ORIGINAL ARTICLE

Myeloperoxidase enzymatic activity is increased

in patients with different levels of dental
crowding after initial orthodontic activation

pez,b Alejandra Meza-Rios,c Juan Armendariz-Borunda,d
Alejandra Navarro-Palacios,a Eliezer Garc
a-Lo
e


and Ana Sandoval-Rodrguez
Guadalajara, Jalisco, Mexico
Introduction: Orthodontic tooth movement implies application of forces that generate an inammatory process.
The myeloperoxidase (MPO) enzyme is found inside neutrophil granules. MPO activity indirectly reects the
level of inammation. The aim of this study was to measure MPO activity in gingival crevicular uid (GCF)
and whole saliva in orthodontic patients with different levels of dental crowding at the alignment phase of orthodontic treatment with the same archwires. Methods: Twenty patients were classied according to the irregularity
index into 2 groups: severe and minimum crowding (10 in each group). MPO activity was evaluated in GCF and
saliva at 0 and 2 hours, and 7 and 14 days after the orthodontic appliances were activated. MPO activity was
measured using the modied Bradley-Bozeman technique. Results: In both groups, the maximum activity
was at 2 hours (P \0.05) after activation. MPO activity remained elevated until day 7, and values similar to baseline were found at day 14 in the GCF and saliva samples. Enzymatic activity did not show statistical differences
between the groups. Conclusions: The amount of dental crowding does not seem to inuence MPO activity,
which showed similar patterns in GCF and saliva, but the values in GCF reected the inammatory changes
more accurately than did the values in saliva. The quantication of MPO activity is a useful biologic marker as
an indirect measurement of inammation generated with tooth movement independent of the amount of crowding. (Am J Orthod Dentofacial Orthop 2014;146:92-7)

rthodontic tooth movement involves 2 interrelated processes: deection or bending of the

alveolar bone and remodeling of the periodontal
tissues.1 This bone remodeling is used by orthodontists
when forces transmitted to the surrounding tissues of
the periodontium initiate the remodeling process.2 The
mechanical stimulus causes an inammatory response
in the periodontal tissues, and inammatory mediators
that trigger the biologic processes associated with
From the University of Guadalajara, Guadalajara, Jalisco, Mexico.
a
Postgraduate student, Department of Orthodontics.
b
Professor and chairman, Department of Orthodontics.
c
Postgraduate student, Department of Molecular Biology and Genomics,
Institute of Molecular Biology and Gene Therapy.
d
Head, Department of Molecular Biology and Genomics.
e
Professor and researcher, Department of Molecular Biology and Genomics,
Institute of Molecular Biology and Gene Therapy.
All authors have completed and submitted the ICMJE Form for Disclosure of
Potential Conicts of Interest, and none were reported.
Address correspondence to: Ana Sandoval-Rodr
guez, Department of Molecular
Biology and Genomics, Institute for Molecular Biology and Gene Therapy,
University of Guadalajara, Sierra Mojada 950, Colonia Independencia,
Guadalajara, Jalisco 44340, M
exico; e-mail, anasol44@hotmail.com.
Submitted, February 2013; revised and accepted, April 2014.
0889-5406/$36.00
Copyright 2014 by the American Association of Orthodontists.
http://dx.doi.org/10.1016/j.ajodo.2014.04.015

92

alveolar bone resorption and apposition are released.3

It has been proven that orthodontic forces cause acute
inammatory reactions, vascular changes, and migration of leucocytes.4,5
Inammation is characterized by inltration of leucocytes, among them neutrophils. These polymorphonuclear
cells have granules that contain myeloperoxidase (MPO);
this enzyme produces oxidant molecules that can cause
lipid peroxidation. The level of MPO activity is proportional to the number of polymorphonuclear cells in a tissue, reecting the degree of inammation.6,7
MPO enzymatic activity analysis of saliva or gingival
crevicular uid (GCF) is a useful method for monitoring
periodontal inammation.7-9 This is particularly
interesting because collection of the sample is not
invasive, and the determination method is simple and
accessible to standard laboratories.9
Dental crowding is a disparity in the relationship between tooth size and jaw size.10 It occurs when the space
required for alignment of the permanent teeth exceeds
the space available in the dental arch. This usually results
in rotated, ectopic, or impacted teeth.11,12
We expected that MPO activity and then inammation could differ in patients exposed to orthodontic

Navarro-Palacios et al

forces with different levels of dental crowding at the

alignment phase of orthodontic treatment. The aim of
this study was to examine changes in MPO activity in
saliva and GCF in patients with different amounts of
dental crowding after their rst orthodontic activation
with the same archwire size.
MATERIAL AND METHODS

Patients attending the orthodontic clinic of the

University of Guadalajara in Mexico were invited to
participate in the study. All patients gave written
informed consent. The study was approved by the
Ethics and Biosecurity Board of the Health Sciences
University Center of the University of Guadalajara.
Twenty orthodontic patients (5 male, 15 female;
mean age, 19 6 1.3 years) were enrolled and classied
as having severe (7-9 mm) or minimum (1-3 mm)
crowding of the mandibular teeth according to the
irregularity index.13 Each group included 10 patients.
All patients met the following criteria: clinically
healthy, no anti-inammatory drugs taken during
the month before the study, no antibiotics taken in
the previous 6 months, nonsmokers, healthy periodontal tissues with generalized probing depths
3 mm or less, no periodontal bone loss according to
their radiographs, and no supragingival plaque on
the teeth.
The subjects were told not to eat or drink for 1 hour
before the examination. The GCF and saliva samples
were collected immediately before the colocation of
the orthodontic xed appliances at baseline, 2 hours, 7
days, and 14 days after the initial orthodontic activation.
The initial activation consisted of placement of superior
and inferior bands with 0.022-in tubes (3M Unitek,
Monrovia, Calif) and bonded xed orthodontic appliances with a 0.022-in slot (3M Unitek) using a direct
technique in all teeth with a light-cured adhesive (Transbond XT, 3M Unitek). To ensure the same activation in
both groups, the initial archwire for all patients was a
0.012-in nickel-titanium alloy (Nitinol Classic; 3M Unitek) with an elastomeric ligature in each bracket. The
wire was fully engaged in each bracket. GCF samples
were taken from the mesiobuccal or distobuccal aspect
of the mandibular incisors that received active forces
during the initial activation. Samples were always taken
from the same site at the 4 time points. After isolating
each tooth with a cotton roll, the crevicular site was dried
with an air syringe. The GCF samples were collected
using an absorbent paper strip (periopaper, Pro-Flow;
Interstate Drug Exchange, Amityville, NY) placed into
the sulcus until some resistance was felt and left in for
30 seconds.14 The paper strips were completely lled.

93

The samples were not contaminated with saliva or blood.

The lled strips were individually placed in sterile Eppendorf tubes containing 100 mL of buffer (50 mmol/L
of Tris-hydrogen chloride, pH 7.4; 200 mmol/L of
sodium chloride; 10 mmol/L of calcium chloride; and
0.02% of triton X-100). The samples were kept on ice
for half an hour and then centrifuged at 13,000 g for
10 seconds at 4
C. Supernatants were collected and
stored at 70
C until further analysis. Saliva was
collected from under the right side of the tongue after
the patients rinsed their mouths with water.
The amount of saliva that was collected was 5 mL
using a 10-mL sterile syringe; it was placed in a 15-mL
sterile Falcon tube (Corning Inc, Corning, NY). The samples were immediately centrifuged at 10,000 g for 10
seconds at 4
C, and the supernatants were stored at
70
C until MPO analyses.
MPO activity was measured by Bradley-Bozeman
modied technique.15,16 Briey, 100 mL of the sample
(GCF, saliva, or MPO standard) was mixed intensively
using a vortex with 2 mL of 0.5% of hexadecyltrimethylammonium bromide (Sigma-Aldrich, St Louis,
Mo) solution. After that, 0.9 mL of solution containing
1.6 mmol/L of 3,30 ,5,50 -tetramethyl benzidine (SigmaAldrich), 50 mmol/L of o-Dianisidine dihydrochloride
(Sigma-Aldrich), and 0.5 mmol/L hydrogen peroxide
were added. An intense vortex was applied for 15
seconds, and the reaction was incubated in darkness
for 10 minutes. Subsequently, 100 mL of the reaction
solution was placed in a 96-well plate (Costar; Corning
Inc), and 50 mL of stop solution (sulphuric acid
[H2SO4], 2 mol/L) was added. A spectrophotometrical
reading was accomplished at a wavelength of 450 nm
using a mQuant microplate reader (Bio-TekInstruments,
Winooski, Vt). A standard curve with MPO reagent
(Sigma-Aldrich) was generated. MPO activity was
expressed as units per 100 mL.
Statistical analysis

Data were expressed as means and standard deviations. Statistical signicances were calculated with the
Friedman test for intergroup and intragroup differences,
followed by the Wilcoxon test for related samples and the
Kruskal-Wallis test for independent samples in both
crowding groups. Differences in the comparisons between the groups were considered signicant at P\0.05.
RESULTS

The mean for dental crowding in the minimum

crowding group was 1.3 6 0.6 mm according to irregularity index. As shown in Figure 1, A, after initial activation, patients with minimum dental crowding showed

American Journal of Orthodontics and Dentofacial Orthopedics

July 2014  Vol 146  Issue 1

Navarro-Palacios et al

94

Fig 1. MPO activity in saliva and GCF in patients with

minimum dental crowding: A, MPO activity in saliva; B,
MPO activity in GCF. *P .0.05; **P .0.01.

increased MPO activity in their saliva at 2 hours (0.30 6

0.11; P \0.05) compared with baseline (0.23 6 0.09)
and 14 days (0.22 6 0.09), and this increase remained
at day 7 (0.29 6 0.18) but without statistical difference.
MPO activity in the GCF showed increased activity at 2
hours (0.04 6 0.03; P \0.05) and 7 days (0.03 6
0.02; P \0.05) compared with baseline (0.02 6 0.01)
and 14 days (0.03 6 0.02), as depicted in the saliva
(Fig 1, B).
In the group with severe dental crowding, the mean
was 8.3 6 0.8 mm according to the irregularity index.
As shown in Figure 2, A, after initial activation, the
patients showed a tendency for increased MPO activity
in their saliva at 2 hours (0.31 6 0.26) and 7 days
(0.31 6 0.20; P \0.05) compared with baseline
(0.18 6 0.10) and 14 days (0.20 6 0.12). There was
no statistical signicance for the data at 2 hours. In
Figure 2, B, MPO enzymatic activity in the GCF increased
at 2 hours (0.06 6 0.04; P \0.05) and at day
7 (0.05 6 0.03; P \0.05) compared with baseline
(0.02 6 0.01) and day 14 (0.03 6 0.01).
As shown in Figure 3, A, MPO activity in the saliva
showed similar patterns in patients with minimum and
severe dental crowding; the levels tended to be increased
at 2 hours and 7 days, compared with the data at baseline and 14 days. However, at 2 hours and 7 days, the

July 2014  Vol 146  Issue 1

Fig 2. MPO activity in saliva and GCF in patients with

severe dental crowding: A, MPO activity in saliva; B,
MPO activity in GCF. *P .0.05.

mean values for the patients with severe dental crowding

were higher than those of the patients with minimum
crowding, perhaps indicating an effect during the acute
phase of inammation. In the GCF, the maximum MPO
activity was detected at 2 hours; this activity was maintained at day 7 and reached levels similar to baseline at
day 14 (Fig 3, B). Also, when all patients were grouped
without taking into account their dental crowding,
MPO activity increased at 2 hours (0.31 6 0.19;
P \0.05) and 7 days (0.31 6 0.18; P \0.05) after the
initial activation in saliva and reached levels similar to
the baseline (0.22 6 0.09) at day 14 (0.21 6 0.01) (Fig
4, A). This pattern was similar for GCF, but in this biologic uid the statistical signicance was even greater;
by day 7, a decrease in MPO activity had started (Fig 4,
B). MPO activity increased at 2 hours (0.04 6 1.01;
P \0.01) and 7 days (0.03 6 0.01; P \0.05) compared
with baseline (0.02 6 0.01) and 14 days (0.02 6 0.01).
Also, the levels of MPO activity were about 10-fold
higher in saliva than in GCF, indicating that GCF is probably a more sensitive marker for inammation caused by
tooth movement.
DISCUSSION

The patients in this study were evaluated using

MPO activity to detect initial inammation caused by

American Journal of Orthodontics and Dentofacial Orthopedics

Navarro-Palacios et al

95

Fig 3. Comparison of MPO activity in saliva and GCF of

patients with different dental crowding: A, MPO activity
in saliva; B, MPO activity in GCF. MDC, Minimum dental
crowding; SDC, severe dental crowding.

orthodontic tooth movement during the alignment

phase after using a 0.012-in nickel-titanium wire tied
to the orthodontic xed appliances. The type of tooth
movement was presumably uncontrolled tipping as expected. Dental crowding denes the range of orthodontic force to be applied in the initial alignment phase. The
rationale was to evaluate the inuence of dental crowding in the initial inammation process according to the
differences in the deection of the initial wire. Since 2
groups with different amounts of crowding and the
same archwire size were evaluated, a difference of the
force range applied in each group was also assumed.
In orthodontics, the source of the force is usually a
deection spring or an archwire. The amount of force
generated is dictated by the spring rate and by the distance of the clinical activation of the wire.17 Furthermore, the 0.012-in archwire used for the initial
leveling phase was a nickel-titanium classic that has a
linear strain delivery curve with a relatively small slope;
therefore, the stress-strain relationship varied with the
amount of activation.18
Since all patients were in the initial alignment and
leveling phase with the same archwire, differences in
inammation were expected to inuence alignment
according to dental crowding. In contrast to our
expectations, when we compared the values of MPO
activity at all monitored times, there was no difference

Fig 4. MPO activity in saliva and GCF in all patients

independent of their dental crowding: A, MPO activity in
saliva; B, MPO activity in GCF. *P \0.05; **P \0.01;
***P .0.001.

between the subjects with minimum and severe dental

crowding as shown in Figure 3. This indicated that the
initial alignment of the teeth according to dental
crowding, when using an archwire that delivers a low
range of force, did not signicantly inuence the level
of inammation. However, the samples from patients
with severe dental crowding tended to show that
mean MPO activity levels increased more than in the
subjects with minimum crowding. Probably, if a higher
archwire than the one we used had been placed, differences in MPO activity (inammation) would have been
found between the groups. The collection times were
based on several studies indicating that inammation
begins immediately after the application of orthodontic forces; this happens just after activation of the
xed appliances.9,19,20 In our study, the base sample
collection indicated the normal level of MPO activity
because the patients had not yet had any
orthodontic tooth movement. Then, subsequent
samples reected the acute and native responses to
an orthodontic force; to our knowledge, this has not
been measured before.
Also, MPO activity in saliva and GCF showed the same
pattern independent of the patients' dental crowding (Figs

American Journal of Orthodontics and Dentofacial Orthopedics

July 2014  Vol 146  Issue 1

Navarro-Palacios et al

96

2 and 3). MPO activity in saliva remained elevated at 2

hours and day 7, but MPO activity in the GCF increased
at 2 hours; by day 7, a diminution was observed,
indicating that this uid can be a marker that accurately
reects inammatory changes. GCF is produced directly
in the gingival sulcus and by extravasation of circulating
plasma.21 Saliva, in contrast, is produced in the salivary
glands. Although saliva contains substances similar to
GCF, it reects the buccal environment more than the
tooth environment. Therefore, GCF likely reects local
tooth inammation caused by orthodontic movement
more accurately than saliva. This could explain the
different patterns of MPO activity that we observed between GCF and saliva.22
This study shows that MPO activity in GCF more
accurately reects inammation due to orthodontic
tooth movement than MPO activity in saliva. These
data disagree with those of Marcaccini et al,9 who
found no difference in the values of MPO activity in
GCF and saliva. We believe that this is because
Marcaccini et al used another MPO standard (polymorphonuclear leucocytes) that does not have the same
accuracy as the isolated enzyme. Also, it is possible
that we detected this peak in MPO activity in saliva
at day 7 because we monitored the rst orthodontic
activation that we thought would be stronger than
the subsequent ones. Nevertheless, with these discrepancies, we believe that both studies reinforce the fact
that MPO activity can be considered an inammation
marker produced by an orthodontic force. Furthermore, MPO activity values in saliva were around
10-fold higher than those in GCF. We suppose that
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. We believe that MPO activity can be
measured with a quick method that is inexpensive
and accessible to most laboratories. 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.
There are many studies of inammatory markers
during tooth movement that were measured using
enzyme-linked
immunosorbent
assay9,19,20,23-25;
however, MPO activity is a simple and easy
spectrophotometric assay from a noninvasive sample
that can aid in the development of tests for most
orthodontists. Nevertheless, further studies by our
group and other groups using MPO activity are needed
to validate it as a reliable orthodontic inammatory
marker.

July 2014  Vol 146  Issue 1

CONCLUSIONS

Dental crowding did not seem to inuence MPO

activity as determined from GCF or saliva after application of the initial orthodontic activations in patients
with minimum and severe dental crowding who were
using the same archwire size. Also, these results suggest
that MPO activity in GCF reects inammatory changes
more accurately than MPO activity in saliva. MPO enzymatic activity seems to be a useful biologic marker to
monitor the inammation caused by orthodontic tooth
movement, especially in GCF.

REFERENCES
1. Murray C. The tissue, cellular and molecular regulation of
orthodontic tooth movement: 100 years after Carl Sandstedt.
Eur J Orthod 2008;28:221-40.
2. Davidovitch Z. Tooth movement. Crit Rev Oral Biol Med 1991;2:
411-50.
3. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level
reactions to orthodontic force. Am J Orthod Dentofacial Orthop
2006;129:469.e1-32.
4. Perinetti G, Serra E, Paolantonio M, Brue C, Meo SD, Filippi MR,
et al. Lactate dehydrogenase activity in human crevicular uid
during orthodontic treatment: a controlled, short-term longitudinal study. J Periodontol 2005;76:411-7.
5. Burke JC, Evans CA, Crosby TR, Mednieks MI. Expression of
secretory proteins in oral uid after orthodontic tooth movement.
Am J Orthod Dentofacial Orthop 2002;121:310-5.
6. Mathy-Hartert M, Bourgeois E, Gr
ulke S, Deby-Dupont G, Caudron I,
Deby C, et al. Purication of myeloperoxidase from equine polymorphonuclear leucocytes. Can J Vet Res 1998;62:127-32.
7. Cao CF, Smith QT. Crevicular uid myeloperoxidase at healthy,
gingivitis and periodontitis sites. J Clin Periodontol 1989;16:17-20.
8. Yamalik N, Caglayan F, Kilinc K, Kilinc A, Tumer C. The importance
of data presentation regarding gingival crevicular uid myeloperoxidase and elastase-like activity in periodontal disease and health
status. J Periodontol 2000;71:460-7.
9. Marcaccini AM, Amato PA, Le~ao FV, Gerlach RF, Ferreira JT.
Myeloperoxidase activity is increased in gingival crevicular uid
and whole saliva after xed orthodontic appliance activation.
Am J Orthod Dentofacial Orthop 2010;138:613-6.
10. Howe R, McNamara JA, O'Connor K. An examination of dental
crowding and its relationship to tooth size and arch dimension.
Am J Orthod 1983;83:363-73.
11. Van der Lieden FP. Theoretical and practical aspects of crowding in
the human dentition. J Am Dent Assoc 1974;89:139-53.
12. Bernab
e E, Del Castillo C, Flores-Mir C. Intra-arch occlusal
indicators of crowding in the permanent dentition. Am J Orthod
Dentofacial Orthop 2005;128:220-5.
13. Little R. The irregularity index: a quantitative score of mandibular
anterior alignment. Am J Orthod 1975;68:554-63.
14. Lamster IB, Hartley LJ, Vogel RI. Development of a biochemical
prole for gingival crevicular uid. Methodological considerations
and evaluation of collagen-degrading and ground substancedegrading enzyme activity during experimental gingivitis.
J Periodontol 1985;56(11 Suppl):13-21.
15. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement
of cutaneous inammation: estimation of neutrophil content with
an enzyme marker. J Invest Dermatol 1982;78:206-9.

American Journal of Orthodontics and Dentofacial Orthopedics

Navarro-Palacios et al

16. Bozeman PM, Learn DB, Thomas EL. Assay of the human leukocyte
enzymes myeloperoxidase and eosinophil peroxidase. J Immunol
Methods 1990;126:125-33.
17. Johnson E. Relative stiffness of beta-titanium archwires. Angle
Orthod 2003;73:253-69.
18. Burstone CJ. Variable modulus orthodontics. Am J Orthod 1981;
80:1-16.
19. Lee KJ, Park YC, Yu HS, Choi SH, Yoo YJ. Effects of continuous and
interrupted orthodontic force on interleukin-1b and prostaglandin
E2 production in gingival crevicular uid. Am J Orthod Dentofacial
Orthop 2004;125:168-77.
20. Tuncer BB, Ozmeric N, Tuncer C, Teoman I, Cakilci B, Y
ucel A, et al.
Levels of interleukin-8 during tooth movement. Angle Orthod
2005;75:631-6.
21. Weinsteing E, Mandel ID, Salkind A, Oshrain HI, Pappas GD.
Studies of gingival uid. Periodontics 1967;5:161-6.

97

22. Akalin FA, Baltacio

glu E, Alver A, Karabulut E. Lipid peroxidation
levels and total oxidant status in serum, saliva and gingival
crevicular uid in patients with chronic periodontitis. J Clin
Periodontol 2007;34:558-65.
23. Tzannetou S, Efstratiadis S, Nicolay O, Grbic J, Lamster I.
Interleukin-1 beta and beta-glucuronidase in gingival crevicular
uid from molars during rapid palatal expansion. Am J Orthod
Dentofacial Orthop 1999;115:686-96.
24. Tzannetou S, Efstratiadis S, Nicolay O, Grbic J, Lamster I. Comparison of levels of inammatory mediators IL-1b and bG in gingival
crevicular uid from molars, premolars, and incisors during rapid
palatal expansion. Am J Orthod Dentofacial Orthop 2008;133:
699-707.
25. Karacay S, Saygun I, Bengi AO, Serdar M. Tumor necrosis factoralpha levels during two different canine distalization techniques.
Angle Orthod 2007;77:142-7.

American Journal of Orthodontics and Dentofacial Orthopedics

July 2014  Vol 146  Issue 1