Antiosteoclastogenesis Activity of CO2 laser Antagonizing Receptor
Activator for Nuclear Factor KappaB Ligand–induced Osteoclast
Differentiation of Murine Macrophages
Chun-Liang Kuo1,2,a, Chia-Tze Kao3,4,a, Hsin-Yuan Fang5,6,7, Tsui-Hsien Huang3,4, YiWen Chen7, Ming-You Shie7
1
Department of Orthodontics, Chi Mei Medical Center, Tainan City, Taiwan
2
Center for General Education, Southern Taiwan University of Science and Technology,
Tainan City, Taiwan
3
School of Dentistry, Chung Shan Medical University, Taichung City, Taiwan
4
Department of Stomatology, Chung Shan Medical University Hospital, Taichung City,
Taiwan
5
Department of Thoracic Surgery, China Medical University Hospital,Taichung, Taiwan
6
School of Medicine, College of Medicine, College of Public Health, Taichung, Taiwan
7
3D Printing Medical Research Center, China Medical University Hospital, Taichung
City, Taiwan
Short title: Antiosteoclastogenesis activity of murine macrophage by CO2 laser
Classification numbers: 87
a
: Both authors contributed equally to this work.
Correspondence:
Ming-You Shie, 3D Printing Medical Research Center, China Medical University
Hospital, Taichung City, Taiwan (E-mail: eviltacasi@gmail.com; tel: +886-4-22052121;
fax: +886-4-24759065)
1
Abstract
Macrophage cells were the important effector cells in the immune reaction, which
are indispensable for osteoclastgenesis and their heterogeneity, plasticity, render
macrophages a primer target for immune system modulation. In recent years, there are
very few studies about the effects of macrophage cells on laser treatment-regulated
osteoclastgenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL
to osteoclastgensis. We used CO2 laser as a model biostimulation to investigate the role
of osteoclastogenic. We also evaluated cell viability, cell death, and cathepsin K
expression. The CO2 laser inhibited receptor activator of the NF-ƘB ligand (RANKL)
induced formation of osteoclasts during the osteoclast differentiation process. It was also
found that irradiation for 2 times reduced RANKL-enhanced TRAP activity in a dosedependent manner. Furthermore, CO2 laser-treatment diminished the expression and
secretion of cathepsin K elevated by RANKL, and was concurrent with the inhibition of
TRAF6 induction and NF-ƘB activation. The current report demonstrates that CO2 laser
abrogated RANKL induced osteoclastogenesis by retarding osteoclast differentiation.
The CO2 laser can modulate every cell through dose-dependent in vitro RANKLmediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts, and
the maturation of osteoclasts. Therefore, the current results serve as an improved
explanation of the cellular roles of macrophage cells populations in osteoclastogenesis as
well as in alveolar bone remodeling by CO2 laser-treatment.
Keywords: CO2 laser, macrophage, receptor activator for nuclear factor kappa B,
tartrate-resistant acid phosphatase, osteoclastgenesis.
2
1. Introduction
Laser is classified as light amplification by stimulated involves the modification
of the environment to affect existing bacteria capable of bioremediation [1,2]. The
application of low level laser light was in many fields including medical, industrial, and
the military [3,4]. There are several commercial product lasers, including CO2, diode, and
erbium (Er):yttrium aluminum garnet (YAG) lasers in the clinical [5]. The potential
safety of laser regeneration is connected with the nondestructive character of laser effect
on tissue matrix and with arrangement of favorable conditions for cell proliferation and
functioning [6,7]. The practical and physical characteristics of these devices and sources
were possible their application in therapies such as efficient fiber-optic coupling to
irradiate interior body parts, easy wavelength tenability, easily of use, and electrical
safety [8,9]. Several studies proved that the antibacterial effect of CO2 laser treatment on
bacteria was high efficiency when bacteria were embedded in biofilm, due to a photothermal mechanism [1,5,9,10]. However, some researches shown that lasers affect
fibroblast proliferation and collagen synthesis and reduce inflammation [11]. In addition,
the laser light can promote periodontal cell differentiation and it has potentially be used
to enhance periodontal tissue regeneration [5,12]. Faster hard tissue formation on the
orthodontic therapy might promote orthodontic tooth stability [13]. Thus, laser-irradiation
can promote cell proliferation and might be helpful for increasing orthodontic tooth
stability.
Bone remodeling is regulated by the replacement of old bone with new bone through
sequential bone formation and resorption [14]. Several immune cytokines, growth factors,
and hormones result in an imbalance between osteoblast and osteoclast activities and can
3
result in skeletal abnormalities, such as osteoporosis and osteopetrosis [15]. Bone
remodeling has been shown to participate in peripheral blood circulating with precursor
cells containing osteoclasts and osteoblasts [14,16]. In several studies affirmed receptor
activators of nuclear factor-ƘB (NF-ƘB) ligand (RANKL)/RANK/osteoprotegerin (OPG)
has been shown to be the important regulator of osteoclasts and pre-osteoclast cells
differentiation, activation, and maturation [15,17,18]. Thus, the importance role of
RANKL in osteoclastgensis underlines the central role played by stromal cells and
osteoblasts in the process [19-21]. Interesting, the macrophages can change their
physiology and phenotype through the environmental signals [5,22]. In addition, the
inflammation macrophages are also known to affect bone pathology and physiology
[9,23,24].Given the important roles of macrophages in the bone formation, some studies
have analyzed the interactions between laser treatment and macrophages. However, these
studies are focused on either the inflammatory contributions or the differentiation into
osteoclasts under various laser treatment [5,25]. Very few reporters have been made on
the effects of macrophages in regulating laser-treatment stimulated osteoclastgenesis.
The CO2 laser treatment is well recognized as the osteoconductive stimulation and
has been widely used for clinical dentistry regeneration application. The effect of CO2
laser treatment on the osteoclastgenesis of macrophages is unclear. Therefore, this study
investigated CO2 laser-treatment with different times induced anti-osteoclastogenic
actions in murine RAW 264.7 macrophages cultured with RANKL. The activity of TRAP
and the induction of osteoclastogenic markers were examined in CO2 laser-treated
differentiating RAW 264.7 macrophages. This study attempted to show how CO2 laser-
4
irradiation manipulated the sequential molecular events induced by RANKL during
osteoclast differentiation.
5
2. Materials and Methods
2.1 RAW 264.7 cell culture
RAW 264.7 macrophage cells (American Type Culture Collection, Manassas, VA)
were cultured in DMEM (Caisson Laboratories, North Logan, UT) containing 10% fetal
bovine serum (FBS; GeneDireX, Taipei, Taiwan), 100 U mL-1 penicillin, and 100 μg mL1
streptomycin (Caisson Laboratories) at 37°C in a humidified atmosphere of 5% CO2 in
air. For osteoclast differentiation, RAW 264.7 cells were cultured in DMEM containing
10% FBS and 50 ng/mL RANKL (ProSpec, Rehovot, Israel) and the culture medium was
changed every 2 days. In addition, the NF-ƘB inhibitory effect on several genes and
proteins production were analysis by treatment with 10 μM SN50 (Sigma-Aldrich, St.
Louis, MO).
2.2 Macrophage cell viability
The macrophage cells were cultured on 96-well for 1 day, than treatment with
carbon dioxide (CO2) laser (Yoshida Dental Laser, Tokyo, Japan) for 1 w with different
times (1 time: 0.002s, 2 times: 0.002s x 2, and 3 times: 0.002s x 3). After different culture
times, cell viability was evaluated by the PrestoBlue® assay (Invitrogen, Grand Island,
NY). Briefly, at the end of the culture period, the medium was discarded and the wells
were washed with cold PBS twice. Each well was filled with medium with a 1:9 ratio of
PrestoBlue® in fresh DMEM and incubated at 37°C for 30 min. The solution in each well
was transferred to a new 96-well plate. Plates were read in a multiwell spectrophotometer
(Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results
were obtained in triplicate from three separate experiments for each test.
6
2.3 TRAP Activity
After culture for 5 days, the cells were fixed with 4% formalin for 15 min and 95%
ethanol for 3 min for measuring TRAP activity. Than, the cells were immersed in 10 mM
citrate buffer (pH 4.6) containing 10 mM sodium tratrate and p-nitrophenylphosphate.
After incubation for 1 h, the reaction mixtures were transferred to a new 24-well. The
reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405
nm. All experiments were done in triplicate from three separate experiments for each test.
2.4 Immunofluorescent stain
To further clarify the effects of tensile force on TRAP activity was analyzed using
fluorescence microscopy. After 5 days of cultured, the unbound cells were rinsed with
cold PBS three times, and the adherent cells were fixed in 4% p-formaldehyde (SigmaAldrich) for 30 min at room temperature and permeabilized with 0.1% Triton X-100
(Sigma-Aldrich) in PBS. The cells were then blocked in PBS supplemented with 5%
bovine serum albumin (Gibco) for 1 h and washed three times with PBS-T (PBS
containing 0.1% Tween 20). After this, the cells were incubated with rabbit anti-mouse
TRAP (GeneTex, San Antonio, TX) for 2 h, followed by a mixture of goat anti-rabbit
IgG antibodies conjugated to Alexa Fluor 488 (Invitrogen). Next, the nuclei were stained
with 300 nM DAPI (Invitrogen) for 1 h. After washing three times with PBS-T, the cells
were viewed under indirect fluorescence using a Zeiss Axioskop2 microscope (Carl Zeiss,
Thornwood, NY, USA) at 200x magnification.
7
2.4 Effects of CO2 laser on the osteoclastgenic differentiation gene expression of
macrophage cells
Macrophage cells were seeded in a 96-well plate at a density of 5 x 104 cells per
well. After 24 h of incubation, the culture medium was removed and treatment with CO2laser with different times. For the detection of osteoclastgenesis-related genes (MMP9,
integrin β3 and cathepsin K), total RNA of all groups was extracted using TRIzol reagent
(Invitrogen) after 5 days and analyzed by RT-qPCR. Total RNA (500 ng) was used for
the synthesis of complementary DNA using cDNA Synthesis Kit (GeneDireX) following
the manufacturer’s instructions. RT-qPCR primers (table 1) were designed based on
cDNA sequences from the NCBI sequence database. SYBR Green qPCR Master Mix
(Invitrogen) was used for detection and the target mRNA expressions were assayed on
the ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City,
California, USA). Each sample was performed in triplicate.
2.5 Protein expression analysis
The enzyme-linked immunosorbent assay (ELISA) was carried out using cell
lysates and a culture medium prepared with cultured RAW 264.7 cells for 5 days. Cells
were lysed in NP-40 lysis buffer (Invitrogen) at 4°C for 30 min and the lysates were
centrifuged at 13,000 g. The culture medium and cell lysates (10 μg protein) were
analysis by using a Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA).
For the detection of protein (cathepsin K, TRAF6, phospho-IƘB, nuclear factor of
activated T cells cytoplasmic (NFATc) 1, MITF and β-actin), we followed the
manufacturer’s instruction, and all ELISA were from Abnova (Abnova, Taipei, Taiwan).
8
The protein concentration was measured by correlation with a standard curve. Protein
expression levels were normalized to the β-actin band for each sample. The results were
obtained in triplicate from three separate samples for each test.
2.6 Statistical Analysis
A one-way variance statistical analysis was used to evaluate the significance of the
differences between the groups in each experiment. Scheffe’s multiple comparison test
was used to determine the significance of the deviations in the data for each specimen. In
all cases, the results were considered statistically significant with a p value < 0.05.
9
3. Results
3.1 Macrophage cell viability
After CO2 laser irradiation, the macrophage cell viability was similar (p > 0.05)
between the CO2 laser-treatment for 1 and 2 times groups and Ctl (Fig. 1). Cell viability
assay showed that the overall metabolic activity of most groups with laser-treatment
increased in a time-dependent manner. However, the number of macrophage cells in the
presence of laser-treatment for 3 times significantly decreased than other groups (p <
0.05).
3.2 TRAP activity
We also investigated the inhibitory effect of nontoxic condition of CO2 lasertreatment on TRAP activity of RAW 264.7 cells stimulated with 50 ng/mL RANKL (Fig.
2A). RANKL induced TRAP activity in RAW 264.7 macrophages. In contrast, this
activity was dose-dependently reduced in CO2 laser-treated macrophages exposed to
RANKL. The results showed that CO2 laser-treated at 2 times, it caused an approximately
50% reduction in TRAP expression. In Fig 2B, RANKL stimulated Raw 264.7 cell
without CO2 laser-treatment produces numerous TRAP-positive cells. After CO2 lasertreated for 5 days, the amount of TRAP-positive cells decreased more than in the
RANKL-treated cells.
3.3 Inhibition of bone resorption by CO2 laser
MMP-9 is responsible for bone resorption mediated by osteoclasts. To test with
CO2 laser expedited RANKL-induced MMP-9 secretion, Raw 264.7 cells were exposed
10
to 50 ng/mL RANKL for 5 d. RANKL considerably elevated the formation of MMP-9,
which was dose-dependent with CO2 laser-treatment times (Fig. 3A). It is known that
integrin αvβ3 plays a role in the regulation of cell migration and the maintenance of the
sealing zone required for effective osteoclastic bone resorption. Cellular integrin β3 was
raised by 50 ng/mL RANKL treatment for 5 days. In addition, CO2 laser reinforced its
induction in a times-dependent manner (Fig. 3B)
3.4 Inhibition of cathepsin K production of osteoclast
RANKL promote the production of cathepsin K from macrophages and such
secretion is significantly inhibited by treating with CO2 laser (Fig. 4A). Moreover, the
RANKL-induced cellular expression of cathepsin K will decrease in the presence of CO 2
laser-treatment (Fig. 4B). A significant (p < 0.05) decrease of 28% and 50% was found
for cathepsin K synthesis in cells cultured under medium contained RANKL compared
with CO2 laser-treatment for 1 and 2 times, respectively. Therefore, CO2 laser-treatment
is effective in retarding osteoclast maturation.
As shown in Figs. 5A and B, as with CO2 laser-treatment 2 times, the NF-ƘB
inhibitor SN50 inhibits the formation of TRAP-positive cells through RANKL. In
addition, RANKL-induced cathepsin K production is inhibited by 10 µM SN50. A
significant decrease (p < 0.05) in cathepsin K levels in the macrophage cells was
measured for CO2 laser-treatment compared with RANKL alone. In addition, the effect of
SN50 against cells on cathepsin K levels was similar to CO2 laser-treatment. Accordingly,
the CO2 laser may affect the cellular secretion of cathepsin K by disturbing an NF-ƘBresponsive mechanism.
11
3.5 Down-regulation of TRAF6 and NF-ƘB transactivation by CO2 laser
During osteoclast differentiation, RANKL induced the activation of the NF-ƘB
signaling pathway. When Raw 264.7 cells are treated with 50 ng/mL RANKL, the
TRAF6 expression is significantly greater (p < 0.05) than in the untreated control cells
(Fig. 6A). In addition, TRAF6 induction was notably inhibited when cells are treated by
CO2 laser. IƘB phosphorylation decreases in cells after irradiation for 1 and 2 times (Fig.
6B). No significant differences (p > 0.05) in IƘB protein expression were detected
between the cells cultures under control condition and laser-treatment 2 times with
RANKL.
3.6 Blockade of RANKL-induced NFATc1 and MITF
NFATc1 has been characterized as a master regulator of NF-ƘB ligand-induced
osteoclast differentiation. MITF is a master regulator of osteoclast bone resorption and a
key regulator of osteoclast function by activating proteins such as cathepsin K and TRAP.
When macrophage cells are exposed to 50 ng/mL RANKL for 3 d, the transcription of
NFATc1 is induced (Fig. 7A). Similarly, CO2 laser-irradiation inhibits the downregulated protein levels of NFATc1 and MITF (Fig. 7A and B). In addition, laserrestrained NFATc1 and MITF production is suppressed by 10 μM SN50. Thus, CO2 laser
may retard osteoclast function inhibition osteoclastogenic protein of TRAP and cathepsin
K.
12
4. Discussion
Macrophage cells play an important role in the interaction between bone modeling
and remodeling. Such effects are commonly attributed to an inflammatory response and
laser treatment. In addition, it is well known that laser-treatment exhibit good
biocompatibility towards cells in the bone remodeling process [26]. However, lasertreatment not only promote cell differentiation [5], but also reduce inflammation in
primary pulp cells [12,26]. However, how CO2 laser affect osteoclastogenic behavior is
not yet clear. The present study reveals a systematic understanding of the RAW 264.7
cells differentiated into osteoclast-like cells and the inhibition of CO2 laser-treated with
different times. In previous study, we study revealed that in response to the CO2-laser
treatment, leading to the release of osteoinductive molecules and anti-inflammatory
cytokines from macrophage cells, which enhanced the osteogenesis of hPDLs through the
BMP2 pathway [26]. In addition, several studies demonstrate immune response was
considered by measuring the expression of IL-10, TNF-α, and IL-1, and macrophage cells
decreased IL-1 expression when irradiated with CO2-laser as compared to those in the
control environment [26]. Similar studies have shown that inflammatory responses to
laser treatment was shown to decrease prostaglandin E2 and IL-1β analyzed and protein
expression; the inhibition of the prostaglandin E2 and IL-1β might be of therapeutic value
[27].
Although macrophages have been confirmed to be involved in the osteogenesis,
there is still no consensus on which phenotype is more useful for the osteogenic
differentiation. The classically activated inflammatory macrophages cells were affected
osteogenic differentiation in hMSCs [28]. Bone reconstitution research must consider
13
molecular interactions and several pathways [29]. Inhibiting osteoblast secreted RANKL
or antagonizing RANKL actions on osteoclasts might be a defended mechanism for
preventing excessive osteoclast differentiation [30]. The CO2 laser has the potential to
antagonize osteoclastogenic effects and bone resorption by promoting osteoblast
differentiation and secretion of anti-osteoclastogenic cytokines [26]. The present study
revealed that the CO2 laser-irradiation inhibited RANKL-induced osteoclastogenesis by
suppressing the secretion of cathepsin K proteolytic enzymes. Anti-osteoclastogenesis
drug inhibited RANKL-induced osteoclast differentiation, and cathepsin K protein
induction, which is associated with reduced NF-ƘB nuclear translocation [31]. In the
RANKL stimulation, the CO2 laser-irradiation hindered TRAF6-NF-ƘB-dependent
transcriptional signaling transduction. There was a inhibition in TRAF6 caused by the
CO2 laser-damaged osteoclast bone resorbing activity. The CO2 laser disturbed osteoclast
maturation and bone resorption by suppressing protein expression of key osteoclast
marker genes through the inhibition of RANK-mediated NF-ƘB signaling [32]. It was
found in the experiments with SN50 that the CO2 laser-irradiation mitigated the secretion
of TRAP and cathepsin K by deterring NF-ƘB transactivation.
In the clinical, the energy fluence range from 1 to 10 Jcm−2, and frequently, the
photochemical interactions can cause the biostimulation of several tissues at very lowpower densities. In contemporary dental practice, the orthodontist treatment is an
increasingly daily occurrence.
To discuss possible ways of using lasers for tissue regeneration, it is important to
know what effect laser parameters have on (a) different types of the cells; (b) different
components of the ECM; (c) signaling molecules produced by the cells and accumulated
14
in the ECM; (d) intercellular and cell-matrix inter-actions. The laser radiation may
provide controllable thermal and mechanical effects (as on the cells, as on the matrix)
resulting in activation of the cellular biosynthesis. From our present study, the CO2 laser
can be evaluated a painless, non-invasive and the thermal therapy that restores tissue
functionality through its bio-stimulation, anti-inflammatory and regenerative effects. The
application of LLLT as a post-orthodontic tooth treatment might increase tooth stability
[13]. In addition, we demonstrate that although most CO2 laser inhibits the
osteoclastgenic gene and protein expressions on Raw 264.7 cells. Consequently, CO2
laser lead to cell signal transduction and outcomes through similar mechanoreceptors and
signaling effectors in cells and tissues, and it is therefore suggested that common
signaling mechanisms are involved in laser-transduction pathways.
15
5. Conclusion
In summary, the current report demonstrates that CO2 laser weakened RANKLinhibits osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser is able to
modulate
every
cell
through
dose-dependent
in
vitro
RANKL-mediated
osteoclastogenesis, such as the function of osteoclasts. However, CO2 laser decreases
TRAF6 expression. Although these are in vitro findings and were gained under a
condition of CO2 laser, the current results serve as an improved explanation of the
cellular roles of macrophage cells populations in osteoclastogenesis as well as in alveolar
bone remodeling by CO2 laser-treatment.
16
Acknowledgements
The authors acknowledge receipt of a grant from the Chung Shan Medical
University Hospital and Chi Mei Medical Center under the project CSMU-CMMC-10204 and the National Science Council grants (NSC 102-2314-B-040-007-MY3) of Taiwan.
The authors declare that they have no conflicts of interest.
.
17
Author disclosure statement
The authors declare no competing financial interests.
18
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Figure Legends
Figure 1. The PrestoBlue® assay performed for viability macrophage cells treated with
CO2 laser for different times. *p < 0.05, compared with Ctl.
Figure 2. (A) The TRAP activity of Raw 264.7 cells cultured with DMEM contained
RANKL (50 ng/mL) and treated with CO2 laser for different times for 5 days. Values not
sharing a common letter are significant difference at p < 0.05. (B) Immunofluorescence
analysis showed promotion of RANKL-induced TRAP formation by tensile force. TRAP
was visualized with a FITC-conjugated secondary antibody and nuclei were blue.
Figure 3. . (A) MMP-9, and (B) integrin β3 gene expression were inhibited by CO2 lasertreatment. Cells were cultured with DMEM contained RANKL (50 ng/mL) and treated
with CO2 laser for different times for 5 days. To measure RANKL-induced MMP-9 and
integrin β3 expression by RT-PCR. Representative data were obtained from three
independent experiments, and β-actin gene was used as an internal control. The bar
graphs (means ± SEM, n = 3) represent quantitative results of the upper bands obtained
from a densitometer. Values not sharing a common letter are significantly different at p <
0.05.
Figure 4. RT-PCR data showing CO2 laser decreasing of RANKL-induced cathepsin K
(A) secretion and (B) gene expression. The bar graphs (means ± SEM, n = 3) represent
quantitative results of the upper bands obtained from a densitometer. Values not sharing a
common letter are significantly different at p < 0.05.
Figure 5. CO2 laser-treatment decreasing of RANKL-induced (A) TRAF6 expression,
and (B) IƘB phosphorylation in cells. The bar graphs (means ± SEM, n = 3) represent
quantitative results of the upper bands obtained from a densitometer. Values not sharing a
common letter are significantly different at p < 0.05.
Figure 6. (B) Inhibitory effects of SN50 on cathepsin K secretion and (C) gene
expression. The bar graphs (means ± SEM, n = 3) represent quantitative results of the
upper bands obtained from a densitometer. Values not sharing a common letter are
significantly different at p < 0.05.
22
Figure 7. Attenuation of NFATc1 and MITF formation were measure in CO2 lasertreated Raw 264.7 cell. The bar graphs (means ± SEM, n = 3) represent quantitative
results obtained from a luminometer. Respective means without a common letter differ, p
< 0.05.
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Fig 1
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Fig 2
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Fig 3
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Fig 4
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Fig 5
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Fig 6
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Fig 7
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