Correlation of IL-17 level in gingival crevicular fluid of orthodontically induced inflammatory root resorption

Objective To investigate IL-17 expression in orthodontic tooth movement and orthodontic nickel-titanium spring-induced inflammatory root resorption. Methods Orthodontic nickel-titanium springs were ligated between the bilateral maxillary first molar and the incisors of the rats to establish a rat model of orthodontic tooth movement (OTM), each rat was subjected to two cycles of near-GCF and peripheral blood serum collection before and after force application, and IL-17 levels in GCF and serum were measured quantitatively by ELISA. Morphological changes in periodontal tissue and root of the experimental dentine were evaluated by hematoxylin and eosin staining. Tartrate-resistant acid phosphatase staining and immunohistochemistry were used to determine the osteoclast number and expression changes in IL-17, receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) in the periodontal tissues, respectively, on the pressure side of the experimental tooth. Results IL-17 was detected in GCF and serum. The pressure area exhibited alveolar bone resorption only at a force of 20g. Additionally, a force of 60g led to root resorption. IL-17, RANKL/OPG and osteoclast number showed similar trend that all expressed increasing high level at early stage, then significantly decreased from days 5 to 14, and revealed 60g group the highest expression level while 0g group the lowest. The change in the IL-17 level in the GCF was strongly correlated with IL-17 and RANKL/OPG expression levels and osteoclast numbers in the periodontal ligament. Conclusions The results indicated that measuring IL-17 level in GCF can predict the risk of alveolar bone and root resorption induced by orthodontic treatment.

IL-17 can activate neutrophils to mediate inflammation, 1 participate in bone resorption in periodontitis, 2 induce the differentiation of monocyte osteoclasts (OCs), 3 and induce osteoblasts (OBs) to express receptor activator of nuclear factor κB ligand (RANKL), which generates osteoclasts through the osteoprotegerin (OPG)/RANKL/RANK signal transduction pathway. 4 Orthodontic treatment can induce root resorption, which is characterized by inflammation and termed orthodontically induced inflammatory root resorption (OIIRR). 5 Animal studies have demonstrated increased IL-17 expression in gingival crevicular fluid (GCF) on the pressured side. 6 The positive expression of IL-17 is highly correlated with the relative root resorption area. 6,7,8 GCF measurements can help confirm the reconstructed state of the periodontium following tooth removal.
Enzyme-linked immunosorbent assay (ELISA) to detect IL-17 in GCF and peripheral blood may be useful to confirm that the IL-17 level in peripheral blood correlates with that in GCF. 9,10 We hypothesized that the force activation of IL-17 production in GCF may contribute to OIIRR. To test this hypothesis, we determined whether orthodontic force induced the activation of IL-17 production in GCF and peripheral blood, and consequently promoted OIIRR by enhancing osteoclastogenesis in a rat model of orthodontic tooth movement (OTM). We investigated whether the IL-17 level in GCF could be indicative of OIIRR.

Animals and Treatments
Sixty-five 8-10-week-old male Wistar rats were used in this study. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the university where the study was performed.

Force Application
The rats were divided randomly into four groups (0g, 20g, 60g, and control). Twenty rats were included in each of the three force groups, and five rats were included in the control group (Table 1).
To establish the OTM animal model, orthodontic nickel-titanium coiled springs 1 mm in length (AOJIE, Hangzhou, China) were ligated between the bilateral maxillary first molar and the incisors of the rats using flowable restorative resin to deliver a force of 0g, 20g, or 60g.
The OTM rats were evaluated at different time points (Table 1) to simulate different OIIRR states. Before and after the forces were applied, GCF was collected from the mesio-bilateral maxillary first molar using absorbent paper and orbital venous plexus blood was drawn for use as peripheral blood.

Tissue Specimen Preparation
The animals were euthanized under deep anesthesia. We selected the intact bilateral maxillary first molar and periodontium as tissue specimens. They were fixed in 4% paraformaldehyde for 24 h (Bio Basic, Markham, Ontario, Canada), decalcified in 10% EDTA-disodium salt for 6-8 weeks (Kelong, Chengdu, China), dehydrated in an alcohol gradient, and embedded in paraffin.

GCF Sampling
GCF samples from the mesial side of bilateral maxillary first molar were collected before and after force application. The study sites were gently dried using an air syringe and were isolated by cotton rolls. Filter paper strips (GAPADENT CO., LTD) were placed gently into the gingival sulcus until a minimum of resistance was felt, left there for 30 s, then put strips into sterilized EP tube. Repeat for eight times at the same site with 1 min interval for each collection. Strips visibly contaminated with blood were discarded. 150 μL phosphate-buffered saline (PBS) 0.05 % (w/v)-Tween-20 buffer was added to each EP tube containing strips and stored −80 °C until the laboratory analyses.

Serum Sampling
Blood (1 mL) was obtained from the orbital venous plexus and serum was separated by centrifugation (3000 rpm, 15 min, cool).

H&E and TRAP Staining
To observe the changes of the root, alveolar bone and periodontal ligament, H&E staining were conducted following the manufacture instruction, images were taken from the pressure side of first molar mesial buccal root with a light microscope(200x).

Statistical Analyses
Data were analyzed using t-tests, one-way analysis of variance, or least significant difference t-tests. Spearman rank correlation analysis was performed. A p-value < 0.05 was considered significant.

Force-mediated changes in GCF
In general, changes in GCF quantity were significantly different between the 0g, 20g, and 60g groups at all time points except at 1 d (p < 0.05). The changes in GCF levels were significantly different between the 0g and 20g groups at 3 and 5 d (p < 0.05), and between the 0g and 60g groups at 3, 5, 7, and 14 d (p < 0.05).

Changes in IL-17 levels in GCF after force application
In general, changes in the GCF IL-17 levels were significantly different between the 0g, 20g, and 60g groups at all time points except at 1 d (p < 0.05). Changes in the GCF IL-17 levels were significantly different between the 0g and 20g groups at 3, 5, and 7 d (p < 0.05), and between the 0g and 60g groups at 3, 5, 7, and 14 d (p < 0.05). The differences between the 20g and 60g groups reached statistical significance at 5, 7, and 14 d (p < 0.05) (Fig. 2).

Detection of IL-17 in the Peripheral Blood
The changes in IL-17 levels in peripheral blood were consistent with those for IL-17 levels in GCF (Fig. 3). Changes in the peripheral blood IL-17 levels in the 20g and 60g groups were significantly different than those in the 0 group at 5 and 7 d (p < 0.05), and between the 20g and 60g groups at 7 d (p < 0.05) (Fig.   3).

Non-force pressure
The surfaces of the tooth and alveolar bone in the control and 0g groups were smooth, and osteoclasts were not detected ( Fig. 4a and b).

IL-17 expression
The 0g group displayed weak positive IL-17 staining in individual cells; in contrast, there was a strong positive expression of IL-17 in the 20g and 60g groups in the cytoplasm of fibroblasts and osteoblasts (Fig. 5a-p).
Statistical analysis of the IL-17 MOD values revealed that IL-17 protein expression in the 20g and 60g groups were significantly different than that of the 0g group at each time point (p < 0.05).
The MOD values of IL-17 were significantly different between the 60g and 20g groups at 5 and 7 d (p < 0.05). There were no significant changes in MOD values throughout the study period in the 0g group (Fig. 5q).

RANKL expression
On the pressure side, RANKL was positively expressed in the cytoplasm of fibroblasts, osteoblasts, and osteoclast ( were significantly different between the 60g and 20g groups at 5, 7, and 14 d (p < 0.05) (Fig. 6q).

OPG expression
OPG was positively expressed in the cytoplasm and nucleus of fibroblasts and osteoblasts at all time points in the 0g force group.
In the 20g and 60g groups, the expression of OPG showed a tendency to gradually decrease and then increase (Fig. 7a-p).
Statistical analysis of the OPG MOD values revealed that OPG expression in periodontal tissues was not significantly changed in the 0g group. Comparison of the OPG MOD values between the 0, 20, and 60g groups revealed that OPG expression was generally not significantly different at all time points (p < 0.05), except for the 20g group on the first day. The MOD values of OPG were significantly different between the 60g and the 20g groups at 1 and 5 d (p < 0.05) (Fig. 7q).

TRAP Staining Results
For the TRAP staining, a PBS negative control group was included to show the background staining (Fig. 8a). Osteoclasts were observed as individual cells in the 0g group. In the 20g and 60g groups, the number of osteoclasts increased at 1 d, peaked at 3 and 5 d, respectively, and then decreased gradually (Fig. 8b-p).
Statistical analysis of the osteoclast count revealed that the number of osteoclasts in the 0g group did not significantly change.
Generally, the osteoclast numbers in the 20g and 60g groups were significantly different than those in the 0g group at all time points (p < 0.05), except at 1 d for the 20g group. Osteoclast numbers were significantly different between the 60g and the 20g groups at 5, 7, and 14 d (p < 0.05) (Fig. 8q).

Correlation Analysis Results
Spearman rank correlation analysis revealed that the level of  Table 2).

DISCUSSION
This study aimed to determine whether IL-17, RANKL, and The level of IL-17 in GCF after orthodontic treatment has been measured in clinical studies. IL-17 in GCF at the orthodontic-treated tooth was reportedly higher than that before treatment and on the tension side. 7 The concentration of IL-17 was shown to correlate with the magnitude of the orthodontic force. 8 When the level of IL-17 in GCF changes, the relevant components in peripheral blood also change. 13 The levels of IL-17 in GCF and peripheral blood were affected