Introduction: The aim of the study was to investigate the caries-preventive effect of fluoride-free toothpastes, containing either herbal agents or (nano-)hydroxyapatite. Methods: Bovine dentin specimens each having a sound (ST) and a demineralized area (DT) were prepared and randomly allocated to eleven groups (n = 187). Treatments during pH cycling (28 days; 6 × 120 min demineralization/day) were brushing 2×/day with 0 ppm F [NaF0], 500 ppm F [NaF500], 1,100 ppm F [NaF1100], grape seed extract [GSE], (nano-)hydroxyapatite, melaleuca oil [MO1, MO2, MO-CU], and propolis + myrrh [PM1, PM2] containing dentifrices. Dentifrice slurries were prepared with deionized water (1:3 w/w). Differences in integrated mineral loss (∆∆Z) and lesion depth (∆LD) were evaluated before and after pH cycling using transversal microradiography. Results: The correlation between ΔΔZ/ΔLD and F concentration (NaF0, NaF500, NaF1100) was strong for the DT (rΔΔZ, DT = 0.681; p < 0.001) and very strong for ST (rΔΔZ, ST = 0.861; p < 0.001), indicating a fluoride dose-response for both baseline substrate conditions. For ΔΔZDT and ΔLDDT, only NaF1100 and GSE revealed significant differences compared with NaF0 (p < 0.001; ANOVA). For ΔΔZST and ΔLDST, significant differences could be found for NaF1100 and NaF500 compared to all fluoride-free groups (p ≤ 0.002; ANOVA), without significant difference between fluoride-free groups (p = 1.000; ANOVA). For DT and ST, a hypermineralized surface layer and no surface loss could only be observed when fluoride was present. Conclusion: A dose-response for fluoride concentrations was observed in this mild demineralization pH-cycling model. Fluoride-free dentifrices containing GSE or melaleuca oil showed certain preventive effect against further progression of root caries lesions. However, surface loss was observed for all fluoride-free dentifrices.

A wide range of pH-cycling models have been used to analyze a variety of hard tissue substrates and fluoride concentrations [1]. Several reviews analyzed potentials and limitations of these models [1, 2]. All reviews highlighted that the models should be capable to demonstrate a (significant) fluoride dose-response similar to the anticipated clinical effect. While comparing numerous different experiments with similar settings [3], different methods of initial demineralization [4], or different lowly and highly cariogenic pH-cycling conditions [5], these fluoride dose-responses were observed. However, only a few studies presented data on the correlations between mineral loss and fluoride concentrations [5‒7] and only in one of the abovementioned pH-cycling models (human or bovine) dentin has been analyzed [5]. In this study, a significant dose-response characteristic for different baseline substrate conditions and for dentifrices containing fluoride in the range 1,450–12,500 ppm (as NaF) in a lowly and highly cariogenic pH-cycling model could be revealed. However, although pH-cycling models should be capable of demonstrating clinically compatible responses, this has only been observed for enamel [1] but not for dentin.

Few studies have indicated a remineralizing effect for dentifrices containing hydroxyapatite (HA) [8, 9] or nano-hydroxyapatite (nHA) [10, 11]. In a remineralizing in vitro model, a HA-containing dentifrice resulted in significantly less mineral gain compared to brushing with 1,400 ppm F [8]. However, the results for nHA-containing dentifrice are not that uniform. Some studies show that enamel mineral gain is not significantly different between a 10 wt% nHA dentifrice and an amine fluoride dentifrice (1,450 ppm F) [10], or a NaF dentifrice (1,100 ppm F) in situ [11]. However, in both these studies even the control groups showed remineralization. Contrastingly, under net-demineralizing conditions, negative results have been observed in vitro [12, 13] and in situ [14]. In fact, experimental and commercial pastes containing nHA were not significantly different to the “no treatment” group or the placebo treatment (paste without nHA and F) when enamel subsurface demineralization was analyzed [12]. However, the anticaries effect of products containing HA or nHA using initially demineralized or sound dentin have not been analyzed so far on dentin.

Products with natural compounds have gained attention of the public. Accordingly, dentifrices with plant extracts have been developed to have healthier and more sustainable properties. The plant extracts contain, e.g., polyphenols, which have been reported to have antimicrobial effect, to cross-link proteins, as well as to inhibit enzymes, such as metalloproteinases and glycosyltransferases [15]. Specifically, the polyphenols that can bind to the dentin collagen increase its mechanical resistance and, in turn, reduce demineralization [16]. Moreover, the inhibition effect of metalloproteinases should also reduce dentin degradation, increasing dentin protection against demineralization [17]. Grape seed extract (GSE) has also shown protective results, probably associated to its high concentration of proanthocyanidin. It has been shown that the GSE can not only modify the dentin substrate but also act on dentin remineralization [18]. Furthermore, plant-based oils can be found in newly oral care products. For instance, melaleuca oil (MO) is known for its antimicrobial and anti-inflammatory effect, which could impact the progression of dental caries lesions [19]. However, to the present moment no study has specifically tested dentifrices containing this type of oil on root caries formed in vitro using a pH-cycling model.

Thus, the aim of the present study was, firstly, to reveal a dose-response for dentifrices containing 0, 500, and 1,100 ppm F for sound dentin surfaces as well as initially demineralized dentin caries lesions and, secondly, to compare the root caries preventive effect of fluoride-free dentifrices containing different (herbal) antibacterial agents and nHA. The first hypothesis was that a significant correlation between mineral loss and fluoride concentration (0, 500, 1,100 ppm F) can be seen for sound surfaces as well as artificial dentin caries lesions. The second hypothesis was that all fluoride-free dentifrices containing (herbal) antibacterial agents or nHA inhibit further demineralization significantly more than a fluoride-free dentifrice without these agents.

Specimen Preparation

Bovine incisors were obtained from freshly slaughtered cattle (negative BSE test) and stored in 0.08% thymol. Teeth were cleaned and 400 dentin blocks (5 mm × 4 mm × 3 mm) were cut (Exakt 300; Exakt Apparatebau, Norderstedt, Germany) from the roots (Fig. 1). The dentin blocks were embedded in epoxy resin (Technovit 4071; Heraeus Kulzer, Hanau, Germany), ground flat and polished (4,000 grit; silicon carbide, Phoenix Alpha, Wirtz-Buehler, Düsseldorf, Germany; Mikroschleifsystem Exakt, Exakt Apparatebau, Norderstedt, Germany) [20, 21].

Fig. 1.

Specimen preparation. a Frontal view of bovine incisor and separation of crown and root. b Lines for cutting perpendicular and parallel to the long axis of the crown. c Obtained specimens (5 mm × 4 mm × 3 mm). d Specimen covered with acid resistant nail varnish (sound control [SC] area [red]). e Initially demineralized specimen (demineralized treatment area [DT] [gray]). f Preparation of the thin sections after initial demineralization for transversal microradiographic analysis. g Specimen covered with resin (sound treatment area [ST]). h, i Preparation of the thin sections after pH cycling for transversal microradiographic analysis.

Fig. 1.

Specimen preparation. a Frontal view of bovine incisor and separation of crown and root. b Lines for cutting perpendicular and parallel to the long axis of the crown. c Obtained specimens (5 mm × 4 mm × 3 mm). d Specimen covered with acid resistant nail varnish (sound control [SC] area [red]). e Initially demineralized specimen (demineralized treatment area [DT] [gray]). f Preparation of the thin sections after initial demineralization for transversal microradiographic analysis. g Specimen covered with resin (sound treatment area [ST]). h, i Preparation of the thin sections after pH cycling for transversal microradiographic analysis.

Close modal

Lesion Formation

Two-thirds of the surface of each specimen was covered with nail varnish (sound control and sound treatment area [ST]) in order to assure enough mechanical and acid resistance. The other third remained uncovered (initially demineralized treatment area [DT]). To create artificial dentin caries lesions in the uncovered area, specimens were stored in a demineralization solution for 7 days (2.0 mL solution/mm2 dentin surface) [20]. The solution contained 50 mm acetic acid, 2.2 mm CaCl2·H2O, 2.2 mm KH2PO4, 47.6 μm NaF, and traces of thymol (pH 5.00; 37°C). During that period, the pH was monitored daily and, if necessary, adjusted with small amounts of either 10% HCl or 10 m KOH to maintain a constant pH value. In this way, from the 400 specimens originally prepared, we selected 187 specimen with a standardized demineralized area with mean (95% confidence interval) baseline mineral loss (ΔZbaseline) of 1,088 (1,056; 1,120) vol% × µm and a lesion depth of 109 (106; 12) µm [13].

Experimental Groups

The specimens were randomly allocated into 11 groups, according to the different toothpastes: 0 ppm F [NaF0], 500 ppm F [NaF500], 1,100 ppm F [NaF1100], GSE, nHA, HA, MO [MO1, MO2], MO + curcuma, and propolis + myrrh [PM1, PM2]. Table 1 shows details of the ingredients of the toothpastes.

Table 1.

Description of groups, toothpastes fluoride content, and active ingredients

GroupDentifriceFluoride content (ppm F)Free fluoride content (SD) (ppm F)(In)active ingredients
NC (negative control) Crest cavity protection (without fluoride), Procter & Gamble, Schwalbach am Taunus, Germany None: sorbitol, aqua, hydrated silica, sodium lauryl sulfate, trisodium phosphate, flavor, sodium phosphate, cellulose gum, carbomer, sodium saccharin, titanium dioxide, blue 1 
NaF500 (dose response) Signal kids 0–6, Kariesschutz & Zuckersäurenschutz, Unilever, Rotterdam, Netherlands 500 494.0 (20.8) (98.8 [4.2] %) Sodium fluoride: aqua, hydrogenated starch, hydrolysate, hydrated silica, cellulose gum, aroma, decyl glucoside, sodium saccharin, CI 16255 
NaF1100 (standard therapy) Crest cavity protection, Procter & Gamble, Schwalbach am Taunus, Germany 1,100 1,042.2 (50.0) (94.7 [4.5] %) Sodium fluoride: sorbitol, water, hydrated silica, sodium lauryl sulfate, trisodium phosphate, flavor, sodium phosphate, cellulose gum, carbomer, sodium saccharin, titanium dioxide, blue 1 
GSE Naturäl com extratos de uva, melissa e camomila, Contente/Suavetex, Uberlândia, Brazil GSE, chamomile, and lemon balm: stevioside, xylitol, carrageenan, xanthan gum, glycerin, lauryl glucoside, calcium carbonate, hydrated silica, polyglyceryl-3-caprylate, aqua, aroma 
nHA Biorepair/Bioniq, Dr. Kurt Wolf GmbH & Co. KG, Bielefeld, Germany nHA: aqua, hydrated silica, glycerin, sorbitol, aroma, cellulose gum, sodium myristoyl sarcosinate, silica, sodium methyl cocoyl taurate, tetrapotassium pyrophosphate, zinc PCA, sodium saccharin, phenoxyethanol, benzyl alcohol, propylparaben, methylparaben, citric acid, sodium benzoate, limonene 
HA KAREX Dental Caries Prevention Toothpaste, Dr. Kurt Wolf GmbH & Co. KG, Bielefeld, Germany HA: aqua, glycerin, hydrogenated starch hydrolysate, xylitol, hydrated silica, tetrapotassium pyrophosphate, silica, aroma (menthol and eucalyptol), cellulose gum, sodium methyl cocoyl taurate, phosphoric acid, sodium cocoyl glycinate, zinc chloride, cetylpyridinium chloride 
MO1 TEBODONT® Zahnpasta toothpaste without fluoride, Dr. Wild & Co. AG, Muttenz, Switzerland MO: glycerin, silica, aqua, xylitol, propylene glycol, cocamidopropyl betain, PEG-40-hydrogenated castor oil, PEG-8, cellulose gum, dipotassium phosphate, titanium dioxide, sodium chloride, saccharin, aroma, limonene 
MO2 Boni Natural Menta & Melaleuca®, Boni Brasil, São Bernardo do Campo, Brasil MO: calcium carbonate, sorbitol, water, glycerin, sodium lauryl sarcosinate, hydrated sílica, xanthan gum, mentha piperita oil, xylitol, benzyl alcohol, menthol, sodium benzoate, Calendula officinalis leaf extract, eucalyptus globulus oil, citrus grandis peel oil, citrus aurantifolia oil, sucralose, potassium sorbate, d-limonene citral 
PM1 Spearmint Fluoride-Free Propolis & Myrrh Toothpaste, Tom’s of Maine, Colgate-Palmolive, New York City, USA Commiphora myrrha (myrrh) resin extract, propolis extract: calcium carbonate, water, glycerin, hydrated silica, xylitol, sodium lauryl sulfate, xanthan gum, natural flavor (cinnamon oil and other natural flavor), benzyl alcohol 
PM2 Dentífrico Mirra, Própolis e Fennel – Corpore Sano (https://www.planetahuerto.es/venta-dentifrico-mirra-propolis-e-hinojo-corpore-sano_24542) Propolis extract, Foeniculum vulgare (fennel) fruit extract, Commiphora myrrha resin extract: dicalcium phosphate dihydrate, water, sorbitol, silica, kaolin, sodium lauryl sulfate, cellulose gum, aroma (flavor), limonene, propanediol, glycerin, sodium saccharin, phenoxyethanol, potassium sorbate, sodium benzoate, CI 42090, CI 19140 
MO-CU Naturäl com extratos cúrcuma, cravo, Melaleuca, Contente/Suavetex, Uberlândia, Brazil Curcuma longa (turmeric) callus extract, eugenia caryophyllus bud extract, Melaleuca alternifolia leaf extract: stevioside, xylitol, xanthan gum, sodium benzoate, glycerin, lauryl glucoside, calcium carbonate, hydrated silica, aqua, aroma 
GroupDentifriceFluoride content (ppm F)Free fluoride content (SD) (ppm F)(In)active ingredients
NC (negative control) Crest cavity protection (without fluoride), Procter & Gamble, Schwalbach am Taunus, Germany None: sorbitol, aqua, hydrated silica, sodium lauryl sulfate, trisodium phosphate, flavor, sodium phosphate, cellulose gum, carbomer, sodium saccharin, titanium dioxide, blue 1 
NaF500 (dose response) Signal kids 0–6, Kariesschutz & Zuckersäurenschutz, Unilever, Rotterdam, Netherlands 500 494.0 (20.8) (98.8 [4.2] %) Sodium fluoride: aqua, hydrogenated starch, hydrolysate, hydrated silica, cellulose gum, aroma, decyl glucoside, sodium saccharin, CI 16255 
NaF1100 (standard therapy) Crest cavity protection, Procter & Gamble, Schwalbach am Taunus, Germany 1,100 1,042.2 (50.0) (94.7 [4.5] %) Sodium fluoride: sorbitol, water, hydrated silica, sodium lauryl sulfate, trisodium phosphate, flavor, sodium phosphate, cellulose gum, carbomer, sodium saccharin, titanium dioxide, blue 1 
GSE Naturäl com extratos de uva, melissa e camomila, Contente/Suavetex, Uberlândia, Brazil GSE, chamomile, and lemon balm: stevioside, xylitol, carrageenan, xanthan gum, glycerin, lauryl glucoside, calcium carbonate, hydrated silica, polyglyceryl-3-caprylate, aqua, aroma 
nHA Biorepair/Bioniq, Dr. Kurt Wolf GmbH & Co. KG, Bielefeld, Germany nHA: aqua, hydrated silica, glycerin, sorbitol, aroma, cellulose gum, sodium myristoyl sarcosinate, silica, sodium methyl cocoyl taurate, tetrapotassium pyrophosphate, zinc PCA, sodium saccharin, phenoxyethanol, benzyl alcohol, propylparaben, methylparaben, citric acid, sodium benzoate, limonene 
HA KAREX Dental Caries Prevention Toothpaste, Dr. Kurt Wolf GmbH & Co. KG, Bielefeld, Germany HA: aqua, glycerin, hydrogenated starch hydrolysate, xylitol, hydrated silica, tetrapotassium pyrophosphate, silica, aroma (menthol and eucalyptol), cellulose gum, sodium methyl cocoyl taurate, phosphoric acid, sodium cocoyl glycinate, zinc chloride, cetylpyridinium chloride 
MO1 TEBODONT® Zahnpasta toothpaste without fluoride, Dr. Wild & Co. AG, Muttenz, Switzerland MO: glycerin, silica, aqua, xylitol, propylene glycol, cocamidopropyl betain, PEG-40-hydrogenated castor oil, PEG-8, cellulose gum, dipotassium phosphate, titanium dioxide, sodium chloride, saccharin, aroma, limonene 
MO2 Boni Natural Menta & Melaleuca®, Boni Brasil, São Bernardo do Campo, Brasil MO: calcium carbonate, sorbitol, water, glycerin, sodium lauryl sarcosinate, hydrated sílica, xanthan gum, mentha piperita oil, xylitol, benzyl alcohol, menthol, sodium benzoate, Calendula officinalis leaf extract, eucalyptus globulus oil, citrus grandis peel oil, citrus aurantifolia oil, sucralose, potassium sorbate, d-limonene citral 
PM1 Spearmint Fluoride-Free Propolis & Myrrh Toothpaste, Tom’s of Maine, Colgate-Palmolive, New York City, USA Commiphora myrrha (myrrh) resin extract, propolis extract: calcium carbonate, water, glycerin, hydrated silica, xylitol, sodium lauryl sulfate, xanthan gum, natural flavor (cinnamon oil and other natural flavor), benzyl alcohol 
PM2 Dentífrico Mirra, Própolis e Fennel – Corpore Sano (https://www.planetahuerto.es/venta-dentifrico-mirra-propolis-e-hinojo-corpore-sano_24542) Propolis extract, Foeniculum vulgare (fennel) fruit extract, Commiphora myrrha resin extract: dicalcium phosphate dihydrate, water, sorbitol, silica, kaolin, sodium lauryl sulfate, cellulose gum, aroma (flavor), limonene, propanediol, glycerin, sodium saccharin, phenoxyethanol, potassium sorbate, sodium benzoate, CI 42090, CI 19140 
MO-CU Naturäl com extratos cúrcuma, cravo, Melaleuca, Contente/Suavetex, Uberlândia, Brazil Curcuma longa (turmeric) callus extract, eugenia caryophyllus bud extract, Melaleuca alternifolia leaf extract: stevioside, xylitol, xanthan gum, sodium benzoate, glycerin, lauryl glucoside, calcium carbonate, hydrated silica, aqua, aroma 

pH-Cycling Condition

A computer-controlled pH cycling and brushing machine developed by our group and previously described [7, 22] was used to simulate oral pH-fluctuation patterns and daily oral care. The pH cycling lasted 28 days, each day consisting of intermittent 6 demineralization periods of 2 h each (total 12 h/day) and 6 remineralization periods of at least 1 h during the day and a longer 6 h overnight period (total 12 h/day).

The remineralization solutions contained 1.5 mm CaCl2, 0.9 mm KH2PO4, and 20 mm N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid as buffer, pH 7.0 (37°C). The demineralization solution contained 50 mm acetic acid, 2.2 mm CaCl2.H2O, 2.2 mm KH2PO4, 23.8 μm NaF, pH 5.0 [23]. For both solutions, the pH was adjusted with small amounts of 10% HCl or 10 m KOH to maintain a constant pH.

New pH-cycling solutions were used for every cycle. The amounts of each solution were large enough to prevent the solutions from becoming saturated with or depleted of mineral ions (0.7 mL solution/mm2 dentin surface).

Surface Treatment and Dentifrice Slurries

Before the first and last remineralizing phase of each day, the specimens were exposed to the respective dentifrice slurries (Table 1) for a total of 120 s, during which they were brushed for 10 s (Oral-B Indicator toothbrush; Procter & Gamble, Schwalbach am Taunus, Germany), which simulates the recommended brushing time of 2 min [24]. Dentifrice slurries were prepared with deionized water in a ratio of 1:3 parts by weight. The slurries were freshly prepared every 2 days and during this period all slurries were stable. Subsequently, the specimens were rinsed with distilled water to remove the slurry. The machine was adjusted to a constant brushing frequency of 60 strokes/min and a constant brushing load of 0.5 N [23].

Determination of Free Fluoride and pH in the Slurries

The total soluble fluoride concentration (F) in the dentifrice slurries was determined using a F-specific electrode (Orion 960900, Boston, MA, USA) as described previously [25].

Transversal Microradiography Analysis

After the pH cycling, a slice of approximately 300 μm thickness (Exakt GmbH, Norderstedt, Germany) was obtained from each specimen and subsequently ground flat and polished to a thickness of 100 μm (±10 μm) using waterproof silicon carbide papers (FEPA grit sizes: 800, 1,200, 2,400, 4,000; Struers). The parallelism of the specimens was tested with a digital micrometer with a precision of 0.001 mm (Mitutoyo, Japan). Contact microradiographs of the dentin specimens were obtained with a nickel-filtered copper (CuKa) X-ray source (PW 1730; Philips, Kassel, Germany) operating at 20 kV and 20 mA. The radiation source-to-film distance was 28 cm. The exposure time was 10 s and a high-resolution film (motion picture fine grain positive film 71337; FUJIFILM Corporation, Japan) was used and developed under standardized conditions according to the manufacturer’s recommendations. Microradiographs were digitalized by an image-analyzing system (Diskus software, version 4.80; Königswinter, Germany) that is interfaced to a universal microscope (Leica DMRX; Germany) and a personal computer.

Calculation of Integrated Mineral Loss, Lesion Depth

A transversal microradiography (TMR) software (version 5.25 by Joop de Vries, Groningen, Netherlands) was used to calculate the mineral loss (ΔZbaseline/ΔZpH-cycle) and lesion depth (LDbaseline/LDpH-cycle) before and after the pH cycling [26]. This is based on the gray levels of the image, as described previously [13, 23]. Mineral losses and lesion depths after pH cycling of initially demineralized surfaces were subtracted from the respective values before pH cycling [7, 25]. Changes in mineral loss (ΔΔZ = ΔZbaseline − ΔZpH-cycle) and lesion depth (ΔLD = LDbaseline − LDpH-cycle) were then calculated. For a more intuitive reading, ΔΔZST and ΔLDST for the sound treatment areas were calculated as well, although values were measured only after the pH cycling. For this, baseline values were assumed to be zero. Furthermore, graphics of mean mineral density profiles were prepared for all groups with the TMR/WIM calculation program.

Statistical Analysis

Data were analyzed using SPSS statistical software (SPSS 29; SPSS, Munich, Germany). Variables were tested for normal distribution (Shapiro-Wilk test). Changes in mineral loss and lesion depth before and after pH cycling (ΔZbaseline vs. ΔZpH cycle and LDbaseline vs. LDpH cycle) were analyzed using two-tailed paired t tests. Analysis of variance (ANOVA) for de- and remineralizing models was used to detect differences in changes of mineral loss (∆∆ZDT, ∆∆ZST) and lesion depth (ΔLDDT, ΔLDST). Correlation between ΔΔZ and [F] as well as between ΔLD and [F] were assessed using the Spearman’s rank correlation coefficient. For this, only groups NC, NaF500, and NaF1100 were used. All tests were performed at a 5% level of significance.

Power Calculation

The number of specimens per group was calculated on the basis of pretests (non-published data). The α-error was set at 5%. Considering the differences between the 0 ppm and 1,100 ppm F dentifrice, the statistical power calculated for ∆∆Z was 85% (mean difference of 450 [SD: 600]) and for ∆LD was 89% (mean difference of 4 [SD: 5]). Dropout rate was assumed not to exceed 10%. Approximately 17 specimens should have been included into the study for analyses of at least 15 specimens per group. Since the retro-perspective power analysis with 12 specimens has still provided a power of at least 100% for the comparison NaF0 and NaF1100 (∆∆ZDT mean difference of 979 [SD: 235]), 99% for NaF0 and NaF500 (∆∆ZDT mean difference of 418 [SD: 235]), and 100% for NaF500 and NaF1100 (∆∆ZDT mean difference of 561 [SD: 235]), no additional specimen was involved in the study. For ∆∆ZST, the retrospective power analysis also revealed a power of at least 100% for all three comparisons.

Mineral Loss and Lesion Depth

After initial demineralization, treatment groups did not differ significantly in mineral loss (p = 1.000; ANOVA) and lesion depth (p = 1.000; ANCOVA). Mean (95% confidence interval) ΔZbaseline, DT was 1,088 (1,056; 1,120) vol% × µm and LDbaseline, DT was 109 (106; 12) µm. Due to losses during preparation, final TMR analysis was performed with at least 12 specimens per group.

For mineral loss, the NaF0 and NaF500 groups presented significantly increased values after pH cycling, indicating further demineralization for these groups. The NaF1100, nHA, HA, MO1, and MO2 groups showed significantly decreased values, indicating remineralization (p ≤ 0.049; two-tailed paired t test).

For initially demineralized treatment areas (Fig. 2a), NaF1100, GSE, MO1, and MO2 presented significantly greater values of mineral content (∆∆ZDT) compared to NaF0 (p ≤ 0.025; ANOVA; Fig. 2), suggestive of a remineralization in these groups compared to a demineralization in the NaF0 group.

Fig. 2.

Means with CIs (95%) of the changes in mineral loss (∆∆Z; a, b) and lesion depths (∆LD; c, d) of initially demineralized surfaces (ΔΔZDT/∆LDDT) and sound surfaces (ΔΔZST/∆LDST). Different letters indicate significant differences between treatments (p < 0.05; ANOVA). Negative ΔΔZ values indicate demineralization; positive ΔΔZ values indicate remineralization. For a more intuitive reading, ΔΔZST/∆LDST values were calculated as well, although the values were measured only after pH cycling. For this, the baseline values were assumed to be zero.

Fig. 2.

Means with CIs (95%) of the changes in mineral loss (∆∆Z; a, b) and lesion depths (∆LD; c, d) of initially demineralized surfaces (ΔΔZDT/∆LDDT) and sound surfaces (ΔΔZST/∆LDST). Different letters indicate significant differences between treatments (p < 0.05; ANOVA). Negative ΔΔZ values indicate demineralization; positive ΔΔZ values indicate remineralization. For a more intuitive reading, ΔΔZST/∆LDST values were calculated as well, although the values were measured only after pH cycling. For this, the baseline values were assumed to be zero.

Close modal

For sound treatment areas (Fig. 2b), all groups presented negative ΔΔZST values, denoting demineralization in all groups, but NaF1100 and NaF500 presented significantly greater ΔΔZST values compared to all other fluoride-free groups (p ≤ 0.002; ANOVA), suggesting significantly less demineralization in these two groups. As for the NaF0, no significant difference was observed with other fluoride-free groups (p = 1.000; ANOVA).

The respective values for lesion depths (ΔLD) can be found in Figure 2c and d. A significant difference in the change of lesion depth (ΔLDDT) could only be observed for NaF1100 GSE and nHA.

Mineral Density Profiles

The mean mineral density profiles after initial demineralization and pH cycling of all groups can be seen in Fig. 3. For ST, a hypermineralized surface area could only be observed for fluoridated groups (NaF500 and NaF1100). For DT, the hypermineralized surface area decreased in groups NaF0, nHA, HA, and PM2. Furthermore, for DT and ST no surface loss was observed only when fluoride was present during pH cycling (NaF500 and NaF1100) or when a hypermineralized surface was present before pH cycling (all initially demineralized specimens).

Fig. 3.

Mean mineral density profiles of the initially demineralized dentin surfaces before (baseline, DT) and after pH cycling (pH cycle, DT) as well as the profiles of sound surfaces (pH cycle, ST). Lesions were assessed using the TMR/WIM calculation program. For DT and ST, a hypermineralized surface layer and no surface loss could only be observed when fluoride was present.

Fig. 3.

Mean mineral density profiles of the initially demineralized dentin surfaces before (baseline, DT) and after pH cycling (pH cycle, DT) as well as the profiles of sound surfaces (pH cycle, ST). Lesions were assessed using the TMR/WIM calculation program. For DT and ST, a hypermineralized surface layer and no surface loss could only be observed when fluoride was present.

Close modal

Correlation Analyses

For mineral changes, a strong (rDT = 0.681) as well as a very strong (rST = 0.861) correlation was found between F concentrations (NaF0, NaF500, NaF1100) and ΔΔZDT as well as ΔΔZST, respectively (Table 2). For changes in lesion depths, moderate (rDT = 0.462) and strong (rST = 0.809) correlations were observed between F concentrations and ΔLDDT and ΔLDST, respectively. All correlations were significant (p < 0.001).

Table 2.

Spearman’s rank correlation coefficient and p values for the relation between the changes in mineral loss and in lesion depth and fluoride concentration

Fluoride concentration (ppm F)DTST
r valuep valuer valuep value
∆∆Z ↔ ppm F 0.681 <0.001 0.861 <0.001 
∆LD ↔ ppm F 0.462 <0.001 0.809 <0.001 
Fluoride concentration (ppm F)DTST
r valuep valuer valuep value
∆∆Z ↔ ppm F 0.681 <0.001 0.861 <0.001 
∆LD ↔ ppm F 0.462 <0.001 0.809 <0.001 

Spearman’s rank correlation coefficient and p values for the correlations between change in mineral loss (ΔΔZ) and lesion depth (ΔLD) and fluoride concentration (F).

Fluoride Analysis

The mean free fluoride content in the dentifrice slurries and the percentage of free fluoride in relation to given fluoride content are given in Table 1. The fluoride concentrations of all fluorides were within the range specified by the manufacturer.

The present in vitro study compared the caries preventive effect of fluoride-free dentifrices containing (herbal) antibacterial agents and nHA for sound dentin surfaces as well as artificial dentin caries lesions. A significant (very) strong correlation between mineral change (∆∆Z) as well as lesion depth (∆LD) and fluoride concentration (0–1,100 ppm F) was observed for both baseline substrate conditions, confirming the first hypothesis. This fluoride dose-response aligns with previous findings in both enamel [27] and dentin [28] studies, where fluoride’s ability to inhibit demineralization and enhance remineralization has been demonstrated in vivo [27, 28], in situ [14, 29, 30], and in vitro [4, 5, 7]. Conversely, the second hypothesis was partially rejected as no significant difference in the change of mineral loss was observed between the regular fluoride-free dentifrice and those containing nHA or (herbal) antibacterial agents, such as propolis and fennel extracts, curcuma, and clove extracts. Interestingly, regarding initially demineralized specimens, GSE revealed a significant difference in ΔΔZDT and ΔLDDT compared to the negative control. This effect could be explained by the carbonate/phosphate ratio and/or the high concentration of proanthocyanidin in the toothpaste. First, the carbonate/phosphate ratio was similar for the GSE and 1,000 ppm F solutions and a remineralizing effect of the GSE was shown for enamel [31, 32] or dentin [18] caries lesion, in non-biofilm and biofilm-induced caries models. Additionally, GSE contains high concentration of proanthocyanidin, which has been shown to inhibit matrix metalloproteinases and cross-link collagen, reducing demineralization [16, 33].

When solely regarding ΔΔZDT, MO1 and MO2 also revealed a significant difference compared to the negative control. These groups contain MO as active ingredient, which is known for its antimicrobial and anti-inflammatory effect [19, 34]. However, in the present study, the progression of artificial caries lesions and the initiation of new caries lesions were analyzed in a non-biofilm chemical model to prepare these specimens, fluoride was present in the pre-demineralization solution, and there was already an established hypermineralized layer on the surface of these specimens even before the pH cycling [31]. When this surface layer is present, it works as a barrier for, firstly, the dissolution of minerals and, secondly, for the diffusion of acids into deeper parts of the lesions [35]. So, it can be also speculated that the inhibitory effect of GSE, MO1, and MO2 on these initially demineralized specimens might be based on an interaction of the proanthocyanidins and the melaleuca with the hypermineralized surface layer, which was established before pH cycling.

For sound surfaces, no significant differences were observed in the change of mineral loss between NaF0 and all the others fluoride-free dentifrices, although greater demineralization was observed in all these groups compared to the fluoride-containing dentifrices (NaF500 and NaF1100). Furthermore, in all fluoride-free groups, surface loss was observed, but no surface loss was observed in fluoridated groups. This finding is in line with the speculation above and indicates that fluoride – either in the dentifrice slurries or in the demineralization solution used to prepare the specimens before the pH cycling – leads to a hypermineralized surface layer, and this, in turn, results in less mineral loss as discussed above. It must be highlighted that this effect is more pronounced when fluoride is also present in the toothpastes.

To simulate brushing procedures, controlled and constant linear strokes with brushing forces of 0.5 N were used. Linear strokes have been observed in vivo [24] and were used in vitro [22, 36], but the brushing force applied in the present study was lower than those measured clinically (1.5–3 N [24]) or in vitro (1.5–2.5 N [36]). This was done since previous studies from our group on enamel [23] and dentin [5, 23] have already indicated that they effectively prevent brushing abrasion. This, however, only occurs depending on the toothpaste used [5, 23]. When fluoride is present, surface loss cannot be observed, but contrary to the expectations and findings of previous studies, this model resulted in surface loss in all fluoride-free groups. This highlights the increased protective effect of fluoride-containing dentifrices.

In the present study, no significant difference in the change of mineral loss between NaF0, HA, and nHA for sound surfaces (and initially demineralized lesions) was observed. Although the anticaries effect of HA in dentin has not been previously analyzed, and the anticaries effect of nHA has only been analyzed once by our working group [14], the results seem to be in agreement with previous in vitro studies on enamel [8‒13]. Under demineralizing conditions, no significant differences between nHA and the negative control were observed [12, 13]. Contrastingly, a more positive remineralizing effect for nHA was observed under net-remineralizing conditions [10, 11]. As speculated previously, the presence of available fluoride might shift the critical pH for demineralization approximately 0.5–1.0 units lower, accelerating the demineralization, at least for enamel [37]. Consequently, fluoride-free dentifrices are expected to be less effective in inhibiting demineralization compared to fluoridated ones [37]. Under constant remineralizing conditions (without any demineralization periods) [10], this (favorable) effect cannot be observed. Thus, the positive remineralizing effects of HA/nHA might be related to the type of (pH-cycling) model being used.

Recent studies observed that baseline substrate condition, particularly ∆Zbaseline and LDbaseline, significantly affects the potential for re- or demineralization during an in vitro or in situ study [5, 26]. Additionally, factors such as the mineral density (“R” value) or the reaction rate of the specimens may also influence de- and remineralization characteristics. In the present study, all specimens underwent identical demineralization times (7 days) and were selected to ensure no difference in ∆Zbaseline and LDbaseline and “R” value [38]. However, compared to previous studies using dentin specimens and a 7-day demineralization period [5, 23], the specimens in the present study revealed rather shallow lesions. This “slower” reaction rate can presumably be seen during pH cycling as well. In contrast to previous studies employing the same pH-cycling regimen, which resulted in substantial net-demineralization models, only minor changes were noted in the present study. Thus, it might be speculated that the observed differences may be even greater with specimens exhibiting faster reaction rates. Furthermore, specimens with slower reaction rates might, consequently, better simulate patients with low caries risk compared to those with high caries risk. Therefore, the present study presumably simulated caries progression and the initiation of new dentin caries lesions in low caries risk patients.

The biofilm-free pH-cycling model used in this study mimics the dynamics of dentin caries formation and offers a controlled environment to separately analyze the anticaries effects of certain antibacterial agents in a dose-response pH-cycling model. However, pH-cycling models have several limitations. First, the demineralizing and remineralizing challenges occur much faster than expected under in vivo conditions, potentially exaggerating the rate of mineral loss or gain compared to natural processes [1]. Second, while brushing was simulated in this study, the procedure may not fully replicate the topical application and clearance of products in the oral cavity, which are influenced by factors such as saliva flow and eating behavior. Third, the complex intraoral environment – comprising bacterial biofilms, saliva, and dietary factors – could not be simulated. Moreover, the surface area/solution ratio and the composition of saliva and plaque fluid found in vivo were not simulated here. Also, bacterial acid production nor change in volume of extracellular polysaccharide was simulated, and therefore no changes in de-/remineralization process were present in our model [39]. These factors are crucial in determining how agents interact with the tooth surface and biofilm, potentially impacting their efficacy. Despite these limitations, this study is among the first to analyze the anticaries effects of certain antibacterial agents, such as GSE and MO, on dentin caries progression. While these agents showed promising results in preventing further demineralization, the findings should be further validated in clinical settings, where biofilm models could better reflect the complex biological environment of the oral cavity.

Moreover, considering the differences between human and bovine dentin, the use of bovine teeth could be considered a limitation of this study. However, the use of bovine teeth for this study model is widely accepted, and the results are in accordance with previous studies [5, 14, 23, 40]. Furthermore, bovine teeth allow for bigger surface areas, which is more suitable for obtaining the different surface conditions in the same specimen. Besides, bovine teeth are easier to obtain, present the same mechanism of caries formation, and have a more homogeneous mineralization pattern, which results in a more consistent experimental response [41, 42].

Within the limitations of this in vitro study, it can be concluded that for specimens with a slow reaction rate, a significant dose-response characteristic for fluoride dentifrices could be revealed in a mild chemical caries model for sound dentin and for artificial dentin caries lesions. Furthermore, the daily use of a fluoride dentifrice containing 1,100 ppm F showed the highest anticaries effect for both initially demineralized and sound surfaces. For fluoride-free dentifrices, dentifrice containing GSE or MO could reveal a significant mineral gain and reduced lesion depth for initially demineralized specimens when compared to a regular fluoride-free dentifrice. In contrast, for fluoride-free toothpastes containing HA, nHA or propolis and fennel extracts, curcuma and clove extracts (PM1, PM2, MO + curcuma), no significant differences in the change of mineral loss and lesion depth were observed compared to a regular fluoride-free dentifrice. Furthermore, after pH cycling, surface loss was observed for all fluoride-free dentifrices.

This study was conducted as part of the doctoral theses of M.K. and A.R. We thank Procter & Gamble (Schwalbach am Taunus, Germany) for providing the fluoride-free dentifrice (NaF0). The manufacturers had no role in the design, conduct, evaluation, or interpretation of the study or in writing the manuscript.

Ethical approval is not required for this study in accordance with local or national guidelines.

The authors declare no potential conflicts of interests with respect to the authorship and/or publication of this article.

This study was funded by the authors and their institution.

R.J.W., H.M.-L., and S.H.N. designed and planned the study; M.K. and A.R. prepared the samples and performed the measurements; R.J.W., M.K., and A.R. performed the statistical analysis; R.J.W., S.H.N., and T.S.C. wrote the manuscript; and H.M.-L., M.K., and A.R. commented on and all authors revised the manuscript.

Additional Information

Mowliharan Kuruparan and Abinaya Ruthiraswaran contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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