The Laryngoscope

V
C 2012 The American Laryngological,
Rhinological and Otological Society, Inc.

Clinical Trial of Regeneration of Aged Vocal Folds With

Growth Factor Therapy

Shigeru Hirano, MD, PhD; Ichiro Tateya, MD, PhD; Yo Kishimoto, MD;
Shin-ichi Kanemaru, MD, PhD; Juichi Ito, MD, PhD

Objectives/Hypothesis: Aged vocal folds are characterized by atrophy of the mucosa, which causes mucosal wave defi-
ciency and glottal insufficiency. This clinical trial examined the regenerative effects and safety considerations of basic fibro-
blast growth factor (bFGF) on restoration of aged vocal folds.
Study Design: Institutional review board–approved human clinical trial.
Methods: Ten patients (6 men and 4 women; mean age, 70.1 years) were recruited in the trial. Ten micrograms of bFGF
was injected into each treated vocal fold. Injection was performed unilaterally or bilaterally according to each patient’s need
and repeated up to seven times if necessary. Patient follow-up continued for at least 6 months. The effectiveness of the treat-
ment was assessed by stroboscopic, acoustic, and aerodynamic measurements.
Results: All patients showed improvement of voice. Significant improvements in maximum phonation time, mean flow
rate, jitter, shimmer, and noise-to-harmonic ratio lasted for at least 1 year. No allergic or long-term adverse effects were
noted.
Conclusions: This clinical trial suggests that bFGF may be effective and safe as a regenerative agent for aged vocal
folds.
Key Words: Aged vocal fold, basic fibroblast growth factor, regeneration, clinical trial.
Level of Evidence: 1b.
Laryngoscope, 122:327–331, 2012

INTRODUCTION been evaluated for their ability to improve aged voice,8,9

Age-related change of voice is characterized as hav-
ing a weak, harsh, and breathy voice.1 This change is
caused by histologic alteration of the lamina propria of
the vocal fold mucosa as well as atrophy of the thyroary-
tenoid muscle. Reorganization of the extracellular
matrix (ECM) in the lamina propria leads to excessive
deposition of disorganized collagen, reduction of elastic
and reticular fibers, and reduction of hyaluronic acid
(HA).2–5 These histologic alterations result in fibrotic
and atrophic changes in the mucosa. Stroboscopic exami-
nation often reveals bowing of the vocal fold, reduction
of the mucosal wave, and glottic insufficiency.6,7
Several therapeutic strategies involving injection
laryngoplasty and laryngeal framework surgery have
From the Department of Otolaryngology–Head and Neck Surgery,
Graduate School of Medicine (S.H., I.T., Y.K., J.I.), Kyoto University, Kyoto,
Japan; and Department of Otolaryngology, Kitano Hospital (S.-I.K.), Osaka,
Japan.
Editor’s Note: This Manuscript was accepted for publication
August 24, 2011.
Given as an oral presentation at the American Laryngological
Association 132nd Annual Meeting Combined Otolaryngology Spring
Meetings, Chicago, Illinois, U.S.A., April 27–28, 2011.
This study was supported in part by the National Institute of
Biomedical Innovation. The authors have no other funding, financial
relationships, or conflicts of interest to disclose.
Send correspondence to Shigeru Hirano, MD, Department of
Otolaryngology–Head and Neck Surgery, Graduate School of Medicine,
Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
E-mail: hirano@ent.kuhp.kyoto-u.ac.jp
DOI: 10.1002/lary.22393
but these showed limited effectiveness. These medializa-
tion procedures can improve glottic closure, which may
contribute to a reduction in effort of voicing. However,
because they do not address the histologic changes of
the mucosa, recovery of vibratory function is limited,
and the quality of voice rarely shows sufficient
improvement.
To achieve recovery of vocal fold–tissue properties,
it is essential to address these histologic alterations in
the aged vocal fold. Regenerative medicine has the
potential to accomplish this aim. Since 2001, we have
been researching the regenerative effects of basic fibro-
blast growth factor (bFGF) on aged vocal folds. We
initially reported that bFGF stimulates growth and pro-
liferation of fibroblasts in aged vocal fold lamina propria
in a rat model and also found that it stimulates cells to
produce more HA and reduce collagen production.10
These effects seemed promising for restoring the histol-
ogy of aged vocal fold mucosa. Subsequent in vivo study
using aged rats demonstrated that transoral injection of
bFGF into the vocal fold increased the amount of HA in
the lamina propria.11 Because HA has been regarded as
the key molecule for maintaining optimal viscoelasticity
of the vocal fold,12 increased deposition of HA was
expected to improve recovery of vibratory function.
Based on the results of this foundational research,
a clinical trial with institutional review board approval
was set up in 2007 in the Department of Otolaryngology,
Kyoto University, to examine the regenerative effects of

Laryngoscope 122: February 2012 Hirano et al.: Regeneration of Aged Vocal Fold
327

Fig. 1. Injection of basic fibroblast growth factor with topical an-
esthesia. (Left) Placement of needle at the vocal fold mucosa.
(Right) Injection of basic fibroblast growth factor into the lamina
propria.
bFGF on recovery of vocal function in an elderly popula-
tion. We reported the preliminary results of the first
case, which indicated remarkable improvement of vocal
fold vibratory properties with improvement in aerody-
namic and acoustic aspects.13 The trial was closed in
2009, and data analysis was completed. Here we report
the full results of the clinical trial.
MATERIALS AND METHODS
Inclusion Criteria
Patients with dysphonia due to vocal fold atrophy who
met the following criteria were recruited in this study: unilat-
eral or bilateral vocal fold atrophy diagnosed by stroboscopic
examination; 50 years of age or older; no organic or neurogenic
lesions on the vocal folds; no history of malignant tumors, he-
patic diseases, kidney dysfunction, or other severe systemic
disorders; and no psychiatric disease. Patients who were judged
inappropriate for the study for any other reason were excluded.
Growth Factor Drug Information
A commercially available form of human recombinant
bFGF (Fiblast; Kaken Co., Tokyo, Japan) was prepared. The
active ingredient of this drug is trafermin (a recombinant genet-
ically engineered form of human bFGF). The supplied drug
information indicates that this drug stimulates proliferation of
endothelial cells and fibroblasts and improves wound healing by
stimulating angiogenesis and formation of proper granulation
tissue. Adverse effects were reported in 1.5% of cases, including
pain, rash, and itching at the application site. Application of
the drug to the site of malignant tumors is not recommended,
as it is possible that it may stimulate growth of the tumor. The
drug half-life is not provided.
Fiblast was approved for the treatment of skin ulcers and
bed sores by the Japanese Ministry of Health in 1991 and has
been widely used on human patients in spray form.
Treatment Protocol
Ten micrograms of bFGF dissolved in 0.5 mL saline was
injected transorally into one side of the vocal fold. The pharynx
and larynx were completely anesthetized with 4% Lidocaine
administered with an atomizer; following anesthetization, the
injection was performed using a curved injection needle (Fig. 1).
Laryngoscope 122: February 2012
328
TABLE I.
Demographic Data of Patients (N 5 10).
Male/female 6/4
Age, yr 70.1 6 5.3
History of vocal abuse 1
History of smoking 0
History of steroid inhaler 0
Follow-up period, mo 11.6 6 4.5
Possible allergic response was checked 1 hour after the injec-
tion. The injection was performed unilaterally or bilaterally and
repeated, at most, four times with an interval of 1 week
between each injection for each treatment cycle. Another cycle
of injections was considered 3 months after the first cycle in
cases in which the initial effect was insufficient.
Assessment
Patient follow-up continued for at least 6 months after the
final injection. Aerodynamic, acoustic, and stroboscopic examina-
tions were performed every 3 months. Stroboscopic examination
was done using a Digital Video Stroboscopy System, Model 9295
(KayPentax, Lincoln Park, NJ). Aerodynamic assessment included
maximum phonation time (MPT) and mean flow rate (MFR) exam-
ined with a phonation analyzer (PA-500; Nagashima Co., Osaka,
Japan). Acoustic analyses evaluated amplitude perturbation quo-
tient (APQ; shimmer), pitch perturbation quotient (PPQ; jitter),
and noise-to-harmonic ratio (NHR) using a Multi-Dimensional
Voice Program (Model 5105; KayPentax). Statistical tests were
completed for each parameter of aerodynamic and acoustic exami-
nation using a repeated-measures analysis of variance with post
hoc Scheffe’s test. P < .05 was considered significant.
RESULTS
Ten patients were enrolled, including four females
and six males. The mean age was 70.1 6 5.3 years (CI).
One patient, a 63-year-old male, was a traditional Japa-
nese singer, but no others showed a history of vocal
abuse. All were nonsmokers with no history of steroid
inhalation. Table I indicates the demographic data of the
patients. The total number of injections varied from one
to seven (mean, 2.7 injections) (Fig. 2). Laryngoscopic ex-
amination showed complete absorption of the injected
Fig. 2. Total number of injections.
Hirano et al.: Regeneration of Aged Vocal Fold
 ciency. Figures 6 and 7 indicate stroboscopic findings at 3
and 14 months after the injection. Vibratory status
improved with complete glottic closure after the injection.

DISCUSSION
The aim of regenerative medicine is to recreate new
tissue using cells, growth factors, and scaffolds.14 These
three elements can be used separately or in combination.
Anti-aging is one of the goals of regenerative medicine,
and the current study was targeted at aged voice. The
intent of the growth factor therapy used in this study

Fig. 3. Aerodynamic assessment. Maximum phonation time (MPT)

and was significantly improved at 3, 9, and 12 months (P ¼ .049,
.037, and .045, respectively). Mean flow rate (MFR) was signifi-
cantly improved at 1 and 3 months (P ¼ .035 and .019, respec-
tively). *P < .05.

solution into the vocal fold 1 hour after injection. No al-

lergic response was found in any patient. Patient follow-
up was 11.6 6 4.5 months (CI). Long-term adverse
effects were not reported within the 2 years of follow-up.

Aerodynamic Examination
Figure 3 shows the mean value, including standard
deviation, of MPT and MFR at each time point. MPT sig-
nificantly increased 3 months after injection (P ¼ .049),
and this effect was maintained for up to 12 months (P ¼
.045). MFR was significantly decreased at 1 and 3
months (P ¼ .035 and 0.019, respectively).

Acoustic Analysis
Figure 4 indicates the temporal changes in PPQ,
APQ, and NHR. The PPQ improved after the injection
and reached a significant level at 12 months (P ¼ .004).
APQ significantly improved at 6 months through 12
months (P ¼ .036 and .02, respectively). NHR was also
significantly improved at 6 months through 12 months
(P ¼ .014 and .01, respectively).
Fig. 4. Acoustic assessment. Pitch perturbation quotient (PPQ)
Representative Cases significantly improved at 12 months (P ¼ .004). Amplitude pertur-
bation quotient (APQ) significantly improved at 6 and 12 months
A 67-year-old male underwent two injections. (P ¼ .036 and .02, respectively), and noise-to-harmonic ratio
Figure 5 shows preinjection stroboscopic findings, which (NHR) significantly improved at 6 and 12 months (P ¼ .014 and
showed bilateral vocal fold atrophy with glottic insuffi- .01, respectively). *P < .05, **P < .01.

Laryngoscope 122: February 2012 Hirano et al.: Regeneration of Aged Vocal Fold
329
Fig. 5. A 67-year-old male. The preinjection images demonstrated bilateral vocal fold atrophy with glottic insufficiency.

Fig. 6. A 67-year-old male. Stroboscopic findings at 3 months after injection indicated improved vibratory status with complete glottis
closure.

Fig. 7. A 67-year-old male. Stroboscopic findings at 14 months after injection still indicated improved vibratory status with complete glottis
closure.
was to transform tissues by stimulation of endogenous
cells with ectopically applied growth factor.
Age-related changes in vocal fold histology have
been well documented, including the accumulation of ex-
cessive collagen and a reduction in HA, elastin, and
reticular fibers. Fibroblasts are the main producers of
these ECM components and thus are most responsible for
alterations and disorganization of the ECM in the aged
lamina propria. Histologic examination of aged human
vocal folds has indicated a decrease in the total number of
cells, reduction in the intracellular organelles responsible
for protein synthesis, and reduced production of ECM
from these cells.15,16 Ding and Gray examined ECM gene
expression in vocal fold tissues of aged rats and found
decreased expression levels of collagen type I and V, as
well as reduced proteinase expression, which suggested a
slowdown of collagen turnover.17 These studies demon-
strated that, as cells in the vocal fold age, changes in
phenotype are associated with a loss of function.
Growth factors affect various cells and their func-
tion and have the potential to stimulate geriatric cells to
function like younger cells. Previous studies have
revealed that bFGF stimulates proliferation and migra-
tion of fibroblasts and keratinocytes, and thus it has
been used for re-epithelialization of intractable skin
ulcers.18 It has also been suggested that exogenous
bFGF improved wound healing of postirradiated skin in
an animal study utilizing pigs.19 Moreover, a prelimi-
nary study of clinical application in the otolaryngologic
field reported that topical administration of bFGF pro-
moted regeneration of the tympanic membrane in
patients with a large perforation of the ear drum.20
The molecular mechanism of bFGF activity was
examined by several in vitro studies. They reported that
bFGF reduced collagen type I mRNA expression in gin-
gival fibroblasts21 and that bFGF stimulated HA
synthesis in skin fibroblasts.22 Our group also has
reported that bFGF increased gene expression of HA
synthase and matrix metalloproteinase in aged rat vocal
fold tissues, demonstrating that it might be suitable for
the regeneration of vocal fold mucosa.23
The results of the present clinical trial were encour-
aging. All cases showed regenerative effects, indicated
by the significant improvements in aerodynamic and
acoustic function (Fig. 3 and 4). Stroboscopic findings
also demonstrated better mucosal vibration with reduc-
tion of glottic gap (Fig. 5 – Fig. 7).
The number of injections required varied by individ-
ual. A single injection was sufficient in two cases, and
seven injections were required in one case. Growth fac-
tor therapy is considered to be a ‘‘trigger’’ for jump-
starting biological processes24; this may explain why
only a few injections of bFGF solution achieved such
drastic effects in most cases.
During the follow-up period, no patients showed a
recurrence of atrophy of the vocal fold mucosa, including
the longest-tracked case, which was followed up for 24
months. However, given that cells in the aged vocal fold
are, by definition, biologically ‘‘aged,’’ their life span can-
not be extended by administration of growth factors. It
is reasonable to assume that the effects of growth factors
Laryngoscope 122: February 2012
would be temporary; therefore, future study will be
needed to examine how long these effects last. It will
also be important to explore more effective ways of
administering the growth factors that may enable these
effects to be strengthened, such as the exploration of
various drug delivery systems.
CONCLUSION
This clinical trial examined the regenerative effects
of bFGF on aged vocal folds in 10 patients. The results
showed good recovery of vibratory properties, as well as
aerodynamic and acoustic function, with no major
adverse effects. Although the number of subjects is still
small and future study is in need, the current trial sug-
gests that bFGF may have a strong regenerative effect
on aged-related atrophy of the vocal fold.
BIBLIOGRAPHY
1. Ramig LO, Gray S, Baker K, et al. The aging voice: A review, treatment
data and familial and genetic perspectives. Folia Phoniatr Logop 2001;
53:252–265.
2. Hirano M, Kurita S, Sakaguchi S. Ageing of the vibratory tissue of human
vocal folds. Acta Otolaryngol (Stockh) 1989;107:428–433.
3. Sato K, Hirano M. Age-related changes of elastic fibers in the superficial
layer of the lamina propria of vocal folds. Ann Otol Rhinol Laryngol
1997;106:44–48.
4. Sato K, Hirano M, Nakashima T. Age-related changes of collagenous fibers
in the human vocal fold mucosa. Ann Otol Rhinol Laryngol 2002;111:
15–20.
5. Butler JE, Hammond TH, Gray SD. Gender-related differences of hyal-
uronic acid distribution in the human vocal fold. Laryngoscope 2001;111:
907–911.
6. Woo P, Casper J, Colton R, Brewer D. Dysphonia in the aging: physiology
versus disease. Laryngoscope 1992;102:139–144.
7. Bloch I, Behrman A. Quantitative analysis of videostroboscopic images in
presbylarynges. Laryngoscope 2001;111:2022–2027.
8. Slavit DH. Phonosurgey in the elderly: a review. Ear Nose Throat J 1999;
78:505–509, 512.
9. Tucker HM. Laryngeal framework surgery in the management of the aged
larynx. Ann Otol Rhinol Laryngol 1988;97:534–536.
10. Hirano S, Bless DM, Munoz del Rio A, Connor NP, Ford CN. Therapeutic
potential of growth factors for aging voice. Laryngoscope 2004;114:
2161–2167.
11. Hirano S, Nagai H, Tateya I, Tateya T, Ford CN, Bless DM. Regenera-
tion of aged vocal folds with basic fibroblast growth factor in a rat
model: a preliminary report. Ann Otol Rhinol Laryngol 2005;114:
304–308.
12. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteoglycans and
their influence on biomechanics. Laryngoscope 1999;109:845–854.
13. Hirano S, Kishimoto Y, Suehiro A, Kanemaru K, Ito J. Regeneration of
aged vocal fold: First human case treated with fibroblast growth factor.
Laryngoscope 2008;118:2254–2259.
14. Vacanti JP, Langer R. Tissue engineering. Science 1993;260:920–926.
15. Hirano M, Sato K, Nakashima T. Fibroblasts in geriatric vocal fold mu-
cosa. Acta Otolaryngol 2000;120:336–340.
16. Sato K, Hirano M. Age-related changes of the macula flava of the human
vocal fold. Ann Otol Rhinol Laryngol 1995;104:839–844.
17. Ding H, Gray SD. Senescent expression of genes coding collagens, colla-
gen-degrading metalloproteinases, and tissue inhibitors of metalloprotei-
nases in rat vocal folds: Comparison with skin and lungs. J Gerontol A
Biol Sci Med Sci 2001;56:B145–152.
18. Rumalla VK, Borah GL. Cytokines, growth factors, and plastic surgery.
Plast Reconstr Surg 2001;108:719–733.
19. Hom DB, Unger GM, Pernell KJ, Manivel JC. Improving surgical wound
healing with basic fibroblast growth factor after radiation. Laryngoscope
2005;115:412–422.
20. Hakuba N, Taniguchi M, Shimizu Y, Sugimoto A, Shinomori Y, Gyo
K. A new method for closing tympanic membrane perforations
using basic fibroblast growth factor. Laryngoscope 2003;113:
1352–1355.
21. Hong HH, Trackman PC. Cytokine regulation of gingival fibroblast lysyl
oxidase, collagen, and elastin. J Periodontol 2002;73:145–152.
22. Heldin P, Laurent TC, Heldin CH. Effect of growth factors on hyaluronan
synthesis in cultured human fibroblasts. Biochem J 1989;258:919–922.
23. Ohno T, Jin Yoo M, Swanson ER, Hirano S, Ossoff RH, Rousseau B. Regen-
erative effects of basic growth factor on extracellular matrix production
in aged rat vocal folds. Ann Otol Rhinol Laryngol 2009;118;559–564.
24. Hom DB, Thatcher G, Tibesar R. Growth factor therapy to improve soft
tissue healing. Facial Plast Surg 2002;18:41–52.
Hirano et al.: Regeneration of Aged Vocal Fold
331