The Laryngoscope

C 2016 The American Laryngological,

V
Rhinological and Otological Society, Inc.

Outcome of Regenerative Therapy for Age-Related Vocal Fold

Atrophy With Basic Fibroblast Growth Factor

Satoshi Ohno, MD, PhD; Shigeru Hirano, MD, PhD; Akiyoshi Yasumoto, MD; Hiroki Ikeda, MD, PhD;
Shinji Takebayashi, MD, PhD; Makoto Miura, MD, PhD

Objectives/Hypothesis: Age-related vocal fold atrophy has become a significant voice disorder as the elderly population
grows. However, several therapeutic challenges have limited attempts to improve voice quality. We reported that basic fibro-
blast growth factor (bFGF) stimulates fibroblasts to produce extracellular matrices such as hyaluronic acid in the lamina
propria, leading to a regeneration of pliable vocal folds in animal models. The aim of this study was to determine the efficacy
of bFGF for the treatment of age-related vocal fold atrophy.
Study Design: Prospective study.
Methods: Six patients with age-related vocal fold atrophy underwent injection of bFGF in their vocal folds. Vocal out-
comes and stroboscopic examinations were evaluated 1, 3, and 6 months after the injection. The outcome measures included
the Voice Handicap Index-10 (VHI-10), GRBAS (grade, roughness, breathiness, asthenia, strain) scale, maximum phonation
time (MPT), the amplitude perturbation quotient (APQ), and the pitch perturbation quotient (PPQ).
Results: The VHI-10 was significantly improved 6 months after bFGF injection. The GRBAS scale, MPT, APQ, and PPQ
were also improved. Stroboscopic examinations showed significant improvement of glottic closure and better mucosal wave.
Conclusions: This is the first study to evaluate the regenerative effects of bFGF injection for the treatment of age-
related vocal fold atrophy using the VHI-10. Injection of bFGF significantly improved VHI-10 scores and glottal insufficiency
for at least 6 months.
Key Words: Basic fibroblast growth factor, vocal fold atrophy, Voice Handicap Index-10, aging.
Level of Evidence: 4.
Laryngoscope, 00:000–000, 2016

INTRODUCTION examination of aged human vocal folds revealed dense,

Recently, the elderly population has been growing
in developed countries. According to the 2010 Revision
of United Nations World Population Prospects, individu-
als over 65 years of age account for approximately 20%
of the population in developed countries and 7.6% of the
population worldwide.1 Thus, age-related disorders are
estimated to become more important, especially in devel-
oped countries.
Age-related voice problems are characterized by
hoarseness, harshness, breathiness, and increased vocal
effort.2 Stroboscopic examination shows atrophy and
bowing of the vocal folds, glottal insufficiency, and
reduced mucosal wave during phonation.3,4 Histologic
From the Department of Otolaryngology, Head and Neck Surgery
(S.O., A.Y.), Kokura Memorial Hospital, Fukuoka, Japan; Department of
Otolaryngology (S.O., A.Y., H.I., S.T., M.M.), Japanese Red Cross Wakayama
Medical Center, Wakayama, Japan; Department of Otolaryngology, Head
and Neck Surgery (S.H.), Graduate School of Medicine, Kyoto University,
Kyoto, Japan.
Editor’s Note: This Manuscript was accepted for publication July
20, 2015.
Presented at the Triological Society Annual Meeting at COSM,
Boston, Massachusetts, U.S.A., April 22–26, 2015.
The authors have no funding, financial relationships, or conflicts
of interest to disclose.
Send correspondence to Satoshi Ohno, MD, Department of Otolar-
yngology, Head and Neck Surgery, Kokura Memorial Hospital, Fukuoka,
802-8555, Japan. E-mail: s_ohno@ent.kuhp.kyoto-u.ac.jp
DOI: 10.1002/lary.25578
disorganized, and increased collagenous fibers5 and
reduction of hyaluronic acid (HA),6 which plays a major
role in maintaining the viscoelasticity of the vocal folds.7
These changes in extracellular matrix (ECM) compo-
nents disrupt the viscoelasticity of the vocal folds, lead-
ing to the aforementioned voice problems. The number
of patients with vocal fold atrophy is reported to
increase from their 50s.1 Voice therapy and/or surgery,
including injection augmentation or medialization lar-
yngoplasty, does not result in satisfactory results.8–10
The failure of these approaches is because the ECM
components of the lamina propria of the vocal folds were
not altered.
Basic fibroblast growth factor (bFGF) is a member
of the fibroblast growth factor family. bFGF is a positive
regulator of angiogenesis and plays a significant role in
wound healing. It has powerful proliferative effects on
fibroblasts and induces production of ECM compo-
nents.11 Hirano et al. reported the regenerative effects of
bFGF on age-related vocal fold atrophy in rats and
humans. Administration of bFGF into the lamina prop-
ria of the vocal folds improves objective measurements,
including aerodynamic and acoustic parameters.12,13
Self-assessment is also an important factor in the treat-
ment of voice disorders. Gartner-Schmidt and Rosen
examined the outcome of voice therapy and surgery for
the treatment of age-related vocal fold atrophy with the

Laryngoscope 00: Month 2016 Ohno et al.: Therapy for Vocal Fold Atrophy With bFGF
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Fig. 1. Voice Handicap Index-10 (VHI-10) scores at preoperation
and 1, 3, and 6 months (M) postoperation. Boxes indicate first
and third quartiles. Median values are denoted by the line in each
box. Whisker caps indicate smallest and largest nonoutlier values.
Outlier is indicated by an X. VHI-10 was significantly improved at
each time point. The scores improved more than five points in all
cases. *P < .05. **P < .01: one-way repeated measures analysis of
variance with post hoc Tukey test.
Voice Handicap Index-10 (VHI-10), which is one of the
self-assessment tools used to evaluate voice problems.8
In the current study, we examined the outcome of
bFGF injection into the lamina propria of the vocal folds for
the treatment of human age-related vocal fold atrophy,
with particular attention to the improvement of both objec-
tive and subjective parameters, including the VHI-10.
MATERIALS AND METHODS
Patients
From 2013 to 2014, six patients (five men and one woman)
diagnosed with age-related vocal fold atrophy were recruited.
The ages of the patients ranged from 52 to 78 years (mean 5 68
years). All patients had no history of malignant tumors in the
larynx or the pharynx, no organic or neurological problem of
the larynx, and no severe systemic disorders. All procedures
were approved by the institutional review board.
Drug Information
A commercial form of human recombinant bFGF (Fiblast;
Kaken Co., Tokyo, Japan) was prepared. The active ingredient
of this drug is trafermin (a recombinant genetically engineered
human bFGF). The supplied drug information shows that this
drug stimulates the growth and proliferation of endothelial cells
and fibroblasts, and contributes to the improvement of wound
healing by stimulating angiogenesis and the formation of proper
granulation tissue. Adverse effects, including pain, rash, and
itching at the application site, were reported in 1.5% of cases.
Application of the drug to the site of malignant tumors is not
recommended due to concerns that the drug might stimulate
tumor growth. Fiblast has been approved for the treatment of
pressure ulcers and burn scars by the Japanese Ministry of
Health and has been widely used on human patients in spray
form for over 20 years. No serious adverse effects have been
reported.
Injection Procedures
The pharynx and larynx were completely anesthetized with
4% lidocaine administered with a nebulizer and a spray. Ten
micrograms of bFGF dissolved in 0.5 mL saline were injected
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transorally into the superficial layer of the lamina propria on
both sides of the vocal folds. The injection was performed using a
curved injection needle under transnasal fiberscopic monitoring
of the larynx. Swelling of the lamina propria by the injected liq-
uid was observed. Patients were instructed to rest their voices
while being monitored for side effects, such as allergic response
and edema of the larynx, on the day of the injection and were
allowed to phonate the next day.
Assessment
Vocal outcomes and stroboscopic examinations were eval-
uated 1, 3, and 6 months after the injection. Stroboscopic exam-
ination was performed using a laryngeal stroboscope (U-Medical
Co. Ltd., Busan, Korea). As a subjective voice assessment, the
VHI-10 was evaluated. Gartner-Schmidt and Rosen proposed
that more than a five-point decrease in the VHI-10 score after
treatment indicates treatment success in age-related vocal fold
atrophy.8 As a perceptual voice assessment, the GRBAS scale
(grade, roughness, breathiness, asthenia, strain) was evaluated.
This scale was first developed by the Japanese Society of Logo-
pedics and Phoniatrics and has become popular worldwide.14 As
an aerodynamic assessment, maximum phonation time (MPT)
was evaluated with a phonation analyzer (PA-1000; Minato
Medical Science Co. Ltd., Osaka, Japan). As an acoustic assess-
ment, amplitude perturbation quotient (APQ; shimmer) and
pitch perturbation quotient (PPQ; jitter) were evaluated with a
Visi-Pitch IV Multi-Dimensional Voice Program (Model 3950B;
KayPENTAX, Montvale, NJ). The amplitude of the mucosal
wave was measured to evaluate the mucosal vibration and elas-
ticity of the vocal fold structures, namely the lamina propria,
using ImageJ software (National Institutes of Health, Bethesda,
MD).15 The distance (d1) from the midline of the glottis to the
free edge of the vocal fold was measured at the anteroposterior
middle portion of the vocal fold during the closed phase. The
closed phase was determined based on the motion of the upper
and lower lips of the vocal folds. The same distance (d2) was
measured at the maximum open phase. The mucosal wave
amplitude was defined by subtracting d1 from d2, and was nor-
malized by dividing this number by the anteroposterior length
of the glottis, from the anterior commissure to the vocal process
(L). The following formula was used: normalized mucosal wave
amplitude (NMWA) 5 (d22d1)/L. Normalized glottal gap (NGG)
was calculated to evaluate glottic closure during the closed
phase. The glottal gap area (a) was measured using ImageJ
software, and the glottal gap area was normalized by dividing it
by L2. The following formula was used: NGG 5 a/L2 3 100.
Assessments for the GRBAS scale, NMWA, and NGG were
performed in a blinded fashion, in which the examiners were
not informed of the group to which the data belonged. Corre-
spondence rates were examined between examiners, revealing
good correspondence rates of more than 95%.
Statistical Analysis
Statistical comparison of the data was performed between
the groups with one-way repeated measures analysis of var-
iance with a post hoc Tukey test or the Kruskal-Wallis test, fol-
lowed by the Steel-Dwass test after comparing their variances
with the F test. A probability (P) value .05 was considered
statistically significant.
RESULTS
The VHI-10 score was significantly improved at
each time point (Fig. 1). The scores improved more than
five points in all cases. The GRBAS scale was improved
Ohno et al.: Therapy for Vocal Fold Atrophy With bFGF
 went bilateral injection of bFGF. Six months after the
injection, all vocal parameters were improved (VHI-10:
13 to 1, GRBAS: 11100 to 10010, MPT: 8 to 10 seconds,
APQ: 2.723 to 2.137, PPQ: 1.041 to 0.59.).
No major side effects, such as an allergic reactions,
were observed.

Fig. 2. GRBAS (grade, roughness, breathiness, asthenia, strain)
scale at preoperation and 1, 3, and 6 months (M) postoperation.
G, R, B, and A were improved at each time point. S was 0 in all
cases and at all time points.
at each time point, except for S, which was 0 in all cases
and at all time points (Fig. 2). MPT, APQ and PPQ were
improved; however, the differences did not reach statisti-
cal significance (Fig. 3A–C). Stroboscopic examinations
showed better mucosal wave and improved glottic
closure. NMWA and NGG were significantly improved at
each time point (Fig. 4A,B). Fig. 5 shows stroboscopic
results of a representative case. The patient was a 62-
year-old male presenting with hoarseness for several
years. The stroboscopic examination revealed bilateral
vocal fold atrophy with glottic insufficiency. He under-
DISCUSSION
bFGF is a small polypeptide growth factor that pro-
motes angiogenesis and wound healing by stimulating
fibroblasts to proliferate and produce ECM compo-
nents.11 Fibroblasts are a major source of ECM compo-
nents in the lamina propria of the vocal folds; these
include HA, collagen, and elastin. In aged vocal folds,
disruption of ECM components, including HA reduction,
is characteristic. Thus, a treatment strategy to stimulate
fibroblasts to produce ECM components by administra-
tion of bFGF makes sense. bFGF induces the upregula-
tion of HA synthase and HA production from the vocal
fold fibroblasts of rats and canines.16–18 Administration
of bFGF into vocal folds improved the restoration of
hyaluronic acid in the lamina propria of the vocal folds
in rat and canine models.12,19,20
The half-life of bFGF is 9 hours in vivo,21 which is
likely too short to regenerate ECM components in the
lamina propria of the vocal folds. However, exogenous
bFGF induces the upregulation of endogenous bFGF and

Fig. 3. Aerodynamic and acoustic parameters at preoperation and 1, 3, and 6 months (M) postoperation. Boxes indicate first and third quar-
tiles. Median values denoted by the line in each box. Whisker caps indicate smallest and largest nonoutlier values. Outlier is indicated by
an X. All parameters showed improvements after the operation; however, the differences did not reach statistical significance. (A) Maximum
phonation time (MPT). (B) Amplitude perturbation quotient (APQ). (C) Pitch perturbation quotient (PPQ).

Fig. 4. (A) Normalized mucosal wave amplitude (NMWA) at preoperation and 1, 3, and 6 months (M) postoperation. **P < .01: one-way
repeated measures analysis of variance with post hoc Tukey test. (B) Normalized glottal gap (NGG) during the closed phase at preoperation
and 1, 3, and 6 months postoperation. *P < .05: Kruskal-Wallis test, followed by Steel-Dwass test. Boxes indicate first and third quartiles.
Median values are denoted by the line in each box. Whisker caps indicate smallest and largest nonoutlier values. Outlier is indicated by an
X. NMWA and NGG were significantly improved after the operation at each time point.

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Fig. 5. Stroboscopic findings of a representative case. Stroboscopy showed complete glottic closure and better mucosal wave at 6 months
after the operation. (A) Before the operation. (B) Six months after the operation.
hepatocyte growth factor from fibroblasts, which is
known to have a strong antiscarring effect22; that is to
say, administration of bFGF becomes a growth factor
cascade trigger that results in long-lasting regenerative
effects. Long-term results over 1 year were not exam-
ined, but the results indicate that further studies of
bFGF are warranted.
The effects of exogenous growth factors are known to
depend on the dosage used. For example, transforming
growth factor b3 (TGFb3) is a strong antiscarring growth
factor that is expressed at high levels in fetal wounds,
which heal without scarring. Administration of TGFb3
prevents scarring in buccal mucosa wounds; however,
negative effects are observed at higher concentrations.23
The dosage of bFGF used in this study was based on pre-
viously reported effective concentrations.13,19,24 However,
because no study has investigated optimal dosage in the
vocal folds, further examination of optimal bFGF dosage
is warranted. For mild atrophy, one injection is consid-
ered sufficient. However, the optimal number of injections
should be considered in each case.
The Voice Handicap Index is a measure of subjec-
tive voice handicap developed by Jacobson et al.25 It is a
patient-based self-assessment tool that consists of 30
items addressing voice problems from functional, physi-
cal, and emotional perspectives. Rosen et al. proposed to
reduce these items from 30 to 10 (VHI-10).26 The VHI-10
has been widely accepted to assess patient’s perception
of their voice handicap. In this study, the VHI-10 scores
improved more than five points after the operation in all
cases.
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Judging from the results of preoperative aerody-
namic and acoustic examination, the patients in this
study consisted of mainly mild atrophy cases. Even mild
atrophic change of the vocal folds that does not affect
aerodynamic and acoustic examination can cause voice
problems and worsen the subjective perception of voice.
The VHI-10 was shown to be a useful tool for such cases
to assess vocal problems and make treatment decisions.
CONCLUSION
The present study demonstrated that administra-
tion of bFGF into the lamina propria of the vocal folds
significantly improved VHI-10 scores in the patients of
age-related vocal fold atrophy. The GRBAS scale, MPT,
APQ, and PPQ scores were also improved. Stroboscopic
examination showed significant improvement of glottic
closure and mucosal wave. The VHI-10 was useful for
making treatment decisions and assessments of treat-
ment success. bFGF injection is a promising therapy for
treating age-related vocal fold atrophy.
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