Relationship between toothpaste brushing, surface roughness, and bacterial adherence using contemporary tooth-colored restorative materials: a laboratory study

INTRODUCTION

Streptococcus mutans is one of the primary colonizing agent in the oral cavity. In addition to the hard tissues of the teeth, surfaces of restorative materials may also experience bio-film formation. The adhesion of micro-organisms to teeth and restorative materials, plays a major role in dental diseases. Bacterial adherence is influenced by several physico-chemical properties of both the material and bacterial surfaces [1]. Bio-film growth and maturity are impacted by surface properties, such as surface roughness (SR) or surface free energy (SFE). A more intricate bio-film might form faster over rougher substrates, since they have more surface area and more SFE [2]. Bollen et al. [3] reported that materials with SR larger than 0.2 μm exhibit higher levels of bacterial adhesion. This SR of restorative materials is influenced by various factors, of which one of the important influents is toothbrushing. The amount of brushing time, brushing force, and abrasiveness of toothpaste might affect the SR of restorations [4], while the SR affects longevity, esthetics, and ability to resist discoloration and stains [5, 6].
At present, with the growing awareness of esthetic rehabilitation, the choice of restorative material is based on esthetics, strength, durability, bio-compatibility, and cost-effectiveness. Moreover, the use of esthetic materials, such as zirconia, zirconomer, cention N, etc., had gained popularity.

OBJECTIVES

In the literature, SR and bacterial adherence of these restorative materials has been investigated individually; however, no prior research has examined the relationships between the effect of toothbrushing with paste on SR and microbial adhesion of these restorative mate­rials. Therefore, this study aimed to evaluate and compare the effect of toothbrushing on SR and bacterial adhe­rence of cention N, zirconomer, zirconia, and enamel. The null hypothesis of the study was that toothbrushing with paste would have no significant effect on SR and bacterial adherence of the tested restorative materials.

MATERIAL AND METHODS

A total of 18 samples were prepared, with sample size calculated based on data from previous study by Kantorski et al. [2]. Therefore, 20 rectangular-shaped samples of 6 × 3 × 3 mm were considered for each material.
Group 1 – Enamel (control group): enamel blocks were prepared by cutting the extracted, free of any defect or restoration, permanent human molar teeth, using micro-motor and diamond disc.
Group 2 – Cention N (Ivoclar, Vivadent, Liechtenstein): powder and liquid was mixed according to the manufacturer’s instruction, placed into a mold, and light-cured for 20 sec using 1,200 mW/cm2 LED light curing unit (Woodpecker Mini S Light Cure LED). Afterwords, the samples were checked visually for imperfections.
Group 3 – Zirconomer (SHOFU Inc., Kyoto, Japan): powder and liquid of zirconomer was mixed as per the manufacturer’s instruction, and placed into a mold. After 3-5 min of hardening, the samples were checked for imperfections.
Group 4 – Zirconia (API Zirconia Dental Ceramics Blank White HT Series): samples were cut from a pre-sintered block using diamond disc, and were subjected to sintering, leading to 20% of volumetric shrinkage of these samples [7].
Group 2 and 3 samples were finished and polished with Super-Snap kit (SHOFU Inc.) according to the manufacturer’s instruction, with each disc from the kit being used for ten seconds. After that all the samples were subjected for SR test using a profilometer (Surface Roughness Tester TIME^® 3200). A spectrophotometer was employed to standardize bacterial culture of Streptococcus mutans (106 cells/ml) using a reference strain of the species. Each sample was submerged into 20 μl of cell suspension and 2 ml of BHI broth in a test tube, followed by incubation (48 h, 37^oC, 5% CO₂). In order to dispose non-adhesive cells, the samples were washed thrice with 5 ml of sterile saline solution. Each sample was kept in a glass tube with 1.5 ml of sterile saline solution, and the tube was vortexed for 2 min to separate the attached bacteria from the tube. 10-fold serial dilutions of the original solution were created in sterile saline solution, and the homogenization process was repeated for 15 sec on a vortex. After plating an aliquot of 0.1 ml, each of the dilutions 10² and 10³ were kept on BHI agar plates, and the plates were incubated (48 h, 37^oC, 5% CO₂). For every sample, the mean number of colony-forming units (CFU/ml) was determined to assess bacterial adherence.

Toothbrushing simulation

A homogeneous solution for tooth brushing simulation using Dant Kanti toothpaste and water in ratio 1 : 3 was prepared. The toothpaste suspension was poured into the cylinders, and the samples were subjected to brushing using a powered toothbrush (Colgate 360 charcoal battery powered electric toothbrush) with a speed of 11,500 strokes/min for 10 sec, twice-daily, for 30 days. In total, each sample received brushing of 10 min. Post-brushing, SR and bacterial adherence of the samples were re-assessed similarly (as mentioned before).

Statistical analysis

Independent t-test was employed to conduct the analysis. Using SPSS 25.0 software, the level of statistical significance was set at p < 0.01.

RESULTS

Both before and after toothpaste brushing, the highest SR was observed on enamel, while the lowest was found on zirconia samples. The maximum and minimum bacterial adherence pre- and post-brushing was observed on cention N and zirconia, respectively (Table 1). Brushing increased the SR of all the tested materials significantly (p < 0.01) (Figure 1); however, bacterial adherence decreased post-brushing (p < 0.01) (Figure 2). On inter-group comparison, zirconia showed significantly less (p < 0.01) SR and bacterial adherence compared with zirconomer, cention N, and enamel (Table 2). The diffe­rence between the SR and bacterial adherence of cention N and zirconomer/enamel was significant pre-brushing; however, post-brushing, the SR difference was significant for enamel but non-significant (p > 0.01) for zirconomer. Both pre- and post-brushing, the SR of zirconomer was significantly lesser than that of enamel. The enamel samples showed significantly less bacterial adherence compared with zirconomer and cention N restorative materials.

DISCUSSION

In the present study, it was observed that toothbrushing with paste increases the SR and decreases the bacterial adherence of restorative materials and enamel, demonstrating negative correlation of the SR and bacterial adherence. The investigated null hypothesis was therefore rejected.
Both extrinsic and intrinsic factors can influence material’s SR. The composition of material, particularly size, form, volume, and distribution of in-organic filler particle, are intrinsic factors. Whereas medications and liquids to which the materials are exposed, kind of polishing system used, and operator-related parameters, are extrinsic factors [8]. Previous investigators [9, 10] have revealed that factors affecting toothbrush abrasion include the type of brush, filament stiffness, brushing method, force, duration, and frequency of brushing. In the current study, Dant Kanti toothpaste was chosen, as it is one of the most widely used herbal toothpaste in India, particularly in rural areas [11, 12].
The minimum SR values before toothpaste brushing in the present study were observed in the zirconia group. This finding is similar to the results of Kozmos et al. [1], and might be due to fracture toughness and grain size of zirconia, which may influence the wear resistance of zirconia, according to a study by Zhang et al. [13]. In Roulet et al. [14], the manual mixing of cention N resulted in larger pores that may cause increased SR. Kaptan et al. [15] reported that SR of cention N was lesser than glass-ionomer-based cements, which might explain why the SR of cention N was less than that of zirconomer. In this study, it was shown that brushing with toothpaste enhanced the SR of enamel and restorative materials statistically. This conclusion is consistent with findings of Pertiwi et al. [16]. The toothpaste’s abrasive components was the cause of increase in the SR value.
It was found that brushing increased the SR of zirconia. Our findings corroborate with a previous study by Pinelli et al. [17], who stated that the use of dentifrice increased the SR of zirconia. Priyam et al. [18] and Aggarwal et al. [19] also concluded that the SR of enamel increased after brushing with Dant Kanti toothpaste due to its abrasiveness. Additionally, it can be because of the loss of minerals in teeth after brushing, as suggested by Pertiwi et al. [16]. It was observed that cention N showed more SR than zirconomer post-brushing; however, the difference was not significant. According to Adsul et al. [20], the glass portion of formulation undergoes precisely controlled micronization to provide the ideal particle size and properties. To further reinforce the composition for longer durability and greater occlusal load resistance, homogeneous zirconia particles were added. Arsath et al. [21] stated that filler technology of cention N provides sufficient strength to endure the stresses and strains of the oral cavity. Additionally, the material has exceptional flexural force due to its extensively cross-linked polymer structure. Filler particles in cention N range in size from 0.1 to 35 μm [22]. This might be the reason for its higher SR. Furthermore, Kaur et al. [23] concluded that enamel shows the highest SR than composite, as toothbrush abrasion has a synergistic effect with erosion on substance loss of enamel.
One exceptional finding of this study was that before toothpaste brushing, zirconia showed the least bacterial adherence as compared with cention N, zirconomer, and enamel. The reasons for the observed least bacterial adherence to zirconia have been abundantly addressed in the literature. Hao et al. stated that the chemical composition, SR, SFE, surface topography, ions release, and other surface features of dental materials, all have a significant impact on bacterial adhesion. In particular, zirconia exhibited a low bio-film accumulation [24]. Research has demonstrated that micro-organisms have a tendency to adhere to surfaces with a high SFE [25]. Bislimi et al. [26] reported that the SFE of zirconia was lower than the composite. This could be the possible explanation of why zirconia demonstrated the least bacterial adherence when compared with other restorative materials and enamel. This was in accordance with a research by Jalalian et al. [27], who discovered that when compared with enamel, zirconia had the least amount of bacterial adherence. S. mutans’s hydrophobic characteristics, which increase their adherence to hydrophobic surfaces, might be one explanation. According to Kozmos et al. [1], ceramic is less hydrophilic than enamel, and this factor may account for zirconia’s lower bacterial adhesion than enamel. As clearly evident from our study, zirconomer showed less bacterial adherence than cention N. The possible reason may be that zirconomer has the highest fluoride release and maximum antibacterial effect, as reported by Feiz et al. [28] and Paul et al. [29].
In the current study, all materials showed a comparable reduction in bacterial adhesion following toothpaste brushing. The antimicrobial compounds in Dant Kanti toothpaste, which exhibit antimicrobial activity against S. mutans, may be the source of this reduced bacterial adherence. In this study, even though the SR of the enamel and restorative material increased, bacterial adhesion reduced after brushing with herbal toothpaste. This study’s findings are consistent with those of Sakul et al. [30] and Somacal et al. [31]. The composition of toothpaste that includes triclosan or fluoride functions as antibacterial agents, might be the reason. Palekar et al. [32] and Vyas et al. [33] found that Dant Kanti toothpaste showed superior antibacterial activity against S. mutans, which helps to explain the outcome of the study. In the present study, bacterial adherence of all the materials used decreased after toothpaste brushing in similar order.
In our study, there are few limitations to acknowledge. The condition of a complex oral environment was not replicated, as it was laboratory-based study. Since just one strain was examined, there was no contact with other micro-organisms present in the oral cavity. A machine that simulates a toothbrush would have been a better option for creating steady, pressure-free strokes. Numerous other factors can also impact the roughness of a restorative material in the oral environment, including the restoration’s location, brushing frequency, age, changes in oral micro-organisms, salivary flow and quality, dietary habits, para-functional habits, and occlusion changes. Future research should assess several herbal, whitening, and fluoridated toothpaste variants.

CONCLUSIONS

Toothbrushing increases the SR and decreases the bacterial adherence of all the experimental materials and enamel. The SR did not display any direct relation to bacterial adherence after toothbrushing. The results of this study demonstrate that individuals with poor oral hygiene would benefit more from receiving zirconia restorative material.

Disclosures

1. Institutional review board statement: Not applicable.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: None.

References