Summary

Introduction:

Silver nanoparticles are currently utilized in the fields of dentistry. The aim of this study was to evaluate the antibacterial properties and ion release of nanosilver coated orthodontic brackets compared to conventional brackets.

Methods:

Nanosilver coating process was applied to standard orthodontic brackets placed on the mandibular incisors of Wistar Albino rats in the study group and conventional brackets in the control group. Dental plaque, mucosal vestibular smears, saliva, and blood samples were collected from rats at various days. The amounts of nanosilver ions in blood and saliva were measured and microbiological evaluation was made for Streptococcus mutans. For testing cariogenicity, all rats were sacrificed at the end of 75 days under anaesthesia. Teeth were stained using a caries indicator, then the caries ratio was assessed.

Results:

Nanosilver coated orthodontic bracket favoured the inhibition of S.mutans on Day 30 and reduction of caries on the smooth surfaces. The nanosilver amounts in the saliva and serum samples were significantly higher in the study group on Day 7.

Conclusion:

It is suggested that nanosilver coated orthodontic brackets, as an antibacterial agent without patient compliance, could be helpful for the prevention of white spot lesions during fixed orthodontic treatment.

Introduction

The purpose of orthodontic treatment with fixed appliances is to improve function and aesthetics. After the placement of fixed orthodontic appliances, patients are at higher risk for caries due to increased plaque and bacteria retention around brackets and bands. Orthodontic bands and brackets make it difficult for patients to achieve proper brushing and oral hygiene maintenance, which increases caries incidence (1–3). Streptococcus mutans and Lactobacilli are the most common bacteria that can produce significant levels of lactic acid causing demineralization of teeth. Demineralization of the enamel results in a displeasing appearance, which is called white spot lesions (WSL) (1, 4). WSL can become noticeable around the brackets within 1 month of bonding and the prevalence of WSL has been reported to be 38% in 6 months, 46% in 12 months after the initiation of the treatment, and 50% at the end of the fixed orthodontic treatment (2, 5, 6).

Fluoride is known as the most appropriate agent that prevents the formation of WSL during orthodontic treatment. In order to prevent WSL, fluoride is applied regularly in orthodontic patients in many different ways including topical fluoride preparations, varnish, gel, and solution implementation around brackets. In addition, when orthodontic patients use a fluoride mouth rinse and tooth brushing with fluoride toothpaste, WSL has been shown to reduce around fixed braces and bands; however, this application is dependent on patient cooperation. Unfortunately, only less than 15% of orthodontic patients follow instructions (7–10). However, there is still a need to prevent WSL by acid-resistant anti-caries agents needing no patient cooperation during orthodontic treatment.

The antimicrobial effects of silver ion or salts are known well since ancient times (11). Silver is currently used to control bacterial growth in a variety of applications, including dental work, catheters, and burn wound dressings (12–15). The term ‘nanotechnology’ was introduced by Professor Norio Taniguchi of Tokyo Science University in 1974 and the word ‘nano’ is used to indicate one billionth of a meter. Silver nanoparticles with their unique chemical and physical properties have been proved as an alternative for the development of new antibacterial agents (12, 16).

The purpose of this study was to assess the antibacterial properties and ion release of nanosilver coated orthodontic brackets, to compare the effects of silver regarding the amount of cariogenic streptococci adhesion, and to provide preliminary information for the prevention of WSL in comparison with conventional orthodontic brackets. The hypothesis tested was that silver nanoparticles coated to orthodontic bracket would decrease the adherence of S.mutans and caries during fixed orthodontic treatment.

Materials and methods

All animal procedures were conducted with the approval of Gazi University Animal Experiments Local Ethics Committee. All animal experiments were performed by one researcher who has a certificate according to the guidelines for proper conduct of animal experiments. Microbiological analyses were performed in Gazi University, Faculty of Dentistry, Department of Medical Microbiology.

Coating procedure

Mandibular incisor orthodontic brackets (Gemini Roth; 3M Unitek, Monrovia, California, USA) were used in this study. All brackets were cleaned sonically with alcohol for 15 minutes. The coating of the brackets was made via physical vapour deposition (Midas Thermal Evaporator, Vaksis, Ankara, Turkey). Brackets were fixated onto the substrate of the e-beam evaporator device using double-sided bonding tapes. Electron beam evaporation method was performed under <2×10−6 Torr vacuum pressure with oil-free pumping for 8 hours and brackets were coated to 1 μm thickness by nanosilver particles.

Rat caries experiments

Twelve male Wistar rats (4 months old) were used in this study. The animals were divided randomly into two groups of six rats each. All rats were dosed by gavage with amoxicillin (25mg/kg of rat) for two consecutive days (on Days −4 and −3) in order to enable the inoculated organisms to establish themselves in the oral cavity to suppress the indigenous flora of the rats. After 2 days, all rats were started to be fed with soft biscuit and water free of antibiotics for inoculation of S.mutans (on Days −2 and −1).

On Day 0, nanosilver coated brackets in the study group and conventional brackets in the control group were bonded using a fluorine-free light-cured composite resin and adhesive (Transbond XT, 3M Unitek) in accordance with the manufacturer’s instructions on the vestibular surface of the mandibular incisors as close as possible to the gum. In addition to bonding the bracket, a ligature wire was used to ligate the bracket to the teeth in order to prevent the loss of the bracket.

Immediately after the bonding process, the mouth of the rat was infected with 0.1ml of a cell suspension containing 6×108 cells of S.mutans ATCC#700610 serotype c (American Type Culture Collection) using a micropipette. Additionally, 1ml of a cell suspension was mixed into the drinking water of the rats in each cage and this mixed drinking water was accessible to the rats until the next morning. Then, this water mix was removed and replaced with regular tap water. During the test period, the animals were kept in plastic cages with free access to food (soft biscuits) and water ad libitum. The rats were kept under an artificial 12-hour light/darkness cycle. Lights were turned on at 7 AM and off at 7 PM. Room temperature varied between 22°C and 24°C, and appropriate room ventilation was maintained.

Collection of samples

All animals were anesthetized with 10mg/kg xylazine hydrochloride and 90mg/kg ketamine hydrochloride by intraperitoneal injection before all mentioned procedure. Saliva, blood, vestibular smear from the lower lip epithelium, and the plaque sample of the incisor teeth were collected from all rats on Day 0 (before inoculation of S.mutans cell suspension), and on Days 1, 3, 7, 14, 30, 45, and 75 (after inoculation).

Saliva was collected by placing a filter paper strip (Periopaper, Oraflow, Smithtown, New York, USA) under the tongue and the volume was measured by means of a precision balance (aeADAM, Kinston, UK). Samples were put into glass capped tubes containing 0.2ml concentrated nitric acid. Blood samples were collected from the tail vein and serum samples were obtained from coagulated and centrifuged blood, which were then stored at −20°C.

Silver analysis was performed by direct injection into the inductively coupled plasma-mass spectrometer (ICP-MS) for saliva and serum samples. The total silver concentration (ppb) was determined for each sample.

Vestibular smear was collected by swabs on sterile coverslips and S.mutans was counted microscopically using the Gram-staining method. Then, the number of S. mutans was counted at randomly chosen 20 different microscopic areas, and it was calculated according to arithmetic mean numbers.

The plaque sample of the incisor teeth was collected by dental floss. Dental floss was put into a sterilized test tube containing 1ml Trypticase Soy Broth medium (TSB Merck, Germany), the test tubes were vortexed for 60 seconds and the suspensions were diluted to 10–1, 10–2, and 10–3 serially. Then, 100 µl amounts of samples were put onto tryptone-yeast extract-cysteine-sucrose-bacitracin agar (TYCSB) for 48–72 hours in a 5% CO2 incubator to culture S.mutans. All plaque samples were investigated for bacterial colonization and the total numbers of viable S.mutans colonies were calculated as colony forming units (CFU). Therefore, the data analysis and statistics were performed according to the 1ml amount of volume as CFU/ml.

All animals were sacrificed on Day 75 by overdose anaesthetic solution. The left jaws were removed and the soft tissue was stripped off. The left jaws were stored in plastic flasks containing 10% formaldehyde solution.

Caries assessment

Caries evaluation was performed on smooth and occlusal surfaces of the maxillary molars and the sulcus of the mandibular molar teeth on the left side of each animal. To evaluate the sulcal caries, mandibular molar teeth were cut off through the sagittal plane. Before the pictures of the samples were taken using a digital camera, all sample teeth were stained using caries indicator-Sable Seek (Ultradent Products, Utah, USA) (Figure 1). The images were loaded in ArcGIS 10.2 for Desktop (ESRI, California, USA). The areas of caries were determined according to the extent of staining with Sable Seek (Ultradent Products) and the rates of caries (CR) were calculated for each surface (CR = Caries area/Tooth area).

Figure 1.

Rats teeth decay. (a) occlusal, (b) smooth surface, and (c) sulcal.

Figure 1.

Rats teeth decay. (a) occlusal, (b) smooth surface, and (c) sulcal.

Statistical analysis

The statistical significance was determined using SPSS 20.0 software for Windows (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk test was used to verify the normality of the data distribution. Differences between the study and the control groups were tested by the Mann–Whitney U-test and intra-group differences were tested by the Wilcoxon test. Values were considered statistically significant at P < 0.05.

Results

In the present study, with a one-sided significance level of 0.05 and a power of 75%, a minimum of six animals per group were included.

After the placement of the brackets, the animals did not display any behavioural or weight changes, and no mortality was observed. During the experiment, brackets were checked every day and, if dropped, a new bracket was bonded immediately, which occurred 17 and 15 times for the study and the control groups, respectively.

Saliva samples

There were not any significant differences between the study and the control groups (Figure 2), whereas on Day 7, the salivary concentration of nanosilver was found significantly higher than the other days in the study group (Table 1). Intra-group examination for the control group showed that there were no significant differences between salivary concentrations of nanosilver on the other days.

Figure 2.

Silver saliva level in the study and the control groups.

Figure 2.

Silver saliva level in the study and the control groups.

Table 1.

Statistical comparison of saliva silver level in the study group.

Days Saliva silver level (ppb) Wilcoxon test 
n Mean Median Min. Max. SD z P 
0.03 0.02 0.00 0.08 0.03 −0.631 0.528 
0.04 0.03 0.00 0.11 0.04 
0.03 0.02 0.00 0.08 0.03 −0.946 0.344 
0.05 0.04 0.00 0.13 0.05 
0.03 0.02 0.00 0.08 0.03 −2.201 0.028* 
0.35 0.08 0.03 1.75 0.68 
0.03 0.02 0.00 0.08 0.03 −0.135 0.892 
14 0.08 0.00 0.00 0.46 0.18 
0.03 0.02 0.00 0.08 0.03 −0.105 0.917 
30 0.03 0.01 0.00 0.09 0.04 
0.03 0.02 0.00 0.08 0.03 −1.782 0.750 
75 0.07 0.07 0.02 0.12 0.04 
Days Saliva silver level (ppb) Wilcoxon test 
n Mean Median Min. Max. SD z P 
0.03 0.02 0.00 0.08 0.03 −0.631 0.528 
0.04 0.03 0.00 0.11 0.04 
0.03 0.02 0.00 0.08 0.03 −0.946 0.344 
0.05 0.04 0.00 0.13 0.05 
0.03 0.02 0.00 0.08 0.03 −2.201 0.028* 
0.35 0.08 0.03 1.75 0.68 
0.03 0.02 0.00 0.08 0.03 −0.135 0.892 
14 0.08 0.00 0.00 0.46 0.18 
0.03 0.02 0.00 0.08 0.03 −0.105 0.917 
30 0.03 0.01 0.00 0.09 0.04 
0.03 0.02 0.00 0.08 0.03 −1.782 0.750 
75 0.07 0.07 0.02 0.12 0.04 

*P < 0.05.

Serum samples

There were no meaningful differences between the study and the control groups (Figure 3). On Day 7, the serum concentration of nanosilver was found significantly higher than the other days in the study group (Table 2). Intra-group examination for the control group showed that there were no differences between the serum concentrations of nanosilver on the other days.

Figure 3.

Silver serum level in the study and the control groups.

Figure 3.

Silver serum level in the study and the control groups.

Table 2.

Statistical comparison of serum silver level in the study group.

Days Serum silver level (ppb) Wilcoxon test 
n Mean Median Min. Max. SD z P 
0.04 0.04 0.03 0.05 0.01 −0.314 0.753 
0.04 0.03 0.01 0.07 0.02 
0.04 0.04 0.03 0.05 0.01 −1.753 0.080 
0.13 0.08 0.03 0.29 0.11 
0.04 0.04 0.03 0.05 0.01 −2.201 0.028* 
0.16 0.07 0.05 0.39 0.15 
0.04 0.04 0.03 0.05 0.01 −1.572 0.116 
14 0.20 0.14 0.02 0.49 0.19 
0.04 0.04 0.03 0.05 0.01 0.314 0.753 
30 0.05 0.03 0.02 0.14 0.05 
0.04 0.04 0.03 0.05 0.01 −1.367 0.172 
75 0.02 0.01 0.00 0.08 0.03 
Days Serum silver level (ppb) Wilcoxon test 
n Mean Median Min. Max. SD z P 
0.04 0.04 0.03 0.05 0.01 −0.314 0.753 
0.04 0.03 0.01 0.07 0.02 
0.04 0.04 0.03 0.05 0.01 −1.753 0.080 
0.13 0.08 0.03 0.29 0.11 
0.04 0.04 0.03 0.05 0.01 −2.201 0.028* 
0.16 0.07 0.05 0.39 0.15 
0.04 0.04 0.03 0.05 0.01 −1.572 0.116 
14 0.20 0.14 0.02 0.49 0.19 
0.04 0.04 0.03 0.05 0.01 0.314 0.753 
30 0.05 0.03 0.02 0.14 0.05 
0.04 0.04 0.03 0.05 0.01 −1.367 0.172 
75 0.02 0.01 0.00 0.08 0.03 

*P < 0.05.

Plaque samples of incisor teeth

The nanosilver coated bracket group exhibited significantly lower S.mutans counts compared to the control group on Day 30 (Table 3). In the study group, the number of S.mutans was found to be smaller on Days 3, 7, 14, 30, and 45 than on Day 0 (Table 4). There were no significant differences in the control group between the numbers of S.mutans counted on different days.

Table 3.

Statistical comparison of groups for Streptococcus mutans counts on the plaque sample of the incisor.

Days Groups S. mutans counts on the plaque sample of the incisor (CFU/ml) Mann–Whitney U-test 
n Mean Median Min. Max. SD Mean rank U P 
Study 226.67 160.00 0.00 640.00 238.22 6.92 15.5 0.699 
Control 246.67 80.00 0.00 880.00 351.83 6.08 
Total 12 236.67 140.00 0.00 880.00 286.65  
Study 53.33 0.00 0.00 320.00 130.64 7.00 15.0 0.699 
Control 0.00 0.00 0.00 0.00 0.00 6.00 
Total 12 26.67 0.00 0.00 320.00 92.38  
Study 0.00 0.00 0.00 0.00 0.00 5.50 12.0 0.394 
Control 86.67 0.00 0.00 480.00 193.36 7.50 
Total 12 43.33 0.00 0.00 480.00 137.99  
Study 0.00 0.00 0.00 0.00 0.00 6.50 18.0 1.000 
Control 0.00 0.00 0.00 0.00 0.00 6.50 
Total 12 0.00 0.00 0.00 0.00 0.00  
14 Study 0.00 0.00 0.00 0.00 0.00 5.50 12.0 0.394 
Control 30666.67 0.00 0.00 96000.00 47575.90 7.50 
Total 12 15333.33 0.00 0.00 96000.00 35851.55  
30 Study 0.00 0.00 0.00 0.00 0.00 4.00 3.00 0.015* 
Control 553.33 580.00 0.00 920.00 381.51 9.00 
Total 12 276.67 0.00 0.00 920.00 386.86  
45 Study 0.00 0.00 0.00 0.00 0.00 4.50 6.00 0.065 
Control 586.67 160.00 0.00 2600.00 1010.87 8.50 
Total 12 293.33 0.00 0.00 2600.00 747.23  
75 Study 1586.67 960.00 0.00 5360.00 2113.22 6.42 17.5 0.937 
Control 3440.00 700.00 0.00 14240.00 5634.47 6.58 
Total 12 2513.33 700.00 0.00 14240.00 4170.99  
Days Groups S. mutans counts on the plaque sample of the incisor (CFU/ml) Mann–Whitney U-test 
n Mean Median Min. Max. SD Mean rank U P 
Study 226.67 160.00 0.00 640.00 238.22 6.92 15.5 0.699 
Control 246.67 80.00 0.00 880.00 351.83 6.08 
Total 12 236.67 140.00 0.00 880.00 286.65  
Study 53.33 0.00 0.00 320.00 130.64 7.00 15.0 0.699 
Control 0.00 0.00 0.00 0.00 0.00 6.00 
Total 12 26.67 0.00 0.00 320.00 92.38  
Study 0.00 0.00 0.00 0.00 0.00 5.50 12.0 0.394 
Control 86.67 0.00 0.00 480.00 193.36 7.50 
Total 12 43.33 0.00 0.00 480.00 137.99  
Study 0.00 0.00 0.00 0.00 0.00 6.50 18.0 1.000 
Control 0.00 0.00 0.00 0.00 0.00 6.50 
Total 12 0.00 0.00 0.00 0.00 0.00  
14 Study 0.00 0.00 0.00 0.00 0.00 5.50 12.0 0.394 
Control 30666.67 0.00 0.00 96000.00 47575.90 7.50 
Total 12 15333.33 0.00 0.00 96000.00 35851.55  
30 Study 0.00 0.00 0.00 0.00 0.00 4.00 3.00 0.015* 
Control 553.33 580.00 0.00 920.00 381.51 9.00 
Total 12 276.67 0.00 0.00 920.00 386.86  
45 Study 0.00 0.00 0.00 0.00 0.00 4.50 6.00 0.065 
Control 586.67 160.00 0.00 2600.00 1010.87 8.50 
Total 12 293.33 0.00 0.00 2600.00 747.23  
75 Study 1586.67 960.00 0.00 5360.00 2113.22 6.42 17.5 0.937 
Control 3440.00 700.00 0.00 14240.00 5634.47 6.58 
Total 12 2513.33 700.00 0.00 14240.00 4170.99  

*P < 0.05.

Table 4.

Statistical comparison of Streptococcus mutans counts in the study group.

Days S. mutans counts on the plaque sample of the incisor (CFU/ml) Wilcoxon test 
n Mean Median Min. Max. SD z P 
226.67 160.00 0.00 640.00 238.22 −1.367 0.172 
53.33 0.00 0.00 320.00 130.64 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
14 0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
30 0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
45 0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −0.946 0.344 
75 1586.67 960.00 0.00 5360.00 2113.22 
Days S. mutans counts on the plaque sample of the incisor (CFU/ml) Wilcoxon test 
n Mean Median Min. Max. SD z P 
226.67 160.00 0.00 640.00 238.22 −1.367 0.172 
53.33 0.00 0.00 320.00 130.64 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
14 0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
30 0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −2.032 0.042* 
45 0.00 0.00 0.00 0.00 0.00 
226.67 160.00 0.00 640.00 238.22 −0.946 0.344 
75 1586.67 960.00 0.00 5360.00 2113.22 

*P < 0.05.

Vestibular smear

The number of S.mutans in the vestibular smear did not differ significantly between the study and the control groups. In the study group, the number of S.mutans was found to be greater on Day 3 than on Day 0. In the control group, the number of S.mutans was greater on Day 3, 7, and 30 than on Day 0 (Figure 4).

Figure 4.

Counts of vestibular Streptococcus mutans in the study and the control groups.

Figure 4.

Counts of vestibular Streptococcus mutans in the study and the control groups.

Caries assessment

The rates of teeth with caries in the mandibular and maxillary molars are shown in Table 5. Significant differences were observed between groups only on the smooth surface.

Table 5.

Statistical comparison of groups for caries ratio.

Caries surface Groups Caries ratio Mann–Whitney U-test 
n Mean Median Min. Max. SD Mean rank U P 
Occlusal Study 4.31 4.39 3.41 5.12 0.60 5.17 10.00 0.240 
Control 6.05 7.06 1.68 8.83 2.66 7.83 
Total 12 5.18 4.60 1.68 8.83 2.05  
Smooth Study 1.46 1.40 0.29 3.37 1.11 4.42 5.50 0.041* 
Control 3.83 3.25 1.23 9.12 2.82 8.58 
Total 12 2.65 1.80 0.29 9.12 2.39  
Sulcal Study 1.82 1.47 0.05 5.34 1.92 6.83 16.00 0.818 
Control 1.28 1.42 0.79 1.59 0.35 6.17 
Total 12 1.55 1.42 0.05 5.34 1.35  
Caries surface Groups Caries ratio Mann–Whitney U-test 
n Mean Median Min. Max. SD Mean rank U P 
Occlusal Study 4.31 4.39 3.41 5.12 0.60 5.17 10.00 0.240 
Control 6.05 7.06 1.68 8.83 2.66 7.83 
Total 12 5.18 4.60 1.68 8.83 2.05  
Smooth Study 1.46 1.40 0.29 3.37 1.11 4.42 5.50 0.041* 
Control 3.83 3.25 1.23 9.12 2.82 8.58 
Total 12 2.65 1.80 0.29 9.12 2.39  
Sulcal Study 1.82 1.47 0.05 5.34 1.92 6.83 16.00 0.818 
Control 1.28 1.42 0.79 1.59 0.35 6.17 
Total 12 1.55 1.42 0.05 5.34 1.35  

*P < 0.05.

Discussion

The sample size in animal studies of nanosilver cytotoxicity per group varies considerably in the literature (17–21). In the present study, the sample size (n = 6) per group was determined depending on the decision of the ethics committee, and the sample size was calculated by considering the mean differences of the serum concentration of nanosilver between the study group and the control group.

Metallic silver is a rare noble metal in the environment. In general medicine, silver is used as medication in the form of silver sulfadiazine in treatments due to its antibacterial activity, especially for burn treatment. In addition, silver comes first among the topics of interest in the field of orthopaedics (silver-coated megaendoprostheses), for the prevention of catheter-related infection (silver-impregnated external ventricular drainage catheter, silver-impregnated haemodialysis catheter), and for the inhibition of prosthetic valve endocarditis (silver-coated prosthetic valve), and also metallic silver is used in dental fillings materials since 1970s. Metallic silver is inert under in vivo conditions, and when it contacts with moisture, like body fluids or secretions, it turns to an active form which is the silver ion (Ag+). Metallic silver shows very few antibacterial effects in its inert state while silver ions have stronger features against a wide spectrum of bacteria (22–27).

Nanosilver has already been in use for the treatment of burn wounds in clinical practice (15, 28). Currently, nanosilver is a leading subject at the field of dentistry and orthodontics (14, 29–31).

The silver nanoparticles show efficient antimicrobial properties compared to other salts due to their extremely large surface area, which provides better contact with microorganisms. When nanosilver is evaluated for its antimicrobial activity, it has been observed that the nanosilver particles get attached to the cell membrane and can penetrate inside the bacteria. Cell death occurs because it disturbs the respiratory chain and leaks through the holes in the cell wall (12, 32).

Therefore, we decided to apply nanosilver for the prevention of WSL, which is a side-effect of the fixed appliance therapy and a major issue that is needed to be solved in orthodontics. Orthodontic brackets coated with nanosilver may lead to a new approach and be a novel solution. Most of the in vitro studies showed that nanosilver is in interaction with the inhibition of S.mutans when supplemented with composite or coated wire, but there is no in vivo study on its effectiveness on S.mutans (14, 29, 33, 34).

In the present in vivo study, the inhibited growth of S.mutans for the prevention of WSL via infection of nanosilver coated bracket was shown. Our results showed that the number of S.mutans around the nanosilver coated brackets was significantly less, especially on Day 30, compared to the control group. This is a very important finding as WSL can become noticeable around the brackets within 1 month after bonding (2). In our study, one bracket was used on each rat due to the narrow width of the mandibular teeth. Nanosilver concentration of the saliva was found significantly higher on Day 7 than the other days in the study group, however, there were no meaningful differences in comparison to the control group.

Previous studies suggested that the antibacterial effectiveness of nanosilver is dependent on the silver ion release and the direct contact to the bacteria (30, 31, 35). Studies showed that materials with higher nanosilver loading release more nanosilver ions and have a higher inhibitory effect (29, 36).

In our study, an analysis of the caries showed that maxillary smooth caries in the study group was significantly less compared to the control group. However, occlusal and sulcal caries scores in the study and the control group did not differ. This suggests that nanosilver ions may be more effective for superficial bacteria compared to the bacteria located in occlusal fissures and deep cavities. This is beneficial with regard to the occurrence of WSL on the vestibular smooth surfaces of teeth in contact with orthodontic attachments.

Antibacterial effectiveness of nanosilver coated materials has been shown with (36) and without (37) the release of nanosilver ions in in vitro studies. In addition, long-term inhibitory effects against S.mutans (38) have been found while no nanosilver ions were released. Li et al. (31) showed that bonding agents with nanosilver were effective against bacterial growth inhibition, not only for S.mutans present on surface, but also for the S.mutans away from the surface in the culture medium.

We found that S.mutans counts were significantly less on Day 30 in the study group than the control group. However, in the intra-group comparisons, the concentration of nanosilver in the saliva was found significantly higher only on Day 7, whereas S.mutans counts were significantly less on Day 3, 7, 14, 30, and 45 in the study group. This indicates that the antibacterial activity of nanosilver has contact-inhibition characteristics more than the release of nanosilver ions, which leads us to prefer this recently invented bracket.

Even if nanosilver ions released from the nanosilver coated orthodontic brackets decrease during the prolonged orthodontic treatment, the antibacterial activity will continue via contact-inhibition as shown above. The smooth surface caries was reduced at the end of the experiment, in spite of the concentration of nanosilver in the saliva, which did not show significant differences between the study and the control group. One of the most important reasons of this condition may be S.mutans counts were insignificantly smaller in the study group than the control group from Day 3 to Day 75 and significantly smaller on Day 30. Also we suppose that contact-inhibition feature of nanosilver is efficient especially for the prevention of WSL around the orthodontic bracket.

Bacteria have a highly developed recognition system capable of recognizing and interacting with specific macromolecules on tissue surfaces. Adhesions of the oral pathogens to the oral mucosa take place via the glycoproteins and glycolipids, which are receptors on the surface of the epithelial cells. There are complementary structures having molecular architecture on the surface of the bacterial cell for adhesion. Bacteria can hold easily, strongly, fast, and persistently to epithelium using these surface molecules (39).

In the present study, counts of S.mutans in the vestibular smear were proliferated quickly after inoculation, and vice versa, in the plaque sample on the teeth. It was found significantly higher on Day 3 in the study group and on Day 3, 7, 30 in the control group. These findings may be an indication of S.mutans being accumulated easily on the oral epithelium. However, the number of S.mutans on the oral epithelium was found to be normal after Day 3 in the study group, whereas it was normalized after Day 30 in the control group. We can predict that the growth of S.mutans was inhibited by the nanosilver coated bracket.

Nanosilver particles have been reported to be cytotoxic in in vitro studies for several types of cells, however, the toxicity mechanism of nanosilver is not clear yet. The common view is that it is similar to bacterial death, which changes the permeability of the cell membrane by blocking the ion channels, causing a mitochondrial dysfunction, and producing oxidative stress (40–43). On the contrary, it has been reported that nanosilver does not lead to significant cytotoxicity in mouse fibroblasts, human osteoblasts (44), and human gingival fibroblasts cell lines (14). The most important factor in the formation of different test results is size and concentration of nanosilver particles and also testing with nanosilver particles alone or with a medical device. It should be noted that in vitro tests cannot entirely predict the overall biocompatibility of a material and in vivo use of the material must be questioned (45).

Animal studies for nanosilver cytotoxicity were conducted via respiratory and gastrointestinal tracts because these are the main entry portals of nanosilver into the human body. Nanosilver (5000mg/kg) was given at once via gavage to mice (46) and, for rats (17), daily intake oral doses of up to 9mg/kg were found to be safe according to chemical parameters and histopathological evaluations at the end of 14 days. The lowest adverse effects were observed in the rats (47) given high doses of nanosilver particles in the long-term (90 days).

We assume that even if the patient swallows the bracket during orthodontic treatment, it is not possible to reach the above-mentioned daily dose.

Argyria, a permanent blue grey discoloration, is the most commonly side-effect of silver, which is stored in skin, nails, and mucous membranes (48, 49). Especially, burn patients are treated by nanosilver coated wound dressings (15, 28, 50). Vlachou (15) and Moiemen (50) noted that there were no signs of argyria while nanosilver serum concentration was 226 and 436 µg/L, respectively. However, Trop (48) showed that the blood nanosilver level was 107 µg/L in a patient who suffered from acute silver toxicity. So, the lethal dose or the blood nanosilver level causing the argyria symptom is not yet known.

In the present study, the serum concentration of nanosilver was found as 0.00175 µg/L in rats. This value could increase during orthodontic treatment due to the use of 20 nano-coated brackets. However, it is clear that the serum nanosilver count still remains far below the levels, as shown in other clinical trials (15, 50).

The antibacterial effects in inhibition of S.mutans, low levels of nanosilver ion release, and reduction of smooth surface caries have been shown by the nano-coated orthodontic bracket as applied to the mandibular incisors of rats in this study. This initial study will be a guide for further advanced work.

Conclusion

Our results show that nano-coated orthodontic bracket is effective in the inhibition of S.mutans and reduction in smooth surface caries. This newly introduced nano-coated orthodontic bracket may be an essential non-compliant appliance in avoidance of WSLs for patients especially with poor oral hygiene during fixed orthodontic treatment and may provide solution to patients with immune deficiency, diabetics or in the need of protection from infections such as with subacute bacterial endocarditis. We need more detailed research to predict the blood silver level during orthodontic treatment to estimate contingent side effects of nanosilver.

Funding

Gazi University Scientific Research Committee (03/2013-01).

Acknowledgements

We thank National Nanotechnology Research Center (UNAM) for kind assistance.

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Author notes

Correspondence to: Gamze Metin Gürsoy, Department of Orthodontics, Faculty of Dentistry, Gazi University, Bişkek cad. 1. Sok. No: 4 Emek, 06510 Ankara, Turkey. E-mail: gamgursoy@gmail.com