Vocal Cord / Fold Development

Newborn vocal folds

Newborns have a uniform monolayered lamina propria, which appears loose with no vocal ligament. The monolayered lamina propria is composed of ground substances such as hyaluronic acid and fibronectin, fibroblasts, elastic fibers, and collagenous fibers. While the fibrous components are sparse, making the lamina propria structure loose, the hyaluronic acid (HA) content is high.

HA is a bulky, negatively charged glycosaminoglycan, whose strong affinity with water procures HA its viscoelastic and shock absorbing properties essential to vocal biomechanics. Viscosity and elasticity are critical to voice production. Chan, Gray and Titze, quantified the effect of HA on both the viscosity and the elasticity of vocal folds (VF) by comparing the properties of tissues with and without HA. The results showed that removal of HA decreased the stiffness of VF by an average of 35%, but increased their dynamic viscosity by an average of 70% at frequencies higher than 1 Hz. Newborns have been shown to cry an average of 6.7 hours per day during the first 3 months, with a sustained pitch of 400-600 Hz, and a mean duration per day of 2 hours. Similar treatment on adult VF would quickly result in edema, and subsequently aphonia. Schweinfurth and al. presented the hypothesis that hyaluronic acid high content and distribution in newborn VF is directly associated with newborn crying endurance. These differences in newborn vocal fold composition would also be responsible for newborns inability to articulate sounds, besides the fact that their lamina propria is a uniform structure with no vocal ligament. The layered structure necessary for phonation will start to develop during the infancy and until the adolescence.

The fibroblasts in the newborn Reinke’s space are immature, showing an oval shape, and a large nucleus-cytoplasm ratio. The rough endoplasmic reticulum and Golgi apparatus, as shown by electron micrographs, are not well developed, indicating that the cells are in a resting phase. The collagenous and reticular fibers in the newborn VF are fewer than in the adult one, adding to the immaturity of the vocal fold tissue.

In the infant, many fibrous components were seen to extend from the macula flava towards the Reinke’s space. Fibronectin is very abundant in the Reinke’s space of newborn and infant. Fibronectin is a glycoprotein that is believed to act as a template for the oriented deposition of the collagen fibers, stabilizing the collagen fibrils. Fibronectin also acts as a skeleton for the elastic tissue formation. Reticular and collagenous fibers were seen to run along the edges of the VF throughout the entire lamina propria. Fibronectin in the Reinke’s space appeared to guide those fibers and orient the fibril deposition. The elastic fibers remained sparse and immature during infancy, mostly made of microfibrils. The fibroblasts in the infant Reinke’s space were still sparse but spindle-shaped. Their rough endoplasmic reticulum and Golgi apparatus were still not well developed, indicating that despite the change in shape, the fibroblasts still remained mostly in a resting phase. Few newly released materials were seen adjacent to the fibroblasts. The ground substance content in the infant Reinke’s space seemed to decrease over time, as the fibrous component content increased, thus slowly changing the vocal fold structure.

Adult vocal folds

Human VF are paired structures located in the larynx, just above the trachea, which vibrate and are brought in contact during phonation. The human VF are roughly 10-15 mm in length, and 3-5 mm thick. Histologically, the human VF are a laminated structure composed of five different layers. The vocalis muscle, main body of the VF, is covered by the mucosa, which consists of the epithelium and the lamina propria. The latter is a pliable layer of connective tissue subdivided into three layers: the superficial layer (SLP), the intermediate layer (ILP), and the deep layer (DLP). Layer distinction is either made looking at differential in cell content or ECM content. The most common way being to look at the ECM content. The SLP has fewer elastic and collagenous fibers than the two other layers, and thus is looser and more pliable. The ILP is mostly composed of elastic fibers, while the DLP has fewer elastic fivers, and more collagenous fibers. In those two layers, which form what is known as the vocalis ligament, the elastic and collagenous fivers are densely packed as bundles that run almost parallel to the edge of the vocal fold.

The extracellular matrix (ECM) of the VF LP is composed of fibrous proteins such as collagen and elastin, and interstitial molecules such as HA, a non-sulfated glycosaminoglycan. While the SLP is rather poor in elastic and collagenous fibers, the ILP and DLP are mostly composed of it, with the concentration of elastic fibers decreasing and the concentration of collagenous fibers increasing as the vocalis muscle is approached. Fibrous proteins and interstitial molecules play different roles within the ECM. While collagen (mostly type I) provides strength and structural support to the tissue, which are useful to withstanding stress and resisting deformation when subjected to a force, elastin fibers bring elasticity to the tissue, allowing it to return to its original shape after deformation. Interstitial proteins, such as HA, plays important biological and mechanical roles in the VF tissue. In the VF tissue, HA plays a role of shear-thinner, affecting the tissue viscosity, space-filler, shock absorber, as well as wound healing and cell migration promoter. The distribution of those proteins and interstitial molecules has been proven to be affected by both age and gender, and is maintained by the fibroblasts.

Vocal fold maturation

Vocal fold structure in adults is quite different from that in newborns. Exactly how the VF mature from an immature monolayer in newborns to a mature three layer tissue in adults is still unknown, however a few studies have investigated the subjects and brought some answers.

Hirano et al. previously found that the newborns did not have a true lamina propria, but instead had cellular regions called maculae flavae, located at the anterior and posterior ends of the loose vocal fold tissue. Boseley and Hartnick examined at the development and maturation of pediatric human vocal fold lamina propria. Hartnick was the first one to define each layer by a change in their cellular concentration. He also found that the lamina propria monolayer at birth and shortly thereafter was hypercellular, thus confirming Hirano’s observations. By 2 months of age, the vocal fold started differentiating into a bilaminar structure of distinct cellular concentration, with the superficial layer being less densely populated than the deeper layer. By 11 months, a three-layered structure starts to be noted in some specimens, again with different cellular population densities. The superficial layer is still hypocellular, followed by an intermediate more hypercellular layer, and a deeper hypercellular layer, just above the vocalis muscle. Even though the VF seem to start organizing, this is not representative of the trilaminar structure seen in adult tissues, where the layer are defined by their differential elastin and collagen fiber compositions. By 7 years of age, all specimens show a three-layered vocal fold structure, based on cellular population densities. At this point, the superficial layer was still hypocellular, the middle layer was the hypercellualr one, with also a greater content of elastin and collagen fibers, and the deeper layer was less cellularly populated. Again, the distinction seen between the layers at this stage is not comparable to that seen in the adult tissue. The maturation of the VF did not appear before 13 years of age, where the layers could be defined by their differential fiber composition rather than by their differential cellular population. The pattern now show a hypocellular superficial layer, followed by a middle layer composed predominantly of elastin fiber, and a deeper layer composed predominantly of collagen fibers. This pattern can be seen in older specimens up to 17 years of age, and above. While this study offers a nice way to see the evolution from immature to mature VF, it still does not explain what is the mechanism behind it.

Macula flavae

Maculae flavae are located at the anterior and posterior ends of the membranous parts of the VF. The histological structure of the macula flava is unique, and Sato and Hirano speculated that it could play an important role in growth, development and aging of VF. The macula flava is composed of fibroblasts, ground substances, elastic and collagenous fibers. Fibroblasts were numerous and spindle or stellate-shaped. The fibroblasts have been observed to be in active phase, with some newly released amorphous materials present at their surface. From a biomechanical point of view, the role of the macula flava is very important. Hirano and Sato studies suggested that the macula flava is responsible for the synthesis of the fibrous components of the VF. Fibroblasts have been found mostly aligned in the direction of the vocal ligament, along bundles of fibers. It then was suggested that the mechanical stresses during phonation were stimulating the fibroblasts to synthesize those fibers.

Impact of phonation

The viscoelastic properties of human vocal fold lamina propria are essential for their vibration, and depend on their ECM composition and structure. Adult VF have a layered structure which is based on the layers differential in ECM distribution. Newborns on the other hand, do not have this layered structure. Their VF are uniform, and immature, making their viscoelastic properties most likely unsuitable for phonation. HA plays a very important role in the vocal fold biomechanics. In fact, HA has been described as the ECM molecule that not only contributes to the maintenance of an optimal tissue viscosity that allows phonation, but also of an optimal tissue stiffness that allows frequency control. CD44 is a cell surface receptor for HA. Cells such as fibroblasts are responsible for synthesizing ECM molecules. Cell surface matrix receptors in return, feed back to the cells through cell-matrix interaction, allowing the cell to regulate its metabolism.

Sato et al. conducted a very interesting study recently. They carried out a histopathologic investigation of unphonated human VF. Vocal fold mucosae, which were unphonated since birth, of three young adults (17, 24, and 28 years old) were looked at using light and electron microscopy. Interestingly, the results show that the vocal fold mucosae were hypoplastic, and rudimentary, and like newborns, did not have any vocal ligament, Reinke’s space, or layered structure. Like newborns, the lamina propria appeared as a uniform structure. Some stellate cells were present in the macula flava, but started to show some signs of degeneration. The stellate cells synthesized fewer ECM molecules, and the cytoplasmic processes were shown to be short and shrinking, suggesting a decreased activity. Those results confirm the hypothesis that phonation stimulates stellate cells into producing more ECM.

Furthermore, using a specially designed bioreactor, Titze et al. showed that fibroblasts exposed to mechanical stimulation have differing levels of ECM production than fibroblasts that are not exposed to mechanical stimulation. The gene expression levels of ECM constituents such as fibronectin, MMP1, decorin, fibromodulin, HA synthase 2, and CD44 were altered. All those genes are involved in ECM remodeling, thus suggesting that mechanical forces applied to the tissue, alter the expression levels of ECM related genes, which in turn allow the cells present in the tissue to regulate the ECM constituent synthesis, thus affecting the tissue’s composition, structure, and biomechanical properties. In the end, cell-surface receptors close the loop by giving feedback on the surrounding ECM to the cells, affecting also their gene expression level.

Impact of hormones

Other studies suggest that hormones play also an important role in vocal fold maturation. Hormones are molecules secreted into the blood stream to be delivered at different targeted sites. They usually promote growth, differentiation and functionality in different organs or tissues. Their effect is due to their ability to bind to intracellular receptors, modulating the gene expression, and subsequently regulating protein synthesis. The interaction between the endocrine system and tissues such as breast, brain, testicles, heart, bones, etc, is being extensively studied. It has clearly been seen that the larynx is somewhat affected by hormonal changes, but surprisingly, very few studies are working on elucidating this relationship. The effect of hormonal changes in voice is clearly seen when hearing male and female voices, or when listening to a teenage voice changing during puberty. Actually, it is believed that the number of hormonal receptors in the pre-pubertal phase is higher than in any other age. Menstruation has also been seen to influence the voice. In fact, singers are encouraged by their instructors not to perform during their pre-menstrual period, because of a drop in their voice quality.

Vocal fold phonatory functions are known to change from birth to old age. The most significant changes occur in development between birth and puberty, and in old age. Hirano et al. previously described several structural changes associated with aging, in the vocal fold tissue. Some of those changes are: a shortening of the membranous vocal fold in males, a thickening of the vocal fold mucosa and cover in females, and a development of edema in the superficial lamina propria layer in both sexes. Hammond et al. observed that the HA content in the vocal fold lamina propria was significantly higher in males than in females. Although all those studies did show that there are clear structural and functional changes seen in the human VF which are associated with gender and age, none really fully elucidated the underlying cause of those changes. In fact, only a few recent studies started to look at the presence and role of hormone receptors in the VF. Newman et al. found that hormone receptors are indeed present in the VF, and show a statistical distribution difference with respect to age and gender. They have identified the presence of androgen, estrogen, and progesterone receptors in epithelial cells, grandular cells and fibroblasts of VF, suggesting that some of the structural changes seen in the VF could be due to hormonal influences. In this specific study, androgen and progesterone receptors were found more commonly in males than in females. In others studies, it has been suggested that the estrogen/androgen ratio be partly responsible for the voice changes observed at menopause. As previously said, Hammond et al. showed than the HA content was higher in male than in female VF. Bentley et al. demonstrated that sex skin swelling seen in monkey was due to an increase in HA content, which was in fact mediated by estrogen receptors in dermal fibroblasts. An increase in collagen biosynthesis mediated by the estrogen receptors of dermal fibroblasts was also observed. A connection between hormone levels, and ECM distribution in VF depending on age and gender could be made. More particularly a connection between higher hormone levels and higher HA content in males could exist in the human vocal fold tissue. Although a relationship between hormone levels and ECM biosynthesis in vocal fold can be established, the details of this relationship, and the mechanisms of the influence has not been elucidated yet.

Further Reading

This article is licensed under the Creative Commons Attribution-ShareAlike License. It uses material from the Wikipedia article on "Vocal folds" All material adapted used from Wikipedia is available under the terms of the Creative Commons Attribution-ShareAlike License. Wikipedia® itself is a registered trademark of the Wikimedia Foundation, Inc.

Last Updated: Feb 1, 2011

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