|Year : 2020 | Volume
| Issue : 4 | Page : 568-573
Comparison of biochemical markers of bone metabolism between conventional labial and lingual fixed orthodontic appliances
AN Gujar1, HA Baeshen2, A Alhazmi3, MS Ghoussoub4, AT Raj5, S Bhandi6, SC Sarode7, KH Awan8, D Birkhed9, S Patil10
1 Department of Orthodontics, KLES Institute of Dental Sciences, Bangalore, Karnataka, India
2 Department of Orthodontics, College of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia
3 Department of Preventive Dental Science, Division of Orthodontics, College of Dentistry, Jazan University, Jazan, Saudi Arabia
4 Department of Orthodontics, School of Dental Medicine, Lebanese University, Hadath, Lebanon
5 Department of Oral Pathology and Microbiology, Sri Venkateswara Dental College and Hospital, Chennai, Tamil Nadu, India
6 Department of Restorative Dental Sciences, Division of Operative Dentistry, College of Dentistry Jazan University, Jazan, Saudi Arabia
7 Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune, India
8 College of Dental Medicine, Roseman University of Health Sciences, South Jordan, Utah, USA
9 Professor Emeritus at the University of Gothenburg, Gothenburg, Sweden
10 Department of Maxillofacial Surgery and Diagnostic Sciences, Division of Oral Pathology, College of Dentistry Jazan University, Jazan, Saudi Arabia
|Date of Submission||19-Sep-2019|
|Date of Acceptance||13-Dec-2019|
|Date of Web Publication||4-Apr-2020|
Dr. S Patil
Department of Maxillofacial Surgery and Diagnostic Sciences, Division of Oral Pathology, College of Dentistry Jazan University, Jazan - 45412
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: The applied orthodontic force causes remodeling of the periodontium through the selective release of cytokines causing resorption of bone, enabling controlled movement of the tooth. This study compared the cytokine profile between patients treated with conventional labial and lingual fixed orthodontic appliances. Patients and Methods: The study included 80 patients in need of orthodontic treatment, out of which 40 patients were treated by the labial fixed appliance and 40 by the lingual fixed appliance. Gingival crevicular fluid (GCF) specimens were gathered from both the groups using a microcapillary pipette. The samples were collected at the beginning of the treatment and after 21 days. Enzyme-linked immunosorbent assay was performed to evaluate the cytokine levels. Results: Interleukin (IL)-1α, 1β, 2, 8, and tumor necrosis factor-alpha (TNF-α) levels were significantly high (P < 0.001) in GCF of participants treated with conventional labial fixed appliance. IL-1α, 1β, 2, 6, 8, and TNF-α levels were significantly high (P < 0.001) in GCF of participants treated by the lingual fixed appliance. The concentrations of TNF-α and IL-1β were increased higher than other cytokines in both the treatment groups. Conclusion: Overall, the lingual fixed appliance had higher cytokine levels than a labial fixed appliance. Analyzing the GCF cytokine levels during orthodontic treatment could provide an ideal platform for monitoring the progress of the treatment.
Keywords: Biomarkers, bone remodeling, cytokines, gingival crevicular fluid, interleukins, orthodontic appliances
|How to cite this article:|
Gujar A N, Baeshen H A, Alhazmi A, Ghoussoub M S, Raj A T, Bhandi S, Sarode S C, Awan K H, Birkhed D, Patil S. Comparison of biochemical markers of bone metabolism between conventional labial and lingual fixed orthodontic appliances. Niger J Clin Pract 2020;23:568-73
|How to cite this URL:|
Gujar A N, Baeshen H A, Alhazmi A, Ghoussoub M S, Raj A T, Bhandi S, Sarode S C, Awan K H, Birkhed D, Patil S. Comparison of biochemical markers of bone metabolism between conventional labial and lingual fixed orthodontic appliances. Niger J Clin Pract [serial online] 2020 [cited 2021 Sep 18];23:568-73. Available from: https://www.njcponline.com/text.asp?2020/23/4/568/281923
| Introduction|| |
During orthodontic treatment, the force applied by the appliance induces the periodontium to remodel through the release of selective cytokine allowing controlled tooth movement.,, Because bone metabolism is the prime factor for tooth movement, recent studies have focused on delineating the molecular factors responsible for bone remodeling during orthodontic treatment.,,,,,,, Cytokines levels in the gingival crevicular fluid (GCF) are used as biochemical markers for bone metabolism during orthodontic treatment. Comparison of the cytokine levels between different orthodontic appliances has provided vital information on the differential effect induced on bone metabolism by varying the appliance design., The demand for aesthetic in orthodontic treatments has led to the increasing use of lingual appliances. Unlike labial fixed appliance and aligners, not much information is available on the bone metabolism markers involved in the lingual fixed appliance. Given the increasing demand for lingual orthodontic appliances, it is vital to study the molecular factor responsible for tooth movement in lingual fixed appliances. Hence, this study was formulated to assess and compare the GCF cytokine levels between patients undergoing orthodontic treatment with the lingual fixed appliance and labial fixed appliance.
| Patients and Methods|| |
The study included patients indicated for orthodontic treatment with labial or lingual fixed appliance. The patients with same clinical presentation (mild malocclusion between 2.1 and 4.0 mm) were included in the study to avoid the treatment bias and random allocation of the included cases into two treatment groups was done to avoid the selection bias., Forty patients (20 with the labial and 20 with the lingual fixed appliance) with an age range of 12–32 years were included. The inclusion criteria were that the patients should have good general health, no history of antibiotic/anti-inflammatory drugs in the last 6 months, and have a healthy periodontium [gingival index (GI) score of less than 1, absence of periodontal pockets with the general probing depths being less than or equal to 3 mm, and absence of radiographic evidence of periodontal bone loss]. Exclusion criteria included the history of smoking, the presence of gingivitis or periodontitis, and systemic disease. The institutional ethics committee approval (IEC No. LE/IEC/APR-18/3). was obtained for the study. Consent was obtained from all the participants in the study. All included patients were advised on maintaining good oral hygiene during orthodontic treatment. The oral hygiene levels of the participants were periodically assessed through their GI score. A GI score of less than 1 was considered as good oral hygiene.
Twenty patients were bonded with conventional MBT metallic labial orthodontic brackets (0.022 × 0.028-inch slot; 3M Unitek, Monrovia, CA, USA) using orthodontic light-cured adhesive (Transbond XT; 3M Unitek) in both maxillary and mandibular arches. Maxillary and mandibular archwires (0.016-inch nickel titanium) were placed for initial alignment and leveling. Twenty patients were bonded with lingual metallic brackets (Ormco Corporation, Glendora, CA, USA) using orthodontic light-cured adhesive (Transbond XT) in both maxillary and mandibular arches. Maxillary and mandibular archwires (0.012-inch nickel titanium) were placed for initial alignment and leveling. All the patients receiving metallic labial and lingual brackets were ligated using elastomeric ligatures (3M Unitek).
The site where the gingival inflammation was minimal/absent was selected. Selection of sample site was done according to convenience. The sample site was standardized for all subjects to the proximal region of either of the maxillary canines.,,,,, Contamination at the sampling site was prevented by isolating the area with sterile gauze. A microcapillary pipette (Sigma-Aldrich, Inc., St. Louis, Missouri, United States) was placed at the gingival sulcus entrance to collect the GCF [Figure 1] and [Figure 2]. A calibrated color-coded (0.2–2 μL) volumetric microcapillary pipette was used to collect a standardized volume (1 μL) of the sample. Samples with blood or saliva contamination were discarded. Eppendorf tubes (0.5 mL) were used to transfer the collected samples. The tubes with the samples were centrifuged for 10 min at 3000 rpm. The centrifuged samples were stored until further procedure at −80°C. Samples were collected at two time points: before the orthodontic treatment – T0 and 21 days after the start of the orthodontic treatment – T1. The 21st day was selected as the second collection point (T1) because the orthodontic treatment-induced indirect resorption starts on the 21st day. When orthodontic force is applied, there is formation of hyalinized tissue which should subside for tooth movement to take place. This occurs through indirect (undermining) resorption where the osteoclasts present within the adjacent bone marrow space resorb the bone adjacent to the cell-free area. This lag phase can last for several days to several months.,,,,,,,,,, A total of six cytokine molecules were to be evaluated from each patient using enzyme-linked immunosorbent assay (ELISA). Thus, from each patient, six samples (one for each cytokine) were collected at T0 and T1. Patient's identification was prevented by coding the samples.
|Figure 1: Mean clinical parameters of plaque index (PI), gingival index (GI), and bleeding on probing (BOP) within labial fixed appliance and lingual fixed appliance groups at T0 and T1 (all the values are statistically insignificant)|
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|Figure 2: Mean and range levels of different cytokines within labial fixed appliance (n = 20) and lingual fixed appliance groups (n = 20) at T0 and T1. Range values of IL-1β in labial fixed appliances are T0 = 0.255 and T1 = 32.80, and in lingual fixed appliances are T0 = 0.255 and T1 = 4.539 (all the values are statistically significant)|
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GCF analysis using ELISA
The stored samples were thawed before being subjected to ELISA. Commercial test systems (Raybiotech, Inc. Norcross, GA, USA) were used to analyze the cytokine levels in the samples.
Assay procedure summary
Solid-phase sandwich ELISA was used for cytokine evaluation in the GCF. Samples were loaded and incubated in each well at room temperature for 2.5 h, followed by consequent addition of biotin antibody (1-h incubation at room temperature), streptavidin solution (45 min incubation at room temperature), one-step substrate reagent – tetramethylbenzidine (TMB) (30 min incubation at room temperature), and stop solution. Immediately after the addition of the stop solution, ELISA reader was used to reading the solution's optical density at 450 nm.
Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) for Windows (Version 22.0; Released 2013; IBM Corp., Armonk, NY, USA). For each of the study groups, interleukin (IL)-1a, IL-1β, IL-2, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-a) mean levels were calculated.
The differences in the mean value of the biomarker levels (in pg/mL) between the baseline and follow-up of the different groups were assessed using the paired t-test. Unpaired t-test was used to assess the difference in the mean cytokine levels between the study groups. P < 0.05 was set as the level of significance.
| Results|| |
Results with no significant difference
There was no statistically significant difference in the GI, plaque index, and bleeding on probing at T0 and T1 of both groups (labial and lingual fixed appliances) as depicted in [Figure 1].
Results with significant difference
IL-1α, IL-1β, IL-2, IL-8, and TNF-α GCF levels significantly increased from T0 to T1 (P < 0.001) in patients treated with labial fixed appliance, while IL-1α, IL-1β, IL-2, IL-6, IL-8, and TNF-α levels significantly increased from T0 to T1 (P < 0.001) in patients treated with lingual fixed appliance as depicted in [Figure 2] and [Figure 3].
|Figure 3: Mean and range values of TNF-α values with labial fixed appliance (n = 20) and lingual fixed appliance groups (n = 20) at T0 and T1 [all the values are statistically significant]|
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Most active cytokines
IL-1β and TNF-α GCF levels were increased to a higher level than other cytokines in both labial and lingual fixed appliances as illustrated in [Figure 2] and [Figure 3].
Least active cytokine
IL-1α GCF levels increased from T0 to T1 in both labial and lingual fixed appliances, but the increase was substantially minimal compared with the increase seen in other cytokines as depicted in [Figure 2] and [Figure 3].
Overall, both the groups (labial and lingual fixed appliance) showed an increase in cytokine levels from T0 to T1, although the levels were significantly higher in lingual fixed appliance than in the labial fixed appliance, except for IL-1α which was more in labial fixed appliances, as shown in [Table 1].
|Table 1: Comparison of the mean differences in the biomarker levels between labial and lingual bracket groups using independent Student's t-test|
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| Discussion|| |
Application of optimal force on the tooth stimulates the release of selective cytokines causing alveolar bone remodeling, allowing controlled tooth movement. GCF has proven to be an ideal source for studying several physiological and pathological biomarkers.,,,,,,,,,,,,,,,,,,, Recent studies have shown GCF to be an ideal source for studying bone metabolism during orthodontic treatment.,,,,,,,,,,,,,,,,, Cytokines are low-molecular-weight signaling molecules (proteins), often referred to as immune-modulating mediators (biomarkers) of various activities involved in changes in PDL tissues, bone, and so on. Some of the most commonly analyzed biomarkers in GCF include the ILs, prostaglandin E2, TNF-α, receptor activator of nuclear factor Kappa-B, osteoprotegerin, acid phosphatase, alkaline phosphatase, osteocalcin, matrix metalloproteinases, receptor activator of nuclear factor Kappa-B ligand, transforming growth factor-β 1, aspartate aminotransferase, interferon-gamma, IL-1RA, and others.
Before exploring into the expression of cytokines in orthodontic treatment, first, it is vital to understand the origins and functions of these bone metabolic mediators. The proinflammatory cytokines include TNF-α, IL-8, IL-6, IL-2, AND IL-1, while the anti-inflammatory cytokines include IL-13, IL-4, and IL-10.,,, Macrophages and endoepithelial cells produce TNF-α, IL-6, IL-2, and IL-1. IL-1 activates the osteoclast, thereby initiating bone resorption. The major physiologic form of IL-1 is IL-1β., T-helper lymphocytes produce IL-2. IL-2 induces bone resorption by stimulating osteoclastic activities and is also implicated in periodontal diseases., IL-6 causes bone resorption by inducing osteoclast formation, stimulating preformed osteoclasts, and regulating inflammatory immune response., Monocytes secrete IL-8 which plays a major role in regulating inflammation-mediated bone resorption through neutrophil recruitment and activation.
A major aspect of any investigative study would be sample collection. Selecting the optimal site of collections is essential to ensure the results are reliable. It is also important to maintain patient compatibility during sample collections by reducing any potential complication and discomfort. As mentioned earlier, in this study samples were collected from the gingival sulcus given its easy accessibility. Also, given their close proximity to periodontal ligament cells, analyzing its samples would more likely be a close representation of the underlying remodeling process. Further sampling from gingival sulcus is noninvasive, allowing multiple sampling for a long period of time. The sites which could provide better information on the alveolar bone remodeling than gingival sulcus would be the periodontal ligament and the resorbing bone tissue, but examining these tissues would need invasive procedures. Thus, this study used gingival sulcus for sampling. Studies have shown that variations in the GCF levels might be in different collection sites., Thus, in this study, GCF collection was standardized to the gingival sulcus of the distal aspect of the maxillary canines.
Periodontal diseases have multifactorial etiologies, with several well-established risk factors including the microbes, associated habits, and host and environmental factors. Orthodontic appliances are a form of environment factor implicated in the development of the periodontal disease. The orthodontic force results in the release of cytokines, and depending on their levels, the result could be controlled tooth movement or gross resorption resulting in compromised periodontium. As the cytokine levels have shown to vary depending on the type of the orthodontic appliance,, it is vital to monitor the cytokine profile of each type of orthodontic appliance to ensure optimal treatment outcome. The variations in the cytokine levels between difference appliances can be attributed to several factors including the force applied and plaque accumulation within the brackets. Although the orthodontist controls the force to be applied, the presence of excess plaque in the retentive areas of the brackets could cause irregularities in cytokine levels by inducing inflammation.
The recent demand for aesthetics in orthodontic treatment has led to the increasing use of lingual fixed appliances, which due to their site of placement have a greater risk of plaque accumulation compared with labial fixed appliances. TNF-α and IL-1 are shown to be major factors in the development of periodontitis. In addition to being prominent inflammatory mediators, TNF-α and IL-1 have shown to induce other mediators of inflammation including matrix metalloproteinases, IL-8, IL-6, and prostaglandin-E2.,, Distalization of periodontal tissues has shown to increase the GCF levels of TNF-α and IL-1. In contrary, few studies have shown only a mild increase in the IL-1 levels.,,, The bacterial challenge often leads to inflammation mediated by TNF-α released from mast cells. Demling et al. have shown that lingual orthodontic therapy has a higher risk of microbial accumulation and damage to the periodontium than the labial orthodontic therapy. In this study, there was no statistically significant difference in the periodontal index, GI, and bleeding on probing between the labial and lingual fixed appliances. Thus, the difference in the cytokine levels between the two groups cannot be attributed to a plaque-mediated inflammation. IL-1β levels have shown to increase during orthodontic treatment on the pressure side, which can be attributed to the mechanical stress exerted by the orthodontic force on the periodontium. Thus, an increase in the IL-1β levels could be an indicator of active bone resorption. Based on the above data, higher levels of IL-1β and TNF-α noted in the lingual fixed appliance could be the result of greater mechanical stress applied on the periodontium by lingual fixed appliance.
| Conclusion|| |
All the cytokines (IL-1α, IL-1β, IL-2, IL-6, IL-8, and TNF-α) analyzed in the study exhibited an increase in their GCF levels after 3 weeks of treatment. The increase in GCF cytokine levels was noted in patients treated with both labial and lingual fixed appliances. TNF-α and IL-β levels were increased to a higher level compared with other cytokines in both treatment groups. Furthermore, the cytokine levels were higher in lingual fixed appliance compared with the labial appliance, indicating that the lingual appliance could potentially be exerting greater mechanical stress on the periodontium than the labial fixed appliance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]