orthodontic bonding materials

1DDS, MSD, School of Dentistry, Department of Orthodontics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 2Biologist and Technical Chemistry, Institute of Chemistry, Department of Analytical Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 3MSD, PhD, Professor, Institute of Chemistry, Department of Analytical Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 4PhD, Professor, Institute of Microbiology Prof. Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 5MSD, PhD, Associate Professor, School of Dentistry, Department of Orthodontics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil.


INTRODUCTION
,Q VSLWH RI WKH YDULRXV EHQH¿WV SURYLGHG E\ orthodontic treatment, the retentive surfaces of the appliance make them difficult to clean DQG DUH GLUHFWO\ UHVSRQVLEOH IRU IRRG DQG ELR¿OP accumulation. Imbalances such as gingivitis, gingival hyperplasia and caries may occur during orthodontic treatment 6,17,28 , especially when patients do not cooperate with regard to performing the recommended cleaning protocols. In this context, there is an increase in the acidogenic bacterial populations, such as Streptococcus mutans and Lactobacillus sp in the plaque and saliva 25, 28 . The set formed by retentive niches, DGGHG WR WKH GH¿FLHQW RUDO K\JLHQH DQG D GLHW rich in fermentable carbohydrates also favors the increase in the Candida albicans population in the oral cavity, particularly if there is caries activity 19 .
There are reports in the literature that microorganisms of the oral microbiome adhere ¿UPO\ WR UHVLQ PDWHULDOV It is also known that components present in the matrix of adhesive materials may accelerate bacterial growth, increasing the levels of pathogens extremely harmful to tissues 3 . Therefore, the ideal bonding material must be capable of withstanding salivary biochemistry, constant changes in pH, different temperatures, and especially the resident oral microbiota. Thus, the importance must be pointed out of maintaining essential properties, such as: KDUGQHVV URXJKQHVV DQG ÀXRULGH UHOHDVH FDSDFLW\ since these are intimately related to the retention of microorganisms and the maintenance of oral health. ,Q YLHZ RI WKH ELR¿OPFRPSRVLWH LQWHUDFWLRQ which is capable of negatively altering the surfaces of materials for orthodontic bonding, these must have adequate resistance and antimicrobial activity 3 .
Fluoridated bonding materials have been inserted outstandingly in Orthodontics because of WKHLU ÀXRULGH UHOHDVH FDSDFLW\ LUUHVSHFWLYH RI WKH patients' cooperation. Nevertheless, it is important to point out that these materials are recommended as supplementary to and not as substitutes for conventional prophylactic alternatives 6 , since their anticariogenic capacity is still being widely discussed 2,6, 23 . In view of the context presented, the aim of this study was to evaluate the microbial inhibition capacity of three orthodontic bonding materials, and their fluoride release in vitro. Thus, the hypothesis considered in the present study was that bonding materials play a biological LQÀXHQFH WR RUWKRGRQWLF WUHDWPHQW RXWFRPHV 7KH FDSDFLW\ RI WKH PDWHULDO WR UHOHDVH ÀXRULGH DQG prevent microbial contamination can be noted in the prevention and maintenance of the integrity of the oral tissues.

Preparation of specimens
To conduct the study, three materials extensively applied in bonding orthodontic accessories were selected: Transbond TM XT (XT Group), a traditional light cure adhesive (3M Unitek, Monrovia, CA, USA), Transbond TM Plus Color Change (PLUS Group), a moisture tolerant light cure adhesive (3M Unitek, Monrovia, CA, USA) and Fuji Ortho TM LC (FUJI Group), a resin reinforced glass ionomer cement (GC America Corporation, Tokyo, Japan) ( Figure  1). For fabricating the specimens with standardized GLPHQVLRQV D SUHIDEULFDWHG WHÀRQ PDWUL[ ZLWK perforations measuring 5 mm in diameter and 2 mm in thickness was used. The materials were manipulated in accordance with the manufacturers' instructions and then inserted into the matrix in a single increment, applying the pressure required for producing specimens with smooth surfaces (Figure 2). Light polymerization was performed for 40 seconds on each surface, using a halogen light with 550 mW/cm 2 (Foto Optilight LD Max, Gnatus -50/60 Hz, Ribeirão Preto, SP, Brazil), with the active tip of the equipment touching directly on the glass slides 26 .

Microbiologic test
Microbiologic analyses were performed in two stages. Firstly, the antimicrobial activity of the materials was evaluated by means of spectrophotometry (quantitative aspect). For this, 18 specimens were prepared, divided in duplicate  for each tested microorganism.
With regard to the qualitative aspect of analyses, after fabricating one control specimen for each material (baseline), the initial surface (contamination-free) record was made by scanning electron microscopy (SEM). After evaluation on the spectrophotometer, one specimen per microorganism was randomly selected from each group, to be submitted to a SEM analysis to verify microbial growth on its surfaces after contamination, and therefore, the inherent antimicrobial potential of each material. For each specimen evaluated by the SEM, various images were obtained in order to contemplate a large scanning area.

Quantitative analysis of antimicrobial activity
The following strains were used in the experiment: S. mutans (ATCC 25175), L. casei (ATCC 4646) and C. albicans (ATCC 10231), kept in BHI broth (Brain Heart Infusion, Himedia, São Paulo, SP, Brazil). Initially, the BHI broth contaminated with the microorganisms of choice (positive control) was placed into 24 well plates, interspersed with wells containing sterile medium (negative control) and inoculated medium in conjunction with the specimens that would have their antimicrobial capacity tested. The quantity of cells in the experiment was standardized in order to attain the desired concentration of 5x10 3 cells per well (500 μl). The plates were incubated in an oven at 37°C, and the plate with S. mutans, was placed in an anaerobic jar. After 24 h, the optical density (OD) readout of each well was taken, starting by calibrating the spectrophotometer to 550 nm (Beckman Coulter DU 530 Spectrophotometer, Fullerton, CA, USA) by taking the sterile medium readout. The inoculated medium readout consisted of the positive control of growth for each microorganism 1,13 .
To obtain the percentage of microbial growth inhibition of the different groups studied, the following equation was used, with: (M) mean of optical densities of each material tested; (C) positive control of growth: % growth inhibition =100-(M/C)x100.

Qualitative analysis of antimicrobial activity
In addition to quantifying the antimicrobial activity by spectrophotometry, the specimens were analyzed by SEM, in order to visualize the microbial proliferation on the surface of each studied material. To obtain the initial record of the material surfaces (baseline), one specimen from each group was prepared on aluminum stubs, and gold sputtered for examination by scanning electron microscopy (JEOL-JSM; 5800LV, Tokyo, Japan). A voltage of 30 kV and low vacuum mode (45 Pa) was used. Immediately after the quantitative analysis of the antimicrobial activity, one specimen per microorganism from each group was randomly selected and ¿[HG stored in 2.5% glutaraldehyde and 0. The daily time when the specimens were moved into their new tubes was standardized. The specimens were manipulated with tweezers and they had been delicately dried with absorbent paper before they were reinserted into the sequence of the cycle. As the changes occurred, the solutions in which the specimens had been immersed were sent for laboratory analysis. It should be pointed out that the ultrapure water was the renewed vehicle, since the same specimen was kept from the beginning to the end of the experiment. The purpose of this ZDV WR LGHQWLI\ WKH GDLO\ ÀXRULGH UHOHDVH FDSDFLW\ by exhaustion of this same material throughout the cycle. During the experiment, no ionic supplement whatsoever was inserted. The ultrapure water was renewed in an attempt to eliminate any possible saturation of the medium.
This study generated a total of 270 accumulated fluoride release readouts, performed by ion Chromatography (DX 80 Ion Analyzer, Dionex, Sunnyvale, CA, USA), in order to perform the VHSDUDWLRQ DQG GHWHUPLQDWLRQ RI ÀXRULGH FRQWHQW in each solution. Before the readouts, the ion Chromatograph was calibrated with 5 specific patterns, these being: 1-ultrapure water; 2-pattern 1 (F -concentration=0.02 mg/L); 3pattern 2 (F -concentration=0.2 mg/L); 4-pattern 3 (F -concentration=1.0 mg/L); 5-pattern 4 (F -concentration=2.0 mg/L). The data obtained were transferred and processed by an integrator, automatic module Dionex 4400 that promotes complete automation of the system. The time that HODSVHG IRU VSHFL¿F GHWHUPLQDWLRQ RI WKH GHVLUHG ion was approximately 12 minutes for each 1.5 mL sample of ultrapure water.

Statistical analysis
The percentage of growth inhibition data for each specimen of the XT, PLUS and FUJI groups obtained in the microbiologic analysis, as well as WKH ÀXRULGH UHOHDVH WHVWV ZHUH VXEPLWWHG WR WKH analysis of variance ANOVA and Tukey Test in the SPSS 17.0 software (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA). The difference between the means was considered VLJQL¿FDQW ZKHQ YDOXHV RI S ZHUH REWDLQHG The results obtained for the qualitative analysis of antimicrobial capacity were descriptively recorded. Table 1 shows the results obtained in the microbiologic test, specifying the activity of the materials in relation to the microorganisms analyzed, with respect to the microbial growth and inhibition.

Quantitative analysis of antimicrobial activity
To evaluate the microbiologic action of materials against S. mutans and L. casei, it was shown that RQO\ WKH 3/86 SUHVHQWHG D VLJQL¿FDQW UHGXFWLRQ LQ growth (p<0.05). With regards to action against C. albicans, the materials showed significant differences among them (p<0.05), with emphasis on the PLUS, since it was the material that SUHVHQWHG VLJQL¿FDQW LQKLELWLRQ SRWHQWLDO DJDLQVW the fungus (p<0.001).

Qualitative analysis of antimicrobial activity
The SEM photomicrographs shown in Figure  3 (1A, 2A, 3A) reveal the images of the negative control specimen surfaces from the XT, PLUS and FUJI groups, respectively. Images 1B and 3B denote S. mutans growth on XT and FUJI specimens, in this order. Visually, the greatest microbial growth occurred in the FUJI group (3B), whereas the PLUS group was found to have a contamination-free surface (2B), which also occurred in image 2C, in which no L. casei was observed on the surface of the specimen from the PLUS group. Images 1C (XT) and 3C (FUJI) also point out microbial growth, which was more expressive in the FUJI group. Lastly, it is possible to visualize great C. albicans proliferation on the    Table 2, and are expressed comparatively in Figure 4. One observes that the FUJI was the most outstanding material for fluoride release, especially in the first 24 hours, followed by the PLUS and XT, respectively. Although the latter characteristically does not UHOHDVH ÀXRULGH WKH UDWHV IRXQG ZHUH YHU\ ORZ DQG LQVLJQL¿FDQW EHLQJ GLUHFWO\ UHODWHG WR WKH DFFXUDF\ of the technique. On the tenth day of the cycle, the values obtained for the FUJI and PLUS were similar (p>0.05).

DISCUSSION
There has been much discussion about the maintenance of the oral health in patients submitted to orthodontic treatment, such as the formation of initial caries lesions, which even today, is a potential risk. In a study recently conducted with 230 individuals, 71.7% developed from 1 to 12 white spot lesions at the end of treatment, and 69.8% presented adequate oral hygiene conditions at the beginning of orthodontic therapy 17 . These data are indicative of the importance of instituting a good preventive program in these patients. Should there be clinical signs of disease progression, efforts must be redoubled in an attempt to   The results obtained demonstrated the greater HI¿FDF\ RI WKH 7UDQVERQG TM Plus Color Change as an antimicrobial material, since it showed the best percentages of growth inhibition for all the species evaluated. The Fuji Ortho TM LC presented the most unfavorable result with regards to antimicrobial FDSDFLW\ KRZHYHU LW VKRZHG WKH JUHDWHVW ÀXRULGH release, which raises questions about the direct relationship of fluoride with microbial growth LQKLELWLRQ ,I RQO\ ÀXRULGH UHOHDVH ZDV UHVSRQVLEOH for the antimicrobial potential of the material, WKH )8-, ZKLFK UHOHDVHG WKH PRVW ÀXRULGH LQ comparison with the other groups with regards to antimicrobial activity, which was not demonstrated LQ WKH ¿QGLQJV RI WKLV UHVHDUFK Considering physicochemical characteristics 10 , Lee, et al. 16 (2009) in their study, concluded that there is greater microbial adherence to the surfaces of the bonding materials than to the orthodontic appliance accessories, and associated this fact with the free surface energy of these materials. Greater adhesion of S. mutans to the Fuji Ortho TM LC was also found than on the composites, corroborating WKH TXDQWLWDWLYH DQG TXDOLWDWLYH ¿QGLQJV RI WKH study. It is also known that the surface roughness of materials is directly proportional to the increase in microbial adhesion 5,20 . Therefore, the fact that glass ionomer cement is a material with low resistance and greater roughness, when compared ZLWK FRPSRVLWHV DOVR MXVWL¿HV WKH EHKDYLRU RI the FUJI in the present study. With regard to chemical composition, the Fuji Ortho TM LC contains the co-monomer TEGDMA, described in the literature as being capable of stimulating microbial proliferation 13 Although the cytotoxicity of the material may be related to microbial growth inhibition, it cannot EH WDNHQ DV WKH RQO\ MXVWL¿FDWLRQ IRU WKH UHVXOWV found in this research, because all the materials tested released substances such as bisphenol A, Bis-EMA, EGDMA, TEGDMA, HEMA, among others, capable of promoting adverse biologic reactions 15,18 . Therefore, it would not be conclusive to attribute the bests results found for the PLUS exclusively to cytotoxicity.
:LWK UHVSHFW WR ÀXRULGH UHOHDVH WKH PDMRULW\ RI studies with the goal of quantifying it use the ionselective electrode in their methodology 2,7 , since there is a scarcity of the use of ion chromatography in the literature to proceed with such an analysis. This technique is frequently used when one desires to obtain the other components in the sample, because of its efficient ion detector system. In the present study, the chosen methodology for measuring fluoride ions was shown to be adequate and to have excellent precision because it determined anions and cations at trace levels 4 .
During the 15 time intervals of analysis for ÀXRULGH TXDQWL¿FDWLRQ LQ DGGLWLRQ WR WKH FODVVLF increase in release in the 24 h 7 , there were some PRPHQWV RI VOLJKW LQFUHDVH LQ ÀXRULGH UHOHDVH EXW QR FRQWLQXDOO\ GHFUHDVLQJ UHOHDVH ZDV YHUL¿HG ZLWK the passage of the time considered (Figure 4). 5HGXFWLRQ LQ WKH TXDQWLW\ RI ÀXRULGH LV H[SODLQHG by the fact that the components of the external layer of the material became exhausted, and were dissolved in the water. With regard to the PLUS JURXS WKH UHOHDVH RI ÀXRULGH FRQWDLQHG ZLWKLQ WKH matrix is a little limited by the resin components WKDW PDNH LW GLI¿FXOW WR GLVSODFH +RZHYHU VLQFH WKH matrix of the FUJI is more sensitive to hydration, there is less limitation of ion displacement 8 . This MXVWL¿HV WKH KLJKHU YDOXHV LQ WKLV JURXS DV ZHOO DV the more recurrent peaks, in spite of it having been equivalent to the PLUS on the 10 th day of the cycle.
,W LV SRVVLEOH WR DVVRFLDWH WKH LQLWLDO ÀXRULGH release from the surface and the later release from the subsurface of the material matrix. The initial DQDORJ EHLQJ D ³VXSHU¿FLDO ZDVKLQJ´ ZKHUHDV the release afterwards, said to be late, is allowed through the micropores and from the matrix of the material itself 7,11 .
2WKHU VWXGLHV KDYH DOVR YHUL¿HG D VPDOO TXDQWLW\ RI ÀXRULGH GHWHFWLEOH LQ WKH 7UDQVERQG TM XT, and the better performance of the Fuji Ortho TM LC when compared with the Transbond TM Plus Color Change. 7KH JUHDWHU ÀXRULGH UHOHDVH IURP UHVLQ modified glass ionomer cements (RMGIC) is attributed to the acid-base reaction between the DOXPLQRVLOLFDWH JODVV SRZGHU FRQWDLQLQJ ÀXRULGH and polyacid liquid, which results in the release of ÀXRULGH LRQV 2,7 . The porosity of the material also has D VWURQJ UHODWLRQVKLS ZLWK WKH TXDQWLW\ RI ÀXRULGH UHOHDVHG DQG WKH JUHDWHU ÀXRULGH UHOHDVH E\ WKH FUJI in comparison with the composites is therefore MXVWL¿DEOH 29 . Ahn, et al. 2 (2011) observed that 50*,&V WHQG WR KDYH JUHDWHU ÀXRULGH LRQ UHOHDVH and recharge capacity, and these ions penetrate precisely into the spaces previously occupied by WKH ÀXRULGH WKDW ZDV UHOHDVHG ,W LV LPSRUWDQW WR emphasize that the process of matrix erosion is GLUHFWO\ UHODWHG WR WKH GLIIXVLRQ RI LRQV DQG ÀXRULGH release 27 .
In spite of the more expressive fluoride UHOHDVH RFFXUULQJ LQ WKH ¿UVW IHZ KRXUV RQH FRXOG DWWULEXWH WKH UDSLG IRUPDWLRQ RI FDOFLXP ÀXRULGH on the enamel surface to this factor, which is of clinical importance in protecting the tooth against demineralization 2,7,21 . In a previous study by Gorton and Featherstone 12 (2003), it was proved that irrespective of the antimicrobial potential, the ÀXRULGH FRQWDLQHG LQ WKH )XML 2UWKR TM LC, used for the bracket bonding, was capable of promoting a cariostatic effect in vivo, when compared with the Transbond TM XT.
In view of the above discussion, the presence of fluoride available to patients submitted to orthodontic treatment is of extreme importance in preventing the appearance of caries lesions 9 , highlighting the bonding material capable of exercising this function 22 . In a similar manner, the interesting factor with regards to the antimicrobial activity of the material also refers to the protection against its surface degradation. The material capable of reducing microbial growth, and particularly microorganism adhesion on its surface, is able to prevent a cycle of deterioration, since bacterial growth on the material is capable of LQFUHDVLQJ LWV URXJKQHVV DQG DFFHOHUDWLQJ ELR¿OP accumulation 3 .

CONCLUSION
This study supports the hypothesis that bonding materials can contribute positively to orthodontic treatment outcomes, with an essential role in the maintenance of health and balance in the oral cavity. The importance is emphasized of the material being able to withstand microbial attacks in order to maintain its integrity and that of the adjacent tissues. In this respect, the Transbond TM Plus Color Change was outstanding among the others, and the Fuji Ortho TM LC presented less satisfactory results. However, with regard to the ÀXRULGH UHOHDVH FDSDFLW\ WKH )8-, JURXS JXDUDQWHHG more expressive results, emphasizing its relevant role in anticariogenic activity. Therefore, one must consider that there is no ideal material, and the FKRLFH VKRXOG EH EDVHG RQ VFLHQWL¿F ¿QGLQJV DQG clinical experience, respecting the individuality of each case.