Markers in blood and saliva for prediction of orthodontically induced inflammatory root resorption: a retrospective case controlled-study
Yashin et al. Progress in Orthodontics
Markers in blood and saliva for prediction of orthodontically induced inflammatory root resorption: a retrospective case controlled-study
Dilara Yashin 1
Oyku Dalci 1
Mohammed Almuzian 0 1 3
Jenkin Chiu 1
Rajiv Ahuja 1
Apurv Goel 2
M. Ali. Darendeliler 1
0 John Radcliffe Hospital, Oxford University Hospitals NHS Foundation Trust , Oxford , UK
1 Discipline of Orthodontics, Faculty of Dentistry, University of Sydney , Sydney , Australia
2 Australian Proteome Analysis Facility (APAF) , Sydney , Australia
3 Eastman Dental Hospital, University College London Hospitals NHS Foundation Trust , London , UK
Background: Hormonal and enzymatic factors may render certain individuals more susceptible to orthodontically induced inflammatory root resorption (OIIRR). The objectives of this study are (1) to identify biochemical key markers in blood and saliva that may be correlated to the trend of extensive OIIRR and (2) to utilise these markers to predict a susceptible patient-receiving orthodontic treatment. Methods: Nine patients (mean age 23 + 2.9 years) who had moderate to severe OIIRR that assessed via orthopantomograms and met the inclusion criteria were classified as the root resorption group (RRG). Blood chemistry was evaluated using the collection of fasting blood and unstimulated saliva samples. Multiplex enzyme-linked immunosorbent assay (ELISA) arrays were used to screen blood and saliva samples for human cytokines, chemokines and several key enzymes that may play a role in root resorption following orthodontic force application. Biochemical findings from 16 matching subjects were used as the control (CG) for comparative measurements. Results: Patients with moderate to severe OIIRR showed a significant increase in salivary cytokines including interleukin (IL) 7, IL-10, IL-12p70 and interferon-gamma (IFN-γ) level as well as a significant decrease in IL-4 level. Osteocalcin and procollagen type I N-terminal peptide (P1NP) appeared to be the only blood factors that showed a significant difference, more in the CG than the RRG. Conclusions: Saliva might be a more valuable way of measuring changes in cytokine expression than blood secondary to orthodontic treatment. Although the increased expression of pro-inflammatory and anti-inflammatory cytokines may be determinants in the development of moderate to severe OIIRR, cytokine expression may be affected by several potential inflammations in another part of the body. Future research could investigate the cause/effect relationship of different cytokines, in a larger group of patients and at different time intervals, using digital subtraction radiography techniques and microfluidic biosensors.
Orthodontically induced inflammatory root resorption
(OIIRR) is not an uncommon iatrogenic consequence of
orthodontic treatment [
]. The inflammatory component
of OIIRR comes from force application inducing local
biological changes that are essential for tooth movement
and also the cause of root resorption process [
in most patients the degree of root resorption is minor
and barely noticeable, moderate to severe resorption can
be exhibited in several teeth, especially in the aesthetic
]. In their 1988 paper, Lavender and Malmgren
quantified root resorption with an index in which grade
1 is an irregular root contour; grade 2 is apical root
resorption of less than 2 mm; grade 3 is apical root
resorption greater than 2 mm to one third of the original
root length; and grade 4 is root resorption exceeding one
third of the original root length [
There are limitations in finding clinical markers (factors)
of root resorption during active orthodontic treatment.
Although the anatomical risk factors have been investigated
], they account for only 20–30% of the
expected variation in severity of OIIRR, indicating that
they are not definitive predictors [
]. Some studies
indicated that progress radiographs, taken at 6 to 12 months
into treatment, could detect early OIIRR [
there is difficulty in using conventional radiography in
detecting early levels of root demineralisation [
it takes approximately 30–60% of mineral content loss in
order to visualise changes radiographically [
conventional radiography should be used as an aid for
comparison of treatment outcome as opposed to clinical
markers for monitoring early changes in OIIRR.
Orthodontic movement is a micro-trauma to the
periodontal ligament associated with a cascade of
local periodontal inflammatory cycle [
to orthodontic movement, several local anti-resorptive and
pro-resorptive cytokines have been observed [
Pro-resorptive cytokines such as interleukin (IL)-1 family
(i.e. IL-1β), IL-6, IL-7, IL-8 and TNF-α. IL-1β which directly
induce osteoclastogenesis and promote osteoclast function
]. Similarly, IL-6 acts synergistically with IL-1 and
TNF-α on osteoclastogenesis and promotes 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
] while IL-8 enhances RANKL expression
], both increase osteoclast generation and activates
osteoclasts. On the other hand, the anti-resorptive
cytokine such as IL-4 and interferon-gamma (IFN-γ)
suppresses osteoclastogenesis and T-cell of
RANKLinduced osteoclastogenesis, respectively [
Granulocyte macrophage colony-stimulating factor
(GMCSF) is another anti-resorptive cytokine that inhibits
bone resorption along with IL-4, IL-10, IL-13, IL-18
and IFN-γ .
There are several systemic biomarkers that could be
related to OIIRR. For instance, the relationship between
OIIRR and thyroxine hormone is still controversial;
however it was found that the level of alkaline phosphatase rises
significantly in patients with OIIRR [
]. Calcitonin has
been proven to reduce orthodontic movement and thus
may also affect root resorption [
]. On the other hand,
an animal study showed that administration of osteocalcin
could accelerate movement and subsequently might
induce severe OIIRR [
]. Parathyroid hormone (PTH) acts
synchronisingly with calcitonin to control the level of
calcium in bone. Increased level of PTH is associated with
skeletal calcium deficiency, faster tooth movement and
may be increased OIIRR [
]. There is also some
evidence indicating that T-lymphocytes are contributor
to the inflammatory response secondary to orthodontic
force, OIIRR and bone remodelling . Brezniak et al.
stated that consumption of alcohol during orthodontic
treatment is related to high risk of OIIRR due to vitamin D
hydroxylation in the liver [
]. Similarly, Collins and
Sinclair showed that the number of mononuclear
osteoclasts boosted following administration of vitamin D in
the periodontal ligament of rats; this was associated with
faster tooth movement and may be related to higher
]. The elevated level of IgE in patients with an
allergy has association, but not statistically significant, with
an increased risk of OIIRR [
The primary aim of this pilot study is to identify key
local, in saliva, and systemic, in blood, biological markers
that are associated with OIIRR in patients with moderate
to severe root resorption. Secondly, this paper aims to use
these findings to develop a predictive model for identifying
patients susceptible to extensive OIIRR in order to allow
for primary prevention of this serious complication. The
null hypothesis of this project stated that there are no
correlations between biological markers found in blood
or saliva and the severity of OIIRR.
This is a single centre study approved by the Ethics
Review Committee of the Sydney Local Health District
(Protocol No X11-0028). All participants who had had
their treatment completed between 2010 and 2012 at
the Orthodontic Department of Sydney Dental Hospital
(SDH) had been screened. Thirty-five participants had
been identified of having moderate to severe root
resorption (Grades 3 and 4) of at least three or more teeth at the
completion of their treatment. The diagnostic criteria
by Levander and Malmgren were used for assessing the
severity of OIIRR seen in digital and non-digital
The 35 identified participants were re-evaluated
against specific inclusion and exclusion criteria. The
inclusion criteria involved participants who were (a)
willing to participate in the research; (b) finished their
orthodontic treatment within 2 years and not receiving
any active orthodontic treatment at the time of study
and (c) the duration of their previous active orthodontic
treatment should be less than 24. Participants with (a)
active systemic disease; (b) history of allergy; (c) history
of dental injury including but not limited to endodontic,
periodontal, and physical; (d) morphological variation of
resorbed teeth at the start of treatment; and (e) recent
oral infections within 1 month such as flu, colds, herpes
zoster and other oral ulcerations were excluded. The
justification for this strict criteria was based on the fact that
literature had shown that oral infection and dental
trauma, and orthodontic treatment at the time of sample
collection, could alter cytokine expression and induce
root resorption not only to the individual tooth but
surrounding teeth [
]. Following the application of these
criteria, 25 participants had been excluded. Henceforth,
only nine participants (mean age of 23 + 2.9 years)
remained, namely root resorption group (RRG). Three
subjects of the RRG had moderate OIIRR and the
remaining had severe OIIRR, the RRG consisted of six
females (mean age 22+ 2.2 years) and three males (mean
age 25+ 3.6 years). Data from 16 participants, who met
the previously mentioned inclusion/exclusion criteria and
had not developed significant OIIRR, were used as a
control (CG) (Fig. 1).
Assessment of OIIRR
Although a periapical radiograph is considered the gold
standard for assessment of OIIRR, as this study was
retrospective in nature, OPGs were available and hence used in
this study. The degree of OIIRR was assessed from the OPG
using the equation (Fig. 2) as proposed by Linge and Linge
in 1991 [
OIIRR¼ ðr1‐r2Þ ðC1=C2Þ
Magnification error (C1/C2) developed using the ratio
of the pre- (C1) and post-treatment (C2) radiographical
crown height. The non-adjusted degree of OIIRR (r1-r2)
was calculated as the difference between pre-treatment
(r1) and post-treatment (r2) radiographical root length.
Blood and saliva sample collection and analysis
Fasting for 12 h before blood and saliva collection had
been requested from participants. Blood chemistry
had been assessed through the collection of 33 mL of
fasting blood by a phlebotomist at Douglass Hanly
Moir Pathology (DHM) in St Vincent’s Clinic.
Participants were also asked to brush their teeth in the clinic
without toothpaste and to rinse with de-ionised water
prior to collection of 10 mL of unstimulated saliva by
expectoration into polypropylene tubes with protease
Blood and saliva samples were stored in a −80 °C
freezer until transported to Australian Proteome
Analysis Facility (APAF) at Macquarie University for
analysis. Multiplex enzyme-linked immunosorbent assays
Erythrocyte sedimentation rate (ESR) Interleukin-10 (IL-10)
Procollagen type I N-terminal peptide Interleukin-5 (IL-5)
(ELISA) was used to screen the samples’ saliva for
cytokines, chemokines and several key enzymes that may
play a role in OIIRR. Blood chemistry results were
obtained from DHM pathology and analysed at APAF
for significance. Table 1 shows blood chemistry factors
and blood/saliva cytokines that were analysed by
DHM and APAF individually.
Cytokine expression analysis
Fluorescence intensity (FI) was used as the outcome
measure for the concentration of cytokine expression in
CG and RRG. Boxplots plotting FI versus cytokine
expression for each diagnosis group allowed for a visual
indication of data distribution and trends. A two-way
analysis of variation (ANOVA) was conducted to
investigate the effect of diagnosis on fluorescence through
the use of the following mixed model calculation:
LogðF 1Þ∼Diagnosis CytokineþSampleþGender
This accounts for the random effects of patients, age
and ethnicity, and the fixed effects of diagnosis, sample,
gender and cytokines. Besides, a one-way ANOVA was
conducted to investigate the effect of diagnosis on blood
and saliva cytokine. All statistical analysis was conducted
using R Statistical Software (Foundation for Statistical
Computing, Vienna, Austria) and the statistical
significance was set at the p < 0.05 level.
Blood chemistry analysis
Chi-square tests were conducted to clarify the association
of blood proteins’ levels and diagnosis. Both CG and RRG
groups had been subclassified into three categories:
increased, decreased and no difference group, according
to the level of the significantly changed blood proteins.
Cytokine expression, as expressed by FI values, revealed
significant findings in saliva samples of RRG patients.
There was a statistically significant decrease in cytokine
expression of IL-4 (p = 0.05) and a significant increase in
cytokine expression of IFN-γ (p = 0.01), IL-10 (p = 0.03),
IL-12p70 (p = 0.02), and IL-7 (p = 0.0001) in RRG
compared to CG (Fig. 3a). However, there was no significant
difference between moderate and severe RRG itself
Figure 4 showed that cytokine expression from blood
samples according to RRG versus CG revealed no
significant difference (p > 0.05). Likewise, there was no
significant difference in cytokine expression between
male and female subjects (p > 0.05).
Blood chemistry results showed normal level for all proteins
factors in both CG and RRG, except for osteocalcin
(p = 0.039) and procollagen Type I N-Terminal Peptide
(P1NP) (p = 0.033) concentrations. The proportion of
participant who showed an increased osteocalcin
concentration was almost six times more in CG (62.5%) than RRG
(11.1%). The proportion of participants who presented
with an elevated P1NP concentration was higher in CG
(75%) than those in RRG (22.2%). None of the participants
in both groups had experienced a drop in P1NP and
osteocalcin concentration. Furthermore, blood
chemistry’s trend tended to fit more closely with gender than
with diagnosis; osteocalcin and P1NP levels were
significantly elevated in male’s subgroup of the controlled
sample compared to the counterpart (p < 0.05). On the
other hand, there was no significant gender difference in
blood factors among moderate and severe RRG (p > 0.05).
However, subdividing the main groups by their gender
further reduced sample numbers, and hence findings
should be evaluated with caution.
This pilot study identified that cytokine expression between
different diagnostic groups is more sensitive using saliva
than in blood samples. In comparison to gingival crevicular
fluid (GCF), saliva as a source to identify cytokines is
considered remote from the root inflammatory zone, which
means least overlapping of cytokines expressions [
Thus, sampling saliva, as a chair-side screening test, is both
affordable and easy compared to collecting blood or GCF.
However, this study also showed that taking saliva samples
might provide some indication of systemic reaction to
The concept that pro-inflammatory cytokines are
resorptive factors and anti-inflammatory cytokines are
anti-resorptive factors is an oversimplified view and one
that is changing. In our study, although the significant
increase in IL-7 and a decrease in IL-4 could be linked
specifically to root inflammatory phase, the increase in
anti-inflammatory cytokines IL-10, IL-12p70 and IFN-γ
was also significant. The anti-inflammatory cytokines may
be upregulated to stimulate a constant state of
remodelling in the bone akin to the body’s defence mechanism.
Cytokines mediate bone damage by driving the
differentiation and activation of the bone-resorbing cell, the
], as well as playing an essential role in immune
cell development and immune-regulation. More recent
evidence suggested that the effect of cytokine levels’
alterations is not a singular effect, that is, cytokines that
promote inflammation can also have anti-inflammatory and
immunosuppressive actions [
]. O’Shea et al. suggested
that the combination of cytokines exerts variable effects at
different times during autoimmune disease processes.
IFN-γ, for example, has been shown to have multiple roles
in autoimmunity; one as a mediator in autoimmune
disease and another as having a protective role [
based on the findings of our study, it was difficult to
precisely define the link between cytokines in OIIRR based
on the small sample size.
A lack of significant difference in cytokine expression
between genders is similar to the findings of Linge and
]. They noted that there was no connection between
sex and severity of root resorption; therefore, if elevated
levels of certain cytokines are linked to root resorption,
gender may not play a role in affecting cytokine expression.
Osteocalcin and P1NP seemed to be the only blood
factors that showed a significant difference between CG
and RRG. However, gender appeared to have a more
significant effect on osteocalcin and P1NP levels than
diagnosis. It is important to isolate the effects of age and
gender on blood osteocalcin and P1NP levels since bone
turnover can be upregulated as a natural process of
human growth. Previous research has shown that the
distribution of osteocalcin at different sites of remodelling
may be age- and gender-related changes [
study has revealed pubertal increases in P1NP level in
both sexes [
]. Therefore, blood osteocalcin and P1NP
may not be useful clinical markers to predict the
susceptibility of patients to OIIRR as often these patients are
receiving treatment at stages of natural growth and
This study applied computations to estimate
radiographical magnification differences (RMD) between pre- and
post-treatment OPGs taken by different operators and
]. Nevertheless, it was limited in its
capacity to account for tilting of teeth especially maxillary
and mandibular anterior teeth which are the most
commonly affected teeth with extensive OIIRR [
factor that should be considered in this study is the
radiographical judgemental errors (RGE) in equating roots’
length from the OPGs. RGE is the result of film-related
factors and/or radiographical conspicuity. Film-related
factors include variation in radiographical brightness and
contrast between pre- and post-treatment radiographs
. While conspicuity is a radiographic term which
describes the background ‘noise’ caused by adjacent
anatomical structures causing difficulty in assessing the
examination area [
], conspicuity arose when root
apices were outside the narrow focal trough ,
incompletely developed roots or when the cervical
vertebrae and other anatomical structures obscured the root
apices. To overcome RGE and IGE, digital subtraction
radiography (DSR) is recommended in prospective
researches. DSR is a technique that requires the subtraction
of one radiographic image from another, provided these
two images have identical and reproducible projections of
the same anatomic region [
8, 9, 38
]. For reproducibility
purpose of DSR, bite registration with polyvinylsiloxane
silicon impression material, invested into the bite block of
a beam-guiding device, is used [
]. After a series of
radiographs are obtained at different time intervals, stable
horizontal and vertical landmarks can be selected and
eliminated in order to ensure measurement accuracy of
the periapical regions between radiographs .
It is worth mentioning that many eligible participants
deterred from consenting to participate in this pilot
study and a repeated measurement of the saliva and
blood sample were not undertaken to decrease the risk
of bias. The main reason was that this study required
large volumes of saliva and blood sample to be sent for
laboratory testing. Recently, the use of microfluidic
biosensors (MFB) as part of point-of-care diagnostic
portable devices (PoC) was recommended in the monitoring
of cytokine expression in a clinical setting [
detection times of MFB are relatively faster than
conventional laboratory testing, taking only from a few seconds
to tens of seconds to process the sample. Another
advantage of MFB is the small sample volume that could
be used, ranging from micro-litres to nano-litres [
Prospect adoption of PoC devices in orthodontic
practices could be used for accurate detection of cytokine
expression and level changes. Thus, it would be possible to
‘red-flag’ patients that are more at risk of extensive OIIRR
earlier. Subsequently, patients at risk can receive adequate
modification in their orthodontic treatment, perhaps even
pausing treatment which would allow the regenerative
properties of cementum to repair the root surface, thereby
minimising irreversible loss of root structure [
Finally, as the relationship between cytokines and OIIRR
in our study was based on the small sample size, future
prospective studies with larger samples may yield interesting
results, in particular the pre- and post-orthodontic level of
This study found that saliva might be more efficient in
measuring changes in cytokine expression than blood.
Osteocalcin and P1NP appeared to be the only blood
factors that showed a significant difference between groups.
The weak association between treatment variables and
patient characteristics with the degree of root resorption
suggests that increased expression of pro-inflammatory,
as well as anti-inflammatory cytokines, may be
determinants in the development of moderate to severe OIIRR.
However, the expression of cytokines may also be affected
by any other potential inflammation in the body.
Further investigations could utilise digital subtraction
radiography techniques and microfluidic biosensors, in a
larger group of patients, to provide a more accurate and
detailed insight into the mechanisms of OIIRR. Cytokine
expression at different time intervals, during and after
orthodontic treatment, may also give more information
about the inflammatory nature of OIIRR.
The authors would like to acknowledge the Australian Society of
Orthodontics Foundation for Research and Education, the Australian
Proteome Analysis Facility at Macquarie University, Dr. Terry Whittle, Dr.
Edmond Breen, Mr. Alamgir Khan and Mr. Torren Carter for their great
supports and assistances.
DY: Participated in the design and coordination of the study, carried out the
sample collection and made substantial contributions to analysis and
interpretation of data. JC and RA: Participated in the design and
coordination of the study, carried out the sample collection and measured
the volume of OIIRR. AG: Performed the data analysis and performed the
statistical analysis. OD: Conceived the study, participated in the design of the
study and made substantial contributions to analysis and interpretation of
data. MA: Made substantial contributions to interpretation of data and
involved in revising the study critically. AD: Conceived the study, participated
in the design and coordination of the study and made substantial
contributions to analysis and interpretation of data. All authors drafted the
manuscript, read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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