A preliminary investigation of short-term cytokine expression in gingival crevicular fluid secondary to high-level orthodontic forces and the associated root resorption: case series analytical study
Ahuja et al. Progress in Orthodontics
A preliminary investigation of short- term cytokine expression in gingival crevicular fluid secondary to high-level orthodontic forces and the associated root resorption: case series analytical study
Rajiv Ahuja 1
Moahmmed Almuzian 0 1 2
Alamgir Khan 3
Dana Pascovici 3
Oyku Dalci 1
M. Ali Darendeliler 1
0 Oxford University Hospitals NHS Foundation Trust , Oxford , UK
1 Discipline of Orthodontics, Faculty of Dentistry, University of Sydney , Sydney , Australia
2 Eastman Dental Hospital, UCLH NHS Foundation Trust , London , UK
3 Australian Proteome Analysis Facility, Macquarie University , Sydney , Australia
Background: Orthodontically induced iatrogenic root resorption (OIIRR) is an unavoidable inflammatory process. Several factors claimed to be related to the severity of OIIRR. Orthodontic forces cause micro-trauma to the periodontal ligament and activate a cascade of cellular events associated with local periodontal inflammation. The purpose of this split-mouth study were (1) to investigate the changes in cytokine profile in the gingival crevicular fluid (GCF) secondary to heavy orthodontic forces and (2) to compare the cytokine expression between participants showing high and low root resorption. Methods: Eight participants requiring maxillary first premolar extractions involved in this study. The teeth on the tested side (TS) received 225 g of controlled buccal tipping force for 28 days, while the contralateral teeth act as a control (CS). GCF was collected from both TS and CS teeth at 0 h (prior to application of force) and 3 h, 1 day, 3 days, 7 days and 28 days after the application of force, and analysed with multiplex bead immunoassay to determine the cytokine levels. Results: Statistically significant temporal increase was found in the TS teeth for tumour necrosis factor alpha (TNF-α) at 3 h and 28 days (p = 0.01). Interleukin 7 (IL-7) significantly peaked at the 28th day. Comparing cytokine profile for participants with high and low root resorption (>0.35 and <0.15 mm3, respectively), the levels of GM-CSF was significantly greater in low root resorption cases (p < 0.05). The amounts of root resorption which craters on mesial, distal surfaces and middle third region were significant in the TS teeth (p < 0.05). Conclusions: IL-7 and TNF-α (pro-resorptive cytokine) increased significantly secondary to a high-level of orthodontic force application. Significantly high levels of granulocyte macrophage colony-stimulating factor (anti-resorptive cytokine) were detected in mild root resorption cases secondary to high-level orthodontic force application. A future long-term randomised clinical trial with larger sample taking in consideration gender, age and growth pattern distribution would be recommended.
Orthodontically induced iatrogenic root resorption
(OIIRR) is an unavoidable inflammatory process that
results in a loss of substance from mineralized cementum
during orthodontic tooth movement, and it occurs when
resorption outpaces healing [
]. OIIRR ranges from mild
(0–2 mm) to severe form (more than 4 mm) [
OIIRR is common in almost 90% of orthodontic
], while, luckily, the severe form develops in
approximately 4% of participants and more frequent in
adults than in adolescents [
Several factors claimed to be related to the severity of
OIIRR such as race, gender, age and genetic and local
factors such as habits, traumatised teeth, pre-existing
root resorption, hypo-functional periodontium, duration
of treatment and magnitude and direction of force [
A systematic review by Weltman et al. showed
insufficient conclusive findings in the clinical management of
root resorption, still, there is evidence to support the use
of light forces, especially with incisor intrusion .
Orthodontic forces cause micro-trauma to the
periodontal ligament and activate a cascade of cellular
events associated with local periodontal inflammation
]. The upregulation of cytokines such as interleukin
(IL)-1 beta (IL-1β), IL-8, tumour necrosis factor alpha
(TNF-α) and prostaglandin E2 (PGE2) following
orthodontic force application has been observed in many
Cytokines were sub-classified into two main groups:
pro-resorptive and anti-resorptive. Pro-resorptive
cytokines such as interleukin-1 superfamily (i.e. IL-1β), IL-6,
IL-7, IL-8 and TNF, TNF-α and IL-1β directly induce
osteoclastogenesis and promote osteoclast function [
]. Similarly, IL-6 acts synergistically, with IL-1 and
TNF-α, on osteoclastogenesis to promote osteoclast
]. IL-7 works indirectly through the
induction of TNF-α, an important augmenter of receptor
activator of nuclear factor kappa-B ligand
(RANKL)-mediated osteoclastogenesis [
], while IL-8 enhances
RANKL expression [
]; both increase osteoclast
generation and activate osteoclasts. On the other hand,
an anti-resorptive cytokines such as IL-4 and interferon
gamma (IFN-γ) suppress osteoclastogenesis and T cell of
RANKL-induced osteoclastogenesis respectively [
Granulocyte macrophage colony-stimulating factor
(GM-CSF) is another anti-resorptive cytokine that
inhibits bone resorption along with IL-4, IL-10, IL-13,
IL18 and IFN-γ . Cementoclasts have similar precursor
] and enzymatic and metabolic properties to an
] and share the common regulatory
mechanism of cellular resorption of mineralized tissues such as
bone and dentine [
]. The differentiation and activation of
pre-cementoblasts under both physiologic and pathologic
OIIRR conditions require the expression of RANKL,
osteoclastogenesis inhibitory factor (OPG) and macrophage
colony-stimulating factor (M-CSF) by the dental cells [
Cellular and tissue reactions start in the initial phase
of tooth movement, immediately after force application.
The rate of connective tissue turnover during tooth
movement is dependent on the individual difference in
anatomic structures, mineral density, the level of
cytokines and variations in metabolic capacity [
variability in the levels of pro-resorptive and
antiresorptive cytokines in the micro-environment
(cytokines’ profile) of periodontal ligament (PDL) may also
lead to a difference of OIIRR in response to a similar
amount of orthodontic force. The use of gingival
crevicular fluid allows a non-invasive means of detecting OIIRR
during tooth movements.
The aims of this study were to investigate the change
in the cytokine profile in gingival crevicular fluid (GCF),
during the initial and lag phases of tooth movement
after applying heavy orthodontic forces, and to compare
the cytokine expression between participants showing
the high and low volume of root resorption. The null
hypothesis is that there is no change in the cytokine
expression and no correlation with the degree of OIIRR
secondary to orthodontic force application.
Ethical approval was obtained from South Western Sydney
Local Health Network Ethics Review Committee (protocol
no. X11-0028 and HREC/11/RPAH/37). The study sample
consisted of eight consecutive participants (6 males, 2
females; mean age = 16.4 years; range 13.9–22.9).
All participants required orthodontic treatment with
extraction of the maxillary first premolars and met
clearly defined inclusion criteria including (1) class I
malocclusion, (2) class I skeletal base, (3) average vertical
height, (4) absence of obvious facial asymmetry, (5)
normal growth and development of the dentition and (6)
radiographical signs of complete apexogenesis of the
upper first premolars. All participants with a history of
medical problem(s) that related to abnormal dental
development, history of trauma or bruxism and caries or
dental or orthodontic treatment were excluded. Clinical
periodontal examinations including periodontal probing
pocket depth, bleeding on probing and attachment level
of the upper first premolars was undertaken before and
during treatment to exclude any periodontal disease.
Informed consents from all subjects were obtained at the
A split-mouth study design with the similar
experimental setup described by Srivicharnkul et al. was adopted
]. SPEED orthodontic brackets (Strite Industries
Cambridge, Ontario, Canada), 0.022″ × 0.028″ slot size,
were bonded to the upper first permanent premolars
and the upper first permanent molar. A 0.017 ×
0.025inch TMA (Beta III titanium, 3M Unitek, Monrovia,
California) cantilever spring was used to apply 225 g of
buccally directed force to the first premolars in the test
side (TS) for 28 days. The orthodontic force was
measured to the nearest gram with a strain gauge
(Dentaurum, Ispringen, Germany). To disocclude the
occlusion and prevent occlusal forces, light cured glass
ionomer cement (Transbond; 3M Unitek, Monrovia,
Calif ) was bonded bilaterally onto the occlusal surfaces of
the lower first molars (Fig. 1).
Gingival crevicular fluid collection and determination of cytokine levels
After isolating the teeth, GCF was collected from the
mesiobuccal aspect of both TS and control side (CS)
teeth at the following time periods: 0 h (prior to
application of force), 3 h, 1 day, 3 days, 7 days and 28 days after
the application of force.
The protocol for GCF collections involved gentle
removal of visible plaque at both sites followed by washing
and drying the premolar regions using air syringe to
avoid contaminating the paper strip (Periopaper, Harco,
Tustin, CA, USA) by the plaque. Then, paper strips were
inserted for 1 mm into the gingival crevice and left there
for 30 seconds. Periotron 8000 (Periotron 8000, Oraflow
Inc., New York, USA) was used to measure the volume
of GCF in each paper strip. Microcentrifugee tubes
containing paper strips were thawed, and GCF was eluted
from the paper strips using 50 μL of phosphate-buffered
saline (pH 7.2) containing protease inhibitor cocktails
(Sigma, St Louis, USA) and 0.05% (v/v) Tween 20
(Sigma, St Louis, USA). Following micro-centrifuging,
the paper strips were immediately frozen at −80 °C for
further immunoassay analysis to determine cytokine
]. Multiplex bead immunoassay (product no.
HSCYTMAG-60SK EMD Millipore corporation) was
used to determine the levels of IL-1β, 2, 4, 5, 6, 7, 8, 10,
12 and 13, GM-CSF, INF-γ and TNF-α simultaneously
using Bio-Plex 200 systems (Bio-Rad, CA, USA).
Processing of the extracted teeth
At the end of day 28, the TS and CS teeth were
extracted atraumatically to avoid damage to the root
cementum. The teeth were processed by immersing
extracted them in a solution of sterilised deionised water
(Milli-Q; Millipore, Bedford, Mass). Then, the teeth were
treated for 10 min in an ultrasonic bath to remove
residual PDL and soft tissue fragments [
remaining visible PDL was manually cleaned using a
damp gauze swab before final disinfection using 70%
alcohol for 30 min. Finally, the disinfected teeth were
stored in Milli-Q at room temperature (23 ± 1 °C) with
50% ± 10% relative humidity before scanning.
Micro-CT scanning procedure
The extracted teeth were scanned individually for 60
minutes per tooth using SkyScan 1172 desktop X-ray
micro-tomograph system (SkyScan, Aartselaar, Belgium)
which allows 180° of rotation. Scanning extended from
the root apex to 2 millimetres below the cementoenamel
junction (CEJ) with a rotation step of 0.45° and an
exposure time of 1.904 seconds. This scanning procedure
produced a total of 420 X-ray images per tooth, which
were stored as 16 bit TIFF (Tagged Image File Format)
files before being reconstructed.
Reconstruction was performed using SkyScan’s
volumetric reconstruction software (NRecon, version 1.6.8,
Aartselaar, Belgium) [
]. An ImageJ variant (FIJI) was
used to isolate and export individual lacunae from axial
slices. An ImageJ macro (ACMM), the University of
Sydney (Enigma), was utilised to measure the volume of
each root lacuna uniformly. The lacunae were separated
according to their location in the coronal, sagittal and
axial plane. One operator carried out volumetric
measurements of OIIRR twice at an interval of 2 weeks in
order to measure intra-operator variability.
To normalise the raw cytokine data, the concentration
of each cytokine was considered. An analysis of variance
was undertaken separately for each cytokine to check for
overall changes with time. ANOVA p values were adjusted
for multiple testing using the Benjamini and Hochberg
The non-parametric alternative to ANOVA
(Kruskal-Wallis test) was also run side by side for
reference, using the raw data to evaluate the significance
of the difference between the groups and within each
time point. Cytokine changes between particular time
points were calculated together by using Tukey’s
Honest Significant Differences as implemented in
Tukey’s HSD function of the R statistical package. In
a similar fashion, a one-way ANOVA was run to
ascertain cytokine changes over time only.
Intra-operator reliability showed that cytokines IL-1β,
IL-4, IL-6, IL-7, IL-8, IFN-γ, GM-CSF and TNF-α were
detected reliably by the immunoassay (p > 0.05), while
IL-2, IL-5, IL-10, IL-12 and IL-13 were not reliably
detected as they were missing in more than 50% (Table 1).
Figure 2 illustrates the overall look of the raw cytokine
data with time and treatment factors. Upward
timedependent changes were detected for IL-7 and TNF-α,
both by ANOVA and the non-parametric version
(Kruskal-Wallis test), while treatment-dependent changes
were detected for IL-4. Additionally, the analysis of
variance identified the cytokine that showed overall
temporal differences without identifying precisely which
pairs of points exhibit a significant difference (Table 1).
The results in Table 2 summarise the time points that
show significant differences in overall cytokine
abundance. The increase in cytokine abundance after day 28
(IL-7 increased at day 28 compared to that at hour 3 ;
TNF-α increased at day 28 compared to that at hour 0,
3 and 24) confirms the visual pattern of the increase in
the data. When the same post hoc analysis was repeated
for the TS teeth, the time differences for TNF-α were
found to be significant. This finding is consistent with a
broad trend of noticeable increase at day 28 (Fig. 2).
Cytokine comparison in high and low OIIRR cases
Of the eight participants enrolled in this study, (i) three
participants were identified as having high OIIRR
(>0.35 mm3), (ii) three showed low OIIRR (<0.15 mm )
and (iii) two participants presented with OIIRR between
0.15 and 0.35 mm , who were excluded from the
analysis. This categorisation was based on the mean
difference of total volume of OIIRR between the TS and CS
A t test for cytokines that only detected in more than
50% of the participants (i.e. IL-1β, IL-4, IL-6, IL-7, IL-8,
IFN-γ, GM-CSF and TNF-α) were undertaken. Average
values of those individual cytokines were generated
separately for participants with high and low OIIRR (Fig. 3).
Though the level of anti-resorptive cytokine GM-CSF
level was significantly higher in low OIIRR cases (p =
0.03), other cytokines showed no differences (p > 0.05).
Micro-CT assessments of OIIRR at different root surface and positions
Table 3 shows the OIIRR at different surfaces and
vertical position. It is obvious that total OIIRR in TS
is significantly higher than in CS, in particular, at the
Cytokines with more than 50% missing observations are presented in italics
middle third of the roots and at both mesial and
distal surface of the roots (p < 0.05).
The null hypothesis of the study, stating that there is no
change in the cytokine expression and no correlation
with the degree of OIIRR secondary to orthodontic force
application, was rejected.
In the present study, the concentration of IL-1β in the
TS teeth increased initially, peaked at days 1 and 7 and
then decreased. This result indicates that IL-1β is
expressed in the GCF during orthodontic tooth
movement similar to that in the previous findings [
Though no statistically significant differences were noted
in IL-1β levels between the TS and CS teeth at any time
points, this indicates that the increase in IL-1β level
might be associated with inflammatory response
secondary to orthodontic mechanical stress .
Similarly, the change in the IL-6 level was statistically
insignificant in agreement with Basaran and colleagues
]. Ren et al. [
] reported an opposite finding; they
observed an increase in levels of IL-6 at early hours of
tooth movement which peaked after hour 24. Moreover,
the concentration of IL-8 in TS fluctuated during the
experimental phase, but there was no considerable change
between the TS and CS. This finding is parallel to the
outcomes of Tuncer et al. [
] but contradicting with
those by Basaran et al. [
] and Ren et al. [
]. In fact,
the latter two studies reported a significant decrease in
the IL-8 level at days 7 and 30 of force application
respectively. These dissimilarities, in the detected levels of
IL-6 and IL-8, in comparison to other studies may be due
to the variation in the used appliance systems, force levels,
small sample size and/or some individual variations.
On the other hand, there was a statistically significant
difference (p = 0.01) in the level of IL-7 and gingival
TNF-α between TS and CS that peaked after day 28,
similar to that in another study [
]. The increase in the
level of TNF-α at day 28 indicates a presence of localised
inflammation secondary to force application; this
correlates with the levels of IL-6 and IL-7 that also
peaked at day 28.
The boost in the pro-resorptive cytokines signifies
their crucial roles in stress-induced inflammation
through stimulating precursor scavenger cells that
required for the removal of hyalinized tissue [
the increase in the levels of pro-resorptive cytokines,
IL6, 7 and TNF α, might indicate a continuous periodontal
remodelling during the lag phase of tooth movement
and a cellular prohibiting mechanism.
The time-dependent change of IL-4 concentration
followed that of IL-1β, i.e. peaked during the period
from day 1 to day 3 but then declined. There were also
no significant differences between the TS and CS teeth
concerning IL-4 concentration. Similarly, the change in
the level of IFN-γ among the TS and CS teeth was
insignificant. IFN-γ concentration in the TS teeth fluctuated,
increasing to its peak at 72 h. The increase in the levels
of IL-4 and IFN-γ closely follows that in IL-1β. This
unique style of cytokine expression is the result of
combined active periodontal remodelling during initial stages
of tooth movement and the cellular prohibiting
mechanism that prevents additional differentiation and
activation of osteoclastic cells.
With regard to GM-CSF level secondary to orthodontic
force application, Ren and colleagues identified a
significant elevation of anti-resorptive cytokine GM-CSF in
juveniles after application of orthodontic force [
However, in the present study, the amounts of GM-CSF
for the TS teeth dropped immediately after the start of the
experimental phase, increased at day 7 and then reached
its peak at day 28, but there was no significant difference
between the TS and CS teeth at different time scale.
Cytokines in high and low OIIRR group
Comparing cytokine profile for participants with high
OIIRR (>0.35 mm3) and those with low OIIRR
(<0.15 mm3), GM-CSF was increased in low OIIRR cases
(p = 0.03), while other cytokines showed no significant
differences (p > 0.05). These results confirm the link between
the high levels of anti-resorptive cytokines such as
GMCSF and the reduced root osteoclastic differentiation.
Micro-CT assessments of OIIRR at different root surface and positions
In this study, the amounts of OIIRR lacunae on the lingual
and buccal surface and at apical and cervical third were
statistically insignificant; however, it was significant at the
pure tension site (mesial and distal surfaces and middle
third region) in the TS teeth. These findings are similar to
some of the previous studies [
] but contradicting
with many others [
]. It will be expected that the
amount of OIIRR at the compression sites (bucco-cervical
and palate-apical regions) would be higher as in the other
], however, this was not confirmed in the current
study. The differences may be attributed to two reasons.
The first reason is called masking effect which is due to
the fact that the measurement of the OIIRR in our study
was not surface-region-specific, i.e. the whole surface
(mesial, distal, buccal or palatal) and the whole radicular
regions (cervical, middle or apical third) were assessed. As
each of the root regions and surfaces included an
overlapped compression and tension site, theoretically, the
metabolic changes between the compression and tension
sides masked each other and were not distinguishable.
Secondly, the differences in the adopted appliance
systems, force levels, small sample size and/or some
individual variations may explain these dissimilarities.
Clinical implications, limitations and future considerations
In this study, two of the pro-resorptive cytokines,
namely, IL-7 and TNF-α, increased significantly in the
TS teeth secondary to heavy force application. This
trend might represent the crucial roles of these cytokines
in active OIIRR following heavy orthodontic forces.
Moreover, as the teeth with low OIIRR demonstrated
significantly high level of GM-CSF (anti-resorptive
cytokines) secondary to heavy force application, gingival
GM-CSF measurement used in our project might be
considered in the future as a non-invasive and useful
biomarker to identify participants’ susceptibility to severe
However, the findings of this study should be taken as
an intriguing hypothesis, not as evidence for some
reasons. Firstly, the sensitivity of cytokine detections had
shown a wide range of variability (approximately ranged
from 1 to 96%). Secondly, the lack of method error
analysis in our small sample size study indicates that GCF
sampling and quantifications of biomarkers are
subjected to comparably significant errors [
Furthermore, it should be acknowledged that there were some
gender imbalance and age heterogeneity in our study
which could be considered as one of the main
confounders. While it would be ideal to assess the
correlation between cytokine expression and OIIRR on various
parts of the tooth root, the gingival sulcus is a pool for
CGF, and it would be impossible to identify cytokine
expression on each surface.
Based on the findings of this study, authors
recommend a long-term future randomised clinical trial
with a large sample size taking in consideration
general factors such as gender, age and growth pattern
distribution as well as local factors such as habits,
traumatised teeth, pre-existing root resorption and
IL-7 and TNF-α (pro-resorptive cytokine) increased
significantly secondary to high-level orthodontic force
application. Significantly high levels of GM-CSF
(antiresorptive cytokine) were detected in low OIIRR cases
secondary to high-level orthodontic force application.
As this study is a preliminary, a future study with
larger sample taking in consideration gender, age and
growth pattern distribution would be recommended.
The study received a grant from the Australian Society of Orthodontists
Foundation for Research and Education.
The authors state that the views expressed in this article are their own and
not an official position of the institution or funder.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
RA participated in the design and coordination of the study, carried out the
sample collection, measured the volume of OIIRR and performed the statistical
analysis. AK carried out the cytokine analysis. DP performed the statistical analysis
and participated in the design and coordination of the study. OD conceived the
study and made substantial contributions to the analysis and interpretation of the
data. MA made substantial contributions to interpretation of data and involved in
revising the study critically. MD conceived of the study, made substantial
contributions to the analysis and interpretation of the data and participated in the
coordination of the study. All authors drafted the manuscript and read and
approved the final manuscript.
The authors declare that they have no competing interests.
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