Evaluation of Antibacterial Effect of Silver Nanoparticle Coated Stainless Steel Band Material – An In vitro Study

Introduction: Decalcification, caries, inflammatory periodontal disease are the most common iatrogenic effects of orthodontic treatment because of failure to maintain proper oral hygiene. Although various methods have been tried to minimize the incidence of white spot lesions, none of them proved to be effective. The purpose of this study was to develop a hard coating of silver nanoparticles on stainless steel band material and to evaluate the antibacterial efficacy against most common cariogenic pathogens. Materials & Method: Stainless steel band material was cut into 45 pieces of about 0.5 x 1 cm in dimension, of these 25 band material strips were coated with silver nanoparticles using thermal evaporation technology in a Vacuum coating unit (Indovision, India) at a vacuum of 4.5 ×10−5 millibar at 961°C for 5 minutes and remaining strips served as control. Scanning electron microscopy (SEM) study of coated band material showed a uniform deposition of silver nanoparticles of about 18.63 percent by weight. Five coated and five uncoated band material strips were utilized for each test to evaluate the antibacterial effect of the coated band material against Streptococcus mutans, Lactobacillus acidophilus using zone of inhibition and direct contact test. In zone of inhibition test, the bacterial growth inhibition zone was measured after a period of 24-48 hours, where as in direct contact test, the number of bacterial colonies were counted after 24 hours, 48 hours and1 week. Five coated band materials were immersed separately in a container having 5 ml of artificial saliva and the amount of silver nanoparticles released from coated samples was evaluated after 24 hrs, 48 hrs, and 1 week using atomic absorption spectrophotometer. Result: A stable uniform coating of silver nanoparticles on the band material was obtained by physical vapor deposition. The coated band material showed a potent antibacterial activity against L.acidophilus and S.mutans. The maximum amount of silver nanoparticles released from the silver nanoparticle coated band material was 0.0236 ± 0.0067 ppm, which is below the maximum permissible level set by WHO [0.1 mg /l], proving it as biocompatible. Conclusion: Silver nanoparticle coating on orthodontic band surfaces can provide suitable antimicrobial activity during active orthodontic treatment.


INTRODUCTION
Fixed orthodontic treatment involves placement of bands and brackets on the teeth, and orthodontic bands are often seated in supra and subgingival areas.Banding of molar teeth has more tendencies for retention of plaque due to the greater area of band material compared to bonded attachments and also due to the obstruction caused by band material to oral prophylactic measures.Accumulation of plaque around bands results in decalcification, white spot lesions and gingival inflammation which may progress literature include direct impregnation with antibiotics and the use of antibiotics or silver doped polymer coatings. 3Among various metals, silver, zinc, copper exhibits high antibacterial property. 4Nanoparticles (NP) have a greater surface-to-volume ratio (per unit mass) compared to non-nanoscale particles, interacting more closely with microbial membranes and provide considerably larger surface area for antimicrobial activity. 5[8][9] As silver nanoparticles (SNP) were known to have good antibacterial activity and biocompatibility, 10 stainless steel band material was coated with silver nanoparticles.Various methods of coating were described in the literature, which includes thermal vacuum evaporation 1,3,8 spray pyrolysis, 13 high vacuum magnetron sputtering equipment [14][15][16] silanization, 9 electroplating, 11 dip coating method. 17In the present study, stainless steel bands are coated with silver nanoparticles using thermal vacuum evaporation technology because this method is capable of producing very high purity thin film with high deposition rate and damage to the substrate can be minimized.

Antibacterial efficacy of silver nanoparticle coated
band material against most common cariogenic pathogens was evaluated.

MATERIALS AND METHOD
Stainless steel band material (0.005'' x 0.018'') was cut into 45 pieces of about 0.5 x1 cm in dimension.
Twenty five stainless steel strips were coated with silver nanoparticles and included in the test group; the remaining strips served as controls.[Table1] The coating was done by thermal evaporation technique using -Vacuum coating unit (Indovision, India) at a vacuum of 4.5 ×10−5 millibar at 961°C for 5 minutes.The substrate (band material) temperature was maintained at 100° C. The silver wire was kept over tungsten filament at a distance of 15 cm from the band material and vaporized to form a uniform coating of silver nanoparticles on the band material.

Characterization of the coated surface
The surface morphology of the coated and uncoated samples was examined by scanning electron microscopy (SEM) and the atomic composition was determined by energy-dispersive x-ray spectroscopy (EDS).

Antimicrobial activity test
The antibacterial properties of the samples were evaluated against Gram-positive S.mutans and L.acidophilus using zone of inhibition test and direct contact test.

Zone of inhibition test
The medium (brain heart infusion agar for Lactobacillus and mutans sanguis agar for S.mutans) (HiMedia, Mumbai) was prepared from dehydrated media as per manufacturer's instructions.Medium at pH 7.2  2]  Bindu SH, Vani SVK, Nirisha G, Madhuri N, Deepa BS, Hemadri S : Evaluation of antibacterial effect of silver nanoparticle coated stainless steel band material -An In vitro study

Direct contact test
The mean number of Lactobacillus colonies grown after 24 and 48 hours and 1 week continued to increase in the control group but has decreased in the test group which was statistically significant.(Fig. 4, 5) [Table 4] The mean number of Streptococcus colonies also showed a similar pattern and the difference between control and test groups after 24 hours, 48 hours and 1 week was statistically significant.(Fig. 6, 7) [Table 5]

Measurement of silver ion released
There was a significant decrease in the concentration of silver ions released from coated samples at 24 hours, 48 hours and 1 week with maximum release of 0.0236 ± 0.0067 ppm at 24 hours, which is less than maximum permissible levels set by WHO (0.1mg/L).The mean concentration of silver ions released at 24 hours, 48 hours, and 1 week were 0.0236 ± 0.0067 ppm, 0.0221 ± 0.0056 ppm, 0.016 ± 0.0032 ppm respectively.[Table 6] The maximum silver ion release of about 0.0236 ± 0.0067 ppm was noted at 24 hrs which is less than maximum permissible level set by WHO (0.1 ppm ). 19

DISCUSSION
White spot formation or demineralization is a prevalent unwanted side-effect of orthodontic therapy.
Orthodontic appliances can affect the self-cleaning ability of teeth, alter the oral microflora and increase the levels of acidogenic plaque bacteria, i.e. mutans streptococci and lactobacilli in saliva and dental biofilm during active wear of the appliance.
In the oral cavity, the antibacterial properties of NPs have been used through two broad mechanisms of combining dental materials with NPs or coating surfaces with NPs to prevent microbial adhesion, with the overall aim of reducing the biofilm formation. 6 antibacterial coating on orthodontic appliances offers a possible strategy for reducing such bacterial damage.
The present study was designed with reference to the unique antimicrobial properties of silver nanoparticles.
In this study, stainless steel band material used to band molar teeth were coated with silver nanoparticles to the literature reported the antibacterial effect of silver nanoparticles against Lactobacillus using disk diffusion assay.There was no difference in the antibacterial effect of silver nanoparticles against L.acidophilus and S.mutans using disk diffusion assay.[Table .3] In the direct contact test, the number of bacterial colonies continued to decrease in the test group,which was attributed to the potent antibacterial effect of silver nanoparticles against both S.mutans and L.acidophilus.The results were similar to the study by Arash et al 11 in which S.mutans colonies increased slightly after six hours and subsequently decreased against silver (8 -10 μm) coated SS brackets when evaluated for a period of 30 days.A study by Mhaske et al 18 showed a significant decrease in the total number of lactobacillus colonies around silver coated SS wire (10 nm thick) compared with the control group after 24 hours when evaluated using direct contact test.The results of our study showed that there is no significant difference in the number of L.acidophilus and S.mutans colonies in the test group at 24 hours and 1 week.This implies that silver nanoparticles exhibited similar antibacterial effect against both L.acidophilus and S.mutans.
In this study, the amount of silver ions released from coated band material continued to decrease over a period of 1 week, with a maximum release of 0.023 ± 0.0067ppm at 24 hours.This was less than the maximum permissible levels set by the WHO (0.1 mg/L). 19No studies in the literature evaluated the concentration of silver ions released from the coated band material over a period of 1 week.However, there are other studies in literature, in which the silver ions release were evaluated from silver nanoparticles coated retainers and stainless steel sheets.They reported that the silver ion concentration on the 8thday was more than the 2nd day, as the solution was not replaced after the 2nd day.
Although nanoparticle coating was done on different materials for different purposes, in studies reported so far, only one study reported silver nanoparticle coating on stainless steel band material.Our data cannot be compared with other studies because of difference in the amount of silver nanoparticle deposited on the surface, the method of coating used, size and surface characteristics of a sample on which coating was done.
As the orthodontic treatment lasts for about 12-24 months, it is pertinent to carry out long term studies to establish standard protocols which would facilitate continuous release of nanoparticles from coated band material in order to maintain prolonged antibacterial activity.
Since the in vitro studies can never simulate oral conditions precisely.Further clinical trials are required to study the antibacterial effect, biocompatibility.

CONCLUSION
In this short term study, band material coated with silver nanoparticles showed a good antibacterial effect against L.acidophilus and S.mutans for a period of one week.This silver nanoparticle coated band material is especially useful in cases where orthodontic treatment lasts for longer duration such as cleft cases and orthognathic surgery and in individuals who lacks manual dexterity.
Financial support and sponsorship: Nil.

Conflicts of interest:
There are no conflicts of interest. OJN

to 7 .
4 was transferred to 9 cm diameter Petri dishes and stored at 2-8° C. Lactobacillus and S.mutans suspensions were prepared in 0.5 McFarland standard concentration (108 bacteria per mL) and transferred to culture medium.Each coated (test sample) and uncoated (control sample) band were placed in the culture using sterile forceps at specific millimeter distance, and all five plates were incubated for 24-48 hours at 37° C. Then the bacterial growth inhibition zone was measured in millimeters.Direct contact test Five coated and five uncoated band materials were placed in separate micro-tubes containing 1mL of brain heart infusion broth for Lactobacillus and mutans sanguis broth for S.mutans.When the strains reached Bindu SH, Vani SVK, Nirisha G, Madhuri N, Deepa BS, Hemadri S : Evaluation of antibacterial effect of silver nanoparticle coated stainless steel band material -An In vitro study Amount of silver ion released -5 the standard 0.5 McFarland concentration, they were diluted in 1:10 ratio and 5 μL of this suspension were then poured into each tube containing 1 mL of the culture medium.The microtubes were incubated at 37° C and after a period of 24 hours, 48 hours and 1 week, ten μL of the suspension were taken from each microtube (five coated, and five uncoated) using a micropipette and cultured on a separate culture medium.These cultures were incubated at 37°C for 24-48 hours and the colonies, each composed of a single set of cultures, were counted.Amount of silver ion released Five coated band materials were immersed separately in a container having 5 ml of artificial saliva with gentle shaking.The saliva was replaced and analyzed after 24 hours, 48 hours, and 1 week.The amount of silver ions leached from the coated samples were measured using atomic absorption spectrophotometer.Data obtained from the results were analyzed statistically using the paired t-test for intragroup comparison of test and control and independent t-test for intergroup comparison.RESULT Surface morphology and atomic composition SEM images showed a uniform deposition of silver nanoparticleson coated band material, whereas uncoated sample showed only generic metal striations (Fig.1a,1b).The EDS spectra of the uncoated sample showed elemental ion content of typical bands such as Cr, Fe, and Ni.However, no silver ions were detected.EDS spectra of the coated stainless steel band material showed the presence of silver ions in addition to the typical composition of the steel.The amount of Ag nanoparticles deposited on the coated band material was about 18.63 percent by weight.(Fig. 2a, 2b) Antibacterial effect Zone of inhibition test A clear zone of inhibition of about 4 mm was observed around the coated samples against both L.acidophilus and S. mutans.Whereas, inhibition zone was not observed around the uncoated SS strips.(Fig. 3a,3b) [Table

Figure 2a :Figure 1 :Figure 3 :
Figure 2a: Image showing elemental composition of coated band material

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produce a potentially antimicrobial band material effective against Gram-positive bacteria.The coating technique used in this study was vacuum evaporation technology.Vacuum evaporation is a physical deposition method that uses resistive heating to produce a metallic thin film of solid on a suitable substrate.The SEM images of coated and uncoated band materials were compared to assess the presence of surface SNPs.SEM images of coated band material showed uniform deposition of SNPs, whereas uncoated band material showed only generic metal striations.EDS for coated surface showed the presence of silver nanoparticles.The amount of silver nanoparticles deposited as reported in various studies ranged from 4.26 % -100 % by weight.Juan et al9 deposited 4.26 % of silver on the titanium surface using silanization method, they found negligible zones of inhibition against E.coli and S.aureus, whereas study by Prabha et al deposited 28.22 % of silver on the band material surface using thermal vacuum evaporation technology and evaluated cytotoxicity with 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay which shows that more than 80 percent viability in all coated samples. 1 In our present study, approximately 18.63 % of silver nanoparticles by weight was deposited on the stainless steel band material, which is thought to have better antibacterial effect and biocompatibility.The results of our study showed a clear zone of inhibition of 4.3 mm and 4.0 mm diameter around coated band material against L.acidophilus and S.mutans respectively, whereas there is no growth inhibition zone around uncoated band material.In contrast, Prabha et al 1 noted no growth inhibition around the silver coated band material against S.mutans.Arash et al 11 evaluated the antibacterial effect of silver (10 μm) coated bracket against S.mutans using disk diffusion assay.They showed that the inhibition halo was absent around the silver coated bracket, which was attributed to smaller physical contact between the silver particles in the bracket and the surrounding culture medium.Morita et al 10 noted a clear zone of inhibition of more than 4.2 mm diameter around Ag ion-coated retainer against S.mutans indicating that Ag ions on the coated wires diffused into the culture medium and inhibited bacterial growth.No studies in

Table 6 : Comparison of silver nanoparticles release in the test group at different time intervals.
Rahmani et al 8 noted that the amount of silver ions