Fluoride concentration in saliva and biofilm fluid following the application of three fluoride varnishes

Abstract

Objective

Most of the commercially available fluoride varnishes (FV) have not been evaluated for their cariostatic properties. Consequently, the aim of this in vivo study was to investigate intra-oral fluoride retention and clearance patterns from three different FV.

Methods

Eighteen subjects (7–11 years) participated in a laboratory analyst-blinded, randomized, crossover study comparing the ability of 5% sodium fluoride varnishes (CavityShield-CS, Enamel Pro-EP, Vanish-V) to enhance fluoride concentrations in biofilm fluid, centrifuged and whole saliva over a period of 48 h after a single FV application.

Results

Similar fluoride concentration × time patterns were noted for all investigated FV and studied variables, with the highest fluoride concentrations observed for the first biological sample collected after FV application (30 min). Mean ± SE (area under fluoride clearance curve) values were (μg F/g or ml × min): biofilm fluid − CS (472 ± 191), EP (423 ± 75), V (1264 ± 279); centrifuged saliva − CS (42 ± 7), EP (19 ± 3), V (41 ± 8); whole saliva − CS (68 ± 11), EP (64 ± 10), V (60 ± 7). V delivered more fluoride to biofilm fluid than CS (p = 0.0116) and EP (p = 0.0065), which did not differ (p = 0.27). For centrifuged saliva, CS and V were not significantly different (p = 0.86), but resulted in higher fluoride retention than EP (p < 0.0008). No significant differences among FV were observed for whole saliva (p = 0.79).

Conclusion

The present study has shown that FV vary in their ability to deliver fluoride intra-orally potentially related to formulation differences. To what extent the present findings relate to clinical efficacy remains, however, to be determined.

Clinical significance

Clinical research that investigates fluoride release patterns into saliva and biofilm fluid from different FV products is insufficient. More research is needed to investigate different FV formulations for their efficacy in order to help clinicians make better evidence based treatment choices.

Introduction

Topical fluorides have been shown to be efficacious in the prevention of caries with fluoride varnishes (FV) containing 5% sodium fluoride being recommended as the primary choice for in-office dental caries prevention . A large number of FV with varying fluoride concentrations and compounds, vastly different formulations and often containing ingredients being attributed cariostatic properties in their own right (e.g. xylitol, calcium compounds) are now commercially available. Comparative caries clinical trials of these different FV, however, have not been conducted to date and only very few FV (three according to the present authors’ review) have ever been evaluated for their efficacy clinically .

As caries clinical trials are not only time- and resource-consuming but also put an unnecessary burden on participants, the study of clinically validated surrogate measures for efficacy is now commonly used by researchers. Current models on the cariostatic action of fluoride emphasize the significance of maintaining cariostatic levels of fluoride in oral fluids, namely saliva and dental biofilm . Therefore, studying fluoride concentration increase in saliva and dental biofilm following the administration of topical fluoride is one way to relate to potential clinical efficacy as it can be indicative of fluoride levels in the aqueous phase available for interaction with the tooth structure during de- and remineralization .

Very few studies on the kinetics of fluoride in saliva and biofilm following FV application have been reported. Two studies reported significant elevations in whole saliva fluoride concentration that persisted for up to 6 h or 24 h , respectively. A fluoride dose-response was also established . The sole study concerned with fluoride in biofilm was able to demonstrate slightly increased fluoride concentrations for up to 30 days after application which depended on the type of varnish. This study also suggested different patterns of fluoride retention for different varnishes . The two comparative studies , however, investigated FV of different fluoride concentrations and compounds (i.e. 5% sodium fluoride [2.26% F], difluorosilane [0.1% F], sodium and calcium fluoride [6% F]), thereby limiting conclusions that can be drawn.

As there is a considerable gap in our understanding of how different FV formulations of identical fluoride concentration affect intra-oral fluoride retention, the present study aimed to investigate fluoride concentrations in biofilm, centrifuged and whole saliva as surrogate measures for cariostatic efficacy after a single application of three inherently different 5% sodium fluoride FV over a period of 48 h after a single application.

Subjects, materials and methods

Ethical aspects and subjects

The study protocol for this laboratory analyst-blind, randomized, cross-over, three-period study was reviewed and approved by the Indiana University Institutional Review Board, #1409221212. The study was conducted at Little Flower Catholic School, Indianapolis, IN, USA, in children age seven to eleven years. Informed consent (parents) and assent (children) were obtained from all study subjects prior to screening. All subjects received oral soft and hard tissue examinations throughout the study. In order to participate in the study, subjects had to meet the following inclusion criteria: be between seven and eleven years old, have good general and oral health, have at least 16 teeth with no cavitated carious lesions, and have no oral soft tissue lesions or active periodontal disease including severe gingivitis. Subjects also had to understand and be willing and able to comply with the instructions provided during the study which included abstention from eating for one hour prior to and for the two-hour duration of the test visit. Potential subjects were excluded if they had known or suspected allergy or hypersensitivity to FV or any of their listed ingredients, were taking fluoride supplements or other fluoride products for medical purposes (except for fluoride naturally occurring in the diet), and were taking prescription antibiotics.

Power calculation

Based on a previous study on adults , with a sample size of 16 subjects, the study had a 80% power to detect a difference of 1.5 for log (area under the curve [AUC]) between any two treatments, assuming two-sided tests each conducted at a 5% significance level, the within-subject correlation is 0.5, and the standard deviation was 2.0. To account for 10% dropout, the study enrolled 18 subjects.

Fluoride varnishes and washout toothpaste

No experimental fluoride varnish was used in this study. Fluoride varnishes contained a standard fluoride level of 5% sodium fluoride, were supplied in single dose packages, purchased from a provider of professional healthcare products and used at least six months prior to expiration. Fluoride concentration of the FV was not determined prior to use. FV packages were weighed before and after treatment application in order to calculate the amount of varnish applied. Pertinent information about the three FV used in this study can be found in Table 1 . Subjects were asked to use fluoride-free toothpaste (Fluoride Free Children’s Toothpaste, Tom’s of Maine, Kennebunk, ME, USA) for a washout period of two weeks prior to the administration of the first treatment and for the duration of the study. A two-week washout period is common in the literature for studies involving FV. A study reported that baseline fluoride values returned to values that are very close to baseline following a washout period of two weeks .

Table 1
Test products.
Fluoride Varnish Manufacturer Fluoride Source and Concentration Carrier Other Active Ingredient
Enamel Pro Premier Dental 5% NaF Rosin Amorphous calcium phosphate (ACP), Xylitol
CavityShield 3 M ESPE 5% NaF Rosin, Polyamide Resin N/A
Vanish 3 M ESPE 5% NaF Pentaerythritol glycerol ester of Rosin Functionalized tri-calcium phosphate (fTCP), Xylitol

Randomization procedures and blinding

A unique screening number was used for all subjects screened for study participation. In addition, the study statistician created a randomization schedule to determine the order of treatment application for each subject. Due to the uniqueness of each FV (color, flavor, and handling properties) the study investigator had the capability to discriminate between varnishes and was therefore blinded only to sample analysis rather than varnish application.

Clinical procedures and methodologies

The investigator completed an oral soft and hard tissue (OSHT) examination at a screening visit to ensure only eligible subjects were enrolled into the study. At all other visits an oral soft tissue (OST) exam only was performed. Subjects were instructed not to brush their teeth or perform any oral hygiene at home on any morning a biofilm sample was collected.

Subjects provided a baseline (BL) five-minute, non-stimulated saliva sample, followed by collection of an interproximal/buccal biofilm sample from all teeth immediately prior to assigned FV treatment. FV treatment was then applied on all teeth surfaces including buccal occlusal third/lingual/occlusal of posterior teeth and facial incisal third/lingual of anterior teeth. The exact amount applied clinically was not standardized per se, but care was taken to cover all aforementioned tooth surfaces and the weight of applied FV determined (see above). The FV treatment was allowed to set and immediately thereafter, saliva samples were collected at 30, 60, and 120 min, and at 24 h and 48 h following the treatment. Approximately 1 mg of interproximal/buccal biofilm was collected immediately after each saliva sample. Subjects remained at school throughout the treatment visits. At the end of each visit, the study investigator brushed the occlusal surfaces of the child’s teeth with water and a new tooth brush (Oral B Indicator Soft, Procter

Saliva collection procedure

Unstimulated whole saliva samples were collected at baseline and immediately following treatment at 30, 60, and 120 min and at 24 h and 48 h. Saliva collection was initiated by having the subjects swallow all the residual saliva in their mouth, and then let saliva pool in their mouths for the five-minute period while their heads were tilted forward. As the subjects felt the need to swallow, they expectorated into a plastic re-sealable collection vial. At the end of the five-minute collection period all remaining saliva was expectorated into the plastic vial. Saliva samples were stored at −20 °C for later fluoride analysis.

Biofilm collection procedure

Immediately before dental biofilm collection, subjects were instructed to swallow all remaining saliva and keep their mouth open. Approximately 1 mg of dental biofilm was collected from the interproximal and buccal surfaces of teeth of all four quadrants. Biofilm samples were collected using a standardized protocol. Pooled biofilm samples were collected using a stainless steel periodontal scaler (S. McCall 17/18, Hu- Friedy, Illinois, USA) from each interproximal area from buccal aspect and buccal area starting from the upper right quadrant to the upper left, lower left and ending in lower right quadrant. The pooled biofilm sample was transferred into a plastic strip.

Biofilm sample preparation

Prior to biofilm sample collection, special centrifuge tubes were constructed by heat sealing 10 microliter (μl) micropipette tips. They were filled with heavy mineral oil (Mineral Oil, Heavy (USP/FCC) Fisher Chemical, Fisher Scientific, USA). Microcentrifuge tubes containing the plastic strip and biofilm sample were centrifuged for 10 min at 10,000 rpm (4,000 g ) at 4 °C . Partially oil-filled fine glass micro pipettes were used to recover small aliquots (approx. 5 nl) from the centrifuged tube under a microscope.

Biofilm fluid fluoride analysis

The micro analytical method was used to analyze biofilm fluid samples for fluoride content . Samples were placed, under mineral oil, on the surface of a specially constructed inverted F electrode. Mineral oil was used to prevent evaporation. Total ionic strength adjusting solution (TISAB III) was added to the samples in a ratio of 9:1. The tip of a micro-reference electrode was placed in contact with the sample to complete the circuit.

Triplicate analyses were performed on each pooled biofilm fluid sample. Biofilm fluid fluoride was expressed as μg F/ml which was calculated by comparison to a standard fluoride curve, constructed the same day of the analysis.

Saliva analysis

Each saliva sample was analyzed as whole and centrifuged saliva for fluoride concentration. A 1.4 ml aliquot of each thawed and vortexed saliva sample was centrifuged for 10 min at 10,000 rpm (4,000 g ) at 7 °C (centrifuged saliva) or analyzed for fluoride content without centrifugation (whole saliva). Analysis of saliva was conducted using a modification of the hexamethyl-disiloxane (HMDS) microdiffusion method as modified recently . One ml of centrifuged saliva sample was pipetted into plastic Petri dishes (Falcon 15-cm plastic Petri dishes), adding enough deionized water to bring final volume in each Petri dish to 3.0 ml. A 0.05 sodium hydroxide analytical reagent (NaOH), 50 microliters (μl) trap solution was placed in five drops on the Petri dish lid and after the addition of 1 ml of sulfuric acid (H 2 SO 4 ) saturated with HMDS through a small hole in the lid of the Petri dish, each dish was immediately tightly sealed with petroleum jelly. During overnight diffusion, fluoride was released by acid hydrolysis and was trapped in NaOH. The trap was recovered and buffered to pH 5.2 with 25 μl acetic acid (CH 3 COOH.). The recovered solution was adjusted to a final volume of 100 ml with deionized water. Analyses were performed in sets of approximately 40 samples. Fluoride was measured using a fluoride combination electrode (Model 9609BNWP, Orion Research, Boston, MA, USA) and meter. The fluoride content (μg F) of the samples was calculated from a standard curve constructed from fluoride standards and microdiffused at the same time as the samples.

The amount of total fluoride in the samples was calculated based on the amount of fluoride divided by the volume of the sample and expressed as μg F/ml of sample.

Subjects, materials and methods

Ethical aspects and subjects

The study protocol for this laboratory analyst-blind, randomized, cross-over, three-period study was reviewed and approved by the Indiana University Institutional Review Board, #1409221212. The study was conducted at Little Flower Catholic School, Indianapolis, IN, USA, in children age seven to eleven years. Informed consent (parents) and assent (children) were obtained from all study subjects prior to screening. All subjects received oral soft and hard tissue examinations throughout the study. In order to participate in the study, subjects had to meet the following inclusion criteria: be between seven and eleven years old, have good general and oral health, have at least 16 teeth with no cavitated carious lesions, and have no oral soft tissue lesions or active periodontal disease including severe gingivitis. Subjects also had to understand and be willing and able to comply with the instructions provided during the study which included abstention from eating for one hour prior to and for the two-hour duration of the test visit. Potential subjects were excluded if they had known or suspected allergy or hypersensitivity to FV or any of their listed ingredients, were taking fluoride supplements or other fluoride products for medical purposes (except for fluoride naturally occurring in the diet), and were taking prescription antibiotics.

Power calculation

Based on a previous study on adults , with a sample size of 16 subjects, the study had a 80% power to detect a difference of 1.5 for log (area under the curve [AUC]) between any two treatments, assuming two-sided tests each conducted at a 5% significance level, the within-subject correlation is 0.5, and the standard deviation was 2.0. To account for 10% dropout, the study enrolled 18 subjects.

Fluoride varnishes and washout toothpaste

No experimental fluoride varnish was used in this study. Fluoride varnishes contained a standard fluoride level of 5% sodium fluoride, were supplied in single dose packages, purchased from a provider of professional healthcare products and used at least six months prior to expiration. Fluoride concentration of the FV was not determined prior to use. FV packages were weighed before and after treatment application in order to calculate the amount of varnish applied. Pertinent information about the three FV used in this study can be found in Table 1 . Subjects were asked to use fluoride-free toothpaste (Fluoride Free Children’s Toothpaste, Tom’s of Maine, Kennebunk, ME, USA) for a washout period of two weeks prior to the administration of the first treatment and for the duration of the study. A two-week washout period is common in the literature for studies involving FV. A study reported that baseline fluoride values returned to values that are very close to baseline following a washout period of two weeks .

Table 1
Test products.
Fluoride Varnish Manufacturer Fluoride Source and Concentration Carrier Other Active Ingredient
Enamel Pro Premier Dental 5% NaF Rosin Amorphous calcium phosphate (ACP), Xylitol
CavityShield 3 M ESPE 5% NaF Rosin, Polyamide Resin N/A
Vanish 3 M ESPE 5% NaF Pentaerythritol glycerol ester of Rosin Functionalized tri-calcium phosphate (fTCP), Xylitol
Only gold members can continue reading. Log In or Register to continue

Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Fluoride concentration in saliva and biofilm fluid following the application of three fluoride varnishes
Premium Wordpress Themes by UFO Themes