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 Table of Contents  
Year : 2019  |  Volume : 53  |  Issue : 1  |  Page : 10-13

Variations of salivary levels of osteoprotegerin during orthodontic tooth movement

1 Post Graduate Trainee, Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India
2 Principal and Dean, Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India
3 Consultant Orthodontist, Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India
4 Prof., Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India

Date of Submission16-Sep-2018
Date of Acceptance28-Nov-2019
Date of Web Publication04-Feb-2019

Correspondence Address:
Dr. Upasak Mukherjee
AB Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jios.jios_175_18

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Aim: The study aimed to determine the variations of salivary levels of osteoprotegerin (OPG) during the different stages of orthodontic tooth movement. Materials and Methods: A sample of 24 patients was taken up for the study. Salivary samples were collected from each of the patients at the following intervals: T(0) – before start of the appliance therapy, T(1) – 24–48 h following treatment initiation, T(2) – after 2 weeks of appliance insertion, and T(3) – 5 weeks of appliance insertion. At all times, whole unstimulated salivary samples were collected. ELISA-based assay was used to determine the levels of OPG in the salivary samples. The results were statistically analyzed using ANOVA with Bonferroni t-test. The significance value of P< 0.05 was considered as statistically significant. Results: According to the results, there was an insignificant decrease and increase of OPG levels. Salivary levels in the alternate stages of treatment. P value for this particular study was found to be 0.05. Conclusion: The findings indicate that the variations in salivary concentrations of OPG might be linked to various stages of orthodontic tooth movement. The site specificity of OPG collection requires further studies in the long run.

Keywords: ELISA, Osteoprotegerin, Saliva

How to cite this article:
Mukherjee U, Nayak U S, Nayak U S, Adarsh N K, Kuttappa M N, Shetty A. Variations of salivary levels of osteoprotegerin during orthodontic tooth movement. J Indian Orthod Soc 2019;53:10-3

How to cite this URL:
Mukherjee U, Nayak U S, Nayak U S, Adarsh N K, Kuttappa M N, Shetty A. Variations of salivary levels of osteoprotegerin during orthodontic tooth movement. J Indian Orthod Soc [serial online] 2019 [cited 2019 Jun 17];53:10-3. Available from: http://www.jios.in/text.asp?2019/53/1/10/251545

  Introduction Top

Bone is primarily made of four major components namely the collagen fibres, polysaccharides, cells and calcium which together forms a constituent tissue. Bone continuously undergoes remodeling and mineralization. Remodeling can be physiological, pathological or mechanical. On application of an orthodontic force, areas of compression and tension develops following the stretch of periodontal ligament fibres. Areas under compression show osteoclastic activity wheresas areas under tension show osteoblastic activitites.[1]

Thus the events leading to orthodontic tooth movement are complex and there still lacks sufficient knowledge regarding the culmination of biochemical events at a molecular level.[2]

Research at the molecular level has lead to the findings of osteoblast derived factors such as receptor activator of nuclear factor kappa B ligand (RANKL) and Osteoprotegerin (OPG) competitively binding to the same receptor site RANK and playing regulatory effects on osteoclastogenesis.[3] Osteoprotegerin belongs to the tumor necrosis factor receptor superfamily and it encodes for type I transmembrane glycoproteins.[4] A study conducted by Florez-Moreno et al[5] concluded that variations of salivary concentrations of RANKL and OPG can be linked to various phases of orthodontic treatment.

OPG also known as osteoclast inhibitory factor is found in the body as soluble decoy receptor. OPG concentrations are high in developing bone and its expression is increased by bone morphogenetic protein(BMP), IL-1, TNF, TGF and estrogen.[6],[7] Subsequently, the OPG levels have been found to decrease with PGE2, Vitamin D3 and parathormones according to Horowitz et al.[8] Study by Lin et al[9] stated that RANKL and OPG not only stimulate osteoclast differentiation, but also play a major role in osteoblastic proliferation.

Thereby the sequence of events surrounding the biomarker and their role in Orthodontic tooth movement is substantially important. The activity of biomarkers in turn dictate the metabolic pathways of the involved tissues.[10] A study conducted by Tuncer et al[11] to measure the RANKL and OPG levels in GCF during canine retraction concluded that OPG levels were significantly higher on the mesial side of retraction without any significant changes for RANKL levels. So OPG was taken as the biomarker of choice. A study done by Nishijima et al[12] on the levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement concluded that the changes of these biomarkers may be linked to bone resorption as a response to compression force.

So the purpose of the study was to determine variations in the salivary levels of Osteoprotegerin as a biomarker during different stages of orthodontic tooth movement.

The Institutional ethical committee clearance was obtained for the above mentioned study from A B Shetty Memorial Institute of Dental Sciences, Mangalore. An informed consent was obtained from each of the participant before sample collection.

  Materials and Methods Top

Source of data

The data were obtained from patients visiting the Department of Orthodontics, A B Shetty Memorial Institute of Dental Sciences, Mangalore. The sample size was determined at 20 with power of the study set at 80%.

Patients between the age range of 18–30 years were taken irrespective of gender variation.

Inclusion criteria

  1. Samples with full set of teeth
  2. Nonsurgical and nonextraction cases
  3. Good general and periodontal health (probing depth <3 mm, no evidence of radiographic bone loss, <10% sites with bleeding on probing, and no gingival inflammation)
  4. Samples taken up for the study were classified under Angle's Class I malocclusion type.

Exclusion criteria

  1. Pregnant/lactating women
  2. Patients with a previous history of alcohol/tobacco use
  3. Patients undergoing orthodontic therapy
  4. Systemic diseases which can affect bone metabolism such as osteoporosis and endocrine diseases
  5. Patients taking antibiotics/steroids/anti-inflammatory for the past 3 months.

The appliance used for this particular study was preadjusted edgewise mechanotherapy (MBT system) using 022 slot (product marketed by American Orthodontics, Wisconsin, USA). All the samples were treated without extraction using preadjusted edgewise mechanotherapy. The archwire sequence used for treating the experimental study group was 0.014” Round NiTi, 0.016” round NiTi, 0.017 X 0.025 “ Rectangular NiTi, 0.017 X 0.025” rectangular stainless steel, 0.019 X 0.025 rectangular stainless steel. The salivary samples were collected at four different stages with round nickel titanium in place (0.014” round NiTi).

Sample preparation

  1. Five milliliters of unstimulated whole saliva sample was collected in 50 ml of sterile plastic centrifuge tube from each patient. The sample collection was done before breakfast at 8 a.m. and any further dental cleaning procedure
  2. Immediately following collection, the samples were centrifuged for 5 min at ×800 g. Supernatants were collected and stored at −75°C unless processed.

Sample collection

Salivary samples were collected at four different stages as follows:

  • Stage 1: Before the initiation of treatment
  • Stage 2: 24-48 hrs after appliance insertion
  • Stage 3: 2 weeks after appliance insertion
  • Stage 4: 5 weeks through treatment.

During salivary sample collection no reactivations were done.

ELISA-based capture assay was used. Undiluted form of clarified salivary samples was used. The optical density for OPG (measured in picograms per milliliter (pg/ml) was determined within 15 min after adding 50 μl of stop solution using microplate reader.

  Results Top

The participants taken up for the study maintained good oral hygiene before the start and during the course of the treatment. The sample size stood at 20 with a total of 80 salivary samples which were assayed using the levels of OPG.

Statistical analysis

The statistical analysis was done through SPSS Inc., Chicago, IL, USA by using ANOVA test and post-hoc Bonferroni method. The statistical calculation showed significant differences in the salivary levels of OPG with P value of 0.05 at 95% confidence level.

The variations in the enzymatic findings indicate fluctuating trends in the level of OPG with time. At baseline, the mean value of OPG was 1.0169 pg/ml with standard deviation (SD) of 0.39 which rose to 1.3214 pg/ml with SD of 0.38 at 24–48 hr after appliance insertion. Two weeks postappliance insertion showed a decrease of OPG value to 1.1644 pg/ml with a SD of 0.45. The OPG values 5 weeks after appliance treatment showed an upward trend to 1.2497 pg/ml with a SD of 0.38. Thus, the OPG values increased from baseline to 48 h whereas it decreased 2 weeks following postappliance insertion. The values showed a sudden increase after the appliance was in place for 5 weeks [Table 1]. The results were found to be statistically insignificant.
Table 1: Osteoprotegerin levels along with statistical values measured at different time intervals of appliance therapy

Click here to view

  Discussion Top

RANK, RANKL and OPG are the key molecules for osteoclast differentiation supported by osteoblasts.[13],[14] OPG is produced by human periodontal cells, pulp cells and the gingival fibroblasts. OPG binding to RANK site competitively inhibiting the binding of RANKL prevents the differentiation of preosteoclasts into mature osteoclasts.[15],[16]

OPG has also been suggested in the treatment of bone arthropathies such as osteoporosis, crippling arthritis and osteopenic disorders.[17] Kanzaki et al[18] concluded that OPG gene transfer prolonged OPG expression and thereby inhibited RANKL mediated osteoclastogenesis.

So this study aimed to check the salivary concentration of Osteoprotegerin at various intervals of orthodontic tooth movement in samples collected at various time intervals. The orthodontic tooth movement involving alveolar process remodeling is time dependent which requires an observation period of atleast 1 month. Therefore the levels of OPG was investigated over a period of 5 weeks.

The detection of OPG and RANKL levels in Gingival Crevicular fluid and their ratio may help the clinician to effectively apply the optimum orthodontic force and to avoid pathologic bone resorption and hyalinization in cases with bone metabolic disorders.[19],[20] Since different bone remodeling mediators are washed into the saliva from gingival crevicular fluid, whole saliva was considered an easy alternative to crevicular fluid for sampling.[21] The ELISA kit used for the study was manufactured by ELABSCIENCES LTD GERMANY. The minimum detectable dose of human OPG is 0.094 ng/ml. The sampling intervals were chosen according to the phases of tooth movement. Initially from the day of force application to 2 days, there is rapid displacement of tooth in the PDL space and alveolar bone bending. This phase is followed by a lag phase spanning from 4 days to 20 days which is mostly due to hyalinization; and finally acceleration phase due to removal of the hyalinized tissue.[22]

Comparing the 48 hour values to the baseline the OPG concentration increased with time. The expression of OPG upregulated under intermittent stresses. Kusumi et al[23] found an increase in OPG synthesis following application of tensile stress to human osteoblasts. These reports seem to correlate with our results of OPG levels within 48 hours of treatment initiation.

Although Florez-Moreno et al[5] had led to contrary findings of decreasing OPG levels immediately after mechanical loading (24-48 hours) attributed to ischaemia and hypoxia due to mechanical compression of microvasculature. The authors perhaps might not have considered that orthodontic tooth movement is a combination of compressive and tensile stress taken together to measure the levels of various biomarkers.

In this particular study, the lag phase corresponded to decrease in OPG levels following 2 weeks of appliance insertion. The pressure site demonstrates decreased cellular activity and tension site shows increased cellular activity.[24],[25] During lag phase, due to the presence of hyalinized zone the cellular activity even at the tension site comes to a standstill correlating to decreased levels of OPG following 2 weeks when compared to 48 hours of appliance insertion.

The post lag phase involves removal of hyalinized tissue thereby restoring the cellular activity at the tension and pressure sites. Thus at 5 weeks of appliance insertion, the OPG levels gained a sharp increase.

Thus our values correlate and corresponds with the findings of the study that when stretch is induced in the Periodontal ligament fibres there causes an upregulation of OPG levels during various stages of orthodontic tooth movement.[26]

Though we have noticed these experimental findings at various intervals, our values are not statistically significant. In most of the previous studies, sample collection was from gingival crevicular fluid instead of washed saliva as taken up for this study. Thereby the site specificity of the GCF was lost and so the results obtained were not statistically significant.

  Conclusion Top

  1. The findings indicate that variations in salivary concentrations of OPG might be linked to various stages of orthodontic tooth movement. Levels of OPG increased following 48 h of appliance insertion and after 5 weeks of appliance treatment whereas decreased after 2 weeks of treatment initiation
  2. Site-specific GCF would have been a better alternative to whole saliva for studying specific biomarkers like OPG and their expression during various stages of orthodontic tooth movement.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Masella RS, Meister M. Current concepts in the biology of orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2006;129:458-68.  Back to cited text no. 1
Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop 2006;129:1-32.  Back to cited text no. 2
Arai F, Miyamoto T, Ohneda O, Inada T, Sudo T, Brasel K, et al. Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-fms and receptor activator of nuclear factor kappaB (RANK) receptors. J Exp Med 1999;190:1741-54.  Back to cited text no. 3
Yasuda H, et al. Osteoclast differentiation factor is a ligand for OPG/osteoclastogenesis -inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998;95:3597-602.  Back to cited text no. 4
Flórez-Moreno GA, Isaza-Guzmán DM, Tobón-Arroyave SI. Time-related changes in salivary levels of the osteotropic factors sRANKL and OPG through orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2013;143:92-100.  Back to cited text no. 5
Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ, et al. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999;20:345-57.  Back to cited text no. 6
Tang L, Lin Z, Li YM. Effects of different magnitudes of mechanical strain on osteoblasts in vitro. Biochem Biophys Res Commun 2006;344:122-8.  Back to cited text no. 7
Horowitz MC, Xi Y, Wilson K, Kacena MA. Control of osteoclastogenesis and bone resorption by members of the TNF family of receptors and ligands. Cytokine Growth Factor Rev 2001;12:9-18.  Back to cited text no. 8
Lin JM, Callon KE, Lin CQ, Bava U, Zheng MH, Reid IR, et al. Alteration of bone cell function by RANKL and OPG in different in vitro models. Eur J Clin Invest 2007;37:407-15.  Back to cited text no. 9
Kearns AE, Khosla S, Kostenuik PJ. Receptor activator of nuclear factor kappaB ligand and osteoprotegerin regulation of bone remodeling in health and disease. Endocr Rev 2008;29:155-92.  Back to cited text no. 10
Tuncer, et al. OPG-RANKL levels after continuous orthodontic force. GMJ 2013;24:33-6.  Back to cited text no. 11
Nishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai K, et al. Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro. Orthod Craniofac Res 2006;9:63-70.  Back to cited text no. 12
Hasegawa T, Yoshimura Y, Kikuiri T, Yawaka Y, Takeyama S, Matsumoto A, et al. Expression of receptor activator of NF-kappa B ligand and osteoprotegerin in culture of human periodontal ligament cells. J Periodontal Res 2002;37:405-11.  Back to cited text no. 13
Hofbauer LC, Heufelder AE. Role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology. J Mol Med (Berl) 2001;79:243-53.  Back to cited text no. 14
Reitan K. Clinical and histologic observations on tooth movement during and after orthodontic treatment. Am J Orthod 1967;53:721-45.  Back to cited text no. 15
Krishnan V, Davidovitch Z. On a path to unfolding the biological mechanisms of orthodontic tooth movement. J Dent Res 2009;88:597-608.  Back to cited text no. 16
Leibbrandt A, Penninger JM. RANK/RANKL: Regulators of immune responses and bone physiology. Ann N Y Acad Sci 2008;1143:123-50.  Back to cited text no. 17
Kanzaki H, Chiba M, Arai K, Takahashi I, Haruyama N, Nishimura M, et al. Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement. Gene Ther 2006;13:678-85.  Back to cited text no. 18
Henneman S, Von den Hoff JW, Maltha JC. Mechanobiology of tooth movement. Eur J Orthod 2008;30:299-306.  Back to cited text no. 19
Theoleyre S, Wittrant Y, Tat SK, Fortun Y, Redini F, Heymann D, et al. The molecular triad OPG/RANK/RANKL: Involvement in the orchestration of pathophysiological bone remodeling. Cytokine Growth Factor Rev 2004;15:457-75.  Back to cited text no. 20
Frodge BD, Ebersole JL, Kryscio RJ, Thomas MV, Miller CS. Bone remodeling biomarkers of periodontal disease in saliva. J Periodontol 2008;79:1913-9.  Back to cited text no. 21
Oppenheim A. Human tissue response to orthodontic intervention of short and long duration. AM J Orthod Dentofacial Orthop 1942;28:263-301.  Back to cited text no. 22
Kusumi A, Sakaki H, Kusumi T, Oda M, Narita K, Nakagawa H, et al. Regulation of synthesis of osteoprotegerin and soluble receptor activator of nuclear factor-kappaB ligand in normal human osteoblasts via the p38 mitogen-activated protein kinase pathway by the application of cyclic tensile strain. J Bone Miner Metab 2005;23:373-81.  Back to cited text no. 23
Oppenheim A. Tissue changes particularly of the bone incident to tooth movement. American Orthodontist 1911;3:56-67.  Back to cited text no. 24
Schwarz AM. Tissue changes incidental to orthodontic tooth movement. International Journal of Orthodontia, Oral Surgery and Radiography 1932;18:331-352.  Back to cited text no. 25
Heller IJ, Nanda R. Effect of metabolic alteration of periodontal fibers on orthodontic tooth movement, An experimental study. Am J Orthod 1979;75:239-58.  Back to cited text no. 26


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