| |

Received: 15 March 2019    Revised: 7 June 2019    Accepted: 7 June 2019

DOI: 10.1111/ocr.12329

REVIEW ARTICLE

Biomarkers in the gingival crevicular fluid used to detect root

resorption in patients undergoing orthodontic treatment: A
systematic review

Francesco Tarallo1 | Claudio Chimenti1 | Giordano Paiella1 | Massimo Cordaro2 |

Michele Tepedino1

1
Department of Biotechnological and
Applied Clinical Sciences, University of
L’Aquila, L’Aquila, Italy
2
Fondazione Policlinico Universitario
A. Gemelli IRCCS, Istituto di Clinica
Odontoiatrica e Chirurgia Maxillo‐
Facciale, Roma‐Università Cattolica del
Sacro Cuore, Rome, Italy
Correspondence
Michele Tepedino, Dipartimento di Scienze
Cliniche Applicate e Biotecnologiche,
Università degli Studi dell’Aquila, Clinica
Odontoiatrica, V.le S.Salvatore, Edificio
Delta 6, 67100 L’Aquila (AQ), Italy.
Email: m.tepedino@hotmail.it
Abstract
Objectives: To evaluate whether changes in the concentration of different biomark‐
ers in the gingival crevicular fluid (GCF) can be used to detect the root resorption
process in adult or adolescent patients undergoing treatment with a fixed appliance,
in comparison with untreated subjects or treated patients not showing signs of root
resorption.
Material and Methods: The following databases were analysed in the period be‐
tween June 2017 and March 2018, without any language and initial date restrictions:
PubMed, EMBASE, Scopus, Web of Science and Cochrane Library. A quality assess‐
ment instrument (QAI) was developed to establish the risk of bias.
Results: A total of 1127 articles were analysed. Based on the inclusion and exclusion
criteria, seven studies qualified for the final review. The QAI tool revealed that five
articles were at a moderate risk of bias and two articles were at a low risk of bias.
Conclusion: Dentine phosphoprotein (DPP) may be considered a relatively useful
marker for root resorption. Dentinal sialoprotein (DSP) could be a potential biomarker
but is not highly helpful at detecting root shortening. Inflammatory cytokines (pro‐
and anti‐resorption), osteopontin (OPN), osteoprotegerin (OPG), RANKL and alkaline
phosphatase (ALP) are useful biomarkers to explain the biological mechanisms that
occur during orthodontic movement but are not specific enough. Further studies are
required to clarify the role of GM‐CSF as a potential biomarker to distinguish sub‐
jects at a risk of severe root resorption in the early phase.

KEYWORDS
gingival crevicular fluid, orthodontics, root resorption

1 |  I NTRO D U C TI O N treatment.2,3 It can be defined as an iatrogenic process that results

Orthodontic tooth movement is a continuous balanced process be‐
1
tween bone formation and bone resorption ; it is not a risk‐free
process, and root resorption is one of the main undesired effects.
Orthodontically induced inflammatory root resorption (OIIRR) is a com‐
mon and unavoidable, yet unexplained, consequence of orthodontic
in the loss of substance from the mineralized cementum and dentine
during orthodontic tooth movement, wherein the resorbed root por‐
tion is replaced with normal bone.2 The estimated rates of orthodontic
patients with root resorption vary extensively among different studies:
the range extends from as low as 26%4 to as high as 100%.5 Generally,
root resorption may be described as mild, moderate or severe.6,7

Orthod Craniofac Res. 2019;00:1–12. wileyonlinelibrary.com/journal/ocr   © 2019 John Wiley & Sons A/S. |  1
Published by John Wiley & Sons Ltd
 |
2       TARALLO et al.

TA B L E 1   Quality assessment instrument (QAI) for the evaluation of risk of bias in individual studies

Study design Score

1. Objective—clearly formulated Y N Unclear

2. Sample size—considered adequate Y N Unclear
3. Spectrum of patients, representative of patients Y N Unclear
receiving the test in practice
4. Ethical clearance mentioned Y N Unclear
5. Selection criteria—clearly described Y N Unclear
6. Adequate method to diagnose root resorption Y N Unclear
7. Baseline characteristics—clearly defined Y N Unclear
8. Control—clearly defined Y N Unclear
9. Orthodontic mechanics explained in sufficient detail Y N Unclear
to permit replication of experiment
10. Orthodontic force—clearly specified Y N Unclear
11. Description of execution of index test sufficient to Y N Unclear
permit replication of test
12. Absence of time difference between index test and Y N Unclear
control—mentioned
13. Index test executed at specified time and environ‐ Y N Unclear
mental conditions
14. Use of proper indices for assessment of gingival and Y N Unclear
periodontal status: pre‐treatment assessment
15. Use of proper indices for assessment of gingival and Y N Unclear
periodontal status: at each observation time
16. Oral hygiene regime—mentioned Y N Unclear
17. Prophylaxis done pre‐treatment Y N Unclear
18. Prophylaxis done at each observation time Y N Unclear

Measurements Score

19. GCF handling characteristic—explained Y N Unclear

20. Measurement method—appropriate to the objective Y N Unclear
21. Reliability—adequate level of agreement Y N Unclear
  Statistical analysis Score
22. Dropouts—dropouts included in data analysis Y N Unclear
23. Statistical analysis—appropriate for data Y N Unclear
24. Confounders—confounders included in analysis Y N Unclear
25. Statistically significant level—P‐value stated Y N Unclear
26. Confidence intervals provided Y N Unclear

Study results and conclusion Score

27. Index test compared to baseline Y N Unclear

28. Index test compared to control Y N Unclear
29. Conclusion—specific Y N Unclear

Abbreviation: GCF, gingival crevicular fluid.

Furthermore, mild OIIRR is a common finding in almost 90% of ortho‐
dontic patients,8 while the severe form develops in approximately 4%
of patients and more frequently in adults than in adolescents.9
Biologically, the interaction between bone formation and resorp‐
tion results in the release of various molecules that can be identified
as potential biomarkers.10 These biomarkers and their correlation with
orthodontic movement can be analysed through the collection of gin‐
gival crevicular fluid (GCF), an inflammatory exudate found in the gin‐
gival sulcus whose variations in composition are representative of the
dynamic and metabolic status of the whole periodontium.11,12
The clinical diagnosis of root resorption is largely obtained through
bi‐dimensional radiographs, but several limitations are present: the
TARALLO et al. |
      3

demanding methods of standardization, limited projection views

and periodic radiation exposure. Bi‐dimensional radiographs do not
allow the accurate identification of root resorption at an early stage
(approximately 60%‐70% of the mineralized tissue is lost prior to the
identification)13,14 and are not able to reveal surface resorption on the
oral and buccal aspects of the roots.15,16 Unlike X‐rays, GCF is a non‐
invasive, relatively simple and easily repeatable diagnostic method;
the other advantages are no exposure to ionizing radiation, the possi‐
bility of obtaining information on the stage of root resorption and on
its activity, the possibility of identifying patients at risk, to obtain an
early diagnosis, to predict the consequences and the clinical course in
high‐risk patients. In the light of these considerations, new diagnostic
tools involving the use of GCF biomarkers should be investigated as a
reliable alternative method for the early diagnosis of root resorption.
This present study is the first systematic review carried out to
answer the following PICOS (patients, intervention, comparator,
outcome and study design) question: to determine whether changes
in the concentration of different biomarkers in the gingival crevicular
fluid can be used to detect the root resorption process in adult or
adolescent patients undergoing treatment with a fixed appliance, in
comparison with untreated patients or treated patients not showing
any signs of root resorption.

2 | M ATE R I A L S A N D M E TH O DS
F I G U R E 1   PRISMA flow chart for the study selection process

2.1 | Protocol

This systematic review was designed according to the guidelines of
the Cochrane Handbook for Systematic Reviews of Interventions
and is reported following the PRISMA guidelines.17,18 The methods
of the analysis and inclusion criteria were specified in advance and
documented in a protocol registered in the National Institute of
Health Research database (https​://www.crd.york.ac.uk/prosp​ero/
Registration number: CRD42018105137). No funding was received
for carrying out the present systematic review.
Otherwise, based on the exclusion criteria, all studies carried
out in vitro, animal studies, meta‐analyses, mini‐reviews, confer‐
ence proceedings, narrative revisions, systematic reviews regarding
the analysis of the crevicular gingival fluid and studies in which the
analysis of the cytokine concentration was explored in the crevicular
fluid taken from the peri‐implant region were excluded.
2.3 | Information sources and search

The following databases were analysed in the period between June

2.2 | Eligibility criteria
According to the formulated PICOS question, this systematic review
focused on all types of human studies (studies), on adolescent and
adult patients (population) undergoing orthodontic treatment with
fixed appliances (intervention), reporting changes in the concentra‐
tion of biomarkers in the GCF as a consequence of external root re‐
sorption following the application of orthodontic force (outcome),
compared with untreated patients or treated patients not showing
any signs of root resorption (comparator). The inclusion criteria
were randomized trials and non‐randomized prospective studies,
retrospective studies and clinical trials, studies performed on either
adults or adolescents, subjects undergoing orthodontic treatment
with fixed multibracket appliances, detection of radiographic signs
of root resorption and GCF samples collected from the gingival sul‐
cus using micropipettes or absorbent papers (Periopaper).
2017 and March 2018, without language and initial date restric‐
tions: MEDLINE via PubMed, EMBASE, Scopus, Web of Science
and Cochrane Library. A search strategy was finalized utilizing
MESH terms, Boolean operators and free‐text terms, as follows:
root resorption AND (GCF OR gingival crevicular fluid). In addition,
a manual search of the reference list of the potential studies was
performed to retrieve any additional articles. Duplicate articles
were removed.
2.4 | Study selection
Eligibility was assessed independently by two authors (FT and GP)
who screened the title and abstract of the articles initially. Full
texts were accessed whenever it was not clear whether the ab‐
stract should be included or not. Any disagreement was resolved by
 |
4       TARALLO et al.

TA B L E 2   Characteristics of the GCF collection and studies included in the systematic review—part I

Ahuja (2017) Balducci (2007) George (2009) Kereshanan (2008)

Sampling methods GCF + Micro‐CT scan Radiographs + GCF Radiographs + GCF Panoramic radiograph + GCF

3
Resorption Low OIIRR (<0.15 mm ), High
assessment OIIRR (>0.35 mm3)
Orthodontic force 250 g
Cytokine IL‐1β, IL‐4, IL‐6, IL‐7, IL‐8,
IFN‐ϒ, TNF‐α, IL‐2, IL‐5,
IL‐10, IL‐12, IL‐13, GM‐CSF
Time intervals 0 h, 3 h, 1 d, 3 d, 7 d, 28 d
Teeth 1st upper premolar
Mild (<2 mm) and Mild (<2 mm) and The apical group (Ra) from the apex up
severe (>2 mm) root severe (>2 mm) root the half of the root length; the coronal
resorption resorption group (Rc) from halfway up the root to
the amelocemental junction
— — —
DMP‐1, DPP, DSP OPN, OPG, RANKL DSP
— — 0 h and 12 wk after
1st and 2nd upper 1st and 2nd upper Premolar, canine and a central incisor in
incisors incisors maxilla and mandible

Collection site Mesiobuccal site Mesial and distal site Mesial and distal site Distogingival site
GCF collection Filter paper strips Filter paper strips Filter paper strips Micropipettes
method

Abbreviations: ALP, alkaline phosphatase; DMP‐1, dentinal matrix protein; DPP, dentinal phosphoprotein; DSP, dentinal sialoprotein; GCF, gingival
crevicular fluid; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; IFN, interferon; IL, interleukin; OIIRR, orthodontically induced
inflammatory root resorption; OPG, osteoprotegerin; OPN, osteopontin; RANKL, RANK‐Ligand; Rx, 2D periapical radiographs;
TNF, tumour necrosis factor.

discussion and consensus or by a third experienced author who was were considered at moderate risk of bias and scores of 20‐29 were
requested to arbitrate (MT). considered at low risk of bias.

2.5 | Data collection 2.7 | Summary measures and approach to synthesis

Two authors (FT and GP) independently extracted data (authors,
year of publication, study design, sample size, sample composition
by sex and age, presence of control group, type of orthodontic treat‐
ment, primary and secondary outcomes, method of assessment, use
of a fixed appliance, grade of root resorption, magnitude of ortho‐
dontic force (if mentioned), cytokines analysed, site of withdrawal,
intervals of collection, inclusion and exclusion criteria) from the
selected studies using a pre‐determined extraction form. Any disa‐
greement between the two authors was resolved by discussion and
consensus or by a third experienced author who was requested to
arbitrate (MT).
Due to the heterogeneity among the studies included in this system‐
atic review, particularly in the different types of cytokines analysed,
the orthodontic force magnitude was not always expressed. Due to
a small sample size and individual biological variations, it was not
possible to perform a meta‐analysis. A narrative synthesis was per‐
formed by illustrating the results from individual studies according
to the group evaluated.
3 | R E S U LT S
3.1 | Study selection

An electronic database search provided a total of 1,127 results. Thirty‐

2.6 | Risk of bias in individual studies
The quality and risk of bias assessment of the articles included in
the review was done using a Quality Assessment Instrument (QAI)
modified and developed from the relevant articles in the literature
(Table 1).19,20
To evaluate the quality of the included studies, the QAI was
based on 29 stringent criteria divided into four domains: study
design (N  =  18), study measurements (N  =  3), statistical analysis
(N = 5), and study results and conclusion (N = 3). For the objective
assessment of the quality, a scoring system was incorporated, where
scores of 1‐9 were considered at a high risk of bias, scores of 10‐19
five articles in PubMed, 978 in Scopus, 87 in Web of Science and 27 in
EMBASE were retrieved. One additional article was retrieved through
manual searching. After adjusting for duplicates, 987 entries were left.
Of these, 905 titles were discarded because they were clearly not rel‐
evant, and 82 abstracts were screened. Seventy‐three abstracts were
discarded due to the methodology not corresponding to the inclusion
criteria; thus, nine full‐text papers were accessed for detailed exami‐
nation. Two articles were excluded because their aim was not corre‐
spondent to that of the present systematic review. Seven studies were
included in the systematic review.16,21-26 A PRISMA flow chart diagram
for the study selection process is reported (Figure 1).
TARALLO et al.       5|

Kunii (2013) Mah (2004) Wahab (2013)

Radiographs + GCF Radiographs + GCF Radiographs + GCF

Severe root resorption involving more than one‐ Teeth with half of the root resorbed, teeth with mild Apical root resorption, lateral root
third of the original root length root resorption resorption (grade from 0 to 3 each)

— — 100 and 150 g

IL‐6 DPP ALP

0 h, 1 h, 4 h, 8 h, 1 d, 3 d, 5 d and 7 d
Maxillary central and lateral incisors
Mesial and distal site
Filter paper strips
— 0 h, 1 wk, 2 wk, 3 wk, 4 wk, 5 wk
Central incisors of untreated subjects (controls), Maxillary canines (study group) and
primary second molar (primary group and positive mandibular canines (control group)
controls), permanent central incisors (orthodontic
group)
— Mesial and distal site
Filter paper strips Filter paper strips

used to apply 225 g of buccally directed force to the first pre‐

3.2 | Study characteristics
The included studies consisted of five clinical trials and two split‐
mouth studies. One study did not mention the age, 23
the sex dis‐
tribution and the type of the patients involved. Kereshanan et al
evaluated root resorption using a panoramic radiograph instead of
periapical radiographs and collected GCF with micropipettes instead
of filter paper strips which were used in all the other studies. Only
two studies clearly expressed the magnitude of the orthodontic
force applied. 21,26 The studies were categorized based on the popu‐
lation and study characteristics and GCF collection characteristics
(Tables 2, 3 and 4).
3.3 | Risk of bias in individual studies
The assessment of the risk of bias, using the customized QAI tool,
revealed that five articles were at moderate risk of bias16,22-25 and
that two articles were at low risk of bias. 21,26 None of the studies fell
in the score between 1 and 10 (Table 5).
3.4 | Qualitative synthesis
Ahujia et al, 21
in their split‐mouth study, investigated the changes
in the cytokine profile in GCF following the application of heavy
orthodontic forces and compared the cytokine expression differ‐
encing mild and severe root resorption in eight young participants.
The authors classified cytokines as pro‐resorptive (IL‐6, IL‐7, IL‐8,
IL‐1β, TNF and TNF‐α) and anti‐resorptive (IL‐4, IFN‐γ and GM‐
CSF) cytokines. A fixed appliance with a cantilever spring was
molars in the test side and no forces in the control side. GCF was
collected from the mesiobuccal aspect of both the test and con‐
trol side using paper strips. At the end of 28  days, the test and
control teeth were extracted atraumatically and analysed with a
3D micro‐CT system. The authors found that, although the level
of some anti‐resorptive cytokines was significantly higher in low
OIIRR cases, other cytokines showed no differences.
The clinical trial of Balducci et al22 identified and quantified ex‐
tracellular matrix proteins and dentinal proteins such as DMP‐1, DSP
and PP analysing the GCF of sixty patients. The subjects were di‐
vided into three groups: two groups with signs of mild and severe
root resorption after orthodontic treatment with a fixed appliance
and a control group who had not started treatment yet and demon‐
strated no radiographic signs of root shortening. GCF was collected
from the mesial and distal side of the upper central and lateral inci‐
sors using paper strips. The DMP showed a higher concentration in
the study groups compared with the control group, while no differ‐
ence was observed between the mild and the severe root resorp‐
tion groups. The PP and DSP concentration in the three groups was
different, with higher values as the severity of the root resorption
increases.
George et al23 conducted a clinical trial to evaluate whether cyto‐
kines are released in the GCF and to verify differences in their levels.
The authors analysed OPG, OPN and RANKL in patients undergo‐
ing treatment for at least 1 year that showed radiographic signs of
mild or severe root resorption. Sixty patients were divided into three
groups: two study groups with respectively mild and severe radio‐
graphic signs of root shortening and one control group. GCF was
 |
6      

TA B L E 3   Characteristics of the studies included in the systematic review—part II

Gender distribution
Authors (publica‐ Patients' age (mean ± SD
tion year) Study design Study sample Control group Type of patients or range) M (n) F (n)

Ahuja (2017) Split‐mouth 8 patients (maxillary first Contralateral first Mixed 16.4 (13.9‐22.9) 6 2
premolar) premolars
Balducci (2007) Clinical trial 60 patients (2 groups of 20 1 (20 participants) Mixed 12‐44 (group 1 = 14‐40; Group 1 = 9; group Group 1 = 11; group 2 = 15;
participants each) group 2 = 16‐44; control 2 = 5; control group = 7 control group = 13
group = 12‐34)
George (2009) Clinical trial 60 patients (2 groups of 20 1 (20 participants) — — — —
participants each)
Kereshanan Clinical trial 50 (33 coronal group, 17 1 (20 participants Adolescent 9‐14 (study groups), 8‐14 — —
(2008) apical group); 20 patients with erupted (control group)
undergoing treatment with mandibular sec‐
fixed appliances ond premolar)
Kunii (2013) Clinical trial 20 patients (1 study group of 1 (15 participants) Adult 28.9 ± 6.1 (study group), Study group = 0; control Study group = 5; control
5 participants) 28.0 ± 5.3 (control group = 2 group = 13
group)
Mah (2004) Clinical trial 60 (20 patients in orthodon‐ 1 (20 participants) Adolescent 12‐16 y (orthodontic Ortho group = 7; pri‐ Ortho group = 13; primary
tic group, 20 patients in group), 9‐12 y (primary mary group = 5; control group = 15; control
primary group) group), 12‐16 y (control group = 8 group = 12
group)
Megat Abdul Split‐mouth 12 patients (maxillary Mandibular Adult 24.7 ± 3.0 y — 12
Wahab, (2013) canines) canines

Abbreviations: GCF, gingival crevicular fluid; OIIRR, orthodontically induced inflammatory root resorption.
TARALLO et al.
TA B L E 4   Characteristics of the studies included in the systematic review—part III
Authors (publication
year) Primary outcome
Ahuja (2017) Quantify cytokine profiles in gingi‐
val crevicular fluid (GCF)
Balducci (2007) Quantify dentin matrix protein 1
(DMP1), dentin phosphophoryn
(PP) and dentin sialoprotein (DSP)
in GCF
George (2009) Quantify cytokine profiles in GCF
Kereshanan (2008) Quantify the dentine sialoprotein
(DSP) in GCF
Kunii (2013) Quantify interleukin (IL)‐6 in GCF
of patients with severe root
resorption
Mah (2004) Verify that during the process of
root resorption, organic matrix
proteins are released into the
GCF
Megat Abdul Wahab Compare the effect of different
(2013) orthodontic forces on specific
alkaline phosphatase (ALP) activi‐
ties in GCF
Abbreviations: GCF, gingival crevicular fluid; OIIRR, orthodontically induced inflammatory root resorption.
Secondary outcome
Compare the cytokine expression between
participants showing low and high volume
of root resorption
— Treatment ≥1 y with mild or severe radio‐
Differences between levels of cytokines in
GCF of subjects with mild and severe root
resorption evaluated by radiographs
— Second primary molars and fully erupted
Investigate the effects of different static
compressive forces (CFs) on IL‐6 produc‐
tion by human periodontal ligament
(hPDL) cells
Verify whether there is a difference in the
levels of cytokines between a group of
patients with mild root resorption and a
control group
Compare the relationship between ALP
levels and the rate of canine movement
during 5 wk of retraction
Inclusion criteria
Class I malocclusion, class I skeletal base,
average vertical height, absence of obvi‐
ous facial asymmetry, normal growth and
development of the dentition and radio‐
graphical signs of complete apex genesis of
the upper first premolars
graphic signs of root resorption
Treatment ≥1 y with mild or severe radio‐
graphic signs of root resorption
mandibular second premolar undergoing
physiological root resorption as confirmed
by dental panoramic tomograms (DPTs).
Class I malocclusion with mild crowding
(≤6 mm; mean arch length discrepancy,
5.4 ± 0.55 mm), extraction of all the four
premolars, the probing depths were ≤ 3 mm
and periodontal bone loss was not evident
radiographically
Good general health, absence of medication,
excellent oral hygiene and no evidence of
caries, abscess or gingivitis
Good general and periodontal health, mild‐
to‐moderate crowding of the maxillary and
mandibular arches, need at least maxillary
first premolar extractions, canine relation‐
ship of class II 1/2 unit or more, class II/1
incisal relationship with an overjet of more
than 6 mm, overbite not more than 50%
TARALLO et al.
Exclusion criteria
History of medical problem(s), history of
trauma or bruxism and caries or ortho‐
dontic treatment
History of medical problem(s), history
of trauma or bruxism and caries or
orthodontic treatment, had received any
anti‐inflammation or antibiotics therapy
6 mo before
—
History of medical problem(s), history of
trauma or bruxism and caries or ortho‐
dontic treatment
History of medical problem(s), history
of trauma or bruxism and caries or
orthodontic treatment, had received any
anti‐inflammation or antibiotics therapy
6 mo before
—
Pregnancy, history of previous orthodontic
treatment or orthopaedic treatment, use
of antibiotics of anti‐inflammatory during
the study
|
      7
 |
8       TARALLO et al.

TA B L E 5   Risk of bias for individual studies calculated using the Quality Assessment Instrument (QAI)

Balducci George
Ahuja et et al et al Kereshanan Kunii et Mah et al Wahab et
QAI question al (2017) (2007) (2009) et al (2008) al (2013) (2004) al (2013)

1. Objective clearly formulated Y Y Y Y Y Y Y

2. Sample size—considered adequate N Y Y Y N N N
3. Spectrum of patients, representative of Y Y Y Y Y Y Y
patients receiving the test in practice
4. Ethical clearance mentioned Y Y Y Y Y Y Y
5. Selection criteria clearly described Y Y Unclear Y Y Y Y
6. Adequate method used to evaluate root Y N N N N N N
resorption
7. Baseline characteristics clearly defined Y N N Y N N Y
8. Control—clearly defined Y N N Y N N Y
9. Orthodontic mechanics explained in Y N N Y N N Y
sufficient detail to permit replication of
experiment
10. Orthodontic force—clearly specified Y N N N N N Y
11. Description of execution of index test Y Y Y Y Y Y Y
sufficient to permit replication of test
12. Absence of time difference between Y Unclear Unclear Unclear Unclear Unclear Y
index test and control—mentioned
13. Index test executed at specified time Y N N Y N N Y
and environmental conditions
14. Use of proper indices for assessment of Y Y N Unclear Y Y Y
gingival and periodontal status: pre‐
treatment assessment
15. Use of proper indices for assessment of Y N N Unclear Y N N
gingival and periodontal status: at each
observation time
16. Oral hygiene regime—mentioned Y Y N Y N Y Y
17. Prophylaxis done: pre‐treatment Unclear Unclear Unclear Unclear Unclear N Y
18. Prophylaxis done: at each observation Unclear Unclear Unclear Unclear Unclear N N
time
19. GCF handling characteristics —explained Y Y Y Y Y Y Y
20. Measurement method—appropriate to Y Y Y Y Y Y Y
the objective
21. Reliability—adequate level of agreement Y Y Y Y Y Y Y
22. Dropouts—dropouts included in data N Unclear N Unclear N N N
analysis
23. Statistical analysis—appropriate for data Y Y Y Y Y Y Y
24. Confounders—confounders included in Y N N N N Y Y
analysis
25. Statistical significance level—P‐value Y Y Y Y Y Y Y
stated
26. Confidence intervals provided N N N Y N N N
27. Index test compared to baseline Y N N N N N Y
28. Index test compared to control Y Y Y Y Y Y Y
29. Conclusions—specific Y Y Y Y Y Y Y
Total 24 15 12 19 14 15 23  
score
TARALLO et al.
collected from the mesial and distal sides of the upper central and
lateral incisors using paper strips. No differences were found in the
OPG and OPN levels. The concentration of RANKL in the GCF was
significantly higher (P < 0.05) in subjects with mild and severe root
resorption than in the controls, although there was a small differ‐
ence between the mild and severe resorption groups.
Kereshanan et al16 conducted a clinical study to investigate the
potential of DSP in GCF as a biomarker of root resorption. Fifty
subjects, which had second primary molars yet, were divided into
two groups depending on the degree of root resorption that had
taken place (apical or coronal group) and twenty patients in the
control group with fully erupted second molars. The second ex‐
perimental group involved twenty patients who were undergoing
orthodontic treatment with fixed appliances. The samples of GCF
were collected from the distal side of a premolar, a canine and a
central incisor in both dental arches immediately prior to ortho‐
dontic intervention and 12 weeks after the placement. Each sample
was obtained using micropipettes by capillary action. The compar‐
ison of the mean relative DSP levels in GCF showed a difference
(P < 0.05) between the two study groups and the control group.
However, there was no significant difference in the levels when
comparing the two groups with the resorbing primary molar group.
24
Kunii et al carried out a clinical study to determine the IL‐6 lev‐
els in the GCF of patients with severe root resorption after ortho‐
dontic treatment. Twenty subjects were divided into a study group
with radiographic signs of severe root resorption and a control group
without signs of root shortening. The GCF was collected from the
mesial and distal side of the maxillary central and lateral incisors
using filter paper strips. The IL‐6 level was statistically higher in the
study group when compared to the controls.
25
Mah et al conducted a clinical trial to verify whether during
the process of root resorption, the organic matrix proteins are
released in the GCF and whether there is a difference between
the levels of DPP in the GCF of primary, orthodontic and con‐
trol groups. The GCF was collected using filter paper strips from
the permanent central incisors, a primary second molar with half
of the root resorbed and permanent central incisors with radio‐
graphic evidence of mild root resorption. The results revealed that
there was a statistically significant difference among the ortho‐
dontic and primary group in comparison with the control group,
but no significant differences were observed between the primary
group and the orthodontic group.
26
Wahab et al in their split‐mouth study compared the effects of
different orthodontic forces (100 and 150 g) on specific ALP activ‐
ities in GCF and their relationship to the rate of canine movement
during the 5  weeks of retraction. GCF was collected on a weekly
basis for six consecutive weeks, starting with the first withdrawal
done before the application of the force. The GCF collection in‐
volved the mesial and distal side of the maxillary canines on both the
test and control teeth. The peaks of ALP activity were at week 1 in
the 150 g group and at week 2 in the 100 g group; however, in the
five weeks of observation, the overall ALP activity increased with
greater rates of tooth movement with 150 g of force reflecting the
|
      9
contribution of osteoblastic activities during both bone formation
and resorption.
4 | D I S CU S S I O N
4.1 | Summary of evidence
The large heterogeneity among the studies did not allow a quanti‐
tative synthesis and a solid evaluation of the diagnostic abilities of
the GCF biomarkers for the early diagnosis of OIIRR. However, the
summary of the results presented here is a useful foundation for the
development of new sound clinical trials on this important topic.
Ahuija et al found that cytokines, such as IL‐1β, IL‐6 and IL‐8, did
not undergo any statistically significant differences between the test
(TS) and the control groups (CS). The result agrees with some studies
but disagrees with many others; the dissimilarities in the detected
levels of IL‐6 and IL‐8 may be due to the variation in the used appli‐
ance systems, force levels, small sample size and/or some individual
variations. On the other hand, there was a statistically significant
difference in the level of IL‐7 and TNF‐α between the TS and CS. The
peak in the level of TNF‐α, IL‐6 and IL‐8 at day 28 indicates a correla‐
tion with the localized inflammation secondary to the application of
orthodontic force. The boost in the pro‐resorptive cytokines signi‐
fies their crucial role in the removal of hyalinized tissue27 and might
indicate continuous periodontal remodelling during the lag phase of
tooth movement. 21
The fluctuating trend of the anti‐resorptive cytokines, IL‐4 and
IFN‐γ, closely follows IL‐1β and may result in preventing the addi‐
tional differentiation and activation of osteoclastic cells during the
initial stages of tooth movement. 21 There were also no significant
differences between the TS and CS teeth concerning their concen‐
tration. The GM‐CSF level dropped immediately on the TS teeth, in‐
creased at day 7 and then reached its peak on day 28, but there was
no significant difference between the TS and CS teeth. However, it
increased in low OIIRR cases, while other cytokines showed no sta‐
tistically significant differences. These results may link the high levels
of anti‐resorptive cytokines, such as GM‐CSF, with the reduced root
osteoclastic differentiation. By evaluating the results, inflammatory
cytokines play a primary role in remodelling tissue, but they may not
be suitable as root resorption biomarkers; their variation is linked to
the physiological inflammatory mechanism that occurs during ortho‐
dontic treatment. Hence, GM‐CSF could be considered, in the future,
as a potential biomarker to distinguish subjects at risk of severe root
resorption in the early phase, but further studies may clarify its role.
Kunii et al24 evaluated the concentration of the cytokine IL‐6.
The results demonstrate a close correlation between the application
of the orthodontic force and the presence of the cytokine whose
values were significantly higher than the control site. The data are in
opposition to the study conducted by Ahuja et al while in line with
Ren et al. 28 This difference in the results could be due to the timing
factor. In fact, Ahuja et al analysed the concentrations of the cyto‐
kine with samples collected at well‐established intervals; Kunii et al,
on the other hand, made a single withdrawal in subjects undergoing
 |
10       TARALLO et al.
orthodontic treatment, not specifying its duration. Although both
the studies were respectively considered at low and moderate risk
of bias, the small sample size in the study groups may be a remark‐
able limitation. Other issues are the different methods of analysis
used and the applied orthodontic force. Therefore, IL‐6 might not be
considered a marker for the diagnosis of OIIRR because, although it
induces or, at least, facilitates the process, it is also produced during
the physiological inflammatory mechanisms.
Dentinal matrix protein (DMP‐1), dentinal sialoprotein (DSP)
and dentine phosphoprotein (DPP) were analysed by Balducci et al,
Kereshanan et al and Mah et al. It emerged that DMP‐1 is present
both in the dentinal structure and in the bone tissue. 29 Furthermore,
although there are statistically significant differences between the
study groups and the control group, the same does not occur be‐
tween the mild and severe root resorption groups. Hence, the pres‐
ence of this non‐collagenous protein in the crevicular fluid may not
be entirely a result of ongoing resorption activity but also due to
increased bone remodelling during orthodontic tooth movement.
Therefore, DMP‐1 cannot be considered a useful marker because
it is not possible to distinguish between its physiological and patho‐
logical activity.
Dentinal sialoprotein was considered by Balducci et al and
Kereshanan et al. The first study noted a considerable increase in
the concentration of this cytokine in both severe and mild root re‐
sorption groups compared with the control group. In particular, the
peak is higher in severe root resorption. The analysis of Kereshanan
et al, on the other hand, is based on a comparison of the concentra‐
tion of the DSP both in subjects undergoing orthodontic treatment
and in subjects with second primary molars undergoing physiological
root resorption. There was a statistically significant difference be‐
tween the study groups and the control group. However, there was
no statistical difference in the concentrations when comparing the
two study groups with the group still exhibiting the deciduous molars
during the reabsorption phase. Although it is known that there are
microscopic differences in the structure of dentine in the primary and
permanent dentitions,30 there are no available data in the literature
characterizing the structure of non‐collagenous proteins in the human
primary dentition. Based on a number of studies on the resorption of
dentine,31-34 researchers have accepted the use of physiological root
resorption as a suitable model to study pathological root resorption. It
is accepted that although the initiation process may differ, the actual
biochemical mechanism that takes place is largely similar.
Additionally, there are some suggestions that DSP may not be
entirely dentine‐specific.35 Genetically, DSP and DPP are expressed
as a single mRNA that encodes for a large precursor protein termed
DSPP traditionally considered to be dentine‐specific. Qin et al found
that the dspp gene was expressed in odontoblasts but at a much
lower level, as well as in osteoblasts.35 The data indicated that differ‐
ent regulatory mechanisms governing DSPP expression are involved
in teeth and bone. The presence of DSP in the bone, although at a
minimal level, may reflect the presence of GCF in the control sam‐
ples. Therefore, it is plausible that the cementum contains the den‐
tinal sialoprotein within its matrix such that it is released into GCF
as a normal consequence of the resorption/repair process of cemen‐
tum both during the physiological resorption of the root and during
the movement induced by orthodontic treatment.16 It emerged that
DSP could be a potential biomarker, but it is not helpful in detecting
root resorption because it is also produced during bone turnover.
The last protein analysed in these studies is the dentine
phosphoprotein (DPP). It seems that the best markers for the diag‐
nosis of root resorption are the proteins of dentine because some
areas of the cementum are resorbed and subsequently repaired
during orthodontic movement.36 Thus, proteins of cementum are
not highly indicative of the loss of root structure. Dentine also has
the capability to repair after resorption, but larger dentine defects
and those at the apex do not repair, causing a loss of dentine to be a
significant part of the loss of root structure. Mah et al evaluated the
concentrations of DPP in the crevicular fluid of subjects undergoing
orthodontic treatment, subjects with the roots of the second molars
undergoing resorption and a control group. The results showed that
there is a statistically significant difference not only between the
control group and the primary group but also between the control
group and the orthodontic group. On the other hand, the difference
between the primary group and the orthodontic group is not signif‐
icant. The comparison among the three groups showed the highest
concentration of DPP in the primary group as could be expected be‐
cause the root resorption of primary teeth involves large portions
of the root. The orthodontic group, on the other hand, had radio‐
graphic signs of resorption involving only the apical portion: also, in
this case, the results reflected the hypothesis because the root por‐
tion resorbed is lower than that of primary teeth. Balducci et al also
achieved similar results: the highest concentrations of DPP were
detected in the group with severe root resorption followed by the
mild resorption group and the control group, which presented the
lowest concentrations. Otherwise, the presence of the DPP in the
control groups was an unexpected result and is clearly more difficult
to explain, as apparently, these teeth were not subject to structural
changes. According to Balducci et al and Mah et al, the most plausible
explanation could be credited to the sensitivity of the ELISA method.
The antibody used was a monoclonal antibody developed from rat
dentine phosphoprotein, with which the human shares many com‐
mon features but also many differences. Therefore, it is an epitope
that could react to other proteins. The goal for the future would be
to develop a monoclonal antibody specific for human DPP. Although
these studies resulted in a moderate risk of bias, other causes are
not excluded, such as that dentine is not a homogeneous tissue and
its protein components change with age as the root matures.37 In
conclusion, DPP may be an excellent biomarker of root resorption as
it is a mainly organic, non‐collagenous constituent of dentine and is
likely to be more indicative of the permanent loss of root structure
compared with cementum proteins.
George et al analysed the organic matrix proteins: osteopontin
(OPN), osteoprotegerin (OPG) and RANKL. Osteopontin is a major
glycosylated protein in the bone and dentin matrix and is produced
by osteoblasts, odontoblasts, osteoclasts and macrophages. The
presence of OPN in the GCF of the study groups may be derived
TARALLO et al.
from enzymatic activity in the neighbouring tissues such as alveolar
bone, cementum, dentin, macrophages in periodontal tissue, blood
and salivary glands during bone resorption. On the other hand, the
concentrations of OPG and RANKL detected were higher in the
study groups than in the control group, but only that of RANKL was
statistically significant. This could be due to the insufficient number
of samples analysed. Therefore, the increase in the RANKL/OPG
ratio confirms the increase in osteoclastic activity that occurs in
subjects undergoing orthodontic treatment. Therefore, markers of
the bone matrix may be used to evaluate the biological resorption
process that occurs during dental movement, but as they are also
produced by many tissues surrounding the dental root; they should
not be considered as highly reliable markers for root resorption.
Alkaline phosphatase (ALP) was analysed by Wahab et al. The
results state that the concentration of the ALP reached its peak in
the first week at the site with 150 g and in the second week at the
site with 100 g. However, in both cases, it began to decrease from
the week following the peak, indicating the removal of the hyalinized
tissue by osteoclasts. The fluctuating activity of ALP reflects the
activity of osteoclasts during both resorption and bone formation
processes. The results indicate that the application of a force equal
to 150 g produces a 25% faster dental movement as shown by the
significant increase in alkaline phosphatase compared with the 100 g
group. In addition, a study showed that any root resorption could be
identified within 6 months.38 In this study, the application of intense
forces did not show a side effect as important as root resorption
after 6 months of treatment. Although there is a low risk of bias, pre‐
cautions may be required in considering the results as only females
were recruited. Therefore, ALP may be a useful marker to evaluate
the bone remodelling process, but it is not suitable to evaluate early
root resorption.
4.2 | Limitations
Two studies21,26 included a small sample size without sample size cal‐
culation. Despite the fact that 2D images do not allow for accurate
identification of root resorption at an early stage and are not able to
reveal surface resorption on the oral and buccal aspects of the root,
only one study21 considered the use of 3D radiographs. Different
teeth were also considered for GCF collection, and the orthodontic
forces applied were not always specifically expressed. The methods
used in the analysed studies were not homogeneous also regarding
the timing of GCF sampling, thus hindering the possibility of a clear
comparison of the results. In addition, the lack of homogeneity in the
results may depend on the numerous cytokines analysed and/or the
genetic variations. Standardization in the method may influence the
outcome of future studies.
5 | CO N C LU S I O N
Based on the results of the present systematic review, it is possible
to conclude the following:
|
      11
1. All the studies have found an increase in the marker con‐
centrations following the application of orthodontic forces,
fluctuating similarly to the inflammatory processes of which
they are represented;
2. The levels of the cytokines are different depending on the sam‐
pling sites and the momentum in which it occurs;
3. Regarding the use of cytokines:
• Dentine phosphoprotein (DPP) may be considered a relatively
useful marker for root resorption because it is the major or‐
ganic and non‐collagenous component of dentine and seems
more indicative of the loss of root structure than cementum
proteins;
• Dental sialoprotein (DSP) in addition to being one of the major
proteins of the organic component of dentine, it is also con‐
tained in the root cement matrix and is released into the GCF
as a normal consequence of the resorption and/or repair pro‐
cess both during the physiological resorption of the root and
the movement induced by orthodontic treatment;
• The protein of the dentinal matrix (DMP‐1) is also a non‐collag‐
enous protein: its presence within the GCF may not be due ex‐
clusively to the process of root resorption in progress but also
to the normal process of bone remodelling; therefore, it cannot
be considered a useful biomarker because it is not possible to
distinguish between its physiological and pathological activity;
• Inflammatory cytokines (pro‐ and anti‐resorption), osteopon‐
tin (OPN), osteoprotegerin (OPG), RANKL and alkaline phos‐
phatase (ALP) are not suitable to diagnose root resorption,
especially at an early stage.
4. Further studies are required to prove the diagnostic ability of the
mentioned biomarkers and to clarify the possible uses of GCF col‐
lection by the orthodontist to detect early root resorption and to
monitor the ongoing process. Based on the results of the present
systematic review and with all its limitations, it can be suggested
to design new clinical trials focused on the ability of GCF to diag‐
nose OIIRR early and to prefer the DPP as a biomarker.
C O N FL I C T O F I N T E R E S T
None to declare.
ORCID
Michele Tepedino  https://orcid.org/0000-0002-8646-9824
REFERENCES
1. Keeling SD, King GJ, McCoy EA, Valdez M. Serum and alveolar bone
phosphatase changes reflect bone turnover during orthodontic tooth
movement. Am J Orthod Dentofac Orthop. 1993;103(4):320‐326.
2. Brezniak N, Wasserstein A. Orthodontically induced inflammatory
root resorption. Part I: the basic science aspects. Angle Orthod.
2002;72(2):175‐179.
 |
12       TARALLO et al.
3. Brezniak N, Wasserstein A. Root resorption after orthodontic
treatment: part 1. Literature review. Am J Orthod Dentofac Orthop.
1993;103(1):62‐66.
4. Kennedy DB, Joondeph DR, Osterberg SK, Little RM. The effect
of extraction and orthodontic treatment on dentoalveolar support.
Am J Orthod. 1983;84(3):183‐190.
5. Harry MR, Sims MR. Root resorption in bicuspid intrusion. A scan‐
ning electron microscope study. Angle Orthod. 1982;52(3):235‐258.
6. Levander E, Malmgren O. Evaluation of the risk of root resorp‐
tion during orthodontic treatment: a study of upper incisors. Eur J
Orthod. 1988;10(1):30‐38.
7. Kaley J, Phillips C. Factors related to root resorption in edgewise
practice. Angle Orthod. 1991;61(2):125‐132.
8. Weltman B, Vig K, Fields HW, Shanker S, Kaizar EE. Root resorption
associated with orthodontic tooth movement: a systematic review.
Am J Orthod Dentofac Orthop. 2010;137(4):462‐476.
9. Linge BO, Linge L. Apical root resorption in upper anterior teeth.
Eur J Orthod. 1983;3:173‐183.
10. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue‐level
reactions to orthodontic force. Am J Orthod Dentofac Orthop.
2006;129(4):469.e1‐32.
11. Alfano MC. The origin of gingival fluid. J Theor Biol.
1974;47(1):127‐136.
12. Kavadia‐Tsatala S, Kaklamanos EG, Tsalikis L. Effects of ortho‐
dontic treatment on gingival crevicular fluid flow rate and compo‐
sition: clinical implications and applications. Int J Adult Orthodon
Orthognath Surg. 2002;17(3):191‐205.
13. Andreasen FM, Sewerin I, Mandel U, Andreasen JO. Radiographic
assessment of simulated root resorption cavities. Dent Traumatol.
1987;3(1):21‐27.
14. Chapnick L, Endo D. External root resorption: an experimen‐
tal radiographic evaluation. Oral Surg Oral Med Oral Pathol.
1989;67(5):578‐582.
15. Acar A, Canyürek U, Kocaaga M, Erverdi N. Continuous vs. dis‐
continuous force application and root resorption. Angle Orthod.
1999;69(2):159‐163.
16. Kereshanan S, Stephenson P, Waddington R. Identification of den‐
tine sialoprotein in gingival crevicular fluid during physiological
root resorption and orthodontic tooth movement. Eur J Orthod.
2008;30(3):307‐314.
17. Liberati A, Altman DG, Tetzlaff J, et al. The PRISMA statement for
reporting systematic reviews and meta‐analyses of studies that
evaluate health care interventions: explanation and elaboration.
PLoS Medicine. 2009;6(7):e1000100.
18. Higgins J, Altman DG, Gotzsche PC, et al. The Cochrane
Collaboration's tool for assessing risk of bias in randomised trials.
BMJ. 2011;343:d5928.
19. Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The de‐
velopment of QUADAS: a tool for the quality assessment of studies
of diagnostic accuracy included in systematic reviews. BMC Med Res
Methodol. 2003;3(1):25.
20. Kuijpers M, Chiu Y‐T, Nada RM, Carels C, Fudalej PS. Three‐di‐
mensional imaging methods for quantitative analysis of facial soft
tissues and skeletal morphology in patients with orofacial clefts: a
systematic review. Kerkis I, editor. PLoS ONE. 2014;9(4):e93442.
21. Ahuja R, Almuzian M, Khan A, Pascovici D, Dalci O, Darendeliler
MA. A preliminary investigation of short‐term cytokine expres‐
sion in gingival crevicular fluid secondary to high‐level orthodon‐
tic forces and the associated root resorption: case series analytical
study. Prog Orthod. 2017;18(1):23.
22. Balducci L, Ramachandran A, Hao J, Narayanan K, Evans C, George
A. Biological markers for evaluation of root resorption. Arch Oral
Biol. 2007;52(3):203‐208.
23. George A, Evans CA, George A, Evans CA. Detection of root re‐
sorption using dentin and bone markers. Orthod Craniofacial Res.
2009;12(3):229‐235.
24. Kunii R, Yamaguchi M, Tanimoto Y, et al. Role of interleukin‐6 in
orthodontically induced inflammatory root resorption in humans.
Korean J Orthod. 2013;43(6):294‐301.
25. Mah J, Prasad N. Dentine phosphoproteins in gingival crevicular
fluid during root resorption. Eur J Orthod. 2004;26(1):25‐30.
26. Megat Abdul Wahab R, Md Dasor M, Senafi S, et al. Crevicular al‐
kaline phosphatase activity and rate of tooth movement of female
orthodontic subjects under different continuous force applications.
J Int J Dent. 2013;2013:245818.
27. Yamaguchi M, Yoshii M, Kasai K. Relationship between substance
P and interleukin‐1 in gingival crevicular fluid during orthodontic
tooth movement in adults. Eur J Orthod. 2005;28(3):241‐246.
28. Ren Y, Hazemeijer H, de Haan B, Qu N, de Vos P. Cytokine profiles
in crevicular fluid during orthodontic tooth movement of short and
long durations. J Periodontol. 2007;78(3):453‐458.
29. George A, Sabsay B, Simonian PA, Veis A. Characterisation of a
novel dentin matrix acidic phosphoprotein. Implications for induc‐
tion of biomineralization. J Biol Chem. 1993;268(17):12624‐12630.
3 0. Ten Cate AR. Oral histology: development, structure, and function
(ed 3). J Oral Maxillofacial Surg. St Louis, MO, Mosby. 1998;48(1):497.
31. Ten Cate AR, Anderson RD. An ultrastructural study of tooth re‐
sorption in the kitten. J Dent Res. 1986;65(8):1087‐1093.
32. Sahara N. Cellular events at the onset of physiological root resorp‐
tion in rabbit deciduous teeth. Anat Rec. 2001;264(4):387‐396.
33. Fukushima H, Kajiya H, Takada K, Okamoto F, Okabe K. Expression
and role of RANKL in periodontal ligament cells during physio‐
logical root‐resorption in human deciduous teeth. Eur J Oral Sci.
2003;111(4):346‐352.
3 4. Sasaki T. Differentiation and functions of osteoclasts and odon‐
toclasts in mineralised tissue resorption. Microsc Res Tech.
2003;61(6):483‐495.
35. Qin C, Brunn JC, Cadena E, et al. The expression of dentin sialo‐
phosphoprotein gene in Bone. J Dent Res. 2002;81(6):392‐394.
36. Owman‐Moll P, Kurol J, Lundgren D. Repair of orthodonti‐
cally induced root resorption in adolescents. Angle Orthod.
1995;65(6):403‐408, discussion 409‐410.
37. Clarkson BH, Chang SR, Holland GR. Phosphoprotein analysis of
sequential extracts of human dentine and the determination of the
subsequent remineralisation potential of these dentine matrices.
Caries Res. 1998;32(5):357‐364.
38. Artun J, Smale I. Behbehani F, Doppel D, Van't Hof M, Kuijpers‐
Jagtman AM. Apical root resorption six and 12 months after ini‐
tiation of fixed orthodontic appliance therapy. Angle Orthod.
2005;75(6):919‐926.
How to cite this article: Tarallo F, Chimenti C, Paiella G,
Cordaro M, Tepedino M. Biomarkers in the gingival crevicular
fluid used to detect root resorption in patients undergoing
orthodontic treatment: A systematic review. Orthod Craniofac
Res. 2019;00:1–12. https​://doi.org/10.1111/ocr.12329​