The interplay between toothbrush stiffness and charcoal-containing dentifrice on the development of enamel topography changes
Changes in enamel surface roughness could promote plaque formation and bacterial proliferation . There are a variety of factors that contribute to the changes in enamel surface roughness, including acid exposure, which dissolves hydroxyapatite found in the enamel surface, and exposure to an abrasive agent contained in different dental dentifrices . The tooth can be protected from these challenges by the formation of salivary pellicle and bacterial biofilms . The salivary pellicle formed over the tooth surface is composed of salivary proteins and glycoproteins that adhere to the enamel, granting a barrier against mechanical abrasion and chemical attacks . Besides, bacterial biofilms act as a protective barrier, preventing direct contact between the tooth surface and abrasive substances . Despite these protective factors, the tooth structure is not completely immune against chemical and abrasive attacks. A wide range of toothpaste is available today in the market, with different abrasive particles, detergents, and therapeutic agents . It is critical to realize that some of the available toothpastes may cause harmful effects to dental tissues . As a result, acknowledging the content and function of each toothpaste allows for the proper choice of the desired target. Ideal toothpaste provides maximum dental cleaning and teeth protection with minimum abrasion . Nowadays, charcoal-containing toothpaste is one of the trendiest items to whiten and clean teeth. Charcoal particle characteristics help remove extrinsic staining, biofilm, and food debris . However, a concern has been raised about using charcoal particles in toothpaste, as these particles’ star and fractal shape may increase teeth’ roughness . This is critical as increased enamel roughness may allow the accumulation of plaque and stains, leading to discolorations and secondary caries . Additionally, enamel wear can lead to tooth sensitivity, which negatively impacts patients’ quality of life . A controversy has been found in the literature concerning the use of charcoal-containing toothpastes . In one study, increased enamel surface roughness has been significantly seen after frequent usage of charcoal-containing toothpaste compared to the control . In another investigation, three charcoal-based toothpastes were found to induce increased the surface roughness of the teeth following 2,000 cycles of brushing simulation . Opposingly, another investigation found that charcoal-containing toothpaste did not affect the surface topographies of tooth enamel . Same findings were observed in another study where different charcoal-based dentifrices were found to induce less enamel changes compared to the control following erosion-toothbrushing abrasion cycling . The conflicting results in the existing literature may stem from variations in brushing force, the number of cycles, and the types of dentifrices used. However, it is worth mentioning that the previous investigations did not take into consideration the abrasivity of the toothbrush itself, as it has been demonstrated that the toothbrush stiffness may modulate the abrasivity of such toothpaste . As a result, investigating the interaction between the use of charcoal-based toothpaste and different bristles’ stiffness may provide more insight concerning the factors leading the enamel abrasion. Therefore, our study aimed to determine the abrasiveness of charcoal toothpaste on enamel using different toothbrush stiffnesses (soft, medium, and hard). Our results may help filling the gap between the controversial studies due to the insufficiency of mentioning the toothbrush bristles type while using activated charcoal. We hypothesize that charcoal-containing toothpaste when used with hard bristles could demonstrate greater enamel topography changes compared to the use of conventional toothpaste or soft bristles. 

Ethical approval, study design, and characterization The use of extracted teeth and the design of this study were approved by the Institutional Review Board at Imam Abdulrahman bin Faisal University (IRB-2023-02-414). In this study, four main groups with three subgroups for each were investigated (Fig. ). The first independent variable was toothpaste at four levels: (i) conventional fluoridated toothpaste without coal particles (Conventional TP), (ii) fluoridated toothpaste with charcoal particles (Charcoal TP), (iii) whitening toothpaste without coal particles (Whitening TP), and (iv) distilled water as a control. Details concerning the used toothpastes and their composition are described in Table . The second independent variable was the toothbrush bristles’ stiffness at three levels: soft, medium, and hard (Table ). The diameter and length of the bristles were determined using a 4.5× magnification device (LUXO, Elmsford, New York, USA) and ImageJ Software (The National Institutes of Health, Bethesda, USA). The toothbrushes (Tara Toothbrush Company LLC, Dammam, Saudi Arabia) were obtained from a local pharmacy store. The dependent variable investigated in the study was enamel surface roughness, which was measured at two time-point. Sample size calculation A priori sample size calculation was carried out using G*Power 3, a statistical tool for computing power analyses for a variety of statistical tests. This calculation established the sample size necessary for two-ANOVA test comparing the mean scores of the twelve groups at a significance level of p < 0.05. The results indicated a minimum of 19 samples in each group to detect medium effect sizes (f = 0.25). The sample size was increased to 22 samples per group to account any type of errors that could occur. Power was set at 80%. Samples preparation After getting the ethical approval, human-extracted premolars were collected and stored in 0.1 wt% of thymol solution at 4 °C till the time of use. Teeth with carious lesions or cracks were excluded. A total of 132 enamel specimens were prepared from extracted premolars using an IsoMet 4000 water-cooled precision saw (Buehler, Lake Bluff, IL, USA). Each specimen was then split into two, resulting in a total of 264 samples . An acrylic resin block was used to hold each specimen, and the outer enamel surface was flattened with silicon carbide paper of #600-, #1200-, and 2000-grit (Wirtz-Buehler, Düsseldorf, Germany) and diamond pastes to produce a flattened window of the enamel at 3 ⋅ 3 mm . Based on a list generated by RANDOM.ORG, all specimens were assigned unique numbers and distributed randomly among the twelve groups, where both the type of toothpaste and the bristles’ stiffness were randomized. Toothbrushing simulation model A custom-made V-8 toothbrushing machine (model ZM-3.8, SD Mechatronik, Feldkirchen-Westerham, Germany) was used to position the samples with their long axis perpendicular to the brushes’ long axis. Each toothpaste group was prepared by diluting 60 mL of dentifrice with distilled water at a 1:3 ratio, ensuring consistent application across samples. Custom-designed plastic trays were utilized to protect reference areas on the teeth, preventing unintended abrasion . Then, teeth were brushed for 1,250 and then for another 1,250 (total 2,500) double strokes, using the same toothbrush, at brushing load of 200 g to simulate typical brushing pressure. Following each specimen, the toothbrush was replaced by another one to exclude any impact related to bristles deformation. After finishing the brushing strokes, the specimens were thoroughly rinsed with distilled water to remove any residual toothpaste and debris . Evaluating the surface roughness The average surface roughness value for each group before and after the brushing simulation was determined through a non-contact optical profilometer (Contour GT-K1 optical profiler; Bruker Nano, Tucson, AZ, USA). With the aid of a regular camera, an area measuring approximately (0.43 × 0.58 mm 2 ) on three locations (center, right side, and left side) of the same specimen was scanned and documented the average Ra. The center of the sample was initially identified by a mark made in the acrylic material . The difference in Ra between before and after the brushing simulation challenge was determined by subtracting the post-brushing value from the pre-brushing value (baseline). Statistical analysis Sigma Plot recorded and analyzed the data. Descriptive statistics (mean, standard deviation, frequency, and percentages) were used to summarize the information. In addition, two-way ANOVA followed by Tukey multiple comparisons were used to compare the outcomes. A P-value of < 0.05 was considered statistically significant. 

The use of extracted teeth and the design of this study were approved by the Institutional Review Board at Imam Abdulrahman bin Faisal University (IRB-2023-02-414). In this study, four main groups with three subgroups for each were investigated (Fig. ). The first independent variable was toothpaste at four levels: (i) conventional fluoridated toothpaste without coal particles (Conventional TP), (ii) fluoridated toothpaste with charcoal particles (Charcoal TP), (iii) whitening toothpaste without coal particles (Whitening TP), and (iv) distilled water as a control. Details concerning the used toothpastes and their composition are described in Table . The second independent variable was the toothbrush bristles’ stiffness at three levels: soft, medium, and hard (Table ). The diameter and length of the bristles were determined using a 4.5× magnification device (LUXO, Elmsford, New York, USA) and ImageJ Software (The National Institutes of Health, Bethesda, USA). The toothbrushes (Tara Toothbrush Company LLC, Dammam, Saudi Arabia) were obtained from a local pharmacy store. The dependent variable investigated in the study was enamel surface roughness, which was measured at two time-point. 

A priori sample size calculation was carried out using G*Power 3, a statistical tool for computing power analyses for a variety of statistical tests. This calculation established the sample size necessary for two-ANOVA test comparing the mean scores of the twelve groups at a significance level of p < 0.05. The results indicated a minimum of 19 samples in each group to detect medium effect sizes (f = 0.25). The sample size was increased to 22 samples per group to account any type of errors that could occur. Power was set at 80%. 

After getting the ethical approval, human-extracted premolars were collected and stored in 0.1 wt% of thymol solution at 4 °C till the time of use. Teeth with carious lesions or cracks were excluded. A total of 132 enamel specimens were prepared from extracted premolars using an IsoMet 4000 water-cooled precision saw (Buehler, Lake Bluff, IL, USA). Each specimen was then split into two, resulting in a total of 264 samples . An acrylic resin block was used to hold each specimen, and the outer enamel surface was flattened with silicon carbide paper of #600-, #1200-, and 2000-grit (Wirtz-Buehler, Düsseldorf, Germany) and diamond pastes to produce a flattened window of the enamel at 3 ⋅ 3 mm . Based on a list generated by RANDOM.ORG, all specimens were assigned unique numbers and distributed randomly among the twelve groups, where both the type of toothpaste and the bristles’ stiffness were randomized. 

A custom-made V-8 toothbrushing machine (model ZM-3.8, SD Mechatronik, Feldkirchen-Westerham, Germany) was used to position the samples with their long axis perpendicular to the brushes’ long axis. Each toothpaste group was prepared by diluting 60 mL of dentifrice with distilled water at a 1:3 ratio, ensuring consistent application across samples. Custom-designed plastic trays were utilized to protect reference areas on the teeth, preventing unintended abrasion . Then, teeth were brushed for 1,250 and then for another 1,250 (total 2,500) double strokes, using the same toothbrush, at brushing load of 200 g to simulate typical brushing pressure. Following each specimen, the toothbrush was replaced by another one to exclude any impact related to bristles deformation. After finishing the brushing strokes, the specimens were thoroughly rinsed with distilled water to remove any residual toothpaste and debris . 

The average surface roughness value for each group before and after the brushing simulation was determined through a non-contact optical profilometer (Contour GT-K1 optical profiler; Bruker Nano, Tucson, AZ, USA). With the aid of a regular camera, an area measuring approximately (0.43 × 0.58 mm 2 ) on three locations (center, right side, and left side) of the same specimen was scanned and documented the average Ra. The center of the sample was initially identified by a mark made in the acrylic material . The difference in Ra between before and after the brushing simulation challenge was determined by subtracting the post-brushing value from the pre-brushing value (baseline). 

Sigma Plot recorded and analyzed the data. Descriptive statistics (mean, standard deviation, frequency, and percentages) were used to summarize the information. In addition, two-way ANOVA followed by Tukey multiple comparisons were used to compare the outcomes. A P-value of < 0.05 was considered statistically significant. 

The impact of the type of toothpaste, bristles’ stiffness, and their interaction after 1,250 and 2,500 cycles of brushing simulation are described in Tables and . The two-way ANOVA analysis revealed that after 1,250 brushing simulation cycles (Table ), the toothpaste type ( P = 0.017) and bristles’ stiffness ( P = 0.022) were significant factors in modulating the enamel surface roughness. Besides, there was a significant interaction ( P = 0.013) when these two factors were analyzed. Table illustrates the two-way ANOVA results following 2,500 brushing simulation cycles. Similarly, both the toothpaste type (P = < 0.001) and the bristles’ stiffness (P = < 0.001) were significant factors in the enamel surface roughness with no significant interaction ( P = 0.227). Following 1,250 brushing simulation cycles (Table ; Fig. ), the whitening toothpaste (39.24 ± 17.07) significantly ( P = 0.037) increased the enamel surface roughness compared to the negative control (22.20 ± 18.34) when soft bristles toothbrushes were used. When bristles with medium stiffness were used, the whitening toothpaste also was associated with the highest value of surface roughness change (47.68 ± 19.90), which was significantly ( P = 0.003) higher than the charcoal toothpaste (25.93 ± 16.09). Table ; Fig. illustrate the enamel roughness values after 2,500 cycles of brushing simulation. The whitening and charcoal toothpastes were associated with increased surface roughness change compared to the conventional toothpaste and the negative control, especially when bristles with medium and hard bristles were used. Using bristles with soft stiffness revealed that charcoal toothpaste was associated with the higher value of surface roughness change (55.86 ± 41.18), which was significant ( P = 0.024) compared to the negative control. Using bristles with medium stiffness showed that the whitening (68.23 ± 48.58) and charcoal (73.62 ± 34.66) toothpastes significantly (P = < 0.05) increased the enamel surface roughness compared to the conventional toothpaste (36.53 ± 22.56). Figure. illustrates the scan images for the investigated groups recorded by the non-contact profilometer. In general, more topographic changes were observed when bristles with hard stiffness were used and when whitening and charcoal toothpastes were applied. 

The alternative hypothesis of this study was supported as charcoal toothpaste increased enamel surface roughness compared to the conventional toothpaste and the negative control, especially when the bristles’ stiffness was hard. The enamel surface roughness change following the application of the charcoal toothpaste was comparable to the whitening toothpaste, suggesting that both may induce harmful effects against the enamel surface. The results of our findings suggest that people should be cautious when using dental products containing charcoal or whitening ingredients for an elongated period as they may abrade the enamel. The number of toothbrushing cycles applied in this study served as a prolonged representation of toothbrushing. In real-life situations, toothbrushing usually occurs at a rate of 4.5 strokes per second . Since 2 min is the suggested brushing time to eliminate plaque , it is expected that around 90 strokes are needed in each sextant. This equates to 15 brushing strokes per tooth or 5 brushing strokes per surface (buccal, lingual, occlusal) . Consequently, if patients brush their teeth on three occasions a day, a total of 5475 strokes would be accomplished on a particular surface in a year. For this study, simulating approximately three and six months, the number of brushing cycles was set at 1,250 and 2,500, respectively. These durations are expected to induce initial changes in the enamel surface. However, it is important to note that the parameters used in this study, as well as the number of cycles, are limited by the absence of saliva, which serves as a potential remineralizing reservoir, and the salivary pellicle, which acts as a protective layer. In several in vitro studies, the brushing force used was between 0.2 and 4.2 N, with an average between 2 and 3 N . In this study, we applied a force equivalent to 2 N to be convenient with the current literature. The Charcoal and whitening toothpastes experienced the highest increase in the Ra value, which is a measure of surface roughness. This increase was mainly attributed to the presence of abrasive materials in the toothpaste. Apart from silica and hydrated silica, Charcoal Formula toothpaste also contains activated carbon or charcoal. Charcoal particles in the toothpaste have a star-shaped or fractal shape, which may contribute to its abrasivity . We intended in this study to use toothpastes with comparable ingredients to emphasize the possible impact of the charcoal powder in the charcoal toothpaste. Statistical analysis comparing the conventional toothpaste (positive control) and the charcoal toothpaste, which have similar types of abrasive materials, revealed significant differences in the changes of Ra values (surface roughness) after three months of brushing. This suggests that the charcoal component in the toothpaste may have played a role in altering the surface roughness of the tooth enamel despite the similar types of abrasive materials used in both toothpastes. It is also possible that the size and quantity of silica particles differ between the two toothpastes. Larger and more abundant silica particles may induce greater surface changes . This is more likely when comparing the ingredients of the conventional and whitening toothpastes, as the only difference between them is the number of pigments, which probably have little to do with the abrasivity of the toothpastes. The relative dentin abrasion values (RDA) of the conventional and whitening toothpastes used in this study were found to be 70 and 124, respectively . This suggests that the conventional toothpaste used here has low abrasivity, while the whitening toothpaste has high abrasivity, mainly related to the size and number of silicas. No available information concerning the RDA value of the charcoal toothpaste used in this study, but it is expected to be close to the whitening toothpaste based on the obtained results. This study also suggests that the use of medium and hard bristles may lead to greater enamel wear compared to soft bristles. While dental practitioners typically recommend soft-bristled toothbrushes to their patients, it is essential to investigate the extent of enamel wear associated with medium and hard bristles. Additionally, the potential interactions between these bristles and toothpastes with varying ingredients and abrasivity warrant exploration. Interestingly, the effects of medium and hard bristles may be comparable to those of soft bristles when used with low-abrasive toothpaste, which was also demonstrated in the study by Turssi et al. . Therefore, a key objective of this article was to examine the interactions between the various toothpastes investigated and the stiffness of different bristles. The results of our study agree with previous investigations showing that charcoal toothpaste may induce enamel surface roughness . However, our results contraindicate other findings, revealing no difference in enamel wear subjected to conventional and charcoal toothpastes . The conflicting results in the existing literature may arise from several methodological differences, including variations in brushing force applied during the experiments, which can significantly influence enamel wear. Additionally, the number of brushing cycles used in each study varies, potentially leading to differing levels of abrasion severities. Furthermore, the types of dentifrices utilized, each with distinct formulations, active ingredients, and abrasive properties, can also contribute to the discrepancies observed in the outcomes. Besides, this controversy could be attributed to the use of one type of bristles stiffness, which could be an important modulator in the wear of enamel . These factors underscore the need for standardized methodologies to allow for more reliable comparisons across studies. Therefore, we intended to use different bristles’ stiffness in this study with clinically relevant parameters to investigate the interaction between the charcoal toothpaste and the stiffness of the bristles. This study found a significant difference in enamel surface roughness between conventional toothpaste versus charcoal and whitening toothpastes when used with hard and medium bristle toothbrushes. However, only a slight difference was observed when using soft bristles. These findings suggest that the risk of enamel wear may be greater when using charcoal and whitening toothpastes in combination with hard or medium bristle toothbrushes, especially with long-term use. While some may wonder if it is safe to use charcoal toothpaste even for a short period, it should be noted that despite the abrasiveness of charcoal toothpaste, which can remove extrinsic stains, it does not provide any intrinsic whitening benefits and can still harm the enamel surface, as indicated by the results of this study. Additionally, there is growing evidence that charcoal toothpastes are not particularly effective at improving teeth color . Therefore, it may be advisable to avoid the use of charcoal-based toothpastes and instead opt for home or professional bleaching/whitening treatments for safe and effective teeth whitening. Our results suggest that individuals should be cautious when using highly abrasive toothpastes and stiff-bristled toothbrushes, as they may have significant adverse effects on an individual’s oral health. One of the primary concerns is the gradual wear down of the tooth enamel over time. Tooth enamel is the outermost, hardest layer of the tooth, and it plays a crucial role in protecting the underlying dentin and pulp. However, the aggressive abrasive action of these dental products can gradually abrade the enamel, leading to increased sensitivity . This increased sensitivity can make everyday tasks like eating and drinking a challenging and unpleasant experience. Furthermore, the loss of enamel can also compromise the aesthetic appearance of the teeth, potentially leading to an aged or unattractive look. Another significant consequence of using abrasive toothpastes and stiff-bristled toothbrushes is the increased risk of gingival recession . To mitigate these issues, it is recommended that individuals use a soft-bristled toothbrush and a non-abrasive, fluoride-containing toothpaste with proper brushing technique. Besides, seeking professional advice from dental practitioners can ensure using the most effective tools for oral hygiene practice. This laboratory study yielded significant findings regarding the impact of charcoal toothpaste and toothbrushes with varying bristle stiffness on enamel surface topography. However, there are some limitations that should be acknowledged. While efforts were made to select and polish the teeth within specific criteria, it is impossible to perfectly standardize the baseline surface roughness and mineral content of the enamel samples . This variability in the starting conditions of the samples could have introduced some inherent differences. Furthermore, various patient-related factors, such as individual differences in oral health status, overall physiological conditions, and general health, can significantly influence the enamel’s response to the wear challenge. These clinical variables are not easily replicated in a controlled laboratory setting. Besides, in the oral environment, the formation of salivary pellicles over the tooth surface may potentially reduce the degree of wear induced by toothbrushing . Putting all these factors into consideration in addition to the need to investigate other commercially available charcoal-based toothpastes, the results obtained here can not be generalized. It is crucial to validate the findings of this in vitro study through well-designed in-vivo models. Despite these limitations, the current study provides valuable insights into the potential abrasive effects of charcoal toothpaste and different bristle stiffnesses on enamel topography. Further clinical research is warranted to fully understand the implications for dental health and to establish safe and effective oral hygiene practices. 

The results of this study suggest that the type of toothpaste used, and the stiffness of the toothbrush bristles can impact the surface roughness of dental enamel. Specifically, the investigated whitening and charcoal toothpastes were associated with increased enamel roughness. Further research, including well-designed clinical studies and investigating a broader sample of commercially available products, is warranted to corroborate these in vitro findings and develop evidence-based recommendations for dental consumers and practitioners. 

