A common orthodontic bonding procedure includes etching the tooth surface, priming the tooth surface, applying a bracket with a bonding adhesive to the tooth surface, and curing the adhesive between the tooth and bracket. The etched surface provides increased surface area and hydrophilic properties; priming protects the etched enamel surface and enhances the bond with the adhesive ; and the bonding adhesive provides adequate physical strength between the bracket base and etched and primed enamel surface, resists displacement by oral function, and transfers requisite orthodontic force to the tooth . Previous in vitro studies have evaluated the relationship between orthodontic bonding and biofilm formation, which is important to prevent common orthodontic complications, such as enamel demineralization and gingival inflammation [3, 5, 6, 9, 12, 14]. However, most studies only used a single bacterial species, mainly S. mutans [9, 12, 14]. The single-species method cannot represent interactions of microorganisms associated with oral biofilms. In this study, a multi-species biofilm model was used under dynamic culture conditions to assess the effects of the orthodontic bonding procedure on biofilm formation and compositional changes in two main oral pathogens, S. mutans and P. gingivalis.
Bovine teeth were used to examine the effects of surface properties on biofilm formation in this study because they are the most widely used alternative for human teeth in dental research. They are easy to obtain in good condition and have a relatively large flat surface. Although the physicochemical characteristics of bovine teeth are not identical to those of human teeth , many studies have reported that there are no significant differences in micro-morphology, physical properties, and chemical composition [20, 21]. In addition, there is no significant difference in biofilm composition between human and bovine teeth .
SR and SW are two main surface properties that influence bacterial adhesion and biofilm formation [3, 4, 9,10,11,12,13]. A rough surface provides a favorable environment for bacterial adhesion and biofilm maturation, because a rough surface plays a protective role against shear force and increases the area available for biofilm formation [11, 12]. On the other hand, higher SW facilitates biofilm formation on dental materials [10, 13] due to its relation to surface free energy and hydrophilicity .
SW is measured by contact angle, which is formed when a droplet of a liquid is placed on a surface . Water is a common liquid to use for measurement of the contact angle because it has no chemical reaction with the underlying surface . We measured the water contact angle of all the specimens to determine the SW prior to starting biofilm experiments, because other probe liquids with different hydrophobicity may affect the surface properties of the underlying material, react with primer or adhesive components, and influence biofilm experiments.
This study demonstrated that surface treatment during orthodontic bonding significantly influences SR and water contact angle. There were significant differences in SR among the surface types (Table 3). The order of SR was AD < PR < BI < ET, which is partly consistent with the results of a previous study showing that etched hydroxyapatite surface is rougher and adhesive surface is smoother than those of other surfaces . Higher SRs of BI and ET than PR and AD might be due to the presence of grooves and ridges on bovine enamel and increased surface irregularities by acid etching , respectively (Fig. 1). Although the wrinkled surface of PR showed a smoother texture (Fig. 1c, g) than BI and ET, wrinkle structures may cause PR to be more irregular than AD, resulting in minor flaws (Fig. 1d, h).
There were also significant differences in water contact angle among the four surface types (Table 3). AD exhibited the greatest value followed by BI, PR, and ET (ET < PR < BI < AD). Because of the inverse relationship between water contact angle and SW , the order of SW may be AD < BI < PR < ET. These findings indicate that both SR and SW have the highest value in ET and the lowest value in AD.
This study demonstrated higher adhesion of S. mutans to BI and ET than to AD (Table 4), which could be explained by the higher SR and SW for BI and ET than for AD (Table 3). Various bacteria are involved in biofilm formation in the oral cavity, which begins with early colonizers, including streptococci and Actinomyces spp., followed by middle-colonizing Porphyromonas spp. and Fusobacteria spp., and late-colonizing Gram-negative anaerobes [1, 2]. Because S. mutans initially adheres to the underlying surface as an early colonizer, adhesion of S. mutans may be more significantly affected by surface properties. Previous microscopic examination of biofilms revealed that bacterial adhesion to the enamel surface starts from surface irregularities, such as grooves, perikymata, and cracks  (Fig. 1), because rough surfaces can act as a buffer against shear forces, which ease the change from reversible to irreversible bacterial attachment and increase the area available for initial bacterial adhesion. In addition, hydrophilic and wettable surfaces are known to collect more plaque by attracting specific bacteria [26, 27]. Since hydrophilic bacteria preferentially adhere to a hydrophilic surface , hydrophilic oral bacteria, such as S. mutans, easily adhere to the hydrophilic and wettable surface . In this regard, the rougher and wetter surfaces of BI and ET may provide more a favorable surface for adhesion of S. mutans than AD.
This study showed that P. gingivalis and total bacteria also showed greater adhesion to BI and ET than to AD (Table 4). After colonization by early colonizers, a combination of bacterial proliferation and recruitment leads to a bacterial mass increase during biofilm maturation . Therefore, successful adhesion of early colonizers such as S. mutans leads to sequential co-adhesion and proliferation of middle and late colonizers and results in increase and maturation of the biofilm, which may explain the similar adhesion tendency of P. gingivalis and total bacteria to that of S. mutans. This hypothesis is supported by the findings of this study demonstrating that SR was positively correlated and water contact angle was negatively correlated with adhesion of P. gingivalis and total bacteria as well as S. mutans (Table 5). Several studies have also demonstrated that SR has a positive correlation with bacterial adhesion and biofilm formation [3, 11, 12] and the significant effects of SW on biofilm formation are widely accepted [10, 13]. All these findings suggest that changes in SR and SW during orthodontic bonding procedures may significantly affect bacterial adhesion and biofilm composition.
Biofilm formation can be influenced not only by changes in SR and SW but also by other surface factors during orthodontic bonding procedures. Bacterial adhesion to ET was expected to be higher than to BI, because of its rougher and wetter properties. However, there was no significant difference in adhesion of any bacteria between BI and ET (Table 4). Cytotoxicity of the phosphoric acid used for acid etching may influence bacterial adhesion. A previous study demonstrated that 37% phosphoric acid has antimicrobial activity by increasing the concentration of hydrogen ions in the microorganism . During the experiment, the remaining phosphoric acid on the irregular surface of the bovine tooth may have influenced the bacterial viability. Although the rougher and wetter surface caused by acid etching could be favorable for bacterial adhesion, the cytotoxic action of phosphoric acid may offset the surface properties. In addition, a previous study reported that an SR over a certain level (over 0.35 μm) might not significantly influence biofilm formation . Although ET was rougher than BI, BI may be rough enough (average 1.61 μm of SR, Table 3) to demonstrate no difference in bacterial adhesion.
Bacterial adhesion to PR was different than that to other surfaces. Because PR was rougher and more wettable than AD, but smoother and less wettable than ET (Table 3), bacterial adhesion to PR was expected to be lower than that to ET and higher than that to AD. However, adhesion of the two oral pathogens and total bacteria to PR was not significantly different from that to ET or AD. This result may be due to chemical properties of the primer. The primer is present in a chemically unstable state in the oral environment because of its lower degree of conversion . In particular, bisphenol A-glycidyl methacrylate (bis-GMA), one of the main components of Transbond XT primer, has two opposing characteristics that influence bacterial adhesion and biofilm formation. One is to facilitate biofilm formation of S. mutans by increased adhesion capacity, enhanced glucan synthesis, and promotion of sugar transport activity . The other is a toxic effect on oral bacteria, such as inhibiting bacterial growth and decreasing cell viability . The leachable components of the primer with these opposing characteristics may differently influence bacterial adhesion to PR.
This study showed that adhesion of S. mutans and total bacteria significantly increased with extended incubation time (T1 < T2, Table 4). Since streptococci are facultative anaerobes, they can successfully adhere to the surface and continue to proliferate well in our aerobic culture condition, which led to subsequent maturation of biofilm and eventually resulted in an increase in total bacteria. In contrast to S. mutans and total bacteria, the amount of P. gingivalis significantly decreased from T1 to T2 (T1 > T2). P. gingivalis is a late colonizer and obligate anaerobe. P. gingivalis is sensitive to an oxidative aerobic environment, possibly hindering its growth. These results are consistent with a previous study that examined biofilm formation on orthodontic adhesive under similar culture conditions to our study .
This in vitro study showed the lowest bacterial adhesion to AD. In particular, the two main oral pathogens showed less adhesion to AD than to BI and ET. Considering that plaque accumulation and enamel demineralization mainly occur at the interface between tooth and adhesive in clinical practice , these findings indicate that when acid etching is wider than intended, covering the etched surface with adhesive may be helpful to reduce biofilm formation around orthodontic appliances. However, it is difficult to maintain a smooth adhesive surface and the remaining adhesive remnant around orthodontic appliances may be difficult to clean properly in the clinical situation. Therefore, clinicians should uniformly apply adhesive, carefully remove adhesive remnants, and perform periodic cleaning around orthodontic appliances to avoid enamel demineralization.
The present study has some limitations. This study showed that there was no significant difference in bacterial adhesion between BI and ET, even though ET had a rougher and more wettable surface than BI. However, the effects of acid etching on bacterial adhesion may be different between human and bovine teeth, because bovine teeth are more irregular and undulating than human teeth . In addition, the multi-species biofilm model used in this study does not simulate the actual oral environment. Further study using an in situ model is needed to evaluate the effects of orthodontic bonding procedures on biofilm formation and to find approaches to minimize the risk of pathologic side effects in orthodontic patients.