https://doi.org/10.52973/rcfcv-e34332
Received: 07/10/2023 Accepted: 02/01/2024 Published: 27/02/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34332
ABSTRACT
In this study, it was aim to examine the local application of bovine
amniotic uid on bone fracture healing in rats. Twenty female sprague
dawley rats included in the study were divided into 2 groups of 10.
The sham group (n=10): Bone fractures were created in the right tibia
bones of the rats and xed with kirschner wire. After a four–week
recovery period, the subjects were sacriced. Local bovine amniotic
uid group (n=10): Bone fractures were created in the right tibia bones
of the rats and local bovine amniotic fuid was applied during xation
with kirschner wire. After a four–week recovery period, the subjects
were sacriced. Samples from all subjects were decalcied, stained
with hematoxylin and eosin, and new bone formation and brosis
were analyzed. When the groups were evaluated in terms of new bone
regeneration, it was determined that the new bone regeneration in
the subjects treated with local bovine amniotic uid were statistically
signicantly higher than sham group (P<0.05). When the groups were
evaluated in terms of brosis, the brosis value in the sham group
was found to be statistically signicantly higher when compared
with the local bovine amniotic uid group (P<0.05). It can be stated
that local bovine amniotic uid application may positively affect the
healing of bone fractures.
Key words: Bovine amniotic uid; bone fracture; local application;
bone fracture healing
RESUMEN
En este estudio, el objetivo fue examinar la aplicación local de líquido
amniótico bovino en la curación de fracturas óseas en ratas. Veinte
ratas hembra Sprague Dawley incluidas en el estudio se dividieron
en 2 grupos de 10. El grupo simulado (n=10): se crearon fracturas
óseas en la tibia derecha de las ratas y se fijaron con alambre
de Kirschner. Después de un período de recuperación de cuatro
semanas, los sujetos fueron sacricados. Grupo de líquido amniótico
bovino local (n = 10): se crearon fracturas óseas en los huesos de la
tibia derecha de las ratas y se aplicó líquido amniótico bovino local
durante la jación con alambre de Kirschner. Después de un período
de recuperación de cuatro semanas, los sujetos fueron sacricados.
Se descalcicaron muestras de todos los sujetos, se tiñeron con
hematoxilina y eosina y se analizó la formación de hueso nuevo y
la brosis. Cuando los grupos fueron evaluados en términos de
regeneración ósea nueva, se determinó que la regeneración ósea
nueva en los sujetos tratados con líquido amniótico bovino local
fue estadísticamente signicativamente mayor que en el grupo
simulado (P<0,05). Cuando los grupos fueron evaluados en términos
de brosis, se encontró que el valor de brosis en el grupo simulado
era estadísticamente signicativamente mayor en comparación con
el grupo de líquido amniótico bovino local (P<0,05). Se puede armar
que la aplicación local de líquido amniótico bovino puede afectar
positivamente la curación de las fracturas óseas.
Palabras clave: Líquido amniótico bovino; fractura ósea; aplicación
local; curación de fracturas óseas
Effects of local application of bovine amniotic uid on fracture healing in
rats (Rattus norvegicus)
Efectos de la aplicación local de líquido amniótico bovino en la
curación de fracturas en ratas (Rattus norvegicus)
Murat Tanrısever
1
* , Ozmen Istek
2
, Hatıce Eroksuz
3
, Burak Karabulut
3
, Erhan Cahıt Ozcan
4
,Muhammet Bahattın Bıngul
5
, Rıdvan Guler
6
,
Serkan Dundar
7
1
2
3
4
5
6
7
*
Bovine Amniotic Fluid for Fracture Healing in Rats / Tanrisever et al. _______________________________________________________________
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INTRODUCTION
Treatment of bone loss due to many diseases including
osteoarthritis, osteogenesis imperfecta, osteoporosis, traumatic
injury, cystic and tumoral lesions is dicult and requires a long
process [1]. Delay in bone healing occurs with a high incidence and this
is an important health problem [2]. About 5–10% of fractures undergo
delayed healing or result in nonunion [3]. The most important step in
the treatment of nonunion problems of broken bones is autologous
bone grafting, in which bone fragments are taken from a secondary
site and transferred to the fracture line in order to accelerate the
healing process. Although autogenous bone grafting is accepted as
the gold standard in bone healing, it can be faced with disadvantages
such as morbidity in the donor area where the graft is taken and low
quality autologous bone graft material [4, 5]. Researchers have been
developing alternative treatment methods for delayed fracture healing
for many years. Numerous approaches have been investigated to
accelerate maturation of regenerated bone, such as growth factors
[6], calcitonin [7], calcium sulfate [8], bisphosphonates [9], electronic
[10] and ultrasonic [11] stimulation. Various factors are dened that
support healing after fractures such as interleukins (IL); IL–1, IL–6,
some growth factors; broblast, transformed and platelet–derived
growth factors [12, 13].
Some researchers have explored the effects of human amniotic
uid, which is rich in growth factors, on fracture healing [13, 14, 15].
In addition, there is no literature on the effects of bovine amniotic
uid (BAF) on fracture healing. In the content of bovine amniotic
uid; mesenchymal cells, growth factors, macromolecules such as
hyaluronic acid (HA) and hyaluronic acid activating agent have been
reported [16, 17, 18, 19]. It is stated that HA suppresses the migration,
proliferation and chemotaxis of lymphocytes, and also reduces scar
formation by suppressing granulocyte phagocytosis and degranulation
and macrophage motility [20].
The aim of this study was to investigate the effects of local use of
bovine amniotic uid which is thought to have a positive stimulating
effect on fracture healing on fracture healing in a rat (
) tibia fracture model.
MATERIAL AND METHODS
This study was approved by Firat University (Protocol Number:
24/02/2020–380123) Local Animal Experiments Ethics Committee. It
was carried out at the Firat University Experimental Research Center,
and the Helsinki Declaration rules were strictly followed during the
experiments. The rats used in the experiments were obtained from
Firat University Experimental Research Center.
This study aimed to evaluate the effect of bovine amniotic uid on
fracture healing in the tibia of rats and histopathologically examine
the bone tissues.
In this study, a total of 20 female Sprague–Dawley rats weighing
300–320 g were used. Rats were kept in 12 hours dark, 12 hours light
environment and temperature throughout the study period. During the
experiment, all animals were allowed easy access to feed and water.
The rats used in the experiment were randomly divided into two
groups. In the sham group (n=10); rats tibias were cut with an electric
bone saw and then repaired with kirschner wire intramedullary and
no additional application was used. In the bovine amniotic uid (BAF)
(n=10) group; rats tibias were cut with an electric bone saw and then
repaired with kirschner wire intramedullary and bovine amniotic uid
was applied to the fracture line.
BAF was taken from healthy pregnant cow by injector (ClickZip,Medical
Device Manufacturer, Thailand) under aseptic conditions during
cesarean delivery at Firat University Animal Hospital. The amniotic
uid was kept cold and transported to the laboratory. It was stored in a
deep freezer (Arçelik, 2533D, Turkiye) at -20 °C until the day of surgery.
It was used after waiting for 15 min to dissolve at room temperature.
Surgical procedures
The rats used in the experiments were administered intramuscular
injection of 50 mg·kg
-1
Xylazine hydrochloride and 5 mg·kg
-1
dose of
Ketamine hydrochloride for general anesthesia. All surgical operations
on the subjects were performed in accordance with the required sterile
surgical procedures. After general anesthesia, the tibial skin was shaved
and cleaned. Before the incision, the knees were washed with 10%
Povidone iodine. A linear incision of 1.5–2 cm in length was made on
the skin of the tibia diaphyseal region. After passing the subcutaneous
connective tissue, the tibiae were cut from the midline of the bone
with an electric bone saw and divided into two parts. Then, the bones
were reconnected with the retrograde intramedullary method with
kirschner wires by using an orthopedic drill. While no extra agent was
used in the sham group, 0.3 mL bovine amniotic uid was applied to
the surfaces of the fracture line in the BAF group. after the integration
of the titanium implants the skins of the rats were then closed with
suturing in their original position with polyglactin 4/0 suture material.
As an antibiotic, all subjects recieved intramuscular 50 mg·kg
-1
Cefazolin
sodium for postoperative 3 days to prevent infection. As a pain reliever,
1 mg·kg
-1
Tramadol hydrochloride was given intramuscularly for 3 days
postoperatively. In this study, the recovery period was determined
as four weeks, and at the end of this period, all rats were sacriced.
Histopathological analysis
After the euthanasia procedure, the tibias removed as a whole
were xed in 10% buffered formalin solution for 3 days. The muscle
tissues around the bones were then cleaned with the microtome
(Leica RM2125, Wetzlar, Germany) blade and placed in a decalcication
solution. Five days later, when the desired exibility was achieved in
the bone samples, the decalcication process was completed and
the bone samples were placed in separate tissue tracking cassettes.
After washing the samples under running tap water for about two
hours, they were passed through alcohol, xylene and paran series
in an automatic tissue tracking device and blocked with paran in a
tissue blocking device. Serial sections 3–5 microns thick were taken
from the paran blocks with a rotary microtome on positively charged
slides. The prepared sections were stained with the hematoxylin–
eosin staining method in an automatic tissue staining machine. The
samples were semi–quantitatively evaluated in terms of fracture
healing in the prepared preparations. The examinations were made
with a trinocular light microscope (Olympus BX43, Tokyo, Japan) with a
camera (Olympus DP72, Tokyo, Japan) and an imaging analysis system.
Fibrosis numbers were scored as followed: No brosis; 0, low-visible
brosis; 1, mild–visible brosis; 2, and dense visible brosis; 3. Bone
formation (BF) was scored as followed: No bone formation, 0; mild
visible bone formation, 1; moderate visible bone formation, 2; and
dense visible bone formation, 3 [21].
FIGURE 1. Decalcied histologic images of the Sham Control Group; (A:2X, B:10X magnication, Hematoxylin Eosine, C: 4X, D: 10X, Masson
Trichrome). *: Newly Regenerated Bone, ¥: Fibrosis
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Statistical analysis
The SPSS 23.0 for Windows program (IBM SPSS Statistics for
Windows, Armonk, NY, USA) was used for statistical analysis. The
data for each group was expressed as mean ± standart deviation. The
Shapiro Wilks test was used to evaluate whether the data showed
normal distribution. The data was found to be normally distributed
and differences between groups were determined using the Student
Test. Signicance was evaluated at the P<0.05 level (TABLE I).
TABLE I
Histologic New Regenerated Bone (NRB) and Fibrosis (FRB)
parameters of the groups after the histological staining
Parameters Groups N Mean SD P*
NRB
Amnion 10 2,1 0,32
0,044
Control 10 1,7 0,48
FRB
Amnion 10 1,8 0,42
0,048
Control 10 2,2 0,42
SD: Standard deviation, *:Student
t Test (P<0,05). Statistically signicant dierence
detected between the groups
RESULTS AND DISCUSSION
No bacterial infection was detected in any of the rats included
in the study during the experimental process. No fatal or nonfatal
complications were detected during the experimental period. All rats
succesfully completed the experiment. When the bone formation
between the groups was examined, it was seen that bone formation
was found to be statistically signicantly higher in the group in which
local bovine amniotic uid was used compared to the results of the
sham group (P<0.05) (TABLE 1). When the brosis values of the groups
were examined, the brosis value of the sham group was detected
statistically signicantly highly than the group in which local bovine
amniotic uid was applied (P<0.05) (FIGS. 1 A,B,C,D and 2 A,B,C,D).
Amniotic fluid, contains cells from the amniotic membrane,
decidual cells, decidual formations, and shed cells from the skin,
digestive, respiratory, and excretory systems of the fetus. It consists
of 99% water, inorganic and organic substances, salts and epithelial
cells shed from the fetus. Half of the organic compounds are proteins
and the other half are carbohydrates, fats, enzymes, hormones and
pigments [22, 23].
FIGURE 2. Decalcied histologic images of the Local Bovine Amniotic Fluid Group; (A:2X, B:10X magnication, Hematoxylin Eosine, C: 2X,
D: 10X, Masson Trichrome). *: Newly Regenerated Bone, ¥: Fibrosis
Bovine Amniotic Fluid for Fracture Healing in Rats / Tanrisever et al. _______________________________________________________________
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Amniotic uid accelerate cell epithelialization and prevent protein
and uid loss on the wound surface, thus reducing adhesion formation,
while increasing broblastic activity with antibacterial and non–
immunological power. Therefore, they contribute to collagen synthesis
and reduce pain and inammation through angiogenesis. In addition,
amniotic uid is a biological product that can be obtained easily and
quickly, and its value is increasing [24, 25].
When the literature is examined, bovine amniotic fluid (BAF)
contains macromolecules such as HA and HA activating agent, as
well as insulin–like growth factor with activating on chondrocytes and
mesenchymal cells, as well as other growth factors [19, 26, 27, 28, 29].
Karaçal . in an study, they reported that amniotic uid can
increase bone healing when applied to experimentally created rabbit
() calvarial defects by subperiosteal method [14].
According to the results of the study Karaçal it was reported
that the local human amniotic uid increased the bone healing in a
statistically signicant way compared to the sham group [14]. Due
to bone regenerative capacity of bovine aminotic uid, the present
study aimed to investigate the effect of the usage of local bovine
amniotic uid on bone healing in experimentally created fractures of
rat tibias. In the present study, when the new bone formation between
the groups was examined, it was seen that new bone formation was
found to be statistically signicantly higher in the group in which local
bovine amniotic uid was used compared to the sham group (P<0.05).
This shows that the present study is parallel with the literature.
Kerimoglu showed in a study that human amniotic uid had
a positive effect on fracture healing in rat tibias [13]. In the study, it
was stated that brosis was higher in the sham group compared to
the other groups. In addition, Kerimoglu evaluated bone healing
with the scintigraphic method. It was suggested that human amniotic
uid may have positive effects on bone healing [13]. In another study,
Gokce reported that local human amniotic uid increases bone
healing in distraction osteogenesis studies in rabbit mandibles [15].
Researchers have reported that locally applied human amniotic uid
can improve new bone formation around the bone in maxillofacial
operations such as distraction osteogenesis [15]. In the present study,
when the brosis values of the groups were examined, the brosis
value of the sham group was found to be signicantly higher in the
statistical evaluation when compared to the group in which local
bovine amniotic uid was applied. (P<0.05). Additonally new bone
formation values in the experimental group; local bovine amniotic
uid group, detected higher compared with the sham group. The
mechanisms of distraction osteogenesis and fracture healing are
very similar. Considering this aspect, it can be stated that bovine
amniotic uid may have a similar effect on bone healing according
to the results of the study carried out [15].
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CONCLUSION
Within the limitaitons of this study, it is concluded that local
application of bovine amniotic uid may be effective in fracture
healing. It is seen that there have not been any studies on fracture
healing using bovine amniotic uid in the literature before, and it can
be thought that more detailed studies are necessary.
Conict of interest
There is no conict of interest.
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