Properties Of Vocal Fold Tissue Research Articles

Preliminary investigation of a novel technique for the quantification of the ex vivo biomechanical properties of the vocal folds

The human vocal fold is a complex structure made up of distinct layers that vary in cellular and extracellular matrix composition. Elucidating the mechanical properties of vocal fold tissues is critical for the study of both acoustics and biomechanics of voice production, and essential in the context of vocal fold injury and repair. Both quasistatic and dynamic behavior in the 10–300Hz range was explored in this preliminary investigation. The resultant properties of the lamina propria were compared to that of the nearby thyroarytenoid muscle. Er, quantified via quasistatic testing of the lamina propria, was 609±138MPa and 758±142MPa in the muscle (p=0.001). E′ of the lamina propria as determined by dynamic testing was 790±526MPa compared to 1061±928MPa in the muscle. Differences in E′ did not achieve statistical significance via linear mixed effect modeling between the tissue types (p=0.95). In addition, frequency dependence was not significant (p=0.18).

Formulation and characterization of a porous, elastomeric biomaterial for vocal fold tissue engineering research.

Biomaterials able to mimic the mechanical properties of vocal fold tissue may be particularly useful for furnishing a 3-dimensional microenvironment allowing for in vitro investigation of cell and molecular responses to vibration. Motivated by the dearth of biomaterials available for use in an in vitro model for vocal fold tissue, we investigated polyether polyurethane (PEU) matrices, which are porous, mechanically tunable biomaterials that are inexpensive and require only standard laboratory equipment for fabrication. Rheology, dynamic mechanical analysis, and scanning electron microscopy were performed on PEU matrices at 5%, 10%, and 20% w/v mass concentrations. For 5%, 10%, and 20% w/v concentrations, shear storage moduli were 2 kPa, 3.4 kPa, and 6 kPa, respectively, with shear loss moduli being 0.2 kPa, 0.38 kPa, and 0.62 kPa, respectively. Storage moduli responded to applied frequency as a linear function. Mercury intrusion porosimetry revealed that all 3 mass concentrations of PEU have a similar overall percentage porosity but differ in pore architecture. Twenty-µm diameter pores are ideal for cell seeding, and a range of mechanical properties indicates that the lower [corrected] mass concentration PEU formulations are best suited for mimicking the viscoelastic properties of vocal fold tissue for in vitro research.

High-Frequency Viscoelastic Shear Properties of Vocal Fold Tissues: Implications for Vocal Fold Tissue Engineering

The biomechanical function of the vocal folds (VFs) depends on their viscoelastic properties. Many conditions can lead to VF scarring that compromises voice function and quality. To identify candidate replacement materials, the structure, composition, and mechanical properties of native tissues need to be understood at phonation frequencies. Previously, the authors developed the torsional wave experiment (TWE), a stress-wave-based experiment to determine the linear viscoelastic shear properties of small, soft samples. Here, the viscoelastic properties of porcine and human VFs were measured over a frequency range of 10-200 Hz. The TWE utilizes resonance phenomena to determine viscoelastic properties; therefore, the specimen test frequency is determined by the sample size and material properties. Viscoelastic moduli are reported at resonance frequencies. Structure and composition of the tissues were determined by histology and immunochemistry. Porcine data from the TWE are separated into two groups: a young group, consisting of fetal and newborn pigs, and an adult group, consisting of 6-9-month olds and 2+-year olds. Adult tissues had an average storage modulus of 2309±1394 Pa and a loss tangent of 0.38±0.10 at frequencies of 36-200 Hz. The VFs of young pigs were significantly more compliant, with a storage modulus of 394±142 Pa and a loss tangent of 0.40±0.14 between 14 and 30 Hz. No gender dependence was observed. Histological staining showed that adult porcine tissues had a more organized, layered structure than the fetal tissues, with a thicker epithelium and a more structured lamina propria. Elastin fibers in fetal VF tissues were immature compared to those in adult tissues. Together, these structural changes in the tissues most likely contributed to the change in viscoelastic properties. Adult human VF tissues, recovered postmortem from adult patients with a history of smoking or disease, had an average storage modulus of 756±439 Pa and a loss tangent of 0.42±0.10. Contrary to the results of some other investigators, no significant frequency dependence was observed. This lack of observable frequency dependence may be due to the modest frequency range of the experiments and the wide range of stiffnesses observed within nominally similar sample types.

Effects of Dehydration on the Viscoelastic Properties of Vocal Folds in Large Deformations

Dehydration may alter vocal fold viscoelastic properties, thereby hampering phonation. The effects of water loss induced by an osmotic pressure potential on vocal fold tissue viscoelastic properties were investigated. Porcine vocal folds were dehydrated by immersion in a hypertonic solution, and quasi-static and low-frequency dynamic traction tests were performed for elongations of up to 50%. Digital image correlation was used to determine local strains from surface deformations. The elastic modulus and the loss factor were then determined for normal and dehydrated tissues. An eight-chain hyperelastic model was used to describe the observed nonlinear stress-stretch behavior. Contrary to the expectations, the mass history indicated that the tissue absorbed water during cyclic extension when submerged in a hypertonic solution. During loading history, the elastic modulus was increased for dehydrated tissues as a function of strain. The response of dehydrated tissues was much less affected when the load was released. This observation suggests that hydration should be considered in micromechanical models of the vocal folds. The internal hysteresis, which is often linked to phonation effort, increased significantly with water loss. The effects of dehydration on the viscoelastic properties of vocal fold tissue were quantified in a systematic way. A better understanding of the role of hydration on the mechanical properties of vocal fold tissue may help to establish objective dehydration and phonotrauma criteria.

Stress relaxation in human true and false vocal folds.

Stress relaxation behavior of the human true and false vocal folds were measured in vitro and modeled for viscoelastic parameter determination. Vibrations of the vocal folds are greatly influenced by the elasticity and viscosity of its mucosal layer. When there is a change in the viscoelastic properties of vocal fold tissue due to disease or trauma such as scarring, voice change may occur. Voice simulation models are in need of viscoelastic properties of human vocal folds for more reliable results. Due to the shortage of human data, these properties have been extrapolated from animal data such as canine vocal fold tissues. In this study, samples are made from human larynges acquired from NDRI, which were snap frozen 24–48 h post mortem and stored in −82 °F. Samples were made from medial portion of true and false vocal folds without any muscle, with a piece cartilage at each end for mounting. Stepwise elongation of 20 and 30% stretch was applied the force response was measured with an ergometer and modeled with a quasi‐linear viscoelastic model. The preliminary results suggest the strong relaxation behavior in both true and false vocal folds are indicative of abundant collagen material. [Work supported by NIDCD Grant # DC00956.]

Biomechanical modeling of the three-dimensional aspects of human vocal fold dynamics

Human voice originates from the three-dimensional (3D) oscillations of the vocal folds. In previous studies, biomechanical properties of vocal fold tissues have been predicted by optimizing the parameters of simple two-mass-models to fit its dynamics to the high-speed imaging data from the clinic. However, only lateral and longitudinal displacements of the vocal folds were considered. To extend previous studies, a 3D mass-spring, cover-model is developed, which predicts the 3D vibrations of the entire medial surface of the vocal fold. The model consists of five mass planes arranged in vertical direction. Each plane contains five longitudinal, mass-spring, coupled oscillators. Feasibility of the model is assessed using a large body of dynamical data previously obtained from excised human larynx experiments, in vivo canine larynx experiments, physical models, and numerical models. Typical model output was found to be similar to existing findings. The resulting model enables visualization of the 3D dynamics of the human vocal folds during phonation for both symmetric and asymmetric vibrations.

Nonlinear viscoelastic response of the vocal fold lamina propria under large‐amplitude oscillatory shear.

Viscoelastic properties of the vocal fold lamina propria have been reasonably quantified beyond the linear range for describing tissue behavior under uniaxial stretch or tensile deformation, but the same cannot be said for oscillatory shear deformation, which is more relevant for understanding the mechanics of large‐amplitude vocal fold vibration. Previous studies reporting the viscoelastic shear properties of vocal fold tissues have been limited to characterization of the small‐amplitude viscoelastic response in the linear viscoelastic region, partly due to the fact that derivation of the elastic and viscous shear moduli is based on the assumption of linearity, and also because of the lack of a coherent framework for describing any nonlinearities beyond the linear viscoelastic region. Based on a recently proposed rheological framework for quantifying such nonlinearities, this study examined the viscoelastic response of human vocal fold cover specimens subjected to large‐amplitude oscillatory shear, up to a shear strain amplitude of around 1.0. Results indicated that the linear viscoelastic moduli (G′ and G″) cannot adequately describe the tissue response under large‐strain shear deformation, whereas geometrical interpretations of Lissajous–Bowditch curves could unveil nonlinearities that are obscured by the use of G′ and G″, such as the phenomenon of strain stiffening.

Modeling of the transient responses of the vocal fold lamina propria

The human voice is produced by flow-induced self-sustained oscillation of the vocal fold lamina propria. The mechanical properties of vocal fold tissues are important for understanding phonation, including the time-dependent and transient changes in fundamental frequency ( F 0 ) . Cyclic uniaxial tensile tests were conducted on a group of specimens of the vocal fold lamina propria, including the superficial layer (vocal fold cover) (5 male, 5 female) and the deeper layers (vocal ligament) (6 male, 6 female). Results showed that the vocal fold lamina propria, like many other soft tissues, exhibits both elastic and viscous behavior. Specifically, the transient mechanical responses of cyclic stress relaxation and creep were observed. A three-network constitutive model composed of a hyperelastic equilibrium network in parallel with two viscoplastic time-dependent networks proves effective in characterizing the cyclic stress relaxation and creep behavior. For male vocal folds at a stretch of 1.4, significantly higher peak stress was found in the vocal ligament than in the vocal fold cover. Also, the male vocal ligament was significantly stiffer than the female vocal ligament. Our findings may help explain the mechanisms of some widely observed transient phenomena in F 0 regulation during phonation, such as the global declination in F 0 during the production of declarative sentences, and local F 0 changes such as overshoot and undershoot.

High Frequency Measurements of Viscoelastic Properties of Hydrogels for Vocal Fold Regeneration

This report describes a torsional wave experiment used to measure the viscoelastic properties of vocal fold tissues and soft materials over the range of phonation frequencies. A thin cylindrical sample is mounted between two hexagonal plates. The assembly is enclosed in an environmental chamber to maintain the temperature and relative humidity at in vivo conditions. The bottom plate is subjected to small oscillations by means of a galvanometer driven by a frequency generator that steps through a sequence of frequencies. At each frequency, measured rotations of the top and bottom plates are used to determine the ratio of the amplitudes of the rotations of the two plates. Comparisons of the frequency dependence of this ratio with that predicted for torsional waves in a linear viscoelastic material allows the storage modulus and the loss angle, in shear, to be calculated by a best-fit procedure. Experimental results are presented for hydrogels that are being examined as potential materials for vocal fold regeneration.

Dependence of phonation threshold pressure on vocal tract acoustics and vocal fold tissue mechanics

Analytical and computer simulation studies have shown that the acoustic impedance of the vocal tract as well as the viscoelastic properties of vocal fold tissues are critical for determining the dynamics and the energy transfer mechanism of vocal fold oscillation. In the present study, a linear, small-amplitude oscillation theory was revised by taking into account the propagation of a mucosal wave and the inertive reactance (inertance) of the supraglottal vocal tract as the major energy transfer mechanisms for flow-induced self-oscillation of the vocal fold. Specifically, analytical results predicted that phonation threshold pressure (Pth) increases with the viscous shear properties of the vocal fold, but decreases with vocal tract inertance. This theory was empirically tested using a physical model of the larynx, where biological materials (fat, hyaluronic acid, and fibronectin) were implanted into the vocal fold cover to investigate the effect of vocal fold tissue viscoelasticity on Pth. A uniform-tube supraglottal vocal tract was also introduced to examine the effect of vocal tract inertance on Pth. Results showed that Pth decreased with the inertive impedance of the vocal tract and increased with the viscous shear modulus (G") or dynamic viscosity (eta') of the vocal fold cover, consistent with theoretical predictions. These findings supported the potential biomechanical benefits of hyaluronic acid as a surgical bioimplant for repairing voice disorders involving the superficial layer of the lamina propria, such as scarring, sulcus vocalis, atrophy, and Reinke's edema.

Viscoelastic properties of the false vocal fold

The biomechanical properties of vocal fold tissues have been the focus of many previous studies, as vocal fold viscoelasticity critically dictates the acoustics and biomechanics of phonation. However, not much is known about the viscoelastic response of the ventricular fold or false vocal fold. It has been shown both clinically and in computer simulations that the false vocal fold may contribute significantly to the aerodynamics and sound generation processes of human voice production, with or without flow-induced oscillation of the false fold. To better understand the potential role of the false fold in phonation, this paper reports some preliminary measurements on the linear and nonlinear viscoelastic behavior of false vocal fold tissues. Linear viscoelastic shear properties of human false fold tissue samples were measured by a high-frequency controlled-strain rheometer as a function of frequency, and passive uniaxial tensile stress-strain response of the tissue samples was measured by a muscle lever system as a function of strain and loading rate. Elastic moduli (Young’s modulus and shear modulus) of the false fold tissues were calculated from the measured data. [Work supported by NIH.]

Biomechanical Effects of Hydration in Vocal Fold Tissues

It has often been hypothesized, with little empirical support, that vocal fold hydration affects voice production by mediating changes in vocal fold tissue rheology. To test this hypothesis, we attempted in this study to quantify the effects of hydration on the viscoelastic shear properties of vocal fold tissues in vitro. Osmotic changes in hydration (dehydration and rehydration) of 5 excised canine larynges were induced by sequential incubation of the tissues in isotonic, hypertonic, and hypotonic solutions. Elastic shear modulus (G'), dynamic viscosity eta' and the damping ratio zeta of the vocal fold mucosa (lamina propria) were measured as a function of frequency (0.01 to 15 Hz) with a torsional rheometer. Vocal fold tissue stiffness (G') and viscosity (eta) increased significantly (by 4 to 7 times) with the osmotically induced dehydration, whereas they decreased by 22% to 38% on the induced rehydration. Damping ratio (zeta) also increased with dehydration and decreased with rehydration, but the detected differences were not statistically significant at all frequencies. These findings support the long-standing hypothesis that hydration affects vocal fold vibration by altering tissue rheologic (or viscoelastic) properties. Our results demonstrated the biomechanical importance of hydration in vocal fold tissues and suggested that hydration approaches may potentially improve the biomechanics of phonation in vocal fold lesions involving disordered fluid balance.

Effect of hydration on the dynamic mechanical properties of vocal fold tissues

Previous research has shown that vocal fold hydration level is an important physiological variable affecting vocal fold vibration and vocal health. For instance, unbalanced laryngeal water transport has been associated with benign vocal fold lesions such as nodules and polyps, while hydration treatment is frequently used as a preventive or therapeutic strategy for the management of a variety of voice disorders. The theoretical basis for the role of hydration in voice production is that it may affect phonation threshold pressure or the ease of phonation, presumably by changing the viscous properties of vocal fold tissues. However, there is little empirical evidence showing how hydration might actually affect the biomechanical properties of vocal fold tissues. This study attempted to assess the effect of hydration on the viscoelastic shear properties of canine vocal fold mucosa. Osmotic changes in the hydration level of excised canine larynges were induced by incubation in hypertonic, isotonic, and hypotonic solutions. Viscoelastic shear properties of vocal fold mucosal tissues were measured with a rheometer. Significant changes in both issue elasticity and viscosity were observed with the induced changes in hydration. These findings supported the hypothesis that hydration affects voice production by altering tissue viscoelastic properties.

Using time-temperature superposition to estimate the elastic shear modulus and dynamic viscosity of human vocal fold tissues at frequencies of phonation

The principle of time-temperature superposition [J. D. Ferry, Viscoelastic Properties of Polymers (Wiley, New York, 1980), pp. 264–320] has been widely used by rheologists to estimate the viscoelastic properties of polymeric materials at time or frequency scales not readily accessible experimentally. This principle is based on the observation that for many polymeric systems molecular configurational changes that occur in a given time scale at a low temperature correspond to those that occur in a shorter time scale at a higher temperature. Thus the viscoelastic properties of these systems empirically measured at various temperatures at a certain frequency are indicative of measurements at different frequencies at a single reference temperature. Using a rotational rheometer, the elastic shear modulus and dynamic viscosity of human vocal fold mucosal tissues were measured at relatively low temperatures (5–25<th>°C) at 0.01–15 Hz. Data were empirically shifted based on this principle to yield a composite ‘‘master curve’’ which gives a prediction of the shear properties at higher frequencies at 37<th>°C. Results showed that the time-temperature superposition principle may be used to estimate the viscoelastic shear properties of vocal fold tissues at frequencies of vocal fold vibration, on the order of 100 Hz. [Work supported by NIH Grant No. P60 DC00976.]

Dynamic shear modulus of vocal-fold tissues and phonosurgical biomaterials

Knowledge of the mechanical properties of vocal fold tissues is necessary for the constitutive modeling of vocal-fold mechanics. Assuming transverse isotropy in a linear, elastic continuum model of vocal-fold tissues, five material constants are needed to solve the constitutive equation [D. A. Berry and I. R. Titze, J. Acoust. Soc. Am. 100, 3345–3354 (1996)]. Among these constants, Poisson’s ratios can be estimated by assuming tissue incompressibility, while the shear moduli and Young’s moduli are related to one another. A parallel-plate rotational rheometer was used to examine the dynamic shear behavior of human vocal fold tissues and three commonly used phonosurgical biomaterials (bovine collagen suspension, absorbable gelatin suspension, and human subcutaneous fat). In oscillation at 0.1–10 Hz and at 37 °C, the magnitude of dynamic shear modulus of vocal fold mucosa was on the order of 100 Pa, close to that of fat. The shear modulus magnitudes of collagen and gelatin were an order of magnitude higher. These results suggest that the use of fat for vocal-fold augmentation surgery is more conducive to phonation, because of its similarity to the vocal-fold mucosa in shear stiffness. [Work supported by NIH.]

Development of an in vitro technique for measuring elastic properties of vocal fold tissue.

The purpose of this investigation was to develop an in vitro technique for measurement of elastic properties of isolated vocal fold tissue. Larynges were excised from anesthetized dogs, immediately submerged in a Krebs-Ringer solution, and aerated with 95% oxygen and 5% carbon dioxide. The tissue was maintained in the aerated electrolyte solution throughout the experiment. The vocalis muscle was carefully dissected, with attachments to the arytenoid and thyroid cartilages maintained. The preparation was then subjected to isometric (constant length) force-elongation measures, which were converted to stress-strain values. Stage I of the investigation identified the dog model with the least intrastudy variability relative to the age, sex, and breed of the research animal, as well as to the effects of curare. Stage II investigated the effects of different instrumentation, definitions of reference length, methods of elongation, and effects of electrical stimulation. Once the procedure had been refined, the effects of age and sex were retested. There was a significant interaction between sex and strain and between age and strain. The least variability was obtained with curarized tissue from one sex of young, mixed-breed dogs, where an arbitrary 1 gram of initial force was the criterion for establishing "zero" strain. Problems associated with determination of reference length and various approaches to this problem are discussed.

Discussions on the application of ultrasound (in the textile industry): Textil 'naia prom., 23, No. 1, p. 69 (1963)

The human vocal folds are complex structures made up of distinct layers that vary in cellular and extracellular composition. The mechanical properties of vocal fold tissue are fundamental to the study of both the acoustics and biomechanics of voice production. To date, quantitative methods have been applied to characterize the vocal fold tissue in both normal and pathologic conditions. This review describes, summarizes, and discusses the most commonly employed methods for vocal fold biomechanical testing. Force–elongation, torsional parallel plate rheometry, simple-shear parallel plate rheometry, linear skin rheometry, and indentation are the most frequently employed biomechanical tests for vocal fold tissues and each provide material properties data that can be used to compare native tissue to diseased or treated tissue. Force–elongation testing is clinically useful, as it allows for functional unit testing, while rheometry provides physiologically relevant shear data, and nanoindentation permits micrometer scale testing across different areas of the vocal fold as well as whole organ testing. Thoughtful selection of the testing technique during experimental design to evaluate a hypothesis is critical to optimize biomechanical testing of vocal fold tissues.