INTRODUCCIÓN

Dental caries is a pathology that progresses chronically due to the lack of control of the factors that produce it. Mutans streptococci is one of the main etiological agents, these bacteria have long filamentary structures on their surface, which allow adhering and persisting in biofilms where acids originate that generate demineralization of the enamel due to the alteration of pH, buffer capacity and salivary flow, resulting in carious lesions or white spots, the main compli-cation that appears after the continuous use of fixed appliances during orthodontic treatment^1-3.

The use of appliances in orthodontics is a factor that predisposes carious lesions because it is a biofilm accumulation factor. White spot is associated with orthodontic treatment when oral hygiene is poor. Additionally, the adhesive systems of fixed orthodontic appliances increase plaque retention capacity and potential de-mineralization; therefore it is relevant to investigate options that allow its prevention and control^4-6.

In addition to a higher incidence of caries, with fixed appliances, an inflammatory reaction of the gingival tissues is evident, when the accumulation of bacterial plaque around the appliances attached to the surface of the teeth is accompanied by poor oral hygiene4,7. Orthodontic treatment produces changes in the oral environment due to the increase in the number of retentive surfaces for the biofilm, increasing the levels of Mutans streptococci⁸.

Nanotechnology includes the study, synthesis and operation of materials at molecular scales of so-called nanomaterials9. Bionanotechnology studies the effects and interactions between nanostructured materials and biological systems. Research in the field of dental nanomaterials has allowed new advances aimed at preserving and improving dental health, the concept of "nanodentistry" has become relevant as the science and technology of the use of nanostruc-tured materials in the diagnosis, prevention and treatment of oral pathologies such as caries, periodontal disease, malocclusions, soft tissue injuries^6,10,11.

The functionality of the AgNPs is due to the fact that the shape, size, agglomeration state, area and surface charge, increase their solubility which favors a better diffusion, distribution, absorption and availability; Many benefits in medicine have been attributed, mainly in the con-trol of infectious diseases. The use of AgNPs in different materials for dental use is very func-tional due to the low probability of developing bacterial resistance and high efficacy against biofilms, they can attack multiple sites within the cell in a very low concentration (0.5 - 1.0 %), altering the permeability and respiratory functions of the cell. Some studies show its effec-tiveness in stopping the advancement of caries and periodontal disease; they can also be used in aqueous irrigating solutions for root canal treatment, such as nanofilled particles for filling materials, adhesives and composites, in the coating of titanium implants and in drug formula-tions including mouthwashes^12-14.

To evaluate the antimicrobial activity of AgNPs, it is necessary to guarantee their stability as biocompatible agents and their biological effects through biofunctionalization processes15. The synthesis requires a control of the size and shape to obtain a group of free particles with certain properties. It is common for the synthesis in solution to be carried out using a metal precursor (AgNO3), a reducing agent (NaBH4 or Ac Ascorbic acid) and a stabilizer (CTAB Ce-tyl trimethyl ammonium bromide)^16,17.

The experiment evaluated the antimicrobial activity of discs soaked up with AgNPs in ortho-dontic adhesives at different concentrations on Mutans streptococci, by measuring inhibition haloes, based on the Duraf-fourd scale of antimicrobial sensitivity.

 

Materials y methods

In vitro experimental study; Solutions of AgNPs less than 100 nm in concentrations of 25, 75, 125 and 175 ppm were prepared, diluted in an orthodontic adhesive, the dilutions were soaked up in disks and evaluated on an infinite population of Mutans streptococci ATCC 25175 bacteria.

Prior to the experiment, AgNP solutions were synthesized and prepared in the Nanostructures laboratory of the Faculty of Chemical Sciences of the UCE, using silver nitrate and collagen in solution, in addition to NaBH4. The nanoparticles were characterized with the technique of analysis of hydrodynamic diameter of very small particles, by means of "Dynamic Light Scat-tering" (DLS Horiba Nano Particle Analyzer SZ-100); the size, shape and type of the AgNPs were determined by Atomic Force Microscopy (Park Systems AFM NX10). Based on the inclusion criteria, AgNPs less than 100 nm were added at the study concentrations to the or-thodontic adhesive (Transbond® XT, 3M. Following the same procedure for concentrations at 25, 75 and 125 ppm, for 175 ppm they were used the following parameters:

1,5 mL x 0,01 m.mol / mL = 0,015 m.mol ------> 4,5 mL = 0,0033 M Ag
0, 0033 M Ag x 107, 8682 g/ mol x 1000mg / g = 359, 56 mg / L = ppm Ag Sol madre
4,87 mL + 0,13 mL agua tipo 1 = 5 mL ---> 350 ppm
1 mL Sol + 1 mL adhesivo = 2 mL / 0,350 mg Ag x 1000 mL = 175 ppm

For the activation of the bacteria, the strain of Mutans streptococci ATCC 25175 was used, in a laminar flow chamber, following the manufacturer's instructions, it was inoculated with a handle in sterile test tubes with 2 ml of BHI broth (Brain Heart Infusion), incubating until growth in the broth is equivalent to a turbidity of 0.5 on the Mc Farland scale, equivalent to 1 x 108 colony forming units per milliliter.

Later, the microorganism was inoculated in 20 Petri dishes with Müller-Hinton agar supple-mented with 5% blood. In each box, 6 paper discs were placed, soaked respectively with the AgNP solutions in the orthodontic adhesive at 25, 75, 125 and 175 ppm, chlorhexidine 0.12% and distilled water. The 20 boxes were incubated in anaero-biosis conditions at a temperature of 37 ° C for 48 hours. The inhibitory effect was determined by measuring the halos of inhibi-tion by means of a millimeter ruler (Microbial Sensitivity Calibrator, MSD - Merck Sharp & Dohme).

With the inhibition halos generated, a database was made and the percentages of length of halos were compared based on the tables of Duraffourd antimicrobial activity, which estab-lishes the diameter of the bacterial growth inhibition halo and places it within the following parameters: Null: (-) less than or equal to 8 mm; Limit sensitivity: = (+) from 9 to 14 mm; Av-erage sensitivity: = (++) from 15 to 19 mm; Highly sensitive: = (+++) equal to or greater than 20 mm¹⁸.

 

Results

The results of the inhibition halos produced with each concentration of AgNPs, showed that in the qualitative evaluation of sensitivity it is evident that 100% of the samples at 25 ppm have activity | null on Mutans streptococci; at 75 ppm 50% (10; 20) shows limit sensitivity; samples at 125 ppm and 175 ppm have antibacterial power with 100% limit sensitivity (20; 20); Chlorhexidine 0.12% exists 60% (12; 20) with medium sensitivity and 40% (8; 20) highly sensitive. table 1. figure 1.

 

Tabla 1. Sensibilidad de las sustancias; Sensitivity of substances

Fuente: Base de datos de la Investigación
Elaborado: Los autores

Figura 1. Sensibilidad de las muestras; Sample sensitivity Fuente: Base de datos de la Investigación
Elaborado: Los autores

Discussion

According to the methodology used, it was possible to observe that the orthodontic adhesive with dilutions of 125 and 175 ppm can generate an inhibitory effect, although the efficacy has been less than that of chlorhexidine, the long-term effect of AgNPs could be decisive in inhibition on Mutans streptococci.

Tristan and colls.7, in their study "Future impact of nanotechnology on medicine and dentistry", they state that there are more and more specific applications such as silver nanoparticles in medicine and dentistry, they are used as a safer alternative for dental fillings as they have antiwear, antifungal and antibacterial properties. For Cardoso⁸ there is an increasing number of reports on the bactericidal activity of Ag-NPs and on their activity against bacterial bio-films.

The efficacy of AgNPs on S. mutans has already been demonstrated, in a similar study Murga and colls.1 evaluated in vitro the antimicrobial efficacy of nanoparticles by incorporating them into the adhesive placed in the dental enamel adjacent to the fixed orthodontic appliances (brackets). in 40 premolars inoculated with Mutans streptococci to count colony-forming units, finding that on day 15 a decrease in the presence of Mutans streptococci in the samples.

According to Krzyściak and colls.2 Silver nanoparticles (Nps Ag), have high efficacy against biofilms because they can attack multiple sites within the cell in a very low concentration (0.5-1.0%) to prevent bacterial growth. In the present study, inhibitory efficacy was found on a specific strain, the data should be confirmed in new studies testing other concen-trations and the long-term antibacterial effect.

Gómez⁹ analyzed the antimicrobial effect of these NPsAg on Gram (-) bacteria (Pseudomonas aeruginosa) and Gram (+) bacteria (Staphylococcus aureus), their results allow us to conclude that NPsAg have a high bactericidal capacity, since it produces a mortality of 99.9% with only small doses of them. In addition, NPsAg-modified Ti / TiO2 substrates were pre-pared, which were shown to be effective in inhibiting the formation of biofilms of both bacte-ria (Pseudomona aeruginosa and Staphylococcus aureus); highlighting the importance of the results of this work in relation to implantable titanium materials.

Our results coincide with previous studies that show an inhibitory effect on Mutans strepto-cocci, these concentrations are lower than those expressed by Padovani and colls.², in his analysis. Therefore, the research hypothesis is verified: There is antimicrobial activity of the silver nanoparticles at different concentrations on the Mutans streptococci in orthodontic adhesive systems with a significant difference with respect to 0.12% chlorhexydine.

Castaneda and colls.⁶ states that research in the field of dental nanomaterial has allowed new advances in nanotechnology aimed at preserving and improving dental health. In this sense, Cardoso19 indicates that there is an increasing number of reports on the bactericidal activity of AgNPs and on their activity against bacterial biofilms, and Padovani and colls.¹² reported that there is a low probability of developing bacterial resistance and that the high efficacy against biofilms must be that they can attack multiple sites within the cell in a very low con-centration (0.5-1.0%). Flores20 analyzed the antimicrobial effect of AgNPs on Aeruginous pseudomonas and Staphylococcus aurous, the results verified the high bactericidal capacity, producing a 99.9% mortality with only small doses of nanoparticles.

The results obtained in this research show that there is a coincidence with the aforementioned approaches, when observing that there is an inhibitory effect on Mutans streptococci, however, that the concentrations of AgNPs are lower than those studied by Padovani and colls.7. The research hypothesis is verified: There is antimicrobial activity in AgNPs at different concentra-tions on Mutans streptococci in orthodontic adhesive systems with a significant difference with respect to 0.12% chlorhexidine. However, new studies should demonstrate long-term efficacy and evaluate adhesives to ensure that their properties would not be altered.

 

Conclusions

Silver nanoparticles smaller than 100 nm, included at 125 and 175 ppm in orthodontic adhe-sives, having antimicrobial activity on Mutans streptococci, however, at study time they do not reach the sensitivity of chlorhexidine to 0.12%.

 

Bibliografía

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	Luis Martin Guevara Ruiz; https://orcid.org/0000-0002-7958-6918
	Pablo Bonilla; https://orcid.org/0000-0003-13711920
	María Fernanda Caicedo Breedy; https://orcid.org/0000-0002-3118-1196