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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 51  |  Issue : 2  |  Page : 75-80

Assessment of myeloperoxidase activity at different force levels in gingival crevicular fluid during initial phase of orthodontic tooth movement


1 PG Student, Department of Orthodontics and Dentofacial Orthopedics, VSPM Dental College and Research Centre, Nagpur, Maharashtra, India
2 HOD Prof. and Guide, Department of Orthodontics and Dentofacial Orthopedics, VSPM Dental College and Research Centre, Nagpur, Maharashtra, India
3 Reader, Department of Orthodontics and Dentofacial Orthopedics, VSPM Dental College and Research Centre, Nagpur, Maharashtra, India
4 Senior Lecturer, Department of Orthodontics and Dentofacial Orthopedics, VSPM Dental College and Research Centre, Nagpur, Maharashtra, India

Date of Submission02-Aug-2016
Date of Acceptance09-Feb-2017
Date of Web Publication17-Apr-2017

Correspondence Address:
Honey Gurbaxani
Lakham Niwas, Kadbi Chowk, Nagpur - 440 004, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jios.jios_154_16

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  Abstract 


Background: Orthodontic movements promote remodeling of the alveolar bone, which is mediated by inflammatory reactions such as characterized by vascular changes and infiltration of leukocytes. Changes in the periodontium occur, depending on the magnitude, duration, and direction of applied force. These changes are often seen in the saliva and gingival fluids through the various substances secreted in them. Aim: The present study aimed to assess myeloperoxidase (MPO) activity at different force levels in gingival crevicular fluid (GCF) during the initial phase of orthodontic tooth movement by varying the effective force levels to 50, 75, 100, and 150 g. Materials and Methods: A total of thirty participants between the age groups of 18–25 years requiring upper first premolar extractions were included in the study. They were divided into three groups (I, II, and III) of ten individuals each, again subdivided into two Subgroups A and B depending on the amount of force applied to the canine. Subgroup A of all the three groups used 150 g, whereas Subgroup B used 50, 75, and 100 g of force, respectively. GCF was collected at 2 h, 7 days, and 14 days of force application. Statistical Analysis: Paired t-test and ANOVA test were used to provide the descriptive statistics of mean optical density to detect the presence of MPO in GCF. Results and Conclusion: There was a highly significant increase in the MPO levels in the GCF at 14th day after force application which can be correlated to the onset of inflammatory reactions in the periodontium.

Keywords: Gingival crevicular fluid, myeloperoxidase, optimum orthodontic force


How to cite this article:
Gurbaxani H, Shenoy U, Banerjee S, Hazarey A, Karia H. Assessment of myeloperoxidase activity at different force levels in gingival crevicular fluid during initial phase of orthodontic tooth movement. J Indian Orthod Soc 2017;51:75-80

How to cite this URL:
Gurbaxani H, Shenoy U, Banerjee S, Hazarey A, Karia H. Assessment of myeloperoxidase activity at different force levels in gingival crevicular fluid during initial phase of orthodontic tooth movement. J Indian Orthod Soc [serial online] 2017 [cited 2017 Jul 23];51:75-80. Available from: http://www.jios.in/text.asp?2017/51/2/75/204603




  Introduction Top


Orthodontic tooth movement occurs upon the application of a controlled mechanical force that results in biologic reactions which model and remodel the surrounding dental and periodontal tissues. Different opinions can be found about the force levels that result in optimal mechanical conditions within the periodontal ligament for orthodontic tooth movement. It is assumed that an optimal force system is important for an adequate biological response in the periodontal ligament.[1],[2] The early phase of orthodontic tooth movement involves an acute inflammatory response, characterized by periodontal vasodilation and migration of polymorphonuclear leukocytes (PMN) out of periodontal ligament capillaries. Locally, PMNs release antimicrobial and inflammatory mediators in the gingival crevicular fluid (GCF). Myeloperoxidase (MPO), is one of such active substances and due to its importance during inflammatory processes and for being an indicator of PMN presence in tissues, has been widely used as an inflammatory marker of both acute and chronic conditions.[3],[4],[5] GCF is an exudate, the constituents of which are derived from a variety of sources, including microbial dental plaque, host inflammatory cells, host tissue, and serum. It is the result of interplay of the bacterial biofilm adherent to the tooth surfaces and cells of the periodontal tissues. Thus, the analysis of specific constituents in the GCF provides a quantitative biochemical indicator for the evaluation of local cellular metabolism.[4],[5],[6],[7]

The present study aims at assessing the activity of MPO enzyme in the GCF by varying the effective force levels and in establishing the efficiency of MPO as a strong indicator of orthodontic tooth movement assessment.

Aims and objectives

The present study aimed to assess MPO activity at different force levels in GCF during the initial phase of orthodontic tooth movement. Furthermore, in addition to the aim was the objective of assessing the MPO activity in GCF by varying the effective force levels to 50, 75, 100, and 150 g.


  Materials and Methods Top


A total of 30 participants, between the age group of 18–25 years requiring upper first premolar extraction, were selected from those visiting the Department of Orthodontics and Dentofacial Orthopaedics of our institute. The study was initiated after the clearance from the Institutional Ethics Committee of our institute. GCF MPO levels were measured using ELISA kit. Patients with good general health, nonsmokers with probing depth ≤3 mm, on no antibiotic and/or anti-inflammatory therapy preceding the study, and requiring upper first premolar extractions were included in the study.

The study comprised three groups with two subgroups as follows:

  • Group I: (10 individuals)


    1. Subgroup A: 150 g of force applied to canine for retraction in maxillary right quadrant
    2. Subgroup B: 50 g of force applied to canine for retraction in maxillary left quadrant


  • Group II: (10 individuals)


    1. Subgroup A: 150 g of force applied to canine for retraction in maxillary right quadrant
    2. Subgroup B: 75 g of force applied to canine for retraction in maxillary left quadrant


  • Group III: (10 individuals)


    1. Subgroup A: 150 g of force applied to canine for retraction in maxillary right quadrant
    2. Subgroup B: 100 g of force applied to canine for retraction in maxillary left quadrant.


Subgroup A of Groups I, II, and III was used as the control group. For the application and assessment of force, nickel–titanium (Ni-Ti) closed coil springs and Richmond Orthodontic Stress and Tension Gauge, i.e., Dontrix gauge were used, respectively. Two Ni-Ti closed coil springs, one on each side of maxillary quadrant, extending from molar tube hook to the power arm of canine bracket were used to retract the canines into the extraction spaces. Force levels were varied using the Dontrix gauge [Figure 1].
Figure 1: Force application using Dontrix gauge

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Gingival crevicular fluid collection

All the selected participants were briefly informed about the procedure to be done and were seated comfortably in the dental chair. Using the Dontrix gauge, a force of 150 g and 50 g was applied to the maxillary right and left canines, respectively.

Using the closed coil Niti springs for the first group, GCF collection was done 2 h, 7 days, and 14 days after application of force. Similarly, a force of 150 and 75 g and a force of 150 and 100 g for the next two consecutive groups were applied to the maxillary right and left canines, respectively. GCF samples were then collected at 2 h, 7 days, and 14 days of force application for these two groups as well.

The GCF sample collection was done using a calibrated microcapillary pipette (1–5 μl) [Figure 2]. Before collection of GCF, any supragingival soft deposits were removed without causing trauma to the gingival crevice. If any hemorrhage was evident after this procedure, no fluid was collected. The area was then thoroughly irrigated with distilled water, isolated by cotton rolls, and dried by steam of air. Suction was also used to frequently aspirate the collected saliva to avoid contamination of GCF. About 15–20 min of isolation of maxillary canines, a ring of clear GCF was seen at the gingival margin. The microcapillary pipette was then placed extracrevicularly in the accumulated fluid and collected in the pipette. From both the maxillary canines for the three groups, a standardized volume of 3 μl was collected. Sites which did not express the appropriate volume of fluid and micropipettes which were contaminated with blood and saliva were not included in the study. Collected GCF samples were immediately transferred to airtight plastic vials and were stored at −70° until assayed.
Figure 2: Micropipette

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Estimation of myeloperoxidase using ELISA kit

For quantitative estimation of MPO, all reagents of the kit were allowed to warm to room temperature for at least 30 min before opening. All reagents, standards, and samples were prepared, according to manufacturer's instructions [Figure 3]. Assay diluent was diluted 5-fold with deionized or distilled water before use. Dilution of sample to 100 μl was carried out using assay diluent.
Figure 3: ELISA kit

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  Results Top


It was found that at 2 h, the mean optical density was maximum for Group C, i.e., 0.177 (0.018), followed by control group with value 0.048 (0.021), whereas it was similar for Group A and B [Graph 1]. After 7 days, the mean optical density was same for control group and Group B, whereas in Group A, it was 0.186 (0.019), followed by 0.244 (0.12) for Group C. Further, at 14th day, the mean optical density shows maximum for control group and Group A, i.e., 0.674 (0.048) as compared to remaining two groups.



The test suggested that at 2 h, the difference of means between groups was statistically significant with P = 0.0001 [Graph 2]. Further, after 7 days, the mean optical density in both the groups increased compared to 2 h. However, the difference in the means between groups was statistically insignificant with P = 0.2971. After 14 days, the mean optical densities in both groups were nearly same as revealed by P = 0.9999, but their means were higher as compared to above two time points.



On similar lines, analysis was performed for control and experimental subgroups of Group B. At 2 h, the mean OD between the groups differed significantly as indicated by P = 0.0001. Moreover, at 7th day, the means between the groups were almost similar as indicated by P = 0.9999. However, after 14 days, the mean value in control group was higher than the mean for Group B, but the difference was insignificant (P = 0.3096) [Graph 3]. For Group C at 2 h, the test suggested that the difference of means was highly significant with P< 0.0001 [Graph 4]. Further, after 7 days, the mean optical density in both the groups increased compared to 2 h. However, the difference in the means between groups was statistically insignificant with P = 0.1461. After 14 days, the mean optical densities in both the groups were higher compared to 7 days, but the difference between the groups was highly insignificant as revealed by P = 0.7957.



Intergroup comparison shows that at 2 h, the mean optical density was maximum for Group C (0.177), followed by control group (0.048), whereas it was similar for Group A and B (0.001) [Graph 5]. The difference in the means was highly significant with P< 0.0001. After 7 days, the mean difference across four groups was insignificantly different with P = 0.0591. Further, P value after 14 days suggested an insignificant difference in the means across four groups as P = 0.7941. The mean optical density for Group C was higher than remaining groups at each time point.




  Discussion Top


According to Quinn and Yoshikawa,[8] most clinical strategies to move teeth are based on the assumption that a force magnitude or range of magnitudes exists, that, when delivered to the periodontal tissue, will yield the most rapid rate of tooth movement. The classic concept of optimum force, as proposed by Schwarz,[2] defines it as the force leading to a change in tissue pressure that approximates the capillary vessel's blood pressure, thus preventing their occlusion in the compressed periodontal ligament. During orthodontic tooth movement, the early response of periodontal tissues to mechanical stress is an acute inflammatory reaction characterized by infiltration of neutrophils which have granules that contain MPO. The level of MPO activity is proportional to the number of polymorphonuclear cells in a tissue, reflecting the degree of inflammation.[3],[4],[5],[6]

In literature, various studies have demonstrated the presence of numerous biomarkers in the GCF during orthodontic tooth movement. These include MPO,[4],[5] acid phosphatase,[9] alkaline phosphatase,[9],[10] cathepsin B,[11] prostaglandin E2,[12] interleukin 1-beta and tumor necrosis factor-alpha,[13] leptin,[14] osteocalcin and N-telopeptides.[15] MPO was the choice of biomarker for the present clinicobiochemical study since it is the earliest marker to monitor the degree of periodontal inflammation.

In the current study, the difference between the presence of MPO in the GCF between the control and the experimental subgroups at 2 h for all the three groups is statistically significant. However, at 7 days and 14 days, difference between the presence of MPO in the GCF between the control and the experimental subgroups for all the three groups is not significant. These findings are consistent with those of Marcaccini et al.[4] with respect to GCF whose study showed that MPO activity is highly increased 2 h after appliance activation in both GCF and saliva, and that it decreases to baseline levels after 7 days. In their study, there was no statistically significant difference between MPO levels collected at 7 and 14 days although a lower value was observed on day 14 in both saliva and GCF. The present study, however, did not aim to assess the MPO levels in the saliva. In contrast to the above results, investigation done by Navarro-Palacios et al.[5] showed that the MPO activity in the saliva remained elevated at 2 h and day 7, but MPO activity in the GCF increased at 2 h; by day 7, a diminution was observed. This indicated that GCF can be a more confirmatory medium that accurately reflects inflammatory changes than saliva. GCF is produced directly in the gingival sulcus and by extravasation of circulating plasma. Saliva, in contrast, is produced by the salivary glands. Although saliva contains substances similar to GCF, it reflects the buccal environment more than the tooth environment. Therefore, GCF likely reflects local tooth inflammation caused by orthodontic movement more accurately than saliva. This could explain the different patterns of MPO activity that the author observed between GCF and saliva.

Investigations by Navarro-Palacios et al.[5] show that MPO activity in GCF more accurately reflects inflammation due to orthodontic tooth movement than MPO activity in saliva. These data disagree with those of Marcaccini et al.[4] who found no difference in the values of MPO activity in GCF and saliva. This is because the author used another MPO standard (PMN) that does not have the same accuracy as the isolated enzyme. Furthermore, it is possible that the authors detected this peak in MPO activity in saliva at day 7 because they monitored the first orthodontic activation that was thought would be stronger than the subsequent ones. Nevertheless, with these discrepancies, it was believed that both studies reinforce the fact that MPO activity can be considered an inflammation marker produced by an orthodontic force. Furthermore, MPO activity values in saliva were around 10-fold higher than those in GCF. This difference can be attributed to the fact that GCF was diluted in the buffer solution used to collect it from the paper strips, whereas the saliva was tested undiluted. MPO activity can be measured with a quick method that is inexpensive and accessible to most laboratories. Considering the basic fact that neutrophils form the first line of defense mechanism for inflammation following application of orthodontic tooth movement, MPO exhibited by the neutrophils' granules would be the first to be exhibited in the GCF. Collecting GCF samples is not invasive; then, MPO activity can rapidly monitor possible deleterious effects if an excessive orthodontic force has been applied, and adjustments can be made according to the individual response to orthodontic forces.

No statistically significant difference was found between the experimental subgroup (50 g) of Group A and that of Group B (75 g, P = 0.9999). Highly statistically significant difference was found between the experimental subgroup (75 grams) of Group B and that of Group C (100 g, P = 0.0001). Highly statistically significant difference was also found between the experimental subgroup (100 g) of Group C and that of Group A (50 g, P = 0.001). Boester and Johnston [16] in their clinical investigation suggested that the two-ounce force level (about 55 g) produced significantly less tooth movement than did the 5, 8, and 11 ounces (140, 225, and 310 g, respectively). The authors also stated that no statistically significant difference was found between the 5, 8, and 11 ounce groups; each produced about the same amount of space closure. Tanne et al.[17] in their experimental research work concluded that before tooth movement, tooth mobility exhibited a substantial increase when forces ranging from 50 to 150 g were applied; the rate of increase gradually decreased as the force rose to 500 g. These findings are consistent with those of Burrstone and Grooves,[18] who looking for the lowest possible force value to retract anterior teeth by tipping movement in 22 children, found no threshold value but observed optimal rates between 50 and 75 g. In contrast to the above findings, Smith and Storey [19] in their study on tooth movement in eight patients concluded that optimal lower canine movement occurred in the 150–250 g range. At higher force levels of 400–600 g, the anchor unit of the second premolar and the first molar moved more than the canine. The present study, however, did not subject the patients to such higher force levels. Furthermore, anchorage was not an assessment factor.

The GCF collection was done using a micropipette, for it facilitates volume-specific collection of GCF, simplifies the entire procedure, and rules out the requirement of additional apparatus associated with other methods of GCF collection.[20]


  Conclusion Top


The observations of the study have led to the following conclusions:

  • There is a highly significant increase in the MPO levels in the GCF at the 14th day after application of 150 g of force
  • The increase in MPO levels can be correlated to the onset of inflammatory reaction in the periodontium preceding orthodontic tooth movement
  • There is also a highly significant increase in the MPO levels in the GCF at 14th day after the application of 50, 75, and 100 g
  • This finding can lead to the conclusion that a minimum of 14 days are required for the beginning of orthodontic tooth movement to take place.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Burstone CJ. The biophysics of bone remodeling during orthodontics-optimal force considerations. In: Norton LA, Burstone CJ, editors. The Biology of Tooth Movement. Boca Raton, Florida: CRC Press; 1989. p. 321-34.  Back to cited text no. 1
    
2.
Schwarz AM. Tissues changes incident to orthodontic tooth movement. Int J Orthod 1932;18:331-52.  Back to cited text no. 2
    
3.
Faith M, Sukumaran A, Pulimood AB, Jacob M. How reliable an indicator of inflammation is myeloperoxidase activity? Clin Chim Acta 2008;396:23-5.  Back to cited text no. 3
    
4.
Marcaccini AM, Amato PA, Leão FV, Gerlach RF, Ferreira JT. Myeloperoxidase activity is increased in gingival crevicular fluid and whole saliva after fixed orthodontic appliance activation. Am J Orthod Dentofacial Orthop 2010;138:613-6.  Back to cited text no. 4
    
5.
Navarro-Palacios A, García-López E, Meza-Rios A, Armendariz-Borunda J, Sandoval-Rodríguez A. Myeloperoxidase enzymatic activity is increased in patients with different levels of dental crowding after initial orthodontic activation. Am J Orthod Dentofacial Orthop 2014;146:92-7.  Back to cited text no. 5
    
6.
Lamster IB, Oshrain RL, Gordon JM. Enzyme activity in human gingival crevicular fluid: Considerations in data reporting based on analysis of individual crevicular sites. J Clin Periodontol 1986;13:799-804.  Back to cited text no. 6
    
7.
Pender N, Samuels RH, Last KS. The monitoring of orthodontic tooth movement over a 2-year period by analysis of gingival crevicular fluid. Eur J Orthod 1994;16:511-20.  Back to cited text no. 7
    
8.
Andreasen GF, Zwanziger D. A clinical evaluation of the differential force concept as applied to the edgewise bracket. Am J Orthod 1980;78:25-40.  Back to cited text no. 8
    
9.
Insoft M, King GJ, Keeling SD. The measurement of acid and alkaline phosphatase in gingival crevicular fluid during orthodontic tooth movement. Am J Orthod Dentofacial Orthop 1996;109:287-96.  Back to cited text no. 9
    
10.
Perinetti G, Paolantonio M, D'Attilio M, D'Archivio D, Tripodi D, Femminella B, et al. Alkaline phosphatase activity in gingival crevicular fluid during human orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2002;122:548-56.  Back to cited text no. 10
    
11.
Griffiths GS, Moulson AM, Petrie A, James IT. Evaluation of osteocalcin and pyridinium crosslinks of bone collagen as markers of bone turnover in gingival crevicular fluid during different stages of orthodontic treatment. J Clin Periodontol 1998;25:492-8.  Back to cited text no. 11
    
12.
Lee KJ, Park YC, Yu HS, Choi SH, Yoo YJ. Effects of continuous and interrupted orthodontic force on interleukin-1beta and prostaglandin E2 production in gingival crevicular fluid. Am J Orthod Dentofacial Orthop 2004;125:168-77.  Back to cited text no. 12
    
13.
Basaran G, Ozer T, Kaya FA, Kaplan A, Hamamci O. Interleukine-1beta and tumor necrosis factor-alpha levels in the human gingival sulcus during orthodontic treatment. Angle Orthod 2006;76:830-6.  Back to cited text no. 13
    
14.
Dilsiz A, Kiliç N, Aydin T, Ates FN, Zihni M, Bulut C. Leptin levels in gingival crevicular fluid during orthodontic tooth movement. Angle Orthod 2010;80:504-8.  Back to cited text no. 14
    
15.
Alfaqeeh SA, Anil S. Osteocalcin and N-telopeptides of type I collagen marker levels in gingival crevicular fluid during different stages of orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2011;139:e553-9.  Back to cited text no. 15
    
16.
Boester CH, Johnston LE. A clinical investigation of the concepts of differential and optimal force in canine retraction. Angle Orthod 1974;44:113-9.  Back to cited text no. 16
    
17.
Tanne K, Inoue Y, Sakuda M. Biomechanical behavior of the periodontium before and after orthodontic tooth movement. Angle Orthod 1995;65:123-8.  Back to cited text no. 17
    
18.
Burrstone CJ, Grooves MH. Threshold and optimum force values for maxillary anterior tooth movement. J Dent Res 1961;39:695.  Back to cited text no. 18
    
19.
Smith R, Storey E. The importance of force in orthodontics: The design of cuspid retraction springs. Aust Dent J 1952;56:291-304.  Back to cited text no. 19
    
20.
Kettle AJ, Winterbourn CC. Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs. Biochem Pharmacol 1991;41:1485-92.  Back to cited text no. 20
    


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