Treatment of Acute Vocal Fold Injury With

Platelet-Rich Plasma
€ rk, †Selcuk Duman, ‡Hasan Esen, †Tahsin Murad Aktan,
*Serap Bulut Cobden, *Kayhan Oztu
‡Mustafa Cihat Avunduk, and *Cagdas Elsurer, *yzKonya, Turkey

Summary: Objectives. Platelet-rich plasma (PRP) is a reliable and has low side-effect profile and has beneficial ef-
fects on wound healing. Its investigatory effects on wound-healing process were shown on various tissues. The aim of
the present study was to evaluate effectiveness of PRP application on scar tissue of acute vocal fold injury.
Materials and methods. Twenty-four Wistar rats were used in the study. The entire layer of the lamina propria down
to the thyroarytenoid muscle of 10 subjects was unilaterally injured by with a microscissor. Gelfoam-absorbed PRP was
applied on the injured area for 10 minutes. Control group consisted of rats unilaterally injured using a microscissor, and
gelfoam with normal saline was applied on the injured area. Following sacrifice, the larynxes were carefully dissected
and removed for histopathologic examination. After excised larynx experiments, serial sections were prepared from
vocal fold. Hematoxylin eosin and immunohistochemical staining were done for epithelial growth factor receptor
(EGFR), fibroblast growth factor receptor (FGFR1), and vascular endothelial growth factor (VEGF) staining for histo-
pathologic examinations.
Results. There was not a significant difference between the two groups for lymphocyte. Although collagen and VEGF
were higher in the study group, there was not a significant difference between the groups (P > 0.05). There was a sig-
nificant difference between control and study groups for EGFR and FGFR1(P < 0.05).
Conclusions. PRP has beneficial effects on wound healing. PRP accelerates epithelization of injured rat vocal folds
by inducing EGFR secretion. PRP is an autogenous, reliable, low side-effect profile, easily harvested material. PRP may
be useful to prevent scar formation.
Key Words: EGFR–FGFR1–Injury–PRP–Scar–VEGF–Vocal fold.

INTRODUCTION fold function by impeding the mucosal wave movement. Because

The vocal folds of the larynx are highly specific and sensitive
structures. They are able to self-sustain oscillation for produc-
tion of voice for speech and singing. The symmetry of the adja-
cent vocal folds and similarity of their microstructure result in
complementary cycle-to-cycle contact, and emittance of rhyth-
mic air pulses. Scar formation disrupts these multilayered sen-
sitive structures and causes significant change in vocal fold
tissue biomechanics in a result of voice problem, which may
have an effect on the life quality or his/her profession.
Vocal fold scarring has many causes, including inflammation,
irritation, or iatrogenic injury.1 Scar formation is the major cause
of dysphonia after vocal cord surgery. Scarring disrupts the extra-
cellular matrix (ECM) of the vocal fold lamina propria, resulting
in a dysphonia that is difficult to treat.2,3 Lamina propria consists
of ECM and cells producing the components of the matrix.
Components of ECM determine the biomechanical properties
of the vocal fold. A balanced distribution of these components
provides an ideal viscoelasticity to vocal fold. During the
wound-healing process, fibrotic tissue replaces the original tis-
sue. Changing in the composition of ECM disturbs the vocal
Accepted for publication July 21, 2015.
The study was presented at Fall Voice Conference, The Westin Riverwalk, San Antonio,
TX, USA, October 23–25, 2014.
From the *Department of Otolaryngology, Selcuklu Faculty of Medicine, Selcuk Uni-
versity, Konya, Turkey; yDepartment of Human Histology and Embryology, Faculty of
Medicine, Necmettin Erbakan University, Konya, Turkey; and the zDepartment of Pathol-
ogy, Faculty of Medicine, Necmettin Erbakan University, Konya, Turkey.
Address correspondence and reprint requests to Serap Bulut Cobden, Department of
Otolaryngology, Selcuklu Faculty of Medicine, Selcuk University, Konya 42080, Turkey.
E-mail: serapbulut88@mynet.com
Journal of Voice, Vol. 30, No. 6, pp. 731-735
0892-1997/$36.00
Ó 2016 The Voice Foundation
http://dx.doi.org/10.1016/j.jvoice.2015.07.012
of the difficulty in remediating chronic vocal fold scar, treat-
ments that target wound-healing events during the acute phase
of injury may be useful in minimizing the effects of eventual
scar formation.4
Platelets play an important role in hemostasis and wound
healing. Alpha granules within platelets contain several growth
factors that have potent effects on wound healing. Platelet-rich
plasma (PRP) is a reliable and has low side-effect profile and
has beneficial effects on wound healing. Recently, the applica-
tion of PRP has been popularized. There are many articles in the
present literature for PRP ranging in fields from orthopedics,
sports medicine, dentistry, otolaryngology, neurosurgery,
ophthalmology, urology, wound healing, cosmetic, cardiotho-
racic, and maxillofacial surgery.5–8 Its investigatory effects on
wound-healing process were shown on many tissues but have
not been studied on vocal folds yet. Thus, the aim of the present
study was to evaluate effectiveness of PRP application on acute
vocal fold injury.
MATERIALS AND METHODS
Twenty-four male pathogen-free Wistar rats (weighing 300–
350 g) were used in the study. All experimental protocols
were approved by the Animal Research Committee of the Nec-
mettin Erbakan University (2011/136). Four subjects of 24 sub-
jects were used for preparing PRP. Remaining 20 subjects were
randomized divided into two groups as PRP group (n ¼ 10) and
control group (n ¼ 10).
PRP preparation
Four subjects of 24 subjects were used for preparing PRP. Four
milliliters of intracardiac blood from each rat were collected in
732
the test tubes containing acid citrate dextrose. The PRP prepa-
ration procedure consisted of two centrifugation steps. After the
first centrifugation (10 minutes, 3000 rpm), the whole plasma
above the buffy coat was collected, separating platelets from
red blood cells and leukocytes. The sample was divided into
1 mL fractions and after the second centrifugation step
(8 min, 2500 rpm). It was activated using 20 mM CaCl2. All
samples were incubated at 37 C for 1 hour.
Vocal fold injury and PRP application procedures
Each subject of both groups was sedated with intramuscular in-
jection of ketamine hydrochloride (15 mg/kg) and xylazine hy-
drochloride (6 mg/kg). The glottis was visualized with a 30 ,
2.7-mm telescope, and the right vocal folds were unilaterally
injured by entire layer of the lamina propria down to the thyro-
arytenoid muscle with a microscissor.
In the PRP group, gelfoam-absorbed PRP was applied on the
injured area for 10 minutes. Control group consisted of unilat-
erally injured using a microscissor, and gelfoam with normal
saline was applied on the injured area. All animals were sacri-
ficed by overdose of intraperitoneal pentobarbital on the 21st
day after treatment. Following sacrifice, the larynxes were care-
fully dissected and removed for histopathologic examination.
Histopathologic examination
After harvesting specimens, immediately they were fixed in
10% formaldehyde for later examination. Subsequently, they
were embedded in paraffin. The specimens were prepared in
an autotechnicon and 5-mm-thick serial sections were done by
a microtome. The paraffin-embedded sections were immuno-
stained with vascular endothelial growth factor (VEGF)
(500 ml), epithelial growth factor receptor (EGFR) (150 mL),
and fibroblast growth factor receptor (FGFR) (125 mg) alpha
antibodies (GeneTex, USA) as follows. After the sections
were deparaffinized, they were rehydrated using methanol
and then incubated with 6% peroxide to block endogenous
peroxidase. The sections were then immersed in 10-mM so-
dium citrate buffer with 0.05% Tween 20 at pH 6.0 to retrieve
the antigen, followed by incubation with 1% bovine serum al-
bumin for 30 minutes at room temperature to block nonspecific
binding of the antibody. The blocked sections were then incu-
bated with VEGF, EGFR, or FGFR1 alpha at dilutions of 1/
FIGURE 1. (A) Collagen, lymphocytes, fibroblasts, and vascular are seen around the injured tissue of PRP group. (B) Control group. C, collagen;
L, lymphocytes; F, fibroblasts; V, vascular structure; hematoxylin and eosin (H&E) staining.
Journal of Voice, Vol. 30, No. 6, 2016
100 ratio and were washed three times in phosphate-buffered
saline. Large volume DAB substrate system (Thermo Scientific,
USA) containing secondary antibody, streptavidin enzyme con-
jugate, and a DAB substrate chromogen mixture was used to
localize the antigen in the tissue specimens. The incubation
times and washes were performed according to the manufac-
turer’s instructions. To visualize the principal stain more easily,
a counterstain (hematoxylin and eosin) was applied.
Hematoxylin and eosin (H&E) staining was applied to
analyze lymphocyte and collagen on the sections obtaining
from the similar area.
The stained sections were investigated by a Nikon Eclipse
E400 light microscope by a blinded pathologist. For each spec-
imen, the same area was photographed after staining by using a
Nikon Coolpix 5000 photograph attachment. The photograph
of Nikon micrometer microscope slide (Stage Micrometer
Type A, MBM11100) was also taken during the procedure.
All photographs were then transferred into PC environment
and analyzed by using Clemex Vision Lite 3.5 Image Analysis
program (Longueuil, Canada). The length was calibrated by
comparing the photograph of specimen with the photograph
of Nikon micrometer microscope slide, which was taken under
the same magnification. Area of 125427.4 mm2 was designated
with using Clemex Vision Lite 3.5 Image Analysis program;
then, lymphocyte, collagen fibers (Figure 1A and B), EGFR
(Figure 2), FGFR1 (Figure 3), and VEGF (Figure 4) positive-
stained cells were marked with the same image analysis pro-
gram in 125427.4 mm2 area. Damaged cells were not evaluated.
The marked cells were counted automatically with the same im-
age analysis Program.
RESULTS
Mean values and standard deviations are given in Table 1. The
mean collagen was calculated for both groups. In the control
group, the mean collagen value was 4.90 ± 0.99, whereas it
was 7.30 ± 0.95 in the study group. Although collagen was
higher in the study group, there was not a significant difference
between groups (P > 0.05). In the control group, the mean lym-
phocytes value was 24.90 ± 4.23, whereas it was 26.70 ± 5.12 in
the study group. There was not a significant difference between
the two groups for lymphocyte (P > 0.05).
Serap Bulut Cobden, et al Effectiveness of PRP Application 733

FIGURE 4. Arrows show vascular endothelial growth factor positive

FIGURE 2. Arrows show epithelial growth factor receptor positive cells, 192 3 139 mm (300 3 300 DPI).
cells, 194 3 139 mm (300 3 300 DPI).

in restoring these structural changes. Different substances

In the control group, the mean EGFR, FGFR1, and VEGF have been studied to assess this subject. Steroid injection,
positive cells were 13.30 ± 2.36, 7.70 ± 1.95, and collagen and autologous fat injection,12,13 fascia implants,14
6.60 ± 2.12, respectively, whereas they were 16.20 ± 2.39, mitomycin-C,15 hyaluronic acid,16 hepatocyte growth factor
9.60 ± 2.07, and 7.90 ± 1.45, respectively, in the study group. (HGF),17 fibroblast growth factor (FGF),18 and stem cell19
There was a significant difference between control and study have been studied in vivo or in vitro vocal fold scar treatment.
groups for EGFR and FGFR1 (P < 0.05). Although VEGF pos- It was found that they have some beneficial effects on the man-
itive cells mean value was higher in the study group than control agement of scar formation.
group, the difference was not significant (P > 0.05). Chhetri et al20 investigated the use of cultured autologous
fibroblast for human vocal fold scars. They injected three doses
DISCUSSION of cells at 4-week intervals. They evaluated mucosal wave grade,
The causes of vocal fold scar are multifactorial. Phonotrauma, voice handicap index, voice quality questionnaire and they re-
chronic inflammation, or surgical trauma will likely disrupt the ported improved outcomes. However, the number of clinical
balance within the ECM. Disruption of this balance will lead to studies on this subject is limited. In the human studies, it is not
scarring in the vocal fold. Scar formation is the major cause of possible to observe the wound healing histopathologically. We
dysphonia after phonosurgery. Histologically, vocal fold scar selected rats to evaluate histopathologic effects of PRP. We did
has been characterized by increased collagen, decreased
hyaluronic acid, and decreased elastin.9,10 The most extreme
response of injury and repair in the vocal folds is scarring, a
response that represents the final stage of wound healing– TABLE 1.
wound contraction and remodeling, which is characterized by Mean Value and Standard Deviations of Lymphocyte,
Collagen, EGFR, FGFR1, VEGF Positive Cells
poor reconstitution of the epidermal/dermal tissue.11 Effective
treatments for vocal fold scar are lacking because of difficulty Groups n Mean Value Standard Deviation
Collagen
1 10 4.90 0.99
2 10 7.30 0.95
Lymphocyte
1 10 24.90 4.23
2 10 26.70 5.12
EGFR
1 10 13.30 2.36
2 10 16.20 2.39
FGFR
1 10 7.70 1.95
2 10 9.60 2.07
VEGF
1 10 6.60 2.12
2 10 7.90 1.45
Abbreviations: Group 1, platelet-rich plasma group; group 2, control
group; EGFR, epithelial growth factor receptor; FGFR, fibroblast growth
FIGURE 3. Arrows show fibroblast growth factor receptor 1 factor receptor; VEGF, vascular endothelial growth factor.
(FGFR1) positive cells, 193 3 139 mm (300 3 300 DPI).
734
not inject PRP because injection can cause some scar formation.
We choosed PRP which is autogenous, reliable, cheap, has a low
side-effect profile, easy to prepare, and can easily be used in the
human beings. Suehiro et al18 investigated the effects of basic
fibroblast growth factor (bFGF) for treatment of acute vocal
fold scar in a canine model. Vocal folds of eight beagles were
unilaterally injured by removal of the mucosa under direct laryn-
goscopy. Four beagles received local injections of bFGF deliv-
ered to the scarred vocal fold at 1 month after injury. The
remaining four beagles received local injections of saline and
served as a sham-treatment group. They found that bFGF group
had less scar tissue. At the present study, we found that PRP pro-
vided an increase of FGF level. Therefore, PRP may have a po-
tential for the treatment of vocal fold scar by increasing FGFR
level. Hirano et al17 studied the effects of HGF for treatment of
acute vocal fold injury in a canine model. Histological data re-
vealed reduced collagen deposition and decreased tissue contrac-
tion of the lamina propria in vocal folds treated with HGF. They
concluded that HGF may be effective in the treatment of acute
vocal fold injury. Stem cell researches to treat vocal fold scars
were made by some authors. Kanemaru et al19 investigated the
use of implanted mesenchymal stem cells for treatment of vocal
fold scar in a canine model. Mesenchymal stem cells were har-
vested from bone marrow and cultured. Cultured mesenchymal
stem cells were then injected into the injured vocal fold using
an atelocollagen scaffold. Histologic and morphologic assess-
ment of treated vocal folds revealed improved healing after
2 months. However, harvesting of mesenchymal stem cells
used in these studies requires an invasive procedure. Many clin-
ical and experimental studies revealed that EGF has positive ef-
fects in several stages of wound healing including inflammation,
wound contraction, proliferation, migration, and angiogen-
esis.21–23 In the present study, we observed that PRP provides
an increase of EGFR level.
The major functions of platelets are preventing acute blood
loss and repairing vascular walls and adjacent tissues after injury.
During wound healing, platelets are activated by contact with
collagen, exposed to the bloodstream after endothelial injury.
Platelets secrete stored intercellular mediators and cytokines
from the cytoplasmic pool and release their a-granule content
after aggregation. This secretion is intense in the first hour, and
platelets continue synthesizing more cytokines and growth
factors from their mRNA reserves for at least another 7 days.
More than 800 different proteins are secreted into the surround-
ing media, having a paracrine effect on different cell types:
myocytes, tendon cells, mesenchymal stem cells from different
origins, chondrocytes, osteoblasts, fibroblasts, and endothelial
cells. Cell proliferation, angiogenesis, and cell migration are
stimulated, resulting in tissue regeneration. There are also reports
confirming that platelets secrete antimicrobial peptides, suggest-
ing an antibiotic effect. It was revealed through a number of
studies that various bioactive substances, including platelet-
derived growth factor, transforming growth factor—beta, and
epidermal growth factor, were discharged from the a-granules
of platelets into plasma when the platelets were destroyed and
activated. These growth factors are contained in wound exudates
from injured subcutaneous tissue and are important in the early
Journal of Voice, Vol. 30, No. 6, 2016
phase of the wound-healing process.21–24 Specifically, they
promote stromal stem cell proliferation and angiogenesis and
are regarded as key signals in tissue repairing and regeneration.
PRP has been used before in many tissues but has not been
tested in wound healing on the vocal folds. The aim of using
PRP is to transfer maximum level of platelets biological
molecules that increase in tissue regeneration to the damaged
tissue. In addition, PRP has been shown to secrete bioactive
proteins that attract macrophages, platelets, and mesenchymal
stem. These cells clean degenerated and necrotic tissue and
contribute to tissue regeneration and healing process.21–24 PRP
may provide beneficial effects not only by growth factors and
cytokines contained in platelets, but also by increasing growth
factor receptors.
CONCLUSIONS
PRP has beneficial effects on wound healing. It accelerates
epithelization of injured rat vocal folds by inducing EGFR
secretion. PRP is an autogenous, reliable, cheap, low side-
effect profile and easily harvesting material. PRP may be useful
to prevent scar formation. New clinical studies are needed to
evaluate the effect of these findings on the functional outcomes.
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