ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 55
OBJECTIVE: To investigate whether low-frequency
electromagnetic fields (EMFs) have any harmful effects
on alveolar bone and whether melatonin (MLT) and
Ganoderma lucidum (GL) have protective effects against
those harmful effects.
STUDY DESIGN: A total of 56 rats were divided into
7 groups and exposed to EMF produced from high volt-
age for 8 hours/day: groups 3 and 6, EMF+MLT (10
mg/kg/day); groups 1 and 4, just EMF; groups 2 and
5, EMF+GL (20 mg/kg/day); group 7, controls. Half of
the groups were sacrificed on day 26 and the rest on day
52. Histopathological and immunohistochemical analy-
ses of the specimens were performed.
RESULTS: Bone resorption, osteoblastic activity, bone
matrix, and osteocyte formation were found by histo­
pathological analysis to be statistically significant in the
EMF, EMF+MLT, and EMF+GL groups as compared
to the control group at day 26 (p<0.05). Bone resorp-
tion, osteoblastic activity, bone matrix, and osteocyte
formation were statistically significant in the EMF and
EMF+GL groups on day 52 (p<0.05). In the immu-
nohistochemical analyses there was a statistically sig-
nificant difference in the expression of osteopontin and
osteonectin of the EMF+MLT and EMF+GL groups
on days 26 and 52 as compared to the control and EMF
groups (p<0.05). There was a statistically significant
increase in the EMF+MLT group on day 52 as com-
pared to the other groups.
CONCLUSION: Exposure to EMF caused histological
changes in the alveolar bone of the rats. The use of MLT
and GL in treatment may be protective against these
harmful effects. (Anal Quant Cytopathol Histpathol
2019;41:55–66)
Keywords: alveolar bone; DNA damage; electro-
magnetic fields; electromagnetic radiation; EMF;
Ganoderma lucidum; lingzhi; low-frequency elec-
tromagnetic fields; mandible; medicine, traditional
Chinese; melatonin; mushrooms; plant extracts,
Chinese; reishi.
Electromagnetic radiation can be classified in 2
forms: ionizing radiation and non-ionizing radia­
tion. Non-ionizing electromagnetic radiation, both
natural and artificial and which humans are ex­
posed to continuously throughout their lives, can
present a variety of biological hazards.1
Strong electromagnetic fields (EMFs) with low
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4102-0055/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Protective Effect of Melatonin and Ganoderma
Lucidum Against the Negative Effects of
Extremely Low Frequency Electromagnetic
Fields on Alveolar Bone in Rats
An Experimental Animal Study
Abdulsamet Tanık, D.D.S., Ph.D., and Mehmet Cihan Yavas
, D.D.S., Ph.D.
From the Department of Periodontology, Faculty of Dentistry, Adıyaman University, Adıyaman, and the Department of Biophysics,
Faculty of Medicine, Ahi Evren University, Kırs
ehir, Turkey.
Dr. Tanık is Assistant Professor, Department of Periodontology, Faculty of Dentistry, Adıyaman University.
Dr. Yavas
 is Assistant Professor, Department of Biophysics, Faculty of Medicine, Ahi Evren University.
Address correspondence to: Abdulsamet Tanık, D.D.S., Ph.D., Department of Periodontology, Faculty of Dentistry, Adıyaman Univer-
sity, Siteler Mahallesi Ataturk Bulvari No. 411 PK: 02200 Center, Adıyaman, Turkey (samet.120a@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. frequencies between 50 and 60 cycles per second
are very harmful. It is possible to protect from an
electrical field produced by nearby electrical lines,
but it is difficult to protect against the magnet-
ic field from them.1 Long-term exposure to low-
frequency magnetic fields resulting from electri­
cal/electronic devices and electrical transmission
lines that we use frequently in daily life can cause
various health problems such as fatigue, irritabil-
ity, aggression, hyperactivity, sleep disturbances,
and emotional instability.2,3
Various studies have shown that exposure to
EMF affects the nervous system, body weight, tis-
sue morphology and histology, circulatory system,
hormonal system, and immune system.4
Exposure to EMF was shown to result in in-
creased phagocytic activity in macrophages and
electrolyte levels of brain tissue and to affect
embryonic development, enzyme, and cellular
changes in different experimental animal studies.5-7
In some studies, long-term exposure to EMF has
been reported to cause cancer formation, DNA
degradation, and changes in cell and tissues.8,9 In
addition to those studies, it is also noteworthy to
find studies that indicate that EMF exposure did
not cause any change in DNA synthesis.10,11 The
main cause of contradictory results may be due to
cellular and molecular changes in tissue depend-
ing on the exposure time of the EMF.12
Low-frequency EMFs have been reported to
cause oxidative and DNA damage by increasing
the production of oxidative products.13 In addi-
tion, this oxidative damage was found to cause a
decrease in the activity of some protein and anti-
oxidant enzymes that play an important role in
the cell cycle and apoptotic pathways, resulting
in increased cancer and childhood leukemia.14,15 In
addition, EMF exposure caused by mobile phones
has been reported to lead to attention deficit,
depression, decreased reflex speed, blurred vision,
and behavior impairment.16 High-frequency EMF
has been reported to have a cytotoxic and geno-
toxic effect on tissues.17
Melatonin (n-acetyl-5-methoxytrypamine) is an
important antioxidant and endogenous hormone
released into the blood mostly from the pineal
gland and other extra-pineal organs.18 Antioxidants
stimulate the release of enzymes and free radicals
in the body.19 It is accepted that melatonin has
anti-inflammatory properties and inhibits cancer
progression.20 Exposure to EMFs causes a decrease
in melatonin concentration.21 Ganoderma lucidum
is a traditional Chinese medicinal mushroom that
has been used for centuries to treat various dis-
eases in Eastern Asia. This mushroom species,
known as Ganoderma lucidum, is effective for the
prevention of immunological disorders, inflam-
mation, free radical production, and hypertension
and cancer treatment.22,23
Bone is a specialized connective tissue and forms
the most important building block of the biological
organism. Bone tissue is composed of osteoblast
and osteoclast cells, collagen fibers, and inorganic
salts. Bone structurally consists of cortical bone
on the outside and trabecular bone on the inside.24
Alveolar bone is made of a thin cortical bone that
forms a primary support structure for the man­
dibular and maxillary teeth and contains many
perforated regions. The thickness of the alveolar
bone differs in the mandible and maxilla.25 Ac-
cording to clinical data, the distance between ad-
jacent teeth, the morphology and quality of the
alveolar bone, the concavity, and the fissures on
the root surface can affect the shape of the alveo-
lar bone.26
Biochemical, biophysical, and biomechanical
changes affect bone cells. For this reason, bone
cells are very sensitive to changes in the EMF
around them. Many studies have reported that
low-frequency EMF exposure has biological effects
on teeth, periodontal tissues, and bone cells of jaw
bones.27-30
Recent studies have shown that electromagnet-
ic field application can stimulate osteoblasts. It
has been shown that this effect is via ion trans-
port canals.31 In some studies, low-frequency EMF
caused bone loss, but some studies reported that
low-frequency EMF increased trabecular bone
maturation, volume, and formation.32-35 In some
studies, however, low-frequency EMF had no ef-
fect on bone tissue. However, the interactive mech-
anisms between low-frequency EMF and bone tis-
sue are still not fully understood. In this study we
aimed to investigate whether low-frequency EMF
has any harmful effects on alveolar bone and
whether melatonin and Gano­
derma lucidum have
a protective effect against those harmful effects
histopathologically and immunohistochemically.
Materials and Methods
Dicle University Animal Experiments Ethics Com-
mittee approved the study (protocol number
2013/13). Operations on the experimental animals
were performed in the operating room at the
56 Analytical and Quantitative Cytopathology and Histopathology®
Tanık and Yavas


3. Prof. Dr. Sabahattin Payzın Health Sciences Re­
search and Application Center of Dicle University.
In this study, 56 healthy Wistar male albino rats
with an average weight of 250–300 g and aged
4 months were used. A 12-hour light/dark cycle
was provided to the experimental animals. The
rats were given free access to water and food,
and the room temperature was kept constant at
22±2°C. The air of the room was filtered, and the
risk of contamination was avoided, with the hu­
midity of the room maintained between 30–40%.
It was ensured that the rats were placed separately
in cages with sawdust so that they did not harm
each other.
Groupings of Experimental Animals
The rats used in the study were planned for 2 dif-
ferent time periods: 26 days and 52 days. These 2
applications were divided into 7 different groups,
each including 4 subgroups:
Group 1: 8 rats were exposed to EMF for 26 days
and sacrificed at day 26.
Group 2: 8 rats were exposed to EMF for 26
days and Ganoderma lucidum was administered
(EMF+GL). The rats were sacrificed at day 26.
Group 3: 8 rats were exposed to EMF for 26
days and melatonin was administered (EMF+
MLT). The rats were sacrificed at day 26.
Group 4: 8 rats were exposed to EMF for 52
days and sacrificed at day 52.
Group 5: 8 rats were exposed to EMF for 52
days and GL was administered (EMF+GL). The
rats were sacrificed at day 52.
Group 6: 8 rats were exposed to EMF for 52
days and melatonin was administered (EMF+
MLT). The rats were sacrificed at day 52.
Group 7: 8 rats were not exposed to any treat-
ment and were used as the control group.
Formation of EMF
To create EMF, 2 transformers and 10 kV (10,000
V) high voltage were used. For transformer 1, in-
put was 220 V and output was 10 kV. For trans-
former 2, the input was 10 kV and the output
was 220 V and 5,000 VA. Both 26-day and 52-day
experiment groups (groups 1, 2, 3, 5, and 6) were
exposed to EMF 8 hours a day (Figure 1). We mea-
sured the average magnetic field density (2.48 µT)
and electric field density (80.3 V/m) in the plexi-
glass cage. EMF was measured with the help of
a Spectran NF-5035 device (Aaronia AG, Strick­
scheid, Germany) using a 6-minute measure-
ment method (International Commission on Non-
Ionizing Radiation Protection).
Melatonin and Ganoderma lucidum were pre-
pared according to the animal weights and appro-
priate standards. For each rat in the 2 melatonin
groups, 10 mg/kg melatonin (Merck KGaA, Darm-
stadt, Germany) dissolved in pure ethanol and
then diluted with distilled water was adminis-
tered intraperitoneally daily. For each rat in the 2
GL groups, 20 mg/kg Ganoderma lucidum (Gano
Excel Industries Sdn. Bhd., Kedah, Malaysia) di-
luted with distilled water was administered by
oral gavage. At the end of the study, rats were
anesthetized using ketamine hydrochloride (Keta­
sol, Ricter Pharma, Welsh, Austria) and 3 mg/kg
2% xylazine hydrochloride (Xylazinbio, Bioveta,
Intermed Medicine, Ankara, Turkey) intramuscu-
larly. The rats were then euthanized by intracar-
diac lethal injection, and the alveoli bone was
surgically cut and removed and then alveolar
Volume 41, Number 2/April 2019 57
Effects of EMF on Alveolar Bone
Figure 1
Schematic view of the
experimental set-up.

4. bone samples were immediately placed in a 10%
formaldehyde solution in closed plastic boxes at
ambient temperature.
Histological Analysis
The samples were directly fixed in a neutral buff-
ered formalin solution in the histology laboratory.
After complete fixation the samples were held
for 12 hours to be rinsed under water. They were
then stored for 12 hours at a graduated increasing
alcohol concentration for dehydration. After trans-
parency within xylol, the tissues were infiltrated
and embedded in blocks. Paraffin blocks were
stained with hematoxylin-eosin for routine stain-
ing after receiving 5-μm-thick sections with a mi-
crotome (Rotatory Microtome RM 2265, Leica, Ger-
many).
Immunohistochemical Analysis
Osteopontin and Osteonectin Immunostaining Method.
The sections from the paraffin blocks were placed
on poly-L-lysine coated slides and held at room
temperature, then kept in a 60°C incubator for 1
night. After the sections cooled, they were kept
in the xylene 2 times for 5 minutes, then put on
ethyl alcohol for 5 minutes. After passing through
the concentrations of alcohol, they were kept in
distilled water for 5 minutes. Then they were put
to ethylenediamaminetetraacetic acid (EDTA) so-
lution to dissolve the bone tissue. The samples
taken on slides were surrounded by a dark open
pen (Huiyou, China) and kept in citric acid (pH
6.0) in a 700 W microwave oven for 7+5 minutes,
so the antigen masking was removed. They were
then cooled for 20 minutes at room temperature,
washed with phosphate-buffered saline (PBS) so-
lution for 3×5 minutes, and kept in 3% hydrogen
peroxide (H2O2) for 20 minutes to provide endog-
enous peroxide blockage. The sections were again
held in PBS for 3×5 minutes and taken into the
incubation vessel. All subsequent operations were
carried out in this incubation vessel. Blocking so-
lution was dripped and kept for 1 hour, and then
osteonectin (mouse monoclonal, 1/200, Santa Cruz
Biotechnology) and osteopontin (mouse monoclo-
nal, 1/200, Santa Cruz Biotechnology) antibodies
were applied to the sections. Primary antibody was
dripped and kept for 1 hour, then washed with
PBS solution for 3×5 minutes. After dripping the
secondary antibody (Histostain-Plus Kit, Invitro-
gen, Carlsbad, California), which is compatible
with the primary antibody, they were left in the
closed moist box at room temperature for 30 min-
utes. The secondary antibody (Zymed, Histostain-
Plus Kit, California, USA) marked with strepta-
vidin, immediately after 3×5 minutes of washing
with PBS solution, was distilled and rested for
30 minutes at room temperature and washed with
PBS solution for 3×5 minutes. Aminoethyl car-
bazole solution (AEC, Invitrogen) was dripped
as chromogen. They were washed with distilled
water to prevent the reaction of antigen-antibody.
Contrast staining was made with Mayer’s hema-
toxylin. The sample was again distilled with water
and closed with a cover slide. In the final stage,
the sections were viewed immunohistopathologi-
cally with a photomicroscope (Nikon Eclipse i50,
Japan) for blind evaluation.
Statistical Analysis
Statistical analysis was performed using SPSS
(IBM v. 21.0 Windows, USA) statistical program.
Histological data were presented as numerical
values, mean arithmetic values (M), and stan-
dard deviation (SD). The Mann-Whitney U test
was used to compare the data with nonnormal
distributions for binary comparisons, and the
Kruskal-Wallis test was used to compare multi­
ple comparisons. The Bonferroni-corrected Mann-
Whitney U test was used to compare more than
2 groups. P<0.05 was considered statistically sig­
nificant for all tests.
Results
The experimental animals smoothly passed the
26- and 52-day healing cycles in cages where ap-
propriate ambient conditions were prepared be-
fore surgery.
Histological Results
As a result of the hematoxylin-eosin staining pro-
cedure, the following results were obtained. His-
tological analysis on day 26 of group 1 revealed
small areas of bone resorption sites (yellow ar-
row) and local inflammation. Dilation and hemor-
rhage (blue arrow) in blood vessels in Haversian
canals in the lamellar bone, pyknosis, and apopto­
tic changes (green arrow) in osteocyte cell nuclei
were observed (Figure 2A). In Group 2, small
inflammatory cell infiltrations in bone matrix
areas (black arrow), hemorrhage in vessels in
Haversian canals, increased osteoclast in bone re-
sorption regions (red arrow), and osteoblast ac-
tivity along with the formation of a new matrix
58 Analytical and Quantitative Cytopathology and Histopathology®
Tanık and Yavas


5. in some areas (green arrow) were observed (Fig-
ure 2B). In Group 3 there was light hemorrhage
in the blood vessels of the Haversian lamella,
osteocytes in the lacunar structure showed regu-
lar settlement while the matrix development was
evident, and new bone trabeculae and osteoblas-
tic activity began to appear (red arrow) (Figure
2C). On histological analysis at day 52 in group
4, bone resorption sites (yellow arrow) were en-
larged and inflammatory cell infiltrations and in-
creased osteoclast (red arrow) were observed. Lo-
cal degeneration sites due to bone resorption were
seen (Figure 2D). In group 5, hemorrhage (yellow
arrow) and matrix formation areas due to osteo-
blastic activity (red arrow) in vessels between
bone lamella and lacunar structured osteocytes
were observed (Figure 2E). In group 6, Haversian
canals and lamellar structure were regular (yel­
low arrow), matrix areas were enlarged due to in-
creased osteoblastic activity (green arrow), and os-
teocytes formed radial alignment around Haver-
sian canals (Figure 2F). In Group 7 the Haversian
lamellar system, matrix structure, and osteocytes
in alveolar bone were uniformly distributed (Fig-
ure 2G).
Immunohistochemical Findings
As a result of the osteopontin immunostaining
procedure, the following results were obtained.
On the 26th day of osteopontin staining in Group
1, there was osteopontin-positive reaction (yel-
low arrow) in osteoclast cells in resorbed bone
areas (Figure 3A). On the 26th day of osteopon-
tin staining in group 2, there were osteoblasts
Volume 41, Number 2/April 2019 59
Effects of EMF on Alveolar Bone
Figure 2 (A) Histopathological examination at day 26 for group 1 displayed bone resorption sites (yellow arrow) and dilation and
hemorrhage (blue arrow) in blood vessels within Haversian canals of lamellar bones, and pyknosis and apoptotic changes (green arrow) in
the osteocyte cell nucleus. (B) A histopathologic section taken from group 2 on day 26; small inflammatory cell infiltrates in bone matrix
areas (black arrow), osteoclast increase in bone resorption regions (red arrow), and in some areas osteoblast activity (green arrow) with
new matrix formation were observed. (C) A histopathologic section taken from group 3 on day 26 displayed new bone trabeculae and
osteoblastic activity beginning to become evident (red arrow). (D) A histopathologic section taken from group 4 on day 52 displayed that
bone resorption zones (yellow arrow) were enlarged and osteoclasts were increased (red arrow). (E) A histopathologic section taken on
day 52 from group 5 displayed that hemorrhage (yellow arrow) and matrix formation areas (red arrow) due to osteoblastic activity were
clearly observed in the veins between the bone lamellae. (F) A histopathologic section taken from group 6 on day 52. Haversian canals
and lamellar structure were regular (yellow arrow), and enlarged matrix areas due to increased osteoblastic activity (green arrow) were
observed. (G) A Histopathologic section taken from group 7 displayed Haversian lamellar system and matrix structure in alveolar bone
and regularly distributed osteocytes. Hematoxylin-eosin staining. Bar=100 μm.

6. in bone matrix and osteopontin-positive expres-
sion in osteocytes (yellow arrow) (Figure 3B). On
the 26th day of osteopontin staining in group 3,
there were osteoblast and osteocyte cell density
due to increased osteoblastic activity and oste-
opontin expression (yellow arrow) in regular
lamella (Figure 3C). There were expanding areas
of bone resorption (red arrow) on the 52nd day
of osteopontin staining in group 4 (Figure 3D).
Osteopontin-positive expression (black arrow) in
osteoclast cells in bone resorbed areas (arrow)
on day 52 of osteopontin staining in group 5
was observed (Figure 3E). Osteopontin expres-
sion (arrow) due to the increase in osteoblast
cells and osteocytes on the 52nd day of osteo­
pontin staining in group 6 was seen (Figure 3F).
In group 7, osteopontin staining showed that ma-
trix distribution in the lamellar bone and osteocyte
cell arrangement were regular, and areas of osteo-
pontin expression (black arrow) were seen (Figure
3G).
As a result of the osteonectin immunostaining
procedure, the following results were obtained. Im-
munohistochemical analysis of osteonectin stain-
ing on day 26 of group 1 showed osteonectin
expression (red arrow) in the matrix area along
the bone lamellae (Figure 4A). Immunohistochem-
ical analysis of osteonectin staining on day 26 of
group 2 showed osteonectin expression (yellow
arrow) in the matrix and osteocytes around the
Haversian lamellae (Fig­
ure 4B). On the 26th day
of osteonectin staining in group 3, osteonectin-
positive expression (yellow arrow) was clearly
observed in the cells in the matrix areas around
the resorption bone sites (Figure 4C). On the
52nd day of osteonectin staining in group 4,
osteonectin-positive expression (red arrow) in
the matrix around the dilated Haversian lamel-
60 Analytical and Quantitative Cytopathology and Histopathology®
Tanık and Yavas

Figure 3 (A) An immunohistochemical section taken from group 1 on day 26. Osteopontin-positive reaction (yellow arrow) in osteoclast
cells in resorbing bone areas is shown. (B) The immunohistochemical section from group 1 on day 26. Osteoblast in bone matrix and
osteopontin-positive expression in osteocytes (yellow arrow) are shown. (C) An immunohistochemical section taken from group 3 on day
26. Osteoblast and osteocyte cell density due to increased osteoblastic activity and osteopontin expression (yellow arrow) at regular
lamellar structures are shown. (D) The immunohistochemical section on day 52 from group 4. Expanding bone resorption areas are seen
(red arrow). (E) Immunohistochemical section on day 52 from group 5. Osteopontin-positive expression (black arrow) in osteoclast cells
in resorbed bone areas are seen. (F) An immunohistochemical section taken on day 52 from group 6. Osteopontin expression due to an
increase in osteoblast cells and osteocytes (arrow) are seen. (G) Immunohistochemical section from group 7. Matrix distribution in
lamellar bone and areas of osteocyte cell lineage and osteopontin expression (black arrow) are seen. Osteopontin immunostaining.
Bar=50 μm.

7. lae and in periodontic fibroblasts outside the re-
sorbed bone were seen (Figure 4D). On the 52nd
day of osteonectin staining in group 5, osteo-
nectin expression (red arrow) outside the bone
resorption sites and osteonectin-positive expres-
sion (yellow arrow) in the area other than the
Haversian lamellae was clearly observed (Fig-
ure 4E). On the 52nd day of osteonectin stain-
ing in group 6, osteocytes displayed osteonectin-
positive reaction (black arrow) along with lacunae
(Figure 4F). On the 52nd day of osteonectin stain-
ing in group 7, the Haversian lamellae and osteo-
cytes showed positive osteonectin expression (blue
arrow) (Figure 4G).
Statistical Results
Histopathological Results (Statistical). For histopatho-
logical examination of the alveolar bone of the rats
taken at day 26 and day 52, the values of bone
resorption, osteoblastic activity, bone matrix, and
osteocyte formation scores were compared be-
tween all groups using the Kruskal-Wallis test.
A statistically significant difference was found
between all groups (Table I) (p<0.001).
The Mann-Whitney U test with Bonferroni cor­
rection was applied to 26-day groups that were
found to be significant using the Kruskal-Wallis
test. There was a significant difference in bone re-
sorption scores between groups 1 and 3, groups 1
and 7, groups 2 and 7, and groups 3 and 7. Com-
parison of osteoblastic activity scores resulted in
the difference between groups 1 and 2, groups 1
and 3, groups 1 and 7, groups 2 and 7, and
groups 3 and 7. Comparison of bone matrix scores
showed a significant difference between groups 1
and 3, groups 1 and 7, groups 2 and 7, and groups
Volume 41, Number 2/April 2019 61
Effects of EMF on Alveolar Bone
Figure 4 (A) An immunohistochemical section taken from group 1 on day 26. Osteonectin expression in the matrix area along the
bone lamellae (red arrow) are seen. Osteonectin immunostaining. Bar=50 μm. (B) An immunohistochemical section taken from group 2
on day 26. Osteonectin expression in the matrix and osteocytes around the Haversian lamellae (yellow arrow) are shown. (C) An
immunohistochemical section taken from group 3 on day 26. Positive expression of osteonectin (yellow arrow) in cells in matrix areas
around the resorbed bone. (D) The immunohistochemical section on day 52 from group 4. Osteonectin-positive expression (red arrow)
around the dilated Haversian lamellae, osteonectin-positive expression (yellow arrow) in the periodontal fibroblasts outside resorbed
bone. (E) The immunohistochemical section on day 52 from group 5. Osteonectin expression (red arrow) in the area other than the bone
resorption sites outside and osteonectin-positive expression (yellow arrow) in the area other than the Haversian lamellae. (F) An
immunohistochemical section taken at day 52 from group 6. Osteocyte-positive reaction of osteocyte cells with lacunae are seen (black
arrow). (G) Immunohistochemical section from group 7. Haversian lamellae and osteonectin-positive expression of osteocytes are seen
(blue arrow). Osteonectin immunostaining. Bar=100 μm.

8. 3 and 7. As a result of comparison of osteocyte
formation scores between groups, statistically, a
significant difference was found between groups
1 and 7, between groups 2 and 7, and between
groups 3 and 7 (Table I).
The Mann-Whitney U test with Bonferroni cor-
rection was applied to the 52-day rats which were
found significant using the Kruskal-Wallis test.
Bone resorption scores were compared, and there
was a significant difference between all groups.
There was a significant difference in the osteoblas-
tic activity scores between all groups except for
groups 4 and 5. In the comparison of osteoblastic
activity scores there was a significant difference
between groups 4 and 5. In the comparison of
bone matrix scores, there was a significant differ-
ence between groups 5 and 6 and between groups
6 and 7. In the comparison of osteocyte formation
scores there was a statistically significant differ-
ence between all groups except for between groups
6 and 7 (Table II).
Statistical Findings of Immunohistochemical Analyses.
The Mann-Whitney U test was performed for the
examination of the number of cells that were os-
teopontin- and osteonectin-positive in the sections
of samples taken from the alveolar bone of the
rats on day 26. There were significant differences
between all groups except for between groups 1
and 4, as well as groups 2 and 3. Also, in the 52-day
rats, there were significant differences between all
groups except for between groups 4 and 7 (Figure
5) (p<0.05).
Discussion
As a result of the increase in the use of tech-
nological electronics devices, exposure to low-
frequency EMF is increasing. It is thought that
due to the excess of this exposure, the health risk
will be higher. Depending on exposure to EMF,
some studies reported cases of cancer, neurode­
generative diseases, hypersensitivity and heart dis-
eases, and congenital anomalies.36-38
In some experimental animal studies, low doses
and controlled applications of EMFs did not have
any negative effects, although high-intensity EMF
exposure resulted in hematological disorders, func-
tional disorders in nervous and digestive systems,
and increased cancer frequency in childhood.15,39
EMFs form an electromagnetic field at 50–60
Hertz (Hz) frequency in an electromagnetic spec-
trum (between 3 and 3000 Hz) at an extremely
62 Analytical and Quantitative Cytopathology and Histopathology®
Tanık and Yavas

Table I Comparison of Histopathological Values of Rats on Day 26
Group 1 Group 2 Group 3 Control p Value p Value p Value p Value p Value p Value
(mean±SD) (mean±SD) (mean±SD) (mean±SD) p Value 1-2 1-3 1-7 2-3 2-7 3-7
Bone resorption 3.29±0.49 2.71±0.49 1.86±0.69 0.14±0.38
<0.001** 0.054 0.003* 0.001* 0.026* 0.001* 0.001*
Osteoblastic activity
0.43±0.53 1.86±0.70 2.14±0.69 3.86±0.38
<0.001** 0.004* 0.002* 0.001* 0.431 0.001* 0.001*
Bone matrix 1.29±0.76 1.86±0.69 2.29±0.49 3.71±0.48
<0.001** 0.184 0.015* 0.001* 0.202 0.002* 0.002*
Osteocyte formation
2.14±0.38 2.00±0.58 2.57±0.54 3.71±0.48
<0.001** 0.593 0.107 0.001* 0.081 0.002* 0.005*
Values are given as M (arithmetic mean)±SD (standard deviation).
*p<0.05, significant between groups.
**p≤0.001, very significant between groups.
p: Kruskal-Wallis test. p1-2, p1-3, p1-7, p2-3, p2-7, and p3-7: Mann-Whitney U test.
Table II Comparison of Histopathological Values of Rats on Day 52
Group 4 Group 5 Group 6 Control p Value p Value p Value p Value p Value p Value
(mean±SD) (mean±SD) (mean±SD) (mean±SD) p Value 4-5 4-6 4-7 5-6 5-7 6-7
Bone resorption 1.86±0.69 2.43±0.54 1.43±0.53 0.14±0.38
<0.001**
0.004* 0.001* 0.001* 0.010* 0.001* 0.002*
Osteoblastic activity
0.71±0.76 1.43±0.79 2.71±0.48 3.86±0.38
<0.001** 0.104 0.002* 0.001* 0.005* 0.001* 0.002*
Bone matrix 1.29±0.48 2.29±0.76 3.14±0.69 3.71±0.48
<0.001** 0.020* 0.002* 0.001* 0.053 0.004*
0.100
Osteocyte formation
1.57±0.53 2.57±0.54 3.29±0.48 3.71±0.48
<0.001**
0.010* 0.001* 0.001* 0.030* 0.005*
0.122
Values are given as M (arithmetic mean)±SD (standard deviation).
*p<0.05, significant between groups.
**p≤0.001, very significant between groups.
p: Kruskal-Wallis test. p4-5, p4-6, p4-7, p5-6, p5-7, and p6-7: Mann-Whitney U test.

9. Volume 41, Number 2/April 2019 63
Effects of EMF on Alveolar Bone
and frequency of pulsed EMF (5000 V/m at 1590
V and 60 Hz) for 14 days and reported that EMF
delayed bone healing in histological evaluation.46
Leisner et al47 used a generator with a 20 cm di-
ameter bobbin as a power source that could gener-
ate EMF, for rats, using the ulnar osteotomy model.
They also stated that low intensity and frequency
pulsed electromagnetic field (DFDEMA) does not
increase or accelerate the recovery of newly formed
ulnar fractures but rather tends to delay the onset
of formation of callus tissue and causes it to be
smaller after it begins to develop. Our study also
showed a significant decrease in bone matrix and
osteocyte formation in the EMF group as com-
pared to the control group at 26 and 52 days,
consistent with findings from other studies.
Hannay et al48 examined the responses of cells
similar to the EMF osteoblasts using 4 different
time protocols and reported that they did not
support bone development. De Barros Filho et
al49 reported that they did not find any signifi-
cant radiological, clinical, or histological differ-
ences in view of bone development. In our study,
both on the 26th and 52nd day, the EMF group
showed a significant decrease in osteoblastic ac-
tivity as compared to the control group.
Experimental studies on the effects of 900 and
1800 MHz radio frequency EMFs from mobile
phones and similar sources on bone tissue have
reported minimal changes in bone mineral den-
sity.50 Çiçek et al51 reported that, in rats exposed
to radio frequency EMF, bone fracture strength,
bending resistance, and total fracture energy de-
creased. In our study, a high degree of increase
in bone resorption in the EMF group as com-
pared to the control group at day 26 and day 52
was in agreement with the result of the mentioned
study.
There are many studies in the literature report-
ing that EMFs have positive or negative effects on
bone tissue and fracture healing, as well as stud-
ies that report no effect. According to several of
these studies, EMFs inhibited bone tissue miner­
alization and osteoclastic activity. It also contrib-
utes to the healing of bone fractures by affecting
the formation of fibrosis tissue and granulation
and helping to heal the wound.40,52 However,
some studies have reported that the EMFs have
a detrimental effect on the differentiation of peri-
odontal tissues, mineral density, and osteoblasts in
jaw bones.45,53 Melatonin modulates bone markers
associated with protection against bone loss and
low frequency.40 The International Commission on
Non-Ionizing Radiation Protection accepts general
exposure limits at 50 Hz frequency as 5000 V/m
for the electric field and as 100 µT for the magnetic
field.41 Ijiri et al42 used pulsating EMF for 10 hours
in their study. However, Matsumoto et al43 used
pulsating EMFs in 2 groups, 4 and 8 hours per
day, in 2 different time zones. We also used 2.48
µT intensity of the magnetic field of 10,000 V and
the electric field intensity was 80.3 V/M, and EMF
was applied for 8 hours each day.
There are very limited clinical and laboratory
studies of EMF on the oral tissues. In addition to
traditional periodontal therapy, EMF stimulation
was found not to provide improvement in clinical
attractor gain or alveolar bone healing.44 In another
study, it was reported that direct electrical current
is a powerful biological agent and accelerates the
turnover of periodontal tissues and alveolar bone
initially by affecting cellular enzymatic phosphor-
ylation activity.45
Marino et al46 studied rats with fibular osteoto-
my that were externally exposed to low intensity
Figure 5 Values of the immunohistochemical analysis of rats
on day 26 and day 52. The osteopontin- and osteonectin-positive
expression in the alveolar bone tissues increased significantly in
the EMF+MLT group on day 52. The values are given in the form
of average positive-stained osteopontin and osteonectin cell
numbers in immunohistochemical sections.
*p<0.05, the difference is significant between groups.
**p≤0.001, the difference is very significant between groups.
p, Mann-Whitney U test.

10. 64 Analytical and Quantitative Cytopathology and Histopathology®
Tanık and Yavas

veolar bone tissue allowed bone to repair itself and
to form bone matrix.
Conclusion
In this study, it can be concluded that EMFs are
harmful and may have negative effects on the
alveolar bone, depending on the intensity and
frequency of EMFs. Changes in the structure of
the alveolar bone are important for tooth loss
and oral health. These changes are indispensable
for raising the standard of life and nutrition. Mel-
atonin and Ganoderma lucidum, with antioxidant
properties, are able to reduce damage to the al-
veolar bone resulting from exposure to EMFs.
However, to protect against the harmful effects of
EMF in individuals who work in environments
that have high EMF emission, it may be advisable
to consume foods and beverages that increase the
amount of melatonin. In addition, treatment pro-
cedures involving melatonin can be useful. Further
work is needed to determine certain treatment pro-
cedures in this regard.
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