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TC
ACT a TC b TC c TC
AMF ACT ACT ACT
AMF AMF AMF
RS
LP LP
VL LP
PMF PMF
PMF
PMF VP VP
VP
VP
Fig. 1. Human adult MFe and VFM. TC = Thyroid cartilage; ACT = anterior commissure tendon; AMF = an-
terior macula flava; PMF = posterior macula flava; VP = vocal process of arytenoid cartilage; RS = Reinkes
space; VL = vocal ligament.
Fig. 2. Human newborn (a), infant (b), and child (c) MFe and growth and development of the human VFM.
LP = Lamina propria of the VFM.
vocal fold stellate cells (VFSCs) and extracellular matri- [2, 1820]. The lamina propria of the newborn VFM is a
ces (EM) such as collagenous, reticular, and elastic fibers, loose structure composed of ground substances and
glycoprotein, and hyaluronic acid (glycosaminoglycan). sparse fibers. Ground substance is present and glycopro-
These EM are essential for the human VFM as a vibrating teins (fibronectin) are abundant in the lamina propria
tissue. The VFSCs in the human adult MFe form an in- [19]. The EM structure of the newborn VFM is incom-
dependent cell category that should be considered a new plete.
category of cells in the human VF. Newborns have MFe at the same sites as adult VFM,
On the basis of the results of our current study, human but they are immature. The MFe of the newborn VFM are
MFe located at both ends of the VFM are inferred to be formed by dense masses of VFSCs (fig. 3, 4) and are situ-
involved in the metabolism of EM, essential for the vis- ated bilaterally at the anterior and posterior ends of the
coelastic properties of the lamina propria of the human newborn VFM. They are round in shape and measure ap-
adult VFM [23]. Human adult MFe are inferred to be proximately 1 ! 1 ! 1 mm. In comparison with adult
responsible for maintaining the characteristic layered MFe, the relative size is almost the same in both age
structure of the human VFM [23]. Human adult MFe are groups. The density of cells in the MFe is about 5 times
also considered to be an important structure in the aging that in the adult MFe (fig. 5). On the other hand, fibro-
of the human VFM [23]. blasts are sparse in the lamina propria of the newborn
Human VF grows and develops, and its layered struc- VFM. The density of fibroblasts in the newborn lamina
ture matures during adolescence [24, 25]. The purpose of propria is about one eighth that of VFSCs in the newborn
the present paper is to summarize the results of our cur- MFe (fig. 5). The MFe are composed of VFSCs, elastic fi-
rent morphological investigations into the MFe during bers, reticular fibers, collagenous fibers, and ground sub-
growth and development of the human VF. stances. Cellular components are markedly abundant
and fibrous components are sparse. More collagenous fi-
Fine Structure of Newborn MFe and VFM (fig. 2a) bers are present than elastic fibers.
[1820] VFSCs in the newborn MFe are stellate or oval in shape,
The structures of the newborn VFM are immature and possess cytoplasmic processes (fig. 6a). The newborn
and differ from those of adults. In newborn VF, the entire VFSCs are smaller in size than those of the adult, and
lamina propria appears as a uniform structure with no some cells form gap junctions with each other (fig. 6a). A
vocal ligament, no Reinkes space or no layered structure few lipid droplets are present in the cytoplasm but they are
a b
much fewer than those found in adults. The lipid droplets possess some features of mesenchymal cells. The morpho-
are 0.60.7 m in diameter, and are thus smaller than logical findings of the newborn VFSCs mentioned above
those of adults. The nucleus-cytoplasm ratio is high, and are recognized to various degrees.
intracellular organelles, such as rough endoplasmic retic- In many types of tissue and cultured cells, the interiors
ulum and Golgi apparatus, are not very well developed. of adjacent cells communicate with each other through
Free ribosomes are well developed in the cytoplasm. Along cell-to-cell channels [26]. The fine structure of the cell-
the periphery of the cytoplasm of the newborn VFSCs that to-cell channel has been well studied and defined as a gap
have developed intracellular organelles, a number of ves- junction [26]. Cell communication is proposed to play an
icles are present. Occasionally, a basal body (fetal rudi- important role in cell growth and differentiation [26].
ments) is noted in the cytoplasm. These findings indicate The VFSCs in the newborn MFe may communicate with
that the VFSCs in the newborn MFe are immature and each other for their growth and differentiation.
Role of Human Newborn, Infant, and Folia Phoniatr Logop 2010;62:263270 265
Child Vocal Fold Macula Flava
300 p < 0.05 300
250 250
p < 0.05
200 200
Number/field
Number/field
p < 0.05
150 150
100 100
NS
NS
NS p < 0.05
50 50 NS
0 0
Newborn Infant Child Adult Aged Newborn Infant Child Adult Aged
a b
Fig. 5. Cell density in the human VFM [data from ref. 12, 21]. NS = Not significant. a VFSC density in the hu-
man macula flava. b Fibroblast density in the lamina propria.
a b
Fig. 6. Transmission electron microscopy of VFSCs in the human newborn macula flava (tannic acid stain).
a Synthesized collagenous and elastic fibers are detected around VFSCs. N = Nucleus; GJ = gap junction; CP =
cytoplasmic process; rER = rough endoplasmic reticulum; M = mitochondrion; CF = collagenous fibers; EF =
elastic fibers. b Synthesis of collagenous and elastic fibers by VFSCs. v = Vesicle; am = amorphous material;
mf = microfibril; cf = collagen fibril; el = elastin.
There are many collagenous fibers around the VFSCs There are some vesicles along the periphery of the cyto-
in the adult MFe in contrast with the newborn MFe plasm, and newly released amorphous materials are pres-
(fig. 4b). Few reticular fibers can be detected around the ent on the cell surface of newborn VFSCs (fig. 6b). Micro-
newborn VFSCs. Electron microscopy indicates that the fibrils 1015 nm in width are observed around the amor-
newborn VFSCs have started to synthesize collagenous phous material. Collagen fibrils are detected near the
and reticular fibers. Synthesis of collagenous and reticu- microfibrils (fig. 6b). Collagenous fibers are made up of
lar fibers occurs in the same way as in the adult MFe. several collagen fibrils. Thus, the VFSCs have started to
Role of Human Newborn, Infant, and Folia Phoniatr Logop 2010;62:263270 267
Child Vocal Fold Macula Flava
The infant membranous portion of the VFM is stained their cytoplasm. The nucleus-cytoplasm ratio is relative-
light blue with Alcian blue at pH 2.5; in particular, the ly small, and intracellular organelles, such as rough en-
MFe are strongly stained. The material that stains with doplasmic reticulum, are not very well developed. Free
Alcian blue (pH 2.5) is digested by hyaluronidase. Hyal- ribosomes are present in the cytoplasm. Along the pe-
uronic acid appears in the VFM and much hyaluronic riphery of the cytoplasm of the childs VFSCs, vesicles are
acid is produced around the VFSCs in the infant MFe. present.
Most of the VFSCs in the infant MFe are stained with There are many collagenous, reticular, elastic fibers
CD44. The percentage of CD44-positive cells in the in- around the VFSCs in the child MFe. Synthesis of these
fant macula flava becomes larger, and 87.2 8 3.0% of the fibers occurs in the same way as in adult MFe. The elec-
stellate cells are stained with CD44 [12]. However, CD44- tron microscopic study indicates that the VFSCs in the
positive fibroblasts become sparse (1.9 8 2.0%) in the child MFe continue to constantly synthesize collagenous,
infant lamina propria of the VFM [12]. The expression of reticular, and elastic fibers.
CD44 and the distribution of hyaluronic acid are the Hyaluronic acid is present in the lamina propria,
same as in the adult VFM. VFSCs and CD44 coopera- in particular, in the MFe of the child VFM. Most of the
tively start to play important roles in the maintenance of VFSCs (94.7 8 1.9%) in the child MFe are stained with
hyaluronic acid in the human VFM during infancy. CD44 [12]. Almost all of the VFSCs in the child MFe
To summarize, during infancy, EM synthesized in the show CD44 expression and a large amount of hyaluronic
MFe appear in the lamina propria of the VFM between acid is present immediately adjacent to VFSCs in the
the anterior and posterior MFe at different times to initi- child MFe. On the other hand, CD44-positive fibroblasts
ate the formation of three-dimensional extracellular ma- are sparse (5.6 8 3.0%) in the lamina propria of the child
trix structure of the VFM that has the viscoelastic prop- VFM [12]. These findings are the same as those in adults.
erties of a vibrating structure. The VFSCs in the MFe and CD44 both continue to play
roles in the metabolism of hyaluronic acid in the human
Fine Structure of Child MFe and VFM (fig. 2C) [21] VFM during childhood.
There are collagenous, reticular and elastic fibers in To summarize, the child MFe are composed of dense
the lamina propria of the child VFM. Vocal ligament and VFSCs and EM. The morphological characteristics of the
layered structure are not present in the lamina propria of VFSCs in the child MFe are not completely the same as
the child VFM. The ground substance throughout the those of adults. However, the VFSCs in the child MFe
VFM is stained light blue with Alcian blue at pH 2.5. The have features characteristic of adult VFSCs and continue
material that stains in the lamina propria of the mucosa to constantly synthesize EM, essential for viscoelastic
with Alcian blue (pH 2.5) is digested by hyaluronidase. properties of the lamina propria of the human VFM as a
Hyaluronic acid is present in the lamina propria of the vibrating tissue. The child MFe including VFSCs con-
child VFM. tinue to synthesize EM and EM have increased. MFe have
MFe are located bilaterally at the anterior and poste- a role to play in the metabolism of EM of the VFM in the
rior ends of the child VFM. They are approximately 1 ! stage of VF growth and development. The VFSCs in the
1 ! 1 mm in size and consist of dense masses of VFSCs. MFe are inferred to be involved in the growth and devel-
Many more VFSCs are distributed in the child MFe than opment of human VFM.
in those of adults. The density of VFSCs in child MFe is
about twice that in adult MFe, and about half that in new- Growth and Development of the Human VFM [22]
born MFe (fig. 5). On the other hand, fibroblasts are Among mammals, only humans can speak and only
sparse in the lamina propria of child VFM. The density human adult VF has a vocal ligament, Reinkes space, and
of fibroblasts is about one fourth that of VFSCs in child a layered structure [28]. Why do only human adults have
MFe. The childs MFe are composed of VFSCs, collage- such a characteristic VF structure? Why and how does
nous fibers, reticular fibers, elastic fibers, and ground newborn VFM grow, develop and mature? What are the
substances. The fibrous components have increased by factors for initiating and continuing the growth of the
comparison with those of the infant MFe. human VFM?
The VFSCs are stellate and possess cytoplasmic pro- Tension is the most important factor which influences
cesses. A few lipid droplets are present in the cytoplasm, the synthesis of collagenous fibers by fibroblasts [29, 30].
but they are much fewer and smaller (0.61 m in diam- The bending stresses on the VF associated with phona-
eter) than those found in adults. Vitamin A is stored in tion are greatest in the region of the MFe [31]. We hypoth-
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