Objectives:

To measure the levels of pentraxin-3 (PTX-3) in gingival crevicular fluid (GCF) in orthodontic young and adult patients in the first 2 weeks after the orthodontic appliance to determine whether those changes occur during orthodontic treatment and if those values could be the expression of an inflammatory state.

Materials and Methods:

GCF samples were collected with paper strips from 16 orthodontic young patients and 13 orthodontic adult patients from an upper canine requiring distalization as a test tooth. A contralateral canine was used as a control tooth. The absorbed volume was eluted in 100 µL phosphate-buffered saline (pH  =  7.2). PTX-3 levels in GCF were determined using a commercial enzyme-linked immunosorbent assay kit, and the results were expressed in ng/mL.

Results:

The results showed an increase of GCF levels of PTX-3 from 1 hour before the orthodontic appliance to a maximum at 24 hours, followed by a decrease in both groups of adult and young patients.

Conclusions:

The results suggest PTX-3 involvement in periodontal orthodontic remodeling and the aseptic inflammation induced by the orthodontic forces.

Gingival crevicular fluid (GCF) is an exudate that precisely reflects the biologic events in the periodontium and may be used to detect the levels of certain biomarkers.1 Expression of biologically active substances such as cytokines,24 matrix metalloproteinases and their inhibitors,58 osteoprotegerin,9 tumor necrosis factor (TNF),10 neuropeptides,11 lactate dehydrogenase,12 aspartate aminotransferase,13 and leptin14 were studied in GCF during orthodontic tooth movement.

Pentraxin-3 (PTX-3), also known as TNF-stimulated gene 14 (TSG-14), is a 45-kDa glycoprotein with a 202 amino-acids C-terminal pentraxin domain, which is longer than that found in other pentraxins such as C-reactive protein (CRP) and serum amyloid P (SAP). Thus, PTX-3 is a “long” pentraxin produced especially by fibroblasts,15 neutrophils,16 and macrophages,15 cells abundantly found in periodontal tissues during orthodontic movements.17 To our knowledge, there are no data in the literature about PTX-3 activity in the GCF during periodontal remodeling in orthodontic teeth movement, so far. Recently, the levels of PTX-3 in GCF were determined in healthy and diseased periodontium18 but not in the healthy periodontium under mechanical stress from orthodontic appliance.

The aim was to measure the levels of PTX-3 in GCF in orthodontic young and adult patients, 1 hour before and at 4, 8, 24, and 72 hours, 1 week, and 2 weeks after the orthodontic appliance and to determine whether those changes occur during orthodontic treatment.

Subjects

The study was undertaken in the Orthodontics and Periodontology Departments of the University of Medicine and Pharmacy, Faculty of Dental Medicine of Craiova, Romania, and was approved by the Ethics Committee of this University.

The patients were enrolled after meeting the following criteria: general good health status, nonsmoking, clinically and radiological healthy periodontal tissues (no gingival bleeding, probing depths <3 mm, and no radiographic evidence of periodontal bone loss), no antibiotic therapy in the past 3 months, no use of anti-inflammatory drugs in the previous 30 days, good oral hygiene, and requiring upper canine distalization with first premolar extraction.

GCF samples were collected from 16 orthodontic young patients (7 girls and 9 boys, mean age 13.81 ± 0.98 years) and 13 orthodontic adult patients (8 women and 5 men, mean age 28.23 ± 3.37).

At the beginning of the study, the patients were given information about the purpose of the orthodontic treatment, period of time, and frequency of presentations. A formal detailed consent was obtained from adult patients or the tutors of patients under legal age. An observation chart was filled out. For the canine distalization, a fixed orthodontic device with stainless-steel direct-bonding 0.018″ Roth brackets (Dentsply GAC International, Bohemia, NY), 0.012″ NiTi archwire (Dentsply GAC International, Bohemia, NY), and laceback19 made from 0.010″ ligature wire was applied to the patients (Figure 1). A transpalatal arch was placed. The canine undergoing a distalization was used as test tooth (TT), and the contralateral canine served as control (CT). The control tooth was free of orthodontic force. TT and CT were selected from the same patient to avoid individual variability.

Figure 1

Intraoral view of orthodontic appliance on the test tooth.

Figure 1

Intraoral view of orthodontic appliance on the test tooth.

Close modal

The selected patients were informed about further keeping good oral hygiene. Motivation was kept during the entire study. At each presentation of the patient, the plaque index (PI) was measured.20 PI < 1 was considered an expression of good oral hygiene.

GCF Sampling

The sampling was performed 1 hour before and at 4, 8, 24, 72 hours, 1 week, and 2 weeks after the orthodontic appliance was activated.

GCF samples were collected from the distal side of the upper canines used as CT and TT using a technique previously described,21 prior to any other clinical procedures to avoid blood contamination.

The tooth was isolated with cotton rolls, and supragingival plaque was removed with a curette (HU-Friedy, Chicago, IL), without touching the marginal gingiva. The sites were gently dried with an air syringe, and a saliva ejector was put in place to avoid any salivary contamination. The GCF was collected with paper strips (PerioPaper, Oraflow, Amityville, NY) inserted into the crevice until mild resistance was felt and left there for 30 seconds. The paper strips contaminated with saliva or blood were discarded. The absorbed GCF was measured with a precalibrated device (Periotron 8000, Oraflow) and then was eluted from the paper strips in 100 µL phosphate-buffered saline (pH  =  7.2). The eluted samples were stored in polypropylene tubes at −20°C prior to analysis.

PTX-3 Immunological Analysis in GCF

PTX-3 levels in GCF were determined using a commercial ELISA kit (Human Pentraxin 3/TSG-14 Quantikine ELISA kit, R&D Systems, Minneapolis, Minn) after the centrifugation of tubes at 2500 g at 4°C. The results were expressed in ng/mL.

Every kit component was used according to the manufacturer's indications. Reading was performed at 450 nm with a correction at 540 nm to reduce optical imperfections on the reading plate.

Statistical Methods

The mean ± standard deviation (SD) were used to express the values of PTX-3 levels. After Friedman's test, a Wilcoxon test was performed to compare the means within each group and Mann-Whitney test to compare the means between groups (P < .05 for statistically significant differences) using professional software (SPSS 16.0, Chicago, Ill).

Throughout the study, the patients' periodontal health was very good, with no accumulation of plaque (PI < 1) and no indication for antibiotic or anti-inflammatory therapy; therefore, no patients were excluded from the study.

The results obtained for the TT in the group of young patients showed an increase of GCF levels of PTX-3 from 1 hour before the orthodontic appliance (−1 hour as baseline level) to a maximum at 24 hours (approximately 2.5-fold compared with the baseline level), followed by a decrease reaching the baseline level at 2 weeks. Statistically significant differences (P < .05) were found between mean levels of PTX-3 at 4, 8, 24, and 72 hours, 1 week, and the baseline level. There was no statistically significant difference between the levels at 2 weeks and the baseline level (Table 1).

Table 1

Pentraxin-3 (PTX-3) Levels in Gingival Crevicular Fluid (GCF) of the Test Tooth (TT) at Different Moments of Orthodontic Treatment

Pentraxin-3 (PTX-3) Levels in Gingival Crevicular Fluid (GCF) of the Test Tooth (TT) at Different Moments of Orthodontic Treatment
Pentraxin-3 (PTX-3) Levels in Gingival Crevicular Fluid (GCF) of the Test Tooth (TT) at Different Moments of Orthodontic Treatment

In the group of orthodontic adult patients, the results showed an increase of PTX-3 values in GCF of TT from 1 hour before application of orthodontic device (−1 hour at baseline level) to a maximum at 24 hours (approximately twofold compared with the baseline level) followed by a decrease reaching the baseline level at 1 week. Statistically significant differences (P < .05) were found between the levels of PTX-3 in GCF at 8, 24, and 72 hours and baseline. No significant difference was found between the values at 4 hours, 1 week, 2 weeks, and baseline (Table 1).

PTX-3 levels in TT in young patients at 4, 8, 24, and 72 hours and 1 week were greater than in adults, reaching a statistically significant difference at 8, 24, and 72 hours (P < .05). In the group of young patients, the values grew more rapidly than in adults, recording at 4 hours a statistically significant difference from baseline, while in adult patients, a significant difference appeared at 8 hours. In young patients, the values returned to the baseline levels within 2 weeks, but in adults, the decrease was faster, within 1 week (Figure 2).

Figure 2

Comparison between evolution of pentraxin-3 levels in gingival crevicular fluid of the test tooth at different moments of orthodontic treatment in young and adult patients.

Figure 2

Comparison between evolution of pentraxin-3 levels in gingival crevicular fluid of the test tooth at different moments of orthodontic treatment in young and adult patients.

Close modal

There were no statistically significant differences between levels of PTX-3 in CT at any moment during the study in both groups, young and adult patients, with the values being similar to baseline levels in TT. Also, there were no significant differences between volumes of GCF sampled between CT and TT. The findings showed no statistically significant differences between the baseline levels of the PTX-3 in adults and children, suggesting that the GCF levels of PTX-3 in the healthy periodontium without mechanical stimulation are not influenced by patient age. There were no statistically significant differences between men and women, indicating that gender does not influence the GCF levels of PTX-3 during orthodontic movements (data not shown).

The pentraxin superfamily is a group of proteins divided in two subgroups, according to the length of the amino acids chain: long pentraxins (including PTX-3) and short pentraxins such as CRP and SAP. PTX-3 is an acute-phase protein, and its involvement in some general diseases characterized by an inflammatory condition was already proven.

Very elevated plasma levels of PTX-3 (119.3 ng/mL) were found in dengue virus infection22 and also in severe meningococcal disease.23 High values of PTX-3 (26.0 ng/mL) were found in association with sepsis, and the effect of antioxidant treatment on PTX-3 levels was demonstrated.24 The implication of PTX-3 in innate immunity25 was studied, and levels of PTX-3 are also considered a marker of inflammation in psoriasis.26 

These studies determined the PTX-3 circulating levels in plasma or serum in a systemic disease. Except for a recent study that evaluated PTX-3 levels in plasma and GCF in patients with gingivitis and periodontitis,18 there are no available data so far about the levels of PTX-3 in GCF related to periodontal activity. There is more information about levels in GCF of a short pentraxin, CRP, in patients with periodontal disease. Correlations were established between the GCF levels of CRP and cardiovascular disease (CVD), and it was demonstrated that high GCF levels of CRP in periodontal disease correlate with the risk of CVD.27 The CRP serum levels were determined also in periodontal orthodontic remodeling,28 and it was shown that those values are not the expression of a systemic inflammation.

To our knowledge, the present study is the first that aimed to measure the GCF levels of PTX-3 in patients with fixed orthodontic treatment to see whether orthodontic tooth movement induces its change and if those values could be the expression of an inflammatory state.

Increased levels of GCF of a neuropeptide (substance P) were found at the same time intervals from the orthodontic appliance application in adult patients, with a peak at 24 hours (2.9-fold compared with baseline), but elevated levels were found also at 8 and 72 hours (2.1- and 1.6-fold, respectively).11 The authors correlated the expression of this biomarker to the mechanical stress and pain reported by the patients in the first 24 hours following the orthodontic treatment,29 with the perception of pain decreasing after 72 hours.30 In the same study by Yamaguchi et al.,11 it was shown that GCF levels of IL-1β also increase at 8, 24, and 72 hours, reaching a maximum at 24 hours (2.2-fold compared with baseline).

IL-6, epidermal growth factor, and TNF-α are elevated also in GCF during the first 24 hours, followed by a decrease to the baseline levels.3 

In the study by Apajalahti et al.,5 increased levels of MMP8 were reported immediately after the activation of the orthodontic appliance, and a significantly higher level of MMP8 in GCF was reported at 8 hours following orthodontic force initiation as an expression of periodontal remodeling. The same study indicated that MMP1 does not seem to contribute to the periodontal ligament remodeling during the first hours of initial tooth movement, but an elevated mRNA expression for MMP1 was found after 12 hours, which may suggest that MMP1 still plays an important role in periodontal remodeling along the deployment of the orthodontic treatment.6 

PTX-3 levels in TT indicated a greater reactivity in young patients, similar to the expression of other biomarkers. Ren et al.31 showed that elevated levels of IL6, prostaglandin E2, and granulocyte macrophage colony–stimulated factor may be reached after more than 24 hours in adults, while in juvenile patients, the increase is faster, in the first 24 hours. Kyomen and Tanne32 found a significantly greater proliferative activity of the periodontal ligament cells in young rats compared with adults with a fixed orthodontic device. Those results show that in the case of orthodontic tooth movement, the inflammatory reaction is faster and more intense in juveniles, perhaps because their immune system is in a continuous state of alert.

In this context, the findings of the present study seem to indicate that PTX-3 participates in the very complex chain of mediators that modulate aseptic inflammation18 as a response to the mechanical stress induced by orthodontic forces. PTX-3, being an acute-phase protein, reflects with fidelity the inflammatory condition. PTX-3, as all other biologically active substances described in the previously cited studies, is produced by cells that are abundantly present in periodontal tissues during orthodontic movements,18 serving as a reasonable explanation why the levels of PTX-3 increase rapidly in GCF.

The maximum GCF levels of PTX-3 at 24 hours in both groups are inferior to the aforementioned19 level (2.83 ng/mL) that corresponds to gingivitis or chronic periodontitis (3.77 ng/mL), which are periodontal diseases contrary to the orthodontic treatment. These values of PTX-3 determined by our team might be an expression of the complex biological activity in the periodontium taking place during the initial orthodontic treatment without any pathological consequences. This theory is supported by the self-decreasing of those values and the good periodontal health of these patients during the 2-week period of the study. Gingivitis is a superficial form of periodontal disease characterized by the presence of inflammation due to bacterial accumulation because of poor oral hygiene. In the present study, all patients had good oral hygiene, favored also by frequent visits for monitoring and sampling in a short-term study, while in a long-term study, with rare presentations, bacterial plaque has accumulated and colonies of Actinobacillus actinomycetemcomitans were identified.33 

With no other available data in the literature about the PTX-3 levels in GCF, our possibilities to compare are limited. Even with this lack of information and in the limitations of this study, we may say that those results reflect the aseptic inflammatory events occurring in the marginal periodontium during the orthodontic mechanical stress. The orthodontic appliance used develops light forces; thus, it could be interesting in future research to measure PTX-3 levels in GCF during orthodontic treatment with other methods of distalization, to see whether the expression of this biomarker correlates with force and velocity of tooth movement.34 Future studies might help clarify the exact place for PTX-3 in this complex network of substances in the GCF during orthodontic treatment.

  • PTX-3 levels in the GCF of orthodontic patients increase during the initial phase, and orthodontic young patients are more reactive than adults.

  • These findings suggest that PTX-3 is involved in the aseptic inflammation and periodontal remodeling in response to orthodontic forces.

This work was supported by a research project of Ministry of Education and Research from Romania, PN CDI grant 573/2008. The authors report no conflicts of interest related to this study.

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