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Current research approaches employ traditional tissue engineering strategies to promote vocal fold (VF) tissue regeneration, whereas recent novel advances seek to use principles of developmental biology to guide tissue generation by mimicking native developmental cues, causing tissue or allogenic/autologous progenitor cells to undergo the regeneration process. To address the paucity of data to direct VF differentiation and subsequent new tissue formation, we characterize structure-proliferation relationships and tissue elastic moduli over embryonic development using a murine model. Growth, cell proliferation, and tissue biomechanics were taken at E13.5, E15.5, E16.5, E18.5, P0, and adult time points. Quadratic growth patterns were found in larynx length, maximum transverse diameter, outer dorsoventral diameter, and VF thickness; internal VF length was found to mature linearly. Cell proliferation measured with EdU in the coronal and transverse planes of the VFs was found to decrease with increasing age. Exploiting atomic force microscopy, we measured significant differences in tissue stiffness across all time points except between E13.5 and E15.5. Taken together, our results indicate that as the VF mature and develop quadratically, there is a concomitant tissue stiffness increase. Greater gains in biomechanical stiffness at later prenatal stages, correlated with reduced cell proliferation, suggest that extracellular matrix deposition may be responsible for VF thickening and increased biomechanical function, and that the onset of biomechanical loading (breathing) may also contribute to increased stiffness. These data provide a profile of VF biomechanical and growth properties that can guide the development of biomechanically-relevant scaffolds and progenitor cell differentiation for VF tissue regeneration.

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In the present study, we investigated the role of mechanical load as generated by amniotic fluid in the vocal fold embryogenesis. In utero, amniotic fluid flows through the laryngeal inlet down into the lungs during fetal breathing and swallowing. In a mouse model, the onset of fetal breathing coincides with epithelial lamina recanalization. The epithelial lamina is a temporal structure that is formed during early stages of the larynx development and is gradually resorbed whereby joining the upper and lower airways. Here, we show that a temporary decrease in mechanical load secondary to drainage of amniotic fluid and subsequent flow restoration, impaired timing of epithelial lamina disintegration. Moreover, re-accumulation of fluid in the laryngeal region led to VF tissue deformation triggering remodeling of the epithelium and pressure generated changes in the elastic properties of the lamina propria, as measured by atomic force microscopy. We further show that load-related structural changes were likely mediated by Piezo 1 -Yap signaling pathway in the vocal fold epithelium. Understanding the relationship between the mechanical and biological parameters in the larynx is key to gaining insights into pathogenesis of congenital laryngeal disorders as well as mechanisms of vocal fold tissue remodeling in response to mechanotransduction.

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INTRODUCTION: Dysphonia is a known consequence of premature birth, and is usually associated with endotracheal intubation in the neonatal period or surgical ligation of persistent patent ductus arteriosus. Recently, cases of dysphonia, in the absence of these causative factors, have been reported. OBJECTIVES: This review seeks to identify literature pertaining to those aspects of laryngeal development that may potentially be disrupted by premature birth. The purpose of the review is to determine whether there is any possible anatomical or physiological explanation for dysphonia to arose solely from premature birth. METHODS: This scoping review was conducted in accordance with the guidelines prescribed by Arskey and O'Malley (2005). Fifteen relevant papers were identified. Results were categorized into age-related categories, to identify changes in the developmental trajectory. Based on the results of the literature search, a further category of unphonated larynges was added. RESULTS: Potential differences in the laryngeal framework (e.g., the development of the cricoid cartilage and the shape of the glottis) and vocal fold histology, depending on gestational age and post-natal phonation were identified. Much literature focused on the macula flavae, however, the layers of the lamina propria were also discussed. DISCUSSION: It is unclear whether the process of differentiation of the layers of the lamina propria, which commences in the second to third months of life in term-born infants, is disrupted by prematurity. Further, development of the macula flavae continues until at least 28 weeks' gestation. Preterm children may not phonate immediately after birth, which may also affect laryngeal development.

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BACKGROUND: The anterior commissure (AC) of the human larynx is usually understood as an area of the glottis anteriorly situated between the two vocal folds inserting to the thyroid cartilage (TC). The pattern of spread of AC carcinoma could hypothesize that AC could include other structures of the ventral larynx as developmental anatomy could demonstrate. METHODS: Ten adult larynx specimens from donation program cadavers and 15 selected fetal specimens (from 11 to 34 weeks of amenorrhoea) were studied using microdissection and histological serial sections stained with hematoxylin-eosin and reticuline. RESULTS: In adult specimens, internal perichondrium of the TC was easily detached from the entire lateral lamina but not from an intermediate lamina superiorly marked by the superior thyroid notch. On this median band of TC is inserted the ventral connective tissue of the three levels of the larynx: the ventral part of the vocal folds with the anterior macula flava, the Broyle's epiglottic ligament, and the subglottic part of the conoid ligament. In young fetuses (11-12 weeks), intermediate lamina of TC joined at the glottic level but not at the supraglottic level; in fetuses at 22-25 weeks, a meshwork of bundles of connective fibers inserted to the intermediate lamina of TC. In fetuses at 33-34 weeks, organization is practically identical to adult specimens. CONCLUSION: According to the adult anatomical features and the organogenesis, the AC of the human larynx could be anatomically defined ventrally as being made up of the intermediate lamina of TC beneath the superior thyroid notch and dorsally the ventral insertions of vocal folds with macula flava, supraglottic Broyle's ligament, and subglottic conoid ligament leading to a "developmental" AC definition which could better explain specific spreading of AC carcinoma.

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This investigation provides the first systematic determination of the cellular and molecular progression of vocal fold (VF) epithelium development in a murine model. We define five principal developmental events that constitute the progression from VF initiation in the embryonic anterior foregut tube to fully differentiated and functional adult tissue. These developmental events include (1) the initiation of the larynx and vocal folds with apposition of the lateral walls of the primitive laryngopharynx (embryonic (E) day 10.5); (2) the establishment of the epithelial lamina with fusion of the lateral walls of the primitive laryngopharynx (E11.5); (3) the epithelial lamina recanalization and separation of VFs (E13.5-18.5); (4) the stratification of the vocal folds (E13.5-18.5); and (5) the maturation of vocal fold epithelium (postnatal stages). The illustration of these morphogenetic events is substantiated by dynamic changes in cell proliferation and apoptosis, as well as the expression pattern of key transcription factors, FOXA2, SOX2 and NKX2-1 that specify and pattern the foregut endoderm. Furthermore, we documented the gradual conversion of VF epithelial cells from simple precursors expressing cytokeratins 8 and 18 in the embryo into mature stratified epithelial cells also expressing cytokeratins 5 and 14 in the adult. Interestingly, in the adult, cytokeratins 5 and 14 appear to be expressed in all cell layers in the VF, in contrast to their preferential localization to the basal cell layer in surrounding epithelium. To begin investigating the role of signaling molecules in vocal fold development, we characterized the expression pattern of SHH pathway genes, and how loss of Shh affects vocal fold development in the mutant. This study defines the cellular and molecular context and serves as the necessary foundation for future functional investigations of VF formation.

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This study is the first detailed qualitative morphologic description of the vocal fold and its associated structures (false vocal fold, larynx ventricle, epithelium, mucous glands, blood vessels, and vocal ligament) of a human fetus aged 25 weeks. In addition, a quantitative analysis of thyroarytenoid (TA) muscle fiber orientation is presented to investigate similarities with adult TA. Histologic cross sections from the vocal fold and the anterior, middle, and posterior regions of the TA muscle were examined bilaterally, and both qualitative and quantitative analyses show that the vocal fold and most of the associated structures are completely established in the studied sample.

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BACKGROUND: Since the seminal work of M. Hirano, which defined the three-layered lamina propria of the human vocal fold, there has been confusion in the labeling of each layer. Recent studies described the composition of fibers and interstitial molecules within the lamina propria leading to various biomechanical properties. However, collagen fibers appear as the most important structure component. METHODS: We used an optical analysis and the picrosirius-polarization method to describe collagen fibers from six adult and two fetal human larynges fixed in formalin and frontally sectioned in the middle part of the vocal fold. RESULTS: The deep layer of the lamina propria is the most densely organized band of collagen fibers penetrating the superficial muscle bundles of the vocal muscle. The mean thickness of this layer is about 36% of the lamina propria and shows a network of strongly birefringent fibers (collagen type I and III). The superficial layer of the lamina propria is a narrow band of collagen fibers immediately below the basement membrane of the epithelium. The mean thickness of this layer is about 13% of the lamina propria and shows strong birefringent fibers. The intermediate layer is the less densely organized band between the deep and superficial layers. The mean thickness of this layer is about 51% of the lamina propria and shows clear, green weakly birefringent fibers characterized as collagen type III. The fetal lamina propria contains only a monolayer distribution of loose collagen fibers between the epithelium and the vocal muscle. CONCLUSION: These results help describe the distribution of collagen fibers within the lamina propria of the human vocal fold and have implications to understand the cover-body theory of voice production both in the adult and newborn.

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Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert's resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

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OBJECTIVES: We undertook (1) to measure the cell density within the lamina propria of the neonatal vocal folds and (2) to examine changes in cell density in the lamina propria with increasing gestational age of the neonatal vocal folds. METHODS: Intact neonatal larynges were obtained from fresh cadaveric specimens. Hematoxylin and eosin-stained slides were used to visualize the laryngeal structures, and photomicrographs of the vocal folds were taken at 100x magnification. The cell density of the lamina propria was calculated by counting the cells within each of five 100-microm2 regions within the study area, and the totals were then averaged for each area. RESULTS: A total of 62 sections from 14 larynges with gestational ages of 19 to 36 weeks were examined. Histologic analysis revealed a uniform appearance of the vocal fold without apparent layers. The cell density of the lamina propria was 30 or more cells per 100 microm2 for 51.2% of larynges with less than 27 weeks of gestation. However, only 14.3% of the larynges with 27 or more weeks of gestation had an average cell density of 30 or more cells per region (p < 0.005). CONCLUSIONS: As described by previous studies, the lamina propria of the neonatal vocal folds is a hypercellular monolayer. The process of vocal fold maturation appears to occur earlier than previously thought, with decreasing cell density in the lamina propria by 27 weeks' gestation.

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OBJECTIVE: The aim of the present study was to investigate morphometric growth patterns of the cartilaginous components and vocal cords in human fetal larynx. METHODS: This study was performed in the Anatomy Department of Meram Medicine Faculty in 2007. The fetuses were obtained from the Gynaecology Department of the Meram Medicine Faculty of Selcuk University, and Dr. Faruk Sükan Maternity Hospital (Konya, Turkey). Forty spontaneously aborted fetuses (25 males and 15 females) without detectable malformations were evaluated. The dimensions of the larynx and its cartilaginous components were measured and the relationship between the obtained data was statistically assessed with Student's t-test. RESULTS: Correlations were found between the cartilaginous components, size of the larynx, and gestational age. No gender differences were identified. The anatomical variations in the epiglottis cartilage dimensions and types were found in 15% of cases. The foramen thyroideum was observed in 12.5% of cases. CONCLUSION: The data of this study may contribute to the knowledge of the fetal larynx regarding laryngomalacia.

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OBJECTIVES: Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. METHODS: Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. RESULTS: Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor a in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. CONCLUSIONS: These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage.

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Conservation laryngeal surgery is an increasingly available alternative for the treatment of laryngeal cancer. Understanding anatomy of laryngeal lymph drainage is essential for clinicians to diagnose, grade and surgically manage the laryngeal cancer. Although the lymphatic drainage of the larynx has been extensively studied, few studies revealed the relationship of the lymphatic drainage between various parts of the larynx. The distribution of lymphatic vessels in the inferior surface of the vocal cord also remains unclear. The aim of this study was to investigate the communication of the lymphatic networks between the vocal cord, the supraglottic and subglottic parts of the larynx, paying special attention to the lymphatic drainage of the inferior surface of the vocal cord. Eighteen larynx specimens from 18 fresh fetal cadavers were manually injected with prassion blue solution into the mucosal or submucosal layer of the larynx in order to reveal the lymphatic vessels in the inner larynx. We found that a rich lymphatic network is present in the inferior surface of the vocal cord, and the lymphatic networks in the superior and inferior surfaces of the vocal cord appear as two different patterns. These findings provide an anatomical basis for the design of a partial or conservation laryngeal surgery, particularly when considering the precise resection margin.

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The lamina propria of the human vocal fold consists of a superficial, intermediate, and deep layer. This stratified structure is thought to facilitate phonation. Each layer has different physical properties based on different alignment and distribution of collagen and elastic fibers. In the present study, developmental changes in vocal fold structure were studied in human fetuses, infants, and children, with special reference to the pattern of distribution of collagen and elastic fibers. Vocal fold specimens were obtained at autopsy from 5 fetuses, 7 neonates, 3 infants, 3 children at the age of 1 year, 3 children at 3 years, 3 children at 5 years, 3 children at 12 years, and 5 subjects at ages ranging from 15 to 22 years. Prior to the examination of collagen fibers, elastic fibers and cells were dissolved with 10% sodium hydroxide treatment. Prior to the examination of elastic fibers, collagen fibers and cells were dissolved by treatment with 90% formic acid. The specimens were then dehydrated, dried, ion-coated with platinum, and examined with a scanning electron microscope. In fetuses and infants, thin, coiled fibers were found distributed densely in the anterior, posterior, and deep parts of the lamina propria, while irregular thick fibers were sparsely seen in the superficial layer of the vocal fold. In children aged 1 to 3 years, the dense fibers in the deep part decreased, and the longitudinal fibers in the superficial layer increased. In children at 5 years of age, longitudinal collagen and elastic fibers were noted in all of the layers of the vocal fold. The distribution of fibers was uniform irrespective of the depth. At 12 years of age, thin, coiled fibers were noted in the superficial layer, while thin, irregular fibers were found in the deep layer. At 17 years, differentiation of the superficial and deep layers was more evident. In male subjects after adolescence the curvature of curly collagen fibers decreased, and the diameter of fibers increased. The present findings suggest that the development of the vocal fold in childhood occurs in two steps. In the first step, dense fibers in the anterior, posterior, and deep parts of the lamina propria found in fetuses and infants shift to the anterior and posterior ends of the vocal fold, between which longitudinal fibers appear. During this step only simple phonation is possible. In the second step, differentiation of the superficial and deep layers occurs, and the stratified structure of the vocal fold appears in the teens. This step is probably related to the complicated modality of phonation in this age group. In males, the development of the vocal fold is completed after changes in collagen fibers during the mutation.

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Histological analysis of the human vocal folds, focused particularly on the lamina propria of the vocal muscle of fetuses, infants, and adults in horizontal laryngeal sections, allowed us to show the differences in morphology and composition according to the laryngeal development. Our results indicate that the origin of the vocal fold is independent of the origin of the other portions of the larynx, and the influence of the zone known as macula flava in the maturation of the vocal fold is an age-related process.

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In this paper, a method to detect the vocal activities of the fetus is reported. An estimation of the intensity and frequency of the voice was made on the assumption that the fetus can vocalize. Based upon these results, experiments to detect the voice were made. From the analysis of the results, it was made clear that the voice seemed to be buried in the noise and difficult to detect.

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The morphogenesis of elastic fibers in human, fetal and adult vocal cords was studied by light microscopy and transmission electron microscopy. The elastic system includes elastic, elaunin and oxytalan fibers at different stages. The development of elastic-system fibers in human vocal cord is characterized by every stage of maturation, whether normal, stifled or accelerated, according to areas.

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Odontocetes (toothed whales) vocalize for communication and echolocation. The mechanisms of sound production, however, remain unclear. Their larynx has long been thought to lack vocal folds and, thus, was considered incapable of generating sounds. This study investigates internal anatomy of the odontocete larynx to: 1) describe the morphology of any folds found, 2) determine any structural homologies between these folds and the vocal folds of terrestrial mammals, and 3) assess their possible function in sound production. Larynges of 24 odontocetes representing ten genera (Delphinus, Stenella, Lagenorhynchus, Tursiops, Grampus, Delphinapterus, Globicephala, Kogia, Mesoplodon, and Phocoena) were studied post mortem. Nine specimens were cut midsagittally, and the remainder were dorsally opened to reveal internal anatomy. Results show that, contrary to established belief, vocal folds are consistently present. They are not isolated bands or "cords," but appear continuous with the internal laryngeal membrane. The attachments of these folds are the same as in terrestrial mammals, thus indicating homology with true mammalian vocal folds. These folds extend from the midline of the thyroid cartilage to the base of the arytenoid cartilages, sometimes to a discrete process. The vocal folds are elongated and oriented in a vertical plane, parallel to airflow direction. Vocal fold morphology varies, appearing as true bifurcated structures, a trifurcated fold, or a single midline fold. Laryngeal ventricles and vestibular folds are also consistently found lateral to the vocal folds. The vocal folds may divide the airstream within the larynx into three separate air currents. Fold vibrations may produce initial laryngeal sound used in echolocation or communication.

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The syrinx, the vocal organ in birds, shows sexual dimorphism in the duck, Anas platyrhynchos. At the cellular level, to examine the role of estrogen in sexually dimorphic and laterally asymmetric development of duck syrinx, cells dissociated from the right and left halves of sexually monomorphic and primitive masculine syrinxes from 10 2/3-day embryos of male and female ducks were cultured by the micromass culture method, with or without estrogen. In the absence of estrogen, primary cell cultures from either side of male syrinx revealed identical abilities in both cell proliferation and chondrogenesis. In the female cell cultures, the right- and left-side cells proliferated equally but the right-side cells accumulated a larger amount of sulfated proteoglycans than the left-side cells did. Both proliferation and chondrogenesis in the male cell cultures were more active than those in the female cell cultures. Estradiol inhibited significantly cell proliferation as well as chondrogenesis in cell cultures from either side of female syrinx. Cultures from right-side cells of male syrinx were less inhibited by estradiol in cell proliferation and especially in chondrogenesis than the other three cultures. Generally, in both sexes the left-side cells of syrinx were more responsive to estrogen than the right-side cells in diminishing proliferation and chondrogenesis. The present results suggest that estrogen inhibits both cell proliferation and chondrogenesis in the female syrinx and that this process may contribute to the development of sexual dimorphism in the duck syrinx.

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Previous investigations of vocal fold lamina propria in adult human larynx have revealed a highly specific organization and distribution of the connective fibres. The authors tried to elicit the developmental characteristics of that process using a material of foetal larynxes, ranging in foetal age from 13 to 23 weeks. The specimens were embedded in paraffin and then serially sectioned in both horizontal and vertical plane. The specific technique of differentiating elastic fibres from collagen fibres was utilized. The distribution and organization of connective tissue in foetal vocal fold lamina propria differed from that in adults and no straight elastic fibres were demonstrated as the first sign of later differentiation.

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