Efficacy of CPP-ACP fluoride varnish applied with and without acid etching in preventing enamel demineralization compared to light-curable fluoride varnish
To compare efficacy of casein phosphopeptide (CPP)–amorphous calcium phosphate (ACP) fluoride varnish and light-curable resin modified glass ionomer fluoride varnish (FV) in preventing white spot lesions and evaluating acid etching prior to CPP-ACPFV application on its efficacy. Molars and premolars were transected and halves divided into four groups (n = 18/group): (1) resin-modified glass ionomer FV: etched and Clinpro-XT varnish (3M ESPE, Pymble, New South Wales, Australia) application; (2) CPP-ACPFV: MI varnish (GC America, Alsip, IL) application; (3) Etch+CPP-ACPFV: etched and MI varnish application; (4) Control: etched and no surface treatment. To simulate 12 weeks in an intraoral environment, samples were subjected to thermocycling, brushing, and pH cycling. Enamel surface microhardness was evaluated at baseline and after the simulated 12 weeks. Representative samples were also assessed using scanning electron microscopy (SEM). At baseline there was no significant difference in microhardness among groups. After the simulated 12 weeks, all groups showed significant within-group differences (P < .001). Control showed the highest percentage loss of surface microhardness (89%), followed by CPP-ACPFV (58%), RMGIFV (51%), and Etch+CPP-ACPFV (24%). The control group had a significant decrease in microhardness compared to all experimental groups (P < .001). No difference was found between the RMGIFV and CPP-ACPFV varnish groups. The Etch+CPP-ACPFV group had significantly less decrease in microhardness compared to the RMGIFV (P < .001) and CPP-ACPFV groups (P < .001). With SEM, control samples showed signs of enamel surface damage, while experimental groups showed spherical particles on a relatively intact surface. RMGIFV and CPP-ACPFV are effective in reducing enamel demineralization. Acid etching the enamel surface prior to CPP-ACPFV varnish application increased its efficacy.ABSTRACT
Objectives
Materials and Methods
Results
Conclusions
INTRODUCTION
White spot lesions (WSL) are a common side effect of comprehensive orthodontic treatment affecting approximately one-third of orthodontic patients.1 These demineralized enamel lesions, appearing as white milky spots, are considered an initial stage of dental caries and may develop rapidly, for example within a month after bonding orthodontic appliances.1 Although behavioral methods, such as optimizing diet and oral hygiene, can help prevent enamel demineralization, these are dependent on patient compliance.
Various formulations of professionally applied topical agents have been developed for prevention of dental caries. Fluoride varnish (FV) has proven effective;2,3 however, frequent repeated application is required to retain efficacy.2,4 To improve longevity of FV, a light-curable resin modified glass ionomer FV (RMGIFV) has been advocated.5–7 Because WSLs are a result of progressive enamel demineralization, to reverse this process, the rate of enamel remineralization must exceed that of demineralization.8–10 Casein phosphopeptide-stabilized amorphous calcium phosphate complex (CPP-ACP) is thought to be particularly effective as CPP stabilizes calcium and phosphate ions, preventing their precipitation, while ACP acts as a reservoir of these remineralizing ions.10
Acid etching of enamel is a procedure used in bonding fixed orthodontic appliances. Beyond increasing bond strength, the etching of enamel exposes enamel crystals that have a high affinity for calcium and phosphate ions.11 When available, fluoride incorporated in remineralized enamel increases enamel hardness and resistance to acid attacks.12,13 Other studies have shown that acid etching increases enamel susceptibility to WSL; however, the high concentrations used and longer etching times assessed in the studies are not clinically applicable.14,15
No previous study compared the efficacy of the light-curable RMGIFV with the CPP-ACPFV nor the effect of etching enamel prior to the application of CPP-ACPFV. The current study, using measurements of surface microhardness (SMH) as an indicator of enamel demineralization, tested the null hypotheses that (1) light-curable RMGIFV provides a similar preventive effect on enamel demineralization as CPP-ACPFV, and (2) acid etching the enamel surface prior to application of CPP-ACPFV does not improve the preventive effects.
MATERIALS AND METHODS
Study Design
The study design is illustrated in Figure 1. The protocol was submitted to the University at Buffalo's Institutional Review Board and received an exempt determination.
Citation: The Angle Orthodontist 92, 2; 10.2319/050121-345.1
Power Analysis
Using software (G-power), assuming an effect size of 1.21 as calculated from a previous study,9 a sample size of n = 16/group was adequate to achieve a type I error rate of α = 0.05 and a power of 90%.16 To compensate for potential damage or loss of samples, two extra teeth were added to each group for an n = 18/group.
Selection of Teeth
Thirty-six extracted human permanent molars and premolars with intact enamel surfaces were collected from the University at Buffalo's oral surgery and periodontal clinics. For disinfection, the teeth were maintained for 1 week in 0.5% Chloramine-T solution and then stored in distilled water at 4°C.
Sample Preparation
The roots were removed and crowns were sectioned mesiodistally to obtain buccal and lingual halves. Each tooth section was embedded in acrylic resin blocks with the enamel surface exposed. A polishing machine (AutoMet 250, Buehler, Lake Bluff, IL) with 600- and 1200-grit silicon-carbide sandpaper was used to create standardized flat surfaces (60 revolutions/minute, 10 seconds).9 The enamel was then covered with an acid-resistant nail polish (Revlon, New York, NY), except for a 3 × 4 mm window of exposed enamel, created by use of adhesive cellophane templates.
Surface Microhardness—Baseline (SMH1)
SMH was obtained using a Vickers microhardness tester (MicroMet 5104, Buehler, Lake Bluff, IL) with a load of 100 g. Six indentations at least 100 μm apart were made, and the microhardness values were calculated.9
Surface Treatments
Samples were divided into four groups (n = 18/group) with varying treatments of the enamel surfaces:
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RMGIFV: Preconditioned with 35% phosphoric acid (15 seconds), water rinse (15 seconds), air dried, one application of light-curable RMGIFV (Clinpro XT Varnish; 3M ESPE, Pymble, New South Wales, Australia).
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CPP-ACPFV: Water rinse (15 seconds), air dried, one application of CPP-ACPFV (MI Varnish, GC America, Alsip, IL).
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Etch+CPP-ACPFV: Preconditioned with 35% phosphoric acid (15 seconds), water rinse (15 seconds), air dried, one application of CPP-ACPFV.
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Control: Preconditioned with 35% phosphoric acid (15 seconds), water rinse (15 seconds), air dried, no further surface treatment (to resemble the orthodontic procedure used in bonding brackets).
Thermocycling
A thermocycling machine (Thermocycler, SD Mechatronik, Feldkirchen-Westerham, Germany) was used to simulate thermal changes in the oral cavity. The teeth were subjected to 15-second dwell times, alternating between 5°C and 55°C baths of distilled water. The thermocycling ran for 900 cycles to simulate the passage of 12 weeks.17
Brushing
A tooth brushing simulator (V-8 Cross Brushing Machine, Sabri Dental Enterprises, Downers Grove, IL) and soft bristled toothbrushes were used to simulate the effect of brushing forces. Using a pressure of 280 g, each tooth was subjected to 1800 strokes, simulating 12 weeks of brushing.18
pH Cycling
The teeth were subjected to 9 days of pH cycling.19 Each day, the specimens were placed in 50 mL demineralizing solution (1.28 mmol/L calcium nitrate, 0.74 mmol/L sodium dihydrogen phosphate, 0.05 mol/L acetate buffer, 0.03 μg F m/L, pH 5.0) for 4 hours, then rinsed with distilled water, dried with absorbent paper, and immersed in 25 mL of remineralizing solution (1.5 mmol/L Ca, 0.9 mmol/L P, 150 mmol/L KCl, 0.05 μg F m/L in 0.1 mol/L Tris buffer, pH 7.0) for 20 hours. The solutions were maintained in an incubator at 37°C and were replaced every 4 days. On day 9, specimens were stored in remineralizing solution for 24 hours. pH cycling was repeated three times (27 days total) to simulate the passage of 12 weeks.19
Surface Microhardness—Final (SMH2)
SMH2 measurements were obtained and the percentage loss of surface microhardness calculated.9 For the RMGIFV group, sites where the resin coating was missing were identified and selected for microhardness testing.
Scanning Electron Microscope (SEM)
Three representative samples per group were prepared for SEM evaluation. Specimens were dried in a vacuum evaporator for 4 days and then sputter coated with gold. Assessments of areas close to an indentation were made using a Hitachi SU70 (Hitachi Ltd, Tokyo, Japan) field emission SEM operated at 2.0 keV. Images were captured and used for descriptive analysis.
Statistical Analysis
Baseline and final SMH data were tested for normality using the Shapiro-Wilk test. Between-group differences were analyzed using one-way analysis of variance (ANOVA) followed by Tukey honest significant difference post-hoc test. Within-group differences were assessed using paired t-test. To evaluate the systematic error and reliability of the microhardness testing, 10 samples from baseline and final were measured again and the results analyzed using paired t-test and interclass correlation coefficient (ICC), respectively.
RESULTS
Paired t-test showed no systematic error in the SMH repeat measurements (P = .81) and the ICC of 0.97 showed the measurements were highly reliable. The Shapiro-Wilk test found SMH1 and SMH2 data were normally distributed.
Table 1 shows the means of SMH1and SMH2 measurements and the changes. At baseline, there was no statistically significant difference among the four groups. After simulating 12 weeks (SMH1 to SMH2), all the groups showed statistically significant within-group differences (P < .001). The control group showed the highest percentage loss of SMH (89%), followed by the CPP-ACPFV (58%), the RMGIFV (51%), and the Etch+CPP-ACPFV group (24%; Figure 2).
Citation: The Angle Orthodontist 92, 2; 10.2319/050121-345.1
Table 2 summarizes the pair-wise comparisons between the groups. ANOVA showed a significant difference among groups (P < .001). Tukey HSD analysis showed statistically significant differences between the control and each experimental group (P < .001) and between the Etch+CPP-ACPFV group and both the RMGIFV (P < .001) and CPP-ACPFV groups (P < .001). There was no significant difference between the RMGIFV and CPP-ACPFV groups.
SEM images of the representative samples are shown in Figure 3. The untreated control showed pronounced features of enamel demineralization including areas of cracks and discontinuity in the enamel surface, whereas the experimental groups showed intact enamel with no clear signs of demineralization. The CPP-ACPFV image showed an intact enamel surface with scattered spherical particles. The RMGIFV sample showed areas where the resin was worn off. The exposed enamel surface appeared relatively sound with occasional spherical particles attached to its surface. In contrast to other experimental groups, the Etch+CPP-ACPFV samples showed abundant spherical particles covering much of the enamel surface.
Citation: The Angle Orthodontist 92, 2; 10.2319/050121-345.1
DISCUSSION
This study used measurements of microhardness as a means of assessing enamel demineralization. Featherstone and colleagues previously demonstrated the validity of this method.20 Results indicated that enamel surfaces after receiving challenges representing the passage of 12 weeks intraorally had significant reductions in microhardness, while FV-treated enamel had notably less change. These findings were consistent with those of a double-blind, randomized controlled trial by Stecksén-Blicks et al. showing the incidence of WSL in orthodontic patients treated with FV (0.1% F) was 7.4% compared to 25.3% in a placebo group (P < .001).21 A systematic review also highlighted the benefit of routine use of FV during fixed orthodontic therapy.4
When using RMGIFV, it was found that, while most of the resin remained on the enamel surfaces, areas where the resin had worn off were observed. Although no study was found testing the effect of brushing forces on the durability of Clinpro XT FV applied on enamel, Canali and associates, using dentin specimens, reported a gradual degradation of the resin matrix with detachment of glass particles 4 days after varnish application.22 Although that finding cannot be compared directly to the current study due to differences in the specimens, both findings differ from the manufacturer's claim that Clinpro-XT varnish layer remains intact for more than 5000 brushing strokes and can resist toothbrush abrasion and normal wear for 6 months or longer. When areas where the RMGIFV had worn off were assessed in the current study, there was a statistically significant loss of microhardness, although the changes were reduced compared to the control group. This indicated that the varnish provided a preventive effect to the enamel even though the material was partially lost. RMGIFV provides a fluoride-releasing coating and pumps fluoride to the underlying and adjacent enamel surfaces, promoting more remineralization than a conventional FV.5 Consistent with the current data, a recent split-mouth randomized clinical trial found that a single application of the RMGIFV at the beginning of comprehensive orthodontic treatment prevented enamel demineralization for a longer duration (4 months) compared to the conventional varnish (45 days).7
Although enamel surfaces treated with CPP-ACPFV had a significant loss in microhardness, the change was notably less than in the control group. A previous study showed that one application of CPP-ACPFV prevented enamel demineralization for at least 4 weeks and limited demineralization up to 12 weeks, suggesting that reapplication should be administered every 4–6 weeks.23 The deterioration in the effect of varnish could be attributed to the depletion of the calcium fluoride compound (CaF2). CaF2 is a major reaction product found after topical application of high fluoride concentrations and is loosely bound to the enamel surface.5 At low pH, CaF2 acts as a reservoir, releasing calcium and fluoride and promoting remineralization of enamel crystals.5 Once the loose CaF2 is consumed, reapplication of the varnish is indicated, especially in high-risk patients. The present study also showed that surface microhardness values obtained with the CPP-ACPFV and RMGIFV groups were not significantly different and, therefore, the first null hypothesis, that light-curable RMGIFV provides a similar preventive effect on enamel demineralization as CPP-ACPFV, was accepted. Although Verma et al. compared the remineralizing effects of CPP-ACPFV and RMGIFV and concluded that CPP-ACPFV remineralized demineralized enamel more effectively, their study was for a short duration (7 days) relative to the 12-week simulation in the current study.24
A unique finding of the present study was the significant difference in microhardness values between Etch+CPP-ACPFV (259 ± 59) vs CPP-ACPFV without etching (148 ± 38; P < .001), indicating that acid preconditioning markedly improved performance of the varnish. These findings lead to a rejection of the second null hypothesis that etching would have no impact on the efficacy of the CPP-ACPFV. This could be attributed to the effect of etching, increasing the enamel's surface area and exposing underlining crystals that have been shown to have increased reactivity for remineralization.11 CPP-ACPFV, containing high levels of calcium, phosphate, and fluoride (5% NaF) ions, would be capable of providing the main elements needed for rebuilding the partially demineralized hydroxyapatite (HA) crystals of enamel.11 When fluoride ions become incorporated in the deeper crystal lattice, fluorohydroxyapatite (FHA) is formed that has a critical pH for demineralization below 4 compared to 5.5 for HA.12,13 Because acid etching is a requisite step for bonding orthodontic appliances, the current results indicated that applying CPP-ACPFV at the bonding appointment, as opposed to later appointments when enamel would not normally be etched, will help maximize varnish efficacy in preventing WSLs. Whether re-etching is necessary at later visits is a question for future research.
Surface microhardness values of the Etch+CPP-ACPFV were significantly higher than the RMGIFV group, even though both varnishes were preceded by acid etching and both varnishes release calcium, phosphate, and fluoride ions. The different outcomes could be explained by the concentration of fluoride ion-released soon after application. Cochrane et al. found that CPP-ACPFV released significantly more fluoride, calcium, and phosphate ions than a conventional FV.8 After 1 and 24 hours, CPP-ACPFV released 276.5 ± 39.2 and 1139.9 ± 90 μmol/g, respectively, while the conventional FV released 18.1 ± 0.7 and 74.7 ± 7.5 μmol/g, respectively.8 Another study reported that RMGIFV released significantly less fluoride compared to a conventional FV.25 In the present study, it was likely that the immediate high fluoride-release of CPP-ACP after acid etching potentially increased formation of the more acid-resistant FHA.
SEM showed notably increased surface roughness in the untreated control sample. In contrast, the experimental groups had enamel surfaces that appeared largely intact, although microhardness testing revealed that some demineralization had occurred. WSL have a relatively intact surface layer (remineralization site) and most of the demineralization occurs in the subsurface or body of the lesion, rendering the enamel softer than normal.26 SEM images in the RMGIFV and the CPP-ACPFV samples showed relatively scarce spherical bodies on the enamel. Zhou et al. found that similar-appearing spherical bodies were composed of CaF2 and were present for at least 6 weeks after FV application.5 In the current study, the limited presence of spherical particles after the simulated 12 weeks suggested that the enamel had consumed most of its CaF2 and that reapplication was required. For the Etch+CPP-ACPFV sample, although the enamel surface was etched at baseline, it revealed no signs of the “honeycomb pattern” of etched enamel, suggesting a remineralized surface. In contrast to the appearance of the other experimental groups, the Etch+CPP-ACPFV sample had abundant spherical bodies on the enamel surface, consistent with potentially having a longer preventive effect.
This investigation had some limitations. Although the microhardness and SEM assessments could only be performed in vitro, a primary limitation was that the conditions used in the current study only approximated the actual intraoral environment. However, the in vitro setting was well suited to minimize confounders when comparing the treatment groups. Another limitation was that the remineralizing solution was not agitated, which may have led to less remineralization, therefore favoring demineralization. Nonetheless, the extensive effects of demineralization found in the control group helped demonstrate the potential benefits of the varnishes in patients undergoing orthodontic treatment.
CONCLUSIONS
Within the limitations of this in vitro study, the following conclusions can be drawn:
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CPP-ACPFV and light-curable RMGIFV were both effective in maintaining enamel microhardness, and thus decreasing the risk of WSL development, for at least 12 weeks.
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Acid etching the enamel surface prior to CPP-ACPFV application significantly enhanced the effect of varnish and prolonged its efficacy in minimizing changes in enamel microhardness and surface texture.
Flow chart illustrating the experimental design.
Percentage of surface microhardness loss of the control and experimental groups.
SEM images of enamel samples at the end of the experimental protocol. Control shows multiple cracks (black arrows) and discontinuity of the surface. CPP-ACPFV shows an intact enamel surface with few spherical particles, most likely CaF2 (white arrows). RMGIFV shows intact enamel surface with resin remnants (white stars) and few particles. Etch+CPP-ACPFV shows numerous spherical particles. Original magnification: 100,000×, left; 500,000×, right.
Contributor Notes