Antibacterial properties and abrasion-stability: Development of a novel silver-compound material for orthodontic bracket application

Dental plaque, which can be found on tooth surfaces in the oral cavity, is a three-dimensional, matrix-enclosed structure, comprising up to 70% bacteria of several hundred different species that interact with each other [23, 35, 44]. If this biofilm is not continuously removed by daily oral care, it promotes the development of severe oral diseases such as caries or periodontitis [14]. Bacteria in dental plaque take up carbohydrates from food and metabolize them to inorganic acids. These acids irreversibly dissolve components from the dental enamel, such as calcium and phosphate, resulting in a roughened tooth surface. The process additionally causes macroscopically visible decalcifications underneath the plaque, called white spot lesions (WSL). WSL are considered active initial caries lesions, which can progress to caries, the most common oral disease [4, 6, 61].

In Germany, up to 45% of children and 58% of young adults receive orthodontic treatment [12]. Multibracket appliances impede daily oral care and reduce the self-cleaning effect in the oral cavity, thus, providing ideal retention surfaces for bacteria [15, 20, 35, 45]. Roughness and the kind of material of the orthodontic brackets can also have an influence on bacterial colonization [50]. The prevalence of caries in 15 year olds was significantly greater compared to 12 year olds due to the eruption of permanent teeth and the closing of the dentition [12]. Furthermore, there is a correlation between the onset of puberty and neglecting oral hygiene [12]. Dental plaque accumulation frequently results in circular WSL around brackets, which significantly increase the frequency of caries [7, 60, 65, 68]. The incidence of WSL and cavitated lesions correlates with the duration of orthodontic treatment and the personal oral hygiene standards. Despite preventive oral hygiene, WSL in orthodontic treatments are still prevalent in up to 72.9% of patients [25, 57]. In addition, biofilm accumulation on orthodontic brackets increases the probability of supragingival plaque, which may lead to gingivitis or, in the worst case, even periodontitis with loss of surrounding bone structure [18, 49, 56]. These data underline the clinical necessity for effective dental plaque prevention in orthodontic treatment [25, 27, 57].

A potential approach for preventing bacterial biofilm accumulation could be the specific modification of the bracket surface—the initial attachment site for bacteria [17]. In this context, using silver has emerged as an interesting material modification as it shows strong intrinsic antibacterial properties and is already frequently used in various areas of dentistry [8, 26, 46‐48, 59, 69, 75]. Silver ions interfere with bacterial metabolism by impairing DNA replication, reducing certain protein activities and inhibiting important enzymes of the respiratory chain [28, 53]. In implantology, the antibacterial effect of silver is already applied in coatings composed of porous silica and silver nanoparticles [46], or silver nanocoatings on implants. Thereby, the bacterial growth in the surrounding tissue can be successfully inhibited, preventing peri-implantitis [8]. In recent years, orthodontic research also investigated materials with silver-based antibacterial effects [22]. Several studies focusing on coatings of conventional stainless steel bracket material with silver compounds have been conducted. The application of a silver–platinum electroplate coating exerted a strong antibacterial effect against the colonization with Streptococcus mutans and Aggregatibacter actinomycetemcomitans [59]. Antiadhesive and antibacterial properties were further demonstrated against S. mutans and Porphyromonas gingivalis on titanium oxide silver coatings [26]. This characteristic was also detected for nano silver–titanium dioxide surfaces [75]. In addition, silver nanocoatings were shown to prevent WSL independently of patient cooperation in an in silico study over 75 days [47].

However, all silver-modifications examined in these previous studies were surface material coatings. Orthodontic brackets are exposed to severe mechanical friction by daily tooth brushing and it was shown that pure material coatings, e.g., polytetrafluoroethylene (PTFE), are not able to withstand this abrasion [19, 31]. Since similar results can be expected for silver coatings, this issue has already been addressed by the authors in a previous study by means of silver implantation via plasma immersion ion implantation and deposition (PIIID) [48]. Although this study demonstrated bactericidal properties in addition to antibacterial effects, the calculated penetration depth of silver ions was only up to 9 nm, which is most likely too low to withstand long-term abrasion [48, 69]. Therefore, a potential abrasion-stable antibacterial material bracket material has not yet been developed.

To overcome these limitations a material that is comparably hard and stable, but also completely silver-penetrated is required. Such material would resist most abrasion but at the same time expose silver on its surface even if slightly abraded. A possibility for creating this material could be using a stable metallic framework, e.g., generated by sintering, with subsequent silver infiltration. As tungsten sintered pellets were available, this material was used for the investigation. The sintered metal exhibits high tensile strength and density [62, 76]. It has already been used as coils for transcatheter embolization of pathological blood vessels and for microwire neural electrodes implanted in animals. Furthermore, tungsten is frequently used in alloys for cardiovascular stents, orthopedic joint replacements and dental prosthetics [62, 74].

In this study, a promising, first-of-its-kind material for orthodontic brackets, which exploits a sintered porous tungsten matrix completely infiltrated with silver to ensure long-term stable antibacterial effects, was investigated. In order to analyze long-term stability, the novel material was exposed to mechanical friction and degradation using a toothbrush simulation machine. Before and after abrasion, the novel material was characterized using energy dispersive X‑ray (EDX) analysis as well as roughness and wall thickness quantification. The antibacterial effect was examined in situ by exposing the material on an occlusal splint intraorally for 48 h in 12 subjects. Biofilm volume and live/dead ratio was quantified using confocal laser-scanning microscopy (CLSM).

White spot lesions are one of the most common complications of modern orthodontic treatment [4, 6, 61]. They are caused by bacterial accumulation and acid production around the brackets [15, 20, 35, 45]. The aim of this study was to investigate the antibacterial effect of a novel silver-infiltrated material for orthodontic brackets. In contrast to previous studies, a specific focus was additionally laid on its abrasion stability.

For fabrication, silver was vacuum-infiltrated into a sintered porous tungsten matrix. To the best of our knowledge, this is the first study to describe the combination of sintering and vacuum-infiltration for the generation of silver-modified materials. The success of the manufacturing process was examined by EDX scanning electron microscopy analysis revealing that the sintering process yielded a tungsten matrix completely interspersed with pores. In these pores of different volumes, silver was infiltrated under vacuum-melting. The results did not yield the expected silver content of 15 und 20%, but were comparably lower. If higher silver proportions would be necessary in the compound material, the infiltration process would need to be adjusted in further experiments. However, even this lower concentrated silver was distributed in all three dimensions throughout the matrix as confirmed by cross-section analyses. This differs from previous silver-modified bracket materials coated or penetrated only to a low depth. In prior modifications, such as silver-platinum electroplate coatings, titanium oxide silver coatings, nano silver–titanium dioxide surfaces, silver nanocoatings and PTFE coatings, silver was only located on the surface [26, 31, 47, 59, 75]. In a PIIID-modified bracket material, the calculated penetration depth of silver ions was also only up to 9 nm [48]. The new material developed in this study provided a clearly improved silver distribution within the material and, thus, fulfilled the prerequisites for an abrasion-stable antibacterial bracket material. Tungsten is already applied in neuroscience, vascular medicine, radiology, orthopedics and prosthetics due to high density, radiopacity, tensile strength and yield strength. When implanted, it has no long-term resistance to corrosion, which is accelerated by the immune system [62, 74]. The mechanisms of the corrosion process have not yet been thoroughly researched [62, 76]. However, in studies with nanoparticle coatings of tungsten disulfide on orthodontic wires, low attrition and reduced friction have been shown [32, 55]. In addition, cytotoxicity analysis of tungsten nanoparticles indicated biocompatibility. For the use as bracket material, the use of tungsten is considered innocuous, as the new material is not implanted but only bonded on the surface of the teeth for a limited period of time [52].

To test the abrasion stability of the new material, a self-constructed tooth brushing simulation machine was used. The set-up simulates realistic tooth brushing movements and flow of saliva and toothpaste. The brushing time was calculated to resemble 2 years of tooth brushing, since treatment with fixed orthodontic appliances usually takes about 1.5–2 years [52]. Thus, the experimental abrasion in this study reflects the natural exposure of bracket materials. The saliva-like mixture was produced from mucin, a glycosylated protein that is a major component of human saliva [38]. In contrast to collecting native saliva from volunteers, this allows for more reproducible experimental conditions and a larger liquid volume. The set-up furthermore applies the recommendations for daily oral care, to be specific, using an electric toothbrush and toothpaste. Electric toothbrushes with an oscillating-rotating motion significantly reduce the incidence of gingivitis compared to manual toothbrushes [33]. Fluoride toothpastes are recommended for their acceptability and for caries prevention [33]. By means of a micrometer, the material thickness was quantified before and after abrasion. The toothbrush simulation machine reduced the wall thickness of the specimens by 30–50 µm only. This reduction was equal between the newly developed material and conventional stainless steel bracket material. As the latter is known for its high abrasion-stability, the new material confirmed to be abrasion-stable as well.

In order to create equal baseline conditions for biofilm analyses, the roughness of each specimen was analyzed. Material roughness has a major influence on bacterial adhesion with increasing values promoting bacterial colonization due to more available surface [64]. There were no significant differences in roughness within and between the groups. The roughness of the materials did not change due to toothbrushing, verifying their abrasion-stability. In addition, a major influence of roughness of either specimen on bacterial adhesion can be excluded. Therefore, in the following in situ examination, the antibacterial effect should be primarily related to the different material compositions.

The antibacterial properties of the novel silver-modified material were tested in situ by participants wearing occlusal splints equipped with the test specimens. To confirm healthy oral conditions, the participants underwent a periodontal screening prior to this study, whereby SBI did not exceed 10%, the PD did not extend beyond 3 mm and the API was less than 25% [14, 70, 72]. Thus, based on the data collected, healthy oral condition of the test subjects could be verified. In addition, further risk factors were excluded that could alter the composition of the microbial flora, such as general diseases, antibiotic use, pregnancy, alcohol consumption and smoking [2, 10, 12, 16, 30, 39, 54, 73]. The splint design has already been used in previous studies of fix specimens [5, 21, 31, 43, 48]. The test specimens are not fixed directly to the enamel, which could lead to enamel cracks when being removed [1, 41]. In this experimental set-up, the splint design of Meyer-Kobbe et al. [48] was used, including laterally attached shield-like plastic plates to prevent the cheek and tongue from contacting the specimens. This prevents the biofilm formation on the specimens from being affected by shear forces resulting in reduced biofilm accumulation. In addition, saliva can circulate unimpededly through the gap between the plastic plates and the specimens [48]. Precluding the possibility of position-dependent biofilm coverage [3, 5], the specimens were fixed in a random order on the individual occlusal splints. Each material was attached to each position on the splint at least once. Thus, position-specific effects could be excluded. The wearing time was set to 48 h, since formation of individual initial biofilm usually occurs within this period of time and this wearing time has already been successfully used in previous studies [3, 5, 43, 48].

For the analysis of the biofilm, an established CLSM method for the quantification of initial biofilm formation was employed. This allows a nearly native detection of biofilm morphology and simultaneous examination of live/dead distribution [22, 29, 51, 66]. In addition to bacterial cells, human cells, presumably gingival epithelial cells, appeared on the surfaces of the stained biofilms. Human gingival epithelial cells can be colonized by oral bacteria and be integrated into the biofilm [67]. This phenomenon has already been observed in other studies, where the human cells were likewise regarded as components of the biofilm [48].

The quantification of biofilms revealed a significant reduction in plaque accumulation in terms of biofilm volume on all silver-infiltrated material surfaces compared to conventional stainless steel bracket material. As an influence of the material’s roughness was excluded, this effect can be attributed to the silver content of the novel material. Interaction between silver and components of the bacterial membrane leads to membrane damage and disruption of intracellular metabolic activity. Protein activity is reduced and bacterial DNA loses its ability to replicate. Consequently, important enzymes of the respiratory chain are inhibited and cell death ensues [28, 40, 53]. The biofilm-reducing effect has already been described using silver coatings such as silver–platinum electroplate coating, titanium oxide–silver coatings, nano silver–titanium dioxide surfaces and silver nanocoatings [26, 47, 59, 75]. Besides their strong antibacterial effect, it should be taken into account that using nano silver-coated orthodontic brackets in an animal model resulted in elevated nano silver levels in saliva and serum [47]. Nano silver is also claimed to induce silver resistance in pathogenic germs as it is used more and more frequently in consumer goods [13]. The exact mechanism as well as the cytotoxic concentration are still under investigation and seem to depend on the specific size of the applied silver [13]. Thus, it might be that ionic silver, which should be responsible for the antibacterial effect observed in this study, is less toxic than nano silver coatings. However, before applying the material developed in this study for orthodontic treatment, toxicity analysis for this specific setup is inevitable.

In the present study, the biofilm volume was reduced by 60% to almost 80% on the newly developed material. This is lower than in a recent study, where a PIIID silver-modified surface, an electroplated silver layer and a physical vapor deposition (PVD) silver coating led to a biofilm reduction of up to 95% [48]. Most probably, this is due to the different types of material modification: Whereas the recent study led to almost continuous silver layers on the surfaces, the material analyzed here was a compound of silver and tungsten. Interestingly, the low Ag and high Ag group of the novel material did not differ in the antibacterial effect. It has already been shown that even low concentrations of silver (5%) are sufficient for effective biofilm prevention [36]. Therefore, for the silver concentrations used in this study (7–15%) the saturation level may have been reached with no further effect with increasing content. With regard for a prospective clinical application as bracket material, this would require lower amounts of silver for the fabrication process. Future studies could evaluate whether even lower concentrations may maintain the same antibacterial effect. In addition, the silver concentration in the novel material will be distinctly lower than in an already accredited product for tooth conservation with silver content of approximately 50% (Ketac silver Aplicap, 3M Deutschland, Neuss, Germany). These are promising preconditions for a potential medical device approval of the novel material [58].

Importantly, there was also no difference when comparing the initial biofilm formation on untreated and abraded specimens. In both cases, the biofilm volume was significantly lower compared to the control material. Thus, the antibacterial properties of the novel material were maintained after simulating 2 years of mechanical tooth brushing abrasion. Most probably, this is due to the abrasion-stability and successful continuous silver-infiltration throughout the material [27, 52]. Dental surfaces are exposed to high shear forces in the mouth. Surface coatings used in previous studies, such as PTFE coatings, have been demonstrated to become partially abraded and rubbed off upon exposure [19]. Long-term stability could, thus, not be guaranteed. To the best of our knowledge, the antibacterial effect of the new material of this study is the first that demonstrated long-term stability over the period of a conventional orthodontic treatment.

To analyze for an additional antibacterial effect of the novel material, as detected for the previously analyzed PIIID silver-modified surfaces [48], the biofilm live/dead ratio was determined. For this purpose, a staining with two fluorescent dyes with different membrane permeability was applied. All bacteria are stained green, but only those with impaired membrane—defined as dead—take up the red dye, which ideally replaces the green dye [11, 37]. An almost unchanged 50/50 distribution of living and dead cells was detected in all groups. Accordingly, the novel material does not exhibit an additional bactericidal effect. These results may be biased by the human cells. The mucosal cells on the specimens, due to possessing different membrane properties than bacteria, also absorb the red dye of the live/dead staining even when they are still alive. As they are several folds greater than bacterial cells, they will account for the major proportion of red staining. Therefore, only a major bactericidal effect would have been detectable. In fact, in the previous study by Meyer-Kobbe et al. [48] the bactericidal effect was detected in a similar experimental set-up. To examine the properties of the novel silver-infiltrated material in more detail, future studies should apply further methods to analyze the bacterial composition and molecular pathways of the antibacterial effect.

In this study, a quantitative analysis of the initial biofilm formation on a novel silver-infiltrated material was successfully performed under in situ conditions. The material exploits for the first time the combination of sintering and silver vacuum-infiltration to ensure a long-term antibacterial effect as required for bracket materials in orthodontics. A significant antibacterial effect was demonstrated, which was maintained after prolonged abrasion using a tooth brushing simulation machine due to its high stability. In further studies, consideration must be given to the clinical implementation of this novel material for orthodontic brackets regarding production and specific application. The fabrication for this study was a pilot process and will require more research on economic feasibility and up-scaling for translation to orthodontic application. Furthermore, it needs to be considered whether the current framework metal tungsten should be substituted in favor of the established orthodontic stainless steel. Nevertheless, the novel material of this study exhibits most promising characteristics to significantly reduce intraoral biofilm formation on orthodontic brackets. It could contribute to fewer WSL in patients receiving orthodontic treatment and reduce the risk of subsequent diseases, such as caries and periodontitis.