). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of calcium reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Calcium concentration in the dental biofilm solids acid extract was measured using a colorimetric reaction (Arsenazo III); protein concentration in the alkali extract was determined using the Lowry colorimetric method. Calcium concentration in the biofilm was expressed as micrograms of calcium/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Dental Biofilm Solids at Baseline	5.74 micrograms calcium/miligram protein Interval 3.78 to 8.72	4.67 micrograms calcium/miligram protein Interval 3.01 to 7.23

SECONDARY outcome

Timeframe: 15 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 15 minutes after mouthwash(es). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of calcium reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Calcium concentration in the dental biofilm solids acid extract was measured using a colorimetric reaction (Arsenazo III); protein concentration in the alkali extract was determined using the Lowry colorimetric method. Calcium concentration in the biofilm was expressed as micrograms of calcium/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Dental Biofilm Solids at 15 Minutes	4.48 micrograms calcium/miligram protein Interval 3.02 to 6.65	8.88 micrograms calcium/miligram protein Interval 6.8 to 11.6

SECONDARY outcome

Timeframe: 60 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 60 minutes after mouthwash(es). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of calcium reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Calcium concentration in the dental biofilm solids acid extract was measured using a colorimetric reaction (Arsenazo III); protein concentration in the alkali extract was determined using the Lowry colorimetric method. Calcium concentration in the biofilm was expressed as micrograms of calcium/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=15 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Dental Biofilm Solids at 60 Minutes	6.64 micrograms calcium/miligram protein Interval 3.4 to 13.0	12.4 micrograms calcium/miligram protein Interval 8.22 to 18.8

SECONDARY outcome

Timeframe: 120 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Dental biofilm was collected 120 minutes after mouthwash(es). The fluid phase of the biofilm was collected after centrifugation, and the remaining solids were treated with strong acid for dissolution of calcium reservoirs. Solids remaining after acid extraction were treated with strong alkali for total dissolution of the biofilm and estimation of biofilm mass by protein concentration. Calcium concentration in the dental biofilm solids acid extract was measured using a colorimetric reaction (Arsenazo III); protein concentration in the alkali extract was determined using the Lowry colorimetric method. Calcium concentration in the biofilm was expressed as micrograms of calcium/milligrams of protein.

Outcome measures

Measure	Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=15 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Dental Biofilm Solids at 120 Minutes	5.08 micrograms calcium/miligram protein Interval 2.94 to 8.77	9.66 micrograms calcium/miligram protein Interval 6.91 to 13.5

SECONDARY outcome

Timeframe: Baseline

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at baseline (before mouthwash(es)), for 5 minutes. Calcium concentration in unstimulated saliva was measured using a colorimetric reaction (Arsenazo III).

Outcome measures

Measure	Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Saliva at Baseline	1.57 milimolar Standard Deviation 0.64	1.76 milimolar Standard Deviation 0.64

SECONDARY outcome

Timeframe: 16-21 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at 16 minutes after mouthwash(es), for 5 minutes. Calcium concentration in unstimulated saliva was measured using a colorimetric reaction (Arsenazo III).

Outcome measures

Measure	Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Saliva at 16 Minutes	1.14 milimolar Interval 0.92 to 1.42	2.38 milimolar Interval 1.65 to 3.43

SECONDARY outcome

Timeframe: 30-35 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at 30 minutes after mouthwash(es), for 5 minutes. Calcium concentration in unstimulated saliva was measured using a colorimetric reaction (Arsenazo III).

Outcome measures

Measure	Fluoride Mouthwash n=16 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Saliva at 30 Minutes	1.12 milimolar Interval 0.87 to 1.45	2.03 milimolar Interval 1.62 to 2.53

SECONDARY outcome

Timeframe: 61-66 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at 61 minutes after mouthwash(es), for 5 minutes. Calcium concentration in unstimulated saliva was measured using a colorimetric reaction (Arsenazo III).

Outcome measures

Measure	Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Saliva at 61 Minutes	1.62 milimolar Standard Deviation 0.60	2.01 milimolar Standard Deviation 0.60

SECONDARY outcome

Timeframe: 90-95 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at 90 minutes after mouthwash(es), for 5 minutes. Calcium concentration in unstimulated saliva was measured using a colorimetric reaction (Arsenazo III).

Outcome measures

Measure	Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Saliva at 90 Minutes	1.50 milimolar Interval 1.21 to 1.88	1.74 milimolar Interval 1.47 to 2.07

SECONDARY outcome

Timeframe: 121-126 minutes after mouthwash(es)

Population: A smaller number of participants analyzed for this variable means that they did not have a sample collected at this time point, or that the amount collected was not enough for the analysis.

Unstimulated saliva was collected at 121 minutes after mouthwash(es), for 5 minutes. Calcium concentration in unstimulated saliva was measured using a colorimetric reaction (Arsenazo III).

Outcome measures

Measure	Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=19 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Calcium Concentration in Saliva at 121 Minutes	1.60 milimolar Standard Deviation 0.57	1.80 milimolar Standard Deviation 0.59

SECONDARY outcome

Timeframe: Baseline, 16, 30, 61, 90 and 121 minutes after mouthwash(es)

Population: The area under the curve was calculated only for participants with all time points collected. This accounts for the smaller number of participants analyzed for this variable.

Fluoride concentration in unstimulated saliva collected at baseline (before mouthwash(es)), 16, 30, 61, 90 and 121 minutes after mouthwash(es) was expressed as the area under the curve of fluoride concentration in saliva as a function of time.

Outcome measures

Measure	Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=18 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Area Under the Curve of Fluoride Concentration in Saliva Overtime	186 parts per million x minutes Interval 111.0 to 312.0	1415 parts per million x minutes Interval 1170.0 to 1713.0

SECONDARY outcome

Timeframe: Baseline, 15, 60 and 120 minutes after mouthwash(es)

Population: Due to the small amount of biofilm collected from participants, resulting in a reduced biofilm fluid volume, there was not enough sample left for calcium measurement after fluoride concentration in the fluid was determined. Therefore, the area under the curve could not be estimated for this variable.

Calcium concentration in dental biofilm fluid collected at baseline (before mouthwash(es)), 15, 60 and 120 minutes after mouthwash(es) will be expressed as the area under the curve of calcium concentration in dental biofilm fluid as a function of time (millimolar x min).

Outcome measures

Outcome data not reported

SECONDARY outcome

Timeframe: Baseline, 15, 60 and 120 minutes after mouthwash(es)

Population: The area under the curve was calculated only for participants with all time points collected. This accounts for the smaller number of participants analyzed for this variable.

Calcium concentration in dental biofilm solids collected at baseline (before mouthwash(es)), 15, 60 and 120 minutes after mouthwash(es) was expressed as the area under the curve of calcium concentration in dental biofilm solids as a function of time.

Outcome measures

Measure	Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=10 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Area Under the Curve of Calcium Concentration in Dental Biofilm Solids Overtime	780 micrograms calcium x time/milligram prot Interval 424.0 to 1436.0	1165 micrograms calcium x time/milligram prot Interval 732.0 to 1854.0

SECONDARY outcome

Timeframe: Baseline, 16, 30, 61, 90 and 121 minutes after a mouthwash(es)

Population: The area under the curve was calculated only for participants with all time points collected. This accounts for the smaller number of participants analyzed for this variable.

Calcium concentration in unstimulated saliva collected at baseline (before mouthwash(es)), 16, 30, 61, 90 and 121 minutes after mouthwash(es) was expressed as the area under the curve of calcium concentration in saliva as a function of time (millimolar x min).

Outcome measures

Measure	Fluoride Mouthwash n=14 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the fluoride mouthwash: Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), was used to rinse the mouth for 1 minute and expectorated.	Calcium Mouthwash Before Fluoride Mouthwash n=17 Participants Participants with normal salivary flow to dry mouth were treated, in a crossover design, with two interventions: a fluoride mouthwash alone, or a fluoride mouthwash preceded by a calcium mouthwash. Participants were randomized to determine which intervention they used in the first experimental phase; the other intervention will the tested in the second phase. This arm refers to the use of the calcium mouthwash before the fluoride mouthwash: Calcium lactate, at a concentration of 150 millimolar, was used to rinse the mouth for 1 minute and expectorated; immediately after, a sodium fluoride rinse, at a concentration of 0.05% (226 ppm of fluoride), was used to rinse the mouth for 1 minute, and expectorated.
Area Under the Curve of Calcium Concentration in Saliva Overtime	176 milimolar x minutes Standard Deviation 46.5	242 milimolar x minutes Standard Deviation 63.8

Adverse Events

Fluoride Mouthwash

Serious events: 0 serious events

Other events: 0 other events

Deaths: 0 deaths

Calcium Mouthwash Before Fluoride Mouthwash

Serious events: 0 serious events

Other events: 0 other events

Deaths: 0 deaths

Serious adverse events

Adverse event data not reported

Other adverse events

Adverse event data not reported

Additional Information

Dr. Livia Tenuta

University of Michigan School of Dentistry

Phone: 734-763-3703

Email: litenuta@umich.edu

Results disclosure agreements

• Principal investigator is a sponsor employee
• Publication restrictions are in place