A Study to Evaluate Il1 And Il6 Gingival Crevicular Fluid Levels in Adolescents and Young Adults During the Early Phase of Orthodontic Tooth Movement.

Background: Evaluating the biomarkers during orthodontic therapy allows us to monitor the effectiveness of the treatment, anticipate the future outcome, and also individualize the treatment according to the response of tissue to different force intensities. The crevicular fluid analysis is a valuable diagnostic method that can be used to examine noninvasive cellular metabolic activity. The goal of this study was to compare the levels of interleukin (IL)-1 and IL-6 in the gingival crevicular fluid (GCF) of clinically healthy adolescents and young adults throughout orthodontic treatment.
Methods: GCF was obtained from 20 orthodontic patients ranging in age from 11 to 28 years old. Measurements were done before (T0) and after (T24) the activation of distalization forces (T1). Enzyme-linked immunosorbent assay (ELISA) testing was used to look at IL-1 and IL-6. Two-sided t-tests were utilized as statistical testing.
Results: The crevicular fluid rate (0.57 L at T0 vs. 0.95 L at T1, P = 0.001) and IL-1 levels (15.67 pg/L at T0 vs. 27.94 pg/L at T1, P = 0.009) both increased significantly between the two time periods. Only a third of the sites tested positive for IL-6. At T1, adolescents have a considerably higher amount of IL than young adults (42.96 pg/L vs. 17.93 pg/L, P = 0.006).
Conclusion: Adolescent periodontal tissues are more susceptible to orthodontic forces than young adult periodontal tissues in the early stages of orthodontic treatment. Copyright:

INTRODUCTION

Orthodontic forces applied onto the tooth cause increased tissue pressure, leading to accelerated bone remodeling. Thus, alterations appear at the extracellular matrix, with alterations of the cell shape and cytoskeletal conformation. As a result of these changes, there are variations in the gingival fluid flow rate and composition. Nevertheless, the orthodontic-induced inflammation and inflammatory markers are transient. The most significant changes appear during the initial acute phase. The initial phase occurs 24–48 h after the orthodontic forces have been applied. This phase is characterized by a rapid and short movement of the tooth. Due to the compression of the periodontal ligament on the one hand, and the stretching, on the other hand, this phase is characterized by an acute inflammatory response, represented by vasodilation, chemo-attraction of the inflammatory cells, osteoblast and osteoclast progenitor cells. The orthodontically induced inflammation is accompanied by an increase of the inflammatory mediators (cytokines, prostaglandins, and leukotrienes), enzymes (metalloproteinase, alkaline phosphatase, and aspartate aminotransferase), growth factors, and neuropeptides, suggesting the fact that there are many factors involved in bone remodeling (immune, endocrines, and nervous). Cytokines are the first factors that influence the stimulation or inhibition of the biomechanical reactions that appear during orthodontically induced inflammation.[1234] The cytokines involved in the bone remodeling process are interleukin (IL)-1 (α, β), IL-6, and tumor necrosis factor (TNF) α. The study's goal was to compare the levels of inflammatory mediators such as cytokines in clinically healthy teenagers and young adults during orthodontic treatment's acute phase. Therefore, the null hypothesis (H0) underlying this study is that there is no statistically significant difference between the inflammatory mediators (such as the cytokines) between groups of healthy adolescents and young adults, respectively, during the acute phase of orthodontic treatment.

METHODOLOGY

The study consisted of 20 patients (10 females, 10 males), aged between 11 and 16 (8 patients) and 17 and 28 (12 patients) years, in our department. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee. Written informed consent was obtained from all patients, before the investigations.

Inclusion criteria

Indication for orthodontic treatment, with premolar extraction (all malocclusion classes were considered)

No systemic affections

Periodontal health

Periodontal health was assessed using the basic periodontal examination (BPE), using a World Health Organization probe (WHO probes 550B, LM-Instruments, Parainen, Finland). The study included only patients with scoring codes 0 (no bleeding on probing, probing pocket depths <3.5 mm, no calculus or overhanging restorations). Moreover, no evident radiographic bone loss was present.

Exclusion criteria

Systemic diseases that have a major role in the loss of periodontal tissues were excluded, such as genetic, metabolic, and endocrine diseases, inflammatory diseases, acquired immunodeficiency diseases

Periodontal diseases (BPE scoring >0, radiographic bone loss)

Systemic treatment with antibiotics or anti-inflammatory treatment in the month preceding the study.

The orthodontic treatment consisted of the application through bonding of metallic brackets (3M, Unitek, CA, USA) with a 0.22-inch slot and orthodontic tubes (3M, Monrovia, CA, USA), using the MBT technique on the maxillary and mandibular arches of young patients with different dento-maxillary anomalies. Brackets were applied through a self-etching technique using Transbond XT material (3M, Unitek), due to its hybrid filling and high resistance to compression and bending. Medium forces were applied, using closed coil springs and 0.19 × 0.25 SS wires (Forestadent, Pforzheim, Germany). All patients were clinically (basic periodontal examination) and radiologically evaluated, monitoring the quality of periodontal tissues to detect incipient signs of periodontal diseases. The crevicular fluid was collected from the distal zone of the canine, before the activation of the orthodontic treatment (control group) and 24 h after its activation (study group). The quantity of crevicular fluid volume was measured using a Periotron 8000 device (Pro-Flow Inc., Amityville, New York, NY, USA), which measures the capacitance modification on the filter paper. The gingival fluid volume was measured using Periotron. The strips contaminated with blood were not used but discarded. After reading the strips, they were introduced in an Eppendorf safe-lock cryo-tube, which contained 250 microliters (μL) of phosphate buffer solution. The immuno-enzymatic analysis of IL-1β and IL-6 was done following the protocol as indicated by the kit manufacturer, both for IL-1β and IL-6 (i.e., different protocols are required by the manufacturer for each of the two cytokines). The statistical software used was IBM SPSS Statistics Base version 26 (International Business Machines Corporation, Armonk, NY, USA). Statistical significance was considered at a 95% confidence level in all analyses, corresponding to P values of < 0.05. We have performed an a priori power analysis using IBM SPSS native power analysis tool to better ground the relevance of our study. We utilized a standard alpha measure of 0.05, the generally utilized level of alpha in medical studies. The power analysis was tailored to our two-sided test chosen for our methodology.

RESULTS

Gingival crevicular fluid (GCF) analysis revealed detectable values for IL-1β at both periods. We were able to identify IL-6 only on a third of the sites, the other values being below the test detection threshold. Thus, the mean value identified for IL-6 at T0 was 0.14 pg/μL, and for T1 1.27 pg/μL, values that were not taken into consideration in the statistics. Moreover, the identified values for IL-6 were lower than those identified for IL-1β. The inflammation caused by the orthodontic movement is followed by an increase in both gingival crevicular levels and inflammatory mediators. Table 1 presents the values 24 h before (T0) and after (T1) the initiation of the orthodontic treatment for gingival crevicular volume and IL-1β levels. Both these parameters showed increased values at T1. Between the two periods, there were significant changes both in the crevicular fluid rate (P = 0.001) and in IL-1β levels (P = 0.009).

Table 1

Gingival crevicular volumes (μL), IL1β values (pg/μL) before and after 24 h after the initiation of orthodontic treatment

	Gingival crevicular volume T0 (μL)	Gingival crevicular volume T1 (μL)	IL1β-T0 (pg/μL)	IL1β-T1 (pg/μL)
Mean	0.55	0.94	15.67	27.94
SE	0.05	0.13	2.21	3.92
SD	0.29	0.46	9.89	17.54
Minimum	0.28	0.37	6.42	13.09
Maximum	0.90	1.53	27.64	69.91
Confidence level (95%)	0.15	0.22	4.62	8.21
P-Value	0.001		0.009

Tables 2 and 3 show a comparison of IL-1β values by age groups in the two periods. Our findings show that adolescents had higher levels of IL-1 than young adults before and after starting orthodontic treatment. The mean value for IL-1 in adolescents increased from 23.50 pg/L at T0 to 45.96 pg/L at T1, whereas the concentration of IL-1 in young adults increased from 13.28 pg/L at T0 to 19.20 pg/L at T0. The level of IL-1 in adolescents was statistically significantly greater 24 h before (P = 0.031) and after the start of orthodontic treatment (P = 0.006).

Table 2

IL1β values (pg/μL) in adolescents (11-16 years old) and young adults (17-28 years old) at T0

Age	17-28 years	11-16 years
Mean	13.28	23.50
SE	1.01	4.30
SD	3.66	12.17
Minimum	6.42	6.47
Maximum	17.58	37.64
Confidence level (95%)	2.21	10.17
P-Value	0.031

Table 3

IL1β values (pg/μL) in adolescents (11-16 years old) and young adults (17-28 years old) at T1

Age	17-28 years	11-16 years
Mean	19.20	45.96
SE	1.53	6.71
SD	5.311	18.99
Minimum	13.09	20.28
Maximum	27.62	20.28
Confidence level (95%)	3.37	15.88
P-Value	0.006

DISCUSSION

Out of the wide range of inflammatory mediators, we chose IL-1β and IL-6 pro-inflammatory cytokines, due to their role in the destructive process of the marginal periodontium. IL-1β and IL-6 are among the first cytokines that modified the concentration of the crevicular fluid at the moment of application of orthodontic forces.[56789] In the study led by Emel Sari and Cihan Ucar,[2] the authors compared the concentration of IL-1β for patients undergoing orthodontic treatment with mini-implants, comparing the concentration of cytokine in the mini-implant sulcus, and at the level of the distalized canine, respectively. Due to the extended methodological similarities with our study, the results obtained by Emel Sari and Cihan Ucar[2] in their work are highly comparable.Firstly, the authors opted for an identical reporting of results, identifying the IL concentration relative to the gingival fluid volume (pg/μL). Furthermore, the dilution volume used (250 μL) was also identical. In our study, the average crevicular volume 24 h after activating the orthodontic apparatus was 0.955 μL. The average value obtained before activation was 0.57 μL. The results are similar to the ones obtained by Drummond et al.[5] They identified a crevicular fluid volume 24 h after the activation of 0.54 ± 19 μL, whereas the volume collected level at the control site was 0.66 ± 21μL. Periodontal tissues' metabolic activity is also influenced by their physiological features. As a result, the patient's age is an important element to consider when it comes to periodontal tissue remodeling during orthodontic therapy. Because alveolar bone density increases with age, teen/adult patients will have a different physiologic response than children. A study conducted by Grzibovskis[7] shows that the reduced level of pro-inflammatory cytokines in adult patients is due to the decreased metabolic activity of the periodontal ligament for this age group. Therefore, the periodontal tissue remodeling and the release of inflammatory molecules decrease with an increase in age. In our study, we observed an increased IL-1β concentration for young patients (45.96 pg/μL) compared to adult patients (19.20 pg/μL)) in the first 24 h after the activation of orthodontic therapy. Similar results were also obtained by Vujacic et al.[8] The authors compared the concentration of IL-1β and IL-6 in the crevicular fluid for adults and children during the orthodontic treatment, at different time intervals. Their results were reported in a different manner from ours (mass of interleukin per 60 s). They identified a similar concentration of IL-1β at the control teeth for both adults and children, with higher concentrations identified at the IL-1β level. With regard to the teeth affected by orthodontic forces, IL-1β had higher values for both adults and children, at 24 and 168 h after the time of application of the device. Overall, the concentration of IL-1β was higher for children compared to adults, with statistically significant differences at the initial time of the device activation and a few hours later. The main limitation of this study is represented by the periods of the measurement of the GCF levels, with the study having been conducted only at T0 and T1.

CONCLUSION

Our study concludes that through the bone remodeling biomarker analysis in the crevicular fluid, the efficiency of the orthodontic treatment can be monitored, evaluating the tissue response to the intensity of the orthodontic force. The volume of crevicular fluid and the level of IL-1 both increased 24 h after the orthodontic therapy began. The GFC levels of IL-1 24 h after the orthodontic treatment was activated differed between adolescents and young adults, implying that the two groups had different tissue reactions. As a result, teenage tooth mobility is faster than that of young adults.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.