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.     

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.     

Mechanical stress during phonation in a self-oscillating finite-element vocal fold model

The stress information during phonation in the vocal folds is helpful in understanding the etiologies of vocal trauma and its related vocal diseases, such as nodules. In this paper, a self-oscillating finite-element model, which combines aerodynamic properties, tissue mechanics, airflow-tissue interactions, and vocal fold collisions, was used to simulate the vocal fold vibration during phonation. The spatial and temporal characteristics of mechanical stress in the vocal folds were predicted by this model. Temporally, it was found that mechanical stress periodically undulates with vibration of the vocal folds and that vocal fold impact causes a jump in the normal stress value. Spatially, the normal stress is significantly higher on the vocal fold surface than inside of the vocal folds. At the midpoint of the medial surface, the peak-to-peak amplitude of the normal stress reaches its maximum value. Using different lung pressures (0-1.5kPa) to drive the self-oscillating model, we found that lower lung pressure can effectively decrease the mechanical stress in the vocal folds. This study supports the fatigue damage hypothesis of vocal trauma. With this hypothesis and the numerical simulation in this study, the clinical observations of vocal fold trauma risk can be explained. This implies the mechanical stress predicted by this self-oscillating model could be valuable for predicting, preventing, and treating vocal fold injury.     

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.     

Effects of Vocal Fold Nodules on Glottal Cycle Measurements Derived from High-Speed Videoendoscopy in Children

The goal of this study is to quantify the effects of vocal fold nodules on vibratory motion in children using high-speed videoendoscopy. Differences in vibratory motion were evaluated in 20 children with vocal fold nodules (5–11 years) and 20 age and gender matched typically developing children (5–11 years) during sustained phonation at typical pitch and loudness. Normalized kinematic features of vocal fold displacements from the mid-membranous vocal fold point were extracted from the steady-state high-speed video. A total of 12 kinematic features representing spatial and temporal characteristics of vibratory motion were calculated. Average values and standard deviations (cycle-to-cycle variability) of the following kinematic features were computed: normalized peak displacement, normalized average opening velocity, normalized average closing velocity, normalized peak closing velocity, speed quotient, and open quotient. Group differences between children with and without vocal fold nodules were statistically investigated. While a moderate effect size was observed for the spatial feature of speed quotient, and the temporal feature of normalized average closing velocity in children with nodules compared to vocally normal children, none of the features were statistically significant between the groups after Bonferroni correction. The kinematic analysis of the mid-membranous vocal fold displacement revealed that children with nodules primarily differ from typically developing children in closing phase kinematics of the glottal cycle, whereas the opening phase kinematics are similar. Higher speed quotients and similar opening phase velocities suggest greater relative forces are acting on vocal fold in the closing phase. These findings suggest that future large-scale studies should focus on spatial and temporal features related to the closing phase of the glottal cycle for differentiating the kinematics of children with and without vocal fold nodules.     

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.     

Hyaluronic acid-based microgels and microgel networks for vocal fold regeneration

Vocal fold scarring disrupts the viscoelastic properties of the lamina propria that are critical for normal phonation. There is a clinical need for the development of advanced biomaterials that approximate the mechanical properties of the lamina propria for in vivo vocal fold regeneration. We have developed hyaluronic acid (HA)-based microgels and cross-linked microgel networks with tunable degradation and mechanical properties. HA microgels were prepared by cross-linking HA derivatives carrying hydrazide (HAADH) and aldehyde (HAALD) functionalities within the inverse emulsion droplets. Alternatively, poly(ethylene glycol) dialdehyde (PEGDiALD) was employed in place of HAALD. Microgels based on HAADH/HAALD are more resistant to enzymatic degradation than those generated from HAADH/PEGDiALD. In vitro cytotoxicity studies using vocal fold fibroblasts indicate that microgels synthesized from HAADH/HAALD are essentially nontoxic, whereas microgels derived from HAADH/PEGDiALD exhibit certain adverse effects on the cultured cells at high concentration (> or =2 mg/mL). These microgels exhibit residual functional groups that can be used as reactive handles for covalent conjugation of therapeutic molecules. The presence of residual functional groups also allows for subsequent cross-linking of the microgels with other reactive polymers, giving rise to doubly cross-linked networks (DXNs) with tunable viscoelasticity. Mechanical measurements using a torsional wave apparatus indicate that HA-based DXNs exhibit elastic moduli that are similar to those of vocal fold lamina propria at frequencies close to the range of human phonation. These HA-based microgel systems are promising candidates for the treatment of vocal fold scarring, not just as biocompatible filler materials, but as smart entities that can repair focal defects, smooth the vocal fold margin, and potentially soften and dissolve scar tissue.     

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.     

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.     

Vocal fold tissue failure: preliminary data and constitutive modeling

In human voice production (phonation), linear small-amplitude vocal fold oscillation occurs only under restricted conditions. Physiologically, phonation more often involves large-amplitude oscillation associated with tissue stresses and strains beyond their linear viscoelastic limits, particularly in the lamina propria extracellular matrix (ECM). This study reports some preliminary measurements of tissue deformation and failure response of the vocal fold ECM under large-strain shear The primary goal was to formulate and test a novel constitutive model for vocal fold tissue failure, based on a standard-linear cohesive-zone (SL-CZ) approach. Tissue specimens of the sheep vocal fold mucosa were subjected to torsional deformation in vitro, at constant strain rates corresponding to twist rates of 0.01, 0.1, and 1.0 rad/s. The vocal fold ECM demonstrated nonlinear stress-strain and rate-dependent failure response with a failure strain as low as 0.40 rad. A finite-element implementation of the SL-CZ model was capable of capturing the rate dependence in these preliminary data, demonstrating the model's potential for describing tissue failure. Further studies with additional tissue specimens and model improvements are needed to better understand vocal fold tissue failure.     

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.     

A Cervid Vocal Fold Model Suggests Greater Glottal Efficiency in Calling at High Frequencies

Male Rocky Mountain elk (Cervus elaphus nelsoni) produce loud and high fundamental frequency bugles during the mating season, in contrast to the male European Red Deer (Cervus elaphus scoticus) who produces loud and low fundamental frequency roaring calls. A critical step in understanding vocal communication is to relate sound complexity to anatomy and physiology in a causal manner. Experimentation at the sound source, often difficult in vivo in mammals, is simulated here by a finite element model of the larynx and a wave propagation model of the vocal tract, both based on the morphology and biomechanics of the elk. The model can produce a wide range of fundamental frequencies. Low fundamental frequencies require low vocal fold strain, but large lung pressure and large glottal flow if sound intensity level is to exceed 70 dB at 10 m distance. A high-frequency bugle requires both large muscular effort (to strain the vocal ligament) and high lung pressure (to overcome phonation threshold pressure), but at least 10 dB more intensity level can be achieved. Glottal efficiency, the ration of radiated sound power to aerodynamic power at the glottis, is higher in elk, suggesting an advantage of high-pitched signaling. This advantage is based on two aspects; first, the lower airflow required for aerodynamic power and, second, an acoustic radiation advantage at higher frequencies. Both signal types are used by the respective males during the mating season and probably serve as honest signals. The two signal types relate differently to physical qualities of the sender. The low-frequency sound (Red Deer call) relates to overall body size via a strong relationship between acoustic parameters and the size of vocal organs and body size. The high-frequency bugle may signal muscular strength and endurance, via a ‘vocalizing at the edge’ mechanism, for which efficiency is critical.     

Mechanisms of Sound Production by Echolocating Bats

The echolocative pulses emitted at high repetition rates bybats pose a number of questions regarding the mechanism of theirproduction. We have investigated some physiologic parametersof vocalization in the North American Vespertilionid bat Eptesicusfuscus, including pulse insertion within the respiratory cycleand subglottic pressure changes accompanying these intense sounds.Subglottic pressures are considerably higher than those characteristicof human speech and, therefore, require a substantial structureto act as a glottal stop. We suggest that this is the role ofthe vocal fold and that the thin, paired vocal and ventricularmembranes are the ultrasonic generators. The cricothyroid muscle,which is thought to influence sound frequency by altering thetension on these membranes, contracts just prior to each ultrasonicvocalization and relaxes during phonation. Cricothyroid musclerelaxation may gradually decrease the tension on the membranesand create the downward frequency sweep characteristic of mostpulses.     

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.     

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.     

Regulation of vocal fold transepithelial water fluxes.

Vocal fold hydration is critical to phonation. We hypothesized that the vocal fold generates bidirectional water fluxes, which are regulated by activity of the Na(+)-K(+)- ATPase. Western blots and immunohistochemistry demonstrated the presence of the alpha-subunit Na(+)-K(+)-ATPase in the canine vocal fold (n = 11). Luminal cells, basal and adjacent one to two layers of suprabasal cells within stratified squamous epithelium, were immunopositive, as well as basolateral membranes of submucosal seromucous glands underlying transitional epithelia. Canine (n = 6) and ovine (n = 14) vocal fold mucosae exhibited transepithelial potential differences of 8.1 +/- 2.8 and 9.3 +/- 1.3 mV (lumen negative), respectively. The potential difference and short-circuit current (ovine = 31 +/- 4 microA/cm(2); canine = 41 +/- 10 microA/cm(2)) were substantially reduced by luminal administration of 75 microM acetylstrophanthidin (P < 0.05). Ovine (n = 7) transepithelial water fluxes decreased from 5.1 +/- 0.3 to 4.3 +/- 0.3 microl x min(-1) x cm(-2) from the basal to luminal chamber and from 5.2 +/- 0.2 to 3.9 +/- 0.3 microl x min(-1) x cm(-2) from the luminal to basal chamber by luminal acetylstrophanthidin (P < 0.05). The presence of the Na(+)-K(+)-ATPase in the vocal fold epithelium and the electrolyte transport derived from its activity provide the intrinsic mechanisms to regulate cell volume as well as vocal fold hydration.