Journal of Photochemistry and Photobiology B: Biology 135 (2014) 65–74
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Histological analysis of the periodontal ligament and alveolar bone
during dental movement in diabetic rats subjected to low-level laser
therapy
Luiz Guilherme Martins Maia a,1, Angela Valéria Farias Alves b,2, Talita Santos Bastos b,2,
Lucas Sandes Moromizato b,2, Isabel Bezerra Lima-Verde b,2, Maria Amália Gonzaga Ribeiro c,3,
Luiz Gonzaga Gandini Júnior d,4, Ricardo Luiz Cavalcanti de Albuquerque-Júnior a,b,⇑
a
School of Dentistry, University Tiradentes (UNIT), Rua Terêncio Sampaio, 309, Grageru, Aracaju 49025700, Sergipe, Brazil
Laboratory of Morphology and Structural Biology, Science and Technology Institute (ITP), Avenida Murilo Dantas, 300, Prédio do ITP, Farolândia, Aracaju 49032-490, Sergipe, Brazil
c
Department of Dentistry, Federal University of Sergipe (UFS), Avenida Cláudio Batista, 54, Sanatório, Aracaju 49000-000, Sergipe, Brazil
d
Department of Dentistry, State University Júlio de Mesquita (UNESP), Rua Humaitá, Centro, 1680, Araraquara 14801-903, São Paulo, Brazil
b
a r t i c l e
i n f o
Article history:
Received 14 January 2014
Received in revised form 30 March 2014
Accepted 31 March 2014
Available online 12 April 2014
Keywords:
Diabetes mellitus
Periodontal ligament
LLLT
a b s t r a c t
Objective: The purpose of this research was to evaluate the histological changes of the periodontal ligament and alveolar bone during dental movement in diabetic rats subjected to low level laser therapy
(LLLT).
Methods: The movement of the upper molar was performed in 60 male Wistar rats divided into four
groups (n = 15): CTR (control), DBT (diabetic), CTR/LT (irradiated control) and DBT/LT (irradiated diabetic).
Diabetes was induced with alloxan (150 mg/kg, i.p.). LLLT was applied with GaAlAs laser at 780 nm (35 J/
cm2). After 7, 13 and 19 days, the periodontal ligament and alveolar bone were histologically analyzed.
Results: The mean of osteoblasts (p < 0.01) and blood vessels (p < 0.05) were significantly decreased in DBT
compared with CTR at 7 days, whereas the mean of osteoclasts was lower at 7 (p < 0.001) and 13 days
(p < 0.05). In DBT/LT, only the mean of osteoclasts was lower than in CTR (p < 0.05) at 7 days, but no difference was observed at 13 and 19 days (p > 0.05). The collagenization of the periodontal ligament was
impaired in DBT, whereas DBT/LLT showed density/disposition of the collagen fibers similar to those
observed in CTR.
Conclusions: LLLT improved the periodontal ligament and alveolar bone remodeling activity in diabetic
rats during dental movement.
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
Diabetes mellitus (DM) is one of the most common endocrine
disorders. It is characterized by persistently raised blood glucose
⇑ Corresponding author at: Laboratory of Morphology and Structural Biology,
Science and Technology Institute (ITP), Avenida Murilo Dantas, 300, Prédio do ITP,
Farolândia, Aracaju 49032-490, Sergipe, Brazil. Tel.: +55 79 32182115/32170192;
fax: +55 79 32182190.
E-mail addresses: [email protected] (L.G.M. Maia), angela.
[email protected] (A.V.F. Alves), [email protected] (T.S. Bastos),
[email protected] (L.S. Moromizato), [email protected]
(I.B. Lima-Verde), [email protected] (M.A.G. Ribeiro), [email protected]
(L.G. Gandini Júnior), [email protected] (R.L.C. Albuquerque-Júnior).
1
Tel.: +55 79 32170192.
2
Tel.: +55 79 32182115; fax: +55 79 32182190.
3
Tel.: +55 79 21051823.
4
Tel.: +55 16 33016300; fax: +55 16 33016328.
http://dx.doi.org/10.1016/j.jphotobiol.2014.03.023
1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
levels (hyperglycaemia), resulting from deficiencies in insulin
secretion, insulin action, or both [1]. Chronic hyperglycaemia is
associated with long-term damage, dysfunction, and failure of
various organs, such as retinopathy, nephropathy, peripheral
neuropathy, and autonomic neuropathy, causing gastrointestinal,
genitourinary, and cardiovascular symptoms, as well as sexual dysfunction [2].
It has been reported that moderate to severe damage to the glucose metabolism directly affects the response of bone and connective tissues to injury [3,4]. Orthodontic treatment in adult diabetic
patients is thus usually complicated by oral problems such as periodontal degradation and bone loss [5]. Although only a few studies
have assessed the histological changes that take place in the periodontal ligament and alveolar bone during dental movement in
diabetic experimental animals, they provide evidence that chronic
hyperglycaemic status might impair the periodontal ligament
response and bone remodeling during orthodontic treatment [6].
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Lasers emit a highly concentrated, non-invasive, non-ionizing
radiation that, when in contact with different tissues, promotes
thermal, photochemical and nonlinear effects [7]. Several studies
have indicated that C (LLLT) modulates different biological
activities, such as anti-inflammatory activity [8,9], angiogenesis
[10,11] and collagen synthesis [12,13]. In particular, the acceleration of bone regeneration by laser treatment has been a focus of
studies [14,15].
It has previously been demonstrated that LLLT can accelerate
tooth movement, as well as alveolar bone remodeling in experimental studies [16,17] and clinical trials [18,19]. However, little
is known about the effect of laser irradiation on the dynamics of
dental movement in diabetic subjects. Therefore, the present study
was designed to assess the histological changes taking place in the
periodontal ligament and alveolar bone during dental movement
in diabetic rats subjected to laser irradiation.
2.4. Experimental tooth movement
At the end of 2 months, anaesthesia was induced with intraperitoneal administration of ketamine–xylazine (100 mg/kg–5 mg/kg),
and an appliance exerting force to widen the space between the
upper central incisors was fitted to both groups. For mesial movement of the upper left 1st molar, the wire end of a 7.0-mm length
of NiTi closed-coil spring (wire size: 0.7 mm, diameter: 1/12 in.,
Orthometric, Marilia, SP, Brazil) was ligated with the maxillary
1st molar cleat using a 0.010-in. stainless steel ligature wire (Morelli, Sorocaba, SP, Brazil). The other side of the coil spring was also
ligated, with the holes in the maxillary incisors drilled laterally just
above the gingival papilla with a #1/4 round bar, using the same
ligature wire (Fig. 1). The orthodontic force exerted by the appliance was 50 g at the start of the experiment. Tooth movement
was performed for 19 days (day 0–19).
2.5. Low-level laser therapy procedures
2. Material and methods
2.1. Ethical perspectives
The ethical principles of the COBEA (Brazilian College for Animal Experimentation) for experiments in animals were applied in
this study. The institutional review board approved the study
(approval n° 341208). The study was carried out at the biotherium
and the Laboratory of Morphology and Structural Biology of Tiradentes University (Aracaju/SE, Brazil).
2.2. Biological assay
Sixty adult male rats (Novergiculs albinus, Wistar lineage),
weighing 250 ± 30 g, were randomly assigned into four experimental groups (n = 15) (Table 1). Animals were kept in plastic cages
with wood shaving bedding (replaced daily), at a controlled temperature of 22 °C, and a 12 h light/dark cycle, with water and food
(diet LabinaÒ, Purina, Sao Paulo, Brazil).
2.3. Alloxan-induced diabetes model
Diabetes status was induced by a single intraperitoneal injection of 150 mg/kg monohydrated alloxan (Sigma, St. Louis, MO,
USA) dissolved in sterile 0.9% saline. After 12 h, a 10% glucose solution was offered to the animals to prevent hypoglycaemia. Blood
samples were collected from the tail vein of the animals after
72 h in order to assess the plasma glucose levels via the glucoseoxidase enzymatic method, using Accu-Chek Advantage (Boehringer, Germany). Animals with glucose levels above 200 mg/dL were
included in the diabetic group. The examinations were repeated
every 7 days to confirm the maintenance of glucose levels. Any animals showing reversion of the signs of diabetes (glucose levels
below 200 mg/dL) were excluded from this study. The animals in
the nondiabetic group (CTR and CTR/LT) received an equivalent
volume of citrate buffer. The orthodontic device was applied
6 weeks after diabetes was induced.
Table 1
Distribution of the animals in the experimental groups according to treatment.
Groups
Pre-treatment
Low level laser therapy (energy density)
CTR
DBT
CTR/LT
DBT/LT
Citrat buffer
Alloxan (150 mg/kg)
Citrat buffer
Alloxan (150 mg/kg)
0 J/cm2
35 J/cm2
0 J/cm2
35 J/cm2
Animals were subjected to transcutaneous irradiation using a
previously calibrated semi-conductor diode laser GaAlAs (Twin
Laser, MMOptics, São Paulo, Brazil) with continuous emission at
780 nm wavelength for 60 s (20 s each point). The output power
used was 70 mW, with a focal spot of 0.04 cm2, and a power density of 1.75 W/cm2. The total energy per session was estimated as
4.2 J (1.4 J/point) and the energy density was 35 J/cm2 distributed
between three different equidistant points in the root portion.
The first irradiation was performed immediately after the activation procedures, and then performed every 48 h over the course
of 7 days.
2.6. Procedures for histomorphological analysis of the specimens
After 7, 13 and 19 days, animals were euthanized in a CO2
chamber for post-mortem removal of the maxillae. Tissue specimens were fixed in buffered formaldehyde (10%, pH 7.4) for 48 h,
decalcified in 5% nitric acid for 72 h, dehydrated in increasing ethyl
alcohol solutions, and diaphanized in xylol for inclusion in paraffin.
Subsequently, ten histological sections (5 lm thick) were obtained
and stained in hematoxylin-eosin for analysis using a light microscope (Olympus CX31 optic microscope) by three trained
observers.
2.7. Histomorphological analysis of the periodontal ligament and
alveolar bone
The intensity of the inflammatory response was assessed in histological sections as follows: 0 (lack of inflammatory reaction); 1
(inflammatory cells representing less than 10% of the cell population observed within the wound area); 2 (inflammatory cells representing between 10% and 50% of the cell population observed
within the wound area); and 3 (inflammatory cells representing
more than 50% of the cell population observed within the wound
area). Moreover, the inflammatory profile (IP) was classified as
acute (predominance of polymorphonuclear cells) or chronic (predominance of mononuclear cells), and graded as slight/absent,
moderate or severe.
2.8. Quantitative analysis of the osteoblast (OsTB)/osteoclast (OsTC)
and blood vessel (BvC) count
Counting of osteoblasts, osteoclasts and blood vessels was performed using an image analysis system (Imagelab). All images
were sent to a PC using an analogue video camera (PAL system),
after being converted to the RGB (red–green–blue) system necessary for digitizing and processing the sections. Five histological
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Fig. 1. Installation of the orthodontic appliance in the experimental animals. (a), (b), (c) Image of the operative field, showing the first molar (d).
fields (200 magnification) of the tension and pressure areas of the
periodontal ligament of the 1st molar were used to determine the
mean number of osteoblasts and osteoclasts, respectively. To count
the blood vessels, images of the tension and pressure areas were
analyses. The images were recorded and automatically processed
to find the cell density (CD) in each reference area (RA). Data were
expressed as mean ± SD.
2.9. Quantitative analysis of collagenization rate (CR)
The collagen deposition rate (CR) in the periodontal ligament
was determined by optical density in the image analysis system
in different randomly selected fields. The system used consisted
of a CCD Sony DXC-101 camera, attached to an Olympus CX31
microscope, from which the images were sent to a monitor (Trinitron Sony). By means of a digitizing system (Olympus C-7070
WIDEZOOM) the images were loaded onto a computer (Pentium
133 MHz), and processed using ImageLab software. Ten fields per
case were analyzed at a magnification of 1000. The thresholds
for collagen fibers were established for each slide, after enhancing
the contrast up to a point at which the fibers were easily identified
as birefringent (collagen) bands. The area occupied by the fibers
was determined by digital densitometric recognition, by adjusting
the threshold level of measurement up to the different color densities of the collagen fibers. The area occupied by the fibers was
divided by the total area of the field. The results were expressed
as the percentage of the periodontal ligament area occupied by
the collagen fibers.
2.10. Statistical analysis
Data obtained in the IP and HC analysis were analysed using the
Kruskal–Wallis test, followed by the post hoc Dunńs test. Data
obtained in the OSTB, OsTC, BvC and CR counts were analysed by
ANOVA followed by the post hoc Tukey’s test. Differences between
groups were regarded as significant when p < 0.05.
3. Results
3.1. Descriptive analysis of histological changes in the periodontal
ligament
After 7 days (Fig. 2), exuberant granulation tissue and multiple
erosive alterations, consistent with howship lacunae, most of them
containing osteoclasts, were seen along the alveolar wall in CTR,
CTR/LT and DBT/LT, particularly in the cervical thirds of the periodontal ligament. However, intense oedematous changes in the
connective tissue associated with chronic inflammatory infiltrate,
and with sparse vascularization, were observed in DBT. It should
be emphasized that the collagen fibers were more apparent and
were well distributed in a parallel organization in the LLLT-treated
groups (CTR/LT and DBT/LT). In addition, coagulative necrosis of
the periodontal ligament was seen in one case of DBT, but not in
the other groups.
After 13 days (Fig. 3), all groups showed a remarkable reduction
in the vascular network and a substantial increase in the fibrous
component. Osteoclastic activity was still present, but was lower
than at 7 days. The collagen fibers had a gross and thick appearance, as well as a parallel arrangement in CTR, CTR/LT and DBT/
LT. In the non-irradiated diabetic animals (DBT), however, the collagen fibers appeared to be thinner with a non-uniform distribution throughout the periodontal ligament.
After 19 days (Fig. 4), the periodontal ligament of both CTR and
CTR/LT presented similar morphological features, in the form of a
cell-rich and moderately vascular connective tissue, associated
with thick, but delicate, parallel collagen fibers. In diabetic animals,
there were parallel bundles of thin collagen fibers associated with
a high content of flat spindle-shaped cells (consistent with fibroblasts), and this fibrous component was more extensive in DBT/
LT than in DBT.
Data on the intensity of the inflammatory response (IR) found
throughout the periodontal ligament is presented in Table 2. The
IR values observed in irradiated non-diabetic animals (CTR/LT) were
lower than in all other groups at 7 and 13 days (p < 0.05). Although
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Fig. 2. Histological changes of the periodontal ligament in the studied groups. (A) CTR, (B) CTR/LT, (C) DBT and (D) DBT/LT. Note the well-developed fibrovascular granulation
tissue in CTR, CTR/LT and DBT/LT, and inflammatory infiltrate and oedema of the connective tissue in DBT. (E) Coagulative necrosis in DBT (7 days – HE, 200).
at 7 days diabetic animals (DBT) showed a significantly more
intense IR than the control group (CTR) (p < 0.05), irradiated diabetic rats (DBT/LT) showed no significant difference in comparison
with CTR. After 13 and 19 days, leukocyte infiltration progressively
decreased, and none of the groups presented significant differences
regarding the intensity of the inflammatory response (p > 0.05).
3.2. Assessment of the mean number of osteoblasts (OsTB)
The results of the histomorphometric study of osteoblast
number (OsTB) in the periodontal cortex of the alveolar wall are
presented in Fig. 5. The values of OsTB were significantly lower
in DBT in comparison with CTR at 7 (p < 0.001) and 13 days
(p < 0.01), but not at 19 days (p > 0.05). Irradiated diabetic animals
presented a significant increase in OsTB compared to non-irradiated animals at 7 and 13 days (p < 0.05), and although DBT/LT still
had OsTB values lower than CTR at 7 days (p < 0.05), no significant
difference was observed between these two groups at 13 days
(p > 0.05). At 19 days, no significant differences in OsTB were
observed between the experimental groups (p > 0.05).
3.3. Assessment of the mean number of osteoclasts (OsTC)
The results of the histomorphometric study of osteoclast
number (OsTC) in the periodontal cortex of the alveolar wall are
presented in Fig. 6. After 7 days, a significant decrease in OsTC
was observed in diabetic compared with control animals
(p < 0.001). Although the application of LLLT in diabetic animals
(DBT/LT) promoted a slight increase in OsTC, it was not statistically
significant in relation to non-irradiated animals (p > 0.05). In
addition, the increase in OsTC in CTR/LT compared with CTR was
not significant (p > 0.05). At 13 days, the OsTC in diabetic animals remained significantly lower than in the control group
(p < 0.01), although in laser-irradiated animals (DBT/LT) this difference was not significant (p > 0.05). At 19 days, no significant differences in OsTC were observed between the experimental groups
(p > 0.05).
3.4. Assessment of the mean number of blood vessels
The results of the histomorphometric study of blood vessel
count (BVC) in the periodontal ligament on the pressure side are
presented in Fig. 7. At 7 days, the blood vessel count was significantly lower in diabetic animals compared with the control group
(p < 0.05). LLLT promoted a significant increase in the BVC in nondiabetic animals (p < 0.01). In addition, there was no difference
between the BVC observed in laser-irradiated diabetic animals
and control animals (p > 0.05). Lower numbers of blood vessels
were observed at 13 and 19 days, and no significant differences
were found between the experimental groups (p > 0.05).
L.G.M. Maia et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 65–74
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Fig. 3. Histological changes of the periodontal ligament in the studied groups. (A) CTR, (B) CTR/LT, (C) DBT and (D) DBT/LT. Note the parallel disposition of the collagen
fibersin CTR, CTR/LT and DBT/LT, and the disorganization of the collagen architecture in DBT (13 days – HE, 200).
Fig. 4. Histological changes of the periodontal ligament in the studied groups. (A) CTR, (B) CTR/LT, (C) DBT and (D) DBT/LT. Note the recovery of the normal appearance of the
periodontal ligament in CTR and CTR/LT, whereas it appears less remodelled in DBT and fibrotic in DBT/LT (19 days – HE, 200).
3.5. Quantitative analysis of collagenization rate (CR)
Collagen fibers were identified due to their intense golden
birefringence under polarized light (Fig. 8), and presented as
fibrous structures arranged in a parallel fashion, connected on
one side to the dental cement and to the alveolar bone on
the other. As shown in Fig. 9, the collagenization rate (CR)
was significantly lower in diabetic animals (DBT) than in
controls (CTR) at 7 (p < 0.001), 13 (p < 0.001) and 19 days
(p < 0.01). Laser irradiation induced a significant increase in
CR in CTR/LT and DBT/LT in comparison with the respective
controls (CTR and DBT) at 7 (p < 0.01 and 0.01) and 13 days
(p < 0.05 and 0.001). In addition, the CR values observed in irradiated diabetic animals (DBT/LT) did not differ significantly
from those in non-diabetic animals (CTR) throughout the experimental period (p > 0.05).
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Table 2
Assessment of the mean scores of the histological grading of the intensity of the
inflammatory response.
Experimental period
(days)
Experimental groups
CTR
CTR/LT
DBT
DBT/LT
7
13
19
2.50 ± 0.5a
0.75 ± 0.3a
0.00 ± 0.0a
1.16 ± 0.4b
0.70 ± 0.2b
0.00 ± 0.0a
3.00 ± 0.0c
1.00 ± 0.4a
0.00 ± 0.0a
2.20 ± 0.6a
0.90 ± 0.4a
0.00 ± 0.0a
Different letters (a, b, c) in the same line represent significantly different values
(p < 0.05). All values are mean ± SD.
4. Discussion
In this study, we found substantial changes in the alveolar bone
and periodontal ligament of diabetic animals in response to the
application of an orthodontic force. Such changes were represented
by persistence of the inflammatory response, impairment of granulation tissue formation (development of neoformed capillary vessels and deposition of type I collagen fibers), a reduction in both
osteoblastic/osteoclastic differentiation and a reduced number of
howship lacunae. Similar morphological and morphometric findings were recently reported by Villarino et al. [6].
The mechanisms responsible for the enhanced inflammatory
response in diabetic animals have not been conclusively established. A possible association with higher levels of TNF, a product
of adipose tissue, which is increased in both humans and animal
models of type II diabetes, has been proposed. Thus, the enhanced
production of TNF dysregulates the cytokine networks and potentiates the inflammatory response. In addition, the production of
advanced glycation end products that are present at higher levels
in diabetic individuals seems to enhance oxidative stress and
amplify inflammatory events in tissues [20].
The impairment of angiogenesis in diabetic animals has previously been reported [21]. Although the precise cause of such
impairment has not been fully clarified, it may be the result of a
decrease in the amount of growth factors that are essential for
wound healing, including FGF-2 and PDGF [22].
On the other hand, the existence of a bone–pancreas endocrine
loop through which insulin signaling in osteoblasts ensures osteoblast differentiation and stimulates osteocalcin production, which
in turn regulates insulin sensitivity and pancreatic insulin secretion, has recently been proposed [23]. Therefore, it is possible that
the significant decrease in osteoblast number observed in this
study might have been a result of the substantial reduction in insulin levels associated with the aloxan-induced pancreatic damage.
Interestingly, the same mechanism would secondarily lead to
reduced levels of calcitonin, an inhibitor of bone resorption, and
should result in increased osteoclastic activity. However, it has
recently been reported that osteoblasts are one the major sources
of the receptor activator for nuclear factor kappa ligand (RANKL)
[24], a molecule widely required for osteoclast formation from
the peripheral blood-derived mononuclear precursors. Hence, the
decrease in osteoblast differentiation would be ultimately responsible for reduced osteoclast formation, as observed in this study.
We found that the application of LLLT during dental movement
reversed some of the deleterious effects associated with diabetic
status. The major biological events modulated by laser irradiation
were closely associated with a less intense inflammatory response,
enhanced stromal cell proliferation, involving osteoblasts and
osteoclasts, improved angiogenesis and granulation tissue formation, and better architectural reorganization of the periodontal collagen fibers.
It has previously been reported that laser irradiation causes an
increase in the number of more differentiated osteoblastic cells
Fig. 5. Assessment of the mean of osteoblast number (OsTB) in the studied groups over the time course of the experiment. Columns marked with different letters represent
significantly different values (ANOVA, p < 0.05).
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71
Fig. 6. Assessment of the mean of osteoclast number (OsTC) in the studied groups over the time course of the experiment. Columns marked with different letters represent
significantly different values (ANOVA, p < 0.05).
and bone nodule formation in vitro, by stimulating the cellular proliferation of osteoblast lineage nodule-forming cells and cellular
differentiation [25]. On the other hand, increased osteoblastic differentiation might promote RANKL release by osteoblasts, resulting
in improved osteoclast formation [24]. Moreover, it has also been
suggested that laser irradiation might stimulate the biological
events associated with osteoclast formation, such as bone marrow-derived mononuclear osteoclast precursors of (preosteoclast)
fusion to mature osteoclasts [16]. As the speed of tooth movement
is highly dependent on the dynamics of bone remodeling, as a
result of the bone resorption and formation balance, it is possible
that LLLT improved tooth movement in diabetic rats as a result
of increased osteoblast/osteoclast differentiation. However, it is
essential to emphasize that, despite the laser-induced histological
improvement in the osteoblast and osteoclast number, the application of LLLT was not able to fully reverse the deleterious effects of
the diabetic status on the differentiation of such cells, since at
7 days, both OsTC and OsTB remained lower than in CTR.
Angiogenesis and consequently granulation tissue formation is
one the major biological events related to the healing process of
the periodontal ligament during tooth movement. Although the formation of blood vessels was impaired in diabetic rats, LLLT
promoted the functional recovery of angiogenesis, and allowed a
suitable granulation tissue formation comparable to that in nondiabetic rats. The stimulatory role played by LLLT in angiogenesis
and granulation tissue during wound healing has previously been
reported during wound healing [26,11] and bone repair [27]. Laser
light irradiation increases the production of nitric oxide (NO),
which in turn can modulate the production and secretion of several
cytokines, such as vascular endothelial growth factor (VEGF) and
platelet-derived growth factor (PDGF), both of which are able to
mediate endothelial proliferation and blood vessel formation, [28]
which could support the histological findings regarding the periodontal vascular content observed in this study. The fact that significant results were limited to 7 days is consistent with the dynamics
of wound healing, since the proliferative vascular events occur in
the early stages of the connective tissue healing process [29].
Finally, improved collagenization and better spatial organization of the collagen fibers were also observed in the periodontal ligament of diabetic rats after laser irradiation. Consistent with our
findings, previous reports have demonstrated that LLLT is able to
improve collagen deposition and remodeling during wound healing
[13,11,30], likely as a result of biomodulatory effects on fibroblast
function. It has been previously demonstrated that increased
expression of MMP-1 and Col-III and decreased expression of
Col-I in periodontal dental ligament (PDL) of diabetic rats. After
the orthodontic induction, osteoclast action was delayed, and
higher Col-III/Col-I and MMP-1/TIMP-1 ratios persisted in the diabetes group compared with the normal group [31]. As observed in
the current study, those data suggest that under mechanical forces,
diabetes prolonged duration of degradation of PDL and remodeling
of PDL and resorption of alveolar bone. More recently, molecular
studies have pointed at a possible role played by LLLT on collagen
(Col-I) and tissue inhibitor of metalloproteinase (TIMP-1) on the
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Fig. 7. Assessment of the mean number of blood vessels (BVC) in the studied groups over the time course of the experiment. Columns marked with different letters represent
significantly different values (ANOVA, p < 0.05).
Fig. 8. Collagen fibers of the periodontal ligament seen under polarized light (arrows) in seven (A–D), 13 (E–H) and 19 days (I–J). Control group (CTR) is expressed in the first
column (A, E, I), non-diabetic irradiated group (CTR/LT) in the second (B, F, J), diabetic group (DBT) in the third (C, G, K) and irradiated diabetic group (DBT/LT) in the fourth
ones (D, H, L) (sirius red, 200 magnification; white bar – 250 lm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)
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73
Fig. 9. Assessment of the mean of collagenization rates (CR) in the studied groups over the time course of the experiment. Columns marked with different letters represent
significantly different values (ANOVA, p < 0.05).
compression and tension sides of orthodontically moved teeth.
Overexpression of five MMP mRNAs was observed in both relapse
and retention groups. However, TIMP-1 immunoreactivity was
inhibited by LILT in both groups, whereas Col-I immunoreactivity
was increased by LILT only in the retention group. These data suggest that, although LILT might act differently on the stability after
orthodontic treatment according to additional retainer wearing or
not, the photobiomodulation process might improve collagen
remodeling [32]. Therefore, it is possible to suggest that LLLT is able
to stimulate collagen deposition and improve the architectural
arrangement of the periodontal fibers in diabetic rats during tooth
movement.
5. Conclusion
The present study suggests that LLLT leads to a substantial
improvement in vascularization and collagenization on the pressure side of the periodontal ligament of diabetic rats subjected to
experimental tooth movement. Notwithstanding these findings,
the impairment of osteoblast and osteoclast differentiation was
only partially reversed by LLLT, suggesting that diabetic patients
who are not strictly monitored should not receive orthodontic
treatment until their metabolic status normalizes, since the application of strong orthodontic forces has been shown to cause
unwanted effects on bone remodeling.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References
[1] L. Bensch, M. Braem, K. Van Acker, G. Willems, Orthodontic treatment
considerations in patients with diabetes mellitus, Am J Orthod Dentofac
Orthop 123 (2003) 74–78.
[14]
American Diabetes Association, Diagnosis and classification of diabetes
mellitus, Diabetes Care 32 (2009) S62–S67.
L.G. Maia, A.C. Monini, H.B. Jacob, L.G. Gandini Jr., Maxillary ulceration
resulting from using a rapid maxillary expander in a diabetic patient, Angle
Orthod 81 (2011) 546–550.
L. Bensch, M. Braem, V.A. Kristien, G. Wiliems, Orthodontic treatment
considerations in patients with diabetes mellitus, Am J Orthod Dentofac
Orthop 123 (2003) 74–78.
M. Skamagas, T.L. Breen, D. LeRoith, Update on diabetes mellitus: prevention,
treatment, and association with oral diseases, Oral Dis 14 (2008) 105–114.
M.E. Villarino, M. Lewicki, A.M. Ubios, Bone response to orthodontic forces in
diabetic Wistar rats, Am J Orthod Dentofac Orthop 139 (2011) S76–S82.
M.R. Hamblin, T.N. Demidova, Mechanisms of low-level light therapy, Proc
SPIE 6140 (2006) 1–12.
F. Correa, R.A. Lopes Martins, J.C. Correa, V.V. Iversen, J. Joenson, J.M. Bjordal,
Low-level laser therapy (GaAs lambda = 904 nm) reduces inflammatory cell
migration in mice with lipopolysaccharide-induced peritonitis, Photomed
Laser Surg 25 (2007) 245–249.
E.S. Boschi, C.E. Leite, V.C. Saciura, E. Caberlon, A. Lunardelli, S. Bittencourt, D.A.
Melo, J.R. Oliveira, Anti-Inflammatory effects of low-level laser therapy
(660 nm) in the early phase in carrageenan-induced pleurisy in rat, Lasers
Surg Med. 40 (2008) 500–508.
A.V. Corazza, J. Jorge, C. Kurachi, V.S. Bagnato, Photobiomodulation on the
angiogenesis of skin wounds in rats using different light sources, Photomed
Laser Surg 25 (2007) 102–106.
M.D. Dantas, D.R. Cavalcante, F.E. Araújo, S.R. Barretto, G.T. Aciole, A.L.
Pinheiro, M.A. Ribeiro, I.B. Lima-Verde, C.M. Melo, J.C. Cardoso, R.L.
Albuquerque Júnior, Improvement of dermal burn healing by combining
sodium alginate/chitosan-based films and low level laser therapy, J Photochem
Photobiol B 105 (2011) 51–59.
A.P. Medrado, A.P. Soares, E.T. Santos, S.R. Reis, Z.A. Andrade, Influence of laser
photobiomodulation upon connective tissue remodeling during wound
healing, J Photochem Photobiol B 92 (2008) 144–152.
M.A. Gonzaga Ribeiro, R.L. Cavalcanti de Albuquerque, A.L. Santos Barreto, V.G.
Moreno de Oliveira, T.B. Santos, C.D. Freitas Dantas, Morphological analysis of
second-intention wound healing in rats submitted to 16 J/cm2 lambda 660-nm
laser irradiation, Indian J Dent Res. 20 (2009) 390.
L.A. Merli, M.T. Santos, W.J. Genovese, F. Faloppa, Effect of low-intensity laser
irradiation on the process of bone repair, Photomed Laser Surg 23 (2005) 212–
215.
74
L.G.M. Maia et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 65–74
[15] A.L. Pinheiro, M.E. Gerbi, Photoengineering of bone repair processes, Photomed
Laser Surg 24 (2006) 169–178.
[16] k. Kawasaki, N. Shimizu, Effects of low-energy laser irradiation on bone
remodeling during experimental tooth movement in rats, Lasers Surg Med 26
(2000) 282–291.
[17] M. Yamaguchi, M. Hayashi, S. Fujita, T. Yoshida, T. Utsunomiya, H. Yamamoto,
K. Kasai, Low-energy laser irradiation facilitates the velocity of tooth
movement and the expressions of matrix metalloproteinase-9, cathepsin K,
and alpha(v) beta(3) integrin in rats, Eur J Orthod 32 (2010) 131–139.
[18] D.R. Cruz, E.K. Kohara, M.S. Ribeiro, N.U. Wetter, Effects of low-intensity laser
therapy on the orthodontic movement velocity of human teeth: a preliminary
study, Lasers Surg Med 35 (2004) 117–120.
[19] W. Limpanichkul, K. Godfrey, N. Srisuk, C. Rattanayatikul, Effects of low-level
laser therapy on the rate of orthodontic tooth movement, Orthod Craniofac Res
9 (2006) 38–43.
[20] H. Lu, M. Raptis, E. Black, M. Stan, S. Amar, D.T. Graves, Influence of diabetes on
the exacerbation of an inflammatory response in cardiovascular tissue,
Endocinology 145 (2004) 4934–4939.
[21] N. Chbinou, J. Frenette, Insulin-dependent diabetes impairs the inflammatory
response and delays angiogenesis following Achilles tendon injury, Am J
Physiol Regul Integr Comp Physiol 286 (2004) R952–R957.
[22] A. Tellechea, E. Leal, A. Veves, E. Carvalho, Inflammatory and angiogenic
abnormalities in diabetic wound healing: role of neuropeptides and
therapeutic perspectives, Open Circ Vasc J 3 (2010) 43–55.
[23] I. Kanazawa, Osteocalcin possesses hormonal function linking bone to glucose
metabolism, Diabetes Metab 2 (2011) 1–3.
[24] B. Siddhartha Kumar, R. Ravi Sankar, A. Sachan, D. Prabath Kumar, D.T.
Katyarmal, K.V.S. Sarma, Effect of oral hypoglycaemic agents on bone
metabolism in patients with type 2 diabetes mellitus, J Clin Sci Res 1 (2012)
83–93.
[25] Y. Ozawa, N. Shimizu, G. Kariya, Y. Abiko, Low-energy laser irradiation
stimulates bone nodule formation at early stages of cell culture in rat calvarial
cells, Bone 22 (1998) 347–354.
[26] V.A. Melo, D.C.S. Anjos, R. Albuquerque Júnior, D.B. Melo, F.U.R. Carvalho, Effect
of low level laser on sutured wound healing in rats, Acta Cir Bras 26 (2011)
129–134.
[27] I. Garavello, V. Baranauskas, M.A. Cruz-Holing, The effects of low laser
irradiation on angiogenesis in injured tibiae, Histol Histopathol 19 (2004)
43–48.
[28] L. Gasparyan, G. Brill, A. Makela, Activation of angiogenesis under influence of
red low level laser radiation, Laser Florence (2004) 1–8.
[29] R.F. Diegelmann, M.C. Evans, Wound healing: an overview of acute, fibrotic
and delayed healing, Front Biosci 1 (2004) 283–289.
[30] L.S. Pugliese, A.P. Medrado, S.R. Reis, A. Andrade Zed, The influence of low-level
laser therapy on biomodulation of collagen and elastic fibers, Pesqui Odontol
Bras 17 (2003) 307–313.
[31] X. Li, L. Zhang, N. Wang, X. Feng, L. Bi, Periodontal ligament remodeling and
alveolar bone resorption during orthodontic tooth movement in rats with
diabetes, Diabetes Technol Ther 12 (2010) 65–73.
[32] S.J. Kim, Y.G. Kang, J.H. Park, E.C. Kim, Y.G. Park, Effects of low-intensity laser
therapy on periodontal tissue remodeling during relapse and retention of
orthodontically moved teeth, Lasers Med Sci 28 (2013) 325–333.