Caries Preventive Effects of Theobromine Containing Toothpaste on Early Childhood Caries: Preliminary Results.

OBJECTIVE: Enamel remineralizing effects of theobromine have received much attention from the clinicians. The aim of this study was to investigate the caries preventive effect of theobromine containing toothpaste on children with early childhood caries (ECC).
MATERIAL AND METHODS: Salivary pH, buffering capacity and frequency of Streptococcus mutans (SM) levels were measured. Each child was assigned either fluoridated or theobromine containing toothpaste. The changes were analyzed using Laser Fluorescence system. Statistical analyses were performed.
RESULTS: We included 13 children (mean age 4.25) with 145 teeth in the fluoridated toothpaste (Colgate Kids toothpaste) group; 13 children (mean age 4.46) with 115 teeth in theobromine containing toothpaste (TheodentTMKids toothpaste) group. Both toothpastes demonstrated enamel remineralization and were effective in increasing the buffering capacity and pH (p < 0.05). A statistically significant decrease in S. mutans levels was found in both toothpaste groups (p<0.05).
CONCLUSION: Both toothpaste group showed a statistically significant amount of enamel remineralization. Since theobromine had the added benefits of increasing the salivary pH and decreasing the S.mutans levels, theobromine containing toothpastes can be considered effective agents in remineralizing white spot lesions and can be used in prevention of early enamel lesions.

Introduction

Dental caries is one of the most significant public health problems (). Early childhood caries is a complex disease affecting children under 3 years of age. It is defined as the presence of one or more decayed (non- cavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces. However, caries is particularly evident in upper central incisors (). Caries affects primary teeth more often than permanent teeth because both enamel and dentin structures of primary teeth are thinner than those of permanent teeth. Also, children are fond of sweetened food and drinks. Additionally, the capability of oral hygiene maintenance in early stage of childhood is not satisfactory; therefore, deciduous teeth are more severely attacked by caries than permanent teeth ().

Fluoride is an agent used extensively for the prevention of dental caries. The use of fluoride-containing toothpastes has been shown to be an effective way for children to prevent permanent teeth from tooth decay (). It is also a well-known fact that fluoride has a beneficial effect on slowing down the caries progression (). While the use of fluoride is beneficial for strengthening the enamel, overexposure to fluoride carries the risk of fluorosis. An increased risk of dental and skeletal fluorosis is due to the ingestion of fluoride from different sources and from swallowing toothpaste with fluoride (). Additionally, it has been disputed whether fluorides interact with ameloblasts, thus having adverse effects on the enamel formation ().

In recent years, different biomimetic agents have been discussed as potential alternatives to fluoride for the prevention of ECC. Some studies showed that it is best to use toothpastes that mainly contain remineralizing agents for children's oral care such as calcium phosphates, that is, CPP-ACP or hydroxyapatite (HAP), (). A variety of agents containing different calcium phosphate compounds that have already been on the market have been well studied (). Previous in vitro and in vivo studies suggested that crystallite size was increased and crystallinity of teeth improved by growing HAP in an apatite-forming system. Therefore, toothpaste containing synthetic hydroxyapatite crystals has been developed to improve remineralization on the basis of structural similarity of the crystals to those contained in human teeth.

Another promising alternative, theobromine, was discovered accidentally during studies of dental caries. Consumption of chocolate as a sugar-containing confectionery has long been linked to dental caries (). However, a series of studies demonstrated that cocoa powder in chocolate by itself might have some caries-inhibitory effect (-). Studies with hamsters showed that pure cocoa powder inhibited dental caries in correlation with the percentage consumed in diet, and that reduced fat cocoa exhibited a higher significant anti-caries effect than the fat-containing cocoa ().

Cocoa solids also contain both theobromine and caffeine. While studying the effect of cocoa on the mineralization of teeth, it was discovered that cocoa has both demineralizing and remineralizing effects because of the coexistence of theobromine and caffeine. On one hand, caffeine reduces the crystal size on the enamel surface when children are exposed to caffeine at a critical growth period (). In a study conducted to investigate the effect of caffeine on crystallization of enamel and formation of caries, it was observed that rats exposed to caffeine had a significantly higher caries score when compared to the decaffeinated control group (). Caffeine reduced crystallization of the enamel and a smaller crystal size increased dissolution of the minerals leading to dental carries (). Along with caffeine, cocoa contains substantial amounts of polyphenolic substances, such as tannin and chlorogenic acid, which tend to produce darker colors (). Therefore, recognizing and investigating the products that can be applied to teeth to decrease the absorption of demineralizing substances and to reduce discoloration of the teeth is important. On the other hand, theobromine was shown to have an entirely opposite effect, by enhancing crystallinity of enamel (, ), binding calcium and phosphate to remineralize the tooth enamel 21. Theobromine is a naturally occurring substance, making it a potential alternative to fluoride as the active ingredient of toothpaste (, ). It is the principle alkaloid from the family of methylxanthines, which also includes theophylline and caffeine, and the main source is Theobroma cacao (cacao tree) plant (). When added to dentifrices, it protects the enamel surface of the tooth in a positive dose-response relationship (). A large number of in-vitro and some in-vivo studies, from the literature, demonstrated that the application of theobromine can result in remineralization and prevention of surface caries in enamel. However, to the best of our knowledge, no study investigated the effects of theobromine containing dentifrices on children.

The purpose of this study was to evaluate the efficacy of theobromine containing toothpaste (THEODENT Kids) on remineralization of white spot lesions, short term effect on S. mutans, buffering capacity and pH of saliva in children with ECC. The null hypothesis to be tested was that no statistically significant difference exists between the fluoride containing toothpaste group and theobromine containing toothpaste group.

Material and Methods

The study was reviewed and approved by The Human Research Ethics Committee of Yeditepe University, School of Medicine on January 07, 2014 with the protocol number of 392.

The power was set at 80%, alfa-level was set as 0.05, and the allocation ratio was equal to 1. The study was conducted with a total of 26 children between the ages of 4-5 presented with ECC who were equally assigned at random to the two study groups. None of the baseline values showed statistical differences among the groups. The parents completed a general health questionnaire form and gave informed consent on behalf of their children to participate in the study. Based on the completed questionnaires, the subjects were assessed for eligibility to undergo the baseline clinical evaluation. The children who had one or more white spot lesions were included to the study. The children with systemic medical conditions, history of prolonged use of antibiotics or medication were not included. The children who were not able to cooperate in the course of clinical examination were excluded from the study.

Clinical evaluation: Oral examination of children was performed under the standard dental operatory light. The surfaces of the teeth were cleaned thoroughly with water, and later air dried with a three-way syringe for 10 seconds. Children were randomly allocated to 2 groups: a control group with 13 children to use a 500 ppm Fluoridated toothpaste (Colgate Kids, Colgate-Palmolive Company, NY) and a test group of 13 children to use a theobromine containing toothpaste (THEODENT Kids, New Orleans) (Table 1). Oral health education was provided to both groups to improve their knowledge, attitude, and practice regarding oral health. Toothpastes were delivered to the parents and they were informed to encourage the children to brush their teeth with the assigned toothpastes two times a day for 1 month.

Table 1

Active and Inactive Ingredients of the toothpastes

	Active Ingredients	Inactive Ingredients
Colgate Kids, Colgate-Palmolive Company,	Sodium Fluoride (0.05% w/v Fluoride Ion).	Sorbitol     Water     Hydrated Cilica     PEG-12     Cellulose Gum     Sodium Lauryl Sulfate     Flavor     Sodium Saccharin     Mica     Blue 1     Titanium Dioxide
THEODENT Kids, New Orleans	Rennou: theobromine, calcium acetate, & sodium hydrogen phosphate	purified water,hydrated silica,sorbitol, xylitol, (plant based)glycerin, xanthan gum,titanium dioxide (non-nano particle form),citric acid,sodium benzoate,stevia extract,sodium bicarbonate,sugar-free chocolate extract,sugar-free vanilla extract

Scoring using Laser Fluorescence (LF): A trained and calibrated pediatric dentist performed the clinical examinations. After calibration with a ceramic standard, the baseline value for each individual was calibrated by measuring which was performed on the sound buccal surface of the tooth. A single examiner made all measurements for 3 times to eliminate the operator effect for each lesion, and the operator was blinded to intervention.

White spot lesions (WSLs) were categorized according to the International Caries Detection and Assessment System (ICDAS II; codes 0–3). (0: sound ;1: first visual changes in enamel;

2: distinct visual changes in enamel; 3: Localized enamel breakdown (without clinical signs of dentinal involvement), ().

A portable LF system (DIAGNODent (DD) pen, KaVo, Biberach, Germany) was used. Standard DD recordings were taken at this stage according to the manufacturer’s instructions. The depths of demineralization were subjected using LF threshold. The manufacturer suggested the presence and depth of a caries lesion at the occlusal surfaces by LF using the following scale: 0–13 = initial caries lesion; 14–20 = enamel caries; 21–30 = initial dentine caries; 31–99 = advanced dentine caries ().

The status of WSLs was assessed using LF at 4 weeks recall and the changes in the amount of the values were recorded.

Saliva samples were also used to assess buffering capacity utilizing the Saliva-Check Buffer test kit (GC Corporation, Tokyo; Japan). The test started at least 2 hours after the last meal and at least 1 hour after brushing of the teeth. Saliva was stimulated by paraffin wax, and then saliva samples were used to measure salivary pH and its buffering capacity. Patients were instructed to spit any pooled saliva into the collection cup. After this procedure, an enclosed pH strip was placed into the sample of collected saliva for 10 seconds. Color change on the pH strip was checked while the paper was still moist. The pH reading was noted and the results were recorded as 5.0- 5.8 (high acidic); 6.0-6.6 (moderately acidic) and 6.8-7.8 (healthy saliva).

5 minute stimulated saliva was used to assess buffering capacity. After that, the test pads color changes were evaluated as green 4; green/blue 3; blue 2; Red/Blue 1 and Red 0 points according to the manufacturer’s instructions.

Testing Saliva Streptococcus mutans

The salivary samples were checked for S. mutans count by GC Saliva-Check Mutans™ test kit (GC Corporation, Tokyo; Japan). Stimulated saliva was tested to measure S. mutans levels at baseline. Technique guidelines were followed according to manufacturer’s instructions to detect S. mutans in saliva via a highly accurate and specific immunochromatography process by employing monoclonal antibodies (MAbs) which can detect small numbers of S. mutans in small salivary samples. A positive result was obtained if a thin red line appeared in the T window high levels of S. mutans (> 5 X 105 CFU/mL); however, if no line appeared (negative result), it indicated low levels of S. mutans (< 5 X 105 CFU/mL). Saliva collection for testing was done at the same time of day for the children.

Statistical analyses were performed using SPSS Statistics Version 19.0 (SPSS Inc., Chicago, IL, USA). In-group comparisons to determine the extent of remineralization was done using the paired t-test. In order to perform between-group comparisons, the difference of means (mean of pre-treatment values minus mean of post-treatment values) for each group was calculated. Between-group comparisons for the difference of means were done using the Mann Whitney U Test. For all the statistical analyses, the significance level was set at <0.05.

Results

We included 13 healthy children with mean age 4.25±0.50 with 145 teeth in the fluoride toothpaste group as the control group, and 13 healthy children with mean age 4.46±0.52 with 115 teeth in theobromine containing toothpaste group as the test group. After 1-month of experimental period, 26 children with a total of 260 study teeth (13 children, 145 teeth in control group; 13 children, 115 teeth in theobromine containing toothpaste group) were re-examined.

Laser Fluorescence (LF)

The mean LF reading was 20.71 ± 1.50 in the control group and 14.60 ± 1.66 in the theobromine containing toothpaste group at baseline, which decreased to 17.00 ±1.27 and 9.75 ±1.16, respectively at 1-month follow-up visit. The use of both of the toothpastes showed a highly significant (p< 0.0001) remineralizing potential at the end of treatment period of 1-month (Figure 1). The remineralizing potential of fluoride containing toothpaste was not significantly higher than that of theobromine containing toothpaste with the mean difference in pre- and post-intervention readings being only 1.22 (p = 0.339) (Table 2).

Figure 1

LF values before and after using Fluoride toothpaste and Theobromine containing Toothpaste

Table 2

Mean Difference of group comparison of DD values

ChangeDIAGNOdent values	N	Mean(SE)	Mean Difference	t value	Pvalue
Fluoride toothpastes	170	3.71 ± 0.82	1.22 0.967 0.339
TheodentTM Kids Toothpaste	115	4.93 ± 0.97

Salivary pH / buffering capacity

There was a significant increase between the baseline and 1-month readings of the salivary pH measurements in Theobromine containing Toothpaste (p=0.013) group, compared to the Fluoride containing toothpaste group (p=0.222)

A statistically significant increase in salivary buffering capacity was detected with the use of both toothpastes, Fluoride Toothpaste and Theobromine containing Toothpaste respectively (p=0.003 and p<0.0001) (Table 3).

Table 3

Saliva pH / buffer before and after using F Toothpaste and TheodentTM Kids Toothpaste

		Fluoride Toothpasten=13mean(SE)	TheodentTM Kids Toothpasten=13mean(SE)
Saliva pH	baseline	7.08± 0.40	6.83 ± 0.44
	after	7.22±0.43	7.17±0.43
	p	0.222	0.013
Saliva buffer	before	7.08±0.61	6.85±0.49
	after	9.46±0.62	9.69 ± 0.53
	p	0.003	0.000

Saliva pH and buffer changes of the subjects in the fluoride toothpaste assigned group showed no difference from those of Theobromine containing Toothpaste assigned group, with the mean difference at the baseline and after intervention being only 0.19 and 0.46 respectively (p = 0.248, p= 0.571) ().

Table 4

Intergroup comparison of saliva pH and Saliva Buffer

	Fluoride Toothpasten=13mean(SE)	TheodentTM Kids Toothpasten=13mean(SE)	Mean Difference	tvalue	Pvalue
Saliva pH	0.15 ± 0.11	0.33± 0.12	0.19	1.18	0.248
Saliva buffer	2.38 ± 0.65	2.38± 0.65	0.46	0.57	0.571

Microbiological analysis

The frequency of S. mutans was 8/13 at the baseline and decreased to 5/13 after using fluoride containing toothpaste for a month. The changes of frequency of S. mutans were 62.5%. In the theobromine containing toothpaste group, the frequency of S. mutans was 12/13 at the baseline and it decreased to 7/13 after using toothpaste for a month. The changes of the frequency of S. mutans were 58.33%. These changes were not found to be statistically significant in both the Fluoride and the Theobromine toothpaste groups (p>0.05) (Figure 2).

Figure 2

The frequency of Saliva S. mutans before and after interventions

Discussion

The observation of a significant net remineralization has revealed that the treatment modalities in this study, theobromine and sodium fluoride (NaF), enhanced the resistance of the remineralized surface to subsequent acid challenge. Therefore, remineralization generated by the use of theobromine as a remineralizing agent demonstrated an increase in resistance of the enamel crystals to further acid attacks ().

Amaechi BT. et al investigated the effects of both theobromine and NaF containing toothpastes. The surface microhardness showed a significant remineralization only when theobromine toothpaste was used. Although it exhibited comparable levels of remineralization on molar level, the amount of theobromine (0.0011 mol/l) required to produce a cariostatic effect was 71 times smaller than that of fluoride (0.0789 mol/l) to produce a comparable effect in theobromine and NaF containing toothpastes. They concluded that theobromine has a remineralization enhancing effect due to its ability to formation of apatite in comparison with the NaF containing toothpastes ().

Lippert F. (2017) investigated the effects of fluoride, strontium, theobromine and their combinations on caries lesion remineralization under pH cycling conditions ().

Kargül et al. observed the effect of theobromine in preserving the enamel surface in their study as well. The objective of this in-vitro study was to investigate the efficacy of theobromine treatment on the surface hardness and topography of human enamel at two different concentrations. The analyses of the microhardness of the enamel surface values at the baseline and after treatment with theobromine showed the protective effect of theobromine treatment (). Further investigations using human teeth were conducted to evaluate how theobromine exposure modified the enamel surface in vitro (, , ). By supporting the findings in the in-vitro study, the results of the in-vivo study have shown that the microhardness of the enamel surface of the theobromine group was greatly enhanced compared to the fluoride group ().

Qasthari A.I. et. al. (2018) showed that theobromine containing toothpastes maintained the enamel surface roughness better than sodium monofluorophosphate toothpastes ().

No studies till date have evaluated the remineralization potential of theobromine containing toothpaste with DIAGNOdent. According to this study, the values decreased for both groups following remineralization. However, no significant difference was found between fluoride containing toothpastes and theobromine containing toothpastes.

This preliminary initiative of insight into the effects of theobromine in preventing ECC in pediatric population demonstrated that after one month of experimental period, there was no significant difference in the values of remineralization of fluoride and theobromine containing toothpaste assigned groups. However it would have been possible to observe a significant difference between the remineralization potentials of the two toothpastes, if a long term study had been conducted.

Several studies comparing theobromine and hydroxyapatite were also conducted and the following results were obtained: a study which aimed to analyze and compare the effects of toothpastes containing theobromine and hydroxyapatite on enamel microhardness has found that enamel microhardness was increased significantly after brushing with both Theodent (theobromine) and Pepsodent (HAP) toothpastes. However, the enhancement of enamel hardness in specimens treated with HAP-containing toothpaste was superior to those treated with theobromine toothpaste ().

Toothbrushing is considered the most common oral hygiene habit. Oral hygiene is very important in protecting teeth against WSLs (). The aim of modern dentistry is to manage initial caries lesions non-invasively through remineralization to prevent disease progression ().

The contents of the toothpaste used have an impact on salivary functions and composition. Therefore, good toothpaste does not only contribute to remineralization directly, but also indirectly by enhancing the role of saliva in remineralization. Saliva is a major contributor to the protection of hard and soft intra oral tissues. Different properties of saliva such as facilitating the clearance of food, gathering and elimination of microorganisms, along with its buffering capacity to neutralize acids, contribute to remineralization of tooth enamel. Apart from that, saliva has antimicrobial properties. Therefore, salivary composition has been found to be associated with dental caries to varying degrees. Salivary properties can be used as biomarkers for indicating the risk of future disease and could potentially inform interference to deal with this risk ().

The importance of caries preventative role of theobromine toothpaste by means of controlling S.mutans levels in saliva was evident compared to fluoride containing toothpastes. Chi et. al. assessed the results of toothbrushing with xylitol toothpaste in the prevention of ECC and the reduction of S.mutans. In the six month follow up, brushing with xylitol/fluoride toothpaste was found to be no more efficacious in reducing ECC than fluoride-only toothpaste in a high caries-risk child population (). However, when Cakır et al. examined the antibacterial effect of Theodent Kids in an in vitro study, they found no antibacterial activity against S. mutans ().

The primary outcomes of this short term follow up study were as follows: after 4 week use of theobromine containing toothpaste, salivary pH, buffering capacity and S.mutans counts were significantly altered. The evidence reflects the important role of theobromine in impeding acid production by bacteria, which, in turn, increases salivary pH and disrupts the acid tolerance of cariogenic bacteria. These findings underline the anti-cariogenic effects associated with oral bioavailability of theobromine in toothpastes.

However, some limitations of our study are sample sizes of the groups and the F concentration of the control toothpaste. Therefore, this study is an observational study with results that deviate from the general failure patterns of the control group. Yet, being one of the first clinical studies on a novel toothpaste, we believe that the results of this study are worth noting.

Conclusions

The present study investigated the caries-preventive effect of Theobromine containing Toothpaste by examining its capacity for remineralizing caries lesions and inducing mineral deposition. The preliminary results suggest that theobromine containing toothpastes might be a choice for the prevention of ECC in children. There is still a clear need for further studies to investigate the mechanism by which theobromine induces mineral deposition and crystal growth, its potential advantages over other enamel protective compounds, its impacts on salivary composition, buffering capacity and clearance, and effects of long term theobromine administration.

This study can offer a novel toothpaste alternative to early childhood oral hygiene habits.