Biofilm volume and acidification within initial biofilms formed in situ on buccally and palatally exposed bracket material

Biofilms are structured microbial communities that are attached to a surface and surrounded by an extracellular matrix. In the mouth, they are responsible for many diseases, like caries and periodontal diseases, the two most prevalent medical threats in industrialized societies [10, 18, 43]. Dental caries is defined as a multifactorial, biofilm-mediated, diet-modulated, noncommunicable, dynamic disease resulting in mineral loss of dental hard tissues [21, 41, 48].

Biofilm formation begins shortly after the tooth surface has been cleaned. A pellicle composed of salivary proteins forms. This pellicle serves oral bacteria as attachment point to colonize the tooth surface [30, 39, 54]. When further bacteria colonize, the plaque grows into a three-dimensional structure surrounded by a matrix of self-produced extracellular polysaccharides [28]. In dental biofilms, the pH value of the extracellular matrix is the main virulence factor for the development of caries [57]. In order to gain energy, microorganisms in the plaque metabolize low-molecular-weight carbohydrates, thereby creating organic acids [10, 67]. This lowers the pH value, and minerals of the enamel such as calcium and phosphate can be dissolved. For tooth enamel, the critical pH value is 5.2–5.7 [28].

Enamel demineralization is also a significant risk factor and one of the greatest challenges during fixed orthodontic treatment [65]. The insertion of brackets, bands and arches complicates oral hygiene and promotes the accumulation of plaque [19].

The local acidification can subsequently lead to white spot lesions (WSL), which represent the first stage of caries formation [40]. WSL are milky white opacities of the enamel surface without cavity formation [59], which can already occur one month after the start of orthodontic treatment [46, 47]. The incidence of WSL is given in the literature up to 72.9% [53]. A 2015 meta-analysis concluded that the incidence of WSL during orthodontic treatment was 45.8% and the prevalence was 68.4% [59].

A significant reduction in the occurrence of WSL during orthodontic treatment was observed with a completely customized lingual multibracket appliance. The global incidence of new WSL for this treatment was determined to be only 3.19% for the entire dentition (teeth 17–47) [69]. When focusing on the upper incisors, it was shown that the subject-related incidence was 9.59% and the teeth-related incidence was 4.1% [36]. A comparison with previous studies on WSL incidence following conventional buccal appliances indicated a reduction by a factor 6.35 for the subject-related and a reduction by a factor 14 for the teeth-related WSL incidence [36]. Another study comparing WSL between lingual and buccal multibracket appliances found that the number of newly developing or progressive WSL was 4.8 times lower lingually than buccally [66]. In addition, the integrated fluorescence loss, measured using the quantitative light-induced fluorescence, was 10.6 times lower for lingual than for buccal surfaces [66].

According to these results, one could hypothesize that there is less biofilm formation on lingual than on buccal orthodontic brackets. As a consequence, acidification would be lower and fewer WSLs would occur. However, studies comparing biofilm formation and acidification lingually/palatally and buccally at the bracket–tooth interface have not yet been carried out.

Therefore, the objective of this in situ study was the three-dimensional investigation of initial intraoral biofilm formation and pH values on buccal and palatal bracket material. For this purpose, splints equipped with test specimens were exposed to the oral cavity of 12 volunteers for a total of 48 h. To quantify biofilm volume and live/dead distribution, the specimens were fluorescently stained and examined by confocal laser scanning microscopy (CLSM). The pH value within the biofilms was measured using pH ratiometry by the pH-sensitive ratiometric dye seminaphthorhodafluor-4F 5‑(and -6)-carboxylic acid (C-SNARF-4). Depending on the protonation, the dye shows a shift in its fluorescence emission detected by CLSM and can, thus, measure the extracellular pH value in the range from 4.5 to 7.0.

By combining the results of biofilm volume and pH value quantification, biofilm formation and acidification on buccally and palatally exposed bracket materials in the upper jaw were compared.

To analyze bacterial biofilm pH at the orthodontic bracket/tooth interface, test specimens consisting of cylindric bracket material on a hydroxyapatite disc were exposed in the upper jaw of 12 participants. To verify healthy oral conditions, initial periodontal screening was performed and revealed the following: PD of 1.5 ± 0.12 mm, SBI of 1 ± 0.02%, and API of 10.64 ± 0.03%. After 48 h of in situ biofilm growth, test specimen of 11/12 subjects (one subject withdrew from the study due to an acute temporomandibular disorder [TMD]) could be included in the study and were stained and analyzed by CLSM. With this experimental design, effects with an effect size >0.4 could be detected ensuring a statistical power of 0.8.

Half of the specimens were analyzed for biofilm volume and live/dead distribution at the bracket/hydroxyapatite disc interface. Representative reconstructions of the biofilms are shown in Fig. 3a, b. They exhibited three-dimensional morphology and consisted of bacterial as well as human cells. As a specific position effect (Fig. 2c) could be excluded by statistical analysis, average values for buccally and palatally exposed specimens were compared. The average buccal biofilm volume was 8.79 × 10⁵ ± 3.31 × 10⁵ μm³ and the average palatal biofilm volume was 8.32 × 10⁵ ± 2.73 × 10⁵ μm³ (Fig. 3c). No statistically significant difference could be detected. The live/dead distribution was also almost similar between buccally and palatally exposed specimens with approximately 54% living and 45% dead cells (Fig. 3d).

The other specimens were analyzed for pH values at different biofilm layers (Fig. 1a). For each position on the specimen (Fig. 2c), three-dimensional images of the biofilm were taken. From these images, four levels were selected for pH analysis (Fig. 4a). Level 1 was defined as bottom layer and level 4 as top layer of the biofilm. Level 2 and 3 were evenly distributed in between. As for biofilm volume a position specific effect (Fig. 2c) of the pH value could be excluded by statistical analysis, average values per level were calculated (Fig. 4b).

For buccally exposed specimens, the pH value steadily increased from pH 6.46 at the bottom layer (level 1) to pH 6.89 at the top layer (level 4). In contrast, for palatally exposed specimens, the pH was 6.40 at the bottom layer, increased up to pH 6.74 at level 3 and slightly reduced to pH 6.68 at level 4.

For biofilms grown on both oral positions, the pH value at the bottom layer was significantly lower compared to all other layers. When comparing both bottom layers (1 and 2) between buccal and palatal specimens, no differences could be detected. In contrast, the pH values of the top layers (3 and 4) were significantly lower palatally than buccally.

Common complications of orthodontic treatment are local demineralizations of the enamel that could lead to the formation of white spot lesions (WSL) and caries. They are caused by acid production of increased amounts of dental plaque around the brackets, due to inadequate oral hygiene during orthodontic treatment [7, 8, 59]. Interestingly, it has been shown that biofilm growth depends on intraoral location [5, 58]. In line with this, WSL showed a decreased clinical prevalence for lingual brackets [66, 69]. Therefore, the hypothesis of this study was that there is a difference in initial intraoral biofilm formation on buccally and palatally bonded brackets, resulting in different acidification. To address this hypothesis, this study quantified volume, live/dead distribution and pH value of initial biofilms formed on buccally and palatally exposed test specimens in the upper jaw of volunteers.

Prior to the study, healthy periodontal conditions of each participant were assured by an initial periodontal screening including probing depths (PD), modified sulcus bleeding index (SBI), and modified approximal plaque index (API) [28]. All values were within the normal range. Further risk factors influencing oral microflora, such as general diseases, antibiotic treatment 6 weeks before participation, alcohol consumption, smoking or pregnancy were also excluded [3, 9, 12, 24, 31, 49, 52, 55]. The selected sample size was set to 12 participants with specimens for each analysis in duplicates, which corresponds to studies with a similar study design and allows for the detection of large effect sizes >0.4 [4, 5, 27, 44, 61]. As the clinically observed lingual and buccal WSL formation upon orthodontic treatment differs by more than 60% [59], this experimental setup was considered sufficient. For biofilm collection, individual occlusal splints of the upper jaw were equipped with test specimens. The test specimens were placed buccally and palatally in the premolar and molar region in the first and second quadrants. This ensured an increased wearing comfort compared to the lower jaw and no impairment of aesthetics and phonetics for this initial study, but needs to be taken into account when evaluating the data. To avoid additional oral position-specific effects [4, 5], specimens were block-wise randomly assigned to the different evaluation groups. Instead of real brackets with geometries varying between different companies and especially between lingual and buccal appliances, uniform cylindric shaped bracket material was used. This ensures a higher reproducibility and comparability between the different positions. The test specimens were also not directly fixed to the splints, but to discs made of pure hydroxyapatite. This mineral is the main component of human enamel [15, 50] and the discs, thus, could be considered biomimetic alloplastic. The advantages of this construction are that it provided a standardized and uniform shape according to the desired dimensions, allowed for the analysis of the bracket/tooth interface, but avoided the use of allogeneic [35, 64, 67, 70] or xenogeneic material [2, 25, 33, 62]. This reduced ethical concerns regarding the study and increased participants acceptance. The decision not to use shielding in the area of biofilm collection—as was done in previous studies [16, 44, 63]—was based on the idea of simulating clinical reality as precisely as possible, i.e., allowing shear forces of the tongue and cheeks. Taken together, this splint design ensured almost natural biofilm formation that was close to the clinical orthodontic situation and, thus, can serve as the basis for further studies.

The splints were worn for 48 h by the participants. This time was set as other studies have demonstrated that it was appropriate for in situ biofilm growth [4, 5, 27, 35, 44, 51, 61, 64]. Due to the fact that oral hygiene was suspended, accumulation of biofilm formation was increased but it still should be considered as an initial biofilm. Even if the study design was not directly comparable with multiyear orthodontic treatment, it was sufficient for an initial study. However, it would be important for future studies to analyze the obtaining findings over the entire period of orthodontic treatment.

For the final analysis, half of the specimens were live/dead fluorescent stained and CLSM was used to quantify biofilm volume and live/dead distribution at the bracket/hydroxyapatite disc interface. This method is well established and allows for biofilm quantification with almost native morphology [17, 22, 29, 45, 60].

The detected biofilm consisted of bacterial and also human cells. These cells were most likely human gingival epithelial cells that are integrated into the biofilm through bacterial colonization, as has been demonstrated in various studies [16, 44, 63].

Compared to other studies, the biofilm volume was low in this study [16, 44]. This is most probably due to a different splint design in which the specimens were exposed to the natural conditions of the oral cavity, including shear forces by cheek and tongue, and were not protected by shield-like structures. However, the live/dead distribution was almost similar [44, 62].

When comparing the biofilms formed buccally and palatally, interestingly no statistically significant differences in volume or the live/dead distribution in this initial intraoral biofilm could be detected. This is in contrast to the study of Auschill et al. [5], which revealed a lower palatal biofilm thickness compared to the examined buccal locations. However, in their study the specimens were differently positioned buccally and palatally. Unlike the buccal specimens, the palatal specimens were placed in the area of the palatal mucosa at a greater distance from the teeth and the dental plaque. Other studies, which did not include palatal or lingual specimens, have shown similar vitality patterns at different locations on the buccal side of the upper and the lower jaw [4]. Taken together, there might be differences in biofilm formation capability at different locations in the oral cavity. However, for bracket-like specimens placed buccally and palatally in the upper jaw, the biofilm formed within the initial period seemed to be similar regarding its general morphology.

To gain deeper insight into acidification properties of biofilms, an analysis of the biofilm pH was carried out with a pH-sensitive ratiometric dye, followed by CLSM and digital image analysis software based on the protocol by Schlafer and Dige [57]. This quantitative fluorescence microscopic examination enables the determination of vertical and horizontal pH gradients in microscopic images without changing the biofilm mechanically, for example, with microelectrodes [57]. The pH was analyzed in different layers of the biofilm, due to the fact that the distribution of nutrients and metabolites in the biofilm is not even [57]. The heterogeneous biofilm has diffusion-modifying properties that cause chemical/nutrient gradients, which lead to microenvironments within the biofilm. Thereby, niches with different pathogenic potentials, such as pH, redox, and nutrient availability can develop [10, 23].

In line with this, in both oral positions the lowest pH value could be detected at the bottom layer. Buccally as well as palatally, the pH dropped likewise from 7.0 to approximately 6.45. The exact value strongly depends on the incubation conditions and the biofilm maturation state. The biofilm in this study was comparable young and incubated only for 45 min and with 0.4% glucose as sole nutrient source. This is most probably the reason for the likewise small drop in pH. An increase in specimen exposure time in the oral cavity, incubation time in glucose solution or additional nutrients available would most probably result in different outcomes. An increased biofilm volume and metabolism would lead to an increase in acid metabolic products that in turn would cause lower pH values, reaching the critical pH value for tooth enamel demineralization [1, 56]. Even though the biofilms in both oral positions showed similar pH values at the bottom layer, statistically significant differences could be observed between the pH values in the top layers 3 and 4. The biofilm grown on palatally exposed specimens showed a lower pH value than that grown buccally, which indicates stronger acidification by the bacteria present. With regard to the aforementioned results, this could not be due to greater palatal biofilm volume or higher viability. Most probably, the buffering effect of saliva from the parotid gland was responsible for the lower acidification buccally [42]. The excretory duct of the parotid gland enters the oral cavity opposite the second upper molar [38] and, thus, in closer proximity to the buccally placed specimens than to the palatal ones. A repetition of this study in the lower jaw could confirm the influence of salivary flow due to the different anatomical conditions. The excretory ducts of the sublingual gland are located close to the incisors and could potentially create a buffering effect that could lead to lower pH values buccally compared to lingually. Other studies have shown that WSL more frequently occurred in the maxillary than in the mandible arch [32, 34]. Gorelick et al. observed that WSL did not occur even after prolonged use of mandibular canine-to-canine bonded retainer and suggested salivary flow as a possible cause [26].

Another reason to consider for the lower acidification detected on the buccal side could be a different bacterial composition between the biofilms of the two oral positions. A consequence of different bacterial composition could be different metabolic activities and products and, thus, to differences in acidification [56]. To address this in more detail, future approaches should quantify bacterial metabolic activity buccally and palatally and identify specific species composition by genomic sequencing.

Regarding the initial hypothesis, it could be shown that the initial oral biofilms form similarly on bracket-like specimens if exposed buccally or palatally in the upper jaw. At both oral positions, the lowest pH value could be detected at the bottom layer of the biofilm, accordingly at the bracket/tooth interface. However, differences were determined at higher levels of the biofilm, which might be due to the specific position in the oral cavity. Further studies should address whether these observations can be confirmed also in the lower jaw with different anatomic properties and, most importantly, over longer periods of time close to the duration of orthodontic treatment. Changing the duration of the specimens in the oral cavity would most likely also result in different, lower pH values that would reach the critical pH for enamel demineralization [1, 56]. Another possibility to study especially the biofilm pH-caused demineralization in more detail would be to use the splint design of this study with bovine enamel instead of hydroxyapatite discs. Here, demineralization could be examined directly using digital transverse microradiography (TMR), a highly appropriate method for validating mineral losses [14]. Furthermore, previous etching of enamel, which is routinely done to increase surface energy, surface area, and porosity for efficient bracket adhesion [6, 11], could influence biofilm accumulation and should be considered in further studies. For example, Knösel et al. have shown that surplus etching of the entire labial surface results in a higher likelihood of developing WSL [37]. Therefore, the design and setup of this study could serve as a blueprint for multiple further analyses to examine pH development at the tooth–bracket interface in more detail.

Regarding site specific effects of demineralization, within the limitations of this study, there is no evidence for an intrinsic reduced biofilm formation capability at the palatal position. Therefore, further factors should also be considered for the clinically observed reduced WSL prevalence in lingual orthodontic treatment. For example, due to the custom-made manufacturing process [68], lingual brackets have a different geometry and the advantage of fitting precisely on the tooth surface [66]. In addition, they have a large base, which almost covers the entire lingual/palatal surface, thereby creating an inherent seal [66]. The specific effect of bracket geometry could be analyzed in further studies.

In this study, a successful comparison between initial biofilm formation on palatally and buccally placed bracket-like material in the upper jaw with regard to acidification, biofilm volume, and live/dead distribution could be achieved. It could be shown that there were no differences in the general biofilm morphology regarding volume and viability. Interestingly, in biofilms from both positions a similarly decreased pH value at the deeper layers of the biofilm could be detected. Towards the top layers of the biofilms, the pH value increased at both positions. Only for palatally exposed specimens did the pH value slightly decrease from level 3 to level 4. Overall, a gradient from low pH values at the bottom layer to higher pH values at the top layer was discernible. Based on the results of this study, initial biofilm formation and acidification is similar on buccally and palatally placed bracket material in the upper jaw. As lingual brackets exhibit reduced WSL formation clinically, future studies should investigate further factors like bracket geometry.