Adapted to roar: functional morphology of tiger and lion vocal folds

Vocal production requires active control of the respiratory system, larynx and vocal tract. Vocal sounds in mammals are produced by flow-induced vocal fold oscillation, which requires vocal fold tissue that can sustain the mechanical stress during phonation. Our understanding of the relationship between morphology and vocal function of vocal folds is very limited. Here we tested the hypothesis that vocal fold morphology and viscoelastic properties allow a prediction of fundamental frequency range of sounds that can be produced, and minimal lung pressure necessary to initiate phonation. We tested the hypothesis in lions and tigers who are well-known for producing low frequency and very loud roaring sounds that expose vocal folds to large stresses. In histological sections, we found that the Panthera vocal fold lamina propria consists of a lateral region with adipocytes embedded in a network of collagen and elastin fibers and hyaluronan. There is also a medial region that contains only fibrous proteins and hyaluronan but no fat cells. Young's moduli range between 10 and 2000 kPa for strains up to 60%. Shear moduli ranged between 0.1 and 2 kPa and differed between layers. Biomechanical and morphological data were used to make predictions of fundamental frequency and subglottal pressure ranges. Such predictions agreed well with measurements from natural phonation and phonation of excised larynges, respectively. We assume that fat shapes Panthera vocal folds into an advantageous geometry for phonation and it protects vocal folds. Its primary function is probably not to increase vocal fold mass as suggested previously. The large square-shaped Panthera vocal fold eases phonation onset and thereby extends the dynamic range of the voice.     

Effect of the ventricular folds in a synthetic larynx model

Within the human larynx, the ventricular folds serve primarily as a protecting valve during swallowing. They are located directly above the sound-generating vocal folds. During normal phonation, the ventricular folds are passive structures that are not excited to periodical oscillations. However, the impact of the ventricular folds on the phonation process has not yet been finally clarified.An experimental synthetic human larynx model was used to investigate the effect of the ventricular folds on the phonation process. The model includes self-oscillating vocal fold models and allows the comparison of the pressure distribution at multiple locations in the larynx for configurations with and without ventricular folds.The results indicate that the ventricular folds increase the efficiency of the phonation process by reducing the phonation threshold level of the pressure below the vocal folds. Two effects caused by the ventricular folds could be identified as reasons: (1) a decrease in the mean pressure level in the region between vocal and ventricular folds (ventricles) and (2) an increase in the glottal flow resistance.The reason for the first effect is a reduction of the pressure level in the ventricles due to the jet entrainment and the low static pressure in the glottal jet. The second effect results from an increase in the glottal flow resistance that enhances the aerodynamic energy transfer into the vocal folds. This effect reduces the onset threshold of the pressure difference across the glottis.     

Modeling viscous dissipation during vocal fold contact: the influence of tissue viscosity and thickness with implications for hydration

The mechanics of vocal fold contact during phonation is known to play a crucial role in both normal and pathological speech production, though the underlying physics is not well understood. Herein, a viscoelastic model of the stresses during vocal fold contact is developed. This model assumes the cover to be a poroelastic structure wherein interstitial fluid translocates in response to mechanical squeezing. The maximum interstitial fluid pressure is found to generally increase with decreasing viscous dissipation and/or decreasing tissue elasticity. A global minimum in the total contact stress, comprising interstitial fluid pressure and elastic stress in the tissue, is observed over the studied dimensionless parameter range. Interestingly, physiologically reasonable estimates for the governing parameters fall within this global minimum region. The model is validated against prior experimental and computational work, wherein the predicted contact stress magnitude and impact duration agree well with published results. Lastly, observations of the potential relationship between vocal fold hydration and increased risk of tissue damage are discussed based upon model predictions of stress as functions of cover layer thickness and viscosity.     

Non-invasive in vivo measurement of the shear modulus of human vocal fold tissue

Voice is the essential part of singing and speech communication. Voice disorders significantly affect the quality of life. The viscoelastic mechanical properties of the vocal fold mucosa determine the characteristics of the vocal folds oscillations, and thereby voice quality. In the present study, a non-invasive method was developed to determine the shear modulus of human vocal fold tissue in vivo via measurements of the mucosal wave propagation speed during phonation. Images of four human subjects' vocal folds were captured using high speed digital imaging (HSDI) and magnetic resonance imaging (MRI) for different phonation pitches, specifically fundamental frequencies between 110 and 440 Hz. The MRI images were used to obtain the morphometric dimensions of each subject's vocal folds in order to determine the pixel size in the high-speed images. The mucosal wave propagation speed was determined for each subject and at each pitch value using an automated image processing algorithm. The transverse shear modulus of the vocal fold mucosa was then calculated from a surface (Rayleigh) wave propagation dispersion equation using the measured wave speeds. It was found that the mucosal wave propagation speed and therefore the shear modulus of the vocal fold tissue were generally greater at higher pitches. The results were in good agreement with those from other studies obtained via in vitro measurements, thereby supporting the validity of the proposed measurement method. This method offers the potential for in vivo clinical assessments of vocal folds viscoelasticity from HSDI.     

Modeling mechanical stresses as a factor in the etiology of benign vocal fold lesions

Vocal fold tissue lesions such as nodules and polyps are thought to develop in response to mechanical stress that occurs during vocal fold collision. Two computational models of vocal fold collision during voice production are used to investigate this hypothesis. A one-dimensional lumped mass model, whose parameters are derived from vocal fold tissue dimensions and material properties, predicts stress perpendicular to the direction of impact (normal stress). A previously published three-dimensional finite element model that incorporates the same dimensions and properties predicts the entire stress tensor. The hypothesis is supported by predictions from the finite element model that three components of normal stress and one component of shear stress are increased during collision in the typical location of lesions (i.e. the center of the superior medial edge of the vocal fold in the middle of the vibrating and contact region). The lumped mass model predicts that mechanical stress is negatively correlated with mucosal thickness (increased by voice warm-up and hydration), is positively correlated with driving force (proportional to voice intensity), and is affected by voice production method. These relationships are consistent with clinical observations of vocal fold lesion risk factors and have implications for improving prevention and treatment of benign vocal fold lesions.     

Influence of asymmetric stiffness on the structural and aerodynamic response of synthetic vocal fold models

The influence of asymmetric vocal fold stiffness on voice production was evaluated using life-sized, self-oscillating vocal fold models with an idealized geometry based on the human vocal folds. The models were fabricated using flexible, materially-linear silicone compounds with Young's modulus values comparable to that of vocal fold tissue. The models included a two-layer design to simulate the vocal fold layered structure. The respective Young's moduli of elasticity of the “left” and “right” vocal fold models were varied to create asymmetric conditions. High-speed videokymography was used to measure maximum vocal fold excursion, vibration frequency, and left–right phase shift, all of which were significantly influenced by asymmetry. Onset pressure, a measure of vocal effort, increased with asymmetry. Particle image velocimetry (PIV) analysis showed significantly greater skewing of the glottal jet in the direction of the stiffer vocal fold model. Potential applications to various clinical conditions are mentioned, and suggestions for future related studies are presented.     

Elasticity and stress relaxation of a very small vocal fold

Across mammals many vocal sounds are produced by airflow induced vocal fold oscillation. We tested the hypothesis that stress-strain and stress-relaxation behavior of rat vocal folds can be used to predict the fundamental frequency range of the species' vocal repertoire. In a first approximation vocal fold oscillation has been modeled by the string model but it is not known whether this concept equally applies to large and small species. The shorter the vocal fold, the more the ideal string law may underestimate normal mode frequencies. To accommodate the very small size of the tissue specimen, a custom-built miniaturized tensile test apparatus was developed. Tissue properties of 6 male rat vocal folds were measured. Rat vocal folds demonstrated the typical linear stress-strain behavior in the low strain region and an exponential stress response at strains larger than about 40%. Approximating the rat's vocal fold oscillation with the string model suggests that fundamental frequencies up to about 6 kHz can be produced, which agrees with frequencies reported for audible rat vocalization. Individual differences and time-dependent changes in the tissue properties parallel findings in other species, and are interpreted as universal features of the laryngeal sound source.     

Mechanism of initiation of oscillatory motion in human glottis

Phonatory onsets of 17 normal subjects under usual speech conditions were investigated by measuring the cross-sectional area of the glottic aperture, using a photoelectric device. During a normal soft or breathy onset of phonation, the vocal fold vibration begins with an open glottis; the pattern of the optical signal is a sine wave of increasing amplitude with one to eight cycles before steady state oscillation is achieved. The first deflection of the base-line is either towards adduction or towards abduction. The classically assumed--since van den Berg et al. (1957)--retro-aspiration phenomenon, consisting in a narrowing of the glottic chink due to the accelerated air flow, according to Bernoulli's law, is incompatible with these observations. An aerodynamic study, with accurate calibration of the photoelectric transducer in one trained subject, as well as flux and subglottic pressure measurements, suggests that the gas flux (air as well as a helium-oxygen mixture) reaches the condition of turbulence at the level of the glottic nozzle, just before vocal folds are set into oscillatory motion. The setting in motion of the free edge of the vocal folds in normal soft or breathy onset of phonation can be explained by a sudden modification of flow conditions within the expiratory gas : the flow is laminar in the trachea and suddenly becomes turbulent at the level of the glottic nozzle. On the other hand, approximately normal atmospheric pressure values are attained due to a Bernoulli-effect at that level, allowing the vocal folds to oscillate sinusoidally about their vibration axes, corresponding to their virtual resting position, like a forced oscillator.     

Synthetic,Multi-Layer,Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties ¹. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone ² and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry ^3,4, clinical instrument development ⁵, laryngeal aerodynamics ^6-9, vocal fold contact pressure ¹⁰, and subglottal acoustics ¹¹ (a more comprehensive list can be found in Kniesburges et al. ¹²)Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds ¹ that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages ^3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers ¹. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.     

Phase relationship between dynamics of the subglottic pressure and oscillatory movement of the vocal folds. I. Sustained phonation

The phase relationship between subglottic pressure and vocal fold length has been studied during sustained phonation in five subjects with normal larynx. Pressure was measured by tracheal puncture and vocal fold length was deduced from simultaneous measurement of translaryngeal impedance in the horizontal plane and transglottal light flux in the vertical plane. The pressure sine wave shows a phase lead of slightly less than 90 degrees relative to the length sine wave. Thus during sustained phonation the vocal apparatus behaves like a harmonic oscillator; the frequency of oscillation is determined by the mechanical parameters of the vibrating system; the source of periodic energy supply is the subglottal pressure wave.     

Optical measurements of vocal fold tensile properties: implications for phonatory mechanics

In voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress-stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects.     

A numerical analysis of phonation using a two-dimensional flexible channel model of the vocal folds.

A two-dimensional flexible channel model of the vocal folds coupled with an unsteady one-dimensional flow model is presented for an analysis of the mechanism of phonation. The vocal fold is approximated by springs and dampers distributed in the main flow direction that are enveloped with an elastic cover. In order to approximate three-dimensional collision of the vocal folds using the two-dimensional model, threshold values for the glottal width are introduced. The numerical results show that the collision plays an important role in speech sound, especially for higher resonant frequency components, because it causes the source sound to include high-frequency components.     

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.     

Cervids with different vocal behavior demonstrate different viscoelastic properties of their vocal folds

The authors test the hypothesis that vocal fold morphology and biomechanical properties covary with species‐specific vocal function. They investigate mule deer (Odocoileus hemionus) vocal folds, building on, and extending data on a related cervid, the Rocky Mountain elk (Cervus elaphus nelsoni). The mule deer, in contrast to the elk, is a species with relatively little vocal activity in adult animals. Mule deer and elk vocal folds show the typical three components of the mammalian vocal fold (epithelium, lamina propria and thyroarytenoid muscle). The vocal fold epithelium and the lamina propria were investigated in two sets of tensile tests. First, creep rupture tests demonstrated that ultimate stress in mule deer lamina propria is of the same magnitude as in elk. Second, cyclic loading tests revealed similar elastic moduli for the vocal fold epithelium in mule deer and elk. The elastic modulus of the lamina propria is also similar between the two species in the low‐strain region, but differs at strains larger than 0.3. Sex differences in the stress–strain response, which have been reported for elk and human vocal folds, were not found for mule deer vocal folds. The laminae propriae in mule deer and elk vocal folds are comparatively large. In general, a thick and uniformly stiff lamina propria does not self‐oscillate well, even when high subglottic pressure is applied. If the less stiff vocal fold seen in elk is associated with a differentiated lamina propria it would allow the vocal fold to vibrate at high tension and high subglottic pressure. The results of this study support the hypothesis that viscoelastic properties of vocal folds varies with function and vocal behavior. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Regeneration of Vocal Fold Mucosa Using Tissue-Engineered Structures with Oral Mucosal Cells

Objectives

Scarred vocal folds result in irregular vibrations during phonation due to stiffness of the vocal fold mucosa. To date, a completely satisfactory corrective procedure has yet to be achieved. We hypothesize that a potential treatment option for this disease is to replace scarred vocal folds with organotypic mucosa. The purpose of this study is to regenerate vocal fold mucosa using a tissue-engineered structure with autologous oral mucosal cells.

Study Design

Animal experiment using eight beagles (including three controls).

Methods

A 3 mm by 3 mm specimen of canine oral mucosa was surgically excised and divided into epithelial and subepithelial tissues. Epithelial cells and fibroblasts were isolated and cultured separately. The proliferated epithelial cells were co-cultured on oriented collagen gels containing the proliferated fibroblasts for an additional two weeks. The organotypic cultured tissues were transplanted to the mucosa-deficient vocal folds. Two months after transplantation, vocal fold vibrations and morphological characteristics were observed.

Results

A tissue-engineered vocal fold mucosa, consisting of stratified epithelium and lamina propria, was successfully fabricated to closely resemble the normal layered vocal fold mucosa. Laryngeal stroboscopy revealed regular but slightly small mucosal waves at the transplanted site. Immunohistochemically, stratified epithelium expressed cytokeratin, and the distributed cells in the lamina propria expressed vimentin. Elastic Van Gieson staining revealed a decreased number of elastic fibers in the lamina propria of the transplanted site.

Conclusion

The fabricated mucosa with autologous oral mucosal cells successfully restored the vocal fold mucosa. This reconstruction technique could offer substantial clinical advantages for treating intractable diseases such as scarring of the vocal folds.     

Design of a mechanical larynx with agarose as a soft tissue substitute for vocal fold applications

Mechanical and computational models consisting of flow channels with convergent and oscillating constrictions have been applied to study the dynamics of human vocal fold vibration. To the best of our knowledge, no mechanical model has been studied using a material substitute with similar physical properties to the human vocal fold for surgical experimentation. In this study, we design and develop a mechanical larynx with agarose as a vocal fold substitute, and assess its suitability for surgical experimentation. Agarose is selected as a substitute for the vocal fold as it exhibits similar nonlinear hyperelastic characteristics to biological soft tissue. Through uniaxial compression and extension tests, we determined that agarose of 0.375% concentration most closely resembles the vocal fold mucosa and ligament of a 20-year old male for small tensile strain with an R(2) value of 0.9634 and root mean square error of 344.05±39.84?Pa. Incisions of 10 mm lengthwise and 3 mm in depth were created parallel to the medial edge on the superior surface of agar phantom. These were subjected to vibrations of 80, 130, and 180 Hz, at constant amplitude of 0.9 mm over a period of 10 min each in the mechanical larynx model. Lateral expansion of the incision was observed to be most significant for the lower frequency of 80 Hz. This model serves as a basis for future assessments of wound closure techniques during microsurgery to the vocal fold.     

Differential cell adhesion to vocal fold extracellular matrix constituents.

The human vocal folds are a complex layering of cells and extracellular matrix. Vocal fold extracellular matrix uniquely contributes to the biomechanical viscoelasticity required for human phonation. We investigated the adhesion of vocal fold stellate cells, a novel cell type first cultured by our laboratory, and fibroblasts to eight vocal fold extracellular matrix components: elastin, decorin, fibronectin, hyaluronic acid, laminin and collagen types I, III and IV. Our data demonstrate that these cells adhere differentially to said substrates at 5 to 120 min. Cells were treated with hyaluronidase and Y-27632, a p160ROCK-specific inhibitor, to test the role of pericellular hyaluronan and Rho-ROCK activation in early and mature adhesion. Reduced adhesion resulted; greater inhibition of fibroblast adhesion was observed. We modulated the fibronectin affinity exhibited by both cell types using Nimesulide, an inhibitor of fibronectin integrin receptors alpha5beta1 and alphavbeta3. Our results are important in understanding vocal fold pathologies, wound healing, scarring, and in developing an accurate organotypic model of the vocal folds.     

Songbirds use pulse tone register in two voices to generate low-frequency sound

The principal physical mechanism of sound generation is similar in songbirds and humans, despite large differences in their vocal organs. Whereas vocal fold dynamics in the human larynx are well characterized, the vibratory behaviour of the sound-generating labia in the songbird vocal organ, the syrinx, is unknown. We present the first high-speed video records of the intact syrinx during induced phonation. The syrinx of anaesthetized crows shows a vibration pattern of the labia similar to that of the human vocal fry register. Acoustic pulses result from short opening of the labia, and pulse generation alternates between the left and right sound sources. Spontaneously calling crows can also generate similar pulse characteristics with only one sound generator. Airflow recordings in zebra finches and starlings show that pulse tone sounds can be generated unilaterally, synchronously or by alternating between the two sides. Vocal fry-like dynamics therefore represent a common production mechanism for low-frequency sounds in songbirds. These results also illustrate that complex vibration patterns can emerge from the mechanical properties of the coupled sound generators in the syrinx. The use of vocal fry-like dynamics in the songbird syrinx extends the similarity to this unusual vocal register with mammalian sound production mechanisms.