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Lasers in Medical Science
ISSN 0268-8921
Volume 30
Number 3
Lasers Med Sci (2015) 30:1061-1068
DOI 10.1007/s10103-015-1710-0
Effect of low-level laser therapy on
bone repair: a randomized controlled
experimental study
Valéria Regina Gonzalez Sella, Fernando
Russo Costa do Bomfim, Paula Carolina
Dias Machado, Maria José Misael da
Silva Morsoleto, et al.
1 23
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ORIGINAL ARTICLE
Effect of low-level laser therapy on bone repair: a randomized
controlled experimental study
Valéria Regina Gonzalez Sella &Fernando Russo Costa do Bomfim &
Paula Carolina Dias Machado &Maria José Misael da Silva Morsoleto &
Milton Chohfi &Helio Plapler
Received: 23 September 2014 /Accepted: 5 January 2015 /Published online: 18 January 2015
#Springer-Verlag London 2015
Abstract The aim of this study was to investigate the effect of
low-level laser therapy (LLLT) on bone repair in femoral frac-
tures. Sixty adult Wistar rats were randomly assigned into one
of two groups: group A (ostectomy + LLLT) or group B
(ostectomy + sham laser). An experimental model of complete
bone fracture was surgically created by removing a 2-mm
fragment from the middle third of the femoral shaft. Data were
analyzed on days 8, 13, and 18 after the fracture (subgroups 1,
2, and 3). Samples were assessed for changes in inflammatory
infiltration; trabecular bone matrix, periosteal, and new bone
formations; and changes in the expression of particular
osteogenic-related proteins (osteocalcin, osteopontin, and
osteonectin). Microscopic analysis revealed a significant de-
crease in inflammatory infiltration, intense trabecular bone
matrix and periosteal formation, and an increase in newly
formed bone after laser irradiation. We also found an increase
in the expression of bone matrix proteins with LLLT, with a
significant difference measured for osteocalcin in the LLLT
group at day 8 (p= 0.007). We show that LLLT plays an im-
portant role in augmenting bone tissue formation, which is
relevant to fracture healing. LLLT may therefore be indicated
as an adjunct therapeutic tool in clinical practice for the treat-
ment or recovery of nonunion injuries.
Keywords Bone remodeling .Femoral fracture .Low-level
laser therapy
Introduction
According to the World Health Organization, there are more
than 150 diseases and syndromes related to skeletal and joint
problems [1]. Approximately six million long-bone fractures
are reported annually in the USA. Although progress has been
made in treatment methods over the past decades, approxi-
mately 5–10 % of fractures still result in delayed union or
nonunion. Moreover, 600,000 individuals experience
prolonged pain and discomfort associated with fracture non-
union every year [2–6]. Among the efforts aimed at minimiz-
ing these complications, clinical solutions using energy emis-
sion (ultrasound, electrical stimulation, and laser irradiation)
have been investigated [2,5,7]. Laser therapy is accessible,
does not require the concomitant use of drugs, does not pro-
mote thermal damage to the tissue [1], and may be applied in
the presence of the types of metal devices [4]commonlyused
to stabilize open or displaced fractures. Thus, various experi-
mental studies have sought to define the role of low-level laser
therapy (LLLT) in fracture healing [1,3,8,9].
The investigation of bone formation and resorption pro-
cesses involves the identification of products that are synthe-
sized by osteoblasts and osteoclasts [10]. Noncollagenous
proteins found in the organic matrix of bone tissue
(osteocalcin, osteopontin, and osteonectin) are commonly
used as bone mineralization markers. Because the exact mech-
anisms involved in the healing process of laser-irradiated bone
tissues have yet to be elucidated and there are no standardized
protocols for LLLT research, we sought to investigate both of
these issues in an in vivo model and analyze the results
through microscopy and immunohistochemistry. Therefore,
V. R. G. Sella :F. R. C. do Bomfim :P. C. D. Machado:
M. J. M. da Silva Morsoleto :H. Plapler
Department of Surgery, Division of Operative Technique and
Experimental Surgery, Universidade Federal de São Paulo [Federal
University of São Paulo] –UNIFESP, São Paulo, SP, Brazil
M. Chohfi
Department of Orthopedics, Universidade Federal de São Paulo
[Federal University of São Paulo] –UNIFESP, São Paulo, SP, Brazil
V. R. G. Sella (*)
R. Botucatu, 740, São Paulo, SP, Brazil CEP 04023-900
e-mail: valsella@uol.com.br
Lasers Med Sci (2015) 30:1061–1068
DOI 10.1007/s10103-015-1710-0
Author's personal copy
the aim of this study was to verify the effect of LLLT on bone
repair at the interface of a nonunion femoral fracture (simulat-
ed by ostectomy) by measuring changes in major bone matrix
proteins that are associated with bone formation.
Materials and methods
Group allocations
Sixty adult (12 weeks, 350 g) male Wistar rats were
provided with regular standard rat food and water ad
libitum throughout the experiment and were housed
one animal per cage in a room with a 12-h light–dark
cycle. The rats were randomly assigned into one of two
groups: group A (n=30), or the LLLT group (ostectomy
+ LLLT), and group B (n= 30), or the sham group
(ostectomy + laser irradiation simulation). The rats in
these groups were respectively divided into three sub-
groups (1–3) according to the day of death after sur-
gery: at day 8 (subgroup 1), day 13 (subgroup 2), and
day 18 (subgroup 3): A1/B1 (n=20), A2/B2 (n=20),
and A3/B3 (n=20).
Surgery
Rats were anesthetized with a mixture of 0.4 mL of 10 %
ketamine (114 mg/kg), 0.2 mL of 2 % xylazine (11.4 mg/kg),
and 0.1 mL of fentanyl citrate (1.4 mg/kg) by intramuscular
injection. Rats were then placed securely on the operating
table in the ventral decubitus position. An incision was first
made through the skin and subcutaneous tissue in the antero-
lateral region of the right thigh. The fascia was then opened,
and the femur was accessed at the space between the rectus
femoris and the vastus lateralis muscles. The middle third of
the femoral shaft was exposed and a straight titanium plate
(22 mm× 3 mm×0.5 mm) with central space and four holes
(Synthes Ind., Rio Claro, Brazil) was fixed to it with 1.5- and
1.7-mm stardrive cortex screws, according to the femur diam-
eter. The bone was sectioned using an oscillating saw
(Implantek Lase, DMC Equipment, São Carlos, Brazil) at
500–800 rpm, with continuous irrigation with saline solution
to the site. An experimental model of complete bone fracture
was created by removing a 2-mm fragment from the middle
third of the femoral shaft (Fig. 1). A precision pachymeter was
used to measure the size of the gap to ensure the distance
between the fragments. After fracture creation, the muscles
were approximated and the skin was closed with continuous
sutures. Enrofloxacin (30 drops) was added to the rats’drink-
ing water daily. Analgesics were not administered and splint
elements were not used.
Low-level laser therapy
Rats in group Awere exposed to LLLT once daily (from day 1
through day 8 after surgery) using a gallium aluminum arse-
nide laser device (model Magnus Plus, DMC Equipment) by
direct contact to the skin at two points on the inner side of the
right hind limb. The device was set according to the following
parameters: continuous mode, λ=808 nm, power density of
0.2 W/cm
2
, fluency of 37 J/cm
2
per site, nominal dose of 2 J,
spot size of 0.02 mm
2
, energy per point=1 J, and exposure
time of 5 s per site. For exposure, each rat was positioned
(Fig. 2) at its back without any restraints and placed in the
hand of one of the experimenters, and the laser was applied on
the opposite side of a fixed plate. The animals of group B were
Fig. 1 Aspect of the femur after ostectomy and plate fixation
Fig. 2 Rat exposure to LLLT procedure
1062 Lasers Med Sci (2015) 30:1061–1068
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similarly prepared; however, they were treated with a sham
laser.
On determined days, the animals were sacrificed by injec-
tion of lethal dose of anesthetics.
Microscopic analysis
Bone tissue samples were fixed in a 10 % formalin solution for
24 h. They were then individually immersed into a rapid
decalcifier (0.7 g of tetrasodium EDTA, 0.14 g of sodium
tartrate, 5 g of sodium/potassium tartrate; 120 mL of hydro-
chloric acid; 900 mL of distilled water) until total decalcifica-
tion was achieved, and the samples were then washed in run-
ning water. Samples were then successively dehydrated in 70,
80, 90, and 100 % alcohol, cleared in an alcohol/xylene bath
(1:1) and then in xylene until translucent. Samples were then
paraffin-embedded to obtain a tissue block, cut into 6-μm-
thick sections using a Microtome (Leica Microsystems,
Wetzlar, Germany), and stored in a drying oven for later
staining.
Histological sections were stained with hematoxylin-eosin,
examined using optical microscopy, and evaluated via image
digitization and computational analysis (Image-Pro Plus ver-
sion 6.3.1, Media Cybernetics, Inc., Rockville, MD, USA).
Inflammatory infiltration and trabecular bone matrix forma-
tion were examined across five quadrants (four peripheral
quadrants and one central quadrant) on each slide at a magni-
fication of ×1000. New bone formation was assessed using
osteocyte counting within the same five quadrants in each
slide at ×100 magnification. A score from 0 to 4 according
to the presence of characteristic cells was used to evaluate
inflammatory infiltration, trabecular bone matrix, and new
bone formation, where 0 = none (no characteristic cells), 1=
minimal (range 1–65 characteristic cells), 2 = mild (range 66–
135 characteristic cells), 3= moderate (range 136–200 charac-
teristic cells), and 4=intense (>200 characteristic cells).
Periosteal growth was determined as the presence or absence
of the tissue found on each slide, for each animal.
Immunohistochemistry
Cross sections (6 μm) of the cut edges of the fracture site were
placed on silanized slides, fixed, deparaffinized, and incubat-
ed in sodium citrate buffer (0.01 M; pH 6.0) for antigen re-
trieval. Sections were then washed with phosphate-buffered
saline (PBS) and immersed in methanol with 0.3 % hydrogen
peroxide for endogenous peroxidase blocking. Sections were
again washed with PBS and then incubated with one of three
primary antibodies: anti-osteocalcin polyclonal antibody, di-
luted 1:250 (Santa Cruz Biotechnology, Dallas, TX, USA)
[11]; anti-osteopontin monoclonal antibody, diluted 1:250
(Santa Cruz Biotechnology) [12], or anti-osteonectin mono-
clonal antibody, diluted 1:250 (Santa Cruz Biotechnology)
[13]. All antibodies were diluted in 0.01 M PBS with 1 %
bovine serum albumin for 18 h at 4 °C. After this first incu-
bation, the sections were washed three times with PBS and
then incubated with a biotinylated secondary antibody solu-
tion provided in the Universal LSAB+ Kit/HRP, Rabbit/
Mouse Kit (code KO675, Dako, Glostrup, Denmark).
Sections were washed and then incubated with streptavidin-
biotin-peroxidase complex solution and working substrate-
chromogen solution both provided in the kit, with three PBS
washes in between these two steps. Finally, the sections were
washed with distilled water, counterstained with 5 % methyl
green, washed again with distilled water, dehydrated, and
mounted in Entellan
®
(Merck Millipore, Darmstadt
Germany). All immunohistochemical reactions occurred in a
light-protected environment.
Immunostaining was assessed using light microscopy
(Leica Microsystems), and the obtained images were analyzed
using Image-Pro Plus. The intensity of the expression of pro-
teins associated with bone formation was defined according to
a score from 0 to 3 (0=no reddish-brown color, 1=light color
(color code X156), 2=medium intense color (color code
E118), and 3= intense color (color code R121) (http://www.
suvinil.com.br/pt/familias/2/600/tijolo.aspx)[14]. Analysis
was performed across five fields of view from each slide,
and a positive control for each antibody was used to
ascertain staining. This positive control staining was
performed in specific cells/tissues: kidney tissue for
osteocalcin and osteopontin, and lung carcinoma cells for
osteonectin [11–13]. The colorimetric method was compared
against a standard scale. This method is well established in the
literature [15,16].
All slides for either microscopic and immunohistochemical
evaluation were examined in duplicate by a biomedical pro-
fessional without prior knowledge of the aim of the study.
Statistical analysis
Mann–Whitney and Kruskal–Wallis nonparametric tests were
performed to determine statistical significance between
groups A and B and among the subgroups (p<0.05).
Results
With regard to microscopic parameters (Fig. 3), better
results were found for group A than for group B
(Fig. 4a, b). The fracture sites from all rats sacrificed
at day 8 (subgroups A1 and B1) showed similar degrees
of inflammatory infiltration, whereas only the rats of
subgroup A2 (on day 13) showed a significantly
lowered inflammatory infiltration response (p=0.015),
asshowninFig.5a, b. This lower degree of
Lasers Med Sci (2015) 30:1061–1068 1063
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inflammation was maintained at day 18 (subgroup A3;
p=0.028). LLLT rats in subgroups A1, A2, and A3
showed an emergence of trabecular bone formation over
time, with significant differences among the three sub-
groups measured (p<0.01 for all 3 days). In contrast, no
trabecular matrix formation was observed for any of the
control rats (subgroups B1, B2, or B3). Periosteal for-
mation was not seen in any of the rats after 8 days of
healing (subgroup A1 and B1), but a high percentage of
animals with periosteal formation were found by day 13
in the LLLT group (subgroup A2) as compared with
control rats at the same time point (subgroup B2; p=
0.001). This increased periosteal response at day 13 was
maintained to day 18 for LLLT rats (subgroup A3)
whereas control rats at day 18 had only just started to
show periosteal differences (subgroup B3 p=0.005).
With regard to bone formation, significant differences
were measured among the groups at each time point,
with better results identified for LLLT rats (subgroups
A1, A2, and A3) as compared with their respective
control counterparts (subgroups B1, B2, and B3;
p<0.001 for all periods).
Immunohistochemistry results (Fig. 6)showedhigherex-
pression of osteocalcin in the tissue samples from LLLT rats
(subgroup A1) at day 8 as compared with that from the control
rats at the same time point (subgroup B1; p=0.007). Whereas
expression of osteocalcin increased for the control rats by day
13 (subgroup B2), osteocalcin expression remained higher for
LLLT rats (subgroup A2). These results indicate that LLLT
rats (group A) had an anticipation on bone-remodeling re-
sponse as compared with control rats (group B). By day 18,
rats in both groups showed no changes in osteocalcin expres-
sion from day 13. Osteopontin showed the expected behavior
(increase on days 8 and 18 and decrease on day 13) in both
groups. The difference between the LLLT and the control
group was significant only on day 8 (p=0.033). LLLT and
control rats showed similar expression changes for
osteonectin. Control rats at day 13 (subgroup B2) showed a
lower expression of osteonectin as compared with the LLLT
rats at that same time point (p= 0.018), and no significant
Fig. 3 Morphometric analysis. Similar inflammatory infiltration was
observed for LLLT and control rats at day 8 (subgroups A1 and B1). A
significant reduction in inflammatory infiltration was only observed for
LLLT rats at day 13 (subgroup A2). Trabecular matrix formation was
found in all of the LLLT rats (group A). Periosteal formation was not
seen at day 8 in either group (subgroups A1 and B1) but was present inthe
LLLT rats at days 13 and 18 (subgroups A2 and A3). A significant
difference in new bone formation was observed for LLLT rats at all
time points (p<0.001)
1064 Lasers Med Sci (2015) 30:1061–1068
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difference was observed for osteonectin expression by day 18
between the two groups.
Discussion
When creating a clinical bone fracture model, it is crucial to
produce a gap that prevents contact between the bone frag-
ments, as contact might provide a more favorable environment
for the union process and facilitate bone growth. Besides this,
bone defects of small diameters are not sufficiently reliable to
demonstrate the efficacy of lasers as biomodulatory therapies
for bone repair [1,17]. In our model, the stability provided by
the plate allowed ambulation and weight bearing [18].
The 808-nm wavelength of the laser used for bone remod-
eling is within the infrared range and also within the so-called
optical window that spans the red and near-infrared wave-
lengths; this wavelength ensures proper penetration of the
laser light into biological tissues. Once light absorption and
scattering (which is dependent on the wavelength and its max-
imal penetration) are obtained in a range between red and
near-infrared lights, this interval will provide the ideal pene-
tration into the biological tissue [5,19–21]. Indeed, it has been
shown that 808-nm light penetrates as much as 54 % deeper
than 980-nm light [22].
Real power was chosen to provide the best energy level in
the least possible time (5 s) in unrestrained animals, and the
choice of this regime was based on previous studies [23–25].
We chose an irradiation spot on the opposite side of the fixed
plate to prevent absorption and scattering losses. Furthermore,
the use of eight laser irradiation sessions and the dates chosen
for killing the animals were defined so as to stimulate and
observe bone growth on proliferative process. The time points
were chosen based on previous studies, where growth inhibi-
tion was observed within a stimulation period longer than 7–
8 days after surgery, with evidence of bone formation between
7 and 15 days and a reduction in the rate of bone formation
from day 21 [2,26–28].
The reduction in inflammatory infiltration observed in at
days 13 and 18 in the LLLT rats led us to assume that these rats
were possibly less affected by the inflammatory phase of bone
repair, thus allowing for an earlier reparative phase and, con-
sequently, earlier new bone formation, as previously observed
[29]. Inflammatory infiltration is part of the bone remodeling
Fig. 4 Histological sections after 13 days. aLLLT rats; it is possible to
observe trabecular bone matrix formation (cross), inflammatory
infiltration (arrow), chondrocytes presence (triangle), and periosteum
formation (sphere). bControl rats; fibroblast re-composition (star)and
inflammatory infiltration (thick arrow) can be observed
Fig. 5 Inflammatory infiltration cells after 13 days. aAscoreof1was
found for LLLT rats. bA score of 2 was determined for the control rats.
The red arrows correspond to inflammatory cells
Lasers Med Sci (2015) 30:1061–1068 1065
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process, and it is useful if not drawn out, as longer inflamma-
tory periods compromise the final bone quality. Laser irradia-
tion did not eliminate the inflammation; rather, it expedited all
of the steps involved in bone formation, including this inflam-
matory phase. The reduction in inflammatory infiltration, to-
gether with the increase in periosteal development and con-
siderable increase in trabecular matrix formation, showed that
rats in the LLLT group underwent an earlier and more orga-
nized process of bone formation. In the absence of LLLT,
however, no significant trabecular matrix was formed, which
is indicative of a slower and less-organized process.
At the cut edge of the fracture, both osteocalcin and osteo-
pontin—two proteins associated with extracellular matrix for-
mation and osteoblast activity [30]—were detected early in
LLLT rats, which is consonant with the microscopic results.
This is very important, as these matrix factors contribute to the
growth, shape, and size of the bone matrix, and affect the
quality of the matrix that is produced [31]. Indeed, LLLT
and its potent effect on increasing proliferation and cell via-
bility may significantly contribute to many biomedical re-
sources that augment tissue formation and repair in regenera-
tive medicine [32]. Thurner’s study [31] on osteopontin
deficiency showed a 30 % decrease in fracture toughness in
the absence of the protein, suggesting an important role for
osteopontin in impeding crack propagation. Its presence on
the surface of samples in “in vivo”bone experiments also
suggests its involvement in the process of cell-matrix adhe-
sion, and matrix-matrix modeling and remodeling [33]. Our
results also suggest a possible connection between the emer-
gence of osteonectin and the formation of the periosteum, both
of which are involved in callus formation. A significant
amount of osteonectin during this stage adds to the progress
of bone formation, because it incorporates collagen, an impor-
tant protein required for the acquisition of the tensile strength
of the bone. The mineralization and subsequent completion of
the repair process of a fracture can be achieved only when
proteins that have an affinity for calcium (such as osteocalcin
and osteopontin) promote mineral deposition, and those with
an affinity for collagen (such as osteonectin) promote bone
strength.
Previous studies have investigated the effect of laser thera-
py on fracture healing [1,5,29,34,35] using injury models
that allow contact between the bone fragments. This led pro-
fessionals in the field to question the efficacy of LLLT in
Fig. 6 Immunohistochemical analysis. Osteocalcin and osteopontin
were detected early in LLLT rats (group A) with statistical significance
observed at day 8 (subgroup A1) as compared with the control rats at that
same time point. Osteonectin expression was significantly higher in
LLLT rats at day 13 (subgroup A2), as was periosteum formation. None
of the proteins showed any difference in expression at day 18 between the
two groups (groups A and B)
1066 Lasers Med Sci (2015) 30:1061–1068
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conditions that would not be resolved without bone-
remodeling aids. Our investigation confirms the beneficial
effects of LLLT in fracture healing [36], not only in reducing
the inflammatory phase but also in intensifying the reparative
phase of repair.
Nonunions represent a treatment challenge for orthopedic
surgeons and a serious socioeconomic problem for the patient.
Our study provides a solution to these concerns and show that
LLLT may be a useful therapeutic tool for bone repair and for
the treatment of impaired bone healing in clinical practice.
Limitations
In future studies, we should measure the reduction of the gap
using radiological images, to provide some insight into the
potential use of LLLT in bone-lengthening surgeries.
Conclusions
We show that LLLT is effective in enhancing bone healing in a
rat nonunion femoral fracture model in vivo. LLLT has an
effect on all phases of this repair process by increasing the
expression of bone formation proteins in vivo and by shorten-
ing all phases of bone remodeling.
Ethical approval Animal manipulation was performed in accordance
with the animal testing guide (in agreement with the Brazilian Legislation
no. 11.794/2008 for Procedures for the Scientific Use of Animals). This
randomized controlled experimental study was previously approved by
the Research Ethics Committee of Federal University of São Paulo under
no. 1101/09.
Conflict of interest All authors have no conflicts of interest.
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