Original Paper Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Received: September 29, 2013 Accepted after revision: January 7, 2014 Published online: July 3, 2014
Dentifrice Fluoride and Abrasivity Interplay on Artificial Caries Lesions Hani M. Nassar a, b Frank Lippert b George J. Eckert c Anderson T. Hara b a
Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; b School of Dentistry, Indiana University, and c Indiana University School of Medicine, Indianapolis, Ind., USA
Key Words Abrasion · Abrasives · Dentifrice · Fluoride · Incipient caries · Remineralization
Abstract Incipient caries lesions on smooth surfaces may be subjected to toothbrushing, potentially leading to remineralization and/or abrasive wear. The interplay of dentifrice abrasivity and fluoride on this process is largely unknown and was investigated on three artificially created lesions with different mineral content/distribution. 120 bovine enamel specimens were randomly allocated to 12 groups (n = 10), resulting from the association of (1) lesion type [methylcellulose acid gel (MeC); carboxymethylcellulose solution (CMC); hydroxyethylcellulose gel (HEC)], (2) slurry abrasive level [low (REA 4/ RDA 69); high (REA 7/RDA 208)], and (3) fluoride concentration [0/275 ppm (14.5 mM) F as NaF]. After lesion creation, specimens were brushed in an automated brushing machine with the test slurries (50 strokes 2×/day). Specimens were kept in artificial saliva in between brushings and overnight. Enamel surface loss (SL) was determined by optical profilometry after lesion creation, 1, 3 and 5 days. Two enamel sections (from baseline and post-brushing areas) were obtained and analyzed microradiographically. Data were analyzed by analysis of variance and Tukey’s tests (α = 5%). Brushing with high-abrasive slurry caused more SL than brushing with low-
© 2014 S. Karger AG, Basel 0008–6568/14/0486–0557$39.50/0 E-Mail [email protected] www.karger.com/cre
abrasive slurry. For MeC and CMC lesions, fluoride had a protective effect on SL from day 3 on. Furthermore, for MeC and CMC, there was a significant mineral gain in the remaining lesions except when brushed with high-abrasive slurries and 0 ppm F. For HEC, a significant mineral gain took place when low-abrasive slurry was used with fluoride. The tested lesions responded differently to the toothbrushing procedures. Both slurry fluoride content and abrasivity directly impacted SL and mineral gain of enamel caries lesions. © 2014 S. Karger AG, Basel
Bacteria present in the dental plaque metabolize sucrose, producing acids as byproducts [Marsh, 1995; Kleinberg, 2002]. As a result, plaque fluid becomes undersaturated with respect to the tooth structure, leading to demineralization and development of incipient caries lesions [Cury and Tenuta, 2009]. Although these lesions have been generally described as subsurface demineralization with a mineralized surface layer, their mineral distribution profile has been shown to vary considerably [Cochrane et al., 2012] potentially due to different clinical factors, including location, stage of development, progression speed, remineralization level and fluoride exposure, among others. At this early stage, there is no need for surgical intervention and the treatment usually consists of elimination of causative factors (plaque and ferAnderson T. Hara Oral Health Research Institute Indiana University School of Dentistry 415 Lansing Street, Indianapolis, IN 46202-2876 (USA) E-Mail ahara @ iu.edu
mentable carbohydrates) and use of fluoride, which has shown the ability to enhance the remineralization of demineralized enamel [Ogaard and Rolla, 1992; Ten Cate and Mundorff-Shrestha, 1995]. Among the different vehicles for fluoride delivery, dentifrices stand out due to their widespread use and easy access to the public [Ripa, 1991]. However, dentifrices contain abrasive components necessary to provide adequate cleaning during toothbrushing [Stookey et al., 1982; Joiner et al., 2002]. Although most dentifrices are not abrasive enough to harm sound enamel [Addy and Hunter, 2003; Hooper et al., 2003; Philpotts et al., 2005], they may cause damage to demineralized enamel. Incipient lesions have softer surfaces [Arends et al., 1987] and lower mechanical properties [Arends and Christoffersen, 1986] compared to sound enamel. Kielbassa et al. [2005] showed that surface loss (SL) values of enamel carious lesions were twice as high as those of sound enamel, indicating that lesion surface layer removal could take place under the effect of toothpaste abrasives. However, the influence of different mineral profiles of the lesion and the interaction between fluoride and abrasive effects remain unknown. This can be an important factor for the arrest of incipient caries lesions [Fejerskov et al., 2008; Cury and Tenuta, 2009], as the dynamics of remineralization during toothbrushing with fluoride dentifrice may be modified by its abrasive potential. It has been suggested that the reversal of early caries lesions may be related to their mechanical removal by abrasion [Artun and Thylstrup, 1989; Fejerskov et al., 2008; Cury and Tenuta, 2009]. However, enamel lesions may greatly differ in depth, mineral profile and chemical composition [Arends and Christoffersen, 1986], which may impact their response to remineralization and surface abrasion, deserving further investigation. In the present study, we created enamel caries lesions in vitro with three distinct mineral contents/profiles and investigated the interplay between fluoride and dentifrice abrasives on their SL and remineralization. The main objective was to better understand the dynamics involved in the arrest of incipient lesions. We hypothesized that toothbrushing abrasion could modulate the fluoride remineralization effect, regardless of the type of lesion studied. Materials and Methods Experimental Design This study followed a factorial 3 × 2 × 2 × 3 design (lesion type × abrasive level × fluoride content × time). Three demineralization protocols were used to create enamel lesions: methylcellulose acid
558
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
gel (MeC), carboxymethylcellulose solution (CMC) and hydroxyethylcellulose gel (HEC). The created lesions were brushed with slurries, simulating dentifrice-saliva mixtures containing low or high abrasives, with or without fluoride, for specified time intervals. A total of 120 specimens of bovine enamel were prepared and randomly distributed among the groups (n = 10). SL values were measured after lesion creation (baseline) and after 1 (day 1), 3 (day 3) and 5 days of testing (day 5) by optical profilometry. The SL difference from baseline was calculated after each brushing time and expressed in micrometers. Mineral changes were measured by transversal microradiography (TMR) after lesion creation and day 5 and expressed as overall mineral loss (ΔZ) and mean lesion depth. Specimen Preparation Enamel slabs (5 × 5 mm) obtained from bovine teeth free from white spots, cracks and other defects were used in this study. After collection and during the preparation process, the teeth were stored in 0.1% thymol solution. The bottom and top (enamel) sides of the slabs were sequentially ground flat using silicon carbide grinding papers (RotoPol 31/RotoForce 4 polishing unit; Struers, Cleveland, Ohio, USA). A uniform thickness of approximately 2 mm was created. Slabs were then embedded in acrylic resin (Varidur acrylic system; Buehler, Lake Bluff, Ill., USA) utilizing a custom-made silicon mold, leaving the enamel surfaces exposed. The embedded blocks were serially ground and polished up to a 4,000-grit grinding paper followed by 1-μm diamond polishing suspension. Lesion Creation Adhesive UPVC tapes were used to cover the enamel surface of each specimen, leaving a 2 × 5 mm central area exposed (fig. 1). 120 specimens were randomly assigned to three groups, according to the lesion type and submitted to one of the three demineralization protocols: (1) 7-day exposure to MeC (modification of the method by Ten Cate et al. [1996]): 5% methylcellulose covered with an equal mass of a solution containing 0.1 M lactic acid at a pH of 4.6 (adjusted using KOH). (2) 10-day exposure to CMC (as described by Lippert et al. [2011]): 0.1 M lactic acid, 4.1 mM Ca (as CaCl2 ∙ 2H2O), 8 mM PO4 (as KH2PO4) and 1% w/v carboxymethylcellulose (high viscosity; Sigma-Aldrich, St. Louis, Mo., USA), pH-adjusted to 5.0 using KOH. (3) 7-day exposure to HEC (modification of the method by Amaechi et al. [1998]): 0.05 M lactic acid at a ratio of 140 g HEC per liter of lactic acid solution, pH-adjusted to 5.0 using KOH. For all demineralization protocols, the demineralizing agent was not stirred or replaced throughout the demineralization period. Procedures were performed at 37 ° C.
Surface Loss Measurement SL was measured using an optical profilometer (Proscan 2000; Scantron, Taunton, UK) after creation of the lesions as well as on days 1, 3 and 5. Tapes were removed from specimens and an area of 3 × 1 mm in the center of the specimen (covering both exposed and tape-covered areas) was scanned. The SL was calculated by subtracting the height of the exposed area from the two reference (tape-covered) areas using a dedicated software (Proscan 2000, Scantron). Profilometric analysis readings from each treatment group were used as baseline for subsequent SL analyses after the brushing-remineralizing procedures. In addition, baseline SL measurements were used for stratified randomization of the specimens into the experimental groups for each lesion type.
Nassar /Lippert /Eckert /Hara
Baseline
3 × 1 mm area used for profilometric analysis scanned in this direction
5m m
2m m
sh
ing
dir ec tio n
1.5 mm
To o
th b
ru
Post
Tape
©/C
5 mm Baseline demineralization area covered with adhesive tape
² © ©= CC
Demineralization area exposed to toothbrushing
SL
Fig. 1. Diagram illustrating the specimen setup used in the study.
Fig. 2. Graph showing the rationale for deriving Δ(ΔZ)C and ΔLC using the formulas Δ(ΔZ)C = ΔZbase – (ΔZpost + SL × 87) and ΔLC = Lbase – (Lpost + SL).
Daily Brushing-Remineralizing Procedure Specimens were brushed with soft toothbrushes (Oral-B 40; Procter & Gamble, Cincinnati, Ohio, USA) mounted in a custommade automated brushing machine, delivering reciprocal brushing strokes under 150 g of load [Ganss et al., 2009]. Four slurry variants (table 1) were used with two levels of abrasives with 275 ppm (14.5 mM) fluoride as NaF (representing 1,100 ppm F of regular toothpaste at a ratio of 1:3) or without fluoride (0 ppm F). Abrasive slurries were prepared by mixing the ingredients above with an aqueous suspension containing 0.5% (w/w) Blanose 7MF carboxymethylcellulose (CMC; Ashland Inc., Covington, Ky., Ohio, USA) and 10% (w/w) glycerol. 60 g of the slurry were used in each slot of the brushing machine. Specimens (n = 10) were brushed with their respective assigned slurry for 50 strokes (15 s) and then stored in artificial saliva (1.45 mM CaCl2 ∙ 2H2O, 5.4 mM KH2PO4, 0.1 M Tris buffer, 2.2 g/l porcine gastric mucin, pH 7.0) for 4 h under stirring at 150 rpm. Then, specimens were brushed for an additional 50 strokes and stored in artificial saliva overnight. The brushing protocol was repeated for 5 days, resulting in a total of 500 brushing strokes per specimen.
Table 1. Abrasive slurry compositions
RDAa
Slurry NaF
Abrasives
1 2 3 4
3.0 g Zeodent® 113c 69 (2.6) 69 (2.6) 3.0 g Zeodent® 113 9.0 g Zeodent® 103 208 (9.4) 9.0 g Zeodent® 103 208 (9.4)
– 0.036 gd – 0.036 g
REAb 4.01 (0.28) 4.01 (0.28) 7.14 (0.69) 7.14 (0.69)
All slurries also contained 2.5 g carboxymethylcellulose (Blanose 7MF, Ashland Inc.), 5.0 g glycerol and were made up to 60.0 g with deionized water. a Relative dentin abrasivity [mean (standard deviation)]. b Relative enamel abrasivity. c Zeodent abrasives are precipitated silica particles (J.M. Huber Corporation, Atlanta, Ga., USA). d This amount is equivalent to 275 ppm F in the prepared slurry.
Microradiographic Analysis Specimens were mounted on plastic rods and sectioned with a hard tissue microtome (Silverstone-Taylor Hard Tissue Microtome, Series 1000 Deluxe; Silverstone-Taylor, SciFab, Lafayette, Colo., USA). Two 100-μm sections were obtained from each specimen; one section was acquired through the baseline demineralized area and the other one was obtained from the demineralized area that was subjected to toothbrushing (fig. 1). Sections were mounted on X-ray-sensitive plates (Microchrome Technology Inc., San Jose, Calif., USA) and subjected to X-ray, along with an aluminum step wedge. Microradiographic images were analyzed with dedicated software (Inspektor TMR 2000, ver.1.25) with 87% set as a reference level, for the average mineral content of sound enamel by volume. The mineral content in bovine specimens is comparable to that of human enamel [Edmunds et al., 1988] and
Fluoride and Abrasives Effect on Artificial Caries Lesions
was found to be equivalent to 87% [Angmar et al., 1963; de Josselin de Jong et al., 1987]. Three parameters were obtained: ΔZ, mean lesion depth and degree of surface zone mineralization (SZmax). For each specimen, the difference in these outcomes between the two sections (baseline and after abrasion) was expressed as Δ(ΔZ), ΔL and ΔSZmax. Since this project involved structural loss due to toothbrushing abrasion, a modification of the TMR parameters was made to compensate for SL (fig. 2). This produced two additional terms: corrected change in mineral loss of the lesion (Δ(ΔZ)C) and corrected change in depth of the lesion (ΔLC) calculated using the following equations: Δ(ΔZ)C = ΔZbase – (ΔZpost + SL × 87) ΔLC = Lbase – (Lpost + SL) Statistical Analysis Repeated measures analysis of variance (RM-ANOVA) was used to test the effects of lesion type (MeC, CMC, HEC), slurry abrasive-
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
559
Table 2. Cumulative SL [mean (standard
deviation)] as a function of lesion type (MeC, CMC, HEC) and slurry fluoride content and abrasive level
Lesion type
Abrasive F (ppm) n level
SL (μm) day 1
day 3
MeC
low high
CMC
low high
HEC
low high
day 5
0 275 0 275
10 10 9 10
0.28 (0.06)a 0.35 (0.10)a 1.70 (0.28)a 1.07 (0.10)a
0.84 (0.13)b 0.55 (0.15)b 6.32 (1.22)b 2.14 (0.35)b
1.13 (0.14)c 0.78 (0.23)c 10.97 (1.43)c 3.90 (0.56)c
0 275 0 275
10 10 9 9
0.23 (0.07)a 0.07 (0.05)a 1.21 (0.17)a 0.97 (0.13)a
0.55 (0.14)b 0.13 (0.05)b 2.11 (0.29)b 1.36 (0.22)b
0.65 (0.16)b 0.21 (0.06)b 2.73 (0.43)c 1.72 (0.32)c
0 275 0 275
10 10 10 10
0.14 (0.06)a 0.14 (0.04)a 0.33 (0.03)a 0.30 (0.06)a
0.22 (0.06)a 0.16 (0.05)a 0.50 (0.07)a,b 0.41 (0.06)a,b
0.26 (0.05)a 0.19 (0.05)a 0.66 (0.08)b 0.50 (0.08)b
Same superscript letters indicate non-significant effect of time (p > 0.05) within rows. Groups connected by brackets are not significantly different (p > 0.05).
Table 3. Means (standard deviation) of TMR parameters
Lesion type
Abrasive F (ppm) n level
Δ(ΔZ)C (vol.%×μm)
ΔLC (μm)
MeC
low high
CMC
low high
HEC
low high
0 275 0 275
10 10 9 10
617 (220)* 915 (166)* 520 (240) 946 (154)*
– 9.4 (3.8)* – 5.2 (2.7) – 8.0 (5.9) – 8.4 (4.7)
0 275 0 275
8 10 9 10
746 (185)* 436 (165)* 260 (164) 573 (223)*
–13.5 (5.2)* – 7.8 (3.4) – 4.7 (5.3) – 7.0 (4.3)
0 275 0 275
9 10 9 8
204 (92) 372 (103)* 191 (96) 48 (209)
– 4.4 (4.0) – 9.4 (5.1) – 1.0 (7.2) – 0.1 (5.6)
* Statistically significant change vs. lesion baseline (p < 0.05).
ness (low, high), slurry fluoride (0 ppm F, 275 ppm F) and time (1, 3, 5 days) on SL [Jennrich and Schluchter, 1986]. An unstructured variance/covariance matrix was used to model the variances and correlations within a specimen over time. Pairwise comparisons among the treatment combinations were made using Tukey’s multiple comparisons procedure to control the overall significance level at 5%. The analyses were performed after a natural logarithm transformation of the data to satisfy the RM-ANOVA assumptions. For TMR, ANOVA was used to test the effects of lesion type (MeC, CMC, HEC), slurry abrasiveness (low, high) and slurry fluoride (0 ppm F, 275 ppm F) on Δ(ΔZ)C and ΔLC. Pairwise com-
560
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
parisons among the treatment combinations were made using Tukey’s multiple comparisons procedure. To test changes in the mineral content within each group, a paired t test was used to compare ΔZbase and ΔZpost.
Results
Specimen Loss Some specimens were excluded from statistical testing (tables 2, 3) either due to the presence of artifacts in the profilometric scans (SL) or for being broken during preparation (TMR). Profilometric Analysis Effect of Lesion Type. MeC had significantly more SL than HEC (p < 0.0001). MeC had significantly more SL than CMC for day 3 (p < 0.0001) and day 5 (p < 0.0001), but not for day 1 (p = 0.06). CMC had significantly more SL than HEC for high-abrasive slurry (p < 0.0001), but not for low-abrasive slurry (p = 0.91). Effect of Abrasive Level. Overall, more SL was associated to the high-abrasive slurries compared to the lowabrasive slurries (p < 0.0001). Effect of Fluoride. For MeC and CMC, 0 ppm F had significantly more SL than 275 ppm F after day 3 (p < 0.05) and day 5 (p < 0.05). However, there was no fluoride effect for day 1 (p > 0.05). Fluoride had no effect on the HEC specimens (p = 0.54).
Nassar /Lippert /Eckert /Hara
Comparisons of mineral loss between baseline and post-brushing are illustrated in figure 4. For the MeC and CMC groups, there was a significant mineral gain in all groups except for the combination of high-abrasive slurries with 0 ppm F. For HEC, only the group brushed with low-abrasive slurries and 275 ppm F showed a significant mineral gain (p = 0.006).
Mineral content (vol.%)
100 80 60 40 MeC
CMC
HEC
Discussion
20 0
0
50
100 Depth (μm)
150
200
Fig. 3. Graph showing the average mineral distribution for MeC, CMC and HEC lesions at baseline.
In this study, it was observed that the interaction between the fluoride content and the abrasive level of dentifrices can potentially affect the abrasion resistance and remineralization of incipient enamel caries lesions. In addition, the different characteristics (mineral content of the lesion, surface layer and mean mineral distribution profile) of the lesions were shown to play an important role.
Transverse Microradiography Baseline comparisons of the lesions created using the three demineralization protocols are shown in figure 3. Overall, MeC lesions had the least mineral content in the surface layer. CMC lesions were significantly deeper compared to the other two lesion types, while HEC had the smallest ΔZbase values. Both abrasive and fluoride levels did not have a significant effect on Δ(ΔZ)C or ΔLC (table 3). However, when comparing lesion types, HEC had significantly lower Δ(ΔZ)C than CMC (p = 0.0162) and MeC (p < 0.0001). CMC and MeC were not significantly different from each other (p = 0.19). The type of lesion had no effect on ΔLC.
Model Justification To investigate the study question under clinically relevant conditions, we chose to use a 5-day remineralization protocol, using artificial saliva, and incorporated the brushing sessions within it. The average brushing time was reported to be approximately 90 s [Ganss et al., 2009]. However, the recommended brushing time to achieve adequate plaque removal is estimated to be 2 min [Van der Weijden et al., 1993]. From multiple pilot studies, we found that these values correspond to 15–17 s of brushing per surface or approximately 50 brushing strokes. This gave a total of 500 brushing strokes at the end of the study that would be representative of 5 days of brushing. Although the duration of the protocol was relatively short, it represents the daily scenario in which the teeth would be exposed to the toothpaste twice per day and remain soaked in saliva for the remainder of the day and while the individual is sleeping. In addition, due to the lower hardness values of the lesions we were able to observe general trends in the behavior of the three tested lesion types in this study. Artificially created enamel lesions can be considered as a suitable alternative in studying enamel lesions in vitro. The protocols used in the present study have been used previously to create subsurface lesions [Ten Cate et al., 1996; Amaechi et al., 1998; Lippert et al., 2011] and were chosen to resemble the variability seen in natural lesions [Cochrane et al., 2012]. This variability could lead to different responses to the toothbrushing abrasion and fluoride effects. Among the three lesion types used in the study, MeC lesions tended to have the least mineralized surface layer, followed by CMC and HEC lesions. For
Fluoride and Abrasives Effect on Artificial Caries Lesions
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Effect of Time. For MeC, day 5 had more SL compared to day 1 for all combinations of abrasive level and fluoride content (p < 0.0001; table 2). Day 5 had significantly more SL than day 3 (p < 0.00001), with a larger difference for high-abrasive slurry level. Day 3 had significantly more SL than day 1 (p < 0.0001). For CMC, day 5 had significantly more SL than day 1 for all abrasive and fluoride combinations (p < 0.0001). Day 5 had significantly more SL than day 3, but only for high-abrasive slurry level (p = 0.0007). Day 3 had significantly more SL than day 1 (p < 0.0001). For HEC, no difference in SL was seen among at the studied days for low-abrasive slurry. When high-abrasive slurry was used, more SL was seen on day 5 compared to day 1 for both fluoride levels; day 3 did not differ from either day 1 or day 5.
561
0 ppm F + low abrasives
100
275 ppm F + low abrasives 0 ppm F + high abrasives
*
80
100
*
80
80
60
60
60
40
40
40
20
20
20
0
0
100
200
100
0
0
100
200
100
*
80
0
*
80
60
40
40
40
20
20
20
0
100
200
0
0
100
200
0
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
100
200
100
0
0
100
200
100
*
80
100
200
0
100
200
0
100
200
100 HEC
200
*
80
60
0
0
100
60
0
275 ppm F + high abrasives
100
0
100
*
80
80
60
60
60
40
40
40
20
20
20
0
0
100 MeC
200
0
0 0
100 CMC
200
0
Fig. 4. Graphs showing the average mineral distribution at baseline (grey lines) and post-brushing (black lines) of the various groups tested in the study. Depth (μm) and mineral content (vol.%) are presented on the x and y axis, respectively. Asterisks indicate significant difference (p < 0.05) for the paired t test within each group.
562
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Nassar /Lippert /Eckert /Hara
Table 4. Baseline data for microradiographic analysis of the three
lesion types [mean (standard deviation)] Lesion type
n
ΔZbase (vol.%×μm)
Lbase (μm)
SZmax (vol.%×min)
MeC CMC HEC
39 37 36
2,576.7 (504.7)a 2,505.7 (476.9)a 1,313.3 (272.2)b
80.9 (11.2)a 108.1 (13.8)b 79.4 (12.8)a
49.2 (4.3)a 60.8 (5.0)b 73.1 (2.8)c
Same superscript letters indicate statistically non-significant difference (p > 0.05) for a particular parameter (in the same column).
HEC, this could translate into a more compact surface that could hinder the penetration of minerals into the body of the lesion. Most of the mineral gain was observed at the surface layer, with minor mineral changes in the body of the lesion. The remineralization potential of fluoride could be seen more clearly in the other lesion types, possibly due to the less mineralized surface layer, in the range of 35 and 55 vol.%, which could invite more minerals leading to more resistance to abrasion [Lippert et al., 2011, 2012]. Therefore, the association between mineral content and mechanical properties, such as abrasion resistance, was shown in the present study.
responses across the lesion types could be explained by the inherent differences in the structure of the surface layer between those lesions. HEC lesions were the most resistant to abrasion, followed by CMC and MeC lesions. This can be attributed to differences in the mineral composition of the surface layer, as deeper parts of the lesion were not directly involved in the abrasive process. Therefore, our data seem to support the hypothesis that toothbrushing abrasion leads to enamel structural loss, playing a role in the arrest of white spot lesions [Stookey and Muhler, 1968; Holmen et al., 1987; Artun and Thylstrup, 1989; Kielbassa et al., 2005; Fejerskov et al., 2008]. As previously mentioned, brushing with the more abrasive slurries could remove the most superficial layers of the lesion, which could potentially enhance its remineralization, especially in the presence of fluoride.
Brushing Effects on Lesion Surface Loss Overall, slurries with high abrasive levels led to more SL compared to low abrasive ones. Slurries with higher REA/RDA values have shown a higher abrasive potential on the natural tooth structure [Philpotts et al., 2005]; consequently, this effect is expected to be more pronounced on the relatively softer lesions. This trend was observed for all three lesion types and is in accordance with results reported by Kielbassa et al. [2005], who found a direct correlation between SL values of artificial enamel lesions and the abrasivity of toothpaste slurries. However, the abrasive effect on the HEC lesions was less pronounced, which could be possibly related to the higher mineral content of the surface layer (table 4). The protective effect of fluoride against SL has been reported previously, especially in relation to softened erosive lesions [Attin et al., 1998]. In the present study, the fluoride effect was dependent on the lesion type and the time effect. In general, MeC and CMC showed a similar trend, with the fluoride effect detected after day 3 and being more pronounced on day 5. However, for HEC, no fluoride protective effect could be observed even after 5 days of exposure. The difference between the fluoride
Brushing Effects on Lesion Remineralization MeC and CMC lesions showed significant mineral gain throughout the study, except when brushed with high abrasives in the absence of fluoride. In this situation, we suspect that the effect of structural loss due to the high abrasivity of the slurry accelerated the removal of the surface layer. A similar finding was reported previously by Kielbassa et al. [2005]. In the presence of fluoride, it was speculated that brushing with high-abrasive slurries could show some benefits by preventing the formation of a highly mineralized surface layer and by facilitating the access of minerals and subsequent remineralization of the body of the lesion. However, this was not observed, possibly due to the excessively harsh toothbrushing abrasion compared to the limited remineralization by fluoride. Use of fluoride-containing slurries with intermediate abrasive levels may allow us to find this effect, deserving further investigation. Likewise, it must be noted that HEC lesions had the least mineral loss at baseline, thereby allowing for comparatively less mineral gain than MeC and CMC lesions. Within groups showing significant mineral gain, those brushed with fluoride-containing slurries showed more mineral gain in the surface layer, as seen in the mineral profiles (fig. 4). Contrary to this, HEC lesions showed significant mineral gain only in the group brushed with low-abrasive slurries in the presence of fluoride. This could be explained by the fact that a higher driving force for mineral deposition of the fluoride was needed to allow some mineral gain past the highly mineralized (∼70 vol.%) and less porous surface layer of the HEC lesion, leading to more resistance to abrasion and consequently hindering further remineralization [Ten Cate, 1990].
Fluoride and Abrasives Effect on Artificial Caries Lesions
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
563
It has been reported that higher values of ΔZbase are associated with a higher tendency of lesions to remineralize [Strang et al., 1987; Schafer et al., 1992]. Although all lesions showed some remineralization in the present study, such a relationship between remineralization and ΔZbase could not be observed in the Δ(ΔZ)C analysis. HEC lesions had significantly lower ΔZbase values compared to MeC and CMC lesions, yet no differences in mineral gain [Δ(ΔZ)C] could be observed. One explanation could be the differences in the remineralization protocols of the cited studies, which were conducted under in situ conditions and involved periods of acid challenge. Acid challenges can dissolve minerals at the surface, forming a more porous layer that could allow minerals to penetrate deeper and enhance remineralization. Another difference is the duration of remineralization. In the present study, remineralization was performed for 5 days, which is shorter than the duration of most in situ studies (2–5 weeks). As with other laboratory reports, this study had some limitations. The artificial lesions used might not represent natural white spot lesions completely. Unfortunately, this limitation cannot be solved until a comprehensive characterization of white spot lesions has been performed. Hence, we chose three different demineralization protocols that resemble the variability associated with natural white spots. Also, the remineralization protocol involved the use of artificial saliva. Natural saliva was not considered due to the large amount needed and potential infection control issues. Still, components of natural saliva, such as salivary proteins, could affect remineralization and SL by acting as a protective layer and/or a lubricating agent [West et al., 1998].
Conclusions
The effect of higher abrasive content could be seen in all three lesion types tested in this study, whereas the effect of fluoride was dependent on the type of lesion tested, with MeC and CMC lesions shown to be more responsive than HEC lesions. More abrasive slurries were not able to allow improved remineralization for the artificially created lesions within the adopted studied conditions.
Acknowledgements Dr. H.M. Nassar was sponsored by a scholarship from King Abdulaziz University. This research was part of his thesis submitted in partial fulfilment of the Doctor of Philosophy in Preventive Dentistry degree awarded in 2012 from Indiana University. The authors would like to thank Dr. Carlos González-Cabezas, Dr. Margherita Fontana and Dr. Tien-Min Chu for their insightful comments and feedback. This project was supported by the Dental Erosion-Abrasion Research Program of the Oral Health Research Institute, Indiana University School of Dentistry.
Author Contribution H.M. Nassar: study design, lab phase conduction, data interpretation, manuscript writing. F. Lippert: study design, data interpretation, manuscript review. G.J. Eckert: study design, statistical analysis, data interpretation, manuscript review. A.T. Hara: study idea, study design, data interpretation, manuscript review.
Disclosure Statement The authors declare no conflict of interest.
References Addy M, Hunter ML: Can tooth brushing damage your health? Effects on oral and dental tissues. Int Dent J 2003;53(suppl 3):177–186. Amaechi BT, Higham SM, Edgar WM: Factors affecting the development of carious lesions in bovine teeth in vitro. Arch Oral Biol 1998;43: 619–628. Angmar B, Carlström D, Glas JE: Studies on the ultrastructure of dental enamel. IV. The mineralization of normal tooth enamel. J Ultrastruct Res 1963;8:12–23. Arends J, Christoffersen J: The nature of early caries lesions in enamel. J Dent Res 1986; 65: 2– 11.
564
Arends J, Jongebloed W, Ogaard B, Rolla G: SEM and microradiographic investigation of initial enamel caries. Scand J Dent Res 1987;95:193– 201. Artun J, Thylstrup A: A 3-year clinical and SEM study of surface changes of carious enamel lesions after inactivation. Am J Orthod Dentofacial Orthop 1989;95:327–333. Attin T, Zirkel C, Hellwig E: Brushing abrasion of eroded dentin after application of sodium fluoride solutions. Caries Res 1998; 32: 344– 350.
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Cochrane NJ, Anderson P, Davis GR, Adams GG, Stacey MA, Reynolds EC: An X-ray microtomographic study of natural white-spot enamel lesions. J Dent Res 2012;91:185–191. Cury JA, Tenuta LM: Enamel remineralization: controlling the caries disease or treating early caries lesions? Braz Oral Res 2009; 23(suppl 1):23–30. de Josselin de Jong E, ten Bosch JJ, Noordmans J: Optimised microcomputer-guided quantitative microradiography on dental mineralised tissue slices. Phys Med Biol 1987;32:887–899.
Nassar /Lippert /Eckert /Hara
Edmunds DH, Whittaker DK, Green RM: Suitability of human, bovine, equine, and ovine tooth enamel for studies of artificial bacterial carious lesions. Caries Res 1988; 22: 327– 336. Fejerskov O, Nyvad B, Kidd E: Dental Caries: The Disease and Its Clinical Management, ed 2. Oxford, Blackwell Munksgaard, 2008. Ganss C, Schlueter N, Preiss S, Klimek J: Tooth brushing habits in uninstructed adults – frequency, technique, duration and force. Clin Oral Investig 2009;13:203–208. Holmen L, Thylstrup A, Artun J: Surface changes during the arrest of active enamel carious lesions in vivo. A scanning electron microscope study. Acta Odontol Scand 1987;45:383–390. Hooper S, West NX, Pickles MJ, Joiner A, Newcombe RG, Addy M: Investigation of erosion and abrasion on enamel and dentine: a model in situ using toothpastes of different abrasivity. J Clin Periodontol 2003;30:802–808. Jennrich RI, Schluchter MD: Unbalanced repeated-measures models with structured covariance matrices. Biometrics 1986;42:805–820. Joiner A, Pickles M, Matheson J, Weader E, Noblet L, Huntington E: Whitening toothpastes effects on tooth stain and enamel. Int Dent J 2002;52:412–430. Kielbassa AM, Gillmann L, Zantner C, MeyerLueckel H, Hellwig E, Schulte-Monting J: Profilometric and microradiographic studies on the effects of toothpaste and acidic gel abrasivity on sound and demineralized bovine dental enamel. Caries Res 2005; 39: 380– 386.
Fluoride and Abrasives Effect on Artificial Caries Lesions
Kleinberg I: A mixed-bacteria ecological approach to understanding the role of the oral bacteria in dental caries causation: an alternative to Streptococcus mutans and the specificplaque hypothesis. Crit Rev Oral Biol Med 2002;13:108–125. Lippert F, Butler A, Lynch RJ, Hara AT: Effect of fluoride, lesion baseline severity and mineral distribution on lesion progression. Caries Res 2012;46:23–30. Lippert F, Lynch RJ, Eckert GJ, Kelly SA, Hara AT, Zero DT: In situ fluoride response of caries lesions with different mineral distributions at baseline. Caries Res 2011;45:47–55. Marsh PD: The role of microbiology in models of dental caries. Adv Dent Res 1995; 9: 244–254; discussion 255–269. Ogaard B, Rolla G: The in vivo orthodontic banding model for vital teeth and the in situ orthodontic banding model for hard-tissue slabs. J Dent Res 1992;71(Spec No):832–835. Philpotts CJ, Weader E, Joiner A: The measurement in vitro of enamel and dentine wear by toothpastes of different abrasivity. Int Dent J 2005;55:183–187. Ripa LW: A critique of topical fluoride methods (dentifrices, mouthrinses, operator-, and selfapplied gels) in an era of decreased caries and increased fluorosis prevalence. J Public Health Dent 1991;51:23–41. Schafer F, Raven SJ, Parr TA: The effect of lesion characteristic on remineralization and model sensitivity. J Dent Res 1992;71(Spec No):811– 813.
Stookey GK, Burkhard TA, Schemehorn BR: In vitro removal of stain with dentifrices. J Dent Res 1982;61:1236–1239. Stookey GK, Muhler JC: Laboratory studies concerning the enamel and dentin abrasion properties of common dentifrice polishing agents. J Dent Res 1968;47:524–532. Strang R, Damato FA, Creanor SL, Stephen KW: The effect of baseline lesion mineral loss on in situ remineralization. J Dent Res 1987; 66: 1644–1646. Ten Cate JM: In vitro studies on the effects of fluoride on de- and remineralization. J Dent Res 1990; 69(Spec No):614–619; discussion 634–636. Ten Cate JM, Dundon KA, Vernon PG, Damato FA, Huntington E, Exterkate RA, Wefel JS, Jordan T, Stephen KW, Roberts AJ: Preparation and measurement of artificial enamel lesions, a four-laboratory ring test. Caries Res 1996;30:400–407. Ten Cate JM, Mundorff-Shrestha SA: Working group report 1: Laboratory models for caries (in vitro and animal models). Adv Dent Res 1995;9:332–334. Van der Weijden GA, Timmerman MF, Nijboer A, Lie MA, Van der Velden U: A comparative study of electric toothbrushes for the effectiveness of plaque removal in relation to toothbrushing duration. Timerstudy. J Clin Periodontol 1993;20:476–481. West NX, Maxwell A, Hughes JA, Parker DM, Newcombe RG, Addy M: A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel. J Dent 1998;26:329–335.
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
565
Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: September 29, 2013 Accepted after revision: January 7, 2014 Published online: July 3, 2014
Dentifrice Fluoride and Abrasivity Interplay on Artificial Caries Lesions Hani M. Nassar a, b Frank Lippert b George J. Eckert c Anderson T. Hara b a
Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; b School of Dentistry, Indiana University, and c Indiana University School of Medicine, Indianapolis, Ind., USA
Key Words Abrasion · Abrasives · Dentifrice · Fluoride · Incipient caries · Remineralization
Abstract Incipient caries lesions on smooth surfaces may be subjected to toothbrushing, potentially leading to remineralization and/or abrasive wear. The interplay of dentifrice abrasivity and fluoride on this process is largely unknown and was investigated on three artificially created lesions with different mineral content/distribution. 120 bovine enamel specimens were randomly allocated to 12 groups (n = 10), resulting from the association of (1) lesion type [methylcellulose acid gel (MeC); carboxymethylcellulose solution (CMC); hydroxyethylcellulose gel (HEC)], (2) slurry abrasive level [low (REA 4/ RDA 69); high (REA 7/RDA 208)], and (3) fluoride concentration [0/275 ppm (14.5 mM) F as NaF]. After lesion creation, specimens were brushed in an automated brushing machine with the test slurries (50 strokes 2×/day). Specimens were kept in artificial saliva in between brushings and overnight. Enamel surface loss (SL) was determined by optical profilometry after lesion creation, 1, 3 and 5 days. Two enamel sections (from baseline and post-brushing areas) were obtained and analyzed microradiographically. Data were analyzed by analysis of variance and Tukey’s tests (α = 5%). Brushing with high-abrasive slurry caused more SL than brushing with low-
© 2014 S. Karger AG, Basel 0008–6568/14/0486–0557$39.50/0 E-Mail [email protected] www.karger.com/cre
abrasive slurry. For MeC and CMC lesions, fluoride had a protective effect on SL from day 3 on. Furthermore, for MeC and CMC, there was a significant mineral gain in the remaining lesions except when brushed with high-abrasive slurries and 0 ppm F. For HEC, a significant mineral gain took place when low-abrasive slurry was used with fluoride. The tested lesions responded differently to the toothbrushing procedures. Both slurry fluoride content and abrasivity directly impacted SL and mineral gain of enamel caries lesions. © 2014 S. Karger AG, Basel
Bacteria present in the dental plaque metabolize sucrose, producing acids as byproducts [Marsh, 1995; Kleinberg, 2002]. As a result, plaque fluid becomes undersaturated with respect to the tooth structure, leading to demineralization and development of incipient caries lesions [Cury and Tenuta, 2009]. Although these lesions have been generally described as subsurface demineralization with a mineralized surface layer, their mineral distribution profile has been shown to vary considerably [Cochrane et al., 2012] potentially due to different clinical factors, including location, stage of development, progression speed, remineralization level and fluoride exposure, among others. At this early stage, there is no need for surgical intervention and the treatment usually consists of elimination of causative factors (plaque and ferAnderson T. Hara Oral Health Research Institute Indiana University School of Dentistry 415 Lansing Street, Indianapolis, IN 46202-2876 (USA) E-Mail ahara @ iu.edu
mentable carbohydrates) and use of fluoride, which has shown the ability to enhance the remineralization of demineralized enamel [Ogaard and Rolla, 1992; Ten Cate and Mundorff-Shrestha, 1995]. Among the different vehicles for fluoride delivery, dentifrices stand out due to their widespread use and easy access to the public [Ripa, 1991]. However, dentifrices contain abrasive components necessary to provide adequate cleaning during toothbrushing [Stookey et al., 1982; Joiner et al., 2002]. Although most dentifrices are not abrasive enough to harm sound enamel [Addy and Hunter, 2003; Hooper et al., 2003; Philpotts et al., 2005], they may cause damage to demineralized enamel. Incipient lesions have softer surfaces [Arends et al., 1987] and lower mechanical properties [Arends and Christoffersen, 1986] compared to sound enamel. Kielbassa et al. [2005] showed that surface loss (SL) values of enamel carious lesions were twice as high as those of sound enamel, indicating that lesion surface layer removal could take place under the effect of toothpaste abrasives. However, the influence of different mineral profiles of the lesion and the interaction between fluoride and abrasive effects remain unknown. This can be an important factor for the arrest of incipient caries lesions [Fejerskov et al., 2008; Cury and Tenuta, 2009], as the dynamics of remineralization during toothbrushing with fluoride dentifrice may be modified by its abrasive potential. It has been suggested that the reversal of early caries lesions may be related to their mechanical removal by abrasion [Artun and Thylstrup, 1989; Fejerskov et al., 2008; Cury and Tenuta, 2009]. However, enamel lesions may greatly differ in depth, mineral profile and chemical composition [Arends and Christoffersen, 1986], which may impact their response to remineralization and surface abrasion, deserving further investigation. In the present study, we created enamel caries lesions in vitro with three distinct mineral contents/profiles and investigated the interplay between fluoride and dentifrice abrasives on their SL and remineralization. The main objective was to better understand the dynamics involved in the arrest of incipient lesions. We hypothesized that toothbrushing abrasion could modulate the fluoride remineralization effect, regardless of the type of lesion studied. Materials and Methods Experimental Design This study followed a factorial 3 × 2 × 2 × 3 design (lesion type × abrasive level × fluoride content × time). Three demineralization protocols were used to create enamel lesions: methylcellulose acid
558
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
gel (MeC), carboxymethylcellulose solution (CMC) and hydroxyethylcellulose gel (HEC). The created lesions were brushed with slurries, simulating dentifrice-saliva mixtures containing low or high abrasives, with or without fluoride, for specified time intervals. A total of 120 specimens of bovine enamel were prepared and randomly distributed among the groups (n = 10). SL values were measured after lesion creation (baseline) and after 1 (day 1), 3 (day 3) and 5 days of testing (day 5) by optical profilometry. The SL difference from baseline was calculated after each brushing time and expressed in micrometers. Mineral changes were measured by transversal microradiography (TMR) after lesion creation and day 5 and expressed as overall mineral loss (ΔZ) and mean lesion depth. Specimen Preparation Enamel slabs (5 × 5 mm) obtained from bovine teeth free from white spots, cracks and other defects were used in this study. After collection and during the preparation process, the teeth were stored in 0.1% thymol solution. The bottom and top (enamel) sides of the slabs were sequentially ground flat using silicon carbide grinding papers (RotoPol 31/RotoForce 4 polishing unit; Struers, Cleveland, Ohio, USA). A uniform thickness of approximately 2 mm was created. Slabs were then embedded in acrylic resin (Varidur acrylic system; Buehler, Lake Bluff, Ill., USA) utilizing a custom-made silicon mold, leaving the enamel surfaces exposed. The embedded blocks were serially ground and polished up to a 4,000-grit grinding paper followed by 1-μm diamond polishing suspension. Lesion Creation Adhesive UPVC tapes were used to cover the enamel surface of each specimen, leaving a 2 × 5 mm central area exposed (fig. 1). 120 specimens were randomly assigned to three groups, according to the lesion type and submitted to one of the three demineralization protocols: (1) 7-day exposure to MeC (modification of the method by Ten Cate et al. [1996]): 5% methylcellulose covered with an equal mass of a solution containing 0.1 M lactic acid at a pH of 4.6 (adjusted using KOH). (2) 10-day exposure to CMC (as described by Lippert et al. [2011]): 0.1 M lactic acid, 4.1 mM Ca (as CaCl2 ∙ 2H2O), 8 mM PO4 (as KH2PO4) and 1% w/v carboxymethylcellulose (high viscosity; Sigma-Aldrich, St. Louis, Mo., USA), pH-adjusted to 5.0 using KOH. (3) 7-day exposure to HEC (modification of the method by Amaechi et al. [1998]): 0.05 M lactic acid at a ratio of 140 g HEC per liter of lactic acid solution, pH-adjusted to 5.0 using KOH. For all demineralization protocols, the demineralizing agent was not stirred or replaced throughout the demineralization period. Procedures were performed at 37 ° C.
Surface Loss Measurement SL was measured using an optical profilometer (Proscan 2000; Scantron, Taunton, UK) after creation of the lesions as well as on days 1, 3 and 5. Tapes were removed from specimens and an area of 3 × 1 mm in the center of the specimen (covering both exposed and tape-covered areas) was scanned. The SL was calculated by subtracting the height of the exposed area from the two reference (tape-covered) areas using a dedicated software (Proscan 2000, Scantron). Profilometric analysis readings from each treatment group were used as baseline for subsequent SL analyses after the brushing-remineralizing procedures. In addition, baseline SL measurements were used for stratified randomization of the specimens into the experimental groups for each lesion type.
Nassar /Lippert /Eckert /Hara
Baseline
3 × 1 mm area used for profilometric analysis scanned in this direction
5m m
2m m
sh
ing
dir ec tio n
1.5 mm
To o
th b
ru
Post
Tape
©/C
5 mm Baseline demineralization area covered with adhesive tape
² © ©= CC
Demineralization area exposed to toothbrushing
SL
Fig. 1. Diagram illustrating the specimen setup used in the study.
Fig. 2. Graph showing the rationale for deriving Δ(ΔZ)C and ΔLC using the formulas Δ(ΔZ)C = ΔZbase – (ΔZpost + SL × 87) and ΔLC = Lbase – (Lpost + SL).
Daily Brushing-Remineralizing Procedure Specimens were brushed with soft toothbrushes (Oral-B 40; Procter & Gamble, Cincinnati, Ohio, USA) mounted in a custommade automated brushing machine, delivering reciprocal brushing strokes under 150 g of load [Ganss et al., 2009]. Four slurry variants (table 1) were used with two levels of abrasives with 275 ppm (14.5 mM) fluoride as NaF (representing 1,100 ppm F of regular toothpaste at a ratio of 1:3) or without fluoride (0 ppm F). Abrasive slurries were prepared by mixing the ingredients above with an aqueous suspension containing 0.5% (w/w) Blanose 7MF carboxymethylcellulose (CMC; Ashland Inc., Covington, Ky., Ohio, USA) and 10% (w/w) glycerol. 60 g of the slurry were used in each slot of the brushing machine. Specimens (n = 10) were brushed with their respective assigned slurry for 50 strokes (15 s) and then stored in artificial saliva (1.45 mM CaCl2 ∙ 2H2O, 5.4 mM KH2PO4, 0.1 M Tris buffer, 2.2 g/l porcine gastric mucin, pH 7.0) for 4 h under stirring at 150 rpm. Then, specimens were brushed for an additional 50 strokes and stored in artificial saliva overnight. The brushing protocol was repeated for 5 days, resulting in a total of 500 brushing strokes per specimen.
Table 1. Abrasive slurry compositions
RDAa
Slurry NaF
Abrasives
1 2 3 4
3.0 g Zeodent® 113c 69 (2.6) 69 (2.6) 3.0 g Zeodent® 113 9.0 g Zeodent® 103 208 (9.4) 9.0 g Zeodent® 103 208 (9.4)
– 0.036 gd – 0.036 g
REAb 4.01 (0.28) 4.01 (0.28) 7.14 (0.69) 7.14 (0.69)
All slurries also contained 2.5 g carboxymethylcellulose (Blanose 7MF, Ashland Inc.), 5.0 g glycerol and were made up to 60.0 g with deionized water. a Relative dentin abrasivity [mean (standard deviation)]. b Relative enamel abrasivity. c Zeodent abrasives are precipitated silica particles (J.M. Huber Corporation, Atlanta, Ga., USA). d This amount is equivalent to 275 ppm F in the prepared slurry.
Microradiographic Analysis Specimens were mounted on plastic rods and sectioned with a hard tissue microtome (Silverstone-Taylor Hard Tissue Microtome, Series 1000 Deluxe; Silverstone-Taylor, SciFab, Lafayette, Colo., USA). Two 100-μm sections were obtained from each specimen; one section was acquired through the baseline demineralized area and the other one was obtained from the demineralized area that was subjected to toothbrushing (fig. 1). Sections were mounted on X-ray-sensitive plates (Microchrome Technology Inc., San Jose, Calif., USA) and subjected to X-ray, along with an aluminum step wedge. Microradiographic images were analyzed with dedicated software (Inspektor TMR 2000, ver.1.25) with 87% set as a reference level, for the average mineral content of sound enamel by volume. The mineral content in bovine specimens is comparable to that of human enamel [Edmunds et al., 1988] and
Fluoride and Abrasives Effect on Artificial Caries Lesions
was found to be equivalent to 87% [Angmar et al., 1963; de Josselin de Jong et al., 1987]. Three parameters were obtained: ΔZ, mean lesion depth and degree of surface zone mineralization (SZmax). For each specimen, the difference in these outcomes between the two sections (baseline and after abrasion) was expressed as Δ(ΔZ), ΔL and ΔSZmax. Since this project involved structural loss due to toothbrushing abrasion, a modification of the TMR parameters was made to compensate for SL (fig. 2). This produced two additional terms: corrected change in mineral loss of the lesion (Δ(ΔZ)C) and corrected change in depth of the lesion (ΔLC) calculated using the following equations: Δ(ΔZ)C = ΔZbase – (ΔZpost + SL × 87) ΔLC = Lbase – (Lpost + SL) Statistical Analysis Repeated measures analysis of variance (RM-ANOVA) was used to test the effects of lesion type (MeC, CMC, HEC), slurry abrasive-
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
559
Table 2. Cumulative SL [mean (standard
deviation)] as a function of lesion type (MeC, CMC, HEC) and slurry fluoride content and abrasive level
Lesion type
Abrasive F (ppm) n level
SL (μm) day 1
day 3
MeC
low high
CMC
low high
HEC
low high
day 5
0 275 0 275
10 10 9 10
0.28 (0.06)a 0.35 (0.10)a 1.70 (0.28)a 1.07 (0.10)a
0.84 (0.13)b 0.55 (0.15)b 6.32 (1.22)b 2.14 (0.35)b
1.13 (0.14)c 0.78 (0.23)c 10.97 (1.43)c 3.90 (0.56)c
0 275 0 275
10 10 9 9
0.23 (0.07)a 0.07 (0.05)a 1.21 (0.17)a 0.97 (0.13)a
0.55 (0.14)b 0.13 (0.05)b 2.11 (0.29)b 1.36 (0.22)b
0.65 (0.16)b 0.21 (0.06)b 2.73 (0.43)c 1.72 (0.32)c
0 275 0 275
10 10 10 10
0.14 (0.06)a 0.14 (0.04)a 0.33 (0.03)a 0.30 (0.06)a
0.22 (0.06)a 0.16 (0.05)a 0.50 (0.07)a,b 0.41 (0.06)a,b
0.26 (0.05)a 0.19 (0.05)a 0.66 (0.08)b 0.50 (0.08)b
Same superscript letters indicate non-significant effect of time (p > 0.05) within rows. Groups connected by brackets are not significantly different (p > 0.05).
Table 3. Means (standard deviation) of TMR parameters
Lesion type
Abrasive F (ppm) n level
Δ(ΔZ)C (vol.%×μm)
ΔLC (μm)
MeC
low high
CMC
low high
HEC
low high
0 275 0 275
10 10 9 10
617 (220)* 915 (166)* 520 (240) 946 (154)*
– 9.4 (3.8)* – 5.2 (2.7) – 8.0 (5.9) – 8.4 (4.7)
0 275 0 275
8 10 9 10
746 (185)* 436 (165)* 260 (164) 573 (223)*
–13.5 (5.2)* – 7.8 (3.4) – 4.7 (5.3) – 7.0 (4.3)
0 275 0 275
9 10 9 8
204 (92) 372 (103)* 191 (96) 48 (209)
– 4.4 (4.0) – 9.4 (5.1) – 1.0 (7.2) – 0.1 (5.6)
* Statistically significant change vs. lesion baseline (p < 0.05).
ness (low, high), slurry fluoride (0 ppm F, 275 ppm F) and time (1, 3, 5 days) on SL [Jennrich and Schluchter, 1986]. An unstructured variance/covariance matrix was used to model the variances and correlations within a specimen over time. Pairwise comparisons among the treatment combinations were made using Tukey’s multiple comparisons procedure to control the overall significance level at 5%. The analyses were performed after a natural logarithm transformation of the data to satisfy the RM-ANOVA assumptions. For TMR, ANOVA was used to test the effects of lesion type (MeC, CMC, HEC), slurry abrasiveness (low, high) and slurry fluoride (0 ppm F, 275 ppm F) on Δ(ΔZ)C and ΔLC. Pairwise com-
560
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
parisons among the treatment combinations were made using Tukey’s multiple comparisons procedure. To test changes in the mineral content within each group, a paired t test was used to compare ΔZbase and ΔZpost.
Results
Specimen Loss Some specimens were excluded from statistical testing (tables 2, 3) either due to the presence of artifacts in the profilometric scans (SL) or for being broken during preparation (TMR). Profilometric Analysis Effect of Lesion Type. MeC had significantly more SL than HEC (p < 0.0001). MeC had significantly more SL than CMC for day 3 (p < 0.0001) and day 5 (p < 0.0001), but not for day 1 (p = 0.06). CMC had significantly more SL than HEC for high-abrasive slurry (p < 0.0001), but not for low-abrasive slurry (p = 0.91). Effect of Abrasive Level. Overall, more SL was associated to the high-abrasive slurries compared to the lowabrasive slurries (p < 0.0001). Effect of Fluoride. For MeC and CMC, 0 ppm F had significantly more SL than 275 ppm F after day 3 (p < 0.05) and day 5 (p < 0.05). However, there was no fluoride effect for day 1 (p > 0.05). Fluoride had no effect on the HEC specimens (p = 0.54).
Nassar /Lippert /Eckert /Hara
Comparisons of mineral loss between baseline and post-brushing are illustrated in figure 4. For the MeC and CMC groups, there was a significant mineral gain in all groups except for the combination of high-abrasive slurries with 0 ppm F. For HEC, only the group brushed with low-abrasive slurries and 275 ppm F showed a significant mineral gain (p = 0.006).
Mineral content (vol.%)
100 80 60 40 MeC
CMC
HEC
Discussion
20 0
0
50
100 Depth (μm)
150
200
Fig. 3. Graph showing the average mineral distribution for MeC, CMC and HEC lesions at baseline.
In this study, it was observed that the interaction between the fluoride content and the abrasive level of dentifrices can potentially affect the abrasion resistance and remineralization of incipient enamel caries lesions. In addition, the different characteristics (mineral content of the lesion, surface layer and mean mineral distribution profile) of the lesions were shown to play an important role.
Transverse Microradiography Baseline comparisons of the lesions created using the three demineralization protocols are shown in figure 3. Overall, MeC lesions had the least mineral content in the surface layer. CMC lesions were significantly deeper compared to the other two lesion types, while HEC had the smallest ΔZbase values. Both abrasive and fluoride levels did not have a significant effect on Δ(ΔZ)C or ΔLC (table 3). However, when comparing lesion types, HEC had significantly lower Δ(ΔZ)C than CMC (p = 0.0162) and MeC (p < 0.0001). CMC and MeC were not significantly different from each other (p = 0.19). The type of lesion had no effect on ΔLC.
Model Justification To investigate the study question under clinically relevant conditions, we chose to use a 5-day remineralization protocol, using artificial saliva, and incorporated the brushing sessions within it. The average brushing time was reported to be approximately 90 s [Ganss et al., 2009]. However, the recommended brushing time to achieve adequate plaque removal is estimated to be 2 min [Van der Weijden et al., 1993]. From multiple pilot studies, we found that these values correspond to 15–17 s of brushing per surface or approximately 50 brushing strokes. This gave a total of 500 brushing strokes at the end of the study that would be representative of 5 days of brushing. Although the duration of the protocol was relatively short, it represents the daily scenario in which the teeth would be exposed to the toothpaste twice per day and remain soaked in saliva for the remainder of the day and while the individual is sleeping. In addition, due to the lower hardness values of the lesions we were able to observe general trends in the behavior of the three tested lesion types in this study. Artificially created enamel lesions can be considered as a suitable alternative in studying enamel lesions in vitro. The protocols used in the present study have been used previously to create subsurface lesions [Ten Cate et al., 1996; Amaechi et al., 1998; Lippert et al., 2011] and were chosen to resemble the variability seen in natural lesions [Cochrane et al., 2012]. This variability could lead to different responses to the toothbrushing abrasion and fluoride effects. Among the three lesion types used in the study, MeC lesions tended to have the least mineralized surface layer, followed by CMC and HEC lesions. For
Fluoride and Abrasives Effect on Artificial Caries Lesions
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Effect of Time. For MeC, day 5 had more SL compared to day 1 for all combinations of abrasive level and fluoride content (p < 0.0001; table 2). Day 5 had significantly more SL than day 3 (p < 0.00001), with a larger difference for high-abrasive slurry level. Day 3 had significantly more SL than day 1 (p < 0.0001). For CMC, day 5 had significantly more SL than day 1 for all abrasive and fluoride combinations (p < 0.0001). Day 5 had significantly more SL than day 3, but only for high-abrasive slurry level (p = 0.0007). Day 3 had significantly more SL than day 1 (p < 0.0001). For HEC, no difference in SL was seen among at the studied days for low-abrasive slurry. When high-abrasive slurry was used, more SL was seen on day 5 compared to day 1 for both fluoride levels; day 3 did not differ from either day 1 or day 5.
561
0 ppm F + low abrasives
100
275 ppm F + low abrasives 0 ppm F + high abrasives
*
80
100
*
80
80
60
60
60
40
40
40
20
20
20
0
0
100
200
100
0
0
100
200
100
*
80
0
*
80
60
40
40
40
20
20
20
0
100
200
0
0
100
200
0
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
100
200
100
0
0
100
200
100
*
80
100
200
0
100
200
0
100
200
100 HEC
200
*
80
60
0
0
100
60
0
275 ppm F + high abrasives
100
0
100
*
80
80
60
60
60
40
40
40
20
20
20
0
0
100 MeC
200
0
0 0
100 CMC
200
0
Fig. 4. Graphs showing the average mineral distribution at baseline (grey lines) and post-brushing (black lines) of the various groups tested in the study. Depth (μm) and mineral content (vol.%) are presented on the x and y axis, respectively. Asterisks indicate significant difference (p < 0.05) for the paired t test within each group.
562
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Nassar /Lippert /Eckert /Hara
Table 4. Baseline data for microradiographic analysis of the three
lesion types [mean (standard deviation)] Lesion type
n
ΔZbase (vol.%×μm)
Lbase (μm)
SZmax (vol.%×min)
MeC CMC HEC
39 37 36
2,576.7 (504.7)a 2,505.7 (476.9)a 1,313.3 (272.2)b
80.9 (11.2)a 108.1 (13.8)b 79.4 (12.8)a
49.2 (4.3)a 60.8 (5.0)b 73.1 (2.8)c
Same superscript letters indicate statistically non-significant difference (p > 0.05) for a particular parameter (in the same column).
HEC, this could translate into a more compact surface that could hinder the penetration of minerals into the body of the lesion. Most of the mineral gain was observed at the surface layer, with minor mineral changes in the body of the lesion. The remineralization potential of fluoride could be seen more clearly in the other lesion types, possibly due to the less mineralized surface layer, in the range of 35 and 55 vol.%, which could invite more minerals leading to more resistance to abrasion [Lippert et al., 2011, 2012]. Therefore, the association between mineral content and mechanical properties, such as abrasion resistance, was shown in the present study.
responses across the lesion types could be explained by the inherent differences in the structure of the surface layer between those lesions. HEC lesions were the most resistant to abrasion, followed by CMC and MeC lesions. This can be attributed to differences in the mineral composition of the surface layer, as deeper parts of the lesion were not directly involved in the abrasive process. Therefore, our data seem to support the hypothesis that toothbrushing abrasion leads to enamel structural loss, playing a role in the arrest of white spot lesions [Stookey and Muhler, 1968; Holmen et al., 1987; Artun and Thylstrup, 1989; Kielbassa et al., 2005; Fejerskov et al., 2008]. As previously mentioned, brushing with the more abrasive slurries could remove the most superficial layers of the lesion, which could potentially enhance its remineralization, especially in the presence of fluoride.
Brushing Effects on Lesion Surface Loss Overall, slurries with high abrasive levels led to more SL compared to low abrasive ones. Slurries with higher REA/RDA values have shown a higher abrasive potential on the natural tooth structure [Philpotts et al., 2005]; consequently, this effect is expected to be more pronounced on the relatively softer lesions. This trend was observed for all three lesion types and is in accordance with results reported by Kielbassa et al. [2005], who found a direct correlation between SL values of artificial enamel lesions and the abrasivity of toothpaste slurries. However, the abrasive effect on the HEC lesions was less pronounced, which could be possibly related to the higher mineral content of the surface layer (table 4). The protective effect of fluoride against SL has been reported previously, especially in relation to softened erosive lesions [Attin et al., 1998]. In the present study, the fluoride effect was dependent on the lesion type and the time effect. In general, MeC and CMC showed a similar trend, with the fluoride effect detected after day 3 and being more pronounced on day 5. However, for HEC, no fluoride protective effect could be observed even after 5 days of exposure. The difference between the fluoride
Brushing Effects on Lesion Remineralization MeC and CMC lesions showed significant mineral gain throughout the study, except when brushed with high abrasives in the absence of fluoride. In this situation, we suspect that the effect of structural loss due to the high abrasivity of the slurry accelerated the removal of the surface layer. A similar finding was reported previously by Kielbassa et al. [2005]. In the presence of fluoride, it was speculated that brushing with high-abrasive slurries could show some benefits by preventing the formation of a highly mineralized surface layer and by facilitating the access of minerals and subsequent remineralization of the body of the lesion. However, this was not observed, possibly due to the excessively harsh toothbrushing abrasion compared to the limited remineralization by fluoride. Use of fluoride-containing slurries with intermediate abrasive levels may allow us to find this effect, deserving further investigation. Likewise, it must be noted that HEC lesions had the least mineral loss at baseline, thereby allowing for comparatively less mineral gain than MeC and CMC lesions. Within groups showing significant mineral gain, those brushed with fluoride-containing slurries showed more mineral gain in the surface layer, as seen in the mineral profiles (fig. 4). Contrary to this, HEC lesions showed significant mineral gain only in the group brushed with low-abrasive slurries in the presence of fluoride. This could be explained by the fact that a higher driving force for mineral deposition of the fluoride was needed to allow some mineral gain past the highly mineralized (∼70 vol.%) and less porous surface layer of the HEC lesion, leading to more resistance to abrasion and consequently hindering further remineralization [Ten Cate, 1990].
Fluoride and Abrasives Effect on Artificial Caries Lesions
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
563
It has been reported that higher values of ΔZbase are associated with a higher tendency of lesions to remineralize [Strang et al., 1987; Schafer et al., 1992]. Although all lesions showed some remineralization in the present study, such a relationship between remineralization and ΔZbase could not be observed in the Δ(ΔZ)C analysis. HEC lesions had significantly lower ΔZbase values compared to MeC and CMC lesions, yet no differences in mineral gain [Δ(ΔZ)C] could be observed. One explanation could be the differences in the remineralization protocols of the cited studies, which were conducted under in situ conditions and involved periods of acid challenge. Acid challenges can dissolve minerals at the surface, forming a more porous layer that could allow minerals to penetrate deeper and enhance remineralization. Another difference is the duration of remineralization. In the present study, remineralization was performed for 5 days, which is shorter than the duration of most in situ studies (2–5 weeks). As with other laboratory reports, this study had some limitations. The artificial lesions used might not represent natural white spot lesions completely. Unfortunately, this limitation cannot be solved until a comprehensive characterization of white spot lesions has been performed. Hence, we chose three different demineralization protocols that resemble the variability associated with natural white spots. Also, the remineralization protocol involved the use of artificial saliva. Natural saliva was not considered due to the large amount needed and potential infection control issues. Still, components of natural saliva, such as salivary proteins, could affect remineralization and SL by acting as a protective layer and/or a lubricating agent [West et al., 1998].
Conclusions
The effect of higher abrasive content could be seen in all three lesion types tested in this study, whereas the effect of fluoride was dependent on the type of lesion tested, with MeC and CMC lesions shown to be more responsive than HEC lesions. More abrasive slurries were not able to allow improved remineralization for the artificially created lesions within the adopted studied conditions.
Acknowledgements Dr. H.M. Nassar was sponsored by a scholarship from King Abdulaziz University. This research was part of his thesis submitted in partial fulfilment of the Doctor of Philosophy in Preventive Dentistry degree awarded in 2012 from Indiana University. The authors would like to thank Dr. Carlos González-Cabezas, Dr. Margherita Fontana and Dr. Tien-Min Chu for their insightful comments and feedback. This project was supported by the Dental Erosion-Abrasion Research Program of the Oral Health Research Institute, Indiana University School of Dentistry.
Author Contribution H.M. Nassar: study design, lab phase conduction, data interpretation, manuscript writing. F. Lippert: study design, data interpretation, manuscript review. G.J. Eckert: study design, statistical analysis, data interpretation, manuscript review. A.T. Hara: study idea, study design, data interpretation, manuscript review.
Disclosure Statement The authors declare no conflict of interest.
References Addy M, Hunter ML: Can tooth brushing damage your health? Effects on oral and dental tissues. Int Dent J 2003;53(suppl 3):177–186. Amaechi BT, Higham SM, Edgar WM: Factors affecting the development of carious lesions in bovine teeth in vitro. Arch Oral Biol 1998;43: 619–628. Angmar B, Carlström D, Glas JE: Studies on the ultrastructure of dental enamel. IV. The mineralization of normal tooth enamel. J Ultrastruct Res 1963;8:12–23. Arends J, Christoffersen J: The nature of early caries lesions in enamel. J Dent Res 1986; 65: 2– 11.
564
Arends J, Jongebloed W, Ogaard B, Rolla G: SEM and microradiographic investigation of initial enamel caries. Scand J Dent Res 1987;95:193– 201. Artun J, Thylstrup A: A 3-year clinical and SEM study of surface changes of carious enamel lesions after inactivation. Am J Orthod Dentofacial Orthop 1989;95:327–333. Attin T, Zirkel C, Hellwig E: Brushing abrasion of eroded dentin after application of sodium fluoride solutions. Caries Res 1998; 32: 344– 350.
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
Cochrane NJ, Anderson P, Davis GR, Adams GG, Stacey MA, Reynolds EC: An X-ray microtomographic study of natural white-spot enamel lesions. J Dent Res 2012;91:185–191. Cury JA, Tenuta LM: Enamel remineralization: controlling the caries disease or treating early caries lesions? Braz Oral Res 2009; 23(suppl 1):23–30. de Josselin de Jong E, ten Bosch JJ, Noordmans J: Optimised microcomputer-guided quantitative microradiography on dental mineralised tissue slices. Phys Med Biol 1987;32:887–899.
Nassar /Lippert /Eckert /Hara
Edmunds DH, Whittaker DK, Green RM: Suitability of human, bovine, equine, and ovine tooth enamel for studies of artificial bacterial carious lesions. Caries Res 1988; 22: 327– 336. Fejerskov O, Nyvad B, Kidd E: Dental Caries: The Disease and Its Clinical Management, ed 2. Oxford, Blackwell Munksgaard, 2008. Ganss C, Schlueter N, Preiss S, Klimek J: Tooth brushing habits in uninstructed adults – frequency, technique, duration and force. Clin Oral Investig 2009;13:203–208. Holmen L, Thylstrup A, Artun J: Surface changes during the arrest of active enamel carious lesions in vivo. A scanning electron microscope study. Acta Odontol Scand 1987;45:383–390. Hooper S, West NX, Pickles MJ, Joiner A, Newcombe RG, Addy M: Investigation of erosion and abrasion on enamel and dentine: a model in situ using toothpastes of different abrasivity. J Clin Periodontol 2003;30:802–808. Jennrich RI, Schluchter MD: Unbalanced repeated-measures models with structured covariance matrices. Biometrics 1986;42:805–820. Joiner A, Pickles M, Matheson J, Weader E, Noblet L, Huntington E: Whitening toothpastes effects on tooth stain and enamel. Int Dent J 2002;52:412–430. Kielbassa AM, Gillmann L, Zantner C, MeyerLueckel H, Hellwig E, Schulte-Monting J: Profilometric and microradiographic studies on the effects of toothpaste and acidic gel abrasivity on sound and demineralized bovine dental enamel. Caries Res 2005; 39: 380– 386.
Fluoride and Abrasives Effect on Artificial Caries Lesions
Kleinberg I: A mixed-bacteria ecological approach to understanding the role of the oral bacteria in dental caries causation: an alternative to Streptococcus mutans and the specificplaque hypothesis. Crit Rev Oral Biol Med 2002;13:108–125. Lippert F, Butler A, Lynch RJ, Hara AT: Effect of fluoride, lesion baseline severity and mineral distribution on lesion progression. Caries Res 2012;46:23–30. Lippert F, Lynch RJ, Eckert GJ, Kelly SA, Hara AT, Zero DT: In situ fluoride response of caries lesions with different mineral distributions at baseline. Caries Res 2011;45:47–55. Marsh PD: The role of microbiology in models of dental caries. Adv Dent Res 1995; 9: 244–254; discussion 255–269. Ogaard B, Rolla G: The in vivo orthodontic banding model for vital teeth and the in situ orthodontic banding model for hard-tissue slabs. J Dent Res 1992;71(Spec No):832–835. Philpotts CJ, Weader E, Joiner A: The measurement in vitro of enamel and dentine wear by toothpastes of different abrasivity. Int Dent J 2005;55:183–187. Ripa LW: A critique of topical fluoride methods (dentifrices, mouthrinses, operator-, and selfapplied gels) in an era of decreased caries and increased fluorosis prevalence. J Public Health Dent 1991;51:23–41. Schafer F, Raven SJ, Parr TA: The effect of lesion characteristic on remineralization and model sensitivity. J Dent Res 1992;71(Spec No):811– 813.
Stookey GK, Burkhard TA, Schemehorn BR: In vitro removal of stain with dentifrices. J Dent Res 1982;61:1236–1239. Stookey GK, Muhler JC: Laboratory studies concerning the enamel and dentin abrasion properties of common dentifrice polishing agents. J Dent Res 1968;47:524–532. Strang R, Damato FA, Creanor SL, Stephen KW: The effect of baseline lesion mineral loss on in situ remineralization. J Dent Res 1987; 66: 1644–1646. Ten Cate JM: In vitro studies on the effects of fluoride on de- and remineralization. J Dent Res 1990; 69(Spec No):614–619; discussion 634–636. Ten Cate JM, Dundon KA, Vernon PG, Damato FA, Huntington E, Exterkate RA, Wefel JS, Jordan T, Stephen KW, Roberts AJ: Preparation and measurement of artificial enamel lesions, a four-laboratory ring test. Caries Res 1996;30:400–407. Ten Cate JM, Mundorff-Shrestha SA: Working group report 1: Laboratory models for caries (in vitro and animal models). Adv Dent Res 1995;9:332–334. Van der Weijden GA, Timmerman MF, Nijboer A, Lie MA, Van der Velden U: A comparative study of electric toothbrushes for the effectiveness of plaque removal in relation to toothbrushing duration. Timerstudy. J Clin Periodontol 1993;20:476–481. West NX, Maxwell A, Hughes JA, Parker DM, Newcombe RG, Addy M: A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel. J Dent 1998;26:329–335.
Caries Res 2014;48:557–565 DOI: 10.1159/000358401
565
Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
