In Situ Comparison of the Remineralization Potential of Optimized Fluoride Dentifrice With Control Fluoride Dentifrice

This will be a double blind, single center, 3-way crossover design study. Two to three days before the start of each treatment period the subjects will have their teeth cleaned to remove all accessible plaque and calculus and will be provided with a non-fluoride dentifrice to use until their next visit. At the beginning of each testing period, two gauze-covered 4 mm round partially demineralized bovine enamel specimens will be placed in the buccal surface of two posterior denture teeth (the specimen site may extend into the buccal flange area, if needed) of the same side of the partial denture. Once specimens are placed, subjects will wear their partial dentures twenty-four hours a day and use their assigned toothpaste twice daily, as instructed, until their next visit. Specimens will be removed after two weeks, and the subjects will undergo at least a four- to five-day washout period followed by another cleaning and two to three day lead in period. This process will be repeated until all subjects have used all three test products. Changes in the mineral content of the enamel specimens will be assessed using surface microhardness (SMH) and transverse microradiography (TMR). Enamel fluoride uptake (EFU) will be determined using the microdrill enamel biopsy technique. In addition, the net acid resistance (NAR) and the comparative acid resistance (CAR) of the demineralized enamel specimens will be determined.

• 0 ppm F (placebo, negative control) (Drug), also known as Tom's of Maine Silly Strawberry: • Each subject will use this product during one of the three treatment periods in the crossover study design.
• 1100 ppm F as sodium fluoride (positive control) (Drug), also known as Crest Cavity Protection Toothpaste: • Each subject will use this product during one of the three treatment periods in the crossover study design.
• 1100 ppm F as sodium fluoride Test Product (Drug), also known as Pronamel Daily Protection: • Each subject will use this product during one of the three treatment periods in the crossover study design.

Primary outcomes

• Percent Surface Microhardness Recovery (%SMH Recovery) [at the end of each two-week treatment per]: The SMH test was used to assess changes in the mineral status of partially demineralized enamel specimens. %SMH Recovery = (D1-R)/(D1-B) ×100 B = indentation length (µm) of sound enamel specimen at baseline D1 = indentation length (µm) after in vitro demineralization R = indentation length (µm) after intra-oral exposure.

Secondary outcomes

• Enamel Fluoride Uptake (µg F/cm2) [at the end of each two-week treatment period]: The microdrill enamel biopsy technique will be used to analyze the fluoride content of the partially demineralized enamel specimens. Each enamel specimen will be mounted perpendicular to the long axis of a drill bit attached to a specially designed microdrill and drilled to a depth of \~100 µm through the entire lesion (four cores per specimen). The diameter of the drill hole will be determined using a calibrated microscope interfaced with an image analysis system. The amount of fluoride-uptake by enamel will be calculated based on the amount of fluoride divided by the area of the enamel cores and expressed as µg F/cm2
• Percent Net Acid Resistance [at the end of each two-week treatment period]: % Net Acid Resistance = \[(D1-D2) / (D1-B)\] * 100 B= Indentation length (µm) of sound enamel at baseline D1= Indentation length (µm) after first in vitro demineralization D2= Indentation length (µm) after second in vitro demineralization
• Percentage Comparative Acid Resistance [at the end of each two-week treatment period]: % Comparative Acid Resistance = \[(D2-R) / (D1-B)\] * 100 B= Indentation length (µm) of sound enamel at baseline R= Indentation length (µm) of enamel after in situ remineralization D1= Indentation length (µm) after first in vitro demineralization D2= Indentation length (µm) after second in vitro demineralization
• Integrated Mineral Loss (∆Z) [at the end of each two-week treatment period]: ∆Z= \[(lesion depth x 87) - area under the curve*\] calculated using Transverse Microradiography software program
• Lesion Depth (µm) [at the end of each two-week treatment period]: Lesion Depth - L (83% mineral i.e. 95% of the mineral content of sound enamel) determined using Transverse Microradiography software program
• Maximum Mineral Density at the Surface-zone (SZmax) [at the end of each two-week treatment period]: SZmax was determined using Transverse Microradiography software program

Sex: All, ages: 18 Years to 85 Years, accepts healthy volunteers

Inclusion Criteria:

1. provide voluntary, written informed consent;
2. be between 18 and 85 years old;
3. understand and be willing, able and likely to comply with all study procedures and restrictions;
4. be wearing a removable mandibular partial denture with sufficient room to accommodate two 4 mm round specimens in the buccal surface of two posterior denture teeth on the same side;
5. be willing and capable of wearing their removable partial denture 24 hours a day for three (3), two-week treatment periods;
6. be willing to allow study personnel to drill specimen sites in two denture teeth in the posterior section of one side of their lower partial denture, which may extend into the buccal flange area below the teeth;
7. be in good medical and dental health with no active caries or periodontal disease; NOTE: subjects presenting at screening with caries may continue in the study if their carious lesions are restored prior to beginning treatment 1; and
8. have a salivary flow rate in the range of normal values (unstimulated whole saliva flow rate ≥ 0.2 mL/min; gum base stimulated whole saliva flow rate ≥ 0.8 mL/min).

Exclusion Criteria:

1. currently be pregnant, intending to become pregnant during the study period or breast feeding;
2. currently have any medical condition that could be expected to interfere with the subject's safety during the study period;
3. currently be taking antibiotics or have taken antibiotics in the two weeks prior to the beginning treatment 1;
4. known or suspected intolerance or hypersensitivity to the study materials (or closely related compounds) or any of their stated ingredients;
5. have participated in another clinical study or receipt of an investigational drug within 30 days of beginning treatment 1; or
6. be taking fluoride supplements, required to use a fluoride mouthrinse or have received a professional fluoride treatment in the two weeks preceding specimen placement.

Participant flow

Outcome results

• Percent Surface Microhardness Recovery (%SMH Recovery) (Primary), Least squares mean, Percent Surface Microhardness Recovery: 0 Ppm F (Placebo) 24.4 (19.3 to 29.5); 1100 Ppm F as Sodium Fluoride (Positive Control) 51.6 (46.5 to 56.7); 1100 Ppm F as Sodium Fluoride Test Product 51.5 (46.5 to 56.6) (p 0.986)
• Enamel Fluoride Uptake (µg F/cm2) (Secondary), Geometric least squares mean, µg F/cm2: 0 Ppm F (Placebo, Negative Control) 1.99 (1.72 to 2.30); 1100 Ppm F as Sodium Fluoride (Positive Control) 10.32 (8.93 to 11.93); 1100 Ppm F as Sodium Fluoride Test Product 11.45 (9.92 to 13.22) (p 0.230)
• Percent Net Acid Resistance (Secondary), Least squares mean, Percent Net Acid Resistance: 0 Ppm F (Placebo, Negative Control) 2.6 (-2.3 to 7.4); 1100 Ppm F as Sodium Fluoride (Positive Control) 34.3 (29.5 to 39.1); 1100 Ppm F as Sodium Fluoride Test Product 37.4 (32.6 to 42.1) (p 0.272)
• Percentage Comparative Acid Resistance (Secondary), Geometric least squares mean, Percentage Comparative Acid Resistance: 0 Ppm F (Placebo, Negative Control) 20.27 (16.26 to 24.43); 1100 Ppm F as Sodium Fluoride (Positive Control) 16.40 (12.55 to 20.39); 1100 Ppm F as Sodium Fluoride Test Product 13.51 (9.78 to 17.36) (p 0.191)
• Integrated Mineral Loss (∆Z) (Secondary), Geometric least squares mean, Integrated Mineral Loss (∆Z): 0 Ppm F (Placebo, Negative Control) 998 (917 to 1087); 1100 Ppm F as Sodium Fluoride (Positive Control) 703 (646 to 765); 1100 Ppm F as Sodium Fluoride Test Product 708 (651 to 770) (p 0.905)
• Lesion Depth (µm) (Secondary), Geometric least squares mean, µm: 0 Ppm F (Placebo, Negative Control) 40.5 (37.2 to 44.1); 1100 Ppm F as Sodium Fluoride (Positive Control) 31.2 (28.7 to 33.9); 1100 Ppm F as Sodium Fluoride Test Product 30.6 (28.1 to 33.2) (p 0.721)
• Maximum Mineral Density at the Surface-zone (SZmax) (Secondary), Least squares mean, Maximum mineral density at the surface-z: 0 Ppm F (Placebo) 59.26 (57.40 to 61.12); 1100 Ppm F as Sodium Fluoride (Positive Control) 66.17 (64.32 to 68.01); 1100 Ppm F as Sodium Fluoride Test Product 65.61 (63.79 to 67.44) (p 0.647)

Adverse events

Time frame: 5 months

Serious adverse events by term

Other adverse events by term

• Clinical Aspects of De/Remineralization of Teeth. Proceedings of Models Conference 1994. Rochester, New York, June 11-14, 1994. Adv Dent Res. 1995 Nov;9(3):169-340. No abstract available. PMID 8615941
• Corpron RE, Clark JW, Tsai A, More FG, Merrill DF, Kowalski CJ, Tice TR, Rowe CE. Intraoral effects of a fluoride-releasing device on acid-softened enamel. J Am Dent Assoc. 1986 Sep;113(3):383-8. doi: 10.14219/jada.archive.1986.0202. PMID 3463611
• Featherstone JD, Mellberg JR. Relative rates of progress of artificial carious lesions in bovine, ovine and human enamel. Caries Res. 1981;15(1):109-14. doi: 10.1159/000260508. No abstract available. PMID 6937246
• Cate JM, Arends J. Remineralization of artificial enamel lesions in vitro. Caries Res. 1977;11(5):277-86. doi: 10.1159/000260279. No abstract available. PMID 18285
• Koulourides T, Phantumvanit P, Munksgaard EC, Housch T. An intraoral model used for studies of fluoride incorporation in enamel. J Oral Pathol. 1974;3(4):185-96. doi: 10.1111/j.1600-0714.1974.tb01710.x. No abstract available. PMID 4219029
• Lippert F, Butler A, Lynch RJ. Characteristics of methylcellulose acid gel lesions created in human and bovine enamel. Caries Res. 2013;47(1):50-5. doi: 10.1159/000343164. Epub 2012 Oct 25. PMID 23108261
• Lippert F, Hara AT. Fluoride dose-response of human and bovine enamel caries lesions under remineralizing conditions. Am J Dent. 2012 Aug;25(4):205-9. PMID 23082383
• Lippert F, Lynch RJ. Comparison of Knoop and Vickers surface microhardness and transverse microradiography for the study of early caries lesion formation in human and bovine enamel. Arch Oral Biol. 2014 Jul;59(7):704-10. doi: 10.1016/j.archoralbio.2014.04.005. Epub 2014 Apr 21. PMID 24798979
• Marinho VC, Higgins JP, Sheiham A, Logan S. Fluoride toothpastes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev. 2003;2003(1):CD002278. doi: 10.1002/14651858.CD002278. PMID 12535435
• Mellberg JR. Hard-tissue substrates for evaluation of cariogenic and anti-cariogenic activity in situ. J Dent Res. 1992 Apr;71 Spec No:913-9. doi: 10.1177/002203459207100S25. PMID 1592986
• Proskin HM, Chilton NW, Kingman A. Interim report of the ad hoc committee for the consideration of statistical concerns related to the use of intra-oral models in submissions for product claims approval to the American Dental Association. J Dent Res. 1992 Apr;71 Spec No:949-52. doi: 10.1177/002203459207100S31. PMID 1592992
• Sakkab NY, Cilley WA, Haberman JP. Fluoride in deciduous teeth from an anti-caries clinical study. J Dent Res. 1984 Oct;63(10):1201-5. doi: 10.1177/00220345840630100601. PMID 6592201
• Twetman S, Axelsson S, Dahlgren H, Holm AK, Kallestal C, Lagerlof F, Lingstrom P, Mejare I, Nordenram G, Norlund A, Petersson LG, Soder B. Caries-preventive effect of fluoride toothpaste: a systematic review. Acta Odontol Scand. 2003 Dec;61(6):347-55. doi: 10.1080/00016350310007590. PMID 14960006
• Walsh T, Worthington HV, Glenny AM, Appelbe P, Marinho VC, Shi X. Fluoride toothpastes of different concentrations for preventing dental caries in children and adolescents. Cochrane Database Syst Rev. 2010 Jan 20;(1):CD007868. doi: 10.1002/14651858.CD007868.pub2. PMID 20091655
• White DJ. Use of synthetic polymer gels for artificial carious lesion preparation. Caries Res. 1987;21(3):228-42. doi: 10.1159/000261026. No abstract available. PMID 3471346
• Zero DT. In situ caries models. Adv Dent Res. 1995 Nov;9(3):214-30; discussion 231-4. doi: 10.1177/08959374950090030501. PMID 8615944
• Zero DT, Rahbek I, Fu J, Proskin HM, Featherstone JD. Comparison of the iodide permeability test, the surface microhardness test, and mineral dissolution of bovine enamel following acid challenge. Caries Res. 1990;24(3):181-8. doi: 10.1159/000261263. PMID 2364403

• Study Protocol and Statistical Analysis Plan (PDF, 2023-07-19)

• Domenick Zero, DDS, MS, Principal investigator, Indiana University
• Anderson Hara, DDS, PhD, Principal investigator, Indiana University
• Frank Lippert, PhD, Principal investigator, Indiana University