Iran J Ortho. In Press(In Press):e7281.

Published online 2016 November 8.

doi: 10.17795/ijo-7281.

Original Article

Effect of Chitosan Nanoparticles Incorporation on Antibacterial

Properties and Shear Bond Strength of Dental Composite Used in

Orthodontics

Ahmad Sodagar,1 Abbas Bahador,2 Yasamin Farajzadeh Jalali,1 Fatemeh Gorjizadeh,1 and Pedram

Baghaeian1,*

1Orthodontics Department, Tehran University of Medical Sciences, Tehran, IR Iran2Microbiology Department, Tehran University of Medical Sciences, Tehran, IR Iran

*Corresponding author: Pedram Baghaeian, Orthodontics Department, Tehran University of Medical Sciences, Tehran, IR Iran. Tel: +98-2188015950, E-mail:pedrambaghaeian@gmail.com

Received 2016 May 29; Accepted 2016 September 18.

Abstract

Background: Plaque accumulation is a drawback of orthodontic treatment, which requires composite for bonding of brackets. Asthe antimicrobial properties of chitosan nanoparticles (NPs) have been proven, the aim of this study was to evaluate the antimicro-bial and mechanical properties of composite resins modified by the addition of chitosan NPs.Methods: Orthodontics composite containing 0%, 1%, 5% and 10% NPs were prepared. 180 composite disks were prepared for ElutedComponent Test, Disk Agar Diffusion Test and Biofilm Inhibition Test to collect the counts of microorganisms on three days, measurethe inhibition diameter and quantify the viable counts of colonies consequently. For shear bond strength (SBS) test 48 intact bovineincisors were divided into 4 groups. For (SBS) test 48 intact bovine incisors were divided into 4 groups. Composites containing 0%,1%, 5% and 10% NPs were used for bonding of bracket. The bracket/tooth SBS was measured by Universal testing machine.Results: All concentration of chitosan NPs had a significant effect on creation and extension of inhibition zone. For S.mutans andS.sanguinis all concentration of chitosan NPs caused reduction of the colony counts. Composite containing 10% chitosan NPs hadsignificant effect on reduction of colony counts for S.mutans and S.sanguinis in all three days. The highest mean shear bond strengthbelonged to the 1% NPs composite while the lowest value was seen in control group.Conclusions: Incorporating chitosan nanoparticles into composite resins confer antibacterial properties of adhesives, while themean shear bond of composite containing NPs don’t change dramatically.

Keywords: Shear Bond Strength, Nanochitosan, Antimicrobial Effect, Stepotococcus, Mutans, Lactobacillus Acidophius.

1. Background

Bonding technique with resin based composite as anadhesive agent has been primarily used in orthodontics forsecuring orthodontics brackets to the surface of the teeth.Unfortunately, in spite of the fact that bonding techniquehave many advantages such as high esthetic and simpleprocedure, still have some drawbacks such as plaque accu-mulation, development of white spot lesions and bond fail-ure, which cause prolonging the treatment course, impos-ing high cost, consuming more chair time and a less thanoptimal esthetic result occurs after treatment due to dem-ineralization of enamel adjacent to the brackets speciallyaround the bracket margin, because of the remaining ex-posed composite surface (1-4).

Several methods have been used to inhibit biofilmgrowth that contributes to dental caries. For instance, one

group of such efforts has been evaluation of effectivenessof incorporating different antimicrobial agents in the ad-hesives; two of the most common examples are fluorideand chlorhexidine (5-9).

Today, one of the most important advances in dentalmaterial field is the application of nanotechnology to resincomposites. Many studies investigated the effect of antimi-crobial nanoparticles incorporated into composite resinsto prevent plaque accumulation and bacterial adhesion.Nanoparticles are believed to penetrate into the cell wall ofbacteria efficiently due to their smaller size, exerting theirantibacterial properties effectively (9, 10). A study evalu-ated the antibacterial effect of composite with differentconcentration of incorporated nanosilver, which has beenused in medicine as an antimicrobial agent (11). The resultof the study showed that nanosilver containing composite

Copyright © 2016, Iranian Journal of Orthodontics. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided theoriginal work is properly cited.

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could confer surface antibacterial activity without signifi-cant difference on shear bond strength. One of the mostimportant acquisition of nanotechnology is nanoparticlesof chitosan, a linear polysaccharide gained by the deacety-lation of chitin, that is a structural biopolymer existing inthe exoskeletons of the crustaceans and mollusks (12). Chi-tosan is widely used in many fields including nutrition,pharmaceutical, cosmetics, and agriculture for its remark-able antiviral, antifungal, and antimicrobial activity. Theantibacterial activity of chitosan is particularly interestingand has been observed against various bacteria (salmonellalyphimurium, pneumoniea, E.coli, S. aureus) (4, 13, 14).

The antibacterial mechanism of chitosan is due to thebonding of amino groups NH3 with positive charge andcarboccilic group with negative charge on the membraneof bacteria. The bonding changes the permeability of thecell membrane resulting in a weakness and tear in mem-brane that is followed by cellular leakage (15).

Regarding the necessity of manufacturing of com-posites with antibacterial properties and the capacity ofan appropriate banding strength, and also consideringmentioned properties of nanochitosan the main goal ofthis study is to investigate the effect of nanochitosan NPsagainst bacteria and shear band strength of adhesive com-posite used in orthodontic treatment.

2. Methods

2.1. Preparation of Nano Chitosan

Chitosan bulk (Acros company USA) with a low molec-ular weight was dissolved in acetic acid (1%) then was ad-justed to PH = 10 by adding NaOH (N = 10). 3 milliliter ofthis milky solution was added to 1 milliliter of Tripolyphos-phates followed by rapid magnetic string mixing at thehighest power. Then Nanoparticles were deposited using acentrifuge. The particles were washed using DI water. Afterfreezing and crashing particles were prepared to use (Fig-ure 1).

2.2. Nanocomposite Preparation

3600 mg of Transbond XT (3M Unitek, USA) compos-ite was blended with 400 mg nano powder (containing50/50 w/w NPs), using a mixing spatula on a glass slab ina semi-dark environment, until a uniform consistency wasachieved. Then 1200 mg of the original composite wasmixed with 1200 mg of 10% w/w blended composite to ob-tain a composite with 5% w/w NPs. Similarly, 240 mg of 10%w/w composite was blended with 2160 mg of original com-posite to obtain 1% w/w NP composite.

Figure 1. SEM Photo of Chitosan Nanoparticle

2.3. Shear Bond Strength Test

48 bovine central incisors with no visible cracks orcaries were disinfected in 0.5% Chloramine-T solution(4°C) for one week. Specimens were randomly divided intofour groups (N = 10) of composites with 0%, 1%, 5% and 10%NP content. Buccal surfaces were cleaned with a prophy-laxis brush without powder, rinsed and dried; 35% phos-phoric acid (Ultra etch, Ultradent, USA) was then appliedto the buccal surfaces for 30 seconds, followed by 30 sec-onds of rinsing and gentle air drying. A uniform, thin layerof adhesive (3 M Unitek, USA) was applied to the buccal sur-faces and light cured (Woodpecker, UK) for 10 seconds afterplacing the stainless steel orthodontic brackets (Standardedgewise, 0.18 slot, 12.62 mm2 base area).

All specimens were thermocycled (Vafaei Industrial,Iran) for 1000 rounds in 24 hours to simulate oral environ-ment. Each cycle consisted of 15 seconds of immersion in5°C water bath, 10 seconds of dwell time and 15 secondsof immersion in 55°C water bath. The thermocycled teethwere then fixed to the corners of 2.5 cm diameter metalmolds using rectangular wire and then the molds werefilled with self-cured acrylic resin (Acropars, Iran) up to thelevel of the cementoenamel junction.

The SBS was measured using Roell-7060 universal test-ing machine (Zwick/Roell, Germany). Specimens were po-sitioned in such way that the bracket base was parallel tothe direction of the applied force. A 0.6 mm metal bladewas used in an inciso-gingival direction at a crossheadspeed of 0.5 mm/min to apply shear force to the compos-ite interface. The obtained value (N) was divided by thebracket surface area (mm2) to calculate the SBS in MegaPascal. Each tooth and bracket complex was then checkedunder a stereomicroscope (Nikon, SMZ800, Japan) at 10Xmagnification to score the amount of remaining adhesive

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using the ARI as follows: 0 = no adhesive on bracket, 1 = <25% adhesive on bracket, 2 = 25 - 50 - % adhesive on bracket,3 = 50 - 75% adhesive on bracket and 4 = 75 - 100% adhesiveon bracket.

2.4. Antimicrobial Test

Preparation of bacterial suspensions: StreptococcusMutans ATCC25175, Streptococcus Sanguinis ATCC10556and Lactobacillus Acidophilus ATCC4356 were supplied inlipophilised form and incubated in broth, in anaerobicand 37°C conditions for 48 hours. 108 CFU/mL microorgan-ism suspensions were prepared by spectrophotometer fordetermining the antimicrobial effect of TiO2 NPs.

Optical density for L. acidophilus in 600 nm is 1, (OD = 1);which is 108 cells per ml. this density was then diluted tentimes and inoculated on BHI (brain heart infusion) agar.OD = 0.2 for the other two organisms was equivalent to 108

cells per mL.Composite disc preparation: 5 mm diameter standard

ring-shaped molds were placed between glass slides, for at-taining a smooth surface, after being filled with compos-ite. Visible light cure (470 nm) was applied for 30 seconds(Bluphase® 16i, Ivoclar Vivadent AG,Australia). Discs werecured another 10 seconds after application of a thin layerof bonding.

All specimens (n = 180) were sterilized in Iran’s Nu-clear Science and Tech gamma radiation center with 25 KGydosages.

2.5. Biofilm Inhibition

Three-day biofilms were generated on composite discs(n = 36) using 24-well plates. Each well was inoculatedwith adjusted bacterial inoculum. Biofilms were grownat 37°C. At the end of the third day, each disc was rinsedwith sterile saline to remove loosely absorbed proteins andbiofilm matrix residues. To count the colony forming units(CFUs) responsible for biofilm formation, specimens weresonicated in sterile saline and then vortexed. CFU/mL ofthe microorganism present in the suspension was countedwith drop-plate method using rapid dilution in microtiterplates.

2.6. Disc Agar Diffusion Test (DAD)

Antibacterial activity of discs via solubility and diffu-sion of chitosan NPs was examined by this test. Compositediscs (n = 36) were placed, 2 Cm apart, on BHI agar plates,which were inoculated with a 200µL bacterial solution (~108 CFU/mL) by a sterile swap. After 48-hour incubation, thebacterial growth inhibition diameter was optically mea-sured.

2.7. Antibacterial Properties of Eluted ComponentsAntibacterial activity of possible Eluted components

from nanoparticle comprising composite discs was alsoevaluated. Composite discs (n = 108) were placed in tubescontaining 5 mL BHI media. BHI media was then aspiratedfrom the tubes on days 3, 15 and 30 and placed in other15 mL plastic tubes; tubes were inoculated with 50 µL ofbacterial culture (final ~ 25 × 105 CFU in 5 mL media) andshaken at 300 rpm rate in 37°C for 24 hours.

2.8. Statistical AnalysisShear bond strength test results were analyzed us-

ing one-way ANOVA followed by Post Hoc Tukey’s HSDtest. The Kruskal-Wallis test was also applied to analyzethe ARI results. Antimicrobial test results were analyzedwith multiple statistical tests. One-way ANOVA was firstused for biofilm inhibition test followed by Tukey HSDtest. Kruskal Wallis Test was used to analyze data attainedfrom DAD test. Two-way ANOVA was first used to ana-lyze day×concentration relation in Eluted componentstest. For those groups with significant difference one-wayANOVA was used.

3. Results

3.1. Biofilm Inhibition TestMature biofilm on four different composite groups

were recorded after 3 days. Descriptive results are shownin Table 1. All tests were carried out three times for eachgroup. Kruskal-Wallis Test showed that S. mutans and S.sanguis colonies were meaningfully lowered in all threegroups of composites containing NPs (P < 0.05). How-ever, for L. acidophilus colonies counts reduced as CS-NPsnanoparticles concentration was increased but this differ-ence was significant only in 10% NPs (Figure 2-4).

3.2. DAD TestIn all three repeated tests, in the all three groups of

composites containing NPs had a significant diameter ofbacterial growth inhibition for all three microorganisms.Kruskal-Wallis Test did not show any significant differencebetween 1%, 5% and 10% NPs groups. The results are illus-trated in Table 2.

3.3. Eluted ComponentsFor S. mutans no significant difference was found for

any of the groups in any day (P = 0.144). Groups of compos-ites containing more than 5% NPs on all days significantlyreduced colony counts of S. mutans (P < 0.001). Statisticallydifferences in all three days were found for S. sanguis (P =0.004) and L. acidophilus (P = 0.020) so each of the cate-gories were analyzed by One-way ANOVA, which showed inFigures 5 and 6.

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Table 1. Colony Counts (CFU/mm2) in 1%, 5% and 10% NPs Containing Composite and Control Group in Biofilm Inhibition Test

Percent N Minimum Maximum Mean Std. Deviation

0

s. mutans 3 761 981 889.67 115.140

s. sanguis 3 523 701 630.6667 94.69072

l.asidophil 3 72 110 89.00 19.313

Valid N (listwise) 3

1

s.mutans 3 391 472 435.33 41.041

s. sanguis 3 168 201 181.6667 17.21434

l.asidophil 3 39 66 54.00 13.748

Valid N (listwise) 3

5

s.mutans 3 112 158 133.00 23.259

s. sanguis 3 41 52 47.00 5.5677

l.asidophil 3 27 29 28.00 1.000

Valid N (listwise) 3

10

s.mutans 3 11 18 14.00 33.606

s. sanguis 3 1.30 2.20 1.733 4.5092

l.asidophil 3 5 9 7.00 2.00

Valid N (listwise) 3

Table 2. Microorganism Inhibition Diameter (Millimeter) by Nanoparticles Diffusion

Microorganism N Minimum Maximum Mean Std. Deviation

Streptococcusmutans

1% 3 6 7 6.67 0.577

5% 3 7 8 7.67 0.577

10% 3 10 13 11.33 1.528

Lactobacillus.ac

1% 3 6 8 7.33 1.155

5% 3 6 9 8 1.732

10% 3 12 13 12.77 0.577

Streptococcussanguis

1% 3 6 9 7.67 1.528

5% 3 6 9 7.67 1.528

10% 3 9 11 10.33 1.155

3.4. Shear Bond Strength

The result of shear bond strength can be seen in Table3 shows that maximum mean of the shear band strengthbelongs to the group with Nanochitosan concentration of1% and the least belongs to the control group. One-samplekolmogrove-simirnove tests showed a normal distributionamong all groups. According to the result of one wayANOVA difference was not significant between groups (P =0.730).

The Kruskal-Wallis test did not show any significant dif-ference among groups in terms of the ARI (P = 0.823).

4. Discussion

The application of nanotechnology in dental compos-ite resins has been introduced to enhance the long-termantimicrobial properties and provide superior mechanicalstrength simultaneously (9, 16).

On the basis of studies that have shown antibacterialeffect of chitosan particles and according to the fact thatnanoparticles have greater effect on membrane of bacte-ria, it seems like the chitosan NPs has induced an antibac-terial activity in resin composite (17).

The antibacterial effect of chitosan NPs against Strepto-

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Table 3. Descriptive Data of Shear Bond Strength in Groups

Nano.percentage N Minimum Maximum Mean Std. Deviation

0.00Strength 12

9.37 28.92 18.6031 5.92000Valid N (listwise) 12

1.00strength 12

11.33 27.66 21.1420 5.15022Valid N (listwise) 12

5.00Strength 12

13.29 31.23 20.4970 5.74314Valid N (listwise) 12

10.00Strength 12

11.41 28.50 20.0349 5.31111Valid N (listwise) 12

Microorganism: 1.00

.00 1.00 5.00 10.00

Nano

95%

CI B

iofi

lm

2000000.00

1500000.00

1000000.00

500000.00

.00

Figure 2. Viable Counts of Streptococcus Mutans Biofilms on the Following Compos-ites: 0%, 1%, 5% and 10% Nanoparticles-Containing Composite Discs

coccus mutans ATCC 25175, Streptococcus sanguis ATCC10556and Lactobacillus acidophilus ATCC4356 was evaluated inthis study. Criteria for selection of these types of mi-croorganisms were their presence as a normal flora of themouth, their large population and their important role incausing oral disease (18, 19). 1%, 5% and 10% concentrationsof NPs were used for purpose of comparing this NP with thepreviously studied documents (4).

The result demonstrated that bacterial biofilm inhi-bition in composite containing NPs is significantly morethan conventional composite resins. This effect increasedas the percentage of NPs in the composites increased insuch a way that the composite containing 1% NPs signifi-cantly reduced S. mutans and S. sanguis. However this hadno effect on biofilm inhibition of L. acidophilus unless 10%NPs. L. acidophilus help the development of caries and arefound in more advanced lesions. Therefore, the biofilm

Microorganism: 2.00

.00 1.00 5.00 10.00

Nano

95%

CI B

iofi

lm1250000.00

1000000.00

750000.00

500000.00

250000.00

.00

Figure 3. Viable Counts of Streptococcus sanguis Biofilms on the Following Compos-ites: 0%, 1%, 5% and 10% Nanoparticles-Containing Composite Discs

Microorganism: 3.00

.00 1.00 5.00 10.00

Nano

95%

CI B

iofi

lm

150000.00

100000.00

50000.00

.00

50000.00

Figure 4. Viable Counts of Lactobacillus acidophilus Biofilms on the Following Com-posites: 0%, 1%, 5% and 10% Nanoparticles-Containing Composite Discs

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Day31530

1 5 10

Percent

95%

CI s

.mu

stan

s.lo

g

8.60

8.40

8.20

8.00

7.60

7.60

Figure5. Colony Counts of Streptococcus Sanguinis on the Following Composites: 0%,1%, 5% and 10% NPs Containing Composite Discs in Eluted Component Test

Day31530

1 5 10

Percent

95%

CI s

.san

guin

is.lo

g

8.40

8.20

8.00

7.60

7.60

7.40

Figure 6. Colony Counts of Lactobacillus Acidophilus on the Following Composites:0%, 1%, 5% and 10% NPs Containing Composite Discs in Eluted Component Test

that containing these microorganisms are very resistantand is not effected by adding NPs to the composite resinsin low concentration. This finding is in agreement withPoosti who investigated the inhibition of bacterial biofilmonly on S. mutans and showed that the inhibition is moreprominent with composite containing 1% TiO2NPs (20).

The results of Eluted Component Test showing conti-nuity of antibacterial effect indicated significant reduc-tion of the colony count of S. mutans and S. sanguis, onlyfor 10%NPs group. Similar results were showed in other NPssuch as ZnO, and TiO2 compared to chitosan (4, 21). Theyshowed NPs maintain its properties for a long time up tothree weeks. Because just a slight increase in the number ofcolonies was observed unlike L. acidophilus that by increas-ing time the number of colonies remains constant.

Presence of S. sanguis in the mouth, reducing the popu-lation of S. mutans, these bacteria are in balance (22). If thenumber of colonies remain constant or not significantlydecreased in the presence of chitosan NPs the desired re-sult is achieved. However, due to the reduced number ofcolonies of S. sanguis in this study can be concluded thatthe chitosan NPs reduce both S. sanguis and S. mutans uns-elected. But since S. mutans have reduced more than S. san-guis Finally, create a favorable effect.

Mirhashemi et al. also reported the antimicrobial ef-fect of chitosan NPs in combination with ZnO-NPs, resultsshowed that ZnO-NPs and Chitosan NPs cause inhibitionof bacterial biofilm, which is more prominent with higherNP concentrations. The 1% group was almost similar tothe control group while the 5% group significantly reducedS. mutans and the 10% group inhibited S. sanguis and allthree organisms significantly (4). This result supposedlycaused by the addition of Chitosan NPs because the stud-ies on ZnO-NPs solely decreased significantly only the L. aci-dophilus on day 30 for the Eluted components test whichshows an enhanced antibacterial activity in the combina-tion of NPs compared to ZnO-NPs used alone, since the lat-ter showed no significant difference on any day for any con-centrations (21). Pinto et al. in which examined the an-timicrobial effect of chitosan NPs in combination with Sil-ver NPs against microorganisms commonly found in food,showed that the effect of chitosan matrix is depend on sil-ver containing; and chitosan NPs alone have a bacterio-static effect and inhibits growths of microorganisms, al-though the choice of microorganisms was different (23).Ahn et al. also demonstrated silver NPs have antimicrobialeffect against refractory bacteria. But Silver NPs in com-bination with composite caused color modification whichdefies the esthetic purposes of composites (24).

For other NPs such as TiO2 the biofilm inhibition wassimilar in the 1% group with present study, Poosti et al.showed that the Light cure orthodontic composite con-

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taining 1% (w/w) TiO2 nanoparticles was quite effective ininhibiting bacterial growth in long term (20).

Properties of composites change by adding NPs to it.So that is important by adding chitosan NPs by means ofdecrease plaque accumulation, another properties of com-posite such as mechanical and physical properties remainapproximately without changes (25, 26). The aim here is toevaluate the SBS modification caused by the addition of CS-NPs to resin composite used in orthodontics.

Results of this study show that addition of chitosanNPs increase SBS of composite. So that the Transbond XTcomposite as a gold standard has the lowest strength andthe 1% NPs containing composite has the highest strengthand it is within the acceptable range 6 - 8 MPa. The bondstrength is decreased as the percentage of NPs increase incomposite, So that with increasing concentration of 1% to5% and from 5% to 10% shear bond strength are reduced.However, there is no significant difference between any ofthe groups. According to the bond strength and scatteringof data to use of 1% weight of NP might be advisable withmechanical modifications only in the acceptable range.

Compared with other studies examining the effects ofother nanoparticles on shear bond strength of resin com-posite, in one study, Poosti et al. showed that there is nosignificant difference between TiO2 NPs containing com-posite and Transbond XT, Which is similar to the presentstudy (20). Results revealed that adding TiO2 nanoparticleto composite in long term enhance its antibacterial effectswithout compromising the physical properties.

In another study Akhavan and sodagar concluded thatthe 1% Silver/hydroxyapatite NPs containing composite hashigher SBS than the control group, while in 5% and 10%groups it was decreased. The similar results with this studywere obtained that the bond strength is decreased as thepercentage of NPs increase in composite (25).

4.1. ConclusionFinally, the findings of the present study indicate that,

dental composite containing chitosan NPs had a quiteantimicrobial effect by means of decreasing the numberof the colonies, in long term without compromising theshear bond strength.

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