Comparison of intraoral biofilm reduction on silver-coated and silver ion-implanted stainless steel bracket material

The formation of biofilm in the oral cavity is one of the major problems in dentistry [13, 27, 58]. Microorganisms trigger caries or periodontopathies, which represent the main conditions that require therapy in the field of dentistry [16, 37, 38]. In addition, it has been shown that periodontal disease is closely associated with systemic diseases, such as arteriosclerotic changes in the blood-conducting vessels [23, 41, 42, 54, 57, 65].

Daily thorough cleaning of the teeth (multiple times per day) can counteract the formation of biofilm but does not eliminate it completely [33]. Immediately after cleaning the teeth, a 0.1–1 µm-thick pellicle is formed from adsorbed proteins on the enamel [34]. Within a few hours, this pellicle enables the adherence of early bacterial colonizers (e. g. Streptococcus salivarius, Streptococcus oralis) [34]. The plaque continues to grow by accumulation of late colonizers (e. g. Aggregatibacter actinomycetemcomitans, Treponema denticola), thereby forming a three-dimensional structure. The components of a mature biofilm are 60–70 vol.% bacteria, embedded in a matrix of extracellular polysaccharides [34]. If a plaque has reached this stage, it can no longer be eliminated by self-cleaning effects in the oral cavity. The bacteria produce organic acids from supplied carbohydrates. A bacterial shift toward an acidic anaerobic environment occurs and the organic acids diffuse into the enamel, thereby releasing calcium and phosphate ions from the crystal lattice. These decalcifications of enamel are subsequently visible as “white spot lesions”. In the event of a prolonged acidic environment and progressive demineralization processes, the initial enamel caries lead to dentin caries [12, 34, 36].

Orthodontic appliances, which are used to treat up to 58% of children and adolescents in Germany [12], represent a particular problem regarding teeth cleaning. Due to their geometry, fixed braces in particular have various recesses that are difficult to clean. This impacts oral hygiene and reduces the self-cleaning effect of the teeth [14]. It can subsequently result in increased plaque accumulation, which can, in turn, cause gingivitis and increased probing depths [18, 20]. As mentioned above, mature plaque has a high acidogenic potential and increases the risk of enamel demineralization and the formation of caries in the area around the bracket [5, 60]. As part of an increasing awareness of health-related issues and for reasons of clinical necessity, preventive treatment concepts are gaining increasing significance [25].

Despite numerous and extensive prevention concepts (e. g. bracket ligating materials containing fluoride [47], enamel sealing in the immediate proximity of the bracket [4, 24, 32], polytetrafluorethylene-coated bracket surfaces [19, 29]), increased plaque accumulation and the subsequent appearance of “white spot lesions” can still be clinically proven [56, 64]. For this reason, additional approaches are required, which would ideally prevent the formation of the initial intraoral biofilm on brackets from the outset. This could be achieved by antibacterial properties of the orthodontic appliances themselves—an approach that is already being pursued in the field of dental implant research [17, 22]. For this purpose, silver is a material that is frequently examined for bracket appliances and implants in the field of dentistry. The precious metal exhibits an antibacterial effect through inhibition of enzymes that are involved in the respiratory chain, thereby disrupting the bacterial metabolism [53]. There are also studies that show that silver particles prevent the replication capability of the deoxyribonucleic acid (DNA) of microorganisms [26]. In vitro investigations of a physical vapor deposition (PVD) coated silver-platinum layer in combination with a subsequent heat treatment, a reduced biofilm adherence of Streptococcus mutans and Aggregatibacter actinomycetemcomitans with simultaneous good biocompatibility was observed [55]. Similar effects were also demonstrated for an alloy of silver and gold nanoparticles, a silver titanium dioxide coating and a silver layer enclosed by plasmapolymers [6, 7, 31, 67]. In addition, an animal study showed that silver-coated surfaces of orthodontic appliances have an antibacterial effect on Streptococcus mutans without additional oral hygiene and patient compliance, thereby reducing the risk of caries [48]. However, there is a decrease in the silver particles released as the orthodontic treatment duration with fixed appliances increases [48]. In addition, the low abrasion resistance of coatings applied on top of the surface poses a problem. The surface layer can be quickly worn down under the prevailing oral conditions [29]. The antibacterial effect of a coating only applied on top of the surface is therefore non-permanent as it loses its effect due to either partial delamination or surface abrasions.

A possibility for increasing the abrasion resistance of silver-modified surfaces is to use the plasma immersion ion implantation and deposition (PIIID) procedure. This technique is a vacuum process during which a metallic sacrificial cathode (here the silver target) is heated until it reaches red heat. By applying negative pulsed high-voltage potentials, silver ions are released from the cathode and accelerated in the direction towards the material (here the bracket material) submerged in a plasma. The high energy metal ions are implanted in the surface of the material [1, 45, 49]. According to manufacturer’s data, an additional silver top layer is built up on the surface. Through a heat treatment following the implantation process, diffusion of the silver ions into deeper lattice structures of the bracket material are achieved and the penetration depth is increased [49]. In theory, the PIIID procedure promises improved abrasion stability of the implanted silver ions compared to silver layers only applied on top of the surface. As the antibacterial effect of silver-implanted bracket material has not yet been described in the field of orthodontic appliances, the objective of this clinical study is the initial investigation of plaque accumulation on PIIID-modified surface. After being worn in the mouth for 48 h, the plaque accumulation was examined for untreated bracket material as well as PVD and galvanically silver-coated bracket material and compared with the antimicrobial effect of PIIID silver-implanted surface. It was analyzed if the null hypothesis, stating no statistical significant differences (p ≤ 0.05) between the four various groups, could be rejected.

The objective of this study was to determine whether implanting silver ions into the surface of stainless steel bracket material using the PIIID procedure has an antibacterial effect on accumulating oral plaque and how comparable this effect is to untreated bracket material, a PVD and galvanic silver coating. In the field of orthodontics, this is the first study to use the PIIID procedure—which is already used in other branches of the medical engineering industry [50].

In order to test the PIIID procedure under normal, periodontally healthy oral flora conditions, the test subjects underwent a periodontal screening examination in advance, which revealed no unusual values regarding API, SBI and PD. In addition, the test subjects did not present any other risk factors that could adjust the composition of the microbial flora, such as pregnancy or smoking [9, 15, 30].

The roughness of all surfaces was initially examined. All bracket samples were below a threshold value of Ra = 0.2 µm, where the formation of biofilm is not substantially influenced by the surface roughness [8, 52, 62]. The differences in biofilm formation can therefore be attributed solely to the type of layer. For the PIIID-modified samples, the calculated penetration depth of the implanted silver ions was theoretical analyzed, and, contrary to the data provided by the manufacturer, was only in the low nm range. Due to a discussion with the manufacturer one reason for this could be the surface structure of the bracket material, as the roughness prevents the vertical entry of the ion beam into the surface layer and therefore reduces the penetration depth measured perpendicular to the surface. As a result of the calculated low penetration depth of the silver ions, oversaturation with silver ions quickly occurs at the material surface and, as a consequence, silver deposits as an additionally top layer on the surface. In the event of persistent ion radiation, a µm-thick silver top layer forms without increased ion implantation occurring.

As documented in the literature there are position-dependent variations in biofilm coverage within the oral cavity [2, 3]. The fastening of the samples on the occlusal splints was randomized for each test subject with the result that each sample was ultimately fastened to each fixation point at least once. This made it possible to rule out an effect of the sample position. An experiment set-up with splints used to fasten the test specimens has already been used in various studies [3, 21, 29, 43]. The splint was used to protect the enamel, as removing brackets or test specimens fixed directly to the teeth can lead to enamel tears on the surface of the tooth [40, 46, 59]. In contrast to previous studies, shield-like plastic plates were also fitted to the occlusal splint in order to hold off the cheek and tongue [29]. The existing shear forces in the oral cavity could reduce the forming biofilm and therefore falsify the result. The circulation of saliva was not affected by an existing gap between the test specimens and the shield. The period of 48 h, for which the splints were worn, was evaluated in preliminary experiments. This period enables the individually formed initial biofilm to be analyzed in a reproducible manner and has already been selected as the examination period in many other studies [3, 10, 21, 29, 44].

The biofilm growth was analyzed by live/dead fluorescent staining and subsequent CLSM. This well-established method for quantifying initial biofilms enables biofilm morphology to be recorded in an almost native manner [28, 35, 51, 61]. In addition to bacterial cells, in the microscopic image of the stained biofilm, human cells, potentially gingival epithelial cells, can be detected on the samples surfaces. It has already been demonstrated that oral bacteria are able to colonize human gingival epithelial cells and thereby integrate them into the biofilm formation [63]. The quantification of the biofilms showed a significant reduction in plaque accumulation with regard to biofilm volume and surface coverage on all silver-modified surfaces compared to untreated bracket material. Silver has an antibacterial effect due to silver particles inducing destruction of the respiratory chain by inhibiting important enzymes [53]. In addition, they inhibit the DNA replication of the microorganisms [26]. No significant differences between the individual silver surface modifications were observed. In contrast to this, with regard to the live/dead distribution, the PIIID procedure was the only examined surface modification that showed a significant increase in dead bacteria compared to untreated bracket steel and the galvanic coating. This indicates that the implanted silver ions in stainless steel bracket material lead to an improved antimicrobial effect. Therefore, despite the low implantation depth of a few nm, the PIIID procedure presented a significant antibacterial effect.

The bacteria stained with fluorescent dyes show up in color when stimulated accordingly. Bacteria that fluorescent red indicate cells with a destroyed membrane whose nucleic acid has been stained by the penetration of the dye into the cell [11]. Future studies should aim to investigate whether the bacteria dyed red by fluorescent dye can nevertheless achieve growth under optimum oral conditions. If this is the case, it can be assumed that the surface exerts a purely bacteriostatic effect on the biofilm formation. However, if the bacteria cannot multiply even under optimum growth conditions, it can be assumed a bactericidal and therefore more effective action of PIIID silver-modified surfaces. An additional analysis of the composition of the bacteria could also shed light on whether the different silver surface layer modifications lead to a shift in bacterial diversity and potentially to a reduction in pathogenic bacteria species. In addition to the antibacterial effect demonstrated here, the potentially improved abrasion behavior due to the implantation of the silver ions is an advantage of the PIIID procedure. Testing the long-term abrasion resistance of PIIID-modified brackets would be an important future aspect. For this purpose, a splint design without the shield-like plastic plates could be used. Furthermore, higher penetration depths of the silver ions are desirable and could be achieved by expending more energy during the ion bombardment of the bracket material. As silver ions located deep in the material surface would have to have a higher abrasion resistance, a long-lasting antibacterial effect could potentially be expected.

A comparative analysis of the initial quantitative biofilm formation on bracket material that has been silver-modified in different ways was successfully performed in this study under in situ conditions. The PIIID procedure with silver ions, which was used for the first time in orthodontic materials has a demonstrable antibacterial effect, which is comparable to a PVD or galvanic silver coating with regard to reducing the biofilm volume and the percentage surface coverage. With regard to the live/dead distribution, however, the PIIID is the only surface modification with a significantly higher proportion of dead cells compared to the untreated control and the galvanic coating. It can therefore be concluded that the PIIID procedure has a positive effect with regard to a reduction in initial intraoral biofilm and the live/dead distribution of bacteria and that an investigation into long-term effectiveness and abrasion stability would be of great interest in future studies.