n Feature Article
Evaluation of the Effect of Platelet-Rich
Fibrin on Long Bone Healing:
An Experimental Rat Model
Turan Cihan Dülgeroglu, MD; Hasan Metineren, MD
abstract
Pseudoarthrosis, or nonunion, of the long bones is a challenging medical condition for orthopedic surgeons to treat. Therefore, healing enhancer materials
are commonly used. The authors investigated whether platelet-rich fibrin accelerates long bone healing by comparing radiological and histological findings in a rat model of open femoral fracture. Platelet-rich fibrin is a current
biomaterial that contains many growth factors and platelets. There are no studies in the literature investigating the effects of platelet-rich fibrin on fracture
healing. Sixteen mature male rats were divided into 2 groups. In both groups,
an open femoral fracture was created. The platelet-rich fibrin was obtained
by centrifuging blood collected from the rats. Rats in the study group were
treated with sterile platelet-rich fibrin, and those in the control group were
administered saline. The rats were killed at the end of 4 weeks and examined
histologically and radiologically. The radiographic and histological scores of
the 2 groups differed significantly (P<.05). These results indicate that plateletrich fibrin is an efficient biomaterial in fracture healing and that it increases
the amount of osseous tissue formation. Platelet-rich fibrin does not cause an
allergic reaction, is cost-effective, and is easy to obtain. Additional studies are
necessary to determine whether platelet-rich fibrin accelerates the fracture
healing process or induces a better quality of fracture healing. [Orthopedics.
2017; 40(3):e479-e484.]
T
he rebuilding of lost or damaged
tissues remains an important medical issue. The limitations of current strategies for the treatment of fracture
have caused difficulties in the bone healing
process. Bone is a metabolically dynamic
biological tissue that comprises active cells
MAY/JUNE 2017 | Volume 40 • Number 3
integrated into a rigid framework.1,2 Bone
healing is a unique regenerative process
that includes mesenchymal cell condensation, chondrogenesis, angiogenesis, and
bone formation.3 It begins with an inflammatory reaction followed by formation of
a soft and then a hard callus. Interactions
among the extracellular matrix, growth
factors, and osteoprogenitor cells contribute to bone remodeling.4 The 3 distinct but
overlapping phases of fracture healing are
the early inflammatory phase, the repair
phase, and the late remodeling phase.5,6
During this last phase, the hard callus is
transformed to yield cortical and trabecular bone.7 Fracture healing is also affected
by different systemic and local variables
that influence restoration of the original
physical and mechanical properties of the
injured tissue. It requires the interaction of
several physiological and biomechanical
steps that reflect a wide range of activities
at the molecular and cellular levels. Understanding the multiple complex processes
underlying fracture healing will ensure
an effective therapeutic approach.8,9 Bone
healing, and specifically the biochemical,
biomechanical, cellular, hormonal, and
The authors are from the Department of Orthopaedics and Traumatology, Dumlupınar University School of Medicine, Kütahya, Turkey.
The authors have no relevant financial relationships to disclose.
Correspondence should be addressed to:
Hasan Metineren, MD, Department of Orthopaedics and Traumatology, Dumlupınar University School of Medicine, 43080 Kütahya, Turkey
([email protected]).
Received: November 1, 2016; Accepted: January 30, 2017.
doi: 10.3928/01477447-20170308-02
e479
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Figure 2: Radiographs of the fractured femora in the experimental platelet-rich fibrin study group (a, b)
and the control group (c, d).
Figure 1: Platelet-rich fibrin.
pathological components, can be studied in
a fracture healing model.
Several alternative healing methods
have been developed during the past few
decades. Of these, platelet-rich plasma
(PRP) and platelet-rich fibrin (PRF) have
been used to accelerate the healing process. Platelet concentrates, prepared from
peripheral blood, were introduced in the
1990s; since then, their use in clinical practice has steadily increased. The positive
results obtained with PRP and PRF correlate with the fact that platelets are active
growth factor–secreting cells that initiate
the wound-healing process,10,11 specifically
connective tissue healing, bone healing,
fibroblast mitogenesis, angiogenesis, macrophage activation, and cell proliferation.12
Platelet-rich fibrin is a second-generation
platelet-rich biomaterial13,14 that was developed in France by Choukroun et al15,16
in 2001 and introduced by Dohan et al17,18
in 2006. It is rich in leukocytes; cytokines;
fibrin; circulating stem cells; the proin-
e480
flammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-a; the
anti-inflammatory cytokines interleukin-4
and interleukin-10; vascular endothelial
growth factor; insulin-like growth factor-I
and insulin-like growth factor-II; and platelet-derived growth factor.18 The successful
preparation of PRF depends on the speed of
blood collection and centrifugation. Slower
processes will result in the polymerization
of fibrin, leading to a smaller fibrin clot.
Unlike PRP, PRF contains the fibrin clot
matrix (PRF-M) and does not immediately
dissolve but rather takes shape slowly, similar to the process of blood clot formation.
The potential of PRF-M in bone and soft
tissue regeneration, without induction of
an inflammatory reaction, has been demonstrated in several studies.19-22 However, few
studies have investigated the effects of PRF
on long bone healing. Thus, in this study,
the authors analyzed the histopathological
and radiological results of PRF application
in a rat model of femoral fracture.
Materials and Methods
Surgical Procedure
The experimental protocols were approved by the Dumlupınar University
Animal Care Committee and the study
was approved by the Ethics Committee
of Dumlupınar University. The study was
conducted in accordance with National
Institutes of Health guidance for the care
and use of laboratory animals. The study
included 16 mature male Wistar albino rats
weighing 325±25 g. The rats were housed
in individual cages in a controlled environment at a room temperature of 23°C±2°C
and a humidity of 40% to 70% in a 12-hour
light–dark cycle. The rats had access to
food and water ad libitum. Antibiotic prophylaxis consisting of 40 mg/kg of cefamezin was administered before anesthesia.
The rats were randomly divided into
experimental and control groups. Animals
in both groups were taken to the operating
room following the necessary monitoring
and preparation processes. The dose of anesthesia was adjusted for each rat according
to body weight, as determined by measurements on an electronic scale. Intraperitoneal anesthesia consisted of 50 mg/kg of
ketamine and 10 mg/kg of xylazine. The
surgeries were performed by one surgeon
(T.C.D.). The left thigh of each rat was prepared with povidone-iodine and the surgical area was covered with a sterile cover.
A 2-cm incision was made on the lateral
aspect of the femur and an open mid-shaft
femoral fracture was created with an oscillatory saw. The femoral canal was cavitated
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Figure 3: Histological and immunohistological assessments of the plateletrich fibrin group: hematoxylin-eosin (a, original magnification ×40; b, original
magnification ×100), Masson’s trichrome (c, original magnification ×100; d,
original magnification ×40), and CD34 (e, original magnification ×400; f, original magnification ×400) staining.
with an 18-gauge needle, after which a motor-driven drill was used to place an 0.8-mm
K-wire into the medullary canal, close to
the upper end of the femur. Then the K-wire
was cut and advanced such that it was close
to the articular surface. Previously obtained
sterile PRF was applied to the bone, the
muscle structures were closed with absorbable suture, and the skin incision was closed
with a 4-0 nonabsorbable suture. The same
procedure was used in the control rats, but
saline was administered instead of PRF.
Postoperative pain was managed by the
subcutaneous application of buprenorphine
hydrochloride. The rats were given water
and standard food ad libitum. The study
was completed at the end of 28 days, at
which time all of the rats were killed. The
left femurs were prepared for radiological
and histological evaluations. The left limb
was removed entirely, without damaging
MAY/JUNE 2017 | Volume 40 • Number 3
Figure 4: Histological and immunohistological assessments of the control
group: hematoxylin-eosin (a, original magnification ×100; b, original magnification ×400), Masson’s trichrome (c, original magnification ×100; d, original
magnification ×100), and CD34 (e, original magnification ×40; f, original magnification ×100) staining.
the callus tissue, to avoid altering the results
of the experiment.
PRF-M Preparation
Blood taken from 4 rats was centrifuged
at 3000 rpm for 10 minutes without added
anticoagulant. The PRF matrix was removed with a thin, flat-ended tool and separated from the lower layer. The obtained
PRF consisted of a yellow fibrin section
(Figure 1) mainly constituting the shaft,
and a red section located under the yellow
fibrin and the buffy coat section, with a
large number of platelets trapped between
these 2 structures.17
Radiological Evaluation
Anteroposterior radiographs were obtained and evaluated by 2 independent
orthopedic surgeons. The findings were
classified according to the method of
Goldberg et al23: 1, no fracture healing; 2,
moderate healing; and 3, complete healing (Figure 2).
Histological Evaluation
Histological evaluations were performed in the clinical pathology unit of
the authors’ hospital. Following the radiological analysis, the entire femur was
fixed in 10% formalin, decalcified, and
then fixed in 10% acetic acid, 0.85% sodium chloride, and 10% formalin for 72
hours. After extraction of the K-wire,
samples from the distal and proximal sections of the fracture line were embedded
in paraffin blocks and cut to yield 4- to
5-mm longitudinal sections, which were
stained with hematoxylin-eosin, Masson’s
trichrome, and CD34 for immunohistochemical examination (Figures 3-4). The
slides were examined at different magni-
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scores between the groups was statistically significant (P<.05) (Figure 5).
Table
Comparison of Histological and Radiological Scores
Median (Maximum–Minimum Range)
Control (n=7)
Platelet-Rich
Fibrin (n=8)
Total (N=15)
Pa
3.50 (4 to 2)
7.29 (8 to 6)
6 (8 to 2)
<.001
Orthopedic
surgeon 1
1.29 (2 to 1)
2.75 (3 to 2)
2.10 (3 to 1)
.002
Orthopedic
surgeon 2
1.43 (2 to 1)
2.75 (3 to 2)
2.18 (3 to 1)
.003
-0.14 (0 to -1)
0 (1 to -1)
-0.08 (1 to -1)
.944
1
1
1
Score
Histological
Goldberg classification
Diff. (1–2)
Pb
a
Mann Whitney U test and Monte Carlo simulation.
Wilcoxon signed rank test and Monte Carlo simulation.
b
fications by 2 pathologists using a light
microscope (Olympus BX51; Olympus,
Tokyo, Japan). Both pathologists were
blind to the therapy and control groups.
The findings were scored using the scale
described by Huo et al.24
Statistical Analysis
SPSS version 22.0 software (IBM
Corporation, Armonk, New York) was
used to evaluate the data. Conformity
of the data to a normal distribution was
tested with the Shapiro-Wilk test. Data
from 2 independent groups were compared using the Mann-Whitney U test and
Monte Carlo simulation. The Wilcoxon
signed rank test was used with the Monte
Carlo simulation for 2 repeated measurements of dependent variables. The Bland
and Altman and concordance coefficient
methods were used to evaluate the conformity and concordance of and the differences between the evaluations of the
orthopedic surgeons. Quantitative data
were expressed as the median (maximum–minimum range) and categorical
data were expressed as number and percentage. The data were analyzed at a 95%
confidence level. P<.05 was considered
statistically significant.
Results
Radiological Data
The median Goldberg classification
scores determined by the 2 orthopedists
were 1.29 and 1.43 in the control group
and 2.75 and 2.75 in the PRF group
(Table). The difference in radiographic
Histological Data
The median histological score was 3.50
in the control group and 7.29 in the PRF
group (Table). The difference was statistically significant (P<.05) (Figure 5).
Discussion
The authors used an animal model of
femoral fracture to demonstrate that PRF
promotes long bone healing. Methods to
improve bone healing, including following
fracture, that accelerate healing as well as
fusion have been examined in several studies.25,26 The complex, immune-mediated
process of fracture healing involves many
local, systemic, and environmental factors, such as the type and size of the wound
caused by the trauma, the involvement of
vascular tissue, and the amount of bleeding.
Angiogenesis is also an important process
in fracture healing, as it affects endochondral and intramembranous repair pathways. Athanasopoulos et al27 showed that
anti-angiogenesis agents impaired bone
healing by causing defective granulation
tissue formation, cartilage tissue differentiation, and endochondral ossification. Vascular endothelial growth factor plays a role
in bone trauma by stimulating bone mineralization and callus formation. Both PRP
and PRF promote bone healing. Gassling et
al28 showed that PRF supports the immune
system, consistent with the current finding
of a lack of infection in the experimental
Figure 5: Descriptive statistical analysis of the histological (a) and radiological (b, c) scores of rats in the control and platelet-rich fibrin (PRF) groups.
e482
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rats. The antimicrobial effects can be attributed to the presence of leukocytes and
interleukins in PRF. In a study by Singh et
al,29 PRF was shown to promote bone and
soft tissue healing in 40 surgical areas, and
to also improve the healing of nerve tissue.
It is unclear whether PRP or PRF is
more efficient. Although PRP contains
more platelets, PRF provides a more
continuous effect, ensuring the slow and
sustained release of growth factors for 14
days. With PRP, there is a surge in growth
factor release, followed by a rapid decrease as the growth factors enter the tissue environment. The longer duration of
action of PRF may improve its efficacy in
bone and tissue regeneration. Moreover,
obtaining PRP is expensive and requires
the use of thrombin. Platelet-rich fibrin is
prepared in a single-stage centrifugation
step and does not require the use of anticoagulants, making its preparation easier
and cost lower. In addition, PRF graft material is in the form of a membrane, which
is often preferred. Platelet-rich fibrin can
be used for many applications, including
dental surgery and the repair of calvarial
bone defects.30 It is an autogenous material that allows the slow and appropriate
release of growth factors.22,30 Importantly,
it can be used to promote natural healing
in patients with a suppressed immune system and a history of radiotherapy.
However, given the scarcity of published studies on the use of PRF in long
bone healing, in this study, the authors
evaluated femoral fracture healing in rats,
both histologically and radiologically, 4
weeks after surgery and PRF application.
The histological and radiological scores of
the PRF group were significantly higher
than those of the control group. The results
of this study indicate that, by increasing the
amount of osseous tissue formation, PRF is
an efficient biomaterial in fracture healing.
Additional studies are necessary to determine the precise mechanisms of its actions,
and whether PRF accelerates the fracture
healing process or ensures a better quality
of fracture healing.
MAY/JUNE 2017 | Volume 40 • Number 3
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