Editing Vocal cords (section)

===Adulthood===
Human vocal cords are paired structures located in the larynx, just above the trachea, which vibrate and are brought in contact during phonation. The human vocal cords are roughly 12 – 24&nbsp;mm in length, and 3–5&nbsp;mm thick.<ref>{{cite journal |vauthors=Hahn MS, Teply BA, Stevens MM, Zeitels SM, Langer R |title=Collagen composite hydrogels for vocal fold lamina propria restoration |journal=Biomaterials |volume=27 |issue=7 |pages=1104–9 |date=March 2006 |pmid=16154633 |doi=10.1016/j.biomaterials.2005.07.022 }}</ref> Histologically, the human vocal cords are a laminated structure composed of five different layers. The vocalis muscle, main body of the vocal cords, is covered by the mucosa, which consists of the epithelium and the lamina propria.<ref name="Hirano, M. 1981">Hirano, M., S. Kurita, and T. Nakashima. Vocal fold physiology : contemporary research and clinical issues. in Vocal Fold Physiology, Conference. 1981. San Diego, Calif.: College-Hill Press.</ref> The latter is a pliable layer of connective tissue subdivided into three layers: the superficial layer (SL), the intermediate layer (IL), and the deep layer (DL).<ref name="Gray00">{{cite journal|author=Gray SD|date=August 2000|title=Cellular physiology of the vocal folds|journal=Otolaryngol. Clin. North Am.|volume=33|issue=4|pages=679–98|doi=10.1016/S0030-6665(05)70237-1|pmid=10918654}}</ref> Layer distinction is either made looking at differential in cell content or [[extracellular matrix]] (extracellular matrix) content. The most common way being to look at the extracellular matrix 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 fibers, and more collagenous fibers.<ref name="Hirano, M. 1981"/> In those two layers, which form what is known as the vocalis ligament, the elastic and collagenous fibers are densely packed as bundles that run almost parallel to the edge of the vocal fold.<ref name="Hirano, M. 1981"/>

There is a steady increase in the elastin content of the lamina propria as humans age (elastin is a yellow scleroprotein, the essential constituent of the elastic [[connective tissue]]) resulting in a decrease in the ability of the lamina propria to expand caused by cross-branching of the elastin fibers. Among other things, this leads to the mature voice being better suited to the rigors of opera.<ref>{{Cite web |title=https://catalyst.library.jhu.edu/discovery/fulldisplay?&context=PC&vid=01JHU_INST:JHU&search_scope=All_except_EU&tab=Everything&docid=cdi_proquest_journals_2451167727 |url=https://catalyst.library.jhu.edu/discovery/fulldisplay?&context=PC&vid=01JHU_INST:JHU&search_scope=All_except_EU&tab=Everything&docid=cdi_proquest_journals_2451167727 |access-date=2025-01-30 |website=catalyst.library.jhu.edu |language=en}}</ref>

The extracellular matrix of the vocal cord LP is composed of fibrous proteins such as collagen and elastin, and interstitial molecules such as [[hyaluronic acid|HA]], a non-sulfated [[glycosaminoglycan]].<ref name=Gray00/> 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.<ref name="Hirano, M. 1981"/> Fibrous proteins and interstitial molecules play different roles within the extracellular matrix. 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.<ref name=Gray00/> Interstitial proteins, such as HA, plays important biological and mechanical roles in the vocal cord tissue.<ref name=Ward02/> In the vocal cord tissue, hyaluronic acid 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]].<ref name=Gray00/><ref name=Hammond97>{{cite journal |vauthors=Hammond TH, Zhou R, Hammond EH, Pawlak A, Gray SD |title=The intermediate layer: a morphologic study of the elastin and hyaluronic acid constituents of normal human vocal folds |journal=J Voice |volume=11 |issue=1 |pages=59–66 |date=March 1997 |pmid=9075177 |doi=10.1016/s0892-1997(97)80024-0}}</ref><ref name=Ward02/><ref>{{cite journal |vauthors=Hammond TH, Gray SD, Butler J, Zhou R, Hammond E |title=Age- and gender-related elastin distribution changes in human vocal folds |journal=Otolaryngol Head Neck Surg |volume=119 |issue=4 |pages=314–22 |date=October 1998 |pmid=9781983 |doi=10.1016/s0194-5998(98)70071-3|s2cid=71920488 }}</ref>

====Maturation====
Vocal fold structure in adults is quite different from that in newborns. Exactly how the vocal cord 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.<ref name="Sato, K. 2001"/><ref>{{cite journal |vauthors=Sato K, Hirano M |title=Histologic investigation of the macula flava of the human newborn vocal fold |journal=Ann. Otol. Rhinol. Laryngol. |volume=104 |issue=7 |pages=556–62 |date=July 1995 |pmid=7598369 |doi=10.1177/000348949510400710|s2cid=32824702 }}</ref> Boseley and Hartnick examined at the development and maturation of pediatric human vocal fold lamina propria.<ref>{{cite journal |vauthors=Boseley ME, Hartnick CJ |title=Development of the human true vocal fold: depth of cell layers and quantifying cell types within the lamina propria |journal=Ann. Otol. Rhinol. Laryngol. |volume=115 |issue=10 |pages=784–8 |date=October 2006 |pmid=17076102 |doi=10.1177/000348940611501012|s2cid=21613826 }}</ref> Hartnick was the first one to define each layer by a change in their cellular concentration.<ref>{{cite journal |vauthors=Hartnick CJ, Rehbar R, Prasad V |title=Development and maturation of the pediatric human vocal fold lamina propria |journal=Laryngoscope |volume=115 |issue=1 |pages=4–15 |date=January 2005 |pmid=15630357 |doi=10.1097/01.mlg.0000150685.54893.e9 |s2cid=6024918 }}</ref> 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 vocal cords 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 hypercellular 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 vocal cords 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 vocal cords, 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 vocal cords.<ref>{{cite journal |vauthors=Sato K, Hirano M |title=Histologic investigation of the macula flava of the human vocal fold |journal=Ann. Otol. Rhinol. Laryngol. |volume=104 |issue=2 |pages=138–43 |date=February 1995 |pmid=7857016 |doi=10.1177/000348949510400210|s2cid=12529469 }}</ref> 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 the vocal cords. 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 vocal cords. 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 [[viscoelasticity|viscoelastic]] properties of human vocal fold lamina propria are essential for their vibration, and depend on the composition and structure of their [[extracellular matrix]]. Adult vocal cords have a layered structure which is based on the layers differential in extracellular matrix distribution. Newborns on the other hand, do not have this layered structure. Their vocal cords are uniform, and immature, making their viscoelastic properties most likely unsuitable for phonation. Hyaluronic acid plays a very important role in the vocal fold biomechanics. In fact, hyaluronic acid has been described as the extracellular matrix 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.<ref name=Chan01/> [[CD44]] is a cell surface receptor for HA. Cells such as [[fibroblast]]s are responsible for synthesizing extracellular matrix 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.<ref>{{cite journal |vauthors=Sato K, Nakashima T, Nonaka S, Harabuchi Y |title=Histopathologic investigations of the unphonated human vocal fold mucosa |journal=Acta Otolaryngol. |volume=128 |issue=6 |pages=694–701 |date=June 2008 |pmid=18568507 |doi=10.1080/00016480701675643 |s2cid=21410937 }}</ref> carried out a histopathologic investigation of unphonated human vocal cords. 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. 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 cell]]s were present in the macula flava, but started to show some signs of degeneration. The stellate cells synthesized fewer extracellular matrix 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 extracellular matrix.

Furthermore, using a specially designed bioreactor, Titze et al. showed that fibroblasts exposed to mechanical stimulation have differing levels of extracellular matrix production from fibroblasts that are not exposed to mechanical stimulation.<ref>{{cite journal |vauthors=Titze IR, Hitchcock RW, Broadhead K, et al |title=Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses |journal=J Biomech |volume=37 |issue=10 |pages=1521–9 |date=October 2004 |pmid=15336927 |doi=10.1016/j.jbiomech.2004.01.007 }}</ref> The gene expression levels of extracellular matrix constituents such as fibronectin, MMP1, decorin, fibromodulin, hyaluronic acid synthase 2, and [[CD44]] were altered. All those genes are involved in extracellular matrix remodeling, thus suggesting that mechanical forces applied to the tissue, alter the expression levels of extracellular matrix related genes, which in turn allow the cells present in the tissue to regulate the extracellular matrix 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 extracellular matrix 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.<ref name=Rios08>{{cite journal |vauthors=Rios OA, Duprat Ade C, Santos AR |title=Immunohistochemical searching for estrogen and progesterone receptors in women vocal fold epithelia |journal=Braz J Otorhinolaryngol |volume=74 |issue=4 |pages=487–93 |year=2008 |pmid=18852972 |doi=10.1016/S1808-8694(15)30593-0 |pmc=9442059 |doi-access=free }}</ref> 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, 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.<ref name=Rios08/> 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.<ref name=Rios08/>

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.<ref name="Hirano, M. 1981"/><ref name=Newman00>{{cite journal |vauthors=Newman SR, Butler J, Hammond EH, Gray SD |title=Preliminary report on hormone receptors in the human vocal fold |journal=J Voice |volume=14 |issue=1 |pages=72–81 |date=March 2000 |pmid=10764118 |doi=10.1016/s0892-1997(00)80096-x}}</ref>  Hirano et al. previously described several structural changes associated with aging, in the vocal fold tissue.<ref>{{cite journal |vauthors=Hirano M, Kurita S, Sakaguchi S |title=Ageing of the vibratory tissue of human vocal folds |journal=Acta Otolaryngol. |volume=107 |issue=5–6 |pages=428–33 |year=1989 |pmid=2756834 |doi=10.3109/00016488909127535}}</ref> 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 hyaluronic acid content in the vocal fold lamina propria was significantly higher in males than in females.<ref name=Hammond97/> Although all those studies did show that there are clear structural and functional changes seen in the human vocal cords 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 vocal cords. Newman et al. found that hormone receptors are indeed present in the vocal cords, and show a statistical distribution difference with respect to age and gender.<ref name=Newman00/> They have identified the presence of [[androgen]], [[estrogen]], and [[progesterone]] receptors in [[epithelial cell]]s, [[granular cell]]s and [[fibroblast]]s of the vocal cords, suggesting that some of the structural changes seen in the vocal cords could be due to hormonal influences.<ref name=Newman00/> 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.<ref>{{cite journal |last=Nelson |first=J.F. |title=The potential role of selected endocrine systems in aging processes |year=1995 |journal=Comprehensive Physiology |pages=377–394 |publisher=Wiley Online Library |url=http://www.comprehensivephysiology.com/WileyCDA/CompPhysArticle/refId-cp110115.html |doi=10.1002/cphy.cp110115 |url-status=live |archive-url=https://web.archive.org/web/20140809215612/http://www.comprehensivephysiology.com/WileyCDA/CompPhysArticle/refId-cp110115.html |archive-date=2014-08-09 |isbn=9780470650714 }}</ref> As previously said, Hammond et al. showed than the hyaluronic acid content was higher in male than in female vocal cords. Bentley et al. demonstrated that sex skin swelling seen in monkey was due to an increase in hyaluronic acid content, which was in fact mediated by estrogen receptors in dermal fibroblasts.<ref>{{cite journal |vauthors=Bentley JP, Brenner RM, Linstedt AD, etal |title=Increased hyaluronate and collagen biosynthesis and fibroblast estrogen receptors in macaque sex skin |journal=J. Invest. Dermatol. |volume=87 |issue=5 |pages=668–73 |date=November 1986 |pmid=3772161 |doi=10.1111/1523-1747.ep12456427|doi-access= }}</ref> An increase in collagen biosynthesis mediated by the estrogen receptors of dermal fibroblasts was also observed. A connection between hormone levels, and [[extracellular matrix]] distribution in the vocal cords depending on age and gender could be made. More particularly a connection between higher hormone levels and higher hyaluronic acid content in males could exist in the human vocal fold tissue. Although a relationship between hormone levels and extracellular matrix biosynthesis in vocal fold can be established, the details of this relationship, and the mechanisms of the influence has not been elucidated yet.