https://doi.org/10.52973/rcfcv-e34372
Received: 30/12/2023 Accepted: 31/01/2024 Published: 11/05/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34372
ABSTRACT
Resveratrol (3,4,5–trihydroxystilbene), an antioxidant compound, has
a natural phytoalexin structure and also has many properties such
as anti–inammatory, antineoplastic and antiplatelet. In addition,
mesenchymal stem cells isolated from various tissues are considered
as a potential cell source for bone regenerative therapies. The present
study aims to examine the effects of Resveratrol and dental pulp–
derived mesenchymal stem cells on new bone formation in rats,
both isolated and combined, by immunohistochemical methods.
Twenty eigth Spraque Dawley male rats were used in the study. The
rats were divided into four groups with seven rats in each group; the
control group (Group 1) (n=7), the Systemic Resveratrol group (Group
2) (n=7), the Stem cell group (Group 3) (n=7), the Stem cell + Systemic
Resveratrol group (Group 4) (n=7). A defect was opened on the tibia
bones of the rats in all groups with a trephane bur (diameter of 3 mm
and a length of 4 mm). After the 4–week experiment, all rats were
sacriced following the experimental protocols specic to each
group. The specimens of tibia were subjected to histomorphological
examination in xative solutions. Values of inammation, connective
tissue formation, osteoclastic activity, osteoblast values, new bone
formation, BMP2 and BMP4 expression levels obtained for all groups
were evaluated by statistical analysis. Compared to the control
group, new bone formation and osteoblastic activity were found to
be signicantly higher in the Stem cell group and Stem cell + Systemic
Resveratrol group. (P=0.001) Additionally, new bone formation in the
Systemic Resveratrol group was found to be signicantly lower than in
the Stem cell + Systemic Resveratrol group. (P=0.006) No signicant
difference was observed between other groups. (P>0.05) According to
the results of the study, it was observed that Stem cell + Resveratrol
treatment was more effective than isolated Resveratrol or isolated
stem cell treatment applications, it induced the development of more
bone trabeculae, decrease inammation and increased the number
of osteoblasts involved in bone formation. In the light of these data, it
was concluded that the combined use of Resveratrol and Stem cells is
more effective on the healing of bone defects than their isolated use.
Key words: Bone defect; Resveratrol; mesenchymal stem cell; rat;
experimental study
RESUMEN
El Resveratrol (3,4,5–trihidroxiestilbeno), un compuesto antioxidante,
tiene una estructura de toalexina natural y además tiene muchas
propiedades tales como antiinamatoria, antineoplásica y antiagregante
plaquetaria. Asi mismo, las células madre mesenquimales aisladas de
diversos tejidos se consideran una fuente potencial de células para
terapias regenerativas óseas. El presente estudio tuvo como objetivo
examinar los efectos del Resveratrol y las células madre mesenquimales
derivadas de la pulpa dental sobre la formación de hueso nuevo en
ratas, tanto de forma aisladas como combinadas; evaluadas mediante
métodos inmunohistoquímicos. En el estudio se utilizaron 28 ratas
macho Spraque Dawley. Las ratas se dividieron en cuatro grupos con
siete ratas en cada grupo; el grupo de control (Grupo 1) (n=7), el grupo
de Resveratrol sistémico (Grupo 2) (n=7), el grupo de células madre
(Grupo 3) (n=7), el grupo de células madre + Resveratrol sistémico
(Grupo 4) (n=7). Se creo un defecto en los huesos de la tibia de las ratas
de todos los grupos con una fresa de trefano (diámetro de 3 mm y una
longitud de 4 mm). Después del experimento de cuatro semanas, todas
las ratas fueron sacricadas siguiendo los protocolos experimentales
especícos de cada grupo. Los tejidos provenientes tibias de animales
sacricados se sometieron a examen histomorfológico en soluciones
jadoras. Los valores de inamación, formación de tejido conectivo,
actividad osteoclástica, valores de osteoblastos, formación de hueso
nuevo, niveles de expresión de BMP2 y BMP4 obtenidos para todos los
grupos se evaluaron mediante análisis estadístico. En comparación
con el grupo de control, se observó que la formación de hueso nuevo
y la actividad osteoblástica eran signicativamente mayores en el
grupo de células madre y en el grupo de células madre + Resveratrol
sistémico. (P=0,001) Además, se encontró que la formación de hueso
nuevo en el grupo de Resveratrol sistémico era signicativamente
menor que en el grupo de células madre + Resveratrol sistémico
(P=0,006) No se observaron diferencias signicativas entre otros
grupos (P>0,05). Según los resultados del estudio, se observó que el
tratamiento con células madre + Resveratrol era mucho más efectivo
que las aplicaciones de tratamiento con Resveratrol aislado o células
madre aisladas, ya que, inducía el desarrollo de más trabéculas óseas,
suprimía más la inamación y aumentaba el número de osteoblastos
involucrados. En la formación ósea. A la luz de estos datos, se concluyó
que el uso combinado de Resveratrol y células madre es más ecaz en
la curación de defectos óseos que su uso aislado.
Palabras clave: Células madre mesenquimales; defecto óseo; rata;
Resveratrol; estudio experimental
A comparative investigation of the effects of Resveratrol and dental pulp
delivered mesenchimal stem cells on rat tibia bone defect healing
Una investigación comparativa de los efectos del Resveratrol y la pulpa dental proporcionó
células madre mesenquimales en la curación de defectos óseos de la tibia de rata
Hatice Demircan Agin
1
, Nedim Gunes
2
, Ridvan Guler
2
*
1
Ministry of Health, Oral and Dental Health Hospital, Department of Oral and Maxillofacial Surgery. Diyarbakır, Türkiye.
2
Dicle University, Faculty of Dentistry, Department of Oral and Maxillofacial Surgery. Diyarbakir, Türkiye.
*Correspondence author: ridvanguler06@gmail.com
Effects of Resveratrol and dental pulp on rat / Agin et al. ___________________________________________________________________________
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INTRODUCTION
In the treatment of defects resulting from previous operations in
oral and maxillofacial reconstructive surgery, congenital defects and
especially trauma, the lost bone tissue must be reconstructed [1]. In
order to accelerate healing after bone defects, mechanical methods
such as electrical and electromagnetic therapy, hyperbaric oxygen
therapy, ultrasound application and low–level laser therapy (LLLT)
are applied. Local/systemic medications can be applied as chemical
methods [2]. Nowadays, especially since chemical methods can have
many side effects, alternative methods with fewer side effects and
similar benets have begun to be used in studies instead of these agents.
The use of antioxidant agents and compounds obtained from plants
has become widespread. Resveratrol (RSVL, 3, 4', 5 trihydroxystilbene),
one of these compounds, has become important for use in clinical and
pharmacological studies [3]. RSVL is a compound with antioxidant
properties in the natural phytoalexin structure, which is found in high
amounts in vegetables and fruits such as grapes, peanuts, raspberries,
mulberries, plums, blackberries; red wine; and the roots of the plant
called Polygonum Cuspidatum [4]. In vitro and animal studies have
shown that RSVL has biological activities such as anti–inammatory,
anti–arthritic, anti–carcinogenic, anti–diabetic, anti–oxidant, anti–
aging effects and estrogenic activity [5]. In vitro studies have shown
that RSVL treatment prevents osteoclastogenesis while activating
osteoblastogenesis on bone metabolism [6, 7].
Stem cells are important in bone healing due to their ability to
self–renew and differentiate into various cells [8]. In particular,
mesenchymal stem cells (MSCs) isolated from different tissues
are used as a potential cell source for cell replacement and bone
regenerative therapies, in addition to their osteoblastic activities [9,
10]. Although MSCs are obtained from many different sources, stem
cells from dental pulp have advantages such as being easily obtained
without the need for a different surgical procedure and having a very
rich stem cell population, compared to stem cells from bone marrow
and other tissues [11]. It has been shown that stem cells obtained
from dental pulp (DPSC) can transform into odontoblast, myocyte,
osteoblast, chondrocyte, adipocyte and corneal epithelial cells [12, 13].
The present study aims to examine the effects of RSVL and DPSC
on new bone formation in rats (Rattus norvegicus), both isolated and
combined, by immunohistochemical methods.
MATERIALS AND METHODS
Ethics and animals
All surgical and experimental procedures were performed at the
Dicle University Experimental Research Center. The content of the
study was approved at the Dicle University Prof. Dr. Sabahattin Payzın
Health Sciences Research and Application Center Animal Experiments
Local Ethics Committee with the date 26.02.2020, protocol 01/2020
and number 02. The study was conducted in accordance with
the principles of the Declaration of Helsinki for the protection of
laboratory animals. 28 male Sprague–Dawley rats (3–5 months old,
250–300 g) were obtained from the Dicle University Experimental
Animal Production and Research Center and experimental procedures
were carried out in the same center. Obtaining stem cells was carried
out at Muğla Sıtkı Kocman University, Department of Genetics and
Bioengineering. The selected animals were kept in plastic cages in
a room with a temperature of 21 ± 10°C. During the experiment, the
rats were kept in an environment that provided a 40–60% humidity
standard with a 12–hour daylight cycle. After the experiment, seven
subjects were kept in the same metal cage. Experimental animals
were fed with standard rat chow containing 21% protein and tap water.
Study design
The 28 male Sprague–Dawley rats were randomly divided into four
groups of seven. Block randomization procedure was not used to
assign rats to different groups. For standardization, a homogeneous
experimental pool was created by looking at the weight and age of all
rats. Randomization sequence concealment and masking was taken
into consideration when grouping the rats.
1.
Control Group (Group 1): The defect area (diameter of 3 mm and
a length of 4 mm) created in the tibia of the rats was allowed
to heal on its own. The rats were kept under observation for
four weeks and then sacriced (n=7).
2.
Systemic Resveratrol Group (Group 2): A defect (diameter of
3 mm and a length of 4 mm) was created in the rat tibias and
RSVL was given systemically via oral gavage at 10 mg·kg
-1
every
day for four weeks. The rats were observed for four weeks and
then sacriced (n=7).
3.
Stem cell Group (Group 3): After creating a defect (diameter
of 3 mm and a length of 4 mm) in the rat tibia and suturing the
periosteum and skin, the rats to which DPSC were applied
locally were kept under observation for four weeks and then
sacriced (n=7).
4.
Stem cell + Systemic Resveratrol Group (Group 4): After creating
a defect (diameter of 3 mm and a length of 4 mm) in the rat tibia
and suturing the periosteum and skin, the rats were administered
DPSC locally and RSVL was administered systemically via oral
gavage at 10 mg·kg
-1
every day for four weeks, and were kept
under observation for four weeks and then sacriced. (n=7).
Preparation and dosage of Resveratrol
RSVL (Sigma, catalog number: R5010, USA) was weighed at 10 mg·kg
-
1
·day
-1
for each rat. RSVL was dissolved in ethanol and diluted with
physiological saline (1:3) and the solution was prepared daily. Starting
from the day of operation, 10 mg·kg
-1
RSVL solution per day was applied
to the Resveratrol and Resveratrol + Stem cell applied rat groups by
oral gavage method, once a day and at the same time every day.
Obtaining dental pulpa mesenchymal stem cells and way of application
Tooth extraction was performed from the lower molars of two adult
male experimental animals of the Sprague Dawley lineage under
anesthesia (Ketamine: 75–90 mg·kg
-1
+ xylazine 5–8 mg·kg
-1
, IP). To
DPSC from extracted teeth, the teeth were cut into small pieces with a
scalpel, then suspended in phosphate buffered saline (PBS) containing
0.1% collagenase and 0.25% trypsinethylenediaminetetraacetic acid
and was incubated at 37°C for 30–60 min. DPSCs in the suspension
passed through 70 mm cell lters were examined morphologically
under a microscope (Soptop ICX41, Ningbo, China), and the cells were
counted using a hemocytometer (Merck Bright–Line
TM
, Darmstadt,
Germany). 2 × 10
6
cells were planted in a 100 mm plastic petri dish.
DPSCs were taken to the incubator for growth and incubated at
37°C, saturated humidity and 5% CO
2
conditions. The cells to be
incubated were grown in alpha–modied Eagle medium containing
5.5 mmol·L
-1
glucose, 20% fetal bovine serum and 1% penicillin
streptomycin. When the cells reached 70–80% conuency, they were
A B
C D
FIGURE 1. Defect area of the A) Control group (Bar scale: 50 μm), B) Systemic
Resveratrol group (Bar scale: 50 μm), C) Stem cell group (Bar scale: 50 μm), D)
Stem cell + systemic Resveratrol group (Bar scale: 50 μm) groups tibial bone in
the fourth week view. (hematoxylin and eosin stain; magnication, 10x)
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passaged two more times and in the third passage (P3), the cells
were characterized for use in experiments. The presence of MSCs
was detected by FACS method with CD45 PC5, CD44 PE, CD105 FITC
and STRO–1 PE antibodies. DPSC were expected to be CD44, CD105
and STRO–1 positive and CD45 negative. Since DPSC are capable of
differentiation, osteogenic differentiation was tested. OriCellTM
osteogenesis differentiation kit (Cyagen, Guangzhou, China) was
used to induce osteogenic differentiation. The obtained stem cells
were administered locally to the defect area with syringe applicator.
The animals were returned to normal feeding and rearing conditions
(fed with standard rat chow containing 21% protein and tap water).
Histologic study
All rats were sacriced on the 28th day by applying 30 ml isourane
(Isourane–USP, Adeka Ilac, Turkiye). The left tibia of the rats was
removed and dissected. For histological analysis, samples were xed in
10% neutral buffered formalin for 24 hours. Then, they were decalcied
in 10% diluted formic acid at room temperature for one month and then
embedded in paran blocks. With the help of a microtome (Catalog no:
Leica RM2265, Wetzlar, Germany), 4–6 μm thick sections were taken
from the blocks for Hematoxylin–Eosin and immunohistochemical
staining. The sections were examined using a light microscope (Nikon
Optiphot–2; Nikon Corporation, Tokyo, Japan) by researchers who
were blind to the treatment assignments. Values of inammation,
connective tissue formation, osteoclastic activity, osteoblast values,
new bone formation, Bone Morphogenic Protein 2 (BMP2) and Bone
Morphogenic Protein 4 (BMP4) expression levels were determined
semi–quantitatively scored.
Semi–quantitative Histologic Scoring
Scoring was performed using a 0 to 4 scoring system (0, no; 1,
minimal; 2, moderate; 3, abundant).
Immunohistochemistry study
Sections taken from the paran blocks were placed in a double
boiler set at 37°C and then onto polylysine slides. The sections were
deparanized in xylene for 3×15 min. The sections were placed in
an ethylenediamine tetraacetic acid (EDTA) buffer solution (pH: 8.0,
catalog no: ab93680, Abcam, Cambridge, USA) and heat–induced
epitope retrieval was performed.
Hydrogen peroxide 71 solution (catalog no: TA–015–HP,
ThermoFischer, Fremont, CA, USA) was dropped onto the sections.
Sections were processed by anti–BMP–2 (catalog no: sc–137087, Santa
Cruz Biotechnology, 10410 Finnell St, Dallas, Texas 75220, US) and
anti–BMP–4 (catalog no: sc–393329, Santa Cruz Biotechnology, 10410
Finnell St, Dallas, Texas 75220, US) overnight at +4°C with antibodies.
Biotin secondary antibody (catalog no: TP–015–BN, ThermoFischer,
Fremont, CA, USA) was dropped onto the sections washed with PBS
and incubated for 14 min. Then, Streptavidin–peroxidase (catalog
no: TS–015–HR, ThermoFischer, Fremont, CA, USA) was added and
washed with PBS after waiting for 15 min. Diaminobenzidine (DAB)
(catalog no: TA–001–HCX, ThermoFischer, Fremont, CA, USA) was
dropped onto the washed sections, the reaction was monitored under
a microscope and stopped with PBS. After counterstaining with Harris
hematoxylin, the sections were covered with Entellan (catalog no:
107961, Sigma–Aldrich, St. Louis, MO, USA) and evaluated and viewed
with a Zeiss Imager A2 photomicroscope (CarlZeiss, Jena, Germany).
Statistical analysis
While evaluating the ndings from the study, SPSS for Windows
version 24.0 package program was used for statistical analysis.
While evaluating the study data, the suitability of the data to normal
distribution was tested with the Shaphiro Wilk test, and Kruskal
Wallis and Dunn Multiple Comparison tests were used to compare the
features that did not comply with normal distribution in more than
2 independent groups. Signicance was evaluated at P<0.05 level.
RESULTS AND DISCUSSION
FIG. 1 shows the alveolar bone sections of all groups that were
stained with hematoxylin and eosin on days 28 after experimental.
In Group 1, mild degenerations were observed in some osteocyte
cells located especially close to the endosteal region in the compact
bone region. It was determined that osteoclastic activity began to
become particularly evident in areas close to this area (FIG. 1A). In
Group 2, osteoblastic activity was observed to start especially in
areas close to the endosteal area (red arrow). Osteocytes have not yet
been observed very clearly in the endosteal region (FIG. 1B). In Group
3, it was observed that especially the newly formed bone trabeculae
pieces, towards the outside of the endosteal region, were rich in
osteoblasts (red arrow), started to develop and expand. A decrease
in the number of osteoclasts was also detected (FIG. 1C). In Group
4, bone trabeculae of different sizes began to be seen in the defect
area (black arrows). A increase in osteoblasts, especially in bone
trabeculae (red arrow), a decrease in osteoclasts, and occasionally
osteocyte cells with distinct lacunae (yellow arrows) were observed
within the bone trabeculae. It was determined that the matrix increase
was intense and the bone trabeculae widened (FIG. 1D).
Immunohistochemistry results
BMP–2 (Bone Mineral Protein–2): In Group 1, in the longitudinal section
of the tibial bone, there was a increase in inammatory cells (asterisk),
especially with an increase in dense connective tissue between the
broken bone pieces, and intense osteoclastic activity (blue arrows),
especially in the periphery of the bone trabeculae. showed a positive
reaction with BMP–2 (FIG. 2A). In Group 2, it was observed that
osteogenic activity turned in favor of bone formation, especially in the
A B
C D
FIGURE 2. Defect area of the A) Control group (Bar scale: 50 μm), B) Systemic
Resveratrol group (Bar scale: 50 μm), C) Stem cell group (Bar scale: 50 μm), D)
Stem cell + systemic Resveratrol group (Bar scale: 50 μm) groups tibial bone in
the fourth week view. (Immunohistochemical staining) (BMP–2)
A B
C D
FIGURE 3. Defect area of the A) Control group (Bar scale: 50 μm), B) Systemic
Resveratrol group (Bar scale: 50 μm), C) Stem cell group (Bar scale: 50 μm), D)
Stem cell + systemic Resveratrol group (Bar scale: 50 μm) groups tibial bone in
the fourth week view. (Immunohistochemical staining) (BMP–2)
TABLE I
Obtained for all groups; values of inammation, connective tissue formation,
osteoclastic activity, osteoblast values, new bone formation, BMP2 and
BMP4 expression levels
Control
(n=7)
Stem Cell
(n=7)
Resveratrol
(n=7)
Stem Cell
Resveratrol
(n=7)
Variables
Median
[25%–75%]
Median
[25%–75%]
Median
[25%–75%]
Median
[25%–75%]
P
Inammation 3 (3 –4) 3 (2 –3) 3 (2 –3) 2 (2 –3) 0,099
Connective tissue
formation
1 (0 –1) 2 (2 –2) 2 (2 –3) 3 (3 –3) 0,001*
Osteoclastic
activity
3 (3 –4) 3 (2 –3) 3 (3 –3) 2 (2 –3) 0,043*
Osteoblastic
activity
1 (1– 2) 2 (2 –2) 3 (2 –3) 3 (2 –3) 0,001*
New bone
formation
0 (0 –0) 1 (1 –2) 2 (2 –3) 3 (3 –3) 0,001*
BMP–2 0 (0 –1) 2 (2 –3) 2 (2 –3) 3 (3 –3) 0,001*
BMP–4 0 (0 –1) 2 (2 –3) 2 (2 –2) 3 (3 –3) 0,001*
*: signicant at 0.05 level; Kruskal Wallis test mean and standard deviation values
Effects of Resveratrol and dental pulp on rat / Agin et al. ___________________________________________________________________________
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osteoblast direction, BMP–2 showed a positive reaction in osteoblasts
(red arrows), and osteocyte cells were not yet fully evident. While
there was decrease in the number of osteoclast cells, an increase in
BMP–2 activity was observed in the osteoclast cells here (blue arrows)
(FIG. 2B). In Group 3, it was observed that bone trabeculae began to
form in the tibial bone, especially in the area close to the actual bone
where the defect area was located (black circle), osteoblastic activity
increased, and a few osteocyte cells began to become evident. A
decrease in osteoclast cells was observed (blue arrow).
The most important feature was that osteocytes in the new bone
trabeculae clearly show the BMP–2 reaction and contribute to new
bone formation (FIG. 2C). In Group 4, effect on ossication due to
the occasional formation and expansion of new bone trabeculae, the
intense expression of osteoblast cells (red arrows) in the periphery,
the increase in matrix formation and the positive BMP–2 reaction of
osteocytes (yellow arrows) was observed (FIG. 2D).
it was not signicant in terms of inammation may be due to the
numerical difference between the groups.
For all other measurements, a signicant difference was found in
at least one group (P<0.05) (TABLE II). The analysis result regarding
the difference between the groups in terms of osteoblast and new
bone formation values is shown in TABLE II. Compared to the control
group, new bone formation and osteoblastic activity were found to
be signicantly higher in Group 3 and Group 4 (P=0.001). Additionally,
new bone formation was found to be signicantly lower in Group
2 compared to Group 4 (P=0.006). No signicant difference was
observed between other groups (P>0.05) (TABLE II).
It was determined that there was a statistically signicant difference
between Group 2, Group 3 and Group 4 when compared with the Control
group in terms of BMP–2 and BMP–4 values (P<0.05). In addition, the
difference in the values of the 'Stem Cell' group (Group 3) in terms
of BMP–4 was found to be signicantly lower than the 'Stem Cell –
Resveratrol' (Group 4) group (P<0.05) (TABLE III).
BMP–4 (Bone Mineral Protein–4): In Group 1, BMP–4 expression was
observed to be positive in some leukocytes (arrowhead), along with
intense inammatory cell inltration around the vessel. Although
there were signs of ossication in some areas, BMP–4 expression
was seen at a high rate in osteoclast cells (blue arrows) due to the
density of osteoclast cells (FIG. 3A). In Group 2, only osteoblastic (red
arrows) activity was positive in small bone trabeculae. The BMP–4
reaction was evaluated as positive, with also a partial decrease in
osteoclasts (blue arrows) (FIG. 3B). In Group 3, the BMP–4 reaction
was observed to be positive, especially in osteoblasts (red arrow) and
osteocytes (yellow arrows), which were enlarged within the defect
area. BMP–4 reaction was also evaluated as positive in a small number
of osteoclasts (blue arrow). Improvement was noted in new bone
formation in terms of matrix development and lacunar structure
(FIG. 3C). In Group 4, a mature bone trabecula located close to the
endosteal region within the defect area is observed to begin to form
(black circle). Osteoblasts located in the periphery were also seen to
be prominent. BMP–4 reaction was found to be positive in osteoblasts
(red arrow) and osteocyte cells (yellow arrow) (FIG. 3D).
According to TABLE I, there was no signicant difference between
the groups in terms of inammation values (P=0.099). The fact that
TABLE II
Analysis result regarding the dierence between groups in terms
of osteoblastic activity and new bone formation values
Group comparisons Osteoblastic Activity New Bone Formation
Group 1 / Group 2 0,110 0,101
Group 1 / Group 3 0,001* 0,001*
Group 1 / Group 4 0,001* 0,001*
Group 2 / Group 3 0,071 0,066
Group 2 / Group 4 0,071 0,006*
Group 3 / Group 4 1,000 0,366
*: signicant at 0.05 level; Kruskal Wallis test and Dunn’s multiple comparison test
TABLE III
Analysis result regarding the dierence between
groups in terms of BMP–2 and BMP–4 values
Group comparisons BMP–2 BMP–4
Group 1 / Group 2 0,013* 0,004*
Group 1 / Group 3 0,005* 0,020*
Group 1 / Group 4 0,001* 0,001*
Group 2 / Group 3 0,718 0,587
Group 2 / Group 4 0,102 0,075
Group 3 / Group 4 0,203 0,020*
*: signicant at 0.05 level; Kruskal Wallis test and Dunn’s multiple comparison test
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Since the oral and maxillofacial region is in constant function, it is
constantly exposed to chemical and mechanical forces. Therefore,
studies are being carried out on many factors that stimulate recovery
in order to restore function to patients as soon as possible [14].
Especially today, systemic and local agents such as bisphosphonate
and parathyroid hormone, which reduce resorption and increase
deposition in bone, are preferred [15].
These agents used have
many side effects. For this reason, alternative drugs with similar
benets but less potential for side effects have begun to be used in
studies. While RSVL treatment stimulates osteoblastogenesis on
bone metabolism, it also inhibits osteoclastogenesis [4, 6, 7]. In the
current study, antioxidant RSVL was administered systemically to
accelerate bone healing and increase stem cell activity.
The rst in vitro study examining the effects of RSVL on the bone
formation and destruction mechanism was conducted by Mizutani
et al. [16] In this study, it was reported that RSVL activated the
proliferation and differentiation of osteoblastic MC3T3–E1 cells in
vitro. In a different study, it was reported that RSVL activates the
proliferation and differentiation of osteoblast cells by increasing the
expression of genes that have a supportive effect on osteogenesis,
and that it acts on bone resorption by suppressing the activation of
genes responsible for the formation of osteoclastogenesis [17, 18].
Mobasheri and Shakibaei concluded in their study where they
evaluated the ndings obtained from in vitro studies, that RSVL
increased bone mass by activating bone formation while inhibiting
bone resorption [6]. In the in vitro study conducted by Backesjo et
al. [19], it was shown that RSVL increased osteoblast differentiation
resulting in new bone formation and inhibited adipocyte formation
and development. In the experimental study of rapid maxillary
expansion conducted to examine the effectiveness of RSVL in local
application by Uysal et al. [20], it was observed that the application
of RSVL activated new bone formation and shortened the required
retention time after the operation. Based on this, it is thought that
RSVL can be used in the treatment of bone fractures and distraction
osteogenesis. In this study, unlike the literature, it was observed that
there was no signicant difference in terms of new bone formation
and osteoblastic activity when isolated use of RSVL was compared
with the control group. In addition, it was observed that new bone
formation and osteoblastic activity were signicantly lower in the
Stem cell + Resveratrol group than in the stem cell groups.
In their studies investigating the effectiveness of RSVL on the
proliferation and differentiation of human bone marrow–derived
MSCs, Ornstrup et al. [21]
have shown that bone marrow–derived
MSCs obtained from adult donors in long–term interaction with RSVL
increased the osteoblastic activities. Although we used DPSC in our
own study, our results are similar.
In the study where Song et al. [22] examined the effects of MSCs
obtained from rat bone marrow on osteoblastic proliferation and
differentiation with the use of RSVL and cyclosporine (CsA), the
enhancing effect of RSVL on osteoblast cell differentiation and
proliferation through the nitric oxide/cyclic guanosine monophosphate
(NO/cGMP) signaling pathway, it has been shown that CsA has an
inhibitory effect on osteoblastic proliferation and differentiation of MSCs
obtained from rat bone marrow. In the present study, unlike the literature,
no signicant difference was found in osteoblastic proliferation and new
bone formation in RSVL compared to the control group.
The effectiveness of RSVL on bone resorption was investigated by
Liu et al. [17]
in a rat model that underwent ovariectomy. Based on
these ndings, it was concluded that RSVL has an increasing effect on
bone density and inhibits the loss of bone calcium amount. In addition,
according to the ndings, it is thought that RSVL may play a protective
role against bone destruction caused by estrogen deciency. In order
to measure the effectiveness of RSVL on bone density in their study
on ovariectomized rats, Lin et al. [23] have shown that daily RSVL
intake activated bone formation in ovariectomized rats.
In the study conducted by Casarin et al. [3] to investigate the
effectiveness of RSVL on bone defects, the aim was to evaluate the
effects of RSVL application on the defect created in the calvarium
and on bone healing around the titanium implant. As a result, it was
reported that RSVL application increased critical size defect repair
and biomechanical resistance of titanium implants, and also positively
affected BMP–2, BMP–7 and osteopontin expression levels. Based
on these results, we think that regular use of RSVL may be a useful
supportive agent on the bone healing mechanism and in the treatment of
edentulous individuals with dental implants. Similarly, in our study, it was
determined that there was a signicant difference in BMP–2 (P=0.013)
and BMP–4 (P=0.004) expression values in the groups in which the critical
size defect opened in the tibia in which RSVL was administered at a dose
of 10 mg·kg
-1
via oral gavage for 1 month, compared to the control group.
MSCs can be isolated from many tissues such as adipose tissue,
umbilical cord blood, peripheral blood, dental pulp, dermis, amniotic
fluid and even tumors. They can differentiate into osteoblasts,
adipocytes, chondrocytes, myoblasts and neurons [24, 25]. In particular,
their osteogenic differentiation potential is superior to other stem
cell types and therefore they are used to improve bone regeneration.
Effects of Resveratrol and dental pulp on rat / Agin et al. ___________________________________________________________________________
6 of 7
Stem cells obtained from dental pulp were rst used clinically in 2009
by d'Aquino et al. [26]
and were applied to 17 patients. According to the
results of the study, it was observed that the stem cells taken from the
patient were more satisfactory in terms of new bone height and tissue
organization on the side where they were placed [26].
It has been shown
in many studies that stem cells obtained from dental pulp can transform
into osteoblasts under in vitro conditions with stimulation. There are
even articles arguing that DPCH cells have greater differentiation
ability under in vitro conditions than bone marrow stem cells, which
are considered the standard in stem cell research [27].
In the study by Jing–hui et al. [28], it was reported that stem
cells transplanted into a gelatin sponge could show osteogenic
differentiation even in an ectopic location in mice (Mus musculus) and
that DPCH cells could be used as a suitable and effective source in bone
tissue engineering studies. DPCH is a rich resource that provides good
results not only for the eld of dentistry but also for other branches
of medicine. Studies have shown that it can be used successfully in
the treatment of myocardial infarction, nerve tissue regeneration,
muscular dystrophy, cerebral ischemia, improvement of angiogenetic
properties and corneal regeneration [29].
In our own study where we
used DPCH, the results are consistent with the literature.
Two methods are frequently used for stem cell applications today.
The rst is intravenous injections (direct application of cells), the
second is cell encapsulation systems (indirect application of cells with
the help of a carrier) [30]. Accordingly, in our study, local transplantation
of MSCs into the defect with the help of scaffolding was applied. The
cell density of the MSCs to be used for tissue regeneration is also an
important factor. After MSC application, sucient tissue uid and blood
supply must be provided so that the cells can maintain their viability
and differentiate into osteoblastic cells [31]. In studies using MSC
concentrations between 2×10
6
– 2×10
7
cells·ml
-1
, it has been reported
that effective tissue regeneration occurs in various defects, including
bone [32].
In the present study, MSCs at a density of 2×10
7
cells/ml,
which is between these values, were used.
Looking at the literature, there are many studies examining
the effects of systemically applied RSVL as a herbal agent and
mesenchymal stem cell application, which is very promising in the
eld of molecular biology, on bone healing separately. However, there
are very few studies comparing the effectiveness of RSVL in bone
healing when used in combination with stem cells. Therefore, we
think that the results of this study will contribute to the literature.
CONCLUSION
As a result of this study, it was observed that isolated RSVL and
isolated DPSC application partially stimulated bone development after
the defect and supported the development of new bone trabeculae.
After the defect was created, it was observed that Stem cell + RSVL
treatment was much more effective than isolated RSVL or isolated
Stem cell treatment, it induced the development of more bone
trabeculae, suppressed inammation more, and increased the number
of osteoblasts involved in bone formation.
Funding
This study is supported by Dicle University Scientic Research
Projects Coordination Oce with project number DİŞ.20.007.
Conict of interest
The authors declare that they have no conicting interest.
ACKNOWLEDGEMENT
The authors thank Dicle University for his contribution to the
histopathological study and Muğla Sıtkı Kocman University for his
contribution to the obtaining mesenchymal stem cells.
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