Bacterial endotoxin adhesion to different types of orthodontic adhesives

Abstract Bacterial endotoxin (LPS) adhesion to orthodontic brackets is a known contributing factor to inflammation of the adjacent gingival tissues. Objective The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Material and Methods Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. Results There was endotoxin adhesion to all materials (p<0.05). No statistically significant difference was found between composites/bonding agents and acrylic resin (p>0.05). There was no significant difference (p>0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). Conclusions LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials.


Introduction
Orthodontic appliances are composed of different materials and accessories with irregular surfaces like brackets, ligatures, bands and wires that create additional sites that harbor dental plaque and oral microorganisms 21 , changing chemical properties of the oral medium 10 . Fixed orthodontic therapy inevitably predisposes patients to an increased risk of dental problems, as fixed appliances make an effective oral hygiene challenging and limit the mechanical cleansing of saliva flow, tongue and oral muscles 24 .
The use of orthodontic appliances can also increase the levels of periodontal pathogens in the supragingival and subgingival, associated with gingival inflammation that can occur during orthodontic treatment 14 .
Periodontopathogenic microbiota is predominantly composed of anaerobic microorganisms 9 , especially Gram-negative bacteria 18 , which contain endotoxin in their cell wall 23 . Bacterial endotoxin, also referred to as LPS due to its lipopolysaccharide nature, is released during bacterial multiplication or death, causing a series of important biological effects 23 that lead to inflammatory reaction and bone resorption in the periapical region 25 .
In vitro and in vivo studies 11,19 have shown that bacterial endotoxin adheres to metal brackets and such affinity affects endotoxin concentration in the gingival sulcus, contributing to inflammation of tissues adjacent to the brackets. By analogy, a similar process could occur on the surface of adhesive systems used for fixation of orthodontic brackets to the dental enamel. To the best of our knowledge there is not a previous study assessing bacterial endotoxin affinity for orthodontic adhesive systems. Therefore, the aim of this study was to assess whether LPS adheres to the components of orthodontic adhesive systems (bonding agent and composite resin), comparing two commercial brands.

Fabrication of specimens
In order to obtain the test specimens, it was used a circular Teflon matrix, manufactured at the Precision Workshop of the University of São Paulo, Ribeirão Preto, SP, Brazil. The matrix consisted of two nested parts: an outer portion and an inner portion in the form of a 3-mm-diameter plunger. Accompanying the matrix there was a 2-mm-thick spacer, which was engaged in the plunger between the two portions so that the outer potion was 2 mm higher than the inner portion, providing adequate thickness to the specimen. Therefore, forty disc-shaped specimens ( Table 1 shows the distribution of relative and absolute endotoxin levels (by scores) in experimental groups. Since no statistically significant difference (p>0.05) was found between the two composites or the two bonding agents, the materials were compared regardless of their brand. Figure 1 is a graphical representation of scores distribution between composites and bonding agents, and it shows a significantly higher endotoxin adhesion to bonding agents than to composites (p=0.0025).

Score
Transbond Endotoxin has high affinity for a variety of dental materials 1,7,13 , including acrylic resin commonly used as a temporary material 4 , and a high affinity for titanium (present in dental implants) with a significant decrease in titanium corrosion resistance 30 . Also in Dentistry, endotoxin is present in necrotic root canals 15 where its presence has been associated with periapical inflammation and bone resorption 6 .
Our option of using endotoxin derived from E. coli in this study was based on its broad indication, based on its proven toxicity, to evaluate the biological activity of LPS at different research levels 12,25 . In addition, the molecular structure of E. coli, according to Mattison, et al. 16 (1987), is representative of most endotoxins.
Moreover, this endotoxin is easier to obtain and cheaper.
The results of this in vitro study showed that bacterial endotoxin has affinity for adhesives frequently used in orthodontics. Previous studies have demonstrated that LPS also adheres to metallic brackets, contributing to the inflammation of tissues adjacent to the brackets 11,19 . However, the lack of studies assessing endotoxin adhesion to other orthodontic adhesive systems does not allow to compare our findings.
Numerous orthodontic adhesive systems are  According to the manufacturers, the bonding agents evaluated in this study do not contain inorganic fillers, while both composites have over 80% of inorganic particles by volume.
Considering the higher affinity of endotoxin for orthodontic bonding agents, additional care is recommended to orthodontists in the sense of avoiding "overwetting" and limiting the application of these materials to the bracket base. Excess material on dental enamel should be carefully removed to avoid leaving areas of bonding agent/composite exposed to oral medium, which could favor the adhesion of LPS to the materials and stimulate the occurrence of inflammation in the gingival tissues adjacent to the brackets.
Further laboratory research and clinical studies are necessary to compare and substantiate these findings.

Conclusion
The results of this study revealed that bacterial endotoxin (LPS) adhered to orthodontic adhesive systems. The bonding agents of both systems presented greater affinity for endotoxin than for composites.