Esthetic elastomeric ligatures: Quantification of bacterial endotoxin in vitro and in vivo

Introduction

The objective of this study was to evaluate in vitro and in vivo bacterial endotoxin (LPS) adhesion in polyurethane and silicone esthetic elastomeric orthodontic ligatures. The null hypotheses tested were: (1) there is no LPS adhesion in esthetic elastomeric orthodontic ligatures; and (2) there is no difference in the LPS adhesion between different brands of these ligatures.

Methods

For the in vitro study, 4 types of esthetic elastomeric ligatures were used (Sani-Ties and Sili-Ties [Dentsply GAC, Islandia, NY;] and Mini Single Case Ligature Stick and Synergy low-friction ligatures [Rocky Mountain Orthodontics, Denver, Colo]), contaminated or not with endotoxin solution. Replicas of twisted wire and cast stainless steel ligatures were used as control. For the in vivo study, 10 male and 10 female patients, aged 15-30 years, received the same 4 types of ligatures, 1 of each inserted in the maxillary and mandibular canines, randomly. Twenty-one days later, the ligatures were removed, and endotoxin quantification was performed using the Limulus amebocyte lysate test. Data were analyzed (α = 0.05) using the Kruskal-Wallis test and Dunn’s posttest or analysis of variance and Tukey’s posttest.

Results

GAC silicone group had the lowest median contamination (1.15 endotoxin units/mL; P <0.0001) in vitro. In the in vivo study, the GAC silicone group had the lowest mean contamination (0.577 endotoxin units/mL; P <0.001). In both studies, the other groups did not present a significant difference when compared with each other ( P >0.05).

Conclusions

LPS exhibited an affinity for all the tested polyurethane and silicone elastomeric ligatures. GAC silicone ligatures presented with lower amounts of LPS attached to their surfaces. Thus, both null hypotheses were rejected.

Highlights

  • In groups submitted to contamination with LPS, it adhered to all tested materials.

  • GAC silicone group had the lowest median of LPS contamination in vitro.

  • GAC silicone group had the lowest mean of bacterial endotoxin contamination.

Orthodontic appliances consist of various materials, such as brackets, ligatures, bands, and wires, that may cause bacterial biofilm retention , and hinder hygiene. , Therefore, during orthodontic therapy, there is a greater accumulation of microorganisms, leading to an increased risk of diseases, such as dental caries and periodontal problems. ,

The use of orthodontic appliances can also increase the levels of periodontal pathogens in the supragingival and subgingival biofilm, associated with gingival inflammation that can occur during orthodontic treatment.

Periodontopathogenic microbiota predominantly consists of anaerobic microorganisms, especially Gram-negative bacteria, , which contain endotoxin in their cell wall. Bacterial endotoxin, also referred to as LPS because of its lipopolysaccharide nature, is released in the bacterial multiplication or death, causing a series of biological effects that lead to the occurrence of an inflammatory reaction and bone resorption , by stimulating the expression of several proinflammatory cytokines and chemokines that induce osteoclastogenesis. , Because of these factors, even a small amount of released endotoxin can cause inflammatory responses and tissue damage, such as bone resorption, which has great biological significance.

Endotoxin has a high affinity for different materials such as metals, , silica, zirconium, , acrylic resins, ceramics, , and even titanium and titanium alloys. In vitro and in vivo studies have shown that bacterial endotoxin (LPS) adheres to metal brackets, , and such affinity can affect the 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 esthetic ligatures used for fixation of orthodontic wires engaged into brackets.

To the best of our knowledge, there are no previous studies assessing bacterial endotoxin affinity for orthodontic ligatures. Therefore, this study aimed to evaluate , in vitro and in vivo, LPS adhesion in polyurethane and silicone esthetic elastomeric orthodontic ligatures. The null hypotheses tested were (1) there is no LPS adhesion in esthetic elastomeric orthodontic ligatures, and (2) there is no difference in the LPS adhesion between different brands of esthetic elastomeric orthodontic ligatures.

Material and methods

In vitro study

Four different types of esthetic elastomeric ligatures were used and divided into 4 groups ( Table I ).

Table I
Esthetic elastomeric ligatures used
Product name Manufacturer Material Code Lot Expiration date
Sani-Ties Dentsply GAC, Islandia, NY Polyurethane 59-200-35 60759 2018/06
Sili-Ties Dentsply GAC, Islandia, NY Silicone 59-950-00 48993 2018/03
Mini Single Case Ligature Stick RMO, Denver, Colo Polyurethane J-00345 55399 2019/4/27
Synergy low-friction ligatures RMO, Denver, Colo Silicone J-00151 44764 2018/12/17

Five units of each type of ligature, taken from their original packages, were contaminated with the endotoxin solution (Groups I-IV, experimental groups) or were immersed in pyrogen-free water (Groups V-VIII, negative control groups).

Replicas of the same size and shape of the elastomeric ligatures were made with twisted GAC 0.10-in orthodontic wire ligature and cast stainless steel (Dentsply GAC, Islandia, NY), the latter being made in the Precision workshop of the University of São Paulo, Campus Ribeirão Preto, São Paulo, Brazil. They were used as an additional control, after undergoing sterilization in a dry depyrogenation oven at 200°C, for 2 hours. Five replica units of each metal material were used as an additional positive control, contaminated with endotoxin: (1) Group IX (replicas of twisted orthodontic wire ligature), (2) Group X (cast stainless steel replicas). As additional negative control, five replica units of each metal material were immersed in pyrogen-free water: (1) Group XI (replicas of twisted orthodontic wire ligature), and (2) Group XII (cast stainless steel replicas). Table II shows the in vitro groups.

Table II
In vitro groups
Group Material Contamination with LPS
I GAC silicone Yes
II GAC polyurethane Yes
III RMO silicone Yes
IV RMO polyurethane Yes
V GAC silicone No
VI GAC polyurethane No
VII RMO silicone No
VIII RMO polyurethane No
IX Twisted orthodontic wire ligature replicas Yes
X Cast stainless steel replicas Yes
XI Twisted orthodontic wire ligature replicas No
XII Cast stainless steel replicas No

In a laminar flow chamber, 350 mg of lyophilized endotoxin from Escherichia coli (Lipopolysaccharide B E.coli 055:B5; Sigma-Aldrich, Saint Louis, Mo) was suspended into 4.7 mL of pyrogen-free water in a Falcon tube, resulting in a 25 ng/mL concentration endotoxin solution. For contamination, specimens were immersed in 1 mL of the solution in plastic tubes for microcentrifuge, pyrogen-free (pyrogen-free tubes, BioWhittaker; Cambrex Corp, East Rutherford, NJ, USA) and placed under agitation (126 rpm) in an incubator at 37°C for 1 hour. The negative control specimens were immersed in pyrogen-free water.

After contamination with LPS, the specimens were individually placed in new nonpyrogenic plastic tubes (pyrogen-free tubes, BioWhittaker, Cambrex Corp) containing 1 mL of pyrogen-free water (recovery solution) and taken to an ultrasonic cleaner (Ultracleaner USC 1600A; Unique Indústria e Comércio de Produtos Eletrônicos Ltda, Indaiatuba, São Paulo, Brazil) for 15 minutes to release endotoxin from the material.

Endotoxin quantification in the ligatures and replicas was performed using the QCL-1000 test (Limulus amebocyte lysate [LAL], QCL-1000; Lonza, Walkersville, Md, USA) following the manufacturer’s instructions. LAL is a quantitative test for the detection of endotoxin with a sensitivity range of 0.1-1.0 endotoxin units (EU)/mL. A standard curve of known endotoxin levels was used to determine the amount of endotoxin in the samples. Specifically, 50 μL of solution of each known standard concentration (1.0 EU/mL, 0.5 EU/mL, 0.25 EU/mL, 0.1 EU/mL, and 0 EU/mL) were dripped in the wells of a nonpyrogenic 96-well polystyrene plate (Corning Incorporated, Corning, NY, USA). Next, 50 μL of the samples were diluted in pyrogen-free water at a ratio of 1:1 and added to the remaining wells. Then 50 μL of LAL solution were added to all wells containing samples or standards. The microplate was then incubated at 37°C for 10 minutes. After that, 100 μL of a chromogenic substrate, preheated to 37°C, was added to the wells, stirred, and incubated at 37°C for 6 minutes in the dark, following the same dripping protocol and maintaining a constant dripping rate. Subsequently, 100 μL of the blocking reagent (25% v/v glacial acetic acid in water) was added to stop the reaction.

The absorbance of each sample was determined using an enzyme-linked immunosorbent assay reader (Ultramark; Bio-Rad Laboratories, Hercules, Calif, USA) at 405 nm. Absorbance was considered to be directly proportional to endotoxin levels in the wells, and it correlated directly to the endotoxin concentration in the range from 0.1 to 1.0 EU/mL. The amount of endotoxin in each sample was expressed in EU/mL and calculated from the solution absorbance values with known endotoxin levels (standard) multiplied by the dilution factor.

In vivo study

The present study was approved by the Ethics Committee in Research of Dentistry School of Ribeirão Preto, University of São Paulo, Brazil (CAAE: 64421417.0.0000.5419). The sample calculation was performed by the SPSS SamplePower software (version 20.0; IBM Software; SPSS Inc, Chicago, Ill, USA), indicating that in a sample of 20 patients per group, there would be an 80% probability of a statistically significant effect being evidenced.

Twenty patients were selected for the study. Patients were all aged 15 to 30 years, with no specific ethnicity or gender, with good general health status, good oral hygiene, and having complete permanent dentition. All of the patients had initiated orthodontic treatment at the University of São Paulo, Ribeirão Preto, São Paulo, Brazil. The following inclusion criteria were considered in this study: no smoking, no use of antibiotics and/or mouthwashes with antimicrobial solutions within 3 months before the start of the study, no periodontal treatment within the previous 3 months, and no clinical signs of gingivitis.

All patients received verbal and written standardized oral hygiene instructions (tooth brushing with modified Bass brushing technique and flossing) from a single researcher (L.S.P.). A toothbrush (Colgate Professional Extra Clean; Colgate-Palmolive, New York, USA) and a fluoridated dentifrice (Colgate Máxima Proteção Anticáries; Colgate-Palmolive) were provided to each patient to minimize the interference of confounding factors on the results. The subjects were asked to brush 3 times daily, after meals, and were instructed not to use any hygiene products other than toothpaste and dental floss.

The patients received metal brackets (Morelli; Sorocaba, São Paulo, Brazil) on the maxillary and mandibular permanent teeth, from the maxillary right 5 to the maxillary left 5, fixed with orthodontic light-cured adhesive (Transbond XT System; 3M Unitek, Monrovia, Calif, USA) following the manufacturer’s instructions. After bonding the brackets, a 0.014-in orthodontic stainless steel wire (Dentsply, GAC, NY) was inserted and fixed with the 4 different types of esthetic elastomeric ligatures used in the in vitro study ( Table I ).

All of the 20 patients received esthetic ligature of each type on the canines (UR3, UL3, LR3, LL3), which were randomly placed with a split-mouth design, following the protocol conducted in a crossover-way to eliminate confounding factors. This procedure was performed with the aid of SAS for Windows (version 9.1.3; SAS Institute Inc, Cary, NC, USA). On the other teeth, conventional gray elastomeric ligatures of the Morelli brand (Morelli; Sorocaba, São Paulo, Brazil) were inserted. Twenty-one days later, the esthetic elastomeric ligatures were removed and placed individually in 1.5 mL plastic tubes for microcentrifuge (Eppendorf, Hamburg, Germany), apyrogenic, containing 1 mL of pyrogen-free water. Afterward, each tube was encoded and placed in an ultrasonic incubator (Ultracleaner USC 1600A) for 15 minutes to desorb the material adhered to its surface. The ligatures were then removed with a clinical clamp, and the tubes containing the suspension were stored at −20°C until the time of endotoxin quantification. This procedure was performed in the same manner as described in the in vitro study.

Statistical analysis

In the in vitro study, data obtained from the quantification of LPS from ligatures were submitted to the Kruskal-Wallis test and Dunn’s posttest. In the in vivo study, the data were submitted to the repeated-measures analysis of variance test and Tukey’s posttest. All analyzes were performed using the Graph Pad Prism 4.0 program (Graph Pad Software Inc, San Diego, Calif, USA), with a significance level of 5%.

Results

In vitro study

No LPS was observed in the ligatures that were not subjected to previous contamination (ie, ligatures did not present contamination by LPS from the manufacturing process [Groups V-VIII]). In addition, no contamination was found in the metal replicas belonging to Groups XI and XII.

In the groups submitted to contamination, it was observed that the LPS adhered to all tested materials (Groups I-IV and IX and X). Group GAC silicone was the one that showed the lowest median of endotoxin contamination (1.15 EU/mL; Q1 = 1.13; Q3 = 1.16) than all other groups: GAC polyurethane (1.17 EU/mL; Q1 = 1.15; Q3 = 1.19), Rocky Mountain Orthodontics (RMO) silicone (1.19 EU/mL; Q1 = 1.18; Q3 = 1.19), RMO polyurethane (1.22 EU/mL; Q1 = 1.20; Q3 = 1.22), cast stainless steel (1.19 EU/mL; Q1 = 1.18; Q3 = 1.20) and twisted orthodontic metal ligature (1.20 EU/mL; Q1 = 1.19; Q3 = 1.21) ( P <0.0001). It was not possible to find statistically significant difference among the other groups ( P >0.05). Figure 1 illustrates the results obtained in vitro.

Jun 12, 2021 | Posted by in Orthodontics | Comments Off on Esthetic elastomeric ligatures: Quantification of bacterial endotoxin in vitro and in vivo
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