Tokai J Exp Clin Med., Vol. 33, No. 3, pp. 119-123, 2008

Intracordal Injection Technique: Materials and Injection Site
Etsuyo TAMURA1), Hiroyuki FUKUDA2), Yasuhiko TABATA3)
1)

Department of Otorhinolaryngology, Tokai University School of Medicine
Tokai University Tokyo Hospital
2)
Tokyo Voice Center, International University of Health and Welfare
3)
Institute for Frontier Medical Sciences, Kyoto University(Received June 20, 2008; Accepted July 23, 2008)

Objective. Various materials have been used for intracordal injection to treat deficiencies in glottal closure.
However, specific injection sites and materials are desirable for various conditions. Here, we reviewed injection sites and materials.
Methods. By considering the characteristics of injection materials used in Japan to date as well as the normal structure of the vocal cord, we have determined injection materials and sites suitable for different typeof disease.
Results. Collagen obtained commercially for injection consists mainly of type I collagen, as does autologous fascia. When this material is injected into the superficial layer of the lamina propria mucosae of thevocal cord, the wave motion of the mucosa during phonation is disturbed. This material therefore should
be injected deeper into the lamina propria. Indications for collagen injection include unilateral recurrent
laryngeal nerve paralysis. Even when injected into the lamina propria, fat tissue does not disturb mucosal
wave motion, and is useful for treating vocal cord atrophy or sulcus vocalis.
Conclusion. When intracordal injection is used in vocal rehabilitation, material and site should be selected,
based on the cause of glottal dysfunction.
Key words: mucosal wave motion, deficiency of glottal closure, lamina propria mucosae, recurrent laryngeal nerve paralysis, vocal cord atrophy, sulcus vocalis

INTRODUCTION
Intracordal injection therapy has been used in vocal
rehabilitation to treat deficiency of glottal closure fromcauses such as unilateral recurrent laryngeal nerve
paralysis, vocal cord atrophy and sulcus vocalis. This
procedure is intended to correct position and volumeof the vocal cord and to attain glottal closure.
Since intracordal injection first was first describedby Brünings (1) in 1911, injection techniques and injection materials have changed.
In Japan, silicone (2) and collagen (3) have beenused for intracordal injection.
Silicone, which has been in use since the 1960s, isa polymer regarded as both suitable for injection andinactive within the human body. When this material
is injected into the vocal cord, the tissue reaction isslight and injection effects are sustained and stable.
Generally, connective tissue capsules are reported
to form around silicone, and then persist stably (4).
However, the injection site should be limited to within
the muscle layer. When silicone is injected into thelamina propria mucosae, mucosal vibration is disturbed and voice quality is worsened. Moreover, since areport by the US Food and Drug Administration (FDA)
in 1992 concerning the induction of human adjuvant
diseases, room-temperature vulcanized silicone (RTVS)
or silicone oil, which were used in the past, are now
difficult to obtain.

Collagen for injection, extracted from bovine dermis, consists mainly of type I collagen. When collagen
initially came to be recognized as an intracordal injection material, its characteristics were reported to differ
from those of injection materials used in the past (5),possibly undergoing replacement by host tissue afterinjection. Furthermore, collagen has been reported to
disturb vocal cord vibration less frequently than silicone, even when injected into the lamina propria (6).
In recent years, autologous tissues such as fat (7) or
fascia (8) have also been used.
Technique using autologous fat for intracordal
injection was first reported by Mikaelian (9) in 1991,
and has been used clinically in Japan (8).
In augmentation using autologous fascia, clinicalapplication preceded systematic study, and results havebeen reported (10). Histologic evaluation after injection was then carried out in experimental models, but
no consensus has been reached regarding the changeof volume from absorption after injection (11).
According to Arnold (12), the requirements forinjection materials include minimal tissue response,
absence of oncogenicity, ease of injection, nonabsorbability, and absence of migration. These conditions
alone may not be sufficient if our aim is, to both correct vocal cord position as in patients with unilateral
recurrent laryngeal nerve paralysis, and correct mucosal volume as in patients with vocal cord atrophy or
sulcus vocalis.

Etsuyo TAMURA, Department of Otorhinolaryngology, Tokai University School of Medicine, Tokai University Tokyo Hospital, 1-2-5 Yoyogi, Shibuya-ku,
Tokyo 151-0053, Japan Tel: 81-3-3370-2321, Fax: 81-3-5354-5366, E-mail: otamura@tok.u-tokai.ac.jp― 119 ―

E. Tamura et al. /Intracordal Injection TechniqueFig. 1. Histologic findings in a dog after
injection of 6.5% noncrosslinking-type
collagen.
HE: HE -stained preparation (x66).
Capsules consisting of collagen fibers
formed around the gel, and infiltrationof fibroblasts was present.
FGF: Immunostaining for FGF (x132).
Cytoplasm of fibroblasts in the gel
reacted with anti-FGF antibody.
TGF-β: Immunostaining for TGF-β(x132). Cytoplasm of fibroblasts in thegel reacted with anti-TGF-βantibody.
PD GF: Immunostaining of PD GF(x132). Cytoplasm of fibroblasts in thegel reacted with anti-PDGF antibody.
From reference 14.

Fig. 2. Histologic findings in a dog after
injection of 6.5% crosslinking-type collagen.
HE: HE-stained preparation (x 66). No
capsule formation is evident surrounding the gel.
FGF: Immunostaining for FGF (x132). Cytoplasm of fibroblasts in thegel showed no reaction with anti-FGF
antibody.
TGF-β: Immunostaining for TGF-β(x132).
Cytoplasm of fibroblasts in the gelshowed a mild reaction with anti-TGFβantibody.
PDGF: Immunostaining of PDGF (x132).
Cytoplasm of fibroblasts in the gelshowed no reaction with antiPDGF antibody. From reference 14.

We have studied the influence of materials on
choice of injection site and on mucosal vibration during phonation, based on the immunological study ofmucosae. Specifically, the desirable properties of injection materials should be defined while considering theanatomy of the vocal cord, which must vibrate at highspeed.
We determined the injection materials and sites suitable for each underlying condition, such as unilateral
recurrent laryngeal nerve paralysis, vocal cord atrophyand sulcus vocalis.
STRUCTURE OF LAMINA PROPRIA MUCOSAE
Immunohistochemical study of the lamina propria
mucosae of the human vocal cord has shown that type
III collagen is distributed widely in the superficial layerof the lamina propria, while type I collagen is present
less superficially, deep in the laryngeal glands, andalso near the anterior and posterior macular flava (13).
This appears to correspond to the conditions of vocal
cord vibration as evaluated by stroboscopic coronal
imaging during phonation. Type III collagen, withhigh elasticity, is present in the superficial layer of thelamina propria mucosae where vocal cord vibration
during phonation is greater, while type I collagen isprevalent at the fulcrum of vocal cord vibration and atsites with less vibration, representing function-relateddistribution.
COLLAGEN FOR INJECTION
The duration of beneficial effects after injection
was studied indirectly in canine models by examining
growth factors associated with wound healing (14). In
dogs with recurrent laryngeal nerve paralysis, 6.5%
noncrosslinking-type collagen or 6.5% crosslinkingtype collagen (Koken) was injected into the paralyzed
vocal cord, after which growth factors assessed
immunohistochemically. These included fibroblast
growth factor (FGF), platelet-derived growth factor(PDGF), and transforming growth factor (TGF)-β.
Figure 1 shows the gel in tissue at the 9th week after
injection of noncrosslinking-type collagen. Similarly,
a histologic preparation at 9 weeks after injection ofcrosslinking-type collagen is shown in Figure 2. In thenoncrosslinking type collagen, immunoreactive growth
factor was present in fibroblasts within the gel, while
no such activity was seen with crosslinking-type collagen. As opposed to the noncrosslinking type, woundhealing had ended by the 9th week after injection ofcrosslinking-type collagen, which appeared to remain― 120 ―

E. Tamura et al. /Intracordal Injection TechniqueFig. 3. Pre- and post-operative stroboscopic images during phonation (one cycle).
Left: Pre-operative image.
Amplitude of vocal cord vibration issmall, and glottal closure is not seen.
Right: Post-operative image .
Glottal closure is observed at short-term
follow-up. From reference 7.

Fig. 4. Coronal section of vocal cord from a dog at
8 weeks after fat grafting.
Arrow, graft (HE staining x 2).
From the reference 7.

Fig. 5. Canine fascia lata.
A: Fresh fascia (HEstaining x50).
B: Freeze-dried fascia (HE staining x 50).
C: Fascia at 3 weeks after subcutaneous injection (HEstaining x 50). From reference 16.

within the host tissues. Collagen without crosslinking
was susceptible to host reaction after injection, and apparently difficult to retain in host tissues.
AUTOLOGOUS TISSUE
We have used autologous materials both in patientswith unilateral recurrent laryngeal nerve paralysis andin patients with sulcus vocalis, and the results weresatisfactory without disturbance of mucosal vibration
after the procedure.
Figure 3 shows the results of autologous fat injection in a 67-year-old woman with sulcus vocalis.
Stroboscopic images during phonation are shown
before and at 8 weeks after the procedure are shown.
Glottal closure was confirmed, and improvement wasnoted not only in voice quality but also in an aerodynamic examination.
However, the fat injection technique remains controversial because the degree of postoperative absorption
differs according to the condition of fat injected (11).
We therefore performed an experiment in dogs. The
upper surface of the vocal cord was incised under alaryngomicroscope, and adipose tissues were grafted asa mass immediately above the muscle layer. In a coronal section of the vocal cord obtained at 8 weeks aftergrafting, the fat remained at the graft site, maintaining the same volume as at the time of grafting (Fig. 4)(15). This suggests that absorption after augmentation
may be decreased by grafting as opposed to injection.
We then developed a method of preservation thatuses freeze-drying, and absorption was evaluated after
injection (15). Dogs were used in the experiment.
After harvesting fascia lata specimens were processedby freeze-drying for 24 h at -65℃, and the minced
in a food processor. Figure 5 shows specimens offresh fascia, fascia after freeze-drying, and fascia at 3weeks after subcutaneous injection in a mixture with
1% hyaluronic acid. The fascia before freeze-drying
consisted mainly of collagen fibers, with few cellularcomponents; fibroblasts were sparsely distributed. In
the specimen of fascia after freeze-drying, no cellularcomponents were recognized, but the collagen fiber
representing the main component showed no apparent
degeneration. After the fascia was injected subcutaneously, ingrowth of fibroblasts and new blood vessels, aswell as infiltration by lymphocytes, was seen. However,― 121 ―E. Tamura et al. /Intracordal Injection TechniqueFig. 6. Coronal section of canine
vocal cord after injection
into the muscle layer (HEstaining x 2).
A: Three weeks after injection of freeze-dried fascia.
B: Four weeks after injectionof fresh fascia.
From reference 16.

Fig. 7. Coronal section of canine vocal cord after injection into the lamina propria mucosae (HE staining; x 2) From reference 16.
A: After injection of freeze-dried fascia (A-5, A-7, A-9 : after 5, 7, and 9 weeks respectively).
B: After injection of fresh fascia (B-3, B-12, B-24: after 3, 12, and 24 weeks respectively)

neutrophils and necrosis were not seen. In an intracordal injection experiment in a dog with recurrent
nerve paralysis, fascia remained present within themuscle layer as a mass, similar to findings after injection of fresh fascia (15).
Furthermore, 0.5 ml of freeze-dried fascia and0.8 ml of fresh fascia were injected into the muscle
layer and the lamina propria mucosae respectively.
Assuming that the remaining fascial tissues took theform of a sphere, measurements were taken using ameasuring ocular with a light microscope, and thevolume was calculated. Figure 6 shows coronal sectionsof vocal cords at 3 and 4 weeks after injection into
the muscle layer. Respective remaining volumes were40% and 31% of injected volume. On the other hand,when freeze-dried fascia was injected into the lamina
propria mucosae, the volume of the fascia decreased
markedly over time (Fig. 7). Simple comparison is uninformative because the period of observation differed
between lamina propria mucosae and the muscle layer,
but the amount remaining in tissues tended to behigher when the injection was directed into the muscle
layer, for both freeze-dried and fresh fascia (16).
CONCLUSION
The vocal cord has a layered structure. Waves ofmucosal vibration reportedly originate mainly in the
superficial layer of the lamina propria mucosae (17).
Based on immunohistochemical results for collagen fibers in the lamina propria mucosae of the vocal cord,― 122 ―

E. Tamura et al. /Intracordal Injection Techniquewe reported that the main component of collagenfibers in the superficial layer is type III collagen, which
has high elasticity, while type I collagen is present at
the fulcrum of vocal cord vibration and at deep sites
showing less vibration (13). This suggests that, consideration of injection materials for intracordal injection
should focus not only on histologic reaction after injection but also on vocal cord vibration.
We evaluated the histologic changes in injection
materials after injection and examined injection sites
based on the relationship between vocal cord constituents and vocal cord vibration.
Silicone, collagen, or fascia should be injected intoa deep layer rather than into the intermediate layerof the lamina propria mucosae. These materials are
suitable for patients with unilateral recurrent laryngeal
nerve paralysis. Autologous fat does not disturb vocal
cord vibration, even when injected into the superficial
layer of the lamina propria.
We propose that autologous fat is superior to othermaterials for vocal rehabilitation in patients with unilateral recurrent laryngeal nerve paralysis and in patients with sulcus vocalis, as it gives satisfactory results
without disturbing mucosal vibration.
REFERENCES
1) Brünings W. Uber eine neue Behandelungs Methodeder
Rekurrens-lamung. Verhandl Ver Deutch Laryngol 1911; 18:93-98.
2) Fukuda H. Vocal rehabilitation with injectable silicone. J
Otolaryngl Jpn 1970; 73: 1506-1526.
3) Okamoto K. A fundamental study of injectable collagen in vocal
rehabilitation. J Otolaryngl Jpn1987; 90: 394-403.
4) Tsuzuki T, Fukuda H, Fujioka T, Takayama E, Kawaida M,Ohki K, Kawasaki J., Follow-up study of patients treated withsilicone injection into the vocal fold. Practica Oto-Rhinolarygologica(Jpn), 1990; 83: 601-605.
5) Ford CN, Martin DW, Warner TF. Injectable collagen in laryngeal rehabilitation. Laryngoscope 1984; 94: 513-518
6) Ford CN, Bless D., Preliminary study of injectable collagen inhuman vocal fold augmentation. Otolaryngol Head Neck Surg
1986; 94: 104-112
7) Tamura E, Kitahara S, Kohno N. et al. Autogenous fat injectionfor sulcus vocalis. Jpn J Logop Phoniatr 2000; 41: 389-3948) Tsunoda K, Takanosawa M, Niimi S. Autologous transplantationof fascia into the vocal fold-A new phonosurgical technique for
glottal imcometence-. Laryngoscope 1999; 109: 504-508
9) Mikaelian DO, Lowry LD and Sataloff RT. Lipoinjection forunilateral vocal cord paralysis. Laryngoscope1991; 101: 465-468.
10) Rihkanen, H.: Vocal fold augmentation by injection of autologous fascia. Laryngoscope 1998; 108: 51-54.
11) Duke SG, Salmon J, Blalock PD Postman GN, et al. Fascia augmentation of vocal
fold: Graft yield in the canine and preliminary clinical experience. Laryngoscope 2001; 111: 759-764
12) Arnold GE. Vocal rehabilitation of paralytic dysphonia: IX,Technique of intracordal injection. Arch Oto-laryngol, 1962; 76:358-36813) Tamura E, Kitahara S, Satoh M, Inouye T, Collagen types distribution in human vocal fold lamina propria: immunohistochemical investigation. Larynx Jpn. 1993; 5: 17-2214) Tamura E, Kitahara S, Nakanoboh M, Satoh M, Furukawa
T, Nohara O, Inouye T, Basic study of implants for vocal foldaugmentation: Immunohistochemical investigation. Larynx Jpn.
1994; 6: 122-129.
15) Tamura E, Kitahara S., Comparison of histological findings
from vocal fold augmentation Materials. Jpn J Logo Phoniat
2002; 43: 450-457.
16) Tamura E, Kitahara S., Autogenous tissue augmentation forunilateral vocal fold paralysis. Jpn J Logo Phoniat 2003; 44:327-332.
17) Hirano M. Morphological structure of the vocal cord as a vibrator and its variations. Folia phoniat 1974; 26: 89- 94― 123 ―