International Orthodontics 2019; 17: 227–234

Websites:
www.em-consulte.com
www.sciencedirect.com

Original article
Influence of low-level laser (LLL) on
interleukin 6 (IL-6) levels in gingival
crevicular fluid (GCF) during orthodontic
tooth movement of periodontally affected
rabbits

Essam Abdelalim Nassar 1,2, Ahmed Maher Fouda 2, Khalid Sadiaq Hassan 3

Available online: 30 April 2019 1. Imam Abdurahman Bin Faisal University, Preventive Dental Science Department,
Saudi Arabia
2. Mansoura University, Orthodontic Department, Faculty of Dentistry, Egypt
3. Al-Azhar University, Assiut branch, Faculty of Dentistry, Egypt

Correspondence:
Essam Abdelalim Nassar, Preventive Dental Sciences Department, College of
Dentistry, Imam Abdulrahman Bin Faisal University, 1982 Dammam, Saudi Arabia.
e_abdelalim@yahoo.com

Keywords Summary
Tooth movement
Cytokines Aim > To evaluate the effects of LLL on IL-6 levels in the GCF and the Probing Pocket Depth
Laser measurements (PPD) during orthodontic tooth movement in periodontally affected rabbits.
Methods > Twenty-four rabbits were divided into 3 groups: Group 1(G1), healthy rabbits with
orthodontic movement, Group 2(G2) periodontally affected rabbits and orthodontic movement,
Group 3(G3) periodontally affected rabbits with orthodontic movement and LLL therapy. A 0.014
stainless steel spring was inserted in the upper central incisors to produce 60gm force. Laser CAT
500 was applied for 3 min/day for 2 weeks. PPD measurements were obtained at base line and
after 14 days with electronic periodontal probe.
Results > IL-6 levels increased gradually after application of orthodontic force, afterwards the 8th
day, a significant difference in the Il-6 levels between G1 vs. G2 and G2 vs. G3 was observed. PPD
measurements showed significant difference between the three groups at base line and after
14 days.
Clinical Significance > LLL application can enhance periodontal ligament regeneration and decrease
the periodontal tissue destruction through suppression of IL-6 levels.
227

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https://doi.org/10.1016/j.ortho.2019.03.004
© 2019 CEO. Published by Elsevier Masson SAS. All rights reserved.
 E.A. Nassar, A.M. Fouda, K.S. Hassan
Original article

Mots clés Résumé

Mouvement dentaire
Cytokines Influence du laser de faible intensité (LLL) sur les taux d'interleukine 6 (IL-6) dans le
Laser fluide gingival sulculaire (GCF) pendant le mouvement orthodontique chez les lapins
atteints de maladie parodontale

Objectif > Évaluer les effets de la LLL sur les taux d'IL-6 dans le GCF et les mesures de la profondeur
des poches au sondage (PPD) pendant le mouvement orthodontique dentaire chez les lapins
atteints de maladie parodontale.
Matériel et méthodes > Vingt-quatre lapins ont été divisés en 3 groupes : Groupe 1(G1) : lapins en
bonne santé sous traitement orthodontique ; groupe 2(G2) : lapins avec atteinte parodontale sous
traitement orthodontique ; groupe 3(G3) lapins avec atteinte parodontale, traitement orthodon-
tique et thérapie LLL. Un ressort en acier inoxydable 0,014 a été inséré dans les incisives centrales
supérieures pour produire une force de 60 g. Le LaserCat500 a été appliqué pendant 3 min/jour
pendant 2 semaines. LaserCat500 Les mesures PPD ont été obtenues au départ et après 14 jours
avec une sonde parodontale électronique.
Résultats > Les taux d'IL-6 ont augmenté progressivement après l'application de la force ortho-
dontique, après le 8e jour, une différence significative dans les taux d'IL-6 entre G1 et G2 et G2 et
G3 a été observée. Les mesures de PPD ont montré une différence significative entre les trois
groupes au départ et après 14 jours.
Signification clinique > L'application de LLL peut améliorer la régénération du ligament parodontal
et diminuer la destruction du tissu parodontal par la suppression des niveaux d'IL-6.

Introduction ligament might be destroyed during tooth tipping or intrusion in

Orthodontic tooth movement is achieved by means of alveolar
bone and periodontal ligament remodeling as a result to the
applied forces [1]. Force application changes the equilibrium
between collagen synthesis and degeneration [2] through a
group of proteolytic enzymes called metalloproteinases (MMPs)
such as MMP-1, MMP-8, and MMP-13, which control periodontal
tissue remodelling [3,4]. The MMP-1 levels in the peridonuim of
rats and humans were increased after application of orthodontic
forces [5,6]. Cytokines such as Interleukins, prostaglandin and
tumour necrosis factor-alpha (TNF-ɑ) were considered as the
regulators of MMP-1 expression in humans [7,8].
Cytokines have an important role in intercellular signalling and
have been involved in the pathology of periodontal diseases,
bone resorption, and bone reaction to orthodontic treatment.
The role of inflammatory mediators such as interleukins, pros-
taglandins and glycosaminoglycan has been investigated during
orthodontic tooth movement [9,10]. Changes in the amount and
concentration of Interleukin -6 (IL-6) in the periodontal tissues
reflect the status of periodontal tissue inflammation. Higher
levels of IL-6 in the crevicular fluid are shown after application
of orthodontic forces [11,12].
Periodontal diseases are characterized by bone loss and
decreased tissue support around the teeth due to inflammation
of the periodontal supporting structures. Orthodontic appliances
are considered as modifying factors for periodontal supporting
tissues [13]. Artun and Urbye suggested that the periodontal
228
periodontally affected teeth [14].
LLL application was associated with healing of the damaged
bone tissue following tooth extraction proved by radiological
analysis [15]. In addition, LLL irradiation has been shown to
increase the number of feasible osteocytes in the irradiated
areas significantly by encouraging the production of bone matrix
and creating an extremely reactive and vital bone tissue. Osteo-
cytes have a principal role in coordinating osteoblast and oste-
oclast recruitment and activity [16]. Moreover, Nissan et al.
reported that the LLL enhanced the bone-healing procedure
in artificially formed osseous cavities through triggering calcium
transfer for the period of new bone formation [17].
The aim of this study was to assess the effects of LLL on IL-6
levels in the gingival crevicular fluid and the probing pocket
depth throughout orthodontic tooth movement in periodontally
affected teeth in rabbit.
Materials and methods
Sample preparation
Sample size was calculated with EpiCalc 2000 version 1.02
(Brixton Books, Brixton, UK) software and indicated 8 rabbits
for each group to be reliable at 80% power and 95% confi-
dence interval. A flow chart for the experimental design is
shown in (figure 1). A total of 24 New Zealand male rabbits, 3.0
to 3.5 Kg each (mean age was 16 weeks) were used in the
study. The animals were randomly divided into 3 equal groups.

tome 17 > n82 > June 2019

Influence of low-level laser (LLL) on interleukin 6 (IL-6) levels in gingival crevicular fluid (GCF) during orthodontic
tooth movement of periodontally affected rabbits
Figure 1
Flow chart of the experimental design
In group 1 (control group), healthy animals received ortho-
dontic appliances. In group 2, periodontally affected animals
with orthodontic appliance. Group 3 represented the peri-
odontally affected animals with orthodontic appliance and
LLL application. The weight of each animal was recorded at
the beginning and checked throughout the experiment, there
was no significant weight loss. All rabbits were left for one
week before starting the experiment to accommodate to the
new environments. Dietary pellets and water were given to
the rabbits at standard times daily.
Production of experimental periodontal diseases
Rabbits were anesthetized by a single intraperitoneal injection
mixture of ketamine and xylazine (1:4). After the anesthesia, a 3-
0 black silk ligature was applied around the maxillary incisors in a
sub-marginal position. The ligature knot was placed on the buccal
side of the teeth. Afterwards, a topical application of
tome 17 > n82 > June 2019
Original article
Porphyromonas gingivalis three times/week for 6-weeks to
develop experimental periodontal diseases [18]. Porphyromonas
gingivalis was prepared as described by Jain et al. Periodontal
diseases was confirmed by the grade of inflammation in the
gingival and alveolar bone loss quantification at the experimen-
tal sites [19].
Orthodontic tooth movement
Two holes were drilled in the labial surface of the two central
incisors of each rabbit with low speed round bur number
10 under irrigation with normal saline. A 0.014 stainless steel
spring was adjusted to produce 60 gm. force according to Kilic
et al. [20]. The forces produced by the spring were measured
with force gauge (Correx Co, Bern, Switzerland). Each arm of the
spring was inserted in a hole and the ends were bent distally.
Any excess wire was trimmed to avoid any harm for the animal
(figure 2).
229
 E.A. Nassar, A.M. Fouda, K.S. Hassan
Original article
Figure 2
Collection of the GCF sample
The LLL application
Laser CAT 500 with a contentious wavelength 904 nm was pre-
adjusted to deliver a laser beam with an output power of 30 mW,
and a frequency of 10000 Hz for 3 minutes according to the
manufacturer's recommendation (MedSolution, Germany). The
LLL was applied once a day for two weeks starting after the
activation of the orthodontic appliance.
Probing pocket depth measurements
PPD measurements were obtained from 4 sites at the upper
incisors (mesial, mid-buccal, distal and mid-lingual). PPD were
measured with an electronic, pressure-calibrated probe (PA-ON
probe [Orange Dental, Biberach, Germany]. The tip of the probe
was flexible and fitted with a ball 0.5 mm in diameter. The tip
yields at a pressure of 20 Ncm according to the manufacturer
guidelines. PPD measurements were shown on the graphic
display of the probe and the average PPD measurements for
each group at the base line and after 14 days were recorded.
IL-6 analysis
Samples of GCF were taken from the upper incisors (figure 2) as
described by Rudin et al. [21]. Paper strips (Periopaper, Pro Flow,
Amityville, NY) were placed into the sulcus after the teeth were
dried with the air. GCF volume was determined after by using
Periotron 8000 (Ora Flow, Plainview, NY). The collected paper
strips were stored in sterile tubes containing 200 mL of sterile
sodium chloride solution at -20 8C until experimentation. The IL-
6 levels were assessed at the base line, 2nd, 4th, 6th, 8th, 10th,
12th and 14th days. IL-6 levels were measured using the
immunoassay system (Immulite, Diagnostic Products, Los
Angeles, Calif).
230
Statistical analysis
Data analysis was performed using SPSS 20.0 (IBM product
Chicago, USA). The results were presented in terms of mean
and standard deviation. Repeated measures-ANOVA was applied
to compare intergroup and intragroup comparison of IL-6 levels
among the three groups on various time intervals. Post-Hoc least
significance test was applied for pairwise comparison of the
studied groups. Unpaired t-test was applied to compare the
mean PPD between baseline and after 14 days observations.
Analysis of variance (ANOVA) compared the mean PPD and Post-
hoc Tukey test was performed to compare the difference of
means between the groups. P-value  0.05 was considered a
statistically significant result.
Results
Means and standard deviations of IL-6 levels in GCF of the
investigated animals at different days were plotted in (figure 3,
table I). In G1 and G2, the concentration of IL-6 increased
gradually from the base line and reached its highest level on
day 8 (20.2 pg/ml in G1, and 39.7 pg/ml in G2). On day
10 however, the Il-6 concentration started to decrease with
the lowest level on day 14 (13.1 pg/ml in G1, and 29.1 pg/
ml in G2). On the other hand, in G3 the highest level was
recorded in the second day (24.3 pg/ml) and it started to
decrease toward the experiment end. A statistically significant
difference in IL-6 at the base line among the 3 groups was found.
Subsequently on the day 2, there was a significant difference in
the IL-6 between G1 vs. G2 and G1 vs. G3. In contrast, no
statistically significant difference was observed between G2
and G3. Additionally, at the 4th and 6th days a significant
difference in IL-6 levels was recorded among the 3 groups
(G1 vs. G2, G2 vs. G3 and G1 vs. G3). Afterward from the 8th
day, there was a significant difference between G1 vs. G2 and G2
vs. G3 while there was no significant difference between G1 and
G3. ANOVA showed a significant difference between PPD of the
Figure 3
Comparing levels of interlukin-6 at different time points in the
three groups
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Influence of low-level laser (LLL) on interleukin 6 (IL-6) levels in gingival crevicular fluid (GCF) during orthodontic
tooth movement of periodontally affected rabbits

Original article
TABLE I
Comparison of mean, S.D and results of repeated ANOVA of IL-6 levels in the three groups at 8 various time intervals.

Days Group-1 Group-2 Group-3

T0 5.14  0.35 6.95  0.30 5.05  0.36

1,2
T2 7.14  0.36 24.81  0.72 24.38  0.49
1,2,3
T4 12.20  0.38 27.98  0.65 22.99  0.66
1,2,3
T6 17.98  0.15 35.93  0.72 21.25  0.69
1,3
T8 20.26  0.92 39.70  0.94 21.00  0.24
1,3
T10 18.63  1.18 34.26  0.58 19.44  1.12
1,3
T12 16.08  0.50 32.08  0.57 17.70  1.23
1,3
T14 13.18  0.51 29.18  0.78 15.09  0.56

T: Time T0: base line, T2: 2nd day, T4: 4th, T6: 6th, T8: 8th, T10: 10 th, T12: 12th, T14: 14th day.
1
Statistically significant difference group-1 vs. group-2 at P  0.05.
2
Statistically significant difference group-1 vs. group-3 at P  0.05.
3
Statistically significant difference group-2 vs. group-3 at P  0.05.

three tested groups at base line as well as on the 14th day. Tukey
test showed a non-significant difference between G2 and G3 at
the base line. In contrast there was a significant difference
between both groups and G1. Meanwhile, after 14 days there
was no significant difference between G1 and G3 but there was
a significant difference between the aforementioned groups
and G2 (figure 4 and table II).
Discussion
Orthodontic tooth movement can be damaging to the periodon-
tal tissues if the orthodontic forces applied on periodontally
affected teeth. Therefore search for a new method to enhance
the periodontal regeneration during orthodontic tooth move-
ment is mandatory. The current study was designed to evaluate
the impact of LLL application on the IL-6 levels in gingival
Figure 4
PPD at the baseline and after 14 days in the experimental
groups
crevicular fluid and the PPD during orthodontic tooth movement
in animal models having periodontally affected teeth. The LLL
effect on orthodontic tooth movement and assessment of Il-6
levels in healthy animals were not included in our study design
since they were evaluated earlier [22,23].
Animal experiment is an important tool to evaluate periodontal
tissue remodelling resulted from orthodontic force and rabbits
were chosen because they were recognized as a good small
animal model to study the periodontal diseases [24]. In order to
get a predictable and reproducible periodontitis,
Porphyromonas gingivalis was topically applied one time each
successive day for 6 weeks around teeth that were ligated
before. Ligature wire alone was not capable of producing tissue
damage in rabbits [16].
In this study, IL-6 expression was examined in the GCF in order to
investigate the relation between IL-6 levels, the pathogenesis of
the periodontal diseases and LLL therapy. It is generally accepted
that the GCF reflects the immune and inflammatory reactions
obtained from host–parasite interactions [25] and bio-mechani-
cal stress [26]. Therefore, GCF constituents, including microbial
products and inflammatory mediators, can be useful for the
determination of the periodontal condition during periodontal
inflammation [25] or orthodontic tooth movement [27]. Our
findings showed a gradual increase in the IL-6 expression in the
GCF after orthodontic force application. These findings were in
accord with Uematsu et al. [11], who reported an increase in the
concentration of IL-1ß, IL-6, tumour necrosis factor (TNF) and
other inflammatory mediators after application of orthodontic
forces in humans. In the current study, IL-6 levels were gradually
increased and reached a maximum on day 8 in G1 and G3. A
significantly higher concentration of IL-6 was noticed in G3
231

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 E.A. Nassar, A.M. Fouda, K.S. Hassan
Original article
TABLE II
Periodontal depth measurements at base line and after 14 days in the three studied groups.
Column1 PPD
Groups Baseline
Group-1 1.30  0.13
Group-2 1.60  0.11
Group-3 1.70  0.20
P value (1 vs. 2 vs. 3) < 0.001
P value (2 vs. 3) P = 0.399
1
Significance at 5% level following the comparison into horizontal direction.
2
Significance at 5% level following the comparison into vertical direction.
compared to G1. The periodontal condition augmented the
expression of IL-6 in addition to the applied orthodontic force
and then IL-6 expression declined in both groups. These findings
are in accordance with other studies that reported a decrease in
the levels of inflammatory cytokines on the 7th and 10th days
following force activation [28,29].
On the other hand, IL-6 levels were more significantly increased
in group 2 compared to G2 and G3. In group 3, IL-6 reached its
highest level in the 2nd day and then decreased after LLL
application. IL-6 is considered a major mediator of the host
response for the tissue injury and infection. It plays a major
role in B-cell differentiation in the immune system. It also has
multiple biological activities, such as the enhancement of cell
proliferation and bone resorption acceleration [30,31]. There-
fore, IL-6 is involved in the pathogenesis of various inflamma-
tory diseases and among them is the periodontal disease. Such
findings indicated that the LLL could inhibit or reduce IL-6
production. Similarly, a significant inhibition of prostaglandin
E2 and IL-1b production after application of LLL were reported
earlier [32].
The current study showed a significant improvement in PPD
measurements in G3 after application of the LLL. Improvement
of the attachment level indicated periodontal tissue regenera-
tion during orthodontic tooth movement. These finding are in
agreement with Alazzawi et al. [33] and Gunji et al. [34], who
concluded that laser irradiation can enhance the velocity of
tooth movement and improve the quality of bone remodelling.
They inferred that, laser irradiation increased NF-KB ligands
(RANKL) gene expression and accelerate osteoclast differentia-
tion facilitating bone resorption on the pressure side whereas on
the tension side, irradiation led to enhance alkaline phosphatase
(ALP) and proliferating cell nuclear antigen (PCNA) release that
induced bone formation. According to the report of Kunimatsu
et al. [35], laser irradiation increased cell division and migration
of mouse calvarial cells (MT3T3-E1) through mitogen-activated
protein kinase (MARK/ERK) signalling that enhance proliferation
232
Column 2 Column 3
14 days P value
1.40  0.12 0.214
1,2
1.80  0.16 0.012
1.50  0.09 0.059
< 0.001
(1 vs. 3) P = 0.279
and migration of osteoblasts to improve regeneration of bone
tissue. LLL was effective when used in healing areas of bone
fracture, but on the other hand it has been unsuccessful when
applied to normal tissue [36]. In addition, LLL therapy resulted in
a better quality of bone, increased the production of differenti-
ated osteoclasts, as well as accelerated tooth movement [37].
Furthermore LLL can enhance cellular production and differenti-
ation of osteoblast lineage nodule-forming cells, especially in
devoted precursors, resulting in a raise in the number of differ-
entiated osteoblastic cells [38]. Gomes et al. [39], reported that
laser irradiation led to continuous reorganization of the soft
periodontal tissue, leading to the maintenance and integrity
of the periodontal microstructure under the orthodontic force
especially in uncontrolled diabetic rats. In G2 the impaired
condition of periodontal tissue plus the orthodontic forces
aggravated the inflammatory process and induced further
destruction of periodontal tissue. Collagen degradation and
deposition occurs normally in the absence of inflammation
during normal physiological PDL and bone remodelling [40].
Conclusions and recommendations
The application of low-level laser reduced PPD and decreased
the inflammatory process through suppression of IL-6 levels and
promoted healing during orthodontic tooth movement in peri-
odontally affected animals. Future histopathological evaluation
of the PDL to assess the changes after LLL therapy, additional
examination of other clinical parameters such as gingival index
and probing attachment level should be done. Further, compa-
rative studies are recommended to compare the effects of LLL
therapy on orthodontic tooth movement among treated and/or
untreated periodontally compromised teeth.
Disclosure of interest
The authors declare that they have no competing interest.
tome 17 > n82 > June 2019
Influence of low-level laser (LLL) on interleukin 6 (IL-6) levels in gingival crevicular fluid (GCF) during orthodontic
tooth movement of periodontally affected rabbits
Ethical approval
This study was approved from Ethical approval committee at Al-
Azhar University, Assiut branch, Egypt.
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tome 17 > n82 > June 2019