J Clin Periodontol 2017; 44: 31–41 doi: 10.1111/jcpe.

12636

Contribution of biomechanical Andressa Vilas Boas Nogueira1,

Rafael Scaf de Molon1,
Marjan Nokhbehsaim2,

forces to inflammation-induced James Deschner2 and

Joni Augusto Cirelli1
1
Department of Diagnosis and Surgery,

bone resorption School of Dentistry at Araraquara, Univ

Estadual Paulista (UNESP), Araraquara, Sa
Paulo, Brazil; 2Section of Experimental
~o

Dento-Maxillo-Facial Medicine, Center of

Dento-Maxillo-Facial Medicine, University of
Nogueira AVB, de Molon RS, Nokhbehsaim M, Deschner J, Cirelli JA. Contribution Bonn, Bonn, Germany
of biomechanical forces to inflammation-induced bone resorption. J Clin Periodontol
2017; 44: 31–41. doi: 10.1111/jcpe.12636.

Abstract
Aim: This study aimed to evaluate the contribution of biomechanical loading to
inflammation-induced tissue destruction.
Materials and Methods: A total of 144 adult Holtzman rats were randomly
assigned into four experimental groups: control (C), ligature-induced periodontal
disease (P), orthodontic movement (OM), and combination group (OMP). On
days 1, 3, 7, and 15, following baseline, nine animals from each experimental
group were killed. Bone volume fraction (BVF) and bone mineral density (BMD)
were measured using micro-computed tomography. Expression and synthesis pro-
file of cytokines and receptors of inflammation in gingival tissues were evaluated
by PCR array assay and multiplex immunoassay.
Results: At 15 days, the OMP group presented a significantly (p < 0.05) lower
BVF and BMD levels when compared to all the other groups. The OMP group
presented the highest number of upregulated protein targets in comparison to the
other groups. Furthermore, the gene expression and protein levels of CCL2,
CCL3, IL-1b, IL1-a, IL-18, TNF-a, and VEGF were significantly (p < 0.05)
higher in the OMP group when compared to the P group.
Key words: bone resorption; chemokines;
Conclusions: In summary, mechanical loading modulates the inflammatory cytokines; periodontal diseases;
response of periodontal tissues to periodontal disease by increasing the expression pro-inflammatory mediators; tooth movement
of several pro-inflammatory mediators and receptors, which leads to increased
bone resorption. Accepted for publication 3 October 2016

Biomechanical forces can contribute to addition, some studies have demon-
the progression of an inflammatory dis- strated that mechanical loading sig-
ease or condition, like tendinopathy nificantly modulates immunoin-
(Spiesz et al. 2015), arthritis (Sun flammatory reactions present in some
2010), and intervertebral disc degen- inflammatory diseases such as
eration (Walter et al. 2015). In atherosclerosis, rheumatoid arthritis,
osteoarthritis, ventilator-induced lung
Conflict of interest and source of
injury, and periodontal disease (PD)
funding statement (Lionetti et al. 2005, Ferretti et al.
The authors declare that there are no 2006, Chatzizisis et al. 2007, Boas
conflicts of interest in this study. Nogueira et al. 2013). According to
This study was supported by a grant some studies, the positive or negative
from the Research Funding Organ effects of mechanical forces seem to
Sa~o Paulo Research Foundation depend on the force type (compres-
(FAPESP), Grants: 2010/07771-4, sion, tension, shear stress) and magni-
2011/13752-5. tude (low, high, physiological). In this
context, some in vitro studies (Long
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
et al. 2001, Deschner et al. 2003,
Nokhbehsaim et al. 2010) have found
that tensile strain can exert anti-
inflammatory effects when applied at
low magnitude and pro-inflammatory
effects when applied at high magni-
tude (Nokhbehsaim et al. 2010,
Jacobs et al. 2014). Interestingly, the
exposure of cells to compression strain
leads to the increase in pro-inflamma-
tory molecules (Lu et al. 2015). Fur-
thermore, in vivo studies have shown
pro-inflammatory effects of experi-
mental orthodontic forces (Bletsa
et al. 2006). Moreover, shear stress
induced by pulsatile fluid flow was
demonstrated to increase the
31
32 Nogueira et al.
intracellular calcium concentration
and nitric oxide production (Bakker
et al. 2009).
Herein, biomechanical loading
represented by orthodontic force was
evaluated in the presence and
absence of PD. As the ageing popu-
lation is expanding worldwide and
so is the prevalence of PD (Susin
et al. 2004, Albandar 2011), the
search for orthodontic treatment has
increased among adult subjects espe-
cially due to aesthetic and functional
reasons. Periodontal patients com-
monly present occlusal and aesthetic
sequelae such as missing teeth, flar-
ing of the anterior maxilla teeth,
spaces, rotations, tilting, and teeth
over eruption. In order to restore
the original position of the teeth
and, therefore, to improve mastica-
tion, dental hygiene, and life quality,
orthodontic treatment is needed,
leading orthodontists to often deal
with an increased number of patients
affected by moderate-to-advanced
PD (Krishnan & Davidovitch 2009).
In such cases, a multidisciplinary
approach involving periodontics and
orthodontics has shown to be the
best option of treatment. However,
some adverse effects on the peri-
odontium of those patients have
been associated with the orthodontic
treatment, such as increased peri-
odontal support destruction induced
by an association of PD and hyper-
occlusal forces (Wennstrom et al.
1993, Harrel & Nunn 2001), and
changes in the subgingival microbio-
logical environment favoured by
orthodontic devices, resulting in an
exacerbation of PD activity and
pocket depth (Sallum et al. 2004,
Lee et al. 2005). On the other hand,
the mechanisms of how biomechani-
cal loading modulates PD progres-
sion are still unclear.
The orthodontic tooth movement
(OTM) occurs in response to
mechanical force by remodelling the
periodontal ligament and alveolar
bone (Krishnan & Davidovitch
2009). Tissue changes are regulated
and maintained by an aseptic acute
inflammatory response characterized
by the release of pro-inflammatory
mediators (Ren et al. 2002, Bletsa
et al. 2006, Krishnan & Davidovitch
2006, 2009). Clinical studies have
shown high levels of cytokines and
chemokines in gingival crevicular
fluid during orthodontic therapy
(Uematsu et al. 1996, Bletsa et al.
2006, Ren & Vissink 2008). More-
over, according to our previous
in vivo study (Boas Nogueira et al.
2013), orthodontic forces can aggra-
vate periodontal destruction by
intensifying periodontal inflamma-
tion. In this study, the novelty was
to explore critical molecules (cytoki-
nes, chemokines, and their receptors)
involved in inflammatory reactions
that have not been evaluated previ-
ously in animals subjected to peri-
odontal disease and biomechanical
force. Therefore, inflammatory medi-
ators are produced and released dur-
ing the progression of both, PD and
OTM. This could be the reason that
orthodontic forces applied in peri-
odontal diseased tissues exacerbates
periodontal breakdown. However, it
is still unclear as to how orthodontic
loading and inflammation co-regu-
late these mediators. Hence, this
study aimed to evaluate the contri-
bution of biomechanical loading to
inflammation-induced tissue destruc-
tion by analysing the inflammatory
protein profiles of the periodontal
tissues.
Materials and Methods
Experimental protocol
The experimental protocol was
approved by the local Ethical Com-
mittee on Animal Experimentation
and followed all recommendations of
the ARRIVE guidelines. A total of
144 male adult Holtzman rats, aver-
age weight of 300 g, were used. Ani-
mals were maintained in the animal
facilities of the School of Dentistry
at Araraquara under controlled tem-
perature (22–25°C) with a 12-h light/
dark cycle. Animals were housed in
plastic cages receiving standard labo-
ratory diet and water ad libitum.
After 1 week of acclimatization, gen-
eral anaesthesia was induced with
intramuscular injections of ketamine
chlorhydrate 10% (0.08 ml per 100 g
body weight) and xylazine chlorhy-
drate 2% (0.04 ml per 100 g body
weight). The animals were randomly
assigned into four experimental
groups: 1-control (sham-operated),
2-ligature-induced PD (P), 3-ortho-
dontic movement (OM), 4-ligature-
induced PD and orthodontic
movement (OMP). After 1, 3, 7, and
15 days of the OTM start (baseline),
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
nine animals from each experimental
group were killed in each period by
anaesthetic overdose. After killing,
the maxillary jaws were hemisected,
and half of the block samples were
fixed in 4% paraformaldehyde for
48 h and stored in 70% ethanol for
bone resorption analysis by micro-
computed tomography (micro-CT)
and later these samples were decalci-
fied in EDTA for stereometry analy-
sis. The other half of the blocks had
the gingival tissue around the maxil-
lary first molars carefully dissected
for extraction of total RNA for
PCR array and RT-qPCR
and extraction of total protein for
multiplex immunoassay.
Ligature-induced PD
Five days before baseline, animals
were anaesthetized and cotton liga-
tures were placed around the cervical
area of the upper first molars bilater-
ally and knotted mesially, aiming to
induce experimental PD. Therefore,
ligature was maintained in this study
from 6 to 20 days after ligature place-
ment, according to killing time
points. According to previous studies,
periodontal inflammation (Xie et al.
2011, Boas Nogueira et al. 2013,
Kirschneck et al. 2016) and alveolar
bone loss are observed after 5 days of
ligature placement, Holzhausen et al.
2002, Boas Nogueira et al. 2013).
Orthodontic tooth movement
To induce OTM, a previously estab-
lished rat tooth movement model and
orthodontic appliance design were
adopted with some changes (Boas
Nogueira et al. 2013). At baseline, a
closed coil nickel-titanium spring
(SentalloyÒ, GAC, Dentsply), which
provides a relatively constant force of
25 g, was connected between the
maxillary first molar and maxillary
central incisor teeth. For spring place-
ment around the central incisor teeth,
grooves were prepared on their sur-
faces to prevent displacement of the
0.20 mm steel wire (CrNi, 55.01.208,
Morelli, Brazil) and a thin layer of
composite resin was placed covering
it. For spring placement on the maxil-
lary first molars, composite resin was
placed at the occlusal surface, and
then the spring was positioned over
it. To avoid occlusal interference,
lower first molars were extracted.

Stereometric analysis
A single and blinded examiner, who
was previously trained and calibrated
to the purpose of the experiment, per-
formed the stereometric analysis using
a point-counting technique. Tissue
blocks were fixed in 4%
paraformaldehyde for 48 h, decalci-
fied in EDTA (10%, 0.5 M) for
3 months, and embedded in paraffin.
Serial sections of 5 lm were obtained
in the buccal–palatal direction and
stained with haematoxylin and eosin.
The images from the histological sec-
tions were taken using a light micro-
scope (LEICA microsystem GmbH,
Wetzlar, Germany) under 2009 mag-
nification. A 60 mm 9 135 mm
(8100 mm2) grid with 50 squares was
constructed using an image editing
software (Adobe Photoshop CS5, San
Jose, CA, USA) and overlaid on the
digital images obtained from the his-
tological sections. The region of inter-
est for the analysis was represented by
two grids, which were positioned at
the mesial and distal roots in the fur-
cation region of the upper first
molars. Intense tissue remodelling
occurs in the furcation region. After
orthodontic movement induction,
bone formation and resorption activ-
ity occurs in the furcation region at
the tension and compression sides of
the mesial and distal roots respec-
tively. The following structures
observed on each intersection point of
the grid were recorded: fibroblast cells
(elongated morphology), inflamma-
tory cells (rounded morphology), vas-
cular structures, and other structures
in accordance to de Souza et al.
(2011) and de Aquino et al. (2009).
To make sure the corrected structures
were being recorded, this analysis
was performed near the microscopy
to confirm in greater magnification
any doubt the examiner might have.
Despite this, if the doubt persisted,
the structure was considered as other
structure. The presence of each struc-
ture was expressed as a percentage of
the total area analysed in accordance
with Odze et al. (1996) and de Souza
et al. (2011). The total number of
each structure was divided by the
total number of intersection points
(50) and multiplied by 100 to obtain
the percentage of each structure in
the region of interest: mesial and dis-
tal sides of the roots in the furcation
region.
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Biomechanical forces and bone resorption
Micro-computed tomography analysis
(micro-CT)
Dissected maxillae of six rats per
group were carefully harvested, fixed
in 4% paraformaldehyde for 48 h,
and stored in 70% ethanol before
micro-CT scanning. Prior to micro-
CT measurement, bone samples were
washed in distilled water and stored
in 0.9% saline solution overnight for
rehydration. The bone samples were
wrapped in humid paper to prevent
artefacts as a consequence of dehy-
dration during the scanning process.
The maxillae were scanned using a
high-resolution micro-CT imaging
system (Skyscan 1076; Bruker, Kon-
tich, Belgium). The chosen scanning
parameters were: X-ray generator
operated at 50 kVp, beam current at
500 lA, 0.5 mm aluminium filtration
at an image resolution of 12.45 lm,
rotation step: 0.5°, and averaging
frame 5. The samples were placed into
the micro-CT surrounded by cylindri-
cal foam bedding under the carbon
bed. For reconstruction (NRecon
1.6.1.5; Bruker), the following param-
eters were used: defect-pixel masking:
10%; beam-hardening: 20%, smooth-
ing: 1, and individually calculated
misalignment compensation values. A
region of interest (ROI) was delimi-
tated from the root apices to the alve-
olar crest, and from the mesial root of
the first molar to the distal root of the
third molar. Teeth roots and peri-
odontal ligaments were excluded
from the ROI, as depicted in exem-
plary axial-section images in Fig. 2a.
In detail, the ROI consisted of 80
slices in the maxilla from the apical
portion of the tooth to the cement-
enamel junction. The bottom limit
started when root apices appeared
and finished at the cement-enamel
junction. The bone was manually
drawn at regular intervals with a
slice-based method every 10 planes
using an interpolated tool. Subse-
quently, roots were manually drawn
in the 80 slices selected and were
excluded from the bone. Thus, the
entire bone area without roots was
included in the ROI. Greyscale
thresholds for quantitation of struc-
tural parameters were determined
using the thresholding algorithm
(255-80). Using custom processing,
white speckles (smaller than 30 voxels)
were removed, and software-based
morphometric analysis of ROIs was
33
performed using CTAnalyzer Soft-
ware (CTAn; Bruker). The microar-
chitecture parameter evaluated was
bone volume (BV), tissue volume
(TV), and bone volume fraction (BV/
TV, %). Bone mineral density was
also measured. The calibration for
bone mineral density (BMD) mea-
surements was performed with the
two calcium hydroxyapatite (CaHA)
phantom rods containing two differ-
ent concentrations of 0.25 and 0.75 g
of CaHA/cm3 respectively. The phan-
tom rods were scanned and recon-
structed using the same parameters
for the maxilla, as described above.
The attenuation coefficient was
entered into the calibration dialogue
window in CTAn for determination of
BMD (mg/cm3). All micro-CT mea-
surements were performed according
to previous published studies (Boux-
sein et al. 2010, de Molon et al. 2015,
2016, Behrendt et al. 2016).
PCR array
PCR array was used to screen mRNA
of 84 different genes involved in the
expression of inflammatory cytokines
and their receptors (RT2ProfilerTM
PCR Array Rat Inflammatory
Cytokines & Receptors, SABio-
sciences, Qiagen, Hilden, Germany)
in gingival tissues of 1-day samples
from all experimental groups. All the
steps followed the manufacturer0 s
protocol. Data analysis was per-
formed by a Web-Based PCR Array
Data Analysis accessed by the online
link: http://www.sabiosciences.com/
pcrarraydataanalysis.php, which con-
verts values from Ct to fold change.
Six significantly upregulated genes
expressed in the OMP group in com-
parison to the C group were randomly
selected and validated by qPCR.
Quantitative real-time PCR
A total RNA of 500 ng was reverse
transcribed using High Capacity
cDNA Reverse Transcription Kit
(Life Technologies, Carlsbad, CA,
USA) and quantitative PCR was
performed using a StepOne thermo-
cycler (Life Technologies) and Taq-
Man gene expression assays
(Table 1) following the manufac-
turer’s protocol. The determination
of the relative levels of gene expres-
sion was performed using the cycle
threshold method and normalized to
34 Nogueira et al.
Table 1. Inventoried TaqMan primers and probes (TaqMan Gene Expression Assays,
Applied Biosystems)
Target gene Assay ID
GAPDH Rn 99999916-s1
CCL2 Rn 00580555-m1
CCR2 Rn 01637698-s1
IL-1b Rn 00580432-m1
IL-6 Rn 99999011-m1
ITGAM Rn 00709342-m1
TNF-a Rn 01525859-g1
the reference gene GAPDH. Results
are represented as the mean mRNA
expression from duplicate measure-
ments of four samples from different
animals at 3 days, expressed as fold
change over the expression levels of
the normalized target gene deter-
mined in cDNA samples prepared
from healthy control gingival tissues.
Multiplex immunoassay
Gingival tissue samples from day 3
had their total proteins extracted using
a detergent-based extraction buffer T-
PER – Tissue Protein Extraction
Reagent (Pierce, Rockford, IL, USA)
containing a protease inhibitor cock-
tail (Complete Protease Inhibitor
Cocktail Tablets; Roche Diagnostics,
Indianapolis, IN, USA). A panel of
cytokines were detected and quantified
using Bio-Plex ProTM Rat Cytokine 24-
plex Assay system (#171-K1001M;
Bio-Rad Laboratories, Hercules CA,
USA) to quantify the levels of EPO,
G-CSF, GM-CSF, GRO/KC, IFN-c,
IL-1a, L-1b, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-10, IL-12p70, IL-13, IL-17A,
IL-18, M-CSF, CCL2, CCL3, CCL20,
RANTES, TNF-a, and VEGF. Sam-
ples were processed and analysed fol-
lowing the instructions of the
manufacturer using a Luminex LX200
instrument (Life Technologies).
Data analysis
Statistical analyses were performed
with the software GraphPad Prism 5
(GraphPad Software Inc., San
Diego, CA, USA). For the PCR
array analysis, the p values were cal-
culated based on Student’s t-test of
the replicates 2( Delta Ct) values for
each gene in the control and treat-
ment groups. For all the other anal-
yses: qPCR, multiplex, stereometry,
bone volume fraction (BVF), and
bone mineral density (BMD), data
Accession # Amplicon (bp)
NM_017008.3 87
NM_031530.1 95
NM_021866.1 103
NM_031512.2 74
NM_012589.1 90
NM_012711.1 76
NM_012675.3 92
were synthesized using mean  stan-
dard deviation. One-way analysis of
variance test (ANOVA) followed by
the Tukey’s post hoc test was used
to determine the presence of any sig-
nificant difference between groups.
The statistical analysis was conducted
for each time point data separately.
Significant differences were consid-
ered when p < 0.05.
Results
Severity of inflammation and bone loss
There was a significant increase in the
number of vascular structures and
inflammatory cells in the OMP group.
Also, there was a decrease in the
number of fibroblasts and other struc-
tures in the diseased groups in all
experimental periods (Fig. 1). Cor-
roborating these findings, reduced
BVF and BMD level were obvious in
the OMP and P group after 1 day
because periodontitis was already
induced 5 days before baseline. Nev-
ertheless, there was no difference
between both groups at day 1. How-
ever, from day 3 on, the OMP group
exhibited smaller BVF and BMD
level as compared to P, and a signifi-
cant difference (p < 0.05) was
obtained after 15 days. The three-
dimensional sagittal micro-CT views
of the maxillary molars from each
group at 15 days are shown in
Fig. 2b. Graphs of all experimental
periods with the results of BVF and
BMD are shown in Fig. 2c.
Gene expression regulation
First, we aimed to screen the expres-
sion of 84 rat inflammatory cytoki-
nes and receptors transcripts in
gingival tissues of teeth, subjected or
not to experimental PD and/or
OTM, by performing a PCR array-
based approach. In the OMP group,
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
52 genes were upregulated out of the
84 genes evaluated, and among
those, 17 genes presented a signifi-
cant difference compared to C group
at day 1. For the P group, 44 genes
were upregulated and 17 had signifi-
cant difference compared to C. For
the OM group, 17 genes were upreg-
ulated but only one had a significant
difference compared to C (Fig. 3
and Table 2). Figure 3 shows the
heat-map with the expression of the
84 genes evaluated according to each
group and Table 1 shows the fold
regulation for all the genes signifi-
cantly (p < 0.05) upregulated in P,
OM, and OMP groups in compar-
ison to the C group. Six significantly
(p < 0.05) upregulated genes in the
OMP group as compared to the C
group out of 17 were randomly
selected for qPCR validation: CCL2,
CCR2, IL-1b, IL-6R, ITGAM, and
TNF-a. The qPCR results (Fig. 4),
confirmed that the combination of
both conditions, that is, PD and
biomechanical loading, induced a
significant upregulation of the
mRNA expression of the previously
cited genes. At day 3, the OMP
group demonstrated a significant
(p < 0.05) increase in the mRNA
expression levels of CCL2 (32.3-fold)
and IL-6 (21.9-fold) genes compared
to the C group, CCR2 (6.9-fold)
gene compared to C and OM
groups, IL-1b (57.0-fold), ITGAM
(8.1-fold), and TNF-a (4.3-fold)
genes compared to all the other
groups.
Protein synthesis regulation
In addition to gene expression analy-
sis, we sought to evaluate protein
synthesis for some upregulated genes
detected by PCR array at 3 days. A
multiplex immunoassay kit in which
a panel of 24 proteins could be
detected at the same time was used.
Gingival tissue samples produced 13
out of the 24 proteins: EPO, GRO/
KC, IL-1a, L-1b, IL-2, IL-7, IL-10,
IL-18, CCL2, CCL3, CCL20, TNF-
a, and VEGF (Fig. 5). Significant
increase in the production of nine
proteins – GRO/KC, IL-1a, L-1b,
IL-18, CCL2, CCL3, CCL20, TNF-
a, and VEGF – was detected in the
OMP group. From these nine pro-
teins, seven were significantly
increased (p < 0.05) in the OMP
group compared to P: IL-1a, L-1b,
 Biomechanical forces and bone resorption 35

Fig. 1. (a) Representative photomicrograph section with the stereometric grid positioned at the furcation region of first upper molar.
(b) Representative photomicrographs of the furcation region of first upper molars of control and experimental groups stained with
haematoxylin and eosin at 15 days. (c and d) Graphs showing the presence of vascular structures (c) and inflammatory cells (d) in
all experimental groups at 1, 3, 7, and 15 days. (e and f) Graphs showing the presence of fibroblasts (e) and other structures (f) in
all experimental groups at 1, 3, 7, and 15 days. Results are expressed as mean and  SD. *Significant difference compared to all
other groups (p < 0.05), h Significant difference compared to OM-Pressure group (p < 0.05), & Significant difference compared to
C group (p < 0.05), D Significant difference compared to OMP-Tension group (p < 0.05), / Significant difference compared to
OMP-Pressure and OMP-Tension groups, and  Significant difference compared to P, OMP-Pressure, and OMP-Tension groups.

CCL2, VEGF, CCL3, TNF-a, and
IL-18, suggesting that biomechanical
force contributes to PD in terms of
bone resorption.
Discussion
Our in vivo study provides original
evidence that mechanical loading
modulates the inflammatory response
of periodontal tissues to PD through
an increased expression of several
pro-inflammatory mediators and
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
receptors. This is the first study that
shows the involvement of some new
molecules such as C-reactive protein
(CRP), integrin alpha M (ITGAM),
integrin beta-2 (ITGB2), lymphotoxin
beta (LTB), and platelet factor 4
(PF4). These molecular changes were
associated with the increase in inflam-
matory cells and vascular structures
at the earlier periods of analysis and
more expressive bone loss at later
time points. Understanding this
mechanism will help explain and
define strategies to prevent the
aggravation of the inflammation and
bone resorption detected in the com-
bination of those clinical conditions.
In this study, after screening 84
inflammatory mediators and receptor
genes and 24 proteins, the results
demonstrated significant increase in
some cytokines, chemokines and their
receptors in the OMP group in com-
parison to all the other groups. Dif-
ferences between the OMP and P
groups deserve to be highlighted, as
36 Nogueira et al.

evaluated in this study, which is a

limitation, those results suggest that
NF-kB, p38 MAPK, Jnk, and JAK/
STAT3 signalling pathways seem to
be more activated when biomechani-
cal force is applied in a bacterial envi-
ronment. Further studies should be
conducted to verify the signalling
pathways involved in this context.
Taken together, the results strongly
suggest that the upregulated pro-
inflammatory cytokines at day 1 were
induced by experimental PD and by
its association with mechanical force,
characterizing an inflammatory con-
dition which progressed to bone
destruction at later time points, as
already expected.
Bone volume fraction and bone
mineral density levels were signifi-
cantly lower for the diseased
groups, during all time points. The
OMP group reached significant
reduction in those levels compared
to all the other groups at 15 days.
In addition, the OM group has not
presented any difference compared
to the control group with regard to
bone loss and increase in inflamma-
tion, suggesting that the orthodon-
tic appliance did not cause any
damage to the periodontal tissues
by itself.
Several new molecules were sig-
nificantly upregulated in the OMP
group (Table 2), as CRP, LTB and
PF4. CRP is a plasma protein con-
sidered an acute-phase marker for
inflammation (Black et al. 2004),
regulated by cytokines (Ebersole &
Cappeli 2000). High levels of CRP
have been found in the serum or gin-
gival crevicular fluid of periodontal
Fig. 2. (a) Representative axial-section images of the region of interest (ROI) showing patients (Fredriksson et al. 1999,
the presence of teeth roots and periodontal ligament on the left and the absence of Slade et al. 2000, Noack et al. 2001,
teeth roots and periodontal ligament on the right. (b) Three-dimensional sagittal Kumar et al. 2013). LTB is a mole-
micro-CT views of maxillary molars of different animals from each experimental cule produced by T lymphocytes and
group (C, OM, P, and OMP) at 15 days. Tooth movement is shown in the OM and has potent pro-inflammatory activ-
OMP groups. (c) Comparison of bone volume fraction (BVF) percentage and bone ity, playing key roles in periodontal
mineral density (BMD) measured in a selected ROI of the maxillary first molars using tissue breakdown. PF4, known as
micro-CT. Results are expressed as mean  SD. *Significant difference compared to CXCL4, is a cytokine released by
all other groups (p < 0.05), h Significant difference compared to C and OM groups
(p < 0.05), & Significant difference compared to C group (p < 0.05).
activated platelets, which has the
function to attract neutrophils,
monocytes, and lymphocytes (Gleiss-
they suggest that the association of IL-1b, IL-6R, and ITGAM were even ner et al. 2010).
both clinical situations, periodontitis more upregulated when biomechani- Another group of mediators eval-
and OTM, results in an increase cal force was associated with peri- uated by PCR array was the inte-
in periodontal damage by the mod- odontitis in the OMP group. This grins, proteins expressed on cells
ulation of inflammatory mediators. suggests that biomechanical force surface. ITGAM, also known as
Table 2 shows that several pro-in- modulated the increase in some macrophage-1 antigen (Mac-1), is an
flammatory molecules were upregu- pro-inflammatory molecules during innate immune receptor expressed on
lated in the P group alone, and some periodontitis progression. Although leucocytes. Increase in the number of
of them such as CCR5, CRP, IL-1a, signalling pathways were not ITGAM-positive cells has been
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Fig. 3. Heat map analysis showing the gene expression profile of gingival tissues in
response to biomechanical forces and periodontal inflammation driven by orthodontic
movement and experimental periodontal disease respectively. Gene expression magni-
tude was identified by colours: from light to dark-green represented genes with mini-
mum to average expression, and from dark to light red, represented genes with
average to maximum expression.
detected in rat PDL subjected to
experimental OTM (Vandevska-
Radunovic et al. 1997). Further-
more, ITGAM expression was
correlated with the increased bone
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Biomechanical forces and bone resorption
loss and increased expression of pro-
inflammatory mediators (Liang et al.
2010). Our data showed significantly
higher levels of ITGAM in the OMP
group compared to the other groups.
37
The chemokines CCL2 and
CCL3 were significantly upregulated
in the OMP group. The CCL2, also
known as monocyte chemotactic
protein-1 (MCP-1), is secreted by
osteoblasts in response to mechani-
cal force application and to inflam-
matory processes in vivo (Rahimi
et al. 1995, Kurtis et al. 2005, Lu &
Kang 2009, Pradeep et al. 2009a,
Liou et al. 2013), playing an impor-
tant regulatory role in bone remod-
elling. CCL3, also known as
macrophage inflammatory protein 1
a (MIP1a), recruits and activates leu-
cocytes during acute inflammatory
response and, osteoclast precursor
cells and osteoblasts for bone
remodelling (Graves & Cochran
2003, Andrade et al. 2009). CCL3
expression is increased in bone and
soft periodontal tissues under
mechanical force (Yu et al. 2004,
Yano et al. 2005).
Several cytokines were evaluated
in this study, which were highly
upregulated in the OMP group. IL-
1a, IL-1b, and TNF-a are the most
abundant pro-inflammatory cytoki-
nes detected in human gingival
crevicular fluid and animal periodon-
tal tissues during OTM and PD pro-
gression (Gorska et al. 2003, Graves
& Cochran 2003, Bletsa et al. 2006,
Dudic et al. 2006, Krishnan & Davi-
dovitch 2006). In this study, a pro-
longed inflammatory process in the
animals from OMP group initiated
by PD and extended by biomechani-
cal forces, was probably responsible
for the failure to form new bone fol-
lowing bone resorption. Evidence for
this was demonstrated in in vivo
studies (Al-Mashat et al. 2006, Liu
et al. 2006). They showed that a
brief event of inflammatory process
leads to bone resorption followed by
bone formation. However, when the
inflammatory process persists for
longer period by another condition
or disease, the ability to form an
adequate amount of new bone is
reduced. Also, the injection of IL-1b
and TNF-a result in bone resorption
and uncoupling bone formation
from bone resorption (Nguyen et al.
1991). Therefore, inflammation
changes osteoblasts’ function and
limits bone formation in addition to
stimulation of osteoclasts production
and bone resorption. Production of
VEGF was significantly increased in
the OMP group. This cytokine not
38 Nogueira et al.

Table 2. Fold regulation compared to control group of significantly expressed genes evalu- presented significant increase in the
ated by PCR array in rat gingival tissues from 1-day experimental groups OM, P, and OMP production of VEGF in periodontal
Gene Fold regulation P value tissues consistent with the histologi-
cal increase in vascular structures,
OM P OMP OM P OMP when compared to the other groups.
VEGF has been considered to play a
CXCR5 1.67 3.05 3.50 0.23 0.09 0.03 role in osteoarthritis pathogenesis
CASP1 2.30 3.32 2.60 0.07 0.0008 0.051 (Oh et al. 2014) and in cartilage
CCL12 3.06 13.43 9.98 0.22 0.01 0.03
pathologies (Beckmann et al. 2014),
CCL2 2.67 24.19 32.35 0.50 0.05 0.03
CCL21 1.65 2.58 1.41 0.053 0.004 0.25
suggesting that our results could be
CCL5 1.80 5.62 15.48 0.32 0.0002 0.13 extrapolated to other clinical situa-
CCL7 2.11 23.37 7.31 0.34 0.001 0.07 tions involving inflammatory
CCR1 6.90 13.29 12.84 0.01 0.02 0.01 reactions.
CCR2 1.17 4.52 6.85 0.58 0.12 0.001 The ligature-induced PD experi-
CCR5 1.65 1.78 14.20 0.34 0.30 0.0004 mental model used in this study has
CRP 2.08 1.18 6.43 0.19 0.68 0.04 been extensively used for a better
CX3CR1 1.60 4.62 1.52 0.16 0.01 0.18 understanding of inflammatory dis-
CXCL5 1.37 3.47 3.17 0.35 0.04 0.01 ease pathogenesis, highlighting the
IL-10 1.06 4.35 2.67 0.71 0.01 0.05
1L-1a 1.02 7.03 41.75 0.52 0.20 0.03
molecular mechanisms involved. It is
IL-1b 1.53 11.32 57.01 0.35 0.11 0.04 well known that after 5 days of
IL-2RB 1.19 7.69 8.01 0.88 0.003 0.13 experimental PD induction in rats, a
IL-2RG 1.09 2.76 1.23 0.96 0.01 0.54 significant difference in bone loss
IL-6R 2.46 9.29 21.84 0.45 0.10 0.03 compared to control animals is
ITGAM 1.61 5.42 8.12 0.42 0.06 0.002 observed (Holzhausen et al. 2002,
ITGB2 3.18 26.27 13.30 0.71 0.04 0.04 Xie et al. 2011, Boas Nogueira et al.
LTB 1.33 3.46 2.12 0.39 0.01 0.04 2013). In our study, the experimental
PF4 4.89 5.19 4.38 0.09 0.01 0.008 periodontitis was present 5 days
SPP1 2.16 8.34 9.53 0.55 0.04 0.08
before the induction of orthodontic
TNF-a 0.95 2.21 4.30 0.65 0.03 0.02
movement until the day of killing, in
Bold values mean significantly difference compared to control group. a total of 20 days.
This study was conducted in an
only mediates angiogenesis and been detected in periodontal tissues attempt to identify molecules
increases vascular permeability, but during OTM (Kaku et al. 2008, involved in the destructive mechanism
also has a role in bone resorption Miyagawa et al. 2009) and PD (R in diseased tissues submitted to
and formation. Increased VEGF has et al. 2014). The OMP group mechanical forces. Relevant

Fig. 4. Regulation of gene expression in rat gingival tissues by inflammatory and/or mechanical stimulus at 3 days, as analysed by
qPCR. Graphs with the mRNA expression of CCL2 (a), CCR2 (b), IL-1b (c), IL-6 (d), ITGAM (e), and TNF-a (f) are shown.
Results are expressed as mean and  SD. *Significant difference compared to all other groups (p < 0.05), h Significant difference
compared to C and OM groups (p < 0.05), & Significant difference compared to C group (p < 0.05).
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
 Biomechanical forces and bone resorption 39

Fig. 5. Regulation of protein synthesis in rat gingival tissues by inflammatory and/or mechanical stimulus at 3 days, as analysed by
multiplex immunoassay. Graphs with the protein levels (pg/ml) are shown. Results are expressed as mean and  SD. *Significant
difference compared to all other groups (p < 0.05), M Significant difference compared to C and P groups (p < 0.05), ♦ Significant
difference compared to P group (p < 0.05), & Significant difference compared to C group (p < 0.05).

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
40 Nogueira et al.
inflammatory mediators involved in a
destructive process were highlighted
and evidenced, suggesting a role in
the pathomechanism of the intense
inflammation and bone resorption
presented in the OMP group. There-
fore, the results suggest the need of
periodontal treatment before starting
orthodontic therapy, as well as main-
taining the periodontal health
throughout the course of this therapy,
since a significant increase in inflam-
mation and destruction of periodon-
tal tissues has been demonstrated
when orthodontic force is applied in
periodontal diseased tissues.
In summary, this study demon-
strates that mechanical loading mod-
ulates the response of periodontal
tissues to PD by increasing the
expression of pro-inflammatory
mediators, which leads to increased
bone resorption.
Acknowledgements
The authors thank FAPESP (São
Paulo Research Foundation, Grants:
2010/07771-4, 2011/13752-5) for
financial support and the research
technicians Ana Claudia G. C. Mir-
anda and Leandro Alves dos Santos
for technical support. The authors
declare no potential conflicts of
interest with respect to the author-
ship and/or publication of this
article.
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Address:
Joni Augusto Cirelli
Department of Diagnosis and Surgery
School of Dentistry at Araraquara
Univ Estadual Paulista (UNESP)
Rua Humait a, 1680, 2° andar
CEP 14801-903
Araraquara, SP, Brazil
E-mail: cirelli@foar.unesp.br
compared to each of those clinical
conditions alone due to the increased
expression and synthesis of proin-
flammatory mediators and receptors.
Practical implications: Periodontal
health is a prerequisite for
orthodontic treatment in order to
avoid periodontal destruction.