Successful and failed mini-implants: microbiological evaluation and quantification of bacterial endotoxin

Abstract Objectives Using two groups of mini-implants (successful and failed) the objectives of this in vivo study were: to evaluate the microbial contamination by the checkerboard DNA-DNA hybridization technique and to quantify the bacterial endotoxin by the limulus amebocyte lysate assay. Material and Methods The 15 successful and 10 failed mini-implants (1.6 mm diameter × 7.0 or 9.0 mm long), placed in the maxilla and/or mandible, were obtained from 15 patients undergoing orthodontic treatment. Data were analyzed statistically by the Wilcoxon rank-sum test using the SAS software (a=0.05). Results All 40 microbial species were detected in both groups of mini-implants, with different frequencies. No differences were observed between the groups with respect to microbial complexes (blue, purple, yellow, green, orange, red and other species) and endotoxin quantification (p>0.05). Conclusion Neither microbial contamination nor endotoxin quantification was determinant for the early loss of stability of the mini-implants.


Introduction
In the last decades, mini-implants have been widely used in Orthodontics as temporary bone anchorage devices to provide greater mechanical control with no need of patient cooperation 1  It is known that the periodontopathogenic microbiota predominantly consists of anaerobes 8 , mostly Gram-negative microorganisms 9 , which have endotoxin (also known as LPS due to its lipopolysaccharide nature) in their cell wall 10 . Endotoxin is released after the death or multiplication of these bacteria and represents a major virulence factor by acting as a potent stimulus for proinflammatory cytokine expression and amplification of the host immune response 11 , resulting in the development of inflammatory reaction and bone resorption [10][11][12] .
Microbial contamination and persistent peri-implant inflammation are two potential causes to be considered and thus microbiological analyses and detection of endotoxin on mini-implants with and without stability should be performed. This knowledge could lead to the development of strategies that can guarantee the long-term success of mini-implants.
Using two groups of mini-implants -stable (successful) and unstable (failed) -the objectives of this in vivo study were: 1) to evaluate the microbial contamination, using DNA probes for 40 bacterial species, by the checkerboard DNA-DNA hybridization (CDDH) biomolecular technique and 2) to quantify the bacterial endotoxin in both groups of mini-implants by the limulus amebocyte lysate assay.

Material and methods
After the research protocol was approved by the institutional Ethics Committee (Process #19866013.0.0000.5419), the patients or their legal representatives signed a written informed consent form for participation. The Declaration of Helsinki guidelines were followed in this investigation.
Initially, sample calculation was performed using SAS Power software and Sample Size 3.1 software for the Wilcoxon two-sample test and a test power between 0.6 and 0.887, respectively, with differences of medians between groups of 200,000 to 400,000 bacteria.
A total of 15 patients, aged between 11 and 49 years, of both genders, who were under corrective orthodontic treatment with fixed appliances at the Orthodontics Clinic were enrolled in the study within a period of 12 months. The participants had good general and oral health, were nonsmokers and had not used antibiotics or anti-inflammatory drugs within 3 months before the mini-implant removal. Two groups of miniimplants were obtained: 15 well-fixed mini-implants (successful), which were removed after completion of orthodontic mechanics or at the end of the treatment, and 10 unstable mini-implants, which were removed early because of excessive mobility and became loose before the desired tooth movement could be achieved

Results
The descriptive analysis of patient data showed no statistically significant difference between groups of successful and failed mini-implants with respect to sex and age. Only the mean time of permanence in the mouth of the mini-implants presented significant difference between groups (p<0.05).
All 40 microbial species of the Actinomyces group, purple, yellow, green, orange, red complexes and other species (100%) were observed in both groups, although with different frequencies (Figure 1). No significant difference (p=0.2824) was found between successful and failed mini-implants considering the frequency of the microbial complexes.
Regarding the semi-quantitative analysis (bacterial cell count), the median of number of microorganisms of the 40 species in the group of successful and failed mini-implants was 12,950,000 and 8,490,000, respectively. No significant difference was found between the groups regarding the total number of microorganisms (p=0.75480). Considering the bacterial species alone, although there was an increase for P. micra, T. denticola and E. saburreum, it was not statistically significant (p>0.05), after adjusting for multiple comparison test (FDR) ( Table   1). No significant difference was observed among the bacterial complexes in the semi-quantitative analysis (  It has been used in Orthodontics to evaluate the contamination of metallic and ceramic brackets 12,[17][18][19] and the subgingival microbiota in patients undergoing orthodontic treatment 20 .
In this study there was no significant difference in the frequency of complexes between the groups. In a