Received: 30/10/2024 Accepted: 26/12/2024 Published: 24/02/2025 1 of 10
https://doi.org/10.52973/rcfcv-e35556 RevistaCientíca,FCV-LUZ/Vol.XXXV
ABSTRACT
Osseointegration is a challenge in the dental implant treatment of
individuals with osteoporosis. Genistein is a phytoestrogen with
benecial effects in the prevention of osteoporosis. This study aims
to evaluate the protective effects of genistein supplementation on
the osseointegration level of titanium implants in ovariectomized
rats. The rats in this study were randomly divided into 5 groups
with 8 rats in each group: Control, Implant, Ovariectomy–Implant,
Ovariectomy–Implant–Genistein, Implant–Genistein. The implants
were surgically integrated tibial bones of rats. Genistein was
administered at 2 mg·kg
-1
by oral gavage three times a week. All
rats were sacriced at the end of 3 months. Biochemical analyses
were made from the blood serum of the rats, histomorphometric
analyses from the implant and surrounding tissues placed in the
tibia, and bone mineral density analyses from the mandibles.
The bone implant connection (BIC) ratio of the Control–Implant
group was higher than the other groups (P<0.05). The BIC
ratio of the Ovariectomy–Implant group was lower than the
Ovariectomy–Implant–Genistein and Implant–Genistein groups
(P<0.05). In terms of thread lling, no statistically signicant
difference was found between the groups (P>0.05). The jaw bone
mineral density (BMD) of the control group was higher than the
Ovariectomy–Implant and Implant–Genistein groups (P<0.05). In
ovariectomy–Implant–Genistein group jaw BMD was higher than
the Ovariectomy–Implant and Implant–Genistein groups (P<0.05).
In conclusion, it can be stated that genistein can improve the
negative effects of ovariectomy on the bone and increase implant
osseointegration. Genistein consumption may increase implant
osseointegration in osteoporotic cases.
Key words: Osseointegration; ovariectomy; osteoporosis;
phytoestrogen; genistein
RESUMEN
La osteointegración es un desafío en el tratamiento con implantes
dentales de individuos con osteoporosis. La genisteína es un
toestrógeno con efectos beneciosos en la prevención de la
osteoporosis. Este estudio tiene como objetivo evaluar los efectos
protectores de la suplementación con genisteína en el nivel de
osteointegración de implantes de titanio en ratas ovariectomizadas.
Las ratas en este estudio se dividieron aleatoriamente en 5 grupos
con 8 ratas en cada grupo: Control, Implante, Ovariectomía–
Implante, Ovariectomía–Implante–Genisteína, Implante–
Genisteína. Los implantes fueron huesos tibiales de ratas integrados
quirúrgicamente. La genisteína se administró a 2 mg·kg
-1
por sonda
oral tres veces por semana. Todas las ratas fueron sacricadas
al nal de los 3 meses. Se realizaron análisis bioquímicos del
suero sanguíneo de las ratas, análisis histomorfométricos del
implante y los tejidos circundantes colocados en la tibia y análisis
de densidad mineral ósea de las mandíbulas. La relación de
conexión ósea del implante (BIC) del grupo Control–Implante
fue mayor que los otros grupos (P<0,05). La relación BIC del grupo
Ovariectomía–Implante fue menor que los grupos Ovariectomía–
Implante–Genisteína e Implante–Genisteína (P<0,05). En cuanto
al relleno de ranura, no se encontró diferencia estadísticamente
signicativa entre los grupos (P>0,05). La densidad mineral ósea
de la mandíbula (BMD) del grupo control fue mayor que los grupos
Ovariectomía–Implante e Implante–Genisteína (P<0,05). Al igual
que los grupos Ovariectomía–Implante e Implante–Genisteína
(P<0,05). En conclusión, se puede afirmar que la genisteína
puede mejorar los efectos negativos de la ovariectomía sobre el
hueso y aumentar la implante osteointegración. El consumo de
genisteína puede aumentar la osteointegración del implante en
casos osteoporóticos.
Palabras clave: Osteointegración; ovariectomía; osteoporosis;
toestrógenos; genisteína
The effects of Genistein on osseointegration of Titanium implants in
experimental ovariectomized model
Efectos de la Genisteína en la oseointegración de implantes de
Titanio en un modelo experimental ovariectomizado
Cansu Busra Uzun
1
, Serkan Dundar
2,3
* , Mustafa Kom
4
, Tuba Talo Yildirim
2
, Alihan Bozoglan
2
, Tansel Ansal Balci
5
,
Cemal Orhan
6
, Kazim Sahin
6
1
Ministry of Health, Atasehir Dental and Oral Health Hospital, Department of Peridontology. Istanbul, Türkiye.
2
Firat University, Faculty of Dentistry, Department of Periodontology. Elazig, Türkiye.
3
Firat University, Faculty of Science, Department of Statistics. Elazig, Türkiye.
4
Firat University, Faculty of Veterinary Medicine, Depatment of Surgery. Elazig, Türkiye.
5
Firat University, Faculty of Medicine, Department of Nuclear Medicine. Elazig, Türkiye.
6
Firat University, Faculty of Veterinary Medicine, Depatment of Animal Nutrition. Elazig, Türkiye.
Corresponding author: sdundar@rat.edu.tr
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INTRODUCTION
In treating partial and complete edentulism, osseointegrated
dental implant–supported prosthetic treatment is a scientically
accepted method. Osseointegration is the healing process in which
clinically asymptomatic rigid xation of alloplastic materials are
achieved and maintained during functional loading [1, 2]. There are
several factors that affect the osseointegration of dental implants,
including surgical considerations, bone quality and quantity and
host–related factors such as the patients’ nutritional status.
Many micronutrients play an important role in dental implant
osseointegration by affecting a number of alveolar bone parameters
such as bone healing after tooth extraction [3].
Osteoporosis has been dened as a systemic skeletal disease
characterized by low bone mass and deterioration of the
microarchitecture of bone tissue, resulting in increased bone
fragility and susceptibility to fracture. Osteoporosis, which causes
massive bone loss in trabecular and cortical bones, affects the
jaw bones as well as other body bones. Studies have shown
that osteoporosis adversely affects bone–implant connection,
especially in the trabecular bone. Therefore, osteoporosis is
considered a relative contraindication for dental implants [4].
Estrogen deciency decreases osteoblast activity and increases
osteoclast activity, thus reducing bone mass. In addition, estrogen
deciency causes an increase in the release of proinflammatory
cytokines such as the tumor necrosis factor–alpha (TNF–a),
interleukin (IL-6) and (IL-1) from osteoblasts, monocytes and
macrophages [5]. These cytokines stimulate stromal cells and
preosteoblasts and secrete factors such as macrophage colony–
stimulating factor, receptor activator of nuclear factor κ–β ligand
(RANKL), IL-6, IL-1, which stimulate the proliferation of osteoclast
precursors or osteoclastogenesis [6, 7].
RANKL also plays an
important role in cases of malignancy or in postmenopausal
osteoporosis, where pathological bone loss is seen [8]. In estrogen
deciency, RANKL production increases and osteoprotegerin (OPG)
decreases in osteoblasts. Osteoprotegerin plays a role in reducing
the production and activity of RANKL. IL-1α, TGF–β, IL-1β, TNF–α,
estrogen and 1,25(OH)2 vitamin D3 stimulate the secretion of
osteoprotegerin from osteoblasts [1]. This event shortens the
life span of osteocytes and osteoblasts and plays a role in the
pathogenesis of senile osteoporosis by causing a decrease in
osteoblastogenesis [9].
Phytoestrogens are a large group of heterocyclic phenols with
a chemical structure similar to estrogen. Phytoestrogens which
are abundant in plants, have received increasing attention as a
dietary component that can affect various aspects of human
health. It has been recently demonstrated that phytoestrogens
can prevent osteoporosis. The positive effect of synthetic
phytoestrogen application on bone tissue has been shown in the
bones of postmenopausal women and osteoporotic experimental
animals. Genistein is a typical soybean isoflavone that acts as a
phytoestrogen. Genistein is the major phytoestrogen in soybeans.
In 1946 the discovery that the cause of infertility in sheep living
in Australia was due to genistein in ground clover eaten by sheep
supported the estrogenic effect of genistein. In 1987, genistein
was found to be a potent and specic repressor of protein kinase.
Since most cancer gene codes are tyrosine kinase–dependent, this
discovery offers hope for cancer treatment. Genistein competes with
estrogen due to defect that it resembles estrogen in structure [5, 6
,7]. The bone–retaining effects of genistein are due to the low doses
of the agonistic effect of this phytoestrogen on estrogen receptors in
osteoblasts. Osteoblasts or their precursors can secrete cytokines
with inhibitory or stimulating effects on osteoblasts in response
to genistein or parathyroid hormone (PTH). Genistein stimulates
the transformation of osteoblast progenitor cells into osteoblasts
while suppressing osteoclast progenitor cells. In addition, genistein
interacts with estrogen receptors in osteoblasts and creates a
suppressive effect on osteoclasts. Therefore, genistein can indirectly
prevent or suppress bone resorption [5, 6, 7, 8].
Genistein offers an alternative treatment option to estrogen
supplementation for perimenopausal and postmenopausal women.
It acts as a selective estrogen receptor modulator (SERM) that
provides an alternative treatment for menopausal signs and
symptoms. In addition to treating osteoporosis, its properties
such as improving cardiovascular health, bone density and skin
quality make genistein a good treatment option for perimenopausal
and postmenopausal women [7, 8].
The aim of this study was to examine the effect of genistein, a
phytoestrogen, on the level of bone–implant connection in titanium
implants applied to the tibia of rats that underwent an ovariectomy.
MATERIAL AND METHODS
Animals and study design
All experimental and surgical procedures in this study were
performed in Firat University Experimental Research Center in
Elazig, Turkiye. The study was approved by the Firat University
Animal Experimental Ethics Committee (Protocol Number: 2018/43).
The recommendations of the Helsinki Declaration regarding the
protection of laboratory research animals were followed.
In this study, 40 healthy adult female SpragueDawley rats (Rattus
norvegicus) of 240–260 g; (Balance Shimadzu, Japan) aged 3–3.5
months were used.
All rats included in the study were obtained from Firat University
Experimental Research Center, Elazig, Turkiye. The rats were kept
in plastic containers and their temperatures were checked daily.
During the experiment, the rats were not limited in terms of food
and water, and the light cycle in the lab was adjusted to rotate
through 12 hours (h) of darkness and 12 h of light. All rats were
selected in the same estrus period to ensure standardization
throughout the experimental protocols. The rats were divided
into 5 groups with 8 rats in each group.
Control group (n=8): No additional procedures were applied to
the rats in this group during the 3–month experimental setup. At the
end of the 3–month experimental period, the rats were sacriced.
Control–implant group (n=8): Under general anesthesia, TiAl
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implants with a 2.5 mm diameter and a 4 mm length were placed
in the corticocancellous bone in the metaphyseal parts of the right
tibia bones of the rats, and no additional application was done
until the end of the study. At the end of the 3–month experimental
period, following the surgical placement of the implants, the rats
were sacriced.
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Ovariectomy–implant group (n=8): Ovariectomy was performed
on the subjects in this group under general anesthesia; TiAl
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implants with a 2.5 mm diameter and a 4 mm length were placed
into the corticocancellous bone in the metaphyseal parts of the
right tibia bones of the rats in the same session. At the end of the
3–month experimental period, following the surgical placement
of the implants, the rats were sacriced.
Ovariectomy–implant–genistein group (n=8): Ovariectomy was
performed on the rats under general anesthesia; TiAl
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4
implants
with a 2.5 mm diameter and a 4 mm length were placed into the
corticocancellous bone in the metaphyseal parts of the right tibia
bones of the rats in the same session. Following this procedure,
2 mg·kg
-1
genistein was given to the rats by oral gavage 3 times
a week for 3 months. At the end of the 3–month experimental
period, following the surgical placement of the implants, the rats
were sacriced [8].
Implant–genistein group (n=8): TiAl
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4
implants with a 2.5 mm
diameter and a 4 mm length were placed into the corticocancellous
bone in the metaphyseal parts of the right tibia bones of the rats.
Following this procedure, 2 mg·kg
-1
genistein was given to the rats
by oral gavage 3 times a week for 3 months. At the end of the 3–
month experimental period, following the surgical placement of
the implants, the rats were sacriced [8].
Surgical procedures
Surgical procedures applied to the rats in the study groups were
performed under general anesthesia by intramuscular injection
of anesthetics (10 mg·kg
-1
xylazine, 40 mg·kg
-1
ketamine). After
shaving the surgical areas of the subjects that were administered
general anesthesia, antisepsis of the surgical areas was provided
with povidone–iodine. In the rats that underwent an ovariectomy,
the abdominal cavity was opened with a 2 cm transverse incision
in the midline of the abdomen (FIGS. 1 A,B). The ovaries were
dissected and excised bilaterally; the abdominal wall, subcutaneous
tissues and skin were sutured separately with 5/0 vicryl; the wound
was closed primarily (FIGS. 1 C,D) [9].
In this study, specially produced titanium implants were used
(TiAl
6
V
4
) and a specially produced application set was used to apply
them. The implants were 2.5 mm in diameter, 4 mm in length and
in screw form with an Resorbable Blast Material (RBM) surface
structure (Implance Implant Systems, AGS Medical Corporation,
Istanbul, Türkiye). An incision was made from the proximal cranial
part of the right tibial bone to access the surgical eld. The soft
tissues and periosteum were dissected with a periosteal elevator
and bone tissue was reached. The corticocancellous bone platform
was exposed in the metaphyseal region of the right tibias, and the
implant sockets were prepared perpendicular to the bone surface.
Irrigation was performed with sterile saline in the surgical area to
prevent heating while opening the implant sockets [1].
FIGURE 1. Shaving the surgical (A) area and opening the abdominal cavity with a transverse incision (B). Bilateral excision of the
ovaries by dissection (C, D)
Osseointegration in ovariectomy model with Genistein / Uzun et al.__________________________________________________________________
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The implant sockets were prepared with an initial bur (AGS
Medical Implance Implants, Istanbul, Türkiye) with a diameter of
1.8 mm at a revolution speed of 500-600 rpm with physiodispenser
(WH DENTAL İmplantmed Classic, Bürmoos, Austria), then an
intermediate bur (AGS Medical Implance Implants, Istanbul,
Türkiye) with a diameter of 2.2 mm and a nal bur (AGS Medical
Implance Implants, Istanbul, Türkiye) with a diameter of 2.5 mm.
The implantation process was completed by placing the implants
in the sockets with the special carrying parts. After the implants
were placed, the periosteum and soft tissues were repositioned in
their original positions with 4-0 silk sutures. Antibiotics (40 mg·kg
-1
cephalosporin) and analgesics (0.1 mg·kg
-1
tramadolhydrochloride)
were administered intramuscularly for 3 d to prevent infection and
pain after surgical procedures [1].
Genistein administration
Genistein, (DSM Nutritional Products Ltd, Research and
Development, Switzerland) containing at least 98% genistein,
was used as genistein. Genistein was weighed (WL 603 Digital
Precision Scale, USA) at a precision of 2 mg·kg
-1
for each subject
and diluted with physiological saline. Rats in the Ovariectomy–
Implant–Genistein and Implant–Genistein groups were fed with
this prepared solution 3 times a week for 3 months by oral gavage.
The genistein dose used in this study was selected based on a
previous study [8].
Collecting blood samples and biochemical analysis
Just before the sacrication procedure of the rats, blood was
taken from their hearts with 10 mL injectors. The blood taken was
placed in 10 mL gel tubes and kept for 10 min, and centrifuged
(NF 200, NÜVE, Ankara, Turkiye) 1007 g force. After the blood
samples were centrifuged serum samples were obtained; alkaline
phosphatase (ALP), calcium (Ca), phosphorus (P), aspartate
aminotransferase (AST), alanine aminotransferase (ALT) rates
were analyzed with the photometric method (Advia Chemistry
XPT, Siemens, Healthineers, Erlangen, Germany) in Firat University
Faculty of Medicine Biochemistry Laboratory.
Nondecalcied histomorphometric analysis
Histological examination of the titanium implants were analyzed
according to the undecalcied preparation method after they were
removed together with the surrounding bone. After the implants
and the surrounding bone tissue were removed as a block from
the soft tissue, they were kept in a 4% buffered formalin solution
for at least 24 h. The samples were dehydrated and embedded in
photopolymerized methylmethacrylate. The implants were cut with
a precision cutting (EXACT Technologies Inc., USA) device from
the middle with the surrounding bone tissue. After this process,
the samples were abraded with a precision sander and sections
with a 50 µm thickness were obtained from each sample. These
sections were stained with toulidine blue for histomorphometric
analysis and the samples surfaces were covered with a coverslip
using methyl methacrylate. Digital images of all samples at
4×, 10× optical magnications were taken and recorded with a
digital camera connected to a light microscope (Nikon, Japan).
Histological bone–implant connection (BIC) (%) and thread lling
ratios (%) measurements were made for each sample using the
Image Analysis Program (Nikon, Japan). The ratio of the total
surface length in contact with the bone to the circumference of
the implant was evaluated as the percentage (%) of bone–implant
connection (BIC) for each implant. The thread lling (TF) ratio for
each implant; was calculated as the percentage (%) of bone–lled
thread area to the total thread area [10, 11].
Evaluation of bone mineral density
Densitometric evaluation was made using the software in Dual
Energy X–Ray Absorptiometry (DEXA) (Hologic® QDR 4500C
Acclaim Series Elite/N:49458, USA) device of Firat University
Faculty of Medicine, Department of Nuclear Medicine, Version 12.3
package. In the evaluation of the mineral density (BMD), bones of
the jaws and right femurs of the rats were recorded.
Statistical analysis
The IBM SPSS Statistics 22 (IBM SPSS, Turkiye) program was
used for statistical analysis. While evaluating the study data, the
conformity of the parameters to normal distribution was evaluated
with the Shapiro Wilks test. While evaluating the study data, in
addition to descriptive statistical methods (mean, standard deviation,
frequency), in comparison of quantitative data, parameters that did
not show normal distribution were found between groups. The
Kruskal Wallis test was used in comparisons and the Mann Whitney
U test was used to determine the group that caused the difference.
Signicance was evaluated at the P<0.05 level.
RESULTS AND DISCUSSION
Biochemical parameters as can be seen in TABLE I. ALP levels
in the implant–genistein group were lower compared to all other
groups (P<0.05). While serum Ca levels in the control group were
higher than the Ovariectomy implant and Ovariectomy implant
genistein groups (P<0.05), there was no difference in Ca levels
between the other groups (P>0.05). P levels in the control group
were detected to be higher than the Ovariectomy implant and
Ovariectomy implant genistein groups (P:0.001; P<0.05). P levels
of the Ovariectomy implant group were lower than those of the
Ovariectomy implant genistein and Implant genistein groups
(P<0.05). On the other hand, there was no difference in AST levels
between any groups (P>0.05). However, ALT levels in the implant
genistein group was lower compared to Control, Control implant,
Ovariectomy implant and Ovariectomy implant genistein groups
(P<0.05). There was no difference between the other groups in
terms of ALT levels (P>0.05).
As seen in TABLE II; there was a difference between the groups
in BIC levels (P<0.05). The highest BIC level was detected in the
controls (FIG. 2) (P<0.05).
The BIC levels in the ovariectomy implant group (FIG. 3) were
lower than the ovariectomy implant genistein (FIG.4) and implant
genistein group (FIG. 5) (P<0.05).
When the groups were evaluated in terms of thread lling, no
statistically signicant difference was found between the groups
(P>0.05). As seen in the TABLE III, there was a difference in
mandibular bone mineral density (BMD) between the groups
(P<0.05), while the highest BMD was found in the control
group (P<0.05).
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TABLE I
Statistical data of ALP, Ca, Phosphorus, AST and ALT parameters of the groups
Groups
ALP (U·L
-1
) Ca (mg·dl
-1
) Phosphorus (mg·dl
-1
) AST (U·L
-1
) ALT (U·L
-1
)
Mean ± SD (median) Mean ± SD (median) Mean ± SD (median) Mean ± SD (median) Mean ± SD (median)
Control(N=8) 78.88 ± 14.97(74) 10.91 ± 0.28(10.8) 5.85 ± 0.32(5.8) 206.75 ± 32.01(199.5) 73.88 ± 7.53(73)
ControlImplant(N=8) 99.25 ± 50.39(87.5) 10.92 ± 1.04(10.6) 5.56 ± 0.53(5.6) 210.5 ± 28.14(203) 74.5 ± 13.31(68.5)
OvariectomyImplant(N=8) 109 ± 52.39(98) 10.42 ± 0.39(10.4) 5.05 ± 0.22(5.1) 216.25 ± 41.17(230.5) 79.75 ± 13.53(78)
OvariectomyImplantGenistein(N=8) 109.5 ± 28.07(120.5) 10.26 ± 0.26(10.3) 5.35 ± 0.14(5.4) 239.88 ± 39.23(247.5) 86.13 ± 14.59(88)
ImplantGenistein(N=8) 53.88 ± 12.9(52) 10.57 ± 0.52(10.5) 5.56 ± 0.28(5.6) 194.5 ± 45.85(185.5) 64.5 ± 22.7(57.5)
P–value 0.009* 0.020* 0.001* 0.163 0.015*
KruskalWallisTest *
P<0.05
TABLE II
Bone implant connection (BIC) and Thread Filling
(TF) parameters of the groups
Groups
BIC (%) TF (%)
Mean ± SD (median) Mean ± SD (median)
ControlImplant(N=8) 67.6 ± 9.44(68) 48.25 ± 8.83(48.8)
OvariectomyImplant(N=8) 43.26 ± 6.09(44) 35.73 ± 15.46(29.4)
OvariectomyImplantGenistein(N=8) 53.99 ± 9.02(52.8) 49.57 ± 19.79(44.5)
ImplantGenistein(N=8) 54.57 ± 10.95(54.6) 43.3 ± 9.24(41.9)
P–value 0.002* 0.214
KruskalWallisTest *
P<0.05
FIGURE 2. Non–Decalcied histologic images of the Control–Implant Group; (A:4×, B:10× magnication, Methylene blue). Implant surface not contacting bone (brown
line), Implant surface contacting with bone (green line).*: Area without bone lling, ¥: Area with bone lling. Total implant surface: £, Bone Implant Contact Ratio (%):
£–α(β)/£. Thread lling detected by measuring the ratio of the bone lled areas in total thread areas. Thread lling areas (à), non–bone areas (ĕ), total area (Ă). Thread
lling Ratios (%):Ă–ĕ/Ă (à/Ă)
TABLE III
Mandibular and femur bone mineral density (BMD) of the groups
Group
Mandibular (BMD) Femur (BMD)
Mean ± SD (median) Mean ± SD (median)
Control(N=8) 0.36 ± 0.03(0.4) 0.24 ± 0.01(0.2)
ControlImplant(N=8) 0.35 ± 0.03(0.4) 0.25 ± 0.03(0.3)
OvariectomyImplant(N=8) 0.33 ± 0.01(0.3) 0.23 ± 0.03(0.2)
OvariectomyImplantGenistein(N=8) 0.37 ± 0.02(0.4) 0.23 ± 0.01(0.2)
ImplantGenistein(N=8) 0.33 ± 0.03(0.3) 0.25 ± 0.02(0.2)
P–value 0.008* 0.322
KruskalWallisTest *
P<0.05
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Mandibular and femur bone mineral density of the groups.
BMD: Bone mineral density. Mandibular BMD in the ovariectomy
implant genistein group was higher than the ovariectomy implant
and implant genistein groups (P<0.05). There was no statistically
difference between the other groups in terms of mandibular BMD
(P>0.05). There was no statistical difference between the groups
in terms of femoral BMD (P>0.05).
It has been shown that genistein, a soybean isoflavone that
acts as a phytoestrogen, may treat osteoporosis [7, 8]. Genistein
improves bone health in bone loss due to estrogen deciency [7, 8].
Changes in the bone after osteoporosis affects the bone–implant
connection negatively; therefore, implant treatment in patients with
osteoporosis is approached with suspicion [4]. Compounds used
in the treatment of osteoporosis which give positive results may
have a positive effect on the bone–implant connection [4]. This
study suggests that genistein may positively affect osteointegration
in patients with osteoporosis.
Osteoporosis, in rats is conrmed by lower BMD and lower
trabecular numbers and thickness, as well as higher trabecular
detachment, by changes observed in the proximal tibia, lumbar
FIGURE 3. Non–Decalcied histologic images of the Ovariectomy–Implant Group; (A:4×, B:10× magnication, Methylene blue). Implant surface not contacting bone
(brown line), Implant surface contacting with bone (green line).*: Area without bone lling, ¥: Area with bone lling. Total implant surface: £, Bone Implant Contact
Ratio (%): £–α(β)/£. Thread lling detected by measuring the ratio of the bone lled areas in total thread areas. Thread lling areas (à), non–bone areas (ĕ), total area
(Ă). Thread lling Ratios (%):Ă–ĕ/Ă (à/Ă)
FIGURE 4. Non–Decalcied histologic images of the Ovariectomy–Implant–Genistein Group; (A:4×, B:10× magnication, Methylene blue). Implant surface not contacting
bone (brown line), Implant surface contacting with bone (green line).*: Area without bone lling, ¥: Area with bone lling. Total implant surface: £, Bone Implant
Contact Ratio (%): £–α(β)/£. Thread lling detected by measuring the ratio of the bone lled areas in total thread areas. Thread lling areas (à), non–bone areas (ĕ),
total area (Ă). Thread lling Ratios (%):Ă–ĕ/Ă (à/Ă)
Osseointegration in ovariectomy model with Genistein / Uzun et al.__________________________________________________________________
_________________________________________________________________________________________________Revista Cientica, FCV-LUZ / Vol.XXXV
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20, 21]. Like estrogen, genistein plays an important protective role
against experimentally induced bone resorption in tissue cultures in
vitro and stimulates osteoblast–mediated bone formation; that is, it
exhibits anabolic effects. Animal model studies with ovariectomized
rats (deprived of endogenous estrogens) have proven that genistein
is as active as estrogens in maintaining bone health. Studies have
shown that phytoestrogens after menopause are more effective than
hormone therapy in maintaining bone mineral density in women
[20, 21, 22]. Similar studies have shown that phytoestrogens bind
to estrogen receptors in the bone and exert an estrogenic effect,
reducing bone destruction in menopause [21, 22]. Results of a
clinical study involving 136 postmenopausal women which received
a soy isoflavone–enriched diet with walking exercise for six months
found signicant increases in bone mineral content and density in the
lumbar spine [23]. In line with this scientic data, this study aimed
to investigate the effect of genistein on bone–implant connection
in the presence of osteoporosis.
Keikhosravi et al. [24] investigated the effects of high–intensity
interval training and genistein on serum osteocalcin and ALP levels
in female elderly rats. It was found that the serum ALP levels were
signicantly increased in the groups given genistein [24]. In this
study, the ALP levels were found to be statistically lower in the
implant genistein group than in the other groups, which contradicts
this study. On the other hand the results of a study by Qi et al [25].
which investigated the effect of genistein and silicon on bone
loss due to ovariectomy; genistein and/or silicon was found to
reduce serum ALP levels [25]. In this study, the lower ALP levels
in the implant genistein group, compared to the ovariectomized
groups, supports this study. ALP values may increase in metabolic
bone diseases. The low ALP values in the genistein group can be
evaluated in this respect, genistein may have suppressed the
bone–destructive effect of ovariectomy.
In a study by Park et al. [26] on female rats that underwent
ovariectomy, it was reported that there was a decrease in serum
vertebrae, and femur at 14, 30, and 60 days (d) after ovariectomy,
respectively. Current data shows that the response of trabecular
bones of the proximal tibia, lumbar vertebrae, and femur to
ovariectomy is similar in rats to that in humans [12].
In this study, implants were placed in the proximal tibia of rats,
and the study was planned as 90 d. Rats are frequently preferred
animals in osseointegration studies in the literature. In line with
this data, rats were preferred in this study [13, 14, 15]. In a study
by Şahin et al. [8] in that investigated the suppressive effects of
lycopene and genistein on breast cancer, 2 mg·kg
-1
genistein was
administered to rats by oral gavage three times a week [8]. In this
study, the rats in genistein groups were administered genistein
in powder form containing at least 98% genistein, mixed with
physiological saline, at 2 mg·kg
-1
, by oral gavage, 3 d a week
throughout the entire experimental period.
Density and volume decrease in bones seen in osteoporosis
occurs in jawbones as well as in other bones. The relationship
between osteoporosis and jawbones was rst studied in 1960. In
osteoporotic patients, the loss of alveolar bone tissue was found
to be greater than that of the body of the mandible. Osteoporosis
gives its rst signs in the alveolar bone due to the fact that the
rate of bone remodeling is higher in the alveolar bone than in other
bones [16]. It was reported in a rewiev that women have lower
mandibular bone mineral content than men, and women over 50
have greater bone loss than men of the same age. In this study,
the mandibular BMD of the rats that underwent an ovariectomy
was found to be lower than the control group, and this result is
consistent with the literature [16, 17, 18].
In the present study, genistein, a phytoestrogen, was used in the
rat ovariectomy model. Genistein is a natural compound belonging
to the isoflavonoid group [7, 8, 19]. Genistein soy isoflavonoids
resemble the molecular structure of estrogen, which is known to
stimulate osteogenesis in bone cells, and acts like estrogen [19,
FIGURE 5. Non–Decalcied histologic images of the Implant–Genistein Group; (A:4×, B:10× magnication, Methylene blue). Implant surface not contacting bone (brown
line), Implant surface contacting with bone (green line).*: Area without bone lling, ¥: Area with bone lling. Total implant surface: £, Bone Implant Contact Ratio (%):
£–α(β)/£. Thread lling detected by measuring the ratio of the bone lled areas in total thread areas. Thread lling areas (à), non–bone areas (ĕ), total area (Ă). Thread
lling Ratios (%):Ă–ĕ/Ă (à/Ă)
Osseointegration in ovariectomy model with Genistein / Uzun et al.__________________________________________________________________
8 of 10 9 of 10
Ca levels due to the decline in estrogen levels, and increased Ca
reabsorption in the estrogen receptors in the kidneys. Yang et al. [27]
found a signicantly lower serum calcium level in ovariectomized
rats in a study using hispidulin, icariin and genistein to compare the
effect of estrogen on bone tissue. In addition, serum Ca levels of
all groups given hispidulin, icariin and genistein were higher than
those of the rats that underwent an ovariectomy. According to the
results of the reviewed literature, the results regarding serum Ca
levels are controversial [12].
Despite this, the fact that serum Ca
levels were found to be signicantly lower in the ovariectomy group
compared to the control group in this study supports the results of
the study of Park et al. and Yang et al. [26, 27].
Qi et al. [25] investigated the effects of genistein and silicon on
bone loss due to ovariectomy in rats with a study where the rats
were randomly divided into 4 groups after the ovariectomy. As
a result of the 10 week treatment of genistein silicone serum P
levels were increased in the ovariectomy group [25]. In this study,
phosphorus levels of the ovariectomy implant group were found
to be statistically signicantly lower than those of the ovariectomy
implant genistein and implant genistein groups, which supports
the current study.
Aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) levels in the blood are strong indicators of liver damage [26,
27, 28]. In a study by Huang et al. [28] which was investigated
the protective effect of genistein on chronic alcohol–induced liver
damage and brosis in rats, found a signicant decrease in AST
and ALT enzyme activities in the subjects treated with genistein
but showed that genistein did not affect basal plasma AST and
ALT activities [26, 27, 28] . In this particular study, the blood AST
and ALT levels of the subjects were examined in order to see the
effect of genistein on liver function. While there was no statistically
signicant difference between the AST levels between the groups,
the ALT level in the genistein group was found to be signicantly
lower than the other groups. This result suggests that genistein
does not have a negative effect on liver function. In addition, the
low ALT values in rats given genistein in this study can be explained
by the liver–protective effect of genistein and supports the studies
of Huang et al. [28].
In a study by Duarte et al. [29] evaluating the effect of estrogen
deciency on the bone tissue around the implants integrated
into ovariectomized rats, found the bone–implant connection
and bone lling values around the implant to be lower in the
ovariectomy group. Giro et al. [30] reported that estrogen
deciency reduced trabecular bone density in a radiographic study
in female rats that investigated the effect of estrogen deciency
treatment with alendronate and estrogen on the bone density
around the osseointegrated implant. In a study by Cui et al. [31]
investigating the osseointegration levels of titanium implants
placed in the cancellous and cortical bones of ovariectomized
animals, areas without signicant bone–implant contact were
detected in ovariectomized rats.
In this study, when BIC levels
were examined, the BIC levels of the ovariectomy–implant group
and the ovariectomy–implant–genistein group were found to
be signicantly lower than those of the control implant group
as supported by the literature and revealed that ovariectomy
adversely affects BIC [30, 31]. In addition, the fact that the BIC
level of the ovariectomy–implant–genistein group was signicantly
higher than that of the ovariectomy implant group suggests that
genistein may positively affect the BIC levels in rats that underwent
an ovariectomy. Although there was no statistically signicant
difference between TF levels in this study, when evaluated
numerically, it was seen that the mean TF level of the ovariectomy
implant group was lower than the mean TF level of the ovariectomy
implant genistein group [29]. This shows that the results of this
study are in accordance with the literature.
Lee et al. [32] reported that when examining the changes in the
microarchitecture of the jaws of ovariectomized rats, the bone
mineral density of the ovariectomized rats were lower compared
to the control group. These authors have found that there is a
decrease consistent with osteoporosis [32] . While there was no
signicant difference in terms of femoral BMD in this study, when
the numerical femoral BMD values were examined, the values of
the group that underwent an ovariectomy were controlled and
lower than the treatment groups. The fact that the jaw BMD levels
were higher in the control group than in the ovariectomy implant
group supports the literature.
Qi and Zheng [25], reported in a study investigating the effect of
genistein supplementation on markers related to the bone mineral
density and bone metabolism in ovariectomized rats, that lumbar
spine and femur bone mineral density decreased signicantly after
ovariectomy in rats, and this decrease was inhibited with genistein
supplementation. According to histomorphometric analyses in the
same study, investigators stated that genistein supplementation
restored bone volume and trabecular thickness of the femur bone
in ovariectomized rats [25]. In this study, the mandibular BMD of
the ovariectomy–implant–genistein group was signicantly higher
than the jaw BMD of the implant–genistein group. Again in this
study, the fact that the mandibular BMD of the control group was
signicantly higher than the Implant–Genistein group suggests that
genistein does not affect the healthy bone tissue, which supports
the literature.
Some limitations were identied while evaluating the ndings
in this study. The rst of these limitations was that only genistein
was evaluated in this study. The second was that the effect of
genistein at different dosages has not been investigated. Third, this
study did not evaluate the survival rate of titanium implants or the
long–term success of bone implant connection. Fourth, long bones
such as the tibia and femur have different osteogenic properties
than the jaw bones (mandible–maxilla) and may therefore respond
differently to genistein [33].
CONCLUSION
Within the limits of this study, it has been observed that
ovariectomy may adversely affect bone tissue and reduce
osseointegration. When the datas of this study are examined,
it can be stated that genistein can improve the negative effects
of ovariectomy on bone tissue and increase osseointegration.
It can be stated that genistein consumption in patients with
postmenopausal osteoporosis may contribute to the treatment
of the skeletal system, as well as the success of implant treatment
in such patients who receive dental implant treatment. Further
studies are needed to examine the relationship between genistein
and bone tissue–osteoporosis.
Osseointegration in ovariectomy model with Genistein / Uzun et al.__________________________________________________________________
_________________________________________________________________________________________________Revista Cientica, FCV-LUZ / Vol.XXXV
9 of 10
ACKNOWLEDGMENT
This study was supported by the Firat University Scientific
Research Projects Department with the project number DHF.18.05
(Cansu Busra Uzun Thesis). The authors wish to thank Implance
Dental Implant Systems, AGS Medical Corporation, Istanbul, Turkiye
for the manufacturing of the implants which used in this study.
Conflict of interest
The authors declarate there is no conflict of interest.
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