Eur J Oral Sci 2008; 116: 89–97  2008 The Authors.

Printed in Singapore. All rights reserved Journal compilation  2008 Eur J Oral Sci
European Journal of
Oral Sciences

Review
Yijin Ren1, Arjan Vissink2
Cytokines in crevicular fluid and 1
Department of Orthodontics and 2Department
of Oral and Maxillofacial Surgery, University
orthodontic tooth movement Medical Center of Groningen, University of
Groningen, Groningen, the Netherlands

Ren Y, Vissink A. Cytokines in crevicular fluid and orthodontic tooth movement. Eur J
Oral Sci 2008; 116: 89–97.  2008 The Authors. Journal compilation  2008 Eur J Oral
Sci

This review aimed to evaluate studies on cytokines in the gingival crevicular fluid
(GCF) during orthodontic treatment, summarizing the regulation patterns of the most
commonly studied cytokines and exploring their clinical implications. To achieve this,
a number of key databases were searched using MESH terms and free text terms. An
additional search was made by reference tracking. The procedures suggested by the
QUOROM statement were followed. Data from the included studies were extracted
into orthodontic mechanics, GCF sampling/handling methods, and cytokine mea-
surements. From the 85 relevant studies identified, 23 studies could be included. Yijin Ren, Department of Orthodontics,
Common drawbacks consisted mainly of inadequacies in the study design (e.g. short University Medical Center of Groningen
(UMCG), Triadegebouw 24, Hanzeplein 1,
duration and small number of study subjects). The most consistent result was a peak of
9700RB Groningen, the Netherlands
cytokine levels at 24 h. Associations existed between prostaglandin E2 (PGE2) and
interleukin-1b (IL-1b) and pain, velocity of tooth movement, and treatment Telefax: +31–50–3619712
E-mail: y.ren@dmo.umcg.nl
mechanics. Interleukin-1b and PGE2 showed different patterns of up-regulation, with
IL-1b being more responsive to mechanical stress and PGE2 more responsive to Key words: gingival crevicular fluid (GCF);
synergistic regulation of IL-1b and mechanical force. The results might be taken to orthodontics; systematic review; interleukin-1b;
prostaglandin E2
support, at the cellular level, the use of light continuous forces for orthodontic
treatment. Accepted for publication October 2007

The analysis of specific constituents in the gingival cre-
vicular fluid (GCF) may provide quantitative biochemi-
cal indicators for using to evaluate the local cellular
metabolism, reflecting the periodontal health status and
bone remodeling process during orthodontic treatment
(1). In recent years, the presence/expression of regulatory
proteins in the GCF has been recognized as a promising
diagnostic tool for monitoring orthodontic treatment
outcome. The interest of most reports was focused on the
presence of new mediators and on the up-regulation/
down-regulation of the levels of these regulatory proteins
in different treatment designs. Currently, the information
from individual studies on the relationship between
regulatory proteins in GCF and orthodontic tooth
movement is still limited. Moreover, several narrative
reviews have extensively illustrated the involvement of
regulatory proteins in periodontal remodeling provoked
by orthodontic stimuli (2, 3), but little effort has been
made to investigate systematically the time-related reg-
ulations and production mechanisms of these regulatory
proteins stimulated by mechanical stress.
Among the more than 100 regulatory proteins detected
in GCF, this review will focus on cytokines. Cytokines
are regulatory proteins secreted by white blood cells and
a variety of other cells in the body. The pleiotropic
actions of cytokines include numerous effects on cells of
the immune system and modulation of inflammatory
responses. In this review, cytokines refer to non-hormone
signaling factors, including lymphocyte-derived factors,
monocyte-derived factors, colony-stimulating factors,
growth factors, and chemotactic chemokines (4). Cyto-
kines are produced by periodontal tissue cells, such as
fibroblasts and osteoblasts, and are involved in normal
physiological turnover and remodeling of bone (5–7).
Many characteristic features of cytokines make them
particularly interesting to orthodontics, as cytokines in
GCF reflect the local microenvironment of periodontal
tissues, the area whereupon the effect of orthodontic
forces is exerted. For example, cytokines act locally over
short distances as autocrine or paracrine intercellular
signals in tissues and are, in general, not constitutively
produced but are expressed in response to local signals
(8). In addition, numerous studies have shown that
cytokines play pivotal roles in cell–cell signaling in bone
(9–11) and in mediating mechanically induced bone
remodeling (e.g. by orthodontic forces) (12–14).
Clinical studies on the up-regulation/down-regulation
of cytokines in GCF collected from patients have been
carried out to provide a non-invasive way to show the
involvement of cytokines in orthodontic tooth move-
ment. However, no report in the literature has systemi-
cally evaluated these studies. Thus, it was the aim of this
systematic review to evaluate studies on cytokine
expression in the GCF during orthodontic treatment, to
summarize the time-related regulation patterns of the
cytokines most commonly studied and to explore their
clinical implications. We focused on the following ques-
tions. Is it possible to perform a meta-analysis on the role
90 Ren & Vissink
of GCF cytokines in orthodontic tooth movement? If not,
is it possible to identify some associations between GCF
cytokines and tooth movement? And, finally, is it possible
to generate a time-related regulation pattern of the pro-
duction of GCF cytokines that may provide evidence
at the cellular level for a certain clinical regimen?
Material and methods
The present review was conducted according to the QUO-
ROM statement suggestions (15). A number of key data-
bases (MEDLINE, PubMed, Embase, Cochrane library,
Web of Science) were searched in January 2007 using the
MESH terms of ÔorthodonticsÕ, Ôtooth movementÕ, and Ôcre-
vicular fluidÕ, and using the free text terms of ÔGCF, gingival
crevicular fluid, cytokines, inflammatory factors, regulatory
proteins, tooth displacement, canine distalization, and root
resorptionÕ. An additional search was performed using ref-
erence tracking. The search results from each database were
combined. Duplicate results were eliminated (Table 1). The
inclusion criteria were: human studies on GCF, cytokines
(including prostaglandins, tumor necrosis factors, interleu-
kins, growth factors, colony-stimulating factors, and inter-
ferons) or their receptors as study mediators, orthodontic
mechanics, GCF sampling and handling methods, cytokine
measurements (if clearly described), controls (either internal
or external controls included), number of study subjects ‡ 5
or number of study teeth ‡ 10, proper oral hygiene control,
no use of antibiotic/anti-inflammatory drugs before tooth
movement and GCF sampling. The exclusion criteria were:
animal studies, in vitro studies, narrative reviews, expert
reviews (non-systematic reviews), and studies on human
GCF enzymes or any metabolic products other than men-
tioned in the inclusion criteria.
Two reviewers (YR and AV) evaluated the potentially
relevant studies identified and screened for retrieval inde-
pendently. Their evaluation results were compared. The
different results were discussed and re-evaluated until con-
sensus was reached. From each study that remained after
application of the inclusion criteria, the data were extracted
into three categories.
1. Orthodontic mechanics: number of study subjects (not
number of teeth or sites studied), age of study subjects
(range whenever possible, otherwise mean age ± stan-
dard deviation (SD)], control systems, and tooth and
treatment mechanics (appliances, force magnitudes, force
re-activations) studied.
Table 1
Search results from different databases
Search restrictions Results
PubMed Publication type: article 50
MEDLINE All publication types 52
Embase Publication type: article 49
Cochrane All Cochrane libraries 0 in force magnitudes was applied, ranging from 18 to
Web of Science Publication type: article 53
Reference tracking Potentially relevant studies 0
not detected by the
database search
Total number of potentially relevant studies 85
2. GCF sampling and handling methods: sampling methods
(paper strips, volume/weight measuring, repeated sam-
pling), storage system (buffer, temperature), and cytokine
measurements (assays, total amount/concentration,
measurement units).
3. Cytokine measurements: duration of the study, number
of samples, time-points and sites (pressure/tension), tar-
get cytokines, up-regulation or down-regulation, and
peak levels.
As cytokine expression in the GCF during orthodontic
treatment is an emerging issue in orthodontic research, we
expected to find (at most) controlled studies. Therefore, we
chose to include, next to randomized clinical trials (RCT),
controlled studies. The data-reporting methods of the in-
cluded studies were evaluated and the (im)possibility of
performing a meta-analysis was assessed. Quantitative data
synthesis was performed only on cytokines that had been
reported in at least five different studies.
Results
The literature search identified 85 potentially relevant
studies. An overview of the search results is listed in
Table 1. From these studies, examination of the titles,
abstracts, and articles in full text revealed that 23 were
relevant for the present review. A summarizing flow
chart showing the papers that were excluded is given in
Fig. 1. All studies included (references 16–38) were
identified as controlled studies (no RCTs were avail-
able). Two types of controls were used in the various
studies (Table 2): internal controls (the baseline level of
the assessed cytokines of the study teeth in that study);
or external controls (the level of the assessed cytokines
in the contralateral or antagonistic teeth). All studies
had in common that the sampling procedures were not
blinded, whereas data analysis was. These studies are
listed in Table 2 (orthodontic mechanics), Table 3
(GCF sampling methods), and Table 4 (cytokine mea-
surements).
Orthodontic mechanics
Most studies included a mixture of adolescents and
young adults, whereas two studies reported on the GCF
cytokine levels in different age groups (Table 2). In only
two studies were more than 20 subjects included. Internal
and external controls were applied at almost equal fre-
quencies. Sixteen out of 23 studies used the upper canine
as the study tooth, and the force system was distalization
with either sectional wires or continuous arch wires. In
the other seven studies, either upper incisors with a labial
tipping movement, or movement of upper molars with
separation elastics or a rapid maxillary expansion
(RME) appliance, were used. Moreover, a great diversity
250 cN. Only two studies investigated the effect of force
re-activations on the expression of tumor necrosis factor-
a (TNF-a), prostaglandin E2 (PGE2), and interleukin
(IL)-1b (16, 30; the RME procedure was not considered
as force re-activation, 34).

Potentially relevant studies identified
and screened for retrieval (n = 85)
In vitro study (n = 4)
Animal study (n = 8)
Review (n = 3)
Not relevant (n = 10)
Human study not on GCF (n = 3)
Human GCF study not on
cytokines (n = 24)
Human GCF study not on
orthodontic tooth movement but
on periodontitis (n = 8)
Studies retrieved for more
detailed evaluation (n = 25)
Orthodontic mechanics, GCF
sampling or cytokine
measurements NOT clearly
described (n = 2)
Study subjects <5, or study
teeth <10 (n = 0)
Study with no controls (n = 0)
Study with no oral hygiene/use
of drugs control (n = 0)
Studies included in the
systematic review (n = 23)
Study with no indication on
peak level, or <3 observation
points (n = 4)
Fig. 1. Flow chart of the literature search. The number of pa-
pers that were excluded for inclusion in the review is listed in
the right column. GCF, gingival crevicular fluid; IL-1b, inter-
leukin-1b, PGE2, prostaglandin E2.
GCF sampling and handling methods
Seventeen out of 23 studies used Periopaper for sampling
(Table 3). Thirteen studies measured GCF volumes using
Periotron; two studies weighed GCF; and eight studies
did not examine the amount (volume or weight) of GCF
collected. Most studies had a sampling time of 30 s
(n = 13). Sixteen studies sampled GCF repeatedly with
either no time interval (n = 3), or with intervals of 1 min
(n = 11), 5 min (n = 1) or 10 min (n = 1). In three
studies, protease inhibitors were added to the storage
buffer. The storage temperature was )20–30C (n = 9)
or )70–80C (n = 3). Cytokine assays were enzyme-
linked immunosorbent assays or radioimmunoassays,
with the exception of one study that captured TNF-a
with coated beads. The cytokine levels (see Table 3 for
the cytokines assessed in the various studies) were pre-
sented differently and expressed in terms of weight,
concentration of total GCF protein or concentration of
total GCF volume.
GCF cytokines and tooth movement 91
Cytokine measurements
Active study durations varied from 5 min to 84 d (Ta-
ble 4). The number of sampling time-points varied from
study to study, and typically 1 and 24 h were included.
When mesial–distal movement forces were applied, most
studies sampled GCF only at pressure sides; otherwise no
discrimination was made between pressure or tension
sides. It was thought to be necessary to make such a
discrimination because different biological processes are
involved on pressure and tension sides (bone resorption
and bone formation, respectively). Three studies com-
pared cytokine levels at pressure and tension sides, of
which only one reported a significant difference (higher
cytokine levels at the tension side). Cytokine levels were
up-regulated in most studies and many cytokines showed
peaks at 24 h, irrespective of treatment mechanics.
However, the down-regulation of cytokines [e.g. IL-8,
insulin-like growth factor (IGF), and osteoprotegerin
(OPG)], was also reported.
Quantitative data synthesis
Significant heterogeneity in study set-up and data
reporting was found (Tables 2–4). Based on these results,
it was concluded that a meta-analysis was not justified.
Not all known cytokines were analyzed in the various
studies. Among the 23 studies, the most commonly
studied cytokines were IL-1b (n = 12), PGE2 (n = 6),
TNF-a (n = 5), and IL-6 (n = 4) (Fig. 2). An evalua-
tion of the data showed that only those on IL-1b and
PGE2 had more than five studies available providing the
time-related scales of cytokine production. Four studies
(17, 28, 32, 33) had to be excluded from quantitative data
synthesis because of insufficient relevant data or fewer
than three observation time-points (Fig. 1).
Regulation patterns of IL-1b and PGE2
Based on the scale (baseline, significant increase, peak)
of cytokine up-regulation, the time-related regulation
patterns of various cytokines could be schematically
assessed. Eight studies on IL-1b and five studies on PGE2
(among which there were four studies on both) were in-
cluded in these assessments. The four studies on canine
distalization (24, 25, 30, 35), applying various force mag-
nitudes, all showed similar patterns of IL-1b up-regulation
with time. All four studies clearly indicated peaks in the
early phase of tooth movement, either at 24 h (24, 30, 35)
or at 72 h (25). This peak was followed by a decrease to-
wards insignificant/baseline levels at 168 h. By compari-
son, the studies applying other treatment mechanics
showed different regulation patterns of IL-1b: Tzannetou
et al. (34) showed continuous significant elevations from
24 h during maxillary expansion, and Dudic et al. (18)
and Giannopoulou et al. (23), using separation elastics,
showed IL-1b peak levels at 24 h, which remained at a high
level thereafter. By contrast, all studies on PGE2 (Fig. 3B)
pointed to a peak at 24 h, despite the various treatment
mechanics applied; one study was on canine distalization
(30); two were on molar separation (18, 23); and two were
92 Ren & Vissink

Table 2
Gingival crevicular fluid (GCF) cytokines and orthodontic tooth movement: orthodontic mechanics

Reference n Age (yr) Control Tooth Force system Force (cN) Re-activations

16 10 15 ± 1 Baseline UC Hybrid retractor ? No

Distraction ? (heavy) Yes, 2/d
17 10 15 ± 4 Antagonistic UC Sectional, distalizing 60,120,240 No
18 18 9–14 Antagonistic UM Separation elastics ? No
19 18 16–19 Baseline UC 0.014 NiTi wire, aligning ? No
Coil spring, distalizing 150 No
20 15 15 ± 3 Con. and antago. UC Elastic chain, distalizing 250 No
15 31 ± 4 Con. and antago. UC Elastic chain, distalizing 250 No
21 10 15 ± 2 Con. and antago. UC Elastic chain, distalizing 250 No
22 15 17 ± 2 Baseline UC 0.014 NiTi wire, aligning ? No
Coil spring, distalizing 150 No
23 18 9–14 Antagonistic UM Separation elastics ? No
24 9 22 ± 2 Contralateral UC Elastic chain, distalizing 250 No
25 15 11–15 Contralateral UC Elastic chain, distalizing 100 No
26 6 10–23 Baseline UCI 0.012 NiTi wire, aligning 70 No
27 10 23 ± 3 Con. and antago. UC Elastic chain, distalizing 250 No
28 10 10–30 Antagonistic UC Sectional, distalizing 18, 60 No
29 10* 15–17 Baseline LC Sectional, distalizing 90 No
UC Sectional, distalizing 115 No
30 10 18–22 Antagonistic UC Sectional, distalizing 100 No
UC Sectional, distalizing ? Yes, 1/wk
31 50 10–26 Contralateral ULI Labial tipping, aligning 70 No
32 43 10–14 Contralateral ULI 0.012 NiTi wire, aligning 70 No
41 21–27 Contralateral ULI 0.012 NiTi wire, aligning 70 No
33 7 12–16 Antagonistic UC Sectional, distalizing 18, 60 No
34 9 10–18 Baseline UM Hyrax RME ? Yes, 2/d
35 12 14 ± ? Con. and antago. UC Elastic chain, distalizing 250 No
36 12 14 ± ? Con. and antago. UC Elastic chain, distalizing 250 No
37 20 13–36 Baseline UC Elastic ligature, distalizing 150 No
UC T-spring, distalizing 150 No
38 10 24–27 Contralateral ULI 0.014 NiTi wire, aligning 100 No

*10 canines, exact number of subjects unknown.

Age (in yr) expressed as range whenever possible, otherwise the mean age ± standard deviation is given.
Control: ; antagonistic, antagonistic teeth; baseline, the baseline level of the study teeth as a control; con. and antago., contralateral
and antagonistic teeth; contralateral, contralateral teeth. Tooth: LC, lower canine; UC, upper canine; UCI, upper central incisor;
UM, upper molar; ULI, upper lateral incisor.
Force system: sectional, canine were moved with sectional wires, which differs from the full arch wire system; ?, not mentioned in the
article; re-activations, re-activation of the orthodontic force system; wk, weeks.

on labial tipping of lateral incisors (31, 38). Moreover,
none of these five studies on PGE2 showed a significant
increase of this cytokine at 1 h, which is different from IL-
1b that often showed an increase at the 1-h time-point.
These results indicated different regulation patterns of the
production of the two cytokines, with IL-1b being more
responsive to the treatment mechanics, although not to
force magnitudes per se, and PGE2 being less dependent on
orthodontic mechanics.
Discussion
This review systematically evaluated studies on cytokines
that are expressed in GCF during orthodontic tooth
movement. In the literature various cytokines were
studied, but unfortunately with a great heterogeneity in
study design and data reporting. Common drawbacks
were identified in clinical and laboratory set-ups when
deciding which papers should be included. Regarding the
time dependency of levels of the assessed cytokines in
GCF, the most consistent results were the peak levels of
the various cytokines measured 24 h after applying an
orthodontic force, irrespective of treatment mechanics.
Associations have been reported between the intensity
of pain experienced and GCF cytokine levels. Gian-
nopoulou et al. (23) reported that the intensity of pain at
1 h was associated with PGE2 levels, whereas pain
intensity at 24 h was associated with IL-1b levels. Yao
et al. (31) found a correlation between pain intensity and
PGE2 levels at 12, 24, and 72 h of tooth movement.
Recently, Yamaguchi et al. (24) showed a significant
correlation between IL-1b and substance P, a peptide
involved in pain and neurotransmission, during ortho-
dontic treatment in adults. This indirectly supported an
association between pain and IL-1b levels.
With respect to the velocity of tooth movement, Iwa-
saki et al. (17, 28, 33) found a positive correlation with
the IL-1b : IL-1 receptor antagonist (IL-1ra) ratio. These
authors suggested that the speed of tooth movement is
related to stress and to IL-1 gene cluster polymorphisms.
Interleukin-6 and granulocyte–macrophage colony-
 GCF cytokines and tooth movement 93

Table 3
Gingival crevicular fluid (GCF) cytokines and orthodontic tooth movement: GCF sampling and handling methods

Handling
Sampling Repeated storage Measuring total Expressed
Reference Year duration (s) sampling temp. (C) amount/concentration in

16 2007 30 4/1 min )80 Concentration pg ll)1

17 2006 30 2/1 min )70 Protein conc. ng ml)1
18 2006 20 No )70 Total amount pg
19 2006 30 No )20 Concentration pg ml)1
20 2006 60 2/0 s )30 Concentration pg ll)1
21 2006 60 2/0 s )30 Concentration pg ll)1
22 2006 30 No )20 Total amount pg
23 2006 20 No )70 Total amount pg
24 2006 60 2/0 s )30 Protein conc. pg lg)1
25 2006 30 2/10 min )70 Total amount pg
26 2005 30 2/1 min )20 Concentration pg ll)1
27 2005 60 2/1 min )30 Protein conc. ng ml)1
28 2005 30 2/1 min )70 Protein conc. ng ml)1
29 2005 30 2/1 min )70 Total amount pg
Concentration pg ml)1
30 2004 30 4/1 min )70 Protein conc. pg lg)1
31 2003 30 No )70 Concentration pg ll)1
32 2002 30 2/5 min )80 Total amount pg
Concentration pg ll)1
33 2001 30 2/1 min )70 Protein conc. IL-1b in ng g)1
IL-ra in lg g)1
34 1999 30 No )70 Total amount pg
35 1996 30 2/1 min )30 Protein conc. pg lg)1
36 1996 30 2/1 min )30 Protein conc. pg lg)1
37 1995 ? No – Total amount ng
38 1994 30 2/1 min )80 Total amount pg

Repeated sampling, times of sampling/time interval between consecutive sampling; temp., temperature.
Total amount/concentration: concentration, cytokine levels expressed as the concentration in total amount
(weight or volume) of the GCF; protein conc., cytokine levels expressed as the concentration in total GCF
proteins; total amount, cytokine levels expressed as the total amount (weight) in GCF samples; ?, not
mentioned in the article; –, not applicable.

stimulating factor (GM-CSF) showed less responsiveness
in adults than in juveniles (20, 32). Because receptor
activator of nuclear factor-jB ligand (RANKL) is an
important regulatory molecule of osteoclastogenesis, and
OPG inhibits this process (39), the decreased RANK-
L : OPG ratio (20) in GCF may explain the decrease in
cytokine production during tooth movement that occurs
with age. Low IL-1b levels in young adults were related
to less-pronounced clinical signs of gingivitis (40). As the
studies in this review all included a proper oral hygiene
control and no use of antibiotic/anti-inflammatory drugs
preceding the experiment, the contributory influence of
gingivitis on the expression of cytokines in subjects of
different ages could be eliminated.
The data on IL-1b suggested that its production was
responsive to different treatment mechanics (Fig. 3A).
Lee et al. (30) compared the production of PGE2 and IL-
1b to continuous or interrupted force and found that
these regulatory proteins showed different responses:
PGE2 levels showed less consistent changes with different
force regimes than IL-1b and the response of PGE2 to
appliance re-activation was not evident. These results
implied that treatment mechanics might be a decisive
factor in the regulation of cytokine productions and that
different cytokines may respond differently to similar
treatment mechanics.
Based on the different expression patterns of IL-1b
and PGE2, we speculate that during orthodontic tooth
movement, force activation might initially trigger the
production of IL-1b. Then, increased IL-1b levels, to-
gether with mechanical stress, synergistically stimulate
PGE2 production, reflected by its rapid elevation and
peak at 24 h. Subsequently, prolonged up-regulation of
PGE2 triggers a feedback mechanism resulting in a
decrease of IL-1b levels, and this in turn results in an
even more rapid decrease of PGE2, irrespective of sus-
tained mechanical stress. In addition, IL-1b was also
shown to be responsive to the intensity of mechanical
interventions. Interleukin-1b levels may reach a signifi-
cant elevation between 1 h (18, 23, 38) and 48 h (25).
The peak of IL-1b has been observed at 24 h (24, 30,
35), 72 h (37) or not at all (34). Without force re-acti-
vation, IL-1b has been shown to decrease significantly
at 168 h to baseline level, but with force re-activation it
reached an even higher peak (30). Moreover, a heavy
decaying force (24, 25, 35), tended to result in a sig-
nificant decrease of the IL-1b level at 168 h, which may
suggest a need for timely re-activation to maintain
sufficient IL-1b production. Light continuous force (28,
30, 33) tended to maintain relatively high IL-1b levels
for a longer period and thus may reduce the frequency
of re-activations. These results have important clinical
94 Ren & Vissink

Table 4
Gingival crevicular fluid (GCF) cytokines and orthodontic tooth movement: cytokine measurements

Reference Year Duration n Sampling points p/t Cytokine U/D Peak Main results

16 2007 7d 4 0, 1, 24, 168 h p TNF-a – – Hybrid: no change with time

Up 24 h Distraction: higher than hybrid
17 2006 84 d 10 0, 1, 3, 7, 14, 28, 42, 56, p IL-1b – – Tooth movement rate relate
70, 84 d IL-1ra – – To IL-1 gene polymorphisms
18 2006 14 d 6 )7 d, 0, 1 min, 1 h, 1 d, p, t IL-1b Up 24 h Higher levels at tension sites
7d PGE2 Up 24 h Higher levels at tension sites
19 2006 6 mns 6 0, 7 d, 21 d, 6 mns, +7 d, ? IL-1b – – Tend to increase
+21 d TNF-a – – Tend to increase
20 2006 7d 4 0, 1, 24, 168 h p RANKL Up 24 h Lower ratio in adults
OPG Down 24 h Lower ratio in adults
21 2006 7d 4 0, 1, 24, 168 h p RANKL Up 24 h No change at 1 h or 168 h
OPG Down 24 h No change at 1 h or 168 h
22 2006 6 mns 6 0, 7 d, 21 d, 6 mns, +7 d, p, t IL-2 – – Tend to increase at 7 d, back at 21 d
+21 d IL-6 – – Tend to increase at 7 d, back at 21 d
IL-8 Down 7d Decrease at 7 d, back to baseline at 21 d
23 2006 7d 4 0, 1, 24, 168 h p IL-1b Up 24 h Associated with pain at 24 h
PGE2 Up 24 h Weak association with pain at 1 h
24 2006 7d 8 0, 1, 4, 8, 24, 72, 120, 168 h p IL-1b Up 24 h Correlated with P-substance
25 2006 7d 6 0, 1, 24, 48, 72, 168 h p IL-1b Up 72 h Back to baseline at 168 h
TNF-a Up 72 h Back to baseline at 168 h
26 2005 10 d 3 0, 4 h, 10 d ? IGF-I Up 4h Decreased 10 d after
IGFBP-3 Down – Time-dependent decrease
27 2005 7d 4 0, 1, 24, 168 h p t-PA Up 24 h Increased only at 24 h
PAI Up 24 h Increased only at 24 h
28 2005 84 d 11 )28, )14, 0, 1, 3, 14, 28, p IL-1b ? ? The ratio of the two correlate with
42, 56, 70, 84 d IL-1ra ? ? The rate of tooth movement
29 2005 30 d 6 0, 1, 24 h, 6 d, 10 d, 30 d p, t IL-8 – – Tend to increase in early phase
30 2004 3 wk 10 0, 1, 24, 168 h, repeat twice IL-1b Up 24 h Continuous: 168 h back to baseline
Interrupted: higher level at reactivation
PGE2 Up 24 h Continuous: 168 h back to baseline
Interrupted: remain high for 1 wk
31 2003 3d 5 0, 12, 24, 48, 72 h – IL-6 Up – Stay at high level
GM-CSF Up – Stay at high level
PGE2 Up 24 h Pain intensity correlated with PGE2
32 2002 24 h 2 0, 24 h – PGE2 Up – Upregulated in both age groups
IL-6 Up/– – Increased only in youngsters
GM-CSF Up/– – Increased only in adults
33 2001 84 d 11 )28, )14, 0, 1, 3, 14, 28, p, t IL-1b Up < 3 d 28 d periodic changes, no site difference
42, 56, 70, 84 d IL-1ra ? < 3 d No periodicity, no site difference
34 1999 80 d 11 0, 24 h, 7 d, 14 d, 21 d, – IL-1b Up ? Increased during active RME; remain
before and after RME high in retention phase
35 1996 7d 4 0, 1, 24, 168 h p IL-1b Up 24 h No change at 1 h or 168 h
IL-6 Up 24 h No change at 1 h or 168 h
EGF Up 24 h No change at 1 h or 168 h
TNF-a Up 24 h No change at 1 h or 168 h
36 1996 7d 4 0, 1, 24, 168 h p TGF-b1 Up 24 h No change at 1 h or 168 h
37 1995 5 min 2 0, 5 min – TNF-a Up – More than twofold increase
38 1994 7d 5 0, 1, 24, 48, 168 h – IL-1b Up 24 h Increased at 1 h, back to baseline
at 168 h
PGE2 Up 24 h Stay high at 48 h, back to baseline
at 168 h

Dur., durations and sampling points; mns, months; RME, rapid maxillary expansion; –, not applicable.
Cytokine: EGF, epidermal growth factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IGF-1, insulin-like growth
factor 1; IGFBP-3, insulin-like growth factor binding protein; IL-2, interleukin 2; IL-6, interleukin 6; IL-8, interleukin 8; IL-1b,
interleukin 1b; IL-1ra, interleukin 1 receptor antagonist; OPG, osteoprotegerin; PAI, plasminogen activator inhibitor; PGE2,
prostaglandin; RANKL, receptor activator of nuclear factor-jB ligand; TGF-b1, transforming growth factor b1; TNF-a, tumor
necrosis factor a; t-PA, tissue-type plasminogen activator; U/D, up-regulation or down-regulation.

implications for orthodontic treatment and provide
evidence at the cellular level for the preference of light
continuous force in orthodontic practice (41), because a
light continuous force induces the relatively longer-
lasting levels of cytokines that are needed for continu-
ous periodontal remodeling. Heavy decaying force not
only may increase the risks for root resorption and
hyalinization of the periodontal ligament (PDL) (42,
43), but also tends to induce sharp up-and-downs in the
cytokine levels, which may result in undesirable tissue

Fig. 2. Frequency of the studied cytokines. Interleukin-1b (IL-
1b) was the cytokine most frequently studied (n = 12), followed
by prostaglandin E2 (PGE2) (n = 6), tumor necrosis factor-a
(TNF-a) (n = 5), and interleukin-6 (IL-6) (n = 4). EGF, epi-
dermal growth factor; GM-CSF, granulocyte–macrophage col-
ony-stimulating factor; IGF, insulin-like growth factor; IL,
interleukin; OPG, osteoprotegerin; RANKL, activator of nu-
clear factor-jB ligand; TGF-b, transforming growth factor-b.
reactions and a need for more re-activation of the
appliance system.
Aspects that remain to be clarified
Many studies either did not discriminate between tension
and pressure sides or merely took samples at pressure
sides. Those studies that sampled at both sides did not
observe a significant or consistent difference (17, 29).
Only one study (18) reported higher cytokine levels at the
tension side at 1 min and 7 d after the placement of
separation elastics, but not at 1 h and 24 h. Therefore,
there might be no clear-cut difference in the assessment
of cytokines in GCF samples collected at the tension or
pressure areas. The side-independency of cytokine levels
in GCF is probably a result of continuous circulation of
the GCF in the periodontal ligament. This is supported
by finite element analyses showing that the compression
and tension zones were observed only when the PDL was
modeled as a linear material (44, 45). A recent study
applying real-time polymerase chain reaction indeed
showed different cytokine expressions at different areas
of the PDL (46). Therefore, the debate seems to involve
A B
Fig. 3. Schematic illustration of cytokine up-regulation as a function of time (in h). (A) Gingival crevicular fluid interleukin-1b
(GCF-IL-1b); (B) gingival crevicular fluid prostaglandin E2 (GCF-PGE2. The y-axis indicates different levels of up-regulation:
baseline level, significant elevation (dotted line), and the peak. The numbers in the figure refer to the reference numbers.
GCF cytokines and tooth movement 95
more factors than the present investgation addresses. So
far, we can only conclude that cytokine levels in the GCF
may not be specific indicators of periodontal remodeling
at a tension area or a pressure area.
Controversial results were reported regarding the
preference of total amount or concentration of GCF
cytokine levels (32, 47). Several studies reported similar
results using both variables (29, 38). Other studies
reporting on protein concentrations showed that the
GCF total protein was not a constant variable with time
(21, 24, 35). Moreover, the same authors were not always
consistent in their reports (19–22, 24). Although the local
cytokine concentrations seem to be more important to
the intensity of cellular reactions than the total amount
of cytokines present, the inherent variability of the GCF
volumes may risk the reliability of concentration values
(36, 48). It would be preferable if all studies used the
same outcome parameters to facilitate comparison of the
results.
No significant difference was found between the cyto-
kine levels at contralateral and antagonistic teeth (35,
36), although contralateral sites tended to show slightly
higher concentrations (Table 1). The studies included
reported neither changes of cytokine levels with time at
control teeth, nor a difference between control and
experimental teeth. Therefore, it seems practical to use
the baseline levels at experimental teeth as ÔcontrolsÕ.
It has been suggested that the cellular response is part
of a generalized increase in metabolic activity rather than
a specific response to mechanical strain (49). Moreover, a
major difficulty in understanding cytokine biology is the
number and complexity of these factors, and their
overlapping activities and multiple biological effects (50,
51). Thus, only the profile of a group of regulatory
proteins may reflect a cascade of cellular activities during
a treatment process (52). Up-regulation of certain cyto-
kines and their specificity to orthodontic stimuli both
have to be interpreted with caution.
Common drawbacks in study set-ups
Most studies concerned reported short-term changes
without re-activation of the appliance system. Thus, it is
too early to draw a general conclusion on the role of
96 Ren & Vissink
GCF cytokines in orthodontic treatment, which, on
average, takes a much longer time. Another difficulty is
the variation in age of the study subjects among the
various reports. As a result, the effects of age on cytokine
production and tooth movement cannot reliably be
assessed in humans at this stage based on the results
currently available in the literature (32, 53). In the vari-
ous studies, considerable fluctuations in cytokine levels
were reported, even in controls, which, apart from the
intrinsic individual differences, could be a result of
treatment and/or laboratory procedures. The large fluc-
tuations in cytokine levels, together with the relatively
small number of study subjects included in the various
studies, weakened the power of analysis.
A further problem concerned repeated sampling of a
particular side. Repeated sampling was often necessary
to collect a sufficient amount of GCF. The protein con-
centration of the initial GCF sample was comparable to
that of interstitial fluid, while repeated sampling resulted
in protein concentrations comparable to serum (54).
Added to this was the problem that often dissimilar
measurement scales and styles were used, which gener-
ates confusion and makes it difficult to compare results
from different studies.
Concluding remark
In conclusion, the results from this review showed
important clinical implications for orthodontic treat-
ment. The results probably provide evidence at the cel-
lular level for the current preferential use of a light
continuous force in daily orthodontics. In addition, GCF
studies offered the advantage of being non-invasive and
potentially diagnostic and prognostic. However, signifi-
cant heterogeneity exists in the literature. Therefore,
future studies should focus on, amongst others, refine-
ment of the GCF sampling and measuring protocols, the
relationship between cytokine production and force
re-activations, and the expression of cytokine receptors,
in order to provide a better illustration of the high
potential of GCF as a diagnostic tool for using to
monitor clinical outcome in orthodontics.
Acknowledgements – This study was supported by a Research
Grant from the European Orthodontic Society (UK) and by a
VENI Research Grant from Netherlands Organization for
Scientific Research (the Netherlands) to the first author.
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