Kiminori Sato (Auth.) - Functional Histoanatomy of The Human Larynx-Springer Singapore (2018) PDF

Kiminori Sato
Functional
Histoanatomy of
the Human Larynx
123
Functional Histoanatomy of the Human Larynx
Kiminori Sato
Functional Histoanatomy of
the Human Larynx
Kiminori Sato
Department of Otolaryngology-Head and
Neck Surgery
Kurume University School of Medicine
Kurume-shi
Fukuoka
Japan
ISBN 978-981-10-5585-0 ISBN 978-981-10-5586-7 (eBook)

https://doi.org/10.1007/978-981-10-5586-7
Library of Congress Control Number: 2017964266
© Springer Nature Singapore Pte Ltd. 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed
to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore
Dr. Hirano and Dr. Sato (left to right)
This book is dedicated to my teacher, mentor and
outstanding role model, Emeritus Professor Minoru
Hirano, the consummate academic surgeon. He has had
the greatest impact not only on my medical knowledge
but also on my approach to learning, teaching and
academic life as a physician. He continues to serve as a
source of inspiration to me.
Sincerely and with the deepest gratitude I dedicated this
book to him.
Kiminori Sato, M.D., Ph.D.
Kurume-shi, Fukuoka
May 2017
Preface
The basic functions of the human larynx are to act as a protective sphincter, to act as a pas-
sageway for air, and to produce sound. The human larynx has a complex structural organiza-
tion with a framework characterized by an external cartilaginous skeleton and internal
connective tissues in a variety of arrangements in the different regions of the larynx. This
framework composed of cartilage, ligaments and muscles contributes to the physiologic func-
tions of the human larynx.
Histology and histoanatomy reflect the organ’s functions very well. Functional histoanat-
omy (physiological histoanatomy, morphophysiology) is a histoanatomy studied in its relation
to functions.
Among mammals, only humans can speak and only the human adult vocal fold has a vocal
ligament, Reinke’s space, and a layered structure. Why do only human adults have such a
characteristic vocal fold structure? Why and how does the newborn vocal fold mucosa grow,
develop and mature? What are the factors for initiating and continuing the growth and develop-
ment of the human vocal fold mucosa? Why does the voice change with age?
Vocal folds are comparable to the strings of a musical instrument. The strings must be
changed from time to time, because they become old and do not vibrate well. However, human
vocal folds maintain their viscoelasticity and produce good vibration for many decades. The
renewal of extracellular matrices in the vocal folds is believed to occur continuously to main-
tain viscoelasticity.
Recent advances in molecular biology shed light on the metabolism of extracellular matri-
ces that are essential for the viscoelastic properties of the human vocal fold mucosa. The
manipulation, not only of cells but also their microenvironment, is one of the strategies in
regenerative medicine. Artificial manipulation of these cells could lead to advanced develop-
ment in vocal fold regeneration. Understanding the mechanisms responsible for microenviron-
mental regulation of the cells in the maculae flavae of the human vocal fold will provide the
tools needed to manipulate cells through their microenvironment for the development of thera-
peutic approaches to diseases and tissue injuries of the human vocal fold. Translational medi-
cine focused on how to regulate cells and extracellular matrices (microenvironments) contained
in the maculae flavae of the human vocal folds will contribute to our ability to restore and
regenerate human vocal fold tissue.
Phonosurgery is a surgery performed on the human larynx to treat phonatory functions and
to improve quality of voice. It is very important to be able to visualize the internal laryngeal
structures by looking at the laryngeal cartilage when performing phonosurgery via an extrala-
ryngeal approach. It is also important to be able to visualize the internal laryngeal structures
including histoanatomy by observing the mucosa of the lumen when performing phonosurgery
via an intralaryngeal approach. Knowledge of the three-dimensional structure, histology and
histoanatomy of the larynx is indispensable to performing phonosurgery.
This book provides essential “functional histoanatomy of the human larynx” information of
which the laryngeal surgeon must have mastery. I feel strongly that a true surgeon is not only
a physician but also a scientist who always approaches each patient with a deep understanding
vii
viii Preface
of the basic medicine, such as essential histoanatomy and pathophysiology of voice disorders.
Understanding of the histologic structures related to laryngeal functions and the h istopathology
of the vocal fold and larynx are vital for understanding the concepts behind phonosurgery.
Kurume-shi, Japan Kiminori Sato, M.D., Ph.D.

Acknowledgements
I would like to thank Emeritus Professor Tadashi Nakashima and my colleagues in the
Department of Otolaryngology-Head and Neck Surgery, Kurume University School of
Medicine.
I would also like to express my deepest appreciation to Ms. Ikuko Tsuda, a technician in our
laryngeal research laboratory. Her efforts and dedications over the three decades ultimately
have resulted in the highest quality histological and histopathological specimens of the human
larynx to date.
I also wish to express thanks to Mr. Edward Martin Kellerman III for his English advise in
the making of this book.
The careful review of the articles by the tireless staff at Springer publishing company is
much appreciated. Without their support, this book would not have been published. Thank you
very much again.
Finally, to my wife, Kaori, to my sons, Kiminobu and Fumihiko and to my daughter, Riko,
thank you for your forbearance over the years of work-related absences.
Kiminori Sato, M.D., Ph.D.
ix
Contents
Part I Whole-Organ Serial Sections of the Human Larynx
1 Whole-Organ Midsagittal Section of the Human Adult Larynx �� 3

2 Whole-Organ Serial Transverse Sections of the Human Adult Larynx�� 5
3 Whole-Organ Serial Coronal Sections of the Human Adult Larynx�� 23
4 Whole-Organ Midsagittal Section of the Human Newborn Larynx �� 35
5 Whole-Organ Serial Transverse Sections of the Human Newborn Larynx �� 37
6 Whole-Organ Serial Coronal Sections of the Human Newborn Larynx�� 49
Part II Functional Histoanatomy of the Human Larynx
7 Clinical Anatomy of the Human Larynx �� 61

7.1 Introduction�� 61
7.2 Clinical Anatomy of the Human Larynx for an Extralaryngeal Approach �� 61
7.3 Clinical Anatomy of the Human Larynx for an Intralaryngeal Approach�� 63
7.3.1 Endoscopic Surgery�� 63
7.3.2 Endolaryngeal Microsurgery�� 63
References�� 67
8 Compartments of the Human Larynx�� 69
8.2 Anterior Commissure Tendon�� 69
8.3 Physiological and Clinical Significance of the Anterior Commissure Tendon�� 72
8.3.1 Laryngeal Functions�� 72
8.3.2 Anterior Commissure Carcinoma �� 72
8.3.3 Midline Lateralization Thyroplasty (Type II Thyroplasty)�� 73
8.4 Cricothyroid Ligament�� 73
8.5 Physiological and Clinical Significance of Cricothyroid Ligament �� 74
8.6 Vocal Ligament and Conus Elasticus (Crico-vocal Membrane) �� 75
8.7 Physiological and Clinical Significance of the Vocal Ligament
and Conus Elasticus�� 75
8.8 Reinke’s Space�� 75
8.9 Physiological and Clinical Significance of Reinke’s Space�� 78
8.10 Thyroglottic Ligament�� 78
8.11 Physiological and Clinical Significance of the Thyroglottic Ligament�� 80
8.12 Quadrangular Membrane and Ventricular Ligament�� 80
8.13 Physiological and Clinical Significance of the Quadrangular Membrane
and Ventricular Ligament�� 82
xi
xii Contents
8.14 Thyrohyoid Membrane �� 82

8.15 Physiological and Clinical Significance of the Thyrohyoid Membrane �� 83
8.16 Spaces of the Larynx�� 83
8.17 Physiological and Clinical Significance of the Spaces of the Larynx�� 83
8.18 The Laryngeal Cartilages�� 83
8.19 Physiological and Clinical Significance of the Laryngeal Cartilages�� 84
8.20 Compartments and Laryngeal Inflammatory Diseases �� 85
8.20.1 Acute Epiglottitis�� 85
8.20.2 Subglottic Laryngitis (Croup) �� 87
References�� 88
9 Histoanatomy of the Human Glottis�� 89
9.2 The Structures Around the Human Adult Glottis�� 92
9.3 The Dimensions and Morphological Characteristics
of the Human Adult Glottis�� 92
9.4 The Dimensions and Morphological Characteristics
of the Human Newborn Glottis�� 93
9.5 The Functions of the Human Newborn Glottis�� 94
9.6 The Clinical Significance of the Dimensions of the Human Glottis�� 96
9.7 Prolonged Endotracheal Intubation in Infants and Adults�� 97
9.7.1 Histopathologic Changes in the Laryngeal Mucosa of
Low-Birth-Weight Infants After Endotracheal Intubation�� 97
9.7.2 Histopathologic Changes of Infant Larynges
After Intubation�� 98
9.7.3 Correlation Between Duration of Intubation,
Degree of Laryngeal Injury, and Weight at Birth�� 102
9.8 Histoanatomy of the Arytenoid Cartilage �� 103
9.8.1 Distribution of Elastic and Hyaline Cartilages
in the Arytenoid Cartilage of Adult Larynges�� 103
9.8.2 Distribution of Elastic and Hyaline Cartilages
in the Arytenoid Cartilage of Newborn Larynges�� 105
9.8.3 Behavior of the Elastic Cartilage Portion
of the Arytenoid Cartilage During Abduction and Adduction�� 105
9.9 Distribution of Elastic Cartilage in the Arytenoid Cartilage
and Its Physiologic Significance �� 108
9.10 Ultrastructure of the Vocal Process of the Arytenoid Cartilage�� 110
9.10.1 Chondrocytes of the Vocal Process �� 110
9.10.2 Collagen Fibers of the Vocal Process�� 111
9.10.3 Elastic Fibers of the Vocal Process �� 111
9.11 Microstructure of the Vocal Process of the Arytenoid Cartilage
and Its Physiologic Significance �� 113
9.11.1 Tip of the Vocal Process�� 113
9.11.2 Posterior Portion of the Vocal Process�� 115
9.11.3 Biomechanical Properties of the Human Vocal Process �� 116
9.12 Histoanatomy of the Posterior Glottis�� 116
9.12.1 Neutral Condition�� 116
9.12.2 Vocal Fold Abduction�� 116
9.12.3 Vocal Fold Adduction�� 117
9.13 The Posterior Glottis and Its Physiologic Significance�� 118
9.14 Cricoarytenoid Joint (Articulation)�� 118
9.15 Geriatric Changes of the Cricoarytenoid Joint (Articulation)�� 119
9.16 Age-Related Changes of the Cricoarytenoid Joint (Articulation)
and Their Biomechanical Properties �� 119
References�� 123
Contents xiii
10 Cells and Extracellular Matrices in the

Human Adult Vocal Fold Mucosa�� 125
10.2 Layered Structure of the Human Vocal Fold�� 126
10.3 Epithelium of the Human Vocal Fold�� 127
10.4 Microstructure of the Epithelium of the Human Vocal
Fold and Its Physiologic Significance�� 127
10.5 Basal Lamina (Basement Membrane) of the Human Vocal Fold�� 128
10.6 Microstructure of the Basal Lamina (Basement Membrane)
of the Human Vocal Fold and Its Physiologic Significance�� 128
10.7 Langerhans Cells of the Human Vocal Fold�� 130
10.8 Langerhans Cells of the Human Larynx and
Their Physiologic Significance �� 130
10.9 Extracellular Matrices in the Human Vocal Fold Mucosa�� 132
10.9.1 Extracellular Matrices in the Superficial Layer
of the Lamina Propria (Reinke’s Space) of the
Human Vocal Fold Mucosa�� 135
10.9.2 Extracellular Matrices in the Intermediate and Deep Layers
of the Lamina Propria of the Human Vocal Fold Mucosa�� 144
10.10 Interstitial Cells in Reinke’s Space of the Human Vocal Fold Mucosa�� 144
10.10.1 Fibroblasts�� 144
10.10.2 Myofibroblasts �� 144
11 Macula Flava and Vocal Fold Stellate Cells of the
Human Adult Vocal Fold �� 147
11.2 Maculae Flavae in the Human Adult Vocal Fold�� 148
11.3 Morphological Characteristics of the Human Adult
Vocal Fold Stellate Cells �� 154
11.4 Synthesis of Extracellular Matrices by the
11.4.1 Collagen Fibers�� 156
11.4.2 Elastic Fibers�� 156
11.4.3 Glycosaminoglycan�� 156
11.5 Vitamin A-Storing Stellate Cells in the Human Maculae Flavae�� 158
11.6 Vocal Fold Stellate Cells as a Diffuse Stellate Cell System�� 159
11.7 Irradiated Macula Flava in the Human Vocal Fold Mucosa�� 160
11.7.1 Morphological Changes of Irradiated Macula Flava
in the Human Vocal Fold Mucosa�� 160
11.7.2 Functional Morphology of the Irradiated
Vocal Fold Stellate Cells�� 162
12 Tissue Stem Cells and the Stem Cell
Niche of the Human Vocal Fold Mucosa�� 165
12.2 Intermediate Filaments of the Cells in the Human Adult Maculae Flavae �� 166
12.3 Radiosensitivity of the Cells in the Human Adult Maculae Flavae�� 168
12.4 Telomerase of the Cells in the Human Adult Maculae Flavae�� 168
12.5 Cell Cycle of the Cells in the Human Adult Maculae Flavae �� 168
12.6 Transition Area Between the Human Adult Maculae
Flavae and Surrounding Tissue �� 169
xiv Contents
12.7 Cell Division of Cells in the Human Maculae Flavae�� 169

12.8 Hierarchy of Tissue Stem Cells in the Human Maculae Flavae�� 171
12.9 Microenvironment of Maculae Flavae as a Stem Cell Niche
in the Human Vocal Fold�� 171
12.9.1 Hyaluronan-Rich Matrix�� 171
12.9.2 A Proper Microenvironment in the Maculae Flavae
as a Stem Cell Niche�� 173
12.10 Origin of Cells in the Human Maculae Flavae�� 174
12.11 Side Population Cells in the Vocal Fold Mucosa�� 176
12.12 Vocal Fold Stem Cells and Their Niche in the
Human Newborn Vocal Fold Mucosa �� 176
13 Cells and Extracellular Matrices in the Human
Newborn Vocal Fold Mucosa�� 179
13.2 Epithelium of the Newborn Vocal Fold�� 179
13.3 Basal Lamina (Basement Membrane) of the
13.4 Lamina Propria of the Newborn Vocal Fold Mucosa�� 180
13.4.1 Fibroblasts�� 181
13.4.3 Reticular Fibers�� 182
13.4.5 Ground Substances�� 183
13.4.6 Blood Vessels �� 183
13.5 Epithelium and Basal Lamina of the Newborn
Vocal Fold Mucosa as a Vibrating Tissue �� 183
13.6 Lamina Propria of the Newborn
Vocal Fold Mucosa as a Vibrating Tissue �� 184
14 Macula Flava of the Human Newborn Vocal Fold�� 185
14.2 Macula Flava in the Human Newborn Vocal Fold Mucosa�� 186
14.3 Cells in the Macula Flava of the Human Newborn Vocal Fold Mucosa �� 186
14.4 Morphological Comparison of Newborn
and Adult Cells Including Vocal Fold Stellate
Cells in the Maculae Flavae�� 191
14.5 Morphological Comparison Between Cells Including
Vocal Fold Stellate Cells in the Maculae Flavae
and Fibroblasts in the Lamina Propria of the
14.6 Cell Origin in the Macula Flava of the Human Newborn
Vocal Fold Mucosa�� 191
14.6.1 Intermediate Filaments of the Cells in the
Newborn Macula Flava�� 192
14.6.2 Telomerase of the Cells in the Newborn Macula Flava �� 193
14.6.3 The Relationship between Bone Marrow-Derived
Cells and Cells in the Human Newborn Macula Flava�� 193
14.6.4 Pluripotency of the Cells in the Newborn Maculae Flavae�� 194
Contents xv
14.7 Cells Including Vocal Fold Stellate Cells in the

Newborn Maculae Flavae�� 195
14.8 Extracellular Matrices in the Macula Flava of the
14.8.1 Collagen and Reticular Fibers�� 195
14.8.3 Glycosaminoglycan�� 196
14.9 Microenvironment in the Macula Flava of the
15 Growth and Development of the Human Vocal Fold Mucosa�� 199
15.2 Cells and Extracellular Matrices in the Human Infant
15.3 Maculae Flavae of the Human Infant Vocal Fold�� 203
15.4 Growth Initiation of the Human Vocal Fold Mucosa in Infancy�� 205
15.5 Cells and Extracellular Matrices in the Human Child
15.6 Maculae Flavae of the Human Child Vocal Fold�� 207
15.7 Morphological Characteristics of the Human Child
15.8 Growth and Development of the Human Vocal Fold Mucosa and Vocal Fold
Vibration �� 210
15.9 Growth and Development of the Human Vocal Fold Mucosa�� 211
16 Mechanical Regulation (Cellular Mechanotransduction)
of the Human Vocal Fold Mucosa�� 213
16.2 Human Adult Vocal Fold Unphonated Since Birth �� 214
16.2.1 Macroscopic Findings of the Human Adult Vocal Fold
Unphonated Since Birth�� 214
16.2.2 Light and Electron Microscopic Findings of the Lamina Propria
of the Human Adult Vocal Fold Mucosa Unphonated Since Birth�� 214
16.2.3 Light and Electron Microscopic Findings of the Maculae Flavae
of the Human Adult Vocal Fold Mucosa Unphonated Since Birth�� 216
16.3 Human Child Vocal Fold Unphonated Since Birth �� 219
16.3.1 Macroscopic Findings of the Human Child
Vocal Fold Unphonated Since Birth�� 219
of the Human Child Vocal Fold Mucosa Unphonated Since Birth�� 219
16.3.3 Light and Electron Microscopic Findings of the Maculae Flavae
of the Human Child Vocal Fold Mucosa Unphonated Since Birth�� 220
16.4 Human Adult Vocal Fold Unphonated for Over a Decade�� 224
16.4.1 Macroscopic Findings of the Human Adult
Vocal Fold Unphonated for Over a Decade �� 224
of a Human Adult Vocal Fold Mucosa
Unphonated for Over a Decade�� 224
xvi Contents
16.4.3 Light and Electron Microscopic Findings

of the Macula Flava of a Human Adult
Vocal Fold Mucosa Unphonated for Over a Decade�� 225
16.5 Cytoskeletons (Mechanoreceptor of Cells) of the
Cells in the Human Macula Flava Unphonated since Birth�� 227
16.6 Comparison Between Vocal Fold Mucosae Phonated
and Unphonated (Non-Vibrated) Since Birth or
Unphonated for Over a Decade�� 228
16.7 Expression and Distribution of Hyaluronic Acid and CD44 in Unphonated
Human Vocal Fold Mucosa �� 230
16.8 Mechanotransduction in the Human Vocal Fold Mucosa �� 231
16.9 The Role of Intermediate Filaments in the Vocal Fold Stellate Cells �� 231
16.10 Mechanical Regulation of Human Vocal Fold Stellate Cells�� 232
17 Geriatric Changes of Cells and Extracellular Matrices
in the Human Vocal Fold Mucosa�� 235
17.2 Reticular and Collagen Fibers in the Superficial
Layer of the Lamina Propria (Reinke’s Space) of the
17.3 Age-Related Changes of Reticular and Collagen Fibers in the
Superficial Layer of the Lamina Propria (Reinke’s Space) of the
17.4 Age-Related Changes of Collagen Fibers in the Intermediate
and Deep Layers of the Lamina Propria (Vocal Ligament) of the
17.5 Age-Related Changes of Reticular and Collagen Fibers
in the Lamina Propria of the Human Vocal Fold Mucosa
and their Biomechanical Properties�� 238
17.5.1 Superficial Layer of the Lamina Propria (Reinke’s Space)
of the Aged Vocal Fold Mucosa �� 238
17.5.2 Intermediate and Deep Layer of the Lamina Propria
(Vocal Ligament) of the Aged Vocal Fold Mucosa�� 240
17.5.3 Destruction of the Layered Structure in the
Aged Adult Vocal Fold Mucosa �� 240
17.6 Age-Related Changes of Elastic Fibers in the Superficial Layer
of the Lamina Propia (Reinke’s Space) of the Human Vocal Fold Mucosa�� 241
17.7 Age-Related Changes of Elastic Fibers in the Intermediate
and Deep Layers of the Lamina Propia (Vocal Ligament) of the
17.8 Age-Related Changes of Elastic Fibers in the Lamina Propia of the
Human Vocal Fold Mucosa and their Biomechanical Properties�� 243
17.9 Age-Related Changes of Ground Substances in the Superficial
Layer of the Lamina Propia (Reinke’s Space) of the
17.10 Bowing of the Aged Vocal Folds�� 245
17.11 Age-Related Changes of Epithelium of the Human Vocal Fold Mucosa�� 245
17.12 Age-Related Changes of Epithelium of the
Human Vocal Fold Mucosa and their Biomechanical Properties�� 247
17.13 Age-Related Changes of Extracellular Matrices and Aging Voice �� 248
17.14 Age-Related Changes of Interstitial Cells in Reinke’s Space of the
Contents xvii
17.15 Age-Related Changes of Cells including Vocal Fold Stellate Cells in

Maculae Flavae of the Human Vocal Fold Mucosa�� 249
18 Geriatric Changes of the Macula Flava of the Human Vocal Fold�� 251
18.2 Maculae Flavae in the Human Aged Vocal Fold �� 252
18.3 Morphological Characteristics of the Human Aged
18.4 Synthesis of Extracellular Matrices by the Aged
18.4.3 Ground Substance�� 259
18.5 The Microenvironment of the Maculae Flavae in the Aged
Human Vocal Fold�� 259
18.6 Age-Related Changes of the Cells Including Vocal Fold Stellate Cells
in the Maculae Flavae and Aging of the Voice�� 259
19 Comparative Histoanatomy of the Vocal Fold Mucosa�� 263
19.2 Canine Vocal Fold: Lamina Propria of the Vocal Fold Mucosa�� 264
19.3 Canine Vocal Fold: Macula Flava of the Vocal Fold Mucosa �� 265
19.3.1 Interstitial Cells�� 266
19.4 Rat Vocal Fold: Lamina Propria of the Vocal Fold Mucosa�� 268
19.5 Rat Vocal Fold: Macula Flava of the Vocal Fold Mucosa �� 268
19.5.1 Interstitial Cells�� 268
19.6 Lamina Propria of the Animal Vocal Fold Mucosa�� 270
19.7 Maculae Flavae of the Animal Vocal Fold Mucosa�� 270
19.8 Unique Structure of the Human Vocal Fold Mucosa�� 271
20 Spaces of the Larynx�� 273
20.2 Distribution of the Preepiglottic Space (PES)�� 273
20.3 Three-Dimensional Reconstruction of the Preepiglottic Space (PES) �� 276
20.4 Physiological and Clinical Significance of the Preepiglottic Space (PES)�� 276
20.5 Distribution of the Paraglottic Space (PGS) �� 278
20.6 Three-Dimensional Reconstruction of the Paraglottic Space (PGS)�� 278
20.7 Physiological and Clinical Significance of the Paraglottic Space (PGS)�� 279
20.8 Distribution of the Cricoid Area (CA)�� 279
20.9 Three-Dimensional Reconstruction of the Cricoid Area (CA) �� 282
20.10 Physiological and Clinical Significance of the Cricoid Area (CA)�� 283
21 Blood Vessels of the Larynx and Vocal Fold�� 287
21.2 Blood Supply of the Larynx�� 287
xviii Contents
21.3 Physiologic Significance of the Blood Supply of the Larynx�� 289

21.4 Vascular Network of the Human Vocal Fold�� 289
21.5 Microstructure of the Blood Vessels in the Human Vocal Fold Mucosa�� 291
21.5.1 Arterioles�� 292
21.5.2 Venules�� 294
21.5.3 Capillaries�� 295
21.6 Physiologic Significance of the Vascular Network in the Human
21.7 Physiologic Significance of Pericytes of Capillaries in the Human
21.8 Microstructure of the Blood Vessels in the Human Vocal Fold Mucosa
with Reinke’s Edema�� 298
21.9 Transendothelial Exchange and Permiability of the Capillaries�� 301
21.9.1 Fenestra Transport�� 301
21.9.2 Vesicular Transport (Transcellular Transport via Vesicles)�� 301
21.9.3 Junctional Tranport (Intercellular Transport)�� 301
21.10 Hemorrhage in Reinke’s Space�� 301
21.11 Microvascular Lesions of the Vocal Fold�� 301
22 The Laryngeal Glands �� 305
22.2 Distribution of the Laryngeal Glands�� 306
22.3 Three-Dimensional Microstructure of Younger Adult Laryngeal Glands�� 307
22.4 Microstructure of Younger Adult Serous Cells in the Laryngeal Glands�� 307
22.5 Microstructure of Younger Adult Mucous Cells in the Laryngeal Glands�� 309
22.6 Distribution of the Laryngeal Glands in the Aged�� 309
22.7 Three-Dimensional Microstructure of Age-Related Changes
in the Laryngeal Glands�� 309
22.8 Microstructure of Age-Related Changes of Serous Cells
22.9 Microstructure of Age-Related Changes of Mucous Cells
22.10 Physiologic and Pathologic Significances of Age-Related Changes
22.10.1 Laryngeal Gland Function �� 311
22.10.2 Effects on Phonatory Function�� 311
22.10.3 Effects on Local Immune Function�� 311
22.10.4 Effects on Local Mucociliary Transport Function �� 311
22.11 Effect of Irradiation on Human Laryngeal Glands �� 311
22.12 Microstructure of the Serous Cells in Irradiated Laryngeal Glands �� 312
22.12.1 Serous Cells in Irradiated Laryngeal Glands with a Short
Duration After Radiotherapy�� 312
22.12.2 Serous Cells in Irradiated Laryngeal Glands with a Long
22.13 Microstructure of the Mucous Cells in Irradiated Laryngeal Glands �� 313
22.13.1 Mucous Cells in Irradiated Laryngeal Glands with a Short
22.13.2 Mucous Cells in Irradiated Laryngeal Glands with a Long
Contents xix
22.14 Physiologic and Pathologic Significances of Irradiated Laryngeal Glands�� 314

22.14.1 Effects on Phonatory Function�� 314
22.14.2 Effects on Local Immune Function�� 314
22.14.3 Effects on Local Mucociliary Transport Function �� 314
23 Atrophy of the Vocal Fold�� 317
23.2 Definition of Vocal Fold Atrophy�� 317
23.3 Recurrent Laryngeal Nerve Paralysis �� 318
23.4 Sulcus Vocalis �� 320
23.5 Irradiated Vocal Fold�� 321
23.6 Geratric Vocal Fold �� 323
23.7 Laryngeal Augmentation Surgery (Injection Laryngoplasty) �� 323
23.8 Injection Material for Laryngeal Augmentation Surgery (Injection
Laryngoplasty)�� 326
Glossary �� 329

About the Author
Kiminori Sato, M.D., Ph.D. is a Professor of the Department

of Otolaryngology-Head and Neck Surgery at the Kurume
University School of Medicine, the Director of Sato Clinic &
Hospital and a committed academic surgeon as well.
Dr. Sato graduated from the Kurume University School of
Medicine and obtained his medical degree in 1983 and
received his Ph.D. from the Graduate School of Medicine at
the Kurume University in 1987. While at the Graduate School
of Medicine, he worked in the Department of Pathology and
specialized in the morphology of the larynx and general
pathology. His postgraduate work and residency training were
done at the Department of Otolaryngology-Head and Neck
Surgery at the Kurume University School of Medicine and the Kurume University Hospital
under the chairmanship of Professor Minoru Hirano. Now as a Professor of the Kurume
University School of Medicine, Dr. Sato continues to train otolaryngology residents and laryn-
gology research fellows while maintaining a private practice. Dr. Sato is a member of the
American Academy of Otolaryngology-Head and Neck Surgery, the American Triological
Society, the American Laryngological Association and the American Broncho-Esophagological
Association.
Dr. Sato has received a number of honors and awards for his achievement. His longstanding
interest and experience in basic research of the larynx are reflected by the Casselberry Award
from the American Laryngological Association and the Seymour R. Cohen Award from the
American Broncho-Esophagological Association.
His major research interests include laryngeal morphology and molecular biology, espe-
cially as it relates to laryngeal function. Dr. Sato has contributed more than 300 articles and
book chapters in English and Japanese, and presents frequently at regional, national and inter-
national conferences.
xxi
Part I
Whole-Organ Serial Sections of the Human Larynx
Whole-Organ Midsagittal Section
of the Human Adult Larynx 1
Abstract
This chapter presents the anatomical structure of the human adult larynx using a whole-
organ midsagittal histological section.
The whole laryngeal midsagittal section is from an autopsy case of a 54-year-old female.
The larynx had no history of laryngeal diseases or voice disorders and appeared normal
macroscopically.
Two types of stains were employed, hematoxylin and eosin (HE) and Elastica van Gieson
(EVG) stains. And a schema of the specimen presenting the anatomical terms of the struc-
tures is shown.
© Springer Nature Singapore Pte Ltd. 2018 3

K. Sato, Functional Histoanatomy of the Human Larynx, https://doi.org/10.1007/978-981-10-5586-7_1
4 1 Whole-Organ Midsagittal Section of the Human Adult Larynx
Fig. 1.1 Midsagittal section of the human adult

larynx. (a) HE stain, (b) EVG stain, (c) schema of
a
the specimen presenting the anatomical terms
c
Whole-Organ Serial Transverse Sections
Abstract
This chapter presents the anatomical structure of the human adult larynx using whole-organ
transverse histological serial sections and facilitates close and precise understanding of the
spatial relationships of the component parts of the larynx to one another as well as the struc-
ture of each component.
The whole laryngeal transverse serial sections are from an autopsy case of a 57-year-old
female. The larynx had no history of laryngeal diseases or voice disorders and appeared
normal macroscopically.
(EVG) stains. For each section level, a specimen stained by HE stain, a specimen stained by
EVG stain, and a schema of the specimen presenting the anatomical terms of the structures
are shown.
It is very important to be able to visualize the internal laryngeal structures by looking at
the laryngeal cartilage when performing phonosurgery via an extralaryngeal approach. It is
also important to be able to visualize the internal laryngeal structures including histoanat-
omy by observing the mucosa of the lumen when performing phonosurgery via an intrala-
ryngeal approach. Knowledge of the three-dimensional structure, histology, and functional
histoanatomy of the larynx is indispensable for laryngology and laryngeal surgery.

6 2 Whole-Organ Serial Transverse Sections of the Human Adult Larynx
Fig. 2.1 Transverse section of the human adult larynx at 13 mm above the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen present-
ing the anatomical terms
2 Whole-Organ Serial Transverse Sections of the Human Adult Larynx 7
Fig. 2.9 Transverse section of the human adult larynx at the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting the ana-
tomical terms
Fig. 2.10 Transverse section of the human adult larynx at 1 mm below the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen pre-
senting the anatomical terms
Fig. 2.15 Transverse section of the human adult larynx at 10 mm below the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen
presenting the anatomical terms
Fig. 2.16 Transverse section of the human adult larynx at 12 mm below the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen
Whole-Organ Serial Coronal Sections
Abstract
This chapter presents the anatomical structure of the human adult larynx using whole-organ
coronal histological serial sections and facilitates close and precise understanding of the
spatial relationships of the component parts of the larynx to one another as well as the struc-
ture of each component.
The whole laryngeal coronal serial sections are from an autopsy case of an 81-year-old
female. The larynx had no history of laryngeal diseases or voice disorders and appeared
are shown.
It is very important to be able to visualize the internal laryngeal structures by looking at
the laryngeal cartilage when performing phonosurgery via an extralaryngeal approach. It is
also important to be able to visualize the internal laryngeal structures including histoanat-
omy by observing the mucosa of the lumen when performing phonosurgery via an intrala-
ryngeal approach. Knowledge of the three-dimensional structure, histology, and functional
histoanatomy of the larynx is indispensable for laryngology and laryngeal surgery.

24 3 Whole-Organ Serial Coronal Sections of the Human Adult Larynx
Fig. 3.1 Coronal section of the human adult larynx at the anterior commissure tendon. (a) HE stain, (b) EVG stain, (c) schema of the specimen
3 Whole-Organ Serial Coronal Sections of the Human Adult Larynx 25
Fig. 3.2 Coronal section of the human adult larynx at the anterior macula flava. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting
the anatomical terms
Fig. 3.3 Coronal section of the human adult larynx at the junction between the anterior one-fourth and the posterior three-fourths of the membra-
nous vocal fold. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting the anatomical terms
Fig. 3.4 Coronal section of the human adult larynx at the midportion of the membranous vocal fold. (a) HE stain, (b) EVG stain, (c) schema of
Fig. 3.5 Coronal section of the human adult larynx at the junction between the anterior three-fourths and the posterior one-fourth of the membra-
nous vocal fold. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting the anatomical terms
Fig. 3.6 Coronal section of the human adult larynx at the posterior macula flava. (a) HE stain, (b) EVG stain, (c) schema of the specimen present-
Fig. 3.7 Coronal section of the human adult larynx at 2 mm posterior to the posterior macula flava. (a) HE stain, (b) EVG stain, (c) schema of the
specimen presenting the anatomical terms
Fig. 3.10 Coronal section of the human adult larynx at 8 mm posterior to the posterior macula flava. (a) HE stain, (b) EVG stain, (c) schema of
Whole-Organ Midsagittal Section
of the Human Newborn Larynx 4
Abstract
This chapter presents the anatomical structure of the human newborn larynx using a whole-
organ midsagittal histological section.
The whole laryngeal midsagittal section is from an autopsy case of a male 3160 g in
weight. The larynx had no history of laryngeal diseases or voice disorders and appeared
(EVG) stains. And a schema of the specimen presenting the anatomical terms of the struc-
tures is shown.

36 4 Whole-Organ Midsagittal Section of the Human Newborn Larynx
Fig. 4.1 Midsagittal section of the human newborn

larynx. (a) HE stain, (b) EVG stain, (c) schema of
a
c
Whole-Organ Serial Transverse Sections
Abstract
This chapter presents the anatomical structure of the human newborn larynx using whole-
organ transverse histological serial sections and facilitates close and precise understanding
of the spatial relationships of the component parts of the larynx to one another as well as the
structure of each component at birth.
The whole laryngeal transverse serial sections are from an autopsy case of a female
3164 g in weight. The larynx had no history of laryngeal diseases or voice disorders and
appeared normal macroscopically.
are shown.
It is important to understand the three-dimensional structure, histology, and functional
histoanatomy of the newborn larynx before growth and development.

38 5 Whole-Organ Serial Transverse Sections of the Human Newborn Larynx
Fig. 5.1 Transverse section of the human newborn larynx at 5 mm above the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen
5 Whole-Organ Serial Transverse Sections of the Human Newborn Larynx 39
Fig. 5.6 Transverse section of the human newborn larynx at the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting the
anatomical terms
Fig. 5.7 Transverse section of the human newborn larynx at 1 mm below the glottis. (a) HE stain, (b) EVG stain, (c) schema of the specimen
Whole-Organ Serial Coronal Sections
Abstract
This chapter presents the anatomical structure of the human newborn larynx using whole-
organ coronal histological serial sections and facilitates close and precise understanding of
the spatial relationships of the component parts of the larynx to one another as well as the
structure of each component.
The whole laryngeal coronal serial sections are from an autopsy case of a male 3850 g
in weight. The larynx had no history of laryngeal diseases or voice disorders and appeared
are shown.
It is important to understand the three-dimensional structure, histology, and functional
histoanatomy of the newborn larynx before growth and development.

50 6 Whole-Organ Serial Coronal Sections of the Human Newborn Larynx
Fig. 6.1 Coronal section of the human newborn larynx at the anterior commissure tendon. (a) HE stain, (b) EVG stain, (c) schema of the specimen
6 Whole-Organ Serial Coronal Sections of the Human Newborn Larynx 51
Fig. 6.2 Coronal section of the human newborn larynx at the anterior macula flava. (a) HE stain, (b) EVG stain, (c) schema of the specimen pre-
Fig. 6.3 Coronal section of the human newborn larynx at the junction between the anterior one-fourth and the posterior three-fourths of the mem-
branous vocal fold. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting the anatomical terms
Fig. 6.4 Coronal section of the human newborn larynx at the midportion of the membranous vocal fold. (a) HE stain, (b) EVG stain, (c) schema
of the specimen presenting the anatomical terms
Fig. 6.5 Coronal section of the human newborn larynx at the junction between the anterior three-fourths and the posterior one-fourth of the mem-
branous vocal fold. (a) HE stain, (b) EVG stain, (c) schema of the specimen presenting the anatomical terms
Fig. 6.6 Coronal section of the human newborn larynx at the posterior macula flava. (a) HE stain, (b) EVG stain, (c) schema of the specimen
Fig. 6.7 Coronal section of the human newborn larynx at 2 mm posterior to the posterior macula flava. (a) HE stain, (b) EVG stain, (c) schema
Part II
Functional Histoanatomy of the Human Larynx
Clinical Anatomy of the Human Larynx
7
Abstract
1. Phonosurgery is a surgery performed on the human larynx to treat phonatory functions
and to improve the quality of voice.
2. There are two major categories of phonosurgery. One surgical option is open-neck laryn-
geal surgery, which is performed via an extralaryngeal approach. Another is endolaryn-
geal microsurgery and endoscopic surgery, which are performed via an intralaryngeal
approach.
3. It is very important to be able to visualize the internal laryngeal structures by looking at
the laryngeal cartilage when performing phonosurgery via an extralaryngeal approach.
It is also important to be able to visualize the internal laryngeal structures including
histoanatomy by observing the mucosa of the lumen when performing phonosurgery via
an intralaryngeal approach.
4. Knowledge of the three-dimensional structure, histology, and histopathology of the lar-
ynx is indispensable to performing phonosurgery.
7.1 Introduction approach. Additionally, understanding the histological struc-

tures of the vocal fold is essential when performing
Phonosurgery is a surgery performed on the human larynx to phonosurgery.
treat phonatory functions and to improve the quality of voice
[1]. Phonosurgery includes phonomicrosurgery, laryngoplas-
tic phonosurgery, laryngeal injection, and reinnervation of 7.2 linical Anatomy of the Human
C
the larynx. Larynx for an Extralaryngeal
Knowledge of the three-dimensional structure, histology, Approach
and histopathology of the larynx is indispensable to perform-
ing phonosurgery [2]. It is important to visualize the inner three-dimensional struc-
Many surgical procedures have been established for pho- ture by observing the laryngeal cartilages. There are four
nosurgery. There are two major categories of phonosurgery. major cartilages of the human larynx: the thyroid, cricoid,
One surgical option is open-neck laryngeal surgery, which is arytenoid, and epiglottic cartilages (Fig. 7.1).
performed via an extralaryngeal approach. Another is endol- When looking at the larynx from the front, the anterior
aryngeal microsurgery, which is performed via an intralaryn- commissure is located halfway between the superior thyroid
geal approach. notch and the inferior border of the thyroid cartilage (inferior
It is very important to be able to visualize the internal thyroid notch), and this point is an external key landmark for
laryngeal structures by looking at the laryngeal cartilage the level of the vocal fold (Fig. 7.1) [3, 4].
when performing phonosurgery via an extralaryngeal When looking at the larynx from the side, the tip of the
approach. It is also important to be able to visualize the inter- vocal process is located at the midpoint between the anterior
nal laryngeal structures by observing the mucosa of the and posterior borders of the thyroid cartilage at the glottic
lumen when performing phonosurgery via an intralaryngeal level (Fig. 7.1) [4]. The vocal ligament runs parallel to the

62 7 Clinical Anatomy of the Human Larynx
Fig. 7.1 Lateral and frontal epiglottic cartilage epiglottic cartilage

views of the human larynx
thyroid superior thyroid notch thyroid

cartilage cartilage
arytenoid vocal process arytenoid

cartilage cartilage
anterior
commissure
vocal
ligament
inferior thyroid notch

cricoid cartilage cricoid cartilage
Lateral view Frontal view
(Fig. 7.4) [4]. The piriform sinus (piriform fossa, piriform

recess) of the hypopharynx is located in the posterior one-
thyroid cartilage
third of the thyroid cartilage (Fig. 7.4).
When laryngeal framework surgery such as arytenoid adduc-
tion is performed, these external key landmarks are useful.
thyroarytenoid The corniculate cartilage is located at the same level as
muscle the upper border of the thyroid lamina (Fig. 7.4). The epi-
glottic cartilage is connected to the superior thyroid notch
anteroinferiorly via the intervening thyroepiglottic ligament.
arytenoid cartilage
The lamina of the cricoid cartilage is about four times higher
than the arch of cricoid cartilage. The inferior border of the
thyroid cartilage is located at the same height as the midpoint
of the cricoid lamina.
The arytenoid is located at the same level as the upper
border of the thyroid lamina. The ventricular fold (vestibular
fold, false vocal fold) is located between the superior thyroid
notch and the anterior commissure level. The vocal fold is
located between the anterior commissure and the inferior
Fig. 7.2 Transverse CT scan of the larynx with left vocal fold paralysis
at the glottis level
border level of the lamina of the thyroid cartilage.
The preepiglottic space is located anterior and lateral to
inferior border of the lamina of the thyroid cartilage. the thyroepiglottic ligament and epiglottic cartilage
Consequently, the vocal ligament runs from the anterior (Fig. 7.5). The paraglottic space is located on the inside sur-
commissure (located halfway between the superior thyroid face of the thyroid lamina.
notch and the inferior thyroid notch) to the tip of the vocal At the supraglottic level, the descending branch of the
process (located at the midpoint between the anterior and superior laryngeal artery is located in the posterior part of the
posterior borders of the thyroid cartilage). paraglottic space. The ventral branch that branches out of the
When laryngeal framework surgery such as thyroplasty is descending branch is located in the anterior portion of the
performed, these external key landmarks are useful (Figs. 7.2 paraglottic space. At the glottic level, the descending branch
and 7.3). of the superior laryngeal artery is divided into the anterior
Regarding the clinical anatomy around the posterior por- and posterior divisions, both being located in the paraglottic
tion of the larynx, the cricoarytenoid joints, the muscular space. The anterior division of the descending branch of the
processes of the arytenoid cartilage, and the upper margin of superior laryngeal artery anastomoses with the cricothyroid
the cricoid lamina are located at the level of the vocal fold branch of the superior thyroid artery in the anterior portion
7.3 Clinical Anatomy of the Human Larynx for an Intralaryngeal Approach 63
a inferior superior During operation, hemostasis of all these laryngeal arter-

thyroid notch thyroid notch ies can be easily performed by electrocoagulation.
elevated
perichondrium
7.3 linical Anatomy of the Human
C
level of
Larynx for an Intralaryngeal
vocal fold Approach
The glottis consists of two parts: an intermembranous por-

tion or anterior glottis and an intercartilaginous portion or
window posterior glottis. The border is defined as a line between the
placement
tips of the bilateral vocal processes. The tip of the vocal pro-
cesses is located at the midpoint between the anterior and
posterior borders of the thyroid cartilage at the glottic level
b (Fig. 7.6).
window 7.3.1 Endoscopic Surgery
When phonosurgery is performed by endoscopic surgery, it is

inner
perichondrium important to visualize the internal laryngeal structures through
observation of the mucosa of the lumen. Additionally, under-
standing the histological structures of the vocal fold is
essential.
Visualizing the internal laryngeal structures through
observation of the mucosa is useful when performing
endoscopic surgery such as injection laryngoplasty
(Fig. 7.7).
c
silicon block 7.3.2 Endolaryngeal Microsurgery
When phonosurgery is perform by endolaryngeal microsur-

gery, it is also important to understand the internal laryngeal
structures by observing the mucosa of the lumen (Fig. 7.8).
Additionally, understanding the histological structures of the
vocal fold is essential.
Main arteries run in the paraglottic space, which is located
deep inside tissue away from the laryngeal lumen.
Regarding the microanatomy of the vocal fold
(Fig. 7.8c), the vibratory portion of the vocal fold is con-
Fig. 7.3 Medialization laryngoplasty (thyroplasty, type I). (a) Design nected to the thyroid cartilage anteriorly via the intervening
of the window placement of the left lamina of the thyroid cartilage. The anterior macula flava and anterior commissure tendon.
vocal fold is located halfway between the superior and inferior thyroid Posteriorly, it is joined to the vocal process of the arytenoid
notch of the thyroid cartilage, and it runs parallel to the inferior border
of the lamina of the thyroid cartilage. (b) Window placement of the left
cartilage via the intervening posterior macula flava. The
lamina of the thyroid cartilage. (c) Insertion of the silicon block. A shim vocal ligament runs between the anterior and posterior
made of silicone, which fits the size of the window and the desired maculae flavae. It can be seen as white fibrous tissue under
depth of displacement of the window, is inserted a microscope. Reinke’s space is the portion that vibrates the
most during phonation.
of the paraglottic space. The posterior division of the The anterior and posterior maculae flavae are dense
descending branch of the superior laryngeal artery anasto- masses of cells and extracellular matrices and are observed
moses with the inferior laryngeal artery in the posterior por- at each end of the membranous portion of the vocal fold dur-
tion of the paraglottic space. ing endolaryngeal microsurgery. They form conspicuous
Fig. 7.4 Lateral and frontal views of the epiglottic cartilage

human larynx
corniculate thyroepiglottic
cartilage ligament
superior thyroid notch
thyroid
lamina
piriform sinus
of hypopharynx vocal process
ventricular fold
anterior
cricoarytenoid commissure vocal fold
joint
muscular process
cricoid lamina
cricoid arch
Lateral view Frontal view
Fig. 7.5 Human larynx viewed from the side paraglottic space
and above.  Ventral branch of superior descending branch of
paraglottic space superior laryngeal artery preepiglottic space
laryngeal artery,  anterior division of
descending branch of superior laryngeal preepiglottic space
artery,  posterior division of descending
branch of superior laryngeal artery, Piriform sinus
superior
of hypopharynx
 ascending branch of cricothyroid branch of laryngeal artery
superior thyroid artery,  cricothyroid branch
of superior thyroid artery
piriform sinus
of hypopharynx
inferior
laryngeal artery
cricothyroid branch of
superior thyroid artery
posterior wall of glottis interarytenoid notch

posterior glottis
(intercartilaginous portion) lamina of cricoid cartilage
arytenoid
Piriform sinus arytenoid cartilage
of hypopharynx
aryepiglottic fold
vocal process
of arytenoid
cartilage
cartilaginous portion
of vocal fold thyroid cartilage
ventricular fold
membranous portion epiglottis

of vocal fold
anterior glottis anterior commissure
Fig. 7.6 Videoendoscopic view of the glottis (intermembranous
(the larynx view from above) portion)
7.3 Clinical Anatomy of the Human Larynx for an Intralaryngeal Approach 65
mucosal bulges. They are visible through the mucosa as

whitish-yellow masses and are very hard or stiff when pal-
pated using forceps.
Viscoelasticity of the lamina propria of the vocal fold
mucosa, especially in Reinke’s space, is required for vibra-
tion and phonation. The viscoelastic properties of the lamina
propria depend on extracellular matrices. The three-
dimensional structure composed of extracellular matrices is
indispensable to the viscoelastic properties of the human
vocal fold mucosa; therefore, the microstructure of the vocal
injection needle
fold should be preserved as much as possible during endol-
aryngeal microsurgery.
When microsurgery for Reinke’s space (Fig. 7.9) such as
cyst extirpation (Fig. 7.10) or Reinke’s edema (Fig. 7.11) is
performed, the approach to Reinke’s space is extremely
Fig. 7.7 Transendoscopic injection laryngoplasty important.
ascending branch of cricothyroid branch

a of superior thyroid artery
anterior commissure
membranous portion
of vocal fold
thyroarytenoid muscle
vocal process of
arytenoid cartilage
paraglottic space
thyroid cartilage
anterior division of
descending branch
of superior laryngeal
artery
muscular process
of arytenoid
cartilage
laryngoscope posterior division of

descending branch
intubation tube lamina of artery
cricoid cartilage
epiglottis
b
vocal fold preepiglottic space
anterior division of
ventricular fold descending branch
vocal process of artery
arytenoid cartilage
paraglottic space
posterior division of
descending branch
artery
thyroid
cartilage
piriform sinus
Fig. 7.8 Overview during endolaryngeal laryngoscope of hypopharynx
microsurgery. (a) Glottic level. (b) Supraglottic
intubation tube arytenoid cartilage
level. (c) Vocal fold. Red line: incision to approach
Reinke’s space arytenoid muscle
Fig. 7.8 (continued) c anterior commissure tendon

anterior commissure
anterior macula flava
superficial layer of membranous portion
lamina propria of vocal fold
(Reinke’s space) incision line
vocal ligament
posterior macula flava

vocal process of cartilaginous portion
arytenoid cartilage of vocal fold
laryngoscope
Reinke’s space way to approach

vocal ligament Reinke’s space incision line
cyst
vocalis muscle
Fig. 7.9 Coronal section of the human adult vocal fold showing the
way to approach Reinke’s space (red line) (27-year-old male, Elastica
van Gieson stain
Fig. 7.10 Endolaryngeal microsurgery for vocal fold cyst. An incision

along the vocal fold is made on the upper surface of the vocal fold, and
then the cyst is removed
References 67
When approaching Reinke’s space, the mucosa of the free

edge of the vocal fold must be preserved. An incision along
incision line the vocal fold must be made on the upper surface of the vocal
fold, and then lesions in Reinke’s space can be removed
(Figs. 7.8c and 7.9). This method results in minimal distur-
bance of voice quality and prevents granulation or scar tissue
formation.
When lesions of the vocal fold are difficult to expose, a resec-
tion of the ventricular fold (vestibular fold, false vocal fold)
makes sufficient exposure possible. The resection of the ven-
tricular fold does not significantly influence laryngeal function.
References
1. Hirano M. Phonosurgery. Basic and clinical investigations. Otologia

(Fukuoka). 1975;21(Suppl. 1):239–440.
2. Hirano M, Sato K. Histological color atlas of the human larynx. San
Diego, CA: Singular Publishing Group Inc.; 1993.
3. Isshiki N. Phonosurgery. theory and practice. Tokyo: Springer-
Verlag; 1989.
Fig. 7.11 Endolaryngeal microsurgery for Reinke’s edema. An inci- 4. Sato K. Three dimensional anatomy of the larynx: investiga-
sion along the vocal fold is made on the upper surface of the vocal fold, tion by whole organ sections. Otologia Fukuoka. 1987;33(Suppl.
and then the material in Reinke’s space is squeezed and sucked out 1):153–82.
Compartments of the Human Larynx
8
Abstract
1. The human larynx has a complex structural organization with a framework characterized
by an external cartilaginous skeleton and internal connective tissues in a variety of
arrangements in the different regions of the larynx.
2. This framework composed of cartilages, ligaments, and muscles contributes to the phys-
iologic functions of the human larynx.
3. This framework also determines, at least initially, the growth and spread of inflammation
and neoplasms of the larynx.
4. Dense compact tissues with few interstices such as cartilage or dense fibrous tissue act
more or less as effective barriers to invading carcinomas.
5. It is very important to understand the internal laryngeal structures and compartments as
well as the histoanatomy by looking from the outside and from the inside of the larynx.
6. Knowledge of the three-dimensional structure, compartments, histology, and histopa-
thology of the larynx is indispensable for laryngology and phonosurgery.
8.1 Introduction structure, compartments, histology, and histopathology of the

larynx is indispensable for laryngology and phonosurgery.
The roles of the human larynx are protection of the lower air-
way, respiration, and phonation. The human larynx has a com-
plex structural organization with a framework characterized 8.2 Anterior Commissure Tendon
by an external cartilaginous skeleton and internal connective
tissues in a variety of arrangements in the different regions of Ridpath noted that there was a small fibrous projection, situ-
the larynx. This framework is composed of cartilages, liga- ated on the median line of the inner surface of the thyroid
ments, and muscles and contributes to the physiologic func- cartilage, which serves for the insertion of the vocal cords [3].
tions of the human larynx. In addition, the framework Since Broyles described this fibrous tissue as an anterior com-
determines, at least initially, the growth and spread of inflam- missure tendon [4], this term (not contained in Terminologia
mation and neoplasms of the larynx. Dense compact tissues Anatomica) is commonly used among physicians.
with few interstices such as cartilage or dense fibrous tissue A tendon is defined as a nondistensible fibrous cord or
act more or less as effective barriers to invading carcinomas. band of variable length that connects the fleshy (contractile)
Pressman has pointed out the existence of submucosal part of muscle with its bony attachment or other structure
connective tissue compartments of the human larynx and has [5]. By this definition, the anterior commissure tendon is not
investigated them by injection of dyes and radioactive iso- a real tendon.
topes [1, 2]. The anterior commissure tendon originates from the mar-
It is very important to understand the internal laryngeal gin of the superior thyroid notch caudalward for approxi-
structures and compartments as well as the histoanatomy by mately 9 to 11 mm, comprising the upper three-fourths of the
looking from the outside and from the inside (the mucosa of midline of the posterior surface of the thyroid cartilage
the lumen) of the larynx. Knowledge of the three-dimensional (Fig. 8.1) [6, 7]. The upper one-fourth of the anterior

70 8 Compartments of the Human Larynx
Fig. 8.1 (a) Midsagittal

section of the human adult a
larynx and anterior epiglottic cartilage
commissure tendon (Elastica
van Gieson stain). (b) Region
B in (a)
thyroepiglottic
ligament
superior
thyroid notch
B
thyroid cartilage
lamina of cricothyroid ligament

cricoid cartilage
arch of
cricoid cartilage
thyroepiglottic thyroid cartilage

ligament
blood
vessels
ossification
anterior commissure
anterior commissure
tendon
8.2 Anterior Commissure Tendon 71
c ommissure tendon is thick. And it is also thick at the level dihedral angle in the upper three-fourths of the midline of the
of the glottis. Its width is 1.2–4 mm, and it is wider at the thyroid cartilage. There are blood vessels in the anterior
upper one-fourth and at the level of the glottis [6, 7]. The commissure tendon (Fig. 8.3).
anterior commissure tendon is composed of collagen fibers. Epiglottic cartilage is connected to the thyroid cartilage
The fibrous tissue blends with the underlying thyroid carti- anteroinferiorly via the intervening thyroepiglottic ligament
lage (Fig. 8.2). An inner perichondrium does not exist at the and anterior commissure tendon (Figs. 8.1 and 8.2b).
a d thyroid cartilage
superior
thyroid notch perichondrium
perichondrium
ndriu
ium
thyroid
cartilage
B
C
D
E
F
G anterior
anteriorr macula
macula flava
flava
b thyroid cartilage
perichondrium perichondrium
e thyroid cartilage
perichondrium
hondrium
perichondrium
thyroepiglottic
ligament
thyro-
arytenoid
glands muscle
thyro-
arytenoid
glands muscle
c thyroid cartilage a nteriorr

anterior
macula
flava
perichondrium perichondrium
vocal
ligament
laryngeal mucosa
laryngeal
ventricle
Fig. 8.2 (a) Transverse sections of the human adult larynx and anterior level of the glottis (level D in a). (e) At the level of the glottis (level E
commissure tendon (area encircled by yellow dotted line, Elastica van in a). (f) At the level of the subglottis (level F in a). (g) At the level of
Gieson stain). (b) At the level of the superior thyroid notch (level B in the subglottis (level G in a)
a). (c) At the level of the laryngeal ventricle (level C in a). (d) At the
At the level of the laryngeal ventricle (Fig. 8.2c), the mid-

f thyroid cartilage
line of the posterior surface of the thyroid cartilage is only
covered with thin laryngeal mucosa.
At the level of the glottis (Fig. 8.2d and e), the vibratory
portion (membranous portion) of the vocal fold is firmly
thyro-
arytenoid connected to the thyroid cartilage anteriorly via the inter-
muscle vening anterior macula flava and anterior commissure
thyro- tendon.
arytenoid At the level of the subglottis (Fig. 8.2f and g), the conus
muscle
elasticus is connected to the thyroid cartilage anteriorly. As
the level goes caudally, the anterior commissure tendon dis-
appears. On the other hand, the inner perichondrium exists at
conus elasticus
the dihedral angle of the midline of the thyroid cartilage, and
laryngeal glands the conus elasticus attaches to the inner perichondrium of the
thyroid cartilage.
g
thyroid cartilage 8.3 hysiological and Clinical Significance
P
perichondrium of the Anterior Commissure Tendon
perichondrium
perichondrium
8.3.1 Laryngeal Functions
The internal connective tissues which form the lumen of the

larynx, such as ligaments, are firmly connected to the thy-
roid cartilage via the intervening anterior commissure ten-
don. In addition, the vocal fold is firmly connected to the
thyroid cartilage anteriorly by the anterior commissure
conus elasticus tendon.
laryngeal glands From the physiological point of view, these structures can
stand intense subglottic pressure during coughing, phona-
tion, etc.
Fig. 8.2 (continued)
8.3.2 Anterior Commissure Carcinoma
Laryngeal carcinoma often breaks through into the ante-

anterior commissure
tendon rior midline of the thyroid cartilage (Fig. 8.4). There are
some anatomical reasons for this. First, if a tumor occu-
pies the anterior commissure, it is closer to the cartilagi-
nous framework than anywhere else on the vocal fold;
elsewhere muscles and perichondrium intervene [8].
Second, in the midline only the fibrous anterior commis-
sure tendon separates the mucosa from the cartilage [8].
blood vessels Anterior commissure carcinoma spreads along the ante-
rior commissure tendon into the thyroid cartilage where
the perichondrium does not intervene. Third, if the tumor
spreads subglottically it can easily penetrate the cricothy-
roid ligament, one of the weak points in the laryngeal
framework [8]. In addition, the median portion of the thy-
roid cartilage is often ossified (Fig. 8.1). Carcinoma inva-
Fig. 8.3 Blood vessels in the anterior commissure tendon (hematoxy- sion takes place preferentially at ossified areas of the
lin and eosin stain) cartilage.
8.4 Cricothyroid Ligament 73
a 8.3.3 Midline Lateralization Thyroplasty

(Type II Thyroplasty)
When midline lateralization thyroplasty for adductor spas-

hyoid bone modic dysphonia is performed [9, 10], surgical management
thyroid cartilage of the dihedral angle of the midline of the thyroid cartilage is
very important.
B In order to make the anterior commissure the proper
width, it is important to split the anterior commissure tendon
at the midline and to spread the glottis while making sure the
tendons remain attached to the bilateral thyroid cartilages
(Fig. 8.5) [11].
cricoid cartilage
8.4 Cricothyroid Ligament
b The cricothyroid ligament runs between the median portion

of the thyroid cartilage and cricoid cartilage (Fig. 8.6). It is
important to note that the ligament does not run through all
aspects of the gap between the thyroid and cricoid cartilages.
Occasionally, the incorrect term “cricothyroid membrane” is
carcinoma used and reinforces an incorrect anatomical understanding of
the ligament.
The cricothyroid ligament is composed of collagen and
elastic fibers which run vertically. Both ends of the ligament
gradually transit into the perichondrium of the thyroid and
cricoid cartilages. The cricothyroid branch of the superior
thyroid artery penetrates at the median portion of the crico-
thyroid ligament (Fig. 8.7).
At the upper portion of the cricothyroid ligament, the
conus elasticus attaches behind the ligament. Moving inferi-
ossified thyroid cartilage
orly, the border between them is not clearly delineated.
Fig. 8.4 (a) Coronal section of the human adult larynx with anterior com-
missure carcinoma (hematoxylin and eosin stain). (b) Carcinoma has
invaded the ossified portion (arrows) of the thyroid cartilage (region B in a)
thyroid cartilage thyroid cartilage
anterior
anterior
commissure
commissure shim
tendon
tendon
epithelium
Fig. 8.5 Midline lateralization anterior

thyroplasty and surgical management
macula
of the dihedral angle of the midline of
the thyroid cartilage flava
Fig. 8.6 Schema of the

cricothyroid ligament
paraglottic space
A
thyroid cartilage
cricothyroid ligament conus elasticus
A
B B
C
cricoid
cartilage conus elasticus
conus elasticus
C
conus elasticus
Fig. 8.7 Transverse section

of the cricothyroid ligament prelaryngeal lymph node
(hematoxylin and eosin stain).
Blood vessels such as the
cricothyroid branch of the
superior thyroid artery and
lymphatic vessels penetrate at
the median portion of the cricothyroid branch
cricothyroid ligament of superior thyroid
artery
cricothyroid cricothyroid
ligament ligament
8.5 hysiological and Clinical Significance

P ment. The motion of cricothyroid joint (articulation) is lim-
of Cricothyroid Ligament ited by the cricothyroid ligament.
The blood vessels and lymphatic vessels penetrate at
The thyroid cartilage is connected to the cricoid cartilage by the median portion of the cricothyroid ligament. Laryngeal
the cricothyroid joint (articulation) and cricothyroid liga- carcinoma, especially anterior commissure carcinoma,

8.8 Reinke’s Space 75
often breaks through into this portion of the cricothyroid 8.7 hysiological and Clinical Significance
P
ligament. of the Vocal Ligament and Conus
The cricothyroid ligament is present at the median por- Elasticus
tion, and there is no barrier (ligament) on either side. The
lateral portions of the cricothyroid ligament are directly In mammals, only human adults have the layered structure, and
connected to the paraglottic space. Consequently, carci- this structure is necessary for vocal fold vibration for phonation.
noma usually spreads outside the larynx by way of this area Adult vocal folds have a layered structure consisting of the epi-
which is one of the weak points in the laryngeal thelium; the superficial, intermediate, and deep layers of the
framework. lamina propria; and the vocalis muscle [12]. The intermediate
and deep layers of the lamina propria form the vocal ligament.
The conus elasticus efficiently concentrates airflow on the
8.6 ocal Ligament and Conus Elasticus
V membranous vocal fold during phonation. In addition, the
(Crico-vocal Membrane) conus elasticus and vocal ligament support the vocal fold
during phonation and withstand intense subglottic pressure
The conus elasticus is a fan-shaped submucosal fibrous during coughing, phonation, etc.
membrane radiating from the anterior commissure tendon The conus elasticus is a barrier against carcinoma inva-
(Fig. 8.8). From this origin it runs to the superior border of sion (Figs. 8.9 and 8.10). Carcinoma is contained within the
the cricoid cartilage and vocal process of the arytenoid carti- space bounded by the conus elasticus.
lage. The bilateral conus elasticus form a cone shape; conse-
quently, this fibrous membrane is designated as the conus
elasticus. The bilateral conus elasticus fuses with the median 8.8 Reinke’s Space
cricothyroid ligament. The conus elasticus is composed of
collagen and elastic fibers. Reinke’s space is a potential space between the epithelium
In mammals, only humans have a vocal ligament. The and vocal ligament [13]. This space was previously
vocal ligament runs between the anterior commissure tendon described by Reinke and has been known as “Reinke’s
and the vocal process of the arytenoid cartilage via the inter- pouch.” Pressman observed that injected dyes remain in the
vening anterior and posterior maculae flavae (Fig. 8.8). The region of the margin of the vocal fold and described “a
free superior margin of the conus elasticus thickens to form vocal cord bursa” there [2]. Reinke’s space is referred to as
the vocal ligament. The vocal ligament laterally continues to the superficial layer of the lamina propria of the vocal fold
the fascia of the thyroarytenoid muscle. mucosa.
anterior macula flava anterior commissure tendon

vocal ligament
posterior
conus elasticus
macula flava
arch of vocal process
cricoid cartilage
thyroid cartilage
Fig. 8.8 Schema of the vocal

ligament and conus elasticus lamina of cricoid cartilage
viewed from above arytenoid cartilage
Fig. 8.9 Coronal section of the a

human adult larynx with
supraglottis carcinoma
(a) Carcinoma has invaded the
vocal fold and ventricular fold.
(b) Carcinoma destroys most of
the thyroarytenoid muscle.
C
Carcinoma is contained within
the space bounded medially by ventricular fold
the conus elasticus (arrows).
Submucosal separation of
carcinoma from overlying vocal fold
mucosa shows that the
subglottis is still intact (region B
in a). (c) Carcinoma has thyroid cartilage
extended the laryngeal ventricle
and has invaded the ossified B
portion of the thyroid cartilage
(arrows) (region C in a)
cricoid cartilage
carcinoma
conus elasticus
c ossified thyroid cartilage
carcinoma
8.8 Reinke’s Space 77
Fig. 8.10 Coronal section of the human adult larynx with

glottic carcinoma (hematoxylin and eosin stain). (a) Glottic a
carcinoma has invaded the subglottis and laryngeal
ventricle. (b) Carcinoma superficially invades the subglottis
along the conus elasticus (region B in a). (c) Superior
laryngeal artery and vein penetrate the thyroid foramen
(region C in a) C
thyroid foramen
laryngeal ventricle
vocal fold
thyroid cartilage
B
cricoid cartilage
lymphocytes
conus
con
cco
onu
on conus
uss e
el
ela
elasticus
lla
assti uelasticus
sttiticcu
cus
us
s
carcinoma
cricoid area
cricoid
cartilage
Fig. 8.10 (continued) c
thyroid cartilage
thyroid foramen
superior laryngeal
artery and vein
thyroid cartilage
Reinke’s space extends along the full length of the vocal of the membranous vocal fold becomes edematous and swol-
fold except at the anterior and posterior ends where the mac- len. Histopathologically, the primary feature is edema in
ulae flavae exist. The relative thickness of Reinke’s space Reinke’s space. The mechanism for the onset and develop-
(the superficial layer of the lamina propria) varies along the ment of the disease remains unclear. However, fragility of
length of the vocal fold. This layer is thickest at the midpoint and alteration in the permeability of the blood vessels are
of the membranous vocal fold and becomes thinner toward presumed to cause edema of Reinke’s space, which likely
the anterior and posterior portions [14]. progresses to Reinke’s edema [15]. See Chap. 21, “Blood
Vessels of the Larynx and Vocal Fold.”
8.9 hysiological and Clinical Significance

P
of Reinke’s Space 8.10 Thyroglottic Ligament
The viscoelastic properties of the lamina propria of the human The thyroglottic ligament is not included among standard
vocal fold mucosa, especially Reinke’s space (the superficial anatomical terms (Terminologia Anatomica) but was
layer of the lamina propria), determine vibratory behavior. The described by Tucker and Smith [16].
three-dimensional structure of extracellular matrices in Reinke’s The thyroglottic ligament fans out from the vocal liga-
space is indispensable to the viscoelastic properties of the ment to the thyroid cartilage (Fig. 8.11). It transits into the
human vocal fold mucosa. In addition, not only the three- inner perichondrium of the thyroid cartilage and into the fas-
dimensional structure of the extracellular matrices but also their cia of the thyroarytenoid muscle and vocal ligament
qualitative and quantitative properties in Reinke’s space have an (Fig. 8.12). The preepiglottic space is adjacent to the para-
effect on the physical properties of the human vocal fold mucosa glottic space posteroinferiorly and is separated from it by the
as a vibrating tissue. See Chap. 10, “Cells and Extracellular thyroglottic ligament; however, the thyroglottic ligament
Matrices in the Human Adult Vocal Fold Mucosa.” disappears posterosuperiorly, and the two spaces are not
Reinke’s edema is a common disease of the vocal fold clearly delineated from each other. The thyroglottic ligament
ultimately causing changes in voice quality. The entire length is composed of collagen and elastic fibers.
8.10 Thyroglottic Ligament 79
Fig. 8.11 Schema of anterior commissure tendon

thyroglottic ligament viewed anterior macula flava
from above vocal ligament
posterior
macula flava thyroglottic ligament
vocal process
arch of
cricoid cartilage
thyroid cartilage
lamina of cricoid cartilage

arytenoid cartilage
a
epiglottic cartilage
preepiglottic space preepiglottic space
thyroglottic ligament thyroglottic ligament

B
thyroarytenoid muscle thyroarytenoid muscle
paraglottic space paraglottic space
b perichondrium thyroid
cartilage
preepiglottic
space
thyroglottic
ligament
paraglottic
space
Fig. 8.12 (a) Coronal section of the human adult larynx at the
anteroposterior midpoint of the vocal fold (Elastica van Gieson
stain). (b) Thyroglottic ligament is a border between preepi-
glottic space and paraglottic space (region B in a). It transits
into the perichondrium of the thyroid cartilage
8.11 P
hysiological and Clinical Significance 8.12 Quadrangular Membrane
of the Thyroglottic Ligament and Ventricular Ligament
The vocal folds are suspended on the thyroglottic ligament The quadrangular membrane is not a sheet but a connective
from the thyroid cartilage. The thyroglottic ligament rein- tissue composed of collagen and elastic fibers running in the
forces the floor of the laryngeal ventricle [16]. ventricular folds (Figs. 8.13 and 8.14).
Carcinoma is contained within the space bounded by the The quadrangular membrane is attached anteriorly to the
thyroglottic ligament. The thyroglottic ligament is a barrier lateral border of the thyroepiglottic ligament (Fig. 8.13b),
against carcinoma invasion from the preepiglottic space to runs posteriorly around the glands in the ventricular fold
the paraglottic space and vice versa. See Chap. 20, “Spaces (Fig. 8.13c), and passes through the ventricular fold and the
of the Larynx.” aryepiglottic fold to continue to the perichondrium of the
Fig. 8.13 (a) Transverse section of

a thyroepiglottic ligament
the human adult larynx at the
ventricular fold level (Elastica van ventricular fold quadrangular membrane
Gieson stain). (b) Quadrangular
preepiglottic space
membrane transits into the B
thyroepiglottic ligament (region B in thyroid cartilage thyroglottic
a). (c) Quadrangular membrane runs ligament
between the laryngeal glands in the C
ventricular fold (region C in a). (d)
Quadrangular membrane transits
into the perichondrium of the medial
and anterior surface of the arytenoid
cartilage (region D in a)
D
arytenoid cartilage
b thyroepiglottic ligament
preepiglottic
space
quadrangular
gland membrane
gland
gland
8.12 Quadrangular Membrane and Ventricular Ligament 81
preepiglottic space c
quadrangular
gland membrane
gland
gland
gland
1000 µm
1000 µm d
quadrangular gland
membrane
gland
gland
gland
arytenoid cartilage
Fig. 8.14 (a) Coronal section

a
of the human adult larynx
(Elastica van Gieson stain). ventricular fold
(b) Quadrangular membrane
runs between the laryngeal thyroid cartilage
paraglottic space
glands in the ventricular fold
and is not clearly delineated
from the ventricular ligament
(region B in a)
B
thyroarytenoid
muscle
cricoid cartilage
gland
quadrangular
membrane
gland
gland
thyroarytenoid
muscle
medial and anterior surface of the arytenoid cartilage The quadrangular membrane is a connective tissue run-
(Fig. 8.13d). It is often reported that the quadrangular mem- ning around the glands in the ventricular fold; consequently,
brane continues to the aryepiglottic ligament above and to it is a barrier against carcinoma invasion and spread of
the ventricular ligament below. However, their borders are inflammation.
not clearly delineated from each other (Fig. 8.14b).
8.14 Thyrohyoid Membrane

8.13 P
hysiological and Clinical Significance
of the Quadrangular Membrane The thyrohyoid membrane is a connective tissue running
and Ventricular Ligament between the superior surface of the lamina of the thyroid car-
tilage and the inferior surface of the hyoid bone. The thyro-
The quadrangular membrane reinforces the ventricular fold. hyoid membrane is composed of collagen and elastic fibers.
8.18 The Laryngeal Cartilages 83
Both ends of the membrane transit into the perichondrium of a

the thyroid cartilage and periostium of the hyoid bone. The hyoid bone
preepiglottic space is surrounded by the thyrohyoid mem-
brane and thyroid cartilage anteriorly. hyoepiglottic ligament
The superior laryngeal artery and vein and lymphatic ves-
sels penetrate the thyrohyoid membrane anterior to the supe-
rior cornu of the thyroid cartilage. They run into the larynx epiglottic cartilage
and hypopharynx.
piriform sinus
8.15 P
of the Thyrohyoid Membrane B
The thyrohyoid membrane surrounds the preepiglottic space

and allows the supraglottis to more effectively play a role carcinoma
during swallowing.
Carcinoma sometimes initially grows and spreads out of superior cornu
the larynx via the blood and lymphatic vessels penetrating
the thyrohyoid membrane (Fig. 8.15). b carcinoma
8.16 Spaces of the Larynx
A space is defined as any demarcated portion of the body,

either an area of the surface, a segment of tissues, or a cavity
[17]. The spaces of the human larynx are a loose areolar area
composed of adipose tissue and loose elastic and collagen thyrohyoid
fibers [6, 18, 19]. membrane
The chief laryngeal spaces of the human larynx are superior cornu of
the preepiglottic space, the paraglottic space, and the thyroid cartilage
cricoid area. These spaces are very important clinically
and are commonly used terms; however, they are not 1000 mm
included among standard anatomic terms (Terminologia
Anatomica).
Distributions of these laryngeal spaces are important in Fig. 8.15 (a) Transverse section of the human adult larynx with hypo-
pharyngeal carcinoma (hematoxylin and eosin stain). (b) Carcinoma
following the spread of laryngeal cancer, and their possible penetrates the thyrohyoid membrane and spread out from the larynx via
physiological and functional significance is also of interest. the blood and lymphatic vessels (arrow). The thyrohyoid membrane
See Chap. 20, “Spaces of the Larynx.” cannot be clearly delineated because of the fibrous tissue around the
carcinoma (region B in a)
8.17 P
of the Spaces of the Larynx and hyaline cartilage. The thyroid and cricoid cartilages
are composed of hyaline cartilage. The epiglottic carti-
See Chap. 20, “Spaces of the Larynx.” lage is composed of elastic cartilage. Hyaline cartilage
can ossify in the elderly, but elastic cartilage does not
ossify in a lifetime.
8.18 The Laryngeal Cartilages A thyroid foramen, a laryngeal anomaly, is a congenital
linear opening located in the lamina of the posterosuperior
The laryngeal cartilages are the frameworks and compart- portion of the thyroid cartilage (Fig. 8.10a and 8.10c). The
ments of the larynx. superior laryngeal artery and vein and the inner branch of
The major laryngeal cartilages are the thyroid, cri- superior laryngeal nerve pass through the thyroid foramen. A
coid, arytenoid, and epiglottic cartilages. Among the thyroid foramen is not a rare laryngeal anomaly and was
laryngeal cartilages, only the arytenoid cartilage is com- found in 39% (47/121) of coronally sectioned specimens in
posed of two types of cartilages, i.e., elastic cartilage Kirchner’s collection [20].
a a
hyoid bone
thyroid cartilage
preepiglottic paraglottic space

thyroglottic
space
B ligament
epiglottic cartilage thyroid B
foramen carcinoma
carcinoma piriform sinus
b thyroid
paraglottic
space
cartilage
b
1000 mm
thyroid
foramen
carcinoma
epiglottic cartilage carcinoma
1000 mm
carcinoma Fig. 8.17 Transverse section of the human adult larynx with hypopha-
ryngeal carcinoma (hematoxylin and eosin stain). (a) Carcinoma of the
Fig. 8.16 (a) Transverse section of the human adult larynx with supra- piriform sinus invades the paraglottic space along the thyroid cartilage.
glottic carcinoma (hematoxylin and eosin stain). (b) Carcinoma inva- The thyroglottic ligament becomes a barrier against carcinoma inva-
sion does not penetrate the elastic cartilage. Carcinoma extends to the sion. (b) Carcinoma can spread out of the larynx via the thyroid
periphery of the epiglottic cartilage or through the foramen in the epi- foramen
glottic cartilage and spreads into the preepiglottic space (region B in a)
Carcinoma invasion commonly takes place at the ossified

areas of hyaline cartilage (Fig. 8.9). On the other hand inva-
8.19 P
hysiological and Clinical sion of the non-ossified areas of hyaline cartilage and elastic
Significance of the Laryngeal cartilage are rare (Fig. 8.16).
Cartilages The paraglottic space connects anteroinferiorly with
extralaryngeal tissues by way of the gap between the thyroid
The larynx is a complex structural organization with a frame- and cricoid cartilages lateral to the cricothyroid ligament.
work characterized by an external cartilaginous skeleton Consequently, carcinoma usually spreads outside the larynx
which suspends internal connective tissues in a variety of by way of this portion which is one of the weak points in the
arrangements in the different regions. laryngeal framework.
The elastic cartilage portion in the arytenoid cartilage plays There is a possibility that carcinoma can invade through
an important role in the physiologic functions of the human the thyroid foramen and spread outside the larynx
glottis. See Chap. 9. “Histoanatomy of the Human Glottis”. (Fig. 8.17).
8.20 Compartments and Laryngeal Inflammatory Diseases 85
a posterior wall of pharynx

lingual tonsil
epiglottis
A
B
aryepiglottic fold
C
D
E
epiglottis arytenoid
vallecula
Fig. 8.19 Pathway of inflammation spread in acute epiglottitis. The

inflammatory exudate spreads from the lingual surface of the epiglottis
(pharynx) downward in the direction of the arrows outside the quadran-
gular membrane
arytenoid
vocal fold
aryepiglottic
fold (Fig. 8.20f). The mucosa is surrounded by the lingual tonsil
ventricular anteriorly, by the hyoepiglottic ligament inferiorly, and by
fold the epiglottic cartilage posteriorly. The preepiglottic space is
bordered by hyoepiglottic ligament.
epiglottis Once severe inflammation occurs around the anteroinfe-
rior portion of the epiglottis (vallecula) (Figs. 8.21 and
Fig. 8.18 Endoscopic view of the larynx with acute epiglottitis 8.22a), the permeability of the capillaries increases.
(59-year-old female). (a) Lingual surface of the epiglottis shows Consequently, edema with fibrin occurs (Fig. 8.22b). Since
edematous inflammation and swelling. (b) Edematous inflammation
the inflammatory exudate cannot extend into the preepi-
spreads from the epiglottis to the aryepiglottic fold and arytenoid,
and the supraglottis (vestibule of the larynx) has become narrow. glottic space inferiorly (Fig. 8.22c) or into the epiglottic
However, the compartments (quadrangular membrane, etc.) of the cartilage posteriorly (Fig. 8.22d), it extends downward to
larynx prevent its extension to the ventricular fold and vocal fold the periphery of the lingual surface of the epiglottis and
mucosa
supraglottis, including the loose connective tissues of the
aryepiglottic fold and arytenoids (Figs. 8.19 and 8.20). In
addition, it affects the oropharynx and hypopharynx. The
8.20 C
ompartments and Laryngeal exudate is also present in the deep loose connective tissue
Inflammatory Diseases of the larynx, extending downward deep to the thyroepi-
glottic, thyroarytenoid, aryepiglottic, and arytenoid mus-
8.20.1 Acute Epiglottitis cles. However, the compartments (quadrangular membrane,
etc.) of the larynx prevent its extension to the laryngeal sur-
Acute epiglottitis (Fig. 8.18) is a fatal disease, and the face of the epiglottis, ventricular fold (Fig. 8.20d), and
patient’s condition progresses rapidly, leading to severe vocal fold mucosa (Fig. 8.20e). Therefore, obstruction of
respiratory obstruction (Fig. 8.19). the laryngeal airway takes place at the supraglottic region
The anteroinferior portion of the epiglottis (vallecula) by pressure from the outside.
(Fig. 8.20a) is composed of loose connective tissue with a The actual airway obstruction is related to the deep
large number of blood vessels as well as capillaries extension of acute inflammation in the suplagottis (supra-
a hyoid bone e vocal ligament

thyroid cartilage thyroarytenoid muscle
arytenoid cartilage
lingual surface epiglottic cartilage cricoid cartilage
of epiglottis
f
b hyoepiglottic ligament
loose connective tissue

preepiglottic space epiglottic cartilage
aryepiglottic fold
laryngeal surface
of epiglottis
c 500 mm
preepiglottic space epiglottic cartilage
thyroid cartilage vestibule of larynx
g
aryepiglottic fold
gland
G
hypopharynx
loose connective tissue

d preepiglottic space
thyroepiglottic ligament
ventricular fold quadrangular membrane

thyroid cartilage
H
1000 mm
arytenoid cartilage
h quadrangular membrane
arytenoid muscle
Fig. 8.20 Transverse sections of the human adult larynx and supraglot-
tic compartments (Elastica van Gieson stain). (a) At the level of the gland
vallecula (level A in Fig. 8.19). (b) At the level of the aryepiglottic fold
(level B in Fig. 8.19). (c) At the level of the arytenoid (level C in
Fig. 8.19). (d) At the level of the ventricular fold (level D in Fig. 8.19). loose connective
tissue
(e) At the level of the vocal fold (level E in Fig. 8.19). (f) Anteroinferior
portion of the epiglottis (vallecula) is composed of loose connective
tissue, and there are a great number of blood vessels as well as capillar-
ies (arrows) (region F in a). (g) The inflammatory exudate extends into
the loose connective tissue in the aryepiglottic fold (arrows) (region G
in c). (h) The inflammatory exudate extends into the loose connective
tissue outside of the quadrangular membrane of the ventricular fold arytenoid cartilage
1000 mm
(arrows) (region H in d)
8.20 Compartments and Laryngeal Inflammatory Diseases 87
b
aryepiglottic fold
epiglottis arytenoid
fibrin
trachea
tongue larynx fibrin
hyoepiglottic
ligament
preepiglottic
space
Fig. 8.21 Acute epiglottitis in an adult (autopsy specimen, 49-year-old c

male) (Photograph courtesy of Dr. Seiich Nakata). The lingual surfaces
of the epiglottis, aryepiglottic folds, and arytenoid are edematous and
swollen (arrows). On the other hand, the preepiglottic space is intact.
hyoepiglottic ligament
Laryngeal surfaces of the epiglottis, ventricular fold, and vocal fold are
mildly inflamed
preepiglottic space
epithelium
lamina propria
of mucosa
Fig. 8.22 Histopathology of acute epiglottitis (hematoxylin and eosin

stain). (a) Mucosa of the lingual surface of the epiglottis. There is a
dense accumulation of inflammatory cells such as neutrophils beneath
the epithelium (original ×200). (b) Mucosa of the lingual surface of the
epiglottis. An acute inflammatory exudate with fibrin infiltrates the
mucosa and forms edema (original ×200). (c) Hyoepiglottic ligament
and preepiglottic space. Inflammatory exudate extends above the hyoepiglottic cartilage
epiglottic ligament. However, the ligament, dense fibrous tissue, acts as
a barrier to inflammatory exudate invasion, and the preepiglottic space
remains intact (original ×25). (d) Mucosa of the laryngeal surface of the
epiglottis. Inflammatory exudate does not penetrate the epiglottic carti-
lage posteriorly. Consequently, the mucosa of the laryngeal surface of
the epiglottis is only mildly inflamed (original ×200) along the compartment of the supraglottis in acute
epiglottitis.
glottitis) and not to the swelling of the epiglottis itself
[21]. Consequently, from the pathological point of view,
the term “acute supraglottitis” is preferable to “acute epi- 8.20.2 Subglottic Laryngitis (Croup)
glottitis.” Acute epiglottitis (supraglottitis) occurs because
of the histological structure of the anteroinferior portion The anatomical compartment of glandular and areolar tissue
of the epiglottis. Acute inflammation and edema extends below the conus elasticus and within the lumen is correlated
Subglottic swelling occurring in cases of acute subglottic

laryngitis is due to edema of this loose connective tissue
below the conus elasticus.
References
1. Pressman JJ, Dowdy A, Libby R, Fields M. Further studies upon
the submucosal compartments and lymphatics of the larynx by the
injection of dyes and radioisotopes. Ann Otol Rhinol Laryngol.
1956;65:963–80.
trachea 2. Pressman JJ, Simon MB, Monell C. Anatomical studies related
to the dissemination of cancer of the larynx. Trans Am Acad
Ophthalmol Otolaryngol. 1960;64:628–38.
3. Ridpath RF. Anatomy of the larynx. The nose, throat, and ear and
vocal fold their diseases. Philadelphia: WB Saunders Co.; 1930. p. 737.
4. Broyles EN. The anterior commissure tendon. Ann Otol.
ventricular fold
1943;52:342–5.
5. Stedman’s medical dictionary. 7th ed. Philadelphia: Wolters
Kluwer/Lippincott Williams & Wilkins; 2012. p. 1658.
Fig. 8.23 Endoscopic view of a larynx with acute subglottic laryngitis 6. Sato K. Three dimensional anatomy of the larynx: investiga-
(61-year-old female). The subglottis is swollen (arrows); however, the tion by whole organ sections. Otologia Fukuoka. 1987;33(Suppl.
vocal fold (Reinke’s space) is intact 1):153–82.
8. Olofsson J, Williams GT, Rider WD, Bryce DP. Anterior commis-
sure carcinoma. Primary treatment with radiotherapy in 57 patients.
thyroid Arch Otolaryngol. 1972;95:230–3.
cartilage 9. Isshiki N. Phonosurgery. Theory and practice. Tokyo: Springer-
Verlag Tokyo; 1989.
10. Isshiki N, Tsuji DH, Yamamoto Y, Iizuka Y. Midline lateralization
vocal fold thyroplasty for adductor spasmodic dysphonia. Ann Otol Rhinol
Laryngol. 2000;109:187–93.
11. Sato K, Matsushima K, Isshiki N, Taname M, Watanabe Y,

conus elasticus Edamatsu H. Clinical histoanatomy around anterior commissure
for type II thyroplasty success. Larynx Japan. 2014;26:1–5.
cricoid area 12. Hirano M. Phonosurgery. Basic and clinical investigation. Otologia
(Fukuoka). 1975;21(Suppl. 1):239–60.
cricoid 13. Dräger DL, Branski RC, Wree A, Sulica L. Friedrich Berthold
cartilage Reinke (1862–1919): anatomist of the vocal fold. J Voice.
2011;25:301–7.
14. Kurita S. Layer structure of the human vocal fold. Morphological
investigation. Otologia (Fukuoka). 1980;26(Suppl. 6):973–97.
15. Sato K, Hirano M, Nakashima T. Electron microscopic and immu-
Fig. 8.24 Coronal section of the human adult larynx (Elastica van nohistochemical investings of Reinke’s edema. Ann Otol Rhinol
Gieson stain). Subglottic swelling (arrows) occurring in cases of acute Laryngol. 1999;108:1068–72.
subglottic laryngitis is due to edema of the loose connective tissue 16. Tucker GF, Smith HR. A histological demonstration of the develop-
below the conus elasticus ment of laryngeal connective tissue compartments. Trans Am Acad
17. Stedman’s medical dictionary for the health professions and nurs-
ing. Illustrated 7th ed. Philadelphia, PA: Lippincott Williams &
with the clinical phenomenon described as subglottic laryn- Wilkins; 2012. p. 1559.
gitis or croup (Fig. 8.23). 18. Sato K, Kurita S, Hirano M. Location of the preepiglottic space and
its relationship to the paraglottic space. Ann Otol Rhinol Laryngol.
The cricoid area of the human larynx is a loose connective 1993;102:930–4.
tissue (areolar tissue) area in the subglottis composed of adi- 19. Sato K, Umeno T, Hirano M, Nakashima T. Cricoid area of the
pose tissue and loose elastic and collagen fibers (Fig. 8.24) larynx: its physiological and pathological significance. Acta
[19]. The cricoid area is observed to be a triangular area sur- Otolaryngol. 2002;122:882–6.
20. Kirchner JA. Atlas on the surgical anatomy of laryngeal cancer. San
rounded by the perichondrium of the cricoid cartilage (cri- Diego, CA: Singular Publishing Group Inc.; 1998.
coid arch), the conus elasticus, and the fibrous layer of the 21. Michaels L. Acute inflammation. Pathology of the larynx. Berlin:
subglottic mucosa [19]. Many blood vessels are present in Springer-Verlag; 1984. p. 68–77.
the cricoid area.
Histoanatomy of the Human Glottis
9
Abstract
1. The human glottis consists of two parts, the intermembranous portion (anterior glottis)
and intercartilaginous portion (posterior glottis). The border of the two parts is defined
by a line between the tips of the bilateral vocal processes.
2. The anterior glottis plays the most important role in phonation and is covered with
stratified squamous epithelium. On the other hand, the posterior glottis appears to have
an equally important role in respiration and is covered with respiratory epithelium
(pseudostratified ciliated epithelium).
3. The posterior glottis is a respiratory glottis, while the anterior glottis a phonatory
glottis.
4. In adults, the area of the posterior glottis occupies approximately 50–60% of the entire
glottic area.
5. The absolute values of the length and area ratios of the newborn posterior glottis are
larger than those of the adult. The epithelium in the newborn posterior glottis is also a
respiratory epithelium (pseudostratified ciliated epithelium), whereas it is stratified
squamous epithelium in the anterior glottis.
6. The newborn posterior glottis occupies approximately 70% of the entire glottic area.
The newborn glottis appears to be favored for respiration over phonation.
7. One reason why prolonged intubation is somewhat better tolerated in infants than adults
is postulated to be the dimensions of the infant larynx. There is no correlation between
the degree of laryngeal injury and the weight at birth.
8. Elastic cartilage is distributed not only at the tip of the vocal process but also at the
superior portion of the arytenoid cartilage from the vocal process to the apex.
9. The vocal process bends at the elastic cartilage portion during adduction and abduction,
and bilateral arytenoid cartilages come into contact mainly at the elastic cartilage
portion.
10. The posterior glottis closes completely at the level of the supraglottis (the tip of the
vocal process and the superior portion of the arytenoid cartilage from the vocal process
to the apex). The epithelium at the contact area is stratified squamous epithelium.
11. The degree of vocal fold approximation can be affected by age-related changes of the
cricoarytenoid joint.
9.1 Introduction two parts is defined by a line between the tips of the bilateral
vocal processes [2].
The human glottis consists of two parts, the intermembra- The anterior glottis plays the most important role in pho-
nous portion (anterior glottis) and intercartilaginous portion nation. Thus, voice disorders are usually caused by lesions of
(posterior glottis) (Figs. 9.1 and 9.2) [1]. The border of the the anterior glottis. The anterior glottis is covered with

90 9 Histoanatomy of the Human Glottis
Fig. 9.1 The structure around thyroid cartilage

the human adult glottis (from anterior commissure tendon
above)
anterior glottis vocal ligament

(intermembranous Reinke’s space
portion)
posterior glottis vocal process

(intercartilaginous (arytenoid cartilage)
portion)
Fig. 9.2 The structure around posterior end of laryngeal ventricle

the human adult glottis (from
the side) tip of vocal process
membranous portion of vocal fold
anterior commissure cartilaginous portion of vocal fold
lateral wall of posterior glottis
posterior wall of glottis
stratified squamous epithelium (Fig. 9.3a). On the other Where is the posterior end of the vocal fold? No defini-
hand, the posterior glottis appears to have an equally impor- tion had been given to the cartilaginous portion of the
tant role in respiration and is covered with respiratory epithe- vocal fold until Hirano and Kurita proposed to define it as
lium (pseudostratified ciliated epithelium) (Fig. 9.3b) [1]. the foldlike structure between the tip of the vocal pro-
In anatomy, the vocal fold is defined as the structure cesses and the posterior end of the laryngeal ventricle
between the anterior commissure and the tip of the vocal pro- (Fig. 9.2) [3].
cesses (Fig. 9.2). No cartilaginous structure is included in the Moreover, there is considerable disagreement about the
vocal fold. However, many clinicians often use such mis- terminology used to refer to some structures around the pos-
leading terms as cartilaginous portion of the vocal fold and terior glottis [1], while yet other structures have not been
posterior glottis. named, identified, or described.
9.1 Introduction 91
Fig. 9.3 The epithelium of

a
the adult glottis (hematoxylin
and eosin stain). (a) The
anterior glottis is covered with
stratified squamous stratified squamous epithelium
epithelium. (b) The posterior
glottis is covered with
respiratory epithelium
(pseudostratified ciliated
epithelium)
lamina propria of mucosa
b
pseudostratified ciliated epithelium

9.2 he Structures Around the Human

T 9.3 he Dimensions and Morphological
T
Adult Glottis Characteristics of the Human Adult
Glottis
The anterior glottis is triangular and the posterior glottis is
trapezoidal in shape (Fig. 9.1). Dimensions of the adult glottis (neutral condition) such as
The anatomical term “commissure” refers to a junction in length and area have been measured (Figs. 9.4 and 9.5) [1].
the anatomy, a site where two things join. Regarding the The average length of the glottis (Fig. 9.4, Lag + Lpg) is
anterior glottis (intermembranous portion), the anterior com- 24.5 ± 1.9 (average ± SD) mm in males and 16.3 ± 1.4 mm in
missure is a junction at which the bilateral vocal folds join. females [1]. The average length of the posterior glottis
Regarding the posterior glottis (intercartilaginous portion), (Fig. 9.4, Lpg) is 9.5 ± 0.9 mm in males and 6.8 ± 0.9 mm in
since the bilateral vocal folds never join at their posterior females [1]. It accounts for 38.6 ± 2.0 (average ± SD) % of the
ends, the term posterior commissure is not proper as an ana- entire glottic length in males and 41.6 ± 4.2% in females [1].
tomical term. The length of the posterior glottis and the total length of the
The posterior aspect of the glottis is a wall; therefore, Dr. glottis differ significantly between the two sexes. However, the
Hirano has proposed the term “posterior wall of the glottis” ratio of the length of the posterior glottis to the total length of
(Fig. 9.2) [1]. The base of this structure is the upper portion the glottis does not differ significantly between the two sexes.
of the lamina of the cricoid cartilage. The average area of the glottis (Fig. 9.4, Aag + Apg) is
The lateral aspects of the posterior glottis can be divided 79.6 ± 23.2 mm2 in males and 51.9 ± 15.6 mm2 in females
into two portions. The border between the two portions is [1]. The average area of the posterior glottis (Fig. 9.4, Apg)
located at the posterior end of the laryngeal ventricle. The is 44.6 ± 13.3 mm2 in males and 31.0 ± 9.7 mm2 in females
posterior part of the lateral aspect of the posterior glottis is [1]. It accounts for 56.5 ± 5.6% of the entire glottic area in
also a wall; therefore, Dr. Hirano has proposed the term “lat- males and 59.8 ± 3.7% in females [1]. The area of the poste-
eral walls of the posterior glottis” (Fig. 9.2) [1]. The base of rior glottis and the total area of the glottis differ significantly
this structure is the medial facet of the arytenoid cartilage. between the two sexes. However, the ratio of the area of the
“Cartilaginous portion of the vocal fold” has been used in posterior glottis to the total area of the glottis, like the ratio
laryngology without precise definition. In anatomy, the vocal of the length, does not differ significantly between the two
fold is defined as the structure which extends from the ante- sexes.
rior commissure to the tip of the vocal process; hence, no The length of the posterior glottis (Lpg on Fig. 9.4)
cartilaginous structure is included in this definition. The accounts for approximately 35–45% of the entire glottic
anterior portion of the lateral aspect of the posterior glottis length (Lag + Lpg) [1]. The area of the posterior glottis
assumes a liplike or foldlike shape similar to the membranes (Apg) occupies approximately 50–65% of the entire glottic
portion of the vocal fold. Therefore, Dr. Hirano has proposed area (Aag + Apg) [1]. This indicates that more respiratory air
the term “cartilaginous portion of the vocal fold” to call this passes through the posterior glottis than through the anterior
portion (Fig. 9.2) [1]. The base of this structure is the vocal glottis, which very likely means that the main role of the
process. posterior glottis is to act as an airway [1].
intermembranous
portion Lag Aag anterior glottis
intercartilaginous
portion Lpg Apg posterior glottis
Lag: length of anterior glottis Aag: area of anterior glottis

Lpg: length of posterior glottis Apg: area of posterior glottis
Fig. 9.4 Dimensions of the Lg: length of glottis (Lag+Lpg) Ag: area of glottis (Aag+Apg)
human glottis
9.4 The Dimensions and Morphological Characteristics of the Human Newborn Glottis 93
Fig. 9.5 The proportional a b

dimensions of the newborn p<0.01 p<0.01
and adult posterior glottis. (a) 70 80
The absolute values of the
length of the posterior glottis.
(b) The absolute values of the 60 70
area of the posterior glottis
60
50
50
40
40
30
30
20
20
10
10
0 0
Newborn Adult Newborn Adult
9.4 he Dimensions and Morphological

T between the two sexes. Additionally, the ratio of the
Characteristics of the Human length of the posterior glottis to the total length of the
Newborn Glottis glottis does not differ significantly between the two
sexes.
Human adult vocal folds have a layered structure with a The average area of the glottis (Fig. 9.4, Aag + Apg) is
vocal ligament [2, 4]. This structure is characterized by the 7.5 ± 2.5 mm2 in males and 8.2 ± 1.7 mm2 in females [7]. The
differences of extracellular matrix distribution and is essen- average area of the posterior glottis (Fig. 9.4, Apg) is
tial for vocal fold vibration and phonation. On the other 5.3 ± 2.0 mm2 in males and 5.5 ± 1.4 mm2 in females [7]. It
hand, at birth, there is no structure corresponding to the vocal accounts for 69.2 ± 4.6% of the entire glottic area in males
ligament and no layered structure like that found in adult and 67.3 ± 5.8% in females [7]. It also accounts for
vocal folds [2, 5, 6]. Development of the vocal ligament and 68.0 ± 5.3% of the entire glottic area in both sexes [7]. The
layered structure of the vocal fold is complete by the end of area of the posterior glottis and the total area of the glottis do
adolescence [6]. Dimensions and morphological characteris- not differ significantly between the two sexes. Additionally,
tics of the human newborn glottis are different from those of the ratio of the area of the posterior glottis to the total area of
the adult. the glottis does not differ significantly between the two
Dimensions of the newborn glottis (cadaveric position) sexes.
such as length and area of the newborn glottis have been The proportional dimensions of the newborn and adult
measured (Figs. 9.4 and 9.5) [7]. glottis have been statistically compared using our data [1, 7].
The average length of the glottis (Fig. 9.4, Lag + Lpg) When we compare the proportional dimensions of the new-
is 5.4 ± 0.6 (average ± SD) mm in males and 5.7 ± 0.4 mm born and adult glottis, the proportional length and area of the
in females [7]. The average length of the posterior glottis newborn posterior glottis are statistically larger than that of
(Fig. 9.4, Lpg) is 3.1 ± 0.5 mm in males and 3.2 ± 0.4 mm the adult (Figs. 9.5 and 9.6).
in females [7]. It accounts for 57.5 ± 3.2% of the entire The epithelium in the newborn anterior glottis is stratified
glottic length in males and 55.5 ± 5.3% in females [7]. It squamous epithelium (Fig. 9.7a), whereas it is respiratory
also accounts for 56.3 ± 4.6% of the entire glottic length epithelium (pseudostratified ciliated epithelium) in the pos-
in both sexes [7]. The length of the posterior glottis and terior glottis (Fig. 9.7b). There is no histologically signifi-
the total length of the glottis do not differ significantly cant difference between the genders.
Fig. 9.6 Glottis of the

newborn (Elastica van Gieson
stain). The newborn posterior
glottis occupies
approximately 70% of the
entire glottic area in the anterior macula flava
cadaveric position, and the
anterior glottis
ratio is larger compared with
that of the adult posterior macula flava
vocal process
(arytenoid cartilage) posterior glottis
9.5 he Functions of the Human Newborn

T the vocal fold stellate cells in the maculae flavae of the
Glottis human vocal fold [5, 8–12].
There is a growing evidence to suggest that the cells includ-
In order of functional priority, the functions of the human ing the vocal fold stellate cells in the human maculae flavae
larynx are (1) protection of the lower airway, (2) respiration, are tissue stem cells or progenitor cells in the human vocal fold
and (3) phonation. The larynx must immediately serve as mucosa [13–15]. There is also a growing evidence to suggest
both an airway and protector of the lower airway during that the human maculae flavae located at both ends of the
swallowing at birth. vocal fold mucosa are a candidate for a stem cell niche, which
The newborn posterior glottis occupies approximately is a microenvironment nurturing a pool of stem cells [13–15].
70% of the entire glottic area in cadaveric position (Fig. 9.6) Current scientific findings suggest that the magnitude and
and was covered with the respiratory epithelium (Fig. 9.7b). frequency of tensile strain are particularly important in deter-
It is extremely likely that the posterior glottis becomes larger mining the type of mechanically induced differentiation that
during respiration (vocal fold abduction). Consequently, the stem cells will undergo [16]. The newborn anterior glottis occu-
newborn glottis appears to be favored for respiration over pies approximately 30% of the entire glottic area and is covered
phonation. with squamous epithelium. Consequently, the newborn glottis
Our previous studies have supported the hypothesis that appears to be favored for respiration over phonation. However,
the tension caused by phonation (vocal fold vibration) after short vocal folds undergo a greater magnitude and frequency of
birth stimulates vocal fold stellate cells in the anterior and tensile strain during vocal fold vibration such as crying and pho-
posterior maculae flavae to accelerate production of extracel- nation. Mechanotransduction of the vocal fold stellate cells in
lular matrices and form the vocal ligament, Reinke’s space the newborn maculae flavae caused by vocal fold vibration is
and the characteristic layered structure [5, 8–12]. The ten- probably an important factor in the growth and development of
sion caused by phonation seems to regulate the behavior of the human vocal fold mucosa (Fig. 9.8).
9.5 The Functions of the Human Newborn Glottis 95
Fig. 9.7 The epithelium of

the newborn glottis
a
(a) The newborn anterior
glottis is covered with
stratified squamous
epithelium. (b) The newborn stratified squamous epithelium
posterior glottis is covered
with respiratory epithelium
(pseudostratified ciliated
epithelium)

9.6 he Clinical Significance

T
of the Dimensions of the Human
Glottis
Posterior glottis Arytenoid Common criteria employed to select the size of an endotracheal
tube are age, height, weight, and diameter of the trachea.
Anterior glottis Anesthesiologists usually select an appropriate endotracheal
tube on the basis of the tracheal diameter appearing on chest
X-rays and select an endotracheal tube whose size is close to that
of the trachea. Since the narrowest portion of the upper airway is
the larynx, post-intubation complications occur in this area.
The mean diameter of the glottis (G on Fig. 9.9) is
4.3 ± 1.0 (average ± SD) mm in adult males, 3.9 ± 0.4 mm in
adult females, 3.0 ± 0.6 mm in newborn males, and
2.3 ± 0.1 mm in newborn females [17, 18]. Thus, the width
of the glottis is largest in adult males than adult females,
newborn males, and newborn females [17, 18].
Epiglottis
The mean diameter of the subglottis (SG on Fig. 9.9) is
13.7 ± 2.2 mm in adult males, 9.0 ± 1.4 mm in adult females,
4.0 ± 0.5 mm in newborn males, and 3.9 ± 0.1 mm in new-
Fig. 9.8 Child’s glottis viewed from above (3-year-old male). The area born females [17, 18]. The diameter of the subglottis of adult
of the intercartilaginous portion (posterior glottis) relative to the entire males is larger than that of adult females (p < 0.05), whereas
glottic area is greater than in adults the difference in newborns is negligible (p < 0.05) [17, 18].
Fig. 9.9 Diameter of the

glottis, subglottis, and trachea
glottic level
subglottic level
SG
tracheal level
T
9.7 Prolonged Endotracheal Intubation in Infants and Adults 97
Fig. 9.10 (a) The ratio of a b

subglottic to tracheal
diameter. (b) The ratio of 1.0 1.0
glottic to tracheal diameter
0.9
0.8
0.7
0.5
0.6
0.5
~
~
0 0
M F M F M F M F
M : male
F : female newborns adults M : male
newborns adults
F : female
The mean diameter of the trachea (T on Fig. 9.9) is 9.7.1 Histopathologic Changes

15.0 ± 1.5 mm in adult males, 10.2 ± 1.3 mm in adult females, in the Laryngeal Mucosa of Low-Birth-
4.3 ± 0.6 mm in newborn males, and 4.3 ± 0.3 mm in new- Weight Infants After Endotracheal
born females [17, 18]. The tracheal diameter is close to that Intubation
of the subglottis (p < 0.05) [17, 18].
The ratio of subglottic to tracheal diameter does not differ The development of modern medicine has improved the sur-
between adults and newborns (p < 0.05) (Fig. 9.10a) [17, vival of babies with low gestational ages and birth weights.
18]. The diameter of the subglottis is 80–100% of the tra- Advances in neonatology have improved the survival rates of
cheal diameter. The ratio of the diameter of the glottis to that preterm and critically ill newborns. In the neonatal intensive
of the trachea is larger in newborns than in adults (p < 0.05) care unit (NICU), newborns with extremely low (less than
(Fig. 9.10b). In adults, particularly in males, the glottis is 1000 g in weight) and very low (less than 1500 g in weight)
much narrower than the trachea (Fig. 9.10b) [17, 18]. birth weights undergo intensive care with endotracheal
In adults, particularly in males, the glottis is narrower intubation. Consequently, there is an increasing concern

than the trachea even though the diameter of the glottis regarding complications, such as subglottic stenosis, in new-
increases by 11% in males and by 5% in females when the borns subjected to prolonged intubation.
vocal folds are abducted [19]. To avoid laryngeal injury, we There have been several studies of intubation-related
should be careful not to select an endotracheal tube with a laryngeal injury in infants [20, 22–28]. However, histopatho-
diameter too close to that of the trachea. logic investigations of the laryngeal mucosa and cartilage in
preterm or extremely immature infants, including extremely
low-birth-weight and very low-birth-weight infants, after
9.7 rolonged Endotracheal Intubation
P endotracheal intubation have been insufficient. Furthermore,
in Infants and Adults whether the potential hazards of intubation-related laryngeal
injury in extremely low-birth-weight infants (less than
The endotracheal tube is commonly inserted into the trachea via 1000 g) are greater than those in infants of other birth weights
the posterior glottis (intercartilaginous portion) [20]. The area of has been controversial.
the newborn posterior glottis (intercartilaginous portion) rela- The histopathologic changes in the laryngeal mucosa of
tive to the entire glottic area is greater than that of adults. In preterm or extremely immature infants, including extremely
newborns, the diameter of the posterior glottis is closer to that of low- and very low-birth-weight infants, after endotracheal
the trachea than in adults [17, 18]. Therefore, insertion into the intubation have been assessed [21].
trachea via the posterior glottis of a non-cuffed endotracheal A descriptive classification of the degree of laryngeal injury
tube similar in size to the trachea is relatively easy. has been devised to evaluate the correlation between the degree
The difference in the three-dimensional laryngeal struc- of laryngeal injury and birth weight. The classification is based
ture between newborns and adults is one reason that p rolonged on the following grades: Grade 0, no injury; Grade 1, injury of
intubation is better tolerated in infants than in adults and why the epithelium; Grade 2, injury of the superficial lamina propria
subglottic injury is relatively severe in infants [21]. of mucosa; Grade 3, injury of the deep lamina propria of
mucosa without cartilage exposure; and Grade 4, injury of the and the supraglottic level. The degree of injury is greater at
deep propria mucosa with cartilage exposure (Fig. 9.11). the subglottis beneath the arytenoid cartilage and posterior
glottis compared with the degree of injury of other laryngeal
regions (Fig. 9.11). The lesions at the glottic level are focal,
9.7.2 H
istopathologic Changes of Infant whereas at the subglottis, they are usually more extensive.
Larynges After Intubation
(a) Ten minutes to twelve hours of continuous intubation.
Significant injuries are confined to the lateral wall of the pos-
terior glottis at the glottic level and lateral and posterior The injured portions have complete or partial loss of epi-
aspects of the subglottis beneath the arytenoid cartilage. In thelium. The focal loss of mucosal epithelium is noted after
some cases, all of the aspects are injured at the cricoid ring. just 10 min intubation (Fig. 9.12a). The lamina propria of the
There is slight injury to the anterior glottis at the glottic level mucosa is not disrupted or injured. The injured portion at the
Fig. 9.11 Correlation a
between degree of laryngeal Degree of injury
injury and birth weight. (a) At
subglottis, (b) at posterior
glottis. Grade 0, no injury; 4
2,500 g to 3,160 g (n=7)
Grade 1, injury of epithelium;
1,500 g to less than 2,500 g (n=10)
Grade 2, injury of superficial
lamina propria of mucosa; 1,000 g to less than 1,500 g (n=6)
Grade 3, injury of deep less than 1,000 g (n=21)
3
without cartilage exposure;
Grade 4, injury of deep
with cartilage exposure 2
0.1 1 10 100 1000

Duration of Intubation (hours)
b
Degree of injury
4 2,500 g to 3,160 g (n=7)

1,500 g to less than 2,500 g (n=10)
1,000 g to less than 1,500 g (n=6)
3 less than 1,000 g (n=21)
0.1 1 10 100 1000

Duration of Intubation (hours)
complete loss of epithelium
necrotic lamina
propria of mucosa
Fig. 9.12 (a) The injured portions have complete or partial loss of epi- tional age, 31 weeks; hematoxylin and eosin stain, original ×50). (e)
thelium after 10 min. of continuous intubation (birth weight, 2976 g; Perichondrium of the cricoid lamina is exposed after 14 days and 12 h
gestational age, 39 weeks; hematoxylin and eosin stain, original ×100). of continuous intubation (birth weight, 830 g; gestational age, 26
(b) Superficial subglottic stroma of lamina propria is necrotic with weeks; hematoxylin and eosin stain, original ×20). (f) Regenerated
minimal inflammatory response after 18 h of continuous intubation stratified squamous epithelium covers fibrous mucosa after 24 days of
(birth weight, 2055 g; gestational age, 34 weeks; hematoxylin and eosin continuous intubation (birth weight, 572 g; gestational age, 23 weeks;
stain, original ×100). (c) One-third of the lamina propria of the subglot- hematoxylin and eosin stain, original ×50). (g) Transverse section of the
tic mucosa is ulcerated after 52 h of continuous intubation (birth weight, cricoid cartilage after 102 days of continuous intubation (birth weight,
1544 g; gestational age, 39 weeks; hematoxylin and eosin stain, original 1890 g; gestational age, 39 weeks; hematoxylin and eosin stain). (h)
×50). (d) Broad and deep ulcer is present on all sides of subglottis after Healing ulcer extends into cricoid cartilage and cricoid lamina is exca-
4 days and 11 h of continuous intubation (birth weight, 1420 g; gesta- vated (region H in g, hematoxylin and eosin stain, original ×20)
c
ulcerated mucosa
bacterial colony
ulcerated mucosa
subglottis is beneath the posterior glottis and, less often, the (c) Twenty-four to forty-eight hours of continuous intubation.
anterior glottis. Little inflammatory response is present in the
lamina propria of the mucosa. The superficial stroma of the lamina propria of the mucosa
is necrotic in many cases. A strong inflammatory response
(b) Twelve to twenty-four hours of continuous intubation. characterized by congestion, hemorrhage, and/or infiltration
of cells is present in some cases. The injured portion of the
The injured portion has a complete loss of epithelium in all larynx is the same as mentioned above.
cases, and the superficial stroma of the lamina propria of the
mucosa becomes necrotic in some cases (Fig. 9.12b). Little (d) Forty-eight to ninety-six hours (4 days) of continuous
inflammation response is present in the subjacent stroma. intubation.
e
exposed cricoid lamina
f regenerated stratified
squamous epithelium
fibrous mucosa
No epithelium or basement membrane remains, and the (f) Seven to thirty days of continuous intubation.
superficial stroma of the lamina propria is necrotic in most
cases, and mucosal hemorrhage and inflammatory response Ulceration of the mucosa becomes deeper, and the peri-
are observed (Fig. 9.12c). chondrium of the cartilage is often exposed after 8 days
(Fig. 9.12e). In larynges intubated for extended duration,
(e) Four to seven days of continuous intubation. fibrosis is found just beneath the intact epithelium. In cases,
which are intubated more than 20 days, regenerated stratified
As the duration of intubation increases, ulceration of the squamous epithelium covers healed ulcers (Fig. 9.12f). Not
mucosa is found to be broader and deeper (Fig. 9.12d). A only injury but also the healing process occurs in these cases
strong inflammatory response is present at the ulcer. with long-term intubation.
g thyroid cartilage
arch of cricoid cartilage
fibrous mucosa
(g) Thirty to one hundred thirty-eight days of continuous healing ulcers extend into the cricoid cartilage and the cartilagi-
intubation. nous rim of the cricoid lamina is excavated (Fig. 9.12g and h).
The perichondrium of the cricoid lamina is exposed in some

cases, and the perichondrium of the arytenoids cartilage is also 9.7.3 C
orrelation Between Duration
exposed in some cases. Some cases show bacterial infestation in of Intubation, Degree of Laryngeal
the ulcerated sites. Regenerated stratified squamous epithelium Injury, and Weight at Birth
covers healing ulcers and the exposed perichondrium of the car-
tilage. Not only injury but also the healing process occurs in Clinically, it is commonly accepted that prolonged intubation
most of these cases with long-term intubation. Occasionally, is somewhat better tolerated in infants than adults [20, 21].
9.8 Histoanatomy of the Arytenoid Cartilage 103
One factor that must contribute to the neonate’s tolerance the degree of laryngeal injury and the weight at birth [21].
of intubation is the relative immaturity of their laryngeal car- Additionally, not only injury but also the healing process
tilage and its associated plasticity [22]. The laryngeal carti- occurs in cases of long-term intubation, even in extremely
lage in the neonate, which is hypercellular with a scant low-birth-weight and very low-birth-weight infants [21].
gel-like matrix, is a pliable substance [22]. With growth as The development of intubation-related late complications
matrices increase, it becomes less hydrated, more fibrous, such as subglottic stenosis occurs after extubation. The
and more rigid [22]. This may be one of the reasons there is dimensions of the larynx are small and the structure is frail in
no correlation between the degree of laryngeal injury and the extremely low-birth-weight and very low-birth-weight
weight at birth in our study. infants. Consequently, a small amount of granulation and
Another reason for the neonate’s tolerance of intubation is scar tissue formation is critical for the airway of extremely
postulated to be the dimensions of the infant larynx, which is low-birth-weight and very low-birth-weight infants.
based on the fact that subglottic injury is greatest in infants. Therefore, other risk factors should be minimized. The
In newborns, the area of the intercartilaginous portion (pos- risk factors implicated in the development of granulation and
terior glottis) relative to the entire glottic area is greater than scar tissue formation, such as infection and laryngopharyn-
in adults [7]. In a newborn, the diameter of the posterior glot- geal reflux, should be reduced. And factors that promote the
tis is closer to the trachea than in an adult [17]. healing process, such as antibiotics for preventing infection
When the degrees of injury in each region are compared, and steroids preventing cicatrices, should be applied.
the injuries in the subglottic region beneath the arytenoid Minimizing other risk factors plays an important role in
cartilage and in the posterior glottis are found to be greatest reducing complications that arise at the injured sites after
in infants (Fig. 9.11). There are few injuries to the anterior long-term intubation in extremely low- and very low-birth-
glottis and supraglottis. weight infants [21].
No correlation between the sex of the infants and laryngeal
damage is found [21]. There is no correlation between the
degree of laryngeal injury and the weight at birth (extremely 9.8 istoanatomy of the Arytenoid
H
low-birth-weight infants, very low-birth-weight infants, low- Cartilage
birth-weight infants, and mature infants) (Fig. 9.11) [21].
The duration of intubation is the most important factor The basic functions of the larynx are to act as a protective sphinc-
related to laryngeal injury. As the duration of intubation ter and as a passageway for air and to act in sound production.
increases, the degree of laryngeal injury also increases During swallowing, respiration, and phonation, the vocal pro-
(Fig. 9.11). While prolonged intubation in neonates can be cesses always move and form the shape of the glottis (Fig. 9.13).
measured in weeks, in adults it should be measured in days Among the laryngeal cartilages, only the arytenoid
[22]. Even though it is commonly accepted that prolonged cartilage is composed of two types of cartilages, i.e.,
intubation is better tolerated in infants than adults, there is no elastic cartilage and hyaline cartilage. These two types of
consensus regarding the limits for safe periods of intubation. cartilage have very different properties, and so the distri-
Strong and Passy reported that after 10 days, the fre- bution of elastic and hyaline cartilages in the arytenoid
quency of complications in neonate endotracheal intubation cartilage and its possible functional significance are of
rises [23]. Dankle et al. reported that the risk of developing interest.
subglottic stenosis increases after 50 days of intubation [29].
In addition, whether the potential risk of intubation-related
laryngeal injury of extremely low-birth-weight (less than 9.8.1 D
istribution of Elastic and Hyaline
1000 g in weight) and very low-birth-weight (less than Cartilages in the Arytenoid Cartilage
1500 g in weight) infants is higher than that of infants of of Adult Larynges
other weights has been controversial [29].
Many risk factors, such as duration of intubation, low birth The posterior macula flava is observed at the posterior end of
weight, and endotracheal tube size, have been reported as the membranous portion of the adult vocal fold. Posterior to
being involved in the development of intubation-related injury it, there is the tip of the vocal process.
[20, 29]. Regarding birth weight, low birth weight (≦1500 g) In a transverse section of the vocal process of the adult
is reported to be a very important determinant of susceptibility arytenoid cartilage (Fig. 9.14), the chondrocytes and ground
to acquired subglottic stenosis [29]. However, there have been substance (elastic fibers) reveal that the tip of the vocal pro-
few reports regarding histopathological investigations of cess is composed of elastic cartilage (Fig. 9.14b). The num-
laryngeal injury of extremely low-birth-weight and very low- ber of elastic fibers decreases toward the posterior portion of
birth-weight infants. The histopathologic investigation which the vocal process (Fig. 9.14c). More posteriorly, the chon-
has been reported shows that there is no correlation between drocytes and ground substance indicate that this portion is
composed of hyaline cartilage (Fig. 9.14d). The transition are followed posteriorly, the superior portion of the arytenoid
between the elastic cartilage portion and hyaline cartilage cartilage is composed of elastic cartilage, and the inferior por-
portion is gradual, and the border between them is not clearly tion of it is composed of hyaline cartilage (Fig. 9.15b–d).
delineated. More posteriorly, only a small part of the apex is composed of
In a coronal section of the adult arytenoid cartilage elastic cartilage (Fig. 9.15e). As stated previously, the transi-
(Fig. 9.15), the tip of the vocal process is elliptic in shape and tion between the elastic and hyaline cartilage portions is grad-
composed only of elastic cartilage (Fig. 9.15a). As the sections ual, and the border between them is not clearly delineated.
Fig. 9.13 Vocal process of the arytenoid

cartilage. (a) Lateral view, (b) superior view, a
(c) frontal view
apex
vocal process of
arytenoid cartilage muscular process of
arytenoid cartilage
cricoid cartilage
b
cricoid arch
vocal process of
arytenoid cartilage
oblong fovea apex
triangular fovea
muscular process of
arytenoid cartilage
cricoid
lamina
apex
triangular fovea
oblong fovea vocal process of

arytenoid cartilage
muscular process of
arytenoid cartilage
cricoid lamina
cricoid arch
The tip of the vocal process and the superior portion of the sections are followed posteriorly, the superior portion of
the adult arytenoid cartilage are composed of elastic carti- the arytenoid cartilage is composed of elastic cartilage, and
lage (Fig. 9.16). In cases of arytenoid cartilage ossification, the inferior portion of it is composed of hyaline cartilage
the hyaline cartilage portion ossifies, but the elastic cartilage (Fig. 9.18b).
portion does not ossify. Similar to the adult arytenoid cartilage, the tip of the vocal
process and the superior portion of the arytenoid cartilage
are composed of elastic cartilage at birth. The transition
9.8.2 D
istribution of Elastic and Hyaline between the elastic and hyaline cartilage portions is gradual,
Cartilages in the Arytenoid Cartilage and the border between them is not clearly delineated.
of Newborn Larynges
Posterior to the posterior macula flava, there is the tip of the 9.8.3 B
ehavior of the Elastic Cartilage
vocal process. Portion of the Arytenoid Cartilage
In a transverse section of the vocal process of the newborn During Abduction and Adduction
arytenoid cartilage (Fig. 9.17), the chondrocytes and ground
substance (elastic fibers) reveal that the tip of the vocal process A transverse section of the vocal process during abduction
is also composed of elastic cartilage (Fig. 9.17b). The number shows that the vocal process bends concavely at the elastic
of elastic fibers decreases toward the posterior portion of the cartilage portion during abduction (Fig. 9.19).
vocal process (Fig. 9.17c). More posteriorly, the chondrocytes On the other hand, the transverse section of the vocal pro-
and ground substance indicate that this portion is composed of cess during adduction shows that the vocal process bends
hyaline cartilage (Fig. 9.17d). The transition between the elas- convexly at the elastic cartilage portion during adduction
tic cartilage portion and hyaline cartilage portion is gradual, (Fig. 9.20).
and the border between them is not clearly delineated. The coronal section of the vocal process during adduction
Coronal sections of the newborn arytenoid cartilage shows that the superior portion of the vocal process (the elas-
(Fig. 9.18) show that the tip of the vocal process is elliptic in tic cartilage portion) protrudes more medially than the infe-
shape and composed only of elastic cartilage (Fig. 9.18a). As rior portion of the vocal process (the hyaline cartilage portion)
hyaline cartilage portion
elastic cartilage portion

C
vocal process
chondrocytes of
elastic cartilage
elastic fibers
Fig. 9.14 (a) Transverse section of the vocal process of the adult ary- sition between the elastic and hyaline cartilage portions is gradual and
tenoid cartilage (Elastica van Gieson stain). (b) The tip of the vocal the border between them is not clearly delineated. (d) Hyaline cartilage
process is composed of elastic cartilage. (c) Transition area between portion of the vocal process
elastic cartilage and hyaline cartilage portions (region C in a). The tran-
hyaline cartilage
elastic cartilage
cartilage lacuna
chondrocytes of
hyaline cartilage
a
tip of
vocal process
elastic
cartilage
thyroarytenoid
Muscle
elastic
cartilage
thyroarytenoid hyaline
Muscle cartilage
Fig. 9.15 Coronal sections of the vocal process of the adult arytenoid cartilage (Elastica van Gieson stain). The tip of the vocal process (a) and
the sections are followed posteriorly from (a) to (e)
(Fig. 9.21). The bilateral arytenoid cartilages come into con- gradual, and the border between them is not clearly delin-
tact mainly at their superior (elastic cartilage) portions. eated [30]. This phenomenon is observed at birth [30].
The vocal process bends at the elastic cartilage portion
during adduction and abduction, and each side of arytenoid
9.9 istribution of Elastic Cartilage
D cartilages comes into contact mainly at the elastic cartilage
in the Arytenoid Cartilage and Its portion (Fig. 9.22) [30, 31]. The epithelium at the contact
Physiologic Significance area (the tip of the vocal process and the superior portion of
the arytenoid cartilage from the vocal process to the apex) is
Elastic cartilage is distributed not only at the tip of the vocal covered with stratified squamous epithelium both in adults
process but also at the superior portion of the arytenoid car- and newborns (Fig. 9.23) [32].
tilage from the vocal process to the apex (Fig. 9.16) [30]. The Elastic and hyaline cartilages have very different quali-
transition between elastic and hyaline cartilage portions is ties. Consequently, their relative distributions in the aryte-
9.9 Distribution of Elastic Cartilage in the Arytenoid Cartilage and Its Physiologic Significance 109
elastic cartilage
thyroarytenoid
Muscle hyaline cartilage
elastic
cartilage
thyroarytenoid
Muscle
hyaline
cartilage
apex
elastic
cartilage
hyaline
cartilage
Fig. 9.16 Schema of elastic

Ap
cartilage portion of right
arytenoid cartilage. VP vocal
process; MP muscular elastic cartilage
e
process; OF oblong fovea; TF
triangular fovea; Ap apex of hyaline cartilage d
arytenoid cartilage TF
thyroarytenoid muscle c
stratifed squamous epithelium

b
a
MP
VP
OF
e d c b a
noid cartilage are of interest in relation to their physiologic 9.10 U

ltrastructure of the Vocal Process
significance. of the Arytenoid Cartilage
Hyaline cartilage, such as the thyroid or cricoid carti-
lage, is firm and plays a role as a framework for the larynx. As mentioned above, the vocal process, when observed by
Thus, the hyaline cartilage portion of the arytenoid carti- light microscopy, reveals that the elastic cartilage portion of
lage is considered to function as a framework for the poste- the vocal process plays an important role in the physiologic
rior glottis. function of the arytenoid cartilage [30, 31].
Elastic cartilage, such as epiglottic cartilage, is soft and Electron micrography shows that the elastic and hyaline
pliable. The tip of the vocal process bends at the elastic car- cartilages of the vocal process are composed of chondro-
tilage portion during adduction and abduction [30, 31]. The cytes, collagen fibers, elastic fibers, and ground substance
arytenoid cartilages come into contact mainly at their supe- (Fig. 9.24).
rior portions, i.e., the elastic cartilage portions. From the
physiologic point of view, the elastic cartilage located at the
tip of the vocal process appears to facilitate movement of the 9.10.1 Chondrocytes of the Vocal Process
vocal process during adduction and abduction, and the elas-
tic cartilage located at the superior portion of the arytenoid The chondrocytes are relatively small in size at the tip of the
cartilage appears to act as a cushion whose purpose is to pro- vocal process (the elastic cartilage portion of the vocal pro-
tect the arytenoid cartilage and covering mucosa from cess) and have small cartilage lacunae (Fig. 9.24a). However,
mechanical damage caused when the bilateral arytenoids the density of chondrocytes is high at these portions, and
come into contact [30]. Furthermore, the elastic cartilage they synthesize fibrous proteins and ground substance.
never ossifies; hence, the elastic cartilage portion of the The size of chondrocytes increases and their density
arytenoid cartilages is able to perform its role throughout an decreases toward the posterior portion of the vocal process.
individual’s lifetime. The chondrocytes are relatively large in size at the posterior
In these ways, the elastic cartilage portion in the arytenoid portion of the vocal process (the hyaline cartilage portion of
cartilage appears to play an important role in the physiologic the vocal process) and have large cartilage lacunae (Fig. 9.24b).
function of the arytenoids. The density of chondrocytes is low in these portions.
9.10 Ultrastructure of the Vocal Process of the Arytenoid Cartilage 111
a b

chondrocytes of
posterior elastic cartilage
macula flava
C
elastic fibers
vocal process
c d
hyaline cartilage
cartilage lacuna
chondrocytes of
elastic cartilage
hyaline cartilage
Fig. 9.17 (a) Transverse section of the vocal process of the newborn The transition between the elastic and hyaline cartilage portions is grad-
arytenoid cartilage (Elastica van Gieson stain). (b) Tip of the newborn ual, and the border between them is not clearly delineated. (d) Hyaline
vocal process is composed of elastic cartilage. (c) Transition area cartilage portion of the newborn vocal process
between elastic cartilage and hyaline cartilage portions (region C in a).
The transition between the elastic and hyaline cartilage 9.10.3 Elastic Fibers of the Vocal Process
area is gradual, and the border between them is not clearly
delineated. Elastic fibers are slender and run among the collagen fibers
(Fig. 9.26). The elastic fibers are cylindrical or elliptical in
shape. The spaces among the fibers are relatively large.
9.10.2 Collagen Fibers of the Vocal Process Elastic fibers are dense at the tip of the vocal process (the
elastic cartilage portion) (Figs. 9.24a and 9.26). Elaunin
At the tip of the vocal process (the elastic cartilage portion), fibers are the predominant elastic fibers there (Fig. 9.27).
collagen fibers are thin and run in various directions Elaunin fibers are elastic-related fibers in which the fibrillary
(Fig. 9.25a). The thickness of the collagen fibers increases, component (microfibril) is quite prominent but the elastic
and they form thick bundles toward the posterior portion of component (elastin, stained black with tannic acid stain) is
the vocal process (Fig. 9.25b). not as abundant as in the typical elastic fibers. The number of
a a thyroid cartilage
B
tip of
vocal process glottis
thyroarytenoid arytenoid cartilage

muscle
elastic cartilage
b
posterior
macula flava

of vocal process
b

of vocal process
elastic cartilage

c (pseudo stratified
ciliated epithelium)
thyroarytenoid
muscle
hyaline cartilage arytenoid muscle
Fig. 9.18 Coronal sections of the vocal process of the newborn aryte- Fig. 9.19 (a) Transverse section of the vocal process during abduction
noid cartilage (Elastica van Gieson stain). (a) Tip of the vocal process. (Elastica van Gieson stain). (b) Tip of the vocal process during abduc-
(b) Posterior portion of the vocal process tion (region B in a). The vocal process bends concavely at the elastic
cartilage portion during abduction. (c) Posterior wall of the glottis dur-
ing abduction (region C in a)
9.11 Microstructure of the Vocal Process of the Arytenoid Cartilage and Its Physiologic Significance 113
glottis epiglottic cartilage

a thyroid cartilage a
B
thyroid cartilage
C
vocal process
of arytenoid
arytenoid cartilage cartilage
B
posterior
macula flava
b
elastic cartilage
portion of elastic cartilage
vocal process portion of
vocal process
hyaline cartilage hyaline cartilage

portion of portion of
vocal process vocal process
posterior arytenoid
c glottis cartilage
lamina
propria Fig. 9.21 (a) Coronal section of the vocal process during adduction
of mucosa (Elastica van Gieson stain). (b) The vocal process during adduction
glands (region B in a). The bilateral arytenoid cartilages come into contact
mainly at their superior (elastic cartilage) portions
elastic fibers decreases toward the posterior portion of the

vocal process. Even in the hyaline cartilage portion of the
vocal process, elastic fibers are found (Figs. 9.24b and 9.28).
cricoid 9.11 M
icrostructure of the Vocal Process
cartilage
of the Arytenoid Cartilage and Its
Physiologic Significance
Fig. 9.20 (a) Transverse section of the vocal process during adduction
(Elastica van Gieson stain). (b) Tip of the vocal process during adduc-
tion (region B in a). The vocal process bends convexly at the elastic 9.11.1 Tip of the Vocal Process
cartilage portion during adduction. (c) Posterior wall of the glottis dur-
ing adduction (region C in a) The chondrocytes are relatively small in size and have small
cartilage lacunae, and their density is high at the tip of the vocal
process (elastic cartilage portion). In this area, many chondro-
cytes synthesize fibrous proteins and ground substance.
Fig. 9.22 Schema of bilateral adduction neutral condition abduction

arytenoid cartilages during
adduction, neutral position,
and abduction. The vocal
process bends at the elastic
cartilage portion during
adduction and abduction, and
each side of the arytenoid
cartilages comes into contact
mainly at the elastic cartilage
portion (shaded area)
a elastic
epiglottic cartilage fibers
collagen fibers
chondrocyte
ventricular chondrocyte
fold stratified squamous
epithelium
thyroid cartilage
vocal fold
lamina of
cricoid cartilage cartilage lacuna
arch of
cricoid cartilage
pseudostratified ciliated
epithelium
b
cartilage
Fig. 9.23 Distribution of stratified squamous epithelium of the laryn- lacuna
geal mucosa. The anterior glottis (intermembranous portion) and the
superior portion (the tip of the vocal process and the superior portion of
the arytenoid cartilage from the vocal process to the apex) of the poste-
rior glottis (intercartilaginous portion) are covered with stratified squa- elastic
chondrocyte fibers
mous epithelium
From the functional point of view, the chondrocytes syn- collagen fibers
thesize fibrous protein and ground substance at the tip of the
vocal process to maintain a pliable and strong structure [33].
cartilage lacuna
The numerous but small chondrocytes and the small spaces
of the cartilage lacunae do not hinder pliable movement
there [33].
At the tip of the vocal process (elastic cartilage portion), Fig. 9.24 Transmission electron micrograph of the vocal process of the
arytenoid cartilage (tannic acid stain). (a) Elastic cartilage portion. (b)
the fibrous components are dense. The collagen fibers are Hyaline cartilage portion
thin and run in various directions. The elastic fibers are
slender and run among the collagen fibers. The presence of
three-dimensional structure demonstrates that the tip of the cess, elaunin fibers are the predominant elastic fibers.
vocal process is not only pliable but also has a relatively Elaunin fibers, which are elastic-related fibers having
strong framework [33]. small amounts of elastin, provide mechanical resistance
Elastic fibers are dense at the tip of the vocal process and a support mechanism [34, 35]. In fact, these fibers
(the elastic cartilage portion). At the tip of the vocal pro- have been found in other areas in which they have that
9.11 Microstructure of the Vocal Process of the Arytenoid Cartilage and Its Physiologic Significance 115
collagen fibers
elastic fiber
collagen fiber
b Fig. 9.26 Scanning electron micrograph of elastic and collagen fibers

in the vocal process of the arytenoid cartilage (sodium hydroxide mac-
eration method) (only elastic and collagen fibers remain following
collagen fibers treatment by this method)
collagen fiber microfibrils
microfibrils
elastin
elastin
collagen fiber
Fig. 9.25 Scanning electron micrograph of collagen fibers in the vocal

process of the arytenoid cartilage (sodium hydroxide maceration Fig. 9.27 Transmission electron micrograph of elaunin fibers in the tip
method) (collagen fibers alone remain following treatment by this of the vocal process of the arytenoid cartilage (tannic acid stain)
method). (a) Tip of the vocal process. (b) Posterior portion of the vocal
process
9.11.2 Posterior Portion of the Vocal Process
function [34, 35]. During abduction and adduction, the tip The thickness of the collagen fibers increases, and they
of the vocal process bends at the elastic cartilage portion. form thick bundles toward the posterior portion of the
There is a great deal of mechanical stress at the tip of the vocal process. The three-dimensional structures show that
vocal process where the elaunin fibers are suggested to the collagen fibers play a role as a strong framework for
provide mechanical resistance and serve as a support the posterior glottis at the posterior portion of the vocal
mechanism [33]. process [33].
9.12 Histoanatomy of the Posterior Glottis
Recently, fiberscopy is a routine procedure at laryngology

clinics. As a result, laryngologists have frequent opportuni-
ties to examine the morphology and physiology of the poste-
rior glottis in daily practice.
collagen fibers elastic fiber
9.12.1 Neutral Condition
Histologically, the structure around the posterior glottis con-

sists of the cartilage and overlying mucosa. The mucosa has
pseudostratified ciliated epithelium in the posterior glottis
(Fig. 9.29). This is in contrast to the stratified squamous epi-
thelium in the anterior glottis (intermembranous portion).
Fig. 9.28 Transmission electron micrograph of elastic fibers in the Regarding the posterior wall of the glottis, the lamina
posterior portion (hyaline cartilage portion) of the vocal process of the propria of the mucosa can be divided into two layers: the
arytenoid cartilage (tannic acid stain)
superficial and the deep layers (Fig. 9.29b). The former is
loose in structure, whereas the latter consists of dense elas-
tic and collagen fibers and many laryngeal glands. Many
The number of elastic fibers decreases toward the poste- fibers in the deep layer run vertically at the posterior wall of
rior portion of the vocal process, though, even in the hyaline the glottis (Fig. 9.29b), whereas most fibers run in an oblique
cartilage portion of the vocal process, elastic fibers are found. direction at the lateral wall of the posterior glottis
Usually, hyaline cartilage does not contain elastic fibers [36]. (Fig. 9.29c).
The hyaline cartilage of the vocal process has a more pliable The underlying cartilage is the cricoid cartilage at the
and stronger structure than other hyaline cartilages found in posterior wall of the glottis and arytenoid cartilage at the lat-
other parts of the body [33]. eral wall of the posterior glottis. The cricoid cartilage and
most of the arytenoid cartilage consist of hyaline cartilage,
whereas a part of the tip of the vocal process of the arytenoid
9.11.3 Biomechanical Properties of the Human cartilage is composed of elastic cartilage.
Vocal Process The bilateral posterosuperior portions of the cricoid area
are surrounded by the arytenoid cartilage, cricoid cartilage,
The posterior macula flava is attached to the tip of the cricoarytenoid joints (articulations), and the mucosa of the
vocal process posteriorly [37]. The transition between the posterior glottis (Fig. 9.29a).
posterior macula flava and the elastic cartilage portion of
the vocal process is gradual, and the transition between its
elastic and hyaline cartilage portions is also gradual [30]. 9.12.2 Vocal Fold Abduction
The borders between them are not clearly delineated.
There are gradual changes in stiffness between the vocal When the vocal fold is abducted, the vocal process moves
fold and the vocal process [30, 33]. The vocal process of not only laterally but also superiorly and posteriorly [31].
the arytenoid cartilage is firm, forming a framework for Bilateral vocal fold abduction forms a pentagonal glottis
the glottis, but is more pliable toward the tip. Gradual (Fig. 9.30). The five corners of the pentagon are located at
changes in stiffness between the vocal fold and the vocal the anterior commissure near the bilateral vocal processes
process may absorb shock during phonation and prevent and at the junctions of the posterior wall of the glottis and the
mechanical damage to the tip of the vocal process. lateral walls of the posterior glottis.
Additionally, the tip of the vocal process bends easily dur- During abduction, the tip of the vocal process pulls over-
ing adduction and abduction. lying mucosa laterally. As a result, a small canopy of mucosa
The gradual changes in stiffness of the three-dimensional forms above the vocal process [31]. Under the canopy a
structures between the vocal fold and the vocal process make corner of the pentagonal glottis is formed near the tip of the
a functional contribution to the roles of the vocal folds and vocal process.
vocal processes [33].
9.12 Histoanatomy of the Posterior Glottis 117
a
anterior glottis
membranous
C portion of
vocal process anterior glottis vocal fold
posterior glottis
(arytenoid cartilage)
B canopy
a* *a
posterior glottis hyaline cartilage

cricoid area portion of
lateral wall of vocal process
pseudostratified
b ciliated epithelium posterior glottis
b* *b
Fig. 9.30 Glottis during deep inspiration viewed from below (through
superficial layer tracheostoma). A tiny canopy is formed at the edge of the vocal fold.
Bilateral vocal fold abduction forms a pentagonal glottis. The five cor-
glands
ners of the pentagon are located at the anterior commissure, near the
bilateral vocal processes (asterisk a), and at the junctions of the poste-
deep layer glands rior wall of the glottis and the lateral walls of the posterior glottis
(asterisk b)
anterior glottis
cricoid cartilage (membranous portion
of vocal fold)
c

of vocal process
arytenoid cartilage hyaline cartilage portion
(vocal process) of vocal process
glands
posterior glottis
pseudostratified
ciliated epithelium
500 µm Fig. 9.31 Glottis during phonation viewed from below (through tra-
cheostoma). Note a conic space in the posterior glottis
Fig. 9.29 (a) Transverse section of the glottis (Elastica van Gieson

stain). (b) The posterior wall of the glottis (region B in a). (c) The lat-
eral wall of the posterior glottis (region C in a) When the bilateral vocal folds are adducted, the anterior
glottis and a small region of the anterior portion of the
posterior glottis close. The main portion of the posterior
The mucosa of the posterior wall of the glottis is thinned glottis does not close at the level of the glottis. However,
and stretched during vocal fold abduction (Fig. 9.19c). it closes completely at the level of the supraglottis (the tip
of the vocal process and the superior portion of the aryte-
noid cartilage from the vocal process to the apex). The
9.12.3 Vocal Fold Adduction epithelium at the contact area is stratified squamous epi-
thelium (Fig. 9.23) [32]. As a result, a conic space forms
When the vocal fold is adducted, the vocal process moves in the posterior glottis during vocal fold adduction
not only medially but also inferiorly and posteriorly [31]. (Fig. 9.31).
The mucosa of the posterior glottis becomes thicker and a

folds during vocal fold adduction, especially at the posterior apex of arytenoid cartilage
wall (Fig. 9.20c). Since many fibers in the deep layer run

vertically at the posterior wall of the glottis, the direction of
fibers in the posterior wall appears to be suitable for the fold-
ing of the mucosa [1].
9.13 T
he Posterior Glottis and Its
vocal process
Physiologic Significance
The anterior glottis plays the most important role for phona-
tion. On the other hand, the posterior glottis appears to play
an equally important role in respiration [1].
The epithelium in the posterior glottis is pseudostratified cricoarytenoid joint
ciliated epithelium (respiratory epithelium), whereas it is
stratified squamous epithelium in the anterior glottis. b cricoarytenoid cricoarytenoid
joint joint
Pseudostratified ciliated epithelium is not suitable for vibra- axis of joint
tion but for respiration. Furthermore, the area of the posterior
glottis occupies more than half of the entire glottic area.
The posterior glottis is a respiratory glottis, while the
anterior glottis is a phonatory glottis [1]. Clinically, diseases
of the anterior glottis usually cause voice disorders. They
disturb respiration when they present a very large obstruc-
tion to the airway. On the other hand, diseases of the poste- lamina of
cricoid cartilage
rior glottis often result in respiratory distress. They do not
affect phonation until they become very extensive and inhibit
vocal fold closure [1].
9.14 Cricoarytenoid Joint (Articulation) arch of cricoid cartilage
During swallowing, respiration, and phonation, the vocal c

processes always move and form the shape of the glottis. The axis of joint
cricoarytenoid joint
biomechanics of the cricoarytenoid joint (articulation) con-
trol abduction and adduction of the vocal fold.
Von Leden and Moore describe the cricoarytenoid joint
as a shallow ball-and-socket joint on the lateral aspect of
the cricoid rim (Fig. 9.32) [38]. The structural arrange-
ment of the cricoarytenoid joint permits two principal
types of motion: a rocking or rotating movement around
the axis of the joint (inward and outward rocking) and a lamina of
linear glide parallel to this axis (lateral and medial slid- cricoid cartilage
ing) [38]. In addition, there is limited motion (a pivot)
around the posterior cricoarytenoid ligaments (Figs. 9.33
and 9.34) and the joint capsule (Fig. 9.35) which permits arch of
a very restricted rotary motion around the attachment of cricoid cartilage
this ligament to the lamina of the cricoid cartilage (lim-
ited pivoting) [38].
The cricoarytenoid joint is comprised of the cricoid and
arytenoid cartilages, which are composed of hyaline carti-
Fig. 9.32 Cricoarytenoid joints of arytenoid and cricoid cartilage. (a)
lage. Just under the surface of the joint cartilage, the lacunae
Right arytenoid cartilage, (b) frontal view of cricoid cartilage, (c) lat-
of chondrocytes are elliptical and the chondrocytes are flat eral view of cricoid cartilage. The principal axis of rotation extends in a
and lay between collagen fibers with their long axis parallel dorsomediocranial and ventrolaterocaudal direction
9.16 Age-Related Changes of the Cricoarytenoid Joint (Articulation) and Their Biomechanical Properties 119
ventricular fold The joint capsule is composed of fibrous membrane and

a
laryngeal ventricle synovial membrane (Fig. 9.35b). Fibrous membrane is
thyroid cartilage chiefly composed of collagen fibers and continues into the
perichondrium of the cricoid and arytenoid cartilage.
Synovial villi protrude into the articular cavity (Fig. 9.35b).
The dominant collagen of the cartilage matrix is type II
[36]. In contrast to type I fibers, which average 75 nm in
B thickness, type II collagen forms faint crossbanded fibers
15–45 nm in diameter that do not assemble into coarse bun-
dles [36]. The smaller fibers form a loose three-dimensional
network throughout the matrix [36].
arytenoid cartilage Proteoglycans are among the largest molecules produced
arytenoid muscle by cells. The principal proteoglycan of the cartilage is
b aggrecan.
arytenoid
cartilage
9.15 Geriatric Changes
of the Cricoarytenoid Joint
(Articulation)
cricoid Geriatric changes are observed both on the surface and in the
area inner portion of the articular cartilage.
One of the common age-related changes of the joint sur-
face is the prominence of the collagen fibers within the carti-
lage matrix (Fig. 9.37). There is significant loss of cartilage
matrix and many of the collagen fibers are exposed on the
joint surface. Other age-related changes are unevenness of the
posterior joint surface and cracks or fissure-like defects (Fig. 9.38).
cricoarytenoid Changes in joint facets are more extensive in the cricoid facet.
ligament In the inner portion of the articular cartilage, calcification
and ossification of the matrix appear in the hyaline cartilage
(Fig. 9.39). The ossified portion consists of bone matrix,
bone marrow, and osteocytes (Fig. 9.40).
Ultrastructurally, prominent collagen fibers stand out in the
ground substance (Fig. 9.41). The collagen fibers form bundles
and the density becomes high. The collagen fibril diameters
arytenoid muscle begin to differ and their outline becomes irregular (Fig. 9.42).
Twisted collagen fibrils are present. Few intracellular organ-
Fig. 9.33 Posterior cricoarytenoid ligament (57-year-old female, elles such as rough endoplasmic reticulum and Golgi apparatus
Elastica van Gieson stain). (a) Transverse section of the human adult
larynx at the upper portion of the lamina of the cricoid cartilage. (b) are present in the cytoplasm of the chondrocytes (Fig. 9.43),
Region B in (a). The posterior cricoarytenoid ligament runs between the indicating that protein synthesis is not occurring within them.
upper rim of the cricoid lamina and medial facet of the arytenoid carti- Some chondrocytes are degenerated (Fig. 9.44).
lage. The ligament continues into the perichondrium of the cricoid and
arytenoid cartilages. The posterior cricoarytenoid ligament prevents
anterior displacement of the arytenoid cartilage
9.16 Age-Related Changes
of the Cricoarytenoid Joint
(Articulation) and Their
to the articular surface (Fig. 9.36). Moving deeper into the Biomechanical Properties
cartilage, chondrocytes become hemispherical or angular.
More intense staining of the capsular or territorial matrix Age-related changes, resulting from remodeling of cartilage
immediately surrounding the isogenous cells is observed. or changes in the mechanical properties of the ground sub-
The shape of the chondrocytes and that of their lacunae stance or the collagen networks within them may prove to be
change moving deeper into the cartilage (Fig. 9.36). important factors in assessing articular surface topography.
Fig. 9.34 Schema of adduction abduction

posterior cricoarytenoid
ligament and arytenoid
adduction and abduction. Red
line: Posterior cricoarytenoid
ligament
glottis
vocal
process
arytenoid lamina of
cartilage cricoid cartilage
thyroarytenoid cricoid joint cavity arytenoid

cricoarytenoid cartilage cartilage
muscle
joint
synovial villi
adipose tissue
synovial
membrane
muscular
cricoid process joint capsule
cartilage of
arytenoid
cartilage b fibrous membrane
B joint surface
posterior
cricoarytenoid
muscle
collagen fibers
chondrocytes
joint
a capsule
Fig. 9.35 Cricoarytenoid joint and joint capsule (43-year-old female,

Elastica van Gieson stain). (a) Transverse section of the human adult
cricoarytenoid joint. (b) Joint capsule (region B in a) arytenoid cartilage
Fig. 9.36 Transverse section of arytenoid cartilage and joint surface

(43-year-old female, Elastica van Gieson stain)
9.16 Age-Related Changes of the Cricoarytenoid Joint (Articulation) and Their Biomechanical Properties 121
Irregularities (cracks or fissure-like defects) and increased

prominence of collagen fibers in the matrix are seen on the
articular surface of the cricoarytenoid joint in the aged.
These observations appear to have substantial implications
for cricoarytenoid joint function. Irregularities in surface
architecture become the cause of wear on the joint surface
and the subsequent exposure of collagen fibers [39]. Such cracks of
irregularities interfere with the smoothness of movement
across the joint surface and prevent a continuous film of
joint surface
synovial fluid to be distributed over the surface. These may
affect the precision of positioning the vocal folds during pre-
phonatory adjustments and/or the responsiveness of the joint
to muscular forces acting on it during frequency change,
while at the same time producing voiceless phonemes,
pauses, and other suprasegmental features [39]. The degree
of vocal fold approximation can be affected by age-related
changes of the cricoarytenoid joint.
Fig. 9.38 Scanning electron micrograph of the cracks or fissure-like

collagen fibers defects of the joint surface of the cricoid cartilage in the aged (90-year-
old female)
joint surface
cartilage matrix
chondrocytes ossification
calcification
Fig. 9.37 Scanning electron micrograph of the joint surface of the cri-
coid cartilage in the aged (90-year-old female)
Fig. 9.39 Transverse section of cricoid cartilage and joint surface in
the aged (72-year-old male, Elastica van Gieson stain)
joint surface chondrocyte collagen fibers
nucleus
bone marrow
osteocyte
bone matrix
Fig. 9.43 Transmission electron micrograph of a chondrocyte in the

cricoid cartilage of the cricoarytenoid joint in the aged (84-year-old
Fig. 9.40 Transverse section of cricoid cartilage and joint surface in
male, uranyl acetate and lead citrate stain)
the aged (97-year-old male, Elastica van Gieson stain)
chondrocyte
collagen fibers
nucleus
collagen fibers degenerated

chondrocytes
Fig. 9.41 Transmission electron micrograph of the cricoid cartilage of

the cricoarytenoid joint in the aged (84-year-old male, uranyl acetate
and lead citrate stain) Fig. 9.44 Transmission electron micrograph of a degenerated chon-
drocyte in the cricoid cartilage of the cricoarytenoid joint in the aged
(84-year-old male, uranyl acetate and lead citrate stain)
collagen fibers
collagen fibers
Fig. 9.42 Transmission electron micrograph of the collagen fibers in

the cricoid cartilage of the cricoarytenoid joint in the aged (84-year-old
male, uranyl acetate and lead citrate stain)
References 123
References 19. Kiyokawa K. Effects of intrinsic laryngeal muscles to the glottis.

Otologia (Fukuoka). 1987;33(Suppl. 1):183–217.
20. Benjamin B. Prolonged intubation injuries of the larynx: endo-
1. Hirano M, Kurita S, Kiyokawa K, Sato K. Posterior glottis.
scopic diagnosis, classification, and treatment. Ann Otol Rhinol
Morphological study in excised human larynges. Ann Otol Rhinol
Laryngol Suppl. 1993;160:1–15.
Laryngol. 1986;95:576–81.
21. Sato K, Nakashima T. Histopathologic changes in laryngeal mucosa
of extremely low-birth weight infants after endotracheal intubation.
Ann Otol Rhinol Laryngol. 2006;115:816–23.
3. Hirano M, Kurita S. Location of the posterior end of the vocal folds:
22. Hawkins DB. Hyaline membrane disease of the neonate. Prolonged
an unrecognized controversy. J Otolaryngol Jpn. 1982;85:432–5.
intubation in management: effects on the larynx. Laryngoscope.
1978;88:201–24.
(Fukuoka). 1975;21(Suppl. 1):239–60.
23. Strong RM, Passy V. Endotracheal intubation. Complications in
5. Sato K, Hirano M, Nakashima T. Fine structure of the human new-
neonates. Arch Otolaryngol. 1977;103:329–35.
born and infant vocal fold mucosae. Ann Otol Rhinol Laryngol.
24. Freeman GR. A comparative analysis of endotracheal intubation in
2001;110:417–24.
neonates, children, and adults: complications, prevention and treat-
6. Hirano M, Kurita S, Nakashima T. Growth, development and aging
ment. Laryngoscope. 1972;82:1385–98.
of human vocal folds. In: Bless DM, Abbs JH, editors. Vocal fold
25. Rasche RF, Kuhns LR. Histopathologic changes in airway mucosa
physiology. San Diego, CA: College-Hill Press; 1983. p. 22–43.
of infants after endotracheal intubation. Pediatrics. 1972;50:632–7.
7. Sato K, Chitose S, Umeno H. Dimensions and morphologi-
26. Lindholm CE. Prolonged endotracheal intubation. Acta Anaesth
cal characteristics of human newborn glottis. Laryngoscope.
Scand. 1969;1(Suppl. 33):1–131.
2015;125:E186–9.
27. MacDonald RE, Smith C, Mitchell D. Airway problems in chil-
8. Sato K, Hirano M. Historogic investigation of the macula flava
dren following prolonged endotracheal intubation. Ann Otol Rhinol
of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
Laryngol. 1966;75:975–86.
1995;104:556–62.
28. Allen TH, Steven IM. Prolonged endotracheal intubation in infants
9. Sato K, Nakashima T, Nonaka S, Harabuchi Y. Histopathologic
and children. Br J Anaesth. 1965;37:566–73.
investigations of the unphonated human vocal fold mucosa. Acta
29. Dankle SK, Schuller DE, McClead RE. Prolonged intubation of
Otolaryngol. 2008;128:694–701.
neonates. Arch Otolaryngol Head Neck Surg. 1987;113:841–3.
10. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi Y. Expression
30. Sato K, Kurita S, Hirano M, Kiyokawa K. Distribution of elastic
and distribution of hyaluronic acid and CD44 in unphonated human
cartilage in the arytenoids and its physiologic significance. Ann
vocal fold mucosa. Ann Otol Rhinol Laryngol. 2009;118:773–80.
Otol Rhinol Laryngol. 1990;99:363–8.
11. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi

31. Hirano M, Yoshida T, Kurita S, Kiyokawa K, Sato K, Tateishi
Y. Histopathologic investigations of the unphonated human child
O. Anatomy and behavior of the vocal process. In: Baer T, Sasaki C,
vocal fold mucosa. J Voice. 2012;26:37–43.
Harris K, editors. Laryngeal function in phonation and respiration.
12. Sato K, Kurita T, Chitose S, Umeno H, Nakashima T. Mechanical
San Diego, CA: Singular Publishing Group Inc.; 1991. p. 3–13.
regulation of human vocal fold stellate cells. Ann Otol Rhinol
32. Sato K. Three dimensional anatomy of the larynx: investigation
Laryngol. 2015;124:49–54.
by whole organ sections. Otologia (Fukuoka). 1987;33(Suppl.
13. Sato K, Umeno T, Nakashima T. Vocal fold stem cells and their
1):153–82.
niche in the human vocal fold. Ann Otol Rhinol Laryngol.
33. Sato K, Hirano M, Nakashima T. Ultrastructure of the vocal

2012;121:798–803.
process of the arytenoid cartilage. Ann Otol Rhinol Laryngol.
14. Kurita T, Sato K, Chitose S, Fukahori M, Sueyoshi S, Umeno
2000;109:650–3.
H. Origin of vocal fold stellate cells in the human macula flava.
34. Cotta-Pereira G, Kattenbach WM, Rodrigo G. Elastic-related fibers
in basement membranes. In: Robert L, editor. Frontiers of Matrix
15. Sato K, Chitose S, Kurita T, Umeno H. Microenvironment of mac-
Biology, vol. 7. Basel: Karger; 1979. p. 90–100.
ula flava in the human vocal folds as a stem cell niche. J Laryngol
35. Cotta-Pereira G, Rodrigo G, Bittencourt-Sampaio S. Oxitalan,

Otol. 2016;130:656–61.
elaunin, and elastic fibers in the human skin. J Invest Dermatol.
16. Kurpinski K, Janairo R, Chien S, Li S. Mechanical regulation of
1976;66:143–8.
stem cells: implications in tissue remodeling. In: Mofrad M, Kamm
36. Fawcett DW. Cartilage. A textbook of histology. New York, NY:
R, editors. Cellular Mechanotransduction. Diverse perspectives
Chapman & Hall; 1994. p. 182–93.
from molecules to tissues. New York, NY: Cambridge University
37. Sato K, Hirano M. Histologic investigation of the macula flava of the
Press; 2010. p. 403–16.
human vocal fold. Ann Otol Rhinol Laryngol. 1995;104:138–43.
17. Sato K, Tateishi O, Muteki T, Hirano M. Morphological study as
38. Von Leden H, Moore P. The mechanics of the cricoarytenoid joint.
to the appropriate size of an endotracheal tube size. Jpn J Anaesth.
Arch Otolaryngol. 1961;73:541–50.
1988;37:1388–93.
39. Kahane JC, Hammons J. Developmental changes in the articular
18. Sato K, Tateishi O, Muteki T, Hirano M. Morphological study
cartilage of the human cricoarytenoid joint. In: Baer T, Sasaki C,
regarding the appropriate size of an endotracheal tube. Jpn Anaesth
Harris K, editors. Laryngeal function in phonation and respiration.
J Rev. 1990;4:103–5.
San Diego, CA: Singular Publishing Group Inc.; 1991. p. 14–28.
Cells and Extracellular Matrices
in the Human Adult Vocal Fold Mucosa 10
Abstract
1. Adult vocal folds have a layered structure consisting of the epithelium; the superficial,
intermediate, and deep layers of the lamina propria; and the vocalis muscle (Hirano,
Otologia (Fukuoka) 21(Suppl. 1): 239–60, 1975).
2. The free edge of the membranous vocal fold is covered with stratified squamous epithe-
lium. Microvilli (microridges) of the stratified squamous epithelium facilitate distribu-
tion and retention of minute amounts of mucus on the vocal fold surface, which is
essential for normal vibration and phonation.
3. Langerhans cells with dendritic shape are situated in the suprabasal region of the strati-
fied squamous epithelium in the larynx.
4. The viscoelastic properties of the lamina propria of the human vocal fold mucosa are
very important for the vibratory behavior of the structure. They greatly depend on the
extracellular matrices. Not only the three-dimensional structure of the extracellular
matrices but also their qualitative and quantitative properties have an effect on the physi-
cal properties of the human vocal fold mucosa.
5. In the human vocal fold mucosa, the extracellular matrices are composed of two families
of macromolecules: fibrillar proteins (collagen and elastin) which provide the fibrous
scaffolding of the lamina propria of the mucosa and the interstitium between the fibrous
scaffolding—the proteoglycans (glycosaminoglycans) and structural glycoproteins.
6. As fibrillar proteins, collagens, and reticular fibers are required for structural mainte-
nance. They are responsible for tensile strength and resilience and serve as stabilizing
scaffolds in the extracellular matrices.
7. The three-dimensional structure of reticular fibers (type III collagen) in the vocal fold
mucosa appears to be one of the key components to maintain the structure and viscoelas-
ticity of the vibrating tissue. The complex of reticular fibers and other extracellular
matrices seems to be very important for the viscoelastic properties of the vocal fold
mucosa.
8. The functions of the microfibril-associated macromolecules are likely to be specific to
some aspect of the mechanical biology of the human vocal fold mucosa.
9. The hyaluronic acid glycosaminoglycan (hyaluronan) is a key molecule influencing tis-
sue viscosity of the lamina propria of the human vocal fold mucosa.

126 10 Cells and Extracellular Matrices in the Human Adult Vocal Fold Mucosa
10.1 Introduction cover consisting of the epithelium and superficial layer of the
lamina propria, a transition zone consisting of the intermedi-
The viscoelastic properties of the lamina propria of the human ate and deep layers of the lamina propria, and a body consist-
vocal fold mucosa determine its vibratory behavior and depend ing of the vocalis muscle [1, 2]. The superficial layer of the
on extracellular matrices, such as collagen fibers, reticular lamina propria is referred to as Reinke’s space. The interme-
fibers, elastic fibers, proteoglycans, glycosaminoglycans, and diate and deep layers of the lamina propria form the vocal
glycoproteins. The three-dimensional structures of these extra- ligament. The vocal ligament runs between the anterior and
cellular matrices are indispensable to the viscoelastic properties posterior maculae flavae. This layered structure is very
of the vocal fold mucosa. The fine structures of the vocal fold important for vibration.
mucosa influence vibrating behavior and voice quality. The relative thickness of the superficial layer of the
lamina propria varies along the length of the vocal fold.
This layer is thickest at the midpoint of the membranous
10.2 L
ayered Structure of the Human vocal fold and becomes thinner toward the anterior and
Vocal Fold posterior portions [3]. Conversely, the intermediate layer
of the lamina propria is thinnest at the midpoint of the
Adult vocal folds have a layered structure consisting of the membranous vocal fold and becomes thicker toward
epithelium; the superficial, intermediate, and deep layers of the anterior and posterior portions [3]. The deep layer of
the lamina propria; and the vocalis muscle (Figs. 10.1 and the lamina propria is thickest at the posterior portion of
10.2) [1, 2]. These layers are grouped into three sections: a the vocal fold [3].
pseudostratified
ciliated epithelium stratified squamous epithelium

• superficial layer Reinke’s space
• intermediate layer
vocal ligament
• deep layer
vocalis muscle
laryngeal gland
Fig. 10.1 Coronal section of

the human younger adult
vocal fold (27-year-old male, pseudostratified ciliated epithelium
Elastica van Gieson stain)
(Photograph courtesy of Dr.
Minoru Hirano, from the
Department of
Otolaryngology-Head and
Neck Surgery)
10.4 Microstructure of the Epithelium of the Human Vocal Fold and Its Physiologic Significance 127
Fig. 10.2 Schema of the

pseudostratified
layered structure of the
ciliated epithelium
human vocal fold [1]
stratified squamous epithelium
(Photograph courtesy of Dr.
Minoru Hirano, from the
Department of
Otolaryngology-Head and epithelium
Neck Surgery) lamina propria
∗ cover of mucosa
• superficial layer
• intermediate layer
∗ transition
zone • deep layer
∗ body vocalis muscle
10.3 Epithelium of the Human Vocal Fold 200 nm in height, of various patterns (Figs. 10.4 and 10.5). The
microvilli (microridges) are already present on the surface cell
The free edge of the membranous vocal fold (anterior glot- membrane of the stratified squamous epithelium at birth.
tis) is covered with stratified squamous epithelium The epithelium of the vocal fold is contiguous with the
(Figs. 10.3 and 10.4). There are no glands or glandular duct pseudostratified ciliated epithelium (Fig. 10.6) of the laryn-
openings at the free edge of the membranous portion of the geal ventricle, ventricular fold, epiglottis, and subglottis
vocal fold. (Fig. 10.7).
The stratified squamous epithelium consists of seven to
eight cell layers of squamous cells. The cells of the basal lay-
ers are columnar or polyhedral. Epithelial cells have cyto- 10.4 M
icrostructure of the Epithelium
plasmic processes and form interdigitation with adjacent of the Human Vocal Fold and Its
cells. Desmosomes at the junction of two adjacent epithelial Physiologic Significance
cells make firm intercellular adhesion. Spaces between the
epithelial cells are relatively large. Near the surface, the The epithelium protects and contains the underlying lamina
number of desmosomes decreases and the cells are more flat- propria of the vocal fold mucosa.
tened. The superficial layer is composed of thin squamous The larynx is lubricated by secretions from the upper
cells. respiratory tract [4]. Lubrication by a thin mucous coating of
The apical cell membrane (surface of the stratified squamous the vocal folds is essential for normal vibration and phona-
epithelium) is furnished with microvilli (microridges), about tion [5].
Fig. 10.3 Stratified
squamous epithelium of the
human vocal fold
Microvilli (microridges) of the stratified squamous epi- the lamina lucida, the lamina densa, and the zona reticularis.
thelium are considered to facilitate distribution and retention The lamina lucida is a low-density clear zone adjacent to the
of minute amounts of mucus on the vocal fold surface [6]. In basal cell membrane. The lamina densa is a high-density
addition, the microvilli (microridges) provide a better surface area of filaments adjacent to the lamina propria. The lamina
for contact, minimizing slippage, such as the tread of a tire densa contains type IV collagen.
provides traction [7, 8]. Hemidesmosomes bind basal cells to the basal lamina.
At birth, intercellular spaces in the stratified squamous Anchoring fibrils tether the basal lamina to the underlying
epithelium are extremely small [9]. On the other hand, inter- connective tissue, the lamina propria of the vocal fold
cellular spaces in the stratified squamous epithelium are rela- mucosa [10, 11]. Anchoring fibrils are composed of type
tively large in adults. However, the epithelial cells have VII collagen. Anchoring fibrils loop from the lamina densa
cytoplasmic processes and form interdigitation with adjacent of the basement membrane into the lamina propria, looping
cells. Desmosomes at the junction of two adjacent epithelial around type III collagen fibers (reticular fibers) and then
cells make intercellular adhesion firm. The epithelium pro- back into the lamina densa [11].
vides a pliable and firm structure as a vibrating tissue.
10.6 M
icrostructure of the Basal Lamina
10.5 B
asal Lamina (Basement Membrane) (Basement Membrane) of the Human
of the Human Vocal Fold Vocal Fold and Its Physiologic
Significance
At the boundary between the epithelium and the underlying
lamina propria, a supporting structure known as the basal The basal lamina mainly provides physical support to the
lamina (basement membrane) is present (Fig. 10.8). The epithelium [12] and is essential for repair of the epithe-
basal lamina of the vocal fold is composed of three zones: lium [13].
10.6 Microstructure of the Basal Lamina (Basement Membrane) of the Human Vocal Fold and Its Physiologic Significance 129
Fig. 10.4 Transmission microvilli

electron micrograph of the a
stratified squamous
epithelium of the human
vocal fold. (a) The cells of
epithelium near the superficial
layer. (b) The cells of
epithelium near the basal
layer
intercellular space keratinocyte
cytoplasmic processes
keratinocyte
nucleus
b intercellular space
nucleus
keratinocyte
nucleus
keratinocyte
desmosome
nucleus
a b
microvilli
Fig. 10.5 Scanning electron micrograph of the surface of the stratified squamous epithelium of the human vocal fold. (a) The apical cell mem-
brane is furnished with microvilli (microridges) which show various patterns. (b) Higher magnification of (a)
10.7 L
angerhans Cells of the Human Vocal 10.8 L
angerhans Cells of the Human
Fold Larynx and Their Physiologic
Significance
Langerhans cells with dendritic shape are situated in the
suprabasal region of the squamous epithelium in the larynx Langerhans cells are important in the human immune system
and the hypopharynx (Fig. 10.9) [14]. as a part of the dendritic cell system [15]. Dendritic cells are
Langerhans cells possess clear cytoplasm containing no a system of antigen-presenting cells and have an ability to
tonofilaments (Figs. 10.10 and 10.11). Desmosomes are capture antigens and initiate T cell-mediated immunity [16].
absent between keratinocytes. The nuclei are markedly Thompson and Griffin noted Langerhans cells in normal and
folded and the nucleoplasm is relatively clear. Golgi appara- pathological vocal fold mucosa using the S-100 polyclonal
tus, mitochondria, and rough endoplasmic reticulum are seen antibody [17].
in the cytoplasm. Some cytoplasmic granules (Langerhans There is immunological resistance within the mucosa,
granules or Birbeck’s granules), rod-shaped with median i.e., local, humoral, and cellular immunity. Laryngeal secre-
striated lines, can be seen in the cytoplasm (Fig. 10.12). The tions contain IgG, IgA, and IgE secretory components and
size of these granules is approximately 40 nm in width and lactoferrin. Secretory IgA in the laryngeal mucosa is espe-
200 nm in length. Occasionally lysosomes containing mela- cially important in the local immune system [18]. Langerhans
nin granules are present in the Langerhans cells (Fig. 10.11b). cells, macrophages, and T cells are important components of
In the larynx, the Langerhans cells are present in squa- cellular immunity. Langerhans cells in the larynx and the
mous epithelium in the epiglottis, aryepiglottic folds, aryte- hypopharynx may possibly be essential to immune regula-
noid regions, the interarytenoid notch, and the membranous tion required for the defense of the mucosa of the airway and
portion of the vocal folds. These cells surround the vestibule the passageway of food.
and glottis of the larynx (Fig. 10.13) [14]. Basic functions of the human larynx are to act as a protec-
In the hypopharynx, the Langerhans cells are situated in tive sphincter, to act as a passageway for air, and to produce
squamous epithelium in the postcricoid area, the piriform sound. In addition, the larynx is suggested to be essential for
sinus, and the posterior wall of the hypopharynx. These cells immune response at the entrance of the trachea in the upper
surround the entrance of the esophagus [14]. respiratory passages.
10.8 Langerhans Cells of the Human Larynx and Their Physiologic Significance 131
Fig. 10.6 Transition area

between stratified squamous a stratified squamous transition area
epithelium (a) and epithelium
pseudostratified ciliated
epithelium (b)
b transition area pseudostratified

ciliated epithelium

10.9 E
xtracellular Matrices in the Human
Vocal Fold Mucosa
In general, extracellular matrices are composed of organized

networks of macromolecules which determine the physical
properties of the tissue. The viscoelastic properties of the
lamina propria of the human vocal fold mucosa are very
important for the vibratory behavior of the structure. They
greatly depend on the extracellular matrices. Not only the
three-dimensional structure of the extracellular matrices but
also their qualitative and quantitative properties have effect
on the physical properties of the human vocal fold mucosa.
cilia The extracellular matrices of the human vocal fold
mucosa are composed of fibrous proteins (collagen, reticular
and elastic fibers, etc.), proteoglycans, glycosaminoglycans
(hyaluronic acid, etc.), glycoproteins (fibronectin, etc.), and
other interstitial molecules (Fig. 10.14).
Fig. 10.7 Scanning electron micrograph of the surface of pseudostrati-

fied ciliated epithelium of the human subglottis
basal cell
hemidesmosome
lamina lucida
lamina densa
zona reticularis
anchoring
fibril
reticular fibers
Fig. 10.8 Transmission
electron micrograph of the
basal lamina (basement
membrane) of the human
vocal fold
10.9 Extracellular Matrices in the Human Vocal Fold Mucosa 133
Fig. 10.9 Langerhans cells

in the squamous epithelium at
the arytenoid of the larynx
(S-100, immunohistochemical stratified squamous epithelium
staining, original ×100)
Langerhans cells
electron micrograph of
Langerhans cell
Langerhans cell in the
squamous epithelium at the
postcricoid area of the
hypopharynx
keratinocytes
Fig. 10.11 (a) Transmission

a keratinocyte
Langerhans cell in the
squamous epithelium at the
vocal fold of the larynx. (b) Langerhans cells keratinocyte
Lysosomes containing melanin
granules in the Langerhans
cells (region B in a) B
nucleus
mitochondrion
lysosomes
melanin granules
nucleus
cytoplasmic granules cytoplasmic granule
(Birbeck’s granules) in (Birbeck’s granule)
Langerhans cells
cytoplasmic granule
(Birbeck’s granule)
epiglottis 10.9.1 Extracellular Matrices in the Superficial

Layer of the Lamina Propria (Reinke’s
Space) of the Human Vocal Fold
Mucosa
10.9.1.1 Collagen and Reticular Fibers

As fibrous proteins, collagen and reticular fibers are required
for structural maintenance. They are responsible for tensile
strength and resilience and serve as stabilizing scaffolds in
the extracellular matrices.
Collagen fibers are thin and sparse in the superficial layer
vocal fold
of the lamina propria (Reinke’s space) (Fig. 10.15) and are
aryepiglottic fold dense in the deep layer of the lamina propria of the human
vocal fold mucosa (Fig. 10.16). They form bundles.
arytenoid Reticular fibers are now known to be simply one of the
several molecular species of collagen [19]. However, it is
useful for descriptive purposes to retain the term “reticu-
interarytenoid notch lar fiber,” because their fibrous elements are smaller than,
and arranged differently from, the more prevalent colla-
Fig. 10.13 Distribution of Langerhans cells in the larynx (shaded
area)
gen fibers [19].
Fig. 10.14 Schema of

extracellular matrices in the
superficial layer of the lamina
propria (Reinke’s space) of a
younger adult human vocal glycosaminoglycan
fold mucosa (proteoglycan)
elastic fiber
glycoprotein
collagen fiber
reticular fiber
mucosa (Fig. 10.16). They are wavelike and do not form

bundles. They are most abundantly seen around the edge
of the vocal fold, and they decrease toward the superior
and inferior portions of the vocal folds. At the superior
and inferior portions of the vocal fold mucosa, there are
no reticular fibers in the lamina propria of the mucosa,
capillary
only collagen fibers (Fig. 10.17). They are not detected in
the deep layer of the lamina propria of the mucosa
(Fig. 10.16b). Reticular fibers are located at the portion of
the vocal fold mucosa which vibrates the most during
elastic fibers phonation [20].
There are many slender reticular fibers in the superficial
collagen fibers layer of the lamina propria (Reinke’s space) of the vocal fold
mucosa (Fig. 10.18). Reticular fibers are made up of a single
unit fibril (Fig. 10.19). They are about 40 nm in diameter and
have crossbands with a periodicity of about 67 nm. Reticular
fibers branch and anastomose. They do not form bundles, but
Fig. 10.15 Coronal section of the superficial layer of the lamina pro-
pria of the human vocal fold mucosa (37-year-old female, Elastica van form delicate three-dimensional networks. Spaces among
Gieson stain). Collagen fibers (stained red) and elastic fibers (stained the reticular fibers are relatively large. Some reticular fibers
black) are round or oval in shape, indicating that they run roughly paral- fuse with fibrils of collagen fibers. Some reticular fibers sur-
lel to the vocal fold edge round bundles of collagen fibers.
There are glycoproteins around the reticular fibers
The predominant type of collagen in the lamina propria of (Fig. 10.20). Glycoproteins are distributed in the spaces
the human vocal folds is type III [10, 20]. Type III collagen among the reticular fibers. There are also glycosamino-
appears to be a major constituent of the slender, 50 nm or less glycans (proteoglycans) around the reticular fibers
fibers that have traditionally been called reticular fibers [19]. (Fig. 10.21). Glycosaminoglycans (proteoglycans) are
The reticular fibers, which are thin and are stained situated in the spaces among the reticular fibers. Elastic
black with silver stain, are found in the superficial and fibers are also located in the spaces among the reticular
intermediate layer of the lamina propria of the vocal fold fibers (Fig. 10.22).
a c
collagen fibers
reticular fibers
B
b superficial Intermediate deep layer Fig. 10.16 (continued)

layer layer
collagen fiber stratified squamous epithelium
collagen fibers
Fig. 10.16 (a) Coronal section of the lamina propria of the human

laryngeal glands
vocal fold mucosa (32-year-old male, silver stain). (b) Transition areas
between superficial, intermediate, and deep layers of the lamina propria
of the human vocal fold mucosa (region B in a). The reticular fibers Fig. 10.17 Lamina propria of the inferior portion of the vocal fold
stained black are found in the superficial and intermediate layers of the mucosa (32-year-old male, silver stain). There are no reticular fibers at
lamina propria. They are not detected in the deep layer of the lamina the superior and inferior portions of the vocal fold mucosa, and only
propria. The collagen fibers stained red are found in the deep layer of collagen fibers stained red are observed
the lamina propria. (c) Superficial layer of the lamina propria of the
human vocal fold mucosa (region C in a). Many reticular fibers stained
black with silver stain are observed 10.9.1.2 C ollagen and Reticular Fibers
in the Superficial Layer of the Lamina
Propria of the Human Vocal Fold
Relatively dense clusters of reticular fibers are observed Mucosa and Their Biomechanical
in close association with the basal lamina of the vocal fold Properties
epithelium (Fig. 10.23). They are located at the lamina retic- It is now generally accepted that reticular and collagen
ularis of the basal lamina (Fig. 10.24). fibers are essentially identical biochemically and have
Relatively dense clusters of reticular fibers are observed now been found to be different morphological forms of
in close association with the tissue surrounding the endothe- the same fibrous protein, collagen [19]. The size and pat-
lial cells and pericytes of the vessels in the vocal fold mucosa tern of the reticular fibers are different from those of more
around the edge (Figs. 10.25 and 10.26). typical collagen fibers [19]. It is useful for descriptive
a collagen fibers
67 nm
reticular fibers
B
42 nm
reticular fibers cross-band
Fig. 10.19 Transmission electron micrograph of reticular fibers in the

superficial layer of the lamina propria of the vocal fold mucosa
b (34-year-old male, uranyl acetate and lead citrate stain)
reticular fibers
glycoprotein
reticular fibers
reticular fibers
Fig. 10.18 (a) Scanning electron micrograph of reticular fibers in the

superficial layer of the lamina propria of the vocal fold mucosa
(45-year-old male, NAOH maceration method). Reticular and collagen Fig. 10.20 Transmission electron micrograph of reticular fibers and
fibers alone remain following treatment by the modified sodium glycoprotein in the superficial layer of the lamina propria of the vocal
hydroxide maceration method. (b) Region B in (a) fold mucosa (34-year-old male, tannic acid stain). The reticular fibers
are covered with glycoprotein and the crossbands of the reticular fibers
cannot be seen
purposes to retain the term reticular fiber for fibrils com-

posed of collagen 50 nm or less in diameter and the term of type III collagen reticular fibers is structural maintenance
collagen fiber for bundles consisting of unit fibrils in expansible organs [19].
50–150 nm in diameter [19]. Reticular fibers in the human vocal fold mucosa are com-
Type III collagen is one of the fibrillar collagens and posed of slender fibrils, and those fibrils do not form bundles
appears to be a major constituent of the slender 50 nm fibers but form delicate three-dimensional networks. The delicate
that have traditionally been called reticular fibers [19]. The three-dimensional structure of the reticular fibers contributes
collagen component of reticular fibers is principally type III to maintaining the structure of the vocal fold mucosa during
collagen [19]. The predominant type of collagen in the lam- phonation without disturbing vibration [20].
ina propria of the vocal folds is type III [10, 20]. In addition, the three-dimensional structure of the reticu-
Collagen fibers are flexible but offer great resistance to lar fibers, whose slender fibrils form delicate three-
any pulling force [19]. Type III collagen reticular fibers are dimensional networks, possesses innumerable potential
thin and result in a more compliant tissue [21]. The function spaces, and the spaces among the fibers are relatively large in
filament
proteoglycan
reticular fibers
reticular fibers
proteoglycan collagen fibers
Fig. 10.23 Reticular fibers at the basal lamina of the vocal fold epithe-
Fig. 10.21 Transmission electron micrograph of reticular fibers and lium (silver stain)
glycosaminoglycan (proteoglycans) in the superficial layer of the lam-
ina propria of the vocal fold mucosa (34-year-old male, ruthenium red
stain). The small dots associated with reticular fibers represent granules
of proteoglycan. Proteoglycan granules are attached to the reticular
fibers and are connected by ruthenium red-staining filaments
basal cell
reticular fibers
elastic fibers
reticular fibers
zona reticularis
elastic fibers
Fig. 10.24 Transmission electron micrograph of reticular fibers at the

basal lamina of the vocal fold epithelium (uranyl acetate and lead citrate
glycoprotein
stain)
Fig. 10.22 Transmission electron micrograph of reticular fibers and

elastic fibers in the superficial layer of the lamina propria of the vocal cosaminoglycan (proteoglycan) is very important for the vis-
fold mucosa (34-year-old male, tannic acid stain)
coelastic properties of the lamina propria of the human vocal
fold mucosa, which are very important for the vibratory
the human vocal fold mucosa. These extracellular interstitial behavior of the structure.
spaces are made up of minute chambers or compartments In fact, type III collagen reticular fibers are most abun-
occupied by other extracellular matrices. The reticular fibers dantly seen around the vocal fold edge [20]. Consequently,
are covered with glycoprotein and glycosaminoglycan (pro- type III collagen reticular fibers are located at the portion of
teoglycan). Glycoprotein and glycosaminoglycan (proteo- the vocal fold mucosa which vibrates the most during phona-
glycan) are also situated among the spaces of reticular fibers, tion [20].
and elastic fibers run through the spaces. One of the roles of Reticular fibers are also found in close association with
glycosaminoglycan (proteoglycan) is to give viscosity to tis- the basal lamina of the vocal fold epithelium and the blood
sues [22]. The main role of the elastic fibers is to give elastic- vessels. The portion of the vocal folds which vibrates the
ity and resilience to the tissues [21]. The complex of type III most is the cover, consisting of the epithelium and the
collagen reticular fibers, elastic fibers, glycoprotein, and gly- superficial layer of the lamina propria. The epithelium and
elastic fibers
capillary
reticular fibers
Fig. 10.25 Reticular fibers around the blood vessels in the vocal fold
mucosa (silver stain)
endothelial cell elastic fibers
pericyte
reticular fibers
Fig. 10.26 Transmission electron micrograph of reticular fibers

around the blood vessels in the vocal fold mucosa (tannic acid stain)
the blood vessels are attached to the superficial layer via the
basal lamina. One of the most highly stressed portions in the
lamina propria of the vocal fold mucosa is the junction Fig. 10.27 (a) Scanning electron micrograph of elastic fibers in the
between the epithelium and lamina propria, and another is superficial layer of the lamina propria of the vocal fold mucosa
the junction between the basal lamina of the blood vessels (39-year-old male, NAOH maceration method). (b) Higher magnifica-
tion of (a). Elastic fibers alone remain following treatment by the modi-
and lamina propria. The delicate three-dimensional networks fied sodium hydroxide maceration method
of reticular fibers distributed at these portions maintain the
structure during vibration [20].
The three-dimensional structure of reticular fibers (type 10.9.1.3 Elastic Fibers (Elastic System Fibers)
III collagen) in the human vocal fold mucosa appears to be In the superficial layer of the lamina propria (Reinke’s space)
one of the key components in the structural maintenance and of the human vocal fold mucosa, elastic fibers are slender,
viscoelasticity of the vibrating tissue [20]. The complex of run in various directions, and branch and anastomose to form
type III collagen reticular fibers and other extracellular loose networks (Fig. 10.27).
matrices seems to be very important for the viscoelastic Ultrastructurally, elastic system fibers are composed of
properties of the human vocal fold mucosa [20]. various amounts of amorphous substances (elastin) and
a amorphos substance (elastin)

amorphos substance
(elastin)
microfibrils
elastic fibers
microfibrils
b
Fig. 10.29 Transmission electron micrograph of elaunin fibers (area
encircled with red dotted line) in the superficial layer of the lamina
amorphos substance propria of the vocal fold mucosa (34-year-old male, tannic acid stain)
(elastin)
elastic fibers
microfibrils microfibrils
Fig. 10.28 Transmission electron micrograph of elastic fibers in the

superficial layer of the lamina propria of the vocal fold mucosa. (a) A
60-year-old male, uranyl acetate and lead citrate stain. (b) A 34-year-
old male, tannic acid stain. Elastin (amorphous substance) is stained
black
microfibrillar components. Elastic fibers are composed of

amorphous substances (elastin) and microfibrils (Fig. 10.28). Fig. 10.30 Transmission electron micrograph of oxytalan fibers (area
Elaunin fibers have microfibrils and a small amount of amor- encircled with red dotted line) in the superficial layer of the lamina
propria of the vocal fold mucosa (32-year-old male, ruthenium red stain
phous substances (elastin) (Fig. 10.29). Oxytalan fibers are
and tannic acid stain)
composed of only microfibrils 10–12 nm in diameter
(Fig. 10.30).
In the shallow portion of the superficial layer of the lam- 10.9.1.4 E lastic Fibers in the Superficial Layer
ina propria of the vocal fold, the thin fibers (oxytalan and of the Lamina Propria of the Human
elaunin fibers) are present (Fig. 10.31). Mature elastic fibers Vocal Fold Mucosa and Their
are observed in the deep portion of the superficial layer, in Biomechanical Properties
the intermediate layer, and in the deep layer of the lamina Elastic fibers are also fibrillar proteins and are a major com-
propria of the vocal fold (Fig. 10.31a). A delicate network of ponent of the extracellular matrices in the human vocal fold
thin and undulated fibers that branch and anastomose with mucosa. The main roles of the elastic fibers are to give elas-
each other run parallel to the epithelial basal lamina ticity and resilience to the tissues [21]. Elastic fibers stretch
(Fig. 10.31b). In addition, thin fibers are present just below even under small forces and easily return to their original
the basal lamina (Fig. 10.31b). These fibers are arranged dimensions when the forces are released [12].
transversally between the basal lamina and lamina propria of Elastic fibers do not consist solely of elastin. The
the mucosa. elastin core is covered with a sheath of microfibrils, each
a
microfibrils
filament
oxytalan fibers proteoglycan
reticular
fibers
proteoglycan
microfibrils
elastic fibers
filament
Fig. 10.32 Transmission electron micrograph of microfibrils and

b other extracellular matrices in the superficial layer of the lamina propria
of the vocal fold mucosa (32-year-old male, ruthenium red stain and
tannic acid stain). Ruthenium red-stained small dots and filaments rep-
stratified squamous epithelium resent proteoglycan
10.9.1.5 Microfibril-Associated
Macromolecules
oxytalan fibers
There are many slender microfibrils (approximately 10 nm in
diameter) in the superficial layer of the lamina propria
(Reinke’s space) of the human vocal fold mucosa (Fig. 10.32).
Microfibrils are made up of a single unit fibril approximately
10 nm in diameter. Some microfibrils are observed to be
alone, while others are observed with other extracellular
matrices (e.g., reticular fibers, elastic fibers, collagen fibers,
proteoglycan, and glycoprotein).
There are glycoproteins around the microfibrils. The
Fig. 10.31 (a) Transverse section of the superficial layer of the lamina
propria of the human vocal fold mucosa (50-year-old male, Weigert’s fibrils of microfibrils are covered with glycoprotein.
resorcin-fuchsin stain with previous oxidation performed using oxone). Glycoproteins are situated around the microfibrils and in the
(b) Higher magnification spaces among the microfibrils. There are also gly
cosaminoglycans (proteoglycans) around the microfibrils.
of which has a diameter of about 10 nm. The microfibrils Glycosaminoglycans (proteoglycans) are situated around the
appear before elastin in developing tissues and seem to microfibrils and in the spaces among the microfibrils.
provide scaffolding to guide elastin deposition. This The microfibrils form delicate three-dimensional net-
amorphous modification is a result of the cross-linking works with other extracellular matrices.
of elastin in previously deposited microfibrils and is the
first step of elastogenesis [23]. Oxytalan and elaunin 10.9.1.6 Microfibril-Associated
fibers are believed to be interrupted states of elastic fiber Macromolecules in the Superficial
maturation differing only in the amount of cross-linking Layer of the Lamina Propria
present [23]. of the Human Vocal Fold Mucosa
In the elastic system fibers, oxytalan and elaunin fibers and Their Biomechanical Properties
are less stretchable and are found in tissues that experi- There is growing evidence that fibrillin-containing microfi-
ence greater stress. Elastic fibers are the most elastic. The brils are not just fibrillin polymers but that a variety of addi-
function of the different types and stages of elastin is tional macromolecules may be associated with these
important in regulating the mechanics of the superficial structures [25]. The interface between the microfibrils and
layer of the lamina propria of the human vocal fold other extracellular matrices has not been adequately identi-
mucosa [24]. fied in the human vocal fold mucosa.
The functions of these molecules are envisioned to include effect on the physical properties of the extracellular matrices
(a) structural support to stabilize the interaction of fibrillin [26]. Interstitial proteins such as the proteoglycans fill in the
molecules within the microfibril; (b) mediation of the interac- spaces between the fibrous proteins and therefore probably
tion of adjacent microfibrils within bundles; (c) assembly of have a strong effect on biomechanical performance [27].
elastin on the surface of the microfibrils; (d) interfacing Further study will allow precise quantitative analysis of mac-
between the microfibrils and other structural elements of dif- romolecules as well as elucidation of specific vocal fold dis-
ferent material; (e) modulation of the interaction of the micro- ease states that exhibit distinctive changes in the proteoglycan
fibrils with cells to influence the deposition, orientation, and content and distribution in the lamina propria of the vocal
organization of microfibrils and elastic fibers in different tis- fold mucosa [26].
sue environments; (f) provision and modulation of nonstruc-
tural functions of the microfibrils, e.g., TGF-beta storage; (g) 10.9.1.8 Hyaluronic Acid (Hyaluronan)
enzymatic activity, e.g., lysyl oxidase; and (h) specific interac- Although most of the glycosaminoglycans found in the
tions with fibrilin-2-containing microfibrils [25]. extracellular matrix exist only as components of proteogly-
The microfibril-associated macromolecules have associa- can and not as free glycosaminoglycans, hyaluronic acid
tions with additional matrix components and thus are likely (hyaluronan) is an exception. Hyaluronic acid is distributed
to possess some of the microfibril-independent functions in the lamina propria of the human vocal fold from birth
listed above [25], indicating that the functions of the (Fig. 10.33).
microfibril-associated macromolecules are likely to be spe- After the discovery of hyaluronic acid (hyaluronan), it
cific to some aspect of the mechanical biology of the human was assumed that its major functions were in the biophysical
vocal fold mucosa. and homeostatic properties of tissues. Indeed, hyaluronic
Further studies are required for the role of microfibril- acid has a marked effect on tissue viscosity, tissue flow, tis-
associated macromolecules in relation to the biomechanical sue osmosis, tissue dampening (shock absorption), and space
properties and regeneration of the human vocal fold as a filling [28]. Hyaluronic acid is a key molecule influencing
vibrating tissue. the tissue viscosity of the lamina propria of the human vocal
fold mucosa [27]. Hyaluronic acid also contributes to opti-
10.9.1.7 Proteoglycan and Glycosaminoglycans mal tissue stiffness, important for vocal fundamental fre-
In the human vocal fold mucosa, the extracellular matrices quency control [29].
are composed of two families of macromolecules: fibrillar
proteins (collagen and elastin), which provide the fibrous
scaffolding of the lamina propria of the mucosa, and the sub-
stances found in the interstitium between the fibrous scaf-
folding—the proteoglycans (glycosaminoglycan) and
structural glycoproteins (Fig. 10.14).
Proteoglycans are composed of glycosaminoglycan
chains covalently attached via a linkage protein to a protein fibroblasts
core [26]. Glycosaminoglycans are linear polymers and
include keratan sulfate, chondroitin sulfate, dermatan sul-
fate, heparan sulfate, and hyaluronic acid. Hyaluronic acid is
not covalently attached to proteins and differs from other
glycosaminoglycans. hyaluronic acid
Three subgroups within the extracellular matrix proteo-
glycan family have been identified: (1) small, interstitial
matrix proteoglycans, decorin, biglycan, and fibromodulin;
(2) large, aggregating chondroitin sulfate proteoglycans,
aggrecan and versican; and (3) heparan sulfate proteoglycans
[26]. This classification is based on the similarities of the
protein cores and glycosaminoglycan chains [26]. All three
types of proteoglycans have been identified within the lam- Fig. 10.33 Transverse section of the vocal fold mucosa (Alcian Blue
ina propria of the vocal fold mucosa [26]. Pawlak et al. stain). Lamina propria of the vocal fold mucosa is stained light blue
with Alcian Blue at pH 2.5. Material stained with Alcian Blue (pH 2.5)
reported that decorin and probably fibromodulin have a lam- is digested by hyaluronidase. One of the glycosaminoglycans, hyal-
ina propria layer specificity [26]. Alteration in the concentra- uronic acid (hyaluronan), is present in the lamina propria of the vocal
tion or production of the proteoglycans could have a profound fold mucosa
10.9.1.9 Glycoprotein 10.10 I nterstitial Cells in Reinke’s Space

Another extracellular matrix substance found in the intersti- of the Human Vocal Fold Mucosa
tium between the fibrous scaffolding is structural glycopro-
teins (Fig. 10.14). The structural glycoproteins are distributed 10.10.1 Fibroblasts
in the human vocal fold mucosa (Figs. 10.20 and 10.22).
Fibronectin, the most common of the glycoproteins in the Fibroblasts are one of the interstitial cells that produce
extracellular matrices, is ubiquitous in the lamina propria of fibrous proteins and other extracellular matrices. However,
the vocal fold mucosa [30]. Fibronectins are a family of fibroblasts in the superficial layer of the lamina propria
closely related adhesive glycoproteins. They serve to help (Reinke’s space) of the vocal fold mucosa synthesize few
bind proteins together and provide molecular strength and extracellular matrices; they are inactive and quiescent under
adhesion between molecules. Fibronectin is present in nor- normal conditions [35].
mal vocal folds, and it seems to be in greater concentration in Some fibroblasts are present throughout the human vocal
some disease states such as nodules and scaring [31, 32]. fold mucosa (Figs. 10.35 and 10.36) but they are sparse.
A great deal of fibronectin is present in the lamina propria Fibroblasts are spindle-shaped and the nuclei elliptic. The
of the newborn vocal fold mucosa [33]. Fibronectin is a gly- nucleus cytoplasm ratio is large with poorly developed rough
coprotein that serves as a template for the oriented deposi- endoplasmic reticulum and Golgi apparatus.
tion of collagen [34]. It acts as an interfibrillar stabilizing
factor between collagen fibrils, as a skeleton for elastic tissue
formation, and is also involved with the aggregation of pro- 10.10.2 Myofibroblasts
teoglycans, elastic fibers, and glycosaminoglycans in the
human newborn and infant vocal fold mucosae [33]. The term “myofibroblasts” was coined by Majno et al. to
define cells which exhibit some of the ultrastructural features
of both smooth muscle cells and fibroblasts [36]. The myofi-
10.9.2 Extracellular Matrices in the broblasts bear resemblance to smooth muscle cells in that
Intermediate and Deep Layers they contain tracts of filaments with focal densities [37].
of the Lamina Propria of the Human Fibroblasts lack an external lamina but a lamina ensheaths
Vocal Fold Mucosa smooth muscle cells and myofibroblasts [37].
The intermediate layer is primarily made up of elastic fibers,

and the deep layer is primarily made up of collagen fibers
[1]. Both layers comprise the vocal ligament (Fig. 10.34).
fibroblast
Fig. 10.34 Scanning electron micrograph of the vocal ligament of the

human vocal fold mucosa (54-year-old male, NaOH maceration Fig. 10.35 Scanning electron micrograph of a fibroblast in the human
method). Fibers run roughly parallel to the vocal fold edge vocal fold mucosa (NaOH maceration method)
References 145
c
nucleus
collagen fiber
pinocytotic vesicles
basal lamina
fibroblast
elastic fiber
proteoglycan
Fig. 10.36 Transmission electron micrograph of a fibroblast in the
human vocal fold mucosa (uranyl acetate and lead citrate stain)
Myofibroblasts are seen in inflamed tissue, injured tissue,
repaired tissue, scars, fibroblastic tumors or tumorlike lesions
a
(fibromatosis), and tumors or lesions containing fibroblasts and
rough endoplasmic
reticulum histiocytes [37]. Myofibroblasts are seen under pathological
conditions in the human vocal fold mucosa (Fig. 10.37).
nucleus
References
1. Hirano M. Phonosurgery. Basic and clinical investigation. Otologia
(Fukuoka). 1975;21(Suppl. 1):239–60.
smooth-walled
3. Kurita S. Layer structure of the human vocal fold. Morphological
vacuole
investigation. Otologia (Fukuoka). 1980;26(Suppl. 6):973–97.
4. Pressman JJ. Physiology of the vocal cords in phonation and respi-
mitochondria ration. Arch Otolaryngol. 1942;35:355–98.
5. Ichikawa T. Basic studies on the lubrication of the larynx during
b phonation. Otologia (Fukuoka). 1982;28:38–52.
6. Tillmann B, Pietzsch I-R, Huenges HL. The human vocal cord sur-
face. Cell Tissue Res. 1977;185:279–83.
7. Gray SD, Titze I, Lusk RP. Electron microscopy of hyperphonated
filaments canine vocal cords. J Voice. 1987;1:109–15.
8. Gray S, Titze I. Histologic investing of hyperphonated canine vocal
cords. Ann Otol Rhinol Laryngol. 1988;97:381–8.
9. Sato K, Kashiwagi S, Hirano M. Ultrastructure of the mucous
membrane of the human newborn vocal folds. J Otolaryngol Jpn.
1997;100:479–83.
10. Gray SD, Hirano M, Sato K. Molecular and cellular structure of
basal lamina vocal fold tissue. In: Titze IR, editor. Vocal fold physiology. San
Diego, CA: Singular Publishing Group, Inc; 1993. p. 1–35.
11. Gray SD, Pignatari SSN, Harding P. Morphologic ultrastructure of
anchoring fibers in normal vocal fold basement membrane zone. J
Voice. 1994;8:48–52.
12. Fawcett DW. Epithelium. A textbook of histology. Philadelphia,
PA: W.B. Saunders Co; 1986. p. 57–72.
13. Fawcett DW. The cell. Philadelphia, PA: W.B. Saunders Co; 1981.
Fig. 10.37 (a) Transmission electron micrograph of a myofibroblast in 14. Sato K, Hirano M. Langerhans cells in the larynx and the hypophar-
the vocal fold mucosa (ruthenium red stain and uranyl acetate and lead ynx. Kurume Med J. 1997;44:297–303.
citrate stain). (b) Myofibroblasts contain tracts of filaments with focal 15. Romani N, Schuler G. The immunologic properties of epidermal
densities. The basal lamina ensheaths myofibroblasts. (c) Pinocytotic Langerhans cells as a part of the dendritic cell system. Springer
vesicles are observed at the periphery of the cytoplasm Semin Immunopathol. 1992;13:265–79.
16. Steinman RM. The dendritic cell system and its role in immunoge- 27. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteo-
nicity. Annu Rev Immunol. 1991;9:271–96. glycans and their influence on biomechanics. Laryngoscope.
17. Thompson AC, Griffin NR. Langerhans cells in normal and patho- 1999;109:845–54.
logical vocal cord mucosa. Acta Otolaryngol. 1995;115:830–2. 28. Laurent TC, Laurent UB, Fraser JR. Functions of hyaluronan. Ann
18. Mogi G, Watanabe N, Maeda S, Umehara T. Laryngeal secre- Rheum Dis. 1995;54:429–32.
tions. An immunochemical and immunohistological study. Acta 29. Chan RW, Gray SD, Titze IR. The importance of hyaluronic

Otolaryngol. 1979;87:129–41. acid in vocal fold biomechanics. Otolaryngol Head Neck Surg.
19. Fawcett DW. Connective tissue proper. A textbook of histology. 2001;124:607–14.
Philadelphia, PA: WB Saunders; 1986. p. 136–51. 30. Hirschi SD, Gray SD, Thibeault SL. Fibronectin: an interesting
20. Sato K. Reticular fibers in the vocal fold mucosa. Ann Otol Rhinol vocal fold protein. J Voice. 2002;16:310–6.
Laryngol. 1998;107:1023–8. 31. Gray SD, Hammond E, Hanson DF. Benign pathologic responses of
21.
Ayad S, Boot-Handford RP, Humphries MJ, Kadler KE, the larynx. Ann Otol Rhinol Laryngol. 1995;104:13–8.
Shuttleworth CA. The extracellular matrix facts book. London: 32. Courey MS, Shohet JA, Scott MA, Ossoff RH. Immunohistochemical
Academic Press; 1994. characterization of benign laryngeal lesions. Ann Otol Rhinol
22. Hay ED. Cell biology of extracellular matrix. 2nd ed. New York: Laryngol. 1996;105:525–31.
Plenum Press; 1991. 33. Sato K, Hirano M, Nakashima T. Fine structure of the human new-
23. Schwartz E, Fleischmajer R. Association of elastin with oxytalan born and infant vocal fold mucosae. Ann Otol Rhinol Laryngol.
fibers of the dermis and with extracellular microfibrils of cultured 2001;110:417–24.
skin fibroblasts. J Histochem Cytochem. 1986;34:1063–8. 34. Anderson JC. Glycoproteins of the connective tissue matrix. In:
24. Hammond TH, Zhou R, Hammond EH, Pawlak A, Gray SD. The Hall DH, Jackson DS, editors. International review of connec-
intermediate layer: a morphologic study of the elastin and hyal- tive tissue research, vol. 7. New York: Academic Press; 1976.
uronic acid constituents of normal human vocal folds. J Voice. p. 251–322.
1997;11:59–66. 35. Hirano M, Sato K, Nakashima T. Fibroblasts in human vocal fold
25. Gibson MA. Microfibril-associated glycoprotein-1 (MAGP-1) and mucosa. Acta Otolaryngol. 1999;119:271–6.
other non-fibrillin macromolecules which may possess a functional 36.
Majno G, Gabbiani G, Hirschel BJ, Ryan GB, Statkov
association with the 10 nm microfibrils. In: Madame curie biosci- PR. Contraction of granulation tissue in vitro: similarity to smooth
ence database (internet). Austin, TX: Landes Bioscience; 2000.– muscle. Science. 1971;173:548–50.
2013. (http://www.ncbi.nlm.nih.gov/books/NBK6448/). 37.
Ghadially FN. Myofibroblasts and myofibroblastoma.
26.
Pawlak AS, Hammond T, Hammond E, Gray SD. Ultrastructural pathology of the cell and matrix. London:
Immunocytochemical study of proteoglycans in vocal folds. Ann Butterworths; 1988. p. 872–81.
Macula Flava and Vocal Fold Stellate
Cells of the Human Adult Vocal Fold 11
Abstract
1. Human adult maculae flavae are dense masses of cells and extracellular matrices located
at the anterior and posterior ends of the membranous portion of the bilateral vocal folds.
2. Human maculae flavae are most likely involved in the metabolism of extracellular
matrices essential for the viscoelasticity of the human vocal fold mucosa and are con-
sidered to be an important structure in the growth, development, and aging of the
human vocal fold mucosa.
3. The extracellular matrices of human adult maculae flavae are composed of glycopro-
teins and glycosaminoglycans (hyaluronan) and fibrillar proteins such as collagen
fibers, reticular fibers, and elastic fibers.
4. Vocal fold stellate cells contained in the human adult maculae flavae were discovered
(Sato, Ann Otol Rhinol Laryngol 110: 319-25, 2001). They are stellate in shape and
possess vitamin A-storing lipid droplets.
5. There are a number of morphological differences between vocal fold stellate cells and
fibroblasts in the human vocal fold mucosa.
6. Along the surface of the vocal fold stellate cells, a number of vesicles are present and
constantly synthesize extracellular matrices which are essential for the viscoelastic
properties of the human vocal fold mucosa.
7. Vocal fold stellate cells possess cytoplasmic processes and are stellate in shape, des-
min-positive cells with perinuclear vitamin A lipid droplets; therefore, the vocal fold
stellate cells show the morphological features of hepatic stellate cells. These results are
consistent with the concept that the vocal fold stellate cells are a member of the pro-
posed diffuse stellate cell system.
8. Radiosensitivity of the vocal fold stellate cells is higher than that of fibroblasts, and
radiation induces dysfunction of the vocal fold stellate cells.
9. As a result of the heterogeneity seen between vocal fold stellate cells and other inter-
stitial cells, it is uncertain whether they derive from the same embryonic source as
fibroblasts in the human vocal fold mucosa.
10. The vocal fold stellate cells in the maculae flavae form an independent cell category
that should be considered a new category of cells in the human vocal fold mucosa.
11.1 Introduction conspicuous mucosal bulges, and they are visible through the
mucosa as whitish-yellow masses.
Clinically, the anterior and posterior maculae flavae are The anterior macula flava of the vocal fold has already
observed at each end of the membranous portion of the vocal been described as the nodulus elasticus in the anatomic text-
fold during endoscopy of the larynx (Fig. 11.1). They form book by Lanz and Wachsmuth [1]. They described the mac-

148 11 Macula Flava and Vocal Fold Stellate Cells of the Human Adult Vocal Fold
11.2 M
aculae Flavae in the Human Adult
Vocal Fold
The vibratory portion (membranous portion) of the vocal

vocal fold is connected to the thyroid cartilage anteriorly via the
process intervening anterior macula flava and anterior commissure
of arytenoid
cartilage tendon. Posteriorly, it is joined to the vocal process of aryte-
glottis posterior noid cartilage via the intervening posterior macula flava
macula flava (Figs. 11.2 and 11.3) [3].
membranous Human adult maculae flavae are dense masses of cells and
vocal fold extracellular matrices (Figs. 11.3, 11.4, 11.5, 11.6, and 11.7)
anterior [12, 13]. The maculae flavae are located at the anterior and
macula flava posterior ends of the membranous portion of the bilateral
anterior commissure vocal folds. They are elliptical in shape and their size is
approximately 1.5 mm × 1.5 mm × 1 mm [12]. The border
between the maculae flavae and the surrounding soft tissue is
relatively clearly delineated (Figs. 11.3, 11.4, 11.8, and 11.9)
Fig. 11.1 Laryngeal endoscopic view of the glottis. Anterior and pos- [14]. The vocal ligament runs between the anterior and pos-
terior maculae flavae can be observed at each end of the membranous
portion of the vocal fold as whitish-yellow masses and conspicuous
terior maculae flavae (Fig. 11.3a).
mucosal bulges The extracellular matrices of human adult maculae flavae
are composed of glycoproteins and glycosaminoglycan and
fibrillar protein such as collagen fibers, reticular fibers, and
ula flava as elastic nodules located at the anterior end of the elastic fibers (Figs. 11.6 and 11.7). These extracellular matri-
vocal fold with numerous elastic fibers [1]. Hirano noted ces in the maculae flavae extend to those in the lamina pro-
the maculae flavae to be masses of dense elastic fibers at the pria (Reinke’s space and vocal ligament) of the human vocal
anterior and posterior ends of the vocal fold, referring to the fold mucosa [12].
former as the anterior macula flava and the latter as the pos- Interstitial cells with a starlike appearance in the human
terior macula flava [2]. adult maculae flavae were discovered in our laboratory in 2001
The histological structure of maculae flavae in the human (Figs. 11.10 and 11.11) [8]. These cells had no nomenclature
adult vocal fold mucosa is unique, and their roles in the vocal and were thus designated vocal fold stellate cells in the series
fold as a vibrating tissue are very interesting. However, their
roles in the human vocal fold have not been clarified until
recently [1–7]. thyroid cartilage
anterior commissure tendon
Vocal fold stellate cells contained in the human macu-
lae flavae were discovered in our laboratory in 2001 [8].
They are considered to be a new category of cells in the
human vocal fold mucosa. Recently, there is growing evi- Reinke’s space
dence to suggest that the cells including vocal fold stellate
vocal ligament
cells in the human maculae flavae are tissue stem cells or
progenitor cells of the vocal fold mucosa, and that the
maculae flavae are a candidate for a stem cell niche, that is, posterior macula flava
a microenvironment nurturing a pool of cells including elastic cartilage portion
of the vocal process
vocal fold stellate cells [9–11]. On the other hand, the
question arises whether the vocal fold stellate cells are tis- of the vocal process
sue stem cells or progenitor cells (transit-amplifying cells).
The vocal fold stellate cells are possibly transit-amplifying
cells, that is, progenitor cells [11]. However, at the present
state of our investigation, it is difficult to determine
whether the vocal fold stellate cells are tissue stem cells or
progenitor cells. Fig. 11.2 Schema of human adult vocal fold and maculae flavae
11.2 Maculae Flavae in the Human Adult Vocal Fold 149
a c
thyroid cartilage vocal ligament
Reinke’s space
posterior
vocal ligament macula flava
vocal process of
vocal process of arytenoid cartilage
arytenoid cartilage
thyroid cartilage
b
anterior
commissure
tendon
anterior
macula flava
vocal ligament
Fig. 11.3 (a) Transverse section of human adult vocal fold (Elastica van Gieson stain). (b) Transverse section of human adult anterior macula
flava (Elastica van Gieson stain). (c) Transverse section of human adult posterior macula flava (Elastica van Gieson stain)
of our study. Many vocal fold stellate cells are present in the But fibroblasts are sparse in Reinke’s space. The density of
human adult maculae flavae, and their density is high [8]. cells in the adult maculae flavae is about 2.5 times that in
However, none are found in Reinke’s space [8]. Fibroblasts Reinke’s space [15].
can be seen throughout the human adult vocal fold mucosa.
vocal fold
stellate cells
fibers lipid droplets
posterior
thyroarytenoid macula flava
muscle
vocal fold stellate cell
conus elasticus
collagen fibers
elastic fibers
Fig. 11.4 Coronal section of posterior macula flava (Elastica van
Gieson stain)
vocal fold stellate cells

collagen fibers
Vocal fold stellate cells
reticular fibers
Fig. 11.5 Macula flava of the human adult vocal fold (hematoxylin
and eosin stain). Human adult maculae flavae are dense masses of cells Fig. 11.6 Macula flava of the human adult vocal fold. (a) Human adult
maculae flavae are dense masses of cells and extracellular matrices
(toluidine blue stain, original ×400). (b) There are many collagen fibers
stained red and elastic fibers stained black around the vocal fold stellate
The latest research shows human maculae flavae con- cells in the human adult maculae flavae (Elastic van Gieson stain). (c)
taining vocal fold stellate cells to be involved in the There are many collagen fibers stained red and reticular fibers stained
black around the vocal fold stellate cells in the human adult maculae
metabolism of extracellular matrices essential for the vis- flavae (silver stain, original ×400). (d) Much glycosaminoglycan (hyal-
coelasticity of the human vocal fold mucosa. They are uronan, hyaluronic acid) is situated around the vocal fold stellate cells
considered to be an important structure in the growth, in the human adult maculae flavae (Alcian Blue stain, pH 2.5). Maculae
development, and aging of the human vocal fold mucosa flavae are strongly stained light blue with Alcian Blue at pH 2.5.
Material in the maculae flavae that is strongly stained with Alcian Blue
[13, 16–19]. (pH 2.5) is digested by hyaluronidase. (e) Coronal section of the poste-
rior macula flava (Alcian Blue stain, pH 2.5)
d a
collagen fibers
elastic fibers
hyaluronic acid (hyaluronan)

b
e
reticular fibers
collagen
fibers
elastic fibers
posterior
macula flava
Fig. 11.7 Transmission electron micrograph (a) and scanning electron

micrograph (b) of the macula flava of the vocal fold mucosa (a: tannic
acid stain, b: NaOH maceration method)
Fig. 11.8 Scanning electron

a
micrograph of the sagittal
section of the anterior macula
flava (a, arrows) and the
border (asterisks) between the
macula flava and lamina
B anterior
propria of the vocal fold lamina propria of
mucosa (b: region B in a) vocal fold mucosa commissure
tendon
anterior
macula flava
thyroid
cartilage
b
lamina propria of
vocal fold mucosa
anterior
macula flava
The histological structures of other mammalian vocal fold flavae are found to differ from those of human adult maculae
mucosa are different from human vocal folds [20]. The macu- flavae [21]. There have been some investigations of the macu-
lae flavae are also present at the anterior and posterior ends of lae flavae using animal samples [22–24]. Since the human
the membranous portion of animal vocal folds, but there is no vocal fold differs in histology, physiology, and pathology from
structure equivalent to the maculae flavae, vocal ligament, and those of other mammals, maculae flavae for investigation
layered structure of the human vocal fold [20]. For example, should ideally be collected from human samples. See Chap.
the structure and morphological functions of canine maculae 19, “Comparative Histoanatomy of the Vocal Fold Mucosa.”
a a
B
B
elastic fibers
anterior collagen fibers

nucleus
macula
flava
thyroid
cartilage
cytoplasmic
processes
C
b lamina propria of
vocal fold mucosa
epithelium b
vesicles
vesicles
rough endoplasmic
anterior reticula
mitochondrion
macula flava
nucleus
Fig. 11.9 Scanning electron micrograph of the coronal section of the ante-
rior macula flava (a, arrows) and the border (asterisks) between the macula
flava and lamina propria of the vocal fold mucosa (b: region B in a)
elastic fibers collagen
fibers
lipid droplet
lipid droplet
Fig. 11.10 Transmission electron micrograph of vocal fold stellate

cell in the human macula flava (uranyl acetate and lead citrate stain). (a)
Vocal fold stellate cells are irregular and stellate in shape and possess
vesicles
slender cytoplasmic processes. (b) Intracellular organelles in the vocal
fold stellate cell (region B in Fig. 11.10a). (c) Lipid droplets in the vocal
fold stellate cell (region C in Fig. 11.10a)
Table 11.1 Morphological comparison between vocal fold stellate

cells in the macula flava and fibroblasts in the human vocal fold mucosa
Vocal fold
stellate cells Fibroblasts
Location Macula flava Membranous portion
cytoplasmic of vocal fold mucosa
process
Cell density High Low
Shape Stellate Spindle, oval
Cytoplasmic process Present Not present
Nucleus-cytoplasm ratio Small Large
Intracellular organelle Well Poorly developed
developed
Rough endoplasmic Well Poorly developed
reticulum and Golgi developed
apparatus
Vesicles Many Few
Lipid droplets Many Few
vocal fold
Vitamin A storing Stored Not stored
stellate cells
Synthesis of extracellular Much Little
matrices
Collagen fibers around Many Not very many
cells
Reticular fibers around Many Not very many
Fig. 11.11 Scanning electron micrograph of vocal fold stellate cell in
cells
the human macula flava (NaOH maceration method)
Elastic fibers around cells Many Not very many
Hyaluronic acid around Much Not much
cells
11.3 Morphological Characteristics Radiosensitivity Higher than
fibroblasts
of the Human Adult Vocal Fold
Stellate Cells
The vocal fold stellate cells possess lipid droplets in the
There are a number of morphological differences between cytoplasm and are 1–2 μm in diameter (Figs. 11.10, 11.12,
vocal fold stellate cells and fibroblasts in the human vocal and 11.14), while fibroblasts have few lipid droplets. They
fold mucosa (Table 11.1). are distributed not only in the cell body but also in the cyto-
Many vocal fold stellate cells are distributed in the human plasmic processes and are of two types: membrane bounded
adult maculae flavae and their density is high (Fig. 11.5). Vocal (Fig. 11.12a) and non-membrane bounded (Fig. 11.12b). The
fold stellate cells are irregular and stellate in shape and possess former are each surrounded by a unit membrane. The rough
slender cytoplasmic processes (Figs. 11.10 and 11.11) [8]. endoplasmic reticulum and membrane bounded lipid drop-
The nucleus in vocal fold stellate cells is oval. The nucleus- lets are closely associated with each other (Fig. 11.12a).
cytoplasm ratio is small, and well-developed intracellular Vesicles and lipid droplets are also present in close proximity
organelles such as rough endoplasmic reticulum and Golgi (Fig. 11.12a). The unit membrane becomes indistinct near
apparatus are present (Fig. 11.10), indicating that protein syn- the vesicles (Fig. 11.12a).
thesis is occurring within them [8]. The vesicular and tubular The vocal fold stellate cells show strong cytoplasm stain-
elements of the rough endoplasmic reticulum frequently expand ing with periodic acid-Schiff (Fig. 11.15) which stains few
to form flat saccular structures called cisternae (Fig. 11.12), components in the cytoplasm of fibroblasts. The cytoplasm
indicating the rough endoplasmic reticulum is moderately dis- of vocal fold stellate cells appears to contain a great deal of
tended with proteinaceous secretary product. The Golgi appa- glycogen and glycoprotein [8].
ratus consist of lamellae associated with vesicles (Fig. 11.12a). The vocal fold stellate cells show cytoplasmic staining
Microfilaments are distributed in the cytoplasm, and 10-nm- with type I (Fig. 11.16a) and type III collagen (Fig. 11.16b),
thick intermediate filaments are also present (Fig. 11.12a). while type I and type III collagens stain few components in
Lysosomes are few. Mitochondria are small and few. the cytoplasm of fibroblasts. The meaning of these findings
Microtubules and basal bodies composed of microtubules are is unclear but possibly may be related to the production of
noted in the cytoplasm (Fig. 11.13). No basal lamina is found. collagen fibrils.
11.4 Synthesis of Extracellular Matrices by the Vocal Fold Stellate Cells 155
a
membrane-
bounded vesicles
lipid droplet
lipid droplets
indistinct membrane
near vesicles

filaments
Golgi apparatus rough endoplasmic
reticula
Fig. 11.14 Lipid droplets in the vocal fold stellate cells are stained
reddish brown (Sudan III stain, original ×400)
b
lipid droplet
lipid droplets
lipid droplet
non-membrane- lipid droplet

bounded
lipid droplet vocal fold stellate cells
Fig. 11.12 Transmission electron micrograph of lipid droplets in a

vocal fold stellate cell. (a) Membrane-bounded lipid droplet and intra- Fig. 11.15 Vocal fold stellate cells show strong cytoplasmic staining
cellular organelles (uranyl acetate and lead citrate stain). (b) Non- with periodic acid-Schiff stain (original ×400)
membrane-bounded lipid droplets (tannic acid stain)
11.4 S
ynthesis of Extracellular Matrices by
the Vocal Fold Stellate Cells
Along the surface of the vocal fold stellate cells, a number of

vesicles are present and constantly synthesize extracellular
matrices which are essential for the viscoelastic properties of
the human vocal fold mucosa [8]. The extracellular matrices
synthesized by the vocal fold stellate cells maintain the vis-
basal bodies coelastic properties of the human adult vocal fold. The fibro-
blasts in Reinke’s space are inactive and produce few
extracellular matrices.
The vocal fold stellate cells in the human adult maculae
flavae have some role in the metabolism of the extracellular
matrices under normal conditions. Fibroblasts in Reinke’s
space may be activated in a pathological state such as injury
Fig. 11.13 Transmission electron micrograph of basal bodies in the of the vocal fold tissue.
vocal fold stellate cell (uranyl acetate and lead citrate stain)
a 11.4.2 Elastic Fibers
collagen fibers (type I) There are many elastic fibers around the vocal fold stellate
cells in the human adult maculae flavae (Figs. 11.6 and
11.7).
The vocal fold stellate cells constantly synthesize elas-
tic fibers in the human adult maculae flavae (Fig. 11.18)
[8]. There are many vesicles at the periphery of the cyto-
plasm, and newly released amorphous materials are pres-
ent on the cell surface of the vocal fold stellate cells [8].
Microfibrils 10–15 nm wide are situated around the amor-
phous material. There are microfibril assemblies on which
elastin appears to be deposited. The amorphous substances
of the elastic fibers are produced by the fusion of microfi-
brils. The elastic fibers consist of amorphous substances
and microfibrils.
b On the other hand, fibroblasts in Reinke’s space have few
vesicles at the periphery of the cytoplasm, and newly released
amorphous materials are not present on the cell surface,
according to electron microscopy.
11.4.3 Glycosaminoglycan
Human adult maculae flavae are strongly stained light blue
with Alcian Blue at pH 2.5 (Fig. 11.6d), and relatively sparse
staining with Alcian Blue at pH 1 is noted. The ground sub-
stances around vocal fold stellate cells are stained in the
same way. Material in the maculae flavae that is strongly
Fig. 11.16 Type I and type III collagen in the cytoplasm of vocal fold
stained with Alcian Blue (pH 2.5) is digested by hyaluroni-
stellate cells, shown by immunohistochemical staining. (a) Type I col- dase. A great deal of glycosaminoglycan (hyaluronan, hyal-
lagen. (b) Type III collagen (original ×400) uronic acid) is situated around the vocal fold stellate cells in
the human adult maculae flavae.
Hyaluronic acid (hyaluronan), one of the glycosamino-
11.4.1 Collagen Fibers glycans, plays an important role in the viscoelasticity of the
human vocal fold mucosa [25–27]. A great deal of hyal-
There are many collagen and reticular fibers (type III colla- uronic acid is situated around the vocal fold stellate cells in
gen) around the vocal fold stellate cells in the human adult the human adult maculae flavae (Fig. 11.6d) [8]. CD44 is a
maculae flavae (Figs. 11.6 and 11.7). cell membrane-localized receptor for hyaluronic acid. The
The vocal fold stellate cells constantly synthesize fibrous number of CD44-positive cells in the maculae flavae of the
protein not only for collagen fibers but also for reticular younger adults is large, and most of the vocal fold stellate
fibers in the human adult maculae flavae (Fig. 11.17) [8]. cells express CD44 (Fig. 11.19) [15]. On the other hand,
There are many vesicles at the periphery of cytoplasm, and CD44-positive fibroblasts in Reinke’s space are sparse [15].
newly released amorphous materials are present on the cell The vocal fold stellate cells constantly synthesize hyaluronic
surface of the vocal fold stellate cells. Microfibrils 10–15 nm acid in the human adult maculae flavae. It is interesting how
wide are observed around the amorphous material. Collagen cells organize their extracellular matrices and how these
fibrils are detected near the microfibrils. Collagen fibers are matrices feed back to cell metabolism involving specific cell-
made up of collagen fibrils. matrix interactions, which are mediated by cell surface
On the other hand, electron microscopic studies indicate matrix receptors. The vocal fold stellate cells in the maculae
that there are few vesicles at the periphery of the cytoplasm, flavae and CD44 cooperatively play important roles in the
and newly released amorphous materials are not present on metabolism of hyaluronic acid in the human vocal fold
the cell surface of the fibroblasts in Reinke’s space. mucosa [15].
11.4 Synthesis of Extracellular Matrices by the Vocal Fold Stellate Cells 157
Fig. 11.17 (a) Synthesis of a

collagen fibers by vocal fold
stellate cells (transmission
electron micrograph, tannic collagen fibril
acid stain). (b) Schematic collagen fiber
diagram of collagen and
reticular fiber formation microfibril amorphous
material
vesicles
rough
endoplasmic
reticulum
b rough endoplasmic
Golgi apparatus reticulum
cytoplasm
vesicle procollagen
collagen molecule
(tropocollagen)
assemble assemble
reticular fibers collagen fibrils collagen fibers
elastic fiber
microfibril
amorphous
material
vesicles
elastin microfibril vocal fold stellate cells
elastin
rough endoplasmic
reticulum
Fig. 11.18 Synthesis of elastic fibers by vocal fold stellate cells (trans- Fig. 11.19 CD44 (a cell membrane-localized receptor for hyaluronic
mission electron micrograph, tannic acid stain) acid) on the cytoplasm of the vocal fold stellate cells shown by immu-
nohistochemical staining
11.5 V
itamin A-Storing Stellate Cells
in the Human Maculae Flavae
The vocal fold stellate cells possess lipid droplets and store
vitamin A in the cytoplasm [28].
Two methods were employed for the detection of vitamin
A. The gold chloride method involves a reduction of gold
chloride by vitamin A [29]. The autofluorescence method
involves an autofluorescence emission by the excitation of
vitamin A by ultraviolet rays at a wavelength of around
340 nm [29].
Only scattered vocal fold stellate cells in the maculae
flavae are stained black with the gold chloride method
(Fig. 11.20a), and no black-stained elements can be seen in
Fig. 11.21 Fluorescence microscopy of the macula flava of the human

a vocal fold. Vitamin A fluorescence (arrows) is observed in the vocal
fold stellate cells distributed in the macula flava
cytoplasmic the cells or connective tissues outside of the maculae flavae

process
(Fig. 11.20b). This gold reaction is therefore highly spe-
cific for vocal fold stellate cells in the maculae flavae. Thus,
vocal fold
vocal fold stellate cells store vitamin A in their
stellate cell
cytoplasms.
lipid droplet The vitamin A autofluorescence from vocal fold stellate
cells in the macula flava of the human adult vocal fold is
shown in Fig. 11.21. Thus, vocal fold stellate cells possess
vitamin A in their cytoplasms. Autofluorescence of vitamin
A from vocal fold stellate cells disappears little by little as
b vocal fold stellate cells are exposed to ultraviolet
anterior macula flava radiation.
The results of both light microscopy by the gold chlo-
ride method and fluorescence microscopy indicate vitamin
A, an essential liposoluble vitamin, is stored in the lipid
droplets of vocal fold stellate cells in the human adult
maculae flavae [28].
It is generally accepted that vitamin A (retinoid) is an
essential liposoluble vitamin, used as a morphogen, which
surrounding tissue controls the differentiation and morphogenesis of cells.
Vitamin A strongly influences the activity of adenosine tri-
phosphate (ATP) sulfurylase and is related to the synthesis of
glycosaminoglycan [30–32]. Retinoid up-regulates the pro-
duction of transforming growth factor-beta (TGF-β) and
Fig. 11.20 Vitamin A in the vocal fold stellate cells (gold chloride induces activation of TGF-β [33]. In the porcine vocal fold,
method, no counterstaining). (a) The cytoplasm of vocal fold stellate
cells contains numerous fine grains of reduced gold, and the nuclei are
increased exposure to retinol induces sequential reorganiza-
stained reddish brown. Vocal fold stellate cells contain vacuoles in the tion of actin cytoskeleton in activated vocal fold stellate cells
cytoplasm, which are in fact vitamin A-containing lipid droplets (origi- [22]. On the other hand, vitamin A is not essential to main-
nal ×200). (b) Border (asterisks) between the cells of the adult anterior taining the extracellular matrices of the rat vocal fold [23].
macula flava containing vocal fold stellate cells and the surrounding
tissue. The border between macula flava and surrounding tissue is
The role of vitamin A (retinoid) in the human vocal fold stel-
clearly delineated (original ×50) late cells is still ambiguous.
11.6 Vocal Fold Stellate Cells as a Diffuse Stellate Cell System 159
11.6 V
ocal Fold Stellate Cells as a Diffuse and is a characteristic of neural crest cells. It is heavily and
Stellate Cell System specifically expressed in astrocytes and certain other astroglia
in the central nervous system. In addition, neural stem cells
Hepatic stellate cells play an important role in liver fibrogen- frequently strongly expressed GFAP.
esis [34]. After liver injury, quiescent hepatic stellate cells All stellate cells are desmin-positive cells with perinu-
become activated, lose their vitamin A stores, and develop clear vitamin A droplets [34]. Desmin belongs to the inter-
into contractile myofibroblast-like cells, which secrete extra- mediate filament protein family. Desmin is a characteristic of
cellular matrix proteins [34]. It is widely accepted that myogenic crest cells and is found in muscle cells.
hepatic stellate cell-derived myofibroblast-like cells contrib- The vocal fold stellate cells in the human maculae flavae
ute to liver fibrosis [34]. possess perinuclear vitamin A lipid droplets, and they
Morphologically similar cells have been found at many express the neural (GFAP)- (Fig. 11.22) and muscle-
extrahepatic sites such as the pancreas, lungs, kidney, spleen, associated (desmin) proteins (Fig. 11.23) seen in the hepatic
and intestine [35–37]. All these stellate cells are desmin-
positive cells with perinuclear vitamin A droplets [34].
Consequently, the concept of a “diffuse stellate cell system”
has been proposed [35–37]. However, the true relationship
between these populations at different sites remains
uncertain.
The concept of a diffuse stellate cell system was previ-
ously proposed as a classification for these cells under the
name “vitamin A-storing cell system” [35]. According to
Yamada and Hirosawa [35], the vitamin A-storing cells
showed the following morphological features: The cells are
irregular in shape and have extended, slender, often branched
cell processes. The cells possess several vitamin A-containing
lipid granules. The number of granules increases in hypervi-
taminosis. No basal lamina is found around the cell surfaces.
Well-developed rough endoplasmic reticulum and Golgi
apparatus are found, and the former is frequently dilated in
cisternal form. Filaments of about 50 Å (5 nm) diameter are
usually seen along the plasma membrane. Microtubules are Fig. 11.22 Glial fibrillary acidic protein (GFAP) in the cytoplasm of
frequently present, especially in the cell processes. Cells are the vocal fold stellate cells shown by immunohistochemical staining
located in the connective tissue spaces and have a close rela-
tionship to both the endothelium of the vascular vessel and
various epithelial tissues.
According to Wake [36], vitamin A-storing cells also have
the following morphological features: Along the surface of the
cytoplasm, a number of micropinocytotic vesicles or caveolae
are observed. Microfilaments are distributed in the cytoplasm
and microtubules and 10 nm filaments are also contained. vocal fold stellate cells
It has not been demonstrated whether the number of lipid
granules (droplets) in the vocal fold stellate cells increases in
hypervitaminosis or whether the vocal fold stellate cells
become activated, lose their vitamin A stores, and develop
into contractile myofibroblast-like cells. However, micro-
scopic studies have revealed that the vocal fold stellate cells
in the maculae flavae of the human vocal folds are similar to
the cells of the vitamin A-storing cell system [28].
A growing list of neural markers has been identified in the
hepatic stellate cells, including RhoN, Glial fibrillary acidic
protein (GFAP), nestin, and neurotrophin receptors [34]. Fig. 11.23 Desmin in the cytoplasm of the vocal fold stellate cells
GFAP is a member of the intermediate filament protein family shown by immunohistochemical staining
stellate cells. These results suggest that the vocal fold stellate a
cells show the morphological features of the hepatic stellate
cells. These results are consistent with the concept that the
vocal fold stellate cells are a member of the proposed diffuse
stellate cell system. As a result of this heterogeneity, it is
uncertain whether the vocal fold stellate cells derive from the
same embryonic source as fibroblasts in the human vocal vocal fold stellate cells
fold mucosa.
11.7 I rradiated Macula Flava in the Human

Vocal Fold Mucosa
b
Voice disorders are one of the complications seen after radio-
therapy, and they are caused by radiation-induced tissue
damage. Radiotherapy may be a double-edged sword with
the capacity to cure tumors but also possibly causing fibrosis,
necrosis, chronic edema, atrophy, and even secondary carci-
nomas. Despite the beneficial tumoricidal effects of radia-
tion, whenever doses of radiation sufficient to kill cancer elastic fibers
cells are used, normal tissues are permanently affected. The collagen fibers
effects of radiation are brought about by the passage of vari-
ous charged particles through cells with resultant disruption
at the molecular level.
Human maculae flavae are most likely involved in the c
metabolism of extracellular matrices in the vocal fold mucosa
and are also most likely responsible for maintaining the char-
collagen fibers
acteristic layered structure of the human vocal fold mucosa vocal fold stellate cell
[13]. Therefore, it is very important to study radiation-

induced damage on the human maculae flavae containing the
vocal fold stellate cells.
reticular fibers
11.7.1 Morphological Changes of Irradiated
Macula Flava in the Human Vocal Fold
Mucosa
d
Like normal human maculae flavae, irradiated maculae fla-
vae are dense masses of cells and extracellular matrices com- hyaluronic acid
posed of collagen fibers, reticular fibers, and elastic fibers,
ground substances, and vocal fold stellate cells (Fig. 11.24).
However, irradiated maculae flavae are rather deficient in
fibrous components compared to those of normal human
vocal folds. Less fibrous proteins are produced around the
vocal fold stellate cells in irradiated maculae flavae. Irradiated
maculae flavae are only slightly stained light blue with
Alcian Blue at pH 2.5 (Fig. 11.24d). The material that stains
in the maculae flavae with Alcian Blue at pH 2.5 is digested
by hyaluronidase. Thus, less hyaluronic acid, one of the gly- Fig. 11.24 Macula flava in the irradiated vocal fold mucosa (2 months
cosaminoglycans, is produced around the vocal fold stellate after radiotherapy, 54.8 Gy). (a) Hematoxylin and eosin stain (original
cells in the irradiated maculae flavae than around those in ×400), (b) Elastica van Gieson stain (original ×400), (c) silver stain
normal human vocal folds. (original ×400), (d) Alcian Blue stain (pH 2.5) (original ×400). There
are few elastic, collagen, or reticular fibers or hyaluronic acid immedi-
ately surrounding the vocal fold stellate cells
11.7 Irradiated Macula Flava in the Human Vocal Fold Mucosa 161
a C
vacuolar vocal
fold stellate cells elastic fibers
B lipid droplets
vacuole
nucleus
degenerated
vocal fold
degenerated vocal stellate cell
fold stellate cells
elastic fibers
b Fig. 11.26 Transmission electron micrograph of degenerated vocal

fold stellate cell in the irradiated macula flava (5 months after radio-
elastic fibers therapy, 70 Gy) (tannic acid stain)
Electron microscopy shows that many vocal fold stel-

late cells are distributed in the maculae flavae; however,
the maculae flavae are rather deficient in fibrous proteins
cleaved nucleus
intracellular (Fig. 11.25a) compared to those of the normal human
organelles vocal folds.
vocal fold
lipid droplets stellate cell In the irradiated maculae flavae, the vocal fold stellate
cells are stellate in shape and possess cytoplasmic pro-
cesses (Fig. 11.25a). No basal lamina is found. Lipid drop-
lets are present in the cytoplasm (Fig. 11.25). Lipofuscin
granules can be detected around the lipid droplets. The
c nucleus-cytoplasm ratio is relatively small, but there are
elastic fibers
lipid droplets few intracellular organelles, such as rough endoplasmic
reticulum and Golgi apparatus. Cytoplasms of some vocal
lipofuscin granules fold stellate cells are light (Fig. 11.25a, b) but most are dark
(Fig. 11.25a, c). Vacuolar degeneration is seen in the cyto-
plasm (Fig. 11.25a). Some vocal fold stellate cells are
nucleus vocal fold
degenerated (Fig. 11.26). The nuclei of the vocal fold stel-
stellate cell late cells are cleaved. There are not as many vesicles at the
periphery of the cytoplasm of irradiated vocal fold stellate
cells. Newly released amorphous materials from the vesi-
cytoplasmic
process
cles are present on the surface of the vocal fold stellate cells
but not as much as in the normal ones. There are no elastin
or collagen fibrils immediately surrounding the irradiated
vocal fold stellate cells with dark cytoplasm (Fig. 11.25a,
Fig. 11.25 (a) Transmission electron micrograph of macula flava in c), but elastic fibers and collagen fibers composed of irreg-
the irradiated vocal fold mucosa (5 months after radiation therapy, ular collagen fibrils can be detected at a distance from the
70 Gy) (tannic acid stain). (b) Vocal fold stellate cell with light cyto- cells (Figs. 11.27 and 11.28).
plasm in the irradiated macula flava (region B in a). (c) Vocal fold stel-
late cell with dark cytoplasm in the irradiated macula flava (region C in
The phenomena mentioned above cannot be seen in the
a). There are few elastin or collagen fibrils immediately surrounding vocal fold stellate cells in non-irradiated macula flava or in
(asterisk) the vocal fold stellate cell the fibroblasts in non-irradiated or irradiated Reinke’s space.
a 11.7.2 Functional Morphology

of the Irradiated Vocal Fold Stellate
elastic fibers Cells
Individual collagen polypeptide chains are synthesized on

membrane-bound ribosomes and injected into the lumen of
collagen fibers the endoplasmic reticulum as larger precursors, called pro-α
chains [38]. Each pro-α chain then combines with two others
to form procollagen in the endoplasmic reticulum, and
procollagens are secreted to be converted to collagen mole-
cules in the extracellular spaces (Fig. 11.17b) [38]. After
elastic fibers
secretion, the propeptides of the fibrillar procollagen mole-
cules are removed by specific proteolytic enzymes outside
the cell [38]. This converts the procollagen molecules to col-
lagen molecules, which assemble in the extracellular space
b
to form much larger collagen fibrils (Fig. 11.17b) [38].
Collagen fibrils have characteristics cross-striations every
67 nm [38]. After the collagen fibrils have formed in the
collagen fibers extracellular space, they are greatly strengthened by the for-
mation of covalent cross-links between lysine residues of the
elastic constituent collagen molecules (Fig. 11.17b) [38].
fibers Some vocal fold stellate cells are degenerated (Fig. 11.26),
and cytoplasms of many vocal fold stellate cells are dark
(Fig. 11.25c) in the irradiated maculae flavae. The cells in
which the cytoplasmic matrices are darker than in normal
cells are often thought to be effete or metabolically inactive
cells [39]. There are not as many vesicles at the periphery of
irradiated vocal fold stellate cells with dark cytoplasm.
Newly released amorphous materials from the vesicles are
Fig. 11.27 (a)(b) Transmission electron micrograph of elastic fibers present on the surface of such vocal fold stellate cells but not
and collagen fibers with irregular collagen fibrils in the irradiated mac- as much as in normal ones. There are no elastic fibers and
ula flava (5 months after radiotherapy, 70 Gy) (tannic acid stain)
collagen fibers immediately surrounding the irradiated vocal
fold stellate cells with dark cytoplasm, but elastic fibers and
collagen fibers composed of irregular collagen fibrils can be
elastic fibers
detected at a distant from the cells. The phenomena men-
tioned above cannot be seen in the vocal fold stellate cells in
the normal macula flava, indicating that radiation inhibits the
synthesis of the precursors that are involved in the formation
of collagen and elastic fibers by the vocal fold stellate cells.
collagen fibers Additionally, irregularly assembled collagen fibers are
observed (Fig. 11.28). It indicates that radiation also inhibits
the extracellular events involved in the formation of collagen
and elastic fibers around the vocal fold stellate cells. It has
been corroborated that the vocal fold stellate cells constantly
synthesize fibrous protein, and collagen and elastic fibers are
constantly formed around the cells.
The phenomena mentioned above also cannot be seen in
Fig. 11.28 Transmission electron micrograph of irregularly assem- fibroblasts in irradiated Reinke’s space, indicating that the
bled collagen fibers in the irradiated macula flava (5 months after radia- radiosensitivity of vocal fold stellate cells is higher than that
tion therapy, 70 Gy) (tannic acid stain) of fibroblasts and that radiation appears to decrease the level
References 163
of activity in vocal fold stellate cells. Radiation-induced 19. Sato K, Hirano M. Age-related changes of the macula flava of
the human vocal fold. Ann Otol Rhinol Laryngol. 1995;104:
changes in the vocal fold stellate cells of the maculae flavae
839–44.
influence the metabolism of extracellular matrices in the 20. Kurita S, Nagata K, Hirano M. Comparative histology of mamma-
vocal fold mucosa, as well as the viscoelasticity of the irradi- lian vocal folds. In: Kirchner JA, editor. Vocal fold histopathology.
ated vocal fold mucosa, and are one of the causes of voice A symposium. San Diego, CA: College Hill Press; 1986. p. 1–10.
21. Sato K, Hirano M, Nakashima T. Comparative histology of the
disorders after irradiation.
maculae flavae of the vocal folds. Ann Otol Rhinol Laryngol.
2000;109:136–40.
22. Fuja TJ, Probst-Fuja MN, Titze IR. Changes in expression of extra-
References cellular matrix genes, fibrogenic factors, and actin cytoskeletal
organization in retinol treated and untreated vocal fold stellate cells.
Matrix Biol. 2006;25:59–67.
1. Lanz TV, Wachsmuth W. Praktische anatomie hals. Berlin:
23. Tateya T, Tateya I, Surles RL, Tanumihardjo S, Bless DM. Roles of
Springer-Verlag; 1955. p. 282.
vitamin A and macula flava in maintaining vocal folds. Ann Otol
Rhinol Laryngol. 2008;117:65–73.
(Fukuoka). 1975;21(Suppl. 1):239–60.
24. Fuja TJ, Probst-Fuja MN, Titze IR. Transdifferentiation of vocal-
fold stellate cells and all-trans retinol-induced deactivation. Cell
Tissue Res. 2005;322:417–24.
4. Subotic R, Vecerina S, Krajina Z, Hirano M, Kurita S. Histological
25. Sato K. Reticular fibers in the vocal fold mucosa. Ann Otol Rhinol
structure of vocal fold lamina propria in foetal larynx. Acta
Laryngol. 1998;107:1023–8.
Otolaryngol. 1984;97:403–6.
26. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteo-
5. Vecerina-Volic S, Hirano M, Karovic-Krzelj V. Macula flava in the
glycans and their influence on biomechanics. Laryngoscope.
vocal fold of human fetus. Acta Otolaryngol. 1988;105:144–8.
1999;109:845–54.
6. Campos Banales ME, Perez Pinero B, Rivero J, Ruiz Casal E,
27. Chan RW, Gray SD, Titze IR. The importance of hyaluronic

Lopez Aguado D. Histological structure of the vocal fold in the
acid in vocal fold biomechanics. Otolaryngol Head Neck Surg.
human larynx. Acta Otolaryngol. 1995;115:701–4.
2001;124:607–14.
7. Fayoux P, Devisme L, Merrot O, Chevalier D, Gosselin B. Histologic
28. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in
structure and development of the laryngeal macula flava. Ann Otol
the human vocal fold. Acta Otolaryngol. 2003;123:106–10.
29. Senoo H, Wake K. Histochemical methods for detection of vitamin
8. Sato K, Hirano M, Nakashima T. Stellate cells in the human vocal
A. Vitamins (Japan). 1985;59:465–9.
fold. Ann Otol Rhinol Laryngol. 2001;110:319–25.
30. Sundaresan PR. Vitamin A and the sulfate-activating enzymes.

9. Sato K, Umeno H, Nakashima T. Vocal fold stem cells and
Biochim Biophys Acta. 1966;113:95–109.
their niche in the human vocal fold. Ann Otol Rhinol Laryngol.
31. DeLuca L, Wolf G. Effect of vitamin A on the mucopolysaccha-
2012;121:798–803.
rides of lung tissue. Arch Biochem Biophys. 1968;123:1–8.
32. Levi AS, Geller S, Root DM, Wolf G. The effect of vitamin A and
other dietary constituents on the activity of adenosine triphosphate
sulphurylase. Biochem J. 1968;109:69–74.
33. Kojima S, Rifkin DB. Mechanism of retinoid-induced activation of
ula flava in the human vocal folds as a stem cell niche. J Laryngol
latent transforming growth factor-beta in bovine endothelial cells. J
Otol. 2016;130:656–61.
Cell Physiol. 1993;155:323–32.
12. Sato K, Hirano M. Histologic investigation of the macula flava of the
34. Eng FJ, Friedman SL, Fibrogenesis I. New insights into hepatic
stellate cell activation: the simple becomes complex. Am J Physiol
13. Sato K, Umeno H, Nakashima T. Functional histology of the mac-
Gastrointest Liver Physiol. 2000;279:G7–G11.
ula flava in the human vocal fold. Part 1. Its role in the adult vocal
35. Yamada E, Hirosawa K. The possible existence of a vitamin

fold. Folia Phoniatr Logop. 2010;62:178–84.
A-storing cell system. Cell Struct Funct. 1976;1:201–4.
14. Sato K, Hirano M, Nakashima T. 3D Structure of the macula flava
36. Wake K. Perisinusoidal stellate cells (fat-storing cells, interstitial
in the human vocal fold. Acta Otolaryngol. 2003;123:269–73.
cells, lipocytes), their related structure in and around the liver sinu-
15. Sato K, Sakamoto K, Nakashima T. Expression and distribution of
soids, and vitamin A-storing cells in extrahepatic organs. Int Rev
CD44 and hyaluronic acid in human vocal fold mucosa. Ann Otol
Cytol. 1980;66:303–53.
37. Zhao L, Burt AD. The diffuse stellate cell system. J Mol Histol.
16. Sato K, Hirano M. Histologic investigation of the macula flava
2007;38:53–64.
38. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. The
1995;104:556–62.
extracellular matrix of animal connective tissues. In: Molecular
biology of the cell. 5th ed. New York, NY: Garland Science; 2008.
p. 1178–204.
2001;110:417–24.
39. Ghadially FN. Cytoplasmic matrix and its inclusions. In:

18. Sato K, Umeno H, Nakashima T. Functional histology of the macula
Ultrastructural pathology of the cell and matrix, vol. 2. 3rd ed.
flava in the human vocal fold. Part 2. Its role in the growth and devel-
London: Butterworths; 1988. p. 953–9.
opment of the vocal fold. Folia Phoniatr Logop. 2010;62:263–70.
Tissue Stem Cells and the Stem Cell
Niche of the Human Vocal Fold Mucosa 12
Abstract
1. The latest research shows that the vocal fold stellate cells in the human maculae flavae
are involved in the metabolism of extracellular matrices that are essential for the
viscoelastic properties of the human vocal fold mucosa. Additionally, vocal fold
stellate cells are considered to be important interstitial cells in the growth, develop-
ment, and aging of the human vocal fold mucosa.
2. Vocal fold stellate cells are considered to be a new category of cells in the human
vocal fold. It is uncertain whether the vocal fold stellate cells are derived from the
same embryonic source as fibroblasts.
3. There is growing evidence to suggest that the cells including vocal fold stellate cells
in the maculae flavae are tissue stem cells of the human vocal fold, and the maculae
flavae are a candidate for a stem cell niche.
4. The cells including the vocal fold stellate cells in the human adult maculae flavae
possess proteins of all three germ layers. This suggests that the cells are undifferenti-
ated and have the ability of multipotency.
5. The radiosensitivity of the cells in the maculae flavae is high, indicating that the cells
are not yet as fully differentiated as fibroblasts.
6. Telomerase resides in the cells in the maculae flavae. They are resting cells
(G0-phase).
7. The cell division in the human adult maculae flavae is reflective of asymmetric self-
renewal and cultured cells form a colony-forming unit. Therefore, the phenomenon
gives rise to the strong possibility that the cells in the human maculae flavae are tissue
stem cells.
8. Recent research suggests that the cells in the human maculae flavae arise not from
resident interstitial cells of the vocal fold mucosa but from the differentiation of bone
marrow cells via peripheral circulation.
9. At birth, the cells have already been supplied from the bone marrow into the maculae
flavae in the newborn vocal fold and are ready to start the growth and development of
the human vocal fold mucosa as a vibrating tissue.
10. The hyaluronan concentration in the maculae flavae is high and contains cells which
possess hyaluronan receptors, indicating that the maculae flavae are hyaluronan-rich
matrix, which is required for a stem cell niche.
11. A proper microenvironment in the maculae flavae of the human vocal fold mucosa is
necessary to be effective as a stem cell niche maintaining the stemness of the contained
tissue stem cells.

166 12 Tissue Stem Cells and the Stem Cell Niche of the Human Vocal Fold Mucosa
12.1 Introduction Another of our studies [8] revealed that the vocal fold
stellate cells in the human maculae flavae are desmin-positive
Stem cells are a subset of cells that have the unique ability to cells that have perinuclear vitamin A droplets and show the
replenish themselves through self-renewal and the potential morphological features of cells included in the proposed
to differentiate into different types of mature cells [1]. These “diffuse stellate cell system” [9].
characteristics therefore play essential roles in organogene- It is uncertain whether the vocal fold stellate cells are
sis during embryonic development and tissue regeneration derived from the same embryonic source as fibroblasts in the
[1]. There are two main types of stem cells: embryonic and human vocal fold mucosa.
adult [1]. As development proceeds, the need for organogen- As a result of latest research, there is a growing evidence
esis arises, and embryos form germ-line stem cells for repro- to suggest that the cells including vocal fold stellate cells in
duction and somatic stem cells for organogenesis [1]. the human maculae flavae are adult multipotent stem cells,
After birth, adult stem cells, including both germ-line tissue stem cells, or progenitor cells in the human vocal fold
stem cells and somatic stem cells, reside in a specific micro- mucosa and that the human maculae flavae are a candidate
environment termed a “niche,” which varies in nature and for a stem cell niche, which is a microenvironment nurturing
location depending on the tissue type [1]. These adult stem a pool of stem cells [10–12].
cells are an essential component of tissue homeostasis; they Investigations concerning how to regulate these cells con-
support ongoing tissue regeneration, replacing cells lost due tained in the human maculae flavae are challenging but
to natural cell death (apoptosis) or injury [1]. important in the field of regenerative medicine of the human
In another classification system, stem cells are divided into vocal fold.
two main groups, pluripotent and multipotent, based on their The manipulation, not only of cells but also their microen-
potential to differentiate. Pluripotent (embryonic) stem cells vironment, is one of the strategies in regenerative medicine.
can differentiate into every kind of cell in the body, while mul- Artificial manipulation of these cells using cutting-edge meth-
tipotent (adult) stem cells can differentiate into multiple, but ods (e.g., via chemical biology) could lead to advanced devel-
not all, cell lineages [1]. opment in vocal fold regeneration. Understanding the
Adult tissue-specific stem cells (tissue stem cells, somatic mechanisms responsible for microenvironmental regulation of
stem cells) have the capacity to self-renew and generate func- the cells including vocal fold stellate cells in the human macu-
tionally differentiated cells that replenish cells lost throughout lae flavae will provide the tools needed to manipulate cells
an organism’s lifetime. Tissue-specific stem cells reside in a through their microenvironment for the development of thera-
niche, whereby a complex microenvironment maintains their peutic approaches to diseases and tissue injuries of the vocal
multipotency. fold. Translational medicine focused on how to regulate cells
Interstitial cells with a starlike appearance in the human and extracellular matrices (microenvironments) contained in
maculae flavae were discovered in our laboratory in 2001 the maculae flavae of the vocal folds will contribute to our
[2]. These cells possess lipid droplets and store vitamin A ability to restore and regenerate human vocal fold tissue.
[2–7]. They have many morphological differences from
the fibroblasts in the human vocal fold mucosa and con-
stantly synthesize extracellular matrices that are essential 12.2 I ntermediate Filaments of the Cells
for the viscoelasticity of the human vocal fold mucosa in the Human Adult Maculae Flavae
[2–7]. These cells had no nomenclature and were thus des-
ignated vocal fold stellate cells in our series of previous The expression of proteins in the intermediate filaments of
studies. the cytoplasm is specific to cell type and differentiation [13].
The latest research confirms that the vocal fold stellate Because of the tissue specificity of intermediate filaments,
cells in the human maculae flavae are involved in the metab- cells from different tissues can be distinguished on the basis
olism of extracellular matrices that are essential for the vis- of the intermediate filament protein present [13].
coelastic properties of the human vocal fold mucosa [2–7]. Microfilaments are distributed in the cytoplasm of the
Vocal fold stellate cells are considered to be a new category cells in the human maculae flavae, and 10-nm-thick fila-
of cells in the human vocal fold and are considered to be ments (intermediate filaments) are also present (Fig. 12.1).
important interstitial cells in the growth, development, and Proteins in the intermediate filaments including cyto-
aging of the human vocal fold mucosa [6, 7]. keratin (Fig. 12.2a), vimentin (Fig. 12.2b), glial fibrillary
12.2 Intermediate Filaments of the Cells in the Human Adult Maculae Flavae 167
cells in the macula flava
microfilaments
lipid droplet
vesicles
nucleus microfilaments
Fig. 12.1 Transmission electron micrograph of microfilaments (inter-

mediate filaments) in the cytoplasm of cells in the human maculae fla-
vae (uranyl acetate and lead citrate stain) cells in the macula flava
acidic protein (GFAP) (Fig. 12.2c), and desmin (Fig. 12.2d)

are distributed in the cytoplasm of the cells including the
vocal fold stellate cells in the adult maculae flavae.
Cytokeratin is a protein of the intermediate filaments of
epithelial cells, and vimentin is a major subunit protein of
the intermediate filaments of mesenchymal cells. Glial
fibrillary acidic protein, a member of the intermediate fila-
ment protein family and characteristic of neural crest cells, c
is heavily and specifically expressed in astrocytes and cer- cells in the macula flava
tain other astroglia in the central nervous system. In addi-
tion, neural stem cells frequently and strongly express glial
fibrillary acidic protein. Desmin is a protein of the interme-
diate filament and is characteristic of myogenic crest cells
and is found in muscle cells.
Additionally, cells in the human maculae flavae express
SOX17 (Fig. 12.3), which is an endodermal cell marker.
Consequently, the cells in the human adult maculae flavae
express proteins of all three germ layers. This suggests that
they are undifferentiated cells and have the ability of
multipotency.
It is uncertain whether the cells in the human adult macu-
d
lae flavae are derived from the same embryonic source as
fibroblasts in the human vocal fold.
Fig. 12.2 Immunohistochemical staining of cells in the human macu-

lae flavae. (a) cytokeratin, (b) vimentin, (c) glial fibrillary acidic pro-
tein, (d) desmin
12.5 C
ell Cycle of the Cells in the Human
Adult Maculae Flavae
The antigen defined as Ki-67 is a human nuclear protein, the

expression of which is strictly associated with cell prolifera-
tion and which is widely used in routine pathology as a pro-
liferation marker to measure the growth fraction of cells
cells in the macula flava [16]. Cells express Ki-67 during proliferation (G1-, S-, G2-,
M-phase) in the cell cycle, but the cells that are in an arrested
state (G0-phase) do not express Ki-67.
The cells including vocal fold stellate cells in the maculae
flavae do not express Ki-67 (Fig. 12.5), indicating that they
are resting cells (G0-phase), as are stem cells.
Fig. 12.3 SOX17 is detected in cells in the human maculae flavae,

shown by immunohistochemical staining
12.3 R
adiosensitivity of the Cells
in the Human Adult Maculae Flavae cells in the macula flava
The radiosensitivity of the cells including vocal fold stellate

cells in the maculae flavae is morphologically higher than
that of fibroblasts in Reinke’s space of the human vocal fold
mucosa (Chap. 11), indicating that the cells are not yet as
fully differentiated as fibroblasts [14].
12.4 T
elomerase of the Cells in the Human Fig. 12.4 Telomerase reverse transcriptase is detected in cells in the
Adult Maculae Flavae human maculae flavae, shown by immunohistochemical staining
A special DNA polymerase called telomerase can catalyze

the formation of additional copies of the telomeric repeat
sequence, thereby compensating for the gradual shortening
that occurs at both ends of the chromosome during DNA
replication [15]. In multicellular organisms, telomerase
resides mainly in the germ cells that give rise to sperm and
eggs and in a few other kinds of proliferating normal cells
such as stem cells [15]. Because telomerase is not found in cells in the macula flava
most cells, their chromosomal telomeres get shorter and
shorter with each cell division [15]. The presence of telom-
erase allows cells to divide indefinitely without telomere
shortening [15].
The cells including vocal fold stellate cells in the maculae
flavae express telomerase reverse transcriptase (Fig. 12.4),
indicating the special DNA polymerase called telomerase
resides in the cells in the maculae flavae. This further sug-
gests that the cells in the human maculae flavae are a tissue
stem cell in the human vocal fold mucosa. Fig. 12.5 Cells in the human maculae flavae do not express Ki-67
(immunohistochemical staining)
12.7 Cell Division of Cells in the Human Maculae Flavae 169
12.6 T
ransition Area Between the Human a hyaline cartilage portion
Adult Maculae Flavae
and Surrounding Tissue
The transition area between the maculae flavae and their sur-
rounding tissue is interesting.
The posterior macula flava is attached to the vocal process
B
of the arytenoid cartilage posteriorly. Elastic cartilage located
at the tip of the vocal process facilitates movement of the vocal process
vocal process during adduction and abduction [17]. The tran-

sition of cells and extracellular matrices between the poste-
rior macula flava and the elastic cartilage portion of the vocal posterior
process is gradual, and the border between them is not clearly macula flava
delineated (Fig. 12.6). The cells in the posterior macula flava
appear to differentiate into chondrocytes in the tip of the elastic cartilage posterior
vocal process. macula flava
b
The cells in the human maculae flavae express CD44
(mesenchymal stem cell marker). Most of the fibroblasts in
the tissue surrounding the maculae flavae do not express
CD44. However, CD44-positive fibroblasts are observed at
the periphery of the maculae flavae (Fig. 12.7). The cells in
the macula flava appear to differentiate into fibroblasts in the
surrounding tissue.
These findings raise the possibility that the cells including
vocal fold stellate cells in the maculae flavae generate func-
tionally differentiated cells, such as chondrocytes and fibro- c
blasts in the human vocal fold mucosa. Additional
investigations are needed to determine whether the cells
including vocal fold stellate cells in the maculae flavae have
the capacity to self-renew and generate functionally differen-
tiated cells (multipotency) that replenish lost cells through-
out an organism’s lifetime.
cells in the posterior
macula flava
12.7 C
ell Division of Cells in the Human
Maculae Flavae
In vitro culturing of the human maculae flavae yields inter-

esting results.
After extraction of the maculae flavae of the human vocal d
fold mucosae from surgical specimens under the microscope,
the maculae flavae are minced, cultured, and proliferated in
culture medium [12].
chondrocytes of
elastic cartilage
Fig. 12.6 Transition area between human adult posterior macula

flava and tip of vocal process of the arytenoid cartilage. (a)
Posterior macula flava and tip of vocal process. (b) Transition
between posterior macula flava and tip of vocal process. (c)
Posterior macula flava. (d) Elastic cartilage portion of vocal
process
a
posterior macula flava macula flava fragment
CD 44 (+) cells cobblestone-like

squamous cells
CD 44 (+) fibroblasts
fibroblast-like
spindle cells
CD 44 (-) fibroblasts
b
Fig. 12.7 Border between the human adult posterior macula flava and
surrounding tissue (CD44, immunohistochemical staining). The border
(asterisks) between the dense mass of macula flava containing vocal
fold stellate cells and surrounding tissue is clearly delineated. The
CD44-positive fibroblasts are observed at the periphery of the human
maculae flavae
fibroblast-like spindle cells
After a few weeks of primary culture in an MF-start pri-
mary culture medium (Toyobo, Osaka, Japan), two types of
cells, fibroblast-like spindle cells (Group A) and cobblestone-
like squamous cells (Group B), grow from the macula flava
fragments (Fig. 12.8). The cobblestone-like squamous cells
are polygonal in shape and have oval-shaped nuclei
(Fig. 12.8c). The nucleus-cytoplasm ratio is high.
After removing the two types of cells by a cell scraper,
each type of cell is individually subcultured in an MF-medium c
(mesenchymal stem cell growth medium) (Toyobo, Osaka,
Japan) to proliferate the cells.
After a week of first subculture, subcultured Group A
cells become stellate in shape and possess slender cytoplas-
mic processes (Fig. 12.9a). Small lipid droplets are present
in the cytoplasm. The nuclei are oval in shape and their cobblestone-like squamous cells
nucleus-cytoplasm ratios are low. These cells are morpho-
logically similar to vocal fold stellate cells.
After a week of second subculture, subcultured Group B
cells form a colony-forming unit (Fig. 12.9b), indicating these
cells are mesenchymal stem cells or stromal stem cells in the
bone marrow.
Colony formation is one of the characteristics of stem
cells. The colony-forming unit in vitro was first described by
Friedenstein et al. [18]. They established that adherent fibro- Fig. 12.8 Primary culture of macula flava with MF-start primary culture
medium (Toyobo, Osaka, Japan) (phase-contrast microscopy). (a) Two
blastic cells that form cell colonies in vitro culture can be types of cells, cobblestone-like squamous cells and fibroblast-like spindle
isolated from the bone marrow stroma. This colony-forming cells, grow from the macula flava fragments in the primary culture. (b)
unit can differentiate into cartilage, bone, and adipose tissue Fibroblast-like spindle cells. (c) Cobblestone-like squamous cells
[19]. Such a colony is also observed in embryonic stem cells
(ES cells) [20], induced pluripotent stem cells (iPS cells) the cells, including the vocal fold stellate cells, in the human
[21], and such tissue stem or progenitor cells as hepatic stem maculae flavae are tissue stem cells.
cells [22] and renal progenitor cells [23]. Therefore, the As mentioned above, the cell division in the human adult
colony-forming phenomenon gives rise to the possibility that maculae flavae with mesenchymal stem cell growth medium
12.9 Microenvironment of Maculae Flavae as a Stem Cell Niche in the Human Vocal Fold 171
Both colony-forming subcultured cells (cobblestone-like

a
squamous cells) (Fig. 12.11) and non-colony-forming sub-
cultured cells (fibroblast-like spindle cells) (Fig. 12.12)
express cytoplasmic cytokeratin (epithelium-associated pro-
tein), vimentin (mesenchymal cell-associated protein), glial
stellate cells fibrillary acidic protein (GFAP) (neural-associated protein),
and desmin (muscle-associated protein) [11]. Consequently,
both colony-forming cells (cobblestone-like squamous cells)
and non-colony-forming cells (fibroblast-like spindle cells)
express ectoderm and mesoderm germ layers. This suggests
that they are undifferentiated cells and have the ability of
multipotency.
The vocal fold stellate cells are possibly transit-amplifying
lipid droplets
cells, that is, progenitor cells [12]. However, at the present
state of our investigation, it is difficult to clarify the stem cell
b system and hierarchy of stem cells in the human maculae
flavae and determine whether the vocal fold stellate cells are
tissue stem cells or progenitor cells.
12.9 M
icroenvironment of Maculae Flavae
as a Stem Cell Niche in the Human
colony-forming unit
Vocal Fold
12.9.1 Hyaluronan-Rich Matrix
The structural and biochemical microenvironment that con-

fers stemness upon cells in multicellular organisms is
referred to as the stem cell niche. A stem cell niche is com-
posed of a group of cells in a special tissue location for the
Fig. 12.9 Individual subculture of each type of cell in an MF-medium maintenance of stem cells [1].
(mesenchymal stem cell growth medium) (Toyobo, Osaka, Japan) to Hyaluronan serves as an important niche component for
proliferate the cells (phase-contrast microscopy). (a) Stellate cells.
numerous stem cell populations [25, 26]. After the discovery
Fibroblast-like cells in the primary culture become stellate in shape and
possess slender cytoplasmic processes and small lipid droplets in the of hyaluronan, it was assumed that its major functions were in
cytoplasm in the subculture. (b) Colony-forming unit. Cobblestone-like the biophysical and homeostatic properties of tissues.
squamous cells in an MF-medium form a colony-forming unit However, current studies lead to understanding that hyaluro-
nan also plays a crucial role in cell behavior [27]. A
is reflective of asymmetric self-renewal (Fig. 12.10) [12]. hyaluronan-rich matrix, which is composed of the glycosami-
Asymmetry in the stem cell niche refers to the notion that noglycan hyaluronan and its transmembrane receptors (a cell
daughter cells are different from each other [24]. There is sig- surface hyaluronan receptor), is able to directly affect the cel-
nificant evidence that many stem cell divisions result in one lular functions of stem cells in a stem cell niche [25, 26].
daughter cell that is similar to the parent cell and, hence, nec- The maculae flavae in the human adult vocal fold are
essarily allows for self-renewal of the stem cell phenotype, strongly stained light blue with Alcian Blue at pH 2.5
whereas the other daughter cell is a differentiated or commit- (Fig. 12.13), and relatively sparse staining with Alcian Blue
ted cell type [24]. Asymmetry in cell division gives rise to the at pH 1.0 is noted. The ground substances around the cells
possibility that the maculae flavae in the human adult vocal including vocal fold stellate cells are stained in the same
fold is a stem cell niche containing tissue stem cells [12]. way. The materials in the maculae flavae that are strongly
stained with Alcian Blue (pH 2.5) are digested by hyaluroni-
dase. A great deal of glycosaminoglycan (hyaluronan) is
12.8 H
ierarchy of Tissue Stem Cells situated around the cells, and hyaluronan concentration in
in the Human Maculae Flavae the human adult maculae flavae is high. The border between
dense masses of hyaluronan (macula flava) and surrounding
Here, the question arises whether the vocal fold stellate cells tissue is clearly delineated (Fig. 12.13b). Additionally, most
are tissue stem cells or progenitor cells (transit-amplifying of the cells including vocal fold stellate cells in the maculae
cells). flavae express CD44 (a cell surface hyaluronan receptor)
Fig. 12.10 Asymmetric cell Stellate Cells

division of cells in the
maculae flavae with
MF-medium. Cell division in
the human maculae flavae
reflects an asymmetric Transit-amplifying cell
self-renewal. One type of cell (Progenitor cell)
is tissue stem cells (vocal fold
stem cells), which form a
colony-forming unit. The
other type is transit-
amplifying cells (progenitor
cells), whose shape is stellate
and similar to the vocal fold Transit-amplifying cell
stellate cells (Progenitor cell)
Tissue stem cell

Stellate Cells
Tissue stem cell
Colony-
forming unit
Tissue stem cell
Colony-forming cells
Cytokeratin Vimentin
GFAP Desmin
Fig. 12.11 Immunohistochemistry of the colony-forming cells (cobblestone-like squamous cells)

12.9 Microenvironment of Maculae Flavae as a Stem Cell Niche in the Human Vocal Fold 173
Non-colony-forming
cells (Vocal fold
stellate cells)
Cytokeratin Vimentin
GFAP Desmin
Fig. 12.12 Immunohistochemistry of the non-colony-forming cells (fibroblast-like spindle cells)
(Fig. 12.14). This indicates that the human maculae flavae

a
are a hyaluronan-rich pericellular matrix [10].
Since the cells including vocal fold stellate cells in the
human maculae flavae have cell surface hyaluronan recep-
tors and are surrounded by a high concentration of hyaluro- cells in the macula flava
nan, the maculae flavae are a candidate for a stem cell niche,
a microenvironment nurturing a pool of tissue stem cells
including vocal fold stellate cells [10]. hyaluronan (hyaluronic acid)
12.9.2 A Proper Microenvironment

in the Maculae Flavae as a Stem Cell
Niche
On the other hand, cells from the maculae flavae cultured

with Dulbecco’s modified Eagle’s medium (DMEM) (Nissui,
hyaluronan (hyaluronic acid)
Tokyo, Japan), instead of mesenchymal stem cell growth b
medium, are stellate in shape and possess slender cytoplas-
mic processes (Fig. 12.15). Cobblestone-like squamous cells *
cells in the macula flava *
do not appear [28]. The nuclei of stellate-shaped cells are
oval, and the nucleus-cytoplasm ratio is low. In the primary *
*
Fig. 12.13 (a) Human adult macula flava (Alcian Blue stain, pH 2.5).
A great deal of hyaluronan (glycosaminoglycan) (light blue stained *
material) is situated around the cells in the macula flava, and hyaluro-
nan concentration in the human adult maculae flavae is high. (b) Border
*
between human adult macula flava and surrounding tissue (Alcian Blue *
stain, pH 2.5). Border (asterisks) between dense mass of the macula
flava containing hyaluronan and surrounding tissue is clearly * fibroblasts
delineated
Stellate Cells
Stellate Cells
Stellate Cells
Stellate Cells
Fig. 12.14 CD44 on cytoplasm of cells including vocal fold stellate
cells in the human macula flava, shown by immunohistochemical
staining
Fig. 12.16 Symmetric cell division with Dulbecco’s modified Eagle’s

medium. During the subculture period, each cell continues to exhibit
stellate cells
stellate shape
human vocal fold mucosa is necessary to be effective as a

stem cell niche maintaining the stemness of the contained
tissue stem cells [12].
lipid droplets 12.10 O

rigin of Cells in the Human Maculae
Flavae
Bone marrow-derived cells have received a great deal of

attention with regard to tissue development and regeneration.
Fig. 12.15 Primary culture of macula flava with Dulbecco’s modified Bone marrow-derived cells contain bone marrow-derived
Eagle’s medium. Stellate cells grow from the macula flava fragments
mesenchymal stem cells, which are multipotent cells capable
of self-renewal [19, 29] and are considered to be the origin of
culture, small lipid droplets are present in the cytoplasm; circulating fibrocytes, which are associated with wound
however, these disappear in the second culture. The stellate healing and tissue fibrosis [30]. They circulate in the periph-
cells proliferate by attaching their cytoplasmic processes to eral blood and are distributed to organs under normal condi-
each other (Fig. 12.15). These cells are morphologically sim- tions. When tissue is injured, they contribute to tissue repair
ilar to vocal fold stellate cells. During the subculture period, by cell differentiation and migrate into injured tissue as
each cell continues to exhibit the same morphological needed [31, 32].
characteristics. Circulating fibrocytes were first described as blood-borne
These phenomena suggest that cell division in the fibroblast-like cells by Bucala et al. [30] They were found to
human maculae flavae with Dulbecco’s modified Eagle’s be unique cells because they co-expressed hematopoietic
medium is reflective of symmetric self-renewal (Fig. 12.16) markers as well as collagen type I and other mesenchymal
[12, 28]. In contrast, the cell division in the human macu- markers. CD34 (hematopoietic stem cell marker), CD45
lae flavae with mesenchymal stem cell growth medium is (leukocyte common antigen), and collagen type I are major
reflective of asymmetric self-renewal (Fig. 12.10) [12]. A markers for circulating fibrocytes derived from the bone
proper microenvironment in the maculae flavae of the marrow [33].
12.10 Origin of Cells in the Human Maculae Flavae 175
b
Fig. 12.18 CD45 is detected in cells in the human maculae flavae,
shown by immunohistochemical staining
collagen fibers
a
ucos
the m cells in the macula flava
ia of
a propr
lamin
epithelium
c posterior macula flava
CD 34 (+) cells
Fig. 12.19 Collagen type I detected in cells and ground substance in
the human maculae flavae, shown by immunohistochemical staining
The cells including vocal fold stellate cells in the human

CD 34 (+) fibroblasts maculae flavae express CD34 (a hematopoietic stem cell
marker) (Fig. 12.17). They also express CD45 (leukocyte
common antigen) (Fig. 12.18) and collagen type I (Fig. 12.19).
CD 34 (-) fibroblasts
These proteins (CD34, CD45, and collagen type I), which are
major markers of bone marrow-derived circulating fibrocytes,
are present in the cells in the human maculae flavae.
Consequently, cells including vocal fold stellate cells in
the human maculae flavae quite possibly arise not from resi-
Fig. 12.17 Immunohistochemical staining of cells in the human mac- dent interstitial cells of the vocal fold mucosa but from the
ulae flavae. (a) CD34 is detected in cells. (b) Macula flava contains
differentiation of bone marrow cells via peripheral circula-
CD34-positive cells. (c) Border between the human adult posterior
macula flava and surrounding tissue (CD34, immunohistochemical tion [11].
staining). The border (asterisks) between a dense mass of macula flava
containing vocal fold stellate cells and surrounding tissue is clearly
delineated. The CD34-positive fibroblasts are observed at the periphery
of the human maculae flavae
12.11 S
ide Population Cells in the Vocal References
Fold Mucosa
1. Li L, Xie T. Stem cell niche: structure and function. Annu Rev Cell
Dev Biol. 2005;21:605–31.
Side population cells are regarded as a cell population 2. Sato K, Hirano M, Nakashima T. Stellate cells in the human vocal
enriched with stem cells or progenitor cells and recognized fold. Ann Otol Rhinol Laryngol. 2001;110:319–25.
as a candidate for tissue stem cells. 3. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in
In one recent study, side population cells were identified in the human vocal fold. Acta Otolaryngol. 2003;123:106–10.
4. Sato K, Hirano M, Nakashima T. Age-related changes in vitamin
the epithelium and subepithelial tissue including the anterior A-storing stellate cells of human vocal folds. Ann Otol Rhinol
and posterior maculae flavae [34]. In another recent study, Laryngol. 2004;113:108–12.
side population cells increased significantly in Reinke’s space 5. Sato K, Nakashima T. Vitamin A-storing stellate cells in the human
of an injured vocal fold starting on day 3, with a peak at day newborn vocal fold. Ann Otol Rhinol Laryngol. 2005;114:517–24.
7, followed by a decrease back to baseline values on day 14 ula flava in the human vocal fold. Part 1: its role in the adult vocal
[35]. These cells in the maculae flavae participated in the fold. Folia Phoniatr Logop. 2010;62:178–84.
early stages of wound healing [35]. 7. Sato K, Umeno H, Nakashima T. Functional histology of the macula
The two investigations cited here suggest that the ante- flava in the human vocal fold. Part 2: its role in the growth and devel-
rior and posterior maculae flavae contain stem cells or pro- 8. Sato K, Umeno H, Nakashima T. Vocal fold stellate cells in the
genitor cells, and these cells have the capacity to play human macula flava and the diffuse stellate cell system. Ann Otol
essential roles in tissue regeneration. On the other hand, Rhinol Laryngol. 2012;121:51–6.
there is a report which proposes that the vocal fold fibro- 9. Zhao L, Burt AD. The diffuse stellate cell system. J Mol Histol.
2007;38:53–64.
blasts are mesenchymal stem cells resident in the vocal fold 10. Sato K, Umeno H, Nakashima T. Vocal fold stem cells and

mucosa [36]. their niche in the human vocal fold. Ann Otol Rhinol Laryngol.
2012;121:798–803.
12.12 V
ocal Fold Stem Cells and Their Niche Ann Otol Rhinol Laryngol. 2015;124:698–705.
in the Human Newborn Vocal Fold 12. Sato K, Chitose S, Kurita T, Umeno H. Microenvironment of mac-
Mucosa ula flava in the human vocal fold as a stem cell niche. J Laryngol
Otol. 2016;130:656–61.
13. Becker WM, Kleinsmith L, Hardin J. Intermediate filament. In: The
Newborns have maculae flavae at the same sites as in adult world of the cell. 6th ed. San Francisco, CA: Benjamin Cummings;
vocal folds [5, 37, 38]. The newborn maculae flavae are com- 2006. p. 446–50.
posed of relatively dense masses of cells and situated at the 14. Sato K, Shirouzu H, Nakashima T. Irradiated macula flava in the
anterior and posterior ends of the bilateral vocal fold muco- human vocal fold mucosa. Am J Otolaryngol. 2008;29:312–8.
15. Becker WM, Kleinsmith LJ, Hardin J. The cell cycle, DNA replica-
sae. The cells in the newborn maculae flavae possess some tion, and mitosis. In: The world of the cell. 6th ed. San Francisco,
features of mesenchymal cells [5, 38]. The cells including CA: Benjamin Cummings; 2006. p. 554–71.
the vocal fold stellate cells in the human newborn maculae 16. Schlüter C, Duchrow M, Wohlenberg C, Becker MH, Key G,

flavae possess proteins of all three germ layers [39]. They are Flad HD, Gerdes J. The cell proliferation-associated antigen
of antibody Ki-67: a very large, ubiquitous nuclear protein with
very likely undifferentiated cells which arise not from resi- numerous repeated elements, representing a new kind of cell cycle-
dent interstitial cells but from the differentiation of bone maintaining proteins. J Cell Biol. 1993;123:513–22.
marrow cells [39]. The results of our studies are consistent 17. Sato K, Kurita S, Hirano M, Kiyokawa K. Distribution of elastic
with the hypothesis that the cells including the vocal fold cartilage in the arytenoids and its physiologic significance. Ann
stellate cells are tissue stem cells or progenitor cells of the 18. Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF,

human newborn vocal fold mucosa [39]. Rudakowa SF, Luriá EA, Ruadkow IA. Precursors for fibroblasts
The maculae flavae in the human newborn vocal fold are in different populations of hematopoietic cells as detected by the
only slightly stained light blue with Alcian Blue at pH 2.5, in vitro colony assay method. Exp Hematol. 1974;2:83–92.
19. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,
and the percentage of CD44-positive cells in the newborn Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak
maculae flavae is not very large [40]. A newborn’s macula DR. Multilineage potential of adult human mesenchymal stem
flava is in the process of acquiring a hyaluronan-rich matrix cells. Science. 1999;284(5411):143–7.
making it a candidate for a stem cell niche. 20. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel
JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from
At birth, the cells have already been supplied, likely human blastocysts. Science. 1998;282(5391):1145–7.
from the bone marrow, into the maculae flavae in the new- 21. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda
born vocal fold, and are ready to start the growth and devel- K, Yamanaka S. Induction of pluripotent stem cells from adult
opment of the human vocal fold mucosa as a vibrating human fibroblasts by defined factors. Cell. 2007;131:861–72.
22. Suzuki A, Zheng Y, Kondo R, Kusakabe M, Takada Y, Fukao K,
tissue [39]. Nakauchi H, Taniguchi H. Flow-cytometric separation and enrich-
References 177
ment of hepatic progenitor cells in the developing mouse liver. 32. Brittan M, Hunt T, Jeffery R, Poulsom R, Forbes SJ, Hodivala-
Hepatology. 2000;32:1230–9. Dilke K, Goldman J, Alison MR, Wright NA. Bone marrow deriva-
23. Osafune K, Takasato M, Kispert A, Asashima M, Nishinakamura tion of pericryptal myofibroblasts in the mouse and human small
R. Identification of multipotent progenitors in the embryonic intestine and colon. Gut. 2002;50:752–7.
mouse kidney by a novel colony-forming assay. Development. 33. Abedi M. Hematopoietic origin of fibrocytes. In: Bucala R, edi-
2006;133:151–61. tor. Fibrocytes in health and disease. Singapore: World Scientific
24. Deasy BM. Asymmetric behavior in stem cells. In: Rajasekhar VK, Publishing; 2012. p. 1–15.
Vemuri MC, editors. Regulatory networks in stem cells. New York, 34. Yamashita M, Hirano S, Kanemaru S, Tsuji S, Suehiro A, Ito
NY: Humana Press Inc.; 2009. p. 13–25. J. Side population cells in the human vocal fold. Ann Otol Rhinol
25. Haylock DN, Nilsson SK. The role of hyaluronic acid in hemopoi- Laryngol. 2007;116:847–52.
etic stem cell biology. Regen Med. 2006;1:437–45. 35. Gugatschka M, Kojima T, Ohno S, Kanemaru S, Hirano

26. Preston M, Sherman LS. Neural stem cell niches: roles for
S. Recruitment patterns of side population cells during wound heal-
the hyaluronan- based extracellular matrix. Front Biosci. ing in rat vocal folds. Laryngoscope. 2011;121:1662–7.
2011;3:1165–79. 36. Hanson SE, Kim J, Johnson BH, Bradley B, Breunig MJ, Hematti
27. Toole BP. Proteoglycans and hyaluronan in morphogenesis and dif- P, Thibeault SL. Characterization of mesenchymal stem cells from
ferentiation. In: Hay E, editor. Cell biology of extracellular matrix. human vocal fold fibroblasts. Laryngoscope. 2010;120:546–51.
2nd ed. New York, NY: Plenum Press; 1991. p. 305–41. 37. Hirano M, Sato K. Histological color atlas of the human larynx. San
28. Sato K, Miyajima Y, Izumaru S, Nakashima T. Cultured stellate cells Diego, CA: Singular Publishing Group Inc.; 1993.
in human vocal fold mucosa. J Laryngol Otol. 2008;122:1339–42. 38. Sato K, Hirano M. Histological investigation of the macula flava
29. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoi- of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
etic tissues. Science. 1997;276:71–4. 1995;104:556–62.
30. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating 39. Sato K, Chitose S, Kurita T, Umeno H. Cell origin in the mac-
fibrocytes define a new leukocyte subpopulation that mediates tis- ula flava of the human newborn vocal fold. J Laryngol Otol.
sue repair. Mol Med. 1994;1:71–81. 2016;130:650–5.
31. Forbes SJ, Russo FP, Rey V, Burra P, Rugge M, Wright NA, Alison 40. Sato K, Sakamoto K, Nakashima T. Expression and distribution of
MR. A significant proportion of myofibroblasts are of bone marrow CD44 and hyaluronic acid in human vocal fold mucosa. Ann Otol
origin in human liver fibrosis. Gastroenterology. 2004;126:955–63. Rhinol Laryngol. 2006;115:741–8.
Cells and Extracellular Matrices
in the Human Newborn Vocal Fold 13
Mucosa
Abstract
1. The epithelium of the edge of the newborn vocal fold consists of stratified squamous
epithelium.
2. At birth, the epithelium and basal lamina of the membranous portion of the human vocal
fold resemble that of adults and are ready to start the growth and development of the
human vocal fold as a vibrating tissue.
3. The lamina propria of the newborn vocal fold mucosa is a loose structure composed of
ground substances and sparse fibers, and no structure corresponding to the vocal liga-
ment can be found. The layered structure in adult vocal folds is not present at birth.
4. The lamina propria of the newborn vocal fold lacks not only a vocal ligament and lay-
ered structure but also the characteristic complex of extracellular matrices seen in adults.
The viscoelasticity of the newborn vocal fold mucosa is morphologically not sufficient
for phonation.
5. Ground substances are abundant and glycoproteins (fibronectin) are present in the lam-
ina propria of the newborn vocal fold mucosa.
6. The newborn vocal fold mucosa is not suitable for vibration but is in the process of acquir-
ing the viscoelastic properties of the human vocal fold mucosa as a vibrating tissue.
13.1 Introduction for the growth and development of the human vocal fold
mucosa as a vibrating tissue [4].
In adults, the vocal fold has a layered structure consisting of
the epithelium, the lamina propria (superficial, intermediate
and deep layers) and the vocalis muscle [1, 2]. These layers 13.2 Epithelium of the Newborn Vocal Fold
are comprised of a cover consisting of the epithelium and the
superficial layer of the lamina propria, a transition area con- The epithelium of the edge of the newborn vocal fold consists
sisting of the intermediate and deep layers of the lamina pro- of stratified squamous epithelium (Fig. 13.1) [5]. There are
pria or vocal ligament, and a body consisting of the vocalis approximately three to four cell layers with a thickness of only
muscle [1, 2]. These structures are required for phonation [1]. 20 μm (Fig. 13.2). The most superficial layer consists of cells
Hirano et al. have reported light microscopic studies of that are squamous. The cells of the basal layer are polyhedral.
newborn vocal folds [2, 3]. The structures are found to differ There are many microvilli (microridges), about 200 nm in
from those of adults. In newborns, the entire lamina propria length, on the surface of the epithelial cells (Fig. 13.3).
of the vocal fold mucosa appears as a uniform structure with The epithelial cells are attached to each other with interdigi-
no vocal ligament [2, 3]. The layered structure of the vocal tation, and intercellular spaces are few (Fig. 13.4). Numerous
fold matures during adolescence [3]. desmosomes show firm attachment between cells (Fig. 13.4).
The maculae flavae are situated at the anterior and poste- Components in the cytoplasm such as mitochondria and rough
rior ends of the human newborn vocal fold mucosa [4]. The endoplasmic reticulum can be seen. Many tonofilaments (cyto-
newborn maculae flavae are considered important structures keratin) are present in the cytoplasm (Fig. 13.4).

180 13 Cells and Extracellular Matrices in the Human Newborn Vocal Fold Mucosa
keratin filaments
interdigitation
desmosome
tonofilaments
capillary
lamina propria of the mucosa
Fig. 13.1 Stratified squamous epithelium of the newborn membranous

vocal fold
Fig. 13.4 Transmission electron micrograph of interdigitations of the
epithelial cells of the newborn membranous vocal fold (uranyl acetate
and lead citrate stain)
nucleus
13.3 B
asal Lamina (Basement Membrane)
of the Newborn Vocal Fold Mucosa
The basal lamina (basement membrane) is composed of the

lamina lucida, lamina densa and lamina reticularis (Fig. 13.5).
The lamina lucida is an electron lucent area about 25 nm in
basal cell
thickness. The lamina densa is an electron dense layer about
nucleus 50 nm in thickness. The lamina densa consists of a dense
meshwork of randomly oriented filaments. Type IV collagen
is found exclusively in the basal lamina. Fibrous components
in the lamina reticularis are sparse.
lamina propria of the mucosa Hemidesmosomes and anchoring fibrils attach epithelium
to the lamina propria of the vocal fold mucosa (Fig. 13.5).
Fig. 13.2 Transmission electron micrograph of the stratified squa-
mous epithelium of the newborn membranous vocal fold (uranyl acetate
The basement membrane zone of the newborn membra-
and lead citrate stain) nous vocal fold resembles that of adults [5].
13.4 L
amina Propria of the Newborn Vocal
Fold Mucosa
Microvilli (microridges)
In the adult vocal fold mucosa, there are many elastic and
collagen fibers in the lamina propria. Elastic fibers are par-
ticularly dense in the intermediate layer and collagen fibers
in the deep layer of the lamina propria [1, 2].
The entire lamina propria of the newborn vocal fold mucosa
except for the anterior and posterior maculae flavae is loose in
structure, and no structure corresponding to vocal ligament
can be seen (Figs. 13.6 and 13.7) [2, 3]. The layered structure
tonofilaments (cytokeratin) in adult vocal folds is not present at birth. The entire lamina
propria is roughly uniform in structure and resembles the
superficial layer of the lamina propria of the adult vocal fold
Fig. 13.3 Transmission electron micrograph of the surface of stratified
mucosa. The lamina propria of the newborn vocal fold mucosa
squamous epithelium of the newborn membranous vocal fold (uranyl is abundant in ground substances, but fibrous components are
acetate and lead citrate stain) not well developed. The lamina propria of the newborn vocal
13.4 Lamina Propria of the Newborn Vocal Fold Mucosa 181
epithelium
keratin filaments
thyroarytenoid lamina propria

muscle
lamina lucida
lamina densa
hemidesmosome lamina reticularis
anchoring fibril
lamina propria of the mucosa

conus elasticus
Fig. 13.5 Transmission electron micrograph of the basal lamina (base-
ment membrane) of the newborn vocal fold mucosa
anterior posterior
macula flava macula flava Fig. 13.7 Coronal section of membranous portion of the newborn
epithelium vocal fold (Elastica van Gieson stain) (Photograph courtesy of Dr.
lamina propria Minoru Hirano, from the Department of Otolaryngology-Head and
Neck Surgery, Kurume University)
rough endoplasmic
cytoplasm reticulum
nucleus
Fig. 13.6 Transverse section of the newborn vocal fold (Elastica van
Gieson stain, original ×25). The lamina propria of the newborn vocal
fold mucosa is a loose structure composed of ground substances and
sparse fibers, and no structure corresponding to the vocal ligament can
be found. The layered structure in adult vocal folds is not present at
birth
fold mucosa has no special structure and is similar to the lam-

ina propria of other laryngeal areas [6]. Fig. 13.8 Transmission electron micrograph of the fibroblast in the
lamina propria of the newborn vocal fold mucosa (uranyl acetate and
Cells and extracellular matrices of the lamina propria of
lead citrate stain)
the newborn vocal fold mucosa, except for the anterior and
posterior maculae flavae, are chiefly composed of fibroblasts,
collagen fibers, reticular fibers, elastic fibers, and ground one-eighth that of cells in the newborn maculae flavae [7].
substances, however, fibrous components are sparse. Some Fibroblasts are not spindle but oval in shape and about 3–5 by
blood vessels are present in the lamina propria. 5–10 μm in size. The nucleus-cytoplasm ratio is high and the
cytoplasm occupies a small area around the nucleus (Fig. 13.8).
Free ribosomes are present in the cytoplasm. The rough
13.4.1 Fibroblasts endoplasmic reticulum and Golgi apparatus are not well
developed, indicating that these cells are in the resting phase
Fibroblasts are sparse in the lamina propria of the newborn [5]. Few vesicles are present along the periphery of the cyto-
vocal fold mucosa, but their density is greater than in adults. plasm. Newly released amorphous material is sparse on the
The density of fibroblasts in the newborn lamina propria is cell surface. Few fibers can be seen close to fibroblasts.
13.4.2 Collagen Fibers 13.4.3 Reticular Fibers
There are collagen fibers throughout the entire lamina pro- Reticular fibers (Type III collagen) are less numerous in the
pria of the newborn vocal fold mucosa, running roughly par- lamina propria of the newborn vocal fold mucosa than in
allel to the vocal fold edge, however, collagen fibers are adults. Reticular fibers are about 38 nm in diameter and have
fewer in the lamina propria of the newborn vocal fold mucosa cross-bands with a periodicity of about 55 nm (Fig. 13.11).
than in that of adults. Collagen fibers are made up of numer-
ous collagen fibrils, approximately 40 to 50 nm in width
(Figs. 13.9 and 13.10). Their structures are nearly mature, 13.4.4 Elastic Fibers
morphologically.
Elastic fibers can be seen in the lamina propria of the new-
born vocal folds mucosa but their density is low (Fig. 13.9).
The number of elastic fibers is small compared to the number
of collagen fibers. The elastic fibers are composed of micro-
fibrils and amorphous substances (elastin) (Fig. 13.12).
collagen fibers
elastic fibers
reticular fiber
glycoproteins
reticular fiber
Fig. 13.9 Transmission electron micrograph of the lamina propria of
the newborn vocal fold mucosa (tannic acid stain). Fibrous components
are sparse. A great deal of glycoprotein is present in the lamina propria
of the newborn vocal fold mucosa Fig. 13.11 Transmission electron micrograph of the reticular fibers in
the lamina propria of the newborn vocal fold mucosa (uranyl acetate
collagen fibrils
collagen fibers
microfibrils
amorphous substances
Fig. 13.10 Transmission electron micrograph of the collagen fibers in
the lamina propria of the newborn vocal fold mucosa (uranyl acetate
and lead citrate stain). Collagen fibers are made up of numerous colla- Fig. 13.12 Transmission electron micrograph of the elastic fibers in
gen fibrils the lamina propria of the newborn vocal fold mucosa (tannic acid stain)
13.5 Epithelium and Basal Lamina of the Newborn Vocal Fold Mucosa as a Vibrating Tissue 183
pericyte
fibroblasts
fibronectin
fibronectin
erythrocyte
endothelial cell
Fig. 13.13 Ground substance, immunohistochemically stained brown
with fibronectin, in the lamina propria of the newborn vocal fold
mucosa (original ×200) Fig. 13.15 Transmission electron micrograph of the capillaries in the
lamina propria of the newborn vocal fold mucosa (uranyl acetate and
lead citrate stain)
a The former are abundant and the latter are sparse and reticular
in shape, indicating that the elastic fibers are immature [6].
fibroblasts 13.4.5 Ground Substances
The ground substances contain a flocculent material that stains

with tannic acid and is abundant in the lamina propria of the
glycosaminoglycan
newborn vocal fold mucosa (Fig. 13.9). Thus, a great deal of
glycoprotein can be seen by electron microscopy [5]. The
ground substances in the lamina propria of the newborn vocal
fold mucosa are stained with fibronectin immunohistochemi-
cally (Fig. 13.13). A great deal of fibronectin (glycoprotein) is
present in the lamina propria of the newborn vocal fold mucosa.
The ground substances are stained light blue with Alcian
blue not only at pH 2.5 but also at pH 1 (Fig. 13.14). Hyaluronan
b
(hyaluronic acid) and other glycosaminoglycans are situated
in the lamina propria of the newborn vocal fold mucosa.
fibroblasts
13.4.6 Blood Vessels
In the lamina propria of the newborn vocal fold mucosa capil-

laries about 10 μm in diameter are present (Fig. 13.15). There
are many pericytes around the capillaries. The cell bodies and
processes of the pericytes encircle the capillaries.
13.5 E
pithelium and Basal Lamina
glycosaminoglycan
of the Newborn Vocal Fold Mucosa
as a Vibrating Tissue
Fig. 13.14 Ground substances are stained light blue with Alcian blue
not only at pH 2.5 (a) but also at pH 1 (b). Hyaluronan and other gly- The number of cell layers of the stratified squamous epithe-
cosaminoglycans are observed in the lamina propria of the newborn lium in the newborn vocal fold mucosa are fewer and the
vocal fold mucosa epithelium is thinner than in adults. Cells of the e pithelium
appear more strongly attached to each other than in adults mucosa has no special structures such as those noted in
since intercellular spaces are few and numerous desmosomes adults. The delicate three-dimensional structure of reticular
are firmly attached between cells in the newborn. fibers seen in the adult vocal fold mucosa is not present. No
The primary function of the basal lamina is to provide a complex of reticular fibers and other extracellular matrices,
physical support for the epithelium. Its structural framework such as elastic fibers and glycosaminoglycan (proteoglycan)
of type IV collagen gives it considerable tensile strength and can be detected.
at the same time it is flexible enough to permit stretch and Collagen fibers have supportive functions and elastic
recoil in the epithelia [8]. The basal lamina also provides for fibers have sufficient resilience to restore themselves to their
cell attachment [8]. original state [12]. Both collagen fibers and elastic fibers are
At birth, the epithelium and basal lamina of the membra- sparse in the lamina propria of the newborn vocal fold
nous portion of the human vocal fold resemble those of mucosa, though collagen fibers are essentially mature while
adults and are ready to start the growth and development of elastic fibers are immature.
the human vocal fold as a vibrating tissue. The viscoelasticity of the newborn vocal fold mucosa is
morphologically not sufficient for phonation. The lamina
propria of the newborn vocal fold lacks not only a vocal liga-
13.6 L
amina Propria of the Newborn Vocal ment and layered structure but also the characteristic com-
Fold Mucosa as a Vibrating Tissue plex of extracellular matrices seen in adults, and therefore,
viscoelasticity for vibration is inadequate.
In adults, the superficial layer of the lamina propria vibrates The newborn vocal fold mucosa is not suitable for vibra-
most markedly during phonation and its viscoelasticity is tion but is in the process of acquiring the viscoelastic proper-
essential for vibration. The delicate three-dimensional struc- ties of the human vocal fold mucosa as a vibrating tissue.
ture of reticular fibers in the vocal fold mucosa is essential
for the structural maintenance and viscoelasticity of the
human vocal fold mucosa as a vibrating tissue [9]. The com- References
plex of reticular fibers and other extracellular matrices, such
as elastic fibers and glycosaminoglycan (proteoglycan), is 1. Hirano M. Phonosurgery. Basic and clinical investigation. Otologia
(Fukuoka). 1975;21(Suppl 1):239–60.
required for the viscoelasticity of the vocal fold mucosa [9]. 2. Hirano M, Sato K. Histological color atlas of the human larynx. San
At birth, there is no structure to be found corresponding to Diego, CA: Singular Publishing Group Inc.; 1993.
the vocal ligament or the layered structure of the adult vocal 3. Hirano M, Kurita S, Nakashima T. Growth, development and aging
fold [2, 3]. Under a light microscope, the entire lamina propria of human vocal folds. In: Bless DM, Abbs JH, editors. Vocal fold
physiology. San Diego: College-Hill Press; 1983. p. 22–43.
of the newborn vocal fold mucosa appears as a roughly uni- 4. Sato K, Hirano M. Histologic investigation of the macula flava
form structure and the lamina propria resembles the superfi- of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
cial layer of the lamina propria of the adult vocal fold [3]. 1995;104:556–62.
Electron microscopy and immunohistochemical investi- 5. Sato K, Kashiwagi S, Hirano M. Ultrastructure of the mucous
membrane of the human newborn vocal folds. J Otolaryngol Jpn.
gation show the lamina propria of the newborn vocal fold to 1997;100:479–83.
be abundant in glycoprotein, especially fibronectin [6]. 6. Sato K, Hirano M, Nakashima T. Fine structure of the human new-
Fibronectin is a glycoprotein that serves as a template for the born and infant vocal fold mucosae. Ann Otol Rhinol Laryngol.
oriented deposition of collagen [10]. It acts as an interfibril- 2001;110:417–24.
lar stabilizing factor between collagen fibrils and as a skele- CD44 and hyaluronic acid in human vocal fold mucosa. Ann Otol
ton for elastic tissue formation and is involved with Rhinol Laryngol. 2006;115:741–8.
aggregation of proteoglycans [10]. 8. Fawcett DW. Epithelium. In: A textbook of histology. Philadelphia,
After the discovery of hyaluronan (hyaluronic acid), it PA: WB Saunders Co.; 1986. p. 57–82.
was assumed that its major functions were related to the bio- Laryngol. 1998;107:1023–8.
physical and homeostatic properties of tissues. However, 10. Anderson JC. Glycoproteins of the connective tissue matrix. In:
current studies have lead to the understanding that hyaluro- Hall DH, Jackson DS, editors. International review of connec-
nan also plays a crucial role in cell behavior [11]. Hyaluronan tive tissue research, vol. 7. New York, NY: Academic Press; 1976.
p. 251–322.
and other glycosaminoglycan are distributed in the lamina 11. Toole BP. Proteoglycans and hyaluronan in morphogenesis and dif-
propria of the newborn vocal fold mucosa. It is able to ferentiation. In: Hay E, editor. Cell biology of extracellular matrix.
directly affect the cellular functions of the human newborn 2nd ed. New York: Plenum Press; 1991. p. 305–41.
vocal fold mucosa. 12. Fawcett DW. Connective tissue proper. In: A textbook of histology.
Philadelphia, PA: WB Saunders Co.; 1986. p. 136–73.
Fibrous components such as reticular fibers are not well
developed. The lamina propria of the newborn vocal fold
Macula Flava of the Human Newborn
Vocal Fold 14
Abstract
1. The newborn maculae flavae are composed of relatively dense masses of cells and situ-
ated at the anterior and posterior ends of the bilateral vocal fold mucosae.
2. The cells in the newborn maculae flavae possess some features of mesenchymeal cells.
3. The vocal fold stellate cells in the newborn maculae flavae are immature, but some of
them have already started the synthesis of extracellular matrices, which are essential for
the viscoelastic properties of the lamina propria of the human newborn vocal fold
mucosa as a vibrating tissue.
4. The cells including the vocal fold stellate cells in the human newborn maculae flavae
possess proteins of all three germ layers. They are undifferentiated cells which arise not
from resident interstitial cells but from the differentiation of bone marrow cells.
5. The results of our studies are consistent with the hypothesis that the cells including the
vocal fold stellate cells in the human newborn maculae flavae are tissue stem cells or
progenitor cells of the human newborn vocal fold mucosa.
6. At birth, these cells have already been supplied from the bone marrow into the maculae
flavae of the newborn vocal fold and are ready to start the growth and development of
the human vocal fold mucosa as a vibrating tissue.
7. The extracellular matrices such as collagen fibers, reticular fibers, elastic fibers and
ground substances are not abundant in the newborn maculae flavae.
8. A newborn’s macula flava is in the process of acquiring a hyaluronan-rich matrix mak-
ing it a candidate for a stem cell niche.
14.1 Introduction viscoelastic properties of the lamina propria of the human

adult vocal fold [4]. Human adult maculae flavae are thought
The membranous portion of the human adult vocal fold to be responsible for maintaining the characteristic layered
mucosa is connected to the thyroid cartilage anteriorly via the structure of the human adult vocal fold mucosa [4]. Human
intervening anterior macula flava and anterior commissure newborn, infant and child maculae flavae are also considered
tendon [1]. Posteriorly, it is joined to the vocal process of the to be responsible for forming the characteristic layered struc-
arytenoid cartilage via the intervening posterior macula flava ture of the human vocal fold mucosa [5–7]. Human maculae
[1]. The vocal ligament runs between the anterior and poste- flavae are considered to be an important structure in the
rior maculae flavae [1]. Many vocal fold stellate cells, which growth, development and aging of the human vocal fold
are stellate in shape and store vitamin A in their lipid droplets, mucosa [4–8].
are distributed in the human adult maculae flavae [2, 3]. Vocal fold stellate cells in the human adult maculae flavae
Human adult maculae flavae, located at both ends of the are vitamin A storing cells and a member of the “Diffuse
vocal fold mucosa, are most likely be involved in the metab- Stellate Cell System” [9]. Vocal fold stellate cells are consid-
olism of extracellular matrices, which are essential for the ered a new category of cells in the human vocal fold mucosa.

186 14 Macula Flava of the Human Newborn Vocal Fold
Adult tissue-specific stem cells (tissue stem cells) have macula flava is attached to the vocal process of the arytenoid
the capacity to self-renew and to generate functionally dif- cartilage posteriorly (Fig. 14.4). The transition of cells and
ferentiated cells that replenish lost cells throughout an organ- extracellular matrices between the posterior macula flava and
ism’s lifetime. There is growing evidence to suggest that the elastic cartilage portion of the vocal process is gradual and the
cells including vocal fold stellate cells in the human maculae border between them is not clearly delineated (Fig. 14.5).
flavae are tissue stem cells or progenitor cells in the human The maculae flavae in the human newborn vocal fold
vocal fold mucosa [10, 11]. The human maculae flavae are a mucosa is composed of cells, elastic fibers, collagen fibers,
candidate for a stem cell niche, which is a microenvironment reticular fibers and ground substances. Cellular components
nurturing a pool of tissue stem cells [10, 11]. (Fig. 14.6a) are more abundant than fibrous components
(Fig. 14.6b) compared with adults.
14.2 M
acula Flava in the Human Newborn
Vocal Fold Mucosa 14.3 C
ells in the Macula Flava
of the Human Newborn Vocal Fold
Newborns have maculae flavae at the same sites as in adult Mucosa
vocal folds [1, 12]. The newborn maculae flavae are com-
posed of relatively dense masses of cells and situated at the Many cells are present in the newborn maculae flavae and
anterior and posterior ends of the bilateral vocal fold muco- their density is great, causing the maculae flavae to appear as
sae (Fig. 14.1). No structure that could be considered to be a a dense mass of cells (Figs. 14.1, 14.2, 14.6, and 14.7). The
vocal ligament is present (Fig. 14.1). The lamina propria of density of cells in the newborn maculae flavae is about five
the mucosa between the anterior and posterior maculae fla- times that of the adult maculae flavae [13]. The density of
vae is a loose structure. cells in the newborn maculae flavae is about eight times that
The newborn maculae flavae are round in shape and mea- of fibroblasts in the lamina propria of the newborn vocal fold
sure approximately 1 × 1 × 1 mm (Figs. 14.1 and 14.2). The mucosa [13].
anterior macula flava is connected to the thyroid cartilage via The cells in the newborn maculae flavae are about
the anterior commissure tendon (Fig. 14.3). The posterior 5–10 μm in size. The cells are oval or stellate in shape and
a b
anterior thyroarytenoid anterior thyroarytenoid

macula muscle macula muscle
flava flava
lamina lamina
propria propria
posterior posterior
macula macula
flava flava
vocal vocal
Fig. 14.1 Horizontal process process
sections of the newborn vocal of arytenoid of arytenoid
fold. (a) Hematoxylin and cartilage cartilage
Eosin stain. (b) Elastica van
Gieson stain
14.3 Cells in the Macula Flava of the Human Newborn Vocal Fold Mucosa 187
a they have cytoplasmic processes (Figs. 14.8 and 14.9). The

nucleus-cytoplasm ratio is large and cytoplasm occupies a
small area around the nucleus. Cells in some cases form gap
posterior junctions with each other (Fig. 14.10). Free ribosomes are
macula
well developed in the cytoplasm (Fig. 14.11). Occasionally,
flava
a basal body is seen in the cytoplasm (Fig. 14.11). These
findings indicate that cells in the newborn maculae flavae
possess some features of mesenchymeal cells.
Intracellular organelles such as rough endoplasmic reticu-
lum and Golgi apparatus in the cytoplasm are not very well
developed (Fig. 14.12). There are few mitochondria and they
are small. No basal lamina is found. Along the periphery of
the cytoplasm of the cells that have developed intracellular
organelles, a number of vesicles are present.
thyroid cartilage
posterior
macula
flava
thyroarytenoid muscle anterior commissure tendon
anterior anterior
macula macula
flava flava
Fig. 14.2 Coronal sections of the newborn posterior macula flava Fig. 14.3 Horizontal section of the newborn anterior macula flava
(arrows). (a) Hematoxylin and Eosin stain. (b) Elastica van Gieson stain (arrows) (Elastica van Gieson stain)
a b
posterior
macula posterior
flava macula
flava
Fig. 14.4 Horizontal sections of

the newborn posterior macula flava vocal vocal
(arrows) and the vocal process of process process
of arytenoid of arytenoid
the arytenoid cartilage. (a)
cartilage cartilage
Hematoxylin and eosin stain. (b)
Elastica van Gieson stain
Fig. 14.5 Transition area of posterior macula flava posterior macula flava
a b
the newborn posterior macula
flava and the vocal process of cells
the arytenoid cartilage. (a)
The cells in the posterior
macula flava (top) gradually
change to chondrocytes in the
vocal process (bottom)
(Hematoxylin and Eosin
stain). (b) The posterior
macula flava (top) gradually
changes to the elastic
cartilage portion of the
arytenoid cartilage (bottom)
(Elastica van Gieson stain)
chondrocytes
elastic cartilage portion of vocal process elastic cartilage portion of vocal process
cells
b collagen fibers
Fig. 14.6 Higher magnifications of the newborn

macula flava. (a) Cellular components are more
abundant (Hematoxylin and eosin stain). (b) Fibrous elastic fibers
components are sparse. Collagen fibers are stained
red and elastic fibers are stained black (Elastica van
Gieson stain)
14.3 Cells in the Macula Flava of the Human Newborn Vocal Fold Mucosa 189
nucleus
cell
collagen fibers
elastic fibers
gap junction
cell
nucleus
Fig. 14.10 Transmission electron micrograph of a gap junction

Fig. 14.7 Transmission electron micrograph of the newborn macula between cells in the newborn macula flava (tannic acid stain)
flava (tannic acid stain)
basal bodies
nucleus
free ribosomes
Fig. 14.11 Transmission electron micrograph of basal bodies and

Fig. 14.8 Transmission electron micrograph of a cell in the newborn ribosomes in the cytoplasm of a cell in the newborn macula flava (ura-
macula flava (tannic acid stain) nyl acetate and lead citrate stain)
gap junction
nucleus nucleus
gap junction
Golgi apparatus
rough endoplasmic
reticulum
cytoplasmic process
Fig. 14.9 Transmission electron micrograph of cells in the newborn Fig. 14.12 Transmission electron micrograph of a cell in the newborn
macula flava (tannic acid stain). The Cells form gap junctions with each macula flava (tannic acid stain). Intracellular organelles such as rough
other endoplasmic reticulum and Golgi apparatus in the cytoplasm are observed
membrane- A few lipid droplets are present in the cytoplasm, but they
bounded lipid are much fewer in number than those found in adults
droplet
(Figs. 14.13 and 14.14). The lipid droplets are 0.6–0.7 μm in
diameter and are thus smaller than those of adults. They are
of two types: membrane-bounded (Fig. 14.13) and non-
membrane-bounded (Fig. 14.14). The former are each sur-
rounded by a unit membrane.
A few cells in the newborn maculae flavae show cyto-
nucleus
plasm staining with periodic acid-Schiff stain (Fig. 14.15).
The cells in the human newborn maculae flavae express type
III collagen (Fig. 14.16).
Fig. 14.13 Transmission electron micrograph of a cell in the newborn

macula flava (tannic acid stain). Membrane-bounded lipid droplets are
observed in the cytoplasm of a cell
a cytoplasmic process
cells
nucleus
Fig. 14.15 Periodic acid-Schiff staining of the cells in the newborn

macula flava. A few cells show cytoplasmic staining with periodic acid-
Schiff stain
non-membrane- cells
bounded lipid
droplet
Fig. 14.14 Transmission electron micrograph of a cell in the newborn

macula flava (uranyl acetate and lead citrate stain). (a) Non-membrane-
bounded lipid droplet is observed in the cytoplasm of a cell. (b) Region Fig. 14.16 Type III collagen immunohistochemical staining of the
B in a cells in the newborn macula flava
14.6 Cell Origin in the Macula Flava of the Human Newborn Vocal Fold Mucosa 191
The morphological findings of the cells in the newborn phous materials are present on and around the cells. The
maculae flavae mentioned above are recognized to various newborn vocal fold stellate cells have started to synthesize
degrees. fibrillar proteins and ground substances.
Newborn vocal fold stellate cells are immature, but some of
them have already started the synthesis of extracellular matrices,
14.4 Morphological Comparison which are essential for the viscoelastic properties of the lamina
of Newborn and Adult Cells Including propria of the human vocal fold mucosa as a vibrating tissue.
Vocal Fold Stellate Cells
in the Maculae Flavae
14.5 M
orphological Comparison Between
The distributions of newborn and adult vocal fold stellate Cells Including Vocal Fold Stellate
cells is the same, and in both cases they are located in the Cells in the Maculae Flavae
maculae flavae, but none are found in the lamina propria of and Fibroblasts in the Lamina Propria
the mucosa between anterior and posterior maculae flavae. of the Human Newborn Vocal Fold
Newborn vocal fold stellate cells are stellate or oval in shape Mucosa
and possess cytoplasmic processes. The morphological char-
acteristics of newborn vocal fold stellate cells are not com- Fibroblasts are present throughout the newborn vocal fold
pletely the same as those of adults, but some of them show mucosa.
the characteristic features of adult vocal fold stellate cells. As noted above the cells in the newborn maculae flavae are
Adult vocal fold stellate cells possess lipid droplets and immature and possess some features of mesenchymal cells.
store vitamin A [2, 3]. Vitamin A strongly influences the The cells in the newborn maculae flavae differ most from
activity of adenosine triphosphate (ATP) sulphurylase and is fibroblasts in shape, being stellate and possessing cytoplasmic
related to the synthesis of glycosaminoglycan [14–16]. Only processes. The cells in the newborn maculae flavae possess a
a few lipid droplets are present in the cytoplasm of newborn few lipid droplets in the cytoplasm, whereas fibroblasts have
vocal fold stellate cells, and they are smaller than those of the no lipid droplets. Intracellular organelles are noted in the cells
adult vocal fold stellate cells. in the newborn maculae flavae, whereas the newborn fibro-
Free ribosomes are well developed but intracellular blasts have a high nucleus-cytoplasm ratio, and fewer intracel-
organelles are not very well developed in the cytoplasm of lular organelles, and thus are inactive and at rest.
newborn vocal fold stellate cells. The newborn vocal fold A few cells in the newborn maculae flavae show strong
stellate cells possess cytoplasmic processes and some cells cytoplasm staining with periodic acid-Schiff stain and the
form gap junctions with each other. These findings indi- cells in the human newborn maculae flavae express type III
cated that the vocal fold stellate cells in the newborn macu- collagen, whereas few components are stained in the cyto-
lae flavae are immature and possess some features of plasm of fibroblasts.
mesenchymal cells. The most outstanding features of some cells in the new-
In many types of tissue and cultured cells, the interiors of born maculae flavae are that they have already started the
adjacent cells communicate with each other though cell-to- synthesis of extracellular matrices. The fibroblasts in the
cell channels [17]. The fine structure of the cell-to-cell chan- lamina propria of the newborn vocal fold mucosa are inac-
nels has been well studied and defined as a gap junction [17]. tive and produce few extracellular matrices. The cells includ-
Cell communication is proposed to play an important role in ing vocal fold stellate cells in the newborn maculae flavae
cell growth and differentiation [17]. The newborn vocal fold have already started the synthesis of extracellular matrices
stellate cells possess cytoplasmic processes and some cells and have some role in the metabolism of extracellular matri-
form gap junctions with each other. Cells in the newborn ces in the newborn vocal fold mucosa.
maculae flavae may communicate with each other for their
growth and differentiation.
The adult vocal fold stellate cells show strong cytoplasm 14.6 C
ell Origin in the Macula Flava
staining with periodic acid-Schiff stain, whereas only some of the Human Newborn Vocal Fold
of the newborn vocal fold stellate cells are stained. The cyto- Mucosa
plasm of newborn vocal fold stellate cells appears to contain
less glycogen than adult vocal fold stellate cells. As a result of the heterogeneity of the cells in the macula
Adult vocal fold stellate cells constantly synthesize amor- flava of the human vocal fold, it is uncertain whether the
phous materials [2]. Newborn vocal fold stellate cells which cells including vocal fold stellate cells derive from the same
have developed intracellular organelles have vesicles along embryonic source as fibroblasts in the lamina propria of the
the periphery of the cytoplasm and newly released amor- human newborn vocal fold mucosa.
14.6.1 Intermediate Filaments of the Cells the newborn maculae flavae express SOX17, which is the
in the Newborn Macula Flava endodermal cell marker (Fig. 14.18).
Cytokeratin is a protein of the intermediate filaments of
The expression of proteins in the intermediate filaments of epithelial cells, and vimentin is a major subunit protein of
the cytoplasm is specific to cell type and differentiation [18]. the intermediate filaments of mesenchymal cells. Glial
Because of the tissue specificity of intermediate filaments, fibrillary acidic protein, a member of the intermediate fila-
cells from different tissues can be distinguished on the basis ment protein family and characteristic of neural crest cells,
of the intermediate filament protein present [18]. is heavily and specifically expressed in astrocytes and cer-
Cytoplasmic cytokeratin (epithelium-associated protein), tain other astroglia in the central nervous system. In addi-
vimentin (mesenchymal cell-associated protein), glial fibril- tion, neural stem cells frequently and strongly express glial
lary acidic protein (neural-associated protein), and desmin fibrillary acidic protein. Desmin, a protein of the intermedi-
(muscle-associated protein) immunoreactivity are present in ate filaments, is characteristic of myogenic crest cells and
the cells including vocal fold stellate cells in the human new- is found in muscle cells.
born maculae flavae (Fig. 14.17). Additionally, the cells in
a
c
cells
cells
b d
cells cells
Fig. 14.17 Immunohistochemical staining of the cells in the newborn macula flava. Cytokelatin (a), vimentin (b), glial fibrillary acidic p rotein
(c) and desmin (d) immunoreactivity are present
14.6 Cell Origin in the Macula Flava of the Human Newborn Vocal Fold Mucosa 193
cells
cells
Fig. 14.18 SOX17 immunohistochemical staining of the cells in the

newborn macula flava Fig. 14.19 Telomerase reverse transcriptase immunohistochemical
staining of the cells in the newborn macula flava
Consequently, the cells in the human newborn maculae to its hematopoietic component, the mesenchymal compo-
flavae express proteins of all three germ layers. This suggests nent of the hematopoietic organs include fibroblast-like cells
that they are undifferentiated and multipotent. (stromal cells), myofibroblasts, adipocytes, and endothelial
cells [20]. Some marrow-derived cells, such as circulating
fibrocytes and pericytes, have been suggested to contribute to
14.6.2 Telomerase of the Cells in the Newborn tissue fibroblasts [20]. Fibroblast-related cells, such as
Macula Flava hepatic stellate cells [21] and myofibroblasts in wounded
skin [22], are also derived from bone marrow. It is interesting
A special DNA polymerase called telomerase can catalyze that the morphological features of the vocal fold stellate cells
the formation of additional copies of the telomeric repeat in the human maculae flavae are similar to the hepatic stel-
sequence, thereby compensating for the gradual shortening late cells and included in the proposed diffuse stellate cell
that occurs at both ends of the chromosome during DNA system [9].
replication [19]. In multicellular organisms, telomerase Marrow-derived circulating fibroblast precursors have
resides mainly in the germ cells that give rise to sperm and been suggested to originate from marrow cells, circulate into
eggs, and in a few other kinds of proliferating normal cells blood cells and, after homing to the tissue, differentiate into
such as stem cells [19]. Because telomerase is not found in fibroblasts [20]. Circulating fibrocytes were first identified
most cells, their chromosomal telomeres get shorter and by Bucala et al. in 1994 [23]. They were found to be unique
shorter with each cell division [19]. The presence of telom- cells because they co-express hematopoietic markers as well
erase allows cells to divide indefinitely without telomere as collagen type I and other matrix proteins (mesenchymal
shortening [19]. markers). Circulating fibrocytes are specifically defined by
The cells in the newborn maculae flavae express telomer- the expression of CD34 (cluster of differentiation 34), CD45
ase reverse transcriptase (Fig. 14.19), indicating telomerase and collagen type I [20].
resides in the cells in the newborn macula flava. This finding The cells in the human newborn maculae flavae express
raises the possibility that the cells including vocal fold stellate hematopoietic markers (CD34, CD45) and type I collagen
cells are tissue stem cells (tissue-specific resident stem cells). (Fig. 14.20), which are the major makers of bone marrow
derived circulating fibrocytes [24]. Most of the fibroblasts in
the tissue surrounding the macula flava do not express CD34
14.6.3 The Relationship between Bone and CD45 (Fig. 14.21).
Marrow-Derived Cells and Cells These observations are consistent with the hypothesis
in the Human Newborn Macula Flava that the cells including vocal fold stellate cells in the
human newborn maculae flavae arise not from resident
Bone marrow has two major components: a hematopoietic interstitial cells but from the differentiation of bone mar-
component and a mesenchymal component [20]. In contrast row cells [24].
a a
cells in the posterior macula flava
*
* *
cells * * * *
fibroblasts
b b
cells
fibroblasts
* *
* *
* *
*
cells in the posterior macula flava
c Fig. 14.21 Border (asterisks) between the cells of the newborn poste-
rior macula flava including vocal fold stellate cells and the surrounding
cells tissue. Cells in the posterior macula flava express CD34 (a) and CD45
(b). The border between posterior macula flava and surrounding tissue
is clearly delineated. CD34 positive and CD45 positive fibroblasts are
observed at the periphery of macula flava
14.6.4 Pluripotency of the Cells

in the Newborn Maculae Flavae
Stage-specific embryonic antigen-3 (SSEA-3) is present on

both cell surface glycolipids and glycopeptides of human
teratocarcinoma cells, on embryonic germ cells and on
embryonic stem cells. SSEA-3 expression decreases as those
cells differentiate.
The cells in the human newborn maculae flavae express
Fig. 14.20 Immunohistochemical staining of the cells in the newborn SSEA-3 (Fig. 14.22). This suggests that many cells includ-
macula flava. CD34 (Cluster of differentiation 34) (a), CD45 (b) and ing vocal fold stellate cells in the human newborn maculae
type I collagen (c) immunoreactivity, which are the major makers of
flavae are stem cell related cells and possibly have
bone marrow derived circulating fibrocytes, are present
pluripotency.
14.8 Extracellular Matrices in the Macula Flava of the Human Newborn Vocal Fold Mucosa 195
cells
collagen fibers
reticular fibers
Fig. 14.22 Stage-specific embryonic antigen 3 immunohistochemical Fig. 14.23 Collagen and reticular fibers in the newborn macula flava
staining of the cells in the newborn macula flava (silver stain). Fibrous components are sparse. Collagen fibers are
stained red and reticular fibers are stained black
14.7 C
ells Including Vocal Fold Stellate
Cells in the Newborn Maculae Flavae
Many cells including the vocal fold stellate cells in the

human newborn maculae flavae are suggested to be undif-
ferentiated cells which arise not from resident interstitial
elastin
cells but from the differentiation of bone marrow cells [24].
collagen fibrils
The results of our study are consistent with the hypothesis
that cells including the vocal fold stellate cells in the maculae
microfibrils
flavae are tissue stem cells or progenitor cells of the human
newborn vocal fold mucosa [24]. amorphous
materials
At birth, these cells have already been supplied from the
vesicles
bone marrow into the maculae flavae in the newborn vocal
fold, and are ready to start the growth and development of
the human vocal fold mucosa as a vibrating tissue [24].
Fig. 14.24 Transmission electron micrograph of the synthesis of col-

lagen and elastic fibers by a cell in the newborn macula flava (tannic
14.8 E
xtracellular Matrices in the Macula acid stain)
Flava of the Human Newborn Vocal
Fold Mucosa
Collagen fibers are made up of collagen fibrils (Fig. 14.25).
14.8.1 Collagen and Reticular Fibers The cells in the maculae flavae synthesize not only collagen
fibers but also reticular fibers (Type III collagen) (Fig. 14.26)
There are many collagen fibers around the cells in adult mac- in the newborn vocal fold mucosa. At birth, the cells in the
ulae flavae, whereas there are not very many collagen fibers macula flava start to synthesize fibrous proteins for collagen
around cells in newborn maculae flavae (Fig. 14.6). Few and reticular fibers.
reticular fibers can be detected around cells in the newborn
maculae flavae (Fig. 14.23).
There are vesicles at the periphery of the cytoplasm, and 14.8.2 Elastic Fibers
newly released amorphous materials are present on the cell
surface in the newborn macula flava (Fig. 14.24). Microfibrils There are many elastic fibers around the cells in the adult
10–15 nm wide are observed around the amorphous materi- maculae flavae, whereas not very many elastic fibers are
als. Collagen fibrils are present near the microfibrils. present around cells in the newborn macula flava (Fig. 14.6).
Figure 14.24 shows electron microscopic evidence of the 14.8.3 Glycosaminoglycan

synthesis of elastic fibers by cells in the newborn macula
flava. There are some vesicles at the periphery of the The ground substances in the adult maculae flavae strongly
cytoplasm, and newly released amorphous materials are seen stain light blue with Alcian Blue at pH 2.5, but sparsely stain
on the cell surface. Microfibrils 10–15 nm wide are situated with Alcian Blue at pH 1. Material in the adult maculae fla-
around the amorphous materials and are microfibril assem- vae that is strongly stained with Alcian Blue (pH 2.5) is
blies on which elastin is deposited (Figs. 14.24 and 14.25). digested by hyaluronidase.
The number of elastic fibers is small compared to that of The ground substances in the newborn maculae flavae
collagen fibers. The elastic fibers are composed of microfi- slightly stain light blue with Alcian Blue at pH 2.5
brils and amorphous substances. The former are abundant, (Fig. 14.27) and at pH 1 (Fig. 14.27). Glycosaminoglycan is
and the latter are sparse and reticular in shape, indicating that present but not abundant in the newborn maculae flavae. A
the elastic fibers are immature. Electron microscopic studies glycosaminoglycan other than hyaluronan (hyaluronic acid)
indicate that the cells in the newborn maculae flavae have is present in the newborn maculae flavae.
already started to synthesize elastic fibers.
microfibrils
a
collagen
fibril
collagen fiber
elastic fiber
elastin glycosaminoglycan
Fig. 14.25 Transmission electron micrograph of collagen and elastic

fibers in the newborn macula flava (tannic acid stain)
reticular fiber
nucleus
glycosaminoglycan
Fig. 14.26 Transmission electron micrograph of a reticular fiber in the Fig. 14.27 Glycosaminoglycan in the newborn macula flava (Alcian
newborn macula flava (uranyl acetate and lead citrate stain). Reticular blue stain). (a) Newborn maculae flavae are slightly stained light blue
fibers are about 38 nm in diameter and have cross-bands with a period- with Alcian blue at pH 2.5. (b) Newborn maculae flavae are slightly
icity of about 55 nm stained light blue with Alcian blue at pH 1
References 197
14.9 M
icroenvironment in the Macula growth and development of the vocal fold. Folia Phoniatr Logop.
2010;62:263–70.
Flava of the Newborn Vocal Fold 8. Sato K, Hirano M, Nakashima T. Age-related changes in vitamine
Mucosa A-storing stellate cells of human vocal folds. Ann Otol Rhinol
Laryngol. 2004;113:108–12.
The structural and biochemical microenvironment that con- 9. Sato K, Umeno H, Nakashima T. Vocal fold stellate cells in the
human macula flava and the diffuse stellate cell system. Ann Otol
fers stemness upon cells in multicellular organisms is Rhinol Laryngol. 2012;121:51–6.
referred to as a stem cell niche. A stem cell niche is com- 10. Sato K, Umeno T, Nakashima T. Vocal fold stem cells and their
posed of a group of cells in a special tissue location for the niche in the human vocal fold. Ann Otol Rhinol Laryngol.
maintenance of stem cells [25]. 2012;121:798–803.
There is growing evidence to suggest that the human maculae H. Origin of vocal fold stellate cells in the human macula flava.
flavae are a candidate for a stem cell niche, which is a microenvi- Ann Otol Rhinol Laryngol. 2015;124:698–705.
ronment nurturing a pool of stem cells [10, 11, 26]. A proper 12. Sato K, Hirano M. Histological investigation of the macula flava
microenvironment in the maculae flavae of the human vocal fold of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
1995;104:556–62.
mucosa is necessary to be effective as a stem cell niche that main- 13. Sato K, Sakamoto K, Nakashima T. Expression and distribution of
tains the stemness of the contained tissue stem cells [25]. CD44 and hyaluronic acid in human vocal fold mucosa. Ann Otol
Hyaluronan serves as an important niche component for Rhinol Laryngol. 2006;115:741–8.
numerous stem cell populations [27, 28]. After the discovery 14. Sundaresan PR. Vitamin A and the sulfate-activating enzymes.

Biochim Biophys Acta. 1966;113:95–109.
of hyaluronan, it was assumed that its major functions were 15. DeLuca L, Wolf G. Effect of vitamin A on the mucopolysaccha-
related to the biophysical and homeostatic properties of tis- rides of lung tissue. Arch Biochem Biophys. 1968;123:1–8.
sues. However, current studies lead to the understanding that 16. Levi AS, Geller S, Root DM, Wolf G. The effect of vitamin A and
hyaluronan also plays a crucial role in cell behavior [29]. A other dietary constituents on the activity of adenosine triphosphate
sulphurylase. Biochem J. 1968;109:69–74.
hyaluronan-rich matrix, which is composed of the glycosami- 17. Kanno Y. Modulation of cell communication and carcinogenesis.
noglycan hyaluronan and its transmembrane receptors (a cell Jpn J Physiol. 1985;35:693–707.
surface hyaluronan receptor), is able to directly affect the cel- 18. Becker WM, Kleinsmith L, Hardin J. Intermediate filament. In: The
lular functions of stem cells in a stem cell nich [27, 28]. world of the cell. 6th ed. San Francisco, CA: Benjamin Cummings;
2006. p. 446–50.
A great deal of glycosaminoglycan (hyaluronan) is situ- 19. Becker WM, Kleinsmith LJ, Hardin J. The cell cycle, DNA replica-
ated in the adult maculae flavae and most of the cells in the tion, and mitosis. In: The world of the cell. 6th ed. San Francisco,
adult maculae flavae express CD44 (a cell surface hyaluro- CA: Benjamin Cummings; 2006. p. 554–71.
nan receptor) [10, 13]. Consequently, adult macula flava is a 20. Abedi M. Hematopoietic origin of fibrocytes. In: Bucala R, edi-
tor. Fibrocytes in health and disease. Singapore: World Scientific
hyaluronan-rich matrix and a candidate for a stem cell niche Publishing; 2012. p. 1–15.
[10]. On the other hand, hyaluronan is not so abundant in the 21. Forbes SJ, Russo FP, Rey V, Burra P, Rugge M, Wright NA, Alison
newborn maculae flavae and only one-third of the cells in the MR. A significant proportion of myofibroblasts are of bone marrow
newborn macula flava express CD44 [13]. A newborn’s mac- origin in human liver fibrosis. Gastroenterology. 2004;126:955–63.
22. Brittan M, Hunt T, Jeffery R, Poulsom R, Forbes SJ, Hodivala-Dilke
ula flava is in the process of acquiring a hyaluronan-rich K, Goldman J, Alison MR, Wright NA. Bone marrow derivation of
matrix making it a candidate for a stem cell niche. pericryptal myofibroblasts in the mouse and human small intestine
and colon. Gut. 2002;50:752–7.
23. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating
fibrocytes define a new leukocyte subpopulation that mediates tis-
References sue repair. Mol Med. 1994;1:71–81.
24. Sato K, Chitose T, Kurita T, Umeno H. Cell origin in the mac-
1. Hirano M, Sato K. Histological color atlas of the human larynx. San ula flava of the human newborn vocal fold. J Laryngol Otol.
Diego, CA: Singular Publishing Group Inc.; 1993. 2016;130:650–5.
2. Sato K, Hirano M, Nakashima T. Stellate cells in the human vocal 25. Li L, Xie T. Stem cell niche: structure and function. Annu Rev Cell
fold. Ann Otol Rhinol Laryngol. 2001;110:319–25. Dev Biol. 2005;21:605–31.
3. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in 26. Sato K, Chitose T, Kurita T, Umeno H. Microenvironment of mac-
the human vocal fold. Acta Otolaryngol. 2003;123:106–10. ula flava in the human vocal fold as a stem cell niche. J Laryngol
4. Sato K, Umeno H, Nakashima T. Functional histology of the mac- Otol. 2016;130:656–61.
ula flava in the human vocal fold. Part 1: its role in the adult vocal 27. Haylock DN, Nilsson SK. The role of hyaluronic acid in hemopoi-
fold. Folia Phoniatr Logop. 2010;62:178–84. etic stem cell biology. Regen Med. 2006;1:437–45.
5. Sato K, Hirano M, Nakashima T. Fine structure of the human new- 28. Preston M, Sherman LS. Neural stem cell niches: roles for

born and infant vocal fold mucosae. Ann Otol Rhinol Laryngol. the hyaluronan- based extracellular matrix. Front Biosci.
2001;110:417–24. 2011;3:1165–79.
6. Sato K, Nakashima T. Vitamin A-storing stellate cells in the human 29. Toole BP. Proteoglycans and hyaluronan in morphogenesis and dif-
newborn vocal fold. Ann Otol Rhinol Laryngol. 2005;114:517–24. ferentiation. In: Hay E, editor. Cell biology of extracellular matrix.
7. Sato K, Umeno H, Nakashima T. Functional histology of the 2nd ed. New York: Plenum Press; 1991. p. 305–41.
macula flava in the human vocal fold. Part 2: Its role in the
Growth and Development of the Human
Vocal Fold Mucosa 15
Abstract
1. Newborns already have immature maculae flavae at the same sites as adults. They are
composed of dense masses of cells including vocal fold stellate cells, whereas extracel-
lular matrix components are sparse. Vocal fold stellate cells in the newborn maculae
flavae have already started synthesizing extracellular matrices.
2. During infancy, the extracellular matrices synthesized in the maculae flavae extend to
the lamina propria of the vocal fold mucosa to initiate the formation of the three-
dimensional extracellular matrix structure of the human vocal fold mucosa.
3. During childhood, maculae flavae containing vocal fold stellate cells continue to synthe-
size extracellular matrices such as collagen, reticular, and elastic fibers and hyaluronic
acid (glycosaminoglycan), which are essential for the viscoelasticity of the human vocal
fold mucosa as a vibrating tissue.
4. The human vocal fold grows and develops and its layered structure matures during
adolescence.
5. Human maculae flavae containing vocal fold stellate cells are involved in the metabo-
lism of extracellular matrices essential for the viscoelasticity of the human vocal fold
mucosa and are considered to be an important structure in the growth and development
of the human vocal fold mucosa.
15.1 Introduction sparse fibers, and no structure corresponding to the vocal

ligament can be found (Fig. 15.2). Ground substances are
In adults, the human vocal fold has a layered structure con- abundant and glycoproteins (fibronectin) are present in the
sisting of the epithelium, the lamina propria (superficial, lamina propria of the newborn vocal fold mucosa. The lam-
intermediate and deep layers) and the vocalis muscle [1, 2]. ina propria of the newborn vocal fold lacks not only a vocal
The superficial layer is referred to as Reinke’s space. The ligament and layered structure but also the characteristic
structure consisting of the intermediate and deep layers of complex of extracellular matrices seen in adults. The visco-
the lamina propria is called the vocal ligament (Fig. 15.1). elasticity of the newborn vocal fold mucosa is morphologi-
The vocal ligament runs between the anterior and posterior cally not sufficient for phonation.
maculae flavae (Fig. 15.1). The ability of the mucosal por- Newborns have maculae flavae at the same sites of the
tion of the layered structure to act as a vibrating tissue is vocal fold (the anterior and posterior ends of the membra-
based on the differences of extracellular matrix distribution; nous portions of bilateral vocal fold mucosae) as adults
it is essential for vocal fold vibration and phonation [1]. (Fig. 15.2). Newborn maculae flavae are formed by dense
The human vocal fold grows and develops and its layered masses of cells including vocal fold stellate cells, whereas
structure matures during adolescence [3, 4]. At birth, the lay- extracellular matrix components, especially fibrous compo-
ered structure seen in adult vocal folds is not present [2, 3, nents, are sparse. The morphological characteristics of the
5–8]. The lamina propria of the newborn vocal fold mucosa newborn maculae flavae are not completely the same as those
is a loose structure composed of ground substances and of adults, and they are immature. The vocal fold stellate cells

200 15 Growth and Development of the Human Vocal Fold Mucosa
in the newborn maculae flavae are also immature, but some 15.2 C
ells and Extracellular Matrices
of them have already started the synthesis of extracellular in the Human Infant Vocal Fold
matrices, such as collagen fibers, reticular fibers, elastic Mucosa
fibers and glycosaminoglycan, which are essential for the
viscoelastic properties of the lamina propria of the human No structure corresponding to the vocal ligament can be
newborn vocal fold mucosa. found in the infant vocal fold mucosa (Figs. 15.2 and 15.3).
The maculae flavae in the newborn vocal fold are ready to During infancy, many reticular fibers (type III collagen)
start the growth and development of the human vocal fold and collagen fibers extend from the anterior and posterior
mucosa as a vibrating tissue. The newborn vocal fold mucosa maculae flavae toward the middle of the lamina propria of
is not suitable for phonation but is in the process of acquiring the vocal fold mucosa, in which glycoprotein (fibronectin) is
the viscoelastic properties of the human vocal fold mucosa abundant (Fig. 15.3). The number of reticular and collagen
as a vibrating tissue. fibers have increased throughout the entire lamina propria of
the infant vocal fold mucosa as compared to newborns.
Reticular and collagen fibers run roughly parallel to the vocal
fold edge (Fig. 15.4). The collagen fibers are made up of
numerous collagen fibrils approximately 50 nm in diameter.
thyroid cartilage
Their structure is nearly mature morphologically. The reticu-
lar fibers (Type III collagen) are made up of unit fibrils and
are about 40 nm in diameter with cross-bands with a period-
icity of about 67 nm (Fig. 15.5). Their slender fibrils of the
reticular fibers do not form bundles (Fig. 15.5).
Reinke’s space
Elastic fibers can be seen in the lamina propria of the
vocal ligament infant vocal fold mucosa at low density (Fig. 15.6) and are
composed of abundant microfibrils and sparse reticular
amorphous substances. The elastic fibers are immature, but
increase in amount over time in the lamina propria after col-
lagen and reticular fibers appear.
Fibronectin is a glycoprotein that serves as a template for
the oriented deposition of collagen [9]. It acts as an interfi-
vocal process of brillar stabilizing factor between collagen fibrils and as a
arytenoid cartilage
skeleton for elastic tissue formation and is also involved with
aggregation of proteoglycans [9].
Reticular and collagen fibers synthesized in infant ante-
rior and posterior maculae flavae extend toward the middle
of the membranous portion of the vocal folds, in which fibro-
nectin (glycoprotein) is abundant. Fibronectin in the lamina
propria appears to direct the oriented deposition of reticular
Fig. 15.1 Human adult maculae flavae and vocal fold mucosa and collagen fibers. Reticular and collagen fiber formation is
Newborn infant child
TC TC TC
ACT ACT ACT
Fig. 15.2 Human newborn, AMF AMF AMF
infant, and child maculae
flavae and growth and LP LP LP
development of the human
vocal fold mucosa. TC thyroid PMF PMF
cartilage, ACT anterior PMF
commissure tendon, AMF VP VP
VP
anterior macula flava, PMF
posterior macula flava, VP
vocal process of arytenoid
cartilage, LP lamina propria
of the vocal fold mucosa
15.2 Cells and Extracellular Matrices in the Human Infant Vocal Fold Mucosa 201
Fig. 15.3 Transverse sec-

a
tions of vocal fold from anterior
6-month-old girl (original commissure
×12.5). (a) Hematoxylin and tendon
Eosin stain, (b) Elastica van anterior posterior
Gieson stain, (c) silver stain. macula flava macula flava
No structure corresponding to lamina propria of
the vocal ligament can be vocal fold mucosa
found in the infant vocal fold
mucosa
b
anterior
commissure
tendon
anterior posterior
macula flava macula flava
lamina propria of
vocal fold mucosa
c
anterior
commissure
tendon
anterior posterior
lamina propria of
vocal fold mucosa
epithelium
reticular fibers
reticular fibers
collagen fibers
Fig. 15.5 Transmission electron micrograph of reticular fibers in the

Fig. 15.4 Transverse section of the lamina propria of the infant vocal lamina propria of the infant vocal fold mucosa (uranyl acetate and lead
fold (6-month-old girl, silver stain). Reticular (stained black) and col- citrate stain). The reticular fibers are unit fibrils about 40 nm in diame-
lagen (stained red) fibers run roughly parallel to the vocal fold edge ter and have cross-bands with a periodicity of about 67 nm
induced in the lamina propria of infant vocal fold mucosa Lamina propria of the infant vocal fold mucosa is stained
with growth. Fibronectin in the lamina propria of the vocal light blue with Alcian Blue at pH 2.5, and the material that
fold mucosa decreases over time with the increase of fibrous stains with Alcian Blue (pH 2.5) is digested by hyaluroni-
components. Fibronectin also acts as an interfibrillar stabiliz- dase (Fig. 15.7). Hyaluronic acid (hyaluronan) has appeared
ing factor between collagen fibrils and is involved in the in the lamina propria of the infant vocal fold mucosa
aggregation of elastic fibers and glycosaminoglycan, and (Fig. 15.7).
acts as a skeleton for elastic tissue formation in the human Fibroblasts are sparse in the lamina propria of the infant
infant vocal fold mucosa. vocal fold mucos (Fig. 15.8). The density of fibroblasts is
15.3 Maculae Flavae of the Human Infant Vocal Fold 203
epithelium
epithelium
collagen fibers
lamina propria
of vocal fold fibroblasts
elastic fibers mucosa
Fig. 15.6 Transverse section of the lamina propria of the infant vocal Fig. 15.8 Fibroblasts in the lamina propria of the infant vocal fold
fold (6-month-old girl, Elastica van Gieson stain). Collagen (stained mucosa (Hematoxylin and Eosin stain)
red) and elastic (stained black) fibers run roughly parallel to the vocal
fold edge
15.3 M
aculae Flavae of the Human Infant
Vocal Fold
epithelium The maculae flavae of infant vocal fold mucosa are also
dense masses of cells including vocal fold stellate cells
(Fig. 15.10). The density of cells including vocal fold stellate
cells in the infant maculae flavae is about three times that of
the adult maculae flavae and about two-thirds that of the
newborn maculae flavae [10] (Fig. 15.9).
The infant maculae flavae are composed of vocal fold
stellate cells (Fig. 15.10), collagen fibers (Figs. 15.11 and
hyaluronic acid
15.12), reticular fibers (Fig. 15.12), elastic fibers (Fig. 15.11),
and ground substances. Cellular components are more abun-
dant than fibrous components. However, fibrous components
have increased by comparison with those of the newborn
maculae flavae. More reticular and collagen fibers are pres-
ent than elastic fibers.
Fig. 15.7 Transverse section of the lamina propria of the infant vocal Many vocal fold stellate cells in the infant maculae flavae
fold (6-month-old girl, Alcian blue stain, pH 2.5). Hyaluronic acid (gly- are stellate in shape, possess cytoplasmic processes, and
cosaminoglycan) appears in the lamina propria of the infant vocal fold have a small nucleus-cytoplasm ratio. Vocal fold stellate
mucosa
cells show cytoplasmic staining with periodic acid-Schiff
stain (Fig. 15.13), type I collagen and type III collagen.
about one-sixth that of the cells including vocal fold stellate Rough endoplasmic reticulum and Golgi apparatus in the
cells in the infant maculae flavae [10] (Fig. 15.9). Many fibro- cytoplasm are developed. Newly released amorphous mate-
blasts are spindle- shaped. The nucleus-cytoplasm ratio is rial is present on the cell surfaces. Collagen, reticular and
large and the cytoplasm occupies a small area around the elastic fibers are seen close to vocal fold stellate cells.
nucleus. The rough endoplasmic reticulum and Golgi appara- The infant membranous portion of the vocal fold mucosa is
tus are not well developed, and few vesicles are present along stained light blue with Alcian Blue at pH 2.5; in particular, the
the periphery of the cytoplasm, indicating the fibroblasts are maculae flavae are strongly stained (Fig. 15.14). The material
quiescence and produce few extracellular matrices. that stains with Alcian Blue (pH 2.5) is digested by hyaluroni-
Cell density in the Cell density in the

human macula flava lamina propria of the mucosa
Number/field
300
p < 0.05
250
p < 0.05
200
p < 0.05
150
100
N.S.
N.S. N.S.
50 p < 0.05 N.S.
0
Newborn Infant Child Adult Aged Newborn Infant Child Adult Aged
Fig. 15.9 Cell density in the macula flava and lamina propria of the human vocal fold mucosa. N.S. not significant
elastic fibers
collagen fibers
Fig. 15.10 Macula flava of the infant vocal fold (6-month-old girl, Fig. 15.11 Macula flava of the infant vocal fold (6-month-old girl,
Hematoxylin and Eosin stain) Elastica van Gieson stain). Collagen (stained red) and elastic (stained
black) fibers have increased by comparison with those of the newborn
maculae flavae
dase. A great deal of hyaluronic acid is produced around the
vocal fold stellate cells in the infant maculae flavae. Most of in numbers, and 87.2 ± 3.0% of the vocal fold stellate cells
the cells including vocal fold stellate cells in the infant macu- immunohistochemically stain with CD44 (Fig. 15.16) [10].
lae flavae are stained with CD44 (a cell surface hyaluronan However, CD44 positive fibroblasts have become sparse
receptor) immunohistochemically (Fig. 15.15). The percent- (1.9 ± 2.0%) in the infant lamina propria of the vocal fold
age of CD44-positive cells in the infant macula flava is larger mucosa [10]. The expression of CD44 and distribution of
15.4 Growth Initiation of the Human Vocal Fold Mucosa in Infancy 205
reticular fibers
hyaluronic acid
collagen fibers
Fig. 15.12 Macula flava of the infant vocal fold (6-month-old girl, Fig. 15.14 Human Infant macula flava (6-month-old girl, Alcian blue
silver stain). Collagen (stained red) and reticular (stained black) fibers stain, pH 2.5). A great deal of the glycosaminoglycan hyaluronan (hyal-
have increased by comparison with those of the newborn maculae uronic acid) (light blue stained material) is situated around the cells in
flavae the macula flava and hyaluronan concentration in the human infant
maculae flavae is high
Fig. 15.13 Macula flava of the infant vocal fold (6-month-old girl,
periodic acid-Schiff stain)
Fig. 15.15 CD44 on cytoplasm of vocal fold stellate cells in the

hyaluronic acid are the same as in the adult maculae flavae human infant macula flava, shown by immunohistochemical staining
[10]. The vocal fold stellate cells and CD44 cooperatively start
to play important roles in maintaining hyaluronic acid in the
human maculae flavae during infancy [10].
During infancy, the hyaluronan (hyaluronic acid) concen- 15.4 G
rowth Initiation of the Human Vocal
tration in the maculae flavae becomes high and most of the Fold Mucosa in Infancy
cells in the maculae flavae possess transmembrane receptors
(a cell surface hyaluronan receptor), indicating that the mac- No structure corresponding to the vocal ligament and
ulae flavae are a hyaluronan-rich matrix, which is required Reinke’s space can be found in the infant vocal fold mucosa
for a stem cell niche. (Figs. 15.2 and 15.3).
Percentages of CD44-positive Percentages of CD44-positive

cells in the human macula flava cells in the lamina propria of
the human vocal fold mucosa
P < 0.05 N.S. N.S.

100 P < 0.05 100
90 90
80 80
P < 0.05
70 70
60 60
50 50
40 40
30 30
20 20 N.S. N.S.
P < 0.05
10 10
0 0
Newborn Infant Child Adult Aged Newborn Infant Child Adult Aged
Fig. 15.16 Percentages of CD44-positive cells in the macula flava and lamina propria of the human vocal fold mucosa N.S. not significant
During infancy, many cells in the macula flava become 15.5 C

ells and Extracellular Matrices
stellate in shape and constantly synthesize extracellular in the Human Child Vocal Fold
matrices, especially reticular and collagen fibers. Mucosa
Glycoproteins (fibronectin) in the lamina propria appear to
determine the orientation of the deposition of reticular and The vocal ligament and layered structure are not present in
collagen fibers synthesized in the maculae flavae. Many the lamina propria of the child vocal fold mucosa.
reticular and collagen fibers extend from the maculae flavae There are collagen, reticular and elastic fibers in the lam-
towards the middle of the lamina propria of the vocal fold ina propria of the child vocal fold mucosa (Figs. 15.17 and
mucosa. Fibrillar collagen, such as reticular and collagen 15.18). Those fibers run roughly parallel to the vocal fold
fibers, serve as stabilizing scaffolds and possess innumerable edge. The elastic fibers are composed of microfibrils and
potential spaces. The extracellular interstitial spaces are amorphous substances (Fig. 15.19). The former are abun-
made up of minute chambers or compartments occupied by dant, and the latter are sparse and reticular in shape.
other extracellular matrices such as elastic fibers and glycos- The ground substances throughout the child vocal fold
aminoglycan including hyaluronic acid. mucosa are stained light blue with Alcian Blue at pH 2.5
During infancy, extracellular matrices synthesized in the (Fig. 15.20). The material that stains in the lamina propria of
maculae flavae appear in the lamina propria of the membra- the mucosa with Alcian Blue (pH 2.5) is digested by hyal-
nous portion of the vocal fold mucosa between the anterior uronidase. Hyaluronic acid is present around the fibroblasts
and posterior maculae flavae, each at a different time, so as in the lamina propria of the child vocal fold mucosa.
to initiate the formation of the three-dimensional extracellu- However, CD44-positive fibroblasts are sparse in the lamina
lar matrix structure of the vocal fold mucosa that has the vis- propria of the child vocal fold mucosa (Fig. 15.16). These
coelastic properties of a vibrating structure [7, 8]. findings are the same as those in adults.
15.6 Maculae Flavae of the Human Child Vocal Fold 207
epithelium
collagen fiber
microfibrils
elastic fiber
reticular fibers collagen fibers elastin
collagen fibril
Fig. 15.19 Transmission electron micrograph of collagen and elastic

Fig. 15.17 Transverse section of the lamina propria of the child vocal fibers in the lamina propria of the child vocal fold mucosa (tannic acid
fold (9-year-old boy, silver stain). Reticular (stained black) and colla- stain)
gen (stained red) fibers run roughly parallel to the vocal fold edge
fibrocyte
fibroblasts
elastic fibers
collagen fibers hyaluronic acid
Fig. 15.20 Transverse section of the lamina propria of the child vocal
fold (9-year-old boy, Alcian blue stain). Hyaluronic acid (glycosamino-
Fig. 15.18 Transverse section of the lamina propria of the child vocal glycan) is present in the lamina propria of the child vocal fold mucosa
fold (9-year-old boy, Elastica van Gieson stain). Collagen (stained red)
and elastic (stained black) fibers run roughly parallel to the vocal fold
edge blasts, few vesicles can be seen. The fibroblasts in the lamina
propria of child vocal fold mucosa are quiescent and produce
few extracellular matrices.
Fibroblasts are sparse in the lamina propria of the child
vocal fold mucosa. The density of fibroblasts is about one-
fourth that of the cells including vocal fold stellate cells in 15.6 M
aculae Flavae of the Human Child
the child maculae flavae [11] (Fig. 15.9). The fibroblasts are Vocal Fold
oval or spindle-shaped, with no cytoplasmic processes and
no lipid droplets (Fig. 15.21). The nucleus-cytoplasm ratio is The maculae flavae are located at the anterior and posterior
high and poorly developed rough endoplasmic reticulum and ends of the bilateral child vocal fold mucosa. They are
Golgi apparatus are apparent. Along the surface of the fibro- approximately 1 × 1 × 1 mm in size and consist of dense
fibroblasts
nucleus
collagen fibers
elastic fibers
Fig. 15.21 Transmission electron micrograph of a fibroblast in the

lamina propria of the child vocal fold mucosa (tannic acid stain) Fig. 15.23 Macula flava of the child vocal fold (9-year-old boy,
Elastica van Gieson stain). Collagen (stained red) and elastic (stained
black) fibers are greater in number when compared with those of the
infant maculae flavae

reticular fibers collagen fibers
Fig. 15.22 Macula flava of the child vocal fold (9-year-old boy,
Hematoxylin and Eosin stain)
Fig. 15.24 Macula flava of the child vocal fold (9-year-old boy, silver
masses of cells including vocal fold stellate cells (Fig. 15.22). stain). Collagen (stained red) and reticular (stained black) fibers have
Many more cells including vocal fold stellate cells are dis- increased by comparison with those of the infant maculae flavae
tributed in the child maculae flavae than in those of adults.
The density of cells including vocal fold stellate cells in the
child maculae flavae is about twice that of adult maculae fla- 15.7 Morphological Characteristics
vae, and about half that of newborn maculae flavae [10, 11] of the Human Child Vocal Fold
(Fig. 15.9). Stellate Cells
The child maculae flavae are composed of vocal fold stel-
late cells (Fig. 15.22), collagen fibers (Figs. 15.23 and 15.24), The vocal fold stellate cells are stellate in shape and possess
reticular fibers (Fig. 15.24), elastic fibers (Fig. 15.23), and cytoplasmic processes (Fig. 15.25). A few lipid droplets are
ground substances. The fibrous components have increased present in the cytoplasm but they are much fewer in number
by comparison with those of the infant maculae flavae. and smaller (0.6–1 μm in diameter) than those found in
15.7 Morphological Characteristics of the Human Child Vocal Fold Stellate Cells 209
a adults. They are of two types: membrane-bounded

(Fig. 15.25b) and non-membrane-bounded. The former are
elastic fibers each surrounded by a unit membrane. Vitamin A is stored in
their cytoplasm (Fig. 15.26). The nucleus in the child vocal
vocal fold stellate cells fold stellate cell is oval. The nucleus-cytoplasm ratio is rela-
tively small, and intracellular organelles, such as rough
endoplasmic reticulum, are not very well developed. Ten-
nucleus nucleus nanometer-thick filaments (intermediate filaments) are pres-
B ent in the cytoplasm (Fig. 15.27). Mitochondria are small
collagen fibers and few. Free ribosomes are present in the cytoplasm. No
basal lamina is found. Vesicles are present along the periph-
ery of the cytoplasm of the child vocal fold stellate cells.
Newly released amorphous material is present on the cell
surfaces (Fig. 15.28). Collagen, reticular and elastic fibers
b nucleus
collagen fiber
Intermediate
filaments
lipid droplet
lipid droplet
elastic fiber lipid droplet

cytoplasmic
process
Fig. 15.25 (a) Transmission electron micrograph of vocal fold stellate

cells in the macula flava of the child vocal fold mucosa (tannic acid
stain). (b) Lipid droplet in a child vocal fold stellate cell (region B in a) Fig. 15.27 Transmission electron micrograph of microfilaments
(intermediate filaments) in the cytoplasm of the cells in the human child
maculae flavae (uranyl acetate and lead citrate stain)
microfibril
elastin elastin
collagen fibril
vesicles
microfibril
vocal fold stellate cells nucleus
microfibril
amorphous
material
rough
endoplasmic
Fig. 15.26 Vitamin A in the vocal fold stellate cells in the child macula reticulum
flava (gold chloride method, no counterstaining. original ×1000). The
cytoplasm of vocal fold stellate cells contains numerous fine grains of
Fig. 15.28 Synthesis of collagen and elastic fibers by vocal fold stel-
reduced gold and the nuclei are stained reddish-brown
late cells in the child macula flava (Transmission electron micrograph,
tannic acid stain)
are seen close to vocal fold stellate cells (Fig. 15.25). 15.30) [11]. Almost all of the vocal fold stellate cells in the
Synthesis of these fibers occurs in the same way as in adult child maculae flavae show CD44 expression and a large
maculae flavae. Electron microscopic study indicates that the amount of hyaluronic acid is present immediately adjacent to
vocal fold stellate cells in the child maculae flavae continue the vocal fold stellate cells. On the other hand, CD44-positive
to constantly synthesize collagen, reticular, and elastic fibers. fibroblasts are sparse (5.6 ± 3.0%) in the lamina propria of
Vocal fold stellate cells show cytoplasmic staining with the child vocal fold mucosa (Fig. 15.16) [11]. These findings
periodic acid-Schiff (PAS) stain, type I collagen and type III are the same as those of adults. The vocal fold stellate cells
collagen. in the maculae flavae and CD44 cooperatively continue to
Hyaluronic acid is present in the lamina propria, in par- play roles in the metabolism of hyaluronic acid in the human
ticular, in the maculae flavae of the child vocal fold mucosa child vocal fold mucosa.
(Fig. 15.29). Most of the vocal fold stellate cells (94.7 ± 1.9%) During childhood, hyaluronan (hyaluronic acid) concen-
in the child maculae flavae stain with CD44 (Figs. 15.16 and tration in the maculae flavae is still high and most of the cells
in the maculae flavae possess transmembrane receptors (a
cell surface hyaluronan receptor) just as in adults indicating
that the maculae flavae are a hyaluronan-rich matrix, which
is required for a stem cell niche.

15.8 G
rowth and Development
of the Human Vocal Fold Mucosa
and Vocal Fold Vibration
Among mammals, only humans can speak and only the

human adult vocal fold has a vocal ligament, Reinke’s space,
and a layered structure [12, 13]. Why do only human adults
hyaluronic acid have such a characteristic vocal fold structure? Why and how
does the newborn vocal fold mucosa grow, develop and
mature? What are the factors for initiating and continuing the
growth of the human vocal fold mucosa?
Tension is the most important factor which influences syn-
Fig. 15.29 Human child macula flava (9-year-old boy, Alcian blue thesis of collagen fibers by fibroblasts [14, 15]. The bending
stain, pH 2.5). A great deal of the glycosaminoglycan hyaluronan (hyal- stresses on the vocal fold associated with phonation are great-
uronic acid) (light blue stained material) is situated around the vocal
fold stellate cells in the macula flava and hyaluronan concentration in est in the region of the anterior and posterior maculae flavae
the human child maculae flavae is high [16]. We hypothesize that the tension caused by phonation
(vocal fold vibration) after birth stimulates vocal fold stellate
cells in the anterior and posterior maculae flavae to accelerate
production of extracellular matrices and form the vocal liga-
ment, Reinke’s space, and the layered structure [5, 7].
Human adult vocal fold mucosa that has remained unpho-
nated (non-vibrated) since birth is hypoplastic and rudimen-
tary, and does not have a vocal ligament, Reinke’s space or a
layered structure [17]. The maculae flavae are atrophic and
vocal fold stellate cells vocal fold stellate cells show decreased activity [17]. This
result supports the hypothesis that the tensions caused by
phonation (vocal fold vibration) after birth stimulates the
vocal fold stellate cells in the anterior and posterior maculae
flavae to accelerate production of extracellular matrices and
form the vocal ligament, Reinke’s space and the characteris-
tic layered structure of the human vocal fold.
Vocal fold vibration (phonation) after birth is one of the
important factors in the growth and development of the
human vocal fold mucosa. See Chap.16, “Mechanical
Fig. 15.30 CD44 on cytoplasm of vocal fold stellate cells in the
human child (9-year-old boy) macula flava, shown by immunohisto-
Regulation (Cellular Mechanotransduction) of the Human
chemical staining Vocal Fold Mucosa.”
References 211
15.9 G
rowth and Development 2. Hirano M, Sato K. Histological color atlas of the human larynx. San
of the Human Vocal Fold Mucosa 3. Hirano M, Kurita S, Nakashima T. Growth, development and aging
of human vocal folds. In: Bless DM, Abbs JH, editors. Vocal fold
Newborns already have immature maculae flavae which are physiology. San Diego, CA: College-Hill Press; 1983. p. 22–43.
composed of dense masses of cells including vocal fold stellate 4. Ishii K, Yamashita K, Akita M, Hirose H. Age-related develop-
ment of the arrangement of connective tissue fibers in the lam-
cells at the same sites as in adults. Their cellular components are ina propria of the human vocal fold. Ann Otol Rhinol Laryngol.
markedly abundant and extracellular matrix components are 2000;109:1055–64.
sparse. Vocal fold stellate cells in the newborn maculae flavae 5. Sato K, Hirano M. Historogic investigation of the macula flava
have already started the synthesis of extracellular matrices. of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
1995;104:556–62.
During infancy, the extracellular matrices synthesized in 6. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in
the maculae flavae extend to the vocal fold mucosa to initiate the human vocal fold. Acta Otolaryngol. 2003;123:106–10.
the formation of the three-dimensional extracellular matrix 7. Sato K, Hirano M, Nakashima T. Fine structure of the human new-
structure of the human vocal fold mucosa. born and infant vocal fold mucosae. Ann Otol Rhinol Laryngol.
2001;110:417–24.
Vocal fold stellate cells in the child maculae flavae con- 8. Sato K, Umeno H, Nakashima T. Functional histology of the macula
tinue to synthesize extracellular matrices made up of colla- flava in the human vocal fold. Part 2: its role in the growth and devel-
gen, reticular, and elastic fibers, glycoprotein and hyaluronic opment of the vocal fold. Folia Phoniatr Logop. 2010;62:263–70.
acid (glycosaminoglycan), which are essential for the visco- 9. Anderson JC. Glycoproteins of the connective tissue matrix. In: Hall
DH, Jackson DS, editors. International review of connective tissue
elasticity of the human vocal fold mucosa as a vibrating tis- research, vol. 7. New York: Academic Press; 1976. p. 251–322.
sue. The maculae flavae in newborns, infants and children 10. Sato K, Sakamoto K, Nakashima T. Expression and distribution of
are related to the growth and development of the human CD44 and hyaluronic acid in human vocal fold mucosa. Ann Otol
vocal fold mucosa. Rhinol Laryngol. 2006;115:741–8.
11. Sato K, Nakashima T. Stellate cells in the human child vocal fold
Human maculae flavae located at both ends of the mem- macula flava. Laryngoscope. 2009;119:203–10.
branous portion of the vocal fold are involved in the metabo- 12. Kurita S, Nagata K, Hirano M. Comparative histology of mamma-
lism of extracellular matrices essential for the viscoelastic lian vocal folds. In: Kirchner JA, editor. Vocal fold histopathology.
properties of the lamina propria of the human vocal fold San Diego: College Hill Press; 1986. p. 1–10.
13. Sato K, Hirano M, Nakashima T. Comparative histology of the
mucosa. Human newborn, infant, and child maculae flavae maculae flavae of the vocal folds. Ann Otol Rhinol Laryngol.
are most likely responsible for forming the characteristic lay- 2000;109:136–40.
ered structure of the human vocal fold mucosa. Human mac- 14. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson

ulae flavae before adolescence are also considered to be an JD. Molecular biology of the cell. 3rd ed. New York: Garland
Publishing, Inc.; 1994.
important structure in the growth and development of the 15. Stopak D, Harris AK. Connective tissue morphogenesis by fibroblast
human vocal fold mucosa. traction. Tissue culture observations. Dev Biol. 1982;90:383–98.
16. Titze IR, Hunter EJ. Normal vibration frequencies of the vocal liga-
ment. J Acoust Soc Am. 2004;115:2264–9.
17. Sato K, Nakashima T, Nonaka S, Harabuchi Y. Histopathologic
References investigations of the unphonated human vocal fold mucosa. Acta
Otolaryngol. 2008;128:694–701.
(Fukuoka). 1975;21(Suppl 1):239–60.
Mechanical Regulation (Cellular
Mechanotransduction) of the Human 16
Vocal Fold Mucosa
Abstract
1. Mechanotransduction caused by vocal fold vibration (phonation) after birth is one of the
important factors in the growth and development of the human vocal fold mucosa as a
vibrating tissue.
2. Mechanotransduction caused by vocal fold vibration (phonation) after the layered struc-
ture has been completed is one of the important factors to maintain the extracellular
matrices and the layered structure of the human adult vocal fold mucosa as a vibrating
tissue.
3. Human vocal fold mucosae that have remained unphonated since birth are hypoplastic
and rudimentary, the maculae flavae are hypoplastic and the cells including vocal fold
stellate cells in the maculae flavae show decreased activity.
4. The latest studies have supported the hypothesis that the tension caused by phonation
(vocal fold vibration) after birth stimulates cells including vocal fold stellate cells in the
anterior and posterior maculae flavae to accelerate production of extracellular matrices
and form the vocal ligament, Reinke’s space and the characteristic layered structure.
5. Vocal fold vibration seems to affect cell morphology and structure in the human maculae
flavae, such as cytoskeletal structure and organization. This supports the hypothesis that
vocal fold vibration regulates cell behavior in the human maculae flavae. In addition to
chemical factors, mechanical factors also appear to modulate cell, including vocal fold
stellate cell, behavior.
16.1 Introduction fold mucosa. Maculae flavae are also considered to be an

important structure in the growth, development and ageing of
Human adult vocal folds have a layered structure with a the human vocal fold mucosa [5–8]. An interstitial cell with a
vocal ligament [1, 2]. The layered structure is essential for star-like appearance in the human maculae flavae, located at
vibration and is required for phonation [1]. The structure’s both ends of the human vocal fold mucosa, was discovered in
ability to act as a vibrating tissue is based on the differences 2001 [9–12]. The cells possess lipid droplets and store vita-
of extracellular matrix distribution. On the other hand, in min A [9, 10]. They also have many morphological differ-
human newborn vocal folds, the entire lamina propria ences from fibroblasts in the vocal fold and constantly
appears as a uniform structure with no vocal ligament [2–4]. synthesize extracellular matrices that are essential for the vis-
The layered structure of the human vocal fold matures dur- coelasticity of the human vocal fold mucosa as a vibrating
ing adolescence [3]. tissue [8, 9, 12]. The latest research shows that the vocal fold
Human maculae flavae located at both ends of the human stellate cells in the maculae flavae form an independent cell
vocal fold mucosa are most likely to be involved in the metab- category that should be considered a new category of cells,
olism of extracellular matrices in the vocal fold mucosa and and are involved in the metabolism of extracellular matrices
form the characteristic layered structure of the human vocal in the human vocal fold mucosa [5–12].

214 16 Mechanical Regulation (Cellular Mechanotransduction) of the Human Vocal Fold Mucosa
As a result of the latest research, there is growing evi- On the other hand, the anterior and posterior maculae fla-
dence to suggest that the cells including vocal fold stellate vae of the human adult vocal folds which have remained
cells in the human maculae flavae are adult multipotent stem unphonated since birth do not form conspicuous mucosal
cells, tissue stem cells or progenitor cells in the human vocal bulges (Fig. 16.1). The membranous portions of the vocal
fold mucosa and that the human maculae flavae are a candi- folds are concave and atrophic (Fig. 16.1).
date for a stem cell niche, which is a microenvironment nur-
turing a pool of stem cells including vocal fold stellate cells
[13–15]. 16.2.2 Light and Electron Microscopic
Current scientific findings suggest that the magnitude and Findings of the Lamina Propria
frequency of tensile strain are particularly important in deter- of the Human Adult Vocal Fold Mucosa
mining the type of mechanically induced differentiation that Unphonated Since Birth
stem cells will undergo [16]. The cells including vocal fold
stellate cells reside in the macula flava, which is a microen- The membranous portion of the vocal folds in these cases is
vironment where the magnitude and frequency of tensile concave (Fig. 16.2). The thickness of the epithelium and
strain during vocal fold vibration are greatest. The function lamina propria ranges from approximately 0.6 to 0.67 mm
and fate of the cells in the human maculae flavae are regu-
lated by various microenvironmental factors. In addition to
chemical factors, mechanical factors also modulate the a
behavior of cells including vocal fold stellate cells in the
human maculae flavae. anterior
We hypothesize that the tensions caused by phonation commissure ventricular fold
(vocal fold vibration) after birth stimulate cells including
vocal fold stellate cells in the anterior and posterior maculae
flavae to accelerate production of extracellular matrices and
form the vocal ligament, Reinke’s space and the layered
structure [4, 6]. If our hypothesis is fact, some morphologic
differences should be detected between adult vocal fold membranous
vocal fold
mucosae that have been phonated and those that have
anterior
remained unphonated since birth [17, 18]. macula flava posterior
We also hypothesized that after the layered structure is macula flava
completed, the tensions caused by phonation (vocal fold
vibration) stimulate cells including vocal fold stellate cells in
b
the anterior and posterior maculae flavae to accelerate pro-
duction of extracellular matrices and maintain the layered
structure of the human adult vocal fold mucosa as a vibrating
anterior
tissue. If our hypothesis is correct, some morphologic differ- commissure
ences should be detected between adult vocal fold mucosae ventricular fold
that have remained phonated and those that have been unpho-
nated for a long period [19].
16.2 H
uman Adult Vocal Fold Unphonated membranous
vocal fold
Since Birth anterior posterior
16.2.1 Macroscopic Findings of the Human
Adult Vocal Fold Unphonated Since
Birth Fig. 16.1 Macroscopic findings of right human adult vocal fold which
have remained unphonated since birth due to cerebral palsy. (a) 17-year-
In adults, the anterior and posterior maculae flavae can be old male, (b) 28-year-old female. Diseases that commonly affect the
observed at each end of the membranes portion of the vocal tissue of the vocal fold were not observed. Bilateral vocal folds were
mobile and vocal fold paralysis was not detected. The anterior and pos-
fold. They form conspicuous mucosal bulges and they are terior maculae flavae do not form conspicuous mucosal bulges. The
visible through the mucosa as whitish-yellow masses. membranous portions of the vocal folds are concave
16.2 Human Adult Vocal Fold Unphonated Since Birth 215
B membranous vocal fold

a a
epithelium
thyroarytenoid muscle arytenoid
cartilage
thyroid cartilage collagen fibers

lamina propria
of mucosa
elastic fibers
anterior anterior
commissure macula flava b adipose tissue
tendon
lamina propria of
vocal fold mucosa thyroarytenoid muscle
b epithelium
collagen fiber lamina propria

elastic fiber of mucosa
thyroid cartilage
Fig. 16.2 (a) Transverse section of the human adult vocal fold unpho-
nated since birth (24-year-old male, Elastica van Gieson stain). (b)
Region B in a
and they are thinner than those of the normal human adult Fig. 16.3 Transverse section of the lamina propria of the human adult
vocal fold mucosa. The vocal fold mucosae are atrophic vocal fold mucosa unphonated since birth (Elastica van Gieson stain).
(a) 24-year-old male. The lamina propria of the vocal fold mucosa
(Figs. 16.2, 16.3, and 16.4). The vocalis muscle shows disuse appears as a uniform structure and collagen fibers (stained red) are
muscle atrophy (Fig. 16.5). dense and elastic fibers (stained black) are sparse. Adipose tissue is
The lamina propria of the vocal fold mucosa appears as a observed in the deep portion of the lamina propria of the vocal fold
uniform structure, and the vocal fold mucosa does not have a mucosa. (b) 17-year-old male. The lamina propria of the vocal fold
mucosa appears as a uniform structure and both collagen fibers stained
vocal ligament, Reinke’s space or a layered structure red and elastic fibers stained black are sparse
(Figs. 16.3 and 16.4). Histologically, adult vocal fold muco-
sae unphonated since birth are hypoplastic and rudimentary,
rather than atrophic [17].
Collagen fibers are dense in some cases (Fig. 16.3a) and There are a large number of collagen fibers, and there are
are sparse in other cases (Fig. 16.3b). Elastic fibers (Fig. 16.3) few elastic fibers or reticular fibers (Fig. 16.8). The fibro-
and reticular fibers (Fig. 16.4) are sparse. The vocal fold blasts in the lamina propria of the vocal fold mucosa are
mucosa is weakly stained with Alcian blue at pH 2.5 spindle-shaped or oval with no cytoplasmic processes
(Fig. 16.6). There is little hyaluronic acid in the lamina pro- (Fig. 16.8). The fibroblasts nuclei are elliptic. The nucleus
pria of the vocal fold mucosa. Adipose tissue is detected in cytoplasm ratio is large, and poorly developed rough
the deep portion of the lamina propria of the vocal fold endoplasmic reticulum and Golgi apparatus are apparent.
mucosa in some case (Fig. 16.7). Along the surface of the fibroblasts, few vesicles can be
Electron microscopy shows that the lamina propria of seen, and few newly released amorphous materials are
the vocal fold mucosa appears as a uniform structure. seen.
anterior anterior
epithelium
commissure macula flava a
tendon
lamina propria of
vocal fold mucosa
collagen fibers
reticular fibers
thyroid cartilage
Fig. 16.4 Transverse section of the lamina propria of the human adult b
vocal fold mucosa unphonated since birth (24-year-old male, silver epithelium
stain). The lamina propria of the vocal fold mucosa appears as a uni-
form structure. Collagen fibers stained red are dense and reticular fibers
stained black are sparse
lamina propria of vocal fold mucosa
muscle fibers
Fig. 16.6 Transverse section of the human adult vocal fold unpho-
nated since birth (24-year-old male, Alcian blue stain, pH 2.5). (a)
Anterior macula flava of the vocal fold mucosa. (b) Lamina propria of
the vocal fold mucosa. There is little hyaluronic acid in the macula flava
and lamina propria of the vocal fold mucosa
The maculae flavae are composed of cells including vocal

Fig. 16.5 Disuse muscle atrophy of the thyroarytenoid muscle of the fold stellate cells (Fig. 16.9), collagen fibers (Figs. 16.10 and
human adult vocal fold unphonated since birth (24-year-old male,
Hematoxylin and Eosin stain). Random distribution of atrophic muscle
16.11), reticular fibers (Fig. 16.11), elastic fibers (Fig. 16.10)
fibers is observed and ground substances. However, the maculae flavae have
fewer fibrous components than those of the normal human
vocal fold. Less fibrous proteins are produced around the
16.2.3 Light and Electron Microscopic vocal fold stellate cells in the maculae flavae. The maculae
Findings of the Maculae Flavae flavae are slightly stained light blue with Alcian blue at
of the Human Adult Vocal Fold Mucosa pH 2.5 (Fig. 16.12). The material stained in the maculae fla-
Unphonated since Birth vae with Alcian blue at pH 2.5 is digested by hyaluronidase.
Less hyaluronic acid is situated around the vocal fold stellate
The maculae flavae exist at the anterior and posterior ends of cells in the maculae flavae. The vocal fold stellate cells show
the bilateral vocal fold. The sizes of the maculae flavae are cytoplasmic staining with periodic acid-Schiff (PAS).
0.8–1 × 0.6–0.7 mm and are smaller than those of the normal Electron microscopy shows that the maculae flavae of the
vocal fold. The maculae flavae are atrophic [17]. vocal fold mucosa are composed of collagen fibers, reticular
16.2 Human Adult Vocal Fold Unphonated Since Birth 217
adipose tissue
Fig. 16.7 Adipose tissue in the lamina propria of the human adult Fig. 16.9 Macula flava of the human adult vocal fold mucosa unpho-
vocal fold mucosa unphonated since birth (24-year-old male, nated since birth (24-year-old male, Hematoxylin and Eosin stain)
Hematoxylin and Eosin stain). There is no adipose tissue in the normal
human vocal fold mucosa. In other mammals, monkeys and horses have
adipose tissue in the intermediate layer of the vocal fold mucosa
collagen fibers
collagen fibers
elastic fiber fibroblast
nucleus
elastic fiber collagen fibers

elastic fibers
Fig. 16.10 Macula flava of the human adult vocal fold mucosa unpho-
Fig. 16.8 Transmission electron micrograph of the lamina propria of nated since birth (24-year-old male, Elastica van Gieson stain). Less
the human adult vocal fold mucosa unphonated since birth (24-year-old fibrous proteins, collagen fibers (stained red) and elastic fibers (stained
male, tannic acid stain) black), are produced around the vocal fold stellate cells in the maculae
flavae
fibers, elastic fibers, ground substances, and cells including

vocal fold stellate cells (Fig. 16.13). However, the maculae The nuclei of the stellate cells are cleaved. Some components
flavae have fewer fibrous proteins than those of the normal in the cytoplasm and some vocal fold stellate cells have degen-
human vocal fold. erated. Accumulations of glycogen p articles (glycogen gran-
Many vocal fold stellate cells are distributed in the maculae ules) are seen in the cytoplasm. In tannic acid stained material,
flavae. The vocal fold stellate cells are irregular and stellate in the glycogen particles are electron-dense and approximately
shape and possess cytoplasmic processes, however, they are 15–30 nm in diameter (Fig. 16.13b). In materials stained with
not slender but short and shrunken. No basal lamina is found. uranyl acetate and lead citrate, a collection of glycogen parti-
Lipid droplets are present in the cytoplasm (Fig. 16.13), how- cles is seen as a lucent stained area (glycogen lake) within the
ever, their number is small. The nucleus-cytoplasm ratio is cytoplasm (Fig. 16.14). There are few microfilaments in the
relatively small, but there are a few intracellular organelles cytoplasm. In some cases, a large number of vacuolar degen-
such as rough endoplasmic reticulum and Golgi apparatus. erations are seen in the cytoplasm (Figs. 16.14 and 16.15).
a
vocal fold
stellate cells
B
collagen fibers
elastic fiber
reticular fibers
collagen fibers
b collagen
fibers
nated since birth (24-year-old male, silver stain). Less fibrous proteins, glycogen particles
collagen fibers (stained red) and reticular fibers (stained black), are pro-
duced around the vocal fold stellate cells in the maculae flavae
lipid droplet
elastic
fibers
collagen
cleaved
fibers
nucleus

Fig. 16.13 (a) Transmission electron micrograph of the macula flava
of the human adult vocal fold unphonated since birth (17-year-old male,
Tannic acid stain). (b) Region B in a
hyaluronic acid
elastic fiber
glycogen lake
collagen cleaved
nucleus cytoplasmic
fibers
process
nated since birth (24-year-old male, Alcian blue stain, pH2.5). Less vocal fold
hyaluronic acid, slightly stained light blue, is situated around the vocal lipid droplet
stellate cells
fold stellate cells in the maculae flavae vacuole
There are not as many vesicles at the periphery of the

cytoplasm of the vocal fold stellate cells (Fig. 16.16). Newly
released amorphous materials from the vesicles are present
on the surface of the vocal fold stellate cells, but not as much vacuole
as in normal ones.
The vocal fold stellate cells in the maculae flavae of the Fig. 16.14 Transmission electron micrograph of the macula flava of
adult vocal fold mucosa unphonated since birth appear to the human adult vocal fold unphonated since birth (28-year-old female,
have decreased their level of activity morphologically [17]. uranyl acetate and lead citrate stain)
16.3 Human Child Vocal Fold Unphonated Since Birth 219
16.3.2 Light and Electron Microscopic

glycogen Findings of the Lamina Propria
particles
of the Human Child Vocal Fold Mucosa
cleaved
Unphonated Since Birth
nucleus
Membranous portions of the vocal fold are slightly concave
degenerated
vacuole vocal fold (Fig. 16.18).
stellate cell The lamina propria of the vocal fold mucosa appears as a
uniform structure, and the vocal fold mucosa does not have a
vocal ligament, Reinke’s space or a layered structure
vacuole (Fig. 16.19). Collagen, elastic and reticular fibers are sparse
(Figs. 16.19 and 16.20). The vocal fold mucosa is weakly
Fig. 16.15 Transmission electron micrograph of the macula flava of

a
the human adult vocal fold unphonated since birth (28-year-old female,
tannic acid stain)
anterior
ventricular fold commissure
collagen fibrils
microfibrils
elastin
amorphous membranous
materials vocal fold
posterior anterior
amorphous macula flava macula flava
materials
glycogen granules b
anterior
commissure
ventricular fold
Fig. 16.16 Synthesis of fibrous protein by vocal fold stellate cell in the
macula flava of the human adult vocal fold unphonated since birth
(28-year-old female, transmission electron micrograph, tannic acid
stain)
membranous
16.3 H
uman Child Vocal Fold Unphonated vocal fold
Since Birth posterior anterior
16.3.1 Macroscopic Findings of the Human

Child Vocal Fold Unphonated Since
Birth Fig. 16.17 Macroscopic findings of left human child vocal folds
which have remained unphonated since birth due to cerebral palsy. (a)
12-year-old male, (b) 7-year-old female. Diseases that commonly affect
The membranous portions of the vocal folds are slightly con- the tissue of the vocal fold were not observed. Bilateral vocal folds were
cave and atrophic (Fig. 16.17). mobile and vocal fold paralysis was not detected. The membranous por-
tions of the vocal folds were slightly concave
Fig. 16.18 Transverse membranous vocal fold

section of the child vocal fold
unphonated since birth posterior anterior anterior
(12-year-old male, Elastica macula macula commissure
van Gieson stain) flava flava tendon
arytenoid cartilage thyroid cartilage
epithelium
epithelium
collagen fibers
collagen fibers reticular fibers
elastic fibers
Fig. 16.19 Transverse section of the lamina propria of the human Fig. 16.20 Transverse section of the lamina propria of the human
child vocal fold mucosa unphonated since birth (12-year-old male, child vocal fold mucosa unphonated since birth (12-year-old male, sil-
Elastica van Gieson stain). The lamina propria of the vocal fold mucosa ver stain). Collagen fibers (stained red) and reticular fibers (stained
appears as a uniform structure. Collagen fibers (stained red) and elastic black) are sparse
fibers (stained black) are sparse
and poorly developed intracellular organelles, such as rough

stained with Alcian blue at pH 2.5 (Fig. 16.21). There is little endoplasmic reticulum and Golgi apparatus are apparent.
hyaluronic acid in the lamina propria of the vocal fold Along the surface of the fibroblasts, few vesicles can be seen
mucosa. Histologically, child vocal fold mucosae unpho- and few newly released amorphous materials are seen on the
nated since birth are hypoplastic and rudimentary [18]. cell surface of the fibroblasts. The fibroblasts in the lamina
Fibroblasts are sparse in the lamina propria of the child vocal propria of the vocal fold mucosa unphonated since birth are
fold mucosa unphonated since birth. Cell density of the fibro- quiescent and do not show morphological changes [18].
blasts in the lamina propria of the child vocal fold mucosa
unphonated since birth is the same as in normal ones (Fig. 16.22).
Electron microscopy shows that the lamina propria of the 16.3.3 Light and Electron Microscopic
vocal fold mucosa unphonated since birth appears as a uni- Findings of the Maculae Flavae
form structure. There are a few collagen, elastic and reticular of the Human Child Vocal Fold Mucosa
fibers. The fibroblasts in the lamina propria of the vocal fold Unphonated Since Birth
mucosa unphonated since birth are spindle-shaped or oval
with no cytoplasmic processes (Fig. 16.23). The nuclei of the The maculae flavae exist at the anterior and posterior ends of
fibroblasts are elliptic. The nucleus-cytoplasm ratio is large the bilateral vocal folds. The sizes of the anterior maculae
collagen
epithelium fibers
fibroblast
hyaluronic acid nucleus
Fig. 16.21 Transverse section of the lamina propria of the human Fig. 16.23 Transmission electron micrograph of the lamina propria of
child vocal fold unphonated since birth (12-year-old male, Alcian Blue the human child vocal fold mucosa unphonated since birth (12-year-old
stain, pH 2.5). There is hyaluronic acid, stained light blue, in the lamina male, Uranyl acetate and lead citrate stain)
propria of the vocal fold mucosa
Fig. 16.22 Cell density of

the child vocal fold mucosae
which have remained
unphonated since birth. NS:
not significant
flavae are 1.0 × 0.9–1.0 mm and are the same size as those of the maculae flavae unphonated since birth is about 3.5 times that
the normal child vocal fold. The sizes of the posterior macula of the lamina propria [18] (Fig. 16.22). However, cell density of
flava are 0.8–0.7 × 0.6–0.5 mm and are smaller than the ante- the vocal fold stellate cells in the maculae flavae of the child
rior macula flava. The sizes of the posterior macula flava are vocal fold mucosa unphonated since birth is lower than that of
also smaller than those of the normal child vocal fold. normal ones [18] (Fig. 16.22). The vocal fold stellate cells show
The maculae flavae of the child vocal fold mucosa unpho- cytoplasmic staining with periodic acid-Schiff (PAS).
nated since birth are composed of dense masses of cells includ- The macula flava is composed of collagen fibers, reticular
ing vocal fold stellate cells (Fig. 16.24). The density of cells in fibers, elastic fibers, ground substances and cells including
collagen fibers
reticular fibers
Fig. 16.24 Macula flava of the human child vocal fold mucosa unpho- Fig. 16.26 Macula flava of the human child vocal fold mucosa unpho-
nated since birth (12-year-old male, Hematoxylin and Eosin stain) nated since birth (12-year-old male, silver stain). Less fibrous proteins,
collagen fibers (stained red) and reticular fibers (stained black), are pro-
duced around the vocal fold stellate cells in the maculae flavae

collagen fibers
elastic fibers
hyaluronic acid
Fig. 16.25 Macula flava of the human child vocal fold mucosa unpho-
nated since birth (12-year-old male, Elastica van Gieson stain). Less
fibrous proteins, collagen fibers (stained red) and elastic fibers (stained Fig. 16.27 Macula flava of the human child vocal fold mucosa unpho-
black), are produced around the vocal fold stellate cells in the maculae nated since birth (12-year-old male, Alcian blue stain, pH 2.5).
flavae Hyaluronic acid, stained light blue, is situated around the vocal fold
stellate cells in the maculae flavae
vocal fold s tellate cells (Figs. 16.25 and 16.26). However, the

maculae flavae have fewer fibrous components than those of vocal fold stellate cells in the maculae flavae than in the nor-
the normal human child vocal folds. Less fibrous proteins are mal child maculae flavae.
produced around the vocal fold stellate cells in the maculae Electron microscopy shows that the maculae flavae of
flavae. The maculae flavae are slightly stained light blue with the child vocal fold mucosa unphonated since birth are
Alcian blue at pH 2.5 (Fig. 16.27). The material in the macu- composed of collagen fibers, reticular fibers, elastic fibers,
lae flavae that is stained with Alcian blue at pH 2.5 is digested ground substances, and cells including vocal fold stellate
by hyaluronidase. Less hyaluronic acid is situated around the cells (Fig. 16.28). The maculae flavae have fewer fibrous
glycogen particles vocal fold

stellate cell
cleaved nucleus
nucleus
vocal fold
stellate cell
lipid droplet cytoplasmic
process
elastic fibers
collagen fibers
Fig. 16.28 Transmission electron micrograph of the macula flava of

the child vocal fold mucosa unphonated since birth (12-year-old male,
tannic acid stain)
Fig. 16.30 Transmission electron micrograph of vocal fold stellate
cells in the macula flava of the child vocal fold mucosa unphonated
since birth (7-year-old female, Uranyl acetate and lead citrate stain).
a The nucleus is crenated and the nuclear contents have a homogenous
appearance
cytoplasmic process proteins than those of normal human child vocal folds.
Many vocal fold stellate cells are distributed in the maculae
flavae. The vocal fold stellate cells are irregular and stellate
in shape and possess cytoplasmic processes. No basal lam-
cleaved
nucleus ina is found. Lipid droplets are present in the cytoplasm,
however, their number is small. The nucleus-cytoplasm
ratio is relatively small, but there are a few intracellular
organelles such as rough endoplasmic reticulum and Golgi
apparatus. The nuclei of the stellate cells are cleaved. Some
components in the cytoplasm and some vocal fold stellate
cells are degenerated. Accumulations of glycogen particles
B
(glycogen granules) are seen in the cytoplasm (Fig. 16.28).
nucleus In tannic acid stained material, the glycogen particles are
b electron-dense and approximately 15–30 nm in diameter. In
materials stained with uranyl acetate and lead citrate, a col-
lection of glycogen particles is seen as a lucent stained area
(glycogen lake) within the cytoplasm (Fig. 16.29). There
glycogen lake
are few microfilaments in the cytoplasm.
The nucleus of some vocal fold stellate cells is crenated
amorphous
and the nuclear contents have a homogeneous appearance
material (Fig. 16.30). No chromatin masses or nucleoli are discern-
vesicle
ible (homogenization of the nucleus).
There are not as many vesicles at the periphery of the
cytoplasm of the vocal fold stellate cells (Fig. 16.29b).
Newly released amorphous materials from the vesicles are
present on the surface of the vocal fold stellate cells but not
as much as in the normal ones.
Fig. 16.29 (a) Transmission electron micrograph of vocal fold stellate The vocal fold stellate cells in the maculae flavae of the
cells in the macula flava of the child vocal fold mucosa unphonated child vocal fold mucosa unphonated since birth appear to
since birth (12-year-old male, Uranyl acetate and lead citrate stain). (b) have decreased their level of activity morphologically [18].
Region B in a
16.4 H
uman Adult Vocal Fold Unphonated fibers were dense in the vocal fold mucosa (Figs. 16.33 and
for Over a Decade 16.34). Elastic fibers and reticular fibers were sparse in the
vocal fold mucosa (Figs. 16.33 and 16.34). The vocal fold
16.4.1 Macroscopic Findings of the Human mucosa was weakly stained with Alcian blue at pH 2.5.
Adult Vocal Fold Unphonated for Over There was little hyaluronic acid in the lamina propria of the
a Decade vocal fold mucosa (Fig. 16.35).
Electron microscopy showed that the lamina propria of
In adults, the anterior and posterior maculae flavae can be the vocal fold mucosa appeared as a uniform structure.
observed at each end of the membranes portion of the vocal There were many collagen fibers (Fig. 16.36), and
fold. They form conspicuous mucosal bulges and they are there were a few elastic and reticular fibers. The fibro-
visible through the mucosa as whitish-yellow masses. blasts in the lamina propria of the vocal fold mucosa were
On the other hand, the anterior and posterior maculae fla- spindle-shaped or oval with no cytoplasmic processes
vae of a human adult vocal fold, which had been unphonated (Fig. 16.36). The fibroblasts nuclei were elliptic. The
for 11 years and 2 months after the layered structure of vocal characteristic findings of the fibroblasts were that the
fold had been completed, did not form conspicuous mucosal nucleus-cytoplasm ratio was relatively small and rough
bulges (Fig. 16.31). The membranous portion of the vocal endoplasmic reticulum and Golgi apparatus were appar-
fold was concave and atrophic (Fig. 16.31). ent (Fig. 16.37). Many vesicles could be seen along the
surfaces of the fibroblasts, and newly released amorphous
materials were seen on the cell surface of the fibroblasts
16.4.2 Light and Electron Microscopic (Fig. 16.37b).
Findings of the Lamina Propria
of a Human Adult Vocal Fold Mucosa membranous vocal fold B
Unphonated for Over a Decade
a
arytenoid
The thickness of the epithelium and lamina propria of the cartilage thyroarytenoid muscle
vocal fold mucosa was approximately 0.4 mm and it was thin-
ner than that of the normal human adult vocal fold mucosa
thyroid cartilage
(Fig. 16.32). The vocal fold mucosa was concave and atro-
phic. The vocalis muscle showed disuse muscle atrophy.
The lamina propria of the vocal fold mucosa appeared as
a uniform structure, and the vocal fold mucosa did not have
the vocal ligament, Reinke’s space or the layered structure.
Histologically, the vocal fold mucosa was atrophic. Collagen
b
anterior
ventricular fold commissure
membranous
posterior vocal fold anterior
thyroid cartilage
Fig. 16.32 Transverse section of the human adult vocal fold unpho-
Fig. 16.31 Macroscopic findings of the left human adult vocal fold nated for 11 years and 2 months after the layered structure of vocal fold
unphonated for 11 years and 2 months after the layered structure of had been completed (64-year-old male, Elastica van Gieson stain). (b)
vocal fold had been completed (64-year-old male) Region B in a
16.4 Human Adult Vocal Fold Unphonated for Over a Decade 225
epithelium
epithelium
collagen fibers elastic fibers
Fig. 16.33 Transverse section of the lamina propria of the human Fig. 16.35 Transverse section of the lamina propria of the human
adult vocal fold mucosa unphonated for over a decade (64-year-old adult vocal fold mucosa unphonated for over a decade (64-year-old
male, Elastica van Gieson stain). Collagen fibers (stained red) are dense male, Alcian blue stain, pH 2.5). There is little hyaluronic acid in the
and elastic fibers (stained black) are sparse in the vocal fold mucosa lamina propria of the vocal fold mucosa
epithelium
collagen fibers
nucleus
reticular fibers
collagen fibers fibroblast
Fig. 16.34 Transverse section of the lamina propria of the human the human adult vocal fold mucosa unphonated for over a decade
adult vocal fold mucosa unphonated for over a decade (64-year-old (64-year-old male, uranyl acetate and lead citrate stain)
male, silver stain). Collagen fibers stained red are dense and reticular
fibers stained black are sparse in the vocal fold mucosa
However, the macula flava had fewer fibrous components than
that of the normal human adult vocal fold. Fewer fibrous pro-
16.4.3 Light and Electron Microscopic teins were produced around the vocal fold stellate cells in the
Findings of the Macula Flava macula flava (Figs. 16.39 and 16.40). The macula flava was
of a Human Adult Vocal Fold Mucosa slightly stained light blue with Alcian blue at pH 2.5
Unphonated for Over a Decade (Fig. 16.41). The material that was stained in the macula flava
with Alcian blue at pH 2.5 was digested by hyaluronidase.
The macula flava was 1.5 × 0.5 mm in size. It was atrophic and Less hyaluronic acid was situated around the vocal fold stel-
smaller than that of the normal human adult vocal fold late cells in the macula flava. The vocal fold stellate cells
(Fig. 16.32b). The macula flava was composed of collagen showed cytoplasmic staining with periodic acid-Schiff (PAS).
fibers, reticular fibers, elastic fibers, ground substances and the Electron microscopy showed that the macula flava of
vocal fold stellate cells (Figs. 16.38, 16.39, and 16.40). the vocal fold mucosa was composed of collagen fibers,
a
B
nucleus
fibroblast
b
nated for over a decade (64-year-old male, Hematoxylin and Eosin
stain)
amorphous material
vesicle
vesicle
nucleus
rough endoplasmic collagen fibers

reticulum
Fig. 16.37 (a) Transmission electron micrograph of the fibroblast in vocal fold stellate cell
the lamina propria of the human adult vocal fold mucosa unphonated
for over a decade (64-year-old male, uranyl acetate and lead citrate
stain). (b) Region B in a. elastic fibers
reticular fibers, elastic fibers, ground substances and the

vocal fold stellate cells (Fig. 16.42a). The maculae flavae
had fewer fibrous proteins than those of normal human nated for over a decade (64-year-old male, Elastica van Gieson stain).
vocal folds. Many vocal fold stellate cells were distrib- Less fibrous proteins, collagen fibers (stained red) and elastic fibers
uted in the maculae flavae. They were irregular and stel- (stained black), are produced around the vocal fold stellate cells in the
late in shape and possessed cytoplasmic processes. The maculae flavae
nuclei of vocal fold stellate cells were cleaved. No basal
lamina was found. Lipid droplets were present in the cyto- were few vesicles at the periphery of the cytoplasm of the
plasm. The nucleus-cytoplasm ratio was relatively small, vocal fold stellate cells (Fig. 16.42b). Newly released
but there were few intracellular organelles such as rough amorphous materials from the vesicles were present on
endoplasmic reticulum and Golgi apparatus. There were the surface of the vocal fold stellate cells, but not as much
few microfilaments in the cytoplasm. Some components as those of normal ones (Fig. 16.42b).
in the cytoplasm of the vocal fold stellate cells had degen- As mentioned above, the vocal fold stellate cells in the
erated. Accumulations of glycogen particles (glycogen macula flava appeared to have decreased their level of activ-
granules) were seen in the cytoplasm (Fig. 16.42a). There ity morphologically. On the other hand, the fibroblasts in the
16.5 Cytoskeletons (Mechanoreceptor of Cells) of the Cells in the Human Macula Flava Unphonated Since Birth 227
a elastic fibers
B
collagen fibers glycogen

vocal fold
particles
reticular fibers stellate cell
nucleus
vocal fold stellate cells lipid droplet

collagen
fibers
b
elastic fibers
nated for over a decade (64-year-old male, Silver stain). Less fibrous
proteins, collagen fibers (stained red) and reticular fibers (stained
black), are produced around the vocal fold stellate cells in the maculae amorphous
flavae materials
Fig. 16.42 (a) Transmission electron micrograph of the macula flava

of the vocal fold mucosa unphonated for over a decade (tannic acid
hyaluronic acid stain). (b) Region B in a. There are few intracellular organelles and are
few vesicles at the periphery of the cytoplasm of vocal fold stellate cells
16.5 Cytoskeletons (Mechanoreceptor

of Cells) of the Cells in the Human
Macula Flava Unphonated Since Birth
nated for over a decade (64-year-old male, Alcian blue stain, pH 2.5). It is generally accepted that tensile and compressive
Less hyaluronic acid, slightly stained light blue, is situated around the strains have direct effects on cell morphology and struc-
vocal fold stellate cells in the maculae flavae
ture including changes in cytoskeletal structure and orga-
nization [16]. Cytoskeletons play the role of the
lamina propria appeared to have increased their level of mechanoreceptor of the cells. Our previous studies have
activity morphologically. The reason for this phenomenon in supported the hypothesis that the tension caused by pho-
the vocal fold mucosa unphonated for over a decade is nation (vocal fold vibration) after birth stimulates cells
ambiguous. Instead of the inactivated vocal fold stellate cells including vocal fold stellate cells in the anterior and pos-
in the macula flava, fibroblasts in the lamina propria may terior maculae flavae to accelerate production of extracel-
take part in the metabolism of extracellular matrices and lular matrices and form the vocal ligament, Reinke’s space
fibrogenesis in the vocal fold mucosa. and the characteristic layered structure [4, 6, 17–20]. The
B cytoplasmic
a process c
C
vocal fold
stellate cell
cleaved
nucleus
lipid droplet
D
cytoplasmic process mitochondria

rough endoplasmic
reticula
b amorphous d nucleus
vesicle material
amorphous
material
intermediate filaments
vesicles
elastic fiber
nucleus
cytoplasmic process
Fig. 16.43 (a) Transmission electron micrograph of the macula flava region C in a, (d) region D in a. The intermediate filaments have disap-
of the human vocal fold mucosa unphonated since birth (28-year-old peared in some parts of the cytoplasm
female, uranyl acetate and lead citrate stain), (b) Region B in a, (c)
tension caused by phonation seems to regulate the behav- 16.6 C

omparison Between Vocal Fold
ior of the cells including vocal fold stellate cells in the Mucosae Phonated and Unphonated
maculae flavae of the human vocal fold. (Non-Vibrated) Since Birth or
Electron microscopy shows that many vocal fold stel- Unphonated for Over a Decade
late cells are distributed in the maculae flavae unphonated
since birth. They are stellate in shape and possessed cyto- Atrophy is a decrease in the size and function of a cell.
plasmic processes (Fig. 16.43). Lipid droplets are present Clinically, it is often recognized as a diminution in the size
in the cytoplasm. The nucleus-cytoplasm ratio is rela- and function of an organ. Vocal fold atrophy can therefore be
tively small, but the intermediate filaments of the vocal defined as a decrease in the size and function of the vocal
fold stellate cells have disappeared in some parts of the fold [22]. Vocal fold atrophy can also be defined as a diminu-
cytoplasm. tion in the size of each portion of the layered structure and
The intermediate filaments (mechanoreceptors of cells) of function of the vocal fold [22]. Clinically, the membranous
the vocal fold stellate cells in the maculae flavae unphonated portion of the atrophic vocal fold becomes concave and rec-
since birth are fewer in number and the expression of their ognized as glottal incompetence.
characteristic proteins (vimentin, desmin, glial fibrillary The mean thickness of the epithelium and lamina propria
acidic protein) is also reduced compared with vocal fold stel- of the vocal fold mucosae of Japanese people in their twen-
late cells in normal vocal folds (Fig. 16.44) [21]. ties is approximately 1.08 mm at the middle, 1.05 mm at the
16.6 Comparisson Between Vocal Fold Phonated and Unphonated Since Birth or for Over a Decade 229
Fig. 16.44 The area of the

stained by immunoreactivity
against vimentine, desmin and
glial fibrillary acidic protein
(GFAP). Normal: Vocal fold
stellate cells in the maculae
flavae in the normal vocal fold
mucosa, Unphonated: Vocal
fold stellate cells in the
maculae flavae in the vocal
fold mucosa which has
remained unphonated since
birth
anterior one-quarter point, and 1.02 mm at the posterior one- Adipose tissue is detected in the deep portion of the lam-
quarter point. [23] The size of the mean Japanese macula ina propria of the vocal fold mucosa unphonated since birth.
flava is approximately 1.5 × 1.5 × 1 mm [5]. Vocal fold There is no adipose tissue in the normal human vocal fold
mucosae that have remained unphonated since birth show a mucosa [26, 27]. In other mammals, monkeys and horses
diminution in the size of the lamina propria in the vocal fold have adipose tissue in the intermediate layer of the vocal fold
mucosa and macula flava, that is to say, the vocal fold mucosa mucosa [26, 27].
appears to be in a state of atrophy. The components of the maculae flavae in the human adult
Hypoplasia is an incomplete development or under- devel- and child vocal fold mucosa unphonated since birth are the
opment of an organ or tissue. The most outstanding histologi- same as those in the macula flava of the normal vocal fold.
cal features of adult vocal fold mucosae unphonated since However, maculae flavae unphonated since birth have fewer
birth are that they are not only atrophic but also they lack the fiber and ground substance components than those of the
vocal ligament, Reinke’s space, and the layered structure. normal human vocal fold mucosa.
Consequently, vocal fold mucosae unphonated since birth are Regarding the cytoplasm, accumulations of glycogen par-
in fact hypoplastic and rudimentary, rather than atrophic. ticles are seen in the cytoplasm of vocal fold stellate cells
The lamina propria of the unphonated vocal fold mucosa unphonated since birth. There is an inverse relationship
appears as a uniform structure, which is mainly composed of between metabolic activity and glycogen deposits, and gly-
collagen fibers. There is little hyaluronic acid in the lamina cogen tends to accumulate in atrophic cells and in cells that
propria of the vocal fold mucosa. Hyaluronic acid or hyaluro- are less active [28]. The vocal fold stellate cells show vacu-
nan, one of the glycosaminoglycans and extracellular matri- olar degeneration in the cytoplasm, and there are not as many
ces, contributes to tissue viscosity and is an important molecule vesicles at the periphery of the cytoplasm. Newly released
for maintaining optimal tissue properties. It also plays an amorphous materials from the vesicles are present on the
important role in the viscoelasticity of the human vocal fold surface of the vocal fold stellate cells, but not as much as on
mucosa [24]. Hyaluronic acid also contributes to optimal tis- those of the normal ones. The vocal fold stellate cells in the
sue stiffness, important for fundamental frequency control. maculae flavae unphonated since birth appear to have a
[25] Consequently, the viscoelasticity is inadequate for vibra- decreased level of activity.
tion of adult vocal fold mucosae unphonated since birth, and Regarding the nucleus, it is crenated or cleaved, nuclear
the structures are not suitable for vibration or phonation. contents are homogenous and no chromatin masses or
n ucleoli (homogenization of the nucleus) are discernible The percentages of CD44-positive cells in the maculae
in some vocal fold stellate cells in the maculae flavae flavae of the human vocal fold mucosa unphonated since
unphonated since birth. Two forms of chromatin are birth are far lower both in children (1.2 ± 0.9%) and adults
known to occur in the interphase nucleus. One is hetero- (6.4 ± 4.8%) than in normal subjects (Fig. 16.45a) [20].
chromatin and the other is euchromatin. Metabolically The density of fibroblasts in the lamina propria of the
active cells have a paler-staining nucleus with fewer and human vocal fold mucosa unphonated since birth is the same
smaller heterochromatin masses [29]. Euchromatin is in children and higher in adults than in normal subjects
active in RNA and DNA synthesis but heterochromatin (Fig. 16.45b) [20].
shows little or no activity as a template for replication and The percentage of CD44-positive fibroblasts in the lamina
transcription [29]. Homogenization of the nucleus in the propria of the unphonated human vocal fold mucosa is
vocal fold stellate cells in the human vocal fold unpho- extremely low. It is the same both in the children (3.7 ± 4.1%)
nated since birth indicates that these cells are not meta- and the adults (6.2 ± 3.3%) and contrasts with that of normal
bolically active [18]. subjects (Fig. 16.45b) [20].
Both cells in the maculae flavae and the fibroblasts in the
lamina propria in the vocal fold mucosa unphonated since
16.7 E
xpression and Distribution birth express little CD44. And in children (during the stage
of Hyaluronic Acid and CD44 of vocal fold development), there is a decreased level of
in Unphonated Human Vocal Fold hyaluronic acid and there is little hyaluronic acid in the adult
Mucosa vocal fold mucosa as well.
CD44 is known to participate in a wide variety of cellular
Hyaluronic acid is abundant in the lamina propria, in particu- functions, including cell to cell aggregation, retention of the
lar in the maculae flavae, of the child and adult normal vocal pericellular matrix, matrix to cell and cell to matrix signal-
fold mucosa [8, 30]. ing, receptor-mediated internalization and degradation of
The density of cells including vocal fold stellate cells in the hyaluronic acid, and cell migration. However, how cells reg-
maculae flavae of the human vocal fold mucosa unphonated ulate their usage of CD44 remains a mystery.
since birth is lower in the children and higher in the adults than The fact that CD44 is a transmembrane receptor with an
in normal subjects (Fig. 16.45a) [20]. The difference of cell extensive cytoplasmic domain automatically suggests that CD44
density between children and adults in the maculae flavae of can communicate cell-matrix interactions into the cell (outside-
the human vocal fold mucosa unphonated since birth is smaller in signaling) and can alter the matrix in response to intracel-
than that of the normal vocal fold mucosae [20]. lular signals (inside-out signaling). Cell membrane-localized
Fig. 16.45 Cell density and a

percentage of CD44 positive
cells in the macula flava (a)
and lamina propria (b) of
normal and unphonated vocal
fold mucosa. NS not
significant
16.9 The Role of Intermediate Filaments in the Vocal Fold Stellate Cells 231
Fig. 16.45 (continued) b
receptors initiate intracellular signaling cascades. The local- “Mechanotransduction” is the term for the ability of liv-
ized concentration of cytoskeletal tensions is a major mediator ing tissues to sense mechanical stress and respond by tissue
of mechanical signaling [31, 32]. Disruption of the cytoskel- remodeling. Cellular mechanotransduction is the mechanism
etal organization caused by the unphonated state may disperse by which cells convert mechanical stimuli into biomechani-
CD44 in the membranes, in turn modifying the capacity of cal responses. More recently, mechanotransduction has
CD44 to bind or otherwise organize extracellular hyaluronic expanded to include the sensation of stress, its translation
acid or to initiate intracellular signaling cascades. Reduced into a biochemical signal and the sequence of biological
intracellular signaling cascades and cross-talk between them responses it produces. Mechanical stress has become increas-
may ultimately alter the cell behavior, including vocal fold stel- ingly recognized as one of the primary and essential factors
late cell behavior, in the maculae flavae of the human vocal fold controlling biological functions, ultimately affecting the
unphonated since birth. functions of the cells, tissue, and organs [35].
It is suggested that the mechanical stress caused by pho-
nation (vocal fold vibration) is one of the primary and essen-
16.8 M
echanotransduction in the Human tial factors controlling biological functions and ultimately
Vocal Fold Mucosa affecting the function of the cells in the vocal fold mucosa.
The bending stresses on the vocal fold associated with
Collagen fibrils form structures that resist tensile forces [32]. phonation (vocal fold vibration) are greatest in the region of
Cells help organize the collagen fibrils they secrete by exert- the maculae flavae located at both ends of the vocal fold
ing tension on the matrix [33]. Collagen fibers have been mucosa [36]. However, the role of mechanotransduction in
shown to align their orientation to withstand longitudinal the vibrating vocal fold mucosa remains unclear.
stress associated with vocal fold oscillation [34]. The vocal
ligament composed of collagen and elastic fibers runs
between the anterior and posterior maculae flavae. 16.9 T
he Role of Intermediate Filaments
Tension caused by phonation seems to regulate the behav- in the Vocal Fold Stellate Cells
ior of the cells including vocal fold stellate cells (mechanical
regulation) in the maculae flavae of the human vocal fold. It Intermediate filaments have a diameter of about 8–12 nm,
is of interest whether the mechanical forces caused by vocal which makes them intermediate in size between microtu-
fold vibration from outside the cells in the maculae flavae bules and microfilaments [37]. They serve as a scaffold to
through cell-matrix contacts influence intracellular signaling support the entire cytoskeletal framework and play a struc-
cascades that ultimately alter many cellular behaviors. tural role [37]. They are also thought to have a tension-bearing
role because they are often found in areas of cells that are chemical factors may be involved in more complicated sig-
subject to mechanical stress [37]. naling mechanisms, and assessment of the relative impor-
The intermediate filaments are distributed in the cyto- tance of each factor needs further investigation.
plasm of the vocal fold stellate cells [38]. The vocal fold
stellate cells are present in the maculae flavae which are sub-
ject to mechanical stress, that is, vocal fold vibration. References
Therefore, the intermediate filaments of the vocal fold stel-
late cells may have a tension-bearing role. 1. Hirano M. Phonosurgery. Basic and clinical investigations. Otologia
(Fukuoka). 1975;21(Suppl 1):239–60.
The intermediate filaments of the vocal fold stellate cells 2. Hirano M, Sato K. Histological color atlas of the human larynx. San
in the human maculae flavae unphonated since birth disap- Diego, CA: Singular Publishing Group Inc.; 1993.
pear in some parts of the cytoplasm and the expression of 3. Hirano M, Kurita S, Nakashima T. Growth, development and aging
their characteristic proteins is also reduced [21]. Vocal fold of human vocal folds. In: Bless DM, Abbs JH, editors. Vocal fold
physiology. San Diego, CA: College-Hill Press; 1983. p. 22–43.
vibration has effects on cell morphology and structure, 4. Sato K, Hirano M, Nakashima T. Fine structure of the human new-
including cytoskeletal structure and organization. born and infant vocal fold mucosae. Ann Otol Rhinol Laryngol.
Consequently, these physical changes could be converted 2001;110:417–24.
into changes in cell signaling and transcriptional activities in 5. Sato K, Hirano M. Histologic investigation of the macula flava of the
the nucleus to cause alterations in differentiation, prolifera- 6. Sato K, Hirano M. Historogic investigation of the macula flava
tion, and migration of the cells including vocal fold stellate of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
cells in the maculae flavae [21]. 1995;104:556–62.
7. Sato K, Hirano M. Age-related changes of the macula flava of the
16.10 M
echanical Regulation of Human CD44 and hyaluronic acid in human vocal fold mucosa. Ann Otol
Vocal Fold Stellate Cells Rhinol Laryngol. 2006;115:741–8.
There is growing evidence that the cells including vocal fold 10. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in
stellate cells in the human maculae flavae are tissue stem the human vocal fold. Acta Otolaryngol. 2003;123:106–10.
cells or progenitor cells in the human vocal fold mucosa 11. Sato K, Hirano M, Nakashima T. Age-related changes in vitamine
[13–15]. The human maculae flavae located at both ends of A-storing stellate cells of human vocal folds. Ann Otol Rhinol
Laryngol. 2004;113:108–12.
the vocal fold mucosa can be a candidate for a stem cell 12. Sato K, Nakashima T. Vitamin A-storing stellate cells in the human
niche, which is a microenvironment nurturing a pool of stem newborn vocal fold. Ann Otol Rhinol Laryngol. 2005;114:517–24.
cells [13, 15]. 13. Sato K, Umeno H, Nakashima T. Vocal fold stem cells and

It is readily apparent that tensile and compressive strains their niche in the human vocal fold. Ann Otol Rhinol Laryngol.
2012;121:798–803.
can have direct effects on cell morphology and structure, 14. Kurita T, Sato K, Chitose S, Fukahori M, Sueyoshi S, Umeno
including changes in the cell membrane, shape, and volume H. Origin of vocal fold stellate cells in the human macula flava.
as well as cytoskeletal structure and organization [16]. These Ann Otol Rhinol Laryngol. 2015;124:698–705.
physical changes can be converted into changes in cell sig- 15. Sato K, Chitose S, Kurita T, Umeno H. Microenvironment of mac-
ula flava in the human vocal fold as a stem cell niche. J Laryngol
naling and transcriptional activities in the nucleus to cause Otol. 2016;130:656–61.
alterations in cellular differentiation, proliferation, and 16. Kurpinski K, Janairo R, Chien S, Li S. Mechanical regulation of
migration [16]. stem cells: implications in tissue remodeling. In: Mofrad M, Kamm
The function and fate of stem cells are regulated by vari- R, editors. Cellular mechanotransduction. Diverse perspectives
from molecules to tissues. New York, NY: Cambridge University
ous microenvironmental factors [16]. In addition to chemical Press; 2010. p. 403–16.
factors, mechanical factors can also modulate stem cell sur- 17. Sato K, Nakashima T, Nonaka S, Harabuchi Y. Histopathologic
vival, organization, migration, proliferation, and differentia- investigations of the unphonated human vocal fold mucosa. Acta
tion [16]. Stem cells are potentially one of the main players Otolaryngol. 2008;128:694–701.
18. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi

in the phenotype determination of a tissue in response to Y. Histopathologic investigations of the unphonated human child
mechanical loading [16]. vocal fold mucosa. J Voice. 2012;26:37–43.
The cells including vocal fold stellate cells in the human 19. Sato K, Umeno H, Ono T, Nakashima T. Histopathologic study
maculae flavae may be sensing the mechanical forces, and of human vocal fold mucosa unphonated over a decade. Acta
Otolaryngol. 2011;131:1319–25.
these tissue-specific mechanical forces (vocal fold vibration) 20. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi Y. Expression
could promote cell differentiation toward the phenotype of and distribution of hyaluronic acid and CD44 in unphonated human
the cells residing within the vocal fold tissue. However, little vocal fold mucosa. Ann Otol Rhinol Laryngol. 2009;118:773–80.
is known about how force affects biological signaling. It is 21. Sato K, Kurita T, Chitose S, Umeno H, Nakashima T. Mechanical
regulation of human vocal fold stellate cells. Ann Otol Rhinol
suggested that the combination of multiple mechanical and Laryngol. 2015;124:49–54.
References 233
22. Sato K. Histopathology of vocal fold atrophy. Jpn J Logop Phoniatr. 31. Chen CS, Tan J, Tien J. Mechanotransduction at cell-matrix and
2002;43:432–7. cell-cell contracts. Annu Rev Biomed Eng. 2004;6:275–302.
23. Kurita S. Layer structure of the human vocal fold: morphological 32. Chen CS. Mechanotransduction-a field pulling together? J Cell Sci.
investigation. Otologia (Fukuoka). 1980;26(Suppl 6):973–97. 2008;121:3285–92.
24. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteo- 33. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. The
glycans and their influence on biomechanics. Laryngoscope. extracellular matrix of animals. Molecular biology of the cell. 3rd
1999;109:845–54. ed. New York, NY: Garland Publishing Inc.; 1994. p. 971–95.
25. Chan RW, Gray SD, Titze IR. The importance of hyaluronic
34. Gray SD, Titze IR, Alipour F, Hammond TH. Biomechanical and
acid in vocal fold biomechanics. Otolaryngol Head Neck Surg. histologic observations of vocal fold fibrous proteins. Ann Otol
2001;124:607–14. Rhinol Laryngol. 2000;109:77–85.
26. Nagata K. A comparative study of the layer structure of the vocal 35. Mofrad M, Kamm R, editors. Cellular mechanotransduction.

fold. Otologia (Fukuoka). 1982;28(Suppl 2):699–738. Diverse perspectives from molecules to tissues. New York, NY:
27. Kurita S, Nagata K, Hirano M. Comparative histology of mamma- Cambridge University Press; 2010.
lian vocal folds. In: Kirchner JA, editor. Vocal fold histopathology. 36. Titze IR, Hunter EJ. Normal vibration frequencies of the vocal liga-
A symposium. San Diego, CA: College Hill Press; 1986. p. 1–10. ment. J Acoust Soc Am. 2004;115:2264–9.
28. Ghadially FN. Glycogen. Cytoplasmic matrix and its inclusions. 37. Becker W, Kleinsmith L, Hardin J. Intermediate filament. The

Ultrastructural pathology of the cell and matrix, vol. 2. 3rd ed. world of the cell. 6th ed. San Francisco, CA: Pearson Education
London: Butterworths; 1988. p. 962–9. Inc. p. 446–50.
29. Ghadially FN. Nucleus. Chromatin in normal, neoplastic and
38. Sato K, Umeno T, Nakashima T. Vocal fold stellate cells in the
necrotic cells. Ultrastructural pathology of the cell and matrix, vol. human macula flava and the diffuse stellate cell system. Ann Otol
1. 3rd ed. London: Butterworths; 1988. p. 14–27. Rhinol Laryngol. 2012;121:51–6.
30. Sato K, Nakashima T. Stellate cells in the human child vocal fold
macula flava. Laryngoscope. 2009;119:203–10.
Geriatric Changes of Cells
and Extracellular Matrices in the Human 17
Vocal Fold Mucosa
Abstract
1. The viscoelastic properties of the lamina propria of the vocal fold mucosa are very
important for the vibratory behavior of the structure. They greatly depend on the extra-
cellular matrices.
2. Not only geriatric changes in the three-dimensional structure of collagen and reticular
fibers but also their qualitative and quantitative changes have effect on the three-dimen-
sional structure of the extracellular matrices.
3. In addition, age-related changes in the other extracellular matrices situated among the
collagen and reticular fibers, such as elastic fibers and glycosaminoglycans (proteogly-
cans), influence the three-dimensional structure of the extracellular matrices. Thus, vis-
coelasticity changes and this change in viscoelasticity explain one component of aging
of the voice.
17.1 Introduction The supportive functions and viscoelasticity of connec-

tive tissue depend largely on the properties of their extracel-
Adult vocal folds have a layered structure consisting of the lular matrices. The main extracellular matrices of the vocal
epithelium; the superficial, intermediate, and deep layers of fold mucosa are reticular fibers, collagen fibers, elastic fibers,
the lamina propria; and the vocalis muscle [1, 2]. These layers glycoprotein, and glycosaminoglycan (proteoglycan) [3, 4].
can be divided into three sections: a cover consisting of the Not only geriatric changes in the three-dimensional struc-
epithelium and the superficial layer of the lamina propria, a ture of the extracellular matrices but also their qualitative
transition zone consisting of the intermediate and deep layers and quantitative changes have effect on the viscoelastic
of the lamina propria (vocal ligament), and a body consisting properties of the lamina propria of the human aged vocal fold
of the vocalis muscle [1, 2]. The superficial layer is referred mucosa. Thus, viscoelasticity must change to ensure normal
to as Reinke’s space and has ground substance, sparse fibro- phonation, and this change in viscoelasticity explains one
blasts, and sparse elastic and collagen fibers [1, 2]. The inter- component of the aging of the voice.
mediate layer is primarily made of elastic fibers, whereas the
deep layer is chiefly composed of collagen fibers [1, 2]. These
layered structures are very important in vibration [1]. 17.2 R
eticular and Collagen Fibers
The viscoelastic properties of the lamina propria of the in the Superficial Layer of the Lamina
human vocal fold mucosa are important for the vibratory Propria (Reinke’s Space) of the Human
behavior of the structure. It is the superficial layer of the Vocal Fold Mucosa
lamina propria (Reinke’s space) of the human vocal fold
mucosa that vibrates most markedly during phonation. The Reticular and collagen fibers are essentially identical bio-
viscoelastic properties of the lamina propria, especially the chemically [5] and have been found to have different mor-
superficial layer of the lamina propria, are very important for phological forms of the same fibrous protein, collagen [5].
the vibratory behavior of the vibrating tissue. They greatly Collagen fibers are flexible but offer great resistance to any
depend on the extracellular matrices. pulling force [5]. The fibrils of fibrillar collagen, such as

236 17 Geriatric Changes of Cells and Extracellular Matrices in the Human Vocal Fold Mucosa
reticular and collagen fibers, provide tensile strength and [3]. Glycoprotein and glycosaminoglycan (proteoglycan) are
resilience and serve as stabilizing scaffolds in the extracel- situated among the spaces of the reticular fibers, and elastic
lular matrices [6]. fibers run through the spaces as well (Fig. 17.1) [3]. The deli-
The predominant type of collagen in the lamina propria of cate three-dimensional structure of reticular fibers contrib-
the vocal fold is type III [3, 7]. Type III collagen is one of the utes to maintaining the structure of the vocal fold mucosa
fibrillar collagens [6, 8, 9] and appears to be a major con- during vibration without disturbing vibration [3]. See
stituent of the slender 50 nm fibers that have traditionally Chap.10, “Cells and Extracellular Matrices in the Human
been designated as reticular fibers [5]. Reticular fibers are Adult Vocal Fold Mucosa.”
found in the superficial and intermediate layer of the lamina
propria of the vocal fold mucosa [3]. In particular, reticular
fibers are most abundantly seen around the vocal fold edge 17.3 A
ge-Related Changes of Reticular
[3]. These distributions of reticular fibers indicate that the and Collagen Fibers in the Superficial
reticular fibers are located at the portion of the vocal fold Layer of the Lamina Propria (Reinke’s
mucosa that vibrates the most [3]. Reticular fibers are thin Space) of the Human Vocal Fold
and result in a more compliant tissue [9]. The function of Mucosa
reticular fibers is to maintain the structure in expansible
organs [5]. The reticular fibers in the vocal fold mucosa are A schema of the reticular and collagen fibers and other extra-
composed of slender fibrils, and those fibrils do not form cellular matrices in the superficial layer of the lamina propria
bundles but form delicate three-dimensional networks [3]. (Reinke’s space) of an aged adult vocal fold mucosa are
Spaces among the fibers are relatively large and possess shown in Fig. 17.2.
innumerable potential spaces [3]. These extracellular inter- As the vocal fold mucosae age, reticular fibers decrease
stitial spaces are made up of minute chambers or compart- and collagen fibers increase, especially in men in the super-
ments occupied by other extracellular matrices (Fig. 17.1) ficial layer of the lamina propria (Reinke’s space) (Figs. 17.3
and 17.4). The collagen fibers form bundles and their density
becomes high. The collagen fibril diameters begin to differ
(40–80 nm) and their outline becomes irregular (Fig. 17.5).
glycosaminoglycan Twisted collagen fibrils are present.
(proteoglycan) As the collagen fibers increase, spaces among the colla-
elastic fiber gen fibers and interstitial spaces available for other extracel-
lular matrices decrease (Figs. 17.6 and 17.7).
glycoprotein
collagen fiber
reticular fiber glycosaminoglycan

(proteoglycan)
elastic fiber
Fig. 17.1 Schema of extracellular matrices in the superficial layer of
the lamina propria (Reinke’s space) of a younger adult human vocal glycoprotein
fold mucosa. In the younger adults’ vocal fold mucosa, collagen fibers
are sparse in the superficial layer of the lamina propria. Reticular fibers, collagen fiber
one of the several molecular species of collagen, are found in the super-
ficial layer of the lamina propria of the vocal fold mucosa. Reticular
fibers are made up of unit fibrils that are about 40 nm in diameter. The reticular fiber
slender fibrils are wavy and do not form bundles. They form delicate
three-dimensional networks. Spaces among the reticular and collagen
fibers for other extracellular matrices are relatively large. Other extra-
cellular matrices, such as glycosaminoglycans, elastic fibers, and glyco- Fig. 17.2 Schema of extracellular matrices in the superficial layer of
proteins, are distributed in the spaces among the reticular and collagen the lamina propria (Reinke’s space) of the human aged vocal fold
fibers mucosa
17.4 Age-Related Changes of Collagen Fibers in the Vocal Ligament of the Human Vocal Fold Mucosa 237
elastic fibers
collagen fibers
collagen fibers
Fig. 17.4 Transmission electron micrograph of the superficial layer of the

b lamina propria of the aged vocal fold mucosa (85-year-old male, tannic
acid stain). The collagen fibers form bundles and the density becomes high
elastic fibers
collagen fibers
twisted collagen fibril
collagen fibril
(40 nm)
collagen fibril
Fig. 17.3 Light microscopic findings of the superficial layer of aged
(80 nm)
adult lamina propria (70-year-old female, (a) hematoxylin and eosin
stain, (b) Elastica van Gieson stain)
Fig. 17.5 Transmission electron micrograph of the collagen fibrils in

17.4 A
ge-Related Changes of Collagen the superficial layer of the lamina propria of the aged vocal fold mucosa
(83-year-old male, tannic acid stain). Diameters of collagen fibrils vary
Fibers in the Intermediate and Deep from 40 to 80 nm and the outlines are irregular
Layers of the Lamina Propria (Vocal
Ligament) of the Human Vocal Fold
Mucosa seen in the deep layer of the lamina propria of the vocal fold
mucosa (Fig. 17.8 and 17.9). Sometimes, the collagen fibers
The number of collagen fibers increases in the deep layer of have increased in all layers from the deep layer to the super-
the aged lamina propria of the vocal fold mucosa (Fig. 17.8 ficial layer of the lamina propria of the aged vocal fold
and 17.9). The deep layer tends to become thicker with age mucosa, and consequently, there is no layered structure
as a result of the increase in collagen fiber density. Sometimes, (Figs. 17.6, 17.7, and 17.10).
masses of dense collagen fibers and fibrous tissue can be
epithelium a a
epithelium
collagen fibers lamina propria lamina propria collagen fibers

of mucosa of mucosa reticular fibers
b b
epithelium
epithelium
hyaluronic acid
lamina propria
hyaluronic acid of mucosa
Fig. 17.6 Transverse section of the vocal fold mucosa (85-years-old Fig. 17.7 Transverse section of the vocal fold mucosa (82-year-old
male, a silver stain, b Alcian Blue stain). (a) The number of collagen female, a silver stain, b Alcian Blue stain). (a) Collagen fibers have
fibers has increased from the deep to superficial layer of the vocal fold increased in the lamina propria of the vocal fold mucosa, but there are
mucosa. There are few spaces among collagen fibers. (b) Spaces among spaces between collagen fibers. (b) There are spaces among collagen
collagen fibers and interstitial spaces have decreased. Little glycosami- fibers and interstitial spaces. Glycosaminoglycan (hyaluronic acid),
noglycan (hyaluronic acid), stained light blue, can be detected stained light blue, can be detected
17.5 A
ge-Related Changes of Reticular 17.5.1 Superficial Layer of the Lamina Propria
and Collagen Fibers in the Lamina (Reinke’s Space) of the Aged Vocal Fold
Propria of the Human Vocal Fold Mucosa
Mucosa and their Biomechanical
Properties The delicate three-dimensional structure of reticular fibers
(type III collagen) in the human vocal fold mucosa appears
In the aged lamina propria of the human vocal fold mucosa, to contribute to maintaining the structure and viscoelasticity
not only changes in the three-dimensional structures of the of the vibrating tissue [3]. And the complex of reticular fibers
collagen and reticular fibers but also their qualitative and and other extracellular matrices seems to be very important
quantitative changes have effect on the three-dimensional for the viscoelastic properties of the human vocal fold
structure of the extracellular matrices. mucosa [3]. The number of reticular fibers (type III collagen)
17.5 Age-related Changes of Collagen Fibers in the Vocal Ligament of the Human Vocal Fold Mucosa 239
a B a
C
epithelium
b epithelium b
superficial layer
of lamina propria
fibrous tissue
collagen fibers
thyroarytenoid
muscle
capillary
c Fig. 17.9 (a) Coronal section of the aged vocal fold (97-year-old male,
Elastica van Gieson stain). (b) Region B in a. Collagen fibers have
increased, and their density is high in the superficial layer of the lamina
propria of the vocal fold mucosa. The superficial layer is thin. (c)
Region C in a. Masses of dense collagen fibers and fibrous tissue can be
seen in the deep layer (arrowed area). (d) Region D in c. Dense collagen
fibers and fibrous tissue are observed
fibrous tissue
decreases in the superficial layer of the lamina propria of the

aged vocal fold mucosa [10]. Consequently, with aging, the
change in the complex of the reticular fibers and other extra-
cellular matrices in the superficial layer of the lamina propria
changes the viscoelastic properties of the human vocal fold
mucosa as a vibrating tissue.
The number of collagen fibers increases in the superficial
Fig. 17.8 (a) Coronal section of the aged vocal fold (92-year-old
layer of the lamina propria of the aged vocal fold mucosa
female, Elastica van Gieson stain). (b) Region B in a. Masses of dense
collagen fibers and fibrous tissue can be seen in the deep layer (arrowed [10, 11, 12]. Collagen fibers form bundles and the density is
area). (c): Region C in b. Dense collagen fibers and fibrous tissue are high, especially in men [10]. Additionally, masses of dense
observed collagen fibers and fibrous tissue can be seen in the vocal
c epithelium
epithelium
elastic fiber
collagen fibers
fibrous tissue
D
collagen fibers
collagen fibers
d Fig. 17.10 Transverse section of the aged vocal fold (78-year-old

female, Elastica van Gieson stain). The collagen fibers form bundles,
and their density becomes high in the lamina propria of the aged focal
fold mucosa
With aging, the changes in the complex of collagen fibers

and other extracellular matrices in the superficial layer of the
fibrous tissue lamina propria of the vocal fold mucosa change the visco-
elastic properties of the lamina propria of the human vocal
fold mucosa. Consequently, geriatric changes in the fine
structure of the extracellular matrices in the superficial layer
of the lamina propria are one of the important causes of
changes in the voice with aging [10].
17.5.2 Intermediate and Deep Layer

Fig. 17.9 (continued) of the Lamina Propria (Vocal Ligament)
of the Aged Vocal Fold Mucosa
fold mucosa [10]. These three-dimensional structures cannot The deep layer of the lamina propria, especially in males,
serve as flexible stabilizing scaffolds in the extracellular tends to become thicker with age, being associated with an
matrices, and fibrous masses disturb vocal fold vibration. increase in collagen fiber density [11, 14]. The collagen
Under transmission electron microscopy, the collagen fibril fibers increase and form bundles, and the density is high in
diameters differ, and their outlines appear irregular. Twisted the deep layer of the lamina propria of the aged vocal fold
collagen fibrils are present. These findings indicate that not mucosa. The collagen fibril diameters differ, and the outlines
only changes in the three-dimensional structures of collagen are irregular. Twisted collagen fibrils are present. Sometimes,
fibers but also qualitative and quantitative changes have masses of dense collagen fibers and fibrous tissue can be
occurred in the aged vocal fold mucosa [10]. seen. These masses seem to disturb the vibration of the aged
As a result of increased collagen fibers, the spaces among vocal fold mucosa.
collagen fibers and interstitial spaces for other extracellular
matrices, such as glycosaminoglycan (proteoglycan) and
elastic fibers, decrease with age in the superficial layer of the 17.5.3 Destruction of the Layered Structure
vocal fold mucosa. One of the roles of glycosaminoglycan in the Aged Adult Vocal Fold Mucosa
(proteoglycan) is to give viscosity to tissues [13]. Therefore,
the aged vocal fold mucosae have less viscosity because of In the aged vocal fold mucosa, the number of collagen
the decrease of glycosaminoglycan (proteoglycan). fibers has sometimes increased all the way from the deep
17.6 Age-Related Changes of Elastic Fibers in the Superficial Layer of the Lamina Propia (Reinke’s Space) 241
layer to the superficial layer of the lamina propria, and

consequently, there is no layered structure. These vocal epithelium
fold mucosae do not have proper viscoelasticity as a vibrat-
ing tissue.
masses of elastic
17.6 A
ge-Related Changes of Elastic Fibers fibers
in the Superficial Layer of the Lamina
Propia (Reinke’s Space) of the Human
Vocal Fold Mucosa
In the aged, the elastic fibers run in various directions, are

branched, and form a complicated network in the superficial
layer of the lamina propria of the vocal fold mucosa
(Fig. 17.11). The spaces between the elastic fibers are smaller collagen fibers
than in younger adults. The surfaces of the fibers are rough
and the fibers appear to vary in size. Sometimes, elastic Fig. 17.12 Light microscopic findings of the superficial layer of the
fibers form a mass (Fig. 17.12). aged adult lamina propria (78-year-old female, Elastica van Gieson
Under scanning electron microscopy, some elastic fibers stain). Masses of dense elastic fibers can be seen
unite to form a sheet with a rough surface (Fig. 17.13) or
form masses (Fig. 17.14).
Under transmission electron microscopy, the elastic fibers
are comprised of microfibrils and amorphous substances. elastic fiber
Many microfibrils are present along the periphery of elastic
fibers in the superficial layer of the lamina propria of the
younger adult vocal fold mucosa.
In the aged vocal fold mucosa, the fibers are larger and
rougher than those in the younger adult. The amorphous sub-
stances have increased in amount, and microfibrils have
become less numerous (Fig. 17.15). Some of the elastic
fibers in the aged vocal folds have become fragmented
(Fig. 17.16).
epithelium
Fig. 17.13 Scanning electron micrograph of elastic fibers in the super-

ficial layer of the lamina propria of the aged vocal fold mucosa (60-year-
old female). Only elastic fibers alone remain following treatment by the
modified sodium hydroxide (NaOH) maceration method. Some elastic
fibers have united to form a sheet with a rough surface (arrow)
elastic fibers
Table 17.1 shows the results for the elastase digestion

study of elastic fibers. The elastic fibers undergo digestion by
elastase (Fig. 17.17). The amorphous substances show a
Fig. 17.11 Light microscopic findings of the superficial layer of the marked affinity for tannic acid and stain black. Staining
aged adult lamina propria (91-year-old male, Elastica van Gieson stain) becomes reticular from the periphery of the fibers at the start
elastic fiber b
elastic fiber
amorphous
substances
microfibrils
elastic fiber
amorphous
substances
fragmented
elastic fibers
Fig. 17.14 Scanning electron micrograph of elastic fibers in the super-
ficial layer of the lamina propria of the aged vocal fold mucosa (77-year-
old male, modified sodium hydroxide (NaOH) maceration method).
Some elastic fibers have united to form masses (arrow)
elastic fiber
elastic fiber amorphous
substances
amorphous Fig. 17.16 Transmission electron micrograph of elastic fibers in the

substances superficial layer of the lamina propria of the aged vocal fold mucosa
microfibrils (74-year-old male, tannic acid stain) Some of the elastic fibers in the
aged vocal fold have become fragmented
Table 17.1 The results of elastase digestion study

30 min 1 h 2 h 3 h 4 h
Elastic fibers in aged adults − − + ++ +++
Elastic fibers in younger adults + ++ +++
Fig. 17.15 Transmission electron micrographs of elastic fibers in the Elastic fiber elastin from the younger adults started to undergo diges-
superficial layer of the lamina propria of the aged vocal fold mucosa tion by elastase within 30 min, and more than 75% was digested within
(a 60-year-old male, uranyl acetate and lead citrate stain, b 74-year- 2 h. Aged specimens did not undergo digestion uniformly. Digestion
old male, tannic acid stain). Amorphous substances have increased in started within 2 h and was more than 75% complete at the end of 4 h
amount and microfibrils have become less numerous −, no digestion; +, less than 25% digestion; ++, 25–75% digestion;
+++, more than 75% digestion
of digestion and works its way toward the interior. The retic- The elastin in the elastic fibers in the superficial layer of
ularly stained potions (elastin, amorphous substances) the lamina propria of the aged vocal fold is difficult to digest
increase, and digestion finally proceeds to completion. compared with the younger adults.
17.8 Age-Related Changes of Elastic Fibers in the Lamina Propia of the Human Vocal Fold Mucosa 243
elastic fibers
microfibrils
digested elastin
(amorphous
substances)
Fig. 17.17 Transmission electron micrograph of elastic fibers under-

going digestion by elastase (34-year-old male, tannic acid stain). Elastin Fig. 17.18 Light microscopic findings of the intermediate layer of the
(amorphous substances) is digested by elastase, and staining by tannic aged adult lamina propria (83-year-old male). The elastic fibers are
acid (black) becomes reticular from the periphery of the elastic fibers branched and form a complicated network, the surfaces of the fibers are
rough, and the fibers vary in size
17.7 A
ge-Related Changes of Elastic Fibers substances (elastin), and microfibrils are fewer in number.
in the Intermediate and Deep Layers The microfibrils constitute the predominant structure of
of the Lamina Propia (Vocal developing elastic fibers [16]. Elastic fibers in the superficial
Ligament) of the Human Vocal Fold layer of the aged lamina propria have thus ceased to develop
Mucosa and have changed morphologically.
Elastic fibers are composed of elastin protein. Elastase
The elastic fibers in the intermediate layer of the lamina pro- decomposes the elastin and is related to the metabolism of
pria are branched and form a complicated network, the sur- elastin and elastic fibers [17, 18]. The elastin in elastic fibers
faces of the fibers are rough, and the fibers vary in size in the superficial layer of the aged lamina propria cannot be
(Fig. 17.18). digested easily by elastase compared with that of younger
Elastic fibers in the intermediate layer of the lamina pro- adults, indicating the metabolism of elastic fibers has
pria become less dense and become atrophic in males, and changed [16]. The activity of serum elastase decreases with
such changes are less marked in females [14]. aging [19]. This suggests that the turnover and metabolism
of elastic fibers in the superficial layer of the lamina propria
of the aged vocal fold mucosa occur more slowly.
17.8 A
ge-Related Changes of Elastic Fibers The main role of elastic fibers is to give elasticity and
in the Lamina Propia of the Human resilience to tissues [9]. The elastic fibers in the superfi-
Vocal Fold Mucosa and their cial layer of the lamina propria of the vocal fold mucosa
Biomechanical Properties change morphologically with age, and the metabolism of
these fibers is disturbed [15]. Consequently, their turn-
The elastic fibers in the superficial layer of the aged lamina over and repair become slow. The elastic fibers appear to
propria change morphologically with age [15]. The elastic lose their function, no longer giving sufficient elasticity
fibers run in various directions, are branched, and form a and resilience to the human vocal fold mucosa as a
complicated network, the surfaces of the fibers are rough, vibrating tissue [15]. The morphologic and metabolic
and the fibers vary in size. Sometimes elastic fibers unite to changes of elastic fibers in the tissue most important for
form a sheet with a rough surface or form masses. These vibration, the superficial layer of the lamina propria of
findings show elastic fibers cannot give sufficient resilience the aged vocal fold, contribute partially to the aging of
to the tissue [15]. The elastic fibers in the superficial layer of the voice.
the aged vocal folds consist of greater amounts of amorphous
17.9 A
ge-Related Changes of Ground
c
Substances in the Superficial
Layer of the Lamina Propia
(Reinke’s Space) of the Human
Vocal Fold Mucosa
Edematous change occurs, especially in females, in the

superficial layer of the lamina propria (Reinke’s space) of the
aged vocal fold mucosa (Figs. 17.19 and 17.20) [14].
As the collagen fibers increase, spaces among the colla-
gen fibers and interstitial spaces for other ground sub-
stances, such as hyaluronic acid (hyaluronan), decrease
with age in the superficial layer of the vocal fold mucosa
(Figs. 17.6 and 17.7).
a lamina propria epithelium

of mucosa
vocal process a
anterior posterior
anterior
commissure
tendon B
thyroarytenoid
muscle
thyroid cartilage
thyroarytenoid
muscle
b lamina propria epithelium epithelium b

of mucosa
lamina propria
of mucosa
thyroarytenoid
muscle
Fig. 17.19 Coronal sections of the aged vocal fold (92-year-old Fig. 17.20 (a) Transverse section of the aged vocal fold (71-year-old
female, a hematoxylin and eosin stain, b and c Elastica van Gieson female, Elastica van Gieson stain). (a) Membranous vocal fold has not
stain). Superficial layer of the lamina propria (Reinke’s space) of the become bowed. (b) Region B in a. Lamina propria of the vocal fold mucosa
vocal fold mucosa is edematous has become loose and edematous. Vocal ligament has disappeared
17.11 Age-Related Changes of Epithelium of the Human Vocal Fold Mucosa 245
17.10 Bowing of the Aged Vocal Folds
In elderly people, vocal folds are bowed without clinically

noticeable organic or functional lesions (Fig. 17.21). anterior
The degree of bowing of the vocal fold (Fig. 17.22) macula flava
increases with age, particularly in males (Fig. 17.23). Bowing
of the vocal fold is much more significant in aged males than
females.
A decrease in the thickness of the lamina propria of the
vocal fold mucosa is one cause of bowing of the vocal fold in b
both sexes (Fig. 17.24).
A decrease in the thickness of the superficial layer of the vocal ligament
lamina propria of the vocal fold mucosa is another cause of
bowing of the vocal fold in both sexes (Fig. 17.25a).
The bowing of the vocal fold is not significantly affected posterior
macula flava
by the decrease in the thickness of the intermediate layer
(Fig. 17.25b) and deep layer (Fig. 17.25c) of the lamina pro-
pria of the vocal fold mucosa in both sexes.
vocal process
a anterior posterior
anterior
commissure
tendon
B
Fig. 17.22 Degree of bowing of the vocal fold. The degree of bowing
of the vocal fold was measured on the whole-organ laryngeal sections.
thyroid cartilage The line running from the anterior to posterior ends of the membranous
thyroarytenoid
vocal fold, the anteroposterior length (l), and the distance (b) from the
muscle deepest bowed portion to the anteroposterior line was measured. The
ratio (b/l) shows the degree of bowing of the vocal fold. The thickness
of each layer of the lamina propria of the vocal fold mucosa is measured
b at the middle portion of the membranous vocal fold. The cross-sectioned
epithelium area of the thyroarytenoid muscle is determined at the midpoint of the
membranous vocal fold
Reinke’s Therefore, not only the age-related atrophy of the thyro-

space
arytenoid muscle but also the decrease in the thickness of the
lamina propria of the aged vocal fold mucosa, especially the
superficial layer, is the cause of the bowing [20]. Bowing of
vocal ligament aged vocal folds is a geriatric change causing glottic incom-
petence with consequent voice disorders and is, at least, one
of the etiologies of the aging of the voice [20].
17.11 A
ge-Related Changes of Epithelium
Fig. 17.21 Transverse section of the aged vocal fold (90-year-old
male, Elastica van Gieson stain). (a) Membranous vocal fold has The free edge of the human vocal fold is covered with non-
become bowed. (b) Region B in a. The epithelium has become thick
(acanthosis). The lamina propria of the vocal fold mucosa, especially
keratinizing stratified squamous epithelium. The cells in the
the superficial layer (Reinke’s space) of the vocal fold mucosa, has basal lamina are columnar or polyhedral, becoming increas-
become thin. The vocal ligament has become thick ingly flattened toward the surface, and in the superficial
b
I a
0.2
males
females
0.1
0
70 80 90 100 age
Fig. 17.23 Relationship between the degree of bowing of the vocal

fold and age. Sixty-four normal human larynges from 30 males and 34
females obtained from autopsy cases were examined. The ages ranged
from 70 to 104 years old
b b
l l
males females
0.2 0.2
r=–0.36 r=–0.29
0.1 0.1
c
0 0
0.5 1 1.5 2 1 1.5
mm mm
Fig. 17.24 Relationship between the degree of bowing of the vocal

fold and thickness of the lamina propria
layers, they are thin squamous cells. The epithelium is com-

posed of seven to eight epithelial cell layers.
Geriatric changes in the stratified squamous epithelium of
the human vocal fold are hyperkeratosis (thickening of the
horny layer of the mucous membrane, Fig. 17.26a), abnor- Fig. 17.25 Relationship between the degree of bowing of the vocal
mal differentiation (the epithelial cells do not become fold and thickness of each layer of the lamina propria. (a) Superficial
increasingly flattened toward the surface, Fig. 17.26b), pro- layer, (b) intermediate layer, (c) deep layer of the lamina propria
longed rete ridge (downward thickening of the stratified
squamous epithelium, Fig. 17.26c), acanthosis (an increase Fig. 17.26d), and atrophy (a diminution in the size and func-
in the thickness of the stratified squamous epithelium, tion of the stratified squamous epithelium, Fig. 17.26e).
17.12 Age-Related Changes of Epithelium of the Human Vocal Fold Mucosa and their Biomechanical Properties 247
hyperkeratosis
a d
epithelium epithelium
lamina propria of mucosa lamina propria of mucosa
b e
epithelium
epithelium

parakeratosis
c Fig. 17.26 (continued)
Under scanning electron microscopy, microvilli of the

aged stratified squamous epithelium of the human vocal fold
epithelium have disappeared (Figs. 17.27 and 17.28).
17.12 A
ge-Related Changes of Epithelium
and their Biomechanical Properties
rete ridges Age-related changes in the stratified squamous epithelium of

the human vocal fold do not provide a pliable and firm struc-
lamina propria of mucosa ture as a vibrating tissue.
The vocal fold is lubricated by secretions from the upper
respiratory tract [21]. Lubrication by a thin mucous coating is
Fig. 17.26 Geriatric changes in the stratified squamous epithelium of
the human vocal fold. (a) hyperkeratosis (81-year-old female), (b)
essential for normal vibration and phonation [22]. Microvilli
abnormal differentiation (81-year-old female), (c) prolonged rete ridge (microridges) of the stratified squamous epithelium are con-
(81-year-old female), (d) acanthosis (91-year-old male), (e) atrophy sidered to facilitate distribution and retention of minute
(91-year-old male)
b
Fig. 17.28 Scanning electron micrograph of the surface of the epithe-
lium of the aged vocal fold (83-year-old male). Microvilli of the strati-
fied squamous epithelium have disappeared
vocal fold mucosa disturbs distribution and retention of min-

ute amounts of mucus on the vocal fold surface. Thus, less
lubrication would explain one of the causes of the voice’s
changes with aging.
In addition, age-related changes in the laryngeal glands
influence not only the amount but also the quality and viscos-
ity of secretions [26]. Geriatric changes of the laryngeal
glands lessen lubrication of the vocal folds, thus causing
aging of the voice to some extent [26]. See Chap. 22, “The
Laryngeal Glands.”
17.13 A
ge-Related Changes of Extracellular
Matrices and Aging Voice
Fig. 17.27 (a) Scanning electron micrograph of the surface of the epi- Stiffness, mass, homogeneity, obstacles, and the layered
thelium of the aged vocal fold (83-year-old male). (b) Higher magnifi- structure of the vocal fold and the symmetry of the bilateral
cation of a. Microvilli of the stratified squamous epithelium have
vocal folds, glottic closure, and subglottal pressure influence
disappeared
vocal fold vibration and phonation.
Not only the three-dimensional structure of the extracel-
amounts of mucus on the vocal fold surface [23]. In addition, lular matrices but also their quality and quantity have effect
the microvilli (microridges) provide a better surface for con- on the viscoelastic property of the lamina propria of the
tact, minimizing slippage, much as the tread of a tire provides human vocal fold mucosa as a vibrating tissue.
traction [24, 25]. See Chap. 10, “Cells and Extracellular Due to age-related changes of extracellular matrices in
Matrices in the Human Adult Vocal Fold Mucosa. the aged vocal fold mucosa, the following geriatric changes
The apical cell membrane (surface of the stratified squa- occur in the aged vocal fold mucosa: (1) Stiffness (elastic
mous epithelium) of the human aged vocal fold loses micro- constant, viscosity coefficient, viscoelasticity) and mass
villi (microridges). Consequently, the surface of the aged have changed. (2) The aged vocal fold mucosa is not
References 249
homogenous along its longitudinal axis. (Consequently,

elastic fiber
differences in biomechanical properties of the bilateral
vocal folds result in asymmetric vocal fold vibration.) (3) rough endoplasmic
reticulum
Obstacles due to organized and scared tissue disturb vocal
fold vibration. (4) There is no layered structure essential
for vocal fold vibration and phonation. (5) Glottal incom-
nucleus
petence occurs due to vocal fold atrophy. In addition, age- fibroblast
related changes of pulmonary functions influence the
subglottal pressure.
Vocal fold vibration is a physical phenomenon. Thus,
age-related changes of extracellular matrices explain geriat-
ric changes of voice. mitochondrion
For example, the stiff and thin vocal fold mucosa of the collagen fiber
aged male vibrates fast with a small amplitude resulting in
fundamental frequency increase. This phenomenon is con-
sistent with the reports that the aged men tend to show a Fig. 17.29 Transmission electron micrograph of a fibroblast in
higher speaking fundamental frequency than young men Reinke’s space of the aged vocal fold (74-year-old male, tannic acid
[27, 28]. Edematous and thick vocal fold mucosa of the stain)
aged female vibrates slow with a large amplitude resulting
in fundamental frequency decrease. This phenomenon is slightly more active in geriatric vocal folds than those in
consistent with the reports that the aged women tend to younger ones [31].
show a lower speaking fundamental frequency than young Fibroblasts in Reinke’s space are thought to be activated in
women [29]. a pathological condition like vocal fold injury [30]. Geriatric
Age-related changes in the three-dimensional structure of changes of extracellular matrices are considered to be one of
the extracellular matrices and their qualitative and quantita- the pathological conditions of the human vocal fold.
tive changes influence vocal fold vibration resulting in tone
changes of the voice.
Significant differences in the histological changes of the 17.15 A
ge-Related Changes of Cells
aged vocal fold are found from one individual to another, just including Vocal Fold Stellate Cells in
as there are individually recognizable differences in geriatric Maculae Flavae of the Human Vocal
changes in the voice. Fold Mucosa
See Chap. 18, “Geriatric Changes of the Macula Flava of the

17.14 A
ge-Related Changes of Interstitial Human Vocal Fold.”
Cells in Reinke’s Space of the Human
Vocal Fold Mucosa
References
Fibroblasts are one of the interstitial cells that produce
fibrous proteins and other extracellular matrices. However, 1. Hirano M. Phonosurgery. Basic and clinical investigation. Otologia
(Fukuoka). 1975;21(Suppl 1):239–98.
fibroblasts in the superficial layer of the lamina propria 2. Hirano M, Sato K. Histological color atlas of the human larynx. San
(Reinke’s space) of the vocal fold mucosa synthesize few Diego, CA: Singular Publishing Group Inc.; 1993.
extracellular matrices; they are inactive and quiescent under 3. Sato K. Reticular fibers in the vocal fold mucosa. Ann Otol Rhinol
normal conditions [30]. Laryngol. 1998;107:1023–8.
4. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteo-
The density of fibroblasts in the aged Reinke’s space is glycans and their influence on biomechanics. Laryngoscope.
the same as in the younger adult [31]. Fibroblasts are 1999;109:845–54.
spindle-shaped and the nuclei are elliptic. The nucleus- 5. Fawcett DW. Connective tissue proper. A textbook of histology.
cytoplasm ratio is large with rough endoplasmic reticulum Philadelphia, PA: WB Saunders; 1986. p. 136–51.
6. Kreis T, Vale R. Guidebook to the extracellular matrix and adhesive
and Golgi apparatus (Fig. 17.29). The degree of develop- proteins. Oxford: Oxford University Press; 1993.
ment of intracellular organelles in the fibroblasts in Reinke’s 7. Gray SD, Hirano M, Sato K. Molecular and cellular structure of
space is greater in the geriatric vocal fold compared with vocal fold tissue. In: Titze IR, editor. Vocal fold physiology. San
younger adults [31]. Glycogen particles are not noted in the Diego, CA: Singular Publishing Group; 1993. p. 1–35.
8. Linsenmayer TF. Collagen. In: Hay ED, editor. Cell biology
cytoplasm of fibroblasts in Reinke’s space of the geriatric of extracellular matrix. 2nd ed. New York: Plenum Press; 1991.
vocal fold [31]. The fibroblasts in Reinke’s space are p. 7–44.
9. Ayad S, Boot-Handford RP, Humphries MJ, Kadler KE, 19. Hall DA. Age changes in the levels of elastase and its inhibitor in
Shuttleworth CA. The extracellular matrix. Facts book. London: human plasma. Gerontologia. 1968;14:97–108.
Academic Press; 1994. 20. Sato K, Sakaguchi S, Hirano M. Histologic investigation of bowing
10. Sato K, Hirano M, Nakashima T. Age-related changes of collag- of the aged vocal folds. Larynx Jpn. 1996;8:11–4.
enous fibers in the human vocal fold mucosa. Ann Otol Rhinol 21. Pressman JJ. Physiology of the vocal cords in phonation and respi-
Laryngol. 2002;111:15–20. ration. Arch Otolaryngol. 1942;35:355–98.
11. Sato K, Sakaguchi S, Kurita S, Hirano M. A morphological study 22. Ichikawa T. Basic studies on the lubrication of the larynx during
of aged larynges. Larynx Jpn. 1992;4:84–94. phonation. Otologia (Fukuoka). 1982;28:38–52.
12. Hammond TH, Gray SD, Butler JE. Age- and gender-related col- 23. Tillmann B, Pietzsch I-R, Huenges HL. The human vocal cord sur-
lagen distribution in human vocal folds. Ann Otol Rhinol Laryngol. face. Cell Tissue Res. 1977;185:279–83.
2000;109:913–20. 24. Gray SD, Titze I, Lusk RP. Electron microscopy of hyperphonated
13. Wight TN, Heinegard DK, Hascall VC. Proteoglycans. Structure canine vocal cords. J Voice. 1987;1:109–15.
and function. In: Hay ED, editor. Cell biology of extracellular 25. Gray S, Titze I. Histologic investigation of hyperphonated canine
matrix. 2nd ed. New York: Plenum Press; 1991. p. 45–78. vocal cords. Ann Otol Rhinol Laryngol. 1988;97:381–8.
14. Hirano M, Kurita S, Sakaguchi S. Ageing of the vibratory tissue of 26. Sato K, Hirano M. Age-related changes in the human laryngeal
human vocal folds. Acta Otolaryngol (Stockh). 1989;107:428–33. glands. Ann Otol Rhinol Laryngol. 1998;107:525–9.
15. Sato K, Hirano M. Age-related changes of elastic fibers in the 27. Mysak ED. Pitch and duration characteristics of older males. J
superficial layer of the lamina propria of vocal folds. Ann Otol Speech Hear Res. 1959;2:46–54.
Rhinol Laryngol. 1997;106:44–8. 28. Hollien H, Shipp T. Speaking fundamental frequency and chrono-
16. Rhodin JA. Connective tissue. Histology. A text and atlas.
logic age in males. J Speech Hear Res. 1972;15:155–9.
New York: Oxford University Press; 1974. p. 140–71. 29. Honjo I, Isshiki N. Laryngoscopic and voice characteristics of aged
17. Ross R, Bornstein P. The elastic fiber. 1. The separation and partial persons. Arch Otolaryngol. 1980;106:149–50.
characterization of its macromolecular components. J Cell Biol. 30. Hirano M, Sato K, Nakashima T. Fibroblasts in human vocal fold
1969;40:366–81. mucosa. Acta Otolaryngol. 1999;119:271–6.
18. Bodley HD, Wood RL. Ultrastructural studies on elastic fibers using 31. Hirano M, Sato K, Nakashima T. Fibroblasts in geriatric vocal fold
enzymatic digestion of thin sections. Anat Rec. 1972;172:71–88. mucosa. Acta Otolaryngol. 2000;120:336–40.
Geriatric Changes of the Macula Flava
of the Human Vocal Fold 18
Abstract
1. Human aged maculae flavae are dense masses of cells and extracellular matrices located
at the anterior and posterior ends of the membranous portion of the bilateral vocal folds.
2. The extracellular matrices of human aged maculae flavae are composed of glycosamino-
glycan (hyaluronan), glycoproteins, and fibrillar protein such as collagen fibers, reticular
fibers, and elastic fibers.
3. Vocal fold stellate cells in the aged maculae flavae are stellate in shape and possess slen-
der cytoplasmic processes. Aged vocal fold stellate cells also possess lipid droplets in
the cytoplasm and store vitamin A. Basically the morphological characteristics of the
aged vocal fold stellate cells are the same as those of younger adults.
4. The cells including vocal fold stellate cells in the aged maculae flavae appear to have a
decreased level of activity, to have abnormal metabolism, and to have undergone
degeneration.
5. In the aged vocal fold stellate cells, there are fewer intracellular organelles than in younger
adults. Some components of the cytoplasm have degenerated. The nuclei of the vocal fold
stellate cells are dense and cleaved. There are few vesicles at the periphery of the cyto-
plasm and few newly released amorphous materials. Some aged vocal fold stellate cells
have degenerated, and an accumulation of glycogen particles is seen in the cytoplasm.
There is a decrease in the number of extracellular matrices synthesized by these inacti-
vated cells. These age-related changes are found to various degrees in the cells in the
maculae flavae.
6. The hyaluronan concentration in the aged maculae flavae is high and contains cells
which possess hyaluronan receptors, indicating that the aged maculae flavae are also a
hyaluronan-rich matrix, which is required for a stem cell niche.
7. Age-related changes of the cells including vocal fold stellate cells in the maculae flavae
most likely influence the metabolism of extracellular matrices in the vocal fold mucosa
as well as the viscoelasticity of the aged vocal fold mucosa and are one of the causes of
the aging of the voice.
18.1 Introduction qualitative and quantitative changes in the extracellular

matrices and in their three-dimensional structure have an
The viscoelastic properties of the lamina propria of the effect on the viscoelasticity of the human vocal fold mucosa
human vocal fold mucosa are indispensable to its vibrating [1–5]. Consequently, these changes in viscoelasticitvy
behavior and depend on extracellular matrices. Age-related explain one component of aging of the voice.

252 18 Geriatric Changes of the Macula Flava of the Human Vocal Fold
Human adult maculae flavae are dense masses of cells and 18.2 M
aculae Flavae in the Human Aged
extracellular matrices located at the anterior and posterior Vocal Fold
ends of the membranous portion of the bilateral vocal folds.
Human maculae flavae are involved in the metabolism of Human aged maculae flavae are dense masses of cells and
extracellular matrices essential for the viscoelasticity of the extracellular matrices (Figs. 18.1 and 18.2) [12, 13]. The
human vocal fold mucosa and maintain the layered structure maculae flavae are located at the anterior and posterior ends
of the human vocal fold [6, 7]. Human maculae flavae are of the membranous portion of the bilateral vocal folds. They
considered to be an important structure in the growth, devel- are elliptical in shape, and their size is approximately 1.5
opment, and aging of the human vocal fold mucosa [6, 7]. × 1.5 × 1 mm [12]. The size of the macula flava of the aged
Vocal fold stellate cells contained in the human maculae vocal fold is the same as in younger adults. The border
flavae were discovered in our laboratory in 2001 [8]. They are between the maculae flavae and the surrounding soft tissue is
considered to be a new category of cells in the human vocal relatively clearly delineated. The vocal ligament runs
fold mucosa. Recently, there is growing evidence to suggest between the anterior and posterior maculae flavae (Fig. 18.1).
that the cells including vocal fold stellate cells in the human The vibratory portion (membranous portion) of the aged vocal
maculae flavae are tissue stem cells or progenitor cells of the fold is connected to the thyroid cartilage anteriorly via the inter-
vocal fold mucosa and that the maculae flavae are a candidate vening anterior macula flava and anterior commissure tendon
for a stem cell niche, that is, a microenvironment nurturing a [12]. Posteriorly, it is joined to the vocal process of arytenoid car-
pool of cells including vocal fold stellate cells [9–11]. tilage via the intervening posterior macula flava (Fig. 18.1) [12].
Age-related changes in the cells including vocal fold stel- The extracellular matrices of human aged maculae flavae
late cells in the maculae flavae are most likely involved in are composed of glycosaminoglycan, glycoproteins, and
geriatric changes in the extracellular matrices of the vocal fibrillar protein such as collagen fibers, reticular fibers, and
fold mucosa. elastic fibers (Figs. 18.3 and 18.4).
a vocal process
anterior posterior
anterior
commissure
tendon B C
thyroid cartilage
thyroarytenoid
muscle
b anterior macula flava
anterior
commissure
tendon
Fig. 18.1 (a) Horizontal section of the aged vocal fold

(90-year-old male, Elastica van Gieson stain). (b) Anterior
macula flava (region B in a). (c) Posterior macula flava thyroarytenoid muscle
(region C in a)
18.2 Maculae Flavae in the Human Aged Vocal Fold 253
c posterior macula flava vocal process
posterior
macula flava
thyroarytenoid
muscle
laryngeal glands
Fig. 18.2 Coronal section of the aged vocal fold

(91-year-old female, Elastica van Gieson stain)
Fig. 18.3 Macula flava of the

human aged vocal fold
(81-year-old female,
hematoxylin and eosin stain).
Human aged maculae flavae
are dense masses of cells
extracellular matrices
b
collagen fibers
Fig. 18.4 Macula flava of the human aged vocal fold. (a)
Human adult maculae flavae are dense masses of cells and
extracellular matrices (83-year-old male, toluidine blue stain, elastic fibers
original ×400). (b) There are many collagen fibers (stained
red) and elastic fibers (stained black) with Elastica van
Gieson stain around the vocal fold stellate cells in the human
aged maculae flavae (90-year-old female, Elastic van Gieson
stain). (c) There are many collagen fibers (stained red) and
reticular fibers (stained black) with silver stain around the
vocal fold stellate cells in the human aged maculae flavae
(82-year-old female, silver stain)
18.3 Morphological Characteristics of the Human Aged Vocal Fold Stellate Cells 255
c in the cytoplasm (Fig. 18.6). They are distributed not only in

the cell body but also in the cytoplasmic processes and are
of two types: membrane bounded and non-membrane
vocal fold stellate cells bounded (Fig. 18.7). The former are each surrounded by a
unit membrane. The rough endoplasmic reticulum, vesicles,
and membrane-bounded lipid droplets are not as closely
associated with each other as are those in younger adults.
Lipofuscin granules can be detected around the lipid drop-
lets (Fig. 18.6).
When the gold chloride method is employed for the detec-
collagen fibers tion of vitamin A, the cytoplasm of the vocal fold stellate
reticular fibers cells contains numerous fine grains of reduced gold, and the
nuclei are stained reddish brown (Fig. 18.8). Vocal fold
stellate cells have lipid droplets in the cytoplasm that contain
vitamin A. Occasionally, a large vacuole can be detected in
the cytoplasm.
Fig. 18.4 (continued) Vitamin A is stored in the lipid droplets of the vocal fold
stellate cells in the macula flava of the human aged vocal
fold.
18.3 Morphological Characteristics The nucleus-cytoplasm ratio is relatively small, but there
of the Human Aged Vocal Fold are fewer intracellular organelles, such as rough e ndoplasmic
Stellate Cells reticulum and Golgi apparatus, than in younger adults. Few
vesicles are present along the periphery of the cytoplasm of
Many vocal fold stellate cells are distributed in the human the vocal fold stellate cells. Some components in the cyto-
aged maculae flavae, and their density is high (Fig. 18.3). plasm and some vocal fold stellate cells are in a state of
Their number and density are the same as in younger degeneration (Fig. 18.9). There are some lysosomes. The
adults (Fig 15.9 in Chap. 15). Basically, the morphologi- nuclei of the vocal fold stellate cells are dense and cleaved
cal characteristics of the aged vocal fold stellate cells are (Fig. 18.6).
the same as those of younger adults. However, there are Accumulations of glycogen particles (glycogen granules)
some geriatric changes. The geriatric changes mentioned are seen in the cytoplasm. In tannic acid-stained material, the
below are recognized in various degrees in the cells in the glycogen particles are electron-dense and approximately
aged maculae flavae. 15–30 nm in diameter (Fig. 18.10). In material stained with
Vocal fold stellate cells are irregular and stellate in shape uranyl acetate and lead citrate, collections of glycogen par-
and possess slender cytoplasmic processes (Figs. 18.5 and ticles present as lucent or faintly stained areas (glycogen
18.6). No basal lamina is found. Lipid droplets are present lakes) within the cytoplasm (Fig. 18.5).
Fig. 18.5 (a) Transmission

electron micrograph of a
a
vocal fold stellate cell in the cytoplasmic
human aged macula flava process
(83-year-old male, uranyl B
acetate and lead citrate stain).
(b) A collection of glycogen
particles presents as a lucent
or faintly stained area
(glycogen lake) stained with
uranyl acetate and lead citrate
within the cytoplasm (region nucleus
B in a) vocal fold
stellate cell
vesicle
rough
endoplasmic
reticula
glycogen lake
nucleus
lipid droplet
cytoplasmic
process nucleus lipofuscin granules
vocal fold stellate cell in the
human aged macula flava
(83-year-old male, uranyl
acetate and lead citrate stain)
18.3 Morphological Characteristics of the Human Aged Vocal Fold Stellate Cells 257
electron micrograph of lipid
membrane-bounded
droplets in a vocal fold lipid droplet
stellate cell in the human aged
macula flava (83-year-old
mitochondrion
male, uranyl acetate and lead
citrate stain)
non-membrane-
bounded lipid
droplet
glycogen lake
vesicle
Fig. 18.8 Light micrograph

of vocal fold stellate cells in
the human aged macula flava
cytoplasmic process
(71-year-old male, gold
chloride method, no
counterstaining, original
×400). Numerous scattered
star-shaped vocal fold stellate
cells in the macula flava are
stained black against the pale
red background, and no
black-stained elements can be
seen in the connective tissue
in the maculae flavae. The
intercellular connective tissue,
such as collagen and elastic vocal fold stellate cell
fibers, has a pale red color
collagen fibers
elastic fiber
degenerated vocal fold stellate
cell in the human aged macula
flava (79-year-old male,
tannic acid stain)
Fig. 18.10 (a) Transmission electron micrograph of a a

vocal fold stellate cell in the human aged macula flava collagen fibers
(79-year-old male, tannic acid stain). (b) Glycogen
particles and lipofuscin granules in the cytoplasm (region
B in a)
elastic fiber
nucleus
cytoplasmic process
b mitochondrion
Glycogen particles
nucleus
lipid droplet
lipofuscin granules
18.4 S
ynthesis of Extracellular Matrices by Reticular fibers, stained black with silver (Fig. 18.4c), can be
the Aged Vocal Fold Stellate Cells detected around vocal fold stellate cells. Newly released
amorphous materials from the vesicles are present on the cell
There are not as many vesicles at the periphery of the cyto- surface of the vocal fold stellate cells (Fig. 18.11).
plasm of the aged vocal fold stellate cells, and very few vesicles Microfibrils 10–15 nm in width are observed around the
can be detected at the periphery of the cytoplasm of degener- amorphous materials (Fig. 18.11). Collagen fibrils are
ated vocal fold stellate cells (Fig. 18.9). Newly released amor- detected near microfibrils (Fig. 18.11). However, the number
phous materials from the vesicles are present on the cell surface of collagen and reticular fibers synthesized by the inactivated
of vocal fold stellate cells, but not as much as in younger adults. cells has decreased.
18.4.1 Collagen Fibers 18.4.2 Elastic Fibers
There are collagen fibers that are stained red with Elastica Elastic fibers, stained black with Elastica van Gieson stain
van Gieson (Fig. 18.4b) and silver stain (Fig. 18.4c) around (Fig. 18.4b), are present around the vocal fold stellate cells in
vocal fold stellate cells in the aged adult maculae flavae. the aged maculae flavae. Newly released amorphous materi-
18.6 Age-Related Changes of the Cells Including Vocal Fold Stellate Cells in the Maculae Flavae and Aging of the Voice 259
als are seen on the cell surface of the vocal fold stellate cells. good vibration for many decades. The renewal of extracel-
Microfibrils 10–15 nm in width are situated around the lular matrices in the vocal folds is believed to occur continu-
amorphous materials (Fig. 18.12). There are microfibril ously to maintain viscoelasticity.
assembles on which elastin appears to be deposited The voice changes with aging. In terms of the musical
(Fig. 18.12). However, the number of elastic fibers synthe- instrument analogy, its strings become old and do not vibrate
sized by the inactivated cells has decreased. well. Actually, the extracellular matrices change in the aged
vocal fold mucosa [1–5]. Age-related qualitative and quantita-
tive changes as well as changes in the three-dimensional struc-
18.4.3 Ground Substance ture of the extracellular matrices influence the viscoelasticity
of the aged vocal fold mucosa. Thus, this change in viscoelas-
Ground substances around the vocal fold stellate cells are ticity would explain one component of aging of the voice.
stained light blue with Alcian Blue at pH 2.5 (Fig. 18.13), The latest research shows human maculae flavae contain-
and relatively sparse staining with Alcian Blue at pH 1.0 is ing vocal fold stellate cells to be involved in the metabolism
noted. The material around the vocal fold stellate cells of extracellular matrices essential for the viscoelasticity of
stained with Alcian Blue (pH 2.5) is digested by hyaluroni- the human vocal fold mucosa and in maintaining the layered
dase. A large amount of the glycosaminoglycan hyaluronan structure of the human vocal fold [6]. The human maculae
(hyaluronic acid) is situated around the vocal fold stellate flavae are considered to be an important structure in the
cells in the aged maculae flavae just as in younger adults. growth, development, and aging of the human vocal fold
The border between dense masses of hyaluronan (macula mucosa [7, 12–15].
flava) and surrounding tissue is clearly delineated. Basically, the morphological characteristics of the aged
vocal fold stellate cells are the same as those of younger
adults. However, there are fewer intracellular organelles than
18.5 The Microenvironment in the younger adults, indicating that the vocal fold stellate
of the Maculae Flavae in the Aged cells in the aged maculae flavae do not constantly synthesize
Human Vocal Fold extracellular matrix protein. Consequently, there are few
vesicles at the periphery of the cytoplasm of the aged vocal
Hyaluronan (hyaluronic acid) concentration in the human fold stellate cells and few newly released amorphous materi-
aged maculae flavae is high. Additionally, most of the cells als, indicating the amount of extracellular matrix proteins
including vocal fold stellate cells in the aged maculae fla- synthesized by these inactivated cells has decreased.
vae express CD44 (a cell surface hyaluronan receptor) Some aged vocal fold stellate cells have degenerated, and
(Fig. 18.14). This indicates that the human aged maculae an accumulation of glycogen particles is seen in the cyto-
flavae are also a hyaluronan-rich pericellular matrix just as plasm. There is an inverse relationship between the meta-
in younger adults [9]. bolic activities of cells and glycogen deposits, and glycogen
Since the cells including vocal fold stellate cells in the tends to accumulate in atrophic cells and in cells that are
human aged maculae flavae have cell surface hyaluronan presumably less active [16]. For instance, accumulation of
receptors and are surrounded by a high concentration of glycogen occurs in cultured human fibroblasts as they
hyaluronan, the aged maculae flavae are also a candidate for approach senescence [17].
a stem cell niche which is a microenvironment nurturing a Many lipofuscin granules are noted around the lipid drop-
pool of tissue stem cells including vocal fold stellate cells. lets in the aged vocal fold stellate cells. Lipofuscin granules are
regarded as residual bodies left behind in the cell after lyso-
somal activity, and they are derived from the degradation of
18.6 A
ge-Related Changes of the Cells endogenous, and not exogenous, material in the lysosome [16].
Including Vocal Fold Stellate Cells The cells including vocal fold stellate cells in the aged
in the Maculae Flavae and Aging macula flava thus appear to have decreased their activity, to
of the Voice have abnormal metabolism, and to have undergone degener-
ation. Age-related changes of the cells in the maculae flavae
Vocal folds are comparable to the strings of a musical instru- most likely influence the metabolism of extracellular matri-
ment. The strings should be changed from time to time ces in the vocal fold mucosa as well as the viscoelasticity of
because they become old and do not vibrate well. However, the aged vocal fold mucosa and are one of the causes of
human vocal folds maintain their viscoelasticity and produce aging of the voice.
Fig. 18.11 Synthesis of

collagen fibers by a vocal fold
stellate cell in the human aged microfibrils
male, tannic acid stain)
amorphous
materials
collagen fibrils
vesicle
vesicle
microfibrils
amorphous
materials elastin
vesicle
Fig. 18.12 Synthesis of
elastic fibers by a vocal fold
stellate cell in the human aged vesicles
male, tannic acid stain)
18.6 Age-Related Changes of the Cells Including Vocal Fold Stellate Cells in the Maculae Flavae and Aging of the Voice 261
Fig. 18.13 Macula flava of

the human aged vocal fold
(82-year-old female, Alcian
Blue stain, pH 2.5). Much
glycosaminoglycan
(hyaluronan, hyaluronic acid)
is situated around the vocal
fold stellate cells in the
human aged maculae flavae
hyaluronic acid
Fig. 18.14 Macula flava of

the human aged vocal fold
(82-year-old female). CD44
(a cell membrane-localized
receptor for hyaluronic acid)
on the cytoplasm of the aged
vocal fold stellate cells is
shown by
immunohistochemical
staining
References 9. Sato K, Umeno H, Nakashima T. Vocal fold stem cells and

2012;121:798–803.
1. Sato K, Hirano M, Nakashima T. Age-related changes of collag-
enous fibers in the vocal fold mucosa. Ann Otol Rhinol Laryngol.
2002;111:15–20.
2. Sato K, Hirano M. Age-related changes of elastic fibers in the
superficial layer of the lamina propria of vocal folds. Ann Otol
Otol. 2016;130:656–61.
3. Hirano M, Kurita S, Sakaguchi S. Ageing of the vibratory tissue of
12. Sato K, Hirano M. Age-related changes of the macula flava of the
human vocal folds. Acta Otolaryngol. 1989;107:428–33.
4. Sato K, Sakaguchi S, Kurita S, Hirano M. A morphological study of
13. Sato K, Hirano M, Nakashima T. Age-related changes in vitamin
aged larynges. Larynx Jpn. 1992;4:84–94.
A-storing stellate cells of human vocal folds. Ann Otol Rhinol
5. Sato K, Sakaguchi S, Hirano M. Histologic investigation of bowing
Laryngol. 2004;113:108–12.
of the aged vocal folds. Larynx Jpn. 1996;8:11–4.
ula flava in the human vocal fold. Part 1. Its role in the adult vocal
1995;104:556–62.
flava in the human vocal fold. Part 2. Its role in the growth and devel-
2001;110:417–24.
16. Ghadially FN. Ultrastructural pathology of the cell and matrix.
London: Butterworths; 1988. p. 962–9.
17. Robbins E, Levine EM, Eagle H. Morphologic changes accom-
panying senescence of cultured human diploid cells. J Exp Med.
1970;131:1211–22.
Comparative Histoanatomy of the
Vocal Fold Mucosa 19
Abstract
1. In mammals, only humans have a layered structure, vocal ligament, and Reinke’s space
of the vocal fold. The layered structure of the human vocal fold is the most suitable for
vocal fold vibration.
2. The absolute values of the length and area ratios of the animal posterior glottis (intercar-
tilaginous portion) are larger than those of the human adult glottis. The animal glottis
appears to be favored for respiration over phonation.
3. The histological structure of the lamina propria of the vocal fold mucosa varies signifi-
cantly among animals.
4. Maculae flavae are also located at anterior and posterior ends of the animal adult vocal
fold. However, the histological structure of the maculae flavae of the vocal fold mucosa
varies significantly among animals. Consequently, their morphological functions are dif-
ferent from those of human maculae flavae. Evolved human maculae flavae contribute to
the development and maintenance of the characteristic layered structure of the human
vocal fold.
5. Any researcher conducting experiments on the vocal fold should know the differences in
vocal fold structure between human beings and animals to be used for experiments, and
histological differences must be taken into account for the experimental results and
conclusions.
19.1 Introduction Human maculae flavae are involved in both the metabolism of
extracellular matrices essential for the viscoelasticity of the
Adult human vocal folds have a layered structure consisting of human vocal fold mucosa and in the maintenance of the lay-
the epithelium; the superficial, intermediate, and deep layers ered structure of the human vocal fold [3]. Human maculae
of the lamina propria; and the vocalis muscle [1]. The vocal flavae are considered to be an important structure in the
ligament, consisting of the intermediate and deep layers of the growth, development, and aging of the human vocal fold
lamina propria, is a transition zone [1]. The layered structure mucosa [4].
is essential for vibration and is required for phonation [1]. Maculae flavae are also present at the anterior and poste-
The various layers of the layered structure of the human rior ends of the membranous portion of animal vocal folds
vocal fold have different mechanical properties [1]. Thus, the [5, 6]. However, the histological structures of the maculae
accepted concept of the layered structure is that the different flavae of the vocal fold mucosa vary significantly among ani-
mechanical properties of each layer of the human vocal fold mals. Additionally, there is no structure equivalent to the
contribute in different ways to the vibratory patterns [1]. The human vocal ligament and layered structure [5, 6]. If the
structure’s ability to act as a vibrating tissue is based on the maculae flavae control the extracellular matrices in places
differences in extracellular matrix distribution. such as the vocal ligament and Reinke’s space of the human
The maculae flavae are located at the anterior and posterior vocal folds, the structure and/or functions of the human mac-
ends of the membranous portion of the human vocal fold [2]. ulae flavae should differ from those of animals.

264 19 Comparative Histoanatomy of the Vocal Fold Mucosa
The use of animal larynges for experimental studies is 19.2 C

anine Vocal Fold: Lamina Propria
commonly performed because they are easier to obtain than of the Vocal Fold Mucosa
human larynges, and the precise timing of the studies can be
controlled. In gross anatomy, the laryngeal structure differs The macroscopic structure of the canine glottis is the most
from animal to animal [5]. In addition, the histological struc- similar to the human glottis [8]. As in humans, the epithelium
tures of the lamina propria of the vocal fold mucosa vary in the canine posterior glottis is a respiratory epithelium (pseu-
significantly among animals [6, 7]. dostratified ciliated epithelium), whereas it is stratified squa-
In mammals, only humans have the layered structure, mous epithelium in the anterior glottis (intermembranous
Reinke’s space, and vocal ligament of the vocal fold. The portion) [8].
layered structure of the human vocal fold is the most suitable In the canine vocal fold mucosa, there is no structure
for vocal fold vibration. Any researcher conducting experi- equivalent to the vocal ligament of the human vocal fold
ments on the vocal fold should know the differences in vocal (Fig. 19.1). The conus elasticus is connected to the superfi-
fold structure between human beings and animals to be used cial layer of the canine vocal fold mucosa (Fig. 19.1b).
for experiments. Histological differences must be taken into The lamina propria of the canine vocal fold mucosa can
account for the experimental results and conclusions. be divided into two layers (Fig. 19.2). The superficial layer is
Comparative histoanatomy of the vocal fold is intriguing dense with collagen and elastic fibers. The deep portion of
from several points of view [6]. It is useful for elucidating the lamina propria is sparse in fibrous components and
the evolution of phonatory functions. Differences tell us appears to be more pliable than the superficial layer. The
more about the mechanism of phonatory functions. They are fibrous components gradually decrease from the superficial
useful in helping researchers determine which animal is layer to the deep layer. The boundary of the two layers is not
most appropriate for experimental studies of the vocal fold. clearly delineated.
laryngeal ventricle
a
a
thyroid cartilage
vocal fold
B
epithelium
conus elasticus
cricoid cartilage
lamina propria
of vocal fold mucosa
b lamina propria b superficial layer

of vocal fold of lamina propria
mucosa
deep layer
of lamina
propria
t hyroarytenoid muscle
conus elasticus
Fig. 19.1 (a) Coronal section of the canine vocal fold (Elastica van
Gieson stain). (b) There is no structure equivalent to the vocal ligament Fig. 19.2 Coronal section of the canine vocal fold mucosa (a, hematoxylin
in the canine vocal fold mucosa (region B in a) and eosin stain; b, Elastica van Gieson stain)
19.3 Canine Vocal Fold: Macula Flava of the Vocal Fold Mucosa 265
19.3 C
anine Vocal Fold: Macula Flava In canine adult vocal fold mucosa, the maculae flavae are
of the Vocal Fold Mucosa vague masses of fibrous tissue (Fig. 19.3). They are tortuous
in shape and approximately 1.5 × 1.5 × 1 mm in size. No
Maculae flavae are located at the anterior and posterior ends vocal ligaments can be detected between the anterior and
of the canine adult vocal folds. The anterior macula flava is posterior maculae flavae.
connected to the thyroid cartilage via an intervening anterior The canine adult maculae flavae are composed of cells
commissure tendon. The posterior macula flava is attached to and extracellular matrices such as collagen fibers, reticular
the vocal process of the arytenoid cartilage posteriorly. fibers, elastic fibers, and ground substance (Fig. 19.4).
a laryngeal ventricle
epiglottic
cartilage
thyroid cartilage a
posterior
macula flava
conus elasticus
cells in macula flava
cricoid cartilage
epithelium
b posterior macula flava
b
elastic fibers
collagen fibers
conus elasticus
Fig. 19.3 (a) Coronal section of the canine posterior macula flava

(Elastica van Gieson stain). (b) Maculae flavae are vague masses of Fig. 19.4 Cells and extracellular matrices in the canine adult maculae
fibrous tissue in canine adult vocal fold mucosa (region B in a) flavae (a, hematoxylin and eosin stain; b, Elastica van Gieson stain)
19.3.1 Interstitial Cells maculae flavae. However, the synthesized collagen and
reticular fibers are less numerous in the canine adult macu-
Numerous cells, which are spindle in shape, can be seen in lae flavae.
the maculae flavae of the canine adult vocal fold mucosa
(Fig. 19.4a). The density of cells in the canine adult macu-
lae flavae is lower than that of the human adult maculae 19.3.3 Elastic Fibers
flavae.
In the canine adult maculae flavae, the cells are spindle Elastic fibers are noted to consist of microfibrils and
or oval in shape and 3 × 5 μm in size, i.e., smaller than the amorphous substances in canine adult maculae flavae
human adult maculae flavae, and the nucleus-cytoplasm (Fig. 19.10). The density of elastic fibers in canine adult
ratio is larger (Fig. 19.5). Intracellular organelles such as maculae flavae is lower than in the human adult maculae
rough endoplasmic reticulum and Golgi apparatus in the flavae (Fig. 19.4b).
cytoplasm, which are less active than in human adult mac- The cells in the canine maculae flavae synthesize elastic
ulae flavae, are poorly developed (Figs. 19.6 and 19.7). fibers (Fig. 19.11). Some vesicles are present along the
Lipid droplets cannot be seen in the cytoplasm of the periphery of the cytoplasm. Newly released amorphous
cells. materials are present on the cell surfaces. Microfibrils can be
seen situated around the amorphous materials. Microfibrils
have assembled together, and elastin appears to have been
19.3.2 Collagen Fibers deposited on them.
The synthesis of elastic fibers in the canine adult maculae
There are collagen and reticular fibers (type III collagen) flavae occurs in the same manner as in the human adult mac-
(Fig. 19.8) around the cells in the canine adult maculae ulae flavae. However, the synthesized elastic fibers are less
flavae. The collagen fibers are composed of numerous col- numerous in the canine adult maculae flavae.
lagen fibrils which are 40–50 nm in width (Fig. 19.6). The
density of collagen fibers in canine adult maculae flavae is
lower than in the human adult maculae flavae (Fig. 19.4b). 19.3.4 Ground Substance
The cells in the canine maculae flavae synthesize collagen
and reticular fibers (Fig. 19.9). Some vesicles are present The canine maculae flavae are stained light blue with Alcian
along the periphery of the cytoplasm. Newly released amor- Blue at pH 2.5 (Fig. 19.12). However, stained canine macu-
phous materials are present on the cell surfaces. Microfibrils lae flavae are tortuous in shape (Fig. 19.12a). Material in the
10–15 nm in width are situated around amorphous material. maculae flavae that is stained with Alcian Blue (pH 2.5) is
The collagen fibrils are near the microfibrils. digested by hyaluronidase. A great deal of glycosaminogly-
The synthesis of collagen and reticular fibers in the can (hyaluronan, hyaluronic acid) is situated around the cells
canine maculae flavae is the same as in the human adult in the canine adult maculae flavae.
collagen fibers
elastic fibers
elastic fiber
nucleus
cell in macula flava

collagen fibers
rough
endoplasmic
reticula
Fig. 19.5 Transmission electron micrograph of the macula flava of the Fig. 19.6 Transmission electron micrograph of the cells in the canine
canine vocal fold (tannic acid stain) macula flava (tannic acid stain)
19.3 Canine Vocal Fold: Macula Flava of the Vocal Fold Mucosa 267
a B amorphous
materials
collagen fibril
nucleus
microfibrils
collagen
fibers
elastic fibers
b Fig. 19.9 Synthesis of collagen fibers in the canine adult macula flava
(uranyl acetate and lead citrate stain)
rough mitochondrion
endoplasmic
reticulum
elastic fiber
microfibrils
microfibrils
nucleus
amorphous
substances
Fig. 19.7 (a) Transmission electron micrograph of the cells in the
canine macula flava (uranyl acetate and lead citrate stain). (b)
Intracellular organelles in the cytoplasm (region B in a)
Fig. 19.10 Transmission electron micrograph of the elastic fibers and

microfibrils in the canine macula flava (uranyl acetate and lead citrate
stain)
reticular fibers microfibrils
elastin
elastin
vesicle
amorphous
materials
Fig. 19.8 Transmission electron micrograph of reticular fibers (type III Fig. 19.11 Synthesis of elastic fibers in the canine adult macula flava
collagen) in the canine macula flava (uranyl acetate and lead citrate stain) (tannic acid stain)
epiglottis
arytenoid

membranous
vocal fold
b posterior anterior
glottis glottis
Fig. 19.13 The structure around the rat adult glottis (from the inside).
The absolute values of the length and area ratios of the rat posterior
hyaluronic acid (hyaluronan) glottis are large
19.5 R
at Vocal Fold: Macula Flava
of the Vocal Fold Mucosa
Fig. 19.12 Macula flava of the canine adult vocal fold (Alcian Blue Maculae flavae are located at anterior and posterior ends of
stain, pH 2.5). (a) Stained canine maculae flavae are tortuous in shape. the rat adult vocal folds. The anterior macula flava is con-
(b) Much glycosaminoglycan (hyaluronan, hyaluronic acid) is situated
around the cells in the canine adult maculae flavae
nected to thyroid cartilage via an intervening anterior com-
missure tendon. The posterior macula flava is attached to
the vocal process of the arytenoid cartilage posteriorly.
In rat adult vocal fold mucosa, the maculae flavae are
vague masses of cells and fibrous tissue (Fig. 19.16) approxi-
19.4 R
at Vocal Fold: Lamina Propria mately 0.2 × 0.2 × 0.25 mm in size. No vocal ligament can be
of the Vocal Fold Mucosa detected between the anterior and posterior maculae flavae.
The rat adult maculae flavae are composed of cells and
The absolute values of the length and area ratios of the rat extracellular matrices such as collagen fibers, elastic fibers,
posterior glottis (intercartilaginous portion) are larger than and ground substance.
those of the human adult glottis (Fig. 19.13). As in humans,
the epithelium in the rat posterior glottis is a respiratory epi-
thelium (pseudostratified ciliated epithelium), whereas it is 19.5.1 Interstitial Cells
stratified squamous epithelium in the anterior glottis
(intermembranous portion) (Fig. 19.14). The rat glottis
Numerous cells can be seen in the maculae flavae of the rat
appears to be favored for respiration over phonation. adult vocal fold mucosa (Fig. 19.16a). The density of cells in
In the rat vocal fold mucosa, there is no structure equiva- the rat adult maculae flavae is relatively high.
lent to the vocal ligament of the human vocal fold (Fig. 19.15). The cells in the rat maculae flavae are stellate or spindle
The lamina propria of the rat vocal fold mucosa is sparse in in shape, have lipid droplets, and store vitamin A in the cyto-
fibrous components. plasm (Fig. 19.17) [9].
19.5 Rat Vocal Fold: Macula Flava of the Vocal Fold Mucosa 269
posterior anterior a
glottis glottis epithelium
arytenoid
cartilage
hypopharynx
thyroid cartilage
Fig. 19.14 Transverse section of the rat vocal fold (hematoxylin and
b
epithelium
eosin stain). The absolute values of the length and area ratios of the rat
posterior glottis are large
epithelium cells in macula flava

a
collagen fibers
elastic fibers
lamina propria of
vocal fold mucosa
fibroblasts
Fig. 19.16 Cells and extracellular matrices in the rat adult posterior
maculae flavae (red encircled area) (a, hematoxylin and eosin stain; b,
Elastica van Gieson stain). The density of cell components in the rat
adult maculae flavae is relatively high. However, the density of fibrous
components is low
b epithelium
vitamin A-storing cells
lamina propria of
collagen fibers
vocal fold mucosa
vitamin A-storing cells
elastic fibers
Fig. 19.17 Vitamin A in the cells in the rat maculae flavae (gold chlo-
ride method, counterstaining with hematoxylin). The cytoplasm of the
Fig. 19.15 Transverse section of the rat vocal fold mucosa (a, hema- cells in the rat maculae flavae contains numerous fine grains of reduced
toxylin and eosin stain; b, Elastica van Gieson stain). There is no struc- gold. The cells in the rat maculae flavae contain vitamin A-containing
ture equivalent to the vocal ligament in the rat vocal fold mucosa lipid droplets
19.5.2 Collagen Fibers

epithelium
There are collagen fibers around the cells in the rat adult
maculae flavae (Fig. 19.16b). The density of collagen fibers
in the rat adult maculae flavae is lower than in the human
adult maculae flavae.

19.5.3 Elastic Fibers
Elastic fibers are noted around the cells in the rat adult macu-
lae flavae (Fig. 19.16b). The density of elastic fibers in the rat hyaluronic acid (hyaluronan)
adult maculae flavae is lower than in the human adult macu-
lae flavae.
19.5.4 Ground Substance Fig. 19.18 Posterior macula flava of the rat adult vocal fold (Alcian
Blue stain, pH 2.5). The stained rat maculae flavae (red encircled area)
The rat maculae flavae are stained light blue with Alcian are round in shape. Much glycosaminoglycan (hyaluronan, hyaluronic
Blue at pH 2.5 (Fig. 19.18). Material in the maculae flavae acid) is situated around the cells in the rat adult maculae flavae
that is stained with Alcian Blue (pH 2.5) is digested by hyal-
uronidase. A great deal of glycosaminoglycan (hyaluronan, 19.7 M
aculae Flavae of the Animal Vocal
hyaluronic acid) is situated around the cells in the rat adult Fold Mucosa
maculae flavae.
Maculae flavae are present at the anterior and posterior ends
of the membranous portion of animal vocal folds [5, 6, 8].
19.6 L
amina Propria of the Animal Vocal However, the histological structure of the maculae flavae
Fold Mucosa varies among animal species. They are composed of cells
and extracellular matrices such as collagen fibers, elastic
The glottis consists of two parts: the intermembranous fibers, and ground substances such as glycosaminoglycan
portion (anterior glottis) and intercartilaginous portion (hyaluronan/hyaluronic acid).
(posterior glottis) [10]. The border between the two parts In canine adult vocal fold mucosa, maculae flavae are
is defined by a line between the tips of the bilateral vocal vague masses of fibrous tissue and tortuous in shape. In rat
processes [2]. adult vocal fold mucosa, maculae flavae are vague masses of
The anterior glottis plays the most important role in pho- cells and fibrous tissue, and cell density is relatively high.
nation. Thus, voice disorders are usually caused by lesions of Human newborn vocal folds have no structure that could
the anterior glottis. The anterior glottis is covered with strati- be considered to be a vocal ligament [1, 2, 11], and the
fied squamous epithelium. On the other hand, the posterior fibrous components are fewer in the maculae flavae than in
glottis appears to have an equally important role in respira- adults [11]. The structure of the lamina propria of the new-
tion and is covered with respiratory epithelium (pseudostrati- born vocal fold is similar to that of the animal adult vocal
fied ciliated epithelium) [10]. fold. And the structure of the newborn maculae flavae is
The absolute values of the length and area ratios of the similar to that of the animal adult maculae flavae. But the cell
animal posterior glottis (intercartilaginous portion) are larger density in the newborn maculae flavae is much greater than
than those of the human adult glottis. Therefore, the animal in animals, and numerous cells start to form extracellular
glottis appears to favor respiration over phonation. matrices such as collagen and elastic fibers [11].
Even though the macroscopic structure of the canine glot- The maculae flavae are composed of cells and extracellular
tis is the most similar to the human glottis, the vocal fold matrices such as collagen fibers, elastic fibers, and ground sub-
structure is different [6]. In the animal vocal fold mucosa, stances such as glycosaminoglycan (hyaluronan/hyaluronic
there is no structure equivalent to the vocal ligament of the acid) in both humans and animals. However, the histological
human vocal fold. In mammals, only humans have the lay- structures of the maculae flavae are different. The human adult
ered structure, vocal ligament, and Reinke’s space of the maculae flavae are composed of dense cells and extracellular
vocal fold. The layered structure of the human vocal fold is matrices. The structure and roles of the maculae flavae of
the most suitable for vocal fold vibration [1]. human vocal fold differ from those of animal vocal folds.
References 271
The most notable difference is the cells in the maculae Maculae flavae are also located at the anterior and posterior
flavae. Vocal fold stellate cells contained in the human adult ends of the animal adult vocal fold. However, their histologi-
maculae flavae were discovered in 2001 [12]. They are stel- cal structure varies significantly from species to species, and
late in shape and possess vitamin A-storing lipid droplets their morphological functions are different from those of
[12, 13]. The vocal fold stellate cells in the maculae flavae human maculae flavae. Evolved human maculae flavae are
form an independent cell category that is considered a new considered to contribute to the development and maintenance
category of cells in the human vocal fold mucosa. Recently, of the characteristic layered structure of the human vocal fold.
there is growing evidence to suggest that the cells including Any researcher conducting experiments on the vocal fold
vocal fold stellate cells in the human maculae flavae are tis- should know the differences in vocal fold structure between
sue stem cells or progenitor cells of the vocal fold mucosa human beings and the animals to be used for experiments.
and that the maculae flavae are a candidate for a stem cell And histological differences must be taken into account for
niche, that is, a microenvironment nurturing a pool of cells the experimental results and conclusions.
including vocal fold stellate cells [14–17].
Human maculae flavae are involved in both the metabo-
lism of extracellular matrices essential for the viscoelasticity References
of the human vocal fold mucosa and in the maintenance of
the layered structure of the human vocal fold [3]. And human 1. Hirano M. Phonosurgery. Basic and clinical investigation. Otologia
maculae flavae are considered to be an important structure in (Fukuoka). 1975;21(Suppl 1):239–45.
the growth, development, and aging of the human vocal fold Diego, CA: Singular Publishing Group; 1993.
mucosa [4]. 3. Sato K, Umeno H, Nakashima T. Functional histology of the mac-
The cells in canine adult maculae flavae are oval and ula flava in the human vocal fold. Part 1. Its role in the adult vocal
spindle in shape, and the nucleus-cytoplasm ratio is large. fold. Folia Phoniatr Logop. 2010;62:178–84.
The density of cells in the canine maculae flavae is lower flava in the human vocal fold. Part 2. Its role in the growth and develop-
than in the human adult maculae flavae. The intracellular ment of the vocal fold. Folia Phoniatr Logop. 2010;62:263–70.
organelles are poorly developed in the cytoplasm, indi- 5. Nagata K. A comparative study of the layer structure of the vocal
cating that the cells do not constantly produce extracel- fold. A morphological investigation of 11 mammalian species.
Otologia (Fukuoka). 1982;28(Suppl 2):699–738.
lular matrices. Consequently, synthesized collagen and 6. Kurita S, Nagata K, Hirano M. Comparative histology of mamma-
elastic fibers are less numerous in the canine maculae lian vocal folds. In: Kirchner JA, editor. Vocal fold histopathology.
flavae. The canine maculae flavae do not appear to pro- A Symposium. San Diego, CA: College Hill Press; 1986. p. 1–10.
duce extracellular matrices in amounts sufficient to 7. Negus VE. The comparative anatomy and physiology of the larynx.
London: William Heinemann Medical Books; 1949.
develop vocal ligaments and the layered structure of the 8. Sato K, Hirano M, Nakashima T. Comparative histology of the
vocal folds [8]. maculae flavae of the vocal folds. Ann Otol Rhinol Laryngol.
The cells in the rat maculae flavae are stellate and spindle 2000;109:136–40.
in shape, have lipid droplets, and store vitamin A in the cyto- 9. Tateya T, Tateya I, Munoz-del-Rio A, Bless DM. Postnatal
development of rat vocal fold. Ann Otol Rhinol Laryngol.
plasm, which are similar to the vocal fold stellate cells in the 2006;115:215–24.
human maculae flavae [9]. However, at the present state of 10. Hirano M, Kurita S, Kiyokawa K, Sato K. Posterior glot-

our investigation, it is difficult to determine whether the cells tis. Morphological study in excised larynges. Ann Otol Rhinol
in the rat maculae flavae are the same category of vocal fold Laryngol. 1986;95:576–81.
stellate cells as in the human maculae flavae. of the human newborn vocal fold. Ann Otol Rhinol Laryngol.
1995;104:556–62.
19.8 U
nique Structure of the Human Vocal fold. Ann Otol Rhinol Laryngol. 2001;110:319–25.
13. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in
Fold Mucosa the human vocal fold. Acta Otolaryngol. 2003;123:106–10.

The absolute values of the length and area ratios of the human their niche in the human vocal fold. Ann Otol Rhinol Laryngol.
anterior glottis (intermembranous portion) are larger than 2012;121:798–803.
those of the animal adult glottis. Therefore, the human glottis H. Origin of vocal fold stellate cells in the human macula flava.
appears to be favored not only for respiration but also for pho- Ann Otol Rhinol Laryngol. 2015;124:698–705.
nation. See Chap. 9, “Histoanatomy of the Human Glottis.” 16. Sato K, Chitose S, Kurita T, Umeno H. Microenvironment of mac-
In mammals, only humans have the layered structure, ula flava in the human vocal folds as a stem cell niche. J Laryngol
Otol. 2016;130:656–61.
vocal ligament, and Reinke’s space of the vocal fold. The 17. Sato K, Chitose T, Kurita T, Umeno H. Cell origin in the mac-
layered structure of the human vocal fold is the most suitable ula flava of the human newborn vocal fold. J Laryngol Otol.
for vocal fold vibration [1]. 2016;130:650–5.
Spaces of the Larynx
20
Abstract
1. The chief laryngeal spaces of the human larynx are the preepiglottic space (PES), the
paraglottic space (PGS), and the cricoid area (CA).
2. The PES, PGS, and CA are a loose connective tissue (areolar tissue) areas composed of
adipose tissue and loose elastic and collagen fibers.
3. The PES exists not only anterior to but also posterolateral and inferolateral to the
epiglottis.
4. The posterior end of the PES is located in the vicinity of the anteroposterior midpoint of
the thyroid lamina.
5. The PGS exists medial to the thyroid lamina.
6. The PES is adjacent to the PGS posteroinferiorly and is separated by fibrous tissue (the
thyroglottic ligament). Posterosuperiorly, the PES and the PGS are not clearly delin-
eated from each other.
7. The CA is located along the superior portion of the cricoid arch on both sides.
8. Cancer invasion into the CA and intravascular tumor invasion facilitate metastasis to the
prelaryngeal, pretracheal, and/or paratracheal regions and facilitate stomal recurrence.
20.1 Introduction 20.2 D

istribution of the Preepiglottic
Space (PES)
A space is defined as any demarcated portion of the body, either
an area of the surface, a segment of tissues, or a cavity [1]. The PES of the human larynx is a loose connective tissue
The chief laryngeal spaces of the human larynx are the (areolar tissue) area composed of adipose tissue and loose
preepiglottic space (PES), the paraglottic space (PGS), elastic and collagen fibers [3].
and the cricoid area (CA). These spaces are very impor- Since Boyer [4, 5] first described the PES as a prelaryngeal
tant clinically and are commonly used terms; however bursa anterior to the thyrohyoid membrane (actually, the PES
they are not included among standard anatomic terms exists posterior to the thyrohyoid membrane), several authors
(Terminologia Anatomica). [6–9] have investigated the PES and have defined its extension.
Distributions of these laryngeal spaces are important in However, a clear indication of its exact location and shape,
following the spread of laryngeal cancer, and their physio- especially its posteroinferior extension, has not been given.

logical and functional significance is also of interest. In addi- Furthermore, there has been no agreement on the border between
tion, cancer invasion into the PES and PGS was one of the the PES and the PGS though distribution of these spaces is impor-
factors affecting TNM classification of malignant tumors tant in following the spread of carcinoma in the larynx.
(UICC, 2009) [2]. There is confusion in the literature regard- Clerf noted that as the lower part of the epiglottis narrows,
ing the definition of these spaces, and there is no agreement the remainder of the posterior boundary of the PES is formed
on their distribution. These spaces are investigated using by an elastic membrane [6]. Tucker and Smith described this
whole-organ serial section technique and computer-aided space as the loose areolar area in front of and alongside the
three-dimensional reconstruction. epiglottis [7]. Norris et al. defined the PES as the midline space

274 20 Spaces of the Larynx
anterior to the epiglottis bounded superiorly by the hyoepiglot- cartilage anteriorly; and by the thyroepiglottic ligament and
tic ligament [8]. Its lateral limits are those fibers that travel epiglottic cartilage posteroinferiorly (Fig. 20.1a) [3].
more vertically from the hyoid to the lateral edge of the epiglot- Transverse sections of the human larynx show that the PES is
tis [8]. All these authors noted that the PES exists not only ante- located anterior, lateral, and posterolateral to the epiglottic carti-
rior to but also lateral to the epiglottis. There is no general lage near the upper edge of the thyroid lamina (Fig. 20.2). The
agreement on the posterior and inferior limits of this space. posterior end of this space is near the anteroposterior midpoint of
Previous reports have indicated that the PES and PGS are con- the thyroid lamina. Condensation of fibrous tissue is observed in
tinuous [7, 9]; however, our investigation using whole-organ serial the midline of the PES. The upper portion of the PGS is situated
section technique with Elastica van Gieson stain revealed that the anterior to the piriform sinus. It is adjacent to the PES, and the two
PES is adjacent to the PGS posteroinferiorly and is separated spaces are not clearly delineated from each other.
from it by fibrous tissue that has been called the thyroglottic liga- At the upper portion of the thyroid cartilage, the PES extends
ment (not included among standard anatomic terms <Terminologia not only anterior to but also posterolateral to the epiglottic carti-
Anatomica>) by Tucker and Smith [7], whereas the two spaces lage (Fig. 20.3). The posterior projection of this space is located
are not clearly delineated from each other posterosuperiorly [3]. in the vicinity of the anteroposterior midpoint of the thyroid
A midsagittal section of the human larynx shows that the lamina. A concentration of fibrous tissue is found around the
PES is a loose connective tissue area and mainly composed of midline of the PES. The upper portion of the PGS is observed
adipose tissue and loose elastic and collagen fibers (Fig. 20.1). anterior to the piriform sinus. The PES and PGS are clearly
The PES is surrounded by the hyoepiglottic ligament superi- delineated from each other by thin fibrous tissue (thyroglottic
orly; by the hyoid bone, thyrohyoid membrane, and thyroid ligament, See Chap. 8, “Compartment of the Human Larynx”).
At the ventricular fold level, the inferolateral portion of the
PES is placed lateral and posterolateral to the thyroepiglottic
a ligament (Fig. 20.4a). This space extends not only anterior to
hyoepiglottic ligament
hyoid bone
thyroid lamina thyroid lamina
preepiglottic space
paraglottic space
paraglottic space
thyrohyoid
membrane
thyroid cartilage thyroepiglottic

ligament
piriform sinus piriform sinus
Fig. 20.2 Transverse section of the human adult larynx near the upper
edge of the thyroid lamina (Elastica van Gieson stain)
elastic fibers
adipose tissue epiglottic cartilage


thyroglottic ligament
paraglottic space
paraglottic space
collagen fibers
Fig. 20.1 (a) Midsagittal section of the human adult larynx (Elastica

van Gieson stain). (b) Higher magnification of the PES. The PES is a
loose connective tissue area and mainly composed of adipose tissue and Fig. 20.3 Transverse section of the human adult larynx at the upper
loose elastic and collagen fibers (Elastica van Gieson stain) portion of the thyroid cartilage (Elastica van Gieson stain)
20.2 Distribution of the Preepiglottic Space (PES) 275
but also inferolateral to the thyroepiglottic ligament. The PGS glottic cartilage at the anteroposterior midpoint of the vocal
exists medial to the thyroid lamina. The PES and PGS are bor- fold (Fig. 20.6a). A concentration of fibrous tissue is
dered by a thick fibrous structure that has been called the thy- observed around the midline of the PES. The PGS is situated
roglottic ligament by Tucker and Smith [7] (Fig. 20.4b).
At the vocal fold level (Fig. 20.5), the PES is no longer thyroarytenoid muscle thyroarytenoid muscle
observed. The PGS is placed medial to the thyroid lamina thyroid lamina thyroid lamina
paraglottic space
along its entire length. paraglottic space
A coronal section of human larynx shows that the PES is
located not only superior to but also inferolateral to the epi-
a thyroepiglottic ligament

thyroarytenoid muscle thyroarytenoid muscle Fig. 20.5 Transverse section of the human adult larynx at the vocal
paraglottic space
B
paraglottic space
fold level (Elastica van Gieson stain)
a epiglottic cartilage

aryepiglottic muscle
b thyroid cartilage
B
15A
preepiglottic preepiglottic
space space
thyroglottic
ligament
paraglottic
space
paraglottic space
12
Fig. 20.4 (a) Transverse section of the human adult larynx at the ven-
tricular fold level (Elastica van Gieson stain). (b) Higher magnification
of the border between PES and PGS (region B in a). The PES is adja- Fig. 20.6 (a) Coronal section of the human adult larynx at the anteropos-
cent to the PGS posteroinferiorly and is separated by fibrous tissue (the terior midpoint of the vocal fold (Elastica van Gieson stain). (b) Higher
thyroglottic ligament) magnification of the border between PES and PGS (region b in a)
medial to the lower two-thirds of the thyroid lamina. The The PES is adjacent to the PGS posteroinferiorly, and they
PES and PGS are clearly bordered by the fibrous structure are separated from each other by the fibrous tissue which has
known as the thyroglottic ligament as named by Tucker and been called the thyroglottic ligament.
Smith [7] (Fig. 20.6b).
20.4 P
20.3 Three-Dimensional Reconstruction of the Preepiglottic Space (PES)
of the Preepiglottic Space (PES)
From the physiologic point of view, the PES bends the epi-
The PES exists around the epiglottis and extends not only glottis posteriorly during swallowing. Fink used the term
anterior to but also posterolateral and inferolateral to the epi- “preepiglottic body” instead of PES [10]. In laryngeal clo-
glottis (Fig. 20.7). Its posterior end is in the vicinity of the sure the preepiglottic body and tubercle are applied to the top
anteroposterior midpoint of the thyroid lamina (Fig. 20.7b). of the adducted ventricular folds and are pressed against
them by approximation of the hyoid bone and thyroid carti-
lage [10].
a
As mentioned above, PES exists astride the epiglottis
[3]. The distribution of the PES allows the epiglottis to
more effectively play the role of retroflexion during swal-
preepiglottic space lowing [3]. In addition, the PES appears to act as a cushion
* *
whose purpose is to protect the epiglottic cartilage from
* * mechanical damage that might otherwise be caused during
* *
swallowing [3].
* *
From the oncological point of view, the histoanatomical
components of the human larynx determine the way cancer
spreads. Additionally, the PES is related to the TNM classifi-
cation of laryngeal cancer (UICC, 2009) [2]. Laryngeal cancer
is classified as T3 when the tumor invades the PES [2].
A tumor has completely invaded the PES in the transverse
section at the ventricular fold level in Figs. 20.8 and 20.9
b shows a larynx with supraglottic carcinoma in coronal sec-
preepiglottic space tions at the anteroposterior midpoint of the vocal fold.
Fibrous tissue (the thyroglottic ligament) blocks the tumor
invasion, and the PGS is intact. This finding supports the
view that the PES and PGS represent separate spaces.
Considering this extent and border of the PES, an invasive
supraglottic laryngeal carcinoma in this space can be resect-
able with a supraglottic horizontal laryngectomy [3]. If the
*
*
* *
preepiglottic space

paraglottic space
tumor paraglottic space
tumor
Fig. 20.7 Reconstructed images of the PES viewed from the anterosu-
perior (a) and the superolateral (b). Red, PES; blue, thyroid cartilage; Fig. 20.8 Transverse section at the ventricular fold of a larynx with
yellow and green, lumen of the larynx and hypopharynx; asterisks, bor- supraglottic laryngeal carcinoma (Elastica van Gieson stain). The thy-
der between PES and PGS roglottic ligament blocks tumor invasion (white arrows)
20.4 Physiological and Clinical Significance of the Preepiglottic Space (PES) 277
Fig. 20.9 Coronal sections at

a preepiglottic space hyoepiglottic ligament
the anteroposterior midpoint
of the vocal fold of a larynx
with supraglottic carcinoma
(a, Elastica van Gieson stain; thyroid cartilage thyroid cartilage
b, hematoxylin and eosin
tumor
stain). The specimen with
Elastica van Gieson stain
shows that the thyroglottic
ligament blocks tumor tumor
invasion (red arrows in a)

tumor
tumor
vocal fold vocal fold
PES and PGS were continuous [7, 9], a supraglottic horizon- sinus and has invaded the upper portion of the PGS
tal laryngectomy would be an unreliable surgical procedure. (Fig. 20.10). Fibrous tissue (the thyroglottic ligament)
A transverse section at the upper portion of the thyroid blocks the tumor invasion, and the PES is intact. This
cartilage of a larynx with hypopharyngeal carcinoma finding also indicates that the PES and PGS are separate
shows that the tumor is mainly located at the piriform spaces.
Fig. 20.10 Transverse thyroepiglottic ligament

section at the upper portion of thyroid cartilage thyroid cartilage
the thyroid cartilage of a preepiglottic space
larynx with hypopharyngeal preepiglottic space
carcinoma (Elastica van
Gieson stain). The thyroglottic ligament
thyroglottic ligament blocks
tumor invasion (red arrows) paraglottic space
tumor
piriform sinus
20.5 D
istribution of the Paraglottic
preepiglottic space
Space (PGS)
thyroid lamina
The PGS exists medial to the thyroid lamina (Figs. 20.3, thyroarytenoid
muscle
20.4, 20.5, 20.6). Laterally, the PGS is surrounded by the paraglottic space
thyroid lamina. Posteriorly, the PGS is surrounded by lateral
cricoarytenoid
the mucosa of the hypopharynx (piriform sinus). muscle
Medially, the PGS is surrounded by the thyroglottic liga- thyroarytenoid muscle
ment, thyroarytenoid muscle, and aryepiglottic muscle paraglottic space
at the supraglottic level (Fig. 20.4a), by the thyroaryte-
noid muscle at the glottic level (Fig. 20.5), and by the
cricothyroid muscle
thyroarytenoid muscle, lateral cricoarytenoid muscle,
conus elasticus
and conus elasticus at the subglottic level (Fig. 20.11).
Inferiorly, the PGS is surrounded by the cricothyroid Fig. 20.11 Coronal section of the human adult larynx (Elastica van
muscle (Fig. 20.11). Gieson stain)
The PGS of the human larynx is a loose connective tissue
(areolar tissue) area composed of adipose tissue and loose
elastic and collagen fibers (Fig. 20.12). Superior laryngeal 20.6 Three-Dimensional Reconstruction
arteries run in this space (Fig. 20.12). of the Paraglottic Space (PGS)
Our investigation using whole-organ serial section
technique with Elastica van Gieson stain revealed that the The PGS is placed on the inside surface of the thyroid lamina
PGS is adjacent to the PES anterosuperiorly and is sepa- (Fig. 20.13). At the supraglottic level, the PGS exists pos-
rated from it by fibrous tissue (thyroglottic ligament) teroinferiorly to the PES and medial to the lamina of the thy-
(Figs. 20.4b and 20.6b), whereas the two spaces are not roid cartilage. At the glottic level, the PGS is present
clearly delineated from each other posterosuperiorly alongside the medial surface of the lamina of the thyroid
(Fig. 20.2) [3]. cartilage.
20.8 Distribution of the Cricoid Area (CA) 279
a
artery
* *
adipose tissue
* *
vein * *
thyroglottic
ligament
paraglottic space
b
Fig. 20.12 Higher magnification of the PGS (region 12 in Fig. 20.6 b)
(Elastica van Gieson stain). The PGS is a loose connective tissue area
and mainly composed of adipose tissue and loose elastic and collagen
fibers
20.7 P
of the Paraglottic Space (PGS)
*
From the physiologic point of view, the PGS is a space for *
the contraction and relaxation of the intrinsic laryngeal mus- *
cles and for the movement of the arytenoid cartilage accom-
panied with adduction and abduction [11]. The PGS is also a
space for the main laryngeal arteries [11]. The PGS does not
interrupt the flow of blood vessels within it (Fig. 20.12) [11].
From the oncological point of view, the spaces of the
human larynx determine the way cancer spreads.
paraglottic space
Additionally, the PGS is related to the TNM classification of
laryngeal cancer (UICC, 2009) [2]. Laryngeal cancer is clas-
Fig. 20.13 Reconstructed images of the PGS viewed from the antero-
sified as T3 when the tumor invades the PGS [2]. superior (a) and the oblique anterosuperior (b). Red, PGS; blue, thyroid
The PGS serves as a pathway leading hypopharyngeal cartilage; yellow and green, lumen of the larynx and hypopharynx;
cancer involving the piriform sinus in an intralaryngeal asterisks, border between PGS and PES
direction (Fig. 20.10). Additionally, the PGS is one of the
routes for prelaryngeal metastasis of laryngeal and hypopha- The CA is one of the laryngeal connective tissue compart-
ryngeal carcinoma (Fig. 20.14), because many blood vessels ments initially described by Pressman et al. [13]. According
are present in the PGS. to the definition by Tucker and Smith, the CA is the region of
areolar tissue medial to the internal perichondrium of the cri-
coid [7]. The CA is bounded by the subglottic area, and it is
20.8 Distribution of the Cricoid Area (CA) fused with the conus elasticus above and the first tracheal
ligament below [7].
The CA of the human larynx is a loose connective tissue The CA is observed to be a triangular area surrounded by
(areolar tissue) area in the subglottis composed of adipose the perichondrium of the cricoid cartilage (cricoid arch), the
tissue and loose elastic and collagen fibers [12]. conus elasticus, and the fibrous layer of the subglottic
mucosa (Fig. 20.15a) [12]. The medial border is a fibrous
a a
C
thyroid lamina
paraglottic space conus elasticus
B
fibrous layer
of the mucosa B
the cricoid cartilage

perichondrium of
tumor
piriform sinus
b
b
cricoid area
conus elasticus
artery thyroid lamina
adipose tissue
vein
vein artery
thyroglottic
ligament
paraglottic space
Fig. 20.15 Higher magnification of the CA (region 15A in Fig. 20.6 a)

Fig. 20.14 (a) Transverse section of a T2N0 hypopharyngeal cancer (Elastica van Gieson stain). (a) The CA is a loose connective tissue area
involving the piriform sinus with a micrometastasis in the PGS. (b) and mainly composed of adipose tissue and loose elastic and collagen
Tumor embolus in the blood vessel (arrow) of the paraglottic space fibers. (b) Blood vessels in the CA (region B in a). (c) Blood vessels
(region B in a) running into the CA from the PGS (region C in a)
tissue for which there is no anatomical term. Tucker used the and on the anteromedial portion of the cricoid cartilage in
fibroglandular layer [14]. Reidenbach used the medial layer transverse sections (Fig. 20.16a).
of the conus elasticus [15]. In this book, the term “fibrous The bilateral anteroinferior portions of the CA are located
layer of the mucosa” is used. near the cricothyroid ligament at the subglottic level
The CA is located on the superomedial portion of the cri- (Fig. 20.16). The bilateral posterosuperior portions of the
coid cartilage in coronal sections (Figs. 20.6a and 20.15a) CA are located near the cricoarytenoid joints (articulations)
20.8 Distribution of the Cricoid Area (CA) 281
c b
paraglottic space blood vessel
conus elasticus
cricoid area
blood fibrous layer
vessel of the mucosa
conus cricoid
elasticus cartilage
blood vessel
conus elasticus
cricoid area
glands
cricothyroid ligament
cricoid area
a
cricoid
area
B (Fig. 20.17) and at the level of the glottis and the upper mar-
gin of the cricoid lamina.
Many blood vessels are present in the CA, and the super-
ficial branch of the cricothyroid artery (the cricothyroid
branch of the superior thyroid artery), which is not included
in standard anatomical reference works (Terminologia
cricoid cartilage Anatomica), runs through it (Fig. 20.15b). Blood vessels in
the PGS pierce the conus elasticus and connect to blood ves-
sels in the CA (Fig. 20.15c). Blood vessels in the CA pene-
trate the anteroinferior portion of the conus elasticus and
extend to the prelaryngeal region (Fig. 20.16).
Fig. 20.16 (a) Transverse section at the subglottis of the human adult
larynx (Elastica van Gieson stain). (b) Blood vessels in the CA pene-
trate the anteroinferior portion of the conus elasticus and extend to the
prelaryngeal region (region B in a). (c) Region C in b
a
a
vocal process of
arytenoid cartilage
crycoarytenoid
B joint
muscular process
of arytenoid
cartilage
lamina of
cricoid cartilage
arytenoid cartilage
epithelium
laryngeal glands
cricoid
area
cricoid cartilage b
Fig. 20.17 (a) Transverse section at the glottis of the human adult lar-
ynx (Elastica van Gieson stain). (b) Posterosuperior portions of the CA
are located near the cricoarytenoid joint (region B in a)
20.9 Three-Dimensional Reconstruction

of the Cricoid Area (CA)
Three-dimensional structural imaging of the CA (Fig. 20.18)

contributes to the understanding of its location, especially
when diagnosis of cancer invasion into the CA is needed.
Each bilateral CA is located along the superomedial por-
tion of the cricoid arch on both sides without connecting at
the midline and distributed in the oblique (anteroinferior to
posterosuperior) direction (Fig. 20.18).
The cricoid area is an anatomical compartment enclosed
by a connective tissue membrane and connected with the Fig. 20.18 Reconstructed images of the CA viewed from the anterosu-
adjacent laryngeal regions by blood vessels. perior (a) and the oblique anterosuperior (b). Red, CA; blue, cricoid
cartilage; yellow and green, lumen of the larynx
20.10 Physiological and Clinical Significance of the Cricoid Area (CA) 283
20.10 P
a
of the Cricoid Area (CA)
From the physiologic point of view, the CA is a flexible

structure that does not disturb the movement of the conus
elasticus, the intrinsic laryngeal muscles (thyroarytenoid and
lateral cricoarytenoid muscles), or arytenoid cartilage during
phonation or respiration [12]. Furthermore the CA does not
interrupt the flow of blood vessels within it [12].
paraglottic space
The anatomical compartment of glandular and areolar tis- tumor
sue below the conus elasticus and within the lumen is corre- thyroid cartilage
lated with the clinical phenomenon described as subglottic

laryngitis or croup. Subglottic swelling occurring in cases of
acute subglottic laryngitis is due to edema of this loose con-
nective tissue below the conus elasticus (CA). See Chap. 8 B
“Compartments of the Human Larynx.”
From the oncological point of view, the CA is related to cricoid cartilage
the growth pattern of laryngeal cancer. The CA is one of the
routes for prelaryngeal, pretracheal, and/or paratracheal
metastasis of laryngeal carcinoma [12]. Many blood vessels,
including the superficial branch of the cricothyroid artery
(the cricothyroid branch of the superior thyroid artery), are b
thyroid cartilage
tumor
present in the CA. These blood vessels penetrate the
anteroinferior portion of the conus elasticus and extend into
the prelaryngeal region (Fig. 20.16). On the basis of histo- conus elasticus
anatomical findings and the investigation of larynges with
laryngeal carcinoma, cancer invasion into the CA and intra- C
vascular tumor invasion facilitate metastasis to the prelaryn- cricoid area
geal, pretracheal, and/or paratracheal regions [12].
cricoid cartilage
The most important pathologic finding is that glottic and
supraglottic carcinomas, without subglottic extension but
with cricoid area invasion (intravascular tumor invasion), can
metastasize to the prelaryngeal, pretracheal, and/or paratra-
cheal lymph nodes [12].
Cancer invasion and/or intravascular tumor invasion into c
the CA are present in most of the cases which develop stomal
recurrences [12].
A coronal section of a T3N0 glottic cancer which devel-
oped stomal recurrence is shown in Fig. 20.19. Cancer has
not invaded either the subglottic region or the deep portion of
the glottis (the PGS). The CA is involved with laryngeal can-
cer, and intravascular cancer invasion is also noted in the CA
(Fig. 20.19).
Cancer invasion and/or intravascular tumor invasion into
the CA are noted in most of the cases with prelaryngeal, pre-
tracheal, and/or paratracheal metastasis [12].
A coronal section of a T3N0 glottic cancer with a micro-
metastasis in the prelaryngeal region is shown on Fig. 20.20. Fig. 20.19 (a) Coronal section of a T3N0 glottic cancer which devel-
Cancer has not invaded either the subglottic region or the oped stomal recurrence. (b) Cricoid area (region B in a). (c) Intravascular
deep portion of the glottis (the PGS). Cancer has invaded the cancer invasion (arrow) (region C in b)
CA, and intravascular cancer invasion is observed

(Fig. 20.20b). A tumor embolus is noted in a vessel in the c
prelaryngeal region (Fig. 20.20c, d).
A schematic illustration of the route of cancer invasion
into the CA is shown on Fig. 20.21. The posterosuperior por-
tion of the CA is located near the glottis and is the portion
most frequently invaded by laryngeal cancer. Glottic cancer
extends in the caudal direction and invades the
CA. Supraglottic carcinoma invades the posterosuperior por- thyroid cartilage
tion of the CA by way of the posterior glottis.
Stomal recurrence after laryngectomy usually indicates
that the patient’s life cannot be saved [16]. Stomal recurrence
occurs as a result of metastasis to the paratracheal lymph thyroid gland D
nodes [16]. The incidence of stomal recurrence is higher in
subglottic carcinomas and in glottic and supraglottic carci- cricoid cartilage
nomas with a subglottic extension [16, 17].
thyroid cartilage d
paraglottic space
thyroid cartilage thyroid gland
tumor
cricoid cartilage
lateral cricoarytenoid Fig. 20.20 (continued)

muscle b
conus elasticus
What constitutes a subglottic extension is not clearly
defined. According to the AJCC (American Joint Committee
on Cancer) cancer staging manual, the glottis occupies a
cricoid area horizontal plane 1 cm in thickness, extending inferiorly from
the lateral margin of the ventricle [18]. The subglottis is the
region extending from the lower boundary of the glottis to
the lower margin of the cricoid cartilage [18]. Kleinsasser
defined glottic carcinoma that extends more than 15 mm
below the free edge of the vocal fold as a subglottic exten-
sion [16]. The Japan Society for Head and Neck Cancer once
cricoid cartilage
defined the immobile portion of the inferior glottis (histo-
logically the portion where the conus elasticus is attached to
the cricoid arch) as the border between the glottis and sub-
glottis [19]. Our investigations are consistent with the crite-
Fig. 20.20 (a, c) Coronal section of a T3N0 glottic cancer with a
micrometastasis in the prelaryngeal region. (b) Cricoid area (region B
ria that the superomedial portion of the cricoid arch, where
in a). Arrow, intravascular cancer invasion. (d) Tumor embolus in the the CA is present, is the border between the glottis and
vessel (arrow) of the prelaryngeal region (region D in c) subglottis.
References 285
3. Sato K, Kurita S, Hirano M. Location of the preepiglottic space and

its relationship to the paraglottic space. Ann Otol Rhinol Laryngol.
1993;102:930–4.
4. Boyer A. Traité complet d’anatomie ou descryiption de toutes les
parties du corps humain. 4th ed. Paris: Chez l'auteur; Migneret;
1815. p. 1797–9.
5. Dobson J. Anatomical eponyms. 2nd ed. Edinburgh: E and S
Livingstone; 1962. p. 33.
6. Clerf LH. The preepiglottic space. Its relation to carcinoma of the
epiglottiss. Arch Otolaryngol. 1944;40:177–9.
7. Tucker GF, Smith HR. A histological demonstration of the develop-
thyroid cartilage ment of laryngeal connective tissue compartments. Trans Am Acad
8. Norris CM, Kuo BF, Tucker GF Jr, Pitser WF. A correlation of
clinical staging, pathological findings and five year end results in
lamina of surgically treated cancer of the larynx. Ann Otol Rhinol Laryngol.
cricoid cartilage 1970;79:1033–48.
arch of 9. Maguire A, Dayal VS. Supraglottic anatomy the pre- or the peri-
cricoid cartilage
epiglottic space? Can J Otolaryngol. 1974;3:432–45.
10. Fink BR. The human larynx. A functional study. New York, NY:
Raven Press; 1975. p. 16–30.
11. Sato K. Three dimensional anatomy of the larynx: investiga-

Fig. 20.21 Schema of the route of cancer invasion into the CA. Shaded tion by whole organ sections. Otologia Fukuoka. 1987;33(supple
area, CA; SGC, supraglottic cancer; GC, glottic cancer 1):153–82.
12. Sato K, Umeno T, Hirano M, Nakashima T. Cricoid area of the
larynx: its physiological and pathological significance. Acta
Otolaryngol. 2002;122:882–6.
Cases of laryngeal carcinoma with a CA extension should
13. Pressman JJ, Simon MB, Monell C. Anatomical studies related
be considered to be at high risk for stomal recurrence [12, to the dissemination of cancer of the larynx. Trans Am Acad
17]. In order to prevent stomal recurrence, it is important to Ophthalmol Otolaryngol. 1960;64:628–38.
dissect the paratracheal soft tissue bilaterally in high risk 14. Tucker GF. Human larynx coronal section atlas. Armed Forced
Institute of Pathology: Washington D.C; 1971.
cases [12, 17].
15. Reidenbach MM. Borders and topographic relations of the cricoid
area. Eur. Arch Otolaryngol. 1997;254:323–5.
16. Kleinsasser O. Tumors of the larynx and hypopharynx. Stuttgart,
References Germany: Georg Thieme Verlag; 1988.
17. Sato K, Kurita S, Matsuoka H, Hirano M. Stomal recurrence follow-
ing total laryngectomy. A clinico-pathological study. J Otolaryngol
1. Stedman’s medical dictionary for the health professions and nurs-
Jpn. 1989;92:1–6.
ing. Illustrated 7th ed. Philadelphia, PA: Lippincott Williams &
18. American Joint Committee on Cancer. AJCC cancer staging man-
Wilkins; 2012. p. 1559.
ual. 7th ed. New York, NY: Springer-Verlag; 2010. p. 57–62.
2. UICC International Union Against Cancer. In: Sobin LH,
19. Japan Society for head and Neck Cancer. General rules for clinical
Gospodarowicz MK, Wittekind Ch, editors. TNM classifica-
and pathological studies on head and neck cancer. Tokyo, Japan:
tion of malignant tumours. 7th ed. Chichester, UK: WILEY-
Kanehara shuppan; 1982. p. 73–7.
BLACKWELL; 2009. p. 39–45.
Blood Vessels of the Larynx
and Vocal Fold 21
Abstract
1. The arteries that supply the human larynx are the superior laryngeal artery, the cricothy-
roid branch of the superior thyroid artery, and the inferior laryngeal artery.
2. The main laryngeal arteries run between the intrinsic laryngeal muscles and thyroid
cartilage and in the paraglottic space. The structure does not interrupt the flow of blood
vessels.
3. The structure of the blood vessels is unique at the vocal fold edge, where only small ves-
sels, including arterioles, venules, and capillaries, are present. The capillaries are distrib-
uted in the superficial layer of the lamina propria (Reinke’s space).
4. The vessels enter the vocal fold edge from the anterior or posterior end of the membra-
nous vocal fold and run essentially parallel to the vocal fold edge.
5. The vascular structures of the human vocal fold that have the capacity to vibrate require
a specific structure suitable for vibration, and these structures minimize hypoxia of the
vocal fold tissue.
6. Vascular structures and their permeability are related to the specific diseases of the
human vocal fold mucosa.
21.1 Introduction 21.2 Blood Supply of the Larynx
The human vocal fold is a vibrating tissue. The portion of the The arteries that supply the human larynx are the superior
vocal fold which vibrates the most during phonation is the laryngeal artery, the cricothyroid branch of the superior thy-
superficial layer of the lamina propria (Reinke’s space) of the roid artery, and the inferior laryngeal artery (Fig. 21.1). The
vocal fold mucosa. Vascular structures of organs which have former two arteries are usually arborized from the superior
the capacity to vibrate require a specific structure suitable for thyroid artery and the latter from the inferior thyroid artery [1].
vibration, and such structures minimize hypoxia of the The superior laryngeal artery enters the larynx through
tissue. the lateral portion of the thyrohyoid membrane. The crico-
From the pathological point of view, vascular structures thyroid branch of the superior thyroid artery enters the lar-
and its permeability are related to the specific diseases of the ynx through the lateral portion of the cricothyroid ligament.
human vocal fold mucosa. The inferior laryngeal artery enters the larynx posteriorly.

288 21 Blood Vessels of the Larynx and Vocal Fold
Fig. 21.1 Arteries of the

SUPERIOR THYROID ARTERY
larynx (Modified from
Pearson, 1975) [1]
Ascending
branch
Ascending
branch Superficial CRICOTHYROID
branch BRANCH
Dorsal
branch
SUPERIOR
LARYNGEAL Deep
Medial
ARTERY branch
branch
Ventral
branch Anterior Medial
division division
Descending
INFERIOR
branch
LARYNGEAL
Posterior ARTERY
division Lateral
division
INFERIOR THYROID ARTERY

Anastomoses
superior laryngeal artery

superior thyroid artery epiglottis
ascending branch
hyoid bone
dorsal branch thyroid cartilage

descending branch ventral branch
descending branch
descending branch (anterior division)
(posterior division)
superior thyroid artery,
inferior laryngeal artery cricothyroid branch
cricoid cartilage
thyroid gland
Fig. 21.2 Right selective

superior thyroid angiogram
(lateral view)
21.4 Vascular Network of the Human Vocal Fold 289
Fig. 21.3 Schema of arteries

in the human larynx. PES
preepiglottic space, EC
epiglottic cartilage, STN
superior thyroid notch, ACT
anterior commissure tendon,
VP vocal process of arytenoid
cartilage, MP muscular
process of arytenoid cartilage,
VF vocal fold, FF false vocal
fold (ventricular fold), TAM
thyroarytenoid muscle, LCAM
lateral cricoarytenoid muscle,
PCAM posterior
cricoarytenoid muscle, AM
arytenoid muscle, PS piriform
sinus of hypopharynx, C
corniculate cartilage
These three arteries make direct anastomoses with each other 21.3 P
hysiologic Significance of the Blood
(Figs. 21.1 and 21.2). Supply of the Larynx
There is a relationship between arteries and spaces in the
human larynx. The preepiglottic space is located anterior and The main laryngeal arteries run between the intrinsic laryngeal
inferolateral to the thyroepiglottic ligament and epiglottic car- muscles and thyroid cartilage and in the paraglottic space [2, 3].
tilage, and the paraglottic space is posteroinferiorly placed on The structure does not interrupt the flow of blood vessels [2].
the inside surface of the thyroid lamina (Fig. 21.3). The main
laryngeal arteries run between the intrinsic laryngeal muscles
and thyroid cartilage in the paraglottic space (Fig. 21.4). 21.4 V
ascular Network of the Human
At the supraglottic level, the descending branch of the Vocal Fold
superior laryngeal artery is located in the posterior part of the
paraglottic space (Fig. 21.4a). The ventral branch that branches The structure of the blood vessels is unique at the vocal
out of the descending branch is placed in the anterior portion fold edge, where only small vessels, including arterioles,
of the paraglottic space (Fig. 21.4a). At the glottic level, the venules, and capillaries, are present [4, 5]. The capillaries
descending branch of the superior laryngeal artery is divided are distributed in the superficial layer of the lamina pro-
into the anterior and posterior divisions, both located in the pria (Reinke’s space) of the vocal fold mucosa. The arte-
paraglottic space (Fig. 21.4a). The anterior division of the rioles and venules are distributed in the intermediate and
descending branch of the superior laryngeal artery anastomo- deep layer of the lamina propria (vocal ligament) of the
ses with the cricothyroid branch of the superior thyroid artery vocal fold mucosa.
in the anterior portion of the paraglottic space. The posterior The vessels enter the vocal fold edge from the anterior or
division of the descending branch of the superior laryngeal posterior end of the membranous vocal fold and run essen-
artery anastomoses with the inferior laryngeal artery in the tially parallel to the vocal fold edge (Figs. 21.5, 21.6, 21.7,
posterior portion of the paraglottic space. 21.8). Away from the edge of the vocal fold, vessels increase
Fig. 21.4 Schema of arteries

in the human larynx. (a)
transverse sections; (b)
coronal sections. SLAd
descending branch of superior
laryngeal artery, SLAv ventral
branch of superior laryngeal
artery, SLAda descending
branch (anterior division) of
superior laryngeal artery,
SLAdp descending branch
(posterior division) of
superior laryngeal artery, ILA
inferior laryngeal artery, CTA
superior thyroid artery, CTAa
ascending branch of
superior thyroid artery, CTAd
deep branch of cricothyroid
branch of superior thyroid
artery, CTAs superficial
branch of cricothyroid branch
of superior thyroid artery, PS
piriform sinus of a
hypopharynx, TGL
thyroglottic ligament, LV
laryngeal ventricle, CA
cricoid area, CTL cricothyroid
ligament, CTM cricothyroid
muscle, PES preepiglottic
space, PGS paraglottic space,
TAM thyroarytenoid muscle,
LCAM lateral cricoarytenoid
muscle, PCAM posterior
cricoarytenoid muscle, VP
vocal process of arytenoid
cartilage, MP muscular
process of arytenoid cartilage
in number, and large vessels run in various directions at the blood vessels in the lamina propria of the mucosa around the
superior and inferior portions of the vocal fold (Figs. 21.5 vocal fold edge are clearly separated from and do not net-
and 21.6). The blood vessels in the lamina propria of the work with those in the vocalis muscle (Figs. 21.8 and 21.9).
mucosa around the vocal fold edge are clearly separated At the midpoint of the vocal fold, especially at the lower
from and do not network with those in the superior and infe- surface of the vocal fold, there is a reticulated vascular net-
rior surfaces of the vocal fold. work [5]. Direct anastomosis between the arterioles and
In the muscle layer of the vocal fold, the blood vessels venules is observed sporadically [5].
enter from the deep portion of the vocal fold (Fig. 21.9). The
21.5 Microstructure of the Blood Vessels in the Human Vocal Fold Mucosa 291
edge of the vocal fold
anterior
capillary
posterior fiber
fiber
inferior
capillaries
Fig. 21.5 Medial aspect of the human vocal fold (silicone rubber com-
pound injection and clearing technique) (Photograph courtesy of Dr.
Shigejiro Kurita, from the Department of Otolaryngology-Head and
Neck Surgery, Kurume University). Around the vocal fold edge, the
blood vessels are small and run roughly parallel to the edge. The vessels
around the vocal fold edge come from the anterior and the posterior end
of the membranous vocal fold. Large vessels run in various directions at
the inferior portion of the vocal fold capillary
collagenous fiber
anterior
elastic fiber
capillaries
edge of the
vocal fold
lateral
Fig. 21.7 Coronal section of the superficial layer of the lamina propria
(Reinke’s space) of the human vocal fold. Capillaries in the superficial
layer of the lamina propria (Reinke’s space) of the vocal fold are round
or oval in shape, indicating that they run roughly parallel to the vocal
fold edge. (a) hematoxylin and eosin stain; (b) Elastica van Gieson
stain
21.5 M
icrostructure of the Blood Vessels
in the Human Vocal Fold Mucosa
posterior
There are only small vessels, including arterioles (Figs. 21.10
Fig. 21.6 Superior aspect of the human vocal fold (silicone rubber and 21.11), capillaries (Figs. 21.12 and 21.13), and venules
compound injection and clearing technique) (Photograph courtesy of (Fig. 21.14), in the mucosa of the vocal fold edge, running
Dr. Shigejiro Kurita, from the Department of Otolaryngology-Head and roughly parallel to the vocal fold edge. The main blood ves-
Neck Surgery, Kurume University). Around the vocal fold edge, the
blood vessels are small and run roughly parallel to the edge. The vessels sels in the superficial layer of the lamina propria (Reinke’s
around the vocal fold edge come from the anterior and the posterior end space) of the vocal fold mucosa are the capillaries.
of the membranous vocal fold. There are large vessels that run in vari-
ous directions at the lateral portion of the vocal fold

a superficial layer of
the human vocal fold (silicone superior
rubber compound injection the lamina propria
and clearing technique) (Reinke’s space) of
(Photograph courtesy of Dr. the vocal fold
Shigejiro Kurita, from the
Department of
Neck Surgery, Kurume
University). Blood vessels in
the superficial layer of the muscle layer of
lamina propria (Reinke’s the vocal fold
space) of the vocal fold are
very small (a). Most of them
are round, oval, or rodlike in
shape, indicating that they run
roughly parallel to the vocal
fold edge (b). The vessels in
the mucosa near the vocal
fold edge are clearly
separated from those in the
vocalis muscle (a, b)
inferior
superficial layer of
the lamina propria
vocalis muscle
21.5.1 Arterioles The terminal arterioles pass through a short transitional

region in which scattered smooth muscle cells persist around
Generally, arterioles range in diameter from 300 μm the blood vessel (Fig. 21.11). Arterial capillaries, the area of
down to less than 50 μm [6]; however, the arterioles in transition from an arteriole to a capillary, are intermediate in
the vocal fold mucosa are relatively thin (Fig. 21.10). form between smooth muscle cells and pericytes (Fig. 21.11).
Smooth muscle cells completely encircle the blood ves- The vessels then continue as capillaries.
sels (Fig. 21.10).

the midpoint of a human vocal fold edge vocal fold edge
vocal fold (Elastica van
Gieson stain and Softex
contact microangiogram)
(Photograph, Softex contact
microangiogram, courtesy of
Dr. Shigejiro Kurita, from the
Department of
Neck Surgery, Kurume
University). The blood vessels
in the mucosa near the edge
of the vocal fold are clearly vocalis muscle
separated from those in the
mucosa on the upper and
lower surfaces of the vocal
fold as well as from those in
the vocalis muscle
smooth muscle cells

smooth muscle
cell
arteriole blood vessel
Fig. 21.11 Scanning electron micrograph of the transition area from

Fig. 21.10 Scanning electron micrograph of arteriole in the vocal fold
arteriole to capillary (arterial capillaries) (Modified NaOH maceration
mucosa (Modified sodium hydroxide (NaOH) maceration method)
method)
pe
ric
capillaries yte
cytoplasmic
cell body process
capillary
pe
ric
y
te
pericytes
Fig. 21.12 Scanning electron micrograph of capillaries in the human Fig. 21.13 Scanning electron micrograph of capillary and pericytes in
vocal fold mucosa (Modified NaOH maceration method) the human vocal fold mucosa (Modified NaOH maceration method)
Fig. 21.14 Scanning
electron micrograph of the
transition area from capillary
to venule (venous capillaries)
(Modified NaOH maceration
method)
pericyte smooth muscle cell
21.5.2 Venules appear around the blood vessel (Fig. 21.14). Venous capil-
laries, the area of transition from a capillary to a venule, are
Several capillaries unite and form a venule, which is a cylin- intermediate in form between pericytes and smooth muscle
drical blood vessel 15–20 μm in diameter [6]. cells (Fig. 21.14). The blood vessels then continue as
The terminal capillaries pass through a short transitional venules.
region in which scattered smooth muscle cells begin to
21.5.3 Capillaries a stratified squamous epithelium
The capillary wall consists of a layer of endothelial cells,

basal laminae, and a sparse network of reticular fibers
(Fig. 21.15). The diameter of capillaries in the human
vocal fold mucosa averages about 8–12 μm, which per-
mits unimpeded passage of the cellular elements of the
basement
blood. The luminal surface of the endothelium is gener- membrane
ally smooth, but the thin margins of the adjacent cells
overlap slightly and a thin marginal fold projects a short
distance into the lumen (Fig. 21.15c). Some endothelial
capillary
cells of the capillaries are interrupted by circular fenestra-
tions or pores [7], 60–70 nm in diameter, each closed by a
very thin pore diaphragm.
Many pericytes can be seen around the capillaries in the
human vocal fold mucosa (Figs. 21.12, 21.13 and 21.15) [8]. B
Each pericyte has a cell body and branching cytoplasmic
processes (Fig. 21.13). The cell bodies are bulged fusiform b
or polygonal. Branching cytoplasmic processes consist of endothelial cell
short circumferential processes and long and relatively thick
longitudinal processes that are parallel to the axis of the ves-
sel. The pericytes are 5–10 μm by 15–30 μm, and cell bodies
are 5–10 μm by 10–15 μm in size. The cell bodies of the
pericytes attach to capillary endothelial cells (Fig. 21.16), cell body
erythrocyte
and the branching processes attach to the capillary endothe-
lial cells at the tips (Fig. 21.16b) and encircle the capillaries capillary nucleus
(Fig. 21.15b). The processes of the pericytes are fingerlike or
clawlike in appearance and, just like the cell bodies, appear
to grasp the vessels. cytoplasmic
Components in the cytoplasm including rough endoplas- C process pericyte
mic reticulum, mitochondria, and free ribosomes are present.
Many cytoplasmic filaments can be seen, not only in the cell c
bodies but in the processes as well (Fig. 21.17). The fila- erythrocyte
ments come together to form dense bodies (Fig. 21.17).
Adjacent to the endothelial cells, a dense meshwork of
cytoplasmic filaments in the processes of pericytes is noted endothelial junction
(Fig. 21.18c). On the outside of the processes, many pinocy- marginal fold
totic vesicles are observed in the cytoplasm (Fig. 21.18c). vesicles
Cell bodies and processes of pericytes encircle the capillaries
and attach to their walls. The cell bodies of pericytes and
endothelial cells remain separated by a gap of 300–500 nm
(Figs. 21.15b and 21.18a). The processes of pericytes are in basal lamina
close contact with endothelial cells, sharing a common base- pericyte
ment membrane with them (Fig. 21.18c). The tips of the pro- reticular fibers
cesses form intercellular tight junctions with endothelial
cells (Fig. 21.18c). These regions correspond to the scanning
electron micrograph (SEM) in Fig. 21.16b. Fig. 21.15 Transmission electron micrographic cross section of capil-
lary in the superficial layer of the lamina propria (Reinke’s space) of the
The cell bodies and processes of pericytes encircle the human vocal fold (uranyl acetate and lead citrate stain). A single endo-
capillaries in the newborn vocal fold mucosa (Fig. 21.19). At thelial cell extends all around the lumen and the pericyte encircles the
birth, the same microstructure of capillaries in the vocal fold capillary (b, region B in a; c, region C in b)
mucosa as in the adult vocal fold mucosa is present.
dense body
pericyte
cell body
B
cytoplasmic filaments
cytoplasmic
processes
endothelial cell
capillary Fig. 21.17 Transmission electron micrograph of the cytoplasm of a
pericyte (uranyl acetate and lead citrate stain). Many cytoplasmic fila-
ments come together to form dense bodies
endothelial
cell
cytoplasmic
processes capillary
capillary pericyte
cell body
B cytoplasmic
process
Fig. 21.16 Scanning electron micrograph of a pericyte around the cap-

illary in the human vocal fold mucosa (Modified NaOH maceration Fig. 21.18 Transmission electron micrograph of an endothelial cell
method) (b: region B in a) and pericyte. (b, region B in a; c, region C in b) (uranyl acetate and lead
citrate stain). The processes of pericytes are in close contact with endo-
thelial cells (a, b), sharing a common basement membrane with them,
and the tips of the processes form intercellular tight junctions with
21.6 Physiologic Significance endothelial cells (c)
of the Vascular Network in the Human
Vocal Fold Mucosa
blood vessels in the vocalis muscle. The structure of the vas-
Only small blood vessels enter the vocal fold edge from the cular network in the mucosa of the vocal fold edge is well
anterior or posterior end of the membranous vocal fold and suited for vibration [3–5]. The structure of the vascular net-
run essentially parallel to the vocal fold edge. And these work in the mucosa of the vocal fold edge is also well suited
small blood vessels are clearly differentiated from blood ves- to prevent circulatory disturbance caused by vocal fold
sels in the upper and lower vocal fold mucosa as well as from vibration.
21.7 Physiologic Significance of Pericytes of Capillaries in the Human Vocal Fold Mucosa 297
b endothelial There is a direct anastomosis between the arterioles and

cell venules in the vocal fold mucosa. When the arteriovenous
anastomosis is contracted, blood passes along the arteriole
into the capillary network. When it relaxes, blood can bypass
the capillaries and go directly into a venule. The arteriove-
nous anastomoses are therefore considered important struc-
tures for regulating the supply of blood to the vocal fold
cytoplasmic mucosa.
process of The vascular structures of the human vocal fold mucosa
pericyte
that have the capacity to vibrate have a specific structure suit-
C able for vibration and also minimize hypoxia of the vocal
fold tissue.
The arterioles form an important segment of the circula-
tion, because they constitute the principle component of the
peripheral resistance to flow that regulates blood pressure
c [6]. The exchange between the blood and the tissue takes
endothelial cell place in the capillaries [6]. The venules also have a role in
the exchange between the blood and the tissue, and they are
particularly important in the changes associated with
shared basement membrane tight junction inflammation [6].
21.7 P
hysiologic Significance of Pericytes
cytoplasmic
process of of Capillaries in the Human Vocal Fold
pericyte Mucosa
cytoplasmic filaments
tight junction
Zimmermann studied the capillary pericytes using light
microscopy with silver staining [9]. Electron microscopic
pinocytotic vesicle studies have been conducted on capillary pericytes in other
organs [10–15]. The number and shape of capillary pericytes
differ according to the organs and tissue [10–15]. The num-
ber of capillary pericytes is related to the density of the capil-
lary bed [14]. Their shape and distribution are related to
organ function.
pericyte The functions of pericytes remain unclear. Synthesis,
endothelial cell mechanical support, protection, detection, differentiation,
and capillary contraction have been suggested [15].
Cytoplasmic filaments were previously noted in pericytes,
and thus they are considered contractile cells that modulate
microvascular blood flow [11, 12]. Pericytes are critical cells
pericyte in vascular biology, especially angiogenesis. They intervene
at different levels of blood vessel formation, being involved
in endothelial cell stimulation and guidance as well as endo-
thelial stabilization and maturation [16].
capillary Pericytes have been previously noted around capillaries in
the vocal fold mucosa [7, 8, 17]. From the morphological
point of view, the pericytes in the human vocal fold mucosa
are essentially the same as those in other organs [8]. Many
pericytes can be seen around capillaries, arterial capillaries,
Fig. 21.19 Transmission electron micrographic cross section of a cap- and venous capillaries in the human vocal fold mucosa [8].
illary in the superficial layer of the lamina propria of the newborn vocal
fold (uranyl acetate and lead citrate stain). A single endothelial cell
The most noteworthy finding concerning the pericytes in the
extends all around the lumen and the pericytes encircle the capillary at human vocal fold mucosa is the presence of processes thicker
birth than those in other organs [8].
Pericytes in the human vocal fold mucosa encircle the cap- space. The most frequent etiologic factors of Reinke’s edema
illary walls. The tips of the processes form tight intercellular are considered smoking and aging. The mechanism for the
junctions with endothelial cells. The cell bodies and processes onset and development of the disease remains unclear.
appear to grasp the vessels and support and protect capillary During endolaryngeal microsurgery for Reinke’s edema,
walls [8]. It is this thickness and firm connection with endo- subepithelial vascularization is seen in the vocal fold mucosa
thelial cells which render them particularly suitable for such (Fig. 21.20). The blood vessels are not parallel to the edge of the
support and protection [8]. Many cytoplasmic filaments can be vocal fold, but run in random directions and are particularly
seen to come together to form dense bodies. The pericytes thus conspicuous in severe cases (Fig. 21.20). Edema in Reinke’s
provide great support and protection for the capillary walls space appears to be related to blood vessels in this space.
[8]. As a result, the vessels in the human vocal fold mucosa, Blood vessels in the superficial layer of the lamina propria
which is the vibrating portion of the vocal folds, do not rupture (Reinke’s space) of the human vocal fold mucosa with
easily, even during frequent and strong vibrations. Reinke’s edema are shown in Fig. 21.21. Subepithelial vas-
The blood flow of the vocal fold mucosa is reduced dur- cularization is evident in Reinke’s space [19]. Blood vessels
ing phonation [18], but increases thereafter. The pericytes in are dilated to 20–30 μm in diameter, but capillary diameter
the vocal fold mucosa appear to provide mechanical support varies considerably. The blood vessel walls are thin, as is the
and protection to the capillary walls, particularly during pho- cytoplasm of the endothelial cells (Fig. 21.21). The cytoplasm
nation [7]. The pericytes also appear to regulate the diameter (cell body and branching cytoplasmic processes) of the peri-
of the capillary during and after phonation. cytes is thin (Fig. 21.21). The cell bodies and branching
Pericytes in the human vocal fold mucosa are also thought
to be critical cells in vascular biology and angiogenesis, espe-
cially revascularization following vocal fold tissue injury. a pericyte
The pericytes have already encircled the capillaries in the
newborn vocal fold mucosa. The pericytes appear ready to pro-
vide support and protection of the blood vessels after birth [7].
erythrocyte
21.8 M
icrostructure of the Blood Vessels
in the Human Vocal Fold Mucosa
with Reinke’s Edema
Reinke’s edema is a common disease of the vocal fold ulti-

mately causing changes in voice quality. The entire length of the cytoplasmic
membranous vocal fold is edematous and swollen (Fig. 21.20). process
Histopathologically, the primary feature is edema in Reinke’s capillary
endothelial cell
b
delated blood vessels endothelial cell
in the vocal fold mucosa
capillary
erythrocyte
thickened
basement membrane
cytoplasmic process
of pericyte
Fig. 21.21 Transmission electron micrograph of Reinke’s space of the

vocal fold mucosa with Reinke’s edema (uranyl acetate and lead citrate
stain). The blood vessels are dilated (a) and the blood vessel walls are
Fig. 21.20 Endolaryngeal microscopic view of Reinke’s edema thin (b), as are the endothelial cells and pericytes
21.8 Microstructure of the Blood Vessels in the Human Vocal Fold Mucosa with Reinke’s Edema 299
capillary p rocesses are not attached to the endothelial cells of the ves-
plasmalemmal vesicle
sels (Fig. 21.21). The number of pericytes has decreased.
endothelial cell The pericytes are situated away from the endothelial cells
and share the thickened basement membrane with them
vesicles
(Fig. 21.21). The pericytes appear not to adequately support
and protect the capillary walls, particularly during phona-
tion. Thus, the blood vessels affected by Reinke’s edema are
plasmalemmal vesicle fragile, and this fragility most likely affects the blood circu-
lation in the vocal fold mucosa.
The exchange between blood and tissue takes place in
the capillaries [6]. Many vesicles are present in the cyto-
reticular fibers plasm of the endothelial cells of the vessels (Fig. 21.22). A
conspicuous feature of endothelial cells is the presence of a
large number of vesicles associated with the plasmalemma
basement membrane
on both surfaces of the cell. The endothelial cells possess
many fenestrae or pores (Fig. 21.23a), and plasma exudes
Fig. 21.22 Vesicular transport of endothelial cells in Reinke’s edema from the capillaries into surrounding tissue via the fenes-
(uranyl acetate and lead citrate stain). In addition to the translocation of trae (Fig. 21.23b). A conspicuous feature of fenestrae or
vesicles from one surface of the endothelium to the other, a vesicle opens pores is that they do not have pore diaphragms (Fig. 21.23b).
at adluminal and abluminal surfaces of the cell (plasmalemmal vesicle)
This accounts for the fact that fluid transverses the wall of
capillaries more rapidly. There are intercellular gaps
a
between endothelial cells of the blood vessels (Fig. 21.24).
capillary A thickened basement membrane and dense reticular fibers
fenestra
are noted around the vessels (Figs. 21.21 and 21.22).
Capillary permeability thus appears to be increased with
Reinke’s edema [19].
The endothelial cells and pericytes of some vessels are
fenestra
reticular fibers degenerated, and partial or complete blood vessel occlusion
is seen in some cases (Figs. 21.25 and 21.26).
basement membrane Immunohistochemical findings of vascular endothelial
growth factor (VEGF) in the superficial layer of the lamina
propria (Reinke’s space) of the vocal fold mucosa with
Reinke’s edema are shown in Fig. 21.27. The interstitial cells
(Fig. 21.27a) and/or inflammatory cells (Fig. 21.27b) in the
superficial layer of the lamina propria (Reinke’s space) show
cytoplasm staining with VEGF, while no staining of this
b
endothelial cell
endothelial cell
capillary capillary
intercellular gap
fenestra
erythrocyte
Fig. 21.23 The fenestrae of endothelial cells in Reinke’s space with

Reinke’s edema (uranyl acetate and lead citrate stain). (a) The endothe-
lial cells possess many fenestrae or pores with pore diaphragms. (b)
Plasma exudes from the capillaries into surrounding tissue via the Fig. 21.24 The intercellular gaps between endothelial cells of the
fenestra (pore) lacking a pore diaphragm blood vessels (uranyl acetate and lead citrate stain)
endothelial cell a
pericyte
Reinke’s space
interstitial cells
erythrocyte
b
Reinke’s space
Fig. 21.25 Degenerated endothelial cells and pericytes along with
partial blood vessel occlusion (uranyl acetate and lead citrate stain)
inflammatory cells
complete blood vessel occlusion
Fig. 21.27 Vascular endothelial growth factor (VEGF), shown by

immunohistochemical staining (original ×400). (a) VEGF staining in
the cytoplasm of interstitial cells in Reinke’s space. (b) VEGF staining
in the cytoplasm of inflammatory cells in Reinke’s space
The most frequent etiologic factors of Reinke’s edema are

considered to be smoking and aging. Another possible
Fig. 21.26 Degenerated endothelial cells and pericytes along with etiologic factor is vocal abuse. Smoking causes the blood
complete blood vessel occlusion (uranyl acetate and lead citrate stain) flow through the blood vessels to decrease. It increases plate-
let aggregation [20] and changes blood lipid and lipoprotein
concentrations [21]. It also increases carboxyhemoglobin
growth factor is found in any component of the lamina pro- [22], with a consequent reduction in the ability of hemoglo-
pria of normal vocal folds. bin to deliver oxygen to tissue. Smoking enhances the
VEGF is implicated in the control of angiogenesis due to possibility of thrombosis in the vocal fold mucosa. These
its selective mitogenic stimulation of vascular endothelial deleterious effects of smoking lead to hypoxia and ischemia
cells and enhancement of vascular permeability. The lamina and affect the blood circulation in the vocal fold mucosa.
propria of the normal vocal fold mucosa shows no staining of Age-related changes in the blood vessels increase the possi-
VEGF. Subepithelial vascularization, dilatation of the blood bility of thrombosis and decrease blood flow through blood
vessels, and increased permeability of the blood vessels are vessels, thus also leading to hypoxia and ischemia.
observed in Reinke’s edema [19]. VEGF produced by inter- Even in normal vocal folds, the blood flow of the vocal fold
stitial cells and/or inflammatory cells in Reinke’s space mucosa is reduced during phonation [18]. Fragile blood vessels
likely promotes vascularization and an increase in blood ves- not parallel to the edge of the vocal fold, but running in random
sel permeability [19]. directions in Reinke’s space, are easily injured and collapse
21.11 Microvascular Lesions of the Vocal Fold 301
d uring phonation [19]. In addition, the vibrating patterns of the 21.9.3 J unctional Tranport (Intercellular
edematous vocal folds with Reinke’s edema change bringing an Transport)
additional adverse effect to the fragile vessels [19].
These etiologic factors bring about hypoxia and ischemia Tight junctions between the endothelial cells are released
of the vocal fold mucosa. Hypoxia in vitro and ischemia creating intercellular gaps between the endothelial cells of
in vivo increase VEGF mRNA in normal tissues and certain the blood vessels (Fig. 21.24). Molecules pass through these
human tumors [23]. These disorders may likely increase discontinuities in the intercellular junctions.
VEGF in Reinke’s space, with possibly greater subepithelial
vascularization and capillary permeability as well [19].
Thus, fragility of and alteration in the permeability of the 21.10 Hemorrhage in Reinke’s Space
vessels are presumed to cause edema of the superficial layer
of the lamina propria (Reinke’s space), which likely pro- The frequent violent slapping of the vocal folds against each
gresses to Reinke’s edema [19]. other caused by overuse or abuse of the voice results in exu-
dation of inflammatory blood products into Reinke’s space.
Therefore, vocal fold polyps usually occur at the midpoint of
21.9 Transendothelial Exchange the membranous vocal fold, which vibrates the most during
and Permiability of the Capillaries phonation (Fig. 21.28).
Trauma of the vessels results in exudation from ruptured
The capillaries are the principle site of the exchange of sub- blood vessels. The histopathological appearances of these
stances between blood plasma and tissue fluid. As mentioned lesions show combinations of the exudation (plasma, eryth-
above, transendothelial exchange and permeability of the cap- rocytes, etc.) and interstitial cell and extracellular matrix
illaries in the superficial layer of the lamina propria (Reinke’s reactions (Fig. 21.29).
space) of the mucosa affect physiological and pathological
conditions of the vocal fold. There are three possible transport
systems of the capillary wall in the human vocal fold mucosa. 21.11 Microvascular Lesions
of the Vocal Fold
21.9.1 Fenestra Transport Microvascular lesions, also called varices or capillary ecta-
sias, are relatively small lesions arising from the microcircu-
The capillaries in the shallow portion of the superficial layer lation of the vocal fold (Fig. 21.30) [24]. Microvascular
of the lamina propria (Reinke’s space) of the vocal fold
mucosa are fenestrated capillaries [7]. The endothelial cells
possess fenestrae or pores with diaphragms (Fig. 21.23a).
Occasionally, fenestrae or pores do not have pore dia-
phragms, and plasma rapidly exudes from the capillaries into
surrounding tissue via the fenestrae (Fig. 21.23b).
21.9.2 esicular Transport (Transcellular

V
Transport via Vesicles) vocal fold
polyp
Particles are taken up in vesicles opening onto the adluminal

surface of the endothelium, then ferried across the cyto-
plasm, and discharged into the extravascular space by fusion
of the vesicles with the abluminal plasmalemma (Fig. 21.22)
[6]. The uptake of materials in small vesicles is a form of
endocytosis and is generally referred to as micropinocytosis
[6]. The use of vesicles to ferry fluid and solutes across the Fig. 21.28 Endoscopic view of a left vocal fold polyp (62-year-old
female). The vocal fold polyp is located at the midpoint of the membra-
cell is largely confined to endothelial cells and is an expres- nous vocal fold. The left vocal fold appears yellow, resulting from the
sion of their specialization for transport (transcytosis) [6]. presence of hemosiderin which usually accompanies the hemorrhage
a stratified squamous epithelium
microvascular
lesion
B
forceps
hemorrhage
capillary Fig. 21.30 Microscopic view of a microvascular lesion of the left vocal fold
during endolaryngeal microsurgery (41-year-old female, soprano singer)
a
capillary
hemorrhage
fibrin
Fig. 21.29 Histopathology of an excised vocal fold polyp. (a) b

Hemorrhage into Reinke’s space. (b) There is marked fibrin (hyaline
pink-stained amorphous material) and erythrocyte exudation (hemor-
rhage) into Reinke’s space. Capillaries are dilated. Connective tissue
has been proliferated and inflammatory cells and fibroblasts have infil-
trated (region B in a)
dilated capillary
lesions are most commonly seen in female professional
vocalists.
Microvascular lesions are the result of microvascular
trauma within the superficial layer of the lamina propria
(Reinke’s space) of the vocal fold. Therefore, microvascular
lesions usually occur on the surface at the midpoint of the
membranous vocal fold, which vibrates the most during pho-
nation (Fig. 21.30). The superficial location of microvascular
lesions, just under the basement membrane (Fig. 21.31),
facilitates surgical accessibility without surgical trauma to Fig. 21.31 Histopathology of an excised microvascular lesion. Dilated
the underlying Reinke’s space. capillaries are observed in the lamina propria of the vocal fold mucosa
just beneath the stratified squamous epithelium (b: region B in a).
When cold instruments are used to perform epithelial cordotomy to
access the lesion, there is no postoperative deterioration in vocal func-
tion and mucosal wave flexibility
References 303
References 13. Shimada T, Nakamura M, Kitahara Y, Kawahara H. Microvasculature

of the rat mammary gland using the chemical digestion method. J
Electron Microsc. 1982;31:185–90.
1. Pearson BW. Laryngeal microcirculation and pathways of cancer
14. Shimada T, Kitamura H, Nakamura M. Three-dimensional architec-
spread. Laryngoscope. 1975;85:700–13.
ture of pericytes with special reference to their topographical rela-
2. Sato K. Three dimensional anatomy of the larynx: Investigation
tionship to microvascular beds. Arch Histol Cytol. 1992;55:77–85.
by whole organ sections. Otologia Fukuoka. 1987;33(supple
15. Rhodin JA. Ultrastructure of mammalian venous capillaries, venules,
1):153–82.
and small collection veins. J Ultrastruct Res. 1968;25:452–500.
16. Ribatti D, Nico B, Crivellato E. The role of pericytes in angiogen-
Diego, California: Singular Publishing Group Inc.; 1993.
esis. Int J Dev Biol. 2011;55:261–8.
4. Hirano M. Phonosurgery. Basic and clinical investigation. Otologia
17. Frenzel H, Kleinsasser O. Ultrastructural study on the small

(Fukuoka). 1975;21(Suppl 1):239–442.
blood vessels of human vocal cords. Arch Otorhinolaryngol.
5. Mihashi S, Okada M, Kurita S, Nagata M, Oda M, Hirano M,
1982;236:147–60.
Nakashima T. Vascular network of the vocal fold. In: Stevens KN,
18. Hiroto I, Toyozumi Y, Tomita H, Miyagi T, Kuroki K, Koike Y,
Hirano M, editors. Vocal fold physiology. Tokyo, Japan: University
Matsushita H. An experimental study on the hemodynamics of the
of Tokyo Press; 1981. p. 45–59.
vocal fold during vibration. Jap Jour Otol Tokyo. 1969;72:884–8.
6. Fawcett DW. Blood and lymph vascular systems. A texbook of his-
19. Sato K, Hirano M, Nakashima T. Electron microscopic and immu-
tology. New York: Chapman & Hall; 1994. p. 368–409.
nohistochemical investings of Reinke’s edema. Ann Otol Rhinol
7. Hirano M, Koike Y, Hirose K, Kasuya T. Observation of mucous
Laryngol. 1999;108:1068–72.
membrane of human vocal cords under electron microscopy. Jap
20.
Hawkins RI. Smoking, platelets and thrombosis. Nature.
Jour Otol Tokyo. 1974;77:650–60.
1972;236:450–2.
8. Sato K, Hirano M. Fine three-dimensional structure of pericytes in
21. Craig WY, Palomaki GE, Haddow JE. Cigarette smoking and serum
the vocal fold mucosa. Ann Otol Rhinol Laryngol. 1997;106:490–4.
lipid and lipoprotein concentrations: an analysis of published data.
9. Zimmermann KW. Der feinere Bau der Blutcapillaren. Z Anat
BMJ. 1989;298:784–8.
Entwicklungsgesch. 1923;68:29–109.
22. Brody JS, Coburn RF. Carbon monoxide-induced arterial hypox-
10. Weibel ER. On pericytes, particularly their existence on lung capil-
emia. Science. 1969;164:1297–8.
laries. Microvasc Res. 1974;8:218–35.
23. Sheveiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth
11. Lebeux YJ, Willemot J. Actin- and myosin-like filaments in rat
factor induced by hypoxia may mediate hypoxia-initiated angio-
brain pericytes. Anat Rec. 1978;190:811–26.
genesis. Nature. 1992;359:843–5.
12. Murakami M, Sugita A, Shimada T, Nakamura K. Surface view of
24. Postma GN, Courey MS, Ossoff RH. Microvascular lesions of the
pericytes on the retinal capillary in rabbits revealed by scanning
true vocal fold. Ann Otol Rhinol Laryngol. 1998;107:472–6.
electron microscopy. Arch Histol Jpn. 1979;42:297–303.
The Laryngeal Glands
22
Abstract
1. The laryngeal glands are exocrine and composed of tubuloalveolar and mixed seromu-
cinous types.
2. The distribution of the glands in the human larynx is distinctive. Laryngeal glands are
abundant in the ventricular fold and around the laryngeal ventricle. There are numerous
serous cells in the supraglottis.
3. Age-related morphologic changes in the laryngeal glands influence not only the amount
but also the quality and viscosity of secretions.
4. Age-related changes lessen lubrication of the vocal folds, thus causing aging of the voice
to some extent. Local immunity and mucociliary transport are also affected. Age-related
changes in the laryngeal glands partially alter laryngeal function.
5. Changes in the laryngeal gland caused by irradiation influence not only the amount
but also the quality of secretions; consequently, they affect the lubrication of the
vocal fold, thus causing voice disorders. Local immunity and mucociliary transport
are also affected. Irradiation’s effects on the laryngeal glands partially alter laryngeal
functions.
22.1 Introduction
serous glands
Many glands are present in the human larynx (Fig. 22.1)
[1, 2]. The laryngeal glands are exocrine and composed of serous demilune
tubuloalveolar and mixed seromucinous types.
The basic functions of the larynx are to act as a protective
sphincter, to act as a passageway for air, and to produce
sound. It also serves as a local defense system against viruses
and bacteria. All these functions require fluid secreted from
the laryngeal glands. mucous glands
The larynx is lubricated by secretions from the upper
respiratory tract [3]. Lubrication of the vocal folds is
essential for normal phonation [4]. The amount and qual-
ity, especially viscosity, of secretions influence phona- Fig. 22.1 Laryngeal glands (seromucinous glands)
tion. Additionally, serous-type glandular acinar cells
produce protein material necessary for the local immune duced by the laryngeal glands have an effect on mucocili-
system [5, 6]. The amount and viscosity of mucus pro- ary transport [7].

306 22 The Laryngeal Glands
22.2 Distribution of the Laryngeal Glands At the level of the subglottis, glands are situated in the
lamina propria of the mucosa.
The distribution of the glands in the human larynx is distinc- At the level of glottis, glands are located in the lamina
tive (Fig. 22.2) [1, 2, 8]. propria of the mucosa around the posterior glottis. On the
thyroepiglottic ligament thyroepiglottic
ligament
A
Arytenoid muscle
B
C
conus elasticus D
E
cricothyroid muscle cricothyroid
ligament
F
gland muscle
prepiglottic space thyroepiglottic ligament

b epiglottic cartilage
A B
piriform
sinus
arytenoid cartilage arytenoid muscle
laryngeal ventricle anterior glottis thyroarytenoid muscle

C D
thyroarytenoid posterior glottis
muscle
arytenoid cartilage
thyroarytenoid muscle cricoid cartilage
E conus elasticus F cricothyroid muscle
lateral
cricoarytenoid
muscle
Fig. 22.2 Distribution of the

glands in the human larynx.
(a) Coronal and sagittal
sections. (b) Transverse cricoid cartilage
sections
22.4 Microstructure of Younger Adult Serous Cells in the Laryngeal Glands 307
other hand, there are no glands or glandular duct openings at

the free edge of the membranous portion of the vocal fold
(anterior glottis).
At the level of the supraglottis, the laryngeal glands are
situated not only in the lamina propria but also in the liga-
ments (Fig. 22.3a), in the muscle (Fig. 22.3b), and in the
space (Fig. 22.3c) below the mucosa. Laryngeal glands are
acinus
a
thyroid cartilage
preepiglottic space
thyroepiglottic ligament acinus
glands
glands
glands
l aryngeal cavity
Fig. 22.4 Scanning electron micrograph of younger adult laryngeal

glands (modified NaOH maceration method)
b thyroid cartilage
paraglottic space abundant in the ventricular fold (false vocal fold) and around
the laryngeal ventricle [1, 2, 8]. There are numerous serous
glands
cells in the supraglottis [8].
glands
22.3 Three-Dimensional Microstructure

of Younger Adult Laryngeal Glands
glands aryepiglottic muscle
Three-dimensional findings of the laryngeal glands in a
human ventricular fold (false vocal fold) are shown in
Fig. 22.4. In younger adults, acini of the laryngeal glands are
c preepiglottic space
abundant.
glands 22.4 M
icrostructure of Younger Adult
glands
Serous Cells in the Laryngeal Glands
glands
foramen of The granular endoplasmic reticula, in which protein

epiglottic material has been newly formed by ribosomes and is in
cartilage
epiglottic cartilage storage, are abundant in the basal cytoplasm of serous
cells (Figs. 22.5, 22.6 and 22.7). Golgi apparatus with
epithelium l aryngeal cavity concentrated protein material is also abundant (Fig. 22.7).
Many transport vesicles are present for the carriage of
protein material in the granular endoplasmic reticulum to
Fig. 22.3 Laryngeal glands at the supraglottis (transverse sections). the Golgi apparatus (Fig. 22.7). Vacuoles with condensed
(a) Laryngeal glands distribute in the ligaments. (b) Laryngeal glands presecretory granules have become separated from the
distribute in the muscle. (c) Laryngeal glands distribute in the preepi-
ends of the saccules (Fig. 22.7). Condensing vacuoles
glottic space. The laryngeal glands are situated not only in the lamina
propria but also in the foramen of epiglottic cartilage and preepiglottic and presecretory granules can be seen near Golgi sac-
space below the mucosa cules (Fig. 22.7).
lumen of acinus
mucigen droplet
secretory granules Golgi apparatus granular
endoplasmic
reticulum
mucous cell
serous cell condensing
presecretory
vacuole
granules
transport vesicles
nucleus
Fig. 22.7 Condensing vacuole and presecretory granule in a serous

cell in a younger adult laryngeal gland (uranyl acetate and lead citrate
Fig. 22.5 Transmission electron micrograph of serous and mucous stain)
cells in younger adult laryngeal glands (uranyl acetate and lead citrate
stain)
lumen of acinus
granular endoplasmic
Golgi apparatus reticulum
secretory
transport vesicle granule
secretory
granule
condensing vacuole
presecretory granule
cell membrane
secretory granules exocytosis

Fig. 22.8 Mature secretory granules in a serous cell in a younger adult
laryngeal gland (uranyl acetate and lead citrate stain). Many mature
secretory granules contain homogeneously electron-dense granules and
Fig. 22.6 Synthesis and release of protein secretory products. are surrounded by boundary membranes
Secretory proteins synthesized on polyribosomes associate with granu-
lar endoplasmic reticula. They are sequestered in the lumen of these
organelles and transported through them to the Golgi apparatus of the
cell. Transport vesicles transfer them from the endoplasmic reticulum
to the Golgi complex. The Golgi apparatus concentrates and packages lumen of acinus
the product in granules limited by a membrane (secretory granule).
Secretory granules fuse with the cell membrane to be released by secre-
tion exocytosis cell membrane
secretory
Many mature secretory granules, surrounded by boundary granule
membranes and consisting of homogeneously electron-dense
granules, accumulate in the apical portion of the serous cells
(Fig. 22.8). These secretory granules, 500–1200 nm in diam- secretory
granule
eter, become more electron-dense and less mottled than pre-
secretory granules.
Many secretory materials are discharged through a pro-
cess of exocytosis; the boundary membrane of the secretory
granules fuses with the cell membrane, and granule contents
are subsequently discharged into the lumen of the acinus Fig. 22.9 Acinar lumen with expelled secretory material of a serous cell
(Figs. 22.9 and 22.10). in a younger adult laryngeal gland (uranyl acetate and lead citrate stain)
22.7 Three-Dimensional Microstructure of Age-Related Changes in the Laryngeal Glands 309
lumen of acinus
expelled
secretory
material
expelled
secretory
material
acinus
acinus
Fig. 22.10 Secretory material of a serous cell expelled into the acinar
lumen in a younger adult laryngeal gland (uranyl acetate and lead
citrate stain)
Fig. 22.12 Scanning electron micrograph of aged adult laryngeal

a glands (81-year-old male, modified NaOH maceration method)
adipose tissue
22.5 M
icrostructure of Younger Adult
serous glands Mucous Cells in the Laryngeal Glands
Granular endoplasmic reticula and Golgi apparatus, which syn-

mucous glands
thesize and store mucigens, are abundant in the basal regions of
mucous cells (Fig. 22.5). Presecretory mucigen droplets have
been formed by the Golgi saccules. Mucigen droplets gradu-
excretory duct
ally increase in size and merge with each other. Each is sur-
rounded by a boundary membrane and contains electron-lucent
material. These droplets are 1–2.5 μm in diameter. Many muci-
gen droplets are discharged through a process of exocytosis.
b 22.6 D
istribution of the Laryngeal Glands
excretory duct in the Aged
In the aged, acini are atrophic; the size of acini varies,

and their number has decreased (Fig. 22.11). The con-
mucous glands centration of laryngeal glands decreases in the aged at
the level of the supraglottis but not at the level of the
subglottis [9]. The ratio of mucous glands tends to
increase, and the ratio of serous glands decreases with
age at the supraglottis [9].
serous glands
22.7 Three-Dimensional Microstructure
of Age-Related Changes
Fig. 22.11 Light micrograph of aged adult laryngeal glands at the in the Laryngeal Glands
supraglottis. (a) An 80-year-old male. The acini decrease and become
atrophic. Laryngeal glands have been replaced by adipose tissue. (b) A
90-year-old male. The number of acini has decreased extremely and In the aged, acini are shrunken and atrophic; the size of acini
become atrophic varies, and their number has decreased (Fig. 22.12).
lumen of acinus 22.8 M

icrostructure of Age-Related
Changes of Serous Cells
in the Laryngeal Glands
nucleus
The granular endoplasmic reticula, transport vesicles, and
Golgi apparatus are sparse in the basal cytoplasm of the
serous cells (Fig. 22.13). The nuclei have slightly increased
in size. Vacuoles with condensed presecretory granules have
secretory nucleus
granule become separated from the ends of the saccules of the Golgi
apparatus (Fig. 22.14). Condensing vacuoles, presecretory
granules, and mature secretory granules have decreased in
serous cell
number (Figs. 22.13 and 22.14). Some mature secretory
granules can be seen in the apical portion of serous cells
myoepithelial cell (Figs. 22.13 and 22.15).
As the most notable finding of the aged serous cells, mature
Fig. 22.13 Transmission electron micrograph of serous cells in an secretory granules, presecretory granules, and condensing
aged adult laryngeal gland (83-year-old male, uranyl acetate and lead
citrate stain)
vacuoles are less electron-dense compared to those in younger
adult specimens but are electron-lucent (Figs. 22.14 and
granular
22.15). Secretory products (protein material) of aged serous
endoplasmic transport vesicle cells differ considerably from those in younger adults [10].
reticulum Protein material has decreased, and the quality has changed in
the aged serous cells [10]. Only a slight discharge of secretory
Golgi apparatus
granules through a process of exocytosis is noted.
condensing
vacuole
22.9 M
icrostructure of Age-Related
Changes of Mucous Cells
in the Laryngeal Glands
presecretory
nucleus
granule
The number of granular endoplasmic reticula and Golgi appa-
ratus has decreased in the basal cytoplasm of mucous cells
(Fig. 22.16). Mucigen droplets are not as numerous as in
younger adults. Discharge of mucigen droplets has decreased.
Fig. 22.14 Condensing vacuole and presecretory granule in a serous
cell in an aged adult laryngeal gland (83-year-old male, uranyl acetate
secretory
granule
mucigen droplet
secretory
granule mucous cells
nucleus
Fig. 22.15 Mature secretory granules in a serous cell in an aged adult

laryngeal gland (83-year-old male, uranyl acetate and lead citrate stain). Fig. 22.16 Transmission electron micrograph of mucous cells in an
Secretory granules lack homogeneously electron-dense granules but are aged adult laryngeal gland (83-year-old male, uranyl acetate and lead
electron-lucent compared to those in younger adults citrate stain)
22.11 Effect of Irradiation on Human Laryngeal Glands 311
22.10 P
hysiologic and Pathologic the importance of the local immune function of secretory
Significances of Age-Related Changes IgA in the laryngeal mucosa [5]. In external secretion, secre-
in the Laryngeal Glands tory IgA is the predominant immunoglobulin consisting of
dimeric IgA and a secretory component [12]. The secretory
22.10.1 Laryngeal Gland Function component is produced mainly in serous-type glandular aci-
nar cells [12]. Lactoferrin is an antibacterial iron-binding
In aged laryngeal glands, mature secretory granules, prese- protein in exocrine secretion produced in the serous-type
cretory granules, and condensing vacuoles in serous cells are glandular acini in the larynx [6]. Serous cells in the laryngeal
electron-lucent, and electron-dense granules are absent [10]. glands produce protein material that is essential for the local
Secretory products (protein material) of aged serous cells are immune defense system.
markedly different from those of the younger adults, and Many secretory granules containing electron-dense
there is much less protein material [10]. granules (protein material) are present in the serous cells
In serous cells, granular endoplasmic reticula and of younger adults. In aged laryngeal glands, secretory
Golgi apparatus are required for secretory granule forma- granules have decreased. The secretory products are elec-
tion (Fig. 22.6) [11]. Granular endoplasmic reticulum tron-lucent and markedly different from those in younger
function, such as formation of protein material, and/or adults. Additionally, the ratio of mucous glands tends to
Golgi apparatus function, such as concentration of protein increase, and the ratio of serous glands decreases with
material, would thus appear to be diminished in the age at the supraglottis [9]. The quantity and quality of
elderly. secretory protein material that contribute to the local
Secretory granules in serous cells have decreased in num- immune system are thus shown to be reduced in the
ber, and mucigen droplets in mucous cells are not as numer- elderly [10].
ous as in younger adults. Exocytosis of secretory granules
and mucigen droplets has decreased. Additionally, the ratio
of mucous to serous glands changes with age at the supra- 22.10.4 E
ffects on Local Mucociliary
glottis [9]. These age-related morphologic changes influence Transport Function
not only the amount but also the quality and viscosity of
secretions [10]. Another important defense mechanism of the upper respira-
tory tract is mucociliary transport. Mucociliary transport is
governed by three factors: cilia, mucus, and their interaction
22.10.2 Effects on Phonatory Function [7]. The amount and viscosity of the mucus determine the
effectiveness of this system [7].
Secretions from the laryngeal glands play an important role. In aged laryngeal glands, mucus decreases, and its qual-
The larynx is lubricated by the flow of thin mucus from ity, especially viscosity, appears to change, with consequent
glands throughout the larynx which is essential for phona- effects on mucociliary transport.
tion [3]. Amount and quality of the fluid, especially viscos- Age-related changes in the laryngeal glands influence the
ity, influence phonation [4]. local immunity and mucociliary transport of the larynx. The
Age-related morphologic changes in laryngeal glands larynx is essential for immune response in upper respiratory
reveal that the amount of this fluid has decreased and viscos- passages. These changes partially contribute to reduction of
ity appears to have changed as well. Morphologic changes of the local defense of the larynx.
the aged vocal folds are partially responsible for the aging of
the voice through changes in laryngeal glands that affect the
lubrication of the vocal folds [10]. 22.11 E
ffect of Irradiation on Human
Elderly patients without organic disease of the larynx Laryngeal Glands
complain of voice disorders or abnormal sensations such as
dryness of the throat. Diminished lubrication with aging con- The influence of irradiation on the laryngeal glands deter-
tributes to the cause of this [10]. mines how well the larynx functions.
Irradiated laryngeal glands in a human ventricular
fold (false vocal fold) are shown in Fig. 22.17. The acini
22.10.3 Effects on Local Immune Function of the glands are atrophic, their size varies, and their
numbers have decreased [13]. The excretory duct is
The mucosa has local immunological resistance. Mogi et al. dilated. The average density of the glands has decreased,
studied the biological properties of laryngeal secretion and and the average ratio of serous-type to mucous-type
noted that they contain immunoglobulin (Ig)G, IgA, IgE, glandular cells has also decreased in the irradiated lar-
secretory components, and lactoferrin [5]. They emphasized ynx [14].
a
cleaved nucleus
laryngeal glands excretory duct Golgi apparatus
serous cells
lumen of
acinus
b serous cells
excretory duct
Fig. 22.18 Transmission electron micrograph of serous cells in the
ventricular fold (9 months after radiotherapy, 66 Gy, uranyl acetate and
lead citrate stain)
serous glands
mucous gland
a
excretory duct
secretory granules
Fig. 22.17 Light micrographs of laryngeal glands in the ventricular

fold (false vocal fold) (2 months after radiotherapy, 54.8 Gy, uranyl
acetate and lead citrate stain). (a) original × 20; (b) original × 200 secretory granules
22.12 M
icrostructure of the Serous Cells
in Irradiated Laryngeal Glands
22.12.1 S
erous Cells in Irradiated Laryngeal b nucleus
Glands with a Short Duration After granular endoplasmic
Radiotherapy reticula
The nucleus-cytoplasm ratio is relatively large, and the

nuclei are cleaved. Intracellular organelles, such as granular secretory
granules merging
endoplasmic reticula and Golgi apparatus, are sparse in the secretory
basal cytoplasm of the serous cells. The transport vesicles granules
are sparse in the basal cytoplasm of the serous cells. The
condensing vacuoles, presecretory granules, and mature
presecretory granules
secretory granules have markedly decreased in number
(Fig. 22.18).
Some mature secretory granules can be seen in the apical
Golgi apparatus
portion of serous cells (Fig. 22.18). However, mature secre-
tory granules are small, 500–1000 nm in diameter, and not
Fig. 22.19 Transmission electron micrograph of secretory granules
electron-dense but are electron-lucent (Fig. 22.19). The sur-
(a) and merging secretory granules (b) in serous cells in the ventricular
rounding boundary membrane is irregular and interrupted fold (9 months after radiotherapy, 66 Gy, uranyl acetate and lead citrate
compared to those in non-irradiated specimens (Fig. 22.19). stain)
22.13 Microstructure of the Mucous Cells in Irradiated Laryngeal Glands 313
The secretory products (protein material) of irradiated serous

cells differ considerably from those in non-irradiated speci-
mens (Fig. 22.19) [13]. The secretory granules cannot keep
their shape but rather merge with each other and disappear lumen of
acinus
without accumulating in the apical portion of the serous cells
(Fig. 22.19b). Only slight discharge of secretory granules
through a process of exocytosis is noted. mucigen droplets
22.12.2 S
erous Cells in Irradiated Laryngeal mucous cell
Glands with a Long Duration After
Radiotherapy
The nuclei are cleaved, and nucleus-cytoplasm ratio is rela-

tively large. There are some intracellular organelles, such as
granular endoplasmic reticula and Golgi apparatus, in the Fig. 22.21 Transmission electron micrograph of mucous cells in the
basal cytoplasm of the serous cells. However, transport vesi- ventricular fold (9 months after radiotherapy, 66 Gy, uranyl acetate and
lead citrate stain)
cles are sparse. Condensing vacuoles, presecretory granules,
and mature secretory granules are decreased in number
(Fig. 22.20). Some small mature secretory granules, approximately
600–800 nm in diameter, can be seen in the serous cells
(Fig. 22.20b). The mature secretory granules are not electron-
a
dense but are electron-lucent (Fig. 22.20b). The secretory
B products (protein material) of irradiated serous cells with a
long duration after radiation therapy differ considerably
from those in non-irradiated specimens [13]. Only slight dis-
charge of secretory granules through a process of exocytosis
cleaved
nucleus is noted.
22.13 M
icrostructure of the Mucous Cells
in Irradiated Laryngeal Glands
22.13.1 M
ucous Cells in Irradiated Laryngeal
Glands with a Short Duration After
Radiotherapy
b secretory granules
The granular endoplasmic reticula and Golgi apparatus are
sparse in the basal cytoplasm of the mucous cells (Fig. 22.21).
Golgi Mucigen droplets are small and not as numerous as those in
apparatus non-irradiated specimens. The discharge of mucigen drop-
presecretory
granular granule lets has decreased (Fig. 22.21).
endoplasmic
reticula
lipofuscin granule
nucleus 22.13.2 M
ucous Cells in Irradiated Laryngeal
condensing Glands with a Long Duration After
vacuole
Radiotherapy
lipid droplets There are some intracellular organelles, such as granular
endoplasmic reticula and Golgi apparatus, in the basal cyto-
Fig. 22.20 Transmission electron micrograph of serous cells in the
plasm of the mucous cells (Fig. 22.22). However, mucigen
ventricular fold (2 years and 9 months after radiotherapy, 60 Gy, uranyl droplets are small and not as numerous as in non-irradiated
acetate and lead citrate stain). (b) region B in a specimens (Fig. 22.22).
granular endoplasmic 22.14.2 Effects on Local Immune Function

reticula
The average density of the laryngeal glands decreases, and
mucous cell
the average ratio of serous-type to mucous-type glandular
cells decreases in the irradiated larynx; consequently, not
only the voice function but also the local defense function of
mucigen droplets the larynx will likely be impaired after radiotherapy [14].
In the irradiated laryngeal glands, the number of secretory
granules in serous cells has decreased. The secretory prod-
Golgi apparatus ucts are not electron-dense but are electron-lucent and mark-
edly different from those in the non-irradiated larynx. The
quantity and quality of secretory protein material that con-
tribute to the local immune system are thus shown to be
myoepithelial cells reduced in the irradiated larynx [13].
Fig. 22.22 Transmission electron micrograph of mucous cells in the

ventricular fold (2 years and 9 months after radiotherapy, 60 Gy, uranyl 22.14.3 E
ffects on Local Mucociliary
acetate and lead citrate stain)
Transport Function
The morphological findings show a decrease in the size
In irradiated laryngeal glands, the amount of mucus
and function of the serous and mucous cells consequently,
decreases, and its quality, especially viscosity, changes. The
meaning irradiated serous and mucous cells of the laryngeal
quantity and quality of mucus that contribute to the muco-
glands are atrophic [13].
ciliary transport are thus shown to be reduced in the irradi-
ated larynx [13].
The larynx is essential for immune response in the upper
22.14 P
hysiologic and Pathologic respiratory passages. Changes caused by irradiation in the
Significances of Irradiated Laryngeal laryngeal glands affect the local immunity and mucociliary
Glands transport of the larynx [13]. Radiation therapy contributes to
reduced local defenses of the larynx [13].
Irradiation’s influences on the laryngeal glands determine
how well the larynx functions. The morphological changes
caused by irradiation affect not only the amount but also the
quality and viscosity of the secretions. Irradiation affects the References
laryngeal gland secretory functions for a long period of time
1. Nassar VH, Bridger GP. Topography of the laryngeal mucous
after radiotherapy [13].
glands. Arch Otolaryngol. 1971;94:490–8.
22.14.1 Effects on Phonatory Function 3. Pressman JJ. Physiology of the vocal cords in phonation and respi-
ration. Arch Otolaryngol. 1942;35:355–98.
4. Ichikawa T. Basic studies on the lubrication of the larynx during
The morphological changes in irradiated laryngeal glands phonation. Otologia (Fukuoka). 1982;28:38–52.
show that the amount of the fluid has decreased and its vis- 5. Mogi G, Watanabe N, Maeda S, Umehara T. Laryngeal secre-
cosity appears to have changed. Irradiation-induced tissue tions. An immunochemical and immunohistological study. Acta
Otolaryngol. 1979;87:129–41.
damage of the vocal folds is partially responsible for voice
6. Nakashima T, Komiyama S, Makishima K, Takeda K, Hiroto
disorders after radiotherapy [15]. Morphological changes in I. Immunopathological study of the larynx. IgA distribution and
irradiated laryngeal glands may affect the lubrication of the secretory activity. Ann Otol Rhinol Laryngol. 1980;89:359–65.
vocal folds and may also contribute to voice disorders after 7. Sakakura Y. Pathogenesis of mucociliary dysfunction in the upper
respiratory tract. Pract Otol (Kyoto). 1987;80:1–18.
radiotherapy [13].
8. Sato K. Three dimensional anatomy of the larynx: investiga-
Irradiation-induced xerostomia is known to be a signifi- tion by whole organ sections. Otologia Fukuoka. 1987;33(supple
cant complication in patients who receive radiotherapy [16]. 1):153–82.
Patients who receive radiotherapy complain of voice disor- 9. Tomita H, Nakashima T, Maeda A, Umeno H, Sato K. Age related
changes in the distribution of laryngeal glands in the human adult
ders and abnormal sensations such as dryness of the throat.
larynx. Auris Nasus Larynx. 2006;33:289–94.
Diminished lubrication caused by the dysfunction of irradi- 10. Sato K, Hirano M. Age-related changes in the human laryngeal
ated laryngeal glands contributes to this dryness. glands. Ann Otol Rhinol Laryngol. 1998;107:525–9.
References 315
11. Fawcett DW. Glands and secretion. A texbook of histology.

14. Nakashima T, Tomita H, Tsuda S, Chitose S. Radiotherapy of the
Philadelphia: Saunders Co; 1986. p. 83–99. neck influences the distribution of laryngeal secretory glands. J
12. Brandtzaeg P. Mucosal and glandular distribution of immu-
Laryngol Otol. 2005;119:976–80.
nogloblin components. Differential localization of free and 15. Sato K, Shirouzu H, Nakashima T. Irradiated macula flava in the
bound SC in secretory epithelial cells. J Immunol. 1974;112: human vocal fold mucosa. Am J Otolaryngol. 2008;29:312–8.
1553–9. 16. Jensen AB, Hansen O, Jorgensen K, Bastholt L. Influence of late
13. Sato K, Nakashima T. Effect of irradiation on the human laryngeal side-effects upon daily life after radiotherapy for laryngeal and pha-
glands. Ann Otol Rhinol Laryngol. 2008;117:734–9. ryngeal cancer. Acta Oncol. 1994;33:487–91.
Atrophy of the Vocal Fold
23
Abstract
1. Atrophy is a decrease in the size and function of a cell, a tissue, and/or an organ.
Clinically, it is often recognized as a diminution in the size or function of an organ.
2. Vocal fold atrophy is defined as a decrease in the size and function of the vocal fold.
Therefore, vocal fold atrophy can also be defined as a diminution in the size of each por-
tion of the layered structure and function of the vocal folds.
3. Clinically, the membranous portion of the atrophic vocal fold becomes concave and can
be easily recognized as a glottal incompetence.
4. The portions of the vocal fold tissue which are atrophic are different in each disease with
vocal fold atrophy. Understanding of the histological structures of the vocal fold and the
histopathology of the vocal fold atrophy is important for understanding the concepts
behind treatment of atrophic vocal folds.
23.1 Introduction l ayers of the lamina propria; and the vocalis muscle [3, 4].
The superficial layer is referred to as Reinke’s space. The
Atrophy is a decrease in the size and function of a cell, a tis- vocal ligament consists of the intermediate and deep lay-
sue, and/or an organ [1]. Clinically, it is often recognized as ers. This layered structure is very important in vibration
a diminution in the size or function of an organ [1]. [3].
Vocal fold atrophy can therefore be defined as a decrease in Therefore, vocal fold atrophy can also be defined as a
the size and function of the vocal fold [2]. Clinically, the mem- diminution in the size of each portion of the layered struc-
branous portion of the atrophic vocal fold becomes concave ture and in the function of the vocal folds.
and can be easily recognized as a glottal incompetence. The portions of the vocal fold tissue which are atrophic
Atrophic portions of the vocal fold tissue are different in are different in each disease with vocal fold atrophy: (1)
each disease with vocal fold atrophy. Understanding of the The size of the vocalis muscle decreases with recurrent
histological structures of the vocal fold and the histopathol- laryngeal nerve paralysis. (2) The size of the superficial
ogy of the vocal fold atrophy is important for understanding layer of the lamina propria of the vocal fold mucosa
the concepts behind treatment of atrophic vocal folds [2]. decreases with sulcus vocalis. (3) Vocal fold tissue decreases
in size after irradiation by laser or other radiation sources.
(4) Both the lamina propria of the vocal fold mucosa and
23.2 Definition of Vocal Fold Atrophy vocalis muscle decrease in size in geriatric vocal folds,
though atrophy of the geriatric vocal folds is chiefly related
The human vocal fold has a layered structure consisting of to a decrease in the size of the lamina propria, especially the
the epithelium; the superficial, intermediate, and deep superficial layer [5].

318 23 Atrophy of the Vocal Fold
23.3 Recurrent Laryngeal Nerve Paralysis thyroarytenoid muscle become atrophic and degenerate
(Fig. 23.2). On the other hand, the lamina propria of the
In cases of vocal fold atrophy caused by recurrent laryngeal vocal fold mucosa is intact (Fig. 23.3). This is the reason
nerve paralysis, a diminution in the size of the thyroaryte- why the vocal fold mucosa vibrates after augmentation sur-
noid muscle can be detected (Fig. 23.1). Muscle fibers of the gery of the thyroarytenoid muscle.
Fig. 23.1 Transverse section

of the human larynx with a
bilateral recurrent laryngeal
nerve paralysis at the glottic
level. A diminution in the size thyroid cartilage
of the thyroarytenoid muscle
can be detected
(a, hematoxylin and eosin
stain; b, Elastica van Gieson membranous
stain) vocal fold
thyroarytenoid
muscle
vocal
process
arytenoid
cartilage
cricoid cartilage
b
thyroid cartilage
vocal ligament
thyroarytenoid
muscle
vocal
process
arytenoid
cartilage
cricoid cartilage
23.3 Recurrent Laryngeal Nerve Paralysis 319
a lamina p
propria
p vocalis muscle
C
b Reinke’s space vocal ligament vocalis muscle

b
muscle fibers
Fig. 23.2 (a) Transverse section of the human vocal fold with recur- c
rent laryngeal nerve paralysis (hematoxylin and eosin stain). (b) Muscle
fibers of the thyroarytenoid muscle have become atrophic and degener- muscle fibers
ated (region B in a)
Fig. 23.3 (a) Transverse section of the human vocal fold with recur-
rent laryngeal nerve paralysis (Elastica van Gieson stain). (b) Lamina
propria of the vocal fold mucosa is intact. Collagen fibers are stained
red and elastic fibers are stained black with Elastica van Gieson stain
(region B in a). (c) Muscle fibers of the thyroarytenoid muscle have
become atrophic and degenerated. Interstitial spaces are sparse (region
C in a)
23.4 Sulcus Vocalis
In cases of vocal fold atrophy caused by sulcus vocalis, a

diminution in the size of the superficial layer of the lamina
anterior
propria (Reinke’s space) of the vocal fold mucosa can be macula flava
posterior
detected (Fig. 23.4) [6]. macula flava
vocal fold
Sulcus vocalis is a furrow along the edge of the membra-
nous vocal fold. The sulcus is confined to the stratified squa-
mous epithelial area [6]. The sulcus is situated in the
superficial layer of the lamina propria [6]. Around the bot-
tom of the sulcus, the superficial layer of the lamina propria sulcus vocalis
is thin [6].
Basement membrane thickness increases markedly at the
furrow of the sulcus vocalis (Figs. 23.5 and 23.6). The thick-
ness is about 12.5 μm. It is composed of the lamina lucida,
lamina densa, and lamina reticularis. The lamina densa is an Fig. 23.4 Macroscopic findings of the left human vocal fold with sul-
electron dense layer and about 40–60 nm in thickness. It cus vocalis (59-year-old male)
becomes multilayered. Collagen fibers increase in the lamina
reticularis.
Collagen fibers are dense in the thin superficial layer of
the lamina propria around the bottom of the sulcus
(Figs. 23.7 and 23.8). Collagen fibers are made up of
numerous collagen fibrils approximately 50 nm in width. epithelium
They run in various directions. Fibrous long-spacing col-
lagen is observed in the superficial layer of the lamina pro-
pria around the bottom of the sulcus (Fig. 23.9). Fibrous
long-spacing collagen is about 150 nm in diameter and has basal lamina
broad crossbands with a periodicity of about 120 nm.
Numerous inner filaments running parallel to the long axis
of fibrous long-spacing collagen can be seen. Common
collagen fibrils are present near fibrous long-spacing col- lamina propria of mucosa
lagen. Reticular fibers (type III collagen) are sparse. The
number of elastic fibers has decreased (Figs. 23.7 and Fig. 23.5 Epithelium and basement membrane zone around the bot-
23.8). Some elastic fibers are fragmented (Fig. 23.10). tom of a sulcus (Elastica van Gieson stain). The basement membrane
Changes are noted in the quality and quantity of collagen, zone stained dark red indicating the presence of collagen fibers
reticular, and elastic fibers constituting the vocal fold
mucosa.
The term “fibrous long-spacing collagen” has been
employed to designate collagen fibrils with markedly greater
basal cell
periodicity and thickness than the common collagen fibril. In
the presence of acid mucopolysaccharides, collagen mole-
cules precipitate in vitro to form fibrous long-spacing fibers
[7]. Increased collagenase activity and the presence of reticu-
lar fibers of collagen (composed of type III collagen) are collagen fibers
multilayered
essential to the formation of fibrous long-spacing collagen lamina densa
in vivo [8]. Fibrous long-spacing collagen is observed in the
superficial layer of the lamina propria around the bottom of
the sulcus vocalis. The predominant type of collagen in the
lamina propria of the human vocal fold is reticular fibers
(type III collagen) [9]. Collagenase activity may possibly be
increased at some time in the lamina propria of the sulcus
Fig. 23.6 Transmission electron micrograph of the basement mem-
vocalis. brane zone around the bottom of the sulcus (uranyl acetate and lead
Capillaries are sparse in the thin superficial layer of the citrate stain). The lamina densa becomes multilayered, and collagen
lamina propria around the bottom of the sulcus. fibers increase in the lamina reticularis
23.5 Irradiated Vocal Fold 321
collagen fibers
elastic fibers
fragmented elastic fibers
Fig. 23.7 Superficial layer of the lamina propria around the bottom of
the sulcus vocalis. (Elastica van Gieson stain, original ×400). Collagen Fig. 23.10 Transmission electron micrograph of elastic fibers in the
fibers (stained red) are dense and elastic fibers (stained black) are sparse superficial layer of the lamina propria of the sulcus vocalis (tannic acid
in the thin superficial layer of the lamina propria around the bottom of stain)
the sulcus
There are some fibroblasts in the lamina propria of the

collagen fibers
sulcus vocalis (Fig. 23.8). They are spindle-shaped or ellipti-
cal. The cytoplasm occupies a small area around the nucleus.
Components of the cytoplasm, such as rough endoplasmic
collagen fibers
reticulum and Golgi apparatus, are few. The fibroblasts syn-
thesize few collagen and elastic fibers and are in the resting
phase.
nucleus Voice disorders of sulcus vocalis are caused by incom-
plete glottic closure and significantly increased stiffness of
the vocal fold mucosa [3]. Increased stiffness is due to mor-
elastic fibers
phologic changes of extracellular matrices in the thin super-
ficial layer of the lamina propria of the vocal fold mucosa
around the sulcus.
fibroblast The cause of sulcus vocalis remains unclear. Aging of tis-
sue, repeated inflammation, and congenital factors are indi-
Fig. 23.8 Transmission electron micrograph of the superficial layer of the cated as possibly involved [3]. There is growing evidence to
lamina propria around the bottom of the sulcus vocalis (tannic acid stain) suggest that the cells in the human maculae flavae are adult
multipotent stem cells, tissue stem cells, or progenitor cells
in the human vocal fold mucosa [10–12]. The latest research
fibrous long -spacing confirms that cells in the human maculae flavae are involved
collagen in the metabolism of extracellular matrices that are essential
for the viscoelasticity in the human vocal fold mucosa, and
they are considered to be important cells in the growth,
development, and aging of the human vocal fold mucosa [13,
14]. Therefore, dysfunction of the cells in the human macu-
lae flava may be one of the causes of sulcus vocalis [6].
collagen
g fibers
23.5 Irradiated Vocal Fold
Vocal fold tissue decreases in size after irradiation by laser or

other radiation sources.
Voice disorders are one of the complications after radio-
Fig. 23.9 Transmission electron micrograph of fibrous long-spacing therapy, and they are caused by radiation-induced tissue
collagens (arrows) in the superficial layer of the lamina propria of the
damage. Radiotherapy may be a double-edged sword with
sulcus vocalis (tannic acid stain)
a epithelium
elastic fibers
b Fig. 23.12 Lamina propria of an irradiated vocal fold mucosa

5 months after radiotherapy (66 Gy). (Elastica van Gieson stain). Elastic
fibers have degenerated
elastic
i fibers
f
collagen fibers
fibrosis
Fig. 23.11 Lamina propria of an irradiated vocal fold mucosa

5 months after radiotherapy (66 Gy). (a, original ×50; b, original ×200,
Elastica van Gieson stain). Collagen fibers, stained red with Elastica
van Gieson stain (b), are dense and high in number, and elastic fibers,
stained black with Elastica van Gieson stain (b), are sparse. The lamina
propria of the vocal fold mucosa becomes monotonous
Fig. 23.13 Vocalis muscle of an irradiated vocal fold mucosa 5 months
after radiotherapy (66 Gy) (Elastica van Gieson stain). Interstitial
the capacity to cure tumors but also with the potential to spaces between muscle fibers are fibrotic
cause fibrosis, necrosis, chronic edema, atrophy, and even
secondary carcinomas [15]. Despite the beneficial tumori-
cidal effects of radiation, whenever doses of radiation suffi-
cient to kill cancer cells are used, normal tissues are
permanently affected [15]. fibroblast
The lamina propria of the irradiated vocal fold mucosa collagen fibers
appears as a uniform structure (Fig. 23.11a). Collagen fibers
are dense and elastic fibers are sparse (Fig. 23.11b). Some
elastic fibers are degenerated (Fig. 23.12). Reticular fibers
nucleus
are sparse. There is little hyaluronic acid in the vocal fold collagen fibers
mucosa. Interstitial spaces between thyroarytenoid muscle
fibers become fibrotic (Fig. 23.13).
Electron microscopy shows that the number of collagen
fibers is elevated in the lamina propria of the irradiated vocal
fold mucosa, and elastic fibers and reticular fibers are sparse elastic fibers
(Fig. 23.14). The fibroblasts in the lamina propria of the
vocal fold mucosa are spindle-shaped or oval with no cyto-
plasmic processes and show no morphologic changes. The an irradiated vocal fold mucosa 5 months after radiotherapy (70 Gy)
fibroblast nuclei are elliptic. The nucleus-cytoplasm ratio is (tannic acid stain)
23.7 Laryngeal Augmentation Surgery (Injection Laryngoplasty) 323
large, and poorly developed rough endoplasmic reticulum 23.6 Geratric Vocal Fold
and Golgi apparatus are apparent. Along the surface of the
fibroblasts, few vesicles can be seen. The lamina propria of the vocal fold mucosa and the vocalis
Voice disorders after radiotherapy are caused by radiation- muscle decrease in size in geriatric vocal folds. Atrophy of
induced tissue damage, and normal tissues are permanently the geriatric vocal folds is chiefly related to the decrease in
affected [15]. Usually, these changes are mild and self-limited, the size of the lamina propria, especially the superficial layer,
but in a certain percentage of patients, there is progression to of the vocal fold [5].
chronic edema, fibrosis, atrophy, and even necrosis [15]. The See Chap. 17 “Geriatric Changes of Cells and Extracellular
effects of radiation are brought about by the passage of various Matrices in the Human Vocal Fold Mucosa.”
charged particles through cells with resultant disruption at the
molecular level [15]. Radiation at cancericidal doses produces
predictable changes in surrounding normal tissues [15]. Later 23.7 L
aryngeal Augmentation Surgery
changes in mucosa consist of submucosal atrophy, dilation and (Injection Laryngoplasty)
atrophy of seromucinous glands, and progressive fibrosis [15].
The lamina propria of the irradiated vocal fold mucosa Laryngoplastic phonosurgery is commonly performed to
appears as a uniform structure, which is mainly composed of improve laryngeal incompetence in patients with vocal fold
increased collagen fibers. There is little hyaluronic acid in atrophy. In cases in which laryngeal incompetence causes
the lamina propria of the vocal fold mucosa. From this point voice disorders and/or aspiration, laryngoplastic phonosur-
of view, the viscoelasticity of the irradiated vocal fold muco- gery is expected to improve not only voice disorders but also
sae is inadequate for vibration, and their structures are also aspiration.
not suitable for vibration and phonation [16]. Injection laryngoplasty is one of the procedures for treat-
Radiation induces changes in the three-dimensional struc- ing laryngeal incompetence. The portions of the vocal fold
ture of collagen fibers, reticular fibers, elastic fibers, and gly- tissue which are atrophic are different in each disease with
cosaminoglycans. Radiation also changes their qualitative vocal fold atrophy. Understanding of the histological struc-
and quantitative features and has an effect on the three- tures of the vocal fold and the histopathology of the vocal
dimensional structure of the extracellular matrices in Reinke’s fold atrophy is important for understanding the concepts
space. Thus, viscoelasticity in the tissue (Reinke’s space) of behind treatment of atrophic vocal folds.
the irradiated vocal fold does change, and this change explains Injection into the thyroarytenoid muscle at the membra-
one component of voice disorders after irradiation [16]. nous portion of the vocal fold is performed in conventional
In some cases, after a long duration after irradiation, the injection laryngoplasty to improve vocal fold atrophy and
lamina propria of the vocal fold mucosa is the same light glottal incompetence (Figs. 23.15, 23.16, and 23.17). Injection
microscopically as a normal vocal fold mucosa. In these cases, into the thyroarytenoid muscle at the cartilaginous portion of
radiation-induced tissue damage appears to have improved. the vocal fold (lateral to the oblong fovea or triangular fovea
Fig. 23.15 Injection location

of the larynx for injection
laryngoplasty. E, epiglottis; V,
vocal fold; F, false vocal fold
(ventricular fold); A,
arytenoid; AF, aryepiglottic
fold; P, piriform sinus of the
hypopharynx. Asterisk 1a:
thyroarytenoid muscle at the
membranous portion of the
vocal fold. Asterisk 1b:
thyroarytenoid muscle at the
cartilaginous portion of the
vocal fold (lateral to the
oblong fovea of the arytenoid
cartilage). Asterisk 2:
ventricular fold (false vocal
fold). Asterisk 3: aryepiglottic
fold. Asterisk 4: medial wall
of the piriform sinus of the
hypopharynx
Reinke's space A B
A B C
vocal ligament
triangular fovea
oblong fovea
vocalis muscle
vocal process of
arytenoid cartilage
lamina of
cricoid
cartilage
Fig. 23.16 Injection location of the membranous portion of the vocal

fold. A In Reinke’s space. B Just below the vocal ligament. C In the
a
Fig. 23.18 Injection location of the cartilaginous portion of the vocal
fold. A Thyroarytenoid muscle lateral to the oblong fovea of the aryte-
thyroid cartilage
ventricular fold noid cartilage. B Thyroarytenoid muscle and soft tissue lateral to the
triangular fovea of the arytenoid cartilage
B
vocal fold
injected autologous
fat of the arytenoid cartilage) produces adduction arytenopexy
(Fig. 23.18) [17–20].
The thyroarytenoid muscle extends to the supraglottis
cricoid cartilage
and plays a role in creating a protective sphincter during
swallowing. Whole-organ serial section studies of the lar-
ynx with vocal fold paralysis show that the thyroarytenoid
b thyroarytenoid muscle lamina propria of
vocal fold mucosa
muscles in the ventricular fold and the aryepiglottic muscle
become atrophic (Fig. 23.19) and their role in creating a
protective sphincter is reduced. On the basis of these obser-
vations, injections into the ventricular fold and aryepiglot-
tic fold are performed to enforce laryngeal closure
(Figs. 23.20 and 23.21) [18–20]. The injection into the
injected autologous
fat
medial wall of the piriform sinus of the hypopharynx
reduces its capacity; consequently, the amount of residual
food retained in it is reduced, and pharyngeal clearance on
the affected side is improved (Fig. 23.20 and 23.21)
[18–20].
Knowledge of the three-dimensional structure of the
Fig. 23.17 (a) Coronal section of the human larynx with recurrent larynx is crucial in performing surgical procedures and in
laryngeal nerve paralysis 16 months after injection laryngoplasty
injecting the materials into the proper position and in the
(20-year-old female, hematoxylin and eosin stain). (b) Thyroarytenoid
muscle is atrophic. Injected autologous fat is well tolerated by the tissue proper amount for improving voice disorders and
and is not resorbed with time (region B in a) aspiration.
23.7 Laryngeal Augmentation Surgery (Injection Laryngoplasty) 325
a a
thyroid cartilage
saliva in left
piriform sinus
thyroarytenoid
ventricular fold
muscle
aryepiglottic
muscle B
concaved vocal fold
arytenoid cartilage
arytenoid muscle
b
b
glands
saliva in left
piriform sinus
glottal incompetence
thyroarytenoid
muscle
aryepiglottic muscle
arytenoid cartilage
muscle fibers
Fig. 23.19 (a) Transverse section of the human larynx with bilateral

recurrent laryngeal nerve paralysis at the supraglottic level (hematoxy-
lin and eosin stain). (b) A diminution in the size of thyroarytenoid mus-
cle and aryepiglottic muscle can be detected (region B in a, original
×12.5). (c) Muscle fibers of the thyroarytenoid muscle have become
Fig. 23.20 View of the larynx before and after injection laryngohypo-
atrophic and degenerated (original ×200)
pharyngoplasty. (a) Respiration before surgery; (b) Phonation before
surgery; (c) respiration after surgery; (d) phonation after surgery
a
vocal fold
injected
autologous fat
harvested fat
b
ventricular fold
injected Fig. 23.22 Harvested autologous liposuctioned fat

autologous fat
the tissue. Second, they must not be resorbed with time.

Third, they must be finely dispersed in a harmless vehicle in
order to be injected through a long needle. Under these
requirements, recently, a few injection materials for vocal
fold augmentation surgery have become available. However,
there are few ideal injection materials for Reinke’s space aug-
mentation surgery because injected material prevents vibra-
Piriform sinus tion of the vocal fold.
of hypopharynx
There are a few injectable biomaterials available for vocal
Fig. 23.21 Axial computed tomography scan after autologous fat fold augmentation surgery. Recently autologous fat [23, 24]
injection laryngoplasty. (a) At level of glottis; (b) At level of (Fig. 23.22) or fascia [25, 26] has become popular for use as
supraglottis an injection material.
Autologous fat injection laryngoplasty was introduced
early in the 1990s [23, 24]. The advantages of autologous
23.8 I njection Material for Laryngeal fat injection laryngoplasty are the following: [20] The sur-
Augmentation Surgery (Injection gical procedure can be performed without an external sur-
Laryngoplasty) gical approach. The injected material is well tolerated by
the laryngeal tissue because it is autologous liposuctioned
After Bruning first introduced injection laryngoplasty using fat. The injection location and the amount of injected mate-
hard paraffin in 1911 [21], many investigations concerning rial can be modified on the basis of the pathological condi-
the method of injection, injection location, and injection tions of the larynx as seen under a microscope. The surgical
materials have been reported. Regarding the injection mate- procedure is technically simple, involves minimal surgical
rials, many materials for injection laryngoplasty have been invasion, and has a wide applicability. The degree of accep-
used for several decades. tance by patients is very high because patients understand
Arnold reported three requirements for suitable intracordal that the injected material is their own tissue and not a for-
injection materials [22]. First, they must be well tolerated by eign body.
23.8 Injection Material for Laryngeal Augmentation Surgery (Injection Laryngoplasty) 327
harvested fat
fat cell
fat cell
Fig. 23.23 Scanning electron micrograph of harvested liposuctioned b

fat after passing through a 19-gauge long needle. Autologous fat is not
damaged by harvesting or the microinjection method
reticular fibers
An additional advantage is that the autologous fat is soft

and pliable. It has viscous properties similar to those of the
normal human lamina propria of the vocal fold mucosa [27].
The cytoplasm and basal lamina of the fat cells form a thin
pellicle around the fat droplets and are invested by a mesh-
work of fine reticular fibers (Figs. 23.23 and 23.24). This
morphological characteristic of liposuctioned fat is one of
the reasons why autologous fat has viscous properties similar
to those of the human lamina propria.
One of the problems of autologous fat injection laryn-
goplasty is whether the injected fat maintains the graft
volume. The fate of injected fat continues to be debated
[28]. The size, density, and proliferation of fat cells and
ground substance around them may be related to resorption
and a decrease in surviving graft volume [28]. The prolif-
erative ability of injected autologous fat may also be related
to some degree to the graft volume after injection laryngo- Fig. 23.24 Scanning electron micrograph of harvested liposuctioned
plasty [28]. fat cells. (a) Fat cells. (b) A meshwork of fine reticular fibers around a
fat cell
References 15. Shumrick KA, Shumrick DA, Vietti MJ. Inflammatory diseases of

the larynx. In: Fried MP, editor. The larynx. A multidisciplinary
approach. 2nd ed. St. Louis, MO: Mosby; 1996. p. 283–306.
1. Rubin E, Farber JL. Cell injury. In: Rubin E, Farber JL, editors.
16. Sato K, Shirouzu H, Nakashima T. Irradiated macula flava in the
Pathology. Philadelphia: Lippincott-Raven Publishers; 1999.
human vocal fold mucosa. Am J Otolaryngol. 2008;29:312–8.
p. 1–35.
17. Laccourreye O, Paczona R, Ageel M, Hans S, Brasnu D, Crevier-
2. Sato K. Histopathology of vocal fold atrophy. Jpn J Logop Phoniatr.
Buchman L. Intracordal autologous fat injection for aspiration after
2002;43:432–7.
recurrent laryngeal nerve paralysis. Eur Arch Otorhinolaryngol.
1999;256:458–61.
(Fukuoka). 1975;21(Suppl 1):239–440.
18. Sato K, Umeno H, Nakashima T. Liposuctioned autologous fat
injection into the larynx and hypopharynx with aspiration after
vagal nerve paralysis. J Jpn Bronchoesophagol Soc. 2002;53:353–7.
5. Sato K, Sakaguchi S, Hirano M. Histologic investigation of bowing
19. Sato K, Umeno H, Nakashima T. Injection laryngoplasty according
of the aged vocal folds. Larynx Jpn. 1996;8:11–4.
to the pathologic condition. J Otolaryngol Jpn. 2003;106:808–14.
6. Sato K, Hirano M. Electron microscopic investigation of sulcus
20. Sato K, Umeno H, Nakashima T. Autologous fat injection laryngo-
vocalis. Ann Otol Rhinol Laryngol. 1998;107:56–60.
hypopharyngoplasty for aspiration after vocal fold paralysis. Ann
7. Gross J. The behavior of collagen units as a model in morphogen-
esis. J Biophys Biochem Cytol. 1956;2(4 Suppl):261–74.
21. Bruning W. Uber eine neue behandlungsmethode der rekurrenslah-
8. Kajikawa K, Nakanishi I, Yamamura T. The effect of collagenase
mung. Ver Deutsch Laryng. 1911;18:93–151.
on the formation of fibrous long spacing collagen aggregates. Lab
22. Arnold GE. Vocal rehabilitation of paralytic dysphonia: VI, fur-
Investig. 1980;43:410–7.
ther studies of intracordal injection materials. Arch Otolaryngol.
1962;73:290–4.
Laryngol. 1998;107:1023–8.
23. Mikaelian D, Lowry LD, Sataloff RT. Lipoinjection for unilateral

vocal cord paralysis. Laryngoscope. 1991;101:465–8.
24. Brandenburg JH, Kirkham W, Koschkee D. Vocal cord augmenta-
2012;121:798–803.
tion with autologous fat. Laryngoscope. 1992;102:495–500.
25. Rihkanen H. Vocal fold augmentation by injection of autologous
fascia. Laryngoscope. 1998;108:51–4.
26. Tsunoda K, Takanosawa M, Niimi S. Autologous transplantation of
fascia into the vocal fold. A new phonosurgical technique for glottal
incompetence. Laryngoscope. 1999;109:504–8.
Otol. 2016;130:656–61.
27. Chan RW, Titze IR. Viscosities of injectable biomaterials in vocal
fold augmentation surgery. Laryngoscope. 1998;108:725–31.
ula flava in the human vocal fold. Part 1: its role in the adult vocal
28. Sato K, Umeno H, Nakashima T. Histological investigation of
liposuctioned fat for injection laryngoplasty. Am J Otolaryngol.
2005;26:219–25.
flava in the human vocal fold. Part 2: its role in the growth and devel-
Glossary
The anatomical terms employed are primarily based on those Common term for
described in Terminologia Anatomica (1998). Terminologia structure not
Anatomica is the international standard on human anatomic contained in
Terminologia Anatomica Anatomical Term Terminologia
terminology. It was developed by the Federative Committee (1998) (Latin) (English) Anatomica
on Anatomical Terminology (FCAT) of the International Plica vestibularis Vestibular fold Ventricular fold,
Federation of Associations of Anatomists and was released False vocal fold
in 1998 [1]. It supersedes the previous international standard, Ventriculus laryngis Laryngeal ventricle
Nomina Anatomica. Sacculus laryngis Laryngeal saccule
Some anatomical terms included in Terminologia Glottis Glottis
Anatomica, however, are seldom used by laryngologists, Rima glottidis Rima glottidis
speech pathologists, and speech scientists. Consequently, Plica vocalis Vocal fold
common terms for structures not contained in Terminologia Macula Flava
Anatomica are also employed. Anterior macula
A comparison table between the terms contained in flava
Terminologia Anatomica (Latin), anatomical terms Posterior
macula flava
(English), and common terms for structures not contained in
Pars intermenbranacea Intermembranous part Anterior glottis
Terminologia Anatomica are listed below for the conve-
Pars intercartilaginea Intercartilaginous part Posterior glottis
nience of readers. Cavitas infraglottica Infraglottic cavity Subglottic space
Tunica mucosa Mucosa, Mucous
membrane
Reference Epithelium Epithelium
Lamina propria Propria mucosae
1. Federative Committee on Anatomical Terminology. Terminologia mucosae
anatomica. International Anatomical Terminology. New York: Glandulae laryngeales Laryngeal glands
Thieme Medical Publishers; 1998.
Recessus piriformis Piriform recess, Piriform sinus
Piriform fossa
Common term for
structure not Cartilagines laryngis Laryngeal cartilage
contained in Cartilago thyroidea Thyroid cartilage
Terminologia Anatomica Anatomical Term Terminologia Prominentia laryngea Laryngeal prominence
(1998) (Latin) (English) Anatomica Lamina dextra / sinistra Rright / left lamina Thyroid lamina
Cavitas Laryngis Laryngeal cavity Incisura thyroidea Superior thyroid notch
Aditus laryngis Laryngeal inlet superior
Vestibulum laryngis Laryngeal Vestibule Incisura thyroidea Inferior thyroid notch
Rima vestibuli Rima vestibuli inferior
Epiglottis Epiglottis Tuberculum Superior thyroid
Plica aryepiglottica Aryepiglottic fold thyroideum superius tubercle
Tuberculum cuneiforme Cuneiform tubercle Tuberculum Inferior thyroid
thyroideum inferius tubercle
Tuberculum Corniculate tubercle
corniculatum Linea obliqua Oblique line
Incisura Interarytenoid notch Cornu superius Superior horn
interarytenoidea Cornu inferius Inferior horn

K. Sato, Functional Histoanatomy of the Human Larynx, https://doi.org/10.1007/978-981-10-5586-7
330 Glossary
Common term for Common term for

structure not structure not
contained in contained in
Terminologia Anatomica Anatomical Term Terminologia Terminologia Anatomica Anatomical Term Terminologia
(1998) (Latin) (English) Anatomica (1998) (Latin) (English) Anatomica
Foramen thyroideum Thyroid foramen Lig. hyoepiglotticum Hyoepiglottic
Cartilago cricoidea Cricoid cartilage ligament
Arcus cartilaginis Arch of cricoid Cricoid arch Membrana fibroelastica Fibroelastic
cricoideae cartilage laryngis membrane of larynx
lamina cartilaginis Lamina of cricoid Cricoid lamina Membrana Quadrangular
cricoideae cartilage quadrangularis membrane
Facies articularis Arytenoid articular Lig. vestibulare Vestibular ligament Ventricular
arytenoidea surface ligament
Facies articularis Thyroid articular Lig. vocale Vocal ligament
thyroidea surface Conus elasticus Conus elasticus,
Cartilago arytenoidea Arytenoid cartilage Cricovocal membrane
Facies articularis Articular surface Anterior
commissure
Basis cartilaginis Base of arytenoid
tendon
arytenoideae cartilage
Thyroglottic
Facies anterolateralis Anterolateral surface
ligament
Crista arcuata Arcuate crest
Articulatio Cricothyroid joint
Colliculus Colliculus cricothyroidea
Fovea oblonga Oblong fovea Capsula articularis Capsule of
Fovea triangularis Triangular fovea cricothyroidea cricothyroid joint
Facies medialis Medial surface Articulatio Cricoaryntenoid joint
Facies posterior Posterior surface cricoarytenoidea
Apex cartilaginis Apex of arytenoid Capsula articularis Capsule of
arytenoideae cartilage cricoarytenoidea cricoarytenoid joint
Processus vocalis Vocal process Masculi laryngis Laryngeal muscles
Processus muscularis Muscular process M. aryepiglotticus Aryepiglottic muscle
Cartilago epiglottica Epiglottic cartilage M. cricothyroideus Cricothyroid muscle
Petiolus epiglottidis Stalk of epiglottis Stalk of Pars recta Straight part
epiglottic Pars obliqua Oblique part
cartilage M. cricoarytenoideus Posterior
Tuberculum Epiglottic tubercle posterior cricoarytenoid muscle
epiglotticum M. cricoarytenoideus Lateral cricoarytenoid
Cartilago corniculata Corniculate cartilage lateralis muscle
Cartilago cuneiformis Cuneiform cartilage M. thyroarytenoideus Thyroarytenoid
Cartilago triticea Triticeal cartilage muscle
Cartilagines tracheales Tracheal cartilage M. vocalis Vocalis muscle
Lig. thyrohyoideum Lateral thyrohyoid Ventricular
laterale ligament muscle
Lig. thyrohyoideum Median thyrohyoid M. thyroepiglotticus Thyroepiglottic
medianum ligament muscle
Membrana Thyrohyoid M. arytenoideus Oblique arytenoid
thyrohyoidea membrane obliquus muscle
Lig. cricothyroideum Cricothyroid ligament M. arytenoideus Transverse arytenoid
Lig. cricotracheale Cricotracheal transversus muscle
ligament Arytenoid
Lig. cricopharyngeum Cricopharyngeal muscle
ligament N. laryngeus superior Superior laryngeal
Lig. cricoarytenoideum Posterior nerve
posterius cricoarytenoid N. laryngeus inferior Inferior laryngeal
ligament nerve
Lig. thyroepiglotticum Thyroepiglottic N. laryngeus recurrens Recurrent laryngeal
ligament nerve
Glossary 331
Common term for Common term for

structure not structure not
contained in contained in
Terminologia Anatomica Anatomical Term Terminologia Terminologia Anatomica Anatomical Term Terminologia
(1998) (Latin) (English) Anatomica (1998) (Latin) (English) Anatomica
A. laryngea superior Superior laryngeal Lateral division
artery of inferior
Ascending laryngeal artery
branch Medial division
Dorsal branch of inferior
laryngeal artery
Medial branch
Ramus cricothyroideus Cricothyroid branch
Ventral branch
Ascending
Descending
branch
branch
Superficial
Anterior
branch
division of
descending Deep branch
branch Preepiglottic
Posterior space
division of Paraglottic
descending space
branch Cricoid area
A. laryngea inferior Inferior laryngeal
artery

Kiminori Sato (Auth.) - Functional Histoanatomy of The Human Larynx-Springer Singapore (2018) PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Kiminori Sato (Auth.) - Functional Histoanatomy of The Human Larynx-Springer Singapore (2018) PDF

Uploaded by

Copyright:

Available Formats

Kiminori Sato

ISBN 978-981-10-5585-0 ISBN 978-981-10-5586-7 (eBook)

Library of Congress Control Number: 2017964266

© Springer Nature Singapore Pte Ltd. 2018

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Kurume-shi, Japan Kiminori Sato, M.D., Ph.D.

Kiminori Sato, M.D., Ph.D.

Part I Whole-Organ Serial Sections of the Human Larynx

1 Whole-Organ Midsagittal Section of the Human Adult Larynx ������������������������� 3

Part II Functional Histoanatomy of the Human Larynx

7 Clinical Anatomy of the Human Larynx ��������������������������������������������������������������� 61

8.14 Thyrohyoid Membrane ��������������������������������������������������������������������������������������� 82

10 Cells and Extracellular Matrices in the

12.7 Cell Division of Cells in the Human Maculae Flavae��������������������������������������� 169

14.7 Cells Including Vocal Fold Stellate Cells in the

16.4.3 Light and Electron Microscopic Findings

17.15 Age-Related Changes of Cells including Vocal Fold Stellate Cells in

21.3 Physiologic Significance of the Blood Supply of the Larynx��������������������������� 289

22.14 Physiologic and Pathologic Significances of Irradiated Laryngeal Glands������� 314

Kiminori Sato, M.D., Ph.D. is a Professor of the Department

© Springer Nature Singapore Pte Ltd. 2018 3

Fig. 1.1 Midsagittal section of the human adult

© Springer Nature Singapore Pte Ltd. 2018 5

© Springer Nature Singapore Pte Ltd. 2018 23

© Springer Nature Singapore Pte Ltd. 2018 35

Fig. 4.1 Midsagittal section of the human newborn

© Springer Nature Singapore Pte Ltd. 2018 37

© Springer Nature Singapore Pte Ltd. 2018 49

7.1 Introduction approach. Additionally, understanding the histological struc-

© Springer Nature Singapore Pte Ltd. 2018 61

Fig. 7.1 Lateral and frontal epiglottic cartilage epiglottic cartilage

thyroid superior thyroid notch thyroid

arytenoid vocal process arytenoid

inferior thyroid notch

Lateral view Frontal view

(Fig. 7.4) [4]. The piriform sinus (piriform fossa, piriform

a inferior superior During operation, hemostasis of all these laryngeal arter-

The glottis consists of two parts: an intermembranous por-

window 7.3.1 Endoscopic Surgery

When phonosurgery is performed by endoscopic surgery, it is

silicon block 7.3.2 Endolaryngeal Microsurgery

When phonosurgery is perform by endolaryngeal microsur-

Fig. 7.4 Lateral and frontal views of the epiglottic cartilage

Lateral view Frontal view

posterior wall of glottis interarytenoid notch

membranous portion epiglottis

mucosal bulges. They are visible through the mucosa as

ascending branch of cricothyroid branch

laryngoscope posterior division of

Fig. 7.8 (continued) c anterior commissure tendon

posterior macula flava

Reinke’s space way to approach

Fig. 7.10 Endolaryngeal microsurgery for vocal fold cyst. An incision

When approaching Reinke’s space, the mucosa of the free

1. Hirano M. Phonosurgery. Basic and clinical investigations. Otologia

8.1 Introduction structure, compartments, histology, and histopathology of the

© Springer Nature Singapore Pte Ltd. 2018 69

Fig. 8.1 (a) Midsagittal

lamina of cricothyroid ligament

thyroepiglottic thyroid cartilage

c thyroid cartilage a nteriorr

At the level of the laryngeal ventricle (Fig. 8.2c), the mid-

Kurume-shi, Japan Kiminori Sato, M.D., Ph.D.

1 Whole-Organ Midsagittal Section of the Human Adult Larynx �� 3

7 Clinical Anatomy of the Human Larynx �� 61

8.14 Thyrohyoid Membrane �� 82

10 Cells and Extracellular Matrices in the

12.7 Cell Division of Cells in the Human Maculae Flavae�� 169

14.7 Cells Including Vocal Fold Stellate Cells in the

16.4.3 Light and Electron Microscopic Findings

17.15 Age-Related Changes of Cells including Vocal Fold Stellate Cells in

21.3 Physiologic Significance of the Blood Supply of the Larynx�� 289

22.14 Physiologic and Pathologic Significances of Irradiated Laryngeal Glands�� 314

7.1 Introduction approach. Additionally, understanding the histological struc-

window 7.3.1 Endoscopic Surgery

silicon block 7.3.2 Endolaryngeal Microsurgery

8.1 Introduction structure, compartments, histology, and histopathology of the

a 8.3.3 Midline Lateralization Thyroplasty

8.5 hysiological and Clinical Significance

8.9 hysiological and Clinical Significance

8.14 Thyrohyoid Membrane

8.16 Spaces of the Larynx

9.2 he Structures Around the Human

9.4 he Dimensions and Morphological

9.5 he Functions of the Human Newborn

9.6 he Clinical Significance

The mean diameter of the trachea (T on Fig. 9.9) is 9.7.1 Histopathologic Changes