Fluoride Retention in the Mouth of Older Adults

• Sponsor: University of Michigan (Other)
• Overall Status: Completed
• CT.gov ID: NCT04239872
• Collaborator: (none)
• Enrollment: 20
• Location: 1
• Arms: 2
• Actual Duration (Months): 12.8
• Patients Per Site Per Month: 1.6

Study Details

Study Description

Brief Summary

Individuals with dry mouth (a reduction in the production of saliva) suffer from high rates of oral diseases. Dry mouth is a common problem among older individuals, because many medications to control chronic conditions (such as blood pressure, bladder-control problems and depression) reduce the flow of saliva. For these individuals, fluoride is used to maintain a good oral health. Usually, higher fluoride concentration products, such as prescription toothpastes, are recommended. In this project, the ability to increase fluoride retention in the mouth by using calcium before a conventional fluoride rinse will be tested in older adults with a range of salivary flow rates. This approach was never tested in patients with dry mouth, for whom the treatment with calcium may also be beneficial. A crossover, clinical study with two experimental phases will be conducted, in which patients with a range of saliva flows (from normal to dry mouth) will rinse with a fluoride rinse only (used at over the counter concentration), or a fluoride rinse preceded by a calcium rinse. The concentration of fluoride and calcium in the saliva and dental plaque residues will be determined up to two hours after the rinse(s), to test the effect of the approach to optimize fluoride retention in the mouth of patients with dry mouth.

Condition or Disease	Intervention/Treatment	Phase
Hyposalivation	Drug: Fluoride mouthwash; Drug: Calcium and fluoride mouthwash	Phase 1

Study Design

• Study Type: Interventional
• Actual Enrollment: 20 participants
• Allocation: Randomized
• Intervention Model: Crossover Assignment
• Masking: None (Open Label)
• Primary Purpose: Prevention
• Official Title: Optimizing Fluoride Retention in the Mouth of Older Adults With Distinct Salivary Flow Rates
• Actual Study Start Date: Mar 11, 2020
• Actual Primary Completion Date: Apr 5, 2021
• Actual Study Completion Date: Apr 5, 2021

Arms and Interventions

Arm	Intervention/Treatment
Experimental: Fluoride mouthwash alone, then Calcium mouthwash before a fluoride mouthwash Participants with normal to dry mouth will first test a Fluoride mouthwash containing 226 ppm of fluoride, and fluoride concentration in saliva and dental biofilm will be assessed overtime for up to 2 hours. After a washout period of at least 3 days, they will then test a Calcium mouthwash (150 milimolars of calcium) immediately before the Fluoride mouthwash, and fluoride concentration in saliva and dental biofilm will be assessed overtime for up to 2 hours.	Drug: Fluoride mouthwash; Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), will be used to rinse the mouth for 1 minute and expectorated.; Other Names: Fluoride rinse ; Drug: Calcium and fluoride mouthwash; Calcium lactate, at a concentration of 150 milimolar, will be 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), will be used to rinse the mouth for 1 minute, and expectorated.; Other Names: Calcium and fluoride rinse
Experimental: Calcium mouthwash before a fluoride mouthwash, then Fluoride mouthwash alone Participants with normal to dry mouth will first test a Calcium mouthwash (150 milimolars of calcium) immediately before a Fluoride mouthwash containing 226 ppm of fluoride, and fluoride concentration in saliva and dental biofilm will be assessed overtime for up to 2 hours. After a washout period of at least 3 days, they will then test the Fluoride mouthwash alone, and fluoride concentration in saliva and dental biofilm will be assessed overtime for up to 2 hours.	Drug: Fluoride mouthwash; Sodium fluoride, at a concentration of 0.05% (226 parts per million (ppm) of fluoride), will be used to rinse the mouth for 1 minute and expectorated.; Other Names: Fluoride rinse ; Drug: Calcium and fluoride mouthwash; Calcium lactate, at a concentration of 150 milimolar, will be 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), will be used to rinse the mouth for 1 minute, and expectorated.; Other Names: Calcium and fluoride rinse

Outcome Measures

Primary Outcome Measures

1. Fluoride Concentration in Dental Biofilm Fluid at Baseline [Baseline]

   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.

2. Fluoride Concentration in Dental Biofilm Fluid at 15 Minutes [15 minutes after mouthwash(es)]

   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.

3. Fluoride Concentration in Dental Biofilm Fluid at 60 Minutes [60 minutes after mouthwash(es)]

   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.

4. Fluoride Concentration in Dental Biofilm Fluid at 120 Minutes [120 minutes after mouthwash(es)]

   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.

5. Fluoride Concentration in Dental Biofilm Solids at Baseline [Baseline]

   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.

6. Fluoride Concentration in Dental Biofilm Solids at 15 Minutes [15 minutes after mouthwash(es)]

   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.

7. Fluoride Concentration in Dental Biofilm Solids at 60 Minutes [60 minutes after mouthwash(es)]

   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.

8. Fluoride Concentration in Dental Biofilm Solids at 120 Minutes [120 minutes after mouthwash(es)]

   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.

9. Area Under the Curve of Fluoride Concentration in Dental Biofilm Fluid Overtime [Baseline, 15, 60 and 120 minutes after mouthwash(es)]

   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).

10. Area Under the Curve of Fluoride Concentration in Dental Biofilm Solids Overtime [Baseline, 15, 60 and 120 minutes after mouthwash]

    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).

Secondary Outcome Measures

1. Fluoride Concentration in Saliva at Baseline [Baseline]

   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.

2. Fluoride Concentration in Saliva at 16 Minutes [16-21 minutes after mouthwash(es)]

   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.

3. Fluoride Concentration in Saliva at 30 Minutes [30-35 minutes after mouthwash(es)]

   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.

4. Fluoride Concentration in Saliva at 61 Minutes [61-66 minutes after mouthwash(es)]

   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.

5. Fluoride Concentration in Saliva at 90 Minutes [90-95 minutes after mouthwash(es)]

   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.

6. Fluoride Concentration in Saliva at 121 Minutes [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.

7. Calcium Concentration in Dental Biofilm Fluid at Baseline [Baseline]

   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.

8. Calcium Concentration in Dental Biofilm Fluid at 15 Minutes [15 minutes after mouthwash(es)]

   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.

9. Calcium Concentration in Dental Biofilm Fluid at 60 Minutes [60 minutes after mouthwash(es)]

   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.

10. Calcium Concentration in Dental Biofilm Fluid at 120 Minutes [120 minutes after mouthwash(es)]

    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.

11. Calcium Concentration in Dental Biofilm Solids at Baseline [Baseline]

    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.

12. Calcium Concentration in Dental Biofilm Solids at 15 Minutes [15 minutes after mouthwash(es)]

    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.

13. Calcium Concentration in Dental Biofilm Solids at 60 Minutes [60 minutes after mouthwash(es)]

    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.

14. Calcium Concentration in Dental Biofilm Solids at 120 Minutes [120 minutes after mouthwash(es)]

    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.

15. Calcium Concentration in Saliva at Baseline [Baseline]

    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).

16. Calcium Concentration in Saliva at 16 Minutes [16-21 minutes after mouthwash(es)]

    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).

17. Calcium Concentration in Saliva at 30 Minutes [30-35 minutes after mouthwash(es)]

    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).

18. Calcium Concentration in Saliva at 61 Minutes [61-66 minutes after mouthwash(es)]

    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).

19. Calcium Concentration in Saliva at 90 Minutes [90-95 minutes after mouthwash(es)]

    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).

20. Calcium Concentration in Saliva at 121 Minutes [121-126 minutes after mouthwash(es)]

    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).

21. Area Under the Curve of Fluoride Concentration in Saliva Overtime [Baseline, 16, 30, 61, 90 and 121 minutes after mouthwash(es)]

    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.

22. Area Under the Curve of Calcium Concentration in Dental Biofilm Fluid Overtime [Baseline, 15, 60 and 120 minutes after mouthwash(es)]

    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).

23. Area Under the Curve of Calcium Concentration in Dental Biofilm Solids Overtime [Baseline, 15, 60 and 120 minutes after mouthwash(es)]

    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.

24. Area Under the Curve of Calcium Concentration in Saliva Overtime [Baseline, 16, 30, 61, 90 and 121 minutes after a mouthwash(es)]

    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).

Eligibility Criteria

Criteria

• Ages Eligible for Study: 65 Years and Older
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: Yes

Inclusion Criteria:

• Provision of signed and dated informed consent form

• Stated willingness to comply with all study procedures and availability for the duration of the study

• Good general health as evidenced by medical history

• Good oral health as evidenced by a clinical oral exam

• Having at least 20 teeth in the mouth, being at least 4 (natural or crowned) teeth in all four quadrants of the mouth

• Having salivary flow rate ranging from normal to hyposalivation according to direct flow rate determination methods

• Agreement to adhere to the study protocol

Exclusion Criteria:

• Unable to understand and/or follow study instructions

• Active periodontitis

• Oral pain

• In need of urgent dental care

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	School of Dentistry, University of Michigan	Ann Arbor	Michigan	United States	48109

Sponsors and Collaborators

• University of Michigan

Investigators

• Principal Investigator: Livia M Tenuta, DDS, MS, PhD, Associate Professor, University of Michigan

Study Documents (Full-Text)

• Study Protocol and Statistical Analysis Plan - Aug 20, 2020

More Information

Publications

None provided.

• Responsible Party: Livia Tenuta, DDS, MS, PhD, Associate Professor, University of Michigan
• ClinicalTrials.gov Identifier: NCT04239872
• Other Study ID Numbers: HUM00159624; UL1TR002240
• First Posted: Jan 27, 2020
• Last Update Posted: Jun 21, 2022
• Last Verified: Jun 1, 2022

• Individual Participant Data (IPD) Sharing Statement: No
• Plan to Share IPD: No
• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No
• Product Manufactured in and Exported from the U.S.: No
• Additional relevant MeSH terms: Xerostomia; Calcium, Dietary; Calcium; Fluorides

Study Results

Participant Flow

Arm/Group Title	Fluoride Mouthwash Alone, Then Calcium Mouthwash Before a Fluoride Mouthwash	Calcium Mouthwash Before a Fluoride Mouthwash, Then Fluoride Mouthwash Alone
Arm/Group Description	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.	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.
Period Title: Overall Study
STARTED	11	9
COMPLETED	11	8
NOT COMPLETED	0	1

Baseline Characteristics

Arm/Group Title	Participants With Normal Salivary Flow to Dry Mouth
Arm/Group Description	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.
Overall Participants	20
Age (Count of Participants)
<=18 years	0 0%
Between 18 and 65 years	0 0%
>=65 years	20 100%
Age (years) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [years]	70.9 (4.6)
Sex: Female, Male (Count of Participants)
Female	12 60%
Male	8 40%
Ethnicity (NIH/OMB) (Count of Participants)
Hispanic or Latino	0 0%
Not Hispanic or Latino	18 90%
Unknown or Not Reported	2 10%
Race (NIH/OMB) (Count of Participants)
American Indian or Alaska Native	0 0%
Asian	0 0%
Native Hawaiian or Other Pacific Islander	0 0%
Black or African American	1 5%
White	18 90%
More than one race	0 0%
Unknown or Not Reported	1 5%
Region of Enrollment (participants) [Number]
United States	20 100%
Unstimulated salivary flow rate, mL/min (mL/min) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mL/min]	0.3 (0.33)
Stimulated salivary flow rate, mL/min (mL/min) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mL/min]	1.21 (0.87)

Outcome Measures

1. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Fluid at Baseline
Description	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.
Time Frame	Baseline

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	14	13
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	0.24	0.22

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.8412
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 11 pairs of data, DF=10
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	-0.00778
	Confidence Interval	(2-Sided) 95%; -0.09209 to 0.07653
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

2. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Fluid at 15 Minutes
Description	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.
Time Frame	15 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	14	14
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	15.27	12.29

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.6504
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 12 pairs of data, DF=11
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	-0.06907
	Confidence Interval	(2-Sided) 95%; -0.3955 to 0.2573
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

3. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Fluid at 60 Minutes
Description	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.
Time Frame	60 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	13	15
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	1.57	4.07

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0024
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 11 pairs of data, DF=10
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.5307
	Confidence Interval	(2-Sided) 95%; 0.2372 to 0.8243
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

4. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Fluid at 120 Minutes
Description	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.
Time Frame	120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	16	10
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	0.63	2.58

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0013
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 9 pairs of data, DF=8
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.5643
	Confidence Interval	(2-Sided) 95%; 0.2938 to 0.8348
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

5. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Solids at Baseline
Description	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.
Time Frame	Baseline

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	17	16
Geometric Mean (95% Confidence Interval) [micrograms fluoride/milligram protein]	0.17	0.13

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.2041
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 15 pairs of data, DF=14
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	-0.1135
	Confidence Interval	(2-Sided) 95%; -0.2964 to 0.06929
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

6. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Solids at 15 Minutes
Description	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.
Time Frame	15 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	16	14
Geometric Mean (95% Confidence Interval) [micrograms fluoride/milligram protein]	0.44	5.30

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 13 pairs of data, DF=12
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	1.095
	Confidence Interval	(2-Sided) 95%; 0.7736 to 1.416
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

7. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Solids at 60 Minutes
Description	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.
Time Frame	60 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	15	16
Geometric Mean (95% Confidence Interval) [micrograms fluoride/miligram protein]	0.23	5.98

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 13 pairs of data, DF=12
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	1.469
	Confidence Interval	(2-Sided) 95%; 1.112 to 1.825
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

8. Primary Outcome

Title	Fluoride Concentration in Dental Biofilm Solids at 120 Minutes
Description	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.
Time Frame	120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	18	15
Geometric Mean (95% Confidence Interval) [micrograms fluoride/miligram protein]	0.18	4.84

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 14 pairs of data, DF=13
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	1.549
	Confidence Interval	(2-Sided) 95%; 1.081 to 2.017
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

9. Primary Outcome

Title	Area Under the Curve of Fluoride Concentration in Dental Biofilm Fluid Overtime
Description	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).
Time Frame	Baseline, 15, 60 and 120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	10	8
Mean (Standard Deviation) [parts per million x minutes]	841.6 (543.2)	764.4 (234.1)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.5543
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 6 pairs of data, DF=5
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	-115.6
	Confidence Interval	(2-Sided) 95%; -584.8 to 353.6
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

10. Primary Outcome

Title	Area Under the Curve of Fluoride Concentration in Dental Biofilm Solids Overtime
Description	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).
Time Frame	Baseline, 15, 60 and 120 minutes after mouthwash

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	14	10
Geometric Mean (95% Confidence Interval) [microgram fluoride/milligram prot x min]	37.85	521.2

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0003
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 8 pairs of data, DF=7
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	1.328
	Confidence Interval	(2-Sided) 95%; 0.8420 to 1.814
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

11. Secondary Outcome

Title	Fluoride Concentration in Saliva at Baseline
Description	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.
Time Frame	Baseline

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	19	20
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	0.14	0.11

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.2824
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 19 pairs of data, DF=18
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	-0.09023
	Confidence Interval	(2-Sided) 95%; -0.2613 to 0.08082
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

12. Secondary Outcome

Title	Fluoride Concentration in Saliva at 16 Minutes
Description	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.
Time Frame	16-21 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	17	20
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	4.60	20.36

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0002
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 17 pairs of data, DF=16
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.6269
	Confidence Interval	(2-Sided) 95%; 0.3488 to 0.9050
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

13. Secondary Outcome

Title	Fluoride Concentration in Saliva at 30 Minutes
Description	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.
Time Frame	30-35 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	17	18
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	2.18	17.05

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 17 pairs of data, DF=16
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.8946
	Confidence Interval	(2-Sided) 95%; 0.6671 to 1.122
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

14. Secondary Outcome

Title	Fluoride Concentration in Saliva at 61 Minutes
Description	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.
Time Frame	61-66 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	18	20
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	1.07	9.98

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 18 pairs of data, DF=17
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.9730
	Confidence Interval	(2-Sided) 95%; 0.7597 to 1.186
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

15. Secondary Outcome

Title	Fluoride Concentration in Saliva at 90 Minutes
Description	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.
Time Frame	90-95 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	18	19
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	0.80	7.66

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 18 pairs of data, DF=17
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.9827
	Confidence Interval	(2-Sided) 95%; 0.7758 to 1.19
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

16. Secondary Outcome

Title	Fluoride Concentration in Saliva at 121 Minutes
Description	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.
Time Frame	121-126 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	19	20
Geometric Mean (95% Confidence Interval) [parts per million (ppm) of fluoride]	0.46	3.71

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 19 pairs of data, DF=18
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.9181
	Confidence Interval	(2-Sided) 95%; 0.6573 to 1.179
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

17. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Fluid at Baseline
Description	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.
Time Frame	Baseline

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	0	0

18. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Fluid at 15 Minutes
Description	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.
Time Frame	15 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	0	0

19. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Fluid at 60 Minutes
Description	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.
Time Frame	60 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	0	0

20. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Fluid at 120 Minutes
Description	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.
Time Frame	120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	0	0

21. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Solids at Baseline
Description	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.
Time Frame	Baseline

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	17	16
Geometric Mean (95% Confidence Interval) [micrograms calcium/miligram protein]	5.74	4.67

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.3283
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 15 pairs of data, DF=14
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	-0.05652
	Confidence Interval	(2-Sided) 95%; -0.1762 to 0.06829
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

22. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Solids at 15 Minutes
Description	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.
Time Frame	15 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	16	14
Geometric Mean (95% Confidence Interval) [micrograms calcium/miligram protein]	4.48	8.88

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0058
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 13 pairs of data, DF=12
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.3411
	Confidence Interval	(2-Sided) 95%; 0.1189 to 0.5633
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

23. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Solids at 60 Minutes
Description	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.
Time Frame	60 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	15	16
Geometric Mean (95% Confidence Interval) [micrograms calcium/miligram protein]	6.64	12.4

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0213
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 13 pairs of data, DF=12
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.2976
	Confidence Interval	(2-Sided) 95%; 0.05266 to 0.5426
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

24. Secondary Outcome

Title	Calcium Concentration in Dental Biofilm Solids at 120 Minutes
Description	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.
Time Frame	120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	18	15
Geometric Mean (95% Confidence Interval) [micrograms calcium/miligram protein]	5.08	9.66

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.014
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 14 pairs of data, DF=13
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.3120
	Confidence Interval	(2-Sided) 95%; 0.07454 to 0.5495
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

25. Secondary Outcome

Title	Calcium Concentration in Saliva at Baseline
Description	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).
Time Frame	Baseline

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	17	19
Mean (Standard Deviation) [milimolar]	1.57 (0.64)	1.76 (0.64)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.1393
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 17 pairs of data, DF=16
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.2524
	Confidence Interval	(2-Sided) 95%; -0.09153 to 0.5962
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

26. Secondary Outcome

Title	Calcium Concentration in Saliva at 16 Minutes
Description	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).
Time Frame	16-21 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	16	19
Geometric Mean (95% Confidence Interval) [milimolar]	1.14	2.38

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0009
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 16 pairs of data, DF=15
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.2921
	Confidence Interval	(2-Sided) 95%; 0.1414 to 0.4427
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

27. Secondary Outcome

Title	Calcium Concentration in Saliva at 30 Minutes
Description	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).
Time Frame	30-35 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	16	18
Geometric Mean (95% Confidence Interval) [milimolar]	1.12	2.03

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0008
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 16 pairs of data, DF=15
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.2433
	Confidence Interval	(2-Sided) 95%; 0.1195 to 0.3671
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

28. Secondary Outcome

Title	Calcium Concentration in Saliva at 61 Minutes
Description	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).
Time Frame	61-66 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	18	17
Mean (Standard Deviation) [milimolar]	1.62 (0.60)	2.01 (0.60)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.1515
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 16 pairs of data, DF=15
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.3306
	Confidence Interval	(2-Sided) 95%; -0.1356 to 0.7969
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

29. Secondary Outcome

Title	Calcium Concentration in Saliva at 90 Minutes
Description	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).
Time Frame	90-95 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	17	19
Geometric Mean (95% Confidence Interval) [milimolar]	1.50	1.74

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.1712
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 17 pairs of data, DF=16
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.04851
	Confidence Interval	(2-Sided) 95%; -0.02327 to 0.1203
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

30. Secondary Outcome

Title	Calcium Concentration in Saliva at 121 Minutes
Description	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).
Time Frame	121-126 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	19	19
Mean (Standard Deviation) [milimolar]	1.60 (0.57)	1.80 (0.59)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.1188
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 18 pairs of data, DF=17
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.2789
	Confidence Interval	(2-Sided) 95%; -0.07934 to 0.6371
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

31. Secondary Outcome

Title	Area Under the Curve of Fluoride Concentration in Saliva Overtime
Description	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.
Time Frame	Baseline, 16, 30, 61, 90 and 121 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	14	18
Geometric Mean (95% Confidence Interval) [parts per million x minutes]	186	1415

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	<0.0001
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 14 pairs of data, DF=13
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.8956
	Confidence Interval	(2-Sided) 95%; 0.7205 to 1.071
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

32. Secondary Outcome

Title	Area Under the Curve of Calcium Concentration in Dental Biofilm Fluid Overtime
Description	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).
Time Frame	Baseline, 15, 60 and 120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	0	0

33. Secondary Outcome

Title	Area Under the Curve of Calcium Concentration in Dental Biofilm Solids Overtime
Description	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.
Time Frame	Baseline, 15, 60 and 120 minutes after mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	14	10
Geometric Mean (95% Confidence Interval) [micrograms calcium x time/milligram prot]	780	1165

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups. To obtain normal distribution, data used in the statistical analysis were transformed to the log10.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0475
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 8 pairs of data, DF=7
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	0.3199
	Confidence Interval	(2-Sided) 95%; 0.004686 to 0.6351
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference of transformed (log10) data = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

34. Secondary Outcome

Title	Area Under the Curve of Calcium Concentration in Saliva Overtime
Description	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).
Time Frame	Baseline, 16, 30, 61, 90 and 121 minutes after a mouthwash(es)

Outcome Measure Data

Analysis Population Description

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.

Arm/Group Title	Fluoride Mouthwash	Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.	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.
Measure Participants	14	17
Mean (Standard Deviation) [milimolar x minutes]	176 (46.5)	242 (63.8)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	Fluoride Mouthwash, Calcium Mouthwash Before Fluoride Mouthwash
	Comments	A paired t-test was used to compare the two groups.
	Type of Statistical Test	Superiority
	Comments
Statistical Test of Hypothesis	p-Value	0.0004
	Comments	The threshold for statistical significance was set at p=0.05.
	Method	t-test, 2 sided
	Comments	Paired t-test was used; there were 13 pairs of data, DF=12
Method of Estimation	Estimation Parameter	Mean Difference (Net)
	Estimated Value	50.38
	Confidence Interval	(2-Sided) 95%; 27.46 to 73.30
	Parameter Dispersion	Type: Value:
	Estimation Comments	Treatment difference = Calcium mouthwash before fluoride mouthwash - Fluoride mouthwash

Adverse Events

Time Frame	Until 126 minutes after mouthwash(es) use.
Arm/Group Title	Fluoride Mouthwash		Calcium Mouthwash Before Fluoride Mouthwash
Arm/Group Description	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.		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.
All Cause Mortality
	Fluoride Mouthwash		Calcium Mouthwash Before Fluoride Mouthwash
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/19 (0%)		0/20 (0%)
Serious Adverse Events
	Fluoride Mouthwash		Calcium Mouthwash Before Fluoride Mouthwash
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/19 (0%)		0/20 (0%)
Other (Not Including Serious) Adverse Events
	Fluoride Mouthwash		Calcium Mouthwash Before Fluoride Mouthwash
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/19 (0%)		0/20 (0%)

Limitations/Caveats

The amount of biofilm collected from participants at the different time points was not enough for all the analyses planned in a number of occasions. This accounts for the lack of data for the calcium concentration in the biofilm fluid. This experiment was conducted during the COVID-19 pandemic.

More Information

Certain Agreements

All Principal Investigators ARE employed by the organization sponsoring the study.

There is NOT an agreement between Principal Investigators and the Sponsor (or its agents) that restricts the PI's rights to discuss or publish trial results after the trial is completed.