Mechanisms of larynx and vocal fold development and pathogenesis.

The larynx and vocal folds sit at the crossroad between digestive and respiratory tracts and fulfill multiple functions related to breathing, protection and phonation. They develop at the head and trunk interface through a sequence of morphogenetic events that require precise temporo-spatial coordination. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as specification of the laryngeal field, epithelial lamina formation and recanalization as well as the development and differentiation of mesenchymal cell populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding congenital laryngeal disorders and the evaluation and treatment approaches in human patients. This review highlights recent advances in our understanding of the laryngeal embryogenesis. Proposed genes and signaling pathways that are critical for the laryngeal development have a potential to be harnessed in the field of regenerative medicine.

Heterogeneity of Stem Cells in the Human Vocal Fold Mucosa.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for viscoelasticity in the human vocal fold mucosa as a vibrating tissue and are considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. Recent evidence has indicated that the cells including vocal fold stellate cells in the maculae flavae of the human vocal fold mucosa are a functionally heterogenous population. 4. The cells in the human maculae flavae possess proteins of all three germ layers, indicating that they are undifferentiated and have the ability of multipotency. 5. 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 putative stem cells. 6. Recent research has suggested that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 7. Cultured cell populations in the human maculae flavae are roughly divided into three groups by morphological features: cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells. However, at the present state of our investigation, it is difficult to clarify the stem cell system and hierarchy of stem cells in the human maculae flavae. 8. Subpopulations of cells in the maculae flavae proliferate extremely slowly and retain stem cell properties. 9. Tension caused by phonation seems to regulate the behavior and heterogeneity of the cells (mechanical regulation) in the maculae flavae of the human vocal fold. 10. The putative stem cells in the maculae flavae appear to differentiate into other kind of cells in the surrounding tissue.

Nasotracheal intubation depth in paediatric patients.

BACKGROUND: The aim of this study was to compare intubation depth using the Microcuff paediatric endotracheal tube (PET) placed with the intubation depth mark between the vocal cords with that of different published formulae/recommendations for nasotracheal intubation depth in children. METHODS: Children aged from birth to 10 yr undergoing elective surgery with nasotracheal intubation were included. Tracheal tubes were adjusted according to the intubation depth mark between the vocal cords using direct laryngoscopy. Nasal intubation depth was recorded and the distance 'tube tip to carina' was measured endoscopically. Based on the recorded nasal intubation depth and measured distance 'tube tip to carina', the position of tube tip and cuff was calculated according to six published formulae/recommendations. RESULTS: Seventy-six children were studied. For the Microcuff PET, the median tube tip advancement within the trachea was 52.9% (41.1-73.8%) of tracheal length. The shortest distance from the 'tube tip to carina' was 15 mm for a 3.5 mm internal diameter tube. If the six published formulae/recommendations had been used, this would have resulted in endobronchial tube placement in up to 9.1% of cases, and the tube tip would have been placed above the glottis in up to 2.6% of cases. The upper border of the cuff would have been placed in the subglottic area in up to 42.1% of cases and in a supraglottic position in up to 63.2% of cases. CONCLUSIONS: This study indicates that nasal intubation with the intubation depth mark placed between the vocal cords was superior to formula-based nasotracheal tube positioning. The latter would result in a high rate of endobronchial intubations, excessively high cuff positions and even tracheal extubations.

Quantitative distinction of unique vocal fold subepithelial architectures using optical coherence tomography.

OBJECTIVES: The primary objective of this study was to quantitatively analyze ex vivo porcine, fetal human, and adult human vocal folds by use of optical coherence tomography (OCT). A secondary objective was to quantitatively discriminate among 1-, 2-, and 3-layer lamina propria structures. METHODS: We performed an analysis of the vocal folds of 10 adult pig, 3 adult human, and 2 fetal human vocal fold specimens using OCT and histologic techniques. We present a quantitative comparison of the OCT results and histologic findings. RESULTS: We found that OCT allowed for the visualization of the subepithelial vocal fold architecture of all imaged tissue, and that it revealed distinct characteristic signal intensities for each type of specimen. CONCLUSIONS: Optical coherence tomography was developed for in vivo imaging of biological microstructures. This study demonstrates the ability of OCT to differentiate between the vocal fold architectures of 3 histologically distinct types of vocal folds. Future studies aim to develop a quantitative optical imaging algorithm that can be used to facilitate an in vivo longitudinal clinical investigation of the changes that occur in this layered structure over time and maturation.

Syringeal vocal folds do not have a voice in zebra finch vocal development.

Vocal behavior can be dramatically changed by both neural circuit development and postnatal maturation of the body. During song learning in songbirds, both the song system and syringeal muscles are functionally changing, but it is unknown if maturation of sound generators within the syrinx contributes to vocal development. Here we densely sample the respiratory pressure control space of the zebra finch syrinx in vitro. We show that the syrinx produces sound very efficiently and that key acoustic parameters, minimal fundamental frequency, entropy and source level, do not change over development in both sexes. Thus, our data suggest that the observed acoustic changes in vocal development must be attributed to changes in the motor control pathway, from song system circuitry to muscle force, and not by material property changes in the avian analog of the vocal folds. We propose that in songbirds, muscle use and training driven by the sexually dimorphic song system are the crucial drivers that lead to sexual dimorphism of the syringeal skeleton and musculature. The size and properties of the instrument are thus not changing, while its player is.

Evaluation of the Human Vocal Fold Lamina Propria Development Using Optical Coherence Tomography.

OBJECTIVES/HYPOTHESIS: Identifying distinctive features of the vocal fold (VF) during development could have significant clinical implications for treating voice disorders. This study investigates the structural organization of the VF microanatomy across gender and age groups using optical coherence tomography (OCT). STUDY DESIGN: Prospective clinical trial. MATERIALS AND METHODS: In vivo OCT images were acquired from 97 patients (58 males and 39 females) aged between 6 weeks and 27 years. All patients showed no signs of vocal fold pathology on endoscopy. Morphological features were extracted from OCT images and statistically compared between age groups. This study was performed at Massachusetts Eye and Ear between 2017 and 2019. RESULTS: All OCT acquisitions show a stratified microanatomy across age groups, even in newborns suggesting the presence of a superficial lamina propria (SLP) at birth. Furthermore, the optical scattering in the VF lamina propria changes according to age, suggesting subepithelial maturation. Although the epithelium thickness was relatively constant across age groups, the SLP showed a significant linear relationship between age and thickness (P = .016). Furthermore, a significant difference (P = .002) in SLP thickness was found between young adult males and females. The overall thickness of the entire mucosa did not change significantly with age. CONCLUSION: OCT is a noninvasive imaging modality capable of providing quantitative morphological features to describe the VF development. A stratified structure can be observed in OCT from newborns to young adults. Further investigations could combine OCT, acoustic measurements, and molecular sensitive techniques to provide a complete interpretation of the VF development. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2558-E2565, 2021.

Functional histology of the macula flava in the human vocal fold--Part 2: Its role in the growth and development of the vocal fold.

OBJECTIVE: This study aims to clarify the role of the maculae flavae (MFe) during growth and development of the human vocal fold mucosa (VFM). METHODS: Our current results concerning the MFe in the human newborn, infant, and child VFM are summarized. RESULTS: Newborns already had immature MFe at the same sites as adults. They were composed of dense masses of vocal fold stellate cells (VFSCs), whereas extracellular matrix components were sparse. VFSCs in the newborn MFe had already started synthesizing extracellular matrices (EM). During infancy, the EM synthesized in the MFe appeared in the VFM to initiate the formation of the three-dimensional extracellular matrix structure of the human VFM. During childhood, MFe including VFSCs continued to synthesize EM such as collagenous, reticular, and elastic fibers, and hyaluronic acid (glycosaminoglycan), which are essential for the human VFM as a vibrating tissue. The MFe in newborns, infants and children were related to the growth and development of the human VFM. CONCLUSION: Human MFe including VFSCs were inferred to be involved in the metabolism of EM, essential for the viscoelasticity of the human VFM, and are considered to be an important structure in the growth and development of the human VFM.

Producing American English vowels during vocal tract growth: a perceptual categorization study of synthesized vowels.

PURPOSE: To consider interactions of vocal tract change with growth and perceived output patterns across development, the influence of nonuniform vocal tract growth on the ability to reach acoustic-perceptual targets for English vowels was studied. METHOD: Thirty-seven American English speakers participated in a perceptual categorization experiment. For the experiment, an articulatory-to-acoustic model was used to synthesize 342 five-formant vowels, covering maximal vowel spaces for speakers at 5 growth stages (from 6 months old to adult). RESULTS: Results indicate that the 3 vowels /i u ae/ can be correctly perceived by adult listeners when produced by speakers with a 6-month-old vocal tract. Articulatory-to-acoustic relationships for these 3 vowels differ across growth stages. For a given perceived vowel category, the infant's tongue position is more fronted than the adult's. Furthermore, nonuniform vocal tract growth influences degree of interarticulator coupling for a given perceived vowel, leading to a reduced correlation between jaw height and tongue body position in infantlike compared with adult vocal tracts. CONCLUSION: Findings suggest that nonuniform vocal tract growth does not prevent the speaker from producing acoustic-auditory targets related to American English vowels. However, the relationships between articulatory configurations and perceptual targets change from birth to adulthood.

Registers in Infant Phonation.

The primary vocal registers of modal, falsetto, and fry have been studied in adults but not per se in infancy. The vocal ligament is thought to play a critical role in the modal-falsetto contrast but is still developing during infancy (Tateya and Tateya, 2015).41 Cover tissues are also implicated in the modal-fry contrast, but the low fundamental frequency (fo) cutoff of 70 Hz, shared between genders, suggests a psychoacoustic basis for the contrast. Buder, Chorna, Oller, and Robinson (2008)6 used the labels of "loft," "modal," and "pulse" for distinct vibratory regimes that appear to be identifiable based on spectrographic inspection of harmonic structure and auditory judgments in infants, but this work did not supply acoustic measurements to verify which of these nominally labeled regimes resembled adult registers. In this report, we identify clear transitions between registers within infant vocalizations and measure these registers and their transitions for fo and relative harmonic amplitudes (H1-H2). By selectively sampling first-year vocalizations, this manuscript quantifies acoustic patterns that correspond to vocal fold vibration types not previously cataloged in infancy. Results support a developmental basis for vocal registers, revealing that a well-developed ligament is not needed for loft-modal quality shifts as seen in harmonic amplitude measures. Results also reveal that a distinctively pulsatile register can occur in infants at a much higher fo than expected on psychoacoustic grounds. Overall results are consistent with cover tissues in infancy that are, for vibratory purposes, highly compliant and readily detached.

Vocal state change through laryngeal development.

Across vertebrates, progressive changes in vocal behavior during postnatal development are typically attributed solely to developing neural circuits. How the changing body influences vocal development remains unknown. Here we show that state changes in the contact vocalizations of infant marmoset monkeys, which transition from noisy, low frequency cries to tonal, higher pitched vocalizations in adults, are caused partially by laryngeal development. Combining analyses of natural vocalizations, motorized excised larynx experiments, tensile material tests and high-speed imaging, we show that vocal state transition occurs via a sound source switch from vocal folds to apical vocal membranes, producing louder vocalizations with higher efficiency. We show with an empirically based model of descending motor control how neural circuits could interact with changing laryngeal dynamics, leading to adaptive vocal development. Our results emphasize the importance of embodied approaches to vocal development, where exploiting biomechanical consequences of changing material properties can simplify motor control, reducing the computational load on the developing brain.

Human induced pluripotent stem cell-derived vocal fold mucosa mimics development and responses to smoke exposure.

Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells' limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.

Patterns of acquisition of native voice onset time in English-learning children.

Learning to speak involves both mastering the requisite articulatory gestures of one's native language and learning to coordinate those gestures according to the rules of the language. Voice onset time (VOT) acquisition illustrates this point: The child must learn to produce the necessary upper vocal tract and laryngeal gestures and to coordinate them with very precise timing. This longitudinal study examined the acquisition of English VOT by audiotaping seven children at 2 month intervals from first words (around 15 months) to the appearance of three-word sentences (around 30 months) in spontaneous speech. Words with initial stops were excerpted, and (1) the numbers of words produced with intended voiced and voiceless initial stops were counted; (2) VOT was measured; and (3) within-child standard deviations of VOT were measured. Results showed that children (1) initially avoided saying words with voiceless initial stops, (2) initially did not delay the onset of the laryngeal adduction relative to the release of closure as long as adults do for voiceless stops, and (3) were more variable in VOT for voiceless than for voiced stops. Overall these results support a model of acquisition that focuses on the mastery of gestural coordination as opposed to the acquisition of segmental contrasts.

Stop-consonant voicing and intraoral pressure contours in women and children.

Previous authors have established that stop consonant voicing is more limited in young children than adults, and have ascribed this to immature vocal-tract pressure management. Physical development relevant to speech aerodynamics continues into adolescence, suggesting that consonant voicing development may also persist into the school-age years. This study explored the relationship between stop consonant voicing and intraoral pressure contours in women, 5 year olds, and 10 year olds. Productions of intervocalic /p b/ were recorded from eight speakers at each age. Measures were made of stop consonant voicing and delta, a measure designed to characterize the time course of intraoral pressure increase in stops, following Muller and Brown [Speech and Language: Advances in Basic Research and Practice, edited by N. Lass (Academic, Madison, 1980), Vol. 4, pp. 318-389]. Age effects for stop consonant voicing and delta were not statistically significant, but correlations between delta and stop voicing were less often significant and sometimes reversed in the children, providing some evidence of immature aerodynamic control. The current data, as well as those of Muller and Brown, also show that the delta measure may yield some paradoxical values, indicating that more work is needed on methods of assessing time-varying characteristics of intraoral pressure.

Great talent, excellent voices-no problem for pubertal girls?

This research on 17 girls (aged 9;9 y to 16;11 y) singing in an established choir was focused on two issues: 1) the variety in physical and vocal development using Gackle's model, and 2) the matching of vocal demands and abilities. Developmental and acoustical data on the speaking and singing voice revealed considerable variation between individual girl singers. The model was greatly applicable. However, all girls had a greater total singing range, mainly in favour of the lower tones, and 11 girls used a lower speaking fundamental frequency. A third of the girls met the vocal and developmental features of their stage at a younger age. Next the lower limit of the frequency range of all girls was several semitones below the lowest notes of the pieces being worked on at the time of the experiment. However the upper limit of the pieces coincided with or exceeded their upper frequency limit.

Fundamental Frequency of Crying in Two-month-old Boys and Girls: Do Sex Hormones During Mini-puberty Mediate Differences?

OBJECTIVE: To evaluate whether the puberty-like sex hormone surge during the first months of life (mini-puberty) affects fundamental frequency (fo) in infant crying as one would derive from hormone influences on voice in adults. STUDY DESIGN: Populational prospective study. PARTICIPANTS: Twenty healthy normal-hearing infants (nine boys) were recruited for participation. METHODS: Spontaneously uttered cries were collected from each infant at 8 weeks of age. The cries were acoustically analyzed for mean fo and fo range. The fo properties were correlated to the average serum levels of bioavailable estradiol (E2) (mean E2/sex hormone-binding globulin [SHBG]) and testosterone (T) (mean T/SHBG) across the second month of life. RESULTS: Whereas no significant hormone effect was found for mean fo, a significant negative correlation (r = -0.55) was found between fo range and mean E2/SHBG. No indication for a T influence on fo features was found at this age. Although girls showed a slightly higher mean E2 concentration than boys did, the observed differences in cry fo range were judged to be reflective of an infant's serum concentration of E2 rather than a sex-based difference. CONCLUSION: In the absence of laryngeal size differences between female and male infants, the result was interpreted as indicative of an E2 influence on viscoelastic properties of the vocal folds. In our opinion, the investigation of young infants' vocalizations during the early postnatal surge of sex steroids (mini-puberty) may advance our understanding of the mechanisms mediating average sex differences in vocal development and early communication.

The anatomical evolution of the thyroid cartilage from childhood to adulthood: A computed tomography evaluation.

OBJECTIVE: To enhance knowledge and understanding of the laryngeal framework maturation in different age groups and genders. STUDY DESIGN: Cohort imaging study. SETTING: Tertiary academic referral center. METHODS: Computed tomography neck scans of 283 patients aged 8 to 20 years were studied. The interlaminae angle (ILA) of the thyroid cartilage at the level of the vocal folds, the anterior projection (angulation) of the thyroid cartilage (TC), and the degree of calcifications were evaluated and compared in sequential age groups of both genders. RESULTS: Neck scans of 171 males and 112 females were reviewed. The average ILA was 76.45° ± 14.2 and 94.25° ± 10.2 for males and females, respectively (P < 10-25). In the female group, the mean angle was relatively constant (91-970) in all age groups, whereas in the male groups the angle decreased with age (920-670) (r = -0.9, P < 0.005) The most significant decrease was measured in the 14- to 15-year age group. The thyroid prominence was significantly more anteriorly angulated in males. The angle in the female age groups was constant (170.1°), and the angle in males decreased with age (161.47°) (P = 0.000008). Calcifications were more prominent at the posterior portion of the cartilage in both genders and increased with age. CONCLUSION: Structural diversities of the TC begin in adolescent males because the thyroid cartilage grows anteriorly with a narrower ILA and with a greater anterior angulation. Our study shows that these changes, along with the degree of laryngeal cartilages calcification in both genders, occur as a continuum throughout puberty. LEVEL OF EVIDENCE: 4. Laryngoscope, 127:E354-E358, 2017.

Development of the human true vocal fold: depth of cell layers and quantifying cell types within the lamina propria.

OBJECTIVES: We sought to further describe the development of the 3-layered human vocal fold in children and to quantify macrophage and myofibroblast concentrations in each layer. METHODS: We used an optical analysis software package to examine 8 longitudinally sectioned human vocal folds that had been fixed in formalin (ages 2 days to 14 years). RESULTS: The 2-day-old vocal fold contained only a monolayer of cells. This became a bilayer by 5 months, and a trilayer began to become evident by 7 years. The percent of total depth represented by the superficial layer of the lamina propria (SLP) gradually decreased with age. The SLP made up 22% of the total lamina propria by age 7 years; this percentage approximates that in the adult vocal fold. Macrophages and myofibroblasts were predominately found in the SLP, and began to be apparent by 11 months of age. CONCLUSIONS: These results help describe the development of human voice and may have implications as to when phonosurgical therapy can be considered for children.

Postnatal development of rat vocal folds.

OBJECTIVES: In this study we aimed to determine the feasibility of using a rat model for the study of postnatal vocal fold (VF) development. METHODS: Eighteen male rats that were 3 days old, 3 weeks old, or 8 months old were analyzed histologically with Alcian blue stain used for detecting hyaluronic acid, elastin-van Gieson stain for elastin, Oil Red O and gold chloride stains for vitamin A-containing lipid droplets, and immunohistochemistry for vimentin (general fibroblast marker) and collagen types I and III. RESULTS: The macula flava (MF) was observed as a mass of cells that expressed vimentin intensively in the cytoplasm. The MF showed denser hyaluronic acid and collagen type I than did the midmembranous portion of the VF lamina propria. Clear developmental changes were evident in the MF and other regions. The vimentin-positive cells of the 3-day-old MF were mainly oval-shaped and had less cytoplasm, whereas those of the 8-month-old MF were spindle- and stellate-shaped and had more cytoplasm, similar to that reported in humans. Vitamin A-containing lipid droplets were limited to the 3-week-old and 8-month-old MFs and were not present in the 3-day-old VF. CONCLUSIONS: These results suggest that a rat model is useful in studying VF development and that vitamin A is related to the maturity of the VF.

Using attenuation coefficients from optical coherence tomography as markers of vocal fold maturation.

OBJECTIVES/HYPOTHESIS: Optical coherence tomography (OCT) is a promising technology to noninvasively assess vocal fold microanatomy. The goal of this study was to develop a methodology using OCT to identify quantifiable markers of vocal fold development. STUDY DESIGN: In vivo study. METHODS: A two-step process was developed to reproducibly image the midmembranous vocal fold edge of 10 patients younger than 2 years and 10 patients between 11 and 16 years of age using OCT. An image analysis algorithm was implemented to extract OCT-derived A-lines for each patient. These A-lines were divided into three zones according to apparent changes in slope. Relative attenuation coefficients, or tissue- and system-dependent parameters that describe the rate at which optical signal decays, were calculated for each zone. RESULTS: Young patients had distinct relative attenuation coefficients in zone 1 (P < .0001), whereas zones 2 and 3 were indistinct (P = .1129). Older patients had distinct relative attenuation coefficients in zones 1, 2, and 3 (P < .0370). Between age groups, relative attenuation coefficients were different in zones 2 and 3 (P < .0001, P = .0315, respectively) and indistinct in zone 1 (P = .1438). CONCLUSIONS: Relative attenuation coefficients can be used as markers of vocal fold development. Differences in relative attenuation coefficients likely represent changes in extracellular matrix structure within the lamina propria and may become useful for guiding treatment of voice disorders in the pediatric population. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E218-E223, 2016.

Development and maturation of the pediatric human vocal fold lamina propria.

OBJECTIVE: To identify characteristic patterns of maturation of the human vocal fold lamina propria as it develops into a mature structure. METHODS: Histologic evaluation of sectioned true vocal folds from 34 archived larynges ages 0 to 18 years using hematoxylin-eosin, trichrome, Alcian blue pH 2.5, Weigert reticular, and Miller's elastin stain. LOCATION: Pathology department at a tertiary care children's hospital. RESULTS: At birth and shortly thereafter, there exists a relative hypercellular monolayer of cells throughout the lamina propria. By 2 months of age, there are the first signs of differentiation into a bilaminar structure of distinct cellular population densities. Between 11 months and 5 years, two distinct patterns are seen: 1) this bilaminar structure and 2) a lamina propria where there exists a third more hypocellular region immediately adjacent to the vocalis muscle (this region is similar to the superficial hypocellular region found just deep to the surface epithelium). By 7 years of age, all of the specimens exhibit this transition between the middle and the deeper layers according to differential density of cell populations. A lamina propria structure defined by differential fiber composition (elastin and collagen fibers) is not present until 13 years of age and then is present throughout adolescence. CONCLUSIONS: Using the classic adult model of fiber composition and density to differentiate the layered structure of the lamina propria of the human vocal fold may not adequately allow for a thorough description of the process of maturation and development. Rather, distinct regions of cell density are seen as early as 2 months postpartum, and the model of cellular distribution may serve better to describe the lamina propria as it develops. Cell-signaling processes that shape the formation of the lamina propria appear to produce layered populations of differential cell density that in turn will later produce differential fiber compositions. Early development therefore can be followed by evaluating the maturation of these differing cell populations. Future studies are needed to quantify these cell distribution patterns, to study the cell signaling processes that trigger this maturation, and to correlate these findings with mechanical modeling.