Caries lesion remineralization with fluoride toothpastes and chlorhexidine - effects of application timing and toothpaste surfactant

Abstract Habitual toothbrushing with fluoridated toothpaste followed by rinsing with antibacterial mouthwashes is a method to maintain good oral hygiene and to diminish the occurrence and severity of dental caries and periodontal disease. However, our understanding of how antimicrobial agents in mouthwashes affect fluoride-mediated caries lesion remineralization is still poor. Objective: The objectives of this in vitro study were a) to determine the effects of the waiting period of chlorhexidine (CHX) rinsing after fluoride toothpaste use and b) to further determine the effect of the type of toothpaste surfactant [sodium dodecyl sulfate (SDS) or cocamidopropyl betaine (CAPB)] on caries lesion remineralization associated with CHX rinsing. Material and Methods: Caries lesions were formed in bovine enamel specimens and assigned to 10 treatment groups (n=18) based on Vickers surface microhardness (VHN). Lesions were then pH-cycled for 10 days with daily regimen comprised of twice daily toothpaste slurry treatments (1150 ppm fluoride, with SDS or CAPB), followed by CHX solution treatments [0, 15, 30 or 60 minutes following slurry treatment or no CHX treatment (negative control)]. VHN was measured again and the extent of lesion remineralization calculated (∆VHN). Results: ∆VHN with SDS-toothpaste was significantly lower than with CAPB-toothpaste, indicating more remineralization for the CAPB-toothpaste. ∆VHN with 0-minute waiting time was significantly lower than with 30-minute waiting time and with negative control. Conclusions: The absence of CHX as an adjunct to fluoride toothpastes led to greater remineralization of enamel lesions compared with the immediate use of CHX treatment for both SDS- and CAPB-toothpastes. CAPB-toothpastes indicated significantly greater remineralization than SDS-toothpastes, and can be suggested for patients at high risk of caries. A 30-minute waiting time for CHX treatment is recommended after brushing.


Introduction
Fluoride has long been recognized as it promotes caries lesion remineralization and inhibits demineralization of tooth surfaces subjected to acids related to the caries process 40 . Several systematic reviews have concluded that fluoride toothpastes prevent caries 24, 35  and cocamidopropyl betaine (CAPB; amphoteric). CAPB is less irritating than SLS, although at the expense of foaming ability 13,20 . Little research has been conducted on how surfactants affect fluoride delivery to the dental hard tissues and, ultimately, lesion remineralization. Chlorhexidine (CHX) is often seen as the "gold standard" due to its antimicrobial action against a wide variety of organisms that has been shown to reduce the incidence of plaque-induced gingivitis 4,19,22 .
CHX has a comprehensive spectrum of activity, including some lipophilic viruses, yeasts, grampositive and -negative bacteria, and dermatophytes 14 .

Study design
Demineralized bovine enamel specimens with predetermined surface microhardness (VHN) were submitted to a 10-day pH-cycling model. During the pH-cycling phase, specimens were exposed to fluoride toothpaste slurries; one containing SDS, the other an amphoteric surfactant (CAPB), followed by a 0.12% CHX rinse at different time intervals after fluoride exposure with no CHX or any other rinse as a negative control. After completion of this phase, the extent of remineralization was determined using VHN.

Enamel specimens
Enamel specimens were prepared as described previously 21 : bovine incisor teeth were dissected into 5×5 mm specimens from the buccal surfaces only by means of a Buehler Isomet low speed saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA). The teeth were stored in deionized water saturated with thymol (0.1% w/v) during the sample preparation process.
The superficial enamel was ground to remove surface irregularities and create a flat enamel surface using a Struers Rotopol 31/Rotoforce 4 polishing unit (Struers Inc., Cleveland, PA, USA). The dentin side of the specimens was ground flat to a uniform thickness with 500-grit silicon carbide grinding paper. The enamel side of the specimens was ground in a series of 1200-, 2400-, and 4000-grit paper. The specimens were then polished using a 1 μm diamond polishing suspension on a polishing cloth. This procedure helped to ensure the removal of surface enamel, which can contain high concentrations of impurities (e.g. Only specimens with a VHN demin that was within the range of the mean VHN demin ±2 standard deviation of all specimens were used in this study. Specimens were stratified into treatment groups using VHN demin to ensure no significant differences in the mean VHN demin between groups. Each treatment group contained 18 specimens.

Sample size calculation
Based on prior data, the coefficient of variation estimated was 0.7. With a sample size of 18 per toothpaste-timing of CHX rinse combination, the study was designed to have 80% power to detect a 1.85× difference between any two groups, assuming twosided tests each conducted at a 5% significance level.

Statistical analysis
The effects of toothpaste and timing of CHX rinse on ∆VHN for remineralization of early caries lesions were analyzed using two-way ANOVA, which included fixed factors for toothpaste, timing of CHX rinse, and their interaction. Pair-wise comparisons between treatments were made using Fisher's Protected Least Significant Differences. A 5% significance level was used for all tests. The distribution of the measurements were examined, and no transformation of the data was necessary.

Results
A total of 200 specimens were initially demineralized, of which 180 specimens were selected for this study as described above. VHN demin and ∆VHN data for each study group as well as the results of the statistical analysis can be found in Table 1   phosphate groups are exposed than calcium atoms.
Therefore, enamel has a negative surface charge 10 .
SDS is an anionic molecule 8  CHX is a large dicationic molecule and can therefore adsorb onto negatively charged surfaces 19 , such as enamel. However, CHX also has a great affinity for the negatively charged SDS, leading to its desorption from the enamel surface and "inactivation" of CHX, and therefore, reduces its availability on the tooth surface. In the past, research on CHX was more concerned with demonstrating antimicrobial and direct lesion effects rather than focusing solely on the latter.
Timmons, et al. 34 (2007) fixed artificial caries lesions on crowns that were placed on prepared patient teeth. Patients were then instructed to brush using a placebo toothpaste, a fluoride toothpaste, or a fluoride toothpaste followed by CHX. Their study showed CHX used in combination with fluoride toothpaste was no more effective in reducing dental caries than fluoride toothpaste alone. Altenburger, et al. 1 (2006) evaluated the ability of CHX/NaF and CHX rinses to remineralize demineralized enamel specimens in situ.
No differences were observed; however, their study did not investigate the effect of CHX on fluoride's ability to remineralize lesions, as there was no fluoride only group. An in vivo study on a twice-daily CHX and once-daily fluoride rinse regimen by Ullsfoss, et al. 36 (1994) utilized plaque-retaining bands on premolars planned for extraction. The authors were able to demonstrate additive effects for the combined CHX and fluoride regimen; however, their model was concerned with prevention of demineralization rather than the enhancement of remineralization. While these studies do not provide a rationale for our findings, they show CHX is not only being used for the prevention of periodontal disease but also in caries prevention. This study, however, has shown that care must be taken when applying CHX in relation to fluoride and that the type of toothpaste needs to be chosen carefully to maximize the anticaries benefits of fluoride.
The following study limitations must be borne in mind when interpreting these data. Hardness techniques cannot directly determine the extent of mineral loss or gain; however, the measured increases in surface hardness are due to remineralization.

Disclosure of conflicting interests
The authors declare no conflict of interest.